Peter Lattke

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.

Release of cobalt and chromium ions into the serum following implantation of the metal-on-metal Maverick-type artificial lumbar disc (Medtronic Sofamor Danek).

Study Design. Cross-sectional study of 10 patients to measure the serum levels of cobalt and chromium after TDA. Objectives. To investigate the release of cobalt and chromium ions into the serum following implantation of the metal-on-metal Maverick-type artificial lumbar disc. Summary of Background Data. In total hip endoprosthetics and consequently for TDA (total disc arthroplasty), metal-on-metal combinations are used with the aim of reducing wear debris. In metal-on-metal TDA the release of metal ions has until now been secondary to the main discussion. Materials and Methods. We investigated the serum cobalt and chromium concentration following implantation of 15 Maverick TDAs (monosegmental L5–S1, n = 5; bisegmental L4–L5 and L5–S1, n = 5; average age, 36.5 years). Five healthy subjects (no metal implants) acted as a control group. The measurements of the metals were carried out using the HITACHI Z-8200 AAS polarized Zeeman atomic absorption spectrometer after an average of 14.8 months. Results. The concentrations of cobalt and chromium ions in the serum amounted on average to 4.75 &mgr;g/L (SD, 2.71) for cobalt and 1.10 &mgr;g/L (SD, 1.24) for chromium. Compared with control group, both the chromium and cobalt levels in the serum showed significant increases (Mann-Whitney U test, P = 0.0120). At follow-up,the Oswestry Disability Score was on average significantly decreased by 24.4 points (L5–S1) (t test, P < 0.05) and by 26.8 points (L4–S1) (t test, P < 0.05). The improved clinical situation is also represented by a significant decrease of the Visual Analog Pain Scale of 42.2 points after the follow-up (t test, P < 0.05). Conclusion. Significant systemic release of Cr/Co was proven in the serum compared with the control group. The concentrations of Cr/Co measured in the serum are similar in terms of their level to the values measured in THA metal-on-metal combinations or exceed these values given in the literature. Long-term implication of this metal exposure in unknown and should be studied further.

Determination of cholesterol in atherosclerotic plaques using near infrared diffuse reflection spectroscopy

The aim of this investigation was to examine whether near infrared diffuse reflection spectroscopy is an acceptable tool for the determination of cholesterol content in atherosclerotic plaques. Using an FT-spectrophotometer (lambda=1000-2500 nm) and fiberoptic systems (d=4 mm), the cholesterol content could be determined in mixtures of the primary compounds of the aortic wall with acceptable precision. Considering the inhomogeneous distribution of cholesterol and cholesterol esters in atherosclerotic plaques the determination of total cholesterol using this method is of acceptable efficacy, even though the calibration procedure did not reflect the composition correctly. Using an energy dose of less than 100 mW/cm(2) to avoid damage to endothelial cells, arterial tissue of about 170-200 microm thickness attenuates the reflected NIRS signal by up to 50%. Cholesterol levels could be determined accurately in atherosclerotic lesions in human aortic specimens obtained by autopsy. The correlation coefficient between the NIRS results and those of HPLC analysis calculated in the investigation of 82 different areas of 18 human aortic specimens was 0.926 (y=0.869x+0. 771, external validation). Acceptable results were also achieved by means of a coronary-catheterlike fiberoptic strand (d=l mm), despite the worsened signal/noise ratio. The results show that the development of a coronary catheter using NIRS appears to be possible in principle.

Reference intervals for plasma free metanephrines with an age adjustment for normetanephrine for optimized laboratory testing of phaeochromocytoma

Background Measurements of plasma normetanephrine and metanephrine provide a useful diagnostic test for phaeochro-mocytoma, but this depends on appropriate reference intervals. Upper cut-offs set too high compromise diagnostic sensitivity, whereas set too low, false-positives are a problem. This study aimed to establish optimal reference intervals for plasma normetanephrine and metanephrine. Methods Blood samples were collected in the supine position from 1226 subjects, aged 5-84 y, including 116 children, 575 normotensive and hypertensive adults and 535 patients in whom phaeochromocytoma was ruled out. Reference intervals were examined according to age and gender. Various models were examined to optimize upper cut-offs according to estimates of diagnostic sensitivity and specificity in a separate validation group of 3888 patients tested for phaeochromocytoma, including 558 with confirmed disease. Results Plasma metanephrine, but not normetanephrine, was higher (P < 0.001) in men than in women, but reference intervals did not differ. Age showed a positive relationship (P < 0.0001) with plasma normetanephrine and a weaker relationship (P = 0.021) with metanephrine. Upper cut-offs of reference intervals for normetanephrine increased from 0.47 nmol/L in children to 1.05 nmol/L in subjects over 60 y. A curvilinear model for age-adjusted compared with fixed upper cut-offs for normetanephrine, together with a higher cut-off for metanephrine (0.45 versus 0.32 nmol/L), resulted in a substantial gain in diagnostic specificity from 88.3% to 96.0% with minimal loss in diagnostic sensitivity from 93.9% to 93.6%. Conclusions These data establish age-adjusted cut-offs of reference intervals for plasma normetanephrine and optimized cut-offs for metanephrine useful for minimizing false-positive results.

