Lack of interference of metronidazole with Roche cobas c502 and c702 chemistry tests.

OBJECTIVE: The antibiotic metronidazole has been suggested to absorb light at a wavelength range commonly used in spectrophotometric assays. We sought to determine if any of the spectrophotometric assays used in our core laboratory would be susceptible to clinically significant interference from metronidazole in blood-based patient specimens. METHODS: Following characterization of the absorbance spectrum for metronidazole, spectrophotometric assays involving either main or subtraction wavelengths that might be susceptible to interference from metronidazole were identified. A total of 24 chemistry tests performed on Roche cobas c502 and/or c702 instruments were evaluated for interference from metronidazole. For each assay, two pools of leftover patient serum, plasma, or whole blood specimens containing the analyte of interest at clinically relevant concentrations were prepared. Each pool was spiked with metronidazole at a final concentration of 200 mg/L (1169 µmol/L) or 10 mg/L (58 µmol/L) or the same volume of water as a control, with triplicate samples for each group. The difference in the measured analyte concentration between experimental and control groups was then compared against the total allowable error for each assay to determine whether clinically significant interference had occurred. RESULTS: There was no significant interference observed with Roche chemistry tests due to the presence of metronidazole. CONCLUSION: This study provides reassurance that metronidazole is not interfering with the chemistry assays used in our core laboratory. Interference from metronidazole may be a historical problem and current spectrophotometric assays may not be susceptible due to improvements in assay design.

Lot verification practices in Ontario clinical chemistry laboratories - Results of a patterns-of-practice survey.

Objectives: Verifying new reagent or calibrator lots is crucial for maintaining consistent test performance. The Institute for Quality Management in Healthcare (IQMH) conducted a patterns-of-practice survey and follow-up case study to collect information on lot verification practices in Ontario. Methods: The survey had 17 multiple-choice questions and was distributed to 183 licensed laboratories. Participants provided information on materials used and approval/rejection criteria for their lot verification procedures for eight classes of testing systems. The case study provided a set of lot comparison data and was distributed to 132 laboratories. Responses were reviewed by IQMH scientific committees. Results: Of the 175 laboratories that responded regarding reagent lot verifications, 74% verified all tests, 11% some, and 15% none. Of the 171 laboratories that responded regarding calibrator lot verifications, 39% verified all calibrators, 4% some, and 57% none. Reasons for not performing verifications ranged from difficulty performing parallel testing to high reagent cost. For automated chemistry assays and immunoassays, 23% of laboratories did not include patient-derived materials in reagent lot verifications and 42% included five to six patient materials; 58% of laboratories did not include patient-derived materials in calibrator lot verifications and 23% included five to six patient materials. Different combinations of test-specific rules were used for acceptance criteria. For a failed lot, 98% of laboratories would investigate further and take corrective actions. Forty-three percent of laboratories would accept the new reagent lot in the case study. Conclusion: Responses to the survey and case study demonstrated variability in lot verification practices among laboratories.

Survey of renin and aldosterone testing practices by Ontario laboratories - Providing insight into best practices.

OBJECTIVES: Testing for renin and aldosterone in clinical laboratories is complicated by pre-analytical considerations such as the posture for blood collection and susceptibility to cryoactivation of renin. From an analytical perspective, there are both renin activity and renin mass or concentration assays available. There can also be variability in result reporting practices and the aldosterone-renin ratio (ARR) cut-off applied to screen for primary aldosteronism (PA). The Institute for Quality Management in Healthcare (IQMH) Centre for Proficiency Testing surveyed laboratories on their handling of renin and aldosterone testing to better understand current practices. DESIGN AND METHODS: An online survey was prepared and sent to 134 Canadian laboratories enrolled in endocrinology proficiency testing with IQMH. RESULTS: One hundred twenty Ontario laboratories submitted responses. While only six (5%) laboratories perform testing for both renin and aldosterone, 108 (90%) collect and process specimens to be tested by reference laboratories. The survey revealed considerable variation in practices including the recommended state of patients prior to sample collection (for example, regarding medications or salt intake), the patient posture specifications for sample collection, the precautions taken against cryoactivation of renin, the choice of renin activity or mass assay, and the ARR cut-off used. The available literature on these factors was then reviewed. CONCLUSIONS: Although there is no standardized procedure for specimen collection, analysis, or result reporting for renin or aldosterone testing, we have attempted to summarize the available literature to develop evidence-based recommendations. Where laboratory practice differs from peers and/or recommended protocols, laboratories should review their practices.

