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Neuropeptide Y and nitrite levels in preeclamptic and normotensive gravid women
Neuropeptide Y and nitrite levels in preeclamptic and normotensive gravid women Robert S. Egerman, MD, Richard N. Andersen, PhD, Fazal M. Manejwala, MD, PhD, and Baha M. Sibai, MD Memphis, Tennessee OBJECTIVES: Vascular tone is controlled largely by the sympathetic nervous system and is modulated by neuropeptide Y. Preeclampsia is linked to sympathetic overactivity. Nitric oxide can cause vasorelaxation of vessels or decrease sympathetic outflow by activating the baroreceptor reflex. Our purpose in this study was to compare serum levels of neuropeptide Y and nitrite levels in normotensive and preeclamptic gravid women. STUDY DESIGN: Twelve preeclamptic and 12 normotensive women matched for race, body mass index, parity, and gestational age were studied. Neuropeptide Y was measured by using a commercial radioimmunoassay. Nitric oxide was converted to nitrite by using metallic cadmium, and nitrite levels were determined spectrophotometrically by using a colorimetric assay. Data are presented as mean ± SEM and were compared by using a t test. RESULTS: Neuropeptide Y levels were similar among preeclamptic and normotensive gravid women (33.8 ± 3.0 and 32.2 ± 3 pg/mL, respectively). Similarly, there were no differences in nitrite concentrations between preeclamptic and normotensive patients (11.6 ± 0.8 vs 11.2 ± 0.4 µmol/L, respectively). We also examined the ratios of neuropeptide Y and nitrite and found no correlation between preeclamptic and normotensive women. CONCLUSION: Peripheral levels of neuropeptide Y or nitrite do not correlate with preeclampsia. Assessment of sympathetic overactivity in preeclampsia requires an alternate model. (Am J Obstet Gynecol 1999;181:921-3.)
Key words: Pregnancy, hypertension, preeclampsia, neuropeptide Y, nitrite
Preeclampsia is a frequent cause of maternal and perinatal morbidity and mortality. Attempts at prediction and prevention of this reversible form of hypertension have been limited by the lack of understanding of the mechanism of the underlying vascular perturbation. The sympathetic nervous system has an important function in the control of blood pressure, and sympathetic overactivity has been implicated in preeclampsia.1 Modulating sympathetic tone are neurotransmitters, including norepinephrine and neuropeptide Y. Neuropeptide Y is a 36-amino-acid tyrosine-rich peptide. It has a direct vasopressor effect in human subjects and potentiates the norepinephrine-mediated action of angiotensin II.2 Furthermore, neuropeptide Y is increased in patients with essential hypertension.3 Vascular tone is also controlled by vasorelaxants, including nitric oxide. Decreased nitric oxide synthase, the enzyme responsible for nitric oxide production, has been From the Department of Obstetrics and Gynecology, University of Tennessee, Memphis. Presented at the Nineteenth Annual Meeting of the Society for MaternalFetal Medicine, San Francisco, California, January 18-23, 1999. Reprints not available from the authors. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/6/101126
proposed as a mechanism for vasoconstriction in preeclampsia.4 Nitric oxide acts centrally, reducing baroreflex-controlled sympathetic outflow.5 In this study we sought to determine levels of neuropeptide Y and nitric oxide in preeclamptic and normotensive women. We postulate that neuropeptide Y levels are increased and nitric oxide levels are depressed in pregnancies complicated by preeclampsia. Material and methods The study was approved by the Institutional Review Board at the University of Tennessee, Memphis, and informed consent was obtained from each subject. Pregnant women were recruited from the prenatal clinic during the third trimester. Twelve preeclamptic and 12 normotensive patients were matched for maternal age, race, parity, body mass index, and gestational age. Preeclampsia was defined by using clinical criteria established by The American College of Obstetricians and Gynecologists and included systolic blood pressure of ≥140 mm Hg and diastolic blood pressure of ≥90 mm Hg after 20 weeks’ gestation with either proteinuria or edema or both. Proteinuria was determined by qualitative analysis with a standard urine dipstick. Patients with antecedent chronic hypertension were excluded. 921
922 Egerman et al
October 1999 Am J Obstet Gynecol
Table I. Neuropeptide Y and nitrite levels in pregnancy (mean ± SEM)
Neuropeptide Y (pg/mL) Nitrite (µmol/L) Neuropeptide Y/nitrite
