Diabetic nephropathy is associated with low-grade inflammation in Type 1 diabetic patients

Aims/hypothesis Increased concentrations of C-reactive protein and interleukin-6, a finding suggestive of the presence of inflammation, have been observed in Type 2 diabetes. In such patients, C-reactive protein was predictive of diabetic nephropathy. Studies on low-grade inflammatory markers and nephropathy in Type 1 diabetic patients have shown conflicting results. Therefore we studied whether low-grade inflammation is associated with diabetic nephropathy in Type 1 diabetic patients.Methods We divided 194 Type 1 diabetic patients into three groups from the Finnish Diabetic Nephropathy Study based upon their albumin excretion rate. Patients with normoalbuminuria (n=67) had no antihypertensive medication or signs of cardiovascular disease, while patients with microalbuminuria (n=64) or macroalbuminuria (n=63) were all treated with an angiotensin-converting enzyme inhibitor, a drug that could attenuate low-grade inflammation. As a measure of insulin sensitivity we used estimated glucose disposal rate. C-reactive protein was measured by radioimmunoassay and interleukin-6 by high sensitivity enzyme immunoassay.Results C-reactive protein was higher in micro- and macroalbuminuric patients compared to normoalbuminuric patients (normoalbuminuria 2.0±1.7, microalbuminuria 2.6±1.7, macroalbuminuria 2.9±2.5 mg/l; p=0.016), while interleukin-6 increased in parallel with the severity of the renal disease (1.9±1.5, 2.9±3.3, 3.6±3.1 ng/l; p<0.0001). In multiple regression analysis albumin excretion rate was the only variable independently associated with C-reactive protein (p=0.03), whereas albumin excretion rate (p=0.0003), HDL-cholesterol (p=0.0135) and duration of diabetes (p=0.0176) were independently associated with interleukin-6.Conclusions/interpretation Low-grade inflammatory markers are associated with diabetic nephropathy in Type 1 diabetic patients. The predictive value needs to be assessed.Abbreviations DN diabetic nephropathy - CRP C-reactive protein - eGDR estimated glucose disposal rate - FinnDiane finnish diabetic nephropathy study - MDRD modification of diet in renal disease     

Anticardiolipin antibodies and lupus anticoagulant in patients treated with different methods of renal replacement therapy in comparison to patients with systemic lupus erythematosus

Summary Antiphospholipid antibodies were found to be associated with certain clinical manifestations such as recurrent venous thrombosis or arterial occlusions in a wide spectrum of immune disorders [6]. We analyzed the plasma concentration of two isotypes (IgG, IgM) of anticardiolipin antibodies (ACA) and lupus anticoagulant (LAC) activity in 84 patients with end-stage renal disease. They were receiving different types of renal replacement therapy and had a high frequency of thrombotic vascular complications. The prevalence of positive tests and the mean ACA concentration obtained in the plasma of renal patients were compared with those in patients with systemic lupus erythematosus (SLE) and in healthy controls. When analyzed as a whole group, renal patients maintained on dialysis (n=45: hemodialysis,n=20; peritoneal dialysis) or with a functioning kidney transplant (n=19) did not differ in mean ACA concentration and LAC activity (n=84, ACA: IgG 10±7 U/ml, IgM 2±1 U/ml, LAC ratio: 1.0±0.2) from healthy subjects (n=50, ACA: IgG 10±3, IgM 2±1 U/ml, LAC ratio: 1.0±0.1) but they had a higher incidence of raised IgG-ACA titers (renal replacement 14% vs. normal controls 4%,p<0.05). No significant correlation was found between thrombotic events and raised ACA or LAC activity in dialysis patients. In contrast, the proportion of SLE patients (n=51) with a raised concentration of ACA was significantly higher (IgG: 69%, IgM: 29%) than that among patients with renal replacement therapy (IgG: 14%, IgM: 4%) or normal controls (IgG: 4%, IgM: 2%,p<0.002). Moreover, recurrent manifestations of thrombosis in SLE were associated with very high IgG-ACA (n=11, IgG 117±91 U/ml) and LAC activity (LAC ratio: 2.4±0.7) in comparison to SLE patients without thrombotic events (n=40, ACA: IgG 23±13). The results of our investigations demonstrate that the pathogenetic role of these phospholipid antibodies in end-stage renal disease is far from established.     

