Plasma pyridoxal 5'-phosphate concentrations in relation to apo-aminotransferase levels in normal, uraemic, and post-myocardial infarct sera

The concentrations of pyridoxal 5'-phosphate, and the holoenzyme activities and apoenzyme contents of alanine aminotransferase and aspartate aminotransferase in plasma were determined simultaneously in healthy individuals, patients with renal insufficiency with and without chronic haemodialysis and in patients with acute myocardial infarction. Plasma pyridoxal 5'-phosphate is significantly diminished in uraemic patients and in post-myocardial infarct sera, healthy females have lower pyridoxal 5'-phosphate levels (26.2 +/- 9.0 nmol/l) than healthy males (41.0 +/- 15.1 nmol/l). The stimulation in vitro of the activities of aspartate aminotransferase and alanine aminotransferase by addition of pyridoxal 5'-phosphate (0.1 mmol/l) was found to be independent of the endogenous coenzyme level. In sera of uraemic patients without chronic haemodialysis an inverse statistic correlation between pyridoxal 5'-phosphate-induced stimulation of aspartate aminotransferase activity and the concentrations of urea (r = -0.696) and creatinine (r = -0.715) was found. The respective correlations are much weaker for alanine aminotransferase. The apoenzyme fraction was highest in post-myocardial infarct sera. Follow up of these patients did not reveal any relationship between the fluctuations of pyridoxal 5'-phosphate levels and apoenzyme contents of both alanine aminotransferase and aspartate aminotransferase. The results permit the conclusion that the degree of in vitro stimulation of aminotransferases by pyridoxal 5'-phosphate can not be predicted from the endogenous coenzyme level.     

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in sera of patients after myocardial infarction

Mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase (AST) were studied in the sera of 42 patients following acute myocardial infarction and compared to creatine kinase (CK), lactate dehydrogenase (LDH) and alanine aminotransferase (ALT). Mitochondrial AST( ASTm ) was detected in 93% (39/42) of patients. Maximum recorded ASTm activity was 59.5 +/- 8.8 U/l and was found 39.4 +/- 3.5 hours after the onset of symptoms (chest pain) of myocardial infarction. In contrast the maximum recorded cytoplasmic AST ( ASTc ) activity was greater (327 +/- 23 U/l) and it occurred earlier (33.5 +/- 2.2 hours) after onset of infarction compared to ASTm . ASTm correlated significantly (p less than 0.05) with ASTc , LDH and ALT but not with total CK or CK-MB. ASTc correlated significantly (p less than 0.05) with total CK, CK-MB and LDH but not ALT. Maximum recorded ASTm activity was significantly associated with the clinical assessment of left ventricular failure ( Killip classification) but not with ventricular arrhythmias. In a subset of 15 patients evaluated with invasive hemodynamic measurements of cardiac output and pulmonary capillary wedge pressure. ASTm correlated significantly (p less than 0.05) and better than CK-MB with the hemodynamic assessment of left ventricular dysfunction. Thus ASTSm can be readily identified in sera of patients after acute myocardial infarction and may be of value in the evaluation of patients with acute myocardial infarction.     

Apoenzyme content of serum aminotransferases in patients with a renal allograft treated with cyclosporine A and azathioprine

In 16 patients with a renal allograft the activity concentrations of aspartate aminotransferase and alanine aminotransferase and the percentage stimulation of both enzymes were investigated. After the transplantation the patients received prednisone and cyclosporine A as immunosuppressive therapy, while exactly 3 months after the date of transplantation prednisone and azathioprine were given as immunosuppressives. In the first period, the percentage increase of the activity concentration of aspartate aminotransferase and alanine aminotransferase upon supplementation of pyridoxal-5'-phosphate in vitro were similar to that of healthy individuals. In the second period, however, the percentage increase of the activity concentration of alanine aminotransferase was much higher than that of aspartate aminotransferase. Cyclosporine A given during a period of about 400 days did not influence the percentage increase of both enzymes. It is concluded that the high stimulation of alanine aminotransferase in the second period depends on the presence of azathioprine or its metabolites in serum.     

