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Prostanoid synthesis by human umbilical artery
PROSTAGLANDINS
PROSTANOID
SYNTHESIS
BY
HUMAN UMBILICAL ARTERY
J.M. Ritter I, M-A. Ongari, S.E. Barrow, M.A. Orchard, I.A. Blair & P.J. Lewis Department of Clinical Pharmacoloqy, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, U.K. ABSTRACT A discrepancy between published values of PGI 2 production by human umbilical artery in vitro measured by platelet bioassay, compared with values of 6-oxo-PGFl~ by radioimmunoassay, raised the possibility that another antiaggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI 2 determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGFle in hydrolysed and non-hydrolysed samples. 6-oxoPGFIe, PGF2~ and TXB 2 were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI 2 concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p < O.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI 2 production determined by bioassay (mean 1.21 ng/mg wet weight/h, range O.59-1.53 ng/mg/h) differed from previously reported values (range 70-325 nq/ma/h). 6-oxo-PGFl~ concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI 2 production by this tissue overestimated it by approximately IO0 times. INTRODUCTION Prostacyclin (PGI 2) production by human umbilical artery, measured by bioassay, has been reported (1,2) to range from 70-325 ng/mg/h. In contrast, the production of 6-oxo-PGFle, the stable hydrolysis product of PGI2, measured by radioimmunoassay (RIA) was approximately iOO-fold less (3). The direction,as well as the magnitude of the discrepancy is unexpected; bioassay may underestimate PGI 2 production because of hydrolysis of PGI 2 during incubation (4). Antiaggregatory activity was not generated following incubatfon with cyclOoxygenase inhibitors (I) so the findings might be explained by the production by umbilical arteries of an iDepartment of Medicine, Case Western Reserve University, Lakeside Hospital, Cleveland, Ohio 44106, U.S.A.
DECEMBER 1982 VOL. 24 NO. 6
881
PROSTAGLANDINS
a d d i t i o n a l p r o s t a n o i d w i t h a n t i a g g r e g a t o r y properties. This w o u l d be i n t e r p r e t e d as PGI 2 w h e n m e a s u r e d by bioassay but w o u l d not be d e t e c t e d by RIA. This p o s s i b i l i t y was i n v e s t i g a t e d by incubating u m b i l i c a l artery rings in buffer. A n t i a g g r e g a t o r y activity in the incubate was c o m p a r e d w i t h PGI 2 c o n c e n t r a t i o n , m e a s u r e d by a r e c e n t l y d e s c r i b e d RIA of 6 - o x o - P G F l e in h y d r o l y s e d and n o n - h y d r o lysed samples at alkaline pH (5). The latter m e t h o d is more specific than p l a t e l e t b i o a s s a y and m e a s u r e s only m a t e r i a l which, like PGI 2 itself, u n d e r g o e s m i l d acid h y d r o l y s i s to a substance that d i s p l a c e s [3HI 6 - o x o - P G F l e from a n t i b o d y (5). Thus, if an a n t i a g g r e g a t o r y substance other than PGI 2 were present, greater P G I 2 - 1 i k e a c t i v i t y would be d e t e c t e d by b i o a s s a y than by RIA. In addition, c o n c e n t r a t i o n s of 6 - o x o - P G F l e , TXB 2 and PGF2~ w e r e determ i n e d in the same samples using gas c h r o m a t o g r a p h y negative ion chemical i o n i s a t i o n mass s p e c t r o m e t r y (GC/NICIMS). MATERIALS
AND METHODS
