Copper and selenium status as biomarkers of neonatal infections

Journal Pre-proof Copper and selenium status as biomarkers of neonatal infections Julian Hackler, Monika Wisniewska, Lennart Greifenstein-Wiehe, Waldemar B. Minich, Malte Cremer, Christoph Buhrer, ¨ Lutz Schomburg

PII:

S0946-672X(19)30412-2

DOI:

https://doi.org/10.1016/j.jtemb.2019.126437

Reference:

JTEMB 126437

To appear in:

Journal of Trace Elements in Medicine and Biology

Received Date:

26 June 2019

Revised Date:

16 October 2019

Accepted Date:

12 November 2019

Please cite this article as: Hackler J, Wisniewska M, Greifenstein-Wiehe L, Minich WB, Cremer M, Buhrer ¨ C, Schomburg L, Copper and selenium status as biomarkers of neonatal infections, Journal of Trace Elements in Medicine and Biology (2019), doi: https://doi.org/10.1016/j.jtemb.2019.126437

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Copper and selenium status as biomarkers of neonatal infections Julian Hackler1, Monika Wisniewska1, Lennart Greifenstein-Wiehe1, Waldemar B. Minich1, Malte Cremer2, Christoph Bührer2, Lutz Schomburg1* 1

Institute for Experimental Endocrinology, Charité Universitätsmedizin Berlin, Berlin, Germany 2

Department of Neonatology, Charité Universitätsmedizin Berlin, Berlin, Germany.

*

Corresponding author:

Lutz Schomburg, Institute for Experimental Endocrinology, Südring 10, CVK, CharitéUniversitätsmedizin Berlin, D-13353 Berlin, Germany

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E-mail: [email protected], Tel. +49-30-450524289, Fax. +49-30-450524922

Neonatal infections are a major risk factor for neonatal mortality. A reliable diagnosis of early-

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onset sepsis (EOS) is hampered by the variable clinical presentations of the children. We hypothesized that changes in the Se or Cu status, or the biomarkers selenoprotein P (SELENOP)

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or ceruloplasmin (CP) alone or in combination may be informative of EOS. We generated a new human CP-specific non-competitive immunoassay (ELISA) suitable of

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analysing small sample volumes and validated the method with a commercial CP source. Using this novel CP assay, we analysed a case-control study of EOS (n=19 control newborns, n=18 suspected cases). Concentrations of Se, Cu, SELENOP, CP, interleukin-6 (IL-6), and C-reactive

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protein (CRP) along with the Cu/Se and CP/SELENOP ratios were evaluated by correlation analyses as biomarkers for EOS. Diagnostic value was estimated by receiver operating characteristic (ROC) curve analyses.

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The new CP-ELISA displayed a wide working (0.10 to 6.78 mg CP/L) and low sample requirement (2 µL of serum, EDTA-, heparin- or citrate-plasma). Plasma CP correlated

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positively with Cu concentrations in the set of all samples (Pearson r=0.8355, p<0.0001). Three of the infected neonates displayed particularly high ratios of Cu/Se and CP/SELENOP, i.e., 3.8to 6.9-fold higher than controls. Both the Cu/Se and the CP/SELENOP ratios correlated poorly with the early infection marker IL-6, but strongly and positively with the acute-phase protein CRP (Cu/Se-CRP: Spearman ϱ=0.583, p=0.011; CP/SELENOP-CRP: ϱ=0.571, p=0.013). The ROC curve analyses indicate that a combination of biomarkers for the Se and Cu status do not improve the early identification of EOS considerably.

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This study established a robust, highly precise, partly validated and scalable novel CP sandwich ELISA suitable for basic and clinical research, requiring minute amounts of sample. The ratio of circulating CP/SELENOP constitutes a promising new composite biomarker for detection of EOS, at least in a subset of severely diseased children.

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Introduction

The trace elements copper (Cu) and selenium (Se) are essential micronutrients for human development and health [1, 2]. Adequate nutritional intake and a sufficient supply of both trace

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elements are of central importance for pregnancy, child development and neonatal health [3]. Neonatal infectious diseases remained at an unfortunate high level in the last decades, causing more than 500,000 deaths worldwide in 2010 alone [4]. An early-onset sepsis (EOS) is mainly caused by vertical transmission of pathogenic bacteria. These pathogens can induce maternal chorioamnionitis and constitute relevant risk factors for cerebral palsy and cystic periventricular

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leukomalacia [5] with abnormal development or neuronal damage eventually leading to severe movement disorders. EOS is associated with infection-related systemic inflammatory response

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syndrome (SIRS) of newborns [6] and causes severe clinical symptoms within the first 48 h of life. In the last decade the incidence of EOS remained relatively high, ranging from 0.54/1000

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to 1.1/1000 live births [7-9]. An early diagnosis is hampered by the variable clinical presentations of EOS covering a wide range of unspecific signs [10]. Consequently, on the one hand, overtreatment with antibiotics is frequent [11], but on the other hand, a misdiagnosis of

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ambiguous early clinical signs as uncritical may result in death or permanent damage [12]. In clinical practice, a series of different markers is evaluated for an early identification and

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diagnosis of early-onset neonatal infections, e.g. respiratory rate, core body temperature, Creactive protein (CRP) [13] and interleukin 6 (IL-6) [14] concentrations. Blood cultures are

