523

Fatty Acid Content of Marine Oil Capsules Kew M. C h e e 1, Jun Xian Gong, Deirdre M. G o o d Rees, Mohsen M e y d a n i , Lynne A u s m a n , Julia Johnson, Edward N. Siguel a n d Ernst J. Schaefer* Lipid Metabolism Laboratory, USDAHuman Nutrition Research Center on Aging at TuftsUniversity,711 Washington Street, Boston, MA 02111 T h e u s e o f d i e t a r y ~3 fatty acid c a p s u l e s h a s b e e n a s s o c i a t e d w i t h a d e c r e a s e in p l a s m a triglyceride levels. In addition, p o p u l a t i o n s c o n s u m i n g d i e t s rich in f i s h a p p e a r to h a v e a d e c r e a s e d i n c i d e n c e o f c a r d i o v a s c u l a r d i s e a s e . E i c o s a p e n t a e n o i c acid (EPA, 20:5oJ3) and d o c o s a h e x a e n o i c acid (DHA, 22.'6w3) are m a j o r fatty a c i d s in f i s h oils. It is b e l i e v e d that f i s h oils e x e r t t h e i r b i o l o g i c e f f e c t t h r o u g h t h e s e fatty acids. Many i n d i v i d u a l s are currently t a k i n g f i s h oil c a p s u l e s to l o w e r lipids, i n c r e a s e b l e e d i n g t i m e , and p o s s i b l y d e c r e a s e c a r d i o v a s c u l a r risk. T h e s e c a p s u l e s a l s o h a v e b e e n c l a s s i f i e d a s f o o d a d d i t i v e s w i t h l e s s string e n t c o n t r o l s o n c o n t e n t . We a s s e s s e d t h e fatty acid, c h o l e s t e r o l , and v i t a m i n A and E c o n t e n t o f e i g h t c o m m e r c i a l l y available c a p s u l e s a l o n g w i t h cod fiver off. The c o n t e n t o f E P A w a s f o u n d to range from 8.726.4% ( w t %) w i t h a m e a n o f 17.3% (82.4% o f l a b e l e d c o n t e n t ) , a n d t h a t o f D H A f r o m 8.9-17.4 % w i t h a m e a n o f 11.5% (90.0% o f l a b e l e d c o n t e n t ) a s a s s e s s e d by capillary c o l u m n gas-liquid c h r o m a t o g r a p h y . T h e m e a n c o n t e n t o f t h e p o l y u n s a t u r a t e d o~3 fatty a c i d s w a s 31.9%, a n d that o f t h e ~ 6 fatty a c i d s w a s 1.4%. T h e c o n t e n t o f s a t u r a t e d fatty a c i d s w a s 32.0%, and that o f m o n o u n s a t u r a t e d fatty a c i d s w a s 25.1%. C h o l e s t e r o l c o n t e n t w a s low, w i t h a range o f 0.7-8.3 m g / g , t h e at o c o p h e r o l r a n g e w a s 0.62-2.24 m g / g , and t h e r a n g e o f retinyl e s t e r s w a s 0.4-298.4 p g / g . Cod liver oil h a d s u b s t a n t i a l l y m o r e retinyl e s t e r s (2450.1 # g / g ) t h a n did f i s h oil c a p s u l e s . Our data s e r v e as an i n d e p e n d e n t g u i d e to f i s h oil c a p s u l e fatty acid c o n t e n t u p o n s i n g l e lot analysis, and indicate that these capsules contain as m u c h s a t u r a t e d fat a s t h e y c o n t a i n oJ3 fatty acids. Lipids 25, 523-528 (1990}. The major m3 fatty acids in p l a s m a are a-linolenic acid (18:3oJ3) and its derivatives eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). a-Linolenic acid can be obtained from plant sources ( r a p e s e e d oil a n d soybean oil), while EPA and DHA are a b u n d a n t in fish and fish oil products. Interest in fish oil p r o d u c t s was stimulated by initial observations t h a t Eskimos have a low incidence of atherosclerosis despite having a diet high in total fat. However, m u c h of their excess fat intake is due to increased intake of m a r i n e oils. Subsequent studies showed t h a t increased fmh oil in the diet was associated with substantial decreases in p l a s m a triglycerides as well as an increase in bleeding time (1-4). It is known t h a t o~3 fatty acids serve as p r e c u r s o r s for t h r o m b o x a n e B3 as well as t h r o m b o x a n e A3, prostaglandin E3, I3 and leuko-

*To whom correspondence should be addressed. 1Current Address: Department of Animal Science, Korea University, Seoul, Korea 13200. Abbreviations: EPA, eicosapentaenoic acid; DHA docosahexaenoic acid; FID, flame ionization detector; GC, gas chromatograph; FAME, fatty acid methyl esters.

