0021-972X/91/7204-0819$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 4 Printed in U.S.A.
Sequential Cerebrospinal Fluid and Plasma Sampling in Humans: 24-Hour Melatonin Measurements in Normal Subjects and after Peripheral Sympathectomy JEFFREY BRUCE*, LAWRENCE TAMARKIN, CHARLES RIEDEL*, SANFORD MARKEY, AND EDWARD OLDFIELD Surgical Neurology Branch, National Institute of Neurologic Disorders and Stroke (J.B., C.R., E.O.), and the Clinical Psychobiology Branch (L.T.), and the Laboratory of Clinical Sciences (S.M.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. Simultaneous measurements of plasma and cerebrospinal fluid (CSF) melatonin and urinary excretion of 6hydroxymelatonin were performed in four normal volunteers and one patient before and after upper thoracic sympathectomy for the control of essential hyperhidrosis. For normal individuals, hourly 24-h melatonin concentrations in plasma and CSF exhibited similar profiles, with low levels during the day and high levels at night. Peak plasma levels varied from 122-660 pmol/L, and the peak CSF levels from 94-355 pmol/L. The onset of the nocturnal increase in melatonin did not occur at the same time for each individual. Urinary 6-hydroxymelatonin levels also exhibited a daily rhythm, with peak excretion at
night. The individual with the lowest nocturnal levels of circulating melatonin also had the lowest excretion of 6-hydroxymelatonin. In the patient with hyperhidrosis, a prominent melatonin rhythm was observed preoperatively in the CSF and plasma. After bilateral T1-T2 ganglionectomy, however, melatonin levels were markedly reduced, and the diurnal rhythm was abolished. These results provide direct evidence in humans for a diurnal melatonin rhythm in CSF and plasma as well as regulation of this rhythm by sympathetic innervation. (J Clin Endocrinol Metab 72: 819-823,1991)
A
LTHOUGH melatonin physiology has been studied extensively in animals, comparable studies in humans are lacking (1). It has been shown in a wide variety of animal species that the pineal gland receives sympathetic input and that this innervation is essential for the generation of a daily rhythm of circulating melatonin concentrations (2-4). Indirect anatomical evidence indicates that a similar pineal regulatory pathway exists in humans as well (5, 6). To investigate this pathway directly, we studied the central and peripheral melatonin profile before and after interruption of this sympathetic pathway in a patient undergoing surgical treatment of essential hyperhidrosis. No data are available in humans comparing the 24-h melatonin profile in cerebrospinal fluid (CSF) with that observed in the peripheral circulation. To compare the melatonin profile in CSF and plasma in the hyperhidrosis patient with that in normal subjects, CSF and plasma
samples were taken at similar intervals for 24-30 h from four normal volunteers. In all subjects the 24-h CSF and plasma melatonin profile was determined along with the urinary excretion of its major metabolite, 6-hyrdroxymelatonin (6-OH melatonin). Similar serum and urine studies have been performed in humans (7-10), but continuous CSF sampling has been limited to calves and nonhuman primates (11, 12). The present study demonstrates the utility of this neurosurgical technique for continuous CSF withdrawal (13-15). Materials and Methods Normal subjects Four normal volunteers (three men and one woman, 19-30 yr of age) were admitted to the Clinical Center at the NIH. All subjects were in excellent physical health, with no medical problems and on no medications. The female volunteer was in the 18th day of a normal 28-day menstrual cycle. All subjects participated in the study after informed consent was obtained (NIH protocol 854-N-51).
Address all correspondence and requests for reprints to: Dr. Jeffrey Bruce, Department of Neurosurgery, Neurological Institute, Box 122, Columbia University College of Physicians and Surgeons, New York, New York 10032. * Present address: Department of Neurosurgery, Neurological Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Hyperhidrotic patient A 28-yr-old man with essential hyperhidrosis of the palms, axillae, and feet was occupationally and socially handicapped 819
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BRUCE ET AL.