Normotensive subjects (n = 12)
Preeclamptic subjects (n = 12)
Statistical significance
32.2 ± 3.5 11.2 ± 0.4 2.7 ± 0.3
33.3 ± 3.6 11.6 ± 0.8 2.9 ± 0.4
P = .73 P = .66 P = .75
Blood samples were collected during the clinic visit. Samples were obtained in siliconized collection tubes containing ethylenediaminetetraacetic acid (EDTA) for neuropeptide Y determination and in plain red-top tubes and allowed to clot for nitrite assay. Samples were centrifuged at 350g for 10 minutes in a refrigerated centrifuge (model 2370; Damon/IEC, Needham Heights, Mass). Sera and EDTA-treated plasma were transferred to sterile containers, labeled, and stored at –70°C for batch analysis. Neuropeptide Y was measured by using a commercial immunoassay kit (Peninsula Laboratories, Belmont, Calif). Samples were prepared for assay by acid-ethanol extraction.6 In brief, 0.5 mL of EDTA-treated plasma was mixed with 1 mL of acid ethanol in 12 × 75 mL polypropylene tubes. After centrifugation at 3000 rpm for 15 minutes, the supernatants were decanted and dried under nitrogen. They were then redissolved in 0.5 mL of 0.9% sodium chloride containing 0.5% bovine serum albumin. Standards and unknowns were incubated overnight at 4°C with the rabbit neuropeptide Y antiserum and iodine 125–labeled neuropeptide Y. Bound and free samples were separated by a second antibody (goat antirabbit immunoglobulin G). Samples underwent centrifugation at 3000 rpm for 10 minutes, and the supernatants were decanted. Droplets remaining in the tube were removed, and the pellets were counted. All samples were analyzed in duplicate. A Micro Medic gamma counter and data reduction system (Horsham, Pa) were used to measure radioactivity and carry out the data reduction. Nitric oxide is unstable in aqueous solution undergoing rapid oxidation to nitrite and nitrate. Therefore it was measured spectrophotometrically as nitrite with the Griess reagent after reduction of nitrate to nitrite by metallic cadmium with a colorimetric assay (Oxford Biomedical Research, Oxford, Mich). Briefly, the cadmium pellets were washed sequentially in 0.1-mol/L hydrochloric acid and 0.1-mol/L ammonium hydroxide before use. Standards were diluted in deionized water from a stock solution provided by the vendor to produce standards over the range of 0.5 to 100 µmol/L. Serum samples were deproteinated by zinc sulfoxide, and the supernatants were then transferred to the cadmium pellets for nonenzymatic reduction of nitrate to nitrite. The nitrite levels of standards and samples were quantified colori-
metrically with the Griess reagent in microliter plates, and measurement of the absorbance at 540 nm was performed in a microliter plate reader. The equation for the standard curve was obtained from a curve-fitting function of Cricket Graph (Computer Associates, San Jose, Calif), and the values of the samples were obtained by application of this equation. Data are presented as mean ± SEM. Comparisons were made by using a t test, with P < .05 considered statistically significant. Results Maternal characteristics were similar between preeclamptic and normotensive subjects regarding maternal age (23.7 ± 1.0 vs 22.1 ± 1.1 years), gestational age (35.7 ± 1.4 vs 36.9 ± 1.0 weeks), and body mass index (31.3 ± 1.1 vs 31.3 ± 1.0 kg/m2). Seven primigravid women were present in each group. Preeclamptic patients had significantly higher systolic and diastolic blood pressures in comparison with the normotensive control subjects (155 ± 6 vs 110 ± 3 mm Hg and 102 ± 3 vs 69 ± 4 mm Hg, respectively; P < .05). Neuropeptide Y and nitrite levels are reported in Table I. No significant differences were detected between the hypertensive and control subjects regarding either neuropeptide Y or nitrite levels. We also examined the ratios of neuropeptide Y and nitrite and found no correlation between preeclamptic and normotensive women. Comment Aside from the widespread endothelial dysfunction evident in preeclamptic pregnancies, increased sympathetic outflow and heightened vascular tone have been well known. Even though serum catecholamine levels are not uniformly elevated in preeclamptic patients, increased sympathetic outflow has been demonstrated by direct measurement of peroneal nerve burst activity.1 Neuropeptide Y is a neurotransmitter involved in sympathetic activity and appetite regulation.7 Recently, there has been considerable interest in the role of neuropeptide Y in hypertensive disease. In this preliminary study we demonstrated that peripheral measurements of neuropeptide Y concentrations were similar among preeclamptic and normotensive subjects. These findings are comparable with those of Kokot et al8 who, in 1998, reported similar neuropeptide Y levels among healthy
