Plasma protein carbonyl concentration is not enhanced by chronic intake of high-protein diets in adult rats

We tested the hypothesis of whether high dietary protein intake is linked to oxidative stress as measured by the concentration of reactive carbonyl residues in plasma proteins. Three groups of male Wistar rats ( approximately 230 g, n = 10) were fed either 15% (15C), 30% (30C), or 60% (60C) casein diets over a period of 18 weeks. For comparison, a vitamin E deficient diet (60C-E) based on diet 60C was given to an additional group to provoke oxidative stress. Concentrations of alpha-tocopherol in plasma and of reactive carbonyl residues in total plasma proteins were measured by high performance liquid chromatography using fluorescence and by diode array detection after 2,4-dinitrophenylhydrazine reaction, respectively. After 1 week the concentration of reactive carbonyl residues in plasma proteins was found to be significantly (P < 0.05) higher in the 60C and 60C-E groups ( approximately 2.7 nmol/mg protein) compared with the 15C and 30C groups ( approximately 1.7 nmol/mg protein). After 14 weeks the 15C (3.4 +/- 1.2 nmol/mg protein) and 60C-E groups (3.9 +/- 1.7 nmol/mg protein) showed a significantly increased concentration of reactive carbonyl residues in plasma protein compared with the 30C and 60C groups (2.5 +/- 1.0 nmol/mg protein; 2.6 +/- 0.8 nmol/mg protein). As expected, chronic vitamin E deficiency (60C-E) resulted in significantly decreased alpha-tocopherol concentrations (3.91 +/- 2.42 micromol/mL vs. 31.3 +/- 4.8 micromol/mL) and a higher concentration of reactive carbonyl residues in plasma proteins. These results do not support the hypothesis that a chronic intake of high-protein diets leads to oxidative stress in adult rats. However, in the non-adapted state (1 week) a high protein intake contributes to oxidative modifications of protein-bound amino acid residues.     

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol. 49:1993–2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized ～1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P < 0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.     

Effect of hemodialysis on the antioxidative properties of serum

In patients with chronic renal failure undergoing regular hemodialysis (HD), oxidative stress is involved in the development of dialysis-related pathologies. The aim of the study was to measure the effect of HD treatment on the general antioxidative status of serum with special consideration of the specific oxidizability of lipids and proteins. Indicators for the oxidative/antioxidative status of plasma were monitored at the beginning and at the end of a dialysis session on the arterial and venous side of the dialyzer. A decrease in the antioxidant status was accompanied by an increased oxidizability of proteins as well as lipids during HD treatment. During the first passage of the dialyzer, the lag time of lipid oxidation decreased from 114.0+/-19.8 to 81.5+/-18.9 min, the lag time of protein oxidation decreased from 105.0+/-24.6 to 72.9+/-21.3 min and the total antioxidative status decreased from 518+/-24 to 252+/-124 microM trolox equivalents. The carbonyl content of serum proteins was high in patients with end stage renal disease (ESRD) (3.9+/-1.1 vs. 0.9+/-0.1 nmol/mg in controls) but did not change significantly during dialysis procedure. Our data demonstrate that the susceptibility of serum lipids and proteins to oxidative modification is severely increased by HD treatment.     

Temporal relationship between lens protein oxidation and cataract development in streptozotocin-induced diabetic rats

We compared the progression of lens opacification with the time course of oxidation of lens proteins under conditions of streptozotocin-induced experimental diabetes in rats. By the end of the 17th week, approx. 50% of the diabetic animals developed mature cataracts. During the following month, 95% of the eyes in the diabetic group became cataractous. In the course of lens opacification we observed a time-dependent increase in the content of protein carbonyls and decrease in the concentration of protein sulfhydryls in the lenses of diabetic animals. Significantly higher protein carbonyl (p<0.01) and lower protein sulfhydryl (p<0.001) content was found in lenses with the advanced stage of cataract when compared with the diabetic lenses still transparent. We showed that the values of protein carbonyls exceeding 1.2 nmol/mg protein and of sulfhydryls falling below 60 nmol/mg protein corresponded to an approximately 50% incidence of mature cataract development. At the end of the 34th week, when all lenses of diabetic rats became cataractous, the corresponding values of protein carbonyls and sulfhydryls were 2.5 nmol/mg protein and 27 nmol/mg protein, respectively. The main finding of this study is the disclosure of quantitative relationship between the degree of protein oxidation and the rate of advanced cataract development in the widely used model of streptozotocin-induced experimental diabetes in rats.     

