http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2014; 27(17): 1723–1727 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2013.876621
ORIGINAL ARTICLE
Serum ischemia-modified albumin levels at diagnosis and during treatment of late-onset neonatal sepsis
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Selcuk Universitesi on 12/27/14 For personal use only.
F. Hu¨meyra Yerlikaya1, Sevil Kurban1, Idris Mehmetoglu1, Ali Annagur2, Huseyin Altunhan3, Ekrem Erbay1, and Rahmi O¨rs4 1
Department of Biochemistry, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey, 2Selcuklu Medical Faculty, Division of _ Baysal University, Bolu, Turkey, and 4Meram Neonatology, Selcuk University, Konya, Turkey, 3Medical Faculty, Division of Neonatology, Abant Izzet Medical Faculty, Necmettin Erbakan University, Konya, Turkey Abstract
Keywords
Sepsis is one of the most common infectious conditions in the neonatal period, and continues as a major source of morbidity and mortality. The aim of this study is to determine serum ischemia-modified albumin (IMA) levels in late-onset neonatal sepsis at the time of diagnosis and after therapy, and to show the meaningful on the follow-up. Also, it is aimed to compare serum IMA levels with serum C-reactive protein (CRP), procalcitonin (PCT) levels and white blood cell count. The study was performed on 33 premature babies with sepsis and 21 healthy premature controls at 7–28 days of age. In the sepsis group, biochemical parameters and blood culture samples were obtained from the blood at the onset and on the fifth day of treatment for each patient. Serum IMA, CRP, PCT and white blood cell count were significantly higher in the sepsis group before treatment when compared with the control group. In addition, the levels of IMA were positively correlated with white blood cell count, CRP and PCT in the sepsis group before treatment. In conclusion, serum IMA levels may be useful in late-onset neonatal sepsis at the time of diagnosis and after therapy. As far as we know this is the first report about the assesment of illness diagnosis and after therapy using serum IMA levels, and further studies are needed to confirm our results in larger groups of patients.
C-reactive protein, ischemia-modified albumin, late-onset neonatal sepsis, procalcitonin, white blood cell count
Introduction Sepsis is one of the most common infectious conditions in the neonatal period, and continues as a major source of morbidity and mortality [1]. Early neonatal sepsis occurs within the first six days of life, while late neonatal sepsis occurs after the last six days of life [2]. C-reactive protein (CRP), white blood cell count, total neutrophil count and immature to total neutrophil ratio (I/T) are the most widely used test in the diagnosis of neonatal sepsis [3]. Some laboratory parameters, such as interleukin-6 and procalcitonin, are also used to support the diagnosis and determine the severity of neonatal sepsis [4]. All provide useful information, but none of them has been showed to be reliable in detecting whole septic babies [3]. A few studies, the white blood cell count demonstrated a low detection sensitivity in neonatal infection [5,6]. Even the combination of total neutrophil count, I/T and platelet count failed to reach an appropriate sensitivity and specifity in pathology of neonatal sepsis [7]. In several studies, high serum CRP levels have also proved useful as a sepsis biomarker, in spite of the negative Address for correspondence: Dr F. Hu¨meyra Yerlikaya, Department of Biochemistry, Meram Medical Faculty, Necmettin Erbakan University, Biyokimya AD, 42080, Konya, Turkey. Tel: 0 332/2236315. Fax: 0 332/ 2237761. E-mail: [email protected]
History Received 10 January 2013 Accepted 15 December 2013 Published online 30 April 2014
predictive value and sensitivity of CRP testing are not enough to allow definitive diagnosis of the condition [8,9]. These results have led to the search for new markers at the time of diagnosis and after therapy. Ischemia-modified albumin (IMA) which is a specific and sensitive marker for ischemic process results from oxidative damage [10,11]. Ischemia and generation of reactive oxygen species can alter the ability of N-terminal region of the albumin molecule to bind transitional metallic ions such as cobalt, copper and nickel [12,13]. This modification of albumin called IMA is incapable of binding to metallic ions [10,13]. Nowadays, IMA is accepted as a marker of oxidative stress related to ischemia-reperfusion in different clinical conditions associated with oxidative stress, such as chronic kidney disease, hyperlipidemia and diabetes [14–16]. Also, it has been reported that circulating IMA was associated with inflammation markers because, inflammation reduces the capacity of albumin to bind cobalt [13,14]. In view of the possible association of inflammation and IMA, the aim of this study is to determine serum IMA levels in late-onset neonatal sepsis at the time of diagnosis and after therapy and to show the meaningful on the follow up. Also, we aim to compare serum IMA levels with serum CRP, procalcitonin (PCT) levels and white blood cell count.
