Acute exposure to the nutritional toxin alcohol reduces brain protein synthesis in vivo.

Few studies have measured brain protein synthesis in vivo using reliable methods that consider the precursor pool, and there is a paucity of data on the regional sensitivity of this organ to nutritional or toxic substances. We hypothesized that different areas of the brain will exhibit variations in protein synthesis rates, which might also be expected to show different sensitivities to the nutritional toxin, ethanol. To test this, we dosed male Wistar rats with ethanol (75 mmol/kg body weight) and measured rates of protein synthesis (ie, the fractional rate of protein synthesis, defined as the percentage of the protein pool renewed each day; k(s), %/d) in different brain regions 2.5 hours later with the flooding dose method using L-[4-(3)H] phenylalanine. In the event that some regions were refractory to the deleterious effects of ethanol, we also predosed rats with cyanamide, an aldehyde dehydrogenase inhibitor (ie, cyanamide + ethanol), to increase endogenous acetaldehyde, a potent neurotoxic agent. The results indicated the mean fractional rates of protein synthesis in the cortex was 21.1%/d, which was significantly lower than either brain stem (30.2%/d, P <.025), cerebellum (30.1%/d, P <.01), or midbrain (29.8%, P <.025). Ethanol significantly decreased protein synthesis in the cortex (21%, P < 0.01), cerebellum (19%, P <.025), brain stem (44%, P <.025), but not in the midbrain (not significant [NS]). However, significant reductions in protein synthesis in the midbrain occurred in cyanamide + ethanol-dosed rats (60%, P <.0001). Cyanamide + ethanol treatment also reduced k(s) in the brain stem (66%, P <.001), cortex (59%, P <.001), and cerebellum (55%, P <.001). In conclusion, the applicability of the flooding dose technique to measure protein synthesis in the brain in vivo is demonstrated by its ability to measure regional difference. Impaired protein synthesis rates may contribute to or reflect the pathogenesis of alcohol-induced brain damage.

Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: effects of surgical stress in human patients.

The urinary catabolites, N2,N2-dimethylguanosine (DMG), pseudouridine (PSU) and 7-methylguanine (m7-Gua) are formed from post-transcriptional methylation of RNA bases and are not reincorporated into RNA upon its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species since DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and m7-Gua occurs in all RNA species. Conventional HPLC analysis has several drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-field 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. Urinary concentrations of these metabolites were found to be in agreement with previously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and reconstitution) and was capable of the simultaneous determination of other relevant analytes such as creatinine. This technique was therefore applied to urine samples from patients who had undergone surgical stress and insulin-like growth factor-1 (IGF-I) therapy. Surgical stress increased the excretion of DMG and m7-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compared with controls but degradation rates of rRNA decreased by approx. 30%. However, injection of IGF-I (40 micrograms/kg s.c.) had no significant effect on the excretion of these nucleosides. These data indicated that IGF-I therapy has no marked effects on RNA turnover following trauma. We suggest that this technique can be applied to study of RNA metabolism in any surgical or medical condition. Furthermore, since only 0.6 ml of urine is required, studies in neonates seem to be feasible.

The urinary excretion of tryptophan and tryptophan metabolites in the chronic ethanol-fed rat.

