A comparison of proteins S-thiolated by glutathione to those arylated by acetaminophen.

This study was designed to evaluate whether the same proteins that irreversibly bind reactive electrophiles of drugs also bind glutathione (GSH) under oxidative conditions. Specifically, proteins that can be arylated by acetaminophen were compared to those that form glutathione-protein mixed disulfides (PSSG) after incubation with diamide. Data are presented which suggest that both GSH and acetaminophen bind to a subset of N-ethylmaleimide (NEM)-reactive protein thiols. To evaluate the pattern of proteins that bind GSH, PSSGs were formed in vitro by incubating cytosolic proteins with GSH and diamide. A sensitive procedure was developed in which PSSGs were first reduced with 0.1 mM dithiothreitol (DTT), and the newly exposed protein thiols were labeled with either [3H]NEM (for quantitative analysis) or with fluorescein-5-maleimide (for visual detection). Acetaminophen binding was achieved by incubating cytosolic proteins in vitro with the reactive acetaminophen metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). Proteins from both assays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blot analysis. Acetaminophen binding was detected by immunoblotting with an affinity-purified antibody against acetaminophen, and PSSGs were visualized using anti-fluorescein antibodies. In both instances, binding to proteins was observed to be selective. A comparison of the proteins modified by GSH binding with those that bind acetaminophen indicates that the major cytosolic acetaminophen-binding protein of 58 kDa may also be modified by glutathiolation under oxidative conditions.

Biochemical and genetic analysis of a child with cystic fibrosis and cystinosis.

We have studied a child with cystic fibrosis (CF), nephropathic cystinosis, and manifestations of Bartter syndrome, a finding reported previously in both of these diseases (CF and cystinosis). The chance of an individual inheriting a mutant allele for both CF and cystinosis from each of his parents by independent segregation is very small. Therefore, other mechanisms of inheritance were investigated, including whether his diseases were caused by a chromosome deletion or rearrangement that caused defects in both genes, whether his phenotype was caused by a new mutation or variant of either disease, or whether both diseases were inherited together due to inheritance of 2 copies of the same chromosome from one of the parents (uniparental disomy). An investigation was made of whether having mutations for both CF and cystinosis resulted in a different phenotype for either disease and whether the child was a heterozygote rather than a homozygote for one of the mutations. The results suggest that neither disease influenced the expression of the defect in the other and that this child inherited a mutant allele for both diseases independently from each parent.

A comparison of the effectiveness of cysteamine and phosphocysteamine in elevating plasma cysteamine concentration and decreasing leukocyte free cystine in nephropathic cystinosis.

Cysteamine (beta-mercaptoethylamine, MEA) is currently used to treat children with nephropathic cystinosis. In this study MEA was compared to phosphocysteamine (MEAP), a phosphorothioester that tastes and smells better than MEA, with respect to its ability to elevate plasma MEA and deplete leukocytes of cystine. Studies were performed in six children with nephropathic cystinosis ranging in age from 2 to 10 yr. After equimolar oral doses of either MEA or MEAP plasma cysteamine was determined at various times for 6 h. MEA was determined by sodium borohydride reduction followed by high-performance liquid chromatography separation and electrochemical detection. Leukocyte cystine was measured before and 1 and 6 h after drug administration. Peak plasma MEA was obtained 30 min to 1 h after a dose and was not significantly different when MEA (48.6 +/- 10.7, mean +/- SD) or MEAP (54.1 +/- 20.2) was given. Significant plasma MEA concentrations were seen as early as 15 min after an oral dose, indicating rapid absorption. Analysis of vomitus indicated that hydrolysis of the phosphate group of MEAP occurs in the stomach. The percent decrease in leukocyte cystine content obtained with MEA administration (61.9%) was not significantly different from the decrease observed when MEAP was administered (65.3%). MEA and MEAP appear to be equally effective in their cystine-depleting properties.

Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection.

Cysteamine is currently used to treat children with the inherited disorder nephropathic cystinosis. A method for the quantitative determination of this aminothiol in human plasma is presented. Whole plasma was reduced with sodium borohydride to convert disulfides to thiols. Cysteamine was then separated by high-performance liquid chromatography and detected electrochemically. The recovery of standard cysteamine added to plasma was 96.6 +/- 1.9%. In a patient with cystinosis, an oral dose of cysteamine was absorbed rapidly, with plasma cysteamine reaching a maximum of 56 microM 1 h after the dose. By 1.8 h the plasma cysteamine concentration had decreased to one-half the maximum value.

An improved method for heterozygote detection of cystinosis, using polymorphonuclear leukocytes.

