The influence of changes in the phospholipid pattern of intact fibroblasts on the activities of four membrane-bound enzymes.

Human skin fibroblasts, grown to confluency in the presence of 32P for random labelling of the phospholipids, showed upon 24 h incubation in the presence of either 8 mM L-serine or 4 mM ethanolamine an increased content of phosphatidylserine (150% of control cells) or phosphatidylethanolamine (116% of control cells), respectively. Concomitantly the phosphatidylcholine correspondingly decreased. Upon cell harvesting and gentle enzyme preparation the base-treated cells demonstrated a significantly higher unstimulated, fluoride- and thyrotropin-stimulated activity of adenylate cyclase. The activities of total ATPase, ouabain-sensitive ATPase, 5'-nucleotidase and gamma-glutamyltransferase remained unaltered. When subjecting enzyme preparations from fibroblasts to ultrasonication the activity of adenylate cyclase decreased progressively with energy applied, whereas the activities of the other enzymes were unaltered ((K+ + Na+)-ATPase, 5'-nucleotidase) or even increased (Mg2+-ATPase, gamma-glutamyltransferase). The results have a bearing upon the regulatory function of the phospholipid microenvironment of membrane-bound enzymes.

Effects of the prostaglandin E2 analogue enprostil on the carbon tetrachloride-induced necrosis of liver cells in mice.

Female mice, eight weeks old, were injected with carbon tetrachloride (CCl4) (10 mg subcutaneously). Groups of mice (n = 10-30) were then injected with enprostil (E) 2, 20 or 50 micrograms/kg body weight (bw) intraperitoneally 15 min and two h after, or E 100 micrograms/kg bw two h after the CCl4 injection. The mice were killed after 24, 48 or 72 h. Plasma activity concentrations of alanine aminotransferase (ALAT) were determined in blood specimens from the iliac veins. The extent of liver cell necrosis in histological sections was recorded on a 100 mm Visual Analogue Scale (VAS) and measured using the electronic Mini Mop method. In the group given the highest single dose of E (100 micrograms/kg) a significant lowering of the CCl4-induced liver cell necrosis was found after 24 h. No significant differences were found after 48 and 72 h. In the other groups injected with lower doses of E after CCl4, no significant differences were found compared to groups injected with CCl4 alone.

Gamma-Glutamyl-3-carboxy-14-nitroanilide: the substrate of choice for routine determinations of gamma-glutamyl-transferase activity in serum?

Gamma-Glutamyl-3-carboxy-4-nitroanilide has been tested as donor substrate in the assay of gamma-glutamyltransferase activity in serum, glycylglycine being used as acceptor substrate. This donor substrate is highly solube even in neutral solutions, in contrast to the commonly used gamma-glutamyl-4-nitroanilide. The enzyme which apparently acts accordingly to a ping-pong bi bi kinetic mechanism, shows an absolute KM value for gamma-glutamyl-3-carboxy-4-nitroanilide of about 0.64 mmol/l, and for glycylglycine of about 13.4 mmol/l. The former KM value is significantly lower than that previously found for gamma-glutamyl-4-nitroanilide. The carboxyl derivative exhibits a marked competitive inhibitory effect on the gamma-glutamyltransferase. This effect is more pronounced than that of gamma-glutamyl-4-nitroanilide. The carboxyl derivative has somewhat higher absorbance in the range of wave length (400-420 nm) used to monitor the formation of the product. It is concluded that as donor substrate in the assay of gamma-glutamyltransferase activity of serum, the new derivative is not substantially superior to the gamma-glutamyl-4-nitroanilide conventionally used.

Dopamine-beta-hydroxylase activity in serum following acute myocardial infarction: an evaluation of this parameter for routine use as an index of sympathetic activity.

Dopamine-beta-hydroxylase activity was measured in sera from 114 normal males and from 11 patients on the 1st, 2nd, 3rd, 5th and 10th day following acute myocardial infarction. A significant elevation of dopamine-beta-hydroxylase levels (P less than 0.001) was found during the first two days after infarction when compared with the 10th-day values. Only a few activities were above the reference range. Temporary elevations of glucose and glycerol levels were also found. Assay of serum dopamine-beta-hydroxylase may be a useful parameter of sympathetic activity in longitudinal studies in which each individual is used as his own reference.

