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EFFECT OF ALIMENTARY LIPÆMIA ON THE THROMBOPLASTIC ACTIVITY OF SERUM
421
17’7±4’5% of the dose necessary to produce fibrillation. In the remaining three experiments, after ouabain had already induced fibrillation, nethalide was run cautiously into the vein. 3-4 mg. per kg. of nethalide converted the fibrillation to a regular rhythm (see figure). A total of 5 mg. per kg. nethalide was administered, and the infusion of ouabain was then continued. There was no further fibrillation, and the mean additional ouabain required to produce cardiac arrest was now 199 g. per kg. or 93% of the dose producing the original fibrillation. In two other experiments, when a strong solution of nethalide (10 mg. per ml.) was injected rapidly after fibrillation was established, not only did the fibrillation cease but the heart stopped altogether. or
Dichloroisopropyl noradrenaline had similar effects to nethalide. The P excitatory actions of this drug can be avoided by administering it as a slow infusion during 10 minutes, when it causes only a slowing of the heart-rate. EFFECT OF ALIMENTARY LIPÆMIA ON THE THROMBOPLASTIC ACTIVITY OF SERUM A
Simple Orientation
Test
on
Activity Changes
SERUM is active in accelerating coagulation,1-4 and this activity of serum extruded during clot retraction can cause an increase in size of the original clot.5-’ From the point of view of thrombogenesis it is important to study all the parameters which can change this activity. This paper concerns the finding that, under conditions to be described, this activity can be tested simply by its augmentation effect on alimentary lipasmia. METHODS
The
principle
1. 2. 3. 4. 5. 6. 7. TABLE
of the
test
is based
on
the
finding
Bordet, J., Gengou, O. Ann. inst. Pasteur, 1904, 18, 98. Hayem, G. L’hemoblaste. Paris, 1923. Wessler, S. J. clin. Invest. 1955, 34, 647. O’Brien, J. R. Brit. J. Hœmat. 1955, 1, 223. Quick, A. J. Lancet, 1960, i, 169. Wessler, S. J. clin. Invest. 1960, 39, 262. Alexander, B. Circulation, 1962, 25, 872.
I-TURBIDITY, PHOSPHOLIPIDS,
(PHYSIOLOGICAL SALINE-BLOOD, r2)
of
7-5 mg. per kg. was insufficient to prevent fibrillation; but 15 mg. per kg. was effective, and the lethal dose of ouabain was significantly increased. CONCLUSIONS
The conclusions that may be drawn from these results are that the slowing of the heart-rate caused by cardiac glycosides may be mainly due to a block of sympathetic action, and if this is already blocked no slowing occurs. Secondly, fibrillation can be both prevented and cured by blockade of p sympathetic actions. Nethalide might well be a valuable antidote in glycoside intoxication. E. M. VAUGHAN WILLIAMS D.M.,
Department of Pharmacology, Oxford University
4 HOURS
Oxon.
Soulier and Le Bolloch8 that serum thromboplastic activity inactivates heparin. Relative changes in activity are estimated indirectly from the degree of anticoagulant activity remaining after the reaction of a constant amount of heparin with a given sample of serum. The remaining activity of serum is determined as a function of the prolongation of coagulation-time of whole blood-i.e., such blood is added to serum with heparin after a constant interaction-time, and the effect is measured with the Hellige thromboelastograph (coagulation system no. 1 = serum-heparin-blood, the coagulability of which is denoted by the symbol r1). After simultaneous determination of coagulability of the blood mixed with the same amount of physiological saline instead of serum+heparin (coagulation system no. 2= physiological saline-whole blood, the coagulability of which is denoted r2) it is possible to express thromboplastic activity indirectly as the difference ri—r. 8.
Soulier, J. P., Le Bolloch, A. G. Rev. hémat. 1950, 5, 148.
AND COAGULATION-TIME IN SYSTEM NO. BEFORE AND
D.SC.
A. SEKIYA M.D. Nagoya
AFTER A FAT LOAD BY
MEASURE OF THROMBOPLASTIC ACTIVITY OF SERUM
TABLE II-CONTROL VALUES
1 (SERUM-HEPARIN-WHOLE BLOOD, rl) AND NO. 2 MOUTH, ALONG WITH VALUES OF Ar (rl-r2) AS A
422
The test is
performed as follows:
serum-exogenous
heparin,
cannot
be excluded.
Finally,
After clotting and clot retraction in 5 ml. blood samples it is possible that, under the influence of a fat load, there was at serum withdrawn after 30-40 minutes (i.e., 37°C) is increased production of heparin-neutralising lipids, in 10 minutes’ centrifugation at 5000 r.p.m., and used immethe qenqe of the rptinft*: of Amatuzio et a]. diately. Glassware was not siliconed. Test-tubes were kept in Institute for Cardiovascular Research, M. VAVřÍK a water bath at 37°C. For coagulation system no. 1, 2 ml. of Prague-Krc M.D. saline containing 3-3 units of heparin was added; and for coagulation system no. 2, 3 ml. of physiological saline only was added. To the system-1 tubes 1 ml. of fresh serum was PHOSPHORUS EXCRETION IN ACUTE AND also added, and the serum was allowed to interact with heparin CHRONIC RENAL DISEASE for 1 minute. To the tubes of both systems 1 ml. of freshly THE differentiation between reversible and irreversible drawn blood was then added, and the tubes were stoppered renal failure assumes great importance if the patient is a and shaken. Thromboelastograph cuvettes were next filled by pipette from each series. potential candidate for artificial dialysis, and especially if The blood was withdrawn while the serum was reacting with he gives an inadequate history. The finding of Goldman heparin, so that it could be added at precisely the right time and Bassets 16 that in chronic renal failure there is a special i.e., 1 minute after the addition of heparin. A stopwatch waspattern of excretion of inorganic phosphates as the renal used to count the time-interval taken by blood sampling (from function decreases stimulated us to investigate the the first appearance of blood in the syringe) so as to be able to possibility of a different pattern in cases of acute reversible start the thromboelastograph recording with precision-in this renal failure. The possibility is theoretically understandcase at the 4th minute (interval F=4 minutes). After the coagulation-time of both systems (rl and rz) had been deter- able, since this is a different type of renal lesion and is therefore expected to behave differently from the chronic mined, the rCr2 difference (A) could be calculated.
This
technique has been used to investigate the serum activity in thirteen healthy controls before and 4 hours after the ingestion of 250 ml. of cream or 70 g. of butter fat. Changes in lipaemia were judged by alterations in the turbidity of the serum (using a Unicam photometer at 620 m), and by analysis for phospholipids using the Fiske-Subbarow method. As controls for this test we used seven subjects who had fasted over the 4-hour period in
question. RESULTS
After fat ingestion, all thirteen subjects showed a considerable increase in serum-turbidity. There were no statistically significant changes in phospholipid content. After fat ingestion in twelve of the thirteen subjects, Ar, as a measure of thromboplastic activity, was significantly decreased when compared with the preingestion values. The average Ar before fat ingestion was 76 minutes 10 seconds, while after fat ingestion Ar was 17 minutes 16 seconds (p < 002). Since these values are indirectly proportional to the serum thromboplastic activity, it seems that a fat load increases this activity. The detailed results are presented in table i, and the control values are given in table 11. The antiheparin activity of the serum is not necessarily the same as the thromboplastic activity; but it seems that the former gives some approximation to the latter as Poller9 10 and O’Brien il have found. These findings appear to provide further evidence of lipogenic acceleration of blood coagulation. McGandy et a1.12 have tested the effect of fat on the thromboplastic activity of serum in chickens; they reported no effect of increased dietary fat on the Wessler serum-thrombus formation. Since the basis of this reaction is not yet known,6 it is difficult to explain the mechanism of the action of fat. The increase in serum thromboplastic activity after fat ingestion is not related to the degree of serum turbidity nor to the changes in serum phospholipids; and this agrees with work concerning the relation of alimentary lipaemia and coagulation using other methods.13 14 The possible role of endogenous heparin in the sensitive system bloodPoller, L. J. clin. Path. 1959, 12, 331. Poller, L. ibid. 1960, 13, 226. O’Brien, J. R. ibid. p. 93. McGandy, R. B., Gotsis, A., Hegsted, D. M. Proc. Soc. exp. Biol., N.Y. 1960, 315, 105. 13. Buzina, R., Keys, A. Circulation, 1956, 14, 854. 14. Keys, A., Buzina, R., Grande, F., Anderson, J. T. ibid. 1957, 15, 274.
