Acta Pædiatrica ISSN 0803-5253
REGULAR ARTICLE
Citrulline concentration in routinely collected neonatal dried blood spots cannot be used to predict necrotising enterocolitis A Englund1, R a Rogvi1, L Melgaard2, G Greisen ([email protected])1 1.Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark 2.Danish Center for Neonatal Screening, Clinical Mass Spectrometry, Statens Serums Institut, Copenhagen, Denmark
Keywords Citrulline, Dried blood spot, Necrotising enterocolitis, Neonatal Screening Correspondence Gorm Greisen, Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9 2100 Copenhagen Ø, Denmark. Tel: +45-3545-4320 | Fax: +45 35455025 | Email: [email protected] Received 25 March 2014; revised 2 June 2014; accepted 7 July 2014. DOI:10.1111/apa.12750
ABSTRACT Aim: Low citrulline concentration is a marker of low functional enterocyte mass, which may predispose neonates to necrotising enterocolitis (NEC). We hypothesised that citrulline could be used to assess the NEC risk that could not be accounted for by gestational age and birthweight. This study investigated whether citrulline concentrations routinely measured in neonatal dried blood spots (DBS) could predict NEC. Methods: We used national Danish registries to retrospectively identify all 361 babies born between 2003 and 2009 who were diagnosed with NEC and had a valid citrulline concentration measured from a DBS sample. The control group comprised 1083 healthy newborns, with three controls for every newborn with NEC, matched for birthweight and gestational age. Results: Neonatal dried blood spots were collected between 2 and 21 days of life, with a median of 8 days. The results showed that NEC was not associated with low citrulline concentration, either in a direct comparison between the NEC and control groups or in a multivariate logistic regression (p = 0.73). Conclusion: The findings of this study show that the citrulline concentrations found in routine DBS samples between 2003 and 2009 did not predict NEC in newborn babies.
INTRODUCTION Although necrotising enterocolitis (NEC) is a leading cause of severe morbidity and mortality in premature infants (1), the aetiology is poorly understood and the condition is likely to be multifactorial. The predominant risk factor is prematurity, which is associated with immature development of the intestine, causing limited motility, digestive capacity, perfusion reserve and cell-mediated immunity (2,3). Other risk factors include very low birthweight and low Apgar score (3). Citrulline is a nonprotein amino acid, a metabolic intermediate in the urea cycle (4), and is produced mainly in both liver and intestine. However, as hepatic citrulline becomes catabolised in the intrahepatic urea cycle, intestinal production remains the only quantitative source of systemically circulating citrulline (5,6). The hypothesis is that intestinal necrosis and loss of functional enterocyte mass will result in lower circulating citrulline concentration.
A number of studies have reported an association between low citrulline blood concentration and loss of functional enterocyte mass, as seen in short bowel syndrome (7,8) Crohn’s disease (9) and chronic villous atrophy (10). Recent studies have reported a significantly lower plasma citrulline concentration in premature infants diagnosed with NEC than in healthy controls (11,12). Furthermore, one study pointed towards a tendency of lower serum citrulline concentrations in infants diagnosed with NEC compared to controls, although the difference did not reach statistical significance (13). While no studies have focussed on the association between plasma citrulline concentration
Key notes
Abbreviations CODR, Cause of Death Registry; DBS, Dried blood spot; DMBR, Danish Medical Birth Registry; MSMS, Tandem mass spectrometry; NBS, Biobank – Newborn Screening Biobank; NEC, Necrotising enterocolitis; NPR, National Patient Registry.
©2014 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 2014 103, pp. 1143–1147
Low citrulline concentration is a marker of low functional enterocyte mass, which may predispose neonates to necrotising enterocolitis (NEC). We used national Danish registries to compare the citrulline concentrations found in dried blood spot (DBS) samples from 361 newborn babies with diagnosed NEC and 1083 healthy controls. Our findings indicate that the citrulline concentrations found in routine DBS samples between 2003 and 2009 did not predict NEC in newborn babies.
1143
Citrulline cannot predict necrotising enterocolitis
Englund et al.
and gestational age, Celik et al. (12) did not find a correlation between citrulline levels and birthweight. Goossens et al. (14) have described reference values for plasma and dried blood spot (DBS) citrulline concentrations in healthy newborn and children. In Denmark, DBS samples are obtained in the first days of the infant’s life as a screening tool for detecting metabolic disorders in newborns. In this screening process, the citrulline concentration is measured and registered in The Danish Newborn Screening Biobank (NBS-Biobank) held by the Statens Serum Institut (15). The aim of this retrospective database study was to investigate whether citrulline concentrations in neonatal screening DBS could be used to predict NEC.
