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Linoleic acid and susceptibility to fatal ventricular fibrillation in rats
265
Atherosclerosis, 27 (1977) 265-269 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
LINOLEIC ACID AND SUSCEPTIBILITY FIBRILLATION IN RATS
R.L. LOGAN, Department (Received (Accepted
P. LARKING
TO FATAL VENTRICULAR
and E.R. NYE
of Medicine of the University of Otago Medical School, Dunedin (New Zealand) 30 September, 1976) 26 October, 1976)
Summary
The effect of linoleic acid on the induction of fatal ventricular fibrillation by intravenous CaClz (lo%), was studied in rats fed for a month from weaning on a diet with either a high or low content of linoleic acid. Studies were performed in the basal state and after pretreatment with noradrenaline, which increased the sensitivity to CaClz equally in animals from both diet groups. Despite considerable differences in the linoleic acid levels in the plasma and myocardium, the two groups did not differ in the incidence of fatal ventricular fibrillation. Our conclusions concerning the effect of linoleic acid on cardiac arrhythmias, and sudden death in particular, are compared with those from other studies. Key words:
Animal fat - Linoleic acid - Myocardium oil - Ventricular fibrillation
-Plasma
lipids -Rat-Safflower
Introduction
There have been conflicting experimental and clinical findings concerning the relationship of linoleic acid and cardiac arrhythmias. Electrophysiological studies on the isolated guinea pig papillary muscle [l] indicate that linoleic acid may be arrhythmogenic and support the inference made from a study of patients following myocardial infarction [2], that there was an increased incidence of arrhythmias associated with high serum levels of linoleic acid. By contrast, linoleic acid given intravenously to unanaesthetized rabbits has been shown to have a protective effect against the induction of ventricular arrhythmias by BaCl, [3]. Long term studies in man have failed to show any effect * This study was supported
by Grant 121
from the National
Heart Foundation
of New Zealand.
266
on the incidence of sudden cardiac death as a result of feeding diets rich in linoleic acid [4+] although in one study [ 71, there was a reduction in the total deaths from coronary heart disease. Since the results of the clinical studies may have been due to factors other than linoleic acid, a study was undertaken in rats to investigate the effect of raised tissue levels of linoleic acid on the induction of fatal ventricular fibrillation (VF). The arrhythmia was induced by calcium chloride (CaCl,), which has been used to study anti-arrhythmic agents in this species [ 81. Materials and Methods Male Wistar rats were divided equally into two diet groups at weaning and studied a month later when their weights were 160-255 g. The diets differed only in their fat content (Table 1): 20% safflower oil (Group S) and 20% animal fat (Group B). VF was induced with 10% CaClz solution (90 mg/kg dose) at 37°C given by rapid i.v. injection to animals anaesthetized with urethane (1.5 mg/kg i.p.) after an overnight fast. Atropine sulphate (0.5 mg/kg i.p.) was given 5 min before the CaClz to prevent asystole, which sometimes occurred following these injections. The heart rhythm was recorded on a direct-writing electrocardiograph using bipolar needle electrodes on each forelimb and an indifferent electrode on the anterior abdominal wall. Three minutes after the injection of CaCl? animals were classified as either a survivor or death from sustained VF. Animals whose classification was uncertain or who developed VF after prolonged asystole or a period of apnoea exceeding a minute were rejected from the study. Animals were selected for testing at random. All steps in the experiment, including classification or rejection of animals, was carried out blind with respect to their diet group. In a second study the same procedure was followed except that 1.2 ml of a freshly prepared solution of noradrenaline bitartrate (10.3 X 10e6 M in 0.15 M saline was infused into a jugular vein for 10 min before the injection of CaClz. In order to achieve the same overall mortality of about 50% a smaller dose of CaCl, was given, 50 mg/kg. Plasma and myocardial free fatty acids (FFA) were analyzed using 5 animals from each diet group in both the basal state (mean weights, Group S 240 g, Group B 180 g) and after pretreatment with noradren-
TABLE 1 COMPOSITION
OF THE TWO DIETS USED
Constituent
% by weight
Salt mixture Casein Bran Vitamin mixture Glucose Safflower oil a
4 20 6 2 48 20
Or
Beef fat b
a Group
S:
20
b Group B.
