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The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion
221
Clinica Chimica Acta, 150 (1985) 221-225 Elsevier
CCA 03242
Short
communication
The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion Sanford
P. Markey
a,*, Sadayoshi Higa a, Ming Shih a, David and Lawrence Tamarkin b
N. Danforth’
ULaboratory of Clinical Science and h Clinical Psychobiology Branch, National Institute of Mental Health, ’ SurgeryBranch, National Cancer Institute, National Institutes o/Health, Bethesda, MD 20205 (USA) (Received
January
Key words: Melatonin;
14th, 1985; revision April 29th, 1985)
6 - Hvdroxymelatonin;
Human plasma; Human urine
Summary A significant correlation (0.76) has been found between nighttime peak .plasma melatonin levels and the 24-h urinary excretion totals for conjugated 6-hydroxymelatonin for a group of 22 women. This study validates the comparison of plasma levels of the hormone or urinary levels of its metabolite to assess pineal gland production of melatonin in humans.
Introduction Plasma levels of melatonin have been useful measures of pineal activity in clinical studies in psychiatry [1,2], cancer research [3], endocrinology [4-61 and pharmacology [7] using radioimmunoassay or gas chromatographic-mass spectrometric (gc-ms) assay procedures [9]. Plasma melatonin levels are low in the day and increase at night to a peak between 24:00 and 03:00, requiring the collection of multiple blood samples for clinical studies. The major urinary melatonin metabolites, glucuronide and sulfate conjugates of 6-hydroxymelatonin, have been quantified by gc-ms procedures (9,101 and reported in endocrine [11,12], hypotension [13] and drug function [14] studies. Multiple plasma level measurements are not practical for many clinical studies, especially when a measure of the amplitude of melatonin production is desired. For this reason, we undertook to determine the correlation between plasma melatonin and urinary 6-hydroxymelatonin for a single group of patients and normal volunteers.
* Correspondence
0009-8981/85/$03.30
to: Dr. S.P. Markey,
Bldg. lo-3N-325,
0 1985 Elsevier Science Publishers
NIH. Bethesda,
B.V. (Biomedical
MD 20205, USA.
Division)
222
Methods Experimental subjects Subjects for this study included 18 healthy female volunteers ranging in age from 18-68 yr (mean 36.6 + 5.6) and 4 patients ranging in age from 29-64 yr (mean 44 + 10.4) with clinical stage I or II breast cancer. At the time of study, the normal subjects had no serious illness, were of normal weight, and had a normal complete blood count (CBC), liver and renal function studies. All the patients presented with untreated primary breast cancer, were of normal weight, and had no evidence of other malignancies or major illness. Prior to and during this study the patients were drug-free and of normal nutritional status. All subjects were admitted to the Clinical Center and were requested to conform to normal ward routines with no dietary or activity restrictions. Serial blood samples were drawn from each subject through an intravenous Heparin-Lock needle and urine was collected on this same day. Blood samples were drawn every 3 h for 24 h. Urine samples were pooled from three blocks of time; 14:00-22:00, 22:00-06:00, 06:00-14:O0. Actual collection times varied somewhat depending upon the patient. Melatonin radioimmunoassay Plasma melatonin was analyzed by a radioimmunoassay which has been validated for use in human plasma [5]. Prior to the radioimmunoassay each plasma sample is extracted with chloroform, reconstituted in 0.1% gelatin-phosphate buffered saline, and extracted a second time with petroleum ether. The combined efficiency for both extractions is 85% and the resultant data are not corrected for the 15% loss of melatonin. The limit of sensitivity of the assay is 0.5 pg/ tube and the intra- and interassay coefficients of variation are 10 and 20%, respectively. 6-Hydroxymelatonin assay The assay as previously described by Tetsuo et al [8] was utilized in these studies. Briefly, deuterated 6-hydroxymelatonin sulfate is added as an internal standard to 3 ml of urine, which is enzymatically hydrolyzed and extracted with dichloromethane. The extracts are subjected to a two-step derivatization procedure and the resulting product purified by silica gel chromatography. Finally, the column elute is concentrated and injected into a gas chromatograph-mass spectrometer (gc-ms) operating in a negative chemical ionization mode. Quantification is based upon the ratio of deuterated internal standard to endogenous derivatized 6-hydroxymelatonin, corrected by a standard curve prepared from authentic free 6-hydroxymelatonin and conjugated deuterated internal standard. Assay to assay variation is corrected using a frozen pooled urine standard measured with each sample batch. As previously reported, intra- and interassay variability were < 10% [8]. Results Plasma melatonin Plots of the data from three of the patients are given in Fig. 1. Table I shows the pattern of the three highest melatonin values for each of the 22 patients measured.
