Note: Descriptions are shown in the official language in which they were submitted.
- 1 - I3~~?~9
BAIR ANALYSIS METHOD
FIELD OF THE INVENTION
This invention rs:lates 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 ana7_yte probe directly to the solubilized analyte-
containing solution to determine the 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
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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 itechniques 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 ingeated drugs or chemicals such as drugs-
of-abuse. In ac3dition, 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 ingested a small amount
of the drug or chemical very recently or a larger amount
several days ea~.~lier. Thus, chronic drug use cannot be
determined with these methods without repeated testing.
In response to i~he problems of establishing a reliable
and accurate mei:hod that would measure both the duration
and intensity o~° use of drugs-of-abuse, medications,
toxic chemicals, etc., work performed by Dr. Werner A.
Baumgartner, as reported in "Radioimmunoassay of Hair
for Determining Opiate Abuse Histories", J. Nucl Med
20:749-752 (1979), determined that long-term histories
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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. 7.t was found that heroin, once in the
bloodstream, wi7.1 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. Val.ente, 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 problem~~ in accurately determining the presence
and amount of an ingested analyte. One of these
problems is that: the solvent extraction methods
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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 involve elevated temperatures which may
damage the analyte. Another disadvantage is that
different analytes require different solvents for
extraction. Fo:r example, a hair sample containing
morphine; pheny:Lcyclindine ("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 meathamphetamine 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 hydro~:ide, followed by analysis of the
extracted drug.
A.W. Holmes, in Textile Research Journal, 706-712,
August 1964, di~~closes the degradation of human hair by
papain using sodium sulfite as enzyme activator.
Annette M. Baumc~artner, 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
1~407~9
-5-
effectuate extraction of drugs of abuse as well as the
author's technique of extracting morphine in an acid or
alkaline medium.
A.M. Baumcrartne~r, 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.
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 O.:Z ml 0.1$ SDS/saline solution, and a
sample assayed by radioimmunoassay.
W.A. Baumgartnei~ 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 chromatograph and mass
spectrometry.
K. Puschel, et a1_, 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.
0. Suzuki, et al_, in Journal of Forensic Sciences, Vol.
29, No. 2, April 1984, pp. 611-617, discloses the
detection of mAt!~~amphetamine and amphetamine in a single
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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 a:L-, in Clin. Chem. 31/10, 1598-1600
(1985), discloses the analysis of hair for nicotine and
cotinine, in wh:LCh 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 b~t radioimmunoassay.
Sramek, HaumQari=ner, et al., in A.M.J. Psychiatry 142:8,
August 1985, di;;closes the analysis of hair samples of
psychiatric patients with methanol extraction and
radioimmunoassay.
Baumaartner, 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 stages that "no DNA could be isolated from
hair shafts".
Smith et al., in J. Forensic Sci. 1986, 31(4), 1269-73,
discloses the dE~tection of cocaine in perspiration,
menstrual bood stains and hair using RIA.
M. Margio, et a7~_, 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 24--26. 1986, discloses various methods to
1~40'l0~
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assay morphine from hair samples.
M. Marino, et a,l., 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 HC1 at 37~C for one
hour.
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 t:he 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 dithic>threitol, proteinase K, and 2~ sodium
dodecylsulfate i.n order to extract DNA from the digest
by a complex chemical extraction method.
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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;
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 keratinizced structures of the body, and, where
applicable, of determining the duration and extent of
exposure of the analyte in a subject;
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_g_
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 s~olubilization 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
dithioerythrito:l (DTE), an enzyme suitable for the
dissolution of Iceratinized 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 kE~ratinized structure is hair. The enzyme
may be selected from the group consisting of peptidase,
endopeptidase, <ind protease and preferably is papain,
chymopapain, or proteinase K. In order to accelerate
the analytical process, cupric sulfate or sodium
arsenite (Na2As1)2) may be added to the digest solu~ion
to deactivate interfering excess dithio~hrPitol or
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dithioerythritol in the mixture. Preferably, the
analysis of the solubilized analyte is performed by a
biological analytical method such as an immunoassay.
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 an.alyte 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/substrat~e activator (DTT and DTE). The resultant
solubilized analyte in the hair digest solution may then
be analyzed by itnown 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.
1~~0~1~9
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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 nirst 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 i~han 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 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 carat 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 othE~r 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, fronn Streptomyces caespitosus). Type VIII
(from Bacillus subtilis), Type XI (proteinase K, fungal,
from Tritirachium album), Type XIV (pronase, from
Streptomyces gr~.seus), 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
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(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, disso:Lution 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 anal~~rte probes such as antibodies.
In contrast to these depilatory methods, the method of
the present invention utilizes dithiothreitol ("DTT", or
2,3-dihydroxybui:ane-1,4-dithiol) or its isomer
dithioerythrito:l ("DTE", or 2,3 dihydroxybutane-1,4-
dithiol) as the substrate and sulfhydryl-enzyme
activating ageni~. 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 _Lnto the hair digest solution. This high
activity of the enzyme has been found to be due, at
least in part, i:o the activation of the keratinized
structure substrate itself by DTT and DTE, presumably by
the action of D~~T and DTE in opening up disulfide bonds
1~4U~U~
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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 t:he enzyme by a direct or indirect (enzyme
self-deactivati~~n) 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
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four to five hours after exposure 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 b.e reactivated or regenerated by exposure
to fresh DTT or DTE.
