Some kinetic properties of gamma-glutamyltransferase from rabbit liver.

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.

Discriminant biochemical markers for evaluating the nutritional status of elderly patients in long-term care.

To determine the most discriminant serum markers of protein-energy status in elderly patients, we performed a discriminant analysis of 85 subjects grouped according to triceps skinfold and midarm circumference values as compared with reference percentiles. Results indicated that neither the classic serum indices of nutritional assessment nor retinol-binding protein can predict undernutrition. However, creatinine, urea, carotene, complement C3, and prealbumin included in a function enabled high discrimination between groups: 68% of subjects in 0-5th percentile for triceps skinfold and 75% of subjects in 0-5th percentile for midarm circumference are correctly predicted. Lower serum concentration was found in the lower anthropometric percentiles except for serum carotene, which showed an inverse relation not explained by diet. We found that nutritional alterations exist in hospitalized elderly patients. We emphasize the importance of considering several biochemical markers for detection of mal-nutrition and the pertinency of further exploration of serum carotene profiles in undernourished elderly patients.

Milk- and soy-protein ingestion: acute effect on serum uric acid concentration.

Acute effect of the ingestion of 80 g each of casein, lactalbumin, and soybean isolate on serum and urinary uric acid concentrations was investigated in 10 healthy subjects. Serum and urinary uric acid concentrations were measured before and after the ingestion of proteins. Serum uric acid decreased significantly 3 h after ingestion of lactalbumin and casein but increased after soybean consumption. Urate clearance was significantly increased after ingestion of each of the three proteins. Multivariate analysis of urate clearance during lactalbumin and casein loads showed that independent correlation was obtained for serum alanine and urea concentration. These results demonstrate that, in addition to their known uricosuric effect, milk proteins acutely decrease serum uric acid concentration. Analysis of the effects of lactalbumin and casein on urinary uric acid elimination suggests that the uricosuric effect of proteins is a multifactorial phenomenon.

Determinants of total serum carotene concentrations in institutionalized elderly.

The current analysis was designed to examine further the association between total serum carotenoids and a series of preselected variables (dietary intakes, anthropometric parameters, serum cholesterol, vitamin A, transport proteins, and others) using 85 elderly patients. Simple correlation between serum carotene and carotene intake, as estimated by a three-day weighed method, was 0.19 (P = 0.08), which was substantially increased (r = 0.29, P = 0.01) after adjustments for a selected set of variables. Standardized coefficients of multivariate regression indicated that the most important predictors of serum carotene were serum cholesterol (beta = 0.38), total serum proteins (beta = -0.35), and sex (beta = 0.34), followed by carotene intake (beta = 0.28) and midarm circumference (beta = 0.20). These variables accounted for 46% of the variance. Results suggest that serum carotene may be related to protein-energy status in hospitalized elderly, but further investigation should be directed to serum carotene in undernourished elderly. Nevertheless, total serum proteins could be an important factor in any attempt to correlate carotene intake and blood concentration.

Enzymatic activity patterns in probability population samples. Serum lactate dehydrogenase.

Sex- and age-specific serum lactate dehydrogenase values provided by a natural population of 2,378 individuals selected by a multistage area cluster probability sampling scheme are given. The activity of this enzyme has definite sex and age associations over the age range studied (larger than or equal to 10 years). In both sexes, it is characterized by elevations in preadolescents, by a sharp decrease in adolescent groups, and by a gradual increase in adult and senescent groups, which, however, does not bring it back to earlier levels. Males have higher mean serum lactate dehydrogenase activities than females in most age groups until about 50 years of age. Unusually high activities have been found in this study, compared with those found in two other widely separated areas in North America using the same assay procedures.

An enzyme-linked immunosorbent assay for the detection of complement components on red blood cells.

A new technic using the principle of enzyme-linked immunoassay (ELISA) has been developed for the detection of complement components on red blood cells sensitized in vivo or in vitro. Using a double-antibody technic, anticomplement antisera (anti-C3c or anti-C3c/C3d) produced in rabbits was incubated with the red blood cells, followed by incubation with antirabbit alkaline phosphatase conjugated antiglobulin. The amount of the enzyme fixed was measured spectrophotometrically by the enzymatic hydrolysis of the substrate PNPP. A calibration curve was made from red blood cells on which complement was deposited by the method of Fruitstone . The technic showed a greater sensitivity than the standard antiglobulin tests and allowed simultaneous qualitative and semiquantitative estimates. The technic can be performed in any laboratory equipped with the standard equipment found in a blood bank, including a spectrophotometer. The authors made a modification of Alsever 's solution, which allowed the safe and stable preservation of complement coated red blood cells for 15 days. Significant positive results were obtained clinically using this technic, while negative or weakly positive reactions were obtained by the conventional antiglobulin tests.

Comparative study of SCH 434 and CTM-D in the treatment of seasonal allergic rhinitis.

