U.S. patent application number 11/586781 was filed with the patent office on 2007-02-22 for diagnostic methods for determining susceptibility to convulsive conditions.
This patent application is currently assigned to Queens University at Kingston. Invention is credited to Allyson J. Campbell, John R. Carran, Angela P. Lyon, Donald F. Weaver.
Application Number | 20070042497 11/586781 |
Document ID | / |
Family ID | 26981328 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042497 |
Kind Code |
A1 |
Campbell; Allyson J. ; et
al. |
February 22, 2007 |
Diagnostic methods for determining susceptibility to convulsive
conditions
Abstract
The present invention exploits the discovery that amounts of
uracil and thymine metabolites, especially .beta.-aminoisobutyric
acid, in various bodily fluids, especially urine, are correlated
with the occurrence of epilepsy when compared to matched control
subjects. Analytical and diagnostic protocols, including a novel
high performance liquid chromatography system, for use in the
invention are disclosed.
Inventors: |
Campbell; Allyson J.;
(Kingston, CA) ; Weaver; Donald F.; (Halifax,
CA) ; Lyon; Angela P.; (Kingston, CA) ;
Carran; John R.; (Kingston, CA) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Assignee: |
Queens University at
Kingston
Kingston
CA
|
Family ID: |
26981328 |
Appl. No.: |
11/586781 |
Filed: |
October 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11106369 |
Apr 13, 2005 |
7153692 |
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11586781 |
Oct 26, 2006 |
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10222957 |
Aug 16, 2002 |
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11106369 |
Apr 13, 2005 |
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60318139 |
Sep 7, 2001 |
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60378781 |
May 7, 2002 |
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Current U.S.
Class: |
436/86 |
Current CPC
Class: |
G01N 33/493 20130101;
G01N 33/6812 20130101; G01N 2800/2857 20130101 |
Class at
Publication: |
436/086 |
International
Class: |
G01N 33/50 20070101
G01N033/50 |
Claims
1. A method of diagnosing a convulsive condition or susceptibility
thereto in a subject comprising the steps of a. analyzing a bodily
fluid from a subject for the presence or amount of a neuro-active
molecule, or the relative amounts of neuro-active molecules,
associated with a convulsive condition; and b. ediagnosing the
subject as at risk of a convulsive condition or susceptibility
thereto when the amount of said compound indicates a likelihood of
same in said subject.
2. A method of modulating the onset of a convulsive condition in a
subject comprising the steps of analyzing a bodily fluid from a
subject at risk of a convulsive condition for the presence or
amount of a neuro-active molecule, or the relative amounts of
neuro-active molecules, associated with a convulsive condition;
determining from the amount of said compound in said bodily fluid
whether said subject is at risk of a convulsive condition; and
treating said subject, if at risk of a convulsive condition, to
modulate the onset of said convulsive condition in said
subject.
3. A method of diagnosing a convulsive condition or susceptibility
thereto in a subject comprising the steps of analyzing a bodily
fluid from a subject for the presence or amount of a .beta.-amino
acid, or the relative amount of .beta.-amino acid; and diagnosing
the subject as at risk of a convulsive condition when the amount of
said .beta.-amino acid indicates a likelihood of same in said
subject.
4. (canceled)
5. The method of claim 1, wherein said neuro-active molecule is a
metabolite of uracil or thymine, or a derivative thereof.
6. The method of claim 1, wherein said compound is an amino acid or
a derivative thereof.
7. The method of claim 5, wherein said metabolite is a .beta.-amino
carboxylic acid.
8. The method of claim 5, wherein said .beta.-amino acid is
selected from .beta.-aminoisobutyric acid and derivatives
thereof.
9. The method of claim 1, wherein said convulsive condition is
selected from the group consisting of epileptogenic associated
disorders, epileptogenesis, and non-epileptic convulsions.
10. The method of claim 1, wherein said convulsive condition is an
epileptogenic-associated disorder selected from epilepsy, head
trauma, stroke, multiple sclerosis, amyotrophic lateral sclerosis,
psychoses, cerebral ischemia, motor neuron disease, Alzheimer's
disease, chicken-pox, measles, encephalitis, pertussis
encephalitis, infections of the CNS, meningitis, encephalitis,
subdural haematoma, brain tumour, birth defects, anoxic brain
injury dementia, or other disorders in which altered activity of
neuro-active molecules is a cause, at least in part, of the
disorder.
11. The method of claim 1, wherein said convulsive condition is
selected from the group consisting of epilepsy and non-epileptic
convulsions.
12. The method of claim 1, wherein said subject has not yet
developed seizures.
13. The method of claim 1, wherein said treatment step comprises
administering an effective amount of an anti-convulsive
pharmaceutical composition.
14. The method of claim 1, wherein said treatment step comprises
administering an effective amount of an anti-epileptogenic
pharmaceutical composition.
15. The method of claim 1, further comprising the steps of
deproteinizing said bodily fluid.
16. The method of claim 15, wherein said deproteinizing step is
ultrafiltration, ultracentrifugation, or chemical
precipitation.
17. The method of claim 16, wherein said chemical precipitation
step employs sulfosalicylic acid, perchloric acid, trichloroacetic
acid, picric acid, acetonitrile, ethanol, acetone, or methanol.
18. The method of claim 1, further comprising derivatizing said
amino acid prior to analyzing it.
19. The method of claim 18, wherein said derivatizing step
covalently attaches a chromophore to said amino acid.
20. The method of claim 18, wherein said derivatizing step employs
o-phthalaldehyde, 9-fluorenylmethylchloroformate, phenyl
isothiocyanate, or 1-dimethylaminonaphthalene-5-sulphonyl
chloride.
21. The method of claim 1, wherein said bodily fluid is urine,
blood, plasma, blood serum, cerebrospinal fluid, sweat, lymph,
amniotic fluid, synovial fluid, conjunctival fluid, salivary fluid,
vaginal fluid, stool, seminal fluid, bile, tears, or mixtures
thereof.
22-40. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
patent application 60/318,139, filed Sep. 7, 2001, and U.S.
provisional patent application 60/378,781, filed May 7, 2002. The
contents of each of these aforementioned applications are hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] A variety of clinical methods exist by which a physician is
directed to a diagnosis of the cause of apparent seizures in a
patient as either epilepsy or otherwise. For example, routine blood
studies including electrolyte and glucose measurements, complete
blood counts, and toxin screens may be carried out to assist a
physician in determining a cause of seizures in a patient. Medical
imaging, including CT and MRI, as well as EEG examinations may also
yield valuable clinical information in this regard. There are,
however, no routinely used prospective or predictive clinical tests
which a physician may perform which indicate whether or not a
patient is at risk of developing seizures in the future.
[0003] Retrospective studies have revealed that several factors are
associated with an increased risk of seizure, for example, a
familial history of seizures, meningitis, or a recent head trauma.
An individual's susceptibility to seizure is determined
additionally by the individual's brain chemistry, and consequently
a head trauma of equal magnitude, e.g., may precipitate seizures in
one individual, but not another. Presently, there is no predictive
test to distinguish between these two hypothetical individuals.
[0004] To the contrary, following head trauma or insult to the
brain it is common practice to administer prophylactically
anti-seizure drugs to most patients who fall into an "at risk"
category, without any analysis of the individual's actual risk.
Accordingly, patients who are at lower risk of developing seizures
are subjected to the unnecessary side-effects of various drugs,
such as, e.g., inhibition of neuroplasticity. A need remains,
therefore, for a predictive test which more accurately indicates a
patient's actual risk of developing seizures.
[0005] Distinguishing pseudoseizures from seizures is another
clinical need that such a test may address. Pseudoseizures are
seizure-like spells with no physiological basis. They can either be
intentionally or subconsciously induced. The treatment for
pseudoseizures is often psychological in nature, and patients
undergo unnecessary effects if anticonvulsant medication is
administered due to a misdiagnosis.