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evaluated for identifying the types of microorganisms involved [15]. So far, the markers available do not yet reach the necessary diagnostic precision to safely indicating an early-onset infection before overt clinical manifestations become evident. Infectious diseases are associated with declining Se concentrations as a negative acute phase reactant [16, 17], and increasing circulating Cu concentrations as positive acute phase reactant [18, 19]. We thus hypothesized that analysing changes in both the Se and Cu status in parallel as a combined biomarker may be informative of early-onset neonatal infections in suspected cases, and that the Se transporter selenoprotein P (SELENOP) together with the major Cu-transporting protein ceruloplasmin (CP) may allow a reliable protein-based analysis and fast detection of early onset infections. To 2

this end, we had already succeeded in establishing and validating an ELISA test for SELENOP quantification [20]. Here, we describe our development of a similarly reliable ELISA test for CP to be used in combination for potentially enabling a fast diagnosis of neonatal infections.

2 2.1

Materials and Methods Study design

We conducted a case-control study on the neonatology wards of the CharitéUniversitätsmedizin Berlin, and analysed for differences in total trace element concentrations as described before [21]. The study had been approved by the ethics committee of the Charité

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(application no: EA2/092/12), and was conducted in accordance with the principles of Helsinki. Plasma samples were available from healthy newborns shortly after birth (control), and from potentially infected newborns shortly after birth (0 h) and at day 2 after birth (48 h). 2.2

Study population

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Several clinical signs had been recorded for diagnosis of potential early-onset infection, including pneumonia, respiratory disorders including tachypnoea and apnea, tachycardia,

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increased (>38.5 °C) or decreased (<36.0 °C) body temperature, lethargy, hypotonia, poor feeding and coagulation disorder [21]. To fulfil the inclusion criteria for neonatal infection, the

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newborn had to show at least one clinical sign and laboratory evidence of an infection by clinically diagnosed elevated IL-6 or CRP concentrations (IL-6 >100 ng/L; CRP >10mg/L at day of birth). In case of suspected early-onset infection, newborns were treated immediately

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with antibiotics (ampicillin and gentamicin) for a period of 3 days. Exclusion criteria were defined as described [21], including gestational age <30 weeks, birth weight <1000 g, genetic disease, congenital malformation, parenteral intake of trace elements or missing informed

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consent. Initially, a total of 44 subjects fulfilled the inclusion criteria [21, 22]. However, as the blood samples from newborns are very limited, only 37 of the initially described 44 patients

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had sufficient leftover volumes available to allow the analysis of CP by ELISA as described in this study (Table 1). 2.3

Development and partial validation of a novel CP-sandwich-ELISA

Monoclonal antibodies (mAb) against CP were generated by immunization of mice with the purified human protein, isolation of the spleen and fusion of the lymphocytes with immortalized murine myeloma cells, essentially as described for the generation of SELENOP-specific mAb [20]. Clone identification, expansion and mAb production were conducted by a commercial service supplier (InVivo Biotech Services GmbH, Hennigsdorf, Germany). Out of a set of 3

several CP-specific mAb, two mAb were chosen based on their specificity for human CP and used to develop a two-site non-competitive immunoassay (sandwich ELISA), involving selection and optimization steps essentially as described [23]. The optimized protocol involves the absorption of the capture mAb (CP-Ab1) to the surface of polystyrene plates overnight at 4°C at a mAb concentration of 0.8 µg/ml. Unspecific binding sites were blocked with bovine serum albumin (Carl Roth GmbH). Human serum samples and purified human CP (Lee BioSolutions, Inc, Maryland Heights, USA) were applied as standards and calibrators in serial dilutions (50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0,10 and 0.05 mg/L). A second CP-specific mAb served as detection mAb (CP-Ab2) and was conjugated with horseradish peroxidase (HRP), as described earlier [24]. The resulting precipitate after conjugation was dissolved in 50 µL H2O.

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The enzymatic assay was started by adding 3,3’,5,5’-tetramethylbenzidine (Surmodics IVD, Ballinasloe, Ireland). The reaction was terminated by adding an equal volume of sulphuric acid (Fluka Analytical). Spectrophotometric read out was recorded at 450 nm using a NanoQuant

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Infinite 200 Pro microplate reader (Tecan Group AG, Männedorf, Switzerland).

Four independent runs of CP quantifications in 96-well format with samples of known CP concentrations were performed for regression model analysis, in accordance with the

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recommendations of the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) [25]. The coefficient of determination was calculated using an unweighted four-parameter logistic

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function, and R2 was ranging from 0.9972 to 0.9943. The limits of quantification were determined for a coefficient of variation (CV) passing the 15% mark. The lower limit of quantification (LLOQ) was calculated at a CP concentration of 0.10 mg/L, and the upper limit

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of quantification (ULOQ) at 6.78 mg/L, thereby defining the working range of this novel CPspecific ELISA between 0.10 and 6.78 mg/L [25], i.e., spanning almost two orders of

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magnitude. Inter-assay and intra-assay CV were below 6% as determined using the same commercially available human CP preparation (Lee BioSolutions) at a concentration of 0.1, 1.5 and 6.0 mg/L, respectively. In order to describe the method accuracy (mean bias), the

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cumulative mean percent relative errors of back-calculated concentrations were determined with serial dilution of human CP (50.0 to 0.05 mg/L). 2.4

IL-6, CRP, SELENOP, CP and trace element analyses

IL-6, CRP, SELENOP and trace elements had been determined in this set of samples before as described earlier [21, 22]. Briefly, CRP had been quantified by a turbidometric assay, IL-6 by an electro-chemical luminescence immunoassay, human SELENOP by an immunoassay (Selenotest®; selenOmed GmbH) and trace elements by total reflection X-ray fluorescence 4

spectroscopy (TXRF) using a benchtop device (S2 PICOFOX, Bruker nano, Berlin, Germany). Plasma samples of 37 neonates were now additionally available for CP analysis by the newly generated CP-specific ELISA. To this end, the samples were pre-diluted 1:300 in sample buffer. 50 µL of diluted sample were incubated on precoated sandwich ELISA plates for 30 min at room temperature. A three-times automatic wash step were performed to rinse unbound CP using HydroFlexTM microplate washer (Tecan Group AG). For specific detection, 50 µL of CP-Ab2 (50 ng/ml) was incubated for 30 min. Unbound CP-Ab2 was rinsed and the enzymatic detection assay was started by adding 100 µL of TMB. The reaction was terminated by adding 100 µL of sulphuric acid (0.25 mol/L). Spectrophotometric read out was recorded within 10

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min at 450 nm using a NanoQuant Infinite 200 Pro microplate reader (Tecan Group AG). Statistical analysis

GraphPad Prism 7 software (GraphPad Software Inc., San Diego, USA) and SPSS (Version 24.0. Armonk, IBM Corp. NY, USA) were used for biostatistical computations. For normally distributed and independent data unpaired t test and one-way ANOVA were conducted to

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compare means. Pearson coefficients r were calculated for correlation analyses yielding linear regression, symbolized by solid lines, and 95% confidence intervals, indicated by dashed lines

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in the figures, respectively. Normally distributed and dependent data were analysed with paired t test to compare means. The Mann-Whitney test was conducted for not-normally distributed

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and independent data to compare means. Spearman rank correlation coefficient ϱ was calculated for correlation analysis. Not-normally distributed and dependent data were analysed with Wilcoxon signed-rank test. The SELENOP, CP concentrations and CP/SELENOP were

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analysed with receiver operating characteristic (ROC) curves, calculating the areas under the curve. All values are presented as means ± standard deviations (SD), if not declared separately. All statistical tests are using an α-level of 0.05, and statistical significance was defined as

Results

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p>0.05 (n.s), p<0.05 (*), p<0.01 (**), p<0.001 (***) or p<0.0001(****).

3.1

Establishment and performance parameters of the novel CP-sandwich-ELISA

From a total of ten available mAb against human CP, one pair was selected for sandwich ELISA development based on specificity and production yield from the respective hybridoma cells. The assay performance parameters were determined with a dilution series of human CP 5

standard ranging from 0.05 to 50.00 mg/L. In replicates of four, the non-linear curve-fitting yielded a coefficient of determination of r2 ≥ 0.9943 (Figure 1A). The percent mean relative errors (%RE) of back-calculated standard concentrations (“mean bias”) was <20% in the range of 0.10 to 12.50 mg/L (Figure 1B). The mean coefficient of variation (CV) was set to an upper limit of 15% defining the LLOQ at a CP concentration of 0.10 mg/L, and the ULOQ at 6.78 mg/L (Figure 1C). The ELISA appears suitable for specific determination of CP (recovery 100±20%) in different matrices including human serum, as well as in EDTA, heparin or citrate plasma (Figure 2A), and even the signals detected from hemolytic serum samples were not deviating strongly from

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the expected values (Figure 2B). The recovery of CP from three different serum samples was constant in a dilution range of 1:100 to 1:750 (Figure 2C), indicating its suitability for CP quantification from minute amounts of sample. The stability of CP was assessed by applying several freeze-thaw cycles to serum samples (n= 4, 50 µL each) consisting of a cooling step to

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-80°C for 1 h and a thaw step to shelf temperature (23°C) for 30 min. The mean recovery was acceptable during the first two cycles, but decreased consistently under 80% after three freeze-

Ceruloplasmin and Selenoprotein P concentrations in early-onset neonatal infection

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thaw cycles (Figure 2D).

A total of 37 plasma samples from neonates were available for this study, including n=19

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patients without clinical symptoms (control group) and n=18 patients with signs of early-onset neonatal infection (infected group, with two samples from 0 h and 48 h after birth) (Table 1). Plasma Cu concentrations were elevated in infected neonates as compared to controls (control:

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492.9±131.0 µg/L (0 h); versus infected: 670.7±267.0 µg/L (0 h) and 830.2±240.9 µg/L (48 h), respectively) (Figure 3A), resulting in an average increase of 1.4-times (0 h) or 1.7-times (48 h

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vs. 0 h). The average Se concentrations were not significantly different between the groups (control: 39.1±10.0 µg/L (0 h), versus infected: 35.0±14.4 µg/L (0 h) and 35.1±9.3 µg/L (48 h vs. 0 h), respectively) resulting in an average decrease by 0.9-times (Figure 3B). In parallel, concentrations of the Cu transporter CP were elevated in infected children at 0 h, and significantly increased at 48 h (control: 158.2±64.9 mg/L (0 h); versus infected: 225.1±117.1 mg/L (0 h) and 331.3±94.2 mg/L (48 h), respectively) (Figure 3C). Average CP concentrations increased in infected versus control newborns by a factor of 1.4 (0 h), or 2.1 (48 h vs. 0 h). SELENOP concentrations were decreased in infected neonates by 0.9-fold (0 h), but recovered 6

to 1.2-fold after 48 h (control: 1.1±0.3 (0 h); versus infected: 1.0±0.5 mg/L (0 h), and 1.3±0.5 mg/L (48 h), respectively) (Figure 3D). The two plasma Cu biomarkers increased consistently in infected neonates from 0 h to 48 h in 17 out of 18 patients in case of Cu (Figure 3E), and in 16 out of 18 patients in case of CP (Figure 4F), respectively.