Dynamics of cytochrome P4502E1 activity in man: induction by ethanol and disappearance during withdrawal phase.

BACKGROUND/AIMS: Chronic ethanol consumption results in the induction of hepatic cytochrome P4502E1 (CYP2E1) in man, which is believed to play an important role in the pathogenesis of alcoholic liver disease. However, the amount and duration of alcohol intake associated with CYP2E1 induction is not known but limited information is available on the disappearance of CYP2E1 following alcohol withdrawal. METHODS: To study these questions, five healthy male volunteers received ethanol daily (40 g/day) over 4 weeks. CYP2E1 induction was monitored by using the chlorzoxazone test before and every week following the start of alcohol ingestion. In addition, CYP2E1 was also determined in five alcoholics 1, 3, 8 and 15 days following ethanol withdrawal and in five patients with non-alcoholic liver disease. RESULTS: A significant CYP2E1 induction occurred 1 week following the ingestion of 40 g ethanol per day and increased further after 4 weeks. The disappearance of CYP2E1 was found to be significant 3 days following ethanol withdrawal and further decreased up to day 8. Thereafter, no significant change occurred and CYP2E1 activities were comparable with those in patients with non-alcoholic liver disease. CONCLUSIONS: These data show a significant and quick induction of CYP2E1 activity, already at moderate alcohol consumption, which may be of importance in the pathogenesis of alcoholic liver disease, of ethanol, drug and vitamin A interactions and in alcohol associated carcinogenesis.     

Chronic hepatitis C: Effect of alcohol on hepatic activity and viral titre

Background/Aims: Alcohol and the hepatitis C virus have been postulated to interact to adversely affect the natural history of patients with chronic liver disease. The aim of this study was to examine the effect of alcohol on hepatitic activity and serum HCV RNA levels in patients with chronic hepatitis C.Methods: Forty-five consecutive patients with chronic hepatitis C were classified according to alcohol intake over the 3-month period preceding study entry: group 1 (n=23), >10 g alcohol/day; group 2 (n=22), ≤10 g alcohol/day. Hepatitic activity and alcohol intake were assessed at study entry and, following moderation of alcohol intake, after a mean follow-up period of 4.4±0.2 months.Results: Hepatitic activity was significantly greater in the patients who consumed >10 g of alcohol/day. Moderation of alcohol consumption in patients consuming >10 g/day resulted in a significant decrease in both disease activity (p=0.0002) and viral RNA titre (p=0.018); there was no change over the study period in patients with a consistently low alcohol intake.Conclusion: The results support the hyptheses that, in patients with chronic hepatitis C, alcohol aggravates hepatic injury, increases viral load and adversely affects the natural history of the associated liver disease.     

Expression of P450 and nuclear receptors in normal and end-stage Chinese livers

AIM: To investigate the expression of P450 enzyme genes by using end-stage liver disease samples and trimmed normal Chinese donor livers.METHODS: The end-stage liver disease samples [n = 93, including hepatocellular carcinoma (HCC), peri-HCC tissue, hepatitis B virus cirrhosis, alcoholic cirrhosis, and severe cirrhosis] and trimmed normal Chinese donor livers (n = 35) from The Institute of Organ Transplantation in Beijing, China. Total RNA was extracted, purified, and subjected to real-time RT-PCR analysis.RESULTS: For cytochrome P450 enzymes 1 (CYP1) family, the expression of CYP1A2 was decreased 90% in HCC, 80% in alcoholic cirrhosis, and 65% in severe cirrhosis. For CYP2 family, the expression of CAR was decreased 50% in HCC, but increased 50% in peri-HCC tissues. Similar decreases (about 50%) of CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 were observed in HCC, as compared to peri-HCC tissues and normal livers. CYP2C19 were decreased in all end-stage liver diseases and CYP2E1 also decreased in alcoholic cirrhosis and severe cirrhosis. For CYP3 family, the expression of PXR was decreased 60% in HCC, together with decreases in CYP3A4, CYP3A5, and CYP3A7. In contrast, the expression of CYP3A7 was slightly increased in HBV cirrhosis. The expression of CYP4A11 was decreased 85% in HCC, 7% in alcoholic cirrhosis and severe liver cirrhosis, along with decreases in PPARα. The 93 end-stage livers had much higher inter-individual variations in gene expression than 35 normal livers.CONCLUSION: The expression of CYP enzyme genes and corresponding nuclear receptors was generally decreased in end-stage liver diseases, and significant differences in gene expression were evident between peri-HCC and HCC.     

