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Separate radioimmune measurements of body fluid Δ9-THC and 11-nor-9-carboxy-Δ9-THC
Life Sciences Vol . 19, pp . 1711-1718, 1976 . Printed in the U .S .A .
Pergamon Presa
SEPARATE RADIOIMMUNE MEASUREMENTS OF BODY FLUID ~'-THC AND 11-NOR-9-CARBOXY-D s -THC James R . Soares and Stanley J . Gross Department of Anatomy School of Medicine University of California Los Angeles, California 90024 (Received in final form October 18, 1976) SUMMARY Simultaneous native molecule and discrete metabolite immune assa~s were performed after exposure of subjects to standardized ~ -THC cigarettes . Plasma (and urine) 11-nor-9-carboxy ~ 9 -THC remains elevated long after O 9 -THC becomes scant or undetectable enabling simple radioimmune determination of recent versus distant exposure to multiple cigarettes . Cannabinoid metabolism remains largely unquantitative due to the lack of Classic metabolic work required administrabroadly applicable technology . tion of radiolabeled compounds followed by a search for radioactive derivation in body fluids (1-3) . A radioimmune technique specifically for ~ 9 -THC in plasma (4) has been extended to measure in body fluids separately 11-nor-9 carboxy-D 9 -THC (C-THC), a major cannabinoid metabolite (5) . The present plasma and urine data pertain separately to ~'-THC and C-THC alterations after human subjects smoked standardized (19 .8 mg ~ 9 -THC)cigarettes . Simultaneous but discrete quantification of native molecules and metabolites) requires ex tremely specific antisera (6 ) . Previously such subtle determinations were available only from complex research instrumentation (e .g . mass spectroscopy) . Rea ents : 3 H-D e -THC (50 Ci/mM) and C-THC were obtained from Research Triangle Inst tute, North Carolina and ~'-THC from the National Institute of Drug Abuse . A 2-(4'-carboxyphenylazo)-~'-THC antiserum (4) at a 1 :100 dilution was used to assay A'-THC . A homologous reagent at 1 :150 dilution was used for 11-nor-9carboxy-0 9 -THC . ASSAYS Plasma 0'-THC : Samples from naive subjects were "spiked" with various known amounts o d -THC . Aliquots (0 .5 - 1 ml) were precipitated with two volumes of absolute ethanol, centrifuged at 4oC and the ethanol "supernatant" shaken with 10 mls ether and 1 ml water . The ether layer was separated, blown down under nitrogen and the residue reconstituted in 1 .0 ml phosphate buffer (O .1M, pH 7 .0) . One-tenth ml volumes were incubated (2 hours, 4oC) with 0 .25 ml of antiserum (diluted to bind 50% of added marker in the absence of unlabelled THC) and 40 pg (5000 cpm) of 3 H-D e -THC . Bound and free fractions were separated by incubation (15 minutes) with 1 ml of a dextran-charcoal suspension (0 .25% dextran, 2 .5% charcoal) and centrifugation . Portions of supernate were transferred to scintillation vials to which 5-10 ml Aquasol (R) (New England Nuclear) were added . Samples were counted for 10 minutes in a Beckman LS-230 liquid 1711
9 9 Body Fluid D -THC and 9-Carboxy-D -1TiC
1712
Vol . 19, No . 11
scintillation counter (50% efficiency for 3 H) . Standard inhibition curves were plotted from percent inhibitions produced by added D e -THC . The foregoing procedures were then utilized for plasma samples containing unknown amounts of ~'-THC . Urine 0'-THC : Normal urines from non-users of marihuana do not inhibit 3 H-~ e THC-antiserum binding . Such samples were "spiked" with various known amounts of De-THC . Aliquots (50 - 100 ul) were incubated directly with 0 .25 ml D 9 -THC antiserum and 3 H-~ ° -THC (5000 cpm) to obtain the standard inhibition curve . In the assay, equivalent volumes of unknown samples were substituted for the standards . Plasma C-THC : Plasma spiked with various amounts of C-THC was treated as described above for ~ 9 -THC except that the ethanol supernatant was evaporated under nitrogen and the residue reconstituted in 0 .1 M dibasic phosphate (1 ml) . As before aliquots (0 .1 ml) were incubated and assayed with the corresponding antiserum . Urine C-THC : Urine samples were treated as for D 9 -THC except for the substitut own C-THC and corresponding antiserum .
