Extracellular Matrix Injury of Kidney Allografts in Antibody-Mediated Rejection: A Proteomics Study.

BACKGROUND: Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft loss. Donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium cause AMR while inflammatory cytokines such as TNFα trigger graft injury. The mechanisms governing cell-specific injury in AMR remain unclear. METHODS: Unbiased proteomic analysis of laser-captured and microdissected glomeruli and tubulointerstitium was performed on 30 for-cause kidney biopsy specimens with early AMR, acute cellular rejection (ACR), or acute tubular necrosis (ATN). RESULTS: A total of 107 of 2026 glomerular and 112 of 2399 tubulointerstitial proteins was significantly differentially expressed in AMR versus ACR; 112 of 2026 glomerular and 181 of 2399 tubulointerstitial proteins were significantly dysregulated in AMR versus ATN (P<0.05). Basement membrane and extracellular matrix (ECM) proteins were significantly decreased in both AMR compartments. Glomerular and tubulointerstitial laminin subunit γ-1 (LAMC1) expression decreased in AMR, as did glomerular nephrin (NPHS1) and receptor-type tyrosine-phosphatase O (PTPRO). The proteomic analysis revealed upregulated galectin-1, which is an immunomodulatory protein linked to the ECM, in AMR glomeruli. Anti-HLA class I antibodies significantly increased cathepsin-V (CTSV) expression and galectin-1 expression and secretion in human glomerular endothelial cells. CTSV had been predicted to cleave ECM proteins in the AMR glomeruli. Glutathione S-transferase ω-1, an ECM-modifying enzyme, was significantly increased in the AMR tubulointerstitium and in TNFα-treated proximal tubular epithelial cells. CONCLUSIONS: Basement membranes are often remodeled in chronic AMR. Proteomic analysis performed on laser-captured and microdissected glomeruli and tubulointerstitium identified early ECM remodeling, which may represent a new therapeutic opportunity.

Evaluation of Glomerular Hemodynamic Function by Empagliflozin in Diabetic Mice Using In Vivo Imaging.

BACKGROUND: Sodium glucose cotransporter 2 inhibitors may reduce kidney hyperfiltration, thereby preventing diabetic kidney disease progression, which may in turn reduce cardiovascular risk, including heart failure. However, the mechanisms that regulate renal function responses to sodium glucose cotransporter 2 inhibition are not yet fully understood. We explored the renal protective effects of sodium glucose cotransporter 2 inhibition with empagliflozin, with a focus on glomerular hemodynamic effects and tubuloglomerular feedback using in vivo multiphoton microscopy imaging techniques. METHODS: C57BL/6 mice and spontaneously diabetic Ins2+/Akita mice were studied. The mice were treated with empagliflozin (20 mg·kg-1·d-1) and insulin for 4 weeks, and the single-nephron glomerular filtration rate was measured using multiphoton microscope. A neuronal nitric oxide synthase inhibitor (7-nitroindazole, 20 mg·kg-1·d-1) or a cyclooxygenase-2 inhibitor (SC58236, 6 mg/L), or an A1 adenosine receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, 1 mg·kg-1·d-1) was administered to elucidate the mechanisms of tubuloglomerular feedback signaling and single-nephron glomerular filtration rate regulation. RESULTS: The urinary excretion of adenosine, nitric oxide metabolites, and the prostanoid prostaglandin E2 was also quantified. The single-nephron glomerular filtration rate in the Ins2+/Akita group was higher than in controls (C57BL/6; 4.9±1.3 nL/min versus Ins2+/Akita; 15.8±6.8 nL/min) and lower in Ins2+/Akita /empagliflozin to 8.0±3.3 nL/min (P<0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (P<0.01) and increased glomerular permeability of albumin in the Ins2+/Akita group. Empagliflozin ameliorated these changes (P<0.01). Urinary adenosine excretion in the Ins2+/Akita/empagliflozin group was higher than in Ins2+/Akita (Ins2+/Akita; 3.4±1.4 nmol/d, Ins2+/Akita/empagliflozin; 11.2±3.0 nmol/d, P<0.05), whereas nitric oxide metabolites and prostaglandin E2 did not differ. A1 adenosine receptor antagonism, but not neuronal nitric oxide synthase or cyclooxygenase-2 inhibition, blocked the effect of empagliflozin on renal function. Empagliflozin increased urinary adenosine excretion and reduced hyperfiltration via afferent arteriolar constriction, effects that were abolished by A1 adenosine receptor blockade. CONCLUSIONS: Adenosine/A1 adenosine receptor pathways play a pivotal role in the regulation of the single-nephron glomerular filtration rate via tubuloglomerular feedback mechanisms in response to sodium glucose cotransporter 2 inhibition, which may contribute to renal and cardiovascular protective effects reported in clinical trials.