Fig. 1. Plasma levels of melatonin (solid circles) are plotted vs. clock time for patients A, B, and C (Table I). On the same time axis the urinary excretion of 6-hydroxymelatonin is given in cross-hatched bars, with units of measurement indicated to the right.
Urinary 6-hydroxymelatunin Histograms from the same three patients are given in Fig. 1. Table I gives the daily excretion pattern of conjugated 6-hydroxymelatonin. TABLE Plasma Patient
A B C D E F G H I J K L M N 0 : R S T U V
I melatonin
levels and urinary
6-hydroxymelatonin
excretion
Plasma melatonin
Urinary
(pg/mI)
(81/8
a
93(24X@), 79, 54 52(23:00), 167.88 5.8(19:00), 8.6, 3.1 12.8(24:00), 16.6, 15.2 10.6(24:00), 60.9, 14.4 16.4(01:00), 19.3, 7.0 35.3(22:00), 58.2, 37 34.3(24X@), 35, 35.4 36.2(22:00), 47.9, 54.0 46.1(01:00), 53.9.40.9 36.9(23:00), 58, 73.2 24(24X@), 9, 20 35(01 :OO), 50,47 39(23:00), 32,25 44(24:00), 97.5, 27.6 62(03:00), 46, 14 29(01:00), 45.0, 36 33(01 :OO), 69, 60 7qO2:00), 52,37 68(02:00), 66.58 25(24X@), 50, 33 62(24:00), 101,66
patterns
for 22 patients
6-hydroxymelatonin h) b
0.7, 18.3, 11.5 1.0, 14.7, 3.7
0, 0.0 0.0.8, 0.7 0, 6.1, 5.9 0.0.4, 1.3 0, 4.2, 2.6 0.0.89, 0.45 0, 0, 5.6 0, 0, 9.6 0, 3.7,1.2 0.1, 1.0.0.5 0.1, 1.6, 1.8 5.3, 8.9, 0.88 0, 6.9, 1.3 0, 6.7, 3.0 0, 0, 8.1 0, 0, 14.8 0.1, 1.8, 9
0.1,0.1,11 0, 1.4, 7.5 0, 12, 6.2
’ The three highest contiguous values are given, with the clock time of the first sample. b Eight-hour intervals are given in the approximate sequence 14:00-22:00, 22:00-06:OO. Actual collection times varied f 2 h depending upon the patient.
06:00-14:O0.
1 ,
,b
0
URINARY
6-HYDROXYMELATONIN
2
3; @g
Fig. 2. Unweighted linear regression analysis of integrated 9-h plasma melatonin values (pg) vs. total 24-h excretion of conjugated 6-hydroxymelatonin (pg). The dashed lines near the fitted line are the 95% confidence limits for the fitted line; the outer pair of dashed lines are the 95% confidence limits for a single observation.
