Advances in methods for characterization of hepatic urea cycle enzymatic activity in HepaRG cells using UPLC-MS/MS.

Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.

[(Bio)artificial liver support: ready for the patient?]. / De kunstlever: klaar voor toepassing bij patiënten?

In 2016, an intensive-care physician has at his disposal a number of artificial organs for the support of patients with organ failure. Examples are the artificial kidney and the heart-lung machine. Artificial livers are being developed for patients with severe liver failure whose lives can only be saved at the present time by a transplant with a donor liver. These artificial livers are based either on a device that removes toxic materials from the patient's blood with, for example, albumin dialysis, or make use of bio-reactors filled with functioning liver cells, the so-called bio-artificial liver. In theory, the bio-artificial liver has the greatest potential to increase life expectancy. The results of clinical studies are also very promising. They are not yet sufficient, however, to permit general clinical use.

In vitro comparison of two bioartificial liver support systems: MELS CellModule and AMC-BAL.

Clinically applied bioartificial liver (BAL) support systems are difficult to compare with regard to overall hepatocyte-specific function and clinical outcome. We compared two clinically applied BAL systems, the Modular Extracorporeal Liver Support (MELS) CellModule and the AMC-bioartificial liver (AMC-BAL) in an in vitro set-up. Both BAL systems were loaded with 10 billion freshly isolated porcine hepatocytes, cultured for 7 days and tested on days 1, 2, 4 and 7. Average decrease in hepatocyte-specific functions over 7 days was 9.7%. Three parameters differed between both bioreactors: lidocaine elimination at days 1 and 2 was significantly higher in the AMCBAL, ammonia elimination showed a significantly higher trend for the AMC-BAL over 7 days and LDH release was significantly lower at day 7 for the MELS CellModule. In conclusion, this first in vitro comparison of two clinically applied BAL systems shows comparable functional capacity over a period of 7 days.

[Recent developments in the treatment of chronic hepatitis B and C]. / Recente ontwikkelingen in de behandeling van chronische hepatitis B en C.

Chronic hepatitis B and C are life-threatening diseases, treated with variable success. Peginterferon-alpha is one of the standard therapies for chronic hepatitis B as well as C. To prevent the development of resistant viruses, combination treatment is preferable to monotherapy. Therefore, in chronic hepatitis B virus peginterferon-alpha combined with nucleoside inhibitors is used. The treatment of chronic hepatitis C virus with a combination of peginterferon-alpha and ribavirine can be improved by new protease inhibitors.

Subnormothermic preservation maintains viability and function in a porcine hepatocyte culture model simulating bioreactor transport.

Bioartificial liver (BAL) systems have been developed to bridge patients with acute liver failure (ALF) to liver transplantation or liver regeneration. Clinical application of BAL systems is dependent on the supportive quality of cells used and direct availability of the whole system. Reliable transport of BAL systems from the laboratory to remote treatment centers is therefore inevitable. Subsequently, preservation conditions play a crucial role during transport of a BAL, with temperature being one of the most determining factors. In this study, we assessed the effect of subnormothermic preservation on freshly isolated porcine hepatocytes cultured in monolayer under oxygenation. Additionally, the effect of the University of Wisconsin (UW) preservation solution was compared with Williams' E (WE) culture medium at 4 degrees C. The control group was cultured for 3 days at 37 degrees C, whereas the transport groups were cultured at 4 degrees C, 15 degrees C, 21 degrees C, or 28 degrees C for 24 h at day 2. All groups were tested each day for cell damage and hepatic functions. Subnormothermic culture (i.e., 15 degrees C to 28 degrees C) for a period of 24 h did not reduce any hepatic function and did not increase cellular damage. In contrast, culture of hepatocytes in WE medium and preservation in UW solution at 4 degrees C significantly reduced hepatic function. In conclusion, freshly isolated porcine hepatocytes can be preserved for 24 h at subnormothermic temperatures as low as 15 degrees C. Future research will focus on the implementation of the AMC-BAL in an oxygenated culture medium perfusion system for transport between the laboratory and the hospital.

