FAD

cross_nutrient

B2-derived cofactor bridges NADPH and the glutathione system.

evidence_location

Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes

experimental_model

Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls.

exposure

Purified-enzyme assay

limitations

Mechanism integrates natural-substrate structures with prior kinetic work; transient intermediates were not all directly trapped here.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

FAD relays electrons through enzyme cysteines.

primary_references

[berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

tissue_or_cell_type

Purified human GSR

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1318–1330

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. · source_derived_draft · unverified_draft

### b2-gsr-flavin-disulfide-relay The GSR catalytic cycle transfers reducing equivalents from flavin to the Cys58-Cys63 active-site disulfide before glutathione-disulfide reduction. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: FAD relays electrons through enzyme cysteines. organism: Homo sapiens tissue_or_cell_type: Purified human GSR experimental_model: Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. limitations: Mechanism integrates natural-substrate structures with prior kinetic work; transient intermediates were not all directly trapped here. exposure: Purified-enzyme assay cross_nutrient: B2-derived cofactor bridges NADPH and the glutathione system. evidence_location: Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes [berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}]

experimental_model

Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays.

exposure

Direct FAD addition; independent MCAD activity and immunoblot readouts.

limitations

Activity and abundance are related observations, not proof that protein loss is the only cause of incomplete rescue.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

For MCAD, supplying cofactor alone could not promptly restore the missing enzyme capacity.

primary_references

[peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

tissue_or_cell_type

Skeletal-muscle homogenates

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 944–955

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. · source_derived_draft · unverified_draft

### b2-met-mouse-mcad-rescue-limit FAD added to muscle assays only mildly improved depressed MCAD activity in Slc25a32 mutant mice; MCAD protein abundance was also lower in K235R homozygotes. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: For MCAD, supplying cofactor alone could not promptly restore the missing enzyme capacity. organism: Mus musculus tissue_or_cell_type: Skeletal-muscle homogenates experimental_model: Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. limitations: Activity and abundance are related observations, not proof that protein loss is the only cause of incomplete rescue. exposure: Direct FAD addition; independent MCAD activity and immunoblot readouts. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}] [peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

Flavin supply supports a distinct branched-chain amino-acid oxidation step.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}]

experimental_model

Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays.

exposure

FAD added directly to separate acyl-CoA dehydrogenase assays.

limitations

In vitro rescue differs from oral riboflavin response; the most pronounced enzyme defects occurred in K235R homozygotes.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

One amino-acid oxidation enzyme retained activity that could be restored by adding its cofactor.

primary_references

[peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

tissue_or_cell_type

Skeletal-muscle homogenates

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 930–942

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. · source_derived_draft · unverified_draft

### b2-met-mouse-sbcad-fad-rescue Adding FAD directly to muscle enzyme assays fully restored the depressed SBCAD activity in the Slc25a32 mutant-mouse experiments. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: One amino-acid oxidation enzyme retained activity that could be restored by adding its cofactor. organism: Mus musculus tissue_or_cell_type: Skeletal-muscle homogenates experimental_model: Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. limitations: In vitro rescue differs from oral riboflavin response; the most pronounced enzyme defects occurred in K235R homozygotes. exposure: FAD added directly to separate acyl-CoA dehydrogenase assays. cross_nutrient: Flavin supply supports a distinct branched-chain amino-acid oxidation step. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}] [peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}]

experimental_model

Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays.

exposure

Direct FAD addition; VLCAD measured separately from MCAD.

limitations

This does not establish the response of genetic ACADVL disease to riboflavin.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

The long-chain oxidation enzyme also had limitations beyond immediately available free cofactor.

primary_references

[peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

tissue_or_cell_type

Skeletal-muscle homogenates

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 957–968

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. · source_derived_draft · unverified_draft

### b2-met-mouse-vlcad-rescue-limit FAD added to muscle assays only mildly improved depressed VLCAD activity in Slc25a32 mutant mice; VLCAD abundance was lower in K235R homozygotes. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The long-chain oxidation enzyme also had limitations beyond immediately available free cofactor. organism: Mus musculus tissue_or_cell_type: Skeletal-muscle homogenates experimental_model: Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. limitations: This does not establish the response of genetic ACADVL disease to riboflavin. exposure: Direct FAD addition; VLCAD measured separately from MCAD. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 50, "char_start": 0, "char_end": 1147, "evidence_access": "full-text"}] [peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

cross_nutrient

B2-FAD supports a folate-processing enzyme.

evidence_location

Results: kinetics, FAD occupancy and SAM inhibition; Table 1; Figs 2-4, 6

experimental_model

Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays.

exposure

Purified-enzyme assay

limitations

Biochemical evidence does not establish a dietary threshold or supplementation benefit.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

MTHFR carries a B2-derived FAD cofactor.

primary_references

[froese2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition. (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 996–1008

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays. · source_derived_draft · unverified_draft

### b2-mthfr-fad-occupancy Native mass spectrometry and crystallography identified FAD bound to recombinant human MTHFR catalytic domains. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: MTHFR carries a B2-derived FAD cofactor. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays. limitations: Biochemical evidence does not establish a dietary threshold or supplementation benefit. exposure: Purified-enzyme assay cross_nutrient: B2-FAD supports a folate-processing enzyme. evidence_location: Results: kinetics, FAD occupancy and SAM inhibition; Table 1; Figs 2-4, 6 [froese2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition. (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

cross_nutrient

B2-FAD and nicotinamide cofactors provide the upstream side of a selenium-containing reductase.

evidence_location

Abstract

experimental_model

Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation.

exposure

Purified-enzyme assay

limitations

Structure used a Sec-to-Cys mutant and oxidized NADP+; direct atom-level conclusions about wild-type selenium chemistry are limited.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rattus norvegicus protein expressed recombinantly

plain_language

Thioredoxin reductase has a flavin input side.

primary_references

[sandalova2001] Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. (2001). https://pubmed.ncbi.nlm.nih.gov/11481439/ DOI: 10.1073/pnas.171178698

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1346–1358

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation. · source_derived_draft · unverified_draft

### b2-txnrd1-fad-nadph-architecture The rat TXNRD1 Sec498Cys structure showed conserved FAD- and NADPH-binding architecture around the N-terminal redox center. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Thioredoxin reductase has a flavin input side. organism: Rattus norvegicus protein expressed recombinantly tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation. limitations: Structure used a Sec-to-Cys mutant and oxidized NADP+; direct atom-level conclusions about wild-type selenium chemistry are limited. exposure: Purified-enzyme assay cross_nutrient: B2-FAD and nicotinamide cofactors provide the upstream side of a selenium-containing reductase. evidence_location: Abstract [sandalova2001] Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. (2001). https://pubmed.ncbi.nlm.nih.gov/11481439/ DOI: 10.1073/pnas.171178698

evidence_location

Abstract; intestinal fractionation and EDTA competition experiments

experimental_model

Rat mucosal fractionation, hydrolase assays and radiolabeled-riboflavin uptake in everted intestinal rings

exposure

Flavin coenzymes with or without EDTA during tracer uptake.

limitations

Rat ex vivo evidence; molecular identities of the hydrolases were not established.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rattus norvegicus

plain_language

Dietary FAD can supply absorbable riboflavin after intestinal cleavage.

primary_references

[transport-diet-1982] FMN phosphatase and FAD pyrophosphatase in rat intestinal brush borders: role in intestinal absorption of dietary riboflavin. (1982). https://doi.org/10.1093/jn/112.2.263 DOI: 10.1093/jn/112.2.263

tissue_or_cell_type

Small-intestinal mucosa

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 124–135

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat mucosal fractionation, hydrolase assays and radiolabeled-riboflavin uptake in everted intestinal rings · source_derived_draft · unverified_draft

### transport-dietary-fad-hydrolysis Rat brush-border preparations released free riboflavin from FAD; inhibiting coenzyme hydrolysis reduced its competition with tracer riboflavin uptake. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Dietary FAD can supply absorbable riboflavin after intestinal cleavage. organism: Rattus norvegicus tissue_or_cell_type: Small-intestinal mucosa experimental_model: Rat mucosal fractionation, hydrolase assays and radiolabeled-riboflavin uptake in everted intestinal rings limitations: Rat ex vivo evidence; molecular identities of the hydrolases were not established. exposure: Flavin coenzymes with or without EDTA during tracer uptake. evidence_location: Abstract; intestinal fractionation and EDTA competition experiments [transport-diet-1982] FMN phosphatase and FAD pyrophosphatase in rat intestinal brush borders: role in intestinal absorption of dietary riboflavin. (1982). https://doi.org/10.1093/jn/112.2.263 DOI: 10.1093/jn/112.2.263

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Figure 5

experimental_model

FAD-saturated purified FADS2 variants

exposure

Molar-excess FAD reconstitution followed by catalytic assays.

limitations

The two variants differ in binding/stability responses; the result concerns maximal in vitro activity.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens protein

plain_language

Stabilizing a variant does not necessarily restore normal enzyme output.

primary_references

[transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

tissue_or_cell_type

Purified protein

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 412–423

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · FAD-saturated purified FADS2 variants · source_derived_draft · unverified_draft

### transport-flad1-residual-catalytic-defect FAD-saturated p.Ser495del and p.Arg530Cys FADS2 remained substantially less catalytically active than wild type. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Stabilizing a variant does not necessarily restore normal enzyme output. organism: Homo sapiens protein tissue_or_cell_type: Purified protein experimental_model: FAD-saturated purified FADS2 variants limitations: The two variants differ in binding/stability responses; the result concerns maximal in vitro activity. exposure: Molar-excess FAD reconstitution followed by catalytic assays. evidence_location: Figure 5 [transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Figure 4A-B

experimental_model

Recombinant variant FAD reconstitution and proteolysis

exposure

Molar excess FAD during in vitro reconstitution.

limitations

Direct FAD exposure in vitro; does not prove universal rescue by dietary riboflavin.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens protein

plain_language

FAD helped stabilize this particular altered enzyme.

primary_references

[transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

tissue_or_cell_type

Purified protein

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 399–410

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant variant FAD reconstitution and proteolysis · source_derived_draft · unverified_draft

### transport-flad1-s495del-stabilization Excess FAD reconstitution restored bound flavin and increased proteolytic stability of recombinant p.Ser495del FADS2. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: FAD helped stabilize this particular altered enzyme. organism: Homo sapiens protein tissue_or_cell_type: Purified protein experimental_model: Recombinant variant FAD reconstitution and proteolysis limitations: Direct FAD exposure in vitro; does not prove universal rescue by dietary riboflavin. exposure: Molar excess FAD during in vitro reconstitution. evidence_location: Figure 4A-B [transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

evidence_access

Primary full text

experimental_model

Bacterial structural enzymology; FAD-supplemented enzyme assays.

limitations

Does not show that human riboflavin intake limits or increases microbial imidazole-propionate production.

nutrient_topic

L-Histidine collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Histidine

plain_language

This microbial reaction uses a riboflavin-derived chemical tool.

primary_references

Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production. · 2021 · https://pubmed.ncbi.nlm.nih.gov/33649331/ · DOI 10.1038/s41467-021-21548-y

L-Histidine: supply, catabolism, histamine, receptors and cross-nutrient mechanisms (2026-09-19) · lines 274–280

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Bacterial structural enzymology; FAD-supplemented enzyme assays. · source_derived_draft · unverified_draft

## histidine-urda-flavin This microbial reaction uses a riboflavin-derived chemical tool. The UrdA catalytic-domain structures contained FAD alongside substrate or product, and assays supported flavin-associated urocanate reduction. Model: Bacterial structural enzymology; FAD-supplemented enzyme assays. Limitations: Does not show that human riboflavin intake limits or increases microbial imidazole-propionate production. Evidence access: Primary full text Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production. · 2021 · https://pubmed.ncbi.nlm.nih.gov/33649331/ · DOI 10.1038/s41467-021-21548-y

evidence_access

Primary indexed abstract and figure descriptions.

experimental_model

Purified human sulfide:quinone oxidoreductase; transient kinetics and flavin spectroscopy.

interpretation_status

Source-derived research curation; not independent raw-data verification.

limitations

Enzyme-bound FAD is not extracellular FMN. This study did not test dietary B2 deficiency or combined B2 and ergothioneine treatment.

nutrient_topic

Ergothioneine mitochondrial supplement; shared molecular requirements are not demonstrated dietary interactions. · L-Ergothioneine

plain_language

Sulfur disposal has a flavin-dependent electron relay.

primary_references

Mishanina et al. Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase. DOI 10.1074/jbc.M115.682369; PMID 26318450; https://pubmed.ncbi.nlm.nih.gov/26318450/

source_locator

Abstract and Figure 1

Ergothioneine: mitochondrial transport, MPST and sulfur-handling dependencies (2026-10-02) · lines 129–129

Original AI-assisted curation of five primary studies with publication identifiers, experimental locators and access limitations. Additive chapter supplement, not publisher full text. · supports · Purified human sulfide:quinone oxidoreductase; transient kinetics and flavin spectroscopy. · source_derived_draft · unverified_draft

Bound FAD cycles between reduction by sulfide and oxidation by ubiquinone during human SQOR catalysis.

evidence_access

Primary abstract

experimental_model

Purified human DASPO/DDO structural and kinetic comparison with human DAO.

limitations

Does not show that dietary riboflavin limits DDO in vivo or that DAO inhibitors also inhibit DDO.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Two similarly named enzymes handle different D-amino-acid pathways and bind their cofactor differently.

primary_references

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain. · 2020 · https://pubmed.ncbi.nlm.nih.gov/31914658/ · DOI 10.1096/fj.201901703R

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 96–102

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified human DASPO/DDO structural and kinetic comparison with human DAO. · source_derived_draft · unverified_draft

## d-aspartate-ddo-fad Two similarly named enzymes handle different D-amino-acid pathways and bind their cofactor differently. Human D-aspartate oxidase structure and kinetics showed a FAD-containing enzyme with higher FAD affinity than human D-amino-acid oxidase. Model: Purified human DASPO/DDO structural and kinetic comparison with human DAO. Limitations: Does not show that dietary riboflavin limits DDO in vivo or that DAO inhibitors also inhibit DDO. Evidence access: Primary abstract Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain. · 2020 · https://pubmed.ncbi.nlm.nih.gov/31914658/ · DOI 10.1096/fj.201901703R

evidence_access

Primary full text

experimental_model

Recombinant mouse versus human DDO assays.

limitations

This is not an observed dietary riboflavin-drug interaction in people.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Cofactor conditions altered the mouse drug-enzyme interaction.

primary_references

Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity. · 2017 · https://pubmed.ncbi.nlm.nih.gov/28393897/ · DOI 10.1038/srep46288

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 272–278

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Recombinant mouse versus human DDO assays. · source_derived_draft · unverified_draft

## d-aspartate-olanzapine-fad-species Cofactor conditions altered the mouse drug-enzyme interaction. Lowering assay FAD from 100 to 4 micromolar shifted olanzapine IC50 at mouse DDO from about 5.6 to 1.4 micromolar; the human enzyme did not show this potency shift. Model: Recombinant mouse versus human DDO assays. Limitations: This is not an observed dietary riboflavin-drug interaction in people. Evidence access: Primary full text Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity. · 2017 · https://pubmed.ncbi.nlm.nih.gov/28393897/ · DOI 10.1038/srep46288

evidence_span

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experimental_model

Purified recombinant enzyme activity and cofactor analysis

exposure

Arginine substrate; BH4, FAD, FMN, heme, iron and zinc measurements

limitations

Biochemical cofactor findings do not show that extra dietary cofactors increase NO in healthy people.

nutrient_topic

Citrulline research collection; topical membership is not evidence of a direct dietary effect. · L-Citrulline

organism

Human NOS3 expressed in yeast

plain_language

One vitamin-B2-derived flavin is part of the enzyme.

primary_references

[citrulline-p10608822] Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris. (1999). https://pubmed.ncbi.nlm.nih.gov/10608822/ DOI: 10.1074/jbc.274.53.37658

tissue_or_cell_type

Endothelial NOS enzyme preparation

Citrulline: arginine recycling, nitrogen disposal and nutrient connections (2026-09-17) · lines 528–539

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified recombinant enzyme activity and cofactor analysis · source_derived_draft · unverified_draft

### citrulline-nos-fad The purified human eNOS preparation contained approximately 0.56 FAD equivalents per subunit. Condition category: normal nutrient_topic: Citrulline research collection; topical membership is not evidence of a direct dietary effect. plain_language: One vitamin-B2-derived flavin is part of the enzyme. organism: Human NOS3 expressed in yeast tissue_or_cell_type: Endothelial NOS enzyme preparation experimental_model: Purified recombinant enzyme activity and cofactor analysis limitations: Biochemical cofactor findings do not show that extra dietary cofactors increase NO in healthy people. exposure: Arginine substrate; BH4, FAD, FMN, heme, iron and zinc measurements evidence_span: {"source_cache": "artifacts/citrulline-research/10608822.abstract.txt", "locator": "Primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "1600b193cc63f30b204285729710bb81f34bc2918a8ca7f97aa97bc4acd998fd", "start_char": 0, "end_char": 2100, "text_sha256": "1600b193cc63f30b204285729710bb81f34bc2918a8ca7f97aa97bc4acd998fd"} [citrulline-p10608822] Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris. (1999). https://pubmed.ncbi.nlm.nih.gov/10608822/ DOI: 10.1074/jbc.274.53.37658

evidence_span

{"source_cache": "artifacts/molybdenum-research/30985987.fulltext.txt", "locator": "Exact primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "d187787b0c042c674903c56275b485ee23c2b3545ed1c0ed02ca0cd2e3abb7c0", "start_char": 11532, "end_char": 12101, "text_sha256": "13ae767e495f8efc3a6c44005156563bc471d8aec37486330086d3fed343ef38"}

experimental_model

Human AOX1 variant crystallography and CD/ThermoFAD stability assays

exposure

Wild type compared with SNP variants

limitations

In-vitro structures and thermal stability; not physiological temperature or diet thresholds.

nutrient_topic

Molybdenum research collection; topical membership is not evidence of a direct dietary effect. · Molybdenum

organism

Homo sapiens protein

plain_language

The molybdenum enzyme also contains a riboflavin-derived cofactor.

primary_references

[mo-p30985987] Human aldehyde oxidase (hAOX1): structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms. (2019). https://pubmed.ncbi.nlm.nih.gov/30985987/ DOI: 10.1002/2211-5463.12617

tissue_or_cell_type

Purified AOX1

Molybdenum: cofactor assembly, sulfur metabolism and nutrient interactions (2026-09-17) · lines 859–870

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human AOX1 variant crystallography and CD/ThermoFAD stability assays · source_derived_draft · unverified_draft

### mo-aox-fad AOX1-bound FAD acted as the intrinsic fluorescence reporter in ThermoFAD unfolding assays. Condition category: normal nutrient_topic: Molybdenum research collection; topical membership is not evidence of a direct dietary effect. plain_language: The molybdenum enzyme also contains a riboflavin-derived cofactor. organism: Homo sapiens protein tissue_or_cell_type: Purified AOX1 experimental_model: Human AOX1 variant crystallography and CD/ThermoFAD stability assays limitations: In-vitro structures and thermal stability; not physiological temperature or diet thresholds. exposure: Wild type compared with SNP variants evidence_span: {"source_cache": "artifacts/molybdenum-research/30985987.fulltext.txt", "locator": "Exact primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "d187787b0c042c674903c56275b485ee23c2b3545ed1c0ed02ca0cd2e3abb7c0", "start_char": 11532, "end_char": 12101, "text_sha256": "13ae767e495f8efc3a6c44005156563bc471d8aec37486330086d3fed343ef38"} [mo-p30985987] Human aldehyde oxidase (hAOX1): structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms. (2019). https://pubmed.ncbi.nlm.nih.gov/30985987/ DOI: 10.1002/2211-5463.12617

cross_nutrient

B2-derived FAD affects folate-enzyme stability.

