Jisook Yim

Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress

As a possible marker of oxidative stress, many studies have measured whole blood reduced glutathione (GSH) and oxidized glutathione (GSSG). However, large differences in GSH and GSSG levels reported in different studies, calls for a reliable standardized method. In this study, we validate not only analytical performance of new measurement method for GSH and GSSG, but also the clinical utility of these markers in a mouse model with chronic oxidative stress. Twenty mice were randomized into four treatment groups according to iron burden: 0mg, 5mg, 10mg, or 15 mg of iron were injected into the peritoneum per day over 4 weeks. To prevent artifactual GSH auto-oxidation, we pretreated the sample with N-ethylmaleimide (NEM) immediately after sample collection. After protein precipitation using sulfosalicylic acid, GSSG and GSH-NEM were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mean GSH/GSSG ratios of the mouse model were 163.1, 31.0, 27.9, and 12.8 for control, 5mg, 10mg, and 15 mg injection groups, respectively, showing a decrease in the GSH/GSSG ratios according to the amount of oxidative stress induced. Inter-assay coefficients of variation were 4.1% for GSH-NEM and 7.3% for GSSG. Recoveries were 98.0-105.9% for GSH-NEM and 98.0-107.3% for GSSG. No ion suppression was observed at the retention time for GSH-NEM and GSSG. This study suggests an accurate method that can be used for glutathione measurement using LC-MS/MS, and showed that GSH/GSSG ratio could provide an assessment of the degree of oxidative stress.

Influence of Vitamin C and Maltose on the Accuracy of Three Models of Glucose Meters

Dear Editor, n nGlucose meters are increasingly used in hospitals and home settings. These handheld devices provide instant readings and are therefore, beneficial for glucose-level monitoring in patients with diabetes [1]. Nonetheless, there have been concerns regarding the measurement accuracy of glucose meters. Some substances, such as vitamin C and maltose, are considered responsible for errors in glucose measurements using point-of-care testing glucose meters [1,2,3]. Vitamin C is widely used in the treatment of cancer, viral infections, severe burns, and chronic fatigue syndrome because of the antioxidant effects [4]. Maltose can be upregulated in patients undergoing peritoneal dialysis involving icodextrin as an osmotic agent [3]. High concentrations of vitamin C and maltose in blood can lead to false increase in glucose meter readings, resulting in misdiagnosis of hypoglycemia and potential fatalities [1]. Therefore, there is an urgent need for interference-resistant glucose meters [5]. We evaluated the interference of vitamin C and maltose with glucose readings obtained using the following three meters: Accuchek Inform (Roche Diagnostics, Indianapolis, IN, USA), Starstrip (Nova Biomedical, Waltham, MA, USA), and Barozen H plus (i-SENS Inc., Seoul, Korea). These three models are based on the glucose dehydrogenase-pyrroloquinoline quinone (GDH-PQQ), modified glucose oxidase (GOD), and GDH-flavin adenine dinucleotide (FAD) assays, respectively [5,6]. n nAfter prior depletion of glucose by overnight storage at room temperature, we prepared three pooled blood specimens [4] from EDTA-treated whole blood samples and added glucose (CAS 50-99-7, Duksan Pure Chemicals Co., Ansan, Korea) at final concentrations of 60, 126, or 300 mg/dL. To study the effect of various concentrations of vitamin C (0, 3, 15, or 30 mg/dL) [7] or maltose (0, 10, 40, 200, or 500 mg/dL) [8], we added 0.15 mL of the vitamin C or maltose stock solutions (control: normal saline) to 3 mL of pooled blood specimens. The stock solutions were prepared from L-ascorbic acid (CAS 50-81-7, Sigma-Aldrich, St. Louis, MO, USA) and D (+) -maltose monohydrate (CAS 6363-53-7, Junsei Chemical Co., Tokyo, Japan). Then, we compared the whole blood glucose readings of the specimens, with and without an interfering substance, using three glucose meters. We also measured the plasma glucose levels in each sample with Hitachi 7600 chemistry analyzer (Hitachi, Tokyo, Japan) for a benchmark comparison. Each sample was tested in duplicate. Assessment of interference and accuracy for each glucose meter was performed according to the criteria of the International Organization for Standardization (ISO) 15197:2013(E) [9]. n nAccuchek Inform glucose level readings at all concentrations were subject to significant positive interference (>10% deviation from control sample levels) from vitamin C presence at concentrations of 15 and 30 mg/dL, whereas no significant positive interference was observed for Barozen H plus and Starstrip models (Fig. 1). However, Starstrip failed to produce readings in two out of three measurements in specimens, which contained 30 mg/dL vitamin C (Fig. 1). In contrast to the effect of vitamin C, no significant interference at any maltose concentration was noted for all the three models tested (Fig. 1). Owing to interference from high concentrations of vitamin C (15 and 30 mg/dL), Accuchek Inform showed unacceptable accuracy levels (>15% difference from the value obtained by the chemistry analyzer; Table 1). Starstrip generally showed lower readings than those determined by the chemistry analyzer, especially at higher glucose concentrations: a negative bias >15% was detected in four out of nine measurements (Table 1). n n n nFig. 1 n nGlucose level measurements in different concentrations of interferents. Glucose was added to blood at various concentrations: low (60 mg/dL), medium (126 mg/dL), and high (300 mg/dL). The X-axis represents the level of interferent, and the Y-axis represents ... n n n n n nTable 1 n nAccuracy of whole-blood glucose concentration readings using three glucose meter models compared with the measurements obtained by using a chemistry analyzer n n n nVitamin C is a strong antioxidant that inactivates free radicals and can be oxidized at the surface of electrochemical strips producing electrons and increasing the current [1]. Icodextrin, an osmotic agent used in peritoneal dialysis, is metabolized in the systemic circulation into various glucose polymers, mainly maltose [10]. It can interfere with readings obtained using GDH-PQQ-based method, because GDH-PQQ catalyzes the oxidation of not only glucose but also other sugars [1]. Thus, vitamin C and maltose can cause positive interference resulting in misdiagnosis of true glucose levels. However, GDH currently used in Accuchek Inform was modified by the manufacturer to increase enzyme specificity for glucose and to diminish probability of incorrect high glucose readings [8]. In this study, all three glucose meters showed reliable results in presence of maltose. However, at higher vitamin C concentrations, Accuchek Inform showed a positive bias, while Starstrip occasionally malfunctioned. n nIn conclusion, high concentrations of vitamin C may affect blood glucose measurements depending on the glucose meter used. Therefore, caution is required while monitoring the glucose level, using a glucose meter, of patients receiving high dose of vitamin C. There is a need for continuous technical improvement and further studies for possible interferents of glucose meters.

Trends in Isolation and Antimicrobial Susceptibility of Enteropathogenic Bacteria in 2001-2010 at a Korean Tertiary Care Hospital

Background: Trends in the isolation of enteropatho- genic bacteria may differ depending on environmental sanitation. The aims of this study were to determine trends in the isolation and antimicrobial resistance patterns of enteropathogenic bacteria over the last 10 years. Methods: We analyzed stool cultures of Salmonella spp., Shigella spp., Plesiomonas shigelloides, Yersinia spp., Vibrio spp., and Campylobacter spp. collected at Severance Hospital between 2001 and 2010. Anti- microbial susceptibility testing was performed using the disk diffusion method for nontyphoidal Salmonella (NTS) and Campylobacter. Results: The number of specimens for stool culture significantly increased from 13,412 during 1969-1978 to 60,714 over the past 10 years, whereas the ratio of positive specimens significantly decreased from 12.9% (1,732) to 1.1% (648). The proportion of Sal- monella Typhi decreased from 97.2% in 1969-1978 to 0.8% in 2001-2010, whereas the proportion of NTS increased from 2.8% to 99.2%. The proportion of Shigella among all enteric pathogens was over 50% from 1969 to 1983, while only seven strains were isolated from 2001 to 2010, with the exception of one outbreak. Campylobacter is the second most prevalent organism. The rates of susceptibility to am- picillin and cotrimoxazole were 61% and 92%, respe- ctively, for NTS isolated from 2006 to 2010. The ci- profloxacin susceptibility rate was 79.5% for Campy- lobacter between 2006 and 2010. Conclusion: The number of isolates of Salmonella Typhi and Shigella significantly decreased, while the proportion of NTS and Campylobacter increased. Con- tinuous monitoring of ciprofloxacin-resistant Campylo- bacter isolates is necessary. (Ann Clin Microbiol 2013; 16:45-51)

Effects of two types of medical contrast media on routine chemistry results by three automated chemistry analyzers

