Search

Elevated liver enzymes are a common scenario encountered by physicians in clinical practice. For many physicians, however, evaluation of such a problem in patients presenting with no symptoms can be challenging. Evidence supporting a standardized approach to evaluation is lacking. Although alterations of liver enzymes could be a normal physiological phenomenon in certain cases, it may also reflect potential liver injury in others, necessitating its further assessment and management. In this article, we provide a guide to primary care clinicians to interpret abnormal elevation of liver enzymes in asymptomatic patients using a step-wise algorithm. Adopting a schematic approach that classifies enzyme alterations on the basis of pattern (hepatocellular, cholestatic and isolated hyperbilirubinemia), we review an approach to abnormal alteration of liver enzymes within each section, the most common causes of enzyme alteration, and suggest initial investigations.

Summary Background Plasma adiponectin is decreased in NASH patients and the mechanism(s) for histological improvement during thiazolidinedione treatment remain(s) poorly understood. Aim To evaluate the relationship between changes in plasma adiponectin following pioglitazone treatment and metabolic/histological improvement. Methods We measured in 47 NASH patients and 20 controls: (i) fasting glucose, insulin, FFA and adiponectin concentrations; (ii) hepatic fat content by magnetic resonance spectroscopy; and (iii) peripheral/hepatic insulin sensitivity (by double-tracer oral glucose tolerance test). Patients were then treated with pioglitazone (45 mg/day) or placebo and all measurements were repeated after 6 months. Results Patients with NASH had decreased plasma adiponectin levels independent of the presence of obesity. Pioglitazone increased 2.3-fold plasma adiponectin and improved insulin resistance, glucose tolerance and glucose clearance, steatosis and necroinflammation (all P

Although recognized for more than 150 years, acute acalculous cholecystitis (AAC) remains an elusive diagnosis. This is likely because of the complex clinical setting in which this entity develops, the lack of large prospective controlled trials that evaluate various diagnostic modalities, and thus dependence on a small data base for clinical decision making. AAC most often occurs in critically ill patients, especially related to trauma, surgery, shock, burns, sepsis, total parenteral nutrition, and/or prolonged fasting. Clinically, AAC is difficult to diagnose because the findings of right upper-quadrant pain, fever, leukocytosis, and abnormal liver tests are not specific. AAC is associated with a high mortality, but early diagnosis and intervention can change this. Early diagnosis is the crux of debate surrounding AAC, and it usually rests with imaging modalities. There are no specific criteria to diagnose AAC. Therefore, this review discusses the imaging methods most likely to arrive at an early and accurate diagnosis despite the complexities of the radiologic modalities. A pragmatic approach is vital. A timely diagnosis will depend on a high index of suspicion in the appropriate patient, and the combined results of clinical findings (admittedly nonspecific), plus properly interpreted imaging. Sonogram (often sequential) and hepatic iminodiacetic acid scans are the most reliable modalities for diagnosis. It is generally agreed that cholecystectomy is the definitive therapy for AAC. However, at times a diagnostic/therapeutic drainage via interventional radiology/surgery may be necessary and life-saving, and may be the only treatment needed.

Pioglitazone treatment improves insulin resistance (IR), glucose metabolism, hepatic steatosis, and necroinflammation in patients with nonalcoholic steatohepatitis (NASH). Because abnormal lipid metabolism/elevated plasma free fatty acids (FFAs) are important to the pathophysiology of NASH, we examined the impact of pioglitazone therapy on adipose tissue insulin resistance (Adipo-IR) during the treatment of patients with NASH. To this end, we assessed glucose/lipid metabolism in 47 patients with impaired glucose tolerance/type 2 diabetes mellitus and NASH and 20 nondiabetic controls. All individuals underwent a 75-g oral glucose tolerance test (OGTT) in which we measured glucose tolerance, IR, and suppression of plasma FFAs. We also measured Adipo-IR index (fasting, FFAs × insulin), hepatic fat by magnetic resonance spectroscopy, and liver histology (liver biopsy). Patients were randomized (double-blind) to diet plus pioglitazone (45 mg/day) or placebo for 6 months, and all measurements were repeated. We found that patients with NASH had severe Adipo-IR and low adiponectin levels. Fasting FFAs were increased and their suppression during the OGTT was impaired. Adipo-IR was strongly associated with hepatic fat (r= 0.54) and reduced glucose clearance both fasting (r=0.34) and during the OGTT (r=0.40, all P

Ethanol ingestion during pregnancy elicits damage to the developing brain, some of which appears to result from enhanced apoptotic death of neurons. A consistent characteristic of this phenomenon is a highly differing sensitivity to ethanol within specific neuron populations. One possible explanation for this “selective vulnerability” could be cellular variations in glutathione (GSH) homeostasis. Prior studies have illustrated that ethanol elicits apoptotic death of neurons in the developing brain, that oxidative stress may be an underlying mechanism, and that GSH can be neuroprotective. In the present study, both multiphoton microscopy and flow cytometry demonstrate a striking heterogeneity in GSH content within cortical neuron populations. Ethanol differentially elicits apoptotic death and oxidative stress in these neurons. When neuron GSH content is reduced by treatment with butathione sulfoxamine, the ethanol-mediated enhancement of reactive oxygen species is exacerbated. Sorting of cells into high- and low-GSH populations further exemplifies ethanol-mediated oxidative stress whereby apoptotic indices are preferentially elevated in the low-GSH population. Western blot analysis of the low-GSH subpopulations shows higher ethanol-mediated expression of active caspase 3 and 24-kDa PARP-1 fragments compared with the high-GSH subpopulation. In addition, neuronal content of 4-hydroxynonenal adducts is higher in low-GSH neurons in response to ethanol. These studies suggest that GSH content is an important predictor of neuronal sensitivity to ethanol-mediated oxidative stress and subsequent cell death. The data support the proposition that the differences in proapoptotic responses to ethanol within specific neuron populations reflect a heterogeneity of neuron GSH content.