experimental_model

Recombinant human MTHFR; kinetics and structures.

exposure

FAD before versus after heating

limitations

Heat challenge is not physiological riboflavin deficiency.

nutrient_topic

Folate and folic acid research collection; topical membership is not evidence of a direct dietary effect. · Folate (vitamin B9)

organism

Homo sapiens

plain_language

Flavin protected the enzyme during heat stress.

primary_references

[froese-2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

tissue_or_cell_type

Purified protein

Folate and folic acid: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 447–458

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTHFR; kinetics and structures. · source_derived_draft · unverified_draft

### folate-methyl-fad-heat-protection FAD supplied before a 46 C, five-minute incubation preserved more MTHFR activity than FAD added after heating. Condition category: normal nutrient_topic: Folate and folic acid research collection; topical membership is not evidence of a direct dietary effect. plain_language: Flavin protected the enzyme during heat stress. organism: Homo sapiens tissue_or_cell_type: Purified protein experimental_model: Recombinant human MTHFR; kinetics and structures. limitations: Heat challenge is not physiological riboflavin deficiency. exposure: FAD before versus after heating cross_nutrient: B2-derived FAD affects folate-enzyme stability. [froese-2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

cross_nutrient

true

evidence_location

Primary Results, wild-type structure and cofactor-binding sites; Methods flavin extraction/HPLC.

experimental_model

Human POR structure and flavin-content analysis

exposure

Recombinant N-terminally truncated human POR; X-ray structure and HPLC flavin assays.

limitations

The structural study did not measure vitamin D outcomes after riboflavin dosing; linkage to CYP2R1 comes from the separate direct reconstitution record.

nutrient

Vitamin D2 and D3 · Vitamin D2 and D3

nutrient_topic

Vitamin D2 and D3 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin D2 and D3

organism

Homo sapiens protein

plain_language

POR carries a FAD cofactor derived from vitamin B2.

primary_references

[xia2011] Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency. (2011). https://pubmed.ncbi.nlm.nih.gov/21808038/ DOI: 10.1073/pnas.1106632108

tissue_or_cell_type

POR cofactor-binding domain

Vitamin D2 and D3: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 407–420

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human POR structure and flavin-content analysis · source_derived_draft · unverified_draft

### vd-act-por-fad Crystallography and extracted-flavin analysis identified FAD bound to recombinant human POR. Condition category: normal nutrient_topic: Vitamin D2 and D3 research collection; topical membership is not evidence of a direct dietary effect. plain_language: POR carries a FAD cofactor derived from vitamin B2. organism: Homo sapiens protein tissue_or_cell_type: POR cofactor-binding domain experimental_model: Human POR structure and flavin-content analysis limitations: The structural study did not measure vitamin D outcomes after riboflavin dosing; linkage to CYP2R1 comes from the separate direct reconstitution record. exposure: Recombinant N-terminally truncated human POR; X-ray structure and HPLC flavin assays. cross_nutrient: true evidence_location: Primary Results, wild-type structure and cofactor-binding sites; Methods flavin extraction/HPLC. nutrient: Vitamin D2 and D3 [xia2011] Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency. (2011). https://pubmed.ncbi.nlm.nih.gov/21808038/ DOI: 10.1073/pnas.1106632108

evidence_span

{"source_cache": "artifacts/coq10-research/38425362.fulltext.txt", "locator": "Primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "7d25af4ecd7340649536b8fea3b8a0a308a1611bbf8c57f6a79b362f443f1cba", "start_char": 9067, "end_char": 13813, "text_sha256": "49612ae4d3a59a75a02fe3fb132abd8aeb7ef8b7cae3b0f79b5557503c55ad51"}

experimental_model

Purified reconstructed COQ metabolon with short-chain substrates

exposure

Enzyme combinations, methyl donors, reductants and metal additions

limitations

Ancestral proteins and CoQ1 analogues; no clinical cofactor dose or proof of nutritional rate limitation. Reaction order need not be universal across species.

nutrient_topic

Coenzyme Q10 research collection; topical membership is not evidence of a direct dietary effect. · Coenzyme Q10 / CoQ10 redox system

organism

Reconstructed ancestral tetrapod proteins

plain_language

A riboflavin-derived cofactor is part of the electron-supply machinery for synthesis.

primary_references

[coq10-p38425362] In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis. (2024). https://pubmed.ncbi.nlm.nih.gov/38425362/ DOI: 10.1038/s41929-023-01087-z

tissue_or_cell_type

Stepwise CoQ head-group assembly

Coenzyme Q10: biosynthesis, electron transfer, antioxidant recycling and nutrient interactions (2026-09-17) · lines 593–604

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified reconstructed COQ metabolon with short-chain substrates · source_derived_draft · unverified_draft

### coq10-fdxr-fad Purified reconstructed FDXR retained FAD. Condition category: normal nutrient_topic: Coenzyme Q10 research collection; topical membership is not evidence of a direct dietary effect. plain_language: A riboflavin-derived cofactor is part of the electron-supply machinery for synthesis. organism: Reconstructed ancestral tetrapod proteins tissue_or_cell_type: Stepwise CoQ head-group assembly experimental_model: Purified reconstructed COQ metabolon with short-chain substrates limitations: Ancestral proteins and CoQ1 analogues; no clinical cofactor dose or proof of nutritional rate limitation. Reaction order need not be universal across species. exposure: Enzyme combinations, methyl donors, reductants and metal additions evidence_span: {"source_cache": "artifacts/coq10-research/38425362.fulltext.txt", "locator": "Primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "7d25af4ecd7340649536b8fea3b8a0a308a1611bbf8c57f6a79b362f443f1cba", "start_char": 9067, "end_char": 13813, "text_sha256": "49612ae4d3a59a75a02fe3fb132abd8aeb7ef8b7cae3b0f79b5557503c55ad51"} [coq10-p38425362] In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis. (2024). https://pubmed.ncbi.nlm.nih.gov/38425362/ DOI: 10.1038/s41929-023-01087-z

cross_nutrient

B2 activation and Mg-dependent FAD synthesis provide a biochemical partner to B1-dependent complexes; direct transfer of newly made FAD to DLD was not tested.

evidence

[{"paper_key": "brizio-2006-fad", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["abstract"], "locator": "Primary publication abstract", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}]

experimental_model

Recombinant human FLAD1 isoforms expressed in E. coli.

limitations

No tissue Mg threshold or combined B1/B2 deficiency experiment.

nutrient

Thiamine (vitamin B1) · Thiamine (vitamin B1)

nutrient_topic

Thiamine research collection; topical membership is not evidence of a direct dietary effect. · Thiamine (vitamin B1)

organism

Homo sapiens

plain_language

The flavin used by E3 must first be made from the riboflavin pathway. This distinct magnesium-dependent synthesis reaction should not be confused with magnesium binding to B1 enzymes.

primary_references

[brizio-2006-fad] Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase (2006). https://pubmed.ncbi.nlm.nih.gov/16643857/ DOI: 10.1016/j.bbrc.2006.04.003

tissue_or_cell_type

Purified/expressed proteins

Thiamine: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 745–757

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human FLAD1 isoforms expressed in E. coli. · source_derived_draft · unverified_draft

### b1-fad-b2-cofactor-synthesis Recombinant human FLAD1 isoforms 1 and 2 exhibited FAD synthetase activity; isoform 2 was purified, and activity required MgCl2. Condition category: normal nutrient_topic: Thiamine research collection; topical membership is not evidence of a direct dietary effect. plain_language: The flavin used by E3 must first be made from the riboflavin pathway. This distinct magnesium-dependent synthesis reaction should not be confused with magnesium binding to B1 enzymes. organism: Homo sapiens tissue_or_cell_type: Purified/expressed proteins experimental_model: Recombinant human FLAD1 isoforms expressed in E. coli. limitations: No tissue Mg threshold or combined B1/B2 deficiency experiment. evidence: [{"paper_key": "brizio-2006-fad", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["abstract"], "locator": "Primary publication abstract", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}] cross_nutrient: B2 activation and Mg-dependent FAD synthesis provide a biochemical partner to B1-dependent complexes; direct transfer of newly made FAD to DLD was not tested. nutrient: Thiamine (vitamin B1) [brizio-2006-fad] Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase (2006). https://pubmed.ncbi.nlm.nih.gov/16643857/ DOI: 10.1016/j.bbrc.2006.04.003

cross_nutrient

Nicotinamide-containing NADPH and B2-derived FAD perform distinct functions.

evidence_location

Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes

experimental_model

Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls.

exposure

Purified-enzyme assay

limitations

Structural support for the established catalytic cycle; radiation reduction must be distinguished from natural catalysis.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

NADPH supplies electrons to the B2-derived cofactor.

primary_references

[berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

tissue_or_cell_type

Purified human erythrocyte-type GSR crystals

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1304–1316

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. · source_derived_draft · unverified_draft

### b2-gsr-nadph-to-fad Human GSR substrate structures place NADPH for hydride transfer to bound FAD, the first redox step in glutathione recycling. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: NADPH supplies electrons to the B2-derived cofactor. organism: Homo sapiens tissue_or_cell_type: Purified human erythrocyte-type GSR crystals experimental_model: Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. limitations: Structural support for the established catalytic cycle; radiation reduction must be distinguished from natural catalysis. exposure: Purified-enzyme assay cross_nutrient: Nicotinamide-containing NADPH and B2-derived FAD perform distinct functions. evidence_location: Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes [berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 5, "char_start": 0, "char_end": 502, "evidence_access": "full-text"}, {"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 6, "char_start": 0, "char_end": 1435, "evidence_access": "full-text"}]

experimental_model

Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography.

exposure

G101del mutagenesis; FAD quantification and calibrated size exclusion.

limitations

Low expression and preparation heterogeneity were reported; no riboflavin-rescue experiment.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A different DLD alteration disrupted both flavin retention and protein assembly.

primary_references

[szabo-2023-dld-variants] Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase (2023). https://pubmed.ncbi.nlm.nih.gov/37446004/ DOI: 10.3390/ijms241310826

tissue_or_cell_type

Purified recombinant DLD

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 861–872

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography. · source_derived_draft · unverified_draft

### b2-met-dld-g101del-fad-retention Human DLD G101del retained about 0.07 FAD per monomer in the recombinant preparation; size-exclusion analysis also indicated loss of its normal dimeric assembly. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A different DLD alteration disrupted both flavin retention and protein assembly. organism: Homo sapiens tissue_or_cell_type: Purified recombinant DLD experimental_model: Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography. limitations: Low expression and preparation heterogeneity were reported; no riboflavin-rescue experiment. exposure: G101del mutagenesis; FAD quantification and calibrated size exclusion. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 5, "char_start": 0, "char_end": 502, "evidence_access": "full-text"}, {"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 6, "char_start": 0, "char_end": 1435, "evidence_access": "full-text"}] [szabo-2023-dld-variants] Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase (2023). https://pubmed.ncbi.nlm.nih.gov/37446004/ DOI: 10.3390/ijms241310826

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

DLD is reused from the B1 complexes; its flavin/NAD step is distinct from upstream thiamine diphosphate chemistry.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 19, "char_start": 0, "char_end": 871, "evidence_access": "full-text"}]

experimental_model

Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography.

exposure

I12T mutagenesis, purified-protein FAD and activity assays.

limitations

Purification instability can affect measured occupancy; correlation does not prove FAD loss alone caused all activity loss.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

This DLD variant poorly retained FAD and had very low catalytic activity.

primary_references

[szabo-2023-dld-variants] Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase (2023). https://pubmed.ncbi.nlm.nih.gov/37446004/ DOI: 10.3390/ijms241310826

tissue_or_cell_type

Recombinant DLD expressed in E. coli

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 847–859

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography. · source_derived_draft · unverified_draft

### b2-met-dld-i12t-fad-retention Purified human DLD I12T retained about 0.15 FAD per monomer versus approximately one in wild type, with forward and reverse activities of about 8% and 2%. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: This DLD variant poorly retained FAD and had very low catalytic activity. organism: Homo sapiens tissue_or_cell_type: Recombinant DLD expressed in E. coli experimental_model: Recombinant human DLD variants expressed in E. coli BL21(DE3), FAD quantification, activity, chromatography and crystallography. limitations: Purification instability can affect measured occupancy; correlation does not prove FAD loss alone caused all activity loss. exposure: I12T mutagenesis, purified-protein FAD and activity assays. cross_nutrient: DLD is reused from the B1 complexes; its flavin/NAD step is distinct from upstream thiamine diphosphate chemistry. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10341545", "locator": "XML .//body//p", "paragraph_index": 19, "char_start": 0, "char_end": 871, "evidence_access": "full-text"}] [szabo-2023-dld-variants] Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase (2023). https://pubmed.ncbi.nlm.nih.gov/37446004/ DOI: 10.3390/ijms241310826

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC26136", "locator": "HTML article p", "paragraph_index": 30, "char_start": 0, "char_end": 1354, "evidence_access": "full-text"}]

experimental_model

Recombinant human ETFA/ETFB expressed in E. coli and crystallized at 2.1 Angstrom resolution.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Static structure; not a measurement of cellular cofactor availability.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

ETF is a two-protein electron shuttle carrying one B2-derived cofactor.

primary_references

[roberts-1996-human-etf] Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution (1996). https://pubmed.ncbi.nlm.nih.gov/8962055/ DOI: 10.1073/pnas.93.25.14355

tissue_or_cell_type

Recombinant protein expressed in E. coli

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 728–739

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human ETFA/ETFB expressed in E. coli and crystallized at 2.1 Angstrom resolution. · source_derived_draft · unverified_draft

### b2-met-human-etf-fad-heterodimer Human ETF crystal structure places its single FAD in a cleft shared by ETFA and ETFB, with most FAD contacts in the alpha-chain C-terminal region. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: ETF is a two-protein electron shuttle carrying one B2-derived cofactor. organism: Homo sapiens tissue_or_cell_type: Recombinant protein expressed in E. coli experimental_model: Recombinant human ETFA/ETFB expressed in E. coli and crystallized at 2.1 Angstrom resolution. limitations: Static structure; not a measurement of cellular cofactor availability. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC26136", "locator": "HTML article p", "paragraph_index": 30, "char_start": 0, "char_end": 1354, "evidence_access": "full-text"}] [roberts-1996-human-etf] Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution (1996). https://pubmed.ncbi.nlm.nih.gov/8962055/ DOI: 10.1073/pnas.93.25.14355

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 3, "char_start": 0, "char_end": 2164, "evidence_access": "full-text"}]

experimental_model

Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Structure establishes location, not nutritional sensitivity.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The B2-derived cofactor sits in the catalytic SDHA part of complex II.

primary_references

[du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

tissue_or_cell_type

HEK293F-derived purified complex II

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 636–647

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. · source_derived_draft · unverified_draft

### b2-met-human-sdha-fad Cryo-EM of active human complex II resolved FAD within SDHA and located the succinate-fumarate active site between its FAD-binding and capping domains. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The B2-derived cofactor sits in the catalytic SDHA part of complex II. organism: Homo sapiens tissue_or_cell_type: HEK293F-derived purified complex II experimental_model: Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. limitations: Structure establishes location, not nutritional sensitivity. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 3, "char_start": 0, "char_end": 2164, "evidence_access": "full-text"}] [du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

cross_nutrient

B2-derived FAD and iron-containing cluster are distinct required molecular components; this does not establish supplement interactions.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC1637562", "locator": "HTML article p", "paragraph_index": 10, "char_start": 0, "char_end": 3701, "evidence_access": "full-text"}]

experimental_model

Purified porcine ETF-QO crystal structures with and without bound ubiquinone.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Static structure; the exact electron-entry sequence required additional functional experiments.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Sus scrofa

plain_language

ETF-QO combines flavin and iron-sulfur chemistry in one protein downstream of ETF.

primary_references

[zhang-2006-etf-qo] Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool (2006). https://pubmed.ncbi.nlm.nih.gov/17050691/ DOI: 10.1073/pnas.0604567103

tissue_or_cell_type

Purified porcine mitochondrial ETF-QO

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 767–779

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified porcine ETF-QO crystal structures with and without bound ubiquinone. · source_derived_draft · unverified_draft

### b2-met-porcine-etfdh-cofactors Porcine ETF-QO crystal structures resolved one FAD and one [4Fe-4S] cluster per protein, together with a separate ubiquinone-binding region. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: ETF-QO combines flavin and iron-sulfur chemistry in one protein downstream of ETF. organism: Sus scrofa tissue_or_cell_type: Purified porcine mitochondrial ETF-QO experimental_model: Purified porcine ETF-QO crystal structures with and without bound ubiquinone. limitations: Static structure; the exact electron-entry sequence required additional functional experiments. exposure: No nutrient intervention; structural or biochemical characterization. cross_nutrient: B2-derived FAD and iron-containing cluster are distinct required molecular components; this does not establish supplement interactions. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC1637562", "locator": "HTML article p", "paragraph_index": 10, "char_start": 0, "char_end": 3701, "evidence_access": "full-text"}] [zhang-2006-etf-qo] Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool (2006). https://pubmed.ncbi.nlm.nih.gov/17050691/ DOI: 10.1073/pnas.0604567103

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

Genetic variation modifies retention of the B2-derived cofactor at a folate enzyme.

evidence_location

Results: FAD loss; Figs 1-3

experimental_model

Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays.

exposure

Purified-enzyme assay

limitations

The diluted purified-protein experiment does not measure intracellular cofactor occupancy in every 677TT carrier.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

This variant loses its cofactor more readily in the dilution experiment.

primary_references

[yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1038–1050

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. · source_derived_draft · unverified_draft

### b2-mthfr-ala222val-fad-loss Ala222Val MTHFR released FAD about three times faster than wild type after dilution; concentration dependence supported dimer dissociation before cofactor loss. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: This variant loses its cofactor more readily in the dilution experiment. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. limitations: The diluted purified-protein experiment does not measure intracellular cofactor occupancy in every 677TT carrier. exposure: Purified-enzyme assay cross_nutrient: Genetic variation modifies retention of the B2-derived cofactor at a folate enzyme. evidence_location: Results: FAD loss; Figs 1-3 [yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

cross_nutrient

Bounds extrapolation of B2-cofactor loss across MTHFR variants.

evidence_location

Results: FAD loss; Figs 1-3

experimental_model

Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays.

exposure

Purified-enzyme assay

limitations

Normal result under tested in-vitro conditions does not exclude all cellular effects of A1298C.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The two common MTHFR variants did not share the same cofactor-retention phenotype.

primary_references

[yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1080–1092

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. · source_derived_draft · unverified_draft

### b2-mthfr-glu429ala-retention Glu429Ala MTHFR showed FAD-release behavior indistinguishable from wild type under the tested dilution conditions. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The two common MTHFR variants did not share the same cofactor-retention phenotype. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. limitations: Normal result under tested in-vitro conditions does not exclude all cellular effects of A1298C. exposure: Purified-enzyme assay cross_nutrient: Bounds extrapolation of B2-cofactor loss across MTHFR variants. evidence_location: Results: FAD loss; Figs 1-3 [yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

cross_nutrient

B2-derived flavins form part of the B12-reactivation machinery.

evidence_location

Abstract

experimental_model

Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution.

exposure

Purified-enzyme assay

limitations

Biochemical evidence does not establish a dietary threshold or supplementation benefit.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

MTRR uses both B2-derived cofactors.

primary_references

[olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1094–1106

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. · source_derived_draft · unverified_draft

### b2-mtrr-dual-flavins Purified recombinant human MTRR contained equimolar FAD and FMN, identifying two distinct flavin cofactors in the same reductase. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: MTRR uses both B2-derived cofactors. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. limitations: Biochemical evidence does not establish a dietary threshold or supplementation benefit. exposure: Purified-enzyme assay cross_nutrient: B2-derived flavins form part of the B12-reactivation machinery. evidence_location: Abstract [olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

