Glucocorticoid-treatment does not influence the synthesis of thromboxane B2 and bicyclo-PGE2 in humans

Glucocorticoid-Treatment Does Not Influence the Synthesis of Thromboxane B2 And Bicycle-PGE2 in Humans H. SINZINGER”,

I. VIRGOLINI,

F. RAUSCHA

and P. FITSCHA

Atherosclerosis Research Group (ASF) Vienna and Atherosclerosis and Thrombosis Research Group (ATK) of the Austrian Academy of Sciences, Schwarzspanierstr. 17, A-1090 Vienna, Austria. I* Reprint requests to H.S.).

AbstractIt has been reported that the anti-inflammatory action of glucocorticoids is due to the inhibition of phospholipases. Consequently, after high-dose steroid treatment in humans a decrease in cyclooxygenase products should be expected. In 15 patients (10 males, 5 females, 29-62 y) undergoing 6-methyl-prednisolone-treatment (40-80 mg daily) for various clinical reasons and in 5 healthy volunteers (4 male, 1 female, 28-37 y) receiving 500 mg 6-methyl-prednisolone daily for 3 days plasma- and serum-thromboxane B2 (TXB*), as well as bicycle-prostaglandin E2 (bicycle-PGE& were monitored over 3 weeks. In the entire follow-up period, however, no significant change in either serum- or plasma-TXB2 or bicycle-PGE2 could be measured in either, patients and volunteers, under glucocorticoid-treatment. These findings indicate that even high-dose glucocorticoidtreatment does not affect the serum- and plasma-metabolites of the eicosanoids examined. It is concluded that in humans a significant inhibition of phospholipases by glucocorticoids and subsequently reduced formation of cyclooxygenase products seems to be rather unlikely.

Introduction There is considerable evidence linking the generation of prostaglandins with inflammation, pain and fever (1). Several other fatty acid products such as 12-hydroxy-5,8,14-eicosatetraenoic acid (2) or malondialdehyde (3) may also exert pro-inflammatory effects. Whereas aspirin-like drugs block the generation of cyclooxygenase products (4), the anti-inflammatory glucocorticoids may prevent the biosynthesis of the whole

cascade of lipid mediators. Dexamethasone is known to inhibit the synthesis of prostaglandins by inhibiting the activation of phospholipase AZ (5-7) which cleaves arachidonic acid. the precursor of prostaglandins from membrane phospholipids. This phospholipase-inhibition is caused by macrocortin (lipocortin), a polypeptide selectively released from leucocytes (8-10). The inhibition of the phospholipase-activity should consequently result in a decreased 135

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PROSTAGLANDINS

production of arachidonic acid metabolites. In accordance with this suggestion it has been stated that histamine- or 5-hydroxy-tryptaminestimulated TXAz-release is inhibited by antiinflammatory steroids in the rat lung (11) and that macrocortin (lipocortin) is responsible for the steroid-induced inhibition of TXA?-release in the perfused rat lung (9). On the other hand glucocorticoids failed to modify the concentrations of prostaglandin urinary metabolites in rabbits (12) and in joint fluid from patients with inflammatory arthritis (13). In a number of patients undergoing glucocorticoid-therapy for various reasons, we always found normal or even increased prostaglandin-values in plasma or serum too. We therefore addressed the question as to whether glucocorticoids may be able to interfere at this enzymatic step by measuring serum- and plasma-TXB?, the stable TXAzderivative (14), as well as the bicycle- PGE?metabolite in plasma in patients and in a small group of healthy volunteers before and during the initial phase of high-dose glucocorticoidtreatment. Subjects and Methods Fifteen patients (10 males, 5 females, 29-62 y) and 5 healthy volunteers (physicians; 4 males, 1 female 28-37 y) were involved in the study. The patients received high-dose 6-methyl-prednisolone-therapy (Urbasan, Hoechst, Frankfurt, FRG) for various clinical reasons. The therapy was started with a daily dose of 40 to 80 mg, was continuously reduced and amounted 20 to 40 mg daily after a 3 weeks treatment-period. The healthy volunteers received 500 mg 6-methylprednisolone daily for 3 days only. The patients and volunteers were monitored for 3 weeks.