of
Saliva 0 s -THC and C-THC : Saliva samples were treated (without extraction) as was done in direct plasma assays . Standard inhibition curves were plotted using saliva from control subjects . MARIHUANA SUBJECTS Plasma and urine samples* obtained from marihuana users at various intervals after smoking one to three standard marihuana cigarettes** were assayed for os-THC and C-THC . Assay sensitivity was defined as the point on the standard curve greater by 2 standard deviations than 0 .0 ng . This was 5 ng/ml (1 ng/tube) for plasma and 1 .25 ng/ml fôr urine . All assays were done in duplicate and the mean value reported . The intraassay coefficient of variation (7) for points within the linear region of the standard curve was less than or equal to 10% . Based on the standard curve the interassay variation (7) approximated 15% . RESULTS Urine Ds-THC : None of four subjects had measurable urinary D 9 -THC . consistent wwith previous work (1 .8) .
This is
Urine C-THC : Levels in 6 occasional smokers 1 - 48 hours after smoking a single mar huana cigarette are shown in Figure 1 . Pre-intoxication urine C-THC was very low in all six subjects . A definite elevation occurred 1 - 2 . hours after smoking, persisting for at least 4 hours . Four of the six acute subjects had elevated urinary C-THC at the 6-hours interval . Gradual diminution occurred over the next 18 hours until levels were not significantly different from pre-intoxication values .
* Provided by the Department of Psychiatry, UCLA School of Medicine . ** Each cigarette contained 19 .8 mg D'-THC .
9 Body Fluid A 9 -THC and 9-Carboxy-D -THC
Vol . 19, No . 11
FIG .
1713
1
~o 60
S U2
"
50 40
F
"
Y
"
2O U
10
1
Preintar
"
i
'
" "
"
I-2
3-4
"
" " = ' 5-6 7-8
" ~r~r-~r~ 12
24
48
POST-INTOXICATION (HOURS)
Urine C-THC in occasional marihuana users 1 - 48 hours after smoking a standard Highest levels were at 2 - 4 hours . (19 .8 mg ~'-THC) cigarette .
FIG . 2 zoo ISO
F
G
100
0 300 250 E P 200 I50 100 50
"
"
i
r~- ~T -"r 5-IS 30-60 120 IBO 240 POST-NTOXICATION (MNUTES)
Plasma e'-THC and C-THC levels in occasional marihuana users 5 - 240 minutes after a single smoke . e 9 -THC peaks 15 minutes or before and C-THC at 30 - 60 minutes in 3 subjects and at 15 minutes in 2 subjects .
1714
9 Body Fluid 0 -THC and 9-Carboxy-D 9-THC
Vol . 19, No . 11
Plasma ~'-THC and C-THC : Plasma samples were obtained from five subjects a ter smo ing a sing e cigarette and from three subjects after smoking three consecutive cigarettes . None of the first group and only one of the second group had measurable pre-intoxication levels of D'-THC . In all five single srtwke subjects definitive De-THC incrert(ents (10 130 ng/ml) occurred 15 minutes after smoking, becoming almost undetectable 2 hours later . Plasma C-THC peaked at 30 - 60 minutes in 3 of the sart(e 5 subjects . Significant levels remained in all, at least 2 hours after exposure (Figure 2) . Following repetitive cigarettes there were rapid shifts in plasma ~9THC and C-THC up to 48 hours (Figure 3) . Expectedly plasma o'-THC peaks (100 - 260 ng/ml) were much higher after multiple consecutive cigarettes than a single one, permitting detection of e'-THC as long as 2 - 3 hours after completion of the last cigarette . Plasma C-THC was vastly higher, peaking (158-460 ng/ml) 30 - 60 minutes after the last cigarette with significant artwunts of this rtletabolite persisting in plasma 48 hours after the last cigarette . FIG . 3
_ 300 É
~aoo c U 2 300 F U . 200
POST-INTOXICATION (HOURS)
Plasma A 9 -THC and C-THC in representative subjects after three consecutive standard marihuana cigarettes . Peaks were much higher but did not shift : -THC (e----~) became insignificant at 4 hours while C-THC (o -o, ~-~, ~--)remains appreciable at 48 hours .