U m b i l i c a l cords were o b t a i n e d from 6 w o m e n following uncomplicated pregnancies. No drugs had been taken for at least 2 weeks before delivery. The cords w e r e h a n d l e d as d e s c r i b e d by B a l c o n i et al (6), the arteries sliced into 1 m m rings with a M c I l w a i n tissue chopper and i~ rings i n c u b a t e d in 1 ml of 50 m M Tris buffer (pH 9.0) in a shakina w a t e r bath at 22 °C for 60 m i n u t e s (i). i00 ~i of this incubate was added to 2.5 ~i IM NaOH (to s t a b i l i s e the PGI 2) and stored o v e r n i g h t at -20 oc. A further 200 ~i for GC/ NICIMS analysis was spiked w i t h the a p p r o p r i a t e d e u t e r a t e d internal standards, m a d e up to 20 ml w i t h d i s t i l l e d w a t e r and stored at -20 oc. A n t i a g g r e g a t o r y activity was assayed on a d e n o s i n e d i p h o s p h a t e (ADP) induced p l a t e l e t a g g r e g a t i o n in a P a y t o n a g g r e g o m e t e r and c o m p a r e d w i t h i n h i b i t i o n by a u t h e n t i c PGI 2 as d e s c r i b e d p r e v i o u s l y (4). RIA for 6 - o x o - P G F l ~ was p e r f o r m e d on d u p l i c a t e samples diluted lO0-fold in pH 8.5 Tris b u f f e r (50 mM). One of the d u p l i c a t e s was h y d r o l y s e d by t e m p o r a r y a c i d i f i c a t i o n with 2M HCI, its pH being subs e q u e n t l y restored w i t h 2M NaOH. IM NaCI was added to the control n o n - h y d r o l y s e d sample. The assay was D e r f o r m e d at pH 8.5 in the p r e s e n c e of 1% human serum albumin. Under these c o n d i t i o n s the d i f f e r e n c e between 6 - o x o - P G F l e in the h y d r o l y s e d and n o n - h y d r o l y s e d samples gives the PGI 2 p r e s e n t (5). W o r k u p for the G C / N I C I M S analysis, i n c l u d i n g e x t r a c t i o n and d e r i v a t i s a t i o n , were d e s c r i b e d e l s e w h e r e (7,8). ADP and human serum albumin (essentially fatty acid free) were o b t a i n e d from Sigma C h e m i c a l Co. (Poole, U.K.) ; PGI 2 was a generous gift from W e l l c o m e R e s e a r c h L a b o r a t o r i e s (Beckenham, U.K.) ; a n t i b o d y to 6 - o x 0 - P G F I ~ was a generous
882
DECEMBER 1982 VOL. 24 NO. 6
PROSTAGLANDINS
gift from Dr. L. M y a t t (Institute of O b s t e t r i c s and gynaecology, H a m m e r s m i t h Hospital, London, U.K.). Tritiated 6 - o x o - P G F l e was o b t a i n e d from N e w E n g l a n d N u c l e a r (Southampton, U.K.); p r o s t a g l a n d i n standards (for GC/ NICIMS) were a gift from Dr. John Pike (Upjohn Company, Kalamazoo, Michigan, U.S.A.). S t a t i s t i c a l c o m p a r i s o n s were by S t u d e n t ' s p a i r e d t test (2 tailed). D i f f e r e n c e s and c o r r e l a t i o n s were c o n s i d e r e d s i g n i f i c a n t w h e n p < 0.05. RESULTS The c o n c e n t r a t i o n s of p r o s t a n o i d s d e t e r m i n e d in the i n c u b a t e s are shown in Table i. The c o n c e n t r a t i o n s of PGI 2 d e t e r m i n e d by the two m e t h o d s were s i g n i f i c a n t l y c o r r e l a t e d (r = 0.92, p < 0.01). There was no c o n s i s t e n t d i f f e r e n c e (p > 0.95, n = 6) b e t w e e n PGI 2 p r o d u c t i o n d e t e r m i n e d by b i o a s s a y and by RIA, the m e a n rates of p r o d u c t i o n b e i n g identical: 1.21 + 0.22 n g / m g / h by RIA (mean + S.E.M.) and 1.21 + O.14 n g / m g / h - b y b i o a s s a y (mean + S . E . M . ) ? There was also ~ s i g n i f i c a n t c o r r e l a t i o n between--the 6 - o x o - P G F l e c o n c e n t r a t i o n s m e a s u r e d by RIA and by G C / N I C I M S (r = 0.92, p < 0.01). However, m e a s u r e m e n t s of 6 - o x o - P G F l ~ concentration by RIA w e r e higher than m e a s u r e m e n t s by G C / N I C I M S (p < 0.O1) c o n s i s t e n t w i t h g r e a t e r s p e c i f i c i t y of the GC/ NICIMS method. TXB 2 and PGF2e were d e t e c t e d but the conc e n t r a t i o n s were low c o m p a r e d w i t h 6 - o x o - P G F l ~ (0-7.2% of the 6 - o x o - P G F l e in the case of TXB2; 0-6.4% in the case of PGF2a. DISCUSSION The a g r e e m e n t b e t w e e n PGI 2 m e a s u r e d by RIA and by p l a t e l e t b i o a s s a y (Table i) d i s p r o v e s the h y p o t h e s i s that u m b i l i c a l artery rings p r o d u c e an a n t i a g g r e g a t o r y p r o s t a n o i d in addition to PGI2, that can be d e t e c t e d under these conditions. The PGI 2 p r o d u c t i o n in the p r e s e n t e x p e r i m e n t s d e t e r m i n e d by b i o a s s a y r a n g e d from 0 . 