Research in
ExperimentalMedicine
Res. Exp. Med. (Berl.) 175, 149--153 (1979)
(~ Springer-Verlag 1979
The Influence of Linoleic Acid Intake on the Excretion of Urinary Prostaglandin Metabolites* N. Z611ner, O. Adam, and G. Wolfram Forschergruppe Ern~ihrung, Medizinische Poliklinik der Universit~it Miinchen, Pettenkoferstrage 8a, D-8000 Mfinchen 2, Federal Republic of Germany
Summary. The influence of linoleic acid intake on human prostaglandin formation was investigated by gas chromatographic determination ofdimethyl tetranorprostanedioate, a derivative of the major urinary end products of prostaglandin metabolism. Six healthy female volunteers were put on liquid formula diets. Each person had a linoleic acid supply of 0, 10, and 50 g per day for periods of two weeks. At the end of these periods the corresponding average amounts of tetranorprostanedioic acid in urine were 92, 175, and 326~tg per day. Key words: H u m a n linoleic acid intake - Liquid formula diets - Prostaglandin metabolites in urine Linoleic acid in food is the main precursor for prostaglandin synthesis in man. Therefore, it seemed reasonable to test whether prostaglandin turnover can be influenced by changing linoleic acid supply. Several workers have shown that in animal and in man endogenous prostaglandins are excreted mainly as polar metabolites in the urine [1, 4, 5, 6]; Hamberg et al. [7] could identify tetranorprostanedioic acid derivatives as the major end products of the metabolism of prostaglandins El, E2, F ~ , F2~. Recently methods have been developed to produce joint derivatives from many end products of prostaglandin metabolism found in the urine, thus permitting an estimation of prostaglandin turnover. By measuring the excretion of the mentioned joint chemical derivatives we determined the influence of dietary linoleic acid on the excretion of prostaglandin metabolites in man using the percentage of linoleic acid in the cholesterol esters of serum as an indicator of linoleic acid uptake [15]. * Supported by a grant from the Deutsche Forschungsgemeinschaft Abbreviation used: TNPA: tetranorprostanedioic acid
0300-9130/79/0175/0149/$1.00
150
N. Z611ner et al.
Methods Six healthy females, 22--24 years old, with normal body weight recorded their diets for a period of eight days. Afterwards they were put on isoenergetic formula diets containing no linoleic acid (diet I), 10 g (diet II) or 50 g (diet III) linoleic acid per day. Each of the three different formula diets was given to every subject in a different order for periods of two weeks. The protein content of all diets was 15 energy%. The formula diet without linoleic acid contained 85 energy% as oligopolymeres of glucose, in the other two formula diets carbohydrates supplied 55% and fat 30% of energy. The composition of the formula diets is shown in Table 1. Water was taken ad libitum. 3 g KC1 and 5 g NaC1 were substituted per 3000 kcal formula diet. Cholesterol content was adjusted to 600mg/d. Multibionta, 1 capsule per day, served as a source of vitamins. Water balance, energy uptake, and body weight were recorded throughout the experiment. Samples were taken from every 24-h urine and were deep-frozen at -20°C until the analysis of creatinine and tetranorprostanedioic acid (TNPA). TNPA in urine was determined by gas-liquidchromatography on two packed columns with liquid phases of different polarity [10]. When starting with formula diet and at the end of each two weeks period fasting blood samples were drawn for determination of fatty acids in serum cholesterol esters by gas liquid chromatography [16].
