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Fluorimetric determination of pantothenic acid in foods by liquid chromatography with post-column derivatization.

A method to determine the content of free pantothenic acid in various foods by reverse phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by successive passages through anion and cation exchange cartridges and a post-column derivatization of pantothenic acid as the fluorescent 1-alkylthio-2-alkylisoindole (reaction of beta-alanin, formed by hot alkaline hydrolysis of pantothenic acid, with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid). An enzymatic hydrolysis prior to the purification step (pepsin at 50 degrees C for 3 h, then pantetheinase and alkaline phosphatase at 20 degrees C for 18 h) made it possible to release the bound pantothenic acid and thus to obtain the total Vitamin B5 content of these foodstuffs. The method proposed for the determination of free and bound pantothenic acid gives a good recovery rate (96-101%) and a satisfactory repeatability (R.S.D.r less than 8%). Owing to its low detection limit (0.65 microg g(-1)) and the good resolution of the pantothenic acid peak, it could most probably be applied to the determination of this vitamin in any foodstuff.

Calibration↗

[Urinary excretion of pantothenic acid as a measurement of nutritional requirements].

Several groups of male Wistar-Rats were fed for 15 month with a purified diet containing different amounts of pantothenic acid. The groups received 150, 100, 25 and 0% pantothenic acid of the requirement respectively. A further group was fed without pantothenic acid in the diet but administered with the antagonist omega-methyl-pantothenic acid. The excretion of pantothenic acid in the urine was measured at different times, and a groups specific level of excretion could be established for the different groups. Differences to the controls were highly significant in the test groups after 2-18 weeks. Studies of this type were conducted in order to obtain basic informations for later experiments in human beings.

Animals↗

Pantothenic acid status of pregnant and lactating women.

Pantothenic acid nutritional status was evaluated longitudinally in 26 pregnant women (experimental group) during their third trimester of pregnancy and at 2 weeks and 3 months postpartum. Seventeen nonpregnant and nonlactating women (control group) participated at the same time intervals. All the women were assessed by the intake calculated from a reported 2-day dietary record and by fasted blood, plasma, and 24-hour urinary levels of pantothenate determined by a radioimmunoassay. Estimated daily mean dietary pantothenate intake and the vitamin density for the experimental group were not statistically different from those for the control group. The dietary pantothenate intake averaged 2.75 mg/1,000 kcal. Average pantothenate blood level of the experimental group was lower than that of the control group. No significant difference was found between the two groups in the pantothenate levels of fasting plasma and urinary excretion. When they did not take pantothenic acid supplements, members of the experimental group had intakes less than the Estimated Safe and Adequate Daily Dietary Intake and lower mean blood values than the members of the control group. This suggests that pregnant and lactating women need to consume more pantothenate to maintain a blood vitamin level similar to that of nonpregnant women. This may be achieved by an increased caloric intake, if desirable, or by more careful selection of foods high in the nutrient.

Adult↗

Pantothenic acid, acute ethanol consumption and sulphadimidine acetylation.

The effect of pantothenic acid and acute ethanol loading on the genetically determined N-acetyltransferase activity has been studied using sulphadimidine as a test substance. The administration of 1100 mg pantothenic acid daily (600 mg orally, 500 mg iv) for seven days did not significantly alter sulphadimidine kinetics in the primarily elderly 21 subjects we investigated. Acute ethanol loading (0.73 g/kg pure alcohol at start and 0.11 g/kg pure alcohol hourly for 8 hours afterwards, stock solution: 20% v/v ethanol in fruit juice) did not change sulphadimidine acetylation in 10 healthy male volunteers. It is concluded that despite theoretical assumptions exogenous factors like pantothenic acid and ethanol do not significantly influence the cytosolic N-acetyltransferase activity. Consequently they do not interfere with the acetylator phenotyping procedure.

Acetylation↗

Effects of excess pantothenic acid administration on the other water-soluble vitamin metabolisms in rats.

