Small low-density lipoproteins and vascular cell adhesion molecule-1 are increased in association with hyperlipidemia in preeclampsia

Small Low-Density Lipoproteins and Vascular Cell Adhesion Molecule-1 Are Increased in Association With Hyperlipidemia in Preeclampsia Carl A. Hubel, Fiona Lyall, Lisa Weissfeld, Robin E. Gandley, and James M. Roberts The pregnancy disorder preeclampsia is characterized by endothelial cell dysfunction that may be promoted by abnormal increases in circulating lipids, particularly triglycerides and free fatty acids. Serum triglyceride concentration is a major regulatory determinant of low-density iipoprotein (LDL) size and density distribution. Smaller, denser LDL particles have several intrinsic properties capable of inducing endothelial dysfunction. The present nested, case-control study of gestationally matched preeclamptic and normal pregnant women tested the hypothesis that hypertriglyceridemia in preeclampsia is accompanied by decreases in LDL peak particle diameter (predominant LDL size). Plasma LDL peak particle diameter was determined by nondenaturing 2% to 16% polyacrylamide gel electrophoresis. Correlations of LDL diameter with the concentration of serum triglycerides, free fatty acids, total cholesterol, LDL-cholesterol, and apolipoprotein B (apo B) were determined. In the same individuals, we measured serum concentrations of a marker of vascular dysfunction previously reported to be increased in preeclampsia, soluble vascular cell adhesion molecule-1 (VCAM-1), and examined the association of VCAM-1 with LDL diameter and serum lipids, LDL peak particle diameter was decreased in preeclampsia relative to normal pregnancy (P < .01). The LDL-cholesterol:apo B ratio, which frequently decreases with decreasing LDL diameter, was also decreased (P < .04). Triglyceride concentrations were increased in preeclampsia (P < .0002), and there was a significant inverse relationship between LDL peak particle diameter and triglycerides (r = - . 5 5 , P < .02). Serum soluble VCAM-1 concentrations were markedly increased in preeclampsia (P < .0003). Apo B (P < .004), free fatty acids (P < ,01), total cholesterol (P < .01), and LDL-cholesterol (P < .02) were also increased. VCAM-1 correlated with apo B (r = .50, P < .03) and LDL-cholesterol {r = .50, P < .03), but showed no relationship with the LDL diameter, LDL-cholesterohapo B ratio, or other lipids. We conclude that the predominance of smaller, denser LDL, a potential contributor to endothelial cell dysfunction, is a feature of preeclampsia. However, the serum VCAM-1 level, one indicator of endothelial involvement, may be influenced more by quantitative lipoprotein changes (serum apo B or LDL-cholesterol) than by LDL particle size.

Copyright© 1998by W,B, Saunders Company HE HUMAN PREGNANCY-SPECIFIC disorder preeclampsia is a leading cause of maternal and neonatal death worldwide. Multiple lines of evidence indicate that dysfunction of the maternal vascular endothelium accounts for the altered vascular reactivity, activation of the coagulation cascade, loss of vascular integrity, and multisystem damage that occurs in preeclampsia. 1 However, the agents responsible for endothelial dysfunction in the disorder remain unknown. Serum triglyceride and free fatty acid concentrations are increased in women with preeclampsia relative to normal pregnancy.2,3 These changes occur well before, and thus are not a consequence of, clinically evident preeclarnpsia. 3 There is evidence that oxidative stress (an imbalance favoring oxidative over antioxidative processes) occurs in preeclampsia; the findings include an increased accumulation of lipid peroxidation products and oxidized low-density lipoprotein (LDL) autoantibodies in the circulation.4-7 Accordingly, there is considerable interest in the role of altered lipids in the promotion of oxidative stress and vascular dysfunction in the disorder. 3-9 LDL particles are heterogeneous in size, density, composition, and function. Using nondenaturing gradient gel electrophoresis, individuals can be classified on the basis of particle diameter corresponding to the major LDL band (LDL peak particle diameter). LDL peak particle diameter is frequently categorized into two principal phenotypes: pattern A (diameter --> 255 A) and pattern B (diameter < 255 ~)~0 One important consequence of hypertriglyceridemia relating to increased cardiovascular risk is to shift the spectrum of LDL subfractions toward smaller, denser (pattern B) particles. 11 Compared with larger and more buoyant LDL, small dense LDL particles are intrinsically more prone to oxidative modification upon contact with oxidants, H a characteristic that may contribute to the ability of small dense LDL to adversely affect numerous cellular

