U.S. patent application number 09/852066 was filed with the patent office on 2002-04-04 for enzyme method for detecting lysophospholipids and phospholipids and for detecting and correlating conditions associated with altered levels of lysophospholipids.
This patent application is currently assigned to Atairgin Technologies, Inc.. Invention is credited to Parrott, Jeff, Small, Chris, Xu, Liang Zhong.
Application Number | 20020039757 09/852066 |
Document ID | / |
Family ID | 22645951 |
Filed Date | 2002-04-04 |
United States Patent
Application |
20020039757 |
Kind Code |
A1 |
Small, Chris ; et
al. |
April 4, 2002 |
Enzyme method for detecting lysophospholipids and phospholipids and
for detecting and correlating conditions associated with altered
levels of lysophospholipids
Abstract
The present invention is an enzymatic method and diagnostic kits
for detecting and quantifying the presence of one or more
lysophospholids in a sample of bodily fluid taken from a test
subject. The method uses enzymes in a two step assay and may be
used to detect disease conditions associated with altered levels of
lysophospholipids and to correlate such conditions with altered
levels of lysophospholipids.
Inventors: |
Small, Chris; (Pullman,
WA) ; Parrott, Jeff; (Irvine, CA) ; Xu, Liang
Zhong; (Mountain View, CA) |
Correspondence
Address: |
LYON & LYON LLP
633 WEST FIFTH STREET
SUITE 4700
LOS ANGELES
CA
90071
US
|
Assignee: |
Atairgin Technologies, Inc.
|
Family ID: |
22645951 |
Appl. No.: |
09/852066 |
Filed: |
May 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09852066 |
May 8, 2001 |
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09176813 |
Oct 22, 1998 |
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6248553 |
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Current U.S.
Class: |
435/25 |
Current CPC
Class: |
C12Q 1/34 20130101; C12Q
1/26 20130101; G01N 2333/916 20130101; C12Q 1/28 20130101; G01N
2333/918 20130101; C12Q 1/44 20130101; G01N 2405/04 20130101; G01N
2333/906 20130101; C12Q 2326/96 20130101; G01N 2333/904
20130101 |
Class at
Publication: |
435/25 |
International
Class: |
C12Q 001/26 |
Claims
what is claimed:
1. A method to detect disease in a patient comprising: digesting a
phospholipid in a sample of bodily fluid from the subject with a
first enzyme to produce substrate; reacting the substrate with a
second enzyme in an enzymatic cycling reaction to produce a
detectable product; determining the concentration of phospholipid
by measuring the detectable product; and correlating the
concentration of phospholipid to the disease condition by
comparison to a normal concentration.
2. The method of claim 1, wherein said first enzyme is selected
from the group consisting of phospholipase B, lysophospholipase,
phospholipase A.sub.1, and phospholipase A.sub.2.
3. The method of claim 1, wherein the second enzyme is selected
from the group consisting of glycerol-3-phosphate dehydrogenase,
glycerol-3-phosphate oxidase, glycerokinase and glycerol
dehydrogenase.
4. The method of claim 1, wherein the substrate is
glycerol-3-phosphate.
5. The method of claim 1, wherein the detectable product is
hydrogen peroxide.
6. The method of claim 5, wherein the step of determining the
concentration of phospholipid by measuring detectable product
comprises measuring an increase in hydrogen peroxide by
colorimetry.
7. The method of claim 1, wherein the detectable product is
NADH.
8. The method of claim 7, wherein said step of determining the
concentration of the phospholipid by measuring the detectable
product comprises measuring oxidation of NADH.
9. The method of claim 1, wherein the step of reacting the
substrate in an enzyme cycling reaction comprises reacting G-3-P
with glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate
oxidase.
10. The method of claim 1, wherein, the sample of bodily fluid is
selected from the group consisting of plasma, serum, urine, saliva,
ascites, cerebral spinal fluid and pleural fluid.
11. The method of claim 1, further comprising the step of
extracting lipids from the sample of bodily fluid.
12. The method of claim 1, further comprising the step of comparing
the concentration of phospholipid with an earlier concentration
from the same subject.
13. The method of claim 1, wherein an increase or decrease in the
concentration of phospholipid relative to normal subjects indicates
the presence of the disease condition.
14. The method of claim 1, wherein the disease condition is
gynecological cancer or ovarian cancer.
15. The method of claim 1, wherein the disease condition is a blood
disorder associated with alteration in the level of phospholipid.
Description
RELATED APPLICATION INFORMATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/176,813 filed Oct. 12, 1998. The priority
of the prior application is expressly claimed, and the disclosure
of this prior application is hereby incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to enzyme methods for
detecting lysophospholipids, such as lysophosphatidic acid,
(LysoPA) and lysophosphatidyl choline (LysoPC), in biological
fluids, and for correlating and detecting conditions associated
with altered levels of lysophospholipids.
BACKGROUND OF THE INVENTION
[0003] Phosphatidyl choline (PC), also named lecithin, is one of
the major sources of polyunsaturated fatty acids such as
arachidonic and linoleic acids. The former is a precursor of
eicosanoids which have numerous biological activities. Hydrolysis
of PC yields lysophosphatidyl choline (LysoPC) and constituent
fatty acids, which have been implicated in signal transduction
(Asaoka et al., Proc. Natl. Acad. Sci. USA 90:4917-4921 (1993);
Yoshida et al., Proc. Natl. Acad. Sci. USA 89:6443-6446 (1992)). An
increasing body of evidence indicates that LysoPC, which is present
in high concentrations in oxidized low density lipoproteins may
play a significant role in atherogenesis and other inflammatory
disorders (Steinberg et al., New..Eng. J. Med. 320:915-924 (1989)).
