Pediatric Reference Value Profiling of Essential Trace and Toxic Elements in Healthy Children and Adolescents Using High-Resolution and Triple Quadrupole Inductively Coupled Plasma Mass Spectrometry.

BACKGROUND: Assessment of trace and toxic element status is important for the diagnosis and monitoring of several pediatric conditions. Elemental deficiency and toxicity have serious implications, particularly in pediatrics wherein risk is higher. Pediatric reference intervals (RIs) for trace elements and normal exposure limits for toxic elements are lacking on modern analytical systems. Herein, reference values were established for 13 plasma and 22 whole blood trace elements in the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort of healthy children and adolescents. METHODS: Approximately 320 healthy children and adolescents were recruited with informed consent. Trace elements were measured in whole blood and plasma samples using 2 technologies: (a) triple quadrupole inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) (n = 172) and (b) high-resolution sector field ICPMS (HR-SF-ICPMS) (n =161). RIs and normal exposure limits were then established according to Clinical and Laboratory Standards Institute guidelines. RESULTS: Of all elements assessed, none required sex partitioning and 8 required age partitioning (e.g., copper, manganese, and cadmium). Reference value distributions determined via ICP-MS/MS and HR-SF-ICPMS demonstrated excellent concordance, with few exceptions (e.g., molybdenum, cobalt, and nickel). CONCLUSIONS: These data represent the first study wherein pediatric RIs and normal exposure limits were derived simultaneously on 2 different clinically validated MS platforms which provide urgently needed data to inform clinical decision-making for trace elements in pediatrics. Study findings suggest some trace elements require age-specific consideration for appropriate interpretation. Highly concordant observations across the 2 analytical methods also demonstrate the comparability and reliability of results obtained on both platforms.

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Excess pulmonary iron has been implicated in the pathogenesis of lung disease, including asthma and COPD. An association between higher iron content in sputum macrophages and infective exacerbations of COPD has previously been demonstrated. OBJECTIVES: To assess the mechanisms of pulmonary macrophage iron sequestration, test the effect of macrophage iron-loading on cellular immune function, and prospectively determine if sputum hemosiderin index can predict infectious exacerbations of COPD. METHODS: Intra- and extracellular iron was measured in cell-line-derived and in freshly isolated sputum macrophages under various experimental conditions including treatment with exogenous IL-6 and hepcidin. Bacterial uptake and killing were compared in the presence or absence of iron-loading. A prospective cohort of COPD patients with defined sputum hemosiderin indices were monitored to determine the annual rate of severe infectious exacerbations. RESULTS: Gene expression studies suggest that airway macrophages have the requisite apparatus of the hepcidin-ferroportin axis. IL-6 and hepcidin play roles in pulmonary iron sequestration, though IL-6 appears to exert its effect via a hepcidin-independent mechanism. Iron-loaded macrophages had reduced uptake of COPD-relevant organisms and were associated with higher growth rates. Infectious exacerbations were predicted by sputum hemosiderin index (ß = 0.035, p = 0.035). CONCLUSIONS: We demonstrate in-vitro and population-level evidence that excess iron in pulmonary macrophages may contribute to recurrent airway infection in COPD. Specifically, IL-6-dependent iron sequestration by sputum macrophages may result in immune cell dysfunction and ultimately lead to increased frequency of infective exacerbation.

Association of Urine Platinum With Acute Kidney Injury in Children Treated With Cisplatin for Cancer.

Cisplatin is a chemotherapeutic agent highly excreted in urine and known to cause acute kidney injury (AKI). As AKI diagnosis by serum creatinine (SCr) is usually delayed, endeavors for finding early AKI biomarkers continue. This study aims to determine if urine platinum (UP) concentration 24 hours after cisplatin infusion is associated with AKI, and to evaluate the association between urine platinum and tubular damage biomarkers: neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1). Children treated with cisplatin in 12 Canadian centers (April 2013 to December 2017) were included. Urine from the morning after the first cisplatin infusion of the first or second cisplatin cycle was measured for urine platinum, NGAL, and KIM-1. SCr and serum electrolytes were used to detect AKI by either SCr elevation or urinary electrolyte wasting (potassium, magnesium, phosphate). The associations of urine platinum with AKI, NGAL, and KIM-1 were assessed. A total of 115 participants (54% boys, median age, 8.5 years; interquartile range, 4.0-13.4) were included, of which 29 (25%) and 105 (91%) developed AKI defined by SCr and electrolyte criteria, respectively. Higher urine platinum was associated with higher cisplatin dose (Spearman rho, 0.21) and with younger age (Spearman rho, -0.33). Urine platinum was not associated with postinfusion AKIor KIM-1, but was weakly associated with NGAL, particularly in participants without SCr AKI (Pearson's r, 0.22). Urine platinum may be a marker of mild tubular injury but is not likely to be a useful biomarker of clinically evident AKI.

