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Ischemic stroke and n-3 fatty acids
Ischemic Stroke and n-3 Fatty Acids Kaj N. Seidelin, MD, Benny Jensen, PhD, Steen B. Haugaard, MD, Jakob Reith, MD and Tom S. Olsen, MD, PhD
The content of fatty acids in subcutaneous adipose tissue was measured to determine whether differences of fatty acids correlate with presence or absence of cerebral infarction in individual patients. Adipose tissue microbiopsies was sampled from 10 patients with computed tomography (CT) verified cerebral infarction and 10 matched control subjects, and assayed for content of fatty acids by gas-liquid chromatographic analysis. There were no differences in levels of n-3 fatty acids of marine origin. Patients with cerebral infarction had statistically significant lower levels of the essential fatty acids linoleic acid (mean + SE, 8.9 + 0.4 v 10.7 + 0.5%) (P < .05) and linolenic acid (0.80 + 0.05 v 1.00 + 0.06%) (P < .05) and high levels of palmitoleic acid (8.5 + 0.6% v 5.7 + 0.4%) (P < .005) indicative of increased lipid synthesis de novo, which might explain the depressed levels of fatty acids primarily supplied by the diet. Although significant differences in levels of essential fatty acids were found, no judgement could be made regarding a causal relationship between essential fatty acids and cerebral infarction. The present study does not support the hypothesis of an association between dietary fatty acids (e.g., fish consumption) and ischemic stroke. Key Words: Ischemic stroke--n-3 Fatty acids-Dietary fat.
Dietary fat has been implicated in the pathogenesis of atherosclerosis. Recently, attention has focused on n-3 fatty acids of marine origin. The n-3 fatty acids compete with n-6 fatty acids for the synthesis of prostaglandins and thromboxanes 1,2 and thereby reduce the production of thromboxane A2,2,3 a mediator of thrombosis and vasospasm. 4 Aspirin, which blocks the biosynthesis of prostaglandins and thromboxane A2, s.6 lowers stroke risk. 6,7 Accordingly, a protective effect of consumption of n-3 fatty acids on ischemic stroke has been proposed. 8 Standard dietary surveys do not always yield reliable information concerning fat intake. The fatty acid composition of depot fat reflects that of the diet. 9 The turnover rate (TI/2) of adipose tissue fatty acids is about one and a half years. 1~ Because of the slow turnover rate of depot fat,
analysis of adipose tissue provides a measure of diet over a relative long period. Microbiopsies of fat can be easily obtained n with minimal discomfort to the subject, and is thus a useful tool in epidemiologic investigations. The postulated beneficial effects of n-3 fatty acids in preventing atherosclerosis have led to recommendations for increased dietary fish consumption. 12 In the casecontrol study presented here, we investigated the associations between dietary n-3 fatty acids and ischemic stroke. Specifically, we hypothesized that if consumption of n-3 fatty acids (i.e., fish consumption) is associated with reduced incidence of ischemic stroke, then patients with ischemic stroke might have lower levels of n-3 fatty acids in depot fat than control subjects.
Methods From the Department of Neurology, Bispebjerg Hospital, Copenhagen, and the Danish Institute for Fisheries Research, Technical University of Denmark, Lyngby,Denmark. Received January 6,1997; accepted April 25,1997. Address reprint requests to Kaj N. Seidelin, MD, Department of Neurology, Bispebjerg Hospital, 2400 Copenhagen, Denmark. Copyright 9 1997by National Stroke Association 1052-3057/97/0606-000553.00/0
Subjects Patients admitted (January 1994 to January 1996) to the stroke unit, Department of Neurology, Bispebjerg Hospital, were eligible for the study if they met the following criteria: (1) CT scan verified cerebral infarction; (2) no concurrent use of anticoagulative medication; (3) no
Journal of Stroke and Cerebrovascular Diseases, Vol. 6, No. 6, 1997: pp 405-409
405
406
K.N. SEIDELIN ET AL.