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Metabolism, Vol 47, No 10 (October), 1998:pp 1281-1288

processes relevant to vascular function.9,al Using analytical centrifugation to separate three LDL subfractions by density, Sattar et ala2 have shown that women with preeclampsia have a decreased concentration of more buoyant LDL (LDL I and II) and an increased concentration of dense LDL (LDL III) in association with hypertriglyceridemia. We sought to extend these studies by examining plasma LDL diameter as a continuous variable to test the association of LDL diameter with lipid concentrations and a marker of endothelial dysfunction in normal and preeclamptic pregnancies. Serum concentrations of the soluble form of vascular cell adhesion molecule (VCAM-1) are increased in women with preeclampsia.13-15 Although soluble VCAM- 1 may be produced by cells other than endothelial cells, its elevation is consistent with endothelial dysfunction and adhesion of activated leukocytes as a component mediating this dysfunction in preeclampsia. 15,16 Several lipids can upregulate endothelial cell surface expression of VCAM-1, including native and oxidized forms of LDL. 17,18Given that several significant changes in LDL chemical and physical properties accompany decreases in LDL diameter, 11J9 diameter/density changes may influence endothe-

From the Magee- Womens Research Institute, Pittsburgh; Department of Obstetrics and Gynecology and Reproductive Sciences and Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA; and Institute of Medical Genetics, Yorkhill Hospitals' Campus, Glasgow, UK. Submitted January 9, 1998; accepted March 9, 1998. Supported by National Institutes of Health Grant No. POI HD30367. Address reprint requests to Carl A. Hubel, PhD, Magee-Womens Research Institute, 204 Craft Ave, Pittsburgh, PA 15213. Copyright © 1998 by W.B. Saunders Company 0026-0495/98/4710-0020503.00/0

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lial V C A M - 1 expression and thus serum concentrations o f the soluble form o f this adhesion molecule. Our nested, case-control study was designed to test the hypothesis that, relative to normal pregnancy, preeclampsia is characterized by a qualitative change toward a p r e d o m i n a n c e o f smaller-diameter L D L in association with hypertriglyceridemia. Correlations o f L D L diameter with the concentration o f serum trigtycerides, free fatty acids, total cholesterol, LDL-cholesterol, and apolipoprotein-(apo B) were determined. Additionally, w e measured the serum concentration o f soluble V C A M - 1 in the same individuals and examined its correlation with L D L diameter and serum lipids.

were excluded. No subjects were on medication known to alter lipid metabolism. Blood samples were obtained before labor and administration of intravenous magnesium sulfate and were nonfasting (without standardization of the time interval between the last meal and venipuncture). Whole venous blood was withdrawn into sterile tubes containing 4 mmol/L potassium-EDTA, and the plasma was separated by centrifugation. Serum was obtained using dry sterile tubes in which the blood was allowed to coagulate for 60 minutes at room temperature before centrifugation. Serum and plasma samples were immediately stored at - 7 0 ° C (without thawing) until analysis. All assays were performed by an individual blinded as to the pregnancy classification of samples.