triene B5 (5-7). These substances all have i m p o r t a n t effects with regard to i m m u n e and platelet function. Functionally i m p o r t a n t effects of 0)3 f a t t y acids in h u m a n s t h a t have been r e p o r t e d include an increased bleeding time, a reduction in platelet aggregation stimulated by adenosine d i p h o s p h a t e (ADP) or collagen, and a reduction in blood pressure (8-11). Moreover these latter effects of fish oils have been related to decreased blood pressure responses to angiotensin II and norepinephrine. In addition, fish oil use has been associated with a reduction in p o l y m o r p h o n u c l e a r leukocyte c h e m o t a x i s and a reduced i n f l a m m a t o r y response (12). For these reasons, a large n u m b e r of patients in the United States are currently using fish oil capsules for a variety of disorders including hyperlipidemia, hypertension, arthritis, and a u t o i m m u n e disorders. In some cases controlled clinical studies have d o c u m e n t e d some efficacy offish oil capsules in the t r e a t m e n t of these disorders. The precise fatty acid composition of m a n y of the commercially available p r o d u c t s used to s u p p l e m e n t patients with r fatty acids has not been widely published (13,14). The p u r p o s e of our investigation was to assess the f a t t y acid composition of commercially available fish oil capsules and of cod liver oil using sensitive a n d reproducible capillary-column gas-liquid c h r o m a t o g r a phic techniques. In addition, we assessed the cholesterol and vitamin A and E content of these capsules, as well as of cod liver oil.

METHODS AND ANALYSIS Fish oil capsules were obtained from p h a r m a c i e s and health food stores in the Boston area. All capsules were in a gelatin form. All analyses were carried out in triplicate. Six individual capsules of each b r a n d f r o m the s a m e lot were analyzed for fatty acid content; 9 capsules of each b r a n d , also from the s a m e lot, were used for vitamin and cholesterol analysis. The following capsules were analyzed: Mega EPA-1000, General Nutrition Corporation, Pittsburgh, PA; Promega, P a r k e - D a ~ s Consumer Health P r o d u c t s Group, Warner L a m b e r t Company, Morris Plains, N J; Protochol, E.R. Squibb and Sons, Inc., Princeton, N J; Discovery, Carter Products, Division of CarterWallace Inc., New York, NY; MaxEPA GNC, General Nutrition Corporation, Pittsburgh, PA; Super MaxEPA, Twin Laboratories Inc., Ronkonkoma, NY; MaxEpa, R.P. Scherer Inc., North America, Clearwater, FL; SuperEpa, Bronson Pharmaceuticals, LaCanada, CA; Norwegian Cod Liver Oil, General Nutrition Corporation, Pittsburgh, PA. The retail cost to us in 1988 was $3.39 for 237 mL of cod liver oil, and ranged from $0.09 p e r capsule for S u p e r E p a to $0.28 per capsule for Promega. We did not analyze for the chemical forms (triglycerides, ethyl/methyl esters) in the p r o d u c t s we studied, and this information was not available from p r o d u c t labeling at time of analysis. Fatty acid analysis. A modification of LePage's m e t h o d was used for lipid extraction and transesterification of LIPIDS,Vol. 25, No. 9 (1990)