[0006] Although epileptic seizures are rarely fatal, large numbers
of patients require medication to avoid the disruptive, and
potentially dangerous, consequences of seizures. In many cases,
medication is required for extended periods of time, and in some
cases, a patient must continue to take prescription drugs for life.
Furthermore, drugs used for the management of epilepsy have
side-effects associated with prolonged usage, and the cost of the
drugs can be considerable.
[0007] It has been postulated that free amino acids play a role in
the normal functioning of the central nervous system. Amino acid
concentrations in the brain specifically depend on several factors,
including tissue metabolism, blood flow, transport or exclusion at
the blood brain barrier, and renal or hepatic function. As such,
amino acid imbalances associated with neurological disorders are of
interest and have served as the basis for a variety of
investigations.
[0008] However, the findings of previous studies on amino acid
imbalances in epilepsy, including those by Plum (Journal of
Neurochemistry 1974, 23, 595-600), Mutani et al. (Epilepsia 1974,
15, 595-597), Crawford and Chadwick (Epilepsy Research 1987, 1,
328-338), Haines et al. (Epilepsia 1985, 26, 642-648), Monaco et
al. (Italian Journal of Neurological Sciences 1994, 15, 137-14),
van Gelder et al. (Neurochemical Research 1980, 5, 659-671), and
Ferrie et al. (Epilepsy Research 1999, 34, 221-229), are
inconsistent. In addition to methodological sources of variation,
inter-study variability has been attributed to such factors as
heterogeneity within the sample population being examined,
circadian variation and short-term dietary amino acid intake.
[0009] Anti-epileptic medication may also contribute to inter-study
variability as increases in glycine, serine and alanine, have been
noted upon valproic acid administration, while increases in free
and total .beta.-aminobutyric acid, homocarosine (a conjugate of
.beta.-aminobutyric acid), .beta.-alanine, glycine and
.beta.-aminoisobutyric acid occur upon vigabatrin administration.
Alternatively, administration of carbamazepine, ethosuximide and
mephobarbital leads to decreases in leucine, proline and
phenylalanine, respectively.
SUMMARY OF THE INVENTION
[0010] The present invention exploits the discovery, described
herein, that amounts of uracil and thymine metabolites, especially
.beta.-aminoisobutyric acid, in various bodily fluids, especially
urine, are correlated with the occurrence of epilepsy when compared
to matched control subjects. Analytical and diagnostic protocols,
including a novel high performance liquid chromatography system,
for use in the invention are disclosed.
[0011] Reported experiments with .beta.-alanine in animals relate
to exploiting its neuro-inhibitory effects, e.g. studying how it
mitigates the extent or threshold of seizure when co-administered
with a drug substance known to cause seizures. It has not been
previously recognized, however, that imbalances of endogenous
.beta.-alanine may be indicative of susceptibility to seizure,
especially idiopathic seizures or epilepsy, familial history, and
seizures resulting from head trauma. The present method may be used
with noninvasive (e.g. urine collection) or minimally invasive
techniques (e.g. blood collection). The method of the invention may
be used to analyze neuro-active molecules such as amino acids in
the urine of subjects.
[0012] In particular, the invention relates to methods of diagnosis
of convulsive conditions or susceptibility thereto in a subject,
wherein a bodily fluid from a subject is analyzed for the presence
of a neuro-active molecule associated with a convulsive condition,
and the subject is diagnosed as at risk of a convulsive condition
or susceptibility thereto if the amount of the compound indicates a
likelihood of same in the subject. Preferred neuro-active molecules
include metabolites of uracil and thymine, particularly
.beta.-amino acids, preferably .beta.-aminoisobutyric acid.
[0013] Furthermore, the invention relates to methods of modulating,
including inhibiting or preventing, the onset of a convulsive
condition in a subject, wherein a bodily fluid from a subject is
analyzed for the presence of a neuro-active molecule associated
with a convulsive condition; determining from the amount of the
compound in the bodily fluid whether the subject is at risk of a
convulsive condition; and treating the subject, if at risk of a
convulsive condition, to modulate the onset of the convulsive
condition in the subject. Preferred neuro-active molecules include
metabolites of uracil and thymine, particularly .beta.-amino acids,
preferably .beta.-aminoisobutyric acid.
[0014] Additionally, a method of quantifying neuro-active molecules
such as .beta.-alanine or .beta.-aminoisobutyric acid is described,
comprising collecting and optionally deproteinizing a bodily fluid
sample, e.g. urine, derivatizing the amino acids present in the
(deproteinized) sample, and analyzing the (derivatized) amino acids
by chromatography (such as reversed phase high performance liquid
chromatography), the chromatography system comprising a column,
mobile phases (preferably acetate buffer and methanol), an optional
internal standard (preferably D,L-ethionine) and a set of external
standards of varying concentration, and a separation program which
produces a resolution for each of the neuro-active molecules of
interest with all other amino acids and molecules present in the
bodily fluid of equal to or greater than one.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 illustrates various metabolic pathways implicated in
the medical conditions described herein and related to
.beta.-alanine and .beta.-aminoisobutyric acid.
[0016] FIG. 2 shows a representative chromatogram depicting the
elution profile for a 100 .mu.mol/L standard mixture of 23 amino
acids according to a method of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention entails a method of diagnosing a
convulsive condition or susceptibility thereto in a subject
comprising the steps of analyzing a bodily fluid from a subject for
the presence or amount(s) of one or more neuro-active molecule(s),
or the relative amounts of neuro-active molecules (e.g. ratio),
associated with a convulsive condition; and diagnosing the subject
as at risk of a convulsive condition or susceptibility thereto if
the amount of said compound indicates a likelihood of same in said
subject. Said subject need not have actually developed
seizures.
[0018] According to the invention, a standard against which the
above measure or measures from test bodily fluids are compared may
be data obtained from a data bank corresponding to currently
accepted normal levels of neuro-active molecules under analysis. In
situations such as those where standard data are not available, the
methods of the invention may further comprise conducting
corresponding analyses in a second set of one or more biological
samples known not to be at risk of a convulsive condition or
susceptibility thereto. Such additional biological samples could be
obtained, for example, previously from the subject under
consideration, or from unaffected members of the public.
[0019] According to the methods of the invention, the comparison of
the above measure or measures may be a straight-forward comparison,
such as a ratio, or it may involve weighting of one or more of the
measures, relative to, for example, their importance to the
particular situation under consideration. The comparison may also
involve subjecting the measurement data to any appropriate
statistical analysis. In most diagnostic procedures in accordance
with the invention, one or more biological samples obtained from an
individual will be subjected to a battery of analyses in which any
number of neuro-active molecules are sought to be detected. In any
such diagnostic procedure it is possible that one or more of the
measures obtained will render an inconclusive result; accordingly,
data obtained from a battery of measures is likely to provide for a
more conclusive diagnosis. It is for this reason that an
interpretation of the data based on an appropriate weighting scheme
or statistical analysis is desirable.
[0020] The term "convulsive disorder" or "convulsive condition"
according to the invention includes conditions wherein a subject
suffers from convulsions. Convulsive disorders include, but are not
limited to, epilepsy, ictogenesis, epileptogenesis, and
non-epileptic convulsions, and convulsions due to administration of
a convulsive agent or trauma to the subject.
[0021] A seizure is a single discrete clinical event caused by an
excessive electrical discharge from a collection of neurons through
a process termed "ictogenesis." As such, a seizure is merely the
symptom of epilepsy.
[0022] Epilepsy is a dynamic and often progressive process
characterized by an underlying sequence of pathological
transformations whereby normal brain is altered, becoming
susceptible to recurrent seizures through a process termed
"epileptogenesis." While it is believed that ictogenesis and
epileptogenesis have certain biochemical pathways in common, the
two processes are not identical.
