Blood lipid fractionation and blood clotting in ischaemic heart disease

Blood lipid fractionation and blood clotting in ischaemic heart disease

102 JOURNAL OF ATHEROSCLEROSISRESEARCH BLOOD L I P I D F R A C T I O N A T I O N AND BLOOD CLOTTING IN ISCHAEMIC H E A R T DISEASE J. D. BILLIMORIA...

451KB Sizes 0 Downloads 5 Views

Recommend Documents

Phospholipid fractionation and blood clotting
The separation of natural and synthetic phospholipids by silicic acid column chromatography has been studied and reprodu

ABO blood groups, age and work in ischaemic heart disease
In a series of male survivors of ischaemic heart disease there were fewer patients belonging to the risk-factor blood gr

Drugs affecting blood clotting and fibrinolysis
This chapter discusses several drugs affecting blood clotting and fibrinolysis. A interesting randomized clinical trial

102

JOURNAL OF ATHEROSCLEROSISRESEARCH

BLOOD L I P I D F R A C T I O N A T I O N AND BLOOD CLOTTING IN ISCHAEMIC H E A R T DISEASE

J. D. BILLIMORIA, V. J. IRANI AND ~. F. MACLAGAN Department of Chemical Pathology, Westminster Medical School, London (Great Britain)

(Received March 7th, 1964)

INTRODUCTION I t is well recognised that certain alterations occur in the serum lipids in cases of ischaemic heart disease. The most consistent is the rise in serum cholesterol which is particularly striking in younger patients but even here the degree of overlap with normal values is considerable, so that no diagnostic significance attaches to the testl, 2. Increased visible lipaemia has been recorded, particularly after f a t t y meals3, 4. The f a t t y acid composition has also been studied recently with conflicting results, since JAMESet al. 5 found a small increase in the oleic acid content of the acetone-soluble fraction whilst LAWRIE et al. 6 reported no significant changes in the f a t t y acid composition of the major lipid fractions. Increased clotting activity has also been reported b y many4,7, s but not by all observers 9 and a similar rise has been regularly found after f a t t y meals, particularly with the Stypven techniquel0,11. The present paper deals with the main lipids from platelet-rich plasma in normal subjects and in patients with ischaemic heart disease. The individual phospholipids have been estimated separately and a complete analysis of the f a t t y acid composition has been included. The clotting activity of the various fractions has been investigated. SUBJECTS, METHODS AND MATERIALS Subjects

Twelve patients between the ages of 40-70 who had a previous history of myocardial infarction confirmed b y electrocardiographic changes were chosen for the main study. They were not on anticoagulant therapy. The interval between infarction and testing varied from 1-6 weeks. The control group of 12 subjects was chosen from the hospital staff or from in-patients who were free from any diseases likely to affect lipid metabolism (diabetes mellitus, xanthomatosis, thyroid disease or nephrosis). The mean age of the patient group (61 years) was somewhat higher than that of the control group (47 years). All the subjects investigated were either on a normal selfchosen diet or a normal ward diet. Blood was collected 2 hours after a fat-free breakfast consisting of tea or coffee without milk and an unrestricted quantity of toast and J. Atheroscler. Res., 5 (1965) 102-111

BLOOD LIPID FRACTIONATIONAND BLOODCLOTTING

103

jam. The Stypven clotting times and lipaemia of platelet-poor plasma of 30 patients with ischaemic heart disease and 30 normal subjects from a previous study 4 are also included here in Table I. The total cholesterol values of 89 subjects with ischaemic heart disease and 45 normal subjects investigated during the course of some 3 years and not published elsewhere are reported in Table III.

