Circulating RBP4 Increase and Its Diagnosis of Chronic Kidney Disease.

OBJECTIVE: Sera retinol binding protein 4 (RBP4) has been associated with renal function. We determined the concentration of sera RBP4 in chronic kidney disease (CKD) and found its diagnostic value for CKD. MATERIALS AND METHODS: The sera samples were collected from 51 patients with CKD and 30 healthy control. The presence of circulating RBP4 was quantified by Enzyme-linked immunosorbent assay (ELISA), cystain C (CysC), urea, creatinine (CREA), uric acid (UA), total protein (TP), albumin (ALB) and 24 hours urinary protein (PRO) were examined by automatic biochemical analyzer. RESULTS: Levels of circulating RBP4 was significantly higher in CKD than in control (P<0.05). Serum RBP4 was positively related to serum urea, CREA and CysC (P<0.01). Receiver operating characteristic (ROC) analysis showed that the area under the curve (AUC) of RBP4 was 0.734. When the cutoff was 78.75ng/ml, the sensitivity and specificity were 52.9% and 100%, respectively. CONCLUSION: This is the first study to show that RBP4 could be usable for CKD diagnosis.

In vitro/vivo drug release and anti-diabetic cardiomyopathy properties of curcumin/PBLG-PEG-PBLG nanoparticles.

BACKGROUND: The objective of this study was to survey the therapeutic function of curcumin-encapsulated poly(gamma-benzyl l-glutamate)-poly(ethylene glycol)-poly(gammabenzyl l-glutamate) (PBLG-PEG-PBLG) (P) on diabetic cardiomyopathy (DCM) via cross regulation effect of calcium-sensing receptor (CaSR) and endogenous cystathionine-γ-lyase (CSE)/hydrogen sulfide (H2S). METHODS: Diabetic rats were preconditioned with 20 mg/kg curcumin or curcumin/P complex continuously for 8 weeks. The blood and myocardiums were collected, the level of serum H2S was observed, and the [Ca2+]i content was measured in myocardial cells, and hematoxylin-eosin, CaSR, CSE, and calmodulin (CaM) expression were detected. RESULTS: Both curcumin and curcumin/P pretreatment alleviated pathological morphological damage of myocardium, increased H2S and [Ca2+]i levels, and upregulated the expression of CaSR, CSE, and CaM as compared to DCM group, while curcumin/P remarkably augmented this effect. CONCLUSION: PBLG-PEG-PBLG could improve water-solubility and bioactivity of curcumin and curcumin/PBLG-PEG-PBLG significantly alleviated diabetic cardiomyopathy.

Reduction of leukocyte-derived H2S linked to abnormal glycolipid metabolism in hypertensive subjects.

We deduced that leukocyte-derived H2S would also play a pivotal role regarding nutrition homeostasis in hypertensive subjects. Plasma was obtained from patients with hypertension (n = 151) as well as control (n = 41). Leukocyte-derived H2S speed was determined, and biochemical indices of glucose and lipid metabolism were measured. Western blot analyses of CSE were also performed. Inflammation factors were measured. Leukocyte-derived H2S is produced at a significantly lower rate in overweight or obese patients (p < 0.05). There is a significant negative correlation between H2S and the levels of HOMA-RI and insulin in overweight patients and has a positive relationship with HDL-C only in overweight hypertensive patients (p < 0.05). Patients with high insulin levels showed down-regulation of CSE (p < 0.05). The levels of IL-10 decreased in both the obese and the overweight which showed significant relationship with all metabolism parameters such as HDL-C(r = 0.176, p = 0.031), insulin (r = -0.181, p = 0.027), HOMA-IR (r = -0.166, p = 0.045), and H2S speed (r = 0.995, p = 0.001). Linear regression analysis showed that insulin levels will increase (ß = -1.685, p = 0.041) with the slower speed of H2S. Leukocyte-derived H2S production varied according to the nutritional status of hypertensive subjects, and the H2S/IL-10 signaling pathway may be the junction point among hypertension, disturbance of nutritional status, and inflammation.

