Isolation and identification of prostaglandin I 2 stimulating factor from human plasma

PROSTAGLANDINS

ISOLATION AND IDENTIFICATION OF PROSTAGLANDIN STIMULATING FACTOR FROM HUMAN PLASMA

Masami Masao Ohkuchi, and Yasumi Uchida *

Tsukamoto,

Toru

Yokoyama,

I 2

Masami

Shiratsuchi

Tokyo Research Laboratories, Kowa Co., LTD., 2-17-43 Noguchi-cho, Higashimurayama, Tokyo 189, Japan. * The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-l Hong", Bunkyo-ku, Tokyo 113, Japan

ABSTRACT To isolate and identify the plasma factor which stimulates prostaglandin I 2 production by rat aortic ring, a human plasma fraction which showed a major stimulating activity on prostaglandin I 2 production was purified by ultrafiltrate, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. The purified plasma factor was identified as uric afAd,;; ilz~ y+;r;;;olet and infrared absorption spectroscopy, and spectroscopy. The stimulating activity of the purified plasma factor and that of authentic uric acid coincided with each other. The stimulating potency of uric acid at its physiological concentration in human plasma ( about 50 Ug/ml ) was half of the deproteinized and was about 30 fold stronger than that of human plasma, L-tryptophan, a cofactor of prostaglandin hydroperoxidase. INTRODUCTION (1) and Aemuzzi (2) demonstrated that In 1978, MacIntyre prostaglandin ( PC ) I 2 production by cultured pig arterial endothelial cells or rat aortic ring was stimulated by human plasma, and they suggested that the stimulating factor(s) regulates PC I 2 production and its deficiency results in microangiopathic ischemia and cell damage. It has been shown that the plasma factor is deficient in patients with hemolytic uremic syndrome (2), thrombotic thrombocytopenic purpura (3). sickle cell anemia (4) and acute myocardial infarction (5,6). Despite many trials to purify and identify the plasma factor, the chemical structure of the factor is not known. Therefore, this study was carried out to isolate and identify the stimulating factor in human plasma. METHODS Blood was drawn by venepuncture from 3 healthy male volunteers ( 25-35 years old ) using a plastic Syringe containing sodium citrate ( final concentration 3.8 % 1. The plasma was obtained by

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PROSTAGLANDINS

centrifugation of the blood at 1,000 g for 10 min at room temperature. According to Remuzzi's method (7), stimulating activity was A wistar strain male rat, weighing 150 to 300 g measured as below. and the aorta was removed and was were stunned and decapitated, It was cut into rings ( 1.7 to 2.0 mm in width stored in saline. 1. The rings were incubated in a siliconized glass tubes ( 7 x 50 mm ) buffer, pH 7.4 ( TBS ), and containing 250 ~1 of 50 mM Tris-saline at 37 oc. were stirred at 1,000 rpm and The medium was exchanged The amounts of PG I 2 released during 15 min every 15 min. endogenous arachidonic acid by isolated rat aortic incubation from rings were measured as a stable 6-Keto-PC F la by RIA ( Radio from an isolated rat aorta was chemical Centre ). The PG I 2 level from the 7th incubation period, following an kept quite constant, initial rapid decrease. From this result, in a case of investigations for effects of test samples on PG I 2 production, a sample was added to the media at the 8th and 9th incubation period. In the experimental results as shown in Fig.1, stimulating activity of PC I 2 production was calculated as the following formula: {( b f where a to c and d to f indicate c )/a)x{d/( e+f))xlOO, 6-Keto-PG F 1~ / mg wet weight of aorta / 15 min during 7, 8 and 9 Only b and c are 6-Keto-PG F lo produced respectively. th period,

10 1

5 -

--._ -5. /a --._ OF-0 d

e

f

8

f 9

0 d

I 6

7

Incubation period

of PG I 2 production with TBS Fig. 1. Time course rings and its stimulation caused by a test sample. the amount of PC I 2 produced by 1 mg ( wet weight ring during each 15 min incubation period in TBS or a test sample ( open circle ).

