- Email: [email protected]
Increased plasma adenosine concentrations and the severity of preeclampsia
Increased Plasma Adenosine Concentrations and the Severity of Preeclampsia Yoshio Yoneyama, MD, Shunji Suzuki, MD, Rintaro Sawa, MD, Koichi Yoneyama, MD, Gordon G. Power, MD, and Tsutomu Araki, MD OBJECTIVE: To investigate the relationship between changes in plasma adenosine and the severity of preeclampsia, and norepinephrine and tumor necrosis factor–␣ concentrations. METHODS: Plasma concentrations of adenosine, norepinephrine, and tumor necrosis factor–␣ relating to the pathogenesis of preeclampsia were measured in women with mild (n ⴝ 21) and severe (n ⴝ 21) preeclampsia and normal pregnancies (n ⴝ 21), matched for age, gestational age, and parity, in the third trimester of pregnancy. We then evaluated the relationships among plasma adenosine, norepinephrine, tumor necrosis factor–␣ concentrations, and the severity of preeclampsia. RESULTS: Mean plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations were significantly higher in women with mild and severe preeclampsia than in normal control subjects (P < .05). In women with preeclampsia, plasma adenosine concentrations increased according to the severity of preeclampsia (0.60 ⴞ 0.03 mol/L and 0.72 ⴞ 0.03 mol/L, respectively, versus 0.41 ⴞ 0.03 mol/L for normal subjects), which correlated with increases of norepinephrine and tumor necrosis factor–␣ concentrations (r ⴝ .58, P < .05; r ⴝ .49, P < .05, respectively). In preeclampsia, norepinephrine concentration also correlated with maternal blood pressure (r ⴝ .50, P < .05). CONCLUSION: Adenosine is an established suppressor of the effects of norepinephrine and tumor necrosis factor–␣. The increased plasma concentrations of adenosine in preeclampsia might serve to counteract further progression of the complication. (Obstet Gynecol 2002;100:1266 –70. © 2002 by The American College of Obstetricians and Gynecologists.)
Preeclampsia is characterized by reduced organ perfusion and endothelial cell dysfunction,1 and several neurohumoral factors are reported to deteriorate in preFrom the Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan; and the Center for Perinatal Biology, Loma Linda University, Loma Linda, California. Supported by a Grant-in-Aid (No. 12470349) from the Ministry of Education, Culture, Sports, Science and Technology.
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eclampsia.2,3 Plasma norepinephrine and other catecholamines are increased in preeclampsia, and there is increased sensitivity to infused norepinephrine.2 There are also elevated concentrations of cytokines such as tumor necrosis factor–␣ in plasma and amniotic fluid in severe preeclampsia.3 Increased plasma concentrations and inappropriate responses to these and other unknown agents have been proposed to be involved in the pathogenesis of preeclampsia.4,5 Adenosine has many important physiologic roles, a number of which directly oppose the actions of excessive concentrations of catecholamines and cytokines.6,7 Maternal plasma adenosine concentration is increased in both normal pregnancy8 and preeclampsia,9,10 and the increase is greater in preeclampsia than in normal pregnancy. There has been relatively little study of the mechanisms that influence plasma levels of adenosine and its various physiologic and pathophysiologic roles in preeclampsia.9,10 Because adenosine rapidly attenuates the excessive adrenergic effects6 and the oxidative burst of tumor necrosis factor–␣,7 we hypothesized that the elevation of plasma adenosine concentration in preeclampsia is an endogenous compensatory response that diminishes the deterioration of preeclampsia induced by excessive release of catecholamines and tumor necrosis factor–␣. To seek such support we measured plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations in 21 normal pregnant women, 21 women with mild preeclampsia, and 21 with severe preeclampsia. We then evaluated the relationship among plasma adenosine, norepinephrine, tumor necrosis factor–␣ concentrations, and the severity of preeclampsia.