Treatment of blood and extraction of plasma Thirty to forty ml of blood was drawn with a siliconed syringe (19-gauge needles) into siliconed tubes containing sodium citrate (3.8% w/v, ~th volume of blood} cooled to 0°C. The blood was centrifuged at 750 g for 15 min and the platelet-rich plasma separated leaving about 0.25 cm of plasma above the red cells. Platelet-poor plasma was obtained by centrifugation at 3500 g for 10 min at 4°C. The plasma turbidity was read on a nephelometer with its arbitrary standard set at 100 units lz. Platelet-poor plasma was only used for the experiments shown in Table I where blood was obtained by a siliconed two-syringe technique. All other work was done on platelet-rich plasma treated as follows. Two 5 ml aliquots of plasma were extracted with ethanol-ether (3 : 1 v/v, 125 ml) as described previously; the remaining plasma was used in the Stypven clotting test 12. One extract was used for a study of the separation and estimation of phospholipid fractions and a second extract was used for estimations of cholesterol and triglycerides and for the fatty acid studies.

Separation of major lipid groups The above extract was evaporated to dryness and redissolved in light petroleum ether (b.p. 60-80°C) and applied to a silicic acid (10 g) column (2 cm diameter). The cholesterol esters were eluted with ether-light petroleum (250 ml, 1% v/v), triglycerides with ether-light petroleum (200 ml, 4% v/v), free cholesterol with ether (100 ml) and the phospholipids with methyl alcohol-water (200 ml, 95% v/v). The quantities and concentration of solvents were arrived at after several check runs in which cholesterol, ester groups and organic phosphorus were estimated on each tube (25 ml fraction) to ensure that no overlap occurred. The phospholipids were further fractionated by linear gradient chromatography 12 into cephalins (mainly phosphatidyl-ethanolamine and a small amount of phosphatidyl-serine) and lecithins, sphingomyelins and lysolecithins.

Fatty acids The individual fractions of triglycerides, cholesterol ester, cephalin and the combined lecithin and sphingomyelin fractions, after removal of solvent, were hydrolysed with ethanolic alkali (22.5 ml ethanol containing 2.5 ml 10 N aqueous KOH) for 2 h at 40°C. Each solution was diluted with water (100 ml) and acidified with aqueous sulphuric acid (4 N, until acid to phenolphthalein). The fatty acids liberated were extracted into n-heptane and were recovered after evaporation of the solvent in vacuo. The fatty acids dissolved in ether were esterified with freshly distilled ethereal diazomethane until tile yellow colour of diazomethane was permanent after standing

j. Atheroscler. Res., 5 (1965) 102-111

104

J. D. BILLIMORIA, V. J. IRANI, N. F. MACLAGAN

at 0°C for 1 h and subsequently room temperature for 1.5 h. The methyl-esters were distilled at 10-2 mm Hg pressure at bath temperature 120°C before being subjected to gas-liquid chromatography. The fatty acids from the combined lecithin and sphingomyelin fraction give only the lecithin fatty acids on hydrolysis by the above procedure.

Gas chromatography The fatty acid esters were chromatographed on a 4' glass column in a Pye-Argon chromatogram. Polyethyleneglycoladipate columns (10% w/w) or LAC 296 (17.5% w/w) on celite were both used successfully at 175°C. The separation of stearic and oleic acid was better on the LAC columns which were also found to be more stable over longer periods of use. In certain cases the runs were checked by the use of an Apiezon M column (20 % w/w on celite). After every two test runs an authentic sample of fatty acid methylesters from Cls-C22 including oleic, linoleic, linolenic and arachidonic acid methyl esters (Mann Assayed, fatty acid methyl esters for chromatography) were run at the same flow rate as the test samples. The positions of the acids on the chromatogram were deduced from retention volumes and comparisons with the authentic samples and the quantities were obtained by triangulation of the peaks.

