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Increased Ascorbate Radical Formation and Ascorbate Depletion in Plasma from Women With Preeclampsia: Implications for Oxidative Stress
Free Radical Biology & Medicine, Vol. 23, No. 4, pp. 597–609, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(97)00010-5
Original Contribution INCREASED ASCORBATE RADICAL FORMATION AND ASCORBATE DEPLETION IN PLASMA FROM WOMEN WITH PREECLAMPSIA: IMPLICATIONS FOR OXIDATIVE STRESS
CARL A. HUBEL,* VALERIAN E. KAGAN, † ELENA R. KISIN, † MARGARET K. MCLAUGHLIN,* and JAMES M. ROBERTS * *Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA and the Departments of Obstetrics and Gynecology; † Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15213, USA (Received 10 November 1996; Revised 31 January 1997; Accepted 4 February 1997)
Abstract—There is evidence that oxidative stress accompanies preeclampsia and plasma ascorbate concentrations are reported to be decreased in the disorder. We tested the hypothesis that an ascorbate-oxidizing activity is increased in plasma from women with preeclampsia relative to normal pregnancy. Electron paramagnetic resonance (EPR) spectroscopy was used to determine (1) plasma functional reserves of ascorbate and total thiols, (2) temporal changes in ascorbate and thiol concentrations during incubation of whole blood in vitro, and (3) ascorbate radical signal kinetics in plasma after equalization of ascorbate concentrations. High-pressure liquid chromatography (HPLC) was used to measure plasma a-tocopherol. Ascorbate concentrations were 50 % lower in preeclampsia relative to normal pregnancy plasma but thiols and a-tocopherol did not differ. The elapsed time prior to half-consumption of plasma ascorbate was decreased approximately three-fold during incubation of whole blood from preeclamptics. No concomitant decrease in thiols was evident. The initial ascorbate radical signal amplitude was greater in preeclampsia plasma and then, in contrast to normal pregnancy plasma, decreased progressively. The iron chelator, deferoxamine had no effect on plasma ascorbate radical formation. We conclude that an ascorbate-oxidizing activity is increased in preeclampsia plasma which might contribute to vascular dysfunction in the disorder. q 1997 Elsevier Science Inc. Keywords—Ascorbate, Vitamin E, Plasma, Free radicals, Pregnancy, Preeclampsia
cells in culture in ways relevant to the endothelial pathology of the disease.2 – 5 There also is evidence that overproduction of reactive oxygen and nitrogen species accompanies preeclampsia and it has been postulated that oxidative events contribute to vascular dysfunction in the disorder.6 – 9 Human plasma contains an efficient arsenal of low molecular weight, nonenzymatic antioxidants that serve to protect the vasculature from oxidant damage.10,11 Of these, reduced ascorbate (vitamin C) is of primary importance in protecting plasma lipoproteins from peroxidation during exposure to a wide spectrum of water- or lipid-soluble free radical generators in vitro.10,12 The effectiveness of ascorbate against lipid-soluble radical initiators is due in part to its ability to regenerate a-tocopherol (vitamin E) by reducing tocopherol radicals in lipoproteins at the water-lipid in-
INTRODUCTION
Preeclampsia is the leading cause of maternal mortality in the Western World and is associated with a five-fold increase in perinatal mortality. The clinical markers of this human pregnancy-specific disorder include hypertension and proteinuria, which reverse after delivery.1 Although the cause of preeclampsia remains obscure, several lines of evidence indicate that dysfunction of the vascular endothelium accounts for the altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity which accompany the disorder.2 A number of reports indicate the presence of unidentified materials in plasma or serum from women with preeclampsia that alter the function of endothelial Address correspondence to: Carl A. Hubel, Magee-Womens Research Institute, 204 Craft Ave., Pittsburgh, PA 15213. 597
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terface.10,13,14 The relatively unreactive semidehydroascorbate anion radical (ascorbate radical) is the intermediate during the oxidation of ascorbate to dehydroascorbate and its measurement provides an index of oxidative stress in plasma.15 – 19 It was previously reported that plasma concentrations of ascorbate are decreased in mild and severe preeclampsia whereas a-tocopherol and beta-carotene are decreased only in severe disease compared to normal pregnant controls.20 The oxidized form of ascorbate, dehydroascorbic acid, was not decreased suggesting that oxidative consumption of ascorbate occurred.20 The present study compared plasma reserves of ascorbate, total thiols (protein thiols plus glutathione), and a-tocopherol in primiparous, normal and preeclamptic pregnancies. Assessment of these data prompted us to examine whether endogenous factors in blood and plasma from women with preeclampsia escalate oxidative depletion of ascorbate in vitro. Freshly obtained, EDTA-anticoagulated blood samples from women with normal and preeclamptic pregnancies were incubated and plasma concentration changes in endogenous ascorbate and total thiols were measured over time by electron paramagnetic resonance (EPR) spectroscopy. We also used EPR spectroscopy to measure temporal changes in ascorbate radical signal amplitude in preeclampsia and normal pregnancy plasma after equalization of ascorbate concentrations by addition of exogenous ascorbate. Data presented suggest that an ascorbate-oxidizing activity is increased in plasma from women with preeclampsia and that this activity does not involve chelatable iron. MATERIALS AND METHODS
Patient selection Subjects were recruited at the time of admittance to the labor and delivery ward at Magee-Womens Hospital as part of our ongoing investigation of preeclampsia. The study was approved by the hospital Institutional Review Board and all subjects gave informed written consent. Demographic and clinical data were collected at routine obstetric visits. Fourteen subjects had preeclampsia, defined using the criteria of gestational hypertension, proteinuria, hyperuricemia, and reversal of hypertension and proteinuria after delivery .1,2 Gestational hypertension was defined as an increase of 30 mmHg systolic or 15 mmHg diastolic blood pressure, as compared to values obtained before 20 wk gestation, or having an absolute blood pressure ú 140/90 mmHg after 20 wk gestation if earlier blood pressures were not known. Proteinuria was defined as ú 500 mg/ 24 h urine collection or ú 2/ on a voided or ú 1/ on a catheterized random urine specimen. Hyperuricemia
was defined as ú 1 standard deviation above usual values (at term ú 5.5 mg/dL). Seventeen subjects had uncomplicated pregnancies. These women were normotensive throughout gestation and without proteinuria or hyperuricemia. Patients with cigarette or illicit drug use, chronic hypertension, renal disease, or previous history of metabolic disorders were excluded. All subjects in our study reported daily intake of standard prenatal vitamins, which included vitamins C and E, during pregnancy. Blood samples Plasma was prepared from whole venous blood withdrawn into sterile tubes containing 4 mM potassium-EDTA. Plasma was separated by centrifugation, placed into aliquots under sterile conditions and frozen at 0707C until assayed. Serum samples for uric acid determination were withdrawn into sterile tubes and allowed to coagulate for 60 min at room temperature. The serum was separated by centrifugation and frozen at 0707C until assayed. Concentrations of antioxidants were measured in 12 preeclampsia and 13 normal predelivery plasma samples in which the interval between blood acquisition and plasma freezing was minimized (mean minutes { SD preeclampsia Å 54 { 7; normal Å 57 { 9). These samples were also matched for the duration of sample storage (mean weeks { SD preeclampsia Å 20.5 { 18; normal Å 20.4 { 18). Intravenous magnesium sulphate had been administered to 4 of 12 women with preeclampsia prior to antepartum blood draw. In 4 of 12 preeclamptics and 5 of 13 controls, labor had begun prior to antepartum blood draw. Antioxidant values for each group as a whole and those obtained prior to labor and magnesium sulphate are listed in Tables 2 and 3, respectively. The above samples were from a subset of the patients listed in Table 1 (Table 1 includes an additional 2 preeclamptic and 4 normal patients whose samples contributed to the plasma pools used in ascorbate radical analyses). The kinetics of endogenous ascorbate and thiol depletion in vitro were examined in freshly obtained, EDTA-anticoagulated whole venous blood (10 ml) from 4 women with preeclampsia and 4 with normal pregnancies. Blood samples were incubated at 257C, in the dark, under ambient air in a tissue culture hood. Aliquots (1 ml) were removed at successive time intervals from 40 to 1200 min (post-draw) and the plasma harvested by centrifugation. The plasma was immediately stored at 0707C and assayed for ascorbate and thiol concentrations within 7 days. These samples, as well as plasma assayed for ascorbate radical formation in vitro, were obtained from a subset of Table
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Table 1. Clinical Characteristics of Patient Groups
Maternal age (years) Prepregnant body mass index (kg/m2) Prepregnant BP (mmHg) Predelivery BP (mmHg) Wks. gestation at delivery Hematocrit (vol:vol %) Serum creatinine (mg/dL) Infant birthweight (g) Uric Acid (mg/dL)
Normal (n Å 17)
Preeclampsia (n Å 14)
26.3 { 6.0 22.6 { 2.6 107/66 { 7/8 115/70 { 10/6 39.9 { 0.8 35.8 { 2.4 N.M. 3485 { 509 4.5 { 1.2
26.6 { 6.2 24.0 { 3.9 112/68 { 10/6 150/93 { 10/5 35.1 { 2.9 35.5 { 2.3 0.9 { 0.3 2330 { 569 6.4 { 1.4
p Value N.S. N.S. N.S. õ0.001 õ0.001 N.S. õ0.001 õ0.004
Data are presented as mean { SD and were analyzed using the Student’s t-test. N.S.: not significant, p ú .05. Blood pressures are systolic/diastolic. N.M.: not measured.
1 patients prior to both onset of labor and magnesium sulphate administration.
Plasma ascorbate and thiol reserves Functional reserves of ascorbate and total thiols (protein thiols plus glutathione) were determined by EPR spectroscopy techniques previously described ( 21 and references therein). Briefly, ascorbate and reduced sulfhydryls can donate electrons to phenoxyl radicals, thus regenerating phenols and forming antioxidant radicals. For hindered phenols, this regeneration can be monitored by EPR spectroscopy. The enzyme tyrosinase is used as a catalyst to generate phenoxyl radicals from the hindered phenol, etoposide (VP-16). VP-16 radical can be detected by its characteristic EPR signal for 50–60 min. The duration of ascorbate radical EPR signal (ascorbate activity) is proportional to ascorbate concentration and is calibrated using ascorbate standards. Total thiol concentration is derived from the lag period after ascorbate consumption and before the reappearance of the VP-16 phenoxyl radical signal.21 Measurements were performed on a JEOL-RE1X spectrometer (JEOL, Kyoto, JPN) at 257C in gas-permeable, 0.8 mm internal diameter, 0.013 mm thickness Teflon tubing (from Alpha Wire Corporation, Elizabeth, NJ). Phenoxyl and ascorbate radical spectra were recorded at g Å 2.00, 335.5 mT center field, 10 mW power, 0.05 mT field modulation, 5 mT sweep width, 1600 receiver gain, and 1.0 second time constant. Spectra were collected using EPRMare software (Scientific Software Services, Bloomington, IL). The detection threshold was 3.0 mM for ascorbate and 12 mM for thiols. Further details of the method are provided in Fig. 1. Ascorbate concentrations were also measured in selected plasma samples using high pressure liquid chromatography (HPLC) with UV detection. Plasma proteins were precipitated using 10% acetic acid (CH3CO2H) and centrifugation (2,000 1 g, 10 min.)
and the supernatant used immediately for analysis. We used a Supelcosil LC-8 column (3 mm particle size, 4.6 1 159 mm; Supelco, Bellefonte, PA) and a mobile phase of 1:24 methanol : water adjusted to pH 3.0 by acetic acid at a flow rate of 1.0 ml/min. A Shimadzu LC-10A HPLC system was used with an LC-600 pump and SPD-10AV UV-detector set at 264 nm absorbance. Under these conditions, the retention time for ascorbate was 2.1 min. The peak corresponding to ascorbate concentration was completely eliminated by the addition of ascorbate oxidase to plasma.
