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Melatonin: Current Status and Perspectives: Proceedings of an International Symposium on Melatonin, Held in Bremen, Federal Republic of Germany, September 28-30, 1980

Melatonin: Current Status and Perspectives: Proceedings of an International Symposium on Melatonin, Held in Bremen, Federal Republic of Germany, September 28-30, 1980

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Melatonin: Current Status and Perspectives: Proceedings of an International Symposium on Melatonin, Held in Bremen, Federal Republic of Germany, September 28-30, 1980

Longueur:
871 pages
Sortie:
Oct 22, 2013
ISBN:
9781483158105
Format:
Livre

Description

Advances in the Biosciences, Volume 29: Melatonin – Current Status and Perspectives is a compilation of papers by different authors presented in the Proceedings of an International Symposium on Melatonin, held in Bremen, Federal Republic of Germany on September 28-30, 1980.
This volume is divided into six parts, wherein the first part covers the testing methods of melatonin; the use of the status of assay methods of melatonin; and related studies. Part 2 tracks the developments in melatonin histophysiology, with attempts to clarify cytological aspects of the indoleamine secretory process in the pineal gland; melatonin production by extra-pineal tissues; and other relationships with the pineal gland. Part 3 focuses on advances in melatonin physiology from hypothetical evolutionary function; the biochemistry and pharmacology of melatonin; to melatonin and pigment cell rhythmicity. Part 4 shows the progress made in molecular endocrinology, while Part 5 presents the results of human melatonin research and covers melatonin serum in humans. The last part is comprised of additional papers that are not classified in the former categories: studies of the effects of light on human plasma melatonin; the role of the environmental factors; and the histology melatonin localized in the salivary glands of the rat palate.
This compilation of papers is intended for biochemists, neuroscientists, and researchers in the fields of endocrinology, human genetics, and pharmaceutical chemistry.
Sortie:
Oct 22, 2013
ISBN:
9781483158105
Format:
Livre

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Melatonin - Elsevier Science

Lynen

ASSAY METHODS OF MELATONIN

Current Status of Assay Methods of Melatonin

J. Arendt,     Division of Clinical Biochemistry, Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH, UK

ABSTRACT

Current available methodology for the assay of melatonin is briefly reviewed. Areas of controversy, due probably to technical problems, are identified, together with observations confirmed by reports from different laboratories. Some technical improvements are suggested.

KEYWORDS

Melatonin assay

TEXT

In a rapidly moving field such as the study of pineal indoleamines, it is likely that an assessment of the current status of assay methodology will be out of date long before it appear in print. Nevertheless trends can be recognised and areas of controversy identified.

Wurtman and Axelrod’s (1965) formulation of the pineal gland as a neuroendocrine transducer, and the consequent interest in pineal products as possible humoral effectors of photic signals, led to intensified efforts to measure the methoxyindoleamines in tissues and body fluids. Circulating melatonin in particular was, and is, considered to be largely, if not exclusively (Lewy and coworkers, 1980; Arendt, Forbes, Brown and Marston, 1980) of pineal origin. While several reports demonstrate its presence and synthesis in the retina and the Harderian gland (Cardinali and Wurtman, 1972; Pang and coworkers, 1976), it is possible that from these sites of origin it does not reach the general circulation.

From 1970 to 1975, it was possible to measure melatonin using the tadpole bioassay of Ralph and Lynch (1970). This assay, whilst considered to be specific, was particularly tedious and necessitated a complicated tadpole breeding programme. In addition, the sensitivity was insufficient for determinations in sequential blood samples : of great importance in the study of a rhythmic variable.

During the years 1975-1978 an expolsion of publications concerning the measurement of melatonin occurred, the vast majority using radioimmunoassay (RIA) followed by gas-chromatography-mass-spectrometry (GC-MS) and gas-chromatography (GC). A review of this methodology has appeared (Arendt, 1978). From 1978 until the time of writing there has been little basic change in methodological approach – as assays have been refined, reported values have generally speaking decreased, and areas of controversy have appeared which may or may not be of methodological origin.