Plasma YKL-40: A New Potential Marker of Fibrosis in Patients with Alcoholic Cirrhosis?

Background: YKL-40 (human cartilage glycoprotein-39, or 38-kDa heparin-binding glycoprotein) is a mammalian member of a protein family that includes bacterial chitinases. YKL-40 mRNA is expressed by human liver and may play a role in tissue remodelling. The aims were to assess whether circulating YKL-40 is released or extracted in the hepatosplanchnic system and to localize YKL-40 in liver tissue. Methods: Plasma YKL-40 was determined by radioimmunoassay in 25 patients with liver diseases (alcoholic cirrhosis (n=20), chronic active hepatitis (n = 2), cirrhosis of unknown aetiology (n=2), and fatty liver (H=1)) and in 18 subjects with normal liver function during a haemodynamic investigation with catheterization of liver vein and the femoral artery. Immunohistochemical studies of the localization of YKL-40 in cryostat liver biopsy specimens were obtained from eight other patients with alcoholic liver disease. Results: Plasma YKL-40 was significantly increased in patients with alcoholic cirrhosis (median, 523 μg/l; P< 0.001) compared with controls (106μg/l), and plasma YKL-40 in the hepatic vein was higher (P < 0.01) than that of the artery in both the patients and controls, showing release of YKL-40 from the hepatosplanchnic area. The release rate of YKL-40 from the hepatosplanchnic area was higher in patients with liver disease than in controls (11.0 versus 2.1 μg/min, P < 0.05). Furthermore, the highest plasma YKL-40 levels were found in patients with a moderate or severe degree of liver fibrosis, and immunohistochemical studies showed positive staining for YKL-40 antigen in areas of the liver biopsy with fibrosis. Conclusions: The increased plasma YKL-40 in patients with alcoholic cirrhosis may reflect the remodelling of liver fibrosis.     

Central role for sodium in the pathogenesis of blood pressure changes independent of angiotensin,aldosterone and catecholamines in Type 1 (insulin-dependent) diabetes mellitus

Summary We studied 73 Type 1 (insulin-dependent) diabetic patients, 18 to 50 years of age, with a diabetes duration of more than five years. Group 1: normal urinary albumin excretion below 30 mg per 24 h (n=19); group 2: microalbuminuria, 30–300 mg per 24 h (n=36); and group 3: diabetic nephropathy, above 300 mg per 24 h (n=18). Fifteen non-diabetic persons matched for sex and age served as control subjects. The sodium intake evaluated on the basis of 24-h urine sodium excretion was similar in patients and control subjects. Blood pressure in groups 1 and 2 and control subjects was below 160/95 mmHg. The blood pressure was increased in group 3 as compared with the other groups (systolic/diastolic 161±22/101±9 mmHg vs 131±13/84±10, mean±SD, p<0.0001). Exchangeable sodium was increased in patients (p<0.01) and correlated to the mean blood pressure (n=70, r=0.41, p<0.01). Extracellular volume was increased in patients (p<0.05), whereas plasma volume was normal. Supine serum angiotensin II was suppressed in the patients (p<0.001). A negative correlation was found between mean blood pressure and supine serum aldosterone (n=68, r=-0.24, p<0.05), and exchangeable sodium and aldosterone (n=66, r=-0.36, p<0.002) in all patients. The catecholamine levels were also suppressed or normal in the patients. These data suggest that sodium retention plays a major role and that the aldosterone, angiotensin II and catecholamine levels are suppressed during the blood pressure rise observed in the very early stages of diabetic renal disease.     