Ascorbate free radical signal intensity in plasma supplemented with exogenous ascorbate Plasma (50 mL) was supplemented with ascorbate stock (made in 100 mmol/L TRIS, pH 7.4) to achieve 1 mmol/L final ascorbate concentration (60 ml total volume). Tris buffer was pretreated with Chelex-100 resin (Sigma Chemical Co. St. Louis, MO) by batch method to remove adventitious iron.22 Chelex-100 -pretreated Tris buffer containing 1 mmol/L ascorbate was used as the plasma-free control. EPR measurements were performed on a JEOL-RE1X spectrometer (JEOL, Kyoto, JPN) at 257C in gas-permeable, 0.8 mm internal diameter, 0.013 mm thickness Teflon tubing (from Alpha Wire Corporation, Elizabeth, NJ). After supplementation of samples with ascorbate, ascorbate radical spectra were immediately recorded at g Å 2.00, 335.5 mT center field, 10 mW power, 0.05 mT field modulation, 5 mT sweep width, 1600 receiver gain, and 1.0 second time constant. Spectra were collected using EPRMare software (Scientific Software Services, Bloomington, IL). No exogenous oxidation catalysts were added. We assayed predelivery plasma from 3 women with preeclampsia and 3 with normal pregnancies as well as 1 normal pregnancy and 1 preeclampsia plasma pool, each pool constructed by combining
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C. A. HUBEL et al. Table 2. Plasma Antioxidant Reserves in Women With Preeclampsia and Normal Pregnancy
Preeclampsia (n Å 12) Normal Pregnancy (n Å 13) Significance
Ascorbate nmol/mL
Total Thiols nmol/mL
Vitamin E nmol/mL
Vitamin E nmol/mmol Lipid*
11.0 (9.2 to 15.3) 21.1 (16.8 to 26.4) p õ .002
646 (518 to 794) 516 (476 to 598) N.S. (p Å .05)
25.7 (21.8 to 30.6) 21.3 (16.1 to 22.8) N.S. (p Å .06)
2.8 (2.4 to 2.9) 2.4 (2.0 to 3.0) N.S. (p Å .53)
Data are Medians and Interquartile Range. N.S.: not significant. * Lipid corrected: vitamin E/(cholesterol / triglycerides) in nmol/mmol.
were exported from the detector using Shimadzu EZChrom software ( Shimadzu Scientific Instruments, Colombia, MD ) .
equivalent volumes of 6 different plasma samples. These samples ( 9 per group ) were obtained prior to magnesium sulphate and labor and included all 7 of the preeclampsia and 5 of the 8 normal pregnancy samples of Table 3. An additional 2 preeclampsia and 4 normal samples contributed to the plasma pools and the patient data corresponding to these samples were incorporated into Table 1. Ascorbate radical signal intensity (amplitude) was expressed as a percentage of the initial signal intensity elicited from the ascorbate-supplemented, normal pregnancy plasma pool. In contrast to ascorbate radical, both ascorbate and dehydroascorbate are EPR-silent. Given similar initial ascorbate concentrations, the initial intensity of the ascorbate radical signal is thus directly proportional to the overall rate of ascorbate oxidation, whereas the signal duration is inversely proportional.
Vitamin E is transported in plasma lipoproteins and thus elevated lipid concentrations generally result in elevated vitamin E.24 Plasma lipids, particularly triglycerides, are increased in preeclampsia.7 Because lipid-adjustment may provide a better assessment of status of a-tocopherol, concentrations were compared both as nmol/mL plasma and as nmol/(mmol total cholesterol / mmol triglycerides).25,26 Total cholesterol and triglycerides were measured by enzymatic colorimetric methods on an Abbott VP Supersystem Bichromatic Analyzer (Abbott Laboratories, Irving, TX).27,28
Plasma a-tocopherol
Data Analysis
Extraction and measurement of the reduced form of a-tocopherol from plasma was adapted from Lang et. al.23 A Shimadzu LC-10A HPLC system was used with LC-10 pump, Supelcosil LC-8 column ( 3 mm particle size, 4.6 mm 1 15.9 cm; Supelco ) , and RF551-Fluorescence detector ( 292 nm excitation and 324 nm emission ) . The eluent was methanol and the flow rate was 1 ml /min. Under these conditions, the retention time for a-tocopherol was 12.0 min. Data
The individuals performing each assay were blinded as to the classification of samples. Student’s unpaired t-test (two-tailed) was used to compare the clinical characteristics of patients in Table 1 and these data are expressed as mean { SD. Nonparametric (Mann– Whitney U) analysis was used to compare antioxidant concentrations and also in vitro ascorbate consumption and ascorbate radical data because these data were significantly skewed. Medians and interquartile ranges are
Lipid correction of a-tocopherol concentrations
Table 3. Plasma Antioxidants Reserves: Prior to Labor and Without Magnesium Sulphate
Preeclampsia (n Å 7) Normal Pregnancy (n Å 8) Significance
Ascorbate nmol/mL
Total Thiols nmol/mL
Vitamin E nmol/mL
Vitamin E nmol/mmol Lipid*
11.1 (9.7 to 15.4) 21.7 (16.8 to 30.7) p õ .006
666 (573 to 742) 555 (461 to 598) p õ .03
26.8 (23.2 to 28.6) 21.3 (15.2 to 22.6) p õ .03
2.8 (2.5 to 2.9) 2.4 (2.1 to 2.9) N.S. (p Å .47)
Data are Medians and Interquartile Range. N.S.: not significant. * Lipid corrected Å vitamin E/(cholesterol / triglycerides) in nmol/mmol.
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Fig. 1. Typical electron paramagnetic resonance (EPR) spectra of tyrosinase-induced radical species generated from VP-16 in the presence and absence of plasma. Phenoxyl radicals were generated at constant rate by preincubation of tyrosinase (Sigma Chemical Co., St. Louis, MO) with the antitumor drug, etoposide (VP-16; 500 mmol/L; Sigma Chemical Co., St. Louis, MO) in 50 mmol/L phosphate buffer (PBS) at 257C. For optimization purposes, ascorbate reserves were assayed using 0.028 U/ mL tyrosinase and 50 mL plasma (Fig. 1 A) and total thiols were assayed using 2.8 U/ mL tyrosinase and 5 mL plasma (Fig. 1 B). Plasma was added (time Å 0) and the final volume adjusted to 60 mL with PBS (pH 7.4). Tyrosinase-catalyzed oxidation of VP-16 in PBS results in the generation of an oxygen-centered phenoxyl radical hyperfine EPR signal (top traces Fig. 1A and 1B). When plasma is added to this system the VP-16 EPR signal is not observed initially (lag period). Instead, a characteristic EPR signal of the semidehydroascorbate anion radical (ascorbate radical) can be detected (Fig. 1A, 2 min. through 12 min.). The duration of this signal is proportional to the ascorbate concentration and is quantitated using ascorbate standards. Subsequently, total plasma thiols are responsible for the part of the lag period during which no EPR signals are observed (Fig. 1B, 2 min. through 8 min.) and this interval is quantified as reduced glutathione (GSH) equivalents. The VP-16-dependent oxidation of thiols commences only after complete oxidation of ascorbate. The thiol oxidation-dependent lag period is followed by reappearance of the VP-16 radical signal (Fig. 1B, 12 min). [Reactions: 1) VP-16 / tyrosinaserVP-16i 2) Ascorbate / VP-16i r ascorbate radical / VP-16 3) RSH / VP-16i r RSi / VP-16].
reported as a measure of the central tendency of these data. Correlations were by Spearman rank test. Significance was accepted for p õ .05. RESULTS
Clinical data Table 1 summarizes the clinical characteristics of all patients from whom blood samples were obtained. By
definition, preeclamptic parturients had elevated blood pressures (p õ .001) and increased concentrations of uric acid (p õ .004) relative to normal pregnancy. Gestational age at delivery and infant birthweights were less for preeclamptic than normal pregnancies (p õ .001 each, Table 1). Mean hematocrit, nonpregnant blood pressures, body mass index, and maternal ages did not differ. Clinical characteristics of representative patient subsets used in each assay did not deviate sig-
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nificantly from those of their respective parent group (data not shown). Ascorbate, thiol, and a-tocopherol reserves Table 2 displays predelivery plasma concentrations of ascorbate, total thiols, and a-tocopherol. Median ascorbate reserves were about 50% lower in plasma from women with preeclampsia relative to normal pregnant controls (p õ .002). Ascorbate concentration was also measured by HPLC in 4 normal and 6 preeclampsia samples and these values agreed closely with those obtained by EPR spectroscopy (Fig. 2; r Å .92). The reserve of plasma total thiols was not significantly greater in preeclamptics than controls (Table 2; p Å .050). Concentrations of a-tocopherol did not differ significantly, either when expressed as nmol/mL plasma (p Å .06) or as nmol/(mmol cholesterol / mmol triglyceride) (p Å .53) (Table 2). The correlation coefficient for serum a-tocopherol and the sum of cholesterol plus triglyceride was 0.78 (p õ .001). Group differences in ascorbate remained and higher thiols were apparent in the preeclamptics when comparisons were restricted to patients neither in labor nor receiving magnesium sulphate prior to time of sampling (Table 3). In this subset of patients, a-tocopherol concentrations without lipid correction were increased in preeclampsia (p õ .03) but did not differ after lipid correction (p Å .47; Table 3). Ascorbate concentration and gestational age in the preeclampsia group did not correlate significantly (r Å .20; p Å .51). Median ascorbate reserves in the 5 preeclampsia samples obtained at term (37 to 39 wk gestation) did not differ from preeclampsia samples obtained preterm (32 to 36 wk gestation) [term 12.4 nmol/mL (9.6 to 16.5 nmol/mL interquartile range); preterm 10.8 nmol/mL (9.2 to 13.4 nmol/mL interquartile range); p Å .47]. Ascorbate reserves were lower in term preeclamptics relative to normal pregnant controls (all of whom were term) (p õ .02). This suggests that lesser mean gestational age of the preeclampsia group does not explain ascorbate differences. There was also no correlation between ascorbate concentration and duration of plasma freezer storage (r Å 0.135, p Å .65). There was a significant inverse correlation between gestational age and thiol concentration in preeclamptics (r Å 0.59; p õ .05). Additionally, preeclampsia and normal pregnancy thiols at term gestation were clearly not different [median thiols: preeclampsia 556 nmol/ mL (485 to 730 nmol/mL interquartile range), normal pregnant 516 nmol/mL (461 to 598 nmol/mL interquartile range), p Å .35)]. Thus, lesser gestational age may have tended to increase thiols in the preeclampsia
Fig. 2. The correlation of ascorbate concentrations measured by highpressure liquid chromatography (HPLC) and by electron paramagnetic (EPR) spectroscopy in normal ( s ) and preeclamptic ( l ) pregnancies.
