Plasma Concentrations of Carotenoids,
Retinol and Tocopherols in Preeclamptic
and Normotensive Pregnant Women
Cuilin Zhang
Department of Epidemiology
University of Washington, Seattle, USA
BACKGROUND (1)
• Preeclampsia - pregnancy induced hypertension
and proteinuria
– One of the common medical complications in
pregnancy;
– One of the leading causes of maternal mortality in
pregnancy;
– An important cause of premature delivery, fetal
growth retardation, perinatal mortality.
• Etiology & Pathogenesis
– Unsolved mysteries;
– Endothelial cell dysfunction appears to be the final
common pathway in the pathogenesis of
preeclampsia.
BACKGROUND (2)
Figure 1: Framework of the Biological Background of the Study
Scavenge
Free Radicals
Antioxidants
• Carotenoids
• Retinol
• Tocopherols
Inhibit
Reactive
Oxygen
Species
Promote
Endothelial
Cell
Dysfunction
Evoke
Preeclampsia
BACKGROUND (3)
Results from Previous Studies:
– Imbalances between lipid peroxidation and
antioxidant defenses in preeclampsia;
– lower plasma -carotene, -carotene and retinol
levels in preeclamptic women;
– -tocopherol in preeclampia: parodoxical;
– Reports concerning other lipid-soluble antioxidants
in pregnancies complicated with preeclampsia are
scarce;
HYPOTHESIS OF THIS STUDY
Plasma concentrations of fat-soluble vitamins
or their precursors and related antioxidants are
decreased in women with preeclampsia.
OBJECTIVES
Examine the relation of plasma concentrations
of carotenoids, tocopherols and retinol with the
risk of preeclampsia while the effects of
potential confounding factors were evaluated.
MATERIALS AND METHODS (1)
• Case-control study
• Conducted at the Maternal and Perinatal Hospital of
Lima and the Dos de Mayo Hospital in Lima, Peru, June
1997 - January 1998.
• Approved by the Ethical Committee of the Dos de Mayo
Hospital, the Maternal Perinatal Hospital of Lima, and
the Human Subjects Committee of the University of
Washington Medical Center.
• Potential study subjects - all new admissions to
antepartum wards, emergency room wards, and labor
and delivery wards of the study hospitals. Recruited
during their hospital stay.
MATERIALS AND METHODS (2)
• Definition of Cases
• Cases- women with a diagnosis of preeclampsia.
• Preeclampsia was defined as:
– persistent (i.e. lasting more than 6 hours) 15 mm Hg
diastolic rise or a 30 mm Hg rise in systolic blood
pressure, or
– persistent blood pressures of at least 140/90 mm Hg.
– and urine protein concentration ( 30 mg/dl or more (or
1+ on a urine dipstick) in at least two random
specimens collected at least 4 hours apart.
• 193 (97% of 199 eligible cases approached) cases
agreed to participate the study.
MATERIALS AND METHODS (3)
Definition of Controls
• Controls- pregnant women uncomplicated by
pregnancy-induced hypertension or proteinuria.
• Controls were frequency matched to cases for
gestational age of admission (within 2 weeks) and on
maternal age (within 5 years).
• 196 (96% of 204 eligible controls approached) controls
agreed to participate the study.
MATERIALS AND METHODS (4)
Exclusion Criteria
Subjects with chronic hypertension prior to pregnancy;
Subjects whose blood samples were drawn during the
intrapartum period or after delivery;
• Overall, 125 preeclampsia cases and 179 normotensive
controls comprised our study population .
MATERIALS AND METHODS (5)
Data Collection
Details regarding data collection methods have been
previously described.
1. Sanchez SE, Zhang C, Williams MA, et al. J Repro Immu
2000;47: 49-63.
2. Sanchez SE, Zhang C, Malinow MR, et al. Am J
Epidemiol (in press, 2000)
3. Ware-Jauregui S, Sanchez SE, Zhang C, et al. Int J
Gynecol Obstet. 1999;67:147-55.
