Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2003-02-14
2004-07-20
Snay, Jeffrey R. (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
C436S086000, C436S161000, C436S172000
Reexamination Certificate
active
06764855
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
BACKGROUND OF THE INVENTION
A variety of diseases such as salt sensitive hypertension, toxemia of pregnancy, asthma, hepatorenal syndrome, diabetes and subarachnoid hemorrhage are associated with abnormalities in arachidonic acid (“AA”) metabolism. Recent studies have indicated that AA is primarily metabolized in the brain, kidney, lung, and vasculature by cytochrome P-450 enzymes to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (diHETEs), and 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-HETE). McGiff J C and Quilley J,
Am J Physiol Regulatory Integrative Comp Physiol
277: R607-R623 (1999); Roman R J and Alonso-Galicia M,
News Physiol Sci
14: 238-242 (1999). 20-HETE and EETs are biologically active and have been implicated as paracrine factors and/or second messengers in the regulation of vascular tone, sodium and water excretion in the kidney, and airway resistance. McGiff J C and Quilley J,
Am J Physiol Regulatory Integrative Comp Physiol
277: R607-R623 (1999); Roman R J and Alonso-Galicia M,
News Physiol Sci
14: 238-242 (1999). Despite the importance of P-450 metabolites of AA, very little is known about the regulation of the concentrations of these mediators in tissue and biological fluids. Part of the problem has been the lack of a sensitive, inexpensive, and high-throughput assay to measure the endogenous concentration of these compounds. To date, gas chromatography-mass spectroscopy (GC-MS) with selective ion monitoring and one report of a fluorescent enzyme based immunoassay (“EIA”) for EETs have been the only methods available to measure the concentration of P-450 metabolites of AA in biological samples. Capdevila J H et al.
J Biol Chem
267: 21720-21726(1992); Catella F et al.,
Proc Natl Acad Sci USA
87: 5893-5897 (1990); Prakash C et al.,
Biochem Biophys Res Commun
185: 728-733 (1992); Schwartzman M L et al.,
Biochem Biophys Res Commun
180: 445-449 (1991); Toto R et al.,
Biochem Biophys Acta
191: 132-134 (1987). The EIA requires a specific antibody that is no longer generally available and, therefore, the assay cannot be reproduced in other labs. GC-MS has been successfully used to measure 20-HETE and EETs in the urine of humans and rats, and the reported concentration of these mediators is in the range of 0.5-5 ng/ml. The urinary excretion of EETs has been reported to increase in rats fed a high-salt diet and in patients with toxemia of pregnancy. Capdevila J H et al.,
J Biol Chem
267: 21720-21726 (1992); Oyekan A O et al.,
J Clin Invest
104:1131-1137(1999); Catella F et al.,
Proc Natl Acad Sci USA
87: 5893-5897 (1990). Moreover, the urinary excretion of 20-HETE is elevated in patients with hepatorenal syndrome and in DOCA-salt hypertensive rats. Sacerdoti D et al.,
J Clin Invest
100: 1264-1270 (1997); Oyekan A O et al.,
Am J Physiol Regulatory Integrative Comp Physiol
176: R766-R775 (1999).
Although GC-MS is a reliable method for the measurement of P-450 metabolites of AA, the high cost for the purchase and maintenance of the instrumentation and difficulties in preparing the samples for analysis have limited the use of this technique. Indeed, the preparation of urine samples for GC-MS involves an organic extraction of the lipid fraction, separation of the EETs or HETEs fractions by thin-layer chromatography and reverse-phase HPLC, derivatization of the samples to the methyl or pentabenzylfluoro esters, and conversion of these esters to trimethylsilyl derivatives. Toto R et al.,
Biochem Biophys Acta
191: 132-134 (1987). It also requires the synthesis and addition of a deuterated internal standard to the samples to correct for variable extraction and derivatization efficiencies. The extensive sample preparation reduces sample recoveries and the detection limits of this technique to the nanogram range. Oyekan A O et al.,
J Clin Invest
104: 1131-1137 (1999); Schwartzman M L et al.,
Biochem Biophys Res Commun
180: 445-449 (1991); Toto R et al.
Biochem Biophys Acta
191: 132-134 (1987). GC-MS is also limited to the measurement of a single compound at a time.
In the last few years, many fluorescent HPLC-based methods have been described for the analysis of fatty acids following derivatization of the carboxyl or hydroxyl groups with agents such as anthryldiazomethane (ADAM), pyrenyldiazomethane (PDAM), bromomethyl- and diethylaminocoumarin, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate and other dyes. Brekke O L et al.,
J Lipid Res
38: 1913-1922 (1997); Amet Y et al.,
J Chromatog B Biomed Appl
68: 233-239 (1996); Minkler P E et al.,
Anal Biochem
231: 315-322 (1995); Yasaka Y and Tanaka M,
J Chromatog B Biomed Appl
659:139-155 (1994); Yasaka Y et al.,
J Chromatog
508:133 (1990). Some of these studies have reported detection limits <10 pg for prostaglandins and fatty acids. The main problems associated with these methods have been difficulties in obtaining consistent derivatization for lack of good catalysts, the inability to clearly resolve all of the P450 metabolites of AA found in biologic samples by HPLC after these compounds have been reacted with a fluorescent compound, and the lack of an internal standard with an extraction and labeling efficiency identical to the compounds of interest.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a fluorescent HPLC assay for simultaneously detecting the presence and/or measuring the level of 20-hydroxyeicosatetraenoic acid (20-HETE) and other P-450 metabolites of AA in a sample. P-450 metabolites of AA are first extracted from the sample and then labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate, a fluorescent material. The labeling reaction is catalyzed by N,N-diisopropylethylamine. Next, the labeled P-450 metabolites are separated on a 4.5×250-mm, 5 &mgr;M particle size C18 reverse-phase HPLC column using a mobile phase of methanol:water:acetic acid (82:18:0.1, v/v/v) and an isocratic elution at a rate of about 1.3 ml per minute. Fluorescence intensities of the column eluent are monitored by a fluorescence detector.
When quantitation of P-450 metabolites in a sample is desired, a known amount of an internal standard is added to the sample before the extraction of P-450 metabolites from the sample. The amount of a P-450 metabolite in the sample can be calculated from the ratio of the P-450 metabolite peak to the internal standard peak. When the HPLC assay of the present invention is used for detecting the presence of a P-450 metabolite in a sample, the use of an internal standard is optional.
The HPLC assay of the present invention can be used for clinical tests of urine, blood, plasma, cerebrospinal fluid, bronchiolar lavage fluid and tissues for the diagnosis of diseases associated with abnormalities in the formation and/or levels of P450 metabolites of AA such as salt sensitive hypertension, toxemia of pregnancy, asthma, hepatorenal syndrome, diabetes and subarachnoid hemorrhage.
It is an object of the present invention to provide a method to simultaneously analyze more P-450 metabolites of AA in a sample than prior art methods.
It is a feature of the present invention that the catalyst for the labeling reaction allows the labeling of P-450 metabolites with a high degree of consistency.
It is an advantage of the present invention that the method has a high degree of intra-assay reproducibility.
It is another advantage of the present invention that the method has a high degree of quantitation sensitivity.
It is yet another advantage of the present invention that the method is relatively simple and inexpensive to perform.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying claims and drawings.
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Reynaud, D et al. “Impro
Maier Kristopher G.
Roman Richard J.
MCW Research Foundation
Quarles & Brady LLP
Snay Jeffrey R.
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