Effects of Ca, Mg, and EDTA on creatine kinase activity in cerebrospinal fluid.

For one to obtain a precise estimate of creatine kinase (CK) activity in cerebrospinal fluid, the sample fraction is increased by about 10-fold over that used for serum. This increases the concentration of interfering substances, Ca being especially important. Therefore, the relationship between Ca, Mg, and EDTA was examined. Enzyme activity was maximal with 15 mmol of Mg per liter in the presence of 3 mmol of EDTA per liter, otherwise according to the (Scandinavian) recommended conditions for determination of CK activity in serum. These modifications increased the activity of CK by 35% for CK-MM and by 60% for CK-BB. Counteraction of Ca-induced inhibition was the main reason to this increase. We describe a practical and sensitive method for determining CK in cerebrospinal fluid.

Secondary metabolic changes in fibroblasts from six patients with hereditary lactic acidosis.

Studies on fibroblasts from patients with lactic acidosis of different causes showed secondary metabolic changes in pathways of glucose metabolism. These secondary changes may be important clues to the diagnosis of the many different types of hereditary lactic acidosis.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

Heparin interference in the measurement of gamma-glutamyltransferase activity with the Scandinavian and the IFCC recommended method.

Heparin in heparinized plasma, and when added to serum, is shown to interfere markedly in the assay of gamma-glutamyltransferase when using the method recommended by both the Scandinavian Society for Clinical Chemistry and Clinical Physiology, and the Expert Panel on Enzymes of IFCC. The effect is also revealed as an apparent sample blank reaction (donor substrate omitted). It decreases as the time of first reading is increased, and it can be reduced or nearly abolished by addition of extra NaCl (increasing the ionic strength). Evidence is presented suggesting that this photometric interference is caused by turbidity due to complex formation between heparin and various plasma proteins. Fibrinogen appears to be one of the main proteins involved. It is concluded that in general care should be taken when using heparinized plasma in enzyme assays, and that heparin should only be used when specific testing has ruled out photometric interference.

[Troponins and other biochemical cardiac markers--time for a change]. / Troponiner og andre nye biokjemiske hjertemarkører--tid for et skifte.

BACKGROUND: During the last ten years new, more sensitive and specific cardiac markers in blood for detection of acute myocardial injuries have been characterised and clinically evaluated. Of practical importance is also the fact that rapid and robust methods and equipments suitable for emergency service have been developed. MATERIAL AND METHODS: Based on literature study and on own experiences we give a survey of the most promising cardiac markers with emphasis on the troponin T and troponin I. RESULTS: Methodological and pathobiochemical aspects are discussed. The diagnostic characteristics and advantages of the new markers are in focus, especially with respect to early detection of acute myocardial injuries. Finally, we discuss their potential use for stratification of patients with acute coronary syndromes with respect to optimal treatment, resource-saving monitoring, and more precise prognostication. INTERPRETATION: It is recommended that one of the troponins, either I or T, should be included in the diagnostic regime for detecting acute coronary syndromes in Norwegian hospitals. CKMB, determined as mass and not as activity, ought to be retained, at least until more clinical experience with the troponins have been gained by the hospital. Myoglobin may only be of interest if early exclusion of myocardial injuries has practical consequences. In this connection the transaminases and LDH are no longer of any interest.

Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients.

Beta-glucuronidase activity in the bile may be of importance in the etiology of pigment gallstones. This enzyme is of hepatic or bacterial origin. We have described a method to measure the activity of bacterial beta-glucuronidase in human bile, using 4-nitrophenyl-beta-D-glucopyranosiduronic acid as substrate. The method was used to measure the beta-glucuronidase activity in the bile from 51 patients with gallstone disease. This activity was related to the presence of beta-glucuronidase-producing bacteria in the bile. Escherichia coli, Bacteroides species, and Clostridium perfringens were the only species found to produce beta-glucuronidase. Patients with beta-glucuronidase-producing bacteria had on an average significantly higher enzyme activity in the bile than patients without such bacteria (p less than 0.01). The limitations of using artificial substrates in this type of studies are discussed.

Co-variation of alanine aminotransferase levels with relative weight in blood donors.

