Demonstration of highly specific and sensitive antibodies to a naturally occurring tetrahydroisoquinoline alkaloid, salsolidine.

1. We report for the first time on the production and characterization of antibodies against a naturally occurring tetrahydroisoquinoline, namely salsolidine (6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline). 2. Immunogen synthesis was carried out by coupling the hapten salsolidine to bovine serum albumin (BSA) as carrier protein on the basis of reductive amination. 3. By immunization of rabbits with salsolidine-BSA conjugate antisalsolidine antibodies were produced. 4. At a final dilution of 1:1700 the highest-titre antiserum bound 35% of 0.21 pmol [3H]salsolidine. This antiserum was used to develop a radioimmunoassay for salsolidine. 5. Cross-reactivity studies revealed a high specificity of the antiserum to the hapten. 6. The antibodies had a high affinity to salsolidine (Ka = 1.5 x 10(9) M-1). 7. Standard curves covered a measuring range of 0.5-70 pmol/tube and the detection limit was found to be 0.27 pmol/tube.

Tetrahydroisoquinoline alkaloids mimic direct but not receptor-mediated inhibitory effects of estrogens and phytoestrogens on testicular endocrine function. Possible significance for Leydig cell insufficiency in alcohol addiction.

Possible effects of various tetrahydroisoquinolines (TIQs) on rat testicular endocrine function were tested in vitro in order to prove whether these compounds, some of which have been claimed to accumulate in alcoholics, may be mediators of the development of Leydig cell insufficiency, a well-known side-effect of ethanol ingestion. TIQ effects on different levels of regulation of testis function were compared in vitro with estrogen effects, since both classes of compounds have structural similarities. Gonadotropin-stimulated testosterone production by testicular Leydig cells was inhibited by tetrahydropapaveroline and isosalsoline, the IC50 values (30 microM) being comparable to those of estradiol (3 microM), 2-hydroxyestradiol (10 microM), and the phytoestrogens, coumestrol (15 microM) and genistein (7 microM); salsolinol (85 microM) and salsoline (240 microM) were less effective, and salsolidine was ineffective. None of these TIQs interacted significantly with testicular estrogen receptor as analyzed by estradiol displacement. However, tetrahydropapaveroline, isosalsoline and salsolinol competitively inhibited (Ki 130-150 microM) substrate binding to cytochrome P450XVII, one key enzyme of androgen biosynthesis, with similar efficiency as the estrogens did (Ki 50-110 microM); salsoline and salsolidine were again much less effective. Since the efficient TIQ concentrations in this system are identical with those reported to generate central-nervous effects, it is concluded that certain TIQs may amplify peripheral inhibitory effects of ethanol on testicular endocrine function by their interaction with at least one enzyme of the androgen biosynthetic pathway.

Separation of the catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid by reversed-phase high performance liquid chromatography.

The catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid can be completely separated by reversed-phase high performance liquid chromatography with chemically bonded octadecylsilane as stationary phase and a citrate/ammonium phosphate buffer (pH 4.5) containing 8% methanol as mobile phase. The two isomers can be electrochemically detected and produce different hydrodynamic voltammograms.