[Serum cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE ) and cytochrome c oxidase (COX) in copper mine miners potentially expose d to hydrogen sulfide]. / ß-syntaza cystationiny (CBS), γ-liaza cystationiny (CSE) i oksydaza cytochromu c (COX) w surowicy górników kopalni miedzi potencjalnie narazonych na emisje siarkowodoru.

BACKGROUND: The aim of the study was to evaluate serum levels of the target enzyme for H2S toxicity--cytochrome c oxidase (COX) and enzymes involved in the synthesis of H2S--cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in copper mine miners. MATERIAL AND METHODS: The initial and basic study was conducted respectively in 237 and 88 miners, working in 2 mining shafts: I--no H2S emissions recorded in the last 10 years (study group A) and II--H2S emissions occurred (study group B). A medical examination was performed and 10 ml of blood was collected from miners immediately after exiting the mine. RESULTS: There were no clinical or biochemical changes typical for H2S toxicity. Sulfhemoglobine was undetectable and there were no changes in the red-ox system. However, in group B, regulatory changes were found; a tendency to higher concentration of CBS and CSE, a higher activity of angiotensin converting enzyme (ACE) compared to group A (p<0.05) and a linear relationship between ACE and CSE (r=0.6927; p<0.001). It has been shown that cigarette smoking decreases COX (p<0.05), however, in miners working in shaft II, the decreased level of COX may result also from the presence of H2S in the gaseous emissions. CONCLUSIONS: COX concentration can be a sensitive indicator of exposure to H2S. The measurements of blood H2S concentrations carried out in workplaces should explain the cause of the changes observed in the COX, CBS and CSE activity.

Immune regulation of hydrogen sulfide in children with acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) and chemotherapy can cause immune imbalance, and gaseous molecule hydrogen sulfide (H2S) can participate in the process of immune response. This study aimed to investigate the immune regulation of H2S in pediatric ALL. METHODS: Children (n = 78) with ALL admitted during 2010-2013 were included in this study. Two blood samples were collected in period of before chemotherapy, bone marrow remission and two days after chemotherapy, respectively. Serum contents of H2S and cytokines, including interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10) and macrophage inflammatory protein-1α (MIP-1α), were detected using ELISA method. Stepwise regression was used to analyze the correlation between H2S and cytokines. Furthermore, human Jurkat cells were cultured in vitro, and nucleoprotein of Jurkat cells and peripheral blood mononuclear cells (PBMCs) were collected, contents of cystathionine γ-lyase (CSE) and certain cytokines were measured by Western blotting. RESULTS: Serum concentrations of H2S, IL-1ß, IL-6, IL-10 and MIP-1a in children with ALL were increased significantly (P < 0.01), while concentrations of IL-2, TNF-α, IFN-γ and IL-4 decreased obviously (P < 0.01). In patients after chemotherapy, concentrations of H2S and IL-10 were decreased significantly (P < 0.05), but IL-4 and IFN-γ concentrations increased markedly (P < 0.05). At remission stage, H2S, IL-1ß, IL-4, IL-6, IL-10 and MIP-1α concentrations were further decreased markedly (P < 0.05), but concentrations of IL-2, TNF-α and IFN-γ increased again (P < 0.05). Protein contents of CSE, IL-10, IL-4 and IL-2 of PBMCs also increased markedly in children with ALL. Moreover, changes of CSE protein contents of PBMCs were consistent with serum H2S contents, and there were significant correlation between H2S and certain cytokines based on stepwise regression analysis. Furthermore, compared with those of PBMCs group, in vitro study indicated that Jurkat cells of H2S group expressed IFN-γ, IL-10, IL-4 and IL-2 protein increased obviously (P < 0.05), while IL-4, IL-2 and CSE expression of PPG group decreased markedly (P < 0.05). CONCLUSION: Gaseous molecule H2S might participate in the process of immune regulation in pediatric ALL through modulating transcription and expression of cytokines.

