Digital ulcers in systemic sclerosis: prevention by treatment with bosentan, an oral endothelin receptor antagonist.

OBJECTIVE: Recurrent digital ulcers are a manifestation of vascular disease in patients with systemic sclerosis (SSc; scleroderma) and lead to pain, impaired function, and tissue loss. We investigated whether treatment with the endothelin receptor antagonist, bosentan, decreased the development of new digital ulcers in patients with SSc. METHODS: This was a randomized, prospective, placebo-controlled, double-blind study of 122 patients at 17 centers in Europe and North America, evaluating the effect of treatment on prevention of digital ulcers. The primary outcome variable was the number of new digital ulcers developing during the 16-week study period. Secondary assessments included healing of existing digital ulcers and evaluation of hand function using the Scleroderma Health Assessment Questionnaire. RESULTS: Patients receiving bosentan had a 48% reduction in the mean number of new ulcers during the treatment period (1.4 versus 2.7 new ulcers; P = 0.0083). Patients who had digital ulcers at the time of entry in the study were at higher risk for the development of new ulcers; in this subgroup the mean number of new ulcers was reduced from 3.6 to 1.8 (P = 0.0075). In patients receiving bosentan, a statistically significant improvement in hand function was observed. There was no difference between treatment groups in the healing of existing ulcers. Serum transaminase levels were elevated to >3-fold the upper limit of normal in bosentan-treated patients; this elevation is comparable with that observed in previous studies of this agent. Other side effects were similar in the 2 treatment groups. CONCLUSION: Endothelins may play an important role in the pathogenesis of vascular disease in patients with SSc. Treatment with the endothelin receptor antagonist bosentan may be effective in preventing new digital ulcers and improving hand function in patients with SSc.

Is clubbing a feature of the anti-phospholipid antibody syndrome?

A patient with the anti-phospholipid antibody syndrome and digital clubbing is described. No recognized cause for the clubbing was found. It is suggested that platelet aggregation and microthrombi formation as a result of anti-phospholipid antibody may be involved in the pathogenesis of the digital clubbing. This may be a new feature of the anti-phospholipid antibody.

Effect of 6-phosphogluconate on phosphoglucose isomerase in rat brain in vitro and in vivo.

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of conditions of homogenization on the activity of pyruvate dehydrogenase and its stability in brain homogenates.

The activity of pyruvate dehydrogenase was higher in brain homogenates prepared by using a Potter-Elvehjem type homogenizer (79.0 +/- 2.38 nmol/min/mg of protein) than by using an Ultra-Turrax homogenizer (from 62.39 +/- 1.35 to 74.60 +/- 1.21 nmol/min/mg of protein). Different conditions of homogenization with the Ultra-Turrax had no significant effect on the activity of pyruvate dehydrogenase. Pyruvate dehydrogenase activity decreased significantly in brain homogenates maintained at 0 degrees C for short periods or stored at ?20 degrees C for several days. Marked differences in pyruvate dehydrogenase activities reported in earlier work have been attributed to its instability in brain homogenates. The results showed that pyruvate dehydrogenase is not a rate limiting enzyme in the metabolism of glucose in brain in vivo.

Effect of a deficiency of thiamine on brain pyruvate dehydrogenase: enzyme assay by three different methods.

A simple and rapid method based on the NADH-linked reduction of a tetrazolium dye was described for the determination of pyruvate dehydrogenase activity in rat brain homogenates. The method (method 3) gave a value of 36.06 +/- 1.24 nmol of pyruvate utilised/min/mg of whole brain protein. This value was higher than that obtained by measurement of the rate of decarboxylation of [1-14C]pyruvate (15.10 +/- 0.88 nmol/min/mg of protein; method 1) and was comparable with the rate of transfer of acetyl groups to an arylamine (39.04 +/- 1.32 nmol/min/mg of protein; method 2). A critique of the values reported by others by different methods was given. The pyruvate dehydrogenase activity in the mitochondria isolated from rat brain was in the "active" (nonphosphorylated) form. A deficiency of thiamine in rats was produced by treatment with pyrithiamine, an antagonist of thiamine. This treatment resulted in abnormal neurological signs, such as ataxia and convulsions. The measurement of the total activity of pyruvate dehydrogenase in the brain by all three methods showed no significant change in the enzymic activity in thiamine-deficient rats after treatment with pyrithiamine. The activities of the enzyme in the brains of pair-fed animals were similar to those in the controls.

