Vitamin B2 (RIBOFLAVIN)

VITAMIN RIBOFLAVIN AND ITS METABOLISM

1. VITAMIN B2 DR.KOPPUKONDA RAVIBABU B.SC.,M.B.B.S.,MD BIOCHEMISTRY

2. Vitamin B2 Vitamin B2, also known as riboflavin , is an essential component of coenzymes that are involved in reduction-oxidation ( redox ) reactions in the body.

3. Chemistry Flavins are a family of yellow-colored compounds with the basic structure of 7,8-dimethyl-10-alkylisoalloxazine. Riboflavin, commonly known as vitamin B2, is the precursor of all biologically important flavins , notably flavin mononucleotide (FMN ) (riboflavin-5′-phosphate) and flavin adenine dinucleotide (FAD) . Riboflavin and its related metabolites act as cofactors to several redox enzymes . FMN is formed from riboflavin by flavokinase -catalyze d phosphorylation , and FAD is formed from FMN and ATP by the action of FAD synthetase , also called pyrophosphorylase . FAD is further converted by covalent bonding to form various tissue flavoproteins . Flavins are stable during exposure to heat but are decomposed by light , which causes photodegradation of the D- ribitol side chain at position 10 of the isoalloxazine ring system to ultimately yield lumiflavin .

4. Chemistry Flavins are stable during exposure to heat but are decomposed by light , which causes photodegradation of the D- ribitol side chain at position 10 of the isoalloxazine ring system to ultimately yield lumiflavin (7,8,1 trimethylisoalloxazine ) under alkaline conditions and lumichrome (7,8-dimethylalloxazine) at all pH values, especially in neutral-to-acidic solutions. Flavins are chemically and biologically reduced to nearly colorless compounds that rapidly re-oxidize on exposure to air (O2).

5. Dietary Sources R ich sources of the coenzyme forms of the vitamin include liver , kidney, and heart. Many vegetables are also good sources , but cereals are low in flavin content. Current practices of Fortification and enrichment of cereal products have made these significant contributors to the daily requirement. Milk from cows and from humans is a good source of the vitamin and probably the main source in western diets. L oss can occur from exposure to light during pasteurization and bottling, or as a result of irradiation to increase vitamin D content.

6. Absorption, Transport, Metabolism, and Excretion Most dietary riboflavin is taken in as a complex of proteins with the coenzymes FMN and FAD. These coenzymes are released from noncovalent attachment to proteins as a consequence of gastric acidification. Nonspecific action of pyrophosphatase and phosphatase on the coenzyme occurs in the upper gut. The vitamin is primarily absorbed in the proximal small intestine by a saturable transport system that is rapid and proportional to intake before leveling off at doses near 27 mg/ day of riboflavin/day. Bile salts appear to facilitate uptake, and a modest amount of the vitamin circulates via the enterohepatic system. Active transport at lower concentrations of intake was believed to be Na ion–dependent and to involve phosphorylation , although later work suggested that uptake is independent of Na ions

7. Absorption, Transport, Metabolism, and Excretion The transport of flavins in human blood involves loose binding to albumin and tight binding to numerous globulins, with major binding noted to several classes of immunoglobulins ( IgA , IgG , and IgM ). Pregnancy increases the concentration of carrier protein for riboflavin , which results in a higher rate of riboflavin uptake at the maternal surface of the placenta . Uptake of riboflavin in the liver is facilitated, possibly requiring a specific carrier at physiologic concentrations, but it can occur by diffusion at higher concentrations.

8. Absorption, Transport, Metabolism, and Excretion Conversion of riboflavin to coenzymes occurs within the cellular cytoplasm of most tissues but particularly in the small intestine, liver, heart, and kidney. The obligatory first step is the ATP-dependent phosphorylation of the vitamin catalyzed by flavokinase . The FMN product can be complexed with specific apoenzymes to form several functional flavoproteins , but the larger quantity is further converted to FAD in a second ATP-dependent reaction catalyzed by FAD synthetase ( pyrophosphorylase ). Biosynthesis of flavocoenzymes , particularly at the flavokinase step, is likely tightly regulated . Thyroxine and triiodothyronine stimulate FMN and FAD synthesis in mammalian systems. FAD is the predominant flavocoenzyme present in tissue, where it is complexed mainly with numerous flavoprotein dehydrogenases and oxidases . Some FAD (<10%) can become covalently linked to any of five specific amino acid residues of a few important apoenzymes .