The efficacy and safety of an extended-release combination of loratadine plus pseudoephedrine sulfate (SCH 434) was compared with that of a tablet containing chlorpheniramine maleate plus pseudoephedrine sulfate (CTM-D) in 131 patients with symptomatic seasonal allergic rhinitis. Patients were randomly assigned to receive either SCH 434 (loratadine 5 mg and pseudoephedrine sulfate 120 mg) or CTM-D (chlorpheniramine maleate 12 mg and pseudoephedrine sulfate 120 mg) twice daily for 2 weeks. Evaluations were made after 3, 7, and 14 days of treatment. Demographics (age, race, sex, and duration of seasonal allergic rhinitis) and baseline total symptom scores were comparable between groups. Both combination products were effective in relieving the symptoms of allergic rhinitis. Improvement in total symptom scores was 54% on day 3 and 65% on day 14 in the SCH 434 group versus 57% on day 3 and 64% on day 14 in the CTM-D group. Individual symptom scores (nasal discharge, stuffiness, nasal itching, sneezing, and ocular symptoms) responded similarly. A smaller proportion of patients in the SCH 434 group reported side effects, especially dry mouth (7% vs 19%, P = 0.07), fatigue (6% vs 25%, P < 0.01), and sedation (7% vs 22%, P < 0.03). In conclusion, the combination of loratadine plus pseudoephedrine sulfate was equally as effective as a classic antihistamine (chlorpheniramine maleate) plus pseudoephedrine sulfate but had a lower incidence of side effects.

Onset of action of loratadine and placebo and other efficacy variables in patients with seasonal allergic rhinitis.

In a double-blind study, 185 patients with seasonal allergic rhinitis were randomly assigned to receive 10 mg of loratadine or placebo once daily for three days. On day 1 of treatment, the onset of relief of symptoms within 30 minutes of drug administration was reported by 13% of the loratadine-treated patients and by 4% of the placebo patients (P less than 0.05). At two hours after drug administration, 65% of the loratadine-treated patients and 48% of the placebo patients reported symptom relief. On day 3, the loratadine-treated patients reported a significantly greater relief of symptoms, and according to both physician and patient evaluations, the treatment response was significantly superior in the loratadine-treated than in the placebo patients. The incidence of sedation was 2% in the loratadine group and 1% in the placebo group.

Automated quantification of bone and liver alkaline phosphatase isoenzymes of human serum.

Coupling two Technicon AAII samplers synchronised at 50 per hour with a 2 : 1 sample to wash ratio, sera are denatured and collected automatically. The incubation is done in continuous flow by passage through a U device made of large metallic needles soaked in a water bath at 60 +/- 0.1 degree C. This allows a very quick temperature equilibration and a very reproducible incubation time of 35 sec. Initial and residual activities of alkaline phosphatase (ALP: EC 3.1.3.1) are measured on a Rotochem II (Aminco) with the procedure recommended by the Société Française de Biologie Clinique (SFBC). For a mixture of bone and liver ALP, the initial rate constant of heat denaturation Kapp = (A X Kb) + (B X Kl), where A and B are the fractions of each isoenzyme in the mixture, and Kb and Kl the rate constants for bone (b) and liver (l) experimentally determined as 1.8 min-1 and 0.45 min-1 respectively. An equation was derived which converts the percent residual activity to a percentage of bone and liver isoenzyme: % bone ALP = 183--2.38 X % residual activity. This automated method was applied to 2700 people of both sexes from 4 to 100 years old.

Measurement of plasma gamma-glutamyltransferase in clinical chemistry: kinetic basis and standardisation propositions.

The conditions for the measurement of gamma-glutamyltransferase (EC 2.3.2.2) activity of human plasma were studied at 30 degrees C using the kinetic technique of Szasz [3]. The optimum pH in Tris (hydroxymethylaminomethane) buffer and 2-amino-2-methyl-1.3-propanediol at a concentration of 100 mmol/1 are 8.0 and 8.1. The kinetic characteristics of human plasma gamma-glutamyltransferase were studied using gamma-L-glutamyl p-nitroanilide and its carboxyl derivative as donor substrates. Glycylglycine was chosen as the best acceptor of the gamma-glutamyl radical. Under these conditions, we have shown that the inhibition by the donor substrate was more important at acidic pH and vanished at alkaline pH. This inhibition was obviously related to the presence of the acceptor, but did not vary with glycylglycine concentration. At pH 8.0, by increasing the acceptor concentration some competition occurs at the donor binding site, as reported by other authors in relation to the known ping-pong bi-bi enzyme mechanism for the gamma-glutamyltransferase. Some displacement of donor substrate by increasing amounts of acceptor substrate could be observed at all pH values we studied. However, the influence of glycylglycine on the enzyme's maximum velocity and affinity for the donor substrate was also pH dependent. Studying the kinetic characteristics of the enzyme as a function of the pH suggests that the enzyme works with more than one active site at pH 7.5-8.0. Based on the results of this study, we propose measurement conditions for gamma-glutamyltransferase at 30 degrees C in routine clinical chemistry without preference in the choice of substrate.

Presence of alkaline phosphatase and gamma-glutamyl transpeptidase on the parietal layer of Bowman's capsule.

