557

Supplementation with Evening Primrose Oil in Atopic Dermatitis: Effect on Fatty Acids in Neutrophils and Epidermis Llselotte S c h ~ e r a , * a n d Knud K r a g b a l l e b aAarhus OUefabrik A/S, Research and Development Laboratories, DK-81OO Aarhus C and bMarselisborg Hospital, Department of Dermatology, DK-8OOO Aarhus C, Denmark

We investigated the effect of oral supplementation with evening primrose oil, contaln|ng 72% iinoleic acid (18.'~4}) and 10% y-linolenic acid (18:3n-6), on the epidermal and neutrophil phospholipid fatty acid composition in 15 patients with atopic dermatitis (AD). Three different dose levels, 4, 8 and 12 capsules per day containing 0.5 g oil, were given to three groups of patients. The only n4} fatty acid showing a significant (p < 0.05) dose-related increase was dihomo-y-linolenic acid (20:3n4}) in neutrophil phospholipids. The highest dose increased dihomwy-linolenic acid by 45% in neutrophil phospholipids, by 46% in lesionfree epidermal phosphatidylcholine, and by 15% in lesionfree epidermal phosphatidylethanolamine. In both lesional and lesion-free epidermis, supplementation resulted in a rise in the ratio between n4] and monounsaturated fatty acids, reaching significance (p < 0.05) in lesional epidermi~. This study shows that moderate and favorable fatty acid changes can be obtained in the epidermis of AD patients, when given 6 g per day of oil rich in n4} fatty acids. The abnormal lipid and fatty acid pattern of the atopic epidermis may be involved in the pathogenesis of the disease, and should therefore be the target for future therapeutic approaches with fatty acid supplements. Lipids 26, 557-560 (1991).

In two previous communications (8,9), we reported that our AD patients apparently did not have any abnormalities in fatty acid desaturation; neutrophil phospholipids and adipose tissue triglycerides showed a normal fatty acid pattern. However, the phospholipid content of the epidermis of these patients was abnormally higl~ Ftm thermor~ the level of n-6 polyunsaturated fatty acids was reduced and that of monounsaturated fatty acids (MUFA) increased in the epidermal phospholipids, resulting in a highly abnormal ratio between these two fractions. The disease severity was directly related to the content of MUFA in the epidermis, and inversely related to the content of n-6 fatty acids. We interpreted these findings as being the consequence of incomplete lipid transformation combined with increased phospholipase A2 activity in the epidermis of these AD patients. The objective of the present study was to measure the magnitude of fatty acid changes in neutrophil and epidermal phospholipids attained by supplementation with evening primrose off in doses commonly employed. MATERIALS AND METHODS

