Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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with a riboflavin standard under identical irradiation

conditions for the sample and standard solution.

0009 Although the original lumiflavin method is rarely

used, the principal methodology has been applied to

high-performance liquid chromatography (HPLC)

analyses of riboflavin content in foods with detection

limits of 0.02 ng of riboflavin per injection.

Microbiological Assay

0010 Besides bioassays, as in chicken and curative rat

growth tests, for example, microbial assays are the

first widely used test methods, measuring the bio-

logical activity of preparations containing total vita-

min B

. The most frequently used test organism is

Lactobacillus casei ssp. rhamnosus (ATCC No.

7469). The detection limit is indicated to 0.5 ng of

riboflavin absolutely. Occasionally, other test organis-

mus are proposed such as Enterococcus faecalis

(ATCC No. 10100) or Leuconostoc mesenteroides

(ATCC No. 9135) with an enhanced sensitivity of

0.1 ng per milliliter of riboflavin. Microbiological

assays with L. casei have been approved by the Asso-

ciation of Official Analytical Chemists International

Methods for riboflavin determination in vitamin

preparations. The growth response of bacteria is pro-

portional to the riboflavin content of the medium and

can be measured turbidimetrically. Earlier techniques

determined metabolically formed lactic acid by titra-

tion. However, comparisons of turbidimetrically after

16 h of incubation yielded results that agreed with

those obtained by titration of lactic acid after 72 h.

Thus, turbidimetric measurement is currently pre-

ferred in view of its shorter incubation time and easier

handling.

0011 The growth response of Lactobacillus species

differs significantly between free riboflavin and

FMN and FAD. Acidic or enzymatic treatment is

therefore needed for releasing and converting the fla-

vins into riboflavin, as described in Methods of De-

termination, followed by adjustment of the extract to

pH 4.5 before incubation. In case of enzymatic re-

lease using takadiastase or clarase, it should be noted

that some enzyme preparations contain variable

traces of riboflavin.

0012 Starch, glycogen, free fatty acids and other lipids,

and protein degradation products can interfere with

the test by either stimulating or inhibiting bacterial

growth. Lipids can be removed either by filtration or

by ether or petroleum ether extraction before hy-

drolysis. Proteins are precipitated at pH 4.5 and

starch split by the acidic or enzymatic hydrolysis

step. As with the other analytical procedures, the

microbiological test should be carried out in dim

light. Furthermore, it is important that the glass

vessels used do not release any traces of alkali and

should thus be cleaned with acid before used, because

the flavins are alkali-sensitive. Hence, it is advisable

to dissolve riboflavin standards in 0.02 M acetic acid.

An optimal extraction procedure applicable for

microbiological or HPLC determination of thiamin

and riboflavin has been worked out by the European

Measurement and Testing Program. The steps of the

extraction are detailed in autoclaving food samples in

0.1 N HCI at 121



C for 30 min and adjusting there-

after to pH 4.0 with 4 M sodium acetate buffer (pH

6.1), followed by the addition of takadiastase (0.1 g

per gram of sample), incubation at 37–45



C (18 h),

and then filtration or centrifugation after cooling.

HPLC Procedures

0013HPLC methods enable separate quantitation of indi-

vidual flavins or simultaneous analysis of riboflavin,

including FMN and FAD. A broad variety of HPLC

techniques are described, with variations in the

column material, mobile phase, separation mode,

and detection system as well. Of the numerous ana-

lytical and technical variations communicated during

the last 20 years, the separation techniques can be

categorized as follows.

0014In most cases, reversed-phase (RP) C

-materials

as stationary phases, and aqueous/organic mobile

phases on the basis of water, methanol, acetonitril

with or without phosphate or acetate buffer have

been used in an isocratic mode. Ion-pair chromatog-

raphy with sodium salts of hexane or heptane sulfonic

acid in the mobile phase (ion-interaction chromatog-

raphy) was rarely used. In some cases, the separation

of riboflavin and flavins was accomplished using the

gradient elution technique. Ion-pair HPLC seems

more favorable only when riboflavin is to be deter-

mined simultaneously with other B-vitamins (e.g., B

, niacin, folic acid, B

), because this technique

results in better peak shapes.

0015The retention times of vitamin B

active com-

pounds are between 5 (FAD) and 20 (riboflavin)

min. Fluorescence monitoring is preferred for detec-

tion, and UV or visible absorbance detection at 270 or

446 nm is restricted to early HPLC analyses or meas-

urements of pharmaceuticals and enriched foods.

The limits of fluorescence detection at 450/520 nm

(ex./em.) for the native flavins were found to

be 0.55 pmol (0.21 ng) of riboflavin, 1.96 pmol

(0.89 ng) of FMN, and 14.19 pmol (11.15 ng) of

FAD. In an earlier HPLC method, the detection limit

could be increased to 0.02 ng of riboflavin/injection

by irradiation of the sample extract and conversion of

the extracted riboflavin to lumiflavin prior to HPLC

separation.

