Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 29

Pteridines Vol. 21, 2010, pp. 29 - 65

Abstracts

Twenty-ninth International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines

Held in St.Christoph, Arlberg, Austria, February 28th - March 7th, 2010 Organized by D.Fuchs (Innsbruck), G.Reibnegger (Graz), A. Griesmacher (Innsbruck)

"Mild Encephalitis" - Brain-organic Patholo- es: streptococcal infection associated autoim- gies Between Neurologic and Psychiatric mune disorder; HSV 1,2 infection associated Disease? autoimmune disorder respectively early state of systemic lupus erythematodes; undefined sys- Bechter K temic autoimmune disorder associated with Sjögren syndrom; suggestive chronic Borna- Clinic for Psychiatry and Psychotherapy II, Ulm Disease-Virus-infection. University, Bezirkskrankenhaus Günzburg, Despite some recent progress, the definition of Günzburg, Germany ME remains preliminary, on the other hand with- in the general problems of definition and catego- The term ‚mild encephalitis' (ME) (1) was pro- rization of inflammation: the established defini- posed some years ago to address a new approach tion dates back to Celsus 40 BC, modern defini- to severe psychiatric disorders with unknown eti- tions depend from the options to investigate the ology, the pathogenesis of which appeared not many aspects and factors involved. A recent defi- appropriately understood. ME means a type of nition (2) "inflammation is a complex set of inter- low level CNS inflammation possibly causing a actions among soluble factors and cells that can spectrum of psychiatric disorders in interaction arise in any tissue in response to traumatic, infec- with other contributing factors. In the meanwhile tious, post-ischaemic, toxic or autoimmune more and more findings support such view injury" appears perfect, but is not applied in clin- including recent own studies by modern CSF ical practice. Conventional histopathology diagnostics, considered the diagnostic gold stan- defines inflammation by "visible inflammatory dard in CNS-inflammation, especially acute cellular infiltrates within tissue", a limited view. It meningoencephalitis: we found in 41% of is known, that CNS-inflammation begins with an patients with affective (n=24) or schizophrenic accumulation of immune competent cells within (n=39) spectrum disorders definite CSF abnor- defined brain areas yet without visible cellular malities; in 14% intrathecal humoral immune infiltrates and such may last for longer time. An responses clearly interpreted as inflammatory; in important difference exists between acute and 10% slightly increased CSF cell counts; in 29% chronic inflammation, chronic not being clearly moderate blood CSF barrier dysfunction, in 24% defined nor in histopathology nor in the clinic, in as the only pathological sign; in 20% of affective the latter by simple time cuts. In neurology it is spectrum patients systemic inflammation, in none accepted to consider encephalitis to be chronic of the schizophrenic spectrum patients. In 6% after four weeks. In psychiatry there is little (n=4) we refined the previous differential diagno- agreement about which disorders have to be sis by the results of CSF investigation and diag- termed chronic. When adopting the neurological nosed probable ME due to various specific caus- time frame probably most psychiatric disorders

Pteridines/Vol. 21/No. 2 30 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines should be considered chronic. In the case of a demonstrate the dilemma in the approach to CNS neurobiological basis such as ME, we would deal inflammation in general, and even more to LLCI nearly exclusively with chronic low level neu- situations. The relationship between brain pathol- roinflammation then in psychiatry. ogy and clinical psychiatric symptoms is known What is about the symptoms of psychoses and to be weak. To better understanding causal rela- encephalitis? Classical encephalitis can cause tionships and finding better definitions we need various psychiatric syndromes, rare single cases more parameters and more refined criteria. In vivo observed may go through the range of psychiatric detection of LLCI is especially difficult. Adopting disorders within weeks, e.g. beginning with per- modern CSF-diagnostics from neurology with its sonality disturbance, progressing to affective broad background knowledge is an important but spectrum and later schizophrenic spectrum disor- only a first step to diagnose ME as a possible psy- der and only finally to organic psychoses and pos- chiatric disease. sibly backwards. The rule of non-specificity of Summing up, the present definition of ME psychiatric symptoms holds for various organic remains preliminary, but appears advantageous to causes including encephalitis. Non-classical the alternative of further neglecting the preva- inflammation (classical i. fits acute but does not lence of LLCI situations, which may not rarely fully cover chronic i.) is now more and more rec- causally underlie psychiatric disorders. We pro- ognized to exist though yet lacking clear defini- pose to diagnose ME in severe psychiatric disor- tions. Just recently one new term was proposed ders (schizophrenic or affective spectrum) with that is 'parainflammation', describing chronic the following CSF-findings: probable ME with inflammation with preferential fibrosis (3). Low oligoclonal bands exclusively detected within level CNS- respectively neuro-inflammation CSF not in parallel in blood, or with intrathecal (LLCI) is now often mentioned but lacks clear production of specific immunglobulin subclasses definition. One good exception is the categoriza- (IgA or IgG or IgM or combined); possible ME tion of microgliaactivation in four grades, repre- with exclusive blood-CSF-barrier-dysfunction senting one important aspect of neuro-inflamma- (for details see (6)). tion though not always indicating inflammation (4); the lower grades proposed (1+2) would well References: match with what here is termed LLCI and ME 1 Bechter K. Mild encephalitis underlying psy- respectively. chiatric disorder - A reconsideration and Refined definitions are not an academic point hypothesis exemplified on Borna disease. but are important for clinic and research. The Neurol Psych Brain Res 2001;9:55-70 unmet problem is demonstrated for example with 2 Nathan C. Points of control in inflammation. the many open questions regarding the role of Nature 2002;420:846-52 HIV-brain-infection for related psychiatric distur- 3 Medzhitov R. Origin and physiological roles bances. When taking a strict definition of HIV- of inflammation. Nature 2008;454:428-35 Encephalitis (HIV-E), HIV-E includes the post- 4 Raivich G, Bohatschek M, Kloss CU, Werner mortem finding of three classical inflammatory A, Jones LL, Kreutzberg GW. Neuroglial acti- foci; then postmortem diagnosis matches relative- vation repertoire in the injured brain: graded ly well with clinical HIV-E-diagnosis (5). response, molecular mechanisms and cues to However, LLCI is present in non-HIV-E cases physiological function. Brain Res Brain Res postmortem frequently and associated with vari- Rev 1999;30:77-105 ous psychiatric syndromes, including cognitive 5 Cherner M, Cysique L, Heaton RK, Marcotte decline or psychosis. Even more critical in such TD, Ellis RJ, Masliah E, Grant I. definition of HIV-E, not only from a practical but "Neuropathologic confirmation of definitional especially from a theoretical point of view is, that criteria for human immunodeficiency virus- non-HIV-E cases may present up to two classical associated neurocognitive disorders. J inflammatory foci within the brain which remain Neurovirol 2007;13:23-8. non-diagnostic for HIV-E. Such examples 6 Bechter K, Reiber H, Herzog S, Fuchs D,

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 31

Tumani H, Maxeiner HG. Cerebrospinal fluid higher ATP content in vari-ous brain areas sug- analysis in affective and schizophrenic spec- gesting that their antioxidative mechanisms seem trum disorders: Identification of subgroups to be less pronounced (GR, GPX, GST) than with immune responses and blood-CSF barri- those of adult rats, while the GSSG content is er dysfunction. J Psychiatric Res 2010:44:321- higher in juvenile rats. The application of 30. Haloperidol to adult rats seems to lead to a reduc- tion of antioxidative mechanisms in various brain areas. There is also evidence that Haloperidol Oxidative and Antioxidative Mechanisms in affects ATP. In juvenile rats, Haloperidol seems to Juvenile and Adult Rats after Application of have rather little influence on antioxidative mech- Haloperidol anisms. While our results show that Haloperidol does Berger M, Clement HW, Heiser P, Sommer O, not directly influence radical concentration in Hoinkes A, Schulz E both young and old rats, there is evidence that Haloperidol clearly has noticeable, age-related University Hospital Freiburg, Department Child effects on radical formation occurring on the level & Adolescent Psychiatry, and University Hospital of radical detoxification mechanisms. Freiburg, Department General & Visceral Surgery, Freiburg, Germany Expression of Indoleamine 2,3-dioxygenase-2 Tardive dyskinesia which is presumed to be (IDO2) at the Human Feto-Maternal Interface connected with elevated oxidative stress, is described as a typical side effect of Haloperidol. Blaschitz A, Maschke P, Gauster M, Dohr G, Ball However, this neurotoxic effect of Haloperidol is HJ, Sedlmayr P discussed controver-sially. In this context, previ- ous studies gave little evidence for differences Institute of Cell Biology, Histology and between adults and children as well as adoles- Embryology, Center of Molecular Medicine, cents. The purpose of this study is to examine Medical University of Graz, Austria, and both oxidative mechanisms, substantiated by Department of Pathology, University of Sydney, reactive oxygen species (ROS), and antioxidative Australia mechanisms in juvenile and adult rats after appli- cation of Haloperidol. Tryptophan catabolism has been implicated in Measurement of reactive oxygen species playing a decisive role in feto-maternal immuno- (ROS) was performed using radical scavenger tolerance. Application of 1-methyl-DL-trypto- CMH by means of microdialysis and subsequent phan which is able to block Indoleamine 2,3- electron spin resonance (ESR) spectroscopy. dioxygenase (IDO) induced rejection of hemi- Organ extraction was car-ried out 3 hours after an allogeneic but not syngeneic embryos (Munn et acute application of Haloperidol (2 mg / kg i. p.). al., 1998). The later finding that allogeneic mat- The organ parts were homoge-nized and their ings of mice in which both parents were geneti- amount of ATP, reduced GSH, oxidized glu- cally IDO-deficient do not lead to higher abortion tathione (GSSG), glutathione reductase (GR), rates was enigmatic in this context (1). For this glutathione peroxidase (GPX), glutathione-S- reason, our attention has been drawn to potential transferase (GST), superoxide dismutase (SOD) compensatory or redundant immunosuppressive as well as NADPH oxidase (NOX) was tested by mechanisms. Until recently the ability to convert means of various kits. tryptophan to N-formylkynurenine was regarded No elevated radical formation could be meas- as being confined to IDO and tryptophan 2,3- ured in vivo following an acute dose of dioxygenase (TDO, which is not blocked by 1- Haloperidol - neither in young nor in old animals. methyl-DL-tryptophan). However, we now know Juvenile rats opposed to adult animals showed a there is a third enzyme, indoleamine 2,3-dioxyge-

Pteridines/Vol. 21/No. 2 32 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines nase 2 (IDO2) with high homology to IDO (now Division of Biological Chemistry, Biocenter, named IDO1) and whose tryptophan-degrading Department for Islet Cell Transplantation, activity is comparatively low (2). Innsbruck Medical University, Innsbruck, and By immunohistochemistry we localized IDO2 Institute of Pathology, St. Vinzenz KH, Zams, protein to the trophoblast of placental villi, with Austria clear preponderance of the syncytiotrophoblast to the cytotrophoblast in first trimester samples. In Ischemia-reperfusion-injury (IRI)-related graft placenta villi at term, the syncytiotrophoblast was pancreatitis is a severe complication following positive, the staining was patchy. Expression of pancreas transplantation (PTX). Tetrahydrobio-

IDO2 in extravillous cytotrophoblast of first pterin (H4B), an essential cofactor of nitric oxide trimester decidua was comparatively minor and synthases (NOS), was shown to significantly irregular, in the basal plate of second trimester attenuate IRI and protect from graft pancreatitis if and term placenta IDO2 is clearly positive in injected to the donors prior to organ retrieval. invasive extravillous cytotrophoblast. Other than Since the underlying mechanism is still under that, IDO2 was found to some extent in glandular debate, we herein investigated if eNOS is the epithelium of first trimester decidua. Non-preg- main target of H4B concerning the observed pro- nant endometrium showed hardly any staining. tective effects using an eNOS-/- model. RT-PCR showed expression of IDO2 mRNA in Male syngeneic C57Bl6 (H-2b) mice were placental villi that increased with gestational age. used as size-matched donor and recipient pairs. IDO2 mRNA was also present in first trimester Donors were either C57Bl6 based eNOS-/- or decidua. eNOS wt mice. Murine cervical pancreas trans- In contrast to our previous finding regarding plantation was performed with a modified no- IDO1, the localization of IDO2 at the feto-mater- touch technique. To induce pancreatitis grafts nal interface is more suggestive of a potential role were subjected to 16 h prolonged cold ischemia in feto-maternal immunotolerance. time (CIT) as well as to 45 min warm ischemia time. Donors were either pre-treated with

References: 50mg/kg body weight H4B or were untreated. 1 Baban B, et al., Indoleamine 2,3-dioxygenase Non-transplanted animals served as controls. expression is restricted to fetal trophoblast Microcirculation was analyzed by intravital fluo- giant cells during murine gestation and is rescence microscopy evaluating functional capil- maternal genome specific. J Reprod Immunol lary density (FCD). Parenchymal damage was

2004;61:67-77. assessed by H&E histology. Intragraft H4B levels 2 Ball HJ, et al., Characterization of an were assessed by HPLC. Since ischemia reperfu- indoleamine 2,3-dioxygenase-like protein sion injury of the pancreatic graft in this model found in humans and mice. Gene was found to be lethal, survival analysis of the 2007;396:203-13. different groups was assessed. Prolonged CIT resulted in markedly impaired microcirculation compared to non-transplanted Tetrahydrobiopterin Saves Murine Pancreatic controls. Independently of their genotype, 2 h fol-

Grafts from Ischemia Reperfusion Injury: lowing reperfusion grafts treated with H4B dis- Application in an eNOS-/- Model played improved FCD compared to untreated ani- mals (p = 0.09). As early as 15 min after reperfu- Cardini B, Oberhuber R, Watschinger K, sion, there were no differences between treated Hermann M, Obrist P, Schneeberger S, Werner- and untreated animals. In both genotypes, reduc- Felmayer G, Pratschke J, Brandacher G, Werner tion of IRI-related tissue damage like parenchy- ER, Maglione M mal edema and acinar and fat necrosis was

decreased following H4B-treatment (p <0.05), Center of Operative Medicine, Department of however the difference was more pronounced in Visceral, Transplant and Thoracic Surgery, wt grafts. Following donor pre-treatment with

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 33

H4B, intragraft H4B levels were significantly hours. For SS we observed a decrease of leptin higher compared to non-treated controls concentration after 24 hours only. SS and SB had

(p <0.05). Finally, H4B-treated pancreatic grafts no effect on the secretion of adiponectin. resulted in significantly prolonged recipient sur- Curcumin decreased the adiponectin concentra- vival, independently of the donor genotype tion significantly after 6h. (p <0.001). To analyze the effect of high calory intake on

H4B attenuates graft pancreatitis and hence sig- the secretion of leptin and adiponectin, we per- nificantly improves recipient and graft survival. formed a within-subject crossover study, were we However, eNOS is not the main target of tetrahy- administered a standardized high-fat meal to 24 drobiopterin action concerning its protective healthy subjects. Plasma concentrations of leptin effects during IRI. and adiponectin were measured in the morning after an overnight fast and during postprandial lipemia, i.e. 2, 4 and 6 hours after meal ingestion Role of Food Additives in Addition to Calory (postprandial experiment). To exclude potential Intake in the Pathogenesis of Obesity confounding factors affecting the adipocytokine plasma concentrations, a control experiment with- Ciardi C, Jenny M, Tschoner A, Ueberall F, out meal ingestion was performed over the same Patsch J, Pedrini M, Ebenbichler C, Fuchs D time period (postabsorptive control experiment). Comparing plasma concentrations of leptin and Department of Internal Medicine I, Division of adiponectin between the postprandial and the Biological Chemistry, and Division of Medical postabsorptive control experiments, we found no Biochemistry, Biocenter, Innsbruck Medical significant differences. University, Austria Our experiments show that food additives SS, SB and curcumin affect the leptin release from Changes of lifestyle including physical inactiv- adipocytes in a dose- and time-dependent fashion, ity and overnutrition have led to a rise of severe in an in vitro study. Decreased leptin release dur- obesity. The daily intake of high calory food and ing consumption of nutrition-derived food addi- food additives has increased substantially during tives could decrease the amount of circulating recent decades. Many of compounds used are leptin to which the central nervous system is antioxidants and may thus interfere with redox- exposed and could therefore contribute to an adi- sensitive cellular signalling. pogenic effect. Leptin and adiponectin are adipokines, which The effect of postprandial lipemia after con- are secreted mainly by adipocytes, and have sumption of a high fat meal has no significant important effects on the metabolism and home- effect on the plasma concentrations of leptin and ostasis of energy. The aim of the in vitro study adiponectin in relation to their postabsorptive was to investigate a potential influence of food plasma profiles in healthy subjects. Therefore, we additives on the leptin and adiponectin release of conclude that prolonged states of obesity are murine adipocytes. Leptin and adiponectin con- required to affect plasma concentrations of these centrations were analyzed in supernatants of adipocytokines. murine adipocytes 3T3-L1 after co-incubation with lipopolysaccharide (LPS) and the widely used food additives sodium sulfite (E221, SS), Serum Phenylalanine to Tyrosine Ratio in sodium benzoate (E211, SB) and curcumin Patients with Lung Cancer and Chronic (E100) in various concentrations for 24 hours. Obstructive Pulmonary Disease (COPD) Moreover, the kinetics of secretion of both adipokines were analyzed. Denz H, Kurz K, Schroecksnadel S, Amann A, A significant and dose-dependent decrease of Fuchs D leptin production was observed after incubation of the cells with SB and curcumin after 12 and 24 Division of Oncology, Hospital Natters, Natters,