Acute starvation affects rat adrenal steroidogenesis

To determine how starvation affects adrenal steroidogenesis we measured the activities of 3 adrenal enzymes involved in corticosterone biosynthesis in a group of adult female rats. The animals were either starved for 7 days or fed ad libitum for the same period. Relative adrenal weight and plasma corticosterone levels were increased in the experimental group of animals compared to the control group (40 +/- 2 vs 27 +/- 1 mg/100 g body weight, P less than 0.001, and 45 +/- 4 vs 30 +/- 5 ng/dl, P less than 0.05 respectively). There were no differences in plasma ACTH levels between the groups (34 +/- 5 vs 26 +/- 4 pg/ml). 11-Hydroxylase activity was increased in the starved group of animals (18 +/- 3 vs 8 +/- 2 nmol/mg protein/min, P less than 0.01). 3 beta-Hydroxysteroid dehydrogenase and 21-hydroxylase activities were not different between the groups (19 +/- 2 vs 16 +/- 1 nmol/mg protein/min, and 100 +/- 10 vs 110 +/- 10 pmol/mg protein/min respectively). These results suggest that acute starvation in rats produces an increase in adrenal 11-hydroxylase activity.     

Increase in oxidative damage to lipids and proteins in skeletal muscle of uremic patients

Muscle weakness and reduced exercise capacity are frequent complaints of patients with chronic uremia. Several lines of evidence have suggested that chronic uremia result in a state of increased oxidative stress. Reactive oxygen species (ROS) and free radicals are capable of damaging lipids and proteins but it remains unclear whether oxidative damage plays a role in the skeletal myopathy commonly seen in chronic uremia. In this cross-sectional study, we compared the levels of oxidative damage to proteins and lipids of skeletal muscle from 40 chronic uremic patients and 20 age- and sex-matched healthy subjects. Protein carbonyls were determined by a spectrophotometric method to assess the oxidative damage to proteins. Our results showed that the mean content of protein carbonyls in skeletal muscles was significantly elevated in the hemodialysis patients ( 3.78 ±0.14 nmol of 2,4-dinitrophenyl-hydrazone per mg of protein) as compared to healthy controls (2.97 ±0.28 nmol per mg of protein, p =0.017 vs normal controls). In addition, we found that the mean malondialdehyde (MDA) level was also significantly increased in the uremic patients compared to healthy controls. Further analysis revealed that there was an age-dependent increase in both oxidative damages in these patients. Regression analysis between plasma protein carbonyl and MDA levels showed a significant correlation between these two parameters ( r =0.43, p =0.002). The finding of increased oxidative damage to protein and lipids provide support that oxidative damage may play a role in the pathogenesis of skeletal myopathy in chronic uremic patients on hemodialysis.     

Oxidative protein damage in plasma of type 2 diabetic patients.

