PROSTAGLANDINS
DETERMINATION
OF PROSTAGLANDIN
HUMAN CEREBROSPINAL
F2c AND E2 CONTENTS
FLUID BY THE RADIOISOTOPE
IN
DILUTION
METHOD
Y. Aizawa Department
and K. Yamada Tokyo College
of Pharmacology,
Shinjuku-ku,
Kitashinjuku
of Pharmacy,
3-20-1, Tokyo, Japan
ABSTRACT Prostaglandin Fla and E2 contents in human cerebrospinal fluid were determined by the radioisotope dilution method. The mean values of PGF2c and PGE2 of men were 9.8kO.87 ng/ml and 6.51t1.39 ng/ml, respectively. Those of women were 8.321.4 ng/ml and 6.9k1.72 ng/ml, respectively. The correlation between age and PG was significantly with PGE2 of men and with PGF2, of women.
INTRODUCTION Prostaglandin(PG)-like activity in cerebrospinal fluid of experimental animals has been measured by bioassay and shown to increase in the case of febrile animals(1, 2, 3). Recently PGF2, content in human cerebrospinal fluid has been measured by means of radioimmunoassay(4) and the gaschromatography-mass spectrometric technique(5). This paper describes the determination of PGFzc and EP contents in human cerebrospinal fluid by the radioisotope dilution method(6). MATERIALS
AND METHODS
Cerebrospinal fluid obtained by lumbar puncture was immediately frozen and stored at -2OOC until analysis. One ml of the cerebrospinal fluid was transferred into a test tube containing approx. 60,000 dpm 3H-PGF2C1 (7.5 Ci/m mole, New England Nuclear Corporation) and 3H-PGE2 (125 Ci/ m mole, NEN) and was thoroughly mixed. The mixture was washed once with 4 ml of petroleum ether. Four ml of acetonitrile and 5 ml of ether were added to the washed cerebrospinal fluid. The acetonitrile-ether phase was separated and washed once with 3 ml of 0.01 N HCl and once with
12-29-75 JANUARY
1976
VOL.
11 NO. 1
PROSTAGLANDINS
3 ml of water, and then was evaporated to dryness under a reduced pressure. 0.1 ml of "C-methanol reagent [ 1 mg of 14C-methanol (56 mCi/m mole, Radiochemical Center) and 2 ml of cont. HzS04 in 100 ml of ethylacetate] was added to the residue at a low temperature and allowed to stand for 24 hr at -lo?. Fifteen pg of non-radioactive PGF,c methylester and the same amount of PGE, methylester were added as carriers, and the 3H-PG-14C-methylester was extracted with 0.5 ml of chloroform. In order to remove the excess methanol, 0.1 ml of 0.01 % potassium permanganate was added to the extract and kept for 10 min at room temperature. The resulting formic acid was washed out once with 0.1 ml of 0.01 % sodium carbonate, and three times with 0.5 ml of water. The washed extract was evaporated to dryness. The residue was redissolved in a small amount of chloroform, and was applied to thinlayer chromatography with silica gel as an adsorbent and a solvent system consisting of ethylacetate -methanol-water (160:25:100). The PGFP, methylester and PGE2 methylester on the chromatogram were detected with iodine vapor, and then the radioactivity was measured by a liquid scintillation counter. The amount of PGF,, and E, were calculated from the ratio of 14C and 3H radioactivity. RESULTS AND DISCUSSION Fig. 1 shows the standard curve for PGFz, and PGE2. As little as less than 2 ng of PG was determined quantitatively by this method(6). The contents of PGF2, and PGEz in cerebrospinal fluid of men and of women are shown in Table 1 and 2. The mean values of PGF2, and PGEz in cerebrospinal fluid of men were 9.8kO.87 ng/ml and 6.521.39 ng/ml, respectively. The mean values of PGF2c and PGE 2 of women were 8.3k1.4 ng/ ml and 6.921.72 ng/ml, respectively. The correlation between age and PGF 2c1content was not recognized in cerebrospinal fluid of men. However, there was a significant correlation between age and PGEp content in cerebrospinal fluid of men (Fig. 2). On the contrary, in the case of women, the correlation between PGF2a content and age was significantly recognized, but not between PGE2 content and age (Fig. 3). Some of the cerebrospinal fluid used in this experiment was obtained from patients suffering from central nervous system disoder. Therefore further investigation would be necessary about this element. The results obtained by the radioisotope dilution method gave higher values as compared with those by radioimmunoassay(4) and gas-chromatography-mass spectrometric technique (5). But the results of the radioisotope dilution method is theoretically accurate, because the loss in the extraction process is corrected.