Distinct B2-cofactor and selenium-site functions cannot substitute for one another.

evidence_location

Abstract

experimental_model

Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays.

exposure

Purified-enzyme assay

limitations

Includes Sec498Cys, Sec498Ser and terminal truncation; engineered proteins, not nutrient-depleted animals.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rattus norvegicus protein expressed in Escherichia coli

plain_language

A defective terminal center can leave upstream flavin reduction intact.

primary_references

[zhong2000] Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. (2000). https://pubmed.ncbi.nlm.nih.gov/10849437/ DOI: 10.1074/jbc.m000690200

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1374–1386

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays. · source_derived_draft · unverified_draft

### b2-txnrd-mutants-upstream-retained Engineered rat TXNRD1 terminal mutants retained one FAD per subunit and rapidly formed a thiolate-flavin charge-transfer signal upon NADPH titration. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A defective terminal center can leave upstream flavin reduction intact. organism: Rattus norvegicus protein expressed in Escherichia coli tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays. limitations: Includes Sec498Cys, Sec498Ser and terminal truncation; engineered proteins, not nutrient-depleted animals. exposure: Purified-enzyme assay cross_nutrient: Distinct B2-cofactor and selenium-site functions cannot substitute for one another. evidence_location: Abstract [zhong2000] Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. (2000). https://pubmed.ncbi.nlm.nih.gov/10849437/ DOI: 10.1074/jbc.m000690200

evidence_location

Abstract; bound-FAD measurements

experimental_model

Purified His-tagged human FADS2 binding and denaturation assays

exposure

Isolated enzyme, chaotrope treatment and denaturation.

limitations

Product retention alone does not prove direct delivery to every cellular flavoprotein.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

FAD can remain tightly associated with the enzyme that makes it.

primary_references

[transport-fads2-2011] Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins. (2011). https://pubmed.ncbi.nlm.nih.gov/21951714/ DOI: 10.1111/j.1742-4658.2011.08368.x

tissue_or_cell_type

Purified enzyme

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 373–384

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified His-tagged human FADS2 binding and denaturation assays · source_derived_draft · unverified_draft

### transport-flad1-fad-binding Purified human FADS2 retained approximately one FAD per enzyme noncovalently; complete release required extensive denaturation. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: FAD can remain tightly associated with the enzyme that makes it. organism: Homo sapiens tissue_or_cell_type: Purified enzyme experimental_model: Purified His-tagged human FADS2 binding and denaturation assays limitations: Product retention alone does not prove direct delivery to every cellular flavoprotein. exposure: Isolated enzyme, chaotrope treatment and denaturation. evidence_location: Abstract; bound-FAD measurements [transport-fads2-2011] Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins. (2011). https://pubmed.ncbi.nlm.nih.gov/21951714/ DOI: 10.1111/j.1742-4658.2011.08368.x

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Figure 4B

experimental_model

Purified recombinant human FADS2 variant

exposure

Wild-type versus variant purified enzyme.

limitations

Variant numbering follows FADS1; this is not a finding for all FLAD1 variants.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens protein

plain_language

This FLAD1 variant retains bound FAD less effectively.

primary_references

[transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

tissue_or_cell_type

Purified protein

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 386–397

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified recombinant human FADS2 variant · source_derived_draft · unverified_draft

### transport-flad1-s495del-binding Recombinant FADS2 carrying p.Ser495del retained less bound flavin than wild-type FADS2. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: This FLAD1 variant retains bound FAD less effectively. organism: Homo sapiens protein tissue_or_cell_type: Purified protein experimental_model: Purified recombinant human FADS2 variant limitations: Variant numbering follows FADS1; this is not a finding for all FLAD1 variants. exposure: Wild-type versus variant purified enzyme. evidence_location: Figure 4B [transport-flad1-disease-2016] Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency (2016). https://pmc.ncbi.nlm.nih.gov/articles/PMC4908180/ DOI: 10.1016/j.ajhg.2016.04.006

evidence_access

Primary abstract

experimental_model

Human fibroblasts, sequence analysis and recombinant expression.

limitations

Does not make ACADSB and ACAD8 interchangeable.

nutrient_topic

L-Isoleucine collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Isoleucine

plain_language

Isoleucine and valine split into different enzyme routes after their shared early steps.

primary_references

Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucine and valine metabolism. · 2000 · https://pubmed.ncbi.nlm.nih.gov/11013134/ · DOI 10.1086/303105

L-Isoleucine: transport, translation, catabolism and cross-nutrient mechanisms (2026-09-19) · lines 178–184

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Human fibroblasts, sequence analysis and recombinant expression. · source_derived_draft · unverified_draft

## isoleucine-acadsb-reaction Isoleucine and valine split into different enzyme routes after their shared early steps. Human ACADSB supported oxidation of 2-methylbutyryl-CoA; patient fibroblast assays and expression studies distinguished this isoleucine enzyme from valine-related ACAD8. Model: Human fibroblasts, sequence analysis and recombinant expression. Limitations: Does not make ACADSB and ACAD8 interchangeable. Evidence access: Primary abstract Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenases in isoleucine and valine metabolism. · 2000 · https://pubmed.ncbi.nlm.nih.gov/11013134/ · DOI 10.1086/303105

cross_nutrient

PLP-dependent KYNU follows the FAD-dependent KMO step, connecting B6 and B2 with tryptophan-niacin metabolism.

existing_related_claim_ids

["5570a502-6ebf-5397-bf2a-0148727e7695"]

experimental_model

Recombinant human KYNU crystallography and biochemical characterization

limitations

KYNU does not directly make NAD; subsequent enzymes and dietary niacin also contribute.

nutrient_topic

Vitamin B6 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin B6

organism

Homo sapiens

plain_language

This B6-dependent step processes the product of upstream KMO chemistry.

primary_references

[b6-kynu-2007] Crystal Structure of Homo Sapiens Kynureninase (2007). https://pmc.ncbi.nlm.nih.gov/articles/PMC2531291/ DOI: 10.1021/bi0616697

tissue_or_cell_type

Purified recombinant protein; no intact tissue

Vitamin B6: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 885–896

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human KYNU crystallography and biochemical characterization · source_derived_draft · unverified_draft

### b6-met-kynu-hydrolysis Human KYNU cleaves 3-hydroxykynurenine to 3-hydroxyanthranilate and alanine using PLP. Condition category: normal nutrient_topic: Vitamin B6 research collection; topical membership is not evidence of a direct dietary effect. plain_language: This B6-dependent step processes the product of upstream KMO chemistry. organism: Homo sapiens tissue_or_cell_type: Purified recombinant protein; no intact tissue experimental_model: Recombinant human KYNU crystallography and biochemical characterization limitations: KYNU does not directly make NAD; subsequent enzymes and dietary niacin also contribute. cross_nutrient: PLP-dependent KYNU follows the FAD-dependent KMO step, connecting B6 and B2 with tryptophan-niacin metabolism. existing_related_claim_ids: ["5570a502-6ebf-5397-bf2a-0148727e7695"] [b6-kynu-2007] Crystal Structure of Homo Sapiens Kynureninase (2007). https://pmc.ncbi.nlm.nih.gov/articles/PMC2531291/ DOI: 10.1021/bi0616697

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_span

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experimental_model

Recombinant protein and engineered bacterial coexpression

exposure

POR mutation and added FMN

limitations

In-vitro rescue of one mutant; not a dietary-riboflavin deficiency experiment or evidence that FMN reverses a DIM-drug interaction.

nutrient_topic

Diindolylmethane (DIM) research collection; topical membership is not evidence of a direct dietary effect. · 3,3'-Diindolylmethane / DIM

organism

Human POR Y181D and human CYP1A2 in engineered systems

plain_language

Inducing an enzyme is not enough if its electron-supply machinery fails.

primary_references

[dim-p19884324] Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect. (2010). https://pubmed.ncbi.nlm.nih.gov/19884324/ DOI: 10.1124/dmd.109.030445

tissue_or_cell_type

FMN-dependent electron transfer

trigger_kind

machinery_impairment Imported condition classification; unverified.

Diindolylmethane (DIM): formation, receptor signaling, metabolism and drug interactions (2026-09-17) · lines 272–283

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant protein and engineered bacterial coexpression · source_derived_draft · unverified_draft

### dim-por-fmn-loss POR Y181D lacked bound FMN and poorly supported CYP1A2-catalyzed reactions despite retaining FAD binding and NADPH utilization. Condition category: machinery_impairment nutrient_topic: Diindolylmethane (DIM) research collection; topical membership is not evidence of a direct dietary effect. plain_language: Inducing an enzyme is not enough if its electron-supply machinery fails. organism: Human POR Y181D and human CYP1A2 in engineered systems tissue_or_cell_type: FMN-dependent electron transfer experimental_model: Recombinant protein and engineered bacterial coexpression limitations: In-vitro rescue of one mutant; not a dietary-riboflavin deficiency experiment or evidence that FMN reverses a DIM-drug interaction. exposure: POR mutation and added FMN evidence_span: {"source_cache": "artifacts/dim-research/19884324.abstract.txt", "locator": "Primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "3f702cc9af1528241d8572b8bcafc23dacb5ed3cacf6bb77403e8aa40d21a214", "start_char": 0, "end_char": 1738, "text_sha256": "3f702cc9af1528241d8572b8bcafc23dacb5ed3cacf6bb77403e8aa40d21a214"} [dim-p19884324] Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect. (2010). https://pubmed.ncbi.nlm.nih.gov/19884324/ DOI: 10.1124/dmd.109.030445

cross_nutrient

true

evidence_location

Primary Figure 2C; Methods 2.3 and reconstitution assays.

experimental_model

Purified enzyme and phospholipid-vesicle reconstitution

exposure

0.25 micromolar CYP2R1; varied POR; 30 min at 37 C; Figure 2C.

limitations

Assay optimum is not a tissue expression target or a vitamin dosing requirement.

nutrient

Vitamin D2 and D3 · Vitamin D2 and D3

nutrient_topic

Vitamin D2 and D3 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin D2 and D3

organism

Homo sapiens proteins

plain_language

CYP2R1 needs an electron-supplying partner.

primary_references

[cheng2018] Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3. (2018). https://pubmed.ncbi.nlm.nih.gov/28716760/ DOI: 10.1016/j.jsbmb.2017.07.011

tissue_or_cell_type

reconstituted membrane

Vitamin D2 and D3: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 392–405

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified enzyme and phospholipid-vesicle reconstitution · source_derived_draft · unverified_draft

### vd-act-por-support Human POR supported CYP2R1-mediated cholecalciferol 25-hydroxylation, with maximal measured activity near a 4:1 POR:CYP2R1 molar ratio. Condition category: normal nutrient_topic: Vitamin D2 and D3 research collection; topical membership is not evidence of a direct dietary effect. plain_language: CYP2R1 needs an electron-supplying partner. organism: Homo sapiens proteins tissue_or_cell_type: reconstituted membrane experimental_model: Purified enzyme and phospholipid-vesicle reconstitution limitations: Assay optimum is not a tissue expression target or a vitamin dosing requirement. exposure: 0.25 micromolar CYP2R1; varied POR; 30 min at 37 C; Figure 2C. cross_nutrient: true evidence_location: Primary Figure 2C; Methods 2.3 and reconstitution assays. nutrient: Vitamin D2 and D3 [cheng2018] Properties of purified CYP2R1 in a reconstituted membrane environment and its 25-hydroxylation of 20-hydroxyvitamin D3. (2018). https://pubmed.ncbi.nlm.nih.gov/28716760/ DOI: 10.1016/j.jsbmb.2017.07.011

evidence_span

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experimental_model

Rat DMGDH crystallography and enzyme characterization

exposure

Dimethylglycine turnover and THF-bound structures

limitations

Species and assay context retained. The proposed intramolecular transfer route is not represented as a directly observed moving intermediate.

nutrient_topic

Choline research collection; topical membership is not evidence of a direct dietary effect. · Choline

organism

Rat recombinant enzyme

plain_language

After betaine donates one methyl group, another enzyme handles the remaining dimethylglycine.

primary_references

[choline-p24858690] Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate. (2014). https://pubmed.ncbi.nlm.nih.gov/24858690/ DOI: 10.1016/j.bbrc.2014.05.064

tissue_or_cell_type

Mitochondrial-enzyme preparation

Choline: metabolism, signaling and nutrient connections (2026-09-17) · lines 659–670

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat DMGDH crystallography and enzyme characterization · source_derived_draft · unverified_draft

### choline-dmgdh-sarcosine Rat DMGDH catalyzes oxidative demethylation of dimethylglycine to sarcosine. Condition category: normal nutrient_topic: Choline research collection; topical membership is not evidence of a direct dietary effect. plain_language: After betaine donates one methyl group, another enzyme handles the remaining dimethylglycine. organism: Rat recombinant enzyme tissue_or_cell_type: Mitochondrial-enzyme preparation experimental_model: Rat DMGDH crystallography and enzyme characterization limitations: Species and assay context retained. The proposed intramolecular transfer route is not represented as a directly observed moving intermediate. exposure: Dimethylglycine turnover and THF-bound structures evidence_span: {"source_cache": "artifacts/choline-research/24858690.abstract.txt", "locator": "Primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f", "start_char": 0, "end_char": 1506, "text_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f"} [choline-p24858690] Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate. (2014). https://pubmed.ncbi.nlm.nih.gov/24858690/ DOI: 10.1016/j.bbrc.2014.05.064

evidence_span

{"source_cache": "artifacts/choline-research/24858690.abstract.txt", "locator": "Primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f", "start_char": 0, "end_char": 1506, "text_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f"}

experimental_model

Rat DMGDH crystallography and enzyme characterization

exposure

Dimethylglycine turnover and THF-bound structures

limitations

Species and assay context retained. The proposed intramolecular transfer route is not represented as a directly observed moving intermediate.

nutrient_topic

Choline research collection; topical membership is not evidence of a direct dietary effect. · Choline

organism

Rat recombinant enzyme

plain_language

Folate and a riboflavin-derived cofactor occupy distinct sites in the same enzyme.

primary_references

[choline-p24858690] Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate. (2014). https://pubmed.ncbi.nlm.nih.gov/24858690/ DOI: 10.1016/j.bbrc.2014.05.064

tissue_or_cell_type

Mitochondrial-enzyme preparation

Choline: metabolism, signaling and nutrient connections (2026-09-17) · lines 672–683

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat DMGDH crystallography and enzyme characterization · source_derived_draft · unverified_draft

### choline-dmgdh-thf Rat DMGDH structures located THF in a domain separated from the FAD-containing active center and connected by an internal channel. Condition category: normal nutrient_topic: Choline research collection; topical membership is not evidence of a direct dietary effect. plain_language: Folate and a riboflavin-derived cofactor occupy distinct sites in the same enzyme. organism: Rat recombinant enzyme tissue_or_cell_type: Mitochondrial-enzyme preparation experimental_model: Rat DMGDH crystallography and enzyme characterization limitations: Species and assay context retained. The proposed intramolecular transfer route is not represented as a directly observed moving intermediate. exposure: Dimethylglycine turnover and THF-bound structures evidence_span: {"source_cache": "artifacts/choline-research/24858690.abstract.txt", "locator": "Primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f", "start_char": 0, "end_char": 1506, "text_sha256": "e00bbb8f1c2c805e9d7261c286bace5758abd277bc95b3e0a830bd9cdb82a34f"} [choline-p24858690] Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate. (2014). https://pubmed.ncbi.nlm.nih.gov/24858690/ DOI: 10.1016/j.bbrc.2014.05.064

evidence_span

{"source_cache": "artifacts/coq10-research/38425362.fulltext.txt", "locator": "Primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "7d25af4ecd7340649536b8fea3b8a0a308a1611bbf8c57f6a79b362f443f1cba", "start_char": 9067, "end_char": 13813, "text_sha256": "49612ae4d3a59a75a02fe3fb132abd8aeb7ef8b7cae3b0f79b5557503c55ad51"}

experimental_model

Purified reconstructed COQ metabolon with short-chain substrates

exposure

Enzyme combinations, methyl donors, reductants and metal additions

limitations

Ancestral proteins and CoQ1 analogues; no clinical cofactor dose or proof of nutritional rate limitation. Reaction order need not be universal across species.

nutrient_topic

Coenzyme Q10 research collection; topical membership is not evidence of a direct dietary effect. · Coenzyme Q10 / CoQ10 redox system

organism

Reconstructed ancestral tetrapod proteins

plain_language

The monooxygenase needed its electron-supplying partners.

primary_references

[coq10-p38425362] In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis. (2024). https://pubmed.ncbi.nlm.nih.gov/38425362/ DOI: 10.1038/s41929-023-01087-z

tissue_or_cell_type

Stepwise CoQ head-group assembly

Coenzyme Q10: biosynthesis, electron transfer, antioxidant recycling and nutrient interactions (2026-09-17) · lines 632–643

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified reconstructed COQ metabolon with short-chain substrates · source_derived_draft · unverified_draft

### coq10-fdx2-coq6 COQ6 substrate conversion required the FDXR-FDX2 pair in the reconstructed assay. Condition category: normal nutrient_topic: Coenzyme Q10 research collection; topical membership is not evidence of a direct dietary effect. plain_language: The monooxygenase needed its electron-supplying partners. organism: Reconstructed ancestral tetrapod proteins tissue_or_cell_type: Stepwise CoQ head-group assembly experimental_model: Purified reconstructed COQ metabolon with short-chain substrates limitations: Ancestral proteins and CoQ1 analogues; no clinical cofactor dose or proof of nutritional rate limitation. Reaction order need not be universal across species. exposure: Enzyme combinations, methyl donors, reductants and metal additions evidence_span: {"source_cache": "artifacts/coq10-research/38425362.fulltext.txt", "locator": "Primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "7d25af4ecd7340649536b8fea3b8a0a308a1611bbf8c57f6a79b362f443f1cba", "start_char": 9067, "end_char": 13813, "text_sha256": "49612ae4d3a59a75a02fe3fb132abd8aeb7ef8b7cae3b0f79b5557503c55ad51"} [coq10-p38425362] In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis. (2024). https://pubmed.ncbi.nlm.nih.gov/38425362/ DOI: 10.1038/s41929-023-01087-z

cross_nutrient

B1 performs E1 carbon chemistry; B2-derived FAD and the niacin-related NAD cofactor participate in the separate shared E3 reaction.

evidence

[{"paper_key": "brautigam-2005-dld", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["abstract"], "locator": "Primary publication abstract", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}]

experimental_model

Human DLD crystallography with NAD+ and NADH.

limitations

Cofactor chemistry does not establish dietary B2/B3 limitation or prove rescue of B1 deficiency.

nutrient

Thiamine (vitamin B1) · Thiamine (vitamin B1)

nutrient_topic

Thiamine research collection; topical membership is not evidence of a direct dietary effect. · Thiamine (vitamin B1)

organism

Homo sapiens

plain_language

The shared E3 protein resets reduced lipoyl carriers using the B2-derived flavin and B3-related NAD system, allowing another round of B1-dependent turnover.

primary_references

[brautigam-2005-dld] Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations (2005). https://pubmed.ncbi.nlm.nih.gov/15946682/ DOI: 10.1016/j.jmb.2005.05.014

tissue_or_cell_type

Purified enzyme

Thiamine: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 717–729

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human DLD crystallography with NAD+ and NADH. · source_derived_draft · unverified_draft