To acquire the data concerning the tolerable upper intake level which prevents health problems from an excessive intake of pantothenic acid, an animal experiment was done. Rats of the Wistar strain (male, 3 wk old) were fed on a diet which contains 0%, 0.0016% (control group), 1%, or 3% calcium pantothenate for 29 d. The amount of weight increase, the food intake, and the organ weights were measured, as well as the pantothenic acid contents in urine, the liver and blood. Moreover, to learn the influence of excessive pantothenic acid on other water-soluble vitamin metabolism, thiamin, riboflavin, a vitamin B6 catabolite, the niacin catabolites, and ascorbic acid in urine were measured. As for the 3% addition group, enlargement of the testis, diarrhea, and hair damage were observed, and the amount of weight increase and the food intake were less than those of the control group. However, abnormality was not seen in the 1% addition group. The amount of pantothenic acid in urine, the liver, and blood showed a high correlation with intake level of pantothenic acid. It was only for 4-pyridoxic acid, a vitamin B6 catabolite, in urine that a remarkable difference was observed against the control group. Moreover, the (2-Py+4-Py)/MNA excretion ratio for these metabolites of the nicotinamide also indicated a low value in the 3% pantothenic acid group. As for the calcium pantothenate, it was found that the 3% level in the diet was the lowest-observed-adverse-effect-level (LOAEL) and the 1% level was the no-observed-adverse-effect-level (NOAEL).

Animals↗

Niacin and pantothenic acid excretions of humans fed a low-methionine, plant-based diet.

The addition of a vitamin to a diet of humans has been shown to increase the excretion of that vitamin. The effects of an increase of one vitamin on another have not been investigated. The objective of the current project was to compare the effects of two supplementation patterns on the niacin and pantothenic acid excretion values of humans consuming a peanut butter-based diet. Two groups each received one of two supplementation regimens. One group received niacin, a multi-vitamin, or no supplement. One group received methionine alone, pantothenic acid alone or methionine plus pantothenic acid. The addition of either vitamin resulted in increased excretion of that vitamin. Urinary niacin excretion of the group that received pantothenic acid and/or methionine was greater than that observed with a multi-vitamin or no supplement. Urinary pantothenic acid excretion was suppressed when niacin was a supplement. Urinary pantothenic acid excretion of the methionine supplement group was greater than the excretion of the groups which received either niacin or multi-vitamin supplements. These data suggest some possible dangers in indiscriminate supplementation of food products.

Adult↗

Determination of pantothenic acid, biotin, and vitamin B12 in nutritional products.

Until recently, liquid chromatographic (LC) methodology for pantothenic acid, biotin, and B12 (cyanocobalamin) has been only marginally successful. These vitamins are difficult to determine by conventional LC techniques and UV detection at 254 or 280 nm, because either the chromophore is inadequate for detection or interference from co-eluting vitamins is overwhelming. Biotin and B12 are usually present in pharmaceutical products at concentrations 100-1000 times lower than other commonly occurring water-soluble vitamins. Co-extraction of all water-soluble vitamins results in gross interferences, especially in LC when the interfering vitamins co-elute with biotin or B12. In addition, pantothenic acid and biotin are colorless in solution and do not exhibit strong UV absorption above 240 nm. As a result, they must be quantitated either by using a low UV wavelength for detection or by derivatizing the vitamin to obtain an adequate chromophore. A description of procedures for LC determination of pantothenic acid, panthenol, cyanocobalamin, and biotin in pharmaceutical products is presented. Pantothenic acid has been measured by using both a derivatization technique and low UV wavelength detection. Biotin has been quantitated by using low UV wavelength detection. The limitations of these techniques are also discussed. Chromatographic separation of cyanocobalamin is complicated by co-eluting vitamins such as riboflavin. It is detected by using the 546 nm wavelength where riboflavin does not interfere.

Biotin↗

Studies on the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese.