The effects of oral contraceptive steroids (OCS) on the disposition and elimination of lorazepam, oxazepam, and chlordiazepoxide were examined. Lorazepam and oxazepam are metabolized via glucuronidation while chlordiazepoxide is metabolized by oxidation in the liver. The disposition and elimination of lorazepam, oxazepam, and chlordiazepoxide was studied in females not taking OCS and females taking OCS (norethindrone acetate, 1 mg; ethinyl estradiol, 50 micrograms) for 6 months or more. The t1/2 (beta) for lorazepam was significantly reduced in women taking OCS (6.0 +/- 3.1 vs. 14.0 +/- 6.2 hr) (p less than 0.005) as compared to controls, and the t1/2 (beta) for oxazepam was reduced in women taking OCS (7.71 +/- 3.23 vs. 12.09 +/- 5.08 hr) as compared to controls, but did not reach statistical significance. The plasma clearance of both lorazepam and oxazepam was significantly increased in women taking OCS [(288.9 +/- 165.9 vs. 77.5 +/- 3.29 ml per min) (p less than 0.01) and (251.2 +/- 106.9 vs. 97.86 +/- 69.4 ml per min) (p less than 0.01), respectively] as compared to controls. The volumes of distribution of lorazepam and oxazepam were significantly increased in women taking OCS (p less than 0.05) while plasma binding of these drugs was similar in both groups. In contrast, the t1/2 (beta) of chlordiazepoxide was significantly prolonged (20.58 +/- 8.08 vs. 11.63 +/- 5.91 hr) (p less than 0.05), and the plasma clearance was significantly reduced (13.41 +/- 4.69 vs. 33.22 +/- 12.37 ml per min) (p less than 0.05) in the OCS group as compared to controls. The volumes of distribution of chlordiazepoxide were similar in both groups, and the plasma binding of chlordiazepoxide tended to be lower in the OCS group but did not reach statistical significance. We conclude that OCS exert a differential effect on the elimination of benzodiazepines, whereby oxidation of chlordiazepoxide is impaired while the glucuronidation of lorazepam and oxazepam is enhanced by OCS.

Drug-induced liver injury (DILI) is a problem of increasing significance, but has been a long-standing concern in the treatment of tuberculosis (TB) infection. The liver has a central role in drug metabolism and detoxification, and is consequently vulnerable to injury. The pathogenesis and types of DILI are presented, ranging from hepatic adaptation to hepatocellular injury. Knowledge of the metabolism of anti-TB medications and of the mechanisms of TB DILI is incomplete. Understanding of TB DILI has been hampered by differences in study populations, definitions of hepatotoxicity, and monitoring and reporting practices. Available data regarding the incidence and severity of TB DILI overall, in selected demographic groups, and in those coinfected with HIV or hepatitis B or C virus are presented. Systematic steps for prevention and management of TB DILI are recommended. These include patient and regimen selection to optimize benefits over risks, effective staff and patient education, ready access to care for patients, good communication among providers, and judicious use of clinical and biochemical monitoring. During treatment of latent TB infection (LTBI) alanine aminotransferase (ALT) monitoring is recommended for those who chronically consume alcohol, take concomitant hepatotoxic drugs, have viral hepatitis or other preexisting liver disease or abnormal baseline ALT, have experienced prior isoniazid hepatitis, are pregnant or are within 3 months postpartum. During treatment of TB disease, in addition to these individuals, patients with HIV infection should have ALT monitoring. Some experts recommend biochemical monitoring for those older than 35 years. Treatment should be interrupted and, generally, a modified or alternative regimen used for those with ALT elevation more than three times the upper limit of normal (ULN) in the presence of hepatitis symptoms and/or jaundice, or five times the ULN in the absence of symptoms. Priorities for future studies to develop safer treatments for LTBI and for TB disease are presented.

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron gamma-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of gamma-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrocyte up to 517% (p < 0.05). Ethanol increased both gamma-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the gamma-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.