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo.

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.

Preclinical left ventricular abnormalities in alcoholics are independent of nutritional status, cirrhosis, and cigarette smoking.

M mode echo recordings of the left ventricle (LV) were performed in 33 patients with alcoholic liver disease, 26 patients with various non-alcoholic liver diseases, and in 18 non-alcoholic controls. Groups were well matched for age and overall nutritional status (as assessed by anthropometry) and none of the subjects studied had cardiorespiratory symptoms. Alcoholics had significantly increased LV free wall thickness and LV cavity dimension at end diastole (EDD). Multiple regression analysis of the data identified alcohol abuse as the most important variable affecting EDD, and this relation could not be explained by differences in age, sex, overall nutrition, cigarette smoking, thiamine status (total blood thiamine and thiamine pyrophosphate concentration), presence of liver disease, or severity of liver disease (cirrhotic vs non-cirrhotic). The increase in LV free wall thickness was not significantly related to alcohol abuse. These results suggest that chronic alcohol abuse is an important independent risk factor for cardiac dilatation, and that increase in EDD may be an early marker of alcoholic cardiomyopathy.

Blood thiamine and thiamine phosphate ester concentrations in alcoholic and non-alcoholic liver diseases.

Thiamine state was investigated in patients with alcoholic liver disease, patients with various non-alcoholic liver diseases, and controls using a direct technique (thiochrome assay) to measure thiamine, thiamine monophospate, and the active coenzyme thiamine pyrophosphate in whole blood after isolating the fractions by ion exchange chromatography. Overall nutrition was similar in all groups as assessed by anthropometry, and no patient had clinical evidence of thiamine deficiency. There was no significant difference among the groups in mean concentration of any form of thiamine. The scatter was much greater in patients with alcoholic liver disease but only 8.7% had biochemical thiamine deficiency (defined as a blood concentration of the active coenzyme greater than 2 SD below the mean control value). An unexpected finding was of abnormally high total thiamine concentrations (greater than 2 SD above the mean control value) in 17.4% of patients with alcoholic liver disease, the highest concentrations being found in two patients with severe alcoholic hepatitis and cirrhosis. The ratio of phosphorylated to unphosphorylated thiamine was calculated as an index of phosphorylation and, although the mean did not differ significantly among the groups, the range was greatest in alcoholic liver disease. The lowest ratios occurred in the two patients with severe alcoholic hepatitis, but neither had evidence of thiamine pyrophosphate deficiency. Contrary to studies using indirect assay techniques, these results suggest that thiamine deficiency is unusual in well nourished patients with alcoholic liver disease. The new finding of unexpectedly high thiamine concentrations in some patients may be due to abnormalities of hepatic storage or release in liver disease, particularly in severe alcoholic hepatitis. There was no convincing evidence of impaired thiamine phosphorylation in any patients with liver disease. Conclusions from studies using indirect assays on the prevalence and mechanisms of thiamine deficiency in liver diseases may not be valid.

Decreased labeling of amino acids by inhibition of the utilization of [3H, 14C]glucose via the hexosemonophosphate shunt in rat brain in vivo.

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [3H]acetate. The results of these experiments also gave evidence of the presence of [3H]glucose and [3H]acetate, and an increase in [3H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [3H]glucose formed by gluconeogenesis from [3H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-3H]glucose or [1-3H]glucose relative to that after co-injection of [U-14C]glucose or [2-14C]glucose. In contrast to the expected formation of [1,6-3H]glucose by gluconeogenesis from [3H]acetate, 3H-labeled glucose isolated from brain, blood and liver showed the presence of [6-3H]glucose only. The values corrected for the presence of [6-3H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [3H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-14C]glucose reported previously and again confirmed using [1-3H], [6-3H], [2-14C] or [U-14C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and gamma-aminobutyrate.

