Ferrous iron administration during pregnancy and adaptational oxidative stress (Pilot study)

Transcription

1 547 Ferrous iron administration during pregnancy and adaptational oxidative stress (Pilot study) Aune Rehema, Kersti Zilmer, Ursula Klaar 1, Helle Karro 1, Tiiu Kullisaar, Mihkel Zilmer Department of Biochemistry, Tartu University 1 Clinic of Obstetrics and Gynecology, Tartu University Clinics, Estonia Key words: iron, anemia, pregnancy, oxidative stress, glutathione. Summary. The purpose of this investigation was to measure the effect of ferrous iron therapy on indices of oxidative stress in pregnant women. Material and methods. Nineteen healthy pregnant women with borderline anemia were subjected to per oral ferrous iron in prophylactic doses (36 mg daily, +Fe group, n=13) or did not get any treatment (-Fe group, n=6). The indices measured in two time-points were: conjugated dienes, lag phase of low-density lipoproteins, total antioxidant activity of the serum, total glutathione and its fractions, protein carbonyls, catalase, ferritin, serum iron and unsaturated iron binding capacity. Results. All women had high values of conjugated dienes, total antioxidant activity and short lag phases of low-density lipoproteins in both time points. Elevated values of oxidized glutathione in both groups increased about 2-fold and reached a significant difference level (p=0.02) by the end of the test period. Conclusion. Low doses of per oral ferrous iron did not change deleteriously the physiological pattern of parameters of oxidative stress in our small group of healthy pregnant non-smokers but the elevation of oxidized glutathione might imply on the elevation of risk. Introduction Iron-deficiency anemia is considered the most widespread pregnancy-associated pathological condition. Severe anemia (hemoglobin (Hb) less than 80 g/l) in the first half of pregnancy is proved to be associated with preterm delivery and small-for-gestational-age fetus (1 3). In contrast, the values of borderline anemia ( g/l of Hb) appear to be related to the minimum incidence level of preterm delivery (4). This is attributed to the fact that high (over 120 g/l) values of hemoglobin might indicate the inadequacy of adaptational plasma volume expansion in the third trimester rather than an iron-replete state of the woman (5). Ferrous iron is the form that is mostly used for correction of iron deficiency. About 3 5% of the iron present in alimentary canal in ferrous form is absorbed. Acidic milieu facilitates the absorption by keeping iron in the ferrous form. Ferrous iron is a central pro-oxidant that propagates free radical reactions through Fenton chemistry both locally (in the gastrointestinal tract) and systemically. An excess of pro-oxidants over antioxidants results in oxidative stress (OS). Numerous pathological conditions with endothelial dysfunction (including cancer, diabetes, liver cirrhoses and atherosclerosis) have OS either as an initiating or pathogenetic mechanism (3). Iron medical preparations (also those exceeding Recommended Dietary Allowances (RDA) 10 times or more) are sold over the counter and suggestions for their use vary considerably. There is no scientifically proven and ultimate consensus about using the iron therapy (the criteria for its use as well as the dosage) during pregnancy. We measured the schemes of iron supplementation applied in Estonia today by using the criteria of OS to evaluate their safety. The whole issue becomes more intricate as pregnancy itself induces OS (6, 7) and as preeclampsia is proved to be (at least partially) mediated by OS (8, 9). Even a short-term use of iron medical preparations should be monitored from the point of view of OS to avoid damages in a situation influencing two generations at a time pregnancy. Sub clinical tortuous damages during pregnancy might increase the risk for the mother or the baby as the OS-mediated free radical processes may initiate the pathogenesis of diseases e. g. diabetes. Correspondence to A. Rehema, Department of Biochemistry, Tartu University, Ravila street 19, Tartu, Estonia aune10@hotmail.com

