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. The objective was to perform analytical verification of a busulfan immunoassay on the Abbott Alinity c platform. METHODS: The MyCare Oncology busulfan immunoassay was configured as a third-party reagent on the Abbott Alinity c. Imprecision, linearity, sample carryover, and onboard stability of reagent studies were evaluated. The performance of the busulfan immunoassay using the Abbott Alinity c was compared to the Beckman Coulter AU480 using sodium heparinized plasma, as well as to LC-MS/MS using lithium heparinized plasma. RESULTS: The imprecision goal of 8% was met, and linearity within the analytical measurement range of 240 to 1700 ng/mL was verified. Sample carryover was negligible, and the reagents were stable onboard for at least 84 days. The busulfan immunoassay correlated well with LC-MS/MS (slope = 0.949, y-intercept = -7.8 ng/mL, r2 = 0.9935) and the Beckman Coulter AU480 (slope = 1.090, y-intercept = -34.5 ng/mL, r2 = 0.9988). CONCLUSIONS: This study demonstrated successful analytical verification of a busulfan third-party immunoassay on the Abbott Alinity c platform. The ability to perform TDM of busulfan on a routine clinical chemistry analyzer will positively impact turnaround times to improve patient outcomes.

Primary Hyperoxaluria Screening and Monitoring: Quantitative Measurement of Plasma Oxalate by Gas Chromatography-Mass Spectrometry With High Sensitivity.

Background: Plasma oxalate measurements can be used for the screening and therapeutic monitoring of primary hyperoxaluria. We developed a gas chromatography-mass spectrometry (GC-MS) assay for plasma oxalate measurements with high sensitivity and suitable testing volumes for pediatric populations. Methods: Plasma oxalate was extracted, derivatized, and analyzed by GC-MS. We measured the ion at m/z 261.10 to quantify oxalate and the 13C2-oxalate ion (m/z: 263.15) as the internal standard. Method validation included determination of the linear range, limit of blank, limit of detection, lower limit of quantification, precision, recovery, carryover, interference, and dilution effect. The cut-off value between primary and non-primary hyperoxaluria in a pediatric population was analyzed. Results: The detection limit was 0.78 µmol/L, and the linear range was up to 80.0 µmol/L. The between-day precision was 5.7% at 41.3 µmol/L and 13.1% at 1.6 µmol/L. The carryover was <0.2%. The recovery rate ranged from 90% to 110%. Interference analysis showed that Hb did not interfere with plasma oxalate quantification, whereas intralipids and bilirubin caused false elevation of oxalate concentrations. A cut-off of 13.9 µmol/L showed 63% specificity and 77% sensitivity, whereas a cut-off of 4.15 µmol/L showed 100% specificity and 20% sensitivity. The minimum required sample volume was 250 µL. The detected oxalate concentrations showed interference from instrument conditioning, sample preparation procedures, medications, and various clinical conditions. Conclusions: GC-MS is a sensitive assay for quantifying plasma oxalate and is suitable for pediatric patients. Plasma oxalate concentrations should be interpreted in a clinical context.

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. To help overcome this, we present a simple and accurate LC-MS/MS method for the quantification of both serum and urine aldosterone using a novel enzymatic hydrolysis procedure. METHODS: Serum and urine aldosterone was extracted and measured by LC-MS/MS. Urine-conjugated aldosterone glucuronide was hydrolyzed using a genetically modified glucuronidase enzyme. The assay precision, accuracy, limit of quantification, recovery, and carryover were evaluated and the new assay cut-offs were proposed. RESULTS: The liquid chromatography method allowed for adequate separation of the aldosterone peak from closely eluting peaks. Significant in vitro aldosterone loss was observed during acid-catalyzed hydrolysis of urine, which was corrected with the addition of the internal standard to the urine before the hydrolysis step. Glucuronidase catalyzed hydrolysis of urine aldosterone glucuronide displays good correlation with the corrected acid-catalyzed hydrolysis. Serum aldosterone showed good agreement with reference values and the consensus range reported for external quality assessment specimens. CONCLUSION: A simple, fast, and highly accurate method for the detection of serum and urine aldosterone has been developed. The proposed novel enzymatic procedure allows for short hydrolysis time and compensates for urine aldosterone loss during the hydrolysis step.

Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents.

