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

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Immunologic Assays

0032 Antibodies specific for (phospho)pyridoxyl groups

have been utilized in immunoassays for certain B

vitamers. However, there is a marked variation in

the affinity of the antibodies for the B

vitamers, so

these methods are not widely used.

Gas Chromatographic Assays

0033 Unless B

vitamers are nonphosphorylated and

their polar functional groups derivatized, these

compounds are not well suited for GC analysis.

Nonvolatile components and water are not easily

removed from sample extracts. Methods involving

the formation of acetyl, isopropylidene, trimethylsi-

lyl, trifluoroacetyl, and heptafluorobutyryl deriva-

tives of the nonphosphorylated B

vitamers have

been developed, but other chromatographic methods

are preferred.

HPLC Assays

0034 The physiochemical properties of the B

vitamers

facilitate their assay by HPLC assay. HPLC has the

potential for high resolution and high sensitivity with

regard to the B

vitamers found in biological mater-

ials, without the necessity of complex extraction and

derivatization techniques. If desired, the phosphate

esters of the phosphorylated B

vitamers can be

hydrolyzed using phosphatase yielding nonphos-

phorylated vitamers. Otherwise, the phosphorylated

vitamers can be preserved during extraction and

quantitated as such. All six B

vitamers and 4-PA

can be quantitated at nanogram levels using HPLC

techniques. Alternatively, all forms of the vitamer can

also be converted to PN, followed by HPLC quantita-

tion of total vitamin B

as PN techniques. This

method is rapidly becoming the preferred way of

determining total vitamin B

. Ion-exchange (anion

or cation) and reversed-phase (with or without

paired-ion) HPLC methods for assay of B

vitamers

have been published. Most of the methods utilize

fluorescence detectors, although some methods,

particularly the earlier methods, do utilize ultraviolet

(UV) detectors; the fluorescence detector is far

more sensitive and specific. The use of an internal

standard, most frequently 4-deoxypyridoxine, in-

creases the accuracy and precision of HPLC analysis

of B

vitamers, providing that it is not naturally

present in the samples. External standards (spiking)

are also utilized in proving identities of the peaks.

The identities of the peaks should be confirmed by

other means such as mass spectrometry and fluores-

cence spectra.

0035 Ion-exchange chromatography is accomplished

using packing material that possesses charge-bearing

functional groups. Many of the HPLC separations

reported in the 1970s were cation- or anion-exchange

methodologies, which generally involved determin-

ations of nonphosphorylated B

vitamers in acid

hydrolysates. An HPLC method has been developed

for the separation of all of the B

vitamers using

Aminex A-25 anion-exchange resin, isocratic elution,

and a pH 10 glycine buffer. This anionic method was

successfully applied to the determination of vitamin

components in milk, animal tissues, meats, and

fortified cereals. Unfortunately, the manufacturer

altered the composition of the resin in such a manner

that the method no longer provided satisfactory

separation of the vitamers. Also, a cation-exchange

HPLC method, fluorometric detection, has been

developed, which successfully quantitated all B

vitamers in plasma and other animal tissues.

0036Reversed-phase chromatography is the term given

to chromatographic conditions in which a nonpolar

stationary phase is used in conjunction with a polar

mobile phase. Gregory and Kirk were the first

to separate nonphosphorylated B

vitamers using

reversed-phase HPLC methods, with fluorescence de-

tection, which successfully measured PN in fortified

cereals.

0037Paired-ion (also called ion-pair) chromatography

was developed to deal with separating compounds

that are either very polar, multiply ionized, or strongly

basic. Several paired-ion, reversed-phase HPLC

methods, both gradient and isocratic, have been de-

veloped for determining vitamin B

components in

biological materials and foods. These methods have

also been utilized successfully in quantitating the six

vitamers and 4-PA in plasma. This type of

methodology may also be utilized in quantitating

glycosylated and other forms of the vitamin.

0038Several researchers have reported comparable

results when specific foods were analyzed for total

vitamin B

vitamer content using microbiological

(S. uvarum) and HPLC techniques. Most researchers

in the vitamin B

area believe that HPLC is the pre-

ferred method for determining the vitamin B

content

of food because individual vitamers can be deter-

mined. However, as indicated earlier, vitamin B

con-

tent data for food composition tables are obtained

primarily using microbiological (S. uvarum) methods.

As discussed earlier, the potencies of the B

vitamers

in the human body appear to be rather similar, so one

questions the importance of routinely determining

each of the six B

vitamers in foods. However it

does have academic interest and is of clinical signifi-

cance where drugs or diseases/conditions influence

the metabolism of the B

vitamers. Information as to

the form of the vitamin in food may be of importance

therapeutically.

