Cyclosporin-induced endothelial dysfunction and hypertension: are nitric oxide system abnormality and oxidative stress involved?

Hypertension is a major side effect of cyclosporin (CsA). While the mechanism(s) responsible are unclear, CsA-induced endothelial dysfunction and CsA-induced hypertension have been attributed to the CsA effect on the endothelial-derived factors controlling vasomotor tone. Endothelial nitric oxide (NO) is crucial in the maintenance of a state of basal vasodilation, and recent studies have suggested an NO-mediated counterregulatory mechanism protective from CsA-induced vasoconstriction. Our study evaluates endothelial nitric oxide synthase (ecNOS) gene status (PCR analysis) and plasma levels of NO metabolites (ELISA) in kidney and heart transplant patients under chronic CsA treatment with CsA-induced hypertension. Since CsA increases superoxide production, which metabolises NO, plasma hydroperoxides from cholesterol esters and from triglycerides and peroxynitrite were also evaluated (HPLC) as an index of the presence of superoxides and of "oxidative stress". Quantification of monocyte ecNOS mRNA and NO metabolites plasma levels from patients and controls (C) demonstrated NO system upregulation in patients notwithstanding the hypertension. The mean ecNOS to beta-actin ratio was 1.80 +/- 0.85 in patients vs 0.40 +/- 0.09 in C (P < 0.04). NO metabolites were 34.03 +/- 14.32 microM in patients vs 11.53 +/- 5.64 microM in C (P < 0.001). Hydroperoxides from cholesterol esters and from triglycerides were also increased in patients, 3.4 +/- 1.4 vs 1.3 +/- 0.6 integrated area units (i. a. u.), P < 0.007 and 10.6 +/- 6.4 vs 1.3 +/- 0.8 i. a. u., P < 0.008, respectively, as well as the peroxynitrite plasma level, 0.32 +/- 0.11 microM/l vs undetectable in C. This study confirms a CsA-induced NO system upregulation in transplanted patients. However, the NO-mediated counterregulatory system to CsA-induced vasoconstriction, present in normals, could be canceled in patients by CsA-induced superoxide (O2-) and free radical production which, by increasing NO metabolism, could contribute to CsA-induced vasoconstriction and hypertension and predispose to atherosclerosis.

High plasma levels of alpha- and beta-carotene are associated with a lower risk of atherosclerosis: results from the Bruneck study.

BACKGROUND AND PURPOSE: A large number of studies have contributed to the hypothesis that carotenoids, vitamins A and E are protective against atherosclerosis by acting as antioxidants. The aim of this study was to assess the relationship between plasma levels of carotenoids (alpha- and beta- carotene, lutein, lycopene, zeaxanthin, beta-cryptoxanthin), vitamins A and E, and atherosclerosis in the carotid and femoral arteries. METHODS: This prospective and cross sectional study involved a randomly selected population sample of 392 men and women aged 45-65 years. Carotid and femoral artery atherosclerosis was assessed by high-resolution duplex ultrasound. RESULTS: alpha- and beta- carotene plasma levels were inversely associated with the prevalence of atherosclerosis in the carotid and femoral arteries (P=0.004) and with the 5-year incidence of atherosclerotic lesions in the carotid arteries (P=0.04). These findings were obtained after adjustment for other cardiovascular risk factors (sex, age, LDL (low density lipoproteins), ferritin, systolic blood pressure, smoking, categories of alcohol consumption, social status, C-reactive protein). Atherosclerosis risk gradually decreased with increasing plasma alpha- and beta-carotene concentrations (P=0.004). No associations were found between vitamin A and E plasma levels and atherosclerosis. CONCLUSIONS: This study provides further epidemiological evidence of a protective role of high alpha- and beta- carotene in early atherogenesis.

Idiopathic hypercalciuria: O2(-)NO relationship and altered bone metabolism.