820
by continuous sweat dripping from his hands. Over 14 yr of excessive sweating, he had demonstrated no response to multiple medical therapies. During the physical examination he was sweating continuously from the palms, axillae, and feet. Sweat dripped steadily from the tips of his fingers, and his hands were cold and clammy. Since his complaints predominantly involved his hands, he was considered a candidate for bilateral upper thoracic sympathectomy. Two weeks after basal 30-h plasma, CSF, and urine samples were obtained, he underwent T l and T2 sympathectomy with excision of the T l and T2 sympathetic ganglia. After surgery, the hands and axillae remained dry, he had no sweating in a heated room, and his hands were warm. Since the upper thoracic ganglia were excised bilaterally, his pupils were symmetrical, and his Homer's syndrome was not obvious. Seven days after surgery plasma, CSF, and urine were again collected for 30 h. Sample collection CSF, serum, and urine were collected over one 30-h period in four normal subjects and in one hyperhidrotic patient before and after bilateral upper thoracic sympathectomy. During the entire sampling period all subjects were required to remain flat in bed, but no restrictions were placed on sleep, lighting, or diet. CSF was collected continuously from an indwelling catheter in the lumbar subarachnoid space, as described previously (15). Briefly, after a routine sterile prep, a polyamide epidural catheter was placed in the lumbar subarachnoid space through an 18-gauge spinal needle. After removing the needle, the catheter was attached to gas-sterilized polyethylene tubing and connected to a peristaltic pump for continuous aspiration of CSF at a rate of 6 mL/h, with aliquots collected into polypropylene tubes in a fraction collector. The pump and the fraction collector were housed in bedside refrigerator at 4 C. The CSF remained at room temperature for approximately 10 min, as calculated by the amount of dead space in the tubing. Samples were kept at 4 C for up to 3 h before being stored at -70 C until analysis. Blood samples were collected through an indwelling venous catheter in the upper extremity. Samples were obtained hourly, put into heparinized glass tubes, and placed on wet ice. Within 2 h, the samples were centrifuged to harvest the plasma. Plasma was immediately transferred to polypropylene tubes for storage at -70 C until analysis. Sequential 4-h urine samples were refrigerated at 4 C for up to 24 h. The sample volumes were measured, and aliquots were placed in propylene tubes and stored at -70 C until analysis for 6-OH melatonin.
JCE & M • 1991 Vol72^No4
this stage, while the plasma samples were extracted a second time with petroleum ether. The efficiency of the first extraction was greater than 95%, while the combined efficiency of the two extractions was 85%. The resultant data were not corrected for the loss of melatonin during the extraction procedure. The limit of sensitivity of the assay was 2 pmol/L, and the intraassay coefficient of variation was 10%. All samples were analyzed in one assay. 6-OH melatonin assay. Urinary 6-OH melatonin concentrations were determined by the gas chromatography-negative chemical ionization mass spectrometry (gc-ms) method of Tetsuo et al. (17). Briefly, deuterated 6-OH melatonin was added to 3 mL of each urine sample as an internal sample standard. Each sample was enzymatically hydrolyzed and extracted with dichloromethane. The extract was then derivatized by a two-step procedure, the product was purified by silica gel chromotography, and the resultant eluate was finally concentrated for injection into the gc-ms. Quantitative analysis was based on the ratio of the deuterated internal standard to the endogenous derivatized 6-OH melatonin compared to the ratios observed for the authentic free 6-OH melatonin prepared in a similar manner as the urine samples. Intra- and interassay variations were less than 10%, and the limit of sensitivity was 0.1 ng/mL.
Results Normal subjects Melatonin concentrations exhibited a prominent diurnal rhythm in CSF and plasma, with low daytime levels and nocturnal peaks occurring between 2400-0600 h (Fig. 1). The temporal profiles of melatonin in CSF and plasma were very similar for an individual, but differed among subjects. Daytime plasma melatonin levels ranged from 8.5-31.5 pmol/L, with nocturnal peaks from 122-660 pmol/L. CSF levels ranged from daytime nadirs of 20-38 pmol/ L, with nocturnal peaks from 94-355 pmol/L. The magnitude of the nocturnal peaks were similar in plasma and CSF for three of the four subjects. Urinary 6-OH melatonin, determined at 4-h intervals during the CSF-sampling period, also exhibited a prominent diurnal rhythm (Fig. 2), which reflected CSF and plasma melatonin levels. Peak levels, ranging from 2.66.7 jug/4-h interval, occurred at night, with nadirs during the day from 0.1-0.4 ^g/4-h interval.
Hyperhidrotic patient
Analyses Melatonin RIA. Plasma and CSF melatonin concentrations were determined by a RIA that has been validated and reliable for use in human plasma and primate CSF (11,16). Before the RIA, plasma and CSF samples were extracted with chloroform, dried in a vacuum oven, and reconstituted in 0.1% gelatinphosphate-buffered saline. The CSF samples were analyzed at
Preoperatively, the patient with hyperhidrosis had a prominent diurnal melatonin rhythm in both the CSF and plasma (Fig. 3). The highest recorded plasma levels occurred at 0700 h, with a concentration of 1000 pmol/ L. The plasma nadir of 33 pmol/L occurred at 2200 h. CSF melatonin levels ranged from less than 2 pmol/L to
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PLASMA AND CSF MELATONIN IN HUMANS PLASMA ..„.. before —•- after
1200
PLASMA CSF
o B u, Z
821
1000
CSF
B
--«-• before —•- after
800
H
z
§
600 400
w
"o
2
6 3,
„••%
200 0
Z
z
24
12
18
24
24
12
18
24
TIME OF DAY FlG. 3. Daily profile of plasma and CSF melatonin concentrations in the hyperhidrosis patient before and after sympathectomy.
gw 2
2-
18
24
6
12
18
24
18
24
6
12
18
1
24
TIME OF DAY 1. Daily profile of CSF and plasma melatonin concentrations in four normal subjects. Data are presented for each patient individually, The only female subject is represented in A.
