The combined pituitary function test in children: an evaluation of the clinical usefulness of TRH and LHRH stimulation tests through a retrospective analysis of one hundred and twenty six cases.

OBJECTIVE: The combined pituitary function test is routinely used in the endocrine investigation of short children. The TRH and luteinising hormone-releasing hormone (LHRH) response tests have been shown to be of minimal value in adults. We have evaluated the clinical utility of these tests in the context of combined pituitary function testing in children. DESIGN: A retrospective analysis of basal hormone measurements and pituitary stimulation tests in relation to clinical assessment of pituitary function. PATIENTS: One hundred and twenty-six children, 82 boys and 44 girls, aged 2-17 years, who had undergone pituitary function testing were studied. RESULTS: The TSH response to TRH stimulation correlated directly with basal plasma TSH but not basal plasma total T4. In patients with an impaired response to stimulation, basal TSH concentrations were <2.0 mIU/l and significantly lower than in patients with a normal response (P < 0.0001). An impaired response to TRH stimulation had a positive predictive value of 0.43 and a negative predictive value of 0.90 for the diagnosis of hypopituitarism. A basal TSH concentration of <2.0 mIU/l had a positive predictive value of 0.22 and a negative predictive value of 0.92. A low basal T4 (normal range 60-140 nmol/l) in combination with an inappropriately low or normal basal TSH was always associated with a diagnosis of hypopituitarism. The responses of plasma LH and FSH to LHRH stimulation correlated directly with basal plasma LH and FSH concentrations. Basal gonadotrophin concentrations, basal sex hormone concentrations or response to LHRH stimulation could not distinguish patients with constitutional delay of growth and puberty from those with hypopituitarism. There was no apparent relationship between either basal gonadotrophin concentrations or response to LHRH stimulation and clinical assessment of pituitary function. In patients > or =13 years with constitutional delay of growth and puberty the median and interquartile ranges of basal LH and FSH were 1.4 IU/l (0.7-3.6) and 2.6 IU/l (2.2-5.2) respectively. The three hypopituitary patients in this study with chronological age > or =13 years had undetectable concentrations of both gonadotrophins. The response of LH and FSH to LHRH stimulation was significantly lower in patients > or =13 years with clinical hypopituitarism than in those with intact pituitary function (P <0.02). CONCLUSION: TRH and LHRH tests in children with short stature appear to have little value over and above the baseline hormone measurements. An abnormal response to hormone stimulation is not diagnostic of hypothalamic-pituitary disease. We have demonstrated that neither TRH nor LHRH stimulation tests should be routinely used in the investigation of children with short stature.

Negative association between erythrocyte reduced glutathione concentration and diabetic complications.

1. Multiple logistic regression analysis of biochemical and clinical variables in diabetic patients was performed to identify those associated with the presence of diabetic complications (retinopathy, neuropathy and nephropathy). 2. The presence of diabetic complications correlated positively with duration of diabetes and patients age and negatively with the concentration of reduced glutathione in erythrocytes. Individually, retinopathy, neuropathy and nephropathy correlated with duration of diabetes, but retinopathy also correlated positively with haemoglobin A1C in diabetic patients. In insulin-dependent patients, the concentration of methylglyoxal was also in the logistic model for retinopathy and diabetic complications, but the logistic regression coefficient was not significant. 3. Multiple linear regression analysis indicated that erythrocyte reduced glutathione concentration correlated negatively with D-lactate concentration and positively with duration of diabetes in insulin-dependent patients and correlated negatively with glucose concentration in non-insulin-dependent diabetic patients. 4. In non-diabetic subjects, erythrocyte glyoxalase I activity correlated positively with methylglyoxal concentration. There was no similar correlation in diabetic patients. In insulin-dependent patients, methylglyoxal concentration correlated positively with duration of diabetes. 5. Glyoxal and methylglyoxal are detoxified by the glyoxalase system with reduced glutathione as co-factor. The concentration of reduced glutathione may be decreased by oxidative stress and by decreased in situ glutathione reductase activity in diabetes mellitus. A reduced concentration of reduced glutathione may predispose diabetic patients to oxidative damage and to alpha-oxoaldehydemediated glycation by decreasing the in situ glyoxalase I activity. Recent studies of vascular endothelial cells in vitro have suggested that alpha-oxoaldehydes detoxified by glyoxalase I are the major precursors of advanced glycation end products implicated in the development of diabetic complications. The role of these factors in the development of diabetic complications and the prospective prevention of diabetic complications by supplementation of reduced glutathione and/or alpha-oxoaldehyde-scavenging agents now deserve investigation.

