Systemic inflammation up-regulates serum hepcidin in exacerbations and stabile chronic obstructive pulmonary disease.

OBJECTIVES: Hepcidin is the main regulator of systemic iron homeostasis and its expression is modulated by iron status, hypoxia, erythroid factors and inflammation. The aim of our study was to examine a relationship between level of hepcidin and iron status, erythropoietic activity, hypoxia and inflammation in exacerbations and stable COPD patients. We hypothesized that hepcidin concentration is changed in COPD patients and is substantially influenced by inflammation and/or hypoxia. DESIGN AND METHODS: The study included 40 COPD patients and 30 healthy subjects. We measured haemoglobin, serum level of hepcidin and parameters indicative for inflammation: interleukin-6 (IL-6) and C reactive protein (CRP); hypoxia: partial oxygen pressure and haemoglobin oxygen saturation; iron status: iron, total iron binding capacity (TIBC), transferring saturation and ferritin; and erythropoietic activity: soluble transferrin receptors, reticulocytes, and erythropoietin. RESULTS: Hepcidin was elevated in exacerbations and in a stable phase compared to the control group and we found positive correlations of hepcidin with inflammatory markers IL-6 and CRP. Hepcidin also correlated positively with ferritin and inversely with TIBC. However, in COPD patients reticulocyte count was significantly reduced and negative correlation with hepcidin was established in exacerbation. No correlations were observed with iron, or indices of hypoxia. In the control group, positive associations were observed only with indices of iron status, positive with ferritin and negative one with TIBC. CONCLUSION: Systemic inflammation and elevated values of IL-6 present in exacerbations and stabile COPD might be responsible for the observed increased hepcidin level.

Paraoxonase 1 activity and phenotype distribution in premenopausal and postmenopausal women.

INTRODUCTION: Postmenopausal women have higher risk of cardiovascular disease. One of the contributing factors could be reduced activity of anti-atherogenic enzyme paraoxonase 1 (PON1). The aim of this study was to examine differences in the lipid status, paraoxonase and arylesterase PON1 activities and PON1 phenotype in women with regular menstrual cycle and in postmenopausal women. MATERIALS AND METHODS: The study included 51 women in reproductive age (25 in follicular and 26 in luteal phase of the menstrual cycle) and 23 women in postmenopause. Lipid parameters in sera were determined using original reagents and according to manufacturer protocol. PON1 activity in serum was assessed by spectrophotometric method with substrates: paraoxon and phenylacetate. PON1 phenotype was determined by double substrate method. RESULTS: Compared to the women in follicular and luteal phase, postmenopausal women have significantly higher concentration of triglyceride [0.9 (0.7-1.3), 0.7 (0.6-1.0) vs. 1.5 (0.9-1.7) mmol/L; P = 0.002], cholesterol [5.10 (4.78-6.10), 5.05 (4.70-5.40) vs. 6.30 (5.73-7.23) mmol/L; P < 0.001], LDL [3.00 (2.56-3.63), 3.00 (2.70-3.70) vs. 3.90 (3.23-4.50) mmol/L; P < 0.001], and apolipoprotein B [0.88 (0.75-1.00), 0.79 (0.68-1.00) vs. 1.07 (0.90-1.24) mmol/L; P = 0.002]. PON1 basal [104 (66-260), 106 (63-250) vs. 93 (71-165) U/L; P = 0.847] and salt-stimulated paraoxonase activity [210 (131-462), 211 (120-442) vs. 180 (139-296) U/L; P = 0.857] as well as arylesterase activity [74 (63-82), 70 (54-91) vs. 70 (60-81) kU/L; P = 0.906] and PON1 phenotype (P = 0.810) were not different in the study groups. CONCLUSION: There are no differences in PON1 activity and PON1 phenotype between women with regular menstrual cycle and postmenopausal women.

Paraoxonase 1 activity in patients with chronic obstructive pulmonary disease.