MATERIALS AND METHODS Pregnant women with mild or severe preeclampsia were recruited consecutively between February 1998 and October 2001 at the Nippon Medical School Hospital in Tokyo. Mild preeclampsia was defined as blood pres-
VOL. 100, NO. 6, DECEMBER 2002 © 2002 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
0029-7844/02/$22.00 PII S0029-7844(02)02247-0
sure greater than 140/90 mm Hg on two or more occasions at least 6 hours apart in women during bed rest and proteinuria greater than 300 mg of protein a day. Severe preeclampsia was defined as blood pressure greater than 160/110 mm Hg on two or more occasions at least 6 hours apart in women during bed rest and proteinuria greater than 500 mg of protein a day. Inclusion criteria were mild or severe preeclamptic women whose blood pressure had been normal during the first 20 weeks’ gestation and with no history of cardiovascular disease, primary hypertension, connective tissue disease, diabetes mellitus or renal disease, or medication with antihypertensives or aspirin; singleton fetus; no fetal structural anomaly; normal response to glucose tolerance testing; and no evidence of recent infections (eg, rubella, toxoplasmosis, hepatitis B and C, cytomegalovirus, syphilis). Twenty-four women with mild preeclampsia and 22 women with severe preeclampsia meeting the eligibility criteria volunteered for the study, and 21 in each group gave informed consent after being advised that they would not benefit directly from this research. Pregnant women without preeclampsia, matched for maternal age (within 0.9 years), parity, and gestational age (within 6 days), were selected to serve as a control group. Twentythree patients were offered entry. Twenty-two of these women satisfied the eligibility criteria, and 21 gave informed consent and constituted the control group. The study was approved by the Ethics Committee of Nippon Medical School Hospital. All women were fully informed of the aim of the study, and informed consent was obtained from all participants. One hour before the study, the women entered a quiet room, and were placed at bed rest in a semirecumbent position. An indwelling 18-gauge catheter (Medikit Co., Tokyo, Japan) was placed in an antecubital vein. After the 1-hour period of accomodation, blood was taken for measurement of plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations. Preparations of samples for these measurements were as described.11–13 The plasma adenosine level was assayed with a modified high performance liquid chromatographic method with photodiode-array detection.11 The detection limit was 5 nmol/L, and the intra- and interassay coefficients of variation were 5.7% and 9.8%, respectively. The plasma norepinephrine level was determined using a high performance liquid chromatographic method with electrochemical detection.12 The detection limit was 10 pg/mL, and the intra- and interassay coefficients of variation were 6.2% and 9.1%, respectively. The plasma tumor necrosis factor–␣ level was measured with a specific enzyme-linked immunoassay.13
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The detection limit was 5 pg/mL, and the intra- and interassay coefficients of variation were 4.9% and 8.2%, respectively. Data are presented as means ⫾ standard errors of the mean. Significant differences between groups for continuous variables were evaluated by analysis of variance. If overall significance was observed, then individual group means were compared by the post hoc multiple comparison test with Bonferroni correction. Statistical analyses were also performed using 2 tests as appropriate. Correlations between the variables were evaluated using linear regression analysis with the least-squares method. Differences were considered significant at P ⬍ .05. Linear regression analysis was performed by the leastsquares method. Differences were considered significant at P ⬍ .05.
RESULTS Clinical characteristics of the study population are given in Table 1. In the present study, all subjects were married Japanese women of middle socioeconomic status who were nonsmokers. None of the patients developed eclampsia or the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome. Mean plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations are shown in Table 2. Mean plasma adenosine concentrations in women with preeclampsia were significantly higher than those in women with normal pregnancies, and increased according to the severity of preeclampsia (Figure 1). In women with preeclampsia, plasma adenosine concentrations significantly correlated with norepinephrine (r ⫽ .58, P ⬍ .05) and tumor necrosis factor–␣ (r ⫽ .49, P ⬍ .05); however, the correlations between the increase of plasma adenosine and mean blood pressure or proteinuria were not significant (r ⫽ .23, P ⫽ .18; r ⫽ .11, P ⫽ .72, respectively). Mean plasma norepinephrine and tumor necrosis factor–␣ concentrations were significantly higher in women with preeclampsia than in women with normal pregnancies, and these substances also increased according to the severity of preeclampsia. Plasma norepinephrine also correlated positively with mean blood pressure (r ⫽ .50, P ⬍ .05), but not with proteinuria (r ⫽ .25, P ⫽ .44), and there were no direct correlations between plasma tumor necrosis factor–␣ and mean blood pressure (r ⫽ .38, P ⫽ .52) or proteinuria (r ⫽ .69, P ⫽ .81) in women with preeclampsia. The significant relationships observed in women with preeclampsia did not exist in healthy women with normal pregnancies.
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Table 1. Demographic Data
Age (y) Nulliparous (n) Proteinuria (mg/d) Blood pressure (mm Hg) Systolic Diastolic Gestational age (wk) At study At delivery Cesarean delivery Birth weight (g) Birth weight ⬍ fifth percentile (n)
Normal pregnancy (n ⫽ 21)
Mild preeclampsia (n ⫽ 21)
Severe preeclampsia (n ⫽ 21)
32.2 ⫾ 1.0 13 (62%) —
32.8 ⫾ 1.3 13 (62%) 478 ⫾ 26
31.9 ⫾ 1.2 13 (62%) 759 ⫾ 44
115 ⫾ 2 70 ⫾ 2
155 ⫾ 2* 98 ⫾ 2*
182 ⫾ 5† 112 ⫾ 4†
30.2 ⫾ 0.6 39.1 ⫾ 0.2 2 (9%) 3104 ⫾ 67 0 (0%)
30.9 ⫾ 0.6 35.7 ⫾ 0.8* 6 (29%)* 2112 ⫾ 195* 2 (9%)
30.2 ⫾ 0.6 32.9 ⫾ 1.1† 11 (52%)† 1502 ⫾ 149† 6 (29%)
Data are presented as means ⫾ SEMs or numbers. * Significantly different from normal pregnancy (P ⬍ .05). † Significantly different from normal pregnancy and mild preeclampsia (P ⬍ .05).
