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Clinical Chemistry 48: 737-741, 2002;
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(Clinical Chemistry. 2002;48:737-741.)
© 2002 American Association for Clinical Chemistry, Inc.

Highly Sensitive Cholesterol Assay with Enzymatic Cycling Applied to Measurement of Remnant Lipoprotein-Cholesterol in Serum

Koji Kishi1a, Koji Ochiai1, Yohsuke Ohta1, Yahiro Uemura1, Kazushi Kanatani2, Katsuyuki Nakajima2 and Masakazu Nakamura3

1 International Reagents Corporation, 1-1-2, Murotani, Nishi-ku, Kobe 651-2241, Japan.

2 Japan Immunoresearch Laboratories Co., Ltd., 351-1, Nishiyokete-cho, Takasaki 370-0021, Japan.

3 Osaka Medical Center for Cancer and Cardiovascular Disease, 1-3-3, Nakamichi, Nishinari-ku, Osaka 537-8511, Japan.

aAuthor for correspondence. Fax 81-78-992-1082; e-mail irckojikishi{at}irc-net.co.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Remnant lipoprotein-cholesterol (RLP-C) concentrations in sera of healthy individuals are very low (0.080–0.437 mmol/L), making conventional cholesterol methods poorly suited to this purpose. We have developed a highly sensitive cholesterol assay (CD method) and applied it to the RLP-C assay.

Methods: The CD shuttled cholesterol reversibly between reduced and oxidized forms in the presence of thio-NAD and NADH. The production rate of thio-NADH correlated with the cholesterol concentration and was measured by the absorbance at 404/500 nm. This CD method was combined with an immunoaffinity separation procedure with specific monoclonal antibodies to apolipoprotein (apo) A1 and apo B-100 and used for RLP-C assay. Results were compared with a RLP-C method that uses cholesterol oxidase, peroxidase, and chromogenic substrate.

Results: The CD method could detect 0.10 x 10-3 mmol/L cholesterol and was at least 5 times more sensitive than the conventional enzymatic method. Within- and between-day imprecision (as CVs) of the RLP-C assay with the CD method was <4%. Regression analysis of RLP-C assays with the new (y) and conventional (x) cholesterol methods yielded: y = 1.02x - 0.008 mmol/L (Sy|x = 0.0065 mmol/L; r = 0.997; n = 297).

Conclusions: Serum RLP-C can be measured by the CD method. The CD method may be useful for other assays that require sensitive cholesterol measurements in biological materials.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cholesterol oxidase-peroxidase methods (1)(2) are widely used to measure serum cholesterol, LDL-cholesterol, and HDL-cholesterol. These assays do not require quantification below 0.026 mmol/L (1 mg/dL). In contrast, assays for remnant lipoprotein-cholesterol (RLP-C)1 (3)(4)(5) require lower detection limits. In the RLP-C assay developed by Nakajima and coworkers (3)(4) and evaluated by Leary et al. (5), a detection limit <0.003 mmol/L (0.12 mg/dL) is needed for cholesterol because serum samples are diluted 61-fold with affinity gel solution for the separation of remnant lipoproteins. The conventional methods for cholesterol are not sufficiently sensitive; moreover, some are not linear at low cholesterol concentrations, thus requiring multiple calibrations.

We have developed an enzymatic cycling method (6), using cholesterol dehydrogenase (CD; EC no. not certified) (7), that can detect 0.10 x 10-3 mmol/L cholesterol with one-point calibration. Using the CD method, we determined RLP-C concentrations in sera from healthy controls.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reagents
We used thio-NAD, oxidized form (Oriental Yeast Co. Ltd.), NADH (Oriental Yeast), recombinant CD (8)(9)(10) (Amano Enzyme Inc.), and cholesterol ester hydrolase (CEH; stearyl-ester acylhydrolase; EC 3.1.1.13) derived from Pseudomonas species (11) (Asahi Chemical Industry Co., Ltd.). Synthetic conjugated bilirubin (Porphyrin Products Inc.), free bilirubin (Sigma), and hemoglobin prepared from hemolyzed human erythrocytes were used to test for interference. Other reagents were purchased from Wako Chemical Industries, Ltd.

procedures
Principle of the CD method.
CEH and CD were used in the CD method (Fig. 1 ). The cholesterol esters were hydrolyzed to free cholesterol and fatty acids by CEH. Free cholesterol changed to the oxidized form reversibly, and this cycling reaction was repeated several times in the presence of thio-NAD and NADH. Thio-NADH (reduced form) produced by the CD method was measured by the change in absorbance per minute at 404/500 nm as a measure of the cholesterol concentration.



