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Mass spectral identification of melatonin in blood
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
MASS SPECTRAL IDENTIFICATION OF MELATONIN IN BLOOD Russell W. Pelham, Charles L. Ralph, and lain M. Campbell Departments of Biology and Biochemistry University of Pittsburgh, Pittsburgh, Pa. 15213
R e c e i v e d D e c e m b e r 22, 1971 SUMMARY Although t h e p i n e a l body i s g e n e r a l l y c o n s i d e r e d t o be an e n d o c r i n e organ, and melatonin is thought by many to be its secretion, identification of this substance in the circulation has never been accomplished. In chicken blood we have identified melatonin using mass spectroscopy and also have shown that melatonin detected by the Ran a pipiens bioassay in serum extracts disappears following pinealectomy. These data support the notion that melatonin is released into the circulation and that it may be a pineal hormone. Melatonin (N-acetyl-5-methoxytryptamine) is usually considered to be a hormone of the pineal body (i). The evidence supporting this concept consists of the observation that the pineal makes melatonin (2)9 metabolizes very little of it (3), and shows diurnal variations in synthesizing enzyme activities (4,5) and melatonin levels (6). Therefore, secretion of melatonin appears a likely explanation for the diurnal variations in its content.
Furthermore,
melatonin has been detected in nerve (7) and in urine (8), suggesting that it arrived at these sites via the circulation from the pineal.
However, since
these latter experiments did not demonstrate the disappearance of melatonin in nerve and urine following pinealectomy, the contention that the melatonin was formed and secreted by the pineal rests on circumstantial evidence. We have identified melatonin in the blood and obtained evidence which suggests that the pineal is responsible for secreting this substance into the circulation.
We chose chickens as our experimental animal as they have high
pineal melatonin levels (6), a very active enzyme that is thought to be responsible for melatonin synthesis (9), and a large easily sampled blood volume.
1236 Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND METHODS Blood was collected from birds 8-12 weeks of age by heart puncture (I0) and allowed to clot.
The serum was harvested by centrifuging the clot
at 3000 X i0 g-minutes and decanting the supernate.
Serum was adjusted to
pH I0 with i0 N NaOH, saturated with NaCI, extracted into two volumes of choloroform which was washed with ~ volume of distilled water and then freezedried.
The residue was taken up into 3 ml of borate buffer (pH i0; 0.25 M)
with agitation and sonification.
After centrifuging at 48,000 X 30 g-minutes
at -5°C and discarding the pellet and floating material, the buffer was extracted as described above and the chloroform evaporated under nitrogen. The residue was teken up in 100~ul of ethanol and chromatographed (Analtech silca gel sheets; 2 5 0 ~
thick) with chloroform-methanol-glacial acetic acid
(93-7-1) in one dimension, followed by ethyl acetate in another dimension (Ii). The fluorescent spot with the same mobility as authentic melatonin was eluted into ethanol, which was centrifuged to remove the silica gel and evaporated to a volume of i 0 ~ I . Mass spectroscopy was performed with an LKB 9000 gas chromatograph-mass spectroscope (GC-MS) at 70 eV.
A 6-foot column packed with SE 30 (3%) was
maintained at 210 ° with flash heater and separator at 260 ° and 250 ° respectively. Gas flow (He) was 40 ca/minute.
Under these conditions melatonin (Regis)
standards had a retention time of 8 minutes. Pinealectomy was performed by the method of Shellabarger (12) on oneday-old chicks.
At autopsy, no scar or pineal tissue was observed under a
25 X dissecting microscope. Blood from pinealectomized animals was assayed for melatonin content by the method developed here (13), which utilizes the nucleocentria aggregation of melanin granules in melanophores of larval Rana pipiens effected by melatonin.
This bioassay can detect as little as I00 picograms/ml of this
substance.
Samples of blood were extracted by t~ ~l~ocedure noted above,
and the chloroform evapo~ted and taken up in 2 ml of deionized water for
1237
Vol. 46, No. 3, 1972
bioassay.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Some chloroform extracts were chromatographed on the systems
noted above. RESULTS Mass Spectroscopy A sample of serum (1300 ml), collected from hirds on alternating days between 1600 and 1900 hours was extracted and injected into the GC-MS system. A substance with the same Rf as authentic melatonin and with the same mass spectrum reported for melatonin (14) was detected.
