Rat brain salsolinol and blood-brain barrier

Brain Research, 224 (1981) 446-451 Elsevier/North-Holland Biomedical Press

446

Rat brain salsolinol and blood-brain barrier

THOMAS ORIGITANO, JEROME HANNIGAN and MICHAEL A. COLLINS * Department of Biochemistry and Biophysics, Loyola University Stritch School of Medicine, Maywood, IL60153 (U.S.A.)

(Accepted July 30th, 1981) Key words: salsolinol -- tetrahydroisoquinolines -- capillary gas chromatography -- blood-brain

barrier

The 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (TIQ), salsolinol, is a substantial constituent along with its mono-O-methylated metabolite(s) in urine obtained from alcoholic individuals during early ethanol detoxification 6. These and other TIQs also are present in significantly lower amounts in normal human urine. Plesumably, levels of salsolinol increase in vivo during alcohol abuse due to the condensation of ethanol-derived acetaldehyde with dopamine (DA). The DA-related TIQs have been detected in the brains of rodents exposed to ethanol under certain conditionsS, 9 and various roles have been proposed for TIQs in the alcoholic disease process4,S, I°. The basis for identification and quantitation of TIQs in the above experiments was gas chromatography (GC) with electron capture detection (ECD), as well as high performance liquid chromatography with amperometric detection (LC/AD). Recently, using a gas chromatography/mass spectrometry (GC/MS) technique, investigators in Sweden confirmed with alcoholic patients that salsolinol and O-methyl-salsolinol(s) were not only present in urine but were quantifiable in the cerebrospinal fluid as wellaA 4. A question which now arises is the relationship between TIQs in the periphery and those present in the central nervous system (CNS). Sj6quist and Magnuson have addressed this point in a GC/MS study 15 of salsoJinol and O-methylsalsolinols (isomer distribution unknown) in the liver and brain of rats prior to and after injection with salsolinol intraperitoneally (i.p.). Inexplicably, appreciable levels of salsolinol (ca. 1 nmol or 175 ng per g tissue) and its O-methylated metabolites were stated to be present endogenously in two brain areas, the striatum (a high DA region) and the limbic forebrain (a region of relatively low DA levels). Salsolinol injection increased brain TIQ levels about two-fold (salsolinol) and 60-fold (O-methylated salsolinols) in either region. The authors failed to comment on the apparent descrepancy between their endogenous salsolinol results and those from several earlier

* To whom correspondence should be addressed. 0006-8993/81/0000-0000/$02.50© Elsevier/North-Holland Biomedical Press

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Fig. 1. A: capillary gas chromatogram of HFBA-derivatized amines in extracts of 120 mg striatum from saline-injected (control) rat. B: capillary gas chromatogram of another control rat striatum (100 mg) to which salsolinol (5.7 ng free base, or 0.32 nmol/g) was added during initial homogenization. Chromatographic conditions: 10 m x 0.25 mm OV-17 WCOT capillary, column temperature, 130 °C, detector temperature, 340 °C, injector temperature, 250 °C, N~O~F = 0.8 kg/cm2, splitter setting, 10/l, attenuation, 64 x 10H. rodent studies which f o u n d no indication of the TIQs in normal brain tissueS,V,9,11,13. While investigating the metabolic fate o f injected TIQs in rats using an improved c h r o m a t o g r a p h i c procedures, we specifically examined several brain areas for salsolinol and the O-methyl isomers. We report here that with a sensitive G C / E C D m e t h o d employing a high resolution glass capillary column which effectively separates the two mono-O-methyl-salsolinols 12, as well as with confirmatory assays by reserved phase L C / A D , salsolinol and its O-methyl derivatives were not present in the rat striatium in significant a m o u n t s either before or following administration (i.p.) of salsolinol in various concentrations.

