Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neopterin, a marker of immune response, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress, correlate at high age as determined by automated simultaneous high-performance liquid chromatography analysis of human urine

Using an established high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, fraction collection, and electrochemical detection, the oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH-dG) can be analyzed rapidly and precisely in human urine samples. In addition, by ultraviolet (UV) detection, it was shown recently that it is possible to simultaneously analyze creatinine and 7-methylguanine (m⁷Gua), an RNA degradation product, in urine. By adding a fluorescence detector to the HPLC system, we now report that it is also possible to detect pteridins such as neopterin and biopterin. The fluorescence detection was evaluated in detail for neopterin, an immune response and tumor marker. The urinary content of neopterin, assessed by using the HPLC method, was verified with a commercial neopterin enzyme-linked immunosorbent assay (ELISA) kit as indicated by the high correlation between the two methods (r = 0.98). In urinary samples from 58 young healthy individuals (male and female nonsmokers, ages 19-39 years), it was found that there was no significant correlation (r = −0.04) between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). In contrast, in urinary samples from 60 old healthy individuals (male and female nonsmokers, ages 60-86 years), there was a significant correlation (r = 0.47) found between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). These findings strongly indicate that the higher level of immune response that was correlating with old age contributes significantly to the higher level of oxidative damage as assessed in the form of 8-OH-dG. Using this type of HPLC system, it is possible to evaluate oxidative DNA damage and immune response simultaneously using the respective urinary markers. These data may contribute to understanding of the pathophysiology of diseases such as infections and tumor progression where both oxidative stress and immune response occur simultaneously.     

Concentrations of neopterin and biopterin in the cerebrospinal fluid of patients with Parkinson's disease

The concentrations of neopterin and biopterin in CSF of 18 younger and 10 older, control patients and of 18 patients with Parkinson's disease were measured by high-performance liquid chromatography with fluorescence detection. Both neopterin concentrations and the neopterin to biopterin ratios in CSF were lower in 50-year or younger group than in 51-year or older group. Biopterin concentrations were also decreased but not significantly in the older group. The concentrations of neopterin and biopterin in CSF of patients with Parkinson's disease were lower than those of the age-matched older control group. However, the neopterin/biopterin ratios tended to be lower but not change significantly as compared to the age-matched older control group.     

Dissociation of neopterin and 7,8-dihydroneopterin from plasma components before HPLC analysis

Measurement of plasma neopterin by HPLC with fluorescence detection is used clinically as a marker of immune cell activation in the management of a number of disease pathologies. HPLC analysis of neopterin requires the acidic removal of plasma proteins but we have found that 7,8-dihydroneopterin is oxidised to neopterin with varying yield. Using acetonitrile as the precipitant, we have measured substantially higher quantities of both total neopterin (7,8-dihydroneopterin and neopterin) and neopterin from plasma of healthy and septicemia patient's. Total neopterin concentrations were on average 50% and 200% greater in healthy and septicemia subjects, respectively, when measured after acetonitrile precipitation compared to trichloroacetic acid. Our data suggests that some pterin co-precipitates with proteins during acid treatment.     

Unhealthy Health Policy: A Critical Anthropological Examination

Unhealthy Health Policy:. Critical Anthropological Examination . Arachu Castro and Merrill Singer, eds. Walnut Creek, CA: AltaMira, 2004. 387 pp.     

Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.     

Influence of neopterin on the generation of reactive oxygen species in human neutrophils

Neopterin is synthesized by human monocyte-derived macrophages primarily upon stimulation with the cytokine interferon-gamma. We studied the influence of neopterin on the generation of reactive oxygen species (ROS) in human peripheral blood neutrophils. Radical formation was measured using a biochemiluminometer. Neutrophils were isolated from peripheral blood of healthy donors. The generation of ROS by neutrophils suspended in Earl's solution (pH=7.4) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol and lucigenin as light emitters. Neopterin induced chemiluminescence in suspensions of neutrophils in the presence of luminol, but not of lucigenin. Neopterin affected only adhesive cells. Addition of neopterin into the suspension of the cells involving D-mannitol, L-histidine and diazabicyclo[2.2.2]octane (DABCO) decreased luminol-dependent chemiluminescence (LDCL) of the neutrophils. The action of superoxide dismutase (SOD) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reduced neopterin-induced LDCL of neutrophils. Data suggest that neutrophils respond on exposure to neopterin with additional generation of singlet oxygen, hydroxyl radical and nitric oxide by nicotinamide adenine dinucleotide phosphate (NADPH)-independent pathways.     

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the simultaneous determination of N²-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-5SA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.     

Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using three retention mechanisms

An automated two-dimensional HPLC method for the determination of neopterin in serum is described. Neopterin is separated from proteins on a short octadecylsilica column by size exllusion and from the majority of the other serum components by adsorption. The fraction containing neopterin is transferred by column switching to a solvent-generated cation-exchange column using dodecylsulfonic acid as surface activator. Parameters influencing the separation performance and senstivity of the fluorescence detection are discussed. The efficiency of the cleaning of the first column was optimized. The method was validated. It achieves a precision of 1% (R.S.D.) and a detection limit of about 0.3 nmol/1. The accuracy is nearly 100%. The method allow a high sample throughput, requiring 15 min per sample.     

Mechanism of neopterin-induced myocardial dysfunction in the isolated perfused rat heart

Neopterin is a sensitive marker for diseases involving increased activity of the cellular immune system in humans. Many studies, however, provide evidence for neopterin not only as a marker, but also for its characteristic effects. Recently, we were able to demonstrate a considerable influence of exogenous neopterin at a concentration of 100 mumol/l on cardiac performance in the Langendorff model of isolated perfused rat hearts. The present study was designed to investigate its possible mechanism. During co-infusion of neopterin at a concentration of 100 mumol/l with the unspecific nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine monoacetate, the nitric oxide donor PAPA NONOate, the free radical scavenger N-acetylcysteine, or the pro-inflammatory cytokine tumor necrosis factor-alpha the effects on cardiac contractility parameters and coronary vascular resistance were studied in 67 male Sprague-Dawley rats. The temperature-controlled and pressure-constant Langendorff apparatus was used with retrograde perfusion of the aorta and a Krebs-Henseleit buffer. Neither the unspecific nitric oxide synthase inhibitor nor the nitric oxide donor excludes nitric oxide from playing a mechanistic role in our perfusion studies. Tumor necrosis factor-alpha was without any synergistic or antagonistic effects when co-treated with neopterin. N-acetylcysteine was most effective in abolishing neopterin-dependent effects on cardiac function. The negative effects of neopterin on cardiac performance might be due to an enhancement of oxidative stress by neopterin that can be attenuated by the antioxidant N-acetylcysteine. Neopterin has to be considered a pathogenic factor in the development of cardiac dysfunction in chronic disease states with high neopterin levels secondary to activation of the immune system.     

Book Review Essays

Amerindian Constructions of Otherness: From, the Enemy's Point of View: Humanity and Divinity in an Amazonian Society. Eduardo Viveiros de Castro.
Amerindian Constructions of Otherness: La memoria rituale: Follia e immagine del Bianco in una tradizione sciamanica amerindiana. Carlo Severi.     

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH.

The steps in the biosynthetic transformation of GTP to 7,8-dihydro-D-erythro-neopterin (H2neopterin), the precursor to the modified folates found in the methanogenic archaea, has been elucidated for the first time in two members of the domain Archaea. In Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH, it has been demonstrated that H2neopterin 2':3'-cyclic phosphate is an intermediate in this conversion. In addition, the formation of the pterin ring of the H2neopterin 2':3'-cyclic phosphate is catalyzed not by a single enzyme, as is known to occur with GTP cyclohydrolase I in the Eucarya and Bacteria, but rather by two or more enzymes. A 2,4,5-triamino-4(3H)-pyrimidinone-containing molecule, most likely 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, has been identified as an intermediate in the formation of the H2neopterin 2':3'-cyclic phosphate. Synthetic H2neopterin 2':3'-cyclic phosphate was found to be readily hydrolyzed by cell extracts of M. thermophila via the H2neopterin 3'-phosphate to H2neopterin, a known precursor to the pterin portion of methanopterin.     

Remarks on the Permian dasycladalean alga Sinoporella leei Yabe, 1949

Sinoporella leei was first described and illustrated by Yabe in 1949 as a new species and was ascribed to a new subgenus of the genus Triploporella (Steinmann). This alga was not illustrated again until Mu published 25-years later a photomicrograph of a longitudinal-tangential section. The recent finding of new specimens in Permian strata of the Guangxi region (South China) allows us to better illustrate this alga, and to improve our understanding of its morphology to the extent that we find it necessary to emend the current diagnosis. The new observations focus mainly on the morphology of the second-order laterals, on their relationship to the first-order laterals, and the connection of the first-order laterals to the main stem. These data suggest that Sinoporella represents a self-standing, discrete genus that differs from Triploporella. We also consider the lack of cystophores (cyst containers sensu De Castro) and the possible occurrence of individual cysts in the first order laterals as characteristics that do not accord with Barttolo's emended diagnosis of Triploporella.     