Subjects and design. Fifteen adult patients with AD, 8 premenopausal women and 7 men, attending the outpatient clinic of the Department of Dermatology at MarselisThe importance of using fatty acid supplements as a treat- borg Hospital (Aarhus, Denmark) agreed to participate ment of atopic dermatitis (AD) has been discussed for in the study. The group of patients was the same as that more than half a century (1). In the last ten years, oil from described previously (8,9}. Their age ranged from 19 to 51; the seeds of evening primrose (Oenothera biennis) has at- the mean age was 32.3 + 10.1 years. In 11 of the patients, tracted special attention due to its unusually high con- eczema had commenced before the second year of life In tent (10%) of y-linolenic acid (GLA). In a recently publish- one patient it started at 15, and in three during adulthood. ed meta-analysis of nine double blind trials with evening Five were smokers and four female patients used oral conprimrose oil, Morse et aL (2) found that the overall disease .traceptives. The affected body surface ranged from 3 to severity of AD patients, especially itching, was signifi- 70%. In 10 of the patients it ranged from 10 to 50%. None cantly reduced in a dose-dependent manner by oral admin- of the patients received topical or systemic treatment for istration of this oil The functioning of the A6 desaturase, 2 wk prior to the study. Subjects which were over- or an enzyme which converts linoleic acid (LA) into GLA, underweight {more than 20% from the ideal weight), or was thought to be defective in AD patients (3). If this were which had diabetes or hyperlipidemia were excluded from true, a direct supply of GLA would mean a bypass of this the study. The patients were randomly assigned to three purported metabolic blockade In previous fatty acid SUl~ dose levels of evening primrose off {Quest Vitamins, Lonplementation trials concerning patients with skin disease, don, U.K.), namely 4, 8 or 12 capsules, each containing only the plasma fatty acid composition was analyzed 0.5 g, per day. The fatty acid composition of the oil given (4-6). Supplementation trials with oils containing GLA as % by weight of total fatty acids was: 16:0 6.3%, 18:0 in humans have resulted in the elevation of plasma 1.6%o, 18:1 10.1%, 18:2n-6 71.5%, and 18:3n-6 9.9%. All paphospholipid levels of dihomo~y-linolenic acid (DGLA), tients were instructed to use only their usual emollient which is the precursor of the putative anti-inflammatory during the trial Blood samples were obtained from fasted substances 15-hydroxy-eicesatrienoic acid and prostaglan- patients at week 0 and 10. Neutrophils were isolated by din E 1 (7). the Hypaque~Ficoll technique, according to Boyum (10). Skin biopsies, weighing 50-250 mg, were taken with a keratome set at 0.2 ram, and frozen immediately in liquid *To w h o m correspondence should be addressed at King Gustav V:s Research Institute,P.O. Box 60004, S-10401 Stockholm, Sweden. nitroger~ as previously described (11). Approximately twothirds of the epidermis" was removed. Abbreviations: AA, arachidonicacid (20:4n-6);AD, atopicdermatitis; Analyticalprocedures. Lipid extractior~ separatio~ and DGLA, dihomo-y-linolenicacid (20:3n-6);F A M E , fatty acid methyl ester; GLA, rlinolenic acid (18:3n-6);LA, linoleicacid (18:2n-6); analysis were carried out as previously described (8,9). M U F A , monounsaturated fatty adds: PC, phosphatidylcholine;PE, Briefly, lipids were extracted from nsutrophils and epider~ phosphatidylethanolamine. mal specimens with chloroform/methanol (2:1, v/v). Total LIPIDS,Vol. 26, No. 7 0991)

558 COMMUNICATIONS TABLE 1 F a t t y Acid Composition of Epidermal Phosphatidylcholine from Lesion-Free and Lesional Areas Before and After Supplementation with Three Doses of Evening Primrose Oil for 10 Wk

Fatty acid 18:2n-6 20:3n-6 20:4n-6 Total n-6e Total MUFAf Total SFAg n-6/MUFA

Dosea 4 8 12 4

Lesion-freeepidermal PC Week 0b Week l0b A %c 28.8 29.8 31.9 1.40

8

1.27

12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12

1.10 4.12 5.12 3.67 35.1 37.0 37.5 28.7 25.8 26.0 35.1 36.0 35.5 1.23 1.44 1.49

• 2.8 + 1.9 _+ 4.2 + 0.43 - 0.42 • 0.26 --- 0.40 • 1.66 --- 0.82 • 2.2 --- 1.8 --- 4.5 -----1.6 • 0.8 ----4.0 -- 1.9 • 1.4 -- 1.0

27.3 31.2 31.0 1.53 1.41 1.60 5.90 5.84 5.53 35.6 39.3 39.2 28.2 23.7 23.8 35.0 35.5 35.7 1.29 1.68 1.66

__. 4.2 + 4.6 +_ 1.4 ___ 0.60 ___ 0.43 _ 0.49d --- 1.93 ___ 2.56 • 1.41d • 2.3 + 3.8 + 2.5 + 5.5 + 2.0 • 2.0d • 5.3 • 2.0 • 1.3

-3.4 4.3 -1.2 0.2 15.0 46.3 37.3 10.7 55.7 2.2 6.4 5.3 -0.2 -7.9 --7.3 -1.7 --1.4 0.6