The interrelation of the trace element concentrations of Cu and Se with their corresponding transport proteins as biomarkers, i.e., CP and SELENOP, respectively, was analysed to evaluate their suitability as diagnostic parameters of early onset infection. Both pairs of biomarkers correlated positively and strongly in the full collection of plasma samples analysed: Cu-CP:

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Pearson r=0.8355, p<0.0001, and Se-SELENOP: Pearson r=0.603, p<0.0001 (n=55) (Figure 4A&B). The average Cu/CP ratios did not differ between the groups (control: 6.0±1.6 (0 h);

infected: 6.0±1.6 (0 h) and 4.9±1.0 (48 h), respectively), but the infected neonates showed on average the lowest quotient at the 48 h time point (Figure 5A). Similarly, the Se/SELENOP ratio

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was not different between controls and infected at the 0 h time point, but was significantly lower at the 48 h time point (control: 18.7±4.7 (0 h); infected: 18.3±3.9 (48 h), and 14.5±4.5 (48 h),

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respectively) (Figure 5B).

In order to test for composite biomarkers indicating early onset infection at the time of birth,

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the ratios of Cu/Se and CP/SELENOP were calculated and compared. In this analysis, three infected newborns displayed exceptionally high ratios of Cu/Se in comparison to the group median (elevation factors of 3.8, 4.6 and 4.9, respectively) (Figure 6A), and the same children

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showed even higher ratios of the CP/SELENOP ratio (elevation factors of 4.0, 6.9 and 5.5, respectively) (Figure 6B). Two of these newborns displayed a 1 min Apgar score of ≤ 4 and a 5 min Apgar score of ≤ 7, indicating a reduced health status. Both, the initially high Cu/Se and

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the CP/SELENOP ratios decreased within 48 h to more intermediate level, likely indicating a successful antibiotic treatment (Figure 6C&D). During treatment, the initially reduced Cu/Se

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and CP/SELENOP ratios increased slightly over time in the group of infected neonates. Both, the Cu/Se and CP/SELENOP ratios appeared not to be associated with Il-6 measured directly after birth (Figure 7A-D). The Cu/Se and CP/SELENOP ratios showed a significant correlation at the 48 h time point with the acute-phase reactant C-reactive protein (CRP) (Cu/SeCRP: Spearman ϱ=0.583, p=0.011; CP/SELENOP-CRP: Spearman ϱ=0.571, p=0.013) (Figure 7E&F). A respective ROC-analysis and calculation of the area under the curve (AUC) yielded 0.5775±0.0960 (p=0.4207) for SELENOP, 0.6725±0.0931 (p=0.0730) for CP, and a slightly 7

improved value of 0.6813±0.0893 (p=0.0596) for the composite biomarker CP/SELENOP (supplementary material).

4

Discussion

Early-onset sepsis poses a high diagnostic challenge during the first hours of life on the neonatal ward. In this study, we aimed to elucidate whether changes in the Se and Cu status, measured as total trace elements and transport proteins, are suitable biomarkers for improving and accelerating the identification of diseased newborns. It was speculated that the protein-based biomarkers show a tighter association with infection than the trace elements, as their biosynthesis and secretion is specifically regulated in liver as positive or negative acute phase

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reactants. Especially combining the two inversely regulated biomarkers SELENOP and CP was expected to yielding a novel and highly informative composite index of neonatal sepsis.

Our results indicate that plasma Cu and CP correlate tightly in both control and infected newborns, as well as plasma Se and SELENOP correlate tightly independent of infection. While

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all four biomarkers thus seem suitable for assessing the respective trace element status, the combined analysis, either the total concentrations of both trace elements in serum or their

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transporters, alone or as a ratio, did not prove to provide a sufficiently specific and sensitive readout with added value for early diagnosis. This is an unfortunate finding, as especially the

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protein-based biomarkers would be suitable for bedside testing enabling a fast analysis in a point-of-care format. Nevertheless, this study established a sensitive, precise, partly validated and scalable novel CP sandwich ELISA suitable for high throughput analysis in basic and

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clinical research, especially also for epidemiological analyses where small volumes of the precious samples only are usually available per new biomarker. Importantly, as the ELISA is based on mAbs, a sufficient number of assays can be prepared for optimizing and validating

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the quantitative method to highest analytical standards, a task usually not possible with commercial systems due to proprietary components and matters of accessibility and high costs.