Chemical analysis and Stypven clotting times Amino and quaternary nitrogen, organic phosphorus, esters, sugars, cholesterol were estimated and paper and thin-layer chromatograms and electrophoretic separation of bases carried out as described in an accompanying paper 12. The methods for Stypven clotting times and nephelometric units of turbidity for measuring lipaemia are given in the same reference. RESULTS

Stypven clotting times and visible lipaemia readings of platelet-rich and platelet-poor plasma are shown in Table I. The clotting times of both plasmas are shorter and the lipaemia higher in the ischaemic heart disease group. The platelet-rich plasma was always noticeably turbid due to platelets and buffy layer and the turbidity difference in the preparations was not significant. The individual lipid levels are shown in Table II. The total cholesterol showed the expected rise which was also seen in free and ester cholesterol. The increase in the other lipid fractions was not statistically significant. A comparison of phospholipid fractions shown in Table III indicated no differences in the cephalin or lysolecithin levels of the two groups when these fractions were calculated as percentage of the total phospholipids or as mg lipid/100 ml of whole blood. The sphingomyelin level in the patient was significantly raised and the lecithin level was slightly decreased. The Stypven clotting activity of the lipid fractions from plasma (Table IV)

J. Atherosder. Res., 5 (1965) 102-111

BLOOD LIPID FRACTIONATION AND BLOOD CLOTTING

105

s h o w s t h a t n e u t r a l f a t s ( c o n t a i n i n g t r i g l y c e r i d e s a n d free a n d e s t e r i f i e d c h o l e s t e r o l ) , l e c i t h i n s a n d s p h i n g o m y e l i n s of b o t h g r o u p s of s u b j e c t s w e r e i n a c t i v e in t h e t e s t . B o t h c e p h a l i n s w e r e a c t i v e b u t i n t h e p a t i e n t g r o u p c e p h a l i n (70 u n i t s / r a g ) w a s a p p r o x i m a t e l y 10 t i m e s as a c t i v e as n o r m a l c e p h a l i n (6 u n i t s / r a g ) . T h e l y s o l e c i t h i n s in b o t h g r o u p s w e r e i n h i b i t o r s of c l o t t i n g .

TABLE

I

MEAN STYPVEN CLOTTING TIME AND T U R B I D I T Y OF PLATELET-POOR (PP) AND PLATELET-RICII (PR) PLASMAS IN ISCHAEMIC HEART DISEASE ( 4 - S.E.)

Plasma

Number of patients

Number of normals

PP PR

30 7

PP PR

30 12

Lipaemia (units)

Significance of difference (P)

Normal

Patients

30 7

21.7 4- 1.2 46.3 4- 3.6

31.1 4- 2.9 56.2 4- 7.3

30 12

31.0 ± 1.2 30.0 4- 1.4

< 0.001 < 0.2

Stypven time (sec) 27.4 4- 1.2 26.2 4- 1.2

< 0.02 < 0.05

TABLE II MEAN PLASMA LIPID ANALYSIS IN ISCHAEMIC HEART DISEASE ( 4 - S. E.)

Lipid fraction

Free cholesterol Ester cholesterol Total cholesterol Triglycerides Phospholipids Total lipids

Number of normals

Number of patients

12 12 45 12 12 12

12 12 89 12 12 12

Lipids (mg/lO0 ml) Normal

Patients

53 133 226 86 214 481

68 147 260 90 225 528

444444-

4 9 6 17 9 31

i 44444-

Significance of difference (P)

11 21 5 13 20 55

< < < < < <

0.3 0.5 0.001 0.9 0.7 0.5

TABLE III PHOSPHOLIPID FRACTIONS OF PLASMA IN ISCHAEMIC HEART DISEASE*

Component

Cephalin Lecithin Sphingomyelin Lysolecithin

Number of subjects in each group 12 12 12 9

Normal % of total

mg/lO0 ml

% of total

mg/lO0 ml

Significance of difference (% of total)

8.1 72.5 17.5 1.8

16.3 149.2 35.8 3.8

7.9 68.8 21.6 1.8

15.9 138.8 43.7 4.0

< < < <

± 4i 4-

Patients

0.5 1.6 1.1 0.3

4- 1.3 -V 7.0 4- 2.4 4- 1.4

4444-

0.7 1.4 1.4 0.5

4444-

1.0 6.7 3.4 1.7

0.9 0.1 0.05 0.9

* Mean values are shown for each fraction as a percentage of total phospholipids and as mg of fraction/100 ml whole blood (-4- S.E.)

j . Mtherosder. Res., 5 (1965) 102-111

106

J.D.

