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Long-term heparin infusion and plasma lipoprotein lipase activity
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
LONG-TERM H E P A R I N I N F U S I O N AND PLASMA L I P O P R O T E I N L I P A S E ACTIVITY
J. R. MUIR W. E. Dunn Laboratory for Cardiovascular Metabolism, Institute of Cardiology, and the National Heart Hospital, London, W.J (Great Britain)
(Received July 12th, 1968)
SUMMARY
The effect of a prolonged intravenous infusion of heparin on plasma lipolytic activity has been studied in 5 patients following recent myocardial infarction. The level of iipoprotein lipase in the plasma fell over the first 3-7 days, and then rose to a level higher than that present on the first day of the heparin infusion. This secondary peak was followed by a further fall in plasma activity. The fluctuations in plasma lipoprotein lipase activity m a y have been due to changes in the sensitivity of the patients to heparin. Whether this was the result of acute myocardial infarction is not clear, as no control studies have been performed. The initial low levels of plasma enzyme m a y have been due to suppression of enzyme synthesis b y high levels of plasma catecholamines and free f a t t y acids following the infarction.
Keywords: H e p a r i n - P l a s m a lipoprotein lipase - M y o c a r d i a l infarction
INTRODUCTION
The intravenous injection of heparin in man and m a n y animals is followed b y the rapid appearance of a lipolytic enzyme, lipoprotein lipase, in the plasma1, ~. The speed with which the enzyme appears in the plasma after heparin has led to the hypothesis that it is situated on or near the capillary endothelium, and that heparin causes its release b y combining with the enzyme perhaps in competition with endogenous heparin2, a. Heparin also stabilizes the plasma enzyme 4, and m a y play an
Present address of the author: Edward A. Doisy Department of Biochemistry, School of Medicine, University of St. Louis, 1402 South Grand Boulevard, St. Louis, Mo. 63104 (U.S.A.). j . Atheroscler. Res., 1968, 8:983-987
984
SHORT COMMUNICATIONS
important part in the lipolytic action of lipoprotein lipase by facilitating its combination with the triglycerides of the chylomicra and very low density lipoproteins 3. Following the injection of 35S-labelled heparin, the time course of heparin radioactive decay and plasma lipolytic activity follow a similar course 5, suggesting that there may be a physico-chemical association between circulating heparin and plasma lipoprotein lipase levels. Although studies have shown that it is possible to deplete perfused organs of elutable lipoprotein lipase by repeated perfusions with heparin 6, the effect of longterm heparin infusion in intact animals has not been studied. This is a preliminary report of such a study using patients who were being treated with heparin infusions after recent myocardial infarction. MATERIALS AND METHODS
Five patients, 4 men and 1 woman, were studied. Their ages ranged from 41 to 63, and they had all suffered recent myocardial infarction. Following admission, they were treated by bed rest and an intravenous infusion of heparin (40,000 I.U. in 500 ml 5 ~o dextrose/24 h) which was continued for a period varying from 8 to 14 days. 10 ml of blood were collected from the antecubital vein in a heparinised syringe every morning after an overnight fast. The plasma was separated by centrifuging the blood at 2600 • g for 15 min at 4~ and the plasma lipolytic activity was either measured immediately or within 10 days of its collection, the plasma being stored in the deep freeze in the interim period. This short storage period does not affect plasma lipoprotein lipase activity to any marked extent 7. The plasma lipolytic activity was measured in an assay system, containing 2 ml of 20 % bovine serum albumin (pH 8.5) from which all the free fatty acids had been extracted b y the method of GOODMANS; 1 ml of 0.1 M Tris buffer (pH 8.5); 1 ml of 20 % "Intralipid" and 1 ml of plasma. The assay system was incubated for 20 min in a water bath at 37~ with continuous gentle shaking. 1-ml aliquots were removed after 5 and 20 min incubation for the measurement of free fatty acid concentration by the method of DOLE AND MEINERTZ9. The release of free fatty acids is linear in this system between 5 and 30 min 1~ The plasma lipolytic activity was characterised on each day in all the patients by measuring the degree of inhibition produced by molar saline. Fifty % inhibition by molar saline is characteristic of lipoprotein lipase in this system 10. RESULTS
The plasma lipolytic activities for the 5 patients on each morning following the start of the heparin infusion are shown in Fig. 1. In all the patients the plasma lipolytic activity fell over the first week after the heparin was started. The rate at which the plasma lipolytic activity declined and the level to which it fell varied from case to case. The initial decline was followed b y a rise in the plasma lipolytic activity to levels higher than those found in the first 2 days. This second peak occurred between the J. Atherosder. Res., 1968, 8:983-987
SHORT COMMUNICATIONS
985
0"40
;
0.30
A.N.~ ~_ 0.20
>-"
/ , /
/1t
..o-LY", t \
A~. 9
is.i2~
~.S/ /",
......
i it,5 D,-" ;~t,",,-//
~ !