2 548 Aune Rehema, Kersti Zilmer, Ursula Klaara et al Materials and methods The Ethics Committee of Human Investigation at Tartu University approved the project and all subjects gave their written informed consent. The subjects were clinically healthy pregnant women with hemoglobin values between 97 and 111 g/l at the enrolment. The average age was 24.8±5.9 years in the Fe-supplemented group (+Fe) and 27.2±4.7 years in the noniron-supplemented group ( Fe). There were 13 women in the +Fe group and 6 in Fe group. The mode of the number of pregnancy was 3 in the +Fe group and 2 in the Fe group. All subjects were non-smokers and consumers of normal mixed food. The gynecologist in charge of the patient (3 doctors) made a routine decision (based on the doctor s practice patterns and Hb, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) value) of whether to administer iron supplements as she would have done if no investigation took place. After that venous blood was collected with the Vacutainer system for additional biochemical parameters. The iron preparation Spartocine 100/250 (UCB Pharma, Belgium, containing 18/36 mg of ferrous aspartate) was used. All participants filled in an anamnestic questionnaire. Clinical parameters (Hb, Hematocrit (Ht), MCH) were taken from case histories (measured on a Sysmex clinical analyzer). After four weeks another venous blood sample was collected. Conjugated dienes (CD) were measured according to R. O. Recknagel and E. A. Glende (10). Lowdensity lipoprotein (LDL) lag phase was calculated from the curve that is formed from the results of measuring the protein-adjusted quantity of the non-highdensity lipoprotein (HDL) fraction (prepared from EDTA-plasma) peroxidation in time spectrophotometrically at 234 nm (11, 12). Serum iron (Fe), unsaturated iron binding capacity (UIBC), total iron binding capacity (TIBC) and saturation of iron-binding proteins (%sat) were measured with a manual spectrophotometrical assay (kit 565-A, Sigma St. Louis Mo). Ferritin was analyzed by the chemiluminescence method (on the Immulite analyzer, Diagnostic Products Corporation). Measurements of glutathione are actually measurements of thiols in whole blood with a method described by G. B. Bhat (13). The method is based on the formation of a chromophoric product from the sulfhydryl reagent 5,5-dithiobis- (2-nitrobenzoic acid), Ellmann reagent, in the presence of glutathione (GSH). The contents of total glutathione (tgsh) and oxidized glutathione (GSSG) are directly measured and the content of reduced glutathione is calculated from them. Glutathione was expressed as µg/ml. The GSSG/GSH ratio was calculated: µg GSSG / µg GSH. Catalase (CAT) in serum was measured by a method of L. Góth (14), the method being spectrophotometrically based on hydrogen peroxide forming a stable complex with ammonium molybdate. Total antioxidant activity (TAA) was measured according to original method of our Department of Biochemistry (15). Protein carbonyls (PC) were quantified by a method of R. L. Levine et al (16), protein content (mg/ml) was determined in each sample (parallel) versus guanidine and calculated from a bovine serum albumin standard curve dissolved in guanidine-hcl and read at 280 nm (17). The carbonyl content was expressed as nmol carbonyl/ mg protein. Statistical analysis was performed using Microsoft Excel (average, standard deviations, Student s t-test for unpaired data between groups of +Fe and Fe and for paired data between two time points, correlations). Results No significant differences between groups in the parameters of OS were found before the test period. The only parameter that gave p<0.05 after 4 weeks was GSSG but both groups had a roughly 2-fold increase in GSSG content that also affected tgsh value (Table, Figure). When compared to the endemic normal (0.17±0.08), GSSG/GSH ratio was high from the beginning in both groups. Mostly the value could be attributed to relatively low content of GSH (the endemic normal being 195±17 µg/ml). We found that glutathione redox ratio and the content of CD tend to increase as pregnancy progresses. This is in agreement with previously published data (18, 19). The content of carbonyl groups did not increase and TAA stayed on the same high level (an adult normal range of TAA is 38±3.7%, unpublished data, as for PC, we have measured 1.09±0.53 nmol/mg prot in group of subjects with atherosclerosis and 3.64±1.56 nmol/mg prot in subjects with renal failure). As for CAT there was much diversity but the means did not differ from each other and from the endemic normal (55±23 ku/l). Compared to the endemic normal CD were relatively high (45.4 to 53.9 in different groups versus 38.4±7.5 µm). LDL lag phases in turn were relatively short when compared to the endemic normal ( versus 49±10 min). No significant differences between groups in these parameters could be detected. No statistically significant correlations between individual parameters were found.