group as a whole. This is consistent with a previous report that plasma thiols in gestationally equivalent normal pregnancy and preeclampsia patients are not different but are decreased relative to nonpregnant controls.29 There was no correlation between a-tocopherol concentration and gestational age for preeclampsia subjects (p Å .6, nonlipid corrected; p Å .95, lipid corrected). Blood ascorbate and thiol changes during incubation in vitro Fresh, EDTA-anticoagulated whole blood was incubated and endogenous plasma ascorbate and total thiols assayed by EPR spectroscopy as described in Methods. Figure 3 shows that in vitro consumption of ascorbate was accelerated relative to controls in 3 of 4 preeclampsia blood samples. Initial ascorbate concentrations were below the limit of detection ( õ 3 nmol/ mL) in a fourth preeclampsia sample. This fourth sample was excluded from subsequent analyses. Median initial ascorbate concentrations were 11.1 nmol/mL (11.0 to 15.5 nmol/mL interquartile range) in preeclampsia (n Å 3) and 16.7 nmol/mL (14.8 to 19.9 interquartile range) in normal pregnancy (n Å 4) samples (p Å .16). The median time interval required for half-consumption of ascorbate was markedly less in the case of preeclampsia [preeclampsia: 95 min (69 to 136 interquartile range); normal pregnancy: 360 min (302 to 410 interquartile range); p õ .04]. Figure 4 shows that no apparent decline in thiols occurred during 1200 min incubation of the same samples.
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Fig. 3. Plasma ascorbate concentration after incubation of whole blood. Fresh whole blood (EDTA-anticoagulated) was incubated at 257C in the dark and plasma was harvested from aliquots of each blood sample at the times indicated. The functional reserve of the reduced form of ascorbate was subsequently analyzed by EPR spectroscopy as described in Methods. Values for each patient plasma are connected by a line.
Semidehydroascorbate anion radical signal intensity Ascorbate concentrations in plasma were normalized by addition of exogenous ascorbate and ascorbate free radical signal monitored over time as described in Methods. Figure 5 shows time-dependent changes in ascorbate free radical EPR spectra in representative preeclampsia and normal pregnancy plasma samples. Figure 6 depicts these changes for individual and pooled plasma, expressed as a percentage of the initial signal amplitude measured in the normal pregnancy plasma pool. The initial steady-state signal amplitude in plasma from preeclamptic patients was in every case larger [median percent of normal pool: preeclampsia 165 (121 to 230 interquartile range); normal pregnant 83 (66 to 93 interquartile range); p õ .03)]. The ascorbate radical signal subsequently decayed more rapidly in preeclampsia plasma (Fig. 6). The initial ascorbate radical signal amplitude in ascorbate-supplemented Tris buffer was lower than in the normal pregnancy plasma pool; this signal did not decay appreciably over 300 min (data not shown). We tested whether chelatable iron might be the mediator of increased ascorbate-oxidation in preeclampsia plasma by comparing initial ascorbate radical EPR signal intensities in the presence and absence of the iron chelator, deferoxamine mesylate (Sigma Chemical Co., St. Louis, MO). Deferoxamine was added 7–10 min before EPR spectra were collected and was present during recording. The ascorbate radical signal detected in Tris buffer (not Chelex-100 pretreated) containing ascorbate is virtually eliminated by prior addition of 350 mmol/L deferoxamine (Fig. 7, columns A and B). Addition of 100 mmol/L iron to Tris buffer containing
ascorbate greatly increased the deferoxamine-inhibitable ascorbate radical signal intensity (Fig. 7, columns C-D). In pregnancy plasma supplemented with ascorbate, addition of iron to exceed transferrin iron binding reserve significantly (p õ .05) increases ascorbate radical steady-state concentration (Fig. 7, columns E-F) but addition of deferoxamine before iron prevents this increase (column G). As shown in columns H-K of Fig. 7, deferoxamine had no significant effect on the ascorbate radical signal elicited by ascorbate-supplemented preeclampsia or normal pregnancy plasma. Ascorbate stock was added to plasma in Chelex-100 pretreated Tris buffer as described in Methods.
DISCUSSION
Reactive oxygen species have important functions in normal physiology but their overproduction may result in, or be the result of, a number of disease states.30 Increased lipid peroxidation metabolites and other indicators of increased oxidative stress have been observed in preeclampsia.6,7,31 Certain antioxidant systems are compromised in preeclampsia relative to normal pregnancy, supporting the notion that imbalances favoring prooxidant over antioxidant systems might be involved in progression of the disorder.8,32 Whereas enzymatic antioxidants are of major importance for intracellular defenses, nonenzymatic antioxidants are the primary protectants against oxidative damage in the extracellular compartment.10 Based on both its concentration and its reaction rate with physiologically relevant oxidants, ascorbate is considered the most important small molecular, water soluble an-
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Fig. 4. Plasma total thiol concentration after incubation of whole blood. Fresh whole blood (EDTA-anticoagulated) was incubated at 257C in the dark and plasma was harvested from aliquots of each blood sample at the times indicated. The functional reserve of total plasma thiols (protein thiols plus glutathione) was analyzed by EPR spectroscopy as described in Methods. Samples were the same as those in Fig. 3. Values for each patient plasma are connected by a line.
tioxidant in plasma.10 During its antioxidant action, ascorbate undergoes two consecutive one electron oxidations to dehydroascorbic acid with intermediate formation of the ascorbate radical. In contrast to ascorbate
radical, ascorbate and dehydroascorbate are EPR-silent. The initial ascorbate radical signal amplitude is directly proportional to the overall rate of ascorbate oxidation, whereas the signal duration is inversely pro-
Fig. 5. Representative EPR spectra of ascorbate radical in plasma from preeclamptic (top) and normal (bottom) pregnancies. Plasma containing added ascorbate (1 mmol/L final concentration) was drawn into a gas-permeable Teflon tube and spectra continuously recorded at the time points indicated (post-ascorbate addition), at 257C and with spectrophotometer conditions as described in Methods. The amplitude of the EPR signal is proportional to the steady-state concentration of ascorbate radical.
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Fig. 6. Temporal changes in ascorbate radical signal amplitude in preeclampsia and normal pregnancy plasma after normalization of ascorbate concentrations by addition of exogenous ascorbate. Ascorbate concentrations in plasma were normalized (1 mmol/ L). EPR signals were monitored as described in Fig. 5 and in Methods. Data are expressed as percent of the initial signal intensity measured in a plasma pool comprised of plasma from 6 women with normal pregnancies. 7A: ( ww ) normal pregnancy pool (n Å 6); ( ss nn hh ) individual normal pregnancy plasma samples. 7B: ( l ) preeclamptic pregnancy pool ( n Å 6); ( mm jj ll ) individual preeclampsia samples.
portional. Ascorbate radical thus serves as a marker for the degree of ongoing oxidative stress in plasma.15 – 19 A major finding of the present report is that in vitro oxidative depletion of endogenous ascorbate is accelerated in whole blood of women with preeclampsia relative to normal pregnancy and that at least a portion of this increased activity is present in plasma (not requiring the presence of blood cells). Using EPR spectroscopy we find changes consistent with accelerated oxidation of ascorbate (increased initial ascorbate radical signal followed by its more rapid disappearance) in plasma from women with preeclampsia relative to controls. Exogenous ascorbate was added to these samples to equalize concentrations prior to analysis. Ascorbate’s role as the terminal small-molecular antioxidant 10,19 likely precludes the possibility that the relevant change in preeclampsia plasma is a decreased protectant activity against ascorbate oxidation. The plasma initial ascorbate radical signal amplitude was higher in normal pregnancy plasma relative to ascorbate-supplemented TRIS buffer and was further increased in preeclampsia plasma, a pattern consistent with increased prooxidant activity. The lack of consumption of thiols suggests that ascorbate is the principal, perhaps sole, small molecular reductant undergoing net oxidation in pregnancy plasma under the conditions examined. Our data also suggest the lack of significant thiol-dependent regen-
eration of ascorbate, a mechanism postulated to exist under certain conditions.33 Significant recycling of ascorbate by thiols would be expected to artificially increase our EPR spectroscopy estimates of ascorbate concentration because this assay (unlike HPLC) measures ongoing ascorbate activity (functional reserve). This would cause values to deviate from those obtained by HPLC. However, there was good agreement between EPR and HPLC determinations of ascorbate in the same samples. The instability of ascorbate in plasma must be considered when relating measured plasma ascorbate concentrations to concentrations in vivo. Mikhail et al.20 demonstrated that plasma concentrations of ascorbate are decreased in women with preeclampsia relative to uncomplicated pregnancies. Plasma samples in their study were acidified with metaphosphoric acid to stabilize ascorbate and analyzed on the day of sample collection. Our plasma samples were not stabilized by acidification and were analyzed after a short interval of storage at 0707C. Ascorbate concentrations in our normal pregnancy plasma samples were similar whereas preeclampsia concentrations were about 30 percent lower than those previously reported.20 Ascorbate is stable in untreated plasma samples at 0707 for at least 3 wk.34,35 Median ascorbate levels in the subset of preeclampsia samples stored 7 days or less were not greater than their respective group as a whole (com-
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Fig. 7. Initial ascorbate radical EPR signal intensity in Tris buffer and in ascorbate-supplemented preeclampsia and normal pregnancy plasma. 7A–7D are single determinations. (A) Ascorbate radical signal in 100 mmol/L Tris buffer (not Chelex-100 pretreated) containing 1 mmol/L ascorbate. (B). Same conditions as 7A but with prior addition of 350 mmol/L deferoxamine mesylate. (C). Increased ascorbate radical signal after addition of 100 mmol/L iron (FeSO4) to Tris buffer containing 1 mmol/ L ascorbate. (D). Same conditions as 7C but with addition of 350 mmol/L deferoxamine prior to iron. 7E-7G are means { SEM of plasma from 3 normal pregnant individuals. (E). Ascorbate radical signal elicited by normal pregnancy plasma supplemented with 1 mmol/L ascorbate. (F). Ascorbate radical signal in normal pregnancy plasma is increased after addition of 100 mmol/L iron to exceed plasma transferrin iron binding reserves. (G). Prior addition of 350 mmol/L deferoxamine prevents the increase seen in 7F. 7H-7I are means { SEM of plasma from 3 other normal pregnant individuals. (H). Ascorbate radical signal elicited by normal pregnancy plasma supplemented with 1 mmol/L ascorbate. (I). No effect of deferoxamine on the ascorbyl radical signal elicited by ascorbate-supplemented normal pregnancy plasma. 7J-7K are means { SEM of plasma from 4 women with preeclampsia. (J). Ascorbate radical signal elicited by preeclampsia plasma supplemented with 1 mmol/L ascorbate. (K). No effect of deferoxamine on the ascorbate radical signal elicited by ascorbate-supplemented preeclampsia plasma. * p õ .05 vs. 7E and 7G.