Lesional epidermal PC Week 0 b Week 10b A %c 25.2 + 3.4 28.4 +- 3.4 28.0 • 4.5 1~16 + 0.37 1.24 + 0.31 0.94 -+ 0.22 3.37 - 0.31 4.19 - 1.53 2.98 +- 0.32 30.6 +- 3.5 34.9 - 2.7 32.8 - 4.5 33.0 -+ 3.3 27.9 - 2.8 30.4 -----4.4 35.4 • 1.3 35.8 • 1.2 35.6 -----0.8 0.94 1.27 1.12

27.0 + 3.4 28.6 + 5.8 27.6 + 3.1 1.58 + 0.46 1.37 + 0.64 1.51 • 0.26 d 5.61 + 1.76 4.40 -t- 2.86 5.04 --- 0.80 d 35.4 - 1.9d 35.4 + 2.6 35.2 • 4.0 27.0 + 2.6d 27.7 • 3.3 27.1 ----4.4d 36.3 • 2.3 35.5 • 2.8 36.3 • 1.0 1.33 d 1.29 1.34 d

8.8 1.8 -0.7 40.3 6.6 62.9 68.2 5.7 69.9 16.6 3.5 7.5 --18.0 -3.7 --10.7 2.6 -0.2 1.9

aNumber of capsules (0.5 g) per day; each dose was taken by five subjects. bValues are mean +__S.D. and given as % weight of total fatty acids. CAverage of individual changes in % of initial value. dSignificantly (p < 0.05) different from week 0 by Wilcoxon's signed rank test. e Include 18:2, 18:3, 20:3, 20:4, 22:4, and 22:5. fInclude 14:1, 15:1, 16:1, 17:1, 18:1, 20:1n-11, and 20:1n-9. gInclude 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, and 22:0.

phospholipids were isolated from the neutrophil lipid extract by solid-phase extraction on prepacked silica cartridges. Epidermal phospholipids were isolated from the epidermal lipidextract on an aminopropyl phase packed in a glass column. Epidermal phospholipids were further separated into classes by normal phase high-pressure liquid chromatography on a silica column in a gradient of acetonitrile/water,as previously described (9).Phospholipids were detected at 203 nm. Individual phospholipid classes were identified using external standards. Individual phospholipid fractions were collected manually and taken to dryness in a rotary evaporator at 50~ Fatty acid methyl esters {FAME) were prepared by transesterification with sodium methoxid~ F A M E were analyzed on a 50 m • 0.25 m m capillary fused silicacolumn coated with a 0.2 ~rn film of C P SIL88 (Chrompack, Delft, The Netherlands). T h e c a r r i e r g a s w a s h e l i u m a n d t h e flow r a t e 2 m L / m i ~ I n j e c t i o n w a s o n - c o l u m n a t 90~ T h e oven w a s p r o g r a m m e d t o r i s e t o 2 1 0 ~ w i t h i n 6 rain a n d t h e n r u n i s o t h e r m a l l y for 18 min. A f l a m e - i o n i z a t i o n d e t e c t o r w a s u s e d , a n d p e a k a r e a s were i n t e g r a t e d a u t o m a t i c a l l y . F A M E were i d e n t i f i e d b y c o m p a r i s o n w i t h a s t a n d a r d m i x t u r e of k n o w n c o m p o s i t i o n (68A a n d 68B, N u C h e k Prep, E l y s i a n , MN). T h e r e l a t i v e c o n t e n t s of m a j o r p e a k s of t h e s e s t a n d a r d s a g r e e d w i t h i n 0.2% w i t h t h e v a l u e s stated by the manufacturer. Statistical analysis. All d a t a a r e p r e s e n t e d a s m e a n _+ S.D. D o s e r e s p o n s e r e l a t i o n s h i p s were a n a l y z e d b y LIPIDS,Vol. 26, No. 7 0991)