76%

by rat aortic a to f indicate ) of rat aotiC ( closed circle

)

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PROSTAGLANDINS with TBS. with a test sample, and other are values produced Deproteinized plasma was obtained by ultrafiltration, suspended in potassium phosphate buffer and was applied lyophilized, to Sephadex G-10 and then to QAE-Sephadex A-25 column to purify the The purified factor was examined by ultraviolet plasma factor. absorption spectroscopy ( Hitachi, model 200-20 spectrophotometer ), infrared absofptfon spectrof?opy ( Shimazu, model IR-435' H "mr and C nmr spectroscopy ( JEOL, model FX-200 spectrometer ), spectrometer ). Uric acid in plasma was measured by Uric acid C-Test using uricase-TOOS method. Uric acid was removed from plasma with uricase from Candida sp. ( Wako Pure Chemical Industry LTD. 1. All data were expressed a8 mea" f SEM, and it was considered significant when p was below 0.05 at Student's t-test. RESULTS Purification

and

Identification

of PG I 2 Stimulant

in Human

Plasma.

One hundred and seventy ml deproteteinized plasma was obtained by ultrafiltration of 220 ml human plasma using a membrane of M.W. activity of the deproteinized plasma was 15,000. The stimulating compared with the untreated plasma. The stimulating activity of the

300 -

200 -

loo r x8

1

I

x4

x2

Xl

Dilution factor Fig. 2 Effects of human plasma ( closed circle ) and deproteinized human plasma ( open circle ) on PG I 2 production by exhausted rat aortic"rings. Ordinate: 6-Keto-PC F 1~ production expressed as percent of the control TBS group. Abscissa: dilution factor. n=6., * P < 0.05 and ** p < 0.01.

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769

PROSTAGLANDINS undiluted deproteinized plasma "as about 3-fold that Of TBS, and "as lost when about Z-fold of the undiluted plasma. The activity plasma was diluted 4 fold ( Fig. 2 ). Ninety ml deproteinized lyophilized and it "as suspended in a trace amount of 10 mM potassium phosphate buffer, pH 7.0. Then, it "as applied to a Sephadex G-10 column ( 2.1 x 100 cm ) equilibrated with the same and "as run with the same buffer at a flow rate of 0.7 buffer, ml/min. The void volume of the column was fraction 11, and the inner volume "as fraction 27. A strong peak "as detected in the fractions 63 to 70 by an absorption at 280 nm as shown in Fig. 3, and each fraction showed same UV absorption spectra. Several other weak peaks were also observed in the fractions 11 to 48. The stimulating activity on PC I 2 production "as observed in the fractions 10 to 35 with activity were collected, and 63 to 70. Both groups of fractions and the stimulating activity "as compared. Both groups of fractions showed almost the same activity( the former, 202+20* and the 63 to 70 "as subjected to a latter, The fraction 230*33**). QAE-Sephadex A-25 HCO - form column ( 1.7 x 25 cm ). The column "as then washed with 200 a,1 water, and "as eluted using 1 liter linear ammonium bicarbonate gradient of 0 to 0.5 M. After this maneuver, only one peak could be detected by absorption at 280 nm as shown in of the eluate "as concentrated in vacua, Fig. 4. Each 10 ml fraction and "as lyophilized for elimination of ammonium bicarbonate. The stimulating activity "as recognized only in the fractions having The active fraction "as dissolved in 1 ml of ultraviolet absorption. 1 M acetic acid, and applied to a Sephadex G-10 column ( 2.1 x 100 cm ) equilibrated with 1 M acetic acid, and "as eluated with the same solution at a flow rate of 0.7 ml/min. Each fraction "as evaporated in vacua and lyophilized to remove acetic acid. The lyophilized powder "as dissolved in 1 ml water. Eighty ~1 aliquot "as used for the bioassay and it "as revealed that the peak of ultraviolet absorbance coincided with the stimulating activity. 2.1 mg white powder was obtained. The ultraviolet Finally, trum of the powder "as with maxima at 194, 235 and :~~"~~~i~~ds~y&"ere 1900 , 573 and 719 in water, respectively, and its spectrum shifted in 0.01 N HCl and 0.01 N NaOH._jn the infrared a strong absorption at 1673 cm absorption spectrum, "as a signed B in a study in D20, pD 10 by to c=o. No signal "as detected H "mr of from the observation of the substance in D 0, pD ;;'Jb;H~3CW~;;sasY , signals were recognized at 102.70, 154.68, 168.37, properties coincide 163.49 and 165.83 ppm. These physico-chemical activity of the purified with those of uric acid. The stimulating activity substance "as measured. As shown in Fig. 5, the stimulating of the substance coincided with that of authentic uric acid. The

Stimulating

Activity

of Uric

Acid

As shown in Fig. 5, the potency stronger than that of L-tryptophan,

790

and

L-Tryptophan

of uric acid "as about 30 fold the cofactor of hydroperOXidaSe.

