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Bioavailability of vitamin B6 in nonfat dry milk and a fortified rice breakfast cereal product
Abstract
The results of microbiological and high performance liquid chromatographic (HPLC) methods for total vitamin B-6 were compared with rat bioassay results for biologically available vitamin B-6 in nonfat dry milk and a rice base breakfast cereal product. Excellent agreement was observed between assay values for total and available vitamin B-6 in the nonfat dry milk, indicating full bioavailability. In contrast, the rat bioassay results indicated that the bioavailability of the vitamin B-6 in the cereal product was low. The factors responsible for the apparently poor absorption or utilization of the vitamin B-6 from the cereal product are not known. The results of this study indicate that the bioavailability of vitamin B-6 may be strongly influenced by food composition.
Previous research has suggested that dietary fiber can reduce the bioavailability of certain micronutrients by ionic binding or physical entrapment. In this study, the ability of eight purified polysaccharides, lignin, and wheat bran to bind B-6 vitamers was examined in vitro using equilibrium dialysis under physiological conditions. The polysaccharides used were cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, citrus pectin, xanthan gum, sodium alginate, and gum arabic. No significant binding was detected when pectin was dialyzed to equilibrium at, 0.5, 0.75, and 1% levels in the presence of 0.1 or 0.1 mM pyridoxine, pyridoxamine, or pyridoxal. When 1% wheat bran, lignin, and the test polysaccharides were incubated overnight with 0.1 mM pyridoxine prior to dialysis, to allow for any slow binding process to occur, no in vitro binding of pyridoxine by any of the test materials could be observed on equilibrium dialysis. These results are consistent with findings of in vivo experiments which showed that cellulose, pectin, and bran did not significantly decrease the bioavailability of vitamin B-6 when fed to the rat and/or the chick.
The thermal degradation kinetics of pyridoxine hydrochloride were examined using a dehydrated model food system designed to simulate a ready-to-eat breakfast cereal. This study was carried out to provide information useful in estimating the thermal losses of pyridoxine hydrochloride in the processing of breakfast cereals and other low moisture dehydrated food systems. Portions of the model system which were fortified with pyridoxine hydrochloride were toasted in a conduction oven at 155°, 170°, 185°, and 200°C for a minimum of six heating times at each temperature. Pyridoxine (PN) content was determined in the heat treated model systems by high performance liquid chromatography (HPLC). For each heat treatment, the loss of pyridoxine could be described by a first order kinetics model. The first order rate constants for 155°, 170°, 185°, and 200°C were 1.74 × 10−2 min−1, 5.22 × 10−2 min−1, 16.88 × 10−2 min−1, and 48.95 × 10−2 min−1, respectively. The calculated Arrhenius activation energy was 29.8 Kcal/mol. In comparing the HPLC method to the standard microbiological assay, the HPLC assay gave lower PN values for the toasted model system. To explain this discrepancy, HPLC fractions were collected and analyzed by the microbiological assay. No significant vitamin B6 activity was found in any fraction other than that containing the PN peak. It is possible that the milder extraction procedure used in the HPLC assay is less capable of recovering forms of PN which may become bound during the toasting process. These potentially bound forms may or may not be biologically available.
The effects of cellulose, pectin and bran on the bioavailability of pyridoxine (PN) were examined using rat and chick bioassay methods. Dose-response curves for growth, feed consumption, feed efficiency and either lever pyridoxal 5'-phosphate (PLP) or erythrocyte aspartate aminotransferase (Asp-AT) activity and PLP stimulation in vitro were compared among animals fed experimental diets varying in dietary fiber source and suboptimal levels. of PN. In rats, the observed stimulation of growth and feed efficiency by pectin, as compared to cellulose, at suboptimal PN levels was attributed to increased synthesis of vitamin B-6 intestinal microflora. Diets containing pectin markedly increased the fecal vitamin B-6 content. No difference in liver PLP was detected among rats fed various diets. In chicks, 5% dietary pectin resulted in increased feed consumption, but also diarrhea and depressed growth. Asp-AT activities at the lower level of dietary PN showed a significant (P greater than 0.05) but modest stimulating effect of pectin on the apparent bioavailability of PN. Bran resulted in a 17% bioavailability decrease of PN as indicated by growth and feed consumption data in the chick. However, no difference in Asp-AT activity or in vitro PLP stimulation was detected in comparison of responses from bran-fed chicks with the standard responses. These results suggest that the polysaccharides tested did not have important deleterious effects on the bioavailability of pyridoxine.
