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Direct analysis of human blood (mothers and newborns) by energy dispersive X-ray fluorescence
ARTICLE IN PRESS Journal of
Trace Elements in Medicine and Biology Journal of Trace Elements in Medicine and Biology 19 (2005) 151–158 www.elsevier.de/jtemb
APPLIED METHODOLOGY
Direct analysis of human blood (mothers and newborns) by energy dispersive X-ray fluorescence P.J. Custo´dioa, Maria Luisa Carvalhoa,, F. Nunesb, S. Pedrosob, A. Camposb a
Universidade de Lisboa, Centro Fı´sica Ato´mica, Faculdade de Cieˆncias, Av. Prof. Gama Pinto, 2, 1649-003 Lisboa, Portugal Hospital Garcia de Orta, Almada, Portugal
b
Received 27 December 2004; accepted 17 August 2005
Abstract This work is an application of energy dispersive X-ray fluorescence (EDXRF) as analytical technique for trace element determination in human tissues. Potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) were determined directly in blood samples from 66 mothers at delivery after full-term pregnancies. The corresponding 66 cord-blood samples of the newborns were also analysed, in order to find element correlations between maternal and newborn blood at birth. The studied samples were obtained from mothers aged between 15 and 39 years old, the gestational age being between 35 and 41 weeks and the newborns’ weight between 2.310 and 4.310 kg. Samples were lyophilised and analysed without any chemical treatment. Very low levels of Pb were found both in maternal and fetal cord blood samples. Cu values ranged from 3 to 13 mg g1, both for mothers and children. A correlation between Cu and Fe concentrations in maternal and fetal cord blood was found. Zn is considered as one of the key elements in newborn health. Concentrations between 10 and 40 mg g1 were measured. A positive correlation between Br levels in mothers and children was observed. Positive correlations for mothers were observed between Zn and Rb as well as K and Fe. The corresponding correlations in fetal cord blood samples were not observed, however positive correlations were found between Ca and K; Cu and Fe. The mean concentrations for each element were similar in maternal and in fetal cord blood, except for Cu and Zn, being higher in maternal samples. No correlations between element concentrations and pathologies of the mothers were observed. r 2005 Elsevier GmbH. All rights reserved. Keywords: EDXRF; Biological samples; Human cord blood; Trace elements; Element analysis
Introduction Energy dispersive X-ray fluorescence spectrometry (EDXRF) was used for quantitative analysis of potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) in maternal and newborn cord blood at birth. Corresponding author.
E-mail address: [email protected] (M.L. Carvalho). 0946-672X/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2005.09.002
Blood is the medium for transport of trace elements in the whole human body. The transport of essential elements from mother to fetus varies during gestation and the transport of proteins may change during pregnancy. Trace elements are present in cord blood and reach the fetus. Therefore, whole blood, plasma or serum are convenient samples for determination of trace element concentrations in order to establish a pattern for fetal development. The role of several elements in fetal growth is well documented in literature [1]. A number of
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studies have been carried out in whole cord blood and serum of newborns and element concentrations have been correlated for mother’s and child’s health [2–4]. Zn is known to be essential for normal embryogenesis, fetal growth and protein synthesis, and low Zn levels suggest the existence of fetal growth problems [5,6]. Cu is also of particular importance, especially early in life, for the development and maintenance of myelin [5]. The impact of inadequate or excess intake of certain nutrients during pregnancy on embryonal and fetal development has been studied [7]. Deficiency of trace elements during intrauterine existence is closely related to mortality and morbidity in the newborn [1,7,8]. The mechanism of transport of trace elements from the mother to the newborn is still not well known. Since the transport of several essential elements across membranes is competitive, many divalent ions compete with each other and positive or negative correlations between them may exist. The effect of gestastional age on cord blood plasma Cu, Zn and Mg has been studied by atomic absorption spectrophotometry [9]. An increase of the Zn concentration in maternal plasma with the increase of gestational age was observed, but a decrease with increasing gestational age in cord blood plasma. On the contrary, Cu concentration decreased in maternal plasma with the increase of gestational age, while in cord blood there was an increase with gestational age. Serum of newborns has also been studied by inductively coupled plasma mass spectrometry (ICP-MS) to compare the element concentrations with those of adults and young infants [10]. The X-ray fluorescence technique was also applied to study human tissues and body fluids, including whole blood and serum [11,12]. Blood samples have also been studied by proton-induced X-ray emission (PIXE) [13]. The purpose of this work was to determine the element concentrations of K, Ca, Fe, Cu, Zn, Br, Rb and Pb in maternal and fetal cord blood at birth and to investigate whether the concentrations of these elements in maternal and fetal cord blood depend on gestational age, birth weight and age of the mother. Moreover, we intended to establish correlations between elements in fetal cord blood, in maternal cord blood and between them. This study aims to be a contribution for a better understanding of the mechanism of trace element transport from the mother to the child, and of the effect of excess or deficiency in certain elements in fetus development.
The 66 cord blood samples were collected post-partum, the mothers being aged between 15 and 39 years. Regarding the delivery labours, 50 were normal, 9 were with forceps and 7 were caesareans. Nine mothers were smokers and 57 were not. Thirty-five babies were male and 31 female, and their weight ranged from 2310 to 4310 g. All except 2 newborns were healthy. The gestation period ranged from 35 to 41 weeks. Blood samples were collected from the umbilical cord, which is the structure that connects the placenta to the fetus. The umbilical cord consists in a long tube, with three blood vessels: a vein and two arteries. In the vein, the oxygenated blood with nutrients flows from mother to fetus, and in the arteries, the venous blood and the excretion products flow from the fetus to the mother. In the placenta, the fetal blood and the maternal blood flow very close to each other, separated by the placenta membrane, which allows a nutrient exchange between mother and fetus. The post-partum tissues were removed using normal surgery equipment, always the same for all the samples.
