Note: Descriptions are shown in the official language in which they were submitted.
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HAIR ANALYSIS METHOD
FIELD OF THE INVENTION
This invention relates to an improved analytical method which effectuates the
relatively rapid solubilization and direct analysis of organic analytes, e.g.,
drugs of
abuse, present in hair and other keratinized structures, e.g., fingernails and
toenails,
without effecting the structure of the analyte or being detrimental to
biological analyte
probes, e.g., antibody, RNA/DNA and bio-receptor probes. The analyte can be
directly
analyzed by adding the analyte probe directly to the solubilized analyte-
containing
solution to determine identity of the analyte as well as the extent and
duration of its
consumption by a subject.
BACKGROUND OF THE INVENTION
In the past, hair analysis techniques for the detection of trace metals were
developed that purported to provide information on an individual's nutritional
status. One
objection to the use of these techniques is the difficulty of distinguishing
between trace
metals deposited in hair from the Bloodstream and metals which have become
embedded in hair through external contact with, for example, water and
cosmetic
agents. Consequently, these techniques are not considered useful by the
medical
community for diagnosing nutritional problems, and therefore have not been
considered
sufficiently accurate to determine the level of a particular trace metal
consumed by a
subject.
The problems with previous hair analysis techniques have caused reliance on
urine and blood analysis techniques for the detection of ingested chemicals,
e.g., drugs-
of-abuse, medications and toxic chemicals, in a subject. However, these
techniques are
also known to be disadvantageous in that the duration and intensity of use or
exposure
cannot be ascertained. Urine and blood analysis, at best, can provide short
term
information concerning ingested drugs or chemicals such as drugs-of abuse. In
addition, there are also problems with the interpretation of such results. For
example,
the detection of a low level of ingested chemical in the urine could mean that
a subject
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ingested a small amount of the drug or chemical very recently or a larger
amount
several days earlier. Thus, chronic drug use cannot be determined with these
methods
without repeated testing.
In response to the problems of establishing a reliable and accurate method
that
would measure both the duration and intensity of use of drugs-of abuse,
medications,
toxic chemicals, etc., work performed by Dr. Werner A. Baumgartner, as
reported in
"Radio Immunoassay of Hair for Determining Opiate Abuse Histories", J. Nucl
Med
20:749-752 (1979), determined that long-term histories of exposure to drugs-of
abuse
can be obtained through the analysis of mammalian body hair, since these
substances
are "trapped" within individual hair fibers during their synthesis. In this
respect, hair was
shown to act like a tape recorder, i.e., past exposure histories can be
evaluated through
sectional analysis of hair samples. It was found that heroin, once in the
bloodstream,
will find its way into hair as it is synthesized.
Thus, it was discovered in this study and confirmed by subsequent studies that
a
variety of chemicals, such as drugs-of-abuse, medications, toxic chemicals,
etc.,
hereinafter collectively referred to as "analyte", are trapped by hair during
its synthesis
and that these substances are "locked up" in hair for essentially the duration
of the hair.
This was found to be true for head and body hair as well as for other
keratinized
structures such as fingernails. Suzuki et al., Forensic Sci. International,
24:9-16, 1984.
These entrapped substances cannot be washed out of hair, and are released only
upon
complete destruction of the hair fiber.
Prior art methods of extracting an analyte from hair included subjecting the
hair
to hot methanol solutions (Baumgartner et al., J. Nucl Med 20, 748, 1979) and
by
overnight incubation of hair in an alkaline or acid medium. D. Valente. et
al., Clinical
Chemistry, 1952, Vol. 27, No. 11, 1981. Prior methods also include the use of
a mortar
and pestle to release the entrapped analyte in conjunction with a solvent.
However, solvent extraction procedures suffer from several problems in
accurately determining the presence and amount of an ingested analyte. One of
these
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problems is that the solvent extraction methods frequently remove only a small
unknown and variable fraction of the total analyte present in the hair sample.
Such
methods also tend to be time consuming, and generally involved elevated
temperatures
which may damage the analyte. Another disadvantage is that different analytes
require
different solvents for extraction. For example, a hair sample containing
morphine,
phenylcyclindine ("PCP"), cocaine and marijuana has to be extracted
sequentially with
several different solvents, which is a very time consuming procedure,
particularly since
the solvents have to be evaporated before analysis can proceed.
Other methods and studies pertaining to the degradation of hair and hair
analysis
include:
O. Suzuki, et. al., in a publication by Elsevier Scientific Publishers Ireland
Ltd.,
discloses a method for detecting methamphotamine and amphetamine in nail
clippings
or hair in which the substance was first washed in a mixture of methanol and
water and
dissolved in sodium hydroxide, followed by analysis of the extracted drug.
A.W. Holmes, in Textile Research Journal, 706-712, August 1964 , discloses the
degradation of human hair by papain using sodium sulfite as enzyme activator.
