Chapter 10 vitamin b6

Cellular levels of thymine, one of the four constituent DNA bases,depend on PLP through its involvement as a coenzyme in folate metabolism.Vitamin B6is widely distributed in foods, and a

Pyridoxal phosphate (PLP), the physiologically active B6vitamer, serves

as a coenzyme for over 100 different enzymes involved in almost allaspects of cellular biochemistry and metabolism Vitamin B6 also plays

an important role in the development and maintenance of a competentimmune system Because of this versatility, vitamin B6 is crucial fornormal growth, development, and homeostasis

In amino acid metabolism, vitamin B6is required in a variety of enzymesconcerned with the interconversion of amino acids, the synthesis of non-essential amino acids, and the metabolism of amino acids in excess of theamounts required for protein synthesis Aminotransferases facilitate thetransfer of amino groups between amino acids and keto acids, and thusrepresent an important link among amino acid, carbohydrate, and fatmetabolism Many neurotransmitters are formed by the PLP-dependentdecarboxylation of amino acids PLP is the coenzyme for glycogen phos-phorylase, a key enzyme in the utilization of liver and muscle glycogenreserves In lipid metabolism, PLP is the coenzyme for phosphatidylserinedecarboxylation PLP is also involved in the synthesis of sphingosine, and

a deficiency of vitamin B6leads to impaired development of brain lipidsand incomplete myelination of nerve fibers in the central nervoussystem Cellular levels of thymine, one of the four constituent DNA bases,depend on PLP through its involvement as a coenzyme in folate metabolism.Vitamin B6is widely distributed in foods, and any diet so poor as to beinsufficient in this vitamin would most likely lack adequate amounts ofother B-group vitamins For this reason, a primary clinical deficiency of

B6in the adult human is rarely encountered The administration of theantagonist deoxypyridoxine to adult volunteers receiving diets low invitamin B6 resulted in lesions of the skin and mouth that resembledthose of riboflavin and niacin deficiency These symptoms responded tovitamin B6therapy, but did not respond to thiamin, riboflavin, or niacin.Chronic overdosing with vitamin B6has been reported to cause periph-eral neuropathy in women Acute toxicity of the vitamin, however, is low

10.2 Chemical Structure, Biopotency, and Physicochemical Properties

10.2.1 Structure and Potency

Vitamin B6is the generic descriptor for all 3-hydroxy-2-methylpyridinederivatives that exhibit qualitatively in rats the biological activity of pyr-idoxine Six B6vitamers are known, namely pyridoxine or pyridoxol (PN),pyridoxal (PL), and pyridoxamine (PM), which possess, respectively,alcohol, aldehyde, and amine groups in the 4-position; their respective

50-phosphate esters are designated as PNP, PLP, and PMP (Figure 10.1).Pyridoxine is systematically named as 3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridine and is available commercially as its hydrochloridesalt, PN.HCl (C8H11O3N.HCl, MW¼ 205.6) PN.HCl is the only form ofvitamin B6 used in the fortification of foods The six B6 vitamers areconsidered to have approximately equivalent biopotency on the basis oftheir ultimate conversion to coenzymes

In its role as a coenzyme, PLP is bound tightly to the apoenzyme by

a Schiff base (aldimine) linkage formed through condensation of the4-carbonyl group with the 1-amino group of specific lysine residues.The resultant Schiff base compound may be subject to nucleophilicattack by a neighboring amino, sulfhydryl, or imidazole group to form

6

are reversibly bound and easily dissociated from the apoenzymes

An ubiquitous bound form of PN that occurs in plant tissues is 50side [2] Two minor derivatives, in which an organic acid is esterified tothe C-6 position of the glucose moiety of PN-glucoside, have been

2

3

4 5 6

N

HO

R

CH2O P O H O

OH

CH2OH CHO

a substituted aldamine (Figure 10.2) [1] All these forms of vitamin B

(b-D-glucopyranosyl)-pyridoxine (Figure 10.3), abbreviated to

PN-gluco-identified in legume seedlings [2] A more complex derivative ofPN-glucoside containing cellobiose and 5-hydroxydioxindole-3-aceticacid moieties has been identified as a major form of vitamin B6in ricebran and legumes [3].