Egerman et al 923
Volume 181, Number 4 Am J Obstet Gynecol
pregnant and preeclamptic women (42.3 ± 4.1 and 43.7 ± 8.5 pg/mL, respectively). In the study by Kokot et al,8 body mass index was significantly higher in the hypertensive group than in the normotensive women (26.9 ± 0.4 vs 23.1 ± 0.7 kg/m2), an important variable accounted for in the present study. Regarding neuropeptide Y levels in other tissues, Fried et al9 examined uterine tissue in normal and preeclamptic pregnancies. In this study neuropeptide Y levels were slightly lower in myometrial tissue from pregnancies complicated by preeclampsia when compared with myometrial tissue from normotensive pregnancies. It is interesting that myometrial levels of neuropeptide Y were lower from both hypertensive and normotensive pregnancies when compared with those of nonpregnant subjects. Nitric oxide has received a multitude of interest regarding its role in the mechanism of preeclampsia. Studies comparing peripheral nitric oxide measurements in preeclamptic and normotensive pregnancies have been inconsistent.10-12 Our nitrite concentrations are similar to those previously reported for pregnancy.13, 14 Given that variations in dietary nitrite intake or renal excretion can affect serum nitrite levels, there are inherent limitations to this approach of blood sampling.15 Nevertheless, we anticipated the following sequence among preeclamptic women: Decreased nitric oxide production would lead to down-regulation of the baroreceptor reflex. Decreased vagal tone would lead to an imbalance of sympathetic activity reflected by higher neuropeptide Y levels. Our study does not support this postulate because neither serum nitrite nor neuropeptide Y concentrations differed between the 2 groups. We cannot exclude a possible role for neuropeptide Y in the development or the exacerbation of preeclampsia; however, our data indicate that assessment of the contribution of sympathetic activity to hypertension in the preeclamptic patient requires an alternate model.
REFERENCES
1. Schobel HP, Thorsten F, Heuszer K, Geiger H, Schmieder RE. Preeclampsia—A state of sympathetic overactivity. N Engl J Med 1996;335:1480-5. 2. Pedrazzini T, Brunner HR, Waeber B. Neuropeptide Y and cardiovascular regulation. Curr Opin Nephrol Hypertens 1993;2:106-13. 3. Erlinge D, Ekman R, Thulin T, Edvinsson L. Neuropeptide Ylike immunoreactivity and hypertension. J Hypertens 1992;10:1221-5. 4. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 5. Scrogin KE, Veelken R, Luft FC. Sympathetic baroreceptor responses after chronic NG-nitro-L-arginine methyl ester treatment in conscious rats. Hypertension 1994;23:982-6. 6. Theodorsson-Norheim E, Hemsén A, Lundberg JM. Radioimmunoassay for neuropeptide Y (NPY): chromatographic characterization of immunoreactivity in plasma and tissue extracts. Scand J Clin Lab Invest 1985;45:355-65. 7. Kalra SP, Horvath TL. Neuroendocrine interactions between galanin, opioids, and neuropeptide Y in the control of reproduction and appetite. Ann N Y Acad Sci 1998;21:863-40. 8. Kokot F, Ulman I, Wiecek A, Irzyniec T, Ulman J. Concentrations of leptin and neuropeptide Y in maternal plasma umbilical cord blood and in amniotic fluid in pregnant women with EPH-gestosis. Arch Immunol Ther Exp (Warsz) 1998;46:311-6. 9. Fried G, Hökfelt T, Lundberg JM, Terenius L, Hamberger L. Neuropeptide Y and noradrenaline in human uterus and myometrium during normal and pre-eclamptic pregnancy. Hum Reprod 1986;1:359-64. 10. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA, Caplan MS. Evaluation of nitric oxide as a medicator of severe preeclampsia. Am J Obstet Gynecol 1996;175:1013-7. 11. Smarason AK, Allman KG, Young D, Redman CW. Elevated levels of serum nitrate, a stable end product of nitric oxide, in women with pre-eclampsia. Br J Obstet Gynaecol 1997;104:53843. 12. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 13. Ranta V, Viinikka L, Halmesmaki E, Ylikorkala O. Nitric oxide production with preeclampsia. Obstet Gynecol 1999;93:442-5. 14. Hata T, Hashimoto M, Manabe A, Aoki S, Iida K, Masumura S, et al. Maternal and fetal nitric oxide synthesis is decreased in pregnancies with small for gestational age infants. Hum Reprod 1998;13:1070-3. 15. Roberts JM. Plasma nitrites as an indicator of nitric oxide production: unchanged production or reduced renal clearance in preeclampsia? Am J Obstet Gynecol 1997;716:954-5.