OBJECTIVESnThe use of iodinated contrast media has grown in popularity in the past two decades, but relatively little attention has been paid to the possible interferential effects of contrast media on laboratory test results. Herein, we investigate medical contrast media interference with routine chemistry results obtained by three automated chemistry analyzers.nnnMETHODSnTen levels of pooled serum were used in the study. Two types of medical contrast media [Iopamiro (iopamidol) and Omnipaque (iohexol)] were evaluated. To evaluate the dose-dependent effects of the contrast media, iopamidol and iohexol were spiked separately into aliquots of serum for final concentrations of 1.8%, 3.6%, 5.5%, 7.3%, and 9.1%. The 28 analytes included in the routine chemistry panel were measured by using Hitachi 7600, AU5800, and Cobas c702 analyzers. We calculated the delta percentage difference (DPD) between the samples and the control, and examined dose-dependent trends.nnnRESULTSnWhen the mean DPD values were compared with the reference cut-off criteria, the only uniformly interferential effect observed for all analyzers was in total protein with iopamidol. Two additional analytes that showed trends toward interferential effects only in few analyzers and exceeded the limits of the allowable error were the serum iron and the total CO2. The other combinations of analyzer and contrast showed no consistent dose-dependent propensity for change in any analyte level.nnnCONCLUSIONSnOur study suggests that many of the analytes included in routine chemistry results, except total protein and serum iron, are not significantly affected by iopamidol and iohexol. These results suggest that it would be beneficial to apply a flexible medical evaluation process for patients requiring both laboratory tests and imaging studies, minimizing the need for strict regulations for sequential tests.

Ascorbate Oxidase Minimizes Interference by High-Concentration Ascorbic Acid in Total Cholesterol Assays

Dear Editor, n nAscorbic acid interferes with certain clinical chemistry assays based on peroxidase and redox indicators [1]. Ascorbic acid is reported to interfere with the measurement of glucose, total cholesterol, triglycerides, and uric acid [2,3,4,5]. We recently encountered several cases of interference from ascorbic acid in serum total cholesterol assays based on colorimetric enzymatic reactions. n nA 48-yr-old-female with recurrent ovarian cancer underwent palliative segmental resection surgery of the small intestine. The patient showed progressive renal impairment following surgery, and total cholesterol level was <3 mg/dL, as measured using the OSR6516 total cholesterol assay (Beckman Coulter, Brea, CA, USA) on an AU5800 analyzer (Beckman Coulter). We repeated the test with the same sample, using the Pureauto S CHO-N total cholesterol assay (Sekisui Medical Co., Tokyo, Japan) on a Hitachi7600 analyzer (Hitachi Co., Tokyo, Japan) and observed a concentration of 121 mg/dL. We did not repeat the OSR6516 assay after a certain period. The specimen was checked for lipemic, hemolytic, and icteric indices to rule out other possible interferents. The patient was administered 30 g of ascorbic acid intravenously daily for 22 days prior to total cholesterol measurements. Dipstick analysis of the patients urine was performed by using an URiSCAN SUPER analyzer (YD Diagnostics, Seoul, Korea), which showed a value of 2+ (equivalent to 25 mg/dL) for ascorbic acid. Three additional cases characterized by spuriously low total cholesterol values owing to ascorbic acid interference are summarized in Table 1. Although we could not determine serum ascorbic acid levels, we detected its presence by using a urine dipstick assay in three of the four cases. n n n nTable 1 n nSummary of the four cases with inappropriately low total cholesterol level n n n nSeveral studies recommend the addition of ascorbate oxidase to minimize ascorbic acid interference in assays to assess uric acid, triglyceride, oxalate, and cholesterol [5,6,7,8]. Most cholesterol assays in clinical laboratories use an enzymatic colorimetric method with the Trinder end-point reaction, in which hydrogen peroxide reacts with a chromogen via peroxidase to form a colored product; absorbance of this product is proportional to the concentration of total cholesterol in the sample [1]. Ascorbic acid interferes with peroxidase-based oxidation of the chromogen [6]. Since dehydroascorbic acid is already oxidized and has lost its reducing power, interference via ascorbic acid can be successfully prevented. Commercially available total cholesterol assays are summarized in Table 2. Ascorbate oxidase included in the Sekisui total cholesterol assay effectively converts serum ascorbic acid (up to concentrations of 50 mg/dL) to dehydroascorbic acid. n n n nTable 2 n nSummary of commercially available total cholesterol assays with respect to the presence of ascorbate oxidase n n n nSince ascorbic acid rapidly auto-oxidizes over time, it is difficult to detect its interference in cholesterol measurements [3,9]. Reviewing medical records can also be problematic; some ascorbic acid supplements are not physician-prescribed but are readily available over-the-counter. Furthermore, it is difficult to predict serum ascorbic acid concentrations based on ingested doses of ascorbic acid in patients with impaired renal function. n nArtiss et al. [9] suggested that we can exclude ascorbate oxidase in wet-chemistry reagent systems for cholesterol determination because solvated ascorbic acid is subject to air oxidation. Thus, the interfering substance is oxidized prior to analysis by laboratory instruments. This is partly true for most samples from patients whose renal function is intact and for those consuming typical ascorbic acid doses. It might be particularly problematic for patients with impaired renal function or for those taking high ascorbic acid doses. In the second case (Table 1), we presumed that this low cholesterol level was likely due to ascorbic acid accumulation in the blood owing to impaired renal function, even though the prescribed dose of ascorbic acid was not high. n nAs all of our cases showed extremely low total cholesterol levels, the laboratory technician easily noticed analytical errors and tried to troubleshoot them before reporting the results. These problems occurred in approximately one case bimonthly and were reported to managers and documented. However, interference from low concentrations of ascorbic acid causes a small negative bias in total cholesterol levels, which may go unnoticed. Ascorbic acid is one of the most widely consumed nutritional supplements, and some physicians prescribe megadoses for cancer therapy [10]. Consequently, managers of clinical laboratories should consider the use of cholesterol assays that include ascorbate oxidase. n nIn conclusion, when colorimetric enzymatic assays detect inappropriately low levels of total cholesterol, we should suspect ascorbic acid interference. Addition of ascorbate oxidase to cholesterol reagents can minimize the inference of ascorbic acid in total cholesterol assays.