Influence of ethnicity on biochemical markers of health and disease in the CALIPER cohort of healthy children and adolescents.

Background Accurate pediatric reference intervals (RIs) for laboratory tests determined in a healthy pediatric population are essential for correct laboratory test interpretation and clinical decision-making. In pediatrics, RIs require partitioning by age and/or sex; however, the need for partitioning based on ethnicity is unclear. Here, we assessed the influence of ethnicity on biomarker concentrations in the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort of healthy children and adolescents and compared the results with the National Health and Nutrition Examination Survey (NHANES). Methods A total of 52 biomarkers were measured in a multiethnic population of 846-1179 healthy children (aged 5 to <19 years) upon informed consent. Biomarker concentrations were retrospectively compared between four major ethnic groups (i.e. Black, Caucasian, East Asian, and South Asian, determined by parental ethnicity). Retrospective results were verified prospectively using an additional 500 healthy pediatric samples with equal sample size across ethnicities. Ethnic-specific differences were assessed based on statistical significance and biological and analytical variations. Appropriate age-, sex-, and ethnic-specific RIs were calculated. Results Ethnic-specific differences were not observed for 34 biomarkers examined in the retrospective analysis, while 18 demonstrated statistically significant ethnic differences. Among these, seven analytes demonstrated ethnic-specific differences in the prospective analysis: vitamin D, amylase, ferritin, follicle-stimulating hormone (FSH), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM). Analysis of select NHANES data confirmed CALIPER findings. Conclusions This is the first comprehensive Canadian pediatric study examining ethnic-specific differences in common biomarkers. While the majority of biomarkers did not require ethnic partitioning, ethnic-specific RIs were established for seven biomarkers showing marked differences. Further studies in other populations are needed to confirm our findings.

The impact of environmental cadmium exposure on type 2 diabetes risk: a protocol for an overview of systematic reviews.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a worldwide epidemic, and while its etiology is polygenic, the role of environmental contaminant exposure in T2DM pathogenesis is of increasing importance. However, the evidence presented in systematic reviews on the relationship between cadmium exposure and T2DM development is inconsistent. This overview aims to assess existing evidence from systematic reviews linking cadmium exposure to T2DM and select metabolic disorders in humans. METHODS: Searches will be conducted in Medline, Embase, Web of Science, GEOBASE, BIOSIS Previews, and Cochrane Database of Systematic Reviews. Two reviewers (J.H and S.T.) will independently complete screening, data abstraction, risk of bias evaluation, and quality assessment. The primary outcome will be the association between cadmium exposure and T2DM prevalence. Secondary outcomes will include prediabetes, obesity, dyslipidemia, hypertension, and non-alcoholic fatty liver disease. We will perform a meta-analysis if two or more studies assess similar populations, utilize analogous methods, have related study designs, and evaluate similar outcomes. DISCUSSION: This overview will assess current evidence from systematic reviews for the association between cadmium exposure and risk of T2DM and other metabolic morbidities. This overview may be helpful for policy-makers and healthcare teams aiming to mitigate T2DM risk in populations at risk of cadmium exposure. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42019125956.

Paediatric reference intervals for 17 Roche cobas 8000 e602 immunoassays in the CALIPER cohort of healthy children and adolescents.