5 9 - 1 . 5 3 ng/mg/h, comp a r e d w i t h v a l u e s of 86-177 ng/mg/h (mean 140 ng/mg/h) r e p o r t e d p r e v i o u s l y (i). In a s u b s e q u e n t paper (2) the same group r e p o r t e d even h i g h e r v a l u e s (up to 325 ng/mg/h). The d i s c r e p a n c y cannot be a t t r i b u t e d to d i f f e r e n c e s in s t o r i n g the cords. In our initial e x p e r i m e n t s (3), cords w e r e h a n d l e d fresh; we have s u b s e q u e n t l y (unpublished observations) found no d i f f e r e n c e in 6 - o x o - P G F l e p r o d u c t i o n by secrments of the same artery s t u d i e d fresh and after storage of the cord at -20 °C w i t h s u b s e q u e n t t h a w i n g on ice (6). We c o n s i d e r e d the p o s s i b i l i t y that c o n c e n t r a t i o n s of 6 - o x o - P G F l e and PGI 2 that we m e a s u r e d w e r e s p u r i o u s l y low. However, the m e a s u r e m e n t s w i t h G C / N I C I M S show that the c o n c e n t r a t i o n s of 6 - o x o - P G F l ~ w e r e in fact o v e r e s t i m a t e d
DECEMBER 1982 VOL. 24 NO. 6
883
PROSTAGLANDINS
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884
DECEMBER
1982 VOL. 24 NO. 6
PROSTAGLANDINS by RIA by 56.9 + 15.7% (mean + S.E.M.) consistent with greater specificity of the GcTNICIMS assay. The high values of PGI 2 production reported previously (1,2) could have arisen if the PGI 2 used as the standard for the bioassay had partially hydrolysed. If this were the cause of the discrepancy, the relative changes previously reported between arteries from healthy and pre-eclamptic pregnancies (2) and between adult and umbilical arteries (i) would be unaffected. The absolute values would however be incorrect. We conclude that the rate of production of PGI 2 by human umbilical arteries is approximately iO0-fold lower than previously reported. ACKNOWLEDGEMENTS We thank Mr. Colin Chappell for excellent technical assistance. M-A.O. is a recipient of an EEC Fellowship. M.A.O. was supported by a British Heart Foundation grant. The work was supported in part by a Medical Research Council programme grant. REFERENCES i. Remuzzi, G., R. Misiani, D. Muratore, D. Marchesi, M. Livio, A. Schieppati, G. Mecca, G. de Gaetano, and M.B. Donati. Prostacyclin and human foetal circulation. Prostaglandins 18: 341-348, 1979. 2. Remuzzi, G., D. Marchesi, C. Zoja, D. Muratore, G. Mecca, R. Misiani, E. Rossi, M. Barbato, P. Capetta, M.B. Donati, and G. de Gaetano. Reduced umbilical and placental vascular prostacyclin in severe pre-eclampsia. Prostaglandins 20: 105-110, 1980. 3. Ritter, J.M., M. Ongari, A.P. Gordon-Wright, I.A. Blair, M.A. Orchard, and P.J. Lewis. In: Prostacyclin in Pregnancy. (P.J. Lewis, S. Moncada and J. O'Grady, eds.) Raven Press, New York, in press. 4. Ritter, J.M., M.A. Orchard, I.A. Blair, and P.J. Lewis. The time course and magnitude of prostacyclin (PGI2) production by rat aortic rings incubated in human plasma. Biochem. Pharmacol. 31: 1163-1165, 1982. 5. Orchard, M.A., I.A. Blair, J.M. Ritter, L. Myatt, M. Jogee, and P.J. Lewis. R a d i o i m m u n o a s s a y at alkaline pH: A m e t h o d for the quantitative determination of prostacyclin. Biochem. Soc. Trans. iO: 241, 1982. 6. Balconi, G., A. Pietra, S. Olivieri, M. Vergara-Dauden, M. Busacca, P. Gementi, J. Pangrazzi, M. Recchia, E. Dejana, and G. de Gaetano. Prostacyclin production by umbilical arteries and cultured endothelial cells from umbilical veins. A methodological approach. In: Prostacyclin in Pregnancy. (P.J. Lewis, S. Moncada and J. O'Grady, eds.) Raven Press, New York, in press.