267
aline as already described (mean weights Group S 200 g, Group B 198 g). Blood was taken from each animal for measurement of plasma FFA [ 91. The ventricular portion of the heart was removed while still beating, washed in ice-cold saline, blotted and immediately homogenized in 20 ml 0.25 M sucrose solution in a Sorvall Omnimixer surrounded by ice. 2 ml of the homogenate was transferred to 10 ml of Dole’s solution and, after extraction by a modified Dole’s procedure [lo], the myocardial FFA were measured calorimetrically [ 9 1. Results were expressed relative to the protein content of the homogenate measured by Lowry’s method [ 111. Lipids were extracted from pooled samples of myocardial homogenates, plasma and adipose tissue by the Folch’s method [12]. After separation by thin layer chromatography, the fatty acids in the FFA and triglycerides (TGFA) were converted to their methyl esters [13], and their composition was determined by gas-liquid chromatography (G.L.C.) using a Pye series 104 chromatograph. Student’s t-test was used to compare mean values. Results The two doses of CaCl, used (90 mg ,and 50 mg/kg) were those which had been found in preliminary experiments on rats fed a normal pellet diet to produce a 50% incidence of fatal VF. In our first study reported here, one animal was rejected leaving 11 animals from Group S (mean weight 207 g) and 10 from Group B (mean weight 210 g). In the second study 20 animals were rejected, 12 for technical reasons and 8 because they developed prolonged apnoea
TABLE
2
COMPARISON OF THE LINOLEIC ACID CONTENT OF PLASMA FFA AND AND TGFA AND THE INCIDENCE OF CaClz-INDUCED FATAL VENTRICULAR RATS FED DIETS WITH HIGH OR LOW CONTENTS OF LINOLEIC ACID Diet group a
Levels of total FFA and percentage Plasma
(Cccl2
S B
After pretreatment S B
acid
Myocardium TGFA
FFA
Level
18:2
Level
18:2
18:2
(PEqll)
(S)
(@q/g protein)
(%)
(%)
25 12
3.5 ?r 0.39 3.1 + 0.29
FFA
Basal studies
of linoleic
TGFA
MYOCARDIAL FIBRILLATION
FFA IN
Incidence of fatal ventricular fibrillation (%)
Ne
90 mg/kg i.u.) 424 r 37’ 378 * 49
with noradrenaline 1335 1057
* 136d * 13Qd
17 11
44b 5
45 50
11 10
42 9
40b 4
50 58
34 31
4 pg (CaCl2 50 mg/kg i.u.) 27 8
3.1 f 0.24 3.0 + 0.28
a Diets differed only in their fat content, safflower oil (Group S) and beef fat (Group B). The analysis of FFA composition was carried out on pooled samples from 5 animals (coefficient of variation of method less than 2%). b Significantly greater than .cowesponding values for Group B, P < 0.01. ’ Mean f SEM of results from 5 animals. d Significantly greater than basal levels, P < 0.001. e N = number of experiments.
268
followed by either VF or asystole. There was no significant difference between the numbers rejected from each diet group. After this step, 34 remained from Group S (mean weight 208 g) and 31 from Group B (mean weight 201 g). Only linoleic acid (18:2) will be mentioned in the results of analysis of various lipid fractions (Table 2). The concentrations of linoleic acid in myocardial triglycerides which were similar to those in the adipose tissue in each group, were significantly higher in Group S (P < 0.01). Total levels of FFA and the relative proportion of linoleic acid in the plasma and myocardium are shown along with the incidence of fatal VF in the two diet groups in Table 2. The proportions of linoleic acid in plasma and myocardial FFA were higher in animals fed the safflower oil diet. Although pretreatment with noradrenaline produced similar significant elevation of plasma FFA level in both groups (P < O.OOl), it did not alter myocardial levels. In Group S it did, however, increase the proportion of linoleic acid in the myocardial FFA from 17 to 42%. The incidence of fatal VF did not significantly differ in the two diet groups. Although pretreatment with noradrenaline increased sensitivity to CaCl,, it did not result in any difference in mortality between the two groups. Discussion A dietary-induced increase of linoleic acid in the plasma and myocardium of young rats, as well as a further elevation by noradrenaline, did not effect the incidence of fatal VF induced by CaCl,. As expected the proportions of linoleic acid in the lipids examined differed considerably between the two diet groups (Table 2). It is difficult to demonstrate that these differences were statistically significant as samples were pooled in order to obtain satisfactory G.L.C. analysis of fatty acids. However, as