Urine neopterin concentrations as a marker for successful blastocyst implantation after assisted reproductive technologies.

Successful blastocyst implantation requires intricately orchestrated adaptation processes involving maternal and fetal mediators. The pivotal role of distinct immune response pathways in early pregnancy is widely acknowledged. Pro-inflammatory cytokines, e.g. interferon-gamma (IFN-gamma), are the primary inducers of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and of neopterin biosynthesis by GTP-cyclohydrolase I. IDO activity has been proposed to be of high clinical relevance in the context of pregnancy. To date, insights arising from clinical studies on IDO activity and neopterin concentration during the very early days of pregnancy are still few. Early morning urinary neopterin concentrations in 61 women undergoing assisted reproduction treatment (72 cycles in total) were examined, upon exclusion of infections, daily over a period of 2 weeks after embryo transfer. Twenty of the study participants (28%) became successfully pregnant, and four women experienced abortion. Neopterin concentrations significantly increased after blastocyst transfer when implantation was successful (chi-squared=23.291, P<0.01; Friedman test), opposed to non-significant changes of neopterin in women with unsuccessful treatment (chi-squared=8.203). The steady increase of neopterin concentrations upon blastocyst transfer indicates that heightened production of neopterin in very early phases of pregnancy may serve as an early predictor of successfully progressing pregnancies in humans.

Association between asymmetric dimethylarginine and neopterin in patients with and without angiographic coronary artery disease.

The increase of circulating asymmetric dimethylarginine (ADMA) concentrations, a competitive inhibitor of the nitric oxide synthases, is associated with an increased cardiovascular risk and is considered to play a role in endothelial dysfunction. Recently, ADMA production was observed in stimulated human peripheral mononuclear cells. In this study, we examined a potential relationship between concentrations of ADMA and of the immune activation marker neopterin in patients scheduled for coronary angiography. In a cross-sectional approach, blood concentrations of ADMA, homocysteine, neopterin, folic acid and vitamins B6 and B12 were compared in 2030 patients, which were recruited as participants of the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study. ADMA concentrations did not differ between patients with coronary artery disease (CAD) (mean +/- SD: 0.82 +/- 0.15 micromol/l) and controls (0.81 +/- 0.14 micromol/l; Welch's t-test: P = n.s.). ADMA concentrations correlated with homocysteine (r(s) = 0.207) and vitamin B6 (r(s) = -0.190), and an even stronger correlation with neopterin (r(s) = 0.276; all P < 0.0001) was observed. In conclusion, increased ADMA concentrations in patients at risk for atherosclerosis are associated with increased neopterin concentrations. Data suggest that immune activation may contribute to increased ADMA production in CAD patients.

Purine metabolism of human glioblastoma in vivo.

The aim of this study was to identify targets for rational chemotherapy of glioblastoma. In order to elucidate differences in the biochemistry of tumor and normal human brain, in vivo pool sizes of purine nucleotides, nucleosides, and nucleobases and of purine metabolizing enzymes in biopsy material from 14 grade IV astrocytomas and 4 normal temporal lobe samples were analyzed. Specimens were collected during surgery using the freeze-clamp sampling technique and analyzed by high pressure liquid chromatography. Total purine nucleotides, adenylates, and guanylates in the tumors were 2186, 1865, and 310 nmol/g (wet weight), respectively, which corresponds to 61, 60, and 71% of normal brain tissue concentrations. Relative to normal brain the tumors had significantly lower ATP and GTP levels, essentially normal pool sizes of purine nucleosides and bases, unchanged activities of the salvage enzymes hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and adenosine kinase (659, 456, and 98 nmol/h/mg protein, respectively) and 4-fold higher activities of IMP dehydrogenase (11.6 nmol/h/mg protein); the latter is the rate limiting enzyme for guanylate de novo synthesis. IMP pools in the tumors were 64% of values in normal brain. Modulation of the guanylate pathway in glioblastoma by inhibition of IMP dehydrogenase with tumor specific agents such as tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) appears to be a rational therapeutic approach. Preliminary in vitro experiments with normal and malignant tissue specimens from 2 additional patients revealed that significant amounts of the active metabolite thiazole-4-carboxamide adenine dinucleotide are formed from tiazofurin. At a concentration of 200 microM this drug was able to deplete guanylate pools in the tumors to a median of 54% of phosphate buffered saline treated controls. Flux studies with [14C]formate showed that tiazofurin strongly inhibited de novo synthesis of guanylates in glioblastoma to an average of 10% of controls. This effect was more pronounced in the tumors as compared to normal brain. No inhibition of salvage of [14C]guanine by tiazofurin could be observed in normal and malignant tissues. Supportive measures have to be considered to inhibit the highly active salvage enzyme hypoxanthine-guanine phosphoribosyltransferase that can partly antagonize a tiazofurin induced decrease in guanine nucleotides.

