An ultrasensitive enzymatic method for measuring mevalonic acid in serum

We have developed a simple, precise, and ultrasensitive enzymatic method for measuring serum mevalonic acid (MVA) concentration, which is thought to be a good indicator of the in vivo cholesterol biosynthesis rate. This assay is based on an enzyme cycling reaction and makes use of HMG-CoA reductase (HMGR), thio-NAD, NADH, and CoA. MVA participates in the HMGR cycling reaction, and its level is measured based on the production of thio-NADH, which is determined from the change in absorbance at 405 nm. To achieve high specificity, we used mevalonate kinase (MVK) in addition to HMGR. Only substrates able to participate in both the HMGR cycling reaction and the MVK reaction are measured as MVA. The detection limit for MVA is 0.4 ng/ml (2.7 nmol/l), and the calibration curve for MVA is linear up to 44 ng/ml (300 nmol/l). Regression analysis with 40 serum samples showed the accuracy of quantifying MVA with this enzymatic assay to be comparable to that using LC-MS/MS (correlation: y = 0.83x + 0.24; r = 0.97). This procedure is simple, precise, and robust. It is also rapid and has a high throughput, making it potentially useful for clinical applications. We have developed a simple, precise, and ultrasensitive enzymatic method for measuring serum mevalonic acid (MVA) concentration, which is thought to be a good indicator of the in vivo cholesterol biosynthesis rate. This assay is based on an enzyme cycling reaction and makes use of HMG-CoA reductase (HMGR), thio-NAD, NADH, and CoA. MVA participates in the HMGR cycling reaction, and its level is measured based on the production of thio-NADH, which is determined from the change in absorbance at 405 nm. To achieve high specificity, we used mevalonate kinase (MVK) in addition to HMGR. Only substrates able to participate in both the HMGR cycling reaction and the MVK reaction are measured as MVA. The detection limit for MVA is 0.4 ng/ml (2.7 nmol/l), and the calibration curve for MVA is linear up to 44 ng/ml (300 nmol/l). Regression analysis with 40 serum samples showed the accuracy of quantifying MVA with this enzymatic assay to be comparable to that using LC-MS/MS (correlation: y = 0.83x + 0.24; r = 0.97). This procedure is simple, precise, and robust. It is also rapid and has a high throughput, making it potentially useful for clinical applications. 3-hydroxy-3-methylglutaryl-CoA lyase 3-hydroxy-3-methylglutaryl-CoA reductase mevalonic acid mevalonate kinase mevalono lactone thionicotinamide adenine dinucleotide Cholesterol originates from dietary intake and from de novo synthesis. HMG-CoA reductase (HMGR) has long been established as the rate-limiting enzyme in the cholesterol biosynthetic pathway (1Parker T.S. McNamara D.J. Brown C. Garrigan O. Kolb R. Batwin H. Jr. Ahrens E.H. Mevalonic acid in human plasma: relationship of concentration and circadian rhythm to cholesterol synthesis rates in man.Proc. Natl. Acad. Sci. USA. 1982; 79: 3037-3041Crossref PubMed Scopus (122) Google Scholar), and the level of mevalonic acid (MVA), the product of HMGR, in plasma appears to be a good indicator of de novo cholesterol synthesis (1Parker T.S. McNamara D.J. Brown C. Garrigan O. Kolb R. Batwin H. Jr. Ahrens E.H. Mevalonic acid in human plasma: relationship of concentration and circadian rhythm to cholesterol synthesis rates in man.Proc. Natl. Acad. Sci. USA. 1982; 79: 3037-3041Crossref PubMed Scopus (122) Google Scholar). Two main types of drugs are in use to lower serum cholesterol: HMGR inhibitors and intestinal cholesterol absorption inhibitors. HMGR inhibitors, also called statins, include pravastatin, simvastatin, and atorvastatin; these compounds lower cholesterol mainly by inhibiting cholesterol synthesis. On the other hand, ezetimibe inhibits the intestinal absorption of cholesterol in the diet. Targeting the appropriate therapy for individual patients could potentially be improved by understanding the underlying causes of hypercholesterolemia by monitoring the levels of plasma and/or urinary MVA. Such measurements would also be valuable in monitoring the response to treatment. A number of methods for measuring serum, plasma, or urinary MVA have been reported. These include a radioenzyme assay (2Popják G. Boehm G. Parker T.S. Edmond J. Edwards P.A. Fogelman A.M. Determination