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At least 19 recordsLinked to original sources
PubMed1989

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.

Amniotic Fluid↗
PubMed1986

Simultaneous and direct determination of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in serum by derivative synchronous fluorescence spectroscopy.

Simultaneous and direct assays of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in human serum are described. The method applied is based on the reaction of these compounds with beryllium in an ammoniacal medium to yield highly fluorescent derivatives. Overlapping of conventional fluorescence spectra is resolved by using second-derivative fluorescence spectroscopy, thus making the use of separation techniques unnecessary. The proposed method is simple (only beryllium and an ammoniacal buffer are needed to develop fluorescence), rapid (the derivative formation is instantaneous and serum treatment only requires deproteinization), and inexpensive (no sophisticated detection equipment is necessary, any conventional modern spectrofluorimeter being adequate for use). The analytical recovery achieved was of about 96% for pyridoxal, 97% for pyridoxal-5'-phosphate, and 100% for pyridoxic acid. Measurements were carried out in a single scan.

Beryllium↗
PubMed1969

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.

Azides↗
PubMed1999

Conformational alteration in serum albumin as a carrier for pyridoxal phosphate: a distinction from pyridoxal phosphate-dependent glutamate decarboxylase.

The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyridoxal-P) carrier, was investigated by using uv/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. Upon interacting with pyridoxal-P, the uv/visible absorption spectrum of BSA exhibits peaks at 330 and 392 nm due to the formation of a Schiff base. Pyridoxal-P quenches the fluorescence emission intensity (excited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polarization of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational change appears to be small since circular dichroism showed only a 2-4% decrease in the alpha-helical content of BSA and no change in the beta-sheet content, and differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, an antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitive sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxal-P, the results were compared with those for glutamate decarboxylase (GAD), a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofactor for activity. Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>10(6) M-1), distinct conformational alterations occur between the two proteins upon interacting with pyridoxal-P: a small conformational change for BSA versus a large conformational change for GAD. In contrast to the case of BSA, AET causes significant effects on both the uv/visible spectrum and fluorescence emission intensity of GAD, because GAD contains sensitive sulfhydryl groups. Factors such as disulfide bond and active site sequence were discussed to understand BAS as a carrier for pyridoxal-P and a pyridoxal-P-independent protein.

Amino Acid Sequence↗
PubMed1966

The oxidation of Schiff bases of pyridoxal and pyridoxal phosphate with amino acids by manganous ions and peroxidase.

1. Oxygen was taken up rapidly when pyridoxal or pyridoxal phosphate was added to mixtures of pea-seedling extracts and Mn(2+) ions. 2. The increases in total oxygen uptake were proportional to the pyridoxal or pyridoxal phosphate added and were accompanied by the disappearance of these compounds. 3. In addition to Mn(2+) ions, the reactions depended on two factors in the extracts, a thermolabile one in the non-diffusible material and a thermostable one in the diffusate; these factors could be replaced in the reactions by horse-radish peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7) and amino acids respectively. 4. When pyridoxal phosphate was added to mixtures of amino acids and Mn(2+) ions oxygen uptake was rapid after a lag period of 30-90min.; the lag period was shortened to a few minutes by peroxidase, particularly in the presence of traces of p-cresol, or by light. 5. When pyridoxal replaced pyridoxal phosphate relatively high concentrations were required and peroxidase had only a small activating effect. 6. Pyridoxal or pyridoxal phosphate disappeared during the reactions and carbon dioxide and ammonia were formed. 7. With phenylalanine as the amino acid present, benzaldehyde was identified as a reaction product. 8. It is suggested that the reactions are oxidations of the Schiff bases formed between pyridoxal or pyridoxal phosphate and amino acids, mediated by a manganese oxidation-reduction cycle, and resulting in oxidative decarboxylation and deamination of the amino acids.

Aldehydes↗
PubMed1983

Levels and subcellular localisation of pyridoxal and pyridoxal phosphate in human polymorphonuclear leukocytes and their relationship to alkaline phosphatase activity.

Neutrophil leukocytes, isolated from normal subjects and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation, showed very similar cytosol distributions of pyridoxal and pyridoxal phosphate and lactate dehydrogenase. The small amounts of pyridoxal and pyridoxal phosphate associated with the dense granule fractions were not associated with the alkaline phosphatase containing granules. The levels of pyridoxal and pyridoxal phosphate were determined in neutrophils from control subjects, women in the third trimester of pregnancy and patients with chronic granulocytic leukaemia. Neutrophil pyridoxal phosphate was increased in women in the third trimester of pregnancy compared to controls, but there was little variation in the level of pyridoxal between the groups. There was no consistent correlation between the pyridoxal phosphate and the neutrophil alkaline phosphatase activity in the patient groups. Although in vitro neutrophil alkaline phosphatase rapidly hydrolyses pyridoxal phosphate, it is suggested that in vivo this is unlikely to be the principal function of the enzyme.