### b1-dld-fad-nad-lipoyl-regeneration Human DLD uses bound FAD and transiently bound NAD+ to oxidize dihydrolipoamide; NADH-bound structures place its nicotinamide ring beside FAD. Condition category: normal nutrient_topic: Thiamine research collection; topical membership is not evidence of a direct dietary effect. plain_language: The shared E3 protein resets reduced lipoyl carriers using the B2-derived flavin and B3-related NAD system, allowing another round of B1-dependent turnover. organism: Homo sapiens tissue_or_cell_type: Purified enzyme experimental_model: Human DLD crystallography with NAD+ and NADH. limitations: Cofactor chemistry does not establish dietary B2/B3 limitation or prove rescue of B1 deficiency. evidence: [{"paper_key": "brautigam-2005-dld", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["abstract"], "locator": "Primary publication abstract", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}] cross_nutrient: B1 performs E1 carbon chemistry; B2-derived FAD and the niacin-related NAD cofactor participate in the separate shared E3 reaction. nutrient: Thiamine (vitamin B1) [brautigam-2005-dld] Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations (2005). https://pubmed.ncbi.nlm.nih.gov/15946682/ DOI: 10.1016/j.jmb.2005.05.014

cross_nutrient

B1, B5-derived CoA, B2-derived FAD and niacin-related NAD act at different steps of one complex.

evidence

[{"paper_key": "nemeria-2014-ogdh", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["p-28"], "locator": "The reaction medium contained the following in 1.0 ml", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}]

experimental_model

Reconstituted human multienzyme assay.

limitations

NADH assay measures overall complex turnover, not every intermediate independently.

nutrient

Thiamine (vitamin B1) · Thiamine (vitamin B1)

nutrient_topic

Thiamine research collection; topical membership is not evidence of a direct dietary effect. · Thiamine (vitamin B1)

organism

Homo sapiens

plain_language

B1-dependent E1 feeds a lipoyl/CoA transfer pathway, and E3 transfers the resulting reducing equivalents to NAD.

primary_references

[nemeria-2014-ogdh] Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate (2014). https://pubmed.ncbi.nlm.nih.gov/25210035/ DOI: 10.1074/jbc.m114.591073

tissue_or_cell_type

Purified enzyme complex

Thiamine: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 855–867

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Reconstituted human multienzyme assay. · source_derived_draft · unverified_draft

### b1-ogdh-complex-couples-succinyl-nadh Human OGDH, lipoylated DLST and DLD assembled into an active complex coupling 2-oxoglutarate oxidation to NADH production in the CoA-containing assay. Condition category: normal nutrient_topic: Thiamine research collection; topical membership is not evidence of a direct dietary effect. plain_language: B1-dependent E1 feeds a lipoyl/CoA transfer pathway, and E3 transfers the resulting reducing equivalents to NAD. organism: Homo sapiens tissue_or_cell_type: Purified enzyme complex experimental_model: Reconstituted human multienzyme assay. limitations: NADH assay measures overall complex turnover, not every intermediate independently. evidence: [{"paper_key": "nemeria-2014-ogdh", "source_bundle": "artifacts/thiamine_metabolism_sources.json", "passage_ids": ["p-28"], "locator": "The reaction medium contained the following in 1.0 ml", "preservation": "Exact text retained in the source bundle; full source document retained when openly retrievable."}] cross_nutrient: B1, B5-derived CoA, B2-derived FAD and niacin-related NAD act at different steps of one complex. nutrient: Thiamine (vitamin B1) [nemeria-2014-ogdh] Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate (2014). https://pubmed.ncbi.nlm.nih.gov/25210035/ DOI: 10.1074/jbc.m114.591073

evidence_access

Primary abstract

experimental_model

Purified enzyme crystal structure and biochemical assays.

limitations

Binding beside FAD does not mean resveratrol depletes riboflavin or displaces FAD.

nutrient_topic

Resveratrol collection; molecular form, preparation, species, exposure and manipulation remain explicit. · Resveratrol

plain_language

A riboflavin-derived cofactor sits beside a directly observed binding site.

primary_references

Crystal structure of quinone reductase 2 in complex with resveratrol. · 2004 · https://pubmed.ncbi.nlm.nih.gov/15350128/ · DOI 10.1021/bi049162o

Resveratrol: metabolites, target selectivity and cross-nutrient mechanisms (2026-09-19) · lines 110–116

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified enzyme crystal structure and biochemical assays. · source_derived_draft · unverified_draft

## resveratrol-nqo2-binding A riboflavin-derived cofactor sits beside a directly observed binding site. Resveratrol bound the active-site cleft of human NQO2 beside FAD, with reported dissociation constant 35 nM, and inhibited activity in vitro. Model: Purified enzyme crystal structure and biochemical assays. Limitations: Binding beside FAD does not mean resveratrol depletes riboflavin or displaces FAD. Evidence access: Primary abstract Crystal structure of quinone reductase 2 in complex with resveratrol. · 2004 · https://pubmed.ncbi.nlm.nih.gov/15350128/ · DOI 10.1021/bi049162o

availability_state

biomarker_context Imported condition classification; unverified.

experimental_model

119 healthy controls and 125 critically ill patients at admission; repeat measurements in 60 patients.

exposure

Observational HPLC plasma and red-cell riboflavin, FMN andFAD; no isolated treatment.

limitations

Observational illness comparison cannot diagnose tissue deficiency or establish that supplementation improves outcomes.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A high circulating B2 concentration during critical illness can coexist with a lower measured cellular FAD pool.

primary_references

[b2-ventura2010] Relation between riboflavin, flavin mononucleotide and flavin adenine dinucleotide concentrations in plasma and red cells in patients with critical illness (2010). https://pubmed.ncbi.nlm.nih.gov/20667447/ DOI: 10.1016/j.cca.2010.07.024

tissue_or_cell_type

Human clinical setting

trigger_kind

biomarker_context Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1641–1651

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · 119 healthy controls and 125 critically ill patients at admission; repeat measurements in 60 patients. · source_derived_draft · unverified_draft

### b2-critical-illness-pools Critically ill patients had higher plasma riboflavin and FMN but lower plasma and red-cell FAD than healthy controls; plasma-vitamer relationships were disrupted. Condition category: biomarker_context nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A high circulating B2 concentration during critical illness can coexist with a lower measured cellular FAD pool. organism: Homo sapiens tissue_or_cell_type: Human clinical setting experimental_model: 119 healthy controls and 125 critically ill patients at admission; repeat measurements in 60 patients. limitations: Observational illness comparison cannot diagnose tissue deficiency or establish that supplementation improves outcomes. exposure: Observational HPLC plasma and red-cell riboflavin, FMN andFAD; no isolated treatment. [b2-ventura2010] Relation between riboflavin, flavin mononucleotide and flavin adenine dinucleotide concentrations in plasma and red cells in patients with critical illness (2010). https://pubmed.ncbi.nlm.nih.gov/20667447/ DOI: 10.1016/j.cca.2010.07.024

availability_state

nutrient_deficiency Imported condition classification; unverified.

cross_nutrient

B2 supports a component of glutathione recycling; this is not proof of universal glutathione exhaustion.

experimental_model

Erythrocytes from marginally riboflavin-deficient people and controls separated into nine density fractions.

exposure

Red-cell age-fraction enzyme/cofactor assays, glutathione, hemoglobin species and peroxide susceptibility; observational comparison.

limitations

Observational age-fraction study; reduced glutathione itself was not among the significant between-group differences.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A B2-dependent recycling enzyme was less active in red cells from the deficient group.

primary_references

[b2-powers1981] Riboflavin deficiency in man: effects on haemoglobin and reduced glutathione in erythrocytes of different ages (1981). https://pubmed.ncbi.nlm.nih.gov/7284295/ DOI: 10.1079/bjn19810031

tissue_or_cell_type

Human clinical setting

trigger_kind

nutrient_deficiency Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1692–1703

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Erythrocytes from marginally riboflavin-deficient people and controls separated into nine density fractions. · source_derived_draft · unverified_draft

### b2-deficiency-redcell-gsr Marginally B2-deficient subjects had lower erythrocyte glutathione reductase activity than controls across the study comparison. Condition category: nutrient_deficiency nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A B2-dependent recycling enzyme was less active in red cells from the deficient group. organism: Homo sapiens tissue_or_cell_type: Human clinical setting experimental_model: Erythrocytes from marginally riboflavin-deficient people and controls separated into nine density fractions. limitations: Observational age-fraction study; reduced glutathione itself was not among the significant between-group differences. exposure: Red-cell age-fraction enzyme/cofactor assays, glutathione, hemoglobin species and peroxide susceptibility; observational comparison. cross_nutrient: B2 supports a component of glutathione recycling; this is not proof of universal glutathione exhaustion. [b2-powers1981] Riboflavin deficiency in man: effects on haemoglobin and reduced glutathione in erythrocytes of different ages (1981). https://pubmed.ncbi.nlm.nih.gov/7284295/ DOI: 10.1079/bjn19810031

availability_state

biomarker_context Imported condition classification; unverified.

cross_nutrient

G6PD genotype and distinct flavin pools alter interpretation of B2/B6-linked assays.

experimental_model

Human erythrocytes from G6PD deficiency, heterozygous beta-thalassemia and controls.

exposure

In-vitro FAD stimulation of glutathione reductase and PNP oxidase activity, with oral-riboflavin response observations.

limitations

Human red-cell observation; the proposed faster FMN-to-FAD flux was an interpretation, not a directly measured universal mechanism.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A red-cell FAD saturation test can look adequate while another B2-dependent enzyme system is poorly supplied.

primary_references

[b2-anderson1987] Glutathione reductase activity and its relationship to pyridoxine phosphate activity in G6PD deficiency (1987). https://pubmed.ncbi.nlm.nih.gov/3582603/ DOI: 10.1111/j.1600-0609.1987.tb01417.x

tissue_or_cell_type

Human clinical setting

trigger_kind

biomarker_context Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1666–1677

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human erythrocytes from G6PD deficiency, heterozygous beta-thalassemia and controls. · source_derived_draft · unverified_draft

### b2-g6pd-fad-assay-limit In G6PD-deficient erythrocytes, glutathione reductase was usually already FAD-saturated despite evidence of low FMN-dependent oxidase activity. Condition category: biomarker_context nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A red-cell FAD saturation test can look adequate while another B2-dependent enzyme system is poorly supplied. organism: Homo sapiens tissue_or_cell_type: Human clinical setting experimental_model: Human erythrocytes from G6PD deficiency, heterozygous beta-thalassemia and controls. limitations: Human red-cell observation; the proposed faster FMN-to-FAD flux was an interpretation, not a directly measured universal mechanism. exposure: In-vitro FAD stimulation of glutathione reductase and PNP oxidase activity, with oral-riboflavin response observations. cross_nutrient: G6PD genotype and distinct flavin pools alter interpretation of B2/B6-linked assays. [b2-anderson1987] Glutathione reductase activity and its relationship to pyridoxine phosphate activity in G6PD deficiency (1987). https://pubmed.ncbi.nlm.nih.gov/3582603/ DOI: 10.1111/j.1600-0609.1987.tb01417.x

cross_nutrient

B2-FAD supports GSH recycling; selenium-dependent GPX use of GSH is a separate reaction.

evidence_location

Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes

experimental_model

Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls.

exposure

Purified-enzyme assay

limitations

GSR is not glutathione peroxidase; this record does not show that B2 improves selenium repletion.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

This enzyme recycles glutathione after oxidation.

primary_references

[berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

tissue_or_cell_type

Purified human GSR

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1332–1344

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. · source_derived_draft · unverified_draft

### b2-gsr-gssg-to-gsh GSR reduces one glutathione-disulfide molecule to two reduced glutathione molecules using the NADPH/FAD catalytic relay. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: This enzyme recycles glutathione after oxidation. organism: Homo sapiens tissue_or_cell_type: Purified human GSR experimental_model: Purified human glutathione reductase crystals with natural substrates, 0.95-1.1-A resolution, chemically reduced controls. limitations: GSR is not glutathione peroxidase; this record does not show that B2 improves selenium repletion. exposure: Purified-enzyme assay cross_nutrient: B2-FAD supports GSH recycling; selenium-dependent GPX use of GSH is a separate reaction. evidence_location: Results: NADPH binding; Fig 1 consensus cycle; GSH/GSSG complexes [berkholz2008] Catalytic cycle of human glutathione reductase near 1 A resolution. (2008). https://pubmed.ncbi.nlm.nih.gov/18638483/ DOI: 10.1016/j.jmb.2008.06.083

cross_nutrient

B2-FAD and nicotinamide-containing NADPH support a branch upstream of de novo niacin synthesis.

evidence_location

Fig 2f and Methods: kinetic assays; interpret species separately

experimental_model

Human KMO in Sf9 membrane preparations; mass-spectrometry kinetics; separate P. fluorescens KMO crystallography.

exposure

Purified-enzyme assay

limitations

Human functional assays and bacterial structures are distinct evidence; this experiment did not test dietary B2 restriction or total NAD synthesis.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A flavin enzyme directs tryptophan-derived kynurenine into the hydroxylated branch.

primary_references

[hutchinson2017] Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase. (2017). https://pubmed.ncbi.nlm.nih.gov/28604669/ DOI: 10.1038/ncomms15827

tissue_or_cell_type

Sf9 membrane fraction expressing human KMO

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1234–1246

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human KMO in Sf9 membrane preparations; mass-spectrometry kinetics; separate P. fluorescens KMO crystallography. · source_derived_draft · unverified_draft

### b2-kmo-kynurenine-hydroxylation Human KMO membrane assays measured kynurenine conversion to 3-hydroxykynurenine with NADPH; at 200 micromolar NADPH, kynurenine Km was 2 micromolar. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A flavin enzyme directs tryptophan-derived kynurenine into the hydroxylated branch. organism: Homo sapiens tissue_or_cell_type: Sf9 membrane fraction expressing human KMO experimental_model: Human KMO in Sf9 membrane preparations; mass-spectrometry kinetics; separate P. fluorescens KMO crystallography. limitations: Human functional assays and bacterial structures are distinct evidence; this experiment did not test dietary B2 restriction or total NAD synthesis. exposure: Purified-enzyme assay cross_nutrient: B2-FAD and nicotinamide-containing NADPH support a branch upstream of de novo niacin synthesis. evidence_location: Fig 2f and Methods: kinetic assays; interpret species separately [hutchinson2017] Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase. (2017). https://pubmed.ncbi.nlm.nih.gov/28604669/ DOI: 10.1038/ncomms15827

availability_state

nutrient_deficiency Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC7477094", "locator": "XML .//body//p", "paragraph_index": 21, "char_start": 0, "char_end": 3026, "evidence_access": "full-text"}]

experimental_model

Mouse B16 melanoma cells cultured in riboflavin-free medium for 3 days; quantitative proteomics.

exposure

Riboflavin-free culture for three days; quantitative proteomics.

limitations

Abundance, not direct pathway flux; cancer-cell dependence may differ from normal cells.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

Removing B2 lowered the abundance of respiratory machinery in these tumor cells.

primary_references

[martinez-limon-2020-b2-depletion] Flavin dependency undermines proteome stability, lipid metabolism and cellular proliferation during vitamin B2 deficiency (2020). https://pubmed.ncbi.nlm.nih.gov/32895367/ DOI: 10.1038/s41419-020-02929-5

tissue_or_cell_type

B16 melanoma cells

trigger_kind

nutrient_deficiency Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 970–981

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Mouse B16 melanoma cells cultured in riboflavin-free medium for 3 days; quantitative proteomics. · source_derived_draft · unverified_draft

### b2-met-b16-respiratory-proteome Three-day riboflavin starvation of mouse B16 cells reduced proteomic clusters associated with electron transport and ATP synthesis. Condition category: nutrient_deficiency nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Removing B2 lowered the abundance of respiratory machinery in these tumor cells. organism: Mus musculus tissue_or_cell_type: B16 melanoma cells experimental_model: Mouse B16 melanoma cells cultured in riboflavin-free medium for 3 days; quantitative proteomics. limitations: Abundance, not direct pathway flux; cancer-cell dependence may differ from normal cells. exposure: Riboflavin-free culture for three days; quantitative proteomics. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC7477094", "locator": "XML .//body//p", "paragraph_index": 21, "char_start": 0, "char_end": 3026, "evidence_access": "full-text"}] [martinez-limon-2020-b2-depletion] Flavin dependency undermines proteome stability, lipid metabolism and cellular proliferation during vitamin B2 deficiency (2020). https://pubmed.ncbi.nlm.nih.gov/32895367/ DOI: 10.1038/s41419-020-02929-5

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC2440658", "locator": "HTML article p", "paragraph_index": 11, "char_start": 0, "char_end": 1107, "evidence_access": "full-text"}]

experimental_model

Bovine heart submitochondrial particles; alkaline reductive inactivation and cofactor reconstitution.

exposure

10 micromolar riboflavin, FAD or FMN in the reconstitution comparison.

limitations

Does not test intact-cell conversion of riboflavin into FMN.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Bos taurus

plain_language

Free B2 and FAD could not substitute directly for FMN in this test tube.

primary_references

[gostimskaya-2007-complex-i-fmn] Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I) (2007). https://pubmed.ncbi.nlm.nih.gov/18037377/ DOI: 10.1016/j.febslet.2007.11.048

tissue_or_cell_type

Heart submitochondrial particles

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 596–607

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Bovine heart submitochondrial particles; alkaline reductive inactivation and cofactor reconstitution. · source_derived_draft · unverified_draft

### b2-met-complex-i-flavin-specificity Riboflavin and FAD failed to protect or reactivate alkaline-inactivated bovine complex I under conditions where FMN did. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Free B2 and FAD could not substitute directly for FMN in this test tube. organism: Bos taurus tissue_or_cell_type: Heart submitochondrial particles experimental_model: Bovine heart submitochondrial particles; alkaline reductive inactivation and cofactor reconstitution. limitations: Does not test intact-cell conversion of riboflavin into FMN. exposure: 10 micromolar riboflavin, FAD or FMN in the reconstitution comparison. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC2440658", "locator": "HTML article p", "paragraph_index": 11, "char_start": 0, "char_end": 1107, "evidence_access": "full-text"}] [gostimskaya-2007-complex-i-fmn] Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I) (2007). https://pubmed.ncbi.nlm.nih.gov/18037377/ DOI: 10.1016/j.febslet.2007.11.048

availability_state

nutrient_deficiency Imported condition classification; unverified.

cross_nutrient

Connects B2-dependent DLD abundance to the canonical B1-dependent mitochondrial complexes; this study does not demonstrate failure of thiamine treatment.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10767280", "locator": "XML .//body//p", "paragraph_index": 58, "char_start": 0, "char_end": 905, "evidence_access": "full-text"}]

experimental_model

Human 143B cells and mouse adult fibroblasts, riboflavin-free medium and separate DPI interventions.

exposure

Riboflavin-free medium compared with matched replete medium.

limitations

No direct PDH/OGDH flux or thiamine-response experiment; selective loss must not be generalized to all flavoproteins.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

B2 withdrawal destabilized selected flavoproteins, including the shared DLD enzyme.

primary_references

[curtabbi-2024-fmn-assembly] Regulation of respiratory complex I assembly by FMN cofactor targeting (2024). https://pubmed.ncbi.nlm.nih.gov/38145589/ DOI: 10.1016/j.redox.2023.103001

tissue_or_cell_type

143B cell proteomics

trigger_kind

nutrient_deficiency Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 622–634

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human 143B cells and mouse adult fibroblasts, riboflavin-free medium and separate DPI interventions. · source_derived_draft · unverified_draft

### b2-met-depletion-dld DLD protein abundance decreased in riboflavin-depleted human 143B cells, while many other mitochondrial matrix flavoproteins did not decline. Condition category: nutrient_deficiency nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: B2 withdrawal destabilized selected flavoproteins, including the shared DLD enzyme. organism: Homo sapiens tissue_or_cell_type: 143B cell proteomics experimental_model: Human 143B cells and mouse adult fibroblasts, riboflavin-free medium and separate DPI interventions. limitations: No direct PDH/OGDH flux or thiamine-response experiment; selective loss must not be generalized to all flavoproteins. exposure: Riboflavin-free medium compared with matched replete medium. cross_nutrient: Connects B2-dependent DLD abundance to the canonical B1-dependent mitochondrial complexes; this study does not demonstrate failure of thiamine treatment. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10767280", "locator": "XML .//body//p", "paragraph_index": 58, "char_start": 0, "char_end": 905, "evidence_access": "full-text"}] [curtabbi-2024-fmn-assembly] Regulation of respiratory complex I assembly by FMN cofactor targeting (2024). https://pubmed.ncbi.nlm.nih.gov/38145589/ DOI: 10.1016/j.redox.2023.103001