[0023] Ictogenesis (the initiation and propagation of a seizure in
time and space) is a rapid and definitive electrical/chemical event
occurring over seconds or minutes. Epileptogenesis (the gradual
process whereby normal brain is transformed into a state
susceptible to spontaneous, episodic, time-limited, recurrent
seizures, through the initiation and maturation of an
"epileptogenic focus") is a slow biochemical or histological
process which generally occurs over months to years.
[0024] Epileptogenesis is a two phase process: Phase 1
epileptogenesis is the initiation of the epileptogenic process
prior to the first seizure, and is often the result of stroke,
disease (e.g. meningitis), or trauma, such as an accidental blow to
the head or a surgical procedure performed on the brain. Phase 2
epileptogenesis refers to the process during which a brain that is
already susceptible to seizures, becomes still more susceptible to
seizures of increasing frequency or severity. While the processes
involved in epileptogenesis have not been definitively identified,
some researchers believe that up-regulation of excitatory coupling
between neurons, mediated by N-methyl-D-aspartate (NMDA) receptors,
is involved. Other researchers implicate down-regulation of
inhibitory coupling between neurons, mediated by
.gamma.-aminobutyric acid (GABA) receptors, pre- or
post-synaptically.
[0025] The term "subject" includes animals susceptible to
convulsive disorders, epileptogenesis or capable of suffering from
epileptogenic-associated states, such as warm-blooded animals, more
preferably a mammal, including, e.g. non-human animals such as
rats, mice, cats, dogs, sheep, horses, cattle, in addition to
humans. In a preferred embodiment, the subject is a human. Subjects
with a family history of convulsive conditions, a history of
cerebral hypoxia or ischemia, intracranial hemorrhage, central
nervous system infection or disease, drug or alcohol withdrawal,
fever, trauma, brain tumor, cerebrovascular disease, metabolic
disorder, degenerative central nervous system disease, drug or
alcohol addiction or use, uremia, hepatic dysfunction,
hypoglycemia, epilepsy, or seizure are preferred subjects for
analysis according to the invention because they may be at risk for
convulsions. Additionally, preferred subjects include those who
have recently been administered an antibiotic, anesthetic,
analgesic, immunomodulatory, psychotropic, sedative, radiographic
contrast-enhancing, stimulant or hallucinogenic drug. A
particularly preferred subject according to the invention is one
who has suffered a head trauma and is at risk of developing
post-traumatic epilepsy (PTE).
[0026] A seizure or convulsion, which terms may be used
interchangeably herein, may be complex partial, simple partial,
absence, secondary generalized tonic clonic, primary generalized
tonic clonic, myoclonic, or atonic.
[0027] "Bodily fluid" as used herein includes, e.g., urine, blood,
blood serum, amniotic fluid; cerebrospinal (i.e. CSF) and spinal
fluid, synovial fluid, conjunctival fluid, salivary fluid, vaginal
fluid, stool, seminal fluid, lymph, bile, tears, and sweat. A
bodily fluid is advantageously CSF, urine, or blood or its
components parts, e.g. plasma. A particularly preferred bodily
fluid is urine.
[0028] "Neuro-active molecules" according to the invention include
neurotransmitters, such as amino acid neurotransmitters,
neutrostimulators, and neurodepressants. Such neuro-active
molecules may alter the ability of a nerve cell to depolarize or to
release or take up neurotransmitter molecules. As described herein,
preferred neuro-active molecules of the invention include
metabolites of uracil or thymine, especially .beta.-amino
carboxylic acids (comprising at least the sub-structure
N--C--C--(C.dbd.O)--O) such as .beta.-alanine and
.beta.-aminoisobutyric acid, and derivatives thereof. Such
derivatives may be esters or other bioconjugates (including
glucuronic acid and sterol conjugates).
[0029] The invention relates to convulsive conditions related to
thymine or uracil metabolism, including abnormalities thereof, and
therefore the compounds depicted in FIG. 1 are neuro-active
molecules according to the invention as described further herein
below.
[0030] "Analyzing" as used herein may be any step which either
qualitatively or quantitatively indicates the amount or presence of
a neuro-active molecule. Examples of analyses of the present
invention include chromatography (including high-performance liquid
chromatography, thin layer chromatography, or gas chromatography),
spectroscopy, spectrometry, and colorimetry (such as by use of a
color-changing indicator as in, for example, a "dip stick" or "test
strip" as commonly used in the detection of glucose in urine),
although other functional equivalents may be employed.
[0031] An analysis step may include further steps of preparing a
sample for study, such as removal of interfering compounds (i.e.
non-neuro-active molecules) from the bodily fluid by such means as
precipitation, filtration, and the like. Additionally, neuro-active
molecules may be derivatized prior to analysis to facilitate
detection. For example, in analysis protocols where detection is by
absorption, it may be advantageous to covalently attach a
chromophore to the neuro-active molecules.
[0032] The present invention also relates to a method of modulating
the onset of a convulsive condition in a subject comprising the
steps of analyzing a bodily fluid from a subject at risk of a
convulsive condition for the presence of a neuro-active molecule
associated with a convulsive condition; determining from the amount
of said compound in said bodily fluid whether said subject is at
risk of a convulsive condition; and treating said subject, if at
risk of a convulsive condition, to modulate the onset of said
convulsive condition in said subject.
[0033] "Modulating" means altering the likelihood that a seizure
will occur. Generally, modulating will mean reducing or inhibiting
the likelihood of a future seizure in a subject in accordance with
the invention. Modulating may refer to any convulsive condition or
a precursor thereof.
[0034] The terms "treatment," "treating," or "treat," include the
administration of an agent (e.g. an anticonvulsive or
anti-epileptogenic, prophylactic or therapeutic pharmaceutical
composition) to a subject, who has a disease or disorder, a symptom
of a disease or disorder, or is at risk of suffering from the
disease or disorder in the future, such that the disease or
disorder (or at least one symptom of the disease or disorder) is
cured, healed, prevented, alleviated, relieved, altered, remedied,
ameliorated, improved or otherwise affected, preferably in an
advantageous manner. "Agents" include anti-convulsive,
anti-seizure, or anti-epileptogenic agents, such as described in
U.S. Pat. No. 6,306,909 B1. Such a treatment step may comprise
administering an effective amount of an anti-convulsive,
anti-seizure, or anti-epileptogenic pharmaceutical composition.
[0035] The language "effective amount" of a compound is that amount
necessary or sufficient to treat or prevent a particular condition,
e.g., to prevent the various morphological and somatic symptoms of
an epileptogenic-associated state. The effective amount can vary
depending on such factors as the size and weight of the subject,
the type of condition, or the particular agent. For example, the
choice of the pharmaceutical composition can affect what
constitutes an "effective amount." One of ordinary skill in the art
would be able to study the aforementioned factors and make the
determination regarding the effective amount of the pharmaceutical
composition without undue experimentation.
[0036] The term "anti-epileptogenic agent" includes agents which
are capable of inhibiting epileptogenesis, e.g., suppressing the
uptake of synaptic GABA (e.g., blocking GABA transporters, e.g.
GAT-1, GAT-2 or GAT-3), depressing glutamatergic excitation (e.g.,
interacting with an NMDA receptor, e.g. at the
strychnine-insensitive glycine co-agonist site), binding to a GABA
receptor (e.g. GABA.sub.A), altering (e.g., increasing or
suppressing) the metabolism of GABA (e.g., via inhibition of GABA
transaminase).
