Advantages and disadvantages of serum cholesterol determination by the kinetic vs the end point method.

Enzymatic assays for cholesterol determination can use an end point or a kinetic method. We evaluated and compared the performance of these methods. We constructed user-defined methods on 3 automated analyzers using Streptomyces cholesterol reagents to evaluate the analytic performance of both methods. Linearity (700-900 mg/dL) and stability of reagents (5-11 weeks) depended on the analyzers. The coefficients of variation for imprecision were 2.41% to 2.99% and 3.78% to 5.52% for the end point and kinetic methods, respectively. The end point method showed less bias at decision cut points (-0.8% to 1.3%) than did the kinetic method (-1.1% to 3.6%) but was more affected by interfering substances.The advantages of the end point over the kinetic method are better precision and lower reagent cost. The end point imprecision fell within National Cholesterol Education Program guidelines (

Application of Streptomyces and Brevibacterium cholesterol oxidase for total serum cholesterol assay by the enzymatic kinetic method.

BACKGROUND: Using non-esterified cholesterol standard, Brevibacterium and Streptomyces are found as suitable sources of cholesterol oxidase for kinetic cholesterol assay. For clinical use, we investigated the suitability of these enzymes for cholesterol determination in human serum. METHODS: We compared the performance of reagents containing 2 enzymes for the kinetic determination of total serum cholesterol with the standardized endpoint method. RESULTS: Reagent containing Streptomyces enzyme was more sensitive than that of Brevibacterium, with linearity up to 20.7 and 2.6 mmol/l, respectively. The analytical reaction for Streptomyces showed a shorter lag phase (148 s) and a steeper slope (absorbance vs. time) than that of Brevibacterium (246 s). The assay using Streptomyces reagent was precise and accurate and compared favorably with the endpoint method (y=1.06x-0.15, r=0.996, bias=0.21 mmol/l). Hemoglobin as high as 7.5 g/l did not interfere while turbidity greater than 2+ (absorbance >0.778 at 670 nm) and bilirubin concentrations >171.0 micromol/l did interfere (in a negative interference). Reagent was stable up to at least 8 weeks. CONCLUSIONS: The Streptomyces cholesterol oxidase, with 3,4-dichlorophenol, proved a suitable source for serum total cholesterol determination by the kinetic method.

Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol.

The enzymatic method for cholesterol determination can use either an endpoint or a kinetic method. Not much is known concerning the properties (K(m) and V(max)) of the commercial enzyme for the kinetic method. We measured the K(m) and V(max) of Brevibacterium, Streptomyces, Pseudomonas fluorescens, and Cellulomonas cholesterol oxidase. Brevibacterium gave the highest K(m) value (230.3 x 10(-4) M), followed by Streptomyces (2.17 x 10(-4) M), Cellulomonas (0.84 x 10(-4) M), and Pseudomonas (0.61 x 10(-4) M). The K(m) values and the linearity obtained from Streptomyces (2.6 mmol/L), Pseudomonas (2.1 mmol/L), or Cellulomonas (2.1 mmol/L) were too low. Dichlorophenol isomers, acting as inhibitors, increased the enzyme's K(m). The addition of 3,4-dichlorophenol raised the K(m) of Streptomyces from 2.17 x 10(-4) to 24.89 x 10(-4) M. The linearity was increased from 2.6 to 13.0 mmol/L. The high K(m) of Brevibacterium resulted in an insensitive reaction and low cholesterol linearity (7.8 mmol/L). An increase in the sample-to-reagent ratio from 1:100 to 1:10 enhanced the reaction rate and the linearity from 7.8 to 20.7 mmol/L. We suggest that Brevibacterium and Streptomyces cholesterol oxidase (with the addition of 3,4 dichlorophenol) are good sources for serum cholesterol determination by the kinetic method.

Risk factors for development of decreased kidney function in a southeast Asian population: a 12-year cohort study.

End-stage kidney disease has become an increasing burden in all regions of the world. However, limited epidemiologic data on chronic kidney disease in Southeast Asian populations are available. Therefore, a cohort study over a period of 12 yr (1985 to 1997) in 3499 employees of the Electric Generation Authority of Thailand, aged 35 to 55 yr, was conducted to determine the prevalence of decreased kidney function and risk factors associated with future development of decreased kidney function. The prevalence of decreased kidney function (GFR <60 ml/min) increased from 1.7% (95% confidence interval [CI], 1.3 to 2.1) in 1985 to 6.8% (95% CI, 5.7 to 7.9) in 1997, and the prevalence of elevated serum creatinine was 6.1% (95% CI, 5.3 to 6.9) and 16.9% (95% CI, 15.3 to 18.5) in 1985 and 1997 surveys, respectively. The adjusted odds ratio for future development of decreased kidney function was 2.57 (1.0 to 6.81) for systolic hypertension (>159 mmHg), 1.82 (1.12 to 2.98) for hyperuricemia (>6.29 mg/dl), 1.68 (1.02 to 2.77) for elevated body mass index (>24.9 kg/m(2)) compared with subjects with systolic BP <140 mmHg, serum uric acid <4.5 mg/dl, and body mass index 20.8 to 22.8 kg/m(2). The rising prevalence of decreased kidney function in this population resulted mainly from the increasing prevalence of the risk factors in the population. Screening to detect decreased kidney function and early intervention to modify the associated risk factors should be considered in otherwise healthy individuals. Future studies are also necessary to determine whether implementation of these measures results in a reduction of ESRD incidence in the population.