LysoPC has been reported to increase the transcription of genes
encoding platelet derived growth factor A and B chains, and
heparin-binding epidermal growth factor-like protein (HB-EGF) in
cultured endothelial cells (Kume and Gimbrone, J. Clin. Invest.
93:907-911 (1994)), and to increase mRNA encoding HB-EGF in human
monocytes (Nakano et al., Proc. Natl. Acad. Sci. USA 91:1069-1073
(1994)). These gene products are mitogens for smooth muscle cells
and fibroblasts (Higashiyama et al., Science 251:936-939 (1991);
Ross, Nature (Lond.) 362:801-809 (1993)). LysoPC has also been
shown to activate protein kinase C in vitro (Sasaki et al., FEBS
Letter 320:47-51 (1993)), to potentiate the activation of human T
lymphocytes (Asaoka et al., Proc. Natl. Acad. Sci. USA 89:6447-6451
(1992)) and to potentiate the differentiation of HL-60 cells to
macrophages induced by either membrane-permeable diacylglycerols or
phorbol esters (Asaoka et al., Proc. Natl. Acad. Sci. USA
90:4917-4921 (1993)).
[0004] LysoPC may also provide a source of bioactive
lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate, LysoPA)
(Moolenaar et al., Rev. Physiol. Biochem. Pharmacol. 119:47-65
(1992)) through hydrolysis by lysophospholipase D (Tokumara et al.,
Biochim. Biophys. Acta 875:31-38 (1986)). LysoPA is a naturally
occurring phospholipid with a wide range of growth factor-like
biological activities. It is well established that LysoPA can act
as a precursor of phospholipid biosynthesis in both eukaryotic and
prokaryotic cells (Van den Bosch, Ann. Rev. Biochem. 43:243-277
(1974); Racenis et al., J. Bacteriol. 174:5702-5710 (1992)). The
ability of LysoPA to act as an intercellular lipid mediator has
been noted (Vogt, Arch. Pathol. Pharmakol. 240:124-139 (1960); Xu
et al., J. Cell. Physiol. 163:441-450 (1995); Xu et al.,
Biochemistry 309:933-940 (1995); Tigyi et al., Cell Biol.
91:1908-1912 (1994); Panetti et al., J. Lab. Clin. Med.
129(2):208-(1997)). LysoPA is rapidly generated by activated
platelets and can stimulate platelet aggregation and wound
repair.
[0005] Ovarian cancer activating factor (OCAF), has been isolated
from ovarian cancer ascites fluid (Mills et al., Cancer Res.
48:1066 (1988); Mills et al. J. Clin. Invest. 86:851 (1990) and
U.S. Pat. Nos. 5,326,690 and 5,277,917) and has been identified to
consist of multiple forms of LysoPA (Xu et al., Clin. Cancer Res.
1:1223-1232 (1995)). LysoPA has been identified as a potent tumor
growth factor in the ascites fluid of ovarian cancer patients
(Id.)
[0006] Other lysophospholipids associated with various conditions
include lysophosphatidyl 20 serine (LysoPS), lysophosphatidyl
ethanolamine (LysoPE), lysophosphatidyl glycerol (LysoPG and
lysophosphatidyl inositol (LysoPI). Activated platelets secrete two
kinds of phospholipase: sPLA2 and PS-PLA1. sPLA2 is reported to be
elevated in inflammatory reactions and inhibition of this enzyme
reduced inflammation (Schrier et al., Arthritis Rheum.
39(8):1292-1299 (1996); Tramposch et al., Pharmacol. and
Experimental Therapeutics 271 (2):852-859 (1994)). PS-PLA1
hydrolyzes phosphatidylserine or lysophosphatidyl seine (LysoPS)
specifically to produce LysoPS or Glycerol-3-P serine. LysoPS
strongly enhances degranulation of rat mast cells induced by
concanavalin A and potentiates histamine release (Tamori-Natori et
al., J. Biochem (Tokyo) 100(3):581-590 (1986)), and can stimulate
sPLA2-elicited histamine release from rat serosal mast cells (Hara
et al., Biol. Pharm. Bull. 19(3):474-476 (1996)). LysoPS is an
inflammatory lipid mediator (Lloret et al., J. Cell Physiol.
165(l):89-95 (1995)) and sPLA2 has been implicated in inflammation
processes (Lloret et al., Toxicon 32(11):1327-1336 (1994)). LysoPI
has been shown to stimulate yeast adenylyl cyclase activity with
implications for modulating the activity of downstream effector
molecules and their interaction with RAS proteins (Resnick and
Thomaska, J. Biol. Chem. 269(51):32336-32341 (1994)).
[0007] Methods for separating and semi-quantitatively measuring
phospholipids such as LysoPA using techniques such as thin-layer
chromatography (TLC) followed by gas chromatography (GC) and/or
mass spectrometry (MS) are known. For example, lipids may be
extracted from the test sample of bodily fluid using extraction
procedures such as those described by Bligh and Dyer, Can. J.
Biochem. Physiol. 37:911-917 (1959). Thin-layer chromatography may
be used to separate various phospholipids, for example as described
by Thomas and Holub, Biochim. Biophys. Acta, 1081:92-98 (1991).