LEUKOTRIENES

AND ESSENTIAL FATI?’

acid, Bayer, Leverkusen, enzyme cyclooxygenase.

FRG)

ACIDS

to block the

R/A for plnsrrm-TXB?

After sedimentation at 4°C for 10 minutes plasma was obtained by centrifugation at 1000 X g for 10 minutes at 4°C. Plasma-samples were divided into aliquots, stored at -70°C and radioimmunoassayed within 2 weeks. The RIA was performed in non-extracted samples using a specific antibody (kindly supplied by B. Peskar, M.D., Prof., Dept. of Pharmacology, Bochum, FRG) and the double antibody technique for the separation of free and antibody-bound ligand (15). 0.5 ml of the samples were incubated with 0.4 ml normal rabbit plasma, 0.5 ml [3H]TXB? (128 Ci/mmol, NEN, Dreieich, FRG. 4000 cpmO.5 ml 10 mM tris- HCI-buffer, pH 7.4, containing 1 mg/ml gelatine) and 0.1 ml of antibody (diluted approximately to bind 40% of label in the absence of inhibitors) at 4°C for 2 hours. For the separation of free and antibody-bound ligand, 0.1 ml antibody against rabbit gamma-globuline from the goat (OTOP 15/16; Behring, Marburg, FRG) were added followed by an overnightincubation at 4°C. On the next day the antigenantibody-complex was sedimented by centrifugation (1500 x g, 60 minutes, 4°C). The supernatant was removed and the precipitate washed twice with 0.5 ml ice-cold 0.9% NaCI, taken up in 10 ml Pica-fluor 30 (Packard, Warrenville, Illinois, USA) and the radioactivity was measured in a liquid scintillation counter for 10 minutes. At 25 pg/ml plasma the interassay variation coefficient amounts 6.5%, the intraassay variation coefficient 4.3%, values are given in pg TXB2/ml plasma. RIA for serum-TXB-

Blood-withdrawal

Blood was drawn after an overnight fast from a cubital vein without venous occlusion via an 1.2 mm diameter needle after a 30 minutes rest. Blood withdrawal was done before the first drugdose (prevalue) and on the 2nd, 3rd. 4th, 7th, 14th, and 21st days thereafter. The sampling for the determination of plasma metabolites (TXB2 and bicylo-PGEZ, 9 ml whole blood for both) was done in precooled plastic syringes, for the determination of serum-TXB:! in prewarmed syringes (1.8 ml whole blood). Blood was anticoagulated 1:lO with 2% sodium EDTA and 1 mg/ml aspisol (lysil-acetylsalicyhc

After an incubation for exactly 60 minutes at 37°C in a water bath, serum was obtained by centrifugation for 10 minutes at 1000 X g at 4°C. The serum-samples were divided into aliquots, stored at 70°C and radioimmunoassayed within 2 weeks. The RIA was performed in non-extracted samples: the ingredients were obtained from Amersham International Buckinghamshire, UK. 0.1 ml serum were mixed with 0.1 ml [3H]TXB3 (1 &i/l0 ml) and with 0.1 ml TXBZ-antiserum. The reaction mixture was incubated for 60 minutes at 37°C and then at 4°C for 16 hours. The unbound TXB:! was separated by means of

GLUCOCORTICOID

137

TREATMENT

dextran-coated charcoal. After incubation at 0°C for 10 minutes the antigen-antibody-complex was sedimented (1000 x g, 10 minutes, 4”C), the supernatant was taken up in 10 ml scintillation fluid and the radioactivity counted in a liquid scintillation counter for 10 minutes. At 195 ng/ml plasma, the interassay variation coefficient amounts to 5.9%, the intraassay variation coefficient 4.1%; values are given in ng TXB2/ml serum. RIA for bicycle-PGEZ