Results W a t e r balance w a s a d a p t e d to the s o d i u m intake within five days of the first period of f o r m u l a diet. F r o m then on b o d y weight r e m a i n e d c o n s t a n t within 1.5 kg; the individually chosen energy intake, determined by individual appetite, r e m a i n e d within 10% of the individuals average. U n d e r c o n v e n t i o n a l diet the calculated linoleic acid intake was a b o u t 20 g per day a n d the c o r r e s p o n d i n g excretion of T N P A in urine 306 gg per day o n the average. After two weeks of each f o r m u l a diet with a daily supply of 0, 10, or 50 g linoleic acid the average excretion of T N P A was 92 gg, 175 gg, a n d 326 gg. A t the same time the percentage of linoleic acid in serum cholesterol esters h a d c h a n g e d in relation to the linoleic acid supply with the f o r m u l a diet, as described before [15]. Figure 1 compiles all results w i t h o u t respect to chronological sequence.
Table 1. Composition of conventional diet and formula diet I--III Formula diet
I
II
III
Conventional diet
Energy (kcal/day) Linoleic acid (g/day) Carbohydrates (g/day) Protein (g/day) Saffior oil (g/day) (71 g linoleic acid per 100 g) Butter fat (g/day) (3.6 g linoleic acid per 100 g) Olive oil (g/day) (8.2 g linoleic acid per 100 g)
2200 0 456 80
2200 10 295 80
2200 50 295 80
2200 20 241 91
10
(7.1)
47
(1.7)
14.6 (1.2)
71
Linoleic Acid Intake and Urinary Prostaglandin Metabolites Iconventionat diet
151 [
formula diets I
~
II
III
linoteic acid intake
4O 3O to
o 7060504030 20 ~-"
linoleic acid inserum cholesterol esters
i •
+ i
tetranorprostanedioic
Fig. 1. Correlation between linoleic acid intake, linoleic acid in serum cholesterol esters, and amount of tetranorprostanedioic acid in 24-h urine. The given values are determinations in six healthy volunteers on conventional diet and at the end of periods of two weeks of formula diets
l
acid in 24-h
urine
500-
400300200'lO0-
Discussion
The urinary excretion of recognized metabolites of prostaglandin E [1,7, 12] and F [1,2,5,7] amounts to about 4 0 p g / d , the authors [1,2,5,7,12,13] reported higher quantities in males (40/.tg/d) than in females (15gg/d). However, if the recognized metabolites and other unrecognized substances are degraded chemically to TNPA, the excretion is much higher. Thus, Nugteren found an excretion of 300 gg _+ 100 p g / d of the various TNPA derivates and no difference between the examined 14 males and five females. Our values determined by the same method are identical, 306 p g / d on the average. Several authors have shown that arachidonic acid applied into the renal artery gives rise to prostaglandin formation in the kidneys [4, 17]. An increased effiux of prostaglandins from other perfused organs such as heart [8], lung [9] and gastric mucosa homogenates [!1] was found, when the concentration of precursors in the perfusate was increased. Moreover, the oral intake of ethyl arachidonate in male volunteers augmented the
152
N. Z611ner et al.
excretion of 7-hydroxy-5-11-diketotetranorprostane-l,16-dioic acid, the major urinary metabolite of prostaglandin E in man [14]. Our results demonstrate that endogenous prostaglandin production of the E and F series is influenced by the oral intake of linoleic acid. The urinary excretion of metabolites, which may be converted to TNPA corresponds to 306lag TNPA per day on a conventional diet. After two weeks without dietary linoleic acid this excretion is greatly reduced to 92lag of TNPA per day, i.e., 30%. Ten grams of dietary linoleic acid increased TNPA substantially to nearly double, and the intake of 50 g linoleic acid per day resulted in a further increase of TNPA (Fig. 1). In linoleic acid deficiency a decreased prostaglandin E turnover in infants has been described [3]. Our results show that in man prostaglandin synthesis is influenced by linoleic acid intake after already short periods of changes in linoleic acid supply; they also suggest that overall prostaglandin synthesis can be increased by increasing the linoleic acid uptake. Prostaglandins, prostacyclin and thromboxanes have, beside many other effects, a regulative function on blood pressure and platelet aggregation. The close correlation found between the amount of linoleic acid in the diet and the excretion of prostaglandins in urine may represent a further step toward the elucidation of the reduction of platelet aggregation by diets high in linoleic acid thus opening new approaches toward an explanation of the beneficial effects of linoleic acid on several manifestations of coronary heart disease.
