Vitamins B6, B12 and folate. (Continuing Education).

Continuing education and the APD program

This quiz is an ideal activity for APD members to include in your CPD log, where it relates to personal learning goals. Record the time taken, to the nearest hour, to complete the quiz and any associated research.

Accredited Practising Dietitian

This quiz has been prepared by Dr Philippa Lyons- Wall, Queensland University of Technology and Dr Samir Samman, The University of Sydney. Correspondence should be directed to Philippa Lyons-Wall, School of

Introduction

Vitamins B6, B12 and folate are members of the B group of vitamins. Like other B vitamins, they are water soluble, required in the diet in relatively small quantities (mg or [micro]g) and act as coenzymes or cofactors in a range of metabolic pathways. The integral role of B6, B 12 and folate in pathways of DNA synthesis and methylation has led to recent scientific interest in their optimal intakes in the prevention or management of several chronic diseases (1).

Vitamin B6 occurs as three vitamer forms based on the pyridine ring: pyridoxine is the most stable and found especially in plants, while pyridoxal and pyridoxamine are found mainly in animals; each vitamer can occur as a phosphorylated form. Pyridoxal-phosphate is the active form but all the vitamers are readily interconverted in the body. Vitamin B12 is used to designate a family of compounds called cobalamins, distinguished by a corrin ring, with a central cobalt atom which confers the vitamin's bright red colour. The active coenzyme contains either a deoxyadenosyl or methyl group attached to the cobalt atom. Vitamin B12 is one of the most stable vitamins. Folate is the generic term for a family of compounds with similar chemical structure to folic acid or pteroylmonoglutamic acid. Folic acid has a single glutamic acid residue while conjugated folic acid has between 2 and 8 glutamic acid residues. Both folic acid and conjugated folic acid are required to undergo reduction to the biologically-active tetrahyd rofolic acid.

The following nutritional science quiz will test your knowledge of basic concepts including major food sources and their absorption, utilisation and excretion, in relation to both deficiency and toxicity of the vitamin.

1. Which of the following foods are good sources of vitamin B12?

a. oysters, beef, liver

b. carrot, pumpkin, mango

c. liver, spinach, breakfast cereal

d. wheat bran, sunflower seeds, fish

2. Why has the term 'folic acid equivalents' been suggested to describe the folic acid content of foods?

a. different forms of the vitamin are absorbed with different efficiency

b. components in foods other than folic acid, have folic acid activity

c. to identify the effects of dietary components on neural tube defects 

d. to make terminology consistent with other vitamins

3. Which anaemia is associated with vitamin B6 deficiency?

a. megaloblastic anaemia

b. hypochromic microcytic anaemia

c. pernicious anaemia

d. haemolytic anaemia

4. Deficiency of vitamin B12 is rare due to:

a. low requirement relative to stores in liver

b. low urinary excretion relative to intake

c. presence of specific binding proteins that bind and retain B12

d. all of the above

5. An elevation in plasma homocysteine concentration can result from a deficiency of which vitamin(s)?

a. folate

b. B12

c. B6

d. all of the above

6. Which of the following statements is correct? Pharmacological doses of vitamin B6:

a. are not toxic because vitamin B6 is water soluble and readily excreted

b. can cause unsteady gait and loss of sensation in the feet and hands

c. can mask symptoms of vitamin B12 deficiency 

d. could cause depletion of methyl groups

7. Which of the following is essential for absorption of vitamin B12?

a. hydrolysis of the glucoside moiety 

b. cleavage of the polyglutamyl side chain

c. intrinsic factor

d. simple diffusion

8. Voluntary fortification with folate was introduced originally as a result of convincing evidence which showed:

a. reduction in risk of neural tube defects

b. dietary intake of folate was below the recommended level of intake

c. reduction of plasma homocysteine

d. all of the above

9. What is the main cause of vitamin B12 deficiency in elderly individuals?

a. inadequate intake

b. reduced absorption

c. increased faecal losses due to impaired enterohepatic recycling of B12

d. all of the above

10. Which of the following aspects of vitamin B6 is correct?

a. requirement is linked to intake of protein

b. involved in synthesis of niacin from tryptophan via the hepatic kynurenine pathway

c. is a coenzyme in utilisation of muscle glycogen

d. all of the above

Answers

1. a

Vitamin B12 is synthesised almost exclusively by micro-organisms and dietary sources are animal products that acquire B12 from either bacterial fermentation (e.g. cheeses) or from micro-organisms ingested in the diet (2). Rich sources include oysters which are filter feeders and accumulate B12 from bacteria in water; meat of ruminant animals (e.g. beef, lamb) that obtain B12 from bacteria in the rumen; and liver which provides an excellent storage depot for B12. Small amounts of B12 can occur in plants through contamination from soil or water (e.g. mushrooms, seaweed) or possibly via nitrogen-fixing bacteria in root nodules of legumes. Folate and vitamin B6 occur widely in animals and plants with considerable overlap in supply. Liver and green leafy vegetables (e.g. spinach) provide good sources of endogenous folate and B6; some processed foods (e.g. breakfast cereals) are fortified with folate. Wheat bran, sunflower seeds and fish are good sources of B6; wheat bran is also a notable source of folate.

