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Plasma total homocysteine in uncomplicated pregnancy and in preeclampsia
European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49
Plasma total homocysteine in uncomplicated pregnancy and in preeclampsia$ Eva Lo´pez-Quesadaa, M. Antonia Vilasecab,*, Jose M. Laillaa a
Obstetrics and Gynecology Department, Hospital Sant Joan de De´u, University of Barcelona, 08950-Esplugues de Llobregat, Barcelona, Spain b Biochemistry Department, Hospital Sant Joan de De´u, University of Barcelona, 08950-Esplugues de Llobregat, Barcelona, Spain Received 7 March 2002; accepted 2 September 2002
Abstract Objectives: (a) To establish the reference values for plasma total homocysteine in our pregnant population. (b) To determine the possible association between hyperhomocysteinemia and preeclampsia in our geographical area. Study design: Control–case study with 32 preeclamptic patients and 64 controls without pregnancy complications. Plasma total homocysteine, determined by HPLC (fluorescence detection), was correlated with serum folate and Vitamin B12 (analyzed by competitive protein binding chemiluminescent assay). Statistical analyses: Mann–Whitney, Wilcoxon and Spearman test (SPSS, 10.0). Results: Homocysteine concentrations in the controls were significantly higher while folate was significantly lower in the third trimester of pregnancy when compared with the second (P < 0:0001). Homocysteine and folate values were significantly higher in patients compared with controls in the third trimester (P ¼ 0:005 and 0.005, respectively). The OR for preeclampsia in hyperhomocysteinemia was 7.7 (95% CI: 1.7–34.8). Conclusion: Pregnant women with hyperhomocysteinemia have a 7.7-fold risk for preeclampsia (CI 95%: 1.7–34.8) compared with normal controls. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hyperhomocysteinemia; Pregnancy; Preeclampsia; Folate; Vitamin B12
1. Introduction Elevated plasma homocysteine is a risk factor for endothelial dysfunction and vascular diseases such as atherosclerosis and occlusive vascular disorders [1]. Hyperhomocysteinemia has also been associated with complications in pregnancy such as neural tube defects, repeated miscarriages, abruptio placentae, fetal death, preeclampsia and intrauterine growth retardation [2,3]. Preeclampsia represents one of the most important complications of pregnancy (5–7% of low-risk pregnancies), but little is known about its etiology. It may be defined as a pregnancy-specific occlusive vascular disorder characterized by endothelial cell dysfunction and increased platelet
$ Eva Lo´pez-Quesada is the recipient of a grant from the Hospital Sant Joan de De´u to perform the present study. The study was partially ´ TV3/993431. supported by the MARATO * Corresponding author. Tel.: þ34-93-280-4000; fax: þ34-93-280-3626. E-mail addresses: [email protected] (M.A. Vilaseca), [email protected] (J.M. Lailla).
aggregation. Hyperhomocysteinemia has been hypothesized to be associated with this placental microvascularization disease [4]. Plasma total homocysteine (tHcy; the sum of all homocysteine forms which generate this amino acid by reduction) depends on many physiological, pathological and genetic determinants, which are closely interrelated to each other [1–4]. Plasma tHcy concentrations decrease during pregnancy [5], and are closely dependent on Vitamin B (folate, Vitamins B12 and B6, riboflavin) intake [1–4]. Although changes in tHcy have been reported in northern European countries [5] and America [6], there are few data in the literature about plasma tHcy concentrations during pregnancy, as well as about the occurrence of hyperhomocysteinemia in preeclampsia in our geographical area [7]. Our objectives in the present study were: (a) to establish the second and third trimester reference values for plasma tHcy concentrations in our normal pregnants, and (b) to investigate the possible association between hyperhomocysteinemia and preeclampsia in our geographical area by means of a control–case study.
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 2 ) 0 0 3 6 7 - 6
E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49
46
(SBDF) derivatives [8]. Serum folates and Vitamin B12 were determined by a competitive protein binding chemiluminescent assay (IMMULITE; Diagnostic Products Corporation).