The spectra for serum
extracts and for melatonin are presented on Figure i. A SERUM EXTRACT
I_
z u,i
160 I
6
173
160 228
I
40
Fig. I
8to
,
120
I.160
200
t
IT
240
Mass spectra of serum extract (A) and melatonin standard (B).
As can be seen, the serum extract has the spectrum characteristic of melatonin, namely, peaks at 232, 173, 160 and 145 m/e.
Furthermore, the
relative intensities of the four peaks is the same in blood extracts as in standards.
The standard, in addition to having the 232 m/e parent peak
corresponding to melatonin, also possessed peaks at 228 and 186 m/e. Prior to injection into the GC-MS, an aliquot of the extract was run in the bioassay, and the extract was calculated to contain 120 ng of melatonin. This agrees with the amount determined by comparing the total ion current of extract with that of standards.
1238
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1
Bioassay Activity of Serum Extracts (ng equivalents of melatonin / ml serum J S.E.)
Time
Shams
Pinealectomized
1030
ND* (4)**
ND
2230
0.07|J-0°007 (4)
ND (4)
(4)
Not Detectable, i.e., levels in serum were less than 0.01 ng equivalents/ml serum, the lower limit of detectability by the bioassay **
Number of birds per group
Effect of time of day and pinealectomy on serum me!atonin Serum (15 ml) was collected at two times of day (1030 and 2230 hours) from pinealectomized and sham-operated animals.
Using the bioassay on serum
extracts, melatonin could be detected at 2230 hours in shams, while none was detected at 1030 in shams, or at either time in pinealectomized animals: This data is summarized in Table i.
The concentration of melatonin in the
serum at 2230 hours was 0.071 + 0.007 ng/ml. When serum extracts from intact birds was subjected to two-dimensional thin layer chromatography, bioassay activity was detected only in that Rf which corresponded to authentic melatonin.
DISCUSSION These results offer the first direct evidence that melatonin is in the circulation, and they provide substantial support for the notion that the pineal secretes this substance into the blood.
To our knowledge, mass
spectroscopy has never been used to detect melatonin in any biological material.
However, this method has been used to identify derivatives of
1239
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
melatonin after gas chromatography.
This GC method has been used to quantify
melatonin content in pineal, but mass spectroscopy of pineal melatonin derivatives was not reported. (15) The mass spectral data conclusively establish that the compound in blood is melatonin.
The spectrum is identical to that in the literature (14),
and resembles that obtained by us with standards.
Since the total ion
current associated with melatonin in the blood extract is similar to that obtained from a standard with the same bioassay activity, all the bioassay activity in serum extracts can be accounted for by melatonin. The presence of an unidentified contaminant with peaks at 228 and 186 m/e in the standard melatonin and the absence of melatonin in blood extracts at one time of day and its presence at another time, suggest that the melatonin in serum extracts cannot be due to contamination from standards. The ri6e in melatonin content at 2230
hours (Table i) indicates that
there may be a diurnal rhythm of melatonin titers.
The reality of such
cannot be proven by two sampling times, but a rhythm in melatonin levels in blood would be anticipated considering the rhythmic changes in melatonin content (6) and hydroxyindole-O-methyltransferase activity (9) that have been r~ported in chicken pineal. The effect of pinealectomy on abolishing the evening rise of melatonin in blood supports the contention that the pineal secretes melatonin.
However,
this is not conclusively proven, as it is conceivable that the pineal exerts a t r o p h i e influence on some other organ that actually nmkes and secretes melatonin.
This seems unlikely, although a non-pineal site where melatonin
may be made is known (16).
Considering the magnitude of the melatonin
rhythm in the pineal, it would be surprising if a large portion of the mela tonin synthesized in the pineal was not actually secreted. The exact function of circulating melatonin is unknown, but whatever that might be may be more easily revealed now that melatonin can be measured in serum and the dynamics of its secretion determined.
