Determination Of Serum Cholesterol Research Articles

France — Determination of cholesterol in serum with renewable electrode : In ANAL. CHIM. ACTA (318/1 (89–93) 1995) L. Charpentier & N. El Murr

France — Determination of cholesterol in serum with renewable electrode : In ANAL. CHIM. ACTA (318/1 (89–93) 1995) L. Charpentier & N. El Murr

放電型サーモスプレイ方式高速液体クロマトグラフィー／質量分析法を用いた同位体希釈法による血清中有機成分の定量

We review recently developed isotope dilution methods using discharge-assisted thermospray (plasmaspray) liquid chromatography/mass spectrometry (LC/MS) for determination of total cholesterol, glucose, creatinine, and urea in human serum. We have optimized the analytical conditions and discussed the analytical performances of the LC/MS methods. With an optimal conditions, the relative standard deviation of LC/MS methods was between 0.5 and 1.3%. We could conclude that the LC/MS method has the advantage in its simplicity of sample preparation, compared with the GC/MS, and could be a useful technique in IDMS analysis.

Amperometric determination of cholesterol in serum with use of a renewable surface peroxidase electrode

A sensitive modified carbon paste electrode for the direct determination of cholesterol is described. Both cyclic voltammetric and chronoamperometric techniques are used. The electrode was modified with hydroxymethyl ferrocene and horseradish peroxidase. Cholesterol oxidase and cholesterol ester hydrolase are used in solution. The measured current is linear with cholesterol concentration in the range 1 × 10 −6 to 1.5 × 10 −4 M. The measuring time is 30 s.

High-performance liquid chromatographic determination of cholesterol and cholestanol in human serum by precolumn derivatization with 2-[2-(isocyanate)ethyl]-3-methyl-1,4-naphthoquinone combined with platinum catalyst reduction and electrochemical detection.

The precolumn derivatization reagent, 2-[2-(isocyanate)ethyl]-3-methyl- 1,4-naphthoquinone (IMQ) was applied to the simultaneous determination of cholesterol and cholestanol in human serum by HPLC with electrochemical detection. The compounds extracted with hexane from human serum, to which was added 1-eicosanol as an internal standard, were reacted with IMQ in acetone and converted into the corresponding carbamic acid esters. After chromatographic separation of the derivatives, they were reduced once in a platinum catalyst reduction column connected on-line, then quantified with an electrochemical detector in the oxidation mode. The detection limits at a signal-to-noise ratio of 3 were 6.6 pg (17 fmol) and 7.4 pg (19 fmol) for cholesterol and cholestanol, respectively in a 10 microliter injection volume. This method was successfully applied to the determination of free and total cholesterol and total cholestanol in normal human serum, and the concentrations of free and total cholesterol were compared with those obtained by an enzymatic method.

Microenzymatic Fluorescence Assay for Serum Cholesterol

An enzymatic assay using fluorometric detection for cholesterol determination in serum is described. Results were compared to a conventional enzymatic colorimetric procedure and to the definitive method, which is based on isotope dilution mass spectrometry. Fluorescence detection enhances sensitivity over current colorimetric methods by approximately two orders of magnitude, and the assay response is linear over three orders of magnitude of cholesterol concentration. The reaction is performed in a single step and can be performed with small sample (1 μl) and reaction (200 μl) volumes. The fluorescence intensity is stable after a 30-min sample incubation at room temperature. The sensitivity of this fluorescence assay makes it possible to measure subnanomoles of cholesterol, allowing accurate measurement of total cholesterol in 1 μl of serum or less. This level of sensitivity will also allow measurement of cholesterol in various isolated lipoprotein fractions.

Analysis of cholesterol and cholesteryl esters in human serum using capillary supercritical fluid chromatography

Capillary supercritical fluid chromatography (SFC) using carbon dioxide as a mobile phase was applied for the determination of free cholesterol and cholesteryl esters in human serum. Serum samples were extracted with methanol—chloroform (2:1, v/v), and the extracts were analyzed by pressure programmed capillary SFC—flame-ionization detection (FID) without thermal degradation and derivatization. The total cholesterol concentrations obtained from SFC analysis were compared with those from GC or enzymatic analysis. The capillary SFC—FID method having high resolution gave an acceptable average relative standard deviation of 2.6%, and a detection limit of 4–6 pg. The quantitative results were acceptable for the simultaneous analysis of cholesterol and its esters in biological fluids. The concentration profiles of each compound in various samples, normal Korean human serum, Western human serum, and from high-cholesterol patient plasma, have been compared with this method.