448 Male Sprague-Dawley rats, 100 zE 10 g, were injected with salsolinol in concentrations (free base) of 5,10 or 20 mg/kg in isotonic saline, or with saline only. (Racemic salsolinol, prepared as the HC1 salt from preparative reaction of DA and acetaldehyde, was identified by melting point, nuclear magnetic resonance spectroscopy, and co-chromatography with authentic sample.) The rats were sacrificed by decapitation 0.5, 2, 5 and 10 h after injection. The corpora striata, hypothalami and hippocampi were removed, weighed and homogenized in ice-cold 0.4 N perchloric acid, which, for some samples, contained dihydroxybenzylamine (DHBA), an internal standard. Salsolinol (HC1) also was added to selected brain homogenates from salineinjected rats. The homogenates were centrifuged (30,000 g) for 20 min at 4 °C. The supernatents, after adjustment to pH 5.5, were pipetted onto BioRex 70 columns (2.5 cm × 0.6 cm) and the amine fractions were eluted with 1.0 N HC1. Portions of the acid eluates were lyophilyzed to dryness and the residues were treated with heptafluorobutyryl anhydride in acetonitrile in order to derivatize polar functions for GC/ECD analysis z. A Varian 3700 gas chromatograph equipped with a 10 m WCOT (OV-17) glass capillary column, a 6SNi detector (pulsed mode) and a CDS-I 11 C computing integrator was utilized. The limits of detectability with this GC/ECD procedure were 2-5 ng/g for salsolinol and 6-8 ng/g for the O-methylated salsolinols. The remaining portions of the BioRex 70 eluates were analyzed directly by LC/AD is. The LC system consisted of a precolumn, a BioSil ODS-10 column, and an amperometric detector (Bioanalytical Systems). As little as 20 ng salsolinol per g tissue could be detected. Overall recoveries for salsolinol by either chromatographic technique ranged between 82 and 88 ~. In Fig. 1 is a representative gas chromatogram (A) of the derivatized BioRex 70 eluate of a striatal sample from a saline-treated rat, showing only traces of a component in the region of salsolinol's retention time (10.08 min, labeled unknown). If the trace component in this and other control striatal extracts is salsolinol, it would constitute but a few nanograms per g tissue, and not the levels reported by Sj6quist and Magnuson 15. This is evident from inspection of chromatogram B in Fig. 1, which displayed an easily detectable salsolinol peak in the derivatized amine extracts of a control striatum to which salsolinol (0.3 nmol/g or 1/3 the level estimated in vivo in ref. 15) had been added during homogenization. Endogenous DA concentrations, determined from similar control striatal extracts containing DHBA internal standard, were in the acceptable range (7-10 #g/g). In the same manner, striatal chromatograms obtained at higher GC column temperatures (145 °C) showed no endogenous Omethyl-salsolinols above minimum detectable levels. The absence of appreciable salsolinol in normal rat striatum was confirmed by LC/AD. In Fig. 2, representative chromatograms of two control striatal extracts, one without added salsolinol (solid tracing) and one with the TIQ (1.3 nmol/g)added during homogenization (dashed tracing), were superimposed. It is evident that salsolinol was not present in 1 nmol/g quantities in control striata. Salsolinol also was not apparent by the LC or GC assays in the hippocampus or hypothalamus of control rats. Finally, the three brain regions from rats injected i.p. with salsolinol (5-20 mg/kg) showed no GC/ECD indications of salsolinol or O-methyl-salsolinol accumulation at

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Fig. 2. (Solid tracing) Liquid chromatogram of BioRex 70 acid eluate of striatum from untreated rat. superimposed on (dashed tracing) liquid chromatogram of another eluate of similar striatum to which salsolinol (1.3 nmol/g) had been added during initial homogenization. Chromatographic conditions: BioSiI-ODS 10 reverse phase column (250 × 4 mm), mobile phase, 0.1 M NaHePO4 + 1 mM EDTA, pH 5.0,1 ml/min, detector, 0.75 V, electrometer, 10 nA/V. any timepoint; the striatal c h r o m a t o g r a m s resembled control c h r o m a t o g r a m A in Fig. 1. This finding is also in some disagreement with the G C / M S study 15 which reported very high (9 nmol/g) levels o f O-methyl-salsolinols 120 min after a salsolinol dose o f ca. 50 mg/kg. In our experiments, salsolinol and O-methyl-salsolinols should have been

