Increased Aspartate Aminotransferase Activity of Serum after in Vitro Supplementation with Pyridoxal Phosphate

1973 ◽  
Vol 19 (1) ◽  
pp. 92-98 ◽  
Author(s):  
Robert Rej ◽  
Charles F Fasce ◽  
Raymond E Vanderlinde
Keyword(s):  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Colorimetric Method ◽  
Small Range ◽  
Aminotransferase Activity ◽  
Kinetic Assay ◽  
Serum Supplementation ◽  
Sample Dilution ◽  
Stimulation Of

Abstract We examined the effect of pyridoxal phosphate supplementation on the apparent aspartate aminotransferase (EC 2.6.1.1.) activity of human serum. Supplementation by 25 µmol/liter effected an average increase of 16% in the results for kinetic assay. The increase was not the result of increased enzymatic or nonenzymatic blanks, and, within a small range, sample dilution had no significant effect. Part of the increase was attributable to the enzyme being protected against the loss of activity that occurs during preincubation with L-aspartate. A similar increase was not demonstrated in a two-point colorimetric method, perhaps because of the short reaction time, without preincubation, and the initial presence of both substrates in the assay. We attempted to Correlate such stimulation of aminotransferase activity and the patient’s diagnosis or treatment. Pyridoxal phosphate should be included in the reaction mixture when aspartate aminotransferase activity is being measured clinically

Effects of Buffers on Aspartate Aminotransferase Activity and Association of the Enzyme with Pyridoxal Phosphate

1975 ◽  
Vol 21 (11) ◽  
pp. 1585-1591 ◽  
Author(s):  
Robert Rej ◽  
Raymond E Vanderlinde
Keyword(s):  
Schiff Base ◽  
Aspartate Aminotransferase ◽  
Phosphate Buffer ◽  
Pyridoxal Phosphate ◽  
Human Origin ◽  
Aminotransferase Activity ◽  
Factors Influencing ◽  
Absorbance Spectra ◽  
Stimulation Of

Abstract Using purified enzymes of human origin and patients’ sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 µmol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/liter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Absorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic catalysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of D-asparate substrates

Biological variability in aspartate aminotransferase activity in serum of healthy persons, and effect of in vitro supplemantation with pyridoxal 5-phosphate.

1977 ◽  
Vol 23 (3) ◽  
pp. 551-554 ◽  
Author(s):  
M Horder ◽  
G N Bowers
Keyword(s):  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Reference Interval ◽  
Individual Variability ◽  
Biological Variability ◽  
Healthy Individuals ◽  
Aminotransferase Activity ◽  
Measured Activity ◽  
Phosphate Supplementation

Abstract Aspartate aminotransferase (EC 2.6.1.1) activities in sera from nine healthy individuals were monitored during two weeks, both with and without first supplementing the serum with pyridoxal phosphate. Pyridoxal phosphate supplementation caused a mean increase of 39% (range, 33-55%) in measured activity. The biological variability during the two-week period was independent of pyridoxal phosphate supplemantation. The intra-individual variability (CV) was 5.3% and 5.1% with and without pyridoxal phosphate supplementation, respectively; the corresponding inter-individual variability was 13.2% and 13.6%. We conclude that the reference interval will be insensitive to intra-individual fluctuations in aspartate aminotransferase activity in serum, whether or not the serum is supplemented with pyridoxal phosphate.

Aspartate aminotransferase activity in human serum. Factors to be considered in supplementation with pyridoxal 5'-phosphate in vitro.

1976 ◽  
Vol 22 (11) ◽  
pp. 1876-1883 ◽  
Author(s):  
M Horder ◽  
R E Moore ◽  
G N Bowers
Keyword(s):  
Human Serum ◽  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Normal Activity ◽  
Aminotransferase Activity ◽  
Serum Factors ◽  
Volume Fractions ◽  
Sources Of Error ◽  
Potential Sources

Abstract The pyridoxal phosphate reactivation of the apo form of aspartate aminotransferase (EC 2.6.1.1) in human serum has been studied with "normal" and above-normal activity of this enzyme. The extent of the reactionation did not depend on the presence of the substrates, L-aspartate or 2-oxoglutarate. Reactivation was greatest with 110 mumol of added pyridoxal phsophate present per liter during a preinucation for 7 min in tris(hydroxymethyl)methylamine buffer wit;h serum volume fractions ranging from 0.017 to 0.267. In comparison with measurements prformed with no exogenous pyridoxal phosphate present, we found two potential sources of error when this cofactor was added: (a) reagent and sample blanks in the pyridoxal phosphate-supplemented system were two- to eightfold higher and (b) progress curves were nonlinear when L-aspartate rather than 2-oxoglutarate was used as the startin substrate. Aspartate aminotransferase measurement sith pyridoxal phosphate supplementation was slightly more precise than without.

Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration.