Pteridines/Vol. 21/No. 2 34 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines and Department for Internal Medicine, University with COPD, the latter presenting widely with nor- Hospital Innsbruck, and Division of Biological mal neopterin concentrations, only 16% presented Chemistry, Biocenter, Innsbruck Medical with neopterin concentrations >10 nmol/L). In as University, Innsbruck, Austria similar way tryptophan metabolic parameters behaved with abnormal levels preferentially in Abnormal metabolism of aromatic amino acids cancer patients. Eleven percent of patients with tryptophan (trp) and phenylalanine (phe) accom- cancer compared to 8 % of patient with COPD panies conditions which go along with chronic presented with phe/tyr >1.25 indicating impaired inflammation and immune responses like cancer, conversion of phe. In the patients with cancer, sig- infection, autoimmune syndromes, cardiovascular nificant correlations existed of neopterin levels =− < disorders, neurodegeneration, etc. Such diseases with kyn/trp (rs 0.587, p 0.0001) but also =− < are often accompanied by neuropsychiatric symp- with tyr concentrations (rs 0.266, p 0.001) as =− toms like cognitive impairment, mood changes well as phe/tyr (rs 0,317, p <0.0001). No such and depression. Part of them can be referred to associations existed in patients with COPD in subnormal trp and thus serotonin (5-hydrox- whom only the correlation between neopterin and = ytryptamine, 5HT) availability, but some patients kyn/trp remained significant (rs 0.476,p <0.002). do not respond well to treatment with selective Impaired PAH activity is indicated in a relevant serotonin reuptake inhibitors (SSRI). percentage of patients with lung cancer but not in Alternatively, dopaminergic dysfunction could be COPD. The impaired PAH activity parallels the involved. increased formation of neopterin and the acceler- Earlier it was observed that patients with hepa- ated degradation of trp which is most probably tocellular carcinoma (1), with burns (2) or with due to enhance activity of indoleamine 2,3-dioxy- HIV-1 infection (3) may present with increased genase (IDO). The lower neopterin concentra- phe concentrations. More recent data obtained tions and kyn/trp in COPD indicates that Th1- from patients after trauma (4), with ovarian can- type immunity is less strongly activated in these cer (5) or HIV-1 infection (6) demonstrate signif- patients as compared with the patients with lung icant associations between neopterin production cancer. The correlation between phe/tyr and and decreased activity of phenylalanine-4- neopterin concentrations as well as kyn/trp in hydroxylase (PAH) as is indicated by an increased lung cancer patients points to a possible role of phe to tyrosine (tyr) ratio (phe/tyr), and again chronic immune activation for the abnormal phe/tyr correlated with neopterin concentrations. activity of PAH. A role of ROS in diminishing In this study, 236 patients (48 females, 149 availability of tetrahydrobiopterin (H4bip), the males, aged 62.4 ± 9.3 y) with lung cancer and 39 necessary cofactor of PAH appears likely. patients (18 females, 21 males, aged 63.3 ± 7.8 y) However, any potential relevance for the neu- with chronic obstructive pulmonary disease ropsychiatric abnormalities in patients still needs (COPD) were investigated. Serum concentrations to be shown. The concurrent determination of of phe and tyr were measured by HPLC monitor- phe/tyr and kyn/trp may provide a basis for ing natural fluorescence of compounds. Results improved treatment decision whether SSRI or were compared to neopterin concentrations meas- other neuromodulators are more appropriate for a ured with ELISA (BRAHMS, Hennigsdorf, single patient. Germany), to the degradation of tryptophan (trp) as reflected by the kynurenine (kyn) to trp ratio References: (kyn/trp) and to nitrite + nitrate production apply- 1 Watanabe A, et al. Serum amino acid levels in ing the Griess reaction. patients with hepatocellular carcinoma. Mean neopterin concentrations (lung cancer: Cancer 1984;54:1875-82. 9.5 nmol/L vs. COPD: 7.7 nmol/L; not signifi- 2 Rath T, et al. Phenylalanine : total amino acid cant) and kyn/trp (lung cancer: 40.7 µmol/mmol ratio in 45 burn patients. Scand J Plast vs. COPD: 34.4 µmol/mmol; p = 0.02) were Reconstr Surg Hand Surg 1987;21:297-300. found to be higher in lung cancer than in patients 3 Ollenschläger G, et al. Plasma amino acid pat-

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 35

tern of patients with HIV infection. Clin Chem kynurenine to tryptophan was calculated to esti- 1988;34:1787-9. mate the serum IDO activity. Histopatological 4 Ploder M, et al. Serum phenylalanine in evaluation of tumour grade, lymph node metasta- patients post trauma and with sepsis correlate sis, peritumoral lymphocytic infiltrate, lymphatic to neopterin concentrations. Amino Acids invasion were made by H-E staining. Total 2008;35:303-7. tumour tissue IDO-immunostaining score was 5 Neurauter G, et al. Serum phenylalanine con- calculated with the sum of the proportion and the centrations in patients with ovarian carcinoma intensity staining scores of peritumoral infiltrat- correlate with concentrations of immune acti- ing cells. In carcinoma group a significant vation markers and of isoprostane-8. Cancer increase in serum kynurenine/tryptophan ratio Lett 2008;272:141-7. was observed (p <0.05), while their serum nitrite, 6 Zangerle R, et al. Increased blood phenylala- neopterin and urinary biopterin levels were not nine to tyrosine ratio in HIV-1 infection and significantly altered (p >0.05 for all). IDO high- correction following effective antiretroviral expression in tumour tissue was significantly cor- therapy. Brain Behav Immun 2010;24:403-8. related with the lymphatic invasion and lymph node metastasis. No correlation was found between IDO expression patterns, tumour stage Tumor Invasion Pattern and Related Serum and serum kynurenine/tryptophan ratio (p >0.05). Trytophan and Neopterin Concentrations in IDO expression levels in tumour tissue signifi- Colorectal Carcinomas cantly correlated with the presence of lymph node metastasis and lymphatic invasion. These results Engin AB, Engin A, Gonul II, Karamercan A, suggested that total IDO immunostaining score Sepici-Dincel A, Dursun A has been included in histopathological evaluation of colorectal cancer cases. Department of Toxicology, Faculty of Pharmacy, and Department of General Surgery and Supported by Gazi University, Scientific Research Department of Pathology, Gazi University, Projects Division, 01/2007-62. Faculty of Medicine, Ankara, Turkey

Lymphatic invasion and the dept of submucos- Effects of Rheumatoid Arthritis and Coronary al invasion of tumour cells are risk factors for Artery Disease on the Expression of lymph node metastasis, which is the strongest Chemokines and Neopterin prognostic determinant for colorectal cancer patients. It was suggested that indoleamine (2,3)- Fagerer N, Kullich W dioxygenase (IDO) high-expressed colorectal tumour cells enable cancer subsets to avoid Ludwig Boltzmann Cluster for Rheumatology, immune attack. Aim of this prospective clinical Balneology und Rehabilitation, Saalfelden, study was to investigate the impact of cellular Austria immune response and tryptophan metabolism on peritumoral lymphocytic infiltration, lymphatic In chronic inflammatory processes chemokines invasion score and lymph node metastasis in col- are abundantly present at the site of inflammation orectal cancer patients. Forty-three patients with- (1). Also the pteridine neopterin is elevated in out signs of malign or metabolic diseases and 44 various diseases with inflammatory background patients with new diagnosed colorectal carcinoma like Rheumatoid Arthritis (RA) and Coronary were included in this study. Serum tryptophan, Artery Disease (CAD). Several reports have indi- kynurenine and urine biopterin concentrations cated a correlation of neopterin with inflammato- were determined by high performance liquid ry activity and intensification of systemic symp- chromatography. Serum nitrite and neopterin le- toms of RA (2) and CAD activity score (3). vels were measured by ELISA. The ratio of Neopterin has already been used as a marker of

Pteridines/Vol. 21/No. 2 36 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines inflammation during clinical management in a rheumatoid arthritis. Ter Arkh 1998;70:28-31. number of diseases (4). Monocytes/macrophages, 3 Murr C, et al. Inverse association between which play an important role as infiltrating cells serum concentrations of neopterin and antiox- at the site of inflammation, produce neopterin as idants in patients with and without angio- well as chemokines upon immune activation. graphic coronary artery disease. Atheroscle- Chemokines play an important role in the immune rosis 2009;202:543-9. response and directing lymphocyte movement. 4 Gieseg SP, et al. Potential to inhibit growth of CCL2/MCP-1 is a molecule with powerful mono- atherosclerotic plaque development through cyte chemotactic activity expressed by mono- modulation of macrophage neopterin/7,8- cytes. CX3CL1/fractalkine is a mediator of dihydroneopterin synthesis. Br J Pharmacol inflammation and angiogenesis in RA. The B-cell 2008 153:627-35. attracting chemokine CXCL13/BCA-1 is expres- sed by fibroblasts and endothelial cells within the inflamed synovium of RA patients. The aim of the Determination of Volatile Organic Compounds present study was to demonstrate if RA patients in Exhaled Breath of Patients with Lung with enhanced neopterin levels show activation of Cancer and Healthy Volunteers Using TD-GC- the proinflammatory chemokines CXCL13, MS and PTR-MS CX3CL1 and CCL2. 113 patients with RA (ACR criteria) including Filipiak W, Filipiak A, Bajtarevic A, Ager C, 24.8% suffering from CAD underwent a laborato- Schwarz K, Amann A ry testing for neopterin, CXCL13, CX3CL1, CCL2 and CRP which were all found in high con- Department of Operative Medicine, Innsbruck centrations (neopterin: 11.10 ± 5.78 nmol/l). Medical University; and Breath Research Unit of Neopterin significantly correlated with all the Austrian Academy of Sciences, A-6020 chemokines: CXCL13 (r = 0.368; p<0.0001), Innsbruck CX3CL1 (r = 0.241; p <0.02), CCL2 (r = 231; p <0.02). Further on strong correlations among all At present, diagnosis of lung cancer very often three chemokines were observed. Patients with happens late in the course of the disease since RA plus CAD in comparison to RA patients with- inexpensive, non-invasive, sufficiently sensitive out cardio-vascular problems showed significant- and specific screening methods are not available. ly higher values of neopterin and CXCL13 Such an early non-invasive diagnosis of lung can- (p <0.03). cer which would improve prognosis and enlarge Increased neopterin and chemokines seem to treatment options can be analysis of exhaled interact in the network of cell-mediated immune breath. response. High neopterin levels in RA patients Since many of the compounds observed in the have strong association to the chemokine induced breath have never been described in the biochem- chemotaxis of B-cells (CXCL13). Our results ical literature, it is of utmost importance for fur- indicate too that the high incidence of CAD in ther biochemical investigations to confirm the Rheumatoid Arthritis could be associated with identification of detected compounds with cer- enhanced mononuclear- (neopterin, CCL2) and tainty. Since identification based on spectral B-cell activation (CXCL13), and as a conse- library match only may be misleading, additional quence increased immune activity. chromatographic data e.g. retention time or reten- tion index is necessary. Only such a validated References: identification allows investigating the biochemi- 1 Godessart N, Kunkel. Chemokines in autoim- cal background of the observed compound. mune disease. Current Op Immunol 2001; Therefore, the primary aim of the present work 13:670-5. was to identify the compounds observed in 2 Nasonov EL, et al. Neopterin: laboratory exhaled breath of lung cancer patients (of all dis- marker of cellular immunity activation in ease stages) in a careful manner. For GC-MS

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 37 analyses we have built up a database of over 230 This research was supported by the European compounds based on calibration mixtures of pure Commission (Project BAMOD, project no LSHC- reference materials. Correct identification and CT-2005-019031) We greatly appreciate the gen- quantitation is particularly difficult in case of erous support of the Member of the Tyrolean PTR-MS analyses where the fragmentation of regional government Dr.E.Koler and the Director protonated compounds take place. Since no sepa- of the University Clinic of Innsbruck (TILAK) ration of analytes takes place before ionization, Mag.A.Steiner. different protonated compounds may contribute to the same ion detected in the PTR-MS spec- trum. Thus, considering M+1 ion may be mislead- Evolution of Regulation, Structure and Func- > ing and for larger alcohols ( C2) it gives false tion in Phenylalanine Hydroxylase results. Exhaled breath and inhaled room air samples Flydal MI, Teigen K, Martinez A were analyzed using proton transfer reaction mass spectrometry (PTR-MS) and adsorption on solid Department of Biomedicine, University of sorbents with subsequent gas chromatography Bergen, Norway mass spectrometry measurements (SPE-GC-MS). For the PTR-MS measurements, 220 lung cancer Phenylalanine hydroxylase (PAH) is a patients and 441 healthy volunteers were recruit- tetrameric enzyme which in mammals is mainly ed. For the GC-MS measurements, we collected present in the liver where it converts phenylala- samples from 37 lung cancer patients and 53 nine to tyrosine. In mammals this is the first step healthy volunteers. Lung cancer patients were in in the catabolic degradation of L-Phe. The different disease stages and under treatment with enzyme is dependent on tetrahydrobiopterin as different regimes. Alveolar breath was collected electron donor in the catalyzed reaction. The with the real-time CO2 control into Tedlar bags biopterin cofactor has an inhibitory effect on PAH and transported to laboratory before PTR-MS and activity and at the same time protects the enzyme SPE-GC-MS analyses. from degradation. Strict regulation of the mam- Approximately, one third of the compounds malian enzyme is important to maintain appropri- detected were hydrocarbons. We found aromatic ate level of phenyalanine to (i) avoid the disease hydrocarbons, alcohols, aldehydes, ketones, phenylketonuria (PKU) characterised by mental esters, ethers, sulphur compounds, nitrogen-con- retardation and (ii) provide a continuous supply of taining compounds and halogenated compounds. tyrosine for protein synthesis. One important reg- Acetonitrile, groups of furans and especially ulatory mechanism is the cooperative response to dienes were found to be correlated with smoking phenylalanine binding in the active sites of the behaviour. four subunits. Respective reaction constants and calibration PAHs from bacteria and lower eukaryotes, correction factors elucidated from calibrations which have been associated to the synthesis of L- were used for determination of benzene, isoprene, Tyr derivatives such as melanine-like compounds, acetone and methanol by PTR-MS analyses. do not show allosteric regulation. We have ana- Benzene clearly is a compound related to smok- lyzed the sequence and structure of PAH from an ing behaviour. The respective concentrations of evolutionary perspective in an effort to explain isoprene, acetone and methanol are lower in lung the structural determinants for the incorporation cancer patients as compared to healthy volun- of regulatory properties in mammals. Sites with a teers. GC-MS results shows several compounds high probability of being involved in a functional found at elevated level in breath of lung cancer or regulatory change were identified and structur- patients in comparison to healthy volunteers, al models of PAH from human, and whereas tetramethylurea was found to be signifi- Caenorhabditis elegans have been prepared and cant, regardless of sample collection method used to visualize the changed sites. Important (with or without CO2 control). areas with high probability of being involved in

