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Method to assay coenzzyme q10 in blood plasma or blood serumRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving OxidoreductaseMethod to assay coenzzyme q10 in blood plasma or blood serum description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080090265, Method to assay coenzzyme q10 in blood plasma or blood serum. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority to U.S. patent application Ser. No. 10/444,535 filed May 23, 2003; which claims benefit to U.S. Provisional Application Ser. No. 60/382,943 filed on May 23, 2002, all of which are expressly incorporated by reference in their entireties as part of the present disclosure. BACKGROUND OF THE INVENTION [0002] Coenzyme Q.sub.10 (2,3 dimethyl-5-methyl-6-decaprenyl benzoquinone) ("CoQ.sub.10") levels in whole blood and plasma have been the subject of much inquiry as described, for example, in Tomasetti, M., Alleva, R., Solenghi, M. D., Littarru, G. P., Distribution of antioxidants among blood components and lipoproteins: significance of lipids/CoQ.sub.10 ratio as a possible marker of increased risk for atherosclerosis. BioFactors, 9, 231-240 (1999), the entire content of which is incorporated herein by reference. It is likely that plasma concentrations of CoQ.sub.10 reflect an overall metabolic demand, as discussed in Littarru, G. P., Lippa, S., Oradei, A., Fiorini, R. M., Mazzanti, L., Metabolic and diagnostic implications of human blood CoQ.sub.10 levels, in Biomedical and Clinical Aspects of Coenzyme Q vol. VI, (eds. K. Folkers, G. P. Littarru, T. Yamagami), Elsevier North Holland, pp. 167-178 (1991), the entire content of which is incorporated herein by reference. In addition, together with other lipophilic antioxidants, CoQ.sub.10 plays an intrinsic role in protecting circulating lipoproteins against oxidative damage. Therefore, the concentration of CoQ.sub.10 in lipoproteins and blood plasma could be of clinical importance regarding oxidative stress and antioxidant defense. Increased levels of CoQ.sub.10 enhance its antioxidant protection, even though the potential to act as an antioxidant in vivo probably depends not only on total CoQ.sub.10 concentration, but also on its redox status. The content of CoQ.sub.10 in single classes of lipoproteins has been found to be strictly correlated with CoQ.sub.10 plasma concentration. Previous reports have shown that the LDL-cholesterol/CoQ.sub.10 ratio significantly correlates with the total-cholesterol/HDL-cholesterol ratio which is usually considered a risk factor for atherosclerosis as described, for example in Alleva, R., Tomasetti, M., Bompadre, S., Littarru, G. P., Oxidation of LDL and their subfractions: kinetic aspects and CoQ.sub.10 content. Molec Asp Med, 18, s105-s112 (1997), the entire content of which is incorporated herein by reference. Some effective hypocholesterolemic agents, namely the statins, also lower plasma CoQ.sub.10 concentrations, owing to the common biosynthetic pathway of cholesterol and the isoprenoide side chain of coenzyme Q as described, for example, in Mortensen, S. A., Leth, A., Agner, E., Rohde, M., Dose-related decrease of serum coenzyme Q.sub.10 during treatment with HMO-CoA reductase inhibitors. Molec Asp Med, 18, s137-s144 (1997), the entire content of which is incorporated herein by reference. Therefore, it would be desirable to have an effective, reliable, fast method to measure CoQ.sub.10 concentrations in blood plasma or blood serum to monitor the CoQ.sub.10 levels in patients receiving hypocholesterolemic agents. [0003] CoQ.sub.10 is used as a food supplement or as an adjunctive therapy in several diseases and the blood plasma or blood serum levels achieved upon oral administration of CoQ10 can correlate with clinical efficacy. Tests of blood plasma or blood serum levels of CoQ.sub.10 are useful for monitoring the bioavailability of orally administered coenzyme Q.sub.10. [0004] Several methods have been described for assaying either total CoQ.sub.10 or the reduced (ubiquinol-10, CoQ.sub.10H.sub.2) and oxidized (ubiquinone-10) forms in blood plasma, and several of these methods are described in the following references, the entire contents of each of which are incorporated herein by reference: Lang, J. K, Packer, L., Quantitative determination of vitamin E and oxidized and reduced coenzyme Q by high-performance liquid chromatography with in-line ultraviolet and electrochemical detection, J Chromatogr, 385, 109-117 (1987); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A., High-performance liquid chromatography-coulometric electrochemical detection of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma, in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 341-348, Academic Press, San Diego (1999); Podda, M., Weber, C., Traber, M. G., Milbradt, R., Packer, L., Sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols and ubiquinones in biological samples in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 330-341, Academic Press, San Diego (1999); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A. Monitoring of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma microsamples using high-performance liquid chromatography with coulometric electrochemical detection. Anal Biochem, 232, 210-216 (1985); Okamoto, T., Fukunaga, Y., Ida, Y., Kishi, T., Determination of reduced and total ubiquinones in biological materials by liquid chromatography with electrochemical detection, J Chromatogr, 430, 11-19 (1988); Grossi, G., Bargossi, A. M., Fiorella, P. L., Piazzi, S. Improved high-performance liquid chromatographic method for the determination of coenzyme Q.sub.10 in plasma. J. Chromatogr., 593, 217-226 (1988); Edlund, P. O., Determination of coenzyme COQ.sub.10, .alpha.