Title:  Detection of hypertension using immunoreactive metabolic products

United States Patent:  6,534,282

Issued:  March 18, 2003

Inventors:  Kim; Hyesook (4683 Ravine Dr., Bloomfield Hills, MI 48301); Capdevila; Jorge H. (6549 Brownlee Dr., Nashville, TN 37205); Novak; Raymond R. (4980 Browning Dr., Orchard Lake, MI 48323); Kroetz; Deanna (373 Dellbrook Ave., San Francisco, CA 94131)

Appl. No.:  946644

Filed:  September 4, 2001

A method to assess hypertension by measuring the amount of free and conjugated hydroxyeicosatrienoic acids (DHETs) and metabolites of DHETs, which are metabolites of arachidonic acid (AA) epoxygenases and epoxide hydrolases, in a biological sample which contains the DHETs (using any methods including GC/MS or ELISA) is disclosed. The method further included determining the amount of molecules containing a DHET-specific epitope immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a control sample(s). Hypertension is determined through the comparison wherein the amount of increase of free and conjugated DHETs and metabolites of DHETs in the sample isolated from an organism. The present invention also provides a method to assess catalytic activity of AA epoxygenases using immunoassays by subtracting the amounts of NADPH-independent epoxyeicosatriencic acids (EETs) from total (NADPH-dependent+independent) EETs. The present invention also provides a method to decrease hepatic M epoxygenase expression including 2C23 by treatment of rats with a glucocorticoid including dexamethasone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides, a method to assess hypertension mediated by AA epoxygenase and epoxide hydrolase by measuring the amount of free and conjugated DHETs and metabolites of DHETs, which are metabolites of AA epoxygenases and epoxide hydrolases. in a biological sample which contains the DHETs. The method further includes determining the amount of molecules containing DHET-specific epitopes immunoreactive with antibodies produced against DHETs present in the sample. This amount is compared with a healthy control sample(s). In general, a panel of healthy control samples is used that are within the normal range. The normal range is established as known in the art and is established for each assay method being utilized, e.g. GC/MS and immunoassays. The hypertension is determined through the comparison wherein the amount of the DHETs is increased in the sample isolated from an organism hypertensive compared to controls.

By assessing hypertension, it is meant that the present inventive assay is capable of being an indication of hypertension. In combination with conventional detection methods, the assay indicates a relationship between hypertension and elevated enzyme activity in the AA cascade. Specifically, the activity of the two enzymes, the epoxygenase and hydrolase which product DHETs, are unexpectedly correlated with the hypertension.

The biological sample can be selected from biological fluids which contain the DHETs and can include, but are not limited to, plasma, urine, cerebrospinal fluids, bile and joint fluids. Urine is the preferred sample.

It has been previously shown that AA epoxygenase activities (EETs +DHETs) of kidney cortical microsomes obtained from spontaneously hypertensive rats (SHR) were not different from those of normotensive Wistar Kyoto rats (WKY) (14). Applicants determined DHET levels in urine samples obtained from SHR and compared with those of WKY and found that contrary to the results obtained with kidney cortical microsome study of those rats, urinary DHET levels of SHR were .about.56-fold higher than those of WKY. Thus, measurement of urinary DHETs provides better (more specific and more sensitive) assessment of hypertension mediated by AA epoxygenase and epoxide hydrolase .

The preferred assay of the present invention, as discussed in detail in the Experiment section herein, is sensitive to both conjugate-free and conjugated forms of DHETs. Recovery rate of ³ H 14,15- and ³ H8,9-DHETs spiked in human urine after liquid phase extraction with ethyl acetate was .about.100%. However, 14,15-DHET levels in urine samples obtained from SHR decreased to the levels lower than .about.40% after ethylacetate extraction. This result demonstrated that 14,15-DHET exists in the urine as free (ethyl acetate extractable) and conjugated (ethyl acetate unextractable) forms and antibodies produced in accordance with the present invention against 14,15-DHETs recognize both free and conjugated forms. Cross-reactivity of the antibodies with the conjugated form of 14,15- and 8,9-DHETs was further demonstrated by ELISAs using 14,15- and 8,9-DHETs conjugated to bovine serum albumin. The antisera recognized the conjugated forms whereas preimmune sera showed only a basal level cross-reactivity with the conjugated forms.