INTRODUCTION: Paraoxonase 1 (PON1) is an antioxidative enzyme manly associated with high density lipoproteins (HDL) in the peripheral blood. The aim of this study was to determine the PON1 paraoxonase and arylesterase activities in patients with chronic obstructive pulmonary disease (COPD). We also aimed to determine the concentration of reduced thiol groups as a marker of protein oxidation. MATERIALS AND METHODS: The study included 105 patients with stable COPD and 44 healthy controls. PON1 activities and thiols concentration were assayed in sera by spectrophotometry. RESULTS: PON1 basal (POX) and salt-stimulated paraoxonase activity (POX1) as well as arylesterase activity (ARE) were significantly reduced in COPD patients. In addition, concentration of reduced thiol groups was significantly decreased in COPD group. PON1 activities were similar in patients with different disease severity (GOLD stages). However, a significant reduction in POX, POX1 and ARE was observed already in GOLD II stage when compared to controls. POX and POX1 showed modest while ARE yielded very good power for discrimination between healthy subjects and COPD patients. Univariate and multivariate logistic regression analysis indicated that ARE is a good COPD predictor. CONCLUSION: Reduction of PON1 activity observed in COPD patients could be partly caused by oxidative environment. Lower concentrations of reduced thiol groups in COPD patients suggest that a decrease in PON1 activity could reflect oxidative changes of enzyme free cysteine residues. Furthermore, decreased PON1 arylesterase activity might indicate a down-regulation of PON1 concentration. Our results suggest that ARE could be considered as potential biomarker for COPD diagnosis.

Gamma-glutamyltransferase and C-reactive protein in stable chronic obstructive pulmonary disease.

Systemic inflammation and oxidative stress are the most important features of chronic obstructive pulmonary disease (COPD). The presence of oxidative stress in the airways of smokers, the largest population of COPD patients, is a consequence of direct inhalation of cigarette smoke and increased inflammation-related production of reactive oxygen species. On the other hand, oxidative stress appears to be the key component of many processes associated with chronic inflammation. We intend to examine whether serum C-reactive protein (CRP) concentration and gamma-glutamyltransferase (GGT) activity might be used as auxiliary markers in monitoring level of oxidative stress and inflammation in clinically stable COPD. We also investigated influence of cigarette smoking on these two systemic parameters. Catalytic activity of GGT and concentration of CRP were determined in sera of COPD patients (N = 109) and in healthy controls (N = 51) by using standard spectrophotometric method and immunoturbidimetric method, respectively. Concentration of CRP and activity of GGT were increased in COPD patients, as compared to healthy controls (p < 0.05). We found a significant positive correlation between those two parameters in COPD patients (r = 0.202, p = 0.0371). Our results showed no difference in GGT activity (p = 0.606) or CRP concentration (p = 0.573) between groups of patients when subdivided according to the severity of the disease. Smoking did not have a significant impact on CRP and GGT values in COPD patients and healthy controls. We showed an increase of serum CRP and GGT values in COPD patients, and we suggest that serum GGT activity might also represent an inflammation/oxidative stress marker. It seems that COPD patients present higher serum CRP and GGT values than healthy subjects independently from their smoking habits.

Polymorphisms of pon1 and pon2 genes in hemodialyzed patients.

OBJECTIVES: Q192R, L55M and -108C>T polymorphisms of pon1 gene affect PON1 paraoxonase activity while S311C polymorphism of pon2 gene might be associated with coronary heart disease. The aims of this study were to determine the frequencies of Q192R, L55M, -108C>T and S311C polymorphisms in hemodialyzed patients and to examine the relationship between pon1 gene polymorphisms and PON1 paraoxonase activity in those patients. DESIGN AND METHODS: The study included 238 control subjects and 263 hemodialyzed patients. RESULTS: PON1 paraoxonase activity was lower in patients. Genotype frequencies were different between two compared groups only for L55M polymorphism, with control group having higher frequency of MM genotype. Polymorphisms of pon1 gene were associated with significant variation in PON1 paraoxonase activity in both study groups. CONCLUSION: Our results suggest that Q192R, L55M and -108C>T polymorphisms are not by itself the causal factors leading to the lower PON1 paraoxonase activity in hemodialyzed patients.