9. Absorption, Transport, Metabolism, and Excretion Examples include 8α- N(3)- histidyl FAD within succinate dehydrogenase and 8α- S-cysteinyl FAD within monoamine oxidase , both of mitochondrial localization. Turnover of covalently attached flavocoenzymes requires intracellular proteolysis, and further degradation of the coenzymes involves nonspecific pyrophosphatase cleavage of FAD to FMN and AMP, and further action by nonspecific phosphates on FMN and AMP. Because there is little storage of riboflavin as such, urinary excretion reflects dietary intake. Milk contains reasonable quantities of the vitamin and lesser amounts of coenzyme, principally FMN. Smaller quantities of side chain degradation products such as lumichrome , 10-formylmethylflavin and 10-(2′-hydroxyethyl) flavin , and ring-altered compounds are excreted. This may largely result from the action of intestinal microorganisms. Traces of 8α-flavin peptides and catabolites are found in urine and feces.

10. Functions These derivatives are capable of one- and two-electron transfer processes and play a pivotal role in coupling the two-electron oxidation of most organic substrates to the one-electron transfer of the respiratory chain, thus becoming involved in energy production. They also function as electrophiles and nucleophiles , with covalent intermediates of flavin and substrate frequently involved in catalysis . Flavoproteins catalyze dehydrogenation reactions , hydroxylations , oxidative decarboxylations , deoxygenations , and reductions of O2 to H2O2. Drug metabolism in conjunction with the cytochrome P450 enzymes and lipid metabolism.

11. Functions Flavins also have pro-oxidative and antioxidative functions. They are believed to contribute to oxidative stress through their ability to produce superoxide and to catalyze the production of H2O2. As an antioxidant, FAD is a coenzyme to glutathione reductase in the regeneration of reduced glutathione from oxidized glutathione, which is necessary for the removal of lipid peroxides. Riboflavin deficiency is associated with increased lipid peroxidation . Flavins have also been linked with apoptosi s and have homocysteine -lowering properties. FAD is a cofactor to methylenetetrahydrofolate reductase (MTHFR) in the remethylation of homocysteine . An interaction between folate , riboflavin, and the genotype of MTHFR is apparent, especially in co lorectal cancer.

12. Functions Prophylaxis of migraine attacks and treatment of lactic acidosis caused by the use of nucleoside reverse transcriptase inhibitors in patients with AIDS or by genetic defects in the mitochondrial respiratory chain, as seen in Leigh disease. Riboflavin is also effective in treating the lipid storage myopathy associated with mutations of the ETFDH (electron-transferring flavoprotein dehydrogenase ) gene. Antagonism of riboflavin metabolism has been used as an anti-infective agent, notably in malaria treatment.

13. Requirements and Reference Nutrient Intakes Riboflavin status has been assessed on the basis of the relationship of dietary intake to overt signs of hyporiboflavinosis , urinary excretion of the vitamin , erythrocyte riboflavin content, and erythrocyte glutathione reductase activity. At least 0.5 mg of riboflavin/1000 kcal is required by adults, and 0.6 mg/1000 kcal constitutes the allowance suggested for all ages. RDA has been set at 1.3 mg/day for men 19 to 70 years of age and older, and 1.1 mg/day for women in the same age group. Children 1 to 3 years old have an RDA of 0.5 mg/day, which increases to 0.6 mg/day up to age 8 years.

14. Requirements and Reference Nutrient Intakes Because pregnant women tend to excrete less riboflavin as pregnancy progresses and additionally exhibit FAD stimulation of erythrocyte glutathione reductase activity, recommended allowances call for an additional 0.3 mg/day during pregnancy. During lactation , between 18 and 80 μg of riboflavin is secreted daily into every 100 mL of human milk. An infant will ingest an average of 750 mL of milk per day during its first 6 months and 600 mL / day for the next 6 months, this secretion rate translates into an ingestion of between 100 and 600 μg of riboflavin per day/day. RDA for lactating women should be increased by an additional 400 to 500 μg /day. Accordingly, the RDA for lactating women has been set at 1.6 mg/day.

15. Intravenous Supply The recommended intravenous supply of riboflavin in adults is 3.6 mg/day. Riboflavin in TPN mixtures may be subject to degradation under exposure to ultraviolet light, so bags containing riboflavin should contain fat emulsion or should be covered to provide protection from light.

16. Riboflavin Deficiency Causes: Riboflavin is synthesized by the intestinal flora. Riboflavin deficiency usually accompanies other deficiency diseases such as beriberi, pellagra and Kwashiorkor.

17. Deficiency In addition to poor intake , hypothyroidism and adrenal insufficiency, which inhibit the conversion of riboflavin to its coenzyme derivatives, by drugs such as chlorpromazine, imipramine , and amitriptyline , which have a similar tricyclic structure to riboflavin , by the anticancer drug doxorubicin and the antimalarial quinacrine . Excess ethanol ingestion interferes with both digestion and absorption of riboflavin.

