Plasma Concentrations of Carotenoids, Retinol and Tocopherols in Preeclamptic and Normotensive Pregnant Women

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Plasma Concentrations of Carotenoids, Retinol and
Tocopherols in Preeclamptic and Normotensive
Pregnant Women

• Cuilin Zhang
• Department of Epidemiology
• University of Washington, Seattle, USA

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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.

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BACKGROUND (2)
Figure 1 Framework of the Biological Background
of the Study
Free Radicals
Promote Evoke
Scavenge Inhibit

• Antioxidants
• Carotenoids
• Retinol
• Tocopherols

Endothelial Cell Dysfunction
Preeclampsia
Reactive Oxygen Species
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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

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HYPOTHESIS OF THIS STUDY

• Plasma concentrations of fat-soluble vitamins or
  their precursors and related antioxidants are
  decreased in women with preeclampsia.

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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.

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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.

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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.

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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.

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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 .

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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 200047 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. 199967147-55.

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MATERIALS AND METHODS (6)

• Standardized structured questionnaire, in-person
  interviews
• Maternal and infant records were reviewed
• Maternal anthropometric measures (height, weight
  and mid-arm 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

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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-enzymatica
  lly, 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.

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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.

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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.

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RESULTS (1)
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Table 1. Distribution of Preeclampsia Cases and
Normotensive Control Subjects According to
Selected Characteristics, Lima, Peru 1997-1998

• Cases (N 125) Controls(N 179)
• Characteristic n n
• Maternal Age lt 19 21 16.8 26 14.6
• (years) 19-34 84 57.2 125 69.8
• ? 35 20 16.0 28 16.1
• Maternal Age (years) 26.6 ? 0.6 26.3 ? 0.5
• Maternal Race/Ethnicity
• European Ancestry 10 8.0 17 9.5
• African Ancestry 6 4.8 0 0.0
• Inca Indian Ancestry 109 87.2
  162 90.5
• Unmarried 85 68.0 120 67.0
• No Family Support 7 5.6 13 7.3
• lt 12 years Education 20 16.0 28 15.6
• Nulliparous 59 47.2 57 31.8?
• No Prenatal Vitamins 53 42.4 69 38.5
• Smoked During Pregnancy 1 0.8 4 2.2

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• Table 2. Plasma Concentrations (?mol/l) of
  Antioxidant Nutrients
• among Preeclamptic and Normotensive Pregnant
  Women, Lima, Peru, 1997-1998
• Antioxidan Cases
  Controls
• Nutrients N125 N179
• (?mol/l) Mean SEM (Median) Mean
  SEM (Median) P-value?
• ?-carotene 0.122 0.006 (0.110)
  0.122 0.005 (0.110) 0.977
• ?-carotene 0.261 0.014 (0.212)
  0.258 0.014 (0.220) 0.870
• Lycopene 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) lt0.001
• ? -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 Students t test.

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Table 4. Odds Ratios (OR) and 95 Confidence
Intervals (CI) of Preeclampsia According to
Quartile of Maternal Plasma Antioxidant
Concentrations, Lima, Peru, 1997-1998 Antioxidan
ts Concentrations Cases Controls (?mol)
n n OR (95 CI) OR (95 CI)
P-value ?-tocopherol lt18.337
22 46 1.00 referent 1.00
referent 18.337-22.480 26 44 1.24
(0.61, 2.52) 1.71 (0.75, 3.93) 22.481-26.013
27 45 1.26 (0.62, 2.54) 1.83 (0.70,
4.75) gt26.013 50 44 2.38
(1.23, 4.60) 4.98 (1.77,13.98)
0.003 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
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Table 3. Odds Ratios (OR) and 95 Confidence
Intervals (CI) of Preeclampsia According to
Quartile of Maternal Plasma Antioxidant
Concentrations, Lima, Peru, 1997-1998 Antioxidant
s Concentrations Cases Controls (?mol)
n n OR (95 CI) OR (95 CI)
P-value Retinol lt0.642 57
47 1.00 referent 1.00 referent
0.642-0.855 25 44 0.44 (0.23,
0.82) 0.48 (0.24, 0.95) 0.856-1.082 20
45 0.36 (0.18, 0.70) 0.31 (0.14,
0.66) gt1.082 23 43 0.40 (0.21,
0.75) 0.32 (0.15, 0.69) 0.001 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
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Table 5. Odds Ratios (OR) and 95 Confidence
Intervals (CI) of Preeclampsia According to
Quartile of Maternal Plasma Antioxidant
Concentrations, Lima, Peru, 1997-1998 Antioxidan
ts Concentrations Cases Controls (?mol)
n n OR (95 CI) OR (95 CI)
P-value Ratio of ?-tocopherol
(?mol/l)/ total cholesterol (mmol/l) lt3.31
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) gt4.10 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
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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.

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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 preeclampsia- increased with successively
  higher quartiles
• No clear patterns of preeclampsia risk associated
  with plasma concentrations of ?-carotene,
  ?-carotene, ?-cryptoxanthin, lutein, lycopene,
  zeaxanthin, and ?-tocopherol, respectively.

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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
  (??-tocopherol- ascorbic acid), and antioxidant
  enzymes (i.e. superoxide dismutase and
  glutathione peroxidase).

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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

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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

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?-tocopherol (in vitro)
-- Mild oxidative condition
Prooxidant
Antioxidant
-- Strong oxidative condition or -- Mild
oxidative condition high
concentration of co- antioxidant (ascorbic
acid,..)
TocH (?-tocopherol) R? (free radical)
RH Toc ? ( ?-tocopheroxyl radical)
Toc? R?
Non-radical products
Toc? AscH (Ascorbic acid) TocH
Asc ?
Toc? LH (Lipid) TocH L ?
Anatol Kontush, etc. J. Lipid Research. 1996
37 1436-1448. Bowry, V. W., etal. J. Am. Chem.
Soc. 1993 1156029-6044. Bowry, V. W., etal.
Biochem. J. 1992 288341-344. Bowry, V.W., etal.
J. Biol. Chem. 1995 270 5756-5763. Bisby, R.
H., etal. Arch. Biochem. Biophys. 1995 317
170-178.
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• ?-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.

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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.

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ACKNOWLEDGEMENTS (1)

• This research was supported by awards from the
  National Institutes of Health (T37-TW00049 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.