Conformational investigation of the cofactor (6R,1'R,2'S-)-5,6,7,8-tetrahydrobiopterin.

(6R,1'R,2'S)-5,6,7,8-Tetrahydrobiopterin is an essential cofactor for several enzymes. Different theoretical models (molecular mechanics, semiempirical quantum chemical calculations) investigating its stereostructure have yielded diverging answers. To clarify these issues, combined molecular mechanical and ab initio quantum chemical calculations were performed, investigating both the axial and the equatorial orientation of the dihydroxypropyl side-chain. After geometry optimization, the resulting most stable structures were subjected to systematic variation of two side-chain torsional angles in order to study the conformational flexibility. The axial side-chain orientation is slightly more stable than the equatorial form. Two weak intramolecular hydrogen bonds contribute to stabilization of the axial conformer, while in the equatorial conformer only one hydrogen bond is detected. An 8 ps molecular dynamical simulation at 310 K suggests that, at realistic temperatures, the molecule is flexible enough to undergo internal motions (rotations, vibrations), rendering questionable the biological significance of mere conformational properties.

Neopterin as a marker for immune system activation.

Increased amounts of neopterin are produced by human monocytes/macrophages upon stimulation with the cytokine interferon-y. Therefore, measurement of neopterin concentrations in body fluids like serum, cerebrospinal fluid or urine provides information about activation of T helper cell 1 derived cellular immune activation. Increased neopterin production is found in infections by viruses including human immunodeficiency virus (HIV), infections by intracellular living bacteria and parasites, autoimmune diseases, malignant tumor diseases and in allograft rejection episodes. But also in neurological and in cardiovascular diseases cellular immune activation indicated by increased neopterin production, is found. Major diagnostic applications of neopterin measurements are, e.g. monitoring of allograft recipients to recognize immunological complications early. Neopterin production provides prognostic information in patients with malignant tumor diseases and in HIV-infected individuals, high levels being associated with poorer survival expectations. Neopterin measurements are also useful to monitor therapy in patients with autoimmune disorders and in individuals with HIV infection. Screening of neopterin concentrations in blood donations allows to detect acute infections in a non-specific way and improves safety of blood transfusions. As high neopterin production is associated with increased production of reactive oxygen species and with low serum concentrations of antioxidants like alpha-tocopherol, neopterin can also be regarded as a marker of reactive oxygen species formed by the activated cellular immune system. Therefore, by neopterin measurements not only the extent of cellular immune activation but also the extent of oxidative stress can be estimated.

Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects.

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.

Effects of pteridines on luminol-dependent chemiluminescence induced by chloramine-T.

Pteridines are ubiquitous in living organisms, but little is known about their biological functions. Different pteridines were tested for their ability to modulate luminol-dependent chemiluminescence induced by chloramine-T at pH = 7.5 and at a concentration of 100 microM for each pteridine. We observed striking differences between the compounds; whereas reduced pteridine species were generally potent scavengers, aromatic pteridines were weak to strong enhancers of the chemiluminescence. Taking into account the detailed chemical structure of the molecules, by multiple linear regression analysis a simple index was constructed that allows prediction of the effects of the different pteridines with high accuracy (linear correlation coefficient between predicted and observed values r = 0.89). The effects of different pteridines on free radical-induced chemiluminescence might bear biological significance since, for example, certain pteridines take part in enzymic reactions involving free-radical intermediates, or are related to the activation of macrophages in close relationship with the oxidative burst.

Enhancement of hydrogen peroxide-induced luminol-dependent chemiluminescence by neopterin depends on the presence of iron chelator complexes.

We have previously shown that neopterin, 6-D-erythro-trihydroxypropyl-pteridine, synthesized by human monocytes/macrophages upon stimulation by interferon-gamma, enhances toxicity of reactive oxygen at neutral or slightly alkaline pH (7.5), but not at acidic pH (below 6.5). In the present study, we explored in more detail the necessary requirements for neopterin to modulate the effects of hydrogen peroxide in a luminol-dependent chemiluminescence assay. We demonstrate that neopterin enhances hydrogen peroxide effects only in the presence of iron chelator complexes like iron-(III)- or iron-(II)-EDTA or iron-(III)-DTPA. Thus, iron chelator complexes together with neopterin may play an important role in macrophage-mediated effector mechanisms.