A N O V A . The fatty acid changes within each dosage group were analyzed by Wilcoxon's signed rank test. RESULTS IAnoleic a c i d (LA) c o m p r i s e d 72% (by w e i g h t of t o t a l f a t t y acids) of t h e oil s u p p l e m e n t . However, t h e level of L A d i d n o t s h o w a n y s y s t e m a t i c c h a n g e in e i t h e r n e u t r o p h i l o r e p i d e r m a l p h o s p h o l i p i d s a f t e r 10 w k of s u p p l e m e n t a t i o n (Tables 1 a n d 2). T h e level of G L A , c o m p r i s i n g 10% of t h e oil, r e m a i n e d u n c h a n g e d a t 0.1-0.2% (by w e i g h t of t o t a l f a t t y acids) in e p i d e r m a l a n d n e u t r o p h i l p h o s p h o l i p i d s ( d a t a n o t shown). T h e c h a n g e s in D G L A a n d A A in t h e e p i d e r m i s were p o s i t i v e in all t h r e e d o s a g e g r o u p s , b u t s i g n i f i c a n t (p < 0.05 b y W i l c o x o n ' s s i g n e d r a n k test) o n l y in s u b j e c t s o n t h e h i g h e s t d o s e (Table 1). T h e t o t a l cont e n t of n-6 f a t t y a c i d s t e n d e d t o i n c r e a s e u p o n s u p p l e m e n t a t i o n in all t h r e e d o s a g e g r o u p s in b o t h l e s i o n a l a n d lesion-free e p i d e r m a l p h o s p h a t i d y l c h o l i n e (PC), while t h a t of t o t a l M U F A t e n d e d t o d e c r e a s ~ T h e r a t i o of n - 6 / M U F A s i g n i f i c a n t l y i n c r e a s e d a t w e e k 10 in l e s i o n a l e p i d e r m i s in t h e l o w e s t a n d h i g h e s t d o s a g e g r o u p (Table 1). I n p h o s p h a t i d y l e t h a n o l a m i n e (PE) f r o m lesion-free epidermis, t h e a v e r a g e c h a n g e s r e l a t i v e to w e e k 0 on t h e h i g h e s t d o s e (12 capsules/day) were 8% in L A , 15% in D G L A , 17% in A A , 2% in t o t a l n-6 f a t t y acids, 4% in M U F A , a n d - 2 % in s a t u r a t e d f a t t y acids. N o n e of t h e s e c h a n g e s r e a c h e d

559

COMMUNICATIONS TABLE 2 Fatty A d d Composition of Neutrophil Total Phospholipids Before and After Supplementation With Evening Primrose Oil for 10 Wk

Fatty acid

Dosea

Week 0 b

18:2n-6

4 8 12

12.3 • 1.5 12.3 • I.I 11.9 • 1.3

12.2 + 1.7 12.2 • 1.2 11.7 • 1.6

--0.3 --0.2 --1.3

20:3n-6 20:4n-6

4 8 12 4

2.71 1.91 2.06 15.3

2.83 2.65 2.91 15.9

6.1 d 42.9 d 45.1 d 5.3

8

15.7 • 2.1

16.1 • 1.5

Total n-6f

12 4

16.9 • 2.8 33.6 • 1.7 32.5 • 1.7 34.0 +_ 2.6 32.7 • 2.3 33.5 • 0.8 33.6 • 2.7 29.4 • 1.2 29.4 • 1.8 28.0 • 2.4

16.1 34.8 34.1 34.2 32.4 32.9 32.8 28.9 28.7 28.8

8

Total MUFAg

12 4 8

Total SFAh

12 4 8 12

• • • •

0.81 0.48 0.65 2.0

Week l0 b

• • • •

0.66 0.52e 0.55 e 1.4

• 1.0 • 1.8 • 1.7 • 2.2 + 2.2 • 2.8 • 1.9 • 0.6 • 1.2 _+ 0.7

A %c

4.8 --2.9

3.5 5.3 0.8 --I.I --1.7

--2.3 -1.6 -2.1 3.3

a Number of capsules(0.5g) per day; each dose was taken by five subjects. bVsluesaremean • S.D.and givenas % weightoftotalfattyacids. CAverage of individual changes in % of initial value. dSignificant (p < 0.05) dose-related rise by ANOVA. e Significantly {p < 0.05) different from week 0 by Wflcoxon's signed

rank test. fn-6 fattyacidsinclude18:2,18:3,20:3,20:4,22:4,and 22:5. gMonounsaturated fattyacidsinclude14:1,15:1,16:1,17:1,18:1, 20:1n-11,and 20:1n-9. hSaturatedfattyacidsinclude14:0,15:0,16:0,17:0,18:0,20:0,and 22:0.