DISCUSSION A pronounced elevation of plasma adenosine concentration was found in women with preeclampsia, which correlated with the severity of preeclampsia, and increases in adenosine were accompanied by increases in plasma norepinephrine and tumor necrosis factor–␣ concentrations. A protective action of adenosine has become recognized, notably in the heart14 and in newborns after vaginal delivery.15 Further, we have demonstrated that adenosine tends to prevent excessive platelet activation9 and regulate the T-helper 1/T-helper 2 imbalance10 in preeclampsia. However, a protective action for adenosine in countering the progression of preeclampsia has not previously been proposed. The progressive increases in adenosine with respect to increases in norepinephrine, tumor necrosis factor–␣, and, indirectly, blood pressure observed in the present study highly support this general concept. In the present study, a significant elevation of plasma adenosine concentration was observed in women with preeclampsia, which correlated with the severity of preeclampsia. The elevation could reasonably be predicted because local tissue hypoxia and the formation of micro-
thrombosis are well-established causes for the release of adenosine. These events occur frequently in preeclampsia, and it is reasonable to conclude that plasma adenosine is elevated in response to these events. The elevated adenosine levels observed in women with mild and severe preeclampsia in the present study are known to cause not cytotoxic effects, but near maximal vasodilatation in many vascular beds16 and to suppress metabolic activity.17 In light of the tendency of adenosine to redress local oxygen supply and use imbalance in preeclampsia, one may speculate that an elevation of adenosine in women with preeclampsia protects themselves and the fetuses by helping them to adapt to unfavorable conditions in preeclampsia. Further, the increased adenosine in preeclampsia might compensate for and tend to buffer further excessive platelet activation to maintain vascular integrity,9 and counteracts further imbalance of the T-helper 1/T-helper 2 ratio.10 From that viewpoint, an elevation of plasma adenosine in preeclampsia according to the severity of preeclampsia might compensate for and tend to counteract the further progression of preeclampsia. In the present study, plasma norepinephrine signifi-
Table 2. Plasma Adenosine, Norepinephrine, and Tumor Necrosis Factor–␣ Concentrations in Normal Pregnancy and Mild and Severe Preeclampsia
Adenosine (mol/L) Norepinephrine (pg/mL) Tumor necrosis factor–␣ (pg/mL)
Normal pregnancy (n ⫽ 21)
Mild preeclampsia (n ⫽ 21)
Severe preeclampsia (n ⫽ 21)
0.41 ⫾ 0.03 132 ⫾ 18 2.0 ⫾ 0.1
0.60 ⫾ 0.03* 238 ⫾ 25* 2.9 ⫾ 0.3*
0.72 ⫾ 0.03† 345 ⫾ 36† 3.7 ⫾ 0.4†
Data are presented as means ⫾ SEMs. * Significantly different from normal pregnancy (P ⬍ .05). † Significantly different from normal pregnancy and mild preeclampsia (P ⬍ .05).
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Figure 1. Changes in plasma adenosine concentrations in women with normal pregnancies and women with preeclampsia. Yoneyama. Adenosine in Preeclampsia. Obstet Gynecol 2002.
cantly increased in women with preeclampsia, which also correlated with the severity of preeclampsia. An increase in plasma norepinephrine also correlated positively with mean blood pressure. These findings are in agreement with previous studies.18,19 Because endogenous increases of norepinephrine augment the activity of ecto-5⬘-nucleotidase, the enzyme responsible for adenosine production,20 the increased plasma norepinephrine may be linked to increases in adenosine production. Indeed, such a linkage is supported by results of this study showing a direct correlation between plasma levels of norepinephrine and adenosine. A similar positive correlation has been reported in patients with chronic heart failure14 and newborns after vaginal delivery,15 conditions in which catecholamines are elevated, and thus adenosine may counteract catecholamine-induced effects in a number of biologic systems and as a general defense mechanism. Overall the data permit the conclusion that excessive norepinephrine increase worsens preeclampsia and that adenosine may attenuate the stimulatory effects of norepinephrine.6 In terms of systems
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analysis, adenosine may contribute to negative feedback against the progressive positive loop between norepinephrine and preeclampsia. In the present study, plasma tumor necrosis factor–␣ increased significantly in preeclamptic women, which is in agreement with a previous study.3 This increase was directly correlated with the increase of adenosine. These results suggest that tumor necrosis factor–␣ may be a direct stimulus for plasma adenosine production in preeclampsia. Tumor necrosis factor–␣ increases transcriptional and protein levels of ecto-5⬘-nucleotidase21 and may thereby in part contribute to increasing the production of adenosine in preeclampsia. Tumor necrosis factor–␣ is biologically active and associated with immune disorders, and that may be causally related to the pathogenesis and the progression of preeclampsia.22 Increased levels of tumor necrosis