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Aspirin, prostacyclin and post-occlusive reactive hyperaemia in man
Prostaglandins Leukotrienes and Medicine 9:
379-385, 1982
ASPIRIN, PROSTACYCLIN AND POST-OCCLUSIVE REACTIVE HYPERAEMIA IN MAN J.E. Tooke, V.C. Menys, H. Tindall, M. Feely & J.A. Davies University Department of Medicine, Leeds General Infirmary, Leeds LSl 3EX (reprint requests to JET). ABSTRACT We studied post-occlusive reactive hyperaemia using ecg-triggered mercury strain-gauge plethysmography in eight normal subjects treated The reactive with incremental doses of aspirin (27.5-1200 mg). hyperaemic response was measured in the finger (predominantly skin blood flow) and the calf (predominantly muscle). Concentrations of TXB2 and 6-oxo-PGF were measured in venous effluent blood from the hand by RIA, following'~rteria1 occlusion. Levels of TXB2 were significantly higher at O-10 and 60-70 seconds (p&.01), and 90-100 seconds (~8.05) following release of occlusion compared to pre-occlusion values. However there was no significant change in concentrations of 6-0x0-PGF,, and therefore by this method release of prostacyclin during reactive hyperaemia in the hand. Aspirin had no influence on finger or calf reactive hyperaemia 90 minutes after dosing, despite marked inhibition of platelet MDA production ( 75% after 110 mg, maximal inhibition after These data provide no support for the hypothesis that 1200 mg). prostacyclin is involved in the determination of the post-occlusive reactive hyperaemic response in the finger and calf in man. INTRODUCTION Local production of prostanoids may be an important determinant of the reactive hyperaemic response. Kilbom & Wennmalm (1) demonstrated that indomethacin suppressed the post-occlusive reactive hyperaemic response in the human forearm and that this suppression was accompanied by a reduction in the PGE-like activity in the venous effluent. Several other lines of evidence (2) suggest that prostaglandins are important in local circulatory control. Recent clinical interest has centered on the pathophysiological role and therapeutic potential of the potent vasodilator PGI . Results reported by Neri Serneri et al (3) leased during periods of arterial occlusion and the vasodilatation which follows release. We decided to study some aspects of the proposed inter-relationship between prostaglandin synthesis and blood flow. Initially, we examined
P-
6
379
whether aspirin, a non-competitive inhibitor of cyclooxygenase influenced the reactive hyperaemic response in a manner similar to that described by Kilbom & Wennmalm (1). Subsequently we tried to confirm the findings of Neri Serneri et al (3) that PGI2 is released during arterial occlusion. SUBJECTS AND METHODS Subjects. Eight healthy male non-smokers, age range from 25 to 37 years, mean 29 years were the subjects for the blood flow studies. The subjects for venous sampling studies were six healthy male non-smokers, age range 25 to 37 years, mean 31 years, four of whom participated in the blood flow studies. No subject was receiving any medication or had consumed aspirin-containing compounds or other known inhibitors of cyclooxygenase within two weeks of the experimental period. Blood flow was studied in each subject on seven Study design. occasions. ReSDOnSeS orior to aspirin administration were determined on two consecutive days. Responses were then determined on 5 consecutive days during incremental doses of aspirin. On the first day of dosing the subject was studied 90 minutes after ingestion of 27.5 mg of aspirin, and similarly following 110 mg on day 2, 330 mg on day 3, Vascular studies were performed 600 mg on day 4 and 1200 mg on day 5. at 9.00 a.m. followin8 acclimatization in a constant temperature room maintained at 24 f 0.5 C. Blood flow. Resting blood flow and the reactive hyperaemic response to a four minute Deriod of arterial occlusion were determined in the calf in 5 subjects,'and in the index finger in all subjects using the Janssen With Scientific Instruments Periflow mercury strain plethysmograph. this instrument an estimate of flow was made every 5 heartbeats, cuff This inflation and deflation being triggered by the R wave of the ECG. enabled an accurate semicontinuous assessment of the reactive hyperaemic response. Calf flow was determined in both legs and the average value Results were recorded for rest flow (mean