Amino acids as biomarkers in the SOD1(G93A) mouse model of ALS.

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1(G93A) transgenic mouse model for ALS. Presymptomatic SOD1(G93A) mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70days of age in both the SOD1(G93A) and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1(G93A)-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.

Serum glutamine synthetase has no value as a diagnostic biomarker for Alzheimer's disease.

In order to test whether serum glutamine synthetase (GS) is of potential diagnostic value for Alzheimer's disease (AD), we set up a study to compare serum GS concentrations between AD patients and control subjects. The study population (n = 165) consisted of AD patients (n = 94) and age-matched (n = 41) and age-unmatched (n = 30) control subjects. Serum GS analysis was performed by means of ELISA. No significant differences in serum GS levels were found between the AD group and age-matched controls. Age correlated positively with serum GS concentrations in AD patients and control subjects. This study suggests that serum GS levels have no diagnostic value for AD.

Glutamine synthetase as an early marker for hepatocellular carcinoma based on proteomic analysis of resected small hepatocellular carcinomas.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. Because small HCCs possess most of the characteristics of early HCC, we investigated small HCCs to screen potential biomarkers for early diagnosis. METHODS: Proteins were extracted from 10 sets of paired tissue samples from HBV-infected small-HCC patients. The extracted proteins were well resolved by two-dimensional electrophoresis. These HCC-associated proteins were then identified by MALDI-TOF/TOF MS following image analysis. Western blotting and immunohistochemistry were used to assess glutamine synthetase (GS) and phenazine biosynthesis-like domain-containing protein (PBLD) expression in liver tissue. Enzyme-linked immunosorbent assays in 152 serum samples (from 49 healthy donors, 24 patients with liver cirrhosis, and 79 with HCC) were used to further assess the significance of GS clinically. RESULTS: Fifteen up-regulated and three down-regulated proteins were identified. Western blotting confirmed GS overexpression and decreased PBLD expression in liver tissue. Immunohistochemistry showed that GS was expressed in 70.0% (84/120) of HCCs and 35.8% (43/120) of nontumor tissues; PBLD was expressed in 74.2% (89/120) of nontumor tissues and 40.8% (49/120) of HCCs. The Chi-square test showed significant expression differences between HCCs and adjacent tissues. Consistent with this, serum GS levels in HCC patients were significantly higher than those in liver cirrhosis patients and healthy donors, while the latter two groups were also significantly different. In addition, a diagnostic cutoff value of 2.6 mg/ml was used for GS; it was elevated in 19 (76.0%) of 25 HCC patients with AFP

Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle.

PURPOSE: The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period. METHODS: Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured. RESULTS: The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue(-1). GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue(-1), whereas GS activity fluctuates between 17 and 23 micromol min(-1) g protein(-1). Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle. CONCLUSIONS: The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS.

Effect of olanzapine treatment on platelet glutamine synthetase-like protein and glutamate dehydrogenase immunoreactivity in schizophrenia.

According to contemporary views, the glutamatergic system is implicated in the pathogenesis of schizophrenia, and atypical neuroleptics exert their effects (at least partially) through the glutamatergic system. Immunoreactive glutamate-metabolising enzymes, such as glutamine synthetase-like protein (GSLP) and two glutamate dehydrogenase isoenzymes (GDH), have been discovered in human platelets. The amount of GSLP in the platelets of 40 chronic patients with schizophrenia was found to be significantly higher than in 33 controls (consistent with our previous finding of increased amounts of GSLP in the prefrontal cortex of chronic schizophrenia patients). Moreover, survival analysis of the group of patients treated with olanzapine for 28 weeks showed that the larger amount of GSLP measured in platelets before treatment, the shorter the treatment time needed to achieve a positive clinical response (defined a priori as > or = 20% reduction in PANSS total score from the initial level before the treatment). Hence, GSLP level may serve as a predictor of the treatment duration to achieve a positive outcome with olanzapine. Both GSLP and GDH were found significantly changed in the course of treatment; hence, treatment with olanzapine influences the amounts of glutamate-metabolising enzymes in the platelets of chronic schizophrenia patients.