METHODS Patients Data were extracted in August 2010 from the Danish Medical Birth Registry (DMBR), The National Patient Registry (NPR) and The Cause Of Death Register (CODR) held by The National Board of Health and in February 2013 from the NBS-Biobank. The DMBR provided data on birthweight, gestational age, sex and birth year for all children born in the period from 2003 to 2009. The NPR provided data on all children admitted to a Danish hospital with NEC (primary or secondary ICD-10 diagnosis code P77 or P77.9) as well as all children diagnosed with any congenital kidney disease (ICD10: Q61, Q62, Q63 and Q64 including all subcodes). For each admission, the NPR carries one primary diagnosis code and may hold several secondary codes. All diagnoses codes were included in the analysis. The CODR provided data on time and cause of all fatalities in children born in the period 1 January 2003 to 31
December 2008. The NBS-Biobank provided data on citrulline concentration from DBS from the cases and matched controls as described below. In Denmark, all healthcare information is stored in the National Patient Registry. All registered citizens in Denmark have a Civil Registry System number, which correlate to the information in the National Patient Registry. The data were extracted with regard to the Civil Registry System number of the newborn, with a registered birth in the years from 2003 to 2009. Cases consisted of all children diagnosed with NEC born in the years from 2003 to 2009. Exclusion criteria included birthweight >8 SD or < 8 SD of expected, stillborn children and death within the first 48 h after birth (Fig. 1). Newborns born in the same period, matched for birthweight and gestational age in a ratio of 3:1 were included as controls. Extraction of the registry data is available to scientists, after study approval by the Danish Data Protection Agency, and the study is thus fully reproducible. Blood sampling and citrulline assessment Measurement of citrulline blood concentrations was used as part of the Danish National Neonatal Screening programme on trial basis from 1st of February 2002 to 31st of January 2009 to screen for citrullinemia. Trial participation was optional; in this study, we found a steady rise in coverage from 65% in 2004 to 90% in 2008. Due to the fact that the extended screening was introduced in 2003, only 57–62% of cases and controls had at least one valid citrulline sample. Ultimately, citrullinemia did not become an established part of the routine screening programme as its value depends upon very early detection (16). After January 2009, raw data on citrulline levels were still measured and collected by means of the mass spectrometer
Figure 1 The process of identifying cases and controls.
1144
©2014 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 2014 103, pp. 1143–1147
Englund et al.
kit, although the values were not interpreted as a part of the screening programme, nor reported to clinicians. For the purpose of this study, we did a retrospective extraction of the raw citrulline concentrations. It should be noted that the data originate from screening methods, and that the reliability of the results is only pursued as far as to determine whether or a child has citrullinemia or not. As part of the screening programme, capillary blood was obtained from heel pricks of newborns and dried on filter paper. A spot with a diameter of 3.2 mm was punched, and analytes were extracted in buffer containing deuteriumlabelled internal standards. Detection was then performed by direct infusion tandem mass spectrometry (MSMS). In the period from February 2002 to January 2009, the sample was scheduled for 5–7 day of age, but had to be delayed in case the infant was not yet given milk feeds or were on antibiotics. Analytes were extracted and afterwards derivatised with butanol using PerkinElmers NeoGram MSMS Derivatised AAAC Reagent Kit. A Sciex API 365 mass spectrometer was applied monitoring a neutral loss of 119 Dalton. After January 2009, sampling time was changed to 2–3 day of age, analytes were extracted using NeoBase Non-derivatised MSMS Kit and a Waters Quattro Micro mass spectrometer was used monitoring the transition m/z 176.1 to m/z 113.1. Ethics The data collection for this study was approved by the National Board of Health and the Danish Data Protection Agency. According to Danish legislation, database studies do not normally require approval from a biomedical research ethics committee nor written consent from individuals. Statistics The total cohort was created as listed in Figure 1 and included all children born in the years 2003–2009. Every child diagnosed with NEC was identified and matched in a 3:1 relationship with controls, matched for gestational age and birthweight, using the OneToManyMTCH macro (17). Weight for gestational age SDS scores was calculated using Marsall’s charts of intrauterine growth (18). The mean birthweight, gestational age, SDS score, time of citrulline sampling, citrulline concentration and time of death in days (if applicable) were calculated for cases and controls. The between-group difference was calculated using a two-tailed t-test or chi-square test. A p-value
REGULAR ARTICLE