Clearly an assay has to be useful within the confines of the system it is proposed to investigate, and the potential user must choose a method according to his or her own requirements. For example clinical screening for pathological disturbance should be rapid and preferably cheap. Where gross differences in measured values are diagnostic, then some degree of noise and assay variation is tolerable. Very few literature reports of gross clinical melatonin variations exist, and these are unconfirmed (Barber, Smith and Hughes, 1978; Silman and coworkers, 1979). Where subtle differences are important, for example phase-changes in circadian rhythm, then a high degree of assay precision and accuracy is required. In the case of one application of melatonin assay – assessment of central nervous syst em rhythmic function – a precise measurement of pineal β-receptor stimulation is required, and interference by other methoxy-indoles, whose synthetic control is differently mediated, is not tolerable.

In the case of urinary melatonin assay, it is likely that a system recognising either 6-hydroxymelatonin (Lewy and coworkers, 1980) or both 6-hydroxymelatonin and melatonin, may well provide more information on pineal output than measurement of melatonin separately (Lynch and coworkers, 1975), in view of the small percentage of melatonin thought to be excreted unchanged (Kopin and coworkers, 1961).

Criteria for melatonin RIA acceptability (which, with slight changes in terminology, can also be applied to other assay systems) are generally considered to be as follows:

1) negligible cross-reactivity with analogues, at the concentrations likely to be present in a given sample;

2) parallelism of serial dilutions of sample with the standard curve;

3) good recovery of added radioactive and non-radioactive melatonin;

4) low assay blank (preferably below the limit of detection of the standard curve;

5) acceptable intra- and inter-assay variation (melatonin assays tend to have a rather large assay variation and account should be taken of this, when present, in the interpretation of results);

6) demonstration of the chromatographic identity of immunoreactivity with melatonin (although quite large discrepancies are sometimes found between chromatographically validated assays of similar samples);

7) comparison with as near an absolutely specific method as possible.

Recent reports of low to undetectable melatonin levels following short-term pinealectomy (Lewy and coworkers, 1980; Arendt and coworkers, 1980), or suppression of pineal indoleamine synthesis (Arendt and coworkers, 1980) may provide a more physiologically meaningful assessment of assay performance.

Most published RIA’s fulfil the criteria 1) − 6) (for references see Arendt, 1978).

In view of the prevailing opinion that GC-MS provides the most specific available technique, several authors have made successful, if limited GC-MS/RIA comparisons, however this is clearly a difficult undertaking for the majority of laboratories. It is to be hoped that inter-laboratory comparative studies will include at least one laboratory performing GC-MS assay so that the maximum number of workers may compare their own results with this highly specific but expensive technique.

Clearly, with RIA, every sample cannot be validated : with a good GC-MS system every sample is validated. A compromise solution, both from the point of view of assay specificity, capital investment, running expenses and throughput, may be found in high-performance liquid chromatography (HPLC) separation of melatonin from extracted material, followed by RIA or electrochemical detection (Goldman, Hamm and Erickson, 1980). Whether the sensitivity required for plasma determinations will be achieved by electrochemical detection remains to be seen.

Comparison of results from different assay systems in order to pinpoint the ‘best’ method for a given situation is clearly difficult. In general, for a small molecule obtainable in a pure state, such as melatonin, provided that recovery of added compound from the (so far inevitable) extraction procedures is good, then the lower the reported values, the more specific the assay is likely to be. Amongst the published RIA methods, that of Rollag and Niswender (1976) using iodinated melatonin analogue as tracer has the greatest sensitivity, although not necessarily the lowest values. Unless every sample is validated, it is more correct to refer to measured immunoreactive material as melatonin-like.