Trends in Cardiovascular Disease Risk Factor Clustering in Eastern Finland: Results of 15-Year Follow-Up of the North Karelia Project

Background. The study describes 15-year cardiovascular disease risk factor clustering trends in eastern Finland and assesses the degree to which high levels of the risk factors are clustering in the same individuals. Methods. Four independent cross-sectional surveys in randomly selected population samples (in 1972, 1977, 1982, and 1987) have been carried out in two provinces in eastern Finland (North Karelia and Kuopio) to assess the level of cardiovascular disease risk factors and the impact of a community-based prevention program. Each survey included a self-administered questionnaire, blood pressure measurements, and determination of serum cholesterol. Blood pressure and serum cholesterol values were dichotomized. All subjects were classified as either current smokers or nonsmokers. The total sample size was 30,118 and the participation rate varied between 77 and 94%. The common age range in all four surveys was 30 to 59 years. Results. The proportion of individuals with a combination of two or three risk factors decreased markedly during the survey period. However, smoking tended to cluster increasingly with the other risk factors in 1987 compared with that seen in 1972. Among men, the proportion of smokers having either high blood pressure or high serum cholesterol levels, or both, is still high. Conclusions. The observed clustering of cardiovascular disease risk factors, particularly among men, underlines the importance of assessing other risk factors in persons with a high level of one risk factor and the need for a multifactorial approach in primary prevention. It is possible that high-risk behaviors, like smoking and high consumption of saturated fat and salt, were normal in 1972, but in 1987 they were declining, though they remained clustered among a particular segment of the population.

Epidemiological assessment of hyperlipidaemia management in primary health care

To evaluate the impact of contemporary management of hyperlipidaemia outside well planned and monitored investigations we studied the laboratory records of primary health care patients. The study comprises 14,988 primary health care patients who had been subject to at least one determination of serum-cholesterol during an 18-month period. About half of the group was subject to more than one examination during the period and presumably participated in an intervention programme. Concentration means (S-cholesterol, S-triglycerides and S-HDL-cholesterol) were calculated for all and for those returning for a repeated measurement. An overrepresentation of women over 50 and an underrepresentation of men of the same age and in the National Cholesterol Education Program (NCEP) risk group was found among those being screened. Patients in the highest cholesterol risk group were the most successful in reducing S-cholesterol whereas an increase of S-cholesterol was found in the lowest risk group. Women under 50 in the highest cholesterol risk group were the most successful group in lowering their cholesterol concentrations. Men under 50 were more likely to return for follow-up than women of the same age; people aged 50–70 were the most likely to return, irrespective of gender and risk group. It was found that initial S-cholesterol was the only significant predictor of the S-cholesterol concentration at follow-up. Further attention should be directed to the intermediate cholesterol risk group in an effort to combat preventable IHD and to develop programmes that attract younger women and men to a higher degree than is currently the case.

Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140°C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile—ethanol—water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 μl), respectively. The values of cholesterol and cholestanol in normal human sera were 135–212 mg/dl and 137–928 μg/dl, respectively.

Serum cholesterol determined by liquid chromatography with 6-chlorostigmasterol as internal standard

We describe an accurate and precise method for determining serum cholesterol by high-performance liquid chromatography (HPLC). After addition of 6-chlorostigmasterol as internal standard, serum is treated with alcoholic potassium hydroxide. Subsequently the cholesterol and internal standard are extracted from the mixture into n-hexane and then derivatized to phenylurethanes for measurement by HPLC with ultraviolet detection. The effective chromatographic separation and the use of an appropriate internal standard make this procedure free from interferences by other serum sterols and precise. The mean cholesterol concentration in Standard Reference Material (SRM) 909 (human serum) assayed by this procedure (4.346 mmol.g-1 x L-1) agreed well with the value assigned by the National Institute of Standards and Technology (4.359 mmol.g-1 x L-1). Within-run and total CVs were 0.56% and 0.78%, respectively. Therefore the performance of this procedure is sufficiently good to allow its use as a candidate reference method for serum cholesterol determination.