In this study, we evaluated protein oxidation in 84 patients with Type 2 diabetes with no complications and in 61 healthy volunteers who formed the control group, whose ages matched those of the patients. We determined plasma carbonyl and plasma thiol levels as markers of oxidative protein damage and erythrocyte glutathione, plasma ceruloplasmin and transferrin as markers of free radical scavengers. The concentrations (mean +/- SD) of both of plasma carbonyl (1.24 +/- 0.46 vs. 0.72 +/- 0.17 nmole/mg protein; p < 0.0001) and lipid hydroperoxides (1.8 +/- 0.63 vs. 1.3 +/- 0.21 micromole/l; p < 0.0001) were increased, and the concentration of plasma transferrin (3.85 +/- 0.65 vs. 4.59 +/- 0.79 g/l; p < 0.05) was decreased, respectively, in Type 2 diabetic patients compared with those of the controls. There were no significant differences in the concentrations of plasma thiol (0.0064 +/- 0.001 vs. 0.0068 +/- 0.001 micromole/mg protein), erythrocyte glutathione (2.54 +/- 0.57 vs. 2.65 +/- 0.56 mg/g Hb), plasma ceruloplasmin (548 +/- 107.30 vs. 609 +/- 93.34 mg/l) between the patients and the controls. These changes observed in diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to oxidative protein damage in Type 2 diabetic patients clinically free of complications.     

Oxidative stress is evident in erythrocytes as well as plasma in patients undergoing heart surgery involving cardiopulmonary bypass

OBJECTIVE: The aim of this study was to analyse the level and progression of oxidative stress, in both plasma and erythrocytes, during heart surgery involving cardiopulmonary bypass. MATERIALS AND METHODS: Twenty two patients undergoing cardiac surgery and considered to present a high/severe level of surgical risk were selected. We took five blood samples at different times during the cardiac surgery and analysed TBARS, alpha-tocopherol, coenzyme Q and retinol in plasma and TBARS (baseline levels and induced by Fe2+-ascorbate oxidation), alpha-tocopherol, coenzyme Q and catalase, superoxide dismutase and gluthatione peroxidase activity in erythrocytes. RESULTS: Plasma results shown a decrease in both alpha-tocopherol and retinol concentration after starting CPB with respect to the reference level (13.6 +/- 1.5 nmol ml(-1) vs. 22.0 +/- 3.0 nmol ml(-1) and 1.2 +/- 0.1nmol ml(-1) vs. 1.8 +/- 0.2 nmol ml(-1), respectively (p < 0.05)). In comparison, in erythrocytes, all antioxidants, both enzymatic and non-enzymatic, increased in activity or concentration after starting CPB. Erythrocyte TBARS, both baseline levels and induced levels, followed a similar pattern, with an increase after starting CPB with respect to the reference level (3.9 +/- 0.6 nmol mg(-1) of protein vs. 2.3 +/- 0.2 nmol mg(-1) of protein and 10.6 +/- 0.8 nmol mg(-1) of protein vs. 6.7+/- 0.6 nmol mg(-1) of protein, respectively (p < 0.05)). CONCLUSION: These results reveal an increase in oxidative stress after CPB, both in plasma and erythrocytes, and although the organism is capable of attenuating this stress by means of various antioxidative defence mechanisms, there is an increased possibility of post-CPB complications and thus of mortality.     

Dose-dependent increase of oxidative damage in the testes of rats subjected to acute iron overload

This study describes the in vivo response of rat testes to acute iron overload. Male Wistar rats (250-300 g) were injected ip with iron dextran at doses of 250 (Fe250), 500 (Fe500), or 1000 mg/kg body wt (Fe1000) or with saline (C). Parameters of oxidative stress and iron toxicity were measured 20 h after injection. Total iron content was 3.5-, 5.3-, and 10.4-fold higher in the Fe250, Fe500, and Fe1000 groups, respectively, compared to controls (320 +/- 22 nmol/g tissue). Histological studies showed that: (a) iron accumulated in the sperm and other testes cells, and (b) spermatogenesis was markedly lower in the Fe1000 group. The concentration of alpha-tocopherol, ubiquinol-9, and ubiquinol-10 in the testes was inversely correlated with the extent of oxidation. Testes chemiluminescence was 45% higher in the Fe1000 group compared to controls (41 cps/cm(2)). Endogenous levels of lipid oxidation, evaluated as 2-thiobarbituric acid-reactive substances, were 46, 73, and 82% higher in the groups Fe250, Fe500, and Fe1000, respectively, than in controls (33.6 +/- 1.4 nmol/g tissue). Oxidative damage to DNA evaluated by the presence of 8-oxo-2'-deoxyguanosine (oxo(8)dG), was 26, 39, and 74% higher in the Fe250, Fe500, and Fe1000 groups, respectively, than in the C group (2.3 +/- 0.1 oxo(8)dG/10(5)dG). Protein oxidation was measured as protein thiols and carbonyl content in proteins and glutamine synthase activity. Protein thiols content and glutamine synthase activity were similar in all the groups, while the protein-associated carbonyls content was 96% higher in the Fe1000 group than in the C group (2.1 +/- 0.4 nmol/mg protein). No changes in the activities of superoxide dismutase, catalase, and glutathione peroxidase were observed. The results showed that in vivo iron overload induced oxidative stress and the impairment of spermatogenesis in rat testes that were dependent on the amount of iron supplemented and its accumulation in the tissue.     