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.

Determination of the cholesterol–collagen ratio of arterial atherosclerotic plaques using near infrared spectroscopy as a possible measure of plaque stability

Particular danger associated with an arteriosclerotic plaque consists in the possible rupture of its cap, dependent on the thickness of the cap covering the lipid core, its composition and different inflammatory changes. The purpose of this study was to compare the total cholesterol and collagen contents of arterial walls, both measured by near infrared spectroscopy (NIRS), and to test whether the ratios of cholesterol to collagen correlate with histochemical parameters possibly being indicators for plaque stability. NIR spectra of 118 sections from 36 human aortas were measured at 1000-2500 nm. Evaluation was performed by the partial least squares method (PLS), the chemical reference analysis by HPLC. Acceptable results were achieved for calibrations. With these calibrations 38 further aortic sections taken at autopsy were NIR-spectroscopically analysed and ordered in relation to histological findings of fatty deposits, cap thickness over the lipid core, and the ratio of fatty deposits to cap thickness. Correlations were found to exist between the spectroscopically determined total cholesterol concentrations and the histologically estimated fatty deposits (r=0.887), between the spectroscopically determined collagen concentrations and the cap thickness over the lipid core (r=0.441), and between the ratios total cholesterol to collagen and the ratios fatty deposits to cap thickness (r=0.575).

Impact of concentrations of glycated hemoglobin, α-tocopherol, copper, and manganese on oxidation of low-density lipoproteins in patients with type I diabetes, type II diabetes and control subjects

The late organ complications in diabetic patients are associated with enhanced oxidation of low-density lipoproteins (LDL). The role of vitamin and trace metal concentrations in this process is not clear. Therefore, we compared the oxidative susceptibility and alpha-tocopherol concentration of LDL with the levels of glycated hemoglobin (HbA1c), copper and manganese. Sixty-three diabetic patients (23 female and 40 male; 53 of type II, 10 of type I) and 35 control subjects (17 female and 18 male) were investigated. The in vitro-formation of conjugated dienes in purified LDL preparations in the presence of copper was followed as absorbance at 234 nm. LDL exhibited a shorter lagtime (44.5 +/- 10.1 vs. 67.8 +/- 16.0 and 50.1 +/- 14.3 vs. 68.8 +/- 14.6 min) for type I and type II diabetic patients vs. sex and age-matched controls, P < 0.001. For all subjects together the lagtime was inversely correlated to HbA1c (r = -0.230, P = 0.023) and positively correlated to LDL alpha-tocopherol/LDL (mol/mol). This ratio was lower in diabetic patients (P < 0.01 for type II) than in control subjects. The copper and manganese plasma levels were not different between diabetic and nondiabetic groups. However, parameters of LDL oxidizability (amount and rate of oxidation) were positively correlated with both copper and manganese concentrations. We conclude that in diabetes the resistance of LDL against oxidation is diminished in relation to the quality of glucose control.

Levodopa therapy in Parkinson’s disease: influence on liquid chromatographic tandem mass spectrometric-based measurements of plasma and urinary normetanephrine, metanephrine and methoxytyramine