44
JANUARY
1976 VOL. 11 NO. 1
PROSTAGLANDINS
3H/1
PGE2
PGF2,
10
0
5
10
Fig. 1.
JANUARY
1976
VOL.
20ng Standard
11 NO, 1
0
5
10
20ng
curve for PGFzc( and PGE2.
45
PROSTAGLANDINS
Table 1 Contents of prostaglandin fluid of men No
FPCl
J32
(ng/ml) -
Clinical
diagnosis
1
25
10.8
2
25
9.9
2.9
3
25
6.0
1.1
4
26
5.8
0.8
5
27
15.2
3.4
6
28
13.5
2.4
7
29
9.0
0.2
8
30
8.8
1.2
9
33
9.2
0.4
10
35
11.7
trace
Lumbar sprain
11
41
10.9
4.8
12
47
8.3
5.5
Intervertebral disc hernia II
13
52
15.3
21.0
14
53
7.7
3.6
15
58
12.8
17.2
16
60
3.4
15.7
17
60
19.0
15.6
18
60
6.0
10.0
19
62
4.9
3.6
20
64
5.6
trace
21
65
6.8
7.9
22
66
6.9
14.4
23
72
18.0
4.3
AVE+S.E. 46
Age
F2e and EZ in cerebrospinal
9.8kO.87
0.7
Intervertebral disc hernia II
Cerebral infarction Hypertension Cerebral tion II
infarc-
Cerebral rhage II
hemor-
Intervertebral disc hernia II Vertigo Dizziness Cerebral tion
infarc-
6.5k1.39 JANUARY
1976 VOL. 11 NO. 1
PROSTAGLANDINS
Table 2 Contents of prostaglandin F2c and El in cerebrospinal fluid of women No
Age
F2a
24
20
5.4
4.6
25
21
1.2
19.5
26
30
1.3
1.2
27
35
9.1
3.8
28
42
2.2
trace
II
29
47
8.6
0.4
11
30
47
6.2
7.0
II
31
48
11.8
4.5
32
50
18.8
5.3
33
55
8.4
5.1
Cerebral infarction Cerebral hemorrhage Braintumor
34
60
10.2
7.6
Headache
35
60
10.2
18.3
36
80
14.2
6.0
E2
(w/ml
AVE+S.E.
JANUARY
8.3k1.4
1976 VOL. 11 NO. 1
Clinical diagnosis
1
Intervertebral disc hernia Hypopituitarism Intervertebral disc hernia II
Cerebral hemorrhage Cerebral infarction
6.9k1.72
47
PROSTAGIANDINS
l l l
l
l
e
l
l
l l l
. l l l l
e
l
e
l
l
l l l l
.
48
l
l
le l
e
le
JANUARY
1976
VOL. 11 NO. 1
PROSTAGLANDINS
al
z l
0 03
l l l
l.
_h
In
0 .
. m
l
JANb ARY 1976 VOL. 11 NO. 1
49
PROSTAGIANDINS
REFERENCES 1) Ramwell, P. W. The action of cerebrospinal fluid on the frog rectus abdominis muscle and other isolated tissue preparations. J. Physiol. 170:21, 1974. 2) Holmes, S. W. The spontaneous release of prostaglandins into the cerebral ventricle of the dog and the effect of external factors on this release. Brit. J. Pharmacol. 37:653, 1970. 3) Feldberg, W. and Gupta, K. P. Pyrogen fever and prostaglandin-like activity in cerebrospinal fluid. J. Physiol. 228:41, 1973. 4) Latorre, E., Patrono, C., Fortuna, A. and GrossiBelloni, D. Role of prostaglandin F2u in human cerebral vasospasm. J. Neurosurg. 41:293, 1974. 5) Wolfe, L. S. and Mamer, 0. A. Measurment of prostaglandin F2u levels in human cerebrospinal fluid in normal and pathological conditions. Prostaglandins. 9:183, 1975. 6) Aizawa, Y. and Yamada, K. Radioisotope dilution method for determination of prostaglandin F2c in rat tissue. Japan. J. Pharmacol. 24:647, 1974.