The association between alanine aminotransferase (ALAT) and body mass, sex and age was examined in 6036 consecutively tested blood donors. ALAT, age and body mass were higher in male donors than in female donros. The non-normal ALAT distribution curve normalized after 1n transformation, which made statistical analysis of the data more feasible. Multiple regression analysis demonstrated that ALAT was influenced by sex and relative weight, in that order, and very poorly by age. It is concluded that obesity is a major cause of raised ALAT in this predominantly healthy donor population.

Cytoplasmic creatine kinase isoenzymes quantitated in tissue specimens obtained at surgery.

Because previous reports have given inconsistent results, we re-examined the catalytic concentrations of cytoplasmic creatine kinase (CK) and of CK isoenzymes in 38 biopsies obtained from 19 different tissues. After homogenization and centrifugation many tissues showed high CK catalytic concentrations; 11 of them contained activity exceeding 50 U/g wet weight (Scandinavian recommended method). The highest specific activities were found in skeletal muscle (2400 U/g), brain (530 U/g), and myocardium (460 U/g). The separate isoenzyme activities were estimated by electrophoretic, anion-exchange chromatographic, immunoinhibiting, and radioimmunological methods. CK-BB was present in all tissues and, in fact, was the only cytoplasmic CK isoenzyme in 16 of the 19 tissues examined. CK-MM was the major isoenzyme of skeletal muscle and myocardium and was in addition observed in placenta, in trace amounts. CK-MB was present in high catalytic concentrations in myocardium (20% of total CK) and in low catalytic concentrations in skeletal muscle (1.1% of total CK).

gamma-Glutamyltransferase in human serum: an analysis of kinetic models.

The purpose of the study was to elucidate details of the kinetic model for gamma-glutamyltransferase when assayed with gamma-glutamyl-3-carboxy-4-nitroanilide and glycylglycine as substrates. Data from several sets of initial velocity measurements were fitted by nonlinear regression to a set of different kinetic models. gamma-Glutamyltransferase acts by a "ping-pong bi-bi" mechanism. A model encompassing transfer and autotransfer, competitive inhibition by the acceptor substrate, and no inhibition by the donor substrate gives the best fit to the experimental data. Effects of spectrophotometric nonlinearity may simulate noncompetitive inhibition by the donor substrate. The nonlinearity is dependent on the absorption of the incubation mixture and therefore is related to the concentration of the donor substrate and the wavelength (405-412 nm) used to monitor the reaction. With decreasing pH the autotransfer fraction decreases and the binding of the donor substrate to the acceptor site increases, simulating an increased competitive inhibition by the donor substrate. These results are of importance when elaborating optimum assay conditions for gamma-glutamyltransferase in serum.

Methodological considerations on chromogenic peptide substrate assays and application on automated analyzers.

Chromogenic peptide substrates (CPS) are small peptides with the chromophore p-nitroaniline (pNA) coupled to the terminal carboxyl group in an amide linkage. These substrates are the basis for a new generation of analyses of serine proteases. A short review of established manual methods for determination of key components of the plasma proteolytic enzyme systems as well as other serine proteases is given here. The enzymatic cleavage of the peptide-pNA amide linkage in CPS results in release of pNA. This reaction can be monitored at 405 nm in a spectrophotometer and can easily be automated using equipment found in most clinical chemistry laboratories. In our laboratory we have adapted such assays to the LKB 8600 automated reaction rate analyzer and the Cobas Bio centrifugal analysis system. Using these machines, we have established methods for the determination of proenzymes, protease-alpha 2M complexes and functional inhibitor capacities of several serine protease systems. With these automated assays a rapid system for close monitoring of the coagulation, fibrinolytic and plasma kallikrein-kinin systems in various disease states has become available.

The vitamin D status of man at 70 degrees north.

The overall vitamin D status of man at 70 degrees north has been evaluated by measuring the total 25-hydroxy-vitamin D (25-OHD) in serum samples from seventeen healthy adults from September 1977 to September 1978. The lowest 25-OHD-concentration was found in March (mean 51.5 nmol/l, SD = 19.5). This was significantly lower than in July and September 1977 and 1978 (P less than or equal to 0.0006). Of particular interest was the highly significant rise in the 25-OHD-concentration from March to July, most probably reflecting sun-induced endogenous synthesis in the skin. Despite the sun being below the horizon for two of the winter months, the 25-OHD-concentration remained at a constant and fairly high level (50-55 nmol/l).