Chromatographic, capillary electrophoretic and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of urinary modified nucleosides as tumor markers.

Modified nucleosides are formed posttranscriptionally in RNA. During RNA turnover free modified nucleosides are formed which circulate in the blood stream and are excreted in the urine. Their levels are increased in a number of malignant diseases, and they can be used in clinical chemistry as tumor markers. The analysis includes the isolation of the nucleosides from urine with phenylboronate gel and their separation and quantitation by HPLC on C18 columns or by capillary electrophoresis on uncoated columns applying a sodium dodecyl sulfate-borate-phosphate buffer. Identification of the nucleosides is performed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry including post source decay spectra. In two clinical studies the diagnostic value of urinary modified nucleosides is investigated, in a study on children with leukemia and other malignant diseases and a study on women with breast cancer. Candidate markers are pseudouridine, 1-methylguanosine, N2-methylguanosine, 3-methyluridine and 1-methyl-inosine.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography.

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1,) the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease.

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC-MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1,000 microg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1 +/- 13.5 microg/l for 2-ethylhexanoic acid, 104.8 +/- 80.6 microg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2 +/- 389.5 microg/l for 2-ethyl-3-oxohexanoic acid.

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from uterine myoma and cervical cancer patients.

Capillary electrophoretic (CE) profiling analysis combined with pattern recognition methods is described for the correlation between urinary nucleoside profiles and uterine cervical cancer. Nucleosides were extracted from urine specimens by solid-phase extraction in affinity mode using phenylboronic acid gel. CE separation was carried out with an uncoated fused-silica capillary (570 mm x 50 microm I.D.) maintained at 20 degrees C, using 25 mM borate-42.5 mM phosphate buffer (pH 6.7) containing 200 mM sodium dodecyl sulfate as the run buffer under the applied voltage of 20 kV. A total of 15 nucleosides were positively identified in urine samples (2 ml) from eight uterine myoma (benign tumor group), 10 uterine cervical cancer (malignant tumor group) patients and 10 healthy females (normal group) studied. The star symbol plots drawn based on each mean concentration of nucleosides normalized to that in normal group enabled one to discriminate malignant and benign groups from normal group. In addition, canonical discriminant analysis performed on the nucleoside data of 28 individual urine specimens correctly classified into three separate clusters according to groups in the canonical plot.

Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP-HPLC and factor analysis method.

Modified nucleosides, formed post-transcriptionally in RNA by a number of modification enzymes, are excreted in abnormal levels in the urine of patients with malignant tumors. To test their usefulness as tumor markers, and to compare them with the conventional tumor markers, a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a factor analysis method have been used to study the excretion pattern of nucleosides of breast cancer patients. A clear cut differentiation of the breast cancer group and the healthy individuals in two clusters without overlapping was obtained.

Application of capillary electrophoresis in clinical chemistry: the clinical value of urinary modified nucleosides.

Urinary modified nucleosides were determined by capillary electrophoresis using a 300 mM SDS-25 mM sodium tetraborate-50 mM sodium dihydrogenphosphate buffer. The nucleosides were extracted from urine by phenylboronate affinity gel chromatography. In cancer patients the levels of the modified nucleosides are generally elevated. By an artificial neural network method breast cancer patients were differentiated from normal individuals, which indicates that the modified nucleosides could be of clinical value as tumor markers.

Reversed-phase high-performance liquid chromatographic investigation of urinary normal and modified nucleosides of cancer patients.

Post-transcriptional modifications in RNA give rise to free modified ribonucleosides circulating in the blood stream and excreted in urine. Due to their abnormal levels in conjunction with several tumor diseases, they have been suggested as possible tumor markers. The developed RP-HPLC method has been applied to analyze the urinary nucleosides in 34 urinary samples from 15 kinds of cancer patients. The statistical analyses showed the urinary nucleoside excretion, especially modified nucleoside levels, in cancer patients were significantly higher than those in normal healthy volunteers. Factor analysis was used to classify the patients with cancer and normal healthy humans. It was found that using 15 urinary nucleoside levels or only five modified nucleoside levels as data vectors the factor analysis plot displayed two almost separate clusters representing each group.

Analysis of volatile organic compounds in human urine by headspace gas chromatography-mass spectrometry with a multipurpose sampler.

