Antioxidants reduce oxidative stress in claudicants.

BACKGROUND: Low-grade ischemia-reperfusion in claudicants leads to damage of local tissues and remote organs. Since this damage is partly caused by oxygen-derived free radicals (ODFR), scavenging these ODFR could reduce the local and remote injury. METHODS: Using a new method by which a free radical reaction product (ortho-APOH) of the exogenous marker antipyrine is measured to quantify the oxidative stress, 16 stable claudicants performed a standard walking test before and after administration of vitamin E (200 mg) and vitamin C (500 mg) daily for 4 weeks. FINDINGS: Ortho-APOH was significantly increased during the reperfusion period (P = 0.026) before administration of the vitamins. After 4 weeks of vitamin supplementation no rise was found in the reperfusion period. Malondialdehyde showed no changes in either group. INTERPRETATION: These findings indicate that administering extra antioxidants to claudicants reduces oxidative stress in these patients. This may also have an effect on the remote ischemia-reperfusion damage and reduce cardiovascular morbidity in this group.

Training software for high-performance liquid chromatography.

A computer simulation program of reversed-phase high-performance liquid chromatography was developed for training purposes. Experimental retention values of 75 organic compounds on a reversed-phase column at four different percentages of organic modifiers were reduced to a two-parameter retention model with the modifier content as variable. Modifiers used were acetonitrile, methanol and tetrahydrofuran. Isocratic and programmed solvent composition were included together with the usual experimental parameters available in modern HPLC equipment, such as UV diode array and refractive index detection. Instrument specifications were made variable within wide ranges. Detailed dispersion data were made available as tabulated output.

Determination of methacrylic acid in the drain of a biotrickling filter using isotachophoresis and capillary zone electrophoresis.

The performance of a biotrickling filter for treatment of concentrated waste gases was investigated. The macrokinetics of methylmethacrylate degradation in the biotrickling filter is studied by measuring the degradation product methacrylic acid in the drain of the filter. The drain was analysed using isotachophoresis (ITP) and capillary zone electrophoresis (CZE). The CZE analyses were carried out in an I.D. 75 microm capillary at 20 kV (negative inlet polarity) using a 0.01 M Tris-acetate buffer of pH 4.45. The electroosmotic flow (EOF) was suppressed by addition of CTA and PVA to the buffer. Detection was at 214 nm. After filtration through a 0.45-microm filter, samples were directly injected. The calibration graph was linear between 10 and 800 mg/l methacrylic acid, with an analysis time under 2 min.

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography.

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program HPCESIM in order to achieve an optimal pH. Calibration curves were recorded in the range 10(-6)-10(-4) M and the detection limit was determined. For both CZE and MECC it was 2x10(-7) M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.

Chiral interactions in capillary zone electrophoresis: computer simulation and comparison with experiment.

Chiral interaction in capillary electrophoresis can be modeled using pK values, mobilities of analytes, and their formation constants with the chiral selector. An existing steady-state simulation program for CE (HPCESIM) was recently extended with a chiral submenu involving the chiral parameters listed above. These were experimentally determined in both our laboratories for mandelic acid and terbutaline using hydroxypropylated beta-cyclodextrin as chiral selector. A comparison was made between both sets of parameters and between experimental electropherograms and those obtained from simulation. Error analysis of the results indicate the sensitivity of the obtained results.

Quality control of histamine and methacholine in diagnostic solutions with capillary electrophoresis.

Solutions of histamine and methacholine bromide in different matrices for diagnostic purposes were analyzed for stability and quality control using capillary electrophoresis. Histamine (2,[4-imidazolyl]ethylamine) [CAS No. 51-45-6] was determined using a 0.1 M Tris-borate buffer of pH 8.3 with 5.10(-5) M cetyltrimethylammonium bromide (CTAB) and 0.005% poly(vinyl alcohol) (PVA) and detected at 214 nm using clenbuterol [4-amino-alpha-(tert.-butylaminomethyl)-3,5-dichlorobenzyl alcohol] [37148-27-9] as an internal standard. Metacholine bromide (acetyl-beta-methacholine bromide) [333-31-3] was determined with a 0.01 M creatinine-chloride buffer of pH 4.85 and detected with indirect UV at 230 nm using potassium as an internal standard. Histamine solutions were stable for a prolonged period of time, whereas under enforced degradation conditions methacholine was hydrolyzed, yielding acetic acid and (tentatively) beta-methylcholine as reaction products.

