Determination of iohexol and iothalamate in serum and urine by capillary electrophoresis.

Estimation of glomerular filtration rate (eGFR) is essential to assess kidney function. Iodine-containing contrast agents detection by HPLC has been proposed as a safe alternative for inulin or radioactive compounds. However, HPLC is a time-consuming and labor-intensive method. The aim of this study was to develop an assay for iohexol and iothalamate using capillary electrophoresis. Iohexol and iothalamate were directly analyzed by CE in serum and urine, using photometric detection (246 nm). Serum peak height was proportional to iohexol and iothalamate concentrations. Detection limits for iohexol and iothalamate were 10 and 5 mg/L. Limits of quantification were 13.0 and 15.0 mg/L. Within-run CVs were 4.9 and 6.5%; between-run CVs 3.1-9.9% and 3.8-13.7%. A good correlation was observed between CE and HPLC: y = 1.1703x + 5.017 (iohexol) and y = 0.7807x + 11.01 (iothalamate; (y = concentration obtained by CE [mg/L], x = concentration obtained by HPLC [mg/L]). In addition, CE allowed to determine urinary iohexol concentration. Although the detection limit for CE was higher than for HPLC, CE can still be used for eGFR determination. Advantages of this high-throughput method are the absence of sample pretreatment and a minimal sample volume requirement.

Dual-wavelength recording, a simple algorithm to eliminate interferences due to UV-absorbing substances in capillary electrophoresis.

Analytical interferences have been described due to the presence of various exogenous UV-absorbing substances in serum. Iodine-based X-ray contrast agents and various antibiotics have been reported to interfere with interpretation of serum protein pherograms, resulting in false diagnosis of paraproteinemia. In the present study, we have explored the possibility of measuring UV absorbance at two distinct wavelengths (210 and 246 nm) to distinguish between true and false paraproteins on a Helena V8 clinical electrophoresis instrument. This study demonstrates that most substances potentially interfering with serum protein electrophoresis show UV-absorption spectra that are distinct from those of serum proteins. Scanning at 246 nm allows detection of all described interfering agents. Comparing pherograms recorded at both wavelengths (210 and 246 nm) enables to distinguish paraproteins from UV-absorbing substances. In case of a true paraprotein, the peak with an electrophoretic mobility in the gamma-region decreases, whereas the X-ray contrast media and antibiotics show an increased absorption when compared to the basic setting (210 nm). The finding of iodine-containing contrast media interfering with serum protein electrophoresis is not uncommon. In a clinical series, interference induced by contrast media was reported in 54 cases (of 13 237 analyses), corresponding with a prevalence of 0.4%. In the same series, 1631 true paraproteins (12.3%) were detected. Implementation of the proposed algorithm may significantly improve the interpretation of routine electrophoresis results. However, attention should still be paid to possible interference due to presence of atypical proteins fractions (e.g., tumor markers, C3).

Microheterogeneity of serum ß-hexosaminidase in chronic alcohol abusers in a driver's license regranting program.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is one of the best indicators for chronic alcohol abuse and detection of relapse. In this study, we explore the microheterogeneity of ß-hexosaminidase (ß-HEX) in chronic alcohol abusers in the framework of a driver's license regranting program. Studies have shown that increased serum activity of ß-HEX B (isoforms P, I, and B) may be a sensitive marker for chronic alcohol abuse. Here, we describe methodology, limitations, and correlation of ß-HEX isoforms with CDT. METHODS: CDT was assayed at the central laboratory of the Ghent University Hospital by capillary zone electrophoresis, measured on the Capillarys 2™ system and was expressed as a percentage of total serum transferrin (%CDT). Serum of chronic alcohol abusers was compared to nonheavy drinkers using agarose gel isoelectric focusing (IEF). Total ß-HEX activity was assayed fluorimetrically following preparative IEF in 81 subjects. ß-HEX isoforms were investigated and compared between nonheavy drinkers and heavy drinkers. RESULTS: Agarose gel IEF shows additional cathodal bands in serum of chronic alcohol abusers. Mean total ß-HEX activity between pH 6.8 and 7.7, designated as HEX-7, showed the highest correlation with %CDT (r = 0.70, p < 0.0001, n = 68). In a selected subgroup, where CDT could not be quantified (n = 13) because of an atypical electropherogram, HEX-7 was in concordance with either estimated %CDT value or liver enzyme activities. CONCLUSIONS: In this proof-of-concept study, we introduce a novel approach to quantify ß-HEX isoforms using preparative IEF and fluorimetry. A highly significant correlation of HEX-7 and %CDT has been found. Because of exclusion of the P isoform, HEX-7 could be a useful supplementary marker for detecting chronic alcohol abuse.

Human complement factor 3 polymorphism determination by capillary electrophoresis of serum.

Variability of complement factor 3 (C3) mobility in serum protein electrophoresis was investigated. We found that the migration time of C3 can be reproducibly determined (beween-run CV=0.76%) using clinical capillary electrophoresis (CE) equipment (the Capillarys™ 2 system, Sebia). Moreover, we found a significant difference (p<0.001) in migration times of the major C3 phenotypes FF (fast-fast), FS (fast-slow) and SS (slow-slow). Glycosylation did not significantly affect test results. This is the first report on the migration time of C3 phenotypes on a clinical CE instrument. The presented method allows faster data than agarose-electrophoresis or genotyping. Moreover, reference ranges for serum C3 concentration depend on C3 phenotype, which allows a better tailored clinical interpretation of C3 concentrations.

Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Proteomics of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1, using a matrix-assisted laser desorption/ionization-tandem-time of flight mass spectrometer.

Shewanella oneidensis MR-1 is a gram-negative facultative aerobic bacterium living at oxic-anoxic interfaces in nature. The plasticity of terminal electron-acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear. In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide. By analysis of two-dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins. The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano-high-performance liquid chromatography coupled to an electrospray ionization-quadrupole-time of flight instrument, a recently introduced matrix-assisted laser desorption/ionization (MALDI) tandem-time of flight mass spectrometer. The instrument allows the acquisition of high quality spectra, in both the mass spectrometry and tandem mass spectrometry mode, and is therefore able to identify protein spots unambiguously. Advantageous to electrospray ionization is a minimised sample handling, inherent to MALDI ionization, and the presence of high energy fragmentation ions, generating sequence information that also can differentiate isobaric amino acids. With this strategy, we could point out a regulatory protein that is up-regulated under iron(III) respiration. This protein, the aerobic respiration control protein (ArcA), has been reported as being a regulator during anaerobiosis in other species. To our knowledge, this is the first report of the possible involvement of ArcA from S. oneidensis MR-1 in the reduction of ferric oxide.