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3106343", "locator": "HTML article p", "paragraph_index": 2, "char_start": 0, "char_end": 1789, "evidence_access": "full-text"}]

experimental_model

Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR.

exposure

Site-directed mutants compared with wild type.

limitations

Bacterial variant experiment; mechanism cannot be assigned quantitatively to human disease variants.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rhodobacter sphaeroides; human ETF/MCAD reagents

plain_language

Changing the flavin environment selectively impaired the quinone-reducing step.

primary_references

[swanson-2008-etf-qo] The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein (2008). https://pubmed.ncbi.nlm.nih.gov/18672901/ DOI: 10.1021/bi800507p

tissue_or_cell_type

Purified recombinant proteins

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 781–792

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR. · source_derived_draft · unverified_draft

### b2-met-etf-qo-fad-quinone-function Rhodobacter ETF-QO N338T and N338A lowered FAD redox potentials and quinone-reductase activity while minimally affecting ETF semiquinone disproportionation. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Changing the flavin environment selectively impaired the quinone-reducing step. organism: Rhodobacter sphaeroides; human ETF/MCAD reagents tissue_or_cell_type: Purified recombinant proteins experimental_model: Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR. limitations: Bacterial variant experiment; mechanism cannot be assigned quantitatively to human disease variants. exposure: Site-directed mutants compared with wild type. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3106343", "locator": "HTML article p", "paragraph_index": 2, "char_start": 0, "char_end": 1789, "evidence_access": "full-text"}] [swanson-2008-etf-qo] The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein (2008). https://pubmed.ncbi.nlm.nih.gov/18672901/ DOI: 10.1021/bi800507p

cross_nutrient

Direct mechanistic integration of an iron-containing center with B2-derived FAD.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3106343", "locator": "HTML article p", "paragraph_index": 51, "char_start": 0, "char_end": 1197, "evidence_access": "full-text"}]

experimental_model

Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Mechanistic inference from mutagenesis/EPR and activity; no dietary iron or B2 intervention.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rhodobacter sphaeroides; human ETF/MCAD reagents

plain_language

ETF-QO uses its iron-sulfur center and flavin for different stages of the electron relay.

primary_references

[swanson-2008-etf-qo] The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein (2008). https://pubmed.ncbi.nlm.nih.gov/18672901/ DOI: 10.1021/bi800507p

tissue_or_cell_type

Purified proteins

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 794–806

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR. · source_derived_draft · unverified_draft

### b2-met-etf-qo-iron-sulfur-entry Selective effects of ETF-QO FAD-site mutations supported electron entry from ETF through the [4Fe-4S] center, followed by flavin-mediated transfer to ubiquinone. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: ETF-QO uses its iron-sulfur center and flavin for different stages of the electron relay. organism: Rhodobacter sphaeroides; human ETF/MCAD reagents tissue_or_cell_type: Purified proteins experimental_model: Recombinant Rhodobacter sphaeroides ETF-QO; human ETF and MCAD in mixed-species reconstitution; mutagenesis and EPR. limitations: Mechanistic inference from mutagenesis/EPR and activity; no dietary iron or B2 intervention. exposure: No nutrient intervention; structural or biochemical characterization. cross_nutrient: Direct mechanistic integration of an iron-containing center with B2-derived FAD. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3106343", "locator": "HTML article p", "paragraph_index": 51, "char_start": 0, "char_end": 1197, "evidence_access": "full-text"}] [swanson-2008-etf-qo] The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein (2008). https://pubmed.ncbi.nlm.nih.gov/18672901/ DOI: 10.1021/bi800507p

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}]

experimental_model

Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature.

exposure

Variant expression with supplemented riboflavin, comparing clinical response groups.

limitations

Response-associated molecular comparison; no dose recommendation and no claim that all missense variants respond.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Different inherited changes left different amounts of rescuable protein function.

primary_references

[cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

tissue_or_cell_type

HEK-293 expression system

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 821–832

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. · source_derived_draft · unverified_draft

### b2-met-etfdh-responsive-milder-folding Under the same supplemented culture conditions, ETF-QO variants linked to riboflavin-responsive MADD exhibited milder folding defects than the nonresponsive or partly responsive variants. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Different inherited changes left different amounts of rescuable protein function. organism: Homo sapiens tissue_or_cell_type: HEK-293 expression system experimental_model: Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. limitations: Response-associated molecular comparison; no dose recommendation and no claim that all missense variants respond. exposure: Variant expression with supplemented riboflavin, comparing clinical response groups. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}] [cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}]

experimental_model

Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature.

exposure

Clinical-response-defined ETFDH variants; supplemented culture riboflavin.

limitations

Primary abstract-level claim; variants are grouped by this study, not a universal response classifier.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Some inherited ETF-QO defects remained severe despite greater B2 supply.

primary_references

[cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

tissue_or_cell_type

HEK-293 expression system

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 808–819

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. · source_derived_draft · unverified_draft

### b2-met-etfdh-severe-rescue-boundary ETFDH variants associated with nonresponsive or partially responsive MADD showed severe misfolding in HEK-293 cells even in riboflavin-supplemented medium. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Some inherited ETF-QO defects remained severe despite greater B2 supply. organism: Homo sapiens tissue_or_cell_type: HEK-293 expression system experimental_model: Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. limitations: Primary abstract-level claim; variants are grouped by this study, not a universal response classifier. exposure: Clinical-response-defined ETFDH variants; supplemented culture riboflavin. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}] [cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}]

experimental_model

Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature.

exposure

Riboflavin and temperature variation in cell-expression experiments.

limitations

Does not determine clinical outcomes during fever or response of an untested mutation.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Cofactor support did not make the altered proteins structurally normal.

primary_references

[cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

tissue_or_cell_type

HEK-293-derived variant ETF-QO assays

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 834–845

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. · source_derived_draft · unverified_draft

### b2-met-etfdh-thermal-rescue-limit Residual low thermal stability of the studied ETF-QO variants indicated that FAD availability did not fully correct their structural defects. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Cofactor support did not make the altered proteins structurally normal. organism: Homo sapiens tissue_or_cell_type: HEK-293-derived variant ETF-QO assays experimental_model: Human HEK-293 cells expressing patient-associated ETF-QO variants under varied riboflavin and temperature. limitations: Does not determine clinical outcomes during fever or response of an untested mutation. exposure: Riboflavin and temperature variation in cell-expression experiments. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID22611163", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1899, "evidence_access": "primary-abstract"}] [cornelius-2012-etfdh-rescue] Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency (2012). https://pubmed.ncbi.nlm.nih.gov/22611163/ DOI: 10.1093/hmg/dds175

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 2, "char_start": 0, "char_end": 1105, "evidence_access": "full-text"}]

experimental_model

Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

UQ1/DCIP are assay reagents; the isolated turnover value is not whole-cell ATP production.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The intact complex moves electrons from succinate into a quinone carrier.

primary_references

[du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

tissue_or_cell_type

HEK293F-derived purified complex II

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 663–674

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. · source_derived_draft · unverified_draft

### b2-met-human-complex-ii-succinate-quinone Purified human complex II coupled succinate oxidation to ubiquinone reduction in a UQ1/DCIP assay, with reported kcat 0.67 +/- 0.02 per second. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The intact complex moves electrons from succinate into a quinone carrier. organism: Homo sapiens tissue_or_cell_type: HEK293F-derived purified complex II experimental_model: Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. limitations: UQ1/DCIP are assay reagents; the isolated turnover value is not whole-cell ATP production. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 2, "char_start": 0, "char_end": 1105, "evidence_access": "full-text"}] [du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

cross_nutrient

B2-derived FAD and iron-containing redox centers cooperate within one respiratory complex.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 2, "char_start": 0, "char_end": 1105, "evidence_access": "full-text"}]

experimental_model

Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Structural co-dependence does not show that B2 corrects iron deficiency or that iron supplementation improves this reaction.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Complex II couples a B2-derived flavin with separate iron-sulfur centers.

primary_references

[du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

tissue_or_cell_type

HEK293F-derived purified complex II

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 649–661

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. · source_derived_draft · unverified_draft

### b2-met-human-sdhb-iron-sulfur Human complex II structure and EPR resolved SDHB-associated [2Fe-2S], [4Fe-4S] and [3Fe-4S] redox centers adjacent to its SDHA FAD system. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Complex II couples a B2-derived flavin with separate iron-sulfur centers. organism: Homo sapiens tissue_or_cell_type: HEK293F-derived purified complex II experimental_model: Human complex II purified from HEK293F cells, cryo-EM, EPR and succinate-quinone activity assays. limitations: Structural co-dependence does not show that B2 corrects iron deficiency or that iron supplementation improves this reaction. exposure: No nutrient intervention; structural or biochemical characterization. cross_nutrient: B2-derived FAD and iron-containing redox centers cooperate within one respiratory complex. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC10161127", "locator": "XML .//body//p", "paragraph_index": 2, "char_start": 0, "char_end": 1105, "evidence_access": "full-text"}] [du-2023-human-complex-ii] Structure of the human respiratory complex II (2023). https://pubmed.ncbi.nlm.nih.gov/37098072/ DOI: 10.1073/pnas.2216713120

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID15159392", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1430, "evidence_access": "primary-abstract"}]

experimental_model

Human ETF-MCAD complex crystallography and solution interfacial mutagenesis.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Primary abstract supports mechanism; no inference about clinical MCAD supplementation response.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Fatty-acid oxidation hands electrons from MCAD to a distinct ETF carrier.

primary_references

[toogood-2004-etf-mcad] Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex (2004). https://pubmed.ncbi.nlm.nih.gov/15159392/ DOI: 10.1074/jbc.m404884200

tissue_or_cell_type

Recombinant human protein complex

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 741–752

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human ETF-MCAD complex crystallography and solution interfacial mutagenesis. · source_derived_draft · unverified_draft

### b2-met-mcad-etf-electron-transfer Human MCAD and ETF form an electron-transfer complex in which a recognition loop anchors binding while the ETF FAD domain samples electron-transfer-compatible conformations. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Fatty-acid oxidation hands electrons from MCAD to a distinct ETF carrier. organism: Homo sapiens tissue_or_cell_type: Recombinant human protein complex experimental_model: Human ETF-MCAD complex crystallography and solution interfacial mutagenesis. limitations: Primary abstract supports mechanism; no inference about clinical MCAD supplementation response. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID15159392", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1430, "evidence_access": "primary-abstract"}] [toogood-2004-etf-mcad] Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex (2004). https://pubmed.ncbi.nlm.nih.gov/15159392/ DOI: 10.1074/jbc.m404884200

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID15159392", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1430, "evidence_access": "primary-abstract"}]

experimental_model

Human ETF-MCAD complex crystallography and solution interfacial mutagenesis.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Residue interactions were inferred from combined structural/kinetic evidence; this is not a dietary-deficiency experiment.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Correct contact between the proteins matters as well as having the cofactor.

primary_references

[toogood-2004-etf-mcad] Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex (2004). https://pubmed.ncbi.nlm.nih.gov/15159392/ DOI: 10.1074/jbc.m404884200

tissue_or_cell_type

Recombinant protein interaction assays

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 754–765

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human ETF-MCAD complex crystallography and solution interfacial mutagenesis. · source_derived_draft · unverified_draft

### b2-met-mcad-etf-interface Interface mutagenesis and solution electron-transfer measurements supported a role for MCAD Glu212 and ETF alpha Arg249 interactions in productive electron transfer. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Correct contact between the proteins matters as well as having the cofactor. organism: Homo sapiens tissue_or_cell_type: Recombinant protein interaction assays experimental_model: Human ETF-MCAD complex crystallography and solution interfacial mutagenesis. limitations: Residue interactions were inferred from combined structural/kinetic evidence; this is not a dietary-deficiency experiment. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMID15159392", "locator": "metadata.abstractText", "paragraph_index": 0, "char_start": 0, "char_end": 1430, "evidence_access": "primary-abstract"}] [toogood-2004-etf-mcad] Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex (2004). https://pubmed.ncbi.nlm.nih.gov/15159392/ DOI: 10.1074/jbc.m404884200

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

DLD links B2 cofactor supply to glycine cleavage and thus folate-mediated one-carbon metabolism.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 43, "char_start": 0, "char_end": 1215, "evidence_access": "full-text"}]

experimental_model

Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays.

exposure

Slc25a32-null versus wild-type embryos.

limitations

Knockout affects multiple flavoproteins; DLD-specific genetic rescue was not performed.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

A defect in mitochondrial flavin supply impaired the shared DLD enzyme.

primary_references

[peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

tissue_or_cell_type

Embryo mitochondria

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 874–886

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. · source_derived_draft · unverified_draft

### b2-met-mouse-null-dld-activity DLD activity in Slc25a32-null embryo mitochondria was about 23.9% of wild-type control. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A defect in mitochondrial flavin supply impaired the shared DLD enzyme. organism: Mus musculus tissue_or_cell_type: Embryo mitochondria experimental_model: Slc25a32-null embryos and missense knock-in mice, isolated mitochondria and skeletal-muscle enzyme assays. limitations: Knockout affects multiple flavoproteins; DLD-specific genetic rescue was not performed. exposure: Slc25a32-null versus wild-type embryos. cross_nutrient: DLD links B2 cofactor supply to glycine cleavage and thus folate-mediated one-carbon metabolism. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC11072207", "locator": "HTML article p", "paragraph_index": 43, "char_start": 0, "char_end": 1215, "evidence_access": "full-text"}] [peng-2022-slc25a32] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism (2022). https://pubmed.ncbi.nlm.nih.gov/35727412/ DOI: 10.1007/s00018-022-04404-0

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 22, "char_start": 0, "char_end": 998, "evidence_access": "full-text"}]

experimental_model

Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue.

exposure

Expression from yeast SDH5 promoter; normalized FAD fluorescence.

limitations

Cross-species complementation; not a riboflavin-supplement rescue experiment.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens protein in Saccharomyces cerevisiae

plain_language

The inherited altered assembly factor failed a functional rescue test.

primary_references

[hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

tissue_or_cell_type

Yeast complementation

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 702–713

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. · source_derived_draft · unverified_draft

### b2-met-sdhaf2-g78r-rescue Human SDHAF2 G78R failed to restore Sdh1 flavination in sdh5-null yeast, whereas wild-type human SDHAF2 restored it to about 77% of wild-type yeast. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The inherited altered assembly factor failed a functional rescue test. organism: Homo sapiens protein in Saccharomyces cerevisiae tissue_or_cell_type: Yeast complementation experimental_model: Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. limitations: Cross-species complementation; not a riboflavin-supplement rescue experiment. exposure: Expression from yeast SDH5 promoter; normalized FAD fluorescence. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 22, "char_start": 0, "char_end": 998, "evidence_access": "full-text"}] [hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC5076811", "locator": "HTML article p", "paragraph_index": 20, "char_start": 0, "char_end": 1274, "evidence_access": "full-text"}]

experimental_model

Human MDA-MB-231 breast cancer cells with CRISPR SDHAF2 disruption and independent siRNA knockdown.

exposure

CRISPR knockout, two independent siRNAs; FAD fluorescence/antibody and enzyme activity.

limitations

Cell-line-specific redundancy is not proof that SDHAF2 is dispensable in every tissue; contextual boundary to yeast and tumor findings.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

This breast cancer cell model assembled active, flavinated complex II without SDHAF2.

primary_references

[bezawork-geleta-2016-sdhaf2] The Assembly Factor SDHAF2 Is Dispensable for Flavination of the Catalytic Subunit of Mitochondrial Complex II in Breast Cancer Cells (2016). https://pubmed.ncbi.nlm.nih.gov/27587393/ DOI: 10.1074/jbc.c116.755017

tissue_or_cell_type

MDA-MB-231 breast cancer cells

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 715–726

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human MDA-MB-231 breast cancer cells with CRISPR SDHAF2 disruption and independent siRNA knockdown. · source_derived_draft · unverified_draft

### b2-met-sdhaf2-knockout-context-boundary SDHAF2 knockout and independent siRNA knockdown in MDA-MB-231 cells retained SDHA flavination; knockout cells also retained measurable SDH and succinate-quinone reductase activity. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: This breast cancer cell model assembled active, flavinated complex II without SDHAF2. organism: Homo sapiens tissue_or_cell_type: MDA-MB-231 breast cancer cells experimental_model: Human MDA-MB-231 breast cancer cells with CRISPR SDHAF2 disruption and independent siRNA knockdown. limitations: Cell-line-specific redundancy is not proof that SDHAF2 is dispensable in every tissue; contextual boundary to yeast and tumor findings. exposure: CRISPR knockout, two independent siRNAs; FAD fluorescence/antibody and enzyme activity. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC5076811", "locator": "HTML article p", "paragraph_index": 20, "char_start": 0, "char_end": 1274, "evidence_access": "full-text"}] [bezawork-geleta-2016-sdhaf2] The Assembly Factor SDHAF2 Is Dispensable for Flavination of the Catalytic Subunit of Mitochondrial Complex II in Breast Cancer Cells (2016). https://pubmed.ncbi.nlm.nih.gov/27587393/ DOI: 10.1074/jbc.c116.755017

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 21, "char_start": 0, "char_end": 672, "evidence_access": "full-text"}]

experimental_model

Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue.

exposure

No nutrient intervention; structural or biochemical characterization.

limitations

Coimmunoprecipitation does not by itself establish the chemistry of FAD attachment.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The human assembly factor physically associated with the flavin-bearing subunit.

primary_references

[hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

tissue_or_cell_type

HEK293 cells

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 676–687

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. · source_derived_draft · unverified_draft

### b2-met-sdhaf2-sdha-binding Wild-type human SDHAF2 coimmunoprecipitated SDHA from transfected HEK293 cells. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The human assembly factor physically associated with the flavin-bearing subunit. organism: Homo sapiens tissue_or_cell_type: HEK293 cells experimental_model: Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. limitations: Coimmunoprecipitation does not by itself establish the chemistry of FAD attachment. exposure: No nutrient intervention; structural or biochemical characterization. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 21, "char_start": 0, "char_end": 672, "evidence_access": "full-text"}] [hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_spans

[{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 13, "char_start": 0, "char_end": 660, "evidence_access": "full-text"}]

experimental_model

Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue.

exposure

SDH5 deletion; SDS-PAGE FAD fluorescence and immunoblot.

limitations

Not proof of universal SDHAF2 requirement in mammalian cells.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Saccharomyces cerevisiae

plain_language

An assembly defect removed the cofactor attachment without simply removing all of the target protein.

primary_references

[hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

tissue_or_cell_type

Yeast mitochondria

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 689–700

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. · source_derived_draft · unverified_draft

### b2-met-yeast-sdh5-flavination Deleting yeast SDH5 eliminated detected covalent FAD attachment to Sdh1 even though Sdh1 protein remained present. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: An assembly defect removed the cofactor attachment without simply removing all of the target protein. organism: Saccharomyces cerevisiae tissue_or_cell_type: Yeast mitochondria experimental_model: Yeast genetics, recombinant coexpression, human HEK293 binding assays and familial paraganglioma tissue. limitations: Not proof of universal SDHAF2 requirement in mammalian cells. exposure: SDH5 deletion; SDS-PAGE FAD fluorescence and immunoblot. evidence_spans: [{"source_bundle": "artifacts/riboflavin_metabolism_sources.json", "source_key": "PMC3881419", "locator": "HTML article p", "paragraph_index": 13, "char_start": 0, "char_end": 660, "evidence_access": "full-text"}] [hao-2009-sdh5] SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma (2009). https://pubmed.ncbi.nlm.nih.gov/19628817/ DOI: 10.1126/science.1175689

