Publikationsserver der RWTH Aachen University
Biomarker in Atemkondensat und Serum bei asbestbedingten Lungenfibrosen
Abstract
dc:descriptionBiological monitoring has a high significance for the detection of impurities and their consequences for human health. Therefore the existence of valid methods to get a certain survey is extraordinary important. The development of biomarkers provides a good and important opportunity to detect first effects developed from former exposures. An appropriate biomarker should be able to evaluate the risk of diseases in sufficient time to give a reasonable contribution for prevention and early diagnoses when measured in preventive medical checkup. To constitute a threshold limit value for persons at risk, first a parameter has to be found which can unequivocally distinguish between diseased and non-diseased persons. The exposure to asbestos can cause a number of different diseases, one of these is lungfibrosis. To achieve the objective target of this thesis, to find a parameter able to differentiate between persons with lungfibrosis (cases) and those without (controls), it was necessary to think about processes starting when fibroses appears. Furthermore some basics of cascades starting after asbestos exposure had to be understood. The existence of oxidative and nitrosative stress occurring because of increased formation of radicals due to asbestos exposure and an expected increased collagen turn-over by reason of a developing fibrosis, provide the possible biomarkers. Because oxidative and nitrosative stress are closely related, nitrotyrosin where choosed, which is often debated in literature to be a marker for nitrosative stress. An increased collagen turn-over dues to augmented release of hydroxyproline. Hydroxyproline only occurs in collagen, being made there by hydroxylation of proline after its incorporation. Therefore hydroxyproline seemed to be an appropriate marker, especially it is also used referred to other diseases causing an increased collagen-turnover. Measurement of hydroxyproline and nitrotyrosine should not be done without a standardisation parameter. Proline and tyrosine, both non-essential amino acids and precursors of hydroxyproline and nitrotyrosine, are very suitable for standardisation. The simultaneous detection of these four substances was an additional aim of the thesis. Because all the four substances were of high polarity, the development of a simultaneous, highly sensitive, accurate and specific LC/MS/MS-method with acceptable retention times were challenging and couldn’t be achieved by the use of reversed phase technique. With the exception of nitrotyrosine, none of the other substances showed good retention times. The application of “classic” normal phase technique would have required a lot of organic eluent, which would have complicated the method and gave adverse effect to the HPLC pump seal. As an alternative the hydrophilic interaction liquid chromatography (HILIC) was chosen. This technique is similar to normal phase technique, because its stationary phase is silica-gel modified and therefore also polar. Mobile phases being used for HPLC consist of an aqueous and an organic eluent, whereas the aqueous is the strongest one. Using this technique the simultaneous separation was very successful. Measurements were taken in exhaled breath condensate and serum. In exhaled breath condensate there is a high dilution effect which has to be compensated by relation to the standardisation parameters. The sample volume of 1 ml was still sufficient. In serum proline and tyrosine are high concentrated so a high dilution factor is required. The concentrations of hydroxyproline, but especially the one of nitrotyrosine are much lower, which results in non-detectable amounts of nitrotyrosine in serum due to the high dilution factor. The subjects were grouped by means of their smoking status and their classification into a fibrosis category depending on international CT-classification, into different categories. The smoking status was validated by anamnestic data as well as by determination of cotinine, which is a marker for acute, not older than 3-4 day smoke exposure. The ratio of hydroxyproline to proline and the ratio of nitrotyrosine and tyrosine in exhaled breath condensate and serum respectively were in the ranges known from literature. When fibrosis exist and smoke exposure is also present a tendency can be seen, that the ratio of hydroxyproline to proline is increased, both in exhaled breath condensate and in serum. Lowest concentrations of hydroxyproline based on proline concentration could be found at non-smoking subjects without fibrosis. Anyhow, no direct correlation could be found between ratios of hydroxyproline to proline in exhaled breath condensate and serum. In consideration of all factors, the ratio of hydroxyproline to proline seems to be a promising approach to differentiate smokers into controls and cases. The ratio of nitrotyrosine to tyrosine showed an unambiguous tendency to non-smoking subjects without fibrosis having the highest and smoking subjects with fibrosis having the lowest ratios. The interrelation between oxidative and nitrosative stress including the influence of iron containing asbestos fibres and smoking exposure on the formation of free radicals is very extensive. In the literature it is described that in presents of nitrosative stress the formation of nitrotyrosine will be increased. In this case, it can be speculated that under asbestos exposure there will be cascades starting, leading not to an increased, but to a decreased ratio of nitrotyrosine to tyrosine. The validation of this hypothesis would be a promising approach for the potential use of nitrotyrosine as a biomarker for the detection of asbestos induced lung fibrosis. This theses offers basic ideas and approaches to make hydroxyproline and nitrotyrosine, and their ratio to their precursors proline and tyrosine respectively, suitable as biomarkers for the detection of asbestos induced lung fibrosis. The implementation of the HILIC technique enabled the development of a valid, high sensitive and specific method for the simultaneous detection of proline, hydroxyproline, tyrosine und nitrotyrosine. The developed hypotheses must be proofed within futher systematical investigations. Therefore the “Asbestfrüherkennungsprogramm Aachen” delivers ideal premises.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Conventz, Angela
- Contributors dc:contributor
-
- Kraus, Thomas
Subjects
dc:subject × 14Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger