H. Denz

Determination of volatile organic compounds in exhaled breath of patients with lung cancer using solid phase microextraction and gas chromatography mass spectrometry

Abstract Background: Analysis of exhaled breath is a promising diagnostic method. Sampling of exhaled breath is non-invasive and can be performed as often as considered desirable. There are indications that the concentration and presence of certain of volatile compounds in exhaled breath of lung cancer patients is different from concentrations in healthy volunteers. This might lead to a future diagnostic test for lung cancer. Methods: Exhaled breath samples from 65 patients with different stages of lung cancer and undergoing different treatment regimes were analysed. Mixed expiratory and indoor air samples were collected. Solid phase microextraction (SPME) with carboxen/polydimethylsiloxane (CAR/PDMS) sorbent was applied. Compounds were analysed by means of gas chromatography (GC) and mass spectrometry (MS). Results: The method we used allowed identification with the spectral library of 103 compounds showing at least 15% higher concentration in exhaled breath than in inhaled air. Among those 103 compounds, 84 were confirmed by determination of the retention time using standards based on the respective pure compound. Approximately, one third of the compounds detected were hydrocarbons. We found aromatic hydrocarbons, alcohols, aldehydes, ketones, esters, ethers, sulfur compounds, nitrogen-containing compounds and halogenated compounds. Acetonitrile and benzene were two of 10 compounds which correlated with smoking behaviour. A comparison of results from cancer patients with those of 31 healthy volunteers revealed differences in the concentration and presence of certain compounds. The sensitivity for detection of lung cancer patients based on eight different compounds not seen in exhaled breath of healthy volunteers was 51% and the specificity was 100%. These eight potential markers for detection of lung cancer are 1-propanol, 2-butanone, 3-butyn-2-ol, benzaldehyde, 2-methyl-pentane, 3-methyl-pentane, n-pentane and n-hexane. Conclusions: SPME is a relatively insensitive method and compounds not observed in exhaled breath may be present at a concentration lower than LOD. The main achievement of the present work is the validated identification of compounds observed in exhaled breath of lung cancer patients. This identification is indispensible for future work on the biochemical sources of these compounds and their metabolic pathways. Clin Chem Lab Med 2009;47:550–60.

Dependence of exhaled breath composition on exogenous factors, smoking habits and exposure to air pollutants.

Non-invasive disease monitoring on the basis of volatile breath markers is a very attractive but challenging task. Several hundreds of compounds have been detected in exhaled air using modern analytical techniques (e.g. proton-transfer reaction mass spectrometry, gas chromatography-mass spectrometry) and have even been linked to various diseases. However,the biochemical background for most of compounds detected in breath samples has not been elucidated; therefore, the obtained results should be interpreted with care to avoid false correlations. The major aim of this study was to assess the effects of smoking on the composition of exhaled breath. Additionally, the potential origin of breath volatile organic compounds (VOCs) is discussed focusing on diet, environmental exposure and biological pathways based on others studies. Profiles of VOCs detected in exhaled breath and inspired air samples of 115 subjects with addition of urine headspace derived from 50 volunteers are presented. Samples were analyzed with GC-MS after preconcentration on multibed sorption tubes in case of breath samples and solid phase micro-extraction (SPME) in the case of urine samples. Altogether 266 compounds were found in exhaled breath of at least 10% of the volunteers. From these, 162 compounds were identified by spectral library match and retention time (based on reference standards). It is shown that the composition of exhaled breath is considerably influenced by exposure to pollution and indoor-air contaminants and particularly by smoking. More than 80 organic compounds were found to be significantly related to smoking, the largest group comprising unsaturated hydrocarbons (29 dienes, 27 alkenes and 3 alkynes). On the basis of the presented results, we suggest that for the future understanding of breath data it will be necessary to carefully investigate the potential biological origin of volatiles, e.g., by means of analysis of tissues, isolated cell lines or other body fluids. In particular, VOCs linked to smoking habit or being the results of human exposure should be considered with care for clinical diagnosis since small changes in their concentration profiles(typically in the ppt(v)–ppb(v) range) revealing that the outbreak of certain disease might be hampered by already high background.