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

Folate binding can stabilize a B2-derived cofactor interaction.

evidence_location

Results: FAD loss; Figs 1-3

experimental_model

Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays.

exposure

Purified-enzyme assay

limitations

Protein-stability assay; clinical folate or riboflavin treatment effects were not tested here.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Folate product helped the variant retain FAD in vitro.

primary_references

[yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1052–1064

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. · source_derived_draft · unverified_draft

### b2-methylfolate-mthfr-retention Methyltetrahydrofolate slowed FAD loss from diluted Ala222Val MTHFR in the tested concentration series. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Folate product helped the variant retain FAD in vitro. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. limitations: Protein-stability assay; clinical folate or riboflavin treatment effects were not tested here. exposure: Purified-enzyme assay cross_nutrient: Folate binding can stabilize a B2-derived cofactor interaction. evidence_location: Results: FAD loss; Figs 1-3 [yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

cross_nutrient

B2-FAD, folate and nicotinamide-containing NADPH participate in one reaction.

evidence_location

Results: kinetics, FAD occupancy and SAM inhibition; Table 1; Figs 2-4, 6

experimental_model

Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays.

exposure

Purified-enzyme assay

limitations

Biochemical evidence does not establish a dietary threshold or supplementation benefit.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The enzyme makes the methylfolate used in homocysteine recycling.

primary_references

[froese2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition. (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1010–1022

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays. · source_derived_draft · unverified_draft

### b2-mthfr-methylfolate-production HPLC assays measured human MTHFR reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using NADPH. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The enzyme makes the methylfolate used in homocysteine recycling. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Recombinant human MTHFR expressed in Sf9 cells; mass spectrometry, 2.5-A structure, HPLC activity assays. limitations: Biochemical evidence does not establish a dietary threshold or supplementation benefit. exposure: Purified-enzyme assay cross_nutrient: B2-FAD, folate and nicotinamide-containing NADPH participate in one reaction. evidence_location: Results: kinetics, FAD occupancy and SAM inhibition; Table 1; Figs 2-4, 6 [froese2018] Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition. (2018). https://pubmed.ncbi.nlm.nih.gov/29891918/ DOI: 10.1038/s41467-018-04735-2

cross_nutrient

B2 flavins support reactivation of B12-dependent folate/homocysteine metabolism.

evidence_location

Abstract

experimental_model

Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution.

exposure

Purified-enzyme assay

limitations

Reconstituted biochemistry; the accessible abstract identifies human MTRR but not the target MTR species. No dietary B2/B12 synergy was tested.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

The flavin reductase helps restore an enzyme that uses B12 and methylfolate.

primary_references

[olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1122–1134

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. · source_derived_draft · unverified_draft

### b2-mtrr-mtr-reactivation Reconstituted assays showed human MTRR sufficient to support NADPH-dependent activity of cobalamin-dependent methionine synthase. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The flavin reductase helps restore an enzyme that uses B12 and methylfolate. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. limitations: Reconstituted biochemistry; the accessible abstract identifies human MTRR but not the target MTR species. No dietary B2/B12 synergy was tested. exposure: Purified-enzyme assay cross_nutrient: B2 flavins support reactivation of B12-dependent folate/homocysteine metabolism. evidence_location: Abstract [olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

cross_nutrient

Nicotinamide-containing NADPH supplies electrons to B2-derived flavins.

evidence_location

Abstract

experimental_model

Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution.

exposure

Purified-enzyme assay

limitations

Biochemical evidence does not establish a dietary threshold or supplementation benefit.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

NADPH provides electrons to the flavin-containing reductase.

primary_references

[olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1108–1120

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. · source_derived_draft · unverified_draft

### b2-mtrr-nadph-reduction NADPH reduction of purified MTRR generated an air-stable flavin semiquinone detected spectroscopically. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: NADPH provides electrons to the flavin-containing reductase. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Recombinant human MTRR purification, flavin analysis, NADPH spectroscopy and methionine-synthase reconstitution. limitations: Biochemical evidence does not establish a dietary threshold or supplementation benefit. exposure: Purified-enzyme assay cross_nutrient: Nicotinamide-containing NADPH supplies electrons to B2-derived flavins. evidence_location: Abstract [olteanu2001] Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. (2001). https://pubmed.ncbi.nlm.nih.gov/11466310/ DOI: 10.1074/jbc.m103707200

availability_state

biomarker_context Imported condition classification; unverified.

experimental_model

124 healthy older adults, mean age 69; 46 with EGRAC >=1.20 randomized equally to riboflavin or placebo.

exposure

1.6 mg/day riboflavin versus placebo for 12 weeks; HPLC plasma/red-cell vitamer measurements.

limitations

A result in this population does not invalidate every use of plasma FAD; cellular compartment and clinical state matter.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

A stable plasma FAD reading did not mean that all B2-related pools were unchanged.

primary_references

[b2-hustad2002] Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation (2002). https://pubmed.ncbi.nlm.nih.gov/12194936/ DOI: 10.1093/clinchem/48.9.1571

tissue_or_cell_type

Human clinical setting

trigger_kind

biomarker_context Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1629–1639

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · 124 healthy older adults, mean age 69; 46 with EGRAC >=1.20 randomized equally to riboflavin or placebo. · source_derived_draft · unverified_draft

### b2-plasma-fad-nonresponse Plasma FAD did not respond significantly to supplementation in the older-adult trial although plasma riboflavin, erythrocyte FMN and other measures did. Condition category: biomarker_context nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A stable plasma FAD reading did not mean that all B2-related pools were unchanged. organism: Homo sapiens tissue_or_cell_type: Human clinical setting experimental_model: 124 healthy older adults, mean age 69; 46 with EGRAC >=1.20 randomized equally to riboflavin or placebo. limitations: A result in this population does not invalidate every use of plasma FAD; cellular compartment and clinical state matter. exposure: 1.6 mg/day riboflavin versus placebo for 12 weeks; HPLC plasma/red-cell vitamer measurements. [b2-hustad2002] Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation (2002). https://pubmed.ncbi.nlm.nih.gov/12194936/ DOI: 10.1093/clinchem/48.9.1571

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

Methionine-cycle feedback affects both folate-enzyme activity and B2-cofactor retention.

evidence_location

Results: FAD loss; Figs 1-3

experimental_model

Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays.

exposure

Purified-enzyme assay

limitations

In-vitro effects; stabilization is not equivalent to increased reaction flux.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Cofactor retention and catalytic speed respond differently to SAM.

primary_references

[yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1066–1078

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. · source_derived_draft · unverified_draft

### b2-sam-mthfr-retention SAM slowed FAD dissociation after MTHFR dilution, including Ala222Val, despite its separate reversible inhibition of catalytic activity. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Cofactor retention and catalytic speed respond differently to SAM. organism: Homo sapiens tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Baculovirus-produced purified human wild-type, Ala222Val, Glu429Ala and double-mutant MTHFR; dilution/cofactor-release assays. limitations: In-vitro effects; stabilization is not equivalent to increased reaction flux. exposure: Purified-enzyme assay cross_nutrient: Methionine-cycle feedback affects both folate-enzyme activity and B2-cofactor retention. evidence_location: Results: FAD loss; Figs 1-3 [yamada2001] Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. (2001). https://pubmed.ncbi.nlm.nih.gov/11742092/ DOI: 10.1073/pnas.261469998

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

B2 cofactor supply interacts with folate-cycle enzyme genotype.

experimental_model

Genotype-stratified randomized trial; 35 TT, 26 CT and 28 CC adults selected, ages 18-65.

exposure

1.6 mg/day riboflavin versus placebo for 12 weeks; experimental regimen.

genotype

MTHFR 677TT; common variant rather than complete loss of enzyme

limitations

Small genotype strata; subgroup magnitude is not an expected response for every TT carrier. The trial did not test cardiovascular events.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

In this trial, improving B2 supply lowered a folate-pathway blood marker in people with two copies of the studied MTHFR variant.

primary_references

[b2-mcnulty2006] Riboflavin lowers homocysteine in individuals homozygous for the MTHFR 677C->T polymorphism (2006). https://pubmed.ncbi.nlm.nih.gov/16380544/ DOI: 10.1161/circulationaha.105.580332

tissue_or_cell_type

Human clinical setting

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1486–1498

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Genotype-stratified randomized trial; 35 TT, 26 CT and 28 CC adults selected, ages 18-65. · source_derived_draft · unverified_draft

### b2-tt-homocysteine-response Riboflavin lowered plasma homocysteine in the MTHFR 677TT group by 22% overall; the lower-baseline-B2 subgroup showed a 40% decrease. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: In this trial, improving B2 supply lowered a folate-pathway blood marker in people with two copies of the studied MTHFR variant. organism: Homo sapiens tissue_or_cell_type: Human clinical setting experimental_model: Genotype-stratified randomized trial; 35 TT, 26 CT and 28 CC adults selected, ages 18-65. limitations: Small genotype strata; subgroup magnitude is not an expected response for every TT carrier. The trial did not test cardiovascular events. exposure: 1.6 mg/day riboflavin versus placebo for 12 weeks; experimental regimen. cross_nutrient: B2 cofactor supply interacts with folate-cycle enzyme genotype. genotype: MTHFR 677TT; common variant rather than complete loss of enzyme [b2-mcnulty2006] Riboflavin lowers homocysteine in individuals homozygous for the MTHFR 677C->T polymorphism (2006). https://pubmed.ncbi.nlm.nih.gov/16380544/ DOI: 10.1161/circulationaha.105.580332

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

B2-derived FAD cannot replace the selenium-containing catalytic residue.

evidence_location

Abstract

experimental_model

Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays.

exposure

Purified-enzyme assay

limitations

Assay-substrate species is unresolved in accessible source details; engineered proteins do not establish dietary B2-by-selenium synergy.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rattus norvegicus protein expressed in Escherichia coli

plain_language

Flavin retention did not preserve efficient thioredoxin reduction.

primary_references

[zhong2000] Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. (2000). https://pubmed.ncbi.nlm.nih.gov/10849437/ DOI: 10.1074/jbc.m000690200

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1388–1400

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays. · source_derived_draft · unverified_draft

### b2-txnrd-sec498cys-turnover Replacing rat TXNRD1 Sec498 with cysteine lowered thioredoxin-reduction kcat about 100-fold; serine substitution and terminal truncation lacked detectable activity. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Flavin retention did not preserve efficient thioredoxin reduction. organism: Rattus norvegicus protein expressed in Escherichia coli tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Rat TXNRD1 Sec498Cys, Sec498Ser and C-terminal truncation expressed in E. coli; FAD analysis, NADPH titration and thioredoxin assays. limitations: Assay-substrate species is unresolved in accessible source details; engineered proteins do not establish dietary B2-by-selenium synergy. exposure: Purified-enzyme assay cross_nutrient: B2-derived FAD cannot replace the selenium-containing catalytic residue. evidence_location: Abstract [zhong2000] Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations. (2000). https://pubmed.ncbi.nlm.nih.gov/10849437/ DOI: 10.1074/jbc.m000690200

cross_nutrient

B2-dependent electron input and selenium-containing terminal chemistry are separate serial steps.

evidence_location

Abstract

experimental_model

Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation.

exposure

Purified-enzyme assay

limitations

Human thioredoxin was docked onto the rat Sec498Cys structure (Fig 6); the complex and cross-species turnover were not measured.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Rattus norvegicus reductase and Homo sapiens thioredoxin docking partner

plain_language

A second redox center carries electrons onward to thioredoxin.

primary_references

[sandalova2001] Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. (2001). https://pubmed.ncbi.nlm.nih.gov/11481439/ DOI: 10.1073/pnas.171178698

tissue_or_cell_type

Purified recombinant enzyme; no intact tissue

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 1360–1372

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation. · source_derived_draft · unverified_draft

### b2-txnrd1-terminal-relay A rat TXNRD1 structure and docking model with human thioredoxin support a relay through the opposite subunit and C-terminal tail toward the substrate. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: A second redox center carries electrons onward to thioredoxin. organism: Rattus norvegicus reductase and Homo sapiens thioredoxin docking partner tissue_or_cell_type: Purified recombinant enzyme; no intact tissue experimental_model: Rat TXNRD1 Sec498Cys recombinant mutant with NADP+, 3.0-A crystallography and substrate-docking interpretation. limitations: Human thioredoxin was docked onto the rat Sec498Cys structure (Fig 6); the complex and cross-species turnover were not measured. exposure: Purified-enzyme assay cross_nutrient: B2-dependent electron input and selenium-containing terminal chemistry are separate serial steps. evidence_location: Abstract [sandalova2001] Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. (2001). https://pubmed.ncbi.nlm.nih.gov/11481439/ DOI: 10.1073/pnas.171178698

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Figure 5A and discussion

experimental_model

Lactating knockout/wild-type mouse comparison with HPLC

exposure

Standard-chow lactating knockout and wild-type mice.

limitations

The independent FAD secretion machinery was not identified.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

Milk retains another flavin supply route when free-riboflavin secretion falls.

primary_references

[transport-abcg2-2007] Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk. (2007). https://pmc.ncbi.nlm.nih.gov/articles/PMC1800714/ DOI: 10.1128/MCB.01621-06

tissue_or_cell_type

Mammary milk

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 557–568

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Lactating knockout/wild-type mouse comparison with HPLC · source_derived_draft · unverified_draft

### transport-abcg2-independent-milk-fad Milk FAD concentrations were unchanged in Abcg2-null dams, demonstrating an ABCG2-independent route for milk vitamin B2 equivalents. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Milk retains another flavin supply route when free-riboflavin secretion falls. organism: Mus musculus tissue_or_cell_type: Mammary milk experimental_model: Lactating knockout/wild-type mouse comparison with HPLC limitations: The independent FAD secretion machinery was not identified. exposure: Standard-chow lactating knockout and wild-type mice. evidence_location: Figure 5A and discussion [transport-abcg2-2007] Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk. (2007). https://pmc.ncbi.nlm.nih.gov/articles/PMC1800714/ DOI: 10.1128/MCB.01621-06

evidence_location

FAD synthesis and reverse pyrophosphorolysis assays

experimental_model

Purified recombinant human FADS2 catalytic assays

exposure

ATP and FMN in FAD-synthesis assays.

limitations

Adenylylation is distinct from RFK phosphorylation; reaction products should not be conflated.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

FLAD1 performs the second activation step, making FAD from FMN.

primary_references

[transport-fads2-2011] Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins. (2011). https://pubmed.ncbi.nlm.nih.gov/21951714/ DOI: 10.1111/j.1742-4658.2011.08368.x

tissue_or_cell_type

Purified protein

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 320–331

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified recombinant human FADS2 catalytic assays · source_derived_draft · unverified_draft

### transport-flad1-adenylylation Human FAD synthase isoform 2 adenylylates FMN with ATP to form FAD and pyrophosphate. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: FLAD1 performs the second activation step, making FAD from FMN. organism: Homo sapiens tissue_or_cell_type: Purified protein experimental_model: Purified recombinant human FADS2 catalytic assays limitations: Adenylylation is distinct from RFK phosphorylation; reaction products should not be conflated. exposure: ATP and FMN in FAD-synthesis assays. evidence_location: FAD synthesis and reverse pyrophosphorolysis assays [transport-fads2-2011] Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins. (2011). https://pubmed.ncbi.nlm.nih.gov/21951714/ DOI: 10.1111/j.1742-4658.2011.08368.x

cross_nutrient

Magnesium is a cofactor for a vitamin B2 activation step.

evidence_location

Abstract and activity assay

experimental_model

Recombinant human FADS2 expressed in E. coli and purified

exposure

MgCl2 requirement tested in purified enzyme assays.

limitations

Assay-specific metal dependence does not quantify human dietary magnesium effects on flavins.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens protein

plain_language

Magnesium supports enzymatic conversion of FMN into FAD.

primary_references

[transport-fads2-2007] Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase. (2007). https://doi.org/10.1016/j.pep.2006.09.002 DOI: 10.1016/j.pep.2006.09.002

tissue_or_cell_type

Purified enzyme

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 333–345

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human FADS2 expressed in E. coli and purified · source_derived_draft · unverified_draft

### transport-flad1-magnesium-requirement Purified human FADS2 showed a strict MgCl2 requirement in the reported spectrophotometric FAD-synthesis assay. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Magnesium supports enzymatic conversion of FMN into FAD. organism: Homo sapiens protein tissue_or_cell_type: Purified enzyme experimental_model: Recombinant human FADS2 expressed in E. coli and purified limitations: Assay-specific metal dependence does not quantify human dietary magnesium effects on flavins. exposure: MgCl2 requirement tested in purified enzyme assays. cross_nutrient: Magnesium is a cofactor for a vitamin B2 activation step. evidence_location: Abstract and activity assay [transport-fads2-2007] Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase. (2007). https://doi.org/10.1016/j.pep.2006.09.002 DOI: 10.1016/j.pep.2006.09.002

evidence_location

Figure 3C-E

experimental_model

HEK293 expression; HPLC measurement of flavin uptake

exposure

10 micromolar flavin, pH 7.4, 10 minutes, 37 C.

limitations

Substrate preference under these conditions is not proof of zero FMN transport at every concentration.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Free riboflavin was the preferred transported flavin.

primary_references

[transport-rfvt3-2014] Functional involvement of RFVT3/SLC52A3 in intestinal riboflavin absorption. (2014). https://doi.org/10.1152/ajpgi.00349.2013 DOI: 10.1152/ajpgi.00349.2013

tissue_or_cell_type

HEK293 cells

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 228–239

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · HEK293 expression; HPLC measurement of flavin uptake · source_derived_draft · unverified_draft

### transport-rfvt3-substrate-discrimination RFVT3-expressing HEK293 cells transported riboflavin strongly, FMN weakly, and showed no measurable FAD transport in the reported assay. Condition category: normal nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Free riboflavin was the preferred transported flavin. organism: Homo sapiens tissue_or_cell_type: HEK293 cells experimental_model: HEK293 expression; HPLC measurement of flavin uptake limitations: Substrate preference under these conditions is not proof of zero FMN transport at every concentration. exposure: 10 micromolar flavin, pH 7.4, 10 minutes, 37 C. evidence_location: Figure 3C-E [transport-rfvt3-2014] Functional involvement of RFVT3/SLC52A3 in intestinal riboflavin absorption. (2014). https://doi.org/10.1152/ajpgi.00349.2013 DOI: 10.1152/ajpgi.00349.2013

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Figure 5C-D

experimental_model

Stable-isotope uptake in isolated knock-in/knockout mitochondria

exposure

10 micromolar isotope-labeled FAD; knockout embryo endpoint at 60 minutes.

limitations

Organelle uptake supports carrier dependence but does not establish exchange partner or transport stoichiometry.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Mus musculus

plain_language

The carrier defect restricts access to mitochondrial FAD.

primary_references

[transport-slc25a32-2022] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism. (2022). https://pmc.ncbi.nlm.nih.gov/articles/PMC11072207/ DOI: 10.1007/s00018-022-04404-0

tissue_or_cell_type

Skeletal muscle and embryonic mitochondria

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 490–501

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Stable-isotope uptake in isolated knock-in/knockout mitochondria · source_derived_draft · unverified_draft

### transport-slc25a32-fad-import K235R/K235R and Y174C/K235R mouse muscle mitochondria accumulated less isotope-labeled FAD; Slc25a32-null embryonic mitochondria also had impaired FAD uptake. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: The carrier defect restricts access to mitochondrial FAD. organism: Mus musculus tissue_or_cell_type: Skeletal muscle and embryonic mitochondria experimental_model: Stable-isotope uptake in isolated knock-in/knockout mitochondria limitations: Organelle uptake supports carrier dependence but does not establish exchange partner or transport stoichiometry. exposure: 10 micromolar isotope-labeled FAD; knockout embryo endpoint at 60 minutes. evidence_location: Figure 5C-D [transport-slc25a32-2022] Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism. (2022). https://pmc.ncbi.nlm.nih.gov/articles/PMC11072207/ DOI: 10.1007/s00018-022-04404-0

availability_state

machinery_impairment Imported condition classification; unverified.

evidence_location

Table 3 and Methods/results

experimental_model

Single genetic case; plasma flavin assay

exposure

Pretreatment plasma measurements.

limitations

One case; normal plasma measurements are not direct measurements of neuronal flavin sufficiency.

nutrient_topic

Riboflavin research collection; topical membership is not evidence of a direct dietary effect. · Riboflavin (vitamin B2)

organism

Homo sapiens

plain_language

Normal plasma flavins did not exclude a cellular transport defect.

primary_references

[transport-slc52a2-disease-2012] Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome (2012). https://pmc.ncbi.nlm.nih.gov/articles/PMC3470687/ DOI: 10.1007/s10545-012-9513-y

tissue_or_cell_type

Plasma and clinical nervous-system phenotype

trigger_kind

machinery_impairment Imported condition classification; unverified.

