Different effects of acarbose and glibenclamide on proinsulin and insulin profiles in people with Type 2 diabetes.

AIM: In a double-blind, placebo-controlled study, we compared the effect of acarbose (A) and glibenclamide (G) on post-prandial (pp) and 24-h profiles of proinsulin and insulin. METHODS: Twenty-seven patients with Type 2 diabetes mellitus insufficiently controlled with diet alone were randomised to receive acarbose, 100 mg thrice daily, glibenclamide, 1 mg thrice daily, or placebo. Before and after 16 weeks of treatment, 24-h profiles of proinsulin, insulin and glucose (fasting, 1 h after breakfast and every 3-h for a 24-h period) were measured under metabolic ward conditions with standardised meals. RESULTS: With acarbose, a reduced 24-h level of proinsulin was observed compared with glibenclamide (AUC 1096 +/- 118 vs. 1604 +/- 174 pmol/l per h, P<0.05) at 16 weeks. The breakfast increment of proinsulin was lower with acarbose than glibenclamide (6.8 vs. 19.3 pmol/l, P<0.05) as was the level at that time (37.3 +/- 5.3 vs. 56.4 +/- 7.5 pmol/l, P<0.05). A lower AUC of insulin after treatment was also observed with acarbose than glibenclamide (7.9 +/- 0.9 vs. 14.8 +/- 4.5 nmol/l per h, P<0.05), as also for 1-h increment (81 +/- 26, vs. 380 +/- 120 pmol/l, P<0.01) and 1-h level (325 +/- 30 vs. 621 +/- 132 pmol/l, P<0.01). Acarbose reduced 1-h breakfast glucose increment (baseline 6.3 +/- 0.6, 16-week 3.5 +/- 0.6 mmol/l, P<0.01) and 1-h glucose level (18.1 +/- 1.1 and 14.5 +/- 1.3 mmol/l, P<0.01), whereas glibenclamide did not (6.6 +/- 0.7 vs. 5.4 +/- 0.6 mmol/l and 18.9 +/- 1.5 vs. 15.3 +/- 1.3 mmol/l). CONCLUSIONS: Measurement of circadian excursions of proinsulin and insulin reveals distinct differences in meal-time proinsulin and insulin increment and level between acarbose and glibenclamide whereas fasting levels of these insulin fractions remained unaffected.

[Urinary concentration of collagen metabolites in endoprosthesis loosening]. / Harnkonzentrationen von Kollagenabbauprodukten bei Endoprothesenlockerungen.

Aseptic loosening is the most common long-term complication in arthroplasty. Loosening is in every case associated with bone resorption at the interface that leads to bone defects and complicates the revision. The diagnosis of aseptic loosening is based on clinical and radiological evaluation. Especially in clinically asymptomatic cases an early diagnosis with these methods is difficult. In our study we wanted to evaluate the diagnostic value of biochemical markers of the bone resorption in aseptic loosening. We compared 58 patients with proven implant loosening during surgery with 67 patients without clinical or radiological signs of loosening. We measured the crosslinks pyridinoline and hydroxypyridinoline in urine samples. In contrast to Schneider et al. [increased urinary crosslink levels in aseptic loosening of total hip arthroplasty, J. Arthroplasty 1995; 13 (6): 687-692] we found no significant differences between loose and asymptomatic hip or knee prosthesis. Also no correlation between the size of the acetabular defects of loose hip implants and the urinary crosslink excretion was measurable. Our results show no or only little diagnostic value of the urinary crosslinks pyridinoline and deoxypyridinoline in aseptic loosening of total hip and knee arthroplasty.

In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation.

In the present study a protocol of in situ reverse transcriptase-nested polymerase chain reaction (in situ RT-nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by "DNA repair mechanisms" and "endogenous priming", a two-step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin-labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5(prime, variant)-tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT-nested PCR, which in comparison to the method of in situ RT-PCR-in situ-hybridisation is simpler and less time-consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Interleukin-1, interleukin-6 and myocardial enzyme response after coronary artery bypass grafting - a prospective randomized comparison of the conventional and three minimally invasive surgical techniques.