Gradient Gel Electrophoresis SUBJECTS A N D M E T H O D S

Study Population and Blood Samples Forty nulliparous women were studied, 20 with preeclampsia and 20 with uncomplicated pregnancy. 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 provided informed written consent. Clinical data were collected at routine obstetric visits and are summarized in Table 1. Preeclampsia was defined using the criteria of gestational hypertension, proteinuria, and hyperuricemia, and reversal of hypertension and proteinuria after delivery. Gestational hypertension was defined as an increase of 30 mm Hg systolic or 15 mm Hg diastolic blood pressure compared with values obtained before 20 weeks of gestation, or an absolute blood pressure of at least 140/90 mm Hg after 20 weeks if earlier blood pressure data were not available. Proteinuria was defined as greater than 500 mg/24-hour urine collection at least 2+ (100 mg/dL) on a voided specimen or at least 1+ (30 mg/dL) on a catheterized random urine specimen. Hyperuricemia was defined as greater than 1 standard deviation above the usual values at the time in gestation at which samples were obtained (at term, >5.5 mg/dL). For each individual with preeclampsia, we identified from the same study population an unrelated normally pregnant control matched for gestational age at the time of blood sampling (mean gestational age difference between pairs, 1.8 weeks; maximum, 3 weeks). Both groups had a median gestational age of 34.5 weeks (Table 1). Ten case-control pairs were studied at term (range, 36 to 42 weeks) and 10 pairs were preterm (range, 26 to 35 weeks). Controls were normotensive throughout gestation and were without proteinuria or hyperuricemia, and delivered at term. Patients with cigarette or illicit drug use, chronic hypertension, renal disease, or a previous history of metabolic disorders

Nondenaturing, 2% to 16% gradient polyacrylamide slab gels were cast in our laboratory according to a procedure provided by RJ. Blanche and R.M. Krauss of Lawrence Berkeley Laboratory (Berkeley, CA). Electrophoresis of the plasma was performed as modified from Nichols et al. 2° The gels were preconditioned in electrophoresis buffer (Tris 90 mmol/L, boric acid 81.5 mmol/L, tetrasodium EDTA 2.5 mmol/L, sodium azide 10 mmol/L [pH 8.3]) at 116 V for 20 minutes in mini 2-Gel electrophoretic chambers (Integrated Separation Systems, Natick, MA). Plasma aiiquots were combined (4:1 vol/vol) with 40% sucrose solution containing 0.1% bromophenol blue, and 10 gL of the mixture was loaded onto the gels. Diameter calibration standards were analyzed on separate lanes of each gel. These included (1) ferritin (122 A) and (2) thyroglobulin (170 ~) (Sigma, St Louis, MO) and (3). two plasma samples obtained in our laboratory containing different LDL peak particle diameters (distinct major bands) of 249 and 269.5 ,~. The plasma standards were previously calibrated against two LDL calibration plasma samples provided by RJ, Blanche and R.M. Kranss (Lawrence Berkeley Laboratory). Case-control pairs were thawed simultaneously and analyzed on adjacent lanes and on at least two gels. Care was taken to avoid any lane assignment patterns that might bias the results. Electrophoresis was performed at 22 V for 15 minutes followed by 70 V for 20 minutes and then 116 V for 24 hours (2,784 V-hours). Gels were stained With Oil Red O (Sigma), and the lane containing thyroglobulin and ferritin was additionally stained21 using a strip of filter paper moistened with Coomassie brilliant blue (Sigma). Gel images were recorded and scanned using a Windows-based optical imaging system (Harmony; Zidek, Rochester, NY). Conversion of the migration distance of the major LDL band into the peak particle diameter was made by a quadratic equation 22 (Fig 1), and average values for each individual were used in data analysis. This diameter was also used to classify LDL as predominantly subclass A (diameter --> 255 •~) or subclass B (diameter < 255 *). In our laboratory, interassay and

Table 1, Clinical Characteristics of the Patient Groups Preeclampsia (n = 20) Characteristic Maternal age (yr) Pre-pregnant body mass index (kg/m 2) Weeks gestation at sampling Weeks gestation at delivery Blood pressure (mm Hg) Pre-pregnant systolic Pre-pregnant diastolic Predelivery systolic Predelivery diastolic Infant birthweight (g) Uric acid (mg/dL) Abbreviation: NS, not significant (P > .05).