blood used for analysis. flow in ml/100 ml of tissue/min recorded over 2 minute period prior to occlusion); peak flow (peak blood flow in ml/100 ml of tissue/min recorded within 10 seconds of release of occlusion); and total incremental flow (flow in ml/100 ml of tissue above rest flow recorded in 100 seconds following release of occlusion). On completion of measurements of blood flow, blood was Blood tests. taken tor estimation of plasma salicylate level, and malondialdehyde (MDA) production (4) as a measure of platelet cyclooxygenase inhibition. To assess PGI release during arterial occlusion we measured the level of the stabl$ metabolite 6-oxo-PGF by radioimnunoassay in venous educed retrogradely into a vein on blood. A butterfly cannula was int4* the dorsum of the hand. Two basal blood samples were taken 4 minutes apart and then 4 minutes arterial occlusion at the wrist applied. Following release of arterial occlusion samples were taken at O-10 Each sample seconds, 30-40 seconds, 60-70 seconds and 90-100 seconds. was withdrawn at a constant rate without venous occlusion and mixed immediately with indomethacin (final concentration 5D0~ol/11 and EDTA The samples (final concentration 5 mnol/l) in a numbered tube at 4 C. 380
were coded to avoid identification during assay and concentrations of were measured without extraction by RIA with a TXB and 6-oxo-PGF de&ion limit of '?20 pg/ml. Non-parametric statistical analysis (Friedman's Statistical analysis. method) was used to compare the post-occlusive reactive hyperaemic Plasma concentrations of TxB and 6response pre and post-aspirin. oxo-PGF during reactive hyperaemia were compared with the &ond of the two'gasal values. RESULTS Reactive hyperaemic response. Aspirin had no statistically significant influence on reactive hyperaemic responses in the calf or finger (Table la,b). Table la,b: la:
Group mean (&SEMI reactive hyperaemic responses following incremental aspirin doses.
Calf (mean left t right)
Aspirin Dose Rest Flow Incremental Peak Flow ml/l00ml/min (mg) ’ ml/l00ml/min : 27.5 110 330 600 1200 lb:
4.8 4.5 f 0.6 0.8 5.4 5.2 5.0 4.9 5.1
f + + i f
0.8 1.1 0.9 0.8 0.9
24.9 + 24.6 f 24.6 f 23.0 + 27.5 + 24.3 f 25.6 f
1.4 1.5 1.3 1.6 1.4 1.4 1.5
Total Incremental Flow 0-100s in ml/lOOml 11.8 11.2 12.0 11.3 12.5 11.9 12.3
+ f f + + f f
0.7 0.7 0.6 0.8 0.7 0.7 0.7
Finger
Aspirin Dose Rest Flow Incremental Peak Flow ml/l00ml/min ml/l00ml/min (mg) 0 2745 110 330 l?!
10.7 + 2.9 14.3 16.3 12.5 15.1 15.3 12.6
_L4.0 f 3.9 f 4.3 f 4.3 f 4.2 2.6
18.9 i 5.9 15.8 14.7 11.9 13.8 19.6 20.7
f f f f +
4.8 4.5 3.3 4.7 6.1 7.3
Total Incremental Flow 0-100's in ml/lOOml 8.2 5.5 8.1 5.7 6.1 7.7 7.0
f f + + + + +
2.4 1.5 2.2 1.8 1.9 3.0 2.1
Mean values obtained from the calf were very similar at all dosage levels (Table la). Responses from the finger showed wider variation at any point in time but again there were no discernable differences between values at any dosage level (Table lb). Finger responses on aspirin fell within the range defined by the two pre-aspirin responses. Table 2 surrmaritesthe percentage inhibition of platelet MDA production indicating inhibition of at least 75% following aspirin 110 fag and maximal inhibition after 1200 mg of aspirin. Plasma salicylate levels confirmed subject compliance. 381
Table 2:
Inhibition of platelet MDA production following varying doses ot asDirln exoressed as oercent ot control
Subject ASA 27.5 mg 24 32
:
4” 2 7
223" ia 5 40 62 28
a mean
ASA 110 mg
ASA 330 mg
ASA 600 mg
100
100
100
;! a2
9"8 91
180'0 76 95 92
PO70 91 97 97
;; 100 99 100 94 98 98
ASA 1200 mg 100
2
100 100 100 100 9'9'
TXB
and 6-oxo-PGF1, levels. Table 3 summarizes the results of assay and 6-oxo-PGF in plasma derived from the venous effluent blood of TxB8 6 s bjects followi *)P g release of arterial occlusion. Of Table 3: TX6 and 6-oxo-PGFl.(pg/ml) levels detectable in venous effluent from dorsum of the hand pre and post arterial occlusion Sample
TXB, Subject
Basal 1
: 4 5
487 76
PO
520 496
MeankSEM
3":
272*103
Basal 2
0-10s
30-40s
60-70s
90-100s
291
536 738 36 325 976 683
446 243 42 375 698 570
738 636 307 502 702 546
454 389 409 524 584 611
396k.95
572-t64
495t38
CO.01
co.05
90-100s
/: 28 397 524 213*90
CD.01
'p' value 6-oxo-PGFl,: Basal 2
0-10s
30-40s
60-70s
70 48 ND ND ND
!J: 75 ND ND ND
ND 140 49 45 ND ND
ND 75 60 24 ::
ND 130 51 21 ND ND
33f9
39f12
49*19
41*10
44218
3a+lo
N.S.
N.S.
N.S.
N.S.
Basal 1
1
ND
PO mean+SEM 'p' value
N.S.
Sample
Subject
: 5
549&135
:: 62 N"o" ND
ND denotes not detectable; for purposes of statistical analysis these points have been assigned an arbitrary value of 20 pg/ml. Subjects 1,2,4 and 5.took part in blood flow studies. 382
were low and there was no The basal levels of 6-0x0-PGF In contrast significant increase following releaL!& of occlusion. plasma TXB levels were statistically significantly higher at O-10, 6070 and 90-400 seconds following release compared with the second basal response. DISCUSSION These results suggest that aspirin has no discernable effect on the reactive hyperaemic response in the calf or digit even at doses which maximally inhibited platelet MDA production. It is unlikely that the plethysmographic recording of reactive hyperaemia was too insensitive to have detected an influence of aspirin as the present instrument permits a more faithful representation of the The marked response than that used by Kilbom and Wennmalm (1). physiological variation in finger blood flow that occurs from moment to moment (5) might have masked a significant effect of aspirin on the However as calf flow is reactive hyperaemic response in the digit. relatively stable and pre-aspirin responses were very reproducible this would not explain the lack of effect of aspirin on the calf reactive hyperaemic response. We found a significant rise in plasma TX6 concentrations following It is unlikely that th& was an artefact induced arterial occlusion. by vascular cannulation as there was no change in the two basal levels obtained. The increase in TXB probably indicated activation of platelets by relative ischaemia in zhe occluded segment. We have been unable to confirm the release of prostacyclin (measured by radioimnunoassay of 6-oxo-PGF ) in effluent venous blood following release of arterial occlusion as & scribed by Neri Serneri and co-workers (31. There are several possible explanations for this discrepancy. We took venous samples from the dorsum of the hand whereas Neri Serneri et al (31 presumably sampled from the antecubital fossa. The circulation to the hand is predominantly skin blood flow whereas the antecubital veins receive a significant proportion of their blood flow from the forearm muscles. It is possible that PG12 is involved in the autoregulation of muscle circulation but our results do not indicate any role for PGI in control of skin circulation. In the five subjects in whom we meas2 red calf blood flow (predominantly muscle) aspirin had no influence on the reactive hyperaemic response. Thus if PGI is involved in the autoregulation of muscle blood flow its activit5 is peculiarly resistant to the action of aspirin. There is some evidence that vessel wall cyclooxygenase might be more resistant to the action of aspirin than the enzyme in platelets (6) but findings using vein rings ex-vivo (7) suggest that both enzymes are sensitive to acetylation at comparatively low doses of around 81 mg. The results of our study are equally compatible with the interpretation that PGI is not involved in the determination of the reactive hyperaemic res6 nse. Kilbom & Wennmalm (1) and Neri Serneri (3) et al both used a bioassay to assess prostaglandin activity in the
383
venous effluent blood. We used radioimunoassay, and measured the level of the stable hydrolysis product of PGI Thus methodological differences might account for the apparent d?’screpancy and PGI -like activity might be present in a form undetectable by the assiB y we employed. However in view of the close correlation between infused PGI and recovery as imnunoassayable 6-oxo-PGF, (8) it seems imprgbable that PGI itself is concerned in control 8f the hyperaemic response to arteri4 occlusion. Indomethacin at the doses used by Kilbom and Wennmalm (1) may inhibit enzymes other than cyclooxygenase (9) which may not be influenced by aspirin. These enzymes might govern the generation of metabolites which are active in the determination of the reactive hyperaemic response. Our results do not exclude a role for products of arachidonate metabolism in control of reactive hyperaemia, but they indicate that any function of products dependent on the activity of cycloxoygenase is probably insignificant. Acknowledgements We acknowledge with gratitude support from the British Heart Antisera used in the radioimnunoassays Foundation (grant no. 80/58). were a generous gift of Pfizer Ltd. V.C.M. is supported by a grant from the M.R.C. REFERENCES ..-. - .._..__ 1.
