Calcium metabolism in pre-eclampsia

International Journal of Gynecology & Obstetrics 66 Ž1999. 245]250

Article

Calcium metabolism in pre-eclampsia J. Ray, K. Vasishta, S. Kaur a , S. Majumdar a , H. Sawhney a,U a

Department of Obstetrics and Gynecology, and Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India Received 3 December 1998; received in revised form 3 June 1999; accepted 4 June 1999

Abstract Objecti¨ es: To study calcium metabolism in pre-eclampsia and normotensive gravid women. Method: Ten milliliters of heparinized blood samples and 24-h urine samples were collected from 50 pre-eclamptic and 50 normotensive primigravidae. Blood samples were studied for calcium uptake, intracellular calcium level and calcium-dependent adenosine triphosphatase activity of red blood cell ghost. Urinary calcium excretion was estimated from the 24-h urine samples. These values were compared in the two groups. Results: The mean gestational age at recruitment was similar in both the groups. The mean maternal age was 24.28" 2.41 years in pre-eclamptic and 23.48" 4.16 years in normotensive women. In pre-eclampsia 24-h urinary calcium excretion Ž71.20" 22.95 mgrday. and calcium-dependent ATPase activity Ž10.78" 2.40 nmolrPirmg proteinrmin. was significantly lower compared to normotensive primigravidae Žcalcium excretion s 189.24" 57.06 mgrday; Ca2q-dependent ATPase s 12.64" 2.42 nmolPirmg rprotein per min; P- 0.001.. Intracellular calcium levels and calcium uptake at 10 min by red blood cells were significantly higher in pre-eclampsia Ž P- 0.05.. Calcium uptake by red blood cells at 20 and 30 min was similar in both groups. Conclusion: Pre-eclampsia is associated with increased levels of intracellular calcium, decreased calcium-dependent ATPase activity of erythrocytes and hypocalciuria. Q 1999 Published by International Federation of Gynecology and Obstetrics. Keywords: Pre-eclampsia; Adenosine triphosphatase activity; Hypocalciuria

U

Corresponding author. Fax: q91-0172-744401.

0020-7292r99r$20.00 Q 1999 Published by International Federation of Gynecology and Obstetrics. PII: S 0 0 2 0 - 7 2 9 2 Ž 9 9 . 0 0 0 9 6 - X

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1. Introduction Pre-eclampsia occurs in 7]10% of pregnancies and remains a leading cause of maternal and neonatal mortality and morbidity w1x. This disorder is characterized by generalized arteriolar vasospasm, abnormalities in plasma volume, hypertension, proteinuria and edema w2x. The cause of the altered vascular reactivity in pre-eclampsia is not clear. The levels of free intracellular calcium is a major determinant of vascular smooth muscle tone and consequently vascular resistance. Cooper et al. w3x, have reported elevated erythrocyte sodium and platelet ionized calcium levels in hypertensive patients. Although epidemiologic studies have suggested a role for calcium deficiency in the development of pre-eclampsia w4,5x the published information regarding calcium metabolism in pre-eclampsia is scanty. Sowers et al. w6x and Haller et al. w7x observed an increase in erythrocyte and platelet calcium levels in pre-eclampsia. Nardulli et al. w8x documented a 50% decrease in adenosine triphosphatase activity of red blood cell ŽRBC. ghost in pre-eclamptic women. On the contrary, Zemel et al. w9x did not observe any difference in basal intracellular calcium levels in pre-eclampsia and normotensive gravid women. The study was undertaken to evaluate calcium metabolism Žerythrocyte calcium levels, calcium uptake, calcium-dependent adenosine triphosphatase activity and urinary calcium excretion. in patients with pre-eclampsia and normotensive gravid controls.

2. Material and methods One hundred primigravidae with singleton pregnancy and a certain menstrual period attending the antenatal clinic and labor room of Nehru Hospital, attached to the Postgraduate Institute of Medical Education and Research, Chandigarh, were recruited in the study. They were divided into two groups. The study group comprised of 50 pregnant women with pre-eclampsia. Pre-