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  		Figure 1. Reaction scheme for the enzymatic cycling method (CD method).

Reagents for the CD method and application to an automated analyzer.
The first reagent contained, per liter, 8000 U of CEH, 1500 U of CD, 0.8 mmol thio-NADH, 2.5 g of sodium cholate, 1 mL of Triton X-100, and 10 mmol HEPES buffer (12). The second reagent contained 2.4 mmol/L NADH in 0.3 mol/L diethanolamine buffer (pH 10).

The assay was performed on a TBA-20R automatic analyzer (Toshiba) 2optimized for a rate assay measuring absorbance at 404/500 nm for 5 min at 37 °C with 10 µL of sample, 270 µL of the first reagent, and 90 µL of the second reagent.

Procedure for RLP-C.
Lipoproteins other than RLP in serum were precipitated with an immunoaffinity separation procedure using monoclonal antibodies to apolipoprotein A1 and B-100 (3)(4); the cholesterol in the supernatant was measured by the CD method as RLP-C. The calibration solution (International Reagents Corp.) contained 0.789 mmol/L (30.5 mg/dL) cholesterol.

The conventional RLP-C assay using the peroxidase (PO; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) method (RLP-Cholesterol JIMRO II; Japan Immunoresearch Laboratory Co. Ltd.) was used as the comparison method (4)(5). Five calibration points were used to construct the calibration curve in the PO method.

samples
Fresh serum samples were randomly collected without anticoagulant as part of a mass examination at Kochi prefecture in cooperation with Osaka Medical Center for Cancer and Cardiovascular Diseases. This study was approved by the Committee for Ethical Standards of Osaka Medical Center for Cancer and Cardiovascular Disease. The 297 individuals studied in this research had no history of cardiovascular disease and were apparently healthy.

statistical analysis
Linear regression, reference intervals, and cutoff values were calculated by the least-squares method, the nonparametric method, and the 75th percentile of the population in this study group, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
optimization of cd method
The optimal pH for the CD method at 37 °C was assessed between pH 7.0 and 9.5 with 0.026 mmol/L cholesterol solution. The highest absorbance was between pH 8.5 and 9.5 (Fig. 2A ). This optimal pH was attained by mixing the first reagent (pH 6.5) and the second reagent (pH 10.0).



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  		Figure 2. Optimal conditions for the CD method.

(A), effect of pH on the CD method with 0.6 mmol/L thio-NAD, 0.6 mmol/L NADH, and 1.13 kU/L CD. (B), effect of thio-NAD concentration on the CD method with 0.6 mmol/L NADH and 1.13 kU/L CD at pH 8.5. (C), effect of NADH concentration on the CD method with 0.6 mmol/L thio-NAD and 1.13 kU/L CD at pH 8.5. (D), effect of CD activity on the CD method with 0.6 mmol/L thio-NAD and 0.6 mmol/L NADH at pH 8.5. (E), effect of CEH activity on the CD method with 0.6 mmol/L thio-NAD, 0.6 mmol/L NADH, and 1.13 kU/L CD at pH 8.5. For the experiment in E, we used three different human sera containing 3.54 mmol/L (•), 5.92 mmol/L ({blacksquare}), and 10.81 mmol/L ({circ}) total cholesterol, diluted 1:401 in saline.

The concentrations of thio-NAD and NADH in the CD method at 37 °C were assessed using a 0.026 mmol/L cholesterol solution. The reaction was increased by addition of the coenzymes (Fig. 2, B and CUp ). To achieve the necessary sensitivity and the absorbance range needed for measurement of cholesterol in RLP-C (0–2.587 mmol/L as RLP-C), we chose 0.6 mmol/L for both thio-NAD and NADH.

The optimal CD activity at 37 °C was assessed using a 0.026 mmol/L cholesterol solution. The sensitivity was increased in proportion to the amount of CD (Fig. 2DUp ). To achieve the necessary sensitivity and the absorbance range for the RLP-C assay, we used a CD activity of 1.13 kU/L.