Receptor-mediated endocytic uptake of methylglyoxal-modified serum albumin. Competition with advanced glycation end product-modified serum albumin at the advanced glycation end product receptor.

Methylglyoxal binds and irreversibly modifies arginine and lysine residues in bovine serum albumin (BSA) under physiological conditions, producing a protein with an increased net negative charge at physiological pH. At 4 degrees C, methylglyoxal-modified BSA (MG-BSA) was bound by cell surface receptors on murine P388D1 macrophages. The apparent dissociation constant KD value was 435 +/- 2 nM, and there were 8.89 +/- 0.02 x 10(5) receptors/cell (n = 6), compare with an apparent KD value of 263 +/- 52 nM and 10.17 +/- 0.93 x 10(5) receptors/cell (n = 11) for advanced glycation end product-modified BSA (AGE-BSA). AGE-BSA competed with MG-BSA for binding to a common receptor; however, a component of AGE-BSA receptor binding could not be displaced by MG-BSA, and a component of MG-BSA receptor binding could not be displaced by AGE-BSA, suggesting that there are binding sites for both AGE-BSA and MG-BSA, competitive and noncompetitive, to MG-BSA and AGE-BSA on P388D1 cells at 4 degrees C. At 37 degrees C, receptor binding of AGE-BSA and MG-BSA was followed by endocytosis and lysosomal degradation of the modified protein. Methylglyoxal-modified proteins are ligands for the AGE receptor, and their formation and metabolism may be linked to the development of diabetic complications.

Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin.

The physiological alpha-oxoaldehyde methylglyoxal binds and modifies arginine, lysine, and cysteine residues in proteins. The kinetics and mechanism of these reactions were investigated with N alpha-acetylamino acids and bovine serum albumin at pH 7.4 and 37 degrees C. The reaction of methylglyoxal with N alpha-acetylarginine involved the initial reversible formation of glycosylamine and 4,5-dihydroxy-5-methylimidazolidine derivatives, with further slow irreversible conversion to an imidazolone, N alpha-acetyl-N delta- (5-methyl-4-imidazolon-2-yl)ornithine. The imidazolone was fluorescent with an excitation lambda max value of 320 nm and an emission lambda max value of 398 nm. Methylglyoxal reacted reversibly with N alpha-acetyllysine to form glycosylamine and bisglycosylamine derivatives. Further reaction of these glycosylamines occurred to form brown, fluorescent oligomers that were not characterized. Methylglyoxal reacted rapidly and reversibly with N alpha-acetylcysteine to form the hemithioacetal adduct. The reaction of methylglyoxal with bovine serum albumin (BSA) at pH 7.4 and 37 degrees C involved the reversible and irreversible formation of methylglyoxal-BSA adducts. Irreversible modification of BSA occurred mainly on arginine residues to form imidazolone. The formation of methylglyoxal-modified proteins involves glycoxidation leading to advanced glycation end product-like fluorescence. It is expected to be increased in diabetes mellitus and may be linked to the development of diabetic complications.

Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications.

1. The metabolism of methylglyoxal by the glyoxalase system may be linked to the development of diabetic complications. The glyoxalase system was characterized in blood samples from patients with insulin-dependent diabetes mellitus (n = 43), patients with non-insulin-dependent diabetes mellitus (n = 107) and 21 normal healthy control subjects. 2. The concentrations of glyoxalase metabolites, methylglyoxal, S-D-lactoylglutathione and D-lactate, were increased in diabetic patients, relative to normal control subjects: methylglyoxal [median, range (n) pmol/g], insulin-dependent patients, 470.7, 85.6-1044.3 (42), P < 0.001, non-insulin-dependent patients, 286.8, 54.7-2370 (105), P < 0.001, control subjects, 79.8, 25.3-892.9 (21); S-D-lactoylglutathione [mean +/- SD (n)pmol/10(6) erythrocytes], combined diabetic patients, 3.37 +/- 0.85 (24), control subjects 4.76 +/- 1.95 (8) P < 0.05; D-lactate [mean +/- SD or median, range (n)nmol/g], insulin dependent patients, median 18.3, 5.7-57.4 (42), P < 0.001, non-insulin-dependent patients, 20.0 +/- 8.9, 2.6-48.4 (105), P < 0.001, control subjects 9.7 +/- 4.3, 1.8-19.7 (21). The reduced glutathione concentrations in blood samples from the insulin-dependent and non-insulin-dependent diabetic patient groups were not different from the control group values (P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The assay of S-D-lactoylglutathione in biological systems.

