Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis.

AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic encephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control (n=8) or portacaval shunt (n=8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37 celsius degree with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (mukat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95+/-268.87 mukat/g of protein in PCA rats vs 429.19+/-126.92mukat/g of protein in sham-operated rats; kidneys PAG activity was 1259.18+/-228.79 mukat/g protein in PCA rats vs 669.67+/-400.8 mukat/g of protein in controls, P<0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in sham-operated rats (cortex: 6646.6+/-1870.4 mukat/g of protein vs 3573.8+/-2037.4 mukat/g of protein in control rats, P<0.01; basal ganglia, PAG activity was 3657.3+/-1469.6 mukat/g of protein in PCA rats vs 2271.2+/-384 mukat/g of protein in sham operated rats, P<0.05; In the cerebellum, the PAG activity was 2471.6+/-701.4 mukat/g of protein vs 1452.9+/-567.8 mukat/g of protein in the PCA and sham rats, respectively, P<0.05; content of protein: cerebral cortex: 162%+/-40% vs 100%+/-26%, P<0.009; and basal ganglia: 140%+/-39% vs 100%+/-14%, P<0.05; but not in cerebellum: 100%+/-25% vs 100%+/-16%, P=ns). CONCLUSION: Increased PAG activity in kidney and duodenum could contribute significantly to the hyperammonaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.

Evaluation of platelet function during extended storage in additive solution, prepared in a new container that allows manual buffy-coat platelet pooling and leucoreduction in the same system.

BACKGROUND: A novel and practical storage container designed for manual buffy-coat pooling and leucodepletion was evaluated to assess its filtration performance and to analyse the quality of stored leucoreduced buffy-coat-derived platelet pools. MATERIALS AND METHODS: To analyse the grifols leucored transfer PL system, blood was collected from random donors into standard triple bag systems, and fractionated using standard procedures to obtain buffy-coats. Ten leucodepleted platelet pools were prepared each from five units of buffy-coats in additive solution. Concentrates were stored for 10 days at 22 °C on an end-over-end agitator. On days 0, 5, 7, and 10 of storage, samples were tested using standard in vitro platelet parameters. RESULTS: The use of this novel system for volume reduction and leucodepletion of buffy-coats resuspended in additive solution led to platelet pools that met the European requirements. pH was maintained well, declining from an initial value of 7.11±0.04 to 6.88±0.08 after 10 days. Parameters of cell lysis, response to a hypotonic stimulus and aggregation induced by agonists (arachidonic acid, ristocetin, collagen or thrombin receptor activating peptide) were also well-preserved. During storage, the quality profile of the platelet pools remained very similar to that previously reported in platelet concentrates in terms of metabolism, platelet activation (CD62, CD63, sCD62), expression of glycoproteins Ib and IIb/IIIa, capacity of glycoprotein IIb/IIIa to become activated upon ADP stimulation, and release of biological response modifiers (sCD40L and RANTES). DISCUSSION: This new system allows the preparation of leucodepleted buffy-coat platelet pools in additive solution with good preservation of platelet function. The logistics of the procedure are relatively simple and it results in good-quality components, which may reduce costs and ease the process of buffy-coat pooling and leucocyte reduction in transfusion services.

Intestinal glutaminase activity is increased in liver cirrhosis and correlates with minimal hepatic encephalopathy.

BACKGROUND/AIMS: We performed the current study to assess the intestinal activity of enterocyte phosphate-activated glutaminase (PAG) in cirrhosis. METHODS: Forty-nine cirrhotic patients and 36 control subjects underwent endoscopic duodenal biopsies. Minimal hepatic encephalopathy (MHE) was evaluated using three psychometric tests. Oral glutamine challenge (OGC) was performed and MELD, Child-Pugh and the presence of esophageal varices were recorded. PAG was measured by enzymatic methods. Cerebral magnetic resonance spectroscopy was performed in 10 cirrhotics. RESULTS: PAG was found to be higher in cirrhotics than control subjects 2.4+/-1.51 vs. 0.68+/-0.57IU/mg protein (P<0.001). PAG was also increased in patients with MHE and correlated with MELD, INR, esophageal varices and serum bile acids. A negative correlation was observed between PAG activity and intra-cerebral choline/creatine ratio (r=-0.67; P=0.035) and a positive correlation with glutamine plus glutamate/creatine ratio (r=0.78; P=0.007). In multivariate analysis using backward logistic regression, presence of MHE was the only variable independently related to altered enterocyte PAG. CONCLUSIONS: Enterocyte PAG is increased in cirrhotic patients and correlates with MHE. These data support a possible role for intestinal glutaminase in the pathogenesis of hepatic encephalopathy (HE) and could be a new target for future therapies.