AHMED EL-SHAHAT, M.D.* and ESRAA M.F. EL-SHAHAT, M.D.**

The Department of Plastic & Reconstructive Surgery* and Clinical Pathology**, Ain Shams University.

ABSTRACT

The role of serum neopterin to predict multiorgan failure (MOF) and death in burned patient is a matter of controversy. This study is an attempt to reevaluate its role. Serum samples of 29 burned patients were collected on the 1st, Ycl and 7th post burn days and tested for neopterin. Patients were divided into two groups based on survival to test neopterin as a prognostic marker. Plasma neopterin levels at Ist, 3'-d and 7th post burn days and plasma neopterin delta change between Yd and I St post burn days (Neopterin A 1 k) and between 7th and Yd post burn days (Neopterin A2-1-1) were used to differentiate between the two groups. Delta changes were used to compare between the two groups based on follow up results rather than results at fixed time point. Wilcoxon's rank sum test and multi variant analysis were used for statistical analysis. There was no significant difference in plasma neopterin levels between survivors and non-survivors. But, non-survived patients had significantly high Neopterin dl (p<0.05) and the significance increased when combined with percentage of burn (p<0.01).

Conclusion: Whereas the absolute plasma neopterin level per se is not effective as a prognostic marker, Neopterin-Al can predict development of MOF and death.

INTRODUCTION

  Neopterin is a low molecular weight pteridine compound. Pteridines were first isolated from a biological material in 1889 [1]. But, Neopterin was first discovered in the larvae of bees in 1963 [2].

 It was then discovered in humans in 1967, when isolated from urine [3]. Neopterin was observed to accumulate in supernatant of cultured human peripheral blood mononuclear cells upon antigenic stimulation in 1983 [4]. Numerous investigations performed in vitro and in vivo support the view that neopterin biosynthesis is closely associated with activation of the cellular immune system [5].

 Stimulated T -lymphocytes produce a soluble factor (gamma interferon) that triggers neopterin formation by monocyteslmacrophages [6].

 Neopterin biosynthesis in macrophages in inflammatory state might be caused exclusively by elevated levels of endogenous Interferon Gamma, which was directly related to the extent of systemic Tlymphocyte activation [7,8]. In the setting of acute thermal injury, stimulation of this T lymphocytemacrophage axis could be affected by different antigenic stimuli, such as the bacterial endotoxin (lipopolysaccharides; LPS), lipid protein complex or denatured collagen in the burn eschar [9-11].

 Although gamma interferon is the most potent inducer of neopterin synthesis in human cells, the production of neopterin is co-stimulated by tumor necrosis factor-alpha (TNFoc) [12]. On the other hand, neopterin stimulates TNF-a, gene expression, which enhances TNF-a synthesis [13,14].

 The interaction of inflammatory mediators like TNFa, and neopterin contributes to an excessive release of nitric oxide (NO), the development of septic shock as well as related cellular destruction and multiple organ failure in humans [14].

 Induction of inducible nitric oxide (NO) synthase (iNOS) expression by neopterin was demonstrated in rat vascular smooth muscle cells with a concomitant increase in NO production and NO release [15]. Hoffmann et al. [16] put an explanation for the pathway of iNOS stimulation by neopterin.

 Neopterin, as a pro-oxidant, may also induce oxidative stress causing apoptotic cell death, or superinduce tumor necrosis factor-a mediated apoptosis by destabilizing the equilibrium between reactive oxygen intermediates and antioxidants [17-19].

 Induible Nitric Oxide Synthase (iNOS) - derived nitric oxide (NO) generates peroxynitrite (ON00-) within the burn wound [2()]. This may have further implications for its pathophysiology at both a molecular and functional level peroxynitr ite (ONOO-) can cause DNA strand breakage [21] and lipid peroxtdation [22] and this hers the potential to induce cellular dysfunction and secondary tissue datuaee under such circumstances.

 A close relationship between the intensity of lipid peroxidation and complications alter burns has been demonstrated [231. It documents the role of oxygen free radicals leading to lipid peroxidation as a causative agent in the mechanism of local wound response, development of burn shock and distant organ injury [23].

 Neopterin serum level can be measured by EnzymeLinked ImmunoSorbent Assay (ELISA), Radtoimmunoassay (RIA) [25J and high Performance Liquid Chromatography (HPLC) [26,27]. Serum neopterin concentration of l0nmol/L is accepted as the upper normal limit [28].

