Transferrin microheterogeneity in pregnancies with preeclampsia

Clinica Chimica Acta 332 (2003) 103 – 110 www.elsevier.com/locate/clinchim

Transferrin microheterogeneity in pregnancies with preeclampsia YangHong Wu a, Haruhiko Sakamoto a,*, Kenji Kanenishi b, Juan Li c, Rafiza Khatun a, Toshiyuki Hata b a

Department of Inflammation Pathology, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan b Department of Perinatology and Gynecology, Kagawa Medical University, Kagawa, Japan c Department of Obstetrics and Gynecology, First Central Hospital, Tianjin, China Received 18 December 2002; received in revised form 12 March 2003; accepted 13 March 2003

Abstract Background: It has been reported that concentrations of serum transferrin (Tf) and its highly sialylated subfraction increase in normal pregnancy. This study investigated changes in the concentrations of serum transferrin and its subfractions in preeclampsia. Methods: The serum concentration of transferrin was determined by a standard turbidimetric assay and microheterogeneous transferrin subgroups (low sialylated, 4-sialo and highly sialylated transferrins) were assessed by crossed immuno-isoelectric focusing. Results: Compared to normal pregnancy, the concentrations of total, 4-sialo and highly sialylated transferrins decreased by 27%, 16% and 38%, respectively, in severe preeclampsia, while these values did not significantly decrease in mild preeclampsia. The concentration of low sialylated transferrin involving 2-sialo- and 3-sialo-transferrins significantly decreased both in mild and severe preeclampsia, the value in severe preeclampsia was even significantly lower than that in nonpregnant women. The serum concentrations of total and highly sialylated transferrins in preeclampsia were correlated positively with infant birth weights (r = 0.587 and r = 0.645, respectively). Conclusions: The serum concentrations of total and highly sialylated transferrins in severe preeclampsia decrease significantly. This might have a negative impact on intrauterine growth. Additionally, the serum low sialylated transferrin decreases more sensitively in preeclampsia, although the concentration is low even in normal pregnancy. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Transferrin; Microheterogeneity; Glycosylation; Preeclampsia; Iron; Intrauterine growth retardation

1. Introduction Transferrin (Tf) is an iron-carrying plasma glycoprotein (200 – 340 mg/dl) and plays an important physiological role in transporting iron from the sites of absorption and storage to the various cells in the * Corresponding author. Tel.: +81-87-891-2113; fax: +81-87891-2116. E-mail address: [email protected] (H. Sakamoto).

human body. Sialic acid is a terminal sugar residue and the only charged carbohydrate in N-linked complex carbohydrate chains of the Tf molecule. Tf shows a microheterogeneity based on a difference in the sialic acid content of the carbohydrate chains, which results in a limited number of Tf isotypes that can be distinguished and quantitated electrophoretically [1 – 3]. Alterations in the sialo-Tf variants, reflecting changes in glycosylation, have been described in several (patho)physiological conditions such as preg-

0009-8981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(03)00134-7

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nancy, rheumatoid arthritis, malignancies, alcohol abuse and Carbohydrate Deficient Syndrome [4 –8]. Previous data on the microheterogeneity of Tf suggest that the functional properties of Tf, such as its iron-donating ability, can be modulated by differences in the carbohydrate moiety [9 –11]. Therefore, iron delivery to target organs via Tf might be influenced by glycosylation conditions [2,9 –12]. Preeclampsia is a severe complication of pregnancy and is the most common cause of maternal and infant illness and death. One of the major causes of perinatal morbidity and mortality in preeclampsia is intrauterine growth retardation. It is possible that changes in the total Tf concentration and its microheterogeneous condition in preeclampsia influence iron delivery to the fetus and thus unfavorably affect fetal growth. A decreased serum Tf in women with preeclampsia has been reported [13,14]. However, there have been no reports on the changes in Tf microheterogeneity in pregnancy with preeclampsia. The aim of the present study was to investigate the changes in the microheterogeneity of serum Tf in women with preeclampsia.