OBJECTIVE: In order to evaluate the traumatic effects of median sternotomy and cardiopulmonary bypass (CPB) in conventional and minimally invasive coronary artery bypass grafting, inflammatory response was studied in a prospective randomized trial in patients referred to single-vessel coronary artery bypass grafting. METHODS: Four surgical techniques were compared: group 1, median sternotomy with CPB in ten patients (eight male, two female; aged 59.6+/-11.0 years (mean+/-SD)); group 2, median sternotomy and off-pump in ten patients (seven male, three female; aged 65.1+/-10.0 years); group 3, minithoracotomy with CPB in ten patients (seven male, three female, aged 61.2+/-10.4 years); group 4, minithoracotomy and off-pump in ten patients (nine male, one female, aged 62.9+/-9.8 years). All patients received a left internal mammary artery graft to the left anterior descending artery (LAD). Clinical data, perioperative values of cytokines and cardiac enzymes were monitored. RESULTS: There were no major complications. Troponin-T and creatine kinase isoenzyme MB (CK-MB) levels were significantly higher in CPB procedures (P<0.0056; multivariate general linear model). Interleukin-6 (IL-6) levels were significantly higher in minithoracotomy procedures. Interleukin-1 (IL-1) was significantly increased in all patients compared with the preoperative values. CONCLUSIONS: The use of CPB is combined with higher levels of troponin-T and CK-MB as signs of myocardial damage. Surgical access was identified as a trigger of inflammatory response, as minithoracotomy is related to higher levels of IL-6. IL-1 increased in all procedures and this occurred independently of the surgical access or the use of CPB, which points out a potential relationship between inflammatory response and anesthesia. Neither CPB nor surgical access influenced the clinical outcome in the treatment of coronary artery single-vessel bypass grafting.

Impaired glucose tolerance (IGT) is not associated with disturbed homocysteine metabolism.

Elevated plasma total homocysteine (tHcy) has been suggested to be an additional risk factor for cardiovascular disease in subjects with impaired glucose tolerance (IGT) and Type 2 diabetes (T2D). In order to investigate whether an insulin resistant/chronic hyperinsulinemic situation in male diabetic and prediabetic subjects directly influences the tHcy metabolism, fasting tHcy and post-methionine load tHcy plasma levels (PML-tHcy) were determined in 15 men with IGT, 13 men with newly diagnosed T2D, and 16 normoglycemic controls (NGT). Fasting tHcy (IGT, 13.1 +/- 4.6; T2D, 12.8 +/- 4.0; NGT, 10.7 +/- 4.4 micromol/L) and PML-tHcy (IGT, 46.5 +/-17.39; T2D, 41.1 +/- 6.8; NGT, 38.0 +/- 9.7 micromol/L) showed no differences between the groups. Fasting tHcy and PML-tHcy correlated with fasting proinsulin (r = 0.395, p < 0.05; r = 0.386, p< 0.05) and creatinine (r = 0.489, p < 0.01; r = 0.339, p < 0.05), resp. Multiple regression analysis showed only a relationship between fasting tHcy and creatinine. No relationships have been found between fasting tHcy and PML-tHcy, resp., and indicators of an insulin resistant state, e.g., insulin and proinsulin, as well as serum cobalamin and folate concentrations. In conclusion, our data suggest that the degree of glucose intolerance has no direct impact on the metabolism of homocysteine. However, tHcy levels tend to be elevated with the development of nephropathy, indicating an association between tHcy and renal function in these subjects.

Expression of secretory group IIA phospholipase A(2) in relation to the presence of microbial agents, macrophage infiltrates, and transcripts of proinflammatory cytokines in human aortic tissues.

Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA(2)-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA(2)-IIA. Transcripts specific for sPLA(2)-IIA, interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were identified by reverse transcription-polymerase chain reaction. In 18 of 102 analyzed specimens, DNA of microbial pathogens was found. Thirteen sections were positive for C pneumoniae, whereas 2 specimens were positive either for cytomegalovirus or for herpes simplex virus. One section contained genomic DNA of all 3 pathogens simultaneously. None of the analyzed tissues exhibited nucleic acids specific for H pylori. In addition to macrophage infiltrates, the presence of microbial DNA was closely associated with the occurrence of transcripts specific for proinflammatory cytokines and sPLA(2)-IIA. Pathogens as well as sPLA(2)-IIA and cytokines were found to be present not only in advanced but also in early stages of atherosclerosis. In tissues negative for sPLA(2)-IIA and cytokine expression, none of the pathogens could be identified. Because macrophages exposed to phospholipase A(2)-treated lipoproteins are transformed into foam cells in vitro, the results of this study suggest an alternative mechanism by which microbial infections may act in a proatherogenic fashion in vessel walls.