3 Ferrous iron administration during pregnancy and adaptational oxidative stressa 549 Table. +Fe and Fe group before intervention and after 4 weeks Difference between +Fe group Fe group Parameter groups, p before after p before after p before after Hb, g/l 103.1± ± ± ± * 0.14 Fe, mmol/l 12.5± ± ± ± Ferritin, µg/l 13.9± ± ± ± CD, µm 45.4± ± ± ± TAA, % 47.5± ± ± ± tgsh, µg/ml 190.8± ± * 148.3± ± * GSSG, µg/ml 81.5± ± * 55.8± ± * * GSSG/GSH 0.81± ± ± ± µg/µg PC, 2.03± ± ± ± nmol/mgprot CAT, ku/l 51.6± ± ± ± LDL lag 30.0± ± ± ± phase, min P is calculated between two time points for both groups and between the supplemented and non-supplemented groups (the last two columns). Values are presented as mean ± SD. *p<0.05, the difference is considered significant. Hb hemoglobin, Fe serum iron, CD conjugated dienes, TAA total antioxidant activity, tgsh total glutathione content, GSSG oxidized glutathione, GSSG/GSH glutathione redox-ratio, PC protein carbonyls, CAT catalase, LDL lag phase the time of resistance to oxidation of low-density-lipoprotein phase of serum , Control +Fe before +Fe after Fe before Fe after GSH GSSG Fig. The dynamics of the glutathione system Control means the endemic normal value. Women in both groups (iron-supplemented +Fe and non-iron-supplemented Fe) experienced similar increase in GSSG content and a relatively low GSH, when compared to the endemic normal. GSH and GSSG are given in µg/ml. Serum iron in both groups was about 14 mmol/l after the investigation period, but the starting points appeared to be slightly different (though not significantly). Ferritin was about at the level of anemia criteria (that is 12 ng/ml). There were no changes in protein carbonyl content and catalase activity. As the values of Hb in the supplemented and non-supplemented groups at start were slightly different (probably

4 550 Aune Rehema, Kersti Zilmer, Ursula Klaara et al reflecting the strong urge to administer iron supplements at Hb levels lower than 100 g/l) we performed a correlation analysis between the values of Hb and the values of GSSG/GSH and GSSG (and, to be absolutely certain, between Hb and GSH) and there was no significant correlation, the correlation coefficients being 0.04 and 0.08 and 0.14, respectively. Discussion Although World Health Organization (WHO) has made its recommendations, there is no worldwide consensus concerning iron prophylaxis and therapy during pregnancy. There does exist a contradiction between scientific results and common practice. The hemoglobin content of the mother s blood that correlates with the best outcome is considered to be g/l in the third trimester (4). Most gynecologists reach for iron tablets when the hemoglobin value drops below 110 g/l. In most cases even serum ferritin content or MCV value are not essential in decision-making. There is no conclusive evidence that improvement of the iron status during the first and second trimester would improve the outcome of pregnancy (20 22) and the hemoglobin values in the third trimester are not considered to influence the outcome significantly at all (1). From the point of view of OS, ferrous iron (that is traditionally the form of treatment) is a potent pro-oxidant. Administration of iron causes local OS. Based on animal models the question has been raised if previously iron-deficit pregnant women were more susceptible to therapeutic-iron-induced OS than ironreplete subjects. This further discourages the concept of routine supplementation with ferrous iron (22). More and more recent articles underlie the necessary caution considering iron therapy (23, 24). High-grade oxidative stress (and iron) is widely acknowledged to have a role in the pathogenesis of neurodegenerative diseases, atherosclerosis, diabetes complications, arthritis etc. (25). Pregnancy itself is associated with an increase in OS (6, 7) (both parameters of oxidation and antioxidative defense are elevated). These changes may play an adaptational role and as long as the importance of them is not clearly understood no attempts to correct these changes are advisable. But preeclampsia is associated with even more serious disbalance towards OS (6, 8, 18, 25) and development of this pathological condition is definitely undesirable. In addition to this, speculations have been made (25) that an uncompensated OS experienced during pregnancy could predict or affect some pathological conditions of mother in the future. The effect