Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage Suzuki H, Sat0 S, Suzuki Y, Takekoshi K, Ishihara N, Shimoda S. Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage. Acta Neurol Scand 1990: 81: 553-554.
H. Suzuki, S. Sato, Y. Suzuki,
A considerable quantity of endothelin-like immunoreactivity was demonstrated as present in the CSF of patients with subarachnoid hemorrhage. The endothelin levels in the CSF raised from 0.4 f 0.2 (Mean f SD) pmol/L at Day 0-1 to 2.2 k 0.6 pmol/L at Day 6 and the levels decreased gradually. The result suggest that endothelin may contribute the generation of vasospasm often observed in subarachnoid hemorrhage.
Key words: endothelin-1 ; subarachnoid hemorrhage; CSF; radioimmunoassay
The pathogenesis of cerebral vasospasm often observed following subarachnoid hemorrhage is still a matter of discussion. The importance of catecholaminergic or peptidergic nervous systems has been proposed (1-3). Extensive degeneration or necrosis of endothelial cells in cerebral vessels has, however, been observed after SAH (4). Therefore this vasospasm may also be considered as a response to the cerebral vessels exposed to vasoactive compounds liberated from damaged endothelial cells. Recently, very potent vasoconstrictor peptide, termed endothelin, has been isolated and identified from porcine and human endothelial cells (5,6). In order to elucidate a possible participation of endothelin in the pathogenesis of cerebral vasospasm, we have developed a sensitive and specific radioimmunoassay for endothelin- 1 and investigated the presence of endothelin like immunoreactivity in the CSF from patients with subarachnoid hemorrhage. Material and methods
We have collected 116 consecutive samples of CSF from 26 patients with subarachnoid hemorrhage (10 men and 16 women of mean age 61 f 9 yr), who had operated on 2.2 & 2.0 days after hospitalization. Ten-20 ml of cerebrospinal fluid samples were extracted using Sep-Pak Cl8 cartridges (Waters). The peptide was eluted with 60 % acetonitrile/water with 0.1 % trifluoroacetic acid. The eluate was evaporated using Speed-Vac concentrator (Sarvant) and reconstituted with 1 ml of sodium phosphate buffer (0.06 mol/l, pH 7.4, containing 0.01 mol/l ethylene
K. Takekoshi, N. Ishihara, S. Shimoda Department of Endocrinology, Internal Medicine, Dokkyo University School of Medicine, Mibu, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan
Dr H Suzuki, Department of Endocrinology, Internal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Accepted for publication January 12, 1990
diamine tetraacetate, 0.0075 mol/l NaN, and 3 g/l of bovine serum albumin). An antiserum was raised against synthetic endothelin- 1 (Novabiochem) conjugated with bovine serum albumin with glutarardehyde. The crossreaction of the other endothelin family in a molar basis was 45 % (endothelin-2), 13 (endothelin-3) or 0.12 % (big endothelin-1), respectively (submitted). No other peptide tested crossreacted in the assay. After 5 days incubation at 4" C with '251-porcine endothelin- 1 (Amersham), antibody-bound label was separated from free by the addition of dextran coated charcoal. The sensitivity of the assay was 1 fmol/tube or 0.1 pmol/liter CSF and 50% inhibition of the zero binding was 26 fmol/tube. The mean recovery for the extraction was 72 14% (Suzuki et al. submitted). Results and discussion
The endothelin concentration increased from 0.4 ? 0.2 at day 0-1 to 2.2 k 0.6 (range; 0.1-5.3) pmol/L at day 6 and then declined gradually (Fig.). Seventeen of 26 patients showed the highest endothelin concentration within Day 4 to Day 6. Statistical analysis using paired data showed the significant increase of endothelin concentration at Days 4-6 (0.5 f 0.5 at Day 0-3 vs 2.9 k 1.8 pmol/L at Day (4-6) (n = 10) or 2.2 k 1.8 at Day 4-6 vs 1.1 k 1.1 pmol/l at Day 7-10 (n = 16), both p < 0.01). The mechanism for the increase in the CSF concentration is unknown. However, severe physical stress (7) might induce endothelin production as an "acute-phase reactant". Beside this, local 553
Endothelin and SAH
Suzuki et al. EDT-IR
Acknowledgements
(pmol/ L 1
This research was supported in part by a grant from Mochida Memorial Foundation (1989).