A procedure for the assay of S-D-lactoylglutathione, the physiological intermediate of the glyoxalase system, in biological systems is described, together with sample storage, sample processing, and statistical evaluation. Specimen data are presented. S-D-Lactoylglutathione was assayed by reverse-phase high-performance liquid chromatography (HPLC) with spectrophotometric detection of the thiolester chromophore at 233 nm. The biological sample was deproteinized with perchloric acid and partially purified by strong anion-exchange solid-phase extraction prior to HPLC. The limit of detection was 3.7 nmol, the recovery 49 +/- 4%, and the intra- and interbatch coefficients of variance 0.7 and 12%, respectively. The concentration of S-D-lactoylglutathione in whole blood from normal control human subjects was (mean +/- SD, nmol/ml whole blood) 16.5 +/- 4.4 (n = 8), and from diabetic patients 21.2 +/- 9.2 (n = 25), which is a significant increase (P < 0.05) from normal controls. The assay of S-D-lactoylglutathione is of increasing interest in studies of the elevation of glyoxalase metabolites in diabetes mellitus and in investigations of the antiproliferative activity of S-D-lactoylglutathione.

Fluorimetric assay of D-lactate.

A fluorimetric assay for D-lactate in human blood samples was developed using an endpoint enzymatic assay with D-lactate dehydrogenase from Staphylococcus epidermidis. The intrabatch and interbatch coefficients of variance were 8.7% (n = 4) and 16.6% (n = 4), respectively. The limit of detection in blood was 3.73 nmol/ml. The assay suffers minor interference from S-D-lactoylglutathione, which was also present in the blood samples. The concentration of D-lactate in blood was (mean +/- SE, nmol/ml) normal healthy individuals, 11.0 +/- 1.2 (n = 7); and diabetic patients, 20.0 +/- 1.3 (n = 55) (a significant increase in diabetes mellitus; P < 0.01, Mann-Whitney U test).

The assay of methylglyoxal in biological systems by derivatization with 1,2-diamino-4,5-dimethoxybenzene.

A procedure for the assay of methylglyoxal in biological systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and high-performance liquid chromatography (HPLC) of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extraction, and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biological matrix were 45 pmol (absorbance detection) and 10 pmol (fluorimetric detection), the recovery was 58%, and the intra- and interbatch coefficients of variance were 7.7 and 30.0%, respectively. The concentration of methylglyoxal in whole blood from normal healthy human individuals was (mean +/- SE, nM) 256 +/- 92 (n = 12) and that from diabetic patients was 479 +/- 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous estimates of the concentration of methylglyoxal in biological samples require re-evaluation.

Electrophoretic analysis of isoforms of glyoxalase II in clinical blood samples.

A non-denaturing polyacrylamide gel electrophoresis procedure is described for the analysis of isoforms of human red blood cell glyoxalase II (EC 3.1.2.6). A cathodic, continuous buffer electrophoresis system was developed, using a 60 g/l polyacrylamide gel with 25 g/l N,N1-methylene-bis-acrylamide in 50 mmol/l sodium succinate buffer, pH 5.5 and 1 degrees C. Staining for glyoxalase II activity involved (a) washing the gel with 50 mmol/l Tris-HCl buffer, pH 7.4 and 37 degrees C, and (b) incubation with developing agent (0.5 g/l 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide, 0.1 g/l 2,6-dichlorophenolindophenol, 1 mmol/l S-D-lactoylglutathione in 100 mmol/l Tris-HCl, pH 7.4). Two activity bands were found in all human blood samples studied to date, probably reflecting the presence of two major isoforms. Optimal sample storage and preparation procedures and validation studies are described. This method is currently in use in an ongoing investigation of glyoxalase II isoforms in disease processes.

Glyoxalase activity in human red blood cells fractioned by age.

Human red blood cells were fractionated by density, which correlates with cell age, and the activities of glyoxalase I and glyoxalase II were determined for each fraction. The activity of glyoxalase I and glyoxalase II both significantly increased during maturation of the red blood cells (P less than 0.001), except in the most dense, old cell fraction where both glyoxalase activities decreased. The increase in glyoxalase activity from the reticulocyte-rich fraction to mature erythrocytes was substantial and markedly different from other glycolytic enzymes which typically decrease. This suggests that glyoxalase activity changes markedly during and probably after the maturation of reticulocytes to erythrocytes. The decrease in glyoxalase activity from the mature to old red blood cell fractions may be caused by oxidative inactivation of glyoxalases. The decreased capacity to metabolise methylglyoxal may be an important factor in red blood cell senesence. This is expected to be particularly important in diabetes mellitus where the rate of methylglyoxal formation is increased during hyperglycaemia.