DECEMBER 1982 VOL. 24 NO. 6
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PROSTAGLANDINS
7. Barrow, S.E., K.A. Waddell, M. Ennis, C.T. Dollery, and I.A. Blair. A n a l y s i s of p i c o m o l a r c o n c e n t r a t i o n s of 6 - o x o - p r o s t a g l a n d i n Fle in b i o l o g i c a l fluids. J. Chromatogr. 239: 71-80, 1982. 8. Waddell, K.A., S.E. Barrow, C. Robinson, M.A. Orchard, C.T. Dollery, and I.A. Blair. The s i m u l t a n e o u s analysis of p r o s t a n o i d s in b i o l o g i c a l fluids by c o m b i n e d c a p i l l a r y column gas c h r o m a t o g r a h y n e g a t i v e ion chemical ionisation mass spectrometry. Biochem. Soc. Trans. 1982, in Dress.
Editor: P.W. Ramwell
886
Received: 9-15-82
Accepted: 11-15-82
DECEMBER 1982 VOL. 24 NO. 6
PROSTANOID
SYNTHESIS
BY
HUMAN UMBILICAL ARTERY
J.M. Ritter I, M-A. Ongari, S.E. Barrow, M.A. Orchard, I.A. Blair & P.J. Lewis Department of Clinical Pharmacoloqy, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, U.K. ABSTRACT A discrepancy between published values of PGI 2 production by human umbilical artery in vitro measured by platelet bioassay, compared with values of 6-oxo-PGFl~ by radioimmunoassay, raised the possibility that another antiaggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI 2 determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGFle in hydrolysed and non-hydrolysed samples. 6-oxoPGFIe, PGF2~ and TXB 2 were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI 2 concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p < O.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI 2 production determined by bioassay (mean 1.21 ng/mg wet weight/h, range O.59-1.53 ng/mg/h) differed from previously reported values (range 70-325 nq/ma/h). 6-oxo-PGFl~ concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI 2 production by this tissue overestimated it by approximately IO0 times. INTRODUCTION Prostacyclin (PGI 2) production by human umbilical artery, measured by bioassay, has been reported (1,2) to range from 70-325 ng/mg/h. In contrast, the production of 6-oxo-PGFle, the stable hydrolysis product of PGI2, measured by radioimmunoassay (RIA) was approximately iOO-fold less (3). The direction,as well as the magnitude of the discrepancy is unexpected; bioassay may underestimate PGI 2 production because of hydrolysis of PGI 2 during incubation (4). Antiaggregatory activity was not generated following incubatfon with cyclOoxygenase inhibitors (I) so the findings might be explained by the production by umbilical arteries of an iDepartment of Medicine, Case Western Reserve University, Lakeside Hospital, Cleveland, Ohio 44106, U.S.A.
