Canadian guidance for diagnosis and management of acute hepatic porphyrias.

Acute hepatic porphyrias (AHP) comprise four rare monogenic autosomal conditions. Each is linked to a deficiency of heme metabolizing enzymes. Common manifestations include severe abdominal pain, nausea, confusion, hyponatremia, hypertension, tachycardia, and neuropathy. Diagnosis is challenging due to a non-specific, variable presentation with symptoms mimicking other common conditions. Initial diagnosis of AHP can be made with a test for urinary porphobilinogen, δ-aminolevulinic acid and porphyrins using a single random (spot) sample. However, many patients have complications due to delays in diagnosis and management. A novel small interfering RNA-based agent, givosiran, has demonstrated efficacy in reducing acute attacks in a recent Phase III trial, leading to its approval for the management of AHP. Early diagnosis is crucial for the timely introduction of disease-modifying treatments that reduce impairments, enhance quality of life, and extend survival. In this guidance, we aim to improve awareness and outcomes of AHP by making recommendations about diagnosis, monitoring, and treatment in Canada.

Effective stewardship strategies to enhance appropriateness of refer-out test requests in a Canadian tertiary centre laboratory.

OBJECTIVES: Specialized testing conducted in reference laboratories is costly and often not optimally directed. Since 2016, our institution has worked to ensure the appropriateness of refer-out (RO) tests. We examine the impact of utilization initiatives on the patterns of requests and completed tests. DESIGN AND METHODS: In 2016, 81 RO tests were selected for a more rigorous approval process. Physicians not pre-approved for testing received a prompt to consult with laboratory subject matter experts (SMEs) for further detail. After review, SMEs provided responses, approving or rejecting requests based on clinical relevance. Stewardship activities also included: repatriating tests locally, preferring Canadian over foreign institutions, unbundling tests, distributing educational memos, and introducing staged testing. We collected data on the number of requested (NoR) and number of completed (NoC) tests in 2015, before the implementation of the new vetting procedures, and for the post-implementation phase from 2016-2022. RESULTS: For 62 targeted RO tests (including trace metals, vitamins, antibodies, and endocrine-related tests), there was a 33% reduction in NoR and a 51% reduction in NoC in 2022 compared to 2015. The total savings for the study period based on NoC was $807,736. The NoC rate for Neuronal antibody tests decreased to 48.6% in 2022, with cost savings of $17,123, and an additional $50,000 saved by changing the testing site. Insourcing apolipoprotein B and fecal calprotectin tests resulted in cost savings of $3,380 and $3,371, respectively, in 2022. CONCLUSIONS: Automated messaging followed by a formal review of RO test requests is an effective utilization strategy that prevents redundant or clinically unjustified testing. This approach leads to significant economic savings and is expected to improve the efficiency of patient care.

Off-road vehicle crashes: dangers of alcohol and drug impairment.

BACKGROUND: Impaired driving is a public health issue, yet little is known concerning the prevalence of substance use in drivers involved in off-road vehicle crashes. The goal of the present study was to describe the demographics and prevalence of alcohol and drug use in drivers of off-road vehicle crashes. METHODS: In this observational substudy, we collected clinical and toxicological data on all moderately or severely injured off-road vehicle drivers who had blood samples obtained within 6 h of the crash. Clinical data were extracted from patients' medical charts and toxicology analyses were performed for blood alcohol, cannabinoids, recreational drugs, and impairing medications. RESULTS: Thirty-three injured drivers met the inclusion criteria. The mean age was 37.6 ± 13.4 years and 79% were male. Blood alcohol was detected in 58% of drivers and 42% of these were above the legal limit. Tetrahydrocannabinol was positive in 12% of drivers, and 18% were positive for recreational drugs. Opiates were detected in 21% of drivers. Overall, 85% were positive for at least one substance and 39% tested positive for multiple substances. CONCLUSION: This study presents the first evidence of alcohol and drug use in off-road vehicle drivers after cannabis legalization in Canada. Our results show that over half of drivers in off-road vehicle crashes test positive for alcohol and 30% tested positive for THC, cocaine, or amphetamines. Opiates are also commonly detected in off-road vehicle drivers. Emergency department (ED) visits resulting from drug driving of off-road vehicles serve as an opportunity for screening, initiating treatment, and connecting patients to interventions for substance use disorders.