A multipurpose sampler (Gerstel MPS), designed for liquid large volume, gaseous and headspace samples was used for the GC-MS analysis of organic volatiles in human urine. Headspace sampling with a volume-, temperature- and speed-controlled gas-tight syringe was combined with a temperature-controlled cold injection system (CIS) for cold trapping, enrichment and focusing of analytes. Regular 2-ml GC vials filled with 1 ml acidified urine were used as headspace sampling vials. A 100-vial autosampler tray was equipped with an additional temperature and heating time controlled "preheating station" for five vials. Profiles of organic volatiles in human urine were determined and 34 components identified. Trimethylamine (TMA) and 4-heptanone as two metabolites of medical interest were quantified. Calibration curves and intra assay imprecision for 4-heptanone concentrations in the range of 40 to 800 ng/ml showed a correlation coefficient of r = 0.9980 and a relative standard deviation (RSD) between 3.0 and 3.4%. Calibration curves and intra-assay imprecision for TMA concentrations in the range of medical interest from 0.5 to 20 micrograms/ml showed a correlation coefficient of r = 0.9968 and a RSD between 4.1 and 6.8%. The high practicability of the multipurpose sampler for both gaseous and liquid samples together with the here shown good reproducibility and sensitivity make this single CIS-GC-MS system very attractive for routine clinical use in metabolic profiling of organic volatiles (headspace) and non-volatiles (liquid).

Identification of plasticizers in medical products by a combined direct thermodesorption--cooled injection system and gas chromatography--mass spectrometry.

The combination of a new thermodesorption module with a cooled injection system now provides a powerful system for direct analysis of volatile trace compounds in gaseous, liquid and solid samples by gas chromatography-mass spectrometry (GC-MS). As a cooled injection system is used for the cryofocusing of the desorbed volatiles the GC-MC system still can be used for the regular analysis of liquid samples. Although plasticizers usually are analyzed by GC-MS after solvent extraction, contaminated solvents and glassware are very well known problems. Analysis of plasticizers in plastic materials by direct thermodesorption instead saves time and avoids cross contaminations. Many medical products are made of plasticized polyvinyl chloride. Extraction of the common plasticizer di(2-ethylhexyl) phthalate (DEHP) into blood will occur, and harmful effects of DEHP in the human body have been suggested. We therefore analyzed 21 different plastic devices which are used for various invasive techniques in medicine by direct thermodesorption GC-MS. In some of the plastics up to 30 different components were identified. By far the most common plasticizer found was DEHP, followed by diethyl and dibutyl phthalates.

Profiling of organic acids by capillary gas chromatography-mass spectrometry after direct methylation in urine using trimethyloxonium tetrafluoroborate.

Trimethyloxonium tetrafluoroborate (TMO) is applied as derivatising reagent to transform urinary organic acids into their methyl esters. The method is suggested as an alternative to the use of diazomethane which is carcinogenic and explosive. In contrast to other methods avoiding diazomethane, such as derivatizations with acetyl chloride-methanol and boron trifluoride-methanol, which require an organic reaction medium and therefore an extraction of the organic acids from the urine, TMO efficiently reacts with the acids in an aqueous solution and can therefore be directly applied to native urine. The use of TMO simplifies and improves the sample preparation in the profile analysis of urinary organic acids by capillary GC-MS and hereby increases the speed of analysis. The method gives reproducible results which are comparable with the data obtained using conventional solid-phase extraction with strong anion-exchange cartridges prior to derivatisation.

Upregulation of cholesterol synthesis after acute myocardial infarction--is cholesterol a positive acute phase reactant?

Acute myocardial infarction is associated with profound alterations in the plasma lipoprotein profile. The mechanism of these alterations is not clear, and both cholesterol biosynthesis up- and downregulation could possibly be a consequence of acute myocardial infarction. We determined plasma lipids, lipoproteins, apolipoproteins, and lathosterol-which is regarded as an estimate of whole body cholesterol biosynthesis in humans-concentrations in 34 patients (age 68+/-10 years, 24 male, 10 female) admitted to our hospital with acute MI and with onset of symptoms within the last 12 h. Samples were taken immediately after admission to the hospital, and 1, 2, and 10 days after admission. On the first day after admission there was a decrease in total cholesterol (C) by 14.1%, (P = 0.01), in LDL-C by 14.4% (P = 0.03), in HDL-C by 9.3% (NS), and in triglycerides by 19.5% (NS). Apolipoprotein B100 was reduced by 18.3% (P = 0.008), and apolipoprotein AI by 12.3% (NS). The lathosterol/cholesterol ratio was increased by 23.1% after 1 day, and by 28.7% after 2 days (P = 0.05). After 10 days, all variables except the apolipoproteins had essentially returned to baseline values. In conclusion, the changes in the plasma lipid profile after acute myocardial infarction are associated with a profound increase of whole body cholesterol biosynthesis as judged by the lathosterol/cholesterol ratio. These changes may possibly enhance the delivery of cholesterol to cells involved in tissue repair mechanisms after acute myocardial infarction.