Annette M. Baumgartner. et al., in the Journal of Nuclear Medicine, 20:748-
752,
1979, discloses the extraction of morphine and heroin from hair by pulverizing
hair with
a mortar and pestle followed by treatment with methanol.
D. Valente. et. al., in Clinical Chemistry, Vol. 27, No. 11, 1981, discloses
Dr.
Baumgartner's technique of subjecting hair to a treatment of hot methanol to
effectuate
extraction of drugs of abuse as well as the author's technique of extracting
morphine in
an acid or alkaline medium.
A.M. Baum4artner, et al., in Journal of Forensic Sciences, p. 576-81, July
1981,
discloses the extraction of PCP with mortar and pestle followed by treatment
with
methanol. The extracted PCP was then analyzed with RIA.
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Smith et al., in Journal of Forensic Sciences, Vol. 26, No. 3, July 1981, pp.
582-
586, disclose the testing of hair for the presence of phenobarbitol, in which
a single head hair was washed, dried, cut in 2 mm lengths and added to 0.2 ml
0.1
SDS/saline solution, and a sample assayed by radioimmunoassay.
W.A. Baumgartner. Black, et al., in J. Nucl Med 23:790-892, 1982, discloses
the
extraction of cocaine from hair samples by refluxing the hair samples in
ethanol followed
by RIA analysis.
Ishiyama. et al., in Journal of Forensic Sciences, Vol. 28, No. 2, April 1983,
pp.
380-385, disclose a method whereby hair from methamphetamine addicts was
dissolved using 1.5 N hydrochloric acid at a pH between 1 and 2, followed by
analysis
using a gas chromatography and mass spectrometry.
K. Puschel. et al., in Forensic Science International, 21 (1983) 181-186,
discloses the dissolving of hair samples by exposure to sodium hydroxide and
heat
followed by analysis for the presence of morphine by RIA.
O. Suzuki. et al., in Journal of Forensic Sciences, Vol. 29, No. 2, April
1984, pp.
611-617, discloses the detection of methamphetamine and amphetamine in a
single
human hair by gas chromatography and chemical ionization mass spectrometry.
The
hair sample was first dissolved in a sodium hydroxide solution to which was
added N-
methylbenzylamine.
N.J. Haley et al., in Clin. Chem. 31/10, 1598-1600 (1985), discloses the
analysis
of hair for nicotine and cotinine, in which washed hair samples were dissolved
in a
buffer solution containing gelatin, sodium chloride, Tris and EDTA, and
adjusted to pH
7.4. Samples were then analyzed by radioimmunoassay.
Sramek. Baumaartner et al., in A.M.J. Psychiatry 142:8, August 1985, discloses
the analysis of hair samples of psychiatric patients with methanol extraction
and
radioimmunoassay.
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Baumgartner. et al., in Clinical Nuclear Medicine, Vol. 10, September 1985,
discloses the benefits of extracting entrapped drugs of abuse from hair
followed by RIA
analysis.
Gill. et al., in Nature, Vol. 318, p. 577 (1985) discloses the use of an
SDS/proteinase k/dithiothreital mixture to extract DNA from whole blood, whole
semen,
vaginal fluid, hair roots, bloodstains and semen stains. The article states
that "no DNA
could be isolated from hair shafts".
Smith et al., in J. Forensic Sci. 1986, 31 (4), 1269-73, discloses the
detection of
cocaine in perspiration, menstrual blood stains and hair using RIA.
M. Marqio, et al., in "Determination of Morphine and Other Opioids in the Hair
of
Heroin Addicts by HPLC and MS/MS" at the International Conference, University
of
Verona, June 25-26, 1986, discloses various methods to assay morphine from
hair
samples.
M. Mariao. et al., in the Journal of Analytical Toxicology, Vol. 10,
July/August
1986, discloses a method for the quantitative determination of morphine
contained in
the hair of heroin addicts, by means of heat-acid hydrolysis, pre-column
dansyl
derivatization, straight phase liquid chromatography and fluorescence
detection.
Smith. et al., in Journal of Forensic Sciences, Vol. 31, No. 4, October 1986,
pp.
1269-1273, disclose a method for the analysis of hair for the presence of
drugs whereby
hair samples were first washed, cut into small segments, mechanically
pulverized for six
minutes, refluxed in ethanol and the samples analyzed using radioimmunoassay.
M. Michalodinitrakis, Med.Sci.Law (1987), Vol. 27, No. 1, discloses the
detection
of cocaine in rats from the analysis of hair samples, which were dissolved
upon
exposure to 1.5 N HCL, which brought the pH value to 1-2, following incubation
with
0.01 N HCI at 37°C for one hour.
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Pelli. et al., in Biomedical and Environmental Mass Spectrometry, Vol. 14, 63-
68
(1987) discloses a procedure for the identification of morphine in the hair of
heroin
addicts in which hair is treated with diethylether and hydrochloric acid
followed by
dissolution of the dried extract in methanol.