Pteridines/Vol. 21/No. 2 38 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines allosteric regulation of mammalian PAH are dis- Health Evaluation) scores were investigated. cussed. Molecular dynamics simulations have Kynurenine concentrations were significantly been applied to these models to get insight into increased in multiple organ dysfunction syndrome the conformational changes related to regulation and septic shock groups compared to the sepsis by both the substrate and biopterin cofactor at an group. Although the survivor group had lower atomic level. neopterin concentrations and tryptophan degrada- This work has identified specific amino acid tion, the difference compared to non-survivors residues important for regulation of enzymatic was not statistically significant. Neopterin con- activity. It has also provided insights into the centrations and kynurenine/tryptophan ratio as an basis for the evolution of sophisticated regulatory indicator of tryptophan degradation were both mechanisms in an important metabolic enzyme. correlated with APACHE II score. Previous studies from us and others (1-4) sug- gested that urinary neopterin concentrations are Serum Neopterin Concentrations and promising in terms of determining disease pro- Tryptophan Degradation in Intensive Care gression and the resultant prognoses of the inten- Unit Patients sive care unit patients. Results of the present study confirm and extend the previous data: Girgin G, Sahin TT, Fuchs D, Yüksel O, Sare M, serum neopterin levels and tryptophan degrada- Kurukahvecioglu O, Baydar T tion were well correlated with the severity of the disease in intensive care unit patients. Department of Toxicology, Faculty of Pharmacy, Hacettepe University and Department of Surgery, References: Faculty of Medicine, Gazi University, Ankara, 1 Baydar T, et al. Neopterin as a prognostic bio- Turkey, Department of Surgery II, Graduate marker in intensive care unit patients. J Crit School of Medicine, Nagoya University, Nogaya, Care 2009;24:318-21. Japan, and Division of Biological Chemistry, 2 Strohmaier W, et al. D-erythro-neopterin plas- Innsbruck Medical University, Innsbruck, Austria ma levels in intensive care patients with and without septic complications. Crit Care Med Increase in neopterin concentrations and tryp- 1987;15:757-60. tophan degradation are common events following 3 Pellegrin K, et al. Enhanced enzymatic degra- the activation of cellular immune system. In neu- dation of tryptophan by indoleamine 2,3- rological, cardiovascular and autoimmune disor- dioxygenase contributes to the tryptophan- ders, different types of malignancies and infec- deficient state seen after major trauma. Shock tions, high neopterin levels and tryptophan degra- 2005;23:209-15. dation have been reported to be well correlated 4 Ploder M, et al. Tryptophan degradation in with the severity of the diseases. The present multiple trauma patients: survivors compared study was undertaken to evaluate serum neopterin with non-survivors. Clin Sci (Lond) levels and tryptophan degradation in intensive 2009;116:593-8. care unit patients. Serum neopterin, tryptophan and kynurenine concentrations were determined in patients with Oxygen Radical Absorbance Capacity systemic inflammatory response syndrome (ORAC) of Compounds Compared to the (n = 9), sepsis (n = 8), septic shock (n = 10), and Suppression of Stimulated Peripheral Blood multiple organ dysfunction syndrome (n = 7). Mononuclear Cells Neopterin concentrations and tryptophan degra- dation were tested if they differed among the Gostner J, Klein A, Jenny M, Schennach H, Fuchs patient groups. Furthermore, the relation between D, Ueberall F neopterin concentrations, tryptophan degradation and APACHE II (Acute Physiology and Chronic Division of Medical Biochemistry and Division

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 39 of Biological Chemistry, Biocenter, Innsbruck observed for all compounds within the analysed Medical University, and Central Institute of concentration range (4, 5). Thus, aside differences Blood Transfusion and Immunology, University in intensity, all tested compounds showed a Hospital Innsbruck, Innsbruck, Austria capacity to suppress Th1-type immune activation pathways, which indicate anti-inflammatory Conditions of oxidative stress which may con- properties of these substances in this in vitro sys- tribute to pathophysiological processes arise tem. In summary, substances with high ORAC when pro-oxidant molecules are produced by the values seem to mediate suppression of neopterin body at a rate that exceeds its ability to neutralize and tryptophan degradation in vitro. However this them. Beside antioxidative active enzymes and is not true for the inverse conclusion, as suppres- molecules produced naturally, also dietary intakes sive effects of compounds on PBMCs do not con- and pharmaceuticals are likely to contribute to the sistently correlate with high antioxidant activity antioxidative defence of the body. Therefore, in the ORAC assay. The measurement of ORAC there is an increasing demand for methods that values of food, food additives or pharmaceuticals characterize the total antioxidant capacity of sub- is certainly of interest, but these values do not stances. The cell-free Oxygen Radical Absor- necessarily reflect the final effects on antioxida- bance Capacity Assay (ORAC) has been widely tive levels or the radical-quenching ability after accepted as a standard tool to measure the antioxi- food intake. Furthermore, only peroxyl radical dative activity in the nutraceutical, pharmaceuti- quenching capacities of compounds have been cal and food industries (1). The ORAC assay prin- determined by this method, without consideration ciple is based on the measurement of peroxyl ra- of other relevant radicals or contributors to oxida- dical damage to a photostable fluorescent probe in tive stress formed in vivo. The PBMC model a time and intensity dependent manner. Anti- involves a broader variety of reactive oxygen oxidants are supposed to prevent the decrease of species (ROS) and provides a link to biological fluorescence by inactivating the peroxyl radicals. relevant activities, although suppressive effects Final values are normalized to trolox, a water sol- on neopterin formation and tryptophan degrada- uble derivate of vitamin E, providing a measure tion might be indirect and must not be mediated of potent antioxidative activity. Evaluation of five by blocking ROS activity mandatory. different substances revealed, that the food col- orant curcumin (E100) showed the highest References: antioxidative effect (5.16 ± 0.17µmol trolox 1 Ou B, et al. Development and validation of an equivalents (TE)). Both, the cannabinoid ?9- improved oxygen radical absorbance capacity tetrahydrocannabinol (THC) and the non-psy- assay using fluorescein as the fluorescent chotropic cannabidiol (CBD) demonstrated also probe. J Agric Food Chem 2001;49:4619-26. significant antioxidative properties (0.49 ± 0.05 2 Maier E, et al. Food preservatives sodium ben- and 0.61 ± 0.06µmol TE, respectively), while the zoate and propionic acid and colorant curcum- food preservatives sodium benzoate (E211) and in suppress Th1-type immune response in propionic acid (E280) did not show any consider- vitro. Pteridines 2007;18:46. able activity (2, 3). Furthermore the in vitro influ- 3 Jenny M, et al. 9-tetrahydrocannabinol and ence of these substances on neopterin production cannabidiol modulate mitogen-induced trypto- and tryptophan degradation in mitogen stimulated phan degradation and neopterin formation in and untreated human peripheral blood mononu- peripheral blood mononuclear cells in vitro. J clear cells (PBMC) was analysed in order to mon- Neuroimmunol 2009;207:75-82. itor potential interactions with Th1-type immune 4 Widner B, et al. Simultaneous measurement of response mechanisms, where pro-oxidant species serum tryptophan and kynurenine by HPLC. are produced upon stimulation of mononuclear Clin Chem 1997;43:2424-6. cells. Dose-dependent suppression of tryptophan 5 Weiss G, et al.. Modulation of neopterin for- degradation and neopterin formation in resting mation and tryptophan degradation by Th1- and in mitogen stimulated PBMC could be and Th2-derived cytokines in human mono-

Pteridines/Vol. 21/No. 2 40 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines

cytic cells. Clin Exp Immunol 1999;116:435- controls (301 ± 120 pmol/mg), Trp was signifi- 40. cantly lower in the AD/MCI group (48.9 ± 9.0 µmol/L; p<0.001) than the control group (65.2 ± 10.7 µmol/L) whereas Kyn showed no significant Carbonyl Proteins as a Clinical Marker in difference between AD/MCI group (1.72 ± 0.56 Alzheimer Disease and its Relation to µmol/L) and control group (1.53 ± 0.29 µmol/L). Tryptophan Degradation and Immune Kyn/Trp of AD/MCI group was significantly Activation higher in AD/MCI (35.2 ± 8.8; p<0.001) patients than in the control group (23.7 ± 4.2). The new Greilberger J, Koidl C, Greilberger M, Lamprecht quotient CP/Trp showed a more than 4 fold M, Schroecksnadel K, Leblhuber F, Fuchs D, Oettl increase in the AD/MCI group (19.8 ± 7.76; p K <0.001) compared to control (4.79 ± 2.26). Homocysteine, folate, vitamin B12 and neopterin Institute of Physiological Chemistry, Center for showed no significant difference. Discriminant Physiological Medicine, Medical University analysis provided CP alone as the best clinical Graz; and Institute of Pharmaceutical Sciences, marker with highest sensitivity (93.8%) and high- University of Graz, Graz, Austria; Division of est specificity (100%) for AD/MCI followed by Biological Chemistry, Biocenter, Innsbruck the ratio of CP/Trp. These results support the Medical University, Innsbruck, Austria; hypothesis that oxidative damage to proteins is Department of Neurological and Psychiatric directly connected with Trp degradation and the Gerontology, Linz, Austria Kyn pathway in the systemic immune activation.

Question arises if oxidative stress is connected References: with systemic immune activation in Alzheimer's 1 Leblhuber F, et al. Systemische Veränderung disease (AD) and mildly cognitive impairment des Immunsystems bei Patienten mit (MCI). During immune response interferon-γ Alzheimer-Demenz. Deutsch Med stimulates the production of reactive oxygen Wochenschr 1998;123:787-91. species, of neopterin and also tryptophan-degrad- 2 Widner B et al. Tryptophan degradation and ing enzyme indoleamine 2,3-dioxygenase (IDO) immune activation in Alzheimer's disease. J enzyme which initiates the degradation of essen- Neural Transm 2000;107:343-3. tial amino acid L-tryptophan (Trp) along the 3 Greilberger J, et al. Malondialdehyde, car- kynurenine (Kyn) pathway. Earlier we have bonyl proteins and albumin-disulphide as use- described increased neopterin formation and sig- ful oxidative markers in mild cognitive nificant degradation of Trp in a subgroup of impairment and Alzheimer's disease. Free patients with AD and MCI (1, 2). In this study, we Radic Res 2008;42:633-8. estimated in patients with AD and MCI (AD/MCI) and an aged matched healthy control group the amount of Kyn, Trp and the Kyn to Trp Urinary Neopterin and Serum Vitamins in ratio (Kyn/Trp), but also the content of carbonyl Patients with Head and Neck Carcinoma proteins (CP) as oxidative stress parameter and homocysteine, neopterin, folate and vitamin B12. Holecková P, Masek M, Pála M., Kalábová H, Additionally we related the oxidative stress Zezulová M, Študentová H, Krcmová L, parameter CP with the degradation of Trp creating Kašparová M, Plíšek J, Solichová D, Melichar B a new quotient CP/Trp. We calculated with dis- criminate analysis for CP, Trp, CP/Trp and Institute of Radiation Oncology and Charles Kyn/Trp its sensitivity, specificity and cut-off University First Medical School, Prague, value (3). Department of Analytical Chemistry, Charles CP was significantly higher in AD/MCI (930 ± University School of Pharmacy, Departments of 265 pmol/mg; p <0.001) patients compared to Oncology and radiotherapy and Gerontology and

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 41

Metabolic Care, Charles University Medical University Hospital Innsbruck, Austria. School Teaching Hospital, Hradec Králové, and Department of Oncology, Palacký University Several studies have indicated an increased risk Medical School and Teaching Hospital, Olomouc, of myocardial infarction (MI) in HIV populations. Czech Republic Triant et al. showed, that MI rates were higher in HIV-infected than -uninfected patients in all age Head and neck carcinomas are tumors of pre- groups, with very high rates in older patients (1). dominantly squamous cell histology. In general, Uncontrolled HIV infection is associated with the prognosis of patients with head and neck car- elevated markers of inflammation, including C- cinomas is poor. Comorbid conditions are fre- reactive protein (CRP) and also neopterin (2). quent in this population, and radical therapies are Levels of these markers decline with treatment, associated with considerable risk of morbidity but not to normal levels, and thus pro-inflamma- and mortality. Therefore, identification of prog- tory signals persist, which may contribute to nostic and predictive parameters is of great increased risk of cardiovascular disease (CVD). importance in patients with carcinomas of the The question, if there is an increased risk of MI in head and neck. Increased urinary concentrations HIV-infected patients under treatment, has been of neopterin are associated with poor prognosis in controversially discussed. In a study of Worm et tumors of different primary locations, but the data al., the investigators reported unexpected on prognostic significance of neopterin in patients increased risk of MI with the use of nucleoside with head and neck carcinomas is more limited. reverse transcriptase inhibitors (NRTIs) abacavir Retinol and alpha-tocopherol are important serum and didanosine, which created much controversy, antioxidants. Neopterin, retinol and alpha-toco- because other groups could not confirm their find- pherol were determined by high-performance liq- ings (3). uid chromatography. The survival was analyzed To elucidate a possible contribution of NRTIs using the Kaplan-Meier method, with survival to increase risk of CVD, with respect to their differences being analyzed by the log-rank test. effects on inflammation associated pathways, we The survival of patients with urinary neopterin investigated several NRTIs on their capacity to concentrations equal or above 214 µmol/mol cre- modulate immune activation cascades in vitro. atinine was significantly inferior (p <0.05) com- For this purpose, we used the nucleoside ana- pared to patients presenting with lower neopterin logues Abacavir, Tenofovir, Emtricitabine and levels. Serum retinol below 1 mol/l was also Lamivudine to determine their influence on tryp- associated with statistically significant inferior tophan degradation and neopterin formation in survival, while serum alpha-tocopherol below 19 unstimulated and phytohemagglutinin (PHA) mol/L was of border-line prognostic significance stimulated peripheral blood mononuclear cells (p = 0.07). In conclusion, urinary neopterin and (PBMC). Additionally, we evaluated the capacity retinol are prognostic factors in patients with head of these NRTIs to modulate Toll-like receptor and neck carcinomas. (TLR)-induced nuclear factor (NF-κB) activation in THP-1-blue monocytes, which stably express a reporter plasmid, expressing a secreted embryon- Immune System Modulation by anti-HIV ic alkaline phosphatase gene under the control of Drugs a promoter inducible by NF-κB. In unstimulated PBMC, significant effects of Jenny M, Schroecksnadel S, Kurz K, Schennach these NRTIS on tryptophan degradation could be H, Zangerle R, Fuchs D observed at doses of 100 and 200 µM. While Emtricitabine showed a stimulatory activity, Division of Biological Chemistry, Biocenter; Abacavir and Tenofovir suppressed spontaneous Department of Dermatology and Venerology, IDO activity within this concentration range. Innsbruck Medical University; Central Institute Neopterin levels in the same supernatants were of Blood Transfusion and Immunology, not affected by the treatment with these NRTIs. In

Pteridines/Vol. 21/No. 2 42 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines mitogen stimulated PBMC, Tenofovir showed a References: trend to enhance mitogen stimulated tryptophan 1 Triant VA, et al. Increased acute myocardial degradation at doses of 1 µM and 10 µM, which infarction rates and cardiovascular risk factors however was not significant. Significant effects among patients with human immunodeficien- could only be observed with Tenofofir and cy virus disease. J Clin Endocrinol Metab Abacavir, but again not until doses of 100 µM, 2007;92:2506-12. exerting a suppressive effect on mitogen induced 2 Wirleitner B, et al. Neopterin in HIV-1 infec- tryptophan degradation. Neopterin levels in mito- tion. Mol Immunol 2005:42:183-94. gen stimulated PBMC increased significantly 3 Worm SW, et al. Risk of myocardial infarction only upon co-treatment with Lamivudine, already in patients with HIV infection exposed to spe- at doses of 1 µM, while treatment with Abacavir cific individual antiretroviral drugs from the 3 at 100 µM and Tenofovir at 200 µM, showed a major drug classes: the data collection on capacity to diminish mitogen induced neopterin adverse events of anti-HIV drugs (D:A:D) formation. study. J Infect Dis 2010;201:318-330 Treatment of unstimulated THP-1-blue cells with NRTIs did not affect NF-κB activity, while LPS stimulated NF-κB activity was moderatly but Urinary Neopterin and Prognosis of Patients significantly enhanced by Lamivudine at doses of with Breast Carcinoma 10 nM, by Abacavir at doses of 100 nM and by Emtricitabine at doses of 1 µM. Tenofovir aug- Kalábová H, Študentová H, Zezulová M, mented LPS induce NF-κB activity significantly Holecková P, Krcmová L, Kašparová M, Plíšek J, not until doses of 10 µM. Due to the reported data Solichová D, Melichar B on obtainable concentrations of NRTIs in the plasma of patients under treatment, ranging from Department of Oncology, Palacký University 10 nM to a maximum of 1 µM, a relevant effect Medical School and Teaching Hospital, Olomouc; may be considered for Lamivudine, Abacavir and Department of Analytical Chemistry, Charles Emtricitabine to enhance NF-κB activity and con- University School of Pharmacy, Departments of sequently the production of associated pro- Oncology and Radiotherapy and Gerontology and inflammatory factors such as tumor necrosis fac- Metabolic Care, Charles University Medical tor-α, nitric oxide, interferon-γ and several inter- School Teaching Hospital, Hradec Králové, and leukins. Institute of Radiation Oncology and Charles In summary, this study revealed, that THP-1- University First Medical School, Prague, Czech blue monocytes seem to be more sensitive to the Republic action of NRTIs than PBMCs, since LPS induced NF-κB activity was significantly enhanced by Increased serum or urinary concentrations of Abacavir and Lamivudine at doses in the neopterin have been described in patients with nanomolar concentration range. In contrast, tumors of different primary locations, but reports PBMCs showed significant effects of NRTIs not on neopterin in patients with breast carcinoma are until doses of 100 µM, except Lamivudine, which relatively innumerous. We have evaluated urinary enhanced mitogen stimulated neopterin formation neopterin in 456 patients with breast carcinoma. in PBMC at doses of 1 µM. In conlusion, results Urinary neopterin was determined using high- of this in vitro study provide a rationale that performance liquid chromatography. Neopterin Abacavir and surprisingly also Lamivudine and was increased above the threshold of 205 Emtricitabine may enhance inflammatory path- µmol/mol creatinine that was defined as upper ways induced by NF-κB, and thereby potentially limits of normal in an earlier study, in 113 patients may contribute to increased risk of cardiovascular (25%). Urinary neopterin was significantly high- disease and accordingly also MI in HIV-infected er in 55 patients with metastatic or recurrent patients. breast carcinoma compared to the other patients (mean ± standard deviation 224 ± 203 vs. 176 ±

