Investigation of folic acid, vitamin B12, vitamin B6 and homocysteine levels in preeclamptic pregnancies

Objective

To investigate the relationship between preeclampsia and serum folic acid, serum B12, plasma vitamin B6 (pyridoxal-5’-phosphate) and plasma homocysteine levels.

Methods

100 pregnant women with preeclampsia and 74 healthy pregnant women were included in the study from September
2004 to August 2005. Serum vitamin B12, folic acid and plasma vitamin B6 and homocysteine levels were measured in all patients at their third trimester of pregnancy

Results

Vitamin B12 serum levels in the preeclamptic and the control group were similar (117.44±42.03 pg/ml in the preeclamptic group and 136.07±59.01 pg/ml in the control group, p>0.05 ). Vitamin B6 (pyridoxal-5’-phosphate) plasma levels in preeclampsia group were lower than control group (6.40±2.91µg/l in preeclampsia group and 11.15±5.76 µg/l in the control group p<0.001). Folic acid serum levels in the preeclampsia group were lower (5.43±3.08 ng/ml in the control, 8.00±5.1 ng/ml, in the preeclamptic group, p<0.001). Homocysteine plasma levels were significantly higher in the preeclampsia group than control group (10.50±6.21 µmol/L in the preeclamptic group and 6.54.±2.64 µmol/L in the control group, p<0.001).

Conclusion

 High homocysteine level and low folic acid and vitamin B6 levels play a role in the pathogenesis of preeclampsia. 

Keywords

Preeclampsia, homocysteine, folate, vitamin B6, vitamin B12.

Introduction

Homocysteine is an aminoacid that forms during methionine metabolism. Homocysteine blood levels are affected by genetic or many acquired factors. The lack of enzymes added to the homocysteine metabolism or the cofactors necessary for its metabolism (folate, B6 vitamin, B12 vitamin) cause hyperhomocysteinemia.(1) Mc Cully was the first to suggest that high homocysteine leads to atherosclerotic and thrombotic complications.(2) Further studies showed disorder in endothelium dependant relaxation in various isolated artery preparations incubated with homocysteine.(3) The placental vascular changes in preeclamptic patients: due to similar changes to those of atherosclerotic patients, various studies have been done to investigate the relationship between preeclampsia and homocysteine, vitamin B6 (pyridoxal-5'-phospate), vitamin B12 and folic acid levels, and it was shown in these studies that preeclamptic patients have higher homocysteine levels than healthy pregnant women, and that there are changes in parallel to this in vitamin B6 (pyridoxal-5’-phosphate), vitamin B12 and folic acid levels.(4,5)
The objective of this study is to research whether there is any difference between preeclamptic pregnant women and healthy normotensive pregnant women through the comparison of serum folic acid, serum vitamin B12, plasma vitamin B6 (pyridoxal-5’-phosphate) and plasma homocysteine levels.

Methods

This study was performed prospectively on a total of 174 normotensive healthy pregnant women and pregnant women diagnosed with preeclampsia who do not use any medication other than the iron preparation and do not smoke and who contacted the Clinics of Gynecology and Obstetrics,Faculty of Medicine, Dicle University between September 2004 and August 2005. Ethical approval was received to perform the study. Furthermore,approval was also received from all patients that their participation in the study is voluntary. Preeclamptic pregnant women were classified as the study group and healthy pregnant women as the control group. The preeclampsia classification was done according to ACOG (American Collegeof Obstetricians and Gynecologists) criteria.6 Blood pressure ≥ 140/90 mmHg and proteinuria > 300 mg/dl in the 24 hour urine sample was considered to be mild preeclampsia; blood pressure ≥ 160/110 mmHg and proteinuria (5 gr/more than 24 hours),oliguria (500 ml/less than 24 hours), headache,sight impairment, pain in the right upper quadrant,liver function failure, and thrombocytopenia were considered severe preeclampsia criteria.
In addition to the obstetric evaluation and laboratory examination in the 3rd trimester of gestation,blood was taken from all patients into EDTA tubes for homocysteine and vitamin B6 (pyridoxal-5’-phosphate) analysis, and into plain gel tubes for folic acid and vitamin B12 analysis. After all samples were centrifuged at 3500 rpm for 10 minutes, plasma was allocated for homocysteine and vitamin B6 (pyridoxal-5’-phosphate) and serum for folic acid and vitamin B12. The samples acquired were stored in the dark and at -24ºC until the day of the study.
Homocysteine levels were studied using a DPC brand "Immulate 2000 Homocysteine" kit (reference range 5.0-12 µmol/L), on the Immulate 2000 apparatus (DPC, Los Angeles) using the immunoassay method.
Vitamin B6 (pyridoxal-5’phosphate) levels were studied using a Recipe brand "ClinRep Complete Kit for vitamin B6 (pyridoxal-5’phosphate) in Plasma and Whole Blood" kit (reference range 8.6-27.2 µg/l), on the HPLC apparatus (Schimadzu,Japan) using the chromatographic method.
Folic acid levels were studied using a Roche brand kit (reference range 3.1-17.5 µg/l), on the E170 system (Roche, Los Angeles) using the chemiluminescence immunoassay method.
Vitamin B12 levels were studied using a Roche brand kit (reference range 30-2000 pg/ml), on the E170 system (Roche, Los Angeles) using the chemiluminescence immunoassay method. The student t test SPSS 10.0 for Windows computer program was used in the statistical analysis of data, and p<0.05 was accepted statistically significant.

