1. 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

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

3. 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

10. 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

22. 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

23. Standard Curves for Biotin

24. 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

25. 0 0 1000 2000 3000 4000 5000 0 0 5 5 10 15 20 25 30 35 Radioactivity (dpm) Retention Time (minute) Biotin Bisnorbiotin Biotin Sulfoxide HPLC of Radiolabeled Biotin Analogs

26. Urinary Analogs Analyzed Against Authentic Standards 35 30 25 20 15 10 5 5 0 0 0 0 5 5 15 20 25 Retention Time (min) Avidin-binding Substances (pmol/mg creatinine) unknown #1 unknown #2 biocytin unknown #5 biotin } } } } } ? d & l biotin sulfoxide } bisnorbiotin } } methyl ketone

27. Mole Percentage of Urinary Biotin and Metabolite in 10 Subjects Unk1Unk1 BSOBSOBNBBNBUnk2Unk2Unk3Unk3Unk4Unk4BiotinBiotinUnk5Unk5 00 2020 4040 6060 8080 Biotin Metabolites Biotin or Metabolite (mol/100 mol) Biotin or Metabolite (mol/100 mol)

28. 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 0 0 20 40 60 80 100 Biotin Metabolite

29. GCRC Study Design egg white diet (avidin) (avidin) non-biotin vitamin supplement biotin supplement Blood and urine collection -10 0 3 7 10 14 17 20 30

30. Urinary Excretion of Biotin 0 0 10 20 30 40 50 60 70 80 N. R. Study Day * p< 0.008 by range test after ANOVA Biotin (nmol/24 h) * * * * * * * * * * * * 0 0 3 3 7 7 10 14 17 20 Mean ± SD

31. Serum Concentration of Biotin 0 0 200 400 600 800 Serum Biotin Mean ± SD (pmol/L) 0 0 3 3 7 7 10 14 17 20 N. R. Study Day

32. 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:

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

34. Mechanism of Biotin-dependent Carboxylase (Pyruvate carboxylase)

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

36. 3-methylcrotonyl-CoA 3-Methylcrotonyl-CoACarboxylase3-Methylcrotonyl-CoACarboxylase 3-methylglutaconyl-CoA 3-HIA leucine

37. Urinary Excretion of 3-HIA 0 0 100 200 300 400 500 600 3-Hydroxyisovaleric Acid (µmol/24 h) N. R. * * * * * * * * * * 0 0 3 3 7 7 10 14 17 20 * p< 0.008 by range test after ANOVA * * Mean ± SD Study Day

38. 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: 1832 - 5, 1992. periconceptional vitamin supplementation”NEJM, 326: 1832 - 5, 1992. 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: 1832 - 5, 1992. periconceptional vitamin supplementation”NEJM, 326: 1832 - 5, 1992.

39. Urinary Excretion of Biotin n.s. p < 0.05 p < 0.003 control early late 0 0 20 40 60 80 100 120 140 160 Biotin Excretion (pmol/mg creat) Biotin Excretion (pmol/mg creat)

40. Urinary Excretion of 3-HIA 3HIA Excretion (µmol/24 h) 3HIA Excretion (µmol/24 h) 0 0 50 100 150 200 250 300 control early late p < 0.0001

41. Biotin Treatment Decreases 3-HIA Excretion Change in Urinary 3-HIA (mmol/mol creatinine) -15-15 -10-10 -5-5 00 55 Placebo Biotin p = 0.001 Late Biotin Early Biotin Late Placebo Early Placebo

42. 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

43. Whole Fetal Skeleton

44. Increase in Limb Malformations with Increasing Egg White Content Increase in Limb Malformations with Increasing Egg White Content 0 0 20 40 60 80 100 Forelimb Hypoplasia Hindlimb Hypoplasia Control 0 1 1.3 2 3 5 10 25 Malformations (%) Dietary Egg White (%)