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Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method
Clinica Chimica Acta 339 (2004) 135 – 145 www.elsevier.com/locate/clinchim
Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method Porntip H. Lolekha a,*, Pornpen Srisawasdi a, Patcharee Jearanaikoon b, Nuanchawee Wetprasit c, Busarawan Sriwanthana d, Martin H Kroll e a
Division of Clinical Chemistry, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b Department of Clinical Chemistry, Faculty of Associated Medical Science, Khonkaen University, Khonkaen 40002, Thailand c Department of Biotechnology, Faculty of Science, Ramkhamheng University, Bangkok 10240, Thailand d National Institute of Health, Department of Medical Sciences, Nonthaburi 11000, Thailand e Department of Pathology and Laboratory Medicine, Dallas Veterans Affairs Medical Center and University of Texas Southwestern Medical School, Dallas, TX 75216, USA Received 7 August 2003; received in revised form 3 October 2003; accepted 3 October 2003
Abstract 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 ( f 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. D 2003 Elsevier B.V. All rights reserved. Keywords: Serum cholesterol; Enzymatic endpoint method; Streptomyces; Pseudomonas fluorescens; Cellulomonas; Brevibacterium
1. Introduction
* Corresponding author. Tel.: +66-662-201-1336; fax: +66-662246-8271. E-mail address: [email protected] (P.H. Lolekha). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.10.005
Determination of serum cholesterol by the enzymatic endpoint method involves the use of three enzymes, and is the most popular mean of determining cholesterol. The enzymatic method uses cholesterol
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esterase (EC 3.1.1.13) to hydrolyze cholesterol esters to free cholesterol. Cholesterol oxidase (EC 1.1.3.6) oxidizes the free cholesterol to cholest-4-en-3-one and hydrogen peroxide. Peroxidase (EC 1.11.1.7) catalyzes the oxidative coupling of hydrogen peroxide with 4-aminophenazone/phenol to form a quinoneimine dye, whose absorbance can measure at 500 nm. The method is simple, specific, and uses no corrosive reagents [1]. Different investigators have reported on the use of cholesterol oxidase isolated from Nocardia, Streptomyces, Mycobacteria, Brevibacterium, Schizophyllum, or Pseudomonas for the method [2– 6]. In the past, most commercial enzymatic cholesterol reagents have used cholesterol oxidase isolated from Nocardia. The Clinical Chemistry Standardization Section of Centers for Disease Control uses the enzyme isolated from Streptomyces for cholesterol determination [7]. Lolekha and Jantaveesirirat [8] suggested that cholesterol oxidase isolated from Streptomyces is superior to that obtained from Nocardia and Pseudomonas fluorescens, because of its lower cost and longer stability. Recently, cholesterol oxidases isolated from Brevibacterium and Cellulomonas have become available commercially; however, the performance of Brevibacterium and Cellulomonas enzyme in the endpoint method have not been reported. This study compared performances of these four cholesterol oxidases for the determination of serum cholesterol by the enzymatic endpoint method. Also, by evaluating the characteristics of the reagents for each enzyme, we could determine which sources are best for serum cholesterol determination.
2. Materials and methods 2.1. Equipment We used the Mega chemical analyzer (E. Merck, Darmstadt, Germany) for the assessment of all cholesterol determinations. Mega uses dual wavelengths for the photometric reading. 2.2. Reagents We used the Mega cholesterol reagent kit (Merck No. 1.07114 E) [9] as the comparative method and reference for the comparison studies (commercial
method). The source of cholesterol oxidase used in this reagent is unspecified. Reagent solution R1 and start reagent R2 are prepared, ready-for-use, by the manufacturer. The commercial enzymatic reagent was composed of 4-aminoantipyrine and 2-hydroxybenzyl alcohol for the conversion of hydrogen peroxide. For the experimental reagents, enzymes and chemicals were from Sigma (St. Louis, MO), except the 4aminophenazone, which was from BDH (Dorset, UK). We prepared the stock reagent, absent cholesterol oxidase, by dissolving cholesterol esterase (bovine pancreas, 200 U/l), peroxidase (10000 U/l), sodium cholate (3 mmol/l), 4-aminophenazone (0.5 mmol/l), phenol (20 mmol/l), and Triton X-100 (2 ml/ l) in phosphate buffer (0.1 mol/l, pH 7.0). To prepare the working reagents for optimizing study, we added 0, 100, 200, 300, 400, and 500 U/ l into the stock reagent for each cholesterol oxidase. For Brevibacterium cholesterol oxidase, we increased the enzyme activities to 1000, 1500, and 2000 U/l. We prepared the stock standard of cholesterol (25.9 mmol/l), by dissolving 250 mg of cholesterol (Sigma grade 99%), in 25 ml of an ethanol diluent (absolute ethanol with Triton X-100, 20% v/v). We prepared the cholesterol working standards from the stock and they ranged from 2.6 to 25.9 mmol/l. 2.3. Samples We used the serum-based calibrator (SMT, Product No. 1.19720 E. Merck, Germany), with a cholesterol concentration of 6.18 mmol/l, to calibrate all of the methods. To assess the acceptability of each run, we used Qualitrol HSN and Qualitrol HSP (Merck, Germany) and Dade Moni-Trol Level 1 and 2 Chemistry Control (Dade International, Miami, FL). 2.4. Procedure We operated the Mega analyzer according to the manufacturer’s specifications. The reagent R1 (60 Al) and distilled water (200 Al) were pipetted into the reaction cuvette, mixed and then the sample (2 Al) was added. After incubating the mixture for 298 s, the reagent R2 (10 Al) and distilled water (25 Al) were added. The absorbances at wavelengths 500 and 604
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nm were measured 248– 284 s after the addition of reagent R2. We performed the experimental cholesterol userdefined methods on the Mega analyzer. The userdefined parameters [10] were set as follows: Reaction Mode (End), Test/Blank W.L (500/604 nm), Sample volume (3 Al), Reagent volume (300 Al). The absorbance was read 540 –576 s after the sample addition. 2.4.1. Optimization of cholesterol oxidase Optimizing each cholesterol oxidase was performed by using cholesterol standard solutions ranging from 2.6 to 25.9 mmol/l. We determined
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cholesterol in working standards using the four sets of reagents. The absorbances versus cholesterol concentration obtained from each reagent, with increasing enzyme activities, were plotted. We selected the minimal enzyme activity producing the maximal cholesterol linearity. 2.4.2. Linearity and reportable range To assess the linearity of each method, we used a set of sera with cholesterol concentrations ranging from 2.6 to 25.9 mmol/l, plotting the absorbances versus cholesterol concentration. Linearity was evaluated using the NCCLS EP6-A guideline and the linear region reported as the reportable range [11].