I,
':'
~" o.lo
',
'
.2
o
~
~ "
',,,// ".,........." ~
""" ......... ' TIME
0-05
~+
io'
'":"
14"
(Days)
Fig. 1. The leve]s of lipoprotein lipase in the plasma of 5 subjects on prolonged heparin infusion (40,000 I.U./24 h) following myocardial infarction.
7th and the 10th day. In patient A.M. the heparin infusion had to be stopped after 8 days due to subcutaneous bleeding but in the other 4 patients the secondary peak was followed by a drop in the activity over the next 24-48 h, after which the levels began to rise again. The possibility that the secondary peaks of plasma lipolytic activity were due to the appearance in the plasma of an enzyme other than lipoprotein lipase was considered. However, the plasma lipolytic activity showed approximately 50 % inhibition with molar saline throughout the duration of the heparin infusion. This degree of inhibition is characteristic of lipoprotein lipase in this asay system 10. The changes in plasma lipolytic activity could have been due to the phasic appearance of an inhibitor of lipoprotein lipase in the circulation. This possibility was investigated in subject H.L. Equal volumes of plasma collected on the 8th day, when no lipolytic activity could be detected in the circulation, and on the 10th day when the
TABLE 1 T H E E F F E C T O F P L A S M A C O L L E C T E D ON D A Y 8 O N T H E L I P O L Y T I C A C T I V I T Y O F P L A S M A C O L L E C T E D DAY
l0 IN SUBJECT
ON
H.L.
Sample
Lipolytic activity (t, equiv FFA /ml plasma/rain)
1 ml 8 day plasma 1 ml 10 day plasma 0.5 ml 8 day plasma 0.5 ml 10 day plasma 0.5 ml 8 day plasma + 0.5 ml 10 day plasma
0.00 0.29 0.00 0.15 0.14 J. Atheroscler. Res., 1968, 8:983-987
986
SHORT COMMUNICATIONS
plasma lipolytic activity was again high, were mixed. The activity of the composite plasma was then measured (Table 1). It is clear that the plasma from the 8th day did not have any inhibitory action on lipoprotein lipase. DISCUSSION
The fluctuations in plasma lipoprotein lipase in response to prolonged heparin infusion were surprising. It may be that they were related to the acute myocardial infarction for which the subjects were being treated, but as no studies of this type have been carried out with normal subjects this is conjectural. As yet it has not proved possible to devise a suitable and ethical "control" clinical situation. The changes in the plasma levels could be due to alterations in a number of factors: (a) the rate of synthesis of the enzyme in the tissues, (b) the sensitivity or the availability of the formed enzyme to release by circulating heparin and (c) the rate of destruction or removal of the enzyme from the plasma. The level of the enzyme in the tissues is controlled by a balance between enzyme synthesis and destruction, the balance being under metabolic controlll, 12. The synthesis of lipoprotein lipase in adipose tissue is depressed by adrenaline and noradrenaline 11 and by a rise in free fatty acid concentration 13. As the plasma free fatty acids and catecholamines are elevated following myocardial infarction14,15, it is possible that this could explain the relatively low levels of lipoprotein lipase released by heparin in the first few days after a myocardial infarct. However, subsequent phasic fluctuations in the plasma levels of the enzyme cannot be explained by changes in circulating catecholamines or free fatty acids. It is unlikely that the failure to demonstrate any lipoprotein lipase activity in the plasma of 2 patients (H.R. and H.L.) at several stages during the heparin infusion, was due to the exhaustion of all of the enzyme situated near the capillary endothelium, as it is this fraction of the enzyme that is primarily concerned with the uptake of triglycerides by the peripheral tissues 12. If this fraction was exhausted, the fasting plasma would be expected to be milky due to the presence of excess chylomicra as is the case in fat-induced hypertriglyceridaemia, in which there is a deficiency