The pathogenesis of idiopathic hypercalciuria (IH) has not been elucidated yet, but a correlation between IH and altered bone metabolism has been proposed. Since nitric oxide (NO) regulates osteoclasts' bone resorption, a possible role for NO can be suggested. In this study we evaluated iNOS gene expression by reverse transcription of mRNA from monocytes, followed by polymerase chain reaction in patients with IH subdivided into fasting (FH) and absorptive (AH) hypercalciuria. Since superoxide (O2-), which metabolizes NO, is overproduced by osteoclasts during bone resorption, peroxynitrite plasma level was evaluated as index of O2-. Vertebral BMD in IH as a whole group was lower vs controls (C) (Z score=-1.78+/-0.2 vs 0.51+/-0.25, p<0.001), but only FH patients showed a reduced bone density (2.13+/-0.18 vs 0.51+/-0.25, p<0.0001). PTH and calcitriol were not different. FH showed an increase in b-ALP vs AH and C (41.1+/-2.6 vs 30.1+/-3.9 vs 26.6+/-3.6 U/l p<0.02), and higher uHP, either on NCD (17.7+/-1.6 vs 11.4+/-1.3 mg/g uCr, p<0.04) or after LCD (26.7+/-2.5 vs 16.7+/-1.9, p<0.01). Cells from FH patients, but not from both AH patients and C, expressed iNOS. Peroxynitrite plasma level was elevated in FH (0.30+/-0.07) pmol/l while not detectable in AH and C. This study confirms an altered bone metabolism only in FH which shows an abnormal NO system. The increased iNOS gene expression in FH, in fact, points toward an altered NO system's activity downstream the generation of NO. A possible interaction of NO with O2-, which breaks down NO, and the role of this interaction in the pathophysiology of IH is discussed.

Assessment of package inserts for diagnostic kits.

To assure the quality of service in laboratory medicine, it is necessary to implement a quality system which comprises the entire testing process. The use of quality reagents is an important aspect of the process. Despite the fact that it is the responsibility of the laboratory to ensure the quality of the analytical system (including reagents) and since it is impossible to evaluate all commercial diagnostic kits, the laboratory often depends on statements issued by the manufacturer to select the most appropriate diagnostics for a particular laboratory. In this study we report the results of the analysis of information provided in 887 package inserts enclosed in the more widely used commercial diagnostic kits, following the Standard for the labelling of clinical laboratory materials of the European Committee for Clinical Laboratory Standards (ECCLS). Only a third of these were in agreement with the guidelines of ECCLS Standard, reporting complete and correct information. We believe that it is necessary to implement a constructive cooperation between manufacturers of diagnostic materials and clinical laboratories to produce a more uniform approach to improvements in laboratory quality assurance.

Immunoturbidimetric assay of glycated hemoglobin.

We investigated the performances of HbA1c determination by a latex enhanced turbidimetric immunoassay using the specific monoclonal antibodies (Unimate, Roche) against the beta-N-terminal fragments. The coefficients of variation ranges from 1.7 to 3.8% within assay (n = 30) and from 3.9 to 4.9% between assay (n = 20). The assay was linear from 2.5 to 14.9% of HbA1c. No interferences was found from fetal, carbamylated, or variant (S) hemoglobins and from labile Schiff adduct with glucose. The following relationship was derived from fresh sample comparison between HPLC (Diamat-BioRad) (x) and immunoassay (y) method: y = 0.971 x + 0.87%, r=0.98, n = 115. The immunoassay provides a highly precise and specific method for HbA1c.

Oxidative DNA damage accumulation in gastric carcinogenesis.

BACKGROUND: Gastric carcinogenesis is a multifactorial, multistep process, in which chronic inflammation plays a major role. AIMS: In order to ascertain whether free radical mediated oxidative DNA damage is involved in such a process, concentrations of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic/carcinogenic adduct, and thiobarbituric acid reactive substances (TBARS), as an indirect measure of free radical mediated damage, were determined in biopsy specimens from patients undergoing endoscopy. PATIENTS: Eighty eight patients were divided into histological subgroups as follows: 27 with chronic non-atrophic gastritis, 41 with atrophic gastritis, six with gastric cancer, and 14 unaffected controls. METHODS: Intestinal metaplasia, Helicobacter pylori infection, and disease activity were semiquantitatively scored. 8OHdG concentrations were assessed by HPLC with electrochemical detection, and TBARS concentrations were fluorimetrically assayed. RESULTS: 8OHdG concentrations (mean number of adducts/10(5) dG residues) were significantly higher in chronic atrophic gastritis (p = 0.0009). Significantly higher concentrations were also detected in the presence of severe disease activity (p = 0.02), intestinal metaplasia (p = 0.035), and H pylori infection (p = 0.001). TBARS concentrations were also higher in atrophic gastritis, though not significantly so. In a multiple logistic regression analysis, 8OHdG concentrations correlated best with the presence and severity of H pylori infection (r = 0.53, p = 0.002). CONCLUSION: Chronic gastritis is characterised by the accumulation of oxidative DNA damage with mutagenic and carcinogenic potential. H pylori infection is the major determinant for DNA adduct formation.

Altered lipid peroxidation/glutathione ratio in experimental extrahepatic cholestasis.