Citrulline concentration in routinely collected neonatal dried blood spots cannot be used to predict necrotising enterocolitis A Englund1, R a Rogvi1, L Melgaard2, G Greisen ([email protected])1 1.Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark 2.Danish Center for Neonatal Screening, Clinical Mass Spectrometry, Statens Serums Institut, Copenhagen, Denmark
Keywords Citrulline, Dried blood spot, Necrotising enterocolitis, Neonatal Screening Correspondence Gorm Greisen, Department of Neonatology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9 2100 Copenhagen Ø, Denmark. Tel: +45-3545-4320 | Fax: +45 35455025 | Email: [email protected] Received 25 March 2014; revised 2 June 2014; accepted 7 July 2014. DOI:10.1111/apa.12750
ABSTRACT Aim: Low citrulline concentration is a marker of low functional enterocyte mass, which may predispose neonates to necrotising enterocolitis (NEC). We hypothesised that citrulline could be used to assess the NEC risk that could not be accounted for by gestational age and birthweight. This study investigated whether citrulline concentrations routinely measured in neonatal dried blood spots (DBS) could predict NEC. Methods: We used national Danish registries to retrospectively identify all 361 babies born between 2003 and 2009 who were diagnosed with NEC and had a valid citrulline concentration measured from a DBS sample. The control group comprised 1083 healthy newborns, with three controls for every newborn with NEC, matched for birthweight and gestational age. Results: Neonatal dried blood spots were collected between 2 and 21 days of life, with a median of 8 days. The results showed that NEC was not associated with low citrulline concentration, either in a direct comparison between the NEC and control groups or in a multivariate logistic regression (p = 0.73). Conclusion: The findings of this study show that the citrulline concentrations found in routine DBS samples between 2003 and 2009 did not predict NEC in newborn babies.
INTRODUCTION Although necrotising enterocolitis (NEC) is a leading cause of severe morbidity and mortality in premature infants (1), the aetiology is poorly understood and the condition is likely to be multifactorial. The predominant risk factor is prematurity, which is associated with immature development of the intestine, causing limited motility, digestive capacity, perfusion reserve and cell-mediated immunity (2,3). Other risk factors include very low birthweight and low Apgar score (3). Citrulline is a nonprotein amino acid, a metabolic intermediate in the urea cycle (4), and is produced mainly in both liver and intestine. However, as hepatic citrulline becomes catabolised in the intrahepatic urea cycle, intestinal production remains the only quantitative source of systemically circulating citrulline (5,6). The hypothesis is that intestinal necrosis and loss of functional enterocyte mass will result in lower circulating citrulline concentration.
A number of studies have reported an association between low citrulline blood concentration and loss of functional enterocyte mass, as seen in short bowel syndrome (7,8) Crohn’s disease (9) and chronic villous atrophy (10). Recent studies have reported a significantly lower plasma citrulline concentration in premature infants diagnosed with NEC than in healthy controls (11,12). Furthermore, one study pointed towards a tendency of lower serum citrulline concentrations in infants diagnosed with NEC compared to controls, although the difference did not reach statistical significance (13). While no studies have focussed on the association between plasma citrulline concentration
Key notes
Abbreviations CODR, Cause of Death Registry; DBS, Dried blood spot; DMBR, Danish Medical Birth Registry; MSMS, Tandem mass spectrometry; NBS, Biobank – Newborn Screening Biobank; NEC, Necrotising enterocolitis; NPR, National Patient Registry.
©2014 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 2014 103, pp. 1143–1147
Low citrulline concentration is a marker of low functional enterocyte mass, which may predispose neonates to necrotising enterocolitis (NEC). We used national Danish registries to compare the citrulline concentrations found in dried blood spot (DBS) samples from 361 newborn babies with diagnosed NEC and 1083 healthy controls. Our findings indicate that the citrulline concentrations found in routine DBS samples between 2003 and 2009 did not predict NEC in newborn babies.
1143
Citrulline cannot predict necrotising enterocolitis
Englund et al.
and gestational age, Celik et al. (12) did not find a correlation between citrulline levels and birthweight. Goossens et al. (14) have described reference values for plasma and dried blood spot (DBS) citrulline concentrations in healthy newborn and children. In Denmark, DBS samples are obtained in the first days of the infant’s life as a screening tool for detecting metabolic disorders in newborns. In this screening process, the citrulline concentration is measured and registered in The Danish Newborn Screening Biobank (NBS-Biobank) held by the Statens Serum Institut (15). The aim of this retrospective database study was to investigate whether citrulline concentrations in neonatal screening DBS could be used to predict NEC.
