Influence of salts on Michaelis-constant values for NADH.

We previously observed [Clin. Chem. 22, 1648 (1976)] that values of the Michaelis constant for NADH for the conversion of pyruvate to lactate with lactate dehydrogenase (EC 1.1.1.27) in the presence of 0.1 mol/liter buffers at 25 degrees C showed first-order dependence on enzyme concentration. This is now recognized to be the result of an inhibitory influence exerted by buffers [NH4HCO2, tris(hydroxymethyl)aminomethane, and phosphate] and salts [(NH4)2SO4 and NaCl] present in the reaction mixtures. Inhibition constants for the enzyme/inhibitor complexes formed with these substances are about 0.3 mol/liter for competition of NH4HCO3 with NaOH and 0.4 mol/liter for competition of NH4HCO3 with pyruvate; they are 0.6 mol/liter for NaCl, 1.0 mol/liter for sodium phosphate, 0.3 mol/liter for (NH4)2SO4, and 0.8 mol/liter for tris(hydroxymethyl)aminomethane when these substances compete with NADH. Because of the large molar ratio of buffer to substrate (about 10(9):1) in enzymatic assays, the buffer concentration significantly influences the Michaelis constant, despite the large value for the inhibition constant. Attention to the concentrations of these substances may be required for decreasing variability in clinical assays in which lactate dehydrogenase and possible other enzymes are used.     

Simultaneous separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography.

Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.     

Determination of phosphodiesterase I activity in human blood serum.

Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis.     

Macromolecular alkaline phosphatase and an immunoglobulin G that inhibited alkaline phosphatase in a patient's serum

Macromolecular alkaline phosphatase (EC 3.1.3.1) was found in the serum of a patient suffering from myasthenia gravis (adult type II) complicated with thymoma, and was shown by immunoelectrophoresis to be bound to immunoglobulins A and G (IgG). Placental alkaline phosphatase, complexed with either the patient's serum or IgG purified from the patient's serum, remained at the origin on electrophoresis, with significant loss of activity. Intestinal alkaline phosphatase, complexed with either the patient's serum or the patient's IgG, migrated to a position similar to that of the macromolecular alkaline phosphatase in the patient's serum on electrophoresis. About 50% of the placental alkaline phosphatase activity was inhibited with 0.1-0.2 g of the patient's IgG per liter, but 6.93 g of the IgG per liter was required for about 20% inhibition of the intestinal alkaline phosphatase activity. The complex of intestinal alkaline phosphatase with the patient's IgG was fairly heat stable. From these results, we concluded that the macromolecular alkaline phosphatase in the patient's serum consisted of intestinal alkaline phosphatase and IgG that was specific for placental alkaline phosphatase.     

Angiotensin II depolarizes podocytes in the intact glomerulus of the Rat.

The aim of this study was to examine the effects of angiotensin II (Ang II) on cellular functions of rat podocytes (pod) in the intact freshly isolated glomerulus and in culture. Membrane voltage (Vm) and ion currents of pod were examined with the patch clamp technique in fast whole cell and whole cell nystatin configuration. Vm of pod was -38+/-1 mV (n = 86). Ang II led to a concentration-dependent depolarization of pod with an ED50 of 10(-8) mol/liter. In the presence of Ang II (10(-7) mol/liter, n = 20), pod depolarized by 7+/-1 mV. In an extracellular solution with a reduced Cl- concentration of 32 mmol/liter, the effect of Ang II on Vm was significantly increased to 14+/-4 mV (n = 8). The depolarization induced by Ang II was neither inhibited in an extracellular Na+-free solution nor in a solution with a reduced extracellular Ca2+ (down to 1 micromol/liter). Like Ang II, the calcium ionophore A23187 (10(-5) mol/liter, n = 9) depolarized pod by 10+/-2 mV, whereas forskolin (10(-5) mol/liter), 8-(4-chlorophenylthio)-cAMP and N2,2''-o-dibutyryl-cGMP (both 5 x 10(-4) mol/liter) did not alter Vm of pod. The angiotensin 1 receptor antagonist losartan (10(-7) mol/liter) completely inhibited the Ang II-induced (10(-7) mol/liter) depolarization (n = 5). Like pod in the glomerulus, pod in short term culture depolarized in response to Ang II (10(-8) mol/liter, n = 5). Our results suggest that Ang II depolarizes podocytes directly by opening a Cl- conductance. The activation of this ion conductance is mediated by an AT1 receptor and may be regulated by the intracellular Ca2+ activity.     

Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.     

Affinity electrophoresis of human serum alkaline phosphatase isoenzymes in agarose gel containing lectin

Separation of alkaline phosphatase isoenzymes using affinity electrophoresis in agarose gel containing lectin is described. The bone and biliary isoenzymes precipitate during electrophoresis and are clearly separated from the liver isoenzyme. The liver, intestinal and placental alkaline phosphatases are essentially not affected by the lectin. The migration distances of the precipitating bone and biliary fractions vary with their alkaline phosphatase activity. The bone isoenzyme is more heterogeneous than the biliary isoenzyme with respect to interaction with lectin forming both insoluble and soluble complexes. Affinity electrophoresis in agarose gel containing lectin can be used for quantitation by densitometry of liver and bone isoenzymes in sera containing only these two fractions but must be combined with conventional electrophoresis, preferably in agar gel, if biliary, intestinal, or placental isoenzymes are also present.     

Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation.

I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol.     

PH measurement problems affecting assay of carcinoembryonic antigen.

Measurments with combination pH electrodes of the pH of the dilute buffers used in a commercial kit (CEA-Roche) for assay of carcinoembryonic antigen resulted in pH values 0.1 to 0.3 unit lower than pH values measured on an electrode system with a capillary junction. If the pH values of these buffers were adjusted, based on such measurements, an error in the assay of 0.2 to 0.6 ng/ml in the 1.5-3.0 ng/ml range would result. We recommend that the pH of dialyzed samples and of the working ethylenediaminetetraacetate and ammonium acetate-acetic acid buffers be monitored with pH electrodes that have a capillary junction between sample and saturated KCI, as is true of most blood-pH instruments. We also recommend use of a 1 mol/liter rather than 2.5 mol/liter stock ammonium acetate-acetic acid buffer, because of the closer similarity of the pH of buffers at this molarity to those at 0.01 mol/liter.     

Thermodynamic characterisation of the mutated isoenzyme A of beta-N-acetylhexosaminidase in GM2-gangliosidosis B1 variant.

Here we report the determination of the activation energies of the plasma isoenzymes of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), isolated by chromatography in DEAE-cellulose, using the neutral chromogenic substrate 3,3'dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide. The activation energy of mutated Hex A isoenzyme (Ea approximately 71.5 kJ/mol) from a patient with GM2-gangliosidosis B1 variant, homozygote for the G533-->A (Arg178His) mutation, was significantly higher than that of normal Hex A (Ea approximately 41.8 kJ/mol) and analogous to that of Hex B isoenzyme (Ea approximately 75.1 kJ/mol). The determination of this thermodynamic variable of Hex in different biological specimens could allow for a straightforward biochemical characterisation of the GM2-gangliosidosis B1 variant.     

Quantitative analysis of alkaline phosphatases in serum and amniotic fluid: comparison of biochemical and immunologic assays

An immunoprecipitation method using monoclonal antibodies specific for the three categories of alkaline phosphatases, liver/bone/kidney, placental, and intestinal, is described and compared with the heat denaturation-chemical inhibition method for quantitating the contributions of the various isoenzymes to the total activity in biologic fluids. Activity values for the three categories of isoenzymes present in normal and pregnancy sera as well as in early gestation amniotic fluids were determined by both methods. There is very close correlation between the values obtained by both methods. Thus, two independent procedures are now available for quantitating the contributions of the various alkaline phosphatase isoenzymes in biologic fluids.     