The optimal CEH activity at 37 °C was assessed using three human sera with total cholesterol concentrations of 3.5, 5.9, and 10.8 mmol/L, respectively, which were diluted 1:401 (1 µL of serum in 400 µL of saline) in saline. The amount of CEH for the hydrolysis of cholesterol esters was fixed at 8 kU/L. This CEH activity hydrolyzed cholesterol esters of high-cholesterol sera (Fig. 2EUp ).

detection limit of cd method for cholesterol
The detection limit of the CD method, evaluated with a sequentially diluted cholesterol solution (1.29 mmol/L), was 0.10 x 10-3 mmol/L (mean + 3 SD of zero calibrator; n = 10).

performance of the new rlp-c assay
We measured 0, 0.098, 0.197, 0.336, and 0.789 mmol/L cholesterol solutions determined as RLP-C by the CD and conventional (PO) method (Fig. 3 ). The CD method had a linear calibration curve (n = 5) that passed through the origin, with fivefold higher absorbance (sensitivity) than that of the PO method. The PO method had a nonlinear calibration curve (n = 5) that did not pass through the origin, and the increase in absorbance was disproportional with the concentration of cholesterol.



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  		Figure 3. The linearity of two methods using five calibration points at low cholesterol concentrations.

Data points indicate change in absorbance per minute ({Delta}Absorbance) relative to cholesterol concentrations in calibrators by the CD method (•) and by the conventional method ({circ}). Each point represents the mean of five determinations. The linear regression of the CD method (y) and cholesterol concentrations (x) yielded: y = 0.0027x + 0.0004 (y = 0.993x + 0.0039 mmol/L). On the other hand, the linear regression of the conventional (PO) method (y) and cholesterol concentrations (x) yielded: y = 0.0005x + 0.0009 (y = 1.053x + 0.046 mmol/L).

The linearity of the RLP-C assay with the CD method was evaluated by sequential dilution of a high RLP-C control serum with saline. The curve was linear up to 2.587 mmol/L RLP-C in this method.

Within- and between-day precision was determined by measuring three control sera containing 0.098, 0.341, 1.058 mmol/L cholesterol. Between-day testing was carried out on 10 points over a 2-week period. The assay was calibrated each testing day. The RLP-C assay with the CD method showed good within- and between-day precision [CVs, 0.57–3.1% (n = 20) and 1.0–3.8%, respectively; Table 1 ]. On the other hand, the RLP-C assay with the conventional (PO) method had within- and between-day CVs of 1.8–6.0% and 2.0–7.5%, respectively.


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  		Table 1. Precision of RLP-C method.

The RLP-C assay with the CD method was not affected by hemoglobin (up to 5.0 g/L), bilirubin (up to 0.35 mmol/L), conjugated bilirubin (up to 0.28 mmol/L), or ascorbic acid (up to 2.84 mmol/L; data not shown).

serum rlp-c concentrations in apparently healthy people
The observed RLP-C concentrations in this study group (n = 297) with the CD method were 0.080–0.437 mmol/L, whereas the results obtained by the conventional (PO) RLP-C assay were 0.085–0.419 mmol/L. The 75th percentile was 0.217 mmol/L with the CD method and 0.220 mmol/L by the conventional (PO) assay.

The correlation by linear regression of RLP-C assay with the CD method (y) and conventional RLP-C assay (x) was: y = 1.02x - 0.008 mmol/L (Fig. 4 ). The mean (SD) of x was 0.188 (0.088) mmol/L, and the mean (SD) of y was 0.184 (0.090) mmol/L. The mean difference between methods was 0.004 mmol/L, and the SD of residuals (Sy|x) was 0.0065 mmol/L. Samples with <0.13 mmol/L RLP-C were 40–60% higher by the new method than by the conventional RLP-C assay.