The effects of vitamin C supplementation on protein oxidation in healthy volunteers

We have investigated vitamin C supplementation effects on immunoglobulin oxidation (carbonyls) and total plasma protein sulfhydryls in healthy human volunteers. After receiving placebo, plasma ascorbate and oxidation markers were unchanged. Following 5 weeks supplementation with vitamin C (400 mg/day), plasma ascorbate increased but no significant effect on protein oxidation was observed. At 10 and 15 weeks supplementation, carbonyl levels were significantly reduced (P < 0.01) in subjects with low baseline ascorbate (29.51 +/- 5.3 microM) but not in those with normal baseline ascorbate (51.81 +/- 2.3 microM). To eliminate any effect from seasonal variation in dietary antioxidant intake, a second phase was undertaken. Subjects on vitamin C for 15 weeks were randomly assigned to receive either placebo or vitamin C. No difference in plasma sulfhydryl content was observed. Subjects withdrawn from supplementation showed an increase in immunoglobulin carbonyl content (P < 0.01). This demonstrates that dietary vitamin C supplementation can reduce certain types of oxidative protein damage in subjects with low basal antioxidant.     

Systemic markers of the redox balance in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is highly prevalent and its pathogenesis is still not completely clarified. Clinically stable patients (n=21) and healthy subjects (n=24) were studied for blood markers of oxidative injury and antioxidant status. The plasma concentration of protein carbonyls was significantly increased in COPD patients, both ex-smokers (0.76 +/- 0.28 nmol mg(-1)) and smokers (0.99 +/- 020 nmol mg(-1)) versus controls (0.49 +/- 0.14 nmol mg(-1)) . The concentration of total thiols was slightly enhanced in plasma of the COPD patients (ex-smokers 492 +/- 23 micromol 1(-1) and smokers 505 +/- 36 micromol 1(-1) versus controls 450 +/- 67 micromol 1(-1); p < 0.05). The activity of the antioxidant enzyme superoxide dismutase was increased in erythrocytes (activity in U g(-1) haemoglobin; ex-smokers 4460 +/- 763 and smokers 4114+/- 1060 versus 3015 +/- 851 in controls; p > 0.01), while glutathione peroxidase activity was decreased in total blood (activity in U g(-1) haemoglobin: ex-smokers 27 +/- 9 and smokers 23 +/- 9 versus 47 +/- 25; p < 0.01). Lower levels of selenium in plasma were also found for COPD patients (concentration in mg 1(-1): ex-smokers 0.030 +/- 0.019 and smokers 0.032 +/- 0.024 versus 0.058 +/- 0.023 in controls; p < 0.01), being more evident in those with very low levels of arterial oxygen pressure. In addition, the levels of potassium and rubidium were increased in blood cells of the patient group. All these changes might reflect oxidant damage and an altered electrolytic homeostasis, and can be interpreted as markers of COPD rather than as indicators of smoking habits.     

