Neopterin concentrations in blood donors differ between AB0 blood group phenotypes

Clinical Biochemistry 38 (2005) 916 – 919

Neopterin concentrations in blood donors differ between AB0 blood group phenotypes Christian Murr a, Katharina Schroecksnadel a,b, Diether Scho¨nitzer c, Dietmar Fuchs a,b,*, Harald Schennach c a

Division of Biological Chemistry, Biocentre, Innsbruck Medical University, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria b Ludwig Boltzmann Institute for AIDS Research, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria c Central Institute of Blood Transfusion, University Clinic Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria Received 2 May 2005; received in revised form 9 June 2005; accepted 6 July 2005 Available online 8 August 2005

Abstract Objectives: Neopterin is produced by human monocyte-derived macrophages upon stimulation with interferon-g and is therefore a sensitive indicator for cellular immune activation. Common factors like age, diastolic blood pressure, body mass index, or smoking habits were found to be associated with neopterin concentrations in humans. Design and methods: In order to find possible genetic determinants which might influence neopterin production, we investigated 8288 consecutive blood donors after exclusion of samples suspicious of infections. Results: Donors with blood group phenotype 0 had moderately, but significantly ( P < 0.0001) higher neopterin concentrations (mean T SD: 6.94 T 1.52 nmol/L) than those with phenotype A (6.75 T 1.50 nmol/L), phenotype B (6.73 T 1.48 nmol/L), and phenotype AB (6.68 T 1.57 nmol/L). Conclusions: Neopterin levels are higher in donors with blood group phenotype 0 than in other phenotypes. Data point to a genetic background of different neopterin concentrations. However, alterations of neopterin levels were much less expressed than the changes known to occur during diseases with an activated immune response. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Neopterin; AB0 blood group phenotypes; Immune activation; Blood donors

Introduction Neopterin is produced by human monocyte-derived macrophages upon stimulation with cytokine interferon-g [1]. Measurement of neopterin concentrations in body fluids like serum or urine has been established as a sensitive method to monitor activation of cellular, Th1 cell-driven immune activation [2]. In humans, increased neopterin production is found during infections with viruses including * Corresponding author. Division of Biological Chemistry, Biocentre, Innsbruck Medical University, and Ludwig Boltzmann Institute for AIDS Research, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria. Fax: +43 512 507 2865. E-mail address: [email protected] (D. Fuchs).

human immunodeficiency virus (HIV), infections with intracellular bacteria and parasites, autoimmune diseases, malignant tumor diseases and in allograft rejection episodes, and also in some neurodegenerative and in cardiovascular diseases [2 – 9]. From several studies in healthy controls, it is evident that factors like older age or body mass index are positively associated with neopterin concentrations, whereas diastolic blood pressure and the number of daily smoked cigarettes are negatively associated with neopterin concentrations [10 – 13]. In order to find possible genetic determinants which might influence neopterin production, we investigated consecutive blood donors and compared their serum neopterin concentrations to AB0 blood group phenotypes. Additional neopterin testing of blood donations is manda-

0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.07.002

C. Murr et al. / Clinical Biochemistry 38 (2005) 916 – 919

tory for all Austrian blood transfusion services in order to improve infectious safety of blood donations, in addition to testing for HIV-1/-2 antibodies, hepatitis C virus (HCV) antibodies, HCV RNA, hepatitis B virus (HBV) surface antigen, alanine aminotransferase (ALT), and Treponema pallidum antibodies [14].

