Bmin Research &&e&z, Vol. 14, pp. S-90, 1985. B Ankho Intemation~l Inc. Printed in the USA 0361-9230185 $3.00 + .Mt

Effect of Ag-THC on Brain and Plasma Catecholamine Levels as Measured by HPLC

VANDANA PATEL, MYRIN BORYSENKO AND M. S. A. KUMAR’

Tufts Un~~~r~~ity &hots of redivide and Veterina~ medicine, deportment of Anatomy and Cellular Biology 136 Harrison Avenue, Boston, MA 02111

Received 23 April 1984

PATEL, V., M. BORYSENKO AND M. S. A. KUMAR. Effect of AeTHC on brain andplasma catecholamine levels as measured by HPLC. BRAIN RES BULL 14(l) 85-90, 1985.-The effects of chronic administration of A”- tetrahydrocannabinol (AsTHe) on the plasma and brain catecholamine (CA) levels were measured using high performance liquid chromatography-electrochemical detection (LC-EC) system. Intact male rats were injected daily with vehicle (50 ~1 oil) or with A*-THC (3 mg/kg body wt) over a period of 25 days. Trunk plasma and tissue from preoptic area (POA) and mediobasal hypothalamus (MBH) were collected and catecholamine levels were detected by LC-EC system coupled to an electronic integrator. Alumina extract of tissue and plasma samples, spiked with the internal standard (dihy- droxy~nzylamine), were injected into the LC-EC system; the CA were chromatographed and eluted within 12 minutes using sodium phosphate buffer as the mobile phase. A!+-THC treatment resulted ina significant decrease in plasma and MBH levels of norepinephrine (NE), epinephrine (E), POA levels of NE; and significant increases in MBH levels of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC). Our study indicates for the first time that fl-THC treatment significantly alters not only the POA and MBH CA levels, but also the plasma CA levels.

A!‘-THC Hypothalamus HPLC Dopamine Norepinephrine Epinephrine DOPAC

PREVIOUS work in our laboratory indicates that A.“-THC increases endogenous opiate activity of the diencephalon [ 181 and that naloxone can counteract the inhibitory effects of As-THC on the proestrus LH surge 1203. These results indicate that A*-THC may bring about its ~tigonadotropic effects by altering the endogenous opiate activity. Such an interaction of AI\“-THC with the diencephalic opioid system should also bring about changes in the catecholaminergic neurotransmitter activities because of the morphological [ 16,211 and functional [ 11, 28, 291 integration of these neuro- transmitter systems. Further, there are no published reports concerning the effects of chronic administration of AS-THC on plasma and brain tissue catecholamine levels in the rat. We describe the effects of A!‘-THC on brain tissue and plasma levels of catecholamines as measured by high per- formance liquid chromatography. Until recent years, cate- cholamines in brain and biological fluids were measured by a wide variety of methods, iucluding fluorometric [2,13] and mdioenzymati~ [9,261 analysis. Recently, liquid chromatog- raphy, coupled with electrochemical detection (LC-EC), has been used extensively for catecholamine determinations [ 12,141. The LC-EC method has been demonstrated to be more sensitive, specific, and cost effective compared with either fluorometric or radioenzymatic methods. We have modified the LC-EC system by the incorporation of an elec- tronic integrator instead of a chart recorder. In the present study, we also describe the advantages of LC-EC data analyses with an integrator.

‘Requests for reprints should be addressed to M. S. A. Kumar.

METHOD

Rmqplts

Norepinephrine (NE), epinephrine (E), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), dihydroxybenzyla- mine (DHBA) and alumina were procured from Sigma Chem- ical Company (P.O. Box 14508, St. Louis, MO 63178). Alumina was washed prior to use as described by Anton and Sayre [2]. Methanol and perchloric acid (HCIO.,) were ob- tained from Fisher Scientific Company (P.O. Box 379, Med- ford, MA 02155). The distilled water used was R.O. pure HPLC grade water.