Fig. 1. Optimization curves of enzymes obtained from cholesterol reagents containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium cholesterol oxidase (D). These curves were obtained with using cholesterol dissolved in ethanol. The detail was described as in the text. The enzyme activity enclosed within the rectangle indicates optimal activity.
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2.4.3. Imprecision We selected sera with low, middle and high cholesterol values. The within-run (20 replicates in the same run) and between-day (20 consecutive days) imprecisions were determined in each serum sample. The means, standard deviations, and coefficients of variation (CVs) were calculated. 2.4.4. Recovery We performed the recovery study by mixing sera containing low (L), middle (M), and high (H) cholesterol in the ratio of 1:1 (L + M, L + H, M + H). We calculated the recovery of cholesterol from the test
reagents compared with the commercial’s reagent in percentage. 2.4.5. Comparison We compared cholesterol in 110 hospitalized sera obtained from the test reagents with the Mega’s reagent [9]. Statistical values of the results between the methods were calculated. 2.4.6. Interference study We prepared the sets of hemolyzed (hemoglobin ranging from 0 to 15.0 g/l), icteric (bilirubin ranging from 0 to 1368 Amol/l) and turbid samples (absor-
Fig. 2. Linearity of serum cholesterol obtained from cholesterol reagents containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium cholesterol oxidase (D). Nonlinearity is shown by deviation from the dotted line.
P.H. Lolekha et al. / Clinica Chimica Acta 339 (2004) 135–145 Table 1 The within-run and between-day imprecision of total serum cholesterol obtained from the cholesterol reagent containing Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium cholesterol oxidase Imprecision
E2
c
E3
E4
3.0 1.4 0.8 1.7
3.3 1.2 0.4 1.6
3.6 1.3 0.9 1.9
3.5 1.1 0.9 1.8
Between-day run (n = 20) Low 3.4 Middle 2.7 High 3.0 Average 3.0
3.3 3.0 2.1 2.8
3.1 2.2 3.0 2.8
3.1 2.9 2.3 2.8
Within-run (n = 20) Low Middle High Average
3. Results 3.1. Optimizing of cholesterol oxidase Fig. 1 shows the optimal activities of cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium. The reaction patterns of Streptomyces (Fig. 1A), P. fluorescens (Fig. 1B), and Cellulomonas cholesterol oxidase (Fig. 1C) were hyperbolic curves. Increasing enzyme activities from 100 to 500 U/l slightly increased the cholesterol reactions. The maximal cholesterol linearity in the reagent using Streptomyces, P. fluorescens, or Cellulomonas isolated enzyme were 15.5, 10.4, and 15.5 mmol/l at the enzyme activities of 200, 300, and 300 U/l, respectively. For Brevibacterium cholesterol oxidase (Fig. 1D), sigmoid curves were obtained. Increasing the enzyme activities from 100 to 500 U/l increased the reaction absorbances. Hyperbolic curves, similar to Streptomyces, P. fluorescens, and Cellulomonas enzymes, were seen when Brevibacterium activity was increased up to 1000– 2000 U/l. The reagents containing cholesterol oxidase from Brevibacterium at 1000, 1500, and 2000 U/l were linear up to 7.8, 18.1, and 18.1 mmol/l, respectively.
Coefficient of variation (CV, %) a
E1
b
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d
a
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. b
bance at 670 nm of 0 – 2.334). A 1:2 dilution of each interference sample with pooled serum of low, middle and high cholesterol levels was made to obtain three sets of various degrees of interfering substances. Cholesterol in each sample’s set was determined by using the cholesterol reagents.
3.2. Analytical performances of the four cholesterol oxidases in sera
2.4.7. Stability Stability of each cholesterol reagent was investigated by analyzing the middle (6.0 mmol/l) and high (9.1 mmol/l) cholesterol samples for 3 months. The reagents were stored in a refrigerator (2 – 8 jC).
Fig. 2 illustrates the linearity of serum cholesterol obtained from the reagents containing the optimal activities of each cholesterol oxidase. The reportable
Table 2 The recovery of total serum cholesterol from the reagent containing Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium cholesterol oxidase Level L + M (1 + 1)e L + H (1 + 1) M + H (1 + 1) Average a
Expected value (mmol/l)
Observed value (mmol/l) a
b
Recovery (%) c
d
Mega method
E1
E2
E3
E4
E1
E2
E3
E4
3.85 9.10 10.05
3.70 8.80 9.70
3.73 8.77 9.73
3.67 8.80 9.73
3.70 8.80 9.73
96.1 96.7 96.5 96.4
96.9 96.4 96.8 96.7
95.3 96.7 96.8 96.3
96.1 96.7 96.8 96.5
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. e Cholesterol concentration: low [L] = 2.9 mmol/l; middle [M] = 4.8 mmol/l; high [H] = 15.3 mmol/l. b
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range for the cholesterol reagents containing Streptomyces (2.6 –18.1 mmol/l), Pseudomonas (2.6 –13.0 mmol/l), Cellulomonas (2.6 –18.1 mmol/l), and Brevibacterium (2.6 – 20.7 mmol/l) enzyme was proven by the polynomial method (NCCLS EP6-A) [11]. Table 1 tabulates imprecision obtained from the reagents containing each cholesterol oxidase. The average CVs for within-run imprecisions were 1.7%, 1.6%, 1.9%, and 1.8% and between-day imprecisions were 3.0%, 2.8%, 2.8%, and 2.8% for cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium, respectively. Table 2 presents the analytical recovery of serum cholesterol obtained from Streptomyces, P. fluores-
cens, Cellulomonas, and Brevibacterium reagents. An average recovery was 96.4%, 96.7%, 96.3%, and 96.5%, respectively. Fig. 3 illustrates the correlation of serum cholesterol levels between our experimental reagents and the commercial reagent obtained from the Mega analyzer [9]. The regression lines by least-squares regression analysis produced slopes ranging from 1.03 to 1.05 and negative intercepts ranging from 0.01 to 0.16 mmol/l. The correlation coefficients were greater than 0.99 and the standard deviation of the residuals (Sy/x) ranged from 0.12 to 0.143 mmol/l. The biases (test enzyme mean minus Mega mean) were small, ranging from 0.10 to 0.14 mmol/l.
Fig. 3. Correlation of total serum cholesterol obtained between the cholesterol reagents containing cholesterol oxidase from Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium (D) and the Mega’s reagent. Regression analysis results are shown.
P.H. Lolekha et al. / Clinica Chimica Acta 339 (2004) 135–145 Table 3 Error analysis of 4 cholesterol assays as calculated at clinical decision cut-points (5.2 and 6.2 mmol/l) Cholesterol (mmol/l)
Analytical error (%) E1a
E2b
E3c
E4d
5.2
5.307 (2.06%)* 6.358 (2.54%)
5.327 (2.44%) 6.352 (2.46%)
5.288 (1.69%) 6.322 (1.96%)
5.283 (1.60%) 6.321 (1.96%)
6.2 a
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. * The percentage errors are shown in parentheses. They were calculated from (test value critical value) divided with critical value and multiplied by 100. b
We estimated the systematic error of each cholesterol assays at the clinical decision cut-point of serum cholesterol (5.2 and 6.2 mmol/l) from the regression equations as shown in Table 3. The systematic errors of all cholesterol methods compared with the Mega
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ranged from 1.60% to 2.54%, and were within the allowable bias recommended by the National Cholesterol Education Program (NCEP) guidelines for cholesterol, < 3% at decision levels [12]. The mean and standard deviations of the cholesterol values calculated from each linear regression at the cut-point values of 5.2 and 6.2 mmol/l were 5.301, 0.02 and 6.338, 0.02 mmol/l, respectively. The deviation from mean errors ranged from 0.018 to 0.026 and 0.017 to 0.020 mmol/l, respectively. Table 4 shows the effects from hemoglobin, turbidity, and bilirubin on cholesterol determination for the four enzymes. The interfering substances produced significant effects on cholesterol concentrations when the results were 10% higher or lower than the original cholesterol concentration [13]. Hemoglobin concentrations up to 7.5 g/l gave no interfering effect on serum cholesterol obtained from Streptomyces, Cellulomonas, or Brevibacterium reagents. However, its concentration at 1.9 g/l caused a positive bias on cholesterol results for the reagent
Table 4 Effect of hemoglobin, turbidity and bilirubin on serum cholesterol determined by the cholesterol reagent containing Streptomyces, Pseudomonas, Cellulomonas, and Brevibacterium cholesterol oxidase Degree
Hemoglobin Cholesterol (mmol/l) (g/l) E1a E2b E3c E4d
Turbidity Cholesterol (mmol/l) (Abs at 670 nm) E1 E2 E3 E4
Bilirubin Cholesterol (mmol/l) (Amol/l) E1 E2 E3 E4
Baseline F 1+ 2+ 3+ 4+ Baseline F 1+ 2+ 3+ 4+ Baseline F 1+ 2+ 3+ 4+
0 0.1 0.9 1.9 3.8 7.5 0 0.1 0.9 1.9 3.8 7.5 0 0.1 0.9 1.9 3.8 7.5
0 0.195 0.389 0.778 0.973 1.167 0 0.195 0.389 0.778 0.973 1.167 0 0.195 0.389 0.778 0.973 1.167
0 85.5 171.0 342.0 513.0 684.0 0 85.5 171.0 342.0 513.0 684.0 0 85.5 171.0 342.0 513.0 684.0
a
1.71 1.69 1.73 1.79 1.82 1.94 4.64 4.63 4.66 4.65 4.74 4.76 7.03 7.00 6.91 6.92 7.03 7.04
1.90 1.82 1.95 2.15e 2.39 2.59 4.91 4.89 5.04 5.35e 5.78 6.16 7.13 7.02 7.30 7.79e 8.48 8.90
1.81 1.82 1.88 1.90 1.94 2.06 4.73 4.76 4.85 4.77 4.87 4.86 7.11 7.05 7.13 7.09 7.08 7.07
1.82 1.82 1.79 1.84 1.84 2.03 4.72 4.79 4.78 4.78 4.73 4.81 7.19 6.84 7.19 7.05 7.07 7.09
1.71 1.87 2.04e 2.33 2.45 2.68 4.64 4.86 4.98e 5.37 5.54 5.79 7.03 7.29 7.37e 7.63 7.67 8.05
1.90 2.02 2.18 2.46e 2.64 2.80 4.91 4.98 5.18 5.44e 5.66 5.90 7.13 7.49 7.59 7.92e 7.80 8.24
1.81 2.03 2.17e 2.49 2.63 2.85 4.73 5.02 5.16e 5.48 5.74 5.85 7.11 7.32 7.37e 7.78 7.88 8.05
1.82 1.97 2.08e 2.41 2.54 2.74 4.72 5.06 5.12e 5.38 5.68 5.87 7.19 7.28 7.60e 7.85 7.78 8.21
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. e Starting point of positive interference (>10% increasing from baseline cholesterol concentration) [13]. f Starting point of negative interference (>10% decreasing from baseline cholesterol concentration). b
1.66 1.54 1.35f 1.14 1.00 0.98 4.67 4.41 4.14f 3.68 3.22 2.76 6.87 6.74 6.32f 5.96 5.22 4.97
1.82 1.63 1.48f 1.20 1.06 1.00 4.89 4.52 4.27f 3.84 3.30 3.00 6.86 6.88 6.52f 6.08 5.51 5.17
1.84 1.65 1.46f 1.23 1.08 1.04 4.75 4.39 4.16f 3.76 3.26 2.89 7.05 6.83 6.50f 5.88 5.29 4.98
1.73 1.56 1.42f 1.18 1.05 1.03 4.74 4.49 4.14f 3.77 3.35 2.82 6.96 6.74 6.44f 5.83 5.35 5.10
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containing P. fluorescens cholesterol oxidase. For turbidity of 1 plus (absorbance >0.389 at 670 nm), significant positive biases on cholesterol results were observed in all reagents, except for the reagent with P. fluorescens, where the interference occurred at the turbidity of 2+ (absorbance >0.778 at 670
nm). Unconjugated bilirubin at 171.0 Amol/l produced a negative interference on all cholesterol levels. Fig. 4 demonstrates the stability of the 4 cholesterol reagents. All cholesterol reagents were stable up 6 weeks when kept at 2– 8 jC.
Fig. 4. Stability of cholesterol reagent, containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium (D) cholesterol oxidase, kept at 2 – 8 jC. All reagents were stable up to 6 weeks.