Effect of neopterin and 7,8-dihydroneopterin on tumor necrosis factor-alpha induced programmed cell death.

Tumor necrosis factor-alpha and the formation of reactive oxygen intermediates are central mediators of apoptosis. Recent data indicated a role of neopterin and 7,8-dihydroneopterin in oxygen radical mediated processes. We have therefore investigated the effect of neopterin-derivatives on TNF alpha induced apoptosis of the monocyte-like cell line U937. At an elevated concentration 7,8-dihydroneopterin was found to superinduce TNF alpha mediated programmed cell death due to the formation of reactive oxygen intermediates. Our results imply that in combination with TNF alpha high concentrations of 7,8-dihydroneopterin enhances apoptosis due to oxidative stress on cells.

Histamine suppresses neopterin production in the human myelomonocytoma cell line THP-1.

Histamine, an important inflammatory mediator in allergic diseases and asthma, was reported to have modulatory effects on T cells by down-regulating Th1-type cell cytokines like interleukin 2 (IL-2) and interferon-gamma (IFN-gamma). In this study we examined the effect of histamine and the histamine-receptor antagonists cimetidine and diphenhydramine on the production of neopterin after stimulation with IFN-gamma in the myelomonocytoma cell line THP-1. Increasing concentrations of histamine markedly suppressed IFN-gamma induced neopterin formation. Simultaneous preincubation of THP-1 cells with histamine, IFN-gamma and different concentrations of the H(2)-receptor antagonist cimetidine showed a clear antagonizing effect on neopterin formation. In contrast, the H(1)-receptor antagonist diphenhydramine was not able to abrogate the suppressive effect of histamine on neopterin production. Our results suggest, that histamine may be a potent inhibitor of effects or mechanisms induced by IFN-gamma in monocytes/macrophages. Cimetidine, and possibly other H(2)-receptor antagonists, may reverse down-regulatory actions of endogenously formed histamine on activated monocytic cells.

Urinary neopterin excretion in patients with squamous carcinoma of the oral cavity.

Urinary neopterin concentrations were studied in 30 patients with squamous carcinoma or adenoid cystic carcinoma of the oral cavity. Compared to healthy controls 19 patients (63%) had increased neopterin concentrations. There was a statistically significant correlation between neopterin levels and tumor differentiation but no correlation of neopterin values with tumor size. Longitudinal studies will be necessary to evaluate a potential usefulness of neopterin concentrations to predict prognosis in squamous carcinomas of the oral cavity.

Dehydroepiandrosterone, ageing and immune activation.

The age-related decline in dehydroepiandrosterone (DHEA) production is currently attracting attention because of its possible relevance to the etiology and management of a number of age-related clinical disorders. Various abnormalities of immune system function have been described in the elderly. Among them, increased concentrations of neopterin have been reported, which is produced by human monocytes/macrophages upon stimulation by interferon-gamma. In order to examine the relation of serum DHEA to serum neopterin, we studied 281 otherwise healthy outpatients, who visited the physician's office for a medical health check-up. 10% presented with increased neopterin concentrations, 0.4% had increased DHEA sulfate (DHEAs) concentrations. DHEAs concentrations were significantly higher in patients with lower neopterin concentrations (Mann-Whitney test: U=4793, P<0.0001). There existed a rather strong inverse correlation between DHEAs concentrations and serum neopterin concentrations (Spearman's rank correlation: r(s)=-0.221, P<0.0001). The data support the concept that the decrease of DHEA with increasing age is related to immune system activation. Oxidative stress which accompanies immune response may diminish DHEA synthesis.

Streptococcal erythrogenic toxins induce neopterin formation in human peripheral blood mononuclear cells but not in the human myelomonocytoma cell line THP-1.

We tested whether the exposure of human monocytic cells to streptococcal erythrogenic toxins A, B, C and a streptococcal-derived Mitogert BX is associated with synthesis of neopterin in vitro. Neopterin production was not induced when the human myelomono-cytoma cell line THP-1 was stimulated with these toxins, and there was only a slight co-stimulatory effect of streptococcal erythrogenic toxin A together with interferon-gamma stimulation. However, these toxins induced interferon-gamma and further neopterin production in peripheral blood mononuclear cells of three healthy individuals. This neopterin formation could be blocked by anti-human interferon-gamma. From our investigations we conclude that there is no direct effect of streptococcal erythrogenic toxins on neopterin production by monocytic cells. However, the data obtained in peripheral blood mononuclear cell culture imply that these toxins are able to stimulate neopterin production in humans via the induction of huge amounts of interferon-gamma.