Increased glutamine synthetase but normal EAAT2 expression in platelets of ALS patients.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and glutamate excitotoxicity has been implicated in its pathogenesis. Platelets contain a glutamate uptake system and express components of the glutamate-glutamine cycle, such as the predominant glial excitatory amino acid transporter 2 (EAAT2). In several neurological diseases platelets have proven to be systemic markers for the disease. We compared properties of key components of the glutamate-glutamine cycle in blood platelets of ALS patients and healthy controls. Platelets were analyzed for (3)H-glutamate uptake in the presence or absence of thrombin and for EAAT2 and glutamine synthetase protein expression by Western blotting. Platelets of ALS patients showed a 37% increase in expression of glutamine synthetase, but normal expression of glutamate transporter EAAT2. Glutamate uptake in resting or thrombin-stimulated platelets did not differ significantly between platelets from ALS patients and controls. Thrombin-stimulation resulted in about a seven-fold increase in glutamate uptake. Our data suggest that glutamine synthetase may be a peripheral marker of ALS and encourage further investigation into the role of this enzyme in ALS.

Different biochemical strategies of two neotropical fish to cope with the impairment of nitrogen excretion during air exposure.

The exposure of fish to air is normally expected to interfere with the nitrogen excretion process. Hoplias malabaricus and Hoplerythrinus unitaeniatus, two teleost species, display distinct behaviors in response to decreases in natural reservoir water levels, although they may employ similar biochemical strategies. To investigate this point, plasma levels of ammonia, urea, uric acid, and the two urea cycle enzymes, ornithine carbamoyl transferase (OCT) and arginase (ARG), as well as glutamine synthetase (GS) were determined for both species after exposure to air. Plasma ammonia increased gradually during exposure to air, but only H. malabaricus showed increased concentrations of urea. Plasma uric acid remained very low in both fish. Enzymatic activities (mean +/- SD, micromol min(-1) g protein(-1)) of H. malabaricus showed significant increases (P<0.05, N = 6) in OCT from 0.84 +/- 0.05 to 1.42 +/- 0.03, in ARG from 8.07 +/- 0.47 to 9.97 +/- 0.53 and in GS from 1.15 +/- 0.03 to 2.39 +/- 0.04. The OCT and ARG enzymes remained constant in H. unitaeniatus (N = 6), but GS increased from 1.49 +/- 0.02 to 2.06 +/- 0.03. Although these species are very closely related and share the same environment, their biochemical strategies in response to exposure to air or to increased plasma ammonia are different.

Glutamine synthetase in cerebrospinal fluid, serum, and brain: a diagnostic marker for Alzheimer disease?

OBJECTIVES: To determine whether the glutamine synthetase (GS) level in cerebrospinal fluid (CSF) is a useful biochemical marker in the diagnosis of Alzheimer disease (AD), and to assess the source of GS (brain vs. blood derived) in CSF. METHODS: Sandwich enzyme immunoassay and immunoblotting were applied to detect GS in CSF and in serum from neurologically healthy control subjects and patients with neurodegenerative diseases, including AD. The origin of GS was estimated by the concentration gradients of CSF to serum and ventricular to lumbar CSF. In addition, postmortem brain tissue from controls and patients with AD was analyzed using immunohistochemistry for expression of GS. RESULTS: Levels of GS were significantly increased in lumbar CSF from patients with AD (20+/-12 pg/mL; P = .01) and to a lesser extent in patients with vascular dementia and amyotrophic lateral sclerosis. In CSF of controls, GS levels were 4+/-3 pg/mL. The GS concentration gradients were less than 1:10 for CSF to serum and 2:1 for ventricular to lumbar CSF. Immunoreactivity of GS was most prominent in astrocytes from temporal neocortex of patients with AD, suggesting a relationship between astrocyte reactions and increased GS levels in CSF. CONCLUSIONS: Level of GS in lumbar CSF of patients with AD is increased significantly but nonspecifically, probably related to the strong astrogliosis in brain. Glutamine synthetase in lumbar CSF is mainly brain derived.