Sample preparation The samples were frozen in sealed plastic bags for transportation from the hospital to the laboratory for further analysis. Prior to analysis, blood samples were lyophilised for 72 h at 601 and low pressure (ca. 0.1 atm). The time was optimised for total removal of water. Following this procedure, a powder was obtained of each sample in a polyester mill, and the samples were stored under controlled humidity conditions. For analysis the samples were pressed into pellets of 2.0 cm in diameter and 1 mm thickness. A minimum of three pellets of each sample were produced to reduce the error of analysis. Each pellet, without any substrate, was glued on a Mylar film and put directly on a sample holder in the X-ray beam for element determination. An EDXRF system was used. Mylar film and glue were previously checked for contamination control. During the grinding of the sample preparation, special care has been paid to avoid contamination, as well as during the whole procedure. All the used materials were of polyester, to avoid any contact with metals.
Experimental setup
Material and methods Samples Blood samples were provided by the obstetric department of Garcia de Orta Hospital in Almada, Portugal.
The spectrometer used in this work for EDXRF analysis consisted of a commercial X-ray tube (PW 1140; 100 kV, 80 mA) equipped with a changeable secondary target of molybdenum (Mo) [14]. With this setup it was possible to obtain virtually a monochromatic source, with energies of the Ka and Kb lines of Mo of 17.44 and 19.60 keV, respectively.
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The X-ray tube, the secondary target and the sample were in a triaxial geometry. By thus taking advantage of the effect of polarisation of the incident X-ray beam from the tube, the background was decreased. Both X-ray beams emitted by the secondary target and the sample were collimated throughout two silver apertures, in order to reduce the scattered radiation and improve the detection limits [15]. The characteristic radiation emitted by the elements present in the sample was detected by means of a silicon (lithium) detector, with a 30 mm2 active area and 8 mm beryllium window. The energy resolution was 135 eV at 5.9 keV and the acquisition system was a Nucleus PCA card. The pulse processing and dead time corrections were automatically adjusted by a commercial pulse processor (Oxford). The characteristic radiation from the lighter elements was strongly absorbed in the air and in the beryllium window of the detector, which made the present setup less suitable for light elements. The quantitative evaluation was made by the fundamental parameters methods [16], which makes use of fundamental parameters such as cross-sections for absorption and X-ray production, transition intensities, fluorescence yields, etc. The relation between the measured peak intensity (I i ) and the concentration of an element (C i ) is given by the equation: I i ¼ I o mK i C i Ai , where I o is the intensity of the X-ray beam, m is the sample thickness (g cm2), K is an experimental calibration factor which depends on the spectrometer geometry, detector efficiency, detector solid angle and cross-sections for producing characteristic X-rays, and together with I o is obtained by analysis of standard reference samples. The sample mass is obtained through the information in the coherent and incoherent scattered radiation with a normalising condition, which means that all concentrations within a sample should add up to P unity, i.e. C ¼ 1. A i is the self-attenuation factor, i i which can be obtained iteratively, by constructing a virtual matrix of three elements representing the matrix. In the present study (light element matrix, which accounts for 80% of the absorption in biological samples) the chosen elements were hydrogen, carbon and oxygen. As the coherent cross-sections, the incoherent cross-sections and attenuation coefficients are
153
strongly depending on the atomic number, the equation system is linearly independent and thus the system always has a single-valued solution. The peak shape and background were described by a mathematical Gaussian function. At the low energy side of the peak, this function was replaced by an exponential function. The basic idea of the fitting procedure was to find a set of parameter values minimising the w2 value. In the experimental spectrum, some interferences appeared on the lines of different elements. This was especially important for krypton (Kr) (Ka) and Pb (Lb) lines and between As (Ka) and Pb (La). In the present work, the Kr (Ka) peak area was calculated from the Ar (Ka) peak area, assuming a steady air composition. This Kr (Ka) peak area was then subtracted from the total Kr (Ka)+Pb (Lb) peak area. The Pb (Lb) peak area was used to calculate the Pb concentration. The ratio between the Pb (Lb) and Pb (La) lines was used to obtain the Pb (La) peak area. This new Pb (La) peak area was subtracted from the As (Ka)+Pb (La) area, allowing to get the As (Ka) area. This was checked with a standard reference material containing Pb and As: Orchard leaves, NBS standard reference material 1571. The obtained values are presented in Table 1. In spite of the good agreement obtained for this standard, when As and Pb were very close to the detection limit, the error of the fitting process was relatively high and we disregarded both elements. A check was carried out with reference materials containing both elements in concentrations below 1 mg g1. The X-ray generator was operated at 50 kV and 20 mA, and a typical acquisition time of 1000 s was used. A collimator of silver was placed in front of the detector in order to restrict the effective area of the detector by excluding regions close to the edges. In Fig. 1, a spectrum of a sample obtained with this system is shown.
Detection limits The detection limits (DL) obtained by EDXRF were calculated according to the equation: pffiffiffiffiffiffiffi 3C i N b DL ¼ , Np
Table 1. Comparison of element concentrations (mg g1) in orchard leaves (NBS standard reference material 1571) measured in this work (7standard deviation) and the certified values
Present work Certified value a
K
Ca
Fe
Mn
Cu
Zn
As
Br
Rb
Pb
15,3007900 14,7007300
21,50071000 20,9007300
310720 300720
9575 9174
1371 1271
2672 2573
12.572.5 1472
10.670.8 10a
12.570.8 1271
4272 4573
Non-certified value.