MATERIALS AND METHODS (6)
• Standardized structured questionnaire, in-person interviews
• Maternal and infant records were reviewed
• Maternal anthropometric measures (height, weight and midarm circumference) were taken during participants' hospital
stay
• Gestational age-LMP, confirmed by ultrasound
• Blood samples were stored at the Blood Bank Laboratory of
Dos de Mayo Hospital after collection with standard method
and then shipped to the United States for biochemical
analyses
MATERIALS AND METHODS (7)
Laboratory Analyses
• PHS Core Laboratory, Fred Hutchinson Cancer Research
Center.
• Antioxidant nutrients- high performance liquid
chromatography (HPLC).
• Plasma total cholesterol concentrations-enzymatically,
standardized by the Lipid Standardization Program of the
Centers for Disease Control and Prevention, Atlanta, GA.
• All laboratory analyses were performed without knowledge
of pregnancy outcome.
MATERIALS AND METHODS (8)
Statistical Analyses
• Standard analysis of continuous data (i.e. means, SEM,
Student's t test) was performed.
• Comparisons of categorical variables were made
between case and control subjects using Chi-squared or
Fisher's exact tests.
• Spearman's correlation coefficient was calculated to
estimate the correlation between maternal plasma
concentrations of antioxidant and cholesterol.
MATERIALS AND METHODS (9)
Statistical Analyses
Categorizing each subject according to quartiles determined
by the distribution of antioxidant concentrations in controls.
Using the lowest category as the referent group, odds ratios
(i.e, estimates of relative risk) and their 95% CI were
calculated.
Logistic regression procedures were used to calculate odds
ratios adjusted by multiple confounding.
RESULTS (1)
Table 1. Distribution of Preeclampsia Cases and Normotensive Control
Subjects According to Selected Characteristics, Lima, Peru 1997-1998
Characteristic
Maternal Age < 19
(years)
19-34
 35
Maternal Age (years)†
Maternal Race/Ethnicity
European Ancestry
African Ancestry
Inca Indian Ancestry
Unmarried
No Family Support
< 12 years Education
Nulliparous
No Prenatal Vitamins
Smoked During Pregnancy
Unplanned Pregnancy
Pre-pregnancy BMI*†
Gestational Age (weeks)†
Total Cholesterol (mmol/l)†
•
•
•
p-values < 0.05 for comparisons
*Pre-pregnancy body mass index
†Mean  SEM
Cases (N = 125) Controls(N = 179)
n
%
n
%
21
16.8
84
57.2
20
16.0
26.6  0.6
26
14.6
125
69.8
28
16.1
26.3  0.5
10
8.0
6
4.8
109
87.2
85
68.0
7
5.6
20
16.0
59
47.2
53
42.4
1
0.8
39
31.2
24.1  0.4
36.0  0.3
6.5  0.2
17
9.5
0
0.0
162
90.5
120
67.0
13
7.3
28
15.6
57
31.8
69
38.5
4
2.2
42
23.5
23.4  0.2
37.3  0.3
6.1  0.1
between preeclampsia cases versus controls.
= BMI = weight (kg)/height (m2).
Table 2. Plasma Concentrations (mol/l) of Antioxidant Nutrients
among Preeclamptic and Normotensive Pregnant Women,
Lima, Peru, 1997-1998
Antioxidan
Nutrients
(mol/l)
Cases
N=125
Mean ± SEM (Median)
-carotene
-carotene
Lycopene
Controls
N=179
Mean ± SEM (Median) P-value
0.122 ± 0.006 (0.110)
0.122 ± 0.005 (0.110) 0.977
0.261 ± 0.014 (0.212)
0.258 ± 0.014 (0.220) 0.870
0.184 ± 0.012 (0.141)
0.193 ± 0.009 (0.160) 0.504
-cryptoxanthin 0.347 ± 0.023 (0.282)
0.428 ± 0.030 (0.271) 0.345*
Lutein
0.362 ± 0.014 (0.336)
0.347 ± 0.011 (0.329) 0.390
Zeaxanthin
0.073 ± 0.003 (0.067)
0.073 ± 0.002 (0.069) 0.990
Retinol
0.774 ± 0.038 (0.681)
0.871 ± 0.024 (0.855) <0.001
 -tocopherol
3.274 ± 0.144 (3.005)
2.976 ± 0.089 (2.753) 0.080
-tocopherol
25.448 ± 0.702 (24.526) 22.856 ± 0.464 (22.480) 0.002
-tocopherol ( µmol/l ) / cholesterol ( mmol/l )
3.961 ± 0.060 (3.890)
3.740 ± 0.043 (3.671) 0.033

*
P-values from Student’s t test.
