Walter Fiehn

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM

OBJECTIVE While elevated blood levels of homocyst(e)ine represent an independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular diseases. RESEARCH DESIGN AND METHODS Plasma levels of homocyst(e)ine and thrombomodulin (TM), markers of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS IDDM patients had higher pre- and postload plasma levels of homocyst(e)ine than did healthy control subjects (12.0 vs. 7.7 μmol/l and 27.6 vs. 16.0 μmol/l; P < 0.001). Of 75 IDDM patients, 26 had plasma homocyst(e)ine levels above the normal range (means ± 2 SD of values obtained in the control group). These IDDM patients with hyperhomocyst(e)inemia had higher plasma TM levels (62.2 vs. 38.2 ng/ml, P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l, P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33% retinopathy, 69 vs. 51% neuropathy, 57 vs. 41% and macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells showed an increased release of TM into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications in patients with diabetes. CONCLUSIONS Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Elevated plasma concentrations of homocysteine in antiepileptic drug treatment.

Summary: Purpose: Homocysteine is an experimental convulsant and an established risk factor in atherosclerosis. A nuritional deficiency of vitamin B6, vitamin B12, or folate leads to increased homocysteine plasma concentrations. During treatment with carbamazepine (CBZ), phenytoin, or phenobarbital, a deficiency in these vitamins is common. The objective of the study was to test the hypothesis that antiepileptic drug (AED) treatment is associated with increased homocysteine plasma concentrations.

Plasma levels of asymmetrical dimethyl-L-arginine in patients with congenital heart disease and pulmonary hypertension.

Asymmetrical dimethyl-l-arginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. We hypothesized that plasma levels of ADMA could be increased in patients with congenital heart disease and pulmonary hypertension. Cardiac catheterization was performed in 20 children and young adults with congenital heart disease with a median age of 10 years (range, 4 months to 33 years). The patients were assigned to group I (high flow, low pressure; n = 14) when Qp/Qs was 1.5 or greater and the mean PAP was less than 25 mm Hg or to group II (high pressure, high resistance; n = 6) when the mean PAP was greater than 25 mm Hg and Rp/Rs was greater than 0.3. Blood samples were taken from pulmonary vein or left ventricle. ADMA was measured by high-performance liquid chromatography. In addition, levels of ADMA were measured in peripheral venous blood obtained from eight control patients. Levels of ADMA in control patients (median, 0.21 &mgr;M/l; range, 0.08–0.27 &mgr;M/l) did not differ from levels obtained in group I (median, 0.30 &mgr;M/l; range, 0.06–0.49) &mgr;M/l). Patients in group II showed increased plasma levels of ADMA (median, 0.55; range, 0.25–0.79) (p < 0.01). Inhibition of nitric oxide synthase by increased levels of ADMA might contribute to pulmonary hypertension in patients with congenital heart disease.

Automated flow cytometric analysis of blood cells in cerebrospinal fluid: analytic performance.

We compared the performance of an automated method for obtaining RBC and WBC counts and WBC differential counts in cerebrospinal fluid (CSF) samples with the reference manual method. Results from 325 samples from 10 worldwide clinical sites were used to demonstrate the accuracy, precision, and linearity of the method.nnAccuracy statistics for absolute cell counts showed a high correlation between methods, with correlation coefficients for all reportable absolute counts greater than 0.9. Linearity results demonstrated that the method provides accurate results throughout the reportable ranges, including clinical decision points for WBCs of 0 to 10/μL. Interassay precision and intra-assay precision for the ADVIA 120 (Bayer HealthCare, Tarrytown, NY) method were acceptable at all levels.nnThe ADVIA 120 CSF Assay enumerates and differentiates cells via flow cytometry in a minimally diluted sample, improving the analysis of typically hypocellular CSF samples. Study results demonstrate that the automated ADVIA 120 CSF Assay is an acceptable alternative to the labor-intensive manual method.

Apolipoprotein E polymorphism and renal function in German type 1 and type 2 diabetic patients.

OBJECTIVE To examine the association of renal function in diabetic patients with apolipoprotein (apo) E polymorphism. RESEARCH DESIGN AND METHODS Apo E genotypes, lipid and lipoprotein serum levels, creatinine clearance (CCr), and excretion of marker proteins were determined in German type 1 (IDDM; n = 162) and type 2 (NIDDM; n = 124) diabetic patients. Albumin and immunoglobulin (Ig) G are considered to reflect charge-size permselectivity of the glomerular capillary basement membrane, and increased alpha 1-microglobulin (MG) excretion indicates compromised reabsorptive capacity of the renal tubules. RESULTS Patients with NIDDM had higher lipid levels and lower CCrs than patients with IDDM. In patients with IDDM, age- and sex-adjusted analysis of variance showed an association between apo E genotypes and CCr, and the Jonckheere-Terpstra test demonstrated a decreasing glomerular filtration rate in the following order of genotypes: ε4ε4/ε4ε3 > ε3ε3 > ε2ε2/ε2ε3. Multiple linear regression analyses revealed that in patients with IDDM, the ε2 allele was a negative predictor of CCr and a positive predictor of urinary excretion of albumin, IgG and α 1-MG independent from HDL and LDL cholesterol, TG concentration, age, and sex. CONCLUSIONS Apo E polymorphism influences serum lipoprotein levels in patients with IDDM and NIDDM. Apo E polymorphism may be a renal risk factor of clinical relevance in normolipidemic patients with IDDM.