Higuchi et al., in Nature, Vol. 332, p. 543 (1988) disclose a method for
dissolving
hair at pH 8 by the action of dithiothreitol, proteinase K, and 2% sodium
dodecylsulfate
in order to extract DNA from the digest by a complex chemical extraction
method.
Also noted is the existence of certain patents, e.g., U.S. Patent Nos.
3,986,926,
3,966,551, 3,939,040 and 3,623,950, which pertain to depilatory agents for the
tanning
of hides, and disclose the use of certain enzymes, including papain, in the
dehairing
process.
However, these and other prior art methods have proven disadvantageous for
the reasons noted above and/or because they degrade the analyte probes (e.g.,
antibodies) of biological analytical methods, thereby preventing the use of
such highly
sensitive analytical techniques.
Thus, there exists a need for an analyte detection method that can rapidly and
completely solubilize a certain analyte from keratinized structures of the
body such as
hair, fingernails, toenails and skin of a subject and which permits direct
analysis of the
identity of the analyte and the duration of use of the analyte in, or exposure
to, a
subject, without destroying the analyte of interest and/or an analyte probe of
biological
analytical methods.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a drug and chemical detection
method;
It is another object of the invention to provide a drug and chemical hair
analysis
method;
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It is another object of the invention to provide a reliable method of
solubilizing,
and directly analyzing the identity of, analytes in head and body hair and
other
keratinized structures of the body, and, where applicable, of determining the
duration
and extent of exposure of the analyte in a subject;
It is yet another object of the invention to provide a hair analysis method
that
solubilizes an analyte from the inner core of hair without causing damage to
the analyte;
It is yet another object of the invention to provide a reliable hair
solubilization and
direct analyte detection method that effectively permits the use of highly
accurate
biological analytical methods;
It is yet another object of the invention to provide a reliable hair analysis
method
that may be performed in a much less period of time than known hair analysis
methods.
These and other objects are achieved by a novel keratinized structure analysis
method which comprises preparing a mixture containing a low-redox potential
compound such as dithiothreitol (DTT) or dithioerythritol (DTE), an enzyme
suitable for
the dissolution of keratinized structures and a sample of a keratinized
structure;
permitting DTT or DTE to activate the keratinized structure and/or the enzyme;
permitting the enzyme to at least substantially dissolve the sample of
keratinized
structure to form a keratin digest solution; and subjecting a portion of the
keratin digest
solution to analysis to detect the identity and amount of the analyte, if
present, in the
keratinized structure sample.
The preferred keratinized structure is hair. The enzyme may be selected from
the
group consisting of peptidase, endopeptidase, and protease and preferably is
papain,
chymopapain, or proteinase K. In order to accelerate the analytical process,
copper salt,
cupric sulfate or sodium arsenite (NazAs02) may be added to the digest
solution to
deactivate interfering excess dithiothreitol or dithioerythritol in the
mixture. Preferably,
the analysis of the solubilized analyte is performed by a biological
analytical method
such as an immunoassay.
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In a further aspect of the invention, there is provided a method for the
detection
of an analyte in melanin granules. The method comprises the steps of preparing
a
mixture comprising an agent selected from the group consisting of
dithiothreitol and
dithioerythritol, an enzyme suitable for the dissolution of hair containing
melanin
granules and a sample of hair. The enzyme is permitted to at least
substantially dissolve
the sample of hair to form a hair digest solution containing melanin granules.
The
melanin granules are separated from the hair digest solution and a second
mixture is
prepared comprising the agent, an enzyme suitable for the dissolution of hair,
ethylene
diamine tetraacetic acid and the melanin granules. The dissolution of the
melanin
granules is permitted and the second mixture is subjected to analysis to
detect the
identity and amount of analyte, if present, in the melanin granules.
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DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, a method is provided that permits
the
rapid and complete solubilization of a certain analyte from head or body hair
or other
keratinized structures of an individual who has previously been exposed to the
analyte,
e.g., has ingested the analyte, followed by the identification of the analyte
by known
analytical biological probes, such as the rapid and highly sensitive
immunoassays. The
solubilization of the analyte from the interior of hair is effectuated without
damaging the
analyte trapped within the organic matrix of the hair fiber which is to be
analyzed, nor
does it effect a subsequently-used probe (e.g., antibody) of a biological
analytical
method. The hair analysis method according to the invention also permits the
detection
of past use patterns in a subject over extended periods of time without
performing
repeated testing as is necessary in conventional analyte detection methods
which
measure the content of the analyte in samples of blood or urine.