10.2.2 Physicochemical Properties

10.2.2.1 Appearance and Solubility

PN.HCl is a white, odorless, crystalline powder with a salty tasteand an mp of 204 –2068C (with decomposition) It is readily soluble inwater (1 g/5 ml), sparingly soluble in ethanol (1 g/100 ml), and veryslightly soluble in diethyl ether and chloroform The pH of a 5%aqueous solution is 2.3 –3.5; pKavalues are 5.0 and 9.0 (258C) The freebase is readily soluble in water and slightly soluble in acetone, chloro-form, and diethyl ether

In aqueous solutions, the B6vitamers exist in a variety of equilibriumforms, depending upon the pH [4] The structures of some of these

as the anion in alkaline solutions, and primarily as the electrically neutral

OH HOCH2

CH N R

N

O HO

H3C

CH2OH

CH2H O

OH

H

H HO H

OH H

CH2OH

FIGURE 10.3

Structure of 50-O-(b- D -glucopyranosyl)-pyridoxine.

forms are shown inFigure 10.4 PN exists as the cation in acidic solutions,

dipolar ion at neutral pH PM also exists as the cation in acidic solutionsand the anion in alkaline solutions, but at neutral pH the predominantform is the tripolar ion The situation is more complicated with PLowing to the possibility of hemiacetal formation or hydration Atneutral pH, the predominant form of PL is the hemiacetal dipolar ion.

10.2.2.2 Stability in Aqueous Solution

Saidi and Warthesen [5] observed that no significant degradation ofPN.HCl took place when aqueous solutions protected from light wereheld at 40 and 608C for up to 140 days at pH levels ranging from 4 to

7 Under the same conditions, PM.2HCl showed a trend of increasingloss with increasing pH, while PL.HCl showed a marked loss at pH 5,but only a moderate loss above and below that pH value Ang [6]showed that PN.HCl was the most stable and PM.2HCl the least stable

of the three vitamers after exposure of aqueous solutions to normallaboratory light at different pH values Low-actinic amber glassware orgold fluorescent lighting protected solutions of PLP from photodegrada-tion, but low-ultraviolet “white” fluorescent lamps failed to do so [7].The principal photodegradation product of PLP is 4-pyridoxic acid

50-phosphate [8]

Shephard and Labadarios [9] investigated the degradation of vitamin

B6standard solutions after experiencing difficulties in the reproducibility

of B6vitamer standard determinations in an HPLC method Of the line vitamer standards used (PN.HCl, PM.2HCl, PL.HCl, PLP, andPMP.HCl), all but PLP were stable when stored individually in thedark Solutions of PLP in water were stable when stored frozen (2208C)

crystal-at a concentrcrystal-ation of 1 mg/ml (pH 3.3) However, storage crystal-at room erature in the dark for 24 h of a laboratory working solution (1 mg/ml)either in sodium acetate buffer (pH 5.5) or in distilled water (pH 6.1)resulted in a 20 or 95% loss of PLP, respectively, due to hydrolysis

temp-to PL When prepared in 0.01 M HCl, PLP was stable for at least

2 days at room temperature It was shown that Schiff base formation

CH3

CHOH O

and transamination reactions can occur between the vitamers themselves

at room temperature or below To prevent these reactions, vitamer utions must be stored separately and compound standards must be pre-pared before use in a fairly acid medium (pH 3.0)

10.3.1 Occurrence

Vitamin B6is present in all natural unprocessed foods, with yeast extract,wheat bran, and liver containing particularly high concentrations Otherimportant sources include whole-grain cereals, nuts, pulses, lean meat,fish, kidney, potatoes, and other vegetables In cereal grains, over 90%

of the vitamin B6is found in the bran and germ [10], and 75 – 90% of the

B6 content of the whole grain is lost in the milling of wheat to extraction flour [11] Thus, white bread is considerably lower in vitamin

low-B6content than is whole wheat bread Milk, eggs, and fruits contain

rela-B6content of selected foods

In raw animal and fish tissue, the major form of vitamin B6 is PLP,which is reversibly bound to proteins as Schiff bases and substitutedaldamines PN and PNP are virtually absent in animal tissues, one excep-tion being liver tissue, in which they are detectable at very low levels.Using a nonhydrolytic extraction procedure and HPLC, the vitamin B6

content of whole pasteurized homogenized milk was found to comprisethe following vitamers: PL (53%), PLP (23%), PMP (12%), and PM(12%); PN was not detected [13] Siegel et al [14] estimated the free andtotal vitamin B6 content of milk by assaying aliquots before and afteracid hydrolysis; the difference indicated the amount of bound vitamin,which was found to be 14% of the total PNP does not occur to anymeasurable extent in natural products

