Published on March 6, 2014
Folic Acid and Vitamin B 12 Dr. Abhishek Roy JR I, MD (Biochemistry), GGMC & JJH, Mumbai Email: firstname.lastname@example.org Dr. Abhishek Roy 1
Dealing with the topic • • • • • • • • Introduction Types and basic biochemistry Dietary Sources Absorption, Transport, Metabolism & Excretion RDA Deficiency and Clinical Manifestations Laboratory Assessments of Status Toxicity Dr. Abhishek Roy 2
Types and forms: • Folate (most oxidized form) • Dihydrofolate (FH2) • Tetrahydrofolate (FH4) (most reduced form) Basic Structure of Folate 1. Bicyclic Pteridine Ring 2. PABA( Para-amino-benzoic acid) 3. Polyglutamate Tail Dr. Abhishek Roy 3
Salient Points: • N5-Methyl-FH4 once formed, the step is irreversible. Dr. Abhishek Roy 4
Folate from food Sources Small degradation Liver Urine Folate Entero-hepatic circulation(through bile) Monoglutamates (Absorbed) Folate Conjugases Duodenum Folate N5-methyl FH4 Dr. Abhishek Roy Within Intestinal Cell 5
Vitamin B12 Structure • Contains a CORRIN ring. • Two of the four pyrrole rings are joined directly rather than joined by methylene bridges(heme) • Unusual feature- Cobalt coordinated with Corrin ring. • In the body, Co reacts with the carbon of a methyl group, forming methylcobalamin, or with the 5’carbon of 5’-deoxyadenosine, forming 5’-deoxyadenosylcobalamin. • The form of Vit B12 found in supplements is Cyanocobalamin. Dr. Abhishek Roy 6
Absorption and Transport of Vitamin B12 Dr. Abhishek Roy 7
RDA of FOLATE • Age Male • 0-6months* 65µg DFE* • 7-12months* 80µg DFE* • 1-3 years 150µg DFE • 4-8 years 200µg DFE • 9-13 years 300µg DFE • 14-18 years 400µg DFE • 19+ years 400µg DFE *Adequate Intake (AI) Female Pregnant Lactating 65µg DFE* 80µg DFE* 150µg DFE 200µg DFE 300µg DFE 400µg DFE 600µg DFE 600µg DFE 400µg DFE 600µg DFE 600µg DFE Dr. Abhishek Roy 8
• 1 DFE(Dietary Folate Equivalent) = 1 µg of Folic acid in dietary food. • 1DFE = 0.6µg of Folic acid when consumed along with dietary food. • 1 DFE= 0.5µg of Folic Acid when consumed in empty stomach. Dr. Abhishek Roy 9
RDA OF VITAMIN B12 • Age Male • 0-6months* 0.4µg • 7-12months* 0.5µg • 1-3 years 0.9µg • 4-8 years 1.2µg • 9-13 years 1.8µg • 14+ years 2.4µg • *Adequate Intake (AI) Female 0.4µg 0.5µg 0.9µg 1.2µg 1.8µg 2.4µg Dr. Abhishek Roy Pregnant Lactating 2.6µg 2.8µg 10
Dietary Sources of Folate • Spinach • Green Leafy Vegetables • Liver • Yeast • Legumes Natural Dietary Sources- Reduced Co-enzyme form Vitamin Supplements/Fortified Foods-Abhishek Roy Oxidized form of Pteridine Ring Dr. 11
Dietary Sources of Vitamin B12 Dr. Abhishek Roy 12
One Carbon Pool • The collection of one-carbon groups attached to FH4 is known as the one-carbon pool. • While attached to FH4, these one-carbon units can be oxidized and reduced. • The most oxidized form is N10-formyl FH4. The most reduced form is N5-methyl-FH4. Once the methyl group is formed, it is not readily reoxidized back to N5, N10 methylene FH4, and thus N5-methyl-FH4 will tend to accumulate in the cell. Dr. Abhishek Roy 13
Salient points of Serine as Carbon donor • Serine’s hydroxyl-methyl group is transferred to FH4 in a reversible reaction, catalyzed by the enzyme serine hydroxymethyltransferase. • This reaction produces glycine and N5, N10-methylene-FH4. • Because serine can be synthesized from 3-phosphoglycerate, an intermediate of glycolysis, dietary carbohydrate can serve as a source of carbon for the one-carbon pool. • The glycine that is produced may be further degraded by donation of a carbon to folate Dr. Abhishek Roy 14
• The chief carbon donator is SERINE. • Methionine metabolism is dependent both on the Vitamin B12 as well as Folate. Dr. Abhishek Roy 15
A deficiency of folate results in the accumulation of FIGLU, which is excreted in the urine. A histidine load test can be used for detecting folate deficiencies. Patients were given a test dose of histidine (a histidine load), and the amount of FIGLU that appeared in the urine was measured. Dr. Abhishek Roy 16
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Methyl Group What do you think the structure of Methotrexate resembles to? Folic Acid. Binds to DHFR 1000 times more strongly Dr. Abhishek Roy 19