FIG.
8-1
B
D before • after
X
o 2100 0100 0500 0900 1300 1700
2100
FOUR HOUR COLLECTION INTERVAL FIG. 4. Daily urinary excretion of 6-OH melatonin in the hyperhidrosis patient before and after sympathectomy.
tomies, the patient's diurnal rhythm of melatonin was abolished in both CSF and plasma (Fig. 3). In addition, the levels were significantly decreased. CSF values were all less than 7 pmol/L, while serum levels were less than 240 pmol/L for the 24-h sampling period. The diurnal rhythm of urinary 6-OH melatonin was also abolished after surgery, and this metabolite was barely detectable or undetectable in the urine throughout the day (Fig. 4).
•IHIB 8-1
Discussion J—,MMM,—,T, 2100 0100 0500 0900 1300 1700 2100
2100 0100 0500 0900 1300 1700 2100
FOUR HOUR COLLECTION INTERVAL
FlG. 2. Daily urinary excretion of 6-OH melatonin in four normal subjects. A-D correspond to the same individuals as those presented in Fig. 1.
270 pmol/L, with a peak occurring at 0600 h and a nadir from 1300-1900 h. The peak urinary 6-OH melatonin level was 2.9 /ig/4-h. interval at night, falling to less than 0.1 Mg/4-h interval during the day. After undergoing bilateral upper thoracic sympathec-
Serial measurement of melatonin levels over 30 h demonstrated a clear daily rhythm in both the peripheral (plasma) and central (CSF) circulations of all four normal subjects studied. This rhythm was reflected in the measurement of urinary 6-OH melatonin collected simultaneously over 4-h intervals. Although a circadian pattern of melatonin secretion was always present, the temporal profiles differed among subjects, as did the concentration of melatonin in plasma and CSF. This confirms others studies which have demonstrated that the normal range for daytime and nighttime plasma and CSF levels is large, and the day-night difference for melatonin levels can vary widely (7-9, 16, 18-24). With
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BRUCE ET AL.
the wide range of "normal" melatonin levels, absolute values of human melatonin levels must be cautiously interpreted. Alterations in normal plasma melatonin levels are associated with depression, seizure control, neoplasia, andpubertal development (9,19-22). Assessment of daily melatonin levels have revealed that some patients or normal subjects have augmented nocturnal secretion, or conversely, some patients have nocturnal levels of melatonin that are similar to daytime levels. The significance of these quantitative differences in melatonin is not known; thus, comparisons between groups of individuals are difficult in humans. The patient with hyperhidrosis had normal central and peripheral melatonin rhythms and a normal excretion of 6-OH melatonin before surgery. Although preoperative melatonin levels were higher in this patient than in any of the normal subjects, it is difficult to attribute this to sympathetic nervous system hyperactivity, since the total urinary excretion of 6-OH melatonin over 24 h was less than that of two of the normal subjects (subjects A and C). After bilateral upper thoracic sympathectomy, melatonin and 6-OH melatonin levels were markedly decreased, and the diurnal rhythms were abolished. Essential hyperhidrosis is characterized by excessive sweating, particularly in the hands and axilla. Although the etiology of this condition is vague, it is thought to be caused by hyperactivity of central innervation of the sympathetic nervous system (25). Surgical treatment of hyperhidrosis involves interruption of the sympathetic innervation of the upper limb by resecting upper thoracic sympathetic ganglia (26). Resection of these ganglia also interrupts peripheral sympathetic pathways, which pass through the superior cervical ganglia to reach the head (27). In several animal species, ablation of the superior cervical ganglia disrupts melatonin rhythms by depriving the pineal gland of sympathetic input (2-4). Although indirect evidence for a similar pathway has been reported in humans (5, 6), our study demonstrates that interruption of peripheral sympathetic input to the pineal results in markedly abnormal melatonin patterns. In humans, then, as well as animals, sympathetic input appears to be necessary for regulation of diurnal melatonin rhythms. Further studies to investigate sympathetic influence on melatonin physiology as well as the central vs. peripheral role of melatonin can now more easily be accomplished with the technique of CSF sampling by continuous lumbar CSF drainage in humans (15). Spinal drainage is commonly used in clinical settings for neurosurgical or anesthesia-related purposes. With the modifications and precautions previously described (15), which permit continuous sampling for extended periods, this technique should prove to be a safe and valuable
JCE & M • 1991 Vol72«No4
research tool for the study of melatonin physiology and other CSF neurohormones and metabolites.