1240
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgements RWP is supported as an NIMH Predoctoral Fellow by MH--III4 to the Psychology Department.
This research is supported in part by a grant to
CLR (NIH NS 08554) and to the Biochemistry Department for development of mass spectroscopic techniques (NIH RR 00273).
We wish to thank John Naworal
for technical assistance in mass spectroscopy, and Paula Pelham and Paul Salve for preparation of the figures.
REFERENCES I) 2) 3) 4) 5) 6) 7) 8) 9) i0) II) 12) 13) 14) 15) 16)
Wurtman, R. J., Axelrod, J., and Kelly, D. E., The Pineal, Academic Press, N. Y., p~16 (1968). Wurtman, R. J., Larin, F., Axelrod, J., Shein~ H. M., and Rosasco, K., Nature, 217:953 (1968). Wurtman, R. J., Axelrod, J., and Potter, L- T., J. Pharm. Exp. Ther., 143, 314 (1964). Axelrod, J., Wurtman, R. J., and Snyder, S. H., J. Biol. Chem. 240:949 (1965). Klein, D. C. and Weller, J. C., Science 169:1093 (1970). Lynch, H. J., Life Sci. (If) 10:791 (1971). Lerner, A. B., Case, J. D., Mori, W., and Wright, M. R., Nature, 183:1821 (1959). Barchas, J. D., and Lerner, A. B., J. Neurochem. 11:489 (1959). Pelham, R. W., and Ralph, C. L., Life Sei (II) 11:51 (1972). Armaghanj V., Pavlech, H. M. and Olsen, N. O., Science 117:156 (1953). Klein, D. C. and Notides, A., Anal. Biochem. 31:480 (1969). Shellabarger, C. J., and Breneman, W. R., Indiana Acad Sci, 59:299 (1950). Ralph, C. L., and Lynch, H. J., Gen. Comp.Endocr. i~5:334 (1970). Jamleson, N. D. and Hutzinger, 0., Anal Biochem 31:480 (1969). Degen, P. H. and Barchas, J. D., Proc West Pharm Soc, 13:34 (1970). Quay, W. B., Life Sei ~:983 (1965).
1241
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
MASS SPECTRAL IDENTIFICATION OF MELATONIN IN BLOOD Russell W. Pelham, Charles L. Ralph, and lain M. Campbell Departments of Biology and Biochemistry University of Pittsburgh, Pittsburgh, Pa. 15213
R e c e i v e d D e c e m b e r 22, 1971 SUMMARY Although t h e p i n e a l body i s g e n e r a l l y c o n s i d e r e d t o be an e n d o c r i n e organ, and melatonin is thought by many to be its secretion, identification of this substance in the circulation has never been accomplished. In chicken blood we have identified melatonin using mass spectroscopy and also have shown that melatonin detected by the Ran a pipiens bioassay in serum extracts disappears following pinealectomy. These data support the notion that melatonin is released into the circulation and that it may be a pineal hormone. Melatonin (N-acetyl-5-methoxytryptamine) is usually considered to be a hormone of the pineal body (i). The evidence supporting this concept consists of the observation that the pineal makes melatonin (2)9 metabolizes very little of it (3), and shows diurnal variations in synthesizing enzyme activities (4,5) and melatonin levels (6). Therefore, secretion of melatonin appears a likely explanation for the diurnal variations in its content.
Furthermore,
melatonin has been detected in nerve (7) and in urine (8), suggesting that it arrived at these sites via the circulation from the pineal.
However, since
these latter experiments did not demonstrate the disappearance of melatonin in nerve and urine following pinealectomy, the contention that the melatonin was formed and secreted by the pineal rests on circumstantial evidence. We have identified melatonin in the blood and obtained evidence which suggests that the pineal is responsible for secreting this substance into the circulation.
We chose chickens as our experimental animal as they have high
pineal melatonin levels (6), a very active enzyme that is thought to be responsible for melatonin synthesis (9), and a large easily sampled blood volume.
1236 Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND METHODS Blood was collected from birds 8-12 weeks of age by heart puncture (I0) and allowed to clot.
The serum was harvested by centrifuging the clot
at 3000 X i0 g-minutes and decanting the supernate.
