Polyamine patterns in plasma of patients with systemic lupus erythematosus and fever.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with various clinical manifestations and serologic markers. In this study, we analyzed nine polyamine (PA) profiles of plasma from patients with SLE and healthy controls (HCs), and the relationship between the PA profiles and disease activity. PA alterations in plasma of 44 patients with SLE and fever were investigated using gas chromatography mass spectrometry (GC-MS) in selected ion monitoring mode using N-ethoxycarbonyl/ N-pentafluoropropionyl derivatives, and compared with those of 43 HCs. Patients with SLE and HCs showed differences in five of nine PA profiles. Among five changed PA levels, four PAs, namely N1-acetylcadaverine, spermidine, N1-acetylspermidine, and spermine, were dramatically decreased. However, the level of cadaverine was increased in patients with SLE. In the partial correlation with PA profiles and disease activity markers of SLE, several disease activity markers and nutritional markers were correlated with cadaverine, spermidine, and N 8-acetylspermidine. Thus, our results provide a comprehensive understanding of the relationship between PA metabolomics and disease activity markers in patients with SLE.

Enantiomeric composition analysis of pranoprofen in equine plasma and urine by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode.

The enantioseparation of pranoprofen after its addition in racemic form into equine plasma and urine was conducted by chiral liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. The methods for the assay of both enantiomers were linear (r≥0.9943) in the low range from 0.001 to 0.1µg/mL and high range from 0.01 to 1.0µg/mL with good precision (% RSD≤5.6) and accuracy (% RE=-5.3 to 1.9). When racemic pranoprofen was orally administered to four horses at a single dose of 3.1mg/kg, the median plasma concentrations of (R)-pranoprofen were lower than the levels of (S)-pranoprofen from start to finish. In contrast, the urinary level of (R)-pranoprofen was 2.5 fold higher than (S)-pranoprofen level for the first 6h, followed by its rapid decrease down below (S)-pranoprofen concentration. Monitoring of the R/S ratios in equine urine may be useful for the prevention of false positive in horse doping test.

Altered urinary polyamine patterns of cancer patients under acupuncture therapy.

The reduction of elevated polyamine (PA) levels in biological fluids of cancer patients were known to be correlated with remission following diverse therapeutic treatments. In this study, altered urinary PA levels from three different cancer cases were monitored at different intervals during the long-term weekday acupuncture treatments. Nine urinary PA levels from 16 normal and three cancer patients with different types were measured by gas chromatography-mass spectrometry in selected ion monitoring mode as N-ethoxycarbonyl-N-pentafluoropropionyl derivatives. Their levels measured at three follow-up stages for each patient were then normalized to the corresponding normal group means and plotted into star symbol patterns. Large alterations of PA levels were observed for each patient. Each normalized concentration displayed elevation of the PA levels in multiples (0.0-57.7) of the respective normal mean values. The normalized PA values were transformed into distorted star patterns which were characteristic of each follow-up stage and of cancer type.

Diagnostic patterns of very-long-chain fatty acids in plasma of patients with X-linked adrenoleukodystrophy.

Pattern recognition analysis on the levels of the very-long-chain fatty acids (VLCFAs) in plasma is described for the visual discrimination of X-linked adrenoleukodystrophy (X-ALD) patients from normal healthy group. Plasma VLCFA compositions of 58 normal subjects and 16 X-ALD patients were examined by gas chromatography as their methyl esters to determine the area percentages of behenic acid (C22:0), lignoceric acid (C24:0) and hexacosanoic acid (C26:0) in the total fatty acids, and the concentration (microg/ml) of C26:0. When star symbol plotting was applied to the VLCFA values of C22:0 (%), C24:0 (%), C26:0 (%), C24:0/C22:0, C26:0/C22:0 and C26:0 (microg/ml) after normalization to the corresponding median values in normal group, the resulting deformed hexagonal star pattern was characteristic of each patient. Therefore, simple visual comparison with the equilateral hexagon of normal group average as the control pattern enabled one readily to discriminate X-ALD patients from the normal group. Additionally, canonical discriminant analysis performed on the six unnormalized VLCFA values correctly classified 74 plasma specimens into two separate clusters according to normal subject or X-ALD patient in the canonical plot.