Isolation of up- and down-regulated cDNAs associated with hepatocellular carcinoma by a subtraction-enhanced display technique.

Identification of gene products exclusively or abundantly expressed in hepatocellular carcinoma (HCC) and in normal liver may yield novel tumor markers. We have isolated 36 up- and down-regulated cDNAs from diethylnitrosamine-induced rat hepatocellular carcinoma and normal liver tissue by using the subtraction-enhanced display technique. Nucleotide sequence analysis revealed that the majority of 20 subtraction-enriched cDNA fragments were well-characterized oncogenes and tumor-associated genes, like c-myc, alpha-prothymosin, p21, glutathione-S transferase (G-ST) and alpha 1-acid glycoprotein (AGP). As demonstrated by Northern blot detection, all of them were preferentially expressed either in HCC or in normal liver (2- to 7-fold). As paradigm, G-ST and AGP were shown to be exclusively overexpressed in tumor nodules by in situ hybridization. In addition, 14 of the remaining 16 novel genes were analysed on Northern blot, 10 of which were differentially expressed in HCC.

Lobular patterns of expression and enzyme activities of glutamine synthase, carbamoylphosphate synthase and glutamate dehydrogenase during postnatal development of the porcine liver.

Carbamoylphosphate synthase and glutamine synthase show a complementary distribution in the liver lobule of the rat. In the human liver lobule, which is approximately 2-fold larger than that of the rat, an intermediate, 'empty' zone is present between the periportal carbamoylphosphate synthase-positive and the pericentral glutamine synthase-positive zone. To investigate whether these differences in gene expression can be attributed to the size of the liver lobule, we investigated the patterns of expression of carbamoylphosphate synthase, glutamine synthase and glutamate dehydrogenase during postnatal development of the pig, a species in which the total number of lobules does not increase after birth. We demonstrate that lobular size increases 3-fold between 1 week and 8 months after birth. In the same developmental period the number of hepatocytes on the porto-central axis increases 2-fold, resulting in a 3-fold increase in cellular volume. However, the lobular patterns of expression of carbamoylphosphate synthase, glutamate dehydrogenase and glutamine synthase do not change anymore after 1 month, i.e., when lobular diameter is comparable to that in rat liver, showing that lobular size is not a major determinant of the heterogeneous patterns of expression of these enzymes. The adult patterns of expression of glutamine synthase, glutamate dehydrogenase and, in particular carbamoylphosphate synthase in the porcine liver resemble those of man. Changes in the enzyme activities of glutamate dehydrogenase and carbamoylphosphate synthase are not related to the lobular size. However, the 70% decrease of GS activity in the 8-month-old pigs corresponds with the gradual 2-3-fold decrease in the size of the GS-positive compartment during postnatal development. During adulthood GS activity increases again to values observed 1 week after birth demonstrating a 2-fold increase in cellular glutamine synthase content. The present data show that the pig is an excellent model to study the regulation and functional implication of zonation of gene expression in the human liver.

Outcome of regional and local ablative therapies for hepatocellular carcinoma: a collective review.

BACKGROUND: Transcatheter arterial (chemo) embolization (TACE), cryoablation (CA) and percutaneous ethanol injection (PEI) were the first regional and local ablative techniques that came into use for irresectable HCC. Radiofrequency ablation (RFA) and interstitial laser coagulation (ILC) followed and have now evolved rapidly. It would not be ethical to compare resection with ablation in patients well enough to undergo major surgery. Therefore, hepatic resection and hepatic transplantation remain the only curative treatment options for HCC. METHODS: On the basis of a Medline literature search and the authors' experiences, the principles, current status and prospects of TACE and local ablative techniques in HCC are reviewed. RESULTS: Complete tumour necrosis can be achieved in 60-100% of patients treated with PEI (70-100%), cryoablation (60-85%), RFA (80-90%) or ILC (70-97%). After TACE significant tumour response is achieved in 17-61.9% but complete tumour response is rare (0-4.8%) as viable tumour cells remain after TACE. Five-year survival rates are available for TACE (1-8%), PEI (0-70%) and cryoablation (40%). Only PEI and RFA were compared in one RCT. RFA was associated with fewer treatment sessions and a higher complete necrosis rate. Furthermore, all techniques are associated with low morbidity and mortality, but cryoablation seems to be associated with a higher morbidity rate. CONCLUSION: TACE has shown to be a valuable therapy with survival benefits in strictly selected patients with unresectable HCC. RFA and PEI are now considered as the local ablative techniques of choice for the treatment of, preferably small, HCC. When tumours are located close to bile ducts or large vessels, PEI remains a valuable therapy. Completeness of ablation can be more easily monitored during cryoablation and another advantage of cryoablation is the possibility of edge freezing. The results of ILC are comparable to RFA with only few side effects and high tumour response rates.