Phospholipids and lysophospholipids are then visualized on plates,
for example using ultraviolet light as described by Gaudette et
al., J. Biol. Chem. 268:13773-13776 (1993). Alternatively,
lysophospholipid concentrations can be identified by NMR or HPLC
following isolation from phospholipids or as part of the
phospholipid (Creer and Gross, Lipids 20(12):922-928 (1985) and
Bowes et al., J. Biol. Chem. 268(19) 13885-13892 (1993)). LysoPA
levels have also been determined in ascites from ovarian cancer
patients using an assay that relies on LysoPA-specific effects on
eukaryotic cells in culture (Mills et al., Cancer Res. 48:1066-1071
(1988)). However, these prior procedures are time-consuming,
expensive and variable and typically only semi-quantitative.
[0008] Development of a rapid and sensitive assay for
lysophospholipid species would facilitate use of these
lysophospholipids as markers for cellular activities such as
platelet activation and for conditions associated with altered
levels of lysophospholipid species. Moreover, such assays would
provide a method for determining correlations between altered
levels of a lysophospholipid and conditions associated with such
altered levels.
SUMMARY OF THE INVENTION
[0009] The present invention encompasses enzymatic methods for
determining concentrations of lysophospholipids, such as LysoPA, in
samples of biological fluids such as serum or plasma. The methods
involves a two-step enzymatic digestion of at least one type of
lysophospholipid to produce a substrate for a subsequent enzymatic
reaction which produces a detectable end product that then permits
detection of the concentration of the lysophospholipid.
[0010] The methods are carried out by detecting the concentration
of a lysophospholipid such as LysoPA in a sample of bodily fluid
taken from a subject. The lysophospholipid in the sample is
preferably first enriched through extraction of lipids. For
example, polar lipids are redissolved in aqueous solution and the
concentration of lysophospholipid is determined using a two-step
enzymatic reaction. The lysophospholipid is digested using an
enzyme to generate a product that is then subject to a second
enzymatic reaction. In a specific embodiment, the second reaction
is an enzymatic cycling reaction that amplifies the signal. This
method permits measurement of a lysophospholipid present in small
amounts in the test sample.
[0011] In one embodiment, an enzyme such as lysophospholipase or
phospholipase B is used to liberate G3P from LysoPA. The level of
G3P is determined using an enzymatic cycling reaction that employs
G3P oxidase and glycerol-3-phosphate dehydrogenase in the presence
of NADH. The amount of LysoPA detected is quantitated
spectrophotometrically by measuring the oxidation of NADH.
Alternatively, the amount of LysoPA is determined colorimetrically
by detection of hydrogen peroxide generated by the cycling
reaction.
[0012] In addition to LysoPA, other lysophospholipids such as
LysoPC, lysophosphatidyl serine (LysoPS), lysophosphatidyl inositol
(LysoPI), lysophosphatidyl ethanolamine (LysoPE) and
lysophosphatidyl glycerol (LysoPG), can be detected using the
methods of the invention. For these lysophospholipids, alternative
enzymes for use in the methods include, but are not limited to,
phospholipase A.sub.1, phospholipase A.sub.2, phospholipase C,
phospholipase D, lecithinase B and lysolecithinase,
glycerophosphocholine phosphodiesterase and glycerol kinase.
[0013] The enzymatic methods of the invention can be used to detect
altered levels of lysophospholipid in a subject compared to normal
levels of the lysophospholipid in normal to detect conditions
associated with such altered levels of lysophospholipid. Diagnosis
of a condition using the methods of the invention may also be
performed by determining the rate of change over time of the
concentration of a lysophospholipid in samples taken from the
subject.
[0014] Another embodiment of the invention is use of the assay in a
method to determine whether a correlation exists between the level
of a lysophospholipid and the presence of a condition. In this
embodiment, the concentration of a lysophospholipid is determined
in samples from subjects known to have a specific disease
condition, such as an inflammatory condition, and compared to
concentration of that lysophospholipid in subjects free of such
condition. Altered levels of lysophospholipid in the samples from
the subjects having a condition as compared to samples from normal
subjects suggest a correlation between the levels of the
lysophospholipid and the presence of the condition.
[0015] Yet another embodiment of the methods of the invention is a
diagnostic kit containing enzyme and other reagents for conducting
the enzymatic assays of the invention to measure concentrations of
lysophospholipids in samples of bodily fluids taken from
subjects.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention provides enzymatic methods for
detecting and quantifying altered concentrations of
lysophospholipids, including, but not limited to, lysophosphatidic
acid (LysoPA), lysophosphatidyl choline (LysoPC), lysophophatidyl
serine (LysoPS), lysophosphatidyl inositol (LysoPI),
lysophosphatidyl ethanolamine (LysoPE) and lysophosphatidyl
glycerol (LysoPG) in a sample of bodily fluid from a subject.
[0017] The subject is an eukaryotic organism, preferably a
vertebrae, including, but not limited to, a mammal, a bird, a fish,
an amphibium, or a reptile. Preferably, the subject is a mammal,
most preferably a human. The bodily fluid includes, but is not
limited to, plasma, serum, urine, saliva, ascites, cerebral spinal
fluid or pleural fluid.
[0018] The conditions correlated with altered concentrations of
these lysophospholipids include, but are not limited to,
inflammatory conditions, i.e. conditions associated with platelet
activation. Altered phospholipid metabolism has been reported in a
number of diseases (for review see Gregor Cevc (Ed.), Phospholipids
Handbook, Ch. 28: Gupta, Phospholipids in Disease, pp. 895-908
(1993)) and can lead to altered lysophospholipid and phospholipid
levels in biological fluids. These diseases include, but are not
limited to, sickle cell anemia, diabetes, muscular dystrophy,
ischemia, liver disease, lung disease, heart disease, malaria,
Alzheimer's, Parkinson's and various cancers. In these conditions,
defective cellular functions may directly or indirectly lead to
changes in steady state levels of phospholipids. Other diseases
include bleeding disorders including those associated with abnormal
platelet function resulting in coagulopathy.