Different hemodynamic patterns of alcoholic and viral endstage cirrhosis: Analysis of explanted liver weight,degree of fibrosis and splanchnic Doppler parameters

Objective. In cirrhosis, portal hemodynamics is usually considered independently of the disease etiology. The objective of this study was to investigate the role of the etiology of liver disease on the relationship between liver blood flow and liver pathology in endstage cirrhosis. Material and methods. Portal blood velocity and volume, congestion index of the portal vein, and hepatic and splenic pulsatility indices were evaluated with echo-Doppler in cirrhotic patients immediately before liver transplantation. When a patent paraumbilical vein was present, its blood flow was measured and effective portal liver perfusion was calculated as portal blood flow minus paraumbilical blood flow. The hemodynamic parameters were correlated with liver weight and the pattern of the liver fibrosis morphometrically assessed in explanted livers. A total of 131 patients with alcoholic or viral cirrhosis were included in the study. Results. In alcoholic cirrhosis, liver weight was higher than that in viral disease (1246±295 g versus 1070±254 g, p=0.001), portal liver perfusion per gram of liver tissue was lower (0.49±0.36 ml g^?1 min^?1 versus 0.85±0.56 ml g^?1 min^?1, p=0.004) and hepatic pulsatility indices were higher (1.45±0.31 versus 1.26±0.30, p=0.018). The degree of liver fibrosis was similar in alcoholic and viral cirrhosis (11.7±5.5% versus 11.0±4.4%, p=NS). An inverse relationship between liver weight and Child-Pugh score was disclosed in viral (p<0.001) but not in alcoholic disease. Conclusions. A different hemodynamic pattern characterizes the advanced stage of cirrhosis of alcoholic and viral origin. A more severe alteration of intrahepatic portal perfusion, probably coexisting with a more severe hepatocyte dysfunction, and a higher liver weight can be detected in alcoholic cirrhosis.     

Depressed selenium and vitamin E levels in an alcoholic population

Serum selenium and vitamin E levels have been measured in subjects with established alcoholic liver disease, in alcoholics within the community, and in appropriate controls. Both serum selenium and vitamin E levels were shown to be significantly depressed (P<0.01) in the alcoholic study groups and serum selenium was more markedly depressed in subjects with established liver disease (controls, serum selenium 108±13 g/liter, vitamin E 27.6±7.2 mol/liter; community alcoholics, serum selenium 94±19 g/liter, vitamin E 15.3±3.4 mol/liter; alcoholic liver disease, serum selenium 78±15 g/liter, vitamin E 14.7±5.6 mol/liter). Depressed serum selenium levels correlated closely with poor nutritional status (r=0.91). There were no changes in serum glutathione peroxidase activity. Liver disease activity, as judged by transaminase (AST), was more markedly abnormal in subjects with combined vitamin E and selenium deficiency compared to those with normal levels or isolated deficiencies (no deficiency, AST 48±19 units, combined deficiency, AST 75±21 units,P<0.03). Serum lipid peroxides were elevated in those with combined deficiency and the values correlated significantly with serum transaminases (r=0.40,P=0.03).     

Procoagulant activity and intimal dysfunction in IDDM

Summary The biological activity of thrombin and coagulation factor Xa was assessed in 62 insulin-dependent diabetic patients. A group of non-diabetic subjects of comparable age and urinary albumin excretion rate (<30 mg/24 h) served as control subjects (group 1,n=14). The patients were divided into three groups according to urinary albumin excretion rate. In group 2, albumin excretion rate was less than 30 mg/24 h (n=17), in group 3 albumin excretion rate was in the range 30–300 mg/24 h (n=20) and in group 4 albumin excretion rate was greater than 300 mg/24 h (n=25). Compared to non-diabetic control subjects an increase in the biological activity of factor Xa was observed in all groups of diabetic patients (prothrombin fragment 1+2 levels were 1.14±0.38 nmol/l in group 2,p<0.005; 1.06±0.45 nmol/l in group 3,p<0.05 and 1.03±0.31 nmol/l in group 4,p<0.05 vs 0.75±0.34 nmol/l in group 1). No difference in the level of antithrombin III was seen between the groups. We reconfirmed the presence of intimal dysfunction in diabetic nephropathy demonstrated by elevated transcapillary escape rate of albumin in group 4 compared with group 2 (8.9±2.0% vs 7.0±1.9%,p<0.05). An overall positive correlation between transcapillary escape rate and prothrombin fragment 1+2 was found (r=0.36,p<0.005). However, in the groups with elevated albumin excretion rate such a correlation was not significant (group 3:r=0.15,p=0.54; group 4:r=0.03,p=0.86) while it was sustained in the groups with albumin excretion rate of less than 300 mg/24 h (group 1:r=0.61,p<0.05; group 2:r=0.64,p<0.05). In conclusion, IDDM patients had elevated biological activity of factor Xa, demonstrated by elevated levels of prothrombin fragment 1+2. This increment could not be explained by a deficiency of antithrombin III. [Diabetologia (1995) 38: 73–78]     

