- Email: [email protected]
Iothalamate stimulates hydroperoxide formation by soybean lipoxygenase
PROSTAGLANDINS
IOTHALAMATE
STIMULATES HYDROPEROXIDE SY SOYBEAN LIPOXYGENASE
FORMATION
Aubrey R. Morrison, Wendel arown and Nabil Tauk Department of Medicine and Pharmacology Washington ilniversity School of Medicine St. Louis, Missouri 63110 Abstract Sodium iothalamate produced a dose dependent increase in basal oxygen consumption when soybean lipoxygenase was incubated with arachidonic acid in Tne increase in oxygen consumption was associated O.lM borate buffer pH 9.0. with an increase in absorbance at 234 nm indicating an increased conjugated The stimulation of 02 consumption was demonstrated to be due diene formation. to an increase in 1500H arachidonic formation. The increase in 1500H arachidonate formation could be blocked by mannitol which is an inhibitor of the lipoxygenase enzyme. N-methyl glucamine (megluinine) which is added to some preparations of iothalamate, was also able to suppress tne increase in hydroperoxide formation in a dose dependent fashion. Introduction Intravascular contrast agents have been associated with a variable incidence and degree of renal functional impairment (1,2). In acute experiments infusion of contrast agents into the renal artery has been asiociated with a biphasic response. There.is an initial increase in renal blood flow followed by a later phase of decrease in renal blood flow Recently Workman et al (6) have shown a relationship between the (3,4,51. renal hemodynamic changes following administration of intravascular contrast agents and renal prostacyclin production in the dog. Their experiments showed a transient reduction in arterio-venous levels of 6-keto PGF,a, the stable metabolite of prostacyclin and renal secretion rates of 6-keto PGFlcl which reached a nadir 30 minutes after infusion of contrast agent. This effect of contrast agents on renal prostacyclin synthesis could be due to a direct effect on prostacyclin synthesis or by increasing the synthesis of an inhibitor of prostacyclin. A well known naturally occurring inhibitor of prostacyclin is 15 hydroperoxy eicosatetraenoic acid (7,8). Since blood vessels have the capacity for both prostacyclin synthesis and hydroperoxide syntnesis, an increase in hydroperoxide formation could perhaps inhibit prostacyclin formation. We therefore asked the question if sodium iothalamate could increase hydroperoxide formation in an -_in vitro system using soybean lipoxygenase as a source of enzyme and arachidonate as a substrate. Materials
and rIethods
Arachidonate acid (CZ,,.~) was purchased from NuCheck Elysian, MN. 1-[14C] arachidonic acid (55 m Ci/minol) was obtained fro,n New England Nuclear, Boston Lipoxidase type IV, sodiurn iothalmate, D-mannitol and N-methyl glucamine ;fls . (rneglumine) were purchased from Sigma Chemical Co., St. Louis, No. 02 consumption was measured by a Clark type 02 electrode and lnodel 160 physiological gas analyser from Beckman, Palo Alto, CA., and coupled to a
MAY 1984VOL. 27 NO. 5
753
PROSTAGLANDINS
[ Na+
Iothalamote] mg/ml
Fig.
754
Dose response curve for soidum iothalamate 1. 100 percent is taken as control value.
on oxygen
consumption.
MAY 1984 VOL. 27 NO. 5
PROSTAGLANDINS
t
8
16
24
32
40
48
56 64 7; RETENT
N VOLUME
Fig. 2. HPLC tracing monitoring 235 nm, continous line and l'+C radioactivity, dotted line. 15 HPETE is 15-hydroperoxy eicosatetraenoic acid. control. B plus 100 mg/ml sodium iothalamate. C plus 100 mM mannitol! 3 100 mM mannitol plus 100 cog/ml sodium iothalamate.