BILLIMORIA, V. J. IRANI, N. F. MACLAGAN

TABLE IV MEAN CLOTTING ACTIVITY OF LIPID FRACTIONS OF PLASMA FROM 12 NORMAL SUBJECTS AND 12 PATIENTS WITH ISCHAEMIC HEART DISEASE ( ~ S. E.)

Clotting activity (units~rag)

Lipid fraction

Normal

Patients

N e u t r a l f a t ( t r i g l y c e r i d e s + c h o l e s t e r o l esters) Lecithins Cephalins

0 0 6.2 4- 0.9

0 0 70.2 4- 5.4

Sphingomyelins Lyso]ecithins

0 inhibition P < 0.0001

0 inhibition

TABLE V CLOTTING ACTIVITIES OF NORMAL AND HEART PATIENTS' CEPHALIN* (FRACTIONS FROM PLATELET-RICH PLASMA)

Subject No.

Normal subjects Stypven time (see)

A ctivi(v (units~rag)

1 2 3

23.5 8 8

0.8 8.0 8.0

4 5 6 7 8 9

9 8 17 9 18 8

10 11 12

8 8 8

Mean =t= S . E . P

Subject No.

Patients Stypven time (see)

A ctivity (units~rag)

13 14 15

6 6 7

< 80 < 80 50

6.4 8.0 2.0 8.0 0.8 8.0

16 17 18 19 20 21

6 6 7 9 6 8

< 80 < 80 80 24 < 80 48

8.0 8.0 8.0

22 23 24

6 6 6

< 80 < 80 < 80

6.2 =t= 6.9

70.2 :=[=5.4 < 0.00001

* All c l o t t i n g t i m e s recorded are a m e a n of t r i p l i c a t e d e t e r m i n a t i o n s . U s u a l l y l i p i d c o n t a i n i n g l # g p h o s p h o r u s w a s a d d e d to t h e s y s t e m . W h e r e v a r y i n g q u a n t i t i e s were us e d t h e u n i t s w e re calc u l a t e d f r o m a c a l i b r a t i o n curve. F o r s t a t i s t i c a l c a l c u l a t i o n v a l u e s < 80 u n i t s / m g h a v e b e e n t a k e n as 80.

The Stypven clotting times and activities in units/mg of cephalin from individual subjects are shown in Table V. There was no overlap in the clotting activity of the two groups, the least active patients' cephalin being more active than the most active normal cephalin. Although the apparent difference in the clotting times of the two groups was small this difference represents a 10-fold increase in active lipid concentration. This will be seen from the calculated units and also from a dilution curve j . Atheroscler. Res., 5 (1965) 102-111

107

BLOOD L I P I D F R A C T I O N A T I O N AND BLOOD CLOTTING

of an active synthetic dioleyl cephalin plotted against Stypven clotting times as shown in Fig. 1. The apparent difference is also diminished by taking values > 80 as 80 units/rag.

40

. . . . . . . . . . . c.o2.t£o[. 2ay2,y_?............ 30 E o~ 2O ¢:

o U

10

~-4

i

i

10-3

10-2

Lipid~system

I

10-I

(mg)

Fig. 1. S t y p v e n c l o t t i n g a c t i v i t y of d i l u t i o n of s y n t h e t i c d i o l e y l p h o s p h a t i d y l e t h a n o l a m i n e .

