1.

Riboflavin and the Flavin coenzymes:

+) Riboflavin consist a tricyclic dimethyl-isoalloxazine ring conjugated to the sugar alcohol ribitol.

+) Ribitol is an open chain form of sugar ribose with aldehyde group reduced to alcohol. It emits
yellow fluorescence and it is stable to heat but sensitive to light.

+) The metabolically active coenzymes are riboflavin 5 -phosphate and flavin adenine dinucleotide
(FAD). In some enzymes the prosthetic group is riboflavin, bound covalently at the catalytic site.

+) Riboflavin phosphate and FAD may be either covalently or noncovalently bound at the catalytic
sites of enzymes. Covalent binding of the flavin coenzymes is normally through the 8-αmethyl group.
8-Hydroxymethyl-riboflavin is formed by microsomal mixed-function oxidases, but it is not known
whether or not this is a precursor of covalently bound flavin coenzymes.

+) When exposed to UV rays, it is converted to lumiflavin which exhibits yellow fluorescence;

lactoflavin from milk, hepatoflavin from liver, ovoflavin from eggs.

2. Metabolism of riboflavin

-Absorption, Tissue Uptake, and Coenzyme Synthesis: Apart from milk and eggs, which contain
relatively large amounts of free riboflavin bound to specific binding proteins, most of the vitamin in
foods is as flavin coenzymes bound to enzymes, with about 60% to 90% as FAD. Riboflavin is present
in foods mostly (80–90%) as FAD and FMN cofactors of proteins. Hydrochloric acid from the stomach
readily releases the flavins that are only loosely bound to their proteins. FMN is dephosphorylated to
riboflavin by alkaline phosphatase in the small intestine. FAD is broken up by nucleotide
pyrophosphatase at the brush border of villous tip cells into AMP and FMN from which riboflavin can
then be released. The ribitol is linked to phosphate in FMN. FAD is formed from FMN by transfer of
an AMP from ATP.

- Riboflavin binding protein: Riboflavin-binding proteins (RfBPs) have been identified in the plasma of
the laying hen and pregnant cows, mice, rats, monkeys, and humans. There is a specific plasma
riboflavin binding protein that is induced by estrogens; in female animals, its concentration varies
through the estrous cycle. The same protein is also synthesized in the testes and is found on the
acrosomal surface of spermatozoa. It has been suggested that active or passive immunization
against the binding protein may provide both male and female contraception.

- Homeostasis: There is no evidence of any significant storage of riboflavin; in addition to the limited
absorption, any surplus intake is excreted rapidly; thus, once metabolic requirements have been
met, urinary excretion of riboflavin and its metabolites reflects intake until intestinal absorption is
saturated. There is only a four-fold difference between the minimum concentration of
flavins in the liver in deficiency and the level at which saturation occurs. Control over tissue
concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase,
and the synthesis and catabolism of flavin-dependent enzymes. The 8α-linkage is not cleaved by
mammalian enzymes and 8α-derivatives of riboflavin are not substrates for flavokinase and cannot
be reutilized.
- The Effect of Thyroid Hormones on Riboflavin Metabolism: The activities of a variety of flavin-
dependent enzymes are depressed in hypothyroidism. They are increased by the administration of
thyroxine or triiodothyronine, as a result of increased synthesis of riboflavin phosphate and FAD,
leading to increased saturation of enzyme proteins with coenzymes. Tissue concentrations of flavin
coenzymes in hypothyroid animals may be as low as in those fed a riboflavin-deficient diet. In
hypothyroid patients, erythrocyte glutathione reductase (EGR) activity may be as low, and its
activation by FAD added in vitro normalized by the administration of thyroid hormones, with no
increase in riboflavin intake. The administration of thyroid hormones to hypothyroid animals results
in a rapid increase in flavokinase activity as a result of the activation of an inactive precursor protein;
as flavokinase activity increases, there is a parallel decrease in the tissue content of an apparently
inactive riboflavin binding protein. Hyperthyroidism is not associated with elevated tissue
concentrations of flavin coenzymes, despite increased activity of flavokinase. Riboflavin may also be
involved in the metabolism of thyroid hormones.

- Catabolism and Excretion of Riboflavin: Riboflavin and riboflavin phosphate that are not bound to
plasma proteins are filtered at the glomerulus; the phosphate is generally dephosphorylated in the
bladder. Excretion happens mainly in urine. Under normal conditions, about 25% of the urinary
excretion of riboflavin is as the unchanged vitamin, with a small amount as a variety of glycosides of
riboflavin and its metabolites. Liver cytochrome P450-linked mixed-function oxidases result in the
production of 7- and 8-hydroxymethylriboflavin, both of which are substrates for flavokinase.
Intestinal bacterial cleavage of the ribityl side chain results in the formation