More particularly, the invention comprises the rapid enzymatic digestion of
the
proteins making up samples of hair and other keratinized structures, followed
by the
effective deactivation of the enzyme and associated enzyme/substrate activator
(DTT
and DTE). The resultant solubilized analyte in the hair digest solution may
then be
analyzed by known biological analytical probes, preferably by highly sensitive
protein-
based analytical techniques such as immunoassay. It has been found that the
amount
of analyte entrapped in hair is directly proportional to the amount of analyte
ingested.
In accordance with the invention, a sample of a keratinized structure, e.g.,
hair, is
first collected from a subject suspected of having been exposed to, or having
ingested,
a particular analyte. Preferably, the hair sample is first washed by known
methods to
remove any analyte or other drug or chemical which may have been deposited on
the
surface of the hair by external contact rather than by actual consumption. The
hair
sample is then subjected to treatment with particular enzymes, together with a
particular
enzyme/substrate activator, so as to effectuate the complete or nearly
complete
dissolution of the organic matrix of the hair fiber, known as keratin. The
subject analyte
that has been "entrapped" within the organic matrix of the hair is then
released into
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solution, or even if protein bound, the analyte is accessible to the antibody
used in
protein-based analytical methods. In order to fully and accurately carry out
the method
of the invention, a complete dissolution of the keratinized structure is
desirable.
The enzymes preferred for the dissolution of the hair samples are those of the
enzyme classes peptidase, endopeptidase and protease. Most active, and
therefore
preferred for use in the invention, are the enzymes papain, chymopapain and
proteinase K.
A number of other proteases have been found to be effective in the method
according to the invention at low pH values (e.g., pH 7-9), namely, protease
Type IV
(bacterial, from Streptomyces caespitosus), Type VIII (from Bacillus
subtilis), Type XI
(proteinase K, fungal, from Tritirachium album), Type XIV (pronase, from
Streptomyces
griseus), Type XVI (from Bacillus subtilis) , Type XVIII (Newlase, from
Rhizopus
species), Type XIX (from Aspergillus sojae) , Type XXI (from Streptomyces
griseus),
Type XXIV (bacterial), Type XXVII (Nagarase), Type III (Prolase), Type X
(Thermophilic-
bacterial Protease, thermolysin); and Type XXIII (from Aspergillus Oryzae).
As noted above, certain art-recognized procedures provide for the use of
papain
for use as a hair depilatory. These depilatory methods remove hair from hides
and skin
by softening it sufficiently so as to permit its ready removal by scraping or
other
mechanical means, and utilize inexpensive and less effective sulfhydryl enzyme
and
substrate activators such as thioglycolic acid or cysteine. Thus, these
methods only
partly degrade the hair and do not provide for the complete chemical
dissolution of the
hair. A mere softening of the hair would not be acceptable in a method
providing for the
analysis of hair for the detection of analyte, since only a complete, or
nearly complete,
dissolution of hair is acceptable in order to obtain a complete release of
"entrapped"
analyte. Moreover, the sulfhydryl enzyme activators used in these depilatory
methods
are also harmful to certain biological analyte probes such as antibodies.
In contrast to these depilatory methods, the method of the present invention
utilizes dithiothreitol ("DTT", or 2,3-dihydroxybutane-1,4-dithiol) or its
isomer
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dithioerythritol ("DTE", or 2,3 dihydroxybutane-1,4-dithiol) as the substrate
and
sulfhydryl-enzyme activating agent. Surprisingly, it has been found that DTT
and DTE
produce a highly active enzyme capable of dissolving hair within a relatively
short period
of time, e.g., about three hours, resulting in the release of the analyte into
the hair
digest solution. This high activity of the enzyme has been found to be due, at
least in
part, to the activation of the keratinized structure substrate itself by DTT
and DTE,
presumably by the action of DTT and DTE in opening up disulfide bonds in the
keratinized structure, which facilitates enzymatic attack.
Once the protein of the keratinized structure has been completely or at least
substantially dissolved, thereby releasing the analyte into the solution
mixture, it has
been found to be necessary to deactivate the enzyme and the enzyme/substrate
activators) in order to subject the analyte to biological analytical probes
such as
antibodies, since the enzyme and enzyme/substrate activator(s), as noted
above, can
interfere with the structural integrity of protein substances involved in the
analytical
method.
The task of deactivating the sulfhydryl-dependent enzymes such as papain has
proven difficult since after the hair digestion step, the enzymes are "buried"
in a "sea" of
sulfhydryl groups belonging to the released hair proteins and enzyme/substrate
activating agents. Known sulfhydryl blocking agents are ineffective in
deactivating the
enzymes, since the known sulfhydryl blockers tend to bind to the degraded hair
proteins
and DTT or DTE and not necessarily to the enzyme sulfhydryl sites critical for
blocking
the activity of the enzymes. Thus, it is not possible to effectively utilize
the protein-based
analytical methods if the enzyme sulfydryl sites are still active.