paring data from Fig. 3 and Table 2). Also, there was no correlation between ascorbate concentration and duration of freezer storage. However, the remarkably rapid in vitro oxidation of ascorbate in blood of women with preeclampsia suggests that our measured plasma concentrations might be an underestimate of in vivo concentrations in this group. Rates of ascorbate oxidation are influenced by the summated effects of a variety of blood components.18,36 Metabolism of ascorbate by neutrophils and red cells might contribute to ascorbate depletion in whole blood. There is evidence for neutrophil activation in preeclampsia and increased superoxide production upon activation.37,38 Extracellular ascorbate can be oxidatively depleted by neutrophil-derived reactive oxygen species.10 Red cell disturbances also characterize preeclampsia.39,40 Erythrocytes contribute to ascorbic acid regeneration in whole blood 41 and this activity could be compromised in preeclampsia and ultimately affect plasma ascorbate reserves.
Ascorbate-oxidizing activities in isolated plasma are necessarily independent of the presence of cells. The ascorbate radical signal detectable in plasma from healthy donors is readily modulated by ascorbate concentration, oxygen partial pressure, pH, and catalytic metal concentration.15,19 In the present study, ascorbate radical signal intensity was analyzed under conditions of identical ascorbate concentration and pH. Buffer solutions added to plasma were Chelex-100 pretreated making it unlikely that adventitious trace metals accounted for group differences. It should be noted that ascorbate oxidation rates are oxygen concentration [O2 ]-dependent and thus rates of ascorbate oxidation would likely be less in the lower [O2 ] of the circulation than that presently observed under atmospheric pressure.18 Iron ions are powerful catalysts of ascorbate oxidation in vitro and disorders leading to the presence of nontransferrin-bound iron in the circulation have been associated with decreases in plasma ascorbate, sug-
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gesting increased ascorbate oxidation.16,18 The ability of iron to oxidize ascorbate in solution is markedly potentiated upon complex of iron with EDTA but is suppressed by the iron chelator, deferoxamine.16,18,19 Although an increase in iron-EDTA complexes could increase ascorbate oxidation, we observed no diminution of ascorbate radical signal intensity after deferoxamine was added to preeclampsia plasma. We found previously that percent transferrin saturation by iron is two-fold increased in sera from women with preeclampsia relative to normal pregnancy. However, saturation in most cases was not to the extent that transferrin-unbound iron in the general circulation would be predicted.32 Additionally, bleomycin-detectable (‘‘catalytic’’) iron 42 was previously not found in any of 6 normal pregnancy and 6 preeclampsia serum samples (unpublished data, analysis courtesy of Drs. P. J. Evans and B. Halliwell, University of London King’s College). The implication is that chelatable iron is unlikely to explain differences in ascorbate oxidation between preeclampsia and normal pregnancy plasma. However, free hemoglobin and its oxidation products such as ferrylhemoglobin are potent oxidants capable of depleting plasma ascorbate.43 Although overt hemolysis is usually considered a late event in preeclampsia, subclinical hemolysis leading to increases in free hemoglobin might be an ongoing event.44,45 In plasma or sera exposed to water-soluble radical generators, the major low molecular weight antioxidants are typically consumed in the following order: ascorbate ° thiols ú uric acid ú a-tocopherol.10 However, we find that total plasma thiols are not decreased in women with preeclampsia relative to normal pregnancy and that decreases in ascorbate are not paralleled by decreases in thiols during incubation of blood samples. Also, the capacity of plasma to scavenge aqueous peroxyl radicals is markedly increased in preeclampsia compared to normal pregnancy and this antioxidant activity is primarily due to increased uric acid.46 In contrast to ascorbate, uric acid and protein thiols are not consumed in plasma exposed to lipid-soluble peroxyl radical generators.10 Based upon these observations, ascorbate consumption in preeclampsia plasma could result from radical generation in the lipid as opposed to aqueous phase of plasma. There is no consensus regarding circulating concentrations of the reduced form of a-tocopherol in preeclampsia relative to normal pregnancy. Uotila and coworkers 47 found increased serum a-tocopherol in severe, but not mild, preeclampsia, relative to normal pregnancy but these differences were eliminated after normalization to cholesterol. In contrast, there are reports of decreased vitamin E levels in severe, but not mild, preeclampsia.20,48 On the basis of blood pressure
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criteria for diagnosis of severe preeclampsia ( ú 160 mm Hg diastolic or ú 110 mm Hg systolic) 20,48 only three subjects in our study would qualify. Plasma ascorbate and vitamin E concentrations in these individuals did not deviate from their parent group (data not shown) but numbers are too limited for definitive comparison. Differences in diagnostic criteria for disease or disease severity, demographics, and nutrient intake before or during pregnancy could explain discrepancies regarding vitamin E status in preeclampsia. All patients in our study reported daily intake of prenatal vitamins containing vitamins E and C during pregnancy, a factor likely to diminish the influence of potential dietary biases. In summary, our data demonstrate that an ascorbateoxidizing activity is increased in blood of women with preeclampsia and that at least a portion of this increased activity is present in plasma (not requiring the presence blood cells). Although our study suggests chelatable iron is not the responsible agent, further work is needed to determine the mechanisms (factors) responsible for the activity increase and to determine if it precedes clinically evident illness. It also remains to be seen whether such phenomena contribute to the course of vascular dysfunction in preeclampsia or are merely an effect of the disease. However, involvement is possible in light of recent studies showing that endothelial-dependent vasodilator responses are improved by intraarterial ascorbate infusion in chronic smokers 49 and in patients with noninsulin-dependent diabetes mellitus 50 , conditions associated with increased free radical activity and depleted ascorbate reserves. Acknowledgements—This study was supported in part by National Institutes of Health grant HD30367. We thank Beth A. Hauth, Univ. Pittsburgh Dept. Epidemiology, for cholesterol and triglyceride determinations and Cindy Schatzman and the other nursing staff of Magee-Womens Research Institute for help in blood sample collection. We also thank Drs. Patricia J. Evans and Barry Halliwell, University of London King’s College, UK, for analysis of serum samples for bleomycin-detectable iron.
REFERENCES 1. Chesley, L. C. Hypertensive disorders of pregnancy. New York: Appleton-Century-Crofts; 1978. 2. Roberts, J. M.; Taylor, R. N.; Goldfien, A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am. J. Hypertens. 4:700–708; 1991 3. Taylor, R. N.; Varma, M.; Teng, N. N.; Roberts, J. M. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J. Clin. Endocrinol. Metab. 71:1675–1677; 1990. 4. Taylor, R. N.; Crombleholme ,W. R.; Friedman ,S. A.; Jones, L. A.; Casal, D. C.; Roberts, J. M. High plasma cellular fibronectin levels correlate with biochemical and clinical features of preeclampsia but cannot be attributed to hypertension alone. Am. J. Obstet. Gynecol. 165:895–901; 1991. 5. Baker, P. N.; Davidge, S. T.; Barankiewicz , J.; Roberts, J. M.
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P.; Tornvall, P.; Hamsten, A. Inverse relation between the concentration of low-density-lipoprotein vitamin E and severity of coronary artery disease. Am. J. Clin. Nutr. 63:377–385; 1996. Bucolo, G.; David, H. Quantitative determination of serum triglycerides by the use of enzymes. Clin. Chem. 19:476–482; 1973. Allain, C. C.; Poon, L. S.; Chan, C. G. S.; Richmond, W.; Fu, P. C. Enzymatic determination of total serum cholesterol. Clin. Chem. 20:470–475; 1974. Wisdom, S. J.; Wilson, R.; McKilliop, J. H.; Walker , J. J. Antioxidant systems in normal pregnancy and in pregnancy-induced hypertension. Am. J. Obstet. Gynecol. 165:1701–1704; 1991. Halliwell, B. The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis 23:118– 126; 1993. Walsh, S. W.; Wang, Y. Trophoblast and placental villous core production of lipid peroxides, thromboxane, and prostacyclin in preeclampsia. J. Clin. Endocrinol. Metab. 80:1888 – 1893; 1995. Hubel, C. A.; Kagan, V. E.; Kozlov, A. V.; Evans, R. W.; Davidge, S. T.; McLaughlin, M. K.; Roberts, J. M. Decreased transferrin and increased transferrin saturation in sera of women with preeclampsia: Implications for oxidative stress. Am. J. Obstet. Gynecol. 175:692–700; 1996. Meister, A. Glutathione-ascorbic acid antioxidant system in animals. J. Biol. Chem. 269:9397–9400; 1994. Maiani, G.; Azzini, E.; Ferro–Luzzi, A. Vitamin C. Int. J. Vitamin. Nutr. Res. 63:289–295; 1993. Bradley, D. W.; Emery, G.; Maynard , J. E. Vitamin C in plasma : A comparitive study of the vitamin stabilized with trichloroacetic acid or metaphosphoric acid and the effects of storage at 0707, 0207, 47, and 257 on the stabilized vitamin. Clin. Chim. Acta. 44:47–52; 1973. Sasaki, R.; Kurokawa, T.; Shibuya, D. Factors influencing ascorbate free radical formation. Biochem. Internat. 10:155–163; 1985. Greer, I. A.; Dawes, J.; Johnston, T. A.; Calder, A. A. Neutrophil activation is confined to the maternal circulation in pregnancyinduced hypertension. Obstet. Gynecol. 78:28–32; 1991. Tsukimori, K.; Maeda, H.; Ishida, K.; Nagata, H.; Koyanagi, T.; Nakano, H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. Obstet. Gynecol. 81:536–540; 1993. Entman, S. S.; Kambam, J. R.; Bradley, C. A.; Cousar, J. B. Increased levels of carboxyhemoglobin and serum iron as an indicator of increased red cell turnover in preeclampsia. Am. J. Obstet. Gynecol. 156:1169–1173; 1987. Cunningham, F. G.; Lowe, T.; Guss, S.; Mason, R. Erythrocyte morphology in women with severe preeclampsia and eclampsia. Am. J. Obstet. Gynecol. 153:358–363; 1985. May, J. M.; Qu, Z. C.; Whitesell, R. R. Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes. Biochemistry 34:12721–12728; 1995. Evans, P. J.; Halliwell, B. Measurement of iron and copper in biological systems: Bleomycin and copper-phenanthroline assays. In: Packer, L., ed. Methods in enzymology. vol. 233. New York: Academic Press; 1994:82–92. Everse, J.; Johnson, M. C.; Marini, M. A. Peroxidative activities of hemoglobin and hemoglobin dervatives. In: Everse, J.; Vandegriff, K.; Winslow, R., eds. Methods in enzymology. vol. 231. New York: Academic Press; 1994:547–561. Samuels, P.; Main, E. K.; Mennuti, M. T.; Gabbe, S. G. The origin of increased serum iron in pregnancy-induced hypertension. Am. J. Obstet. Gynecol. 157:721–725; 1987. Sarrel, P. M.; Lindsay, D. C.; Poole–Wilson, P. A.; Collins, P. Hypothesis: Inhibition of endothelium-derived relaxing factor by haemoglobin in the pathogenesis of preeclampsia. Lancet 336:1030–1032; 1990. Uotila, J. T.; Kirkkola, A. L.; Rorarius, M.; Tuimala, R. J.; Metsa–Ketela, T. The total peroxyl radical-trapping ability of plasma and cerebrospinal fluid in normal and preeclamptic parturients. Free Radic. Biol. Med. 16:581–590; 1994.