Changes in mass and catalytic activity concentrations of aspartate aminotransferase isoenzymes in serum after a myocardial infarction

We used an RIA and inhibition of enzyme activity to monitor the changes in mass and catalytic concentrations of the aspartate aminotransferase (EC 2.6.1.1;AST) isoenzymes in serum after myocardial infarction. Cytosolic (c-AST) and mitochondrial (m-AST) forms of AST were present in sera from all 38 of our patients. Although the immunological and catalytic concentrations of both isoenzymes correlated well with the size of the infarct, c-AST gave a better measure than did m-AST. About 20% of the total enzyme activity at peak activity was from the mitochondrial isoenzyme. Both isoenzyme activities peak at very nearly the same time, but m-AST has the longer half-life. Immunological evidence of the mitochondrial isoenzyme can be detected in serum for at least eight days after the infarct. The presence of left ventricular failure produces greater serum isoenzyme activities than in those without failure.     

New automated measurement of mitochondrial aspartate aminotransferase with use of protease 401

Total mitochondrial aspartate aminotransferase (EC 2.6.1.1), the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using a proteolytic method: inactivation of cytosolic aspartate aminotransferase with cytosolic aspartate aminotransferase-inactivating protease 401 from Streptomyces violaceochromogenes. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5'-phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r = 0.994, n = 145) and showed excellent inhibitory activity of the protease for holo- and apo-cytosolic aspartate aminotransferase up to 5000 U/L and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5'-phosphate per liter. Within-run Cvs were good (1.13-7.49%). Mean values for total mitochondrial aspartate aminotransferase and apo-mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n = 154). Various common interferents tested did not affect this assay.     

Difficulties in the normalization of aminotransferase measurement with enzyme standards

Commercial control sera contain varying amounts of pyridoxal-5'-phosphate. Normalization of aminotransferase activity measurement with enzyme "standards" is possible only when pyridoxal-5'-phosphate is added to the reaction medium. Pyridoxal-5'-phosphate addition should be compulsory in national recommendations.     

Measurement of aspartate aminotransferase activity: effects of oxamate.

Oxamate, a potent inhibitor of lactate dehydrogenase, is shown also to inhibit aspartate aminotransferase activity, both in human serum and in purified isoenzymes of human origin. The inhibition was competitive with respect to 2-oxoglutarate for both isoenzymes. The apparent Ki was 29 mmol/L for the cytoplasmic enzyme and 17 mmol/L for the mitochondrial enzyme. Noncompetitive inhibition was found between oxamate and aspartate. At saturating concentrations of substrate (2-oxoglutarate greater than or equal to 15 mmol/L, L-aspartate greater than or equal 150 mmol/L) oxamate inhibited the mitochondrial enzyme but had less effect on the cytoplasmic isoenzyme. Oxamate at 40 mmol/L inhibited the enzyme in serum by 11 and 9% in assays containing 2-oxoglutarate at 6.7 and 15 mmol/L, respectively. This concentration of oxamate inhibited enzyme activity in serum by 5% more than did the same concentration of Cl- (itself an inhibitor). Oxamate (less than or equal to 30 mmol/L) had no measurable effect on the stability or activity of porcine malate dehydrogenase. Until the effects of its inhibitory properties are considered, addition of oxamate to suppress lactate dehydrogenase-mediated side reactions in the assay of aspartate aminotransferase cannot be recommended.     

Evidence of a theophylline-induced vitamin B6 deficiency caused by noncompetitive inhibition of pyridoxal kinase