References 1. Preslock JP. The pineal gland: basic implications and clinical correlations. Endocr Rev. 1984;5:282-308. 2. Moore RY. Neural control of pineal function in mammals and birds. J Neural Trans. 1978;13(Suppl):47-58. 3. Reiter RJ, Rudeen PK, Banks AF, Rollag MD. Acute effects of unilateral or bilateral superior ganglionectomy on rat pineal Nacetyltransferase activity and melatonin content. Experentia. 1979;35:691-2. 4. Lincoln GA, Almeida FX, Klandorf H, Cunningham RA. Hourly fluctuations in the blood levels of melatonin, prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone, triiodothyronine, thyroxine and cortisol in rams under artificial photoperiods, and the effects of cranial sympathectomy. J Endocrinol. 1982;92:237-50. 5. Tetsuo M, Polinsky RJ, Markey SP, Kopin IJ. Urinary 6-hydroxymelatonin excretion in patients with orthostatic hypotension. J Clin Endocrinol Metab. 1981;53:607-10. 6. Kneisley LW, Moskowitz MA, Lynch HJ. Cervical spinal cord lesions disrupt the rhythm in human melatonin excretion. J Neural Transm. 1978;13(Suppl):311-23. 7. Arendt J, Wetterberg L, Heyden T, Sizonenko PC, Paunier L. Radioimmunoassay of melatonin: human serum and cerebrospinal fluid. Horm Res. 1977;8:65-75 8. Pelham RW, Vaughan GM, Sandock KL, Vaughan MK. Twentyfour hour cycle of a melatonin-like substance in the plasma of human males. J Clin Endocrinol Metab. 1973;37:341-4. 9. Vaughan GM. Melatonin in humans. Pineal Res Rev. 1984;2:141201. 10. Tetsuo M, Markey SP, Kopin IJ. Measurement of 6-hydroxymelatonin in human urine and its diurnal variations. Life Sci. 1980;27:105-9. 11. Reppert SM, Perlow MJ, Tamarkin L, Klein DC. A diurnal melatonin rhythm in primate cerebrospinal fluid. Endocrinology. 1979;104:295-301. 12. Hedlund L, Lischko MM, Rollag MD, Niswender GD. Melatonin: daily cycle in plasma and cerebrospinal fluid of calves. Science. 1977;195:686-7. 13. Post KD, Stein BM. Technique for spinal drainage. Neurosurgery. 1979;4:255. 14. McCallum J, Maroon JC, Jannetta PJ. Treatment of postoperative cerebrospinal fluid fistulas by subarachnoid drainage. J Neurosurg. 1975;42:434-7. 15. Bruce JN, Oldfield EH. Method for sequential sampling of cerebrospinal fluid in humans. Neurosurgery. 1988;23:788-90. 16. Tamarkin L, Abastillas P, Chen H-C, McNemar A, Sidbury JB. The daily profile of plasma melatonin in obese and Prader-Willi syndrome in children. J Clin Endocrinol Metab. 1982;55:491-5. 17. Tetsuo M, Markey SP, Colburn RW, Kopin IJ. Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromotography negative chemical ionization mass spectrometry. Anal Biochem. 1981;110:208-15. 18. Smith JA, Mee TJX, Barnes ND, Torburn RJ, Barnes JLC. Melatonin in serum and spinal fluid. Lancet. 1976;2:425. 19. Wetterberg L, Aperia B, Beck-Friis J, et al. Melatonin and cortisol levels in psychiatric illness. Lancet. 1982;2:100. 20. Branchey L, Weinberg U, Branchey M, Linkowski P, Mendlewicz J. Simultaneous study of 24-hour patterns of melatonin and cortisol secretion in depressed patients. Neuropsychobiology. 1982;8:25532. 21. Tamarkin L, Danforth D, Lichter A, et al. Decreased nocturnal plasma melatonin peak in patients with estrogen receptor positive breast cancer. Science. 1982;216:1003-5.
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PLASMA AND CSF MELATONIN IN HUMANS 22. Waldhauser F, Frisch H, Waldhauser M, Weiszenbacher G, Zeitlhuber U, Wurtman RJ. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet. 1984;l:362-5. 23. Markey SP, Higa S, Shih M, Danforth DN, Tamarkin L. The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion. Clin Chim Acta. 1985;150:221-5. 24. Young SN, Gauthier S, Kiely M, Lai S, Brown GM. Effect of oral melatonin administration on melatonin, 5-hydroxyindoleacetic
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acid, indoleacetic acid, and cyclic nucleotides in human cerebrospinal fluid. Neuroendocrinology. 1984;39:87-92. 25. Dohn DF, Zraik 0. Essential hyperhidrosis-pathogenesis and treatment. Cleveland Clin Q 1969;36:79-83. 26. Ray BS. Sympathectomy of the upper extremity. J Neurosurg. 1952;10:624-32. 27. Carpenter MB, Sutin J. Human neuroanatomy. Baltimore: WilHams and Wilkins; 1983;213-4.