Correlation of plasma melatonin and urinary Ghydroxymelatonin Figure 2 shows the correlation between the summed three highest melatonin values for each patient vs. the total daily excretion of conjugated 6-hydroxymelatonin. Least squares analysis gave an absolute multiple correlation coefficient of 0.762 with a probability that this correlation came from a random population of 0.00004. The BRIGHT statistical algorithms were used for these analyses [15]. Discussion The correlation between summed (9 h) plasma melatonin levels and urinary melatonin metabolite levels is good and should be useful for future clinical studies. It demonstrates that subjects having normal liver function with high plasma levels of melatonin excrete relatively high amounts of 6-hydroxymelatonin, and that subjects without a detectable plasma melatonin rhythm excrete low or undetectable levels of urinary metabolite. For subjects with normal liver function this correlation resolves questions regarding major contributions from differences in metabolic turnover, e.g. low plasma levels are not primarily the result of more rapid metabolism and clearance, but are the result of lowered pineal production of melatonin. That the correlation is not higher than 0.76 may be due to effects of metabolic clearance as well as experimental limitations. As seen in Fig. 1 in a comparison of patients A and B, the method for determining the plasma melatonin integral is subject to some error due to the difficulty in sampling a changing function at fixed time intervals. Sampling the urine at only three timps places a high degree of reliance upon each value determined, which must be multiplied by a large urine volume factor. The facile decomposition of 6-hydroxymelatonin conjugates in urine requires
225
immediate refrigeration of each collected specimen; lack of compliance will add to increased variability in reported values. While both of the assays employed in this study have their inherent errors and difficulties, this plasma hormone-urinary metabolite correlation serves to validate future clinical studies using either measure, assuming that future assays are at least at the present level of specificity and accuracy. References 1 Branchey L, Weinberg U, Branchey M, Linkowski P. Mendlewicz J. Simultaneous study of 24-hour melatonin and cortisol secretion in depressed patients. Neuropsychobiology 1982; 8: 225-232. 2 Lewy AJ, Kern H, Rosenthal NE, Wehr TA. Artificial light treatment of a manic-depressive patient with seasonal mood cycle. Am J Psycho1 1982; 139: 1496-1498. 3 Tamarkin L, Danforth D, Lichter A, DeMoss E, Cohen M, Chabner B, Lippman M. Decreased nocturnal plasma melatonin peak in patient with estrogen receptor positive breast cancer. Science 1982; 216: 1003-1005. 4 Penny R. Melatonin excretion in normal males and females: increase during puberty. Metabolism 1982; 31: 816-823. 5 Tamarkin L, Abastillas P, Chen H-C. McNemar A, Sidbury J. The daily profile of plasma melatonin in obese and Prader-Willi syndrome children. J Clin Endocrinol Metab 1982; 55: 491-495. 6 Waldhauser F, Frisch H, Waldhauser M. Weiszenbacher G, Zeithlhuber U, Wurtman RJ. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet 1984: I: 362-365. 7 Thompson C, Checkley SA, Corn T, Franey C, Arendt J. Down-regulation at pineal b-adrenoceptor in depressed patients treated with desipramine. Lancet 1983; I: 1101. 8 Tetsuo M, Markey SP, Colburn RW, Kopin IJ. Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromatography - negative chemical ionization mass spectrometry. Anal B&hem 1981; 110: 208-215. 9 Lynch HJ, Wurtman RJ. Melatonin levels as they relate to reproductive physiology. In: Reiter RJ, ed. The pineal gland, Vol. 2. Boca Raton: CRS Press, 1981: 103-123. 10 Fellenberg AJ. Phillipou G, Seamark RF. Specific quantitation of urinary 6-hydroxymelatonin sulphate by gas chromatography mass spectrometry. Biomed Mass Spectrom 1980; 7: 84-87. 11 Fellenberg AJ, Phillipou G, Seamark RF. Urinary 6-sulphatoxy melatonin excretion during the human menstrual cycle. Clin Endocrinol 1982; 17: 71-75. 12 Tetsuo M, Poth M, Markey SP. Melatonin metabolite excretion during childhood and puberty. J Clin Endocrinol Metab 1982; 55: 311-313. 13 Tetsuo M, Polinsky RJ, Markey SP, Kopin IJ. Urinary 6-hydroxymelatonin excretion in patients with orthostatic hypotension. J Clin Endocrinol Metab 1981; 53: 607-610. 14 Golden RN, Markey SP, Potter WZ. A new marker for noradrenergic function in man? Abstr. 137th Am. Psych. Assoc. Los Angeles, CA, 1984. 15 Cole BR. Rodbard D, Munson PJ. BRIGHT Stat Pack. Bethesda: Dept. Health and Human Resources. 1983.