OBJECTIVES: Monitoring estradiol (E2) is important for determining the onset of pubertal development as well as in the evaluation of girls with precocious puberty. However, E2 measurement remains an analytical challenge in children, who have lower circulating levels. We developed and evaluated a simple and sensitive LC-MS/MS procedure for serum E2 quantification in pediatric populations and established age- and sex-specific pediatric reference intervals. METHODS: Residual patient serum samples were used to evaluate the analytical performance of our in-house LC-MS/MS E2 assay. The evaluation included accuracy, precision, linearity, functional sensitivity (LLoQ), and method comparison. Age- and sex-specific pediatric E2 reference intervals were also established from a cohort of 405 healthy children (birth to 18 years) recruited with informed consent. Age- and sex-specific differences were assessed, and outliers were removed. Reference intervals were established using the robust method. RESULTS: The assay imprecision was <5.3 %. Assay linearity ranged from 13.7 to 1923.3 pmol/L. The LLoQ corresponding to a CV of 20 % was determined to be 8.9 pmol/L. Bland-Altman analysis revealed a mean bias of 29.3 pmol/L or 9.1 % between our LC-MS/MS E2 assay and an external reference laboratory measuring E2 by LC-MS/MS. CONCLUSIONS: Our LC-MS/MS E2 assay shows acceptable accuracy, precision, functional sensitivity (LLoQ), and linearity for E2 quantification. Our LC-MS/MS E2 assay also showed good agreement with an external reference laboratory measuring E2 by LC-MS/MS. In addition, using CALIPER samples, we established robust age- and sex-specific pediatric E2 reference intervals to improve accuracy of test result interpretation and clinical decision making.

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. METHODS: Phenylalanine/tyrosine-free whole blood was used to make accurate and stable DBS calibrators. Six calibrators cover the range of 0-1000 µmol/L. Underivatized phenylalanine and tyrosine were extracted and measured by LC-MS/MS. Precision, accuracy, limit of quantification, recovery and carryover were validated. External quality assurance materials were also used to evaluate performance of multi-point calibrations and single-point calibrations. RESULTS: The run time was 4.5-minute. Accuracy analysis showed good agreement with reference materials. Precision, recovery, and the lower and upper limit of quantification met the criteria. When phenylalanine and tyrosine concentrations were less than 150 µmol/L, the 5-point calibration without the calibrator of 1000 µmol/L had the best performance. When the concentrations were > 250 µmol/L, the single-point calibration of 500 µmol/L had the best performance. CONCLUSION: We developed a simple, fast and highly accurate method for the detection of phenylalanine and tyrosine on DBS, with chromatographic separation and underivatized analysis. Based on the calibration performance, a 6-point calibration method is satisfying for this test. An optional dynamic calibration method, which includes 6-point calibration, 5-point calibration and single-point calibration, can further increase test reliability.

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. Serum samples were used to measure 37 amino acids by ultra-performance liquid chromatography, 32 acylcarnitines, as well as free and total carnitine by tandem mass spectrometry, and ß-hydroxybutyrate and free fatty acids using the Vitros 5.1 chemistry analyzer. P ediatric reference intervals were calculated using non-parametric statistics and partitioned based on age- and sex-distributions. RESULTS: Approximately 80% of all analytes required 2 to 4 age-dependent partitions, with over 50% of amino acids and over 70% of acylcarnitines exhibiting significant physiological changes during the neonatal period. Also, 21% of all analytes required partitioning during puberty and adolescence, half of which produced sex-specific distributions. CONCLUSIONS: A comprehensive reference interval database for metabolic disease biomarkers established in this study will improve detection of IEMs by providing appropriate age- and sex-related information in the pediatric population. It will also aid newborn screening programs and guide the management of patients with known metabolic diseases, especially pubertal and adolescent boys and girls that display sex-specific concentrations.

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. However, the interpretation of serum 25(OH)D levels may be complicated by the presence of the C3-epimer of 25(OH)D. We aimed to quantify C3-epi-25(OH)D3 in pregnant women and fetuses, to explore the relationship of the C3-epimer between maternal and cord samples, and to establish whether infant C3-epimer abundance is explained by prenatal formation. METHODS: In a sub-study of a randomized trial of prenatal vitamin D3, 25(OH)D3 and C3-epi-25(OH)D3 were quantified by LC-MS/MS in 71 sets of mother-fetus-infant serum samples, including maternal delivery specimens, cord blood, and infant specimens acquired at 3-28 weeks of age. RESULTS: Without supplementation, median concentrations of C3-epi-25(OH)D3 were higher in infants (6.80 nmol/L) than mothers (0.45 nmol/L) and cord blood (0 nmol/L). However, there was substantial variation such that C3-epi-25(OH)D3 accounted for up to 11% (maternal), 14% (cord), and 25% (infant) of the total 25(OH)D3. Supplemental vitamin D3 significantly increased maternal-fetal C3-epi-25(OH)D3, and was a preferential source of C3-epi-25(OH)D3 compared to basal vitamin D, possibly due to C3-epi-cholecalciferol in the supplement. Multivariate regression did not suggest transplacental transfer of C3-epi-25(OH)D3, but rather indicated its generation within the fetal-placental unit from maternally-derived 25(OH)D3. Neither maternal nor fetal C3-epi-25(OH)D3 is accounted for the relatively high concentrations of infant C3-epi-25(OH)D3, suggesting rapid postnatal generation. CONCLUSIONS: C3-epi-25(OH)D3 is present in some pregnant women and fetuses, but does not appear to be efficiently transferred transplacentally. High C3-epimer concentrations in infancy are probably due to postnatal formation rather than fetal stores.