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Materials and methods The study was performed on premature patients at 7–28 days of age, hospitalized with a diagnosis of late sepsis in Necmettin Erbakan University, Meram Medical Faculty, Newborn Intensive Care Unit (NICU) between September and November 2011. Thirty-three premature babies with sepsis and 21 healthy premature controls were included in the study. The study was approved by the Clinical Investigations Ethics Committee. Informed consents were obtained from the parents. Demographic and clinical characteristics, and the laboratory findings of each patient included in the study were recorded. Babies diagnosed by clinical and laboratory findings as late neonatal sepsis at the late neonatal period (7–28 days) and those under 36th gestational weeks were included in the study. Babies having acute renal injury, dialysis, abnormal karyotype, suspected congenital metabolic disease, cyanotic congenital heart disease, serious congenital malformation, perinatal asphyxia, maternal diabetes, intracranial hemorrhage, suspected and diagnosed necrotizing enterocolitis, those treated with indomethacin, ibuprofen and amphotericin B, history of or a requirement for surgical intervention, and patent ductus arteriosus (PDA) were excluded from the study. The control group was composed of babies born from a healthy mother (less than 36 weeks of gestational age), having no antenatally or postnatally detected renal pathology, having received no antibiotic treatment, having no history of perinatal asphyxia, with no observed congenital anomalies, and having no postpartum disorders. The To¨llner sepsis scoring was performed on all babies included in the study. In the sepsis group, biochemical parameters and blood culture samples were obtained from the blood at the onset and on the fifth day of treatment for each patient. For the control group, blood samples were obtained once during the routine investigations. The samples were stored frozen at 80 C until the day of serum IMA. The other analytes were measured immediately. Measurement of ischemia-modified albumin levels Ischemia-modified albumin level was measured by a colorimetric assay developed by Bar-Or et al. [17] based on measurement of unbound cobalt after incubation with patient serum. Increased amounts of IMA results in less cobalt binding and more residual unbound cobalt available for complex with a chromogen [dithiothreitol (DDT)], which can be measured photometrically. The procedure was as follows: 50 mL of 0.1% cobalt chloride was added to 200 mL of serum, gently mixed, and waited for 10 min for adequate cobaltalbumin binding. Fifty microliters of DTT, at a concentration of 1.5 mg/mL, was added as a colorizing agent and the reaction was stopped 2 min later by adding 1.0 mL of 0.9% NaCl. The colored product was measured at 470 nm and compared to a serum-cobalt blank without DTT and reported in absorbance units (ABSU). Adjusted IMA was calculated as (individual serum albumin concentration/median serum albumin concentration of the population) IMA value. This formula was applied to correct IMA values for serum albumin (median serum albumin concentration of each group of the subjects were used separately) [18].
J Matern Fetal Neonatal Med, 2014; 27(17): 1723–1727
Table 1. Demographic characteristics of babies included in the study*.
Weight (g) Gestational weeks Age (post-natal days) Sex (female/male)
Sepsis group
Control group
p
2845.8 ± 704.6 35.93 ± 2.53 18.03 ± 8.2 14/19
2813.3 ± 650.3 35.90 ± 2.7 20.47 ± 7.6 9/12
0.864 0.963 0.271 0.598
*All values (except gender) are mean ± standart deviations.
Measurement of the other analytes Serum CRP measurements were performed in the Beckman Coulter instrument (Beckman Coulter, Inc., Fullerton, CA) using the DDS commercial kit by immunoturbidimetric method and values over 10 mg/L were accepted as significant. Serum PCT measurement was performed using the chemiluminescence method with the Kryptor instrument with Sensitive-PCT kit (BRAHMS Diagnostica, Berlin, Germany), and values over 0.5 ng/mL were accepted as significant. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total bilirubin and direct bilirubin were measured by commertially available kits based on routine methods on the Synchron LX System (Beckman Coulter, Fullerton, CA). Statistical analysis Statistical analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL). To compare the ratio of categorical variables, we used the chi-squared test. We performed (inter-group comparisons) independent samples t test to compare the difference in the biochemical parameters and demographic characteristics of babies. In addition, intragroup comparisons (before treatment and after treatment) were performed by paired-sample t test. The correlations between variables were tested by Pearson’s correlation test. Finally, multiple linear regression analysis was performed to analyze the effects of various independent variables on the serum IMA levels. All data are expressed as mean ± standard deviations (SD). Differences were considered significant at a probability level of p50.05.