Four experiments were conducted to examine the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese fed purified diets. Goslings fed diets deficient in either riboflavin, pantothenic acid, nicotinic acid, or choline grew poorly. Feeding a pantothenic acid-deficient diet resulted in 100% mortality. Goslings fed diets containing 530 mg/kg of choline or less developed perosis. Under the conditions of these experiments it was found that: 1) goslings require no more than 3.84 mg/kg of riboflavin and 31.2 mg/kg of nicotinic acid in the diet for rapid growth and normal development, 2) the pantothenic acid requirement of goslings is no more than 12.6 mg/kg of diet, and 3) a dietary choline level of 1530 mg/kg is adequate for both the prevention of perosis and rapid growth of goslings. The levels of vitamins found to support normal growth and development of goslings appear to be similar to requirements of other species that have been examined.

Animal Feed↗

Molecular cloning and application of a gene complementing pantothenic acid auxotrophy of sake yeast Kyokai no. 7.

Kyokai no. 7 is the most widely used yeast in sake brewing. This yeast is a pantothenic acid auxotroph at 35 degrees C, and this phenotype has been used to distinguish Kyokai no. 7 from other sake yeasts. We cloned a DNA fragment complementing the pantothenic acid auxotrophy from a genomic library of a Saccharomyces cerevisiae laboratory strain. DNA sequence analysis revealed that the DNA fragment encodes ECM31, the deletion of which had previously been identified as a calcofluor white-sensitive mutation. The ECM31 product is similar to the Escherichia coli ketopantoate hydroxymethyltransferase. Disruption of ECM31 in a laboratory S. cerevisiae strain resulted in pantothenic acid auxotrophy, indicating that ECM31 is also involved in pantothenic acid synthesis in yeast. A hybrid of a Kyokai no. 7 haploid and the ecm31 disruptant required pantothenic acid at 35 degrees C for its growth, suggesting that Kyokai no. 7 possesses a temperature-sensitive allele of ECM31. Thus, the ECM31 gene can be used as a selective marker in the transformation of Kyokai no. 7.

Journal Article↗

Pantothenic acid deficiency as a factor contributing to the development of hypertension.

In endemic pantothenic acid deficiency of some Japanese populations, increased occurrence of hypertension has been described. However, all attempts to produce hypertension experimentally by means of pantothenic acid deficiency have failed up to now. As a consequence, the observations made in Japan have largely been ignored. In this paper, pantothenic acid deficiency will be shown to be a factor in the experimental origin of hypertension due to adrenal regeneration.

Adrenal Glands↗

Quantification of free and bound pantothenic acid in foods and blood plasma by a stable isotope dilution assay.

A stable isotope dilution assay for quantification of pantothenic acid in food and blood plasma uses a 4-fold labeled isotopomer of the vitamin as an internal standard. Pantothenic acid and its labeled analogue were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry, showing a minimized spectral overlap. In starch a detection limit of 44 microg/kg, an intrasample relative standard deviation of 6.7%, and recovery values ranging between 97.5 and 99.4% were determined. Total pantothenic acid content was determined in rice, milk powder, apple juice, and blood plasma after enzymatic hydrolysis of the vitamin's conjugates; free pantothenic acid was quantified prior to enzyme treatment. Almost all results were found to be in good agreement with literature data.

Carbon Isotopes↗

Preparation of high-specific-activity D-[3-3H]pantothenic acid.

High-specific-activity D-[3-3H]pantothenic acid (5 Ci/mmol) was prepared from commercially available beta-[3-3H]alanine employing Escherichia coli strain DV1 (panD2 pan F1). This strain is defective in beta-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input beta-[3-3H]alanine to extracellular D-[3-3H]pantothenate. The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The overall yield of D-[3-3H]pantothenic acid was 30% and radiochemical purity was greater than 99%.

Alanine↗

The effect of pantothenic acid on the diet of growing chicks on energy utilization and body composition.