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Besides method establishment, the identification of a fraction of infected newborns (n=3 of 18, i.e., 17%) with exceptionally high Cu/Se and CP/SELENOP ratios warrants further analysis. Notably, two of these children were very sick, exhibiting a low 1 min and 5 min Apgar score indicative of an impairment of several vital functions directly after birth [26]. A low Se status of the mother is known to constituting a risk factor for pregnancy complications [27]. Maternal Se concentrations were significantly lower in adolescent pregnant women delivering low weight infants (49.4±3.5 µg/L) compared with those delivering appropriate weighted infants (65.1±2.4 µg/L) [28]. In addition, maternal Se status during pregnancy is positively associated 8

with motor and language development of the newborn [29], and constitutes a relevant risk factor for infections, thereby potentially closing a vicious cycle [30]. Similarly, elevated Cu levels are associated with pregnancy complications, especially preeclampsia [31], and elevated Cu concentrations correlate negatively with intrauterine growth and birth weight [32]. Collectively, these findings indicate the importance of a balanced ratio of the trace elements Se and Cu for a healthy pregnancy, an undisturbed child development and a low neonatal infection risk. In this respect, the newly developed CP assay along with the SELENOP ELISA may facilitate the identification of subjects with a dysbalance in these micronutrients, either in pregnancy with relevance for both mother and the unborn child or

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shortly after birth, using immunological tools that allow sensitive, fast and routine analysis by photometric methods, i.e., less challenging than quantification of the trace elements directly via other more sophisticated, expensive and labour-intensive spectroscopic technology.

In case the CP/SELENOP index is elevated, indicative of infection or inadequate nutritional

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supply, a Se supplementation may be considered in pregnancy or after birth. However, there is considerable uncertainty in the scientific literature with respect to the effects of post-diagnostic

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Se supplementation in infectious diseases, and whether this will ameliorate SIRS outcome and reduce adult and neonatal morbidity or not [33-37]. It was shown that non-survivors of intensive care unit patients with SIRS had significantly lower initial and minimum plasma Se

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concentrations than survivors [38]. Additional observational studies on this issue are needed before respective interventions can be discussed and considered.

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The particular strength of this study is the establishment of a partly validated, precise and high throughput sandwich ELISA for CP quantification suitable for basic and clinical research and capable for assessing the Cu status in newborns by an immunometric method. The ratio of

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circulating CP/SELENOP constitutes a promising new composite biomarker for detection of severe infections. Among the weaknesses of this study is the unfavourable outcome with respect

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to the major hypothesis, i.e., that the CP/SELENOP index may constitute the long sought-for novel biomarker of neonatal infection, and the relatively small study cohort. However, the data obtained in this explorative analysis now allow for a power assessment-based design of a larger follow-up study.

9

Funding Research

is

funded

by

Charité

Medical

School

Berlin

and

the

Deutsche

Forschungsgemeinschaft (DFG Research Unit 2558 TraceAge, Scho 849/6-1).

Conflict of Interest LS holds shares in selenOmed GmbH, a company involved in selenium status assessment and

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supplementation. JH, MW, LG-W, MW, MC, and CB have nothing to declare.

References

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[1] L. Schomburg, Dietary Selenium and Human Health, Nutrients 9(1) (2016). [2] M. Bost, S. Houdart, M. Oberli, E. Kalonji, J.F. Huneau, I. Margaritis, Dietary copper and human health: Current evidence and unresolved issues, Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS) 35 (2016) 107-115. [3] A. Mousa, A. Naqash, S. Lim, Macronutrient and Micronutrient Intake during Pregnancy: An Overview of Recent Evidence, Nutrients 11(2) (2019). [4] R. Lozano, M. Naghavi, K. Foreman, S. Lim, K. Shibuya, V. Aboyans, J. Abraham, T. Adair, R. Aggarwal, S.Y. Ahn, M.A. AlMazroa, M. Alvarado, H.R. Anderson, L.M. Anderson, K.G. Andrews, C. Atkinson, L.M. Baddour, S. Barker-Collo, D.H. Bartels, M.L. Bell, E.J. Benjamin, D. Bennett, K. Bhalla, B. Bikbov, A.B. Abdulhak, G. Birbeck, F. Blyth, I. Bolliger, S. Boufous, C. Bucello, M. Burch, P. Burney, J. Carapetis, H. Chen, D. Chou, S.S. Chugh, L.E. Coffeng, S.D. Colan, S. Colquhoun, K.E. Colson, J. Condon, M.D. Connor, L.T. Cooper, M. Corriere, M. Cortinovis, K.C. de Vaccaro, W. Couser, B.C. Cowie, M.H. Criqui, M. Cross, K.C. Dabhadkar, N. Dahodwala, D. De Leo, L. Degenhardt, A. Delossantos, J. Denenberg, D.C. Des Jarlais, S.D. Dharmaratne, E.R. Dorsey, T. Driscoll, H. Duber, B. Ebel, P.J. Erwin, P. Espindola, M. Ezzati, V. Feigin, A.D. Flaxman, M.H. Forouzanfar, F.G.R. Fowkes, R. Franklin, M. Fransen, M.K. Freeman, S.E. Gabriel, E. Gakidou, F. Gaspari, R.F. Gillum, D. GonzalezMedina, Y.A. Halasa, D. Haring, J.E. Harrison, R. Havmoeller, R.J. Hay, B. Hoen, P.J. Hotez, D. Hoy, K.H. Jacobsen, S.L. James, R. Jasrasaria, S. Jayaraman, N. Johns, G. Karthikeyan, N. Kassebaum, A. Keren, J.-P. Khoo, L.M. Knowlton, O. Kobusingye, A. Koranteng, R. Krishnamurthi, M. Lipnick, S.E. Lipshultz, S.L. Ohno, J. Mabweijano, M.F. MacIntyre, L. Mallinger, L. March, G.B. Marks, R. Marks, A. Matsumori, R. Matzopoulos, B.M. Mayosi, J.H. McAnulty, M.M. McDermott, J. McGrath, Z.A. Memish, G.A. Mensah, T.R. Merriman, C. Michaud, M. Miller, T.R. Miller, C. Mock, A.O. Mocumbi, A.A. Mokdad, A. Moran, K. Mulholland, M.N. Nair, L. Naldi, K.M.V. Narayan, K. Nasseri, P. Norman, M. O'Donnell, S.B. Omer, K. Ortblad, R. Osborne, D. Ozgediz, B. Pahari, J.D. Pandian, A.P. Rivero, R.P. Padilla, F. Perez-Ruiz, N. Perico, D. Phillips, K. Pierce, C.A. Pope, E. Porrini, F. Pourmalek, M. Raju, D. Ranganathan, J.T. Rehm, D.B. Rein, G. Remuzzi, F.P. Rivara, T. Roberts, F.R. De León, L.C. Rosenfeld, L. Rushton, R.L. Sacco, J.A. Salomon, U. Sampson, E. Sanman, D.C. Schwebel, M. Segui-Gomez, D.S. Shepard, D. Singh, J. Singleton, K. Sliwa, E. Smith, A. Steer, J.A. Taylor, B. Thomas, I.M. Tleyjeh, J.A. Towbin, T. Truelsen, E.A. Undurraga, N. Venketasubramanian, L. Vijayakumar, T. Vos, G.R. Wagner, M. Wang, W. Wang, K. Watt, 10

