A SENSITIVE AND SPECIFIC ASSAY FOR
5-METHOXYTRYPTOPHOL IN PLASMA R. M. LEONE, R. E. SILMAN, R. J. L. HOOPER, M. D. A. FINNIE, S. J. CARTER, R. EDWARDS, I. SMITH, P. TOWELL AND P. E. MULLEN
Department of Reproductive Physiology and * Department of Chemical Pathology, St Bartholomew's Hospital Medical College, London, EC1A 7BE, \Courtauld Institute, Middlesex Hospital Medical School, London, W1P 7PN, and \Institute of Psychiatry, De Crespigny Park, London, SE5 8AF
(Received 17 October 1978) SUMMARY
5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography\p=n-\mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable. INTRODUCTION
In man, the pineal gland is active throughout life and the calcification that occurs with maturity does not signify disuse (Wurtman, Axelrod «fe Barchas, 1964). There are two major areas where it is thought to play a significant physiological role. As a neuroendocrine transducer, it responds to the light and dark conditions of the environment and can mediate the hormonal changes of night-time and of sleep (Wurtman «fe Anton-Tay, 1969). As an endocrine organ, it participates in the hypothalamic-pituitary-gonadal axis, and two pineal índoles, melatonin and 5-methoxytryptophol, can act as potent antigonadotrophic hormones (Kinson & Peat, 1971 ; Vaughan, Vaughan «fe Reiter, 1976). Yet, despite being implicated in these general processes, its precise function has remained undefined. In large part, this results from the difficulties encountered in measuring its products. To date, pineal studies have depended on animal experiments in vitro where the enzyme activity within the gland has been related to its supposed output at the time of death. Alternatively, studies in vivo have depended on pinealectomy or on the administration of pineal hormones. But progress has been obstructed by the lack of availability of sensitive and specific assays which would allow the direct measurement of pineal output. In man, attempts to monitor the contribution of the pineal gland to different physiological and pathological states have awaited such
methods. The pineal
gland synthesizes a family of structurally related Índoles (Fig. 1). The enzyme hydroxy-indole-O-methyl transferase converts 5-hydroxyindoles to 5-methoxyindoles and is
cd
D. O
to be found almost exclusively within the pineal gland (Axelrod, McLean, Albers «fe Weissbach, 1961). Thus, the 5-methoxyindoles may be regarded as 'pineal specific' and four have been identified so far. They are, 5-methoxytryptophol (ML; Mclsaac, Farrell, Taborsky «fe Taylor, 1965), JV-acetyl 5-methoxytryptamine (melatonin; Lerner, Case & Takahashi, 1960), 5-methoxytryptamine (Wurtman & Axelrod, 1968), and 5-methoxyindole-acetic acid (Lerner et al. 1960). Of these, only melatonin has received any continuing attention and this is partly
the result of biological accident. It is the most potent lightener of amphibian melanocytes and so is peculiarly suited to measurement by the sensitive frog-skin bioassay (Lerner «fe Wright, 1960). However, since many other substances lighten frog skins, plasma or tissue samples have to be submitted to Chromatographie separation and identification before they can be usefully measured by this means. Recently, immunoassays have been developed for melatonin (Grota
5-METHOXYTRYPTOPHOL IN PLASMA R. M. LEONE, R. E. SILMAN, R. J. L. HOOPER, M. D. A. FINNIE, S. J. CARTER, R. EDWARDS, I. SMITH, P. TOWELL AND P. E. MULLEN
Department of Reproductive Physiology and * Department of Chemical Pathology, St Bartholomew's Hospital Medical College, London, EC1A 7BE, \Courtauld Institute, Middlesex Hospital Medical School, London, W1P 7PN, and \Institute of Psychiatry, De Crespigny Park, London, SE5 8AF
(Received 17 October 1978) SUMMARY
5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography\p=n-\mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable. INTRODUCTION
In man, the pineal gland is active throughout life and the calcification that occurs with maturity does not signify disuse (Wurtman, Axelrod «fe Barchas, 1964). There are two major areas where it is thought to play a significant physiological role. As a neuroendocrine transducer, it responds to the light and dark conditions of the environment and can mediate the hormonal changes of night-time and of sleep (Wurtman «fe Anton-Tay, 1969). As an endocrine organ, it participates in the hypothalamic-pituitary-gonadal axis, and two pineal índoles, melatonin and 5-methoxytryptophol, can act as potent antigonadotrophic hormones (Kinson & Peat, 1971 ; Vaughan, Vaughan «fe Reiter, 1976). Yet, despite being implicated in these general processes, its precise function has remained undefined. In large part, this results from the difficulties encountered in measuring its products. To date, pineal studies have depended on animal experiments in vitro where the enzyme activity within the gland has been related to its supposed output at the time of death. Alternatively, studies in vivo have depended on pinealectomy or on the administration of pineal hormones. But progress has been obstructed by the lack of availability of sensitive and specific assays which would allow the direct measurement of pineal output. In man, attempts to monitor the contribution of the pineal gland to different physiological and pathological states have awaited such
methods. The pineal
gland synthesizes a family of structurally related Índoles (Fig. 1). The enzyme hydroxy-indole-O-methyl transferase converts 5-hydroxyindoles to 5-methoxyindoles and is
cd
D. O
to be found almost exclusively within the pineal gland (Axelrod, McLean, Albers «fe Weissbach, 1961). Thus, the 5-methoxyindoles may be regarded as 'pineal specific' and four have been identified so far. They are, 5-methoxytryptophol (ML; Mclsaac, Farrell, Taborsky «fe Taylor, 1965), JV-acetyl 5-methoxytryptamine (melatonin; Lerner, Case & Takahashi, 1960), 5-methoxytryptamine (Wurtman & Axelrod, 1968), and 5-methoxyindole-acetic acid (Lerner et al. 1960). Of these, only melatonin has received any continuing attention and this is partly
the result of biological accident. It is the most potent lightener of amphibian melanocytes and so is peculiarly suited to measurement by the sensitive frog-skin bioassay (Lerner «fe Wright, 1960). However, since many other substances lighten frog skins, plasma or tissue samples have to be submitted to Chromatographie separation and identification before they can be usefully measured by this means. Recently, immunoassays have been developed for melatonin (Grota
