First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

The Simultaneous measurement of serum testosterone and 5α-dihydrotestosterone by gas chromatography-mass spectrometry (GC-MS).

BACKGROUND: Simultaneous measurement of testosterone (T) and 5α-dihydrotestosterone (DHT) is important for diagnosing androgen deficiency states and hyperandrogenism in males and females, respectively. However, immunoassays used for T and DHT determination suffer from inadequate specificity and sensitivity, while tandem mass spectrometry is expensive and demanding in use. METHODS AND RESULTS: We developed a selective gas chromatography-mass spectrometry (GC-MS) method for parallel T and DHT measurement. The assay showed a linear response up to 46.5nmol/L, intra- and interassay imprecision and inaccuracy <15% and recoveries in spiked samples >90% for both analytes. The limit of quantitation was 0.117nmol/L for T and 0.168nmol/L for DHT. Comparison with immunoassays revealed good agreement for T in males, but a bias in favour of immunoassays at low concentrations for T in females and DHT in both sexes. We established reference ranges for T and DHT and suggest interval partitioning for T according to age in men and menstrual cycle in women. Assay validation in a clinical setting suggests that measuring DHT or T/DHT ratio may help identify patients with polycystic ovary syndrome. CONCLUSION: We developed a selective, simple and inexpensive GC-MS method for parallel measurement of T and DHT with potential use in the clinical laboratory.

Determination of serum cholestane-3ß,5α,6ß-triol by gas chromatography-mass spectrometry for identification of Niemann-Pick type C (NPC) disease.

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3ß,5α,6ß-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography-mass spectrometry (GC-MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC-MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03-200ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03ng/mL and 0.01ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5-10-fold increase in C-triol levels. We developed a quick and sensitive GC-MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing.

Head-To-Head Assessment of Diagnostic Performance of Testosterone Immunoassays in Patients With Polycystic Ovary Syndrome.

BACKGROUND: Determination of plasma testosterone is critical for the proper diagnosis of polycystic ovary syndrome (PCOS), but the interpretation of biochemical tests is hampered by inadequate specificity and precision of available immunoassays. We here compared the diagnostic performance of three testosterone immunoassays (Advia Centaur, Immulite 2000 XPi, Cobas e411) in PCOS patients using receiver operator characteristics curve analysis. METHODS AND RESULTS: Plasma levels of testosterone, androstendione, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, estradiol, progesterone, steroid hormone binding globulin, luteinizing hormone, and follicular stimulating hormone were determined in 188 patients with PCOS and 202 controls. Free testosterone (fT) levels and free androgen index (FAI) were calculated. Testosterone levels measured on Advia Centaur, Immulite 2000 XPi, and Cobas e411 showed clear linear relationship to each other. Testosterone measured with Advia Centaur showed discriminatory performance superior to Immulite 2000 XPi and Cobas e411. Calculation of fT or FAI improved the performance of Advia Centaur and Immulite 2000 XPi, which nevertheless performed better than Cobas e411. The performance of other parameters was inferior to that of testosterone, fT, and FAI. CONCLUSION: Present study documents striking differences between testosterone immunoassays with respect to their capacity to identify PCOS patients and favors the use of calculated parameters reflecting active testosterone in plasma.

Everolimus therapy is associated with reduced lipoprotein-associated phospholipase A2 (Lp-Pla2) activity and oxidative stress in heart transplant recipients.

BACKGROUND: Several studies demonstrated decreased severity and incidence of cardiac allograft vasculopathy (CAV) in heart transplant recipients receiving immunosuppressive therapy with everolimus. However, data regarding the influence of everolimus on risk factors predisposing to CAV are hitherto limited. We here systematically evaluated cardiovascular risk factors in heart transplanted patients, who underwent conversion to everolimus or were maintained on conventional therapy with calcineurin inhibitors (CNI). METHODS: 50 Patients receiving everolimus and 91 patients receiving CNI in addition to mycophenolate mofetil and low-dosed steroids were included in the study. CAV risk factors were determined in plasma or urine using standard enzymatic or immunochemical methods. RESULTS: No significant differences were observed between both groups with regard to lipid (total, LDL- and HDL-cholesterol), metabolic (glucose, insulin), inflammatory (C-reactive protein, IL-6, myeloperoxidase) and cardiac (troponin I, NT-proBNP) risk factors. However, significantly lower activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) and a negative correlation between the Lp-PLA2 activity and the everolimus concentration were observed in plasmas from everolimus-treated patients. Conversion to everolimus significantly lowered Lp-PLA2 activity in heart transplant recipients. Studies in vitro revealed reduced Lp-PLA2 expression in hepatocytes and macrophages pre-exposed to everolimus. In addition, reduced plasma markers of oxidative stress including oxidized LDL, 8-iso-prostaglandin F2α and protein carbonyls were noted in heart transplant recipients receiving everolimus therapy. CONCLUSION: Our results suggest that everolimus specifically lowers plasma activity and cellular production of Lp-PLA2 and thereby dampens oxidative stress. These effects may additionally contribute to the reduced CAV incidence observed in heart transplant recipients receiving everolimus therapy.