References 1. Alm6, B., Hansson, G.: Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger. Prostaglandins 15, 200--217 (1978) 2. Aizawa, Y., Yamada, K., Hata, M.: Double isotope derivative dilution method for the determination of prostaglandin F and E type metabolites in urine. Prostaglandins 14, 1165--1174 (1977) 3. Friedman, Z., Seyberth, H., Lamberth, E., Oates, J.: Decreased prostaglandin E turnover in infants with essential fatty acid deficiency. Pediat. Res. 12, 711--714 (1978) 4. Fr6hlich, T. C., Wilson, T. W., Sweetman, B. J., Smigel, M., Nies, A. S., Cart, K., Watson, J. T., Oates, J. A.: Urinary prostaglandins. Identification and origin. J. Clin. Invest. 55,763-770 (1975) 5. Granstr6m, E., Kindahl, H.: Radioimmunoassay for urinary metabolites of prostaglandin F2~. Prostaglandins 12, 759--783 (1976) 6. Hamberg, M., Samuelson, B.: On the metabolism ofprostaglandins E1 andE2in man. J. Biol. Chem. 246, 6713--6721 (1971) 7. Hamberg, M.: Quantitative studies on prostaglandin synthesis in man. Anal. Biochem. 55, 368--378 (1973) 8. Mentz, P., Forster, W.: The influence of unsaturated fatty acids on prostaglandin release in isolated perfused Guinea pig hearts. Prostaglandins 14, 173--179 (1977) 9. Nijkamp, F. P., Moncada, S., White, H. L., Vane, J. R.: Diversion of prostaglandin endoperoxide metabolism by selective inhibition of thromboxane A2 biosynthesis in lung, spleen, or platelets. Eur. J. Pharmacol. 44, 179--186 (1977) 10. Nugteren, D. H.: The determination of prostaglandin metabolites in human urine. J. Biol. Chem. 250, 2808--2812 (1975) 11. Peskar, B. M.: On the synthesis ofprostaglandinsbyhumangastricmucosaanditsmodification by drugs. Biochim. Biophys. Acta 487, 307--314 (1977)
Linoleic Acid Intake and Urinary Prostaglandin Metabolites
153
12. Seyberth, H. W., Sweetmann, B. J., Fr6hlich, J. C., Oates, J. A.: Quantification of the major urinary metabolite of the E prostaglandins by mass spectrometry: evaluation of the method's application to clinical studies. Prostaglandins 11, 381--388 (1976) 13. Scherer, B., Schnermann, J., Sofroniev, M., Weber, P. C.: Prostaglandin analysis in urin of humans and rats by different radioimmunoassays: effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide. Prostaglandins 15, 255--265 (1978) 14. Seyberth, H. W., Oelz, O., Kennedy, T., Sweetman, B. J., Danon, A., Fr6hlich, J. C., Heimberg, M., Oates, J. A.: Increased arachidonate in lipids after administrationto man: effects on prostaglandin biosynthesis. Clin. Pharmacol. Ther. 18, 521--529 (1975) 15. Wolfram, G., Z611ner,N.: Der Linolsgurebedarf des Menschen. In: Polyenfetts~iuren. Wiss. Ver6ff. DGE 22, S. 51--60. Darmstadt: Dr. D. Steinkopff 1971 16. Z6Uner, N., Eberhagen, D.: Untersuchung und Bestimmung der Lipoide im Blut. BerlinHeidelberg-New York: Springer 1965 17. Zusman, R. M., Keiser, H. R.: Prostaglandin Ez biosynthesis by rabbit renomedullary interstitial cells in tissue culture. J. Biol. Chem. 252, 2069--2071 (1977) Received December 22, 1978 / Accepted February 14, 1979
ExperimentalMedicine
Res. Exp. Med. (Berl.) 175, 149--153 (1979)
(~ Springer-Verlag 1979
The Influence of Linoleic Acid Intake on the Excretion of Urinary Prostaglandin Metabolites* N. Z611ner, O. Adam, and G. Wolfram Forschergruppe Ern~ihrung, Medizinische Poliklinik der Universit~it Miinchen, Pettenkoferstrage 8a, D-8000 Mfinchen 2, Federal Republic of Germany