Results There were no statistical differences with regard to demographic characteristics between the groups (Table 1). The biochemical parameters of the babies are presented in Table 2. As seen from the table, white blood cell count, CRP, PCT, IMA and adjusted IMA were significantly higher in the sepsis group before treatment (p50.001 for the other parameteres, p50.01 for IMA, and p50.05 for adjusted IMA) when compared with the control group. While, serum albumin (p50.001) and total bilirubin (p50.01) levels were significantly lower in the sepsis group before treatment when compared with the control group. In addition, serum CRP (p50.01) and PCT (p50.05) levels were significantly higher in the sepsis group after treatment when compared with the control group. In intra-group comparison, white blood cell count, CRP, PCT, total bilirubin, IMA and adjusted IMA were significantly higher in the sepsis group before treatment
Ischemia-modified albumin and neonatal sepsis
DOI: 10.3109/14767058.2013.876621
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Table 2. Laboratory findings in the sepsis group before and after treatment and the control groups*. Sepsis group
3
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White blood cell count (/mm ) CRP (mg/L) PCT (ng/mL) AST (U/L) ALT (U/L) Albumin (g/dL) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) IMA (ABSU) Adjusted IMA (ABSU)
Before treatment
After treatment
Control group
*p
**p
***p
17393.0 ± 9665.3 33.77 ± 14.18 44.05 ± 31.6 44.54 ± 15.2 31.42 ± 15.2 3.04 ± 0.3 4.46 ± 3.0 0.39 ± 0.2 1.47 ± 0.25 1.47 ± 0.35
10787.0 ± 2218.4 3.99 ± 1.9 0.11 ± 0.1 41.12 ± 14.7 32.18 ± 14.7 3.68 ± 0.2 2.35 ± 1.6 0.26 ± 0.1 1.25 ± 0.25 1.26 ± 0.25
9906.2 ± 3087.8 2.61 ± 1.4 0.04 ± 0.03 41.52 ± 13.5 28.14 ± 12.7 3.58 ± 0.3 2.72 ± 1.2 0.36 ± 0.2 1.23 ± 0.36 1.23 ± 0.37
50.001 50.001 50.001 0.450 0.398 50.001 0.005 0.604 0.016 0.031
0.265 0.005 0.027 0.918 0.292 0.195 0.351 0.071 0.812 0.804
p50.001 p50.001 p50.001 0.278 0.850 p50.001 p50.001 0.002 0.005 0.010
CRP, C-reactive protein; PCT, procalcitonin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; IMA, ischemia-modified albumin; *p, before treatment sepsis group compared with control group; **p, after treatment sepsis group compared with control group; ***p, before treatment sepsis group compared with after treatment sepsis group. *All values (except gender) are mean ± standard deviations.
Figure 1. Correlations between (A) IMA and CRP (r ¼ 0.465, p50.01), (B) IMA and PCT (r ¼ 0.404, p50.05), (C) IMA and white blood cell count (r ¼ 0.527, p50.01).
(p50.001 for the other parameteres, p50.01 for IMA, and p50.05 for adjusted IMA) when compared with the sepsis group after treatment. While, serum albumin (p50.001) and direct bilirubin (p50.01) levels were significantly lower
in the sepsis group before treatment when compared with the sepsis group after treatment. Simple correlation analysis were performed to investigate the association of serum IMA levels with white blood
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cell count, CRP and PCT. As shown in Figure 1, the levels of IMA were positively correlated with white blood cell count (r ¼ 0.527, p50.01), CRP (r ¼ 0.465, p50.01) and PCT (r ¼ 0.404, p50.05) in the sepsis group before treatment. On the other hand, there were no correlations between serum IMA levels with white blood cell count, CRP and PCT in the sepsis group after treatment and the control group. A subsequent multiple linear regression analysis revealed that there remained a significant and positive correlation between the dependent variable being IMA and white blood cell count in the sepsis group before treatment ( ¼ 0.459, p ¼ 0.012).
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Discussion Neonatal sepsis, a life-threatening condition, remains a major problem in neonates admitted to the neonatal intensive care units, with high morbidity and mortality rates in spite of advances in treatment and supportive cares, especially in developing countries [19]. Early recognition and treatment of the disease are vital to improve the outcome [3]. The quest for a highly sensitive and specific ideal marker in this disease is challenge for healthcare professional [2]. The results of the present study indicate that serum IMA levels increase in the sepsis group compared to the control group. Also, this is the first report demonstrating the decrease of serum IMA levels after treatment in the sepsis group. In a study of adults, serum IMA levels of patients with severe sepsis had increased compared to the healthy control subjects [20]. The result of this study supports our results. Reactive oxygen species (ROS), are chemically reactive molecules containing oxygen, are produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms, which remove them via antioxidative systems [21]. Normal human albumin has the N-terminal region. The N-terminal region is also the area to which such transition metals as cobalt, copper and nickel are bound. The generation of ROS damage the N-terminal region. Albumin’s capacity to bind metals such as nickel, cobalt and copper is diminished. The resultant albumin as such is IMA [22]. Sepsis produce systemic tissue damage that is probably due to the result from widespread inflammation and following endothelial injury [23]. It has been reported that ROS play a significant role in the pathogenesis of neonatal sepsis and its complications [24]. The inflammatory response to critical illness involves the activation of leukocytes and other inflammatory cells resulting in the overproduction of ROS [23]. The oxidative stress caused by ROS has been implicated in apoptotic cell death and in turn, can be harmful to the patient when the antioxidant defense systems are not effective [25]. The dysregulation of the inflammatory response that occurs during sepsis may be due to changes in the apoptotic program [24]. The consequences of these oxidative processes during sepsis may be ongoing cell damage that culminates in multisystem organ failure, often leading to death [23,24]. On the other hand, we have found high-serum CRP levels and low-serum albumin levels in the sepsis group before treatment compared to the control group. It has been reported that this is often observed in inflammatory processes due to a discrete acute phase pesponse resulting in the decrease
