Phar 722Pharmacy Practice III

Vitamins-

Riboflavin (B2)

Spring 2006

Riboflavin (B2) Study Guide

• The applicable study guide items in the Vitamin Introduction

• History

• Structure including commercial forms of the vitamin

• Conversion to cofactor forms

• Function of the cofactor including the specific types of reactions

• Deficiency condition

History• Shortly after the discovery of thiamine

from yeast concentrates, the presence of a second nutritional factor in such materials was suggested.

• This second factor also was reported to have a pellagra-preventative activity since it alleviated a deficiency-induced dermatitis in rats.

• It was called Vitamin B2 in England and Vitamin G in the United States.

Chemistry• Riboflavin has a characteristic flavin

ring system which gives it a unique spectroscopic and instability properties.

• Two commercial forms.– Riboflavin, itself, is poorly water soluble (1

gm/10,000 ml).– Riboflavin phosphate’s solubility is

0.1 gm/ml

N

N

NH

NH3C

H3C

O

O

CH2 CH

OH

CH

OH

CH

OH

CH2 OH

Riboflavin (Vitamin B2)

N

N

NH

NH3C

H3C

O

O

CH2 CH

OH

CH

OH

CH

OH

CH2 O P

O

O-

O-

Riboflavin PhosphateFlavin Mononucleotide (FMN)

Solubility: 1 gm/10,000 ml;A commercial form

Solubility: 0.1 gm/ml;A commercial form

Riboflavin Uptake and Metabolism• Because of its poor water soluble, uptake of riboflavin is slow since

the vitamin should be in solution in order to enter the intestinal mucosa cell. Therefore, it is recommended that supplemental administration of riboflavin be done with food in order to delay intestinal emptying.

• In the mucosa cell, a flavokinase phosphorylates the terminal alcohol with a phosphate from an ATP forming riboflavin phosphate.

– The latter, as the sodium salt, also is the water soluble (0.1 gm/1 ml) commercial form of the vitamin.

• The conversion of riboflavin phosphate (FMN) to the more common cofactor, flavin adenine dinucleotide (FAD) requires the conversion of ATP to AMP and the formation of an anhydride linkage.

– The pyrophosphate will be cleaved providing enough energy for this synthesis to go to completion. While it is not clear, this step probably occurs in the tissues requiring FAD.

• While flavin chemistry has been studied extensively, there has been little research of riboflavin biochemistry.

N

N

NH

NH3C

H3C

O

O

CH2 CH

OH

CH

OH

CH

OH

CH2 O P

O

O-

O P

O

O-

O CH2

N

NN

N

NH2

O

OHOH

HH

HH

N

N

NH

NH3C

H3C

O

O

CH2 CH

OH

CH

OH

CH

OH

CH2 O P

O

O-

O-

ATP

ADP

PPi

ATPN

N

NH

NH3C

H3C

O

O

CH2 CH

OH

CH

OH

CH

OH

CH2 OH

Riboflavin (Vitamin B2) Riboflavin PhosphateFlavin Mononucleotide (FMN)

Riboflavin Adenine Dinucleotide (FAD)

Flavokinase

FADpyrophosphorylase

Intestinal mucosa

Tissues whereFAD is required

Biochemical Functions-1• Riboflavin, as FMN/FMNH2 or

FAD/FADH2, is the coenzyme/cofactor of flavin enzymes. – It is required for many oxidation-reduction

reactions generally (but not always) of carbon-carbon bonds.

– Even though there is no defined deficiency syndrome, this vitamin is required for several life-supporting oxidation-reduction biochemical reactions.

• Some examples are on the next slides.

N

N

NH

NH3C

H3C

O

O

R

FAD or FMN (oxidized)

e- + H+

N

N

NH

NH3C

H3C

O-

O

R

FADH. or FMNH.

(semiquinone)

H

e- + H+

N

N

NH

NH3C

H3C

O

O

R

FADH2 or FMNH2 (reduced)

H

H

Biochemical Functions-2

• Oxidation-reduction of carbon-carbon bonds.