Saliva e'-THC and C-THC : Saliva samples (after rinsing) obtained from an occasional marihuana user demonstrated only C-THC after one standard e'-THC cigarette, the peak level at 15 minutes becoming undetectable 2 hours later (Table 1) . The baseline ~'-THC (1 cigarette) would seem to indicate that measured C-THC came from secreted saliva rather than solubilized cigarette or smoke .
9 Body Fluid D 9 -THC and 9-Carboxy-D -THC
Vol . 19, No . 11
1715
TABLE 1 Saliva e'-THC and C-THC Levels 15 - 240 Minutes After Smoking a Single Cigarette Time ~Minute~ Pre-intox .
15
30
60
180
240
e'-THC (ng/ml)
0
0
0
0
0
0
C-THC
0
12
3
2
0
0
(ng/ml)
Assa Validation : Extract assays were corroborated (correlation coefficient, 0 .98, Ta e 2 y direct (unextracted) plasma measurements (9) in which sample, antibody, and tritiated marker were simply incubated 30 minutes prior to charcoal separation (as above) and counting . TABLE 2 e'-THC Measured Using Plasma Extracts and Unextracted Plasma Unextracted Plasma e'-THC (ng/ml) 0 8 240 5 0 156 140 6 19 10 30 50 60 0 120 15 5 22 8 23 0 Correlation Coefficient = 0 .98
Reconstituted Ether Extracts e'-THC (ng/ml) 0 9 300 9 0 150 155 18 31 16 45 54 45 0 100 10 5 28 10 21 8
1716
Body Fluid D 9 -TfiC and 9-Carboxy-0 9-THC
Vol . 19, No . 11
DISCUSSION Although discrimination between closely related molecules is the forte of complex analytical technology, antibodies can also recognize and measure subtle differences in discrete functional groups (10) . The foregoing body fluid measurement separately of A 9 -THC and C-THC constitute the first immunologic quantitation of two discrete cannabinoid metabolites in body fluids . Initial findings outline temporal patterns in plasma and urine following marihuana inhalation . Such discrete ~e-THC and C-THC metabolite measurements should contribute to our understanding of compartment turnover, tissue storage and clinical correlates . After one standard marihuana cigarette, urines from occasional user subjects were negative or marginal for 0s-THC in contrast to C-THC (up to 60 ng/ml at 48 hours) . Concomitantly, plasma D e -THC peaked at 15 - 30 minutes and C-THC at 30 - 60 minutes after a single standard smoke in a majority of subjects . Importantly, Ds-THC became almost undetectable 1 - 2 hours after the solitary exposure contrasting sharply with C-THC which persisted for several hours in plasma . This critical divergence of plasma 4 9 -THC versus C-THC was even greater in subjects who had smoked 3 consecutive cigarettes . Plasma ~'-THC became almost undetectable 4 hours after the last one had been consumed in contrast to persistent (48 hours) significant levels of C-THC . Unfortunately suitable chronic smoker plasma samples were not available for analysis . The apparent slight secondary increment in plasma C-THC in the multiple cigarette group could relate to the assay or reflect intrinsic physiological variation at the blood-alveolar barrier due to the extremely hydrophobic, lipophilic character of marihuana . A plasma sample negative for D 9 -THC would seem to be unambiguous evidence that Marihuana had not been smoked in the preceding hour . Even after 3 consecutive standard marihuana cigarettes ~'-THC drops at 4 hours to the level of current detectability . A large control population can now be studied for Ds-THC and C-THC (ratios) to interpret properly individual subject variants and corresponding behavioral patterns . Although we arbitrarily chose to obtain initial samples 