1. Lipid peroxidation can occur in the presence of a cellular antioxidant-oxidant imbalance, but the role of lipid peroxides in cholestasis is not well understood. 2. This study was undertaken in order to: (i) evaluate the behaviour of a product of lipid peroxidation (thiobarbituric acid-reactive species), and of an important antioxidant tripeptide, reduced glutathione, in the course of experimental extrahepatic cholestasis; and (ii) ascertain whether there was a link between this aspect and the alterations in liver morphology. 3. Forty-five male Sprague-Dawley rats (250-300 g) were double bile duct ligated and followed from 1 to 28 days. At the end of each experimental period, blood and liver samples were collected for thiobarbituric acid-reactive species and glutathione determinations. 4. Bile duct ligated rats showed a marked increase in liver weight which was related to cholestasis duration and to some anatomical alterations such as bile duct proliferation and dilation and liver fibrosis (periportal, perivenular, perineoductular and parenchymal). 5. An increase in serum lipid peroxidation was also observed but this was not linked to hepatic thiobarbituric acid-reactive species. Erythrocyte and hepatic glutathione decreased in relation to cholestasis duration. Serum lipid peroxides and erythrocyte glutathione were correlated with liver cell necrosis. 6. In conclusion, experimental extrahepatic cholestasis determines bile duct proliferation and fibrosis, the degree of which is directly related to the duration of cholestasis itself and to liver cell necrotic phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal functional alterations in extrahepatic cholestasis: can oxidative stress be involved?

Renal function may be compromised by extrahepatic cholestasis. In this context, the nephrotoxic role of bile salts is well known. Recently, however, it has been claimed that other factors, such as lipid peroxides, are involved. We therefore created bile duct ligation in 40 Sprague-Dawley rats. During the follow-up (from 1 to 28 days), significant variations were found in liver histological parameters, but not in renal morphology. Fourteen days after ligation, significant increases were found in serum and urinary thiobarbituric-acid-reactive species and phospholipase A2 (indirect indices of lipid peroxidation), whereas 8-10 days after ligation, a significant decrease was observed in erythrocytic and hepatic GSH levels. The variations in urinary thiobarbituric-acid-reactive species and in phospholipase A2 were not correlated with concomitant variations in the sera. Urinary lipid peroxides were directly correlated with the degree of liver morphological alterations and inversely with circulating GSH. Urinary outputs of lipid peroxides, phospholipase A2 and N-acetyl-glucosaminidase were correlated with each other. These results suggest that there is an imbalance in the oxidative-antioxidant hepatic system in experimental extrahepatic cholestasis. The reduced bioavailability of blood GSH may alter the oxidative equilibrium in other organs, such as the kidney, triggering and favoring the lipoperoxidative cascade.

Noradrenergic and hormonal responses to physical exercise in adolescents. Relationship to anxiety and tolerance to frustration.

Seventy physically healthy 14-year-old adolescents, 40 boys and 30 girls, were evaluated psychologically and endocrinologically. After the psychological tests (Anxiety Score Test for Adolescents, Rosenzweig, Pictures Frustration Test for Children), subjects were divided into group A, with low anxiety/sense of guilt and high self-esteem/tolerance to frustration and group B with the opposite. In both groups, we measured basal plasma levels of noradrenaline (NE), growth hormone (GH), prolactin (PRL), melatonin (MT) and luteinizing hormone (LH) and their response to physical exercise (the Harvard step test). Basal levels of the hormones and of NE were not different in the two groups. After the physical stimulus, NE levels rose significantly more in B girls than in A and significantly less in B than in A boys. GH and PRL levels increased only in A girls and MT in B boys, while LH levels decreased in A boys and girls but not in B subjects.

Measurement of alkaline phosphatase activity in serum with N-methyl-D-glucamine as a buffer: evaluation of the method for routine use.

Some aspects of the measurement of alkaline phosphatase activity concentration in human serum, using N-methyl-D-glucamine as a buffer, were evaluated with a view to the possible routine use of the method. The evaluated characteristics included: the temperature-dependence of the pH of the buffer; the effect of adding the magnesium/zinc ions buffer; the effect of the serum volume fraction; the substrate-starter versus the serum-starter mode; the effect of modifying the formulation of reagents; the within-run and the between laboratory imprecision; the correlation with an alternative routine method. Also, sex- and age-related reference values were produced, based on 2968 values from selected reference sample groups in 7 laboratories. In general, the results demonstrate the robustness of the method, its adaptability to a variety of mechanized analysers, and hence its feasibility as a routine measurement method.

Renal handling of amylase and immunoreactive trypsin in pancreatic cancer and chronic pancreatitis.