J Matern Fetal Neonatal Med, 2014; 27(17): 1723–1727
of albumin synthesis by the liver in favor of acute phase proteins [14]. In addition, we have found a positive correlation between serum IMA and hsCRP levels in the sepsis group before treatment. Also, there are several reports indicating association between IMA and hsCRP [20,26,27]. As a result of these findings, a lower albumin level may lead to a larger amount of free cobalt available to react with DTT resulting in increasing ABSU in the IMA assay [14]. In this study, significantly positive correlation between IMA and PCT found in the sepsis group before treatment. PCT is produced predominantly by monocytes and hepatocytes [28]. It may be used to monitor the activity and prognosis of severe bacterial infections [29]. The concentration of PCT began to rise within 4 h after the patients exposure to bacterial endotoxin, and peaked at 6–8 h and decreased to normal levels after receiving effective treatment within 24 h [4]. Indeed, it has observed the decrease in serum PCT and IMA levels after treatment in this study. Also, this correlation which is found in the sepsis group before treatment not found in the control group and the sepsis group after treatment. Addition of CRP and PCT, hematological parameters such as white blood cell count are used in identifying infected newborn and there is the suggestion that serial normal values can be reassuring for noninfection [30]. However, some studies have shown that screening white blood cell count are poorly predictive of infection in neonates [31,32]. The possible sources of heterogeneity in the published studies were population, age, whether the subjects were at term or preterm, methodological quality, different leukocyte indices, different cutoff values, and interpretation of test results by different laboratory observers [7]. In our study, white blood cell count in the sepsis group before treatment were significantly higher than those of the control group and positively correlated with IMA levels in this group. In conclusion, serum IMA levels may be useful in lateonset neonatal sepsis at the time of diagnosis and after therapy. As far as we know this is the first report about the assesment of illness diagnosis and after therapy using serum IMA levels, and further studies are needed to confirm our results in larger groups of patients.
Declaration of interest The authors stated that there are no conflict of interest regarding the publication of this article.
References 1. Campos DP, Silva MV, Machado JR, et al. Early-onset neonatal sepsis: cord blood cytokine levels at diagnosis and during treatment. J Pediatr (Rio J) 2010;86:509–14. 2. Valerio TA, Cancelier CA, Constantino L, et al. Inflammatory and oxidative cord blood parameters as predictors of neonatal sepsis severity. Rev Bras Ter Intensiva 2012;24:30–4. ¨ rs R, Mehmetog˘lu I. Procalcitonin 3. Altunhan H, Annagu¨r A, O measurement at 24 hours of age may be helpful in the prompt diagnosis of early-onset neonatal sepsis. Int J Infect Dis 2011;15: 854–8. 4. Zhang Y, Xue C, Zhu T, et al. Serum bone alkaline phosphatase in assessing ıllness severity of ınfected neonates in the neonatal ıntensive care unit. Int J Biol Sci 2012;8:30–8.
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DOI: 10.3109/14767058.2013.876621
5. Kite P, Millar MR, Gorham P, Congdon P. Comparison of five tests used in diagnosis of neonatal bacteraemia. Arch Dis Child 1988;63: 639–43. 6. Manroe BL, Weinberg AG, Rosenfeld CR, Browne R. The neonatal blood count in health and disease. I. Reference values for neutrophilic cells. J Pediatr 1979;95:89–98. 7. Kurt AN, Aygun AD, Godekmerdan A, et al. Serum IL-1beta, IL-6, IL-8, and TNF-alpha levels in early diagnosis and management of neonatal sepsis. Mediators Inflamm 2007;2007:31397. doi: 10.1155/2007/31397. 8. Gerdes JS. Clinicopathologic approach to the diagnosis of neonatal sepsis. Clin Perinatol 1991;18:361–81. 9. Caldas JP, Marba ST, Blotta MH, et al. Accuracy of white blood cell count, C-reactive protein, interleukin-6 and tumor necrosis factor alpha for diagnosing late neonatal sepsis. J Pediatr (Rio J) 2008;84:536–42. 10. Dursun A, Okumus N, Zenciroglu A. Ischemia-modified albumin (IMA): could it be useful to predict perinatal asphyxia? J Matern Fetal Neonatal Med 2012;25:2401–5. 11. Gugliucci A, Hermo R, Monroy C, et al. Ischemia-modified albumin levels in cord blood: a case-control study in uncomplicated and complicated deliveries. Clin Chim Acta 2005;362:155–60. 12. Chan B, Dodsworth N, Woodrow J, et al. Site-specific N-terminal auto-degradation of human serum albumin. Eur J Biochem 1995; 227:524–8. 13. Zuwała-Jagiełło J, Warwas M, Pazgan-Simon M. Ischemiamodified albumin (IMA) is increased in patients with chronic hepatitis C infection and related to markers of oxidative stress and inflammation. Acta Biochim Pol 2012;59:661–7. 14. Kaefer M, Piva SJ, De Carvalho JA, et al. Association between ischemia modified albumin, inflammation and hyperglycemia in type 2 diabetes mellitus. Clin Biochem 2010;43:450–4. 15. Mehmetoglu I, Yerlikaya FH, Kurban S, et al. Oxidative stress markers in hemodialysis and peritoneal dialysis patients, including coenzyme Q10 and ischemia-modified albumin. Int J Artif Organs 2012;35:226–32. 16. Kotani K, Caccavello R, Sakane N, et al. Influence of ezetimibe monotherapy on ischemia-modified albumin levels in hypercholesterolemic patients. Pharmacol Rep 2011;63: 1248–51. 17. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt-albumin binding and its potential as marker its potential as a marker for myocardial ischemia-A preliminary report. J Emer Med 2000;19: 311–15.