Plasma sitosterol elevations are associated with an increased incidence of coronary events in men: results of a nested case-control analysis of the Prospective Cardiovascular Münster (PROCAM) study.

BACKGROUND AND AIM: Sitosterolemia, a rare genetic disorder characterized by profoundly elevated plasma sitosterol concentrations, is associated with premature atherosclerosis in some individuals. This study was conducted to evaluate if the modest sitosterol elevations seen in the general population are also associated with the occurrence of coronary events. METHODS AND RESULTS: A nested case-control study using stored samples from male participants in the Prospective Cardiovascular Münster (PROCAM) study was performed. Each of 159 men who suffered a myocardial infarction or sudden coronary death (major coronary event) within 10 years of follow-up in PROCAM was matched with 2 controls (N = 318) by age, smoking status, and date of investigation. Analysis was performed using conditional logistic regression. Plasma sitosterol concentrations were elevated in cases compared with controls (4.94 +/- 3.44 micromol/L versus 4.27 +/- 2.38 micromol/L; P = 0.028). The upper quartile of sitosterol (>5.25 micromol/L) was associated with a 1.8-fold increase in risk (P < 0.05) compared with the lower three quartiles. Among men with an absolute coronary risk > or = 20% in 10 years as calculated using the PROCAM algorithm, high sitosterol concentrations were associated with an additional 3-fold increase in the incidence of coronary events (P = 0.032); a similar, significant relationship was observed between a high sitosterol/cholesterol ratio and coronary risk (P = 0.030). CONCLUSIONS: Elevations in sitosterol concentrations and the sitosterol/cholesterol ratio appear to be associated with an increased occurrence of major coronary events in men at high global risk of coronary heart disease. Further evaluations are warranted to confirm these preliminary findings.

Gas chromatography-mass spectrometry and molecular genetic studies in families with the Conradi-Hünermann-Happle syndrome.

The Conradi-Hünermann-Happle syndrome is an X-linked dominant disease that is due to mutations in the gene for emopamil binding protein. Emopamil binding protein is a Delta8-Delta7 sterol isomerase and plays a pivotal role in the final steps of cholesterol biosynthesis. We wanted to know to what extent this X-linked dominant enzyme defect has functional consequences at the biochemical level and whether it is possible to predict the clinical phenotype from serum sterol measurements. Therefore we performed sterol biochemical studies in 11 Conradi-Hünermann-Happle syndrome families and compared the results obtained to the clinical and molecular genetic findings. To assess disease severity a score considering bone and skin involvement and further features was used. For evaluation of the functional consequences we studied serum samples using gas chromatography-mass spectrometry analysis. For mutation screening we analyzed the emopamil binding protein gene using polymerase chain reaction, heteroduplex analysis of all exons, direct sequencing, and restriction enzyme analysis. Mutations in the emopamil binding protein gene were found in all 11 families including seven novel mutations affecting exons 2, 4, and 5. Gas chromatography-mass spectrometry analysis revealed markedly elevated levels of 8-dehydrocholesterol and of cholest-8(9)-en-3beta-ol and helped to identify somatic mosaicism in a clinically unaffected man. The extent of the metabolic alterations in the serum, however, do not allow prediction of the clinical phenotype, nor the genotype. This lack of correlation may be due to differences in X-inactivation between different tissues of the same patient and/or loss of the mutant clone by outgrowth of proficient clones after some time.