Historical Context of Vitamin B 12 Pernicious anemia –Megaloblastic anemia –Neuropathy: particularly degeneration of spinal cord –Universally fatal –Extrinsic factor from liver Patients were not producing enough –Gastric acid to denature R protein –Intrinsic factor Pernicious anemia –Megaloblastic anemia –Neuropathy: particularly degeneration of spinal cord –Universally fatal –Extrinsic factor from liver Patients were not producing enough –Gastric acid to denature R protein –Intrinsic factor

Structure of Vitamin B 12 Cobalamins –Corrin ring contains central cobalt atom Adenosylcobalamin Methylcobalamin Cobalamins –Corrin ring contains central cobalt atom Adenosylcobalamin Methylcobalamin

Absorption & Transport of Vitamin B 12 Receptors on ileal mucosal cells Transcobalamin II Portal circulation Receptor on the cell surface Endocytosis and lysosomal degradation of the complex Reduction of cobalt Cytosolic methylation Mitochondrial adenosylation Receptors on ileal mucosal cells Transcobalamin II Portal circulation Receptor on the cell surface Endocytosis and lysosomal degradation of the complex Reduction of cobalt Cytosolic methylation Mitochondrial adenosylation

Role of Ascorbate in the Degradation of Tyrosine Oxidation of p-hydroxyphenylpyruvate –Maintain Cu 2+ ? Homogentisate oxidation –Maintain Fe 2+ ? Synthesis of epinephrine Bile acid synthesis (7  -hydroxylase) Enhancement of iron absorption Antioxidant Oxidation of p-hydroxyphenylpyruvate –Maintain Cu 2+ ? Homogentisate oxidation –Maintain Fe 2+ ? Synthesis of epinephrine Bile acid synthesis (7  -hydroxylase) Enhancement of iron absorption Antioxidant

Incubation 2: Transfer to a microtiter well coated with biotin-BSA ( ) Incubation 1: Biotin incubated with HRP-avidin Detection of Biotin ( ) by HRP-Avidin ( ) B B B B

Standard Curves for Biotin

Biotin Catabolites Bisnorbiotin O O C C S S HC H2CH2C H2CH2C HN CH NH Biotin Sulfoxide O O - (CH 2 ) 4 -C O O OH H2CH2C H2CH2C O O C C S S HC HN CH NH - (CH 2 ) 2 - C O O CH 3 Bisnorbiotin methyl ketone H2CH2C H2CH2C O O C C S S HC HN CH NH - (CH 2 ) 4 -C O O OH Biotin sulfone O O O O H2CH2C H2CH2C O O C C S S HC HN CH NH - (CH 2 ) 2 - C O O OH

Radioactivity (dpm) Retention Time (minute) Biotin Bisnorbiotin Biotin Sulfoxide HPLC of Radiolabeled Biotin Analogs

Urinary Analogs Analyzed Against Authentic Standards Retention Time (min) Avidin-binding Substances (pmol/mg creatinine) unknown #1 unknown #2 biocytin unknown #5 biotin } } } } } ? d & l biotin sulfoxide } bisnorbiotin } } methyl ketone

Mole Percentage of Urinary Biotin and Metabolite in 10 Subjects Unk1Unk1 BSOBSOBNBBNBUnk2Unk2Unk3Unk3Unk4Unk4BiotinBiotinUnk5Unk Biotin Metabolites Biotin or Metabolite (mol/100 mol) Biotin or Metabolite (mol/100 mol)

Mole Percentage of Serum Biotin and Metabolites in 15 Subjects Biotin or Metabolite (mol/100 mol) Biotin or Metabolite (mol/100 mol) BSO BNB Biotin All Unknowns Biotin Metabolite

GCRC Study Design egg white diet (avidin) (avidin) non-biotin vitamin supplement biotin supplement Blood and urine collection

Urinary Excretion of Biotin N. R. Study Day * p< by range test after ANOVA Biotin (nmol/24 h) * * * * * * * * * * * * Mean ± SD

Serum Concentration of Biotin Serum Biotin Mean ± SD (pmol/L) N. R. Study Day

Biotin-dependent Carboxylases Acetyl-CoA Malonyl-CoA Cytosol and Mitochondria: Pyruvate Oxaloacetic acid Propionyl-CoA Methylmalonyl-CoA 3-Methylcrotonyl-CoA 3-Methylglutaconyl-CoA MCC PCC PC ACC Mitochondria:

Biosynthesis of Holocarboxylases Apocarboxylase + biotin + ATP Holocarboxylase + AMP + pyrophosphate Holocarboxylase synthetase

Mechanism of Biotin-dependent Carboxylase (Pyruvate carboxylase)

Biotinidase is a Digestion & Salvage Enzyme E-Biotin Biotinidase Proteases Peptides + Biotin Peptides + Biotin

3-methylcrotonyl-CoA 3-Methylcrotonyl-CoACarboxylase3-Methylcrotonyl-CoACarboxylase 3-methylglutaconyl-CoA 3-HIA leucine

Urinary Excretion of 3-HIA Hydroxyisovaleric Acid (µmol/24 h) N. R. * * * * * * * * * * * p< by range test after ANOVA * * Mean ± SD Study Day

Malformation VitaminTrace-Element Malformation VitaminTrace-Element Group Group Group Group Neural-tube defect 0 6 Congenital hydrocephalus 0 2 Cleft palate 0 2 Limb-reduction defect 1 5 Cleft lip 4 3 (with or without cleft palate) Czeizel & Dudas “Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation”NEJM, 326: , periconceptional vitamin supplementation”NEJM, 326: , Malformation VitaminTrace-Element Malformation VitaminTrace-Element Group Group Group Group Neural-tube defect 0 6 Congenital hydrocephalus 0 2 Cleft palate 0 2 Limb-reduction defect 1 5 Cleft lip 4 3 (with or without cleft palate) Czeizel & Dudas “Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation”NEJM, 326: , periconceptional vitamin supplementation”NEJM, 326: , 1992.

Urinary Excretion of Biotin n.s. p < 0.05 p < control early late Biotin Excretion (pmol/mg creat) Biotin Excretion (pmol/mg creat)

Urinary Excretion of 3-HIA 3HIA Excretion (µmol/24 h) 3HIA Excretion (µmol/24 h) control early late p <

Biotin Treatment Decreases 3-HIA Excretion Change in Urinary 3-HIA (mmol/mol creatinine) Placebo Biotin p = Late Biotin Early Biotin Late Placebo Early Placebo

Marginal Biotin Deficiency is Very Teratogenic in ICR Mice No signs or symptoms in the dam Normal dam & fetal weight gain Resorptions < 6% and global disruption was not seen High rates of skeletal malformations No signs or symptoms in the dam Normal dam & fetal weight gain Resorptions < 6% and global disruption was not seen High rates of skeletal malformations

Whole Fetal Skeleton

Increase in Limb Malformations with Increasing Egg White Content Increase in Limb Malformations with Increasing Egg White Content Forelimb Hypoplasia Hindlimb Hypoplasia Control Malformations (%) Dietary Egg White (%)