- Email: [email protected]
Non protein bound iron concentrations in amniotic fluid
Clinical Biochemistry 38 (2005) 674 – 677
Non protein bound iron concentrations in amniotic fluid D. Gazzoloa, S. Perroneb, P. Paffettib, M. Longinib, P. Vezzosib, M. Bruschettinia, M. Lituaniac, G. Buonocoreb,* b
a Department Paediatrics G. Gaslini, Children’s University Hospital, 16148 Genoa, Italy Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico ‘‘Le Scotte’’, V.le Bracci 36, 53100 Siena, Italy c Centre of Fetal and Perinatal Medicine, Galliera Hospital, 16121 Genoa, Italy
Received 20 December 2004; received in revised form 14 March 2005; accepted 21 March 2005 Available online 26 April 2005
Abstract Objectives: To investigate whether amniotic fluid concentrations of non protein bound iron (NPBI) vary with growth in healthy fetuses and also offer a reference curve in the second trimester of pregnancy. Design and methods: Amniotic fluid concentrations of NPBI were measured by HPLC in 118 women with physiological singleton pregnancies, who underwent amniocentesis for fetal karyotype between weeks 15 and 18 of gestation. Results: NPBI increased progressively from weeks 14 – 15 to weeks 15 – 16, peaking at 17 – 18 weeks of gestation. NPBI values regressed positively with gestational age (GA). Multiple linear regression analysis between NPBI, as dependent variable, and various fetal parameters, as independent variables, showed a statistically significant regression coefficient with GA, bi-parietal diameter and transverse cerebellar diameter. Conclusions: The present data constitutes the first quantification of NPBI concentrations in amniotic fluid under physiological conditions. Correlations with GA and ultrasound fetal biometry suggest that NPBI may play a role in fetal growth. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Non protein bound iron; Fetus; Amniotic fluid; Pregnancy; Oxidative stress
Introduction Iron is the most abundant transition metal in the body [1] and an essential factor for the growth and well-being of almost all living organisms [2]. The essentiality of iron is well established in view of its involvement in a large number of metabolic processes, including DNA, RNA and protein synthesis, as cofactor for numerous enzymes and myelin synthesis [3,4]. Iron is involved in oxygen transport, storage, utilization, activation and detoxification, in nitrogen fixation, antibacterial defenses and photosynthesis [5]. Iron deficiency during early development of the brain has been related to behavioral alterations including deficits in learning and memory mediated by the hippocampus [6]. Severe iron deficiency may lead to similar * Corresponding author. Fax: +39 0577 586182. E-mail address: [email protected] (G. Buonocore).
deficits in cell energy metabolism and organ performance in the brain, resulting in a reduced ability to respond to restriction of oxygen and perfusion. Studies on iron uptake by the brain have shown that iron transport and transferrin-binding sites are at a maximum during the period of brain growth, and maximum uptake by the brain occurs in 15-day-old rats [7]. In the human brain, growth spurt begins early in life and continues throughout the first year of life [8]. This is a critical period during which the brain undergoes several fundamental developmental phases such as maturation of axonal and dendritic outgrowth, establishment of neuronal connections, synaptogenesis, multiplication of glia cells with accompanying myelinization and cell, axonal and dendritic death [9]. Normally, iron is safely transported by proteins such as transferrin (Tf) and lactoferrin and stored in proteins such as ferritin (Ft) and hemosiderin [10]. In healthy adults, plasma transferrin is approximately one-third loaded with iron, and the protein retains a considerable ability to bind iron salts.
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.03.010
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677
Transferrin can bind 2 mol of iron per mol of protein. When iron is correctly loaded on its high-affinity binding sites, it is not available as a growth factor for tissues nor is it available as a pro-oxidant factor [11,12]. Under normal conditions, non protein bound iron is undetectable in adults [13]. To be redox cycling active, iron must be released from its macromolecular complexes as ‘‘free iron’’. Since iron ions cannot exist in plasma, the term Ffree iron_ or non protein bound iron (NPBI) has been introduced to indicate a low molecular mass iron form, free of high affinity binding to Tf [11]. Clinical and experimental studies have shown that NPBI is toxic, but in moderate quantities and leashed to protein, is an essential element in all cell aerobic metabolism and growth [14]. The present study was aimed to investigate amniotic fluid NPBI concentration in pregnancies that underwent amniocentesis between 15 and 18 weeks of gestation also providing a reference curve.
675
Materials Disodium nitrilotriacetic acid (NTA) and nonahydrate ferric nitrate were obtained from Aldrich. HPLC grade acetonitrile was obtained from Aldrich. Grade HPLC water was obtained from a Millipore Direct Q-5 system (conductivity 0.055 As cm). PIPES buffer was obtained from Sigma and 1,2-dimethyl-3-hydroxy-4(1H)-pyridone (DHP) from Aldrich. Mobile phase A 3.5 mM solution of DHP was prepared in 5 mM PIPES buffer. After adjusting with sodium hydroxide to pH 7.0 T 0.05 [27], the solution was automatically mixed with 4% of acetonitrile in the HP/1100 HPLC apparatus. Measurements
Materials and methods Patients We studied 118 normal consecutive singleton pregnancies presenting between June 2002 and November 2003 (mean age: 35.2 T 2.8 years; 26 under and 92 over 35 years), all of whom underwent amniocentesis for fetal karyotype between weeks 15 and 18 of gestation (mean 16.3 weeks). Appropriate fetal growth was determined on the basis of ultrasonographic signs (bi-parietal diameter and abdominal circumference between the 10th and 90th percentiles) according to the normograms of Campbell and Thoms [15] and by postnatal confirmation of birth weight between the 10th and 90th percentiles according to our population standards. Fetal biometry was obtained by a single especially trained examiner using an ATL HDI 3000 ultrasound system (Philips Medical Systems, The Netherlands). Exclusion criteria were: multiple pregnancies, intrauterine growth retardation, gestational hypertension, diabetes, infections, fetal malformations, chromosomal abnormalities, maternal exposure to alcohol, cocaine or tobacco smoke, perinatal asphyxia and dystocia. The study protocol was approved by the local ethics committee, and the women gave their informed consent. Assay of alpha Feto Protein (aFP) and NPBI aFP levels were determined using a commercially available immunoassay kit (DPC, Los Angeles, CA). Amniotic fluid samples were obtained from the amniotic cavity and were assayed for NPBI which were analyzed with respect to ultrasound parameters recorded at the time of amniocentesis. Amniotic fluid samples containing detectable traces of hemoglobin were excluded. NPBI plasma levels were detected by HPLC using the method described by Kime R. et al. (1996) [16], partially modified [17].
NPBI was determined with the system operating at a pressure of 90 – 95 bar and a flow rate of 0.75 ml/min. Detection was carried out subtracting the absorption at 620 nm from that at 450 nm. The method is based on preferential chelation of NPBI by a large excess of the low affinity ligand, disodium nitrilotriacetic acid (NTA). NTA captures all iron bound to low molecular weight proteins and non-specifically bound to serum proteins. It does not remove iron bound to transferrin or ferritin. To separate NPBI, a two-step filtration procedure was used: (1) filtration through a 100 kDa MWCO Vecta-Spin Micro-Whatman ultracentrifuge filter; (2) filtration through a 20 kDa MWCO Vecta Spin MicroWhatman ultracentrifuge filter at RCF 16.1 and 4-C. The filtrate was injected directly into an isocratic reverse-phase liquid chromatography system using precolumn derivatization with the high affinity iron ligand, 1,2-dimethyl-3-hydroxy-4(1H)-pyridone (DHP), which forms a colored complex with Fe3+ that absorbs at 450 nm. The complex had a molar extinction coefficient quite similar to 1-propyl-2-methyl-3-hydroxy 4-pyridinone. All glassware and plasticware were treated to minimize iron contamination. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 AM. The DHP – Fe complex eluted with a retention time of about 2.6 min. The standard curve for the DHP – Fe complex was linear between 0.01 and 400 AM in water as well as in amniotic fluid. Detection limit was 0.01 AM for all biological fluids. Statistical analysis NPBI concentrations were expressed as median and interquartile ranges, and clinical, laboratory and ultrasound parameters as means T SD. Amniotic fluid concentrations of NPBI and neonatal parameters were analyzed by the Kruskall – Wallis and Mann – Whitney U two-sided test
676
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677
when data were not normally distributed. Relations between NPBI and AFP amniotic fluid concentrations and between NPBI and weeks of gestation were analyzed by linear regression analysis. Multiple linear regression analysis was performed with NPBI concentrations as the dependent variable against the various clinical parameters (gestational age, fetal weight, head circumference and bi-parietal diameter, transverse cerebellum diameter and abdominal circumference estimated at time of amniocentesis). P < 0.05 was considered significant. Statistical analysis was performed with STATA SE 8 software package (4905 Lakeway Drive College Station, Texas 77845 USA 800).