2. Materials and methods 2.1. Study group and design This control–case study was conducted at the Hospital Sant Joan de De´ u (University of Barcelona), from May 2000 to September 2001. It included 32 patients with preeclampsia and 64 controls without pregnancy complications. Preeclampsia was diagnosed when a blood pressure higher than 140/90 mmHg and a proteinuria higher than 300 mg/24 h were observed on at least two occasions more than 6 h apart, after the 20th week of pregnancy. Controls were 64 pregnant women monitored at the Department of Obstetrics and Gynecology of our hospital from the first trimester and who completed the pregnancy without complications. The controls’ recruitment was randomized: we included all pregnants without previous risk factors who came to our department and who were randomly assigned at consulting room number 1 for pregnancy control. Exclusion criteria were the following: pregnant women who did not stop folate supplementation by the 12th week of gestation, special diets implying a folate consumption higher or lower than normal intakes in our geographical area (assessed by a nutritional questionnaire), altered renal function, diabetes or chronic diseases, treatment with antifolate drugs (antiepileptics, metotrexate), twin pregnancies, maternal age above 40 years old, and non-Caucasian ethnic origin. All participants in this study signed an informed consent agreement in accord with the Helsinki Declaration of 1964, revised in 1996. Our Hospital Ethics Committee approved the study. Biochemical analyses were performed in preeclampsia women at diagnosis, which was always in the third trimester of pregnancy. In controls, a first determination was performed in the 24th week, and a second one in the 34th week of pregnancy. Blood was collected in the fasting state to analyze plasma tHcy, serum folate and Vitamin B12. Renal and hepatic functions were also evaluated. 2.2. Laboratory analyses Plasma tHcy was determined by HPLC with fluorescence detection of the 7-fluorobenzo-2oxa-1,3-diazole-4-sulfonate
2.3. Statistical analysis Plasma tHcy values did not show a Gaussian distribution. Therefore, non-parametric Mann–Whitney U-test for unpaired samples was applied to compare tHcy and vitamin concentrations of patients and controls, while Wilcoxon test for paired samples was used to compare those values of controls in the second and third trimesters of pregnancy, with a 95% confidence interval (CI). Spearman test was applied to study the possible relationship between tHcy and B Vitamins in patients and control groups. Spearman test was also applied to study the possible relationship between tHcy and severity type of preeclampsia (based on proteinuria and blood pressure) and between tHcy and gestational age in patients with preeclampsia. The risk of preeclampsia in hyperhomocysteinemia patients was estimated as the odds ratio (OR) and expressed with the 95% CI. Statistical significance was accepted at P < 0:05. Statistical analyses were performed using the package SPSS (Version 10.0).
3. Results Characteristics of subjects studied in the two groups, cases and controls, are described in Table 1. In the control group, tHcy values were significantly higher while folate levels were significantly lower in the third trimester compared with the second trimester of pregnancy (P < 0:0001) (Table 2). Hyperhomocysteinemia (defined as tHcy concentrations >P95 of the control group) was established as values higher than 7.7 mmol/l in the second trimester, and as values higher than 10.5 mmol/l in the third trimester of pregnancy. Plasma tHcy and folate values were significantly higher in the patient group compared with the control group in the third trimester (P ¼ 0:005 and 0.005, respectively), but not Vitamin B12 values, which were not significantly different (Table 3).
Table 1 Characteristics of the study groups: patients with preeclampsia and controls
Number of women Age (years) Parity (%) (0/1) Body mass index (kg/m2) Urate (mmol/l) Proteinuria (mg/day) Gestational age (weeks)a Fetal weight (g) a
Gestational age at delivery.