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Plasma ascorbate in preeclampsia 47. Uotila, J. T.; Tuimala, R. J.; Aarnio, T. M.; Pyykko, K. A.; Ahotupa, M. O. Findings on lipid peroxidation and antioxidant function in hypertensive complications of pregnancy. Br. J. Obstet. Gynecol. 100:270–276; 1993. 48. Wang, Y.; Walsh, S. W.; Guo, J.; Zhang, J. The imbalance between thromboxane and prostacyclin in preeclampsia is associated with an imbalance between lipid peroxides and vitamin E in maternal blood. Am. J. Obstet. Gynecol. 165:1695–1700; 1991. 49. Heitzer, T.; Just, H.; Munzel, T. Antioxidant vitamin C improves endothelial dysfunction in chronic smokers. Circulation 94:6– 9; 1996. 50. Ting, H. H.; Timimi, F. K.; Boles, K. S.; Creager, S. J.; Ganz,
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P.; Creager, M. A. Vitamin C improves endothelium-dependent vasodilation in patients with noninsulin-dependent diabetes. J. Clin. Invest. 97:22–28; 1996. ABBREVIATIONS
EDTA— [(ethylenedinitrilo)-tertaacetic acid tri-potassium salt] EPR— electron paramagnetic resonance HPLC— high pressure liquid chromatography; mmHg— millimeters of mercury
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PII S0891-5849(97)00010-5
Original Contribution INCREASED ASCORBATE RADICAL FORMATION AND ASCORBATE DEPLETION IN PLASMA FROM WOMEN WITH PREECLAMPSIA: IMPLICATIONS FOR OXIDATIVE STRESS
CARL A. HUBEL,* VALERIAN E. KAGAN, † ELENA R. KISIN, † MARGARET K. MCLAUGHLIN,* and JAMES M. ROBERTS * *Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA and the Departments of Obstetrics and Gynecology; † Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15213, USA (Received 10 November 1996; Revised 31 January 1997; Accepted 4 February 1997)
Abstract—There is evidence that oxidative stress accompanies preeclampsia and plasma ascorbate concentrations are reported to be decreased in the disorder. We tested the hypothesis that an ascorbate-oxidizing activity is increased in plasma from women with preeclampsia relative to normal pregnancy. Electron paramagnetic resonance (EPR) spectroscopy was used to determine (1) plasma functional reserves of ascorbate and total thiols, (2) temporal changes in ascorbate and thiol concentrations during incubation of whole blood in vitro, and (3) ascorbate radical signal kinetics in plasma after equalization of ascorbate concentrations. High-pressure liquid chromatography (HPLC) was used to measure plasma a-tocopherol. Ascorbate concentrations were 50 % lower in preeclampsia relative to normal pregnancy plasma but thiols and a-tocopherol did not differ. The elapsed time prior to half-consumption of plasma ascorbate was decreased approximately three-fold during incubation of whole blood from preeclamptics. No concomitant decrease in thiols was evident. The initial ascorbate radical signal amplitude was greater in preeclampsia plasma and then, in contrast to normal pregnancy plasma, decreased progressively. The iron chelator, deferoxamine had no effect on plasma ascorbate radical formation. We conclude that an ascorbate-oxidizing activity is increased in preeclampsia plasma which might contribute to vascular dysfunction in the disorder. q 1997 Elsevier Science Inc. Keywords—Ascorbate, Vitamin E, Plasma, Free radicals, Pregnancy, Preeclampsia
cells in culture in ways relevant to the endothelial pathology of the disease.2 – 5 There also is evidence that overproduction of reactive oxygen and nitrogen species accompanies preeclampsia and it has been postulated that oxidative events contribute to vascular dysfunction in the disorder.6 – 9 Human plasma contains an efficient arsenal of low molecular weight, nonenzymatic antioxidants that serve to protect the vasculature from oxidant damage.10,11 Of these, reduced ascorbate (vitamin C) is of primary importance in protecting plasma lipoproteins from peroxidation during exposure to a wide spectrum of water- or lipid-soluble free radical generators in vitro.10,12 The effectiveness of ascorbate against lipid-soluble radical initiators is due in part to its ability to regenerate a-tocopherol (vitamin E) by reducing tocopherol radicals in lipoproteins at the water-lipid in-
INTRODUCTION
Preeclampsia is the leading cause of maternal mortality in the Western World and is associated with a five-fold increase in perinatal mortality. The clinical markers of this human pregnancy-specific disorder include hypertension and proteinuria, which reverse after delivery.1 Although the cause of preeclampsia remains obscure, several lines of evidence indicate that dysfunction of the vascular endothelium accounts for the altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity which accompany the disorder.2 A number of reports indicate the presence of unidentified materials in plasma or serum from women with preeclampsia that alter the function of endothelial Address correspondence to: Carl A. Hubel, Magee-Womens Research Institute, 204 Craft Ave., Pittsburgh, PA 15213. 597
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terface.10,13,14 The relatively unreactive semidehydroascorbate anion radical (ascorbate radical) is the intermediate during the oxidation of ascorbate to dehydroascorbate and its measurement provides an index of oxidative stress in plasma.15 – 19 It was previously reported that plasma concentrations of ascorbate are decreased in mild and severe preeclampsia whereas a-tocopherol and beta-carotene are decreased only in severe disease compared to normal pregnant controls.20 The oxidized form of ascorbate, dehydroascorbic acid, was not decreased suggesting that oxidative consumption of ascorbate occurred.20 The present study compared plasma reserves of ascorbate, total thiols (protein thiols plus glutathione), and a-tocopherol in primiparous, normal and preeclamptic pregnancies. Assessment of these data prompted us to examine whether endogenous factors in blood and plasma from women with preeclampsia escalate oxidative depletion of ascorbate in vitro. Freshly obtained, EDTA-anticoagulated blood samples from women with normal and preeclamptic pregnancies were incubated and plasma concentration changes in endogenous ascorbate and total thiols were measured over time by electron paramagnetic resonance (EPR) spectroscopy. We also used EPR spectroscopy to measure temporal changes in ascorbate radical signal amplitude in preeclampsia and normal pregnancy plasma after equalization of ascorbate concentrations by addition of exogenous ascorbate. Data presented suggest that an ascorbate-oxidizing activity is increased in plasma from women with preeclampsia and that this activity does not involve chelatable iron. MATERIALS AND METHODS
Patient selection Subjects were recruited at the time of admittance to the labor and delivery ward at Magee-Womens Hospital as part of our ongoing investigation of preeclampsia. The study was approved by the hospital Institutional Review Board and all subjects gave informed written consent. Demographic and clinical data were collected at routine obstetric visits. Fourteen subjects had preeclampsia, defined using the criteria of gestational hypertension, proteinuria, hyperuricemia, and reversal of hypertension and proteinuria after delivery .1,2 Gestational hypertension was defined as an increase of 30 mmHg systolic or 15 mmHg diastolic blood pressure, as compared to values obtained before 20 wk gestation, or having an absolute blood pressure ú 140/90 mmHg after 20 wk gestation if earlier blood pressures were not known. Proteinuria was defined as ú 500 mg/ 24 h urine collection or ú 2/ on a voided or ú 1/ on a catheterized random urine specimen. Hyperuricemia
was defined as ú 1 standard deviation above usual values (at term ú 5.5 mg/dL). Seventeen subjects had uncomplicated pregnancies. These women were normotensive throughout gestation and without proteinuria or hyperuricemia. Patients with cigarette or illicit drug use, chronic hypertension, renal disease, or previous history of metabolic disorders were excluded. All subjects in our study reported daily intake of standard prenatal vitamins, which included vitamins C and E, during pregnancy. Blood samples Plasma was prepared from whole venous blood withdrawn into sterile tubes containing 4 mM potassium-EDTA. Plasma was separated by centrifugation, placed into aliquots under sterile conditions and frozen at 0707C until assayed. Serum samples for uric acid determination were withdrawn into sterile tubes and allowed to coagulate for 60 min at room temperature. The serum was separated by centrifugation and frozen at 0707C until assayed. Concentrations of antioxidants were measured in 12 preeclampsia and 13 normal predelivery plasma samples in which the interval between blood acquisition and plasma freezing was minimized (mean minutes { SD preeclampsia Å 54 { 7; normal Å 57 { 9). These samples were also matched for the duration of sample storage (mean weeks { SD preeclampsia Å 20.5 { 18; normal Å 20.4 { 18). Intravenous magnesium sulphate had been administered to 4 of 12 women with preeclampsia prior to antepartum blood draw. In 4 of 12 preeclamptics and 5 of 13 controls, labor had begun prior to antepartum blood draw. Antioxidant values for each group as a whole and those obtained prior to labor and magnesium sulphate are listed in Tables 2 and 3, respectively. The above samples were from a subset of the patients listed in Table 1 (Table 1 includes an additional 2 preeclamptic and 4 normal patients whose samples contributed to the plasma pools used in ascorbate radical analyses). The kinetics of endogenous ascorbate and thiol depletion in vitro were examined in freshly obtained, EDTA-anticoagulated whole venous blood (10 ml) from 4 women with preeclampsia and 4 with normal pregnancies. Blood samples were incubated at 257C, in the dark, under ambient air in a tissue culture hood. Aliquots (1 ml) were removed at successive time intervals from 40 to 1200 min (post-draw) and the plasma harvested by centrifugation. The plasma was immediately stored at 0707C and assayed for ascorbate and thiol concentrations within 7 days. These samples, as well as plasma assayed for ascorbate radical formation in vitro, were obtained from a subset of Table
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Table 1. Clinical Characteristics of Patient Groups
Maternal age (years) Prepregnant body mass index (kg/m2) Prepregnant BP (mmHg) Predelivery BP (mmHg) Wks. gestation at delivery Hematocrit (vol:vol %) Serum creatinine (mg/dL) Infant birthweight (g) Uric Acid (mg/dL)
Normal (n Å 17)
Preeclampsia (n Å 14)
26.3 { 6.0 22.6 { 2.6 107/66 { 7/8 115/70 { 10/6 39.9 { 0.8 35.8 { 2.4 N.M. 3485 { 509 4.5 { 1.2
26.6 { 6.2 24.0 { 3.9 112/68 { 10/6 150/93 { 10/5 35.1 { 2.9 35.5 { 2.3 0.9 { 0.3 2330 { 569 6.4 { 1.4
p Value N.S. N.S. N.S. õ0.001 õ0.001 N.S. õ0.001 õ0.004
Data are presented as mean { SD and were analyzed using the Student’s t-test. N.S.: not significant, p ú .05. Blood pressures are systolic/diastolic. N.M.: not measured.