eclampsia was defined as hypertension of 140r90 mmHg Žor more. or a rise of systolic blood pressure of 30 mmHg or diastolic blood pressure of 15 mmHg over the baseline value in addition to urine albumin of 1 q or more. The control group consisted of 50 normotensive primigravidae matched for age and gestational age. Patients with chronic hypertension, renal disease, or any medico-surgical disorder and with a history of intake of calcium channel blockers were excluded from the study. All the women from both groups were on iron, folic acid and calcium Ž1 g. supplementations during pregnancy. Dietary calcium intake was similar in both groups. Ten milliliters of heparinized blood sample and 24-h urine samples were collected from all the women. Blood samples were analyzed for calcium uptake, intracellular calcium levels and calcium-dependent adenosine triphosphatase activity of RBC ghosts. 2.1. ATPase estimation Hemoglobin-free RBC ghosts were prepared from the heparinized blood samples. Ghosts were stored at y20 o C in solution containing 17 mmolrl Tris hydrochloride acid and 0.1 mmolrl ethylene diaminetetracetic acid ŽpHs 7.5 at 0 o C. and used within 7 days after preparation. The calcium adenosine triphosphatase activities of RBC ghosts were determined for each point in triplicate following the method described by Quigley and Gotterer w10x as modified by Roullet et al. w11x. The inorganic phosphate liberated during the reaction was determined according to a slight modification of the method described by Marin et al. w12x. Briefly, the calcium ATPase activity was carried out as follows: 100 ml of ghosts Ž1 mg proteinrml. was incubated for 30 min in 400 ml of a reaction buffer in the presence and absence of calcium. At the end of the incubation period 300 ml of stop solution was added and the tubes placed on ice for 10 min. The color was developed and measured at 705 nm. The Ca2q ATPase activity was calculated as the difference between the quantity of phosphate liberated in the presence and absence of Ca2q.

J. Ray et al. r International Journal of Gynecology & Obstetrics 66 (1999) 245]250

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2.2. Calcium estimation

2.5. Statistical analysis

Serum total calcium as well as urinary calcium and creatinine were determined by standard clinical chemical methods using the SMA II analytic system ŽTechnicon, NY, USA..

Statistical analysis of the data was done by using paired and unpaired t-test, and analysis of variance.

2.3. Intracellular calcium estimation Intracellular-free calcium ion concentration was measured by the fluorescent indicator fura2racetoxymethyl ester ŽFura 2rAM; Sigma Chemical Co., USA. taken up in the cell as its membrane permeate AM form and trapped intracellularly after hydrolysis by the cytosolic esterases w13x. The RBCs were loaded with Fura2rAM by incubation with 2 mmolrml ester for 45 min at 378C. The cells were washed, centrifuged and resuspended to give a final concentration of 8]10 = 10 5 cellsrml. The fluorescence Ž F . was measured at 340r510 nm Žexcitationr emission. in a kostron spectrofluorometer. Fmin and Fmax were measured by permeabilizing the cells with 10 ml of digitonin Ž10 mM. at zero Ca2q concentration with 15 ml of 1 mmol EGTA, pHs 7.4 adjusted with Tris and saturating Ca2q concentrations, respectively. The intracellular-free Ca2q concentration was calculated by the following formula: Ca2q i sFy Fmin = K d rFmax y F, where K d is the dissociation constant of Fura 2rAM and its value is 224 nmolrl. 2.4. Ca 2 q-Uptake Calcium uptake by RBCs was studied by a modified Brass Method w14x as modified by Gulati et al. w15x. Briefly the gel-filtered RBCs were incubated with CaCl 2 for 30 min at 378C. A trace quantity of 45 CaCl 2 ŽBARL, Bombay, India. was added to the RBCs and aliquots of the suspension removed at specified time intervals Ž10, 20 and 30 min.. Each aliquot was diluted with an ice-cold wash buffer and filtered through 0.45-mm millipore HAWP filters. Filters were rinsed twice with a wash buffer and the 45 Ca retained on the RBCs was measured by scintillation counting on a Beta-scintillation counter.

3. Results Subject characteristics are described and compared in Table 1. The maternal age and gestational age at recruitment was similar in both groups. In the study group, the majority of women Ž82%. had proteinuria in the range of 1 q to 2 q and 48% of them had diastolic blood pressure of 100 mmHg or more. The 24-h urinary calcium excretion was significantly lower in women with pre-eclampsia ŽTable 2.. Calcium-dependent ATPase activity of red blood cell membranes was lower in the study group Ž10.78" 2.40 nmolrPirmg protein per min. compared to the control group Ž12.64 " 2.42 nmolrPirmg protein per min.. Intracellular calcium levels were significantly higher in the study group Ž62.82" 22.64 nmolrl = 10 6 RBCs than in the control group Ž50.14" 16.52 nmolrl = 10 6 RBCs P- 0.005.. Calcium uptake by RBCs was studied at 10-, 20- and 30-min intervals. Calcium uptake at 10 min was significantly higher in the study group Ž P- 0.05.. At 20 and 30 min, calcium uptake was similar in both groups ŽTable 3.. Calcium uptake at 20 min was at a maximum in both the preTable 1 Subject characteristics Parameter Žmean " S.D..