PGE? is rapidly metabolized to 15-keto-13,14dihydro-PGEz, which undergoes subsequent chemical reactions in the blood stream, leading to 15 keto-13,14-dihydro-PGAZ, which is partly covalently bound to albumin. By incubation of plasma samples at pH 10 to 11, all metabolites of PGE;! are converted to the same compound 11-deoxy-13,14-dihydro-15 keto-llg-16E-cycloPGE?, i.e. bicycle-PGE (16). Antibodies raised to this stable bicycle-derivate (17) were used to perform a RIA (kindly supplied by Prof. Dr. B. A. Peskar). This approach has circumvented hazards of directly measuring a metabolite that is chemically unstable. Unextracted samples of peripheral venous blood were assayed using the double antibody technique. Standard curves were prepared with unextracted prostaglandinfree plasma to correct for non-specific protein binding (sensitivity 10 pg/ml, maximum binding at 70 pg/ml; cross-reactivity to the corresponding bicycle-PGEl about 25%, to other prostaglandins less than 1%). At 112 pg/ml the intraassay coefficient of variation was 5.2% and the interassay coefficient of variation was 8.1%. Values are given in pg bicycle-PGEz/ml plasma. Statistical analysis

The values are given as x * SD; calculation for significance was performed by means of Student’s t-test.

P!wl

30_

20

IO-‘\

O-1,

P

1

I

1

2

weeks -

I

3

Fig 1. Three weeks follow-up of plasma-TXB2-values in 6methyl-prednisolone treated patients [m] and healthy volunteers [a].

nglml 260

0 P

2

1

3

weeksFig 2. Three weeks follow-up of serum-TXB+alues in 6 methyl-prednisolone treated patients [m] and healthy volunteers [a].

m/ml 60-

40-

Results

The mean prevalue for plasma-TXB;? was significantly (p < 0.01) higher in the patients’ group as compared to the healthy volunteers (19 f 5 vs 12 f 4 pg/ml, Fig 1). The mean prevalue for serum-TXBz, however, was not different between the patients and the volunteers (204 f 29 vs 199 + 15 ng/ml, Fig 2). The mean prevalue for plasma bicycle-PGEZ in the patients’ group amounted 37 + 8 pg/ml; in the healthy volun-

20-

0',

P

I

2

1

1

3

weeks Fii 3. Three weeks follow-up of bicycle-PGE, in 6-methylprednisolone treated patients [m] and healthy volunteers

[*I.

138

PROSTAGLANDINS LEUKOTRIENES AND ESSENTIAL FATTY

teers the actual value was significantly (p < 0.01) lower (29 * 4 pg/ml, Fig 3). During the initial monitoring phase after high-dose glucocorticoidtreatment for up to 3 weeks no effect on either serum- and plasma- TXB2 or bicycle-PGE2 could be found in humans.

References I. Vane J R. Inhibition of prostaglandin

2.

3.

Discussion

Glucocorticoids do not have any anti-cyclooxygenase activity but exert their action by preventing the release of the fatty acid substrates required for prostaglandin-biosynthesis (.5,7,11,18) from phospholipids. However, these studies all have been performed in vitro, either using cultured cells or by means of perfused organ-systems. On the contrary, in vivo, (19) after experimental dexamethasone-treatment (up to 0.4 mg/kg body weight) the ability of fetal rat lung homogenate to convert [ 14C]-arachidonic acid to 6-0x0-PGFi,, the stable PGIz-derivative, and PGE2 was enhanced. These data suggest in addition to accelerating surfactant phospholipid synthesis (20,21) and differentiation of connective tissue (22), that dexamethasone also may accelerate the maturation of the enzymes involved in prostaglandin-synthesis. Even after treatment with the rather high-dose of 12 mg/kg body-weight, dexamethasone stimulated the conversion of [ 14C]-arachidonic acid to 6-ketoPGFi, in fetal or maternal rat lung homogenate (23) indicating that the dose of dexamethasone probably did not reach the concentration required to inhibit prostaglandin-synthesis in organs such as the lung. On the other hand dexamethasone adminstered at a rate of 1 mg/kg/day in rabbits (12) did not affect the basal urinary excretion rates of 6 different cyclooxygenase products confirming our present data that after treatment with 6-methyl-prednisolone in both, patients and volunteers, the levels of plasma- and serum-TXB;! as well as bicycloPGE? are not changed by glucocorticoids. We, therefore, assume that, in humans glucocorticoids do not exert an in vivo inhibitory effect on the phospholipase A2-activity at the dose-ranges and treatment-period studied.

4.

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Acknowledgements We gratefully acknowledge the valuable help by Sonja Reiter in the performance of the radio-immunoassays.

ACIDS

16.

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