of adipose tissue lipoprotein lipase 16. However, the plasma drawn from the 2 patients on these occasions was normal in appearance. It is therefore likely that the sensitivity of the patients to heparin changed during the course of the prolonged infusion. The third factor which could influence the levels of lipoprotein lipase in the plasma is the rate with which the enzyme is destroyed or removed from the plasma. In man, the enzyme is removed from the plasma in both the liver 17 and the lungs is and there is a positive correlation between the levels of endogenous arterial lipoprotein lipase and the pulmonary artery pressure 19. Changes in the plasma levels of lipoprotein lipasein response to a standard heparin infusion might therefore be expected to occur in patients with altering degrees of cardiac failure. However, this is not applicable to the present group of patients, as none of them was in cardiac failure at any time during the study. J. Atherosclev. Res., 1968, 8:983-987
SHORT COMMUNICATIONS
987
REFERENCES 1 SHORE, B., A. V. NICHOLS AND N. K. FREEMAN, Evidence for lipolytic action by h u m a n p l a s m a
2 3
4 5 6 7 8 9
10 11
12
obtained after intravenous administration of heparin, Proc. Soc. exp. Biol. ( N . Y . ) , 1953, 86: 709. ROBINSON, D. S. AND J. E. FRENCH, Heparin, the clearing factor and fat transport, Pharmacol. Rev., 1960, 12: 241. ENGELBERG, H., Heparin and the removal of triglycerides from the blood stream, Amer. J. Cardiol., 1964, 14: 8. ROBINSON, D. S., F u r t h e r studies on the lipolytic s y s t e m induced in p l a s m a by heparin infusion, Quart. J. exp. Physiol., 1956, 14: 195. PORTE, D. AND R. H. WILLIAMS, Post heparin lipolytic activity following intravenous heparin S-35, Proc. Sue. exp. Biol. ( N . Y . ) , 1965, 118: 639. ROBINSON, 2[). S. AND M. A. JENNINGS, Release of clearing factor lipase by the perfused r a t heart, J. Lipid Res., 1965, 6: 222. BOBERG, J. AND L. A. CARLSON, Determination of heparin-induced lipoprotein lipase activity in h u m a n plasma, Clin. chim. Acta, 1964, 10: 420. GOODMAN, D. S., Preparation of h u m a n serum albumin free of long chain fatty acids, Science, 1957, 123: 1296. DOLE, V. P. AND H. MEINERTZ, Microdetermination of long chain f a t t y acids in p l a s m a and tissues, J. biol. Chem., 1960, 235: 2595. MUIR, J. R., A rapid m e t h o d for the assay of plasma lipoprotein in lipase using " I n t r a l i p i d " , Clin. chim. Acta, 1967, 17: 312. WING, D. R., M. R. SALAMANAND D. S. ROBINSON, Clearing factor lipase in adipose tissue. Factors influencing the increase in enzyme activity produced on incubation of tissue from starved rats in vitro, Biochem. J., 1966, 99: 648. ROBINSON, D. S. AND D. R. WING, Role of clearing factor lipase in uptake of triglycerides. In:
Enzyme Activities Related to Lipid Transport, 7 lth Int. Conf. Biochem. Lipids, Jerusalem, 1967. 13 NIKKILA, E. A., H o r m o n a l and metabolic regulations of lipoprotein lipase activity of adipose
tissue. In: Enzyme Activities Related to Lipid Transport, l l t h Int. Conf. Biochem. Lipids, Jerusalem, 7967. 14 OLIVER, M. F., V. A. KURIEN AND T. W. GREENWOOD, Relation between serum free f a t t y acids
and a r r h y t h m i a s and death after acute myocardial infarction, Lancet, 1968, 1: 710. 15 BRAY, C., E. L. McDONALD AND ~N~.RESTIEUX, Personal communication, 1968. 16 HARLAN, W. R., P. S. WINESETT AND A. J. WASSERMAN, Tissue lipoprotein lipase in normal individuals and in individuals with exogenous hypertriglyceridaemia and the relationship of this enzyme to t h e assimilation of fat, J. clin. Invest., 1967, 46: 239. 17 YOSHITOSHI, Y., C. NAITO, H. OKANIWA, M. USUI, T. MOGAMI AND T. TOMONO, Kinetic studies on metabolism of lipoprotein lipase, J. clin. Invest., 1968, 43: 707. is MUIR, J. R., The regional production of lipoprotein lipase, Clin. Sci., 1968, 34: 261. 19 MUIR, J. R., Endogenous p l a s m a lipolytic activity in m a n and its handling by the lungs, In preparation.