It cannot be emphasised too strongly that the establishment of physiological melatonin variations in an adequately large population is an essential prerequisite to pathological studies. However, the complete absence of 24-hour variations is, according to the vast majority of authors an infrequent observation in normal mammals, and would appear to be good evidence of disturbed pineal and/or central nervous rhythmic function. Indeed the areas of least controversy in the measurement of melatonin include its almost uniform dark-phase rise in all species studied to date (Lynch and coworkers, 1975; Kennaway and coworkers, 1976; Rollag and Niswender, 1976; Arendt and coworkers, 1977; Wilkinson and coworkers, 1977; Per low and coworkers, 1980; Vivien-Roëls and coworkers, 1979; Owens, Gern and Ralph, 1980). In addition, several authors have shown a lack of immediate response of human circulating melatonin to artificial light in the dark-phase (Jimerson and coworkers, 1977; Arendt, 1978; Lynch and coworkers, 1978) compared to other species (Rollag and Niswender, 1976; Illnerova and Vanacek, 1979; Perlow and coworkers, 1980) and disturbed plasma melatonin variations in psychiatric disease (Wirz-Justice and Arendt, 1979; Lewy and coworkers, 1979; Wetterberg and coworkers, 1979; Mendlewicz and coworkers, 1979). Most other observations are not yet repeated in competing laboratories. Good general agreement is found amongst different laboratories with respect to pineal and plasma melatonin levels in rats and sheep measured by RIA. Recent results in adult human values show very large (up to 50-fold) variations from the lowest reported daytime levels (Lewy and Markey, 1978 – GC-MS) to the highest (Mendlewicz and coworkers, 1979 – RIA), probably due in part to differences in assay specificity. In addition reports of high spikes of circulating melatonin in man during day and night (Weitzman and coworkers, 1978; Mullen and coworkers, 1980), are unconfirmed. In the authors laboratory daytime melatonin in normal volunteers sampled at hourly intervals, is almost invariably low to undetectable.

It is as yet impossible to revue urinary melatonin assays due to their paucity (Lynch and coworkers, 1975; Akerstedt and coworkers, 1980). A major obstacle has been the apparent lack of specificity of direct RIA for urinary melatonin (Arendt, 1978). We have successfully modified RIA (Arendt and coworkers, 1977) to the determination of melatonin in neonatal urine, with demonstrable chromatographic specificity (Marston and coworkers, 1980). In the course of these investigations it became evident that melatonin is unstable over periods of more than three months in frozen urine. Preliminary observations in neonatal urine, assayed rapidly after collection, indicate that infants develop a 24-hour melatonin rhythm at different rates, even under similar environmental conditions.

Every melatonin assayist’s ideal must be an automated system, not requiring extraction, of sub-picogram sensitivity, absolute specificity, throughput measured in hundreds per day, readily available, and cheap to run. It does not exist yet.

For the moment, future prospects for melatonin assay include greater automation of GC-MS assay, HPLC coupled with sensitive detection methods, and direct plasma RIA without extraction using sufficiently sensitive and specific antibodies.

ACKNOWLEDGEMENT

Some of the work reported here was financially supported by the Medical Research Council and the Agricultural Research Council.

REFERENCES

Âkerstedt, T., Fröberg, J. E., Friberg, Y., Wetterberg, L. Melatonin excretion, body temperature and subjective arousal during 64 hours of sleep deprivation. Psychoneuroendocrinol.. 1979; 4:219–225.

Arendt, J., Wetterberg, L., Heyden, T., Sizonenko, P. C., Paunier, L. Radioimmunoassay of melatonin: human serum and cerebrospinal fluid. Hormone Res.. 1977; 8:65–75.

Arendt, J. Melatonin assays in body fluids. J. Neural Transm. Suppl.. 1978; 13:265–278.

Arendt, J., Forbes, J. M., Brown, W. B., Marston, A. Effect of pinealectomy on immunoassayable melatonin in sheep. J. Endocrinol.. 1980; 85:1–2P.

Arendt, J., Ho, A. K., Laud, C., Marston, A., Nohria, V., Smith, J. A., Symons, A. M. Differential effect of benserazide (Ro4-4602) on the concentrations of indoleamines in rat pineal and hypothalamus. Brit. J. Pharmac.. 1980. [In press].

Axelrod, J. The Pineal Gland: A Neurochemical Transducer. Science. 1974; 184:1341–1348.

Barber, S. G., Smith, J. A., Hughes, R. C. Melatonin as a tumour marker in a patient with pineal tumour. Brit. Med. J.. 1978; ii:328.

Cardinali, D. P., Wurtman, R. J. Hydroxyindole-o-methyltransferase in rat pineal, retina and Harderian gland. Endocrinol.. 1972; 91:247–252.