Serum was adjusted to
pH I0 with i0 N NaOH, saturated with NaCI, extracted into two volumes of choloroform which was washed with ~ volume of distilled water and then freezedried.
The residue was taken up into 3 ml of borate buffer (pH i0; 0.25 M)
with agitation and sonification.
After centrifuging at 48,000 X 30 g-minutes
at -5°C and discarding the pellet and floating material, the buffer was extracted as described above and the chloroform evaporated under nitrogen. The residue was teken up in 100~ul of ethanol and chromatographed (Analtech silca gel sheets; 2 5 0 ~
thick) with chloroform-methanol-glacial acetic acid
(93-7-1) in one dimension, followed by ethyl acetate in another dimension (Ii). The fluorescent spot with the same mobility as authentic melatonin was eluted into ethanol, which was centrifuged to remove the silica gel and evaporated to a volume of i 0 ~ I . Mass spectroscopy was performed with an LKB 9000 gas chromatograph-mass spectroscope (GC-MS) at 70 eV.
A 6-foot column packed with SE 30 (3%) was
maintained at 210 ° with flash heater and separator at 260 ° and 250 ° respectively. Gas flow (He) was 40 ca/minute.
Under these conditions melatonin (Regis)
standards had a retention time of 8 minutes. Pinealectomy was performed by the method of Shellabarger (12) on oneday-old chicks.
At autopsy, no scar or pineal tissue was observed under a
25 X dissecting microscope. Blood from pinealectomized animals was assayed for melatonin content by the method developed here (13), which utilizes the nucleocentria aggregation of melanin granules in melanophores of larval Rana pipiens effected by melatonin.
This bioassay can detect as little as I00 picograms/ml of this
substance.
Samples of blood were extracted by t~ ~l~ocedure noted above,
and the chloroform evapo~ted and taken up in 2 ml of deionized water for
1237
Vol. 46, No. 3, 1972
bioassay.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Some chloroform extracts were chromatographed on the systems
noted above. RESULTS Mass Spectroscopy A sample of serum (1300 ml), collected from hirds on alternating days between 1600 and 1900 hours was extracted and injected into the GC-MS system. A substance with the same Rf as authentic melatonin and with the same mass spectrum reported for melatonin (14) was detected.
The spectra for serum
extracts and for melatonin are presented on Figure i. A SERUM EXTRACT
I_
z u,i
160 I
6
173
160 228
I
40
Fig. I
8to
,
120
I.160
200
t
IT
240
Mass spectra of serum extract (A) and melatonin standard (B).
As can be seen, the serum extract has the spectrum characteristic of melatonin, namely, peaks at 232, 173, 160 and 145 m/e.
Furthermore, the
relative intensities of the four peaks is the same in blood extracts as in standards.
The standard, in addition to having the 232 m/e parent peak
corresponding to melatonin, also possessed peaks at 228 and 186 m/e. Prior to injection into the GC-MS, an aliquot of the extract was run in the bioassay, and the extract was calculated to contain 120 ng of melatonin. This agrees with the amount determined by comparing the total ion current of extract with that of standards.
1238
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1
Bioassay Activity of Serum Extracts (ng equivalents of melatonin / ml serum J S.E.)
Time
Shams
Pinealectomized
1030
ND* (4)**
ND
2230
0.07|J-0°007 (4)
ND (4)
(4)
Not Detectable, i.e., levels in serum were less than 0.01 ng equivalents/ml serum, the lower limit of detectability by the bioassay **
Number of birds per group
Effect of time of day and pinealectomy on serum me!atonin Serum (15 ml) was collected at two times of day (1030 and 2230 hours) from pinealectomized and sham-operated animals.
Using the bioassay on serum
extracts, melatonin could be detected at 2230 hours in shams, while none was detected at 1030 in shams, or at either time in pinealectomized animals: This data is summarized in Table i.
The concentration of melatonin in the
serum at 2230 hours was 0.071 + 0.007 ng/ml. When serum extracts from intact birds was subjected to two-dimensional thin layer chromatography, bioassay activity was detected only in that Rf which corresponded to authentic melatonin.