Subarachnoid hemorrhage, cerebral hemorrhage, and serum cholesterol concentration in men and women

The role of serum cholesterol in predicting the risk of stroke is unclear and may depend on the subtype of the disease. In 1964 to 1965, 54,385 Swedish men and women participated in a health survey with serum cholesterol and diastolic blood pressure determinations. The Swedish mortality register was used to identify causes of death in this cohort during 20.5 years of follow-up (1964 to 1985). A person-year-based Poisson model was used for multivariate analysis. Relative risk increased with decreasing serum cholesterol level for subarachnoid hemorrhage in men and for cerebral hemorrhage in women but not for subarachnoid hemorrhage in women. For cerebral hemorrhage in men, the risk function was U-shaped. Adjustment for diastolic blood pressure did not significantly change the relation between the risk for any of the different stroke types and the cholesterol level. A low cholesterol level predicts death from intracranial bleeding, but the data suggest that there is differing risk pattern for men and women.

Determination of serum cholesterol by stable isotope dilution method using discharge-assisted thermospray liquid chromatography/mass spectrometry

A discharge-assisted thermospray (plasmaspray) liquid chromatography/mass spectrometric method for the determination of total serum cholesterol is described. The method incorporates stable isotope dilution using (3,4-13C)cholesterol as an internal standard. Liquid chromatographic separation is performed using methanol as a flow solvent and effluents are directly introduced to the mass spectrometer. [MH-H2O]+ ions are monitored during liquid chromatography/mass spectrometry using the selected ion monitoring method. Satisfactory agreement between the analytical result and the certified value of the National Institute of Standards and Technology (formerly National Bureau of Standards) standard reference material serum is obtained with a relative standard deviation of 0.6%.

An evaluation of the Reflotron for the determination of plasma cholesterol in capillary blood. Effect of operator variability.

The availability of portable analysers potentially puts the detection of individuals with high serum cholesterol levels on a similar footing to that of high blood pressure. The Reflotron, which can make cholesterol measurements on capillary blood samples without the need for centrifugation, has been evaluated in clinical practice. In 94 consecutive patients capillary blood was used for the determination of serum cholesterol by the Reflotron, by a laboratory technician (n = 37) and by a nursing sister (n = 57). Venous blood was also taken from the same patients within a few minutes of the capillary sample for enzymic determination of serum cholesterol at the hospital laboratory. Overall, the Reflotron values were about 0.6 mmol/l lower than those obtained at the hospital laboratory. The accuracy and precision of the results obtained by the nurse were poorer than those obtained by the technician, even though the nurse had attended a three day training course. Our results indicate that the Reflotron is a suitable instrument for use in screening for serum cholesterol, provided personnel operating the machine have adequate training and experience, and provided it is borne in mind that the results obtained may be slightly lower than standard laboratory methods.

Clinical surveillance of gynecologic cancer patients

A survey of gynecologic oncologists was used to determine the optimum follow-up plan for asymptomatic, disease-free patients previously treated for a gynecologic malignancy. Ninety-four (91%) of 103 questionnaires were completed and returned. The majority of respondents recommended a clinic visit for these patients every 3 months for the first year after the completion of therapy, every 3 or 4 months for the second year, every 6 months for the following 3 years, and then annually after this initial 5-year period. Breast, abdominal, lymph node, and pelvic examinations, as well as a pap smear and stool guaiac, were done at each of these clinic visits. A yearly chest X ray was performed by the majority of the survey respondents for the first 3 years following initial treatment. Determinations of serum CA-125 and ovarian germ cell tumor markers were done at each follow-up appointment during the first 5 years after therapy if they were previously elevated. The patients' smoking habits were discussed with them by the majority of respondents. Mammograms were performed periodically before age 50 and then yearly after age 50 by most respondents. Serum cholesterol determinations were accomplished every 5 years or more frequently if indicated. Calcium supplementation was recommended for postmenopausal patients. The majority of respondents did not recommend routine vitamin supplementation or surveillance sigmoidoscopy.