Goldman, M. E., Hamm, H., Erickson, C. K. Determination of melatonin by high-performance liquid chromatography with electrochemical detection. J. Chromatog.. 1980; 190:217–220.

Illnerova, H., Backström, M., Saaf, J., Wetterberg, L., Vangbo, B. Melatonin in rat pineal gland and serum; rapid parallel decline after light exposure at night. Neurosci. Letters. 1978; 9:189–193.

Jimerson, D. C., Lynch, H. J., Post, R. M., Wurtman, R. J., Bunney, W. E. Urinary melatonin rhythms during sleep deprivation in depressed patients and normals. Life Sci.. 1977; 20:1501–1508.

Kennaway, D. J., Frith, E. G., Phillipou, G., Matthews, C., Seamark, R. F. A specific radioimmunoassay for melatonin in biological tissues and fluids and its validation by gas-chromatography-mass-spectrometry. Endocrinology. 1977; 101:119–123.

Kopin, I. J., Pare, C. M.B., Axelrod, J., Weissbach, H. The fate of melatonin in animals. J. Biol. Chem.. 1961; 236:3072–3075.

Lewy, A. J., Markey, S. P. Analysis of melatonin in human plasma by gas-chromatography negative chemical ionization mass spectrometry. Science. 1978; 201:741–743.

Lewy, A. J., Wehr, T. A., Gold, P. W., Goodwin, F. K. Plasma melatonin in manic-depressive illness. In: Usdin E., Kopin I.J., Barchas J., eds. Catecholamines: Basic and Clinical Frontiers. New York: Pergamon Press; 1979:1173.

Lewy, A. J., Tetsuo, M., Markey, S. P., Goodwin, F. K., Kopin, I. J. Pinealectomy abolishes plasma melatonin in the rat. J. Clin. Endocrinol. Metab.. 1980; 50:204–205.

Lynch, H. J., Wurtman, R. J., Moskowitz, M. A., Ardier, M. C., Ho, M. H. Daily rhythm in human urinary melatonin. Science. 1975; 187:169–171.

Lynch, H. J., Jimerson, D. C., Ozaki, Y., Post, R. M., Bunney, W. E., Jr., Wurtman, R. J. Entrainment of rhythmic melatonin secretion in man to a 12-hour phase shift in the light/dark cycle. Life Sci.. 1978; 23:1557–1563.

Marston, A., Arendt, J., Campbell, C, Armstrong-Esther, C., Hawkins, L. and Goodison, S. (1980). Assay of melatonin in neonatal urine (Abstr.) 4th European Neuroscience Meeting.

Mendlewicz, J., Linkowski, P., Branchey, L., Weinberg, U., Weitzman, E. D., Branchey, M. Abnormal 24-hour pattern of melatonin secretion in depression. Lancet. 1979; ii:1362.

Mullen, P.E., Hooper, J., Leone, R., Linsell, C., McKeon, P., Silman, R. and Smith, I. (1980). The 24-hour pattern of secretion of melatonin and 5-methoxytryptophol in man. (Abstr.) XI International Congress of the International Society of Psychoneuroendocrinoftgy.

Owens, D. W., Gern, W. A., Ralph, C. L. Melatonin in the blood and cerebrospinal fluid of the green sea turtle (Chelonia mydas). Gen. and Comp. Endocrinol.. 1980; 40:180–187.

Pang, S. F., Brown, G. M., Grota, L. J., Rodman, R. L. Radioimmunoassay of melatonin in pineal glands, harderian glands, retinas and sera of rats or chickens. Fed. Proc. Fed. Am. Socs. exp. Biol.. 1976; 35:691.

Perlow, M. J., Reppert, S. M., Tamarkin, L., Wyatt, R. J., Klein, D. C. Photic regulation of the melatonin rhythm: monkey and man are not the same. Brain Res.. 1980; 182:211–216.

Ralph, C. L., Lynch, H. J. A quantitative melatonin bioassay. Gen. Comp. Endocrinol.. 1970; 15:334–338.

Rollag, M. D., Niswender, G. D. Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimes. Endocrinology. 1976; 98:482–489.