6018 VITAMIN B

/Properties and Determination

Summary

0039 The quantitation of vitamin B

components in bio-

logical materials is difficult. The vitamin must be

extracted, generally using acidic media, from plant

and animal products before being analyzed by one

of the many chemical, biochemical or biological

methods. The official AOAC method for determin-

ation of the vitamin B

content of foods involves the

use of the microorganism Saccharomyces uvarum.

Although researchers have developed HPLC methods

that quantitate all six forms of the vitamin, these

methods are rarely utilized in determining the vitamin

content of food. Little difference appears to exist

in bioavailability of the different B

vitamers in

humans so total vitamin B

composition is deter-

mined by conversion to pyridoxine. Researchers are

continuing to seek improved methodologies for the

analysis of vitamin B

involving use of the latest

technology such as mass spectrometry. Additional

research is also needed to determine the importance

of specific forms of the vitamin in humans.

Seealso: Bioavailability of Nutrients; Chromatography:

High-performanceLiquidChromatography;Gas

Chromatography; Food Fortification

Further Reading

Ang CY (1979) Stability of three forms of vitamin B

laboratory light conditions. Journal of the Association of

Official Analytical Chemists 62: 1170–1173.

Association of Official Analytical Chemists (2000) Official

Methods of Analysis, 19th edn. Arlington, VA: AOAC.

Batenhorst JH, Sun J, Giraud DW et al. (1995) Retention of

selected nutrients in pork roasts prepared using different

cookery methods. Journal of Muscle Foods 6: 359–368.

Brin M (1978) Vitamin B

: chemistry, absorption, metabol-

ism, catabolism, and toxicity. In: Human Vitamin B

Requirements, pp. 1–20. Washington, DC: National

Academy of Sciences.

Chrisley BMc, Thye FW, McNair HM and Driskell JA

(1988) Plasma B-6 vitamer and 4-pyridoxic acid concen-

trations of men fed controlled diets. Journal of Chroma-

tography 428: 35–42.

DeRitter E (1976) Stability characteristics of vitamins in

processed foods. Food Technology 30: 48–54.

Driskell JA (1994) Vitamin B-6 requirements of humans.

Nutrition Research 14: 293–324.

Driskell JA (1995) Sensory and nutritive qualities of food

prepared by microwaving as compared to other cooking

methods. Microwave World 16(2): 6–9.

Eitenmiller RR and Landen WO, Jr (1999) Vitamin Analy-

sis for the Health and Food Sciences, Chapter 10. Boca

Raton, FL: CRC Press.

Gregory JF (1980) Effects of e-pyridoxyllysine bound to

dietary protein on the vitamin B-6 status of rats. Journal

of Nutrition 110: 995–1005.

Gregory JF (1988) Methods for determination of vitamin B

in foods and other biological materials: a critical

review. Journal of Food Composition and Analysis 1:

105–123.

Gregory JF (1990) The bioavailability of vitamin B

: recent

findings. Annals of the New York Academy of Sciences,

pp. 86–95. New York: New York Academy of Sciences.

Gregory JF (1997) Bioavailability of vitamin B-6. European

Journal of Clinical Nutrition 51: S43–S48.

Harris SA (1968) Pyridoxine. Encyclopedia of Chemical

Technology 16: 806–824.

International Union of Pure and Applied Chemistry – Inter-

national Union of Biochemistry, Commission on Bio-

chemical Nomenclature (1973) Nomenclature for

vitamin B

and related compounds: recommendations

1973. European Journal of Biochemistry 40: 325–327.

Johnson JA and Driskell J (1987) Vitamin retention and

sensory evaluation of selected foods prepared by boiling,

boiling-in-bag, and steaming methods. Home Econom-

ics Research Journal 16: 136–142.

Leklem JE and Reynolds RD (eds) (1981) Methods in Vita-

min B-6 Nutrition: Analysis and Status Assessment.

New York: Plenum Press.

Leklem JE (1991) Vitamin B

. In: Machlin LJ (ed.) Hand-

book of Vitamins, 2nd edn., Chapter 9. New York:

Marcel Dekker.

McCance RA, Widdowson EM and Holland B (1993) The

Composition of Foods, 5th edn. London: Springer Verlag.

McCormick DB and Chen H (1999) Update on interconver-

sions of vitamin B-6 with its coenzyme. Journal of

Nutrition 129: 325–327.

Orr ML (1969) Pantothenic Acid, Vitamin B

and Vitamin

in Foods. US Department of Agriculture, Home

Economics Research Report No. 36. Washington, DC:

US Government Printing Office.

Reiter LA and Driskell JA (1985) Vitamin B-6 content of

selected foods served in dining halls. Journal of The

American Dietetic Association 85: 1625–1627.