factor–␣ elicit activation of the immune system against the fetal allograft, and adenosine suppresses immune-triggered cytokine production,23 and by this means also, in addition to its anticatecholamine actions, it may be an endogenous compensatory mechanism against the progression of preeclampsia. In summary, we conclude that increases in plasma adenosine in preeclampsia occur in parallel with elevations of catecholamines and tumor necrosis factor–␣. It seems likely that adenosine participates in negative feedback loops that redress elevations of these stimuli, which may eventually worsen preeclampsia. Of course, other factors may be involved in the pathogenesis and progression of preeclampsia. Further, adenosine kinase, adenosine deaminase, and platelet activation may also largely contribute to the regulation of plasma adenosine concentrations in preeclampsia. Further investigation is needed to clarify the possible mechanisms and roles of an increase of plasma adenosine concentrations in women with preeclampsia. REFERENCES 1. Roberts JM, Redman CWG. Pre-eclampsia: More than pregnancy-induced hypertension. Lancet 1993;341: 1447–51. 2. Øian P, Kjeldsen SE, Eide I, Maltau JM. Increased arterial catecholamines in preeclampsia. Acta Obstet Gynecol Scand 1986;65:613– 6. 3. Kupferminc MJ, Peaceman AM, Aderka D, Wigton TR, Rehnberg KA, Socol ML. Tumor necrosis factor-␣ is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol 1994;170: 1752–9. 4. Rogers GM, Taylor RN, Roberts JM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. Am J Obstet Gynecol 1988;159:908 –14.
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5. Schobel HP, Fischer T, Heuszer K, Geiger H, Schmieder RE. Preeclampsia—a state of sympathetic overactivity. N Engl J Med 1996;335:1480 –5. 6. Dobson JG Jr. Mechanism of adenosine inhibition of cathecholamine-induced responses in heart. Circ Res 1983;52: 151– 60. 7. Barnes CR, Mandell GL, Carper HT, Luong S, Sullivan W. Adenosine modulation of tumor necrosis factor-␣induced neutrophil activation. Biochem Pharmacol 1995; 50:1851–7. 8. Yoneyama Y, Suzuki S, Sawa R, Otsubo Y, Power GG, Araki T. Plasma adenosine levels increase in women with normal pregnancies. Am J Obstet Gynecol 2000;182: 1200 –3. 9. Yoneyama Y, Suzuki S, Kiyokawa Y, Power GG, Araki T. Plasma adenosine level and P-selectin expression on platelet in preeclampsia. Obstet Gynecol 2001;97:366 –70. 10. Yoneyama Y, Suzuki S, Sawa R, Yoneyama K, Power GG, Araki T. Relation between adenosine and T-helper 1/Thelper 2 imbalance in women with preeclampsia. Obstet Gynecol 2002;99:641– 6. 11. Maguire MH, Szabo I, Valko IE, Finley BE, Bennett T1. Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversedphase high- performance liquid chromatography with photodiode-array detection and on-line validation of peak purity. J Chromatogr B Biomed Sci Appl 1998;707: 33– 41. 12. Hjemdahl P, Daleskog M, Kahn T. Determination of plasma catecholamines by high-performance liquid chromatography with electrochemical detection. Life Sci 1979; 25:131–5. 13. Kwitakowski D, Hill AVS, Sambou I, Twumasi P, Castracane J, Manogue KR, et al. TNF concentration in fetal cerebral, non-fetal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 1990;336:1201– 4. 14. Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M. Plasma adenosine levels increase in patients with choronic heart failure. Circulation 1997;95:1363–5.
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15. Irestedt L, Dahlin I, Hertzberg T, Sollevi A, Lagercrantz H. Adenosine concentration in umbilical cord blood of newborn infants after vaginal delivery and cesarean section. Pediatr Res 1989;26:106 – 8. 16. Berne RM. Adenosine: An important physiological regulator. New Physiol Sci 1986;1:163–7. 17. Bardenhueser H, Schrader J. Supply-to-demand ratio for oxygen determines formation of adenosine by the heart. Am J Physiol 1986;250:173– 80. 18. Tinkanen H, Rorarius M, Metsa¨-Katela¨ T. Catecholamine concentrations in venous plasma and cerebrospinal fluid in normal and complicated pregnancy. Gynecol Obstet Invest 1993;35:7–11. 19. Kjeldsen SE, Eide I, Aakesson I, Øian P, Maltau JM, Gjesdal K. Increased arterial adrenaline is highly correlated to blood pressure and in vivo platelet function in pre-eclampsia. J Hypertens 1985;3:S93–5. 20. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Sato H, et al. ␣1–adrenoceptor activation increases ecto-5-nucleotidase activity and adenosine release in rat cardiomyocytes by activating protein kinase C. Circulation 1995;91:2226 –34. 21. Savic V, Stefanovic V, Ardaillou N, Ardaillou R. Induction of ecto-5⬘-nucleotidase of rat cultured mesangial cells by interleukin-1 and tumor necrosis factor-␣. Immunology 1990;70:321– 6. 22. Dekker GA, Sibai BM. The immunology of preeclampsia [review]. Semin Perinatol 1999;23:24 –33. 23. Zidek Z. Adenosine-cyclic AMP pathways and cytokine expression. Eur Cytokine Netw 1999;10:319 –28. Address reprint requests to: Yoshio Yoneyama, MD, Nippon Medical School, Department of Obstetrics and Gynecology, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113-8603, Japan; E-mail: [email protected]. Received January 30, 2002. Received in revised form May 6, 2002. Accepted June 19, 2002.