Comparative study of two automated high-sensitivity C-reactive protein methods in a large population.

OBJECTIVES: Increased serum C-reactive protein (CRP) is associated with future risk of coronary heart disease in apparently healthy individuals. Numerous high-sensitivity CRP (hs-CRP) methods are available but their comparability in large populations has not been assessed. This study aimed to evaluate the performance of two CRP methods in a large Asian population. DESIGN AND METHODS: We compared the Tina-quant CRP immunoturbidimetric assay (Roche COBAS INTEGRA) to the N high-sensitivity latex-enhanced immunonephelometric (BN 100 nephelometer, Dade Behring) assay using 4118 serum samples from the International Collaborative Study on Atherosclerosis and Stroke in Asia (Inter ASIA). RESULTS: The median hs-CRP value for the N high-sensitivity CRP method (1.23 mg/L) was significantly lower than that for the Tina-quant method (1.50 mg/L), P < 0.001. The two methods were highly associated (r = 0.9916). Deming regression analysis gave a slope of 0.958 [95% confidence interval (CI): 0.954-0.962] with an intercept of 0.280 [95% confidence interval (CI): 0.268-0.292]. The mean of the method differences was 0.19 mg/L and the limits of agreement (LOA), which encompass 95% of results, were -0.36-0.74 mg/L. We found the percentages of low, average, and high-risk results were 42.9, 33.8, and 23.3 for the N high-sensitivity CRP and 33.2, 41.1, and 25.7 for the Tina-quant method. The percentage of samples concordant by both methods was 87.4%. The Tina-quant CRP classified more subjects into the high-risk group. CONCLUSIONS: The two hs-CRP methods were highly associated and are suitable for screening large populations.

Comparison of two homogeneous HDL cholesterol methods in a large population study.

OBJECTIVES: High-density lipoprotein cholesterol (HDL-C) is an independent risk factor for coronary heart disease. Data are lacking on the performance of homogeneous methods using a large number of samples. DESIGN AND METHODS: We compared the performance of two HDL-C direct assays, the Dimension RxL (the Dade method) and the COBAS INTEGRA (the Roche method), for population screening. Performance was assessed using 4214 sera obtained from the International Collaborative Study on Atherosclerosis and Stroke In Asia (InterASIA) participants. RESULTS: The method comparison results demonstrated that both methods were highly correlated (r = 0.972). Deming regression analysis showed a slope of 1.009 +/- 0.007, an intercept of 0.048 +/- 0.009 and a S(y/x) of 0.08. The means were 1.29 +/- 0.33 and 1.23 +/- 0.33 mmol/l for the Roche and Dade methods, respectively, and the range of observed values were 0.30-3.05 and 0.19-2.86 mmol/l, respectively. The 95% confidence interval for the mean of the method differences was -0.10 to 0.22 mmol/l. Percentages of low (> or = 1.55 mmol/l), normal (1.03-1.54 mmol/l), and high risk (< 1.03 mmol/l) results were 15.5, 55.4, 29.0 for the Dade and 19.3, 59.3, 21.4 for the Roche method. The percentage of concordantly classified subjects at each cut point was 77.1%, 84.4%, and 95.5%. The percentage of overall consistency subjects was 85.4%. Thirteen percent of subjects were discordantly classified into the higher-risk group while the 1.6% of subjects were discordantly classified into the lower-risk group. CONCLUSIONS: Both homogeneous HDL-C methods were correlated and agree well with one another. The percentage of concordantly classified subjects was high. Thus, either method is suitable for large population studies.

Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method.

BACKGROUND: Cholesterol oxidase is used for the determination of serum cholesterol. It can be derived from Streptomyces, Pseudomonas fluorescens, Cellulomonas, and Brevibacterium. This study compared the performance characteristics of four enzymes in the endpoint cholesterol determination. METHODS: Using the Mega analyzer, we studied assay optimization, linearity, precision, recovery, interference, stability, and compared 110 patient samples. RESULTS: The linearity for the four enzymes was up to 13.0 mmol/l at the optimal enzyme activity. The average within-run CVs ranged from 1.6% to 1.9% and between-day ranged from 2.8% to 3.0%, within the NCEP analytical criteria. The analytical recoveries obtained from four reagents ( approximately 96.5%) were excellent. The assays using these enzyme sources compared favorably with the commercial method and appeared accurate near the clinical decision cut-points. Hemoglobin concentration at 1.9 g/l interfered with the P. fluorescens cholesterol oxidase. Bilirubin caused a negative interference while lipemia generated a positive interference with all enzyme sources. Reagents were stable up to 6 weeks. CONCLUSIONS: Streptomyces, Cellulomonas, and Brevibacterium were essentially analytically equivalent. Streptomyces and Cellulomonas cholesterol oxidase are one-quarter as expensive Brevibacterium. Cellulomonas is a new source of cholesterol oxidase for determining serum cholesterol by the endpoint method.