history of drug or alcohol abuse; (4) no use of marine oil supplementation within the last 12 months; (5) no history of coronary heart disease or other major concomitant disease; (6) ability and willingness to give written informed consent. Ten patients with cerebral infarction agreed to participate. The majority of patients had only minor strokes. The Scandinavian Stroke Scale (SSS) score of stroke patients on admission was 42 + 14 ( m e a n _ SD). Seven of the patients had lacunar infarcts, 2 had subcortical infarcts, and 1 had a cortical infarct. One patient had atrial fibrillation. The ECGs of other patients were normal. To minimize bias, because of an inverse relationship between coronary artery disease and n-3 fatty acids, 9 no patient had major signs of atherosclerosis such as leg claudication, angina pectoris, or cardiac insufficiency. We also excluded patients who used dietary supplements of marine oils because these patients have high levels of n-3 fatty acids in depot fat. Patients admitted to the hospital for plastic surgery served as control subjects. No history of stroke was present in the control group. Ten control subjects agreed to participate in the study. Similar exclusion criteria were used for the two groups. The study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee of Copenhagen. Stroke was defined according to the World Health Organisation (WHO) criteria: rapidly developed clinical signs of focal disturbance of cerebral function, lasting more than 24 hours or leading to death, with no apparent
BHT 14:0 16:0 16:1 \
/
\
cause other than vascular origin. 13 CT scanning was performed with a Siemens Somatom DR scanner. Contrast was not given routinely. The SSS was used to assess neurological deficits. 14,1sSSS evaluates level of consciousness; eye movement; power in arm, hand, and leg; orientation, aphasia; facial paresis; and gait. The total score ranges from 0 to 58 points.
Techniques Subcutaneous adipose tissue biopsies were obtained by needle aspiration from the buttock fat by a method previously described and validated in detail by Beynen and Katan. 11 The fat biopsies were stored at -20~ until analysis. The composition of fatty acids in biopsies of adipose tissue was determined by gas-liquid chromatography (GLC) (Fig 1) after saponification of the lipid extract with methanolic sodium hydroxide (0.5 mol/L) and methylation with 20% boron triflourid (BF3) in methanol containing 0.025% hydroquinone. For GLC analysis we used an HP 5890A gas chromatograph (HewlettPackard Co, Sunnyvale, CA) equipped with an Omegawax 320 fused silica capillary column (30 m x 0.32 m m inner diameter; 0.25 ~m film thickness; Supelco Inc, Supelco Park, PA), operating from 160~ to 220~ Fatty acids were detected by flame ionization. For signal integration a HI} 3396A digital integrator was used. Fatty acids were tentatively identified by comparing their retention times with those of known standards (Nu-Chek-Prep Inc, Elysian, MN). Forty-four peaks corresponding to specific
18:1 18:2
/
\
/
14:1 18:3{o3 18:0
12:0
1 1 20:4 22:5{o3
\
"u,....Iul,.
,J~
Figure1. Gas-liquidchromatagramoffatty acid methyl esters in human adipose tissue.
22:6{o3
/
ISCHEMIC STROKE AND n-3 FATTY ACIDS
407
Table 1. Characteristics of control patients and patients
(C 18:3) were significantly lower in the stroke group when compared to controls (P < .05).
with cerebral infarction
Female (n = 10)
Male (n = 10)
Smokers Nonsmokers Smokers Nonsmokers Control subjects Patients with cerebral infarction
3
4
3
0
4
3
2
1
fatty acids were identified. The individual fatty acid methyl esters were quantified as the percentage of the total peak area. The analyses were performed in pairs and blindly. Differences between the percentages of fatty acids were determined by two-tailed Student's t-test. Probability values <.05 were considered significant.
