Urinary excretion of PGI123-M and recent N-6-3 fatty acid intake

PROSTAGLANDINS

URINARY EXCRETION OF PGI 2 / ~-M E AND RECENT N-6/3 FATTY ACID T. H a m a z a k i , S. F i s c h e r , M. U r a k a z e , S. Yano, and T. K u w a m o r i +

S. S a w a z a k i ,

The First Dept. of Internal Medicine, Toy-m- Medical and Pharmaceutical University, 2630 Sugitani, Toyam- City, Toy-m- 930-01, Japan Medizininsche Klinik Innenstadt der Universitat MUnchen, Ziemssentrasse 1, 8000 Mflnchen 2, F.R.G. + Food-Nutrition Dept., Toy-m- Women's College, GankaiJi, Toyama City, Toyam- 930, Japan

Abstract Epidemiological studies were performed in a Japanese fishing village when catches of fish were highest and in a Japanese farming village with usual fish consumption. Intake of eicosapentaenoic, docosahexaenoic and also arachidonic acid were significantly higher in the fishing village during the 3 days of the study than in the farming village. The correlation between eicosapentaenoic acid intake on the day when urine was collected and excretion of Al7-2,3-dinor-6-keto-prostaglandin Fie, the main urinary metabolite of prostaglandin 13, was highly significant, whereas there was no correlation between arachidonic or linoleic acid intake and excretion of 2,3-dinor-6-keto-prostaglandin Fle, the main urinary metabolite of prostaglandin I2. We suggest that the arachidonic acid pool for prostaglandin 12 production is not quickly influenced by dietary linoleic or arachidonic acid b e c a u s e of a large pool size of a r a c h i d o n i c acid and a slow conversion of linoleic acid to arachidonic acid, while prostaglandin 13 formation is directly related to the intake of eicosapentaenoic acid.

APRIL 1989 VOL. 37 NO. 4

417

PROSTAGLANDINS Introduction E p i d e m i o l o g i c a l studies in G r e e n l a n d Eskimos and in Japanese fishing villages have shown a low incidence of coronary heart d i s e a s e (1,2), which might be partly due to a reduced platelet function (3), and a favorable shift in the p r e s t a c y c l i n (PGI) / t h r o m b o x a n e balance (4). High levels of the n-3 p o l y u n s a t u r a t e d fatty acids, e i c o s a p e n t a e n o i c acid (EPA) and d o c o s a h e x a e n o i c acid (DHA), w h i c h are d e r i v e d from the Eskimos" food of maritime origin, are found in their plasma p h o s p h o l i p i d s and blood cells (3,5). It has also been found that pulse wave velocity of the aorta in inhabitants of fishing villages is significantly slower (indicating less sclerosis) than that of farming villages in Japan (2). E p i d e m i o l o g i c a l studies in a J a p a n e s e fishing and a farming v i l l a g e indicated that PGI 3 formation was dependent on the recent intake of EPA, w h i c h happened to be higher in the farming village than in the fishing village due to record low catches of fish in the latter at that season (6). In the following study the intake of n-3 and n-6 p o l y u n s a t u r a t e d fatty acids in a fishing and a farming village in Japan were c o m p a r e d w h e n catches of fish were highest. C o r r e l a t i o n s b e t w e e n intake of EPA, DHA, linoleic acid (LA) and a r a c h i d o n i c acid (AA), and formation of PGI2/3 were calculated.

Material

and Methods

E p i d e m i o l o g y of a f i s h i n g v e r s u s a f a r m i n g village. E p i d e m i o l o g i c a l i n v e s t i g a t i o n s were p e r f o r m e d in the fishing v i l l a g e M i y a z a k i situated at the seaside and the farming v i l l a g e Y o k o g o s h i situated 20 km from the shore n e i g h b o r i n g on a m o u n t a i n o u s area in D e c e m b e r 1985 . Both villages were located in the e a s t e r n part of T o y a m a Prefecture. We asked each m e m b e r w h o was 30-60 years old of a fishers" c o o p e r a t i v e in the fishing v i l l a g e and each member (30-60 years old) of the farming village, w h i c h was a very small village, to p a r t i c i p a t e in the present study. Eight males (45 + 7 yr, mean + SD) and I0 females (41 ~ 6 yr) in the fishing village, and i0 maYes (47 + 6 yr) and 13 females (44 + 8 yr) in the farming v i l l a g e v o T u n t e e r e d and were investigated? A f t e r having been i n s t r u c t e d how to m e a s u r e food with food models, the villagers were asked to record what and how much they ate d u r i n g 3 consecutive days prior to a day of blood sampling for fatty acid analysis of platelet phospholipids. The record of i n g e s t e d food, especially fish, was c h e c k e d at T. Kuwamori's i n t e r v i e w with villagers. The c o n s u m p t i o n of EPA, DHA, LA and AA was c a l c u l a t e d by a Sharp M Z - 8 0 B m i c r o c o m p u t o r (Tokyo) with a n u t r i t i o n - c a l c u l a t i n g p r o g r a m (Nutri Com, JAIS, Tokyo). The p r o g r a m had a memory of fatty acid a n a l y s i s of over 600 food m a t e r i a l s including about 60 fishes with seasonal fluctuations of their fatty acid content and shellfishes, which could cover almost all food material data collected during food analyses. In case that we could not find the food m a t e r i a l which had been