Summary. The influence of linoleic acid intake on human prostaglandin formation was investigated by gas chromatographic determination ofdimethyl tetranorprostanedioate, a derivative of the major urinary end products of prostaglandin metabolism. Six healthy female volunteers were put on liquid formula diets. Each person had a linoleic acid supply of 0, 10, and 50 g per day for periods of two weeks. At the end of these periods the corresponding average amounts of tetranorprostanedioic acid in urine were 92, 175, and 326~tg per day. Key words: H u m a n linoleic acid intake - Liquid formula diets - Prostaglandin metabolites in urine Linoleic acid in food is the main precursor for prostaglandin synthesis in man. Therefore, it seemed reasonable to test whether prostaglandin turnover can be influenced by changing linoleic acid supply. Several workers have shown that in animal and in man endogenous prostaglandins are excreted mainly as polar metabolites in the urine [1, 4, 5, 6]; Hamberg et al. [7] could identify tetranorprostanedioic acid derivatives as the major end products of the metabolism of prostaglandins El, E2, F ~ , F2~. Recently methods have been developed to produce joint derivatives from many end products of prostaglandin metabolism found in the urine, thus permitting an estimation of prostaglandin turnover. By measuring the excretion of the mentioned joint chemical derivatives we determined the influence of dietary linoleic acid on the excretion of prostaglandin metabolites in man using the percentage of linoleic acid in the cholesterol esters of serum as an indicator of linoleic acid uptake [15]. * Supported by a grant from the Deutsche Forschungsgemeinschaft Abbreviation used: TNPA: tetranorprostanedioic acid
0300-9130/79/0175/0149/$1.00
150
N. Z611ner et al.
Methods Six healthy females, 22--24 years old, with normal body weight recorded their diets for a period of eight days. Afterwards they were put on isoenergetic formula diets containing no linoleic acid (diet I), 10 g (diet II) or 50 g (diet III) linoleic acid per day. Each of the three different formula diets was given to every subject in a different order for periods of two weeks. The protein content of all diets was 15 energy%. The formula diet without linoleic acid contained 85 energy% as oligopolymeres of glucose, in the other two formula diets carbohydrates supplied 55% and fat 30% of energy. The composition of the formula diets is shown in Table 1. Water was taken ad libitum. 3 g KC1 and 5 g NaC1 were substituted per 3000 kcal formula diet. Cholesterol content was adjusted to 600mg/d. Multibionta, 1 capsule per day, served as a source of vitamins. Water balance, energy uptake, and body weight were recorded throughout the experiment. Samples were taken from every 24-h urine and were deep-frozen at -20°C until the analysis of creatinine and tetranorprostanedioic acid (TNPA). TNPA in urine was determined by gas-liquidchromatography on two packed columns with liquid phases of different polarity [10]. When starting with formula diet and at the end of each two weeks period fasting blood samples were drawn for determination of fatty acids in serum cholesterol esters by gas liquid chromatography [16].
Results W a t e r balance w a s a d a p t e d to the s o d i u m intake within five days of the first period of f o r m u l a diet. F r o m then on b o d y weight r e m a i n e d c o n s t a n t within 1.5 kg; the individually chosen energy intake, determined by individual appetite, r e m a i n e d within 10% of the individuals average. U n d e r c o n v e n t i o n a l diet the calculated linoleic acid intake was a b o u t 20 g per day a n d the c o r r e s p o n d i n g excretion of T N P A in urine 306 gg per day o n the average. After two weeks of each f o r m u l a diet with a daily supply of 0, 10, or 50 g linoleic acid the average excretion of T N P A was 92 gg, 175 gg, a n d 326 gg. A t the same time the percentage of linoleic acid in serum cholesterol esters h a d c h a n g e d in relation to the linoleic acid supply with the f o r m u l a diet, as described before [15]. Figure 1 compiles all results w i t h o u t respect to chronological sequence.
Table 1. Composition of conventional diet and formula diet I--III Formula diet
I
II
III
Conventional diet
Energy (kcal/day) Linoleic acid (g/day) Carbohydrates (g/day) Protein (g/day) Saffior oil (g/day) (71 g linoleic acid per 100 g) Butter fat (g/day) (3.6 g linoleic acid per 100 g) Olive oil (g/day) (8.2 g linoleic acid per 100 g)
2200 0 456 80
2200 10 295 80
2200 50 295 80
2200 20 241 91
10
(7.1)
47
(1.7)
14.6 (1.2)
71
Linoleic Acid Intake and Urinary Prostaglandin Metabolites Iconventionat diet
151 [
formula diets I
~
II
III
linoteic acid intake
4O 3O to
o 7060504030 20 ~-"
linoleic acid inserum cholesterol esters
i •
+ i
tetranorprostanedioic
Fig. 1. Correlation between linoleic acid intake, linoleic acid in serum cholesterol esters, and amount of tetranorprostanedioic acid in 24-h urine. The given values are determinations in six healthy volunteers on conventional diet and at the end of periods of two weeks of formula diets
l
acid in 24-h
urine
500-
400300200'lO0-
Discussion
The urinary excretion of recognized metabolites of prostaglandin E [1,7, 12] and F [1,2,5,7] amounts to about 4 0 p g / d , the authors [1,2,5,7,12,13] reported higher quantities in males (40/.tg/d) than in females (15gg/d). However, if the recognized metabolites and other unrecognized substances are degraded chemically to TNPA, the excretion is much higher. Thus, Nugteren found an excretion of 300 gg _+ 100 p g / d of the various TNPA derivates and no difference between the examined 14 males and five females. Our values determined by the same method are identical, 306 p g / d on the average. Several authors have shown that arachidonic acid applied into the renal artery gives rise to prostaglandin formation in the kidneys [4, 17]. An increased effiux of prostaglandins from other perfused organs such as heart [8], lung [9] and gastric mucosa homogenates [!1] was found, when the concentration of precursors in the perfusate was increased. Moreover, the oral intake of ethyl arachidonate in male volunteers augmented the
152
N. Z611ner et al.
excretion of 7-hydroxy-5-11-diketotetranorprostane-l,16-dioic acid, the major urinary metabolite of prostaglandin E in man [14]. Our results demonstrate that endogenous prostaglandin production of the E and F series is influenced by the oral intake of linoleic acid. The urinary excretion of metabolites, which may be converted to TNPA corresponds to 306lag TNPA per day on a conventional diet. After two weeks without dietary linoleic acid this excretion is greatly reduced to 92lag of TNPA per day, i.e., 30%. Ten grams of dietary linoleic acid increased TNPA substantially to nearly double, and the intake of 50 g linoleic acid per day resulted in a further increase of TNPA (Fig. 1). In linoleic acid deficiency a decreased prostaglandin E turnover in infants has been described [3]. Our results show that in man prostaglandin synthesis is influenced by linoleic acid intake after already short periods of changes in linoleic acid supply; they also suggest that overall prostaglandin synthesis can be increased by increasing the linoleic acid uptake. Prostaglandins, prostacyclin and thromboxanes have, beside many other effects, a regulative function on blood pressure and platelet aggregation. The close correlation found between the amount of linoleic acid in the diet and the excretion of prostaglandins in urine may represent a further step toward the elucidation of the reduction of platelet aggregation by diets high in linoleic acid thus opening new approaches toward an explanation of the beneficial effects of linoleic acid on several manifestations of coronary heart disease.