2. Materials and methods 2.1. Subjects Thirty-two pregnant women were recruited at admission to the labor and delivery ward at Kagawa

Medical University Hospital: 12 women were normotensive showing no abnormal manifestations, 7 had mild preeclampsia (MPE) and 13 had severe preeclampsia (SPE). Venous blood was taken from normal pregnant women and patients with MPE and SPE at the gestational periods of 32.8 F 3.7, 34.4 F 4.7 and 32.8 F 3.4 weeks, respectively. Normal pregnant women did not receive any medication. The preeclamptic patients received hydralazine treatment for blood pressure. Neither preeclamptic women nor normal pregnant women received iron supplements. Written informed consent to use their blood samples along with their clinical data for this study was obtained from all subjects. The study was approved by the local ethical committee of Kagawa Medical University. Fifteen normal nonpregnant women (26.9 F 4.5 years) with regular menstruation who were volunteers from Kagawa Medical University were also recruited as controls. Preeclampsia was defined as a blood pressure z 140/90 mm Hg on two occasions at least 6 h apart after 20 weeks gestation, and a proteinuria level z 500 mg/24 h or z 2+ by dipstick testing on at least two separate occasions [15]. Severe preeclampsia was defined as a higher blood pressure z 160 mm Hg systolic or z 110 mm Hg diastolic on two occasions at least 6 h apart, and a proteinuria level z 5 g/24 h or z 3+ by dipstick testing on at least two separate occasions [15]. Severe alcoholism and subjects with anemia, chronic hypertension, renal or metabolic diseases were

Table 1 Clinical characteristics of normal pregnant women and preeclamptic patients Normal pregnancy (n = 12) Maternal age (years) Gestational age at the time of taking blood (weeks) Gestational age at delivery (weeks) Primigravida Blood pressure (mm Hg) Cesarean section/vaginal delivery Infant birth weight (g) Apgar score Normal (>6) Abnormal ( < 6) Placental weight (g)

MPE (n = 7)

SPE (n = 13)

28.3 F 3.8 32.8 F 3.7 39.3 F 1.0

31.0 F 4.1# 34.4 F 4.7# 38.8 F 0.7#

29.4 F 4.3# 32.8 F 3.4# 33.6 F 3.0*

7 107/67 F 11/12 3/9 3051.4 F 413.7

3 144/98 F 6/3* 2/5 2789.5 F 483.5#

8 167/108 F 13/7* 11/2 1446.6 F 358.7*

12

5 2

556.3 F 106.4

510.0 F 89.2#

Blood pressures are systolic/diastolic. Data are presented as mean F S.D. and were analyzed with the Student’s t-test. * Significantly different ( P < 0.001) compared with normal pregnancy. # Not significantly different ( P>0.05) compared with normal pregnancy.

13 315.0 F 52.8*

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excluded from the study. Demographic and clinical data, including aspartate aminotransferase (AST) activity were collected at routine obstetric visits. 2.2. Sample pretreatment Five milliliters of venous blood samples was obtained in glass tubes without anticoagulant. After clot retraction, the sample was centrifuged at 1200  g for 20 min at 4 jC. The serum was used immediately or stored below 20 jC until use. After thawing, 100 Al of the serum was mixed with 5 Al of 0.5 mol/l NaHCO3 and 3 Al of 10 mmol/l Fe(III) citrate and then left at room temperature for 1 h to ensure complete iron saturation of the Tf. 2.3. Assessment of Tf microheterogeneity To assess Tf microheterogeneity, crossed immunoisoelectric focusing (CIEF) according to the method of de Jong with a slight modification was carried out [1,4,16]. Briefly, Tf subfractions were separated by isoelectric focusing on polyacrylamide gel strips carrying an immobilized pH-gradient in the first phase. The second phase, which was run perpendicularly to the first, involved a simultaneous rocket immunoelectrophoresis of all Tf fractions. This resulted in a pattern in which nine Tf fractions were separated due to differences in sialic acid number per molecule; increased sialylation per molecule indicated increased branching of the glycans attached to the protein [1,4]. The relative proportions of Tf subfractions were ascertained by measurement of the areas enclosed by the immunoelectrophoretic peaks, and the concentration of each Tf subfraction was calculated from the relative proportion and total Tf concentration determined by a standard turbidimetric assay. Fig. 1. Tf microheterogeneity patterns analyzed by CIEF. (A) Normal nonpregnant women; (B) normal pregnant women; (C) women with mild preeclampsia; (D) women with severe preeclampsia. The indices beneath the patterns indicate the number of sialic acids attached to the N-linked glycans of the corresponding Tf subfractions. The double peaks of the low sialylated Tfs (involving 2-sialo- and 3-sialo-Tfs) observed in B and D indicate that the examined individuals have C1C2 genotype of Tf. TfC1 and TfC2 are two major variants of human Tf. Genetic polymorphism is another determinant of Tf microheterogeneity, which is best expressed in the low sialylated Tfs with a small split on CIEF [1].