418

APRIL 1989 VOL. 37 NO. 4

PROSTAGLANDINS r e p o r t e d by a resident in the memory list, we substituted the most similar material in the list for the unlisted material. In a d i f f e r e n t study we c a l c u l a t e d the a c c u r a c y of this computere s t i m a t e d fatty acid intake based on food records provided by volunteers. The d i f f e r e n c e between the estimated intake and direct biochemical a n a l y s i s of food was within 20% of the biochemical analysis with regard to LA, EPA and DHA, and within 30% with regard to AA. Urine was also c o l l e c t e d for the 24 h prior to the blood s a m p l i n g and soon frozen at -70°C for mass s p e c t r o m e t r i c analyses of 2 , 3 - d i n o r - 6 - k e t o - p r o s t a g l a n d i n Fie (the main urinary m e t a b o l i t e of PGI2, PGI2-M) and A l 7 - 2 , 3 - d i n o r - 6 - k e t o p r o s t a g l a n d i n F I ~ (the main urinary m e t a b o l i t e of PGI3, PGI3-M) , w h i c h was performed by S. Fischer in Munich, Germany. M e a s u r e m e n t and statistics. E D T A - a n t i c o a g u l a t e d blood (1.5 mg/ml) was taken from each p a r t i c i p a n t early in the m o r n i n g w h i l e fasting, and the fatty acid c o m p o s i t i o n of p h o s p h o l i p i d s in p l a t e l e t s was a n a l y z e d as previously r e p o r t e d (7). Briefly, w a s h e d platelets were sonicated. Total lipids were e x t r a c t e d w i t h c h l o r o f o r m / m e t h a n o l (2:1). The total p h o s p h o l i p i d f r a c t i o n was o b t a i n e d by TLC. After t r a n s e s t e r i f i c a t i o n with trifluoride, m e t h y l esters of fatty acids in the p h o s p h o l i p i d fraction were s e p a r a t e d with a c a p i l l a r y column (Advans-AD, 0.24 m m x 25 m, S h i n w a k a g a k u , Kyoto) attached to a GC7A gas c h r o m a t o g r a p h (Shimadzu, Tokyo). About 100 ml of the 24 h urine from each participant was a n a l y z e d for P G I 2 / 3 - M as d e t a i l e d earlier (4) by combined gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y (GC-MS) in the negative ion chemical i o n i z a t i o n mode. The GC-MS system was a Finnigan MAT 44S (Bremen, F.R.G.) equipped with an SE 30 fused silica c a p i l l a r y column (25 m, 0.25 m m i.d.). O p e r a t i o n conditions of the GC-MS were as d e s c r i b e d earlier (4). Data are e x p r e s s e d as means ~ SD and treated by unpaired t-test or least square method

(8).

Results Food intake of the p a r t i c i p a n t s of the two villages is shown in Table I. Higher intake of protein and lipids in the fishing v i l l a g e was due mainly to higher fish intake. Intake of EPA and DHA mostly from fish and also AA intake were c o n s i s t e n t l y higher during the 3 days of the study in the fishing village than in the farming village. The intake of EPA, DHA, LA and AA d u r i n g the 3 days of the study and the average intake are indicated in T a b l e 2. The average ratio of AA to LA is also shown in Table 2. Urinary e x c r e t i o n of PGI3-M and also PGI2-M was higher in the fishing village than in the farming village, although not s i g n i f i c a n t l y (Table 3). C o r r e l a t i o n s b e t w e e n intake of p r e c u r s o r fatty acid EPA or DHA and excretion of PGI3-M, and b e t w e e n AA or LA intake and excretion of PGI2-M during the 3 days of the food study are shown in Table 4. There was highly s i g n i f i c a n t c o r r e l a t i o n between EPA intake of the 3rd day and