Two experiments were conducted to investigate the effects of varying levels of pantothenic acid on the efficiency of energy utilization and changes in body composition of the growing chick. The results indicate that a pantothenic acid deficiency does not interfere with the chick's ability to obtain metabolizable energy from the diet. The data do show that the efficiency of energy utilization and body composition were markedly affected when chicks were fed diets deficient in pantothenic acid. In both experiments, protein, fat and energy stores were significantly reduced when pantothenic acid-deficient diets were fed. Increased estimates of heat increment per gram of diet were observed when levels of pantothenic acid below the N.R.C. (1971) recommended level of 10 mg. per kg of diet were fed.

Animal Feed↗

Development of an ELISA for pantothenic acid (vitamin B5) for application in the nutrition and biological fields.

Immunological assays appear to be the only alternative to the microbiological method for analysis of pantothenic acid in foods and blood. In order to evaluate the influence of the linker on the immunogenicity of the hapten, we have tried to raise antisera against pantothenic acid in rabbits using different conjugates. The hapten was coupled to a carrier protein (BSA or thyroglobulin) using adipoyl dichloride (adipoyl conjugate) or bromoacetyl bromide (acetyl conjugate). Only the acetyl conjugate has induced the production of a specific antibody. With this antibody, an assay on microplate using the ELISA inhibition technique was developed to measure pantothenic acid. The use of pantothenic acid coupled to thyroglobulin with adipoyl dichloride as the capture antigen has improved the sensitivity of the ELISA. This assay was applied to food products and blood.

Animals↗

Pantothenic acid and pantothenol increase biosynthesis of glutathione by boosting cell energetics.

We have previously observed (summarized in BioFactors 17 (2003) 61) that pantothenic acid, pantothenol and other derivatives that are precursors of CoA protect cells and whole organs against peroxidative damage by increasing the content of cell glutathione. The present investigation was aimed to elucidate the mechanism of this increase in human lymphoblastoic (Jurkat) cells. It showed that incubation of the cells with pantothenic acid or pantothenol increased mainly the content of free glutathione, with little effect on protein-bound glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, prevented this increase. Increase of the content of free glutathione, as produced by pantothenic acid or pantothenol, was largely prevented by respiratory chain inhibitor rotenone, inhibitor of mitochondrial ATP synthesis oligomycin and uncoupler of oxidative phosphorylation of carbonyl cyanide 3-chlorophenylhydrazone. These treatments also decreased the cellular content of ATP. Preincubation with pantothenic acid or pantothenol also increased cell respiration with pyruvate as the exogenous substrate. Although no significant increase of total cell CoA content could be found, it is concluded that the increase of the glutathione level was due to increased production of ATP that was, in turn, a result of the increased content of mitochondrial CoA.

Acetylcysteine↗

Myocardial metabolism of pantothenic acid in chronically diabetic rats.

Transport and metabolism of [3H]pantothenic acid ([3H]Pa) was investigated in hearts from control and streptozotocin-induced diabetic rats. In isolated perfused hearts from control animals, the transport of [3H]Pa was linear over 3 h of perfusion when 11 mM glucose was the only exogenous substrate. The in vitro transport of [3H]Pa by hearts from 48-h diabetic rats was reduced by 65% compared to controls and was linear over 2 h of perfusion with no further accumulation of Pa during the third hour. The defect in transport observed in vitro could be corrected by in vivo treatment with 4 U Lente insulin/day for 2 days. In vitro addition of insulin in the presence of 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate had no effect on [3H]Pa transport in hearts from 48-h diabetic rats during 3 h of perfusion. Accumulation of [3H]Pa was not inhibited by inclusion of 0.7 mM amino acids, 1 mM carnitine, 50 microM mersalic acid or 1 mM panthenol, pantoyllactone or pantoyltaurine. Uptake was inhibited by 1 mM nonanoic, octanoic or heptanoic acid, 0.1 mM biotin or 0.25 mM probenecid, suggesting a requirement for the terminal carboxyl group for transport. Transport of pantothenic acid was reduced in hearts from diabetic rats within 24 h of injection of streptozotocin. In vitro accumulation of [3H]Pa decreased to 10% of control 1 week after streptozotocin injection and then remained at 30% of the control value over 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