[Simultaneous determination of three quaternary ammonium muscle relaxants in blood by high performance liquid chromatography-tandem mass spectrometry].

Vecuronium, rocuronium, and pancuronium are widely used as non-depolarizing muscle relaxants. There have been occasional cases of allergic reactions and even death when using such muscle relaxants. Rapid determination of the concentration of these muscle relaxants in blood can provide valuable information for early clinical diagnosis. As quaternary ammonium compounds, these muscle relaxants are highly polar. Hence, they cannot be retained effectively on reversed-phase chromatographic columns with conventional mobile phases. These quaternary ammonium muscle relaxants are mainly separated by ion-pair chromatography. Using an ion-pairing reagent can help improve the retention capabilities of quaternary ammonium muscle relaxants. Nevertheless, the sensitivity of MS detection is significantly decreased because of ionic inhibition caused by the ion-pairing reagent in the mobile phase. Furthermore, ion-pairing reagents can pollute the MS system. A method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the simultaneous determination of the three quaternary ammonium muscle relaxants in blood. The blood samples were diluted and subjected to high-speed centrifugation. The supernatant was purified on a Bond Elut AL-N solid phase extraction column and then filtered through a 0.45 µm microporous membrane. The quaternary ammonium muscle relaxants were separated on a ZIC-cHILIC analytical column (50 mm×2.1 mm, 3.0 µm) with gradient elution. Acetonitrile and 0.1% formic acid aqueous solution were used as mobile phases. The separated compounds were analyzed by tandem MS with an electrospray ionization (ESI) source in positive and multiple reaction monitoring (MRM) modes. The matrix effects of vecuronium, rocuronium, and pancuronium in blood were 88.1% to 95.4%. The calibration curves for vecuronium, rocuronium, and pancuronium showed good linear relationships in each range, and all correlation coefficients (R2) were > 0.996. The limits of detection of vecuronium, rocuronium, and pancuronium were 0.2-0.8 ng/mL, with the corresponding limits of quantification being 0.5-2.0 ng/mL. The recoveries of vecuronium, rocuronium, and pancuronium were 92.8% to 110.6%, with relative standard deviations (RSDs) of 3.2%-9.4%. This method is sensitive, accurate, and easy to operate, and it can be used to rapidly determine vecuronium, rocuronium, and pancuronium in blood.

Probiotics improve the neurometabolic profile of rats with chronic cholestatic liver disease.

Chronic liver disease leads to neuropsychiatric complications called hepatic encephalopathy (HE). Current treatments have some limitations in their efficacy and tolerability, emphasizing the need for alternative therapies. Modulation of gut bacterial flora using probiotics is emerging as a therapeutic alternative. However, knowledge about how probiotics influence brain metabolite changes during HE is missing. In the present study, we combined the advantages of ultra-high field in vivo 1H MRS with behavioural tests to analyse whether a long-term treatment with a multistrain probiotic mixture (VIVOMIXX) in a rat model of type C HE had a positive effect on behaviour and neurometabolic changes. We showed that the prophylactic administration of this probiotic formulation led to an increase in gut Bifidobacteria and attenuated changes in locomotor activity and neurometabolic profile in a rat model of type C HE. Both the performance in behavioural tests and the neurometabolic profile of BDL + probiotic rats were improved compared to the BDL group at week 8 post-BDL. They displayed a significantly lesser increase in brain Gln, a milder decrease in brain mIns and a smaller decrease in neurotransmitter Glu than untreated animals. The clinical implications of these findings are potentially far-reaching given that probiotics are generally safe and well-tolerated by patients.

Longitudinal neurometabolic changes in the hippocampus of a rat model of chronic hepatic encephalopathy.

BACKGROUND & AIMS: The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. METHODS: Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time 1H-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. RESULTS: Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2-4 weeks post-BDL while creatine (-8%) and ascorbate (-12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6-8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. CONCLUSION: Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment. LAY SUMMARY: The sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease.

Erythrocytes as bioreactors to decrease excess ammonium concentration in blood.