Decreased cystathionine-γ-lyase (CSE) activity in livers of type 1 diabetic rats and peripheral blood mononuclear cells (PBMC) of type 1 diabetic patients.

The liver plays a major role in the formation of H2S, a novel signaling molecule. Diabetes is associated with lower blood levels of H2S. This study investigated the activities of cystathionine-γ-lyase (CSE, the enzyme that catalyzes H2S formation) in livers of type 1 diabetic (T1D) animals and in peripheral blood mononuclear cells (PBMC) isolated from T1D patients. T1D is associated with both hyperketonemia (acetoacetate and ß-hydroxybutyrate) and hyperglycemia. This study also examined the role of hyperglycemia and hyperketonemia per se in decreased CSE activity using U937 monocytes and PBMC isolated from healthy subjects. Livers from streptozotocin-treated T1D rats demonstrated a significantly higher reactive oxygen species production, lower CSE protein expression and activity, and lower H2S formation compared with those of controls. Studies with T1D patients showed a decrease in CSE protein expression and activity in PBMC compared with those of age-matched normal subjects. Cell culture studies demonstrated that high glucose (25 mm) and/or acetoacetate (4 mm) increased reactive oxygen species, decreased CSE mRNA expression, protein expression, and enzymatic activity, and reduced H2S levels; however, ß-hydroxybutyrate treatment had no effect. A similar effect, which was also observed in PBMC treated with high glucose alone or along with acetoacetate, was prevented by vitamin D supplementation. Studies with CSE siRNA provide evidence for a relationship between impaired CSE expression and reduced H2S levels. This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE expression and activity, which can contribute to the impaired H2S signaling associated with diabetes.

[Effect of nitric oxide on the preterm neonatal rabbit ductus arteriosus cystathionine-γ-lyase/hydrogen sulfide pathway].

OBJECTIVE: To investigate the impact of sodium nitroprusside (a nitric oxide donor) in the ductus arteriosus in preterm rabbits on hydrogen sulfide (H(2)S)-cystathionine-γ-lyase (CSE) system. METHODS: For 16 Japanese white rabbits pregnant for 21 days were randomly divided into four groups, each of the following groups had 4 rabbits: control group, intraperitoneal injection of sodium nitroprusside 1 mg/kg, 2.5 mg/kg, and 5.0 mg/kg groups. The rabbits in control group had a peritoneal puncture with a simple hollow needle, and those in the other groups were given corresponding dose of intraperitoneal injection of sodium nitroprusside at gestational age 23 and 25 days, respectively. At gestational age 26 days the fetuses of the pregnant rabbits were removed surgically, and 28 fetal rabbits were obtained from the control group, 27 from the sodium nitroprusside small dose group, 29 from the medium dose group, and 26 from the large dose group. The fetal heart blood sample of 1 ml was taken from each fetus, and immediately after sampling the arterial ductal tissues were dissected. Fetal rabbit plasma proteins hydrogen sulfide content was determined by using de-protein method, and real time quantitative RT-PCR was used for determination of arterial tissue CSE gene and western-blotting was used for measuring protein expression of CSE. RESULTS: In control group hydrogen sulfide content of fetal rabbits plasma (55.68 ± 6.57) µmol/L and arterial tissue CSE mRNA expression was 1.07 ± 4.12; the parameters in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (60.02 ± 6.09) µmol/L and 3.46 ± 0.18; in intraperitoneal injection of sodium nitroprusside group 2.5 mg/kg, were (64.71 ± 7.12) µmol/L and 10.95 ± 0.22; and in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (70.63 ± 8.07) µmol/L and 19.56 ± 0.17. Comparison between small dose group and control group, medium dose group and small dose group, high dose group and medium dose group showed that the above data were significantly different P < 0.05, with the injection of sodium nitroprusside CSE protein expression increased gradually with increasing doses. CONCLUSION: Sodium nitroprusside showed an enhancing effect on preterm CSE-H(2)S system in rabbit ductus arteriosus in a certain range of concentration in a dose-dependent manner.