Background: This multicenter prospective, randomized, double-blind placebo-controlled trial was designed to evaluate the effectiveness of polyenylphosphatidylcholine against the progression of liver fibrosis toward cirrhosis in alcoholics. Seven hundred eighty-nine alcoholics with an average intake of 16 drinks per day were enrolled. To control excessive drinking, patients were referred to a standard 12-step–based alcoholism treatment program, but most patients refused to attend. Accordingly, study follow-up procedures incorporated the essential features of the brief-intervention approach. An overall substantial and sustained reduction in drinking was observed. Hepatic histological and other findings are described in a companion article. Methods: Patients were randomized to receive daily three tablets of either polyenylphosphatidylcholine or placebo. Monthly follow-up visits included an extensive session with a medical nurse along with brief visits with a study physician (hepatologist or gastroenterologist). A detailed physical examination occurred every 6 months. In addition, telephone consultations with the nurse were readily available. All patients had a liver biopsy before entry; a repeat biopsy was scheduled at 24 and 48 months. Results: There was a striking decrease in average daily alcohol intake to approximately 2.5 drinks per day. This was sustained over the course of the trial, lasting from 2 to 6 years. The effect was similar both in early dropouts and long-term patients, i.e., those with a 24-month biopsy or beyond. Conclusions: In a treatment trial of alcoholic liver fibrosis, a striking reduction in alcohol consumption from 16 to 2.5 daily drinks was achieved with a brief-intervention approach, which consisted of a relative economy of therapeutic efforts that relied mainly on treatment sessions with a medical nurse accompanied by shorter reinforcing visits with a physician. This approach deserves generalization to address the heavy drinking problems commonly encountered in primary care and medical specialty practices.

Vitamin E and vitamin C treatment improves fibrosis in patients with nonalcoholic steatohepatitis

In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P

Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by diffuse fatty infiltration and inflammation. The exact prevalence of NASH is unclear, but it is becoming more evident that the disease is much more common than previously thought. Although generally a benign, indolent process, it can progress to advanced liver disease in approximately 15-20% of patients. Clinical characteristics associated with NASH include obesity, hyperlipidemia, diabetes mellitus, and hypertension, all of which have been associated with underlying insulin resistance. Typically, this disease becomes evident in the fourth or fifth decade of life with an equal sex predilection. NASH is thought to be caused, in part, by impaired insulin signaling, leading to elevated circulating insulin levels and subsequent altered lipid homeostasis. This process is likely multifactorial and includes both genetic and environmental factors. Treatment options to date are limited and are based on very small clinical trials. Current investigations are focusing on improving the underlying insulin resistance that has been associated with NASH as well as other therapies that decrease oxidative stress or improve hepatocyte survival.

Background 4-Hydroxynonenal (HNE), a toxic lipid peroxidation product, has been implicated in mitochondrial damage in rat liver by ethanol consumption. The present study assessed the effects of short-term in vivo ethanol exposure on HNE detoxification by mitochondrial glutathione S-transferase (GST). Methods Male Sprague Dawley® rats were administered 5 doses of ethanol (4 g/kg) at 12 hr intervals by gavage. Pair-fed rats that received isocaloric dextrose instead of ethanol served as controls. Mitochondrial and submitochondrial fractions were prepared from the livers. Mitochondrial contents of HNE and HNE-glutathione conjugate were measured by high-performance liquid chromatography. GST isoforms were identified by Western blots in submitochondrial fractions. Results Whereas there was an 80% increase in mitochondrial HNE content after ethanol consumption, there was a 42% decrease in the content of HNE-glutathione conjugate, compared with controls (p < 0.05). After ethanol exposure, the GST activities toward HNE in intact mitochondria and in the membranous fraction were decreased by 37% and 45% (p < 0.05), respectively, whereas that in the aqueous fraction was unchanged. Kinetic analysis of HNE conjugation by the membrane-associated GST showed that ethanol decreased the Vmax nearly by half (p < 0.05), whereas it did not affect the Km. HNE conjugation by the aqueous GST demonstrated a higher Km than that of the membrane-associated GST, although its kinetics were not significantly altered by ethanol. Immunochemical analysis with Western blots demonstrated that both the membranous and the aqueous fractions of mitochondria contain GST-α and GST-μ isoforms, whereas GST-π was absent. Conclusions HNE detoxification by mitochondrial GST is compromised by short-term ethanol consumption, which may contribute to elevated mitochondrial HNE content and hence its toxicity in the ethanol-exposed liver.

Background: In utero ethanol exposure causes abnormal fetal brain development that may partly be due to enhanced cell death. The mechanisms underlying this remain to be defined, but ethanol-induced oxidative stress may play a role. The following studies investigated the effects of short-term in utero ethanol exposure on fetal brain mitochondrial events that are known to elicit apoptotic cell death. Evidence is presented suggesting that 4-hydroxynonenal (HNE), a toxic product of lipid oxidation, is a causal factor in the observed mitochondrial damage. Methods: Mitochondria were isolated from control and ethanol-exposed fetal brains (days 17 and 18 of gestation). Permeability transition was determined spectrophotometrically, and cytochrome c and apoptosis-inducing factor (AIF) release were assessed by Western blotting. Caspase-3 activity and DNA fragmentation were determined both as markers for mitochondrially mediated apoptosis and as consequences of cytochrome c and AIF release. Results: Maternal ethanol intake caused an increase in mitochondrial permeability transition, and this was accompanied by cytochrome c and AIF release from fetal brain mitochondria that exceeded control values by 62 and 25%, respectively (p < 0.05). In utero ethanol exposure resulted in a 30% increase in caspase-3 activity and a 25% increase in DNA fragmentation (p < 0.05) in the fetal brain. HNE levels were increased by 23% (p < 0.05) in mitochondria by in vivo ethanol exposure. In vitro treatment of fetal brain mitochondria with HNE (25–100 μM) also caused increases in mitochondrial permeability transition, as well as dose-dependent releases of cytochrome c and AIF. Conclusions: These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.