ARTICLE IN PRESS p2 0.18 [0.15–0.29] 1.970.3 2.3 [1.7–3.1] 2272 22 [19–24] 320720 286 [226–332]
25007200 2400 [2200–2500]
4.870.7 4.3 [3.7–4.8]
1471 13 [12–14]
p2 0.24 [0.15–0.31]
Rb Br Zn
Se
23007200 2500 [2100–2800]
In Tables 4 and 5, the obtained results for the mean element concentrations and standard deviation (mg g1) in fetal cord blood and in maternal cord blood for five ranges of the mother’s age are presented. Numbers in brackets correspond to the number of analyzed samples in each group. The standard deviation was calculated for each case, considering the number of samples and the number of replicates, respectively. The high values of standard deviation obtained in some cases can be attributed to biological factors affecting the different
Present work Certified value
Results and discussion
Cu
The accuracy was checked by analysis of reference materials, prepared in pellets following the same preparation method as for the samples. The element concentrations obtained for the standard reference material orchard leaves (NBS standard reference material 1571) are shown in Table 1, those obtained for the standard reference material freeze dried animal blood (IAEA, A-13) are presented in Table 2. The results obtained in the present work were in very good agreement with the certified values. The standard deviation was obtained considering the error resulting from the fitting process and the differences in concentration for three pellets analysed repeatedly five times, respectively. Statistic treatment for all the results was carried out taking into account the high number of results (Table 3).
Fe
Accuracy tests
Ca
where C i is the concentration of the element i; N b is the counting rate for the background and N p is the counting rate for the corresponding peak. The results for orchard leaves (NBS standard reference material 1571) are presented in Table 3.
K
Fig. 1. Spectrum of a blood sample obtained by EDXRF.
Pb
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Table 2. Comparison of element concentrations (mg g1) in freeze dried animal blood (IAEA, A-13) measured in this work (7standard deviation) and the certified values with the corresponding range
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Table 3.
DL
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Detection limits (DL; mg g1) obtained by EDXRF in orchard leaves (NBS standard reference material 1571) K
Ca
Mn
Fe
Cu
Zn
As
Rb
Sr
Pb
70
50
4.0
3.1
1.2
1.2
0.5
1.2
0.6
1.1
Table 4. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for five ranges of the mother’s age Element
K Ca Fe Cu Zn Br Rb Pb
Mother’s age (years) 15–20
20–25
25–30
30–35
35–40
(N ¼ 5)
(N ¼ 8)
(N ¼ 21)
(N ¼ 17)
(N ¼ 9)
10,48972992 3787119 24707414 4.770.9 1472 1274 1475 p2
938171912 338785 24907314 5.070.7 1572 1376 1374 p2
980071314 386791 29357458 5.671.9 1672 1573 1473 p2
990071349 385766 306771108 5.972.2 1575 1675 1675 p2
978371399 307746 28347346 5.070.6 1372 1474 1573 p2
Numbers in brackets correspond to the number of analysed samples in each group.
Table 5. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for five ranges of the mother’s age Element
K Ca Fe Cu Zn Br Rb Pb
Mother’s age (years) 15–20
20–25
25–30
30–35
35–40
(N ¼ 5)
(N ¼ 8)
(N ¼ 21)
(N ¼ 17)
(N ¼ 9)
772771372 356780 21277307 1172 3875 1573 1373 p2
786371214 397784 20657367 1072 32711 1575 1173 p2
848271000 386795 25057425 1072 3974 1874 1373 p2
85327728 391758 25477373 1072 3577 1874 1373 p2
767371326 373766 22567470 1073 3477 1574 1272 p2
Numbers in brackets correspond to the number of analysed samples in each group.
samples (e.g. dietary habits and specific characteristics for each individual). In fact, the differences in element concentrations obtained for the three replicates were negligible, when compared with the variations between samples. The most important conclusion from these tables is the similarity of the concentration values in maternal and newborn samples with varying age of the mothers. However, differences were observed when element levels in maternal samples were compared with the corresponding levels in fetus samples. There was a slight increase of K, Rb and Fe in fetus blood samples compared to the mothers’ samples; on the other hand,
the Cu and Zn concentrations were lower in the fetus compared to the maternal samples. The Ca and Br levels were very similar in the two sets of samples (Tables 4 and 5). In Tables 6 and 7, the obtained mean element concentrations and standard deviation (mg g1) in fetal cord blood and in maternal cord blood for five ranges of the newborn’s weight, respectively are presented. Numbers in brackets correspond to the number of analyzed samples in each group. From these results, it can be concluded that element concentrations for mother and child are more or less the same for different weight of the newborns. However, the concentrations in fetal cord
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blood were different from the corresponding concentrations in maternal cord blood. Higher values for K and Rb were observed in fetal samples and higher values for Cu and Fe were found in maternal samples. All the other elements were present in analogous concentrations in both sets of samples. These results are in agreement with reported Cu concentrations in full-term newborns [17–19]. A similar study in amniotic fluid and placenta [20] has shown a dependence of Ca, Fe, Cu and Zn levels on the mother’s age and the newborn’s weight. These dependences were not observed in the present work for maternal and fetus cord blood. In Tables 8 and 9, the element concentrations in fetal and maternal cord blood according to gestational age (GA) are shown. A slight increase in K and Fe concentrations with increasing GA was observed for maternal samples, while a similar slight increase was observed not only for K and Fe but also for Cu and Zn in fetus samples. A stronger dependence for Cu and Zn
on GA both for mothers and children has been previously observed [9]. In that study, samples with GA between 24 and 42 weeks were analysed. Strong element variations are indeed expected, taking into account that the transport of essential elements changes at different stages of gestation. The slight increase in the levels of some elements in the present study reflects the fact that only full term pregnancies were considered, and therefore no big variations were expected. Statistical analysis has been carried out. The Spearman correlation coefficients, rs , between element concentrations in fetal cord blood, maternal cord blood and in maternal and fetal cord blood are presented in Tables 10–12, respectively. In these tables, values not corresponding to any correlation between elements have been omitted. The type of correlation (+) or () is also indicated. According to this test, the closer the rs values are to 1, the stronger is the correlation, for a significance of p ¼ 0:05.