Elevated plasma concentrations of lipoprotein(a) in medicated epileptic patients

Abstract Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for vascular diseases. There are no data on Lp(a) levels in patients on long-term medication with carbamazepine, phenytoin, phenobarbital, or valproate. To investigate the effects of such treatment on Lp(a) levels and common carotid artery intima media thickness we studied 51 epileptic outpatients on long-term antiepileptic medication and 51 age- and sex-matched controls. Lp(a) levels above 45 mg/dl were found in 11 of 50 patients, but in only 4 of 51 controls (P<0.05). The mean serum concentration of Lp(a) was 33.0±7.0 mg/dl in patients and 16.9±2.7 mg/dl in controls (P<0.05). Epileptic patients also had a thicker intima media of the common carotid artery (0.79±0.04 mm) than controls (0.69±0.02 mm, P<0.05) as measured by B-mode ultrasonography. Our results suggest an untoward effect of long-term antiepileptic medication on Lp(a) serum concentrations. Elevated Lp(a) levels might be a risk factor for arteriosclerosis in epileptic patients.

Loss of CD95 expression is linked to most but not all p53 mutants in European hepatocellular carcinoma

Abstract. Experimental data have shown p53-dependent CD95 induction to be associated with increased levels of apoptosis after cytostatic treatment in hepatoma cells. A study of Japanese hepatocellular carcinoma (HCC) has reported an inverse correlation between CD95 and p53 expression. To examine the interaction of p53 and CD95 in tumors we investigated which alterations in p53 can be linked to loss of CD95 expression in European HCC. In 39 tumors we analyzed CD95 by immunohistochemistry and assessed the correlation between the findings of the p53 status as determined by immunohistochemistry and single-strand conformation polymorphism with polymerase chain reaction sequencing. In 10 of 14 tumors with evidence of p53 aberration there was also loss of CD95 expression, compared to 6 of 25 samples with apparent wild-type p53 (P<0.01). Three tumors with p53 mutations but sustained CD95 expression showed single base substitutions mapping to a narrow region of 20 codons in p53. A significant correlation with differentiation status of the tumor was found for the p53 aberration but not for CD95 expression. This is the first study to link loss of CD95 expression to specific p53 alterations in HCC. Functional p53 appears to be a major factor for CD95 expression in hepatocytes, the loss of which could contribute to chemoresistance and possibly immune evasion in hepatocellular carcinoma. Sustained CD95 expression in tumors with certain p53 aberrations may indicate functional heterogeneity of p53 mutants.

The Clinical Significance of Measuring Different Anti-dsDNA Antibodies by Using the Farr Assay, an Enzyme Immunoassay and a Crithidia luciliae Immunofluorescence Test

Anti-double-stranded DNA (dsDNA) antibodies are highly specific for the diagnosis of systemic lupus erythematosus (SLE) but are heterogeneous in respect to, for example, avidity, class and cross-reactivity. Sera from 2061 patients were measured by three methods: an enzyme-linked immunosorbent assay (ELISA), an indirect immunofluorescence test with Crithidia luciliae as substrate (CLIF), and the Farr assay, a radioimmunological method based on the ammonium sulfate precipitation of immune complexes. The different anti-dsDNA antibody determinations were evaluated by analysis of patient records. The reason for a reactive Farr assay in 14 patients was predominantly the measurement of antibodies of the IgM class, which are not detected by the ELISA. The detection of additional antibodies to dsDNA of the IgA class, to single-stranded DNA or to histones plays a minor role. In comparison with the Farr assay, we found more positive results with the ELISA, which additionally detects anti-dsDNA antibodies of low avidity. The ELISA might also yield positive values in conditions such as chronic liver diseases, various infections and connective tissue diseases other than SLE. Avoiding the disadvantages of radioactivity, the ELISA is well suited as a screening test for dsDNA antibodies. However, positive results should be confirmed by the CLIF test or preferably by the Farr assay, thus combining sensitivity with specificity.

Alteration of erythrocyte (Na++K+)-ATPase by replacement of cholesterol by desmosterol in the membrane

Cholesterol of red blood cells (RBC) is readily exchanged by desmosterol and vice versa. The resulting alteration in the sterol composition influences the specific (Na++K+)-ATPase activity. It is suggested that this effect is due to an altered membrane fluidity.

Anti-p53 in Breast Cancer: Concordance of Different Assay Procedures and Association with p53 Antigen Expression

Objective: Anti-p53 levels detected by different methods were compared in a predefined group of patients with breast cancer and correlated with p53 antigen expression in the corresponding tumors. Methods: P53 autoantibodies were investigated in 165 patients with primary breast cancer using ELISAs with recombinant or native p53. Immunoblot and indirect immunofluorescence (Huh7) were used for confirmation, p53 antigen expression in the tumor was determined immunohistochemically. Results: Using ELISA, overall 18/165 positives (11%) were detected, with only partly concordant results between the assays. Five positive sera were confirmed by immunoblot, and three also by indirect immunofluorescence. Anti-p53-positive patients detected by more than two assays showed accumulated p53 in the tumor (6/6) and mostly suffered from recurrent tumors (4/6; p = 0.02). In these cases, a trend towards a shortened disease-free interval was found (26 vs. 49 months; n.s.). In patients with a positive or borderline result in only one of the serological methods, there was no increased rate of p53 accumulation compared to anti-p53-negative patients (4/19 versus 35/126). Conclusions: Lack of assay standardization may partly explain the divergence in reports on anti-p53 and its clinicopathological associations. We speculate that, in different groups of patients, anti-p53 might be induced by different mechanisms.