of 10-hydroxyethylflavin (an oxidation product of lumiflavin), lumichrome,

and 7- and 8-carboxy-lumichromes, which are also excreted in the urine

- Biosynthesis of Riboflavin: A number of fungi have a failure of the normal regulation of riboflavin
synthesis and are overproducers of the vitamin. Mutants of Ashbya gossypii can produce and
excrete so much that riboflavin crystallizes in the culture medium. The precursors for riboflavin
biosynthesis in plants and microorganisms are guanosine triphosphate and ribulose 5-phosphate. In
yeasts and fungi, opening of theimidazole ring is followed by reduction of the ribose side chain to
ribitol, deamination, and dephosphorylation to yield amino-ribitylamino-pyrimidinedione; in
bacteria, deamination occurs before reduction of the sugar. The final step is a dismutation reaction
between twomolecules of dimethyllumazine, catalyzed by riboflavin synthase, yielding riboflavin and
aminoribitylamino-pyrimidinedione.

3. METABOLIC FUNCTIONS OF RIBOFLAVIN

- The Flavin Coenzymes: FAD and Riboflavin Phosphate: The majority of flavoproteins have FAD as
the prosthetic group rather than riboflavin phosphate. Some have both flavin coenzymes, and some
have other prosthetic groups.

- Single-Electron–Transferring Flavoproteins: Flavoproteins catalyzing single-electron transfer

provide the link between substrate oxidation catalyzed by dehydrogenases and the mitochondrial
electron transport chain.

- Two-Electron–Transferring Flavoprotein Dehydrogenases: The initial step of the two-electron–

transferring reactions is the removal of a proton from the substrate. Next step is the intermediate
formation of an adduct between the substrate and prosthetic group at N-5 of the flavin, undergoing
cleavage to yield dihydroflavin and the oxidized product, which is commonly a carbon–carbon
double bond. Then, the reduced flavin isreoxidized by reaction with an electron-transferring
flavoprotein, as discussed above, or in some cases by reaction with nicotinamide nucleotide
coenzymes.

- Nicotinamide Nucleotide Disulfide Oxidoreductases: Glutathione reductase, thioredoxin reductase,

and lipoamide dehydrogenase are members of a groupof flavoproteins that contain an active site
disulfide as well as FAD. They catalyze the NAD(P)H dependent reduction of a disulfide substrate to
its dithiol form.

- Flavin oxidases: Flavin oxidases include d- and l-amino acid oxidases, and some amine oxidases,
although others are quinoproteins. In these enzymes, the flavin is reduced by dehydrogenation of
the substrate, by way of an intermediate substrate-flavin adduct, as occurs in the dehydrogenases.

- NADPH Oxidase, the Respiratory Burst Oxidase: NADPH oxidase was originally described in
activated macrophages, whose function is to generate reactive oxygen species and halogen radicals
as part of the cytotoxic action against phagocytosed microorganisms. It catalyzes transfer of
electrons from NADPH onto cytochrome b558, which then reduces oxygen to yield 2 mol of
superoxide and two protons. Activation of the oxidase requires increased formation of NADPH and
hence increased oxidation of glucose through the pentose phosphate pathway, the so-called
respiratory burst.

- Molybdenum-Containing Flavoprotein Hydroxylases: Xanthine oxidoreductase and aldehyde

oxidase represent a distinct class of flavin-dependent oxidases. They both dehydrogenate and
hydroxylate the substrate. The oxygen introduced into the substrate by these enzymes is derived
from water, and the role of molecular oxygen is in the reoxidation of the reduced flavin. Among
other reactions, aldehyde oxidase is important in the oxidation of N1-methyl nicotinamide to methyl
pyridone carboxamide and pyridoxal to 4-pyridoxic acid.

- Flavin Mixed-Function Oxidases (Hydroxylases): The flavin-dependent mixed-function oxidases

include amine N-oxidases and a variety of S-oxidases. They provide an alternative to cytochrome
P450- dependent enzymes in the metabolism of xenobiotics.

- The Role of Riboflavin in the Cryptochromes: One of the mechanisms of DNA repair in bacteria,
acting to reduce cyclobutane dipyrimidines and pyrimidine-pyrimidone dimers formed by exposure
to UV light, is the blue light-activated photolyase. The primary light-trapping pigment is 5,10-
methylene tetrahydrofolate , which then transfers the excitation energy of the trapped photon to
FADH, which reduces the substrate.

4. Riboflavin Deficiency:

Causes:

+) Inadequate intake

+) Impaired absorption due to intestinal diseases

+) Chronic alcoholics are susceptible to B, deficiency

Clinical features:

+) Cheilosis (fissures at the corners of mouth)

+) Glossitis (tongue smooth and purplish)

+) Corneal vasculalarization: includes dryness, burning & itching and lacrimination

+) Measurement of glutathione reductase in erythrocytes is a reliable diagnostic test to assess

riboflavin deficiency

+) Reference interval
+) Serum or plasma level is 4 to 24 ug/ dl