A placebo-controlled, double-blind study indicated that theophylline administration to apparently healthy, young men induced significantly depressed plasma pyridoxal-5'-phosphate levels. Plasma pyridoxal levels were not affected by theophylline therapy. The effect of theophylline on circulating pyridoxal-5'-phosphate levels is explained by the observation that theophylline acts as a noncompetitive inhibitor for erythrocyte pyridoxal kinase (EC 2.7.1.35) with an apparent inhibition constant (Ki) of 1.28 x 10(-5) mol/L. Theophylline did not affect erythrocyte pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) activity. During theophylline therapy, erythrocyte pyridoxal kinase levels increased nearly twofold from an initial mean level of 24.2 +/- 4.0 (+/- SD) nmol to 46.9 +/- 7.3 nmol pyridoxal-5'-phosphate per gram of hemoglobin per hour. This partially counteracted the effect of theophylline on vitamin B6 metabolism. Nevertheless, erythrocyte pyridoxal-5'-phosphate levels in subjects given theophylline decreased significantly (p = 0.03) from pretreatment levels. The oral tryptophan load test resulted in significantly (p = 0.007) increased urinary xanthurenic acid excretion after 4 weeks of theophylline therapy. Both plasma pyridoxal-5'-phosphate levels and the tryptophan load test results normalized after 1 week of pyridoxine supplementation, indicating that 10 mg pyridoxine per day was effective to counteract the antagonistic effect of short-term theophylline therapy on vitamin B6 metabolism.     

Column chromatography and immunoassay compared for measuring the isoenzymes of aspartate aminotransferase in serum.

We compare a column-chromatographic method and a homogeneous immunoassay method for separately measuring the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase. Analytical recovery for the two methods averaged 102% (SD, 2%) and 103% (SD, 4%), respectively, for 11 pools prepared by adding the purified isoenzymes to serum and 102% (SD 8.9%) and 89% (SD, 8.1%) for 26 unaltered specimens of human serum. In comparing the results of the immunoassay method (y) to the chromatographic method (x), our measurements agreed closely for the mitochondrial (y = 0.947 X + 7, r = 0.9991, standard error of estimate = 2.9 U/L) and cytoplasmic (y = 0.92x-6, r = 0.9995, standard error of estimate = 2.1 U/L) isoenzymes in pools prepared from the purified isoenzymes. Similar measurements of the 26 human serum specimens yielded the following results for least-squares evaluation; cytoplasmic isoenzyme y = 1.03x-11, r = 0.994, and standard error of estimate = 6.0 U/L; mitochondrial isoenzyme y = 0.75x+0, r = 0.927, and standard error of estimate = 3.9 U/L.     

Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.     

A new cause of increased serum aspartate aminotransferase activity

Two healthy young women had an unexplained persistent elevation of aspartate aminotransferase (ASAT) activity. In both cases electrophoresis of serum ASAT isoenzymes displayed an abnormally moving fraction that comprised the whole serum activity, while liver and muscle revealed the normal cytoplasmic and mitochondrial isoenzymes. In the first case serum ASAT was found to be bound by serum IgG, in the second case the binding protein remained unidentified.     

Changes in activation of aspartate aminotransferase by pyridoxal 5'-phosphate after experimental liver damage in rabbits

The aspartate aminotransferase activity with and without pyridoxal 5'-phosphate supplementation was examined in mitochondrial and cytoplasmic preparations from fresh human heart and liver samples. The apoenzyme was fully saturated in all cases. Liver cell damage was produced by ischaemia and carbon tetrachloride poisoning in two groups of rabbits. The activity of aspartate aminotransferase with and without pyridoxal 5'-phosphate was measured in the plasma and in cytoplasmic and mitochondrial preparations from both groups. After carbon tetrachloride poisoning the enzyme activity in the plasma increased within 2 h but was not enhanced by pyridoxal 5'-phosphate. Following ischaemia, plasma enzyme activity only increased between 4 and 8 h and was progressively stimulated by pyridoxal 5'-phosphate. Up to 15 h after carbon tetrachloride poisoning the liver cytoplasmic and mitochondrial apo-enzyme remained fully saturated with co-enzyme. In contrast, a pronounced loss of co-enzyme occurred in both fractions of the ischaemic group. These results suggest that the type of injury and not necessarily the organ affected could determine the degree of activation of aspartate aminotransferase by pyridoxal 5'-phosphate observed in human myocardial infarction and liver disease.     

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase

We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at lieu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run <4.45%) and a coefficient of correlation of r = 0.985 (N = 125). © 1993 Wiley-Liss, Inc.     