Background: Recent reports have suggested that food deprivation may contribute to acetaminophen hepatotoxicity by shunting drug detoxification from the conjugative to the potentially toxic oxidative pathways. Methods: This study assessed this concept in a prospective study of food restriction of 500 calories/day over 5 days and also of 1000 calories/day over 13 days. Obese, otherwise normal, individuals received 2 g acetaminophen orally at the start and again after food restriction. Sequential liver tests, as well as serum and urine acetaminophen and its derivatives were measured. Results: In both food-restricted groups there was no evidence of any change in the elimination or in the metabolic pattern of acetaminophen removal. Liver tests remained normal. The average weight loss was about 6 pounds. Conclusions: Our data, with this brief, but major degree of food restriction, and this load of acetaminophen (half-maximal daily dose), do not demonstrate an effect of caloric restriction on acetaminophen disposition.

Aims The primary objective of this study was to describe the single dose pharmacokinetics of ribavirin in subjects with normal liver function and those with various degrees of stable chronic liver disease. Additionally this study assessed the safety and tolerability of ribavirin in this population. Methods Single oral 600 mg doses of ribavirin were administered to healthy male and female volunteers (n = 6) and patients with stable chronic liver disease (n = 17), in a parallel group study. Pharmacokinetic sampling and tolerability assessments were performed up to 168 h post dose. Results Single oral doses of 600 mg ribavirin were well tolerated by healthy volunteers and patients with varying degrees of hepatic dysfunction. Although mean Cmax increased with the severity of hepatic dysfunction, there was no change in extent of absorption or renal clearance of ribavirin. Conclusions There are no pharmacokinetic reasons for initial dose adjustment of ribavirin in patients with hepatic dysfunction.

Background: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. Methods and Results: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 μM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. Conclusions: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.

Trazodone (Desyrel) is a second-generation, nontricyclic antidepressant that has been in use in North America since the early 1980s. It has the advantage of being more sedating and having less anticholinergic side effects than other secondary amines in the piperazine class, namely, desipramine and nortriptyline. Five previous cases of trazodone hepatotoxicity have been reported in the literature, one describing chronic damage and the others, more acute cellular and cholestatic injury. We describe a case of acute reversible liver injury with the use of trazodone. This case is unique in that injury occurred after protracted (18 months) drug use and while the patient was on corticosteroids. Moreover, the diagnosis was confirmed by an inadvertent challenge with trazodone. This case reports not only a well documented instance of trazodone-induced liver injury, but also serves as a basis for a brief discussion of mechanisms, clinical monitoring, and therapy in drug-induced hepatotoxicity.

This study addresses the role of the lipid peroxidation product, 4-hydroxynonenal (HNE), in ethanol-related damage of cytochrome c oxidase (COX) in vivo. It utilizes an animal model with acute ethanol exposure in which HNE levels in liver mitochondria are strikingly increased. Pregnant female Sprague-Dawley rats were administered 5 doses of ethanol (4 gm/kg, po at 12-hour intervals) beginning on day 17 of gestation and were sacrificed on day 19. Controls were pair-fed and received dextrose isocaloric to ethanol. Mitochondria were isolated from maternal and fetal livers and COX activities were measured spectrophotometrically. Compared with the pair-fed controls, COX activity was decreased with exposure to ethanol by 25% in maternal rats and 43% in fetal rats (P

The role of 4-hydroxynonenal (HNE), a major lipid peroxidation product, in oxidative damage to mitochondrial cytochrome c oxidase (COX) was examined. Oxidative stress was induced in mitochondria isolated from livers of male Sprague-Dawley rats by tert-butylhydroperoxide (t-BHP). COX activity was inhibited, with a concomitant increase in endogenous HNE level in mitochondria. COX activity was also inhibited following incubation of mitochondria with 50-450 microM HNE. Blocking HNE degradation intensified COX inhibition by HNE and by t-BHP-induced oxidative stress, the latter accompanied by a simultaneous increase in endogenous HNE production. On the other hand, COX inhibition by HNE was markedly reduced by potentiating HNE degradation via enhancing conjugation of HNE with reduced glutathione (GSH). Incubation of purified COX with 10-400 microM HNE resulted in HNE adduct formation with specific subunits of COX, correlated with inhibition of the enzyme activity. These data suggest that HNE may inhibit mitochondrial COX by forming adducts with the enzyme, and that this could be one mechanism underlying mitochondrial damage caused by oxidative stress. The findings also illustrate a role for GSH in protecting mitochondria from the deleterious effects of HNE.

Fetal ethanol (E) exposure has well documented deleterious effects on brain development, yet it is uncertain if the neurotoxicity of maternal E consumption is generated by E itself, by its primary metabolite acetaldehyde (AcHO), or both. The current studies present evidence that homogenates of immature rat brains can generate AcHO via a catalase (CAT)-mediated reaction and that AcHO may be produced in vivo by this system. Homogenates of day 19 fetal rat brain were incubated with E (50 mM). When incubated with CAT inhibitors (sodium azide or 3-aminotriazole), AcHO formation was blocked, whereas neither the alcohol dehydrogenase inhibitor, 4-methylpyrazole, nor P-450 inhibitors decreased AcHO production. Three hours after one oral dose of E (4 g/kg) to a pregnant dam (gestation day 19), AcHO levels in fetal brain increased to 14.28 +/- 1.82 nM/g tissue. Baseline CAT activity in day 19 fetal brains was 4.5 times adult values (p < 0.05). Western blot analysis determined that CAT protein level in the day 19 fetal brain exceeded that in adult brain by 2.5 times. One hour after a single dose of E, CAT activity in day 19 fetal brain increased by 8.2 units/mg protein. In 5-day-old neonatal brains during the "third trimester" brain growth spurt, baseline CAT activity was twice the adult values (p < 0.05) and a 2-day in vivo E regimen increased AcHO levels to four times the control values, with a concomitant 1.7-fold increase in CAT activity. This was prevented by administration of a CAT inhibitor (3-amino-1,2,4-triazole). Immunohistochemical staining of neonatal brains exposed to E illustrated the presence of acetaldehyde-protein adducts. We conclude that AcHO is likely produced in rat fetal and neonatal brain via CAT-mediated oxidation of E. This phenomenon may be an important factor in the neurotoxic effects of in utero E exposure.

Lipid peroxidation has been implicated in ethanol-induced liver injury and observed in fetal liver and brain after maternal ethanol consumption with mitochondria being the target organelles. This process generates a highly reactive and toxic product, 4-hydroxynonenal (HNE). In the present study, HNE levels and metabolism were assessed in mitochondria of fetal and maternal liver after in vivo ethanol exposure. Female Sprague-Dawley rats received five doses of ethanol (4 g/kg orally at 12-hour intervals) and were killed on day 19 of gestation. The results showed that HNE levels were enhanced in hepatic mitochondria of fetal rats exposed to ethanol, far in excess of that in adult liver mitochondria. Measurement of HNE metabolism showed that fetal mitochondria had a lower capacity for HNE catabolism than adult mitochondria. In adult mitochondria, HNE could be metabolized by nicotine adenine dinucleotide-dependent oxidation, reduced glutathione conjugation, and reduced nicotine adenine dinucleotide-dependent reduction, whereas in fetal liver only the former two pathways were active, but to a lesser degree than in adult mitochondria. On the other hand, mitochondria from fetal liver showed a higher production of HNE when oxidative stress was induced with t-butyl hydroperoxide. Prior in vivo ethanol exposure further potentiated HNE formation in t-butyl hydroperoxide-stimulated fetal liver mitochondria, but not in adult mitochondria. These findings indicate that increased levels of HNE in fetal liver mitochondria after maternal ethanol consumption reflect a higher susceptibility to HNE formation in addition to a lesser capacity to metabolize it. The enhanced accumulation of this toxic aldehyde may contribute to oxidative damage observed in fetal tissues after in utero ethanol exposure.

Sumatriptan (Imitrex), a selective 5-hydroxytryptamine receptor agonist, has been found to be of therapeutic benefit in the acute management of migraine. There is no information on the transfer of this agent across the human placenta. Accordingly, the current study assessed the transport of this drug across the normal term human placenta, using the isolated perfused single cotyledon technique. We found that only about 15% of a single dose of the agent placed in the maternal reservoir crossed into the fetal compartment over 4 hr. Given the average elimination half-life of 2 hr for sumatriptan, it is evident that only very small amounts of the agent will cross from mother to fetus after single doses of Imitrex. Only the parent drug entered the fetal compartment. Metabolites were not detected in the perfusates, but there was evidence of some metabolism of sumatriptan in the placenta. The nature of the metabolites has not been determined. The mechanism of transfer of the drug across the placenta is passive (i.e., the clearance is similar to L-glucose which is passively transported), the rate of transfer is equal in both directions (maternal to fetal and in the reverse), and the drug does not cross into the fetus against a concentration gradient. This passive transport of sumatriptan across the placenta is consistent with its molecular weight, its water solubility, and its slow penetration across the blood-brain barrier in experimental animals.

Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19, 1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 +/- 0.12 SE (nMol/mg protein) to 2.00 +/- 0.08 (p0.05) and conjugated dienes from 0.230 +/- 0.006 SE (OD223/mg lipid) to 0.282 +/- 0.006 (p0.05). In fetal liver, MDA levels increased from 2.39 +/- 0.08 SE (nMol/mg protein) to 2.87 +/- 0.08 (p0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 +/- 0.72 to 16.13 +/- 1.06 (nMol/mg protein) (p0.05). A 10% decrease in GSH was seen in fetal liver (p0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