C C

H

R3H

R1

R2 R4 C C

R4

R3R2

R1FADH2

FADH2

FAD

FAD

Biochemical Functions-3

HN

NNH

N

O

Hypoxanthine

HN

NH

NH

N

O

Xanthine

HN

NH

NH

HN

O

Uric AcidO

O

OO2 H2O2

Xanthine Oxidase

O2 H2O2

Xanthine Oxidase

GuanineAdenine

Severalsteps

Xanthine Oxidase

Biochemical Functions-4

FMN FMNH2

H

H

FADH2FAD

H+

+

+

H+

H+

H+

H

H+

+

Electron (respiratory) transport chain

Biochemical Functions-5

• Oxidation-Reduction of Thiol-Disulfide Systems • Examples: glutathione reductases and lipoic acid in oxidative

decarboxylations of α- ketoacids (pyruvate, α-

ketoglutarate)

FADH2

FADH2

FAD

FAD

R SH R SH+ R S S R

ThiolDisulfide

Biochemical Functions-6

• Monoamine oxidase (MAO)• Examples: Metabolism of dopamine, norepinephrine, epinephrine

and serotonin

CH2 NH2R R C H

OO2 + H2O H2O2 + NH4+

MonoamineOxidase

Biochemical Functions-7• Biochemical Transformations of Other

Vitamins– Folic Acid– Pyridoxine

• NOTE: This illustrates how one vitamin is dependent on there being an adequate concentration of another vitamin. A deficiency of one vitamin may induce a deficiency of another vitamin.

Riboflavin Deficiency• Ariboflavinosis

– Clinical riboflavin deficiency.– There is no deficiency syndrome in humans associated with

this vitamin.– Usually associated with deficiencies of other “B vitamins”.

• Symptoms– Sore throat– Redness and swelling of the lining of the mouth and throat– Cracks or sores on the outside of the lips (cheliosis) and at

the corners of the mouth (angular stomatitis)– Moist, scaly skin inflammation (seborrheic dermatitis)– Vascularization of the cornea

• There is no good assay that correlates a riboflavin deficiency with specific biochemical function.

• It appears that humans do store appreciable amounts (possibly over six months) of the vitamin.

Hypervitaminosis Riboflavin

• Because of its poor solubility, it probably would be difficult to obtain a toxic dose of this vitamin. – Water soluble riboflavin phosphate may be

another matter. Nevertheless, no significant toxicities have been reported.

• Estimated toxic dose: 1,000 mg (1 gm)– There is no UL.

Dosage Forms• Most of the commercial forms of the vitamin are synthetic.

• Stability– Riboflavin is one of the most unstable of the vitamins,

particularly in light. Solutions of the vitamin must be protected from light.

• Riboflavin– This is a neutral molecule. Salt formation is not possible. – Because of its poor water solubility, it is used in dry dosage

forms.

• Riboflavin Phosphate– The sodium salt is very soluble (0.1 gm/1 ml). – It is used in liquid dosage forms.

DRIs-1• AI

– Infants 0.3 - 0.4 mg/day

• EAR– Children (1 - 13 years) 0.4 - 0.8 mg/day– Males (14 - 19 years) 1.1 mg/day– Females (14 - 19 years) 0.9 mg/day– Men (19 - 70+ years) 1.1 mg/day– Women (19 - 70+ years) 0.9 mg/day– Pregnancy 1.2 mg/day– Lactation 1.3 mg/day

DRIs-2

• RDA– Children (1 - 13 years) 0.5 - 0.9 mg/day– Males (14 - 19 years) 1.3 mg/day– Females (14 - 19 years) 1.0 mg/day– Men (19 - 70+ years) 1.3 mg/day– Women (19 - 70+ years) 1.1 mg/day– Pregnancy 1.4 mg/day– Lactation 1.6 mg/day

• UL– None reported

Food Sources• Liver

• Kidney

• Milk

• Yeast

• Plant

• Animal tissue

• Animals have to obtain riboflavin from plants or other animals.