Assessment and improvement of liver specific function of the AMC-bioartificial liver.

UNLABELLED: The variety of methods for measuring bioactive mass and functionality of bioartificial livers (BAL) is confusing and prevents accurate comparison of reported data. Here we present a comparison of different hepatocyte quantification methods and propose that estimation of cell pellet volume after centrifugation generates a reliable, useful and fast method. In addition a correlation is made between several function tests performed in 26 bioreactors to assess their predictive value. The ammonia eliminating capacity was found to be most predictive for other liver functions, except for lidocaine elimination as a measure of mixed function oxidase activity, which should therefore be determined separately. The oxygen consumption test proved to be an easy and predictive parameter as well. The first generation of our BAL system needed further development to assure optimal treatment of acute liver failure (ALF) patients. Changes in the porcine hepatocyte isolation method and bioreactor loading as well as changes in bioreactor configuration, including use of different materials, resulted in a significantly improved level and maintenance of in vitro BAL function. A fourfold increase in ammonia eliminating capacity, which is only reduced to 75% after seven days of culturing, offers promising prospects for further clinical application. CONCLUSION: The current second generation of our BAL and improvement of hepatocyte isolation and testing protocols have led to a significant increase in the level as well as the maintenance of hepatocyte specific function in our BAL. Finally, consensus on definition of the bioactive mass to be loaded in the bioreactor and insight in the variation and reliability of the functional and metabolic parameters enhances comparison of the different types of bioartificial livers presented in literature.

['Artificial support in case of hepatic failure'; a report from the Dutch Health Council]. / 'Kunstmatige ondersteuning bij leverfalen'; een signalement van de Gezondheidsraad.

Acute liver failure has a high mortality (40-95%) depending on the cause. Emergency liver transplantation is the only way to improve survival: a one-year survival of 5o-6o%. In the past, many different modalities of artificial liver support have been studied. None of them appeared to be able to improve survival compared to maximal intensive care treatment. Two rather recent approaches are the development of a bioartificial liver (BAL), charged with billions of porcine liver cells, and albumin dialysis (MARS). A signalling report has been sent to the Dutch Minister of Health to resume the current position of BAL and MARS in the treatment of severe liver failure. The outcome is that no firm conclusions can yet be drawn as to the applicability of these modalities. Only two small-scale controlled clinical trials have been published on the MARS technique and the only published large-scale controlled clinical trial of a BAL in acute liver failure is not conclusive. On theoretical grounds, BAL treatment has more potential than MARS since a BAL will replace not only the failing hepatic detoxification but also the synthetic and metabolic functions. So far, no evidence has been found for transmission ofporcine pathogens to patients despite numerous phase 1 studies of bioartificial livers charged with porcine hepatocytes. More well-designed controlled clinical trials are needed. Therefore, the Dutch moratorium on xenotransplantation should be revised.

Metabolic studies of erythropoietin (Ep): I. A reliable micro assay method for Ep in rat plasma using fetal mouse liver cells (FMLC).

