High-throughput and sensitive quantitation of plasma catecholamines by ultraperformance liquid chromatography-tandem mass spectrometry using a solid phase microwell extraction plate.

Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC-MS/MS analysis run time is 2.0 min per sample. This UPLC-MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50-250 μL, e.g., from children and mice).

New diagnoses of dementia among older patients admitted to postacute care.

Objectives: To determine the prevalence of dementia and the proportion of undiagnosed dementia in elderly patients admitted to postacute care, and to identify patients' characteristics associated with undiagnosed dementia. Design: Cross-sectional study. Setting: Academic postacute rehabilitation facility in Lausanne, Switzerland. Participants: Patients (N = 1764) aged 70 years and older. Measurements: Data on socio-demographic, medical, functional, and affective status were collected upon admission. Data on cognitive performance (Mini-Mental State Exam [MMSE]), and cognition-related discharge diagnoses were abstracted through a structured review of discharge summaries. Results: Overall, 24.1% (425/1764) patients had a diagnosis of dementia, most frequently secondary to Alzheimer's disease (260/425, 61.2%). Among dementia cases, 70.8% (301/425) were newly diagnosed during postacute stay. This proportion was lower among patients referred from internal medicine than from orthopedic/surgery services (65.8% versus 74.8%, P = .042). Compared to patients with already diagnosed dementia, those newly diagnosed were older, lived alone more frequently, and had better functional status and MMSE score at admission (all P < .05). In multivariate analysis, previously undetected dementia remained associated with older age (OR = 2.4 for age 85 years and older, 95% CI 1.5-4.0, P = .001) and normal MMSE at admission (OR = 5.9, 95% CI 2.7-12.7, P < .001). Conclusion: Dementia was present in almost a fourth of elderly patients referred to postacute care, but was diagnosed in less than a third before admission. Oldest old patients appear especially at risk for underrecognition. These results emphasize the high diagnostic yield of systematic cognitive assessment in the postacute care setting to improve these patients' management and quality of life.

Prospective determination of plasma imipenem concentrations in critically ill children.

Plasma imipenem concentrations were measured in 19 critically ill children (median age, 0.8 year; range, 0.02 to 12.9 years). Wide interindividual variations (2 to 4x at peak and >10x at trough concentrations) resulted in unpredictable plasma levels in several children. To avoid subtherapeutic drug levels, we recommend treatment with at least 100 mg/kg of body weight/day of imipenem-cilastatin for critically ill children requiring such therapy.

Variabilité des concentrations plasmatiques de l'angiotensinogène et risque d'hypertension artérielle chez le rat transgénique [Variability of plasma angiotensinogen levels and risk of hypertension in a transgenic rat model].

AIM: Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors. METHODS: Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n=3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction). RESULTS: At baseline, transgenic rats had +18mmHg higher bood pressure and -8% lower body weight compared to non-transgenic rats (P<0.05) without significant changes for the vehicle groups throughout the study (P>0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P<0.05) in transgenic and +25%, +5.3% and +6.7% (P>0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P>0.05). CONCLUSION: Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.

Distribution of plasma levels of adiponectin and leptin in an adult Caucasian population.

OBJECTIVES: Little is known regarding the distribution and the determinants of leptin and adiponectin levels in the general population. DESIGN: Cross-sectional study. PATIENTS: Women (3004) and men (2552) aged 35-74 living in Lausanne, Switzerland. MEASUREMENTS: Plasma levels of leptin and adiponectin (ELISA measurement). RESULTS: Women had higher leptin and adiponectin levels than men. In both genders, leptin and adiponectin levels increased with age. After adjusting for fat mass, leptin levels were significantly and negatively associated with age in women: 18.1 +/- 0.3, 17.1 +/- 0.3, 16.7 +/- 0.3 and 15.5 +/- 0.4 ng/ml (adjusted mean +/- SE) for age groups [35-44], [45-54], [55-64] and [65-75], respectively, P < 0.001. A similar but nonsignificant trend was also found in men. Conversely, the age-related increase of adiponectin was unrelated to body fat in both genders. Post-menopausal women had higher leptin and adiponectin levels than premenopausal women, independently of hormone replacement therapy. Although body fat mass was associated with leptin and adiponectin, the associations were stronger with body mass index (BMI), waist and hip in both genders. Finally, after adjusting for age and anthropometry, no relationships were found between leptin or adiponectin levels with alcohol, caffeine consumption and physical activity, whereas smoking and diabetes decreased leptin and adiponectin levels in women only. CONCLUSIONS: The age-related increase in leptin levels is attributable to changes in fat mass in women and probably also in men. Leptin and adiponectin levels are more related to BMI than to body fat mass. The effects of smoking and diabetes appear to be gender-specific.