Purification and immunocharacterization of human brain glutamine synthetase and its detection in cerebrospinal fluid and serum by a sandwich enzyme immunoassay.

A modified procedure employing a single chromatographic step to purify glutamine synthetase from human brain is described. The enzyme was characterized by native, denaturing, and two-dimensional gel electrophoresis and immunoblotting. Yield and purity were assessed by enzyme activity assay and a newly developed sandwich enzyme immunoassay using a mouse monoclonal antibody against native sheep brain glutamine synthetase. The immunoassay detected glutamine synthetase protein in samples where the enzyme had been inactivated by repeated cycles of freezing and thawing, and in serum and cerebrospinal fluid where glutamine synthetase was undetectable by the enzyme activity assay. Native glutamine synthetase from human brain occurred as an octamer with an estimated molecular weight of 360-400 kDa. Under reducing and denaturing conditions, the enzyme dissociated into monomeric subunits with an estimated molecular weight of 44 kDa. The monomers were recognized by the monoclonal antibody on immunoblots but not in the sandwich enzyme immunoassay, suggesting that the antigenic site occurs once on each subunit. Both human and sheep brain glutamine synthetases were composed of three and four different types of subunits with isoelectric points ranging from 7.0-7.2 and 6.8-7.0, respectively.

Changes in distribution and activity of glutamine synthetase in carbon tetrachloride-induced cirrhosis in the rat: potential role in hyperammonemia.

Cirrhosis induced in rats by carbon tetrachloride was used to study alterations in the activities and lobular distribution of carbamoylphosphate synthetase and glutamine synthetase. Specific activity of carbamoylphosphate synthetase in cirrhotic subjects was decreased to 70% of controls. Staining was homogeneous within micronodular areas, but varied from area to area and generally showed a decreased intensity. Specific activity of glutamine synthetase and the size of the glutamine synthetase-positive area were decreased to 20% and less of controls. Glutamine synthetase-positive hepatocytes were rare and scattered at the periphery of nodular areas and within fibrous septa, the normal association with the central veins being widely lost. Rarely, complete micronodules showed a slight homogeneous staining for glutamine synthetase. Arginase activity was not affected, whereas glutaminase activity was enhanced by 50%. Serum levels of ammonia were elevated more than 2-fold and those of glutamine by 30%. In contrast, urea levels tended to be slightly diminished. Serum ammonia levels showed a clear negative correlation with the specific activity of glutamine synthetase and the size of the glutamine synthetase-positive area. Furthermore, blood urea levels correlated with the sum of ammonia and glutamine concentrations, but not with each of these substrate concentrations alone. These data suggest that the changes in activity and distribution of glutamine synthetase contribute to hyperammonemia in cirrhosis. Despite a reduced activity of the initial enzyme of the urea cycle, urea synthesis is not diminished accordingly. This may be due to an enhanced flux caused by the elevated blood level of ammonia and an increased hydrolysis of glutamine, because of higher levels of glutaminase.

Discrimination between periportal and pericentral necrosis of rat liver by determination of glutamine synthetase and other enzyme activities in serum.

Periportal or pericentral necrosis of rat liver was produced by injection of allyl-alcohol or bromobenzene, respectively. Activities of predominantly periportal and perivenous enzymes were determined in serum during maximal necrosis. Aspartate aminotransferase, which is more or less homogeneously distributed in the liver acinus, exhibited similar activities in serum after periportal and pericentral injury. Serum activities of the mainly periportal enzymes alanine aminotransferase and fructose 1,6-bisphosphatase were 1.5- to 2-fold higher after periportal as compared to pericentral necrosis. Serum activity of the mainly pericentral glutamate dehydrogenase was 3-fold higher after pericentral than after periportal damage. However, due to individual variations necrosis could not be definitively localized in any case by measurement of these enzyme activities. Better discrimination between periportal and pericentral necrosis was achieved by the serum activity of the exclusively pericentral enzyme glutamine synthetase, which was 8-fold higher after pericentral as compared to periportal necrosis. Conclusive discrimination was obtained by the activity ratio fructose 1,6-bisphosphatase/glutamine synthetase in serum.