Roth-Maier et al [15] reported that the vitamin B6in all tested foods ofplant origin occurs in the form of PN and PM, except for corn (maize)where more than 50% of the vitamin B6content occurred as PL A pro-portion of the PN in plant tissues may be present as PN-glucosideand/or more complex derivatives of PN-glucoside No generalizationscan be made as to one group of foods consistently having a high PN-glucoside content Typical sources of PN-glucoside (expressed as a percen-tage of the total vitamin B6 present) are bananas (5.5%), raw broccoli(35.1%), raw green beans (58.5%), raw carrots (70.1%), and orange juice(69.1%) [16]

tively low concentrations of the vitamin.Table 10.1[12] gives the vitamin

10.3.2 Stability

In general, vitamin B6is unstable during prolonged heat treatment, andnot sensitive to oxidation by air The stability of vitamin B6toward foodprocessing and storage depends to some extent on the B6 vitamercontent of the food, because PN is considerably more stable to heatthan PL or PM [17] This means that plant foods (containing mostly PN)are likely to be rather more stable than foods of animal origin (containingmostly PL or PM) Interconversions between the aldehyde (PL and PLP)and amine (PM and PMP) B6 vitamers via reversible interaction withproteins or carbonyl compounds occur during the processing or storage

of meat and dairy products [18] The kinetics of vitamin B6degradationhave been discussed by Gregory [19]

TABLE 10.1

Vitamin B6Content of Various Foods

Food

mg Vitamin B 6 /100 g Edible Portion Cow milk, whole, pasteurized 0.06

Cheese, cheddar, average 0.15

Egg, chicken, whole, raw 0.12

Wheat flour, wholemeal 0.50

Wheat flour, white, plain 0.15

Rice, brown, raw N

Beef, trimmed lean, raw, average 0.53

Lamb, trimmed lean, raw, average 0.30

Pork, trimmed lean, raw, average 0.54

Chicken meat, raw 0.38

Liver, lamb, fried 0.53

Cod, raw, fillets 0.18

Potato, main crop, old, average, raw 0.44

Chick peas, dried, raw 0.53

Soybeans, dried, raw 0.38

Lentils, green and brown, whole, dried, raw 0.93

Red kidney beans, dried, raw 0.40

Note: N, the vitamin is present in significant quantities but there is no

reliable information on the amount.

Source: From Food standards Agency, McCance and Widdowson’s The

composition of foods, 6th summary ed., Royal Society of Chemistry,

Cambridge, 2002 With permission.

Raab et al [20] reported that water-blanching caused a loss of 19 –24%

of vitamin B6 from lima beans, while steam-blanching caused a loss

of 13– 17% Ekanayake and Nelson [21] measured total vitamin B6

(acid-hydrolyzed) and available vitamin B6(enzyme-treated) in a study

of thermally processed limas beans About 20% of the PN content waslost during the blanching of the beans, but the subsequent heat sterili-zation did not affect the total vitamin B6content There was, however, a50% drop in available vitamin B6 The data suggested that the PL andPLP of the heat-processed beans were bound in some way, and this pre-vented the vitamers from being released by digestion A 55% loss ofvitamin B6activity in canned lima beans relative to frozen lima beanswas also reported by Richardson et al [22] using a rat growth assay.Sterilization and subsequent 5-year storage did not affect the vitamin B6

content of canned military rations [23] Thermal processing had littleeffect on the concentration of PN-glucoside in alfalfa sprouts, demonstra-ting the stability of the glycosidic bond as well as the stability of the PNmoiety of the conjugate [24] There is good stability of vitamin B6 inwheat flour during bread making [25]

Most of the research into the effects of food processing upon vitamin B6

bioavailability has been directed to the heat-sterilization of canned orated milk by retort processing The heat-sterilization of milk and unfor-tified infant formula resulted in losses of 36– 67% of naturally occurringvitamin B6 using Saccharomyces cerevisiae as the assay organism, thelosses appearing progressively during the first 10 days after processing[26] The reduction of vitamin activity was due mainly to the loss of thepredominant B6 vitamer, PL In the same study, PM and PL added tomilk were degraded to roughly the same extent as natural vitamin B6,whereas added PN.HCl was not appreciably destroyed by autoclaving.Gregory and Hiner [17] reported that PN.HCl is stable during retortprocessing, whereas PM.2HCl and PL.HCl are 2.5- to 3.5-fold lessstable Vitamin B6 losses are not as great when processing spray-driedmilk products and condensed milk as when heat sterilization is employed[26] Ford et al [27] reported no loss of vitamin B6during the ultra-hightemperature (UHT) processing of milk, but up to 50% of the vitaminwas lost during 90 days’ storage The UHT processing (heating at 110–1128C for 15–20 min) was unlikely to be responsible for these storagelosses since similar losses during storage were found with raw milkheld at2308C Conventional pasteurization of milk has no effect on thevitamin B6content [28]