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M.A. Weinstock, R. Weintraub, J.D. Wilkinson, A.D. Woolf, S. Wulf, P.-H. Yeh, P. Yip, A. Zabetian, Z.-J. Zheng, A.D. Lopez, C.J.L. Murray, Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010, The Lancet 380(9859) (2012) 2095-2128. [5] Y.W. Wu, J.M. Colford, Jr., Chorioamnionitis as a risk factor for cerebral palsy: A metaanalysis, Jama 284(11) (2000) 1417-1424. [6] T. Kawasaki, Update on pediatric sepsis: a review, Journal of intensive care 5 (2017) 4747. [7] J.W. Fjalstad, H.J. Stensvold, H. Bergseng, G.S. Simonsen, B. Salvesen, A.E. Ronnestad, C. Klingenberg, Early-onset Sepsis and Antibiotic Exposure in Term Infants: A Nationwide Population-based Study in Norway, The Pediatric infectious disease journal 35(1) (2016) 1-6. [8] S.J. Schrag, M.M. Farley, S. Petit, A. Reingold, E.J. Weston, T. Pondo, J. Hudson Jain, R. Lynfield, Epidemiology of Invasive Early-Onset Neonatal Sepsis, 2005 to 2014, Pediatrics 138(6) (2016). [9] B.J. Stoll, N.I. Hansen, P.J. Sanchez, R.G. Faix, B.B. Poindexter, K.P. Van Meurs, M.J. Bizzarro, R.N. Goldberg, I.D. Frantz, 3rd, E.C. Hale, S. Shankaran, K. Kennedy, W.A. Carlo, K.L. Watterberg, E.F. Bell, M.C. Walsh, K. Schibler, A.R. Laptook, A.L. Shane, S.J. Schrag, A. Das, R.D. Higgins, Early onset neonatal sepsis: the burden of group B Streptococcal and E. coli disease continues, Pediatrics 127(5) (2011) 817-826. [10] A.L. Shane, P.J. Sanchez, B.J. Stoll, Neonatal sepsis, Lancet (London, England) 390(10104) (2017) 1770-1780. [11] R.H. Clark, B.T. Bloom, A.R. Spitzer, D.R. Gerstmann, Reported medication use in the neonatal intensive care unit: data from a large national data set, Pediatrics 117(6) (2006) 19791987. [12] R. Libster, K.M. Edwards, F. Levent, M.S. Edwards, M.A. Rench, L.A. Castagnini, T. Cooper, R.C. Sparks, C.J. Baker, P.E. Shah, Long-term outcomes of group B streptococcal meningitis, Pediatrics 130(1) (2012) e8-15. [13] A.Q. Ismail, A. Gandhi, Using CRP in neonatal practice, The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet 28(1) (2015) 3-6. [14] H. Kuster, M. Weiss, A.E. Willeitner, S. Detlefsen, I. Jeremias, J. Zbojan, R. Geiger, G. Lipowsky, G. Simbruner, Interleukin-1 receptor antagonist and interleukin-6 for early diagnosis of neonatal sepsis 2 days before clinical manifestation, Lancet (London, England) 352(9136) (1998) 1271-1277. [15] J.P. Buttery, Blood cultures in newborns and children: optimising an everyday test, Archives of disease in childhood. Fetal and neonatal edition 87(1) (2002) F25-28. [16] C. Nichol, J. Herdman, N. Sattar, P.J. O'Dwyer, D.S. O'Reilly, D. Littlejohn, G. Fell, Changes in the concentrations of plasma selenium and selenoproteins after minor elective surgery: Further evidence for a negative acute phase response?, Clin Chem 44(8) (1998) 17641766. [17] K. Renko, P.J. Hofmann, M. Stoedter, B. Hollenbach, T. Behrends, J. Kohrle, U. Schweizer, L. Schomburg, Down-regulation of the hepatic selenoprotein biosynthesis machinery impairs selenium metabolism during the acute phase response in mice, Faseb J 23(6) (2009) 1758-1765. [18] A. Zoli, L. Altomonte, R. Caricchio, A. Galossi, L. Mirone, M.P. Ruffini, M. Magaro, Serum zinc and copper in active rheumatoid arthritis: Correlation with interleukin 1 beta and tumour necrosis factor alpha, Clin Rheumatol 17(5) (1998) 378-382. [19] C. Chiarla, I. Giovannini, J.H. Siegel, Patterns of correlation of plasma ceruloplasmin in sepsis, J Surg Res 144(1) (2008) 107-110. 11