Results All newborn infants were in normal clinical condition at birth, and no overt neurological syndrome was detected at discharge from hospital. Gestational age 39 T 1 week, birthweight 2.880 T 119 g and Apgar scores evaluated at 1 and 5 min 8 T 1 and 9 T 1, respectively, were within normal ranges. Elective Cesarean section was performed in 40/118 cases, and the other babies were delivered vaginally. At sampling, all fetuses showed appropriate growth according to ultrasound scanning parameters, namely head circumference 134.8 T 9.9 mm (25th and 75th percentiles: 127.2 – 142.1), bi-parietal diameter 122.5 T 7.8 mm (25th and 75th percentiles: 118.3– 126.5), transverse cerebellum diameter 15.8 T 0.9 mm (25th and 75th percentiles: 15.1– 16.3) and estimated fetal weight 191 T 25 g (25th and 75th percentiles: 176 –208). aFP was detectable in all the amniotic fluid samples, and results were within normal ranges for our standards 15.6 T 5.2 Ag/ml. No significant correlation was found between amniotic fluid concentrations of aFP and NPBI (r = 0.12; P > 0.05). NPBI levels increased progressively from weeks 14 – 15 to 15 – 16 of gestation (median 0 Amol/l; 5th percentile 0 Amol/l; 95th percentile: 2.36 Amol/l) peaking at weeks 17 –18 (median 3.2 Amol/l; 5th percentile: 0 Amol/l; 95th percentile: 14 Amol/l) (Kruskall–Wallis test P < 0.0001). NPBI showed a positive correlation with gestational age (r = 0.62; P < 0.001) (Fig. 1). Multiple linear regression analysis showed a statistically significant correlation of NPBI with gestational age ( P < 0.001), bi-parietal diameter and transverse cerebellum diameter ( P < 0.05, for both), whereas no significant correlations were found with fetal weight.
Fig. 1. Significant correlation (r = 0.62; P < 0.001) between individual NPBI concentrations in amniotic fluid (mmol/l) of normal fetuses (n = 118) and gestational age (weeks) between weeks 15 and 18.
adults is approximately one-third loaded with iron, retaining a considerable ability to bind iron salts, whereas in preterm babies, it is totally iron saturated [12,14]. These concentrations of NPBI in amniotic fluid are intriguing and may be related to the possibility that high NPBI concentrations are an expression of fetal oxidative stress [15], except that all subjects enrolled in the present study were normal at sampling and birth. Another explanation could be the trophic role of NPBI in nervous system development. Experimental studies on iron uptake by the brain have shown that iron transport and transferrin-binding sites are a maximum during the period of brain growth [18,19]. In the human brain, a growth spurt begins in pregnancy and continues throughout the first year of life [20]. Studies in vitro and in vivo have shown that iron plays a role in myelination processes in the prenatal and postnatal periods. Moreover, iron deficiency during embryo genesis affects glial lineage cells in a tissue-specific manner [18,19]. The present data provide a reference curve for NPBI in amniotic fluid during the second trimester of pregnancy. This could be useful in future study of pathological conditions in early fetal life. The progressive increase in NPBI from weeks 15 – 18 of gestation could reflect enhanced release of NPBI as a trophic factor at an early developmental stage, when growth is especially active. In conclusion, the present findings offer insights into iron metabolism in vivo, especially with regard to a possible role in fetal growth. References
Discussion The present observations are the first data on NPBI concentrations in amniotic fluid in the second trimester of normal pregnancies. Since NPBI is undetectable in maternal blood of normal adults [13], the origin of NPBI in amniotic fluid is presumably the fetus. Indeed, plasma Tf of healthy
[1] Crichton RR, Ward RJ. Iron species in iron homeostasis and toxicity. Analyst 1995;120:693 – 7. [2] Ponka P, Beaumont C, Richardson DR. Function and regulation of transferrin and ferritin. Semin Hematol 1998;35:35 – 54. [3] Janetzky B, Reichmann H, Youdim MBH, Riederer P. Iron and oxidative damage in neurodegenerative diseases. New York’ WileyLiss; 1997.