ABSTRAIT: CONTEXTE: La conduite avec facultés affaiblies est un problème de santé publique, mais on sait peu de choses sur la prévalence de la toxicomanie chez les conducteurs impliqués dans des accidents de la route. L'objectif de la présente étude était de décrire la démographie et la prévalence de la consommation d'alcool et de drogues chez les conducteurs de véhicules hors route. MéTHODES: Dans le cadre de cette sous-étude observationnelle, nous avons recueilli des données cliniques et toxicologiques sur tous les conducteurs de véhicules hors route ayant subi des blessures modérées ou graves qui avaient reçu des échantillons de sang dans les 6 heures suivant l'accident. Les données cliniques ont été extraites des dossiers médicaux des patients et des analyses toxicologiques ont été effectuées pour l'alcool dans le sang, les cannabinoïdes, les drogues récréatives et les médicaments pour les facultés affaiblies. RéSULTATS: Trente-trois conducteurs blessés répondaient aux critères d'inclusion. L'âge moyen était de 37,6 13,4 ans et 79 % étaient des hommes. L'alcool dans le sang a été détecté chez 58 % des conducteurs et 42 % d'entre eux dépassaient la limite légale. Le tétrahydrocannabinol était positif chez 12 % des conducteurs et 18 % étaient positifs aux drogues récréatives. Des opiacés ont été détectés chez 21 % des conducteurs. Dans l'ensemble, 85 % étaient positifs pour au moins une substance et 39 % étaient positifs pour plusieurs substances. CONCLUSION: Cette étude présente les premières preuves de la consommation d'alcool et de drogues chez les conducteurs de véhicules hors route après la légalisation du cannabis au Canada. Nos résultats montrent que plus de la moitié des conducteurs de véhicules hors route ont un résultat positif au test de dépistage de l'alcool et 30 % ont un résultat positif au test de dépistage du THC, de la cocaïne ou des amphétamines. Les opiacés sont également couramment détectés chez les conducteurs de véhicules hors route. Les visites aux services d'urgence (SU) découlant de la conduite de véhicules hors route avec facultés affaiblies par la drogue constituent une occasion de dépistage, d'amorcer un traitement et de mettre les patients en contact avec des interventions pour les troubles liés à la consommation de substances.

The analytical performance of six urine drug screens on cobas 6000 and ARCHITECT i2000 compared to LC-MS/MS gold standard.

BACKGROUND: Immunoassays provide a rapid tool for the screening of drugs-of-abuse (DOA). However, results are presumptive and confirmatory testing is warranted. To reduce associated cost and delay, laboratories should employ assays with high positive and negative predictive values (PPVs and NPVs). Here, we compared the results of urine drug screens on cobas 6000 (cobas) and ARCHITECTi2000 (ARCHITECT) platforms for six drugs against LC-MS/MS to assess the analytical performance of these assays. METHODS: Eighty nine residual urine specimens, which tested positive for amphetamine, THC-COOH, benzoylecgonine, EDDP, opiates and/or oxycodone during routine drug testing, were stored frozen until later confirmation by LC-MS/MS. Immunoassays were performed on cobas and ARCHITECT using a split sample. A third aliquot from these samples was tested by LC-MS/MS to assess the percentage of false positive, false negative, true positive and true negative results and calculate the PPVs and NPVs for each immunoassay. RESULTS: The PPVs of THC-COOH and EDDP assays were 100% on both platforms. Suboptimal PPVs were achieved for oxycodone (cobas, 57.1% vs ARCHITECT, 66.7%), amphetamine (77.8 vs. 100%), opiates (80.0 vs. 84.6%) and benzoylecgonine (88.9 vs. 84.2%) assays. The NPV was 100% for cobas and ARCHITECT oxycodone assays. Lower NPVs were achieved for THC-COOH (cobas, 28.6% vs ARCHITECT, 25.0%), EDDP (72.7% for both assays), benzoylecgonine (74.4% vs 73.8%), amphetamine (83.3% vs 82.8%) and opiates (100% vs 85.3%). CONCLUSION: Overall, cobas and ARCHITECT urine drug screens have comparable analytical performance. Confirmatory testing is warranted for positive test results especially for oxycodone, amphetamine, opiates and cocaine. Negative drug screen results must be interpreted with caution especially for THC-COOH, EDDP, benzoylecgonine, amphetamine and opiates.