Methods Samples of 9081 consecutive blood donors from the Austrian Tyrol were collected from July to August 2004. Blood samples were drawn by venous puncture during blood donation. The blood was allowed to clot at room temperature, and serum was obtained by centrifugation at 3220  g for 15 min. All analyses except PCR testing were performed within 1 day after blood collection. For exclusion of infections hazardous to blood recipients, in all cases serum antibodies against HIV-1 and -2, HCV, T. pallidum, and HBV surface antigen were determined. Absence of viral RNA or DNA (HCV, HBV, hepatitis A virus, HIV-1, Parvovirus B19) was demonstrated by PCR pool testing. Serum neopterin was measured by a commercially available ELISA (ELItest Neopterin, BRAHMS Diagnostica, Berlin, Germany) with a sensitivity of 2 nmol/L neopterin and an interassay coefficient of variation ranging from 3.9 to 8.2%. Upper limits of the normal (95th percentiles) for neopterin concentrations depend on age, ranging from 8.7 nmol/L (19 –75 years) to 13.5 nmol/L (below 19 years) and 19.0 nmol/L (above 75 years), as described earlier [2]. According to results of previous studies [14], all donors with neopterin concentrations above 11.0 nmol/L were excluded from transfusion and also from this study, in order to exclude possible infections. ALT activity was measured by a method optimized according to the criteria of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) at 37-C with a Dade Dimension (AR autoanalyzer and commercial kits (Dade Behring, Marburg, Germany). Individuals with ALT above 86 U/L were regarded as suspicious for hepatic infections and their donations were therefore also excluded from transfusion. AB0 blood group phenotypes were determined from blood samples containing EDTA for anticoagulation by the DiaMed ID-Micro Typing System (DiaMed AG, Cressier sur Morat, Switzerland), which uses a sephadex gel contained in microtubes, that are either neutral or impregnated with specific antibodies.

917

and 4% phenotype AB. Neopterin concentrations increased with age (Bravais– Pearson correlation: r = +0.2597; 95% confidence interval: 0.2395 to 0.2797; P < 0.0001), and females had slightly higher neopterin concentrations (mean T SD: 6.91 T 1.53 nmol/L) than males (6.77 T 1.51 nmol/L; Student’s t test: t = 4,107; P < 0.0001). As calculated by one-way analysis of variance (ANOVA), those with blood group phenotype 0 had higher neopterin concentrations (mean SD: 6.94 T 1.52 nmol/L) than those with phenotype A (6.75 T 1.50 nmol/L), phenotype B (6.73 T 1.48 nmol/L), and phenotype AB (6.68 T 1.57 nmol/L; F = 12.469; P < 0.0001; Fig. 1). Therefore, no significant difference of means between phenotype A and B, A and AB, or B and AB could be detected by the Bonferroni post hoc test. To test the relationship between AB0 blood group phenotypes, serum neopterin concentrations, age, and sex, the effect of AB0 blood group phenotypes and age and sex on serum neopterin concentrations was calculated by threeway ANOVA using the program SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL). Thereby, the factor age was dichotomized by the median of the observed distribution. Since variances in the 16 subgroups formed on the basis of AB0 blood group phenotypes, age and sex were different, a reciprocal transformation of neopterin concentrations was done before analysis. All three factors, namely, AB0 blood group phenotypes ( F = 0.23; P < 0.0001), age ( F = 157.43; P < 0.0001), and sex ( F = 16.11; P = 0.0001) showed an effect on neopterin concentrations. All interaction terms (AB0 blood group phenotypes vs. age, AB0 blood group phenotypes vs. sex, and age vs. sex) were statistically not significant, denoting that interactions are negligible. This is in line with the assumption that all three factors, namely, AB0 blood group phenotypes, age, and sex, showed an independent effect on neopterin concentrations.

Discussion Significantly higher neopterin concentrations with older age observed in this retrospective study of apparently healthy

Results About 8.7% of the blood donors were excluded from statistical analysis because of positive test results. From the remaining 8288 study subjects (4829 males, 3459 females) with a median age of 41.4 years (interquartile range: 32.4 – 50.5, range: 18.0 – 65.6 years), 45% had blood group phenotype 0, 41% were phenotype A, 10% phenotype B,

Fig. 1. Mean values and standard error of the mean (SEM) of serum neopterin concentrations in relationship to AB0 blood group phenotypes (*P < 0.01, compared to blood group phenotype 0).