0016In principle, the extraction techniques and sample

clean-up suitable for HPLC analysis resemble those

4986 RIBOFLAVIN/Properties and Determination

tbl0003 Table 3 HPLC analysis of flavins

Food sample/matrix

(compounds deter-

mined)

Extraction procedure Column Mobile phase Detection

(nm)

Detectionlimit

Dairy products (total

riboflavin)

Disperse in water, clean

up on C

cartridge

Biosil ODS-5S C

O/CH

OH/acetic

acid ¼65/35/1

UV 270 10 ng

Fruits, vegetables

(total riboflavin)

0.1 N HCI (30 min) at

100



C, incubation with

mylase (38



Altex Ultrasphere

ODS 5 mm

O/CH

OH ¼60/

40 þ5mM

heptanesulfonic

acid, pH 4.5

Fluorescence

450/530

(ex./em.)

0.2 ng per

injection

Potatoes,

vegetables (total

riboflavin)

0.1 N HCI (30 min) at

121



C, incubation with

takadiastase (45–50



m-Bondapack C

OH/H

O ¼30/70 Fluorescence

450/510

(ex./em.)

0.1 ng per

injection

Dairy products

(FAD, FMN,

riboflavin)

sorboflavin as

internal standard

Homogenize with 6% ,

containing 2 M urea

pass through C

solid

phase, elute with 10% /

OH ¼4/1

LC-18, 3 mm

(Supelco)

14% acetonitril in

01. M KH

,pH

2.9

Fluorescence

450/530

(ex./em.)

FMN 2.5 nM

FAD 3 nM

Riboflavin

2.5 nM

Dairy products, raw

and cooked

meats, cereals

(FAD, FMN,

riboflavin)

7-ethyl-8-methyl-

riboflavin as

internal standard

Homogenize with CH

OH/

¼9/10 mix with

citrate-phosphate

buffer, pH 5.5

(containing 0.1%

sodium azide),

centrifugation

2PLRP-S, 5 mm

column

temperature 40



Acetonitril/0.1%

sodium azide in

0.01 M citrate-

phosphate buffer,

pH 5.5, gradient

elution

Fluorescence

450/522

(ex./em.)

96–113%

recovery

Cereals, various

foods (thiamin

and total

riboflavin)

0.1 N HCI, autoclave

(15 min) at 125



adjust to pH 4.0–4.5

(2 N NaOAc),

incubation with

claradiastase at 50



(3 h), 50% TCA and

heating at 90



(15 min), adjust to pH

3.5 (2 N NaOAc),

filtration

m Bondapak C

OH/0.005 M

phosphate buffer

pH 7.0 ¼35/65

Fluorescence

440/520

(ex./em.)

80–96%

recovery

Dairy þ meat

products, fruits,

vegetables, flour,

baked products,

beer, coffee (total

riboflavin)

0.05 M (0.1 N) H

autoclave (20 min) at

121



C, adjust to pH 4.5

(2.5 M acetate buffer),

incubation with

claradiastase at 45



(overnight); filtrated

sample passed

through C

Sep-Pak,

elution with 40–70%

Spherisorb ODS

2.5 mm

O/CH

OH ¼65/35 Fluorescence

445/525

(ex./em.)

20 pg

Blood (FAD, FMN,

riboflavin)

10% TCA at 4



C (30 min),

adding NaOAc-buffer,

centrifugation

Hypersil ODS 5 mm 0.3 M KH

OH ¼83.3/

16.7 (pH 2.9)

Fluorescence

470/525

(ex./em.)

FMN 15 nM

FAD 20 nM

Riboflavin

10 nM

Serum, urine

(riboflavin)

isoriboflavin as

internal standard

Homogenize/mix with

TCA (100 gl

1

)

centrifugation, pass

through Sep-Pak C

(serum)

ROSIL C

HL 5 mm CH

OH/H

COOH ¼36.7/

63.7/0.1

Fluorescence

450/530

(ex./em.)

10 mgl

1

Modified according to Ball GFM (1998) Bioavailability and Analysis of Vitamins in Foods, pp. 294–305. New York: Chapman & Hall; Eitenmiller RR and

Landen WO, Jr. (1999) Vitamin Analysis for the Health and Food Sciences, pp. 229–337. Boca Raton, FL: CRC Press; De Leenheer AP, Lambert WE and

Nelis HJ (eds), (2000), Modern Chromatographic Analysis of Vitamins, 3rd edn. New York: Marcel Dekker.