Results
Basic characteristics of patients and control subjects are given in Table 1. The two groups were matched for age (mean • SE; 73.2 • 2.1 and 72.7 • 3.2 years, respectively), gender, and smoking habits. The content of the individual fatty acids expressed as percentage of the total peak area in the chromatograms is given in Table 2. Unidentified compounds did not exceed 2% of the total peak area. No significant differences in percentage proportions of the long-chain n-3 fatty acids, eicosapentaenoic acid (EPA) (C 20:5, n-3), docosapentaenoic acid (DPA) (C 22:5, n-3) and docosahexaenoic acid (DHA) (C 22:6, n-3), representative of fish consumption were found. A highly significant increase in palmitoleic acid (C 16:1) was found in patients with cerebral infarction when compared with control subjects (P < .005). The percentage proportions of stearic acid (18:0), linoleic acid (C 18:2) and linolenic acid
Discussion
The association between dietary fat, atherosclerosis, and stroke remains controversial. Epidemiologic studies of Greenland Eskimos 16 and Japanese 17 populations who consume substantial amounts of n-3 fatty acids have shown low mortality rates from coronary heart disease (CHD), although incidence rates of cerebrovascular disorders are not low. These cross-cultural comparisons, however, are of limited value because populations may be protected by racial factors, and these studies did not take into account numerous other risk factors that differ among populations. The observed association between a low-fat diet, intracerebral hemorrhage, and lacunar stroke in Japan may be explained by other major risk factors; the incidence rates of stroke, salt consumption, and blood pressure have declined markedly during the last decade. 10 Previous case-control studies of fish consumption and stroke have shown conflicting results. Although some studies showed significant inverse associations between fish consumption and stroke incidence, 8,18 others did not. 19,20Stratified analyses according to stroke type were used in only one previous study; no significant association between fish consumption and ischemic stroke was found. 21 Although previous studies have involved thousands of subjects, they have all been based on dietary recall, and discrepancies may be explained by inaccurate assessment of dietary fat. One case-control study on the fatty acid composition in 33 patients with cerebrovascular disease has been published, 22 but did not include measurements of n-3 fatty acids. Lower levels of the essential fatty acid linoleic acid (C 18:2) among male patients with stroke were reported. These results should be interpreted with caution because no distinction was made between hemorrhage and isch-
Table 2. Fatty acid composition in controls and cases of cerebral h,farction* Common name
Fatty acid
Controls (n = 10)
Infarcts (n = 10)
Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Eicosapentaenoic acid Docosapentaenoic acid Docosahexaenoic acid
C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2,n-6 C 18:3,n-3 C 20:5,n-3 C 22:5,n-3 C 22:6,n-3
2.8 - 0.2 18.8 --- 0.8 5.7 • 0.4 3.8 - 0.4 47.6 - 0.7 10.7 --- 0.5 1.00 • 0.06 0.10 • 0.01 0.30 - 0.02 0.34 • 0.03
2.7 - 0.2 19.8 --. 0.8 8.5 • 0.6 2.7 • 0.2 47.6 • 0.9 8.9 --- 0.4 0.80 --- 0.05 0.11 • 0.01 0.31 __.0.02 0.30 • 0.03
*• + SE.