[Protection of CSE/H2S system in hepatic ischemia reperfusion injury in rats].

OBJECTIVE: To study the protective function and pathophysiology of cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) system in hepatic ischemia-reperfusion injury (HIRI) in rats. METHODS: Wistar rats were randomly distributed into sham group (n = 18), ischemia-reperfusion (IR) group (n = 18), IR + NaHS group (n = 18) and IR + DL-propargylglycine (PAG) group (n = 18). The hepatic IR model was established by Pringle's hepatic vascular occlusion. At each of the indicated time points (1, 3 and 6 hours after IR), the serum levels of H(2)S and the hepatic CSE activity were measured. The serum levels of inflammatory factors, including TNF-α, IL-10 were determined by ELISA methods. The expression of apoptotic protein, TNF-α, in liver tissue was tested by Western blot assay, cell apoptosis was examined by TUNEL and the histological changes were examined in each group. RESULTS: The serum levels of H(2)S and CSE activity were significantly increased in group IR compared with group sham at all indicated time points (P < 0.05). The serum level of inflammatory factors (P < 0.01) and the hepatic expression of TNF-α protein (P < 0.05) were elevated obviously in group IR than that in group sham. Administration of NaHS could reduce the production of inflammatory factors in serum (P < 0.01), inhibit hepatic protein expression of TNF-α (P < 0.05) and attenuate the liver histological scores of IR injury (P < 0.05), whereas PAG aggravated them. CONCLUSION: The endogenous CSE/H(2)S system maybe involved in the pathogenesis of hepatic IR injury, which suggests that CSE/H(2)S system can protect liver from IR injury in rats by intervening in inflammatory reaction, attenuating the injury severity and inhibiting expression of apoptotic protein TNF-α.

Rhodanese, but not cystathionine-gamma-lyase, is associated with dextran sulfate sodium-evoked colitis in mice: a sign of impaired colonic sulfide detoxification?

Clinical studies suggest that colonic luminal hydrogen sulfide (H(2)S), produced by sulfate-reducing bacteria or through other pathways, might be involved in the pathogenesis of inflammatory bowel disease (IBD). Nonetheless, this hypothesis has been poorly investigated by basic studies using laboratory animals. We thus focused on two enzymes, cystathionine-gamma-lyase (CSE) that generates H(2)S from l-cysteine, and rhodanese that directly or indirectly detoxifies H(2)S, particularly in relation to the colitis induced by dextran sulfate sodium (DSS) in mice. CSE was a major H(2)S-forming enzyme in colonic and renal homogenates from mice and rats, and the rhodanese activity was also detectable in both tissues. Colitis-related symptoms including decreased body weight gain, diarrhea, hematochezia and shortening of colon length were observed in the mice drinking DSS. Those symptoms were not or only slightly attenuated by repeated administration of a CSE inhibitor. CSE activity and protein levels in the colonic tissue did not notably change in the mice with colitis. In contrast, the activity and protein/mRNA levels of rhodanese in the colon, but not kidney, significantly decreased nearly in parallel with the development of colitis, followed by elevation of rhodanese activity in red blood cells (RBCs). These data show that rhodanese, but not CSE, is associated with DSS-induced colitis in mice, leading to a hypothesis that impaired detoxification of H(2)S due to down-regulation or suppression of colonic rhodanese is involved in IBD. The delayed enhancement of rhodanese activity in RBCs, a possible compensative event, might be available as a disease marker for IBD.

Hydrogen sulfide ameliorates vascular calcification induced by vitamin D3 plus nicotine in rats.

