New Psychoactive Substances: A Canadian perspective on emerging trends and challenges for the clinical laboratory.

The production and use of New Psychoactive Substances (NPS) has skyrocketed over the last decade, causing major challenges for government authorities, public health agencies, and laboratories across the world. NPS are designed to mimic the psychoactive effects of unregulated or controlled drugs, while constantly being modified to evade drug control regulation. Hence, they are referred to as "legal highs", as they are technically legal to sell, possess, and use.

Evaluation of Busulfan as a Third-Party Immunoassay on a Clinical Chemistry Analyzer.

Background: Busulfan is widely used in conditioning regimens to prepare patients for hematopoietic stem cell transplantation. Therapeutic drug monitoring (TDM) is critical due to large inter- and intra-individual variability in busulfan pharmacokinetics, and the risk of adverse consequences of toxicity including hepatic veno-occlusive disease. Busulfan is most commonly measured by liquid chromatography-mass spectrometry (LC-MS/MS), which is not as widely available in clinical laboratories as automated routine clinical chemistry analyzers.

A Novel Enzymatic Hydrolysis Method for Urine Aldosterone Quantification: A Case for Reassessing Clinical Cut-Offs of Primary Aldosteronism.

Background: Primary aldosteronism (PA) is a common endocrine cause of secondary hypertension. The aldosterone/renin ratio is an important tool for PA screening, and dynamic testing in serum or urine is used to confirm the diagnosis. While LC-MS/MS is considered the gold standard for testing, there is significant interlaboratory variability between the extraction procedures, which can impact diagnostic interpretation.

A highly accurate mass spectrometry method for the quantification of phenylalanine and tyrosine on dried blood spots: Combination of liquid chromatography, phenylalanine/tyrosine-free blood calibrators and multi-point/dynamic calibration.

Background: Measurement of quantitative levels of phenylalanine and tyrosine in blood is an essential test for the diagnosis of and monitoring genetic disorders associated with phenylalanine metabolism, such as phenylketonuria (PKU), tyrosinemia, and defects of tetrahydrobiopterin synthesis and recycling. We developed a highly accurate and fast liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantification of phenylalanine and tyrosine on dried blood spot (DBS). We also designed a performance score system to evaluate various calibration methods in matrix matched material.

Dynamic biological changes in metabolic disease biomarkers in childhood and adolescence: A CALIPER study of healthy community children.

Background: Understanding age- and sex-specific biological changes in metabolic disease biomarkers is essential for their appropriate utilization in management of children with inborn errors of metabolism (IEM). The CALIPER program aimed to establish pediatric reference values in healthy community children for common metabolic biomarkers and determine the effects of key covariates including age and sex across the pediatric age.

Methods: A cohort of 500 healthy children and adolescents from birth to 19years were initially recruited to establish pediatric reference intervals according to the CLSI C28-A3 guidelines.

Maternal-fetal-infant dynamics of the C3-epimer of 25-hydroxyvitamin D.

Background: Poor vitamin D status (i.e. low serum 25-hydroxyvitamin D (25(OH)D)) has been associated with adverse clinical outcomes during pregnancy and childhood.

Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines.

Background: Bronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids.

Analytical measurement of serum 25-OH-vitamin D₃, 25-OH-vitamin D₂ and their C3-epimers by LC-MS/MS in infant and pediatric specimens.

Objectives: To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D₃ (25-OH-D₃), 25-OH-vitamin D₂ (25-OH-D₂), and their C3-epimers.

Methods: Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed.

Analytical measurement and clinical relevance of vitamin D(3) C3-epimer.

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D(3) (25(OH)D(3)). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D(3) that has been shown to contribute significantly to 25(OH)D(3) concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D(3) methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D(3) in infant, pediatric, and adult populations.

The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation.

24,25-Dihydroxyvitamin D (24,25VD) is a major catabolite of 25-hydroxyvitamin D (25VD) metabolism, and may be physiologically active. Our objectives were to: (1) characterize the response of serum 24,25VD(3) to vitamin D(3) (VD(3)) supplementation; (2) test the hypothesis that a higher 24,25VD(3) to 25VD(3) ratio (24,25:25VD(3)) predicts 25VD(3) response. Serum samples (n=160) from wk 2 and wk 6 of a placebo-controlled, randomized clinical trial of VD(3) (28,000IU/wk) were analyzed for serum 24,25VD(3) and 25VD(3) by mass spectrometry.

Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry.

Background: Currently there is no reliable method suitable for routine measurement of serum free testosterone (FT).

Aim: To develop such a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/MS) that directly detects and quantifies the FT present in serum.

Methods: Ultrafiltrate testosterone obtained from 0.

Rapid determination of serum testosterone by liquid chromatography-isotope dilution tandem mass spectrometry and a split sample comparison with three automated immunoassays.

Objectives: To develop a rapid convenient-to-implement high performance liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for determination of serum testosterone concentration in routine clinical laboratories.

Methods: Following extraction by organic solvents, an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source was used to separate, detect and quantify serum testosterone. Ion-transitions of m/z 289.

Retinol (vitamin A) and retinol-binding protein levels are decreased in ankylosing spondylitis: clinical and genetic analysis.

