Johan H. Strømme

Practical Points Regarding Routine Determination of γ -Glutamyl Transferase (γ-GT) in Serum with a Kinetic Method at 37°C

Kinetic data at 37°C on γ-GT activity in serum with γ-glutamyl-4-nitroanilide and glycylglycine as substrates are reported. The apparent Km for the two substrates were 0.95 and 9.5 mmol/l, respectively. Without glycylglycine the enzymatic formation of 4-nitroaniline was only 5 per cent of that with both substrates. 4-nitroaniline showed a slight inhibitory effect on the γ-GT activity. On the basis of the results an optimized modification at 37°C sof the traditional method of Szasz (17) is described. The method is well suited for automation, e.g. in LKB 8600 Reaction Rate Analyzer. Somewhat higher reference values for men (9-35 U/l) than women (6-25 U/l) were found. Data on purity, solubility, and stability of the reagents, and on the precision of the method are given.

Reference intervals for eight enzymes in blood of adult females and males measured in accordance with the International Federation of Clinical Chemistry reference system at 37°C: part of the Nordic Reference Interval Project

As part of the Nordic Reference Interval Project we present reference intervals for alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), lactate dehydrogenase (LD), alkaline phosphatase (ALP), gamma‐glutamyltransferase (GT), amylase (AMY) and pancreatic type of AMY in blood of adult males and females. A total of 3036 reference persons, all of whom considered themselves to be in good health, were recruited by 102 Nordic clinical biochemical laboratories. Exclusions were undertaken on the basis of predefined biochemical and clinical criteria. Enzyme activities in serum and plasma were measured in the different laboratories using various commercially available routine measurement systems at 37°C. Only results obtained with the International Federation of Clinical Chemistry (IFCC) compatible measuring systems were selected for estimation of the enzyme reference intervals. The final number of results on each enzyme varied from 459 (LD) to 2300 (ALT). The 2.5 and 97.5 percentile reference limits were calculated by a non‐parametric method in accordance with the IFCC recommendations, using the Refval 4.0 data program. Statistical partitioning testing was undertaken to decide whether the reference intervals ought to be partitioned according to gender and/or age. For most of the enzymes, but not for all, the upper reference limits were found to be higher than those that have been in general use until now.

Fatal lactic acidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase.

Extract: An infant suffering from metabolic acidosis attributable to hyperlactatemia (6.1 mmol/liter) accompanied by hyperalaninemia (1 mmol/liter) and hyperserinemia (0.6 mmol/liter) is described. The urinary excretion of lactate and pyruvate was greatly elevated; the lactate to pyruvate ratio was normal. The urine showed low levels of citrate, isocitrate, and cis-aconitate, and low or normal levels of α-oxoglutarale, succinate, malate, and methylmalonate. Aspartate was slightly elevated in serum and urine, indicating a corresponding increase of its α-ketoacid oxaloacetate. These patterns of organic acids and amino acids suggested an in vivo defect in the oxidation of pyruvate. Fibroblasts cultured from skin biopsy from the patient metabolized radioactive pyruvate (final concentration 0.04–2 mmol/liter) to CO2 at rates from 5 to 17% of that of fibroblasts from normal control subjects. Enzyme studies with fibroblast sonicates revealed a severe deficiency of the pyruvate dehydrogenase complex (about 8% of normal), and this error was localized to the first unit of the complex, i.e., the pyruvate dehydrogenase (about 4% of normal). Fibroblasts from both parents metabolized pyruvate to CO2 at a slightly reduced rate, suggesting parental heterozygosity.Speculation: An exact classification, which may have both prognostic and therapeutic implications, of patients suffering from the heterogenous group of diseases, congenital lactic acidosis, will be possible only when the various underlying defects have been demonstrated. Since we are probably dealing with errors localized close to the center of the energy metabolism, sophisticated in vivo and in vitro studies will be required to reach this goal. However, we expect each defect to give rise to secondary derangements in the patterns of organic acids and amino acids in serum and urine, which are more or less typical for each defect. Once these patterns are known, a precise biochemical diagnosis may be reached by comparatively simple examinations.

Does chronic alcohol consumption really induce hepatic microsomal gamma-glutamyltransferase activity?

Summary Ethanol feeding of rats for 6–7 weeks was accompanied by elevated plasma and liver γ-glutamyltransferase (GT) activities compared to enzyme activities in control animals fed carbohydrates isocalorically. Similar differences in hepatic GT activity were present after feeding these diets for 1 or 4 weeks. In all instances the difference between ethanol-fed and control rats was due to a reduction of GT activity in the animals fed the control diet, while ethanol-fed rats had GT activities close to pre-experimental values. Since neither treatment influenced the subcellular distribution of hepatic GT activity, no evidence for induction of microsomal GT activity from a pretreatment level due to ethanol was present.