A decrease of plasma macrophage migration inhibitory factor concentration is associated with lower numbers of circulating lymphocytes in experimental Plasmodium falciparum malaria.

Macrophage migration inhibitory factor (MIF) has recently been implicated in the pathogenesis of malarial anaemia. However, field studies have reported contradictory results on circulating MIF concentrations in patients with clinically overt Plasmodium falciparum malaria. We determined plasma MIF levels over time in 10 healthy volunteers during experimental P. falciparum infection. Under fully controlled conditions, MIF levels decreased significantly during early blood-stage infection and reached a nadir at day 8 post-infection. A decrease in the number of circulating lymphocytes, which are an important source of MIF production, paralleled the decrease in MIF levels. Monocyte/macrophage counts remained unchanged. At MIF nadir, the anti-inflammatory cytokine interleukin (IL)-10, which is an inhibitor of T-cell MIF production, was detectable in only 2 of 10 volunteers. Plasma concentrations of the pro-inflammatory cytokines IL-8 and IL-1beta were only marginally elevated. We conclude that circulating MIF levels decrease early in blood-stage malaria as a result of the decline in circulating lymphocytes.

Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects.

BACKGROUND: Endothelin-1 is an endothelium-derived potent vasoconstrictor peptide of 21 amino acids. To establish reference values in different models of hypertension and in human subjects an assay for plasma immunoreactive endothelin-1 (ET-1) was optimized. METHODS: ET-1 is extracted by acetone from 1 mL of plasma and subjected to a sensitive enzyme-linked immunosorbent assay. RESULTS: The detection limit for plasma ET-1 is 0.05 fmol/mL. Mean recoveries of the 1, 2, 5, and 10 fmol of ET-1 added to 1 mL of plasma were 66%, 75%, 85%, and 92%, respectively. Within- and between-assay coefficients of variation were < or =12% and < or =10%, respectively. Assay accuracy was demonstrated by consistent recoveries of added ET-1 over the entire physiologic range of plasma concentrations and by the linearity of ET-1 concentrations measured in serially diluted plasma extracts (r = 0.99). No ET-1 was detected when albumin buffer was extracted instead of plasma. Using this method, we found increased ET-1 levels in plasma of three experimental rat models of hypertension: stroke prone spontaneously hypertensive rats (SP-SHR), deoxycorticosterone acetate-salt hypertensive rats, and one kidney-one clip hypertensive rats. In contrast, plasma ET-1 levels of SHR were half those of normotensive Wistar rats. In two kidney-one clip hypertensive rats, plasma ET-1 concentrations were not different from those found in sham-operated control rats. Plasma ET-1 concentrations of 37 healthy men were 0.85 +/- 0.26 fmol/ml (mean +/- SD). CONCLUSIONS: The present assay reliably measures ET-1 levels in rat and human plasma. It allows to discriminate between different forms of hypertension with high or low circulating levels of ET-1.

Diagnostics VIH tardifs en 2009 en Suisse: motivation a un dépistage systématique du VIH [Late diagnoses of HIV infection in 2009 in Switzerland: towards a universal HIV testing?]

Individuals who are unaware of their HIV infection constitute a fragile and a critical population both from a personal and from a community-based point of view: if the infection is diagnosed too late, the outcome can be complicated by AIDS-defining diseases, whose prognosis may remain unfavourable even after the initiation of a potent antiretroviral therapy. These patients contribute to the virus spreading into the community, owing to a high viral load. It is now necessary to recognise the limits of a risk behaviour and disease-driven HIV screening policy. Since 2006, the American guidelines recommend a routine HIV testing for all patients age 13 to 64 years unless they specifically refuse the test (opt-out). In Switzerland, the recommendations remain so far risk-centred.

The validity of clinical diagnoses of dementia in a group of consecutively autopsied memory clinic patients.