Plasma concentrations of glutamate and its metabolites in patients with Alzheimer's disease.

Neurologic and psychologic tests without brain tissue biopsy do not establish the diagnosis of Alzheimer's disease. This pilot study demonstrates significant increases in the activity of plasma glutamate dehydrogenase and the plasma concentrations of aspartate, glutamate, and alpha-ketoglutarate in nursing home residents with previously diagnosed Alzheimer's disease when compared with that in other nursing home residents without Alzheimer's disease who had no complicating conditions. Plasma concentrations of gamma-aminobutyric acid, glutamine, and activities of plasma glutamate decarboxylase, glutaminase, and glutamine synthetase were not significantly different in the two groups. A discriminant analysis number, based on the four significantly different compounds, is obtained that may be used as the basis for an inexpensive, non-invasive, and accurate screening test for Alzheimer's disease.

Glutamine and alanine metabolism in lungs of septic rats.

1. The metabolism of glutamine and alanine in the lung was studied in rats made septic by a caecal ligation and puncture technique. 2. The blood glucose concentration was not significantly different in septic rats, but blood pyruvate, lactate, glutamine and alanine concentrations were markedly increased as compared with sham-operated rats. Conversely, blood ketone body and plasma cholesterol concentrations were significantly decreased in septic rats. Both plasma insulin and plasma glucagon concentrations were markedly elevated in response to sepsis. Sepsis resulted in a negative nitrogen balance. 3. Sepsis increased the rates of production of glutamine (52.5%, P less than 0.001), alanine (38.9%, P less than 0.001) and glutamate (48.6%, P less than 0.001) by lung slices incubated in vitro. 4. Sepsis increased lung blood flow by 27.6% (P less than 0.05). Blood flow and arteriovenous concentration difference measurement across the lung of septic rats showed an increase in the net exchange rates of glutamine (142.5%, P less than 0.001), alanine (129.4%, P less than 0.001), glutamate (100.9%, P less than 0.001) and ammonia (138.0%, P less than 0.001) as compared with sham-operated control rats. 5. Sepsis produced significant decreases in the lung concentrations of glutamine (36.8%), glutamate (20.8%), 2-oxoglutarate (64.8%) and AMP (18.3%). The lung concentrations of alanine (95.9%), ammonia (67.7%) and pyruvate (89.7%) were increased. 6. The maximal activities of glutamine synthetase (20.4%, P less than 0.05), phosphate-dependent glutaminase (18.9%, P less than 0.05) and alanine aminotransferase (25.5%, P less than 0.05) were increased, but there was no marked change in that of glutamate dehydrogenase, in the lungs of septic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Zonation of total and plasma protein turnover in rat liver: effects of chronic ethanol treatment.

To study the acinar distribution of protein turnover, rats were given a tracer dose of 14C-valine intraperitoneally. At 30 min or 20 hr later, livers were perfused in situ and periportal (pp) or perivenous (pv) cell lysates were collected after digitonin pulse infusion. In control rats the post-label radioactivity of cell lysates showed no zonation. The radioactivity of the pp and pv cell lysates was significantly higher in livers of chronically ethanol-fed rats than in the controls, and the increase was more prominent in the pp region. Furthermore, after chronic ethanol feeding, an enrichment of plasma protein in the pv region was observed. The results support the view that in the control rats liver protein turnover is not zonated. Chronic ethanol feeding may, however, induce such zonation, as evidenced by both a slower rate of protein turnover in the pp cells and preferential pv accumulation of plasma proteins.