We describe optimized, ultraviolet spectrophotometric procedures for determination of erythrocyte transketolase, glutathione reductase, and aspartate aminotransferase activity, and their activation by their respective coenzymes--thiamine pyrophosphate, flavin-adenine dinucleotide, and pyridoxal-5-phosphate--as tests for vitamin B1, B2, and B6 deficiency. With these procedures we have investigated healthy subjects on normal and vitamin-supplemented diets, and a series of (mainly) alcoholic hospital in-patients. The enzyme procedures described have good precision and can be readily carried out in the routine laboratory. Abnormal transketolase activation correlated well with clinical evidence of vitamin B1 deficiency.
Nine collaborators studied a method for determining vitamin B6 and pyridoxine, pyridoxal, and pyridoxamine in foods and like materials. The 3 vitamin B6 components in food extracts were eluted in separate fractions from an ion exchange column and determined separately by microbiological assay, using the yeast S. carlsbergensis as the test organism. It is recommended that the method be adopted as official first action.
1. For chicks and rats pyridoxine, pyridoxal and pyridoxamine were equally active in terms of the free bases when given separately from the diet.
2. Under our experimental conditions pyridoxine mixed with the chick diet was stable, but 20% of pyridoxamine, and a variable amount of pyridoxal was lost.
3. The vitamin B 6 activities measured with Saccharomyces carlsbergensis , chicks and rats respectively and expressed as μg. pyridoxine/g. freeze-dried milk were: raw milk 3·4, 3·2 and 4·9; evaporated milk 1·0, 2·1 and 2·7; stored evaporated milk 0·6, 1·4 and 2·0. For the chicks the milks were mixed with the diets; they were given separately to the rats.
4. The microbiological and biological results for raw milk agreed within the limits of experimental error. For the processed milks the differences between biological and microbiological tests were statistically significant.
5. All three methods of assay showed a 45–70% loss of vitamin B 6 activity on processing and a further loss of 30% of the remainder after storage for 6 months at room temperature.
We are indebted to Mr J. Rothwell, Department of Dairying, University of Reading, for preparing the evaporated milk and to Dr B. Record, Ministry of Supply, Microbiological Research Establishment, Porton, for freeze-drying the milk. We should like to thank Dr S. K. Kon for his interest in this work.
Pyridoxine, pyridoxal, pyridoxamine, and total unchromatographed vitamin B6 values for meats, fish, dairy products, and commercial infant formulas were determined microbiologically using S. carlsbergensis. The hydrolyzed food extracts were chromatographically separated using a Dowex 50 resin column prior to microbiological assay. The vitamin B6 values as assayed ranged for meats from 3 to 8 μg. B6/gram, fish from 0.5 to 4 μg. B6/gram, dairy products from 0.01 to 4 μg. B6/gram, and commercial infant formulas from 0.4 to 4 μg. B6/gram.
Free and total vitamin B6 values of a few selected foods determined microbiologically were compared to vitamin B6 values obtained by rat bioassay. Free and total vitamin B6 values were obtained on extracts chromatographically separated into pyridoxine, pyridoxal, and pyridoxamine using a Dowex 50 resin column, as well as on extracts not chromatographed. Bioassay values agree with values obtained microbiologically on the chromatographed extracts of these samples.
Heat sterilization of liquid milk products results in a loss of vitamin B6 as determined by a microbiological assay. A rat growth procedure was adapted for analysis of milk samples by the use of a semisynthetic basal diet with a composition simulating milk. The biological assay of heat-sterilized liquid milk yielded vitamin B6 values that were lower than those obtained by the microbiological method. The bioassay and the microbiological method were in agreement in the assay of spray-dried milk.
ABSTRACTA soluble model system was utilized to study the interactions of pyridoxal (PL) and pyridoxal phosphate (PLP) with proteins during the thermal processing of foods. Heat stable peptides, rather than intact proteins, were used in the model system to maintain solubility and permit subsequent evaluation of the peptide binding of the B6 vitamers. Ultraviolet and visible difference spectra and fluorescence emission spectra confirmed the binding of PL and PLP to the peptides during processing. Total peptide-bound PLP (as the sum of Schiff base, pyridoxylamino, and substituted aldamine derivatives) comprised about 21% of the model system PLP. Approximately 60% of the peptide-bound PLP was bound via nonreducible linkages, suggesting the possible formation of biologically unavailable pyridoxylamino complexes. The acid stability of the PLP complexes, tested to simulate gastric conditions, indicated that most of the nonreducible bound PLP was in the form of pyridoxylamino complexes. Therefore, approximately 10% of the model system PLP was rendered biologically unavailable during these process conditions. The presence of ascorbic acid and/or glucose in the model systems had no significant effect on the extent or manner of peptide-binding of the B6 vitamers; however, glucose-induced browning appeared to slightly inhibit pyridoxylamino complex formation. The results of this study indicate that losses of vitamin B6 bio-availability during thermal processing may be lower than previously reported.