Multiple pre- and post-analytical lean approaches to the improvement of the laboratory turnaround time in a large core laboratory.

BACKGROUND: Core laboratory (CL), as a new business model, facilitates consolidation and integration of laboratory services to enhance efficiency and reduce costs. This study evaluates the impact of total laboratory automation system (TLA), electric track vehicle (ETV) system and auto-verification (AV) of results on overall turnaround time (TAT) (phlebotomy to reporting TAT: PR-TAT) within a CL setting. METHODS: Mean, median and percentage of outlier (OP) for PR-TAT were compared for pre- and post-CL eras using five representative tests based on different request priorities. Comparison studies were also carried out on the intra-laboratory TAT (in-lab to reporting TAT: IR-TAT) and the delivery TAT (phlebotomy to in-lab TAT: PI-TAT) to reflect the efficiency of the TLA (both before and after introducing result AV) and ETV systems respectively. RESULTS: Median PR-TATs for the urgent samples were reduced on average by 16% across all representative analytes. Median PR-TATs for the routine samples were curtailed by 51%, 50%, 49%, 34% and 22% for urea, potassium, thyroid stimulating hormone (TSH), complete blood count (CBC) and prothrombin time (PT) respectively. The shorter PR-TAT was attributed to a significant reduction of IR-TAT through the TLA. However, the median PI-TAT was delayed when the ETV was used. Application of various AV rules shortened the median IR-TATs for potassium and urea. However, the OP of PR-TAT for the STAT requests exceeding 60min were all higher than those from the pre-CL era. CONCLUSIONS: TLA and auto-verification rules help to efficiently manage substantial volumes of urgent and routine samples. However, the ETV application as it stands shows a negative impact on the PR-TAT.

Evaluation of the impact of a total automation system in a large core laboratory on turnaround time.

BACKGROUND: Growing financial and workload pressures on laboratories coupled with user demands for faster turnaround time (TAT) has steered the implementation of total laboratory automation (TLA). The current study evaluates the impact of a complex TLA on core laboratory efficiency through the analysis of the In-lab to Report TAT (IR-TAT) for five representative tests based on the different requested priorities. METHODS: Mean, median and outlier percentages (OP) for IR-TAT were determined following TLA implementation and where possible, compared to the pre-TLA era. RESULTS: The shortest mean IR-TAT via the priority lanes of the TLA was 22min for Complete Blood Count (CBC), followed by 34min, 39min and 40min for Prothrombin time (PT), urea and potassium testing respectively. The mean IR-TAT for STAT CBC loaded directly on to the analyzers was 5min shorter than that processed via the TLA. The mean IR-TATs for both STAT potassium and urea via offline centrifugation were comparable to that processed by the TLA. The longest mean IR-TAT via regular lanes of the TLA was 62min for Thyroid-Stimulating Hormone (TSH) while the shortest was 17min for CBC. All parameters for IR-TAT for CBC and PT tests decreased significantly post- TLA across all requested priorities in particular the outlier percentage (OP) at 30 and 60min. CONCLUSIONS: TLA helps to efficiently manage substantial volumes of samples across all requested priorities. Manual processing for small STAT volumes, at both the initial centrifugation stage and front loading directly on to analyzers, is however likely to yield the shortest IR-TAT.