Background The diagnostic utility of laboratory tests in paediatric medicine relies heavily on the availability of appropriate reference intervals (RIs). The Canadian Laboratory Initiative on Paediatric Reference Intervals (CALIPER) has established a comprehensive database of covariate-stratified RIs for many paediatric laboratory tests using a large, healthy reference population. Several automated analysers in widespread use in clinical laboratories have already been studied. Here, we extend the testing to Roche immunoassays and report, for the first time, comprehensive paediatric RIs for 17 endocrine and special chemistry markers. Methods A total of 741 healthy children and adolescents (1 day to <19 years) were recruited and serum samples were analysed for 17 immunoassays on the Roche cobas 8000 e602 Immunoassay Analyzer. Age and sex-specific RIs were established and corresponding 90% confidence intervals (CIs) were calculated in accordance with Clinical and Laboratory Standards Institute guidelines. Results Reference values for all analytes measured required age partitioning, particularly during early life and throughout adolescence. Of the 17 analytes measured, eight required sex partitioning, including ferritin, thyroid stimulating hormone (TSH), total triiodothyronine (TT3) and all fertility/sex hormones, except prolactin. Conclusions This is the first study to determine accurate paediatric RIs for Roche immunoassays. RIs were generally similar to those previously published by CALIPER on other analytical platforms, highlighting the reproducibility of age- and sex-specific trends in reference values observed across the paediatric age range. The RIs established in this study will improve the accuracy of test result interpretation and clinical decision-making in clinical laboratories utilising Roche immunoassays.

Colorimetric Detection of Uranyl Using a Litmus Test.

Ingestion of water containing toxic contaminants above levels deemed safe for human consumption can occur unknowingly since numerous common contaminants in drinking water are colorless and odorless. Uranyl is particularly problematic as it has been found at dangerous levels in sources of drinking water. Detection of this heavy metal-ion species in drinking water currently requires sending a sample to a laboratory where trained personnel use equipment to perform the analysis and turn-around times can be long. A pH-responsive colorimetric biosensor was developed to enable detection of uranyl in water which coupled the uranyl-specific 39E DNAzyme as a recognition element, and an enzyme capable of producing a pH change as the reporter element. The rapid colorimetric assay presented herein can detect uranyl in lake and well water at concentrations relevant for environmental monitoring, as demonstrated by the detection of uranyl at levels below the limits set for drinking water by major regulatory agencies including the World Health Organization (30 µg/L). This simple and inexpensive DNAzyme-based assay enabled equipment-free visual detection of 15 µg/L uranyl, using both solution-based and paper-based pH-dependent visualization strategies.

Distinguishing reference intervals and clinical decision limits - A review by the IFCC Committee on Reference Intervals and Decision Limits.

Reference Intervals (RIs) and clinical decision limits (CDLs) are a vital part of the information supplied by laboratories to support the interpretation of numerical clinical pathology results. RIs describe the typical distribution of results seen in a healthy reference population while CDLs are associated with a significantly higher risk of adverse clinical outcomes or are diagnostic for the presence of a specific disease. However, as the two concepts are sometimes confused, there is a need to clarify the differences between these terms and to ensure they are easily distinguished, especially because CDLs have a clinical association with specific diseases and risks, thereby implying that effective clinical interventions are available. It is important to note that, because population-based RIs are derived from the range of values expected in a typical community population, laboratory results that fall outside a RI do not necessarily indicate a disease but rather that additional medical follow-up and/or treatment may be warranted. In contrast, CDLs are associated with a risk of specific adverse outcomes, and are commonly used to interpret laboratory test results, including lipid parameters, glucose, hemoglobin A1c (HbA1c), and tumor markers, to determine risk of disease, to diagnose or to treat. In recent years, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee on Reference Intervals and Decision Limits (C-RIDL) has focused primarily on RIs and has performed multicenter studies to obtain common RIs. However, the broader responsibility of the Committee, from its name, includes "decision limits". C-RIDL now aims to emphasize the importance of the correct use of both RIs and CDLs and to encourage laboratories to specify the appropriate information to clinicians as needed. This review discusses RIs and CDLs in detail, describes the similarities and the differences between these two important tools in laboratory medicine, and clearly explains the processes used to define them. C-RIDL encourages the involvement of laboratory professionals in the establishment of both RIs and CDLs.

Pediatric reference intervals for 29 Ortho VITROS 5600 immunoassays using the CALIPER cohort of healthy children and adolescents.

