WO1997013148A1

WO1997013148A1 - Method for measuring free radical cytotoxic degradation

Info

Publication number: WO1997013148A1
Application number: PCT/US1996/015792
Authority: WO
Inventors: Hildegarde L. A. Staninger
Original assignee: Staninger Hildegarde L A
Priority date: 1995-10-02
Filing date: 1996-10-01
Publication date: 1997-04-10
Also published as: AU7385496A

Abstract

A method is described for measuring the malondialdehyde level, and thus the oxidation state, of a body. A fluid sample is analyzed for one or more of a group of indicator compounds, including n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1-(2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile. A comparison of the analysis with an optimal profile also can be used to provide an indication of whether supplemental antioxidants should be administered.

Description

METHOD FOR MEASURING FREE RADICAL CYTOTOXIC DEGRADATION

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to biomedical testing methods, and, more particularly, to methods for assaying the oxidation state of a patient.

Description of Related Art

A free radical is an atom or group of atoms that has an unpaired valence electron. Free radicals can be produced by photolysis or pyrolysis in which a bond is broken without forming ions. Because of the free valency, most free radicals are extremely reactive.

Free radicals in living systems are known to be generated by heavy metals, some toxins, high-energy radiation, pesticides, and excessive exposure to hyperbaric oxygen. Such environmental factors may also create xenobiotics (non-naturally occurring chemicals present in the body) and/or metabolites that are toxic to the system. Within the body free radicals may be produced by the abnormal oxidation and breakdown of fats, the breakdown of hydrogen peroxide produced in the body, and escape from normal metabolic processes.

Peroxides in particular are formed by free radical oxidation of body fats and ingested oils (see FIG. 1). Peroxidized fats are immunosuppressants, mutagens, carcinogens, cross-linkers, and macrophage inhibitors. Cross-linking damage may contribute to disease states such as atherosclerosis and to the degradation of cell membranes, which can lead to cell lysis.

A typical sequence of events leading to the proliferation of free radicals is as follows:

RH + O₂ → R* + H₂O* (superoxide free radical)

R* +O₂ → RO₂*

RO₂* + RH → R* + ROOH (organic peroxide) R* + R* → R:R (cross-linked organic molecule)

R* + H₂O* → ROOH One compound that is known as a byproduct of lipid peroxidation is malondialdehyde (MDA), which has been used as an indicator of free radical damage in the body. Several methods have been disclosed for the quantification of MDA, including high-performance liquid chromatography (HPLC; Largilliere and Melancon, Anal. Biochem. 170, 123, 1988). In addition, the lysine derivative of MDA can be measured in urine samples using HPLC (Ekstrom et al., Chem. -Biol. Interactions 66, 177, 1988). SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a method for assaying the oxidation state of a body.

It is an additional object to provide a method for assaying for malondialdehyde levels in a body.

It is another object to provide a method for assaying for breakdown products of malondialdehyde levels in a body.

It is a further object to provide a method for optimizing the oxidation state of a body.

The method of the present invention is utilized for measuring a level of malondialdehyde in a body, which is believed to provide an indication of the body's oxidation state. The method comprises the steps of collecting a fluid sample from the body and analyzing the fluid sample for the presence of an indicator compound.

As was discussed above, it has proved difficult to assay for malondialdehyde directly, and thus the method of the present invention is directed to the identification and measurement of levels of one or more of a group of indicator compounds. This indicator compound may be chosen from a group consisting of n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1-(2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile.

Typically the method is carried out using either a urine sample or a blood sample, although the use of these fluids are not meant to be limiting, as it would be obvious to a person skilled in the art that testing would also be possible on samples of plasma, lymphatic fluids, perspiration, and breath, among others.

The features that characterize the invention, both as to organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description used in conjunction with the accompanying drawing. It is to be expressly understood that the drawing is for the purpose of illustration and description and is not intended as a definition of the limits of the invention. These and other objects attained, and advantages offered, by the present invention will become more fully apparent as the description that now follows is read. BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of the generation of free radicals and their interaction with organelles and other cell components.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A description of the preferred embodiments of the present invention will now be presented.