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18. Lippi G, Montagnana M, Salvagno GL, Guidi GC. Standardization of ischaemia-modified albumin testing: adjustment for serum albumin. Clin Chem Lab Med 2007;45:261–2. 19. Thaver D, Zaidi AK. Burden of neonatal infections in developing countries: a review of evidence from community-based studies. Pediatr Infect Dis J 2009;28:3–9. 20. Erdem SS, Yerlikaya FH, C ¸ ic¸ekler H, Gu¨l M. Association between ischemia-modified albumin, homocysteine, vitamin B 12 and folic acid in patients with severe sepsis. Clin Chem Lab Med 2012;50: 1417–21. 21. Gutteridge JM, Halliwell B. Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 2000; 899:136–47. 22. Chan B, Dodsworth N, Woodrow J, et al. Sitespecific N-terminal auto-degradation of human serum albumin. Eur J Biochem 1995; 227:524–8. 23. Taylor DE, Piantadosi CA. Oxidative metabolism in sepsis and sepsis syndrome. J Crit Care 1995;10:122–35. 24. Cherian S, Jameson S, Rajarajeswari C, et al. Oxidative stress in sepsis in children. Indian J Med Res 2007;125:143–8. 25. Batra S, Kumar R, Kapoor AK, Ray G. Alterations in antioxidant status during neonatal sepsis. Ann Trop Paediatr 2000;20:27–33. 26. Kaefer M, Piva SJ, De Carvalho JA, et al. Association between ischemia modified albumin, inflammation and hyperglycemia in type 2 diabetes mellitus. Clin Biochem 2010;43:450–4. 27. Duarte MM, Rocha JB, Moresco RN, et al. Association between ischemia-modified albumin, lipids and inflammation biomarkers in patients with hypercholesterolemia. Clin Biochem 2009;42:666–71. 28. Buhaescu I, Yood RA, Izzedine M. Serum procalcitonin in systemic autoimmune diseases – where are we now? Semin Arthritis Rheum 2010;40:176–83. 29. Ertug˘rul S, Annagur A, Kurban S, et al. Comparison of urinary neutrophil gelatinase-associated lipocalin, C-reactive protein and procalcitonin in the diagnosis of late onset sepsis in preterm newborns. J Matern Fetal Neonatal Med 2013;26:430–3. 30. Greenberg DN, Yoder BA. Changes in the differential white blood cell count in screening for group B streptococcal sepsis. Pediatr Infect Dis J 1990;9:886–9. 31. Ottolini MC, Lundgren K, Mirkinson LJ, et al. Utility of complete blood count and blood culture screening to diagnose neonatal sepsis in the asymptomatic at risk newborn. Pediatr Infect Dis J 2003;22: 430–4. 32. Christensen RD, Rothstein G, Hill HR, Hall RT. Fatal early onset group B streptococcal sepsis with normal leukocyte counts. Pediatr Infect Dis 1985;4:242–5.
ORIGINAL ARTICLE
Serum ischemia-modified albumin levels at diagnosis and during treatment of late-onset neonatal sepsis
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Selcuk Universitesi on 12/27/14 For personal use only.
F. Hu¨meyra Yerlikaya1, Sevil Kurban1, Idris Mehmetoglu1, Ali Annagur2, Huseyin Altunhan3, Ekrem Erbay1, and Rahmi O¨rs4 1
Department of Biochemistry, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey, 2Selcuklu Medical Faculty, Division of _ Baysal University, Bolu, Turkey, and 4Meram Neonatology, Selcuk University, Konya, Turkey, 3Medical Faculty, Division of Neonatology, Abant Izzet Medical Faculty, Necmettin Erbakan University, Konya, Turkey Abstract
Keywords
Sepsis is one of the most common infectious conditions in the neonatal period, and continues as a major source of morbidity and mortality. The aim of this study is to determine serum ischemia-modified albumin (IMA) levels in late-onset neonatal sepsis at the time of diagnosis and after therapy, and to show the meaningful on the follow-up. Also, it is aimed to compare serum IMA levels with serum C-reactive protein (CRP), procalcitonin (PCT) levels and white blood cell count. The study was performed on 33 premature babies with sepsis and 21 healthy premature controls at 7–28 days of age. In the sepsis group, biochemical parameters and blood culture samples were obtained from the blood at the onset and on the fifth day of treatment for each patient. Serum IMA, CRP, PCT and white blood cell count were significantly higher in the sepsis group before treatment when compared with the control group. In addition, the levels of IMA were positively correlated with white blood cell count, CRP and PCT in the sepsis group before treatment. In conclusion, serum IMA levels may be useful in late-onset neonatal sepsis at the time of diagnosis and after therapy. As far as we know this is the first report about the assesment of illness diagnosis and after therapy using serum IMA levels, and further studies are needed to confirm our results in larger groups of patients.