Biochemistry tests in hospitalized COVID-19 patients: Experience from a Canadian tertiary care centre.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) has variable clinical presentation, from asymptomatic to severe disease leading to death. Biochemical markers may help with management and prognostication of COVID-19 patients; however, their utility is still under investigation. METHODS: A retrospective study was conducted to evaluate alanine aminotransferase, C-reactive protein (CRP), ferritin, lactate, and high sensitivity troponin T (TnT) levels in 67 patients who were admitted to a Canadian tertiary care centre for management of COVID-19. Logistic, cause-specific Cox proportional-hazards, and accelerated failure time regression modelling were performed to assess the associations of initial analyte concentrations with in-hospital death and length of stay in hospital; joint modelling was performed to assess the associations of the concentrations over the course of the hospital stay with in-hospital death. RESULTS: Initial TnT and CRP concentrations were associated with length of stay in hospital. Eighteen patients died (27%), and the median initial TnT concentration was higher in patients who died (55 ng/L) than those who lived (16 ng/L; P < 0.0001). There were no survivors with an initial TnT concentration > 64 ng/L. While the initial TnT concentration was predictive of death, later measurements were not. Only CRP had prognostic value with both the initial and subsequent measurements: a 20% increase in the initial CRP concentration was associated with a 14% (95% confidence interval (CI): 1-29%) increase in the odds of death, and the hazard of death increased 14% (95% CI: 5-25%) for each 20% increase in the current CRP value. While the initial lactate concentration was not predictive of death, subsequent measurements were. CONCLUSION: CRP, lactate and TnT were associated with poorer outcomes and appear to be useful biochemical markers for monitoring COVID-19 patients.

Vitamin B 12 deficiency and hyperhomocysteinaemia in outpatients with stroke or transient ischaemic attack: a cohort study at an academic medical centre.

OBJECTIVE: We sought to assess the current magnitude of the opportunity for secondary stroke prevention with B vitamins. DESIGN: A cohort study. SETTING: The Urgent TIA (Transient Ischaemic Attack) Clinic at an academic medical centre. MAIN OUTCOME MEASURES: We assessed the prevalence of biochemical vitamin B12 deficiency (B12Def, serum B12 <156 pmol/L), hyperhomocysteinaemia (HHcy; plasma total homocysteine [tHcy] >14 µmol/L) and metabolic B12 deficiency (MetB12Def, serum B12 <258 pmol/L and HHcy) between 2002 and 2017, by age group and by stroke subtype. RESULTS: Data were available in 4055 patients. B12Def was present in 8.2% of patients overall; it declined from 10.9% of patients referred before 2009 to 5.4% thereafter (p=0.0001). MetB12Def was present in 10.6% of patients, and HHcy was present in 19.1% of patients. Among the patients aged ≥80 years, MetB12Def was present in 18.1% and HHcy in 35%. Among the 3410 patients whose stroke subtype was determined, HHcy was present in 18.4% of patients: 23.3% of large artery atherosclerosis, 18.1% of cardioembolic, 16.3% of small vessel disease, 10.8% of other unusual aetiologies and 13.6% of undetermined subtypes (p=0.0001). CONCLUSIONS: Despite a decline in our referral area since 2009, B12Def, MetB12Def and HHcy remain common in patients with stroke/TIA. Because these conditions are easily treated and have serious consequences, all patients with stroke/TIA should have their serum B12 and tHcy measured.

Activation of the dsRNA-Activated Protein Kinase PKR in Mitochondrial Dysfunction and Inflammatory Stress in Metabolic Syndrome.

BACKGROUND: The double stranded RNA (dsRNA)-activated protein kinase PKR is a well-established protein kinase that is activated by dsRNA during viral infection, and it inhibits global protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor 2α (eIF2α). Recent studies have greatly broadened the recognized physiological activities of PKR by demonstrating its fundamental role in inflammatory signaling, particularly in chronic, low-grade inflammation induced by metabolic disorders, known as metaflammation. Metaflammation is initiated by the activation of the NOD-like receptor (NLR), leucine-rich repeat, pyrin domaincontaining 3 (NLRP3) gene by mitochondrial reactive oxygen species (ROS). A protein complex defined as the metaflammasome is assembled in the course of metaflammation. This complex integrates nutritional signaling with cellular stress, inflammatory components, and insulin action and is essential in maintaining metabolic homeostasis. PKR is a key constituent of the metaflammasome and interacts directly with several inflammatory kinases, such as inhibitor κB (IκB) kinase (IKK) and c-Jun N-terminal kinase (JNK), insulin receptor substrate 1 (IRS1), and component of the translational machinery such as eIF2α. CONCLUSION: This review highlights recent findings in PKR-mediated metaflammation and its association with the onset of metabolic syndrome in both human and animal models, with a focus on the molecular and biochemical pathways that underlie the progression of obesity, insulin resistance, and type-2 diabetes.