The retention of biologically available vitamin B6 during storage was examined using dehydrated food systems fortified with various B6 vitamers. Storage conditions of 37°C and 0.6 water activity (aw) were selected to permit estimation of maximum rates of nonenzymatic browning and vitamin degradation typically encountered in food storage. The stability of the vitamers during storage was evaluated by periodic assay with a high performance liquid chromatographic (HPLC) method. Losses of pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and pyridoxal phosphate (PLP) could be described by fist order kinetics. When stored in small volume metal containers, the first order rate constants were 0.015–0.020 day−1 for PL, PM and PLP, and 0.0049 day−1 for PN degradation in food systems. Storage in metal cans which provided a large headspace volume resulted in significantly lower rates of browning and degradation of the B6 vitamers. The only significant degradation mechanism identified was the binding of PLP to proteins to form e-pyridoxyllysine. Correlation of rat bioassay results with microbiological and HPLC assay data indicated that the B6 vitamers remaining after storage for 128 days at 37°C retained full vitamin activity. Determination of the model system vitamin B6 content by the semiautomated fluorometric procedure yielded consistently high results, as found in previous research. These studies indicate that HLPC and microbiological assay results accurately reflect the content of biologically available vitamin B6 in dehydrated food systems after storage at 37°C and 0.6 aw for 128 days. They also demonstrate the potential for application of the HPLC assay to the determination of vitamin B6 in foods.
Dehydrated food systems were employed to study the stability and bioavailability of vitamin B6 as affected by roasting at 180°C for 25 min. The roasting conditions were selected to permit estimation of the maximum effects of the roasting process on the B6 vitamers and are above those which would be normally used for commercial processing. The relative degradation of pyridoxine, pyridoxamine, and pyridoxal phosphate was found to be 50-70% by both microbiological and semi-automated fluorometric assay methods. Estimates of biologically available vitamin B6 in the roasted model systems, as determined by rat bioassay correlated closely with microbiological results. Thus, vitamin B, remaining after roasting at 180°C for 25 min was biologically available and active. Semiautomated fluorometric values for total vitamin B6 in untreated and roasted systems were 1.65-3.63 times higher than corresponding microbiological assay data. Fluorescence spectra studies of samples prepared for the semiautomated assay revealed no detectable interfering compounds. Microbiological assay provided an accurate evaluation of available vitamin B6 in the roasted model food system as shown by the rat bioassay. The consistently high results from the semiautomated fluorometric method indicate that further work is required before this method can be accurately used to quantitatively determine the level of vitamin B6 in foods.
Microbiological assay remains one of the most widely used methods for determination of the vitamin B6 group—pyridoxal, pyridoxamine, and pyridoxine—because of its sensitivity, its high degree of specificity, and its suitability for use in assaying the crude extracts of biological materials. The method depends on the selection of a microorganism that requires vitamin B6 for growth. Such a microorganism is grown in media containing an excess of all nutrients required for growth except vitamin B6. Although several test organisms have been used in the determination of vitamin B6, three tests are preferred (1) Saccharomyces carlsbergensis 4228 (ATCC 9080) for the determination of pyridoxal, pyridoxine, and pyridoxamine, (2) Lactobacillus casei (ATCC 7469) for the determination of pyridoxal, and (3) Streptococcus faecium Φ51 for the determination of pyridoxal and pyridoxamine. To determine how much of each of the three forms of the vitamin is present in a sample, the sample may be reassayed with test organisms of differing specificity. Bain and Williams have described a procedure for separating both phosphorylated and free forms of vitamin B6 on ion-exchange resins.
The relationship of plasma pyridoxal 5'-phosphate (PLP) to PLP content of tissues and activities of PLP-dependent enzymes was examined to establish its value in assessing vitamin B6 nutrition. Weanling rats were fed ad libitum for 9 weeks purified diets which supplied 0, 4, 12, 24, and 100 micrograms of pyridoxine daily. Growth increased with increasing pyridoxine intake, reaching a maximum at 24 micrograms/day. Liver and brain PLP also increased, attaining maximal values at 12 micrograms. By contrast, muscle and plasma PLP did not saturate when vitamin B6 intake was increased to 100 micrograms. Erythrocytic holoenzyme activity of aspartate (Asp) aminotransferase became maximal with 24 micrograms but that of alanine (Ala) aminotransferase did not. Hepatic holoenzyme activities of Ala, Asp and tyrosine aminotransferases reached maximal values with only 4 micrograms vitamin B6 but that of serine dehydratase became maximal with 12 micrograms. Measurement of coenzyme saturability suggested that apoenzyme degradation, coenzyme affinity and PLP transfer determine the activities of these enzymes. It is concluded that plasma and muscle PLP behave as mobilizable storage pools and that plasma PLP is a sensitive and reliable indicator of vitamin B6 nutrition.