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677 [4] Youdim MB, Ben-Shachar D, Riederer P. Iron in brain function and dysfunction with emphasis on Parkinson’s disease. Eur Neurol 1991;31:34 – 40. [5] Touati D. Iron and oxidative stress in bacteria. Arch Biochem Biophys 2000;1:1 – 6. [6] Felt BT, Lozoff B. Brain iron and behavior of rats are not normalized by treatment of iron deficiency anemia during early development. J Nutr 1996;126:693 – 701. [7] Taylor EM, Crowe A, Morgan EH. Transferrin and iron uptake by the brain: effects of altered iron status. J Neurochem 1991;57:1584 – 92. [8] Davison AN, Dobbing J. Myelination as a vulnerable period in brain development. Br Med Bull 1966;22:40 – 4. [9] Kolb B, Whishaw IQ. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog Neurobiol 1989;32:235 – 76. [10] O’Connell M, Halliwell B, Moorhouse CP, Aruoma OI, Baum H, Peters TJ. Formation of hydroxyl radicals in the presence of ferritin and haemosiderin. Is haemosiderin formation a biological protective mechanism? Biochem J 1986;234:727 – 31. [11] Weinberg ED. The iron-withholding defense system. Am Soc Microbiol News 1993;59:559 – 62. [12] Gutteridge JMC, Paterson SK, Segal AW, Halliwell B. Inhibition of lipid peroxidation by the iron-binding protein lactoferrin. Biochem J 1981;199:259 – 61.
677
[13] Ciccoli L, Rossi V, Leoncini S, Signorini C, Paffetti P, Bracci R, et al. Iron release in erythrocytes and plasma non protein-bound iron in hypoxic and non hypoxic newborns. Free Radic Res 2003;37:51 – 58. [14] Buonocore G, Perrone S. Iron: A potent prooxidant. In: Raiha NC, Rubaltelli FF, editors. Infant formula: closer to the reference. Nestle` Nutrition workshop series. Pediatric Program, vol. 47. Philadelphia’ Lippincott Williams and Wilkins; 2002. p. 85 – 96. [15] Campbell S, Thoms A. Ultrasound measurement of the fetal head to abdomen circumference ratio in the assessment of growth retardation. Br J Obstet Gynaecol 1977;84:165 – 74. [16] Kime R, Gibson A, Yong W, Hider R, Powers H. Chromatographic method for the determination of non-transferrin-bound iron suitable for use on the plasma and bronchoalveolar lavage fluid of preterm babies. Clin Sci 1996;91:633 – 8. [17] Buonocore G, Perrone S, Longini M, Paffetti P, Vezzosi P, Gatti MG, et al. Non protein bound iron as early predictive marker of neonatal brain damage. Brain 2003;126:1224 – 30. [18] Morath DJ, Mayer-Proschel M. Iron deficiency during embryogenesis and consequences for oligodendrocyte generation in vivo. Dev Neurosci 2002;24:197 – 207. [19] Rao R, Georgieff MK. Perinatal aspects of iron metabolism. Acta Paediatr, Suppl 2002;91:124 – 9. [20] Davison AN, Dobbing J. Myelination as a vulnerable period in brain development. Br Med Bull 1966;22:40 – 4.
