b12 - bind, distribution, metabolism, excretion rates; grey hair

VITAMIN B12

Vitamin B12 (Cobalamin)

• Vitamin B12 is generic name for group of compounds called corrinoids– corrin nucleus– atom of cobalt in center– attached are one of following

• CN = cyanocobalamin (CNCbl)• OH=hydroxocobalamin (OHCbl)• 5’-adenosyl=adenosylcobalamin*(AdoCbl)• -CH3=methylcobalamin* (MeCbl)

Vitamin B12

P

H2N

N

N O

NH2

NH2

ONH2

O

HO HO

OH

N

HO

OO

NCo

N

O

O O-

O

C

NH

H2N

H2N

NN

Sources• Seldom found in foods from plants• Synthesis is limited to bacteria. Rumen microbial

synthesis depend on the supply of cobalt in the diet

• Animal products– Derive their cobalamin from micro-organisms– Meat, poultry, fish, shellfish, eggs, milk, milk products– Liver is a good source

• Supplements– Cyanocobalamin and hydroxycobalamin

Sources of Vitamin B12 in Foods

Food Vitamin B12

(µg/100g)

Food Vitamin B12

(µg/100g)

Meats Beef 1.94-3.64 Beef brain 7.83 Beef kidney 38.3 Beef liver 69-122 Chicken 0.32 Chicken liver 24.1 Ham 0.8 Pork 0.55 Turkey 0.379Dairy products Milk 0.36 Cheeses 0.36-1.71 Yoghurt 0.06-0.62

Fish and sea food Herring 4.3 Salmon 3.2 Trout 7.8 Tuna 2.8 Clams 19.1 Oysters 21.2 Lobster 1.28 Shrimp 1.9 Other Eggs, whole 1.26 Eggs white 0.09 Eggs yolk 9.26Vegetables,grains,fruits none contain vitamin B12

Digestion/Absorption• Ingested cobalamins must be released from food

matrix – Attached to polypeptides in foods– Release occurs via action of pepsin

• Functions at low pH • Requires adequate HCl production

• Released cobalamin interacts with:– R-binders (R-protein), Transcobalamin (TCI),

Haptocorrin (Hc)• R protein (found in saliva and gastric juice)• Non-specific• Complex moves from stomach into SI• In duodenum, R protein is hydrolyzed by proteases

– Inhibited by pancreatic insufficiency

Absorption• Cobalamins bind to IF in proximal intestine

– Cobalamin-IF complex travel to ileum– Binds to receptors and is slowly absorbed into enterocyte

• Can occur by passive diffusion when pharmacologic amounts are given– Used for people not producing IF

• Malabsorption occurs– Achlorydia– Lack of IF– Pancreatic insufficiency

• Absorption rate decreases with increased intake (80% to 3%)

In Circulation• Cobalamins bound to one of three proteins called

transcobalamins– TCI = binds 90% of vitamin B12 and may function as

circulating storage form (hoptocorrin)– TCII = carries newly absorbed cobalamin to tissues and

helps with uptake of cobalamin• Receptors on cells for TCII

– TCIII = delivers cobalamin from periphery back to liver– Methylcobalamin and adenosylcobalamin found

primarily in blood• Stored in liver (adenosylcobalamin)

Vitamin B12 Absorption in Ileum

Pernicious Anemia Toh, B-H et al. NEJM 337:1441,1991End stage of type A chronic atrophic (autoimmune) gastritis

B12

R + B12

B12

B12IF + B12

IF . B12

TCII . B12

IF

IF

IF

IF

IF

R

R

Pancreatic proteases(degradation of R protein)

Acid pH

Ileal receptor

Stomach

Vitamin B12 absorption

Transport• TC receptors are degraded upon cellular

uptake to release B12.• All vitamin within cell is bound to protein:

– Methionine synthetase (cytosol) and methymalonyl-CoA mutase (mitochondria)

• Distribution in tissues– Total body store 2-5mg– Liver (60%)– Long biological half life: 350-400 days in human.– Low reserve in infant: about 25 μg.– Plasma: methylcobalamin (60-80% of total)

Vitamin B12

• Metabolism– Whole-body turnover ~ 0.1% / day– Turnover rate is about 2.5 μg/day– Loss of vitamin mainly due to fecal loss

• Excretion: 0.1-0.2% of total body reserve

Metabolic Roles of Vitamin B12 in Animals

Enzyme Metabolic function Adenosylcobalamin Methylmalonyl-CoA mutase Conversion of methylmalonyl-CoA to succinyl-CoA in the degradation of propionate.

L-α-Leucine mutase Conversion of L-α-Leucine to aminoisocapronate as the first step in the synthesis/degradation of the amino acid Methylcobalamin Methionine synthetase Methylation of homocysteine to produce methionine, serving as the methyl group carrier between the donor 5-methyl-FH4 and the acceptor homocysteine

Functions• Three known enzymatic reactions require

Vitamin B12– Methylcobalamin required for methionine

synthesis from Hcy (cytosol)• Nitrous oxide inhibits MS• Oxidizes cobalt from 1+ to 3+ state• Must be in +1 state to function as coenzyme

– Adenosylcobalamin• Required for two reactions• Both are mutases and function in mitochondria

Possible role of vitamin B12 in choline and methionine

synthesis

Serine Methionine

Vitamin B6 CH3-cobalamin S-adenosylCO2 transmethylase homocysteine CH3-cobalamin Ethanolamine Choline