BACKGROUND: Epidemiological studies show that up to 10% of individuals aged 65 years and older suffer from dementia, most commonly from dementia of the Alzheimer Type (DAT) (1). Clinicopathological studies are critical to our understanding of this disease and improving the accuracy of clinical diagnoses. OBJECTIVES: Our objectives were to examine the validity of clinical diagnoses of DAT, to determine the prevalence of different forms of dementia in this sample, and to investigate the relationship between age at death and polymorbidity. SUBJECTS AND METHOD: Clinical data were available from 221 patients who had been examined at the Basel Memory Clinic between 1986 and 1996. From this population, 34% (75 patients) were autopsied in the Department of Pathology, University Hospital Basel, and neuropathological examinations were additionally performed on 62 (83%) of these patients. Clinical and neuropathological data were retrospectively compared. RESULTS: 67.8% of the neuropathologically examined patients received a definitive diagnosis of AD (Alzheimer's disease), vascular dementia (VaD) or mixed dementia (AD and VaD). AD alone or with other histopathological hallmarks of dementia was the most prevalent neuropathological diagnosis (63%). VaD was deemed the only cause of dementia in only 4.8% of patients. The sensitivity for DAT was 75.9%, the specificity 60.6%. Increasing age was associated with an increasing number of clinical and neuropathological diagnoses. CONCLUSION: The sensitivity and specificity of the clinical diagnoses of DAT found in our study are similar to previous reports (2-5). Older patients had more etiologies of their dementia than younger patients. This study reaffirms the need for internationally accepted criteria for clinical and neuropathological diagnoses, as well as further clinical-neuropathological investigations to further refine the clinical diagnostic process.

Plasma lipoprotein-associated phospholipase A2 activity in Alzheimer's disease, amnestic mild cognitive impairment, and cognitively healthy elderly subjects: a cross-sectional study.

ABSTRACT: INTRODUCTION: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulating enzyme with pro-inflammatory and oxidative activities associated with cardiovascular disease and ischemic stroke. While high plasma Lp-PLA2 activity was reported as a risk factor for dementia in the Rotterdam study, no association between Lp-PLA2 mass and dementia or Alzheimer's disease (AD) was detected in the Framingham study. The objectives of the current study were to explore the relationship of plasma Lp-PLA2 activity with cognitive diagnoses (AD, amnestic mild cognitive impairment (aMCI), and cognitively healthy subjects), cardiovascular markers, cerebrospinal fluid (CSF) markers of AD, and apolipoprotein E (APOE) genotype. METHODS: Subjects with mild AD (n = 78) and aMCI (n = 59) were recruited from the Memory Clinic, University Hospital, Basel, Switzerland; cognitively healthy subjects (n = 66) were recruited from the community. Subjects underwent standardised medical, neurological, neuropsychological, imaging, genetic, blood and CSF evaluation. Differences in Lp-PLA2 activity between the cognitive diagnosis groups were tested with ANOVA and in multiple linear regression models with adjustment for covariates. Associations between Lp-PLA2 and markers of cardiovascular disease and AD were explored with Spearman's correlation coefficients. RESULTS: There was no significant difference in plasma Lp-PLA2 activity between AD (197.1 (standard deviation, SD 38.4) nmol/min/ml) and controls (195.4 (SD 41.9)). Gender, statin use and low-density lipoprotein cholesterol (LDL) were independently associated with Lp-PLA2 activity in multiple regression models. Lp-PLA2 activity was correlated with LDL and inversely correlated with high-density lipoprotein (HDL). AD subjects with APOE-ε4 had higher Lp-PLA2 activity (207.9 (SD 41.2)) than AD subjects lacking APOE-ε4 (181.6 (SD 26.0), P = 0.003) although this was attenuated by adjustment for LDL (P = 0.09). No strong correlations were detected for Lp-PLA2 activity and CSF markers of AD. CONCLUSION: Plasma Lp-PLA2 was not associated with a diagnosis of AD or aMCI in this cross-sectional study. The main clinical correlates of Lp-PLA2 activity in AD, aMCI and cognitively healthy subjects were variables associated with lipid metabolism.

Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia.

Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

Plasma aldosterone is independently associated with the metabolic syndrome.

The aim of this study was to analyze the associations of plasma aldosterone and plasma renin activity with the metabolic syndrome and each of its components. We analyzed data from a family based study in the Seychelles made up of 356 participants (160 men and 196 women) from 69 families of African descent. In multivariable models, plasma aldosterone was associated positively (P < 0.05) with blood pressure in older individuals (interaction with age, P < 0.05) and with waist circumference in men (interaction with sex, P < 0.05) and negatively with high-density lipoprotein cholesterol, in particular in individuals with elevated urinary potassium excretion (interaction with urinary potassium, P < 0.05); plasma renin activity was significantly associated with triglycerides and fasting blood glucose. Plasma aldosterone, but not plasma renin activity, was associated with the metabolic syndrome per se, independently of the association with its separate components. The observation that plasma renin activity was associated with some components of the metabolic syndrome, whereas plasma aldosterone was associated with other components of the metabolic syndrome, suggests different underlying mechanisms. These findings reinforce previous observations suggesting that aldosterone is associated with several cardiovascular risk factors and also suggest that aldosterone might contribute to the increased cardiovascular disease risk in individuals of African descent with the metabolic syndrome.