on the developing fetus cannot be excluded either (e. g. from the point of view of future diabetes or atherosclerosis). Our aim was to measure the influence of routine practice of iron treatment on the indices of iron metabolism and OS. We considered it important to measure the potential risk in clinically healthy subjects to exclude all possible interactions of risk factors (smoking, previous hypertension, diabetes, dyslipidemia, multiple fetuses, infection, etc.). The aim was to test the most widespread clinical situation (borderline anemia) and supplemental iron doses that did not exceed RDA for non-pregnant women more than 2 times. The upper safe level for per oral iron (hem and nonheme iron together) is agreed to be mg a day. The RDA for pregnant women in many countries is 30 mg (22). The discrepancy might arise in part from the fact, that at 30 mg level 95% of women do not get anemic (do not have Hb below 110 g/l). Not many attempts have been made to measure the potential harm done at these doses. We found no conclusive adverse influence to the situation of OS in pregnant women of our test group (Table). All the babies were born with Apgar scores However, the elevation of GSSG might imply on the elevation of risk and as the study design was limited in terms of enrolment conditions and sample size the results may not describe the situations where any risk factor is present or when the supplemented doses of ferrous iron are higher than 36 mg. Until larger investigations with higher doses and in risk groups are conducted, it would be wise to measure the level of oxidized glutathione and whole blood thiol oxidation ratio (primary and sensitive markers of intracellular high-grade OS), if any increase in risk is suspected or when ferrous iron supplementation is considered during pregnancy. Conclusions In pregnant women with borderline iron-deficiency anemia (defined by Hb and proved by ferritin) we found (compared to the endemic normal) high levels of TAA ( in different time points vs. 38±3.7%) and high levels of CD ( vs. 38.4±7.5 µm). These values indicate both enhanced lipid peroxidation and elevated antioxidative defense. However, the parameters giving the most prominent changes during the 4- week test period and ultimately being statistically different in the tested groups belonged to the glutathione system (especially GSSG that also differed significantly from the endemic normal) (Figure). As the glutathione system has paramount importance in cellular defense in OS the impairment may be clinically (prognostically) significant. In our study group of pregnant nonsmokers we

5 Ferrous iron administration during pregnancy and adaptational oxidative stressa 551 received the results that are in consistency with previously published data: the pregnancy-associated changes in OS parameters both in cellular and serum markers (6, 7) and for the first time documented the possibly problematic increase in GSSG in answer to ferrous iron administration. As our study group had no other high-grade OS risk factors the results imply on the possible clinical importance of oxidized glutathione level and whole blood thiol oxidation ratio measurements (primary and sensitive markers of intracellular high-grade OS) if any increase in risk is suspected or when ferrous iron supplementation is considered during pregnancy. Acknowledgements The authors would like to express their gratitude to Tiiu Vihalemm and Külli Teder for their technical contribution and inspiring comments to this work. The publication of this work was supported by Estonian Science Foundation grant No Dvivalentės geležies vartojimas nėštumo ir adaptacinio oksidacinio streso metu (pilotinis tyrimas) Aune Rehema, Kersti Zilmer, Ursula Klaar 1, Helle Karro 1, Tiiu Kullisaar, Mihkel Zilmer Tartu universiteto Biochemijos katedra 1 Tartu universiteto klinikų Akušerijos ir ginekologijos klinika, Estija Raktažodžiai: geležis, anemija, nėštumas, oksidacinis stresas, gliutationas. Santrauka. Tyrimo tikslas. Įvertinti dvivalentės geležies poveikį nėščiųjų oksidacinio streso indeksams. Tyrimo medžiaga ir metodai. Devyniolika sveikų nėščiųjų, kurioms nustatyta ribinės anemijos požymių, buvo suskirstytos į dvi grupes: trylikos moterų grupei (pirma grupė) buvo skirta geležis profilaktinėmis dozėmis (36 mg per dieną, +Fe grupė), o šešių moterų grupei (antra grupė) geležies neskirta ( Fe grupė). Tyrimo pradžioje ir praėjus keturioms savaitėms, buvo