MED 562 Fall 2017 Water Soluble Vitamins Problem Set 2 1) Cytochrome P450 Reductase (CPR) is an enzyme responsible for the reduction of a large portion of the cytochrome P450 (CYP) enzymes. a. What cofactor is required for CPR activity, and from what vitamin is the cofactor derived? b. Do you expect a purified solution of CPR to be colored, and if so what color would it be (you may assume that all enzyme is in its oxidized state)? c. Suppose CYP3A4 and excess ADPH are added to the solution. What color change (if any) would you expect as a result (ignoring any color contribution from the 3A4)? a. Cofactors involved in CPR activity are FAD and FM, which are derived from riboflavin (B2). b. Assuming that the FAD and FM cofactors are completely oxidized, the solution be yellow. c. ADPH acts as a two-electron donor to FAD and FM, thus the cofactors should be converted to their reduced, colorless forms. 2) What are the main types of reactions that use folates as a cofactor? Methylation reactions where folate as cofactor provides a single carbon: 1. Conversion of homocysteine to methionine, also uses B12 2. ucleic acid synthesis a. Purine synthesis (C2 and C8) b. Pyrimidine synthesis to make thymidine from uridine 3) What is one of the most important concerns with folate deficiency (especially for pregnant women or women who may become pregnant)? Folate deficiency is associated with teratogenicity such as orofacial clefts and neural tube defects. 4) How does Methotrexate affect folates? Describe the therapeutic strategy of Leucovorin rescue. Methotrexate inhibits DHFA reductase, which prevents formation of THFA, the active coenzyme form of folic acid. By supplementing certain patients with leucovorin (5 formyl THFA), a higher dose can be administered because 5-formyl THFA can be readily converted to THFA and other forms of THFA. 5) What makes high dose folate supplements risky? See answer to 6. 6) How does Vitamin B 12 deficiency promote folate deficiency, in biochemical terms? High dose supplementation of Vitamin B9 could mask Vitamin B12 deficiency. B12 recycles methyl-thfa back to THFA, so B12 deficiency could lead to folate deficiency. However, if you supplement with too much folate, the hematological symptoms may go away by increasing levels of folate, while the symptoms related to pernicious anemia caused by B12 deficiency will go unchecked. This is especially dangerous because B12 deficiency can lead to neurological damage. 7) What two classes of enzymes use Vitamin B 12 as a cofactor? What are their main functions? 1. Methionine synthase uses methyl B12 (coenzyme form is 5-methyl cobalamin) to convert homocysteine to methionine and also for folate recycling.
2. Methyl malonyl CoA mutase uses B12 (coenzyme form is 5-deoxyadenosyl cobalamin) in the oxidation of odd-number fatty acid chains. 8) How is Vitamin B 12 absorbed? In cases of B 12 deficiency, what are the major routes of administration and what are the rationalizations for each? Hydrochloric acid in the stomach splits B12 from peptide links in food and intrinsic factor (glycoprotein) transports B12 across the intestinal wall for absorption in the ileum. In the case of pernicious anemia caused by deficiencies in intrinsic factor synthesis, oral supplementation does not work and IM injection is preferred, although sublingual or nasal spray may also work. If pernicious anemia is caused by low stomach acid, oral supplementation could still work to some extent. 9) What type of chemical reactions is Coenzyme A (CoASH) used for and what vitamin is converted to Coenzyme A? Vitamin B5 is converted to Coenzyme A by addition of b-mercaptoethylamine. CoA is primarily used for the transfer of acyl groups (due to its high energy thioester bond) necessary in the metabolism of fatty acids, amino acids, and carbohydrates. 10) What vitamin deficiency can be induced by consumption of raw egg whites? Why? (Bonus: How do biochemists leverage this effect for research?) B7 or biotin. Raw egg whites contain a protein called avidin which will bind strongly to biotin and lead to deficiency state. Biotin-avidin binding is one of the strongest non-covalent interactions in nature, thus can be used by researchers to pull down proteins of interest by tagging the proteins with biotin and then passing through an avidin-affinity column. 11) How to check vitamin B12 deficiency? Schilling s Test with radiolabeled B12, or determination of methyl malonic acid levels in urine. 