Jo

ur

na

lP

re

-p

ro of

[20] S. Hybsier, T. Schulz, Z. Wu, I. Demuth, W.B. Minich, K. Renko, E. Rijntjes, J. Kohrle, C.J. Strasburger, E. Steinhagen-Thiessen, L. Schomburg, Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P, Redox Biol 11 (2017) 403-414. [21] L. Wiehe, M. Cremer, M. Wisniewska, N.P. Becker, E. Rijntjes, J. Martitz, S. Hybsier, K. Renko, C. Buhrer, L. Schomburg, Selenium status in neonates with connatal infection, The British journal of nutrition 116(3) (2016) 504-513. [22] M. Wisniewska, M. Cremer, L. Wiehe, N.P. Becker, E. Rijntjes, J. Martitz, K. Renko, C. Bührer, L. Schomburg, Copper to Zinc Ratio as Disease Biomarker in Neonates with EarlyOnset Congenital Infections, Nutrients 9(4) (2017). [23] E.C. Kuhn, A. Slagman, E.C.D. Kuhn-Heid, J. Seelig, C. Schwiebert, W.B. Minich, C. Stoppe, M. Mockel, L. Schomburg, Circulating levels of selenium-binding protein 1 (SELENBP1) are associated with risk for major adverse cardiac events and death, Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS) 52 (2019) 247-253. [24] D.M. Boorsma, G.L. Kalsbeek, A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 23(3) (1975) 200-207. [25] B. DeSilva, W. Smith, R. Weiner, M. Kelley, J.M. Smolec, B. Lee, M. Khan, R. Tacey, H. Hill, A. Celniker, Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules, Pharm Res-Dordr 20(11) (2003) 1885-1900. [26] L.V. Simon, B.N. Bragg, APGAR Score, StatPearls, Treasure Island (FL), 2019. [27] I.H. Qazi, C. Angel, H.X. Yang, B. Pan, E. Zoidis, C.J. Zeng, H.B. Han, G.B. Zhou, Selenium, Selenoproteins, and Female Reproduction: A Review, Molecules 23(12) (2018). [28] H.D. Mistry, L.O. Kurlak, S.D. Young, A.L. Briley, F.B. Pipkin, P.N. Baker, L. Poston, Maternal selenium, copper and zinc concentrations in pregnancy associated with small-forgestational-age infants, Maternal & child nutrition 10(3) (2014) 327-334. [29] K. Polanska, A. Krol, W. Sobala, J. Gromadzinska, R. Brodzka, G. Calamandrei, F. Chiarotti, W. Wasowicz, W. Hanke, Selenium status during pregnancy and child psychomotor development-Polish Mother and Child Cohort study, Pediatr Res 79(6) (2016) 863-869. [30] M. Ryan-Harshman, W. Aldoori, The relevance of selenium to immunity, cancer, and infectious/inflammatory diseases, Can J Diet Pract Res 66(2) (2005) 98-102. [31] Y.Q. Fan, Y. Kang, M. Zhang, A meta-analysis of copper level and risk of preeclampsia: evidence from 12 publications, Bioscience Rep 36 (2016). [32] H. Shoji, N. Ikeda, C. Kojima, T. Kitamura, H. Suganuma, K. Hisata, S. Hirayama, T. Ueno, T. Miida, T. Shimizu, Relationship between copper and lipids and atherogenic indices soon after birth in Japanese preterm infants of 32-35 weeks, J Dev Orig Hlth Dis 8(2) (2017) 256-260. [33] B.A. Darlow, N.C. Austin, Selenium supplementation to prevent short-term morbidity in preterm neonates, The Cochrane database of systematic reviews (4) (2003) CD003312. [34] R. Aggarwal, G. Gathwala, S. Yadav, P. Kumar, Selenium Supplementation for Prevention of Late-Onset Sepsis in Very Low Birth Weight Preterm Neonates, Journal of tropical pediatrics 62(3) (2016) 185-193. [35] W. Manzanares, M. Lemieux, G. Elke, P.L. Langlois, F. Bloos, D.K. Heyland, High-dose intravenous selenium does not improve clinical outcomes in the critically ill: a systematic review and meta-analysis, Critical care 20(1) (2016) 356. [36] H. Brodska, J. Valenta, K. Malickova, P. Kohout, A. Kazda, T. Drabek, Biomarkers in critically ill patients with systemic inflammatory response syndrome or sepsis supplemented with high-dose selenium, Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS) 31 (2015) 25-32. 12

[37] Y. Sakr, V.P. Maia, C. Santos, J. Stracke, M. Zeidan, O. Bayer, K. Reinhart, Adjuvant selenium supplementation in the form of sodium selenite in postoperative critically ill patients with severe sepsis, Critical care 18(2) (2014) R68. [38] Y. Sakr, K. Reinhart, F. Bloos, G. Marx, S. Russwurm, M. Bauer, F. Brunkhorst, Time course and relationship between plasma selenium concentrations, systemic inflammatory response, sepsis, and multiorgan failure, British journal of anaesthesia 98(6) (2007) 775-784.