DECEMBER 1982 VOL. 24 NO. 6
881
PROSTAGLANDINS
a d d i t i o n a l p r o s t a n o i d w i t h a n t i a g g r e g a t o r y properties. This w o u l d be i n t e r p r e t e d as PGI 2 w h e n m e a s u r e d by bioassay but w o u l d not be d e t e c t e d by RIA. This p o s s i b i l i t y was i n v e s t i g a t e d by incubating u m b i l i c a l artery rings in buffer. A n t i a g g r e g a t o r y activity in the incubate was c o m p a r e d w i t h PGI 2 c o n c e n t r a t i o n , m e a s u r e d by a r e c e n t l y d e s c r i b e d RIA of 6 - o x o - P G F l e in h y d r o l y s e d and n o n - h y d r o lysed samples at alkaline pH (5). The latter m e t h o d is more specific than p l a t e l e t b i o a s s a y and m e a s u r e s only m a t e r i a l which, like PGI 2 itself, u n d e r g o e s m i l d acid h y d r o l y s i s to a substance that d i s p l a c e s [3HI 6 - o x o - P G F l e from a n t i b o d y (5). Thus, if an a n t i a g g r e g a t o r y substance other than PGI 2 were present, greater P G I 2 - 1 i k e a c t i v i t y would be d e t e c t e d by b i o a s s a y than by RIA. In addition, c o n c e n t r a t i o n s of 6 - o x o - P G F l e , TXB 2 and PGF2~ w e r e determ i n e d in the same samples using gas c h r o m a t o g r a p h y negative ion chemical i o n i s a t i o n mass s p e c t r o m e t r y (GC/NICIMS). MATERIALS
AND METHODS
U m b i l i c a l cords were o b t a i n e d from 6 w o m e n following uncomplicated pregnancies. No drugs had been taken for at least 2 weeks before delivery. The cords w e r e h a n d l e d as d e s c r i b e d by B a l c o n i et al (6), the arteries sliced into 1 m m rings with a M c I l w a i n tissue chopper and i~ rings i n c u b a t e d in 1 ml of 50 m M Tris buffer (pH 9.0) in a shakina w a t e r bath at 22 °C for 60 m i n u t e s (i). i00 ~i of this incubate was added to 2.5 ~i IM NaOH (to s t a b i l i s e the PGI 2) and stored o v e r n i g h t at -20 oc. A further 200 ~i for GC/ NICIMS analysis was spiked w i t h the a p p r o p r i a t e d e u t e r a t e d internal standards, m a d e up to 20 ml w i t h d i s t i l l e d w a t e r and stored at -20 oc. A n t i a g g r e g a t o r y activity was assayed on a d e n o s i n e d i p h o s p h a t e (ADP) induced p l a t e l e t a g g r e g a t i o n in a P a y t o n a g g r e g o m e t e r and c o m p a r e d w i t h i n h i b i t i o n by a u t h e n t i c PGI 2 as d e s c r i b e d p r e v i o u s l y (4). RIA for 6 - o x o - P G F l ~ was p e r f o r m e d on d u p l i c a t e samples diluted lO0-fold in pH 8.5 Tris b u f f e r (50 mM). One of the d u p l i c a t e s was h y d r o l y s e d by t e m p o r a r y a c i d i f i c a t i o n with 2M HCI, its pH being subs e q u e n t l y restored w i t h 2M NaOH. IM NaCI was added to the control n o n - h y d r o l y s e d sample. The assay was D e r f o r m e d at pH 8.5 in the p r e s e n c e of 1% human serum albumin. Under these c o n d i t i o n s the d i f f e r e n c e between 6 - o x o - P G F l e in the h y d r o l y s e d and n o n - h y d r o l y s e d samples gives the PGI 2 p r e s e n t (5). W o r k u p for the G C / N I C I M S analysis, i n c l u d i n g e x t r a c t i o n and d e r i v a t i s a t i o n , were d e s c r i b e d e l s e w h e r e (7,8). ADP and human serum albumin (essentially fatty acid free) were o b t a i n e d from Sigma C h e m i c a l Co. (Poole, U.K.) ; PGI 2 was a generous gift from W e l l c o m e R e s e a r c h L a b o r a t o r i e s (Beckenham, U.K.) ; a n t i b o d y to 6 - o x 0 - P G F I ~ was a generous