Preeclamptic patients
Controls
32 30.5 (25–40) 58/42 26.3 (19.3–37.2) 416 (256–529) 819 (313–10,000) 33 (28–40) 1315 (695–4320)
64 30 (19–39) 56/44 25.6 (18.4–38.5) Non-determined Non-determined 39 (34–41) 3330 (1890–4200)
Significance (Mann–Whitney) Non-significant Non-significant Non-significant
P < 0.0001 P < 0.0001
E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49 Table 2 Plasma tHcy, folate and vitamin B12 concentrations in controls, comparing the second and third trimesters of pregnancy Controls
tHcy (mmol/l) median (range)
Folate (nmol/l) median (range)
Vitamin B12 median (range)
Second trimester Third trimester Significance (Wilcoxon test)
5.3 (3.1–10) 6.3 (3.2–13.0) P < 0.0001
21.1 (1.8–54.4) 14.8 (3.2–46.8) P < 0.0001
203 (89–528) 189 (88–562) Non-significant
47
A significant correlation was found between tHcy and urate in the preeclampsia patients (P ¼ 0:004). However, no correlation was observed between tHcy and severity type of preeclampsia (based on proteinuria and blood pressure) or between tHcy and gestational age or fetal weight in patients with preeclampsia.
4. Comment
Table 3 Plasma total homocysteine, folate and vitamin B12 concentrations in preeclampsia patients, compared with third-trimester controls tHcy (mmol/l) Folate (nmol/l) Vitamin B12 median (range) median (range) median (range) Preeclampsia patients 8.2 (3.0–17.0) Controls third trimester 6.3 (3.2–13.0) Significance P ¼ 0.005 (Mann–Whitney)
24.0 (5.8–54.4) 215 (102–503) 14.8 (3.2–46.8) 189 (88–562) P ¼ 0.005 Non-significant
Hyperhomocysteinemia was observed in 6/32 (18.7%) of patients versus 2/64 (3%) of third-trimester controls. The OR for preeclampsia in hyperhomocysteinemia patients was 7.7 (CI 95%: 1.7–34.8). A significantly negative correlation was observed between homocysteine and folate only in the control group (second trimester: r ¼ 0:443; P ¼ 0:001; third trimester: r ¼ 0:391; P ¼ 0:002) (Fig. 1). No correlation was observed between Vitamin B12 and homocysteine levels in any group.
Plasma tHcy concentrations in women in the second and third trimesters of pregnancies without complications are lower than values found in non-pregnant women (median: 8.5; range: 5.8–12.8 mmol/l) [8]. Plasma tHcy decreases during pregnancy, probably due to increased plasma volume, which is responsible for hemodilution, hormonal changes associated with pregnancy, and increased fetal need for methionine [9,10]. The stand up of homocysteine to the fetal metabolism is demonstrated in a number of studies [11]. Folate supplementation during the first trimester further decreases homocysteine values. Although control pregnant women included in this study stopped folate supplementation by the 12th week of gestation, they still showed high serum folate in the 24th week, while values significantly decreased at the 34th week of pregnancy. Conversely, plasma tHcy concentrations significantly increased throughout the gestation, showing an inverse relationship with serum folate levels (Fig. 1). Data reported in the literature about plasma tHcy in uncomplicated pregnancies vary depending on the analytical procedures, the week of pregnancy, the vitamin content or
Fig. 1. Correlation between tHcy and folate in the second and third trimesters of pregnancy in the control group.