evap-Losses of indigenous vitamin B6 in foods during storage have beenobserved [22,29 –31] The decrease in vitamin B6 activity on storage ofbeef liver, boned chicken, cabbage, and green beans was not observed

in lima beans and sweet potatoes using a rat growth assay [22] Usingselective extraction techniques and HPLC, Addo and Augustin [32]

showed that PLP and PM in stored potatoes remained unchanged,whereas the PN-glucoside increased more than fourfold, and PN wasreduced by half These observations indicated a possible synthesis ofvitamin B6 during storage The storage of various foods at 2188C for

5 months resulted in a 19– 60% decrease in total vitamin B6 The losswas significantly greater in foods of animal origin (an average of 55%)than in plant-derived foods [33] Unlike the loss in indigenous vitamin

B6during storage, the stability of PN added to fortify various products

is high Bunting [34] reported the retention of 90 –100% of the PNadded to corn meal and macaroni following storage at 388C and 50% rela-tive humidity for 1 yr There is good stability of vitamin B6 in fortifiedcereal products after storage [35 –37] However, only 18– 44% of thevitamin B6in a commercial rice-based PN-fortified breakfast cereal wasfound to be biologically available using a rat bioassay [38]

In a report on the influence of cooking [39], stewing reduced the content

of total vitamin B6by 56% and braising by 58% in beef and by 58 and 45%,respectively, in pork Part of the loss was due to release of meat juice Withmeat juice included, the percentage of vitamin retained was 80 and 63% inbeef, and 68 and 62% in pork Losses of the total vitamin B6in vegetablesafter boiling in water were between 16 and 61%; losses were lower aftersteaming (between 10 and 24%) due to less leaching

The research effort has led to the general conclusion that the thermalprocessing of milk products and other animal-derived foods promotesthe chemical reduction of protein-bound Schiff base forms of PL andPLP to peptide-linked 1-pyridoxyllysine (Figure 10.5) or its 50-phosphatederivative This reaction has been demonstrated to take place in a modelfood system during thermal processing [40], in evaporated milk, chickenliver, and muscle that were autoclaved to simulate retort processing [18]and in a dehydrated model food system that was stored at 378C and 0.6water activity (Aw) for 128 days [41]

Protein-bound 1-pyridoxyllysine can be phosphorylated by pyridoxalkinase, the enzyme that phosphorylates the free B6vitamers after they

N CH3

OH HOCH2

have been absorbed into the enterocytes [42] The 50-phosphorylated1-pyridoxyllysine can then be oxidized to PLP by pyridoxamine (pyridox-ine) 50-phosphate oxidase, the enzyme responsible for converting PMPand PNP to PLP [42] These two enzyme reactions provide a metabolicbasis for the observed 50% vitamin B6activity of 1-pyridoxyllysine rela-tive to the molar potency of PN [43] When fed to rats at low levels invitamin B6-deficient diets, 1-pyridoxyllysine exhibited antivitamin B6

activity, which could be counteracted by dietary supplementation with

PN [43] This antivitamin B6activity may be at least partly attributable

to the competitive inhibition of pyridoxal kinase This antagonisticeffect of 1-pyridoxyllysine, when present in vitamin B6-deficient diets,may have been responsible for the severe deficiency developed ininfants fed unfortified, heat-sterilized, canned infant formulas [44]

10.3.3 Applicability of Analytical Techniques

In foods of animal origin, PL, PLP, PM, and PMP are found as a result ofinterconversion of aldehyde and amine forms during processing andstorage Plant-derived foods contain mostly PN, a significant proportion

of which may be present as PN-glucoside PNP does not occur to any nificant extent in natural products PN.HCl is used in the fortification offoods The PLP that is bound to the apoenzyme by a Schiff base inanimal tissues is presumed to be totally bioavailable The PN-glucosideand other conjugated forms found in plant-derived foods appear to belargely unavailable to humans

sig-The total available vitamin B6activity of a food or diet containing allforms of the vitamin is measured most conclusively by an animal assay