C-reactive protein, ischemia-modified albumin, late-onset neonatal sepsis, procalcitonin, white blood cell count
Introduction Sepsis is one of the most common infectious conditions in the neonatal period, and continues as a major source of morbidity and mortality [1]. Early neonatal sepsis occurs within the first six days of life, while late neonatal sepsis occurs after the last six days of life [2]. C-reactive protein (CRP), white blood cell count, total neutrophil count and immature to total neutrophil ratio (I/T) are the most widely used test in the diagnosis of neonatal sepsis [3]. Some laboratory parameters, such as interleukin-6 and procalcitonin, are also used to support the diagnosis and determine the severity of neonatal sepsis [4]. All provide useful information, but none of them has been showed to be reliable in detecting whole septic babies [3]. A few studies, the white blood cell count demonstrated a low detection sensitivity in neonatal infection [5,6]. Even the combination of total neutrophil count, I/T and platelet count failed to reach an appropriate sensitivity and specifity in pathology of neonatal sepsis [7]. In several studies, high serum CRP levels have also proved useful as a sepsis biomarker, in spite of the negative Address for correspondence: Dr F. Hu¨meyra Yerlikaya, Department of Biochemistry, Meram Medical Faculty, Necmettin Erbakan University, Biyokimya AD, 42080, Konya, Turkey. Tel: 0 332/2236315. Fax: 0 332/ 2237761. E-mail: [email protected]
History Received 10 January 2013 Accepted 15 December 2013 Published online 30 April 2014
predictive value and sensitivity of CRP testing are not enough to allow definitive diagnosis of the condition [8,9]. These results have led to the search for new markers at the time of diagnosis and after therapy. Ischemia-modified albumin (IMA) which is a specific and sensitive marker for ischemic process results from oxidative damage [10,11]. Ischemia and generation of reactive oxygen species can alter the ability of N-terminal region of the albumin molecule to bind transitional metallic ions such as cobalt, copper and nickel [12,13]. This modification of albumin called IMA is incapable of binding to metallic ions [10,13]. Nowadays, IMA is accepted as a marker of oxidative stress related to ischemia-reperfusion in different clinical conditions associated with oxidative stress, such as chronic kidney disease, hyperlipidemia and diabetes [14–16]. Also, it has been reported that circulating IMA was associated with inflammation markers because, inflammation reduces the capacity of albumin to bind cobalt [13,14]. In view of the possible association of inflammation and IMA, the aim of this study is to determine serum IMA levels in late-onset neonatal sepsis at the time of diagnosis and after therapy and to show the meaningful on the follow up. Also, we aim to compare serum IMA levels with serum CRP, procalcitonin (PCT) levels and white blood cell count.
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J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Selcuk Universitesi on 12/27/14 For personal use only.
Materials and methods The study was performed on premature patients at 7–28 days of age, hospitalized with a diagnosis of late sepsis in Necmettin Erbakan University, Meram Medical Faculty, Newborn Intensive Care Unit (NICU) between September and November 2011. Thirty-three premature babies with sepsis and 21 healthy premature controls were included in the study. The study was approved by the Clinical Investigations Ethics Committee. Informed consents were obtained from the parents. Demographic and clinical characteristics, and the laboratory findings of each patient included in the study were recorded. Babies diagnosed by clinical and laboratory findings as late neonatal sepsis at the late neonatal period (7–28 days) and those under 36th gestational weeks were included in the study. Babies having acute renal injury, dialysis, abnormal karyotype, suspected congenital metabolic disease, cyanotic congenital heart disease, serious congenital malformation, perinatal asphyxia, maternal diabetes, intracranial hemorrhage, suspected and diagnosed necrotizing enterocolitis, those treated with indomethacin, ibuprofen and amphotericin B, history of or a requirement for surgical intervention, and patent ductus arteriosus (PDA) were excluded from the study. The control group was composed of babies born from a healthy mother (less than 36 weeks of gestational age), having no antenatally or postnatally detected renal pathology, having received no antibiotic treatment, having no history of perinatal asphyxia, with no observed congenital anomalies, and having no postpartum disorders. The To¨llner sepsis scoring was performed on all babies included in the study. In the sepsis group, biochemical parameters and blood culture samples were obtained from the blood at the onset and on the fifth day of treatment for each patient. For the control group, blood samples were obtained once during the routine investigations. The samples were stored frozen at 80 C until the day of serum IMA. The other analytes were measured immediately. Measurement of ischemia-modified albumin levels Ischemia-modified albumin level was measured by a colorimetric assay developed by Bar-Or et al. [17] based on measurement of unbound cobalt after incubation with patient serum. Increased amounts of IMA results in less cobalt binding and more residual unbound cobalt available for complex with a chromogen [dithiothreitol (DDT)], which can be measured photometrically. The procedure was as follows: 50 mL of 0.1% cobalt chloride was added to 200 mL of serum, gently mixed, and waited for 10 min for adequate cobaltalbumin binding. Fifty microliters of DTT, at a concentration of 1.5 mg/mL, was added as a colorizing agent and the reaction was stopped 2 min later by adding 1.0 mL of 0.9% NaCl. The colored product was measured at 470 nm and compared to a serum-cobalt blank without DTT and reported in absorbance units (ABSU). Adjusted IMA was calculated as (individual serum albumin concentration/median serum albumin concentration of the population) IMA value. This formula was applied to correct IMA values for serum albumin (median serum albumin concentration of each group of the subjects were used separately) [18].
