International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods.

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Chicago Healthy Aging Study: objectives and design.

Investigators in the Chicago Healthy Aging Study (CHAS) reexamined 1,395 surviving participants aged 65-84 years (28% women) from the Chicago Heart Association Detection Project in Industry (CHA) 1967-1973 cohort whose cardiovascular disease (CVD) risk profiles were originally ascertained at ages 25-44 years. CHAS investigators reexamined 421 participants who were low-risk (LR) at baseline and 974 participants who were non-LR at baseline. LR was defined as having favorable levels of 4 major CVD risk factors: serum total cholesterol level <200 mg/dL and no use of cholesterol-lowering medication; blood pressure 120/≤80 mm Hg and no use of antihypertensive medication; no current smoking; and no history of diabetes or heart attack. While the potential of LR status in overcoming the CVD epidemic is being recognized, the long-term association of LR with objectively measured health in older age has not been examined. It is hypothesized that persons who were LR in 1967-1973 and have survived to older age will have less clinical and subclinical CVD, lower levels of inflammatory markers, and better physical performance/functioning and sleep quality. Here we describe the rationale, objectives, design, and implementation of this longitudinal epidemiologic study, compare baseline and follow-up characteristics of participants and nonparticipants, and highlight the feasibility of reexamining study participants after an extended period postbaseline with minimal interim contact.

Potential problems in the use of commercial preparations of radiolabeled cholesterol.

Through a series of biological and analytical procedures, we demonstrate that a compound purchased from a commercial supplier as [7-3H]cholesterol was not cholesterol. In mouse peritoneal macrophages, this compound was metabolized differently than other radiolabeled cholesterol preparations and was accumulated in the steryl ester pool. In contrast, Fu5AH rat hepatoma cells did not discriminate this compound from cholesterol. Further analysis of the anomalous [7-3H]cholesterol by TLC after cholesterol oxidase treatment and by HPLC indicated that this radiochemical was less polar than cholesterol standard and other radiolabeled cholesterol preparations tested. Mass spectrometry analysis disclosed that the chemical has a similar fragmentation pattern and the same molecular weight (386) as cholesterol.

Cholesterol screening: comparative evaluation of on-site and laboratory-based measurements.

We measured cholesterol in capillary blood samples from 9683 volunteers over a four-day on-site community screening program, using the "Reflotron" desk-top analyzer (Boehringer-Mannheim Diagnostics, Indianapolis, IN). We also measured cholesterol in venous blood samples from 3% of those screened (a) with the Reflotron at the screening sites, (b) in a qualified hospital clinical laboratory, and (c) in a Centers for Disease Control standardized lipoprotein research laboratory. The sensitivity (and specificity) of the Reflotron measurements, with use of the lipoprotein laboratory measurements as the point of reference, was 0.95 (0.73) in capillary blood samples and 0.88 (0.93) in venous blood samples, compared with 0.99 (0.87) in the hospital clinical laboratory. The Reflotron measurements correlated less well with the lipoprotein laboratory values in both venous blood (r = 0.91) and capillary blood (r = 0.89) samples than did the clinical laboratory values (r greater than 0.99). Furthermore, the capillary blood measurements averaged 7% higher than venous measurements when both kinds of samples were analyzed in the Reflotron.

Regional interlaboratory standardization of determinations of cholesterol, high-density lipoprotein cholesterol, and triglycerides.

The Clinical Chemistry Forum of Central Virginia initiated a lipid standardization program to help ensure that its members meet the current National Cholesterol Education Program guidelines for cholesterol testing, and to standardize assays of high-density lipoprotein (HDL) cholesterol and triglycerides so as to provide accurate lipid profiles. We found that freshly collected, never-frozen human sera must be used to assess interlaboratory accuracy for cholesterol, HDL cholesterol, and triglycerides assays, and that at least 23 samples are required to detect a 3% bias with 90% power when the between-laboratory imprecision (CV) is 3%. After recalibration, all 12 laboratories had a mean HDL cholesterol bias less than or equal to 5%, nine of 10 laboratories had a mean HDL cholesterol bias less than or equal to 40 mg/L for samples with values less than or equal to 570 mg/L, and 10 of 12 laboratories had a mean triglycerides bias less than or equal to 10% for fresh human sera split between participants and the Centers for Disease Control. Pools of frozen human sera were shown to have matrix biases greater than 3% for cholesterol in seven of 11 laboratories, and greater than 40 mg/L for HDL cholesterol in six of nine laboratories.

Gas-liquid chromatographic determination of total cholesterol in multicomponent foods.

A method is described for the determination of total cholesterol in multicomponent foods and also other products such as nonfat dry milk, dried whole egg solids, and certain candy bars. The lipid is extracted from the sample by a mixed solvent and saponified. The unsaponifiable fraction which contains the cholesterol and other sterols is extracted with benzene. An aliquot is evaporated to dryness and the residue is dissolved in dimethylformamide. The sterols are derivatized to form trimethylsilyl (TMS) ethers. The TMS-cholesterol derivative is quantitatively determined by gas-liquid chromatography, using 5alpha-cholestane as an internal standard. Nine laboratories participated in a collaborative study of the determination of total cholesterol in deviled ham sandwich spread, vegetable beef stew, corned beef hash, frozen chicken pot pie, pizza pepperoni, fish sticks, breaded shrimp, chocolate-covered candy bars, dried whole egg solids, and nonfat dry milk and the results are reported here. The coefficient of variation ranged from 5.64 to 23.2%, with an average coefficient of variation of 14.8%.