Kilbom A, Wennmalm A. Endogenous prostaglandins as local effect of indomethacin on regulators of blood flow in man: Journal of Physiology reactive and functional hyperaemia. (London), 257: 109, 1976.
2.
Prostaglandins and local Messina EJ, Weiner R, and Kaley G. Federation Proccedings,35 (12): circulatory circulatory control. 2367, 1976.
3.
Release of Neri Serneri GG, Masotti G, Poggesi L, Galanti G. prostacyclin into the bloodstream and its exhaustion in humans after local blood flow changes (ischaemia and venous stasis). 197, 1980. Thrombosis Research, 17:
4.
Simple, sensitive McMillan RM, MacIntyre DE, Gordon JL. fluorimetric assay for malondialdehyde production by blood platelets. Thrombosis Research, 11: 425, 1977.
5.
Burch GE. Digital plethysmography, chapter Stratton, New York, 1954.
6.
Masotti G, Galanti G, Poggesi L, Abbate R, Neri Serneri GG. Differential inhibition of prostacyclin production and platelet aggregation by aspirin. The Lancet,ii: 1213, 1979.
384
IV.
Grune
and
7.
Differential Hanley SP, Bevan J, Cockbill SR, Heptinstall S. inhibition of low-dose aspirin of human venous prostacyclin i: synthesis and platelet thromboxane synthesis. The Lancet, 969, 1981.
8.
Machin SJ, Chamone DAF, Defreyn G, Vermylen J. The effect of clinical rostacyclin infusions in advanced arterial disease on platelet function and plasma 6-keto-PGFla levels. British Journal of Haematology, 47: 413, 1981.
9.
Simon LS, Mills JA. Non-steroidal anti-inflammatory drugs. England Journal of Medicine, 302, 1179, 1980.
385
New
379-385, 1982
ASPIRIN, PROSTACYCLIN AND POST-OCCLUSIVE REACTIVE HYPERAEMIA IN MAN J.E. Tooke, V.C. Menys, H. Tindall, M. Feely & J.A. Davies University Department of Medicine, Leeds General Infirmary, Leeds LSl 3EX (reprint requests to JET). ABSTRACT We studied post-occlusive reactive hyperaemia using ecg-triggered mercury strain-gauge plethysmography in eight normal subjects treated The reactive with incremental doses of aspirin (27.5-1200 mg). hyperaemic response was measured in the finger (predominantly skin blood flow) and the calf (predominantly muscle). Concentrations of TXB2 and 6-oxo-PGF were measured in venous effluent blood from the hand by RIA, following'~rteria1 occlusion. Levels of TXB2 were significantly higher at O-10 and 60-70 seconds (p&.01), and 90-100 seconds (~8.05) following release of occlusion compared to pre-occlusion values. However there was no significant change in concentrations of 6-0x0-PGF,, and therefore by this method release of prostacyclin during reactive hyperaemia in the hand. Aspirin had no influence on finger or calf reactive hyperaemia 90 minutes after dosing, despite marked inhibition of platelet MDA production ( 75% after 110 mg, maximal inhibition after These data provide no support for the hypothesis that 1200 mg). prostacyclin is involved in the determination of the post-occlusive reactive hyperaemic response in the finger and calf in man. INTRODUCTION Local production of prostanoids may be an important determinant of the reactive hyperaemic response. Kilbom & Wennmalm (1) demonstrated that indomethacin suppressed the post-occlusive reactive hyperaemic response in the human forearm and that this suppression was accompanied by a reduction in the PGE-like activity in the venous effluent. Several other lines of evidence (2) suggest that prostaglandins are important in local circulatory control. Recent clinical interest has centered on the pathophysiological role and therapeutic potential of the potent vasodilator PGI . Results reported by Neri Serneri et al (3) leased during periods of arterial occlusion and the vasodilatation which follows release. We decided to study some aspects of the proposed inter-relationship between prostaglandin synthesis and blood flow. Initially, we examined
P-
6
379