P P P P
NS NS < .005 < .05 NS < .05 < .05 NS NS NS
408 emic stroke, and the mean age of patients was 67 years, in comparison to a mean age of 29 years of the control subjects. 22 To the best of our knowledge, ours is the first investigation on content of n-3 fatty acids in adipose tissue in patients with ischemic stroke. No differences were found in content of n-3 fatty acids of marine origin in stroke patients compared with matched controls. The observation of small decreases in concentrations of linoleic acid and linoleic acid should not be interpreted as a sign of chronic deficiency of essential fatty acids. Linoleic acid is the most variable fatty acid in human beings. For example, in New Zealanders, ~ linoleic acid contributes to only 3% of the total fatty acids, compared with 25.6% in Israelis. 24Variation in linoleic acid content of this order of magnitude among populations on no experimental diet does not support those observations in which small differences in linoleic acid concentrations in stroke patients have been interpreted as relative linoleic acid deficiency. The patients with cerebral infarction yielded high levels of palmitoleic acid. Palmitoleic acid usually constitutes about 5% to 7% of the total fatty acids in human adipose tissue. 9 Marginally higher amounts are found in populations subsisting on very-low-fat diets. 9 Higher levels also can be found in conditions of increased de novo lipid synthesis such as in utero 2s or among patients on longterm parenteral nutrition. 26 Alcohol consumption also correlates positively to adipose tissue levels of palmitoleic acid and negatively to linoleic acid. 27 Alcohol is used for de novo lipid synthesis in the liver (before use as energy fuel in peripheral tissues) with production of nonessential fatty acids. Therefore the lower adipose tissue linoleate levels may simply be a dilutional effect of newly synthesized nonessential fatty acids. At present we have no easy explanation for the high levels of palmitoleic acid among the stroke patients in this study; they had minor strokes and did not receive parenteral nutrition. Also patients with known or suspected abuse of alcohol were not included. Case-control studies are sensitive to selection bias. In considering reasons for the differences, it cannot be excluded that volunteers were more conscious of their health but control subject were chosen from a group of patients within the same hospital. Cases and controls were matched for all background variables known to bias the levels of adipose tissue fatty acids. 9 In conclusion, although significant differences in levels of essential fatty acids were found in our study, no conclusions can be made about the possibility of a causal relationship between the intake of essential fatty acids and stroke. The present study does not support the hypothesis of an association between dietary n-3 fatty acids (e.g., fish consumption) and ischemic stroke.
K.N. SEIDELIN ET AL.
References 1. Galli C, Agradi E, Petroni A, Socini A. Modulation of prostaglandin production in tissues by dietary essential fatty acids. Acta Med Scand 1980;642(suppl):171-9. 2. Fischer S, Weber PC. Thromboxane A3 (TXA3) is formed in human platelets after dietary eicosapentaenoic acid (C20:5oJ3). Biochem Biophys Res Commun 1983;116: 1091-9. 3. Dyerberg J, Bang HO, Stoffersen E. Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? Lancet 1978;2:117-9. 4. Kinsella JE, Lokesh B, Stone RA. Dietary n-3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms. Am J Clin Nutr 1990;52:128. 5. Clarke RJ, Mayo G, Price P, Fitzgerald GA. Suppression of thromboxane A2 but not of systemic prostacyclin by controlled-release aspirin. N Engl J Med 1991;325:113741. 6. Oates JA, Fitzgerald GA, Branch RA, Jackson EK, Knapp HR, Roberts LJ. Clinical implications of prostaglandin and thromboxane A2 formation. N Engl J Med 1981;304: 76-9. 7. Antiplatelet Trialists' Collaboration. Secondary prevention of vascular disease by prolonged antiplatelet treatment. Br Med J 1988;296:320-31. 8. Kell SO, Feskens EJM, Kromhout D. Fish consumption and risk of stroke: The Zupten Study. Stroke 1994;25:328332. 9. Seidelin KN. Fatty acid composition of adipose tissue in humans. Implications for the dietary fat-serum cholesterol-CHD issue. Prog Lipid Res 1995;3:199-217. 10. Dayton S, Pearce ML, Hashimoto S, Dixon WJ, Tomiyasu U. A controlled clinical trial of a diet high in unsaturated fat in preventing complications of atherosclerosis. Circulation 1969;40 (suppl.l]):1-63. 11. Beynen AC, Katan MB. Rapid sampling and long-term storage of subcutaneous adipose tissue biopsies for deterruination of fatty acid composition. Am J Clin Nutr 1985;42:317-22. 12. Beilin LJ. Non-pharmacological management of hyper tension: optimal strategies for reducing cardiovascular risk. J Hypertens 1994;12(suppl 10):$71-$81. 13. Report of the WHO task force on stroke and other cerebrovascular disorders. Stroke-1989: recommendations on stroke prevention, diagnosis and therapy. Stroke 1989;20:1407-31. 14. Scandinavian Stroke Study Group. Multicenter trial of hemodilution in ischemic stroke: Background and study protocol. Stroke 1985;16:885-90. 15. Lindenstrom E, Boysen G, Christiansen LW, ~ Rogvi Hansen B, Nielsen PW. Reliability of Scandinavian Stroke Scale. Cerebrovasc Dis 1991;1:103-7. 16. Kromann N, Green A. Epidemiological studies in the Upemavik District, Greenland. Acta Med Scand 1980;208: 401-6. 17. Shimamoto T, Komachi T, Inada H, et at. Trends for coronary heart disease and stroke and their risk factors in Japan. Circulation 1989;79:503-5. 18. Gillum RF, Mussolino ME, Madans JH. The relation between fish consumption and stroke incidence: the NHANES I Epidemiologic Follow-up Study. Arch Intern Med 1996;156:537-42. 19. Morris MC, Manson JE, Rosner B, Buring JE, Willet WC, Hennekens CH. Fish consumption and cardiovascular
ISCHEMIC STROKE A N D n-3 FATTY ACIDS
20.
