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.

Plasma levels of 5alpha-androstane-3alpha,17beta-diol in boys during adolescent growth.

A radioimmunoassay using as antibody against testosterone-3-carboxymethyloxime-BSA is described for the measurement of androstanediol (5alpha-androstane-3alpha,17beta-diol) in human plasma. Sephadex LH 20-column chromatography was used to separate other steroids croos-reaching with the antibody from androstanediol. The sensitivity of the assay was 7 pg and the recovery of labelled androstanediol was 65.1%. The intra-assay coefficient of variation was 9.9%. The existence of 5alpha-androstane-3alpha,17beta-diol in the androstanediol fraction could be demonstrated by gas chromatography and mass spectrometry (GC-MS). Plasma concentration of this substance was measured in 53 normally developing pre-pubertal and pubertal boys and in 13 adult men. The mean concentrations significantly rose from puberty stage 1 to 2, and stage 2 to 3. Although there was no significant difference between the plasma concentrations at stages 3 and 4, an increment from stage 4 and 5 was highly significant. Levels in adult males were significantly higher than those in the stage 5 of normal puberty.

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 excretion patterns of endogenously produced alcohols in type 1 (IDDM) and type 2 (NIDDM) diabetes mellitus compared with healthy control subjects.

Urinary excretion patterns of various endogenously produced alcohols, such as ethanol, propanol, isobutanol, butanol, and isopentanol, were evaluated in 17 type 1 (IDDM) and 15 type 2 (NIDDM) diabetic patients, and in two different groups of healthy control subjects (n = 12, n = 8, respectively) matched for sex, age and weight. In addition to the urinary alcohol excretion determined by gas-chromatography and mass-spectrometry, four cardiovascular reflex tests were performed, and the motor and sensory conduction velocities of three different peripheral nerves were measured. In the type 1 diabetic patients, urinary excretions of ethanol and propanol were significantly higher than in the control subjects (P less than 0.0001, P less than 0.00001, respectively), whereas the control subjects exhibited significantly higher urinary excretion rates of the other three alcohols (P less than 0.007, P less than 0.02 and P less than 0.002, respectively) compared with the type 1 diabetic patients. In the type 2 diabetic patients, only the urinary excretion of propanol was significantly elevated (P less than 0.002) compared with the control subjects, while the urinary excretion rates of butanol and isopentanol were significantly lower (P less than 0.02, P less than 0.05, respectively) than in the controls. Urinary alcohol excretions were not related to diabetic peripheral neuropathy in both groups studied. The clinical meaning of the urinary excretion patterns of different endogenously produced alcohols in diabetes mellitus has to be further evaluated.

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.

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.

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.

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).

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.

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.

Screening and identification of familial defective apolipoprotein B-100 in clinical samples by capillary gel electrophoresis.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder. It is characterized by a decreased affinity of low density lipoprotein (LDL) for the LDL receptor, as a consequence of a substitution of adenine by guanine in exon 26 of the apolipoprotein B-100 gene, coding for the putative LDL receptor-binding domain of the mature protein. This disorder is associated with a strikingly high incidence of arteriosclerosis and tends to cause disease and premature death. In this communication we describe a rapid capillary gel electrophoretic method in combination with molecular biology techniques to facilitate the diagnosis of FDB. Mutation screening for FDB is performed by an allele-specific amplification followed by capillary gel electrophoresis (CGE). For the combined polymerase chain reaction (PCR)-CGE method, a total analysis time of only 3 h is needed, a period that is normally necessary for the run and for staining of the gel only, not including the time for PCR, gel casting, etc. In our pilot study 4 of 43 hypercholesterolemic patients were found to have the predominant apoB 3500 codon mutation. The verification is demonstrated by DNA-sequencing. This pilot study will be followed by a large cohort analysis of the south-west German population to determine the frequency of FDB in this area. The PCR-CGE method on the Dionex capillary electrophoresis system (CES I) allows rapid, fully automated detection of the mutation resulting in the unequivocal diagnosis of FDB.

Capillary electrophoresis, a rapid and sensitive method for routine analysis of apolipoprotein A-I in clinical samples.

A method for the determination of apolipoprotein A-I, the major protein compartment of HDL, in human serum is described. Rapid and easy serum sample preparation, well separated Apo A-I peaks in the human serum electropherogram and good linearity of the peak area vs. concentration plot, covering the range of the clinically relevant Apo A-I serum contents, suggest the introduction of this method routinely in clinical laboratories.

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.

In vivo clearance and elimination of nine marker substances during hemofiltration with different membranes.

The handling of low, middle and high molecular weight markers was examined in seven stable dialysis patients during hemofiltration with different membranes. Four membranes were examined in a randomized, crossover order (polysulfone, polyamide, AN69 polyacrylonitrile, Asahi polyacrylonitrile) by measuring plasma and dialysate concentrations of phosphate, creatinine, vitamin B12, beta 2-microglobulin, furanic acid, hippuric acid, retinol-binding protein, alpha-1-antitrypsin, and albumin. Sieving coefficients and plasma clearances of beta 2-microglobulin or retinol-binding protein were markedly or slightly lower during hemofiltration with the Asahi polyacrylonitrile membrane than with the other membranes (highest removal with polysulfone/AN69 polyacrylonitrile membranes). No differences of obvious clinical relevance could be seen between the four membranes. A high beta 2-microglobulin removal rate might be important to prevent dialysis-associated amyloidosis.

Volatile organic components in the Skylab 4 spacecraft atmosphere.

The volatile organic components in the spacecraft cabin atmosphere of Skylab 4 were trapped on a solid adsorbent at various times during the mission. In post-flight analyses, more than 300 compounds in concentrations from less than 1 ppb up to 8000 ppb could be detected by high-resolution gas chromatography. In the samples of the 11th, 47th, and 77th day of the mission, approximately 100 components in the molecular weight range of 58 to 592 were identified by mass spectrometry. Besides components known from other environments, such as alkanes, alkenes, and alkylated aromatic hydrocarbons, components typical for the human metabolism such as ketones and alcohols were found. Other typical components in the spacecraft atmosphere are fluorocarbons (freons) and various silicone compounds, mostly normal and cyclic methylsiloxanes.

Application of capillary electrophoresis in clinical chemistry--developments from preliminary trials to routine analysis.

Since instruments performing capillary electrophoresis (CE) became commercially available in the late 1980s, information on this relatively new analytical technique has been increasing almost exponentially. At the beginning of the last decade, fundamental discoveries in the field were made mainly in the laboratories of analytical chemists; but now, this separation science is giving increasing impact to the laboratories of clinical chemists. This paper briefly reviews the history, instrumentation, different modes and theory of CE, and the prominent fields of its applications in clinical chemistry.