BACKGROUND: Accurate reference intervals (RIs) based on a healthy pediatric population are essential for pediatric test result interpretation. The CALIPER project has recruited a large healthy cohort and completed a series of a priori studies to address gaps in pediatric RIs. As immunoassays from different manufacturers for endocrine and special chemistry markers are not standardized and show marked intermethod differences, direct RI studies are needed for each major analytical platform. Here, we report age- and sex-specific pediatric RIs for 29 immunoassays on the Ortho Clinical Diagnostics (Ortho) VITROS® 5600 analyzer. METHODS: Health information and blood samples were collected from healthy pediatric subjects. Using the Ortho VITROS 5600 Integrated System MicroWell Technology, 29 biomarkers were measured. Analyte concentrations were partitioned by age and sex according to the Harris and Boyd method. After removing outliers, age- and sex-specific RIs and corresponding 90% confidence intervals were calculated according to CLSI guidelines. RESULTS: All analytes required age partitioning except ß-human chorionic gonadotropin (ß-hCG), cancer antigen 15-3 (CA15-3), rubella immunoglobulin G (rubella IgG), and vitamin D. Several analytes including estradiol, progesterone, testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), free triiodothyronine (FT3), total triiodothyronine (TT3), total thyroxine (TT4), thyroid uptake, ferritin, intact parathyroid hormone (iPTH), total prostate-specific antigen (tPSA), free prostate-specific antigen (fPSA), cancer antigen 125 (CA125), creatine kinase MB (CK-MB), and myoglobin showed sex differences, observed mostly with the onset of puberty. CONCLUSIONS: Complex reference value trends were observed across the pediatric age range for several biomarkers examined on Ortho VITROS immunoassays. The availability of VITROS immunoassay RIs will enable accurate laboratory test interpretation and diagnosis for the pediatric population. As recommended by the CLSI EP28-A3c guidelines, implementation of these RIs should be validated for each laboratory's local pediatric population.

Does the usage of digital chest drainage systems reduce pleural inflammation and volume of pleural effusion following oncologic pulmonary resection?-A prospective randomized trial.

BACKGROUND: Prolonged air leak and high-volume pleural drainage are the most common causes for delays in chest tube removal following lung resection. While digital pleural drainage systems have been successfully used in the management of post-operative air leak, their effect on pleural drainage and inflammation has not been studied before. We hypothesized that digital drainage systems (as compared to traditional analog continuous suction), using intermittent balanced suction, are associated with decreased pleural inflammation and postoperative drainage volumes, thus leading to earlier chest tube removal. METHODS: One hundred and three [103] patients were enrolled and randomized to either analog (n=50) or digital (n=53) drainage systems following oncologic lung resection. Chest tubes were removed according to standardized, pre-defined protocol. Inflammatory mediators [interleukin-1B (IL-1B), 6, 8, tumour necrosis factor-alpha (TNF-α)] in pleural fluid and serum were measured and analysed. The primary outcome of interest was the difference in total volume of postoperative fluid drainage. Secondary outcome measures included duration of chest tube in-situ, prolonged air-leak incidence, length of hospital stay and the correlation between pleural effusion formation, degree of inflammation and type of drainage system used. RESULTS: There was no significant difference in total amount of fluid drained or length of hospital stay between the two groups. A trend for shorter chest tube duration was found with the digital system when compared to the analog (P=0.055). Comparison of inflammatory mediator levels revealed no significant differences between digital and analog drainage systems. The incidence of prolonged post-operative air leak was significantly higher when using the analog system (9 versus 2 patients; P=0.025). Lobectomy was associated with longer chest tube duration (P=0.001) and increased fluid drainage when compared to sub-lobar resection (P<0.001), regardless of drainage system. CONCLUSIONS: Use of post-lung resection digital drainage does not appear to decrease pleural fluid formation, but is associated with decreased prolonged air leaks. Total pleural effusion volumes did not differ with the type of drainage system used. These findings support previously established benefits of the digital system in decreasing prolonged air leaks, but the advantages do not appear to extend to decreased pleural fluid formation.

Testosterone monitoring for men with advanced prostate cancer: Review of current practices and a survey of Canadian physicians.