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Vol. 72, No. 4 Printed in U.S.A.
Sequential Cerebrospinal Fluid and Plasma Sampling in Humans: 24-Hour Melatonin Measurements in Normal Subjects and after Peripheral Sympathectomy JEFFREY BRUCE*, LAWRENCE TAMARKIN, CHARLES RIEDEL*, SANFORD MARKEY, AND EDWARD OLDFIELD Surgical Neurology Branch, National Institute of Neurologic Disorders and Stroke (J.B., C.R., E.O.), and the Clinical Psychobiology Branch (L.T.), and the Laboratory of Clinical Sciences (S.M.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. Simultaneous measurements of plasma and cerebrospinal fluid (CSF) melatonin and urinary excretion of 6hydroxymelatonin were performed in four normal volunteers and one patient before and after upper thoracic sympathectomy for the control of essential hyperhidrosis. For normal individuals, hourly 24-h melatonin concentrations in plasma and CSF exhibited similar profiles, with low levels during the day and high levels at night. Peak plasma levels varied from 122-660 pmol/L, and the peak CSF levels from 94-355 pmol/L. The onset of the nocturnal increase in melatonin did not occur at the same time for each individual. Urinary 6-hydroxymelatonin levels also exhibited a daily rhythm, with peak excretion at
night. The individual with the lowest nocturnal levels of circulating melatonin also had the lowest excretion of 6-hydroxymelatonin. In the patient with hyperhidrosis, a prominent melatonin rhythm was observed preoperatively in the CSF and plasma. After bilateral T1-T2 ganglionectomy, however, melatonin levels were markedly reduced, and the diurnal rhythm was abolished. These results provide direct evidence in humans for a diurnal melatonin rhythm in CSF and plasma as well as regulation of this rhythm by sympathetic innervation. (J Clin Endocrinol Metab 72: 819-823,1991)
A
LTHOUGH melatonin physiology has been studied extensively in animals, comparable studies in humans are lacking (1). It has been shown in a wide variety of animal species that the pineal gland receives sympathetic input and that this innervation is essential for the generation of a daily rhythm of circulating melatonin concentrations (2-4). Indirect anatomical evidence indicates that a similar pineal regulatory pathway exists in humans as well (5, 6). To investigate this pathway directly, we studied the central and peripheral melatonin profile before and after interruption of this sympathetic pathway in a patient undergoing surgical treatment of essential hyperhidrosis. No data are available in humans comparing the 24-h melatonin profile in cerebrospinal fluid (CSF) with that observed in the peripheral circulation. To compare the melatonin profile in CSF and plasma in the hyperhidrosis patient with that in normal subjects, CSF and plasma
samples were taken at similar intervals for 24-30 h from four normal volunteers. In all subjects the 24-h CSF and plasma melatonin profile was determined along with the urinary excretion of its major metabolite, 6-hyrdroxymelatonin (6-OH melatonin). Similar serum and urine studies have been performed in humans (7-10), but continuous CSF sampling has been limited to calves and nonhuman primates (11, 12). The present study demonstrates the utility of this neurosurgical technique for continuous CSF withdrawal (13-15). Materials and Methods Normal subjects Four normal volunteers (three men and one woman, 19-30 yr of age) were admitted to the Clinical Center at the NIH. All subjects were in excellent physical health, with no medical problems and on no medications. The female volunteer was in the 18th day of a normal 28-day menstrual cycle. All subjects participated in the study after informed consent was obtained (NIH protocol 854-N-51).
Address all correspondence and requests for reprints to: Dr. Jeffrey Bruce, Department of Neurosurgery, Neurological Institute, Box 122, Columbia University College of Physicians and Surgeons, New York, New York 10032. * Present address: Department of Neurosurgery, Neurological Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Hyperhidrotic patient A 28-yr-old man with essential hyperhidrosis of the palms, axillae, and feet was occupationally and socially handicapped 819
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BRUCE ET AL.