The renal hemodynamic effects of the SGLT2 inhibitor dapagliflozin are caused by post-glomerular vasodilatation rather than pre-glomerular vasoconstriction in metformin-treated patients with type 2 diabetes in the randomized, double-blind RED trial.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve hard renal outcomes in type 2 diabetes. This is possibly explained by the fact that SGLT2i normalize the measured glomerular filtration rate (mGFR) by increasing renal vascular resistance, as was shown in young people with type 1 diabetes and glomerular hyperfiltration. Therefore, we compared the renal hemodynamic effects of dapagliflozin with gliclazide in type 2 diabetes. The mGFR and effective renal plasma flow were assessed using inulin and para-aminohippurate clearances in the fasted state, during clamped euglycemia (5 mmol/L) and during clamped hyperglycemia (15 mmol/L). Filtration fraction and renal vascular resistance were calculated. Additionally, factors known to modulate renal hemodynamics were measured. In 44 people with type 2 diabetes on metformin monotherapy (Hemoglobin A1c 7.4%, mGFR 113 mL/min), dapagliflozin versus gliclazide reduced mGFR by 5, 10, and 12 mL/min in the consecutive phases while both agents similarly improved Hemoglobin A1c (-0.48% vs -0.65%). Dapagliflozin also reduced filtration fraction without increasing renal vascular resistance, and increased urinary adenosine and prostaglandin concentrations. Gliclazide did not consistently alter renal hemodynamic parameters. Thus, beyond glucose control, SGLT2i reduce mGFR and filtration fraction in type 2 diabetes. The fact that renal vascular resistance was not increased by dapagliflozin suggests that this is due to post-glomerular vasodilation rather than pre-glomerular vasoconstriction.

Urinary adenosine excretion in type 1 diabetes.

In experimental models of diabetes, augmented sodium-glucose cotransport-2 (SGLT2) activity diminishes sodium (Na+) delivery at the macula densa. As a result, less vasoconstrictive adenosine is generated, leading to afferent arteriolar vasodilatation and hyperfiltration. The measurement and significance of urinary adenosine in humans has not been examined extensively in states of renal hemodynamic impairment like that of diabetes. Our aim was to validate a method for urine adenosine quantification in humans and perform an exploratory post hoc analysis to determine whether urinary adenosine levels change dynamically in response to natriuresis in patients with type 1 diabetes (T1D) before and after treatment with the SGLT2 inhibitor (SGLT2i) empagliflozin. We hypothesized that SGLT2i, which reduces renal hyperfiltration through increased Na+ delivery to the macula densa, would increase urinary adenosine excretion. Urine adenosine corrected for creatinine was measured using our validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in 40 healthy participants and 40 patients with T1D. In the T1D cohort, measurements were performed during clamped euglycemic and hyperglycemic conditions before and following 8 wk of SGLT2i therapy. Urinary adenosine was detectable in healthy subjects (0.32 ± 0.11 µmol/mmol Cr) and patients with T1D. In response to SGLT2i, urine adenosine increased during clamped hyperglycemia (0.40 ± 0.11 vs. 0.45 ± 0.12 µmol/mmol Cr, P = 0.005). Similar trends were observed during clamped euglycemia (P = 0.08). In conclusion, SGLT2i increases urinary adenosine excretion under clamped hyperglycemic conditions in patients with T1D. The potentially protective role of SGLT2i against glomerular hyperfiltration and its mediation by adenosine in diabetes merits further study.

Accuracy of testosterone concentrations in compounded testosterone products.