Deactivation of at least certain of the non-sulfhydryl
dependent prote:inases, 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
immmunoassay techniques.
It has also been found that active DTT and DTE present
in the hair digf~st solution constitute a hazard to the
structure and a<:tivity of other proteins to which it is
exposed, e.g., antibodies utilized in radioimmunoassay.
Thus, it was a i°urther 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 i:he 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 t)TT 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
.~a40 ~~9
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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 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 S 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 precipitab:Le 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 analytica:L methods such as radioimmunoassay
"RIA"). Such methods are preferred for use in the
invention becau:ae RIA and related immuno- or ligand
1~40~~~
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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 mE~thods are not protein-based, the step of
deactivation of the enzyme and OTT or DTE is not
necessary when rising non-protein-based analytical
techniques. However, the speed and gentleness of the
extraction method according to the invention and the
ability to quani:itate the extraction efficiency through
the inclusion o!: 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 a:c cocaine, morphine/heroin, 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 di.goxin and amphetamines and toxic
chemicals such ass nicotine. It is contemplated that any
organic snalyte present in the bloodstream of an
individual qnd transferred to the hair during its
13~~~~9
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synthesis can bye 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 m:L water be used, although concentrations
of DTT or DTE o:E 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 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 pFi 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
1'~~0'~0~
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enzyme, it is :preferred to perform the method between
about 20 and 4~0 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 ~:IV (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 ca.n 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
1a40'~09
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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 b:y the method of the 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 enz~~me to be used. Preferably, proteinase K
is utilized as i:he enzyme when such resistance to
digestion is en<:ountered.
Alternatively, when it is not possible to make use of
body hair or in some instance when the use of hair is
not desirable, t:he 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 or 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
1~~~'~09
-20-
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 o:E about 5 mg EDTA/ml of digest solution.
Since certain analytes or drugs of abuse such as PCP
have been disco~~ered to accumulate in these granules,
dissolution of i~he 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, e.g., by centrifugation. The melanin granules
are then contacted with EDTA, the enzyme and DTT or DTE
to release the <~nalyte from the melanin granules, and
the analyte ana:Lyzed 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 consunnption, 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 repu7_sive 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, usefsl for the
~_~40"~09
-21-
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 t':hey do not limit the invention as set
forth in the specification and claims.
EXAMPLE 1
Extraction of Cocaine From Hair Sample
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 dithioth:reitol (2,3-dihydroxybutane-1,4-dithiol,
Cleland's reagent, obtained from Sigma Chemical Co., St.
Louis. MO.), wa:a added. The pH of the solution was
adjusted to pH !~.1 with 15% potassium hydroxide added
dropwise with si.irring of the DTT solution. Stirring
was continued while adding 80 microliters of Type III
papain solution (papainase EC 3.4.22.2) (obtained from
Sigma Chemical Co., 16-40 BAEE units 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-L-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
,....
-22-
200 microliters of a 1 molar phosphate buffer, pH 5.5.
100 microliters of this solution was assayed by RIA for
the presence of cocaine [benzoylecgonine equivalent, or
"BEE"]. RIA analysis revealed 83.6 nanograms BEE/l0 mg
of hair.
EXAMPLE 2
Addition of Dithioerythritol
The hair sample of Example 1 was analyzed using the
digestion and assay procedure set forth 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 RI,A analysis, which revealed 85.0 nanograms
of cocaine (BEE)/10 mg of hair.
.,..-...
140?0~
-23-
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 soluti~~n was added to 1.0 ml of the hair digest
solution prepa red as set forth in Example 1. The
solution was shaken at 37~ C for 30 minutes. 200
microliters of 1M, 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 .92 nanograms of cocaine (BEE) per 10 mg
of hair.
EXAMPLE 5
Substrate Activation By Dithiothreitol (DTT)
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
specimen dissolved within 10 minutes as compared to
within one hour for a control specimen not pretreated
with DTT and digested as in EXAMPLE 1, thereby
demonstrating that DTT activated not only the
sulfhydryl-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
~.~~.~? ~9
-24-
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 7Ø 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 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 Sulfhyd:ryl Compounds in the Activation of Hair
for Proteinase 1:C Digestion
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 tJze hair sample by the substrate
'~~ 1.340~~9
-25-
activator, DTT.
EXAMPLE $
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 <iissolution occurred. The digest
solution was then analyzed by RIA as previously
described.
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 thEa 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 re~iealed morphine content in the three
sections of 13.~i, 5.7 and 0 nanograms/10 mg hair.
EXAMPLE 10
Dissolution of Digestion-Resistant Hair
-26-
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)/lOmg 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 witlaout departing from the invention as
described herein and as defined in the appended claims.