Non protein bound iron concentrations in amniotic fluid D. Gazzoloa, S. Perroneb, P. Paffettib, M. Longinib, P. Vezzosib, M. Bruschettinia, M. Lituaniac, G. Buonocoreb,* b
a Department Paediatrics G. Gaslini, Children’s University Hospital, 16148 Genoa, Italy Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico ‘‘Le Scotte’’, V.le Bracci 36, 53100 Siena, Italy c Centre of Fetal and Perinatal Medicine, Galliera Hospital, 16121 Genoa, Italy
Received 20 December 2004; received in revised form 14 March 2005; accepted 21 March 2005 Available online 26 April 2005
Abstract Objectives: To investigate whether amniotic fluid concentrations of non protein bound iron (NPBI) vary with growth in healthy fetuses and also offer a reference curve in the second trimester of pregnancy. Design and methods: Amniotic fluid concentrations of NPBI were measured by HPLC in 118 women with physiological singleton pregnancies, who underwent amniocentesis for fetal karyotype between weeks 15 and 18 of gestation. Results: NPBI increased progressively from weeks 14 – 15 to weeks 15 – 16, peaking at 17 – 18 weeks of gestation. NPBI values regressed positively with gestational age (GA). Multiple linear regression analysis between NPBI, as dependent variable, and various fetal parameters, as independent variables, showed a statistically significant regression coefficient with GA, bi-parietal diameter and transverse cerebellar diameter. Conclusions: The present data constitutes the first quantification of NPBI concentrations in amniotic fluid under physiological conditions. Correlations with GA and ultrasound fetal biometry suggest that NPBI may play a role in fetal growth. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Non protein bound iron; Fetus; Amniotic fluid; Pregnancy; Oxidative stress
Introduction Iron is the most abundant transition metal in the body [1] and an essential factor for the growth and well-being of almost all living organisms [2]. The essentiality of iron is well established in view of its involvement in a large number of metabolic processes, including DNA, RNA and protein synthesis, as cofactor for numerous enzymes and myelin synthesis [3,4]. Iron is involved in oxygen transport, storage, utilization, activation and detoxification, in nitrogen fixation, antibacterial defenses and photosynthesis [5]. Iron deficiency during early development of the brain has been related to behavioral alterations including deficits in learning and memory mediated by the hippocampus [6]. Severe iron deficiency may lead to similar * Corresponding author. Fax: +39 0577 586182. E-mail address: [email protected] (G. Buonocore).
deficits in cell energy metabolism and organ performance in the brain, resulting in a reduced ability to respond to restriction of oxygen and perfusion. Studies on iron uptake by the brain have shown that iron transport and transferrin-binding sites are at a maximum during the period of brain growth, and maximum uptake by the brain occurs in 15-day-old rats [7]. In the human brain, growth spurt begins early in life and continues throughout the first year of life [8]. This is a critical period during which the brain undergoes several fundamental developmental phases such as maturation of axonal and dendritic outgrowth, establishment of neuronal connections, synaptogenesis, multiplication of glia cells with accompanying myelinization and cell, axonal and dendritic death [9]. Normally, iron is safely transported by proteins such as transferrin (Tf) and lactoferrin and stored in proteins such as ferritin (Ft) and hemosiderin [10]. In healthy adults, plasma transferrin is approximately one-third loaded with iron, and the protein retains a considerable ability to bind iron salts.
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.03.010
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677
Transferrin can bind 2 mol of iron per mol of protein. When iron is correctly loaded on its high-affinity binding sites, it is not available as a growth factor for tissues nor is it available as a pro-oxidant factor [11,12]. Under normal conditions, non protein bound iron is undetectable in adults [13]. To be redox cycling active, iron must be released from its macromolecular complexes as ‘‘free iron’’. Since iron ions cannot exist in plasma, the term Ffree iron_ or non protein bound iron (NPBI) has been introduced to indicate a low molecular mass iron form, free of high affinity binding to Tf [11]. Clinical and experimental studies have shown that NPBI is toxic, but in moderate quantities and leashed to protein, is an essential element in all cell aerobic metabolism and growth [14]. The present study was aimed to investigate amniotic fluid NPBI concentration in pregnancies that underwent amniocentesis between 15 and 18 weeks of gestation also providing a reference curve.
