Effects of folic acid supplementation to rations differing in the concentrate to roughage ratio on ruminal fermentation, nutrient flow at the duodenum, and on serum and milk variables of dairy cows

The present study was undertaken to determine the effects of dietary folic acid (FOL) supplementation on ruminal fermentation, duodenal nutrient flow, serum and milk variables, and on B-vitamin concentration in serum. The study was divided into two experiments: in Exp. 1 the forage to concentrate (F:C) ratio of the diet (DM basis) was 34:66 (high concentrate, HC), while in Exp. 2 the F:C ratio was 66:34 (high forage, HF). In addition, the cows received 0 or 1 g FOL/d. In Exp. 1, two German Holstein cows equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were dry and five were lactating (186 +/- 144 days in milk); in Exp. 2 four cows were dry and four were lactating (165 +/- 57 days in milk). In cows fed the HC diet, FOL supplementation decreased the ruminally-fermented organic matter. Thus, less energy was available for ruminal microorganisms, which resulted in a reduced microbial crude protein flow at the duodenum. Feeding the HF diet, FOL supplementation only increased the apparent ruminal digestibility of acid detergent fibre (ADF). With the HF diet, FOL had no influence on the serum levels of glucose, non-esterified fatty acids, beta-hydroxybutyrate, urea, thiamine, riboflavin, pyridoxal-5'-phosphate, pyridoxic acid, pyridoxal, pyridoxine, pyridoxamine, pantothenic acid, nicotinamide or nicotinic acid, whereas supplementing FOL to the HC diet lowered the serum glucose and riboflavin levels. In both experiments, the supplementation of FOL had no effects on milk composition. Folic acid supplementation to both diets increased the concentrations of serum 5-methyl-tetrahydrofolate. However, no beneficial effects to dairy cows were obvious. Therefore, to achieve certain results, studies with a higher number of non-fistulated cows would be necessary.     

Modification of the spectrophotometric method of determination of pyridoxal-5'-phosphate and pyridoxal in enzymatic preparations]

In quantitative measurements of pyridoxal-5'-phosphate and pyridoxal in enzymes routinely used phenylhydrasine was substituted for 4-nitrophenylhydrasine. This increased the sensitivity of the method by 70%. The modified procedure had another advantage: it allowed measurements of the optic density of resulting 4-nitrophenylhydrasones at 430 nm for acid solutions and at 550 nm for alkaline solutions.     

Determination of pyridoxal phosphate levels in the brains of audiogenic and normal mice

The concentrations of pyridoxal-5-phosphate in the brains of audiogenic and normal mice were measured fluorimetrically. The brain of the audiogenic mouse (DBA/2J) contains 25% more pyridoxal-5-phosphate than the brain of a control mouse. Intraperitoneal injection of this substance causes a transient increase of its concentration in the brain, lasting a few hours. The substance thereafter is degraded to pyridoxal and pyridoxic acid.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).     

Inactivation of avian myeloblastosis virus DNA polymerase by specific binding of pyridoxal 5'-phosphate to deoxynucleoside triphosphate binding site.

Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.     

Spectrophotometric determination of pyridoxal and pyridoxal 5′-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay

A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.     

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate

A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.     

Effects of folic acid supplementation to rations differing in the concentrate to roughage ratio on ruminal fermentation,nutrient flow at the duodenum,and on serum and milk variables of dairy cows

The present study was undertaken to determine the effects of dietary folic acid (FOL) supplementation on ruminal fermentation, duodenal nutrient flow, serum and milk variables, and on B-vitamin concentration in serum. The study was divided into two experiments: in Exp. 1 the forage to concentrate (F:C) ratio of the diet (DM basis) was 34:66 (high concentrate, HC), while in Exp. 2 the F:C ratio was 66:34 (high forage, HF). In addition, the cows received 0 or 1 g FOL/d. In Exp. 1, two German Holstein cows equipped with cannulas in the dorsal sac of the rumen and in the proximal duodenum were dry and five were lactating (186 ± 144 days in milk); in Exp. 2 four cows were dry and four were lactating (165 ± 57 days in milk). In cows fed the HC diet, FOL supplementation decreased the ruminally-fermented organic matter. Thus, less energy was available for ruminal microorganisms, which resulted in a reduced microbial crude protein flow at the duodenum. Feeding the HF diet, FOL supplementation only increased the apparent ruminal digestibility of acid detergent fibre (ADF). With the HF diet, FOL had no influence on the serum levels of glucose, non-esterified fatty acids, beta-hydroxybutyrate, urea, thiamine, riboflavin, pyridoxal-5′-phosphate, pyridoxic acid, pyridoxal, pyridoxine, pyridoxamine, pantothenic acid, nicotinamide or nicotinic acid, whereas supplementing FOL to the HC diet lowered the serum glucose and riboflavin levels. In both experiments, the supplementation of FOL had no effects on milk composition. Folic acid supplementation to both diets increased the concentrations of serum 5-methyl-tetrahydrofolate. However, no beneficial effects to dairy cows were obvious. Therefore, to achieve certain results, studies with a higher number of non-fistulated cows would be necessary.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid.

Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.     

Phospholipase C from Bacillus cereus. Evidence for essential lysine residues.

1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase

The amino acid sequences associated with pyridoxal 5'-phosphate binding sites in chicken liver fatty acid synthase have been determined: a site whose modification causes selective inhibition of the enoyl reductase activity and a site (site I) that is not associated with enzymatic activity. The amino acid sequences of peptides obtained by trypsin hydrolysis of the pyridoxamine 5'-phosphate labeled enzyme were determined. For the site associated with enoyl reductase activity, the sequence similarities between chicken and goose are extensive and include the sequence Ser-X-X-Lys, a characteristic structural feature of pyridoxamine enzymes. In addition, the spatial relationships between the pyridoxal 5'-phosphate binding sites and reductase site(s) have been studied with fluorescence resonance energy-transfer techniques. The distances between site I and the enoyl reductase and beta-ketoacyl reductase sites are greater than 50 and 41-44 A, respectively. The distance between the two reductase sites is greater than 49 A.     

Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

Acid-base, tautomeric and isomeric equilibrium of pyridoxal oximes, pyridoxal-5'-phosphate and some of their analogs

Success has been achieved in detailed understanding of tautomeric and isomeric equilibria and search for the new tautomeric and isomeric forms of oximes of pyridoxal, pyridoxal-5'-phosphate and some of their analogs, their presence is explained. This is due to a careful deconvolution of absorption spectra of different ionic forms of oximes into bands corresponding to separate electronic transitions. The spectroscopical data and the results of quantum-chemical calculations are compared for all the forms of compounds under investigation. As it has been found to be valid for other vitamin B6 derivatives as well, quantum-chemical calculations can be used for analytical purposes.