Experiment

Male Sprague Dawley rats of approximately 230 g body weight (Charles River, Wilmington, MA) were subcutane- ously injected daily with either vehicle (7 rats) or with 3 mg AS-THCYkg body weight (8 rats). Delta-9-THC was provided in pure form in absolute ethanol by the National Institute on Drug Abuse (NIDA). The ArTHC for injection was prepared by evaporating the alcohol under a stream of nitrogen and reconstituting the residue in vegetable oil. Vehicle (oil) or A”-THC in oil were administered in 50 ~1 volume daily. At the end of 26 days of A”-THC treatment, all rats were decapi- tated and the first 3 ml of the trunk blood collected. Hypo- thalamus and POA tissue weighing approximately 15 and 18 mg respectively were dissected out as described elsewhere

85

X6 PATEL, BORYSENKO AND KUMAR

FIG. I. Chromatogram of peak separations for various amines as printed by the electronic integrator. The retention time for each peak is also printed by the integrator. Chromatogram of different standards (A) (200 pgs of each component) and of hypothalamus extract (B).

[19]. Plasma and brain tissue samples were extracted and assayed for catechoiamines as described below.

Instrurrwntation

The High Performance Liquid Chromatography (HPLC) System consisted of a model 110 A pump (Beckman Instru- ments, 2500 Harbor Blvd., Fullerton, CA 92634), model 210 sample injector (Beckman Instruments, 1780 Fourth Street, Berkeley, CA 94710), microsorb reverse phase short C-18,3 pm column (Rainin Instruments, Mack Road, Wobum, MA 01801) connected in series with an electrochemical detection system consisting of a carbon paste electrode and IX-4B amperometric detector (Bioanalytical Systems, 1205 Kent Avenue, West Lafayette, IN 47906) connected to an elec- tronic integrator model 3390A (Hewlett Packard Instru- ments, 32 Hartwell Avenue, Lexington, MA 02173). The working electrode potential was set at +0.75V vs. an Ag/AgCl reference electrode. Integrator was set at electrical zero, chart speed was set at 0.3 cmimin. Calculation of cali- bration curve and sample curves were based on the height of the peaks. Integrator functions 0 (set baseline now) and 2 (set baseline at next valley point) were used to construct the base line. These integrator functions enable the integrator to cor- rect for the baseline shifts during a chromatog~phic run.

The mobile phase used was phosphate buffer 0.05 M, pH 4.0 (containing sodium-octyl sulfate, 0.15 mM; EDTA, 0.01 mM; methanol, %). The mobile phase was filtered under vacuum through a 0.45 pm millipore HP type filter and thor- oughly degassed before use.

Plasma samples (OS-1 ml) were deproteinized by the ad- dition of perchloric acid (0.5 ml of 0.1 M solution) and the plasma catecholamines were purified and concentrated by repeated extractions with alumina as described by Anton and Sayre [Z]. Alumina (20 mg) and 25 ~1 of sodium metabisul~te (10 mM) were added to 0.5-l ml of deproteinized plasma containing I ng of DHBA as an internal standard. The pH of this solution was adjusted to 8.5520.05 with Tris buffer (1.5 M, pH 8.6). The tubes were vortexed for 3 min and cen- trifuged (1200 x g at 4°C for 10 min) and the supematant discarded. The alumina pellet was washed three times with distilled water. After the third wash, care was taken to re- move almost all the water. The catecholamines were eluted with 100 ~1 (SO ~1 x 2) of 0.1 M HCIO,. Of the clear superna- tant, 20 111 were injected into the LC-EC system. Brain tissue, medial basal hypothalamus (MBH) and preoptic area (POA) were homogenized in 0.1 M HClO,. The catecholamines used as standards were treated and eluted similarly to the sample preparation described above. The mobile phase was pumped at a constant flow rate of 1.0 muminute.

The data were analyzed by Student r-test.