statisticalsignificanc~The change in neutrophilphosphc~ lipid fatty acids upon supplementation is shown in Table 2. Here a significantdose-relatedrisein D G L A was found (p < 0.05 by ANOVA). DISCUSSION I n p r e v i o u s s t u d i e s o n t h e effect of f a t t y a c i d s u p p l e m e n tation on patients with AD, emphasis was placed on the clinical effect a n d on t h e p l a s m a p h o s p h o l i p i d f a t t y a c i d p a t t e r n (5,12,13). T h e effect of s u p p l e m e n t a t i o n w i t h n-6 f a t t y a c i d s on e p i d e r m a l p h o s p h o l i p i d s a n d n e u t r o p h i l s has, t o o u r knowledge~ n o t b e e n i n v e s t i g a t e d b e f o r e in h u m a n s . O u r d a t a s h o w t h a t s u p p l e m e n t a t i o n w i t h even i n g p r i m r o s e oil r e s u l t e d in t h e e l e v a t i o n of D G L A in neutrophils and epidermal phopholipids, as has been s h o w n in p l a s m a (4). D e s p i t e t h e h i g h c o n c e n t r a t i o n of L A

and G L A in evening primrose oil,the levelsof these fatty acids remained virtuallyunchanged. This shows that L A and G L A are rapidly desaturated and elongated when taken orally. The effect of supplementation with evening primrose oilon the tissue phospholipid fatty acid composition has been investigated in animals. In mice macrophages, the D G L A concentration was increased 5-foldby feeding mice 25% (% by weight of totalfatty acids)of G L A in the form of borage oil for three weeks {14).In another study with guinea pigs (7),using the same dose for eight weeks, a 10-,

4- and 1.3-foldelevation of GLA, D G L A and arachidonic acid (AA), respectively,and a 2-foldreduction in L A were seen in the epidermal phospholipid fraction,compared to animals fed a safflower oil-enricheddiet. A n analysis of the lipoxygenase products formed in the epidermis of these animals revealed that metabolites of D G L A , such as 15-hydroxy-eicosatrienoic acid and prostaglandin E1 were synthesized. These substances have been proposed to have anti-inflammatory properties (7).Dietary G L A may therefore be a source for endogenous generation of anti-inflammatory metabolites in the epidermis which may suppress localinflammatory reactions {15).However, the highest dose of evening primrose oil normally given to patients is approximately 0.6% (% by weight of total dietary fat),corresponding to 12 X 0.5 g capsules per day. Our data show that this dose only leads to a 50% increase in D G L A in the epidermis after 10 w k of supplementation,reaching levelsfar lessthan in experimental animals. It is questionable whether the level of D G L A in human epidermis, attained by oral supplementation with evening primrose oil,would be large enough to support the synthesis of local anti-inflammatory metabolites,even ifsupplementation were continued for longer periods.The bent~ flcialeffect of n-6 fatty acid supplementation in AD, as reported by others {21,probably relies on other mechanisms. In a previous publication {9) we reported that the epidermis of A D patients had a lower n-6/MUFA ratio than the epidermis of healthy subjects, and that the disease severitycorrelated directlywith the total M U F A content of the epidermal phospholipids. In essentialfatty acid deficiency,which also results in dry scaling skin, an imbalance is found in the ratiobetween L A and M U F A {16).Although A D patients are not deficientin essential fatty acids, the characteristicdry skin could be caused by a partial substitution of polyunsaturated fatty acids by M U F A , leading to a defective structure of the epide~ real water barrier. The present results show that supplementation with n-6 fatty acids results in a rise in the n-6/MUFA ratio,particularly in lesionalepidermis. Sul> jects on the highest dose actually increased their n-6/ M U F A ratio to a value of 1.68 in lesion-freeepidermis. Previously we reported a value of 1.64 for healthy epider~ mis (91.Because this study was not controlled, we cannot relate the clinical outcome of the patients to the observed changes in the fatty acid composition of epidermal phospholipids. If and how these changes are causally related to the disease remains to be established.Because linoleicacid is an essentialcomponent of acylceramides, which constitutethe epidermal water barrier(17),itwould be of considerable interestto study the effectof n-6 fatty acid supplementation on the metabolism of acylceramides in the atopic epidermis. In conclusion, this study shows that supplementation with up to 600 rag/day of evening primrose oil increases the levelof n-6 fatty acids,especiallyD G L A , by 15-60% in neutrophil and epidermal phospholipids. Furthermore, supplementation results in a beneficialshift in the ratio between n-6 and M U F A . However, no clear evidence has yet been presented in favor of G L A as being the active component in evening primrose oil.N e w studies should clarifyifmore conventional vegetable oilscontaining high levels of linoleicacid, such as safflower or sunflower oil, may mediate the desired lipidchanges in the epidermis, and if the dose should be further increased. LIPIDS,Vol. 25, Na 7 (1991)