METHODS Patients Data were extracted in August 2010 from the Danish Medical Birth Registry (DMBR), The National Patient Registry (NPR) and The Cause Of Death Register (CODR) held by The National Board of Health and in February 2013 from the NBS-Biobank. The DMBR provided data on birthweight, gestational age, sex and birth year for all children born in the period from 2003 to 2009. The NPR provided data on all children admitted to a Danish hospital with NEC (primary or secondary ICD-10 diagnosis code P77 or P77.9) as well as all children diagnosed with any congenital kidney disease (ICD10: Q61, Q62, Q63 and Q64 including all subcodes). For each admission, the NPR carries one primary diagnosis code and may hold several secondary codes. All diagnoses codes were included in the analysis. The CODR provided data on time and cause of all fatalities in children born in the period 1 January 2003 to 31
December 2008. The NBS-Biobank provided data on citrulline concentration from DBS from the cases and matched controls as described below. In Denmark, all healthcare information is stored in the National Patient Registry. All registered citizens in Denmark have a Civil Registry System number, which correlate to the information in the National Patient Registry. The data were extracted with regard to the Civil Registry System number of the newborn, with a registered birth in the years from 2003 to 2009. Cases consisted of all children diagnosed with NEC born in the years from 2003 to 2009. Exclusion criteria included birthweight >8 SD or < 8 SD of expected, stillborn children and death within the first 48 h after birth (Fig. 1). Newborns born in the same period, matched for birthweight and gestational age in a ratio of 3:1 were included as controls. Extraction of the registry data is available to scientists, after study approval by the Danish Data Protection Agency, and the study is thus fully reproducible. Blood sampling and citrulline assessment Measurement of citrulline blood concentrations was used as part of the Danish National Neonatal Screening programme on trial basis from 1st of February 2002 to 31st of January 2009 to screen for citrullinemia. Trial participation was optional; in this study, we found a steady rise in coverage from 65% in 2004 to 90% in 2008. Due to the fact that the extended screening was introduced in 2003, only 57–62% of cases and controls had at least one valid citrulline sample. Ultimately, citrullinemia did not become an established part of the routine screening programme as its value depends upon very early detection (16). After January 2009, raw data on citrulline levels were still measured and collected by means of the mass spectrometer
Figure 1 The process of identifying cases and controls.
1144
©2014 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 2014 103, pp. 1143–1147
Englund et al.
kit, although the values were not interpreted as a part of the screening programme, nor reported to clinicians. For the purpose of this study, we did a retrospective extraction of the raw citrulline concentrations. It should be noted that the data originate from screening methods, and that the reliability of the results is only pursued as far as to determine whether or a child has citrullinemia or not. As part of the screening programme, capillary blood was obtained from heel pricks of newborns and dried on filter paper. A spot with a diameter of 3.2 mm was punched, and analytes were extracted in buffer containing deuteriumlabelled internal standards. Detection was then performed by direct infusion tandem mass spectrometry (MSMS). In the period from February 2002 to January 2009, the sample was scheduled for 5–7 day of age, but had to be delayed in case the infant was not yet given milk feeds or were on antibiotics. Analytes were extracted and afterwards derivatised with butanol using PerkinElmers NeoGram MSMS Derivatised AAAC Reagent Kit. A Sciex API 365 mass spectrometer was applied monitoring a neutral loss of 119 Dalton. After January 2009, sampling time was changed to 2–3 day of age, analytes were extracted using NeoBase Non-derivatised MSMS Kit and a Waters Quattro Micro mass spectrometer was used monitoring the transition m/z 176.1 to m/z 113.1. Ethics The data collection for this study was approved by the National Board of Health and the Danish Data Protection Agency. According to Danish legislation, database studies do not normally require approval from a biomedical research ethics committee nor written consent from individuals. Statistics The total cohort was created as listed in Figure 1 and included all children born in the years 2003–2009. Every child diagnosed with NEC was identified and matched in a 3:1 relationship with controls, matched for gestational age and birthweight, using the OneToManyMTCH macro (17). Weight for gestational age SDS scores was calculated using Marsall’s charts of intrauterine growth (18). The mean birthweight, gestational age, SDS score, time of citrulline sampling, citrulline concentration and time of death in days (if applicable) were calculated for cases and controls. The between-group difference was calculated using a two-tailed t-test or chi-square test. A p-value