1979 ◽  
Vol 25 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J C Hafkenscheid ◽  
C C Dijt
Keyword(s):  
Enzymatic Activity ◽  
Serum Sample ◽  
Aspartate Aminotransferase ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Reaction Medium ◽  
Aminotransferase Activity ◽  
Serum Aminotransferases ◽  
Stimulation Of

Abstract To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.

Interlaboratory Proficiency, Intermethod Comparison, and Calibrator Suitability in Assay of Serum Aspartate Aminotransferase Activity

1975 ◽  
Vol 21 (8) ◽  
pp. 1141-1158 ◽  
Author(s):  
Robert Rej ◽  
C F Fasce ◽  
Raymond E Vanderlinde
Keyword(s):  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase ◽  
Continuous Flow ◽  
Pyridoxal Phosphate ◽  
Diazonium Salt ◽  
Assay Method ◽  
Kinetic Assay ◽  
Mean Values ◽  
Cytoplasmic Enzyme ◽  
Calibration Materials

Abstract Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 °C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was > 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, < 10%). However, the automated continuous-flow mAlate Dehydrogenase/Nadh Coupled Method Produced An Average Cv > 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.

scholarly journals Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

2009 ◽  
Vol 297 (1) ◽  
pp. G179-G186 ◽  
Author(s):  
Rachel M. Gwynne ◽  
Joel C. Bornstein
Keyword(s):  
Electrical Stimulation ◽  
Pyridoxal Phosphate ◽  
Enteric Neurons ◽  
Disulfonic Acid ◽  
Myenteric Neurons ◽  
Mrs 2179 ◽  
Neurokinin 1 ◽  
Oxide Synthase ◽  
Stimulation Of

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300–900 ms) or slow (durations 1.3–9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2–4-disulfonic acid (30 μM, N = 3) or the specific P2Y1 antagonist MRS 2179 (10 μM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 ( N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y1, S neurons).

scholarly journals Selective inhibition of alanine aminotransferase and aspartate aminotransferase in rat hepatocytes

1984 ◽  
Vol 220 (3) ◽  
pp. 707-716 ◽  
Author(s):  
N W Cornell ◽  
P F Zuurendonk ◽  
M J Kerich ◽  
C B Straight
Keyword(s):  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Rat Hepatocytes ◽  
Selective Inhibition ◽  
Enoic Acid ◽  
Urea Synthesis ◽  
Aminotransferase Activity ◽  
Glucose Synthesis ◽  
Alanine Aminotransferase Activity

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.

scholarly journals Tissue pyridoxal phosphate concentration and pyridoxaminephosphate oxidase activity in riboflavin deficiency in rats and man

1974 ◽  
Vol 32 (2) ◽  
pp. 249-255 ◽  
Author(s):  
Ambale V. Lakshmi ◽  
M. S Bamji
Keyword(s):  
Human Blood ◽  
Pyridoxal Phosphate ◽  
Reductase Activity ◽  
Human Subjects ◽  
Phosphate Concentration ◽  
Riboflavin Deficiency ◽  
Glutathione Reductase Activity ◽  
Riboflavin Deficient ◽  
Stimulation Of

1. Parenteral administration of pyridoxal 5′-phosphate (PALP) to riboflavin-deficient rats increased the non-FAD component of erythrocyte riboflavin.2. Pyridoxaminephosphate oxidase (EC 1.4.3.5) activity in the livers of riboflavin-deficient animals was only 15% of that of controls. PALP concentration in blood, liver and brain was not affected. Deficient animals had higher levels of pyridoxine in liver.3. Human subjects with lesions of the mouth responded to treatment with either riboflavin or pyridoxine.4. PALP concentration of human blood was not affected by administration of riboflavin but was markedly increased by pyridoxine.5. Erythrocyte glutathione reductase activity (EC 1.6.4.2) in humans was increased and in vitro stimulation of the enzyme with FAD was decreased by treatment with riboflavin, but not by treatment with pyridoxine.

scholarly journals The effect of oral contraceptives on the apparent vitamin B6status in some Sudanese women

1986 ◽  
Vol 56 (2) ◽  
pp. 363-367 ◽  
Author(s):  
Eltayeb Y. Salih ◽  
Asma A. Zein ◽  
Riad A. Bayoumi
Keyword(s):  
Side Effects ◽  
Nutritional Status ◽  
Oral Contraceptives ◽  
Aspartate Aminotransferase ◽  
Low Dose ◽  
Pyridoxal Phosphate ◽  
Vitamin B ◽  
In Vitro Activation ◽  
Dose Oral

1. In vitro activation of erythrocyte aspartate aminotransferase (EC2.6.1.1) activity by pyridoxal phosphate was used to assess vitamin B4nutritional status in forty Sudanese women taking combined, low-dose oral contraceptives (oestrogen-progestogen; OC) and in thirty healthy, non-pregnant women not taking OC.2. Fourteen (35%) out of forty OC users showed apparent vitamin B4deficiency.3. Side-effects associated with OC were more common among the apparently vitamin-B4-deficient OC users than among OC users and non-OC users not deficient in vitamin B4.

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