The biological activity of protein bound epsilon-pyridoxyllysine residues in a phosphopyridoxyl-bovine serum albumin (PP-BSA) preparation was evaluated. Previous studies have demonstrated that pyridoxal phosphate may bind to food proteins as epsilon-pyridoxyllysine complexes during processing and storage. The present research, employing PP-BSA as a model, was initiated to determine the nutritional consequences of epsilon-pyridoxyllysine formation in foods. The concentration of epsilon-pyridoxyllysine residues in the PP-BSA was determined spectrophotometrically and chromatographically. Rat bioassay of the PP-BSA revealed that epsilon-pyridoxyllysine exhibited 60% activity relative to the molar potency of pyridoxine. These results suggest the partial release of bound vitamin B-6 possibly by in vivo enzymatic hydrolysis of epsilon-pyridoxyllysine. The presence of PP-BSA in a test diet containing 0.25 microgram added pyridoxine per g of diet inhibited the utilization of approximately half of the free pyridoxine by the rats. It is postulated that the observed inhibition resulted from an antivitamin B-6 effect of intact epsilon-pyridoxyllysine. This effect requires further investigation.
The vitamin B6 requirement of the male Wistar strain rat fed a 20% casein, 20% corn oil diet, was investigated in experiments designed to provide dose response curves over the range of 5 to 80 μg/day. Pyridoxine hydrochloride was administered by subcutaneous injection, by daily oral dosing and by incorporation in the diet. All routes gave comparable results. A linear relationship was noted between the logarithm of the dose administered and the body weight gain and the erythrocyte transaminase activities over considerable portions of the dose response curves. For maximal weight gain, the requirement was between 40 and 80 μg pyridoxine hydrochloride/day. For maximal erythrocyte glutamic-oxalacetic transaminase activity, the requirement was also in the range of 40 to 80 μg/day; for the glutamic-pyruvic enzyme, the requirement was 80 μg or more/day. The 3 parameters provide comparable estimates of vitamin B6 requirement. It is suggested that rat diets should provide about 100 μg pyridoxine hydrochloride/day, or 60 to 70 μg/10 g diet, to ensure the adequacy of the intake. This is considerably higher than the commonly accepted vitamin B6 requirement of the rat.
To study relationships between the vitamin B«-vitamer content of rat tissues to the activities of 2 transaminase enzymes in those tissues, and the relative biological activity of 3 vitamin Be-vitamers in the rat, we arranged to study, in depth 7 groups of rats that were fed diets containing (/ig/gdiet) 0, 0.5, 1.0, 2.0, and 10.0 of pyridoxol-HCl and 1.0 of pyridoxal-HCl or pyridoxamine-2HC1. Al though a dietary level of 2.0 ¿¿g/g diet of pyridoxol was adequate to support maximal growth, higher tissue levels of total vitamin Be and of the individual vitamers were reached at the lO.Ojug/g diet level, in liver, kidney, brain, muscle, and heart. Of the 3 vitamin Be vitamers, pyridoxamine was depleted to the greatest extent, as percent age, at lower levels of dietary pyridoxol. In studies on glutamic-pyruvic (GPT) and glutamic-oxalacetic transaminase (GOT) activities in the above tissues plus plasma and red blood cells, enzyme activity increased with increased dietary pyridoxol and with increased tissue vitamin Bs content. GPT was depleted to a greater extent than, and GOT to a lesser extent than, the percentage depletion for tissue vitamin B8 at lower levels of pyridoxol intake. It appeared from studies on growth, tissue content of vitamin Be and vitamers, and tissue transaminase activity, that at the level of 1.0/ig/g diet, pyridoxal was essentially equivalent to pyridoxol for vitamin B« activity; the study on pyridoxamine required re-evaluation. A simplified medium was used for the microbiological portion of the assay of the vitamin B6 vitamers.
A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.
Vitamin B-6 enrichment of wheat flour: stability and bioavallability.Report to the Food and Nutrition Board:Committee on Food Protection of the National Academy of Sciences. Technology of Fortification of Cereal-Grain Products
- J E Lecklem
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Standardization in a method for clinical hemoglobinometry
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Comparison of high performance liquid chromatographic and microbiological procedures for the determination of vitamin B‐6 in fortified cereals
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