Circulating matrix metalloproteinase-9 is transiently elevated after colorectal surgery

Background and aims Plasma levels of matrix metalloproteinases (MMPs) may yield important information in patients suffering from colorectal cancer but the effect of surgery, a common treatment modality in these patients, on circulating MMP levels is currently unknown. The aim of this study was to assess whether plasma MMP-2 and MMP-9 levels are affected by operative procedures.Materials and methods In total 128 patients undergoing elective surgery for colorectal cancer (n=66), liver metastases from colorectal origin (n=50) and arthrosis of the hip (n=12) were included in the study. Gelatinase activity was measured, using quantitative gelatin zymography, in plasma obtained before operation and 1 week, 1 month and 3 months postoperatively.Results One week after operation a significant increase in proMMP-9 activity was measured after colorectal surgery (260%, p =0.0038), liver surgery (285%, p <0.0001) and hip surgery (217%, p =0.012) as compared with preoperative levels. After 1 month proMMP-9 activity had returned to preoperative levels. No effect on proMMP-2 activity was measured.Conclusion Operative procedures have a profound but transient effect on plasma MMP-9 activity. If used to assess disease status, postoperative plasma MMP levels should be interpreted with caution.     

Genetic polymorphisms of genes coding to alcohol-metabolizing enzymes in western Mexicans: association of CYP2E1*c2/CYP2E1*5B allele with cirrhosis and liver function

Background: Alcoholic cirrhosis constitutes a major public health problem in the world where ADH1B, ALDH2, and CYP2E1 polymorphisms could be playing an important role. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy control individuals (C) and patients with alcoholic cirrhosis (AC) from western Mexico. Methods: Ninety C and 41 patients with AC were studied. Genotype and allele frequency were determined through polymerase chain reaction‐restriction fragment length polymorphisms. Results: Polymorphic allele distribution in AC was 1.6%ADH1B*2, 0.0%ALDH2*2, and 19.5%CYP2E1*c2; in C: 6.1%ADH1B*2, 0%ALDH2*2, and 10.6%CYP2E1*c2. CYP2E1*c2 polymorphic allele and c1/c2 genotype frequency were significantly higher (p < 0.05 and p < 0.01, respectively) in patients with AC when compared to C. Patients with AC, carrying the CYP2E1*c2 allele, exhibited more decompensated liver functioning evaluated by total bilirubin and prothrombin time, than c1 allele carrying patients (p < 0.05). Cirrhosis severity, assessed by Child’s Pugh score and mortality, was higher in patients carrying the c2 allele, although not statistically significant. Conclusions: In this study, CYP2E1*c2 allele was associated with susceptibility to AC; meanwhile, ADH1B*2 and ALDH2*2 alleles were not. CYP2E1*c2 allele was associated with AC severity, which could probably be attributed to the oxidative stress promoted by this polymorphic form. Further studies to clearly establish CYP2E1*c2 clinical relevance in the development of alcohol‐induced liver damage and its usefulness as a probable prognostic marker, should be performed. Also, increasing the number of patients and including a control group conformed by alcoholic patients free of liver damage may render more conclusive results. These findings contribute to the understanding of the influence of gene variations in AC development among populations, alcohol metabolism, and pharmacogenetics.     