50
JANUARY
1976 VOL. 11 NO. 1
DETERMINATION
OF PROSTAGLANDIN
HUMAN CEREBROSPINAL
F2c AND E2 CONTENTS
FLUID BY THE RADIOISOTOPE
IN
DILUTION
METHOD
Y. Aizawa Department
and K. Yamada Tokyo College
of Pharmacology,
Shinjuku-ku,
Kitashinjuku
of Pharmacy,
3-20-1, Tokyo, Japan
ABSTRACT Prostaglandin Fla and E2 contents in human cerebrospinal fluid were determined by the radioisotope dilution method. The mean values of PGF2c and PGE2 of men were 9.8kO.87 ng/ml and 6.51t1.39 ng/ml, respectively. Those of women were 8.321.4 ng/ml and 6.9k1.72 ng/ml, respectively. The correlation between age and PG was significantly with PGE2 of men and with PGF2, of women.
INTRODUCTION Prostaglandin(PG)-like activity in cerebrospinal fluid of experimental animals has been measured by bioassay and shown to increase in the case of febrile animals(1, 2, 3). Recently PGF2, content in human cerebrospinal fluid has been measured by means of radioimmunoassay(4) and the gaschromatography-mass spectrometric technique(5). This paper describes the determination of PGFzc and EP contents in human cerebrospinal fluid by the radioisotope dilution method(6). MATERIALS
AND METHODS
Cerebrospinal fluid obtained by lumbar puncture was immediately frozen and stored at -2OOC until analysis. One ml of the cerebrospinal fluid was transferred into a test tube containing approx. 60,000 dpm 3H-PGF2C1 (7.5 Ci/m mole, New England Nuclear Corporation) and 3H-PGE2 (125 Ci/ m mole, NEN) and was thoroughly mixed. The mixture was washed once with 4 ml of petroleum ether. Four ml of acetonitrile and 5 ml of ether were added to the washed cerebrospinal fluid. The acetonitrile-ether phase was separated and washed once with 3 ml of 0.01 N HCl and once with
12-29-75 JANUARY
1976
VOL.
11 NO. 1
PROSTAGLANDINS
3 ml of water, and then was evaporated to dryness under a reduced pressure. 0.1 ml of "C-methanol reagent [ 1 mg of 14C-methanol (56 mCi/m mole, Radiochemical Center) and 2 ml of cont. HzS04 in 100 ml of ethylacetate] was added to the residue at a low temperature and allowed to stand for 24 hr at -lo?. Fifteen pg of non-radioactive PGF,c methylester and the same amount of PGE, methylester were added as carriers, and the 3H-PG-14C-methylester was extracted with 0.5 ml of chloroform. In order to remove the excess methanol, 0.1 ml of 0.01 % potassium permanganate was added to the extract and kept for 10 min at room temperature. The resulting formic acid was washed out once with 0.1 ml of 0.01 % sodium carbonate, and three times with 0.5 ml of water. The washed extract was evaporated to dryness. The residue was redissolved in a small amount of chloroform, and was applied to thinlayer chromatography with silica gel as an adsorbent and a solvent system consisting of ethylacetate -methanol-water (160:25:100). The PGFP, methylester and PGE2 methylester on the chromatogram were detected with iodine vapor, and then the radioactivity was measured by a liquid scintillation counter. The amount of PGF,, and E, were calculated from the ratio of 14C and 3H radioactivity. RESULTS AND DISCUSSION Fig. 1 shows the standard curve for PGFz, and PGE2. As little as less than 2 ng of PG was determined quantitatively by this method(6). The contents of PGF2, and PGEz in cerebrospinal fluid of men and of women are shown in Table 1 and 2. The mean values of PGF2, and PGEz in cerebrospinal fluid of men were 9.8kO.87 ng/ml and 6.521.39 ng/ml, respectively. The mean values of PGF2c and PGE 2 of women were 8.3k1.4 ng/ ml and 6.921.72 ng/ml, respectively. The correlation between age and PGF 2c1content was not recognized in cerebrospinal fluid of men. However, there was a significant correlation between age and PGEp content in cerebrospinal fluid of men (Fig. 2). On the contrary, in the case of women, the correlation between PGF2a content and age was significantly recognized, but not between PGE2 content and age (Fig. 3). Some of the cerebrospinal fluid used in this experiment was obtained from patients suffering from central nervous system disoder. Therefore further investigation would be necessary about this element. The results obtained by the radioisotope dilution method gave higher values as compared with those by radioimmunoassay(4) and gas-chromatography-mass spectrometric technique (5). But the results of the radioisotope dilution method is theoretically accurate, because the loss in the extraction process is corrected.