3.01
References 1. LANGR, ZIMMER R. Cerebral blood flow. Exp Neuroll974: 43: 143-161. 2. LEETGF, SU C, BEVANJA. Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. Cir Res 1976: 39: 120-126. 3. ALLEN JM, SCHON F, TODD N, etal. Presence of
0
1 2
6 8 10 12 13-18 day8 after onset Fig 1 Endothelin concentrations in cerebrospinal fluid from patients with subarachnoid hemorrhage. Each point and bar denote mean f SEM. Numbers in parenthesis indicate case numbers studied at each time point Shaded area shows the detection limit of the assay EDT-IR. endothelin immunoreactivity.
0-I
4
accumulation of factors such as adrenalin, thrombin ( 5 ) or transforming growth factor p (TGF-P) (8) may accelerate the production of endothelin- 1 via the induction of the gene expression. The absolute levels of endothelin- 1 in the cerebrospinal fluid were well in accordance with the levels which induced pharmacological effect on cerebral artery (9, 10). Furthermore, considering its long-lasting effect, endothelin- 1 supposed to be the most promissing candidate for inducing the cerebral vasospasm following subarachnoid hemorrhage.
554
neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm. Lancet 1984: ii: 550-552. 4. ALKSNEJF, BRANSONPJ. Pathogenesis of cerebral vasospasm. Neurol Res 1980: 2: 273-282. 5. YANAGISAWA M, KURIHARA H, KIMURAS , et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature: 1988: 332: 411-415. 6. ITOHY, YANAGISAWA M, OHKUBOS , etal. Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBS Lett 1988: 231: 440-444. 7. MIYAUCHIT, YANAGISAWAM, TOMIZAWAT, etal. Increased plasma concentrations of endothelin- 1 and big endothelin-1 in acute myocardial infarction. Lancet 1989: ii: 53-54. 8. KURIHARA H, YOSHIZUMIM, SUGIYAMA T, ef al. Trans-
forming growth factor+ stimulates the expression of endothelin mRNA by vascular endothelial cells. Biochem Biophys Res Commun 1989: 159: 1435-1440. 9. ASANOT, IKEGAKI I, SUZUKIY, et al. Endothelin and the production of cerebral vasospasm in dogs. Biochem Biophys Res Commun 1989: 159: 1345-1351. 10. SAITOA, SHIBAR, KIMURAS , etal. Vasoconstrictor response of large cerebral arteries of cats to endothelin, and endothelium-derived vasoactive peptide. Eur J Pharmacol 1989: 162: 353-358.
H. Suzuki, S. Sato, Y. Suzuki,
A considerable quantity of endothelin-like immunoreactivity was demonstrated as present in the CSF of patients with subarachnoid hemorrhage. The endothelin levels in the CSF raised from 0.4 f 0.2 (Mean f SD) pmol/L at Day 0-1 to 2.2 k 0.6 pmol/L at Day 6 and the levels decreased gradually. The result suggest that endothelin may contribute the generation of vasospasm often observed in subarachnoid hemorrhage.
Key words: endothelin-1 ; subarachnoid hemorrhage; CSF; radioimmunoassay
The pathogenesis of cerebral vasospasm often observed following subarachnoid hemorrhage is still a matter of discussion. The importance of catecholaminergic or peptidergic nervous systems has been proposed (1-3). Extensive degeneration or necrosis of endothelial cells in cerebral vessels has, however, been observed after SAH (4). Therefore this vasospasm may also be considered as a response to the cerebral vessels exposed to vasoactive compounds liberated from damaged endothelial cells. Recently, very potent vasoconstrictor peptide, termed endothelin, has been isolated and identified from porcine and human endothelial cells (5,6). In order to elucidate a possible participation of endothelin in the pathogenesis of cerebral vasospasm, we have developed a sensitive and specific radioimmunoassay for endothelin- 1 and investigated the presence of endothelin like immunoreactivity in the CSF from patients with subarachnoid hemorrhage. Material and methods
We have collected 116 consecutive samples of CSF from 26 patients with subarachnoid hemorrhage (10 men and 16 women of mean age 61 f 9 yr), who had operated on 2.2 & 2.0 days after hospitalization. Ten-20 ml of cerebrospinal fluid samples were extracted using Sep-Pak Cl8 cartridges (Waters). The peptide was eluted with 60 % acetonitrile/water with 0.1 % trifluoroacetic acid. The eluate was evaporated using Speed-Vac concentrator (Sarvant) and reconstituted with 1 ml of sodium phosphate buffer (0.06 mol/l, pH 7.4, containing 0.01 mol/l ethylene