The present invention provides a method for measuring the malondialdehyde level in a body, which, as indicated above, is believed to provide an indication of the oxidation state of the body. For an indication of the formation and mechanisms of action of free radicals within the cell, see FIG. 1. Broadly, the method comprises the steps of collecting a fluid sample from the body, which typically comprises collecting a urine or blood sample, and analyzing the fluid sample for the presence of an indicator compound.

As listed previously, the indicator compound is chosen from the group consisting of n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1-

(2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile.

The structures for malondialdehyde and the group of indicator compounds are as follows:

1. malondialdehyde (MDA)

2. n-(2-propenal)lysine

3, n-alpha-acetyl ester (ethyl malonate)

CH₂(COCC₂H₅)₂ 4. serine ethanolamine

5. cyclized adduct with guanine to yield a guanine-malondialdehyde adduct

6. beta-methoxyacrolain (metagenic side product forMDA)

7. N-(2-propanal) ethanolamine

8. N-(E-2propenal) lysine

9. N-(2-propenal)serine

10. N-alpha-methyl-E-(2-propenal)lysine

11. methanol (methanol sub metabolite offormic acid)

CH₃OH

12. Formic acid

HCOOH

13. Formaldehyde

HCOH

14. 2,4-dinitro phenol hydrazine (DNPH)

16. 2,4-dintro phenyl hydrazone

17. 1,(2,4-dinitro phenyl)pyrazol

18. glutaraldehyde

C₅H₈O₂ (or) OHCCH₂CH₂CH₂CHO

19. glutathione

C₁₀H₁₇N₃O₆S (or)

20. glutaric acid

CDOH(CH₂)₃COOH (or) C₅H₈O₆

21. glutaronitrile

CN(CH₂)₃-CN (or) C₅H₆N₂

Generally, in order to provide a better indication, a plurality of these indicator compounds should be analyzed for.

A number of analytical methods can be used to identify and quantify the indicator compounds. Exemplary methods include, but are not limited to, high-performance liquid chromatography, immunoassay testing, color indicator testing, toxicological pathology specimen monitoring, electromagnetic resonance testing (harmonic reflectivity), and a combination of gas chromatography and mass spectrometry. In a preferred embodiment of the latter, secondary ion mass spectrometry is used.

One particular analytical method, high-performance liquid chromatography (HPLC), would be performed as follows:

1. Activate an HPLC cartridge with 60% methanol.

2. Dilute sample 1:10 in methanol.

3. Add 0.5 ml 2% acetic acid to 11 ml sample.

4. Shake for 30 min.

5. Perform solid-phase extraction of sample.

6. Degas sample.

7. Draw up sample into a syringe, attach syringe filter, and empty sample into vial. 8. Draw up sample into an HPLC syringe and inject 7.5-10 ml of sample onto HPLC column.

The expected run time is 10 minutes. The resulting chromatograph can then be analyzed for the indicator compounds, both qualitatively and quantitatively, by methods known in the art.

In an alternate embodiment of the invention, further steps can be performed to optimize the oxidation state of a body. Specifically, the fluid sample analysis is compared with a predetermined optimal presence and quantity of the indicator compound, from which may be determined the oxidation state of the body and thus whether the oxidation state is suboptimal. Then, if the oxidation state is determined to be suboptimal, antioxidant compounds are administered to the body to improve the oxidation state.

Many applications of this embodiment are envisioned, including monitoring the progress of AIDS, cancer, atherosclerosis, alateral sclerosis, muscular dystrophy, and juvenile diabetes. In addition, patients receiving treatments having potentially dangerous side effects such as chemotherapy can be monitored to determine if the course of treatment should be continued or altered.

In another embodiment, the method of the present invention could be coupled with other assay techniques, such as measurements of enzyme concentrations, to provide an indication of site-specific free radical damage. For example, a measurement of a liver enzyme coupled with the method of the present invention would indicate a level of cytotoxic degradation in the liver.