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The Tf subfractions were divided into three subgroups: low sialylated Tf (the sum of 0-, 1-, 2- and 3sialo-Tfs), 4-sialo-Tf and highly sialylated Tf (the sum of 5-, 6-, 7- and 8-sialo-Tfs). All microheterogeneity patterns were assessed in duplicate. 2.4. Statistical analysis Total Tf concentrations were compared using the two-tailed Student’s t-test, and concentrations of Tf subgroups by the Mann – Whitney U-test. The P values were corrected for multiple comparisons. Correlation coefficients were evaluated by linear regression analysis. P < 0.05 was considered significant.

3. Results 3.1. Clinical data Maternal ages and gestational ages of groups of normal normotensive pregnancies and pregnancies with preeclampsia were in the same ranges when the peripheral blood was obtained for this study (Table 1). By definition, both MPE and SPE groups had elevat-

Table 2 Concentrations (Amol/l) of total Tf and Tf subgroups in NP (nonpregnant women), P (normal pregnant women), MPE and SPE groups

NP (15) P (12) MPE (7) SPE (13)

Low sialylated Tf

4-sialo-Tf

Highly sialylated Tf

Total Tf

3.4 F 1.5 4.4 F 1.4 3.0 F 0.8 2.3 F 1.0

20.9 F 3.2 27.4 F 5.4 25.8 F 3.1 22.9 F 3.7

6.9 F 1.2 16.8 F 4.1 16.3 F 2.0 10.3 F 2.0

31.3 F 5.4 48.6 F 9.7 45.1 F 5.2 35.6 F 6.2

Data are presented as mean (Amol/l) F S.D.

ed blood pressures compared to normal pregnancy ( P < 0.001, respectively; Table 1). Three of thirteen patients in the SPE group had AST activities >42 IU/l, but < 60 IU/l, suggesting mild liver damage (data not shown). In SPE, all of the gestational ages at the time of delivery (33.6 F 3.0 weeks), birth weights ( < 2500 g), placental weights (315.0 F 52.8 g) and Apgar scores ( < 6) were significantly less than those in normal pregnancy ( P < 0.001, respectively; Table 1). 3.2. Tf microheterogeneity Fig. 1 shows the Tf microheterogeneity patterns obtained from the normal nonpregnant, normally pregnant, MPE and SPE groups. 0-sialo-, 1-sialoand 8-sialo-Tfs were not detected in all cases. Compared to nonpregnant control women, the total Tf concentration was 55% greater in normal pregnant women; all of 4-sialo, highly sialylated and low sialylated Tfs significantly increased; and the highly sialylated Tf increased by about 140%, which amounted to about 57% of the total Tf increment (Fig. 2; Tables 2 and 3).

Table 3 P values for comparisons in concentrations of total Tf and Tf subgroups in NP (nonpregnant women), P (normal pregnant women), MPE and SPE groups

Fig. 2. Histogram of the concentrations of Tf subgroups in normal nonpregnant women (control), normal pregnant women (normal pregnancy), women with mild preeclampsia (MPE) and women with severe preeclampsia (SPE).

P vs. NP MPE vs. P SPE vs. P MPE vs. NP SPE vs. NP

Low sialylated Tf

4-sialo-Tf

Highly sialylated Tf

Total Tf

< 0.05 < 0.05 < 0.001 NS < 0.05

< 0.005 NS < 0.05 < 0.005 NS

< 0.0001 NS < 0.0005 < 0.0005 < 0.0001

< 0.0001 NS < 0.001 < 0.0001 NS

NS = not significant.

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Fig. 3. Scatterplot of the concentrations of 2-sialo-Tf (left) and 3-sialo-Tf (right) in the sera of normal nonpregnant women (control), normal pregnant women (normal pregnancy), women with mild preeclampsia (MPE) and women with severe preeclampsia (SPE). Horizontal bars represent the median values.

Compared to normal pregnancy, all of the mean serum concentrations of total, 4-sialo, highly sialylated and low sialylated Tfs in preeclampsia decreased. Significant differences were observed in total Tf, all three Tf subgroups in SPE and only in low sialylated Tf in MPE. In SPE, the decrease in highly sialylated Tf (38%) was most marked, amounting to about 50% of the decrease in total Tf (27%), and 4sialo-Tf decreased by 16%. All of the average serum concentrations of total, 4-sialo and highly sialylated Tfs in preeclampsia still showed some increase compared to nonpregnant control group, although no significant differences were found in total Tf and 4sialo-Tf in SPE. In contrast, the mean values of low sialylated Tf in the preeclampsia groups (both MPE and SPE) were below that in the nonpregnant control group, although no significant difference was found in MPE (Fig. 2; Tables 2 and 3). As for the low sialylated Tf, the median 2-sialo-Tf concentration was significantly higher and 3-sialo-Tf was slightly, but not significantly higher in normal pregnant women than in normal nonpregnant women. Compared to normal pregnancy, the concentrations of both 2-sialo-Tf and 3-sialo-Tf in SPE, and 3-sialo-Tf in MPE significantly decreased. The median concen-