APRIL 1989 VOL. 37 NO. 4

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PROSTAGLANDINS e x c r e t i o n of PGI3-M, but there w a s no c o r r e l a t i o n b e t w e e n AA or LA intake on any of the 3 days and e x c r e t i o n of PGI2-M. EPA c o n t e n t of the total p h o s p h o l i p i d s of p l a t e l e t s was higher in the fishing v i l l a g e (4.04 + 1.79 mol %, n=15) than in the f a r m i n g v i l l a g e (3.16 + 1.15 %, n = 2 2 , not significant). The ratio of E P A / A A in the toatl p h o s p h o l i p i d s of p l a t e l e t s in the fishing v i l l a g e (0.34 + 0.19) w a s s i g n i f i c a n t l y h i g h e r than that of the farming village--(0.22 ~ 0.09, p<0.05).

Table 3. Urinary e x c r e t i o n of P G I 2 / 3 - M 3rd day of the food study in t h e f f ~ i n g Villages Fishing

Farming

(ng/g creatinine) on the and the farming village.

PGI2-M v.

v.

Table 4. excretion

PGI3-M

M

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19 ~ 20

F

125 + 51

15 + 14

Total

127 + 60

17 + 17

M

93 + 16

12 ~

F

113 + 34

12 + ii

Total

105 + 29

12 + I0

C o r r e l a t i o n b e t w e e n p r e c u r s o r fatty a c i d of P G I - M (all p a r t i c i p a n t s , n=41).

Correlation

ist day

2nd day

8

intake

and

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=

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r

=

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AA

vs.

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r

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vs.

PGI2-M

r

= -0.05

-0.14

-0.16

*The day w h e n urine w a s collected, c o r r e l a t i o n (r value) of n-3 f a t t y (or 2nd) day and the 3rd day a m o n g f o l l o w s : E P A (ist vs. 3rd) = 0.42, (Ist vs. 3rd) = 0.52, and D H A 2nd

a, acid all EPA vs.

p<0.01; b, p<0.005. The intake b e t w e e n the ist the p a r t i c i p a n t s is as (2nd vs. 3rd) = 0.61, D H A 3rd) = 0.76.

Discussion usual

422

E p i d e m i o l o g i c a l s t u d i e s in a J a p a n e s e f a r m i n g v i l l a g e w i t h fish c o n s u m p t i o n and in a J a p a n e s e fishing v i l l a g e were

APRIL 1989 VOL. 37 NO. 4

PROSTAGLANDINS p e r f o r m e d soon after the winter fishing season had started. An aim of the study was to c o m p a r e the uptake of n-3 and n-6 p o l y u n s a t u r a t e d fatty acids in these villages and the formation of PGI2/3. As expected, the intake of the n-3 p o l y u n s a t u r a t e d fatty abids EPA and DHA was s i g n i f i c a n t l y higher in the fishing v i l l a g e during the study. Also c o n s u m p t i o n of AA was higher in the fishing village c o n f i r m i n g the finding of Bang et al. that AA is contained in the m a r i t i m e food of Eskimos to a c o n s i d e r a b l e extent (9). F o r m a t i o n of PGI 3 as m e a s u r e d by its main urinary metabolite, PGI3-M , tended to be h i g h e r in the f i s h i n g v i l l a g e and was in betw@en that of E s k i m o s and w e s t e r n societies with an low fish c o n s u m p t i o n (i0). The main finding of the study was that there was no c o r r e l a t i o n between the recent intake of A A or LA and e x c r e t i o n of PGI2-M s u g g e s t i n g that f o r m a t i o n of PGI 2 and s u b s e q u e n t e x c r e t i o n of P G I 2 - M was i n d e p e n d e n t of the A A intake of the last 3 days. Also the c o r r e l a t i o n b e t w e e n the intake of LA, the p r e c u r s o r fatty acid of AA, on any of the 3 days of the study and e x c r e t i o n of P G I 2 - M was not significant. Our finding may be e x p l a i n e d m a i n l y by the large AA pool for P G I 2 - M formation compared to EPA pool and also by the very small d a i I y intake of A A (Table 2). However, the AA pool for PGI 2 formation may be i n f l u e n c e d by q u i t e a large intake of AA, which r e m a i n s to be investigated. In the present study, the p a r t i c i p a n t s ingested an e x t r e m e l y low amount of AA c o m p a r e d to LA (Table 2) as was o b s e r v e d in the nutrition of the w e s t e r n societies of Europe and N o r t h A m e r i c a (ii). One of the reasons why we could not find any s i g n i f i c a n t correlation b e t w e e n LA intake and P G I 2 - M levels may be a slow c o n v e r s i o n rate of LA to AA; experiments w i t h l a b e l e d LA in man have shown that it takes about 3 days before orally a d m i n i s t e r e d LA is t r a n s f o r m e d to AA in a p p r e c i a b l e amounts (12). On the other hand, intake of EPA on the last day of the study, when urine was collected, and e x c r e t i o n of P G I 3 - M on that day were highly correlated, w h e r e a s intake of EPA on the first day and e x c r e t i o n of P G I 3 - M on the 3rd day of the study were not c o r r e l a t e d at all. This f i n d i n g confirms the previous o b s e r v a t i o n s that PGI3-M is already formed on the day of mackerel ingestion (i0) and that EPA intake is highly c o r r e l a t e d to PGI3-M e x c r e t i o n on the same day (another e p i d e m i o l o g i c a l stud F) (6). The intake on the 3rd day of DHA, which can be r e t r o c o n v e r t e d in man to EPA and t r a n s f o r m e d within 4 h to PGI3-M (13), is also c o r r e l a t e d to P G I 3 - M excretion. Of c o u r s e , - w h e n e a t i n g fat fish, a higher intake of DHA does also mean a higher c o n s u m p t i o n of EPA. These findings suggest that the EPA pool for PGI 3 formation is rather small and, therefore, is influenced immediately by dietary EPA, while the AA pool for PGI 2 p r o d u c t i o n is not quickly i n f l u e n c e d by usual dietary LA or AA.