It was surprising that antibodies produced against DHETs

a) recognize free (ethyl acetate extractable) and conjugated (ethyl acetate unextractable) forms of DHETs;

b) are useful for detection of hypertension related to AA oxygenase and epoxyde hydrolase activities and

c) useful for measurement of AA oxygenase and epoxide hydrolase activities.

It was unexpected to find that NADPH-independent (AA-epoxygenase-independent) EET formation with hepatic microsomes. AA-epoxygenase activity analysis using ¹⁴ CAA measures only AA-epoxygenase-dependent EET formation whereas assessment of EETs using immunoassays measures both AA epoxygenase-dependent and independent EET formation. Dexamethasone treatment increased AA epoxygenase-independent EET formation activity in liver microsomes. Subtracting the amounts of NADPH-independent EETs from total (NADPH-dependent+NADPH-independent) EETs after incubation of the enzyme with NADPH provides better assessment of catalytic activity of AA epoxygenases.

Epoxide hydrolase activity also can be measured by the ELISAs against 14,15-DHET because anti-DHET does not recognize 14,15-EET.

It has been previously shown that CYP2C23 is one of AA epoxygenases expressed in rats. So far, CYP2C11, CYP2C23, CYP2B1, CYP2B2, and CYP2J3 are identified as AA epoxygenases expressed in rats (1). Dexamethasone treatment substantially decreased hepatic AA epoxygenase activity which coincided with decrease in CYP2C23 protein levels. This unexpected result showed that 2C23 is a predominant AA epoxygenase isoform in the rat liver and hepatic AA epoxygenase expression is lowered by treatment of rats with chemicals such as glucocorticoids including dexamethasone.

In general the quantification of the sample is done utilizing an immunoassay as described in the Examples herein. However alternative immunoassays or GC/MS can be used as the assay in accordance with the present invention. Most of the techniques used in performing immunoassays are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraph may serve as a guideline.

In general, ELISAs are the preferred immunoassays employed to assess the amount of EETs and DHETs in a specimen. ELISA assays are well known to those skilled in the ag. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIAs) or fluoroimmunoassays (FIAs) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 and may be adapted to be used the method of the present invention.

The free and conjugated forms of the EETs and DHETs are measured utilizing the immunoassay as set forth for example in the Examples herein with an antibody which recognized both forms. Alternatively, antibodies can be utilized which are specific for each form.

Most of the techniques used to produce antibodies are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guideline. Antibody production: Antibodies (immunoglobulins) may be either monoclonal or polygonal and are raised against the immunogen. Such immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies: A laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y., 1988 and Borrebaeck, Antibody Engineering-A practical Guide, W. H. Freeman and Co., 1992. Antibody fragments may also be prepared from the antibodies and include Fab, F(ab')², and Fv by methods known to those skilled in the art.

For producing polycgonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen, generally together with an adjuvant and, if necessary, coupled to a carrier: antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific. That is, the sera can be absorbed against related immunogens, e.g. the free and conjugated forms of EETs and DHETs, so that no cross-reactive antibodies remain in the sera thereby rendering it monospecific. Testing for this specificity can be undertaken as described in Example 1.

For producing monoclonal antibodies the technique involves hyperimmunization of an appropriate donor with the immunogen or immunogen fragment, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.

For producing recombinant antibody (19) (20) (21), messenger RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reverse-transcribed to obtain complimentary DNAs (cDNAs). Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody.

The antibody or antibody fragment can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art to be used in the immunoassay. (For a general discussion of conjugation of fluorescent or enzymatic moieties (22). The binding of antibodies to a solid support substrate is also well known in the art (22) (24). The detectable moieties contemplated with the present invention can include ferritin, alkaline phosphatase, b-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴ C and iodination as needed for the immunoassay.

Claim 1 of 9 Claims

What is claimed is:

1. A method for assessing hypertension in a mammal comprising the steps of only measuring an amount of of free and conjugated dihydroxyeicosatrienoic acids and their metabolites of arachidonic acid converted to dihydroxyeicosatrienoic acid metabolites by both arachidonic acid (AA) epoxygenases and soluble epoxide hydrolases, in a first biological sample; comparing the amount of the free and conjugated dihydroxyeicosatrienoic acids and their metabolites to a second sample from a non-hypertension mammal of a same species and determining if the first mentioned sample contains a comparatively elevated amount of the free and conjugated dihydroxyeicosatrienoic acids and their metabolites as an indication of hypertension mediated by the epoxygenases and epoxide hydrolases.
 

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