Increased blood ammonium concentrations cause neurological complications. Existing drugs are not always sufficiently effective. Alternatively, erythrocytes-bioreactors (EBRs) loaded with enzymes utilizing ammonium, were suggested for ammonium removal from blood. However all they worked only for a short period of time. The reasons for this were not investigated. In this study, EBR mathematical models were developed and analysed based on the reactions of glycolysis and different enzymes utilizing ammonium, which showed that the efficiency and duration of EBRs' functioning could be limited due to low permeability of the cell membrane for some key substrates and products. A new enzyme system including glutamate dehydrogenase and alanine aminotransferase was proposed and realised experimentally, which was not limited by cell membrane permeability for glutamate and α-ketoglutarate due to creating metabolic pathway where these metabolites were produced and consumed cyclically. New bioreactors removed ammonium in vitro at the rate of 1.5 mmol/h × lRBCs (for human bioreactors) and in vivo in a model of hyperammoniemia in mice at the rate of 2.0 mmol/h × lRBCs (for mouse bioreactors), which correlated with model calculations. Experimental studies proved the proposed mathematical models are correct. Mathematical simulation of erythrocyte-bioreactors opens new opportunities for analysing the efficiency of any enzyme included in erythrocytes.

Impairment of cognitive function in ornithine transcarbamylase deficiency is global rather than domain-specific and is associated with disease onset, sex, maximum ammonium, and number of hyperammonemic events.

Beginning in 2006, the Urea Cycle Disorders Consortium (UCDC) has conducted a longitudinal study of eight inherited deficiencies of enzymes and transporters of the urea cycle, including 444 individuals with ornithine transcarbamylase deficiency (OTCD), of whom 300 (67 males, 233 females) received psychological evaluation. In a cross-sectional study (age range, 3-71 years), analysis of covariance (ANCOVA) determined the association between outcomes in five cognitive domains (global intelligence, executive functions, memory, visuomotor integration, visual perception) and sex, age at testing and timing of disease onset defined as early onset (≤28 days; EO), late onset (LO), or asymptomatic (AS). The dataset of 183 subjects with complete datasets (31 males, 152 females) revealed underrepresentation of EO subjects (2 males, 4 females), who were excluded from the ANCOVA. Although mean scores of LO and AS individuals were within 1 SD of the population norm, AS subjects attained significantly higher scores than LO subjects and males higher scores than females. Correlations between cognitive domains were high, particularly intelligence proved to be a distinguished indicator for cognitive functioning. Maximum plasma ammonium concentration and intelligence correlated significantly higher in EO (r = -0.47) than in LO subjects (r = 0.04). Correlation between the number of hyperammonemic events and intelligence scores were similar for EO (r = -0.30) and LO (r = -0.26) individuals. The number of clinical symptoms was significantly associated with intelligence (r = -0.28) but not with scores in other domains. Results suggest that OTCD has a global impact on cognitive functioning rather than a specific effect on distinct cognitive domains.

Beneficial Effects of Dietary Tempeh Prepared with Rhizopus stolonifer on Liver Function in Rats Fed with a High-Fat Diet.

The regional standard for tempeh established by the Codex Alimentarius defines the use of Rhizopus oligosporus, R. oryzae, and/or R. stolonifer as soybean tempeh starters. However, comparative studies on the functions of tempeh prepared with these Rhizopus species are scarce. In the present study, we examined the effects of dietary tempeh prepared with these three Rhizopus species using rats fed with a high-fat diet. Compared to the control diet, consumption of tempeh prepared with R. stolonifer significantly suppressed serum levels of aspartate transaminase, total bilirubin, and ammonium (indices of liver function). However, less or no suppression was observed with tempeh prepared with R. oligosporus or R. oryzae. Serum levels of triglyceride, total cholesterol, HDL cholesterol, and glucose were unaffected. Liver levels of free cholesterol, a parameter relating to liver injury, were significantly decreased by the three types of the tempeh examined; however, there was no difference in the free cholesterol levels among the tempeh groups. We conclude that the ingestion of tempeh prepared with R. stolonifer might have beneficial effects pertaining to the liver function in rats fed with high-fat diets.