Humoral SPARC/osteonectin protein in plasma cell dyscrasias

The matricellular protein SPARC (secreted protein acidic and rich in cysteine)/osteonectin was determined in patients with multiple myeloma and related disease to assess the hypothesized role of SPARC as a possible marker of tumor burden and disease progression. Soluble SPARC was measured by competitive enzyme-linked immunosorbent assay (ELISA) in plasma of 42 patients, including sequential measurements in individual patients, and in 20 healthy controls. SPARC values were heterogeneous in multiple myeloma patients showing a decline from baseline levels recorded in controls (456±195 vs 600±63 ng/ml, p=0.00023). A SPARC showed a significant positive correlation with platelet count (r=0.72, p=0.000000, n=42), hemoglobin (r=0.52, p=0.00037, n=42), and IgG level (r=0.43, p=0.0085, n=42) and negative correlation with ₂-microglobulin (r=–0.46, p=0.0023, n=42), aspartate aminotransferase (AST) (r=–0.42, p=0.0061, n=41), interleukin (IL)-6 (r=–0.41, p=0.008, n=42), lactate dehydrogenase (LDH) (r=–0.36, p=0.02, n=41), and percentage of plasma cells in bone marrow aspirate (r=–0.34, p=0.029, n=42). No correlation was found between SPARC and M component or disease stage. Investigations performed during the course of disease, including sequential measurements in individual patients, showed a trend to downregulation by disease progression, with the lowest level recorded in the terminal stage (217±107 ng/ml, n=11). Patients with established osteolytic lesions had lower plasma SPARC at diagnosis (309±197 vs 581±293, p=0.021), which correlated with osteocalcin by disease progression (r=0.31, p=0.026). The results of this pilot study revealed abnormalities in the level of humoral SPARC in multiple myeloma and an overall trend to downregulation in the advanced stage of the disease. The regulation of SPARC seems to be opposite to the markers of tumor burden and of aggressive multiple myeloma phenotype.     

Hepatic cytosolic oestrogen receptors in patients with liver disease

Abstract. Patients with liver disease present many of the features of ‘feminized’ hepatic metabolism. Oestrogens exert their effects through interaction with specific cellular high-affinity receptors (ER). We measured hepatic ER in 102 needle biopsies from patients with chronic alcoholic and non-alcoholic liver disease using an enzyme immunoassay. Fifteen patients with no or minimal changes in liver histology served as controls. The hepatic ER concentrations were significantly (P = 0.05) lower in the 44 men (median 13 fmol mg^?1 protein, interquartile range 7–17 fmol mg^?1 protein) compared to the 58 women (median 15 fmol mg^?1 protein, interquartile range 10–21 fmol mg^?1 protein). Patients with alcoholic liver disease (n = 63) had significantly (P < 0.05) lower ER concentrations than controls (n = 15) (median 13 fmol mg^?1 protein, interquartile range 7–17 fmol mg^?1 protein vs. median 16 fmol mg^?1 protein, interquartile range 10–26 fmol mg^?1 protein), and compared with patients with non-alcoholic liver disease (n = 24) (P < 0.05, median 20 fmol mg^?1 protein, interquartile range 11–24 fmol mg^?1 protein). ER concentrations were significantly lower (P < 0.05) in patients with alcoholic liver disease and alcoholic hepatitis (n = 21) compared to those without alcoholic hepatitis (n = 42) (medians 10 vs. 14 fmol mg^?1 protein, interquartile ranges 6–15 fmol mg^?1 protein vs. 9–18 fmol mg^?1 protein), while ER concentrations did not differ significantly (P > 0.05) between actively drinking (median 13 fmol mg^?1 protein, interquartile range 7–17 fmol mg^?1 protein) and abstaining alcoholic patients (median 13 fmol mg^?1 protein, interquartile range 7–18 fmol mg^?1 protein). In summary, the small but significant variation in hepatic ER concentrations reflects variation in liver function rather than an effect of ethanol.     

High plasma levels of betaine,a trimethylamine N-Oxide-related metabolite,are associated with the severity of cirrhosis

Background and Aims

The gut microbiome-related metabolites betaine and trimethylamine N-oxide (TMAO) affect major health issues. In cirrhosis, betaine metabolism may be diminished because of impaired hepatic betaine homocysteine methyltransferase activity, whereas TMAO generation from trimethylamine may be altered because of impaired hepatic flavin monooxygenase expression. Here, we determined plasma betaine and TMAO levels in patients with end-stage liver disease and assessed their relationships with liver disease severity.