 Some authors found that it was hardly possible to differentiate survivors from non-survivors among burned patients by means of difference in neopterin .Serum levels [11,291. On the other hand, other authors 1301 found that the mean scrum neopterin was higher in nonsurvivors compared with sursisors. Therefore, the value of using senior neopterin levels to predict mufti-organ failme and mortality related to thermal injury still requires further evaluation.

MATERIAL AND METHODS

1- Patient Groups:

  The study- was carried out at the burn unit of Ain Shams University Hospitals. The local ethical comtntnee appros ed the study. Consents were taken from the patients or their guardians before inclusion in the study. The study was conducted on thirtyone patients of both genders, who were admitted to the hurn unit in the period between October 2003 and October 2004 with total burned surface area ('ITSA) ranging from 9 to 95% (35.9ą21°%%). Two patients died on the Ist and 5th days of admission before collecting the whole samples and were excluded from the study. The patients were classified into two groups based on the survival to test neopterin as a prognostic marker. Survivors were defined as patients discharged from the hospital alive (they were 15 and the age mean ssas 24.5=/-18 years); non-sm-vivors were patients w llo died in the hospital because of any post burn Complications (they were 14 and the age mean was 26.5=/-I 1 years). Ten healthy solunteer individuals were included as control for the nonnal serum neopterin level.

2- Methods:

A- Clinical Assessment: Flistory taking and through physical examination %v ere done. Determination of the percentage of burn ('fBSA) was done. based on Lttnd-Browder chart [311. Percentage of burn was used Lis a co-variable (in IPUIll-\ arrant analysis) to differentiate between survivors and non-survivors.

B- Blood Samples: Fiee Milliliters of Venous blood were collected under aseptic prccautrons at the IM, 3Td and 7111 post-burn days. Samples were left in plain tubes to allow coagulation. and then centrifuged at 1500xg for 15 minutes. The separated serum was divided into two ttliquols, whtclt were stored at -70°C. One aliquote was used lot serum creatinine assay and the other for the assay of scrum neopterin. Repeated thawing and freezing was ttvoidcd.

C-Analytical Methods: Neopterin was assayed by competitive cnzytne linked immunosorbart assay (ELISA) method using reagent supplied by immunobiological laboratories (IBL), Hamburg. Germany (4990-5328-9111). Plasma neopterin lescls at Ist, 3rd and 701 post burn days. plasma neopterin delta change between 3rd and Ist post burn days (Neopterin AI) and plasma neopterin delta change between 7th and 31d post burn days (Neopterin A2) ssere used to differentiate bettyeen the two groups. Delta changes were used to compare between the two groups based on follow up results rather than results at fixed time point.

Assay Procedure: Standard and serum samples were added to rrricroplate wells coated w ith capture monoclonal antineopterin antibody. After incuhation and washing steps, biotin-labeled polyclonal anLihuman neopterin antibody solution was added for further incubation time. The plate was covered with black foil at room temperatme (20-25°C) on an orbital shaker. Following final washing steps, hydrogen peroxide tetramcthylbenzidine was incubated into the \sells, resulting in a colored product. After the addition of oxidic stop solution. the color Ns as measured at 450mn within 15 minutes. the absorbance and color intensity were considered proportional to the concentration of ileopterin in the sample. A standard otuve was determined with a (cast detectable limit of 4nmol/L [32].

Serum creatinine assay was done on synchron CXS Delta autoanaly,er applying a modified rate Jaffe method [331. Serum creatintne values at 1°C 3rd and 7111 post burn days w ere used to differentiate between survivors and nott-survivors.

D- Statistical Methods: Statistical analysis was performed using statistical soft ware package "SPSS" version 9.05. Data were expressed as meanąSD (standard deviation). Statistical comparison between the recorded mean values of the different variables in studied group was done using the Wilcoxon's rank sum test and the multi-variant analysis. The correlation between variables was tested by Sperman's correlation coefficient. The level of significance at p=0.05 was selected. Values >0.05 were considered nonsignificant. Values <0.05 are considered significant. Values <0.01 or 0.001 were considered highly significant.

RESULTS

  The means and standard deviation for Neopterin and Creatinine at different time period for both groups are shown in Tables (1,2). The means and standard deviation for Neopterin Al & A2 and percentage of burn are shown in Table (3). The significance in difference between groups regarding each variable is shown in the footnote of each table. The mean serum neopterin level of the ten healthy controls was 8.2ą2.2nmol, which was not significantly different from the values in all patients within the first day after burn injury (p>0.05). Serum Neopterin level increased in most patients on the 3rd post-burn day. There was no significant difference in plasma neopterin levels between survivors and non-survivors at any of the three tested time periods (1St, 3rd or 7th post burn days). Non-survived patients had significantly high Neopterin Al (p

  There was no significant difference between the two groups as regard serum creatinine value at any tested time.