of mevalonate in blood plasma in man and rat. Mevalonate “tolerance” tests in man.J. Lipid Res. 1979; 20: 716-728Abstract Full Text PDF PubMed Google Scholar), enzyme immunoassay (3Hiramatsu M. Hayashi A. Hidaka H. Ueshima H. Kanno T. Enzyme immunoassay of urinary mevalonic acid and its clinical application.Clin. Chem. 1998; 44: 2152-2157Crossref PubMed Scopus (10) Google Scholar), GC-MS assay (4Scoppola A. Maher V.M. Thompson G.R. Rendell N.B. Taylor G.W. Quantitation of plasma mevalonic acid using gas chromatography-electron capture mass spectrometry.J. Lipid Res. 1991; 32: 1057-1060Abstract Full Text PDF PubMed Google Scholar–7Woollen B.H. Holme P.C. Northway W.J. Martin P.D. Determination of mevalonic acid in human urine as mevalonic acid lactone by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 2001; 760: 179-184Crossref PubMed Scopus (19) Google Scholar), and LC-MS/MS assay (8Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar–13Waldron J. Webster C. Liquid chromatography-tandem mass spectrometry method for the measurement of serum mevalonic acid: a novel marker of hydroxymethylglutaryl coenzyme A reductase inhibition by statins.Ann. Clin. Biochem. 2011; 48: 223-232Crossref PubMed Scopus (14) Google Scholar). Until now, however, the use of enzymatic spectrophotometric methodology has been precluded by the extremely low levels of MVA in human plasma and serum. The normal range of MVA concentration has been reported to be 1.0–11.2 ng/ml (6.7-75.6 nmol/l) in plasma (9Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard.Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar) and 12.28 ± 2.54 ng/ml (82.9 ± 17.1 nmol/l) in serum (11Wani T.A. Samad A. Tandon M. Saini G.S. Sharma P.L. Pillai K.K. The effects of rosuvastatin on the serum cortisol, serum lipid, and serum mevalonic acid levels in the healthy Indian male population.AAPS PharmSciTech. 2010; 11: 425-432Crossref PubMed Scopus (13) Google Scholar). In the present study, we describe a simple, precise, and ultrasensitive enzymatic method for measuring MVA in serum. To achieve high sensitivity to MVA, we used an enzymatic cycling method (14Takahashi M. Ueda S. Misaki H. Sugiyama N. Matsumoto K. Matsuo N. Murao S. Carnitine determination by an enzymatic cycling method with carnitine dehydrogenase.Clin. Chem. 1994; 40: 817-821Crossref PubMed Scopus (98) Google Scholar–18Ueda S. Oda M. Imamura S. Ohnishi M. Kinetic study of the enzymatic cycling reaction conducted with 3alpha-hydroxysteroid dehydrogenase in the presence of excessive thio-NAD+ and NADH.Anal. Biochem. 2004; 332: 84-89Crossref PubMed Scopus (20) Google Scholar) involving the use of HMGR, thionicotinamide adenine dinucleotide (thio-NAD), NADH, and CoA. To achieve high specificity, the HMGR cycling reaction is carried out with and without a preceding mevalonate kinase (MVK) reaction, and the MVA con­cen­tra­tion is calculated from the difference between the two results. The MVK reaction eliminates the participation of possible HMGR cycling reaction substrates other than MVA (e.g., HMG-CoA). Using this HMGR cycling method, we determined MVA concentrations in 40 sera and validated the results by comparison with values obtained using LC-MS/MS. DL-mevalonolactone (DL-MVAL), DL-HMG-CoA sodium salt trihydrate, fluvastatin sodium hydrate, simvastatin, atorvastatin calcium salt trihydrate, mevinolin (lovastatin), mevastatin, pravastatin sodium salt hydrate, and Proclin300 were obtained from Sigma-Aldrich (St. Louis, MO). Deuterated MVAL (d7-MVAL) was from CDN Isotopes (Pointe-Claire, Quebec, Canada). ATP, NADH, and thio-NAD were from Oriental Yeast (Tokyo, Japan). Glycine, Tris, ammonium sulfate, nitro blue tetrazolium chloride, CoA, and ascorbic acid were from Wako Pure Chemicals (Osaka, Japan). HEPES, MES, and EDTA were from Dojindo Laboratories (Kumamoto, Japan). “Interference Check A plus” was from Sysmex (Kobe, Japan). Q Sepharose big beads, Phenyl Sepharose fast flow, Q Sepharose fast flow, DEAE-Sepharose fast flow, and Blue Sepharose fast flow were from GE Health Care (Tokyo, Japan). All other chemicals were of the highest quality commercially available. Glucose-6-phosphate dehydrogenase and diaphorase were from TOYOBO (Osaka, Japan). ADP-specific glucokinase and 3-hydorxybutyrate dehydrogenase were from Asahi Kasei Pharma (Tokyo, Japan). HMGR was purified