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 43

177 µmol/mol creatinine, p <0.05). Increased uri- that contribute to the degradation of collagen in nary neopterin was associated with significantly the fibrous cap. Macrophages activated by inter- inferior overall survival in patients with tumor feron-γ synthesize neopterin (1) which, in turn, expressing hormone receptors, human epidermal can interfere with intracellular signalling path- growth factor receptor-2, but not in patients with ways. triple negative tumors. In conclusion, serum In 1991, Melichar et al. found increased uri- neopterin is increased only in a minority of nary neopterin concentrations in patients with patients with breast carcinoma, but high neopterin acute myocardial infarction (AMI).(2) Several concentrations herald poor prognosis. studies have shown that circulating neopterin concentrations are higher in patients with acute coronary syndromes (ACS) than in patients with Neopterin and Cardiovascular Disease chronic stable angina pectoris (CSA) and healthy persons (3, 4). Schumacher et al. reported elevat- Kaski JC ed neopterin levels in patients with chronic CAD and more pronounced increased levels in patients Division of Cardiac and Vascular Sciences. St with AMI (3) and Gupta et al. showed signifi- George's University of London, London, UK cantly higher neopterin levels in patients with ACS than in patients with a previous AMI (5). Current evidence supports a key role for Increased neopterin is associated with the pres- inflammation in all phases of atherosclerosis, ence of vulnerable or disrupted atheromatous from endothelial damage through to disease pro- plaques (6, 7) and represents a marker of gression and, ultimately, the cardiovascular com- increased risk of further events in patients with plications of atherosclerosis. The growing under- ACS. Recent studies from our group have shown standing of the role of inflammation in atheroge- that increased serum neopterin predicts rapid nesis has focused attention on whether circulating CAD progression (8) and the development of levels of inflammatory biomarkers may help to major adverse coronary events in patients with identify patients at risk of future cardiovascular CSA (9, 10) and in hypertensive persons without events. Of all available biomarkers, the most obstructive CAD (11). widely studied has been C-reactive protein In patients with CSA, García-Moll et al. found (CRP), a nonspecific acute-phase protein, which that women who developed cardiac events during has been shown to be an independent predictor of follow-up had higher baseline neopterin concen- coronary risk in patients with angina and in appar- trations than those without events (10). Studies ently healthy subjects. from Avanzas et al. (9, 11) demonstrated that ele- In recent years, neopterin, an aromatic pteri- vated neopterin levels were an independent pre- dine with low molecular mass (253 Da) has been dictor of major adverse coronary events during a suggested to be a useful tool in the assessment of 1-year follow-up in hypertensive patients with cardiovascular risk. Neopterin is mainly produced typical angina and without significant angio- by activated macrophages and thought to repre- graphic atherosclerotic disease and also in sent a marker of immune activation and patients with CSA, irrespective of CAD severity. macrophage activity. (1) Several studies have In ACS Auer et al. (12) showed that neopterin shown that macrophages play a key role in athero- levels, measured 72 hours after the onset of the matous plaque progression and disruption. index ACS, were significantly higher in patients Indeed, plaque vulnerability has been shown to be with a clinical history of major cardiovascular a function of the increased numbers of events. In a recent large study, Ray et al. demon- macrophages and lymphocytes. Activated lym- strated a relationship between plasma neopterin phocytes produce interferon-γ, which reduces col- levels and acute coronary events (nonfatal MI or lagen synthesis within atheromatous plaques and unstable angina) in patients with ACS enrolled in activates macrophages. Activated macrophages the PRavastatin Or atorVastatin Evaluation synthesize and release matrix metalloproteinases Infection Therapy-Thrombolysis In Myocardial

Pteridines/Vol. 21/No. 2 44 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines

Infarction (PROVE IT-TIMI 22) trial (13). neopterin: A marker of coronary artery disease The Systemic Inflammation Evaluation in activity in women. Heart 2000;83:346-50. Patients with non-ST segment elevation Acute 11 Avanzas P, et al. Prognostic value of neopterin coronary syndromes (SIESTA) study (14) levels in treated patients with hypertension assessed the relationship between plasma and chest pain but without obstructive coro- neopterin levels and adverse clinical outcomes in nary artery disease. Am J Cardiol Mediterranean patients with non-ST segment ele- 2004;93:627-9. vation (NSTE) ACS. In this study, raised 12 Auer J, et al. Prognostic significance of neopterin levels (> 9.56 nmol/L) were a predictor immune activation after acute coronary syn- of increased risk at 180-day follow up. Moreover, dromes. J Am Coll Cardiol 2002;39:1878. the addition of neopterin to TIMI risk score 13 Ray KK, et al. Long-term prognostic value of increased its prognostic ability. These data taken neopterin: A novel marker of monocyte activa- together indicate that neopterin is an independent tion in patients with acute coronary syndrome. marker of cardiovascular risk. Circulation 2007;115: 3071-8. 14 Kaski JC, et al. Elevated serum neopterin lev- References: els and adverse cardiac events at 6 months fol- 1 Huber C, et al. Immune response-associated low-up in Mediterranean patients with non- production of neopterin. Release from ST-segment elevation acute coronary syn- macrophages primarily under control of inter- drome. Atherosclerosis 2008; 201:176-83. feron-gamma. J Exp Med 1984;160:310-6. 2 Melichar B, et al. Increased urinary neopterin in acute myocardial infarction. Clin Chem Metabolism of Fluorescence Labelled-alkyl- 1994;40:338-9. glycerols and Fatty Aldehydes by Cultured 3 Schumacher M, et al. Increased neopterin in Cells patients with chronic and acute coronary syn- dromes. J Am Coll Cardiol 1997;30:703-7. Keller MA, Watschinger K, Golderer G, 4 Schumacher M, et al. Neopterin levels in Hermetter A, Werner-Felmayer G, Werner ER patients with coronary artery disease. Atherosclerosis 1992;94:87-8. Division of Biological Chemistry, Biocenter, 5 Gupta S, et al. Serum neopterin in acute coro- Innsbruck Medical University, Innsbruck; and nary syndromes. Lancet 1997;349:1252-3. Institute of Biochemistry, Graz University of 6 Avanzas P, et al. Markers of inflammation and Technology, Graz, Austria multiple complex stenoses (pancoronary plaque vulnerability) in patients with non-ST In cells ether lipids have important roles in segment elevation acute coronary syndromes. maintaining proper brain function, spermatogene- Heart 2004;90:847-52. sis, mediation of inflammation, anchoring pro- 7 Garcia-Moll X, et al. Serum neopterin and teins to membranes and lens organisation. The complex stenosis morphology in patients with tetrahydrobiopterin-dependent enzyme alkylglyc- unstable angina. J Am Coll Cardiol erol monooxygenase (glycerylether monooxyge- 2000;35:956-62. nase, E.C. 1.14.16.5) is so far the only known 8 Zouridakis E, et al. Markers of inflammation enzyme that is able to cleave the ether bond of and rapid coronary artery disease progression these lipids producing a free fatty aldehyde and a in patients with stable angina pectoris. glycerol derivative. The aldehydes thus formed Circulation 2004;110:1747-53. are converted to the corresponding acids by fatty 9 Avanzas P, et al. Elevated serum neopterin pre- aldehyde dehydrogenase (FALDH; E.C. dicts future adverse cardiac events in patients 1.2.1.48). If this enzyme is deficient, the toxic with chronic stable angina pectoris. Eur Heart aldehydes and related metabolites accumulate and J 2005;26:457-63. cause the autosomal recessive disorder Sjögren 10 Garcia-Moll X, et al. Increased serum Larsson Syndrome (SLS) which is characterised

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 45 by ichthyosis, mental retardation and spasticity. including natural camphor and gypsum. Padma To follow the fate of the fluorescent labelled Basic, a modification of Padma 28, contains the substrates 1-O-pyrenedecyl glycerol, pyrenede- same active ingredients with the exception of canal and pyrenedecanol in different types of aconiti tuber. Ethanolic extracts of Padma 28 and cells, we have developed sensitive methods for Padma Basic were shown to exert significant sup- the quantification of various metabolites of the pressive effects on neopterin production and tryp- applied substrate in the supernatant medium of tophan degradation in mitogen-stimulated periph- cultured adherent cells. eral blood mononuclear cells (PBMC) in vitro. In We found significant differences in metabolism this study, earlier obtained data on the effects of of normal human fibroblasts (HFB) and fibrob- ethanolic extracts of Padma 28 on tryptophan lasts of an SLS patient. After incubation with 10 degradation in phytohaemagglutinin A (PHA)- µM aldehyde substrate for 24h, in normal human stimulated and unstimulated PBMC were con- fibroblast primarily the fatty acid was formed, firmed and also observed with ethanolic extracts while in SLS cells the fatty alcohol was accumu- of Padma Basic and water extracts of both formu- lated. In both cell lines the toxic aldehyde was las. In contrast to the ethanolic extracts, Padma 28 removed almost entirely. and Padma Basic extracted in water, enhanced Incubation with 10 µM alcohol substrate tryptophan degradation in unstimulated cells. revealed that HFB cells are able to metabolise the Additionally, the antioxidative capacity of all alcohol into the corresponding acid while this was extracts were investigated using the cell-free oxy- not the case for SLS cells, in which only margin- gen radical absorbance capacity (ORAC)-Assay al amounts of acid was formed. Experiments with and a cellular fluorescence based antioxidant purified, recombinant rat FALDH demonstrated activity assay, which measures formation of reac- that the enzyme alone is not able to catalyse this tive oxygen species (ROS) upon stimulation with reaction. This suggests that FALDH is essential peroxyl radical generating 2,2`-azobis-2-methyl- but not solely responsible for the degradation of propanimidamide (AAPH). Furthermore, the fatty alcohols. effects of both preparations were tested on their capacity to modulate nuclear factor (NF)- B activ- Supported by the Austrian Research Funds "zur ity and expression of heme oxygenase-1 (HO-1). Förderung der wissenschaftlichen Forschung" Modulation of NF-κB activity was monitored in P19764 and "Tiroler Wissenschaftsfonds" lipopolysaccharide (LPS)-stimulated and unstim- TWF0404_589. ulated THP-1-blue monocytes, stably expressing a reporter plasmid, containing secreted embryon- ic alkaline phosphatase gene under the control of Dose-dependent effects of Tibetan remedies on a promoter inducible by the transcription factor peripheral blood mononuclear cells in vitro NF-κB. Expression of HO-1 was determined in PHA-stimulated PBMC, in unstimulated THP-1- Klein A, Jenny M, Gostner J, Vennos C, blue cells and hepatocarcinoma Hep-G2 cells. Schennach H, Fuchs D, Ueberall F Compared to the effects of Padma 28 and Padma Basic extracted in water, the ethanolic extracts of Division of Biological Chemistry and Division of these formulas showed a stronger antioxidant Medical Biochemistry, Biocenter, Innsbruck, capacity in the ORAC-assay and also a more pro- Austria; and PADMA AG, Schwerzenbach, nounced inhibition of AAPH-induced intracellu- Switzerland lar formation of ROS in Hep-G2 cells. Treatment of LPS-stimulated THP-1-blue cells with both, The Tibetan herbal remedy Padma 28 is used the ethanolic and water extracts of both formulas, for the treatment of disturbances of blood circula- diminished NF-κB activity after six hours of tion, varicosis, arteriosclerosis, rheumatism and treatment to about 70%, at highest concentrations chronic inflammation disorders. Padma 28 is tested (100µg/ml). In contrast, twenty-four hours composed of twenty different powdered plants after treatment, LPS induced NF- B activity was

Pteridines/Vol. 21/No. 2 46 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines enhanced to about 117% and 110% by the addi- In parallel, concentrations of immune activation tion of Padma 28 and Padma Basic extracted in marker neopterin and of cytokines tumor necrosis ethanol. Both formulas extracted in water also factor-α and interleukin-6 (IL-6) and soluble IL-2 enhanced the LPS-induved NF-κB activity to receptor-α and various T-cell subpopulations we- about 127% and 173% respectively. Expression re analyzed. of HO-1 was induced in a dose-dependent manner Patients presented with significant decline of by the ethanolic extracts of both formulas in tryptophan concentrations (56.7 vs. 64.2 µmol/L; PBMC, THP-1-blue cells and Hep-G2 cells. In p <0.01) which was more expressed in patients summary, Padma 28 and Padma Basic extracted with a greater Hamilton depression (HAM-D) =− < in ethanol, generally showed stronger activity score (rs 0.48, p 0.05). Kynurenine concen- compared to the formulas extracted in water, trations and the kynurenine to tryptophan ratio although both formulas exhibit comparable (kyn/trp) were lower in patients, but only the dif- antioxidative properties and a potential to modu- ference of kynurenine levels reached the level of late inflammation induced pathways. statistical significance (1.82 vs. 1.45 µmol/L; p <0.01). Moreover, kynurenine concentrations correlated with weight loss in patients. Changes in LNAA in Patients with Depressive Out of the immunological parameters, only Disorders neopterin differed significantly and was higher in patients (mean: 6.3 nmol/L) than in controls Kohl C, Fuchs D, Birkner Ph, Jakob G, Pröll S, (mean: 5.2 nmol/L; p = 0.01) whereas the investi- Koudouovoh-Tripp P, Sperner-Unterweger B gated cytokines and T-cell subsets were not dif- ferent between patients and controls. Phenyl- Division of Biological Psychiatry, Departement alanine was significantly lower in patients and of Psychiatry, Division of Biological Chemistry, also tyrosine levels showed a tendency of lower Biocenter, Innsbruck Medical University. levels in patients. Results of the General Sleep Innsbruck, Austria Questionnaire demonstrated significant sleep dis- turbances in patients with major depression. Large neutral amino acids (valine, isoleucine, Thereby sleep duration correlated inversely with leucine, tyrosine, phenylalanine; LNAA) compete kyn/trp and difficulty falling asleep was associat- for transport across the blood-brain barrier via the ed with higher neopterin concentrations. L-type amino acid carrier. The molar ratio of total We conclude that there are significant distur- plasma tryptophan to the sum of other LNAA is bances of tryptophan and neopterin metabolism in thought to reflect brain serotonin formation. In patients with major depression, patients present- vitro and in vivo studies suggest a link between ing with higher neopterin and lower tryptophan. immune activation and changes of aromatic However, also kynurenine concentrations were amino acids tryptophan and phenylalanine (1-3) decreased and thus kyn/trp was not altered. Thus, which are of great importance for the proper data do not support a role of IDO in tryptophan biosynthesis of neurotransmitters. In patients suf- metabolic changes in major depression. Pathways fering form cancer and HIV-1 infection as well as involved in the tryptophan metabolic changes in animal model systems a role of tryptophan- observed might be distinct from those involved in degrading enzyme indoleamine 2,3-dioxygenase the pathogenesis of mood disturbances in patients (IDO) in the precipitation of depressive symp- suffering from chronic inflammatory disorders. toms has been reported (4-6). In order to find a possible relevance of these immunobiochemical References: alterations in the development of psychoneuroim- 1 Widner B, et al. Neopterin production trypto- munological alterations in patients with major phan degradation and mental depression: what depression, we compared plasma tryptophan, is the link? Brain Behav Immun 2002;16:590- kynurenine, phenylalanine and tyrosine concen- 5. trations in 27 patients and in 31 healthy controls. 2 Neurauter G, et al. Chronic immune stimula-