An investigation was made into the hypothesis that chronic ethanol ingestion disturbs the metabolism of tryptophan which is reflected by alterations in the urinary excretion of the metabolites 5-hydroxyindoleacetic acid (5-HIAA), anthranilic acid (AA) and indoleacetic acid (IAA). In particular, we investigated whether experimental chronic alcoholism is associated with a decrease in the tryptophan metabolite ratios as suggested in the literature. Male Wistar rats were chronically fed a nutritionally-complete liquid diet in which ethanol comprised 35% of total calories: controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At 6 weeks, 24 h urine samples were collected for the analysis of tryptophan, 5-HIAA, AA and IAA by HPLC. During ethanol-feeding there were reductions in the daily urinary excretion (i.e. mumol/24 h) of tryptophan (-57%, P = 0.026) and concomitant increases in 5-HIAA excretion (62%, P = 0.057). Expression of data in terms of lean tissue mass (i.e. urinary creatinine) revealed identical conclusions. An analysis was performed on the molar ratios of these urinary analytes. The tryptophan: total metabolite ratio was significantly decreased (by -53%), but the AA: total metabolite ratio was not significantly altered (P = 0.102). The ratios 5-HIAA/AA and 5-HIAA/IAA were slightly increased, but they did not attain statistical significance (P > 0.351). It was concluded that chronic ethanol feeding is associated with significant changes in the urinary excretion of tryptophan and its related metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain nucleic acid composition and fractional rates of protein synthesis in response to chronic ethanol feeding: comparison with skeletal muscle.

Brain atrophy is a common feature of chronic alcohol misuse, although the pathogenic mechanisms are unknown. We propose that defects in protein synthesis are contributing events. To test this hypothesis the experimental effects of chronic (i.e., 2 and 3 weeks) ethanol feeding on brain nucleic acid composition and rates of protein synthesis in vivo were investigated. These were compared with those of skeletal muscle (represented by the plantaris). Male Wistar rats, used at mean body weights of either 82 g (first study for 2 weeks ) or 93 g (second study for 3 weeks) were fed a nutritionally complete liquid diet in which ethanol comprised a third of the total calories. Control rats were pair-fed identical amounts of the same diet, in which ethanol was substituted by isoenergetic glucose. At 2 weeks there were small reductions (i.e., approximately 5-10%) in the weight of the whole brain, cortex, and brain stem. Ethanol-induced reductions in the total protein content of the brain stem was found at 2 weeks, although these changes did not achieve significance. At 3 weeks the weights of whole brain were significantly reduced compared to a greater reduction in skeletal muscle weights. Total protein contents were reduced at 3 weeks in the whole brain and skeletal muscle. At 2 weeks there were decreases in the RNA contents of the cortex, brain stem, and entire brain. There were also reductions in cerebellum RNA composition only when expressed relative to DNA. The DNA composition of the brain was relatively unaffected by chronic ethanol feeding. At 3 weeks, total RNA and DNA were reduced in the whole brain and muscle. Fractional rates of protein synthesis (i.e., the percentage of tissue protein pool renewed each day) in the brain were unaltered after 3 weeks of ethanol feeding, but were reduced in skeletal muscles, largely as a consequence of reduced RNA composition. In conclusion, only moderate changes in the brain were found in ethanol feeding. These data can be compared to skeletal muscle, which shows that ethanol induces profound reductions in protein, RNA, and protein synthesis rates.

Lysosomal and nonlysosomal protease activities of the brain in response to ethanol feeding.

The contribution of impaired degradative processes to the cellular changes occurring in the brain as a consequence of chronic ethanol exposure was assessed. Male Wistar rats were fed nutritionally adequate liquid diets containing ethanol as 35% of total dietary calories. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. The results showed that at the end of 3 weeks the activities of neutral protease (nonlysosomal) and cathepsin D (lysosomal) were unaltered. However, there were significant elevations in the activities of the lysosomal enzyme cathepsin B, regardless of whether the activities were expressed relative to wet weight ( p = 0.005), protein (p = 0.006), or DNA (p = 0.045). In addition, we showed that the activities of cathepsin B were not significantly affected by additions of carnosine or acetaldehyde, in vitro. However, neutral protease activities were increased by carnosine additions in vitro. We conclude that selective alterations in brain protease activities may be contributing factors in the genesis of alcoholic brain disorders.

A pilot investigation of the effect of tryptophan manipulation on the affective state of male chronic alcoholics.

A pilot study was conducted to investigate the hypothesis that dietary tryptophan manipulation would influence self-report affective status in alcoholic males. No significant effect of dietary manipulation was observed on the tryptophan/large neutral amino acids ratio or psychological indices of affect. The notion that dietary manipulation may be utilized in improving mood state in alcoholic males was not supported.

Seizures induced by theophylline and isoniazid in mice.

Isoniazid-induced seizures respond poorly to anticonvulsants but well to pyridoxine (Vitamin B6); theophylline produces difficult-to-treat seizures with substantial morbidity and mortality. Theophylline therapy depresses plasma pyridoxal-5'-phosphate (PLP), the active metabolite of pyridoxine, suggesting that theophylline-induced seizures might be amenable to treatment with pyridoxine. Our study established the dose-response relationship for convulsions due to isoniazid and theophylline in mice and determined if pyridoxine antagonized such seizures. Female CD-1 outbred mice weighing 25 to 30 g were used. Clonic seizures had clonic activity lasting 5 sec; tonic seizures had loss of the righting reflex with tonic hindlimb extension. Groups of 10 mice received single doses of 50, 100, 150, 200, 250 or 300 mg aminophylline/kg i.p. or 100, 150, 200, 250, 300 or 350 mg isoniazid/kg i.p. and were observed for seizures or death. Pyridoxine or saline with aminophylline or isoniazid were administered simultaneously. The LD50 for aminophylline was 266 mg/kg; for isoniazid it was 160 mg/kg. Doses of 150 mg aminophylline/kg or 100 mg isoniazid/kg did not induce seizures. Pyridoxine with aminophylline or isoniazid did not alter the frequency or time of onset of seizures or death. This was unexpected because pyridoxine antagonizes theophylline-induced seizures in mice and reverses isoniazid-induced seizures in humans. We found no evidence that PLP depletion in mice is a mechanism for seizures induced by isoniazid or aminophylline in a fashion similar to isoniazid in humans.

Characterization of contractile and non-contractile protein synthesis in the stomach, small and large intestine and caecum of the rat, and response to acute ethanol dosage.

An investigation was made into the relative composition and synthesis rates of smooth muscle contractile proteins in vivo in different regions of the rat gastrointestinal tract. There was considerable homogeneity in the composition of sarcoplasmic proteins in the small bowel (i.e. 54-58 mg/g) but considerable variability in the large bowel, i.e. highest in the caecum (97 mg/g) and lowest in the colon (21 mg/g). The myofibrillary protein concentration was constant throughout the gastrointestinal tract, i.e. 20-34 mg/g. Stromal fractions varied from 6 to 39 mg/g and was highest in the cardiac region of the stomach and lowest in the duodenum. Fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]phenylalanine. In control rats synthesis rates in sarcoplasmic protein fractions were relatively much higher (43-107%/day) than myofibrillar (27-52%/day) or stromal (6-26%/day) proteins. Fractional rates of stromal protein synthesis showed the greatest variability while myofibrillar synthesis rates the least, throughout the alimentary tract. Jejunal seromuscular layer myofibrillar proteins had the highest synthesis rates (49%/day). In response to acute ethanol injection, protein synthesis in all jejunal fractions fell by 20-30%. Contractile and non-contractile proteins from the cardiac region of the stomach, duodenal seromuscular layer and large bowel seromuscular layer were insensitive to ethanol administration. Protein synthesis of sarcoplasmic proteins from the antrum, ileum seromuscular layer and myofibrillar proteins from the ileum seromuscular layer and caecum were also significantly depressed as a result of ethanol treatment.

Ethanol dosage regimes in studies of ethanol toxicity: influence of nutrition and surgical interventions.