Predictability of the Individual Outcome of a Physical Training Program of an Army Special Forces Unit

In a search for parameters that could predict the chances of success in a 3-month basic training program of a Special Forces unit, 293 conscripts were studied before the start of the training period. Physical activities, smoking habits, and dietary and drinking habits were evaluated by questionnaire. Anthropometric values were noted. A blood sample was taken for determination of serum cholesterol, high-density lipoprotein (HDL)-cholesterol and gamma-glutamyltranspeptidase (GGT). Physical capacity was assessed by bicycle ergometry. Sixty-five trainees were discontinued from the program for intercurrent diseases or trauma, 132 went successfully through the whole program (group S), and 96 trainees failed (group F). More candidates of group S stated that they participated in sports (p less than 0.01) and fewer smoked (p less than 0.01) than in group F. Trainees of group F had a higher consumption of spirits (p less than 0.05) than trainees of group S. There was no difference between the two groups in weight, percentage body fat, physical capacity, total serum cholesterol, and GGT values. HDL-cholesterol was higher in group S compared to group F (p less than 0.01). Stepwise discriminant analysis applied to anthropometric, biochemical, and cycloergometric data indicated HDL-cholesterol, height, resting heart rate, heart frequency at maximal exercise, and serum cholesterol as significant discriminating variables (p less than 0.001), giving only a 63% chance of correctly classifying the candidates.

Serum levels of cholesterol and ischemic heart disease mortality. The Värmland Study.

The objective of the Värmland Study was to examine how serum cholesterol can be used to predict short- and long-term ischemic heart disease (IHD) mortality, especially in women aged 65 or older. This prospective cohort study involved about 20 years of follow-up after a single determination of serum cholesterol and included participants in a health screening undertaken from 1962 to 1965 (48,076 men, 48,732 women). The main outcome measures were mortality from IHD, acute myocardial infarction (MI), and chronic ischemic heart disease (CIHD). An IHD mortality trend was associated with increasing cholesterol levels for people younger than 65 years, and was more pronounced for men than women. For people 65 years or older, there was a weak trend for men, but not even a tendency for women. Regarding acute MI, significant trends were observed for males as well as females, for young as well as old people. A mild CIHD mortality trend was observed for young men. Otherwise no significant trend was seen.

Serum Cholesterol as a Prognostic Factor after Myocardial Infarction: The Framingham Study

To determine the relation between serum cholesterol levels and the long-term risk for reinfarction, death from coronary heart disease, and all-cause mortality in persons who recover from myocardial infarction. Prospective, longitudinal study. A geographically defined population-based cohort of adults participating in the Framingham Heart Study. Men (n = 260) and women (n = 114), 33 to 88 years of age (mean age, 62 years), who had a history of myocardial infarction. A complete physical examination, including electrocardiographic evaluation, blood pressure measurement, height and weight measurements, determination of smoking habits, and casual determinations of blood glucose and serum cholesterol, was done approximately 1 year after recovery from initial myocardial infarction. Patients were followed after infarction for the occurrence of reinfarction or death (mean follow-up, 10.5 years; range, 0.8 to 31.6 years). The mean cholesterol level after infarction was 5.21 mmol/L (242.8 mg/dL); 20% of patients had levels below 5.17 mmol/L (200 mg/dL), and 22% had levels of 7.11 mmol/L (275 mg/dL) or more. Compared with patients who had cholesterol levels below 5.17 mmol/L, patients with levels of 7.11 mmol/L or more were at increased risk for reinfarction (relative risk, 3.8; 95% Cl, 1.6 to 8.7), death from coronary heart disease (relative risk, 2.6; Cl, 1.4 to 4.8), and all-cause mortality (relative risk, 1.9; Cl, 1.2 to 2.9) based on multivariate Cox regression analyses adjusted for other coronary risk factors. Intermediate cholesterol levels (5.17 mmol/L to 7.11 mmol/L) were generally not associated with increased risk. The association between elevated serum cholesterol and increased risk was strongest in men; however, elevated cholesterol levels were found to be most strongly related to death from coronary disease and to all-cause mortality in persons who were 65 years of age or more. Patients who have recovered from a myocardial infarction and who have high cholesterol levels are at an increased long-term risk for reinfarction, death from coronary heart disease, and all-cause mortality. Our results confirm the prognostic value of cholesterol levels measured after myocardial infarction and support the role of lipid management in this population.