type. As the glomerular filtration-rate (G.F.R.) decreases with advancing chronic renal disease, the glomerular filtration of phosphate and the urinary phosphate tend to become substantially identical, indicating a quantitative transfer of the phosphorus filtered at the glomeruli to the urine." Therefore, as the G.F.R. decreases the ratio clearance-ofphosphorus/ c1earance-of-creatinine (Cp/Ccr) increases. In other words, as the G.F.R. decreases, the fraction of filtered phosphorus excreted increases.17 Whereas normally the Cp/Ccr is correlated to the serum inorganic phosphorus,18 in chronic renal failure the increase in Cp/Ccr as the G.F.R. decreases is independent of the serum phosphorus." This is possibly a mechanism Amatuzio, D. S., Grande, F., Wada, S., Hay, L. J. J. Lab. clin. Med. 1960, 56, 277. 16. Goldman, R., Bassett, S. H. J. clin. Invest. 1954, 33, 1623. 17. Reiss, E., Bricker, N. S., Kime, S. W., Jr., Morrin, P. A. F. ibid. 1961, 40, 165. 18. Stanbury, S. W. Advanc. intern. Med. 1958, 9, 231. 15.
9. 10. 11. 12.
Fig. I-Creatinine
clearance (ml. per min.).
17’7±4’5% of the dose necessary to produce fibrillation. In the remaining three experiments, after ouabain had already induced fibrillation, nethalide was run cautiously into the vein. 3-4 mg. per kg. of nethalide converted the fibrillation to a regular rhythm (see figure). A total of 5 mg. per kg. nethalide was administered, and the infusion of ouabain was then continued. There was no further fibrillation, and the mean additional ouabain required to produce cardiac arrest was now 199 g. per kg. or 93% of the dose producing the original fibrillation. In two other experiments, when a strong solution of nethalide (10 mg. per ml.) was injected rapidly after fibrillation was established, not only did the fibrillation cease but the heart stopped altogether. or
Dichloroisopropyl noradrenaline had similar effects to nethalide. The P excitatory actions of this drug can be avoided by administering it as a slow infusion during 10 minutes, when it causes only a slowing of the heart-rate. EFFECT OF ALIMENTARY LIPÆMIA ON THE THROMBOPLASTIC ACTIVITY OF SERUM A
Simple Orientation
Test
on
Activity Changes
SERUM is active in accelerating coagulation,1-4 and this activity of serum extruded during clot retraction can cause an increase in size of the original clot.5-’ From the point of view of thrombogenesis it is important to study all the parameters which can change this activity. This paper concerns the finding that, under conditions to be described, this activity can be tested simply by its augmentation effect on alimentary lipasmia. METHODS
The
principle
1. 2. 3. 4. 5. 6. 7. TABLE
of the
test
is based
on
the
finding
Bordet, J., Gengou, O. Ann. inst. Pasteur, 1904, 18, 98. Hayem, G. L’hemoblaste. Paris, 1923. Wessler, S. J. clin. Invest. 1955, 34, 647. O’Brien, J. R. Brit. J. Hœmat. 1955, 1, 223. Quick, A. J. Lancet, 1960, i, 169. Wessler, S. J. clin. Invest. 1960, 39, 262. Alexander, B. Circulation, 1962, 25, 872.