It may be appreciated by one skilled in the art that additional embodiments may be contemplated, including coupling the method with other biomedical and biochemical techniques to provide an indication of the oxidation state of the body. In the foregoing description, certain terms have been used for brevity, clarity, and understanding, but no unnecessary limitations are to be implied therefrom beyond the requirements of the prior art, because such words are used for description purposes herein and are intended to be broadly construed.

Moreover, the embodiments of the apparatus illustrated and described herein are by way of example, and the scope of the invention is not limited to the exact details of construction.

Having now described the invention, the construction, the operation and use of preferred embodiment thereof, and the advantageous new and useful results obtained thereby, the new and useful constructions, and reasonable mechanical equivalents thereof obvious to those skilled in the art, are set forth in the appended claims.

Claims

What is claimed is;

1. A method for measuring a level of malondialdehyde in a body, the method comprising the steps of:

collecting a fluid sample from the body; and

analyzing the fluid sample for the presence of an indicator compound, the indicator compound chosen from a group consisting of n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n- (2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1-(2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile.

2. The method recited in Claim 1, wherein the collecting step comprises collecting a urine sample.

3. The method recited in Claim 1, wherein the collecting step comprises collecting a blood sample.

4. The method recited in Claim 1, wherein the analyzing step comprises the step of performing high-performance liquid chromatography.

5. The method recited in Claim 1, wherein the analyzing step comprises the step of immunoassay testing.

6. The method recited in Claim 1, wherein the analyzing step comprises the step of color indicator testing.

7. The method recited in Claim 1, wherein the analyzing step comprises the step of toxicological pathology specimen monitoring.

8. The method recited in Claim 1, wherein the analyzing step comprises the steps of performing gas chromatography and mass spectrometry.

9. The method recited in Claim 8, wherein the performing mass spectrometry step comprises the step of performing secondary ion mass spectrometry.

10. The method recited in Claim 1, wherein the analyzing step comprises the step of performing electromagnetic resonance testing.

11. A method for measuring an oxidation state of a body, the method comprising the steps of:

collecting a fluid sample from the body; and analyzing the fluid sample for the presence of a plurality of indicator compounds, the indicator compounds comprising breakdown products of malondialdehyde, the indicator compounds chosen from a group consisting of n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guaninemalondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2, 4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1-(2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile.

12. The method recited in Claim 11, wherein the analyzing step comprises the step of performing high-performance liquid chromatography.

13. A method for optimizing an oxidation state of a body, the method comprising the steps of: collecting a fluid sample from the body;

analyzing the fluid sample for the presence and quantity of an indicator compound, the indicator compound chosen from a group consisting of n-(2-propenal) lysine, an n-α-acetyl ester of n-(2-propenal) lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4-dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1- (2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile;

comparing the fluid sample analysis with a predetermined optimal presence and quantity of the indicator compound to determine an oxidation state of the body;

determining from the comparison whether the oxidation state is suboptimal; and

administering, if the oxidation state is determined to be suboptimal, antioxidant compounds to the body to improve the oxidation state.

14. A method for measuring site-specific cytotoxic degradation in a body, the method comprising the steps of:

collecting a fluid sample from the body;

assaying the fluid sample for a level of an enzyme specific to the body site;

analyzing the fluid sample for the presence and quantity of an indicator compound, the indicator compound chosen from a group consisting of n-(2-propenal)lysine, an n-α-acetyl ester of n-(2-propenal)lysine (such as ethyl malonate), serine ethanolamine, a cyclized guanine-malondialdehyde adduct, β-methoxyacrolain, n-(2-propanal)ethanolamine, n-(E-2-propenal)lysine, n-(2-propenal)serine, n-α-methyl-E-(2-propenal)lysine, methanol, formic acid, formaldehyde, 2,4- dinitrophenol hydrazine (DNPH), 2,4-dinitro phenyl hydrazone, 1- (2,4-dinitro phenyl)pyrazole, glutaraldehyde, glutathione, glutaric acid, and glutaronitrile;

comparing the fluid sample analysis and assay with a predetermined optimal presence and quantity of the indicator compound and site-specific enzyme to determine an oxidation state of the body site; and

determining from the comparison whether the oxidation state is suboptimal.