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 47

tion correlates with reduced phenylalanine mins before ART, 30 patients were B-vitamin sup- turn-over. Curr Drug Metabol 2008;9:622-7. plemented during ART, while 40 did not take vita- 3 Neurauter G, et al. Serum phenylalanine con- mins. Neopterin, iron, transferrin, ferritin and centrations in patients with ovarian carcinoma GDF-15 were measured by ELISA, tryptophan correlate with concentrations of immune acti- and kynurenine were determined by HPLC. Viral vation markers and of isoprostane-8. Cancer load was quantified by real time PCR. Lett 2008;272:141-7. Thirty-two HIV-infected patients suffered from 4 Huang A, et al. Serum tryptophan decrease anaemia before ART (25 with anaemia of chronic correlates with immune activation and disease), 12 patients were anaemic after ART. impaired quality of life in colorectal cancer. Significant correlations existed between markers Brit J Cancer 2002;86:1691-6. of immune activation (neopterin, tryptophan 5 Schroecksnadel K, et al. Quality of life and degradation) and parameters of iron metabolism immune activation in patients with HIV infec- (ferritin, GDF-15) before ART. ART was very tion. Brain Behav Immun 2008;22:881-9. effective to decrease viral load, slow down 6 O'Connor JC, et al. Lipopolysaccharide- immune activation and modulate iron metabo- induced depressive-like behavior is mediated lism: Neopterin, kynurenine and ferritin concen- by indoleamine 2,3-dioxygenase activation in trations decreased significantly, while tryptophan mice. Mol Psychiatry 2009;14:511-22. and GDF-15 concentrations raised. Concentrations of vitamin B12 increased under ART, while folic acid levels decreased. Iron Metabolic Changes and Immune Differences were encountered between patients Activation in Patients with HIV 1 Infection who received vitamin supplementation and those before and after Antiretroviral Therapy who did not: Only patients, who received vitamin supplementation during ART, showed a signifi- Kurz K, Schroecksnadel S, Seifert M, Sarcletti M, cant increase of haemoglobin levels. Fuchs D, Zangerle R, Weiss G Disturbed iron metabolism and enhanced tryp- tophan degradation coincide with immune activa- Clinical Immunology and Infectious Diseases, tion in patients with HIV-infection. B vitamin Department of Internal Medicine I, Division of supplementation during ART appears to be bene- Biological Chemistry, Biocenter, and Department ficial for patients with anaemia. of Dermatology and Venerology, Innsbruck Medical University, Innsbruck, Austria

A Murine Model of Human BH4-responsive Anaemia is a frequent complication in HIV- Phenylketonuria infected patients, several studies have shown that anaemia is a major prognostic marker for an Lagler FB, Gersting SW, Zsifkovits C, increased mortality and morbidity of patients. Glossmann H, Muntau AC Cyokine-mediated suppression of haematopoiesis and disturbed iron metabolism appear to play a University, 6020 Innsbruck, Austria; and key role in the development of HIV-related Department of Molecular Pediatrics, Dr. von anaemia. Hauner Children's Hospital, Ludwig-Maximi- In this study we examined markers of immune lians-University, 80337 Munich, Germany activation and iron metabolism in patients with HIV infection undergoing antiretroviral therapy Sapropterin dihydrochloride, the synthetic (ART). form of 6(R)-L-erythro-5,6,7,8-tetrahydrobiop-

105 patients with HIV-infection were recruited terin (BH4) was recently approved for the treat- from the department of Dermatology and ment of phenylketonuria. It corrects phenylala- Venerology (Innsbruck) and were treated with nine hydroxylase (PAH) deficiency and hyper- ART for 12 months. 25 patients received B-vita- phenylalaninemia by a mechanism different from

Pteridines/Vol. 21/No. 2 48 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines its cofactor action in a substantial share of Intensive Care Medicine, Medical University patients. In vitro studies using purified PAH Innsbruck, Austria pointed to a stabilization of the misfolded protein against accelerated proteolytic degradation. In Regular night shift work has been shown to

BH4 deficient and wild-type mice pharmacologi- evoke various adverse effects on employee's cal doses of BH4 increased the activity and con- health, mostly being associated with the disrup- centration of wild-type PAH protein. However, an tion of circadian rhythm. Unlike other profes- animal model of BH4-responsive PAH deficiency sions, medical personnel are scheduled to work was not available so far. on 24-hour shifts several times a month. The Pahenu1 mouse model was generated by Melatonin and neopterin concentrations eviden- germline mutagenesis earlier. It harbors the tially show a 24 h-periodicity, both indicating an V106A mutation in the regulatory domain of acrophase in the early morning hours. It was the PAH. Although the specific activity of purified aim of the present study to investigate whether the V106A PAH was unimpaired in vitro, Pahenu1 physiological diurnal rhythm was altered as a mice showed hyperphenylalaninemia (median, result of a single 24-hour shift in anaesthetists. 189 µmol/L; wild-type 76 µmol/L) and decreased Therefore, urinary neopterin and melatonin levels phenylalanine oxidation in vivo (33% of wild- that might possibly be altered due to desynchro- type) in accordance with low PAH activity in liver nization were measured. lysates. The decrease of PAH amount in Pahenu1 Over three consecutive days, including a 24- liver lysates and eukaryotic expression systems hour shift, urine samples were collected daily at was reversible upon protease inhibitors. This five different time points. Neopterin and aMT6-s pointed to a misfolding-induced instability of (6-sulfateoxymelatonin) courses were assayed V106A, which was confirmed by further in-vitro using high pressure liquid chromatography and a studies. BH4 treatment in vivo significantly low- commercially available competitive immunoas- ered blood phenylalanine concentrations (median: say kit, respectively. Neopterin/creatinine and 396 to 139 µmol/L), normalized the phenylala- melatonin/creatinine ratios were calculated. nine oxidation to wild type levels and increased Ten anaesthetists aged between 29 and 35 and the amount of PAH in liver lysates by conforma- ten medical students aged between 25 and 31 tional stabilization. were included in the study. Urinary neopterin val- In conclusion, PAHenu1 is a model for human ues ranged between morning values of (mean and

BH4 responsive PAH deficiency, that allows to 95% CI) 95.0 (76.3; 113.8) and 91.3 (73.7; 108.8) study misfolding-induced loss-of-function pathol- nmol/mg, late evening values of 77.4 (62.6; 92.1) ogy in vivo. It will facilitate the further elucida- and 77.1 (63.4; 90.8) nmol/mg for anaesthesists tion of disease specific pharmacological effects of and students, respectively. Neither the factor

BH4. Moreover Pahenu1 and the methods estab- "time" nor of the factor "group" was found to lished in this project will enable in vivo evaluation have a significant effect on neopterin courses. of novel therapeutic molecules for the treatment aMT6-s fluctuated between values of 21.6 (17.0; of BH4-responsive PKU. 26.2) and 17.6 (15.1; 20.1) pg/mg in the morning and 7.3 (3.5; 11.1) and 4.7 (2.3; 7.1) pg/mg at late night for anaesthetists and students, respectively. Circadian Neopterin Profiles During a Single A marked circadian rhythm of aMT6-s courses 24-hour Shift in Anaesthetists was observed in both groups, but no effect of the factor "group" could be found calculating analy- Leichtfried V, Putzer G, Perkofer D, ses of variance. Schobersberger W, Benzer A Similar to previous investigations of our group nocturnal urinary neopterin/creatinine ratio did Institute for Sports Medicine, Alpine Medicine & not show diurnal variations over study duration. Health Tourism, UMIT Hall and TILAK We suspect that due to those differing results Innsbruck, Clinic for Anaesthesiology and compared to other studies, our results could give

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 49 evidence for an impact of a single 24-h shift on Nifedipine treated mice, independently of circadian disruption. On the contrary, melatonin dietary iron overload, showed an improved sur- courses did not indicate circadian disturbance. vival and presented with reduced numbers of Total duration of shift work experience did not Salmonella in the liver and the spleen as com- seem to have an effect with showing similar pared to solvent injected animals. Even though neopterin and aMT6-s courses in anaesthetists these effects were more pronounced in the iron and students. diet fed group. Serum iron levels were decreased as a reflection of the iron depleting effects of the drug. This was paralleled by decreased ferritin Nifedipine Affects the Course of Salmonella levels in the liver and spleen and increased ferro- Infection by Pharmacological Modification of portin 1 expression as determined by means of Iron Homeostasis western blot analysis. Interestingly, we did not observe differences in the expression of a panel of Mair SM, Nairz M, Muehlbacher T, Schroll A , pro- and anti-inflammatory cytokines according Sonnweber T, Weiss G to nifedipine treatment. Our data provide evidence that nifedipine may Department of Internal Medicine I, Clinical be a promising adjunctive therapy for the treat- Immunology and Infectious Diseases, Medical ment of infections with intracellular pathogens. University of Innsbruck, Austria Mechanistically the iron depleting effects may be due to limitation of the availability of the essen- Iron overload aggravates the clinical course of tial nutrient iron for intracellular pathogen as a infections by negatively affecting cell mediated consequence of nifedipine mediated induction of immune mechanisms of macrophages and T cellular iron export via ferroportin. helper cell type 1 effector pathways. Recently, we have shown that the calcium antagonist nifedipine enhances DMT1 mediated iron transport in vitro Dynamic Relations between Physical Activity and causes subsequent depletion from liver and and Cellular Immunity in Three Patients with the circulation in iron overloaded mice. Herein, SLE: a Time Series Analysis Approach we thus investigated whether nifedipine may impact on the clinical course of an invasive infec- Mandl M, Fuchs D, Schubert C tion via modulation of iron homeostasis. RAW 264.7 cells were infected with Clinic of Medical Psychology, and Division of Salmonella typhimurium and stimulated with Biological Chemistry, Biocentre, Innsbruck varying concentrations of nifedipine. Mice were Medical University, Innsbruck, Austria fed with a control diet or iron enriched diet for three weeks, infected intraperitoneally with Systemic lupus erythematosus (SLE) is a Salmonella typhimurium and treated with solvent chronic autoimmune disease with various clinical control or nifedipine for three consecutive days. manifestations. Until today, few studies have 24 hours later mice were sacrificed, the bacterial investigated the influence of physical activity on loads were quantified in the liver and the spleen, the immune system in patients with SLE. It is and the expression of iron regulating genes was suggested that these studies produced inconsistent determined by means of RT-PCR and Western findings because of not considering the temporal blot analysis. qualities of the interaction between physical The cell culture model revealed a significant activity and immune activity under real-life con- decrease of the bacterial load upon nifedipine ditions. treatment. In RT-PCR analysis an increase of the Three females (46, 52, and 34 years-old) with iron exporter ferroportin1 expression was SLE in remission participated in this study. observed following treatment with nifedipine Patients collected their entire urine and assessed leading to iron restriction within macrophages. their daily physical activity via a visual analogue

Pteridines/Vol. 21/No. 2 50 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines scale (VAS) ranging from 0 to 10 cm over a peri- 04510, México DF, and Departamento de od of 56 consecutive days in 12-hour intervals Ecología Evolutiva, Museo Nacional de Ciencias (total of 112 consecutive measurements). After Naturales, CSIC. C/José Gutierrez Abascal, 2. the investigation period, urinary neopterin and 28006. Madrid, Spain creatinine were measured applying HPLC. Before cross-correlational analyses, time series were Pterins (2-amino-4(3H)-oxo-pteridine deriva- controlled for serial dependencies applying tives) are common animal colorants which consti- ARIMA-modeling. In all three patients physical tute heterocyclic compounds and have the highest activity positively correlated twice with urinary nitrogen content of any pigment analyzed from neopterin concentrations, meaning that a certain animals. It has been reported that pterins modu- time after exercise two elevations of urinary late oxidative stress as these molecules are able to neopterin followed. In Case 1, increases of phys- scavenge free radicals. Previous reports suggest ical activity were followed by urinary neopterin three possible mechanisms that are responsible increases after 12 and 144 hours, in Case 2 after for scavenging free radicals; these are electron 72 and 144 hours, and in Case 3 after 36 and 132 transfer (ET) reaction, hydrogen atom transfer hours. As elevations of neopterin levels after (HAT) and radical addition. In this paper, the physical engagement have hitherto only been seen capacity to scavenge free radicals (antiradical in athletes after highly intense training (1), this capacity) of pterins is analyzed, using Density study suggests that patients with SLE may react Functional Approximation calculations and con- more sensitively to physical activity than healthy sidering two possible mechanisms: ET and HAT. individuals. Although a multiphased immune For the electron transfer process, the Full Electron response after physical engagement has already Donator Acceptor Map (FEDAM) of pterins indi- been described in literature (2), a rise in cates that the antiradical capacity of those pterins neopterin-levels after 144 hours in response to is lower than the antiradical capacity of any physical activity is a novum. Further integrative carotenoids. In terms of the HAT mechanism, single-case studies combining subjective and there is a correlation between the dissociation objective measurement of physical activity have energy involved in the removal of one hydrogen to follow in order to strengthen the findings of atom from pterins and its antiradical capacity. this study. Pterins are depicted as poorer antiradicals than carotenoids in terms of the HAT mechanism, with References: biopterin, sepiapterin and 7,8-dihydrobiopterin 1 Schobersberger W et al. Increase in immune representing the only exceptions. Further studies activation, vascular endothelial growth factor focusing on the third mechanism (radical addi- and erythropoietin after an ultramarathon run tion) are necessary in order to fully understand the at moderate altitude. Immunobiology antiradical capacity of these substances. For this 2000;201:611-620. reason, work continues in order to clarify these 2 Cunniffe B et al. Time course of changes in aspects. immuneoendocrine markers following an international rugby game. Eur J Appl Physiol 2010;108:113-122. Pharmacological Chaperones for the Aromatic Amino Acid Hydroxylases

Are Pterins Able to Modulate Oxidative Stress? Martinez A, Calvo AC, Scherer T, Pey AL, Ying M, Winge I, McKinney J, Teigen K, Haavik J, Martínez A, Barbosa A Thöny B

Instituto de Investigaciones en Materiales, Department of Biomedicine, University of Universidad Nacional Autónoma de México, Bergen, Bergen, Norway, and Department of Circuito Exterior S. N., Ciudad Universitaria, CP Pediatrics, University of Zürich, Zürich,

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 51

Switzerland Prognostic Significance of Urinary Neopterin in Colorectal Cancer Patients Treated with The aromatic amino acid hydroxylase (AAAH) Cetuximab enzyme family includes phenylalanine hydroxy- lase (PAH), tyrosine hydroxylase (TH), and the Melichar B, Kalábová H, Študentová H, Zezulová tryptophan hydroxylases (TPH-1 and 2). These M, Holecková P, Krcmová L, Kašparová M, enzymes are structurally and functionally related Plíšek J, Solichová D and in mammals they are mainly distributed in liver and kidney (PAH) and neuronal and neu- Department of Oncology, Palacký University roendocrine tissues (TH and TPH-1 and 2). The Medical School and Teaching Hospital, Olomouc; AAAHs share a number of ligands, such as the Department of Analytical Chemistry, Charles cofactor tetrahydrobiopterin (BH4) and inhibitors. University School of Pharmacy, Departments of In fact the AAAHs do not show strict substrate Oncology and Radiotherapy and Gerontology and selectivity, and can utilize all three aromatic Metabolic Care, Charles University Medical amino acids as substrates to some extent. It is thus School Teaching Hospital, Hradec Králové, and important to investigate binding selectivity for Institute of Radiation Oncology and Charles compounds used in therapeutic interventions aim- University First Medical School, Prague, Czech ing the modulation of each AAAH, such as BH4, Republic stabilizers (pharmacological chaperones) and inhibitors. Through high-throughput screening we An increase of urinary neopterin has been recently discovered four compounds (I-IV) that described after administration of different anti- functioned as pharmacological chaperones for cancer agents. Cetuximab is a chimeric antibody PAH (Pey et al. (2008) J Clin Invest 118, 2858- used in the therapy of advanced colorectal carci- 2867). These compounds stabilized the functional noma that may act partly through activation of tetrameric conformation of recombinant wild- host imine response. We have studied urinary type PAH and PKU mutants, and increased neopterin in 44 pre-treated patients with metasta- steady-state PAH protein levels in eukaryote cells tic colorectal carcinoma during therapy with and liver of treated mice. We now investigated the cetuximab, in the majority of cases in combina- effect of these compounds on TH and TPH2 in tion with irinotecan, 5-fluorourcil and leucovorin. vitro, comparative to PAH, by differential scan- Urinary neopterin was determined by high per- ning fluorimetry. For the most effective com- formance liquid chromatography A significant pounds (II-IV) we also measured their in vivo correlation was observed between urinary effect. Only compound III (3-amino-2-benzyl-7- neopterin and peripheral blood leukocyte count = < = nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1- (rs 0.38; p 0.05), hemoglobin (rs -0.34; < = < one) bound and stabilized all three purified p 0.05) and CEA (rs 0.33; p 0.05) concentra- enzymes (PAH, TH (isoform 1) and TPH2), and tion. Seventeen patients had urinary neopterin increased TH activity in treated mice. For the equal or above 214 µmol/mol creatinine. Survival other compounds, different effects were measured of patients with urinary neopterin concentration for their action in vitro and in vivo for each of 214 µmol/mol creatinine or above was signifi- AAAH. Our results will be discussed in the con- cantly inferior compared to patients with initial text of the potential of these compounds as phar- urinary neopterin below 214 µmol/mol creatinine macological chaperones for the treatment of dis- (median 10.1 vs. 17.5 months, p <0.05). Urinary orders associated with AAAH misfolding. neopterin concentrations increased during the therapy only in patients with normal pre-treat- ment neopterin concentrations. In conclusion, an increase of urinary neopterin in patients treated indicates the activation of systemic immune response by the therapy that could be responsible for anti-tumor activity of cetuximab. Urinary

Pteridines/Vol. 21/No. 2 52 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines neopterin is a prognostic factor in patients with increased retinol and alpha-tocopherol concentra- metastatic colorectal carcinoma treated with tions. cetuximab.