OBJECTIVE: This study aims to evaluate the accuracy of the testosterone concentrations within testosterone gels and creams manufactured by compounding pharmacies. METHODS: Ten compounding pharmacies within Toronto area were included. Pharmacies were blinded as to the nature of the study. A standardized prescription for 50 mg of compounded testosterone gel/cream applied once daily was presented to each pharmacy. Two independently compounded batches were analyzed from each pharmacy 1 month apart. Testosterone concentrations in a 5-g sachet of Androgel® 1% (Abbott) and 5-g tube of Testim®1% (Auxilium) were evaluated as controls. Samples were analyzed independently and in a blinded fashion by the Laboratory Medicine Program at the University Health Network. Measurement of testosterone concentration was performed using a modified liquid chromatography tandem mass spectrometry validated for serum testosterone. RESULTS: Compounded formulations included seven gels and three creams with a volume/daily dose ranging from 0.2 mL to 1.25 mL. Product cost ranged from $57.32 to $160.71 for a 30-day supply. There was significant variability both within and between pharmacies with respect to the measured concentration of testosterone in the compounded products. In contrast, the concentration of testosterone within Androgel and Testim was consistent and accurate. Collectively, only 50% (batch 1) and 30% (batch 2) of the compounding pharmacies provided a product with a testosterone concentration within ± 20% of the prescribed dose. Two pharmacies compounded products with >20% of the prescribed dose. One pharmacy compounded a product with essentially no testosterone. CONCLUSIONS: Testosterone concentrations in compounded testosterone products can be variable and potentially compromise the efficacy and safety of treatment.

Serum voriconazole level variability in patients with hematological malignancies receiving voriconazole therapy.

INTRODUCTION: Voriconazole plasma concentrations have been correlated with oral dosing in healthy subjects, but have been poorly characterized in ill patients with hematological malignancies receiving intensive chemotherapy. METHODS: The relationship between orally administered voriconazole, plasma concentrations and liver toxicity was examined in a cohort of 69 primarily acute leukemia patients undergoing intensive chemotherapy. RESULTS: Oral administration of voriconazole was associated with significant interpatient variability, with voriconazole steady-state concentrations ranging from 0 µg/mL to 16.6 µg/mL. Approximately 20% of patients achieved steady-state concentrations <1 µg/mL. When adjusted for weight, patients receiving higher voriconazole doses tended toward higher plasma concentrations; however, there was no significant relationship between the plasma concentration and genotype, age, sex or use of concomitant proton pump inhibitors. Voriconazole concentrations were correlated with higher serum alkaline phosphatase levels at day 6 to 8, and with higher bilirubin and aspartate aminotransferase levels at day 14 to 16, but not with other liver enzyme levels. CONCLUSION: In ill patients with acute leukemia and related disorders undergoing treatment with oral voriconazole, there is a poor correlation between the voriconazole dose and plasma concentrations, and many patients achieve levels that are considered to be subtherapeutic. The findings support the routine use of therapeutic drug monitoring in these patients.

INTRODUCTION: Les concentrations plasmatiques de voriconazole sont corrélées avec les doses orales chez les sujets en santé, mais sont mal caractérisées chez les patients malades atteints d'une hémopathie maligne sous chimiothérapie intensive. MÉTHODOLOGIE: Les chercheurs ont examiné le lien entre le voriconazole administré par voie orale et la toxicité hépatique dans une cohorte de 69 patients atteints surtout de leucémie aiguë sous chimiothérapie intensive. RÉSULTATS: L'administration de voriconazole par voie orale s'associait à une importante variabilité interpatient, les concentrations à l'état stable oscillant entre 0 µg/mL et 16,6 µg/mL. Environ 20 % des patients ont obtenu des concentrations à l'état stable de moins de 1 µg/mL. Après rajustement selon le poids, les patients qui receviaent des doses plus élevées de voriconazole avaient tendance à présenter des concentrations plasmatiques plus élevées. Cependant, on ne constatait aucun lien significatif entre la concentration plasmatique et le géno-type, l'âge, le sexe ou l'utilisation concomitante d'inhibiteurs de la pompe à protons. Les concentrations de voriconazole étaient corrélées avec des taux de phosphatase alcaline sérique plus élevés les jours 6 à 8 et à des taux de bilirubine et d'aspartate aminotransférase plus élevés les jours 14 à 16, mais pas à d'autres taux d'enzymes hépatiques. CONCLUSION: Chez les patients malades atteints d'une leucémie aiguë et de troubles connexes qui suivent un traitement au voriconazole par voie orale, la corrélation entre la dose de voriconazole et les concentrations plasmatiques est faible, et de nombreux patients obtiennent des taux considérés comme subthérapeutiques. Les observations soutiennent une pharmacovigilance systématique chez ces patients.