noradrenaline pretreatment had little effect on the proportions of linoleic acid in the myocardial TGFA, statisticaly analysis was carried out by combining the results from the basal and noradrenaline pretreated groups. The use of atropine is unlikely to have influenced our results as neither vagotomy in rats [ 141 nor pretreatment with atropine in dogs [15] reduces sensitivity to induction of VF in this way. Our findings agree with those of diet studies in man that have failed to show a change in the incidence of sudden cardiac death in men receiving a linoleic acid rich diet [4-6]. They do not support the evidence for an arrhythmogenic action of linoleic acid, inferred from a study in post-infarction patients, where the incidence of ventricular arrhythmias was increased in those with raised serum levels of this fatty acid [ 21. The tissue levels of linoleic acid in the rats of our two diet groups were comparable to those reported in these clinical studies [2,7,16]. The large difference in myocardial levels of linoleic acid between the two groups, particularly after the noradrenaline infusion (Table 2), is clearly of relevance to the investigation of its effect on the induction of VF. It is difficult to compare this study with work showing an anti-arrhythmic action of linoleic acid [ 31, as a different experimental model was used and linoleic acid was given in large intravenous doses. In this study we have not investigated the effect of linoleic acid on arrhythmias other than VF or in the presence of myocardial ischaemia. While there is
269
some evidence that dietary modification which increases tissue levels of linoleic acid may be associated with a reduction in the overall mortality of coronary heart disease [ 71, our study provides no evidence that it specifically affects the incidence of fatal VF. Acknowledgements The authors are grateful to Mr. E. Everest for his technical M.F. Oliver for his helpful comments.
help and to Dr.
References 1 Luderitz, B., Naumann d’Alnoncourt, C. and Steinbeck, G.. Effects of free fatty acids on electrophysiological properties of ventricular myocardium, Klin. Wschr.. 54 (1976) 309. 2 Ravens, K.G. and Jipp, P.. Die freien PlasmafettsLuren in der Friihphase nach einem Myokardinfarkt, Arzneim.-Forsch., 22 (1972) 1831. 3 Me&. H.-J. and F8rster, W., Evidence for anti-arrhythmic efficiency of arachidonic and linoleic acid Preliminary results, Prostaglandins. 4 (1973) 751. 4 Leren, P.. The effect of plasma cholesterol lowering diet in male survivors of myocardial infarction, Acta Med. Stand., (Suppl.) 466 (1966). 6 M.R.C., Controlled trial of soya-bean oil in myocardial infarction, Lance& 2 (1968) 693. 6 Dayton, S., Pearce, M.L.. Goldman, H., Harmish, A., Plotkind, D.. Shickman. M.. Winfield, M.. Zager, A. and Dixon, W., Controlled trial of a diet high in unsaturated fat for prevention of atherosclerotic complications, Lancet, 2 (1968) 1060. 7 Miettinen, M., Turpeinen, 0.. Karvonen, M.J., Elosuo. R. and Paavilsinen, E., Effect of cholesterol lowering diet on mortality from coronary heart disease and other causes. Lancet, 2 (1972) 835. 8 Fiirster. W.. Mest, H.-J. and Mentz, P., The influence of PGFzo, on experimental arrhythmias. Prostaglandins, 3 (1973) 896. 9 Laurell, S. and Tibling, G., Calorimetric micro-determination of free fatty acids in plasma. Clin. Chim. Acta, 16 (1967) 57. 10 Trout. D.L.. Estes, E.H. and Friedberg, S.J., Titration of free fatty acids of plasma-A study of current methods and a new modification, J. Lipid Res.. 1 (1960) 199. 11 Lowry, O.H.. Rosebrough, N.J., Farr. A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem.. 193 (1951) 265. 12 Folch. J., Lees, M. and Sloane Stanley, G.H.. A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 13 Morrison, W.R. and Smith, L.M., Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride - methanol. J. Lipid Res., 5 (1964) 600. 14 Malinow. M.R.. Battle, F.F. and M&mud. B., Nervous mechanisms in ventricular arrhythmias induced by calcium chloride in rats, Circ. Res., 1 (1953) 554. 16 Hoff, H.. Smith, P. and Winkler, A., Electrocardiographic changes and concentration of calcium in serum following intravenous injection of calcium chloride, Amer. J. Physiol.. 125 (1939) 162. 16 Dayton, S.. Hashimoto. S.. Dixon, W. and Pearce, M.L.. Composition of lipids in human serum and adipose tissue during prolonged feeding of diet high in unsaturated fat. J. Lipid Res.. 7 (1966) 103.