Thus, it was surprisingly discovered that DTT and DTE act not only to activate
enzymes and/or the keratinized structure substrate causing unexpectedly high
hair
digestion activity, but that they also spontaneously act to deactivate the
enzyme by a
direct or indirect (enzyme self-deactivation) mechanism after the enzyme
effectuates
the complete, or nearly complete, dissolution of the hair protein. Typically,
the enzyme
deactivation action of DTT or DTE occurs within about four to five hours after
exposure
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to the enzyme, which is a sufficient amount of time for the enzyme to
effectuate the
dissolution of the hair sample. Once the enzyme has been deactivated, it has
been
found that the enzyme cannot be reactivated or regenerated by exposure to
fresh DTT
or DTE.
Deactivation of at least certain of the non-sulfhydryl dependent proteinases,
e.g.,
proteinase K, by its inhibitor, phenylmethyl sulfonyl chloride, is generally
not required
since the enzyme has not been found to be active against the antibodies used
in protein
based immunoassay techniques.
It has also been found that active DTT and DTE present in the hair digest
solution constitute a hazard to the structure and activity of other proteins
to which it is
exposed, e.g., antibodies utilized in radioimmunoassay. Thus, it was a further
surprising
result that DTT or DTE in the reaction mixture will not only spontaneously act
to
deactivate the enzyme, but itself is spontaneously deactivated in the digest
solution.
Typically, the spontaneous deactivation of DTT or DTE will occur about 14
hours after
its first exposure to the enzyme, depending on the various concentrations and
amounts
of the enzyme and DTT or DTE utilized, the pH, temperature, amount of hair
sample,
etc.
Thus, in accordance with the method of the invention, complete hair digestion
can be carried out in a relatively short period of time, e.g., overnight, and
the hair digest
solution, which includes the released analyte of interest, can be directly
subjected,
effectively and accurately, to protein-based ligand assay analysis methods the
next
morning. Typically, the entire method, from the washing of hair samples to the
identification of the analyte, should take no longer than about 16-20 hours.
Little or no
intervention by the individual performing the method is needed to release the
analyte
.from the hair sample once the enzyme and DTT or DTE come into contact with
the hair
sample.
Alternatively, it has been discovered that the addition of cupric sulfate to
the
sulfhydryl group-rich hair digest solution acts to more rapidly deactivate the
sulfhydryl
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groups of DTT or DTE. Thus, the addition of low amounts of the cupric sulfate
to the
hair digest mixture after digestion of the hair sample and the deactivation of
the enzyme
by DTT or DTE significantly accelerates the time in which the hair digest
mixture can be
subjected to the analysis method since it is not necessary to wait for the
self
deactivation of DTT or DTE, which occurs approximately fourteen hours after
its
addition to solution. Typically, about 100 microliters of cupric sulfate (10
mg/ml) is
added to 1 ml of hair digest mixture about 4 to 5 hours after contacting the
enzyme and
DTT or DTE with the hair sample so as to permit the enzyme sufficient time to
dissolve
the hair sample.
Similarly, sodium arsenite (NaAs02) may be utilized in the invention to remove
residual DTT or DTE by formation of a precipitable compound. Typically, 100
microliters
of a 100 mg/ml solution of sodium arsenite is added to 1 ml of hair digest
solution to
effectuate the deactivation of DTT and DTE.
Once the rapid and effective dissolution of hair for the purpose of releasing
entrapped analytes is effectuated as described above, the analyte mixture may
then be
subjected to direct analysis by art recognized protein-based analytical
methods such as
radio immunoassay ("RIA"). Such methods are preferred for use in the invention
because RIA and related immuno- or ligand assays are currently the only known
mass
production procedures having the required sensitivity and convenience for
measuring
the low concentrations of analytes contained in hair samples. The use of these
methods
is preferred because only about 0.5 to 1.0 mg. of hair is necessary for
analysis by RIA
and other protein-based analytical methods. Indeed, for certain drugs-of
abuse, it has
been found that analysis by the method according to the invention can be
effectively
performed on as little as one or two hairs about one inch in length.
Other analytical methods may be utilized in place of the protein-based
analytical
methods, including instrumental means such as chromatography, mass
spectrometry,
etc. Because these methods are not protein-based, the step of deactivation of
the
enzyme and DTT or DTE is not necessary when using non-protein-based analytical
techniques. However, the speed and gentleness of the extraction method
according to
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the invention and the ability to quantitate the extraction efficiency through
the inclusion
of a "spike", i.e., the inclusion of a known amount of analyte, makes the
presently
disclosed extraction method also the method of choice for instrumental
analysis
methods such as gas chromatography and mass spectrometry.