Table 6. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for five ranges of the child’s weight Element
K Ca Fe Cu Zn Br Rb Pb
Child’s weight (kg) 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
(N ¼ 2)
(N ¼ 13)
(N ¼ 29)
(N ¼ 12)
(N ¼ 6)
98477629 342770 28007197 4.970.3 1571 1473 2177 p2
997072118 367794 27967515 5.470.8 1572 1372 1573 p2
960371306 358786 300671298 5.672.1 1574 1574 1474 p2
985471512 387777 28307650 5.371.5 1572 1675 1574 p2
11,26071764 377758 27497262 4.670.5 1572 1478 1473 p2
Numbers in brackets correspond to the number of analysed samples in each group.
Table 7. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for five ranges of the child’s weight Element
K Ca Fe Cu Zn Br Rb Pb
Child’s weight (kg) 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
(N ¼ 2)
(N ¼ 13)
(N ¼ 29)
(N ¼ 12)
(N ¼ 6)
86117542 400771 22917250 1271 3677 1574 1675 p2
810571408 386774 22717412 1073 3876 1573 1273 p2
821571099 369769 24957550 1072 3679 1874 1373 p2
80297762 4117112 23337464 1172 3775 1776 1273 p2
87857625 389717 25217255 1072 3473 1777 1373 p2
Numbers in brackets correspond to the number of analysed samples in each group.
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Table 8. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for three ranges of gestational age Element
(N ¼ 2) K Ca Fe Cu Zn Br Rb Pb
Table 10. Spearman correlation coefficients: element concentrations in fetal cord blood (significant correlation at p ¼ 0:005)
Gestational period (weeks) 35–37
888771711 309720 25277232 5.070.3 1371 1372 1574 p2
37–39 (N ¼ 25) 985671772 356782 29047896 5.571.7 1574 1575 1575 p2
K 39–41 (N ¼ 35) 994871440 378783 292371072 5.471.7 1572 1474 1473 p2
Numbers in brackets correspond to the number of analysed samples in each group.
K Ca Fe Cu Zn Br Rb
Element
K Ca Fe Cu Zn Br Rb Pb
Gestational period (weeks) 35–37
37–39
39–41
(N ¼ 2)
(N ¼ 25)
(N ¼ 35)
730472389 3917162 20417391 8.573.2 3174 1371 1174 p2
818771204 394790 23227408 1073 3777 1775 1373 p2
83107881 374759 25047520 1172 3777 1774 1373 p2
Numbers in brackets correspond to the number of analysed samples in each group.
No strong positive correlations were found. However, some correlations could be observed between elements in fetal cord blood: K and Ca as well as Cu and Fe. In maternal samples, positive correlations were found between K and Fe as well as Rb and Zn. Finally, the element contents in fetal and maternal samples presented some weak positive correlations for K, Ca, Fe, Cu, Br and Rb.
Conclusions In the present work, it has been demonstrated that EDXRF is a powerful multielement technique for the investigation of trace element concentrations in biolo-
Ca
Fe
+0.50
+0.30
+0.50 +0.30
Cu
Zn
Br
Rb
+0.47 +0.47 +0.35 +0.42 0.35
0.42
Table 11. Spearman correlation coefficients: element concentrations in maternal cord blood (significant correlation at p ¼ 0:005) K
Table 9. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for three ranges of gestational age
157
K Ca Fe Cu Zn Br Rb
Ca
Fe
Cu
Zn
Br
+0.56
+0.35
+0.56
+0.35 +0.35
0.35
Rb
+0.36 +0.36
+0.36 +0.61
+0.36
+0.61 +0.51
+0.51
Table 12. Spearman correlation coefficients: element concentrations in fetal and maternal cord blood (significant correlation at p ¼ 0:005)
K Ca Fe Cu Zn Br Rb
K
Ca
Fe
+0.50 +0.51
+0.32
+0.36 +0.51 +0.50
+0.35
Cu
Zn
Br
Rb
+0.44
+0.39 +0.79
+0.38 +0.36 +0.55
+0.40
gical tissues, in a fast and easy way. The samples are not destroyed and no chemical preparation is necessary. All studied elements are detected simultaneously in a single analysis. Moreover, only a small amount of sample – a few grams – is necessary for analysis. However, the method is limited to elements heavier than aluminium and the sensitivity is limited to 1 mg g1 for heavy elements, which is a drawback of this technique. This is the case for lead, chromium and cadmium, which normally are present in blood samples in low levels. This study also found some statistical correlations between element concentrations in the cord blood of mothers and newborns at birth.
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Furthermore, a dependence of element levels on gestational age was confirmed. No substantial changes in the element contents were observed according to the age of the mothers, which might be explained by the fact that those are mostly dependent on environment contamination and dietary habits, which were more or less the same for all subjects investigated.
Acknowledgements The authors express their sincere gratitude to Garcia de Orta Hospital for providing the samples.