Silman, R. E., Leone, R. M., Hooper, R. J.L., Preece, M. A. Melatonin, the pineal gland and human puberty. Nature. 1979; 282:301–303.

Tetsuo, M., Markey, S. P., Kopin, I. J. Measurement of 6-hydroxymelatonin in human urine and its diurnal variations. Life Sci.. 1980. [In press].

Vivien-Roels, B., Arendt, J., Bradtke, J. Circadian and circannual fluctuations of pineal indoleamines (serotonin and melatonin) in Testudo hermanni Gmelin (Reptila Chelonia). Gen. Comp. Endocrinol.. 1979; 37:197–210.

Weitzman, E. D., Weinberg, U., D’Eletto, R., Lynch, H., Wurtman, R. J., Czeisler, Ch., Erlich, S. Studies of the 24-hour rhythm of melatonin in man. J. Neural. Trans. Suppl.. 1978; 13:325–329.

Wetterberg, L., Beck-Friis, J., Aperia, B., Petterson, U. Melatonin / Cortisol ratio in depression. Lancet. 1979; ii:1361.

Wilkinson, M., Arendt, J., Bradtke, J., de Ziegler, D. Determination of dark-induced elevation of pineal N-acetyl transferase activity with simultaneous radioimmunoassay of melatonin in pineal, serum and pituitary of the male rat. J. Endocrinol.. 1977; 72:243–244.

Wirz-Justice, A., Arendt, J. Diurnal, mentrual cycle and seasonal indole rhythms in man and their modification in affective disorders. In: Obiols J., Ballus C., Gonzalez Monclus E., Pujol J., eds. Biological Psychiatry Today. Elsevier/North Holland Press; 1979:294–302.

Radioimmunoassays for Human Melatonin

N. Birau*§, W. Schloot*§, R. Khoory*§, H. Hofbauer*, C. Röver* and M. Birau*,     *Institute of Preventive Endocrinology, Bremen, Federal Republic of Germany; §Centre of Human Genetics and Genetic Counselling, University of Bremen, Bremen, Federal Republic of Germany

Publisher Summary

This chapter discusses radioimmunoassay (RIA) for human melatonin. The chapter presents a study in which the cross-reactivity of used antiserum was determined for 50% displacement of the labeled melatonin. Each melatonin determination starts with the extraction. Synthetic melatonin was used for the preparation of the standards. The head of the dilution serie was a melatonin solution. The assay procedure corresponded to a pipetting and incubation scheme. After the last incubation, all tubes were centrifuged. Immediately after centrifugation, the supernatant was discarded. The tubes stood for some time upside down on some wood pulp. The precipitate was re-suspended in bidestilled water. The mini-vials were capped, were mixed by constant over-turning for one minute, and were stood for equilibration of the phases. The activity of each vial was counted for some minutes in a β-counter.

ANTI-MELATONIN SERUM

The cross-reactivity of the used antiserum (from Gregory M. Brown, McMaster University, Canada) was determined for 50 % - displacement of the labeled melatonin. The results are shown in Table I.

Table I

Cross-reactivity of melatonin antiserum °

°Final dilution 1 : 6,000. Tracer ³H-Melatonin, 26 Ci/mM, 2,000 cpm

They coincide with those of Pang and others (1).

EXTRACTION

Each melatonin determination starts with the extraction : add 2.5 ml dichloromethane to 0.5 ml serum in 15 ml Corex tubes and vortex 30 seconds.

Centrifuge at 15,000 × g and 4°C for 15 minutes and dry the organic phase under N2-stream at 35°C.

Cap the tubes and store at 4°C.

PREPARATION OF REAGENTS

A 0.1 % gelatine phosphate buffer is used : 1.42 g Na2HPO4, 8.76 g NaCl, 9.30 g EDTA and 0.1 g Thimerosal (Fa. Sigma, München) are dissolved in bidestilled water. Make a stock up to 1 liter and adjust the pH to 6.5. Prior to use 1.0 g gelatine / / 1,000 ml buffer is added.

