Br. J. exp. Path. (1978) 59, 390
THE EFFECTS OF CHANGES IN DIET ON LIPID LEVELS AND PLATELET THROMBUS FORMATION IN LIVING BLOOD VESSELS A. J. HONOUR, R. D. CARTER AND J. I. MANN Fromn The Department of the Regius Professor of Medicine and the Department of Social and Community Medicine, University of Oxford, Radcliffe Infirmary, Oxford Received for publication March 10, 1978
Summary.-The work reported here describes an in vivo study over several days in each animal, of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topically applied adenosine diphosphate (ADP) in rabbits fed initially on normal diets, then on experimental "saturated" or "polyunsaturated" diets. The effects of these diets on triglyceride fatty acids and on cholesterol and triglyceride levels are reported. It may be of significance that in each experiment the "polyunsaturated" and "saturated" diets appeared to be associated with an increased sensitivity to ADP greater than observed in rabbits on a normal diet or in normal animals in other studies.
IT HAS BEEN SUGGESTED that in a diet substitution of saturated fat for polyunsaturated fat might lessen the frequency of premature ischaemic heart disease (Royal College of Physicians Report, 1976). Certainly such dietary manipulation causes reduction of blood cholesterol levels in man and experimental animals (Mann and Truswell, 1972). Recently O'Brien et at., using in vitro methods, have shown decreased platelet aggregation in volunteers after they had eaten meals containing predominantly polyunsaturated rather than saturated fat (O'Brien, Etherington and Jamieson, 1976). We have previously studied changes in intra-arteriolar aggregation of platelets in living blood vessels of rabbits in diabetes and on different drug regimes, (Honour and Hockaday, 1976; Honour, Hockaday and Mann, 1976; Honour, Carter and Mann, 1977) and we report now our findings using the same in vivo methods concerning the changes observed on diets differing in their composition of fat. MATERIALS AND METHODS Male New Zealand white rabbits were used throughout. Their usual diet was "Oxoid diet 18"
and water (normal diets). Two experimental diets were employed. One was intended to be high in saturated fatty acids and during this experimental period the pellets were soaked in lard liquified by melting in boiling water ("saturated" diet). The other was intended to be high in polyunsaturated fatty acids and during the "polyunsaturated" dietary periods the pellets were soaked in corn oil. In Experiments 1 and 2 the "saturated" diets were given first and in Experiments 3 and 4 the "polyunsaturated" diets were given first. The proportion of linoleic acid (C18:2) as a percentage of total plasma triglyceride fatty acid strongly reflects the degree of polyunsaturation of the diet in man (Moore et al., 1977) and, using the methods described by these workers, we carried out similar measurements in the rabbits during the different dietary periods. Cholesterol (Searcy and Bergquist, 1960), triglyceride (Eggstein and Kreutz, 1966) and blood glucose (Technicon autoanalyser) measurements were carried out at regular intervals during the experimental diets. Intra-arteriolar aggregation of platelets was studied as follows: Rabbits were anaesthetized with i.p. Nembutal (pentobarbitone sodium). Preparation of cerebral cortex, microscopic observation of pial arteries and the application of a window to the skull in the form of a perspex capsule with a removable lid, have been described elsewhere (Honour and Ross Russell, 1962; Honour and Hockaday, 1976). Briefly, a disc of bone is removed from the skull in the right parietal region, and the membranes excised to expose the
A. J. HONOUR, R. D. CARTER AND J. I. MANN
pial arteries. The capsule consists of 2 parts, a base and a lid. The base is fixed to the skull and filled with warm sterile saline. The lid is screwed into the base of the capsule, closing it and simultaneously ejecting the excess saline through a channel in the side of the lid. The skin is drawn together around the base of the capsule with silk sutures, the cut edges being located within a groove cut around it. Such a preparation can be re-used on several occasions and usually remains good for many weeks, it being possible to monitor the preparation continuously through the lid by inspection at any time. The membranes begin to grow in again from the periphery after a few days, but these can be again removed to expose afresh the pial surface. Previous injury sites show as slight swellings on the pial arteries and are easily recognized and avoided. Alternatively the distribution of the arteries and injury sites may be recorded on film. The arteries used for the experiments are between 100 and 200 ,um in diameter. Their number and distribution is very variable and can be a limiting factor in the number of observations that may be made in any one preparation. Electrical injuries were inflicted using a unipolar system as described by Honour, Pickering and Sheppard (1971). For each series of observations the animal is anaesthetized with Nembutal and with sterile precautions the capsule is opened and a warm sterile saline drip set up to leave a constant film of moisture over the exposed brain surface. The unipolar electrode is a fine platinum wire of negative polarity applied directly to the artery wall, and at a potential of 5V with a current of 10-20 t,A for 45 s replicable injuries are made. Subsequent events are followed with a
391
proportion of linoleic acid than that observed on the normal and "saturated" diets. There did not appear to be a difference between the normal and "saturated" diets.