The method according to the invention has been found to be effective in
detecting the use and prior use of drugs of abuse such as cocaine,
morphine/heroine,
marijuana, phenylcyclidine or "PCP", and methaqualone. Moreover, the method
according to the invention has been found to be effective in determining prior
usage of
prescription drugs such as digoxin and amphetamines and toxic chemicals such
as
nicotine. It is contemplated that any organic analyte present in the
bloodstream of an
individual and transferred to the hair during its synthesis can be extracted
and analyzed
in accordance with the method of the invention.
In carrying out the method according to the invention, it is preferred that an
aqueous solution of about 110 mg DTT or DTE/10 ml water be used, although
concentrations of DTT or DTE of about 50 - 200 mg/10 ml water have been shown
to be
effective in the invention. It is preferred that the weight ratio of DTT or
DTE to papain or
chymopapain be about 110:2 [when enzyme purity is 16-40 BAEE units/mg
protein],
although efficacious results have been observed at weight ratios of DTT or DTE
to
papain or chymopapain ranging between about 110:1 to about 110:4. With respect
to
proteinase K and other proteases, it is preferred that the weight ratio of DTT
or DTE to
proteinase K (or other proteases) be about 1200:1 (when enzyme purity is 10-20
units
per mg. protein), although weight ratios of 1200:0.5 to about 1200:2 will also
be
effective.
The concentration of hair protein is preferably kept constant at about 10 mg
hair/cc of digest solution so as to prevent variable matrix effects in a
subsequently
utilized protein-based analytical method.
It is preferred that the enzymatic digestion of hair, according to the method
of the
invention, be conducted at low temperatures and near neutral pH. In this
regard, it is
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preferred to perform the method, when papain or chymopapain is utilized as the
enzyme, at a temperature of between about 20°C and 40°C, and at
a pH between about
pH 8.8 and 10.5. Preferably, the pH is between about 8.8 and 9.5. In a most
preferred
embodiment, the temperature is about 37°C and the pH about 9.1.
When proteinase K or other proteases are utilized as the enzyme, it is
preferred
to perform the method between about 20 and 40 degrees centigrade and at a pH
between about 7 and 9. In the most preferred embodiment, the temperature is
about 37
degrees centigrade and the pH about 7.0; under these conditions, the risk of
damaging
a particular analyte is at a minimum. Other enzymes which dissolve hair under
neutral
conditions include: Protease Type XIV(Pronase), Type IV, Type VIII, Type XXVII
(Nagarase), Type XXVIII (Newlase), Type XXVIII, Type XVI, Type XXI and Type
XXIII.
In contrast to other available analyte detection methods such as urine and
blood
analysis, the method in accordance with the invention permits detection of
exposure to
an analyte over a period of time, and is therefore quite beneficial in
detecting chronic
drug use. Since hair is known to grow at a rate of about 0.3-0.4 mm/day or
about 1.0-
1.3 cm/month, it is possible to measure consumption or exposure as far back as
the
hair length permits by evaluating snippets of hair of various lengths, and the
use of
highly sensitive protein-based analytical methods permits analysis of small
samples of
analyte contained in the small snippets of hair.
Through sectional analysis, the method of the invention provides a relatively
permanent record and evidence of a pattern of drug use, or the prior ingestion
of other
substances, for periods ranging from several days to months or even years
after last
use. The history of such exposure can be made as detailed as desired by
analyzing
suitably short sections of hair representing different periods of growth. In
this way, prior
usage over time, and the extent of such use, can be determined.
Although the use of head hair is preferred for use in the invention due to its
length and accessability, it is possible to utilize any other body hair in the
method of the
invention. Thus, it is not practically possible to avoid testing by the method
of the
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invention by shaving one's head.
However, treatments such as perming and dyeing may increase the rate of
dissolution of hair subjected to the method according to the invention. In
some cases,
some analyte may be lost prior to performing the procedure due to such
treatments.
When the subject hair has been so altered, an increase in digestion rate is
evident and
an appropriate correction factor may be applied based upon known rates of
normal hair
dissolution.
Certain other cosmetic agents, such as certain relaxing agents, may cause hair
to become resistant to digestion. Such resistance may be overcome by
increasing the
quantity of enzyme to be used. Preferably, proteinase K is utilized as the
enzyme when
such resistance to digestion is encountered.
Alternatively, when it is not possible to make use of body hair or in some
instance
when the use of hair is not desirable, the use of other keratinized tissue
such as
fingernails, toenails, and skin may be used in the invention. In this regard,
the effective
ratio of DTT or DTE to enzyme needed to dissolve fingernails and toenails in
order to
release the analyte is about the same as for use with hair, as discussed
above. Once
the fingernail and toenail samples are dissolved in accordance with the method
described herein, the released analyte may be analyzed by a desired analytical
method.