AIM: To investigate the role of the endogenous cystathionine gamma-synthase (CSE)/hydrogen sulfide (H2S) pathway in vascular calcification in vivo. METHODS: A rat vascular calcification model was established by administration of vitamin D3 plus nicotine (VDN). The amount of CSE and osteopontin (OPN) mRNA was determined by using semi-quantitative reverse-transcription polymerase chain reaction. The calcium content, 45Ca2+ accumulation and alkaline phosphatase (ALP) activity were measured. H2S production and CSE activity were measured. RESULTS: von Kossa staining produced strong positive black/brown staining in areas among the elastic fibers of the medial layer in the calcified aorta. The calcium content, 45Ca2+ accumulation and ALP activity in calcified arteries increased by 6.77-, 1.42-, and 1.87-fold, respectively, compared with controls. The expression of the OPN gene was upregulated (P<0.01). Expression of the CSE gene was downregulated. However, calcium content, 45Ca2+ uptake and ALP activity in the VDN plus NaHS group was lower than that in the VDN group. The content of calcium and 45Ca2+ accumulation and activity of ALP in the aorta were 34.8%, 40.75% and 63.5% lower in the low-dosage NaHS group than in the VDN group, respectively (P<0.01), and the calcium content and deposition of 45Ca2+ and activity of ALP was 83.9%, 37.8 % and 46.2% lower in the aorta in the high-dosage NaHS group than in the VDN group, respectively (P<0.01). The expression of the OPN gene was downregulated. CONCLUSION: The production of H2S, and CSE activity were decreased and CSE gene expression was downregulated in rats with vascular calcification. H2S can ameliorate vascular calcification, suggesting that the H2S/CSE pathway plays a regulatory role in the pathogenesis of vascular calcification.

Methionine transsulfuration is increased during sepsis in rats.

Methionine transsulfuration in plasma and liver, and plasma methionine and cysteine kinetics were investigated in vivo during the acute phase of sepsis in rats. Rats were infected with an intravenous injection of live Escherichia coli, and control pair-fed rats were injected with saline. Two days after injection, the rats were infused for 6 h with [(35)S]methionine and [(15)N]cysteine. Transsulfuration was measured from the transfer rate of (35)S from methionine to cysteine. Liver cystathionase activity was also measured. Infection significantly increased (P < 0.05) the contribution of transsulfuration to cysteine flux in both plasma and liver (by 80%) and the contribution of transsulfuration to plasma methionine flux (by 133%). Transsulfuration measured in plasma was significantly (P < 0.05) higher in infected rats than in pair-fed rats (0.68 and 0.25 micromol. h(-1). 100 g(-1), respectively). However, liver cystathionase specific activity was decreased by 17% by infection (P < 0.05). Infection increased methionine flux (16%, P < 0.05) less than cysteine flux (38%, P < 0.05). Therefore, the plasma cysteine flux was higher than that predicted from estimates of protein turnover based on methionine data, probably because of enhanced glutathione turnover. Taken together, these results suggest an increased cysteine requirement in infection.

Effect of glucose-cysteine adduct on cysteine desulfuration in guinea pig tissues.

Effect of intraperitoneal administration (12 mmol/kg of body weight) of glucose-cysteine adduct 2-(D-gluco-pentahydroxypentyl)-thiazolidine-4-carboxylate, (glc-cys) on the rhodanese, gamma-cystathionase and 3-mercaptopyruvate sulfurtransferase (MPST) activity levels in guinea pig tissues was studied. The rhodanese activity value in liver increased by 41%, 3-mercaptopyruvate sulfurtransferase by 24%, and gamma-cystathionase by 12% after three successive days of the administration. In the kidney, on the contrary, glc-cys administration resulted in about 18% decrease in the gamma-cystathionase activity value, whereas no changes in MPST and rhodanese activity values were observed. In the case of the brain, rhodanese and gamma-cystathionase did not change their activity but the activity of MPST decreased by 21%. MPST level did not change substantially in whole blood after glc-cys treatment. The results seem to indicate that in guinea pig liver but not in kidney and brain, glc-cys has a potential to activate the desulfuration pathway of L-cysteine metabolism.