Effects of diets on cholesterol and triglyceride levels Cholesterol levels were lower on the "polyunsaturated" diet, in each experiment, than on the "saturated" and normal diets. Triglyceride levels did not show any particular trend.
Effects of diets on concentrations of ADP required to produce platelet aggregation Regardless of the stage of each experiment, the "polyunsaturated" and "saturated" diets were associated with an increased sensitivity to ADP than that observed on the normal diet and in normal animals in other studies. These changes were independent of changes in body weight. Blood glucose levels did not change. DISC'USSION
Our results confirm the well known hypocholesterolaemic effect of polyunsaturated fat. In each of the 4 experiments cholesterol levels were at their lowest when diets containing corn oil were fed. Triglyceride levels, however, showed a variable response. The results of the platelet thrombus formation studies were surprising. On normal diets white body formation was noted (as had been the case in all our previous investigations) after the application of ADP in concentrations ranging from 1/4096 to 1/16384 (570-142-5mmol/ 1). On both the saturated and polyRESULTS unsaturated diets, sensitivity of ADP for The results of the 4 experiments are platelet thrombus formation was raised shown in the Table. (1/131072-1/262144; 1758-89 mmol/1) to the levels of increased sensitivity we had Effects of diets on triglyceride fatty acids previously noted in rabbits after the inThe "polyunsaturated" diet was, in all jection of i.v. alloxan. These results could the experiments, associated with a higher not be attributed to changes in body weight stereoscopic microscope. The platelet-aggregating agent used was adenosine diphosphate (ADP) (Sigma), dissolved in sterile normal saline, the solution containing 1 mg/ml 2-34 x 106 mmol/1, which was further diluted 1:1 serially with sterile normal saline to the required test concentrations. All solutions were freshly prepared and kept cool throughout the observations. Application was made dropwise from sterile Pasteur pipettes, the saline drip being interrupted during each challenge with ADP, the end point for sensitivity to ADP being the most dilute solution at which white body formation was seen.
392
DIET AND PLATELET THROMBUS FORMATION ce1 18
4
00
QO
0 4
OX
=
_
O_00
00
CO
ci
4-I
Co
in C> r-
P-,
C.)
0-
C
i 00
10
00 ,-
CO ,w CDOCO Wmr-
co
00
O
00
It
P-
CO
CO
CO
O CO
-
0
C
ll 4
O
00 x CO
)
q
0
=
-r
COto
N
II
10
COm
*.1
.2
oo
C)
I-
14CO
C1-
Z'S
00
Co
I'
11
r-
m
o o;
I
CO CO
co
UrC
Ci CO
00 rl
-4
-I "--
4
*s1
C)
*t
C.)
pq s
41 CD 3333a 5S
sq 4U
C)
CO
CO
aci
Ce
N
N
NN
N
N
N
t_; _;
CO
C6
CO
tc
-
CO CiCO
CO -r
m
aq
-
-_
Ci CO
COO CO ,-
Q
z -
to
o) ws
EH
0
l 00
r
Io
-4OD 114
o~~~~~
bO bo 00 *s1
%.1
-6)
eq
41'.
S °Si
4
10
A. J. HONOUR, R. D. CARTER AND J. I. MANN
0 -I0
0)I I
- 1C) _,
I
I
)
>Ct 1 cq~
~
-
~I o
00
P-
CO
i
IC
m I-
0
O
00
r
(O
CI
0 C 1co
10l
N-
CNI
C0
-
N)
C
10
10
-4
ID
1-
C
CO
0)
1
I
410
eIs
P.-
t-N
t
o
N-
10
0N
10 10
-
oo
10
IN
CO
oo
-
CO0
IN
C 00
0)
IN
Co
I"
--I 171 0)
CO
CO
-4
4
P-
C
P-
oo
Z
0i .4.
C
CO
_P
N-
CO
CO
oz o.) 0
0
0
o
o3 9
C
CS0
Ca
OD
o
0C
0
oCa
9
C5
_4
O4
C
O
N-
t-
r tCC
-4
al
r-tCONtr-00 U: .
.--
-4
_. 1-
I -
)f
.
cq
10 X6
.6 aq
-
1t6 .
Iq
r10 CC
*6 u:
r
'0 t--
N1
IN6
16 a4
-
tN IN
CO t-
6.6
ai.: -
N
0 Co tC6
_-
_
_
393
394
DIET AND PLATELET THROMBUS FORMATION
and were observed regardless of the sequence in which the diets were given. The hypocholesterolaemic effects of polyunsaturated fat are very likely to be beneficial and O'Brien et at. (1976) have claimed that their findings suggesting decreased platelet activation after a single meal high in polyunsaturated fat may also be relevant to preventive dietary therapy in man. Our findings are based on in vivo animal experiments in longer-term studies and although the findings in experimental animals are not necessarily applicable to the human situation, they do suggest that further studies of platelet function are essential before the widespread recommendation of any changes concerning the intake of dietary fat are made.
Financial support came in part from a Medical Research Council grant to A.J.H. REFERENCES EGGSTEIN, M. & KREUTZ, F. H. (1966) Eine neue Bestimmung der Neutralfette in Blut serum und Gewebe. Klin. Wsch., 44, 262. HONOUR, A. J. & HOCKADAY, T. D. R. (1976) Increased Sensitivity of in vivo Platelet Aggrega-
tion after Alloxan on Streptozotocin. Br. J. exp. Path., 57, 1. HONOUR, A. J., HOCKADAY, T. D. R., & MANN, J. I. (1976). Reversibility by Dipyridamole of the increased sensitivity of in vivo platelet aggregation in rabbits after Alloxan. Br. J. exp. Path., 57, 11. HONOUR, A. J., CARTER, R. D. & MANN, J. I. (1977) The Effects of Treatment with Aspirin and Antithrombotic Agent SH 1117 upon Platelet Thrombus Formation in Living Blood Vessels. Br. J. exp. Path., 58, 474. HONOUR, A. J. & Ross RUSSELL, R. W. (1962) Experimental Platelet Embolism. Br. J. exp. Path., 43, 350. HONOUR, A. J., PICKERING, G. W. & SHEPPARD, B. L. (1971) Ultra-structure and Behaviour of Platelet Thrombi in Injured Arteries. Br. J. exp. Path., 52, 482. MANN. J. I. & TRUSWELL, A. S. (1972). Effects of Isocaloric Exchange of Dietary Sucrose and Starch on Fasting Serum Lipids, Post Prandial Insulin Secretion and Alimentary Lipaemia in Human Subjects. Br. J. Nutr., 27, 395. MOORE, R. A., OPPERT, S., EATON, P. & MANN, J. I. (1977) Triglyceride Fatty Acids Confirm a Change in Dietary Fat. Clin. Endocr., 7, 143. O'BREIN, J. R., ETHERINGTON, M. D. & JAMIESON, S. (1976) Acute Platelet Changes after Large Meals of Saturated and Unsaturated Fats. Lancet, 1, 878. ROYAL COLLEGE OF PHYSICIANS (1976) Report on the Prevention of Coronary Heart Disease. Jnl. R. Coll. Phyaiciavs Lond., 10, 213. SEARCY, R. L. & BERGQUIST, L. M. (1960) A new colour reaction for the quantitation of serum cholesterol. Clin. chim. Acta, 5, 192. TECHNICON AUTOANALYSER MANUAL. Glucose oxidase (gum guiac) method).