Figures

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Figure 1: Bioanalytical characterization of human CP-ELISA. (A) Goodness of fit for independent duplicates of human CP standard calibrators (0.05–50.0 mg/L). Coefficient of determination (R2) was calculated using an unweighted four parameter logistic regression with R2=0.9943–0.9972. (B) Mean relative error (RE) of the back-calculated CP concentrations

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(0.1–12.5 mg/L). The mean RE was smaller than ±10% for all standard calibrators within the working range. (C) Precision profile of human CP calibrators and determination of the working

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range with CV<15%. The interceptions are indicating the lower limit of quantification (LLOQ)

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= 0.1 mg/L and upper limit of quantification (ULOQ) = 6.78 mg/L.

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Figure 2: (A) Comparison of different biological sample types and (B) of non-hemolytic versus hemolytic samples. Serum CP-concentrations were defined as 100%. (C) Dilutional linearity of three serum samples. Dilution step 1:300 was set to 100%. * = dilutions were above the ULOQ. (D) Recovery of serum ceruloplasmin after multiple freeze-thawing cycles. Human serum sample (n=4) were stored for 1 h at -80°C and thawed at RT for 30 min. Cycle 1 was defined

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as 100%.

Figure 3: Plasma concentrations of copper, selenium, ceruloplasmin (CP) and selenoprotein P (SELENOP) in control and infected neonates. (A) Copper were slightly elevated in infected neonates at 0 h, as described earlier [22] and differed significantly after 48 h (p<0.0001). (B) Se concentrations and (D) SELENOP concentrations did not differ on average between the two groups, as described [21]. (C) CP plasma concentrations were slightly elevated in infected neonates at 0 h and differed significantly after 48 h (p<0.0001). (E) Plasma copper were 15

significantly elevated in infected neonates at 48 h as compared to the 0 h time point (p=0.0002), as described earlier [22]. (F) CP concentrations were significantly elevated in infected neonates

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at 48 h as compared to the 0 h time point (p<0.0006).

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correlate positively with plasma selenium concentrations, as described earlier [21] (Pearson

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r=0.603, p<0.0001).

Figure 5: Ratio of Cu to CP and Se to SELENOP. (A) The average Cu/CP ratios did not differ

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between the groups (control: 6.0±1.6; infected: 0 h: 6.0±1.6; and 48 h: 4.9±1.0), but the infected neonates showed the lowest quotient after 48 h. (B) The Se/SELENOP ratio was slightly

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reduced in infected neonates at 0 h, and significantly lower (p<0.0043) 48 h after birth (control:

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18.7±4.7; infected: 0 h: 18.3±3.9; and 48 h: 14.5±4.5).

Figure 6: Characterization of neonates in relation to early onset infection. (A) On average (median and IQR), the Cu/Se ratio was significantly increased in infected neonates at 0 h (p<0.0223). Three infected individuals showed particularly increased ratios in comparison to the full groups of neonates. (B) At 0 h the CP/SELENOP ratio did not differ on average. However, the same three patients as in (A) showed again a particularly elevated SELENOP/CP 18

ratio at 0 h, which declined to the 48 h time point. Both, the initially high (C) Cu/Se and (D)

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CP/SELENOP ratios decreased within 48 h.

Figure 7: Correlation analysis of the biomarkers Cu/Se and CP/SELENOP versus IL-6 and CRP in healthy and infected neonates. (A, B) Cu/Se ratios and (C, D) CP/SELENOP ratios

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appeared not to be associated with Il-6. Correlation analysis of (E) Cu/Se and (F) CP/SELENOP yielded a significant positive correlation in relation to CRP concentrations at the 48 h time point

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(Spearman ϱ=0.583, p=0.011 and ϱ=0.571, p=0.013, respectively).

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0.5775±0.09604

(p=0.4207),

CP

0.6725±0.09309

(p=0.0730)

and

CP/SELENOP

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0.6813±0.08928 (p=0.0596).

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Appendix Fig 1: ROC-analysis and calculation of area under curve (AUC): SELENOP

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Tables

Table 1: Group characteristics of neonatal samples available (subset of the children described earlier [21]) Infected Group n=18 n=6 (33%)

p-value (two-sided) -

2486 ± 687 8.4 [4.3-12.5] 9 [7-9] 9 [8-10]

3123 ± 775 12.1 ± 7.9 1983.9 [234.6-3733.3] 8 [4.5-9] 9 [7.5-10]

0.0067 0.0292 n.s n.s

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Participants Preterm infants (gestational age <37 weeks) Birth weight [g] Peak CRP [mg/L] IL-6 [pg/mL], day 1 Apgar 1 min a Apgar 5 min a

Control Group n=19 n=15 (79%)

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a - scoring of Apgar (Appearance, Pulse, Grimace, Activity and Respiration) appearance: 0 – blue, or pale all over; 1 – blue at extremities & body pink, 2 – body & extremities are pink pulse: 0 – absent; 1 – < 100 beats /min; 2 – >100 beats /min grimace: 0 – no response to stimulation; 1 – grimace on (aggressive) stimulation; 2 – cry on stimulation activity: 0 – none; 1 – some flexion; 2 – active motion respiration: 0 – absent; 1 – weak irregular or gasping; 2 – strong or robust cry

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