Analysis of secretory group II phospholipase A2 expression in human aortic tissue in dependence on the degree of atherosclerosis.

Secretory non-pancreatic (group II) phospholipase A2 (sPLA2) releases precursors of important mediators of inflammation from phospholipids. Based on the inflammatory character of atherosclerosis we previously described the identification of sPLA2 in human atherosclerotic plaques. In vitro studies on lipoproteins have shown that sPLA2 is able to favour the formation of foam-like cells representing a typical feature of early atherosclerotic lesions. In the present study the expression of sPLA2 in relation to the degree of atherosclerosis was investigated. Aortic tissue samples of 25 autopsy cases ranging in age from 1 to 77 years were taken from 2 cm above the heart and 3 cm below the renal arteries. The material was classified regarding the degree of atherosclerotic changes based on staining with haemalaun and eosine as well as on staining according to Goldner. Furthermore, immunohistochemical procedures detecting sPLA2, macrophages and smooth muscle cells were performed. The study has shown that in the abdominal aorta the enzyme was present in all advanced atherosclerotic lesions, but only in some preatheromas and precursors of atherosclerosis. However, this correlation did not occur in the thoracic aorta, where sPLA2-positive results showed a similar frequency in all degrees of atherosclerotic lesion. The enzyme was found in all three layers of the vessel wall without significant differences. Round cells, scarcely smooth muscle cells and endothelial cells were identified as sPLA2-positive. However, these data do not allow a conclusion as to which type of cell is responsible for the secretion of sPLA2. In summary, the correlation between the expression of this enzyme and the degree of atherosclerosis underlines the possible importance of sPLA2 in atherogenesis.

Diagnostic significance of prostate-specific antigen velocity at intermediate PSA serum levels in relation to the standard deviation of different test systems.

Serial prostate-specific antigen (PSA) measurements (PSA velocity) as an additional instrument to detect prostatic cancer was introduced in 1992. It has previously been reported that PSA increase per year differed in the last 5 years prior to diagnosis in patients with benign prostatic hyperplasia (0.18 ng/ml/year), locally confined (0.75 ng/ml/year) and metastasized (4.4 ng/ml/year) cancer of the prostate (CaP) in contrast to healthy men (0.04 ng/ml/year). The ability of PSA velocity to detect organ-confined CaP in patients with intermediate PSA serum values depends therefore on a reliable and reproducible PSA result. The present study comprised 85 men with PSA values between 3 and 8 ng/ml (Abbott IMx). PSA measurements were repeated with Abbott IMx (n = 85 patients) and Hybritech Tandem-E (n = 59 patients) assays. The PSA serum values differed from one examination to the other from 0.02 to 2.74 ng/ml with the Abbott IMx. Standard deviation amounted to 0.35 ng/ml with the Abbott IMx PSA assay. Using the Hybritech Tandem-E assay, mean standard deviation was 1.15 ng/ml and therefore higher than with the Abbott IMx assay. The difference from one test to the other ranged from 0.05 to 4.05 ng/ml with the Hybritech Tandem-E. Using the Abbott IMx assay, 10.6% of all repeat measurements exceeded 1 ng/ml whereas in the Hybritech Tandem-E assay 62.7% of the second measurements differed > 1 ng/ml from the first PSA result. An increase in PSA serum values may therefore be due to intratest variation, physiological day-to-day variation as well as prostatic disease. It is important to notice that the intra-assay variation may be greater than the PSA increase per year in a patient with CaP. Therefore, PSA velocity seems to be of limited value.

Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2.