THE EFFECTS OF CHANGES IN DIET ON LIPID LEVELS AND PLATELET THROMBUS FORMATION IN LIVING BLOOD VESSELS A. J. HONOUR, R. D. CARTER AND J. I. MANN Fromn The Department of the Regius Professor of Medicine and the Department of Social and Community Medicine, University of Oxford, Radcliffe Infirmary, Oxford Received for publication March 10, 1978
Summary.-The work reported here describes an in vivo study over several days in each animal, of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topically applied adenosine diphosphate (ADP) in rabbits fed initially on normal diets, then on experimental "saturated" or "polyunsaturated" diets. The effects of these diets on triglyceride fatty acids and on cholesterol and triglyceride levels are reported. It may be of significance that in each experiment the "polyunsaturated" and "saturated" diets appeared to be associated with an increased sensitivity to ADP greater than observed in rabbits on a normal diet or in normal animals in other studies.
IT HAS BEEN SUGGESTED that in a diet substitution of saturated fat for polyunsaturated fat might lessen the frequency of premature ischaemic heart disease (Royal College of Physicians Report, 1976). Certainly such dietary manipulation causes reduction of blood cholesterol levels in man and experimental animals (Mann and Truswell, 1972). Recently O'Brien et at., using in vitro methods, have shown decreased platelet aggregation in volunteers after they had eaten meals containing predominantly polyunsaturated rather than saturated fat (O'Brien, Etherington and Jamieson, 1976). We have previously studied changes in intra-arteriolar aggregation of platelets in living blood vessels of rabbits in diabetes and on different drug regimes, (Honour and Hockaday, 1976; Honour, Hockaday and Mann, 1976; Honour, Carter and Mann, 1977) and we report now our findings using the same in vivo methods concerning the changes observed on diets differing in their composition of fat. MATERIALS AND METHODS Male New Zealand white rabbits were used throughout. Their usual diet was "Oxoid diet 18"
and water (normal diets). Two experimental diets were employed. One was intended to be high in saturated fatty acids and during this experimental period the pellets were soaked in lard liquified by melting in boiling water ("saturated" diet). The other was intended to be high in polyunsaturated fatty acids and during the "polyunsaturated" dietary periods the pellets were soaked in corn oil. In Experiments 1 and 2 the "saturated" diets were given first and in Experiments 3 and 4 the "polyunsaturated" diets were given first. The proportion of linoleic acid (C18:2) as a percentage of total plasma triglyceride fatty acid strongly reflects the degree of polyunsaturation of the diet in man (Moore et al., 1977) and, using the methods described by these workers, we carried out similar measurements in the rabbits during the different dietary periods. Cholesterol (Searcy and Bergquist, 1960), triglyceride (Eggstein and Kreutz, 1966) and blood glucose (Technicon autoanalyser) measurements were carried out at regular intervals during the experimental diets. Intra-arteriolar aggregation of platelets was studied as follows: Rabbits were anaesthetized with i.p. Nembutal (pentobarbitone sodium). Preparation of cerebral cortex, microscopic observation of pial arteries and the application of a window to the skull in the form of a perspex capsule with a removable lid, have been described elsewhere (Honour and Ross Russell, 1962; Honour and Hockaday, 1976). Briefly, a disc of bone is removed from the skull in the right parietal region, and the membranes excised to expose the
A. J. HONOUR, R. D. CARTER AND J. I. MANN
pial arteries. The capsule consists of 2 parts, a base and a lid. The base is fixed to the skull and filled with warm sterile saline. The lid is screwed into the base of the capsule, closing it and simultaneously ejecting the excess saline through a channel in the side of the lid. The skin is drawn together around the base of the capsule with silk sutures, the cut edges being located within a groove cut around it. Such a preparation can be re-used on several occasions and usually remains good for many weeks, it being possible to monitor the preparation continuously through the lid by inspection at any time. The membranes begin to grow in again from the periphery after a few days, but these can be again removed to expose afresh the pial surface. Previous injury sites show as slight swellings on the pial arteries and are easily recognized and avoided. Alternatively the distribution of the arteries and injury sites may be recorded on film. The arteries used for the experiments are between 100 and 200 ,um in diameter. Their number and distribution is very variable and can be a limiting factor in the number of observations that may be made in any one preparation. Electrical injuries were inflicted using a unipolar system as described by Honour, Pickering and Sheppard (1971). For each series of observations the animal is anaesthetized with Nembutal and with sterile precautions the capsule is opened and a warm sterile saline drip set up to leave a constant film of moisture over the exposed brain surface. The unipolar electrode is a fine platinum wire of negative polarity applied directly to the artery wall, and at a potential of 5V with a current of 10-20 t,A for 45 s replicable injuries are made. Subsequent events are followed with a
391
proportion of linoleic acid than that observed on the normal and "saturated" diets. There did not appear to be a difference between the normal and "saturated" diets.