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Trace Elements in Medicine and Biology Journal of Trace Elements in Medicine and Biology 19 (2005) 151–158 www.elsevier.de/jtemb
APPLIED METHODOLOGY
Direct analysis of human blood (mothers and newborns) by energy dispersive X-ray fluorescence P.J. Custo´dioa, Maria Luisa Carvalhoa,, F. Nunesb, S. Pedrosob, A. Camposb a
Universidade de Lisboa, Centro Fı´sica Ato´mica, Faculdade de Cieˆncias, Av. Prof. Gama Pinto, 2, 1649-003 Lisboa, Portugal Hospital Garcia de Orta, Almada, Portugal
b
Received 27 December 2004; accepted 17 August 2005
Abstract This work is an application of energy dispersive X-ray fluorescence (EDXRF) as analytical technique for trace element determination in human tissues. Potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) were determined directly in blood samples from 66 mothers at delivery after full-term pregnancies. The corresponding 66 cord-blood samples of the newborns were also analysed, in order to find element correlations between maternal and newborn blood at birth. The studied samples were obtained from mothers aged between 15 and 39 years old, the gestational age being between 35 and 41 weeks and the newborns’ weight between 2.310 and 4.310 kg. Samples were lyophilised and analysed without any chemical treatment. Very low levels of Pb were found both in maternal and fetal cord blood samples. Cu values ranged from 3 to 13 mg g1, both for mothers and children. A correlation between Cu and Fe concentrations in maternal and fetal cord blood was found. Zn is considered as one of the key elements in newborn health. Concentrations between 10 and 40 mg g1 were measured. A positive correlation between Br levels in mothers and children was observed. Positive correlations for mothers were observed between Zn and Rb as well as K and Fe. The corresponding correlations in fetal cord blood samples were not observed, however positive correlations were found between Ca and K; Cu and Fe. The mean concentrations for each element were similar in maternal and in fetal cord blood, except for Cu and Zn, being higher in maternal samples. No correlations between element concentrations and pathologies of the mothers were observed. r 2005 Elsevier GmbH. All rights reserved. Keywords: EDXRF; Biological samples; Human cord blood; Trace elements; Element analysis
Introduction Energy dispersive X-ray fluorescence spectrometry (EDXRF) was used for quantitative analysis of potassium (K), calcium (Ca), iron (Fe), copper (Cu), zinc (Zn), bromine (Br), rubidium (Rb) and lead (Pb) in maternal and newborn cord blood at birth. Corresponding author.
E-mail address: [email protected] (M.L. Carvalho). 0946-672X/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2005.09.002
Blood is the medium for transport of trace elements in the whole human body. The transport of essential elements from mother to fetus varies during gestation and the transport of proteins may change during pregnancy. Trace elements are present in cord blood and reach the fetus. Therefore, whole blood, plasma or serum are convenient samples for determination of trace element concentrations in order to establish a pattern for fetal development. The role of several elements in fetal growth is well documented in literature [1]. A number of
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studies have been carried out in whole cord blood and serum of newborns and element concentrations have been correlated for mother’s and child’s health [2–4]. Zn is known to be essential for normal embryogenesis, fetal growth and protein synthesis, and low Zn levels suggest the existence of fetal growth problems [5,6]. Cu is also of particular importance, especially early in life, for the development and maintenance of myelin [5]. The impact of inadequate or excess intake of certain nutrients during pregnancy on embryonal and fetal development has been studied [7]. Deficiency of trace elements during intrauterine existence is closely related to mortality and morbidity in the newborn [1,7,8]. The mechanism of transport of trace elements from the mother to the newborn is still not well known. Since the transport of several essential elements across membranes is competitive, many divalent ions compete with each other and positive or negative correlations between them may exist. The effect of gestastional age on cord blood plasma Cu, Zn and Mg has been studied by atomic absorption spectrophotometry [9]. An increase of the Zn concentration in maternal plasma with the increase of gestational age was observed, but a decrease with increasing gestational age in cord blood plasma. On the contrary, Cu concentration decreased in maternal plasma with the increase of gestational age, while in cord blood there was an increase with gestational age. Serum of newborns has also been studied by inductively coupled plasma mass spectrometry (ICP-MS) to compare the element concentrations with those of adults and young infants [10]. The X-ray fluorescence technique was also applied to study human tissues and body fluids, including whole blood and serum [11,12]. Blood samples have also been studied by proton-induced X-ray emission (PIXE) [13]. The purpose of this work was to determine the element concentrations of K, Ca, Fe, Cu, Zn, Br, Rb and Pb in maternal and fetal cord blood at birth and to investigate whether the concentrations of these elements in maternal and fetal cord blood depend on gestational age, birth weight and age of the mother. Moreover, we intended to establish correlations between elements in fetal cord blood, in maternal cord blood and between them. This study aims to be a contribution for a better understanding of the mechanism of trace element transport from the mother to the child, and of the effect of excess or deficiency in certain elements in fetus development.
The 66 cord blood samples were collected post-partum, the mothers being aged between 15 and 39 years. Regarding the delivery labours, 50 were normal, 9 were with forceps and 7 were caesareans. Nine mothers were smokers and 57 were not. Thirty-five babies were male and 31 female, and their weight ranged from 2310 to 4310 g. All except 2 newborns were healthy. The gestation period ranged from 35 to 41 weeks. Blood samples were collected from the umbilical cord, which is the structure that connects the placenta to the fetus. The umbilical cord consists in a long tube, with three blood vessels: a vein and two arteries. In the vein, the oxygenated blood with nutrients flows from mother to fetus, and in the arteries, the venous blood and the excretion products flow from the fetus to the mother. In the placenta, the fetal blood and the maternal blood flow very close to each other, separated by the placenta membrane, which allows a nutrient exchange between mother and fetus. The post-partum tissues were removed using normal surgery equipment, always the same for all the samples.