In vitro-studies have shown that phospholipid hydrolysis of low density lipoproteins (LDL) by bee venom or porcine pancreatic phospholipase A2 (PLA2) leads to an increased uptake of these lipoproteins by macrophages transforming them into foam cells. Recently, a secretory phospholipase A2, group II, was detected in human atherosclerotic plaques. In order to investigate the role of this enzyme in the pathogenesis of atherosclerosis, a structurally identical human secretory PLA2 was purified from the medium of HepG2 cells stimulated with interleukin-6 and tumor necrosis factor-alpha. The activity of the purified enzyme towards the phospholipids of native and modified low density lipoproteins was compared with the activity towards Escherichia coli-membranes and other phospholipid substrates. Compared to E. coli-membranes, native LDL proved to be a poor substrate for group II PLA2. After mild oxidation induced by copper ions or by 2,2-azobis(2-amidinopropane) (AAPH), the susceptibility of LDL to phospholipid hydrolysis was found to be increased by 25 and 23%, respectively, whereas extensive copper-mediated oxidation caused a decreased hydrolysis. Aging of LDL at 6 degrees C for weeks or at 37 degrees C for hours resulted in an increase in PLA2-catalyzed phospholipid hydrolysis of up to 26-fold. LDL protected from oxidation by probucol during aging showed a lesser increase in susceptibility to phospholipid hydrolysis. Our results suggest that PLA2, group II, can increase the atherogenicity of LDL by its ability to hydrolyze the phospholipids of these lipoproteins, especially after modifications that are likely to occur in vivo.

Alterations in the physiochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2. A spin-label study.

Human low and high density lipoproteins (LDL and HDL, respectively) were treated with porcine pancreatic phospholipase A2 (PLA2) in the presence of albumin resulting in hydrolysis of 40-84% of the lipoproteins phospholipids. The resulting PLA2-treated LDL and HDL and concurrent control lipoproteins incubated without PLA2 were reisolated by ultracentrifugation and labelled with 5-doxyl- and 16-doxyl-stearic acid, and with a spin-labelled analogue of maleimide. Analysis of ESR spectra showed that phospholipid hydrolysis both of LDL and HDL resulted in an increase in order, micro-viscosity and polarity of lipid regions in the surface monolayer of the particles. In the temperature range from 3 degrees C to 50-60 degrees C, Arrhenius plots of a spectral parameter of LDL and HDL labelled with 5-doxyl-stearate exhibited alterations which suggest an increase in free cholesterol content near the surface of the lipoproteins after PLA2-treatment. ESR spectra of the maleimide analogue bound covalently to the protein moiety of the lipoproteins have demonstrated that, following phospholipid hydrolysis, the conformation of the apoproteins became more condensed, with more masked domains. The possible implications of the revealed alterations for enhanced delivery of LDL and HDL cholesterol to cells after phospholipolysis of the lipoproteins are discussed.

Secretory group II phospholipase A2 in human atherosclerotic plaques.

Atherosclerotic plaques exhibit a series of features that are similar to those of chronic inflammation. Based on the fact that during inflammation several cell types synthesize and secrete a group II phospholipase A2 (PLA2), an immunohistochemical study was undertaken to explore whether this enzyme can be identified in human atherosclerotic lesions. Tissue specimens obtained from 13 patients who had undergone arteriectomy and three specimens with advanced atherosclerotic plaques obtained at autopsy were analyzed and compared to arteries free of atherosclerosis. The results showed that in all areas with atherosclerotic lesions, a staining with monoclonal antibodies raised against group II PLA2 was evident. In normal arteries without thickened intima, this immunostaining was completely negative. With the use of specific monoclonal antibodies against macrophages (anti-KP-1) and smooth muscle cells (anti-alpha-actin), PLA2-positive cells were identified as foam cells mainly derived from macrophages. In addition to these cells, other regions of the thickened intima gave a partially positive reaction with anti-PLA2 antibodies, but could not be stained with either anti-KP-1 or anti-alpha-actin. Some of these regions were localized on edges of calcification and cell necrosis. Other PLA2-positive regions seem to be associated with extracellular matrix structures. In summary, the findings of this study may be regarded as further evidence to support the link between atherosclerosis and chronic inflammatory processes. In view of the fact that the in vitro modification of lipoproteins by PLA2-treatment induces lipid deposition in macrophages, the results of this study suggest that group II PLA2 may actively be involved in the formation of foam cells in vivo.

Changes in epitope exposition of apolipoprotein A-I on the surface of high density lipoproteins after phospholipase A2 treatment.