Two main reactions of Vitamin B12 • The transfer of a methyl group from N5-methyl FH4 to homocysteine to form methionine. • The rearrangement of the methyl group of L-methylmalonyl CoA to form succinyl CoA Dr. Abhishek Roy 20
The Methyl-Trap Hypothesis • The equilibrium lies in the direction of the N5-methyl FH4 form. • This appears to be the most stable form of carbon attached to the vitamin. • However, in only one reaction can the methyl group be removed from N5-methyl FH4, and that is the methionine synthase reaction, which requires vitamin B12. • Therefore if there is Vitamin B12 deficiency, then most folate forms in body is “trapped” in N5-methyl FH4 form- Functional Folate Deficiency. • This is the Methyl-Trap Hypothesis. Dr. Abhishek Roy 21
How Folate, Vitamin B12 and SAM are related? Dr. Abhishek Roy 22
Tests done Serum Cobalamin • normal range- 160-200ng/L to 1000ng/L. • If megaloblastic anemia then levels are <100ng/L. • Measured by ELISA. Serum Methylmalonate and Homocysteine • Advantage- These can pick up def in early stages even in absence of hematological abnormalities or subnormal levels of serum cobalamin. • Disadvantage- Serum MMA varies with RF. Dr. Abhishek Roy 23
Serum Folate • Measured by ELISA. • Normal range : 2µg/L - 15µg/L Red Cell Folate • Valuable test for body folate stores. • It is though affected by recent diets and traces of hemolysis. • In normal adults, range is 160-640µg/L of packed red cells. Histidine Load test generally not done now a days Dr. Abhishek Roy 24
Detection of cause of Vitamin B12 by Schilling Test RL oral Vit B12 + IM Unlabelled VitB12 STAGE 1 Measure the 24hrs-48hrs urine sample Actual RL Vit B12- Dietary deficiency Decreased RL B12- Absorption problem NOTE: Unlabelled Vit B12 through IM is given only once Dr. Abhishek Roy 25
STAGE 2 RL oral Vit B12 + Intrinsic factor Measure the 24hrs-48hrs urine sample Actual RL Vit B12Pernicious anemia Dr. Abhishek Roy Decreased RL B12- No Dietary Def., No IF def. 26
RL oral Vit B12 + Antibiotics STAGE 3 Measure the 24hrs-48hrs urine sample Actual RL Vit B12- Bacterial Overgrowth Dr. Abhishek Roy Decreased RL B12- No dietary Def, No IF def, No Bact. Overgrowth 27
RL oral Vit B12 + Pancreatic enzymes STAGE 4 Measure the 24hrs-48hrs urine sample Actual RL Vit B12- Pancreatic Insufficiency like Chronic Pancreatitis Dr. Abhishek Roy 28
Deficiency of Vitamin B12 • Pernicious anemia with atrophic gastritis is the most common cause of its deficiency in the western countries, however, in India, alcoholism, malnutrition and ileo-cecal tuberculosis are the common causes. • Two most common manifestation 1. Neurological Manifestation(Caused By Hypomethylation of Nervous system) 2. Hematological Manifestation(Due to adverse effects of Vitamin B12 on Folate Metabolism) Dr. Abhishek Roy 29
Neurological symptoms • Symmetric numbness and tingling of the hands and feet, diminishing vibratory and position sense, and progression to a spastic gait disturbance. • The patient may become somnolent or may become extremely irritable (“megaloblastic madness”). • Blind Spots in Visual field followed by alterations in Gustatory and Olfactory function. • This is believed to be caused by hypomethylation within the nervous system, brought about by an inability to recycle homocysteine to methionine and from there to SAM. • Ultimately both ascending and descending tracts may be affected and get degenerated- SUBACUTE COMBINED DEGENERATION OF SPINAL CORD • The nervous system lacks the betaine pathway of methionine regeneration and is dependent on the B12 system. Dr. Abhishek Roy 30
Other neurological Symptoms • Additional symptoms of vitamin B12 deficiency include difficulty maintaining balance, depression, confusion, dementia, poor memory, and soreness of the mouth or tongue. • The neurological symptoms of vitamin B12 deficiency can occur without anemia, so early diagnosis and intervention is important to avoid irreversible damage. Dr. Abhishek Roy 31
Betaine Pathway of Methionine Regeneration Dr. Abhishek Roy 32
Severe Combined Degeneration of Spinal Cord Dr. Abhishek Roy 33