MAY 1984 VOL. 27 NO. 5
755
PROSTAGLANDINS
strip chart recorder, Linear instruments, Irvine, CA. The polarographic cell had a total volulne of 1.2 ml. Polarographic experiments were carried out using soyoean lipoxiddse 400ng and substrate drachidonic acid 25 @g/ml in a total volume of 1 ml. Buffer used was 0.1 M borate pH 9.0. Na+ iothalarnate, D-mannitol and N-.nethyl glucamine were ddded at various concentration to the polarqraphic cell. For radiochemical experiments, arachidonate 25 Pg/ml and l-14C arachidonate 600,i)OO cpm were incubated in one ml of 0.1 i"lborate buffer pH 9.0 with 400 ng soybean lipoxidase for 5 minutes at roo,n temperature. III other rddiochemical experiments iothdlamate 100 ~my/ml and lnannitol 100 mM were added separately and then together to the incubations. The incubations were stopped with 300 ~1 [methanol and then acidified to pH 3.5 with O.lN HCl. To the methanolic extract 6 vols of diethyl ether and 4 vols of water were added and the ether phase evaporated to dryness and reconstituted in a snall volume of the mobile phase MeOH:HzO:Acetic acid (75:25:0.01). HPLC was carried out on a gaters system and UV absorption Imonitored at 235 nm. Radioactivity was Imeasured using a flow through detector obtained from Radiomatic Instruments (Tampa, FL). Results and Discussion increases in basal 02 consumption Fig. 1 shows the dose dependent obtained in the polarographic experiments. A 50'6 increase in 02 consumption was obtained with concentrations of iothalamate of 45 mg/:nl. The increase in 02 consumption was associated with an increase in hydroperoxide formation determined radiochemicdlly Fig. 2. Fig. 2A shows the production of 15-HPETt under basal condition as described in methods. Fig. 23 shows the increase in 15-HPETE formation when the incubation were carried out in the presence of 100 Ing/lnlisthalamate. '3f note is the almost complete consumption of substrate after 5 minutes of incubation. I*lannitolat 100 ml"1was previously shown to inhibit soybean lipoxygendse by greater than 90.X (9). Fig. 2C shows a similar experiment, indicating the inhibition of 15 OOH formation by mannitol. Fig. 20 shows that at a concentrdtion of 100 mM, mannitol also inhibited 15-HPETE formation in the presence of 100 mg/!nl sodium iothdlamate. Meglumine (Nmethyl glucamine) has structural similarities to mannitol Fig. 3. And is added to some preparations of iothalamate. megldmine like mannitol and other hexoses, was able to inhibit the increase in 02 consumption under basal conditions dnd also when stimulated with sodium iothlamate. Fici. 4 shows the dose dependent inhibition of iothalamate stimulated 02 consumption by N-methyl glucamine. Radiocontrast agents have been shown to decrease renal blood flow. Recently it has been suggested that the decrease in renal blood flow is related to a decreased renal production of 6-keto PGFla the stable dehydration product of PGIz. Stimulation of 15-HPETE ~01~1 d potentially decrease 6-keto PGFla release by inhibition of prostacyclin synthetase. The kidney has been shown to have both 12 and 15 lipoxygenase (10,11,12) pathways thus the enzyme are present for increased fatty acid imetabolism by a lipoxygenase when exposed to sodium iothalamate. If 75 ml of Conray 400 '@ (Malinkrodt) is adlninistered to a patient intravenously the final concentrations of the radio pharmaceutical (assuming a total plasma water of 2.5 Litres) will be about 20 Ing/:nlof sodium iothalamate. In the kidney the concentration is likely to exceed this considerably and thus will be in the range to significantly Imodulate renal lipoxygenases in cortex and medulla. The administration of contrast agents eg. sodium iothalamate directly into the arterial circulation may also produce much higher concentrations of contrast for example during
756
MAY 1984 VOL. 27 NO. 5
PROSTAGLANDINS
MEGLUMINE (N-methyl glucamine) CH,NHCH,
CH,OH
I HCOH
I HOCH
I
HOCH
HOCH
I
HCOH
HCOH
I HCOH
I HCOH
CH,OH
Fig. 3.