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4. Discussion An accurate and precise cholesterol measurement is needed for the uniform interpretation of cholesterol levels to assess a individual’s risk for coronary heart disease and to monitor treatment [14]. Recommended cholesterol cut-points are effective only when the laboratory measurements are reliable. In addition to the measure of total cholesterol, cholesterol oxidase is used in the measurement of HDL- and LDL-cholesterol by both direct and indirect methodologies. Lolekha and Jantaveesirirat [8] suggested Streptomyces cholesterol oxidase as a superior source of enzyme compared with the enzymes obtained from Nocardia and P. fluorescens because of lower cost and longer stability. Cellulomonas and Brevibacterium represent new sources for cholesterol oxidase, but little is known about their performance characteristics. The maximal cholesterol linearity was observed for reagents containing minimum cholesterol oxidase enzyme activities at 200, 300, 300, and 1500 U/l for Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium, respectively. The optimal activities of Streptomyces and P. fluorescens enzymes chosen are similar to those of our previous study [4]. The 4 cholesterol reagents possessed sufficient activities to provide linear responses from 13.0 to 20.7 mmol/l. The reportable ranges obtained from Streptomyces and P. fluorescens cholesterol oxidase are similar to those of our previous study [8]. The average analytical recoveries of serum cholesterol obtained from 4 cholesterol reagents were excellent. The results of linear regression showed good association with the reference commercial method. Biases between the methods were small. Differences between the Mega method and the experimental methods (percentage error of 1.60 – 2.54%) might reflect a small bias in the Mega method. In addition, the errors of 0.83% and 0.59% obtained from the comparison data among methods using each cholesterol oxidase represent the worst-case scenarios of error at cutpoints of 5.2 and 6.2 mmol/l, respectively, and hence, these errors were not analytically significant. One requires the best accuracy at the cholesterol cut-point, because it is around these values where most clinical decisions will be made. Accuracy errors at the cutpoints determine the number and percentage of clinical misdiagnoses. Because the analytical errors
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around 5.2 – 6.2 mmol/l are so small ( < 1%), these errors can be larger at the low or the high end of the scale without causing misdiagnoses. A comparison of results among the experimental methods is more meaningful. All the methods agree and appear accurate compared with the mean. These results imply that the accuracy of cholesterol determination does not depend on the source of enzyme being used. The average CVs of the within-run and between-day imprecision are in the analytical criteria (%CV < 3.0) recommended by the US National Cholesterol Education Program [12]. A common interference may create analytical bias in clinical laboratory analysis. Bilirubin and turbidity caused the greatest problems with all experimental cholesterol determinations while hemoglobin presented a difficulty only for the P. fluorescens source of cholesterol oxidase. Hemoglobin affects the spectrometric measurement either by increasing the absorption around 500 nm or by the modification of the blank value [15]. Using a secondary wavelength enhances the interference of hemoglobin for the P. fluorescens reagent (Table 4). This interference may due to the alterable absorbance of hemoglobin by this enzymatic reaction, at the measuring wavelength rather than that of the absorbance used for the blank. Other sources are not affected by hemoglobin. Lipemia has a direct effect on nearly all photometry analysis involving light scattering or absorption [13,15]. The turbidity (absorbance >0.389 at 670 nm) generated an interference with all cholesterol oxidase sources. Even though the Mega method uses the bichromatic wavelength blanking procedure, the analytical results were still increased. The absorbance by lipid particle is greater at 500 nm than at 600 nm [16]. The effect of turbidity was not seen in the previous study [8]. The different results of interference between our study and the previous study may be due to the principle of photometry measurement. The Mega analyzer (used in our study) measures the absorbance by a horizontal light path using dual wavelengths (500/604 nm) while the Labsystem analyzer (used in previous study) employed a vertical light path and the single wavelength (500 nm). Markedly lipemic sera may produce a concave surface in the cuvette of the reaction mixture altering the absorbance. Such an effect results decreased absorbance less than for a given amount of absorbance by the measurement with
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a vertical light path [17]. The compensation of absorbance obtained between the lipid particle and the concave surface may reduce the effect of turbidity. Mechanisms of bilirubin interference on the enzymatic cholesterol assay are spectral and chemical [13]. Spectral interference gives false positive results related to the strong absorbance of bilirubin between 400 and 540 nm. For the chemical interference, bilirubin competes with the peroxidase for H2O2 in the color reaction system. This competition produces a false negative result proportional to its concentration. Our results show a negative bias for all reagents. Thus, chemical interference prevailed over the optical interference for the endpoint cholesterol assays. The stability of all cholesterol reagents is the same for all 4 enzyme sources with stability up to at least 6 weeks. We found the shelf life of P. fluorescens reagent similar to that of the prior study while the shelf life of Streptomyces reagent was shorter than in the previous study (11 weeks) [8]. The activity of Streptomyces cholesterol oxidase used in our study (200 U/l) is lower than the previous study (250 U/l), implying that the reagent’s stability may depend on the concentration of enzyme being used. To calculate the cost of each cholesterol oxidase, we used the minimal enzyme activity that gave the optimal cholesterol linearity as shown in Fig. 1. We selected activities of cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium at 200, 300, 300, and 1500 U/l, respectively. Although Streptomyces cholesterol oxidase was least expensive (US$ 0.16/ml) compared with Pseudomonas (US$ 0.23/ml), Cellulomonas (US $ 0.19/ml), and Brevibacterium (US $ 0.72/ml), it requires shipment on ice ( 20 jC). Because frozen transport is difficult to guarantee in all countries, Streptomyces cholesterol oxidase may not appropriate for some geographic reasons. For this reason, cholesterol oxidase from Cellulomonas may represent the best source of enzyme for use in cholesterol determination by the endpoint method.
5. Conclusion One can use Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium as the sources of cholesterol oxidase for serum cholesterol determined by the
enzymatic endpoint method. All the methods agree and appear accurate near the clinical decision cutpoints. However, we did find disadvantages. The P. fluorescense source of cholesterol oxidase is susceptible to hemoglobin interference. Even though the performance of Brevibacterium cholesterol oxidase is excellent, its requirement for higher activity made its more costly (>4-fold) compared with the other enzymes. The cost and performances of both Streptomyces and Cellulomonas cholesterol oxidase were quite similar. We suggest that both Streptomyces and Cellulomonas are good sources of cholesterol oxidase for the determination of serum cholesterol by the enzymatic endpoint method. The results found here may also apply to the measurement or development of new methods for HDL- and LDL-cholesterol.
Acknowledgements This study was supported in part by the Thai Research Fund Royal Golden Jubilee PhD program.
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P.H. Lolekha et al. / Clinica Chimica Acta 339 (2004) 135–145 [9] Cholesterol, Merck Mega. Cholesterol reagent product No. 1.07114 E. Merck, 64271 Darmstadt, Germany. [10] Instruction Manual, Merck MEGA, 4th ed. Part No. 9.71007.9221 Merck KgaA D-64271 Darmstadt, Germany. [11] National Committee for Clinical Laboratory Standards. Evaluation of the linearity of quantitative measurement procedures: a statistical approach. Approved Guideline. Document EP6-A23. Wayne, PA: NCCLS; 2003. [12] National Institutes of Health. Recommendations for improving cholesterol measurement: a report from the Laboratory Standardization Panel of the National Cholesterol Education Program. In: NIH Publication, vol. 90-2964. Bethesda, MD: National Institutes of Health; 1990. [13] Kroll MH, Elin RS. Interference with clinical laboratory analyses. Clin Chem 1994;40:1995 – 2005.