675
Materials Disodium nitrilotriacetic acid (NTA) and nonahydrate ferric nitrate were obtained from Aldrich. HPLC grade acetonitrile was obtained from Aldrich. Grade HPLC water was obtained from a Millipore Direct Q-5 system (conductivity 0.055 As cm). PIPES buffer was obtained from Sigma and 1,2-dimethyl-3-hydroxy-4(1H)-pyridone (DHP) from Aldrich. Mobile phase A 3.5 mM solution of DHP was prepared in 5 mM PIPES buffer. After adjusting with sodium hydroxide to pH 7.0 T 0.05 [27], the solution was automatically mixed with 4% of acetonitrile in the HP/1100 HPLC apparatus. Measurements
Materials and methods Patients We studied 118 normal consecutive singleton pregnancies presenting between June 2002 and November 2003 (mean age: 35.2 T 2.8 years; 26 under and 92 over 35 years), all of whom underwent amniocentesis for fetal karyotype between weeks 15 and 18 of gestation (mean 16.3 weeks). Appropriate fetal growth was determined on the basis of ultrasonographic signs (bi-parietal diameter and abdominal circumference between the 10th and 90th percentiles) according to the normograms of Campbell and Thoms [15] and by postnatal confirmation of birth weight between the 10th and 90th percentiles according to our population standards. Fetal biometry was obtained by a single especially trained examiner using an ATL HDI 3000 ultrasound system (Philips Medical Systems, The Netherlands). Exclusion criteria were: multiple pregnancies, intrauterine growth retardation, gestational hypertension, diabetes, infections, fetal malformations, chromosomal abnormalities, maternal exposure to alcohol, cocaine or tobacco smoke, perinatal asphyxia and dystocia. The study protocol was approved by the local ethics committee, and the women gave their informed consent. Assay of alpha Feto Protein (aFP) and NPBI aFP levels were determined using a commercially available immunoassay kit (DPC, Los Angeles, CA). Amniotic fluid samples were obtained from the amniotic cavity and were assayed for NPBI which were analyzed with respect to ultrasound parameters recorded at the time of amniocentesis. Amniotic fluid samples containing detectable traces of hemoglobin were excluded. NPBI plasma levels were detected by HPLC using the method described by Kime R. et al. (1996) [16], partially modified [17].
NPBI was determined with the system operating at a pressure of 90 – 95 bar and a flow rate of 0.75 ml/min. Detection was carried out subtracting the absorption at 620 nm from that at 450 nm. The method is based on preferential chelation of NPBI by a large excess of the low affinity ligand, disodium nitrilotriacetic acid (NTA). NTA captures all iron bound to low molecular weight proteins and non-specifically bound to serum proteins. It does not remove iron bound to transferrin or ferritin. To separate NPBI, a two-step filtration procedure was used: (1) filtration through a 100 kDa MWCO Vecta-Spin Micro-Whatman ultracentrifuge filter; (2) filtration through a 20 kDa MWCO Vecta Spin MicroWhatman ultracentrifuge filter at RCF 16.1 and 4-C. The filtrate was injected directly into an isocratic reverse-phase liquid chromatography system using precolumn derivatization with the high affinity iron ligand, 1,2-dimethyl-3-hydroxy-4(1H)-pyridone (DHP), which forms a colored complex with Fe3+ that absorbs at 450 nm. The complex had a molar extinction coefficient quite similar to 1-propyl-2-methyl-3-hydroxy 4-pyridinone. All glassware and plasticware were treated to minimize iron contamination. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 AM. The DHP – Fe complex eluted with a retention time of about 2.6 min. The standard curve for the DHP – Fe complex was linear between 0.01 and 400 AM in water as well as in amniotic fluid. Detection limit was 0.01 AM for all biological fluids. Statistical analysis NPBI concentrations were expressed as median and interquartile ranges, and clinical, laboratory and ultrasound parameters as means T SD. Amniotic fluid concentrations of NPBI and neonatal parameters were analyzed by the Kruskall – Wallis and Mann – Whitney U two-sided test
676
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677
when data were not normally distributed. Relations between NPBI and AFP amniotic fluid concentrations and between NPBI and weeks of gestation were analyzed by linear regression analysis. Multiple linear regression analysis was performed with NPBI concentrations as the dependent variable against the various clinical parameters (gestational age, fetal weight, head circumference and bi-parietal diameter, transverse cerebellum diameter and abdominal circumference estimated at time of amniocentesis). P < 0.05 was considered significant. Statistical analysis was performed with STATA SE 8 software package (4905 Lakeway Drive College Station, Texas 77845 USA 800).
