Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.     

Effect of ortho-iodo sodium benzoate on hemoglobin-oxygen affinity in normal and ischemic myocardium

The effect of ortho-iodo sodium benzoate (OISB) on the oxyhemoglobin dissociation curve of coronary venous blood was studied in an isolated canine heart preparation perfused at a constant coronary blood flow. Changes in P-50 (millimeters of mercury) [the oxygen tension (pO2) at which hemoglobin is 50% saturated], were used to express hemoglobin-oxygen affinity. Intracoronary infusion of OISB (200, 400 and 800 mg/min) produced a dose-related increase in coronary venous P-50 and a concurrent increase in coronary venous pO2. In addition, OISB produced a significant decrease in heart rate and increase in coronary artery perfusion pressure. During cardiac pacing at 150, 190 and 230 beats/min, OISB (400 mg/min) significantly increased coronary venous P-50, myocardial oxygen exrraction (O2E) and oxygen consumption (MVO2) whereas coronary venous PO2 was not changed. Furthermore, a 5-minute intracoronary infusion of OISB (200 mg/min) during myocardial ischemia produced an increase in O2E, MVO2 and myocardial contractility with little change in coronary venous pO2. These results suggest that acute pharmacological manipulation of the oxyhemoglobin dissociation curve may enhance oxygen release to the myocardium while maintaining the effective driving pressure (as reflected in coronary venous pO2) for diffusion of oxygen to the myocardium.     

Hyperammonemic encephalopathy after chemotherapy. Survival after treatment with sodium benzoate and sodium phenylacetate

A 16-year-old boy had hyperammonemia and encephalopathy develop after high-dose chemotherapy for acute lymphoblastic leukemia. He was treated successfully with the ammonia-trapping agents sodium benzoate and sodium phenylacetate.     

Effect of sodium restriction on urinary excretion of cortisol and its metabolites in humans

The adrenal gland is involved in the control of urinary sodium excretion mainly via the secretion of the mineralocorticoid aldosterone. Although under certain conditions glucocorticoid seem to be also involved in the regulation of sodium homeostasis, there are contradictory reports on the relationship between cortisol secretion and sodium intake. Given recent findings linking regulation of physiological activity of steroids to the activity of specific enzymatic pathways, we have examined changes in urinary excretion of cortisol and its metabolites in eight healthy volunteers on a low sodium diet. Urinary steroids were measured with specific radioimmunoassays after extraction and chromatography (F and E) or after dilution (THF and THE). Excretion of cortisol (124 +/-41 nmol/day) was significantly lower on Day 2 (86 +/- 27 nmol/day, p < 0.01) and Day 7 (85 +/- 25 nmol/day, p < 0.01) of sodium restriction. On the same samples calculated ratios of THF/F (55 +/- 15; 61 +/- 22; 68 +/- 21) and E/F (2.5 +/- 0.6; 2.8 +/- 1.4; 3.0 +/- 1.3) reflecting the activity of 5 beta-reductase and 11 beta-hydroxysteroid dehydrogenase, respectively, showed significant increases in the former on both Days 2 and 7 and for the latter only on Day 7. This study supports the notion that sodium restriction decreases urinary cortisol excretion and provides evidence that increased activity of 5 beta-reductase and lowered metabolism by 11 beta-HSD are presumably the mechanisms of this decrease.     

Effect of dehydroepiandrosterone on brown adipose tissue and energy balance in mice

BACKGROUND: Psychotropic drug dosing regimens are often based on the pharmacokinetic elimination half-life of the compound. This implies that the pharmacokinetic half-life of the drug may be the critical or sole determinant of pharmacodynamic half-life. In the present study, we examined the safety and efficacy of once- versus twice-daily dosing regimens of the immediate-release formulation of venlafaxine, a serotonin and norepinephrine reuptake site blocker with a short elimination half-life. METHOD: Forty-eight patients with a diagnosis of DSM-IV major depressive episode were randomly assigned to once-daily (N = 25) versus twice-daily (N = 23) venlafaxine. Venlafaxine was started at 37.5 mg daily with specified increments up to 225 mg daily. Efficacy was rated using the Hamilton Rating Scale for Depression (HAM-D), the Montgomery-Asberg Depression Rating Scale (MADRS), and the Clinical Global Impressions scale (CGI). RESULTS: Twenty-one patients in each group completed 6 weeks of treatment. We observed a significant reduction in mean weekly HAM-D and MADRS scores at weeks 1 through 6 for both dosing groups (p < .001). There were no statistically significant differences in mean HAM-D or MADRS scores between dosing groups at any time point. There was, however, a nonsignificant trend for a more rapid reduction in the mean HAM-D score at week 2 (p < .06) and in the mean MADRS score at week 1 (p < .07) and week 2 (p < .09) in the b.i.d. dosing group. Similarly, there was a significant decrease in the CGI score at week 2 (p < .02) in the b.i.d. dosing group. The rate of adverse events was similar between treatment groups; the most common adverse events were transient nausea and headaches. CONCLUSION: These results indicate that the immediate-release formulation of venlafaxine may be safe and effective in some patients when used in a once-daily dose regimen. Moreover, the present results suggest that the short elimination half-life of immediate-release venlafaxine should not be the sole determinant for multiple daily dosing and that antidepressant activity may be more profoundly influenced by a drug's pharmacodynamic half-life than by its pharmacokinetic half-life.     