820
by continuous sweat dripping from his hands. Over 14 yr of excessive sweating, he had demonstrated no response to multiple medical therapies. During the physical examination he was sweating continuously from the palms, axillae, and feet. Sweat dripped steadily from the tips of his fingers, and his hands were cold and clammy. Since his complaints predominantly involved his hands, he was considered a candidate for bilateral upper thoracic sympathectomy. Two weeks after basal 30-h plasma, CSF, and urine samples were obtained, he underwent T l and T2 sympathectomy with excision of the T l and T2 sympathetic ganglia. After surgery, the hands and axillae remained dry, he had no sweating in a heated room, and his hands were warm. Since the upper thoracic ganglia were excised bilaterally, his pupils were symmetrical, and his Homer's syndrome was not obvious. Seven days after surgery plasma, CSF, and urine were again collected for 30 h. Sample collection CSF, serum, and urine were collected over one 30-h period in four normal subjects and in one hyperhidrotic patient before and after bilateral upper thoracic sympathectomy. During the entire sampling period all subjects were required to remain flat in bed, but no restrictions were placed on sleep, lighting, or diet. CSF was collected continuously from an indwelling catheter in the lumbar subarachnoid space, as described previously (15). Briefly, after a routine sterile prep, a polyamide epidural catheter was placed in the lumbar subarachnoid space through an 18-gauge spinal needle. After removing the needle, the catheter was attached to gas-sterilized polyethylene tubing and connected to a peristaltic pump for continuous aspiration of CSF at a rate of 6 mL/h, with aliquots collected into polypropylene tubes in a fraction collector. The pump and the fraction collector were housed in bedside refrigerator at 4 C. The CSF remained at room temperature for approximately 10 min, as calculated by the amount of dead space in the tubing. Samples were kept at 4 C for up to 3 h before being stored at -70 C until analysis. Blood samples were collected through an indwelling venous catheter in the upper extremity. Samples were obtained hourly, put into heparinized glass tubes, and placed on wet ice. Within 2 h, the samples were centrifuged to harvest the plasma. Plasma was immediately transferred to polypropylene tubes for storage at -70 C until analysis. Sequential 4-h urine samples were refrigerated at 4 C for up to 24 h. The sample volumes were measured, and aliquots were placed in propylene tubes and stored at -70 C until analysis for 6-OH melatonin.
JCE & M • 1991 Vol72^No4
this stage, while the plasma samples were extracted a second time with petroleum ether. The efficiency of the first extraction was greater than 95%, while the combined efficiency of the two extractions was 85%. The resultant data were not corrected for the loss of melatonin during the extraction procedure. The limit of sensitivity of the assay was 2 pmol/L, and the intraassay coefficient of variation was 10%. All samples were analyzed in one assay. 6-OH melatonin assay. Urinary 6-OH melatonin concentrations were determined by the gas chromatography-negative chemical ionization mass spectrometry (gc-ms) method of Tetsuo et al. (17). Briefly, deuterated 6-OH melatonin was added to 3 mL of each urine sample as an internal sample standard. Each sample was enzymatically hydrolyzed and extracted with dichloromethane. The extract was then derivatized by a two-step procedure, the product was purified by silica gel chromotography, and the resultant eluate was finally concentrated for injection into the gc-ms. Quantitative analysis was based on the ratio of the deuterated internal standard to the endogenous derivatized 6-OH melatonin compared to the ratios observed for the authentic free 6-OH melatonin prepared in a similar manner as the urine samples. Intra- and interassay variations were less than 10%, and the limit of sensitivity was 0.1 ng/mL.
Results Normal subjects Melatonin concentrations exhibited a prominent diurnal rhythm in CSF and plasma, with low daytime levels and nocturnal peaks occurring between 2400-0600 h (Fig. 1). The temporal profiles of melatonin in CSF and plasma were very similar for an individual, but differed among subjects. Daytime plasma melatonin levels ranged from 8.5-31.5 pmol/L, with nocturnal peaks from 122-660 pmol/L. CSF levels ranged from daytime nadirs of 20-38 pmol/ L, with nocturnal peaks from 94-355 pmol/L. The magnitude of the nocturnal peaks were similar in plasma and CSF for three of the four subjects. Urinary 6-OH melatonin, determined at 4-h intervals during the CSF-sampling period, also exhibited a prominent diurnal rhythm (Fig. 2), which reflected CSF and plasma melatonin levels. Peak levels, ranging from 2.66.7 jug/4-h interval, occurred at night, with nadirs during the day from 0.1-0.4 ^g/4-h interval.
Hyperhidrotic patient
Analyses Melatonin RIA. Plasma and CSF melatonin concentrations were determined by a RIA that has been validated and reliable for use in human plasma and primate CSF (11,16). Before the RIA, plasma and CSF samples were extracted with chloroform, dried in a vacuum oven, and reconstituted in 0.1% gelatinphosphate-buffered saline. The CSF samples were analyzed at
Preoperatively, the patient with hyperhidrosis had a prominent diurnal melatonin rhythm in both the CSF and plasma (Fig. 3). The highest recorded plasma levels occurred at 0700 h, with a concentration of 1000 pmol/ L. The plasma nadir of 33 pmol/L occurred at 2200 h. CSF melatonin levels ranged from less than 2 pmol/L to
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 May 2015. at 06:29 For personal use only. No other uses without permission. . All rights reserved.
PLASMA AND CSF MELATONIN IN HUMANS PLASMA ..„.. before —•- after
1200
PLASMA CSF
o B u, Z
821
1000
CSF
B
--«-• before —•- after
800
H
z
§
600 400
w
"o
2
6 3,
„••%
200 0
Z
z
24
12
18
24
24
12
18
24
TIME OF DAY FlG. 3. Daily profile of plasma and CSF melatonin concentrations in the hyperhidrosis patient before and after sympathectomy.
gw 2
2-
18
24
6
12
18
24
18
24
6
12
18
1
24
TIME OF DAY 1. Daily profile of CSF and plasma melatonin concentrations in four normal subjects. Data are presented for each patient individually, The only female subject is represented in A.