[0019] Thus, the methods of the present invention are directed to
the detection of conditions that are known to correlate, or the
identification of conditions to correlate, with altered
concentrations of lysophospholipids in the bodily fluids from a
subject relative to concentrations found in bodily fluids from a
subject lacking a condition associated with altered concentrations
of lysophospholipids (i.e. "normal subjects").
Uses of the Invention
[0020] The methods of the invention provides a rapid and accurate
assay with increased sensitivity for detecting small amounts of
lysophospholipids present in samples of bodily fluids from
subjects. The enzymatic assay can be used to detect conditions
associated with altered levels of lysophospholipids in a sample
from a subject as compared to normal samples. In addition, the
assay permits determination of correlations between various disease
conditions and alterations in the levels of lysophospholipids. The
methods of the invention and test kits thus provide a practical
means to detect conditions associated with altered levels of
certain lysophospholipids.
Enzymatic Methods for Detecting and Ouantifying
Lysophospholipids
[0021] The methods of the invention are carried out as follows. A
biological sample such as whole blood is collected from a subject.
Lipids are extracted from plasma or serum from the sample, for
example, by organic extraction using chloroform:methanol and
centrifugation and enriching for a selected species of
lysophospholipid, e.g. LysoPA, or for total lysophospholipids. The
need for enrichment depends in part on the specificity of the
enzyme used to digest the lysophospholipid to be determined. An
enzyme which hydrolyzes the lysophospholipid is incubated with the
extracted lipid sample producing a smaller metabolite. Next another
enzymatic digestion is performed to produce a detectable product.
In one embodiment an enzyme cycling reaction which consists of two
enzymatic reactions that accumulates detectable products is
performed. In the Examples herein to detect LysoPA levels,
Phospholipase B (PLB) or lysophospholipase (LYPL, EC 3.1.1.5, Asahi
Chemical Industry Co., Ltd., Tokyo, Japan) is used to produce
glycerol-3-phosphate (G-3-P). An enzyme cycling reaction is then
performed using glycerol-3-phosphate dehydrogenase,
glycerol-3-phosphate oxidase and NADH to accumlate H.sub.2O.sub.2
and NAD (U.S. Pat. No. 5,122,454, Ueda et al.)
[0022] The level of LysoPA is detected by monitoring the oxidation
of NADH spectrophotometrically at 340 nm (i.e. disappearance of
OD.sub.340) and the accumulation of H.sub.2O.sub.2 colorimetrically
using peroxidase. Numerical values are obtained from a standard
curve consisting of known C18:1 LysoPA. Typical standard curves
include known amounts of LysoPA from 0 to 3 .mu.M. Assays are
preferably performed in duplicate with both positive and negative
controls. The difference between OD.sub.340 before and after the
enzyme cycling reaction is directly proportional to the amount of
LysoPA present. Background signals in plasma without phospholipase
B are substracted from all samples. LysoPA standard curve values
are plotted and fitted to a linear or second-order polynominal
curve fit. The levels of LysoPA in each sample are determined by
comparing each signal measured to the standard curve.
[0023] Alternatively, the lysophospholipid can be detected using
additional and/or different enzyme combinations. For example,
phospholipase C (BC 3.1.4.3, Sigma Chemical Co., St. Louis, Mo.) is
used to cleave inorganic phosphate (Pi) from LysoPA. Levels of
LysoPA are then determined by measuring the amount of liberated Pi
using established procedures, e.g. using a commercially available
kit (Procedure 670, Sigma Chemical Co., St. Louis, Mo.). For
increased sensitivity, Pi is determined using purine nucleoside
phosphorylase (PNP), xanthine oxidase (XOD) and urate oxidase (UOD)
as previously described (Kawasaki et al., Analytical Biochem.
182:366-370 (1989)). The latter method generates 3 H.sub.2O.sub.2
molecules for every Pi. The accumulation of H.sub.2O.sub.2 is
detected colorimetrically using peroxidase.
[0024] In another embodiment, the lysophospholipid, such as LysoPA,
is incubated with phospholipase B or lysophospholipase to produce
G-3-P. G-3-P is converted to dihydroxyacetone phosphate and
hydrogen peroxide using G-3-P oxidase in the presence of oxygen and
water. In the presence of NADH, G-3-P dehydrogenase converts
dihydroxyacetone phosphate back to G-3-P and oxidizes NADH to NAD.
The disappearance of NADH is monitored spectrophotometrically at
OD.sub.340. Alternatively, the production of hydrogen peroxide may
be measured, for example colorimetrically by fluorometry or
chemiluminescence. For a colorimetric assay any of a number of
chromogenic substrates may be used including 4-aminoantipyrine
(AAP), pyrogallol, 2-(2'-Azinobis (3-ethylbenzthiazoline-sulfonic
acid) (ABTS) and 3,3',5,5'-tetramethylbenzidine) (TMB).
[0025] In yet another embodiment, LysoPC may be determined by
liberating glycerophosphorylcholine (GPC) and fatty acid from
LysoPC using phospholipase B or lysophospholipase. The level of
LysoPC is determined by liberating choline and glycero-3-phosphate
(G-3-P) from GPC using GPC phosphodiesterase (GPC-PDE) followed by
a colorimetric enzymatic determination of choline using choline
oxidase, 4-aminoantipyrine (AAP), 3,5
Dichloro-2-hydroxybenzenesulfonic acid sodium salt (HDCBS) and
peroxidase. Choline is detected by oxidizing to H.sub.2O.sub.2 and
betaine and then using peroxidase to form quinoneimine dye.