Methods

Plasma betaine and TMAO concentrations were measured by nuclear magnetic resonance spectroscopy in 129 cirrhotic patients (TransplantLines cohort study; NCT03272841) and compared with levels from 4837 participants of the PREVEND cohort study. Disease severity was assessed by Child-Pugh-Turcotte (CPT) classification and Model for End-stage Liver Disease (MELD) score.

Results

Plasma betaine was on average 60% higher (p < .001), whereas TMAO was not significantly lower in cirrhotic patients vs. PREVEND population (p = .44). After liver transplantation (n = 13), betaine decreased (p = .017; p = .36 vs. PREVEND population), whereas TMAO levels tended to increase (p = .085) to higher levels than in the PREVEND population (p = .003). Betaine levels were positively associated with the CPT stage and MELD score (both p < .001). The association with the MELD score remained in the fully adjusted analysis (p < .001). The association of TMAO with the MELD score did not reach significance (p = .11). Neither betaine nor TMAO levels were associated with mortality on the waiting list for liver transplantation (adjusted p = .78 and p = .44, respectively).

Conclusion

Plasma betaine levels are elevated in cirrhotic patients in parallel with disease severity and decrease after liver transplantation.     

Lymphocyte apoptosis and macrophage function: correlation with disease activity in systemic lupus erythematosus

Increased lymphocyte apoptosis and defects in macrophage removal of apoptotic cells have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the relationship between peripheral lymphocyte apoptosis, macrophage function as determined by the serum levels of neopterin and interferon- (IFN-), and SLE disease activity. Peripheral apoptotic lymphocytes (AL) were detected by annexin V-fluorescein isothiocyanate (FITC) staining and flow cytometry. Serum levels of neopterin and IFN- were measured by enzyme-linked immunosorbent assay (ELISA). SLE disease activity was determined using the systemic lupus activity measure (SLAM) and the serum titer of anti-dsDNA antibodies. The percentage of AL in the peripheral blood of active SLE patients was significantly higher (13.07±7.39%, n=30) than that of the inactive SLE patients (4.08±3.55%, n=8, p<0.01) and normal controls (5.13±3.37%, n=11, p<0.01). Serum levels of neopterin in active SLE patients were significantly higher (1.39±1.10 g/dl, n=22) than in controls (0.26±0.19 g/dl, n=20, p<0.01). Serum levels of IFN- in active SLE patients were elevated (58.97±34.52 ng/l, n=15) when compared with controls (28.06±2.35 ng/l, n=16, p<0.05). The percentage of AL correlated significantly with serum levels of neopterin (r=0.446, p<0.05, n=22) and SLAM score (r=0.533, p<0.001, n=38), but not with the serum levels of IFN-. The SLAM score also correlated with the serum levels of neopterin (r=0.485, p<0.05, n=22), but not with those of IFN-. Our study supported the hypothesis that increased lymphocyte apoptosis has a pathogenic role in SLE. The increased levels of serum neopterin may suggest an attempt of the patients macrophage system to remove the apoptotic cell excess. Since serum levels of neopterin correlated with the overall lupus disease activity, they may be regarded as an index of SLE disease activity.     

Oxidative stress and regulation of anti‐oxidant enzymes in cytochrome P4502E1 transgenic mouse model of non‐alcoholic fatty liver