Determination of serum cholesterol by isotope dilution mass spectrometry with a benchtop capillary gas chromatograph/mass spectrometer: comparison with the National Reference System's Definitive and Reference Methods

We developed an isotope dilution mass spectrometric cholesterol method with [25,26,27-13C]cholesterol as internal standard and a benchtop gas chromatograph/mass spectrometer (GC/MS) that is much easier and less time consuming than previously described Reference and Definitive Methods for cholesterol. The internal standard, cholesterol standards, and unknown specimen are delivered volumetrically with an automated dilutor and the saponifying reagent. After saponification, extraction, and derivatization, specimens are injected into a benchtop quadrupole MS with an autosampler. Unknown cholesterol concentrations are calculated automatically by comparing the peak area ratio of the m/z = 368, 371 ion pair with the ratios for the cholesterol standards (0 to 12.93 mmol/L). We found within-run and day-to-day (overall) imprecision of 0.44% and 0.95%, respectively, when specimens were assayed singly. In several lyophilized and frozen Standard Reference Material (SRM) pools, cholesterol results with our GC/MS method averaged 0.4% less than the National Institute for Standards and Technology definitive GC/MS result performed about three years earlier. Our GC/MS results averaged 1.3% and 2.0% less than results by the National Reference System (NRS) Abell-Levy-Brodie-Kendall (ALBK) results from clinical specimens and the SRM pools, respectively. These results are consistent with the previously reported bias between the NRS Reference and Definitive Methods and the 0.1% per year decrease in cholesterol concentrations in SRM pools as determined by GC/MS analysis. These results further emphasize the small but consistent bias between cholesterol results by isotope dilution mass spectrometry and the ALBK Reference Method, the latter being the basis for the National Cholesterol Education Program guidelines and population reference values.

(2-Phthalimidyl)benzoyl Azides as Fluorescence Labeling Reagents for Alcohols in High Performance Liquid Chromatography

Two fluorescence labeling reagents, 3- and 4-(2-phthalimidyl)benzoyl azide, were designed for primary and secondary alcohols for high performance liquid chromatography. These reagents, prepared from orthophthalaldehyde and aminobenzoic acid in several steps, react with alcohols at 125°C for 30 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column ERC-ODS-1161 with acetonitrile/water (70:30) or acetonitrile/ethanol (70:30) as eluent. The extent of conversion of cholesterol into fluorescent derivative was 100%. The detection limit for the alcohols labeled with 3-(2-phthalimidyl)benzoyl azide was 0.1-0.4 pmol per injection (10 μl). 3-(2-Phthalimidyl)-benzoyl azide was applied for the determination of cholesterol in human serum by high performance liquid chromatography with fluorescence detection. The values of cholesterol in normal human sera were 4.8-9.4 μmol/ml.

Family History Evaluation as a Predictive Screen for Childhood Hypercholesterolemia

A study was conducted to evaluate the efficacy of family history factors as screening criteria for childhood hypercholesterolemia. When they were seen for routine care at one of eight office practices, 1005 prepubertal children underwent random serum cholesterol determinations. Parental and grandparental histories of cardiovascular risk factors and atherosclerotic complications prior to 55 years of age were also obtained. Of the initial group, 274 children had total cholesterol levels ≥175 mg/dL, and 175 of these children returned for retesting after an overnight fast. A total of 88 children were found to have low-density lipoprotein-cholesterol (LDL-C) values ≥90th percentile for age and sex. Maternal and paternal histories of hypercholesterolemia were significantly associated with elevated LDL-C (odds ratio = 7.3 and 2.9, respectively), but had extremely low sensitivities (0.09, 0.15) despite modest positive predictive values (0.42, 0.22). Grandparental histories of sudden death, peripheral vascular disease, and gout were associated with elevated LDL-C, but sensitivities and positive predictive values for all of these factors were less than 0.22. Family history factors most commonly recommended as criteria for cholesterol screening in children did not identify half of all the children with elevated LDL-C and did not selectively identify the most severely affected children. Adding information concerning the presence of childhood obesity did not result in appreciable improvement in LDL-C detection beyond that achieved by family history factors alone. It was concluded that if thorough identification of young children with elevated LDL-C is desired, inclusive population screening rather than a family history-based strategy would be the most effective approach.