Sample preparation The samples were frozen in sealed plastic bags for transportation from the hospital to the laboratory for further analysis. Prior to analysis, blood samples were lyophilised for 72 h at 601 and low pressure (ca. 0.1 atm). The time was optimised for total removal of water. Following this procedure, a powder was obtained of each sample in a polyester mill, and the samples were stored under controlled humidity conditions. For analysis the samples were pressed into pellets of 2.0 cm in diameter and 1 mm thickness. A minimum of three pellets of each sample were produced to reduce the error of analysis. Each pellet, without any substrate, was glued on a Mylar film and put directly on a sample holder in the X-ray beam for element determination. An EDXRF system was used. Mylar film and glue were previously checked for contamination control. During the grinding of the sample preparation, special care has been paid to avoid contamination, as well as during the whole procedure. All the used materials were of polyester, to avoid any contact with metals.
Experimental setup
Material and methods Samples Blood samples were provided by the obstetric department of Garcia de Orta Hospital in Almada, Portugal.
The spectrometer used in this work for EDXRF analysis consisted of a commercial X-ray tube (PW 1140; 100 kV, 80 mA) equipped with a changeable secondary target of molybdenum (Mo) [14]. With this setup it was possible to obtain virtually a monochromatic source, with energies of the Ka and Kb lines of Mo of 17.44 and 19.60 keV, respectively.
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The X-ray tube, the secondary target and the sample were in a triaxial geometry. By thus taking advantage of the effect of polarisation of the incident X-ray beam from the tube, the background was decreased. Both X-ray beams emitted by the secondary target and the sample were collimated throughout two silver apertures, in order to reduce the scattered radiation and improve the detection limits [15]. The characteristic radiation emitted by the elements present in the sample was detected by means of a silicon (lithium) detector, with a 30 mm2 active area and 8 mm beryllium window. The energy resolution was 135 eV at 5.9 keV and the acquisition system was a Nucleus PCA card. The pulse processing and dead time corrections were automatically adjusted by a commercial pulse processor (Oxford). The characteristic radiation from the lighter elements was strongly absorbed in the air and in the beryllium window of the detector, which made the present setup less suitable for light elements. The quantitative evaluation was made by the fundamental parameters methods [16], which makes use of fundamental parameters such as cross-sections for absorption and X-ray production, transition intensities, fluorescence yields, etc. The relation between the measured peak intensity (I i ) and the concentration of an element (C i ) is given by the equation: I i ¼ I o mK i C i Ai , where I o is the intensity of the X-ray beam, m is the sample thickness (g cm2), K is an experimental calibration factor which depends on the spectrometer geometry, detector efficiency, detector solid angle and cross-sections for producing characteristic X-rays, and together with I o is obtained by analysis of standard reference samples. The sample mass is obtained through the information in the coherent and incoherent scattered radiation with a normalising condition, which means that all concentrations within a sample should add up to P unity, i.e. C ¼ 1. A i is the self-attenuation factor, i i which can be obtained iteratively, by constructing a virtual matrix of three elements representing the matrix. In the present study (light element matrix, which accounts for 80% of the absorption in biological samples) the chosen elements were hydrogen, carbon and oxygen. As the coherent cross-sections, the incoherent cross-sections and attenuation coefficients are
153
strongly depending on the atomic number, the equation system is linearly independent and thus the system always has a single-valued solution. The peak shape and background were described by a mathematical Gaussian function. At the low energy side of the peak, this function was replaced by an exponential function. The basic idea of the fitting procedure was to find a set of parameter values minimising the w2 value. In the experimental spectrum, some interferences appeared on the lines of different elements. This was especially important for krypton (Kr) (Ka) and Pb (Lb) lines and between As (Ka) and Pb (La). In the present work, the Kr (Ka) peak area was calculated from the Ar (Ka) peak area, assuming a steady air composition. This Kr (Ka) peak area was then subtracted from the total Kr (Ka)+Pb (Lb) peak area. The Pb (Lb) peak area was used to calculate the Pb concentration. The ratio between the Pb (Lb) and Pb (La) lines was used to obtain the Pb (La) peak area. This new Pb (La) peak area was subtracted from the As (Ka)+Pb (La) area, allowing to get the As (Ka) area. This was checked with a standard reference material containing Pb and As: Orchard leaves, NBS standard reference material 1571. The obtained values are presented in Table 1. In spite of the good agreement obtained for this standard, when As and Pb were very close to the detection limit, the error of the fitting process was relatively high and we disregarded both elements. A check was carried out with reference materials containing both elements in concentrations below 1 mg g1. The X-ray generator was operated at 50 kV and 20 mA, and a typical acquisition time of 1000 s was used. A collimator of silver was placed in front of the detector in order to restrict the effective area of the detector by excluding regions close to the edges. In Fig. 1, a spectrum of a sample obtained with this system is shown.
Detection limits The detection limits (DL) obtained by EDXRF were calculated according to the equation: pffiffiffiffiffiffiffi 3C i N b DL ¼ , Np
Table 1. Comparison of element concentrations (mg g1) in orchard leaves (NBS standard reference material 1571) measured in this work (7standard deviation) and the certified values
Present work Certified value a
K
Ca
Fe
Mn
Cu
Zn
As
Br
Rb
Pb
15,3007900 14,7007300
21,50071000 20,9007300
310720 300720
9575 9174
1371 1271
2672 2573
12.572.5 1472
10.670.8 10a
12.570.8 1271
4272 4573
Non-certified value.