FIG.
8-1
B
D before • after
X
o 2100 0100 0500 0900 1300 1700
2100
FOUR HOUR COLLECTION INTERVAL FIG. 4. Daily urinary excretion of 6-OH melatonin in the hyperhidrosis patient before and after sympathectomy.
tomies, the patient's diurnal rhythm of melatonin was abolished in both CSF and plasma (Fig. 3). In addition, the levels were significantly decreased. CSF values were all less than 7 pmol/L, while serum levels were less than 240 pmol/L for the 24-h sampling period. The diurnal rhythm of urinary 6-OH melatonin was also abolished after surgery, and this metabolite was barely detectable or undetectable in the urine throughout the day (Fig. 4).
•IHIB 8-1
Discussion J—,MMM,—,T, 2100 0100 0500 0900 1300 1700 2100
2100 0100 0500 0900 1300 1700 2100
FOUR HOUR COLLECTION INTERVAL
FlG. 2. Daily urinary excretion of 6-OH melatonin in four normal subjects. A-D correspond to the same individuals as those presented in Fig. 1.
270 pmol/L, with a peak occurring at 0600 h and a nadir from 1300-1900 h. The peak urinary 6-OH melatonin level was 2.9 /ig/4-h. interval at night, falling to less than 0.1 Mg/4-h interval during the day. After undergoing bilateral upper thoracic sympathec-
Serial measurement of melatonin levels over 30 h demonstrated a clear daily rhythm in both the peripheral (plasma) and central (CSF) circulations of all four normal subjects studied. This rhythm was reflected in the measurement of urinary 6-OH melatonin collected simultaneously over 4-h intervals. Although a circadian pattern of melatonin secretion was always present, the temporal profiles differed among subjects, as did the concentration of melatonin in plasma and CSF. This confirms others studies which have demonstrated that the normal range for daytime and nighttime plasma and CSF levels is large, and the day-night difference for melatonin levels can vary widely (7-9, 16, 18-24). With
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the wide range of "normal" melatonin levels, absolute values of human melatonin levels must be cautiously interpreted. Alterations in normal plasma melatonin levels are associated with depression, seizure control, neoplasia, andpubertal development (9,19-22). Assessment of daily melatonin levels have revealed that some patients or normal subjects have augmented nocturnal secretion, or conversely, some patients have nocturnal levels of melatonin that are similar to daytime levels. The significance of these quantitative differences in melatonin is not known; thus, comparisons between groups of individuals are difficult in humans. The patient with hyperhidrosis had normal central and peripheral melatonin rhythms and a normal excretion of 6-OH melatonin before surgery. Although preoperative melatonin levels were higher in this patient than in any of the normal subjects, it is difficult to attribute this to sympathetic nervous system hyperactivity, since the total urinary excretion of 6-OH melatonin over 24 h was less than that of two of the normal subjects (subjects A and C). After bilateral upper thoracic sympathectomy, melatonin and 6-OH melatonin levels were markedly decreased, and the diurnal rhythms were abolished. Essential hyperhidrosis is characterized by excessive sweating, particularly in the hands and axilla. Although the etiology of this condition is vague, it is thought to be caused by hyperactivity of central innervation of the sympathetic nervous system (25). Surgical treatment of hyperhidrosis involves interruption of the sympathetic innervation of the upper limb by resecting upper thoracic sympathetic ganglia (26). Resection of these ganglia also interrupts peripheral sympathetic pathways, which pass through the superior cervical ganglia to reach the head (27). In several animal species, ablation of the superior cervical ganglia disrupts melatonin rhythms by depriving the pineal gland of sympathetic input (2-4). Although indirect evidence for a similar pathway has been reported in humans (5, 6), our study demonstrates that interruption of peripheral sympathetic input to the pineal results in markedly abnormal melatonin patterns. In humans, then, as well as animals, sympathetic input appears to be necessary for regulation of diurnal melatonin rhythms. Further studies to investigate sympathetic influence on melatonin physiology as well as the central vs. peripheral role of melatonin can now more easily be accomplished with the technique of CSF sampling by continuous lumbar CSF drainage in humans (15). Spinal drainage is commonly used in clinical settings for neurosurgical or anesthesia-related purposes. With the modifications and precautions previously described (15), which permit continuous sampling for extended periods, this technique should prove to be a safe and valuable
JCE & M • 1991 Vol72«No4
research tool for the study of melatonin physiology and other CSF neurohormones and metabolites.