It is a well recognized problem that sample derived transferrin-bound iron (Tf-Fe) interferes with radio-iron incorporation into heme in the in vitro assay of erythropoietin (Ep) using fetal mouse liver cells (FMLC). This paper describes a mathematical procedure to correct for the unknown quantity of "cold" Tf-Fe in a plasma sample, being assayed for Ep in the FMLC. Excellent recovery of Ep activity was obtained with this method of correction, when testing solutions containing Step III sheep Ep with and without human Tf-Fe. The distorted dose response curves, obtained, when testing specimens of rat plasma, showed, after correction, linearity and parallelism to a Step II Ep dose response curve, permitting valid estimation of the potency ratio. Reproducibility of this method was found to be excellent. Accuracy was acceptable with plasma samples containing more than 50 mU Ep/ml. The FMLC and the exhypoxic polycythemic mouse assay were compared using several batches of rat plasma. Good agreement was found between the results in both assays. As only a small amount of sample is needed (0.2 ml plasma) this method enabled us to estimate EP activity in single rats serially without disturbing their plasma volume significantly.

Metabolic studies on erythropoietin (EP): II. The role of liver and kidney in the metabolism of Ep.

Half plasma disappearance time (HDT) of endogenous erythropoietin (Ep) was measured in single rats, using an in vitro assay system for EP. In rats treated with the hepatotoxic agent d-Galactosamine-HCl (GalN), a small but significant elevation of HDT was found as compared with control rats (164 and 105 min, respectively). In bilaterally nephrectomized rats mean HDT was significantly elevated: 266 min. Combination of nephrectomy and GalN treatment did not result in a significant further elongation of HDT (mean = 301 min). In experiments using isolated liver perfusion, rat livers (with and without GalN treatment) were shown to be unable to change perfusate Ep titre during 4 h of perfusion. It is concluded that hepatic degradation of Ep in rats is only minimal. The kidney however is important in the catabolism of Ep.

Blood coagulation in anhepatic pigs: effects of treatment with the AMC-bioartificial liver.

The function of a newly devised bioartificial liver (AMC-BAL) based on viable, freshly isolated porcine hepatocytes has been evaluated in anhepatic pigs. The aim of this study was to assess the contribution of BAL treatment on blood coagulation parameters. Pigs were anesthetized and a total hepatectomy was performed (n = 15). The infrahepatic caval vein and the portal vein were connected to the subdiaphragmatic caval vein using a three-way prosthesis. Animals received standard intensive care (control, n= 5), treatment with an empty BAL (device control, n= 5) or with a cell-loaded BAL (BAL-treatment, n= 5) for a period of 24 h starting 24 h after hepatectomy. Coagulation parameters studied concerned prothrombin time (PT), platelet count, the procoagulant system (factors (F)II, FV, FVII, FVIII and fibrinogen), anticoagulant system (AT III), fibrinolytic system (t-PA, PAI-1) as well as markers of coagulation factor activation (TAT complexes, prothrombin fragment F1 + 2). FII, FV, FVII, AT III and fibrinogen rapidly decreased after total hepatectomy in pigs in accordance with the anhepatic state of the animals. FVIII levels were not influenced by the hepatectomy. A mild drop in platelet count was seen in all groups. Treatment of anhepatic pigs with the cell-loaded BAL did not restore PT or clotting factor levels. TAT and F1 + 2 complexes, however, were significantly increased in this group. Levels of t-PA and PAI-1 were not influenced by cell-loaded BAL treatment. Treatment of anhepatic pigs with the AMC-BAL based on freshly isolated porcine hepatocytes does not result in an improved coagulation state due to extensive consumption of clotting factors. However, increased levels of TAT complexes and prothrombin fragments F1 + 2 during treatment of anhepatic pigs indicate synthesis and direct activation of coagulation factors, leading to thrombin generation. This demonstrates that this bioartificial liver is capable of synthesizing coagulation factors.

Long-term follow-up of antiviral combination therapy in chronic hepatitis B.

For patients with chronic hepatitis B e (HBe)-positive hepatitis, long-term results of pilot studies with lymphoblastoid interferon-alpha, acyclovir, or a combination, and of a randomized controlled trial of interferon/desciclovir combination therapy are presented. HBe seroconversion was observed in more than 40 percent of patients treated with combination therapy, 30 percent with interferon therapy, 18 percent with acyclovir, and 0 percent with no treatment. HBe reactivation occurred in two patients with cirrhosis. Hepatitis B surface seroconversion followed HBe seroconversion in 11 to 30 percent of treated patients. HBe seroconversion was significantly related to initial low levels of viral replication and to transient aminotransferase elevation during the second half of the interferon treatment of 16 weeks. Clinical improvement and persistent normalization of aspartate aminotransferase was observed in all patients with HBe seroconversion. Conversion to a state of virus latency (HBe negative) mostly occurred after therapy, suggesting that the specific immunologic host response had been brought about by the suppression of virus replication through antiviral agents. Recommendations for selection of patients for antiviral combination therapy are made on the basis of these long-term results.