whether aspirin, a non-competitive inhibitor of cyclooxygenase influenced the reactive hyperaemic response in a manner similar to that described by Kilbom & Wennmalm (1). Subsequently we tried to confirm the findings of Neri Serneri et al (3) that PGI2 is released during arterial occlusion. SUBJECTS AND METHODS Subjects. Eight healthy male non-smokers, age range from 25 to 37 years, mean 29 years were the subjects for the blood flow studies. The subjects for venous sampling studies were six healthy male non-smokers, age range 25 to 37 years, mean 31 years, four of whom participated in the blood flow studies. No subject was receiving any medication or had consumed aspirin-containing compounds or other known inhibitors of cyclooxygenase within two weeks of the experimental period. Blood flow was studied in each subject on seven Study design. occasions. ReSDOnSeS orior to aspirin administration were determined on two consecutive days. Responses were then determined on 5 consecutive days during incremental doses of aspirin. On the first day of dosing the subject was studied 90 minutes after ingestion of 27.5 mg of aspirin, and similarly following 110 mg on day 2, 330 mg on day 3, Vascular studies were performed 600 mg on day 4 and 1200 mg on day 5. at 9.00 a.m. followin8 acclimatization in a constant temperature room maintained at 24 f 0.5 C. Blood flow. Resting blood flow and the reactive hyperaemic response to a four minute Deriod of arterial occlusion were determined in the calf in 5 subjects,'and in the index finger in all subjects using the Janssen With Scientific Instruments Periflow mercury strain plethysmograph. this instrument an estimate of flow was made every 5 heartbeats, cuff This inflation and deflation being triggered by the R wave of the ECG. enabled an accurate semicontinuous assessment of the reactive hyperaemic response. Calf flow was determined in both legs and the average value Results were recorded for rest flow (mean blood used for analysis. flow in ml/100 ml of tissue/min recorded over 2 minute period prior to occlusion); peak flow (peak blood flow in ml/100 ml of tissue/min recorded within 10 seconds of release of occlusion); and total incremental flow (flow in ml/100 ml of tissue above rest flow recorded in 100 seconds following release of occlusion). On completion of measurements of blood flow, blood was Blood tests. taken tor estimation of plasma salicylate level, and malondialdehyde (MDA) production (4) as a measure of platelet cyclooxygenase inhibition. To assess PGI release during arterial occlusion we measured the level of the stabl$ metabolite 6-oxo-PGF by radioimnunoassay in venous educed retrogradely into a vein on blood. A butterfly cannula was int4* the dorsum of the hand. Two basal blood samples were taken 4 minutes apart and then 4 minutes arterial occlusion at the wrist applied. Following release of arterial occlusion samples were taken at O-10 Each sample seconds, 30-40 seconds, 60-70 seconds and 90-100 seconds. was withdrawn at a constant rate without venous occlusion and mixed immediately with indomethacin (final concentration 5D0~ol/11 and EDTA The samples (final concentration 5 mnol/l) in a numbered tube at 4 C. 380
were coded to avoid identification during assay and concentrations of were measured without extraction by RIA with a TXB and 6-oxo-PGF de&ion limit of '?20 pg/ml. Non-parametric statistical analysis (Friedman's Statistical analysis. method) was used to compare the post-occlusive reactive hyperaemic Plasma concentrations of TxB and 6response pre and post-aspirin. oxo-PGF during reactive hyperaemia were compared with the &ond of the two'gasal values. RESULTS Reactive hyperaemic response. Aspirin had no statistically significant influence on reactive hyperaemic responses in the calf or finger (Table la,b). Table la,b: la:
Group mean (&SEMI reactive hyperaemic responses following incremental aspirin doses.
Calf (mean left t right)
Aspirin Dose Rest Flow Incremental Peak Flow ml/l00ml/min (mg) ’ ml/l00ml/min : 27.5 110 330 600 1200 lb:
4.8 4.5 f 0.6 0.8 5.4 5.2 5.0 4.9 5.1
f + + i f
0.8 1.1 0.9 0.8 0.9
24.9 + 24.6 f 24.6 f 23.0 + 27.5 + 24.3 f 25.6 f
1.4 1.5 1.3 1.6 1.4 1.4 1.5
Total Incremental Flow 0-100s in ml/lOOml 11.8 11.2 12.0 11.3 12.5 11.9 12.3
+ f f + + f f
0.7 0.7 0.6 0.8 0.7 0.7 0.7
Finger
Aspirin Dose Rest Flow Incremental Peak Flow ml/l00ml/min ml/l00ml/min (mg) 0 2745 110 330 l?!