RIBOFLAVIN/Properties and Determination 4987

used for quantification by the other methods dis-

cussed. Most often, vitamin B

active substances are

released from the food matrix by autoclaving with

0.1 N mineral acid (HCI or H

) followed by

enzymatic digestion with papain, takadiastase, or

claradiastase. Solid-phase clean-up prodecures on

-materials or Florisil are often used for support

prior to injection. The extraction procedure depends

on the type of analysis, whether the total riboflavin

content, i.e. the sum of FAD, FMN, and free ribofla-

vin is to be determined, or whether the separated

quantitation of FAD and FMN in addition to free

riboflavin is required. Treatment with potassium

permanganate can be omitted, because possibly

remaining extraneous fluorescent substances are

chromatographically separated.

0017 Combined acid and enzymatic hydrolysis is advan-

tageous, particularly for foods with a high starch or

protein content, to liberate the bound flavins. Occa-

sionally, autoclaving with dilute mineral acid is in-

complete, particularly for the conversion of FMN to

riboflavin, as indicated by the appearance of an FMN

peak in the following chromatogram. In addition,

FMN may be partially converted during acid hy-

drolysis to biologically active isomeric riboflavin

phosphates, which can be separated from FMN by

HPLC analysis, and are ignored when calculating

total riboflavin. The combination of acidic and en-

zymatic digestion is thus advisable prior to HPLC

analysis for the determination of total riboflavin.

When using this extraction procedure and following

HPLC analysis, the riboflavin content of various

foods such as breakfast cereals, porridges, milk, and

milk products correlates well with the microbio-

logical assay if Lactobacillus casei is used. The simul-

taneous determination of FAD and FMN besides free

riboflavin requires non-degradative techniques, such

as extraction of flavins by methanol/dichloromethane

followed by partitioning with citrate buffer (pH 5.5)

or extraction with 6% formic acid containing 2 M

urea. The HPLC separation and quantitation of the

flavins were carried out using internal standards

(7-ethyl-8-methyl-riboflavin, sorboflavins, isoribo-

flavin, nicotinamide, and others). This analytical

device can be used in the flavin analysis (FAD,

FMN, and riboflavin) of milk and dairy products,

fruits and vegetables, meats, and cereal products. In

many cases, HPLC methods have been developed

for the simultaneous or sequential determination of

riboflavin and thiamin in foods. Both vitamins are

tbl0004 Table 4 Riboflavin content of foods

Content

(mgper100 g)

in ingested food

Milk andmilk products Meat and meat prod-

ucts

Cereals Vegetables and

legumes

Fruitsand nuts Fish

> 3.00 Pig’s liver

2.50–3.00 Beef liver, calf liver

2.00–2.50 Beef kidney, calf

kidney

1.50–2.00 Pig’s kidney

1.00–1.50 Pig’s heart

0.70–1.00 Wheatgerms

0.50–0.70 Camembert type,

cheddar type,

Danish blue type,

Parmesan

Wheat bran

0.30–0.50 Cream Brie (50%),

Edam type, fresh

cheese (skim milk),

Eggs, lamb Eel, mackerel

0.20–0.30 Fresh cheese (50%),

Gouda cheese

(45%), yogurt

(low-fat)

Goose, pork (lean)

veal

Green cabbage,

soybean meal

Cashew nuts,

hazel nuts,

sallow thorn

Flounder,

herring,

pilchard,

plaice

0.15–0.20 Cottage cheese,

cows’ milk (fresh)

Beef (lean), chicken,

duck, turkey

Oat flakes,

wholemeal

Broccoli

(boiled),

mangold,

soybean

sprouts,

spinach

(boiled)

Avocado Haddock,

salmon

0.10–0.15 Asparagus

(boiled)

Peanuts

(roasted)

According to Souci SW, Fachmann W and Kraut H (2000) Food Composition and Nutrition Tables. Stuttgart: Medpham Scientific.

4988 RIBOFLAVIN/Properties and Determination

extracted by a common procedure using acidic and

enzymatic digestion.

0018 For biological samples, such as blood or plasma/

serum, the preferred extraction medium is 5–10%

trichloroacetic acid, which is suitable for denaturat-

ing the relatively weak protein binding, while keeping

the phosphorylated forms intact. In this way, flavins

in whole blood can be analyzed by isocratic RP-

HPLC against an external standard. A list of usual

HPLC procedures is given in Table 3, and an over-

view of the methods of riboflavin determination is

given in Table 2.

Food Content

0019 Vitamin B

is widely distributed in animal and vege-

table foods. Protein-rich foods of animal origin are, as

a rule, considerable sources of this vitamin with good

bioavailability. Particularly rich in riboflavin are

offal, such as heart, liver, and kidney. Because of the

relative heat stability, only minor vitamin losses occur

during preparation. Likewise, pasteurization of milk

causes losses of less than 10%. However, exposure to

day light may result in a remarkable decrease in the

riboflavin content in foods, depending on the surface

area exposed. Vitamin losses occur during the milling

of cereals. White flour with a low extraction rate

contains about one-third of the vitamin content of

whole grain flour. Of special importance for the diet-

ary habits in Western countries are milk and dairy

products despite only a medium riboflavin content. If

these are omitted from the diet, it may be difficult to

achieve an adequate vitamin intake (Table 4).