OBSTETRICS & GYNECOLOGY
Preeclampsia is characterized by reduced organ perfusion and endothelial cell dysfunction,1 and several neurohumoral factors are reported to deteriorate in preFrom the Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan; and the Center for Perinatal Biology, Loma Linda University, Loma Linda, California. Supported by a Grant-in-Aid (No. 12470349) from the Ministry of Education, Culture, Sports, Science and Technology.
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eclampsia.2,3 Plasma norepinephrine and other catecholamines are increased in preeclampsia, and there is increased sensitivity to infused norepinephrine.2 There are also elevated concentrations of cytokines such as tumor necrosis factor–␣ in plasma and amniotic fluid in severe preeclampsia.3 Increased plasma concentrations and inappropriate responses to these and other unknown agents have been proposed to be involved in the pathogenesis of preeclampsia.4,5 Adenosine has many important physiologic roles, a number of which directly oppose the actions of excessive concentrations of catecholamines and cytokines.6,7 Maternal plasma adenosine concentration is increased in both normal pregnancy8 and preeclampsia,9,10 and the increase is greater in preeclampsia than in normal pregnancy. There has been relatively little study of the mechanisms that influence plasma levels of adenosine and its various physiologic and pathophysiologic roles in preeclampsia.9,10 Because adenosine rapidly attenuates the excessive adrenergic effects6 and the oxidative burst of tumor necrosis factor–␣,7 we hypothesized that the elevation of plasma adenosine concentration in preeclampsia is an endogenous compensatory response that diminishes the deterioration of preeclampsia induced by excessive release of catecholamines and tumor necrosis factor–␣. To seek such support we measured plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations in 21 normal pregnant women, 21 women with mild preeclampsia, and 21 with severe preeclampsia. We then evaluated the relationship among plasma adenosine, norepinephrine, tumor necrosis factor–␣ concentrations, and the severity of preeclampsia.
MATERIALS AND METHODS Pregnant women with mild or severe preeclampsia were recruited consecutively between February 1998 and October 2001 at the Nippon Medical School Hospital in Tokyo. Mild preeclampsia was defined as blood pres-
VOL. 100, NO. 6, DECEMBER 2002 © 2002 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.
0029-7844/02/$22.00 PII S0029-7844(02)02247-0
sure greater than 140/90 mm Hg on two or more occasions at least 6 hours apart in women during bed rest and proteinuria greater than 300 mg of protein a day. Severe preeclampsia was defined as blood pressure greater than 160/110 mm Hg on two or more occasions at least 6 hours apart in women during bed rest and proteinuria greater than 500 mg of protein a day. Inclusion criteria were mild or severe preeclamptic women whose blood pressure had been normal during the first 20 weeks’ gestation and with no history of cardiovascular disease, primary hypertension, connective tissue disease, diabetes mellitus or renal disease, or medication with antihypertensives or aspirin; singleton fetus; no fetal structural anomaly; normal response to glucose tolerance testing; and no evidence of recent infections (eg, rubella, toxoplasmosis, hepatitis B and C, cytomegalovirus, syphilis). Twenty-four women with mild preeclampsia and 22 women with severe preeclampsia meeting the eligibility criteria volunteered for the study, and 21 in each group gave informed consent after being advised that they would not benefit directly from this research. Pregnant women without preeclampsia, matched for maternal age (within 0.9 years), parity, and gestational age (within 6 days), were selected to serve as a control group. Twentythree patients were offered entry. Twenty-two of these women satisfied the eligibility criteria, and 21 gave informed consent and constituted the control group. The study was approved by the Ethics Committee of Nippon Medical School Hospital. All women were fully informed of the aim of the study, and informed consent was obtained from all participants. One hour before the study, the women entered a quiet room, and were placed at bed rest in a semirecumbent position. An indwelling 18-gauge catheter (Medikit Co., Tokyo, Japan) was placed in an antecubital vein. After the 1-hour period of accomodation, blood was taken for measurement of plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations. Preparations of samples for these measurements were as described.11–13 The plasma adenosine level was assayed with a modified high performance liquid chromatographic method with photodiode-array detection.11 The detection limit was 5 nmol/L, and the intra- and interassay coefficients of variation were 5.7% and 9.8%, respectively. The plasma norepinephrine level was determined using a high performance liquid chromatographic method with electrochemical detection.12 The detection limit was 10 pg/mL, and the intra- and interassay coefficients of variation were 6.2% and 9.1%, respectively. The plasma tumor necrosis factor–␣ level was measured with a specific enzyme-linked immunoassay.13
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The detection limit was 5 pg/mL, and the intra- and interassay coefficients of variation were 4.9% and 8.2%, respectively. Data are presented as means ⫾ standard errors of the mean. Significant differences between groups for continuous variables were evaluated by analysis of variance. If overall significance was observed, then individual group means were compared by the post hoc multiple comparison test with Bonferroni correction. Statistical analyses were also performed using 2 tests as appropriate. Correlations between the variables were evaluated using linear regression analysis with the least-squares method. Differences were considered significant at P ⬍ .05. Linear regression analysis was performed by the leastsquares method. Differences were considered significant at P ⬍ .05.