Alternatively, G-3-P is measured using G-3-P dehydrogenase and
oxidase in the cycling reaction of the assay of the invention.
[0026] In addition to LysoPA and LysoPC, other lysophospholipids
such as lysophosphatidyl serine (LysoPS), lysophosphatidyl inositol
(LysoPI), lysophosphatidyl ethanolamine (LysoPE) and
lysophosphatidyl glycerol (LysoPG), can be detected using the two
step enzymatic assay methods of the invention.
[0027] Enzymes for use in the first step of the method to digest
lysophospholipids include, but are not limited to,
lysophospholipase, phospholipase B, phospholipase A.sub.1,
phospholipase A.sub.2, phospholipase C, and phospholipase D.
[0028] Enzymes for use in detecting the product of enzymatic
digestion of lysophospholipids in step one include
glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate oxidase,
glycerophosphorylcholine phosphodiesterase (GPC-PDE), choline
oxidase, serine dehydrogenase, serine deaminase, aldehyde
dehydrogenase, ethanolamine deaminase, glycerokinase and glycerol
dehydrogenase.
[0029] For example, to determine LysoPS, the LysoPS is digested by
phospholipase D into serine and LysoPA. The amount of serine
produced is determined by detecting NADH formation (absorbance at
A.sub.340) via serine dehydrogenase. Alternatively, the serine is
deaminated using deaminase to form ammonia (NH.sub.3) and
HOCH.sub.2-CO--COOH. Alternatively, LysoPS can be digested by
lysophospholipase to form Glycerol-3-P serine which is then
digested using glycerol-3-P choline phosphodiesterase (GPC-PDE) to
form Glycerol-3-P and serine. The LysoPS is determined by detecting
NH.sub.3 production or NADPH production via serine dehydrogenase or
by using a Lyso-PS specific lysophospholipase enzyme.
[0030] LysoPE can be determined using the enzyme assay of the
invention by hydrolyzing LysoPE into LysoPA and ethanolamine by
phospholipase D. The ethanolamine is then deaminated by deaminase
and dehydrogenated to produce NADH to produce HOCH.sub.2--CHO and
NH.sub.3 The HOCH.sub.2--CHO is then digested with aldehyde
dehyrogenase to form NADH which is then detected by spectrometry
(e.g. at A.sub.340). Alternatively a LysoPE-specific
lysophospholipase enzyme can be used to hydrolyze LysoPE to
Glycerol-3-P ethanolamine which in turn is hydrolyzed to
Glycerol-3-P by glycerophosphorylcholine phosphodiesterase
(GPC-PDE). Glycerol-3-P is then measured using the cycling reaction
of the invention.
[0031] In the methods of the invention, an alternative to the
liquid organic extraction for enrichment includes the use of solid
phase extraction using, e.g. a Bond-Elut.RTM. column (Varian,
Harbor City, Cailf.) consisting of silica or fluorosil can be used
to enrich for the lysophospholipid and to remove proteins and other
lipids.
[0032] In order to optimize recovery of the desired
lysophospholipid, inhibitors of endogenous enzymes that may be
present in the sample may be used to prevent an increase in
background levels of lysophospholpid or degradation of the
lysophospholipid levels in the sample. Such inhibitors include
specific PLA.sub.2 inhibitors such as Aristolic Acid
(9-methoxy-6-nitrophenanthro- (3,4-d)-dioxole-5-carboxylic acid,
Biomol Research Laboratories, Plymouth Meeting, Pa.); ONO-R-082
(2-(p-Amylcinnamoyl)amino-4-chlorobenzoic acid, Biomol); OBAA
(3-(4-Octadecyl)-benzoylacrylic acid, Biomol), 4-Bromophenacyl
Bromide (Sigma); Quincrine
(6-Chloro-9-(4-diethylamino)-1-methylbutyl)amino-2-met-
hoxycridine, Mepacrine, Sigma); Manoalide (Biomol) and HELSS
(Haloenol lactone suicide substrate, Biomol); phosphodiesterase
inhibitors such as IBMX (3-Isobutyl- 1-methylxanthine, CalBiochem,
La Jolla, Cailf.); Ro-20- 1724 (CalBiochem); Zaprinast (CalBiochem)
and Pentoxifylline (CalBiochem); general protease inhibitors such
as E-64 (trans-Epoxysuccinyl-L-leucylamido-(4-guanidino) butane,
Sigma); leupeptin (Sigma); pepstatin A (Sigma); TPCK
(N-tosyl-L-phenylalanine chloromethyl ketone, Sigma); PMSF
(Phenylmethanesulfonyl fluoride, Sigma); benzamidine (Sigma) and
1,10-phenanthroline (Sigma); organic solvents including chloroform
and methanol; detergents such as SDS; proteases that would degrade
phospholipases such as trypsin (Sigma) and thermostable protease
(Boehringer Mannheim Biochemicals, Indianapolis, Ind.); and metal
chelators such as EDTA (Ethylenediaminetetracetic acid, Sigma) and
EGTA (Ethylene glycol-bis-(beta-aminoethyl ether), Sigma).
[0033] The assay may be performed in a microtiter plate format to
permit small volumes of samples and reagents to be employed and for
monitoring, e.g. using an ELISA reader. These formats facilitate
automating the performance of the assay. Reduced processing times
for the assays using such formats may reduce variability between
results.
[0034] Correlation of Lysophospholipid Levels with Disease
[0035] Initially, physiological ("normal") concentrations of
lysophospholipids and/or specific lysophospholipid species are
determined in subjects not having a disease condition.