18. Deficiency Riboflavin coenzymes are involved in the metabolism of vitamin B12 and folic acid (irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate [5-MTHF]), and therefore, are a determinant of plasma homocysteine concentration, pyridoxine (conversion to pyridoxal 5-phosphate) and niacin (conversion of 5-hydroxytryptamine to tryptophan, which is required for niacin synthesis). Therefore, deficiency will affect enzyme systems other than those requiring flavin coenzymes per se. With increasing riboflavin deficiency, tissue concentrations of FMN and FAD will fall, as does flavokinase activity, thus further decreasing FMN concentrations. FMN concentrations are decreased proportionately more than FAD concentrations. Decreases in the activities of enzymes requiring FMN generally follow the fall in tissue concentrations, whereas FAD-dependent enzymes are more variably affected.

19. Deficiency The deficiency syndrome is characterized by (1) sore throat, (2) hyperemia, (3) edema of the pharyngeal and oral mucous membranes, (4) cheilosis , (5) angular stomatitis , (6) glossitis (magenta tongue ), (7) seborrheic dermatitis , and (8) normochromic , normocytic anemia associated with pure red blood cell (RBC) aplasia of the bone marrow.

20. Deficiency Because riboflavin-derived cofactors are essential for the function of numerous dehydrogenases , they have been linked to genetic defects such as Brown- Vialetto -Van Laere and Fazio- Londe syndromes . Mutations in the electron transferring flavoprotein genes (ETFA/ETFB) and its dehydrogenase (ETFDH) are causative for multiple acyl-CoA dehydrogenase deficiency. *Mutations in ACAD9, which encodes the acyl-CoA dehydrogenase 9 protein, were recently reported in mitochondrial disease with respiratory chain complex I deficiency.

21. Toxicity No adverse effects have been associated with ingestion of riboflavin appreciably above RDA amounts probably because of its limited solubility and limited gastric absorption.

22. Laboratory Assessment of Status Riboflavin status can be assessed by (1) determination of urine riboflavin excretion, (2) by a functional assay measuring the erythrocyte glutathione reductase activation coefficient, which is the ratio between enzyme activity determined with and without the addition of the cofactor, FAD. (3) direct measurement of riboflavin or its metabolites in plasma or erythrocytes. Urinary riboflavin has been measured using fluorometric and microbiological procedures, but for specificity, HPLC combined with fluorometric detection is the method.

23. Laboratory Assessment of Status Under conditions of adequate intake (AI), the amount excreted per day is more than 120 μg or 80 μg /g creatinine . The rate of excretion expressed as micrograms per gram creatinine is greate r for children than for adults. Conditions that cause negative nitrogen balance and the administration of antibiotics and certain psychotropic drugs ( phenothiazine derivatives) increase urinary riboflavin as a consequence of tissue depletion and displacement.

24. Laboratory Assessment of Status Riboflavin status is commonly assessed by the determination of FAD-dependent glutathione reductase activity in freshly lysed erythrocytes. Most Potential problems include that (1 ) in long-standing riboflavin deficiency, apoenzyme activity may be reduced, possibly leading to a misleading activation coefficient calculation. (2) in patients with glucose-6- phosphate deficiency, a misleadingly low activation coefficient may be measured, which is possibly caused by enhanced binding of FAD to the apoenzyme .

25. Laboratory Assessment of Status Direct measurement of riboflavin, FMN, and FAD in plasma or erythrocytes is undertaken by HPLC, usually with fluorescence detection after protein precipitation, or by capillary zone electrophoresis with laser-induced fluorescence detection. All B2 vitamers , except plasma FAD , are potential indicators of vitamin B2 status, and that plasma riboflavin and erythrocyte FMN may be useful in assessment of vitamin B2 status in population studies. In critically ill patients, plasma albumin-bound FAD may decrease as a result of the systemic inflammatory response and due to redistribution of FAD because of the increased tissue requirement. Red cell FAD is unaffected . the measurement of red cell FAD is more sensitive and recommended, especially in critically ill patients.

26. Preanalytical Variables Whole blood collected into containers with the preservatives Li heparin or EDTA Ideally, a fasting sample should be collected, especially if the patient is receiving oral or parenteral vitamin B2 supplementation. Otherwise, sampling should be undertaken at least 8 hours post-supplementation . Because vitamin B2 is light-sensitive , samples should be protected from light by wrapping with foil. A recent study using HPLC with fluorimetric detection reported that vitamin B2 showed good stability up to 72 hours at room temperature.