15 minutes after smoking, mass spectroscopic analyses of plasma samples following exposure to marihuana demonstrate a 5 minute peak (11) reaching 15 minute levels in harmony with ours . Unfortunately mass spectroscopic derivatization and quantitation of C-THC is not yet available . Its proportion of total urinary cannabinoids is uncertain . However, C-THC's metabolic importance (5) is supported by our experimental data pertaining to the relative proportions of D s -THC and C-THC in blood and saliva . Implicit from the foregoing experimental observations is the potential ambiguity of information to be derived from assays in which "specificity" exists only for "the closed 3 ring cannabinoid" moiety (12, 13, 14) . Almost all D 9 -THC metabolites and naturally occurring non-psychoactive cannabinoids (e .g .cannabinol) are in this broad category . Another THC immunogen preparation (15) links Ds-THC to the carrier protein via a 5'-carboxyl site on the pentyl side chain with resultant blockade
Vol . 19, No . 11
9 9 Body Fluid D -THC and 9-Carboxy-D -THC
1717
of the latter . There are potential endogenous metabolites (11) containing substitutents on the pentyl moiety which could be expected to crossreact with the relevant antisera . Of immediate consequence would be the inability to quantify separately the endogenous C-THC metabolite described in the foregoing experiments . While isotope cannabinoid assays are presently hampered primarily by nonhomologous markers, ~'-THC and C-THC can discretely be measured with acSpecific immune assays, especially those without extraction, should curacy . permit large scale kinetic and forensic studies . ACKNOWLEDGMENT Supported in part by National Institute on Drug Abuse Grant #DA-00106 REFERENCES
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
L .Lemberger, S .D .Silberstein, J .Axelrod, and I .J .Kopin, Science 170, 1320 - 1322 (1970) . .Lember L er, J .Axelrod, and I .J .Kopin, Ann . N .Y . Acad . Sci . _191, 142-154 1971) . M .E .Wall, D .R .Brine, C .G .Pitt, and M .Perez-Reyes, J .Am .Chem .Soc . 94, 8579-8581 (1972) . S.J .Gross, J .R .Soares, S-L .R .Wong, and R .E .Schuster, Nature _252, 581-582 (1974) . M .E .Wall, D .R .Brine, and M .Perez-Reyes, Int .Conf .Pharmacol . Cannabis Raven Press, New York (1975) . J .R .Soares, E .Zimmermann, and S .J .Gross, Febs .Lett . 61, 263-266 (1976) . G .E .Abraham, R .Swerdloff, and D .Tulchinsky, J .C1in .E~ocr . Metab . 32, 619-624 (1971) . L.E .Hollister, S .L .Kanter, R .D .Board, and D .E .Green, Res . Comm .Chem . Pathol . Pharmacol . 8, 579-584 (1974) . J .R .Soares, S .J .Gross, and R .A .Bashore, J .C1in .Endocr . Metab . _40, 970-976 (1975) . S .J .Gross, and J .R .Soares, Immunoassays for Drugs Subject to Abuse . (Eds . S .J .Mule, I .Sunshine, M .Braude, and R .E .Willette) p .3, CRC Press, Cleveland, Ohio (1974) . A .Ohlsson, J .E .Lindgreen, K .Leander, and S .Agurell, National Institute on Drug Abuse Research Monograph Series p .48, (1976) . J .D .Teale, J .M .Clough, E .M .Pialli, L .J .Kin , and V .Marks, Res .Comm . Chem . Pathol . Pharmacol, 11, 339-042 (197 V .Marks, J .D .Teale, and D .Fry, British Medical Journal 3, 348-349 (1975) . A .R .Chase, P .R .Kelley, A .Taunton-Rigby, R .T .Jones, and T .Harwood, National Institute on Drug Abuse Research Monograph Series 7, p .l, (1976) . C .E .Cook, M .L .Hawes, E .W .Amerson, C .G .Pitt, and D .Williams, National Institute on Drug Abuse Research Monograph Series 7, p .15, (1976) .
5.