Generally, the parietal layer of Bowman's capsule in the mammal kidney consists of a squamous epithelium resting upon a basement lamina. However, tall cylindrical cells resembling those of the proximal tubules were observed on the outer wall of Bowman's capsule in the mouse and rat kidneys. These cells were provided with an apical brush border and were positive for alkaline phosphatase and gamma-glutamyl transpeptidase suggesting phosphate and amino acid transport at the capsular level.

Simultaneous visualization of alkaline phosphatase and gamma-glutamyl transpeptidase in kidney sections.

An histochemical method is presented to simultaneously localize, for the first time, alkaline phosphatase (ALP) and gamma-glutamyltranspeptidase (gamma-GT) in the kidney. The reaction product of ALP activity appears as a dark brown precipitate of lead sulfide, while a bright red copper chelate of an azo dye (Fast blue BBN salt) final product indicates sites of gamma-GT activity. The amalgamation of Mayahara's (ALP) and Rutenberg's (gamma-GT) techniques resulted in the demonstration of various categories of kidney tubules, according to the staining reaction of the cell brush borders: Black tubules where ALP predominates; Intermediate tubules showing a mixture of brown and red precipitates; Red tubules indicating a prevalence of gamma-GT activity; Negative tubules. A possible relation might exist between the staining characteristics observed and the different proximal tubule segments, thus allowing their distinction. In addition, this technique has the advantage to permit the concomitant study of ALP and gamma-GT distribution on the same tissue section instead of serial sections, so reducing the number of manipulations and observations as well as the amount of tissue required.

Heterogeneous distribution of alkaline phosphatase and gamma-glutamyl transpeptidase in the mouse nephron.

In the mouse nephron, ALP and gamma-GT were found to be heterogeneously distributed along the proximal tubule. For both enzymes, 4 large categories of tubules could be recognized on the basis of the enzymatic activity: intense; intermediate; weak; negative. The localization of ALP and gamma-GT was in opposite gradient along the proximal tubule and it apparently corresponded to the 3 sequential segments S1, S2, and S3. In fact, S1 could be identified with certainty because this first portion was often seen attached to the renal corpuscle. This segment displayed a very intense ALP activity (category 1), but a weak one for gamma-GT (category 3). Intermediate tubules for ALP and gamma-GT activities (category 2) seemingly were parts of S2. Those tubules where ALP activity was weak (category 3) while that of gamma-GT was intense (category 1) probably belonged to S3. As a result, it becomes possible to clearly distinguish the segments S1, S2, and S3, not only on a structural and biochemical basis but as well by the localization of brush border enzymes. Distal tubules showed no enzyme activity (category 4). In other respects, the presence of ALP and gamma-GT on the parietal layer of Bowman's capsule strongly suggests that these tall cylindrical cells are morphologically and enzymatically identical to those of the S1 segment, and that they might have similar functional roles.

Identification of proximal tubule segments in the mouse nephron by simultaneous visualization of alkaline phosphatase and gamma-glutamyl transpeptidase.

ALP and gamma-GT are 2 brush border enzymes that can be individually demonstrated on adjacent sections by the histochemical methods of Mayahara (ALP) and Rutenberg (gamma-GT). On the basis of each enzyme activity, it was possible to recognize different categories of tubules in the mouse nephron. In fact, both enzymes were heterogeneously distributed along the proximal tubule, but in opposite gradients. The various staining intensities probably corresponded to proximal segmentation, but were sometimes difficult to evaluate. A technique was perfected to localize both enzymes in the same tissue section. Since each enzyme produced a distinct type of colored precipitates (ALP: black, gamma-GT: red), 4 categories of tubules could be identified, according to staining characteristics: 1. black tubules where ALP activity was predominant, corresponded to S1 segments, 2. black and red tubules where the 2 activities were about equivalent, were considered as parts of S2, 3. red ones where gamma-GT activity was high, were identified as portions of S3, 4. negative tubules where no activity was apparent, represented distal and straight collecting tubules. In addition to economize time and tissue, this simple technique permits to easily estimate variations in enzyme activities that probably correspond to structural and functional differences in the segments of the proximal tubule.

Patterns of calcium intake among French-Canadians living in Montreal.

In order to determine calcium intake, 1,450 French-Canadians of both sexes, all age groups and various socio-economic status, were recruited in Montreal and Laval using a stratified randomized selection. One thousand and sixty-five (73%) subjects completed food records: 82.4% for a period of seven days, the others for periods varying from one to six days, providing 6,590 days of food records. Subjects ranged in age from one month to 95 years with a mean age of 35.1 years. No seasonal variation in calcium intake was observed. On average, men consumed more calcium than women. This difference was significant for age groups 13 to 15, 19 to 24 and 25 to 49 (p < 0.05). Womens' diets, however, had a higher calcium density than those of men, except among 10-15-year-olds. Mean calcium intake exceeded the RNI for the Canadian population in all age groups except females aged 10 to 15 years, and both men and women aged 50 years and over. The results of this study suggest that those in the study region most at risk for insufficient calcium intake are adolescent females and older men and women.