Urinary organic acid screening by solid-phase microextraction of the methyl esters.

We developed a new sample preparation method for profiling organic acids in urine by GC or GC-MS. The method includes derivatisation of the organic acids directly in the aqueous urine using trimethyloxonium tetrafluoroborate as a methylating agent, extraction of the organic acid methyl esters from the urine by solid-phase microextraction, using a polyacrylate fiber with a thickness of 85 microm and transfer of the methyl esters into the GC or the GC-MS instrument. Desorption of the analytes takes place in the heated injection port. The proposed sample preparation is very simple. There is no need for any evaporation step and for the use of an organic solvent. The risk of contamination and the loss of analytes are minimized. The total sample preparation time prior to GC or GC-MS analysis is about 40 min, and therefore more rapid than other sample preparation procedures. The urinary organic acids are well separated by GC and 29 substances are identified by GC-MS.

Gas chromatographic profiling and pattern recognition analysis of urinary organic acids from uterine myoma patients and cervical cancer patients.

An efficient organic acid profiling and pattern recognition method is described for the correlation between urinary organic acid profiles and uterine cervical cancer. After methoximation of keto acids in alkalinized urine samples, all free organic acids were recovered by a dual solid-phase extraction procedure, followed by conversion to tert.-butyldimethylsilyl derivatives for the profiling analysis by dual-capillary column gas chromatography (GC) with subsequent screening for acids by retention index (I) library matching. A total of 50 organic acids were positively identified in urine samples (0.25 ml) from 12 uterine myoma (benign tumor group) and 14 uterine cervical cancer (malignant tumor group) patients studied. When the GC profiles were simplified to their corresponding organic acid I spectra in bar graphical form, characteristic patterns were obtained for each average of benign and malignant tumor groups. Stepwise discriminant analysis performed on the GC data selected 16 acids as the variables discriminating between the two groups. Canonical discriminant analysis applied to these 16 variables correctly classified 26 urine samples into two separate clusters according to tumor types in the canonical plot.

Analysis of traditional Chinese anticancer drugs by capillary electrophoresis.

The results reported in this communication demonstrate that capillary zone electrophoresis (CZE) can be used easily for the quantitative determination of the potential anticancer drugs berberine and isoguanosine in the extract of the traditional Chinese medicinal herb. Isoguanosine and berberine can be monitored selectively and sensitively at 254 nm within 14 min in the plant extract using a 100-mM sodium citrate running buffer (adjusted to pH 2.7; applied voltage 12 kV). The concentration range of 0.1-50 micrograms ml-1 proved to be sufficient for exact quantification and the peak profile showed good reproducibility [relative standard deviations (n = 32); for the migration time 0.22% (isoguanosine) and 1.32% (berberine); for the peak area 2.8% (isoguanosine) and 3.2% (berberine)]. The measured concentrations in the crude extract were 1.3 micrograms ml-1 for isoguanosine and 8.7 micrograms ml-1 for berberine. In addition to the better separation performance, CZE shows several other remarkable advantages over high-performance liquid chromatography (HPLC), such as rapidity of analysis, small sample volume, no requirement of organic solvent in the running buffer and low cost of reagents.

Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients.

This paper gives a capillary electrophoretic method for the separation of 15 urinary normal and modified nucleosides from cancer patients in less than 40 min. A 500 mm x 50 microns uncoated capillary column (437.5 mm to window) was used. The effects of the voltage and the sodium dodecyl sulfate (SDS) concentration in the buffer on the separation were studied. With reproducibilities of migration times better than 1.2% (R.S.D.) and determined concentrations better than 5-25%, depending on the concentrations of nucleosides in the urine, the analytical characteristics of the method were good. Using this developed method, the concentrations of 13 normal and modified nucleosides, extracted on a phenyl boronic acid affinity chromatography column, in 25 urines from patients of 14 kinds of cancer were determined. The levels (nmol/mumol creatinine) of modified nucleosides in urines from cancer patients were increased as compared with those in normal urines.