An effective utilization management strategy by dual approach of influencing physician ordering and gate keeping.

OBJECTIVES: There is increasing recognition of the importance of appropriate laboratory test utilization. We investigate the effect of a multifaceted educational approach that includes physician feedback on individual test ordering, in conjunction with targeted restriction, on the utilization of selected laboratory tests. DESIGN AND METHODS: Scientific evidence was compiled on the usefulness and limitations of tests suspected of being over utilized in our laboratories. A variety of approaches were used to deliver education on each of the targeted tests, with greater focus on primary care physicians (PCPs). Feedback on requesting behavior of these tests was also communicated to the latter group which included an educational component. Laboratory based restriction of testing was also exercised, including the unbundling of our electrolyte panel. RESULTS: PCP requesting patterns for the selected tests were found to be markedly skewed. The interventions implemented over the study period resulted in a substantial 51% reduction in overall ordering of five of the targeted tests equating to an annual marginal cost saving of $60,124. Unbundling of the electrolyte panel resulted in marginal cost savings that equated annually to $42,500 on chloride and $48,000 on total CO2. CONCLUSIONS: A multifaceted educational approach combined with feedback on utilization and laboratory driven gate-keeping significantly reduced the number of laboratory tests suspected of being redundant or unjustifiably requested. Laboratory professionals are well positioned to manage demand on laboratory tests by utilizing evidence base in developing specific test ordering directives and gate-keeping rules.

Pushing the limits of chemistry point-of-care testing for the management of patients under investigation for Ebola virus disease.

BACKGROUND: With the recent outbreak in West Africa, hospitals worldwide have been developing protocols for suspect of cases of Ebola virus disease. Patients with Ebola virus disease present with a severe gastroenteritis leading to dehydration and electrolyte abnormalities and as such, routine chemistry analysis is essential for patient management. While point-of-care testing can be used with additional precautions for rapid chemistry analyses in a laboratory setting, significant delays could ensue before specimens arrive to the laboratory. This study evaluated the stability of eight chemistry analytes up to 4 h post-collection. METHODS: Blood was collected by venipuncture from 20 healthy volunteers and tested at times 0, 30, 60, 90, 120 and 240 h. Approximately 100 µl of blood was dispensed into a CHEM 8+Cartridge and processed on a model 300 i-STAT 1 Analyzer (Abbott Point of Care Inc.) and ANOVA was used to assess statistical significant difference from the initial time point. RESULTS: While the manufacturer recommends testing within 30 min of collection, no significant variation was observed for most analytes with time points extending up to 4 h. In contrast, glucose concentrations decreased significantly (P < 0.0001) over time at an average rate of 0.0032 mmol/L per min. CONCLUSIONS: This study provides supporting data suggesting that delays up to 4 h can be tolerated, giving ample time for collection and transport of specimens to the clinical laboratory. For glucose, POC testing could still be used, taking into account the collection time and the average rate of decrease.

Loss of expression of DNA mismatch repair proteins is rare in pancreatic and small intestinal neuroendocrine tumors.

CONTEXT: Two recent studies have identified a high rate of microsatellite instability (MSI) in pancreatic neuroendocrine tumors (pNETs). Microsatellite instability is rare in small intestinal neuroendocrine tumors (NETs). It is unclear why there is discordance in the frequency of MSI in the 2 studies of pNETs and why this mechanism is comparatively rare in small intestinal tumors. Loss of expression of DNA mismatch repair (MMR) proteins, which is known to correlate strongly with MSI, is not well studied in pancreatic or small intestinal NETs. OBJECTIVE: To determine if there is loss of expression of MMR protein expression in pancreatic or small intestinal NETs. DESIGN: Sixty-nine patients (31 male, 38 female; mean age, 59.2 years) were identified who had a resection for a primary pancreatic (n  =  35) or primary small intestinal (n  =  34) NET during an 18-year period. Immunohistochemical stains for MLH1, MSH2, MSH6, and PMS2 were applied to archived tissue from all cases. All pNETs with adequate tissue (n  =  32) were also assessed by MSI analysis. RESULTS: There was preserved expression of MLH1, MSH2, MSH6, and PMS2 in all 35 pNETs. Of 32 pNETs tested by polymerase chain reaction, 28 were microsatellite stable and DNA did not amplify in 4. In 34 small intestinal NETs, 2 cases had indeterminate MLH1 and 1 case had indeterminate PMS2 expression. The remainder had intact MMR protein expression. CONCLUSION: Defects in DNA MMR proteins are rare in pancreatic and small intestinal NETs, raising doubt that MSI plays a significant role in the pathogenesis of these tumors.

