Capillary Electrophoresis Laser-induced Fluorescence Research Articles

Sensitive determination of rose bengal in brown sugar by a molecularly imprinted solid-phase extraction monolithic capillary column coupled with capillary electrophoresis

A method employing molecularly imprinted solid-phase extraction (MISPE) coupled with capillary electrophoresis laser-induced fluorescence detection (CE-LIF) was developed for the determination of rose bengal in brown sugar. The rose bengal imprinted monolithic column was prepared by in situ polymerization, utilizing rose bengal as a template, methacrylic acid as a functional monomer and ethylene dimethacrylate as a cross-linker. The capacity and specificity of this column were evaluated by CE-LIF, and the morphology was characterized by scanning electron microscopy. The recoveries ranged from 89.7% to 90.3% (relative standard deviation 3.6–4.5%, n = 5). The capacities of the molecularly imprinted polymer and non-imprinted polymer columns were 1.314 μg mg−1 and 0.531 μg mg−1, respectively. The detection limit (S/N = 3) was 3 ng mL−1. The enrichment factor was higher than 63-fold and the MISPE column managed to remove rose bengal from the matrix of brown sugar effectively. This method can be applied to sensitively and effectively determine rose bengal in brown sugar.

Sialic acid methylation refines capillary electrophoresis laser-induced fluorescence analyses of immunoglobulin G N-glycans of ovarian cancer patients.

Alterations in IgG N-glycosylation coincide with the development of a number of diseases including cancer and could potentially be used as diagnostic markers. CE-LIF of 8-aminopyrene-1,3,6-trisulfonic acid labeled N-glycans is a well-established rapid method to characterize IgG N-glycans that needs only low amounts of starting material. However,sialylated N-glycans have short migration times due to their negative charge. As a result,some of them are not well resolved and co-migrate with neutral glycans. In this work, we neutralized the negative charge of sialic acids by methylation and optimized the protocol using the commercially available disialylated biantennary oligosaccharide (A2G2S2). IgGN-glycans isolated from healthy human serum were then analyzed using this method. We could demonstrate that co-migration of A2, FA2G2S1, and FA2B[3]G1S1 was prevented,which allowed an accurate quantification of these N-glycans. Finally, we investigated the IgG N-glycan profiles of patients suffering from ovarian cancer using the conventional and methylation methods.With both methods, we observed an increase of agalactosylated structures that was accompanied by a decrease in digalactosylated structures. Finally, using the methylation protocol, we could further demonstrate an increase of A2, which was technically impossible with the conventional method.

Simultaneous Detection of Six Urinary Pteridines and Creatinine by High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Clinical Breast Cancer Detection

Recent preliminary studies have implicated urinary pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary pteridines: 6-biopterin, 6-hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the pteridines is between 0.025 and 0.5 μg/L, and for creatinine, it is 0.15 μg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary pteridines as breast cancer biomarkers.

Determination of Ephedrine and Pseudoephedrine by Field-Amplified Sample Injection Capillary Electrophoresis

A simple and rapid capillary electrophoresis method was developed for the separation and determination of ephedrine (E) and pseudoephedrine (PE) in a buffer solution containing 80 mM of NaH2PO4 (pH 3.0), 15 mM of β-cyclodextrin and 0.3% of hydroxypropyl methylcellulose. The field-amplified sample injection (FASI) technique was applied to the online concentration of the alkaloids. With FASI in the presence of a low conductivity solvent plug (water), an approximately 1,000-fold improvement in sensitivity was achieved without any loss of separation efficiency when compared to conventional sample injection. Under these optimized conditions, a baseline separation of the two analytes was achieved within 16 min and the detection limits for E and PE were 0.7 and 0.6 µg/L, respectively. Without expensive instruments or labeling of the compounds, the limits of detection for E and PE obtained by the proposed method are comparable with (or even lower than) those obtained by capillary electrophoresis laser-induced fluorescence, liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. The method was validated in terms of precision, linearity and accuracy, and successfully applied for the determination of the two alkaloids in Ephedra herbs.