Normal Pregnancy(n = 20)

Median

Range

Median

27.0 25.6 34.5 34.5

18-35 16.8-42.2 26-40 26-42

22.0 21.7 34.5 40

120 70 157.5 98 1,961 6.3

98-130 60-81 137-186 81-106 514-4,355 5.3-9.6

112 70 117 71 3,537 4.2

Range 16-30 17.8-31.8 27-41 38-42 100-120 58-80 95-140 60-91 2,927-4,367 2.2-6.6

P <.01 NS NS <.02 NS NS <.0001 <.0001 <.O01 <.0001

LDL SIZE IN PREECLAMPSIA

1283

1

2

3

4

5

5~itin (122,~)

~rge

is

a/ kDt.(269.5 A) Apoferritin

1 0.5

f~x) = 6.694u-5"x^2 + -4 658ff-2*x + 9.20aft+0 R2 2 = 9 623E-1.R1^2 = 9.878E-1.R0~2 = 9.999E-1

0

120

1;0 2;0 210 Diameter (A,lgstroms)

......~

Thyroglobulin

A

B

Fig 1. (A) Representative calibration curve using gel migration distance in cm (y-axis) versus particle diameter of calibration standards (x-axis). (B) Corresponding 2% to 16% polyacrylamide gradient gel. Lane I, ferritin and thyroglobulin bands; lane 2, small LDL standard; lane 3, large LDL standard; lane 4, normal pregnancy LDL; lane 5, preeclampsia LDL

intraassay coefficients of variation for LDL peak particle diameter were 3.4% and 1.8%, respectively.

Lipid and Apolipoprotein Analyses These analyses were performed in single runs to avoid interassay variation. Serum cholesterol and triglyceride concentrations were determined by enzymatic methods. 23'24 Serum apo B concentrations were measured using a variation of the Boehringer-Mannheim procedure in which diluted serum is incubated with anti-human apo B antibodies and the resulting turbidity is read at 340/660 nm. The LDL-cholesterol level was measured directly in plasma by an immunoprecipitation method (Sigma). 25 Serum free fatty acid levels were measured using a commercially available kit (NEFA C; Wako Chemicals USA, Richmond, VA). Analyses were made using an Abbott VP Supersystem Bichromatic Analyzer (Abbott Laboratories, Irving, TX) in the Nutrition Laboratory of the Department of Epidemiology, University of Pittsburgh Graduate School of Public Health. The laboratory has maintained the required levels of proficiency to be included in the Centers for Disease Control-National Heart, Lung, and Blood Institute Lipid Standardization Program since 1982.

Serum VCAM-1 Serum concentrations of the cell adhesion molecule VCAM-1 were measured using a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Oxon, UK). The double-antibody sandwich assay uses two murine monoclonal antibodies directed against different epitopes on the human VCAM-1 molecnle: one coated onto the walls of the microtiter wells and the other conjugated to the enzyme horseradish peroxidase as the detecting system. Samples were diluted 1:50 in diluent supplied with the kit. Concentrations were determined against a standard curve generated using known concentrations of recombinant human VCAM-1. No cross-reactivity has been found with natural human immunoglobnlin G, recombinant human ICAM-1, or E-selectin. The lower limit of detection is 2 ng/mL and all samples exceeded this value. Samples were assayed in duplicate, and the mean of the two measurements was used.

Statistics Data are presented as the median and range. Because the subjects were matched for gestational age, analyses of case-control differences were made primarily by the nonparametric Wilcnxon sign-rank test. The significance level was computed based on an exact method using Monte Carlo simulation. This approach is generally more conservative, since significance levels based on exact methods tend to be larger than significance levels obtained from the standard normal approximations. The chi-square test with the Yates continuity correction was used to test whether the proportion of small dense LDL (subclass B) differed significantly in preeclampsia versus the control group. Pearson correlations were computed by taking the differences between preeclamptic and control values and using this outcome as the variable for correlation. Thus, the correlation between any two variables was computed by taking the difference in the first variable between the controls and preeclamptics and correlating this difference with the corresponding difference in the second variable. This approach was used because file subjects were matched. In this case, the significance levels are exact levels computed from Monte Carlo simulation. The matched pairs were broken to determine Pearson correlations for LDL size and triglycefides and to test (two-tailed t) for differences in the slope of the corresponding regression lines for normal and preeclamptic pregnancies. Significance was accepted at P less than .05. RESULTS

Clinical Data Table 1 s u m m a r i z e s the clinical characteristics o f all patients f r o m w h o m blood s a m p l e s were obtained. B y definition, preeclamptic parturients h a d an elevated blood p r e s s u r e and i n c r e a s e d uric acid concentration relative to n o r m a l parturients. Gestational age at delivery and infant b i r t h w e i g h t were less for preeclamptic v e r s u s n o r m a l pregnancies. M e d i a n gestational age at s a m p l i n g , p r e - p r e g n a n c y blood pressure, and prep r e g n a n c y b o d y m a s s i n d e x did n o t differ b e t w e e n groups. T h e