In view of the higher clotting activity of the patients' cephalin, the fatty acid composition of the cephalins in the two groups of subjects was investigated (Table VI). F a t t y acids with higer carbon numbers than 20 were grouped together and constituted 5-6% of the total in both groups. The only significant difference was found in the oleic acid content of the two groups, the patient group containing some 30% oleic acid as compared with only 19% oleic acid in the normal group (P > 0.02). TABLE MEAN

VI

PERCENTAGE

FATTY

ACID

PATIENTS' PLASMA CEPHALINS ( ~

Carbon No.*

Normal subjects (9)

COMPOSITION

OF NORMAL

AND

S. E.)

Patients (9)

Significance of difference (P)

12 : 0 14 : 0

2.7 4- 1.0 4.8 -g 1.3

1.3 ± 0.5 4.1 4- 0.8

< 0.3 < 0.7

16 : 0 16 : 1

15.1 4- 2.2 8.9 4- 3.0

11.1 ::t: 2.6 11.7 :j: 2.0

< 0.3 < 0.3

16 : 2

1.0 4- 0.6

0.5 ± 0.3

< 0.5

17 : 0 ? 18 : 0

5.7 4- 1.4 7.1 4- 0.8

4.9 4- 2.3 6.6 ± 1.9

< 0.8 < 0.9

18 : 1

19.1 4- 2.5

30.5 4- 3.9

18 : 2 18 : 3

6.7 4- 2.1 8.4 4- 2.7

6.7 4- 2.3 5.7 _u 1.5

N.S. < 0.3

20 : !

8.1 + 2.2

5.7 _u 2.0

< 0.5

20 : 4

7.2 :k 2.0

5.1 i

< 0.5

1.4

< 0.02

* T h e c a r b o n a t o m s a n d n u m b e r of d o u b l e b o n d s a r e r e p r e s e n t e d b e f o r e a n d a f t e r t h e colon.

J. Atheroscler. Res., 5 (1965) 102-111

108

j. D. BILLIMORIA, V. J. IRANI, N. F. MACLAGAN

T h e t o t a l u n s a t u r a t e d to s a t u r a t e d a c i d r a t i o ( c a l c u l a t e d f r o m t h e table) was g r e a t e r for. t h e p a t i e n t g r o u p (2.33 : 1.00) t h a n for t h e n o r m a l g r o u p (1.67 : 1.00) a n d t h e oleic a c i d c o n t e n t a c c o u n t e d for a b o u t h a l f t h e u n s a t u r a t e d acids in i s c h a e m i c h e a r t disease. T h e f a t t y a c i d c o m p o s i t i o n of t h e t r i g l y c e r i d e f r a c t i o n of p l a s m a is s h o w n in T a b l e V I I . A g a i n t h e oleic a c i d f r a c t i o n , w h i c h r e p r e s e n t e d well o v e r half t h e u n s a t u r a t e d a c i d s (36 %) in i s c h a e m i c h e a r t disease, was c o n s i d e r a b l y h i g h e r h e r e t h a n in t h e n o r m a l g r o u p ( 2 3 % ) (P < 0.01). T h e differences in t h e rest of t h e f a t t y a c i d c o m p o s i t i o n w e r e n o t significant. T h e f a t t y a c i d c o m p o s i t i o n of t h e l e c i t h i n a n d c h o l e s t e r o l e s t e r f r a c t i o n s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t in t h e t w o g r o u p s a n d are n o t t a b u l a t e d . T h e oleic a c i d c o n t e n t of t h e f o u r m a i n lipid f r a c t i o n s s t u d i e d are s u m m a r i s e d in T a b l e V I I I .

T A B L E VII MEAN F A T T Y ACID COMPOSITION OF PLASMA TRIGLYCERID]~S ( 4 - S.E.)

Carbon No.

Normal subjects (9)

Patients (9)

Significance of difference ( P)

1.1 4- 0.3

< 0.3 < 0.3

12 : 0

1.6 4-4-0.3

14 : 0 16 : 0

3.7 4- 1.6 17.9 4- 4.2

16 : 1

8.6 4- 1.8

6.1 4- 1.7

< 0.4

16 : 2

1.0 3.0 4- 2.2

0.5 4- 0.9 2.3 4- 0.4

1.9 4- 0.5 16.6 4- 3.0

< 0.9

18 : 0

3.0 4- 0.4

2.4 4- 2.3

18 : 1

22.7 4- 2.1

36.1 4- 3.2

18 : 2

5.3 4- 1.1

5.6 4- 1.4

< 0.5 <0.8 < 0.9 < 0.01 < 0.9

18 : 3

4.9 4- 1.6

3.8 4- 0.6

< 0.6

20 20 20 20 20

1.6 7.4 1.6 2.1 5.2

1.3 4.3 1.7 0.3 3.0

< < < < <

17:0

1.5 4-

?