Heterozygotes for the autosomal recessive disease cystinosis are currently detected by measuring the cystine content of mixed-leukocyte preparations. The present study was designed to reassess the accuracy of this method and to determine whether measuring the cystine content of purified polymorphonuclear leukocytes would improve heterozygote detection. Blood samples were obtained from 29 obligate heterozygotes for nephropathic cystinosis, one obligate heterozygote for benign cystinosis, and 18 individuals presumed to be normal. When the cystine content of mixed-leukocyte preparations was measured, three heterozygote values overlapped the normal range. When polymorphonuclear-leukocyte cystine content was measured, no heterozygote values were within the normal range. Measurement of the cystine content of purified preparations of polymorphonuclear leukocytes affords a simple method that improves the sensitivity of heterozygote detection for cystinosis.

Diet composition and lipoprotein lipase (EC 3.1.1.34) activity in human obesity.

1. Adipose tissue lipoprotein lipase (EC 3.1.1.34; AT-LPL), a rate-limiting enzyme in triglyceride storage in adipose tissue, is hormonally regulated and may be important in the maintenance of obesity. 2. In twelve obese women, AT-LPL activity was measured before weight loss, during weight loss and after 1 and 2 weeks of weight maintenance on either a high-carbohydrate or a high-protein diet. 3. When related to tissue weight, AT-LPL activity during the 2 weeks of weight maintenance was higher than the initial AT-LPL activity; there was no difference when activity was expressed per cell. 4. Changes in AT-LPL activity were not affected by diet composition. AT-LPL activity correlated with insulin levels and a change in insulin sensitivity of AT-LPL was observed after weight loss.

Altered plasma free and protein-bound sulfur amino acid levels in patients undergoing maintenance hemodialysis.

The plasma sulfur amino acid status of maintenance hemodialysis (MHD) patients has not previously been comprehensively investigated. We measured plasma sulfur amino acid levels in 24 MHD patients (12 fasted, 12 nonfasted) both before and after a routine 4-h hemodialysis and in 13 normal individuals (seven fasted, six nonfasted). Plasma free cystine, homocystine, cysteine-homocysteine-mixed disulfide (MDS), methionine, and taurine and protein-bound cysteine and homocysteine were measured. In nonfasted patients, plasma homocystine and MDS were not measured. Fasted patients predialysis had elevated plasma free cystine, homocystine, MDS, methionine, and taurine; all of these, except taurine, fell during dialysis. In nonfasted patients, plasma free cystine was elevated. Protein-bound cysteine and homocysteine were elevated predialysis and postdialysis in all patients; protein-bound cysteine fell during dialysis but remained above normal. In MHD patients predialysis, plasma free cystine levels correlated with plasma MDS, protein-bound cysteine, and age. These findings indicate pervasive alterations in plasma sulfur amino acid levels in MHD patients.

Meal-induced changes in lipoprotein lipase activity in brown fat and other tissues of rats.

Brown fat thermogenesis is increased after a single test meal. This study was conducted to determine whether lipoprotein lipase activity is higher in brown adipose and other tissues after a single large meal. Rats were trained to eat two large meals per day. Two hours after consuming a test meal, lipoprotein lipase activity was measured in interscapular brown adipose tissue, retroperitoneal and epididymal white adipose tissue, gastrocnemius and soleus skeletal muscles and heart. After a high carbohydrate test meal, lipoprotein lipase activity in white adipose tissue pads was higher (P less than 0.05) and that in brown adipose tissue was lower (P less than 0.05) than in these tissues from the meal-deprived group. Muscle lipoprotein lipase did not change significantly. A high fat test meal did not significantly alter lipoprotein lipase activity in brown adipose tissue, white adipose tissue, gastrocnemius or soleus when compared to the meal-deprived control, but heart lipoprotein lipase activity was significantly elevated. These findings indicate that after a single test meal lipoprotein lipase activity in brown adipose tissue is not higher than that from the meal-deprived group and therefore, lipoprotein lipase may not play a rate-limiting role in moving free fatty acids into this tissue in the postprandial state.

The use of cyst(e)ine in the removal of protein-bound homocysteine.

Competition between homocyst(e)ine and cyst(e)ine for binding sites on plasma proteins was examined both in vivo and in vitro. Plasma-free cyst(e)ine concentrations were elevated in rats fed diets adequate or deficient in vitamin B6 containing 2.4% L-cystine; however, plasma protein-bound cysteine was not increased. Feeding high cystine diets did not slow the accumulation or decrease the concentration of plasma protein-bound or free homocyst(e)ine in vitamin B6-deficient rats. An in vitro experiment demonstrated that plasma protein-bound cysteine increased when plasma was incubated with increasing concentrations of cysteine, and decreased with increasing homocysteine concentrations. Plasma protein-bound homocysteine concentration was increased by increasing the concentration of homocysteine in the incubation medium; however, increasing the cysteine concentration failed to decrease bound homocysteine. The affinity of homocysteine for binding sites on plasma proteins appeared to be high because cysteine did not displace the bound homocysteine. Therefore, feeding a high cystine diet is unlikely to cause a decrease in bound homocysteine in homocystinuric patients due to competition for binding sites, but may still be beneficial because plasma cystine concentrations are below normal in individuals with homocystinuria.