21.
22.
23.
disease in the Physicians' Health Study: A prospective study. Am J Epidemiol 1995;142:166-75. Orencia AJ, Daviglus ML, Dyer AR, ShekeUe RB, Stamler J. Fish consumption and stroke in men: 30 years findings of the Chicago Western Electric Study. Stroke 1996;27: 204-9. Jamrozik K, Broadhurst RJ, Anderson CS, StewartWynne EG. The role of lifestyle factors in the etiology of stroke: A population-based case-control study in Perth, Western Australia. Stroke 1994;25:51-9. Antonini FM, Bucalosi A, Petruzzi, Simoni R, Morini PL, D'Alessandro A. Fatty acid composition of adipose tissue in normal, atherosclerotic and diabetic subjects. Atherosclerosis 1970;11:279-89. Shorland FB, Czochanska Z, Prior [AM. Studies on the
409
24. 25. 26. 27.
fatty acid composition of adipose tissue and blood lipids of Polynesians. Am J Clin Nutr 1969;22:594-605. Blondheim SH, Home T, Davidovich R, Kapitulnik J, Segal S, Kaufmann NA. Unsaturated fatty acids in adipose tissue of Israeli Jews. Isr J Med Sci 1976;12:658-61. King KC, Adam PAJ, Laskowski DE, Schwartz R. Sources of fatty acids in the newborn. Paediatrics 1971;47(supp1.II)192-8. Ito Y, Hudgins LC, Hirsch J, Shike M. Adipose tissue fatty acid composition in recipients of long-term total parenteral nutrition (TPN). Am J Clin Nutr 1991;53:1483-92. Arab L, Schellenberg B, Schlierf G. EmKhrung und Gesundheit. Eine Untersuchung bei jungen Frauen und M/innem in Heidelberg. Infusionstherapie 1981;7(suppl.II):l-203.
The content of fatty acids in subcutaneous adipose tissue was measured to determine whether differences of fatty acids correlate with presence or absence of cerebral infarction in individual patients. Adipose tissue microbiopsies was sampled from 10 patients with computed tomography (CT) verified cerebral infarction and 10 matched control subjects, and assayed for content of fatty acids by gas-liquid chromatographic analysis. There were no differences in levels of n-3 fatty acids of marine origin. Patients with cerebral infarction had statistically significant lower levels of the essential fatty acids linoleic acid (mean + SE, 8.9 + 0.4 v 10.7 + 0.5%) (P < .05) and linolenic acid (0.80 + 0.05 v 1.00 + 0.06%) (P < .05) and high levels of palmitoleic acid (8.5 + 0.6% v 5.7 + 0.4%) (P < .005) indicative of increased lipid synthesis de novo, which might explain the depressed levels of fatty acids primarily supplied by the diet. Although significant differences in levels of essential fatty acids were found, no judgement could be made regarding a causal relationship between essential fatty acids and cerebral infarction. The present study does not support the hypothesis of an association between dietary fatty acids (e.g., fish consumption) and ischemic stroke. Key Words: Ischemic stroke--n-3 Fatty acids-Dietary fat.