Atherosclerosis, 27 (1977) 265-269 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
LINOLEIC ACID AND SUSCEPTIBILITY FIBRILLATION IN RATS
R.L. LOGAN, Department (Received (Accepted
P. LARKING
TO FATAL VENTRICULAR
and E.R. NYE
of Medicine of the University of Otago Medical School, Dunedin (New Zealand) 30 September, 1976) 26 October, 1976)
Summary
The effect of linoleic acid on the induction of fatal ventricular fibrillation by intravenous CaClz (lo%), was studied in rats fed for a month from weaning on a diet with either a high or low content of linoleic acid. Studies were performed in the basal state and after pretreatment with noradrenaline, which increased the sensitivity to CaClz equally in animals from both diet groups. Despite considerable differences in the linoleic acid levels in the plasma and myocardium, the two groups did not differ in the incidence of fatal ventricular fibrillation. Our conclusions concerning the effect of linoleic acid on cardiac arrhythmias, and sudden death in particular, are compared with those from other studies. Key words:
Animal fat - Linoleic acid - Myocardium oil - Ventricular fibrillation
-Plasma
lipids -Rat-Safflower
Introduction
There have been conflicting experimental and clinical findings concerning the relationship of linoleic acid and cardiac arrhythmias. Electrophysiological studies on the isolated guinea pig papillary muscle [l] indicate that linoleic acid may be arrhythmogenic and support the inference made from a study of patients following myocardial infarction [2], that there was an increased incidence of arrhythmias associated with high serum levels of linoleic acid. By contrast, linoleic acid given intravenously to unanaesthetized rabbits has been shown to have a protective effect against the induction of ventricular arrhythmias by BaCl, [3]. Long term studies in man have failed to show any effect * This study was supported
by Grant 121
from the National
Heart Foundation
of New Zealand.
266
on the incidence of sudden cardiac death as a result of feeding diets rich in linoleic acid [4+] although in one study [ 71, there was a reduction in the total deaths from coronary heart disease. Since the results of the clinical studies may have been due to factors other than linoleic acid, a study was undertaken in rats to investigate the effect of raised tissue levels of linoleic acid on the induction of fatal ventricular fibrillation (VF). The arrhythmia was induced by calcium chloride (CaCl,), which has been used to study anti-arrhythmic agents in this species [ 81. Materials and Methods Male Wistar rats were divided equally into two diet groups at weaning and studied a month later when their weights were 160-255 g. The diets differed only in their fat content (Table 1): 20% safflower oil (Group S) and 20% animal fat (Group B). VF was induced with 10% CaClz solution (90 mg/kg dose) at 37°C given by rapid i.v. injection to animals anaesthetized with urethane (1.5 mg/kg i.p.) after an overnight fast. Atropine sulphate (0.5 mg/kg i.p.) was given 5 min before the CaClz to prevent asystole, which sometimes occurred following these injections. The heart rhythm was recorded on a direct-writing electrocardiograph using bipolar needle electrodes on each forelimb and an indifferent electrode on the anterior abdominal wall. Three minutes after the injection of CaCl? animals were classified as either a survivor or death from sustained VF. Animals whose classification was uncertain or who developed VF after prolonged asystole or a period of apnoea exceeding a minute were rejected from the study. Animals were selected for testing at random. All steps in the experiment, including classification or rejection of animals, was carried out blind with respect to their diet group. In a second study the same procedure was followed except that 1.2 ml of a freshly prepared solution of noradrenaline bitartrate (10.3 X 10e6 M in 0.15 M saline was infused into a jugular vein for 10 min before the injection of CaClz. In order to achieve the same overall mortality of about 50% a smaller dose of CaCl, was given, 50 mg/kg. Plasma and myocardial free fatty acids (FFA) were analyzed using 5 animals from each diet group in both the basal state (mean weights, Group S 240 g, Group B 180 g) and after pretreatment with noradren-
TABLE 1 COMPOSITION
OF THE TWO DIETS USED
Constituent
% by weight
Salt mixture Casein Bran Vitamin mixture Glucose Safflower oil a
4 20 6 2 48 20
Or
Beef fat b
a Group
S:
20
b Group B.