  Only one of non-survivors had TBSA <30% (=25°0). The death occurred after 40 days of burn and the cause was pulmonary embolism. The other causes of death in non-survivors were septicemic shock, single (excluding renal) or multiple organ failure. Trials to treat these complications failed.

DISCUSSION

 A major cause of immune aberrations following trauma (burn) is an increase in circulating bacterial endotoxin (341. This occurs even in the absence of Gramnegative bacterial sepsis and in burns victims these alterations are related to the extent of the injury X35). Clinical observation demonstrates that circulating endotoxin is definitely associated with adverse outcome such as development of MOF after severe burns [361].

 Burn patients are known to be endotoxaernia both initially, (owing to bacterial translocation or the passage of endotoxin across the gut barrier) and late owing to wound sepsis or other sepsis [35,37,38]. In case of the study done by Yao et al. [36]. Two peaks of circulating endotoxin were identified in patients who developed MOF, the first was observed on the third day after injury and the second in 2-3 weeks post-burn.

 Both in vitro and in vivo studies have shown that endotoxin directly augmented neopterin production from macrophages and that it was also an effective inducer of neopterin biosynthesis under conditions in which the participation of interferon gamma was excluded [9,1o].

 Increased neopterin concentrations are observed in diseases with intensified monocytelmacrophage activity. Neopterin measurements in body fluids provide information on a current state of cellular immune response and frequently help predict disease progression. The release of neopterin begins 3 days before T cells proliferation reaches maximum, and an increase in neopterin production can be observed about one week before the appearance of specific antibodies. Therefore, neopterin may be of clinical use as an early inflammation marker 1281. Neopterin concentrations are found to increase in infectious diseases, burn, malignancies, autoimmune diseases, heart and kidney failure, coronary artery disease and allograft rejection [39].

 Although neopterin is used as a marker of general macrophage activation, and cytokines are known to rise promptly after burn injury, a significant elevation in neopte'rin was not seen within 24 hours post-burn (111. The delayed increase in neopterin might be due to two reasons.. The first is that the strongest inducer of Neopterin, interferon gamma appears relatively late both in vivo and in vitro studies [40,411. The second is due to that the key enzyme of the tetrahydrobiopterin pathway, cyclohydrolase I, is synthesized de novo upon stimulation 1421. In our study we started collecting samples after the Ist 24 hours. But still at the Ist post burn day, neopterin level showed no significant difference from the values in the healthy control.

 Neopterin as other pteridine derivatives is sensitive to photo-decomposition, therefore protection of sample from direct sun light is necessary as mentioned by Fuchs et al. [5]. This can be achieved by covering the sample tubes by tin foil. Neopterin has no catabolism and clearance is through the kidney. Hence biological half-life depends solely on renal clearance 143]. No neopterin receptor is known and it does not undergo extravasation, This makes it a good indicator of the amount of interferon gamma produced [39].

 There was no need to correlate neopterin to creatinine in this current study because there was no renal impairment in any patient included in the study at any of the three tested post burn days and there was no significant difference in serum creatinine between the two groups at any tested time.

 In this current study the role of neopterin change rather than isolated values was important in predicting mortality. There was a significantly higher neopterin data change between 3rd and Ist days in case of nonsurvivors than in survivors (p<0.05). Neopterin delta change between 3rd and I st post burn days >1.5nmollL can predict MOF and death.

 The significance became higher (p<0.01) when the percentage of burn was added as an associated variable (using multivariate analysis). Neopterin delta change between 3rd and I St post burn days > I .Onmol/L combined with % burn >36% can predict MOF and death.

 These findings allow us to predict as early as the 3rd post burn day the possibility of developing mufti organ failure and death unless aggressive intervention is done. This intervention may include: (1) Prevention of hypoperfusion that is followed by ischemia reperfusion injury, which produce free oxygen radicals, (2) early excision of burn eschar to remove the lipid protein complexes, and (3) early feeding to protect the gut mucosal barrier together with protection against infection, which produce endotoxins (the lipopolysaccharide of gram negative bacterial cell wall).