1. Dideoxyinosine (ddI) has recently been approved for the treatment of patients with HIV infection. As increasing numbers of such patients are pregnant, we wished to define the rate and mechanism(s) of ddI transfer by the placenta to the foetus. Using isolated single perfused human term placental cotyledons, the drug was shown to cross the placenta from mother to foetus at a rate of 25% that of a freely diffusible marker, antipyrine, and at about half the rate of zidovudine (AZT). The transfer of ddI was similar in both directions (maternal to foetal and the reverse), equal to that of L-glucose, a passively transported sugar, and was not inhibited by excess inosine or uric acid (structural analogues of ddI). ddI did not cross to the foetus against a concentration gradient. The transport process appeared to be passive and it was not altered by AZT. 2. ddI was not metabolized in the Krebs Ringer buffer/albumin perfusate, and placental homogenates converted only 4% of ddI to hypoxanthine over the 4 h incubation. However, when maternal term or cord blood was incubated with ddI for 3 h, 50% of the drug was converted to hypoxanthine in maternal blood and to hypoxanthine and uric acid in cord blood. 3. Thus, ddI metabolism in maternal blood should decrease its net transfer to the foetus in vivo. In the foetal circulation, ddI will be further metabolized by erythrocytes to hypoxanthine and possibly to uric acid. Hence, the fraction of administered ddI delivered to foetal tissues should be much lower than that of AZT.

Cytomegalovirus is a common cause of intrauterine infection. Ganciclovir is an accepted therapeutic agent for this infection, but is proscribed in pregnancy, except when there is a life-threatening maternal infection, because of its known teratogenic and embryotoxic effects in experimental animals. There are no such data in humans and the human transplacental transfer of this drug has not been studied. This study defines the rate and mechanism of human-placental ganciclovir transport using maternal-facing syncytiotrophoblast vesicles and the perfused, isolated single-cotyledon system and determines further the effects of ganciclovir on fetal tissue, using cultured rat fetal hepatocytes. Ganciclovir was taken up by the maternal-facing placental membrane by a carrier-dependent, Na-independent system inhibited by adenine, guanine, and acyclovir, but not by cytosine and thymine or thymidine and uridine. By contrast, the overall transfer of the drug by the placenta was passive and without drug metabolism. Therefore, the drug is concentrated initially at the maternal placental surface and then crosses passively into the fetal compartment, with the latter process being rate-limiting. There was little or no toxic effect of high concentrations of ganciclovir on cultured fetal-rat hepatocytes.

Biotin, a vitamin essential for many metabolic reactions, is supplied to the fetus exclusively from the mother. Deficiency of biotin in pregnancy leads to impaired fetal growth and development. Alcohol taken in pregnancy likewise may cause fetal growth abnormalities. Normal biotin transport via the placenta and the effects of ethanol on this transport apparently have not been studied. Our aims were to characterize these phenomena for the normal human-term placenta. Using maternal-facing placental membrane vesicles, biotin uptake was sodium- and temperature-dependent, saturable, and inhibited by structural analogs of biotin (desthiobiotin, biocytin, and biotin methyl ester), as well as by 4 and 10 hr exposure to 3 g/liter ethanol. Using the isolated perfused single cotyledon method to measure placental transport of biotin at a perfusion concentration of 1 nM, the overall rate of biotin transport was found to be only 30% that of antipyrine, a freely diffusible marker. Clearance of biotin was approximately 2 ml/hr.g placenta, which was equal to the clearance of passively transferred L-glucose; biotin clearance was similar in both maternal to fetal and fetal to maternal directions. Overall transfer of biotin from maternal to fetal compartments was not inhibited by 500-fold greater concentrations of the three analogs, did not proceed against a biotin concentration gradient, and was not inhibited by 90-240 min exposure to an initial concentration of 4 g/liter ethanol. Concentration of biotin in the fetal compartment at the end of the study was not higher than on the maternal side (after maternal to fetal infusion), but placental concentration was 2- to 3-fold greater. No significant metabolism of biotin was detected. Exposing human placental cultured trophoblast on day 3 to 24 hr of ethanol (2 g/liter) had no effect on the net uptake of biotin by these cells. These studies provide evidence that maternal-facing placental membranes take up biotin by a mediated, carrier-dependent process that is inhibited by ethanol; however, based on the perfusion studies, we conclude that the overall (maternal-fetal) rate-limiting transfer of biotin by the human placenta is most consistent with a passive process, which is not inhibited by short-term exposure to ethanol.

This study defines human placental transport of cocaine and its two minor, but pharmacologically active, metabolites--norcocaine and cocaethylene. The experimental system was the single, isolated perfused cotyledon of a normal term human placenta, and antipyrine served as a freely diffusible marker. Cocaine was transferred rapidly by the placenta at a rate about 80% that of antipyrine. The transfer had characteristics of passive transport consistent with the high lipid solubility of the drug. We found no evidence of significant placental metabolism of cocaine during its rapid placental transfer. Ethanol did not alter the cocaine transfer rate. Norcocaine and cocaethylene were equally as rapidly transferred. Thus the placenta is no barrier to the transfer of cocaine and its derivatives to the fetus.