Familial Hypomagnesemia—A Follow-Up Examination of Three Patients After 9 to 12 Years of Treatment

Summary: Three children with familial hypomagnesemia from infancy were treated perorally with magnesium for 9 to 12 years. Their somatic and intellectual development have since been normal. Without therapy, the serum magnesium fell from subnormal (about 0.5 mmoles/liter) to very low values (0.2 to 0.3 mmoles/liter) within 1 to 4 wk. We observed a secondary fall in serum calcium and potassium and an increase in sodium and phosphate although serum concentrations of PTH, calcitonin, and 25-OH-vitamin D in the blood remained normal. Balance studies confirmed the presence of a defect in the intestinal absorption of magnesium and excluded a defective renal tubular transport system. The subjects continued to require daily magnesium supplements to avoid serious symptoms. Optimal dosage was found to be in the range 0.5 to 0.75 mmoles/kg·day; doses above this caused diarrhoea and a fall in the serum and urine levels of magnesium. Pathophysiologic mechanisms involved in the electrolyte changes that occurred secondarily to the hypomagnesemia are discussed.Speculation: The pathophysiologic basis for the impaired intestinal absorption of magnesium in this condition is unknown. In fact, the mechanism by which magnesium is normally transported across membranes is also largely unknown. A facilitated transport mechanism seems to be involved. It appears reasonable to believe that errors in enzymes, or perhaps more likely, in specific magnesiumbinding proteins that are involved in the membrane transport form the molecular basis for familial hypomagnesemia.

SCE Nordic α-amylase method selection and calibration study. A report by the Committee of Enzymes of the Scandinavian Society for Clinical Chemistry (SCE)

Seventy-six Nordic routine laboratories participated in a joint SCE-NORDKEM study comprising evaluation, selection, and temporary calibration of amylase methods. Human control materials with known fractions of salivary and pancreatic amylase were determined by seven routine amylase assays based on substrates with glucosyl (G) chain lengths G4, G5, G5-6, G7, G9, amylopectin and blue starch polymer (Phadebas). The data were plotted before and after calibration of each method using a human pancreatic calibrator with an assigned value of 390 U/l (37 degrees C, Phadebas). the study led to three conclusions: The analytical overestimation of salivary to pancreatic amylase ratio (S:P) increased with decreasing number of glucosyl units in the substrates. Relative to the S:P value of blue starch polymer (set at 1.00), for example, tetraose mean S:P value was 1.55. The hydrolysis rates relative to that with blue starch polymer decreased with the number of glucosyl units in the substrates. The precalibration values of all methods spread over an approximately six-fold range. Post-calibration values of all methods, except tetraose, showed an acceptable inter-laboratory comparability. The CV values for low, medium, and high controls were about 5.5, and 6% respectively. As a temporary solution to the current problem of diverse amylase assays, the SCE suggests calibration of the methods considered acceptable in this study. The long-term effects will be evaluated in a follow-up study within a year.

Dopamine-β-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.

γ-Glutamyl-3-carboxy-4-nitroanilide: the substrate of choice for routine determinations of 7-glutamyltransferase activity in serum?

Abstract γ-Glutamyl-3-carboxy-4-nitroanilide has been tested as donor substrate in the assay of γ-glutamyltransferase activity in serum, glycylglycine being used as acceptor substrate. This donor substrate is highly soluble even in neutral solutions, in contrast to the commonly used γ-glutamyl-4-nitroanilide. The enzyme which apparently acts according to a ping-pong bi bi kinetic mechanism, shows an absolute km value for γ-glutamyl-3-carboxy-4-nitroanilide of about 0.64 mmol/1, and for glycylglycine of about 13.4 mmol/1. The former KM value is significantly lower than that previously found for γ-glutamyl-4-nitroanilide. The carboxyl derivative exhibits a marked competitive inhibitory effect on the γ-glutamyltransferase. This effect is more pronounced than that of γ-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 γ-glutamyltransferase activity of serum, the new derivative is not substantially superior to the γ-glutamyl-4-nitroanilide conventionally used.

Ethanol elimination-rates determined by breath analysis as a marker of recent excessive ethanol consumption

The rate of ethanol elimination was studied in two groups of men by means of an Alcotest 7010 breath analyser. The experimental group consisted of 15 skid-row alcoholics undergoing detoxification. Their median daily ethanol consumption was 211 (range 26-476) g pure ethanol during the last year. The control group was made up of 12 age-matched healthy social drinkers consuming 9 (range 4-23) g day-1 pure ethanol during the last year. The median ethanol elimination-rate in the elimination phase was 0.25 (range 0.13-0.31) g 1-1 h-1 during the detoxification period in the experimental group. This value was approximately 70% higher than in the control group (0.14(0.12-0.17) g 1-1 h-1). Some correlation was found between reported ethanol intake, and the calculated ethanol elimination-rate, as well as gamma glutamyl transferase (GGT), alanine amino transferase (ALAT), aspartate amino transferase (ASAT), glutamate dehydrogenase (GLDH), mean corpuscular volume (MCV) and HDL-cholesterol. Of these measures, ethanol elimination-rate showed highest sensitivity and efficiency for detection of ethanol consumption above the limit of 50 g per day.

Heparin interference in the measurement of γ-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.