10.7 + 2.9 14.3 16.3 12.5 15.1 15.3 12.6
_L4.0 f 3.9 f 4.3 f 4.3 f 4.2 2.6
18.9 i 5.9 15.8 14.7 11.9 13.8 19.6 20.7
f f f f +
4.8 4.5 3.3 4.7 6.1 7.3
Total Incremental Flow 0-100's in ml/lOOml 8.2 5.5 8.1 5.7 6.1 7.7 7.0
f f + + + + +
2.4 1.5 2.2 1.8 1.9 3.0 2.1
Mean values obtained from the calf were very similar at all dosage levels (Table la). Responses from the finger showed wider variation at any point in time but again there were no discernable differences between values at any dosage level (Table lb). Finger responses on aspirin fell within the range defined by the two pre-aspirin responses. Table 2 surrmaritesthe percentage inhibition of platelet MDA production indicating inhibition of at least 75% following aspirin 110 fag and maximal inhibition after 1200 mg of aspirin. Plasma salicylate levels confirmed subject compliance. 381
Table 2:
Inhibition of platelet MDA production following varying doses ot asDirln exoressed as oercent ot control
Subject ASA 27.5 mg 24 32
:
4” 2 7
223" ia 5 40 62 28
a mean
ASA 110 mg
ASA 330 mg
ASA 600 mg
100
100
100
;! a2
9"8 91
180'0 76 95 92
PO70 91 97 97
;; 100 99 100 94 98 98
ASA 1200 mg 100
2
100 100 100 100 9'9'
TXB
and 6-oxo-PGF1, levels. Table 3 summarizes the results of assay and 6-oxo-PGF in plasma derived from the venous effluent blood of TxB8 6 s bjects followi *)P g release of arterial occlusion. Of Table 3: TX6 and 6-oxo-PGFl.(pg/ml) levels detectable in venous effluent from dorsum of the hand pre and post arterial occlusion Sample
TXB, Subject
Basal 1
: 4 5
487 76
PO
520 496
MeankSEM
3":
272*103
Basal 2
0-10s
30-40s
60-70s
90-100s
291
536 738 36 325 976 683
446 243 42 375 698 570
738 636 307 502 702 546
454 389 409 524 584 611
396k.95
572-t64
495t38
CO.01
co.05
90-100s
/: 28 397 524 213*90
CD.01
'p' value 6-oxo-PGFl,: Basal 2
0-10s
30-40s
60-70s
70 48 ND ND ND
!J: 75 ND ND ND
ND 140 49 45 ND ND
ND 75 60 24 ::
ND 130 51 21 ND ND
33f9
39f12
49*19
41*10
44218
3a+lo
N.S.
N.S.
N.S.
N.S.
Basal 1
1
ND
PO mean+SEM 'p' value
N.S.