2. a

Folic acid in foods occurs mainly in a form conjugated to multiple glutamate residues which must first be hydrolysed to form free folic acid before absorption (2). Hydrolysis is accomplished by conjugases secreted in the upper small intestine although absorption of the different conjugated forms is variable. While almost all of the monoglutamate is available for absorption, only 30% to 80% of the polyglutamates are available due in part to the presence of conjugase inhibitors in certain plant foods (e.g. bananas, oranges, beans) (3). Zinc deficiency and chronic high alcohol intakes can also reduce folate absorption since conjugase is zinc dependent and inhibited by ethanol.

Synthetic folate as a supplement or fortified product is in the free form and well absorbed. The differences in absorbability have led to the concept of dietary folate equivalents (DFE), where 1 DFE = 1 [micro]g food folate = 0.6 [micro]g folic acid from fortified food or supplement (4). Some other vitamins (e.g. niacin, retinol) are expressed in 'equivalents' because the active form is derived from different precursors. However, not all vitamins exist in multiple forms (e.g. riboflavin, thiamin) and therefore the term equivalents is not universally required.

3. b

Deficiency of B6 results in hypochromic microcytic anaemia due to impaired synthesis of the haem moiety of haemoglobin; erythrocytes are small and pale due to the low content of oxygenated haemoglobin. B6 is required as a coenzyme for aminolevulinic acid synthase which catalyses the first step in the synthesis of haem. Deficiency of folate results in megaloblastic anaemia due to impaired synthesis of DNA with failure of normal cell division and maturation of the red blood cells. The reduced cell division results in large red blood cells (megaloblasts) that are immature and, unlike normal red blood cells, retain a nucleus. Since vitamin B12 acts with folate in the pathway of DNA synthesis, B12 deficiency can also result in a megaloblastic anaemia that is morphologically similar to folatedeficiency anaemia but develops more rapidly (3). Pernicious anaemia is B12 deficiency arising from the lack of intrinsic factor required for the enteric absorption of the vitamin. Haemolytic anaemia can be caused by vitamin E deficiency which results in increased destruction of red blood cells. Vitamin E is a lipid-soluble antioxidant that preserves the structural integrity of cellular membranes.

4. d

Vitamin B12 is stored in relatively large amounts mainly in the liver (60%) and muscles (30%), with typical body stores ranging from 2 to 5 mg. A small amount (2 to 5 [micro]g/d) is excreted, mainly into bile, and this provides the basis for the small dietary requirement (RDI is 2 [micro]g/d) (2). The vitamin is well retained in plasma and cells due to binding to specific proteins called transcobalamins or to B12-dependent enzymes, and unlike with most B vitamins, there are minimal losses of B12 in urine. Good storage capacity and minimal excretion together with a long biological half life (350--400 d) mean that B12 deficiency is rare and the timeframe for deficiency to develop is three to six years. Elevated concentrations of urinary methylmalonic acid (MMA) are a biomarker of B12 deficiency; risk groups include vegans and breastfed infants of vegetarian mothers (2).

5. d

Homocysteine is an intermediate in the methylation cycle which utilises all three vitamins, folate, B12 and B6. Folate, in the form of methyl tetrahydrofolate, donates a methyl group to homocysteine to form methionine. This reaction is dependent on the action of vitamin B12 as a coenzyme. Therefore in the absence of folate and /or B12, the conversion of homocysteine to methionine does not occur and homocysteine accumulates. An alternative metabolic fate of homocysteine is its degradation to cystathionine in a B6-dependent reaction. Therefore in the absence of B6, homocysteine accumulates. Raised plasma homocysteine has been implicated in the development of neural tube defects and vascular disease.

Nutritional management of homocysteinaemia can be confounded by the presence of a genetic defect in methylene tetrahydrofolate reductase (MTHFR), which is involved in recycling of the active form of folic acid. A mutation in MTHFR has been associated with reduced enzymatic activity, lowered plasma folate and elevated plasma homocysteine. A significant proportion of the population (5% to 15%) are homozygotes with increased risk of vascular disease and neural tube defects in pregnancy. Folate supplements may be most beneficial in this subset of high-risk individuals. Vitamin B12 deficiency, as indicated by an increased excretion of methylmalonic acid, could also be a primary cause of homocysteinaemia, especially in older individuals (2).