J. Atheroscler. Res., 1968, 8 : 9 8 3 - 9 8 7
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
LONG-TERM H E P A R I N I N F U S I O N AND PLASMA L I P O P R O T E I N L I P A S E ACTIVITY
J. R. MUIR W. E. Dunn Laboratory for Cardiovascular Metabolism, Institute of Cardiology, and the National Heart Hospital, London, W.J (Great Britain)
(Received July 12th, 1968)
SUMMARY
The effect of a prolonged intravenous infusion of heparin on plasma lipolytic activity has been studied in 5 patients following recent myocardial infarction. The level of iipoprotein lipase in the plasma fell over the first 3-7 days, and then rose to a level higher than that present on the first day of the heparin infusion. This secondary peak was followed by a further fall in plasma activity. The fluctuations in plasma lipoprotein lipase activity m a y have been due to changes in the sensitivity of the patients to heparin. Whether this was the result of acute myocardial infarction is not clear, as no control studies have been performed. The initial low levels of plasma enzyme m a y have been due to suppression of enzyme synthesis b y high levels of plasma catecholamines and free f a t t y acids following the infarction.
Keywords: H e p a r i n - P l a s m a lipoprotein lipase - M y o c a r d i a l infarction
INTRODUCTION
The intravenous injection of heparin in man and m a n y animals is followed b y the rapid appearance of a lipolytic enzyme, lipoprotein lipase, in the plasma1, ~. The speed with which the enzyme appears in the plasma after heparin has led to the hypothesis that it is situated on or near the capillary endothelium, and that heparin causes its release b y combining with the enzyme perhaps in competition with endogenous heparin2, a. Heparin also stabilizes the plasma enzyme 4, and m a y play an
Present address of the author: Edward A. Doisy Department of Biochemistry, School of Medicine, University of St. Louis, 1402 South Grand Boulevard, St. Louis, Mo. 63104 (U.S.A.). j . Atheroscler. Res., 1968, 8:983-987
984
SHORT COMMUNICATIONS
important part in the lipolytic action of lipoprotein lipase by facilitating its combination with the triglycerides of the chylomicra and very low density lipoproteins 3. Following the injection of 35S-labelled heparin, the time course of heparin radioactive decay and plasma lipolytic activity follow a similar course 5, suggesting that there may be a physico-chemical association between circulating heparin and plasma lipoprotein lipase levels. Although studies have shown that it is possible to deplete perfused organs of elutable lipoprotein lipase by repeated perfusions with heparin 6, the effect of longterm heparin infusion in intact animals has not been studied. This is a preliminary report of such a study using patients who were being treated with heparin infusions after recent myocardial infarction. MATERIALS AND METHODS
Five patients, 4 men and 1 woman, were studied. Their ages ranged from 41 to 63, and they had all suffered recent myocardial infarction. Following admission, they were treated by bed rest and an intravenous infusion of heparin (40,000 I.U. in 500 ml 5 ~o dextrose/24 h) which was continued for a period varying from 8 to 14 days. 10 ml of blood were collected from the antecubital vein in a heparinised syringe every morning after an overnight fast. The plasma was separated by centrifuging the blood at 2600 • g for 15 min at 4~ and the plasma lipolytic activity was either measured immediately or within 10 days of its collection, the plasma being stored in the deep freeze in the interim period. This short storage period does not affect plasma lipoprotein lipase activity to any marked extent 7. The plasma lipolytic activity was measured in an assay system, containing 2 ml of 20 % bovine serum albumin (pH 8.5) from which all the free fatty acids had been extracted b y the method of GOODMANS; 1 ml of 0.1 M Tris buffer (pH 8.5); 1 ml of 20 % "Intralipid" and 1 ml of plasma. The assay system was incubated for 20 min in a water bath at 37~ with continuous gentle shaking. 1-ml aliquots were removed after 5 and 20 min incubation for the measurement of free fatty acid concentration by the method of DOLE AND MEINERTZ9. The release of free fatty acids is linear in this system between 5 and 30 min 1~ The plasma lipolytic activity was characterised on each day in all the patients by measuring the degree of inhibition produced by molar saline. Fifty % inhibition by molar saline is characteristic of lipoprotein lipase in this system 10. RESULTS
The plasma lipolytic activities for the 5 patients on each morning following the start of the heparin infusion are shown in Fig. 1. In all the patients the plasma lipolytic activity fell over the first week after the heparin was started. The rate at which the plasma lipolytic activity declined and the level to which it fell varied from case to case. The initial decline was followed b y a rise in the plasma lipolytic activity to levels higher than those found in the first 2 days. This second peak occurred between the J. Atherosder. Res., 1968, 8:983-987
SHORT COMMUNICATIONS
985
0"40
;
0.30
A.N.~ ~_ 0.20
>-"
/ , /
/1t
..o-LY", t \
A~. 9
is.i2~
~.S/ /",
......
i it,5 D,-" ;~t,",,-//
~ !