[0037] Further examples of pharmaceutical compositions of the
present invention include carbamazepine, clobazam, diazepam,
lamotrigine, lorazepam, oxazepam, phenobarbital, phenytoin,
primidone, valproate, ethosuximide, topirimate, felbamate,
clonazepam, clobazarn, nitrazepam, vigabatrin, gabapentin,
levetiracetam, or tiagabine, or other pharamceuticals approved for
the treatment of seizures or epilepsy by government regulatory
agencies (such as the United States Food & Drug
Administration), or combinations thereof.
[0038] Generally, a convulsive condition is selected from the group
consisting of epileptogenic associated disorders, epileptogenesis,
and non-epileptic convulsions. Inhibiting epileptogenesis includes
both partial and complete reversal of epileptogenesis. Inhibiting
epileptogenesis includes prevention of epileptogenesis or a
decrease or slowing in the rate of epileptogenesis (e.g. a partial
or complete stop in the rate of epileptogenic transformation of the
brain or central nervous system tissue). It also includes any
inhibition or slowing of the rate of the biochemical processes or
events which take place during Phase 1 or Phase 2 epileptogenesis
and lead to epileptogenic changes in tissue, i e., in tissues of
the central nervous system (CNS), e.g. the brain. Examples of
processes in pathways associated with epileptogenesis are discussed
in more detail herein. Modulating epileptogenesis also includes the
prevention, slowing, halting, or reversing the process of
epileptogenesis, i.e. the changes in brain chemistry which result
in epileptic seizures.
[0039] The term "epileptogenic-associated disorders" includes
disorders of the central and peripheral nervous system which may
advantageously be treated as described in, e.g. U.S. Pat. No.
6,306,909 B1 and PCT publication WO 98/40,055. In an advantageous
embodiment, the nervous system disorders are disorders associated
with or related to the process or the results of epileptogenic
transformation of the brain or other nervous tissue.
[0040] Examples of epileptogenic-associated disorders include
epilepsy, head trauma, stroke, multiple sclerosis, amyotrophic
lateral sclerosis, psychoses, cerebral ischemia, motor neuron
disease, Alzheimer's disease, encephalitis (including encephalitis
arising from chicken-pox, measles or pertussis), infections of the
CNS (meningitis, encephalitis), subdural haematoma, brain tumour,
and birth defects including anoxic brain injury, dementia and other
disorders (in humans or animals) in which altered activity of
neurotransmitters is a cause, at least in part, of the disorder
(see, e.g. Schoepp et al., Eur. J. Pharmacol. 1991, 203, 237-243;
Leeson et al, J. Med. Chem. 1991, 34, 1243-1252; Kulagowski et al,
J. Med. Chem. 1994, 37, 1402-1405; Mallamo et al, J. Med. Chem.
1994, 37, 44384448; and references cited therein). The term
epileptogenic-associated disorders includes both convulsive
disorders and disorders associated with NMDA receptor activity.
[0041] The invention also relates to particular novel methods of
analysis, including a method of quantifying neuro-active molecules,
such as .beta.-alanine or .beta.-aminoisobutyric acid, comprising
the steps of collecting a bodily fluid sample, such as urine;
optionally deproteinizing said sample; optionally derivatizing the
neuro-active molecules present in said (deproteinized) sample; and
analyzing said (derivatized) neuro-active molecules by
chromatography, said chromatography system comprising a column
(preferably a reversed phase C8 or C18 column), mobile phases
(preferably acetate buffer and methanol), an optional internal
standard (preferably D,L-ethionine) and a set of external standards
of varying concentration, and a separation program which produces a
resolution for derivatized neuro-active molecule(s) of interest
present in said sample of equal to or greater than one. This method
is most advantageously applied to the analysis of amino acid
neuro-active molecules, particularly .beta.-aminoisobutyric acid or
another metabolite of uracil or thymine.
[0042] The analysis method may further comprise a step of
deproteinizing said bodily fluid, for example by ultrafiltration,
ultracentrifugation, or chemical precipitation. The chemical
precipitation step may employ a precipitating agent, for example,
sulfosalicylic acid, perchloric acid, trichloroacetic acid, picric
acid, acetonitrile, ethanol, acetone, or methanol.
[0043] The derivatizing step may covalently attach a chromophore to
an amino acid, and preferred reagents for use in such a
derivatizing step include o-phthalaldehyde,
9-fluorenylmethylchloroformate, phenyl isothiocyanate, or
1-dimethylaminonaphthalene-5-sulphonyl chloride, as well as other
commercially available reagents.
[0044] In this invention, levels of .beta.-aminoisobutyric acid are
correlated with the occurrence of epilepsy, as demonstrated by the
following, wherein the concentrations of .beta.-alanine and its
metabolic equivalent .beta.-aminoisobutyric acid in urine collected
from subjects with epilepsy and matched control subjects were
studied. A novel reversed-phase high performance liquid
chromatography (RP-HPLC) program is disclosed for the analytical
separation and quantification of .beta.-alanine and
.beta.-aminoisobutyric acid in urine.
[0045] Protein in physiological fluids is typically inevitable, but
it interferes with amino acid analysis and shortens the lifetime of
chromatographic columns. Several methods have been proposed for the
removal of protein from physiological fluids, including chemical
precipitation, ultrafiltration, and ultracentrifugation, with
chemical precipitation finding the most frequent use.
Sulfosalicylic acid is a common precipitation agent, and is used as
a solution in distilled de-ionized water in concentrations as high
as 20% (w/v). This methodology does, however, tend to lower the
concentrations of aspartic acid and glutamic acid in solution due
to their decreased solubility under strongly acidic conditions.
Once precipitation is complete, the sample is filtered, making it
ready for derivatization and analysis.
[0046] With a few exceptions, free amino acids cannot be detected
using experimental techniques such as UV absorption. A variety of
methods known in the art are therefore available for the
derivatization of amino acids prior to analytical separation and
detection by RP-HPLC. Generally, pre-column derivatization is
preferred, as it results in increased resolution and sensitivity
over the corresponding post-column methodology. With derivatization
using o-phthalaldehyde (OPA), 9-fluorenylmethyl chloroformate
(FMOC--Cl), phenyl isothiocyanate (PITC) or
1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl-Cl), the
automated OPA method is generally the most amenable to routine
analysis of primary amino acids except cysteine.
[0047] With the OPA method, primary amino acids are reacted with
o-phthalaldehyde in the presence of the reducing agent
.beta.-mercaptoethanol. Detection of the corresponding derivatized
amino acid is achieved by the monitoring of absorbance at a
wavelength of 340 nm. This reaction occurs in a 1:1:1 ratio, with
the elimination of two molecules of water, to yield the
corresponding fluorescent 1-alkylthio-2-alkyl-substituted
isoindoles. OPA is inherently non-fluorescent and as such gives low
reagent interference. The derivatization reaction is rapid and
occurs readily at ambient temperature. Typically, detection limits
lie in the low picomole range. Disadvantages, however, include the
instability of the fluorescent isoindoles, the inability to detect
secondary amino acids such as proline and hydroxyproline, and the
poor fluorescent response arising from the derivatization of a few
amino acids, in particular cysteine.
[0048] Several modifications may improve the sensitivity and
reproducibility of this reaction as well as the stability of its
products. The addition of BRIJ-35 (polyoxyethylene lauryl ether
(ICI Americas)) enhances the fluorescent response of lysine and
hydroxylysine. Alternative thiol containing reagents, such as
tert-butyl thiol, also increase the stability of the corresponding
isoindoles. The reproducibility of the derivatization reaction can
be enhanced through the maintenance of constant reaction times and
temperatures, e.g. via automated derivatization at
temperature-controlled conditions. Finally, avoiding the use of
excess o-phthalaldehyde reagent minimizes the degradation of the
isoindole intermediate, which improves the sensitivity of
quantitative amino acid analysis.
[0049] Traditionally, analytical separation and detection of amino
acids in physiological fluids involved ion-exchange chromatography
in combination with post-column ninhydrin derivatization and
subsequent detection using either spectrophotometry or colorimetry.