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HUBEL ET AL

270

double in preeclampsia compared with matched normal pregnancy (P < .0002 and .01, respectively). Apo B was increased and the LDL-cholesterol:apo B ratio was decreased in preeclampsia (P < .004 and .04, respectively). Total cholesterol (P < .01) and LDL-cholesterol (P < .02) concentrations were also increased.

P<0,01

o

2651 AA Oo :A AAAA zssl .........~ .................. ~

LDL P e a k 260 1 Particle Diameter

(A)

250~B ~Ak~k~ 2451 /k Z40! Preeclampsia

O

.....

Soluble VCAM-1 Women with preeclampsia had markedly higher serum VCAM-1 concentrations (preeclampsia: median, 904 ng/mL; range, 666 to 1,443; normal pregnancy: median, 628 ng/mL; range, 424 to 1,018; P < .0003). Two of the normal pregnancy samples were unavailable at the time of assay; analyses were thus performed on 18 case-control pairs. Inclusion of the two preeclampsia samples (with VCAM- 1 values of 831 and 1,413 ng/mL) that lacked corresponding controls would have had no effect on the preeclampsia group median and range (Fig 3).

O Normal P r e g n a n c y

Fig 2. Scatterplot of LDL peak particle diameter measured in EDTA plasma from women with preeclampsia and with uncomplicated pregnancies. Horizontal bars indicate the group median; broken horizontal line separates pattern A (diameter -> 255 A) from pattern B (diameter < 255 A) LDL.

median maternal age for the controls was 5 years less than for the preeclamptics (mean 4.7 years; P < .01). However, maternal age did not correlate with total cholesterol (r = .004, P = .98), LDL-cholesterol (r = .04, P = .82), LDL diameter (r = .05, P = .77), apo B (r = .06, P = .70), LDL-cholesterol: apo B ratio (r = .08, P = .60), triglycerides (r = .11, P = .49), or VCAM-1 (r = .13, P = .43).

LDL Peak Particle Diameter Only one LDL band was evident in approximately 60% of the samples in both groups. The remainder demonstrated one, or less commonly, two, secondary bands usually appearing as shoulders to the major band (data not shown); only the major band was used in data analysis. Figure 2 displays individual LDL peak particle diameters for women in each group. LDL peak particle diameter was significantly decreased in preeclampsia compared with normal pregnancy (preeclampsia: median, 254 A; range, 243 to 261; normal pregnancy: median, 258 range, 248 to 268; P < .01). In 15 of 20 case-control pairs, LDL diameter was smaller in women with preeclampsia. LDL with a diameter less than 255 ,£, (subclass B) predominated in 60% of women with preeclampsia, as compared with 25% in normal pregnancy (P < .05).

Lipid and ApoIipoprotein Concentrations Serum lipid and apo B concentrations are listed in Table 2. Triglyceride and free fatty acid concentrations were nearly

Correlative Data Figure 4 indicates that LDL diameter and triglyceride concentration correlated inversely within both the preeclampsia and control groups (preeclampsia, r = - . 6 5 , P < .002; controls, r = - . 6 1 , P < .005). There was a significant group difference in the regression line slope (P < .05), indicating a smaller decrease in LDL diameter for a given increase in triglycerides in the preeclampsia group (Fig 4). Using differences between matched preeclamptic and control values as the variable for correlation, the LDL peak particle diameter correlated inversely with serum triglyceride concentration (r = - . 5 5 , P < .02). The LDL-cholesterol:apo B ratio (a variable that generally decreases with decreasing particle diameter) also correlated inversely with triglycerides (r = - . 7 6 , P < .004) and positively with LDL diameter (r = .54, P < .02). VCAM-1 concentrations correlated significantly with apo B and LDL-cholesterol (r = .50, P < .03 for each case). VCAM-1 did not correlate with LDL diameter (r = .12, P = .59), the LDL-cholesterol:apo B ratio (r = - . 3 8 , P = . 10), triglycerides (r = .36, P = .11), free fatty acids (r = .34, P = .14), or total cholesterol (r = .24, P = .30). DISCUSSION

