Determination of trace elements in placenta by total reflection X-ray fluorescence spectrometry: effects of sampling and sample preparation

Abstract

Placental elemental composition can serve as an indicator for neonatal health. Medical studies aiming at revealing such cause-and-effect relationships or studies monitoring potential environmental influences consist of large sample series to ensure statistically sufficient data. Several analytical techniques have been used to study trace metals in human placenta. However, most techniques require provision of clear liquid sample solutions and therefore time- and reagent-consuming total digestion of biological tissue is necessary. In total reflection X-ray fluorescence spectrometry (TXRF)-a straightforward multielement analytical technique-in contrast suspensions of minute sample amounts can be analyzed directly. Therefore, herein we report on a valid method to prepare homogenous sample suspensions for sustainable and fast TXRF analysis of large sample series. The optimized method requires only 10 mg of powdered placental tissue and 1 mL nitric acid. Suspensions are readily prepared within 30 min and the found mass fractions of major, minor, and trace elements are in good agreement in comparison to analysis of digests. In addition, possible effects on fixation time and the exact sampling location, i.e., maternal vs. fetal side of the placenta, were studied applying this method. Thereby, significant differences for fetal placenta tissue compared to maternal or intermediate tissue were observed revealing accumulation of trace elements in the fetal side of the placenta. Furthermore, considerable depletion of up to 60% mass fraction with longer fixation duration occurred in particular in fetal placenta tissue. These findings help to understand the large ranges of mass fraction of elements in placenta reported in the literature and at the same time indicate the necessity for more systematic investigation of non-homogenous elements distributed in placenta taking sampling and stabilization methods into account.

Keywords: Fetal and maternal tissue; Placenta; Sample preparation and stabilization; Suspension; TXRF; Trace elements.

Fig. 1

Investigation of sample homogeneity by evaluation of calcium, copper, and iron recovery by TXRF determination in suspensions compared to digestions after (a) simple grinding procedure and random sampling; (b) simple grinding procedure and subsampling of fine or coarse sample substance; and (c) enhanced grinding procedure and random sampling. N = 10, Box plot presentation gives median, 25–75% percentiles and includes all data points for each element. Black diamonds: data points from random sampling (n = 30); blue diamonds: data points from fine powder sampling (n_sub = 10); orange diamonds: data points from coarse powder sampling (n_sub = 10)

Fig. 2

Comparison of results of element determination by TXRF in digests and suspensions. Solid black line indicates bisector. Results are expressed as mean ± SD with n > 3, N = 9

Fig. 3

Effect of sampling location on mass fractions after 12 h (a), 24 h (b), and 36 h (c) of fixation time. Results are expressed as relative mass fractions (fetal, black square; intermediate, black circle; maternal, black triangle) calculated from mean w_Fetal as reference. Error bars represent ± 1 SD with n > 3; N = 1. Values for mass fractions and number of detections n are given in the SI Tables S1-9

Fig. 4

Effect of fixation time on mass fractions for fetal (a), intermediate (b), and maternal (c) tissue. Results are expressed as relative mass fractions (12 h, black square; 24 h, black circle; 36 h, black triangle) calculated from mean w_12h as reference. Error bars represent ± 1 SD with n > 3; N = 1. Values for mass fractions and number of detections n are given in the SI Tables S1-9