Subsequently, the concentration of the lysophospholipids are
measured in a sample of bodily fluid from a test subject to be
screened for the disease and compared to the concentrations
established for normal subjects. Concentrations of lysophospholipid
that are significantly increased or decreased relative to normal
controls, for example one or more standard deviations above normal,
may indicate the presence of a condition associated with altered
levels of the lysophospholipid.
[0036] In addition, the response of a condition to treatment may be
monitored by determining concentrations of lysophospholipid in
samples taken from a subject over time. The concentration of a
lysophospholipid is measured and compared to the concentration
taken at the earlier time from that patient. If there is an
increase in the concentration of lysophospholipid over time, it may
indicate an increase in the severity of the condition in the
subject. Conversely, if there is a decrease in the concentration of
lysophospholipid, it may indicate an improvement in the condition
of the subject.
[0037] Diagnostic Kits
[0038] The methods described herein for measuring concentrations of
lysophospholipids in samples of bodily fluids from a subject may
also be performed, for example, by using pre-packaged diagnostic
kits. Such kits include enzyme reagents for digesting one or more
lysophospholipid, for example phospholipase B. The reagents include
those to perform the enzyme cycling reaction such as
glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate oxidase
and .beta.-nicotinamide adenine dinucleotide (NADH) and ancillary
agents such as buffering agents, and agents such as EDTA to inhibit
subsequent production or hydrolysis of lysophospholipids during
transport or storage of the samples. The kits may also include an
apparatus or container for conducting the methods of the invention
and/or transferring samples to a diagnostic laboratory for
processing, as well as suitable instructions for carrying out the
methods of the invention.
[0039] The following examples are presented to demonstrate the
methods of the present invention and to assist one of ordinary
skill in using the same. The examples are not intended in any way
to otherwise limit the scope of the disclosure or the protection
granted by Letters Patent granted hereon.
EXAMPLES
Example I
Detection and Ouantitation of Lysopa Levels in Human Plasma
[0040] Reagents
[0041] Phospholipase B (PLB), glycerol-3-phosphate oxidase,
glycerol-3-phosphate dehydrogenase, human plasma, human serum,
4-aminoantipyrine (AAP) and calcium chloride were purchased from
Sigma Chemical Co., St. Louis, Mo. Lysopholipase (LYPL) was
purchased from Asahi Chemical Industry, Tokyo, Japan. Peroxidase
and NADH were purchased from Boerhinger Mannheim, Indianapolis,
Ill. All lipid standards, fatty acids and methyl esters were
purchased from Avanti Polar Lipids, Alabaster, Ala. or Sigma
Chemical Co. 3,5 Dichloro-2-hydroxybenzenesulfon- ic acid sodium
salt (HDCBS) was purchased from Biosynth AG, Naperville, Ill.
[0042] Sample Collection and Processing
[0043] Blood was collected in BD vacutainer tubes #6415 or #7714
utilizing a 3.2% buffered citrate (acid citrate) and maintained
capped on ice until processing. Within 1 hour of draw, blood was
centrifuged at 3000.times.g (in a cold centrifuge if possible) for
15 minutes. Plasma was removed and transferred to a plastic tube
and frozen at -20.degree. C. to -80.degree. C. Alternatively, blood
was drawn into EDTA-containing vacutainer tubes and centrifuged at
580.times.g for 5 minutes. The supernatant was transferred to a
siliconized tube and centrifuged again at 8000.times.g for 5
minutes. The supernatant was collected into another siliconized
tube and frozen at -70.degree. C.
[0044] Sample Preparation and Thin Layer Chromatography
[0045] Approximately 0.5 ml of plasma was added to 3.75 ml of
chloroform:methanol (1:2), vortexed and centrifuged at 3000 rpm for
10 minutes. The supernate was decanted into a new tube to which was
added 1.25 ml chloroform and 1.75 ml water. This mixture was
vortexed and centrifuged again to yield a biphasic solution. The
lower layer was saved and the upper layer was collected into
another tube. To this upper layer, 2.5 ml chloroform and 63 .mu.l
concentrated hydrochloric acid were added. The mixture was vortexed
and then centrifuged again. The lower layer resulting from this
acidified chloroform extraction was collected and pooled with the
lower layer that was saved. The pooled extract volume was reduced
to less than 50 .mu.l under a nitrogen stream and spotted onto the
origin of a silica gel G TLC plate (Fisher Scientific, Santa Clara,
Cailf.). Chromatography was performed in a solvent system
containing chloroform:methanol:ammonium hydroxide (65:35:5.5).
[0046] Lipids and standards were visualized by spraying the
developed plate with Rhodamine 6G (Sigma Chemical) in water and the
spot corresponding to LysoPA was scraped from the plate. Each
sample was spiked with heptadecanoic acid as an internal standard.
The fatty acids were hydrolyzed by adding 1 ml of 1N NaOH in
methanol and incubating at 100.degree. C. for 15 minutes. After
cooling, 1 ml of boron triflouride (14% in methanol, Alltech
Associates, Deerfield, Ill.) was added and the sample incubated 30
minutes at room temperature to produce methyl esters. 2 ml hexane
and 1 ml water were added and the mixture was vortexed thoroughly
and centrifuged for 3-5 minutes at 3000 rpm to facilitate phase
separation. The organic (top) layer was collected, dried under
nitrogen, resuspended in 25 .mu.l hexane and sealed in an
autosampler vial.
[0047] Gas Chromatography
[0048] Fatty acid methyl esters (FAMES) were quantified using gas
chromatography (GC) on a Hewlett Packard 5890 Series II GC fitted
with an autosampler and flame ionization detector. 2 .mu.l of
sample in hexane were injected into a Supelco S PB-5 capillary
column (Supelco, Bellefonte, Pa.). The GC program was set as
follows: 170-235.degree. C. at 10.degree. C. per minute and then
held at 235.degree. C. for 13.5 minutes for a total run time of 20
minutes. Retention times for the methyl esters were determined
using known standards and compared to peaks in unknown samples.