Bone turnover markers in the management of postmenopausal osteoporosis.

Osteoporosis is the most common cause of fragility fractures. Bone remodelling is essential for repairing damaged areas within bone to preserve bone strength and for assisting in mineral homeostases. In young adults, bone remodelling is usually balanced with approximately as much bone replaced as is removed during each remodelling cycle. However, when remodelling becomes accelerated in combination with an imbalance that favours bone resorption over formation, such as during menopause, precipitous losses in bone mass occur. Bone turnover markers (BTMs) measure the rate of bone remodelling allowing for a dynamic assessment of skeletal status and hold promise in identifying those at highest risk of rapid bone loss and subsequent fracture. Further, the use of BTMs to monitor individuals administered osteoporosis therapy is attractive as monitoring anti-fracture efficacy with bone mineral density has significant limitations. This review details remodelling biology, pre-analytical and analytical sources of variability for BTMs, describes the most commonly used resorption and formation markers, and offers some guidelines for their use and interpretation in the laboratory and the clinic.

Apolipoprotein E-polymorphism, frailty and mortality in older adults.

Although apolipoprotein E (ApoE) polymorphism is associated with variable risks of several illnesses, and with mortality, no persuasive relationship has been demonstrated with frailty. Here, the clinical examination cohort (n=1452 older adults, aged 70+ years at baseline) of the Canadian Study of Health and Aging was evaluated, with 5-year follow-up data. Frailty was defined using both the phenotypic definition from the Cardiovascular Health Study (Frailty-CHS) and the 'Frailty Index', from which age-specific trajectories of deficit accumulation can be estimated. In age-sex adjusted analyses, people with ApoE 4 allele had a higher risk of death (hazard ratio [HR]=1.20; 95% confidence interval: 1.01-1.45), but this relationship was not significant when adjusted for cognitive impairment (1.06; 95% confidence interval: 0.88-1.27). There was no association between frailty and ApoE polymorphism, defined in age-sex adjusted models either as Frailty-CHS (ApoE4 HR 1.17; 95% confidence interval: 0.98-1.40, frailty HR 1.37; 95% confidence interval: 1.28-1.46) or by the Frailty Index (ApoE4 HR 1.07; 95% confidence interval: 0.90-1.29, frailty HR 35.3; 95% confidence interval: 20.4-61.1). The data do not support an association between ApoE polymorphism and frailty. This result did not depend on how frailty was defined.

Effect of homocysteine concentration in early pregnancy on gestational hypertensive disorders and other pregnancy outcomes.

BACKGROUND: Increased total homocysteine (tHcy) may be associated with placental-mediated adverse pregnancy outcomes, but few prospective studies have measured tHcy before pregnancy outcome. This study was undertaken to determine whether increased tHcy measured in early pregnancy is associated with pregnancy loss, gestational hypertension (GH), preeclampsia, or small for gestational age (SGA) infants. METHODS: We conducted a prospective cohort study between 2002 and 2005. We measured tHcy and serum folate in blood samples from pregnant women (<20 weeks' gestation) and collected detailed pregnancy information through a questionnaire and medical record review. RESULTS: Of the 2119 women included in the study, 103 had a pregnancy loss, 115 had gestational hypertension, 65 had preeclampsia, and 129 had an SGA infant. Subjects with increased tHcy concentrations were at increased risk of pregnancy loss [relative risk (RR) 2.1, 95% CI 1.2-3.6] or preeclampsia (RR 2.7, 95% CI 1.4-5.0) than subjects with lower tHcy concentrations, but increased tHcy concentration was not associated with increased risk of developing GH or having an SGA infant. CONCLUSION: The finding of high tHcy in early pregnancy as a risk factor for pregnancy loss and preeclampsia is consistent with a hypothesis that increased tHcy results in abnormalities of the placental vasculature.