Alkaline Phosphatase↗
PubMed1991

The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues. Fluorescence studies of pyridoxal kinase.

Fluorescence spectroscopy, static and dynamic, was applied to deduce proximity relationships between paramagnetic metal ions chelated to ATP and the inhibitor pyridoxal oxime bound to the site of the substrate pyridoxal. The fluorescence yield of free pyridoxal oxime is considerably reduced in the presence of CoCl2 (1 mM) due to the formation of a non-fluorescent complex. The fluorescence properties of pyridoxal oxime bound to pyridoxal kinase (50 microM, Kd = 1 microM) remain invariant in the presence of CoCl2 (1 mM) and ATP (1 mM). The results are interpreted to mean that the distances are too great for direct or first-coordination-sphere interaction between pyridoxaloxime and Co(II) centers. The bidentate complex Cr(III).ATP is a competitive inhibitor of ATP (Ki = 83 microM) and its binding to pyridoxal kinase (50 microM) complexed to pyridoxal oxime (50 microM) was monitored by static and dynamic fluorescence spectroscopy. The fluorescence yield of bound pyridoxal oxime is reduced by 25% in the presence of Cr(III).ATP (0.8 mM) leading to a decrease in fluorescence lifetime from (tau) = 6.60 ns to (tau) = 4.20 ns. The decrease in fluorescence yield together with a diminution in fluorescence lifetime can be explained in terms of radiationless energy transfer from excited pyridoxal oxime to Cr(III) chelated to ATP. Using Forster's equation and an efficiency of energy transfer of 0.25, it was found that a distance of 1.3 nm separates pyridoxal oxime from the Cr(III) center.

Adenosine Triphosphate↗
PubMed2006

Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

Adenosine Triphosphate↗
PubMed1985

Pyridoxal phosphate-unrelated inhibition of hippocampal glutamic acid decarboxylase by convulsant pyridoxal sulphate.

Previous studies from this laboratory have shown that pyridoxal-5'-sulphate, the synthetic analogue of pyridoxal phosphate, causes epileptic seizures including tonic-clonic convulsions. These seizure activities are prevented or reversed by GABA or muscimol. In an attempt to delineate the biochemical basis of these seizure processes further, we have studied and shown that pyridoxal sulphate is a competitive inhibitor of glutamic acid decarboxylase. In addition, the chronic administration of pyridoxal sulphate was shown to reduce the concentration of pyridoxal phosphate in the cerebellum, the cerebrum, and basal ganglion, but not in the hippocampus. The activity of hippocampal glutamic acid decarboxylase was reduced after 1, 3, and 5 days of chronic application of pyridoxal sulphate. The inhibition was demonstrated, whether glutamic acid decarboxylase was assayed in the presence or absence of its coenzyme pyridoxal phosphate. Unlike findings in the hippocampus, the activity of glutamic acid decarboxylase in other brain regions was unaffected following chronic application of pyridoxal sulphate. The selective toxic effects of pyridoxal sulfate to the hippocampus, a brain area well known for its high susceptibility to seizure discharges, deserve additional indepth investigation.

Animals↗
PubMed1987

Effect of different levels of oral pyridoxine supplementation on plasma pyridoxal-5'-phosphate and pyridoxal levels and urinary vitamin B-6 excretion.

Plasma pyridoxal-5'-phosphate and pyridoxal levels increased significantly (p less than 0.05) when single, oral bolus doses of pyridoxine were increased from 10 to 25 and from 25 to 50 mg in nine female volunteers. However, when the dose was increased to 100 mg, plasma pyridoxal-5'-phosphate levels did not differ significantly from those recorded after the 50 mg dose. Within 3 h plasma pyridoxal levels rose with a factor of 3.85 compared with the 50 mg dose but high pyridoxal levels were eliminated from the circulation. Renal clearance of pyridoxal remained a constant, low percentage (less than 2.0%) of each pyridoxine supplement in spite of the observed very high circulating pyridoxal levels. Pyridoxine supplementation is discussed in relation to circulating pyridoxal-5'-phosphate and pyridoxal availability for cellular metabolism.

Administration, Oral↗
PubMed1982

Modification of intracellular hemoglobin with pyridoxal and pyridoxal 5'-phosphate.