Sauberlich HE (1999) Laboratory Tests for the Assessment

of Nutritional Status. Boca Raton, FL: CRC Press.

Storvick CA, Benson EM, Edwards MA and Woodring MJ

(1964) Chemical and microbiological determination

of vitamin B

. Methods of Biochemical Analysis XII:

183–276.

Ubbink JB (1992) Vitamin B

. In: DeLeenheer AP, Lambert

WE and Nelis HJ (eds) Modern Chromatographic

Analysis of the Vitamins, 2nd edn., Chapter 10. New

York: Marcel Dekker.

US Department of Agriculture, Agricultural Research Ser-

vice (1999) USDA Nutrient Database for Standard Ref-

erence, Release 13. Nutrient Data Laboratory Home

Page, http://www.nal.usda.gov/fnic/foodcomp.

Yang J, Sulaeman A, Setiawan B et al. (1994) Sensory

qualities and nutrient retention of beef strips prepared

by different household cooking techniques. Journal of

The American Dietetic Association 94: 199–201.

Yang J, Sulaeman A, Setiawan B et al. (1994) Sensory and

nutritive qualities of pork strips prepared by three house-

hold cooking techniques. Journal of Food Quality 17:

33–40.

VITAMIN B

/Properties and Determination 6019

Yuan X, Marchello MJ and Driskell JA (1999) Selected

vitamin contents and retentions in bison patties as re-

lated to cooking method. Journal of Food Science 64:

462–464.

Physiology

D A Bender, University College London, London, UK

Introduction

0001 Vitamin B

has a central role in amino acid metabol-

ism, as the coenzyme for a variety of reactions, in-

cluding transamination and decarboxylation. It is also

the coenzyme of glycogen phosphorylase, and acts to

modulate the activity of steroid and other hormones

(including retinoids and vitamin D) which act by

regulation of gene expression.

Dietary Forms, Biological Availability, and

Metabolism

0002 The main form of vitamin B

in foods is pyridoxal

phosphate, bound to enzymes. There is also a small

amount of pyridoxamine phosphate. In plant foods a

significant amount of the vitamin is present as pyrid-

oxine.

0003 A number of plants contain relatively large amounts

of pyridoxine glycosides, which are not biologically

available, since they are not substrates for mamma-

lian glycosidases. They are absorbed (passively) from

the intestinal lumen and are excreted more or less

quantitatively in the urine. Between 5% and 50% of

the total vitamin B

in some foods may be present as

pyridoxine glycosides.

0004 A proportion of the vitamin B

in foods may be

biologically unavailable, especially after heating, as a

result of the formation of (phospho)pyridoxyllysine

by reduction of the aldimine (Schiff base), by which

pyridoxal phosphate is bound to the e-amino groups

of lysine residues in proteins. While some of this

pyridoxyllysine may be useable, since it is a substrate

for pyridoxine phosphate oxidase, it is also a vitamin

antimetabolite, and even at relatively low concen-

trations can accelerate the development of deficiency

in experimental animals maintained on deficient

diets. In the 1950s there was an outbreak of vitamin

deficiency among infants fed on formula that had

been overheated in manufacture, resulting in the for-

mation of relatively large amounts of pyridoxyllysine.

Digestion and Absorption

0005Pyridoxal phosphate bound as a Schiff base to

lysine in dietary proteins is released on digestion

of the protein. The phosphorylated vitamers are

dephosphorylated by membrane-bound alkaline

phosphatase in the intestinal mucosa; pyridoxal, pyr-

idoxamine, and pyridoxine are all absorbed rapidly

by passive diffusion. Intestinal mucosal cells have

pyridoxine kinase, and pyridoxine phosphate oxidase

so that there is net accumulation by metabolic trap-

ping. Much of the ingested pyridoxine is released into

the portal circulation as pyridoxal, after dephos-

phorylation at the serosal surface.

Metabolism and Transport

0006Most of the absorbed vitamin is taken up by the liver,

although other tissues can also take up the unpho-

sphorylated vitamers from the circulation. Uptake is

by carrier-mediated diffusion, followed by metabolic

trapping as phosphate esters. Pyridoxine and pyri-

doxamine phosphates are oxidized to pyridoxal phos-

phate. All tissues have pyridoxine kinase activity, but

pyridoxine phosphate oxidase is only found in liver,

kidney, and brain (Figure 1).

0007Pyridoxine phosphate oxidase is a flavoprotein,

and its activity falls markedly in riboflavin deficiency.

Despite this central role of riboflavin in vitamin B

metabolism, blood and tissue concentrations of

pyridoxal phosphate are not affected by riboflavin

deficiency, and riboflavin nutrition appears to have

no effect on vitamin B

nutritional status.