DISCUSSION These results offer the first direct evidence that melatonin is in the circulation, and they provide substantial support for the notion that the pineal secretes this substance into the blood.
To our knowledge, mass
spectroscopy has never been used to detect melatonin in any biological material.
However, this method has been used to identify derivatives of
1239
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
melatonin after gas chromatography.
This GC method has been used to quantify
melatonin content in pineal, but mass spectroscopy of pineal melatonin derivatives was not reported. (15) The mass spectral data conclusively establish that the compound in blood is melatonin.
The spectrum is identical to that in the literature (14),
and resembles that obtained by us with standards.
Since the total ion
current associated with melatonin in the blood extract is similar to that obtained from a standard with the same bioassay activity, all the bioassay activity in serum extracts can be accounted for by melatonin. The presence of an unidentified contaminant with peaks at 228 and 186 m/e in the standard melatonin and the absence of melatonin in blood extracts at one time of day and its presence at another time, suggest that the melatonin in serum extracts cannot be due to contamination from standards. The ri6e in melatonin content at 2230
hours (Table i) indicates that
there may be a diurnal rhythm of melatonin titers.
The reality of such
cannot be proven by two sampling times, but a rhythm in melatonin levels in blood would be anticipated considering the rhythmic changes in melatonin content (6) and hydroxyindole-O-methyltransferase activity (9) that have been r~ported in chicken pineal. The effect of pinealectomy on abolishing the evening rise of melatonin in blood supports the contention that the pineal secretes melatonin.
However,
this is not conclusively proven, as it is conceivable that the pineal exerts a t r o p h i e influence on some other organ that actually nmkes and secretes melatonin.
This seems unlikely, although a non-pineal site where melatonin
may be made is known (16).
Considering the magnitude of the melatonin
rhythm in the pineal, it would be surprising if a large portion of the mela tonin synthesized in the pineal was not actually secreted. The exact function of circulating melatonin is unknown, but whatever that might be may be more easily revealed now that melatonin can be measured in serum and the dynamics of its secretion determined.
1240
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgements RWP is supported as an NIMH Predoctoral Fellow by MH--III4 to the Psychology Department.
This research is supported in part by a grant to
CLR (NIH NS 08554) and to the Biochemistry Department for development of mass spectroscopic techniques (NIH RR 00273).
We wish to thank John Naworal
for technical assistance in mass spectroscopy, and Paula Pelham and Paul Salve for preparation of the figures.
REFERENCES I) 2) 3) 4) 5) 6) 7) 8) 9) i0) II) 12) 13) 14) 15) 16)
Wurtman, R. J., Axelrod, J., and Kelly, D. E., The Pineal, Academic Press, N. Y., p~16 (1968). Wurtman, R. J., Larin, F., Axelrod, J., Shein~ H. M., and Rosasco, K., Nature, 217:953 (1968). Wurtman, R. J., Axelrod, J., and Potter, L- T., J. Pharm. Exp. Ther., 143, 314 (1964). Axelrod, J., Wurtman, R. J., and Snyder, S. H., J. Biol. Chem. 240:949 (1965). Klein, D. C. and Weller, J. C., Science 169:1093 (1970). Lynch, H. J., Life Sci. (If) 10:791 (1971). Lerner, A. B., Case, J. D., Mori, W., and Wright, M. R., Nature, 183:1821 (1959). Barchas, J. D., and Lerner, A. B., J. Neurochem. 11:489 (1959). Pelham, R. W., and Ralph, C. L., Life Sei (II) 11:51 (1972). Armaghanj V., Pavlech, H. M. and Olsen, N. O., Science 117:156 (1953). Klein, D. C. and Notides, A., Anal. Biochem. 31:480 (1969). Shellabarger, C. J., and Breneman, W. R., Indiana Acad Sci, 59:299 (1950). Ralph, C. L., and Lynch, H. J., Gen. Comp.Endocr. i~5:334 (1970). Jamleson, N. D. and Hutzinger, 0., Anal Biochem 31:480 (1969). Degen, P. H. and Barchas, J. D., Proc West Pharm Soc, 13:34 (1970). Quay, W. B., Life Sei ~:983 (1965).
1241