However, the advantage of reduced analysis times, improved
resolution and enhanced sensitivity, along with the development of
HPLC has prompted a shift away from this classical method of amino
acid analysis. Several manual and automated RP-HPLC procedures
using o-phthalaldehyde derivatization have since been developed for
the separation and detection of amino acids in physiological
fluids, but very few have been specifically designed for the
detection and quantification of .beta.-alanine and
.beta.-aminoisobutyric acid.
[0050] Metabolic pathways involving .beta.-alanine and
.beta.-aminoisobutyric acid are depicted in FIG. 1. As illustrated
in this FIG. 1, .beta.-alanine and .beta.-aminoisobutyric acid are
believed to be endogenously derived via the metabolism of uracil
and thymine, respectively.
[0051] The first of three enzymes involved in this pathway is
dihydropyrimidine dehydrogenase. This enzyme is responsible for
catalyzing the reversible NAPDH-dependent conversion of uracil and
thymine to dihydrouracil and dihydrothymine. This initial step is
rate determining with respect to the overall breakdown of uracil
and thymine to .beta.-alanine and .beta.-aminoisobutyric acid.
[0052] Further transformation of these metabolites, through the
action of dihydropyrimidinase, reversibly yields
.beta.-ureidopropionate and .beta.-ureido-iso-butyric acid.
[0053] Finally, .beta.-alanine synthase, also referred to as
.beta.-ureidopropionase or N-arbamoyl-.beta.-alanine
amidohydrolase, facilitates the irreversible hydrolytic cleavage of
.beta.-ureidopropionate and .beta.-ureido-iso-butyric acid to give
.beta.-alanine and .beta.-aminoisobutyric acid as well as the
release of ammonia (NH.sub.3) and carbon dioxide (CO.sub.2). This
enzyme is of particular importance in animals in that it is
directly responsible for the in vivo biosynthesis of
.beta.-alanine, with its action occurring predominantly in the
liver. It is the R-isomer of .beta.-aminoisobutyric acid that is
formed via this reaction. The corresponding S-isomer is generated
through the metabolism of L-valine.
[0054] Minor sources of .beta.-alanine arise from the actions of
two enzymes, aspartate decarboxylase, found in bacteria of the
intestinal lumen which decarboxylates aspartic acid to give
.beta.-alanine; and carnosinase, which catabolizes carnosine to
give .beta.-alanine and histidine.
[0055] Dihydropyrimidine dehydrogenase is the rate-limiting enzyme
in the catabolic pathway from uracil and thymine to .beta.-alanine
and .beta.-aminoisobutyric acid. A deficiency of this enzyme has
deleterious physiological effects. Dihydropyrimidine dehydrogenase
deficiency results from the autosomal recessive inheritance of a
mutant allele coding for the dihydropyrimidine dehydrogenase enzyme
(Gonzales, et al., T.I.P.S. 1995, 16, 325-327). The presence of
this mutation leads to the loss of a 165 base pair exon, resulting
in the expression of truncated mRNA. This mutation can lead to a
drop in enzyme activity by as much as 98 to 100%.
[0056] Dihydropyrimidine dehydrogenase deficiency has two distinct
clinical forms (Scriver, et al., The Metabolic and Molecular Bases
of Inherited Disease; 7 ed.;. Scriver, et al., Eds.; McGraw-Hill,
Inc.: New York, 1995; Vol. 1). The genetic form, involving an
inborn error of metabolism, is an early onset disorder commonly
associated with neurological signs such as seizures, impaired
cognitive development, hypertonia, hyperreflexia, microcephaly and
dysmyelination. The iatrogenic form, which occurs following
exposure to the cancer chemotherapeutic agent 5-fluorouracil, is
characterized by clinical symptoms such as encephalopathy,
neurotoxicity and neutropenia. Withdrawal of this drug eliminates
all symptoms of this disorder (Tuchman, et al., New Eng. J. Med
1985, 313, 245-249).
[0057] The pathophysiology underlying the association of epilepsy
with dihydropyrimidine dehydrogenase deficiency remains unclear. It
has, however, been suggested that seizure etiology may arise at the
level of the nucleic acids (Braakhekke, et al, J. Neuro. Sci. 1987,
78, 71-77). Uridine, a pyrimidine nucleoside, has been shown to
exhibit anticonvulsant activity in animal models of epilepsy.
Atypical regulation of uridine and its related compounds may
therefore be important in explaining abnormal central nervous
system regulation. A correlation between the lack of .beta.-alanine
and the neurological symptoms of dihydropyrimidine dehydrogenase
deficiency has also been suggested.
[0058] Since its initial detection, several cases of
dihydropyrimidine dehydrogenase deficiency have been documented
(van Gennip, et al., Adv. Exp. Med. Biol. 1989, 253A, 111-118).
Diagnosis of this disorder is normally based on presence of high
levels of uracil, thymine and 5-hydroxymethyluracil (a metabolite
of thymine), in physiological fluids (Valik, et al., Mayo Clin.
Proc. 1997, 72, 719-725; van Gennip, et al, Clin. Chem. 1993, 39,
380-385). In urine, uracil and thymine levels can be elevated by as
much as one hundred fold for the iatrogenic form and one thousand
fold for the genetic form. Definitive diagnosis of this disorder
does, however, require conclusive proof of an enzyme deficiency. To
quantify dihydropyrimidine dehydrogenase activity, cultured
fibroblasts are first incubated with 14C-thymine. The loss of
14C-thymine and the formation of 14C-dihydrothymine are then
quantified using a combination of HPLC and liquid scintillation
counting to give a measure of enzyme activity (Bakkeren, et al.,
Clin. Chim. Acta 1984, 140, 247-256).
[0059] Dihydropyrimidinuria is a disorder resulting from a
deficiency in dihydropyrimidinase, the second of three enzymes
along the catabolic pathway from uracil and thymine to
.beta.-alanine and .beta.-aminoisobutyric acid. Excretion of large
quantities of dihydrouracil and dihydrothymine are therefore
associated with this condition. Although it is believed to be
autosomal recessive, little else is known about this disorder. Only
two cases of dihydropyrimidinuria have been reported. One subject
exhibited convulsions, lowered consciousness and metabolic
acidosis, while the other showed signs of gross microcephaly,
spastic quadriplegia, choreiform movements and severe developmental
retardation (Webster, et al., The Metabolic and Molecular Bases of
Inherited Disease; 7 ed.; Scriver, et al, Eds.; McGraw-Hill, Inc.:
New York, 1995; Vol. 2).
[0060] Catabolism of .beta.-alanine primarily occurs through the
actions of two aminotransferases,
.beta.-alanine-.alpha.-ketoglutarate transaminase and
.beta.-alanine-pyruvate transaminase, and results in the production
of malonic acid semialdehyde. It is a deficiency of the former
enzyme that is believed to underlie hyper-.beta.-alaninemia, a rare
disorder characterized by increased levels of .beta.-alanine and
GABA in cerebrospinal fluid, plasma and urine as well as
.beta.-aminoisobutyric acid in urine (Scriver, et al., New Eng. J.
Med. 1966, 274, 635-643). This postulate is supported by three key
observations. First, the administration of pyridoxine, whose
derivative pyridoxal-5-phosphate acts as an aminotransferase
coenzyme, has been effective in the symptomatic treatment of
hyper-.beta.-alaninemia. Second, .beta.-alanine,
S-.beta.-aminoisobutyric acid, and GABA are all transaminated with
.alpha.-ketoglutarate via the actions of these transaminases in
both the brain and liver. Finally, the fact that both
.beta.-alanine and GABA are elevated in physiological fluids
suggests a lack of involvement of .beta.-alanine-pyruvate
transaminase, whose substrate specificity is for .beta.-alanine
alone.