0008Pyridoxine phosphate oxidase is inhibited by its

product, pyridoxal phosphate. This is not simple

product inhibition, but involves binding at a specific

inhibitor site on the enzyme. The normal intracellular

concentration of free pyridoxal phosphate gives

significant inhibition, which indicates that this is a

physiologically important mechanism in the control

of tissue pyridoxal phosphate.

0009Pyridoxine is phosphorylated rapidly in liver

and other tissues. Pyridoxal phosphate does not

cross cell membranes, and uptake and efflux of the

vitamin in most tissues are as pyridoxal. Pyridoxal

phosphate is exported from the liver bound to

albumin. Much of the free pyridoxal phosphate in

the liver is hydrolyzed to pyridoxal, which is also

exported, and circulates bound both to albumin and

to hemoglobin in erythrocytes. Free pyridoxal

remaining in the liver is rapidly oxidized to 4-pyri-

doxic acid, which is the main excretory product of the

vitamin.

0010Extrahepatic tissues take up pyridoxal from the

plasma. Pyridoxal phosphate is hydrolyzed to

pyridoxal, which can cross cell membranes, by

6020 VITAMIN B

/Physiology

extracellular alkaline phosphatase, then trapped

intracellularly by phosphorylation.

0011 Tissue concentrations of pyridoxal phosphate are

controlled by the balance between phosphorylation

and dephosphorylation. The activity of phosphatases

acting on pyridoxal phosphate is greater than that of

the kinase in most tissues. This means that pyridoxal

phosphate that is not bound to enzymes will be

dephosphorylated and hence leave the cell by diffu-

sion. Thus there is little accumulation of pyridoxal

phosphate in tissues, other than that which is

bound to enzymes and other proteins (e.g., hormone

receptors).

0012 Free pyridoxal either leaves the cell or is oxidized

to 4-pyridoxic acid by aldehyde dehydrogenase,

which is present in all tissues, and also by hepatic

and renal aldehyde oxidase. 4-Pyridoxic acid is the

main excretory product of vitamin B

, and its excre-

tion reflects recent intake more than the state of

underlying tissue reserves of the vitamin. Small

amounts of pyridoxal and pyridoxamine are also

excreted in the urine, although much of the active

vitamin B

which is filtered at the glomerulus is

resorbed in the kidney tubules.

Storage and Body Reserves

0013There is no specific storage of vitamin B

in the body;

as discussed above, pyridoxal phosphate that is

not bound to enzymes is rapidly dephosphorylated,

oxidized to 4-pyridoxic acid, and excreted.

0014The total body pool of vitamin B

is of the order of

60 mmol (250 mg); 15 mmol (3.7 mg) per kg body

weight. About 80% of this is in muscle, associated

with glycogen phosphorylase. This does not seem to

function as a true reserve of the vitamin and is not

released from muscle in times of deficiency.

0015Muscle pyridoxal phosphate is released into the

circulation (as pyridoxal) in starvation, as muscle

glycogen reserves are exhausted, and there is less

requirement for glycogen phosphorylase activity.

Under these conditions it is available for redistribu-

tion to other tissues, especially liver and kidney, to

HOCH

HC O

HOCH

COOH

HOCH

Kinase

Phosphatase

Kinase

Phosphatase

Kinase

Phosphatase

OOP

Pyridoxine

Pyridoxal

Pyridoxine phosphate

Oxidase

4-Pyridoxic acid Pyridoxal phosphate

Transaminases

Oxidase

Pyridoxamine Pyridoxamine phosphate

fig0001 Figure 1 Metabolism of vitamin B

VITAMIN B

/Physiology 6021

meet the increased requirement of transamination of

amino acids for gluconeogenesis.

Metabolic Functions of Vitamin B

0016 The metabolically active vitamer is pyridoxal phos-

phate, which is involved in many reactions of amino

acid metabolism, where the carbonyl group is the

reactive moiety, in glycogen phosphorylase, where it

is the phosphate group that is important in catalysis,

and in the release of hormone receptors from tight

nuclear binding, where again it is the carbonyl group

that is important.

The Role of Pyridoxal Phosphate in Amino Acid

Metabolism

0017 The various reactions of pyridoxal phosphate in amino

acid metabolism (Figure 2) all depend on the same

chemical principle – the ability to stabilize amino acid

carbanions, and hence to weaken bonds about the

a-carbon of the substrate. This is achieved by reaction

of the a-amino group with the carbonyl group of the

coenzyme to form a Schiff base (aldimine).

0018 Pyridoxal phosphate is bound to enzymes, in the

absence of the substrate, by the formation of an

internal Schiff base to the e-amino group of a lysine

residue at the active site. Thus the first reaction be-

tween the substrate and the coenzyme is transfer of

the aldimine linkage from this e-amino group to the

a-amino group of the substrate.