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  		Figure 4. Correlation between the RLP-C assay with the CD method (y) and the conventional (PO) method (x).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD from Nocardia species (13) has high substrate specificity for cholesterol (7) and allows a substrate cycling reaction between the thio-NAD and NADH redox reaction. The number of cycles occurring between oxidized substrate ({Delta}-4-cholesten-3-one) and reduced substrate (cholesterol) was estimated to be 10/min from the molar absorptivity. This CD method showed good linearity even at very low cholesterol concentrations, and the calibration curve passed through the origin of the coordinated axes. The detection limit of the CD method (10-4 mmol/L) was approximately one-tenth that of a PO method reported, for example, by Leary et al. (5) as 0.0013 mmol/L (0.08 mmol/L as serum RLP-C). The change in absorbance (sensitivity) of the CD method was at least 5 times higher than that of the PO method. If the sample volume for the cholesterol assay is increased from 10 µL, further improvement of sensitivity appears possible.

The CD method meets the needs for an RLP-C assay. Serum RLP-C >0.194 mmol/L is a cardiovascular risk factor (4)(5). When serum samples with RLP-C of 0.194 mmol/L were diluted 61-fold with the immunoaffinity gel solution for the separation of RLP, the final cholesterol concentration in the supernatants was <0.003 mmol/L (0.194 divided by 61). The RLP-C assay with the CD method required only one calibrator, had suitable precision and linearity, and was not affected by the potential interferences tested.

In conclusion, the CD method is well suited for accurate RLP-C assays and is expected to provide an easy method for the measurement of very low concentrations of cholesterol. The CD method as a highly sensitive cholesterol assay method also appears suitable for measurement of very low cholesterol in other biological materials.


   Acknowledgments
 
We thank Dr. Tetsunori Akiba (Amano Enzyme Inc., Nagoya, Japan) for kindly supplying recombinant CD.


   Footnotes
 
1 Nonstandard abbreviations: RLP-C, remnant lipoprotein-cholesterol; CD, cholesterol dehydrogenase; CEH, cholesterol ester hydrolase; and PO, peroxidase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470-475.[Abstract]
  2. Roeschlau P, Bernt E, Gruber W. Enzymatic determination of total cholesterol in serum. J Clin Chem Biochem 1974;12:403-407.
  3. Nakajima K, Saito T, Tamura A, Suzuki M, Nakano T, Adachi M, et al. Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I mixed gel. Clin Chim Acta 1993;223:53-71.[ISI][Medline] [Order article via Infotrieve]
  4. Nakajima K, Okazaki M, Tanaka A, Pullinger CR, Wang T, Nakano T, et al. Separation and determination of remnant lipoprotein in serum from diabetes patients using monoclonal antibodies to apo B-100 and apo A-I. J Clin Ligand Assay 1996;19:177-183.
  5. Leary ET, Wang T, Baker DJ, Cilla DD, Zhong J, Warnick GR, et al. Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma. Clin Chem 1998;44:2490-2498.[Abstract/Free Full Text]
  6. Takahashi M, Ueda S, Misaki H, Sugiyama N, Matsumoto K, Matsuo N, et al. Carnitine determination by an enzymatic cycling method with carnitine dehydrogenase. Clin Chem 1994;40:817-821.[Abstract/Free Full Text]
  7. Kishi K, Watazu Y, Katayama Y, Okabe H. Characteristics and applies of recombinant cholesterol dehydrogenase. Biosci Biotechnol Biochem 2000;64:1352-1358.[Medline] [Order article via Infotrieve]
  8. Horinouchi S, Ishizuka H, Beppu T. Cloning, nucleotide sequence, and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp. and its hyperexpression in Streptomyces spp. Appl Environ Microbiol 1991;57:1386-1393.[Abstract/Free Full Text]
  9. Hopwood DA, Kieser T, Wright H M, Bibb MJ. Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol 1983;129:2257-2269.[Medline] [Order article via Infotrieve]
  10. Katz E, Thompson CJ, Hopwood DA. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 1983;129:2703-14.[Medline] [Order article via Infotrieve]
  11. Uwajima T, Terada O. Purification and properties of extracellular cholesterol ester hydrolase of Pseudomonas fluorescens. Agric Biol Chem 1975;39:1511-1512.
  12. Ferguson WJ, Braunschweiger KI, Braunschweiger WR, Smith JR, McCormick JJ, Wasmann CC, et al. Hydrogen ion buffers for biological research. Anal Biochem 1980;104:300-310.[ISI][Medline] [Order article via Infotrieve]
  13. Akiba T (inventor). The method for preparation of NAD(P)-dependent cholesterol dehydrogenase. Japanese patent no. 90-18064, 1990..



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