DECEMBER 1966 VOL. 36 NO. 6

PROSTAGLANDINS

0

50

100

Fraction number

between fractions of eluates of Sephadex C-10 Fig. 3. Relationship gel filtration of deproteinized human plasma ( abscissa ) and ultraviolet absorbance at 280 nm ( closed circle ) or stimulating activity expressed as percent of the control TBS group ( open column ). Each fraction was composed of 10 ml eluate. One hundred fractions were divided into 6 and the stimulating activity was measured.

Fraction number Fig. 4. Relations of fractions of the eluates of QAE-Sephadex ion exchange chromatography ( abscissa ) to ultraviolet absorbance at 280 nm ( ordinate ) and to the active fractions ( shaded column ).

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PROSTAGLANDINS

Concentration ( pgl ml 1

of purified plasma factor ( closed triangle ), uric Fig. 5. Effects acid ( closed square ) and L-tryptophan ( closed circle 1 on PC I 2 measured as 6-Keto-PG F 1 (I / mg wet weight / 15 min. Namely, PG I 2 stimulating activity of these substances was expressed as percent of that of control TBS group. n=4., * p ( 0.05 and l * p < 0.01. Degradation

of Uric

Acid

in Human

Deproteinized

Plasma.

To obtain uric acid free deproteinized plasma, uricase from Degradation was monitored by measuring the Candida sp. was induced. absorption of ultraviolet at 280 "m. Uric acid in deproteinized plasma was abolished 25 mu/ml of uricase. Also, absence of uric acid in the reaction filtrate was confirmed by Uric acid C-Test after The stimulating activity of deproteinized adding 50 mu/ml uricase. plasma and uric acid free deproteinized plasma was compared. As plasma showed shown in Fig. 6, the uric acid - free deproteinized an almost similar stimulating activity to that of 2 fold dilution of untreated deproteinized plasma ( the former, 265+27** and the latter, 253+25** ). Isolation and Other Eluates

Identification from Sephadex

of the Other Active Substances, G-10 Column Chromatography.

the

Fractions 10 to 49 were collected, and were absorbed to 2 ml and washed with 10 ml waterIt was eluted with 50 active carbon ml of 50 percent methanol solution containing 0.1 ml of acetic acid. in vacua and lyophilized. The The", the eluate was concentrated powder thus obtained was dissolved in 90 ml water ( the original was 1.8 activity of the solution plasma volume 1. The stimulating the pass and the washed fractions folds of that of TBS. However,

792

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PROSTAGLANDINS

300 200 100 0 of PG I 2 production by 2 fold dilution of Fig. 6. Acceleration deproteinized human plasma ( open column ) and that of undiluted uric acid free ( shadow column ). n=4., * p < 0.05 and ** p < 0.01. from the active carbon column chromatography showed little activity. Although we tried to purify the active substances by CM-Sephadex, Sephadex G-25 and Diaion HP-20 etc., it was difficult DEAE-Sephadex, to purify them due to dispersions DISCUSSION It has been reported that the stimulating factor of PG I 2 production exists in human plasma (l-6). However, purification, and identification has not been successful. In this study, isolation and purification of the major component of the stimulating factor could be made successfully by ultrafiltration, Sephadex G-10 gel filtration and QAE-Sephadex column chromatography. It was revealed that the purified stimulating plasma factor is uric acid. Uric acid is differrent from the plasma factors described by MacIntyre (1) and Remuzzi (Z), since the latters are of high molecular weight. Also, uric acid was distinguished from platelet-derived serum factor, heat-stable found by Seid et al. (8). Ritter found dialysable stimulating factor which is formed during coagulation, and anti-stimulating factor is excluded during coagulation (9,lO). There is still a possibility that uric acid is Ritter's factor. Although there are many minor stimulating factors which could not the stimulating potency be identified in deproteinized human plasma, of uric acid accounted for about a half that of deproteinized plasma. The uric acid concentration in human serum is 2.0 to 6.5 uric acid actually At this range of concentration, mg/dl (11).