RESULTS AND DISCUSSION

Complete separation of catecholamine standards (NE, E, DA, DOPAC and DHBA 200 pg of each) was achieved by the LC-EC system (Figs. 1 and 2). The NE peak was well re- solved from the solvent front and there was no overfapping of adjacent peaks. At the end of each chromatographic run, a

EFFECT OF AS-THC ON CA LEVELS

JU

FIG. 2. Representative chromatograph of rat plasma extract. The NE, E and DHBA retention times are printed along with the peaks.

report was printed by the integrator which included the peak number, retention time, peak height, percent of height, and ratio of area:height. The amount of each catecholamine in- jected was automati~aIly calculated by the integrator. The calculation of CatechoIamines in the sample by the integrator was based on the ratio of absolute response factor and amount of each component to that of internal standard.

There was good linear relationship between the amount of catecholamines injected and the amount calculated for the different catechoiamines (Fig. 3, Table 1). There was also a good correlation between increasing amounts of injected catecholamines and their respective peak heights as reported by the integrator (Fig. 4). The correlation coefficient (r- value) for each of the catecholamines injected over a concen- tration range of 25-500 pg was as fohows: DHBA-0.997; NE-0.997; E-0.997; DA-0.996; and DOPAC-0.996. The per- cent recovery of all catecholamines measured ranged be- tween SO-95%. Inclusion of the deproteinizing step and re- peated extraction of the alumina may have contributed to the higher recoveries reported here which are significantly better than the recoveries of 50-75% reported by Mefford et al. [22], but comparable to the recoveries reported by Mori [25], Yui et al. [30] and Anton and Sayre [2]. Repeated extractions of plasma perhaps increase the efftciency of catecholamine recovery by increasing the absorption of CA by alumina. Repeated eiution with 0.1 M HCIO, also seen to increase the

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5i 400 -

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50 100 200 200 400 500

FIG. 3. Linear relationship between the amount of norepineph~ne (NE) injected and the amount calculated by the electronic integrator.

FIG. 4. Relationship between the increasing amount of various amines injected and their respective peak heights calculated in arbi- trary units by the electronic integrator.

PATEL, BORYSENKO AND KUMAR

TABLE I

RELATIONSHIP BETWEEN AMOUNT OF CATECHOLAMINES INJECTED AND THE AMOUNT CALCULATED BY THE INTEGRATOR

Amount Added

(P& NE

Amount Calculated (pg) (mean .? SEM

E DA DOPAC

25 23.74 -c 0.2h 26.04 k 0.04 27.66 c 0.08 26.33 + 0.34 50 46.29 + 0.26 51.10 f 0.4x 53.44 _’ 1.73 53.03 f 0.68

100 93.51 L 1.92 99.97 i 2.71 306.23 + 1.59 102.46 2 2.87 200 199.06 !x 7.63 202.69 t 6.74 183.81 “. 10.60 177.52 i 8.59 300 269.04 rt 1.71 303.13 + 1.79 299.16 -)r 3.34 294.68 f 0.76 400 375.47 .L X.93 387.Sl r 4.31 375.47 ?I 1.34 381.3.5 t 4.00 500 461.82 _t. 19.25 498.64 t 1.21 477.02 ‘. 7.41 474.41 t 7.10

TABLE 2 EFFECT OF CHRONIC ii*-THC TREATMENT ON PLSMA AND TISSUE NE. E. DA

AND DOPAC LEVELS (MEAN i SEM)

Plasma (nglml)

MBH ing/mg protein)

WA (ngimg protein f

Control Rats (n=7)

NE 2.33 i- 0.44 56.81 k 7.86 13.34 t 1.27 E 9.16 :F 0.66 3.38 ?r 0.45 0.53 rt 0.07 DA - ~5.84 + 0.62 7.88 -t 0.80 DOPAC - 3.5s i 0.66 3.66 f 0.33

A”-THC Treated Rats (n=X)

NE 3.69 rt ().?I-f 31.37 r 1.99f 10.69 + 0.67+ E 6.83 I 0.30: 2.01 t 0.15.1 0.51 i- 0.05 DA - il. 17 z? 0.x3+ 5.36 f O..sl” DOPAC 5.83 f 0.26$ 2.36 -c 0.27-i

*[><0.05; ?p<O.OOl v’r. corresponding control values. Plasma DA and DOPAC were not measured.

recovery of catecholamines. It has been shown that up to 15% of catecholamines were retained in the alumina after the first elution [2] and a subsequent wash with perchloric acid is needed to recover the residual amounts of catecholamines from the alumina.