By feeding the material directly, both the free and bound forms canexert their combined effect The free vitamers are equally active molefor mole when fed to animals as separate supplements in solution.However, when mixed in the ration, PM and PL are less active than PN[45] Both the rat and the chick are sensitive to influences of diet compo-sition, especially with respect to fermentable carbohydrates, whichprovide the means for vitamin B6production by microflora in the largeintestine The utilization of microbially produced vitamin B6by copro-phagy or direct intestinal absorption can bias quantitative bioassays.Total vitamin B6activity is usually estimated microbiologically using aturbidimetric yeast assay; the radiometric microbiological assay is a morerecent innovation With the aid of HPLC, it is possible to measure simul-taneously all of the B6 vitamers likely to be present in a food extract,together with the inactive metabolite 4-pyridoxic acid For routine foodanalysis, it is customary to hydrolyze the phosphorylated forms and todetermine PN, PL, and PM, each of which represents the sum of the

phosphorylated and nonphosphorylated forms This approach is validbecause, in humans, all six vitamers exhibit equal biological activity on

a molar basis The chromatographic separation of three compounds,rather than six, is obviously less demanding of the HPLC system, andthe data obtained can be compared with data obtained by microbiologicalassay, in which dephosphorylation is obligatory Traditional methodsused to assay vitamin B6in food products may overestimate the bioavail-ability of the vitamin due to the use of extraction techniques that comple-tely liberate PN from glycosylated forms The use of selective extractionprocedures allows the various bound forms of vitamin B6to be deter-mined by HPLC Gregory [2] has critically reviewed methods for determi-nation of vitamin B6in foods and other biological materials

No international unit of vitamin B6 activity has been defined, andanalytical results are expressed in weight units (mg) of pure PN.HCl.All six vitamers are considered to have approximately equivalent biologi-cal activity in humans as a result of their enzymatic conversion to themajor coenzyme form, PLP

In many fruits and vegetables, 30% or more of the total vitamin B6 ispresent as PN-glucoside The binding of PLP to protein through aldimine(Schiff base) and substituted aldamine linkages is reversibly dependent

on pH, the vitamin – protein complexes being readily dissociated undernormal gastric acid (low pH) conditions The release of PLP from itsassociation with protein is an important step in the subsequent absorption

of vitamin B6, as binding to protein inhibits the next step, hydrolysis ofPLP by alkaline phosphatase [47] It would appear, therefore, that thewidespread practice of raising the postprandial gastric and upper smallintestinal pH by the use of pharmaceutical antacids may impair vitamin

B6absorption

Physiological amounts of PLP and PMP are largely hydrolyzed byalkaline phosphatase in the intestinal lumen before absorption of free

PL and PM [48,49] When present in the lumen at nonphysiological levelsthat saturate the hydrolytic enzymes, substantial amounts of PLP andPMP are absorbed intact, but at a slower rate than their non-phosphorylated forms.

The absorption of PN, PL, and PM takes place mainly in the jejunumand is a dynamic process involving several inter-related events The vita-mers cross the brush-border membrane by simple diffusion In humans,

PM is absorbed more slowly or metabolized differently, or both, thaneither PL or PN [50] Within the enterocyte, PN, PL, and PM are converted

to their corresponding phosphates by the catalytic action of cytoplasmicpyridoxal kinase, and transaminases interconvert PLP and PMP The con-version of a particular vitamer to other forms by intracellular metabolismcreates a concentration gradient across the brush border for that vitamer,thus enhancing its uptake by diffusion [51] The phosphorylated vitamersformed in the cell are largely dephosphorylated by nonspecific phospha-tases, thus permitting easy diffusion of B6compounds across the baso-lateral membrane The major form of vitamin B6released to the portalcirculation is the nonphosphorylated form of the vitamer predominant

in the intestinal lumen

Part of the PN-glucoside in plant-derived foods is hydrolyzed to PNand glucose by brush-border and cytosolic intestinal b-glucosidases; the

PN is conveyed to the liver where it is converted to biologically activePLP Another part of dietary PN-glucoside is absorbed intact by simplediffusion, but not metabolized by the liver A small part is unabsorbedand eliminated with the feces

10.5.1 Bioavailability of Vitamin B6in Foods

Gregory [52] discussed factors affecting the bioavailability of vitamin B6infoods and presented a critical assessment of the methodology Inherentproblems with rat bioassays make them unreliable for the determination

of vitamin B6bioavailability of foods and attention has turned to the use

of protocols with human subjects The principal indices of B6ity are urinary excretion of 4-pyridoxic acid and total vitamin B6, andplasma PLP concentration [53]

bioavailabil-The bioavailability of vitamin B6 in foods is highly variable, owinglargely to the presence of poorly utilized PN-glucoside in plant tissues

As expected, vitamin B6 generally has a lower availability from derived foods than from animal tissues [54] In humans, the vitamin B6

plant-from whole wheat bread and peanut butter was 75 and 63%, respectively,