to homogeneity from overproducing recombinant Escherichia coli expressing the HMGR gene from Pseudomonas sp. MV -1 strain as previously described with minor modifications (19Matsuoka, T., 2011. Method and reagent for determining mevalonic acid, 3-hydoroxymethylglutaryl coenzyme A, coenzyme A. European Patent Application EP2380989A1.Google Scholar). Briefly, the cell lysate was centrifuged, after which the supernatant was subjected to Q sepharose big beads chromatography, ammonium sulfate precipitation, Phenyl Sepharose fast flow chromatography, Q Sepharose fast flow chromatography, dialysis, and concentration. The enzyme activity was assayed based on NADH production measured as a change in absorbance at 340 nm. The reaction mixture contained 37.5 mmol/l glycine (pH 10.0), 5 mmol/l DL-MVAL, 0.75 mmol/l NAD, and 0.5 mmol/l CoA at 37°C. One unit of enzyme activity was defined as the amount of enzyme forming 1 μmol of NADH per minute under the above conditions. MVK was purified to homogeneity from overproducing recombinant E. coli expressing the MVK gene from Saccharomyces cereviciae (NBRC1136) as described previously with minor modifications (19Matsuoka, T., 2011. Method and reagent for determining mevalonic acid, 3-hydoroxymethylglutaryl coenzyme A, coenzyme A. European Patent Application EP2380989A1.Google Scholar). Briefly, the cell lysate was centrifuged, after which the supernatant was subjected to Q Sepharose big beads chromatography, ammonium sulfate precipitation, Phenyl Sepharose fast flow chromatography, Blue Sepharose fast flow chromatography, dialysis, and concentration. The enzyme activity was assayed based on NADH formation measured as a change in the absorbance of formazan dye at 550 nm. The reaction mixture contained 50 mmol/l Tris-HCl (pH 7.5), 0.005% nitro blue tetrazolium chloride, 5 mmol/l glucose, 1 mmol/l MgCl2, 1 mmol/l NAD, 1 mmol/l ATP, 5 U/ml ADP-specific glucokinase, 5 U/ml glucose-6-phosphate dehydrogenase, 5 U/ml diaphorase, and 2 mmol/l DL-MVAL at 37°C. One unit of enzyme activity was defined as the amount of enzyme forming 1 μmol of NADH per minute under the above conditions. 3-Hydroxy-3-methylglutaryl-CoA lyase (HMGL) was purified to homogeneity from overproducing recombinant E. coli expressing the HMGL gene from Pseudomonas putida KT2440 strain (ATCC47054) as described previously with minor modifications (19Matsuoka, T., 2011. Method and reagent for determining mevalonic acid, 3-hydoroxymethylglutaryl coenzyme A, coenzyme A. European Patent Application EP2380989A1.Google Scholar). Briefly, the cell lysate was centrifuged, after which the supernatant was subjected to Q Sepharose big beads chromatography, ammonium sulfate precipitation, DEAE Sepharose fast flow chromatography, Phenyl Sepharose fast flow chromatography, dialysis, and concentration. The enzyme activity was assayed based on the oxidation of NADH measured as a decrease in the absorbance at 340 nm. The reaction mixture contained 19.4 mmol/l Tris-HCl (pH 9.5), 0.19 mmol/l NADH, 4.9 U/ml 3-hydorxybutyrate dehydrogenase, and 0.17 mmol/l DL-HMG-CoA at 37°C. One unit of enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol of NADH per minute under the above conditions. Serum samples from 40 volunteers were purchased from Kohjin Bio (Tokyo, Japan). Pooled plasma and serum were from Kohjin Bio (Tokyo, Japan). Control serum, “L-Consera IEX,” was from Nissui Seiyaku (Tokyo, Japan). Solutions of statins were prepared in distilled water (fluvastatin sodium hydrate, simvastatin, atorvastatin calcium salt trihydrate, lovastatin, and mevastatin) or dimethyl sulfoxide (pravastatin sodium salt hydrate) and then added to control serum “L-consera IEX.” Solutions of bilirubin, hemoglobin, and chyle were prepared in “Interference Check A plus” following the manufacturer's instructions and added to control serum “L-Consera IEX” (1:9 vol). Because only (R)-MVA (D-MVA) is biologically active in the mevalonate pathway, we prepared a standard MVA solution in which the (R)-MVA concentration was known. The MVA solution was prepared by dissolving DL-MVAL in water and diluting it with water. The (R)-MVA concentration in a racemic MVA solution was determined using the modified HMGR coupled with HMGL method described previously (19Matsuoka, T., 2011. Method and reagent for determining mevalonic acid, 3-hydoroxymethylglutaryl