MANNITOL
CH,OH
Structures of N-methylglucamineand mannitol.
MAY 1984VOL. 27 NO. 5
757
PROSTAGLANDINS
E c
250 B--w
0
0 150 o\o Control
100% 50
66 mg/ml _
132 mg/ml
Iothalamate
Iotholamate 132mg/ml
1,--1---_ 75
-eV N-methyl
Fig.
100
150
glucamine mM
Effects of sodium iothalamate and sodium iothalamate plus 4. methylglucamine on basal oxygen consumption by soybean lipoxygenase.
renal angiography and significantly indirectly PGI- production.
modify
renal
lipoxygenase
products
N-
and
Acknowledgements The work is supported 32. Dr. Xorrison is an Association.
758
by NIH research grants PO1 Air109976and AM30542established investigator of the American Heart
MAY
1984 VOL. 27 NO. 5
PROSTAGLANDINS
REFERENCES 1.
Bennett, W.bl. Lult, F. Porter, G.A. Pathogenesis of renal failure due to aminoglycosides and contrast media used in roentgenography. Am. J. Med. -1980: 69: 767-774. -
2.
Byrd, L. Sherman, R.L. clinical and pathological
3.
Caldicott, W.J.H., Hollenberg, N.K., Abrams, H.L.: Characteristics of resoonse of renal vascular bed to contrast media: evidence for vasbconstriction induced by renin-angiotenoin system, Invest. Radiol. 1970 -5: 539-547.
4.
Talner, L.B., Davidson, A.J.: Renal hemodynamic media. Invest. Radiol. 1968: -3: 301-309.
5.
Larson, T.S., Hudson, K., Mertz, J.I., Rornero, J.C. and Knox, F.G. vasoconstrictive response to contrast medium. J. Lab. Clin. Med. 101: 385-391
6.
Workman, R.J., Shaff, M.I., Jackson, R.V., Diggs, M.D., Frazier, M.G., Briscoe. W.J. Relationship of Renal hemodvnamic and functional chances following intravascular contrast to the renin-angotension system and reial prostaglandin in the dog. Invest. Radiol. 1983: -18: 160-166.
7.
Moncada, S., Gryglewski, R.J., Bunting, S. and Vane, J.R. A lipid peroxide inhibits the enzyme in blood vessels microsomes that generate from prostaglandin endoperoxides the substance (Prostaglandin X) which prevents platelet aggregation. Prostglandins 1976: -12: 715-737.
a.
Gryglewski, R.J., Bunting, S., Moncada, J., Flower, R.J., and Vane, J.R. Arterial walls are protected aqainst deoosition of platelet thromibi bv a (Prostajlandin X)- which they make' from prostaglanhin substance endoperoxides. Prostaglandins 1976: -12: 685-713.
9.
Morrison, A.R., Winokur, Lipoxygenase by Mannitol.
Radiocontrast induced acute renal failure: review. -Medicine: 1979: 58: 270-279.
effects
T.S., Wendell Brown: Inhibition BBRC 1982: 108: 1757-1762.
of
of
A
contrast
Renal 1983:
Soybean
hlorrison, A.R. Regional 10. Winokur, T.S., synthesis of monohydroxy eicosanoids by the kidney. J. Biol. Chem. 1981: 256: 10221-10223. 11. Sraer, J. Rigaud, M., Bens, M., Rabinovitch, H., Ardaillou. Metabolism of acid in the liooxvqenase oathwav in human and murine arachidonic J. Biol. Chem. 198i: $& 4325i4330.‘ glomeruli. 12. Jim, K., Hassid, A., Sung, F., Dunn, M.J. Lipoxygenase activity in rat kidney glomeruli, glomerular epithetial cells and cortical tubules. J. 9iol. Chem. 1982: __257: 10294-10299.