Epigenotoxicity of environmental pollutants evaluated by a combination of DNA methylation inhibition and capillary electrophoresis–laser-induced fluorescence immunoassay

A variety of environmental pollutants may cause abnormal DNA methylation, which further disturb gene expression. In this work, we developed a rapid and sensitive method for the characterization and identification of the epigenotoxicity of environmental pollutants in terms of DNA methylation. The method combines in vitro inhibition reactions of a model DNA methyltransferase (DNMT) with rapid and sensitive capillary electrophoresis-laser-induced fluorescence (CE-LIF) immunoassays. This method was first assessed using two known DNMT inhibitors, (-)-epigallocatechin-3-gallate and RG108, and then applied to epigenotoxic evaluation of four aldehydes and six benzo-1,4-quinones. It was found that all these electrophilic chemicals could inhibit DNMT activity, probably due to their interactions with the active sites of DNMT. Interestingly, benzo-1,4-quinones displayed more inhibitory effects on DNMT activity than aldehydes. Among the tested six benzo-1,4-quinones, halogenated benzo-1,4-quinone showed higher inhibitory activity than non-halogenated p-benzo-1,4-quinone. Owing to its speed and sensitivity, our method will be potentially applicable for fast epigenotoxic screening of environmental pollutants and mechanistic study of environmental epigenetics.

Rapid and sensitive detection of Cronobacter spp. (previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis–laser-induced fluorescence detector

Cronobacter spp. (Enterobacter sakazakii) is an emerging opportunistic pathogen with a 40-80% mortality rate in infants and immunocompromised crowd resulting from the consumption of contaminated food. A novel method for detecting Cronobacter spp. in food samples by duplex polymerase chain reaction (PCR) in combination with capillary electrophoresis-laser induced fluorescence (CE-LIF) detector has been developed. The specific gene sequences of 16S-23S rDNA internal transcribed spacer (ITS) and the outer membrane protein A (OmpA) of Cronobacter spp. were amplified by duplex PCR. The PCR products were separated and determined sensitively by CE-LIF within 12min. The relative standard deviations of migration time for the detected DNA fragments were 2.01-2.91%. The detection limit was as low as 1.6×10(1)cfu/mL of Cronobacter spp. Besides, the specificity of the method was verified by 24 non-Cronobacter bacterial strains. A total of 120 commercial infant food formula were tested for the presence of Cronobacter spp. by using the proposed method. This current study demonstrates that the combination of CE-LIF method with duplex PCR is rapid, sensitive and environmental friendly, and has the potential to be adapted for the routine detection of Cronobacter spp. in food samples. To the best of our knowledge, this is the first use of CE-LIF for the detection of Cronobacter spp.

Response of Lumbriculus variegatus transcriptome and metabolites to model chemical contaminants

Assessment of the underlying molecular events leading to xenobiotic toxicity is challenging especially when techniques are applied in isolation. We examined transcriptional and metabolic changes in Lumbriculus variegatus exposed to benzo(a)pyrene (B(a)P), cadmium (Cd) or pentachlorophenol (PCP) by DNA microarrays (7422 ESTs) and gas chromatography–mass spectrometry (GC–MS), respectively. In addition, the DNA damage response of worms exposed to B(a)P was assessed by a capillary electrophoresis laser induced fluorescence (CE-LIF) immunoassay. We found elevated expression of oxidative stress responsive genes, which correlated positively with the changes in antioxidant vitamin precursors including alpha-tocopherol and cholecalciferol. Other genes with strong differential expressions were mostly involved in actin related processes and proteolysis, despite an apparent delayed Cd response. Phosphates, sugars and fatty acids were effectively reduced and suggested that chemical treatments may have interfered with energy metabolism. The increased amount of B(a)P diol-epoxide (BPDE)–DNA adducts in exposed worms appeared to correlate with the variability in uridine, inosine and xanthine, which are key components of nucleoside metabolism. This suggests that DNA damage was imminent or peaked within 6h. The results conformed to transcriptional changes in B(a)P exposed worms and compliment other approaches to elucidate underlying molecular changes.