REFERENCES

1. Haavik J.: From butterflies to neurobiology and the diagnosis of AIDS, 1889. The 100th anniversary of the discovery of pteridines. Tidsskrift for den Norske Laegeforening, 109: 1986-1989 (abstract). Quoted from Berdowska A. and Zwirska-Korczala K.: Neopterin measurement in clinical diagnosis. J. Clin. Pharm. Therap., 26: 319-329, 2001.
2. Rernbold H. and Buschmann L.: Struktur and Synthese des Neopterins. Chem. Ber., 96: 1406-10, 1963. Quoted from Fuchs D., Weiss G., Reibnegger G. and Wachter H.: The role of Neopterin as a Monitor of Cellular Immune Activation in Transplantation, Inflammatory, Infectious and Malignant Diseases. Crit. Rev. Clin. Lab. Sci., 29 (3,4): 307-341, 1992.
3. Sakurai A. and Goto M.: Neopterin: isolation from human urine. J. Biochem. (Tokyo), 61: 142-5, 1967. Quoted from Fuchs D., Weiss G., Reibnegger G. and Wachter H.: The role of Neopterin as a Monitor of Cellular Immune Activation in Transplantation, Inflammatory, Infectious and Malignant Diseases. Crit. Rev. Clin. Lab. Sci., 29 (3,4): 307-341, 1992.
4. Huber C., Fuchs D., Hausen A., et al.: Pteridines as a new marker to detect human T cells activated by allogenie or modified self major histocompatibility complex (MHC) determinants. J. Immunol., 130: 1047-50, 1983.
5. Fuchs D., Weis G., Reibnegger G. and Wachter H.: The role of Neopterin as a Monitor of Cellular Immune Activation in Transplantation, Inflammatory, Infectious and Malignant Diseases. Crit. Rev. Clin. Lab. Sci., 29 (3,4): 307-34 t, 1992.
6. Huber C., Batchelor J.R., Fuchs D., et al.: Immune responseassociated production of neopterin. Release from macrophages primarily under control of interferon gamma. J. Exp. Med., 160: 310-6, 1984.
7. Schoedon G., Troppmair J., Adolf D., et al.: Interferongamma enhances biosynthesis of pteridines in peripheral blood mononuclear cells by induction of GTPcyclohydrase I activity. J. Interferon Res., 6: 697-703, 1986.
8. Fuchs D., Hansen A., Reibnegger G., et al.: Neopterin as a marker of activated cell-mediated immunity. Immunol. Today, 9: 150-5, 1988.
9. Troppmair J., Nachbaur K., Herold M., et al.: In vitro and in vivo studies on the induction of neopterin biosynthesis by cytokines, alloantigens and lipopolysaccharide (LPS). Clin. Exp. Immunol., 74: 392-7, 1988.
10. Bloom J.N., Suffredini A.F., Parrillo J.E. and Palestine A.C.: Serum neopterin levels following intravenous en dotoxin administration to normal humans. Immunobiology, 181: 31723, 1990.
11. Yao Y.M., Yu Y., Wang YP., et al.: Elevated serum neopterin level: its relation to endotoxaemia and sepsis in patients with major burns. Eur. J. Clin. Invest., 26: 224-230, 1996.
12. Werner E.R., erner-Felmayer G., Fuchs D., et al.: Biochemistry and function of pteridine synthesis in human and murine macrophages. Pathobiology, 59: 276-279, 1991.
13. Barak M. and Gruener N.: Neopterin augmentation of tumor necrosis factor production. Irnmunol. Lett., 30: 101-106, 1991.
14. Hoffmann G., Frede S., Kenn S., et al.: Neopterin-induced tumor necrosis factor-a synthesis in vascular smooth muscle cells in vitro. International Archives of Allergy and Immunology, 116: 240-245, 1998.
15. Schobersberger W., Hoffmann G., Grote J., et al.: Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells. FEBS Lett., 377: 461464, 1995.
16. Hoffmann G., Schobersberger W., Frede S., et al.: Neopterin activates transcription factor nuclear factor-KB in vascular smooth muscle cells. FEBS Lett., 391: 181-184, 1996.
17. Baier-Bitterlich G., Fuchs D. and Wachter H.: Chronic immune stimulation, oxidative stress and prognosis in HIV infection. Biochern. Pharmacol., 53: 755-763, 1997.
18. Schobersberger W., Hoffmann G., Hobisch-Hagen P., et al.: Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2. FEBS Lett., 397: 263-268, 1996.
19. Fuchs D., Gruber A., Uberall F. and Wachter H.: Oxidative stress and apoptosis. Immunol. Today, 15: 496, 1994.
20. Rawlingson A., Grenacre S.A. and Brain S.D.: Functional significance of iNOS induction, neutrophil accumulation and protein nitration in the thermally injured rat cutaneous microvasculature. Am. J. Pathol., 162 (4): 1373-80, 2003.