Riboflavin: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 451–462

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Single genetic case; plasma flavin assay · source_derived_draft · unverified_draft

### transport-slc52a2-normal-plasma The child with compound SLC52A2 L123P/L339P variants had normal measured plasma riboflavin, FMN and FAD despite a transport-associated neurologic disorder. Condition category: machinery_impairment nutrient_topic: Riboflavin research collection; topical membership is not evidence of a direct dietary effect. plain_language: Normal plasma flavins did not exclude a cellular transport defect. organism: Homo sapiens tissue_or_cell_type: Plasma and clinical nervous-system phenotype experimental_model: Single genetic case; plasma flavin assay limitations: One case; normal plasma measurements are not direct measurements of neuronal flavin sufficiency. exposure: Pretreatment plasma measurements. evidence_location: Table 3 and Methods/results [transport-slc52a2-disease-2012] Impaired riboflavin transport due to missense mutations in SLC52A2 causes Brown-Vialetto-Van Laere syndrome (2012). https://pmc.ncbi.nlm.nih.gov/articles/PMC3470687/ DOI: 10.1007/s10545-012-9513-y

evidence_access

Primary abstract

experimental_model

Truncated purified human HYPDH; quinone analogue and oxygen comparisons.

limitations

CoQ10 as the physiological acceptor is inferred, not directly measured in intact human tissues; this does not demonstrate benefit from CoQ10 supplements.

nutrient_topic

L-Proline collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Proline

plain_language

Hydroxyproline breakdown can feed a quinone electron-transfer route.

primary_references

Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria. · 2015 · https://pubmed.ncbi.nlm.nih.gov/25697095/ · DOI 10.1042/BJ20141159

L-Proline: synthesis, collagen processing, redox metabolism and cross-nutrient mechanisms (2026-09-19) · lines 286–292

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Truncated purified human HYPDH; quinone analogue and oxygen comparisons. · source_derived_draft · unverified_draft

## l-proline-hypdh-electrons Hydroxyproline breakdown can feed a quinone electron-transfer route. Quinone acceptors including CoQ1 reacted more efficiently than oxygen during purified human HYPDH turnover, supporting a dehydrogenase mechanism. Model: Truncated purified human HYPDH; quinone analogue and oxygen comparisons. Limitations: CoQ10 as the physiological acceptor is inferred, not directly measured in intact human tissues; this does not demonstrate benefit from CoQ10 supplements. Evidence access: Primary abstract Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria. · 2015 · https://pubmed.ncbi.nlm.nih.gov/25697095/ · DOI 10.1042/BJ20141159

evidence_access

Primary abstract

experimental_model

Human recombinant catalytic core, residues 157–515; substrate kinetics.

limitations

PRODH2 is not simply a second interchangeable proline oxidase.

nutrient_topic

L-Proline collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Proline

plain_language

Hydroxyproline uses a related but distinct breakdown enzyme.

primary_references

Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria. · 2015 · https://pubmed.ncbi.nlm.nih.gov/25697095/ · DOI 10.1042/BJ20141159

L-Proline: synthesis, collagen processing, redox metabolism and cross-nutrient mechanisms (2026-09-19) · lines 278–284

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Human recombinant catalytic core, residues 157–515; substrate kinetics. · source_derived_draft · unverified_draft

## l-proline-hypdh-specificity Hydroxyproline uses a related but distinct breakdown enzyme. Purified truncated human PRODH2/HYPDH contained one FAD and showed approximately twelvefold higher catalytic efficiency for hydroxyproline than proline. Model: Human recombinant catalytic core, residues 157–515; substrate kinetics. Limitations: PRODH2 is not simply a second interchangeable proline oxidase. Evidence access: Primary abstract Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria. · 2015 · https://pubmed.ncbi.nlm.nih.gov/25697095/ · DOI 10.1042/BJ20141159

evidence_access

Primary full text; background reaction distinguished from new experiments

experimental_model

Established reaction described in a primary mitochondrial proline-oxidation study.

limitations

The reaction description is biochemical background, distinct from the paper-specific ROS-site experiments.

nutrient_topic

L-Proline collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Proline

plain_language

Breaking proline down starts with a flavin-dependent mitochondrial enzyme.

primary_references

Sources of superoxide/H2O2 during mitochondrial proline oxidation. · 2014 · https://pubmed.ncbi.nlm.nih.gov/25184115/ · DOI 10.1016/j.redox.2014.07.003

L-Proline: synthesis, collagen processing, redox metabolism and cross-nutrient mechanisms (2026-09-19) · lines 94–100

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Established reaction described in a primary mitochondrial proline-oxidation study. · source_derived_draft · unverified_draft

## l-proline-prodh-oxidation Breaking proline down starts with a flavin-dependent mitochondrial enzyme. Mitochondrial PRODH catalyzes the FAD-dependent oxidation of proline to P5C, linking proline breakdown to mitochondrial electron transfer. Model: Established reaction described in a primary mitochondrial proline-oxidation study. Limitations: The reaction description is biochemical background, distinct from the paper-specific ROS-site experiments. Evidence access: Primary full text; background reaction distinguished from new experiments Sources of superoxide/H2O2 during mitochondrial proline oxidation. · 2014 · https://pubmed.ncbi.nlm.nih.gov/25184115/ · DOI 10.1016/j.redox.2014.07.003

availability_state

machinery_impairment Imported condition classification; unverified.

cross_nutrient

true

evidence_location

Experimental procedures and Results: CblC Has a Flavin Reductase Fold; Figure 1c

experimental_model

Recombinant human MMACHC deltaC38 biochemical assay

exposure

30–40 micromolar FMN/FAD with 200 micromolar NADPH, or photoreduced FMN

limitations

Engineered truncation and reconstitution; does not establish that riboflavin supplementation rescues MMACHC defects.

nutrient_topic

Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin B12 (cobalamins)

organism

Homo sapiens

plain_language

A truncated B12-processing protein could use supplied flavins to carry out this reaction.

primary_references

[koutmos-2011-flavin] Structural Basis of Multifunctionality in a Vitamin B12-processing Enzyme (2011). https://pubmed.ncbi.nlm.nih.gov/21697092/ DOI: 10.1074/jbc.M111.261370

tissue_or_cell_type

Cell-free assay

trigger_kind

machinery_impairment Imported condition classification; unverified.

Vitamin B12: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 753–765

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MMACHC deltaC38 biochemical assay · source_derived_draft · unverified_draft

### b12-mmachc-free-flavin-decyanation Human MMACHC residues 1–244 supported cyanocobalamin decyanation with FMN or FAD plus NADPH, without a separately supplied flavoprotein reductase; reduced FMN also supported the reaction. Condition category: machinery_impairment nutrient_topic: Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. plain_language: A truncated B12-processing protein could use supplied flavins to carry out this reaction. organism: Homo sapiens tissue_or_cell_type: Cell-free assay experimental_model: Recombinant human MMACHC deltaC38 biochemical assay limitations: Engineered truncation and reconstitution; does not establish that riboflavin supplementation rescues MMACHC defects. exposure: 30–40 micromolar FMN/FAD with 200 micromolar NADPH, or photoreduced FMN cross_nutrient: true evidence_location: Experimental procedures and Results: CblC Has a Flavin Reductase Fold; Figure 1c [koutmos-2011-flavin] Structural Basis of Multifunctionality in a Vitamin B12-processing Enzyme (2011). https://pubmed.ncbi.nlm.nih.gov/21697092/ DOI: 10.1074/jbc.M111.261370

cross_nutrient

true

evidence_location

Results: Figure 4; Materials and Methods: Decyanation of CNCbl

experimental_model

Human purified-protein anaerobic reconstitution

exposure

4 micromolar reductase, 20 micromolar MMACHC/CNCbl, 200 micromolar NADPH

limitations

Reconstituted biochemical system; establishes reaction capability rather than the quantitatively dominant pathway in living humans.

nutrient_topic

Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin B12 (cobalamins)

organism

Homo sapiens

plain_language

This flavoprotein can pass reducing power into B12 processing.

primary_references

[kim-2008-decyanation] Decyanation of vitamin B12 by a trafficking chaperone (2008). https://pubmed.ncbi.nlm.nih.gov/18779575/ DOI: 10.1073/pnas.0805989105

tissue_or_cell_type

Cell-free assay

Vitamin B12: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 683–695

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human purified-protein anaerobic reconstitution · source_derived_draft · unverified_draft

### b12-mtrr-mmachc-electrons Recombinant human MTRR with NADPH supported MMACHC-bound cyanocobalamin conversion to cob(II)alamin; free cyanocobalamin was not detectably reduced without MMACHC. Condition category: normal nutrient_topic: Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. plain_language: This flavoprotein can pass reducing power into B12 processing. organism: Homo sapiens tissue_or_cell_type: Cell-free assay experimental_model: Human purified-protein anaerobic reconstitution limitations: Reconstituted biochemical system; establishes reaction capability rather than the quantitatively dominant pathway in living humans. exposure: 4 micromolar reductase, 20 micromolar MMACHC/CNCbl, 200 micromolar NADPH cross_nutrient: true evidence_location: Results: Figure 4; Materials and Methods: Decyanation of CNCbl [kim-2008-decyanation] Decyanation of vitamin B12 by a trafficking chaperone (2008). https://pubmed.ncbi.nlm.nih.gov/18779575/ DOI: 10.1073/pnas.0805989105

cross_nutrient

true

evidence_location

Results: Figure 4; Materials and Methods: Decyanation of CNCbl

experimental_model

Human purified-protein anaerobic reconstitution

exposure

4 micromolar reductase, 20 micromolar MMACHC/CNCbl, 200 micromolar NADPH

limitations

Reconstituted biochemical system; establishes reaction capability rather than the quantitatively dominant pathway in living humans.

nutrient_topic

Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin B12 (cobalamins)

organism

Homo sapiens

plain_language

This flavoprotein can pass reducing power into B12 processing.

primary_references

[kim-2008-decyanation] Decyanation of vitamin B12 by a trafficking chaperone (2008). https://pubmed.ncbi.nlm.nih.gov/18779575/ DOI: 10.1073/pnas.0805989105

tissue_or_cell_type

Cell-free assay

Vitamin B12: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 697–709

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human purified-protein anaerobic reconstitution · source_derived_draft · unverified_draft

### b12-ndor1-mmachc-electrons Recombinant human NDOR1/NR1 with NADPH supported MMACHC-bound cyanocobalamin conversion to cob(II)alamin; free cyanocobalamin was not detectably reduced without MMACHC. Condition category: normal nutrient_topic: Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. plain_language: This flavoprotein can pass reducing power into B12 processing. organism: Homo sapiens tissue_or_cell_type: Cell-free assay experimental_model: Human purified-protein anaerobic reconstitution limitations: Reconstituted biochemical system; establishes reaction capability rather than the quantitatively dominant pathway in living humans. exposure: 4 micromolar reductase, 20 micromolar MMACHC/CNCbl, 200 micromolar NADPH cross_nutrient: true evidence_location: Results: Figure 4; Materials and Methods: Decyanation of CNCbl [kim-2008-decyanation] Decyanation of vitamin B12 by a trafficking chaperone (2008). https://pubmed.ncbi.nlm.nih.gov/18779575/ DOI: 10.1073/pnas.0805989105

cross_nutrient

true

evidence_location

Indexed primary abstract

experimental_model

Purified human proteins

exposure

Purified MTRR plus MMAB and reductants

limitations

MTRR coupling in vitro does not identify the physiological mitochondrial electron donor.

nutrient_topic

Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. · Vitamin B12 (cobalamins)

organism

Homo sapiens

plain_language

Human MTRR supported B12 activation in a test-tube system; this does not establish its mitochondrial role.

primary_references

[leal-2004-human-mmab] Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase. (2004). https://pubmed.ncbi.nlm.nih.gov/15347655/ DOI: 10.1074/jbc.m405449200

tissue_or_cell_type

Purified protein assay

Vitamin B12: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 991–1003

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified human proteins · source_derived_draft · unverified_draft

### mtrr-supports-mmab-in-vitro Purified human MTRR coupled cob(II)alamin reduction to MMAB-dependent adenosylcobalamin synthesis in vitro. Condition category: normal nutrient_topic: Vitamin B12 research collection; topical membership is not evidence of a direct dietary effect. plain_language: Human MTRR supported B12 activation in a test-tube system; this does not establish its mitochondrial role. organism: Homo sapiens tissue_or_cell_type: Purified protein assay experimental_model: Purified human proteins limitations: MTRR coupling in vitro does not identify the physiological mitochondrial electron donor. exposure: Purified MTRR plus MMAB and reductants cross_nutrient: true evidence_location: Indexed primary abstract [leal-2004-human-mmab] Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase. (2004). https://pubmed.ncbi.nlm.nih.gov/15347655/ DOI: 10.1074/jbc.m405449200

evidence_access

Primary full text

experimental_model

Purified bacterial full-length/truncated proteins, activity assays and ligand-bound crystal structures.

limitations

This species is a structural model; abundance or flux in an individual human microbiome is not measured.

nutrient_topic

L-Histidine collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Histidine

plain_language

A bacterial enzyme sends a histidine-derived intermediate down a different branch.

primary_references

Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production. · 2021 · https://pubmed.ncbi.nlm.nih.gov/33649331/ · DOI 10.1038/s41467-021-21548-y

L-Histidine: supply, catabolism, histamine, receptors and cross-nutrient mechanisms (2026-09-19) · lines 266–272

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified bacterial full-length/truncated proteins, activity assays and ligand-bound crystal structures. · source_derived_draft · unverified_draft

## histidine-urda-product A bacterial enzyme sends a histidine-derived intermediate down a different branch. Full-length and two-domain S. oneidensis UrdA converted urocanate to imidazole propionate and did not show the tested fumarate-reductase activity. Model: Purified bacterial full-length/truncated proteins, activity assays and ligand-bound crystal structures. Limitations: This species is a structural model; abundance or flux in an individual human microbiome is not measured. Evidence access: Primary full text Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production. · 2021 · https://pubmed.ncbi.nlm.nih.gov/33649331/ · DOI 10.1038/s41467-021-21548-y

evidence_access

Primary indexed abstract. Physiological acceptor assignment is a kinetic interpretation, not a direct measurement of flux in a patient.

experimental_model

Purified human SQOR in nanodiscs; steady-state and rapid-kinetic assays.

interpretation_status

Source-derived research curation; not independent raw-data verification.

limitations

Predominant acceptor is a kinetic interpretation; alternative acceptors can work in other assay conditions. Ergothioneine and nutrient depletion were not tested.

nutrient_topic

Ergothioneine mitochondrial supplement; shared molecular requirements are not demonstrated dietary interactions. · L-Ergothioneine

plain_language

Glutathione also participates in disposal of sulfide-derived sulfur.

primary_references

Landry et al. H2S oxidation by nanodisc-embedded human sulfide quinone oxidoreductase. DOI 10.1074/jbc.M117.788547; PMID 28512131; https://pubmed.ncbi.nlm.nih.gov/28512131/

source_locator

Abstract

Ergothioneine: mitochondrial transport, MPST and sulfur-handling dependencies (2026-10-02) · lines 137–137

Original AI-assisted curation of five primary studies with publication identifiers, experimental locators and access limitations. Additive chapter supplement, not publisher full text. · supports · Purified human SQOR in nanodiscs; steady-state and rapid-kinetic assays. · source_derived_draft · unverified_draft

Nanodisc-embedded human SQOR transferred sulfane sulfur to glutathione; kinetics supported GSH as the predominant physiological acceptor.

evidence_access

Primary abstract

experimental_model

Purified native and recombinant porcine kidney enzyme.

limitations

Species-specific enzyme evidence; FAD is recycled rather than consumed once per substrate molecule.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

The first chemical step removes reducing equivalents from D-aspartate.

primary_references

Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 104–110

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified native and recombinant porcine kidney enzyme. · source_derived_draft · unverified_draft

## d-aspartate-ddo-imino The first chemical step removes reducing equivalents from D-aspartate. Porcine kidney DDO oxidized D-aspartate to iminoaspartate while reducing enzyme-bound FAD. Model: Purified native and recombinant porcine kidney enzyme. Limitations: Species-specific enzyme evidence; FAD is recycled rather than consumed once per substrate molecule. Evidence access: Primary abstract Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

evidence_access

Primary abstract

experimental_model

Purified porcine enzyme oxidative half-reaction.

limitations

This chemistry alone does not demonstrate oxidative injury after human oral supplementation.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Clearing D-aspartate also generates a peroxide-handling requirement.

primary_references

Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 112–118

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified porcine enzyme oxidative half-reaction. · source_derived_draft · unverified_draft

## d-aspartate-ddo-peroxide Clearing D-aspartate also generates a peroxide-handling requirement. Reoxidation of reduced FAD by oxygen during porcine DDO turnover produced hydrogen peroxide. Model: Purified porcine enzyme oxidative half-reaction. Limitations: This chemistry alone does not demonstrate oxidative injury after human oral supplementation. Evidence access: Primary abstract Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

evidence_access

Primary abstract

experimental_model

Purified porcine enzyme concentration-response assays.

limitations

Do not extrapolate assay thresholds to dietary dosing.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Different substrates change the enzyme kinetics differently.

primary_references

Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 120–126

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified porcine enzyme concentration-response assays. · source_derived_draft · unverified_draft

## d-aspartate-ddo-substrate-activation Different substrates change the enzyme kinetics differently. Porcine DDO showed substrate activation above approximately 0.2 mM D-aspartate; N-methyl-D-aspartate instead produced substrate inhibition. Model: Purified porcine enzyme concentration-response assays. Limitations: Do not extrapolate assay thresholds to dietary dosing. Evidence access: Primary abstract Functional and structural characterization of D-aspartate oxidase from porcine kidney: non-Michaelis kinetics due to substrate activation. · 2007 · https://pubmed.ncbi.nlm.nih.gov/17234685/ · DOI 10.1093/jb/mvm041

evidence_access

Primary abstract

experimental_model

Recombinant human 341-residue DDO-1.

limitations

Assay kinetic constants are not clinical plasma targets; do not assign these activities to the alternatively spliced deletion isoform.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Humans have an enzyme that breaks down D-aspartate.

primary_references

Structural and functional characterization of the human brain D-aspartate oxidase. · 1997 · https://pubmed.ncbi.nlm.nih.gov/9163533/ · DOI 10.1093/oxfordjournals.jbchem.a021655

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 72–78

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Recombinant human 341-residue DDO-1. · source_derived_draft · unverified_draft