1 patients prior to both onset of labor and magnesium sulphate administration.
Plasma ascorbate and thiol reserves Functional reserves of ascorbate and total thiols (protein thiols plus glutathione) were determined by EPR spectroscopy techniques previously described ( 21 and references therein). Briefly, ascorbate and reduced sulfhydryls can donate electrons to phenoxyl radicals, thus regenerating phenols and forming antioxidant radicals. For hindered phenols, this regeneration can be monitored by EPR spectroscopy. The enzyme tyrosinase is used as a catalyst to generate phenoxyl radicals from the hindered phenol, etoposide (VP-16). VP-16 radical can be detected by its characteristic EPR signal for 50–60 min. The duration of ascorbate radical EPR signal (ascorbate activity) is proportional to ascorbate concentration and is calibrated using ascorbate standards. Total thiol concentration is derived from the lag period after ascorbate consumption and before the reappearance of the VP-16 phenoxyl radical signal.21 Measurements were performed on a JEOL-RE1X spectrometer (JEOL, Kyoto, JPN) at 257C in gas-permeable, 0.8 mm internal diameter, 0.013 mm thickness Teflon tubing (from Alpha Wire Corporation, Elizabeth, NJ). Phenoxyl and ascorbate radical spectra were recorded at g Å 2.00, 335.5 mT center field, 10 mW power, 0.05 mT field modulation, 5 mT sweep width, 1600 receiver gain, and 1.0 second time constant. Spectra were collected using EPRMare software (Scientific Software Services, Bloomington, IL). The detection threshold was 3.0 mM for ascorbate and 12 mM for thiols. Further details of the method are provided in Fig. 1. Ascorbate concentrations were also measured in selected plasma samples using high pressure liquid chromatography (HPLC) with UV detection. Plasma proteins were precipitated using 10% acetic acid (CH3CO2H) and centrifugation (2,000 1 g, 10 min.)
and the supernatant used immediately for analysis. We used a Supelcosil LC-8 column (3 mm particle size, 4.6 1 159 mm; Supelco, Bellefonte, PA) and a mobile phase of 1:24 methanol : water adjusted to pH 3.0 by acetic acid at a flow rate of 1.0 ml/min. A Shimadzu LC-10A HPLC system was used with an LC-600 pump and SPD-10AV UV-detector set at 264 nm absorbance. Under these conditions, the retention time for ascorbate was 2.1 min. The peak corresponding to ascorbate concentration was completely eliminated by the addition of ascorbate oxidase to plasma.
Ascorbate free radical signal intensity in plasma supplemented with exogenous ascorbate Plasma (50 mL) was supplemented with ascorbate stock (made in 100 mmol/L TRIS, pH 7.4) to achieve 1 mmol/L final ascorbate concentration (60 ml total volume). Tris buffer was pretreated with Chelex-100 resin (Sigma Chemical Co. St. Louis, MO) by batch method to remove adventitious iron.22 Chelex-100 -pretreated Tris buffer containing 1 mmol/L ascorbate was used as the plasma-free control. EPR measurements were performed on a JEOL-RE1X spectrometer (JEOL, Kyoto, JPN) at 257C in gas-permeable, 0.8 mm internal diameter, 0.013 mm thickness Teflon tubing (from Alpha Wire Corporation, Elizabeth, NJ). After supplementation of samples with ascorbate, ascorbate radical spectra were immediately recorded at g Å 2.00, 335.5 mT center field, 10 mW power, 0.05 mT field modulation, 5 mT sweep width, 1600 receiver gain, and 1.0 second time constant. Spectra were collected using EPRMare software (Scientific Software Services, Bloomington, IL). No exogenous oxidation catalysts were added. We assayed predelivery plasma from 3 women with preeclampsia and 3 with normal pregnancies as well as 1 normal pregnancy and 1 preeclampsia plasma pool, each pool constructed by combining
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C. A. HUBEL et al. Table 2. Plasma Antioxidant Reserves in Women With Preeclampsia and Normal Pregnancy
Preeclampsia (n Å 12) Normal Pregnancy (n Å 13) Significance
Ascorbate nmol/mL
Total Thiols nmol/mL
Vitamin E nmol/mL
Vitamin E nmol/mmol Lipid*
11.0 (9.2 to 15.3) 21.1 (16.8 to 26.4) p õ .002
646 (518 to 794) 516 (476 to 598) N.S. (p Å .05)
25.7 (21.8 to 30.6) 21.3 (16.1 to 22.8) N.S. (p Å .06)
2.8 (2.4 to 2.9) 2.4 (2.0 to 3.0) N.S. (p Å .53)
Data are Medians and Interquartile Range. N.S.: not significant. * Lipid corrected: vitamin E/(cholesterol / triglycerides) in nmol/mmol.
were exported from the detector using Shimadzu EZChrom software ( Shimadzu Scientific Instruments, Colombia, MD ) .
equivalent volumes of 6 different plasma samples. These samples ( 9 per group ) were obtained prior to magnesium sulphate and labor and included all 7 of the preeclampsia and 5 of the 8 normal pregnancy samples of Table 3. An additional 2 preeclampsia and 4 normal samples contributed to the plasma pools and the patient data corresponding to these samples were incorporated into Table 1. Ascorbate radical signal intensity (amplitude) was expressed as a percentage of the initial signal intensity elicited from the ascorbate-supplemented, normal pregnancy plasma pool. In contrast to ascorbate radical, both ascorbate and dehydroascorbate are EPR-silent. Given similar initial ascorbate concentrations, the initial intensity of the ascorbate radical signal is thus directly proportional to the overall rate of ascorbate oxidation, whereas the signal duration is inversely proportional.
Vitamin E is transported in plasma lipoproteins and thus elevated lipid concentrations generally result in elevated vitamin E.24 Plasma lipids, particularly triglycerides, are increased in preeclampsia.7 Because lipid-adjustment may provide a better assessment of status of a-tocopherol, concentrations were compared both as nmol/mL plasma and as nmol/(mmol total cholesterol / mmol triglycerides).25,26 Total cholesterol and triglycerides were measured by enzymatic colorimetric methods on an Abbott VP Supersystem Bichromatic Analyzer (Abbott Laboratories, Irving, TX).27,28
Plasma a-tocopherol
Data Analysis
Extraction and measurement of the reduced form of a-tocopherol from plasma was adapted from Lang et. al.23 A Shimadzu LC-10A HPLC system was used with LC-10 pump, Supelcosil LC-8 column ( 3 mm particle size, 4.6 mm 1 15.9 cm; Supelco ) , and RF551-Fluorescence detector ( 292 nm excitation and 324 nm emission ) . The eluent was methanol and the flow rate was 1 ml /min. Under these conditions, the retention time for a-tocopherol was 12.0 min. Data
The individuals performing each assay were blinded as to the classification of samples. Student’s unpaired t-test (two-tailed) was used to compare the clinical characteristics of patients in Table 1 and these data are expressed as mean { SD. Nonparametric (Mann– Whitney U) analysis was used to compare antioxidant concentrations and also in vitro ascorbate consumption and ascorbate radical data because these data were significantly skewed. Medians and interquartile ranges are
Lipid correction of a-tocopherol concentrations
Table 3. Plasma Antioxidants Reserves: Prior to Labor and Without Magnesium Sulphate
Preeclampsia (n Å 7) Normal Pregnancy (n Å 8) Significance
Ascorbate nmol/mL
Total Thiols nmol/mL
Vitamin E nmol/mL
Vitamin E nmol/mmol Lipid*
11.1 (9.7 to 15.4) 21.7 (16.8 to 30.7) p õ .006
666 (573 to 742) 555 (461 to 598) p õ .03
26.8 (23.2 to 28.6) 21.3 (15.2 to 22.6) p õ .03
2.8 (2.5 to 2.9) 2.4 (2.1 to 2.9) N.S. (p Å .47)
Data are Medians and Interquartile Range. N.S.: not significant. * Lipid corrected Å vitamin E/(cholesterol / triglycerides) in nmol/mmol.
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Fig. 1. Typical electron paramagnetic resonance (EPR) spectra of tyrosinase-induced radical species generated from VP-16 in the presence and absence of plasma. Phenoxyl radicals were generated at constant rate by preincubation of tyrosinase (Sigma Chemical Co., St. Louis, MO) with the antitumor drug, etoposide (VP-16; 500 mmol/L; Sigma Chemical Co., St. Louis, MO) in 50 mmol/L phosphate buffer (PBS) at 257C. For optimization purposes, ascorbate reserves were assayed using 0.028 U/ mL tyrosinase and 50 mL plasma (Fig. 1 A) and total thiols were assayed using 2.8 U/ mL tyrosinase and 5 mL plasma (Fig. 1 B). Plasma was added (time Å 0) and the final volume adjusted to 60 mL with PBS (pH 7.4). Tyrosinase-catalyzed oxidation of VP-16 in PBS results in the generation of an oxygen-centered phenoxyl radical hyperfine EPR signal (top traces Fig. 1A and 1B). When plasma is added to this system the VP-16 EPR signal is not observed initially (lag period). Instead, a characteristic EPR signal of the semidehydroascorbate anion radical (ascorbate radical) can be detected (Fig. 1A, 2 min. through 12 min.). The duration of this signal is proportional to the ascorbate concentration and is quantitated using ascorbate standards. Subsequently, total plasma thiols are responsible for the part of the lag period during which no EPR signals are observed (Fig. 1B, 2 min. through 8 min.) and this interval is quantified as reduced glutathione (GSH) equivalents. The VP-16-dependent oxidation of thiols commences only after complete oxidation of ascorbate. The thiol oxidation-dependent lag period is followed by reappearance of the VP-16 radical signal (Fig. 1B, 12 min). [Reactions: 1) VP-16 / tyrosinaserVP-16i 2) Ascorbate / VP-16i r ascorbate radical / VP-16 3) RSH / VP-16i r RSi / VP-16].