Training software for electrophoresis.

Computer programs, simulating electrophoretic separations, were evaluated and discussed with respect to their suitability for training purposes. Quite a number of them, mainly those dealing with steady-state phenomena, are sufficiently fast and user-friendly for the purpose of visualization of electrophoretic principles. Transient-state or dynamic models, however, are more suitable for the advanced user, mainly because of their inherent complexity and long calculation times.

Concept of response factor in capillary isotachophoresis. Determination of drugs in solution for intravenous injection.

Twenty-six drugs in solutions for intravenous injection were determined by capillary isotachophoresis with only one calibration point for each component. The maximum deviation of the labelled concentration was 2%. A new calibration constant is introduced, viz., the response factor (RF, dimensionless), which is independent of the diameter of the capillary, the construction of the universal detector and the driving current used during detection. It is shown that the RF can be used on different equipment, using different currents during detection. It appears that the RF is usable for routine analysis when a deviation of 5% is acceptable. Daily one-point recalibration, however, improves this value to 1%.

Composition of the nucleotides pool in a morphogenetic compartment in eggs of Nassarius reticulatus (Mollusca) analysed by capillary isotachophoresis.

The spectrum of low molecular weight compounds, in particular of ribonucleotides, within first cleavage stage embryos of the polar lobe-forming mollusc Nassarius reticulatus and the distribution of the compounds within the embryo at the trefoil stage of first cleavage are analysed by means of capillary isotachophoresis after 0.5 M PCA extraction. The compounds which are found in the whole trefoil embryo (T), the lobeless part (LL), and the polar lobe (PL) respectively, and the mean quantities (nmol. microliter-1; n = 6) are: UTP (11.5, 4.8, 5.6), ITP (8.5, 3.6, 5.0), GTP (10.3, 3.0, 9.0), ATP (29.8, 13.4, 18.8), UDP (11.8, 3.4, 8.7), CTP (8.0, 3.1, 4.5), GDP (5.3, 2.6, 3.4), ADP (16.5, 6.1, 11.6), CDP (4.0, 1.4, 2.6), GMP (4.7, 2.7, 4.3), glucose-6-phosphate (G6P) (53.5, 38.8, 13.0). These compounds appear to be localized in the non-yolk cytoplasmic pool. As the volume ratio of PL/LL for total volume and for non-yolk cytoplasmic volume is about 0.74 and 0.60 respectively, the concentration of all nucleotides in PL as compared to LL is significantly higher (HO, p less than 0.001), both relative to the total volume and to the non-yolk cytoplasmic volume. The G6P concentration is considerably higher in the lobeless part. The morphogenetic role of the vegetal pole compartment of the egg apparently is correlated with a relatively high level of its nucleotide contents.

Isotachophoresis as a candidate reference method in analytical chemistry. Determination of sodium in serum.

Isotachophoresis seems a likely candidate for a reference method, as it is more accurate and precise than common routine methods. The response is highly linear and depends on a well defined transport number of the leading ion, stability of the driving current, mobility of the separand, which is well controlled by the leading electrolyte and the use of a high-resolution detector. The suitability of isotachophoresis as a reference method was investigated for the determination of sodium in human serum. The operational conditions were 0.01 M K+/citrate (leading electrolyte) at pH 5.5 and 0.01 M creatinine X HCl (terminating electrolyte). Both n-butylamine and ammediol could be used as internal standards. The calibration graph constructed from standard solutions, diluted by weight with the internal standard, yielded a correlation coefficient of 0.99994 (n = 50) in the working range. The method seems especially useful for the determination of any ionic solute in, e.g., clinical samples (lithium, calcium, creatinine or drugs).

Isotachophoresis of allergenic extracts.

One aspect of the isotachophoretic determination of protein patterns in biological samples of interest is the characterization of allergens. This group of (glyco) proteins, causing allergic reactions, is used both for diagnosis and in the treatment of allergy. The aim of this investigation was to obtain a maximum amount of information, within one run, on the (glyco)protein composition of a number of allergenic extracts (e.g., from pollen or house dust mites). Commercially available extracts were dialysed prior to analysis to remove disturbing buffer constituents. A high-pH system was chosen in order to obtain a maximum amount of information from the samples (1-2 microliter). The leading electrolyte was 0.01 M C1-, buffered with Tris (pH 8.2), containing 0.2% w/v hydroxyethylcellulose, and the terminating electrolyte was beta-alanine, buffered to pH 10 with Ba(OH)2. The total analysis time was 15-20 min using a PTFE capillary (0.2 mm I.D.). The pre-separation current was 30 microA and the current during detection was 15 microA. UV absorption was measured at 280 nm. For optimal discrimination of the compounds of interest, an ampholyte mixture was used for spacing. The analytical procedure yielded highly reproducible UV patterns. Significant differences between various allergenic extracts were observed. It was concluded that isotachophoresis is a powerful method for the physico-chemical characterization of individual allergenic extracts, e.g., with respect to manufacturing and quality control.