Background Medication-related interferences with measurements of catecholamines and their metabolites represent important causes of false-positive results during diagnosis of phaeochromocytomas and paragangliomas (PPGLs). Such interferences are less troublesome with measurements by liquid chromatography with tandem mass-spectrometry (LC-MS/MS) than by other methods, but can still present problems for some drugs. Levodopa, the precursor for dopamine used in the treatment of Parkinson’s disease, represents one potentially interfering medication. Methods Plasma and urine samples, obtained from 20 Parkinsonian patients receiving levodopa, were analysed for concentrations of catecholamines and their O-methylated metabolites by LC-MS/MS. Results were compared with those from a group of 120 age-matched subjects and 18 patients with PPGLs. Results Plasma and urinary free and deconjugated (free + conjugated) methoxytyramine, as well as urinary dopamine, showed 22- to 148-fold higher (P < 0.0001) concentrations in patients receiving levodopa than in the reference group. In contrast, plasma normetanephrine, urinary noradrenaline and urinary free and deconjugated normetanephrine concentrations were unaffected. Plasma free metanephrine, urinary adrenaline and urinary free and deconjugated metanephrine all showed higher (P < 0.05) concentrations in Parkinsonian patients than the reference group, but this was only a problem for adrenaline. Similar to normetanephrine, plasma and urinary metanephrine remained below the 97.5 percentiles of the reference group in almost all Parkinsonian patients. Conclusions These data establish that although levodopa treatment confounds identification of PPGLs that produce dopamine, the therapy is not a problem for use of LC-MS/MS measurements of plasma and urinary normetanephrine and metanephrine to diagnose more commonly encountered PPGLs that produce noradrenaline or adrenaline.

Increased hepatic cholesterol accumulation in transgenic mice overexpressing human secretory phospholipase A2 group IIA.

It has been demonstrated in transgenic mice that the overexpression of human phospholipase A2 group IIA (sPLA2), an acute-phase reactant, is associated with depressed plasma cholesterol levels, altered lipoprotein compositions, and increased lipid depositions in aortic walls. It was the aim of the present study to investigate whether the reduced plasma cholesterol levels in sPLA2-transgenic mice may be due to an increased transfer of lipids from sPLA2-modified lipoproteins to the liver and/or other nonvascular tissues. Ten sPLA2-transgenic mice and an equal number of nontransgenic littermates were fed a cholesterol-enriched (1%) diet for 13 weeks. After autopsy, cholesterol and triglyceride concentrations were measured in homogenates of liver, spleen, kidney, and myocardial tissues. Compared to the nontransgenic controls, the sPLA2-transgenic mice exhibited signi- ficantly lower plasma cholesterol levels, which was due to a reduction in both HDL and β-lipoprotein (LDL+β-VLDL) cholesterol. Liver tissues from the transgenic mice were found to contain signi- ficantly increased concentrations of free and esterified cholesterol, which was not associated with increased triglyceride concentrations. Spleen, kidney, and heart tissues of the two animal groups showed no significant differences in cholesterol or triglyceride concentrations. The findings suggest that the overexpression of human secretory phospholipase A2 group IIA leads to an enhanced delivery of cholesterol from phospholipolysed lipoproteins to the liver. This mechanism is likely to contribute to the development of hypocholesterolemia observed in patients with inflammatory diseases.

Preservation of urine free catecholamines and their free O-methylated metabolites with citric acid as an alternative to hydrochloric acid for LC-MS/MS-based analyses

Abstract Background: Measurements of urinary fractionated metadrenalines provide a useful screening test to diagnose phaeochromocytoma. Stability of these compounds and their parent catecholamines during and after urine collection is crucial to ensure accuracy of the measurements. Stabilisation with hydrochloric acid (HCl) can promote deconjugation of sulphate-conjugated metadrenalines, indicating a need for alternative preservatives. Methods: Urine samples with an intrinsically acidic or alkaline pH (5.5–6.9 or 7.1–8.7, respectively) were used to assess stability of free catecholamines and their free O-methylated metabolites over 7 days of room temperature storage. Stabilisation with HCl was compared with ethylenediaminetetraacetic acid/metabisulphite and monobasic citric acid. Catecholamines and metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Free catecholamines and their O-methylated metabolites were stable in acidic urine samples over 7 days of room temperature storage, independent of the presence or absence of any stabilisation method. In contrast, free catecholamines, but not the free O-methylated metabolites, showed rapid degradation within 24 h and continuing degradation over 7 days in urine samples with an alkaline pH. Adjustment of alkaline urine samples to a pH of 3–5 with HCl or 4.8–5.4 with citric acid completely blocked degradation of catecholamines. Ethylenediaminetetraacetic acid/metabisulphite, although reducing the extent of degradation of catecholamines in alkaline urine, was largely ineffectual as a stabiliser. Conclusions: Citric acid is equally effective as HCl for stabilisation of urinary free catecholamines and minimises hazards associated with use of strong inorganic acids while avoiding deconjugation of sulphate-conjugated metabolites during simultaneous LC-MS/MS measurements of free catecholamines and their free O-methylated metabolites.