Factors affecting the accumulation of homocyst(e)ine in rats deficient in vitamin B-6.

Factors that may affect the concentration of accumulated homocyst(e)ine in rats deficient in vitamin B-6 were studied. Hepatic cystathionine synthase activity in vitamin B-6 deficient rats was 41.5% of control and plasma protein-bound and free homocyst(e)ine concentrations were significantly increased; however, among deficient rats there was no correlation between the concentration of homocyst(e)ine and the activity of hepatic cystathionine synthase. The plasma protein-bound homocysteine concentration did not fluctuate with time of day, but a decrease in bound homocysteine was found when blood was sampled repeatedly from the same animal during a 24-hour period. A 24-hour fast resulted in a reduction in plasma homocyst(e)ine to concentrations not different from those of control animals. The protein content of the diet affected growth but not food intake of rats. Feeding a 10% versus a 60% casein diet did not change the concentration of accumulated homocyst(e)ine. Of the factors examined, only food restriction significantly affected plasma homocyst(e)ine concentration in rats deficient in vitamin B-6. It is speculated that decreased and sporadic food intake in rats deficient in vitamin B-6 may be responsible for the large fluctuations in homocyst(e)ine concentration observed in these animals over time.

Homocyst(e)ine accumulation in pigs fed diets deficient in vitamin B-6: relationship to atherosclerosis.

The early onset of atherosclerotic lesions in homocystinuric individuals has implicated homocyst(e)ine in the development of atherosclerosis. Two trials were conducted in which diets totally or partially deficient in vitamin B-6 were fed to pigs to investigate the accumulation of homocyst(e)ine in the plasma and the development of vascular lesions. In one trial plasma free homocyst(e)ine levels were 179 and 43 mumol/liter in deficient and adequate pigs, respectively, on day 24, while cysteine levels were 39 and 155 mumol/liter. The concentration of plasma protein-bound homocysteine and cysteine reflected the plasma-free values. Because pigs deficient in pyridoxine could be used only over short time intervals, pigs in trial 2 received 0, 0.03, 0.3 or 3 mg (i.e., 0, 2, 20 or 200% of allowance) of supplemental pyridoxine . HCl per kilogram diet. After 12 weeks pigs deficient and adequate in vitamin B-6 were injected intravenously with Evan's blue dye and the vascular trunk perfused with 2% glutaraldehyde. The aorta and major organs were removed and examined for vascular lesions. Grossly no significant lesions were seen. Light microscopy revealed occasional foci of intimal degeneration and mural thickening in the renal arterioles of pigs deficient in vitamin B-6. An area of focal medial necrosis was observed in one of the pigs deficient in vitamin B-6. Pigs fed diets containing 0.03 mg pyridoxine . HCl per kilogram diet had homocyst(e)ine concentrations not different from pigs fed diets with no added pyridoxine. Animals fed diets containing 0.3 mg pyridoxine . HCl per kilogram had homocyst(e)ine concentrations slightly higher than controls fed 3.0 mg/kg. Feed intake and weight gain increased with increasing pyridoxine in the diet. Swine offer an excellent vascular model for humans. Diets partially deficient in vitamin B-6 which cause the homocyst(e)ine concentration to increase, but allow better growth and feed consumption than diets totally deficient in pyridoxine, could be fed to pigs to study homocyst(e)ine-induced vascular damage over extended period of time.

Accumulation of homocyst(e)ine in vitamin B-6 deficiency: a model for the study of cystathionine beta-synthase deficiency.

The accumulation of homocyst(e)ine in rats deficient in vitamin B-6 was monitored. Homocysteine and cysteine linked by disulfide bonds to plasma proteins, to red blood cells (RBC) membranes, and free in plasma were analyzed by HPLC separation and electrochemical detection. As the vitamin B-6 deficiency progressed, the concentration of plasma protein-bound and RBC membrane-bound homocysteine increased and that of cysteine decreased. Changes in free homocysteine concentration paralleled those seen in protein-bound homocysteine, but free cystein concentration did not fluctuate throughout the deficiency. Refeeding vitamin B-6 to deficient animals resulted in a return of homocysteine and cysteine concentrations to control levels within 2 days. Bound homocysteine and cysteine and plasma free homocyst(e)ine concentrations in rats deficient in vitamin B-6 were in the same concentration range as those seen in patients with homocystinuria due to cystathionine beta-synthase deficiency. Monitoring changes in plasma protein-bound and free homocysteine concentration during vitamin B-6 deficiency in rats may provide a useful system for the study of cystathionine beta-synthase deficiency and its treatment.