Correlation between neopterin, interferon-gamma and haemoglobin in patients with haematological disorders.

Abstract: In this study, we further investigated a possible link between activation of cell‐mediated immunity and anaemia in patients with haematological neoplasias. We compared serum concentrations of interferon‐gamma and neopterin with haemoglobin levels. Significantly increased interferon‐gamma and neopterin concentrations indicated persistent activation of cell‐mediated immunity. Neopterin levels correlated significantly to interferon‐gamma concentrations and inversely to haemoglobin levels. The data indicate an association between activated macrophages and the development of anaemia in patients with haematological neoplasias.

Weight loss in patients with hematological neoplasias is associated with immune system stimulation

SummaryWeight loss is the main symptom of so-called tumor cachexia. The pathogenetic mechanisms underlying cachexia are poorly understood; however, it appears that enhanced formation of cytokines such as interferon-γ and tumor necrosis factor-α are involved. In 94 patients suffering from hematological neoplasias we compared body weight changes with serum neopterin, tryptophan, and kynurenine. Biochemical changes, the formation of neopterin, the degradation of tryptophan are closely related to interferon-γ activity. The majority of our patients had increased neopterin and decreased tryptophan concentrations. Weight loss was seen particularly in patients with higher neopterin and lower tryptophan values. An association between higher neopterin levels and greater weight loss was apparent at study entry and during the follow-up of patients. Our data support the concept that weight loss is closely linked to endogenous interferon-γ activity.

Association between the activation of macrophages, changes of iron metabolism and the degree of anaemia in patients with malignant disorders

Abstract: In this study, we investigated a possible association between the degree of macrophage activation ‐ as measured by serum neopterin concentrations ‐ and disturbances of iron metabolism, determined by the concentrations of ferritin and serum iron, in patients with malignant disorders. Additionally we evaluated correlations between these factors and the degree and type of anaemia. Seventy‐three patients, who suffered from non‐Hodgkins lymphoma (NHL) (n = 43), Hodgkins disease (n = 11), myeloma or monoclonal gammopathy of unknown significance (n = 9), myelodysplastic syndrome (n = l), and solid tumours (n = 9), were examined. Mean neopterin levels were raised in all groups, patients with NHL showing the highest concentrations. Ferritin but not neopterin concentrations were higher in males than in females. A significant correlation was found between neopterin and ferritin concentrations (p<0.01). Considering only female patients the strength of the correlation was the same (p<0.02). In addition, we found inverse correlations of neopterin with haemoglobin and iron concentrations (all p< 0.01). Similar relationships existed in patients during follow‐up. Our results support the hypothesis of an association between the degree of activation of macrophages and the development of anaemia by a shift or iron towards the storage sites.

Value of urinary neopterin in the differential diagnosis of bacterial and viral infections.

SummaryNeopterin is released by stimulated macrophages. In this study we analyzed the diagnostic potential of urinary neopterin concentrations in patients with bacterial and viral infection. All but one of 17 patients with viral infection had increased urinary neopterin concentrations. Patients with bacterial urinary tract infection also showed increased neopterin concentrations, whereas patients with bacterial pneumonia had significantly lower neopterin levels. In addition, patients with acute bacterial pneumonia had lower neopterin levels than patients with protracted infection. A significant inverse correlation between urinary neopterin and hemoglobin concentrations was found. Neopterin concentrations could serve as a helpful additional marker of infectious diseases. Combined with other clinical and laboratory parameters it is a useful parameter for distinguishing between viral and bacterial origins of infection, as was shown by multivariate stepwise linear discriminant analysis.