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E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49
supplementation of the diets, and the genetic background of the women studied. Therefore, the establishment of reference values for each laboratory and country seems advisable in order to make it possible to accurately interpret the results. Our tHcy reference values are lower than those reported by some authors [12,13], but higher than control values reported in other studies [5,6]. According to our results, we may reliably consider hyperhomocysteinemia plasma tHcy values higher than 7.7 mmol/l in the second trimester and 10.5 mmol/l in the third trimester in the conditions of our study. There has been reported to be an association between hyperhomocysteinemia and preeclampsia [13–17], although there is some controversy about this point [18]. The pathophysiology of hyperhomocysteinemia in vascular disease is still under investigation. Endothelial dysfunction, which is thought to play a role in the pathophysiology of preeclampsia, can be mediated by oxidative stress [13,18]. However, some authors questioned whether the mild hyperhomocysteinemia observed in preeclampsia, with tHcy values similar to those found in normotensive non-pregnant women, can damage the vascular endothelium of preeclamptic patients. Vascular endothelium of pregnant women might be more vulnerable to oxidative injury [9]. According to our results, hyperhomocysteinemia in the third trimester of pregnancy is associated with an increased risk of preeclampsia. These results agree with those reported by most authors [13–17], although they are questioned by some others, which attributed hyperhomocysteinemia to fluctuations of plasma volume in preeclampsia [18]. Interestingly, we found high tHcy concentrations in preeclamptic women in spite of folate levels in the high-normal range. In fact, no correlation was found between tHcy and folate values in the patient group, although a close relationship was found in controls either in the second or third trimester. A possible explanation for the high folate levels in preeclamptic patients may be the ingestion of a rich folate diet during pregnancy. However, the high folate levels in our preeclamptic women with hyperhomocysteinemia (range: 5.8–54.4 nmol/l) did not succeed in lowering plasma tHcy concentrations. Other genetic or acquired determinants of hyperhomocysteinemia would account for the high homocysteine concentrations observed in preeclampsia [17]. The prevalence of the common 677C ! T polymorphism of the methylenetetrahydrofolate reductase gene has been determined in some studies with controversial results [17,18]. An important step toward better understanding the implication of increased tHcy values in preeclampsia would be knowledge of the predictive value of hyperhomocysteinemia in early pregnancy. Although some authors recently reported that elevated plasma homocysteine level in early pregnancy might increase the risk of developing severe preeclampsia almost three-fold [19], no changes were found in tHcy levels in other series [20]. Future lines of research may give us more information about this subject, as well as about the etiology of hyperhomocysteinemia in preeclampsia.
In summary, hyperhomocysteinemia was observed in 18.7% of patients versus 3% of third-trimester controls in our series. Our results indicate that pregnant women with hyperhomocysteinemia have a 7.7-fold increased risk for preeclampsia compared with normal controls.
Acknowledgements We are grateful to J. Moreno for skillful technical assistance, to Dr. C. Valls for vitamin determinations, and to M. Herna´ ndez, A. Mestres and J. Moreno for blood collection from controls.
References [1] Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Ann Rev Med 1998;49:31–62. [2] Lo´ pez-Quesada E, Vilaseca MA, Gonza´ lez S. Homocisteı´na y gestacio´ n. Med Clin (Barc) 2000;115:352–6. [3] Vollset SE, Refsum H, Irgens L, Emblem BM, Tverdal A, Gjessing HK, et al. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study. Am J Clin Nutr 2000;71:962–8. [4] Wang J, Trudinger B, Duarte N, Wilcken D, Wang X. Elevated circulating homocysteine levels in placental vascular disease and associated preeclampsia. Br J Obstet Gynecol 2000;107:935–8. [5] Walker M, Smith G, Perkins S, Keely E, Garner P. Changes in homocysteine levels during normal pregnancy. Am J Obstet Gynecol 1999;180:660–4. [6] Hogg BB, Tamura T, Johnston KE, Dubard MB, Goldenberg RL. Second-trimester plasma homocysteine levels and pregnancy-induced hypertension, preeclampsia, and intrauterine growth restriction. Am J Obstet Gynecol 2000;183:805–9. [7] De Falco M, Pollio F, Scaramellino M, Pontillo M, Lieto AD. Homocysteinaemia during pregnancy and placental disease. Clin Exp Obstet Gynecol 2000;27:188–90. [8] Vilaseca MA, Moyano D, Ferrer I, Artuch R. Total homocysteine in pediatric patients. Clin Chem 1997;43:690–2. [9] Powers R, Evans R, Majors A, Ojimba J, Ness R, Grombleholme W, et al. Plasma homocysteine concentration is increased in preeclampsia and is associated with evidence of endothelial activation. Am J Obstet Gynecol 1998;179:1605–11. [10] Malinow MR, Rajkovic A, Duell PB, Hess DL, Upson BM. The relationship between maternal and neonatal umbilical cord plasma homocysteine suggests a potential role for maternal homocysteine in fetal metabolism. Obstet Gynecol 1998;178:228–33. [11] Steegers-Theunissen R, Wathen N, Eskes T, Van Raaij-Selten B, Chard T. Maternal and fetal levels of methionine and homocysteine in early human pregnancy. Br J Obstet Gynecol 1997; 104:20–4. [12] Khong TY, Hague WM. The placenta in maternal hyperhomocysteinaemia. Br J Obstet Gynecol 1999;106:273–8. [13] Raijmakers M, Zusterzell P, Steegers E, Hectors M, Demacker P, Peters W. Plasma thiol status in preeclampsia. Obstet Gynecol 2000;95:180–4. [14] Rajkovic A, Catalano PM, Malinow MR. Elevated homocysteine levels with preeclampsia. Obstet Gynecol 1997;90:168–71. [15] Leeda M, Riyazi N, De Vries J, Jakobs C, Van Geijn H, Dekker G. Effects of folic acid and vitamin B6 supplementation on women with hyperhomocysteinemia and a history of preeclampsia or fetal growth restriction. Am J Obstet Gynecol 1998;179:135–9.