0019The ring nitrogen of pyridoxal phosphate exerts a

strong electron-withdrawing effect on the aldimine,

and this leads to weakening of all three bonds about

the a-carbon of the substrate. In nonenzymic model

systems, all the possible pyridoxal-catalyzed reac-

tions are observed: a-decarboxylation, aminotransfer,

racemization, and relevant side-chain elimination and

replacement reactions. By contrast, enzymes show

specificity for the reaction pathway followed; which

bond is cleaved will depend on the orientation of

the Schiff base relative to reactive groups of the cata-

lytic site. However, a number of decarboxylases and

enzymes that catalyze side-chain elimination reac-

tions of amino acids undergo gradual inactivation as

a result of catalyzing the half-reaction of transamina-

tion, leaving (catalytically inactive) pyridoxamine

phosphate at the catalytic site.

0020a-Decarboxylation If the electron-withdrawing

effect of the ring nitrogen is primarily centered on

OOP

Lysine

COOH

CR COOH

OOP

Amino acid

substrate

Lysine

Decarboxylation

Substrate aldimine

COOH

Substrate ketimine

Pyridoxamine phosphate

Tautomerization

Pyridoxal phosphate internal aldimine

(Schiff base)

R−CH

−NH

Product amine

Transamination

(amino transfer)

Product oxo-acid

(keto-acid)

fig0002 Figure 2 Roles of vitamin B

in amino acid metabolism.

6022 VITAMIN B

/Physiology

the a-carbon-carboxyl bond, the result is decarboxy-

lation of the amino acid with the release of carbon

dioxide. The resultant carbanion is then protonated,

and the primary amine corresponding to the amino

acid is displaced by the lysine residue at the active site,

with reformation of the internal Schiff base.

0021 A number of the products of the decarboxylation of

amino acids are important as neurotransmitters and

hormones: 5-hydroxytryptamine, the catecholamines

dopamine, norepinephrine (noradrenaline), and epi-

nephrine (adrenaline), histamine and g-aminobuty-

rate (GABA), and as the diamines and polyamines

involved in the regulation of DNA metabolism. The

decarboxylation of phosphatidylserine to phospha-

tidylethanolamine is important in phospholipid

metabolism.

0022 Racemization of Amino Acids Deprotonation of the

a-carbon of the amino acid leads to tautomerization

of the Schiff base to yield a quinonoid ketimine. The

simplest reaction that the ketimine can undergo is

reprotonation at the now symmetrical a-carbon. Dis-

placement of the substrate by the reactive lysine resi-

due results in the racemic mixture of d- and l-amino

acid.

0023 Amino acid racemases are important in bacterial

metabolism, since several d-amino acids are

required for the synthesis of cell wall mucopolysac-

charides. There is no evidence that there are any

mammalian amino acid racemases; such utilization

of d-amino acids as occurs is probably due to the

action of renal d-amino acids oxidase to form

the symmetrical 2-oxo-acid which is then a substrate

for transamination.

0024Transamination Hydrolysis of the a-carbon-amino

bond of the ketimine formed by deprotonation of the

a-carbon of the amino acid results in the release of

the 2-oxo-acid corresponding to the amino acid sub-

strate, and leaves pyridoxamine phosphate at the

catalytic site of the enzyme. In this case there is no

reformation of the internal Schiff base to the reactive

lysine residue. This is the half-reaction of transamina-

tion. The process is completed by reaction of pyridox-

amine phosphate with a second oxo-acid substrate,

forming an intermediate ketimine, followed by the

reverse of the reaction sequence shown in Figure 2,

releasing the amino acid corresponding to this second

substrate after displacement from the aldimine by the

reactive lysine residue to reform the internal Schiff

base.

0025Transamination (Figure 3) is of central importance

in amino acid metabolism, providing pathways for

the catabolism of all of the amino acids except lysine,

which does not undergo transamination. Many of

these reactions are linked to the amination of 2-oxo-

glutarate to glutamate or glyoxylate to glycine, which

are substrates for oxidative deamination, reforming

the oxo-acids. Transamination reactions also provide

a pathway for the synthesis of those amino acids for

which there is an alternative source of the oxo-acid

(the nonessential amino acids). Indeed, the nonessen-

tial amino acids can be defined as those whose oxo-

acids can be formed other than from the amino acid

itself.

0026Side-chain elimination and replacement reac-

tions The third bond in the Schiff base aldimine

that can be labilized by the electron-withdrawing

C COOH

Pyridoxal phosphate

Substrate amino acid

Amino donor

C COOH

Product amino acid

C COOH

Pyridoxamine phosphate

Product oxo-acid (keto-acid)

Substrate oxo-acid (keto-acid)

Amino acceptor

fig0003 Figure 3 The role of vitamin B

in transamination reactions.