In another aspect of the invention, it has been surprisingly discovered that
melanin granules contained in hair can be dissolved by the combined action of
the
enzyme (preferably papain), DTT or DTE and ethylene diamine tetraacetic acid
(EDTA), the latter at a concentration of about 5 mg EDTA/ml of digest
solution. Since
certain analytes or drugs of abuse such as PCP have been discovered to
accumulate in
these granules, dissolution of the granules, which are present in the digest
solution of
hair, can be effectuated and the analyte contained in the granule identified.
In accordance with this aspect of the invention, a hair digest solution is
obtained
as described above, and the melanin granules recovered from the hair digest
solution,
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e.g., by centrifugation. The melanin granules are then contacted with EDTA,
the
enzyme and DTT or DTE to release the analyte from the melanin granules, and
the
analyte analyzed by the methods described above.
The benefits to be obtained from use of the method according to the invention
are many. The method provides a prompt and accurate diagnosis of prior
exposure to a
particular analyte. The subject hair and keratinized structure analysis method
can
provide a record of consumption, or non-consumption, over very long periods of
time.
Guess work regarding the true significance of one blood or urine analysis will
be
eliminated. Hair collection is less intrusive and less physically repulsive
than blood or
urine collection, and samples cannot be altered or substituted, nor can
detection be
evaded by short term abstention or "flushing" (excessive fluid intake) prior
to a
scheduled testing, e.g., pre-employment test or annual physical examination.
Samples
may be stored indefinitely without refrigeration.
The methods according to the invention, useful for the dissolution of
keratinized
structures, e.g., hair, can also be used to ascertain the presence and
structure of
naturally occurring components of hair such as DNA.
The following examples illustrate certain aspects of the invention but they do
not
limit the invention as set forth in the specification and claims.
EXAMPLE 1
Extraction of Cocaine From Hair Sample
10 mg of hair was removed from a subject suspected of being a cocaine addict
and washed by shaking in water at 37°C for 30 minutes. To 10 ml of
distilled water, 110
mg of dithiothreitol (2,3-dihydroxybutane-1, 4-dithiol, Cleland's reagent,
obtained from
Sigma Chemical Co., St. Louis, M0.), was added. The pH of the solution was
adjusted
to pH 9.1 with 15% potassium hydroxide added dropwise with stirring of the DTT
solution. Stirring was continued while adding 80 microliters of Type III
papain solution
(papainaseT"" EC 3.4.22.2) (obtained from Sigma Chemical Co., 16-40 BAEE units
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activity per mg protein).The enzyme solution was at a concentration of 30 mg
of enzyme
protein/ml of water, where 1 mg of enzyme protein has an activity of 16-40
BAEE units
[one BAEE unit will hydrolyze 1.0 micromole of sodium benzoyl-I-arginine
ethylester at
pH 6.2 at 25°C].
To 1 ml of this solution was added the 10 mg hair sample in a 13X75 mm
polycarbonate test tube. The solution was incubated in a 37 ° C water
bath with shaking
for 2 hours, and the solution was allowed to stand overnight at 37°C
without shaking.
The solution containing the dissolved hair sample was centrifuged at 2,000 rpm
[Damon
IEC model CRU 5,000 centrifuge] to remove the melanin granules. To 1 cc of the
hair
digest solution was added 200 microliters of a 1 molar phosphate buffer, pH
5.5.
100 microliters of this solution was assayed by RIA for the presence pf
cocaine
[benzoylecgonine equivalent, or "BEE"]. RIA analysis revealed 83.6 nanograms
BEE/10
mg of hair.
EXAMPLE 2
Addition of Dithioerythritol
The hair sample of Example 1 was analyzed using the digestion and assay
procedure set fourth in Example 1, except for the replacement of
dithiothreitol (DTT) by
dithioerythritol (DTE). The sample was assayed by RIA, which revealed 82
nanograms
cocaine (BEE) per 10 mg of hair.
EXAMPLE 3
Addition of Cupric Sulfate
After digesting the hair sample in the water bath for four hours, 100
microliters of
a 10 mg/ml cupric sulfate solution was added to 1 ml of the hair digest
solution prepared
as set forth in Example 1. The solution was shaken at 37°C for about 30
minutes prior
to the addition of phosphate buffer and assay by RIA. One hundred microliters
of the
hair digest solution was subjected to RIA analysis, which revealed 85.0
nanograms of
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cocaine (BEE)/10 mg of hair.
EXAMPLE 4
Addition of Sodium Arsenite
After digesting the hair sample in the water bath for four hours, 100
microliters of
a 100 mg/ml sodium arsenite solution was added to 1.0 ml of the hair digest
solution
prepared as set forth in Example 1. The solution was shaken at 37°C for
30 minutes.
200 microliters of 1 M, pH 6.5, phosphate buffer was added prior to assay by
RIA. One
hundred microliters of the hair digest solution was subjected to RIA analysis,
which
revealed 82 nanograms of cocaine BEE per 10 mg of hair.