See also: Analysis of Food; Chromatography: High-

performance Liquid Chromatography; Enzymes:

Functions and Characteristics; Milk: Dietary Importance;

Spectroscopy: Fluorescence

Further Reading

Ball GFM (1998) Bioavailability and Analysis of Vitamins

in Foods. New York: Chapman & Hall. pp. 294–305.

De Leenheer AP, Lambert WE and van Bocxlaer JF (eds)

(2000) Modern Chromatographic Analysis of Vitamins,

3rd edn. New York: Marcel Dekker.

Eitenmiller RR and Landen WO Jr. (1999) Vitamin Analysis

for the Health and Food Sciences, pp. 229–337. Boca

Raton, FL: CRC Press.

Friedrich W (1988) Vitamin B

: Riboflavin and its bioactive

variants. In: Vitamins. New York: Walter de Gruyter.

Official Methods of Analysis of AOAC International

(1999) 16th edn., vol. II. Gaitherburg, MD: AOAC

International.

Souci SW, Fachmann W and Kraut H (2000) Food Com-

position and Nutrition Tables. Stuttgart: Medpharm

Scientific.

Strohecker R and Henning HM (1965) Vitamin Assay

Tested Methods. Darmstadt: Verlag Chemie.

The Merck Index (1989) 11th edn. Rahway NJ: Merck &

Co.

Physiology

D B McCormick, Emory University School of

Medicine, Atlanta, GA, USA

Introduction

0001Following the earlier identification of riboflavin and

the two most common flavocoenzymes, the more

recent recognition of the greater diversity of natural

flavins has led to a broader appreciation of the mul-

tiple functions and metabolic processing of these im-

portant compounds. Much of the progress in this area

and detail of function of the flavins and flavoproteins

have been given in the periodic symposia held on this

subject. This article reviews the principal features

of the physiological handling of riboflavin and its

natural derivatives in the mammalian body and,

where known, in the human.

Digestion and Bioavailability

0002Riboflavin and lesser amounts of natural derivatives

are released by digestion of complexes, mostly flavo-

proteins, contained within foods. Coenzyme forms

of the vitamin, mainly flavin adenine dinucleotide

(FAD) and flavin mononucleotide (FMN), are re-

leased from noncovalent attachment to proteins as a

consequence of gastric acidification. Nonspecific

hydrolyses of the coenzyme forms by pyrophospha-

tase and phosphatase occur in the upper small intes-

tine. By such actions, FAD is converted to FMN,

which is further converted to riboflavin. Several per

cent of 8a-(amino acid)riboflavins originally in cova-

lent attachment as 8a-FAD linked to certain enzymes,

notably of mitochondrial origin, are also released by

such hydrolases that function together with proteoly-

sis of the attached protein chains, which begins in the

stomach with pepsin and continues in the small intes-

tine with trypsin, chymotrypsin and exopeptidases.

Traces of other ring and side-chain substituted flavins

are similarly released by combinations of the above

actions on non-covalently and covalently bound

flavins. Riboflavin 5

-glycosides, for example, are

cleaved by glycosidases present in the succus enteri-

cus. The digestive processes and locale for release of

RIBOFLAVIN/Physiology 4989

flavins from ingested material is shown in Figure 1.

(See Coenzymes.)

0003 While the efficiency of release of riboflavin from

noncovalently bound forms is essentially complete in

the normal gastrointestinal tract, the vitamin is not

recovered intact from flavin covalently linked to pro-

tein. Since the latter is less than 10% of total flavin

within the diverse foods ingested by people and most

mammals, the average bioavailability of riboflavin is

fairly high. (See Bioavailability of Nutrients.)

Absorption and Transport

0004 Riboflavin and a fraction of flavin metabolites,in-

cluding ring-altered forms, e.g., 8a-(amino acid)ribo-

flavins, and side-chain derivatives, e.g., 7,8-dimethyl-

10-formyl-methylisoalloxazine, are absorbed primar-

ily in the proximal small intestine by a saturable

transport system that is rapid and approximately pro-

portional to dose before levelling off. This saturation

level is achieved with about 25 mg of the vitamin

given in a single bolus to adult humans. Bile salts

appear to facilitate the uptake, and a modest amount

of flavin circulates via the enterohepatic system.

The initial uptake of riboflavin by enterocytes

is Na

-dependent and reflects an adenosine 5

triphosphatase (ATPase)-involved active cotransport

system. Metabolic trapping by conversion to FMN

and FAD occurs before release of the vitamin to cir-

culation by nonspecific pyrophosphatase and phos-

phatase. (See Bile.)