RESULTS Clinical characteristics of the study population are given in Table 1. In the present study, all subjects were married Japanese women of middle socioeconomic status who were nonsmokers. None of the patients developed eclampsia or the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome. Mean plasma adenosine, norepinephrine, and tumor necrosis factor–␣ concentrations are shown in Table 2. Mean plasma adenosine concentrations in women with preeclampsia were significantly higher than those in women with normal pregnancies, and increased according to the severity of preeclampsia (Figure 1). In women with preeclampsia, plasma adenosine concentrations significantly correlated with norepinephrine (r ⫽ .58, P ⬍ .05) and tumor necrosis factor–␣ (r ⫽ .49, P ⬍ .05); however, the correlations between the increase of plasma adenosine and mean blood pressure or proteinuria were not significant (r ⫽ .23, P ⫽ .18; r ⫽ .11, P ⫽ .72, respectively). Mean plasma norepinephrine and tumor necrosis factor–␣ concentrations were significantly higher in women with preeclampsia than in women with normal pregnancies, and these substances also increased according to the severity of preeclampsia. Plasma norepinephrine also correlated positively with mean blood pressure (r ⫽ .50, P ⬍ .05), but not with proteinuria (r ⫽ .25, P ⫽ .44), and there were no direct correlations between plasma tumor necrosis factor–␣ and mean blood pressure (r ⫽ .38, P ⫽ .52) or proteinuria (r ⫽ .69, P ⫽ .81) in women with preeclampsia. The significant relationships observed in women with preeclampsia did not exist in healthy women with normal pregnancies.
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Table 1. Demographic Data
Age (y) Nulliparous (n) Proteinuria (mg/d) Blood pressure (mm Hg) Systolic Diastolic Gestational age (wk) At study At delivery Cesarean delivery Birth weight (g) Birth weight ⬍ fifth percentile (n)
Normal pregnancy (n ⫽ 21)
Mild preeclampsia (n ⫽ 21)
Severe preeclampsia (n ⫽ 21)
32.2 ⫾ 1.0 13 (62%) —
32.8 ⫾ 1.3 13 (62%) 478 ⫾ 26
31.9 ⫾ 1.2 13 (62%) 759 ⫾ 44
115 ⫾ 2 70 ⫾ 2
155 ⫾ 2* 98 ⫾ 2*
182 ⫾ 5† 112 ⫾ 4†
30.2 ⫾ 0.6 39.1 ⫾ 0.2 2 (9%) 3104 ⫾ 67 0 (0%)
30.9 ⫾ 0.6 35.7 ⫾ 0.8* 6 (29%)* 2112 ⫾ 195* 2 (9%)
30.2 ⫾ 0.6 32.9 ⫾ 1.1† 11 (52%)† 1502 ⫾ 149† 6 (29%)
Data are presented as means ⫾ SEMs or numbers. * Significantly different from normal pregnancy (P ⬍ .05). † Significantly different from normal pregnancy and mild preeclampsia (P ⬍ .05).