ARTICLE IN PRESS p2 0.18 [0.15–0.29] 1.970.3 2.3 [1.7–3.1] 2272 22 [19–24] 320720 286 [226–332]
25007200 2400 [2200–2500]
4.870.7 4.3 [3.7–4.8]
1471 13 [12–14]
p2 0.24 [0.15–0.31]
Rb Br Zn
Se
23007200 2500 [2100–2800]
In Tables 4 and 5, the obtained results for the mean element concentrations and standard deviation (mg g1) in fetal cord blood and in maternal cord blood for five ranges of the mother’s age are presented. Numbers in brackets correspond to the number of analyzed samples in each group. The standard deviation was calculated for each case, considering the number of samples and the number of replicates, respectively. The high values of standard deviation obtained in some cases can be attributed to biological factors affecting the different
Present work Certified value
Results and discussion
Cu
The accuracy was checked by analysis of reference materials, prepared in pellets following the same preparation method as for the samples. The element concentrations obtained for the standard reference material orchard leaves (NBS standard reference material 1571) are shown in Table 1, those obtained for the standard reference material freeze dried animal blood (IAEA, A-13) are presented in Table 2. The results obtained in the present work were in very good agreement with the certified values. The standard deviation was obtained considering the error resulting from the fitting process and the differences in concentration for three pellets analysed repeatedly five times, respectively. Statistic treatment for all the results was carried out taking into account the high number of results (Table 3).
Fe
Accuracy tests
Ca
where C i is the concentration of the element i; N b is the counting rate for the background and N p is the counting rate for the corresponding peak. The results for orchard leaves (NBS standard reference material 1571) are presented in Table 3.
K
Fig. 1. Spectrum of a blood sample obtained by EDXRF.
Pb
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Table 2. Comparison of element concentrations (mg g1) in freeze dried animal blood (IAEA, A-13) measured in this work (7standard deviation) and the certified values with the corresponding range
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Table 3.
DL
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Detection limits (DL; mg g1) obtained by EDXRF in orchard leaves (NBS standard reference material 1571) K
Ca
Mn
Fe
Cu
Zn
As
Rb
Sr
Pb
70
50
4.0
3.1
1.2
1.2
0.5
1.2
0.6
1.1
Table 4. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for five ranges of the mother’s age Element
K Ca Fe Cu Zn Br Rb Pb
Mother’s age (years) 15–20
20–25
25–30
30–35
35–40
(N ¼ 5)
(N ¼ 8)
(N ¼ 21)
(N ¼ 17)
(N ¼ 9)
10,48972992 3787119 24707414 4.770.9 1472 1274 1475 p2
938171912 338785 24907314 5.070.7 1572 1376 1374 p2
980071314 386791 29357458 5.671.9 1672 1573 1473 p2
990071349 385766 306771108 5.972.2 1575 1675 1675 p2
978371399 307746 28347346 5.070.6 1372 1474 1573 p2
Numbers in brackets correspond to the number of analysed samples in each group.
Table 5. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for five ranges of the mother’s age Element
K Ca Fe Cu Zn Br Rb Pb
Mother’s age (years) 15–20
20–25
25–30
30–35
35–40
(N ¼ 5)
(N ¼ 8)
(N ¼ 21)
(N ¼ 17)
(N ¼ 9)
772771372 356780 21277307 1172 3875 1573 1373 p2
786371214 397784 20657367 1072 32711 1575 1173 p2
848271000 386795 25057425 1072 3974 1874 1373 p2
85327728 391758 25477373 1072 3577 1874 1373 p2
767371326 373766 22567470 1073 3477 1574 1272 p2
Numbers in brackets correspond to the number of analysed samples in each group.
samples (e.g. dietary habits and specific characteristics for each individual). In fact, the differences in element concentrations obtained for the three replicates were negligible, when compared with the variations between samples. The most important conclusion from these tables is the similarity of the concentration values in maternal and newborn samples with varying age of the mothers. However, differences were observed when element levels in maternal samples were compared with the corresponding levels in fetus samples. There was a slight increase of K, Rb and Fe in fetus blood samples compared to the mothers’ samples; on the other hand,
the Cu and Zn concentrations were lower in the fetus compared to the maternal samples. The Ca and Br levels were very similar in the two sets of samples (Tables 4 and 5). In Tables 6 and 7, the obtained mean element concentrations and standard deviation (mg g1) in fetal cord blood and in maternal cord blood for five ranges of the newborn’s weight, respectively are presented. Numbers in brackets correspond to the number of analyzed samples in each group. From these results, it can be concluded that element concentrations for mother and child are more or less the same for different weight of the newborns. However, the concentrations in fetal cord
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blood were different from the corresponding concentrations in maternal cord blood. Higher values for K and Rb were observed in fetal samples and higher values for Cu and Fe were found in maternal samples. All the other elements were present in analogous concentrations in both sets of samples. These results are in agreement with reported Cu concentrations in full-term newborns [17–19]. A similar study in amniotic fluid and placenta [20] has shown a dependence of Ca, Fe, Cu and Zn levels on the mother’s age and the newborn’s weight. These dependences were not observed in the present work for maternal and fetus cord blood. In Tables 8 and 9, the element concentrations in fetal and maternal cord blood according to gestational age (GA) are shown. A slight increase in K and Fe concentrations with increasing GA was observed for maternal samples, while a similar slight increase was observed not only for K and Fe but also for Cu and Zn in fetus samples. A stronger dependence for Cu and Zn
on GA both for mothers and children has been previously observed [9]. In that study, samples with GA between 24 and 42 weeks were analysed. Strong element variations are indeed expected, taking into account that the transport of essential elements changes at different stages of gestation. The slight increase in the levels of some elements in the present study reflects the fact that only full term pregnancies were considered, and therefore no big variations were expected. Statistical analysis has been carried out. The Spearman correlation coefficients, rs , between element concentrations in fetal cord blood, maternal cord blood and in maternal and fetal cord blood are presented in Tables 10–12, respectively. In these tables, values not corresponding to any correlation between elements have been omitted. The type of correlation (+) or () is also indicated. According to this test, the closer the rs values are to 1, the stronger is the correlation, for a significance of p ¼ 0:05.