Clinica Chimica Acta, 150 (1985) 221-225 Elsevier
CCA 03242
Short
communication
The correlation between human plasma melatonin levels and urinary 6-hydroxymelatonin excretion Sanford
P. Markey
a,*, Sadayoshi Higa a, Ming Shih a, David and Lawrence Tamarkin b
N. Danforth’
ULaboratory of Clinical Science and h Clinical Psychobiology Branch, National Institute of Mental Health, ’ SurgeryBranch, National Cancer Institute, National Institutes o/Health, Bethesda, MD 20205 (USA) (Received
January
Key words: Melatonin;
14th, 1985; revision April 29th, 1985)
6 - Hvdroxymelatonin;
Human plasma; Human urine
Summary A significant correlation (0.76) has been found between nighttime peak .plasma melatonin levels and the 24-h urinary excretion totals for conjugated 6-hydroxymelatonin for a group of 22 women. This study validates the comparison of plasma levels of the hormone or urinary levels of its metabolite to assess pineal gland production of melatonin in humans.
Introduction Plasma levels of melatonin have been useful measures of pineal activity in clinical studies in psychiatry [1,2], cancer research [3], endocrinology [4-61 and pharmacology [7] using radioimmunoassay or gas chromatographic-mass spectrometric (gc-ms) assay procedures [9]. Plasma melatonin levels are low in the day and increase at night to a peak between 24:00 and 03:00, requiring the collection of multiple blood samples for clinical studies. The major urinary melatonin metabolites, glucuronide and sulfate conjugates of 6-hydroxymelatonin, have been quantified by gc-ms procedures (9,101 and reported in endocrine [11,12], hypotension [13] and drug function [14] studies. Multiple plasma level measurements are not practical for many clinical studies, especially when a measure of the amplitude of melatonin production is desired. For this reason, we undertook to determine the correlation between plasma melatonin and urinary 6-hydroxymelatonin for a single group of patients and normal volunteers.
* Correspondence
0009-8981/85/$03.30
to: Dr. S.P. Markey,
Bldg. lo-3N-325,
0 1985 Elsevier Science Publishers
NIH. Bethesda,
B.V. (Biomedical
MD 20205, USA.
Division)
222
Methods Experimental subjects Subjects for this study included 18 healthy female volunteers ranging in age from 18-68 yr (mean 36.6 + 5.6) and 4 patients ranging in age from 29-64 yr (mean 44 + 10.4) with clinical stage I or II breast cancer. At the time of study, the normal subjects had no serious illness, were of normal weight, and had a normal complete blood count (CBC), liver and renal function studies. All the patients presented with untreated primary breast cancer, were of normal weight, and had no evidence of other malignancies or major illness. Prior to and during this study the patients were drug-free and of normal nutritional status. All subjects were admitted to the Clinical Center and were requested to conform to normal ward routines with no dietary or activity restrictions. Serial blood samples were drawn from each subject through an intravenous Heparin-Lock needle and urine was collected on this same day. Blood samples were drawn every 3 h for 24 h. Urine samples were pooled from three blocks of time; 14:00-22:00, 22:00-06:00, 06:00-14:O0. Actual collection times varied somewhat depending upon the patient. Melatonin radioimmunoassay Plasma melatonin was analyzed by a radioimmunoassay which has been validated for use in human plasma [5]. Prior to the radioimmunoassay each plasma sample is extracted with chloroform, reconstituted in 0.1% gelatin-phosphate buffered saline, and extracted a second time with petroleum ether. The combined efficiency for both extractions is 85% and the resultant data are not corrected for the 15% loss of melatonin. The limit of sensitivity of the assay is 0.5 pg/ tube and the intra- and interassay coefficients of variation are 10 and 20%, respectively. 6-Hydroxymelatonin assay The assay as previously described by Tetsuo et al [8] was utilized in these studies. Briefly, deuterated 6-hydroxymelatonin sulfate is added as an internal standard to 3 ml of urine, which is enzymatically hydrolyzed and extracted with dichloromethane. The extracts are subjected to a two-step derivatization procedure and the resulting product purified by silica gel chromatography. Finally, the column elute is concentrated and injected into a gas chromatograph-mass spectrometer (gc-ms) operating in a negative chemical ionization mode. Quantification is based upon the ratio of deuterated internal standard to endogenous derivatized 6-hydroxymelatonin, corrected by a standard curve prepared from authentic free 6-hydroxymelatonin and conjugated deuterated internal standard. Assay to assay variation is corrected using a frozen pooled urine standard measured with each sample batch. As previously reported, intra- and interassay variability were < 10% [8]. Results Plasma melatonin Plots of the data from three of the patients are given in Fig. 1. Table I shows the pattern of the three highest melatonin values for each of the 22 patients measured.