Native and Aadaptive Immune Response: Serum Homocysteine, C-reactive Protein, Serum Neopterin Concentrations in Healthy Retinol and Alpha-tocopherol and Urinary Blood Donors are not Influenced by MBL Neopterin in Patients with Breast Carcinoma Polymorphism Treated with Bevacizumab Muehlbacher A, Scholz L, Gassner C, Grabmer C, Melichar B, Kalábová H, Studentová H, Zezulová Mayersbach P, Schennach H M, Holecková P, Krcmová L, Kasparová M, Plísek J, Solichová D Central Institute for Blood Transfusion and Immunology, General Hospital and University Department of Oncology, Palacký University Clinics, Innsbruck, Austria Medical School and Teaching Hospital, Olomouc; Department of Analytical Chemistry, Charles Mannose-binding lectin (MBL) is an important University School of Pharmacy, Departments of component of the immune defence able to bind to Oncology and Radiotherapy and Gerontology and repeating mannose based structural patterns typi- Metabolic Care, Charles University Medical cal of microbial surface. Importantly, it also acti- School Teaching Hospital, Hradec Králové, and vates the complement system through a distinc- Institute of Radiation Oncology and Charles tive third pathway, independent of both antibody University First Medical School, Prague, Czech and the C1 complex. The serum MBL level is Republic variable in healthy population. The mean serum MBL level in an individual is genetically deter- Increased serum or urinary concentrations of mined by three single nucleotide substitutions in neopterin, an indicator of systemic immune exon-1 of the human MBL2 gene also referred as response, have been described in patients with D, B, and C variants whereas the wild type allele tumors of different primary locations, and further is referred as A. These mutations result in amino increase has been observed during anticancer acid substitution in the collagen-like domain, therapy. An increase of urinary neopterin has been thereby reducing the amount of functional serum described after administration of cytokines, cyto- MBL level. In addition, three pairs of allelic toxic chemotherapy or external beam radiation, dimorphisms can occur in the downstream pro- but much less is currently known about the effects moter. The combination of structural gene and of targeted agents on systemic immune response. promoter polymorphisms results in a dramatic We have studied serum homocysteine, C-reactive variation in MBL concentration in apparently protein, alpha-tocopherol and retinol, and urinary healthy individuals of up to 1000 fold (Caucasian neopterin in patients with metastatic breast cancer <20-10,000ng/ml). The variant alleles are very treated with bevacizumab, taxane and carbo- frequent in normal, healthy populations, where platin. Homocysteine and C-reactive protein were they are present in 20 to 50% of the individuals. determined immunochemically. Alpha-toco- Epidemiological studies have suggested that pherol, retinol and urinary neopterin were deter- genetically determined variations in MBL serum mined by high performance liquid chromatogra- concentrations influence the susceptibility to and phy. Homocysteine, CRP and urinary neopterin the course of different types of infectious, autoim- decreased, while retinol and alpha-tocopherol mune, neoplastic, metabolic and cardiovascular increased during the therapy. In conclusion, a diseases. decrease of urinary neopterin and C-reactive pro- In this study we investigated the influence of tein during bevacizumab therapy was associated MBL polymorphisms on serum neopterin concen- with decreased serum homocysteine and trations.

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 53

Blood samples were obtained from 67 unrelat- the early puerperal period in normal gravidity ed healthy platelet apheresis donors. Genomic without infection, with normal gestation age and DNA was isolated from mononuclear cells using healthy newborns. Nucleon BACC2 kit from Amersham Biosciences Blood specimens were obtained from 46 moth- and screened for the relevant MBL variants with ers (23 with spontaneous delivery/23 Cesarean PCR-SSPs based on the same techniques, as section) together with cord blood aged median applied for routine typing of HLA. Additional 31/30, range 22-37/24-46 years. Delivery was at MBL serum levels were measured using MBL week 40 ± 1, the Apgar scores of newborns Ologomer Elisa Kit from BioPorto. For compari- ranged within 8-10, all were without puerperal son serum neopterin concentrations from the last infection. In the plasma (EDTA) samples we 10 donations of every donor were used. analysed concentrations of neopterin (ELISA, According to the literature donors were BRAHMS, Hennigsdorf, Germany), reference grouped into low, intermediate and high MBL range: 5.3 ± 2.7 nmol/L, PCT (TRACE - Kryptor, levels. BRAHMS Germany) reference range: <0.05 Based on our results 22.4%, 11.9% and 65.7% ug/L and ultrasensitive CRP (CRPus; TRACE - of the donors belonged to the low, intermediate Kryptor, BRAHMS Germany) reference range: and high group, respectively, confirming the find- 0.07 - 8.2 mg/L. In total 5 specimens of each ing of other European studies. There was no dif- mother/child pair were examined, 4 specimens ference in serum neopterin concentrations obtained from the mother (days 1, 3, 4, 5) and between the 3 groups, median ranges from 3.6 to cord blood. Blood cell counts were measured and 6.8 nmol/L in the low, 3.6 to 6.9 nmol/L in the residual plasma was divided into 2-3 aliquots and intermediate, and 3.8 to 6.2 nmol/L in the high frozen at -35 °C. In the early puerperium mean group. There were 2 donations from 2 different neopterin concentrations were 9.3 (S.D.: ±2.7, donors with neopterin levels >10 nmol/L both median: 8.2; d1), 10.1 (±2.9, 10.0; d3), 8.5 (±2.4, belonging to the high group. 7.7; d4) and 8.4 (±2.3, 9.0; d5) nmol/L in samples obtained after normal delivery and slightly but not significantly higher in samples obtained after Inflammatory Markers in Post-partum Period Cesarean section was performed 9.35 (+2.4, 10.0; d1), 10.2 (±3.4, 9.5; d3), 11.0 (±4.0, 9.8; d4) and Parrak V, Malova A, Secnik P, Redecha M, 9.8 (±3.2, 8.4; d5). Neopterin concentrations were Mistrik M, Fuchs D about twice as high in cord blood as compared with concentrations obtained in plasma speci- St. Cyril and Methods Hospital, Bratislava- mens from mothers, again there was no signifi- Petrzalka, and SK-lab, Lucenec, Slovakia; and cant difference between samples obtained after Division of Biological Chemistry, Biocenter, normal delivery (17.4±4.9, 17.9; cord blood) as Innsbruck Medical University, Innsbruck, Austria compared with Cesarean section (20.4±5.7, 20.7; cord blood). PCT concentrations showed a similar The most commonly used marker to detect pattern as neopterin in mothers, but also cord infectious and inflammatory responses in the clin- blood specimens contained a comparable PCT ics today is C-reactive protein (CRP), e.g., clini- concentration as compared with blood specimens cians in gynecology or surgery use CRP, some- of mothers. CRP levels were significantly higher times procalcitonin (PCT) is applied. Other mark- in days 3-5 as compared with day 1, CRP was ers as macrophage product neopterin and some almost undetectable in cord blood specimens. interleukins seem to be only introduced as scien- CRP levels after normal vaginal delivery how tific parameters. Also gravidity can be associated similar dynamic compare to Cesarean section, with inflammatory complications which very with higher levels after Cesarean section most often take place in the puerperal period. The aim probably due to damage of tissue. In both groups of this study is a wide variation scale, which complicates the was to compare some inflammatory markers in use of this marker alone in the diagnostic of early

Pteridines/Vol. 21/No. 2 54 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines puerperal infection. pterin deaminase which could be explored or In sum, there are significant changes of manipulated for the production of a potent anti- neopterin, CRP and PCT in females after delivery. cancer drug. However, to dissect their possible clinical rele- vance further studies on the comparison between healthy normals and newborns with complica- Antioxidant Property Exhibited by Novel tions are still required. Enzyme Pterin Deaminase

Sabarathinam S, Priyadharshini U, Angayarkanni N-Terminal Sequencing of Pterin Deaminase J, Palaniswamy M from Micrococcus luteus BI252 using MALDI- TOF Cancer Therapeutics Laboratory, Department of Microbial Biotechnology, School of Priyadharshini U, Sabarathinam S, Angayarkanni Biotechnology and Genetic Engineering, J, Palaniswamy M Bharathiar University, and Department of Microbiology, Karpagam University, Coimbatore, Cancer Therapeutics Laboratory, Department of Tamil Nadu, India Microbial Biotechnology, School of Biotechno- logy and Genetic Engineering, Bharathiar Univer- With the advent of the enzymes as drugs, can- sity and Department of Microbiology, Karpagam cer therapy has highlighted one to focus and ven- University, Coimbatore, Tamil Nadu, India ture in search of an efficient enzyme as anti- tumour drug. The most unexplored and possibly a The most unexplored and possibly a potent potent drug is the enzyme pterin deaminase, anticancer drug is the enzyme pterin deaminase which deaminates folic acid to products 2- belonging to class of hydrolase, which deami- deamino-2-hydroxy folic acid and ammonia. nates pterin to products lumazine and ammonia. Inspite of the patent and exploration of the pre- The enzyme pterin deaminase has been patented liminary antitumour property of enzyme pterin for the antitumour property against leukaemia. deaminase in prokaryotes and eukaryotes, very Inspite of the patent, very few works has been car- few works has been carried out in elucidating the ried out in exploring the structural properties of various roles or properties of the enzyme. The the enzyme. reasons behind the lack of work are unclear cur- In the present study, an attempt has been made rently and this tempted us to speculate and inves- to purify the enzyme pterin deaminase to homo- tigate the properties of pterin deaminase from rat geneity from Micrococcus luteus BI252 and liver. sequence the N-terminal using MALDI-TOF. The In the present study, we have isolated and char- enzyme was purified to homogeneity in a sequen- acterized the enzyme pterin deaminase from rat tial manner using alcohol precipitation, Gel filtra- liver. The enzyme was purified to homogeneity in tion and Ion exchange column chromatography. a sequence by precipitation and ion-exchange The purified enzyme was run on Non-denaturing chromatography. The purified enzyme was gel electrophoresis and the obtained band was checked for its antioxidant properties. The digested using trypsin. The tryptic digest was sub- enzyme pterin deaminase exhibited antioxidant jected to MADLI-TOF analysis and the spectrum property by scavenging the hydrogen peroxide was predicted using MASCOT software. The N- free radicals compared to the free radical induced terminal sequence was then explored in the pro- by DPPH. tein database for the identification of protein Thus the present work has thrown a lime light using BLAST software and found to be novel on the antioxidant property of the enzyme pterin with less than 50% homogeneity. deaminase apart from the anti-tumour property. Thus the present study would throw light for the structural characterization of the enzyme

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 55

Phenylalanine to Tyrosine Ratio (phe/tyr ratio) ratios were measured in a patient with dihy- in Children with Neuropediatric Abnormali- dropteridine reductase (DHPR)-deficiency (BH4- ties deficiency, CSF 2.82 and plasma 2.28). Additionally, the ratios were elevated in some Scholl-Bürgi S, Karall D, Albrecht U, Haberlandt patients with immunologic or infectious disorders E, Rostásy K, Fuchs D like cerebellitis. We conclude that the parallel monitoring of phe Department of Pediatrics IV, Division of Neona- and tyr and calculating phe/tyr in plasma and CSF tology, Neuropediatrics and Inborn Errors of Me- may not only support diagnosis of phenylke- tabolism and Division of Biological Chemistry, tonurias but also of mild disturbances of phe and Biocenter, Medical University Innsbruck, Austria tyr metabolism in children and juveniles with inflammatory disorders and/or neuropsychiatric Phenylalanine (4)-hydroxylase (PAH) converts abnormalities. phenylalanine (phe) to tyrosine (tyr). An altered phe/tyr ratio is indicative for a disturbed hydrox- References: ylation reaction which can be detected by parallel 1 Neurauter G, et al. Chronic immune stimula- measurements of phe and tyr (1). 5,6,7,8- tion correlates with reduced phenylalanine

Tetrahydrobiopterin (BH4), the cofactor of PAH, turnover. Curr Drug Metabol 2008;9:622-7. is easily oxidized and in situations with oxidative 2 Scholl-Bürgi S, et al. Amino acid cere- stress this will lead to a decreased hydroxylation brospinal fluid/plasma ratios in children: influ- of phe. Consequently, phe/tyr ratio is elevated. ence of age, gender, and antiepileptic medica- In this retrospective study, we calculated tion. Pediatrics 2008;121:e920-6. phe/tyr in cerebrospinal fluid (CSF) and plasma of children with neurologic diseases, who under- went simultaneous lumbar and venous puncture. Serum Neopterin Production and Tryptophan Phe and tyr concentrations were determined using Degradation in Patients with Atopic Dermati- ion exchange chromatography with ninhydrine tis detection (2). From 70 punctures (68 patients with neurologic diseases of unknown origin, see refer- Schroecksnadel S, Fuchs D, Kofler H ence (2) for inclusion and exclusion criteria for this group) 38 were excluded because of therapy Division of Biological Chemistry, Biocenter, with antiepileptic drugs, 3 because of elevated Medical University of Innsbruck, Innsbruck; and neopterin concentrations and 3 because of mean- Private Allergy clinic , Hall in Tyrol, Austria while known diagnosis. The 'reference' values for phe/tyr were calcu- Atopic dermatitis is an eczematous, highly pru- lated in 26 patients (15 girls, 11 boys; mean age ritic chronic and recurrent inflammatory skin dis- 5.1 years, median 4.2 years, range 0.5-17.2 ease starting at young age. Although much years). In CSF phe/tyr was 0.88 ± 0.29 (mean ± research has already been done, its causes and SD), in plasma phe/tyr was 0.95 ± 0.17. Two immunological mechanisms are only partly patients of the 'reference' group had elevated CSF understood. It is generally accepted that besides phe/tyr of 1.60 and 1.62. The corresponding plas- genetic predisposition, a epidermal barrier dys- ma phe/tyr was only slightly elevated 1.28 and function and heightened susceptibility to viral, 1.22. fungal and bacterial infection, increasing inflam- In additional 155 patients with neuropediatric mation ,the increased prevalence of atopic disease disorders and inherited disorders of metabolism is mainly due to a disturbed balance of type-1 and we calculated phe/tyr in CSF and plasma. With a type-2 T-helper cells (Th1 and Th2-type) immuni- cut-off value of 1.15 for both compartments, 17 ty, and as a consequence, pathways triggered by patients had elevated ratios in plasma and 20 Th1-type cytokines might be altered. During Th1- patients had elevated ratios in CSF. The highest type immune response large amounts of neopterin