In order to evaluate the renal metabolism of amylase and immunoreactive trypsin (IRT) in chronic pancreatic disease, we assayed amylase, IRT and creatinine in serum and urine and gamma-glutamyl transferase (GGT) in dialyzed urine as well as alpha-glucosidase (AGL) and ribonuclease (RNase) in 24 control subjects, 34 patients with pancreatic cancer, 52 with chronic pancreatitis and 32 with extra-pancreatic diseases. Urinary amylase and IRT outputs were found to be more elevated in chronic pancreatitis than in control subjects. The levels of serum amylase, its renal inputs and outputs were correlated with the corresponding IRT values. Multiple regression analyses (dependent on amylase or IRT urinary outputs, circulating levels of the two enzymes, creatinine clearance and the excretion of GGT, AGL and RNase predictor variables) showed significant correlations. The standardized partial regression coefficients found to be significant were: GGT, RNase and serum amylase for amylase, and GGT and RNase for IRT. No difference was found between amylase and IRT outputs in patients with chronic pancreatitis, taking the presence or the absence of alcohol abuse, exocrine insufficiency and pancreatic pseudocysts into consideration. Urinary GGT excretion correlated with serum amylase and IRT levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal tubular dysfunction in pancreatic cancer and chronic pancreatitis.

Urinary excretion of alpha-glucosidase (AGL), gamma-glutamyltransferase (GGT) and ribonuclease (RNase), and serum amylase and immunoreactive trypsin (IRT) were determined in 38 control subjects, 48 patients with pancreatic cancer, 77 with chronic pancreatitis and 47 with extrapancreatic diseases in order to ascertain the presence of a renal tubular damage and to investigate its etiology. A significantly increased frequency of pathological results for all urinary enzymes was documented in the various groups of patients as compared to controls. Significant correlations were detected among AGL, GGT and RNase. Considering the subjects as a whole, GGT and RNase excretions correlated with serum IRT and amylase; the two urinary enzymes were found to be higher when jaundice was present. In chronic pancreatic disease enzymuria was related to increased serum pancreatic enzymes; in extrapancreatic diseases it was associated to hyperbilirubinemia. The vast majority of patients with pancreatic cancer and elevated urinary enzymes presented hepatic metastases and/or jaundice. We can conclude that an anatomical and functional tubular impairment is detectable in some patients with chronic pancreatic and extrapancreatic diseases. Tubular damage seems to least in part to be related to pancreatic inflammation and necrosis in chronic pancreatic disease, while jaundice may be found to play an important role in diseases of the hepatobiliary tract. In pancreatic cancer, liver dysfunction (presence of liver metastases and/or extrahepatic cholestasis) also appears to be involved in altering tubular cells.

Influence of isoenzyme patterns on commutability in enzyme determinations.

The use of recommended methods has very much improved the precision and accuracy of enzymatic determinations. The use of 'enzyme reference materials' allows us to overcome the remaining difficulties. The possibility of extending standardization by using calibration materials to convert results obtained by different methods requires an essential property: 'commutability'. The relationship between patients' specimens and reference materials are conditioned by analytical causes (relative to the used methods) and clinical causes (relative to the condition of the human samples to be examined). Among the clinical causes, the different isoenzyme content of patients sera, induced by different pathologies, was considered. The effect of this influence has been investigated with an analytical approach of the isoenzymes in the individual clinical samples. The results obtained on determination of alkaline phosphatase (ALP), creatine kinase (CK), and lactate dehydrogenase (LD), are discussed according to a model of investigation which can be utilized for the experimental protocols in the assessment of commutability.

Molecular size distribution of immunoreactive trypsin and renal tubular dysfunction: role in trypsin plasma-urine transfer.

In order to investigate the role of circulating free trypsinogen and renal tubular dysfunction in affecting trypsin plasma-urine transfer, serum immunoreactive trypsin (IRT), its urinary output, IRT molecular size distribution, filtrable immunoreactive trypsin, gamma-glutamyltransferase and alpha-glucosidase outputs were studied in 6 control subjects, 9 patients with pancreatic cancer and 15 with chronic pancreatitis. The majority of immunoreactivity was always eluted at a molecular weight of about 24,000 and might therefore be considered as free trypsinogen. Variable amounts of IRT at higher molecular weights, possibly represented by trypsin-inhibitor complexes, were also detected. Increasing IRT levels were generally accounted for by free trypsinogen, regardless of the nature of the disease. Unlike serum free trypsinogen levels, renal tubular damage, evaluated by means of the excretion of two high-molecular weight urinary enzymes, seems to play a prominent role in explaining trypsin plasma-urine transfer.