12) What disease commonly occurred in the corn belt of the US in the 1900 s? What could have remedied this situation? Populations within the corn belt suffered from the disease pellagra due to the lack of digestible niacin and tryptophan in corn. This could have been avoided by food fortification or supplementation with niacin or by adopting the ancient Latin American tradition of soaking corn in alkaline lime water to break down the kernels and liberate nutritionally available niacin. 13) What enzymatic reactions does biotin mostly participate in? Carboxylation reactions in lipid and carbohydrate synthesis, where carboxybiotin is the active species. 14) What are the major functions of Vitamin C in the cell? All of its functions relate to its ability to donate electrons: 1. antioxidant (free radical scavenger) 2. enzyme cofactor for numerous reactions, ex. hydroxylation of proline and lysine in collagen synthesis 3. absorption of iron
15) What obel Prize winner claimed that mega-dosing (multiple grams/day) of Vitamin C was How to Live Longer and Feel Better? Do you agree or disagree with the efficacy of high intake of Vitamin C in prevention of disease? Linus Pauling. There is no truly correct answer to the second question however, prevailing research has not shown any benefits to mega-dosing Vitamin C in reducing the risk of cancer and other infections. Additionally, there is an upper limit to the amount of Vitamin C that can be absorbed per day so additional intake would mostly be excreted in the urine. 16) How to check folate deficiency? Folate level in erythrocytes. 17) Why do alcoholics frequently suffer from folate deficiency? Alcohol decreases enterohepatic circulation of folate. 18) The tolerable Upper Limit dose is the: a. highest dose of a vitamin at which no toxicity or insufficiency is observed. b. the lowest does of a vitamin at which benefits are observed with supplementation. c. 1.2 times the RDA. d. none of the above. 19) If the EAR for a vitamin is 0.5 mgs/day then the RDA is: a. 0.6 mg/day b. 0.5 mg/day c. 0.75 mg/day d. 1.0 mg/day e. none of the above 20) Which of the following statement is false? a. Vitamin C is not well retained in our body b. verdosing of vitamin C likely causes toxicity c. verdose of vitamin A likely causes toxicity d. Vitamin B1 and C needs regular replacement 21) Rice polishings containing which vitamin and is responsible for beriberi resulting from overconsumption of polished rice in Ancient Asia?
a. Vitamin C b. Vitamin A c. Vitamin B1 d. Vitamin D 22) Which imine form of pyridoxal below yields an amino acid upon hydrolysis [P= HP 3 - ]: A B C D E Enzyme 2 P R 2 P H 2 2 2 P H 2 2 P R C 2 2 P 23) Which of the following are possible fates for the hydroxyethyl TPP form of thiamine: a. decarboxylation to yield C 2 and pyruvate b. deprotonation to a carbanion followed by attack at an aldehydic carbon to transfer two carbons to a sugar c. attack by ethanol to reduce systemic ethanol levels and benefit alcoholics d. reduce dehydroascorbate back to vitamin C 2 2 P P H S 24) Which vitamin need is proportional to calorie intake? a. Riboflavin b. Folic acid c. Thiamin d. B12 H
25) Tryptophan is the biosynthetic precursor to which vitamin? f. iacin And which vitamin is involved in this transformation? c. Pyridoxal a. Thiamin b. Riboflavin c. Pyridoxal d. Folic acid e. B12 f. iacin 26) Match the test for deficiency of a vitamin, with the vitamin: Vitamin B 2 c. erythrocyte glutathione reductase activity Vitamin B 12 a. urine methylmalonic acid level a. urine methylmalonic acid level b. transketolase assay in red blood cells c. erythrocyte glutathione reductase activity d. erythrocyte transaminase activity 27) To lower the plasma level of homocysteine, the best way is to supplement with: a. Thiamine b. Thiamine, riboflavin and B 6 c. Vitamin B 6, folic acid and cobalamin d. Thiamine, riboflavin and niacin 28) In principle, which drugs or vitamins below could possibly result in iatragenic Vitamin B 6 deficiency if used chronically, based on the structures shown: H H H 2 H a. Phenytoin b. Isoniazid c. Methotrexate d. Trimethoprim
29) Choose the correct description about Leucovorin: a. is contraindicated in levo-dpa therapy b. can cause vitamin B 6 deficiency c. can allow ordinarily lethal dose of methotrexate to be used against tumor d. act as an antibiotic by inhibiting bacterial DHFA reductase 30) An elderly patient with pernicious anemia and low gastric HCl would most likely benefit from: a. oral supplements containing vitamin B 12. b. oral supplements containing vitamin B 9. c. IM injections of vitamin B 12. d. transdermal patches of containing vitamin B 6. 31) Please write the products for the following transformations and specify which vitamins participated as a cofactor. H H C H 2 C 2 2 Prolyl 4-hydroxylase ( Vitamin C ) H H C H 2 C 2 H H