EXAMPLE 5
Substrate Activation By Dithiothreitol (DTT)
10 mg of hair were exposed to 11 mg of DTT at pH 9.1 for a period of 20 hours.
The DTT solution was removed and replaced with DTT and papain as in EXAMPLE 1.
The hair hour for a control specimen not pretreated with DTT and digested as
in
EXAMPLE 1, thereby demonstrating that DTT activated not only the sulfhydyl
dependent enzyme, papain, but the enzyme substrate, hair, as well.
EXAMPLE 6
Digestion and Analysis of Hair Using Proteinase-K
10 mg of hair was removed from a subject suspected of being a cocaine user
and washed by shaking in water at 37 degrees centigrade for 60 minutes. To 10
ml of
0.05 M TRIS buffer, pH 7.0, 60 mg of dithiothreitol (DTT) and 20 mg sodium
dodecyl
sulfate (lauryl sulfate) was added. The pH of the solution was checked to
ensure the
solution was buffered at a pH 7.O.To this solution was added 0.5 mg proteinase
K
[Protease Type XI, from Tritirachium album, obtained from Sigma Chemical Co.;
1 mg
of enzyme protein has an activity of 10-20 units; one unit will hydrolyze
casein to
produce color equivalent to 1.0 umole (181 ug) of tyrosine per minute at pH
7.5 at 37
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degrees centigrade (color by Folin-Ciocalteu reagent)].
To 1 ml of this solution was added the 10 mg hair sample in a 13 x 75 mm
polycarbonate test tube. The solution was incubated in a 37° C water
bath with shaking
for 1 hour and allowed to stand overnight at 37° C without shaking. The
solution
containing the dissolved hair sample was centrifuged at 2,000 RPM to remove
the
melanin granules. To 1 cc of the hair digest solution was added 100
microliters of
Cupric Sulfate (10 mg/ml), and this solution was shaken at 37° C for 30
minutes. 200 ul
of 1 M phosphate buffer, pH 7.0, was then added.
100 microliters of this solution was assayed by RIA for the presence of
cocaine
(benzoylecgonine equivalents, or BEE). RIA analysis revealed 31.2 nanograms
BEE/10
mg of hair.
EXAMPLE 7
Role of Sulfhydryl Compounds in the Activation of Hair for Proteinase K
Digestion
10 mg of hair was incubated in a solution identical to that described in
EXAMPLE
6 except that DTT was omitted. No digestion of hair occurred during 24 hours
of
enzyme exposure, thereby demonstrating the need for the enzyme activation of
the hair
sample by the substrate activator, DTT.
EXAMPLE 8
Dissolution of Fingernails
A 10 mg. sample of fingernail clippings was obtained from a subject, and
subjected to a detergent wash. 220 mg of DTT was added to 10 ml of water in a
test
tube and the pH adjusted to pH 9.1 as in Example 1. A papain suspension, 160
microliters, was then added. 1.0 ml of this solution was then added in a test
tube to 10
mg of fingernail clippings and shaken at 37°C for a period of 24 hours
until dissolution
occurred. The digest solution was then analyzed by RIA as previously
described.
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EXAMPLE 9
Performance of Sectional Analysis
A hair sample, about 6 cm in length, was obtained from an individual suspected
of being a heroin addict. The samples were carefully sectioned into three 2 cm
sections,
with corresponding sections added to three separate test tubes and washed. The
hair
samples were subjected to the process described in Example 1, except that
chymopapain (EC 3.4.22.6 was used in place of papain as the enzyme. The
samples
were agitated overnight as previously described.
RIA analysis revealed morphine content in the three sections of 13.5, 5.7 and
0
nanograms/10 mg hair.
EXAMPLE 10
Dissolution of Digestion-Resistant Hair
Ten milligrams of hair which had been treated with relaxer was incubated
overnight in the solution digest described in Example 6. The hair sample did
not
dissolve in the usual 20-hour period. A greater and additional amount of
proteinase K,
i.e., 1 mg, was then added to the partially digested sample. The sample then
dissolved
within the next 24 hours. The digest was centrifuged and 100 ul CuS04 solution
(10
mg/ml) was added to 1 ml of the supernatant which was then shaken at 37
degrees
centigrade for 30 minutes. 200 ul of 1 M phosphate buffer pH 7 was added. Due
to the
high amount of proteinase K in the resulting digest, 20 ul of the proteinase
inhibitor
phenylmethyl sulfonyl chloride in ethanol was added to the digest prior to
assay by RIA.
RIA analysis revealed 7.4 ng cocaine (BEE)/10mg hair.
While there have been described what are presently believed to be preferred
embodiments of the invention, it will be apparent to one skilled in the art
that numerous
changes can be made in the ingredients, conditions and proportions set forth
in the
foregoing embodiments without departing from the invention as described herein
and as
defined in the appended claims.