Gas chromatographic-mass spectrometric determination of plasma saturated fatty acids using pentafluorophenyldimethylsilyl derivatization.

An improved method for the detection of 11 saturated fatty acids (SFAs) including C12:0-C26:0 (even numbers only), C17:0, C19:0 and C23:0 in human plasma by gas chromatography-mass spectrometry (GC-MS) with a stable isotope internal standard as d3-stearic acid is described. This procedure was based on acidic treatment, liquid-liquid extraction, and chemical derivatization prior to instrumental analysis. Eleven pentafluorophenyldimethylsilyl-SFA derivatives were well separated without any interfering peaks in plasma samples. The characteristic ions at M-15, constituting the base peaks in the electron impact mass spectra for 11 SFAs, permitted their sensitive detection by GC-MS in the selected ion monitoring (SIM) mode. The SIM responses were linear with correlation coefficients varying from 0.993 to 0.999 in the concentration range of 0.05 to approximately 50 microg/ml for the 11 SFAs. The detection limits for SIM of the SFAs varied in the range of 0.05 to approximately 10.0 pg. When applied to the plasma samples of normal subjects and patients with X-linked adenoleukodystrophy, which is one of the hereditary peroxisomal disorders, the present method enabled us to determine the SFAs with good sensitivity and good overall precision and accuracy within the concentration ranges of 0.14 to approximately 82.35 micromol/l.

Determination of very-long-chain fatty acids in serum by gas chromatography-nitrogen-phosphorus detection following cyanomethylation.

A sensitive method for very-long-chain fatty acid analysis was developed by gas chromatography-nitrogen-phosphorus detection by using cyanomethyl derivatization. Bromoacetonitrile as alkylating reagent was used to improve nitrogen phosphorus detection detectability of compounds containing non-nitrogen. The carboxyl group of very-long-chain fatty acid was alkylated to cyanomethyl esters. Reaction conditions were 40 min at 60 degrees C under potassium carbonate base. Heptacosanoic acid was used as an internal standard and hexane was used as a solvent of extraction. The extraction yield was 82.8% or more, relative standard deviation of the precision test was 8.3% or more and the result of linearity test showed a good correlation coefficient of r2=0.999 in the range of 0.1-50 microg/ml. The quantification limits were 10 ng/ml when 0.5 ml of serum was used. The present method proved simple, rapid, inexpensive and resistant to contaminants. When it was applied to serum samples taken from patients with X-linked adrenoleukodystrophy which is a hereditary X-linked disorder characterized by progressive demyelination and adrenal insufficiency during childhood, relative increase of the concentration of hexacosanoic acid and the concentration ratios of hexacosanoic, lignoceric to behenic acid was observed in comparison with control samples.

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.

Rapid gas chromatographic profiling and screening of biologically active amines.

An efficient method is described for the simultaneous determination of 57 amines including volatile aliphatic amines, nonvolatile polyamines and catecholamines present in aqueous samples. The method is based on two-phase isobutyloxycarbonylation (isoBOC) with a pH shift. In 1.0 M phosphate buffer at pH 7.5, phenolic hydroxyl groups were allowed to react with isobutyl chloroformate in the dichloromethane phase, and subsequently pH of the aqueous phase was increased to 12.0 for the reaction of basic amino functions. The resulting N(O)-isoBOC amines were recovered by solid-phase extraction using Chromosorb P in normal phase partition mode, with subsequent tert.- butyldimethylsilylation of the remaining hydroxyl groups for gas chromatographic analysis. Using this combined procedure, linear responses were obtained in the concentration range of 0.2-12 ppm, with correlation coefficients varying from 0.945 to 0.999 for most of the amines studied except for 5-methoxytryptamine (0.864). Temperature-programmed retention index (I) sets as measured on DB-5 and DB-17 dual-capillary columns of different polarity were characteristic of each amine and thus, useful in the screening for amines by computer I matching. When applied to saliva samples, the present method allowed rapid screening for each spiked amine and unspiked polyamines such as 1,3-diaminopropane, putrescine, cadaverine and spermidine.