coenzyme A, coenzyme A. European Patent Application EP2380989A1.Google Scholar). Briefly, all (R)-MVA in the sample was converted, and the amount was calculated based on the increase in NADH. Because the HMGR cycling reaction is run with a preceding MVK reaction, the MVA solution cannot be used as a calibrator because the MVA would be eliminated by the MVK reaction. We therefore adopted HMG-CoA solution as a calibrator, and the levels of the calibrator HMG-CoA were determined using standard (R)-MVA solutions in the HMGR cycling protocol without the MVK reaction, as described below. The HMG-CoA solution was prepared by dissolving DL-HMG-CoA sodium salt trihydrate in water and then diluting with buffer containing 10 mmol/l H2SO4 and 0.1% Tween 80. The enzymatic measurement of MVA is illustrated schematically in Fig. 1. MVA is reversibly converted to HMG-CoA by HMGR, and this cycling reaction is repeated. The MVA concentration is derived from the amount of thio-NADH formed, which is measured based on the change in absorbance at 405/660 nm. The enzymatic assay is illustrated schematically in Fig. 2. 1) Samples are divided into two portions, and one portion is exposed to MVK to convert MVA to phosphomevalonate, which is not to be a substrate for HMGR. 2) The MVK-treated and -untreated samples are then added to the HMGR cycling reaction (the MVK reaction is inactivated by adding EDTA) in which MVA and other possible substrates (e.g., HMG-CoA) are repeatedly oxidized or reduced, causing the accumulation of thio-NADH and NAD. 3) For samples not treated with MVK, the amount of MVA with other possible HMGR substrates is measured based on the increase in thio-NADH monitored as an increase in absorbance at 405 nm. With MVK-treated samples, HMGR substrates other than MVA are measured. 4) The amount of MVA in the sample is calculated from the difference between the substrate levels in the MVK-treated and -untreated samples.Fig. 2Schematic illustration of the enzymatic assay method. The enzyme method contains two series of reactions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two reaction mixtures were used for the enzymatic measurement of MVA. For MVK-treated samples, the first reaction mixture (R-1) contained 0.7 mmol/l NADH, 0.4 mmol/l ATP, 0.4 mmol/l MgCl2, 0.16 U/ml MVK, 0.05% sodium azide, 0.072% Tween80, 18% sucrose, 30 mmol/l sodium bicarbonate, and 50 mmol/l glycine (pH 10.0). The second reaction mixture (R-2) contained 25 mmol/l thio-NAD, 2.4 mmol/l CoA, 10 mmol/l EDTA, 120 U/ml HMGR, 0.02% Proclin300, 18% sucrose, 110 mmol/l HEPES, and 25 mM MES (pH 5.8). For samples not treated with MVK, the mixtures were the same, except that MVK was omitted from R-1. The assay was run on a Hitachi 7170S automatic analyzer. In assays with and without MVK, 150 μl of R-1 were incubated with 5 μl of sample for 10 min at 37°C, after which 50 μl of R-2 were added. The absorbances at 405 nm (main wavelength) and 660 nm (subwavelength) were measured for 12 min after the R-2 was added. The increase in absorbance from min 11 to min 22 was calculated and calibrated using calibration solutions containing known concentrations of HMG-CoA. With MVK treatment, up to a maximum of 74 ng/ml (R)-MVA can be eliminated, and the observed absorbance change for HMG-CoA is more than 95% of that without MVK treatment. Measurement of MVA using LC-MS/MS was carried out after conversion of MVA to MVAL, as described previously with slight modification (10Saini G.S. Wani T.A. Gautam A. Varshney B. Ahmed T. Rajan K.S. Pillai K.K. Paliwal J.K. Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect. J.J. Lipid Res. 2006; 47: 2340-2345Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The system is comprised of an API-3200 mass spectrometer (AB SCIEX; Foster, CA) equipped with a LC20A liquid chromatography system (Shimadzu; Kyoto, Japan). Chromatography was done using a Shodex Asahipak ODP-40 2D column (2.0 mm × 150 mm, 5 μm; Showa Denko; Tokyo, Japan) maintained at 40°C. The mobile phase was a linear gradient of 10 mmol/l ammonium formate and methanol from 90:10 (v/v) to 10:90 (v/v). The flow rate was 0.2 ml/min, and the injection volume was 10 μl. The MS/MS conditions were: duration, 10 min; scan type, positive MRM; GS1, 80 p.s.i.; GS2, 70 p.s.i.; CUR, 10 p.s.i.; ion source voltage, 5000 V; temperature, 