0005Circulatory transport of flavin involves loose asso-

ciation with albumin and tight associations with

some globulins. A subfraction of immunoglobulin G

(IgG) has been found to bind avidly a small portion of

the total free flavin in blood, and several immuno-

globulins contribute significantly to plasma transport

of the vitamin. Some riboflavin-binding proteins in

plasma are pregnancy-specific, including the classic

case of the estrogen-induced egg-white protein. These

proteins have at least some portion of the binding

domain in common and are essential for fetal devel-

opment. Placental transfer of riboflavin in the human

and other mammals involves binding proteins that

help vector the vitamin and enhance supply to the

fetus.

0006Uptake processes for flavins by mammalian cells

have some characteristics in common, but there are

both qualitative and quantitative differences among

different cell types. Entry of riboflavin appears to be

carrier-mediated (facilitated) at physiological concen-

trations of the vitamin, since there is relative specifi-

city to a saturable component that is responsible for

initial rapid uptake. A riboflavin-binding protein has

even been isolated from the plasma membrane of rat

liver cells. The nonepithelial hepatocyte does not

depend on Na

for riboflavin import, as do bipolar

epithelial types such as the enterocyte or renal prox-

imal tubular cell. Slower passive diffusion becomes

more evident when the facilitating transporter is

exceeded by pharmacological levels of the vitamin.

In all cases, metabolic trapping of riboflavin by

Stomach:

glycosides

glycose

glycosidases

Flavinyl glycosides

(glucose, etc.)

Free flavins

(Rb, FMN, FAD)

Flavoprotein complexes

(FAD > FMN > Rb)

(poly) peptides

trypsin,

chymotrypsin,

peptidases

nucleotide

pyrophosphatase

AMP

alkaline

phosphatase

H+,

pepsin

Flavoprotein compounds

(Proteinyl 8α-FAD)

peptidyl 8α-FAD 8α-FAD

8α-FMN 8α-Rb

Rb, FMN, FAD Rb, FMN, FAD Rb, FMN Rb

Small intestine:

fig0001 Figure 1 Digestion of flavins in monogastric mammals. Rb, riboflavin; P

, inorganic phosphate. Reproduced with modifications from

Riboflavin: Physiology, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds),

1993, Academic Press.

4990 RIBOFLAVIN/Physiology

phosphorylation dependent upon cytosolic flavoki-

nase follows passage of the vitamin through the

plasma membrane. Release of riboflavin from cells

requires hydrolysis of FMN by nonspecific phosphat-

ases.

Cellular Interconversions

0007 The metabolic interconversions of riboflavin and

flavocoenzymes are summarized in Figure 2.

0008 Conversion of riboflavin to coenzymes occurs

within the cellular cytoplasm of most tissues, but

particularly in the small intestine, liver, heart, kidney,

and brain. The obligatory first step is the adenosine

-triphosphate (ATP)-dependent phosphorylation of

the vitamin catalyzed by flavokinase, which utilizes

2þ

. The FMN product can be complexed with

specific apoenzymes to form several functional flavo-

proteins, but the major portion is further converted to

FAD in a second ATP-dependent reaction catalyzed

by FAD synthetase, which utilizes Mg

2þ

. It is clear

that the biosynthesis of flavocoenzymes is regulated

by supply of riboflavin (flavin status), competition for

ATP (energy status), and hormonal balances. Thyrox-

ine and triiodothyronine stimulate FMN and FAD

synthesis in mammalian systems. This seems to in-

volve a hormone-mediated increase in an active form

of flavokinase. As a product of the synthetase, FAD is

also an effective inhibitor at this second step and may

help regulate its own formation. FAD is the predom-

inant flavocoenzyme present in tissues where it is

mainly complexed with numerous flavoprotein

dehydrogenases and oxidases. Several percent of the

FAD also becomes covalently attached to specific

amino acid residues of a few important apoenzymes.

Examples for the human include the 8a-N

-histidyl

FAD within the mitochondrial dehydrogenases for

succinate, dimethylglycine and sarcosine, and the

8a-S-cysteinyl FAD within monoamine oxidase,

also of mitochondrial localization. (See Hormones:

Thyroid Hormones.)

0009Turnover of covalently attached flavocoenzymes

requires intracellular proteolysis, and further degrad-

ation of the coenzymes involves nonspecific pyro-

phosphatase and specific 5

-nucleotidase cleavage

of FAD to AMP (adenosine monophosphate) and

FMN and action by nonspecific phosphatases on the

latter.

Storage and Catabolism

0010There is little storage of riboflavin as such, since most

exists within flavocoenzymes that are in relatively

tight associations in holoenzymatic systems. During

such severe deficiency of the vitamin as leads to death

of experimental animals, there is a reduction in the

level of extractable flavin that can approach about

half of that found in an optimally supplemented con-

trol. Hence, there is moderately effective retention of

riboflavin by its metabolic commitment to bound

forms; however, even a modest deficit of the vitamin

is reflected in the decrease in function of certain

flavoproteins well before full-blown symptoms of

deficiency.