DISCUSSION A pronounced elevation of plasma adenosine concentration was found in women with preeclampsia, which correlated with the severity of preeclampsia, and increases in adenosine were accompanied by increases in plasma norepinephrine and tumor necrosis factor–␣ concentrations. A protective action of adenosine has become recognized, notably in the heart14 and in newborns after vaginal delivery.15 Further, we have demonstrated that adenosine tends to prevent excessive platelet activation9 and regulate the T-helper 1/T-helper 2 imbalance10 in preeclampsia. However, a protective action for adenosine in countering the progression of preeclampsia has not previously been proposed. The progressive increases in adenosine with respect to increases in norepinephrine, tumor necrosis factor–␣, and, indirectly, blood pressure observed in the present study highly support this general concept. In the present study, a significant elevation of plasma adenosine concentration was observed in women with preeclampsia, which correlated with the severity of preeclampsia. The elevation could reasonably be predicted because local tissue hypoxia and the formation of micro-
thrombosis are well-established causes for the release of adenosine. These events occur frequently in preeclampsia, and it is reasonable to conclude that plasma adenosine is elevated in response to these events. The elevated adenosine levels observed in women with mild and severe preeclampsia in the present study are known to cause not cytotoxic effects, but near maximal vasodilatation in many vascular beds16 and to suppress metabolic activity.17 In light of the tendency of adenosine to redress local oxygen supply and use imbalance in preeclampsia, one may speculate that an elevation of adenosine in women with preeclampsia protects themselves and the fetuses by helping them to adapt to unfavorable conditions in preeclampsia. Further, the increased adenosine in preeclampsia might compensate for and tend to buffer further excessive platelet activation to maintain vascular integrity,9 and counteracts further imbalance of the T-helper 1/T-helper 2 ratio.10 From that viewpoint, an elevation of plasma adenosine in preeclampsia according to the severity of preeclampsia might compensate for and tend to counteract the further progression of preeclampsia. In the present study, plasma norepinephrine signifi-
Table 2. Plasma Adenosine, Norepinephrine, and Tumor Necrosis Factor–␣ Concentrations in Normal Pregnancy and Mild and Severe Preeclampsia
Adenosine (mol/L) Norepinephrine (pg/mL) Tumor necrosis factor–␣ (pg/mL)
Normal pregnancy (n ⫽ 21)
Mild preeclampsia (n ⫽ 21)
Severe preeclampsia (n ⫽ 21)
0.41 ⫾ 0.03 132 ⫾ 18 2.0 ⫾ 0.1
0.60 ⫾ 0.03* 238 ⫾ 25* 2.9 ⫾ 0.3*
0.72 ⫾ 0.03† 345 ⫾ 36† 3.7 ⫾ 0.4†
Data are presented as means ⫾ SEMs. * Significantly different from normal pregnancy (P ⬍ .05). † Significantly different from normal pregnancy and mild preeclampsia (P ⬍ .05).
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Figure 1. Changes in plasma adenosine concentrations in women with normal pregnancies and women with preeclampsia. Yoneyama. Adenosine in Preeclampsia. Obstet Gynecol 2002.
cantly increased in women with preeclampsia, which also correlated with the severity of preeclampsia. An increase in plasma norepinephrine also correlated positively with mean blood pressure. These findings are in agreement with previous studies.18,19 Because endogenous increases of norepinephrine augment the activity of ecto-5⬘-nucleotidase, the enzyme responsible for adenosine production,20 the increased plasma norepinephrine may be linked to increases in adenosine production. Indeed, such a linkage is supported by results of this study showing a direct correlation between plasma levels of norepinephrine and adenosine. A similar positive correlation has been reported in patients with chronic heart failure14 and newborns after vaginal delivery,15 conditions in which catecholamines are elevated, and thus adenosine may counteract catecholamine-induced effects in a number of biologic systems and as a general defense mechanism. Overall the data permit the conclusion that excessive norepinephrine increase worsens preeclampsia and that adenosine may attenuate the stimulatory effects of norepinephrine.6 In terms of systems
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analysis, adenosine may contribute to negative feedback against the progressive positive loop between norepinephrine and preeclampsia. In the present study, plasma tumor necrosis factor–␣ increased significantly in preeclamptic women, which is in agreement with a previous study.3 This increase was directly correlated with the increase of adenosine. These results suggest that tumor necrosis factor–␣ may be a direct stimulus for plasma adenosine production in preeclampsia. Tumor necrosis factor–␣ increases transcriptional and protein levels of ecto-5⬘-nucleotidase21 and may thereby in part contribute to increasing the production of adenosine in preeclampsia. Tumor necrosis factor–␣ is biologically active and associated with immune disorders, and that may be causally related to the pathogenesis and the progression of preeclampsia.22 Increased levels of tumor necrosis factor–␣ elicit activation of the immune system against the fetal allograft, and adenosine suppresses immune-triggered cytokine production,23 and by this means also, in addition to its anticatecholamine actions, it may be an endogenous compensatory mechanism against the progression of preeclampsia. In summary, we conclude that increases in plasma adenosine in preeclampsia occur in parallel with elevations of catecholamines and tumor necrosis factor–␣. It seems likely that adenosine participates in negative feedback loops that redress elevations of these stimuli, which may eventually worsen preeclampsia. Of course, other factors may be involved in the pathogenesis and progression of preeclampsia. Further, adenosine kinase, adenosine deaminase, and platelet activation may also largely contribute to the regulation of plasma adenosine concentrations in preeclampsia. Further investigation is needed to clarify the possible mechanisms and roles of an increase of plasma adenosine concentrations in women with preeclampsia. REFERENCES 1. Roberts JM, Redman CWG. Pre-eclampsia: More than pregnancy-induced hypertension. Lancet 1993;341: 1447–51. 2. Øian P, Kjeldsen SE, Eide I, Maltau JM. Increased arterial catecholamines in preeclampsia. Acta Obstet Gynecol Scand 1986;65:613– 6. 3. Kupferminc MJ, Peaceman AM, Aderka D, Wigton TR, Rehnberg KA, Socol ML. Tumor necrosis factor-␣ is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol 1994;170: 1752–9. 4. Rogers GM, Taylor RN, Roberts JM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. Am J Obstet Gynecol 1988;159:908 –14.