Normal human pregnancy is characterized by progressive increases in LDL and very-low-density lipoprotein (VLDL) concentrations in the maternal circulation as reflected by increases in triglycerides and cholesterol (approximately 300% and 50% by term, respectively). 26 The normal gestational increase in triglycerides is associated with a significant shift in LDL to subtypes of smaller diameter. 27,2s The principal finding

Table 2. Serum Lipid and Apo B Comparisons Preeclampsia (n = 20)

Normal Pregnancy (n = 20)

Variable

Median

Range

Median

Range

P<

Triglycerides (mg/dL) Free fatty acids (retool/L) Total cholesterol (mg/dL) LDL-cholesterol (mg/dL) Apo B (mg/dL) LDL-cholesterohapo B ratio

321.5 1.07 277 167 156 1.09

191-1,014 0.19-2.12 188-410 105-312 97-255 0.93-1.39

180 0.59 246 152 131 1.21

106-319 0.11-1.49 188-349 95-244 63-197 0.93-2.11

,0002 .01 .01 .02 .004 .04

LDL SIZE IN PREECLAMPSIA

1285

P<0.0003

,8oo 14oo2 VCAM-1 (ng/mL)

1200 ~

,ooo

O

800-

OOoo

• AI~•

6oo2

O

400200

t

Preeclampsia (n=18)

Normal Pregnancy (n=18)

Fig 3. Scatterplot of serum soluble VCAM-1 concentrations in women with preeclampsia and with uncomplicated pregnancies. Horizontal bars indicate the group median.

of the present study is that the LDL peak particle diameter was significantly decreased in nulliparous women with preeclampsia compared with nulliparous controls matched for gestational age. In 15 of 20 case-control pairs, LDL diameter was smaller in individuals with preeclampsia. The prevalence of pattern B LDL (<255 A diameter) was more than double in the preeclampsia group. We also report a decrease in the LDL-cholesterol:apo B ratio in preeclampsia. This change is frequently observed in individuals with smaller, denser LDL because these LDL particles are relatively depleted of cholesteryl esters and enriched in protein. 29 Despite significant group differences, it is evident from Fig 2 that the peak particle diameters overlap substantially. However, gestational progression toward smaller, denser LDL may account for some of this overlap. It is notable that five of the controls had very small LDL (<252 A diameter) and that four of these five samples were from term gestations. Similarly, five of seven preeclampsia samples with pattern A LDL (-->255 A) were preterm. Sattar et all= measured the mass of three LDL subfractions (LDL I, II, and III) isolated on the basis of increasing density

270O ~ O

O normal pregnant I • preeclampsia

260-

I

LDL Peak Particle Diameter

(A)

250-

240 0

200

400

600

800 1000

Triglyceride (mg/dL) Fig 4. Correlation of LDL peak particle diameter and nonfasting serum triglycerides in normal and preeclamptic pregnancies. Preeclampsia, y = 259.3 - 0.015x; normal pregnant, y = 266.7 - 0.053x.