reported as a measure of the central tendency of these data. Correlations were by Spearman rank test. Significance was accepted for p õ .05. RESULTS
Clinical data Table 1 summarizes the clinical characteristics of all patients from whom blood samples were obtained. By
definition, preeclamptic parturients had elevated blood pressures (p õ .001) and increased concentrations of uric acid (p õ .004) relative to normal pregnancy. Gestational age at delivery and infant birthweights were less for preeclamptic than normal pregnancies (p õ .001 each, Table 1). Mean hematocrit, nonpregnant blood pressures, body mass index, and maternal ages did not differ. Clinical characteristics of representative patient subsets used in each assay did not deviate sig-
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nificantly from those of their respective parent group (data not shown). Ascorbate, thiol, and a-tocopherol reserves Table 2 displays predelivery plasma concentrations of ascorbate, total thiols, and a-tocopherol. Median ascorbate reserves were about 50% lower in plasma from women with preeclampsia relative to normal pregnant controls (p õ .002). Ascorbate concentration was also measured by HPLC in 4 normal and 6 preeclampsia samples and these values agreed closely with those obtained by EPR spectroscopy (Fig. 2; r Å .92). The reserve of plasma total thiols was not significantly greater in preeclamptics than controls (Table 2; p Å .050). Concentrations of a-tocopherol did not differ significantly, either when expressed as nmol/mL plasma (p Å .06) or as nmol/(mmol cholesterol / mmol triglyceride) (p Å .53) (Table 2). The correlation coefficient for serum a-tocopherol and the sum of cholesterol plus triglyceride was 0.78 (p õ .001). Group differences in ascorbate remained and higher thiols were apparent in the preeclamptics when comparisons were restricted to patients neither in labor nor receiving magnesium sulphate prior to time of sampling (Table 3). In this subset of patients, a-tocopherol concentrations without lipid correction were increased in preeclampsia (p õ .03) but did not differ after lipid correction (p Å .47; Table 3). Ascorbate concentration and gestational age in the preeclampsia group did not correlate significantly (r Å .20; p Å .51). Median ascorbate reserves in the 5 preeclampsia samples obtained at term (37 to 39 wk gestation) did not differ from preeclampsia samples obtained preterm (32 to 36 wk gestation) [term 12.4 nmol/mL (9.6 to 16.5 nmol/mL interquartile range); preterm 10.8 nmol/mL (9.2 to 13.4 nmol/mL interquartile range); p Å .47]. Ascorbate reserves were lower in term preeclamptics relative to normal pregnant controls (all of whom were term) (p õ .02). This suggests that lesser mean gestational age of the preeclampsia group does not explain ascorbate differences. There was also no correlation between ascorbate concentration and duration of plasma freezer storage (r Å 0.135, p Å .65). There was a significant inverse correlation between gestational age and thiol concentration in preeclamptics (r Å 0.59; p õ .05). Additionally, preeclampsia and normal pregnancy thiols at term gestation were clearly not different [median thiols: preeclampsia 556 nmol/ mL (485 to 730 nmol/mL interquartile range), normal pregnant 516 nmol/mL (461 to 598 nmol/mL interquartile range), p Å .35)]. Thus, lesser gestational age may have tended to increase thiols in the preeclampsia
Fig. 2. The correlation of ascorbate concentrations measured by highpressure liquid chromatography (HPLC) and by electron paramagnetic (EPR) spectroscopy in normal ( s ) and preeclamptic ( l ) pregnancies.
group as a whole. This is consistent with a previous report that plasma thiols in gestationally equivalent normal pregnancy and preeclampsia patients are not different but are decreased relative to nonpregnant controls.29 There was no correlation between a-tocopherol concentration and gestational age for preeclampsia subjects (p Å .6, nonlipid corrected; p Å .95, lipid corrected). Blood ascorbate and thiol changes during incubation in vitro Fresh, EDTA-anticoagulated whole blood was incubated and endogenous plasma ascorbate and total thiols assayed by EPR spectroscopy as described in Methods. Figure 3 shows that in vitro consumption of ascorbate was accelerated relative to controls in 3 of 4 preeclampsia blood samples. Initial ascorbate concentrations were below the limit of detection ( õ 3 nmol/ mL) in a fourth preeclampsia sample. This fourth sample was excluded from subsequent analyses. Median initial ascorbate concentrations were 11.1 nmol/mL (11.0 to 15.5 nmol/mL interquartile range) in preeclampsia (n Å 3) and 16.7 nmol/mL (14.8 to 19.9 interquartile range) in normal pregnancy (n Å 4) samples (p Å .16). The median time interval required for half-consumption of ascorbate was markedly less in the case of preeclampsia [preeclampsia: 95 min (69 to 136 interquartile range); normal pregnancy: 360 min (302 to 410 interquartile range); p õ .04]. Figure 4 shows that no apparent decline in thiols occurred during 1200 min incubation of the same samples.
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Fig. 3. Plasma ascorbate concentration after incubation of whole blood. Fresh whole blood (EDTA-anticoagulated) was incubated at 257C in the dark and plasma was harvested from aliquots of each blood sample at the times indicated. The functional reserve of the reduced form of ascorbate was subsequently analyzed by EPR spectroscopy as described in Methods. Values for each patient plasma are connected by a line.
Semidehydroascorbate anion radical signal intensity Ascorbate concentrations in plasma were normalized by addition of exogenous ascorbate and ascorbate free radical signal monitored over time as described in Methods. Figure 5 shows time-dependent changes in ascorbate free radical EPR spectra in representative preeclampsia and normal pregnancy plasma samples. Figure 6 depicts these changes for individual and pooled plasma, expressed as a percentage of the initial signal amplitude measured in the normal pregnancy plasma pool. The initial steady-state signal amplitude in plasma from preeclamptic patients was in every case larger [median percent of normal pool: preeclampsia 165 (121 to 230 interquartile range); normal pregnant 83 (66 to 93 interquartile range); p õ .03)]. The ascorbate radical signal subsequently decayed more rapidly in preeclampsia plasma (Fig. 6). The initial ascorbate radical signal amplitude in ascorbate-supplemented Tris buffer was lower than in the normal pregnancy plasma pool; this signal did not decay appreciably over 300 min (data not shown). We tested whether chelatable iron might be the mediator of increased ascorbate-oxidation in preeclampsia plasma by comparing initial ascorbate radical EPR signal intensities in the presence and absence of the iron chelator, deferoxamine mesylate (Sigma Chemical Co., St. Louis, MO). Deferoxamine was added 7–10 min before EPR spectra were collected and was present during recording. The ascorbate radical signal detected in Tris buffer (not Chelex-100 pretreated) containing ascorbate is virtually eliminated by prior addition of 350 mmol/L deferoxamine (Fig. 7, columns A and B). Addition of 100 mmol/L iron to Tris buffer containing
ascorbate greatly increased the deferoxamine-inhibitable ascorbate radical signal intensity (Fig. 7, columns C-D). In pregnancy plasma supplemented with ascorbate, addition of iron to exceed transferrin iron binding reserve significantly (p õ .05) increases ascorbate radical steady-state concentration (Fig. 7, columns E-F) but addition of deferoxamine before iron prevents this increase (column G). As shown in columns H-K of Fig. 7, deferoxamine had no significant effect on the ascorbate radical signal elicited by ascorbate-supplemented preeclampsia or normal pregnancy plasma. Ascorbate stock was added to plasma in Chelex-100 pretreated Tris buffer as described in Methods.
DISCUSSION
Reactive oxygen species have important functions in normal physiology but their overproduction may result in, or be the result of, a number of disease states.30 Increased lipid peroxidation metabolites and other indicators of increased oxidative stress have been observed in preeclampsia.6,7,31 Certain antioxidant systems are compromised in preeclampsia relative to normal pregnancy, supporting the notion that imbalances favoring prooxidant over antioxidant systems might be involved in progression of the disorder.8,32 Whereas enzymatic antioxidants are of major importance for intracellular defenses, nonenzymatic antioxidants are the primary protectants against oxidative damage in the extracellular compartment.10 Based on both its concentration and its reaction rate with physiologically relevant oxidants, ascorbate is considered the most important small molecular, water soluble an-
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Fig. 4. Plasma total thiol concentration after incubation of whole blood. Fresh whole blood (EDTA-anticoagulated) was incubated at 257C in the dark and plasma was harvested from aliquots of each blood sample at the times indicated. The functional reserve of total plasma thiols (protein thiols plus glutathione) was analyzed by EPR spectroscopy as described in Methods. Samples were the same as those in Fig. 3. Values for each patient plasma are connected by a line.
tioxidant in plasma.10 During its antioxidant action, ascorbate undergoes two consecutive one electron oxidations to dehydroascorbic acid with intermediate formation of the ascorbate radical. In contrast to ascorbate
radical, ascorbate and dehydroascorbate are EPR-silent. The initial ascorbate radical signal amplitude is directly proportional to the overall rate of ascorbate oxidation, whereas the signal duration is inversely pro-
Fig. 5. Representative EPR spectra of ascorbate radical in plasma from preeclamptic (top) and normal (bottom) pregnancies. Plasma containing added ascorbate (1 mmol/L final concentration) was drawn into a gas-permeable Teflon tube and spectra continuously recorded at the time points indicated (post-ascorbate addition), at 257C and with spectrophotometer conditions as described in Methods. The amplitude of the EPR signal is proportional to the steady-state concentration of ascorbate radical.