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[14] National Institutes of Health. Third report of the Expert Panel on detection, evaluation, and treatment of high blood cholestrerol in adults. In: NIH Publication, vol. 01-3670. Bethesda, MD: National Institutes of Health; 2001. [15] Guder WG, Fonseca-Wollheim FD, Wuppertal WH, Schmitt YM, Gorlitz GT, Wisser H, Zawta B. The haemolytic, Icteric and lipemic sample recommendation regarding their recognition and prevention of clinically relevant interferences. J Lab Med 2000;24:357 – 64. [16] Pesce MA, Bodourian SH. Interfernce with the enzyme measurement of cholesterol in serum by use of five reagent kits. Clin Chem 1977;23:751 – 60. [17] Operation Manual, Labsystem Oy. Pulttitie 9-11, 00810 Helsinki 81, Finland.
Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method Porntip H. Lolekha a,*, Pornpen Srisawasdi a, Patcharee Jearanaikoon b, Nuanchawee Wetprasit c, Busarawan Sriwanthana d, Martin H Kroll e a
Division of Clinical Chemistry, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand b Department of Clinical Chemistry, Faculty of Associated Medical Science, Khonkaen University, Khonkaen 40002, Thailand c Department of Biotechnology, Faculty of Science, Ramkhamheng University, Bangkok 10240, Thailand d National Institute of Health, Department of Medical Sciences, Nonthaburi 11000, Thailand e Department of Pathology and Laboratory Medicine, Dallas Veterans Affairs Medical Center and University of Texas Southwestern Medical School, Dallas, TX 75216, USA Received 7 August 2003; received in revised form 3 October 2003; accepted 3 October 2003
Abstract 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 ( f 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. D 2003 Elsevier B.V. All rights reserved. Keywords: Serum cholesterol; Enzymatic endpoint method; Streptomyces; Pseudomonas fluorescens; Cellulomonas; Brevibacterium
1. Introduction
* Corresponding author. Tel.: +66-662-201-1336; fax: +66-662246-8271. E-mail address: [email protected] (P.H. Lolekha). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.10.005
Determination of serum cholesterol by the enzymatic endpoint method involves the use of three enzymes, and is the most popular mean of determining cholesterol. The enzymatic method uses cholesterol
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esterase (EC 3.1.1.13) to hydrolyze cholesterol esters to free cholesterol. Cholesterol oxidase (EC 1.1.3.6) oxidizes the free cholesterol to cholest-4-en-3-one and hydrogen peroxide. Peroxidase (EC 1.11.1.7) catalyzes the oxidative coupling of hydrogen peroxide with 4-aminophenazone/phenol to form a quinoneimine dye, whose absorbance can measure at 500 nm. The method is simple, specific, and uses no corrosive reagents [1]. Different investigators have reported on the use of cholesterol oxidase isolated from Nocardia, Streptomyces, Mycobacteria, Brevibacterium, Schizophyllum, or Pseudomonas for the method [2– 6]. In the past, most commercial enzymatic cholesterol reagents have used cholesterol oxidase isolated from Nocardia. The Clinical Chemistry Standardization Section of Centers for Disease Control uses the enzyme isolated from Streptomyces for cholesterol determination [7]. Lolekha and Jantaveesirirat [8] suggested that cholesterol oxidase isolated from Streptomyces is superior to that obtained from Nocardia and Pseudomonas fluorescens, because of its lower cost and longer stability. Recently, cholesterol oxidases isolated from Brevibacterium and Cellulomonas have become available commercially; however, the performance of Brevibacterium and Cellulomonas enzyme in the endpoint method have not been reported. This study compared performances of these four cholesterol oxidases for the determination of serum cholesterol by the enzymatic endpoint method. Also, by evaluating the characteristics of the reagents for each enzyme, we could determine which sources are best for serum cholesterol determination.
2. Materials and methods 2.1. Equipment We used the Mega chemical analyzer (E. Merck, Darmstadt, Germany) for the assessment of all cholesterol determinations. Mega uses dual wavelengths for the photometric reading. 2.2. Reagents We used the Mega cholesterol reagent kit (Merck No. 1.07114 E) [9] as the comparative method and reference for the comparison studies (commercial
method). The source of cholesterol oxidase used in this reagent is unspecified. Reagent solution R1 and start reagent R2 are prepared, ready-for-use, by the manufacturer. The commercial enzymatic reagent was composed of 4-aminoantipyrine and 2-hydroxybenzyl alcohol for the conversion of hydrogen peroxide. For the experimental reagents, enzymes and chemicals were from Sigma (St. Louis, MO), except the 4aminophenazone, which was from BDH (Dorset, UK). We prepared the stock reagent, absent cholesterol oxidase, by dissolving cholesterol esterase (bovine pancreas, 200 U/l), peroxidase (10000 U/l), sodium cholate (3 mmol/l), 4-aminophenazone (0.5 mmol/l), phenol (20 mmol/l), and Triton X-100 (2 ml/ l) in phosphate buffer (0.1 mol/l, pH 7.0). To prepare the working reagents for optimizing study, we added 0, 100, 200, 300, 400, and 500 U/ l into the stock reagent for each cholesterol oxidase. For Brevibacterium cholesterol oxidase, we increased the enzyme activities to 1000, 1500, and 2000 U/l. We prepared the stock standard of cholesterol (25.9 mmol/l), by dissolving 250 mg of cholesterol (Sigma grade 99%), in 25 ml of an ethanol diluent (absolute ethanol with Triton X-100, 20% v/v). We prepared the cholesterol working standards from the stock and they ranged from 2.6 to 25.9 mmol/l. 2.3. Samples We used the serum-based calibrator (SMT, Product No. 1.19720 E. Merck, Germany), with a cholesterol concentration of 6.18 mmol/l, to calibrate all of the methods. To assess the acceptability of each run, we used Qualitrol HSN and Qualitrol HSP (Merck, Germany) and Dade Moni-Trol Level 1 and 2 Chemistry Control (Dade International, Miami, FL). 2.4. Procedure We operated the Mega analyzer according to the manufacturer’s specifications. The reagent R1 (60 Al) and distilled water (200 Al) were pipetted into the reaction cuvette, mixed and then the sample (2 Al) was added. After incubating the mixture for 298 s, the reagent R2 (10 Al) and distilled water (25 Al) were added. The absorbances at wavelengths 500 and 604
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nm were measured 248– 284 s after the addition of reagent R2. We performed the experimental cholesterol userdefined methods on the Mega analyzer. The userdefined parameters [10] were set as follows: Reaction Mode (End), Test/Blank W.L (500/604 nm), Sample volume (3 Al), Reagent volume (300 Al). The absorbance was read 540 –576 s after the sample addition. 2.4.1. Optimization of cholesterol oxidase Optimizing each cholesterol oxidase was performed by using cholesterol standard solutions ranging from 2.6 to 25.9 mmol/l. We determined
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cholesterol in working standards using the four sets of reagents. The absorbances versus cholesterol concentration obtained from each reagent, with increasing enzyme activities, were plotted. We selected the minimal enzyme activity producing the maximal cholesterol linearity. 2.4.2. Linearity and reportable range To assess the linearity of each method, we used a set of sera with cholesterol concentrations ranging from 2.6 to 25.9 mmol/l, plotting the absorbances versus cholesterol concentration. Linearity was evaluated using the NCCLS EP6-A guideline and the linear region reported as the reportable range [11].