Homocystinuria: EVIDENCE FOR THREE DISTINCT CLASSES OF CYSTATHIONINE β-SYNTHASE MUTANTS IN CULTURED FIBROBLASTS

We have compared in vivo pyridoxine responsiveness with in vitro cystathionine beta-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5'-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five- and sevenfold stimulation of cystathionine beta-synthase activity with added pyridoxal-5'-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two- and fivefold with the added cofactor. Cystathionine beta-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5'-phosphate were estimated and thermostability (54 degrees C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine beta-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5' phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activity in vitro or with stimulation of in vitro enzyme activity by cofactor.     

Tedisamil Attenuates Ventricular Fibrillation in a Conscious Canine Model of Sudden Cardiac Death

BACKGROUND: The electrophysiologic and antifibrillatory properties of tedisamil (KC-8857;3,7-di-(cyclopropylmethyl)-9,9-tetramethylene-3,7-diazabicyclo[3.3.1]-nonane dihydrochloride) were studied in a conscious canine model of sudden cardiac death. METHODS AND RESULTS: Three to five days after surgically induced myocardial infarction (2-hour occlusion of the left anterior descending coronary artery), animals were subjected to programmed electrical stimulation to identify those at risk for ischemia-induced ventricular fibrillation. Sixty minutes after tedisamil (10 mg/kg, administered orally) PES was repeated. Tedisamil increased the ventricular effective refractory period from 106 +/- 6 to 134 +/- 7 ms (P <.05) compared to placebo treatment, which did not alter the ERP (123 +/- 6 to 116 +/- 5 ms). Tedisamil prolonged the QTc interval, from a predrug value of 308 +/- 14 to 327 +/- 14 ms, postdrug. The extent of the surgically induced anterior wall myocardial infarct did not differ between groups, tedisamil, 29 +/- 2%, and placebo, 28 +/- 2% of the left ventricle. CONCLUSIONS: Tedisamil conferred protection against ischemia induced ventricular fibrillation; 7 of 10 tedisamil-treated dogs survived, compared to 4 of 14 surviving in the vehicle treated group (P <.05). Although we observed instances of vomiting and/or diarrhea in several dogs after a single oral administration of tedisamil, the data indicate that oral administration of tedisamil provides protection from ischemia-induced ventricular fibrillation in the postinfarcted conscious canine. The mechanism by which tedisamil achieves its antifibrillatory effect may be related to its ability to prolong the ERP of the ventricular myocardium without altering ventricular conduction velocity.     

Relative stabilities of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in lyophilized materials.

Eight different pools of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase were prepared, to examine the effects of the following matrix variables: the matrix support material (bovine serum albumin and polyvinylpyrrolidone), endogenous pyridoxal concentration, and azide as an antimicrobial preservation. Storage temperatures of 25 and 37 degrees C were used as a rapid and convenient means of accelerating the degradation process. Activity of the enzyme was measured with and without pyridoxal in the reaction solution. We found that the mitochondrial isoenzyme was consistently more labile than the cytoplasmic isoenzyme under identical storage conditions. Both isoenzymes were more stable in matrixes containing bovine serum albumin than in those containing polyvinylpyrrolidone. No apparent difference in the stability of either isoenzyme was observed at matrix pyridoxal concentrations of 15 micromol/L and 150 micromol/L. Only the mitochondrial isoenzyme in matrixes containing bovine serum albumin and 15 micromol of pyridoxal per liter had increased activity (about 9%) when pyridoxal was added to the enzymatic reagent. The amount of activity in reconstituted specimens did not apparently change after 72 h at 4 degrees C.     

Activities of mitochondrial aspartate aminotransferase and creatine kinase isoenzyme MB in serum following coronary bypass surgery

The mitochondrial isoenzyme of aspartate aminotransferase showed only slight increases in serum of twenty-seven patients after uncomplicated coronary bypass surgery, which contrasted the rapid and substantial increases in creatine kinase MB. In seven patients suffering perioperative infarction or serious complications, substantial increases in mitochondrial aspartate aminotransferase were detected and the elevations in creatine kinase MB were prolonged. Mitochondrial aspartate aminotransferase may appear as a specific marker of myocardial necrosis following coronary bypass surgery. The elevations of creatine kinase and creatine kinase MB were detected as early as 5 minutes after onset of coronary reperfusion and slightly higher activities were measured in coronary sinus blood than in systemic blood sampled simultaneously. Increases in mitochondrial aspartate aminotransferase, however, could first be measured 8 hours after reperfusion.     

Dynamics of creatine kinase shuttle enzymes in the human heart

Myocardial cytoplasmic creatine kinase subunits M and B, mitochondrial CK (CKMIT), and citrate synthase (CS) were determined in 10 locations of the normal human heart (n = 8) and in papillary muscles of patients operated on for mitral regurgitation (n = 6). Compared to atrial biopsies, septal and left ventricular biopsies showed higher activities for CS (P less than 0.0001), total CK (P less than 0.05) and CKMIT (P less than 0.0001). CKM was evenly distributed. CKB activity in the right septum and left ventricular locations were 0.5-1% of total CK and 4-5 times lower than those of the atria and the right ventricular free wall. Activities of CS, CKB and CKMIT in right septal biopsies did not differ from those in left ventricular locations. The activities of CS, total CK, and CKM in papillary muscle from patients operated on for mitral regurgitation did not differ from that of healthy papillary muscle. CKMIT was about 40% lower (P less than 0.02), whereas CKB was 15-20 times higher (P less than 0.0001) than in the healthy heart. In conclusion, adaptations within the creatine kinase system occur in the human heart in health and disease. Small amounts of CKB in the normal left ventricle, as opposed to the right ventricular free wall, might be related to differences in myocardial perfusion during the cardiac cycle. In disease, a decreased CKMIT and dramatically increased CKB may indicate a stressed intracellular energy transfer. CK enzyme activities in right septal biopsy specimens may be used as an indication of metabolic stress on the myocardium of the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of pyridoxal 5'-phosphate on the temperature relationships of aspartate aminotransferase isoenzymes]

The relationship between temperature and the behaviour of aspartate aminotransferase was investigated in the presence of pyridoxal 5'-phosphate. The addition in vitro of pyridoxal 5'-phosphate caused an increase in the activity and altered the thermal behaviour of aspartate aminotransferase. In choosing the temperature for the determination of enzymic activity, the concentration of the coenzyme must therefore also be considered.     

The effect of sustained stellate ganglion stimulation on left ventricular contractility in the dog

Although a progressive reduction in left ventricular contractility during sustained left stellate ganglion stimulation has been well documented, there have been no reports on the contractile state after nerve stimulation. Left ventricular contractility after cessation of 60 min of electrical (10 V. 10 Hz. 1 msec) left stellate ganglion stimulation has been assessed in open chest dogs. Before and 15 min after stimulation, left ventricular contractility was evaluated by the end-systolic pressure-segment length relationship using ultrasonic crystals during a stepwise aortic constriction to increase left ventricular afterload. Restimulation of the left stellate ganglion was also performed 15 min after cessation of the first stimulation. After sustained left stellate ganglion stimulation, the end-systolic points shifted to the right from the control and the slope of multiple pressure-segment length coordinates significantly decreased (102.5 +/- 16.1 to 76.5 +/- 10.2 mmHg/mm, mean +/- S.E., p less than 0.05, n = 5), indicating a depression of left ventricular contractility. Increased left ventricular dP/dt max and norepinephrine level in the coronary sinus gradually returned to near base line during 60 min of stimulation. These reduced responses lasted for at least 15 min after cessation of stimulation. The myocardial norepinephrine content was reduced to 0.59 +/- 0.08 (mean +/- S.E.) ng/mg wet tissue from 0.90 +/- 0.15 of the control level (p less than 0.05). These data suggested that left ventricular contractility decreased after sustained cardiac sympathetic nerve stimulation, probably due to norepinephrine reduction in the myocardium.