Clinical evaluation of a benchtop dry chemistry analyzer for measurement of ammonium concentrations in canine plasma samples.

OBJECTIVE To evaluate the clinical performance of a veterinary benchtop dry chemistry analyzer for measurement of plasma ammonium concentrations in dogs by comparing results with those obtained by a reference standard test method. DESIGN Prospective evaluation study. SAMPLE 32 blood samples from 30 dogs (16 with and 14 without suspected hepatobiliary disease). PROCEDURES Blood samples were collected by jugular venipuncture. A veterinary benchtop dry chemistry analyzer and a reference standard (enzymatic) test method were used to measure ammonium concentrations in plasma collected from heparinized whole blood. Bland-Altman plots were used to assess intermethod agreement. Results were compared by linear regression, and correlation was calculated by the Pearson method. Samples were classified as having high or normal ammonium concentrations on the basis of cutoff data for the relevant test method; results were compared between methods to evaluate diagnostic agreement. RESULTS 31 of 32 (97%) samples were classified correctly with the benchtop analyzer; 1 sample with a high ammonium concentration was classified as having a normal value (ie, false-negative result) by this method. A strong positive correlation (r2 = 0.989) was found between methods, with constant, proportional, negative bias for benchtop analyzer results. The interassay coefficient of variation (ie, precision) for measurement of an internal standard with the benchtop analyzer was 2.74% (n = 20 repetitions). CONCLUSIONS AND CLINICAL RELEVANCE The dry chemistry analyzer used in the study had acceptable clinical performance for detection of high versus normal ammonium concentrations in canine plasma, indicating the method can be used to aid diagnosis in dogs with suspected hepatobiliary disease.

Fragmentation patterns involving ammonium adduct fragment ions: A comparison of the determination of metaldehyde in human blood by HPLC-QqQ-MS/MS and UHPLC-Q-TOF-MS.

This paper presents a rapid, sensitive and precise method for the determination of metaldehyde in human blood, using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Separation was performed with a Poroshell 120 EC-C18 column; 2.7 µm atrazine­d5 (IS) and 200 mg NaCl were added to the blood sample. Proteins in human blood were precipitated using acetonitrile; the supernatant was then analyzed with the UHPLC-Q-TOF-MS or HPLC-QqQ-MS/MS system. The results of selectivity, linearity, accuracy, precision, limits of quantification, recovery, and matrix effects were sufficient to enable the measurement of metaldehyde in human blood samples. In addition, we proposed a fragmentation pathway involving ammonium adduct fragment ions for metaldehyde.

Acute central nervous system complications and ammonium levels in adult patients with acute lymphoblastic leukemia receiving l-asparaginase.

We analyzed ammonium levels and acute central nervous complications in adult patients receiving chemotherapy for acute lymphoblastic leukemia with or without asparaginase (l-asp). Twenty patients with a median age of 40.3 years were included. Ammonium-levels were measured during 88 chemotherapy cycles, 60 cycles (68%) with l-asp, and 28 cycles (32%) without l-asp. Ammonium was elevated in 87% of all l-asp containing cycles, with median ammonium levels of 169 µmol/l (interquartile range (IQR) 91-269 µmol/l). These values were higher when compared to ammonium levels at baseline (31.5 µmol/l, IQR 24-40 µmol/l, p < .001), and when compared to levels in cycles without l-asp (30 µmol/l, IQR 19-41 µmol/l, p < .001). Acute hyperammonemic encephalopathy was diagnosed in one patient, and encephalopathy for other reasons, but with hyperammonemia as a possible contributing factor in four patients. In conclusion, ammonium levels are elevated in all adult patients receiving l-asparaginase, but only some patients will present symptoms of encephalopathy.

A case of hereditary hemorrhagic telangiectasia with hepatic encephalopathy due to portal hepatic venous shunt.