Fig. 1. Plasma levels of melatonin (solid circles) are plotted vs. clock time for patients A, B, and C (Table I). On the same time axis the urinary excretion of 6-hydroxymelatonin is given in cross-hatched bars, with units of measurement indicated to the right.
Urinary 6-hydroxymelatunin Histograms from the same three patients are given in Fig. 1. Table I gives the daily excretion pattern of conjugated 6-hydroxymelatonin. TABLE Plasma Patient
A B C D E F G H I J K L M N 0 : R S T U V
I melatonin
levels and urinary
6-hydroxymelatonin
excretion
Plasma melatonin
Urinary
(pg/mI)
(81/8
a
93(24X@), 79, 54 52(23:00), 167.88 5.8(19:00), 8.6, 3.1 12.8(24:00), 16.6, 15.2 10.6(24:00), 60.9, 14.4 16.4(01:00), 19.3, 7.0 35.3(22:00), 58.2, 37 34.3(24X@), 35, 35.4 36.2(22:00), 47.9, 54.0 46.1(01:00), 53.9.40.9 36.9(23:00), 58, 73.2 24(24X@), 9, 20 35(01 :OO), 50,47 39(23:00), 32,25 44(24:00), 97.5, 27.6 62(03:00), 46, 14 29(01:00), 45.0, 36 33(01 :OO), 69, 60 7qO2:00), 52,37 68(02:00), 66.58 25(24X@), 50, 33 62(24:00), 101,66
patterns
for 22 patients
6-hydroxymelatonin h) b
0.7, 18.3, 11.5 1.0, 14.7, 3.7
0, 0.0 0.0.8, 0.7 0, 6.1, 5.9 0.0.4, 1.3 0, 4.2, 2.6 0.0.89, 0.45 0, 0, 5.6 0, 0, 9.6 0, 3.7,1.2 0.1, 1.0.0.5 0.1, 1.6, 1.8 5.3, 8.9, 0.88 0, 6.9, 1.3 0, 6.7, 3.0 0, 0, 8.1 0, 0, 14.8 0.1, 1.8, 9
0.1,0.1,11 0, 1.4, 7.5 0, 12, 6.2
’ The three highest contiguous values are given, with the clock time of the first sample. b Eight-hour intervals are given in the approximate sequence 14:00-22:00, 22:00-06:OO. Actual collection times varied f 2 h depending upon the patient.
06:00-14:O0.
1 ,
,b
0
URINARY
6-HYDROXYMELATONIN
2
3; @g
Fig. 2. Unweighted linear regression analysis of integrated 9-h plasma melatonin values (pg) vs. total 24-h excretion of conjugated 6-hydroxymelatonin (pg). The dashed lines near the fitted line are the 95% confidence limits for the fitted line; the outer pair of dashed lines are the 95% confidence limits for a single observation.