Endo-β-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: Application to biomarker discovery

Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-β-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55min instead of 21h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis–laser-induced fluorescence (CE–LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker.

Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin and 5 mM HPA-β-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue.

Effects of metoclopramide on mRNA levels of steroid 5α-reductase isozymes in prostate of adult rats

The rising incidence of prostate cancer and benign prostatic hypertrophy in the Western world is a cause of increasing public health concern. The most active androgen in the prostate is 5α-dihydrotestosterone obtained from testosterone (T) by the enzyme 5α-reductase (5α-R), expressed in the prostate as two isozymes, 5α-R1 and 5α-R2. These isozymes are involved in the growth and development of normal prostate and in the onset and progression of prostate disease. Besides androgens, prolactin (PRL) may also play a role, although it is not clear whether its effects on the prostate are in synergism with or independent of those of androgens. We previously demonstrated that sulpiride, an inductor of hyperprolactinemia, increased mRNA levels of 5α-R isozymes in prostate of adult rat. We hypothesized a possible interrelationship between PRL levels and 5α-R, although the effects of sulpiride per se cannot be ruled out. In the present study, one-step quantitative reverse transcription polymerase chain reaction coupled with laser-induced fluorescence capillary electrophoresis was used to quantify mRNA levels of both 5α-R isozymes in prostate of adult rat after administration of metoclopramide (MTC), another inductor of PRL secretion. With the administration regimens studied, MTC produced an increase in prostate weight and mRNA levels of 5α-R1 and 5α-R2 in adult rats. Given our finding that MTC per se or MTC-induced hyperprolactinemia modifies prostate disease-related parameters in animals with reduced plasma T levels, further investigation is warranted into the possibility that MTC use by aging males may increase their risk of prostate disease.

A CE–LIF method to monitor autophagy by directly detecting LC3 proteins in HeLa cells

A fast, simple and quantitative approach was established for monitoring autophagy in HeLa cells by directly detecting the conversion of green fluorescent protein (GFP) labelled autophagy markers, GFP-LC3-I to GFP-LC3-II, in crude cellular extract using capillary electrophoresis (CE) with laser-induced fluorescence (LIF). Compared with the traditional methods, this proposed method is simpler and more reliable. Moreover, high sensitivity, with a limit of detection (LOD) of 0.48 ppt (bovine serum albumin as standard protein), was obtained by an in-capillary derivatization method coupled with a field-enhanced sample injection (FESI) technique, this may allow the success of this technique in the detection of endogenous markers of autophagy in cells.

Capillary electrophoresis–laser-induced fluorescence (CE-LIF) assay for measurement of intracellular d-serine and serine racemase activity

An enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for the analysis of d-serine (d-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5mM hydroxyl propyl-β-cyclodextrin in borate buffer [80mM, pH 9.3]. The method was able to resolve d-Ser and l-Ser with an enantioselectivity (α) of 1.03 and a resolution (Rs) of 1.37. Linearity was established from 0.25 to 100.00μM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular d-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in d-Ser and associated EC50 values produced by l-Ser and the concentration-dependent decreases in d-Ser and associated IC50 values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR.

Universal Primer-Multiplex-Polymerase Chain Reaction (UP-M-PCR) and Capillary Electrophoresis–Laser-Induced Fluorescence Analysis for the Simultaneous Detection of Six Genetically Modified Maize Lines

To meet the labeling and traceability requirement of genetically modified (GM) maize and their products for trade and regulation, it is essential to develop a specific detection method for monitoring the presence of GM content. In this work, six GM maize lines, including GA21, Bt11, NK603, Bt176, Mir604, and Mon810, were simultaneously detected by universal primer-multiplex-polymerase chain reaction (UP-M-PCR), and the amplicons for the six event-specific genes as well as the endogenous Ivr gene were successfully separated by the method of capillary electrophoresis-laser-induced fluorescence (CE-LIF). The UP-M-PCR method overcame the disadvantages in conventional M-PCR, such as complex manipulation, lower sensitivity, amplification disparity resulting from different primers, etc., and in combination with CE-LIF, it obtained a high sensitivity of 0.1 ng for both single and mixed DNA samples. The established method can be widely used for the qualitative identification of the GM maize lines.