No attenuation of ischemic and reperfusion injury in Kupffer cell-depleted, cold-preserved rat livers.

Activated Kupffer cells (KC) have been implicated in the damage sustained by preserved liver grafts during ischemia and reperfusion. The aim of this study was to compare ischemia/reperfusion injury in preserved, KC-depleted rat livers and preserved control livers, with special regard to sinusoidal endothelial cell (SEC) injury. Wistar rats were injected with liposome-encapsulated dichloromethylene diphosphonate, 48 hr before hepatectomy, to eliminate KC, or were withheld this pretreatment (controls). Livers were flushed with cold University of Wisconsin solution and after 0, 8, 16, or 24 hr of storage at 4 degrees C, were reperfused in a recirculation system with 200 ml of oxygenated Krebs-Henseleit solution at 37 degrees C for 90 min. Damage to SEC was measured by the uptake of hyaluronic acid (HA) from the perfusate and release of purine nucleoside phosphorylase (PNP). Perfusate samples were, furthermore, analyzed for aspartate aminotransferase (AST) and tumor necrosis factor-alpha. Carbon particles were infused in the perfusate to determine the phagocytotic capacity of KC. Biopsies were taken for histological examination and sections were stained with ED2 monoclonal antibodies to confirm the absence of KC. After 90 min of reperfusion, immediately after cold flush (t0), the uptake of HA was 72.2+/-2.3% and 69.3+/-1.3% in KC-depleted livers and in control livers, respectively (n.s.). After 8 hr of storage, HA uptake was 21.6+/-4.5% and 34.6+/-8.0%, respectively (n.s.). After 16 and 24 hr of storage and reperfusion, no uptake of HA was found in either KC-depleted or control livers, indicating abolished SEC function. PNP activities in the perfusate were higher in control livers (after 8 and 24 hr of storage), presumably due to release from damaged KC. No difference was found in AST and no tumor necrosis factor-alpha was measured in the perfusates of normal and KC-depleted livers. Electron microscopic studies showed that after 8 and 24 hr of storage and reperfusion, KC were activated and were able to phagocytose colloidal carbon. Our conclusion was that the elimination of Kupffer cells did not result in reduction of ischemic and reperfusion damage in livers preserved up to 24 hr, as assessed in vitro by SEC uptake of HA, PNP release, and AST release.

Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research.

Among the large range of organs involved in the field of tissue engineering (skin, blood vessels, cartilage, etc.) the liver has been given broad attention in the last decade. Liver support systems encompassing artificial and bioartificial systems are applied to treat patients with fulminant hepatic failure (FHF) as a bridge to orthotopic liver transplantation or to liver regeneration. To test safety, technical applicability and therapeutic effect of liver support systems, reliable animal models are needed. Due to the complexity of FHF many diverse attempts have been made to develop an adequate animal model to study liver failure, liver regeneration and liver support systems. In this paper an overview is given of the different models and their advantages and disadvantages are discussed. Suggestions are made for the most suitable large animal model to test liver support systems.

A survey of liver pathology in needle biopsies from HBsAg and anti-HBe positive individuals.