K. Takekoshi, N. Ishihara, S. Shimoda Department of Endocrinology, Internal Medicine, Dokkyo University School of Medicine, Mibu, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan
Dr H Suzuki, Department of Endocrinology, Internal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Accepted for publication January 12, 1990
diamine tetraacetate, 0.0075 mol/l NaN, and 3 g/l of bovine serum albumin). An antiserum was raised against synthetic endothelin- 1 (Novabiochem) conjugated with bovine serum albumin with glutarardehyde. The crossreaction of the other endothelin family in a molar basis was 45 % (endothelin-2), 13 (endothelin-3) or 0.12 % (big endothelin-1), respectively (submitted). No other peptide tested crossreacted in the assay. After 5 days incubation at 4" C with '251-porcine endothelin- 1 (Amersham), antibody-bound label was separated from free by the addition of dextran coated charcoal. The sensitivity of the assay was 1 fmol/tube or 0.1 pmol/liter CSF and 50% inhibition of the zero binding was 26 fmol/tube. The mean recovery for the extraction was 72 14% (Suzuki et al. submitted). Results and discussion
The endothelin concentration increased from 0.4 ? 0.2 at day 0-1 to 2.2 k 0.6 (range; 0.1-5.3) pmol/L at day 6 and then declined gradually (Fig.). Seventeen of 26 patients showed the highest endothelin concentration within Day 4 to Day 6. Statistical analysis using paired data showed the significant increase of endothelin concentration at Days 4-6 (0.5 f 0.5 at Day 0-3 vs 2.9 k 1.8 pmol/L at Day (4-6) (n = 10) or 2.2 k 1.8 at Day 4-6 vs 1.1 k 1.1 pmol/l at Day 7-10 (n = 16), both p < 0.01). The mechanism for the increase in the CSF concentration is unknown. However, severe physical stress (7) might induce endothelin production as an "acute-phase reactant". Beside this, local 553
Endothelin and SAH
Suzuki et al. EDT-IR
Acknowledgements
(pmol/ L 1
This research was supported in part by a grant from Mochida Memorial Foundation (1989).
3.01
References 1. LANGR, ZIMMER R. Cerebral blood flow. Exp Neuroll974: 43: 143-161. 2. LEETGF, SU C, BEVANJA. Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. Cir Res 1976: 39: 120-126. 3. ALLEN JM, SCHON F, TODD N, etal. Presence of
0
1 2
6 8 10 12 13-18 day8 after onset Fig 1 Endothelin concentrations in cerebrospinal fluid from patients with subarachnoid hemorrhage. Each point and bar denote mean f SEM. Numbers in parenthesis indicate case numbers studied at each time point Shaded area shows the detection limit of the assay EDT-IR. endothelin immunoreactivity.
0-I
4
accumulation of factors such as adrenalin, thrombin ( 5 ) or transforming growth factor p (TGF-P) (8) may accelerate the production of endothelin- 1 via the induction of the gene expression. The absolute levels of endothelin- 1 in the cerebrospinal fluid were well in accordance with the levels which induced pharmacological effect on cerebral artery (9, 10). Furthermore, considering its long-lasting effect, endothelin- 1 supposed to be the most promissing candidate for inducing the cerebral vasospasm following subarachnoid hemorrhage.
554
neuropeptide Y in human circle of Willis and its possible role in cerebral vasospasm. Lancet 1984: ii: 550-552. 4. ALKSNEJF, BRANSONPJ. Pathogenesis of cerebral vasospasm. Neurol Res 1980: 2: 273-282. 5. YANAGISAWA M, KURIHARA H, KIMURAS , et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature: 1988: 332: 411-415. 6. ITOHY, YANAGISAWA M, OHKUBOS , etal. Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBS Lett 1988: 231: 440-444. 7. MIYAUCHIT, YANAGISAWAM, TOMIZAWAT, etal. Increased plasma concentrations of endothelin- 1 and big endothelin-1 in acute myocardial infarction. Lancet 1989: ii: 53-54. 8. KURIHARA H, YOSHIZUMIM, SUGIYAMA T, ef al. Trans-
forming growth factor+ stimulates the expression of endothelin mRNA by vascular endothelial cells. Biochem Biophys Res Commun 1989: 159: 1435-1440. 9. ASANOT, IKEGAKI I, SUZUKIY, et al. Endothelin and the production of cerebral vasospasm in dogs. Biochem Biophys Res Commun 1989: 159: 1345-1351. 10. SAITOA, SHIBAR, KIMURAS , etal. Vasoconstrictor response of large cerebral arteries of cats to endothelin, and endothelium-derived vasoactive peptide. Eur J Pharmacol 1989: 162: 353-358.