Study group

Control group

P value

Age Žyears. Gestational age Žweeks. Blood pressure ŽmmHg. Systolic Diastolic

24.28" 2.41 36.62" 2.42

23.48" 4.16 36.81" 2.23

) 0.05 ) 0.05

144.8" 9.74 97.8" 8.47

114.88" 7.64 76.32" 5.23

- 0.001 - 0.001

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J. Ray et al. r International Journal of Gynecology & Obstetrics 66 (1999) 245]250

Table 2 Comparison of urinary calcium excretion Ž24-h., calcium-dependent ATPase activity and intracellular calcium levels of RBCs in study and control groups Parameters Žmean " S.D..

Study group Žmean " S.D..

Range

Control group Žmean " S.D..

Range

P value

24-h urinary calcium excretion Žmgrday. Ca2q-dependent ATPase activity of RBCs membrane ŽnmolrPirmg proteinrmin. Intracellular Ca2q in RBCs Žnmolrl = 106 RBCs.

71.20" 22.95

36]132

189.24" 57.06

65]310

- 0.001

10.78" 2.40

4.6]15.8

12.64" 2.42

7.7]18.2

- 0.001

62.82" 22.64

29]141

50.14" 16.52

23]115

- 0.005

eclamptic and normotensive women ŽStudy group s 0.80" 0.19 nmolrmg protein; control groups 0.79" 0.09 nmolrmg protein.. There was a significant difference in calcium uptake at various time intervals Ž10, 20 and 30 min. in both groups ŽFig. 1..

4. Discussion Cellular abnormalities of pregnancy-induced hypertension ŽPIH. remain poorly understood. Recently it has been postulated that cellular cation metabolism may play a role in the pathogenesis of PIH. Pitkin w16x noticed that calcium absorption increases in normal pregnancy, but serum calcium levels do not change significantly. Urinary calcium excretion in normal pregnancy is 350]620 mgrday compared to 100]250 mgrday in nonpregnant woman. The serum calcium level in pre-eclampsia appears to be no different from the

values in normotensive pregnant women but urinary calcium is considerably reduced in preeclampsia w17,18x. Suraez et al. w19x observed that low urinary calcium excretion Ž- 3.4 mgrkg per 24 h. at 17]20 weeks of gestation is a major risk factor for subsequent development of pre-eclampsia in asymptomatic primigravidae. In the present study urinary calcium excretion was significantly lower in pre-eclamptic women Ž71.20" 22.95 mgrday. compared to normotensive women Ž189.24" 57.06 mgrday.. Calcium transport and calcium-dependent ATPase activity have been shown to decrease in RBCs of hypertensive subjects and this correlates well with increased intracellular calcium levels in erythrocyte and platelets of patients with essential hypertension. Sowers et al. w6x reported that erythrocyte calcium levels were elevated in pre-eclamptic women. This increase was specific for calcium because intracellular concentration of sodium, potassium

Table 3 Comparison of calcium uptake Žnmolrmg protein. by RBCs at different time intervals in study and control groups Time Žmin.

10 20 30

Study group Žmean " S.D..

Calcium uptake and range Žnmolrmg protein. Range

Control group Žmean " S.D..

Range

P value

0.24" 0.04 0.80" 0.18 0.41" 0.10

0.19]0.33 0.11]1.52 0.25]0.72

0.22" 0.03 0.70" 0.09 0.38" 0.05

0.15]0.3 0.46]0.97 0.23]0.52

- 0.05 ) 0.05 ) 0.05

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Fig. 1. Calcium uptake by RBC Žnmolrmg protein at various time intervals in study and control groups..

and magnesium was not different in pre-eclamptic and normotensive gravid women. On the contrary, Zemel et al. w9x, did not observe any significant change in the basal levels of intracellular calcium in pre-eclamptic and normotensive patients during the course of pregnancy. However, they did observe that platelet intracellular calcium levels in pre-eclamptic women were more sensitive to arginine and vasopressin than those of normotensive pregnant women. Discriminant analysis indicated that the platelet intracellular calcium response to arginine and vasopressin during the first trimester was a

significant predictor of subsequent development of pre-eclampsia. Nardulli et al. w8x have demonstrated decreased erythrocyte calcium-dependent ATPase activity in pre-eclamptic patients. This decreased activity of calcium-dependent ATPase may be responsible for the increased intracellular calcium levels. In the present study, increased levels of intracellular calcium and decreased calcium-dependent ATPase activity of RBCs were observed in pre-eclamptic patients ŽFig. 2.. Calcium uptake by RBCs at 10 min was significantly Ž P- 0.05. higher in pre-eclamptic women Ž0.24" 0.04 nmolrmg

Fig. 2. Comparison of calcium levels Ž24-h urinary calcium]ATPase activity and intracellular calcium Ca2q levels in RBCs..