A Novel Pyridoxal 5′-Phosphate-dependent Amino Acid Racemase in the Aplysia californica Central Nervous System

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.

Detection Limit of Monoclonal B-Cells Using Multiplex PCR and Laser-Induced Fluorescence Capillary Electrophoresis

Background: The identification of monoclonality has been widely used for making diagnoses of lymphoproliferative lesions. Awareness of the sensitivity and detection limit of the technique used would be important for the data to be convincing. Methods: We investigated the minimum requirement of cells and sensitivity of gel electrophoresis (GE) and laser-induced fluorescence capillary electrophoresis (LFCE) for identifying IgH gene rearrangement using BIOMED-2 protocols. DNA extracted from Raji cells were diluted serially with peripheral blood mononuclear cells (PBMNCs) DNA. DNA from mixtures of diffuse large B-cell lymphoma (DLBCL) and reactive lymph nodes were also serially diluted. Results: For Raji cells, the detection limit was 62 and 16 cell-equivalents for GE and LFCE, respectively. In the condition with PBMNCs mixture, 2.5% and 1.25% of clonal cells was the minimum requirement for GE and LFCE, respectively. In 23% of DLBCL cells in tissue section, the detection limit was 120 and 12 cell-equivalents for GE and LFCE, respectively. In 3.2% of DLBCL cells, that was 1,200 and 120 cell-equivalents for GE and LFCE, respectively. Conclusions: These results show that LFCE method is more sensitive than GE and the sensitivity of clonality detection can be influenced by the amount of admixed normal lymphoid cells.

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 μm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 μmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.

Quantification of neurotransmitter amino acids by capillary electrophoresis laser-induced fluorescence detection in biological fluids

The role of neurotransmitter amino acids (NAAs) in the functioning of the nervous system has been the focus of increasingly intense research over the past several years. Among the various amino acids that have important roles as neurotransmitters, there are alanine (Ala), glutamic acid (Glu), aspartic acid (Asp), serine (Ser), taurine (Tau) and glycine (Gly). NAAs are present in plasma, cells and--at trace levels--in all biological fluids, but complex components in biological matrices make it difficult to determine them in biological samples. We describe a new capillary electrophoresis (CE) method with laser-induced fluorescence detection by which analytes are resolved in less than 12 minutes in a 18 mmol/L phosphate run buffer at pH 11.6. The use of elevated temperatures during sample derivatization leads to a drastic reduction in the reaction time, down to 20 min, compared to the 6-14 h usually described for reactions between FITC and amino acids at room temperature. In order to demonstrate its wide range of applications, the method was applied to the analysis of NAA in human plasma and in other sample types, such as red blood cells, urine, cultured cells, cerebrospinal fluid, saliva and vitreous humor, thus avoiding the typical limitations of other methods, which are normally suitable for use with only one or two matrix types.