918

C. Murr et al. / Clinical Biochemistry 38 (2005) 916 – 919

blood donors are in good agreement with the previous studies in this field [10 –13,15]. The background of this phenomenon is still unclear; possibly the higher incidence of developing disease processes is associated with immune activation and therefore there is increased neopterin production in the elderly and atherosclerosis [8], dementia [9], or autoimmune disorders may contribute to higher reference values. Data could in fact indicate that in some of the individuals of the reference population, pathological processes have already started but are clinically not yet detectable. In the elderly, the population of CD28 negative CD8+ T lymphocytes increases with older age and was found to correlate with higher neopterin concentrations [16]. In a population at risk for atherosclerosis, individuals with wild type toll-like receptor 4 presented with higher neopterin concentrations than those with toll-like receptor 4 polymorphisms Asp299Gly or THr399Ile, which are associated with impaired lipopolysaccharide signaling in the innate immune response [17]. It is also known that lipopolysaccharide from gram-negative bacteria may induce neopterin production in vitro, possibly via stimulation of toll-like receptors [18]. Likewise, in healthy individuals seropositive for Helicobacter pylori, higher neopterin concentrations were found compared to seronegatives [19]. From these findings, one may conclude that higher neopterin concentrations could result from exposure to E. coli, and possibly other gut bacteria, which may represent a stronger challenge for the different arms of the innate and adaptive immune system in individuals with blood group phenotype 0. Higher neopterin concentrations could indicate a higher degree of macrophage activation, higher baseline interferon-g formation, and greater risk of developing inflammation and oxidative stress in individuals with blood group phenotype 0. Higher neopterin concentrations in otherwise healthy individuals were found to be associated not only with a higher risk of developing coronary heart disease, but were also claimed to indicate an increased risk of cancer [20,21]. Whether these risk categories in some way are associated with different blood group phenotypes remains speculative. Already among the first observations in immunology, it was argued that the naturally occurring antibodies against AB0 blood group determinants, such as anti-A or anti-B isoantibodies, would be of heterogenetic microbial origin [22]. Thus, typical intestinal bacteria like E. coli present surface antigens similar to antigen A and B, and, by feeding blood group-specific bacteria, an increase of such isoantibodies could be shown [23]. Higher neopterin concentrations due to bacterial challenge seem plausible, if intestinal bacteria like E. coli evoke less strong immune activation in individuals with blood group phenotype A, B, or AB than in those with blood group phenotype 0. The frequency of distribution of AB0 blood group phenotypes in our study compares well with literature data for central Europe [24]. Moderately but significantly higher neopterin concentrations of individuals with blood group

phenotype 0 compared to those with blood group phenotype A, B, or AB were found. Various investigations concerning AB0 blood group phenotypes and their possible relation to infectious diseases have been performed during the past decades [25]. Thereby, for example, a higher incidence of urinary tract infection in women with blood group phenotype B was described [26]. Nevertheless, by comparing studies, quite a lot of partly contradicting findings can be found [25]. The fact that many of those investigations were done with smaller populations compared to our study could be important, as the difference of neopterin concentration between blood group phenotypes in our study was rather small in absolute terms, and also the relative frequency of blood group B and AB was low compared to that of blood group 0 and A. From our study, we conclude that large data sets are required to find a reliable statistically significant association between blood group phenotypes and certain infectious diseases. Higher neopterin levels in donors with blood group phenotype 0 point to a genetic background of this difference. Notably, alterations of neopterin levels were much less expressed than changes known to occur during diseases with an activated immune response.

Acknowledgment This work was supported by the Austrian Federal Ministry of Social Affairs and Generations.

References [1] Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al. Immune response-associated production of neopterinR Release from macrophages primarily under control of interferon gamma. J Exp Med 1984;160:310 – 6. [2] Murr C, Widner B, Wirleitner B, Fuchs D. Neopterin as a marker for immune system activation. Curr Drug Metab 2002;3:175 – 87. [3] Fuchs D, Hausen A, Reibnegger G, Werner ER, Dierich MP, Wachter H. Neopterin as a marker for activated cell-mediated immunity: application in HIV infection. Immunol Today 1988;9:150 – 5. [4] Fuchs D, Weiss G, Reibnegger G, 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 1992; 29:307 – 41. [5] Murr C, Bergant A, Widschwendter M, Heim K, Schro¨cksnadel H, Fuchs D. Neopterin is an independent prognostic variable in females with breast cancer. Clin Chem 1999;45:1998 – 2004. [6] Samsonov MY, Tilz GP, Egorova O, Reibnegger G, Balabanova RM, Nassonov EL, et al. Serum soluble markers of immune activation and disease activity in systemic lupus erythematosus. Lupus 1995; 4:29 – 32. [7] Reibnegger G, Aichberger C, Fuchs D, Hausen A, Spielberger M, Werner ER, et al. Posttransplant neopterin excretion in renal allograft recipients: reliable diagnostic aid of acute rejection and predictive marker of long-term survival. Transplantation 1991;52:58 – 63. [8] Weiss G, Willeit J, Kiechl S, Fuchs D, Jarosch E, Oberhollenzer F, et al. Increased concentrations of neopterin in carotid atherosclerosis. Atherosclerosis 1994;106:263 – 71.