Editor: John E. Pike 10=24=83 Received: Accepted: 4-6-84
MAY 1984 VOL. 27 NO. 5
759
IOTHALAMATE
STIMULATES HYDROPEROXIDE SY SOYBEAN LIPOXYGENASE
FORMATION
Aubrey R. Morrison, Wendel arown and Nabil Tauk Department of Medicine and Pharmacology Washington ilniversity School of Medicine St. Louis, Missouri 63110 Abstract Sodium iothalamate produced a dose dependent increase in basal oxygen consumption when soybean lipoxygenase was incubated with arachidonic acid in Tne increase in oxygen consumption was associated O.lM borate buffer pH 9.0. with an increase in absorbance at 234 nm indicating an increased conjugated The stimulation of 02 consumption was demonstrated to be due diene formation. to an increase in 1500H arachidonic formation. The increase in 1500H arachidonate formation could be blocked by mannitol which is an inhibitor of the lipoxygenase enzyme. N-methyl glucamine (megluinine) which is added to some preparations of iothalamate, was also able to suppress tne increase in hydroperoxide formation in a dose dependent fashion. Introduction Intravascular contrast agents have been associated with a variable incidence and degree of renal functional impairment (1,2). In acute experiments infusion of contrast agents into the renal artery has been asiociated with a biphasic response. There.is an initial increase in renal blood flow followed by a later phase of decrease in renal blood flow Recently Workman et al (6) have shown a relationship between the (3,4,51. renal hemodynamic changes following administration of intravascular contrast agents and renal prostacyclin production in the dog. Their experiments showed a transient reduction in arterio-venous levels of 6-keto PGF,a, the stable metabolite of prostacyclin and renal secretion rates of 6-keto PGFlcl which reached a nadir 30 minutes after infusion of contrast agent. This effect of contrast agents on renal prostacyclin synthesis could be due to a direct effect on prostacyclin synthesis or by increasing the synthesis of an inhibitor of prostacyclin. A well known naturally occurring inhibitor of prostacyclin is 15 hydroperoxy eicosatetraenoic acid (7,8). Since blood vessels have the capacity for both prostacyclin synthesis and hydroperoxide syntnesis, an increase in hydroperoxide formation could perhaps inhibit prostacyclin formation. We therefore asked the question if sodium iothalamate could increase hydroperoxide formation in an -_in vitro system using soybean lipoxygenase as a source of enzyme and arachidonate as a substrate. Materials
and rIethods
Arachidonate acid (CZ,,.~) was purchased from NuCheck Elysian, MN. 1-[14C] arachidonic acid (55 m Ci/minol) was obtained fro,n New England Nuclear, Boston Lipoxidase type IV, sodiurn iothalmate, D-mannitol and N-methyl glucamine ;fls . (rneglumine) were purchased from Sigma Chemical Co., St. Louis, No. 02 consumption was measured by a Clark type 02 electrode and lnodel 160 physiological gas analyser from Beckman, Palo Alto, CA., and coupled to a
MAY 1984VOL. 27 NO. 5
753
PROSTAGLANDINS
[ Na+
Iothalamote] mg/ml
Fig.
754
Dose response curve for soidum iothalamate 1. 100 percent is taken as control value.
on oxygen
consumption.
MAY 1984 VOL. 27 NO. 5
PROSTAGLANDINS
t
8
16
24
32
40
48
56 64 7; RETENT
N VOLUME
Fig. 2. HPLC tracing monitoring 235 nm, continous line and l'+C radioactivity, dotted line. 15 HPETE is 15-hydroperoxy eicosatetraenoic acid. control. B plus 100 mg/ml sodium iothalamate. C plus 100 mM mannitol! 3 100 mM mannitol plus 100 cog/ml sodium iothalamate.