Sample
Subject
: 5
549&135
:: 62 N"o" ND
ND denotes not detectable; for purposes of statistical analysis these points have been assigned an arbitrary value of 20 pg/ml. Subjects 1,2,4 and 5.took part in blood flow studies. 382
were low and there was no The basal levels of 6-0x0-PGF In contrast significant increase following releaL!& of occlusion. plasma TXB levels were statistically significantly higher at O-10, 6070 and 90-400 seconds following release compared with the second basal response. DISCUSSION These results suggest that aspirin has no discernable effect on the reactive hyperaemic response in the calf or digit even at doses which maximally inhibited platelet MDA production. It is unlikely that the plethysmographic recording of reactive hyperaemia was too insensitive to have detected an influence of aspirin as the present instrument permits a more faithful representation of the The marked response than that used by Kilbom and Wennmalm (1). physiological variation in finger blood flow that occurs from moment to moment (5) might have masked a significant effect of aspirin on the However as calf flow is reactive hyperaemic response in the digit. relatively stable and pre-aspirin responses were very reproducible this would not explain the lack of effect of aspirin on the calf reactive hyperaemic response. We found a significant rise in plasma TX6 concentrations following It is unlikely that th& was an artefact induced arterial occlusion. by vascular cannulation as there was no change in the two basal levels obtained. The increase in TXB probably indicated activation of platelets by relative ischaemia in zhe occluded segment. We have been unable to confirm the release of prostacyclin (measured by radioimnunoassay of 6-oxo-PGF ) in effluent venous blood following release of arterial occlusion as & scribed by Neri Serneri and co-workers (31. There are several possible explanations for this discrepancy. We took venous samples from the dorsum of the hand whereas Neri Serneri et al (31 presumably sampled from the antecubital fossa. The circulation to the hand is predominantly skin blood flow whereas the antecubital veins receive a significant proportion of their blood flow from the forearm muscles. It is possible that PG12 is involved in the autoregulation of muscle circulation but our results do not indicate any role for PGI in control of skin circulation. In the five subjects in whom we meas2 red calf blood flow (predominantly muscle) aspirin had no influence on the reactive hyperaemic response. Thus if PGI is involved in the autoregulation of muscle blood flow its activit5 is peculiarly resistant to the action of aspirin. There is some evidence that vessel wall cyclooxygenase might be more resistant to the action of aspirin than the enzyme in platelets (6) but findings using vein rings ex-vivo (7) suggest that both enzymes are sensitive to acetylation at comparatively low doses of around 81 mg. The results of our study are equally compatible with the interpretation that PGI is not involved in the determination of the reactive hyperaemic res6 nse. Kilbom & Wennmalm (1) and Neri Serneri (3) et al both used a bioassay to assess prostaglandin activity in the
383
venous effluent blood. We used radioimunoassay, and measured the level of the stable hydrolysis product of PGI Thus methodological differences might account for the apparent d?’screpancy and PGI -like activity might be present in a form undetectable by the assiB y we employed. However in view of the close correlation between infused PGI and recovery as imnunoassayable 6-oxo-PGF, (8) it seems imprgbable that PGI itself is concerned in control 8f the hyperaemic response to arteri4 occlusion. Indomethacin at the doses used by Kilbom and Wennmalm (1) may inhibit enzymes other than cyclooxygenase (9) which may not be influenced by aspirin. These enzymes might govern the generation of metabolites which are active in the determination of the reactive hyperaemic response. Our results do not exclude a role for products of arachidonate metabolism in control of reactive hyperaemia, but they indicate that any function of products dependent on the activity of cycloxoygenase is probably insignificant. Acknowledgements We acknowledge with gratitude support from the British Heart Antisera used in the radioimnunoassays Foundation (grant no. 80/58). were a generous gift of Pfizer Ltd. V.C.M. is supported by a grant from the M.R.C. REFERENCES ..-. - .._..__ 1.
Kilbom A, Wennmalm A. Endogenous prostaglandins as local effect of indomethacin on regulators of blood flow in man: Journal of Physiology reactive and functional hyperaemia. (London), 257: 109, 1976.
2.
Prostaglandins and local Messina EJ, Weiner R, and Kaley G. Federation Proccedings,35 (12): circulatory circulatory control. 2367, 1976.
3.
Release of Neri Serneri GG, Masotti G, Poggesi L, Galanti G. prostacyclin into the bloodstream and its exhaustion in humans after local blood flow changes (ischaemia and venous stasis). 197, 1980. Thrombosis Research, 17:
4.
Simple, sensitive McMillan RM, MacIntyre DE, Gordon JL. fluorimetric assay for malondialdehyde production by blood platelets. Thrombosis Research, 11: 425, 1977.
5.
Burch GE. Digital plethysmography, chapter Stratton, New York, 1954.
6.
Masotti G, Galanti G, Poggesi L, Abbate R, Neri Serneri GG. Differential inhibition of prostacyclin production and platelet aggregation by aspirin. The Lancet,ii: 1213, 1979.
384
IV.
Grune
and
7.
Differential Hanley SP, Bevan J, Cockbill SR, Heptinstall S. inhibition of low-dose aspirin of human venous prostacyclin i: synthesis and platelet thromboxane synthesis. The Lancet, 969, 1981.
8.
Machin SJ, Chamone DAF, Defreyn G, Vermylen J. The effect of clinical rostacyclin infusions in advanced arterial disease on platelet function and plasma 6-keto-PGFla levels. British Journal of Haematology, 47: 413, 1981.
9.
Simon LS, Mills JA. Non-steroidal anti-inflammatory drugs. England Journal of Medicine, 302, 1179, 1980.
385
New