Quantitation of peaks was performed by comparison to a heptadeconic
acid standard curve using calibration against the heptadecanoic
acid internal standard.
[0049] Sample Preparation for the Enzymatic Assay
[0050] Approximately 0.5 ml of plasma were added to 3.75 ml of
chloroform:methanol (1:2), vortexed and centrifuged at 3000 rpm for
10 minutes. The supernate was decanted into a new tube to which was
added 1.25 ml chloroform and 1.75 ml water. This mixture was
vortexed and centrifuged as above to yield a biphasic solution. The
upper layer was collected into another tube and 2.5 ml chloroform
and 63 .mu.l concentrated hydrochloric acid were added, the mixture
vortexed and centrifuged as before. The lower layer was collected
and transferred into a clean tube. The sample was evaporated
completely under nitrogen and the dried lipid extract was
reconstituted in 250 .mu.l of sample buffer containing 2.5% Triton
X-100, 5 mM CaCl.sub.2, and 100 mM Tris (pH 8.0). The sample was
stored at -70.degree. C. until it was assayed.
[0051] Alternatively, a modified extraction procedure was developed
that only utilized 100 .mu.l of sample and significantly reduced
the levels of contaminating lipids such as phosphatidylcholine and
lysophosphatidylcholine. In this extraction, 0.1 ml of plasma was
added to 0.75 ml of chloroform:methanol (1:2), vortexed and
centrifuged at 14,000 rpm for 5 minutes. The supernate was decanted
into a new tube to which was added 0.25 ml of chloroform and 0.35
ml of water. This mixture was vortexed and centrifuged as above to
yield a biphasic solution. The lower layer was discarded and to the
remaining upper layer was added 0.5 ml chloroform. The sample was
vortexed and centrifuged again at 14,000 rpm for 5 minutes. Once
again the lower layer was discarded. To the upper layer, 0.5 ml
chloroform and 12.6 .mu.l concentrated hydrochloric acid were
added, the mixture vortexed and centrifuged as before. The
acidified lower layer was collected and transferred to a clean
tube. The sample was evaporated completely under nitrogen and
reconstituted in 100 .mu.l of sample buffer containing 2.5% Triton
X-100, 5 mM CaCl.sub.2, and 100 mM Tris (pH 8.0). The sample was
stored at -70.degree. C. until assayed.
[0052] Enzyme Assay
[0053] In the well of a 96 well microtiter plate, 5-100 .mu.l of
the extracted lipid sample was incubated with 0.25 units of
phospholipase B or LYPL in 100 mM Tris (pH 8.0) at 37.degree. C.
for 30-60 minutes to produce G-3-P: 100 .mu.l of cycling reaction
enzyme mix containing 1.7 units of G-3-P dehydrogenase, 4 units of
G-3-P oxidase, 0.25 MM NADH and 5 mM CaCl.sub.2 in 50 mM Tris (pH
8.0) was added and the mixture incubated at 37.degree. C. for an
additional 60 minutes. The G-3-P oxidase converts G-3-P to
dihydroxyacetone phosphate and H.sub.2O.sub.2. The dihydroxyacetone
phosphate is in turn converted back to G-3-P by G-3-P
dehydrogenase. This reaction oxidizes NADH to NAD. Therefore, as
cycling continues, both H.sub.2O.sub.2 and NAD accumulate.
[0054] The level of LysoPA was determined by monitoring the
oxidation of NADH (i.e. the reduction of absorbance at 340 nm after
the cycling action compared to A.sub.340 before cycling). In
addition, the accumulation of H.sub.2O.sub.2 was determined
colorimetrically by adding 50 .mu.l of a solution containing 0.5
units peroxidase, 0.5% HDCBS and 0.15% AAP in 100 mM Tris 8.0 to
each well and recording the absorbance at 505 nm.
[0055] Numerical values for concentrations of LysoPA were obtained
from a standard curve constructed from known LysoPA amounts. An
internal standard of extracted plasma was included within each
assay (i.e. each plate) that was measured at different dilutions.
In some cases, this internal standard was used to correct for
variations between different experiments. Internal standards were
also measured in the absence of PLB or LYPL enzyme. This
"no-enzyme" sample provided a background value that was subtracted
from each unknown when calculating the LysoPA levels using the NADH
measurement. When the colorimetric method was used, the plate was
blanked at 505 nm prior to color development.
[0056] Results
[0057] The results of the two-step enzymatic assay of the invention
are shown in Table 1.
1TABLE I ENZYME ASSAY TO DETECT LYSOPA Enzyme Assay TLC/GC Assay
Sensitivity 0.2 .mu.M 1 .mu.M Inter-assay variability 5% 15%
Intra-assay variability <5% 15% Yield 90% 10% Sample Volume 0.1
ml 0.5-1 ml Processing Time 3-4 hours 1-2 days (20 samples)
[0058] These results demonstrate the advantages of the present
enzymatic assay as compared to the TLC/GC assay. The assay is
linear from 0.2 .mu.M to 1 .mu.M of LysoPA concentration. In
addition, the enzymatic assays of the present invention provide
high yield, increased sensitivity and rapid processing time.