MAY 1984 VOL. 27 NO. 5
755
PROSTAGLANDINS
strip chart recorder, Linear instruments, Irvine, CA. The polarographic cell had a total volulne of 1.2 ml. Polarographic experiments were carried out using soyoean lipoxiddse 400ng and substrate drachidonic acid 25 @g/ml in a total volume of 1 ml. Buffer used was 0.1 M borate pH 9.0. Na+ iothalarnate, D-mannitol and N-.nethyl glucamine were ddded at various concentration to the polarqraphic cell. For radiochemical experiments, arachidonate 25 Pg/ml and l-14C arachidonate 600,i)OO cpm were incubated in one ml of 0.1 i"lborate buffer pH 9.0 with 400 ng soybean lipoxidase for 5 minutes at roo,n temperature. III other rddiochemical experiments iothdlamate 100 ~my/ml and lnannitol 100 mM were added separately and then together to the incubations. The incubations were stopped with 300 ~1 [methanol and then acidified to pH 3.5 with O.lN HCl. To the methanolic extract 6 vols of diethyl ether and 4 vols of water were added and the ether phase evaporated to dryness and reconstituted in a snall volume of the mobile phase MeOH:HzO:Acetic acid (75:25:0.01). HPLC was carried out on a gaters system and UV absorption Imonitored at 235 nm. Radioactivity was Imeasured using a flow through detector obtained from Radiomatic Instruments (Tampa, FL). Results and Discussion increases in basal 02 consumption Fig. 1 shows the dose dependent obtained in the polarographic experiments. A 50'6 increase in 02 consumption was obtained with concentrations of iothalamate of 45 mg/:nl. The increase in 02 consumption was associated with an increase in hydroperoxide formation determined radiochemicdlly Fig. 2. Fig. 2A shows the production of 15-HPETt under basal condition as described in methods. Fig. 23 shows the increase in 15-HPETE formation when the incubation were carried out in the presence of 100 Ing/lnlisthalamate. '3f note is the almost complete consumption of substrate after 5 minutes of incubation. I*lannitolat 100 ml"1was previously shown to inhibit soybean lipoxygendse by greater than 90.X (9). Fig. 2C shows a similar experiment, indicating the inhibition of 15 OOH formation by mannitol. Fig. 20 shows that at a concentrdtion of 100 mM, mannitol also inhibited 15-HPETE formation in the presence of 100 mg/!nl sodium iothdlamate. Meglumine (Nmethyl glucamine) has structural similarities to mannitol Fig. 3. And is added to some preparations of iothalamate. megldmine like mannitol and other hexoses, was able to inhibit the increase in 02 consumption under basal conditions dnd also when stimulated with sodium iothlamate. Fici. 4 shows the dose dependent inhibition of iothalamate stimulated 02 consumption by N-methyl glucamine. Radiocontrast agents have been shown to decrease renal blood flow. Recently it has been suggested that the decrease in renal blood flow is related to a decreased renal production of 6-keto PGFla the stable dehydration product of PGIz. Stimulation of 15-HPETE ~01~1 d potentially decrease 6-keto PGFla release by inhibition of prostacyclin synthetase. The kidney has been shown to have both 12 and 15 lipoxygenase (10,11,12) pathways thus the enzyme are present for increased fatty acid imetabolism by a lipoxygenase when exposed to sodium iothalamate. If 75 ml of Conray 400 '@ (Malinkrodt) is adlninistered to a patient intravenously the final concentrations of the radio pharmaceutical (assuming a total plasma water of 2.5 Litres) will be about 20 Ing/:nlof sodium iothalamate. In the kidney the concentration is likely to exceed this considerably and thus will be in the range to significantly Imodulate renal lipoxygenases in cortex and medulla. The administration of contrast agents eg. sodium iothalamate directly into the arterial circulation may also produce much higher concentrations of contrast for example during
756
MAY 1984 VOL. 27 NO. 5
PROSTAGLANDINS
MEGLUMINE (N-methyl glucamine) CH,NHCH,
CH,OH
I HCOH
I HOCH
I
HOCH
HOCH
I
HCOH
HCOH
I HCOH
I HCOH
CH,OH
Fig. 3.