Effect of nicardipine, a Ca2+ channel blocker, on pyruvate dehydrogenase activity and energy metabolites during cerebral ischemia and reperfusion in gerbil brain

The purpose of this study was to determine if nicardipine, a calcium ion channel blocker, affects pyruvate dehydrogenase (PDH) activity and improves energy metabolism during cerebral ischemia and reperfusion. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 60 min followed by reperfusion up to 120 min in gerbils. Nicardipine (1 mg/kg) or saline (vehicle-treated) was given to gerbils 30 min prior to the occlusion of the common carotid arteries. PDH activity and metabolites (ATP, PCr, and lactate) were measured in cortex prior to ischemia, immediately following ischemia, and after each reperfusion period. After 60 min ischemia, PDH activity increased in both groups, and was significantly higher in the nicardipine-treated group. After 20 min reperfusion, PDH activity in the nicardipine-treated group recovered to control levels, whereas, the PDH activity in the vehicle-treated group remained elevated, and was higher than the nicardipine-treated animals. At 60 and 120 min reperfusion, the activities in the vehicle-treated group were significantly below control levels, there were no differences, however, between the two groups. ATP and PCr concentrations were markedly depleted immediately after ischemia in both groups. ATP levels at 20 min reperfusion and PCr levels at 60 min reperfusion were significantly higher in the nicardipine-treated group. Lactate concentrations in both groups increased 7-8 fold, similarly, immediately after ischemia. During reperfusion, the lactate remained elevated in both groups, though the levels in the nicardipine-treated group were lower than those in the vehicle-treated group, but not significantly. Nicardipine treatment normalized PDH activity quickly and improved energy metabolism after reperfusion.     

Effect of dietary sodium phytate on the hepatic and serum levels of lipids and on the hepatic activities of NADPH-generating enzymes in rats fed on sucrose

The effect was investigated of dietary 0.5% sodium phytate on the hepatic and serum lipid status in rats fed on sucrose for 29 or 30 days. The increase in hepatic weight, levels of hepatic total lipids, triglyceride, cholesterol, serum triglyceride and phospholipid, and the activities of hepatic NADPH-generating enzymes due to sucrose feeding were generally depressed by dietary sodium phytate.     

Effect of doxapram on the action of other drugs and the hepatic drug-metabolizing system in mice

Effects of doxapram, a respiratory stimulant, on the action of other drugs and the activity of the hepatic drug-metabolizing enzyme were studied in mice. The hypothermic effect induced by aminopyrine and the muscle relaxant effect induced by meprobamate were potentiated by the pretreatment with doxapram 60 min before. Furthermore, doxapram significantly enhanced the lethalities of picrotoxin and strychnine and the analgesic actions of aminopyrine and morphine. The plasma concentration of aminopyrine or pentobarbital in doxapram-treated mice was higher than those in untreated mice, and the plasma concentration of normustard related to an active metabolite of cyclophosphamide after the administration of cyclophosphamide was lower in doxapram-treated mice. On the other hand, doxapram (50 mg/kg, i.p.) reduced remarkably the activities of aminopyrine N-demethylase and aniline hydroxylase in the hepatic 9,000xg supernatant fraction, and also reduced the cytochrome P-450 contents in hepatic microsomes. However, no significant alteration by doxapram was observed on the activities of NADH-ferricyanide reductase and NADPH-cytochrome c reductase and cytochrome b5 contents. It seems likely that the mechanisms of the interaction between doxapram and combined drugs involved the depression of the hepatic drug-metabolizing system in microsomes and a subsequent variation of drug level in the plasma.     

Clinical and biochemical studies on periodic hyperammonemia with hyperlysinemia and homocitrullinuria

An 18-year-old mentally and physically retarded boy, suffering from episodes of anorexia, vomiting, coma and convulsion which have been severer with advance in age, had periodic hyperammonemia, hyperlysinemia and homocitrullinuria. Blood cell arginase activity of the patient on normal diet was markedly reduced after an oral load of L-lysine. The oral loading tests of L-lysine revealed hyperammonemia, hyperlysinemia, hyperargininemia, hypercitrullinemia and homocitrullinuria. Etiology of metabolic error of our patient was discussed in reference to lysine-urea cycle.     