-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection. J. Chromatogr, 425, 87-97 (1988); Lagendijk, J., Ubbink, J. B., Vermaak, W. J., Measurement of the ratio between the reduced and oxidized forms of coenzyme Q10 in human plasma as a possible marker of oxidative stress, J. Lipid Res, 37, 67-75 (1996); Yamashita, S., Yamamoto, Y., Simultaneous detection of ubiquinol and ubiquinone in human plasma as a marker of oxidative stress, Anal. Biochem, 250, 66-73 (1997). As used herein, ubiquinol means reduced CoQ.sub.10 and ubiquinone means oxidized CoQ.sub.10. [0005] The previous methods of analyzing blood plasma to assay the concentration of CoQ.sub.10 have several disadvantages. Prior methods require that the CoQ.sub.10 be extracted from the plasma, followed by drying, which concentrates the extract. Losses in CoQ.sub.10 can occur during the drying and concentration step. In addition, these methods analyze the oxidized form of COQ.sub.10, while most of the CoQ.sub.10 in the plasma is in the reduced form. These methods rely on oxidation of the CoQ.sub.10 during the extraction procedure. UV methods for assaying CoQ.sub.10 usually quantify the oxidized coenzyme at 275 nm. It is commonly assumed that CoQ.sub.10 is completely oxidized during the extraction and HPLC procedure, but this is not necessarily the case when the sample is fresh and the reduced form of CoQ.sub.10 largely predominates. Accordingly, these methods can result in underestimates of CoQ.sub.10 concentration if all of the CoQ.sub.10 is not oxidized. [0006] The present invention is directed to a new, simplified method for evaluating total CoQ.sub.10 in blood plasma or blood serum. The method of the present invention results in reduced time and cost for the analysis of CoQ.sub.10 in plasma or serum as compared to prior methods, and provides more accurate results. BRIEF DESCRIPTION OF THE DRAWINGS [0007] FIG. 1A shows a typical chromatogram of a plasma sample, before and after spiking with a known amount of standard. A typical chromatogram of the standard alone is also shown. [0008] FIG. 1B shows a diode array analysis of the peak of a standard of CoQ.sub.10 (50 ng). [0009] FIG. 1C shows a diode array analysis of the peak obtained on a plasma sample analyzed by the method of the present invention. [0010] FIG. 2 shows a linear correlation between the results obtained by analysis of a sample for CoQ.sub.10 using a reference electrochemical detection method and the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION [0011] The present invention relates to a method to assay the concentration of CoQ.sub.10 in blood plasma or blood serum. The sample of blood plasma or blood serum is treated with an oxidizing agent having a redox potential higher than the redox potential of COQ.sub.10, followed by extraction with an alcohol and direct injection of the alcohol extract into a High Performance Liquid Chromatography ("HPLC") apparatus. [0012] In a preferred embodiment of the method, the CoQ.sub.10 in the plasma or serum sample is oxidized by adding para-benzoquinone as an oxidizing agent. Extraction is performed using an alcohol, preferably n-propanol. The extract is then assayed by direct injection of the propanol extract into the HPLC apparatus without bringing the extract to dryness. The method can be conducted on fresh plasma or serum samples since CoQ.sub.10 present in the sample is oxidized prior to propanol extraction. [0013] The method may be performed using an oxidizing agent other than benzoquinone. For example, other oxidizing agents having a redox potential higher than the ubiquinone/ubiquinol couple may be used in the method. Also, the extraction of the oxidized CoQ.sub.10 in the plasma or serum sample may be performed using any appropriate alcohol known to those skilled in the art. For example, 1-propanol, butanol or pentanol may be used for the extraction of the oxidized CoQ.sub.10. [0014] A particularly preferred embodiment of the present method is set forth in the Example below. It should be understood that the description set forth below is not intended to limit the invention in any way, and those skilled in the art will readily understand that modifications to the reagents, equipment or other parameters set forth below can be made without departing from the spirit or scope of the invention. Results obtained using the method on various samples are also described below. EXAMPLE Materials and Methods [0015] Reagents [0016] R.S. type methanol and n-propanol were used (obtained from Carlo Erba, Rodano, Milan, Italy). Ethanol was R.S. plus grade (obtained from Carlo Erba, Rodano, Milan, Italy). Benzoquinone was obtained from Sigma (St Louis, Mo., USA). Lithium perchlorate was obtained from Aldrich (Steinheim, Germany). [0017] Solutions for the ECD (electrochemical detection) chosen as a reference method were filtered through a Nylon 66 membrane, 0.2 .mu.m.times.47 mm (Supelco, Bellafonte, Pa., USA) and degassed. Pure Coenzyme Q.sub.10 standard was obtained from Kaneka (Osaka, Japan). Standard solutions were in ethanol. [0018] Samples [0019] Blood was drawn from the cubital vein of laboratory staff, after informed consent, and anti-coagulated with lithium heparin. Plasma obtained after centrifugation at 4,000 g for 15 min., at 4.degree. C. was used fresh, or after storage at -80.degree. C. for the day-to-day precision assay. 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