Androgen-deprivation therapy (ADT) is a standard of care in the treatment of advanced prostate cancer; however, testosterone monitoring practices for men undergoing ADT vary across Canada. Although a testosterone level of 1.7 nmol/L or lower has historically been defined as the accepted castrate level, newer assays with improved sensitivity have shown that both medical and surgical castration can suppress testosterone levels to below 0.7 nmol/L. This review explores the evidence supporting a redefinition of the castrate testosterone level as 0.7 nmol/L or lower, and presents results of a survey of testosterone monitoring practices among 153 Canadian urologists, uro-oncologists, and radiation oncologists who manage the treatment of men with hormone-sensitive prostate cancer.

Comparison of Fecal Calprotectin Methods for Predicting Relapse of Pediatric Inflammatory Bowel Disease.

Background. Pediatric inflammatory bowel disease (IBD) is on the rise worldwide. Endoscopies are necessary for IBD assessment but are invasive, expensive, and inconvenient. Recently, fecal calprotectin (FCal) was proposed as a noninvasive and specific marker of gut inflammation. We evaluated the analytical performance of three FCal assays and their clinical performance in predicting relapse in pediatric IBD. Methods. This study used 40 pediatric IBD and 40 random non-IBD patients' fecal samples. Two automated ELISAs (Bühlmann and PhiCal® Calprotectin-EIA) and an EliA (Phadia 250 EliA-Calprotectin) were used to evaluate the analytical performance. The clinical performance was assessed by PhiCal Calprotectin-EIA, EliA-Calprotectin, and Bühlmann immunochromatographic point-of-care test (POCT). Results. All assays displayed acceptable analytical performance below and above the medical decision cut-off [imprecision (CV < 10% intra-assay; <15% interassay); linearity (overall mean % deviation < 16.5%)]. The agreement with PhiCal Calprotectin-EIA was 100% and 78.6% for Bühlmann (95% CI, 87.5-100; Kappa: 1) and EliA-Calprotectin (95% CI, 60.5-89.8; Kappa: 0.32), respectively, and 63.6% between Bühlmann and EliA-Calprotectin (95% CI, 46.6-77.8; Kappa: 0.16). All assays evaluated had similar clinical performance [AUC: 0.84 (EliA-Calprotectin); 0.83 (POCT and PhiCal Calprotectin-EIA)]. Conclusion. FCal levels determined using the same method and assay together with clinical history would be a noninvasive and useful tool in monitoring pediatric IBD.

Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain Activity: A Pilot Study in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Probiotics can reduce symptoms of irritable bowel syndrome (IBS), but little is known about their effects on psychiatric comorbidities. We performed a prospective study to evaluate the effects of Bifidobacterium longum NCC3001 (BL) on anxiety and depression in patients with IBS. METHODS: We performed a randomized, double-blind, placebo-controlled study of 44 adults with IBS and diarrhea or a mixed-stool pattern (based on Rome III criteria) and mild to moderate anxiety and/or depression (based on the Hospital Anxiety and Depression scale) at McMaster University in Canada, from March 2011 to May 2014. At the screening visit, clinical history and symptoms were assessed and blood samples were collected. Patients were then randomly assigned to groups and given daily BL (n = 22) or placebo (n = 22) for 6 weeks. At weeks 0, 6, and 10, we determined patients' levels of anxiety and depression, IBS symptoms, quality of life, and somatization using validated questionnaires. At weeks 0 and 6, stool, urine and blood samples were collected, and functional magnetic resonance imaging (fMRI) test was performed. We assessed brain activation patterns, fecal microbiota, urine metabolome profiles, serum markers of inflammation, neurotransmitters, and neurotrophin levels. RESULTS: At week 6, 14 of 22 patients in the BL group had reduction in depression scores of 2 points or more on the Hospital Anxiety and Depression scale, vs 7 of 22 patients in the placebo group (P = .04). BL had no significant effect on anxiety or IBS symptoms. Patients in the BL group had a mean increase in quality of life score compared with the placebo group. The fMRI analysis showed that BL reduced responses to negative emotional stimuli in multiple brain areas, including amygdala and fronto-limbic regions, compared with placebo. The groups had similar fecal microbiota profiles, serum markers of inflammation, and levels of neurotrophins and neurotransmitters, but the BL group had reduced urine levels of methylamines and aromatic amino acids metabolites. At week 10, depression scores were reduced in patients given BL vs placebo. CONCLUSION: In a placebo-controlled trial, we found that the probiotic BL reduces depression but not anxiety scores and increases quality of life in patients with IBS. These improvements were associated with changes in brain activation patterns that indicate that this probiotic reduces limbic reactivity. ClinicalTrials.gov no. NCT01276626.