Pteridines/Vol. 21/No. 2 56 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines are released by human monocytes/macrophages patients studied is still small and any conclusion especially upon stimulation by the Th1-type has to be substantiated in further studies with cytokine interferon-γ (IFN-γ). Therefore, determi- higher patient numbers. nation of neopterin concentrations represents an indirect measurement of IFN-γ levels and allows References: to monitor Th1-type immune response (1). In par- 1 Schroecksnadel K, et al. Monitoring trypto- allel, IFN-γ induces indoleamine 2,3-dioxygenase phan metabolism in chronic immune activa- (IDO) which degrades tryptophan to kynurenine. tion. Clin Chim Acta 2006;364:82-90. IDO enzyme activity can be estimated by calcu- 2 Kositz C, et al. Serum tryptophan concentra- lating the ratio of serum/plasma concentrations of tion in patients predicts outcome of specific kynurenine to tryptophan (kyn/trp). immunotherapy with pollen extracts. Int Arch In this study, we evaluated whether neopterin Allergy Immunol 2008;147:35-40. concentrations or tryptophan degradation in atopic dermatitis patients could be markers to reflect disease activity and severity in actually Immunomodulatory Effects of Vitamin K- determined serum neopterin, tryptophan and antagonist Acenocoumarol (Sintrom) kynurenine levels concentrations in 15 patients (9 women, 6 men) with severe atopic dermatitis. Schroecksnadel S, Jenny M, Kurz K, Schennach Results were compared with concentrations in 38 H, Weiss G, Fuchs D serum specimens collected from healthy blood donors. Serum neopterin and tryptophan concen- Department of Internal Medicine and Division of trations did not differ between the two groups, but Biological Chemistry, Biocenter, Medical kynurenine levels and kyn/trp -although still University of Innsbruck, Innsbruck, Austria rather in the low range- were significantly higher in atopic dermatitis patients (2.13 ± 0.16 µM; Vitamin K-antagonists belong to the group of 36.3 ± 3.38 µmol/mmol) than in blood donors anticoagulants and are commonly used for the (1.4 ± 0.06 µM; 24.5 ± 1.02 µmol/mmol) (both treatment and prevention of deep venous throm- p <0.001), respectively. In patients we found a bosis, pulmonary embolism, myocardial infarc- positive correlation between neopterin levels and tion and stroke. Their antithrombotic effects and = < tryptophan degradation (rs 0.58, p 0.05). All the mechanisms involved in blood coagulation neopterin concentrations measured in patients and are well documented, but little is known about healthy controls were <8.7 nM, the upper limit of their interaction with the immune system, though the normal (= 95th percentile). inflammation and immune activation are crucial- Although in our study serum neopterin levels ly involved in the pathogenesis of atherosclerosis did not differ between healthy controls and atopic and cardiovascular disease. Within cell-mediated dermatitis patients, whereas tryptophan degrada- (= Th1-type) immune response pro-inflammatory tion reflected by kynurenine concentrations and cytokines especially interferon-γ activate GTP- kyn/trp ratio was slightly increased, we may con- cyclohydrolase I to form neopterin in human clude that IDO is involved in the tryptophan macrophages and dendritic cells. In parallel, metabolic changes. The significant correlation indoleamine 2,3-dioxygenase (IDO) is induced found between neopterin concentrations and and degrades tryptophan (trp) to kynurenine kyn/trp underlines this conclusion. However, such (kyn), IDO enzyme activity can be estimated by it is in contrast to what was found in pollinosis calculating the ratio of kyn to trp concentrations patients earlier, when a subgroup of patients was (kyn/trp). indentified with highly increased serum trypto- This in vitro study investigated the effects of phan concentrations which predicted unrespon- vitamin K-antagonist acenocoumarol (Sintrom) siveness to hyposensitisation therapy (2). In these on freshly isolated peripheral blood mononuclear patients no association was observed between cells (PBMC) from healthy blood donors. PBMC neopterin and kyn/trp. However, the number of were incubated with accelerating doses of aceno-

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 57 coumarol and were either left unstimulated or To investigate the adhesion of neutrophils we stimulated with T-cell mitogen phytohaemagglu- coated 12-well plates with mouse serum and tinin (PHA) after 30 minutes. Concentrations of allowed cells to adhere for 40min. To study neu- neopterin, trp and kyn were measured in super- trophile function upon microbial challenge a sin- natants after 48 hours of stimulation. gle intradermale injection of Salmonella IDO-activity and neopterin formation were sig- typhimurium (300 CFU) was given to wild-type nificantly higher in PHA-stimulated PBMC than and Lcn2 -/- C57BL6 mice. After 24h, the skin at in unstimulated cells. Acenocoumarol dose- each injection site was excised and fixed in for- dependently inhibited trp degradation and malin for histological analysis. neopterin production in parallel, and at concentra- We found that the migration of human and tions as low as 10 µg/ml a significant effect was murine granulocytes was significantly increased demonstrated in PHA-stimulated cells, whereas in upon the addition of Lcn2, which was not linked unstimulated cells no such effect was observed. to increased formation of IL-8/KC. Mechanisti- Though plasma levels of acenocoumarol in cally, this could be traced back to Lcn2 mediated treated patients are below the concentrations of changes of ERK1/2 signalling in these cells. our in vitro experiments, our data suggests that Accordingly, we found significantly reduced neu- the immunomodulatory capacity of aceno- trophile migration into intradermale bacterial coumarol could be part of its positive effects in lesions of Lcn2 -/- as compared to their wild type antithrombotic treatment and prophylaxis. littermates. In addition to a reduced chemotactic activity, neutrophils from Lcn2-/- mice also pre- sented with an impaired adherence to surfaces. Neutrophil Gelatinase-associated Lipocalin We herein describe a novel role of Lcn2 as an (NGAL) Promotes Granulocytes Emigration important paracrine chemo-attractant and an indispensable factor for neutrophile function in Schroll A, Eller K, Feistritzer C, Moser P, Nairz M, bacterial infections. Sonnweber T, Mair SM, Rosenkranz A, Theurl I, Weiss G The Relationship between Alcohol Intake and Clinical Immunology and Infectious Diseases, Cellular Immune Activity in Systemic Lupus Department of Internal Medicine I, Innsbruck Erythematosus May Change from Inhibitory Medical University, Innsbruck, Austria to Stimulatory within Two Months of Study

Neutrophil gelatinase-asscoiated licpocalin Schubert C, Fuchs D (NGAL, 24p3 or lipocalin-2, Lcn2) is a 21 kDA protein of the lipocalin superfamily. NGAL has Clinic of Medical Psychology, and Division of been shown to affect cellular iron homeostasis Biological Chemistry, Biocenter, Innsbruck transport, cellular apoptosis, and the course of Medical University, Innsbruck, Austria infections with gram negative bacteria via binding of bacterial siderophores. Since lipocalin is pro- In recent years, interest has been growing duced and released in large quantities by granulo- regarding the role of alcohol intake on systemic cytes, we questioned whether this peptide may lupus erythematosus (SLE) disease activity. This affect the immunological function of these cells. integrative single-case study (1) investigated the Human or murine neutrophile granulocytes dynamic interdependencies between alcohol con- were purified and attracted in vitro with varying sumption, various emotional states and cellular concentrations of human interleukin-8 (hIL-8), immune activity in SLE using time series analysis mIL-8 (KC), mrLcn2, hLcn2 and/or an anti- (i.e., ARIMA modeling, cross-correlational analy- mLcn2-Ab. Neutrophil migration assays were sis). We determined neopterin concentrations performed using a modified 48-well Boyden (HPLC) in a 52-year-old woman suffering from microchemotaxis chamber. SLE, who collected her entire urine on a twice-

Pteridines/Vol. 21/No. 2 58 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines daily basis for a period of 56 days (total: 112 erythematosus. Psychoneuroendocrinology time-points). Aside from the central interest of 2003;28:459-473. our studies, i.e., the impact of everyday incidents 2 Winkler C, et al. Beer down-regulates activat- on cellular immune system dynamics, the patient ed peripheral blood mononuclear cells in vitro. also provided daily information on intake of alco- Intern Immunopharmacol 2006;6:390-395. holic beverages (type and quantity) and various 3 Wirleitner B, et al. Resveratrol suppresses emotional state variables (mental activity, irrita- interferon-gamma-induced biochemical path- tion, mood). Cross-correlational analyses revea- ways in human peripheral blood mononuclear led that moderate alcohol consumption had differ- cells in vitro. Immunol Lett 2005;100:159- ent effects on urinary neopterin levels depending 163. on whether time series data were analyzed before or after the occurrence of an inflammatory event (acute paranasal sinusitis) that had taken place Staphylococcus aureus Lipoproteins Affect halfway through the study period (time-points 45 Immune Response and Body Iron Homeostasis - 54). Before sinusitis (1 - 44), increases in alco- hol consumption were parallelized by decreases Sonnweber T, Schmaler M, Theurl I, Schroll A, in urinary neopterin levels (lag0: r =−0.371; Nairz M, Seifert M, Mair S, Landmann R, Weiss p <0.05) whereas after sinusitis (55 - 112), G increases in alcohol intake preceded increases in urinary neopterin levels with a temporal delay of Department for Internal Medicine I, Clinical 12 hours (lag1: r =+0.308; p <0.05). The emo- Immunology and Infectious Diseases, Innsbruck tional states under study did not interfere with the Medical University, Innsbruck, Austria; and associations between alcohol intake and neopterin Division Infection Biology, University Hospital, levels. As to the biochemical mechanisms of the Basel, Switzerland alcohol-immune effects seen in this study, ethanol as well as antioxidative compounds such as Toll-like receptor 2 (TLR2) is a receptor for resveratrol or humulones contained in wine or lipoproteins (lpp) and signals by the adaptor beer may have been responsible for suppression MyD88. Interactions of TLRs with lpps are of inflammation (2, 3). However, the change in known to be crucial for eliciting an adequate host the direction of effect between alcohol consump- response against bacterial infections. As the tion and neopterin levels before and after sinusitis impact of lpp on TLR2-MyD88 activation for S. cannot be explained solely on a biochemical aureus in systemic infection is unknown, we stud- basis. Rather, the clear influence of psychological ied the effects of lpp maturation in two S. aureus factors on stress system activity might suggest strains (SA113 and Newman) deficient in the that the patient's health perception during sinusi- enzyme prolipoprotein diacylglyceryl transferase tis modified the meaning alcohol had for her and, (∆lgt). consequently, how drinking alcohol affected her C57BL/6 mice were injected either 1 x 106 to 1 immune system. Though the underlying mecha- x 108 cfu of S. aureus 113/Newman wt or S. nisms of the current findings remain far from aureus lgt (dissolved in 200 µL of 0.9% NaCl) or clear, this study shows that even within the same 0.9% NaCl into the lateral tail vein. The animals patient with SLE the relationship between alcohol were sacrificed after 24 hours or 7 days after ini- intake and neopterin levels might not always be tiation of the infection. We extracted RNA and stable suggesting a possible reason for inconsis- protein from spleen and liver and studied the tent evidence from conventional group studies. expression of critical genes in iron homeostasis and innate immunity by means of QRT-PCR and Refernces Western blots. Iron content of organs was quanti- 1 Schubert C, et al. Daily psychosocial stressors fied by a colorimetric assay. and cyclic response patterns in urine cortisol The injection of Staphylococcus aureus resul- and neopterin in a patient with systemic lupus ted in a severe sepsis with stimulation of pro-

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 59 inflammatory and anti-inflammatory cytokine sive care unit. Since the field of breath analysis is formation. ∆lgt staphylococcal infection resulted relatively new, and the advances in analytical in impaired immune response, reflected by a technology occur so fast, many compounds in decreased expression of the pro-inflammatory exhaled breath have been detected whose bio- cytokine TNF-α as compared to wt staphylococ- chemical origin has not yet been studied. Even cal infection, along with a modulation in the less is known about the relationship of volatile expression of iron homeostasis genes in the liver compounds to cellular processes like malignant and most pronounced in the spleen which was transformation. most evident when investigating the expression of A particular focus of breath research is on divalent metal transporter-1 and the iron export patients suffering from lung cancer. Investi- protein ferroportin. We further observed signifi- gations of exhaled breath from cancer patients cant differences of iron content in organs between showed that concentrations of specific com- SA113/Newman wt infected mice and animals pounds may be increased or decreased in compar- exposed to SA113/Newman ∆lgt. In addition, we ison with healthy age-matched controls. So far, found an impaired bacterial growth of ∆lgt little is known about the underlying metabolic staphylococci under iron restricted conditions as mechanisms and different sources leading to the compared to wt staphylococci. Finally, we could production of volatile organic compounds demonstrate that excessive iron supply nullified (VOCs). Since tumours are complex and hetero- the growth deficits of ∆lgt S. aureus. geneous systems it is possible that tumorous and In conclusion, these results indicate that lpp also non-tumorous components like immune cells induction of inflammatory cytokines has an and even microorganisms contribute to VOCs. impact on iron transport mechanisms. Changes in The objective of this work was to investigate iron metabolism are known to influence host VOCs released or consumed by various lung cells immune effector functions as well as bacterial of carcinogenic origin as well as healthy control growth rates and survival and may therefore con- cells from lung and dermis in vitro. tribute to the disease severity in S. aureus sepsis. We investigated four lung cancer cell lines (A549, NCI-H2087, NCI-H1666 and CALU-1), primary lung epithelial cells (HBEpC) and TDS-GCMS Analysis of Volatile Metabolites of immortalized fibroblasts originating from human Human Lung Cancer and Non-carcinogenic dermis (hFB, human fibroblast) for the release or Cells in vitro consumption of VOCs by GC-MS after thermal desorption. Cells were trypsinized and up to 100 Sponring A, Filipiak W, Filipiak A, Ager C, x 106 cells were incubated in a sealed fermenter Schubert J, Miekisch W, Amann A, Troppmair J for 18 hours. Prior to gas chromatography mass spectrometry (GC-MS) analyses samples from Center of Operative Medicine, Innsbruck Medical the headspace of the culture vessel were collected University, Breath Research Unit of the Austrian with simultaneous preconcentration by adsorption Academy of Sciences, Daniel-Swarovski on solid sorbents and then VOCs were thermod- Research Laboratory, Department of Visceral-, esorbed for analysis by GC-MS. For comparison Transplant- and Thoracic Surgery, 6020 we analyzed the VOCs present in the headspace Innsbruck, Austria; University of Rostock, of the medium control. Department of Anesthesiology and Intensive Using this approach, we identified VOCs that Care, 18057 Rostock, Germany were similar in a particular transformed and in non-transformed cells independently if the latter Analysis of volatile organic compounds had been derived from lung or skin. For instance, (VOCs) in the exhaled breath for the detection of concentrations of 2-pentanone and 2,4-dimethyl- human diseases is a non-invasive diagnostic 1-heptene were found to be increased in HBEpCs method. Breath can be sampled as often as it is and hFB cells and the A549 lung cancer cell line. desirable, even during operations or at an inten- A similar observation was made for 2,3,3-

Pteridines/Vol. 21/No. 2 60 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines trimetylpentane which appeared in increased con- Metabolic Care, Charles University Medical centrations in control cells and CALU-1 lung can- School Teaching Hospital, Hradec Králové, and cer cells. In addition, the ethers methyl tert-butyl Institute of Radiation Oncology and Charles ether and ethyl tert-butyl ether could be detected University First Medical School, Prague, Czech at elevated levels in A549 cells and one of the Republic untransformed cell lines. However, especially branched hydrocarbons and alcohols were seen The introduction of targeted agents has pro- increased more frequently in untransformed than foundly affected the treatment of metastatic renal transformed cells. Interestingly, a big variety of cell carcinoma (RCC). Increased urinary concen- predominantly aldehydes and the ester n-butyl trations of neopterin are associated with poor acetate were present at decreased concentrations prognosis in tumors of different primary loca- in the headspace of all cell lines tested compared tions. However, the data on prognostic signifi- with medium controls. Again greater variety was cance of neopterin in patients with RCC is more shown by hFB and HBEpC cells compared to car- limited. Neopterin was determined by high-per- cinogenic cell lines. For example 2-butenal was formance liquid chromatography. The survival metabolized only by these two types of control was analyzed using the Kaplan-Meier method, cells, but none of the cancer cell lines. with survival differences being analyzed by the In summary, our findings confirm that several log-rank test. Fifty-five patients with metastatic VOCs like unsaturated hydrocarbons and oxygen- RCC were studied. No differences were observed containing compounds are released, while differ- between previously treated and systemic treat- ent aldehydes and the ester n-butyl acetate are ment-na?ve patients. Patients ineligible for inclu- consumed or metabolized by the cell lines and sion into clinical trial tended to have higher uri- primary cells studied here. The healthy primary nary neopterin. The survival of patients with cells display more compounds with increased or increased urinary neopterin concentrations using decreased concentration (compared to medium) different cutoff points was significantly inferior than the tumorigenic cell lines. (p <0.05) in the whole group as well as in patients with no prior systemic therapy. In conclusion, uri- This research was supported by the European nary neopterin is a prognostic factor in metastatic Commission (Project BAMOD, project no LSHC- RCC. CT-2005-019031) We greatly appreciate the gen- erous support of the Member of the Tyrolean regional government Dr. Erwin Koler and the Clinical Status in Long Term Successfully Director of the University Clinic of Innsbruck Transplanted Patients (TILAK) Mag. Andreas Steiner. Tenschert W, Atanackovic D, Cao Y, Thaiss F

Urinary Neopterin in Metastatic Renal Cell Department of Internal Medicine II and III, Carcinoma Patients Treated with Bevacizu- University Medical Center Hamburg Eppendorf mab-Based Therapy Hamburg, Germany

Studentová H, Kalábová H, Zezulová M, Allogeneic kidney transplantation is a well Holecková P, Krcmová L, Kasparová M, Plísek J, established treatment for endstage renal disease; Solichová D, Masek M, Melichar B early postoperative organ function and survival rates are estimated over 90%, less is known about Department of Oncology, Palacký University the clinical status of patients with graft function Medical School and Teaching Hospital, Olomouc; of more than 10 years. Department of Analytical Chemistry, Charles Our clinical retrospective study in 29 kidney University School of Pharmacy, Departments of transplant recipients (22 males, 7 females, mean Oncology and Radiotherapy and Gerontology and age 56 ± 10 years), mean graft survival of 18 ± 6,