Lipoxygenic micromethod for specific determination of lipase activity in serum and duodenal fluid.

We propose a rapid enzymatic micromethod for the specific determination of lipase (EC 3.1.1.3) activity in serum and duodenal fluid. Free linoleic acid produced during 10-min incubation of 10 mul of sample with 1 ml of substrate (trillinolein emulsion) at 30 degrees C is converted by lipoxygenase (EC 1.99.2.1), in a coupled reaction, to its hydroperoxide, which is measured photometrically after solubilizing the reaction mixture in ethanol. Lipase activity is calculated from the rate of hydroperoxide formation, with linoleic acid as primary standard. The velocity of the reaction is greatest at pH 8.8, 35-37 degrees C, and a deoxycholate concentration of 3.6 mmol/liter. The energy of activation is 6.7 kcal/mol. The differing "apparent" Km values obtained for lipase in undiluted serum (4 X 10(-5) mol/liter) and in albumin-based diluents (1 X 10(-5) mol/liter) indicate the presence of a competitive inhibitor in the serum matrix. We detected no lipase activity in urine. Results by the proposed method correlate well with those by a copper soap extraction method (r = 0.95), but values are significantly higher for pancreatitis patients' sera (slope 1.6). The linear dynamic range extends to 1000 U/liter. Hemolysis, lipemia, and hyperbilirubinemia do not interfere. The normal range is 40-60 U/liter. Lipase activity of pancreatitis patients generally exceed 1000 U/liter during the acute phase and 250 U/liter for as long as 10 days after it.     

Improved electrophoretic resolution of human serum alkaline phsophatase isoenzymes in agarose gel by Triton X-100.

Triton X-100 caused the liver isoenzymes of alkaline phosphatase in some serum samples to separate into two fractions on agarose gel electrophoresis. One of these fractions migrated at the rate of the original one, and one migrated more slowly. The latter fraction corresponded to a fast-moving component on electrophoresis in Cellogel and seems to be identical with a slowly moving isoenzyme in starch and polyacrylamide gel electrophoresis. The migration rates of the bone, placental, and intestinal isoenzymes were unaltered, but the fractions appeared sharper.     

Isoelectric focusing on cellulose acetate membrane: a separation procedure for alkaline phosphatase isoenzymes

An isoelectric focusing technique for the separation of alkaline phosphatase isoenzymes on cellulose acetate membrane is described. Optimal conditions for isoelectric focusing were established by changing ampholine concentration and focusing conditions. Bone, liver, intestinal, and placental isoenzymes can be resolved into vacious sub-bands in a pH range of 4.1 to 5.2. These sub-bands were correlated with the findings of electrophoretic isoenzyme separation. The whole procedure proves very simple to perform and comparatively time saving (4 h). This procedure may help clarify the problems of ALP isoenzyme differention when electrophoretic patterns are unresolved.     

Characteristics of the inhibition of serum alkaline phosphatase by theophylline.

1. The effect of various parameters on the inhibition of alkaline phosphatase activity by theophylline was studied. The influence of pH is great, being optimum for a value of 9.4. The temperature and the magnesium ion concentration have no to low effect. 2. The nature of the substrate and the nature of the buffer in which the enzymatic activity is measured have an effect:by measuring the activity of alkaline phosphatase in serum by a method with phenolphthaleinphosphate in Tris buffer, a negligible interference by therapeutic or toxic levels of theophylline is observed. 3. The inhibition by theophylline varies greatly with the origin of alkaline phosphatase: the liver isoenzyme is strongly inhibited, the intestinal one to a lesser degree and the placental isoenzyme almost not inhibited. 4. The inhibition of the liver and intestinal isoenzymes are uncompetitive and the inhibition constants were measured.     

Measurement of 25-hydroxyvitamin D3 in serum.

We describe a method for measuring 25-hydroxyvitamin D3 in serum. Extraction with dichloromethane/methanol (2/1 by vol), followed by chromatography on a column of Sephadex LH-20, resulted in an overall analytical recovery of 82% +/- 3.5% (SD). Diluted normal rat serum was used as binding protein because it contains a transport protein that has both a high affinity (Ka = 2 X 10(10) liter/mol) and a high capacity (3 X 10(-6) mol/liter) for 25-hydroxyvitamin D3. There is no advantage in using more complex binding proteins derived either from rachitic animals or from cytosol preparations. Concentrations of 25-hydroxyvitamin D3 (13.4 +/- 4 mug/liter) in the serum of apparently normal Belgian subjects are lower than those reported for North Americans, but resemble those reported for the United Kingdom.     

Biphasic effects of prostaglandin E2 on the human fat cell adenylate cyclase.

Adenylate cyclase of human fat cell ghosts shows a biphasic response towards prostaglandin E2 with inhibition occurring at nanomolar concentrations of the hormone and stimulation at concentrations beyond 10(-6) mol/liter. The expression of the inhibitory effect is critically dependent on GTP. Under the conditions employed (1 mmol/liter ATP, 5 mmol/liter Mg2+, 30 degrees C) the inhibitory component of prostaglandin E2 became apparent at GTP concentrations exceeding 10(-6) mol/liter. The prostaglandin E2-induced inhibition displayed characteristic features of prostaglandin action in intact fat cells with respect to the effective concentrations and degree of inhibition. It is concluded that prostaglandin E2 is capable of inducing antagonistic effects upon lipolysis via interaction with the membrane-bound adenylate cyclase.     

Characterization of alkaline phosphatase isoenzymes in serum by agar gel electrophoresis

A simple method of characterizing alkaline phosphatase isoenzymes in serum by agar gel electrophoresis is described. By using sera from cases of obstructive jaundice as markers in the electrophoresis reproducible β₂-phosphatase could be obtained. Combining the electrophoretic tests with heat stability tests at 56° bile, liver, bone, intestinal, and placental isoenzymes could be assessed for routine clinical purposes. Results obtained with 221 normal subjects are given and the clinical use of the method is briefly discussed.     

琼脂糖等电聚焦法测定碱性磷酸酶的同工酶

本文采用琼脂糖等电聚焦法测定了人体组织和健康人血清中的碱性硝酸酶同工酶，发现小肠中至少有一种碱性磷酸酶同工酶的亚型，胎盘中有四种，肝脏中有两种，实验证明，本法的重复性好，分辨率高，灵敏度高，最低检测限要达２５Ｕ／Ｌ，克服了国外报道中常有的区带扩散，分辨率低等弊病。     

Rat plasma clearance rate and organ distribution of beta-hexosaminidase isoenzymes from human serum.

beta-Hexosaminidase (EC 3.2.1.30) is markedly increased in human serum in liver disease, chronic alcoholism, and pregnancy. Knowledge of the clearance rate of plasma/serum beta-hexosaminidase is necessary to evaluate this increase. We studied the plasma clearance of beta-hexosaminidase isoenzymes (purified from human serum and placenta) after their infusion into rat circulation. A recently developed enzyme immunoassay method was used to measure the human beta-hexosaminidase isoenzymes; this method relies on both immunoreactivity and enzyme activity, so intact human isoenzymes were measured. In comparison with the placental isoenzymes (t1/2 less than 2 min), the serum forms showed a considerably slower clearance (t1/2 = 2-4 h). However, desialylation of the serum forms resulted in their rapid clearance (t1/2 less than 2 min). The organ localization of the enzyme eliminated from the circulation was investigated 24 h after infusion. Placental and native serum isoenzymes accumulated mainly in the liver and the spleen. Desialylated serum forms were almost exclusively localized to the liver.