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PROSTAGLANDINS could stimulate PC I 2 production by an exhausted aortic ring. Ogino reported that uric acid activates PC endoperoxide synthetase and its potency is similar to that of L-tryptophan, a cofactor of PG endoperoxide synthetase (12). In this study, however, the stimulating potency of uric acid was 30 times larger than that of L-tryptophan. It is well known that uric acid causes gout. In 1970, however, Proctor (13) found that uric acid has a property similar to ascorbic acid and acts against oxygen toxicity. Since then, facts which indicate beneficial actions of uric acid have been accumulated (14-18). Although sites of action of uric acid in arachidonic the acid cascade were not examined, uric acid could partcipate in PC I 2 production in humans. REFERENCES 1)

2)

3)

4)

5) 6)

7)

8)

9)

10)

11)

794

MacIntyre,D.E., J.D.Pearson and J.L.Gordon, Localisation and stimulation of prostacyclin production in vascular cells. Nature, 271(g), 549-551 (1978) Remuzzi,G., R.Mlsiani, R.Marchesi, M.Livio, G.Mecca, G.de Gaetano and M.B.Donati,Haemolytic-uremic syndrome: Deficiency of plasma factor(s) regulating prostacyclin activity ? The Lancet, (1978) 21, 871-872 Hensby,C.N., P.J.Lewis, P.Hilgard, G.J.Mufti, J.Hows and J.Webster, Prostacyclin deficiency in thrombotic thrombocytopenic purpura. The Lancet, 6, 748 (1979) Stuart,M.J. and R.H.Sills, Deficiency of plasma prostacyclin or PG I 2 regenerating ability in sickle cell anaemia. Br.J.Haematology, 48, 545-550 (1981) Yui,Y and C.Kawai, Prostaglandin metabolism in ischemic heart disease. Coronary, 2(l), 49-52 (1985) Uchida,Y., M.Masuo and T.Sugimoto, Measurement of plasma factors which stimulate prostaglandin I 2 production in patients with acute myocardial infarction. Shinzo (in Japanese), 17(9), 927-935 (1985) Remuzzi,G., M.Livio, A.E.Cavenaghi, D.Marchesi, G.Mecca, M.B.Donati and G.de Gaetano, Unbalanced prostaglandin synthesis and plasma factors in uraemic bleeding: A hypothesis. Thrombosis Res., 13, 531-536 (1978) P.B.B.Jones and R.G.G.Russell, Seid,J.M., The presence in normal plasma, serum and platelets of factors that stimulate the production of prostacyclin (PC I 2) by cultured endothelial Science, 64, 387-394 (1983) cells. Clinical M.A.Orchard and P.J.Lewis, Ritter,J.M., Stimulation of vascular prostacyclin (PC I 2) production by human serum. Biochem. Pharmac., 31, 3047-3050 (1982) Ritter,J.M., M.A.Ongari, M.A.Orchard and P..J.Lewis, Prostacyclin synthesis is stimulated by a serum factor formed during coagulation.Thromb.Haemostas., 49(l), 58-60 (1983) Nishioka,K., Uric acid. Sougou Linsho (in Japanese), 34, 51-55

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(1985 I O.Hayaishi and T.Tokuyama, Isolation of an 12) Ogino,N., S.Yamamoto, activator for prostaglandin hydroperoxidase from bovine vesicular gland cytosol and its identification as uric acid. B.B.R.C., 87(l), 184 (1979) Similar functions of uric acid and ascorbate 13) Proctor,P., in man ? Nature, 228, 868 (1970) 14) Kellogg,III,E.W. and I.Fridovich, Liposome oxidation and erythrocyte lysis by enzymically generated superoxide and hydrogen peroxide. J.Biol.Chem., 252(19), 6721-6728 (1977) E.Schwiers and P.Hochstein, Uric acid 15) Ames,B.N., R.Cathcart, provides antioxidant defense in humans against oxidantand radical-caused aging and cancer: A hypothesis. Proc.Natn. Acad.Sci.USA, 78(11), 6858-6862 (1981) 16) Smith,R.C. and L.Lawing, Antioxidant activity of uric acid and 3-N-ribosyluric acid with unsaturated fatty acids and erythrocyte membranes. Arc.Bioc.Biop., 223(l), 166-172 (1983) 17) Cohen,A.M., R.E.Aberdroth and P.Hochstein, Inhibition of free radical-induced DNA damage by uric acid. FEBS Lett., 174(l), 147-150 (1984) 18) Lam, K.W., D.Fong, A.Lee and K.M.D.Liu, Inhibition of ascorbate oxidation by urate. J.Inorg.Biochem., 22, 241-248 (1984)

Editor: A. Nies

DECEMBER 1988 VOL. 36 NO. 6

Received: 2-25-88

Accepted: 10-19-88