Fig. 1. Optimization curves of enzymes obtained from cholesterol reagents containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium cholesterol oxidase (D). These curves were obtained with using cholesterol dissolved in ethanol. The detail was described as in the text. The enzyme activity enclosed within the rectangle indicates optimal activity.
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2.4.3. Imprecision We selected sera with low, middle and high cholesterol values. The within-run (20 replicates in the same run) and between-day (20 consecutive days) imprecisions were determined in each serum sample. The means, standard deviations, and coefficients of variation (CVs) were calculated. 2.4.4. Recovery We performed the recovery study by mixing sera containing low (L), middle (M), and high (H) cholesterol in the ratio of 1:1 (L + M, L + H, M + H). We calculated the recovery of cholesterol from the test
reagents compared with the commercial’s reagent in percentage. 2.4.5. Comparison We compared cholesterol in 110 hospitalized sera obtained from the test reagents with the Mega’s reagent [9]. Statistical values of the results between the methods were calculated. 2.4.6. Interference study We prepared the sets of hemolyzed (hemoglobin ranging from 0 to 15.0 g/l), icteric (bilirubin ranging from 0 to 1368 Amol/l) and turbid samples (absor-
Fig. 2. Linearity of serum cholesterol obtained from cholesterol reagents containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium cholesterol oxidase (D). Nonlinearity is shown by deviation from the dotted line.
P.H. Lolekha et al. / Clinica Chimica Acta 339 (2004) 135–145 Table 1 The within-run and between-day imprecision of total serum cholesterol obtained from the cholesterol reagent containing Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium cholesterol oxidase Imprecision
E2
c
E3
E4
3.0 1.4 0.8 1.7
3.3 1.2 0.4 1.6
3.6 1.3 0.9 1.9
3.5 1.1 0.9 1.8
Between-day run (n = 20) Low 3.4 Middle 2.7 High 3.0 Average 3.0
3.3 3.0 2.1 2.8
3.1 2.2 3.0 2.8
3.1 2.9 2.3 2.8
Within-run (n = 20) Low Middle High Average
3. Results 3.1. Optimizing of cholesterol oxidase Fig. 1 shows the optimal activities of cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium. The reaction patterns of Streptomyces (Fig. 1A), P. fluorescens (Fig. 1B), and Cellulomonas cholesterol oxidase (Fig. 1C) were hyperbolic curves. Increasing enzyme activities from 100 to 500 U/l slightly increased the cholesterol reactions. The maximal cholesterol linearity in the reagent using Streptomyces, P. fluorescens, or Cellulomonas isolated enzyme were 15.5, 10.4, and 15.5 mmol/l at the enzyme activities of 200, 300, and 300 U/l, respectively. For Brevibacterium cholesterol oxidase (Fig. 1D), sigmoid curves were obtained. Increasing the enzyme activities from 100 to 500 U/l increased the reaction absorbances. Hyperbolic curves, similar to Streptomyces, P. fluorescens, and Cellulomonas enzymes, were seen when Brevibacterium activity was increased up to 1000– 2000 U/l. The reagents containing cholesterol oxidase from Brevibacterium at 1000, 1500, and 2000 U/l were linear up to 7.8, 18.1, and 18.1 mmol/l, respectively.
Coefficient of variation (CV, %) a
E1
b
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d
a
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. b
bance at 670 nm of 0 – 2.334). A 1:2 dilution of each interference sample with pooled serum of low, middle and high cholesterol levels was made to obtain three sets of various degrees of interfering substances. Cholesterol in each sample’s set was determined by using the cholesterol reagents.
3.2. Analytical performances of the four cholesterol oxidases in sera
2.4.7. Stability Stability of each cholesterol reagent was investigated by analyzing the middle (6.0 mmol/l) and high (9.1 mmol/l) cholesterol samples for 3 months. The reagents were stored in a refrigerator (2 – 8 jC).
Fig. 2 illustrates the linearity of serum cholesterol obtained from the reagents containing the optimal activities of each cholesterol oxidase. The reportable
Table 2 The recovery of total serum cholesterol from the reagent containing Streptomyces, P. fluorescens, Cellulomonas, or Brevibacterium cholesterol oxidase Level L + M (1 + 1)e L + H (1 + 1) M + H (1 + 1) Average a
Expected value (mmol/l)
Observed value (mmol/l) a
b
Recovery (%) c
d
Mega method
E1
E2
E3
E4
E1
E2
E3
E4
3.85 9.10 10.05
3.70 8.80 9.70
3.73 8.77 9.73
3.67 8.80 9.73
3.70 8.80 9.73
96.1 96.7 96.5 96.4
96.9 96.4 96.8 96.7
95.3 96.7 96.8 96.3
96.1 96.7 96.8 96.5
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. e Cholesterol concentration: low [L] = 2.9 mmol/l; middle [M] = 4.8 mmol/l; high [H] = 15.3 mmol/l. b
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range for the cholesterol reagents containing Streptomyces (2.6 –18.1 mmol/l), Pseudomonas (2.6 –13.0 mmol/l), Cellulomonas (2.6 –18.1 mmol/l), and Brevibacterium (2.6 – 20.7 mmol/l) enzyme was proven by the polynomial method (NCCLS EP6-A) [11]. Table 1 tabulates imprecision obtained from the reagents containing each cholesterol oxidase. The average CVs for within-run imprecisions were 1.7%, 1.6%, 1.9%, and 1.8% and between-day imprecisions were 3.0%, 2.8%, 2.8%, and 2.8% for cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium, respectively. Table 2 presents the analytical recovery of serum cholesterol obtained from Streptomyces, P. fluores-
cens, Cellulomonas, and Brevibacterium reagents. An average recovery was 96.4%, 96.7%, 96.3%, and 96.5%, respectively. Fig. 3 illustrates the correlation of serum cholesterol levels between our experimental reagents and the commercial reagent obtained from the Mega analyzer [9]. The regression lines by least-squares regression analysis produced slopes ranging from 1.03 to 1.05 and negative intercepts ranging from 0.01 to 0.16 mmol/l. The correlation coefficients were greater than 0.99 and the standard deviation of the residuals (Sy/x) ranged from 0.12 to 0.143 mmol/l. The biases (test enzyme mean minus Mega mean) were small, ranging from 0.10 to 0.14 mmol/l.