VITAMIN B

/Physiology 6023

effect of the ring nitrogen of pyridoxal phosphate is

that between the a-carbon and the side-chain of the

amino acids, resulting in a variety of a–b elimination

and b–g replacement reactions.

The Role of Pyridoxal Phosphate in Glycogen

Phosphorylase

0027 Glycogen phosphorylase catalyzes the sequential

phosphorolysis of glycogen to release glucose 1-phos-

phate; it is thus the key enzyme in the utilization of

muscle and liver reserves of glycogen.

0028 Unlike other pyridoxal phosphate-dependent

enzymes, in which the carbonyl group is essential

for catalysis, the internal Schiff base between pyri-

doxal phosphate and lysine in glycogen phosphory-

lase is not broken during the reaction. The catalytic

region of the coenzyme is the 5

-phosphate group.

The initial stage in the phosphorolysis of glycogen

is protonation of the glycosidic oxygen of the poly-

saccharide by inorganic phosphate. The resultant

oxycarbonium ion is stabilized by the inorganic phos-

phate. The role of pyridoxal phosphate is as a proton

shuttle or buffer to stabilize the oxycarbonium-

phosphate ion pair, permitting covalent binding of

the phosphate to the oxycarbonium ion, to form

glucose 1-phosphate.

The Role of Pyridoxal Phosphate in Steroid

Hormone Action

0029 Pyridoxal phosphate has a role in modulating the

action of those hormones which act by binding to a

nuclear receptor protein, inducing transcription of

DNA, and hence regulating gene expression, leading

to new protein synthesis. Such hormones include an-

drogens, estrogens, progesterone, glucocorticoids,

calcitriol (the active metabolite of vitamin D), reti-

noic acid and other retinoids, and thyroid hormone.

Target-tissue specificity of hormone action is insured

by the presence of characteristic receptor proteins

with a zinc finger motif, which are responsible for

both nuclear uptake of the hormone and the inter-

action with DNA and nucleoproteins to initiate gene

expression.

0030 Pyridoxal phosphate reacts with a lysine residue in

the receptor protein, and displaces the hormone–

receptor complex from tight nuclear binding.

In vitro, reaction with pyridoxal phosphate also in-

hibits the binding of receptor protein to isolated DNA

and chromatin. The effect is specific for the phosphor-

ylated vitamer, suggesting that there may be a specific

pyridoxal-phosphate binding site on the receptor pro-

teins, and it occurs at low concentrations of pyridoxal

phosphate, of the same order of magnitude as occur in

tissues under normal conditions.

0031This suggests that pyridoxal phosphate acts as a

cofactor in the release of hormone–receptor com-

plexes from tight nuclear binding, resulting in release

of the receptor from the nucleus, termination of

hormone action, and recycling receptor protein for

further uptake of hormone.

0032In experimental animals, vitamin B

deficiency

results in increased and prolonged nuclear uptake

and retention of steroid hormones in target tissues,

and there is enhanced sensitivity to hormone action.

Deficient animals show greater induction of uterine

peroxidase, and considerably greater suppression of

the hypothalamic secretion of luteinizing hormone,

by estrogens than do vitamin B

-supplemented con-

trols. In vitamin B

-deficient male animals there is an

increased mitotic response of the prostate to low

doses of testosterone. Deficient male animals have a

higher activity of ornithine decarboxylase (an andro-

gen-induced enzyme) in the liver, and deficient

females have higher renal ornithine transaminase

(an estrogen-induced enzyme). The induction of

hepatic tyrosine transaminase and tryptophan dioxy-

genase by glucocorticoids is also enhanced in vitamin

-deficient animals.

0033In a variety of cells in culture that have been trans-

fected with a glucocorticoid, estrogen, or progester-

one response element linked to a reporter gene, acute

vitamin B

depletion (by incubation with 4-deoxypyr-

idoxine) leads to a twofold increase in expression of

the reporter gene in response to hormone action.

Conversely, incubation of these cells with high con-

centrations of pyridoxal, leading to a high intracellu-

lar concentration of pyridoxal phosphate, results in

a halving of the expression of the reporter gene in

response to hormone stimulation.

Criteria of Adequacy and Assessment of

Nutritional Status

Plasma Concentrations of the Vitamin

0034The fasting plasma concentration of either total vita-

min B

or, more specifically, pyridoxal phosphate, is

widely used as an index of vitamin B

nutritional

status. The generally accepted criteria of adequacy

are shown in Table 1.