The fetal alcohol syndrome is primarily an impairment of growth and development. Zinc deficiency also causes abnormal fetal growth. Moreover, alcohol has been shown in some rodent studies to impair placental transport of zinc. The purpose of this investigation was to define better normal human placental zinc transport and the effects of alcohol on this process. To do this we employed the isolated perfused single cotyledon human term placental model, as well as the cultured human cytotrophoblast. In the perfused placental studies, it was shown that zinc is transferred by the placenta very slowly, about 6% of the rate of transport of antipyrine, a freely diffusible marker. The transfer is comparable in both directions, maternal to fetal and the reverse. Zinc does not cross the placenta against a zinc concentration gradient, in either direction. Rather there is good evidence of significant uptake (storage) of the zinc by the placenta on the recirculating compartment side of gradient studies. Moreover, when the perfusion fluid was low (0.2 g/100 ml) in albumin, about twice as much zinc accumulated in the perfused cotyledon and there was less zinc in the maternal compartment, as compared to perfusion with ten-fold higher (2.0 g/100 ml) albumin concentrations. Thus, ligand binding in the perfusate importantly influences placental zinc uptake. Interestingly, however, the increased placental binding of zinc did not translate into greater transfer of zinc to the fetal compartment. Thus, normal zinc transfer is slow, equal bidirectionally, and dependent on ligand binding in perfusate and placenta. Equal transport in both directions and absence of accumulation of zinc against a concentration gradient at “physiologic” zinc concentrations are not consistent with active (energy dependent) transfer. Alcohol (400 mg/100 ml) added to the perfusate did not impair placental zinc transfer from the maternal to fetal circuits over 4 h. Also, net uptake of zinc by human placental trophoblast exposed to 200 mg/100 ml alcohol concentration for 48 h was comparable to control values. Thus, alcohol exposure over this time in both experimental systems did not impair zinc transport by the term human placenta.

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.

This study reports on the interactive effects of ethanol and caffeine on growth of rat fetal hepatocytes. Exposure of cultured rat fetal hepatocytes (RFH) to ethanol in concentrations above 1 mg/ml, causes a blockade of EGF-dependent cell replication along with an overexpression of surface EGF receptors (EGF-R). However, RFHs exposed for 24 hours to ethanol at a concentration of 1 mg/ml alone had little effect on cell replication. Caffeine, when combined with this concentration of alcohol, progressively impaired RFH growth by up to 100%. Caffeine alone up to 10 micrograms/ml, on the other hand, caused a progressive increase in RFH replication associated with a 69% enhancement of DNA synthesis. Caffeine concentrations in excess of 50 micrograms/ml had no effect on replicative capacity. Concomitant caffeine exposure had no effect on the ethanol-related increase in cell DNA content, yet it caused a further enhancement of the cell protein accural induced by ethanol alone. Caffeine (10 micrograms/ml) alone had no effect on EGF-R expression, while ethanol (2 mg/ml) increased it by almost 200%. Addition of caffeine to ethanol reduced this enhanced EGF binding by 45%. Scatchard analysis indicated that no treatment altered ligand affinity for the receptor, but that the alterations in binding caused by ethanol and the caffeine/ethanol combination reflected changes in binding capacity, in both low and high affinity components. It is concluded that (1) ethanol blocks EGF-mediated replication accompanied by a reduction in DNA synthesis, (2) caffeine alone at low concentrations has the opposite effect and can actually potentiate the EGF-mediated mitogenic response, (3) caffeine in combination with ethanol acts synergistically to reduce RFH replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Prior studies by many laboratories have illustrated that ethanol can elicit a cascade of caspase-dependent apoptotic events in cultured neurons. Studies in our laboratory have connected this to oxidative stress and effects on fetal cortical neuron glutathione homeostasis. Aims: The intent of the following studies is to address mechanisms underlying ethanol-associated DNA damage that may be connected to apoptotic death of neurons. Methods: Cultures of fetal rat cerebral cortical neurons were utilized. Estimates of DNA damage was determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and nuclear condensation; Poly(ADP-ribose) polymerase-1 (PARP-1) expression was determined by immunostaining and Western blotting; and occurrence of parylation and AIF translocations were assessed by Western blotting. Results: Ethanol treatment of the neurons generated increases in DNA damage by 4 hours while nuclear condensation was low at the short exposure period but increased markedly by 24 hours. This was temporally related to a marked up-regulation of PARP-1 expression. Activity of PARP-1, as assessed by PolyADP-ribose (PAR) formation, occurred within 15 minutes and peaked by 6 to 8 hours of ethanol treatment. An almost complete translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus occurred by 24 hours of ethanol treatment (4.0 mg/ml). Ethanol treatment for 4, 12, and 24 hours elicited an increasing caspase-mediated cleavage of PARP-1 to its 24 kDa fragment. Conclusions: These data illustrate the rapid occurrence of DNA damage following ethanol exposure and that PARP-1 pathways may play a role in the subsequent apoptotic death of these neurons.