from the plasma of women with preeclampsia and normal pregnancy. They observed that women with preeclampsia had lower concentrations of LDL I and II (the more buoyant, larger type) and markedly elevated concentrations of LDL III (denser, smaller variant) in conjunction with elevated plasma triglycerides. Thus, our qualitative determination of the predominant diameter is consistent with their assessment of the subfraction mass. Our data suggest that the plasma triglyceride concentration is a major determinant of LDL diameter during late normal pregnancy and preeclampsia; the correlation of these variables is in good agreement with a previous normal pregnancy study,27 and is consistent with the positive correlation between the mass of LDL III and plasma triglyceride reported by Sattar et al.12 Unexpectedly, we find a significant group difference in the slope of the regression line for these variables, indicating a smaller decrease in LDL size for a given increment in plasma triglyceride concentration in the preeclampsia group. Group differences in lipoprotein metabolism, or alternatively, general resistance to further decreases in LDL size at high triglyceride concentrations, could account for differences in slope. Our study was limited by the lack of standardization of meal content and the interval between the last meal and venipuncture, introducing a potential source of error to analyses involving triglycerides. However, recent studies of nonpregnant individuals have revealed strong inverse correlations between nonfasting triglycerides and LDL diameter3°,3~; the latter study pointed out that nonfasting triglycerides may be more indicative of average 24-hour triglyceride levels. Also, reports indicate no significant effect of fasting status on LDL particle diameter. 31,32 The immunoprecipitation method we used to determine LDLcholesterol, unlike the Friedewald calculation, also does not require fasting triglyceride levels. It has been noted that nonfasting serum triglyceride and free fatty acid concentrations are increased in preeclampsia relative to normal pregnancy,33 a finding confirmed by the present study. Since both fasting and nonfasting free fatty acids and triglycerides are increased in preeclampsia, the total exposure of endothelial cells to these lipids must be higher in preeclampsia. The absence of a correlation of maternal age with serum lipids and VCAM-1 suggests that the 5-year median age difference between the two groups is without significant effect. Although LDL particle diameter tends to decrease and triglycerides increase with advancing age in the general population, women may be substantially protected from this trend before menopause. 34,35 The enormous effects of pregnancy per se would likely overshadow any childbearing-age effects on lipids. However, systematic studies of younger versus older pregnant women for such measures are presently lacking. Soluble VCAM-1 levels show little variation in healthy pregnant women regardless of age (SD < 30% of mean in one study). 15 The normal pregnancy decreases in LDL size are driven by increases in serum triglycerides promoted, in turn, by increased adipocyte lipolytic activity secondary to the insulin-resistant condition of late gestation. 27,36 The increased release of free fatty acid and glycerol into the circulation increases the substrate for hepatic triglyceride (VLDL) synthesis. Estrogeninduced increases in the hepatic output of VLDL also likely o c c u r . 36,37 Increased transfer of triglyceride from VLDL to LDL coupled with hydrolysis of triglyceride in LDL may increase the

1286

production of smaller, denser LDL particles during pregnancy.3s Women with preeclampsia display accentuated insulin resistance relative to normal pregnancy.39,4° Heightened insulin resistance in preeclampsia likely increases the mobilization of fatty acids from visceral adipocytes, fueling overproduction of VLDL by the liver; this process, perhaps combined with suppression of lipoprotein lipase, would culminate in elevated serum triglycerides (and smaller, denser LDL). Sattar et alia report that preheparin hepatic lipase activity is increased in preeclampsia plasma, which, by increasing hydrolysis of LDL triglycerides, could partially explain the predominance of small dense LDL in the syndrome. The magnitude of the LDL diameter difference between groups in our study ( ~ 4 A) is similar to that found in several nonpregnancy diseases relative to controls. 30'41 However, our data in comparison to normal pregnancy studies suggest that the decrease in LDL size from early gestation to term in normal pregnancy is usually greater than the additional decrease associated with preeclampsia. We have noted an approximately 10-A average decrease in LDL diameter from earIy (5 to 12 weeks) to term gestation in uncomplicated pregnancies. 2s Silliman et al27 reported similar differences during late pregnancy compared with 6 weeks postpartum. We find that a substantial proportion of term controls manifest a predominance of small (pattern B) LDL, in agreement with the prior report. 27 The implications of the LDL size decrease in normal pregnancy and the further decrease in preeclampsia for maternal and fetal metabolism remain unknown. Given the overlap between groups, it is unlikely that a predominance of small dense LDL is the only agent responsible for vascular dysfunction in preeclampsia. Nevertheless, a number of deleterious effects are plausible. Cell culture experiments indicate that small dense LDL fractions have a greater capacity to stimulate thromboxane synthesis by endothelial cells42 and an increase in intracelhilar calcium in vascular smooth muscle, 43 changes potentially relevant to vasospasm in preeclampsia. 5 Other properties of small dense LDL particles include reduced receptormediated uptake in their native form, which may increase the plasma residence time and ultimately penetration into the arterial wall, and increased adhesiveness to proteoglycans of the artery subeudothelial interstitial matrix.51.44Oxidative modification presumably accounts for many of the adverse effects of small dense LDL on cellular function. H'45 Lipids in the small dense LDL particle are intrinsically more susceptible to oxidative modification (assessed as the lag time before acceleration of lipid peroxidation following incubation with oxidants). H Accumulation of lipid-laden macrophages (foam cells) in the decidual arterioles, a significant pathologic event in preeclampsia,46 and elevated titers of autoantibodies to oxidized LDL in the disorder 4 are suggestive of LDL oxidation. 4 The susceptibility of isolated LDL to Cu2+-mediated oxidation is reportedly increased in preeclampsia, 4v but it is not yet known whether this increased susceptibility is due to the decrease in LDL size. We report that serum soluble VCAM-1 concentrations are significantly increased in women with preeclampsia, in agreement with previous studies.1315 These data, including a report of elevated soluble VCAM-1 levels 3 to t5 weeks before the onset of clinical symptoms) 5 support the notion that disturbed