L-cysteine and glutathione metabolism are impaired in premature infants due to cystathionase deficiency.

There are conflicting reports in the literature as to whether L-cysteine is an essential amino acid in premature infants as the result of the absence of hepatic cystathionase activity. To analyze the physiological importance of the cystathionase deficiency, we studied sulfur amino acid metabolism in human neonates of different gestational ages. Plasma cystathionine concentrations are higher in premature infants < or = 32 wk gestation (group 1) than in premature infants of 33-36 wk gestational age (group 2) or in full-term infants (group 3), whereas plasma cysteine concentrations are much lower in group 1 and 2 premature infants than in mature infants. Furthermore, erythrocytes from group 1 premature infants synthetize glutathione from L-methionine (a process dependent on the cystathionase pathway) at a much lower rate than do erythrocytes from group 2 premature or full-term infants. Thus, the metabolic flow through the transsulfuration pathway may be insufficient to meet the glutathione and cysteine requirements of very premature infants.

Elevation of cystathionine gamma-lyase activity in the serum of rats treated with a single dose of carbon tetrachloride.

Cystathionine gamma-lyase activity in the sera of rats subjected to experimental hepatotoxicity after intraperitoneal administration of carbon tetrachloride (CCl4) was measured and compared with activities of aspartate aminotransferase (GOT) and alanine aminotransferase (GPT), which have been clinically used for detecting liver damage. In the experimental subjects, serum levels of cystathionine gamma-lyase showed a similar behavior to GOT and GPT, increasing markedly with respect to the controls after administration of CCl4 and reaching a maximum at 24 hours. No such cystathionine gamma-lyase activity was detected immunochemically in the control subjects. These data suggest that measurement of serum cystathionine gamma-lyase activity could be used as a sensitive and specific marker of hepatic cytolysis.

Cysteine auxotrophy of human leukemic lymphoblasts is associated with decreased amounts of intracellular cystathionase protein.

A series of human lymphoblastoid cell lines derived from nonleukemic donors are known to be cysteine prototrophs (cys+), while several lymphoblastoid lines derived from leukemic donors are cysteine auxotrophs (cys-). We have tested representative cell lines of each type for their content of cystathionase enzyme activity by a specific catalytic assay and their total cystathionase protein content by immunoprecipitation of in vivo labeled protein. There was a close correlation between the cellular content of the enzyme as determined in the two assays. Specifically, those cys+ lines having readily measureable enzyme by catalytic assay were found to contain significantly higher levels of immunoprecipitable Mr 43 000 cystathionase subunit than those cys- lines tested which were depleted in active enzyme. Thus, the absolute cysteine requirement of the leukemic, cys- cell lines tested is likely due to an intracellular reduction of cystathionase protein.

Methionine adenosyltransferase, cystathionine beta-synthase and cystathionine gamma-lyase activity of rat liver subcellular particles, human blood cells and mixed white cells from rat bone marrow.

1. Methionine adenosyltransferase (ATP:L-methionine-S-adenosyl transferase, EC 2.5.1.6), cystathionine beta-synthase F1L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and cystathionine gamma-lyase [L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] activities were found only in the cytosol fraction of rat liver cells. None was found in the mitochondrial or endoplasmic reticulum fractions as judged by the distribution of marker enzymes on a density gradient after centrifugation of the cytoplasmic fraction of a liver homogenate, or in a preparation of liver cell nuclei. 2. Polymorphs, lymphocytes (with admixed monocytes) and mixed bone marrow white cells contained no methionine adenosyl transferase, cystathionine beta-synthase or cystathionine gamma-lyase activities. 3. The possible bearing of these results on the problem of abnormal cystine storage in cystinosis is briefly discussed.