Validation of a Capillary Electrophoresis Assay for Monitoring Iodine Nutrition in Populations for Prevention of Iodine Deficiency: An Interlaboratory Method Comparison.

BACKGROUND: A capillary electrophoresis (CE) assay was recently introduced as a new method for monitoring iodine nutrition in large-scale epidemiological studies. However, further tests revealed unanticipated matrix-dependent interferences when analyzing submicromolar levels of iodide in human urine as the predominate ionic form of dietary iodine. Herein, we describe a rigorous validation study that was used to identify sources of bias and establish modifications to the original CE method to improve method accuracy. METHODS: An interlaboratory method comparison using CE with UV detection and inductively coupled plasma-mass spectrometry (ICP-MS) was performed to quantify urinary iodide concentrations (n = 71) independently at McMaster University and Hamilton General Hospital, as well as the CDC as part of their quality assurance program. A positive bias in the original CE method was indicated, and buffer conditions were subsequently optimized to overcome matrix interferences for reliable iodine status determination. RESULTS: Positive bias in CE was attributed to variable concentrations of sulfate, a major urinary anion interference with similar mobility to iodide under the conditions originally reported. By increasing the concentration of α-cyclodextrin in the background electrolyte, the CE method was able to tolerate urinary sulfate over its normal physiological range without loss in signal response for iodide. The optimized CE assay generated results that were consistent with ICP-MS using 2 different internal standards (187Re and 130Te) with a median bias under 10%. CONCLUSIONS: CE offers a simple, selective, and cost-effective separation platform for surveillance of the iodine status of a population requiring only small volumes (<10 µL) of biobanked urine specimens, which is comparable to previously validated screening methods currently used in global health initiatives for prevention of iodine deficiency disorders.

Lipoprotein interactions with a polyurethane and a polyethylene oxide-modified polyurethane at the plasma-material interface.

Lipoproteins [high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL)] are present in blood in relatively high concentrations, and, given their importance in cardiovascular disease, the interactions of these species with blood contacting biomaterials and their possible role in thrombogenesis is of interest. In the present communication, quantitative data on the adsorption of apolipoprotein AI, apolipoprotein AII (the main protein components of HDL), and apolipoprotein B (the main protein component of LDL and VLDL), as well as the lipoproteins themselves from plasma to a biomedical grade polyurethane (PU) with and without a copolymer additive that contains polyethylene oxide (PEO) segments, were investigated. Adsorption from some binary solutions was also studied. Significant quantities of the apolipoproteins were found to adsorb from plasma to the PU, while adsorption to the PEO material was more than 90% lower, demonstrating strong protein resistance of the latter material. In contrast, significant quantities of the lipoproteins were found to adsorb to the PEO as well as to the PU material. From these and previously published results, it is concluded that the protein layer formed on the PU surface from plasma (and by extension from blood) contains apolipoproteins and lipoproteins in addition to other plasma proteins; the layer formed on the PEO surface, however, appears to contain minimal quantities of plasma proteins (including free apolipoproteins) but significant quantities of lipoproteins.

The effects of diet on occlusive coronary artery atherosclerosis and myocardial infarction in scavenger receptor class B, type 1/low-density lipoprotein receptor double knockout mice.