Assessing the necessity of including a crossover period with dual reporting when changing total prostate-specific antigen methods.

OBJECTIVES: Options are limited for the laboratory when changing analytical methods for tumor markers. Standardization between methods should alleviate this burden. Our objective was to assess the necessity of dual reporting for total prostate-specific antigen (tPSA) when changing methods that have been calibrated against the World Health Organization PSA reference material. DESIGN AND METHODS: Following an initial validation where 40 patient samples were divided and analyzed on the Roche E-modular (current method) and Abbott ARCHITECT (new method) for tPSA, a crossover period ensued with dual reporting of tPSA over 54days (n=1110 samples with both tPSA results reported). Passing & Bablok regression was performed, with agreement during the crossover period also determined by two methods. Method#1: if the percent difference between the result pair was ≤0.33×the intraindividual biological variation (0.33×CVi=6.0%) for tPSA; and Method#2: if the Roche tPSA result was within an acceptable analytical range based on Abbott's maximum allowable imprecision for the tPSA assay (CV≤8%) to determine if there was a range of optimal agreement that might not require dual reporting. RESULTS: During the crossover period, Passing & Bablok regression yielded the following relationship: Abbott tPSA=1.04(95% CI: 1.03 to 1.04)×(Roche tPSA)-0.02(95% CI: -0.03 to -0.02). Only 52% (95%CI: 49 to55) of the results were in agreement by Method#1 whereas 95% (95% CI: 92 to 97) of the results between 3.3 and 19µg/L were found to have acceptable agreement based on Method#2. CONCLUSIONS: These findings suggest that dual reporting of tPSA is required when changing methods. However, the extent of dual reporting may be limited to specific concentration ranges with future studies required to validate selective dual reporting when changing tPSA assays.

Role of cysteine-protease CGHC motifs of ER-60, a protein disulfide isomerase, in hepatic apolipoprotein B100 degradation.

Apolipoprotein B100 (apoB), the structural component of very low density lipoproteins (VLDL), is susceptible to misfolding and subsequent degradation by several intracellular pathways. ER-60, which has been implicated in apoB degradation, is a protein disulfide isomerase (PDI) that forms or rearranges disulfide bonds in substrate proteins and also possesses cysteine protease activity. To determine which ER-60 function is important for apoB degradation, adenoviruses encoding wild-type human ER-60 or a mutant form of human ER-60 (C60A, C409A) that lacked cysteine protease activity were overexpressed in HepG2 cells. Overexpression of wild-type ER-60 in HepG2 cells promoted apoB degradation and impaired apoB secretion, but mutant ER-60 overexpression did not. In McArdle RH-7777 cells, VLDL secretion was markedly inhibited following overexpression of wild-type but not mutant ER-60, an effect that could be blocked by oleate treatment. Mutant ER-60 was not trapped on apoB as it was with the control substrate tapasin, suggesting that ER-60's role in apoB degradation is likely unrelated to its protein disulfide isomerase activity. Thus, ER-60 may participate in apoB degradation by acting as a cysteine protease. We postulate that apoB cleavage by ER-60 within the ER lumen could facilitate proteasomal degradation of the C-terminus of translocationally-arrested apoB.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors.

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia.

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

Fructose: a highly lipogenic nutrient implicated in insulin resistance, hepatic steatosis, and the metabolic syndrome.