Total serum pentosidine quantification using liquid chromatography-tandem mass spectrometry.

Pentosidine (PEN) is an Advanced Glycation End-product (AGE) that is known to accumulate in bone collagen with aging and contribute to fracture risk. The PEN content in bone is correlated with serum PEN, making it an attractive, potential osteoporosis biomarker. We sought to develop a method for quantifying PEN in stored serum. After conducting a systematic narrative review of PEN quantification methodologies, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying total serum PEN. Our method is both sensitive and precise (LOD 2 nM, LOQ 5 nM, %CV < 6.5 % and recovery 91.2-100.7 %). Our method is also equivalent or better than other methods identified in our review. Additionally, LC-MS/MS avoids the pitfalls and limitations of using fluorescence as a means of detection and could be adapted to investigate a broad range of AGE compounds.

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice.

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

Conversion of methane to methanol: nickel, palladium, and platinum (d9) cations as catalysts for the oxidation of methane by ozone at room temperature.

The room-temperature chemical kinetics has been measured for the catalytic activity of Group 10 atomic cations in the oxidation of methane to methanol by ozone. Ni(+) is observed to be the most efficient catalyst. The complete catalytic cycle with Ni(+) is interpreted with a computed potential energy landscape and, in principle, has an infinite turnover number for the oxidation of methane, without poisoning side reactions. The somewhat lower catalytic activity of Pd(+) is reported for the first time and also explored with DFT calculations. Pt(+) is seen to be ineffective as a catalyst because of the observed failure of PtO(+) to convert methane to methanol.

Salsalate, but not metformin or canagliflozin, slows kidney cyst growth in an adult-onset mouse model of polycystic kidney disease.

BACKGROUND: Multiple preclinical studies have highlighted AMP-activated protein kinase (AMPK) as a potential therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). Both metformin and canagliflozin indirectly activate AMPK by inhibiting mitochondrial function, while salsalate is a direct AMPK activator. Metformin, canagliflozin and salsalate (a prodrug dimer of salicylate) are approved for clinical use with excellent safety profile. Although metformin treatment had been shown to attenuate experimental cystic kidney disease, there are concerns that therapeutic AMPK activation in human kidney might require a higher oral metformin dose than can be achieved clinically. METHODS: In this study, we tested metformin-based combination therapies for their additive (metformin plus canagliflozin) and synergistic (metformin plus salsalate) effects and each drug individually in an adult-onset conditional Pkd1 knock-out mouse model (n = 20 male/group) using dosages expected to yield clinically relevant drug levels. FINDINGS: Compared to untreated mutant mice, treatment with salsalate or metformin plus salsalate improved kidney survival (i.e. blood urea nitrogen <20 mmol/L at the time of sacrifice) and reduced cystic kidney disease severity. However, the effects of metformin plus salsalate did not differ from salsalate alone; and neither metformin nor canagliflozin was effective. Protein expression and phosphorylation analyses indicated that salsalate treatment was associated with reduction in mTOR (mammalian target of rapamycin) activity and cellular proliferation in Pkd1 mutant mouse kidneys. Global gene expression analyses suggested that these effects were linked to restoration of mitochondrial function and suppression of inflammation and fibrosis. INTERPRETATION: Salsalate is a highly promising candidate for drug repurposing and clinical testing in ADPKD.

Catalytic oxidation of H2 by N2O in the gas phase: O-atom transport with atomic metal cations.

Twenty-five atomic cations, M (+), that lie within the thermodynamic window for O-atom transport catalysis of the oxidation of hydrogen by nitrous oxide, have been checked for catalytic activity at room temperature with kinetic measurements using an inductively-coupled plasma/selected-ion flow tube (ICP/SIFT) tandem mass spectrometer. Only 4 of these 25 atomic cations were seen to be catalytic: Fe (+), Os (+), Ir (+), and Pt (+). Two of these, Ir (+) and Pt (+), are efficient catalysts, while Fe (+) and Os (+) are not. Eighteen atomic cations (Cr (+), Mn (+), Co (+), Ni (+), Cu (+), Ge (+), Se (+), Mo (+), Ru (+), Rh (+), Sn (+), Te (+), Re (+), Pb (+), Bi (+), Eu (+), Tm (+), and Yb (+)) react too slowly at room temperature either in their oxidation with N 2O to form MO (+) or in the reduction of MO (+) by H 2. Many of these reactions are known to be spin forbidden and a few actually may lie outside the thermodynamic window. Three alkaline-earth metal monoxide cations, CaO (+), SrO (+), and BaO (+), were observed to favor MOH (+) formation in their reactions with H 2. A potential-energy landscape is computed for the oxidation of H 2 with N 2O catalyzed by Fe (+)( (6)D) that vividly illustrates the operation of an ionic catalyst and qualitatively accounts for the relative inefficiency of this catalyst.