I-TURBIDITY, PHOSPHOLIPIDS,
(PHYSIOLOGICAL SALINE-BLOOD, r2)
of
7-5 mg. per kg. was insufficient to prevent fibrillation; but 15 mg. per kg. was effective, and the lethal dose of ouabain was significantly increased. CONCLUSIONS
The conclusions that may be drawn from these results are that the slowing of the heart-rate caused by cardiac glycosides may be mainly due to a block of sympathetic action, and if this is already blocked no slowing occurs. Secondly, fibrillation can be both prevented and cured by blockade of p sympathetic actions. Nethalide might well be a valuable antidote in glycoside intoxication. E. M. VAUGHAN WILLIAMS D.M.,
Department of Pharmacology, Oxford University
4 HOURS
Oxon.
Soulier and Le Bolloch8 that serum thromboplastic activity inactivates heparin. Relative changes in activity are estimated indirectly from the degree of anticoagulant activity remaining after the reaction of a constant amount of heparin with a given sample of serum. The remaining activity of serum is determined as a function of the prolongation of coagulation-time of whole blood-i.e., such blood is added to serum with heparin after a constant interaction-time, and the effect is measured with the Hellige thromboelastograph (coagulation system no. 1 = serum-heparin-blood, the coagulability of which is denoted by the symbol r1). After simultaneous determination of coagulability of the blood mixed with the same amount of physiological saline instead of serum+heparin (coagulation system no. 2= physiological saline-whole blood, the coagulability of which is denoted r2) it is possible to express thromboplastic activity indirectly as the difference ri—r. 8.
Soulier, J. P., Le Bolloch, A. G. Rev. hémat. 1950, 5, 148.
AND COAGULATION-TIME IN SYSTEM NO. BEFORE AND
D.SC.
A. SEKIYA M.D. Nagoya
AFTER A FAT LOAD BY
MEASURE OF THROMBOPLASTIC ACTIVITY OF SERUM
TABLE II-CONTROL VALUES
1 (SERUM-HEPARIN-WHOLE BLOOD, rl) AND NO. 2 MOUTH, ALONG WITH VALUES OF Ar (rl-r2) AS A
422
The test is
performed as follows:
serum-exogenous
heparin,
cannot
be excluded.
Finally,
After clotting and clot retraction in 5 ml. blood samples it is possible that, under the influence of a fat load, there was at serum withdrawn after 30-40 minutes (i.e., 37°C) is increased production of heparin-neutralising lipids, in 10 minutes’ centrifugation at 5000 r.p.m., and used immethe qenqe of the rptinft*: of Amatuzio et a]. diately. Glassware was not siliconed. Test-tubes were kept in Institute for Cardiovascular Research, M. VAVřÍK a water bath at 37°C. For coagulation system no. 1, 2 ml. of Prague-Krc M.D. saline containing 3-3 units of heparin was added; and for coagulation system no. 2, 3 ml. of physiological saline only was added. To the system-1 tubes 1 ml. of fresh serum was PHOSPHORUS EXCRETION IN ACUTE AND also added, and the serum was allowed to interact with heparin CHRONIC RENAL DISEASE for 1 minute. To the tubes of both systems 1 ml. of freshly THE differentiation between reversible and irreversible drawn blood was then added, and the tubes were stoppered renal failure assumes great importance if the patient is a and shaken. Thromboelastograph cuvettes were next filled by pipette from each series. potential candidate for artificial dialysis, and especially if The blood was withdrawn while the serum was reacting with he gives an inadequate history. The finding of Goldman heparin, so that it could be added at precisely the right time and Bassets 16 that in chronic renal failure there is a special i.e., 1 minute after the addition of heparin. A stopwatch waspattern of excretion of inorganic phosphates as the renal used to count the time-interval taken by blood sampling (from function decreases stimulated us to investigate the the first appearance of blood in the syringe) so as to be able to possibility of a different pattern in cases of acute reversible start the thromboelastograph recording with precision-in this renal failure. The possibility is theoretically understandcase at the 4th minute (interval F=4 minutes). After the coagulation-time of both systems (rl and rz) had been deter- able, since this is a different type of renal lesion and is therefore expected to behave differently from the chronic mined, the rCr2 difference (A) could be calculated.