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protein. compared to normotensive women Ž0.22 " 0.03 nmolrmg protein.. At 20- and 30-min intervals calcium uptake by RBCs was similar in both groups. In conclusion, PIH is associated with abnormal calcium metabolism. Elevated intracellular calcium levels are due to decreased calcium-dependent ATPase activity, as calcium uptake by red blood cells at 30 min was similar in pre-eclamptic and normotensive gravid women. Hypocalciuria in PIH might be a result of abnormal calcium uptake and decreased calcium efflux by the renal tubular cells similar to that of RBCs. Whether abnormal calcium metabolism in PIH is a primary cause or a consequence which leads to further changes in pre-eclampsia, needs further evaluation.

w8x

w9x

w10x

w11x

w12x

References w1x WHO Technical Report Series No. 758. The hypertensive disorders of pregnancy. Geneva: World Health Organization, 1987:93. w2x Sabatini S. Pathophysiology of, and therapeutic strategies for hypertension in pregnancy. Curr Opin Nephrol Hypertens 1993;2:763]774. w3x Cooper RS, Shamsi N, Katz S. Intracellular calcium and sodium in hypertensive patients. Hypertension 1987;9: 224]229. w4x Belizan JM, Villar J. The relationship between calcium intake and edema, proteinuria and hypertension-gestosis; an hypothesis. Am J Clin Nutr 1980;33:2202]2210. w5x Villar J, Belizan JM, Fischer PJ. Epidemiologic observations on the observations on the relationship between calcium intake and eclampsia. Int J Gynecol Obstet 1983;21:271]278. w6x Sowers JR, Zemel MB, Bronsteen RA et al. Erythrocyte cation metabolism in pre-eclampsia. Am J Obstet Gynecol 1989;161:441]445. w7x Haller H, Oeney T, Hauck U, Destler A, Phillip T. Increased intracellular free calcium and altered sensitiv-

w13x

w14x w15x

w16x

w17x

w18x

w19x

ity to angiotensin II in platelets of pre-eclamptic women. Am J Hypertens 1989;2:238]243. Nardulli G, Provabio F, Limongi FG, Marin R, Proverbio T. Pre-eclampsia and calcium adenosine triphosphatase activity of red blood cell ghosts. Am J Obstet Gynecol 1994;171:1361]1365. Zemel MB, Zemel PC, Berry S et al. Altered platelet calcium metabolism as an early predictor of increased peripheral vascular resistance and pre-eclampsia in urban block women. N Eng J Med 1990;323:434]438. Quigley JP, Gotterer GS. Distribution of Naq]Kq stimulated ATPase activity in rat intestine mucosa. Biochim Biophys Acta 1969;173:456]458. Roullet JB, Le Quan Sarg KH, Luft U, Watnabe M, Otsuka K, McCarron DA, Devynck MA. Inhibition of Ca2q uptake into A7r5 vascular smooth muscle cells by farneol: lack of effect on membrane fluidity and Ca2q ATPase activities. J Hypertens 1997;15Ž12.Pt2.:1723] 1728. Marin R, Proverbio T, Proverbio F. Inside out basolateral plasma membrane vesicles from rat kidney proximal to tubular cells. Biochem Biophys Acta 1986;858: 195]201. Grinkiewicz G, Poeni M, Tsein RY. A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440]4350. Brass LF. Ca2q hemostasis in unstimulated platelets. J Biol Chem 1984;259:12560]12570. Gulati S, Khullar M, Sagar S, Ganguly NK, Sharma BK. Ca2q transport in unstimulated platelets of essential hypertensives and their blood relatives. J Hypertens 1992;10:1213]1219. Pitkin RM, Reynolds WA, Williams GA, Horgia F. Calcium metabolism in normal pregnancy: a longitudinal study. Am J Obstet Gynecol 1979;133:781]790. Taufield PA, Ales KL, Resnick LM, Druzin ML, Gertner JM, Laragh JH. Hypocalciuria in pre-eclampsia. N Eng J Med 1987;316:715]718. Sanchez-Ramos L, Sandroni S, Andres FJ, Kaunitz AM. Calcium excretion in pre-eclampsia. Obstet Gynecol 1991;77:510]513. Suarez VR, Trelles JG, Miyahira JM. Urinary calcium in asymptomatic primigravidas who later developed preeclampsia. Obstet Gynecol 1996;87:79]92.