Effect of the peroxisome proliferator ciprofibrate on lipid peroxidation and 8-hydroxydeoxyguanosine formation in transgenic mice with elevated hepatic catalase activity

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens which have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have produced a transgenic mouse line that has elevated catalase activity specifically in the liver. In this study, we have examined if catalase overexpression influences the induction of lipid peroxidation or oxidative DNA damage, two mechanisms which have been hypothesized to be important in the carcinogenesis by peroxisome proliferators. Transgenic mice or non-transgenic litter mates were fed either 0.01% ciprofibrate or a control diet for 21 days. The activities of fatty acyl CoA oxidase and lauric acid hydroxylase were not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. Hepatic lipid peroxidation was estimated by quantifying the concentrations of malondialdehyde and conjugated dienes. Ciprofibrate treatment did not affect either endpoint, but catalase overexpression increased the concentrations of malondialdehyde (in untreated mice only) and conjugated dienes (in both untreated and ciprofibrate-fed mice). Oxidative DNA damage was estimated by quantifying 8-hydroxydeoxyguanosine (8-OHdG) by high-performance liquid chromatography/electrochemical detection. Ciprofibrate treatment significantly increased hepatic 8-OHdG concentrations, in agreement with several previous studies, but catalase overexpression did not significantly affect them, although 8-OHdG concentrations were decreased 50% in untreated mice. These results imply that the metabolism of hydrogen peroxide by catalase is not an important factor in the development of hepatic lipid peroxidation. The decrease in hepatic 8-OHdG in untreated transgenic mice and the increase seen after ciprofibrate administration imply that hydrogen peroxide is important in the formation of 8-OHdG. While the lack of decreased 8-OHdG levels in ciprofibrate-treated transgenic mice does not support this conclusion, it is possible that catalase levels were not sufficiently high to affect this endpoint. Transgenic mice with higher hepatic catalase activities may be required to resolve this issue.     

Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry

Liver toxicity following an overdose of acetaminophen is frequently considered a model for drug-induced hepatotoxicity. Extensive studies over many years have established that such toxicity is well correlated with liver protein arylation by acetaminophen metabolites. Identification of protein targets for covalent modifications is a challenging but necessary step in understanding how covalent binding could lead to liver toxicity. Previous approaches suffered from technical limitations, and thus over the last 10 years heroic efforts were required to determine the identity of only a few target proteins. We present a new mass spectrometry-based strategy for identification of all target proteins that now provides a comprehensive survey of the suite of liver proteins modified. After administration of radiolabeled acetaminophen to mice, the proteins in the liver tissue lysate were separated by two-dimensional polyacrylamide gel electrophoresis. In-gel digestion of the radiolabeled gel spots gave a set of tryptic peptides, which were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Interrogation of data bases based on experimentally determined molecular weights of peptides and product ion tags from postsource decay mass spectra was employed for the determination of the identities of modified liver proteins. Using this method, more than 20 new drug-labeled proteins have been identified.     

Rapid induction of hepatic acetyl-CoA carboxylase activity after estradiol benzoate injection in the quail

The time course of hepatic Acetyl-CoA carboxylase activity as well as hepatic and plasmatic fatty acids concentrations following a single injection of estradiol benzoate (EB, 0.2 mg/kg) was studied in the quail. Acetyl-CoA carboxylase activity increases rapidly and reaches its peak 3 h after the injection of EB. Similarly, hepatic and plasmatic fatty acids concentrations are significantly increased 6 h after the hormonal injection and attain their highest level 18 h later. These results suggest that estrogen affects the hepatic fatty acids biosynthesis by regulating the conversion of acetyl-CoA to malonyl-CoA.     

Enzyme activities and metabolites along the kynurenine pathway in mice with Harding-Passey melanoma

The effects of cyclophosphamide were studied 2, 5 and 9 days after terminating a two-week every-second-day intraperitoneal medication in young, male rats. The cytostatic effect was assessed by counting white blood cells (WBC) in arterial blood. WBC counts were reduced by 72% of control values at 2 days and regained normal values at 9 days after an overshooting leucocytosis at 5 days. Compared to control animals, the treated rats had significantly reduced body weights, metaphyseal bending strength and longitudinal growth of the femur at all time intervals observed. The torsional strength of the femur diaphysis and the strength of wounded skin were not affected at 2 days, but significantly reduced at 5 and 9 days. The tensile strength of intact skin was not found to be affected by the drug. From the 5th to the 9th day after ending medication, the curves for control and treated animals were assuming parallel slopes regarding metaphyseal bone strength, longitudinal bone growth and tensile strength of wounds. This may indicate reversion of the drug effects approximately one week after terminating medication.     