This
technique has been used to investigate the serum activity in thirteen healthy controls before and 4 hours after the ingestion of 250 ml. of cream or 70 g. of butter fat. Changes in lipaemia were judged by alterations in the turbidity of the serum (using a Unicam photometer at 620 m), and by analysis for phospholipids using the Fiske-Subbarow method. As controls for this test we used seven subjects who had fasted over the 4-hour period in
question. RESULTS
After fat ingestion, all thirteen subjects showed a considerable increase in serum-turbidity. There were no statistically significant changes in phospholipid content. After fat ingestion in twelve of the thirteen subjects, Ar, as a measure of thromboplastic activity, was significantly decreased when compared with the preingestion values. The average Ar before fat ingestion was 76 minutes 10 seconds, while after fat ingestion Ar was 17 minutes 16 seconds (p < 002). Since these values are indirectly proportional to the serum thromboplastic activity, it seems that a fat load increases this activity. The detailed results are presented in table i, and the control values are given in table 11. The antiheparin activity of the serum is not necessarily the same as the thromboplastic activity; but it seems that the former gives some approximation to the latter as Poller9 10 and O’Brien il have found. These findings appear to provide further evidence of lipogenic acceleration of blood coagulation. McGandy et a1.12 have tested the effect of fat on the thromboplastic activity of serum in chickens; they reported no effect of increased dietary fat on the Wessler serum-thrombus formation. Since the basis of this reaction is not yet known,6 it is difficult to explain the mechanism of the action of fat. The increase in serum thromboplastic activity after fat ingestion is not related to the degree of serum turbidity nor to the changes in serum phospholipids; and this agrees with work concerning the relation of alimentary lipaemia and coagulation using other methods.13 14 The possible role of endogenous heparin in the sensitive system bloodPoller, L. J. clin. Path. 1959, 12, 331. Poller, L. ibid. 1960, 13, 226. O’Brien, J. R. ibid. p. 93. McGandy, R. B., Gotsis, A., Hegsted, D. M. Proc. Soc. exp. Biol., N.Y. 1960, 315, 105. 13. Buzina, R., Keys, A. Circulation, 1956, 14, 854. 14. Keys, A., Buzina, R., Grande, F., Anderson, J. T. ibid. 1957, 15, 274.
type. As the glomerular filtration-rate (G.F.R.) decreases with advancing chronic renal disease, the glomerular filtration of phosphate and the urinary phosphate tend to become substantially identical, indicating a quantitative transfer of the phosphorus filtered at the glomeruli to the urine." Therefore, as the G.F.R. decreases the ratio clearance-ofphosphorus/ c1earance-of-creatinine (Cp/Ccr) increases. In other words, as the G.F.R. decreases, the fraction of filtered phosphorus excreted increases.17 Whereas normally the Cp/Ccr is correlated to the serum inorganic phosphorus,18 in chronic renal failure the increase in Cp/Ccr as the G.F.R. decreases is independent of the serum phosphorus." This is possibly a mechanism Amatuzio, D. S., Grande, F., Wada, S., Hay, L. J. J. Lab. clin. Med. 1960, 56, 277. 16. Goldman, R., Bassett, S. H. J. clin. Invest. 1954, 33, 1623. 17. Reiss, E., Bricker, N. S., Kime, S. W., Jr., Morrin, P. A. F. ibid. 1961, 40, 165. 18. Stanbury, S. W. Advanc. intern. Med. 1958, 9, 231. 15.
9. 10. 11. 12.
Fig. I-Creatinine
clearance (ml. per min.).