Fig. 3. Correlation of total serum cholesterol obtained between the cholesterol reagents containing cholesterol oxidase from Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium (D) and the Mega’s reagent. Regression analysis results are shown.
P.H. Lolekha et al. / Clinica Chimica Acta 339 (2004) 135–145 Table 3 Error analysis of 4 cholesterol assays as calculated at clinical decision cut-points (5.2 and 6.2 mmol/l) Cholesterol (mmol/l)
Analytical error (%) E1a
E2b
E3c
E4d
5.2
5.307 (2.06%)* 6.358 (2.54%)
5.327 (2.44%) 6.352 (2.46%)
5.288 (1.69%) 6.322 (1.96%)
5.283 (1.60%) 6.321 (1.96%)
6.2 a
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. * The percentage errors are shown in parentheses. They were calculated from (test value critical value) divided with critical value and multiplied by 100. b
We estimated the systematic error of each cholesterol assays at the clinical decision cut-point of serum cholesterol (5.2 and 6.2 mmol/l) from the regression equations as shown in Table 3. The systematic errors of all cholesterol methods compared with the Mega
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ranged from 1.60% to 2.54%, and were within the allowable bias recommended by the National Cholesterol Education Program (NCEP) guidelines for cholesterol, < 3% at decision levels [12]. The mean and standard deviations of the cholesterol values calculated from each linear regression at the cut-point values of 5.2 and 6.2 mmol/l were 5.301, 0.02 and 6.338, 0.02 mmol/l, respectively. The deviation from mean errors ranged from 0.018 to 0.026 and 0.017 to 0.020 mmol/l, respectively. Table 4 shows the effects from hemoglobin, turbidity, and bilirubin on cholesterol determination for the four enzymes. The interfering substances produced significant effects on cholesterol concentrations when the results were 10% higher or lower than the original cholesterol concentration [13]. Hemoglobin concentrations up to 7.5 g/l gave no interfering effect on serum cholesterol obtained from Streptomyces, Cellulomonas, or Brevibacterium reagents. However, its concentration at 1.9 g/l caused a positive bias on cholesterol results for the reagent
Table 4 Effect of hemoglobin, turbidity and bilirubin on serum cholesterol determined by the cholesterol reagent containing Streptomyces, Pseudomonas, Cellulomonas, and Brevibacterium cholesterol oxidase Degree
Hemoglobin Cholesterol (mmol/l) (g/l) E1a E2b E3c E4d
Turbidity Cholesterol (mmol/l) (Abs at 670 nm) E1 E2 E3 E4
Bilirubin Cholesterol (mmol/l) (Amol/l) E1 E2 E3 E4
Baseline F 1+ 2+ 3+ 4+ Baseline F 1+ 2+ 3+ 4+ Baseline F 1+ 2+ 3+ 4+
0 0.1 0.9 1.9 3.8 7.5 0 0.1 0.9 1.9 3.8 7.5 0 0.1 0.9 1.9 3.8 7.5
0 0.195 0.389 0.778 0.973 1.167 0 0.195 0.389 0.778 0.973 1.167 0 0.195 0.389 0.778 0.973 1.167
0 85.5 171.0 342.0 513.0 684.0 0 85.5 171.0 342.0 513.0 684.0 0 85.5 171.0 342.0 513.0 684.0
a
1.71 1.69 1.73 1.79 1.82 1.94 4.64 4.63 4.66 4.65 4.74 4.76 7.03 7.00 6.91 6.92 7.03 7.04
1.90 1.82 1.95 2.15e 2.39 2.59 4.91 4.89 5.04 5.35e 5.78 6.16 7.13 7.02 7.30 7.79e 8.48 8.90
1.81 1.82 1.88 1.90 1.94 2.06 4.73 4.76 4.85 4.77 4.87 4.86 7.11 7.05 7.13 7.09 7.08 7.07
1.82 1.82 1.79 1.84 1.84 2.03 4.72 4.79 4.78 4.78 4.73 4.81 7.19 6.84 7.19 7.05 7.07 7.09
1.71 1.87 2.04e 2.33 2.45 2.68 4.64 4.86 4.98e 5.37 5.54 5.79 7.03 7.29 7.37e 7.63 7.67 8.05
1.90 2.02 2.18 2.46e 2.64 2.80 4.91 4.98 5.18 5.44e 5.66 5.90 7.13 7.49 7.59 7.92e 7.80 8.24
1.81 2.03 2.17e 2.49 2.63 2.85 4.73 5.02 5.16e 5.48 5.74 5.85 7.11 7.32 7.37e 7.78 7.88 8.05
1.82 1.97 2.08e 2.41 2.54 2.74 4.72 5.06 5.12e 5.38 5.68 5.87 7.19 7.28 7.60e 7.85 7.78 8.21
E1: Streptomyces cholesterol oxidase. E2: P. fluorescens cholesterol oxidase. c E3: Cellulomonas cholesterol oxidase. d E4: Brevibacterium cholesterol oxidase. e Starting point of positive interference (>10% increasing from baseline cholesterol concentration) [13]. f Starting point of negative interference (>10% decreasing from baseline cholesterol concentration). b
1.66 1.54 1.35f 1.14 1.00 0.98 4.67 4.41 4.14f 3.68 3.22 2.76 6.87 6.74 6.32f 5.96 5.22 4.97
1.82 1.63 1.48f 1.20 1.06 1.00 4.89 4.52 4.27f 3.84 3.30 3.00 6.86 6.88 6.52f 6.08 5.51 5.17
1.84 1.65 1.46f 1.23 1.08 1.04 4.75 4.39 4.16f 3.76 3.26 2.89 7.05 6.83 6.50f 5.88 5.29 4.98
1.73 1.56 1.42f 1.18 1.05 1.03 4.74 4.49 4.14f 3.77 3.35 2.82 6.96 6.74 6.44f 5.83 5.35 5.10
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containing P. fluorescens cholesterol oxidase. For turbidity of 1 plus (absorbance >0.389 at 670 nm), significant positive biases on cholesterol results were observed in all reagents, except for the reagent with P. fluorescens, where the interference occurred at the turbidity of 2+ (absorbance >0.778 at 670
nm). Unconjugated bilirubin at 171.0 Amol/l produced a negative interference on all cholesterol levels. Fig. 4 demonstrates the stability of the 4 cholesterol reagents. All cholesterol reagents were stable up 6 weeks when kept at 2– 8 jC.
Fig. 4. Stability of cholesterol reagent, containing Streptomyces (A), P. fluorescens (B), Cellulomonas (C), or Brevibacterium (D) cholesterol oxidase, kept at 2 – 8 jC. All reagents were stable up to 6 weeks.