Key words: Pregnancy, hypertension, preeclampsia, neuropeptide Y, nitrite
Preeclampsia is a frequent cause of maternal and perinatal morbidity and mortality. Attempts at prediction and prevention of this reversible form of hypertension have been limited by the lack of understanding of the mechanism of the underlying vascular perturbation. The sympathetic nervous system has an important function in the control of blood pressure, and sympathetic overactivity has been implicated in preeclampsia.1 Modulating sympathetic tone are neurotransmitters, including norepinephrine and neuropeptide Y. Neuropeptide Y is a 36-amino-acid tyrosine-rich peptide. It has a direct vasopressor effect in human subjects and potentiates the norepinephrine-mediated action of angiotensin II.2 Furthermore, neuropeptide Y is increased in patients with essential hypertension.3 Vascular tone is also controlled by vasorelaxants, including nitric oxide. Decreased nitric oxide synthase, the enzyme responsible for nitric oxide production, has been From the Department of Obstetrics and Gynecology, University of Tennessee, Memphis. Presented at the Nineteenth Annual Meeting of the Society for MaternalFetal Medicine, San Francisco, California, January 18-23, 1999. Reprints not available from the authors. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/6/101126
proposed as a mechanism for vasoconstriction in preeclampsia.4 Nitric oxide acts centrally, reducing baroreflex-controlled sympathetic outflow.5 In this study we sought to determine levels of neuropeptide Y and nitric oxide in preeclamptic and normotensive women. We postulate that neuropeptide Y levels are increased and nitric oxide levels are depressed in pregnancies complicated by preeclampsia. Material and methods The study was approved by the Institutional Review Board at the University of Tennessee, Memphis, and informed consent was obtained from each subject. Pregnant women were recruited from the prenatal clinic during the third trimester. Twelve preeclamptic and 12 normotensive patients were matched for maternal age, race, parity, body mass index, and gestational age. Preeclampsia was defined by using clinical criteria established by The American College of Obstetricians and Gynecologists and included systolic blood pressure of ≥140 mm Hg and diastolic blood pressure of ≥90 mm Hg after 20 weeks’ gestation with either proteinuria or edema or both. Proteinuria was determined by qualitative analysis with a standard urine dipstick. Patients with antecedent chronic hypertension were excluded. 921
922 Egerman et al
October 1999 Am J Obstet Gynecol
Table I. Neuropeptide Y and nitrite levels in pregnancy (mean ± SEM)
Neuropeptide Y (pg/mL) Nitrite (µmol/L) Neuropeptide Y/nitrite
Normotensive subjects (n = 12)
Preeclamptic subjects (n = 12)
Statistical significance
32.2 ± 3.5 11.2 ± 0.4 2.7 ± 0.3
33.3 ± 3.6 11.6 ± 0.8 2.9 ± 0.4
P = .73 P = .66 P = .75
Blood samples were collected during the clinic visit. Samples were obtained in siliconized collection tubes containing ethylenediaminetetraacetic acid (EDTA) for neuropeptide Y determination and in plain red-top tubes and allowed to clot for nitrite assay. Samples were centrifuged at 350g for 10 minutes in a refrigerated centrifuge (model 2370; Damon/IEC, Needham Heights, Mass). Sera and EDTA-treated plasma were transferred to sterile containers, labeled, and stored at –70°C for batch analysis. Neuropeptide Y was measured by using a commercial immunoassay kit (Peninsula Laboratories, Belmont, Calif). Samples were prepared for assay by acid-ethanol extraction.6 In brief, 0.5 mL of EDTA-treated plasma was mixed with 1 mL of acid ethanol in 12 × 75 mL polypropylene tubes. After centrifugation at 3000 rpm for 15 minutes, the supernatants were decanted and dried under nitrogen. They were then redissolved in 0.5 mL of 0.9% sodium chloride containing 0.5% bovine serum albumin. Standards and unknowns were incubated overnight at 4°C with the