Folic acid improves endothelial dysfunction in type 2 diabetes--an effect independent of homocysteine-lowering.

Diabetes is associated with endothelial dysfunction, which in part may be related to uncoupling of the endothelial nitric oxide (NO) synthase enzyme, thus reducing the availability of NO. As folates may potentially reverse the uncoupling of NO synthase, we wanted to determine whether folic acid supplementation could modulate endothelial function and markers of inflammation in patients with type 2 diabetes without vascular disease. Nineteen patients with type 2 diabetes were treated with folic acid (10mg/day for 2 weeks) versus placebo in a randomized, placebo-controlled, cross-over study with an 8-week washout period between treatments. Fasting endothelium-dependent flow-mediated dilatation (FMD) of the brachial artery, endothelium-independent nitroglycerin-mediated dilatation (NMD), plasma homocysteine, serum lipids, folate, and inflammatory markers (high-sensitivity C-reactive protein, soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, interleukin-18, tumor necrosis factor-alpha) were assessed after each 2-week treatment period. Folic acid supplementation significantly increased folate levels and lowered plasma homocysteine levels. Folic acid significantly improved FMD compared to placebo (5.8 +/- 4.8% vs 3.2 +/- 2.7%, p = 0.02). There were no significant effects of folic acid supplementation on lipids, NMD, or the inflammatory markers. There was no relationship between the change in homocysteine and the improvement in FMD. Thus, 2 weeks of folic acid supplementation can improve endothelial dysfunction in type 2 diabetics independent of homocysteine-lowering, but does not modulate markers of inflammation.

Improved prediction of early-onset coronary artery disease using APOE epsilon4, BChE-K, PPARgamma2 Pro12 and ENOS T-786C in a polygenic model.

OBJECTIVES: Coronary artery disease (CAD) is often polygenic due to multiple mutations that contribute small effects to susceptibility. Since most prior studies only evaluated the contribution of single candidate genes, we therefore looked at a combination of genes in predicting early-onset CAD [apolipoprotein E (APOE) epsilon4, butyrylcholinesterase (BChE) K, peroxisome proliferator-activated receptor gamma2 (PPARgamma2) Pro12Ala and endothelial nitric oxide synthase (ENOS) T-786C]. DESIGN AND METHODS: We examined the frequencies, individually and in combination, of all four alleles among patients with early-onset CAD (n = 150; <50 years), late-onset CAD (n = 150; >65 years) and healthy controls (n = 150, age range 47-93 years). Differences in the proportion of subjects in each group with the given gene combination were assessed and likelihood ratios (LR) were calculated using logistic regression to combine the results of multiple genes. RESULTS: Early-onset CAD patients had increased, but non-significant, frequencies of PPARgamma2 Pro12/Pro12 (P = 0.39) and ENOS T-786C (P = 0.72), while BChE-K was only significantly higher in early-onset CAD patients compared to controls (P = 0.03). There were significantly more APOE epsilon4 alleles alone (P = 0.02) or in combination with BChE-K (P = 0.02) among early-onset CAD patients compared to late-onset CAD ones or controls. When combined, there was a higher prevalence of all four alleles in early-onset CAD (early-onset CAD patients: 10.7%, late-onset CAD patients: 3.3% and controls: 2.7%, P = 0.01). LR for early-onset CAD for a single allele was relatively small (1.08 for PPARgamma2 to 1.70 for APOE epsilon4). This increased to 2.78 (1.44-5.37) when combining all four alleles, therefore increasing the pre-test probability of CAD from 5% to a post-test probability of 12.7%. CONCLUSIONS: While any single mutation causes only a mildly increased LR (none > 1.7), in combination, the likelihood of early-onset CAD increased to 2.78 with four mutations. The genetics of early-onset CAD appear to be multifactorial, requiring polygenic models to elucidate risk.