Table 6. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for five ranges of the child’s weight Element
K Ca Fe Cu Zn Br Rb Pb
Child’s weight (kg) 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
(N ¼ 2)
(N ¼ 13)
(N ¼ 29)
(N ¼ 12)
(N ¼ 6)
98477629 342770 28007197 4.970.3 1571 1473 2177 p2
997072118 367794 27967515 5.470.8 1572 1372 1573 p2
960371306 358786 300671298 5.672.1 1574 1574 1474 p2
985471512 387777 28307650 5.371.5 1572 1675 1574 p2
11,26071764 377758 27497262 4.670.5 1572 1478 1473 p2
Numbers in brackets correspond to the number of analysed samples in each group.
Table 7. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for five ranges of the child’s weight Element
K Ca Fe Cu Zn Br Rb Pb
Child’s weight (kg) 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
(N ¼ 2)
(N ¼ 13)
(N ¼ 29)
(N ¼ 12)
(N ¼ 6)
86117542 400771 22917250 1271 3677 1574 1675 p2
810571408 386774 22717412 1073 3876 1573 1273 p2
821571099 369769 24957550 1072 3679 1874 1373 p2
80297762 4117112 23337464 1172 3775 1776 1273 p2
87857625 389717 25217255 1072 3473 1777 1373 p2
Numbers in brackets correspond to the number of analysed samples in each group.
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Table 8. Mean element concentrations (mg g1) and standard deviations in fetal cord blood obtained by EDXRF for three ranges of gestational age Element
(N ¼ 2) K Ca Fe Cu Zn Br Rb Pb
Table 10. Spearman correlation coefficients: element concentrations in fetal cord blood (significant correlation at p ¼ 0:005)
Gestational period (weeks) 35–37
888771711 309720 25277232 5.070.3 1371 1372 1574 p2
37–39 (N ¼ 25) 985671772 356782 29047896 5.571.7 1574 1575 1575 p2
K 39–41 (N ¼ 35) 994871440 378783 292371072 5.471.7 1572 1474 1473 p2
Numbers in brackets correspond to the number of analysed samples in each group.
K Ca Fe Cu Zn Br Rb
Element
K Ca Fe Cu Zn Br Rb Pb
Gestational period (weeks) 35–37
37–39
39–41
(N ¼ 2)
(N ¼ 25)
(N ¼ 35)
730472389 3917162 20417391 8.573.2 3174 1371 1174 p2
818771204 394790 23227408 1073 3777 1775 1373 p2
83107881 374759 25047520 1172 3777 1774 1373 p2
Numbers in brackets correspond to the number of analysed samples in each group.
No strong positive correlations were found. However, some correlations could be observed between elements in fetal cord blood: K and Ca as well as Cu and Fe. In maternal samples, positive correlations were found between K and Fe as well as Rb and Zn. Finally, the element contents in fetal and maternal samples presented some weak positive correlations for K, Ca, Fe, Cu, Br and Rb.
Conclusions In the present work, it has been demonstrated that EDXRF is a powerful multielement technique for the investigation of trace element concentrations in biolo-
Ca
Fe
+0.50
+0.30
+0.50 +0.30
Cu
Zn
Br
Rb
+0.47 +0.47 +0.35 +0.42 0.35
0.42
Table 11. Spearman correlation coefficients: element concentrations in maternal cord blood (significant correlation at p ¼ 0:005) K
Table 9. Mean element concentrations (mg g1) and standard deviations in maternal cord blood obtained by EDXRF for three ranges of gestational age
157
K Ca Fe Cu Zn Br Rb
Ca
Fe
Cu
Zn
Br
+0.56
+0.35
+0.56
+0.35 +0.35
0.35
Rb
+0.36 +0.36
+0.36 +0.61
+0.36
+0.61 +0.51
+0.51
Table 12. Spearman correlation coefficients: element concentrations in fetal and maternal cord blood (significant correlation at p ¼ 0:005)
K Ca Fe Cu Zn Br Rb
K
Ca
Fe
+0.50 +0.51
+0.32
+0.36 +0.51 +0.50
+0.35
Cu
Zn
Br
Rb
+0.44
+0.39 +0.79
+0.38 +0.36 +0.55
+0.40
gical tissues, in a fast and easy way. The samples are not destroyed and no chemical preparation is necessary. All studied elements are detected simultaneously in a single analysis. Moreover, only a small amount of sample – a few grams – is necessary for analysis. However, the method is limited to elements heavier than aluminium and the sensitivity is limited to 1 mg g1 for heavy elements, which is a drawback of this technique. This is the case for lead, chromium and cadmium, which normally are present in blood samples in low levels. This study also found some statistical correlations between element concentrations in the cord blood of mothers and newborns at birth.
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Furthermore, a dependence of element levels on gestational age was confirmed. No substantial changes in the element contents were observed according to the age of the mothers, which might be explained by the fact that those are mostly dependent on environment contamination and dietary habits, which were more or less the same for all subjects investigated.
Acknowledgements The authors express their sincere gratitude to Garcia de Orta Hospital for providing the samples.
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