An 86-year-old male who was able to perform all activities of daily living (ADL) was diagnosed with hereditary hemorrhagic telangiectasia (HHT) at 70 years of age. Following his diagnosis, he had been receiving treatment at our hospital. After the sudden onset of a consciousness disorder, he was admitted to our hospital's emergency department with asterixis, a high serum ammonia level, and hepatic encephalopathy. After angiography, he was diagnosed with hepatic encephalopathy due to portal hepatic venous shunts. HHT is characterized by abnormal blood vessel construction and the formation of peripheral vasodilatation and shunt blood vessels. Although rare, portal hepatic venous shunts may sometimes cause hepatic encephalopathy. The extent of this shunt increases with age. As Japan is an increasingly aging society, the number of HHT patients with hepatic encephalopathy is likely to increase markedly in the future.

Association of physical performance and biochemical profile of mice with intrinsic endurance swimming.

We aimed to investigate the potential mediators and relationship affecting congenital exercise performance in an animal model with physical activity challenge from physiological and biochemical perspectives. A total of 75 male ICR mice (5 weeks old) were adapted for 1 week, then mice performed a non-loading and exhaustive swimming test and were assigned to 3 groups by exhaustive swimming time: low exercise capacity (LEC) (<3 hr), medium exercise capacity (MEC) (3-5 hr), and high exercise capacity (HEC) (>5 hr). After a 1-week rest, the 3 groups of mice performed an exhaustive swimming test with a 5% and 7.5% weight load and a forelimb grip-strength test, with a 1-week rest between tests. Blood samples were collected immediately after an acute exercise challenge and at the end of the experiment (resting status) to evaluate biochemical blood variables and their relation with physical performance. Physical activity, including exhaustive swimming and grip strength, was greater for HEC than other mice. The swimming performance and grip strength between groups were moderately correlated (r=0.443, p<0.05). Resting serum ammonium level was moderately correlated with endurance with a 7.5% weight load (r=-0.447, p<0.05) and with lactate level (r=0.598, p<0.05). The pulmonary morphology of the HEC group seemed to indicate benefits for aerobic exercise. Mice showed congenital exercise performance, which was significantly correlated with different physical challenges and biochemical variable values. This study may have implications for interference in intrinsic characteristics.

Shunt porto-sistémico congénito en hígado no cirrótico como causante de síndrome confusional agudo reiterado: report of one case / Portosystemic shunt in a normal liver causing confusional states

Portosystemic shunts are rare vascularization disorders, and an uncommon cause of confusional states. We report an 87-year-old male with a previously normal cognitive status who was repeatedly admitted for sudden symptoms of disorientation and functional limitation. The patient had high ammonium levels which lead to the suspicion of the presence a portosystemic shunt, even in the absence of pre-existing liver disease. A contrast enhanced computed tomography of the abdomen confirmed the presence an abnormal communication of the right portal vein with the suprahepatic veins. The communication was embolized and the confusional states of the patient subsided.

[Portosystemic shunt in a normal liver causing confusional states]. / Shunt porto-sistémico congénito en hígado no cirrótico como causante de síndrome confusional agudo reiterado: Report of one case.

Portosystemic shunts are rare vascularization disorders, and an uncommon cause of confusional states. We report an 87-year-old male with a previously normal cognitive status who was repeatedly admitted for sudden symptoms of disorientation and functional limitation. The patient had high ammonium levels which lead to the suspicion of the presence a portosystemic shunt, even in the absence of pre-existing liver disease. A contrast enhanced computed tomography of the abdomen confirmed the presence an abnormal communication of the right portal vein with the suprahepatic veins. The communication was embolized and the confusional states of the patient subsided.

L-glutamine supplementations enhance liver glutamine-glutathione axis and heat shock factor-1 expression in endurance-exercise trained rats.