The solvent system employed by us resulted in the elution of all catechol~ines within 12 minutes without any overlap. The retention time for DA (the last peak) was 11.44r0.33 min (mean2S.D. of 10 runs). The LC-EC system was able to estimate as little as 25 pgs of NE, E, DOPAC or DA in brain tissue and plasma samples. The values for plasma NE and E and brain tissue NE, E, DOPAC and DA are reported in Table 2 and are comparable to the values reported for plasma [23] and hypothalamus [22]. The elution profile of plasma NE, E, and DHBA was similar to that of tissue CA (Fig. 2). We did not encounter any interfering peaks co-eluting with the internal standard. This was confirmed by lack of peaks corresponding to the retention time of the internal standard in plasma samples not spiked with DHBA. Inclusion of a second internal standard (e.g., ~-methyldopamine) is desir- able but does not seem to be necessary for routine monitor- ing of plasma NE and E.

Analysis of plasma samples from A!‘-THC treated rats showed a significant decrease in NE and E levels (Table 2). This is the first report describing the effects of chronic A!‘-THC treatment on plasma CA in rats. Plasma DA and DOPAC were not measured and hence, not included in the data. It is interesting to note that E levels were higher than NE levels in the plasma of controls and A!‘-THC-treated animals. Similar obse~ations were made by Ben-Jonathan in decapitated adult rats [ 171. The levels of NE and E observed in this study agree with the reported plasma values in decapi- tated rats 1171 and are much higher than the NE and E levels in plasma collected under minimal stress conditions [ 10,231. As has been suggested earlier by Depocas and Behrens [8], a sudden drop in blood pressure associated with decapitation may result in a massive outpouring of catecholamine from the adrenal medulla. This may explain the higher plasma levels of NE and E in the decapitated rats.

In agreement with our results, A”-THC has been shown to have a transient stimulatory effect, followed by a prolonged inhibitory effect on the production of NE and E by the ad- renal medulla [24]. Systemic administ~tion of AS-THC has also been shown to bring about bradycardia, decreased car-

EFFECT OF A*-THC ON CA LEVELS 89

disc output [15], indicating an interaction with autonomic nervous system. It is possible that circulating CA in the plasma may bring about these cardiovascular effects of A”- THC. However, the possibility should be considered that a differential response to stress situations (i.e., decapitation by adult rats) may also contribute to the differences in the plasma CA levels of the rats in this study.

In the MBH, NE and E contents were significantly de- creased whiie those of DA and DOPAC were significantly increased by chronic A”-THC treatment. In the POA, A9THC treatment resulted in significant decreases in NE, DA, and DOPAC levels but E levels were not affected. In a previous study, we have shown that A!LTHC treatment re- sults in elevation of diencephalic met-enkephalin levels [ 181. Opioid peptide-containing neurons have been shown to modulate neurotransmitter release from catecholaminergic nerve terminals [I, 5, 6, 271. Our previous study [20] indi- cated that A!‘-THC-induced changes in the opiate activity are

important in blocking the proestrus LH and prolactin surges. Numerous studies indicate that catecholamine neurons may be closely associated with the modulation of LH [41 and prolactin [7] release, and that opioid peptides generally sup- press the CA neuronal activities [3, 11, 291 in the di- encephalon as well as in the locus coeruleus [ZSJ. Although it seems likely that changes in tissue and plasma catechola- mines levels due to A”-THC treatment may be mediated, in part, by altered endogenous opiate activity, a direct effect of AH-THC on the catecholaminergic cells was not ruled out by this study.

ACKNOWLEDGEMENTS

We wish to thank the National Institute on Drug Abuse (NIDA) for providing the free As-THC sample, and Mrs. Arlene Kronman for excellent secretarial assistance.

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