Background and Aim: Reactive oxygen species produced by cytochrome P4502E1 (CYP2E1) are believed to play a role in pathophysiology of non‐alcoholic fatty liver disease (NAFLD). However, little is known about the expression, protein content and activity of anti‐oxidant enzymes and the role of inducible nitric oxide synthase (iNOS), a source of reactive nitrogen species, in NAFLD. In the present study, we evaluate gene expression, protein content and activity of anti‐oxidant enzymes, and iNOS, in a CYP2E1 overexpressing model of non‐alcoholic fatty liver (NAFL). Methods: Non‐transgenic (nTg) and CYP2E1 transgenic (Tg) mice were fed rodent chow for 8 months. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver triglycerides, malondialdehyde and protein carbonyls were measured. Gene expression of NF‐E2‐related factor (Nrf2), superoxide dismutase‐1, ‐2 (SOD‐1,2), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase‐1 (HO‐1) and iNOS were determined. Protein content, activity and nitrosylation of the enzymes were also measured. Results: Tg mice had greater CYP2E1 activity and histological liver injury. MDA and protein carbonyls were increased, indicating insufficient anti‐oxidant response. Gene expression of Nrf2, CAT, GPx, HO‐1 and iNOS were significantly increased. Protein content and enzyme activities of most anti‐oxidant enzymes were not correspondingly increased. iNOS activity and nitrosylation of CAT and SOD was greater in Tg mice liver. Conclusion: Hepatocyte‐specific CYP2E1 overexpression results in increased oxidative stress and nitrosative stress. Several anti‐oxidant enzymes are upregulated. Failure of corresponding increase in total protein and activity of anti‐oxidant enzymes suggests modification/degradation, possibly by nitrosylation, due to increased iNOS activity in a CYP2E1 overexpressing NAFL mouse model.     

Increased serum adiponectin levels in type 1 diabetic patients with microvascular complications

Aims/hypothesis Low serum adiponectin (ADPN) has been associated with increased risk of cardiovascular disease (CVD) and retinopathy in patients with type 2 diabetes mellitus. In type 1 diabetic patients, the relationship between ADPN and the presence of vascular complications is largely unknown.Methods We investigated the relationship between serum ADPN and the presence of retinopathy, nephropathy and CVD in patients with type 1 diabetes, divided into matched groups with normoalbuminuria and no retinopathy (n=67), simplex retinopathy (n=106) or proliferative retinopathy (n=19), and nephropathy with simplex (n=62) or proliferative retinopathy (n=137). Healthy control subjects (n=25) were included.Results Serum ADPN was increased in subjects with type 1 diabetes compared with control subjects (p<0.0001). Further, serum ADPN was higher in patients with than in those without nephropathy (p<0.0001). It was also higher in normoalbuminuric patients with than in those without proliferative retinopathy (p<0.0001). These differences remained significant after adjustment for known risk factors (p<0.03). CVD was also associated with elevated ADPN levels (p<0.05), but this difference became insignificant after risk factor adjustment. The most important predictor of serum ADPN was sex (r²=19%) in normoalbuminuric patients and GFR in patients with nephropathy (r²=18%).Conclusion/interpretation Patients with type 1 diabetes and microvascular complications have higher serum levels of ADPN than patients without complications. It remains to be clarified whether elevated levels of ADPN are pathogenically related to the development of microvascular complications or represent a beneficial counter-regulatory response.     

Measurement and diagnostic use of hepatic cytochrome P450 metabolism of oleic acid in liver disease

Background: Oleic acid is a major systemically circulating fatty acid in humans with atheroprotective and immunomodulatory properties. As of today, the contribution of individual cytochrome P450 (CYP) mono‐oxygenases to the epoxidation of this fatty acid is unknown. Furthermore, the extent of the oleic acid oxidation product cis‐9,10‐epoxyoctadecanoic acid (cis‐EODA) in humans and its plasma levels in patients with impaired liver function are not known. Patients and methods: We studied cis‐EODA in plasma of patients suffering from chronic liver diseases, a condition that often displays impaired liver CYP enzyme activities. Fifteen CYP mono‐oxygenases were investigated in vitro as a potential source of cis‐EODA. Results: Strikingly, plasma levels of cis‐EODA were significantly repressed (P<0.0005) when patients with liver impairment (n=16) were compared with healthy subjects (n=14). Production of cis‐EODA was catalysed by CYP in the following order: 2C8, 2C9, 2C19, 3A4, 1A2 and CYP3A7. Conclusion: cis‐EODA plasma concentrations are decreased in hepatic disease with impaired liver function. Oleic acid is primarily oxidized to oleic acid oxide (cis‐EODA) by CYP2C and CYP3A mono‐oxygenases. The liver is the major organ responsible for the oxidation of oleic acid to cis‐EODA, and thus, cis‐EODA may be a suitable biomarker to assess liver function.