Wernicke’s encephalopathy (WE) is a potentially reversible metabolic brain dysfunction resulting from thiamine deficiency. It is generally characterized by ataxia, ophthalmoplegia and global confusion. Described in Berlin in 1881 by Carl Wernicke, it was initially known as polioencephalitis hemorrhagica superioris and considered a fatal syndrome. The first reported cases were three patients, two with alcoholism and one with persistent vomiting after the ingestion of sulfuric acid in a suicide attempt. The common feature shared by these cases upon post-mortem exam consisted of punctate hemorrhages in the grey matter of the walls of the third and fourth ventricles and mammillary bodies (Cirignotta et al., 2000; Truswell, 2000). In 1935, Strauss discovered that the cause of Wernicke’s findings was vitamin B1 (thiamine) deficiency (Chiossi et al., 2006). Bonhoeffer posited that Wernicke’s encephalopathy and the psychosis described by Korsakoff actually represented two phases of the same pathological process (Cirignotta et al., 2000). The observation that Wernicke’s encephalopathy and Korsakoff’s psychosis have identical neuropathology supported this belief (Charness, 1999). Thus Wernicke’s encephalopathy (WE) and Korsakoff’s psychosis (KP) are often used interchangeably as the Wernicke-Korsakoff Syndrome (WKS).

The aim of this chapter is to present a contemporary overview of ethanol-induced apoptosis in the brain, with a focus on the potential role of oxidative stress and some new concepts related to glia-mediated neuroprotection and selective vulnerability of neurons to ethanol. While ethanol-related oxidative stress and neuron apoptotic death have been documented in the adult brain, the vast majority of reports have centered on the developing organ (Schenker et al., 1990). We address both settings and offer several potential explanations for the high sensitivity of the fetal brain to these toxic responses to ethanol. Of note is that neurotoxic responses to ethanol have been recognized for several decades yet the mechanisms underlying these often devastating effects remain controversial. The following material abundantly illustrates that the setting is multifactorial with multiple ethanol-related perturbations at play, likely with each impacting to different degrees on various brain areas as well as on different neuron and glia populations. Finally, neuron survival and functions are intimately connected to the glia with which neurons are commingled. Such interactions may often be essential to neuron survival and we include a brief overview of recent studies addressing ethanol effects on neuroprotective glia/neuron interactions.

The purpose of this review is to discuss current information about the mechanism (s) of FAS. This will utilize the two lines of evidence -those obtained in experimental studies, which are more controlled as to specific factors but perhaps not fully applicable to the clinical situation, and those derived from observations in patients with all the complexities of such surveys

After consuming comparable amounts of ethanol, women have higher blood ethanol concentrations than men, even with allowance for differences in size, and are more susceptible to alcoholic liver disease. Recently, we documented significant "first-pass metabolism" of ethanol due to its oxidation by gastric tissue. We report a study of the possible contribution of this metabolism to the sex-related difference in blood alcohol concentrations in 20 men and 23 women. Six in each group were alcoholics. The first-pass metabolism was determined on the basis of the difference in areas under the curves of blood alcohol concentrations after intravenous and oral administration of ethanol (0.3 g per kilogram of body weight). Alcohol dehydrogenase activity was also measured in endoscopic gastric biopsies. In nonalcoholic subjects, the first-pass metabolism and gastric alcohol dehydrogenase activity of the women were 23 and 59 percent, respectively, of those in the men, and there was a significant correlation (rs...

Recent work has shown that colchicine may benefit patients with primary biliary or alcoholic cirrhosis. However, very little is known about its pharmacokinetics in the presence of impaired liver function. To study this we examined the effects of three models of experimental liver dysfunction and one of cytochrome P-450 inhibition on colchicine elimination in the rat. The models of experimental liver dysfunction included bile duct ligation (with sham-operated controls), α-naphthylisothiocyanate-induced intrahepatic cholestasis and galactosamine-induced diffuse hepatocelluar necrosis. The control group had a colchicine clearance of 77.33 ml/min. kg ± 8.27 ml/min kg, a half-life of 16.68 min ± 0.97 min and a volume of distribution of 1.84 L/kg ± 0.15 L/kg. Cimetidine administration, 120 mg/kg intraperitoneally 15 min before colchicine administration, caused clearance to decrease by 32% (p < 0.05) and half-life to increase by 38% (p < 0.05). Volume of distribution did not change. At 48 hr after bile duct ligation, colchicine clearance decreased by 84% (p < 0.05), terminal half-life increased to 513.7 min ± 106.6 min (p < 0.05) and volume of distribution increased by 175% (p < 0.05). Colchicine pharmacokinetics in sham-operated rats were not statistically different from the above mentioned controls. After α-naphthylisothiocyanate administration, colchicine clearance decreased by 55% (p < 0.05), the halflife increased by 56% (p < 0.05) and the volume of distribution decreased by 30% (p < 0.05). After administration of galactosamine, 1,000 mg/kg, colchicine clearance decreased by 62% (p < 0.05), half-life increased to 144.72 min ± 35.30 min (p < 0.05) and volume of distribution increased by 124% (p < 0.05). These data show that experimental hepatic injury in the rat significantly impairs colchicine pharmacokinetics. They also support the hypothesis that the liver is a major route of colchicine elimination.

Alcoholic liver disease (ALD) evolves through various stages, and malnutrition correlates with the severity of ALD. Poor nutrition is caused both by the substitution of calories from alcohol for calories from food and by the malabsorption and maldigestion of various nutrients attributed to ALD. The only established therapy for ALD consists of abstinence from alcohol. Sufficient nutritional repletion coupled with appropriate supportive treatment modalities may be effective in reducing complications associated with ALD---particularly infection. Nutrition makes a significant positive contribution in the treatment of ALD, especially in selected malnourished patients.