Objective: Retinol (vitamin A) plays an important role in bone structure and function. Treatment with retinoids has been associated with bone abnormalities mimicking spondyloarthropathy and diffuse idiopathic skeletal hyperostosis. To determine whether retinol concentrations are altered in patients with ankylosing spondylitis (AS), we examined serum retinol levels in patients with AS and healthy controls.

Levels of 4-hydroxynonenal and malondialdehyde are increased in brain of human chronic users of methamphetamine.

Animal studies suggest that the widely used psychostimulant drug methamphetamine (MA) can harm brain dopamine neurones, possibly by causing oxidative damage. However, evidence of oxidative damage in brain of human MA users is lacking. We tested the hypothesis that levels of two "gold standard" products generated from lipid peroxidation, 4-hydroxynonenal (one of the most reactive lipid peroxidation aldehyde products) and malondialdehyde, would be elevated in post mortem brain of 16 dopamine-deficient chronic MA users compared with those in 21 matched control subjects.

Association of the lymphoid tyrosine phosphatase R620W variant with rheumatoid arthritis, but not Crohn's disease, in Canadian populations.

Objective: A single-nucleotide polymorphism in the PTPN22 gene encoding the lymphoid protein tyrosine phosphatase (Lyp) has recently been identified as a functional variant associated with susceptibility to rheumatoid arthritis (RA), type 1 diabetes, and systemic lupus erythematosus. To determine whether association of this variant (PTPN22 1858T) with RA is reproducible and is also observed in another autoimmune condition, Crohn's disease, we investigated the association between the PTPN22 1858T allele and RA and Crohn's disease in a Canadian population.

Methods: Two RA case-control cohorts representing a total of 1,234 patients and 791 healthy controls as well as a cohort of 455 patients with Crohn's disease and 190 controls were genotyped for the PTPN22 C1858T polymorphism, and genotype frequencies were compared between patients and controls.

SLC22A4 polymorphisms implicated in rheumatoid arthritis and Crohn's disease are not associated with rheumatoid arthritis in a Canadian Caucasian population.

Objective: Single-nucleotide polymorphisms (SNPs) in the SLC22A4 gene encoding the organic cation transporter OCTN1 have been associated with rheumatoid arthritis (RA) in the Japanese population and with Crohn's disease in a Canadian cohort. The RA-associated and Crohn's disease-associated SNPs include, respectively, an intronic variant (slc2F2) and an exonic variant (1672T). We used a case-control approach to investigate the prevalence of these variants in a Canadian RA cohort and to determine whether RA and Crohn's disease share SLC22A4 susceptibility alleles.

A risk haplotype in the Solute Carrier Family 22A4/22A5 gene cluster influences phenotypic expression of Crohn's disease.

Background And Aims: Previously, we identified 2 functionally relevant polymorphisms in the SLC22A4 / 22A5 genes at the IBD5 locus that alter gene/protein function and comprise a 2-allele haplotype ( SLC22A -TC) associated with increased risk for Crohn's disease (CD). Here we examine the contribution of this susceptibility haplotype alone and in combination with CARD15 variants to CD subphenotypes and to susceptibility to ulcerative colitis (UC).

Methods: Phenotype-genotype associations were evaluated in a Canadian cohort including 507 patients with CD, 216 patients with UC, and 352 ethnically matched controls genotyped for SLC22A4 C1672T, SLC22A5 G-207C, and the major CD-associated CARD15 variants.

An improved assay for plasma methylmalonic acid using chemical ionization gas chromatography mass spectrometry.

Objectives: To develop a precise and sensitive assay for methylmalonic acid (MMA) using positive chemical ionization gas chromatography mass spectrometry (CI GC-MS), and to illustrate its clinical utility.

Methods: Using the developed assay, reference intervals were determined with 108 ambulatory individuals, and potential clinical utility examined in 178 consecutive patients with possible cobalamin deficiency (serum B12<200 nmol/L).

Results And Conclusions: Methylmalonic acid measured by CI GC-MS was precise (CV: 4-5%), and sensitive (limit of quantitation: 37 nmol/L).

L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy.

Under conditions of iron overload, which are now reaching epidemic proportions worldwide, iron-overload cardiomyopathy is the most important prognostic factor in patient survival. We hypothesize that in iron-overload disorders, iron accumulation in the heart depends on ferrous iron (Fe2+) permeation through the L-type voltage-dependent Ca2+ channel (LVDCC), a promiscuous divalent cation transporter. Iron overload in mice was associated with increased mortality, systolic and diastolic dysfunction, bradycardia, hypotension, increased myocardial fibrosis and elevated oxidative stress.

Quantitative assay of plasma homocysteine thiolactone by gas chromatography/mass spectrometry.

Enzymatic cyclization of homocysteine forms a reactive thiolactone that may play an important role in its cardiovascular toxicity, but reliable quantitation of the free thiolactone metabolite in physiological fluids has not been reported. We have therefore used a highly selective gas chromatography/mass spectrometry (GC/MS) technique combined with the sensitivity of negative chemical ionization (NCI) to develop a quantitative method for the detection of homocysteine thiolactone (HcyTL) in plasma. To improve accuracy the deuterated isomer d(4)-HcyTL was synthesized and added to plasma as internal standard.