Platelet count, mean platelet volume and smoking status in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), an increasing global health problem, may be complicated by acute atherothrombotic events. Although systemic inflammation plays the leading role in atherothrombotic processes, platelet activation and increased coagulation together with oxidative stress can significantly exacerbate atherosclerosis in COPD patients. In this study we determined platelet count, mean platelet volume (MPV) and classical markers of systemic inflammation - serum C-reactive protein (CRP), white blood cell (WBC) count and the relative proportion of segmented neutrophils in COPD patients, and compared them to those from the healthy controls. The most important and novel finding of this study was that patients with COPD had a significantly increased platelet count, along with a reduced MPV when compared to healthy controls (286 vs. 260 × 10(9)/l; 9.6 vs. 8.7 fL, respectively). Cigarette smoking had no influence on these results. The presence of systemic inflammation was clearly proved by the increase in classical inflammatory markers (CRP, WBC and segmented neutrophil count).

Effect of non-genetic factors on paraoxonase 1 activity in patients undergoing hemodialysis.

OBJECTIVES: Hemodialyzed patients have lower paraoxonase 1 (PON1) activity. Higher mortality risk from cardiovascular disease observed in these patients could be due to the low antiathetrogenic activity of PON1. Understanding the mechanism that causes lower PON1 activity could provide the possibility for modulation of enzyme activity in purpose of preventing and/or decreasing development of atherosclerosis. DESIGN AND METHODS: 87 healthy individuals and 71 hemodialyzed patients were enrolled in this study. RESULTS: Hemodialyzed patients had reduced PON1 paraoxonase and arylesterase activity, concentrations of HDL, HDL(3) and HDL(2) and concentrations of free thiol groups. Distribution of HDL subfractions and distribution of PON1 phenotypes as well as concentrations of MDA were not different between two study groups. In the in vitro experiment high concentrations of urea, creatinine, uric acid and addition of patient's sera ultrafiltrate did not significantly affect PON1 paraoxonase activity. CONCLUSION: Decreased HDL concentration as well as lower PON1 concentration (shown indirectly by the enzyme arylesterase activity) might contribute, at least partly, to the reduced PON1 activity observed in hemodialyzed patients. Decreased concentration of free thiol groups in sera suggest that free thiol group (Cys284) in PON1 might also be oxidized, which can affect PON1 activity.

Glutathione cycle in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidant/antioxidant imbalance. Glutathione is the most abundant cellular low-molecular weight thiol and the glutathione redox cycle is the fundamental component of the cellular antioxidant defence system. Concentration of total glutathione and catalytic activities of glutathione peroxidase and glutathione reductase were determined in peripheral blood of patients (n = 109) and healthy subjects (n = 51). Concentration of total glutathione in patients was not changed in comparison to healthy controls. However, we found statistically significant difference between patients with moderate and severe disease stages. Glutathione reductase activity was increased, while glutathione proxidase activity was decreased in the patients with COPD, when compared to healthy controls. We found no significant difference in glutathione peroxidase and glutathione reductase activities between stages. Patients who smoked had lower concentration of total glutathione compared with former smokers and never-smoking patients. Lung function parameters were inversely associated with glutathione level. Evidence is presented for differential modulation of glutathione peroxidase and glutathione reductase activities in peripheral blood of patients with stable COPD. We suppose that in addition to glutathione biosynthesis, glutathione reductase-dependent regulation of the glutathione redox state is vital for protection against oxidative stress.

A journey through mitogen-activated protein kinase and ochratoxin A interactions.

Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, carcinogenic, and cytotoxic action. It has been proposed that OTA might be involved in the development of Balkan endemic nephropathy, which is associated with an increased risk of urinary tract tumours, and of other forms of interstitial nephritis. Cell susceptibility to OTA mainly depends on mycotoxin concentrations, duration of exposure, and intracellular molecular and genetic context. OTA can affect a cell by stimulating or inhibiting certain signalling pathways such as mitogen-activated protein kinase (MAPK). Three major mammalian MAPKs have been described: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. All MAPKs regulate diverse cellular programmes, but in most cases ERKs have been linked to cell survival, while JNKs, and p38 MAPKs have been implicated in cell death by apoptosis. This review looks into OTA-mediated MAPK activation and its effects.

Levels changes of blood leukocytes and intracellular signalling pathways in COPD patients with respect to smoking attitude.