Results All newborn infants were in normal clinical condition at birth, and no overt neurological syndrome was detected at discharge from hospital. Gestational age 39 T 1 week, birthweight 2.880 T 119 g and Apgar scores evaluated at 1 and 5 min 8 T 1 and 9 T 1, respectively, were within normal ranges. Elective Cesarean section was performed in 40/118 cases, and the other babies were delivered vaginally. At sampling, all fetuses showed appropriate growth according to ultrasound scanning parameters, namely head circumference 134.8 T 9.9 mm (25th and 75th percentiles: 127.2 – 142.1), bi-parietal diameter 122.5 T 7.8 mm (25th and 75th percentiles: 118.3– 126.5), transverse cerebellum diameter 15.8 T 0.9 mm (25th and 75th percentiles: 15.1– 16.3) and estimated fetal weight 191 T 25 g (25th and 75th percentiles: 176 –208). aFP was detectable in all the amniotic fluid samples, and results were within normal ranges for our standards 15.6 T 5.2 Ag/ml. No significant correlation was found between amniotic fluid concentrations of aFP and NPBI (r = 0.12; P > 0.05). NPBI levels increased progressively from weeks 14 – 15 to 15 – 16 of gestation (median 0 Amol/l; 5th percentile 0 Amol/l; 95th percentile: 2.36 Amol/l) peaking at weeks 17 –18 (median 3.2 Amol/l; 5th percentile: 0 Amol/l; 95th percentile: 14 Amol/l) (Kruskall–Wallis test P < 0.0001). NPBI showed a positive correlation with gestational age (r = 0.62; P < 0.001) (Fig. 1). Multiple linear regression analysis showed a statistically significant correlation of NPBI with gestational age ( P < 0.001), bi-parietal diameter and transverse cerebellum diameter ( P < 0.05, for both), whereas no significant correlations were found with fetal weight.
Fig. 1. Significant correlation (r = 0.62; P < 0.001) between individual NPBI concentrations in amniotic fluid (mmol/l) of normal fetuses (n = 118) and gestational age (weeks) between weeks 15 and 18.
adults is approximately one-third loaded with iron, retaining a considerable ability to bind iron salts, whereas in preterm babies, it is totally iron saturated [12,14]. These concentrations of NPBI in amniotic fluid are intriguing and may be related to the possibility that high NPBI concentrations are an expression of fetal oxidative stress [15], except that all subjects enrolled in the present study were normal at sampling and birth. Another explanation could be the trophic role of NPBI in nervous system development. Experimental studies on iron uptake by the brain have shown that iron transport and transferrin-binding sites are a maximum during the period of brain growth [18,19]. In the human brain, a growth spurt begins in pregnancy and continues throughout the first year of life [20]. Studies in vitro and in vivo have shown that iron plays a role in myelination processes in the prenatal and postnatal periods. Moreover, iron deficiency during embryo genesis affects glial lineage cells in a tissue-specific manner [18,19]. The present data provide a reference curve for NPBI in amniotic fluid during the second trimester of pregnancy. This could be useful in future study of pathological conditions in early fetal life. The progressive increase in NPBI from weeks 15 – 18 of gestation could reflect enhanced release of NPBI as a trophic factor at an early developmental stage, when growth is especially active. In conclusion, the present findings offer insights into iron metabolism in vivo, especially with regard to a possible role in fetal growth. References
Discussion The present observations are the first data on NPBI concentrations in amniotic fluid in the second trimester of normal pregnancies. Since NPBI is undetectable in maternal blood of normal adults [13], the origin of NPBI in amniotic fluid is presumably the fetus. Indeed, plasma Tf of healthy
[1] Crichton RR, Ward RJ. Iron species in iron homeostasis and toxicity. Analyst 1995;120:693 – 7. [2] Ponka P, Beaumont C, Richardson DR. Function and regulation of transferrin and ferritin. Semin Hematol 1998;35:35 – 54. [3] Janetzky B, Reichmann H, Youdim MBH, Riederer P. Iron and oxidative damage in neurodegenerative diseases. New York’ WileyLiss; 1997.