trations of 2-sialo-Tf and 3-sialo-Tf in SPE were even below those in nonpregnant women, although no significant difference was found in 2-sialo-Tf (Fig. 3). Significant positive correlations were observed between serum total Tf concentrations and infant birth weights (Fig. 4; r = 0.587, P < 0.04) or serum highly

Fig. 4. Correlation of serum Tf concentration (Amol/l) and infant birth weight (g) in SPE.

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Fig. 5. Correlation of serum highly sialylated Tf concentration (Amol/l) and infant birth weight (g) in SPE.

sialylated Tf concentrations and infant birth weights (Fig. 5; r = 0.645, P < 0.02) in SPE, but not in MPE (r = 0.282, P = 0.540; r = 0.320, P = 0.484, respectively).

4. Discussion In this study, the concentrations of total Tf along with those of the highly sialylated Tf during normal gestation were shown to increase, agreeing well with the previous reports [1 –4]. The concentrations of low sialylated Tf and 2-sialo-Tf were also shown to increase significantly in normal pregnancy. This agrees with the report by Stauber et al. [17], but not with the study of de Jong et al. [1,4]. The reasons for the increases in the concentrations of total and highly sialylated Tfs may be due to increased hepatic synthesis of Tf following decreased iron storage in the liver [18,19] and placental synthesis of Tf. The altered hormonal status should have some effects on total Tf and Tf microheterogeneity during pregnancy, because it has been reported that progesterone can influence glycosylation of Tf and estrogen can stimulate total Tf synthesis [4,20]. Tf synthesized by syncytiotrophoblasts and cytotrophoblasts has been demonstrated to have highly sialylated and branched glycans [21], which may also be one of the reasons for the increased highly sialylated Tf during pregnancy.

Pregnancy causes an extreme increase in the maternal demand for iron. The cumulated demand for iron in pregnancy may reach as much as 8 mg/ day, which exceeds the uptake rate from the gut, although iron absorption from the gut increases in pregnancy [22]. Therefore, mobilization of iron from storage sites such as the liver is an important mechanism to supplement the iron demand in pregnancy, particularly in the later stages. Increased serum total Tf throughout pregnancy can promote serum irontransport capability and thus meet the increased demand of the fetoplacental unit and maternal bone marrow for iron. The increase in the highly sialylated Tf during pregnancy coincides with the increase in iron fluxes to both the placenta and the maternal bone marrow [3,4]. Previous investigations showed that the carbohydrate moiety of Tf can influence the Tf and iron uptake by hepatocytes [10,11]. These findings suggest that increased highly sialylated Tf during pregnancy may facilitate both mobilization of iron from the liver and uptake of iron by hepatocytes. However, the true physiologic significance of the increased production of highly sialylated Tf during pregnancy is unclear. In this study, the serum Tf concentrations in severely preeclamptic women were shown to decrease compared to those in normal pregnant women, as described previously [13,14]. Falling levels of estrogen and progesterone in the serum and urine have been reported during pregnancy with preeclampsia [23 –25], which might be the cause of decreased total Tf and highly sialylated Tf. It has been reported that a reciprocal relationship between serum iron concentration and total Tf commonly exists in disorders affecting iron metabolism [18]. A similar mechanism to maintain the reciprocal relationship might be working in preeclampsia, since the serum iron concentration significantly increases in preeclampsia [13,26]. Proteinuria might also contribute to decreased total Tf in SPE. In the present study, only 3 of 13 severely preeclamptic patients had mildly abnormal AST values, and none showed lowered total Tf relative to other patients, therefore the decreased total Tf in SPE could not be explained by liver damage. Both in pregnant and nonpregnant women, the liver is the site of active production of Tf. Carbohydrate units are added to Tf post-translationally from a dolichol-oligosaccharide precursor by the action of