:0 :1 :2 :3 :4

4- 1.1 4- 2.4 4- 1.3 4- 1.4 4- 1.7

4- 0.6 4- 0.9 4- 0.9 ± 0.3 4- 0.9

0.9 0.3 0.9 0.3 0.3

TABLE V I I I OLEIC ACID C O N T E N T OF PLASMA L I P I D S FROM NORMALS AND P A T I E N T S (MEAN 4- S. F..)

Lipid fraction

Triglycerides Cholesterol esters Cephalins Lecithins

Oleic acid content

Significance (P)

Patients

Normals

35.0 20.0 30.5 18.4

23.0 20.0 19.0 14.2

± 3.0 -t- 2.9 ± 4.0 -4- 2.1

J. Atheroscler. Res., 5 (1965) 102-111

44± ±

2.0 0.9 1.5 1.2

< 0.01 N.S. < 0.02 N.S.

BLOOD L I P I D FRACTIONATION AND BLOOD CLOTTING

109

Other chemical differences

The amino-N/P ratios of the cephalin in normal subjects gave the expected value 0.99 (S.E. 0.2) but the unusually high value of 3.22 (S.E. 0.3) in ischaemic heart disease. No qualitative differences in the two cephalins were observed on thin-layer chromatography or paper electrophoresis of the bases after hydrolysis of the cephalins. However, on high-voltage electrophoresis 12 of the two intact cephalins, patients' cephalin was found to contain some free serine which would account for the highel amino-nitrogen value. The cerebroside fractions which occur with the cephalins were found to contain the same amount of sugar in both normal and patients' plasm~ (54.6 ± 5.1 and 58.7 4-3.5 #moles respectively, calculated as galactose in the orcinol-H2S04 reaction). DISCUSSION

In comparative studies in blood coagulation most workers have chosen to work with platelet-poor plasma mainly because of difficulties arising from platelet breakclown during manipulation of the blood. Nevertheless, platelets are an important source of active lipids and for this reason platelet-rich plasma has been used in the present work. Examination of the lipid fractions of the ischaemic heart disease group showed the expected increase in total cholesterol and in addition a small but significant increase in sphingomyelin with a decrease in the level of lecithin. These results are somewhat similar to the finding of increased cholesterol and sphingomyelin in the arterial wall in Stage III of atherosclerosis 13. The only blood lipid which is active in the Stypven coagulation test is cephalin 1~. Although the plasma cephalin level in ischaemic heart disease showed no increase over the normal, the patients' cephalin was far more active in coagulation than normal cephalin and it therefore seemed likely that a qualitative difference of the two cephalins must exist. Indeed although they contained the same bases (ethanolamine and serine), their fatty acid composition was markedly different, since patients' cephalin contained a definite increase of oleic acid. Our previous finding on the high clotting activity of oleyl cephalin from platelets 12 indicates that the differences between normal and patients' plasma may well reside in their platelet cephalin composition. Palmitoleyl, linoleyl and arachidonyl cephalin have not been tested for clotting activity but are unlikely to be involved since they are present in equal amounts in normal and patients' cephalin. The absence of overlap between the clotting activity of cephalins from normal and patients' platelet-rich plasma was particularly noteworthy and contrasts with the results of lipid estimations. It recalls the reports on platelet stickiness by MCDONALD AND EDGILL 8, and emphasises the importance of platelet investigations in this condition. In the plasma studied here, obtained after a fat-free breakfast, there was negligible turbidity due to fat (since further centrifugation produced a clear plasma) and the J. Atheroscler. Res., 5 (1965) 102-111