0035Conditions that involve increased plasma activity

of alkaline phosphatase may result in reduced plasma

pyridoxal phosphate, without affecting tissue concen-

trations of pyridoxal phosphate or vitamin B

nutri-

tional status, as assessed by other criteria. There is a

compensatory increase in the circulating concentra-

tion of pyridoxal, which is the main form for tissue

uptake of vitamin B

. Despite the fall in plasma

pyridoxal phosphate in pregnancy, which has been

6024 VITAMIN B

/Physiology

widely interpreted as indicating vitamin B

depletion,

the plasma concentration of (pyridoxal phosphate

plus pyridoxal) is unchanged. This suggests that de-

termination of plasma pyridoxal phosphate alone

may not be a reliable index of vitamin B

nutritional

status.

Urinary Excretion of 4-Pyridoxic Acid

0036 About half of the normal dietary intake of vitamin B

is excreted as 4-pyridoxic acid. Urinary excretion of

4-pyridoxic acid will largely reflect recent intake of

the vitamin rather than underlying nutritional status;

the criteria for assessment of 4-pyridoxic acid

excretion are shown in Table 1.

Coenzyme Saturation of Transaminases

0037 Various pyridoxal phosphate-dependent enzymes

compete with each other for the available pool of

coenzyme. Thus the extent to which an enzyme is

saturated with its coenzyme provides a means of as-

sessing the adequacy of the body pool of coenzyme.

This can be determined by measuring the activity of

the enzyme before and after the activation of any

apoenzyme present in the sample by incubation with

pyridoxal phosphate added in vitro. Erythrocyte

aspartate and alanine transaminases are both com-

monly used; the results are expressed as either the

percentage stimulation of activity by added pyridoxal

phosphate, or the activation coefficient – the ratio of

activity with added coenzyme to that without added

coenzyme.

0038 It seems to be normal for a proportion of pyridoxal

phosphate-dependent enzymes to be present as in-

active apoenzyme, without coenzyme. This may be a

mechanism for metabolic regulation. It is possible

that increasing the intake of vitamin B

,soasto

insure complete saturation of pyridoxal phosphate-

dependent enzymes, may not be desirable.

Metabolic Loading Tests

0039A direct test of the adequacy of an individual’s intake

to meet his or her idiosyncratic metabolic require-

ment is the ability to metabolize a test dose of a

substrate whose metabolism is dependent on the vita-

min. For vitamin B

, two metabolic loading tests can

be used, although neither can be considered to be

reliable for population studies of vitamin B

status.

0040The Tryptophan Load Test The oxidative pathway

of tryptophan metabolism is shown in Figure 4.

Kynureninase is a pyridoxal phosphate-dependent

enzyme, and in vitamin B

deficiency its activity is

lower than that of tryptophan dioxygenase. This

means that there is a considerable accumulation of

both hydroxykynurenine and kynurenine, sufficient

to permit greater than usual metabolic flux through

kynurenine transaminase, resulting in increased for-

mation of kynurenic and xanthurenic acids. Although

kynurenine transaminase is also pyridoxal phos-

phate-dependent, it is relatively unaffected in vitamin

deficiency. Kynureninase is exquisitely sensitive to

vitamin B

deficiency because it undergoes a slow

inactivation as a result of catalyzing the half-reaction

of transamination in addition to its normal reaction.

The resultant enzyme with pyridoxamine phosphate

at the catalytic site is catalytically inactive, and can

only be reactivated if there is an adequate concen-

tration of pyridoxal phosphate to displace the

pyridoxamine phosphate.

0041Xanthurenic and kynurenic acids are easy to

measure in urine, so that the ability to metabolize a

test dose of 2 or 5 g of tryptophan has been widely

adopted as a convenient and sensitive index of vita-

min B

nutritional status. However, induction of tryp-

tophan dioxygenase by glucocorticoid hormones will

result in a greater rate of formation of kynurenine and

hydroxykynurenine than the capacity of kynureni-

nase, and will thus lead to increased formation of

kynurenic and xanthurenic acids – an effect similar

to that seen in vitamin B

deficiency. Such results may

be erroneously interpreted as indicating vitamin B

deficiency in a variety of subjects whose problem is

increased glucocorticoid secretion as a result of stress

or illness, not vitamin B

deficiency.