Results

A total of 174 cases were included in the study of which 100 were preeclampsia cases (Group 1) and 74 were healthy pregnancy cases (Group 2).Cases were separated into two groups: preeclamptic and healthy pregnant women. In group 1 cases (n=100), mild preeclampsia was predicted in 74 cases (74%) and severe preeclampsia in 26 cases (26%). The demographic, hematological and biochemical parameters, and newborn weights and first and 5th minute Apgar scores of the cases are shown in Table 1. No statistically significant difference was predicted between the age, gravid, parities
between both groups (p>0.05). The week of delivery, birth weight, first and 5th minute apgar scores between both groups were statistically significant (p<0.001).
No statistical difference could be predicted between the preeclampsia and control groups interms of vitamin B12 serum level (respectively,117.44±42.03 pg/ml, 136.07±59.01 pg/ml, p>0.05) (Diagram 1).
The vitamin B6 (pyridoxal-5’-phosphate) plasma level was found to be low in the preeclampsia group in comparison to the control group (respectively,6.40±2.91 µg/l, 11.15±5.76 µg/l p<0.001) (Diagram 2). 
Folic acid serum level was found to be lower in the preeclampsia group (respectively, 5.43±3.08 ng/ml, 8.00±5.1 ng/ml, p<0.001) (Diagram 3). 
And the homocysteine plasma level was found to be at a significantly high ratio in the preeclampsia group in comparison to the healthy pregnancy group (respectively, 10.50±6.21 µmol/L, 6.54±2.64 µmol/L, p<0.001) (Diagram 4) (Table 2).
Furthermore, no statistical difference could be predicted in terms of the vitamin B12, vitamin B6, folic acid and homocysteine serum levels of mild and severe preeclamptic patients (p>0.05).

Discussion

Homocysteine is methionine’s non-essential amino acid derivative. High plasma homocysteine levels are a risk factor for endothelial dysfunction, vascular disease, aterosclerosis.(1-5) 
Endothelial damage plays a role in the etiology of preeclampsia. It leads to vascular endothelial damage at high homocysteine levels. Therefore, the relationship between high homocysteine levels and preeclampsia has been shown in various studies.(6) De Vries et al have predicted that 24% of preeclamptic patients also have high homocysteine.(8) It is considered that high homocysteine levels may also make vascular endothelia in preeclamptic patients more sensitive towards oxidasive stress.(9) In their study, Kassab et al have predicted that high homocysteine leads to maternal hypotension, proteinuria, renal damage, intrauterine developmental retardation, and increased fetal mortality.(10) Patrick et al have shown that decreased folic acid and B12 levels cause high homocysteine concentrations in preeclamptic patients.(11) In their study, Gürbüz et al have shown that there is a prevalent degree of high homocysteine concentration in preeclamptic patients, and that high homocysteine concentrations are in parallel with the bad prognosis of preeclampsia.(12) Calle et al have stated that vitamin B6 (pyridoxal-5’-phosphate) and vitamin B12 play a role in the homocysteine metabolism, and that the administration of these vitamins during the term of gestation with a diet prevent complications such as spontaneous low intrauterine developmental retardation, preeclampsia,intrauterine morbidity linked to gestation.(13) In their study, while predicting homocysteine levels as higher in the preeclampsia pregnancy group in comparison to the healthy pregnancy group, Üstüner et al have predicted that serum folic acid levels are low and have stated that it is necessary to correct the lack of nutritional folic acid during the antenatal term.(14) The lack of folic acid in pregnant women receiving folic acid treatment in the second and third trimester of gestation was predicted to be less in comparison to those not receiving the treatment. Thence, with the consideration that 30% of preeclamptic gestations will repeat, it is suggested that folic acid treatment be started to observe the homocysteine level in preeclamptic pregnant women and to prevent its repetition in subsequent pregnancies.(15,16)

Conclusion

According to data from this study, plasma homocysteine concentrations being predicted as higher and folic acid and vitamin B6 levels as lower in preeclamptic pregnant women in comparison to healthy pregnant women gives rise to the thought that high homocysteine concentration and low folic acid and vitamin B6 levels may play a role in the pathogenesis of preeclampsia. If preeclampsia and high homocysteine level are predicted together during the gestational term, we believe that it is necessary to inform the patient in terms of possible risks and to closely monitor the gestation.