Effects of diets on cholesterol and triglyceride levels Cholesterol levels were lower on the "polyunsaturated" diet, in each experiment, than on the "saturated" and normal diets. Triglyceride levels did not show any particular trend.
Effects of diets on concentrations of ADP required to produce platelet aggregation Regardless of the stage of each experiment, the "polyunsaturated" and "saturated" diets were associated with an increased sensitivity to ADP than that observed on the normal diet and in normal animals in other studies. These changes were independent of changes in body weight. Blood glucose levels did not change. DISC'USSION
Our results confirm the well known hypocholesterolaemic effect of polyunsaturated fat. In each of the 4 experiments cholesterol levels were at their lowest when diets containing corn oil were fed. Triglyceride levels, however, showed a variable response. The results of the platelet thrombus formation studies were surprising. On normal diets white body formation was noted (as had been the case in all our previous investigations) after the application of ADP in concentrations ranging from 1/4096 to 1/16384 (570-142-5mmol/ 1). On both the saturated and polyRESULTS unsaturated diets, sensitivity of ADP for The results of the 4 experiments are platelet thrombus formation was raised shown in the Table. (1/131072-1/262144; 1758-89 mmol/1) to the levels of increased sensitivity we had Effects of diets on triglyceride fatty acids previously noted in rabbits after the inThe "polyunsaturated" diet was, in all jection of i.v. alloxan. These results could the experiments, associated with a higher not be attributed to changes in body weight stereoscopic microscope. The platelet-aggregating agent used was adenosine diphosphate (ADP) (Sigma), dissolved in sterile normal saline, the solution containing 1 mg/ml 2-34 x 106 mmol/1, which was further diluted 1:1 serially with sterile normal saline to the required test concentrations. All solutions were freshly prepared and kept cool throughout the observations. Application was made dropwise from sterile Pasteur pipettes, the saline drip being interrupted during each challenge with ADP, the end point for sensitivity to ADP being the most dilute solution at which white body formation was seen.
392
DIET AND PLATELET THROMBUS FORMATION ce1 18
4
00
QO
0 4
OX
=
_
O_00
00
CO
ci
4-I
Co
in C> r-
P-,
C.)
0-
C
i 00
10
00 ,-
CO ,w CDOCO Wmr-
co
00
O
00
It
P-
CO
CO
CO
O CO
-
0
C
ll 4
O
00 x CO
)
q
0
=
-r
COto
N
II
10
COm
*.1
.2
oo
C)
I-
14CO
C1-
Z'S
00
Co
I'
11
r-
m
o o;
I
CO CO
co
UrC
Ci CO
00 rl
-4
-I "--
4
*s1
C)
*t
C.)
pq s
41 CD 3333a 5S
sq 4U
C)
CO
CO
aci
Ce
N
N
NN
N
N
N
t_; _;
CO
C6
CO
tc
-
CO CiCO
CO -r
m
aq
-
-_
Ci CO
COO CO ,-
Q
z -
to
o) ws
EH
0
l 00
r
Io
-4OD 114
o~~~~~
bO bo 00 *s1
%.1
-6)
eq
41'.