MANNITOL
CH,OH
Structures of N-methylglucamineand mannitol.
MAY 1984VOL. 27 NO. 5
757
PROSTAGLANDINS
E c
250 B--w
0
0 150 o\o Control
100% 50
66 mg/ml _
132 mg/ml
Iothalamate
Iotholamate 132mg/ml
1,--1---_ 75
-eV N-methyl
Fig.
100
150
glucamine mM
Effects of sodium iothalamate and sodium iothalamate plus 4. methylglucamine on basal oxygen consumption by soybean lipoxygenase.
renal angiography and significantly indirectly PGI- production.
modify
renal
lipoxygenase
products
N-
and
Acknowledgements The work is supported 32. Dr. Xorrison is an Association.
758
by NIH research grants PO1 Air109976and AM30542established investigator of the American Heart
MAY
1984 VOL. 27 NO. 5
PROSTAGLANDINS
REFERENCES 1.
Bennett, W.bl. Lult, F. Porter, G.A. Pathogenesis of renal failure due to aminoglycosides and contrast media used in roentgenography. Am. J. Med. -1980: 69: 767-774. -
2.
Byrd, L. Sherman, R.L. clinical and pathological
3.
Caldicott, W.J.H., Hollenberg, N.K., Abrams, H.L.: Characteristics of resoonse of renal vascular bed to contrast media: evidence for vasbconstriction induced by renin-angiotenoin system, Invest. Radiol. 1970 -5: 539-547.
4.
Talner, L.B., Davidson, A.J.: Renal hemodynamic media. Invest. Radiol. 1968: -3: 301-309.
5.
Larson, T.S., Hudson, K., Mertz, J.I., Rornero, J.C. and Knox, F.G. vasoconstrictive response to contrast medium. J. Lab. Clin. Med. 101: 385-391
6.
Workman, R.J., Shaff, M.I., Jackson, R.V., Diggs, M.D., Frazier, M.G., Briscoe. W.J. Relationship of Renal hemodvnamic and functional chances following intravascular contrast to the renin-angotension system and reial prostaglandin in the dog. Invest. Radiol. 1983: -18: 160-166.
7.
Moncada, S., Gryglewski, R.J., Bunting, S. and Vane, J.R. A lipid peroxide inhibits the enzyme in blood vessels microsomes that generate from prostaglandin endoperoxides the substance (Prostaglandin X) which prevents platelet aggregation. Prostglandins 1976: -12: 715-737.
a.
Gryglewski, R.J., Bunting, S., Moncada, J., Flower, R.J., and Vane, J.R. Arterial walls are protected aqainst deoosition of platelet thromibi bv a (Prostajlandin X)- which they make' from prostaglanhin substance endoperoxides. Prostaglandins 1976: -12: 685-713.
9.
Morrison, A.R., Winokur, Lipoxygenase by Mannitol.
Radiocontrast induced acute renal failure: review. -Medicine: 1979: 58: 270-279.
effects
T.S., Wendell Brown: Inhibition BBRC 1982: 108: 1757-1762.
of
of
A
contrast
Renal 1983:
Soybean
hlorrison, A.R. Regional 10. Winokur, T.S., synthesis of monohydroxy eicosanoids by the kidney. J. Biol. Chem. 1981: 256: 10221-10223. 11. Sraer, J. Rigaud, M., Bens, M., Rabinovitch, H., Ardaillou. Metabolism of acid in the liooxvqenase oathwav in human and murine arachidonic J. Biol. Chem. 198i: $& 4325i4330.‘ glomeruli. 12. Jim, K., Hassid, A., Sung, F., Dunn, M.J. Lipoxygenase activity in rat kidney glomeruli, glomerular epithetial cells and cortical tubules. J. 9iol. Chem. 1982: __257: 10294-10299.
Editor: John E. Pike 10=24=83 Received: Accepted: 4-6-84
MAY 1984 VOL. 27 NO. 5
759