References 1. Alm6, B., Hansson, G.: Analysis of metabolic profiles of prostaglandins in urine using a lipophilic anion exchanger. Prostaglandins 15, 200--217 (1978) 2. Aizawa, Y., Yamada, K., Hata, M.: Double isotope derivative dilution method for the determination of prostaglandin F and E type metabolites in urine. Prostaglandins 14, 1165--1174 (1977) 3. Friedman, Z., Seyberth, H., Lamberth, E., Oates, J.: Decreased prostaglandin E turnover in infants with essential fatty acid deficiency. Pediat. Res. 12, 711--714 (1978) 4. Fr6hlich, T. C., Wilson, T. W., Sweetman, B. J., Smigel, M., Nies, A. S., Cart, K., Watson, J. T., Oates, J. A.: Urinary prostaglandins. Identification and origin. J. Clin. Invest. 55,763-770 (1975) 5. Granstr6m, E., Kindahl, H.: Radioimmunoassay for urinary metabolites of prostaglandin F2~. Prostaglandins 12, 759--783 (1976) 6. Hamberg, M., Samuelson, B.: On the metabolism ofprostaglandins E1 andE2in man. J. Biol. Chem. 246, 6713--6721 (1971) 7. Hamberg, M.: Quantitative studies on prostaglandin synthesis in man. Anal. Biochem. 55, 368--378 (1973) 8. Mentz, P., Forster, W.: The influence of unsaturated fatty acids on prostaglandin release in isolated perfused Guinea pig hearts. Prostaglandins 14, 173--179 (1977) 9. Nijkamp, F. P., Moncada, S., White, H. L., Vane, J. R.: Diversion of prostaglandin endoperoxide metabolism by selective inhibition of thromboxane A2 biosynthesis in lung, spleen, or platelets. Eur. J. Pharmacol. 44, 179--186 (1977) 10. Nugteren, D. H.: The determination of prostaglandin metabolites in human urine. J. Biol. Chem. 250, 2808--2812 (1975) 11. Peskar, B. M.: On the synthesis ofprostaglandinsbyhumangastricmucosaanditsmodification by drugs. Biochim. Biophys. Acta 487, 307--314 (1977)
Linoleic Acid Intake and Urinary Prostaglandin Metabolites
153
12. Seyberth, H. W., Sweetmann, B. J., Fr6hlich, J. C., Oates, J. A.: Quantification of the major urinary metabolite of the E prostaglandins by mass spectrometry: evaluation of the method's application to clinical studies. Prostaglandins 11, 381--388 (1976) 13. Scherer, B., Schnermann, J., Sofroniev, M., Weber, P. C.: Prostaglandin analysis in urin of humans and rats by different radioimmunoassays: effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide. Prostaglandins 15, 255--265 (1978) 14. Seyberth, H. W., Oelz, O., Kennedy, T., Sweetman, B. J., Danon, A., Fr6hlich, J. C., Heimberg, M., Oates, J. A.: Increased arachidonate in lipids after administrationto man: effects on prostaglandin biosynthesis. Clin. Pharmacol. Ther. 18, 521--529 (1975) 15. Wolfram, G., Z611ner,N.: Der Linolsgurebedarf des Menschen. In: Polyenfetts~iuren. Wiss. Ver6ff. DGE 22, S. 51--60. Darmstadt: Dr. D. Steinkopff 1971 16. Z6Uner, N., Eberhagen, D.: Untersuchung und Bestimmung der Lipoide im Blut. BerlinHeidelberg-New York: Springer 1965 17. Zusman, R. M., Keiser, H. R.: Prostaglandin Ez biosynthesis by rabbit renomedullary interstitial cells in tissue culture. J. Biol. Chem. 252, 2069--2071 (1977) Received December 22, 1978 / Accepted February 14, 1979