Totemism, Animism and North Asian Indigenous Ontologies

This article examines the extent to which new theories of animism advanced by Descola and Viveiros de Castro are consistent with the indigenous ontologies of North Asia. Based on a survey of North Asian ethnography and on fieldwork in Mongolia and Siberia, it is proposed that an analytical distinction between animist and totemist modalities will shed light on indigenous ontologies in North Asia. Whereas the ontologies of Northern North Asia (NNA) are predominantly animistic in nature, the ontologies of Southern North Asia (SNA) are predominantly totemistic. This opposition falls in line with established anthropological distinctions concerning North Asian societies, such as the one between 'horizontally' and 'vertically' organized social formations. Finally, adopting Viveiros de Castro's notion of 'perspectivism', I address the question of why, when perspectivist notions seem to thrive in NNA, the societies of SNA do not show them.     

Comparison of the community structure of the marine molluscs of the “Banco D. João de Castro” seamount (Azores,Portugal) with that of typical inshore habitats on the Azores archipelago

D. João de Castro Bank (DJC) is a shallow seamount with hydrothermal activity located between the islands of Terceira and São Miguel, Azores (Portugal). A survey of the molluscan fauna was conducted in this peculiar habitat. We found a smaller number of molluscan taxa and a different community structure compared to shore environments on the Azores. This coincides with impoverished environmental settings at this location. Possible reasons such as the degree of isolation (inshore vs. offshore locations), geological age, and special ecological conditions related to seamounts and hydrothermal vents are discussed.     

Identification of neopterin as a potential indicator of infection in burned patients.

Several fluorescent substances are present in the supernatants of acid-precipitated whole blood or plasma from burned patients. Perchloric acid supernatants of sera from infected, but not uninfected, burned rats contained a fluorescent substance with maximum emission at 420 nm at 355-nm excitation (355 ex/420 em). In this study of serum from burned human patients, several fluorescent substances were resolved by reverse-phase high-pressure liquid chromatography. One of these fluorescent components had an high-pressure liquid chromatography retention time and fluorescent characteristics identical to those of neopterin. The identification of this component as neopterin was verified by thermospray mass spectrometry. Serum neopterin concentrations were then determined in supernatants of patient serum samples having various levels of 355 ex/420 em fluorescence. A correlation was found between the concentrations of neopterin determined by high-pressure liquid chromatography and the presence of bacteremia in burned patients. These findings suggest that neopterin, which is a useful indicator of infection in other clinical settings, may also be an indicator of infection in burned patients.     

A workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experiments

We present and evaluate a strategy for the mass spectrometric identification of proteins from organisms for which no genome sequence information is available that incorporates cross-species information from sequenced organisms. The presented method combines spectrum quality scoring, de novo sequencing and error tolerant BLAST searches and is designed to decrease input data complexity. Spectral quality scoring reduces the number of investigated mass spectra without a loss of information. Stringent quality-based selection and the combination of different de novo sequencing methods substantially increase the catalog of significant peptide alignments. The de novo sequences passing a reliability filter are subsequently submitted to error tolerant BLAST searches and MS-BLAST hits are validated by a sampling technique. With the described workflow, we identified up to 20% more groups of homologous proteins in proteome analyses with organisms whose genome is not sequenced than by state-of-the-art database searches in an Arabidopsis thaliana database. We consider the novel data analysis workflow an excellent screening method to identify those proteins that evade detection in proteomics experiments as a result of database constraints.     

Interference of a methotrexate derivative with urinary oncopterin [N2-(3-aminopropyl)biopterin] measurement by high-performance liquid chromatography with fluorimetric detection

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N²-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 μmollmol creatinine. The present results indicate that the peak found with elution from the C₁₈ column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.     

Serum neopterin levels in liver cirrhosis

Neopterin is a pyrazino-pyrimidine compound which is biosynthesized by macrophages. Increased concentrations of neopterin have been reported in conditions causing a stimulation of cellular immunity, such as viral and other infections, graft versus host disease, autoimmune disease and different malignancies. Recently, urinary neopterin levels have been found increased in patients with acute viral hepatitis and NANB chronic hepatitis. In the present study, neopterin serum levels have been measured in 23 cirrhotic patients (6 HBV related, and 17 cryptogenetic cirrhosis, 7 of them occurring in alcoholic subjects) and in 24 normal subjects. Mean values of serum neopterin were significantly increased in cirrhotics (3.92 +/- 3.28 ng/ml versus 1.24 +/- 0.51 ng/ml in controls, p less than 0.01). Serum neopterin values were not found to be significantly different in cirrhotics assessed in three different clinical classes according to Child's classification and in cirrhotics with and without serological findings of active disease. In fact, in cirrhotic patients, serum neopterin levels did not correlate with the values of serum AST, ALT, ALP, GGT and gamma-globulin. These data show that increased levels of serum neopterin occur in cirrhotic patients, but there is no relation between serum neopterin values and the activity or the clinical severity of the disease. The results are consistent with the hypothesis that activated macrophages are involved in all stages of liver cirrhosis irrespective of its aetiology.