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Fig. 6. Temporal changes in ascorbate radical signal amplitude in preeclampsia and normal pregnancy plasma after normalization of ascorbate concentrations by addition of exogenous ascorbate. Ascorbate concentrations in plasma were normalized (1 mmol/ L). EPR signals were monitored as described in Fig. 5 and in Methods. Data are expressed as percent of the initial signal intensity measured in a plasma pool comprised of plasma from 6 women with normal pregnancies. 7A: ( ww ) normal pregnancy pool (n Å 6); ( ss nn hh ) individual normal pregnancy plasma samples. 7B: ( l ) preeclamptic pregnancy pool ( n Å 6); ( mm jj ll ) individual preeclampsia samples.
portional. Ascorbate radical thus serves as a marker for the degree of ongoing oxidative stress in plasma.15 – 19 A major finding of the present report is that in vitro oxidative depletion of endogenous ascorbate is accelerated in whole blood of women with preeclampsia relative to normal pregnancy and that at least a portion of this increased activity is present in plasma (not requiring the presence of blood cells). Using EPR spectroscopy we find changes consistent with accelerated oxidation of ascorbate (increased initial ascorbate radical signal followed by its more rapid disappearance) in plasma from women with preeclampsia relative to controls. Exogenous ascorbate was added to these samples to equalize concentrations prior to analysis. Ascorbate’s role as the terminal small-molecular antioxidant 10,19 likely precludes the possibility that the relevant change in preeclampsia plasma is a decreased protectant activity against ascorbate oxidation. The plasma initial ascorbate radical signal amplitude was higher in normal pregnancy plasma relative to ascorbate-supplemented TRIS buffer and was further increased in preeclampsia plasma, a pattern consistent with increased prooxidant activity. The lack of consumption of thiols suggests that ascorbate is the principal, perhaps sole, small molecular reductant undergoing net oxidation in pregnancy plasma under the conditions examined. Our data also suggest the lack of significant thiol-dependent regen-
eration of ascorbate, a mechanism postulated to exist under certain conditions.33 Significant recycling of ascorbate by thiols would be expected to artificially increase our EPR spectroscopy estimates of ascorbate concentration because this assay (unlike HPLC) measures ongoing ascorbate activity (functional reserve). This would cause values to deviate from those obtained by HPLC. However, there was good agreement between EPR and HPLC determinations of ascorbate in the same samples. The instability of ascorbate in plasma must be considered when relating measured plasma ascorbate concentrations to concentrations in vivo. Mikhail et al.20 demonstrated that plasma concentrations of ascorbate are decreased in women with preeclampsia relative to uncomplicated pregnancies. Plasma samples in their study were acidified with metaphosphoric acid to stabilize ascorbate and analyzed on the day of sample collection. Our plasma samples were not stabilized by acidification and were analyzed after a short interval of storage at 0707C. Ascorbate concentrations in our normal pregnancy plasma samples were similar whereas preeclampsia concentrations were about 30 percent lower than those previously reported.20 Ascorbate is stable in untreated plasma samples at 0707 for at least 3 wk.34,35 Median ascorbate levels in the subset of preeclampsia samples stored 7 days or less were not greater than their respective group as a whole (com-
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Fig. 7. Initial ascorbate radical EPR signal intensity in Tris buffer and in ascorbate-supplemented preeclampsia and normal pregnancy plasma. 7A–7D are single determinations. (A) Ascorbate radical signal in 100 mmol/L Tris buffer (not Chelex-100 pretreated) containing 1 mmol/L ascorbate. (B). Same conditions as 7A but with prior addition of 350 mmol/L deferoxamine mesylate. (C). Increased ascorbate radical signal after addition of 100 mmol/L iron (FeSO4) to Tris buffer containing 1 mmol/ L ascorbate. (D). Same conditions as 7C but with addition of 350 mmol/L deferoxamine prior to iron. 7E-7G are means { SEM of plasma from 3 normal pregnant individuals. (E). Ascorbate radical signal elicited by normal pregnancy plasma supplemented with 1 mmol/L ascorbate. (F). Ascorbate radical signal in normal pregnancy plasma is increased after addition of 100 mmol/L iron to exceed plasma transferrin iron binding reserves. (G). Prior addition of 350 mmol/L deferoxamine prevents the increase seen in 7F. 7H-7I are means { SEM of plasma from 3 other normal pregnant individuals. (H). Ascorbate radical signal elicited by normal pregnancy plasma supplemented with 1 mmol/L ascorbate. (I). No effect of deferoxamine on the ascorbyl radical signal elicited by ascorbate-supplemented normal pregnancy plasma. 7J-7K are means { SEM of plasma from 4 women with preeclampsia. (J). Ascorbate radical signal elicited by preeclampsia plasma supplemented with 1 mmol/L ascorbate. (K). No effect of deferoxamine on the ascorbate radical signal elicited by ascorbate-supplemented preeclampsia plasma. * p õ .05 vs. 7E and 7G.
paring data from Fig. 3 and Table 2). Also, there was no correlation between ascorbate concentration and duration of freezer storage. However, the remarkably rapid in vitro oxidation of ascorbate in blood of women with preeclampsia suggests that our measured plasma concentrations might be an underestimate of in vivo concentrations in this group. Rates of ascorbate oxidation are influenced by the summated effects of a variety of blood components.18,36 Metabolism of ascorbate by neutrophils and red cells might contribute to ascorbate depletion in whole blood. There is evidence for neutrophil activation in preeclampsia and increased superoxide production upon activation.37,38 Extracellular ascorbate can be oxidatively depleted by neutrophil-derived reactive oxygen species.10 Red cell disturbances also characterize preeclampsia.39,40 Erythrocytes contribute to ascorbic acid regeneration in whole blood 41 and this activity could be compromised in preeclampsia and ultimately affect plasma ascorbate reserves.
Ascorbate-oxidizing activities in isolated plasma are necessarily independent of the presence of cells. The ascorbate radical signal detectable in plasma from healthy donors is readily modulated by ascorbate concentration, oxygen partial pressure, pH, and catalytic metal concentration.15,19 In the present study, ascorbate radical signal intensity was analyzed under conditions of identical ascorbate concentration and pH. Buffer solutions added to plasma were Chelex-100 pretreated making it unlikely that adventitious trace metals accounted for group differences. It should be noted that ascorbate oxidation rates are oxygen concentration [O2 ]-dependent and thus rates of ascorbate oxidation would likely be less in the lower [O2 ] of the circulation than that presently observed under atmospheric pressure.18 Iron ions are powerful catalysts of ascorbate oxidation in vitro and disorders leading to the presence of nontransferrin-bound iron in the circulation have been associated with decreases in plasma ascorbate, sug-
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gesting increased ascorbate oxidation.16,18 The ability of iron to oxidize ascorbate in solution is markedly potentiated upon complex of iron with EDTA but is suppressed by the iron chelator, deferoxamine.16,18,19 Although an increase in iron-EDTA complexes could increase ascorbate oxidation, we observed no diminution of ascorbate radical signal intensity after deferoxamine was added to preeclampsia plasma. We found previously that percent transferrin saturation by iron is two-fold increased in sera from women with preeclampsia relative to normal pregnancy. However, saturation in most cases was not to the extent that transferrin-unbound iron in the general circulation would be predicted.32 Additionally, bleomycin-detectable (‘‘catalytic’’) iron 42 was previously not found in any of 6 normal pregnancy and 6 preeclampsia serum samples (unpublished data, analysis courtesy of Drs. P. J. Evans and B. Halliwell, University of London King’s College). The implication is that chelatable iron is unlikely to explain differences in ascorbate oxidation between preeclampsia and normal pregnancy plasma. However, free hemoglobin and its oxidation products such as ferrylhemoglobin are potent oxidants capable of depleting plasma ascorbate.43 Although overt hemolysis is usually considered a late event in preeclampsia, subclinical hemolysis leading to increases in free hemoglobin might be an ongoing event.44,45 In plasma or sera exposed to water-soluble radical generators, the major low molecular weight antioxidants are typically consumed in the following order: ascorbate ° thiols ú uric acid ú a-tocopherol.10 However, we find that total plasma thiols are not decreased in women with preeclampsia relative to normal pregnancy and that decreases in ascorbate are not paralleled by decreases in thiols during incubation of blood samples. Also, the capacity of plasma to scavenge aqueous peroxyl radicals is markedly increased in preeclampsia compared to normal pregnancy and this antioxidant activity is primarily due to increased uric acid.46 In contrast to ascorbate, uric acid and protein thiols are not consumed in plasma exposed to lipid-soluble peroxyl radical generators.10 Based upon these observations, ascorbate consumption in preeclampsia plasma could result from radical generation in the lipid as opposed to aqueous phase of plasma. There is no consensus regarding circulating concentrations of the reduced form of a-tocopherol in preeclampsia relative to normal pregnancy. Uotila and coworkers 47 found increased serum a-tocopherol in severe, but not mild, preeclampsia, relative to normal pregnancy but these differences were eliminated after normalization to cholesterol. In contrast, there are reports of decreased vitamin E levels in severe, but not mild, preeclampsia.20,48 On the basis of blood pressure
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criteria for diagnosis of severe preeclampsia ( ú 160 mm Hg diastolic or ú 110 mm Hg systolic) 20,48 only three subjects in our study would qualify. Plasma ascorbate and vitamin E concentrations in these individuals did not deviate from their parent group (data not shown) but numbers are too limited for definitive comparison. Differences in diagnostic criteria for disease or disease severity, demographics, and nutrient intake before or during pregnancy could explain discrepancies regarding vitamin E status in preeclampsia. All patients in our study reported daily intake of prenatal vitamins containing vitamins E and C during pregnancy, a factor likely to diminish the influence of potential dietary biases. In summary, our data demonstrate that an ascorbateoxidizing activity is increased in blood of women with preeclampsia and that at least a portion of this increased activity is present in plasma (not requiring the presence blood cells). Although our study suggests chelatable iron is not the responsible agent, further work is needed to determine the mechanisms (factors) responsible for the activity increase and to determine if it precedes clinically evident illness. It also remains to be seen whether such phenomena contribute to the course of vascular dysfunction in preeclampsia or are merely an effect of the disease. However, involvement is possible in light of recent studies showing that endothelial-dependent vasodilator responses are improved by intraarterial ascorbate infusion in chronic smokers 49 and in patients with noninsulin-dependent diabetes mellitus 50 , conditions associated with increased free radical activity and depleted ascorbate reserves. Acknowledgements—This study was supported in part by National Institutes of Health grant HD30367. We thank Beth A. Hauth, Univ. Pittsburgh Dept. Epidemiology, for cholesterol and triglyceride determinations and Cindy Schatzman and the other nursing staff of Magee-Womens Research Institute for help in blood sample collection. We also thank Drs. Patricia J. Evans and Barry Halliwell, University of London King’s College, UK, for analysis of serum samples for bleomycin-detectable iron.
REFERENCES 1. Chesley, L. C. Hypertensive disorders of pregnancy. New York: Appleton-Century-Crofts; 1978. 2. Roberts, J. M.; Taylor, R. N.; Goldfien, A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am. J. Hypertens. 4:700–708; 1991 3. Taylor, R. N.; Varma, M.; Teng, N. N.; Roberts, J. M. Women with preeclampsia have higher plasma endothelin levels than women with normal pregnancies. J. Clin. Endocrinol. Metab. 71:1675–1677; 1990. 4. Taylor, R. N.; Crombleholme ,W. R.; Friedman ,S. A.; Jones, L. A.; Casal, D. C.; Roberts, J. M. High plasma cellular fibronectin levels correlate with biochemical and clinical features of preeclampsia but cannot be attributed to hypertension alone. Am. J. Obstet. Gynecol. 165:895–901; 1991. 5. Baker, P. N.; Davidge, S. T.; Barankiewicz , J.; Roberts, J. M.
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EDTA— [(ethylenedinitrilo)-tertaacetic acid tri-potassium salt] EPR— electron paramagnetic resonance HPLC— high pressure liquid chromatography; mmHg— millimeters of mercury
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