21. Epe B., Ballmaier D., Roussyn I., et al.: DNA damage by peroxynitrite characterized with DNA repair enzymes. Nucleic Acids Res., 24 (21): 4105-10, 1996.
22. Radi R., Beckman J.S., Bush K.M. and Freeman B.A.: Peroxynitrite induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys., 288 (2): 481-7, 1991.
23. Saez J., Ward P., Ganther B. and Vivaldi E.: Superoxide radical involvement in the pathogenesis of burn shock. Circ. Shock, 12: 229239, 1984.
24. Oldham K.T., Gwice K.S., Till G.O. and Ward P.A.: Activation of complernent by hydroxyl radical in thermal injury. Surgery, 104: 272-9, 1988.
25. William J., Saad N., Salib M., et al.: The acute effect of intravenously administered recombinant human erythropoietin on the immune response of uremic patients maintained on regular hemodialysis. Artificial Organs, 22: 192-196, 1998.
26. Werner E.R., Bichler A., Daxenbichler G., et al.: Determination of neopterin in serum and urine. Clinical Chemistry, 33: 62-66, 1987.
27. Weiss G., Kronberger P., Conard F., et al.: Neopterin and prognosis in patients with adenocarcinorna of the colon. Cancer Research, 53: 260265, 1993.
28. Millner M.M., Franthal W., Thalhamrner G.H., et al.: Neopterin concentrations in cerebrospinal fluid and serum as an aid in differentiating central nervous system and peripheral infections in children. Clinical Chemistry, 44: 161-167, 1998.
29. Balogh D., Lamrner H., Kornberger P., et al.: Neopterin plasma levels in burn patients. Burns, 18: 185-8, 1992.
30. Grabosch A. and Rokos H.: Neopterin as parameter of cell-mediated immunity response in thermally injured patients. Burns, 18: 113-6, 1992.
31. Hammond J.S. and Ward C.G.: Transfers from emergency room to burn center: error in burn size estimate. J. Trauma, 27:1161, 1987.
32. Westermann J., Thiemann F., Gerstner L., et al.: Evaluation of a new simple and rapid enzyme linked immunosorbant assay kit for neopterin determination. Clin. Chem. Lah. Med., 38: 345-353, 2000.
33. Vasiliades J.: Reaction of alkaline sodium picrate with creatinine: kinetics and mechanism of formation of the mono-creatinine picric acid complex. Clin. Chern., 22,: 1664-1671, 1976.
34. Munster A.M., Winchurch R.A., Thupari J.N. and Ernest C.B.: Reversal of post burn immuno-suppression with low dose of polymyxin B. J. Traurna, 26: 995-8, 1986.
35. Winchurch R.A., Thupari J.N. and Munster A.M.: Endotoxemia in burn patients: levels of circulating endotoxin are related to burn size. Surgery, 102: 808-12, 1987.
36. Yao Y.M., Bahrami S., Leichtfried G., et al.: Pathogenesis of hemorrhageinduced bacterial/endotoxin translocation in rats: effect of recombinant bactericidal/permeabilityincreasing protein. Ann. Surg., 221: 398-405, 1995.
37. Dobke M.K., Sirrroni J., Ninnemann J.L., et al.: Endotoxemia after burn injury: effect of early excision on circulating endotoxin levels. J. Burn Car. Rehabil., 10: 1071I, 1989.
38. Deitch E.A. and Berg R.: Bacterial translocation from the gut: mechanism of infection. J. Burn Car. Rehabil., 8: 475-82, 1987.
39. Berdowska A. and Zwirska-Korczala K.: Neopterin measurement in clinical diagnosis. J. Clin. Pharm. Therap., 26: 319-329, 2001.
40. Robin B., Fuchs D., Koller W. and Wachter H.: Course of immune activation markers in patients after severe multiple trauma. Pteridines, 2: 95-7, 1990.
41. Hendseron D.C., Sheldon J., Riches P. and Hobbs J.R.: Cytokine induction of neopterin production. Clin. Exp. Immunol., 83: 479-82, 1991.
42. Werner E.R., Werner-Felmayer G., Fuchs D., et al.: Tetrahydrobiopterin biosynthesis activities in human macrophages, fibroblast, THP-1 and T24 cells. J. Biol. Chem., 265: 3189-92, 1990.
43. Roumen R.M.H., Red] H., Schlag G., et al.: Inflammatory mediators in relation to the development of multiple organ failure in patients after severe blunt trauma. Crit. Care Med., 23 (3): 474-480, 1995.

*Neopterin dl = (Neopterin Yd day-Neopterin Ist day)/ Neopterin Ist day.
**Neopterin 02 = (Neopterin 7th day-Neopterin Yd day)/ Neopterin Yd day.