Chemical Stability of Plerixafor after Opening of Single-Use Vial.

BACKGROUND: The addition of the immunostimulant plerixafor to the current standard-of-care regimens of granulocyte colony-stimulating growth factor with or without chemotherapy has improved clinical results in terms of successful stem cell mobilization and the outcomes of stem cell transplant in various settings. With this medical innovation has come an added financial cost for institutions where stem cell transplants are routinely performed, and there may be a further financial burden when the contents of partial vials of the drug are wasted, given that plerixafor vials (Mozobil, Sanofi-Aventis Canada Inc) are currently deemed suitable only for single use. OBJECTIVE: To determine whether the portion of plerixafor remaining in an opened vial of the Mozobil product after administration of a single dose is chemically stable, by comparison with the original product. METHODS: Stability testing of partial drug contents of an opened vial, stored at room temperature or under refrigeration (4°C), was conducted using liquid chromatography-tandem mass spectrometry analysis. The mean concentration of plerixafor (µmol/L), standard deviation, coefficient of variation, and bias were determined on days 2, 3, 11, 17, 24, and 31. Method validation included determination of precision, sensitivity, recovery, dilution linearity, and carryover. RESULTS: Throughout the 4-week testing period, measured plerixafor concentration in aliquots stored at room temperature and under refrigeration, tested in series over time, appeared similar. The mean residual drug concentration after initial opening was slightly, but not significantly, higher for the sample designated for storage at room temperature than the one designated for refrigerated storage (40.4 versus 39.9 µmol/L; p = 0.37). CONCLUSIONS: Residual plerixafor after initial opening of a vial of the Mozobil product remained chemically stable for at least 2 weeks both at room temperature and under refrigeration. The results of this study provide in vitro evidence to support multiple uses, instead of single use, of vials of this drug in an aseptic, controlled environment.

CONTEXTE: L'ajout de l'immunostimulant plérixafor aux traitements reconnus comme la norme de soins actuelle quant au facteur de stimulation des colonies de granulocytes, accompagné ou non de chimiothérapie, a amélioré les résultats cliniques de mobilisation des cellules souches et les résultats de greffe de cellules souches dans différents contextes. Cela dit, avec cette innovation médicale vient un poids financier supplémentaire pour les établissements où l'on exécute couramment des greffes de cellules souches. En outre, comme les fioles de plérixafor (Mozobil, Sanofi-Aventis Canada Inc.) sont présentement jugées adéquates pour un usage unique seulement, l'excédent de médicament gaspillé peut représenter une dépense additionnelle. OBJECTIF: Déterminer si ce qui reste de Mozobil dans une fiole ouverte après l'administration d'une dose unique est chimiquement stable comparativement au produit de départ. MÉTHODES: Une étude de stabilité du contenu partiel d'une fiole de médicament ouverte, entreposé à température ambiante ou conservé au réfrigérateur (4°C), a été réalisée par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem. La concentration moyenne de plérixafor (µmol/L), l'écart-type, le coefficient de variation et le biais ont été établis aux jours 2, 3, 11, 17, 24 et 31. La méthode de validation comprenait la détermination de : la précision, la sensibilité, la récupération, la limite de linéarité et la contamination inter-échantillons. Résultats :Tout au long des quatre semaines d'analyse, les concentrations mesurées des aliquotes de plérixafor, entreposées à température ambiante ou conservées au réfrigérateur et analysées en séries chronologiques, semblaient similaires. Les concentrations moyennes de médicament restant après l'ouverture initiale étaient légèrement plus élevées lorsqu'entreposées à température ambiante (40,4 µmol/L) que lorsque réfrigérées (39,9 µmol/L) (p = 0,37), mais pas de façon significative. CONCLUSIONS: Le plérixafor résiduel, après l'ouverture initiale des fioles de Mozobil, demeurait chimiquement stable pendant au moins deux semaines, qu'il soit entreposé à température ambiante ou conservé au réfrigérateur. Les résultats de la présente étude offrent des données in vitro qui soutiennent une utilisation multiple, plutôt qu'un usage unique, des fioles de ce médicament en milieu aseptique contrôlé.