I,
':'
~" o.lo
',
'
.2
o
~
~ "
',,,// ".,........." ~
""" ......... ' TIME
0-05
~+
io'
'":"
14"
(Days)
Fig. 1. The leve]s of lipoprotein lipase in the plasma of 5 subjects on prolonged heparin infusion (40,000 I.U./24 h) following myocardial infarction.
7th and the 10th day. In patient A.M. the heparin infusion had to be stopped after 8 days due to subcutaneous bleeding but in the other 4 patients the secondary peak was followed by a drop in the activity over the next 24-48 h, after which the levels began to rise again. The possibility that the secondary peaks of plasma lipolytic activity were due to the appearance in the plasma of an enzyme other than lipoprotein lipase was considered. However, the plasma lipolytic activity showed approximately 50 % inhibition with molar saline throughout the duration of the heparin infusion. This degree of inhibition is characteristic of lipoprotein lipase in this asay system 10. The changes in plasma lipolytic activity could have been due to the phasic appearance of an inhibitor of lipoprotein lipase in the circulation. This possibility was investigated in subject H.L. Equal volumes of plasma collected on the 8th day, when no lipolytic activity could be detected in the circulation, and on the 10th day when the
TABLE 1 T H E E F F E C T O F P L A S M A C O L L E C T E D ON D A Y 8 O N T H E L I P O L Y T I C A C T I V I T Y O F P L A S M A C O L L E C T E D DAY
l0 IN SUBJECT
ON
H.L.
Sample
Lipolytic activity (t, equiv FFA /ml plasma/rain)
1 ml 8 day plasma 1 ml 10 day plasma 0.5 ml 8 day plasma 0.5 ml 10 day plasma 0.5 ml 8 day plasma + 0.5 ml 10 day plasma
0.00 0.29 0.00 0.15 0.14 J. Atheroscler. Res., 1968, 8:983-987
986
SHORT COMMUNICATIONS
plasma lipolytic activity was again high, were mixed. The activity of the composite plasma was then measured (Table 1). It is clear that the plasma from the 8th day did not have any inhibitory action on lipoprotein lipase. DISCUSSION
The fluctuations in plasma lipoprotein lipase in response to prolonged heparin infusion were surprising. It may be that they were related to the acute myocardial infarction for which the subjects were being treated, but as no studies of this type have been carried out with normal subjects this is conjectural. As yet it has not proved possible to devise a suitable and ethical "control" clinical situation. The changes in the plasma levels could be due to alterations in a number of factors: (a) the rate of synthesis of the enzyme in the tissues, (b) the sensitivity or the availability of the formed enzyme to release by circulating heparin and (c) the rate of destruction or removal of the enzyme from the plasma. The level of the enzyme in the tissues is controlled by a balance between enzyme synthesis and destruction, the balance being under metabolic controlll, 12. The synthesis of lipoprotein lipase in adipose tissue is depressed by adrenaline and noradrenaline 11 and by a rise in free fatty acid concentration 13. As the plasma free fatty acids and catecholamines are elevated following myocardial infarction14,15, it is possible that this could explain the relatively low levels of lipoprotein lipase released by heparin in the first few days after a myocardial infarct. However, subsequent phasic fluctuations in the plasma levels of the enzyme cannot be explained by changes in circulating catecholamines or free fatty acids. It is unlikely that the failure to demonstrate any lipoprotein lipase activity in the plasma of 2 patients (H.R. and H.L.) at several stages during the heparin infusion, was due to the exhaustion of all of the enzyme situated near the capillary endothelium, as it is this fraction of the enzyme that is primarily concerned with the uptake of triglycerides by the peripheral tissues 12. If this fraction was exhausted, the fasting plasma would be expected to be milky due to the presence of excess chylomicra as is the case in fat-induced hypertriglyceridaemia, in which there is a deficiency of adipose tissue