C. Murr et al. / Clinical Biochemistry 38 (2005) 916 – 919 [9] Leblhuber F, Walli J, Demel U, Tilz GP, Widner B, Fuchs D. Increased serum neopterin concentrations in patients with Alzheimer’s disease. Clin Chem Lab Med 1999;37:429 – 31. [10] Reibnegger G, Huber LA, Ju¨rgens G, Scho¨nitzer D, Werner ER, Wachter H, et al. Approach to define normal aging in man. Immune function, serum lipids, lipoproteins and neopterin levels. Mech Ageing Dev 1988;46:67 – 82. [11] Diamondstone LS, Tollerud DJ, Fuchs D, Wachter H, Brown LM, Maloney E, et al. Factors influencing serum neopterin and h2microglobulin levels in healthy diverse population. J Clin Immunol 1994;14:368 – 74. [12] Ledochowski M, Murr C, Widner B, Fuchs D. Association between insulin resistance, body mass and neopterin concentrations. Clin Chim Acta 1999;282:115 – 23. [13] Schennach H, Murr C, Ga¨chter E, Mayersbach P, Scho¨nitzer D, Fuchs D. Factors influencing serum neopterin levels in a population of blood donors. Clin Chem 2002;48:643 – 5. [14] Schennach H, Mayersbach P, Scho¨nitzer D, Fuchs D. Additional neopterin screening to improve safety of blood donations. Pteridines 2000;11:76 – 80. [15] Ledochowski M, Murr C, Ja¨ger M, Fuchs D. Dehydroepiandrosterone, ageing and immune activation. Exp Gerontol 2001;36:1739 – 47. [16] Murr C, Hainz U, Asch E, Berger P, Jenewein B, Saurwein-Teissl M, et al. Increased neopterin concentration in older age coincides with decline of CD28+ CD45RA+ T-cells. Pteridines 2004;15: 170 – 4. [17] Kiechl S, Lorenz E, Reindl M, Wiedermann CJ, Oberhollenzer F, Bonora E, et al. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002;347:185 – 92.

919

[18] Werner-Felmayer G, Baier-Bitterlich G, Fuchs D, Hausen A, Murr C, Reibnegger G, et al. Detection of bacterial pyrogens on the basis of their effects on gamma interferon-mediated formation of neopterin or nitrite in cultured monocyte cell lines. Clin Diagn Lab Immun 1995;2:307 – 13. [19] Ledochowski M, Widner B, Fuchs D. Helicobacter pylori and chronic immune activation. Am Heart J 2000;139:925 – 6. [20] Bayram M, Bayram O, Boyunaga H, Ozer G. A research on the level of urine neopterin to see if it may provide a vital clue for a provisional diagnosis of breast cancer in menopausal women. Maturitas 2004; 48:432 – 7. [21] Bayram M, Boyunaga H, Diribas K, Ozer G, Akgul EO, Erbil MK. The detection of urinal neopterin concentration increases the efficiency of cervical smear in the diagnosis of cervical cancer. Acta Med (Hradec Kralove) 2004;47:125 – 8. [22] Wiener AS. Origin of naturally occurring hemagglutinins and hemolysins; a review. J Immunol 1951;66:287 – 95. [23] Springer GF, Horton RE. Blood group isoantibody stimulation in man by feeding blood group-active bacteria. J Clin Invest 1969;48:1280 – 91. [24] Kretschmer V, Sonneborn H-H. Blutgruppenantigene und-antiko¨rper. In: Lothar T, editor. Labor und Diagnose. Frankfurt/Main, Germany’ TH-Books; 1998. p. 896 – 955. [25] Berger SA, Young NA, Edberg SC. Relationship between infectious diseases and human blood type. Eur J Clin Microbiol Infect Dis 1989;8(8):681 – 9. [26] Ziegler T, Jacobsohn N, Fu¨nfstu¨ck R. Correlation between blood group phenotype and virulence properties of Escherichia coli in patients with chronic urinary tract infection. Int J Antimicrob Agents 2004;24(Suppl 1):70 – 5.