524 K. CHEE ET AL. fatty acids (15). Briefly, fish oil was removed from capsules, weighed precisely (10-15 mg) and dissolved in 5 mL of isooctane. One h u n d r e d #L samples along with 40#g of internal s t a n d a r d (margaric acid, 17:0) were mixed in 13 • 100 m m glass tubes. Two mL of a m e t h a n o l / benzene (4:1 v/v) solution was added, as was 200 #L of acetyl chloride with slow stirring. The tubes were flushed with nitrogen gas, sealed with Teflon tape, and tightly closed with Teflon-lined caps. The content was then methanolyzed at 100~ for 1 hr in a Reacti-Therm heating/stirring block. Tubes were then cooled in water, 1 mL of isooctane was added, followed by 5 mL of 6% potassium carbonate to neutralize the mixture. Tubes were centrifuged at 1500 g for 15 rain, and the clear u p p e r phase containing fatty acid methyl esters (FAME) was transferred to a 1-mL autosampler vial. After overlayering with anhydrous sodium sulfate (1 m m layer thick), vials were filled with nitrogen, sealed with crimp caps, and readied for injection. An a u t o m a t e d system was used for analysis, which consisted of a Hewlett Packard (HP, Hewlett-Packard Co., Avondale, PA) 7673A automatic sampler, an HP 5890 gasliquid c h r o m a t o g r a p h (GC), an HP 3393A integrator, and a Nelson c h r o m a t o g r a p h y d a t a system (Nelson Analytical, Inc., Cupertino, CA) which collects, reintegrates and stores the data. Samples in the autosampler t r a y were cooled to keep sample t e m p e r a t u r e at approximately 10~ and to minimize FAME oxidation. The column used for analysis was a 0.32-mm (interior diameter), 30-m (length) Supelcowax 10 (Supelco, Inc., Bellefonte, PA) capillary column. A flame ionization detector (FID) was used. The GC was run using H2 as the carrier gas with a head pressure of 9 psi and a flow rate of 4 mL/min. Other conditions were as follows: flame H 2 - 20- psi at 25 m L / min and air - 40 psi at 385 mL/min; make up N2 - 27 m L / min; t e m p e r a t u r e program, first r a m p 150-1900C with increases of 4~ second r a m p 190-2100C for 10 min; third r a m p 210-250 ~ with increases of 10~ and then the t e m p e r a t u r e was held at 250~ for 6 min. Both the injector and detector temperatures were maintained at 2700C. The signal range and attenuation were both 0. Samples (0.5/zL) were injected into the GC in the splitless mode. Standard FAME obtained from Nuchek Prep (Elysian, MN), Sigma (St. Louis, MO), and Supelco were used for collaboration and peak identification. FAME concentrations were calculated b y t h e Nelson d a t a system based on comparisons with the concentration of the internal

s t a n d a r d (17:0, margaric acid), and the percentage report was based on fatty acid a m o u n t / t o t a l oil amount. Sterol analysis. Sterols were extracted from fish oils according to Method B for lipid extracts (with minor modifications) as described by Kovacs et al. (16). About 50 mg of lipid extract was saponified in a tightly capped (Teflon-lined) 16 )< 125 m m screw cap test tube containing 0.5 mL 50% potassium hydroxide (KOH) and 2 mL 95% ethanol. The tube contents were boiled on a hot plate for 1 hr while being continuously stirred with a magnetic bar. After cooling, 1.5 mL of distilled water was added and the unsaponifiable material was extracted 4-times with hexane (2.5 mL each). The combined extracts were dried u n d e r nitrogen, suspended in 500 #L hexane, and analyzed for free sterols by gas chromatography. A Varian 6500 Gas Liquid C h r o m a t o g r a p h (Varian Associates Inc., Walnut Creek, CA) was used. The glass column was 10foot by 4 m m with a 2-mm interior diameter and was packed with 1% OV-210/2% SE-30 on acid-washed GasChrom P. The column t e m p e r a t u r e was 240~ the injector t e m p e r a t u r e was 275~ and the detector (FID) t e m p e r a t u r e was held at 240~ The carrier gas was helium at a flow rate of 30 mL/min. The Nelson (Nelson Analytical, Inc., Cupertino, CA) software package was used to integrate peaks. V i t a m i n analysis. Retinyl esters were measured by normal phase high pressure liquid c h r o m a t o g r a p h y as previously described (17). Vitamin E (a- and ~/-tocopherols) were measured after alkaline hydrolysis by reverse phase high pressure liquid c h r o m a t o g r a p h y as previously described (18).

RESULTS Table 1 shows the b r a n d name and manufacturing c o m p a n y for each p r o d u c t tested, as well as their labeled EPA, DHA, cholesterol, vitamin A, and vitamin E content. According to label information, EPA content ranged from 18% to 30% (wt %), while DHA content ranged from 12% to 20%. According to the label, cholesterol content of fish oil capsules ranged from 0-12 mg per 1 g capsule, vitamin A content from less t h a n 66 to 10,000 IU/g retinol equivalents, and vitamin E from 0-0.6 mg/g. In Table 2 we have listed the concentrations of the major saturated fatty acids found in the various capsules. Many of the capsules contained substantial a m o u n t s of s a t u r a t e d fat. Myristic acid (14:0) content ranged from

TABLE 1 F i s h Oil S u p p l e m e n t L a b e l C o n t e n t a

Brand name

Company

EPA

DHA

MegaEpa Promega Protochol Discovery MaxEpa SuperMaxEpa MaxEpa SuperEpa Cod liver oil

GNC Parke-Davis Squibb Carter GNC TwinLab Scherer Bronson GNC

180 280 180 180 180 187.5 186 300 n.p.

120 120 120 120 120 125 158 200 n.p.

Walues provided in mg/g, unless otherwise indicated. bData not provided. LIPIDS,Vol. 25, No. 9 (1990)

Cholesterol 12