Liver L-glutamine is an important vehicle for the transport of ammonia and intermediary metabolism of amino acids between tissues, particularly under catabolic situations, such as high-intensity exercise. Hence, the aim of this study was to investigate the effects of oral supplementations with L-glutamine in its free or dipeptide forms (with L-alanine) on liver glutamine-glutathione (GSH) axis, and 70 kDa heat shock proteins (HSP70)/heat shock transcription factor 1 (HSF1) expressions. Adult male Wistar rats were 8-week trained (60 min/day, 5 days/week) on a treadmill. During the last 21 days, the animals were daily supplemented with 1 g of L-glutamine/kg body weight per day in either l-alanyl-L-glutamine dipeptide (DIP) form or a solution containing L-glutamine and l-alanine in their free forms (GLN+ALA) or water (controls). Exercise training increased cytosolic and nuclear HSF1 and HSP70 expression, as compared with sedentary animals. However, both DIP and GLN+ALA supplements enhanced HSF1 expression (in both cytosolic and nuclear fractions) in relation to exercised controls. Interestingly, HSF1 rises were not followed by enhanced HSP70 expression. DIP and GLN+ALA supplements increased plasma glutamine concentrations (by 62% and 59%, respectively) and glutamine to glutamate plasma ratio in relation to trained controls. This was in parallel with a decrease in plasma ammonium levels. Supplementations increased liver GSH (by 90%), attenuating the glutathione disulfide (GSSG) to GSH ratio, suggesting a redox state protection. In conclusion, oral administration with DIP and GLN+ALA supplements in endurance-trained rats improve liver glutamine-GSH axis and modulate HSF1 pathway.

Blood ammonium and lactate accumulation response to different training protocols using the parallel squat exercise.

Three parallel squat protocols with equal total work volume were used to determine the metabolic response of resistance exercise with different practical training protocols combining program variables in the way that they are typically prescribed in field. Sixteen men able to back squat 1.5 times their body weight participated in the study. Individualized muscular endurance (ME), strength (STR), and hypertrophy (HYP) squat workouts were developed based on a 1 repetition maximum back squat. Each protocol was performed 3-7 days apart in random order. Venous blood was obtained after 5 minutes of seated rest both before and after each workout for ammonium and lactate analysis. The ME protocol (79.8 µM [SD = 45.4], 95% confidence interval [CI]: 55.7-104.0) produced a greater change of plasma ammonium than both the HYP (45.3 µM [SD = 34.5], 95% CI: 26.9-63.6, p = 0.017) and STR (31.7 µM [SD = 52.3], 95% CI: 3.9-59.6, p = 0.006) protocols. Change of blood lactate concentration from resting levels to postexercise levels was significantly different (p = 0.005) between ME (6.1 mM [SD = 2.9], 95% CI: 4.6-7.7) and STR (3.9 mM [SD = 2.5], 95% CI: 2.6-5.2) protocols. The main finding of this study is that blood ammonium and lactate seem to accumulate in response to an increasing number of repetitions with decreasing rest time between sets. As consequence, a greater number of repetitions should be added to a resistance workout, along with a shorter rest time between sets when training for events that induce a large metabolic load. The metabolic accumulation associated with high repetition exercise may represent the need for longer recovery time between these types of workouts compared with workouts using a low number of repetitions.

Characterizing the plasticity of nitrogen metabolism by the host and symbionts of the hydrothermal vent chemoautotrophic symbioses Ridgeia piscesae.

Chemoautotrophic symbionts of deep sea hydrothermal vent tubeworms are known to provide their hosts with all their primary nutrition. While studies have examined how chemoautotrophic symbionts provide the association with nitrogen, fewer have examined if symbiont nitrogen metabolism varies as a function of environmental conditions. Ridgeia piscesae tubeworms flourish at Northeastern Pacific vents, occupy a range of microhabitats, and exhibit a high degree of morphological plasticity [e.g. long-skinny (LS) and short-fat (SF) phenotypes] that may relate to environmental conditions. This plasticity affords an opportunity to examine whether symbiont nitrogen metabolism varies among host phenotypes. LS and SF R. piscesae were recovered from the Axial and Main Endeavour Field hydrothermal vents. Nitrate and ammonium were quantified in Ridgeia blood, and the expression of key nitrogen metabolism genes, as well as stable nitrogen isotope ratios, was quantified in host branchial plume and symbiont-containing tissues. Nitrate and ammonium were abundant in the blood of both phenotypes though environmental ammonium concentrations were, paradoxically, lowest among individuals with the highest blood ammonium. Assimilatory nitrate reductase transcripts were always below detection, though in both LS and SF R. piscesae symbionts, we observed elevated expression of dissimilatory nitrate reductase genes, as well as symbiont and host ammonium assimilation genes. Site-specific differences in expression, along with tissue stable isotope analyses, suggest that LS and SF Ridgeia symbionts are engaged in both dissimilatory nitrate reduction and ammonia assimilation to varying degrees. As such, it appears that environmental conditions -not host phenotype-primarily dictates symbiont nitrogen metabolism.