[0061] Only two cases of hyper-.beta.-alaninemia have been reported
to date. One subject exhibited somnolence and repeated grand-mal
seizures and died within five months of birth, while the other was
described as having intermittent generalized tonic-clonic seizures,
lethargy and Cohen's syndrome (Higgins, et al., Neurology 1994, 44,
1728-1732). The etiology of these neurological symptoms remains
unclear. Plausible explanations include the inhibition of GABA
transaminase by excess .beta.-alanine, competitive binding of
.beta.-alanine to the GABA receptor, as well as agonism of the
strychinine-sensitive glycine and NMDA receptors by
.beta.-alanine.
[0062] Hyper-.beta.-aminoisobutyric aciduria is a reasonably
prevalent disorder involving a deficiency in
.beta.-aminoisobutyrate-pyruvate transaminase, an enzyme
responsible for the catabolism of .beta.-aminoisobutyric acid.
Subjects with this disorder typically exhibit less than 10% of the
normal enzyme activity and therefore excrete large quantities of
this amino acid. The genetic variant of this disorder is postulated
to be recessive, stemming from a genetic polymorphism at a single
locus. This trait appears to have a nonrandom distribution within
the population, with highest frequencies in the Micronesian
population and lowest frequencies in the Caucasian population.
[0063] Other factors are known to influence the excretion of
.beta.-aminoisobutyric acid. Children are known to have higher
excretion rates than adults, while females tend to have higher
excretion rates than males. Enhanced excretion of
.beta.-aminoisobutyric acid is also a factor to be considered with
neoplastic states and with Down's syndrome as well as during
periods of increased somatic cell growth, when pyrimidine turnover
is significantly elevated.
[0064] Therefore, a variety of neuro-active molecules, such as
amino acids, including .beta.-alanine, .beta.-aminoisobutyric acid,
and those compounds depicted in FIG. 1 are within the scope of the
present invention.
[0065] The invention described herein is exemplified by the
following non-limiting method. Other analytical methods known in
the art may be employed according to the teachings herein. The
method described below may be modified by one skilled in the art
using no more than routine experimentation. Such functionally
equivalent analytical methods are also encompassed by the instant
invention.
[0066] HPLC-grade methanol and HPLC-grade glacial acetic acid were
obtained from Fisher Scientific (Fair Lawn, N.J.), sodium acetate
was obtained from Sigma-Aldrich (Milwaukee, Wis.) and fluoraldehyde
reagent solution was obtained from Pierce Chemical (Rockford,
Ill.). Individual L-amino acids, .beta.-alanine,
.beta.-aminoisobutyric acid, D,L-ethionine and sulfosalicylic acid
were also obtained from Sigma-Aldrich.
[0067] A System Gold liquid chromatographic system was combined
with a Model 125 programmable solvent module fitted with an Altex
210A injection valve, a Model 166 programmable UV-VIS detector
module (Beckman, San Ramon, Calif.); a Dell 489P/33 computer and an
Epson FX-870 printer. A 5 .mu.m Ultrasphere ODS column (250
mm.times.4.6 mm I.D.) with a 5 .mu.m Ultrasphere ODS guard column
(45 mm.times.4.6 mm I.D.), both from Beckman, were used. Solvents
were filtered through 0.22 .mu.m nylon filters (N02SP04700) from
Osmonics (Minnetonka, Minn., U.S.A.). Distilled de-ionized water
was prepared using a Culligan de-ionizer from Structural Fibers
(Chardon, Ohio).
[0068] Human subjects with epilepsy and matched control subjects
were sub-classified into the following five groups:
[0069] Epilepsy Groups: E+D+) subjects whose seizure frequency per
month was greater than zero (mean average seizure frequency per
month 1.23; range 0.24) over the past six months prior to sample
collection and who were taking anti-epileptic medication (8 male
and 7 female), E-D+) subjects whose seizure frequency per month was
zero over the past six months prior to sample collection and who
were taking anti-epileptic medication (13 male and 9 female), and
E-D-) subjects whose seizure frequency per month was zero over the
past six months prior to sample collection and who were not taking
anti-epileptic medication (9 male and 3 female).
[0070] Control Groups: C-D+) subjects without a prior history of
seizures and who were taking anti-epileptic medication (18 male and
6 female) and C-D-) subjects without a prior history of seizures
and who were not taking anti-epileptic medication (22 male and 5
female).
[0071] For those subjects with epilepsy, the etiology of seizures
was infection (chicken-pox, measles encephalitis, pertussis
encephalitis, viral encephalitis, viral meningitis) for 8 subjects,
trauma (subdural hematoma) for 2 subjects, birth complications
(anoxic brain injury) for 11 subjects, central nervous system
defects (cerebral cortical atrophy with hydrocephalus, congenital
brain abnormality with hydrocephalus, spastic quadriplegia, spina
bifida with hydrocephalus) for 7 subjects, prenatal complications
(maternal congenital rubella, maternal eclampsia) for 2 subjects,
miscellaneous (fetal complications due to hyperemesis graviderum,
Rett syndrome) for 2 subjects and unknown for 17 subjects.
[0072] Subjects with epilepsy in groups E+D+ and E-D+ as well as
matched control subjects in group C-D+ were receiving various
combinations of anti-epileptic medication including carbamazepine,
clobazam, diazepam, lamotrigine, lorazepam, oxazepam, phenobarbital
and phenytoin. Eleven subjects with epilepsy (30%) and 21 control
subjects (88%) were taking only one medication, 14 subjects with
epilepsy (38%) and three matched control subjects (12%) were taking
two medications, 10 subjects with epilepsy (27%) were taking three
medications, while 2 subjects with epilepsy (5%) were taking four
medications.
[0073] Subjects with one of primary generalized epilepsy (absence
seizures), chromosomal abnormalities (fragile X syndrome, Down's
syndrome, and Angelman's happy puppet syndrome) or amino acid
disorders (phenylketonuria, amino aciduria) were excluded, as were
subjects receiving the anti-epileptic medication vigabatrin.
[0074] A single urine sample was collected from each subject in the
study population between the hours of 6:00 and 11:00 am on the
morning following a meatless dinner. In this way, it was
anticipated that sample variability arising from circadian
variation as well as the influence of diet on uracil concentrations
might be minimized. Samples were screened for leukocytes, nitrite,
pH, protein, glucose, ketones, urobilinogen, bilirubin and blood
using a CHEM 9 Chemstrip Urine Test Strip from Boehringer Mannheim
(Indianapolis, Ind., U.S.A). All samples used in this study fell
within normal reference ranges for the aforementioned criteria Each
sample was separated into three aliquots and placed in separate
labeled disposable vials. Samples were cooled to 0-4.degree. C. for
transport. For long-term storage, samples were kept at -50 to
-60.degree. C.
[0075] A stock solution of .beta.-alanine and
.beta.-aminoisobutyric acid was prepared by dissolving each
compound in distilled de-ionized water to a final concentration of
0.1 mmol/L. Working external standards were prepared from the stock
solution by dilution with distilled de-ionized water to final
concentrations of 1, 5, 10, 20, 40, 60, 80, 100, 200, 400, and 600
.mu.mol/L. A stock solution of D,L-ethionine, the internal
standard, was prepared by dissolving it in distilled de-ionized
water to a final concentration of 1 mmol/L. A stock solution of 23
amino acids was prepared by dissolving each compound in distilled
de-ionized water to a final concentration of 1 mmol/L. Stock
solutions of the individual L-amino acids were prepared to final
concentrations ranging from 30 .mu.mol/L to 2.5 mmol/L. The
standard amino acid mixture was used for method development while
individual L-amino acid standards were used for peak
identification. For long-term storage, all standards were kept at
-50.degree. C. to -60.degree. C.
