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.

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.

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

INTRODUCTION: In total hip endoprosthetics and consequently for TDA, 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: In order to investigate the ion release following the implantation of the metal-on-metal Maverick type artificial lumbar disc we measured the serum cobalt and chromium concentration following implantation of 15 Maverick TDAs (monosegmental L5/S1, n = 5; bisegmental L4/5 and L5/S1, n = 5; average age 36.5 years). Five healthy subjects (no metal implants) acted as a control group. The two measurements of the metals were carried out using the absorption spectrometry after an average of 14.8 and 36.7 months. RESULTS: In summary, the concentrations of cobalt and chromium ions in the serum at both follow-ups amounted on average to 3.3 microg/l (SD 2.6) for cobalt and 2.2 microg/l (SD 1.5) for chromium. These figures are similar to the figures shown in the literature following the implantation of metal-on-metal THA. After a comparison to the control group, both the chromium and cobalt levels in the serum showed visible increases regarding the first and the second follow-up. DISCUSSION: As there is still a significant release of cobalt and chromium into the serum after an average follow-up of 36.7 months a persistent release of these ions must be taken into consideration. Despite the evaluation of the systemic and local effects of the release of Cr/Co from orthopaedic implants has not yet been concluded, one should take into consideration an explanation given to patients scheduled for the implantation of a metal-on-metal TDA about these results and the benefits/risks of alternative combinations of gliding contact surfaces.

Dehydroepiandrosterone induces a neuroendocrine phenotype in nerve growth factor-stimulated chromaffin pheochromocytoma PC12 cells.

The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex, which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders, a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. Nerve growth factor (NGF)-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein-2. We examined whether DHEA may stimulate the cells toward a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence but not absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells toward a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla.

On interaction of activated protein C with human aortic smooth muscle cells attenuating the secretory group IIA phospholipase A2 expression.

INTRODUCTION: Pharmacological restriction of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) expression is thought to be beneficial in the treatment of inflammatory diseases such as sepsis and septic shock. In this study we investigated the effects of activated protein C (APC) on sPLA(2)-IIA expression, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and on DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and CCAAT box enhancer binding protein-beta (C/EBP-beta) in human aortic smooth muscle cells (HASMC). MATERIALS AND METHODS: To achieve elevated sPLA(2)-IIA production as occurring during inflammation, HASMC were stimulated with interferon-gamma (IFN-gamma) alone and in combination with other inductors, thus modeling the strong sPLA(2)-IIA elevation by inflammation. RESULTS AND CONCLUSIONS: APC inhibited the stimulated expression of sPLA(2)-IIA in HASMC dose-dependently (1-300 nM). At the same time, APC increased the phosphorylation of ERK 1/2 and decreased NF-kappaB and C/EBP-beta DNA-binding activities in these cells, as compared with respective stimulated controls. Reverse transcriptase-polymerase chain reaction and cell-based ELISA reveal an endothelial protein C receptor (EPCR) expression in HASMC. Application of antibodies against EPCR and protease-activated receptor-1 (PAR-1) reduced the APC-induced ERK 1/2 activation and the treatment of cells with a PAR-1 antagonist diminished the sPLA(2)-IIA inhibition. The obtained results show that APC effectively suppresses the up-regulated sPLA(2)-IIA expression, which might contribute to the reported beneficial effects of APC in the treatment of severe inflammatory disorders.

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).

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 significantly lower plasma cholesterol levels, which was due to a reduction in both HDL and beta-lipoprotein (LDL + beta-VLDL) cholesterol. Liver tissues from the transgenic mice were found to contain significantly 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.

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.

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 phaeochromocytoma, 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.

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 microg/L (SD, 2.71) for cobalt and 1.10 microg/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 is unknown and should be studied further.