Dietary fat has been implicated in the pathogenesis of atherosclerosis. Recently, attention has focused on n-3 fatty acids of marine origin. The n-3 fatty acids compete with n-6 fatty acids for the synthesis of prostaglandins and thromboxanes 1,2 and thereby reduce the production of thromboxane A2,2,3 a mediator of thrombosis and vasospasm. 4 Aspirin, which blocks the biosynthesis of prostaglandins and thromboxane A2, s.6 lowers stroke risk. 6,7 Accordingly, a protective effect of consumption of n-3 fatty acids on ischemic stroke has been proposed. 8 Standard dietary surveys do not always yield reliable information concerning fat intake. The fatty acid composition of depot fat reflects that of the diet. 9 The turnover rate (TI/2) of adipose tissue fatty acids is about one and a half years. 1~ Because of the slow turnover rate of depot fat,
analysis of adipose tissue provides a measure of diet over a relative long period. Microbiopsies of fat can be easily obtained n with minimal discomfort to the subject, and is thus a useful tool in epidemiologic investigations. The postulated beneficial effects of n-3 fatty acids in preventing atherosclerosis have led to recommendations for increased dietary fish consumption. 12 In the casecontrol study presented here, we investigated the associations between dietary n-3 fatty acids and ischemic stroke. Specifically, we hypothesized that if consumption of n-3 fatty acids (i.e., fish consumption) is associated with reduced incidence of ischemic stroke, then patients with ischemic stroke might have lower levels of n-3 fatty acids in depot fat than control subjects.
Methods From the Department of Neurology, Bispebjerg Hospital, Copenhagen, and the Danish Institute for Fisheries Research, Technical University of Denmark, Lyngby,Denmark. Received January 6,1997; accepted April 25,1997. Address reprint requests to Kaj N. Seidelin, MD, Department of Neurology, Bispebjerg Hospital, 2400 Copenhagen, Denmark. Copyright 9 1997by National Stroke Association 1052-3057/97/0606-000553.00/0
Subjects Patients admitted (January 1994 to January 1996) to the stroke unit, Department of Neurology, Bispebjerg Hospital, were eligible for the study if they met the following criteria: (1) CT scan verified cerebral infarction; (2) no concurrent use of anticoagulative medication; (3) no
Journal of Stroke and Cerebrovascular Diseases, Vol. 6, No. 6, 1997: pp 405-409
405
406
K.N. SEIDELIN ET AL.
history of drug or alcohol abuse; (4) no use of marine oil supplementation within the last 12 months; (5) no history of coronary heart disease or other major concomitant disease; (6) ability and willingness to give written informed consent. Ten patients with cerebral infarction agreed to participate. The majority of patients had only minor strokes. The Scandinavian Stroke Scale (SSS) score of stroke patients on admission was 42 + 14 ( m e a n _ SD). Seven of the patients had lacunar infarcts, 2 had subcortical infarcts, and 1 had a cortical infarct. One patient had atrial fibrillation. The ECGs of other patients were normal. To minimize bias, because of an inverse relationship between coronary artery disease and n-3 fatty acids, 9 no patient had major signs of atherosclerosis such as leg claudication, angina pectoris, or cardiac insufficiency. We also excluded patients who used dietary supplements of marine oils because these patients have high levels of n-3 fatty acids in depot fat. Patients admitted to the hospital for plastic surgery served as control subjects. No history of stroke was present in the control group. Ten control subjects agreed to participate in the study. Similar exclusion criteria were used for the two groups. The study was conducted in accordance with the Helsinki Declaration and approved by the Ethical Committee of Copenhagen. Stroke was defined according to the World Health Organisation (WHO) criteria: rapidly developed clinical signs of focal disturbance of cerebral function, lasting more than 24 hours or leading to death, with no apparent
BHT 14:0 16:0 16:1 \
/
\
cause other than vascular origin. 13 CT scanning was performed with a Siemens Somatom DR scanner. Contrast was not given routinely. The SSS was used to assess neurological deficits. 14,1sSSS evaluates level of consciousness; eye movement; power in arm, hand, and leg; orientation, aphasia; facial paresis; and gait. The total score ranges from 0 to 58 points.