HUBEL ET AL

endothelial cell function is primary to the pathogenesis of preeclampsia. Increased expression of adhesion molecules by the endothelium could be responsible, in part, for the neutrophil activation that occurs in preeclamp sia. 15,16Circulating free fatty acids and LDL can modulate endothelial cell surface expression of VCAM-1.17'48 Human LDL induces VCAM-1 expression by human umbilical vein endothelial cells in culture in a dosedependent manner but independently of LDL oxidation, t7 VCAM-1 gene expression is frequently coupled to oxidative stress by redox-sensitive transcription factors, particularly NF-KB or NF-KB-like factors. 18,49 Oxidized LDL induces VCAM-I expression by endothelial cells in culture indirectly by potentiation of cytokine-acfivated VCAM-1 gene expression is or by inducing leukocytes to secrete factors that stimulate VCAM-1.so Changes in LDL diameter may impart substantial changes in LDL characteristics, and could thereby influence VCAM-1 expression and circulating concentrations of this adhesion molecule. However, contrary to our hypothesis, the serum soluble VCAM-1 concentration did not correlate inversely with LDL peak particle diameter. VCAM-1 also did not correlate with triglycerides or the LDL cholesterol:apo B ratio (variables associated with LDL size). These data do not support a direct link between a predominance of small dense LDL and increased VCAM-1 in preeclampsia. On the contrary, our correlative data are more consistent with an association between quantitative lipoprotein changes (increased apo B concentration reflecting increased number of apo B-containing particles, or increased LDL-cholesterol) and increased VCAM-1 expression. In summary, relative to normal pregnancy, the hypertriglyceridemia of preeclampsia is accompanied by a qualitative shift toward smaller, denser LDL. For reasons yet unclear, the slope of the regression line of LDL diameter and triglycerides was less steep in the preeclampsia group (smaller decreases in LDL diameter for given increases in triglycerides). Markedly increased serum concentrations of soluble VCAM- 1, indicative of endothelial involvement, were observed in the preeclampsia group. The VCAM-1 concentration correlated with apo B and LDL-cholesterol concentrations, but the hypothesized inverse relationship with LDL peak particle diameter was not observed. Although the cause and effect remains to be established, VCAM-1 levels may be influenced more by the concentration of apo B-containing lipoproteins than by the LDL peak particle diameter (predominant LDL size). The question remains as to whether an increased prevalence of small dense LDL contributes to any of the protean manifestations of vascular dysfunction in preeclampsia. ACKNOWLEDGMENT

We thank the nurses and staff of the Clinical Data Core and the nurses of Magee-Womens Hospital for invaluable assistance in obtaining biological samples, and we thank Marsha J. Gallaher and Yasser Shakir for skillful technical assistance in the gradient gel analyses. The assistance of Susan L. Davis in gel scanning and in preparation of Fig 1 is gratefully acknowledged. We thank Beth A. Hanth and Dr Rhobert W. Evans, University of Pittsburgh Department of Epidemiology, for lipid and apotipoprotein determinations. "Wealso thank Dr Ronald M. Krauss and Dr Patricia J. Blanche, Lawrence Berkeley Laboratory (Berkeley, CA), for providing the plasma LDL calibration standards and gradient gel recipe.

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