[0076] Urine samples were deproteinized via chemical precipitation
using sulfosalicylic acid according to the following procedure,
although other deproteinization procedures as known in the art may
also be employed.
[0077] Urine samples were thawed and vortex mixed for 30 sec. In a
disposable tube, 100 .mu.L 15% (w/v) sulfosalicylic acid was added
to 1 mL of sample. The resulting mixture was vortex mixed and left
standing for 5 min. The treated sample was filtered through a
disposable 5'' P.P. chromatography column fitted with a medium
(45-90 .mu.m) filter from DiaMed Lab Supplies (Mississauga, ON,
Canada) into a second disposable tube. Deproteinization led to
urine sample dilution by a factor of 1.10.
[0078] Fluoraldehyde reagent solution, containing o-phthalaldehyde
(0.8 mg/mL, purchased from Pierce Chemical Co., Rockford, Ill.),
.beta.-mercaptoethanol and BRIJ-35, in a borate buffer
(pH.about.10), was used for the derivatization of primary amino
acids in urine samples and standards according to the following
procedure: In an ice bath, 10 .mu.L of 1 mmol/L D,L-ethionine
(internal standard) was added to 30 .mu.L of a sample, external
standard or blank (distilled de-ionized water) in a disposable
tube. The resulting solution was vortex mixed for 10 sec. To this
was added 20 .mu.L of fluoraldehyde reagent solution and the
resulting solution was vortex mixed for 20 sec. After 1 min, 80
.mu.L of 0.1 mmol/L sodium acetate buffer (pH=7.0) was added and
the solution was vortex mixed for 20 sec. The derivatized solution
was loaded onto the injection loop and 20 .mu.L was injected at 2
min.
[0079] RP-HPLC was used to separate and detect the presence of
amino acids in urine. Eluent A (50 mmol/L sodium acetate buffer, pH
5.7) and eluent B (methanol) were degassed by vacuum filtration
through a nylon filter (0.22 um). The upper and lower pressure
limits were set at 4.00 and 0.00 kPSI. For all samples and
standards, the injection volume was 20 .mu.L. Elution was performed
at ambient temperature at a flow rate of 1.5 mL/min with the
concentration of eluent B as follows: 0-16 min, isocratic elution
at 30%: 16-21 min, linear gradient from 30-36%: 21-28 min,
isocratic elution at 36%: 28-32 min, linear gradient from 36-55%:
32-35 min, isocratic elution at 55%: 35-38 min, linear gradient
from 55-70%: 38-48 min isocratic elution at 70%. The absorbance of
the column eluate was monitored at a wavelength of 340 nm. The
integration parameters, peak threshold and peak width, were set at
3.9.times.10.sup.4 and 0.48, respectively. The column was washed
and reconditioned with the concentration of eluent B as follows:
48-51 min, linear gradient from 70-100%: 51-56 min, isocratic
elution at 100%: 56-59 min, linear gradient from 100-30%: 59-69
min, isocratic elution at 30%. Total analysis time per sample was
48 min, while total analysis time between samples was 69 min.
[0080] The RP-HPLC procedure was optimized to ensure maximal and
reproducible separation of a 23 amino acid mixture with respectable
resolution of taurine, .beta.-alanine, .beta.-aminoisobutyric acid
and .beta.-aminobutyric acid. A representative chromatogram
depicting the elution profile for a 100 .mu.mol/L standard mixture
of 23 amino acids is illustrated in FIG. 2.
[0081] Urinary creatinine concentrations were quantified and used
to standardize amino acid results for the sample population with
respect to renal clearance. .beta.-Alanine and
.beta.-aminoisobutyric acid concentrations were expressed as ratios
relative to the concentrations of creatinine in their respective
samples. TABLE-US-00001 TABLE 1 Levels of .beta.-alanine and
.beta.-aminoisobutyric acid in urine from subjects with epilepsy.
.beta.-Alanine.sup.a .beta.-Aminoisobutyric Acid.sup.a Subject
(.mu.mol/mmol Creatinine) (.mu.mol/mmol Creatinine) 1 1.400 41.85 2
0.5799 39.72 3 1.411 11.80 4 2.061 10.63 5 1.220 16.95 6 0.8061
5.660 7 7.092 101.2 8 0.1816 10.35 9 1.751 <0.3310 10 <0.2967
34.80 11 4.061 125.3 12 2.217 28.31 13 1.371 30.29 14 4.160 6.415
15 <0.1312 7.183 16 1.852 42.52 17 5.307 75.77 18 2.465 16.19 19
1.080 9.256 20 1.143 7.380 21 <0.3580 9.930 22 3.587 140.1 23
0.9659 345.7 24 0.9704 7.048 25 1.017 18.57 26 14.39 43.09 27 2.464
9.337 28 0.4409 13.98 29 1.495 42.77 30 1.233 71.01 31 0.7048 16.58
32 2.008 42.58 33 11.06 12.75 34 3.926 15.36 35 5.954 23.07 36
0.6052 3.428 37 0.2621 19.11 38 0.8235 20.79 39 0.3032 3.030 40
2.453 45.99 41 1.471 36.96 42 0.4948 20.76 43 2.568 81.94 44 0.6136
13.77 45 2.502 17.58 46 10.28 40.65 47 1.042 143.6 48 1.127 6.014
49 0.4841 4.322 Mean.sup.b 2.509 38.07 Median.sup.b 1.406 18.07
Standard 2.970 56.92 Deviation.sup.b Confidence 1.624-3.394
32.80-43.34 Interval.sup.b,c .sup.aValues for .beta.-alanine and
.beta.-aminoisobutyric acid are expressed as a ratio of the amino
acid concentration, measured in units of .mu.mol/L, against
creatinine, measured in units of mmol/L. .sup.bValues in which the
concentration of .beta.-alanine or .beta.-aminoisobutyric acid fell
below the limit of detection were not included in the calculation.
.sup.cConfidence intervals were calculated at the 95% confidence
level.
[0082] TABLE-US-00002 TABLE 2 Levels of .beta.-alanine and
.beta.-aminoisobutyric acid in urine from a matched control group.
.beta.-Alanine.sup.a .beta.-Aminoisobutyric Acid.sup.a Subject
(.mu.mol/mmol Creatinine) (.mu.mol/mmol Creatinine) 1 0.2763 4.567
2 10.44 9.216 3 0.4764 3.560 4 4.736 11.44 5 0.7618 6.124 6 19.38
9.079 7 <0.1075 8.300 8 1.573 37.08 9 0.7881 9.435 10 6.375
8.064 11 0.4085 12.92 12 1.403 7.159 13 3.122 12.92 14 0.2249 8.300
15 1.662 21.04 16 0.8533 3.811 17 0.7743 8.101 18 0.1502 7.979 19
1.806 60.68 20 0.6231 18.14 21 2.518 16.99 22 0.3880 36.44 23 2.921
46.43 24 <0.05104 4.627 25 0.4782 8.229 26 2.480 5.205 27 0.5029
21.74 28 1.248 22.37 29 15.27 14.16 30 0.4514 3.354 31 43.41 25.95
32 0.9628 31.99 33 12.44 9.725 34 11.31 21.30 35 0.2259 3.219 36
0.2200 37.65 37 3.418 14.43 38 0.4532 26.93 39 0.6999 10.26 40
0.04266 18.26 41 0.1145 6.350 42 <0.1147 2.176 43 1.822 3.131 44
0.7089 4.340 45 0.1500 20.69 46 1.390 6.114 47 0.7200 38.42 48
2.612 15.40 49 8.350 15.50 50 <0.07333 9.588 51 8.901 68.91
Mean.sup.b 2.916 16.43 Median.sup.b 8.533 10.26 Standard 4.390
14.63 Deviation.sup.b Confidence 1.626-4.206 12.29-20.57
Interval.sup.b,c .sup.aValues for .beta.-alanine and
.beta.-aminoisobutyric acid are expressed as a ratio of amino acid
concentration, measured in units of .mu.mol/L, against creatinine,
measured in units of mmol/mL. .sup.bValues in which the
concentration of .beta.-alanine or .beta.-aminoisobutyric acid fell
below the limit of detection were not included in the calculation.