882
DECEMBER 1982 VOL. 24 NO. 6
PROSTAGLANDINS
gift from Dr. L. M y a t t (Institute of O b s t e t r i c s and gynaecology, H a m m e r s m i t h Hospital, London, U.K.). Tritiated 6 - o x o - P G F l e was o b t a i n e d from N e w E n g l a n d N u c l e a r (Southampton, U.K.); p r o s t a g l a n d i n standards (for GC/ NICIMS) were a gift from Dr. John Pike (Upjohn Company, Kalamazoo, Michigan, U.S.A.). S t a t i s t i c a l c o m p a r i s o n s were by S t u d e n t ' s p a i r e d t test (2 tailed). D i f f e r e n c e s and c o r r e l a t i o n s were c o n s i d e r e d s i g n i f i c a n t w h e n p < 0.05. RESULTS The c o n c e n t r a t i o n s of p r o s t a n o i d s d e t e r m i n e d in the i n c u b a t e s are shown in Table i. The c o n c e n t r a t i o n s of PGI 2 d e t e r m i n e d by the two m e t h o d s were s i g n i f i c a n t l y c o r r e l a t e d (r = 0.92, p < 0.01). There was no c o n s i s t e n t d i f f e r e n c e (p > 0.95, n = 6) b e t w e e n PGI 2 p r o d u c t i o n d e t e r m i n e d by b i o a s s a y and by RIA, the m e a n rates of p r o d u c t i o n b e i n g identical: 1.21 + 0.22 n g / m g / h by RIA (mean + S.E.M.) and 1.21 + O.14 n g / m g / h - b y b i o a s s a y (mean + S . E . M . ) ? There was also ~ s i g n i f i c a n t c o r r e l a t i o n between--the 6 - o x o - P G F l e c o n c e n t r a t i o n s m e a s u r e d by RIA and by G C / N I C I M S (r = 0.92, p < 0.01). However, m e a s u r e m e n t s of 6 - o x o - P G F l ~ concentration by RIA w e r e higher than m e a s u r e m e n t s by G C / N I C I M S (p < 0.O1) c o n s i s t e n t w i t h g r e a t e r s p e c i f i c i t y of the GC/ NICIMS method. TXB 2 and PGF2e were d e t e c t e d but the conc e n t r a t i o n s were low c o m p a r e d w i t h 6 - o x o - P G F l ~ (0-7.2% of the 6 - o x o - P G F l e in the case of TXB2; 0-6.4% in the case of PGF2a. DISCUSSION The a g r e e m e n t b e t w e e n PGI 2 m e a s u r e d by RIA and by p l a t e l e t b i o a s s a y (Table i) d i s p r o v e s the h y p o t h e s i s that u m b i l i c a l artery rings p r o d u c e an a n t i a g g r e g a t o r y p r o s t a n o i d in addition to PGI2, that can be d e t e c t e d under these conditions. The PGI 2 p r o d u c t i o n in the p r e s e n t e x p e r i m e n t s d e t e r m i n e d by b i o a s s a y r a n g e d from 0 . 5 9 - 1 . 5 3 ng/mg/h, comp a r e d w i t h v a l u e s of 86-177 ng/mg/h (mean 140 ng/mg/h) r e p o r t e d p r e v i o u s l y (i). In a s u b s e q u e n t paper (2) the same group r e p o r t e d even h i g h e r v a l u e s (up to 325 ng/mg/h). The d i s c r e p a n c y cannot be a t t r i b u t e d to d i f f e r e n c e s in s t o r i n g the cords. In our initial e x p e r i m e n t s (3), cords w e r e h a n d l e d fresh; we have s u b s e q u e n t l y (unpublished observations) found no d i f f e r e n c e in 6 - o x o - P G F l e p r o d u c t i o n by secrments of the same artery s t u d i e d fresh and after storage of the cord at -20 °C w i t h s u b s e q u e n t t h a w i n g on ice (6). We c o n s i d e r e d the p o s s i b i l i t y that c o n c e n t r a t i o n s of 6 - o x o - P G F l e and PGI 2 that we m e a s u r e d w e r e s p u r i o u s l y low. However, the m e a s u r e m e n t s w i t h G C / N I C I M S show that the c o n c e n t r a t i o n s of 6 - o x o - P G F l ~ w e r e in fact o v e r e s t i m a t e d