Synthetic melatonin (Fa. Sigma, München) is used for the preparation of the standards. Head of the dilution serie is a melatonin solution of 2,000 pg/ml. Acetyl-5-methoxytryptamine, N-[2-aminoethyl-2-³H]-is used as tracer [New England Nuclear, Inc., Boston (Molecularweight 232.26, ethanol-water liquid 96 : 4, specific activity 26.40 Ci/mM, 0.25 mCi and respectively 0.0022 mg in 250 μl)]. One μl is added to 20 ml buffer and 50μl of this solution is counted. The outcome should be 2,000 cpm.

Additionaly a saturated ammonium sulfate solution of pH 7.0 is prepared.

ASSAY PROTOCOL

The assay procedure corresponds to the pipetting- and incubation-scheme which is shown in Table II (double determinations).

Table II

RIA for human serum melatonin-Pipetting scheme and incubations*

*B0 = 0 standard (i.e. without binding) ; NSB = non specific binding ; TA = total activity.

After the last incubation all tubes are centrifuged for 10 minutes at 3,500 × g and 4°C. Immediately after centrifugation the supernatant is discarded. The tubes stand for 15 minutes upsides down on wood-pulp. The precipitate is resuspended in 1.05 ml bidestilled water. The contents of each tube is transferred to a mini-vial (Fa. Packard, Frankfurt/M) + 5 ml liquid scintillation solvent (Pico Fluor 30, Fa. Packard, Frankfurt/M). The mini-vials are capped, mixed by constant over-turning for one minute and stand for 30 minutes for equilibration of the phases. The activity of each vial is counted for 10 minutes in a ß-counter (Prias PL Tri-Carb, Fa. Packard, Frankfurt/M). The counts are printed by a Teletype maschine. The B/Box100 – calculation and the following logit/log-transformation is carried out by computer and a self developed program.

EVALUATION OF THE ASSAY

In the repeated performance of this R I A in the last two years (more than 18,000 determinations) a mean limit of detection of 10 ± 2.2 pg/ml, i.e. 3-5 pg/ /tube has been determined (2,8-10).

The precision of this RIA for melatonin in human serum has been controlled (2). Table III summarizes the results of this investigations.

Table III

Precision of RIA for melatonin in human serum

n = number of determinations

X = mean values of melatonin in pg/ml

SD = standard deviations

V.K. = variation coefficients in %

The interassay and intraassay coefficients of variation were 9.6 % and 4.8 % respectively.

Tests of parallelism and recovery tests were performed in order to check the specificity of this RIA for melatonin (i.e. assay validation, see Fig. 1).

Fig. 1 Specificity of RIA for melatonin

A = parellelism tests

B = recovery tests

The performance of this RIA for human serum melatonin is comparable with those quoted in literature (1 – 6).

Fig. 2 RIA for melatonin – Evaluation of the data

C = B/B0 × 100 – calculation

D = logit/log – transformation

The feasibility of the assay also includes quantitative determination of the human melatonin in plasma, serum, urin, cerebrospinal fluid, amniotic fluid as well as tissues (1 – 15).

ACKNOWLEDGEMENT

Supported in part by grants from the Deutsche Forschungsgemeinschaft from special funds provided by the Bundesministerium für Forschung und Technologie (4851 / 148 / 79), from Sandoz A.G. – Basel, Isotopen Dienst West GmbH – Dreieich and Hormon – -Chemie GmbH – München.

REFERENCES

1. Pang, S. F., Brown, G. M., Grota, L. J., Chambers, J. W., Rodman, R. L. Neuroendocrinology. 1977; 23:1–13.

2. Birau, N., R. Khoory, H. Hofbauer, and W. Schloot (1980). Lab. Med. Accepted for publication.

3. Arendt, J. J. Neural Transmission, Suppl.. 1978; 13:265–278.

4. Arendt, J., Wilkinson, M.Jaffe B.M., Behrman H.R., eds. Methods of Hormone Radioimmunoassay. Academic Press: New York, San Francisco, London, 1979:101–119. [Chap. 6].