S °Si
4
10
A. J. HONOUR, R. D. CARTER AND J. I. MANN
0 -I0
0)I I
- 1C) _,
I
I
)
>Ct 1 cq~
~
-
~I o
00
P-
CO
i
IC
m I-
0
O
00
r
(O
CI
0 C 1co
10l
N-
CNI
C0
-
N)
C
10
10
-4
ID
1-
C
CO
0)
1
I
410
eIs
P.-
t-N
t
o
N-
10
0N
10 10
-
oo
10
IN
CO
oo
-
CO0
IN
C 00
0)
IN
Co
I"
--I 171 0)
CO
CO
-4
4
P-
C
P-
oo
Z
0i .4.
C
CO
_P
N-
CO
CO
oz o.) 0
0
0
o
o3 9
C
CS0
Ca
OD
o
0C
0
oCa
9
C5
_4
O4
C
O
N-
t-
r tCC
-4
al
r-tCONtr-00 U: .
.--
-4
_. 1-
I -
)f
.
cq
10 X6
.6 aq
-
1t6 .
Iq
r10 CC
*6 u:
r
'0 t--
N1
IN6
16 a4
-
tN IN
CO t-
6.6
ai.: -
N
0 Co tC6
_-
_
_
393
394
DIET AND PLATELET THROMBUS FORMATION
and were observed regardless of the sequence in which the diets were given. The hypocholesterolaemic effects of polyunsaturated fat are very likely to be beneficial and O'Brien et at. (1976) have claimed that their findings suggesting decreased platelet activation after a single meal high in polyunsaturated fat may also be relevant to preventive dietary therapy in man. Our findings are based on in vivo animal experiments in longer-term studies and although the findings in experimental animals are not necessarily applicable to the human situation, they do suggest that further studies of platelet function are essential before the widespread recommendation of any changes concerning the intake of dietary fat are made.
Financial support came in part from a Medical Research Council grant to A.J.H. REFERENCES EGGSTEIN, M. & KREUTZ, F. H. (1966) Eine neue Bestimmung der Neutralfette in Blut serum und Gewebe. Klin. Wsch., 44, 262. HONOUR, A. J. & HOCKADAY, T. D. R. (1976) Increased Sensitivity of in vivo Platelet Aggrega-
tion after Alloxan on Streptozotocin. Br. J. exp. Path., 57, 1. HONOUR, A. J., HOCKADAY, T. D. R., & MANN, J. I. (1976). Reversibility by Dipyridamole of the increased sensitivity of in vivo platelet aggregation in rabbits after Alloxan. Br. J. exp. Path., 57, 11. HONOUR, A. J., CARTER, R. D. & MANN, J. I. (1977) The Effects of Treatment with Aspirin and Antithrombotic Agent SH 1117 upon Platelet Thrombus Formation in Living Blood Vessels. Br. J. exp. Path., 58, 474. HONOUR, A. J. & Ross RUSSELL, R. W. (1962) Experimental Platelet Embolism. Br. J. exp. Path., 43, 350. HONOUR, A. J., PICKERING, G. W. & SHEPPARD, B. L. (1971) Ultra-structure and Behaviour of Platelet Thrombi in Injured Arteries. Br. J. exp. Path., 52, 482. MANN. J. I. & TRUSWELL, A. S. (1972). Effects of Isocaloric Exchange of Dietary Sucrose and Starch on Fasting Serum Lipids, Post Prandial Insulin Secretion and Alimentary Lipaemia in Human Subjects. Br. J. Nutr., 27, 395. MOORE, R. A., OPPERT, S., EATON, P. & MANN, J. I. (1977) Triglyceride Fatty Acids Confirm a Change in Dietary Fat. Clin. Endocr., 7, 143. O'BREIN, J. R., ETHERINGTON, M. D. & JAMIESON, S. (1976) Acute Platelet Changes after Large Meals of Saturated and Unsaturated Fats. Lancet, 1, 878. ROYAL COLLEGE OF PHYSICIANS (1976) Report on the Prevention of Coronary Heart Disease. Jnl. R. Coll. Phyaiciavs Lond., 10, 213. SEARCY, R. L. & BERGQUIST, L. M. (1960) A new colour reaction for the quantitation of serum cholesterol. Clin. chim. Acta, 5, 192. TECHNICON AUTOANALYSER MANUAL. Glucose oxidase (gum guiac) method).