In this review we consider some of the practical facets of acute and chronic drug regimes. In particular, we illustrate our arguments with specific reference to alcohol and draw attention to methodological constraints that might alter biochemical or physiological processes. These includes the imposition of nutritional abnormalities and surgical procedures. These two areas are highlighted because there is evidence to show that they have marked influences on metabolic parameters, exemplified by tissue protein synthesis. In general, there is no controversy as to methods for acute studies with alcohol, although some reports have failed to investigate whether intravenous, intragastric or intraperitoneal regimes more accurately mimic the clinical situation. With regard to chronic feeding regimes, evidence is provided to support the argument that, currently, the most appropriate feeding protocol ensures that both control and treated groups receive identical amounts of nutrients albeit with differences in the calories apportioned to ethanol, which is substituted by isocaloric glucose or other carbohydrates in controls. However, there are still other methods being employed: these are subject to misinterpretation as the principle of ensuring that control and ethanol-fed rats receive identical amounts of nutrients, is ignored. In this review we also draw attention to the fact that surgical procedures, which are often employed to facilitate the measurement of body parameters (for example, implantation of cannulae), themselves alter tissue metabolism. The importance of this relates to the concept of metabolic superimposition, which implicates interacting responses when two or more stresses (i.e. surgery and drug administration) are superimposed.

Osmotic diarrhoea and skeletal muscle protein synthesis in vivo.

The pathogenic nature of the wasting seen in diarrhoea is unknown. This study measured protein synthesis in an established model of diarrhoea using lactose for seven days. Comparisons were also made with data obtained from rats fed an identical diet in which lactose was replaced by isocaloric glucose ad libitum (that is, the control diet). To account for diarrhoea induced anorexia, a third group of rats were included, which were fed identical amounts of the control diet as the rats with diarrhoea inducing diet. Comparisons of the diarrhoea induced group with rats fed the control diet ad libitum showed that diarrhoea caused a significant reduction in body weights. Type I and type II muscles showed significant reductions in protein, RNA, and DNA contents, as well as a fall in the derived parameters, RNA/DNA, protein/DNA, and RNA/protein. Fractional rates of protein synthesis (ks) were also reduced. However, synthesis rates of type I and II muscles relative to RNA (kRNA) were unchanged in these muscles in diarrhoea induced rats compared with ad libitum fed controls. In the jejunum there was an increase in the RNA/DNA ratio, and reductions in ks and kRNA. Comparisons were also made between rats with diarrhoea and rats pair fed the control diet. There were no changes in total muscle protein, RNA or DNA contents. This suggests that an important feature of body wasting in diarrhoea is the element of anorexia, which induces severe metabolic changes. The comparison between rats with diarrhoea and the pair fed group showed that histological features of the plantaris were not overtly changed, though diarrhoea caused significant reductions in RNA/DNA, protein/DNA, ks, and kRNA. Similar changes were seen for the soleus; though the reduction in ks failed to attain statistical significance. In the jejunum a comparison of diarrhoea induced rats with pair fed controls, showed increases in the ratios of RNA/DNA and protein/DNA.

Contractile protein synthesis rates in vivo in the rat jejunum: modulating role of adrenalectomy and thyroidectomy on ethanol-induced changes.

Acute ethanol toxicity has many deleterious effects on the gastrointestinal tract and, in particular, inhibits small intestinal protein synthesis. The mechanism(s) of the ethanol-induced inhibition of protein synthesis are unknown. This study was designed to investigate the role of adrenal and thyroid hormones on the ethanol-induced inhibition of whole jejunal protein synthesis in adrenalectomised and thyroidectomised male Wistar rats. Acute ethanol dosage significantly reduced protein synthesis in all subcellular protein fractions of the whole jejunum, in sham-thyroidectomized, thyroidectomized, sham-ardrenalectomized and adrenalectomized rats. Synthesis rates relative to RNA and DNA were also highly significantly reduced in all treatment groups in ethanol-dosed rats. However in most protein fractions the inhibition of protein synthesis was greater in thyroidectomized compared to sham-thyroidectomized rats, whereas the reverse was true in adrenalectomized rats. Neither adrenalectomy or thyroidectomy completely abolished ethanol-induced inhibition in protein synthesis, suggesting that ethanol or its metabolites, e.g. acetaldehyde, acts directly on the tissue. However, the ethanol-induced inhibition of protein synthesis was greater in thyroidectomized rats demonstrating that contractile and non-contractile proteins synthesis in the jejunum is under control of complex regulatory processes.