The rate of utilization of glucose via hexosemonophosphate shunt in brain.

The concentration of 6-phosphogluconate in the brain increased from 0-24 nmol/g in the controls to 1430 and 1506 nmol/g in rats treated with 50 mg of 6-aminonicotinamide/kg of body weight. A dose-dependent increase in the concentrations of glucose and glucose 6-phosphate as well as of 6-phosphogluconate was found in the brains of 6-aminonicotinamide-treated rats. The biochemical changes and symptoms of neurological disorder in 6-aminonicotinamide-treated rats were not due to hypothermia. The rate of utilization of glucose via the hexosemonophosphate shunt was determined by isolation of gluconate from 6-phosphogluconate and measurement of its [14C]content at short time intervals after injection of [U-14C]glucose into 6-aminonicotinamide-treated rats; it was 16.5 nmol of glucose utilized/min per g of brain, and represented approximately 2.3% of the overall utilization of glucose in the brain. A highly significant correlation was observed between the concentration of 6-phosphogluconate and the concentration of glucose 6-phosphate and free glucose. The validity of this correlation was supported by the results of previous investigations involving several other treatments.

The effect of inhibition of hexosemonophosphate shunt on the metabolism of glucose and function in rat brain in vivo.

Rats treated 4 hr previously with 6-aminonicotinamide showed a twenty-four fold increase of [14C]phosphogluconate in the adult brain at 30 min after injection of [U-14C]glucose indicating a blockade of the hexosemonophosphate shunt. There was a significant increase in the 14C-content of glucose and glucose-6-phosphate, and a decrease in that of amino acids. [14C]Phosphoglycerate content showed no consistent change after 6-aminonicotinamide treatment. The concentration of glucose and glucose 6-phosphate increased significantly without a significant change in the lactate pool in the brain of 6-aminonicotinamide treated rats. The rate of utilization of glucose in the brain of control rats was 0.73 mumol/min per g of brain. It decreased by 16% in rats treated with 6-aminonicotinamide; the results suggested that both glycolysis and pyruvate oxidation were affected. The amount of glucose utilized in the brain by the hexosemonophosphate shunt was approximately 0.0093 mumol/min per g of brain, i.e. 1.3% of the total rate of utilization of glucose. The observed changes were not due to hypothermia. The rate of glucose utilization was higher in animals exposed to higher ambient temperature and to stress caused by handling. The results were explained by postulating a role for the hexosemonophosphate shunt in providing neurotransmitter amino acids glutamate and gamma-aminobutyrate, and interdependence of brain function and glucose utilization.

Uptake and calcium-dependent release of ethylenediamine (1,2-diaminoethane) by rat brain slices.

The uptake of [14C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4 degrees C. This uptake showed a Km of 1.36 mM, was partly sodium-dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 mM potassium, and by electrical stimulation, the release in both cases being calcium-dependent. In view of these findings and the reported interactions of ethylenediamine with gamma-aminobutyric acid-related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.

The effect of 6-aminonicotinamide on the levels of brain amino acids and glucose, and their labeling with 14C after injection of [U-14C] glucose.

The brains of rats paralysed at 4 hr after the administration of 6-aminonicotinamide were found to contain decreased levels of glutamate and gamma-aminobutyrate. The glucose content of the brain of the treated rats was several fold higher than in controls. The incorporation of 14C into brain amino acids at 30 min after the injection of [U-14C]glucose was decreased by 16%: this was attributed to mainly decreased labeling of glutamate and associated amino acids. The results are discussed in the light of previous findings that the administration of 6-aminonicotinamide resulted in the blockade of the direct oxidation of glucose by the pentose phosphate pathway.