vertinami šie rodikliai: konjuguoti dienai, mažo tankio lipoproteinų oksidacijos vėlavimo fazės trukmė, bendrasis antioksidacinis serumo aktyvumas, bendrasis gliutationo ir jo frakcijų kiekis, baltymo karbonilų kiekis, katalazės kiekis, feritino kiekis, serumo geležies kiekis ir neįsotintos geležies sujungimo geba. Rezultatai. Abiejų tyrimų metu visoms moterims nustatytas didelis konjuguotų dienų kiekis, didelis bendrasis antioksidacinis serumo aktyvumas ir trumpos mažo tankio lipoproteinų oksidacijos vėlavimo fazės. Jau ir taip padidėjęs oksiduoto gliutationo kiekis abiejų grupių tiriamosioms tyrimo pabaigoje padidėjo du kartus ir tapo statistiškai reikšmingas (p=0,02). Išvados. Mažos geriamosios geležies dozės neturėjo neigiamos įtakos oksidacinio streso fiziologiniams parametrams nerūkančių moterų grupėje, bet oksiduoto gliutationo kiekio didėjimas gali turėti įtakos rizikos padidėjimui. Adresas susirašinėjimui: A. Rehema, Tartu universiteto Biochemijos katedra, Ravila 19, Tartu, Estija El. paštas: aune10@hotmail.com References 1. Scanlon KS, Yip R, Schieve LA, Cogswell ME. High and low hemoglobin levels during pregnancy: differential risks for preterm birth and small for gestational age. Obstet Gynecol 2000;96: Rasmussen KM. Is there a causal relationship between iron deficiency or iron-deficiency anemia and weight at birth, length of gestation and perinatal mortality? J Nutr 2001; 131S: Schümann K. Safety aspects of iron in food. Ann Nutr Metab 2001;45: Steer PJ. Maternal hemoglobin concentration and birth weight. Am J Clin Nutr 2000;71 Suppl 5: Scholl TO, Reilly T. Anemia, iron and pregnancy outcome. J Nutr 2000;44S: Morris JM, Gopaul NK, Endresen MJ, Knight M, Linton EA, Dhir S, et al. Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia. Br J Obstet Gynaecol 1998;105(11): Geisser P. Iron Therapy with special emphasis on oxidative stress. 1st ed. Vifor Inc.; Hubel CA. Dyslipidemia, iron, and oxidative stress in preeclampsia: assessment of maternal and feto-placental interactions. Semin Reprod Endocrinol 1998;16: Roberts JM, Cooper DW. Pathogenesis and genetics of preeclampsia. Lancet 2001;357(9249): Recknagel RO, Glende EA. Spectrophotometric detection of lipid conjugated dienes. Methods Enzymol 1984;105: Zhang A, Vertommen J, Van Gaal L, De Leeuw I. A rapid and simple method for measuring the susceptibility of low-densitylipoprotein and very-low-density-lipoprotein to copper catalyzed oxidation. Clin Chim Acta 1994;227(1 2): Esterbauer H, Gebicki J, Puhl H, Jurgens J. The role of lipid peroxidation and antioxidants in oxidative modification of

6 552 Aune Rehema, Kersti Zilmer, Ursula Klaara et al LDL. Free Rad Biol Med 1992;13: Bhat GB, Tinsley SB, Tolson JK, Bath JM, Block ER. Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury. J Cell Physiol 1992;152: Góth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 1991; 196: Starkopf J, Zilmer K, Vihalemm T, Kullisaar T, Zilmer M, Samarutel J. Time course study of oxidative stress during open heart surgery. Scand J Thorac Cardiovasc Surg 1995; 29: Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186: Lyras L, Evans PJ, Shaw PJ, Ince PG, Halliwell B. Oxidative damage and motor neurone disease. Difficulties in the measurement of protein carbonyls in human brain tissue. Free Radic Res 1996;24(5): Raijmakers MT, Zusterzeel PL, Roes EM, Steegers EA, Mulder TP, Peters WH. Oxidized and free whole blood thiols in preeclampsia. Obstet Gynecol 2001;97(2): Little RE, Gladen BC. Levels of lipid peroxides in uncomplicated pregnancy: a review of the literature. Reprod Toxicol 1999;13: Walker AR. The remedying of iron deficiency: what priority should it have? Br J Nutr 1998;79: Hollįn S, Johansen KS. Adequate iron stores and the Nil nocere principle. Haematologia (Budap) 1993;25(2): Casanueva E, Viteri FE. Iron and oxidative stress in pregnancy. J Nutr 2003;133 Suppl 5 (Pt 2): Freitas J, Meneghini R. Iron and its sensitive balance in the cell. Mutat Res 2001;475: Drüeke T, Witko-Sarsat V, Massy Z, Descamps-Latscha B, Guerin AP, Marchais SJ, et al. Iron therapy, advanced oxidation protein products, and carotid artery intima-media thickness in end-stage renal disease. Circulation 2002;106: Gratacós E. Lipid-mediated endothelial dysfunction: a common factor to preeclampsia and chronic vascular disease. Eur J Obstet Gynecol Reprod Biol 2000;92:63-6. Received 16 January 2004, accepted 22 April 2004 Straipsnis gautas , priimtas