Trypsin/creatinine clearance ratio and serum immunoreactive trypsin in digestive and pancreatic diseases.

The behavior of trypsin/creatinine clearance ratio (Ctr/Ccr) and serum immunoreactive trypsin (IRT) was evaluated in a total of 168 subjects with pancreatic cancer, chronic pancreatitis and non-pancreatic digestive diseases. Amylase/creatinine clearance ratio (Cam/Ccr) and serum amylase levels were also evaluated in order to establish their possible relationship with Ctr/Ccr and IRT values. Elevated Ctr/Ccr and IRT values were observed in several patients with pancreatic cancer and chronic pancreatitis. Abnormal IRT and Ctr/Ccr values were found in 28.2 and 4% of non-pancreatic digestive diseases, respectively. IRT and amylase serum levels showed consensual modifications, while Ctr/Ccr showed a behavior different from that of Cam/Ccr. Liver damage seems to play a role in increasing serum IRT levels of patients without pancreatic involvement, while the increased Ctr/Ccr seems to depend on other factors, for instance renal tubular dysfunction.

CA 19-9 in the differential diagnosis between pancreatic cancer and chronic pancreatitis.

CA 19-9 serum concentration was determined by a immunoradiometric technique in 130 subjects to evaluate its role in differentiating pancreatic cancer from chronic pancreatitis. Two threshold values were chosen, 17 and 37 U/ml. With the former, sensitivity, specificity and diagnostic accuracy were 86.7, 62.3 and 49.0 respectively, with the latter 73.3, 87.0 and 60.3%. The receiver-operating characteristic curves demonstrated a satisfactory discriminating capacity of CA 19-9 as regards pancreatic cancer and chronic pancreatitis; in contrast, the discrimination was poor for other gastrointestinal diseases, mainly of a malignant nature.

Are urinary enzymes useful markers of kidney damage in obstructive jaundice? An experimental study on Sprague-Dawley rats.

To evaluate the reliability of urinary enzymes as markers of renal tubular damage in obstructive jaundice, research was carried out on 26 Sprague-Dawley rats submitted to bile duct ligation and on 16 sham-operated rats. The fractional clearances of lysozyme (CfrLYS) and of malto-dehydrogenase (CfrMDH)-indices of tubular function-and the fractional excretions of gamma-glutamyltransferase (UfrGGT) and of alpha-glucosidase (UfrAGL)-indices of tubular anatomic damage - were measured 5, 10, 20 and 30 days after operation. Creatinine clearance, urinary sodium excretion, urinary potassium excretion, proteinuria, plasma bilirubin and bile acids were also measured. Kidneys were taken for histology. All rats submitted to common bile duct ligation had high levels of bilirubin and bile acids; proximal tubules were damaged and the extent of the lesions increased with time. However, creatinine clearance, urinary sodium excretion, proteinuria, CfrMDH and UfrAGL gave no indication of renal lesions, whereas CfrLYS and UfrGGT were significantly higher 20 and 30 days after bile duct ligation, respectively. These findings show that CfrLYS and UfrGGT could be useful tests for renal tubular lesions in jaundice.

Clinical significance of beta-thromboglobulin in patients with high platelet count.

The aim of the study was to investigate the relevance of beta-thromboglobulin (beta tg) measurement in patients with thrombocytosis. We have, therefore, studied the level of plasma and platelet beta tg in 74 patients with high platelet count in addition to the evaluation of platelet aggregation and platelet serotonin (5-HT) content. The determinations of platelet serotonin content and aggregation are confirmed to be useful in the differentiation between primary and secondary forms of thrombocytosis. The mean plasma level of beta tg in patients with myeloproliferative disease was significantly raised, but the amount observed in subjects with secondary thrombocytosis is increased too. Considering the amount of beta tg in relation to whole blood platelet count (beta tg ratio), no difference was observed between all patients and controls. The beta tg ratio allowed the identification of a group of patients with an increased ratio and a decreased beta tg platelet content who showed the highest occurrence of thrombosis (66%).

Urinary ribonuclease excretion in pancreatic disease.

Urinary ribonuclease output and indices of renal tubular integrity were evaluated in control subjects and patients with pancreatic cancer, chronic pancreatitis and extrapancreatic diseases. The aim of the study was to ascertain the contribution to such diagnoses of ribonuclease determination in urine, and the possible influence of tubular damage on the extent of ribonuclease excretion. Information from the ribonuclease assay in urine offered no advantage over that obtained by the same determination in serum; tubular damage may contribution in some cases to an elevated ribonuclease excretion.