400°C; declustering potential, 26 V; collision energy, 13 V for MVAL and 15 V for d7-MVAL; collision cell potential, 26 V; reaction monitoring for MVAL and for The calibration curve was using MVAL, and was used as an internal of MVAL from serum samples was as A serum sample was after adding of internal standard solution of distilled and 1 of The mixture was to for 30 min and then to an Tokyo, Japan) with of methanol and of The was then with of and of after which the MVAL was with The was then and the was in 0.2 of The MVAL calibration curve was linear from 0.5 to The of for standard MVAL at 0.5 and ng/ml were and The MVAL, and used in the LC-MS/MS were all the values for serum determined using LC-MS/MS the because to be All MVA concentrations obtained using the ultrasensitive enzymatic cycling assay are (R)-MVA was by making measurements using solutions containing known concentrations of MVA. analysis the relationship y = + = and the curve was linear to at ng/ml (300 nmol/l). The lower detection limit of the assay was to be 0.4 ng/ml by solutions containing or ng/ml MVA with the that the ± cannot of detection of MVA in in a was by measuring control serum samples, control samples containing ng/ml or ng/ml MVA, and a MVA solution containing The from to was by measuring the ng/ml MVA solution on using reagent solutions that been at and calibrated The was The enzymatic assay showed good and of MVA in control measured in a of MVA were added to serum, plasma, and distilled water and measured using enzymatic was observed in both serum and plasma The enzymatic assay was not by to to to chyle to or ascorbic acid to The assay was also not by simvastatin, lovastatin, mevastatin, or pravastatin, at up to ng/ml Because the in plasma or serum is to be 5 ng/ml for atorvastatin and simvastatin, ng/ml for and 10 ng/ml for pravastatin, this assay is to serum from patients study by bilirubin, hemoglobin, and ascorbic in a study by in a MVA levels were measured in 40 individual serum samples using enzymatic assay and the between the two methods was HMGR enzyme cycling method for MVA has two and novel the detection limit of 0.4 ng/ml (2.7 nmol/l) is to lower than the reported limit for the enzyme cycling in this assay has the reported detection limit for spectrophotometric this is the first enzyme cycling method based on a enzyme reaction. A the reaction between MVA and HMG-CoA is illustrated in Fig. 5 M. coenzyme A 2004; PubMed Scopus Google Scholar). The enzyme cycling reaction between MVA and not between MVA and or between MVA and was previously (19Matsuoka, T., 2011. Method and reagent for determining mevalonic acid, 3-hydoroxymethylglutaryl coenzyme A, coenzyme A. European Patent Application EP2380989A1.Google Scholar), the cycling reaction was without CoA, and adding HMGL to the reaction the cycling reaction using MVK, we were able to the HMGR enzyme cycling reaction for MVA. the in the presence and of MVK one to out from substrates other than MVA, as and other possible it the use of a calibrator other than MVA, which is eliminated by We used HMG-CoA solution as drugs not with the HMGR enzyme cycling assay because was more enzyme present in the reaction mixture than is present under conditions and because the of for statins is of lower than human HMGR M. of the HMG-CoA reductase of Pseudomonas Sci. 2004; PubMed Scopus (16) Google Scholar). The between the enzymatic assay and 4) that the accuracy of the enzymatic method is comparable to the sample was = and LC-MS/MS both (R)-MVA and for the than are 1) a sample = and 2) that LC-MS/MS both (R)-MVA and is not known to in human blood or other the presence of and the that this could and could an in one or more other than cholesterol present the measurement of MVA only in serum, this method also appears to be to urine and other We measured MVA in human urine because the MVA concentration is in urine than in serum. this also be to measuring MVA in after and of appropriate samples for the In we have developed a simple, precise, ultrasensitive MVA assay for quantifying MVA in serum without an The results of the study the assay is also to This enzyme cycling method is and of high accuracy is comparable to and it can be used with automatic which would be useful for clinical The Kasei for valuable and and Kasei for

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