Determination of quinine in beverages, pharmaceutical preparations and urine by isotachophoresis.

The suitability of isotachophoresis for the determination of quinine in different samples was investigated. The operational conditions were 0.01 M potassium-morpholinoethanesulphonic acid (MES) (pH 6.0) with 0.05% Mowiol as the leading electrolyte and ca. 0.005 M creatinine-MES as the terminating electrolyte. The analyses were carried out at 25 microA in a 0.2 mm I.D. PTFE capillary with UV and conductivity detection. Quinine-containing beverages were degassed by sonification and directly injected. The limit of detection was 5 mg/l with a 4 microliter injection volume. The allowed concentrations could be determined with sufficient accuracy. Analgesic preparations were dissolved in a solution of 5 X 10(-3) M MES with sonification. The quinine levels found agreed well with the declared values. The other constituents of the pharmaceuticals did not interfere with the analysis. Urine samples from volunteers were analysed after consumption of tonic. The samples were extracted with dichloromethane-isopropanol (95:5), vortexed, centrifuged, evaporated to dryness, the residue dissolved in 5 X 10(-3) M MES and analysed. At a concentration factor of 33, the limit of detection was ca. 60 micrograms in 48-h urine: 2-15% of the quinine consumed was excreted as the parent compound in the first 48 h after consumption. The combination of the extraction procedure and the operational system makes the method suitable for the determination of a number of other alkaloids in physiological samples.

Determination of heavy metals by isotachophoresis.

The suitability of isotachophoresis for the analysis of metals in, e.g., environmental samples was studied. In a cationic operational system the heavy metals Fe, Cu, Ni, Cd, Co, Zn, Pb and Mn were simultaneously determined. The separation was achieved through complex formation with one of the counter ions, hydroxyisobutyric acid. The other counter ion was acetic acid, the leading ion was 0.02 M potassium or sodium (pH 4.1) and the terminator was H+. The analysis time was 15 min at 60 microA in a 0.2 mm I.D. capillary. Aqueous samples containing ppm and ppb amounts were enriched on a cation exchanger with an extremely low affinity for sodium (Chelex 100). Good recovery, linearity, precision and accuracy were obtained even down to the ppb range. Although the sensitivity of the method is not greater than that of some of the more established methods for the individual metals, a great advantage of isotachophoresis is the simultaneous determination of the metals, with equal response factors. An example is given of the determination of metals, including aluminium, in serum.

Determination of theophylline binding to human serum proteins by isotachophoresis.

Free theophylline was isolated from human serum by ultrafiltration and analysed in a leading electrolyte of 7.5 mM morpholinoethanesulphonic acid with ammediol as a counter ion at pH 8.90 and alpha-alanine as a terminator. The UV (280 nm) absorbance of the theophylline spike between serine and bicine as spacers was integrated. Binding percentages to human pool serum, human albumin and alpha 1-acid glycoprotein (orosomucoid) were determined at physiological concentrations, and found to be 55, 44 and 12%, respectively. The calibration lines were straight from 0 to 30 mg/l, with a standard deviation of 0.2 mg/l. The detection limit was 1 mg/l. The time of analysis was 12 min at 40 microA in a 0.2 mm I.D. capillary.

Determination of conjugated bile acids in human bile by isotachophoresis in a non-aqueous solvent using a.c. conductivity and UV detection.

A method for the determination of conjugated bile acids in human bile using isotachophoresis in 95% methanol is described. The leading ion is 0.01 M chloride, the counter ion is hydroxylamine at its pK value and the terminating ion is N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES). The sample preparation consists of C18-silica cartridge adsorption. Microlitre amounts of the methanol eluate are injected and analysed within 20 min in a 0.2 mm I.D. PTFE capillary. The sensitivity of the method is better than 50 ng of each of the conjugated bile acids using a.c. conductivity detection.