267
aline as already described (mean weights Group S 200 g, Group B 198 g). Blood was taken from each animal for measurement of plasma FFA [ 91. The ventricular portion of the heart was removed while still beating, washed in ice-cold saline, blotted and immediately homogenized in 20 ml 0.25 M sucrose solution in a Sorvall Omnimixer surrounded by ice. 2 ml of the homogenate was transferred to 10 ml of Dole’s solution and, after extraction by a modified Dole’s procedure [lo], the myocardial FFA were measured calorimetrically [ 9 1. Results were expressed relative to the protein content of the homogenate measured by Lowry’s method [ 111. Lipids were extracted from pooled samples of myocardial homogenates, plasma and adipose tissue by the Folch’s method [12]. After separation by thin layer chromatography, the fatty acids in the FFA and triglycerides (TGFA) were converted to their methyl esters [13], and their composition was determined by gas-liquid chromatography (G.L.C.) using a Pye series 104 chromatograph. Student’s t-test was used to compare mean values. Results The two doses of CaCl, used (90 mg ,and 50 mg/kg) were those which had been found in preliminary experiments on rats fed a normal pellet diet to produce a 50% incidence of fatal VF. In our first study reported here, one animal was rejected leaving 11 animals from Group S (mean weight 207 g) and 10 from Group B (mean weight 210 g). In the second study 20 animals were rejected, 12 for technical reasons and 8 because they developed prolonged apnoea
TABLE
2
COMPARISON OF THE LINOLEIC ACID CONTENT OF PLASMA FFA AND AND TGFA AND THE INCIDENCE OF CaClz-INDUCED FATAL VENTRICULAR RATS FED DIETS WITH HIGH OR LOW CONTENTS OF LINOLEIC ACID Diet group a
Levels of total FFA and percentage Plasma
(Cccl2
S B
After pretreatment S B
acid
Myocardium TGFA
FFA
Level
18:2
Level
18:2
18:2
(PEqll)
(S)
(@q/g protein)
(%)
(%)
25 12
3.5 ?r 0.39 3.1 + 0.29
FFA
Basal studies
of linoleic
TGFA
MYOCARDIAL FIBRILLATION
FFA IN
Incidence of fatal ventricular fibrillation (%)
Ne
90 mg/kg i.u.) 424 r 37’ 378 * 49
with noradrenaline 1335 1057
* 136d * 13Qd
17 11
44b 5
45 50
11 10
42 9
40b 4
50 58
34 31
4 pg (CaCl2 50 mg/kg i.u.) 27 8
3.1 f 0.24 3.0 + 0.28
a Diets differed only in their fat content, safflower oil (Group S) and beef fat (Group B). The analysis of FFA composition was carried out on pooled samples from 5 animals (coefficient of variation of method less than 2%). b Significantly greater than .cowesponding values for Group B, P < 0.01. ’ Mean f SEM of results from 5 animals. d Significantly greater than basal levels, P < 0.001. e N = number of experiments.
268
followed by either VF or asystole. There was no significant difference between the numbers rejected from each diet group. After this step, 34 remained from Group S (mean weight 208 g) and 31 from Group B (mean weight 201 g). Only linoleic acid (18:2) will be mentioned in the results of analysis of various lipid fractions (Table 2). The concentrations of linoleic acid in myocardial triglycerides which were similar to those in the adipose tissue in each group, were significantly higher in Group S (P < 0.01). Total levels of FFA and the relative proportion of linoleic acid in the plasma and myocardium are shown along with the incidence of fatal VF in the two diet groups in Table 2. The proportions of linoleic acid in plasma and myocardial FFA were higher in animals fed the safflower oil diet. Although pretreatment with noradrenaline produced similar significant elevation of plasma FFA level in both groups (P < O.OOl), it did not alter myocardial levels. In Group S it did, however, increase the proportion of linoleic acid in the myocardial FFA from 17 to 42%. The incidence of fatal VF did not significantly differ in the two diet groups. Although pretreatment with noradrenaline increased sensitivity to CaCl,, it did not result in any difference in mortality between the two groups. Discussion A dietary-induced increase of linoleic acid in the plasma and myocardium of young rats, as well as a further elevation by noradrenaline, did not effect the incidence of fatal VF induced by CaCl,. As expected the proportions of linoleic acid in the lipids examined differed considerably between the two diet groups (Table 2). It is difficult to demonstrate that these differences were statistically significant as samples were pooled in order to obtain satisfactory G.L.C. analysis of fatty acids. However, as