E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49 [16] Sorensen T, Malinow MR, Williams MA, King IB, Luthy DA. Elevated second-trimester serum homocysteine levels and subsequent risk of preeclampsia. Gynecol Obstet Invest 1999;48:98–103. [17] Lachmeijer AM, Arngrimsson R, Bastiaans EJ, Pals G, ten Kate LP, de Vries JI, et al. Mutations in the gene for methylenetetrahydrofolate reductase, homocysteine levels, and vitamin status in women with a history of preeclampsia. Am J Obstet Gynecol 2001;184:394– 402.
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[18] Raijmakers MT, Zusterzeel PL, Steegers EA, Peters WH. Hyperhomocysteinaemia: a risk factor for preeclampsia? Eur J Obstet Gynecol Reprod Biol 2001;95:226–8. [19] Cotter AM, Molloy AM, Scott JM, Daly SF. Elevated plasma homocysteine in early pregnancy: a risk factor for the development of severe preeclampsia. Am J Obstet Gynecol 2001;185:781–5. [20] Hietala R, Turpeinen U, Laatikainen T. Serum homocysteine at 16 weeks and subsequent preeclampsia. Obstet Gynecol 2001;97:527–9.
Plasma total homocysteine in uncomplicated pregnancy and in preeclampsia$ Eva Lo´pez-Quesadaa, M. Antonia Vilasecab,*, Jose M. Laillaa a
Obstetrics and Gynecology Department, Hospital Sant Joan de De´u, University of Barcelona, 08950-Esplugues de Llobregat, Barcelona, Spain b Biochemistry Department, Hospital Sant Joan de De´u, University of Barcelona, 08950-Esplugues de Llobregat, Barcelona, Spain Received 7 March 2002; accepted 2 September 2002
Abstract Objectives: (a) To establish the reference values for plasma total homocysteine in our pregnant population. (b) To determine the possible association between hyperhomocysteinemia and preeclampsia in our geographical area. Study design: Control–case study with 32 preeclamptic patients and 64 controls without pregnancy complications. Plasma total homocysteine, determined by HPLC (fluorescence detection), was correlated with serum folate and Vitamin B12 (analyzed by competitive protein binding chemiluminescent assay). Statistical analyses: Mann–Whitney, Wilcoxon and Spearman test (SPSS, 10.0). Results: Homocysteine concentrations in the controls were significantly higher while folate was significantly lower in the third trimester of pregnancy when compared with the second (P < 0:0001). Homocysteine and folate values were significantly higher in patients compared with controls in the third trimester (P ¼ 0:005 and 0.005, respectively). The OR for preeclampsia in hyperhomocysteinemia was 7.7 (95% CI: 1.7–34.8). Conclusion: Pregnant women with hyperhomocysteinemia have a 7.7-fold risk for preeclampsia (CI 95%: 1.7–34.8) compared with normal controls. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hyperhomocysteinemia; Pregnancy; Preeclampsia; Folate; Vitamin B12
1. Introduction Elevated plasma homocysteine is a risk factor for endothelial dysfunction and vascular diseases such as atherosclerosis and occlusive vascular disorders [1]. Hyperhomocysteinemia has also been associated with complications in pregnancy such as neural tube defects, repeated miscarriages, abruptio placentae, fetal death, preeclampsia and intrauterine growth retardation [2,3]. Preeclampsia represents one of the most important complications of pregnancy (5–7% of low-risk pregnancies), but little is known about its etiology. It may be defined as a pregnancy-specific occlusive vascular disorder characterized by endothelial cell dysfunction and increased platelet
$ Eva Lo´pez-Quesada is the recipient of a grant from the Hospital Sant Joan de De´u to perform the present study. The study was partially ´ TV3/993431. supported by the MARATO * Corresponding author. Tel.: þ34-93-280-4000; fax: þ34-93-280-3626. E-mail addresses: [email protected] (M.A. Vilaseca), [email protected] (J.M. Lailla).