0011Though certain bacteria, especially of the Pseudo-

monas genus, can extensively degrade both the ring

system and side-chains of flavins, mammals are more

limited in their abilities to catabolize the vitamin. The

considerable diversity of flavin metabolites in and

from mammals reflects the composite of reactions of

photochemical processes on the skin, microfloral ac-

tivities in the gastrointestinal tract, as well as somatic

actions both directly on flavin and on derivatives

presented to cells by circulatory recovery from dermal

tissue and by enterohepatic retrieval from the gut.

The diverse flavin-related products identified from

humans and other mammals are summarized in

Figure 3.

0012Cleavage of the d-ribityl side-chain at position 10

is mainly, if not entirely, attributable to light and

intestinal microflora. Both can lead to partial frag-

mentation to form the 10-formylmethylflavin. This

can be oxidized by alimentary bacteria of the rumin-

ant and human to form the 10-carboxymethylflavin,

and a further fraction of the formylmethyl compound

is interconverted with the 10-hydroxymethylflavin as

a result of pyridine-nucleotide-dependent dehydro-

genase in tissue. Lumichrome-level compounds not

only can result from complete removal of the side-

chain by microflora, which can be decreased by anti-

biotic administration, but can accompany lumiflavin

as a photoproduct from action of light on flavin

within the dermal tissue. Catabolites of riboflavin

that primarily derive from oxidations within tissues

ATP

RIBOFLAVIN

FMN

F'MN

AMP

FAD

F'AD

ATP

ADP

flavokinase

(Zn

)

(Mg

)

phosphatase

Pyrophosphatase

synthetase

fig0002 Figure 2 Metabolic interconversions of riboflavin and

flavocoenzymes. Reproduced from Riboflavin: Physiology, En-

cyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

RIBOFLAVIN/Physiology 4991

are the 7- and 8-hydroxymethylriboflavins (7a-and

8a-hydroxyriboflavins). These, and products from

further oxidation of the hydroxymethyl functions to

formyl and carboxyl groups, reflect microsomal

mixed-function oxidase activity. Other flavin catabo-

lites include those from 8a -(amino acid)riboflavins

released from covalently bonded FAD. An 8a-

sulfonylriboflavin may derive from the 8a-cysteinyl-

FAD of monoamine oxidase. A peptide ester and

a glucoside, both linked to the 5

-hydroxymethyl

terminus of the vitamin, have also been found.

Excretion and Secretion

0013Since no isoalloxazine (flavin) can be biosynthesized

within the cells of mammals that lack riboflavin

synthetase, excretion and secretion reflect dietary

intake and catabolic and photodegradative events.

Essentially all known catabolites of riboflavin have

been detected in urine; many of the lumichrome-level

compounds are also in feces. For normal adults

eating varied diets, riboflavin comprises 60–90%

of urinary flavin, 7-hydroxymethylriboflavin 3–7%,

O-C-CH

-R

-CH

Lumichrome + Lumiflavin

- CHO

5⬘

1⬘

-S-CH

R-SO

-CH

αα

-(CHOH)

-CH

10-Hydroxyethyl-

flavin

10-Formylmethyl-

flavin

8α-N

-Histidyl-

FAD

8α-S-Cysteinyl

FAD

8α-Sulfonyl-

riboflavin

(HOCH)

5⬘-Riboflavinyl-peptide/ester

N N - CH

Riboflavin

Catabolites

HOCH

C HOCH

8-Hydroxymethyl-

riboflavin

8-Formyl 8-Carboxy 7-Carboxy 7-Formyl 7-Hydroxymethyl-

riboflavin

8-Carboxylumichrome 7-Carboxylumichrome

fig0003 Figure 3 Photochemical, microfloral, and cellular catabolism of riboflavin within mammals. Reproduced from Riboflavin: Physi-

ology, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

4992 RIBOFLAVIN/Physiology

8a-sulfonylriboflavin 2–15%, 8-hydroxymethylribo-

flavin 1–8%, 10-hydroxyethylflavin 1–7%, riboflavi-

nyl peptide ester up to 5%, with traces of lumiflavin

and, sometimes, the 10-formylmethyl- and carboxy-

methylflavins.

0014 The presence in milk of ‘lactoflavin,’ an early name

for riboflavin, led to the recognition of this food as a

good source of the vitamin. For milk from both cows

and humans, the flavin in highest concentration other

than the free vitamin is FAD, which can comprise

over a third of total flavin. Much of this is hydrolyzed

to FMN by pasteurization. Fairly significant quan-

tities of the 10-(2

-hydroxyethyl)flavin are notable,

since this catabolite has antivitamin activities, as

reflected in competitive inhibition of both cellular

uptake and subsequent flavokinase-catalyzed phos-

phorylation of riboflavin. Hence, this catabolite,

which may reach 10–12% of flavin in cow’s milk,

modestly subtracts from the biological activity of

this food. Several percent of both 7- and 8-hydroxy-

methylriboflavins are also present, with more of the

former. Smaller amounts of other catabolites, includ-

ing the 10-formylmethylflavin and lumichrome,

comprise most of the rest. (See Milk: Dietary

Importance.)