0042Inhibition of kynureninase, e.g., by estrogen me-

tabolites, also results in accumulation of kynurenine

and hydroxykynurenine, and hence increased forma-

tion of kynurenic and xanthurenic acids, again giving

results which falsely suggest vitamin B

deficiency.

tbl0001 Table 1 Indices of vitamin B

nutritional status

Adequate status

Plasma total vitamin B

> 40 nmol (10 mg) l

1

Plasma pyridoxal phosphate >30 nmol (7.5 mg) l

1

Erythrocyte alanine aminotransferase

activation coefficient

<1.25

Erythrocyte asparate aminotransferase

activation coefficient

<1.80

Erythrocyte asparate aminotransferase >0.13 units

(8.4 mkat) l

1

Urine 4-pyridoxic acid >3.0 mmol 24 h

1

>1.3 mmol mol

1

creatinine

Urine total vitamin B

>0.5 mmol 24 h

1

>0.2 mmol mol

1

creatinine

Urine xanthurenic acid after 2 g

tryptophan load

<65 mmol 24 h

1

increase

Urine cystathionine after 3 g methionine

load

<350 mmol 24 h

1

increase

VITAMIN B

/Physiology 6025

This has been widely, but incorrectly, interpreted as

estrogen-induced vitamin B

deficiency – it is in fact

simple competitive inhibition by estrogen metabolites

of the enzyme that is the basis of the tryptophan load

test.

0043 There is normally a considerable excess of either

apokynureninase or kynureninase that has undergone

transamination, and has pyridoxamine phosphate at

the catalytic site, in the liver. This can be activated by

(relatively high concentrations of) pyridoxal phos-

phate. The abnormalities of tryptophan metabolism

associated with increased activity of tryptophan diox-

ygenase, or inhibition of kynureninase by estrogen

metabolites, are thus corrected by the administration

of high doses of vitamin B

, although they are not in

fact due to deficiency.

0044This means that, while the tryptophan load test

may be an appropriate index of status in controlled

depletion/repletion studies to determine vitamin B

requirements, it is not an appropriate index of status

in population studies.

0045The methionine loading test The metabolism of me-

thionine, shown in Figure 5, includes two pyridoxal

phosphate-dependent steps, catalyzed by cystathio-

nine synthetase and cystathionase. In vitamin B

deficiency there is an increase in the plasma concen-

tration of homocysteine, and increased urinary

COOH

Tryptophan

Tryptophan dioxygenase and

formylkynurenine hydroxylase

Kynurenine

aminotransferase

COOH

CH COOH

Kynurenine

aminotransferase

COOH

Kynurenine

Kynurenic acid

3-Hydroxykynurenine

3-Hydroxyanthranilic acid

Kynureninase

(vitamin B

-dependent)

Alanine

Xanthurenic acid

COOH

Acetyl CoA

Quinolinic acid

Nicotinamide

Kynurenine hydroxylase

fig0004 Figure 4 The oxidative pathway of tryptophan metabolism – the basis of the tryptophan load test for vitamin B

status.

6026 VITAMIN B

/Physiology

excretion of cystathionine and homocysteine, both

after a loading dose of methionine and under basal

conditions. The ability to metabolize a test dose of

methionine therefore provides an index of vitamin B

nutritional status. Because measurement of homocys-

teine and cystathionine is technically less easy than

measurement of xanthurenic and kynurenic acids, the

methionine load test has been less widely used than

the tryptophan load test.

0046 Some 10–25% of the population have a genetic

predisposition to hyperhomocysteinemia, which is

a risk factor for atherosclerosis and coronary heart

disease, as a result of polymorphism in the gene for

methylenetetrahydrofolate reductase. As discussed

below, there is no evidence that supplements of vita-

min B

reduce fasting plasma homocysteine in these

subjects and, like the tryptophan load test, the

methionine load test may be an appropriate index

of status in controlled depletion/repletion studies

to determine vitamin B

requirements, but not in

population studies.

Requirements and Recommendations

0047The total body pool of vitamin B

is of the order of

15 mmol (3.7 mg) per kg of body weight. Isotope

tracer studies suggest that there is a turnover of

about 0.13% per day, and hence a minimum require-

ment for replacement of 0.02 mmol (5 mg) per kg of

body weight – some 350 mg day

1

for a 70-kg adult.

0048Most studies of vitamin B

requirements have

followed the development of abnormalities of trypto-

phan (and sometimes also methionine) metabolism

during depletion, and normalization during repletion

with graded intakes of the vitamin. While the trypto-

phan and methionine loading tests are unreliable as

Methylated

product

Acceptor

S-adenosylmethionine S -adenosylhomocysteine

Methyltransferase

Methionine adenosyltransferase

ATP

Adenosine

COOH

Methionine

Tetrahydrofolate

Methyl

tetrahydrofolate

Methionine synthetase

(vitamin B

-dependent)

Homocysteine Homocystine

Nonenzymic

Cystathionine Cysteine

Serine

Cystathionase

(vitamin B

-dependent)

Cystathionine synthetase

(vitamin B

-dependent)

α-Ketobutyrate

fig0005 Figure 5 The pathway of methionine metabolism – the basis of the methionine load test for vitamin B

status.

VITAMIN B

/Physiology 6027