aggregation. Hyperhomocysteinemia has been hypothesized to be associated with this placental microvascularization disease [4]. Plasma total homocysteine (tHcy; the sum of all homocysteine forms which generate this amino acid by reduction) depends on many physiological, pathological and genetic determinants, which are closely interrelated to each other [1–4]. Plasma tHcy concentrations decrease during pregnancy [5], and are closely dependent on Vitamin B (folate, Vitamins B12 and B6, riboflavin) intake [1–4]. Although changes in tHcy have been reported in northern European countries [5] and America [6], there are few data in the literature about plasma tHcy concentrations during pregnancy, as well as about the occurrence of hyperhomocysteinemia in preeclampsia in our geographical area [7]. Our objectives in the present study were: (a) to establish the second and third trimester reference values for plasma tHcy concentrations in our normal pregnants, and (b) to investigate the possible association between hyperhomocysteinemia and preeclampsia in our geographical area by means of a control–case study.
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 2 ) 0 0 3 6 7 - 6
E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49
46
(SBDF) derivatives [8]. Serum folates and Vitamin B12 were determined by a competitive protein binding chemiluminescent assay (IMMULITE; Diagnostic Products Corporation).
2. Materials and methods 2.1. Study group and design This control–case study was conducted at the Hospital Sant Joan de De´ u (University of Barcelona), from May 2000 to September 2001. It included 32 patients with preeclampsia and 64 controls without pregnancy complications. Preeclampsia was diagnosed when a blood pressure higher than 140/90 mmHg and a proteinuria higher than 300 mg/24 h were observed on at least two occasions more than 6 h apart, after the 20th week of pregnancy. Controls were 64 pregnant women monitored at the Department of Obstetrics and Gynecology of our hospital from the first trimester and who completed the pregnancy without complications. The controls’ recruitment was randomized: we included all pregnants without previous risk factors who came to our department and who were randomly assigned at consulting room number 1 for pregnancy control. Exclusion criteria were the following: pregnant women who did not stop folate supplementation by the 12th week of gestation, special diets implying a folate consumption higher or lower than normal intakes in our geographical area (assessed by a nutritional questionnaire), altered renal function, diabetes or chronic diseases, treatment with antifolate drugs (antiepileptics, metotrexate), twin pregnancies, maternal age above 40 years old, and non-Caucasian ethnic origin. All participants in this study signed an informed consent agreement in accord with the Helsinki Declaration of 1964, revised in 1996. Our Hospital Ethics Committee approved the study. Biochemical analyses were performed in preeclampsia women at diagnosis, which was always in the third trimester of pregnancy. In controls, a first determination was performed in the 24th week, and a second one in the 34th week of pregnancy. Blood was collected in the fasting state to analyze plasma tHcy, serum folate and Vitamin B12. Renal and hepatic functions were also evaluated. 2.2. Laboratory analyses Plasma tHcy was determined by HPLC with fluorescence detection of the 7-fluorobenzo-2oxa-1,3-diazole-4-sulfonate
2.3. Statistical analysis Plasma tHcy values did not show a Gaussian distribution. Therefore, non-parametric Mann–Whitney U-test for unpaired samples was applied to compare tHcy and vitamin concentrations of patients and controls, while Wilcoxon test for paired samples was used to compare those values of controls in the second and third trimesters of pregnancy, with a 95% confidence interval (CI). Spearman test was applied to study the possible relationship between tHcy and B Vitamins in patients and control groups. Spearman test was also applied to study the possible relationship between tHcy and severity type of preeclampsia (based on proteinuria and blood pressure) and between tHcy and gestational age in patients with preeclampsia. The risk of preeclampsia in hyperhomocysteinemia patients was estimated as the odds ratio (OR) and expressed with the 95% CI. Statistical significance was accepted at P < 0:05. Statistical analyses were performed using the package SPSS (Version 10.0).