OBJECTIVES: To examine whether the proportions of blood leukocyte subsets are altered in COPD, and to assess the disturbances in signalling pathways in the leukocytes of COPD patients, with respect to smoking history. DESIGN AND METHODS: The study was performed at the Faculty of Pharmacy and Biochemistry and University Hospital for Lung Diseases, Zagreb, Croatia. Leukocyte counts were determined in 28 COPD male patients (11 smokers, 9 ex-smokers, 8 non-smokers) and 42 healthy male subjects (15 smokers, 13 ex-smokers, 14 non-smokers). We assessed activation of MAPKs (ERK, JNK, p38), and expression of Bcl-2 and Bax in the leukocytes by western blotting. RESULTS: Neutrophil and monocyte percentages were significantly increased, and lymphocyte percentage significantly decreased in COPD patients compared with healthy subjects (p<0.05). However, no significant smoking effect regarding leukocyte subsets was observed when compared current or former smokers with non-smokers of each studied group. ERK was activated in non-smokers only, especially in healthy ones. In contrast, strong induction of JNK and p38 phosphorylation, decreased Bcl-2 levels and increased Bax levels were detected in COPD smokers and COPD ex-smokers, but also in healthy individuals who smoke compared with healthy non-smokers (p<0.05). CONCLUSION: These results show that COPD and smoking affect intracellular signalling pathways. Understanding of the basic cellular and molecular mechanisms in COPD is essential for identification of molecules that may serve as targets for diagnosis and therapeutic interventions.

Mycotoxin fumonisin B1 alters cellular redox balance and signalling pathways in rat liver and kidney.

Mycotoxin fumonisin B(1) (FB(1)) is a frequent contaminant of grain, particularly maize, but the mechanism of its toxicity in the kidney and liver is not fully understood. FB(1)-stimulated oxidative stress might disturb cellular redox state and signal transduction pathways of the target cells. In this study we measured total intracellular glutathione (GSH), and assessed mitogen-activated protein kinases (MAPKs) activation and the expression of heat shock proteins (Hsps) Hsp25 and Hsp70 in the liver and kidney of male Wistar rats given 0.5 mg FB(1)/kg b.w. intraperitoneally for 2 or 7 days. The effect of FB(1) on GSH levels, MAPK activation and Hsp expression was found to be related to the type of tissue affected and the length of treatment. In rat liver, cellular GSH content increased, Hsp expression was up-regulated, and ERK and p38 were activated after the 7-day treatment, while even the 2-day treatment sufficed to produce phospho-JNK signal. In rat kidney, GSH levels decreased after the 2- and 7-day treatment with FB(1), while after the 7-day treatment all three MAPKs were activated, Hsp25 expression increased and Hsp70 expression decreased. In conclusion, FB(1) alters cellular redox balance, which leads to tissue-specific activation and expression of redox-sensitive signalling molecules. It seems that kidney cells are more sensitive to adverse effects of FB(1).

Expression of Hsp70 in kidney cells exposed to ochratoxin A.

Ochratoxin A (OTA) is a possible etiological agent of endemic nephropathy, a chronic renal disease with high prevalence in limited geographic areas. Ochratoxicosis has many characteristics of different pathological states in which heat shock proteins (Hsps) are usually induced. The most inducible heat shock proteins belong to the Hsp70 family. We determined the level of expression of Hsp70 by the Western blot analysis in kidneys of rats treated with low doses of OTA and in LLC-PK1 and MDCK cells exposed to OTA. Estimation of cell viability and release of lactate dehydrogenase (LDH) confirmed the toxic effects of OTA on cultured cells. OTA affects the relative distribution of two Hsp70 isoforms (68-kDa and 74-kDa isoforms), but does not change total amount of Hsp70 in rat kidney. No changes in the Hsp70 level were detected in LLC-PK1 and MDCK cells treated with OTA, although the cells were seriously injured, as was seen from the reduced cell viability and increased release of LDH. Both cell lines were capable of having Hsp70 induced following a heat shock. However, exposure of the cells to OTA before the heat shock challenge prevented Hsp70 induction. Results of the study show that OTA does not induce Hsp70 in rat kidney or in cultured kidney cells. The absence of Hsp70 protective effects in the cells and tissues might be a possible explanation for the cumulative destructive effects of OTA and a silent onset of endemic nephropathy in humans and of OTA-induced experimental nephrotoxicity in animals.