Adenosylcobalamin

• Methylmalonyl CoA mutase– Converts L-methylmalonyl CoA generated from

propionyl CoA to succinyl CoA– Propionyl CoA generated from oxidation of

• Odd chain fatty acids• Valine,methionine, isoleucine, threonine

– In cobalamin deficiency• Methylmalonyl CoA and methylmalonic acid (MMA)

accumulate; see rise in blood and urine• Have observed genetic defects in methylmalonyl CoA mutase

and adenosylcobalamin synthesis

Vitamin B12-dependent isomerization of methylmalonyl CoA to succinyl CoA

Valine CO2 methylmalonyl Isoleucine mutase Methionine Propionyl CoA Methylmalonyl CoA Succinyl CoA krebs’Threonine adenosyl Cycle cobalamin

Adenosylcobalamin

• Leucine Aminomutase– Requires adenosylcobalamin as co-enzyme– B leucine generated from bacteria in gut may be

converted to L-leucine in body– Beta leucine may also be generated from L-leucine

and undergo transamination and provide alternate route for leucine catabolism

Signs of Vitamin B12 Deficiency

Organ system Signs

General Growth Decrease Vital organs Hepatic, cardiac and renal steatosis Fetus Hemorrhage, myopathy, death

Circulatory Erythrocytes Anemia

Nervous Peripheral neuropathy

Deficiency• Definite association

– Megaloblastic anemia– Neuropathy

• Possible association– Atheroma

• Hcy• Pernicous anemia : no increase

– NTDs• B12 association

– Hepatic steatosis• Ethanol inhibits methionine synthase• Methionine and choline deficiency

Megaloblastic anemia• Low B12 low methionine and THF high tHcy

and 5-CH3-THF• Low THF low 5,10-CH2-THF low conversion of

dUMP to dTMP low DNA synthesis• Low methionine and SAM: low methylene

reductase– 5,10-CH2-THF 5-CH3-THF (irreversible)– 5-CH3-THF: poor substrate for glutamate synthetase,

poor incorporation into CH3-Cbl (folate deficiency)

Neuropathy• Undetected Vitamin B12 deficiency leads to

neuropathy (10+ years)• Cause

– Related to availability of methionine for SAM??– SAM required for methylation reactions

• Essential for myelin maintenance and thus neural function

• Neuropathy induced in animals by N2O inhalation• Neuropathy associated with N2O inhalation in

Humans• Congenital deficiency

Congenital Disorders of Vitamin B12 Metabolism

Condition Missing/deficient factor

Signs/symptoms

Methylmalonic aciduria

Lack of intrinsic factor

Imerslund-Gräsbeck syndrome

Lack of transcobalamins

Lack of R proteins

Methylmalonyl-CoA mutase

Intrinsic factor

IF receptors

Transcobalamins

R proteins

Methylmalonic aciduria, homocysteinuria, lethargy, muscle cramps, vomiting, mental retardation

Signs consistent with vitamin B12 deficiency

Specific malabsorption of vitamin B12

Severe (fatal) megaloblastic anemia appearing early in life

None

Disorders of Cbl absorption• Malabsorption of food

• Achlorydia: Corrected with synthetic source• Pancreatic Insufficiency

– Low pancreatic enzymes, bicarbonate, affect intestinal pH• Pernicious anemia

– Low If secretion, antibodies (blocking and binding)• Gastrectomy and destruction of gastric mucosa

» Treat with pharmacologic amounts of Vit B12

• Decreased absorptive surface– Ileal resection, celiac and tropical sprue, ileitis

• Parasitic Infections (Tape Worm)• Infestation of the intestinal lumen:

– Competition with bacteria• AIDS: Low B12 (uptake of IF-B12 complex)

Vitamin B12 deficiency

• Disorders of transport– TCII deficiency (1st or 2nd month of life)

• Inherited disorders of Cbl metabolism– Disorders of AdoCbl (CblA and CblB) and

methylmalonyl CoA mutase• OHCbl to AdoCbl!!• No megaloblastic anemia or neuropathy

• Defects in cellular CH3-Cbl and AdoCbl synthesis– Treatment with OH-Cbl: variable response

• Defects in CH3-Cbl syntheis– Failure to thrive, vomiting, anemia, neuropathy

Vitamin B12 (Cobalamin)

Common initial sign of B12 deficiency:

The red sore tongue, with atrophy of the papillae is often present in pernicious anemia and, in the case illustrated, angular stomatitis is also present.

Vitamin B12 (Cobalamin)

Pallor of pernicious anemia:

There is a pronounced lemon-yellowish tint to the skin together with faint icterus of the sclerae due to hyperbilirubinanemia. The skin is often velvety smooth, yet inelastic. It is remarkable how frequently patients have blonde or prematurely grey hair and light-colored irises.

Potential Causes of Vitamin B12 Deficiency

Cause Example

Inadequate intake Plant-derived, unsupplemented diet Impaired absorption Lack of IF Pancreatic insufficiency Intestinal parasitism Drug treatment

Vitamin B12 Deficiency• Inadequate absorption is primarily responsible• Inadequate intake is more common among

vegetarians– Prevent with consumption of fortified cereals

• Occurs in stages– Low serum concentrations– Low cell concentrations– Decreased DNA synthesis

• High Hcy and MMA– Megaloblastic Anemia

Assessment– Serum B12– MMA*– Hcy– Schilling test

• Used to determine problems with absorption• Administer radioactive vitamin B12 and measure its

urinary excretion