44
JANUARY
1976 VOL. 11 NO. 1
PROSTAGLANDINS
3H/1
PGE2
PGF2,
10
0
5
10
Fig. 1.
JANUARY
1976
VOL.
20ng Standard
11 NO, 1
0
5
10
20ng
curve for PGFzc( and PGE2.
45
PROSTAGLANDINS
Table 1 Contents of prostaglandin fluid of men No
FPCl
J32
(ng/ml) -
Clinical
diagnosis
1
25
10.8
2
25
9.9
2.9
3
25
6.0
1.1
4
26
5.8
0.8
5
27
15.2
3.4
6
28
13.5
2.4
7
29
9.0
0.2
8
30
8.8
1.2
9
33
9.2
0.4
10
35
11.7
trace
Lumbar sprain
11
41
10.9
4.8
12
47
8.3
5.5
Intervertebral disc hernia II
13
52
15.3
21.0
14
53
7.7
3.6
15
58
12.8
17.2
16
60
3.4
15.7
17
60
19.0
15.6
18
60
6.0
10.0
19
62
4.9
3.6
20
64
5.6
trace
21
65
6.8
7.9
22
66
6.9
14.4
23
72
18.0
4.3
AVE+S.E. 46
Age
F2e and EZ in cerebrospinal
9.8kO.87
0.7
Intervertebral disc hernia II
Cerebral infarction Hypertension Cerebral tion II
infarc-
Cerebral rhage II
hemor-
Intervertebral disc hernia II Vertigo Dizziness Cerebral tion
infarc-
6.5k1.39 JANUARY
1976 VOL. 11 NO. 1
PROSTAGLANDINS
Table 2 Contents of prostaglandin F2c and El in cerebrospinal fluid of women No
Age
F2a
24
20
5.4
4.6
25
21
1.2
19.5
26
30
1.3
1.2
27
35
9.1
3.8
28
42
2.2
trace
II
29
47
8.6
0.4
11
30
47
6.2
7.0
II
31
48
11.8
4.5
32
50
18.8
5.3
33
55
8.4
5.1
Cerebral infarction Cerebral hemorrhage Braintumor
34
60
10.2
7.6
Headache
35
60
10.2
18.3
36
80
14.2
6.0
E2
(w/ml
AVE+S.E.
JANUARY
8.3k1.4
1976 VOL. 11 NO. 1
Clinical diagnosis
1
Intervertebral disc hernia Hypopituitarism Intervertebral disc hernia II
Cerebral hemorrhage Cerebral infarction
6.9k1.72
47
PROSTAGIANDINS
l l l
l
l
e
l
l
l l l
. l l l l
e
l
e
l
l
l l l l
.
48
l
l
le l
e
le
JANUARY
1976
VOL. 11 NO. 1
PROSTAGLANDINS
al
z l
0 03
l l l
l.
_h
In
0 .
. m
l
JANb ARY 1976 VOL. 11 NO. 1
49
PROSTAGIANDINS
REFERENCES 1) Ramwell, P. W. The action of cerebrospinal fluid on the frog rectus abdominis muscle and other isolated tissue preparations. J. Physiol. 170:21, 1974. 2) Holmes, S. W. The spontaneous release of prostaglandins into the cerebral ventricle of the dog and the effect of external factors on this release. Brit. J. Pharmacol. 37:653, 1970. 3) Feldberg, W. and Gupta, K. P. Pyrogen fever and prostaglandin-like activity in cerebrospinal fluid. J. Physiol. 228:41, 1973. 4) Latorre, E., Patrono, C., Fortuna, A. and GrossiBelloni, D. Role of prostaglandin F2u in human cerebral vasospasm. J. Neurosurg. 41:293, 1974. 5) Wolfe, L. S. and Mamer, 0. A. Measurment of prostaglandin F2u levels in human cerebrospinal fluid in normal and pathological conditions. Prostaglandins. 9:183, 1975. 6) Aizawa, Y. and Yamada, K. Radioisotope dilution method for determination of prostaglandin F2c in rat tissue. Japan. J. Pharmacol. 24:647, 1974.
50
JANUARY
1976 VOL. 11 NO. 1