450 detectable in the brains of rats if they were present in the amounts estimated by GC/MS. The reason for the large difference between the results of our assays and those of the GC/MS study 15 is not obvious. The mass spectometric detector is generally considered to be the most specific available, although it should be noted that the GC/MS assay evidently was less sensitive for salsolinol than our G C / E C D approach, and was unable to separate the two O-methyl-salsolinol isomers. It is possible that components not chromatographically resolved from (traces of) salsolinol were contributing mass fragments to the 602 and/or 617 species; for example, the major ion from 6,7-dihydroxy-TIQ (DA/formaldehyde condensation product), a compound recently identified in the rat CNS 1, would be observed at 602. Other experimental factors to be considered include the use of older rats of a different strain, chloroform administration prior to decapitation, and the alumina rather than cation exchange column isolation step. In summary, we find by both capillary G C / E C D and LC/AD analysis that there is little or no endogenous salsolinol and its O-methyl metabolites in the corpus striatum or other brain regions of the normal rat. Furthermore, salsolinol administered i.p. (to 20 mg/kg) does not result in measurable brain salsolinol or mono-O-methylsalsolinol. A prominent blood-brain barrier seems to exist for salsolinol which must be considered in pharmacological and behavioral experiments with this catecholic TIQ. The results suggest that the salsolinol in the CNS of rats during ethanol intoxication 5 is not of peripheral origin, and that the T1Q reported 3 to be present in the spinal fluid of human alcoholics during acute detoxification is formed centrally. The research was supported by USPHS AA00266. M.A.C. was supported by a Luso-American (Fulbright-Hays) Exchange Commission fellowship in the Laborat6rio de Farmacologia, Porto, during the completion of this manuscript.

1 Barker, S. A., Monte, J. A., Tolbert, L. C., Brown, G. B. and Christian, S. T., Gas chromatographic/ mass spectrometric evidence for the identification of 6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline as a normal constituent of rat brain : its quantification and comparison to rat whole brain levels of dopamine, Bioehem. Pharmacol., 30 (1981) in press. 2 Bigdeli, M. and Collins, M. A., Tissue catecholamines and potential tetrahydroisoquinoline alkaloid metabolites: a gas chromatographic assay method with electron capture detection, Bioehem. Med., 12 (1975) 55-65. 3 Borg, S., Kvande, H., Magnuson, E. and Sj6quist, B., Salsolinol and salsoline in cerebrospinal lumbar fluid of alcoholic patients, ,4ctapsychiat. stand., 62 Suppl. 286 (1980) 171-177. 4 Cohen, G., Alkaloid products in the metabolism of alcohol and biogenicamines, Bioehem. Pharmacol., 25 (1976) 1123-1128. 5 Collins, M. A. and Bigdeli, M., Tetrahydroisoquinolines in vivo. Rat brain formation of salsolinol, a condensation product of dopamine and acetaldehyde, under certain conditions during ethanol intoxication, Life Sci., 16 (1975) 585-592. 6 Collins, M. A., Nijm, W. P., Borge, G., Teas, G. and Goldfarb, C., Dopamine-related tetrahydroisoquinolines: significant urinary excretion by alcoholics followingalcohol consumption, Science, 206 (1979) 1184-I 186. 7 Dean, R. A., Henry, D. P., Bowsher, R. R. and Forney, R. B., A sensitive radioenzymatic, assay for the simultaneous determination of salsolinol and dopamine, Life Sci., 27 (1980) 403-412.

451 8 Dietrich, R. and Erwin, V. E., Biogenic amine-aldehyde condensation products: tetrahydroisoquinolines and tryptolines (B-carbolines), Ann. Rev. Pharmacol. Toxicol., 20 (1980J 55-80. 9 Hamilton, M. G., Hirst, M. and Blum, K., In vivo formation of isoquinoline alk~oids: effects of time and route of administration of alcohol. In H. Begleiter (Ed.), Biological Effects of Alcohol, Plenum, New York, 1980, pp. 73-86. 10 Melchior, C. L. and Collins, M. A., The routes and significance of endogenous synthesis of alkaloids in mammals, CRC Ann. Rev. 7oxicol., 8 (1981) in press. 11 O'Neil, P. J. and Rahwan, R. G., Absence of formation of brain salsolinol in ethanol-dependent mice, J. Pharmacol. Exp. Ther., 200 (1975) 306-313. 12 Origitano, T. and Collins, M. A., Confirmation of an unexpected brain O-methylation pattern for the dopamine-derived tetrahydroisoquinoline, salsolinol, Life ScL, 26 (1980) 2061-2065. 13 Riggin, R. and Kissinger, P. T., Determination of tetrahydroisoquinoline alkaloids in biological materials with high performance liquid chromatography, Anal. Chem., 49 (1977) 530-533. 14 Sj6quist, B., Borg, S. and Kvande, H., Catecholamine-derived compounds in urine and cerebrospinal fluid from alcoholics during and after long-standing intoxication, Substance and Alcohol Actions/Misuse, 3 (1981) in press. 15 Sj6quist, B. and Magnuson, E., Analysis of salsolinol and salsoline in biological samples using deuterium-labeled standards and gas chromatography - - mass spectrometry, J. Chromatogr., 183 (1980) 17-24.