Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Contents of all forms of vitamin B6, pyridoxine-ß-glucoside and 4-pyridoxic acid in mature milk of Japanese women according to 4-pyridoxolactone-conversion high performance liquid chromatography.

The contents of six vitamin B6 forms, pyridoxine-ß-glucoside, and 4-pyridoxic acid in mature milk of 20 Japanese lactating women consuming ordinary Japanese foods were determined by a 4-pyridoxolactone-conversion HPLC method. These compounds were determined with the average recovery rate of 83.9% or more. The average total content of vitamin B6 forms was 1.01 ± 0.32 (µmol/L). Pyridoxal and pyridoxal 5'-phosphate were found in all of the samples, and their average contents were 0.71 ± 0.28 (µmol/L) and 0.16 ± 0.07 (µmol/L), respectively. Pyridoxamine, pyridoxine, pyridoxamine 5'-phosphate, pyridoxine 5'-phosphate, and pyridoxine-ß-glucoside were found in 15, 14, 13, 9, and 7 samples, respectively. The presence of pyridoxine 5'-phosphate was for the first time found in human milk. A method for the determination of 4-pyridoxic acid, which is the excretion form of vitamin B6, was modified to quantitate it by isocratic HPLC. 4-Pyridoxic acid was found in all samples, and its average content was 0.094 ± 0.040 (µmol/L), which was only 12% of its content in cow (Holstein) milk. The total content of vitamin B6 forms, and predominant presence of pyridoxal among other vitamin B6 forms in the Japanese women's milk samples shared similar characteristics with American women's milk samples.

Determination of individual vitamin B(6) compounds based on enzymatic conversion to 4-pyridoxolactone.

A determination method for individual natural vitamin B(6) compounds was developed. The vitamin B(6) compounds were specifically converted into 4-pyridoxolactone (PAL), a highly fluorescent compound, through a combination of enzymatic reactions and HCl-hydrolysis. PAL was then determined by HPLC. Pyridoxal was completely oxidized to PAL with pyridoxal 4-dehydrogenase (PLDH). Pyridoxine and pyridoxamine were totally converted into PAL through a coupling reaction involving pyridoxine 4-oxidase and PLDH, and one involving pyridoxamine-pyruvate aminotransferase and PLDH, respectively. The 5'-phosphate forms and pyridoxine-beta-glucoside were hydrolyzed with HCl, and then determined as their free forms. Pyridoxine 5'-phosphate and pyridoxine-beta-glucoside were not separately determined here. Three food samples were analyzed by this method.

Simultaneous fluorometric resolution of pyridoxal, pyridoxamine and 4-pyridoxic acid using chemometric methods.

An alternatively minimizing covariant matrix error (AMCME) algorithm, newly proposed by the present authors, was applied to the simultaneous fluorometric determination of pyridoxal, pyridoxamine and 4-pyridoxic acid without loss of sensitivity. The experimental results illustrate that the profiles of spectra and concentration can be accurately resolved using the AMCME algorithm with a high sensitivity and stable repeatability. That is to say, the closely overlapping problem of the spectra could be resolved owing to the characteristic features of the AMCME algorithm.

[Connection between changing the vitamin and immune status and the character of the throat microflora in patients with chronic tonsillitis]. / Niedobór witamin oraz zaburzenia ukladu immunologicznego a charakter mikroflory gardla u chorych z przewleklym zapaleniem migdalków podniebiennych.

The author investigated 28 patients with chronic tonsillitis. The fungi sort Candida on the tonsils was discovered at 10 (35.72) patients, the bacterial flora was discovered at the other patients. There were more considerable decrease content of vitamins B2, C, methylnicotinamide and methylmalonic acid in the urine, vitamins B2 and methylnicotinamide in the saliva, vitamins A and B1 in the serum of the blood in the group of the patients with the fungus flora on the tonsils. It was discovered the decrease of numbers of lymphocytes CD8 and level of immunoglobulins G and M, disimmunoglobulinemia in this group. This decrease of the vitamins, cell and humoral immunity level leads to disbacteriosis, decompensation of the disease and development of the complications.

Tissue B-6 vitamer concentrations in rats fed excess vitamin B-6.