Biochemical Functions

0015 In bound coenzymic forms, riboflavin participates in

oxidation–reduction reactions in numerous meta-

bolic pathways and in energy production via the re-

spiratory chain. A variety of chemical reactions are

catalyzed by flavoproteins. The redox functions of a

flavocoenzyme (Figure 4) include one-electron trans-

fers, during which the neutral, oxidized quinone level

of flavin is half reduced to the radical semiquinone,

which can exist within natural pH ranges as neutral

or anionic species. A further electron transfer can lead

to a fully reduced hydroquinone. In addition, a single-

step, two-electron transfer from substrate to flavin

can occur with hydride ion transfer, e.g., from re-

duced pyridine nucleotide, or by base abstraction of

a substrate proton together with carbanion addition.

0016 There are flavoprotein-catalyzed dehydrogenations

that are both pyridine-nucleotide dependent and

independent, reactions with sulfur-containing com-

pounds, hydroxylations, oxidative decarboxylations,

dioxygenations, and reduction of oxygen to hydrogen

peroxide. The intrinsic abilities of flavins – to be

varyingly potentiated as redox carriers upon differen-

tial binding to proteins, to participate in both one-

and two-electron transfers, and to react in reduced

(1,5-dihydro) form with oxygen – permit wide scope

in their operation.

Requirements and Intakes

0017The requirement levels for riboflavin, in contrast to

those for thiamin, are not raised when energy utiliza-

tion is increased. Because of the interdependence of

protein, energy intake, and metabolic body size, how-

ever, allowances calculated on these three bases do

not differ significantly. Clinical signs of deficiency in

adults can be prevented with intakes of riboflavin

above 0.4 mg per 1000 kcal, but over 0.5 mg per

1000 kcal may be required to maintain tissue reserves

in adults and children as reflected in urinary excre-

tion, erythrocyte riboflavin, and erythrocyte glu-

tathione reductase. From these considerations, the

riboflavin allowances are now computed as 0.6 mg

per 1000 kcal for people of all ages. This leads to US

Recommended Dietary Allowances (RDAs) ranging

from 0.4 mg per day for early infants to 1.7 mg per

day for young adult males. However, for elderly

people and others whose daily calorie intake may be

less than 2000 kcal, a minimum of 1.2 mg per day is

recommended in the USA. Since pregnancy imposes

extra demands, reflected by decreased excretion

−

Flavoquinone: yellow, fluorescent,

neutral, oxidized level

Flavosemiquinone:

blue, neutral radical

Flavohydroquinone:

neutral reduced level

Flavohydroquinone:

anionic reduced level

Flavosemiquinone:

red, anionic radical

−

~ 8.4

~ 6.2

fig0004Figure 4 Oxidation–reduction states of flavocoenzymes func-

tioning physiologically. Reproduced from Riboflavin: Physiology,

Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

RIBOFLAVIN/Physiology 4993

and an elevated FAD stimulation of erythrocyte

glutathione activity, an additional 0.3 mg per day is

recommended. The lactating woman secretes ap-

proximately 35 mg per 100 ml of milk for an output

of about 0.26 mg per day (750 ml) during the first 6

months and 0.21 mg per day (600 ml) during the

second 6 months. Since the utilization of the

additional riboflavin for milk production is assumed

to be 70%, an additional intake of 0.5 mg is

recommended for the first 6 months and 0.4 mg for

the second. (See Lactation: Physiology.)

0018 Small amounts of riboflavin, largely as digestible

coenzymes, are present in most plant and animal

tissue. Good sources are eggs, lean meats, milk, broc-

coli, and enriched breads and cereals. Such losses as

occur during cooking are largely attributable to

leaching of the heat-stable but light-sensitive flavins

into water.

0019 When supplementation or therapy with riboflavin

is warranted, oral administration of five to 10 times

the RDA is usually satisfactory.

Deficiency Causes and Symptoms

0020 Pure, uncomplicated riboflavin deficiency is probably

never encountered in patients, but is accompanied by

multiple nutrient deficiencies. Ariboflavinosis can

result from such primary and secondary factors as

commonly affect supply or utilization of other nutri-

ents as well. Inadequate dietary intake most com-

monly related to limited availability of food, but

sometimes exacerbated by poor storage or process-

ing, remains the major cause. In addition, anorexic

persons rarely ingest adequate amounts of riboflavin

and other nutrients.