5. Brown, G. M., Young, S. N., Gauthier, S., Tsui, H., Grota, L. J. Life Sciences. 1979; 25:929–936.

6. Smith, J. A., Padwick, D., Mee, T. J.X., Minneman, K. P., Bird, E. D. Clinical Endocrinology. 1977; 6:219–225.

7. Rollag, M. D., Niswender, G. D. Endocrinology. 1976; 98:482–489.

8. Birau, N., Schloot, W. IRCS J. Med. Sci.. 1979; 7:400.

9. Birau, N., Schloot, W. Acta endocr. (Kbh.), Suppl.. 1979; 225:240.

10. Birau, N., Schloot, W. Ric. Sci. Ed. Perm., Suppl.. 1979; 10:43.

11. Khoory, R., R. Dubbels, W. Schloot, and N. Birau (1980). In this book.

12. Levine, L., Riceberg, L. J. Res. Comm. Chem. Pathol. Pharmacol.. 1975; 10:693–702.

13. Kennaway, D. J., Frith, R. G., Phillipou, G., Matthews, C. D., Seamark, R. F. Endocrinology. 1977; 101:119–127.

14. Arendt, J., Wetterberg, L., Heyden, T., Sizonenko, P. C., Paunier, L. Hormone Res.. 1977; 8:65–75.

15. Arendt, J., Paunier, L., Sizonenko, P. C. J. Clin. Endocrinol. Metab.. 1975; 40:347–350.


†Postgraduate exercise of an International Symposium on Melatonin (September 28-30, Bremen, F.R.Germany) held in Institute of Preventive Endocrinology, Bremen, and University of Bremen, October 1, 1980, Bremen, F.R.Germany. organizer : N. Birau.

Melatonin in Human Serum—A Collaborative Study of Current Radioimmunoassays

L. Wetterberg* and O. Eriksson**,     *Karolinska Institute, Department of Psychiatry, St. Göran’s Hospital, Stockholm, Sweden; **Kabi AB Research Department, Stockholm, Sweden

ABSTRACT

A collaborative study has been performed with 12 participating laboratories. Four levels of melatonin have been assayed. Night and day serum from human donors were used. Eight laboratories reported recoveries in the range 70-130 % at the levels 0.65 and 2.6 nmol/l. The values from the other laboratories are discussed. True values in the day and night serum could not be established as none of the laboratories with GC-MS methods reported any results. Most of the laboratories using RIA-methods report comparable results.

KEYWORDS

Melatonin

radioimmunoassay (RIA)

human

serum

INTRODUCTION

At the pineal meeting in Jerusalem 1977 the first collaborative study was presented. This study used calf serum to which melatonin had been added (Wetterberg 1977). Many investigators suggested that the study should be repeated using human serum sampled both in daytime and at night to provide samples containing endogenous melatonin and also endogenous precursors and metabolites of melatonin as well as possible interfering substances.

Such a study has now been done. Samples were sent to 20 laboratories of which 12 laboratories, all using RIA-methods, have replied. It has not been possible to obtain answers from laboratories using gaschromatography-masspectrometry (GC-MS) methods.

PREPARATION OF SAMPLES AND METHODS

Serum samples were collected at St. Göran’s Hospital and the lyophilized ampoules were prepared by the Pharmaceutical Department at Kabi AB. From a day pool of 1020 ml of serum from five donors, 169 samples of 3 ml aliquots were lyophilized and labeled A.

From a night pool of 520 ml of serum from three donors, 95 samples of 3 ml aliquots were lyophilized and labelled B.

A stock solution was prepared by dissolving 1.44 mg melatonin (FLUKA 63610) in 7.2 ml of phosphate-saline-gelatin buffer (PSG-buffer is 0.05M phosphate; 0.15 M NaCl, 0.1 % gelatin pH 7. By dilution 1:100 (1.00 ml to 100 ml with PSG) and further 1:20 (1.00 ml to 20 ml with PSG) a solution containing 100 ng melatonin/ml was obtained. Of this solution 0.75 ml was added to 500 ml of the day serum pool, mixed for 10 minutes and 163 samples of 3 ml aliquots were lyophilized and labelled C (day serum + 0.65 nmol/1 melatonin).

Of the melatonin solution 1.80 ml was added to 300 ml of the night serum pool, mixed for 10 minutes and 97 samples of 3 ml aliquots were lyophilized and labeled D (night serum + 2.6 nmol/1 melatonin).