As dietary exposure to fructose has increased over the past 40 years, there is growing concern that high fructose consumption in humans may be in part responsible for the rising incidence of obesity worldwide. Obesity is associated with a host of metabolic challenges, collectively termed the metabolic syndrome. Fructose is a highly lipogenic sugar that has profound metabolic effects in the liver and has been associated with many of the components of the metabolic syndrome (insulin resistance, elevated waist circumference, dyslipidemia, and hypertension). Recent evidence has also uncovered effects of fructose in other tissues, including adipose tissue, the brain, and the gastrointestinal system, that may provide new insight into the metabolic consequences of high-fructose diets. Fructose feeding has now been shown to alter gene expression patterns (such as peroxisome proliferator-activated receptor-γ coactivator-1α/ß in the liver), alter satiety factors in the brain, increase inflammation, reactive oxygen species, and portal endotoxin concentrations via Toll-like receptors, and induce leptin resistance. This review highlights recent findings in fructose feeding studies in both human and animal models with a focus on the molecular and biochemical mechanisms that underlie the development of insulin resistance, hepatic steatosis, and the metabolic syndrome.

Apolipoprotein B100 biogenesis: a complex array of intracellular mechanisms regulating folding, stability, and lipoprotein assembly.

Apolipoprotein B100 (apoB) is a large amphipathic lipid-binding protein that is synthesized by hepatocytes and used to assemble and stabilize very low density lipoproteins (VLDL). It may have been derived through evolution from other lipid-associating proteins such as microsomal triglyceride transfer protein or vitellogenin. The correct folding of apoB requires assistance from chaperone proteins in co-translational lipidation, disulfide bond formation, and glycosylation. Any impairment in these processes results in co-translational targeting of the misfolded apoB molecule for proteasomal degradation. In fact, most of the regulation of apoB production is mediated by intracellular degradation. ApoB that misfolds post-translationally, perhaps as a result of oxidative stress, may be eliminated through autophagy. This review focuses on the proposed pentapartite domain structure of apoB, the role that each domain plays in the binding of lipid species and regulation of apoB synthesis, and the process of VLDL assembly. The factors involved in the recognition, ubiquitination, and proteasomal delivery of defective apoB molecules are also discussed.

Glucosamine-induced endoplasmic reticulum stress attenuates apolipoprotein B100 synthesis via PERK signaling.

Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749-1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2alpha. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.

Mechanisms targeting apolipoprotein B100 to proteasomal degradation: evidence that degradation is initiated by BiP binding at the N terminus and the formation of a p97 complex at the C terminus.

OBJECTIVE: In lipid-poor states, the ubiquitin-proteasomal pathway rapidly degrades misfolded apolipoprotein B100 (apoB) cotranslationally, although the mechanism of delivery from the ER to cytosolic proteasomes is poorly understood. Here we demonstrate key roles of BiP, an ER luminal chaperone, and p97, a cytosolic ATPase anchored to the ER membrane, in the targeting of apoB for proteasomal degradation. METHODS AND RESULTS: Using coimmunoprecipitations, we observed associations of apoB with BiP, p97, Derlin-1, VIMP, and the E3 ubiquitin ligase Hrd1 in HepG2 cells. BiP and p97 were found to bind apoB cotranslationally. Expression of C-terminal truncated apoB molecules in COS-7 cells showed an N-terminal region outside apoB15 and a C-terminal region found in apoB72 were required for BiP and p97 binding, respectively. Interestingly, overexpression of dominant negative p97 demonstrated that the ATPase activity of p97 was essential for proteasomal degradation of apoB but not for apoB binding. However, p97 activity did not appear to affect the N terminus of apoB, which may be cleaved before degradation. CONCLUSIONS: These data suggest that p97 and BiP play critical roles in the cotranslational delivery of apoB to proteasomes and formation of a degradative complex. Proteasomal degradation appears to selectively target apoB molecules with large C-terminal domains.

Fructose and the metabolic syndrome: pathophysiology and molecular mechanisms.

Emerging evidence suggests that increased dietary consumption of fructose in Western society may be a potentially important factor in the growing rates of obesity and the metabolic syndrome. This review will discuss fructose-induced perturbations in cell signaling and inflammatory cascades in insulin-sensitive tissues. In particular, the roles of cellular signaling molecules including nuclear factor kappa B (NFkB), tumor necrosis factor alpha (TNF-alpha), c-Jun amino terminal kinase 1 (JNK-1), protein tyrosine phosphatase 1B (PTP-1B), phosphatase and tensin homolog deleted on chromosome ten (PTEN), liver X receptor (LXR), farnesoid X receptor (FXR), and sterol regulatory element-binding protein-1c (SREBP-1c) will be addressed. Considering the prevalence and seriousness of the metabolic syndrome, further research on the underlying molecular mechanisms and preventative and curative strategies is warranted.

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins.

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.