0021 Decreased assimilation results from abnormal di-

gestion, absorption, or both. Lactose intolerance as a

result of lactase insufficiency, mostly encountered

among Blacks and Asians, argues against such people

consuming nonlactase-treated milk, which is a good

source of the vitamin. Malabsorption can occur as a

result of tropical spure, celiac disease, malignancy

and resection of the small bowel, and gastrointestinal

and biliary obstruction. Poor absorption also results

from disorders that increase motility and decrease

gastrointestinal passage time, such as diarrhea, infec-

tious enteritis, and irritable bowel syndrome. (See

Food Intolerance: Lactose Intolerance.)

0022 Rather rarely encountered, but usually significantly

improved by therapeutic treatment with riboflavin,

are certain inborn errors where the genetic defect is

in formation of a normal flavoprotein. Cases in this

category include fatty acid desaturases in which spe-

cific defects have been found for the mitochondrial

FAD-dependent dehydrogenases for short-chain,

long-chain, and multi-chain acyl-CoAs (acyl coen-

zyme As). The young patients have a lipid storage

myopathy, often accompanied by carnitine insuffi-

ciency, and exhibit glutaric aciduria. A low, FMN-

dependent pyridoxine 5

-phosphate oxidase activity

due to an erythrocyte deficiency of FMN, confirmed

by response to oral riboflavin, was reported in the

majority of subjects with d-glucose 6-phosphate

dehydrogenase deficiency. Such cases seem to have

an accelerated conversion of FMN to FAD so that

glutathione reductase is saturated. This contrasts

with heterozygous b-thalassemia, in which there is

an inherited slow erythrocyte conversion of riboflavin

to FMN, a decrease in subsequent FAD, and a high

stimulation of the erythrocyte glutathione reductase

by extraneous FAD.

0023Defective utilization can result from disturbances

in hormonal production, certainly relating to thyroid

hormone, but less likely as a result of taking oral

contraceptives. Phenothiazine derivatives appear to

impair use of riboflavin.

0024Increased destruction of riboflavin occurs during

treatment of neonatal jaundice with phototherapy.

In this case, the side-chain of the vitamin is photo-

chemically destroyed, as it is involved in the photo-

sensitized oxidation of bilirubin to more polar,

excretable compounds.

0025The finding that phenobarbital induces microso-

mal oxidation of the 7-methyl function of the vitamin

lends credence to the belief that long-time use of

barbiturates may jeopardize flavin status.

0026Enhanced excretion of riboflavin occurs in cata-

bolic patients undergoing nitrogen loss. The relation-

ship of the vitamin to protein status has long been

recognized. Also, certain antibiotics and pheno-

thiazine drugs increase excretion of riboflavin. (See

Drug–Nutrient Interactions.)

0027Increased requirements can, of course, be the con-

sequence of one or more of the above-mentioned

factors. For example, protein–calorie malnutrition

commonly accompanies a diminution in both absorp-

tion and utilization of riboflavin. Systemic infections,

even without gastrointestinal involvement, some-

times lead to increased requirements that can result

from decreased intake, defective absorption, poor

utilization, and increased excretion. (See Protein: De-

ficiency.)

0028Clinical deficiency of riboflavin has been reduced

by feeding a riboflavin-deficient diet and/or by the

administration of an antagonist such as galactoflavin.

The deficiency syndrome is characterized by sore

throat, hyperemia and edema of the pharyngeal and

oral mucous membranes, cheilosis, angular stoma-

titis, glossitis (magenta tongue), seborrheic dermatitis,

and normochromic, normocytic anemia associated

4994 RIBOFLAVIN/Physiology

with pure red cell cytoplasia of the bone marrow. As

noted above, some of these symptoms, e.g., glossitis

and dermatitis, when encountered in the field, may

have resulted from other complicating deficiencies.

Severe riboflavin deficiency can also affect the

conversion of vitamin B

to its coenzyme and even

curtail conversion of tryptophan to niacin. (See

Niacin: Physiology; Vitamin B

: Properties and

Determination.)

Toxicity

0029 Toxicity from ingestion of excess riboflavin by

experimental animals or humans is doubtful. The

capacity of the human gastrointestinal tract to absorb

orally administered riboflavin may be less than 30 mg

in a single dose. The limited solubility and absorptiv-

ity of this vitamin as encountered in multivitamin

preparations and natural foodstuffs, and its ready

excretion as typical of water-soluble vitamins, nor-

mally precludes a health risk. There is one report of

EEG (electroencephalogram) abnormalities in two

patients during long-term treatment with riboflavin

and niacin.

See also: Bile; Bioavailability of Nutrients; Coenzymes;

Drug–Nutrient Interactions; Food Intolerance: Lactose

Intolerance; Hormones: Thyroid Hormones; Lactation:

Human Milk: Composition and Nutritional Value;

Physiology; Milk: Dietary Importance; Niacin: Properties

and Determination; Protein: Deficiency; Vitamins:

Overview; Determination; Vitamin B

: Properties and

Determination