6. b

Although vitamin B6 is water soluble and readily excreted in urine, toxicity has been reported with individuals taking gram doses (6 g/d) of pyridoxine for five to 40 months (3). Symptoms include an unsteady gait with impaired joint position and vibratory senses, numbness of feet and hands and decreased sensation of lips and tongue. When pyridoxine ingestion is ceased, recovery is slow and not always complete. The mechanism is unclear but excess pyridoxine appears to cause degeneration of large sensory nerve axons in the peripheral nerves (4). The lowest observed adverse effect level is 500 mg (4) which is approximately 200 to 500 times the RDI (1 to 2 mg/d). Vitamin B12 and folic acid are not toxic in doses that exceed the RDI by several hundred times. Both vitamins need to be bound to proteins to be retained in serum and storage tissues and intakes that exceed the binding capacity in serum and tissues are excreted (3). Unmonitored use of folate supplements to treat megaloblastic anaemia caused by B12 defici ency, may cause short-term improvement in the anaemia, but allows neurological damage to progress irreversibly, due to the underlying B12 deficiency.

7. c

In the stomach, gastric acid and pepsin are required to release vitamin B12 from proteins in the food matrix. The free vitamin then binds to an R protein and moves into the small intestine where the R protein is hydrolysed by pancreatic proteases. The free vitamin then binds with a glycoprotein, intrinsic factor (IF), originating from the gastric parietal cells. The B12-IF complex escapes hydrolytic attack from intestinal proteases and travels to the ileum where specific IF receptors facilitate absorption of the vitamin. It is the requirement for intrinsic factor that makes B12 absorption unique, and both the stomach and ileum (especially the terminal ileum) must be functioning normally for effective absorption to occur.

In some plant but not animal foods vitamin B6 occurs as a pyridoxine glucoside. This form is less bioavailable than other forms of B6, possibly due to the inability of mammalian glucosidase to hydrolyse the sugar moiety (4); 5% of B6 in sunflower seeds, 36% in wheat bran and 50% in spinach exist as the glucoside form (3).

8. a

Evidence from a range of human studies conducted between 1980 and 1995 has shown convincingly that increased intake of folate in the early stages of pregnancy can reduce the incidence of neural tube defects by about 75% (5). The RDI for folate is 0.2 mg/d and increases to 0.4 mg/d during pregnancy. The need for folate is crucial when the neural tube normally closes at days 21 to 28 after conception, often before a woman realises she is pregnant. As a result of these studies voluntary fortification up to 50% RDI per serve is now permitted for selected foods including breakfast cereals, flour, bread, pasta, yeast extracts and fruit and vegetable juices (6). A higher dose is needed to prevent recurrence of neural tube defects in women with a history of the disorder. This is hard to achieve by consuming fortified foods or folate-rich sources alone and a supplement of 4 mg/d is recommended, periconceptually, for women at high risk. Possible reduction in plasma homocysteine is an additional benefit of folate fortif ication but was not the primary reason for fortification.

9. b

Vitamin B12 deficiency in elderly individuals is caused mainly by reduced absorption. This can arise from a number of factors which include: (i) decreased synthesis of intrinsic factor (IF) due to the atrophy of gastric mucosa that can occur with ageing; (ii) interaction with drugs, such as antacids, which reduce secretion of gastric acid needed to release B12 from its binding to proteins in the food matrix; and (iii) surgical procedures, such as gastrectomy or resection of the terminal ileum, which can result in reduced absorption due to loss of secretion of IF or to removal of IF receptors, respectively. Intake of B12 is usually adequate in elderly individuals since it is found widely in all animal foods and requirement is relatively small. Inadequate intake may occur in elderly vegans who do not take supplements, however this group represents a small proportion of the population.

10. d

The RDI for vitamin B6 is 0.02 mg/g protein or 0.9 to 1.4 mg/d for women and 1.3 to 1.9 mg/d for men, assuming a protein intake of 10% to 15% of energy. The requirement is linked to dietary protein because pyridoxal phosphate, the active form of B6, is a coenzyme for almost all reactions that involve amino acid metabolism. Examples of B6 dependent enzymes are the aminotransferases, which are central in amino acid metabolism, and kynureninase, which is required for the conversion of tryptophan to niacin. Pyridoxal phosphate also acts as a coenzyme for glycogen phosphorylase which releases glucose-1-phosphate from glycogen, an important step in the utilisation of glycogen in muscle. Due to the relatively large muscle mass and wide utilisation of glycogen phosphorylase, this role accounts for more than half of B6 in the body (2).

References

(1.) Mann J, Truswell AS, editors. Essentials of human nutrition. 2nd ed. Oxford: Oxford University Press; 2002.

(2.) Combs GF. The vitamins: Fundamental aspects in nutrition and health. 2nd ed. New York: Academic Press; 1998.

(3.) Shils ME, Olson JA, Shike M, Ross AC. Modern nutrition in health and disease. 9th ed. Sydney: Williams & Wilkins; 1999.

(4.) Groff JL, Gropper SS. Advanced nutrition and human metabolism. 3rd ed. California: Wadsworth Thomson Learning; 2000.

(5.) Bower C. Folate and the prevention of birth defects. Aust J Nutr Diet 1996;53(Suppl):5S-8S.

(6.) Truswell AS. Prospects for folic acid fortification and its monitoring. Aust J Nutr Diet 1996;53(Suppl):17S-23S.