AIMS: To use laboratory data and liver biopsies, prospectively obtained from hepatitis B surface antigen (HBsAg) and anti hepatitis B e antigen (anti-HBe) positive patients, for the assessment of: (1) the relation between biopsy length/number of portal tracts and sampling error; (2) the relation between the severity of piecemeal necrosis and the new grading terminology (minimal, mild, moderate, and severe chronic hepatitis); and (3) liver pathology, which has not been studied in patients with this specific serological profile. METHODS: The study group (n = 174) included 104 patients with normal aminotransferase concentrations and no cases with clinically apparent cirrhosis. The specimen length and number of portal tracts were measured at light microscopy examination. Sampling error analysis was related to the discrepancies between aminotransferase concentrations versus histological grade. Detailed histological scorings were undertaken by the reference pathologist and compared with laboratory and hepatitis B virus (HBV) DNA precore sequence data. RESULTS: Sampling error seemed to be a constant feature, even for biopsies > or = 20 mm, but increased dramatically in biopsies < 5 mm long and/or containing less than four portal tracts. Between 25% and 30% of biopsies, graded as "mild" or "moderate" activity showed features of moderate and severe piecemeal necrosis, respectively. Ten per cent of the patients with normal aminotransferase values had stage III-IV hepatic fibrosis, and 20% had piecemeal necrosis. Only cytoplasmic, not nuclear, core antigen expression was a strong predictor of high hepatitis B viraemia. There was no association between precore stop codon mutations, grade/stage of liver disease, and hepatitis B core antigen (HBcAg) expression. CONCLUSIONS: The specimen available for light microscopical examination should be > 5 mm long and should contain more than four portal tracts. In addition, the new grading terminology might give the clinician an inappropriately mild impression of the severity of piecemeal necrosis. Furthermore, even in the presence of normal aminotransferase concentrations, considerable liver pathology can be found in 10-20% of HBsAg and anti-HBe positive individuals; such pathology is not associated with the occurrence of precore stop codon mutations.

Significant improvement of survival by intrasplenic hepatocyte transplantation in totally hepatectomized rats.

The effect of intrasplenic hepatocyte transplantation (HTX) was studied in an experimental model of acute liver failure in rats with chronic liver atrophy. Rats underwent a portacaval shunt operation on Day -14 to induce liver atrophy, and underwent total hepatectomy on Day 0 as a start of acute liver failure. Intrasplenic hepatocyte or sham transplantation was performed on Day -7,-3, or -1 (n = 4 to 6 per group). During the period following hepatectomy, mean arterial blood pressure was maintained above 80 mm Hg and hypoglycaemia was prevented. Severity of hepatic encephalopathy was assessed by clinical grading and EEG spectral analysis, together with determination of blood ammonia and plasma amino acid concentrations, and "survival" time. Histological examination of the spleen and lungs was performed after sacrifice. Intrasplenic hepatocyte transplantation resulted in a significant improvement in clinical grading in all transplanted groups (p < 0.05), whereas a significant improvement in EEG left index was seen only in the group with transplantation on Day -1 (p < 0.05). In contrast to hepatocyte transplantation 1 day before total hepatectomy, rats with hepatocyte transplantation 3 and 7 days before total hepatectomy showed a significant 3- and 2-fold increase in "survival" time compared to sham transplanted controls: HTX at Day -1: 7.5 +/- 0.3 h vs. 5.9 +/- 0.6 h (p > 0.05), HTX at Day -3: 19.7 +/- 3.7 h vs. 6.5 +/- 0.3 h (p < 0.05), and HTX at Day -7: 13.8 +/- 3.2 h vs. 6.3 +/- 0.3 h (p < 0.05). Furthermore, rats with hepatocyte transplantation on Day -3 and -7 showed significantly lower blood ammonia concentrations after total hepatectomy (p < 0.0001). Histological examination of the spleens after sacrifice showed clusters of hepatocytes in the red pulp. Hepatocytes present in the spleen for 3 and 7 days showed bile accumulation and spots of beginning necrosis. The present data show that in a hard model of complete liver failure in portacaval shunted rats, intrasplenic hepatocyte transplantation is able to prolong "survival" time significantly 2- to 3-fold. The relevance of this observation for human application is discussed.

Brain 31P NMR spectroscopy in the conscious rat.

Using a home-built head-body holder (HBH) which enables in vivo 31P NMR spectroscopy measurements on conscious rats, no significant changes were observed in the cerebral relative concentrations of ATP, phosphocreatine, phosphomonoesters, inorganic phosphate and intracellular pH during pentobarbital anesthesia in normal rats as compared to their conscious state.