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a multi-glycosyltransferase system [27]. So the decreased highly sialylated Tf in SPE may be secondary to inhibition of the enzyme responsible for the incorporation of sialic acid into Tf (multi-glycosyltransferase). However, a decreased activity of multi-glycosyltransferase in preeclampsia has not yet been reported. As for the low sialylated Tf, our study revealed that its concentration as well as 3-sialo-Tf concentration decreased sensitively in accordance with the severity of preeclampsia. So low sialylated Tf or 3-sialo-Tf might be better markers for preeclampsia. One of the reasons the low sialylated Tf in preeclampsia decreased might be the same one that caused the decrease in total Tf [28]; however, it is currently not known why it decreased in SPE to the level lower than that in nonpregnant women. The major source of iron in the placental transport system is maternal Tf. Decreased total Tf in SPE might reduce the transplacental transfer of iron resulting in fetal iron deficiency. It has been reported that qualitative changes in the glycosylation of glycoproteins can be accompanied by changes in their functional properties [6,10,11,29– 32]. Therefore, the decrease in Tf glycosylation in SPE might unfavorably affect the transport and delivery of iron to tissues and cells by Tf, including transplacental iron transport, and aggravate fetal iron deficiency. In our study, a positive significant correlation was found between the maternal serum total Tf concentrations and infant birth weights or maternal serum highly sialylated Tf concentrations and infant birth weights in SPE. This suggests that the decreases in maternal total Tf and Tf glycosylation in SPE might have a negative impact on intrauterine growth by influencing the iron supply to the fetus and correlate with the intrauterine growth retardation often observed in preeclampsia. Additionally, serum Tf may limit oxidant induction on tissue injury through its capacity to bind with iron, and thus may correlate with the progressive increase in the serum anti-oxidative activity during normal gestation [13,14]. Decreased total Tf along with increased iron concentration in SPE result in the marked increase in Tf saturation and then may exacerbate oxidative stress by decreasing the serum antioxidant capacity [13,14,26]. This might be another reason for the increased perinatal morbidity and mortality in preeclampsia.

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In conclusion, the serum concentrations of total and highly sialylated Tfs decrease significantly in SPE when compared to those in normal pregnancy. This might have a negative impact on intrauterine growth by suppressing the iron supply to the fetus and thus correlate with intrauterine growth retardation. In addition, the low sialylated Tf involving 2-sialo- and 3sialo-Tfs decreases more sensitively in preeclampsia, although the concentration per se is low even in normal pregnancy. Acknowledgements The authors thank Ms. Sumiko Tanaka and Dr. Masaki Ueno for their technical assistance, and Ms. Yuko Fujiwara and Dr. Koichi Matsumoto for their help with the statistical analysis. References [1] de Jong G, van Eijk HG. Microheterogeneity of human serum transferrin: a biological phenomenon studied by isoelectric focusing in immobilized pH gradients. Electrophoresis 1988; 9:589 – 98. [2] de Jong G, van Dijk JP, van Eijk HG. The biology of transferrin. Clin Chim Acta 1990;190:1 – 46. [3] van Eijk HG, de Jong G. The physiology of iron, transferrin, and ferritin. Biol Trace Elem Res 1992;35:13 – 24. [4] de Jong G, van Noort WL, Feelders RA, de Jeu-Jaspars CM, van Eijk HG. Adaptation of transferrin protein and glycan synthesis. Clin Chim Acta 1992;212:27 – 45. [5] van Eijk HG, van Noort WL, de Jong G, Koster JF. Human serum sialo transferrins in diseases. Clin Chim Acta 1987;165: 141 – 5. [6] Feelders RA, Vreugdenhil G, de Jong G, Swaak AJG, van Eijk HG. Transferrin microheterogeneity in rheumatoid arthritis. Rheumatol Int 1992;12:195 – 9. [7] Yamashita K, Koide N, Endo T, Iwaki Y, Kobata A. Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. J Biol Chem 1989;264:2415 – 23. [8] Stibler H. Carbohydrate-deficient transferrin in serum: a new marker of potentially harmful alcohol consumption reviewed. Clin Chem 1991;37(12):2029 – 37. [9] de Jong G, van Eijk HG. Functional properties of the carbohydrate moiety of human transferrin. Int J Biochem 1989; 21:253 – 63. [10] Hu WL, Chindemi PA, Regoeczi E. Reduced hepatic iron uptake from rat aglycotransferrin. Biol Met 1991;4(2):90 – 4. [11] Hoefkens P, Huijskes-Heins MI, de Jeu-Jaspars CM, van Noort WL, van Eijk HG. Influence of transferrin glycans on receptor binding and iron-donation. Glycoconj J 1997;14(2): 289 – 95.

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