J Matern Fetal Neonatal Med, 2014; 27(17): 1723–1727
Table 1. Demographic characteristics of babies included in the study*.
Weight (g) Gestational weeks Age (post-natal days) Sex (female/male)
Sepsis group
Control group
p
2845.8 ± 704.6 35.93 ± 2.53 18.03 ± 8.2 14/19
2813.3 ± 650.3 35.90 ± 2.7 20.47 ± 7.6 9/12
0.864 0.963 0.271 0.598
*All values (except gender) are mean ± standart deviations.
Measurement of the other analytes Serum CRP measurements were performed in the Beckman Coulter instrument (Beckman Coulter, Inc., Fullerton, CA) using the DDS commercial kit by immunoturbidimetric method and values over 10 mg/L were accepted as significant. Serum PCT measurement was performed using the chemiluminescence method with the Kryptor instrument with Sensitive-PCT kit (BRAHMS Diagnostica, Berlin, Germany), and values over 0.5 ng/mL were accepted as significant. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total bilirubin and direct bilirubin were measured by commertially available kits based on routine methods on the Synchron LX System (Beckman Coulter, Fullerton, CA). Statistical analysis Statistical analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL). To compare the ratio of categorical variables, we used the chi-squared test. We performed (inter-group comparisons) independent samples t test to compare the difference in the biochemical parameters and demographic characteristics of babies. In addition, intragroup comparisons (before treatment and after treatment) were performed by paired-sample t test. The correlations between variables were tested by Pearson’s correlation test. Finally, multiple linear regression analysis was performed to analyze the effects of various independent variables on the serum IMA levels. All data are expressed as mean ± standard deviations (SD). Differences were considered significant at a probability level of p50.05.
Results There were no statistical differences with regard to demographic characteristics between the groups (Table 1). The biochemical parameters of the babies are presented in Table 2. As seen from the table, white blood cell count, CRP, PCT, IMA and adjusted IMA were significantly higher in the sepsis group before treatment (p50.001 for the other parameteres, p50.01 for IMA, and p50.05 for adjusted IMA) when compared with the control group. While, serum albumin (p50.001) and total bilirubin (p50.01) levels were significantly lower in the sepsis group before treatment when compared with the control group. In addition, serum CRP (p50.01) and PCT (p50.05) levels were significantly higher in the sepsis group after treatment when compared with the control group. In intra-group comparison, white blood cell count, CRP, PCT, total bilirubin, IMA and adjusted IMA were significantly higher in the sepsis group before treatment
Ischemia-modified albumin and neonatal sepsis
DOI: 10.3109/14767058.2013.876621
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Table 2. Laboratory findings in the sepsis group before and after treatment and the control groups*. Sepsis group
3
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White blood cell count (/mm ) CRP (mg/L) PCT (ng/mL) AST (U/L) ALT (U/L) Albumin (g/dL) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) IMA (ABSU) Adjusted IMA (ABSU)
Before treatment
After treatment
Control group
*p
**p
***p
17393.0 ± 9665.3 33.77 ± 14.18 44.05 ± 31.6 44.54 ± 15.2 31.42 ± 15.2 3.04 ± 0.3 4.46 ± 3.0 0.39 ± 0.2 1.47 ± 0.25 1.47 ± 0.35
10787.0 ± 2218.4 3.99 ± 1.9 0.11 ± 0.1 41.12 ± 14.7 32.18 ± 14.7 3.68 ± 0.2 2.35 ± 1.6 0.26 ± 0.1 1.25 ± 0.25 1.26 ± 0.25
9906.2 ± 3087.8 2.61 ± 1.4 0.04 ± 0.03 41.52 ± 13.5 28.14 ± 12.7 3.58 ± 0.3 2.72 ± 1.2 0.36 ± 0.2 1.23 ± 0.36 1.23 ± 0.37
50.001 50.001 50.001 0.450 0.398 50.001 0.005 0.604 0.016 0.031
0.265 0.005 0.027 0.918 0.292 0.195 0.351 0.071 0.812 0.804
p50.001 p50.001 p50.001 0.278 0.850 p50.001 p50.001 0.002 0.005 0.010
CRP, C-reactive protein; PCT, procalcitonin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; IMA, ischemia-modified albumin; *p, before treatment sepsis group compared with control group; **p, after treatment sepsis group compared with control group; ***p, before treatment sepsis group compared with after treatment sepsis group. *All values (except gender) are mean ± standard deviations.