## d-aspartate-human-ddo-activity Humans have an enzyme that breaks down D-aspartate. Purified human DDO-1 oxidized D-aspartate; the reported Km was 2.7 mM and kcat 52.5 per second. Model: Recombinant human 341-residue DDO-1. Limitations: Assay kinetic constants are not clinical plasma targets; do not assign these activities to the alternatively spliced deletion isoform. Evidence access: Primary abstract Structural and functional characterization of the human brain D-aspartate oxidase. · 1997 · https://pubmed.ncbi.nlm.nih.gov/9163533/ · DOI 10.1093/oxfordjournals.jbchem.a021655

evidence_access

Primary abstract

experimental_model

Purified recombinant human DDO-1.

limitations

Does not establish endogenous NMDA synthesis or justify conflating NMDA with D-aspartate.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

The enzyme accepts a related chemical that is distinct from the NMDA receptor.

primary_references

Structural and functional characterization of the human brain D-aspartate oxidase. · 1997 · https://pubmed.ncbi.nlm.nih.gov/9163533/ · DOI 10.1093/oxfordjournals.jbchem.a021655

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 80–86

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified recombinant human DDO-1. · source_derived_draft · unverified_draft

## d-aspartate-human-ddo-nmda The enzyme accepts a related chemical that is distinct from the NMDA receptor. Human DDO-1 also oxidized N-methyl-D-aspartate, with reported Km 6.8 mM and kcat 37.7 per second. Model: Purified recombinant human DDO-1. Limitations: Does not establish endogenous NMDA synthesis or justify conflating NMDA with D-aspartate. Evidence access: Primary abstract Structural and functional characterization of the human brain D-aspartate oxidase. · 1997 · https://pubmed.ncbi.nlm.nih.gov/9163533/ · DOI 10.1093/oxfordjournals.jbchem.a021655

evidence_access

Primary full text

experimental_model

Purified human DDO drug concentration-response assay.

limitations

Biochemical potency is not proof of clinically relevant brain inhibition or a reason to change treatment.

nutrient_topic

D-Aspartate collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · D-Aspartate

plain_language

Two drugs differed at a D-aspartate-clearing enzyme.

primary_references

Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity. · 2017 · https://pubmed.ncbi.nlm.nih.gov/28393897/ · DOI 10.1038/srep46288

D-Aspartate: synthesis, clearance, neural and endocrine mechanisms (2026-09-19) · lines 264–270

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Purified human DDO drug concentration-response assay. · source_derived_draft · unverified_draft

## d-aspartate-olanzapine-human-ddo Two drugs differed at a D-aspartate-clearing enzyme. Olanzapine inhibited recombinant human DDO at an IC50 near 23 micromolar under both 4 and 100 micromolar FAD conditions; clozapine did not inhibit the tested enzyme. Model: Purified human DDO drug concentration-response assay. Limitations: Biochemical potency is not proof of clinically relevant brain inhibition or a reason to change treatment. Evidence access: Primary full text Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity. · 2017 · https://pubmed.ncbi.nlm.nih.gov/28393897/ · DOI 10.1038/srep46288

evidence_access

Primary abstract

experimental_model

Human ERO1A structural and biochemical study.

limitations

Protein-residue oxidation is separate from free cystine reduction; dietary cysteine effects were not tested.

nutrient_topic

L-Cysteine collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Cysteine

plain_language

Cysteines already incorporated into proteins are joined and rearranged during folding.

primary_references

Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI. · 2010 · https://pubmed.ncbi.nlm.nih.gov/20834232/ · DOI 10.1038/emboj.2010.222

L-Cysteine: sulfur allocation, redox supply and cross-nutrient mechanisms (2026-09-19) · lines 484–490

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Human ERO1A structural and biochemical study. · source_derived_draft · unverified_draft

## l-cysteine-ero1-pdi-targeting Cysteines already incorporated into proteins are joined and rearranged during folding. Human ERO1A targets PDI through contacts with its b-prime substrate-binding domain, supporting the disulfide-forming pathway for protein folding. Model: Human ERO1A structural and biochemical study. Limitations: Protein-residue oxidation is separate from free cystine reduction; dietary cysteine effects were not tested. Evidence access: Primary abstract Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI. · 2010 · https://pubmed.ncbi.nlm.nih.gov/20834232/ · DOI 10.1038/emboj.2010.222

evidence_access

Primary abstract

experimental_model

Human ERO1A hyperactive and inactive crystal structures.

limitations

This is enzyme regulation, not proof that additional cysteine increases protein folding or antioxidant capacity.

nutrient_topic

L-Cysteine collection; species, compartment, exposure, co-substrates and manipulation remain explicit. · L-Cysteine

plain_language

Cysteine bonds can regulate an enzyme as well as stabilize folded proteins.

primary_references

Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI. · 2010 · https://pubmed.ncbi.nlm.nih.gov/20834232/ · DOI 10.1038/emboj.2010.222

L-Cysteine: sulfur allocation, redox supply and cross-nutrient mechanisms (2026-09-19) · lines 492–498

AI-assisted research curation; primary references, access levels and experimental limitations individually identified. Not publisher full text. · supports · Human ERO1A hyperactive and inactive crystal structures. · source_derived_draft · unverified_draft

## l-cysteine-ero1-regulatory-disulfides Cysteine bonds can regulate an enzyme as well as stabilize folded proteins. Regulatory cysteines in a flexible ERO1A loop change disulfide arrangements and electron transfer, restraining its oxidative activity. Model: Human ERO1A hyperactive and inactive crystal structures. Limitations: This is enzyme regulation, not proof that additional cysteine increases protein folding or antioxidant capacity. Evidence access: Primary abstract Crystal structures of human Ero1α reveal the mechanisms of regulated and targeted oxidation of PDI. · 2010 · https://pubmed.ncbi.nlm.nih.gov/20834232/ · DOI 10.1038/emboj.2010.222

experimental_model

Human GCDH crystallography and substrate-mechanism analysis

limitations

The downstream shared ECHS1/HADH/ACAT1 reactions are separate records; these individual enzyme assays do not measure the full lysine-derived flux in a person.

organism

Homo sapiens

plain_language

GCDH shortens the lysine-derived carbon chain.

primary_references

[fu2004] Crystal Structures of Human Glutaryl-CoA Dehydrogenase with and without an Alternate Substrate: Structural Bases of Dehydrogenation and Decarboxylation Reactions (2004). https://pubs.acs.org/doi/10.1021/bi049290c DOI: 10.1021/bi049290c

tissue_or_cell_type

Mitochondrial matrix

L-Lysine: mechanism-first literature curation (2026-09-17) · lines 136–144

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Human GCDH crystallography and substrate-mechanism analysis · source_derived_draft · unverified_draft

### gcdh-crotonyl-coa FAD-containing human GCDH dehydrogenates glutaryl-CoA through glutaconyl-CoA and decarboxylates it to crotonyl-CoA. Plain language: GCDH shortens the lysine-derived carbon chain. Condition category: normal organism: Homo sapiens tissue_or_cell_type: Mitochondrial matrix experimental_model: Human GCDH crystallography and substrate-mechanism analysis limitations: The downstream shared ECHS1/HADH/ACAT1 reactions are separate records; these individual enzyme assays do not measure the full lysine-derived flux in a person. [fu2004] Crystal Structures of Human Glutaryl-CoA Dehydrogenase with and without an Alternate Substrate: Structural Bases of Dehydrogenation and Decarboxylation Reactions (2004). https://pubs.acs.org/doi/10.1021/bi049290c DOI: 10.1021/bi049290c

experimental_model

Purified LSD1 enzyme / histone assays and cellular RNA interference.

limitations

Do not extend this specific activity to trimethyllysine or every histone site. This reaction modifies lysine already in a protein. It does not show that extra oral lysine increases the reaction or improves a clinical outcome.

organism

Human

plain_language

Some lysine methyl marks can be erased by a flavin enzyme.

primary_references

[lsd1-2004] Histone demethylation mediated by the nuclear amine oxidase homolog LSD1 (2004). https://pubmed.ncbi.nlm.nih.gov/15620353/ DOI: 10.1016/j.cell.2004.12.012

tissue_or_cell_type

Not specified as a whole tissue; see experimental model.

L-Lysine: mechanism-first literature curation (2026-09-17) · lines 557–565

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Purified LSD1 enzyme / histone assays and cellular RNA interference. · source_derived_draft · unverified_draft

### kdm1a-h3k4-demethylation KDM1A/LSD1 oxidatively removes a methyl group from H3K4me2 through a flavin-dependent reaction. Plain language: Some lysine methyl marks can be erased by a flavin enzyme. Condition category: normal organism: Human tissue_or_cell_type: Not specified as a whole tissue; see experimental model. experimental_model: Purified LSD1 enzyme / histone assays and cellular RNA interference. limitations: Do not extend this specific activity to trimethyllysine or every histone site. This reaction modifies lysine already in a protein. It does not show that extra oral lysine increases the reaction or improves a clinical outcome. [lsd1-2004] Histone demethylation mediated by the nuclear amine oxidase homolog LSD1 (2004). https://pubmed.ncbi.nlm.nih.gov/15620353/ DOI: 10.1016/j.cell.2004.12.012

evidence_span

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experimental_model

Recombinant human mARC1/mARC2 biochemical and spectroscopic reconstitution

exposure

N-hydroxylated substrates; cofactor reconstitution

limitations

The 2010 paper could not identify the Mo-ligating cysteine; later structural/mutagenesis work resolves that point. Its earlier inference is not imported as current fact.

nutrient_topic

Molybdenum research collection; topical membership is not evidence of a direct dietary effect. · Molybdenum

organism

Human proteins expressed in Escherichia coli

plain_language

Niacin, riboflavin, iron and molybdenum meet in one electron-transfer chain.

primary_references

[mo-p20861021] Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes. (2010). https://pubmed.ncbi.nlm.nih.gov/20861021/ DOI: 10.1074/jbc.m110.169532

tissue_or_cell_type

Purified enzyme system

Molybdenum: cofactor assembly, sulfur metabolism and nutrient interactions (2026-09-17) · lines 963–974

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human mARC1/mARC2 biochemical and spectroscopic reconstitution · source_derived_draft · unverified_draft

### mo-marc-fad-nadh The reconstituted mARC system used NADH/FAD-dependent cytochrome b5 reductase and heme-containing cytochrome b5. Condition category: normal nutrient_topic: Molybdenum research collection; topical membership is not evidence of a direct dietary effect. plain_language: Niacin, riboflavin, iron and molybdenum meet in one electron-transfer chain. organism: Human proteins expressed in Escherichia coli tissue_or_cell_type: Purified enzyme system experimental_model: Recombinant human mARC1/mARC2 biochemical and spectroscopic reconstitution limitations: The 2010 paper could not identify the Mo-ligating cysteine; later structural/mutagenesis work resolves that point. Its earlier inference is not imported as current fact. exposure: N-hydroxylated substrates; cofactor reconstitution evidence_span: {"source_cache": "artifacts/molybdenum-research/20861021.abstract.txt", "locator": "Exact primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "0f430936dcc4701e5d57040edcab4b0418e3c49c59620e0f7d5f54aff41e7877", "start_char": 0, "end_char": 1557, "text_sha256": "0f430936dcc4701e5d57040edcab4b0418e3c49c59620e0f7d5f54aff41e7877"} [mo-p20861021] Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes. (2010). https://pubmed.ncbi.nlm.nih.gov/20861021/ DOI: 10.1074/jbc.m110.169532

evidence_span

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experimental_model

Recombinant human XDH variants with urate, superoxide and NO assays

exposure

Xanthine, oxygen and inorganic nitrite assays

limitations

Canonical electron-transfer mechanism stated in this primary article; not an experiment on dietary B2 or niacin depletion.

nutrient_topic

Molybdenum research collection; topical membership is not evidence of a direct dietary effect. · Molybdenum

organism

Homo sapiens protein

plain_language

Riboflavin-derived FAD, iron-sulfur centers and niacin-derived NAD work alongside molybdenum.

primary_references

[mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

tissue_or_cell_type

Purified human enzyme

Molybdenum: cofactor assembly, sulfur metabolism and nutrient interactions (2026-09-17) · lines 768–779

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human XDH variants with urate, superoxide and NO assays · source_derived_draft · unverified_draft

### mo-xdh-nad The XDH form transfers purine-derived electrons through its iron-sulfur centers and FAD to NAD+, producing NADH. Condition category: normal nutrient_topic: Molybdenum research collection; topical membership is not evidence of a direct dietary effect. plain_language: Riboflavin-derived FAD, iron-sulfur centers and niacin-derived NAD work alongside molybdenum. organism: Homo sapiens protein tissue_or_cell_type: Purified human enzyme experimental_model: Recombinant human XDH variants with urate, superoxide and NO assays limitations: Canonical electron-transfer mechanism stated in this primary article; not an experiment on dietary B2 or niacin depletion. exposure: Xanthine, oxygen and inorganic nitrite assays evidence_span: {"source_cache": "artifacts/molybdenum-research/37713777.fulltext.txt", "locator": "Exact primary full-text span; zero-based, end-exclusive Unicode character offsets", "file_sha256": "a8092b2aa7328b59f0275dd8a37d2a2dbb5abb2a32236d0cab5fffcc1ddc671d", "start_char": 0, "end_char": 1579, "text_sha256": "18325172be489f8f4beae16c04dbc4f49f94c87186321e32aede61f27089c7c4"} [mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

evidence_span

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experimental_model

Recombinant human XDH variants with urate, superoxide and NO assays

exposure

Xanthine, oxygen and inorganic nitrite assays

limitations

The 2023 Fig. 6E corrigendum corrects a displayed panel; authors state data and conclusions are unchanged. Enzyme activity is not a clinical benefit or dietary response.

nutrient_topic

Molybdenum research collection; topical membership is not evidence of a direct dietary effect. · Molybdenum

organism

Homo sapiens protein

plain_language

The purine-breakdown pathway uses a molybdenum enzyme to make urate.

primary_references

[mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

tissue_or_cell_type

Purified human enzyme

Molybdenum: cofactor assembly, sulfur metabolism and nutrient interactions (2026-09-17) · lines 742–753

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human XDH variants with urate, superoxide and NO assays · source_derived_draft · unverified_draft

### mo-xdh-xanthine Recombinant human XDH/XOR catalyzed conversion of xanthine to urate. Condition category: normal nutrient_topic: Molybdenum research collection; topical membership is not evidence of a direct dietary effect. plain_language: The purine-breakdown pathway uses a molybdenum enzyme to make urate. organism: Homo sapiens protein tissue_or_cell_type: Purified human enzyme experimental_model: Recombinant human XDH variants with urate, superoxide and NO assays limitations: The 2023 Fig. 6E corrigendum corrects a displayed panel; authors state data and conclusions are unchanged. Enzyme activity is not a clinical benefit or dietary response. exposure: Xanthine, oxygen and inorganic nitrite assays evidence_span: {"source_cache": "artifacts/molybdenum-research/37713777.abstract.txt", "locator": "Exact primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "30e93ea24784120403bef1dfe72cf46411f1af88946b9cc1cba3d4c45a367bf9", "start_char": 0, "end_char": 1655, "text_sha256": "30e93ea24784120403bef1dfe72cf46411f1af88946b9cc1cba3d4c45a367bf9"} [mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

evidence_span

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experimental_model

Recombinant human XDH variants with urate, superoxide and NO assays

exposure

Xanthine, oxygen and inorganic nitrite assays

limitations

The 2023 Fig. 6E corrigendum corrects a displayed panel; authors state data and conclusions are unchanged. Enzyme activity is not a clinical benefit or dietary response.

nutrient_topic

Molybdenum research collection; topical membership is not evidence of a direct dietary effect. · Molybdenum

organism

Homo sapiens protein

plain_language

The enzyme can generate oxidants as well as carry out purine breakdown.

primary_references

[mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

tissue_or_cell_type

Purified human enzyme

Molybdenum: cofactor assembly, sulfur metabolism and nutrient interactions (2026-09-17) · lines 781–792

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human XDH variants with urate, superoxide and NO assays · source_derived_draft · unverified_draft

### mo-xor-oxygen In its oxidase activity, XOR can generate superoxide and hydrogen peroxide rather than exclusively transferring electrons to NAD+. Condition category: normal nutrient_topic: Molybdenum research collection; topical membership is not evidence of a direct dietary effect. plain_language: The enzyme can generate oxidants as well as carry out purine breakdown. organism: Homo sapiens protein tissue_or_cell_type: Purified human enzyme experimental_model: Recombinant human XDH variants with urate, superoxide and NO assays limitations: The 2023 Fig. 6E corrigendum corrects a displayed panel; authors state data and conclusions are unchanged. Enzyme activity is not a clinical benefit or dietary response. exposure: Xanthine, oxygen and inorganic nitrite assays evidence_span: {"source_cache": "artifacts/molybdenum-research/37713777.abstract.txt", "locator": "Exact primary indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "30e93ea24784120403bef1dfe72cf46411f1af88946b9cc1cba3d4c45a367bf9", "start_char": 0, "end_char": 1655, "text_sha256": "30e93ea24784120403bef1dfe72cf46411f1af88946b9cc1cba3d4c45a367bf9"} [mo-p37713777] Natural mutations of human XDH promote the nitrite (NO2-)-reductase capacity of xanthine oxidoreductase: A novel mechanism to promote redox health? (2023). https://pubmed.ncbi.nlm.nih.gov/37713777/ DOI: 10.1016/j.redox.2023.102864

experimental_model

Recombinant human MTHFR; cryo-EM and biochemistry.

limitations

Not a whole-body SAM threshold.

nutrient_topic

Folate and folic acid research collection; topical membership is not evidence of a direct dietary effect. · Folate (vitamin B9)

organism

Homo sapiens

plain_language

SAM switches MTHFR into a closed shape.

primary_references

[mthfr-allostery-2024] Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase (2024). https://pubmed.ncbi.nlm.nih.gov/38622112/ DOI: 10.1038/s41467-024-47174-y

tissue_or_cell_type

Purified protein

Folate and folic acid: mechanisms, deficiency and nutrient interactions (2026-09-17) · lines 460–469

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Recombinant human MTHFR; cryo-EM and biochemistry. · source_derived_draft · unverified_draft

### folate-methyl-sam-dual-binding Dual SAM binding rearranged the human MTHFR linker and inserted a loop that blocked catalytic substrate access. Condition category: normal nutrient_topic: Folate and folic acid research collection; topical membership is not evidence of a direct dietary effect. plain_language: SAM switches MTHFR into a closed shape. organism: Homo sapiens tissue_or_cell_type: Purified protein experimental_model: Recombinant human MTHFR; cryo-EM and biochemistry. limitations: Not a whole-body SAM threshold. [mthfr-allostery-2024] Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase (2024). https://pubmed.ncbi.nlm.nih.gov/38622112/ DOI: 10.1038/s41467-024-47174-y

experimental_model

Purified rat liver cytosolic thioredoxin reductase and recombinant active-site variants.

limitations

This experiment-specific relationship does not establish a human dietary-deficiency threshold or supplementation benefit.

organism

Rat protein and recombinant enzyme assays

Selenium: literature corrections and mechanism additions · lines 965–974

Metabolic Ledger literature curation, 17 September 2026; primary papers linked individually · supports · Purified rat liver cytosolic thioredoxin reductase and recombinant active-site variants. · secondary_verified · secondary_verified

## txnrd1-reduces-txn1 TXNRD1 recharges thioredoxin so it can reduce other proteins. Cytosolic TXNRD1 uses NADPH-derived reducing equivalents to reduce oxidized thioredoxin through its flavin and C-terminal redox centers. Experimental model: Purified rat liver cytosolic thioredoxin reductase and recombinant active-site variants. Organism: Rat protein and recombinant enzyme assays Limitations: This experiment-specific relationship does not establish a human dietary-deficiency threshold or supplementation benefit. Primary reference: [Mammalian thioredoxin reductase: C-terminal redox center and selenium-to-sulfur substitution](https://pmc.ncbi.nlm.nih.gov/articles/PMC15961/)