110

j . D . BILLIMORIA,V. J. IRANI, N. F. MACLAGAN

arachidonic acid content of their cephalin fractions was between 6--9~/o of total f a t t y acids. This contrasts with NELSON'S15 findings on lipaemic plasma where as much as 25~/o arachidonic acid was reported. I t should be noted that our patients were not studied in the fasting state and the influence of our fat free meal on their plasma lipids is unknown; however, the control group was tested under identical conditions. Further, the tests were necessarily carried out on the patients within 1-6 weeks of the infarct and the degree of variation in phospholipid levels during this time needs investigation. Our results suggest that the higher oleic acid content of cephalin in ischaemic heart disease is probably responsible fol its higher blood clotting activity. The other minor chemical differences observed and the increased oleic acid content of the triglyceride fraction of patients' plasma cannot directly be releated with coagulation but deserve further investigation.

ACKNOWLEDGEMENTS We are indebted to the medical staff of The Westminster Hospital Group for access to the patients, to Dr. D. C. O. JAMESfor referring cases of ischaemic heart disease and to the normal subjects for their co-operation in the tests. We also thank Miss M. ATTWOOD for valuable technical assistance and the Central Research Fund of the University of London for a grant for the purchase of the gas chromatogram. The work was supported b y generous grants from the endowment funds of Westminster Hospital.

SUMMARY

Lipids from platelet-rich plasma in cases of ischaemic heart disease have been compared with those from normal subjects and the thromboplastic activities of all fractions tested by the Stypven test. The f a t t y acid composition ot the fractions has been determined. Total cholesterol and sphingomyelin were increased in ischaemic heart disease. Increased clotting activity in ischaemic heart disease was found both with platelet-rich and with platelet-poor plasma. Only the cephalin fractions were active in clotting tests. Although the levels of total cephalin were similar in the two groups, the patients' cephalin had uniformly higher clotting activity and a markedly higher oleic acid content. The triglycerides also had a significantly higher oleic acid content in ischaemic heart disease.

J. Atheroscler. Res., 5 (1965) 102-111

BLOOD LIPID FRACTIONATION AND BLOOD CLOTTING

1 ll

REFERENCES 1 E. Y. LAWRY, G. V. MANN, A. PETERSON, A. P. WYSOCKI, R. O'CONNEL AND F. J. STORE, Am. J. Med., 22 (1957) 609. 2 M. F. OLIVER AND G. S. BOYD, Brit. Heart J., 15 (1953) 387. 3 j . R. A. MITCHELL AND B. BRONTE-STEWART,Lancet, i (1959) 167. 4 D. C. O. JAMES, J. DRYSDALE, J. D. BILLIMORIA, D. WHEATLEY, C. J. GAVEY AND N. F. MACLAGAN,Lancet, ii (1961) 798. 5 A. T. JAMES, J. E. LOVELOCK,J. WEBB AND W. S. TROTTER, Lancet, i (1957) 705. 6 T. D. V. LAWRIE, S. G. McALPINE, B. PIERIE AND B. M. RIFKIND, Clin. Sci., 20 (1960) 255. 7 j . R. O'BRIEN, Lancet, i (1958) 410. 8 L. McDONALD AND M. EDGILL, Lancet, i (1959) 1115. 9 C. MERSKEY AND H. L. NOSSEL, Lancet, i (1957) 806. 10 j . R. O'BRIEN, Lancet, ii (1956) 232. 11 N. F. MACLAGAN, J. D. BILLIMORIA AND C. CURTIS, Lancet, ii (1958) 865. 12 j . D. BILLIMORIA, V. J. IRANI AND N. F. MACLAGAN,J. Atherosder. Res., 5 (1965) 90. 13 C. J. F. B6TTCHgR AND C. M. VAN GENT, J. Atheroscler. Res., 1 (1961) 36. i4 L. McDONALD AND M. EDGILL, Lancet, ii (1957) 457. 15 G. J. NELSON, J. Lipid Res., 3 (1962) 71.

J. Atheroscler. Res., 5 (1965) 102-111