EXAMPLE II
[0059] Detection and Ouantitation of Lysopc Levels in Human Plasma
and Serum
[0060] Reagents
[0061] Lysophospholipase (LYPL) was purchased from Asahi Chemical
Industry, Tokyo, Japan. Glycerophosphorylcholine phosphodiesterase
(GPC-PDE), choline oxidase, and 4-aminoantipyrine (AAP) were
purchased from Sigma Chemical Co., St. Louis, Mo. Peroxidase was
purchased from Boerhinger Mannheim, Indianapolis, Ind. 3,5
Dichloro-2-hydroxybenzenesulf- onic acid sodium salt (HDCBS) was
purchased from Biosynth AG, Naperville, Ill. All lipid standards
and fatty acids were purchased from Avanti Polar Lipids, Alabaster,
Ala. or Sigma Chemical Co.
[0062] Sample Collection and Processing
[0063] Blood was collected and plasma was processed as described in
Example I. For serum, blood was collected in silicone-coated
Vacutainer tubes (Red Top) and was centrifuged under normal
conditions. Serum and plasma was transferred to plastic tubes and
stored frozen at -20.degree. C. to -80.degree. C.
[0064] Sample Preparation for the Enzymatic Assay
[0065] Approximately 35 .mu.l plasma or serum was diluted 1:10 in
sample buffer (1% Triton, 10 mM calcium chioride, 50 mM Tris pH
8.0) to a total volume of 350 .mu.l.
[0066] Enzymatic Assay
[0067] In the well of a 96 well microtiter plate, 100 .mu.l of the
diluted lipid is aliquoted in replicate. To each well, 50 .mu.l of
LYPL (0.125 Units) /GPC-PDE (0.0125 Units) is added and incubated
at 37.degree. C. for 10 minutes. This reaction produces
glycerophosphorylcholine as an intermediate through LYPL digestion
of LysoPC. The GPD-PDE then liberates G-3-P and choline from
glycerophosphorylcholine. The plate is then blanked A505 in the
ELISA reader. Next, 50 .mu.l choline detection mix (0.15 Units
choline oxidase, 0.5 Units peroxidase, 0.03% AAP, 0.125% HDCBS, 100
mM Tris pH 8.0) is added and incubated at 37.degree. C. for 15
minutes. The plate is then read at A.sub.505.
[0068] Table II illustrates the results of the assay for LysoPC.
The assay is linear from 5 to 200 .mu.M LysoPC, sensitive to 5
.mu.M LysoPC and exhibits low intra-assay and inter-assay
variability.
2TABLE II ENZYME ASSAY TO DETET LYSOPC IN PLASMA Sensitivity 5
.mu.M Linear Range 5-200 .mu.M Intra-assay variability 3.0%
Inter-assay variability 6.0%
[0069] These results show that LysoPC is easily detected in plasma
or serum using the two-step enzyme assay of the invention. Similar
results were obtained from plasma or serum from the same patient,
demonstrating that the method is applicable to either plasma or
serum. Typical LysoPC levels in plasma or serum ranged from 50
.mu.M to 500 .mu.M. As a result, LysoPC can be determined in a 1:10
diluted sample using this assay.
EXAMPLE III
Detection and Ouantitation of Lysopa in Samples from Patients
having Cancer
[0070] LysoPA levels were determined in plasma of both non-cancer
subjects and patients having ovarian cancer. Blood was collected
from female patients and was processed as described above in
Example I. Plasma from the samples was prepared for the enzymatic
assay of the invention as described above in Example I. The enzyme
assay was performed as described above in Example I.
[0071] Average LysoPA levels for non-cancer and cancer patients as
determined using the enzyme assay shows that average levels of
LysoPA were significantly increased in the plasma of patients
having ovarian cancer as determined using the methods of the
invention.
[0072] In addition, levels of LysoPC and PC were determined from
the plasma of patients with and without ovarian cancer using the
enzyme assay as described above in Examples II and III. These
results were combined and multipled to yield a multi-lipid
diagnostic measurement. Levels of LysoPC and PC determined
independently were 10 to 100% higher in ovarian cancer versus
normal patients. Combining and multiplying LysoPA X LysoPC X PC
levels for each sample yielded a measurement from 400% to 500%
higher in ovarian cancer versus normal patients. These results
suggest that the combinatorial approach may provide a more accurate
assay for detecting conditions such as cancer associated with
altered levels of lysophospholipids and phospholipids by reducing
the number of false positive and false negative results.
EXAMPLE IV
Detection and Ouantification of Lysopa in Patients Having a
Bleeding Disorder
[0073] LysoPA levels were determined as described above in Example
I in 93 plasma samples from male and female patients over an age
range of 1-80 years. Of the 93 samples, 17 of samples came from
patients who were previously diagnosed with bleeding disorders
(i.e. coagulopathy). LysoPA levels were determined. Patients having
a bleeding disorder demonstrated significantly higher average
LysoPA levels than those patients not having cancer or a bleeding
disorder.
[0074] The results from the examples herein demonstrate that the
methods of the invention can be used to detect altered
lysophospholipid and phospholipids such as PC levels in patients
having various disease conditions associated with such altered
levels. Moreover, these results provide a new method for diagnosing
disease conditions associated with altered levels of
lysophospholipids in which levels of different phospholipids such
as LysoPA and LysoPC in plasma or serum are multiplied to detect
the disease condition.
[0075] Various publications are cited herein which are hereby
incorporated by reference in their entirety.
[0076] As will be apparent to those skilled in the art in which the
invention is addressed, the present invention may be embodied in
forms other than those specifically disclosed above without
departing from the spirit or potential characteristics of the
invention. Particular embodiments of the present invention
described above are therefore to be considered in all respects as
illustrative and not restrictive. The scope of the present
invention is as set forth in the appended claims and equivalents
thereof rather than being limited to the examples contained in the
foregoing description.
* * * * *