P-values from Mann-Whitney U test
Table 4.
Odds Ratios (OR) and 95% Confidence Intervals (CI) of
Preeclampsia According to Quartile of Maternal Plasma
Antioxidant Concentrations, Lima, Peru, 1997-1998
Antioxidants
Concentrations Cases Controls
(mol)
n
n
OR (95% CI) OR (95% CI) †
-tocopherol
<18.337
18.337-22.480
22.481-26.013
>26.013
*Adjusted
22
26
27
50
46
44
45
44
1.00 referent
1.24 (0.61, 2.52)
1.26 (0.62, 2.54)
2.38 (1.23, 4.60)
1.00 referent
1.71 (0.75, 3.93)
1.83 (0.70, 4.75)
4.98 (1.77,13.98)
P-value*
0.003
P-value of test of linear trend.
†Adjusted for maternal age, nulliparity, pre-pregnancy body mass index (BMI) (quartile), use of
prenatal vitamins, gestational age at blood collection, education, planned pregnancy, and total
cholesterol concentration.
‡Adjusted for maternal age, nulliparity, pre-pregnancy BMI (quartile), use of prenatal vitamins,
gestational age at blood collection, education, and planned pregnancy
Table 3.
Odds Ratios (OR) and 95% Confidence Intervals (CI) of
Preeclampsia According to Quartile of Maternal Plasma
Antioxidant Concentrations, Lima, Peru, 1997-1998
Antioxidants
Concentrations Cases Controls
(mol)
n
n
OR (95% CI) OR (95% CI) †
Retinol
<0.642
0.642-0.855
0.856-1.082
>1.082
*Adjusted
57
25
20
23
47
44
45
43
1.00 referent
0.44 (0.23, 0.82)
0.36 (0.18, 0.70)
0.40 (0.21, 0.75)
1.00 referent
0.48 (0.24, 0.95)
0.31 (0.14, 0.66)
0.32 (0.15, 0.69)
P-value*
0.001
P-value of test of linear trend.
for maternal age, nulliparity, pre-pregnancy body mass index (BMI) (quartile), use of
prenatal vitamins, gestational age at blood collection, education, planned pregnancy, and total
cholesterol concentration.
‡Adjusted for maternal age, nulliparity, pre-pregnancy BMI (quartile), use of prenatal vitamins,
gestational age at blood collection, education, and planned pregnancy
†Adjusted
Table 5.
Odds Ratios (OR) and 95% Confidence Intervals (CI) of
Preeclampsia According to Quartile of Maternal Plasma
Antioxidant Concentrations, Lima, Peru, 1997-1998
Antioxidants
Concentrations Cases Controls
(mol)
n
n
OR (95% CI) OR (95% CI) †
P-value*
Ratio of -tocopherol (mol/l)/ total cholesterol (mmol/l)
<3.31
18
44
1.00 referent
1.00 referent‡
3.31-3.71
30
46
1.69 (0.82, 3.48)
1.73 (0.76, 3.92)
3.72-4.10
26
45
1.44 (0.68, 3.02)
1.85 (0.81, 4.24)
>4.10
50
44
2.88 (1.44, 5.76)
3.47 (1.60, 7.57) 0.002
*Adjusted
P-value of test of linear trend.
†Adjusted for maternal age, nulliparity, pre-pregnancy body mass index (BMI) (quartile), use of
prenatal vitamins, gestational age at blood collection, education, planned pregnancy, and total
cholesterol concentration.
‡Adjusted for maternal age, nulliparity, pre-pregnancy BMI (quartile), use of prenatal vitamins,
gestational age at blood collection, education, and planned pregnancy
RESULTS (2)
• Summary of results
– Plasma concentrations of retinol and (-tocopherol are
associated with the risk of preeclampsia.
• Retinol:
– A negative relationship between plasma concentrations of
retinol with risk of preeclampsia: mean value lower in
cases; ORs decreased across increasing quartiles of
plasma retinol concentrations.
