Medicina (Kaunas) 2004; 40(6)
547
Ferrous iron administration during pregnancy and adaptational oxidative stress (Pilot study) Aune Rehema, Kersti Zilmer, Ursula Klaar1, Helle Karro1, 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 (95–105 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 con-
ditions 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, 51014 Tartu, Estonia E-mail:
[email protected]
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 mg/ml. The GSSG/GSH ratio was calculated: mg GSSG / mg 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