Gliotoxic effects of alpha-aminoadipic acid isomers on the carp retina: a long term observation.

The glutamate analogue, alpha-aminoadipic acid was intravitreally administered in the D-, DL- and L-forms to carp (Cyprinus carpio) retina in vivo. To make a quantitative assessment of its gliotoxic action, the activity of glutamine synthetase, whose localization was confirmed in glial Müller cells by an immunohistochemical technique, was examined at various intervals over one month. Intravitreal injection of 8 mumol alpha-aminoadipic acids reduced the glutamine synthetase activity within 4 h and maximally by 24 h. The maximum reduction evoked by L-, DL- and D-forms was about 65, 45 and 28% in reduction, and their minimum effective dose was 0.8, 1.5 and 2.0 mumol, respectively. At three to four days after alpha-aminoadipic acids injection, sodium dodecyl sulphate gel electrophoresis suggested that some retinal proteins including glutamine synthetase were significantly reduced, whilst others were increased. These biochemical changes were fully reversed one to two weeks after administration of the D- or DL-forms, but not until one month with the L-form. The electroretinographic b-wave, reflecting glial activity, was completely blocked by 8 mumol alpha-aminoadipic acids within 4 h. The electroretinographic b-wave was recovered first in the case of D- and then of DL-form at two to three weeks after injection, but only 50% recovery was seen in the case of L-form even two months later. A high dose of DL-alpha-aminoadipic acid (16 mumol) induced as long lasting a suppression in the glutamine synthetase and electroretinographic b-wave activities as 8 mumol L-alpha-aminoadipic acid. Therefore, the gliotoxic efficacy of L-alpha-aminoadipic acid at micromol orders was two-fold higher than that of DL-alpha-aminoadipic acid. Differences in the time-course of recovery of the suppression of glutamate synthetase and electroretinographic b-wave activities induced by alpha-aminoadipic acids are discussed in terms of its gliotoxicity.

Presence and steroid inducibility of glutamine synthetase in human leukemic cells.

Glutamine synthetase (EC 6.3.1.2; GS) is present in lymphoblasts from patients with acute lymphoblastic leukemia (ALL) as well as in normal peripheral blood lymphocytes. In 16 out of 20 ALL patients studied exposure of the cells to physiological concentrations of dexamethasone in vitro increased enzyme activity above the control levels. The increase was specific for glucocorticoid receptor ligands. A direct correlation was found between the magnitude of glucocorticoid-mediated increase of GS activity and the cellular levels of specific glucocorticoid receptors assayed in the same cell specimen. Moreover, the basal levels of the enzyme measured in cells prior to exposure to dexamethasone correlated negatively with receptor density. It is suggested that the presence of steroid-inducible GS in ALL cells may prove to be a marker for functional receptor sites.

Erythrocytic enzymes and amino acids related to glutamic acid metabolism in childhood hypoproteinemic states.

The erythrocyte enzymes of glutamic acid metabolism (glutaminase I, glutaminase II, glutamic acid decarboxylase, glutamine synthetase, and transaminases) and related amino acids (glutamine, glutamic acid, aspartic acid, alanine, and gamma-aminobutyric acid) were estimated in 69 children with protein-energy malnutrition, 13 with nephrosis, and 10 with Indian childhood cirrhosis. Twenty-one apparently healthy children served as controls. There was a significant increase in the activities of erythrocytic glutaminase I, glutaminase II, glutamic acid decarboxylase, and glutamine synthetase in all the three hypoproteinemic states, while the activities of the transaminases showed a decrease in all the conditions. The concentrations of all the amino acids were significantly increased in both the varieties of protein-energy malnutrition (edematous and nonedematous). In nephrosis and Indian childhood cirrhosis, aspartic acid, alanine, and gamma-aminobutyric acid showed a significant rise. The concentration of glutamic acid was also significantly increased in nephrosis. The observations of the present study suggest an increase in intracellular production of glutamic acid in hypoproteinemia.