Pergamon Presa
SEPARATE RADIOIMMUNE MEASUREMENTS OF BODY FLUID ~'-THC AND 11-NOR-9-CARBOXY-D s -THC James R . Soares and Stanley J . Gross Department of Anatomy School of Medicine University of California Los Angeles, California 90024 (Received in final form October 18, 1976) SUMMARY Simultaneous native molecule and discrete metabolite immune assa~s were performed after exposure of subjects to standardized ~ -THC cigarettes . Plasma (and urine) 11-nor-9-carboxy ~ 9 -THC remains elevated long after O 9 -THC becomes scant or undetectable enabling simple radioimmune determination of recent versus distant exposure to multiple cigarettes . Cannabinoid metabolism remains largely unquantitative due to the lack of Classic metabolic work required administrabroadly applicable technology . tion of radiolabeled compounds followed by a search for radioactive derivation in body fluids (1-3) . A radioimmune technique specifically for ~ 9 -THC in plasma (4) has been extended to measure in body fluids separately 11-nor-9 carboxy-D 9 -THC (C-THC), a major cannabinoid metabolite (5) . The present plasma and urine data pertain separately to ~'-THC and C-THC alterations after human subjects smoked standardized (19 .8 mg ~ 9 -THC)cigarettes . Simultaneous but discrete quantification of native molecules and metabolites) requires ex tremely specific antisera (6 ) . Previously such subtle determinations were available only from complex research instrumentation (e .g . mass spectroscopy) . Rea ents : 3 H-D e -THC (50 Ci/mM) and C-THC were obtained from Research Triangle Inst tute, North Carolina and ~'-THC from the National Institute of Drug Abuse . A 2-(4'-carboxyphenylazo)-~'-THC antiserum (4) at a 1 :100 dilution was used to assay A'-THC . A homologous reagent at 1 :150 dilution was used for 11-nor-9carboxy-0 9 -THC . ASSAYS Plasma 0'-THC : Samples from naive subjects were "spiked" with various known amounts o d -THC . Aliquots (0 .5 - 1 ml) were precipitated with two volumes of absolute ethanol, centrifuged at 4oC and the ethanol "supernatant" shaken with 10 mls ether and 1 ml water . The ether layer was separated, blown down under nitrogen and the residue reconstituted in 1 .0 ml phosphate buffer (O .1M, pH 7 .0) . One-tenth ml volumes were incubated (2 hours, 4oC) with 0 .25 ml of antiserum (diluted to bind 50% of added marker in the absence of unlabelled THC) and 40 pg (5000 cpm) of 3 H-D e -THC . Bound and free fractions were separated by incubation (15 minutes) with 1 ml of a dextran-charcoal suspension (0 .25% dextran, 2 .5% charcoal) and centrifugation . Portions of supernate were transferred to scintillation vials to which 5-10 ml Aquasol (R) (New England Nuclear) were added . Samples were counted for 10 minutes in a Beckman LS-230 liquid 1711
9 9 Body Fluid D -THC and 9-Carboxy-D -1TiC
1712
Vol . 19, No . 11
scintillation counter (50% efficiency for 3 H) . Standard inhibition curves were plotted from percent inhibitions produced by added D e -THC . The foregoing procedures were then utilized for plasma samples containing unknown amounts of ~'-THC . Urine 0'-THC : Normal urines from non-users of marihuana do not inhibit 3 H-~ e THC-antiserum binding . Such samples were "spiked" with various known amounts of De-THC . Aliquots (50 - 100 ul) were incubated directly with 0 .25 ml D 9 -THC antiserum and 3 H-~ ° -THC (5000 cpm) to obtain the standard inhibition curve . In the assay, equivalent volumes of unknown samples were substituted for the standards . Plasma C-THC : Plasma spiked with various amounts of C-THC was treated as described above for ~ 9 -THC except that the ethanol supernatant was evaporated under nitrogen and the residue reconstituted in 0 .1 M dibasic phosphate (1 ml) . As before aliquots (0 .1 ml) were incubated and assayed with the corresponding antiserum . Urine C-THC : Urine samples were treated as for D 9 -THC except for the substitut own C-THC and corresponding antiserum .
of
Saliva 0 s -THC and C-THC : Saliva samples were treated (without extraction) as was done in direct plasma assays . Standard inhibition curves were plotted using saliva from control subjects . MARIHUANA SUBJECTS Plasma and urine samples* obtained from marihuana users at various intervals after smoking one to three standard marihuana cigarettes** were assayed for os-THC and C-THC . Assay sensitivity was defined as the point on the standard curve greater by 2 standard deviations than 0 .0 ng . This was 5 ng/ml (1 ng/tube) for plasma and 1 .25 ng/ml fôr urine . All assays were done in duplicate and the mean value reported . The intraassay coefficient of variation (7) for points within the linear region of the standard curve was less than or equal to 10% . Based on the standard curve the interassay variation (7) approximated 15% . RESULTS Urine Ds-THC : None of four subjects had measurable urinary D 9 -THC . consistent wwith previous work (1 .8) .