Diets containing 1, 10, 100, 175 or 250 times the NRC recommended level of pyridoxine HCl (7 mg/kg) were fed to rats (218 g, 12 per group) to evaluate the effects on tissue B-6 vitamer concentrations. After 10 wk, food intake and body weights did not differ among groups. Overt toxicity was not observed. Tissues were taken from five rats of each group after overnight food deprivation (unfed rats); the remaining seven rats in each group were allowed access to food (fed rats). In plasma of unfed rats, 4-pyridoxic acid and pyridoxal concentrations increased significantly (P < 0.05) with increasing dietary pyridoxine; pyridoxal phosphate was not affected by dietary pyridoxine. Concentrations of pyridoxal phosphate and pyridoxal increased significantly with increasing dietary pyridoxine in erythrocytes of unfed rats. Excretion of urinary B-6 vitamers and 4-pyridoxic acid in a 24-h period increased with dietary pyridoxine in fed rats. As dietary pyridoxine was increased, kidney pyridoxal concentrations increased significantly in fed rats only. Dietary pyridoxine did not affect vitamer concentration in muscle and liver of either unfed or fed rats, or in brain of unfed rats. Muscle glycogen phosphorylase, which contains pyridoxal phosphate, was not affected by dietary pyridoxine. There was a marginally significant (P = 0.058) increase in erythrocyte alanine, but not in aspartate, aminotransferase activity with increasing dietary pyridoxine. Plasma concentration of pyridoxal phosphate, which is used as a measure of vitamin B-6 status, did not reflect intake of pyridoxine in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.

Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material.

The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.

The fasting B6 vitamer profile and response to a pyridoxine load in normal and cirrhotic subjects.

This study established the fasting plasma and urine profiles of vitamin B6 in cirrhotics and assessed the response to an oral dose of pyridoxine. High-performance liquid chromatography was used to measure all vitameric coenzymatic and degradatory forms. In 31 patients with cirrhosis and 15 healthy controls, fasting plasma and 24-hr urine collection showed: plasma pyridoxal-5'-phosphate, the biologically active form, was significantly (p less than 0.001) reduced in cirrhotics (mean +/- S.D.: 5.7 +/- 3.2 ng per ml) compared to normals (14.2 +/- 7.5 ng per ml); plasma pyridoxal was detected in more cirrhotics (48%) than normals (28%); pyridoxic acid, the end catabolite, was significantly (p less than 0.05) lower in plasma of cirrhotics compared to normals, but 24-hr urine excretion was not different. Administration of 25 mg of pyridoxine to 7 cirrhotics and 5 normals showed the following plasma changes: pyridoxine rapidly peaked at 30 min and was totally cleared from plasma by 3 hr; plasma pyridoxal and pyridoxic acid increased in parallel up to 40-fold over baseline by 1 to 2 hr and rapidly fell toward baseline by 8 hr, and plasma pyridoxal-5'-phosphate, in contrast, increased significantly (p less than 0.05) from baseline by 60 min and was maintained above normal for 24 hr. The area under the plasma concentration vs. time curve (AUC) for pyridoxal-5'-phosphate was significantly (p less than 0.05) less for the cirrhotics than normals and showed a significant negative correlation to hepatocyte function and blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Newer laboratory methods for assessing nutriture of selected B-complex vitamins.

This review is limited to progress in the development of new or improved laboratory procedures to assess the nutriture of thiamin, riboflavin, niacin, vitamin B6, and folic acid. There has been marked progress in this area for the other vitamins as well. The increased availability of radioassay techniques and HPLC methodologies that have application in nutrition assessment is significant. However, for a number of the vitamins, there is still a need for additional methods that provide functionally interpretable preclinical information and give accurate assessment of body reserves of the nutrient. Often the guides used to interpret the information obtained are tentative and require validation or revision. This situation is further complicated by the frequent lack of suitable reference standards for quality controls and interlaboratory validation.

[Methods and their evaluation in estimating the vitamin B6-status in humans. 4. 4-PA: reliability of the parameters]. / Methoden und deren Wertung zur Bestimmung des Vitamin-B6-Versorgungszustandes beim Menschen. 4. Mitteilung: 4-PA: Die Aussagefähigkeit des Parameters.

Effects of physiological factors on 4-PA-excretion of more than 400 industrial workers and students were examined. Borderline values are discussed. With increasing age men as well as women showed significant higher 4-PA-values. After optimizing the vitamin B6-uptake by means of vitamin administration the differences disappear. Age-depending variations in the ability forming 4-PA are not likely. The higher 4-PA-excretion of men is probably due to better dietary supply rather than to sex differences in metabolising the vitamin. There are no considerable influences on the parameter by oral contraceptives Short-term variations of dietary vitamin B6-supply have striking effects on the 4-PA-excretion and restrict the reliability of this parameter. An insufficient vitamin B2-supply can stimulate a marginal vitamin B6-status. Alcohol consumption the day before does not change the 4-PA-excretion.

The conversion of 3-Hydroxy-2,4,5-trihydroxymethylpyridine into pyridoxine by Kloeckera apiculata.

Kloeckera apiculata, a vitamin B-6-dependent yeast, grows in the presence of 3-hydroxy-2,4,5-trihydroxymethylpyridine in vitamin B-6-free media. On a molar basis the growth-promoting activity of this compound is approximately one-tenth that of other forms of vitamin B-6. [G-3H]3-Hydroxy-2,4,5-trihydroxymethylpyridine is converted into radioactive vitamin B-6 compounds of the same specific radioactivity by growing cultures of K. apiculata.