Oxidative damage to proteins and decrease of antioxidant capacity in patients with varicocele

To examine oxidative damage to blood proteins in the spermatic vein and seminal plasma antioxidant capacity of patients with varicocele, 30 young male patients with varicocele (group 1), 25 young male patients with subclinical varicocele (group 2), and 15 normal young males without varicocele (group 3) were recruited in this study. Varicocele and subclinical varicocele were confirmed by physical examination and Doppler ultrasonography. Blood samples were drawn from peripheral and spermatic veins before varicocelectomy. Plasma protein carbonyls were measured by a spectrophotometric assay after reacting with 2,4-dinitrophenylhydrazine. Protein thiols and ascorbic acid of seminal plasma were measured by spectrophotometric methods. We found that plasma protein carbonyls in the spermatic veins were significantly higher than those of corresponding peripheral veins in all 30 patients in group 1 and 12 patients in group 2 receiving varicocelectomy. Protein carbonyls in the spermatic veins of patients with varicocele (3.72 +/- 0.56 nmole/mg protein) and patients with subclinical varicocele (3.50 +/- 0.30 nmole/mg protein) were found to be higher than those of the control (2.35 +/- 0.33 nmole/mg protein). Protein thiols were 0.97 +/- 0.96, 1.50 +/- 0.89, and 3.49 +/- 0.81 nmole/ml, and ascorbic acid levels were 1.87 +/- 0.42, 2.13 +/- 0.24, and 2.38 +/- 0.07 mg/dl, in seminal plasma of the patients in groups 1, 2, and 3, respectively. Seminal plasma protein thiols and ascorbic acid levels in group 1 were significantly lower than those in groups 2 and 3, respectively. These results indicate that oxidative stress in the patients with varicocele and subclinical varicocele was higher than that of the control. We suggest that plasma protein carbonyls, and protein thiols and ascorbic acid of seminal plasma are useful markers for the assessment of oxidative stress in patients with varicocele and subclinical varicocele.     

Altered lipoprotein subclass distribution and PAF-AH activity in subjects with generalized aggressive periodontitis

In this study, we examined whether the documented increase of plasma triglycerides in patients with generalized aggressive periodontitis (GAgP) is associated with changes in lipoprotein subclass distribution and/or LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) activity. Lipoprotein subclasses were analyzed in whole plasma samples using nuclear magnetic resonance methods. Compared with subjects without periodontitis (NP subjects; n = 12), GAgP subjects (n = 12) had higher plasma levels of large, medium, and small VLDL (35.0 +/- 6.7 vs. 63.1 +/- 9.6 nmol/l; P = 0.025), higher levels of intermediate density lipoprotein (24.8 +/- 11.6 vs. 87.2 +/- 16.6 nmol/l; P = 0.006), lower levels of large LDL (448.3 +/- 48.5 vs. 315.8 +/- 59.4 nmol/l; P = 0.098), and higher levels of small LDL (488.2 +/- 104.2 vs. 946.7 +/- 151.6 nmol/l; P = 0.021). The average size of LDL from NP and GAgP subjects was 21.4 +/- 0.2 and 20.6 +/- 0.3 nm, respectively (P = 0.031). Compared with NP subjects, GAgP subjects had a greater number of circulating LDL particles (961.3 +/- 105.3 vs. 1,349.0 +/- 133.2 nmol/l; P = 0.032). Differences in the plasma levels of large, medium, and small HDL were not statistically significant. NP and GAgP subjects had similar plasma levels of total LDL-associated PAF-AH activity; however, LDL of GAgP subjects contained less PAF-AH activity per microgram of LDL protein (1,458.0 +/- 171.0 and 865.2 +/- 134 pmol/min/microg; P = 0.014). These results indicate that, in general, GAgP subjects have a more atherogenic lipoprotein profile and lower LDL-associated PAF-AH activity than NP subjects. These differences may help explain the increased risk of GAgP subjects for cardiovascular disease.     

Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease, but few studies have investigated protein oxidation in the inflamed mucosa. In this study, protein carbonyl formation on colonic mucosal proteins from mice was investigated following in vitro exposure of homogenates to iron/ascorbate, hydrogen peroxide, hypochloric acid (HOCl), or nitric oxide (*NO). Total carbonyl content was measured spectrophotometrically by derivatization with dinitrophenylhydrazine (DNPH), and oxidation of component proteins within the tissue was examined by Western blotting for DNPH-derivatized proteins using anti-dinitrophenyl DNP antibodies. These results were compared with protein carbonyl formation found in the acutely inflamed mucosa from mice with colitis induced by dextran sulfate sodium (DSS) administered at 5% w/v in the drinking water for 7 d. In vitro, carbonyl formation was observed after exposure to iron/ascorbate, HOCl and *NO. Iron/ascorbate (20 microM/20 mM) exposure for 5 h increased carbonyl groups by 80%, particularly on proteins of 48, 75-100, 116, 131, and 142 kDa. Oxidation by 0.1 and 0.5 mM HOCl did not increase total carbonyl levels, but Western blotting revealed carbonyl formation on many proteins, particularly in the 49-95 kDa region. After exposure to 1-10 mM HOCl, total carbonyl levels were increased by 0.5 to 12 times control levels with extensive cross-linking and fragmentation of proteins rich in carbonyl groups observed by Western blotting. In mice with acute colitis induced by DSS, protein carbonyl content of the inflamed mucosa was not significantly different from control mucosa, (7.80 +/- 1.05 vs. 8.43 +/- 0.59 nmo/mg protein respectively, p = .16 n = 8, 10); however, Western blotting analysis indicated several proteins of molecular weight 48, 79, 95, and 131 kDa that exhibited increased carbonyl content in the inflamed mucosa. These proteins corresponded to those observed after in vitro oxidation of normal intestinal mucosa with iron/ ascorbate and HOCl, suggesting that both HOCl and metal ions may be involved in protein oxidation in DSS-induced colitis. Identification and further analysis of the mucosal proteins susceptible to carbonyl modification may lead to a better understanding of the contribution of oxidants to the colonic mucosa tissue injury in inflammatory bowel disease.     

High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein

The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med.23:361-366; 1997 ]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 mug protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0-3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples.     

Effect of verapamil on CRF-induced abnormalities in phospholipid contents of brain synaptosomes

Chronic renal failure is associated with significant reductions in total phospholipids, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine of brain synaptosomes. These derangements in synaptosomal phospholipid metabolism were attributed to the state of secondary hyperparathyroidism of chronic renal failure (CRF) and the parathyroid hormone-induced accumulation of calcium in synaptosomes. This study examined whether a calcium channel blocker, verapamil, would prevent this synaptosomal calcium accumulation and correct the abnormalities in synaptosomal phospholipids in CRF. Verapamil treatment of normal rats for 21 days did not affect synaptosomal content of calcium or phospholipids. CRF of 21 days' duration was associated with a significant (P less than 0.01) increase in synaptosomal calcium (10.2 +/- 0.5 vs 7.4 +/- 0.6 nmol/mg protein) and a significant reduction (P less than 0.01) in total phospholipids (397 +/- 12 vs 529 +/- 19 nmol phospholipid P/mg protein), phosphatidylinositol (2.7 +/- 0.22 vs 4.6 +/- 0.27 nmol phospholipid P/mg protein), and phosphatidylserine (37 +/- 1.9 vs 83 +/- 5.2 nmol phospholipid P/mg protein). Simultaneous treatment of CRF rats with verapamil for 21 days reversed the synaptosomal abnormalities in calcium and phospholipid contents. Our data support the notion that the effect of excess parathyroid hormone of CRF on synaptosomal phospholipids is mainly due to the parathyroid hormone-induced calcium accumulation.     

Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.     

Lipid peroxidation and active calcium transport in inside-out vesicles of red blood cells from preeclamptic women

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside-out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49+/-3% in the preeclamptic women as compared to the gestational controls ( 8.06 +/- 0.11 nmol Ca++/mg protein min, gestational controls; 4.08 +/- 0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05 +/- 0.96 nmol Pi/mg protein min, gestational controls; 8.85 +/- 0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.