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 61 range 10 -30 years) investigated the actual physi- Felmayer G, Berger J, Werner ER cal status, renal function, blood pressure (bp), incidence of malignancies and intake of immuno- Division of Biological Chemistry, Biocenter; suppressive medication. Department of Internal Medicine; and Center of By clinical examination, 27/29 patients were in Operative Medicine, Department of Visceral, a good physical condition ( mean BMI 24.9),all Transplant and Thoracic Surgery, Innsbruck non-smokers, 8 were active in sports, one patient Medical University, Innsbruck; Center for Brain was a well treated type I diabetic, two suffered Research, Medical University of Vienna, Vienna, from a metabolic syndrome, one patient presented Austria; and Institute of Biochemistry, Graz donor specific alloantibodies (35% panel-reactive University of Technology, Graz, Austria antibodies). Mean creatinine values were 1.27 ± 0.4 mg/dl; glomerular filtration rate (GFR) was The degradation of alkylglycerols in mammalia 63 ± 20 ml/min/1.73 m2/MDRD (MDRD = occurs by the consecutive action of two mem- Modification of Diet in Renal Disease). Patients brane bound enzymes. The first step is catalysed treated with calcineurin inhibitors (CNI+), by alkylglycerol monooxygenase (glycerylether showed no different graft function compared to monooxygenase, EC 1.14.16.5), a tetrahydro- those with calcineurin free (CNI-) medication. biopterin-dependent enzyme, and consists in the Mild proteinuria was found in 11/29 (38%) cleavage of the ether bond. The resulting alde- patients. hyde is then oxidized and thereby detoxified by Before transplantation, blood pressure was the NAD-dependent enzyme fatty aldehyde dehy- hypertensive in26/29 patients, after engraftment, drogenase (EC 1.2.1.48). By alternative splicing, two patients presented mild hypertension, two fatty aldehyde dehydrogenase can have two dis- were normotensive without and 25 were nor- tinct C-termini, one localising the enzyme to the motensive under medication. Mean blood pres- endoplasmic reticulum and the other targeting it sure values were 125:78 mm Hg and not statisti- to peroxisomes. As these two enzymes are acting cally different in CNI+ or CNI- treated patients. in such a metabolic proximity, we wanted to Immunosuppressive therapy consisted in CNI- investigate the distribution of alkylglycerol monotherapy in 17, CNI-combination in 4 and monooxygenase in comparison to fatty aldehyde CNI-free medication in 7 patients respectively. dehydrogenase in mouse tissues and subcellular One patient refused any immunosuppressive compartments. For this aim we have measured the medication since 12 years. Malignancies were activities of both enzymes using our established observed in 3 patients (10.3%) (2 skin cancers, 1 HPLC methods based on fluorescent substrates in post-transplant lymphoproliferative disorder = a variety of mouse tissues and in fractions of sub- PTLD); all patients were treated successfully cellular density gradients. without impairment of the graft function. Activity could be detected in almost all tissues In summary, longtime graft survival was with peak levels in liver, fat tissues and in the gas- observed in a cohort of 29 patients with excellent trointestinal tract. Interestingly, even though hav- compliance, careful antihypertensive and ing a similar distribution pattern, fatty aldehyde immunosuppressive treatment. Further investiga- dehydrogenase activity always exceeded alkyl- tions on operative tolerance are in progress. glycerol monooxygenase activity by at least ten fold ensuring clearing of the toxic intermediate product. Also the subcellular distribution was Distribution of Alkylglycerol Monooxygenase similar for the two enzymatic activities with peak and Fatty Aldehyde Dehydrogenase Activities levels found in the endoplasmic reticulum but in Tissues and Subcellular Compartments revealing a potential peroxisomal contribution also for alkylglycerol monooxygenase. In further Watschinger K, Keller MA, Wiesinger C, experiments we also tested for the influence of Maglione M, Duwensee K, Tancevski I, Forss- streptozotocin-induced diabetes in mice, fenofi- Petter S, Hermetter A, Golderer G, Werner- brate feeding and absence of the substrate in

Pteridines/Vol. 21/No. 2 62 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines etherlipid deficient mice on the activities of alkyl- tryptophan metabolism in a cohort of 356 over- glycerol monooxygenase and fatty aldehyde weight and obese (aged 11.3 ± 2.97 years; mean dehydrogenase. ± S.D.), otherwise healthy children and 32 non- In conclusion, both enzymes responsible for obese controls which were recruited at the degradation of saturated etherlipids are similarly University Hospital of Salzburg, Department of regulated and expressed in a wide range of mouse Pediatrics. Distribution of female to male was tissues and subcellular compartments. equal in both groups. Neopterin concentrations were measured using ELISA (BRAHMS, Supported by the Austrian Research Funds "zur Hennigsdorf, Germany), and tryptophan and Förderung der wissenschaftlichen Forschung" kynurenine concentrations were determined by P19764. HPLC. Body mass index (BMI) differed significantly between obese and non obese probands (28 ± Serum Neopterin and Tryptophan Concentra- 5.64 vs. 18 ± 2.19 SD kg/m2 U = 9.24, p <0000.1, tions in Obese Children Mann Whitney U-test). Obese children had sig- nificantly higher serum triglyceride 109 ± 63.9 vs. Weghuber D, Fuchs D, Krösslhuber V, Mangge H, 51 ± S30.5 (U= 6.67 p <0.0001) lower HDL-cho- Zaknun D lesterol (50 ± 12.3 vs. 63 ± 13.1 mg/dl; U = 4.9 p <0.0001), higher fasting glucose levels (88 ± 9.4 Department of Pediatrics, University Hospital of vs. 75 ± 7.6 mg/dl; U = 7.3 p <0.0001) and were Salzburg, Salzburg; Division of Biological significantly more Insulin resistant (HOMA-IR Chemistry, Biocenter, Innsbruck Medical 1.0 vs. 3.5; p <0.0001). Serum neopterin concen- University, Innsbruck, Austria; Clinical Institute trations were low in both groups with an average of Medical and Chemical Laboratory Diagnostics, of 5.13 ± 1.59 in normal weight controls vs. 5.85 Medical University of Graz, Graz; Department of ± 2.86 nmol/L in obese juveniles (not significant). Pediatrics, Medical University of Vienna, Vienna, By contrast, there was no difference in kynure- Austria nine concentrations and the kynurenine to trypto- phan ratio (kyn/trp), although tryptophan concen- Neopterin is synthesized from guanosine trations were significantly lower in the obese triphosphate by enzyme GTP cyclohydrolase I. It (80.7 ± 12.7 µmol/L) vs. non-obese subjects (74.7 is released preferentially from human monocyte- ± 18.8 µmol/L; U = 2.384, p = 0.017). Tryptophan derived macrophages upon stimulation with the concentrations correlated with size and weight in Th1-type cytokine interferon-γ that is the proba- both groups, but neither tryptophan nor kynure- bly most important mediator of antimicrobial and nine concentrations not kyn/trp correlated with antitumor defence in vivo. Thus, neopterin con- neopterin concentrations. centrations in body fluids usually correlate with This study shows that obesity in juveniles is immune activation and are significant predictors not associated with increased neopterin concen- of chronic infections, various types of cancer and trations. Results suggest that obesity at least in the cardiovascular disease (1, 2). In adults, an associ- early course of the disease does not lead directly ation between increased body mass index and to Th1-type immune activation and associated neopterin concentrations was observed (3), but cardiovascular disease at this stage. Only in the although there is evidence for a proinflammatory later course a switch to Th-1 type immune activa- state accompanied by impaired vascular endothe- tion and associated cardiovascular diseases may lial function scarce in childhood and adolescence, take place. Chronic infections or other cofactors such data are still scarce (4). Moreover, an earlier might be important to trigger cytokine production study indicated that neopterin concentrations are and elevated neopterin concentrations at the later decreased rather than increased in juvenile obesi- stage. ty (5). In a cross-sectional approach we investi- gated serum neopterin concentrations as well as

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 63

References: which has not been characterized yet. 1 Grammer TB, et al. Neopterin as a predictor of The aromatic amino acid hydroxylase proteins total and cardiovascular mortality in individu- show significant sequence homology among each als undergoing angiography (The other, and share a common reaction mechanism Ludwigshafen Risk and Cardiovascular Health characterized by the use of a non-heme iron, stoi- Study). Clin Chem 2009;55:115-46. chiometric oxidation of the tetrahydrobiopterin 2 Fuchs D, et al. The role of neopterin in athero- cofactor, use of tetrahydrobiopterin in oxygen genesis and cardiovascular risk stratification. activation and the need for external recycling of Curr Med Chem 2009;16:4644-53. the tetrahydrobiopterin cofactor which leaves the 3 Ledochowski M, et al. Association between reaction as 4a carbinolamine derivative. insulin resistance, body mass and neopterin The nitric oxide synthases show significant concentrations. Clin Chim Acta 1999;282:115- sequence homology among each other, but share 23. no homology with aromatic amino acid hydroxy- 4 Kapiotis S et al. A proinflammatory state is lases. The mechanism of tetrahydrobiopterin detectable in obese children and is accompa- stimulation of nitric oxide synthases is fundamen- nied by functional and morphological vascular tally different from that of aromatic amino acid changes. Arterioscler Thromb Vasc Biol hydroxylases: The tetrahydrobiopterin cofactor is 2006:26:2541-2546. not involved in oxygen activation, is not con- 5 Mangge H, et al. Preatherosclerosis and meta- sumed stoichiometrically and does not need to be bolic abnormalities in obese juveniles during recycled by external enzymes. In contrast, lifestyle intervention - the impact of SAT dis- tetrahydrobiopterin donates a single electron and tribution - The STYrian Juvenile Obesity a proton, thus forming a radical intermediate Study (STYJOBS). Pteridines 2007;18:46-7. which is recycled to the active tetrahydrobiopterin by nitric oxide synthase itself. Another difference to the aromatic amino acid hydroxylases is that Mechanisms of Tetrahydrobiopterin Stimula- nitric oxide synthases contain a catalytically tion of Enzymes - an Overview active heme iron. Alkylglycerol monooxygenase, a membrane Werner ER, Watschinger K, Keller MA, Golderer bound enzyme, has many features similar to the G, Werner-Felmayer G aromatic amino acid hydroxylases in terms of tetrahydrobiopterin cofactor handling. Division of Biological Chemistry, Biocentre, Tetrahydrobiopterin is required stoichiometrical- Innsbruck Medical University, Innsbruck, Austria ly, and is recycled by external enzymes. In addi- tion, alkylglycerol monooxygenase can be inhib- Currently five enzymatic reactions are agreed ited by the iron chelator 1,10-phenanthroline, to require tetrahydrobiopterin a cofactor: Three suggesting that it does also contain a non-heme are the aromatic amino acid hydroxylases pheny- iron. Since no sequence homologous to aromatic lalanine 4-monooxygenase (E.C. 1.14.16.1), tyro- amino acid hydroxylases and nitric oxide synthas- sine 3-monooxygenase (E.C. 1.14.16.2) and tryp- es is found in databases, we assume that alkyl- tophan 5-monooxygenase (E.C. 1.14.16.4), which glycerol monooxygenase may form a third, dis- are encoded by single genes (PAH and TH), and tinct class of tetrahydrobiopterin dependent by two genes (TPH1 and TPH2), respectively. enzyme. The fourth enzymatic reaction is the group of nitric oxide synthases (E.C. 1.14.13.39), which occur in three isoforms encoded by three genes in mammals, NOS1, NOS2 and NOS3. The fifth reaction requiring tetrahydrobiopterin is alkyl- glycerol monooxygenase (glyceryl ether monooxygenase, E.C.1.14.16.5), the sequence of

Pteridines/Vol. 21/No. 2 64 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines

Increased Plasma Phenylalanine to Tyrosine activity. Ratio in HIV-1 Infection and Correction Increase of phenylalanine is probably due to Following Effective Antiretroviral Therapy diminished activity of PAH which is probably due

to a role of ROS in diminishing BH4 appears (5). Zangerle R, Kurz K, Neurauter G, Kitchen M, Any relevance for the neuropsychiatric abnormal- Sarcletti M, Fuchs D (Innsbruck, Austria) ities in patients still needs to be shown. Future HIV Unit, Department of Dermatology and studies might be able to show whether the decline Venerology, Clinical Immunology and Infectious of phe/tyr under ART may concur with the often Diseases, Department of Internal Medicine, and improved neuropsychiatric status in treated Division of Biological Chemistry, Biocenter, patients. Innsbruck Medical University, Innsbruck, Austria References: Abnormal metabolism of aromatic amino acids 1 Widner B, et al. Neopterin production trypto- tryptophan, phenylalanine and tyrosine accompa- phan degradation and mental depression: what nies conditions which go along with chronic is the link? Brain Behav Immun 2002;16: 590- inflammation and immune responses, e.g., cancer, 5. infection, coronary artery disease, neurodegener- 2 Neurauter G, et al. Chronic immune stimula- ation and normal ageing (1, 2). Several years ago, tion correlates with reduced phenylalanine higher serum and plasma concentrations of the turn-over. Curr Drug Metabol 2008;9:622-7. essential amino acid phenylalanine have been 3 Ollenschläger G, et al. Plasma amino acid pat- documented in patients with HIV-1 infection (3). tern of patients with HIV infection. Clin Chem They may relate to a diminished conversion of 1988;34:1787-9. phe to tyrosine (tyr) by the enzyme phenylala- 4 Zangerle R, et al. Increased blood phenylala- nine-hydroxylase (PAH). PAH is rate-limiting in nine to tyrosine ratio in HIV-1 infection and the biosynthesis of dopamine, and impaired PAH correction following effective antiretroviral activity is reflected by an increased phe to tyr therapy. Brain Behav Immun 2010;24:403-8 ratio (phe/tyr). In this study, plasma phe/tyr was 5 Neurauter G, et al. Serum phenylalanine con- measured in 107 patients with HIV-1 infection centrations in patients with ovarian carcinoma before and after 12 months of effective antiretro- correlate with concentrations of immune acti- vial therapy (ART) (4). Results were compared vation markers and of isoprostane-8. Cancer with CD4+ cell counts, HIV-1 RNA levels and Lett 2008;272:141-7. concentrations of immune activation marker neopterin. Before ART, phe/tyr was mean ± S.D.: 0.99 ± Urinary Neopterin in Patients with Gastro- 0.57 µmol/µmol and correlated significantly with intestinal Stromal Tumors Treated with Ima- plasma and urine neopterin concentrations (both p tinib <0.001) and less strongly with HIV-RNA levels and CD4+ counts (both p <0.05). After ART, Zezulová M, Kalábová H, Studentová H, phe/tyr dropped (p = 0.01) which was due to a Holecková P, Krcmová L, Kasparová M, Plísek J, decline of phe concentrations from 73.1 ± 34.0 Solichová D, Masek M, Melichar B mol/L at baseline to 62.9 ± 17.8 µmol/L after ART (p = 0.01) and a concomitant increase of tyr Department of Oncology, Palacký University concentrations (p = 0.01). In parallel, significant Medical School and Teaching Hospital, Olomouc; reductions of plasma and urine neopterin concen- Department of Analytical Chemistry, Charles trations were observed during ART. University School of Pharmacy, Departments of Increased phe/tyr is frequent in patients with Oncology and Radiotherapy and Gerontology and HIV-1 infection and is related to immune activa- Metabolic Care, Charles University Medical tion. ART was found to decrease phe/tyr and this School Teaching Hospital, Hradec Králové, and change could indicate and influence on PAH Institute of Radiation Oncology and Charles

Pteridines/Vol. 21/No. 2 Abstracts: 29th International Winter-Workshop on Clinical, Chemical, and Biochemical Aspects of Pteridines 65

University First Medical School, Prague,Czech µmol/mol creatinine, p = 0.001). No statistically Republic significant changes of urinary neopterin were observed during the therapy with imatinib, but a Increased serum or urinary concentrations of marked decrease was observed occasionally dur- neopterin have been described in patients with ing the first days of imatinib treatment, in associ- tumors of different primary locations, but so far ation with tumor control. The survival of patients there were no reports on neopterin in patients with with increased urinary neopterin was shorter, but gastrointestinal stromal tumors (GIST). We have the difference in survival was not statistically sig- studied urinary neopterin in 26 patients with nificant. In conclusion, urinary neopterin is GIST. Urinary neopterin was determined by high increased in patients with advanced/metastatic performance liquid chromatography Compared to GIST. No significant changes were observed in controls (n = 16), urinary neopterin was increased association with imatinib therapy, but a decreas- in patients with advanced/metastatic GIST (324 ± ing trend of urinary neopterin concentrations was 306, range 78 - 1425 vs. 124 ± 54, range 34 = 229 evident in individual patients.

Pteridines/Vol. 21/No. 2