3. Results Characteristics of subjects studied in the two groups, cases and controls, are described in Table 1. In the control group, tHcy values were significantly higher while folate levels were significantly lower in the third trimester compared with the second trimester of pregnancy (P < 0:0001) (Table 2). Hyperhomocysteinemia (defined as tHcy concentrations >P95 of the control group) was established as values higher than 7.7 mmol/l in the second trimester, and as values higher than 10.5 mmol/l in the third trimester of pregnancy. Plasma tHcy and folate values were significantly higher in the patient group compared with the control group in the third trimester (P ¼ 0:005 and 0.005, respectively), but not Vitamin B12 values, which were not significantly different (Table 3).
Table 1 Characteristics of the study groups: patients with preeclampsia and controls
Number of women Age (years) Parity (%) (0/1) Body mass index (kg/m2) Urate (mmol/l) Proteinuria (mg/day) Gestational age (weeks)a Fetal weight (g) a
Gestational age at delivery.
Preeclamptic patients
Controls
32 30.5 (25–40) 58/42 26.3 (19.3–37.2) 416 (256–529) 819 (313–10,000) 33 (28–40) 1315 (695–4320)
64 30 (19–39) 56/44 25.6 (18.4–38.5) Non-determined Non-determined 39 (34–41) 3330 (1890–4200)
Significance (Mann–Whitney) Non-significant Non-significant Non-significant
P < 0.0001 P < 0.0001
E. Lo´ pez-Quesada et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 108 (2003) 45–49 Table 2 Plasma tHcy, folate and vitamin B12 concentrations in controls, comparing the second and third trimesters of pregnancy Controls
tHcy (mmol/l) median (range)
Folate (nmol/l) median (range)
Vitamin B12 median (range)
Second trimester Third trimester Significance (Wilcoxon test)
5.3 (3.1–10) 6.3 (3.2–13.0) P < 0.0001
21.1 (1.8–54.4) 14.8 (3.2–46.8) P < 0.0001
203 (89–528) 189 (88–562) Non-significant
47
A significant correlation was found between tHcy and urate in the preeclampsia patients (P ¼ 0:004). However, no correlation was observed between tHcy and severity type of preeclampsia (based on proteinuria and blood pressure) or between tHcy and gestational age or fetal weight in patients with preeclampsia.
4. Comment
Table 3 Plasma total homocysteine, folate and vitamin B12 concentrations in preeclampsia patients, compared with third-trimester controls tHcy (mmol/l) Folate (nmol/l) Vitamin B12 median (range) median (range) median (range) Preeclampsia patients 8.2 (3.0–17.0) Controls third trimester 6.3 (3.2–13.0) Significance P ¼ 0.005 (Mann–Whitney)
24.0 (5.8–54.4) 215 (102–503) 14.8 (3.2–46.8) 189 (88–562) P ¼ 0.005 Non-significant
Hyperhomocysteinemia was observed in 6/32 (18.7%) of patients versus 2/64 (3%) of third-trimester controls. The OR for preeclampsia in hyperhomocysteinemia patients was 7.7 (CI 95%: 1.7–34.8). A significantly negative correlation was observed between homocysteine and folate only in the control group (second trimester: r ¼ 0:443; P ¼ 0:001; third trimester: r ¼ 0:391; P ¼ 0:002) (Fig. 1). No correlation was observed between Vitamin B12 and homocysteine levels in any group.