lipoprotein lipase 16. However, the plasma drawn from the 2 patients on these occasions was normal in appearance. It is therefore likely that the sensitivity of the patients to heparin changed during the course of the prolonged infusion. The third factor which could influence the levels of lipoprotein lipase in the plasma is the rate with which the enzyme is destroyed or removed from the plasma. In man, the enzyme is removed from the plasma in both the liver 17 and the lungs is and there is a positive correlation between the levels of endogenous arterial lipoprotein lipase and the pulmonary artery pressure 19. Changes in the plasma levels of lipoprotein lipasein response to a standard heparin infusion might therefore be expected to occur in patients with altering degrees of cardiac failure. However, this is not applicable to the present group of patients, as none of them was in cardiac failure at any time during the study. J. Atherosclev. Res., 1968, 8:983-987
SHORT COMMUNICATIONS
987
REFERENCES 1 SHORE, B., A. V. NICHOLS AND N. K. FREEMAN, Evidence for lipolytic action by h u m a n p l a s m a
2 3
4 5 6 7 8 9
10 11
12
obtained after intravenous administration of heparin, Proc. Soc. exp. Biol. ( N . Y . ) , 1953, 86: 709. ROBINSON, D. S. AND J. E. FRENCH, Heparin, the clearing factor and fat transport, Pharmacol. Rev., 1960, 12: 241. ENGELBERG, H., Heparin and the removal of triglycerides from the blood stream, Amer. J. Cardiol., 1964, 14: 8. ROBINSON, D. S., F u r t h e r studies on the lipolytic s y s t e m induced in p l a s m a by heparin infusion, Quart. J. exp. Physiol., 1956, 14: 195. PORTE, D. AND R. H. WILLIAMS, Post heparin lipolytic activity following intravenous heparin S-35, Proc. Sue. exp. Biol. ( N . Y . ) , 1965, 118: 639. ROBINSON, 2[). S. AND M. A. JENNINGS, Release of clearing factor lipase by the perfused r a t heart, J. Lipid Res., 1965, 6: 222. BOBERG, J. AND L. A. CARLSON, Determination of heparin-induced lipoprotein lipase activity in h u m a n plasma, Clin. chim. Acta, 1964, 10: 420. GOODMAN, D. S., Preparation of h u m a n serum albumin free of long chain fatty acids, Science, 1957, 123: 1296. DOLE, V. P. AND H. MEINERTZ, Microdetermination of long chain f a t t y acids in p l a s m a and tissues, J. biol. Chem., 1960, 235: 2595. MUIR, J. R., A rapid m e t h o d for the assay of plasma lipoprotein in lipase using " I n t r a l i p i d " , Clin. chim. Acta, 1967, 17: 312. WING, D. R., M. R. SALAMANAND D. S. ROBINSON, Clearing factor lipase in adipose tissue. Factors influencing the increase in enzyme activity produced on incubation of tissue from starved rats in vitro, Biochem. J., 1966, 99: 648. ROBINSON, D. S. AND D. R. WING, Role of clearing factor lipase in uptake of triglycerides. In:
Enzyme Activities Related to Lipid Transport, 7 lth Int. Conf. Biochem. Lipids, Jerusalem, 1967. 13 NIKKILA, E. A., H o r m o n a l and metabolic regulations of lipoprotein lipase activity of adipose
tissue. In: Enzyme Activities Related to Lipid Transport, l l t h Int. Conf. Biochem. Lipids, Jerusalem, 7967. 14 OLIVER, M. F., V. A. KURIEN AND T. W. GREENWOOD, Relation between serum free f a t t y acids
and a r r h y t h m i a s and death after acute myocardial infarction, Lancet, 1968, 1: 710. 15 BRAY, C., E. L. McDONALD AND ~N~.RESTIEUX, Personal communication, 1968. 16 HARLAN, W. R., P. S. WINESETT AND A. J. WASSERMAN, Tissue lipoprotein lipase in normal individuals and in individuals with exogenous hypertriglyceridaemia and the relationship of this enzyme to t h e assimilation of fat, J. clin. Invest., 1967, 46: 239. 17 YOSHITOSHI, Y., C. NAITO, H. OKANIWA, M. USUI, T. MOGAMI AND T. TOMONO, Kinetic studies on metabolism of lipoprotein lipase, J. clin. Invest., 1968, 43: 707. is MUIR, J. R., The regional production of lipoprotein lipase, Clin. Sci., 1968, 34: 261. 19 MUIR, J. R., Endogenous p l a s m a lipolytic activity in m a n and its handling by the lungs, In preparation.
J. Atheroscler. Res., 1968, 8 : 9 8 3 - 9 8 7