Effect of cyclooxygenase-2 inhibition with rofecoxib on endothelial dysfunction and inflammatory markers in patients with coronary artery disease.

OBJECTIVES: The aim of this study was to determine whether selective cyclooxygenase-2 (COX-2) inhibition with rofecoxib can modulate endothelial dysfunction and levels of circulating inflammatory markers in patients with established coronary artery disease (CAD). BACKGROUND: Expression of COX-2 is upregulated in atherosclerosis. Thus, it has been hypothesized that COX-2 may contribute to atherogenesis by producing eicosanoids, which mediate vascular inflammation and endothelial dysfunction. METHODS: In a randomized, double-blind, placebo-controlled, parallel-design trial, we studied the vascular effects of rofecoxib on brachial artery vasoreactivity and inflammatory markers in 60 patients with angiographically proven CAD who were taking concomitant low-dose aspirin. Patients were randomly assigned to receive either rofecoxib (25 mg/day; n = 30) or placebo (n = 30) for eight weeks. Brachial artery endothelium-dependent flow-mediated dilation (FMD), endothelium-independent nitroglycerin-mediated dilation (NMD), and inflammatory markers (i.e., high-sensitivity C-reactive protein [CRP], soluble intercellular adhesion molecule-1 [sICAM-1], and soluble interleukin-6 receptor [sIL-6r]) were measured at baseline and after eight-week follow-up. RESULTS: Baseline clinical characteristics were similar in the two groups. After eight weeks of treatment, FMD did not significantly change in either the rofecoxib or placebo group (4.0 +/- 3.0% to 4.0 +/- 3.8% vs. 2.7 +/- 2.7% to 3.1 +/- 2.7%, respectively; p = 0.6 by two-way analysis of variance). Similarly, NMD remained unchanged in both groups. Levels of CRP, sICAM-1, and sIL-6r were not significantly altered in either the rofecoxib or placebo group. CONCLUSIONS: The addition of selective COX-2 inhibition with rofecoxib did not appear to have any favorable or adverse effects on endothelial dysfunction or vascular inflammation in patients with CAD using concomitant low-dose aspirin.

Microsatellite instability in multifocal urothelial carcinoma and effect on BAX and AXIN2.

OBJECTIVES: Urothelial carcinomas have a synchronous or metachronous multifocal pattern of occurrence, questioning their clonal origin. Genetic alterations such as microsatellite instability (MSI) affect various tumors including urothelial cancers. These alterations can affect repeat sequences and cause mutations in coding regions of genes involved in transformation, tumor suppression and apoptosis. Recently, the eight-guanine (G8) and the seven-guanine (G7) repeat sequences of the BAX and AXIN2 genes respectively, were shown altered in different cancers. Since BAX is involved in apoptosis while the AXIN2 is involved in beta-catenin metabolism, a protein involved in cell adhesion and DNA transcription, and due to the multifocal nature of urothelial cancer, we investigated these two genes for alterations in repeat sequences in patients with this cancer. PATIENTS AND METHODS: The eight microsatellites BAT25, BAT26, D2S123, D3S1029, D5S346, D17S588, D17S261, MYCL1 were used to screen 25 tumors from seven patients with eight upper and 17 lower urinary tract carcinomas and compare them to DNA from normal tissue. Regions spanning the G8 and G7 repeat sequences of BAX and AXIN2 were sequenced for mutations including expansion and deletion abnormalities. RESULTS: Six microsatellites were seen altered in one patient with kidney and bladder cancer affecting both tissues when compared to normal DNA albeit not similarly except for MYCL1. There was no change in the BAX G8 or AXIN2 G7 microsatellites. There was no MSI seen in any of the remaining six patients. CONCLUSION: MSI occurs in urothelial cancer, but was not seen to affect the BAX G8 or AXIN2 G7 repeats in this study. However, to determine if MSI affects these genes in these tumors will require a larger study. Moreover, our results suggest that these tumors may have a monoclonal origin with further genetic changes resulting in oligoclonality, or could suggest a similar initiating event leading to a similar initial genetic alteration at different sites with subsequent varying events due to a genetically unstable malignant phenotype.