DECEMBER 1982 VOL. 24 NO. 6
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PROSTAGLANDINS
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1982 VOL. 24 NO. 6
PROSTAGLANDINS by RIA by 56.9 + 15.7% (mean + S.E.M.) consistent with greater specificity of the GcTNICIMS assay. The high values of PGI 2 production reported previously (1,2) could have arisen if the PGI 2 used as the standard for the bioassay had partially hydrolysed. If this were the cause of the discrepancy, the relative changes previously reported between arteries from healthy and pre-eclamptic pregnancies (2) and between adult and umbilical arteries (i) would be unaffected. The absolute values would however be incorrect. We conclude that the rate of production of PGI 2 by human umbilical arteries is approximately iO0-fold lower than previously reported. ACKNOWLEDGEMENTS We thank Mr. Colin Chappell for excellent technical assistance. M-A.O. is a recipient of an EEC Fellowship. M.A.O. was supported by a British Heart Foundation grant. The work was supported in part by a Medical Research Council programme grant. REFERENCES i. Remuzzi, G., R. Misiani, D. Muratore, D. Marchesi, M. Livio, A. Schieppati, G. Mecca, G. de Gaetano, and M.B. Donati. Prostacyclin and human foetal circulation. Prostaglandins 18: 341-348, 1979. 2. Remuzzi, G., D. Marchesi, C. Zoja, D. Muratore, G. Mecca, R. Misiani, E. Rossi, M. Barbato, P. Capetta, M.B. Donati, and G. de Gaetano. Reduced umbilical and placental vascular prostacyclin in severe pre-eclampsia. Prostaglandins 20: 105-110, 1980. 3. Ritter, J.M., M. Ongari, A.P. Gordon-Wright, I.A. Blair, M.A. Orchard, and P.J. Lewis. In: Prostacyclin in Pregnancy. (P.J. Lewis, S. Moncada and J. O'Grady, eds.) Raven Press, New York, in press. 4. Ritter, J.M., M.A. Orchard, I.A. Blair, and P.J. Lewis. The time course and magnitude of prostacyclin (PGI2) production by rat aortic rings incubated in human plasma. Biochem. Pharmacol. 31: 1163-1165, 1982. 5. Orchard, M.A., I.A. Blair, J.M. Ritter, L. Myatt, M. Jogee, and P.J. Lewis. R a d i o i m m u n o a s s a y at alkaline pH: A m e t h o d for the quantitative determination of prostacyclin. Biochem. Soc. Trans. iO: 241, 1982. 6. Balconi, G., A. Pietra, S. Olivieri, M. Vergara-Dauden, M. Busacca, P. Gementi, J. Pangrazzi, M. Recchia, E. Dejana, and G. de Gaetano. Prostacyclin production by umbilical arteries and cultured endothelial cells from umbilical veins. A methodological approach. In: Prostacyclin in Pregnancy. (P.J. Lewis, S. Moncada and J. O'Grady, eds.) Raven Press, New York, in press.
DECEMBER 1982 VOL. 24 NO. 6
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PROSTAGLANDINS
7. Barrow, S.E., K.A. Waddell, M. Ennis, C.T. Dollery, and I.A. Blair. A n a l y s i s of p i c o m o l a r c o n c e n t r a t i o n s of 6 - o x o - p r o s t a g l a n d i n Fle in b i o l o g i c a l fluids. J. Chromatogr. 239: 71-80, 1982. 8. Waddell, K.A., S.E. Barrow, C. Robinson, M.A. Orchard, C.T. Dollery, and I.A. Blair. The s i m u l t a n e o u s analysis of p r o s t a n o i d s in b i o l o g i c a l fluids by c o m b i n e d c a p i l l a r y column gas c h r o m a t o g r a h y n e g a t i v e ion chemical ionisation mass spectrometry. Biochem. Soc. Trans. 1982, in Dress.
Editor: P.W. Ramwell
886
Received: 9-15-82
Accepted: 11-15-82
DECEMBER 1982 VOL. 24 NO. 6