This is
Urine C-THC : Levels in 6 occasional smokers 1 - 48 hours after smoking a single mar huana cigarette are shown in Figure 1 . Pre-intoxication urine C-THC was very low in all six subjects . A definite elevation occurred 1 - 2 . hours after smoking, persisting for at least 4 hours . Four of the six acute subjects had elevated urinary C-THC at the 6-hours interval . Gradual diminution occurred over the next 18 hours until levels were not significantly different from pre-intoxication values .
* Provided by the Department of Psychiatry, UCLA School of Medicine . ** Each cigarette contained 19 .8 mg D'-THC .
9 Body Fluid A 9 -THC and 9-Carboxy-D -THC
Vol . 19, No . 11
FIG .
1713
1
~o 60
S U2
"
50 40
F
"
Y
"
2O U
10
1
Preintar
"
i
'
" "
"
I-2
3-4
"
" " = ' 5-6 7-8
" ~r~r-~r~ 12
24
48
POST-INTOXICATION (HOURS)
Urine C-THC in occasional marihuana users 1 - 48 hours after smoking a standard Highest levels were at 2 - 4 hours . (19 .8 mg ~'-THC) cigarette .
FIG . 2 zoo ISO
F
G
100
0 300 250 E P 200 I50 100 50
"
"
i
r~- ~T -"r 5-IS 30-60 120 IBO 240 POST-NTOXICATION (MNUTES)
Plasma e'-THC and C-THC levels in occasional marihuana users 5 - 240 minutes after a single smoke . e 9 -THC peaks 15 minutes or before and C-THC at 30 - 60 minutes in 3 subjects and at 15 minutes in 2 subjects .
1714
9 Body Fluid 0 -THC and 9-Carboxy-D 9-THC
Vol . 19, No . 11
Plasma ~'-THC and C-THC : Plasma samples were obtained from five subjects a ter smo ing a sing e cigarette and from three subjects after smoking three consecutive cigarettes . None of the first group and only one of the second group had measurable pre-intoxication levels of D'-THC . In all five single srtwke subjects definitive De-THC incrert(ents (10 130 ng/ml) occurred 15 minutes after smoking, becoming almost undetectable 2 hours later . Plasma C-THC peaked at 30 - 60 minutes in 3 of the sart(e 5 subjects . Significant levels remained in all, at least 2 hours after exposure (Figure 2) . Following repetitive cigarettes there were rapid shifts in plasma ~9THC and C-THC up to 48 hours (Figure 3) . Expectedly plasma o'-THC peaks (100 - 260 ng/ml) were much higher after multiple consecutive cigarettes than a single one, permitting detection of e'-THC as long as 2 - 3 hours after completion of the last cigarette . Plasma C-THC was vastly higher, peaking (158-460 ng/ml) 30 - 60 minutes after the last cigarette with significant artwunts of this rtletabolite persisting in plasma 48 hours after the last cigarette . FIG . 3
_ 300 É
~aoo c U 2 300 F U . 200
POST-INTOXICATION (HOURS)
Plasma A 9 -THC and C-THC in representative subjects after three consecutive standard marihuana cigarettes . Peaks were much higher but did not shift : -THC (e----~) became insignificant at 4 hours while C-THC (o -o, ~-~, ~--)remains appreciable at 48 hours .
Saliva e'-THC and C-THC : Saliva samples (after rinsing) obtained from an occasional marihuana user demonstrated only C-THC after one standard e'-THC cigarette, the peak level at 15 minutes becoming undetectable 2 hours later (Table 1) . The baseline ~'-THC (1 cigarette) would seem to indicate that measured C-THC came from secreted saliva rather than solubilized cigarette or smoke .
9 Body Fluid D 9 -THC and 9-Carboxy-D -THC
Vol . 19, No . 11
1715
TABLE 1 Saliva e'-THC and C-THC Levels 15 - 240 Minutes After Smoking a Single Cigarette Time ~Minute~ Pre-intox .