References 1. Preslock JP. The pineal gland: basic implications and clinical correlations. Endocr Rev. 1984;5:282-308. 2. Moore RY. Neural control of pineal function in mammals and birds. J Neural Trans. 1978;13(Suppl):47-58. 3. Reiter RJ, Rudeen PK, Banks AF, Rollag MD. Acute effects of unilateral or bilateral superior ganglionectomy on rat pineal Nacetyltransferase activity and melatonin content. Experentia. 1979;35:691-2. 4. Lincoln GA, Almeida FX, Klandorf H, Cunningham RA. Hourly fluctuations in the blood levels of melatonin, prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone, triiodothyronine, thyroxine and cortisol in rams under artificial photoperiods, and the effects of cranial sympathectomy. J Endocrinol. 1982;92:237-50. 5. Tetsuo M, Polinsky RJ, Markey SP, Kopin IJ. Urinary 6-hydroxymelatonin excretion in patients with orthostatic hypotension. J Clin Endocrinol Metab. 1981;53:607-10. 6. Kneisley LW, Moskowitz MA, Lynch HJ. Cervical spinal cord lesions disrupt the rhythm in human melatonin excretion. J Neural Transm. 1978;13(Suppl):311-23. 7. Arendt J, Wetterberg L, Heyden T, Sizonenko PC, Paunier L. Radioimmunoassay of melatonin: human serum and cerebrospinal fluid. Horm Res. 1977;8:65-75 8. Pelham RW, Vaughan GM, Sandock KL, Vaughan MK. Twentyfour hour cycle of a melatonin-like substance in the plasma of human males. J Clin Endocrinol Metab. 1973;37:341-4. 9. Vaughan GM. Melatonin in humans. Pineal Res Rev. 1984;2:141201. 10. Tetsuo M, Markey SP, Kopin IJ. Measurement of 6-hydroxymelatonin in human urine and its diurnal variations. Life Sci. 1980;27:105-9. 11. Reppert SM, Perlow MJ, Tamarkin L, Klein DC. A diurnal melatonin rhythm in primate cerebrospinal fluid. Endocrinology. 1979;104:295-301. 12. Hedlund L, Lischko MM, Rollag MD, Niswender GD. Melatonin: daily cycle in plasma and cerebrospinal fluid of calves. Science. 1977;195:686-7. 13. Post KD, Stein BM. Technique for spinal drainage. Neurosurgery. 1979;4:255. 14. McCallum J, Maroon JC, Jannetta PJ. Treatment of postoperative cerebrospinal fluid fistulas by subarachnoid drainage. J Neurosurg. 1975;42:434-7. 15. Bruce JN, Oldfield EH. Method for sequential sampling of cerebrospinal fluid in humans. Neurosurgery. 1988;23:788-90. 16. Tamarkin L, Abastillas P, Chen H-C, McNemar A, Sidbury JB. The daily profile of plasma melatonin in obese and Prader-Willi syndrome in children. J Clin Endocrinol Metab. 1982;55:491-5. 17. Tetsuo M, Markey SP, Colburn RW, Kopin IJ. Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromotography negative chemical ionization mass spectrometry. Anal Biochem. 1981;110:208-15. 18. Smith JA, Mee TJX, Barnes ND, Torburn RJ, Barnes JLC. Melatonin in serum and spinal fluid. Lancet. 1976;2:425. 19. Wetterberg L, Aperia B, Beck-Friis J, et al. Melatonin and cortisol levels in psychiatric illness. Lancet. 1982;2:100. 20. Branchey L, Weinberg U, Branchey M, Linkowski P, Mendlewicz J. Simultaneous study of 24-hour patterns of melatonin and cortisol secretion in depressed patients. Neuropsychobiology. 1982;8:25532. 21. Tamarkin L, Danforth D, Lichter A, et al. Decreased nocturnal plasma melatonin peak in patients with estrogen receptor positive breast cancer. Science. 1982;216:1003-5.
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PLASMA AND CSF MELATONIN IN HUMANS 22. Waldhauser F, Frisch H, Waldhauser M, Weiszenbacher G, Zeitlhuber U, Wurtman RJ. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet. 1984;l:362-5. 23. Markey SP, Higa S, Shih M, Danforth DN, Tamarkin L. The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion. Clin Chim Acta. 1985;150:221-5. 24. Young SN, Gauthier S, Kiely M, Lai S, Brown GM. Effect of oral melatonin administration on melatonin, 5-hydroxyindoleacetic
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acid, indoleacetic acid, and cyclic nucleotides in human cerebrospinal fluid. Neuroendocrinology. 1984;39:87-92. 25. Dohn DF, Zraik 0. Essential hyperhidrosis-pathogenesis and treatment. Cleveland Clin Q 1969;36:79-83. 26. Ray BS. Sympathectomy of the upper extremity. J Neurosurg. 1952;10:624-32. 27. Carpenter MB, Sutin J. Human neuroanatomy. Baltimore: WilHams and Wilkins; 1983;213-4.
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