Correlation of plasma melatonin and urinary Ghydroxymelatonin Figure 2 shows the correlation between the summed three highest melatonin values for each patient vs. the total daily excretion of conjugated 6-hydroxymelatonin. Least squares analysis gave an absolute multiple correlation coefficient of 0.762 with a probability that this correlation came from a random population of 0.00004. The BRIGHT statistical algorithms were used for these analyses [15]. Discussion The correlation between summed (9 h) plasma melatonin levels and urinary melatonin metabolite levels is good and should be useful for future clinical studies. It demonstrates that subjects having normal liver function with high plasma levels of melatonin excrete relatively high amounts of 6-hydroxymelatonin, and that subjects without a detectable plasma melatonin rhythm excrete low or undetectable levels of urinary metabolite. For subjects with normal liver function this correlation resolves questions regarding major contributions from differences in metabolic turnover, e.g. low plasma levels are not primarily the result of more rapid metabolism and clearance, but are the result of lowered pineal production of melatonin. That the correlation is not higher than 0.76 may be due to effects of metabolic clearance as well as experimental limitations. As seen in Fig. 1 in a comparison of patients A and B, the method for determining the plasma melatonin integral is subject to some error due to the difficulty in sampling a changing function at fixed time intervals. Sampling the urine at only three timps places a high degree of reliance upon each value determined, which must be multiplied by a large urine volume factor. The facile decomposition of 6-hydroxymelatonin conjugates in urine requires
225
immediate refrigeration of each collected specimen; lack of compliance will add to increased variability in reported values. While both of the assays employed in this study have their inherent errors and difficulties, this plasma hormone-urinary metabolite correlation serves to validate future clinical studies using either measure, assuming that future assays are at least at the present level of specificity and accuracy. References 1 Branchey L, Weinberg U, Branchey M, Linkowski P. Mendlewicz J. Simultaneous study of 24-hour melatonin and cortisol secretion in depressed patients. Neuropsychobiology 1982; 8: 225-232. 2 Lewy AJ, Kern H, Rosenthal NE, Wehr TA. Artificial light treatment of a manic-depressive patient with seasonal mood cycle. Am J Psycho1 1982; 139: 1496-1498. 3 Tamarkin L, Danforth D, Lichter A, DeMoss E, Cohen M, Chabner B, Lippman M. Decreased nocturnal plasma melatonin peak in patient with estrogen receptor positive breast cancer. Science 1982; 216: 1003-1005. 4 Penny R. Melatonin excretion in normal males and females: increase during puberty. Metabolism 1982; 31: 816-823. 5 Tamarkin L, Abastillas P, Chen H-C. McNemar A, Sidbury J. The daily profile of plasma melatonin in obese and Prader-Willi syndrome children. J Clin Endocrinol Metab 1982; 55: 491-495. 6 Waldhauser F, Frisch H, Waldhauser M. Weiszenbacher G, Zeithlhuber U, Wurtman RJ. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet 1984: I: 362-365. 7 Thompson C, Checkley SA, Corn T, Franey C, Arendt J. Down-regulation at pineal b-adrenoceptor in depressed patients treated with desipramine. Lancet 1983; I: 1101. 8 Tetsuo M, Markey SP, Colburn RW, Kopin IJ. Quantitative analysis of 6-hydroxymelatonin in human urine by gas chromatography - negative chemical ionization mass spectrometry. Anal B&hem 1981; 110: 208-215. 9 Lynch HJ, Wurtman RJ. Melatonin levels as they relate to reproductive physiology. In: Reiter RJ, ed. The pineal gland, Vol. 2. Boca Raton: CRS Press, 1981: 103-123. 10 Fellenberg AJ. Phillipou G, Seamark RF. Specific quantitation of urinary 6-hydroxymelatonin sulphate by gas chromatography mass spectrometry. Biomed Mass Spectrom 1980; 7: 84-87. 11 Fellenberg AJ, Phillipou G, Seamark RF. Urinary 6-sulphatoxy melatonin excretion during the human menstrual cycle. Clin Endocrinol 1982; 17: 71-75. 12 Tetsuo M, Poth M, Markey SP. Melatonin metabolite excretion during childhood and puberty. J Clin Endocrinol Metab 1982; 55: 311-313. 13 Tetsuo M, Polinsky RJ, Markey SP, Kopin IJ. Urinary 6-hydroxymelatonin excretion in patients with orthostatic hypotension. J Clin Endocrinol Metab 1981; 53: 607-610. 14 Golden RN, Markey SP, Potter WZ. A new marker for noradrenergic function in man? Abstr. 137th Am. Psych. Assoc. Los Angeles, CA, 1984. 15 Cole BR. Rodbard D, Munson PJ. BRIGHT Stat Pack. Bethesda: Dept. Health and Human Resources. 1983.