15
30
60
180
240
e'-THC (ng/ml)
0
0
0
0
0
0
C-THC
0
12
3
2
0
0
(ng/ml)
Assa Validation : Extract assays were corroborated (correlation coefficient, 0 .98, Ta e 2 y direct (unextracted) plasma measurements (9) in which sample, antibody, and tritiated marker were simply incubated 30 minutes prior to charcoal separation (as above) and counting . TABLE 2 e'-THC Measured Using Plasma Extracts and Unextracted Plasma Unextracted Plasma e'-THC (ng/ml) 0 8 240 5 0 156 140 6 19 10 30 50 60 0 120 15 5 22 8 23 0 Correlation Coefficient = 0 .98
Reconstituted Ether Extracts e'-THC (ng/ml) 0 9 300 9 0 150 155 18 31 16 45 54 45 0 100 10 5 28 10 21 8
1716
Body Fluid D 9 -TfiC and 9-Carboxy-0 9-THC
Vol . 19, No . 11
DISCUSSION Although discrimination between closely related molecules is the forte of complex analytical technology, antibodies can also recognize and measure subtle differences in discrete functional groups (10) . The foregoing body fluid measurement separately of A 9 -THC and C-THC constitute the first immunologic quantitation of two discrete cannabinoid metabolites in body fluids . Initial findings outline temporal patterns in plasma and urine following marihuana inhalation . Such discrete ~e-THC and C-THC metabolite measurements should contribute to our understanding of compartment turnover, tissue storage and clinical correlates . After one standard marihuana cigarette, urines from occasional user subjects were negative or marginal for 0s-THC in contrast to C-THC (up to 60 ng/ml at 48 hours) . Concomitantly, plasma D e -THC peaked at 15 - 30 minutes and C-THC at 30 - 60 minutes after a single standard smoke in a majority of subjects . Importantly, Ds-THC became almost undetectable 1 - 2 hours after the solitary exposure contrasting sharply with C-THC which persisted for several hours in plasma . This critical divergence of plasma 4 9 -THC versus C-THC was even greater in subjects who had smoked 3 consecutive cigarettes . Plasma ~'-THC became almost undetectable 4 hours after the last one had been consumed in contrast to persistent (48 hours) significant levels of C-THC . Unfortunately suitable chronic smoker plasma samples were not available for analysis . The apparent slight secondary increment in plasma C-THC in the multiple cigarette group could relate to the assay or reflect intrinsic physiological variation at the blood-alveolar barrier due to the extremely hydrophobic, lipophilic character of marihuana . A plasma sample negative for D 9 -THC would seem to be unambiguous evidence that Marihuana had not been smoked in the preceding hour . Even after 3 consecutive standard marihuana cigarettes ~'-THC drops at 4 hours to the level of current detectability . A large control population can now be studied for Ds-THC and C-THC (ratios) to interpret properly individual subject variants and corresponding behavioral patterns . Although we arbitrarily chose to obtain initial samples 15 minutes after smoking, mass spectroscopic analyses of plasma samples following exposure to marihuana demonstrate a 5 minute peak (11) reaching 15 minute levels in harmony with ours . Unfortunately mass spectroscopic derivatization and quantitation of C-THC is not yet available . Its proportion of total urinary cannabinoids is uncertain . However, C-THC's metabolic importance (5) is supported by our experimental data pertaining to the relative proportions of D s -THC and C-THC in blood and saliva . Implicit from the foregoing experimental observations is the potential ambiguity of information to be derived from assays in which "specificity" exists only for "the closed 3 ring cannabinoid" moiety (12, 13, 14) . Almost all D 9 -THC metabolites and naturally occurring non-psychoactive cannabinoids (e .g .cannabinol) are in this broad category . Another THC immunogen preparation (15) links Ds-THC to the carrier protein via a 5'-carboxyl site on the pentyl side chain with resultant blockade
Vol . 19, No . 11
9 9 Body Fluid D -THC and 9-Carboxy-D -THC
1717
of the latter . There are potential endogenous metabolites (11) containing substitutents on the pentyl moiety which could be expected to crossreact with the relevant antisera . Of immediate consequence would be the inability to quantify separately the endogenous C-THC metabolite described in the foregoing experiments . While isotope cannabinoid assays are presently hampered primarily by nonhomologous markers, ~'-THC and C-THC can discretely be measured with acSpecific immune assays, especially those without extraction, should curacy . permit large scale kinetic and forensic studies . ACKNOWLEDGMENT Supported in part by National Institute on Drug Abuse Grant #DA-00106 REFERENCES
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
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