Relation between butyrylcholinesterase K variant, paraoxonase 1 (PON1) Q and R and apolipoprotein E epsilon 4 genes in early-onset coronary artery disease.

OBJECTIVES: The common K variant of butyrylcholinesterase (BChE-K), an enzyme which metabolizes acetylcholine and organophosphates, has been associated with Alzheimer's disease, especially in the presence of the apolipoprotein E epsilon 4 allele (APOE-epsilon 4). Although APOE-epsilon 4 has been associated with the development of coronary artery disease (CAD), an association between the BChE-K variant and CAD has not been explored. Paraoxonase 1 (PON1), located within HDL, is an enzyme which also metabolizes organophosphates and may be antiatherogenic. The R192 variant of PON1 (PON1-R) has been associated with CAD. DESIGN AND METHODS: To determine whether BChE-K is also associated with premature CAD, we examined the frequency of BChE-K among patients with early-onset CAD (n = 150; < 50 yr) vs. late-onset CAD (n = 150; > 65 yr) by molecular analysis. We also examined the frequency of the PON1-R allele in both groups, and explored whether there was synergism between BChE-K and APOE-epsilon 4, BChE-K and PON1-R or PON1-R and APOE-epsilon 4. RESULTS: The frequency of the BChE-K allele tended to be greater among early-onset CAD patients compared to late-onset CAD patients (41.3% vs. 31.3%; p = 0.07), but without any significant difference between males and females. There was no difference in the prevalence of the PON1-R allele between those with early- or late-onset CAD (46.0% vs. 52.7%; p = 0.25). Twenty-two patients with early-onset CAD had both the BChE-K plus APOE-epsilon 4 alleles (14.7%) compared to 11 late-onset CAD patients (7.3%) (p = 0.04). There was no such association between BChE-K and PON1-R, nor PON1-R and APOE-epsilon 4. CONCLUSIONS: Our study suggests that there is a minor association between BChE-K and early-onset CAD, especially in the presence of the APOE-epsilon 4 allele.

Stability of the Bax (G)8 microsatellite in localized prostatic adenocarcinoma.

OBJECTIVES: Microsatellite instability has been found in a variety of tumors including prostate cancer. Bax, a pro-apoptotic protein from the Bcl-2 family of proteins, has a microsatellite composed of an eight deoxyguanine [(G)8] tract located in exon 3. Prostate carcinoma cells have increased proliferation indices and lower levels of apoptosis when compared to benign tissue. We investigated whether instability in the Bax (G)8 microsatellite contributes to loss of apoptotic control in localized prostate cancer. PATIENTS AND METHODS: Thirty-eight patients undergoing radical prostatectomy for localized prostate carcinoma participated in this study. Prostate carcinoma was microdissected, and polymerase chain reaction amplification of a region containing the (G)8 microsatellite was performed on DNA from peripheral blood leukocytes and tumors, followed by single strand conformational polymorphism (SSCP) analysis and direct DNA sequencing. RESULTS: SSCP analysis showed no alteration in the number of bands detected upon comparison of tumor tissue to leukocytes, suggesting no alterations in the microsatellite. This was confirmed by direct sequencing, which demonstrated a normal (G)8 sequence in each case. CONCLUSION: We conclude that the Bax (G)8 microsatellite is stable in localized stage T2 and T3 prostate cancer. Our findings argue against a mutator phenotype pathway leading to loss of apoptotic control in localized prostate cancer.