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5. Schobel HP, Fischer T, Heuszer K, Geiger H, Schmieder RE. Preeclampsia—a state of sympathetic overactivity. N Engl J Med 1996;335:1480 –5. 6. Dobson JG Jr. Mechanism of adenosine inhibition of cathecholamine-induced responses in heart. Circ Res 1983;52: 151– 60. 7. Barnes CR, Mandell GL, Carper HT, Luong S, Sullivan W. Adenosine modulation of tumor necrosis factor-␣induced neutrophil activation. Biochem Pharmacol 1995; 50:1851–7. 8. Yoneyama Y, Suzuki S, Sawa R, Otsubo Y, Power GG, Araki T. Plasma adenosine levels increase in women with normal pregnancies. Am J Obstet Gynecol 2000;182: 1200 –3. 9. Yoneyama Y, Suzuki S, Kiyokawa Y, Power GG, Araki T. Plasma adenosine level and P-selectin expression on platelet in preeclampsia. Obstet Gynecol 2001;97:366 –70. 10. Yoneyama Y, Suzuki S, Sawa R, Yoneyama K, Power GG, Araki T. Relation between adenosine and T-helper 1/Thelper 2 imbalance in women with preeclampsia. Obstet Gynecol 2002;99:641– 6. 11. Maguire MH, Szabo I, Valko IE, Finley BE, Bennett T1. Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversedphase high- performance liquid chromatography with photodiode-array detection and on-line validation of peak purity. J Chromatogr B Biomed Sci Appl 1998;707: 33– 41. 12. Hjemdahl P, Daleskog M, Kahn T. Determination of plasma catecholamines by high-performance liquid chromatography with electrochemical detection. Life Sci 1979; 25:131–5. 13. Kwitakowski D, Hill AVS, Sambou I, Twumasi P, Castracane J, Manogue KR, et al. TNF concentration in fetal cerebral, non-fetal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 1990;336:1201– 4. 14. Funaya H, Kitakaze M, Node K, Minamino T, Komamura K, Hori M. Plasma adenosine levels increase in patients with choronic heart failure. Circulation 1997;95:1363–5.
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15. Irestedt L, Dahlin I, Hertzberg T, Sollevi A, Lagercrantz H. Adenosine concentration in umbilical cord blood of newborn infants after vaginal delivery and cesarean section. Pediatr Res 1989;26:106 – 8. 16. Berne RM. Adenosine: An important physiological regulator. New Physiol Sci 1986;1:163–7. 17. Bardenhueser H, Schrader J. Supply-to-demand ratio for oxygen determines formation of adenosine by the heart. Am J Physiol 1986;250:173– 80. 18. Tinkanen H, Rorarius M, Metsa¨-Katela¨ T. Catecholamine concentrations in venous plasma and cerebrospinal fluid in normal and complicated pregnancy. Gynecol Obstet Invest 1993;35:7–11. 19. Kjeldsen SE, Eide I, Aakesson I, Øian P, Maltau JM, Gjesdal K. Increased arterial adrenaline is highly correlated to blood pressure and in vivo platelet function in pre-eclampsia. J Hypertens 1985;3:S93–5. 20. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Sato H, et al. ␣1–adrenoceptor activation increases ecto-5-nucleotidase activity and adenosine release in rat cardiomyocytes by activating protein kinase C. Circulation 1995;91:2226 –34. 21. Savic V, Stefanovic V, Ardaillou N, Ardaillou R. Induction of ecto-5⬘-nucleotidase of rat cultured mesangial cells by interleukin-1 and tumor necrosis factor-␣. Immunology 1990;70:321– 6. 22. Dekker GA, Sibai BM. The immunology of preeclampsia [review]. Semin Perinatol 1999;23:24 –33. 23. Zidek Z. Adenosine-cyclic AMP pathways and cytokine expression. Eur Cytokine Netw 1999;10:319 –28. Address reprint requests to: Yoshio Yoneyama, MD, Nippon Medical School, Department of Obstetrics and Gynecology, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113-8603, Japan; E-mail: [email protected]. Received January 30, 2002. Received in revised form May 6, 2002. Accepted June 19, 2002.
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