The bottles were labelled Melatonin in human serum for the collaborative study 1978. Manuf. date April 78. Storage: Refrigerator (+5 C). Lot No. DcV 9. The four different concentrations of melatonin were labelled A, B, C and D as described above Five bottles of each concentration were sent to the participants. They were told that sample D could be assayed in several dilutions to check if diluted samples gave a parallel line with the standard. They were asked to report their results and where raw data were complete, a recalculation was done with a NIH-RIA computer program. No essential differences were obtained. The original data were transformed to the SI-unit mol/l and are reported.

The participants are listed in Table 1 in a different order than in the Tables with results.

TABLE 1

Participating laboratories, methods and standard substance of melatonin.

RESULTS

The numbers of the laboratories are identical in Table 2-4 but different from the order in Table 1.

TABLE 2

Reported results (recalculated to the same units of concentration) nmol/l on non-diluted samples.

×Note. Vial D. Assayed at 4 occasions, mean value 2.3 nmol/l (SD% 51). At one occasion the obtained value was more than 4 SD lower and at one occasion more than 4 SD higher than the mean. These values were excluded.

TABLE 3

Difference between A – C, B – D and recovery in percent.

TABLE 4

Dilutions of D (night serum +2.6 nmol/l). All values are nmol/l.

The results are listed in Table 2. Vials C and D were prepared by addition of melatonin to day and night serum. The recovery of exogenous melatonin was possible to calculate by the difference C-A and D-B. The results are shown in Table 3. Vial D had such a high melatonin concentration that it was possible to check if the diluted sample gave a parallel line with the standard curve. Five laboratories have reported their results in this respect (Table 4). The treatment of ampoule D has been different, which is reported in Remarks in Table 4.

DISCUSSION

Most laboratories have found low concentration of melatonin in vial A (day serum), usually lower than their lowest standard. Two laboratories (No. 4 and 8) found vial B (night serum) to be below their lowest standard curve.

Laboratory 11 reports very high results and also the recoveries are high (more than 200 %). Laboratory 2 and 12 report very high results in C and D which give too high recovery (130-190 %) and laboratory 8 reports very low results and also low recovery (<30 %).

Laboratory 7 found a high concentration in vial A and B but a low concentration in D indicating a non parallelism between samples and standards.

The other laboratories all have the results within 70-130 % range of the true value in the recovery test. This is what can be expected as the relative standard deviation usually is above 10 % for each determination.

ACKNOWLEDGEMENT

This work was supported by grants from Swedish Medical Research Council No. 3371 and AB KABI-Vitrum, Stockholm, Sweden. All scientists who have collaborated in this study are greatly acknowledged.

REFERENCES

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Wetterberg, L., Eriksson, O., Lynch, H. J., Jimerson, D. C., Ozaki, J., Post, R. M., Bunny, W. E., Jr., Wurtman, R. J. Life Science. 1978; 23:1557.

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DEVELOPMENTS IN MELATONIN HISTOPHYSIOLOGY

Cytological Aspects of Indoleamine Secretion in the Pineal Gland

P. Pévet,     The Netherlands Institute for Brain Research, Amsterdam; Department of Anatomy and Embryology, University of Amsterdam, Amsterdam, The Netherlands

ABSTRACT

The present review, based upon numerous reports published during the last ten years, attempts to clarify some cytological aspects of the indoleamine secretory process in the pineal. Because only a small part of the serotonin can be engaged in the formation of melatonin, serotonin and 5-methoxyindoles have been separately considered. By combined fluorescence histochemical, radioautographic and ultracytochemical data obtained in normal and drug-treated animals, it has been shown that anabolism, catabolism and storage of serotonin takes place in pineal cells belonging to the sensory cell line (photoreceptor cells, secretory rudimentary photoreceptor cells or pinealocytes). The role of the glial cells in indole metabolism is not evident. Although they are probably cells of the sensory line, the scarcity of reports concerned directly with melatonin has made it impossible to identify with certainty the cells implicated in its storage. The concept of melatonin as a pineal hormone is

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