Characterization of circulating antibodies with affinity to an epitope used in antibody-conjugated magnetic immunoassays from a case of falsely elevated cyclosporine A

BACKGROUNDnWe report a case demonstrating falsely elevated cyclosporine A (CsA) levels by using the antibody-conjugated magnetic immunoassay (ACMIA). To determine the cause of the false positive result, we examined the presence of an interferent in the patients sample.nnnMETHODSnA 26-year-old male patient displayed a high CsA concentration eight weeks after discontinuation of CsA treatment when measured by ACMIA. The possibility of assay interference was examined using serial dilution tests, heterophilic blocking tubes, erythrocyte washing, polyethylene glycol (PEG) protein precipitation, and a Protein A sepharose bead immunoabsorption assay.nnnRESULTSnThe serial dilution test showed nonlinearity, suggesting the presence of an interferent. CsA concentrations in the patients plasma pre- and post-heterophilic blocking tube treatment were similar. Erythrocyte washing and PEG protein precipitation tests indicated the existence of a protein interferent in the plasma. Moreover, the Protein A bead immunoabsorption assay identified the interferent as antibodies against a unique epitope on the monoclonal antibody-β-galactosidase (mAb-β-gal) enzyme conjugate used in CsA ACMIA.nnnCONCLUSIONSnWe identified a circulating antibody against the unique epitope of the CsA ACMIA mAb-β-gal conjugate that induces falsely high CsA concentrations. These findings suggest that clinically aberrant CsA levels require immediate re-measurement by reference methods such as mass spectrometry.

Antimicrobial susceptibility of clinical isolates of Bacteroides fragilis group organisms recovered from 2009 to 2012 in a Korean Hospital

Background Periodic monitoring of antimicrobial resistance trends of clinically important anaerobic bacteria such as Bacteroides fragilis group organisms is required. We determined the antimicrobial susceptibilities of clinical isolates of B. fragilis group organisms recovered from 2009 to 2012 in a tertiary-care hospital in Korea. Methods A total of 180 nonduplicate clinical isolates of B. fragilis group organisms were collected in a tertiary care hospital. The species were identified by conventional methods: the ATB 32A rapid identification system (bioMérieux, France) and the Vitek MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (bioMérieux). Antimicrobial susceptibility was determined by the CLSI agar dilution method. Results Imipenem and meropenem resistance rates were 0-6% for B. fragilis group isolates. The rate of resistance to piperacillin-tazobactam was 2% for B. fragilis and 0% for other Bacteroides species, but 17% for B. thetaiotaomicron isolates. High resistance rates to piperacillin (72% and 69%), cefotetan (89% and 58%), and clindamycin (83% and 69%) were observed for B. thetaiotaomicron and other Bacteroides spp. The moxifloxacin resistance rate was 27% for other Bacteroides spp. The MIC50 and MIC90 of tigecycline were 2-4 µg/mL and 8-16 µg/mL, respectively. No isolates were resistant to chloramphenicol or metronidazole. Conclusions Imipenem, meropenem, chloramphenicol, and metronidazole remain active against B. fragilis group isolates. Moxifloxacin and tigecycline resistance rates are 2-27% and 8-15% for B. fragilis group isolates, respectively.