OBJECTIVE: Deficiency of the high-density lipoprotein receptor, scavenger receptor class B, type I (SR-BI), in apolipoprotein E knockout or hypomorphic mice, respectively, results in spontaneous or diet-inducible occlusive coronary artery (CA) atherosclerosis, myocardial infarction, and early death. Here, we examine effects of SR-BI deficiency on cardiovascular phenotypes in low-density lipoprotein receptor (LDLR) knockout mice fed different atherogenic diets. APPROACH AND RESULTS: SR-BI/LDLR double knockout and control LDLR knockout mice were fed atherogenic diets containing different amounts of fat, cholesterol, and sodium cholate. Double knockout mice fed atherogenic diets high in cholesterol exhibited significantly reduced survival compared with LDLR knockout mice fed the same diets. In addition to increased diet-accelerated aortic sinus atherosclerosis, we observed significant diet-induced CA atherosclerosis in double knockout mice and diet-dependent accumulation of platelets in CA atherosclerotic plaques. This was accompanied by substantial myocardial fibrosis in double knockout mice fed high cholesterol diets. Atherogenic diet fed double knockout mice also exhibited higher circulating cytokine levels, monocytosis with increased proportions of Ly6C(hi) and Ly6C(int) monocytes, and higher adhesion molecule expression in CA endothelial cells compared with control LDLR knockout mice. CONCLUSIONS: Diet-accelerated atherosclerosis and occlusive, platelet-rich CA disease in SR-BI/LDLR double knockout mice is affected by amounts of cholesterol and cholate in atherogenic diets and is accompanied by increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CAs and increased Ly6C(hi) and Ly6C(int) monocytes in circulation. The increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in CA endothelial cells in SR-BI-deficient mice likely explains their increased susceptibility to atherosclerosis in CAs.

Strong anion determination in biological fluids by capillary electrophoresis for clinical diagnostics.

New methods for quantitative analysis of strong anions are required for diagnostic testing of human diseases. Current techniques suffer from poor selectivity and/or long analysis times that are not amenable for labile anions in high-saline or volume-restricted samples. We introduce a rapid assay (<5 min) based on capillary electrophoresis (CE) with indirect UV detection for simultaneous analysis of sulfate, sulfite, and chloride in human urine, plasma, and sweat specimens. Remarkable selectivity for strong anions is achieved by using an acidic background electrolyte under reversed polarity that results in electrokinetic rejection of matrix interferences at the capillary inlet. A dual co-ion probe system consisting of 5 mM naphthalene disulfonate (NDS) and 5 mM naphthalene trisulfonate (NTS) in 0.4 M formic acid, pH 2.0 is developed for detection of UV transparent anions (S/N ≈ 3, 60 µM with a 25 µm inner diameter fused-silica capillary) with good peak symmetry and baseline stability. Due to the chemical reactivity of sulfite, dilute formaldehyde is used as a reagent to form an acid-stable hydroxymethylsulfonate adduct. Method validation confirmed excellent linearity (R(2) > 0.999), good accuracy (mean bias ≈7%), and acceptable long-term reproducibility (CV < 10%) over 20 days. The assay allows for artifact-free determination of sulfate and sulfite with consistent results for chloride when compared to standard electrochemical methods (R(2) > 0.975). Preliminary data suggest that kidney-stone formers have lower urinary sulfate excretion relative to non-kidney-stone patient controls (p = 0.0261). CE offers a selective yet robust platform for routine analysis of strong anions that is needed for confirmatory testing of cystic fibrosis, sulfite oxidase deficiency, urolithiasis, and other disorders of sulfur metabolism and/or anion transport.

Evaluation of a commercially available carbohydrate deficient transferrin kit to detect beta-2-transferrin in cerebrospinal fluid using capillary electrophoresis.

OBJECTIVE: The aim of this study was to evaluate the CEofix™ CDT Capillary Electrophoresis (CE) kit for the detection of ß-2-tranferrin (ß-2-Tf) in cerebrospinal fluid (CSF). DESIGN AND METHOD: Evaluation was performed according to CLSI EP5-A and EP12-A guidelines. RESULTS: The method resolved ß-2-Tf from other Tf isoforms. The C50 for ß-Tf was determined to be 0.46 mg/L. Neither hemoglobin nor bilirubin co-migrated with ß-2-Tf. CONCLUSIONS: The CDT kit can be used for detecting ß-2-Tf in CSF by CE.

Proteomic analysis of plasma from Holstein cows testing positive for Mycobacterium avium subsp. paratuberculosis (MAP).

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & ß (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.