Figure 1. Correlations between (A) IMA and CRP (r ¼ 0.465, p50.01), (B) IMA and PCT (r ¼ 0.404, p50.05), (C) IMA and white blood cell count (r ¼ 0.527, p50.01).
(p50.001 for the other parameteres, p50.01 for IMA, and p50.05 for adjusted IMA) when compared with the sepsis group after treatment. While, serum albumin (p50.001) and direct bilirubin (p50.01) levels were significantly lower
in the sepsis group before treatment when compared with the sepsis group after treatment. Simple correlation analysis were performed to investigate the association of serum IMA levels with white blood
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cell count, CRP and PCT. As shown in Figure 1, the levels of IMA were positively correlated with white blood cell count (r ¼ 0.527, p50.01), CRP (r ¼ 0.465, p50.01) and PCT (r ¼ 0.404, p50.05) in the sepsis group before treatment. On the other hand, there were no correlations between serum IMA levels with white blood cell count, CRP and PCT in the sepsis group after treatment and the control group. A subsequent multiple linear regression analysis revealed that there remained a significant and positive correlation between the dependent variable being IMA and white blood cell count in the sepsis group before treatment ( ¼ 0.459, p ¼ 0.012).
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Discussion Neonatal sepsis, a life-threatening condition, remains a major problem in neonates admitted to the neonatal intensive care units, with high morbidity and mortality rates in spite of advances in treatment and supportive cares, especially in developing countries [19]. Early recognition and treatment of the disease are vital to improve the outcome [3]. The quest for a highly sensitive and specific ideal marker in this disease is challenge for healthcare professional [2]. The results of the present study indicate that serum IMA levels increase in the sepsis group compared to the control group. Also, this is the first report demonstrating the decrease of serum IMA levels after treatment in the sepsis group. In a study of adults, serum IMA levels of patients with severe sepsis had increased compared to the healthy control subjects [20]. The result of this study supports our results. Reactive oxygen species (ROS), are chemically reactive molecules containing oxygen, are produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms, which remove them via antioxidative systems [21]. Normal human albumin has the N-terminal region. The N-terminal region is also the area to which such transition metals as cobalt, copper and nickel are bound. The generation of ROS damage the N-terminal region. Albumin’s capacity to bind metals such as nickel, cobalt and copper is diminished. The resultant albumin as such is IMA [22]. Sepsis produce systemic tissue damage that is probably due to the result from widespread inflammation and following endothelial injury [23]. It has been reported that ROS play a significant role in the pathogenesis of neonatal sepsis and its complications [24]. The inflammatory response to critical illness involves the activation of leukocytes and other inflammatory cells resulting in the overproduction of ROS [23]. The oxidative stress caused by ROS has been implicated in apoptotic cell death and in turn, can be harmful to the patient when the antioxidant defense systems are not effective [25]. The dysregulation of the inflammatory response that occurs during sepsis may be due to changes in the apoptotic program [24]. The consequences of these oxidative processes during sepsis may be ongoing cell damage that culminates in multisystem organ failure, often leading to death [23,24]. On the other hand, we have found high-serum CRP levels and low-serum albumin levels in the sepsis group before treatment compared to the control group. It has been reported that this is often observed in inflammatory processes due to a discrete acute phase pesponse resulting in the decrease
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of albumin synthesis by the liver in favor of acute phase proteins [14]. In addition, we have found a positive correlation between serum IMA and hsCRP levels in the sepsis group before treatment. Also, there are several reports indicating association between IMA and hsCRP [20,26,27]. As a result of these findings, a lower albumin level may lead to a larger amount of free cobalt available to react with DTT resulting in increasing ABSU in the IMA assay [14]. In this study, significantly positive correlation between IMA and PCT found in the sepsis group before treatment. PCT is produced predominantly by monocytes and hepatocytes [28]. It may be used to monitor the activity and prognosis of severe bacterial infections [29]. The concentration of PCT began to rise within 4 h after the patients exposure to bacterial endotoxin, and peaked at 6–8 h and decreased to normal levels after receiving effective treatment within 24 h [4]. Indeed, it has observed the decrease in serum PCT and IMA levels after treatment in this study. Also, this correlation which is found in the sepsis group before treatment not found in the control group and the sepsis group after treatment. Addition of CRP and PCT, hematological parameters such as white blood cell count are used in identifying infected newborn and there is the suggestion that serial normal values can be reassuring for noninfection [30]. However, some studies have shown that screening white blood cell count are poorly predictive of infection in neonates [31,32]. The possible sources of heterogeneity in the published studies were population, age, whether the subjects were at term or preterm, methodological quality, different leukocyte indices, different cutoff values, and interpretation of test results by different laboratory observers [7]. In our study, white blood cell count in the sepsis group before treatment were significantly higher than those of the control group and positively correlated with IMA levels in this group. In conclusion, serum IMA levels may be useful in lateonset neonatal sepsis at the time of diagnosis and after therapy. As far as we know this is the first report about the assesment of illness diagnosis and after therapy using serum IMA levels, and further studies are needed to confirm our results in larger groups of patients.
Declaration of interest The authors stated that there are no conflict of interest regarding the publication of this article.
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