560 COMMUNICATIONS

ACKNOWLEDGMENTS The technical assistance of Berit Kristensen is gratefully acknowledged. This study was supported by the Danish Academy of Technical Sciences.

REFERENCES 1. Burton, J.L. (1989) Lancet I, 27-31. 2. Morse, P.E, Horrobin, D.E, Manku, M.S., Stewart, J.CM., Allen, R., Littlewood, S., Wright, S., Burton, J., Gould, D.J., Holt, P.J., Jansen, C.T., Mattila, L., Meigel, W., Dettke, T., Wexler, D., Guenther, L., Bordon~ A., and Patrizi, A. (1989) Br. J. DermatoL 121, 75-90. 3. Manku, M.S., Horrobin, D.F., Morse, N., Kyte, V., and Jenkins, K. (1982) Prostaglandins, Leukotrienes MecL 9, 615-628. 4. Manku, M.S., Horrobin, D.F., Morse, N.L., Wright, S., and Btm ton, J.L. (1984) Br. J. DermatoL 110, 643-648. 5. Schalin-Karrila, M., Mattfla, L., Jansen, C.T.,and Uotila, P. (1987) Br. J. DermatoL 117, 11-19.

LIPIDS,Vol. 26, Na 7 (1991)

6. Nissen, H.P., Wehrmann, W., Kroll, U., and Krsysel, H.W. (1988) Fat Sci. TechnoL 7, 268-271. 7. Miller, C.C, and Ziboh, V.A. {1988)Biochem. Biophys. Res. Commum 154, 967-974. 8. Schtifer, L., Kragballe, K., Jepsen" L.V.J., and Iversen" I. (1991) J. Invest. DermatoL 96, 16-19. 9. Sch~fer, L., and Kragballe, K. (1991) J. Invest. DermatoL 96, 10-15. 10. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 215, 77-89. 11. Fogh, K., Kiil, J., Herlin" T., Ternowitz, T., and Kragballe, K. (1987) Arc& DermatoL Res. 279, 504-511. 12. Wright, S., and Burton, J.L. (1982) Lancet ii, 1120-1122. 13. Bamford' J.T.M., Gibson, R.W., and Reuler, C.M. {1985) J. Am~ Acad. DermatoL 13, 959-965. 14. Chapkin, R.K, Somers, S.D., and Erickson, K.L. (1988) Lipids 23, 766-770. 15. Ziboh, V.A., and Miller, C~C.(1990)Ann~ Pea Nutr. 10, 433-450. 16. Ziboh, V.A., and Chapkin" R.S. {1990)Prog. LipidRes. 27, 81-105. 17. Wertz, RW., Swartzendruber, D.C., Abraham, W., Madison, K.C., and Downing, D.T. (1987) Arch. DermatoL 123, 1381-1384. [Received October 29, 1990; Revision accepted April 30, 1991]

Supplementation with evening primrose oil in atopic dermatitis: effect on fatty acids in neutrophils and epidermis.

We investigated the effect of oral supplementation with evening primrose oil, containing 72% linoleic acid (18:2n-6) and 10% gamma-linolenic acid (18:...
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