.sup.cConfidence intervals were calculated at the 95% confidence
level.
[0083] Nonparametric statistical analysis was used to assess the
statistical significance of differences in urinary .beta.-amino
acid concentrations observed between subjects with epilepsy and
matched control subjects. Using a two-tailed Mann-Whitney test with
a normal approximation, a significant difference for
.beta.-aminoisobutyric acid. (Zc=2.40, 0.01<P<0.02) was found
between subjects with epilepsy and matched control subjects.
[0084] The corresponding one-tailed tests showed that levels of
.beta.-aminoisobutyric acid were higher for those subjects with
epilepsy (Zc=2.40, 0.005<P<0.01). Significant differences in
.beta.-alanine and .beta.-aminoisobutyric acid concentrations were
not observed when comparing male subjects with epilepsy to female
subjects with epilepsy (.beta.-alanine U=338;
.beta.-aminoisobutyric acid U=354) as well as when comparing male
subjects in the control population to female subjects in control
population (.beta.-alanine U=229; .beta.-aminoisobutyric acid
U=267). These results show that gender does not influence the
statistical significance of the observed differences in urinary
.beta.-amino acid concentrations between subjects with epilepsy and
matched control subjects.
[0085] Using a Mann-Whitney test with a normal approximation,
significant differences in urinary .beta.-aminoisobutyric acid
concentrations (Zc=2.37, 0.01<P<0.02) were determined upon
comparing male subjects with epilepsy to male matched control
subjects. Based on the corresponding one-tailed Mann-Whitney test,
these levels were statistically higher for those male subjects with
epilepsy (Zc=2.37, 0.005<P<0.01).
[0086] Using a Krusal-Wallis test with a chi-square approximation,
significant differences in the urinary levels of .beta.-alanine and
.beta.-aminoisobutyric acid were not detected between subgroups
E+D+, E-D+ and E-D- of the population with epilepsy (.beta.-alanine
Hc=0.585; .beta.-aminoisobutyric acid H=0.266). Nor were
differences determined between subgroups C-D+ and C-D- of the
control population, using a two-tailed Mann-Whitney test with a
normal approximation (.beta.-alanine Zc=0.387;
.beta.-aminoisobutyric acid Zc=0.670).
[0087] These results show that seizure frequency and anticonvulsant
medication do not affect the statistical significance of the
observed differences in urinary .beta.-amino acid concentrations. A
comparison of subjects with epilepsy in groups E+D+ and E-D+,
receiving anticonvulsant medication, to matched control subjects in
the group C-D-, not receiving anticonvulsant medication,
demonstrated a significant difference for .beta.-aminoisobutyric
acid (Zc=2.08, 0.02<P<0.05), with higher levels of this amino
acid occurring in the urine of these subjects with epilepsy
(Zc=2.08, 0.01<P<0.025).
[0088] Similarly, a comparison of subjects with epilepsy in groups
E+D+ and E-D+, receiving anticonvulsant medication, to matched
control subjects in the group C-D+, also receiving anticonvulsant
medication, demonstrated significant differences in
.beta.-aminoisobutyric acid levels (Zc=2.06, 0.02<P<0.05).
.beta.-Aminoisobutyric acid concentrations were again found to be
statistically higher for these subjects with epilepsy, as
determined by a one-tailed Mann-Whitney test with a normal
approximation (Zc=2.06, 0.01<P<0.025). These results show
that significant differences in urinary .beta.-aminoisobutyric acid
concentrations are not influenced by anticonvulsant medication
alone.
[0089] The decision to analyze urine rather than cerebrospinal
fluid (CSF) or plasma was taken after careful consideration. It has
long been appreciated that the chemical milieu of the CSF provides
information regarding abnormal cerebral metabolism. CSF is not,
however, a mere ultrafiltrate formed by the choroids plexus, but
arises from the interactions between blood and the CNS (Perry, et
al., Clin. Invest. 1961, 40, 1363-1372). These interactions permit
many substances, especially amino acids, to exhibit similar levels
in plasma as well as in CSF.
[0090] Scriver et al. in their studies of people with
hyper-.beta.-alaninemia, clearly demonstrated that the increased
levels of .beta.-alanine in the CSF are directly reflected in
plasma (op. cit.). They also noted a direct relationship between
plasma levels and urinary excretion, attributing this observation
to the renal tubular transport of .beta.-amino acids. Urinary
levels of .beta.-alanine therefore provide a window of observation
into the metabolism of .beta.-alanine within the CNS.
[0091] From an analytical point of view, urine analysis is
preferred as the control range for .beta.-aminoisobutyric acid in
adult urine is substantially higher when compared to plasma or CSF
(.beta.-aminoisobutyric acid=10-510 .mu.mol/L for urine, 0
.mu.mol/L for plasma, <10 nmol/L for CSF). Urine also represents
an easily accessible biological fluid in which to collect from the
brain-injured individual.
[0092] To validate the notion of screening for seizure
susceptibility based on urinary levels of .beta.-alanine and
.beta.-aminoisobutyric acid, the sensitivity and specificity of
this assay were calculated at defined .beta.-amino acid
concentrations. Optimal results were achieved when the cut-offs for
seizure susceptibility were set at concentrations of 0.8
.mu.mol/mmol creatinine for .beta.-alanine and 10 .mu.mol/mmol
creatinine for .beta.-aminoisobutyric acid in urine samples,
although useful clinical data may be obtained at other cut-off
values. The skilled artisan will appreciate that such cut-off
levels may be different for other bodily fluids. For
.beta.-aminoisobutyric, the sensitivity of this assay, defined as
the probability of testing positive for seizure susceptibility when
a susceptibility is truly present, was determined to be 73%, while
the specificity, defined as the probability of testing negative for
seizure susceptibility when no susceptibility exists, was 47%
[0093] In summary, the results indicate that subjects with seizure
disorders excrete more .beta.-aminoisobutyric acid in their urine
than people who do not have seizure disorders. Urinary levels of
these amino acids were statistically higher for the 49 subjects
with epilepsy relative to the 51 matched control subjects.
Statistical differences are not significantly influenced by gender,
administration of anticonvulsant medication, or seizure frequency.
Accordingly urinary concentrations of .beta.-alanine and
.beta.-aminoisobutyric acid may be used as biological markers for
seizure presence and susceptibility and epileptogenesis.
[0094] The potential clinical applications of measurements of
urinary .beta.-alanine and .beta.-aminoisobutyric acid levels are
multiple. First, such an assay may assist in verifying the presence
of epilepsy. Differentiating seizures from nonepileptic seizures
(psuedoseizures) is a common clinical problem. Urinary
.beta.-aminoisobutyric acid levels augment clinical observation,
EEG studies and serum prolactin measurements as useful clinical
tools in the differentiation between epileptic and nonepileptic
seizures. Secondly, a urinary assay for .beta.-aminoisobutyric acid
has utility in predicting seizure susceptibility. Seizures may
arise from a diversity of CNS insults, including trauma, infection,
ischaemia, and neoplasia. Identifying which subset of patients will
ultimately develop recurrent seizures after such an insult is
currently an unattainable clinical goal. The predictive test
described herein to identify those with a predisposition to
epilepsy therefore has significant clinical value.
Equivalents
[0095] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the following claims. The contents of all
references, issued patents, and published patent applications cited
throughout this application are hereby incorporated by
reference.
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