Plasma tHcy concentrations in women in the second and third trimesters of pregnancies without complications are lower than values found in non-pregnant women (median: 8.5; range: 5.8–12.8 mmol/l) [8]. Plasma tHcy decreases during pregnancy, probably due to increased plasma volume, which is responsible for hemodilution, hormonal changes associated with pregnancy, and increased fetal need for methionine [9,10]. The stand up of homocysteine to the fetal metabolism is demonstrated in a number of studies [11]. Folate supplementation during the first trimester further decreases homocysteine values. Although control pregnant women included in this study stopped folate supplementation by the 12th week of gestation, they still showed high serum folate in the 24th week, while values significantly decreased at the 34th week of pregnancy. Conversely, plasma tHcy concentrations significantly increased throughout the gestation, showing an inverse relationship with serum folate levels (Fig. 1). Data reported in the literature about plasma tHcy in uncomplicated pregnancies vary depending on the analytical procedures, the week of pregnancy, the vitamin content or
Fig. 1. Correlation between tHcy and folate in the second and third trimesters of pregnancy in the control group.
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supplementation of the diets, and the genetic background of the women studied. Therefore, the establishment of reference values for each laboratory and country seems advisable in order to make it possible to accurately interpret the results. Our tHcy reference values are lower than those reported by some authors [12,13], but higher than control values reported in other studies [5,6]. According to our results, we may reliably consider hyperhomocysteinemia plasma tHcy values higher than 7.7 mmol/l in the second trimester and 10.5 mmol/l in the third trimester in the conditions of our study. There has been reported to be an association between hyperhomocysteinemia and preeclampsia [13–17], although there is some controversy about this point [18]. The pathophysiology of hyperhomocysteinemia in vascular disease is still under investigation. Endothelial dysfunction, which is thought to play a role in the pathophysiology of preeclampsia, can be mediated by oxidative stress [13,18]. However, some authors questioned whether the mild hyperhomocysteinemia observed in preeclampsia, with tHcy values similar to those found in normotensive non-pregnant women, can damage the vascular endothelium of preeclamptic patients. Vascular endothelium of pregnant women might be more vulnerable to oxidative injury [9]. According to our results, hyperhomocysteinemia in the third trimester of pregnancy is associated with an increased risk of preeclampsia. These results agree with those reported by most authors [13–17], although they are questioned by some others, which attributed hyperhomocysteinemia to fluctuations of plasma volume in preeclampsia [18]. Interestingly, we found high tHcy concentrations in preeclamptic women in spite of folate levels in the high-normal range. In fact, no correlation was found between tHcy and folate values in the patient group, although a close relationship was found in controls either in the second or third trimester. A possible explanation for the high folate levels in preeclamptic patients may be the ingestion of a rich folate diet during pregnancy. However, the high folate levels in our preeclamptic women with hyperhomocysteinemia (range: 5.8–54.4 nmol/l) did not succeed in lowering plasma tHcy concentrations. Other genetic or acquired determinants of hyperhomocysteinemia would account for the high homocysteine concentrations observed in preeclampsia [17]. The prevalence of the common 677C ! T polymorphism of the methylenetetrahydrofolate reductase gene has been determined in some studies with controversial results [17,18]. An important step toward better understanding the implication of increased tHcy values in preeclampsia would be knowledge of the predictive value of hyperhomocysteinemia in early pregnancy. Although some authors recently reported that elevated plasma homocysteine level in early pregnancy might increase the risk of developing severe preeclampsia almost three-fold [19], no changes were found in tHcy levels in other series [20]. Future lines of research may give us more information about this subject, as well as about the etiology of hyperhomocysteinemia in preeclampsia.
In summary, hyperhomocysteinemia was observed in 18.7% of patients versus 3% of third-trimester controls in our series. Our results indicate that pregnant women with hyperhomocysteinemia have a 7.7-fold increased risk for preeclampsia compared with normal controls.
Acknowledgements We are grateful to J. Moreno for skillful technical assistance, to Dr. C. Valls for vitamin determinations, and to M. Herna´ ndez, A. Mestres and J. Moreno for blood collection from controls.
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