The immunoreactivity of high density lipoprotein (HDL) modified by treatment with porcine pancreatic phospholipase A2 (PLA2) was studied in a competitive radioimmunoassay using 6 different monoclonal apolipoprotein (apo) A-I antibodies. The competition tests have shown that after PLA2 treatment the immunoreactivity of selected epitopes of apo A-I changed in different ways. While the binding behavior of two epitopes remained unchanged, three epitopes exhibited decreased immunoreactivities after phospholipids hydrolysis. In contrast to the latter epitopes, the immunoreactivity of an epitope located on the cyanogen bromide fragment 4 of apo A-I increased with the degree of lipolysis. A loss of apo A-I from HDL as a consequence of PLA2-treatment did not occur as shown by the determination of the apo A-I concentration in HDL before and after treatment with PLA2. Using overlapped synthetic decapeptides it could be shown that the epitope increasingly exposed on the particle surface of PLA2-modified HDL consists of the amino acid residues 162-173 and 212-229. These residues are characterized by high hydrophobic indices as determined by hydropathy analysis. Furthermore, these regions belong partially to the proposed receptor-binding domain of apo A-I. Thus, an increased exposition of this epitope might result in elevated cellular binding affinities of HDL occurring after modification of lipoproteins by PLA2-treatment.

Exposure of macrophages to PLA2-modified lipoproteins leads to cellular lipid accumulations.

After a 24-h exposure of mouse peritoneal macrophages to LDL, of which the phospholipids were reduced to 30% by phospholipase A2 (PLA2)-treatment, the cellular level of free and esterified cholesterol was elevated 1.9- and 5.0-fold, respectively. Furthermore, the activity of the cytosolic acyl-CoA cholesterol acyl-transferase (ACAT), which was calculated from the rate of 14C-oleate incorporation into the cellular cholesteryl esters, increased with the degree of LDL hydrolysis. The incubation of macrophages with native HDL led to decreased labeling of cellular cholesteryl esters. However, after PLA2-treatment of HDL the esterification rate increased with the degree of hydrolysis analogous to PLA2-modified LDL. The formation of numerous intracellular lipid droplets was observed by light microscopy after staining with Sudan-III. These data suggest that phospholipases A2 may play a role in the transformation of macrophages into foam-cells, a hallmark of early atherosclerotic lesions.

Interaction of apolipoprotein B-containing lipoprotein secreted by Hep G2 cells with receptors for low-density lipoprotein.

Radioligand and immunoenzymatic techniques were used to characterize the receptor binding properties of apolipoprotein B-containing lipoprotein produced by HepG2 cell line (H-LpB). It was found that compared to plasma low-density lipoprotein (LDL), the interaction of H-LpB nonseparated from conditioned medium with normal fibroblasts was 6-8-times lower and only slightly exceeded the nonspecific binding of LDL modified by malondialdehyde, while the uptake of the indicated lipoproteins by LDL receptor-negative strain of fibroblasts were identical. The uptake of H-LpB by normal fibroblasts increased 1.5-2-times after isolation from the culture medium by immunoaffinity chromatography. The effect of isolation could be explained by the finding that apolipoprotein E-containing lipoprotein secreted by HepG2 cells effectively competed for the binding with LDL-receptors. The obtained results suggest that H-LpB produced by HepG2 cells is poorly recognized by the LDL-receptors.

[Analysis of the body fluids using SDS-gradient gel electrophoresis]. / Untersuchungen von Körperflüssigkeiten mittels SDS-Gradientengelelektrophorese.

In the present report we describe a horizontal SDS-electrophoresis in thin- or ultrathin-layer polyacrylamide pore-gradient gel polymerized on polyester films for the estimation of proteins in several human body fluids. Up to 25 samples can be analyzed side by side under identical conditions. The combination of Coomassie blue staining with silver-staining allows the analysis of fluids containing low protein content also without concentrative techniques.

The influence of a steroid hormone and of physical exercise on protein metabolism in rats.

The influence of an anabolic steroid hormone preparation and of a physical exercise training program was studied on the nitrogen and protein metabolism in rats with the help of the 15N tracer technique and the emission spectrometric 15N isotope analysis. For the determination of the dynamic parameters of the protein metabolism graphic (stochastic) and computer aided compartmental methods were compared. Using as a stochastic approach the area-method the animals showed significant differences in the protein turnover parameters under the influence of hormone treatment and (or) physical stress by swimming exercise in comparison to the controls.