Quantitative mass spectrometry-based assay development and validation: from small molecules to proteins.

Mass spectrometry (MS) has emerged as a powerful analytical tool for the identification, characterization and quantification of various biomolecules (small molecules, drug metabolites and proteins) in biological specimens. The use of mass spectrometers in the clinical diagnostic laboratories have gained popularity due to its ease of development of new assays, ability to measure multiple analytes in a single analytical run, low volume requirements and low reagent costs. Novel technological advancements in ionization sources, instrumentation and software have increased the popularity of these platforms. Consequently, a number of home-brew assays, utilizing the power of MS, are being developed and validated for clinical diagnostic use. In this review, we will discuss the two phases that precede method implementation: method development and validation for both small molecule analysis and protein quantification using liquid chromatography tandem mass spectrometry (LC-MS/MS). Some of the challenges facing protein quantification will be highlighted and an outlook for the future of laboratory medicine and MS will be provided.

Performance evaluation of Siemens ADVIA Centaur and Roche MODULAR Analytics E170 Total 25-OH Vitamin D assays.

OBJECTIVES: To evaluate the newly developed Roche MODULAR Analytics E170 Total Vitamin D and the Siemens ADVIA Centaur Vitamin D Total assays. MATERIALS AND METHODS: Assays were evaluated using the Clinical and Laboratory Standards Institute protocols. Split patient samples were compared with LC-MS/MS and DiaSorin LIAISON assays (n=79 including 15 specimens with detectable endogenous 25-OH vitamin D(2)). Assay accuracy was also evaluated using the Vitamin D External Quality Assessment Scheme (DEQAS) samples. RESULTS: The ADVIA Centaur and E170 assays demonstrated maximum total CVs of 14.1% and 5.9%, respectively. Both showed excellent linearity (R(2)>0.99). The ADVIA Centaur assay demonstrated interference with bilirubin at 800 µmol/L, hemolysis at 1.25 g/L, and triglycerides at 2.8 mmol/L. Compared to LC-MS/MS, the ADVIA Centaur assay demonstrated a R(2) value of 0.893, average bias of -8.8%; the E170 assay an R(2) value of 0.872, average bias of 14.3% with underestimation of 25-OH vitamin D(2). Compared to the LIAISON assay, the ADVIA Centaur assay demonstrated an R(2) value of 0.781, average bias of -17.3%; the E170 assay an R(2) value of 0.823, average bias of 11.4%. The ADVIA Centaur and E170 assays demonstrated a biases of <20% in 10/10 and 8/10 DEQAS samples, respectively. CONCLUSIONS: The ADVIA Centaur and E170 vitamin D assays demonstrated acceptable linearity, imprecision, and accuracy. The E170 assay demonstrated consistent underestimation of 25-OH vitamin D(2) levels. Compared with LC-MS/MS, the ADVIA Centaur assay demonstrated a higher R(2) value and a smaller average bias than the E170 assay.

Activation of X-H and X-D bonds (X = O, N, C) by alkaline-earth metal monoxide cations: experiment and theory.

Experimental investigations are reported for reactions of MO (+) (M = Ca, Sr, and Ba) with elemental hydrides water, ammonia and methane proceeding in the gas phase at 295 +/- 3 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. Measurements were taken with an inductively-coupled plasma/selected-ion flow tube (ICP/SIFT) tandem mass spectrometer and a novel electrospray ion source/ion selection quadrupole/selected-ion flow tube/triple quadrupole (ESI/qQ/SIFT/QqQ) mass spectrometer. All three alkaline-earth metal oxide ions exclusively abstract a H-atom from the three hydrides with rate coefficients > 1 x 10(-11) cm(3) molecule(-1) s(-1). Formation of metal hydroxide ion was followed by sequential addition of water or ammonia, but not methane. Density functional calculations have provided potential energy surfaces for the X-H bond activations leading to H-atom abstraction as well as those for O-atom transfer and H(2)O elimination (with ammonia and methane). A comparison of experimental and theoretical isotope effects points toward a mechanism involving the direct atom transfer from XH and XD to O in MO (+)via a three-centered transition structure.