noradrenaline pretreatment had little effect on the proportions of linoleic acid in the myocardial TGFA, statisticaly analysis was carried out by combining the results from the basal and noradrenaline pretreated groups. The use of atropine is unlikely to have influenced our results as neither vagotomy in rats [ 141 nor pretreatment with atropine in dogs [15] reduces sensitivity to induction of VF in this way. Our findings agree with those of diet studies in man that have failed to show a change in the incidence of sudden cardiac death in men receiving a linoleic acid rich diet [4-6]. They do not support the evidence for an arrhythmogenic action of linoleic acid, inferred from a study in post-infarction patients, where the incidence of ventricular arrhythmias was increased in those with raised serum levels of this fatty acid [ 21. The tissue levels of linoleic acid in the rats of our two diet groups were comparable to those reported in these clinical studies [2,7,16]. The large difference in myocardial levels of linoleic acid between the two groups, particularly after the noradrenaline infusion (Table 2), is clearly of relevance to the investigation of its effect on the induction of VF. It is difficult to compare this study with work showing an anti-arrhythmic action of linoleic acid [ 31, as a different experimental model was used and linoleic acid was given in large intravenous doses. In this study we have not investigated the effect of linoleic acid on arrhythmias other than VF or in the presence of myocardial ischaemia. While there is
269
some evidence that dietary modification which increases tissue levels of linoleic acid may be associated with a reduction in the overall mortality of coronary heart disease [ 71, our study provides no evidence that it specifically affects the incidence of fatal VF. Acknowledgements The authors are grateful to Mr. E. Everest for his technical M.F. Oliver for his helpful comments.
help and to Dr.
References 1 Luderitz, B., Naumann d’Alnoncourt, C. and Steinbeck, G.. Effects of free fatty acids on electrophysiological properties of ventricular myocardium, Klin. Wschr.. 54 (1976) 309. 2 Ravens, K.G. and Jipp, P.. Die freien PlasmafettsLuren in der Friihphase nach einem Myokardinfarkt, Arzneim.-Forsch., 22 (1972) 1831. 3 Me&. H.-J. and F8rster, W., Evidence for anti-arrhythmic efficiency of arachidonic and linoleic acid Preliminary results, Prostaglandins. 4 (1973) 751. 4 Leren, P.. The effect of plasma cholesterol lowering diet in male survivors of myocardial infarction, Acta Med. Stand., (Suppl.) 466 (1966). 6 M.R.C., Controlled trial of soya-bean oil in myocardial infarction, Lance& 2 (1968) 693. 6 Dayton, S., Pearce, M.L.. Goldman, H., Harmish, A., Plotkind, D.. Shickman. M.. Winfield, M.. Zager, A. and Dixon, W., Controlled trial of a diet high in unsaturated fat for prevention of atherosclerotic complications, Lancet, 2 (1968) 1060. 7 Miettinen, M., Turpeinen, 0.. Karvonen, M.J., Elosuo. R. and Paavilsinen, E., Effect of cholesterol lowering diet on mortality from coronary heart disease and other causes. Lancet, 2 (1972) 835. 8 Fiirster. W.. Mest, H.-J. and Mentz, P., The influence of PGFzo, on experimental arrhythmias. Prostaglandins, 3 (1973) 896. 9 Laurell, S. and Tibling, G., Calorimetric micro-determination of free fatty acids in plasma. Clin. Chim. Acta, 16 (1967) 57. 10 Trout. D.L.. Estes, E.H. and Friedberg, S.J., Titration of free fatty acids of plasma-A study of current methods and a new modification, J. Lipid Res.. 1 (1960) 199. 11 Lowry, O.H.. Rosebrough, N.J., Farr. A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem.. 193 (1951) 265. 12 Folch. J., Lees, M. and Sloane Stanley, G.H.. A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 13 Morrison, W.R. and Smith, L.M., Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride - methanol. J. Lipid Res., 5 (1964) 600. 14 Malinow. M.R.. Battle, F.F. and M&mud. B., Nervous mechanisms in ventricular arrhythmias induced by calcium chloride in rats, Circ. Res., 1 (1953) 554. 16 Hoff, H.. Smith, P. and Winkler, A., Electrocardiographic changes and concentration of calcium in serum following intravenous injection of calcium chloride, Amer. J. Physiol.. 125 (1939) 162. 16 Dayton, S.. Hashimoto. S.. Dixon, W. and Pearce, M.L.. Composition of lipids in human serum and adipose tissue during prolonged feeding of diet high in unsaturated fat. J. Lipid Res.. 7 (1966) 103.