Neopterin--its clinical use in urinalysis.

Increased amounts of neopterin are released during cellular immune response. Neopterin concentrations can be monitored in serum and urine of patients since neopterin is removed from the circulation by renal excretion. In allograft recipients, rising neopterin concentrations indicate rejection episodes early. Neopterin concentrations correlate with the extent and activity of viral infections, malignancies, and autoimmune diseases. We investigated excretion kinetics of neopterin in a rhesus monkey which received a high dose of neopterin intravenously. A sharp increase of urinary neopterin concentrations was observed, and from the data the half-life of neopterin in the circulation was estimated to be 90 minutes. By comparing urine and serum neopterin concentrations in HIV seropositive and seronegative human individuals, a strong correlation and similar diagnostic sensitivity between urine and serum values was observed. Thus, neopterin concentrations in serum or urine seem of equal value for diagnostic application as long as renal function is normal.

Neopterin: a prognostic variable in operations for lung cancer.

BACKGROUND: We studied the prognostic value of preoperatively measured neopterin to predict survival of lung cancer patients. Neopterin is produced and secreted by interferon-gamma-stimulated monocytic cells. High urinary neopterin concentrations are found in patients with viral infections, allograft rejection episodes, and some malignant diseases. In various tumor types high urinary neopterin concentrations are associated with a worse prognosis. METHODS: Preoperative neopterin levels of 110 patients (29 women, 81 men) with lung cancer including 7 patients with small cell lung cancer were measured and related to the time of survival after operation. Patients with clinically suspected stage IIIB lung cancer were not operated and therefore not enrolled in this study. Infectious diseases were not apparent at the time of preoperative urine sampling. Median postoperative follow-up period was 17.4 months. RESULTS: In a univariate analysis, patients with a preoperative neopterin concentration of more than 212 micromol/mol creatinine (4th quartile) were determined to have a significantly lower survival probability. In a multivariate analysis, a neopterin concentration of more than 212 micromol/mol creatinine (p < 0.01) and T-stage status (p < 0.005) were determined to be significantly predictive variables for worse survival prognosis. CONCLUSIONS: Preoperative neopterin proved to be a reliable prognostic factor for survival. Immunology may provide an accurate assessment of tumor aggression and its clinical behavior. In this sense, neopterin can serve as an immunologically based estimation of malignant outgrowth. In patients who are operable by clinical tumor stage but have a high risk for operation, elevated preoperative neopterin may help in the decision for a nonoperative treatment.

Association between insulin resistance, body mass and neopterin concentrations.

Obesity is frequently associated with insulin resistance. Recently an important role of the cytokine tumor necrosis factor-alpha in mediating insulin resistance of obesity through its overexpression in fat tissue has been reported. In order to examine the relation of insulin resistance to obesity and to serum neopterin, as a parameter of immune activation, we studied 1234 otherwise healthy outpatients, who visited the physician's office for a medical health check-up. 7% showed elevated glucose concentrations, 34% elevated body mass indices. There were significant correlations between glucose concentrations and body mass indices and of the latter with serum neopterin concentrations. Neopterin concentrations were significantly higher in patients with elevated body mass indices (Mann-Whitney test, U = 131 358, p = 0.0003) and elevated glucose concentrations (Mann-Whitney test, U = 35 350 p =0.02). The data may indicate that moderate immune stimulation plays a role in the development of insulin resistance, and an influence of tumor necrosis factor-alpha seems to be probable.

Increased serum amylase and lipase in fructose malabsorbers.