Plasma Thiols and Taurine Levels in Central Retinal Vein Occlusion

Purpose: To determine the plasma levels of the sulfur-containing amino-acids homocysteine, cysteine, cysteinylglycine, glutamylcysteine, glutathione, and taurine in patients with central retinal vein occlusion (CRVO) and in healthy subjects and to ascertain whether there are statistically significant differences between patients and controls.Methods: Laser-induced fluorescence capillary electrophoresis was used to measure the plasma levels of homocysteine, cysteine, cysteinylglycine, glutamylcysteine, glutathione, and taurine in 29 patients with CRVO and 80 age- and gender-matched control subjects. Wilcoxon or Student’s t-test was used, when appropriate, to determine differences between groups. Multivariate logistic regression analysis was used to determine the risks for CRVO.Results: CRVO patients showed significantly higher concentrations of cysteine (p = 0.032) and significantly lower concentrations of cysteinylglycine (p = 0.009) and taurine (p = 0.0002) than controls. Conversely, there were no significant differences in plasma homocysteine, glutamylcysteine, and glutathione between CRVO patients and controls. When categorized by CRVO type (ischemic/non-ischemic), taurine was still lower in both subgroups than in controls, whereas cysteine, cysteinylglycine, as well as homocysteine, were significantly higher only in the ischemic subgroup. In non-ischemic CRVO, cysteinylglycine fell just short of statistical significance (p = 0.06). Logistic regression analysis revealed an odds ratio of 1.02 (95% confidence interval (CI): 1.01–1.04, p = 0.001) for cysteine, 0.79 (95% CI: 0.70–0.89, p = 0.0002) for cysteinylglycine, and 0.94 (95% CI: 0.90–0.97, p = 0.002) for taurine.Conclusions: Results suggest that reduced plasma levels of cysteinylglycine and taurine may contribute to the pathogenesis of both CRVO types. Furthermore, this study also demonstrated an association between ischemic CRVO and higher concentrations of homocysteine and cysteine.

Determination of the β-Glucosidase Activity in Different Soils by Pre Capillary Enzyme Assay Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Enzyme activities can provide indication for quantitative changes in soil organic matter (SOM). It is known that the activities of most enzymes increase as native SOM content reflecting larger microbial communities and stabilization of enzymes on humic materials. Beta-glucosidase (beta-Glu) activities have been frequently used as indicators of changes in quantity and quality of SOM. In this study we propose a simple and very sensitive method, which has lower limit of detection compared with classic spectrophotometric method with the aim of determinate the beta-Glu activity in soil samples using Fluorescein mono-beta-D-glucopyranoside (FMGlc) as a substrate. The fluorescein released by the enzymatic reaction was quantified by capillary electrophoresis-laser induced fluorescence (CE-LIF) method. The background electrolyte (BGE) consisted in 40 mM phosphate buffer, pH 6. The LOD and LOQ for fluorescein were 1.3 10(-7) mg mL(-1) and 6.4 10(-6) mg mL(-1), respectively. This work deals with the minimization of the mixture for the enzymatic reaction and with the optimization conditions of CE separation. To the best of our knowledge, this is the first time that an enzymatic activity was detected in soil using CE-LIF system.

Decreased Plasma Cysteinylglycine and Taurine Levels in Branch Retinal Vein Occlusion

Purpose: Our aim was to determine the plasma levels of the sulfur-containing amino acids homocysteine, cysteine, cysteinylglycine, glutamylcysteine, glutathione and taurine in patients with branch retinal vein occlusion (BRVO) and in healthy subjects and to ascertain whether there are statistically significant differences between patients and controls. Methods: Homocysteine, cysteine, cysteinylglycine, glutamylcysteine, glutathione and taurine plasma levels were measured in 40 patients with BRVO and 80 age- and gender-matched control subjects by using laser-induced fluorescence capillary electrophoresis methods. Wilcoxon’s or Student’s t test was used, when appropriate, to determine differences between the groups. Conditional logistic regression analysis was performed to determine the risk factors for BRVO. Results: BRVO patients showed significantly lower plasma concentrations of cysteinylglycine (p = 0.02) and taurine (p < 0.0001) than controls. Conversely, there were no significant differences in plasma homocysteine, cysteine, glutamylcysteine and glutathione between patients with BRVO and controls. Conditional logistic regression analysis revealed an odds ratio of 0.95 (95% confidence interval: 0.92–0.98, p = 0.001) for taurine and 0.86 (95% confidence interval: 0.78–0.96, p = 0.006) for cysteinylglycine. Conclusions: This study failed to demonstrate an association between BRVO and the plasma levels of homocysteine, cysteine, glutamylcysteine and glutathione. Cysteinylglycine and taurine were significantly lower in BRVO patients, thus suggesting that reduced plasma levels of these sulfur-containing amino acids may contribute to the pathogenesis of BRVO.