Cocaine and its metabolites constrict cerebral arterioles in newborn pigs

We examined the effect of cocaine and several of its metabolites on cerebral arterioles in newborn pigs and evaluated the sympathomimetic properties of each of the compounds as a vasoactive mechanism. After piglets were equipped with closed cranial windows, compounds were suffused over the brain surface and pial arteriolar diameter (base line, approximately 100 microns) was recorded. Cocaine, cocaethylene, norcocaine, ecogonine, benzoylecgonine and ecgonine methylester each caused a dose-dependent (10(-8) M to 10(-4) M) decrease in pial arteriolar diameter: maximum percent reductions in diameter induced by each compound (10(-4) M) were, respectively, 12 +/- 1, 12 +/- 2, 11 +/- 1, 7 +/- 1, 7 +/- 2 and 5 +/- 1. In analyzing the dose-response curves, cocaethylene was the most potent vasoconstrictor, followed by cocaine, norcocaine and then ecogonine, benzoylecgonine and ecgonine methylester. Cerebral vasoconstriction induced by topically applied norepinephrine was enhanced by cocaine, norcocaine and cocaethylene, but not by the other three metabolites. Topical application of phentolamine failed to block vasoconstriction elicited by cocaine or its metabolites, although it did block vasoconstriction elicited by norepinephrine. These observations indicate that cocaine and its metabolites constrict the immature cerebrovasculature by a non-sympathomimetic mechanism.     

Regional variations in cerebral proton spectroscopy in patients with chronic hepatic encephalopathy

Regional variations in proton magnetic resonance spectroscopy (MRS) were assessed in 26 patients and 14 healthy volunteers using a two dimensional chemical shift imaging technique. Patients were classified as being neuropsychiatrically unimpaired, or as having subclinical or overt chronic hepatic encephalopathy (CHE). Peak area ratios of choline (Cho), glutamine and glutamate (Glx) and N-acetylaspartate (NAA) relative to creatine (Cr) were measured. Significant reductions in mean Cho/Cr and elevations in mean Glx/Cr were observed in the patient population, which correlated with the severity of CHE. There were significant regional variations in these metabolite ratios with the mean Cho/Cr lowest in the occipital cortex and the mean Glx/Cr highest in the basal ganglia. NAA/Cr remained relatively constant in all areas of the brain analysed. The regional variation in the metabolite ratios suggests that spectral information from more than one voxel may be useful in the assessment of patients with CHE.     

Early effects of sodium iodate injection on ERG in mice

The electroretinogram (ERG) was recorded in 15 ICR albino mice before and 2, 4 and 24 h after injection of 40 mg/kg of NaIO3 and the results were compared with the corresponding ultrastructural findings. Both a- and b-wave amplitudes decreased at 2 h after injection and increased at 4 h to the levels before injection and further increased at 24 h after injection. The b-wave threshold intensity increased by 2.1 log units at 2 h after injection. Thereafter, it decreased once at 4 h and significantly increased at 24 h by 3.4 log units. Electron microscopic examination showed swollen cytoplasmic organelles of the retinal pigment epithelial cells at 4 h after injection. It was thus demonstrated that an early effect of NaIO3 on the electrical response of the mouse retina was more conspicuous than histopathological changes.     

Effects of infused sodium lactate on glucose and energy metabolism in healthy humans

To assess the effects of lactate on glucose metabolism, sodium lactate (20 mumol.kg-1.min-1) was infused into healthy subjects in basal conditions and during application of a hyperinsulinaemic (6 pmol.kg-1.min-1) euglycaemic clamp. Glucose rate of appearance (GRa) and disappearance (GRd) were measured from plasma dilution of infused U- 13C glucose, and glucose oxidation (G(ox)) from breath 13CO2 and plasma 13C glucose. In basal conditions, lactate infusion did not alter G(ox) (8.8 +/- 0.9 vs 9.2 +/- 1.1 mumol.kg-1.min-1), while GRa slightly decreased from 15.2 +/- 0.8 basal to 13.9 +/- 0.9 mumol.kg-1.min-1 after lactate (p < 0.05). During a hyperinsulinaemic clamp, hepatic glucose production was completely suppressed with or without lactate. Lactate decreased G(ox) from 17.1 +/- 0.4 to 13.4 +/- 1.2 mumol.kg-1.min-1 (p < 0.05), whereas GRd was unchanged (39.7 +/- 3.6 vs 45.6 +/- 2.6 mumol.kg-1.min-1. It is concluded that infusion of lactate in basal conditions does not increase GRa or interfere with peripheral glucose oxidation, and that during hyperinsulinaemia lactate decreases glucose oxidation but does not alter hepatic or peripheral insulin sensitivity.