BACKGROUND: Fructose malabsorption is frequently seen in the general population and is characterised by the inability to absorb fructose efficiently. Due to fructose malabsorption, fructose reaches the colon where it is broken down by bacteria to short fatty acids, CO(2) and H(2). Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequence. We recently found that fructose malabsorption is associated with low plasma folic acid concentrations and low serum tryptophan and zinc. Because fructose malabsorption apparently is associated not only with malabsorption of other nutrients, but also with abdominal discomfort, it was of interest to examine whether mild pancreatitis may be involved. METHODS: We retrospectively examined our data in 159 otherwise healthy adults (110 females, 49 males) aged 14-84 years (mean 45.6+/-14.4 S.D.) with gastrointestinal complaints for serum amylase and serum lipase concentrations. The patients have been tested earlier for fructose malabsorption and lactose maldigestion by measuring breath H(2) concentrations after an oral dose of 25 g fructose and 50 g lactose, respectively, 1 week apart. RESULTS: Fructose malabsorption (H(2) concentrations > or =20 ppm over baseline values) was detected in 107 of 159 individuals (67.3%). These subjects with fructose malabsorption presented with significantly higher serum amylase concentrations (73.1 U/l+/-25.7 S.D.) compared to individuals with normal fructose absorption (59.6 U/l+17.9 S.D; p=0.0009). Fructose malabsorbers also presented with higher serum lipase concentrations (122.0 U/l+/-100.3 S.D.) compared to normals (89.5 U/l+/-46.5 S.D.; p<0.05). To determine whether this finding is a consequence of any sort of malabsorption syndrome or whether it is specific for fructose malabsorption, all subjects were screened for lactose maldigestion. Lactose maldigestion (H(2) concentrations>20 ppm over baseline after lactose loading) was found in 50 of 159 individuals (31.4%). There were no significant differences in either amylase or lipase concentrations in lactose maldigestors. CONCLUSION: Serum amylase and lipase concentrations are higher in subjects with fructose malabsorption compared to normals. Therefore, fructose malabsorption should be considered as a differential diagnosis in moderately elevated serum amylase.

Increased neopterin concentrations in patients with cancer: indicator of oxidative stress?

In vitro, large amounts of neopterin are produced by human monocytes/macrophages upon stimulation with interferon-gamma. In vivo increased neopterin concentrations in human serum and urine indicate activation of cell-mediated (Th1-type) immune response, e.g., during virus infections, autoimmune diseases, allograft rejection and in certain types of malignancy. In various groups of patients with malignant diseases neopterin concentrations correlate to the stage of disease, and higher neopterin concentrations in serum, urine or ascitic fluid were shown to significantly predict worse prognosis regarding relapse and survival. The amounts of neopterin produced by activated monocytes/macrophages correlate with their capacity to release reactive oxygen species (ROS). With this background, neopterin concentrations in body fluids can be regarded as an indirect estimate of the degree of oxidative stress emerging during cell-mediated immune response. Moreover, recently neopterin was found itself to be capable of enhancing toxic effects induced by ROS. In vitro, neopterin derivatives were able to interfere with intracellular signal transduction pathways involved in, e.g., programmed cell death and the induction of proto-oncogene c-fos or nuclear factor-chi B. The data support the view that increased production of ROS--indicated by increased neopterin concentrations--could modulate the development, the proliferation and the survival of malignant cells.

Effect of the calcium entry blocker nimodipine on the metabolism of nucleic acids in rat brain ischemia.

The effect of nimodipine, a 1,4 dihydro-piridine calcium entry blocker (200 micrograms/kg), was investigated in rats after definitive ischemia or 2 min of global ischemia (neck tourniquet method). The brains were freeze-clamped at the desired time intervals and subjected to high pressure liquid chromatography analyses for nucleotides and enzymatic lactate estimation. Although in the definitive ischemia (removal of the brain) no difference was observed in the treated versus the untreated animals, there was a statistically significant difference in both groups after global ischemia followed by reperfusion. Thirty minutes after reflow the brains of the treated animals contained 1,690 +/- 62 nmol ATP/g as compared to 765 +/- 259 nmol ATP/g in the untreated animals (p less than 0.05). The normal controls amounted to 1,932 +/- 77 nmol ATP/g. Also the adenylate energy charge returned to normal in the treated animals (treated animals and controls 0.69 and 0.72, respectively). From these preliminary data we conclude that nimodipine is able to restore mitochondrial function after ischemia and to maintain a high level of energy-rich phosphates. Thus, calcium entry blockers may be effective in preserving and protecting cerebral tissue from irreversible injury after ischemia.

Immune reaction links disease progression in cancer patients with depression.

Mood disturbances and depression are supposed to have a negative impact on patients' outcome in malignant tumour disease. On the other hand, poor prognosis in cancer patients is associated with chronic immune challenge which is paralleled by enhanced degradation of the essential amino acid tryptophan and thus decreased plasma tryptophan concentrations. Because tryptophan is precursor for the biosynthesis of the neurotransmitter serotonin (= 5-hydroxytryptamine, 5HT), low tryptophan concentrations will lead to decreased availability of serotonin which finally increases the susceptibility for the development of mood disturbances and depression in the patients. Thus, the development of depression in cancer patients may result from chronic cellular immune stimulation. In conclusion, a more aggressive tumour rather than depression will be responsible for worse outcome of cancer patients and will be associated with a more drastic challenge of the immune system, as a side effect leading to neurotransmitter disturbances.