Techniques Subcutaneous adipose tissue biopsies were obtained by needle aspiration from the buttock fat by a method previously described and validated in detail by Beynen and Katan. 11 The fat biopsies were stored at -20~ until analysis. The composition of fatty acids in biopsies of adipose tissue was determined by gas-liquid chromatography (GLC) (Fig 1) after saponification of the lipid extract with methanolic sodium hydroxide (0.5 mol/L) and methylation with 20% boron triflourid (BF3) in methanol containing 0.025% hydroquinone. For GLC analysis we used an HP 5890A gas chromatograph (HewlettPackard Co, Sunnyvale, CA) equipped with an Omegawax 320 fused silica capillary column (30 m x 0.32 m m inner diameter; 0.25 ~m film thickness; Supelco Inc, Supelco Park, PA), operating from 160~ to 220~ Fatty acids were detected by flame ionization. For signal integration a HI} 3396A digital integrator was used. Fatty acids were tentatively identified by comparing their retention times with those of known standards (Nu-Chek-Prep Inc, Elysian, MN). Forty-four peaks corresponding to specific
18:1 18:2
/
\
/
14:1 18:3{o3 18:0
12:0
1 1 20:4 22:5{o3
\
"u,....Iul,.
,J~
Figure1. Gas-liquidchromatagramoffatty acid methyl esters in human adipose tissue.
22:6{o3
/
ISCHEMIC STROKE AND n-3 FATTY ACIDS
407
Table 1. Characteristics of control patients and patients
(C 18:3) were significantly lower in the stroke group when compared to controls (P < .05).
with cerebral infarction
Female (n = 10)
Male (n = 10)
Smokers Nonsmokers Smokers Nonsmokers Control subjects Patients with cerebral infarction
3
4
3
0
4
3
2
1
fatty acids were identified. The individual fatty acid methyl esters were quantified as the percentage of the total peak area. The analyses were performed in pairs and blindly. Differences between the percentages of fatty acids were determined by two-tailed Student's t-test. Probability values <.05 were considered significant.
Results
Basic characteristics of patients and control subjects are given in Table 1. The two groups were matched for age (mean • SE; 73.2 • 2.1 and 72.7 • 3.2 years, respectively), gender, and smoking habits. The content of the individual fatty acids expressed as percentage of the total peak area in the chromatograms is given in Table 2. Unidentified compounds did not exceed 2% of the total peak area. No significant differences in percentage proportions of the long-chain n-3 fatty acids, eicosapentaenoic acid (EPA) (C 20:5, n-3), docosapentaenoic acid (DPA) (C 22:5, n-3) and docosahexaenoic acid (DHA) (C 22:6, n-3), representative of fish consumption were found. A highly significant increase in palmitoleic acid (C 16:1) was found in patients with cerebral infarction when compared with control subjects (P < .005). The percentage proportions of stearic acid (18:0), linoleic acid (C 18:2) and linolenic acid
Discussion
The association between dietary fat, atherosclerosis, and stroke remains controversial. Epidemiologic studies of Greenland Eskimos 16 and Japanese 17 populations who consume substantial amounts of n-3 fatty acids have shown low mortality rates from coronary heart disease (CHD), although incidence rates of cerebrovascular disorders are not low. These cross-cultural comparisons, however, are of limited value because populations may be protected by racial factors, and these studies did not take into account numerous other risk factors that differ among populations. The observed association between a low-fat diet, intracerebral hemorrhage, and lacunar stroke in Japan may be explained by other major risk factors; the incidence rates of stroke, salt consumption, and blood pressure have declined markedly during the last decade. 10 Previous case-control studies of fish consumption and stroke have shown conflicting results. Although some studies showed significant inverse associations between fish consumption and stroke incidence, 8,18 others did not. 19,20Stratified analyses according to stroke type were used in only one previous study; no significant association between fish consumption and ischemic stroke was found. 21 Although previous studies have involved thousands of subjects, they have all been based on dietary recall, and discrepancies may be explained by inaccurate assessment of dietary fat. One case-control study on the fatty acid composition in 33 patients with cerebrovascular disease has been published, 22 but did not include measurements of n-3 fatty acids. Lower levels of the essential fatty acid linoleic acid (C 18:2) among male patients with stroke were reported. These results should be interpreted with caution because no distinction was made between hemorrhage and isch-