Serum soluble tumour necrosis factor receptor 55 is increased in patients with haematological neoplasias and is associated with immune activation and weight loss

Enhanced concentrations of soluble forms of the receptor for tumour necrosis factor (TNF)-alpha have been detected in the serum of cancer patients. We determined serum concentrations of soluble TNF receptor p55 (sTNF-R55) in patients with haematological neoplasias, 50 patients suffering from non-Hodgkins lymphoma (n = 35), Hodgkins disease (n = 10) and multiple myeloma (n = 5). Compared with healthy controls and with patients with potential thyroid disease, significantly elevated concentrations of sTNF-R55 were found (mean +/- standard error: 2.68 +/- 0.22 vs. 1.23 +/- 0.21 ng/ml, P < 0.0001 and 2.18 +/- 0.32 ng/ml, P = 0.03). Likewise, neopterin concentrations were raised (19.6 +/- 3.66 vs. 5.3 +/- 0.25 nmol/l in controls, P < 0.0001). We found a significant correlation between sTNF-R55 and neopterin concentrations (Rs = 0.544, P < 0.001). Patients with weight loss showed higher sTNF-R55 concentrations than patients with stable weight. Our results confirm the relevance of sTNF-R55 concentrations in serum of patients with cancer.

Successful antiangiogenic combination therapy for pseudomyxoma peritonei with bevacizumab and capecitabine

Effective systemic therapy for advanced pseudomyxoma peritonei (PMP) is the focus of investigation. We describe a case of PMP arising from an adenoma of the appendix in a 58-year-old man. First, the patient underwent explorative laparotomy with ileocoecal resection, but without possibility of major tumor debulking due to adhesive gross tumor masses. Subsequently, six cycles of Folfox IV chemotherapy were administered, without response, but with severe side effects. Upon progressive disease, a combination of bevacizumab and capecitabine led to a long term stabilization of disease and obvious improvement of performance status. Our case suggests that modulation of tumor microenvironment and angiogenesis by bevacizumab, potentially augmented by monochemotherapy, may be beneficial in borderline tumors such as PMP.

Treatment with low dose human recombinant interferon-alpha-2-ARG induces complete remission in patients with hairy cell leukemia.

Five cases with advanced hairy cell leukemia refractory to treatment with splenectomy and chemotherapy as well as one patient presenting with a stage-A response to splenectomy were treated with rhu-IFN-alpha 2-arg. 5 X 10(6) were administered intramuscularly every day. Both patients, with advanced disease resistant to conventional therapy and treated for six or more months with rhu-IFN-alpha 2-arg, achieved complete clinical remissions. Three further cases treated for less than half a year and also with advanced disease achieved partial remission states with marked reduction of circulating hairy cells and with recovery of normal hemopoiesis. Minimal residual disease in the remaining patient during a three-month period of treatment did not respond. Side effects of rhu-IFN-alpha 2 low dose therapy were minimal in 5 cases and comprised a severe leukopenia reversible after dose reduction in one patient.

Hyperhomocysteinaemia and immune activation in patients with cancer

Abstract Background: Recently, homocysteine production was observed in tumour cell lines and homocysteine was proposed as a tumour marker. Furthermore, homocysteine production by activated immunocompetent cells was demonstrated. Methods: In this study, homocysteine metabolism and immune activation status were investigated in 128 patients suffering from various types of cancer (haematological disorders, lung cancer, gastrointestinal tumours, gynaecological cancer and tumours of other localisation) and healthy age-matched controls. Results: A high percentage of patients (39.1%) showed moderate hyperhomocysteinaemia, while cysteine, folate and vitamin B12 concentrations were within reference ranges. Most patients were found to have elevated concentrations of the immune activation and inflammation markers neopterin and C-reactive protein (CRP), as well as a higher erythrocyte sedimentation rate (ESR). Patients of different cancer groups differed significantly regarding vitamin B12 and neopterin concentrations; higher B12 levels were also associated with tumour progression. Univariate regression analysis showed that CRP, ESR and neopterin were suited best to predict death. In multivariate analysis, neopterin was best suited to predicting death, while homocysteine and B vitamins were not associated with patient outcome. Homocysteine concentrations were correlated with folate and cysteine levels. Higher neopterin concentrations coincided with lower folate concentrations, but higher vitamin B12 concentrations. Conclusions: Associations between neopterin and folate concentrations may indicate that cellular immune activation might partly contribute to the development of folate deficiency in cancer patients, thus possibly also impairing homocysteine remethylation. Clin Chem Lab Med 2007;45:47–53.