Fish oil and the pan-PPAR agonist tetradecylthioacetic acid affect the amino acid and carnitine metabolism in rats.

Peroxisome proliferator-activated receptors (PPARs) are important in the regulation of lipid and glucose metabolism. Recent studies have shown that PPARα-activation by WY 14,643 regulates the metabolism of amino acids. We investigated the effect of PPAR activation on plasma amino acid levels using two PPARα activators with different ligand binding properties, tetradecylthioacetic acid (TTA) and fish oil, where the pan-PPAR agonist TTA is a more potent ligand than omega-3 polyunsaturated fatty acids. In addition, plasma L-carnitine esters were investigated to reflect cellular fatty acid catabolism. Male Wistar rats (Rattus norvegicus) were fed a high-fat (25% w/w) diet including TTA (0.375%, w/w), fish oil (10%, w/w) or a combination of both. The rats were fed for 50 weeks, and although TTA and fish oil had hypotriglyceridemic effects in these animals, only TTA lowered the body weight gain compared to high fat control animals. Distinct dietary effects of fish oil and TTA were observed on plasma amino acid composition. Administration of TTA led to increased plasma levels of the majority of amino acids, except arginine and lysine, which were reduced. Fish oil however, increased plasma levels of only a few amino acids, and the combination showed an intermediate or TTA-dominated effect. On the other hand, TTA and fish oil additively reduced plasma levels of the L-carnitine precursor γ-butyrobetaine, as well as the carnitine esters acetylcarnitine, propionylcarnitine, valeryl/isovalerylcarnitine, and octanoylcarnitine. These data suggest that while both fish oil and TTA affect lipid metabolism, strong PPARα activation is required to obtain effects on amino acid plasma levels. TTA and fish oil may influence amino acid metabolism through different metabolic mechanisms.

Parallel changes in intracellular water volume and pH induced by NH(3)/NH(4)(+) exposure in single neuroblastoma cells.

BACKGROUND: Increased blood levels of ammonia (NH3) and ammonium (NH4(+)), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4(+) at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4(+) elicits changes in intracellular pH (pHi), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4(+) has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. METHODS: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4(+) of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm), and pHi was measured ratiometrically. RESULTS: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH4Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH4Cl solutions in which either extracellular [NH3] or [NH4(+)] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4(+)]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca(2+). Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH4Cl-induced swelling. CONCLUSIONS: A quantitative description of the osmotic changes produced by exposure to NH3/NH4(+) in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH4(+) due to rapid permeation and protonation of NH3. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema.

[Preventive and therapeutic effects of extract from Rheum palmatum on hepatic encephalopathy in rats with acute liver failure].

OBJECTIVE: To probe the preventive, therapeutic effect and the possible mechanism of extract from Rheum palmatum (ERP) on hepatic encephalopathy(HE) in rats with acute liver failure. METHODS: HE was induced by the administration of 300 mg thioracetamide per kg body weight by gavage on two consecutive days. The effects of ERP were observed on neurology test, serum ammonium, serum endotoxin and liver impairment. RESULTS: ERP could improve rat neuro-reflexes, decrease the staging of HE and rat serum ammonium, endotoxin concentrations, and reduce the liver impairment. CONCLUSION: ERP significantly prevents and treats HE in rats with thioacetamide-induced acute liver failure.