Hematological Manifestations Megaloblastic Anemia • The presence of red cells that are macrocytic and oval (macro-ovalocytes) is highly characteristic. • There is marked variation in the size (anisocytosis) and shape (poikilocytosis) of red cells. • Neutrophils are also larger than normal (macropolymorphonuclear) and hypersegmented, having five or more nuclear lobules instead of the normal three to four. • NOTE: Whatever the mechanism, lack of folate is the proximate cause of anemia in vitamin B12 deficiency, since the anemia improves with administration of folic acid- Functional Folate Deficiency Dr. Abhishek Roy 34
Hyper-segmented Neutrophils Dr. Abhishek Roy 35
Ineffective Erythropoeisis Dr. Abhishek Roy 36
Other system affected by Vitamin B12 and Folate • Epithelial surfaces: After marrow most frequently affected. Mouth, Stomach,small intestines, respiratory, urinary and female genital tracts- Macrocytosis + Increased multinucleate and dying cells. • Complications of Pregnancy: The gonads are also affected, and infertility is common in both men and women with either deficiency. Maternal folate deficiency has been implicated as a cause of prematurity, and both can cause recurrent fetal loss and neural tube defects. Dr. Abhishek Roy 37
CVS and Hyperhomocysteinemia Dr. Abhishek Roy 38
Neural Tube Defects • Failure of a portion of the neural tube to close, or reopening of a region of the tube after successful closure, may lead to one of several malformations. • Most common –Defect in caudal part of spinal cord. • Types of Spinal dysraphism- Spina bifida occulta, Myelomeningocele, Meningocele, Anencephaly. • Folate deficiency during the initial weeks of gestation has been implicated as a risk factor; differences in rates of neural tube defects between populations can be attributed in part to polymorphisms in enzymes of folic acid metabolism. • Folate deficiency may affect cell division during critical periods that coincide with closure of the neural tube. Dr. Abhishek Roy 39
Myelomeningocele Dr. Abhishek Roy 40
Anencephaly All that is left is small, vascular mass of disorganized neural tissue (cerebrovasculosa), mixed with choroid plexus. Dr. Abhishek Roy 41
Therapy for megaloblastic Anemia Cobalamin deficiency • Lifelong regular cobalamin injections. Surgery if tropical sprue(gut replacement surgeries), fish tapeworm, intestinal stagnant loop. • Replenishment of body store complete with six 1000µg injections of hydroxocobalamin given at 3- to 7-day interval. Maintenance therapy1000µg/3months. • Even in Pernicious Anemia, its documented that very large doses (1000-2000)µg oral doses can lead to absorption of Vit B12 from mucous membranes. Dr. Abhishek Roy 42
Folate Deficiency • Oral doses of 5-15mg folate daily are satisfactory. Its important to continue therapy for around 4months by which time all folate deficient red cells are replaced by folate repleted population. • Always check if the megaloblastic anemia is due to vit b12 def or not else folate will correct the anemia(As methyl trap is bypassed) but not the neurological symptoms of Vit B12. Pregnancy- Folic Acid 400µg daily, as supplement before and throughout pregnancy. Previous cases of NTDs mother, are given 5mg daily dose. Some studies say to give Zinc if folate supplements are to be given during pregnancy. Dr. Abhishek Roy 43
Hypervitaminosis • B12 is commonly given in doses much higher than the RDA (2.4 mcg (micrograms)) without known toxicity. Most B12 supplements will provide at least 2,000 mcg of B12 and some deliver as much as 5,000mcg. There has been no scientific evidence demonstrating any significant toxicty when given at this level. Currently no tolerable upper limit of B12 has been set by The Food and Nutrition Board indicating this lack of toxic effects • Folate: Folate is not considered to be toxic and even high doses of Folic Acid are considered to be safe and non-toxic. However, high intakes of Folic Acid can make it difficult to detect a Vitamin B12 deficiency because Folic Acid also reduces Vitamin B12 deficiency symptoms but without correcting the neurological damage that also occurs. This is why most Folic Acid products also contain Vitamin B12. • Adverse Effects may include: • fever • itching • mental changes • shortness of breath • skin rash • sleep disturbances • wheezing Dr. Abhishek Roy 44
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