Table 2. Fatty acid composition in controls and cases of cerebral h,farction* Common name
Fatty acid
Controls (n = 10)
Infarcts (n = 10)
Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Eicosapentaenoic acid Docosapentaenoic acid Docosahexaenoic acid
C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2,n-6 C 18:3,n-3 C 20:5,n-3 C 22:5,n-3 C 22:6,n-3
2.8 - 0.2 18.8 --- 0.8 5.7 • 0.4 3.8 - 0.4 47.6 - 0.7 10.7 --- 0.5 1.00 • 0.06 0.10 • 0.01 0.30 - 0.02 0.34 • 0.03
2.7 - 0.2 19.8 --. 0.8 8.5 • 0.6 2.7 • 0.2 47.6 • 0.9 8.9 --- 0.4 0.80 --- 0.05 0.11 • 0.01 0.31 __.0.02 0.30 • 0.03
*• + SE.
P P P P
NS NS < .005 < .05 NS < .05 < .05 NS NS NS
408 emic stroke, and the mean age of patients was 67 years, in comparison to a mean age of 29 years of the control subjects. 22 To the best of our knowledge, ours is the first investigation on content of n-3 fatty acids in adipose tissue in patients with ischemic stroke. No differences were found in content of n-3 fatty acids of marine origin in stroke patients compared with matched controls. The observation of small decreases in concentrations of linoleic acid and linoleic acid should not be interpreted as a sign of chronic deficiency of essential fatty acids. Linoleic acid is the most variable fatty acid in human beings. For example, in New Zealanders, ~ linoleic acid contributes to only 3% of the total fatty acids, compared with 25.6% in Israelis. 24Variation in linoleic acid content of this order of magnitude among populations on no experimental diet does not support those observations in which small differences in linoleic acid concentrations in stroke patients have been interpreted as relative linoleic acid deficiency. The patients with cerebral infarction yielded high levels of palmitoleic acid. Palmitoleic acid usually constitutes about 5% to 7% of the total fatty acids in human adipose tissue. 9 Marginally higher amounts are found in populations subsisting on very-low-fat diets. 9 Higher levels also can be found in conditions of increased de novo lipid synthesis such as in utero 2s or among patients on longterm parenteral nutrition. 26 Alcohol consumption also correlates positively to adipose tissue levels of palmitoleic acid and negatively to linoleic acid. 27 Alcohol is used for de novo lipid synthesis in the liver (before use as energy fuel in peripheral tissues) with production of nonessential fatty acids. Therefore the lower adipose tissue linoleate levels may simply be a dilutional effect of newly synthesized nonessential fatty acids. At present we have no easy explanation for the high levels of palmitoleic acid among the stroke patients in this study; they had minor strokes and did not receive parenteral nutrition. Also patients with known or suspected abuse of alcohol were not included. Case-control studies are sensitive to selection bias. In considering reasons for the differences, it cannot be excluded that volunteers were more conscious of their health but control subject were chosen from a group of patients within the same hospital. Cases and controls were matched for all background variables known to bias the levels of adipose tissue fatty acids. 9 In conclusion, although significant differences in levels of essential fatty acids were found in our study, no conclusions can be made about the possibility of a causal relationship between the intake of essential fatty acids and stroke. The present study does not support the hypothesis of an association between dietary n-3 fatty acids (e.g., fish consumption) and ischemic stroke.
K.N. SEIDELIN ET AL.
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