References

1.Kang SS, Zhou J, Wong PWK, Kowalisyn J, StrokoschG. Intermediate homocysteinemia: a termolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988, 43: 414-21. 
2.Mc Kully KS. Vascular pathology of homocysteinaemia: implications for the pathogenesis of arterioslerosis. Am J Pathol 1969; 56: 111-28 
3.Lang D, Hussain SA, Lewis MJ. Homocysteine inhibits endothelium-dependent relaxation in isolated aortic rings. Br J Pharmacol 1997; 120: 145-7.
4.Sanchez SE, Zhang C, Rene Malinow M, Ware-Jauregui S, Larrabure G, Williams MA. Plasma folate, vitamin B(12), and homocyst(e)ine concentrations in preeclamptic and normotensive Peruvian women. Am J Epidemiol 2001; 153: 474-80.
5.Herrmann W, Hubner U, Koch I, Obeid R, Retzke U, Geisel J. Alteration of homocysteine catabolism in pre-eclampsia, HELLP syndrome and placental insufficiency. Clin Chem Lab Med 2004; 42: 1109-16. 
6.The American College of Obstetricians and Gynecologist. ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Int J Gynaecol Obstet 2002; 77: 67-75 
7.Raijmakers MT, Zusterzeel PL, Steegers EA, Peters WH. Hyperhomocysteinaemia: a risk factor for preeclampsia? Eur J Obstet Gynecol Reprod Biol 2001; 95: 226-8. 
8.De Vries JI, Dekker GA, Huijgens PC, Jakobs C, Blomberg BM, van Geijn HP. Hyperhomocysteinemia and protein S deficiency in complicated pregnancies. Brit J Obstet Gynecol 1997; 11: 1248-54. 
9.Powers RW, Evans RW, Majors AK, Ojimba JI, Ness RB, Crombleholme WR, Roberts JM. Plasma homocysteine concentration is increased in preeclampsia and is associated with evidence of endothelial activation. Am J Obstet Gynecol 1998; 179: 1605-11. 
10.Kassab SE, Abu-Hijleh MF, Al-Shaikh HB, Nagalla DS. Hyperhomocysteinemia in pregnant rats: Effects on arterial pressure, kidneys and fetal growth. Eur J Obstet Gynecol Reprod Biol. 2005 Jul 25 Epub ahead of print. 
11.Patrick TE, Powers RW, Daftary AR, Ness RB, Roberts JM. Homocysteine and folic acid are inversely related in black women with preeclampsia. Hypertension 2004; 43: 1279-82. 
12.Gurbuz A, Karateke A, Mengulluoglu M. Elevated plasma homocysteine levels in preeclampsia and eclampsia. International Journal of Gynecology & Obstetrics 2004; 87: 165-6 
13.De Usandizaga R, Sancha M, Magdaleno F, Herranz A, Cabrillo E. Homocysteine, folic acid and B-group vitamins in obstetrics and gynaecology. Eur J Obstet Gynecol Reprod Biol 2003; 107: 125-34. 
14.Üstüner I, Sönmezer M, Cengiz B, Kahraman K, Elhan A, Söylemez F. Serum Folik Asit, Vitamin B12 ve Plazma Homosistein Düzeylerinin Preeklampsi ile İlişkisi. Klinik Bilimler & Doktor Kadın Doğum 2005; 11: 78-82. 
15.De la Calle M. Hiperhomocisteinemia Preeclampsia Tesis Doctoral. Madrid, 2000. 
​16.A. Rajkovic, P.M. Catalano and M.R. Malinow, Elevated homocysteine levels with preeclampsia. Obstet. Gynecol 1997; 90: 168-71.

	File/Dsecription
	Diagram 1. Vitamin B12 levels of the group of preeclampsia and healthy pregnant women.
	Diagram 2. Vitamin B6 levels of the group of preeclampsia and healthy pregnant women.
	Diagram 3. Folic acid levels of the group of preeclampsia and healthy pregnant women.
	Diagram 4. Homocysteine levels of the group of preeclampsia and healthy pregnant women.
	Table 1. The demographic, hematological and biochemical parameters of the groups and the weights of the newborn.