Acknowledgment S.F. was supported by the D e u t s c h e F o r s c h u n g s - g e m e i n s c h a f t . The authors are grateful to Miss Akimi Takashima and Miss Akiya Kizu for their technical and editorial assistance.

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423

PROSTAGLANDINS References i.

Kromann, N. and Green, A. Epidemiological studies in the Upernavik District, Greenland. Acta med Scand 208:401-406, 1980.

2.

Hamazaki, T., Urakaze, M., Sawazaki, S., Yamazaki, K., Taki, H. and Yano, S. Comparison of pulse wave velocity of the aorta between inhabitants of fishing and farming villages in Japan. Atherosclerosis 73:157-160, 1988.

3.

Dyerberg, J. and Bang, H.O. Haemostatic function and platelet polyunsaturated fatty acids in Eskimos. Lancet 2:433-435, 1979.

4.

Fischer, S., Weber, P.C. and Dyerberg, J. The prostacyclin /thromboxane balance is favorably shifted in Greenland Eskimos. Prostaglandins 32:235-241, 1986.

5.

Dyerberg, J., Bang, H.O. and Hj@rne, N. Fatty acid composition of the plasma lipids in Greenland Eskimos. Am J Clin Nutr 2_88:958-966, 1975.

6.

Hamazaki, T., Fischer, S., Urakaze, M., Sawazaki, S. and Yano, S. Comparison of the urinary metabolites of prostacyclin and thromboxane of the 2- and 3-series in a Japanese fishing and a Japanese farming village. Prostaglandins 32:655-664, 1986.

7.

Hamazaki, T., Urakaze, M., Yano, S., Soda, Y., Miyamato, A., Kubota, K. and Ibuki, F. Injection of tridocosahexaenoylglycerol emulsion and fatty acid composition of blood cells. Lipids 22:1031-1034, 1987.

8.

Lentner, C., Lentner, C., and Wink, A., ed., Geigy scientific tables vol. 2. Ciba-Geigy, Basle, 1982.

9.

Bang, H.O., Dyerberg, J. and Sinclair, H.M. The composition of the Eskimo food in north western Greenland. Am J ~lin Nutr 3_33:2657-2661, 1980.

i0.

Fischer, S. and Weber, P.C. Prostaglandin vivo in man after dietary eicosapentaenoic Nature 307:165-168, 1984.

ii.

Goodnight, S.H., Harris, W.S., Connor, W.E. and Illingworth, D.R. Polyunsaturated fatty acids, hyperlipidemia and thrombosis. Arteriosclerosis 2:87-113, 1982.

12.

Nichaman, M.Z., ~ s o n , R.E. and Sweeley, C.C. Metabolism of linoleic acid i- ~C in normolipemic and hyperlipemic humans fed linoleate diets. Am J Clin Nutr 2:1070-1083, 1967.

13.

Fischer, S., Vischer, A., Preac-Mursic, V. and Weber, P.C. Dietary docosahexaenoic acid is retro-converted in man to eicosapentaenoic acid, which can be quickly transformed to prostaglandin 13. Prostaglandins 34:367-375, 1987.

Editor: W. Lands

424

Received: 9-19-88

I3.is formed in aczd.

Accepted: 3-3-89

APRIL 1989 VOL. 37 NO. 4