The aldehyde forms of vitamin B6, pyridoxal and pyridoxal 5'-phosphate (PLP) have aroused interest as antisickling agents because of their ability to modify hemoglobin (Hb) and their low toxicity. To study their rate of formation and stability inside red cells, pyridoxal-Hb and PLP-Hb were measured in lysates from treated normal and sickle erythrocytes using isocratic high pressure liquid chromatography on Bio-Rex 70. The validity of this assay was confirmed by isoelectric focussing, fluorescence scans of reduced globin, and treatment of cells with pyridoxal 14C. Optimal conditions were described for treatment of whole blood with pyridoxal and washed erythrocytes with PLP. Although there was competition between 2,3-DPG and PLP, but not pyridoxal, for binding to Hb, depletion of 2,3-DPG prior to treatment was unnecessary. No special requirements were noted for the anticoagulants or buffers used. Sickle erythrocytes formed PLP-Hb more rapidly than normal erythrocytes, but pyridoxal-Hb appeared at the same rate in both types of erythrocytes. During incubation of treated erythrocytes in untreated plasma, the stability of pyridoxal-Hb varied inversely with the hematocrit, but PLP-Hb was stable at all hematocrits tested. The absence of hemolysis during a 4 day incubation of treated normal red cells implies that treatment with pyridoxal or PLP did not severely impair red cell metabolism.

Anemia, Sickle Cell↗
PubMed1992

Metabolism of [14C]- and [32P]pyridoxal 5'-phosphate and [3H]pyridoxal administered intravenously to pigs and goats.

To gain more information about the kinetics of vitamin B-6 metabolism in vivo, the metabolism of tracer was examined after the simultaneous intravenous administration of [32P] and [14C]pyridoxal phosphate and [3H]pyridoxal in two 93-kg pigs and two 60-kg goats. In the pigs, [14C] removal was monophasic with T1/2 of 16 and 18 min and clearance of 165 and 248 mL/min. In the goats, [14C] removal was biphasic with T1/2 of 49 and 114 min for 0-30 min and 209 and 227 min for 0.5-6 h (clearance 20 and 17 mL/min). Uptake of pyridoxal phosphate by liver and resecretion into the plasma were too small to cause a detectable decrease in the [32P]:[14C] ratio. Pyridoxal removal from plasma was similar in both species, with a half-life of approximately 12 min from 0-30 min and approximately 50 min for 0.5-3 h. Clearance of [3H]pyridoxal in the four animals ranged from 412 to 2258 mL/min. Little [14C] entered the erythrocytes. The [3H] entered readily but was converted to pyridoxal phosphate faster in the pigs than in the goats. [14C] and [3H] were excreted as pyridoxic acid at the same rate. However, during the 54 h after injection the goats excreted approximately 60% of the [14C] doses in the urine compared with approximately 30% in the pigs. About 5-10% of the [14C] and [3H] doses were recovered in goat milk over 54 h. Pyridoxal kinase activity was higher in lactating mammary tissue than in liver, kidney or muscle of goats.

Animals↗
PubMed1983

A fluorimetric method for the measurement of pyridoxal and pyridoxal phosphate in human plasma and leucocytes, and its application to patients with sideroblastic marrows.

A highly sensitive fluorimetric assay for the measurement of pyridoxal and pyridoxal phosphate in biological tissues is described. The method involves the enzymic hydrolysis of pyridoxal phosphate to pyridoxal. The pyridoxal (free or total) is separated on an anion-exchange column, concentrated by cation-exchange chromatography and reacted with potassium cyanide under slightly alkaline conditions to form 4-pyridoxolactone, a highly fluorescent compound. The method is applied to the measurement of pyridoxal, pyridoxal phosphate and total pyridoxal in plasma and neutrophils from control subjects and patients with sideroblastic marrow and identified the patient with pyridoxine-responsive sideroblastic anaemia.

Anemia, Sideroblastic↗
PubMed1999

Plasma pyridoxal phosphate and pyridoxic acid and their relationship to plasma homocysteine in a representative sample of British men and women aged 65 years and over.

Concentrations of pyridoxal phosphate and pyridoxic acid were measured in fasting plasma samples from British men and women aged 65 years and over, participating in a National Diet and Nutrition Survey during 1994-5, selected to be representative of the population of mainland Britain. In this population, the concentration of pyridoxal phosphate declined, whereas pyridoxic acid rose, with increasing age and frailty; however, both status indicators were strongly and directly (with a positive coefficient) correlated with estimates of vitamin B6 intake. This was little affected by the inclusion of food energy and protein intakes in the model. Forty-eight percent of the participants living in the community and 75% of those living in institutions had plasma pyridoxal phosphate concentrations below a range considered normal from other studies. In a univariate regression model, plasma pyridoxal phosphate concentrations were inversely correlated with plasma homocysteine concentrations, consistent with the hypothesis that vitamin B6 status may influence plasma homocysteine levels, and hence vascular disease risk. However, this relationship was partly attenuated in a multiple regression model including age, sex, domicile and biochemical status indices, including those of folate and vitamin B12. There was evidence that plasma pyridoxal phosphate was sensitive to metabolic conditions associated with inflammation and the acute-phase reaction, and that plasma pyridoxic acid was sensitive to renal function. Thus, neither index is an ideal predictor of vitamin B6 status in older people, unless these confounding factors are allowed for. Since poor vitamin B6 status may have health implications, e.g. for immune function, cognition, and for essential intermediary metabolic pathways in older people, it needs to be investigated as a possible public health problem.

Adolescent↗