RESULTS (3)
• -tocopherol, ratio of -tocopherol to total plasma
cholesterol concentrations :
– A strong positive relationship between plasma
concentrations of  -tocopherol with risk of preeclampsia:
mean values- higher in cases; ORs of preeclampsiaincreased with successively higher quartiles
• No clear patterns of preeclampsia risk associated with
plasma concentrations of -carotene, -carotene, cryptoxanthin, lutein, lycopene, zeaxanthin, and tocopherol, respectively.
DISCUSSION (1)
•
Potential Limitations & Strengths
•
Limitations
– Cannot determine cause- effect sequence;
– Lack of information pertaining to maternal dietary habits,
limited our ability to assess maternal dietary intake of
these antioxidants and risk of preeclampsia;
– Unable to measure the co-antioxidants (-tocopherolascorbic acid), and antioxidant enzymes (i.e. superoxide
dismutase and glutathione peroxidase).
DISCUSSION (2)
• Strengths
– Relatively large sample size
– Adjusting for potential confounders, esp. gestational
age at sample collection and plasma lipid level
– Differential misclassification of maternal plasma
antioxidant concentrations - unlikely
DISCUSSION ( 3 )
Explainations for possible pathophysiologic mechanisms
for the observed puzzling association between tocopherol and risk of preeclampsia
-- Compensatory increase in response to the elevated
oxidative stress of preeclampsia
-- -tocopherol: antioxidant, prooxidant?
-- Altered placental physiology in preeclampsia
Preeclampsia-related placental abnormalities the transfer
of maternal nutrients to the fetus
-tocopherol
(in vitro)
-- Mild oxidative condition
Prooxidant
Antioxidant
-- Strong oxidative condition or
-- Mild oxidative condition +
high concentration of coantioxidant (ascorbic acid,..)
TocH (-tocopherol) + R (free radical)
Toc + R
Toc + AscH (Ascorbic acid)
Toc + LH (Lipid)
RH + Toc  ( -tocopheroxyl radical)
Non-radical products
TocH + Asc 
TocH + L 
Anatol Kontush, etc. J. Lipid Research. 1996; 37: 1436-1448.
Bowry, V. W., etal. J. Am. Chem. Soc. 1993; 115:6029-6044.
Bowry, V. W., etal. Biochem. J. 1992; 288:341-344.
Bowry, V.W., etal. J. Biol. Chem. 1995; 270: 5756-5763.
Bisby, R. H., etal. Arch. Biochem. Biophys. 1995; 317: 170-178.
-tocopherol: antioxidant, prooxidant?
• It is possible that under the physiological oxidative
stress (if it is mild), commonly identified in
preeclampsia, after -tocopherol co-antioxidants such
as ascorbate acid are consumed, -tocopherol may act
as a prooxidant rather than an antioxidant.
CONCLUSION & FUTURE STUDIES
• Preeclampsia may not be a state of global antioxidant
deficiency in maternal peripheral circulation.
• Future prospective longitudinal studies involving
measurements of concentrations of antioxidant
nutrients and enzymes in blood and placental tissue and
oxidative condition are needed to confirm and expand
upon our findings.
ACKNOWLEDGEMENTS (1)
•
•
•
•
•
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This research was supported by awards from the National Institutes of Health (T37TW00049 and HD/HL R01-32562).
Michelle A. Williams ScD. Department of Epidemiology, University of Washington,
Seattle, WA, USA;
Irena B. King Ph.D. PHS Core Laboratory, Fred Hutchinson Cancer Research Center,
Seattle, WA, USA.
Wendy M. Leisenring, ScD. Division of Clinical Research, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA.
Suzie Ware-Jauregui. School of Medicine, University of Washington, Seattle, WA,
USA.
Sixto E. Sanchez MD, MPH. Dos de Mayo Hospital, Lima Peru.
Gloria Larrabure MD, Victor Bazul, MD. Materno-Perinatal Institute, Lima Peru.
The authors thank Mirtha Grande, Elena Sanchez, Nelly Toledo, Hong Tang,
Mohammed Adem, and June Hu for their skillful technical assistance.