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4. Discussion An accurate and precise cholesterol measurement is needed for the uniform interpretation of cholesterol levels to assess a individual’s risk for coronary heart disease and to monitor treatment [14]. Recommended cholesterol cut-points are effective only when the laboratory measurements are reliable. In addition to the measure of total cholesterol, cholesterol oxidase is used in the measurement of HDL- and LDL-cholesterol by both direct and indirect methodologies. Lolekha and Jantaveesirirat [8] suggested Streptomyces cholesterol oxidase as a superior source of enzyme compared with the enzymes obtained from Nocardia and P. fluorescens because of lower cost and longer stability. Cellulomonas and Brevibacterium represent new sources for cholesterol oxidase, but little is known about their performance characteristics. The maximal cholesterol linearity was observed for reagents containing minimum cholesterol oxidase enzyme activities at 200, 300, 300, and 1500 U/l for Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium, respectively. The optimal activities of Streptomyces and P. fluorescens enzymes chosen are similar to those of our previous study [4]. The 4 cholesterol reagents possessed sufficient activities to provide linear responses from 13.0 to 20.7 mmol/l. The reportable ranges obtained from Streptomyces and P. fluorescens cholesterol oxidase are similar to those of our previous study [8]. The average analytical recoveries of serum cholesterol obtained from 4 cholesterol reagents were excellent. The results of linear regression showed good association with the reference commercial method. Biases between the methods were small. Differences between the Mega method and the experimental methods (percentage error of 1.60 – 2.54%) might reflect a small bias in the Mega method. In addition, the errors of 0.83% and 0.59% obtained from the comparison data among methods using each cholesterol oxidase represent the worst-case scenarios of error at cutpoints of 5.2 and 6.2 mmol/l, respectively, and hence, these errors were not analytically significant. One requires the best accuracy at the cholesterol cut-point, because it is around these values where most clinical decisions will be made. Accuracy errors at the cutpoints determine the number and percentage of clinical misdiagnoses. Because the analytical errors
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around 5.2 – 6.2 mmol/l are so small ( < 1%), these errors can be larger at the low or the high end of the scale without causing misdiagnoses. A comparison of results among the experimental methods is more meaningful. All the methods agree and appear accurate compared with the mean. These results imply that the accuracy of cholesterol determination does not depend on the source of enzyme being used. The average CVs of the within-run and between-day imprecision are in the analytical criteria (%CV < 3.0) recommended by the US National Cholesterol Education Program [12]. A common interference may create analytical bias in clinical laboratory analysis. Bilirubin and turbidity caused the greatest problems with all experimental cholesterol determinations while hemoglobin presented a difficulty only for the P. fluorescens source of cholesterol oxidase. Hemoglobin affects the spectrometric measurement either by increasing the absorption around 500 nm or by the modification of the blank value [15]. Using a secondary wavelength enhances the interference of hemoglobin for the P. fluorescens reagent (Table 4). This interference may due to the alterable absorbance of hemoglobin by this enzymatic reaction, at the measuring wavelength rather than that of the absorbance used for the blank. Other sources are not affected by hemoglobin. Lipemia has a direct effect on nearly all photometry analysis involving light scattering or absorption [13,15]. The turbidity (absorbance >0.389 at 670 nm) generated an interference with all cholesterol oxidase sources. Even though the Mega method uses the bichromatic wavelength blanking procedure, the analytical results were still increased. The absorbance by lipid particle is greater at 500 nm than at 600 nm [16]. The effect of turbidity was not seen in the previous study [8]. The different results of interference between our study and the previous study may be due to the principle of photometry measurement. The Mega analyzer (used in our study) measures the absorbance by a horizontal light path using dual wavelengths (500/604 nm) while the Labsystem analyzer (used in previous study) employed a vertical light path and the single wavelength (500 nm). Markedly lipemic sera may produce a concave surface in the cuvette of the reaction mixture altering the absorbance. Such an effect results decreased absorbance less than for a given amount of absorbance by the measurement with
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a vertical light path [17]. The compensation of absorbance obtained between the lipid particle and the concave surface may reduce the effect of turbidity. Mechanisms of bilirubin interference on the enzymatic cholesterol assay are spectral and chemical [13]. Spectral interference gives false positive results related to the strong absorbance of bilirubin between 400 and 540 nm. For the chemical interference, bilirubin competes with the peroxidase for H2O2 in the color reaction system. This competition produces a false negative result proportional to its concentration. Our results show a negative bias for all reagents. Thus, chemical interference prevailed over the optical interference for the endpoint cholesterol assays. The stability of all cholesterol reagents is the same for all 4 enzyme sources with stability up to at least 6 weeks. We found the shelf life of P. fluorescens reagent similar to that of the prior study while the shelf life of Streptomyces reagent was shorter than in the previous study (11 weeks) [8]. The activity of Streptomyces cholesterol oxidase used in our study (200 U/l) is lower than the previous study (250 U/l), implying that the reagent’s stability may depend on the concentration of enzyme being used. To calculate the cost of each cholesterol oxidase, we used the minimal enzyme activity that gave the optimal cholesterol linearity as shown in Fig. 1. We selected activities of cholesterol oxidase from Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium at 200, 300, 300, and 1500 U/l, respectively. Although Streptomyces cholesterol oxidase was least expensive (US$ 0.16/ml) compared with Pseudomonas (US$ 0.23/ml), Cellulomonas (US $ 0.19/ml), and Brevibacterium (US $ 0.72/ml), it requires shipment on ice ( 20 jC). Because frozen transport is difficult to guarantee in all countries, Streptomyces cholesterol oxidase may not appropriate for some geographic reasons. For this reason, cholesterol oxidase from Cellulomonas may represent the best source of enzyme for use in cholesterol determination by the endpoint method.
5. Conclusion One can use Streptomyces, P. fluorescens, Cellulomonas, and Brevibacterium as the sources of cholesterol oxidase for serum cholesterol determined by the
enzymatic endpoint method. All the methods agree and appear accurate near the clinical decision cutpoints. However, we did find disadvantages. The P. fluorescense source of cholesterol oxidase is susceptible to hemoglobin interference. Even though the performance of Brevibacterium cholesterol oxidase is excellent, its requirement for higher activity made its more costly (>4-fold) compared with the other enzymes. The cost and performances of both Streptomyces and Cellulomonas cholesterol oxidase were quite similar. We suggest that both Streptomyces and Cellulomonas are good sources of cholesterol oxidase for the determination of serum cholesterol by the enzymatic endpoint method. The results found here may also apply to the measurement or development of new methods for HDL- and LDL-cholesterol.
Acknowledgements This study was supported in part by the Thai Research Fund Royal Golden Jubilee PhD program.
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