Artificial neural network classification based on capillary electrophoresis of urinary nucleosides for the clinical diagnosis of tumors.

Nucleosides in human urine have been studied frequently as a possible biomedical marker for cancers, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. A capillary electrophoretic method that can quantitatively analyze urinary normal and modified nucleosides in less than 40 min with a good resolution and sufficient sensitivity has been developed. Twelve kinds of normal and modified nucleosides were determined in urine samples from 25 healthy persons and 25 cancer patients of 14 kinds of cancers. Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached to 100% and above 85% of the members in the predicting set were correctly classified. In addition, the neural network technique was compared with methods of the principal component analysis and the canonical discriminant analysis. The results demonstrate that the predictive ability of the artificial neural network is stronger than the others in this study.

Gas chromatographic profiling analysis of urinary organic acids from nonsmokers and smokers.

A rapid profiling and screening procedure is described for the comparative analysis of urinary organic acids among the groups of nonsmokers and smokers. The procedure involves solid-phase extraction of organic acids using Chromosorb P in normal-phase partition mode, with subsequent single-step conversion to tert.-butyldimethylsilyl derivatives, followed by direct gas chromatographic (GC) analysis on dual-capillary columns. A total of forty-two organic acids were positively identified by retention index (I) matching in urine samples (0.25 ml) from eleven nonsmokers and fifteen smokers studied. When the GC profiles were simplified to their corresponding organic acid I spectra in bar graphical form, characteristic patterns were obtained for each individual as well as for each average of nonsmoking and smoking groups. When stepwise discriminant analysis was performed on GC data after omitting hippuric acid, seven acids were selected as the variables most discriminating between smokers and nonsmokers. The star symbol plots drawn based on these discriminants were characteristic of each individual and group average, enabling to distinguish smokers from nonsmokers. And canonical plot produced by canonical discriminant analysis using the same variables as the data vectors displayed two separate clusters representing each group.

Capillary electrophoresis in clinical chemistry.

Since its introduction, capillary electrophoresis has diversified, spreading out into different specialized fields covering solutions for almost any analytical questions arising in research laboratories. In the context of clinical chemistry, results must be provided at low costs and in a clinically relevant time frame; however, the attributes which have made capillary electrophoresis such a successful tool in basic research are identical to those attracting clinical laboratories: speed (more efficient, less labor-intensive), low costs (minimal buffer consumption), small sample volume (reduced blood collection volume from patient), increased selectivity (determination of multiple solutes in one run), and versatility (detection of analytes over the wide range of molecular masses and chemical composition). Nevertheless, it should be mentioned that there are still some drawbacks at this stage to be solved in the near future, such as lack of sensitivity for many clinical applications or the constraint to measure in a sequential mode. The aim of this survey is to familiarize clinical chemists, as well as chemists, with a short introduction to capillary electrophoresis, followed by chapters reviewing prominent fields of applications and the latest developments in clinical chemistry.

Quantitation of urinary nucleosides by high-performance liquid chromatography.

It is known that some modified, especially methylated, nucleosides originating from RNA degradation are excreted in abnormal levels in the urine of patients with malignant tumours and they have been proposed as tumour markers. Their measurement could provide a non-invasive diagnostic method, be helpful in the identification of different cancers and in the monitoring of therapeutic effects. In this study, we developed and optimized an analytical procedure to isolate and quantify normal and modified ribonucleosides. The extraction of urinary nucleosides was performed by affinity chromatography on a phenylboronic acid column prior to separation. The reversed-phase high-performance liquid chromatography method allowed a complete separation of sixteen urinary ribonucleosides. The recoveries for the different nucleosides ranged from 83 to 100%, except for xanthosine (66%) and pseudouridine (74%). In normal 24 h urine, the mean levels of thirteen nucleosides (in nmol of nucleoside/mumol of creatinine) were found to be as follows: dihydrouridine (6.37), pseudouridine (25.52), cytidine (0.07), uridine (0.21), 1-methyladenosine (2.19), inosine (0.30), guanosine (0.06), xanthosine (0.59), 3-methyluridine (0.11), 1-methylinosine (1.13). 1-methylguanosine (0.74), adenosine (0.21) and 5'-deoxy-5'-methylthioadenosine (0.12). The first results concerning two kinds of tumours, i.e. breast and floor of mouth tumours, showed some abnormal levels of ribonucleosides. Further experiments are now in progress to measure the modified nucleosides in urine of patients with different forms of cancer.