rabbit neuropeptide Y antiserum and iodine 125–labeled neuropeptide Y. Bound and free samples were separated by a second antibody (goat antirabbit immunoglobulin G). Samples underwent centrifugation at 3000 rpm for 10 minutes, and the supernatants were decanted. Droplets remaining in the tube were removed, and the pellets were counted. All samples were analyzed in duplicate. A Micro Medic gamma counter and data reduction system (Horsham, Pa) were used to measure radioactivity and carry out the data reduction. Nitric oxide is unstable in aqueous solution undergoing rapid oxidation to nitrite and nitrate. Therefore it was measured spectrophotometrically as nitrite with the Griess reagent after reduction of nitrate to nitrite by metallic cadmium with a colorimetric assay (Oxford Biomedical Research, Oxford, Mich). Briefly, the cadmium pellets were washed sequentially in 0.1-mol/L hydrochloric acid and 0.1-mol/L ammonium hydroxide before use. Standards were diluted in deionized water from a stock solution provided by the vendor to produce standards over the range of 0.5 to 100 µmol/L. Serum samples were deproteinated by zinc sulfoxide, and the supernatants were then transferred to the cadmium pellets for nonenzymatic reduction of nitrate to nitrite. The nitrite levels of standards and samples were quantified colori-
metrically with the Griess reagent in microliter plates, and measurement of the absorbance at 540 nm was performed in a microliter plate reader. The equation for the standard curve was obtained from a curve-fitting function of Cricket Graph (Computer Associates, San Jose, Calif), and the values of the samples were obtained by application of this equation. Data are presented as mean ± SEM. Comparisons were made by using a t test, with P < .05 considered statistically significant. Results Maternal characteristics were similar between preeclamptic and normotensive subjects regarding maternal age (23.7 ± 1.0 vs 22.1 ± 1.1 years), gestational age (35.7 ± 1.4 vs 36.9 ± 1.0 weeks), and body mass index (31.3 ± 1.1 vs 31.3 ± 1.0 kg/m2). Seven primigravid women were present in each group. Preeclamptic patients had significantly higher systolic and diastolic blood pressures in comparison with the normotensive control subjects (155 ± 6 vs 110 ± 3 mm Hg and 102 ± 3 vs 69 ± 4 mm Hg, respectively; P < .05). Neuropeptide Y and nitrite levels are reported in Table I. No significant differences were detected between the hypertensive and control subjects regarding either neuropeptide Y or nitrite levels. We also examined the ratios of neuropeptide Y and nitrite and found no correlation between preeclamptic and normotensive women. Comment Aside from the widespread endothelial dysfunction evident in preeclamptic pregnancies, increased sympathetic outflow and heightened vascular tone have been well known. Even though serum catecholamine levels are not uniformly elevated in preeclamptic patients, increased sympathetic outflow has been demonstrated by direct measurement of peroneal nerve burst activity.1 Neuropeptide Y is a neurotransmitter involved in sympathetic activity and appetite regulation.7 Recently, there has been considerable interest in the role of neuropeptide Y in hypertensive disease. In this preliminary study we demonstrated that peripheral measurements of neuropeptide Y concentrations were similar among preeclamptic and normotensive subjects. These findings are comparable with those of Kokot et al8 who, in 1998, reported similar neuropeptide Y levels among healthy
Egerman et al 923
Volume 181, Number 4 Am J Obstet Gynecol
pregnant and preeclamptic women (42.3 ± 4.1 and 43.7 ± 8.5 pg/mL, respectively). In the study by Kokot et al,8 body mass index was significantly higher in the hypertensive group than in the normotensive women (26.9 ± 0.4 vs 23.1 ± 0.7 kg/m2), an important variable accounted for in the present study. Regarding neuropeptide Y levels in other tissues, Fried et al9 examined uterine tissue in normal and preeclamptic pregnancies. In this study neuropeptide Y levels were slightly lower in myometrial tissue from pregnancies complicated by preeclampsia when compared with myometrial tissue from normotensive pregnancies. It is interesting that myometrial levels of neuropeptide Y were lower from both hypertensive and normotensive pregnancies when compared with those of nonpregnant subjects. Nitric oxide has received a multitude of interest regarding its role in the mechanism of preeclampsia. Studies comparing peripheral nitric oxide measurements in preeclamptic and normotensive pregnancies have been inconsistent.10-12 Our nitrite concentrations are similar to those previously reported for pregnancy.13, 14 Given that variations in dietary nitrite intake or renal excretion can affect serum nitrite levels, there are inherent limitations to this approach of blood sampling.15 Nevertheless, we anticipated the following sequence among preeclamptic women: Decreased nitric oxide production would lead to down-regulation of the baroreceptor reflex. Decreased vagal tone would lead to an imbalance of sympathetic activity reflected by higher neuropeptide Y levels. Our study does not support this postulate because neither serum nitrite nor neuropeptide Y concentrations differed between the 2 groups. We cannot exclude a possible role for neuropeptide Y in the development or the exacerbation of preeclampsia; however, our data indicate that assessment of the contribution of sympathetic activity to hypertension in the preeclamptic patient requires an alternate model.
REFERENCES
1. Schobel HP, Thorsten F, Heuszer K, Geiger H, Schmieder RE. Preeclampsia—A state of sympathetic overactivity. N Engl J Med 1996;335:1480-5. 2. Pedrazzini T, Brunner HR, Waeber B. Neuropeptide Y and cardiovascular regulation. Curr Opin Nephrol Hypertens 1993;2:106-13. 3. Erlinge D, Ekman R, Thulin T, Edvinsson L. Neuropeptide Ylike immunoreactivity and hypertension. J Hypertens 1992;10:1221-5. 4. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 5. Scrogin KE, Veelken R, Luft FC. Sympathetic baroreceptor responses after chronic NG-nitro-L-arginine methyl ester treatment in conscious rats. Hypertension 1994;23:982-6. 6. Theodorsson-Norheim E, Hemsén A, Lundberg JM. Radioimmunoassay for neuropeptide Y (NPY): chromatographic characterization of immunoreactivity in plasma and tissue extracts. Scand J Clin Lab Invest 1985;45:355-65. 7. Kalra SP, Horvath TL. Neuroendocrine interactions between galanin, opioids, and neuropeptide Y in the control of reproduction and appetite. Ann N Y Acad Sci 1998;21:863-40. 8. Kokot F, Ulman I, Wiecek A, Irzyniec T, Ulman J. Concentrations of leptin and neuropeptide Y in maternal plasma umbilical cord blood and in amniotic fluid in pregnant women with EPH-gestosis. Arch Immunol Ther Exp (Warsz) 1998;46:311-6. 9. Fried G, Hökfelt T, Lundberg JM, Terenius L, Hamberger L. Neuropeptide Y and noradrenaline in human uterus and myometrium during normal and pre-eclamptic pregnancy. Hum Reprod 1986;1:359-64. 10. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA, Caplan MS. Evaluation of nitric oxide as a medicator of severe preeclampsia. Am J Obstet Gynecol 1996;175:1013-7. 11. Smarason AK, Allman KG, Young D, Redman CW. Elevated levels of serum nitrate, a stable end product of nitric oxide, in women with pre-eclampsia. Br J Obstet Gynaecol 1997;104:53843. 12. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 13. Ranta V, Viinikka L, Halmesmaki E, Ylikorkala O. Nitric oxide production with preeclampsia. Obstet Gynecol 1999;93:442-5. 14. Hata T, Hashimoto M, Manabe A, Aoki S, Iida K, Masumura S, et al. Maternal and fetal nitric oxide synthesis is decreased in pregnancies with small for gestational age infants. Hum Reprod 1998;13:1070-3. 15. Roberts JM. Plasma nitrites as an indicator of nitric oxide production: unchanged production or reduced renal clearance in preeclampsia? Am J Obstet Gynecol 1997;716:954-5.