D. Gazzolo et al. / Clinical Biochemistry 38 (2005) 674 – 677 [4] Youdim MB, Ben-Shachar D, Riederer P. Iron in brain function and dysfunction with emphasis on Parkinson’s disease. Eur Neurol 1991;31:34 – 40. [5] Touati D. Iron and oxidative stress in bacteria. Arch Biochem Biophys 2000;1:1 – 6. [6] Felt BT, Lozoff B. Brain iron and behavior of rats are not normalized by treatment of iron deficiency anemia during early development. J Nutr 1996;126:693 – 701. [7] Taylor EM, Crowe A, Morgan EH. Transferrin and iron uptake by the brain: effects of altered iron status. J Neurochem 1991;57:1584 – 92. [8] Davison AN, Dobbing J. Myelination as a vulnerable period in brain development. Br Med Bull 1966;22:40 – 4. [9] Kolb B, Whishaw IQ. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog Neurobiol 1989;32:235 – 76. [10] O’Connell M, Halliwell B, Moorhouse CP, Aruoma OI, Baum H, Peters TJ. Formation of hydroxyl radicals in the presence of ferritin and haemosiderin. Is haemosiderin formation a biological protective mechanism? Biochem J 1986;234:727 – 31. [11] Weinberg ED. The iron-withholding defense system. Am Soc Microbiol News 1993;59:559 – 62. [12] Gutteridge JMC, Paterson SK, Segal AW, Halliwell B. Inhibition of lipid peroxidation by the iron-binding protein lactoferrin. Biochem J 1981;199:259 – 61.
677
[13] Ciccoli L, Rossi V, Leoncini S, Signorini C, Paffetti P, Bracci R, et al. Iron release in erythrocytes and plasma non protein-bound iron in hypoxic and non hypoxic newborns. Free Radic Res 2003;37:51 – 58. [14] Buonocore G, Perrone S. Iron: A potent prooxidant. In: Raiha NC, Rubaltelli FF, editors. Infant formula: closer to the reference. Nestle` Nutrition workshop series. Pediatric Program, vol. 47. Philadelphia’ Lippincott Williams and Wilkins; 2002. p. 85 – 96. [15] Campbell S, Thoms A. Ultrasound measurement of the fetal head to abdomen circumference ratio in the assessment of growth retardation. Br J Obstet Gynaecol 1977;84:165 – 74. [16] Kime R, Gibson A, Yong W, Hider R, Powers H. Chromatographic method for the determination of non-transferrin-bound iron suitable for use on the plasma and bronchoalveolar lavage fluid of preterm babies. Clin Sci 1996;91:633 – 8. [17] Buonocore G, Perrone S, Longini M, Paffetti P, Vezzosi P, Gatti MG, et al. Non protein bound iron as early predictive marker of neonatal brain damage. Brain 2003;126:1224 – 30. [18] Morath DJ, Mayer-Proschel M. Iron deficiency during embryogenesis and consequences for oligodendrocyte generation in vivo. Dev Neurosci 2002;24:197 – 207. [19] Rao R, Georgieff MK. Perinatal aspects of iron metabolism. Acta Paediatr, Suppl 2002;91:124 – 9. [20] Davison AN, Dobbing J. Myelination as a vulnerable period in brain development. Br Med Bull 1966;22:40 – 4.