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. METHODS: In vitro studies were used for the dose-response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase). RESULTS: Both compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0-80 µM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content. CONCLUSIONS: The combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

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. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

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. The ion-transition 401.2→365.2 was monitored for 25-OH-D3 and C3-epi-25-OH-D3, 407.2→371.3 for d6-25-OH-D3, 413.2→331.2 for 25-OH-D2 and C3-epi-25-OH-D2 and 419.2→337.1 for, d6-25-OH-D2. As a proof-of-principle, 25-OH-D3 and C3-epi-25-OH-D3 were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer. RESULTS: The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D2/3 and <15% for C3-epi-25-OH-D3. C3-epi-25-OH-D3 was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D3 concentrations increased linearly, while C3-epi-25-OH-D3 concentrations remained constant. At 12 months of age, C3-epi-25-OH-D3 concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D3 supplements did not contain appreciable amounts of C3-epi-D3. CONCLUSIONS: The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D3 metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.

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. Serum 24,25VD(3) was highly correlated with 25VD(3) in placebo- and VD(3)-treated subjects at each time point (p<0.0001). At wk 2, the 24,25:25VD(3) ratio was lower with VD(3) than with placebo (p=0.035). From wk 2 to wk 6, the 24,25:25VD(3) ratio increased with the VD(3) supplement (p<0.001) but not with placebo, such that at wk 6 this ratio did not significantly differ between groups. After correcting for potential confounders, we found that 24,25:25VD(3) at wk 2 was inversely correlated to the 25VD(3) increment by wk 6 in the supplemented group (r=-0.32, p=0.02) but not the controls. There is a strong correlation between 24,25VD(3) and 25VD(3) that is only modestly affected by VD(3) supplementation. This indicates that the catabolism of 25VD(3) to 24,25VD(3) rises with increasing 25VD(3). Furthermore, the initial ratio of serum 24,25VD(3) to 25VD(3) predicted the increase in 25VD(3). The 24,25:25VD(3) ratio may therefore have clinical utility as a marker for VD(3) catabolism and a predictor of serum 25VD(3) response to VD(3) supplementation.

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 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS. RESULTS: Total imprecision determined over twenty runs was <6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analogue immunoassay (correlation coefficient 0.667; bias -81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LC-MS/MS (correlation coefficient 0.8996, bias 10%). Agreement was closest with ED method (correlation coefficient 0.9779, bias 2.4%). CONCLUSION: We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum free testosterone. The method is relatively fast, reliable and is suitable for the routine clinical laboratory practice.

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 289.2-->109.1 and 294.2-->113.2 were used to monitor testosterone and testosterone-2,2,4,6,6-d(5), respectively. RESULTS: Functional sensitivity was 0.056 nmol/L (CV 20%). Within-run and total imprecision were 4.6% and 5.2% at 1.3 nmol/L, 2.4% and 4.3% at 11.0 nmol/L, and 1.9% and 1.9% at 23.4 nmol/L respectively. The LC-MS/MS method agreed closely with three automated immunoassays when the concentration of testosterone exceeded 3 nmol/L. However, the immunoassays showed a positive bias at concentrations below 3 nmol/L. CONCLUSION: This method provides a rapid, simple, highly selective and sensitive procedure that can be easily used for determination of serum testosterone in routine clinical laboratories. It measures serum testosterone precisely and accurately at concentrations found in children and adults of both genders.

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. METHODS: Retinol was assessed using mass spectrometry, and retinol-binding protein levels were assessed by ELISA. Retinol levels were correlated with clinical disease activity indices. The CYP26 gene, which plays a key role in retinol metabolism, was examined to define any single-nucleotide polymorphisms (SNP) associations with AS. RESULTS: Retinol levels were significantly lower in the AS cohort than in controls (mean 2.39 +/- 0.88 micromol/l for AS, 3.34 +/- 1.01 micromol/l for controls; p < 0.0001). Retinol-binding protein levels were also lower in AS than controls (AS 4.65 +/- 2.10 microg/l; controls 7.48 +/- 4.87 microg/l; p < 0.001). Serum retinol levels did not correlate with indices of disease activity defined serologically (C-reactive protein, erythrocyte sedimentation rate) or clinically (Bath AS Disease Activity Index, Bath AS Functional Index). Genetic analysis showed that an exonic CYP26C1 SNP (rs11187265) is not associated with AS. CONCLUSION: The hallmark of AS is neo-ossification. AS is associated with abnormal serum levels of retinol, a biochemical factor linked to pathological hyperostosis. Further genetic studies are warranted into the genetic basis of the retinol-AS relationship.

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. Derivatized aldehyde concentrations were determined by gas chromatography-mass spectrometry. In the MA group, we found significantly increased levels of 4-hydroxynonenal and malondialdehyde in the dopamine-rich caudate nucleus (by 67 and 75%, respectively) and to a lesser extent in frontal cortex (48 and 36%, respectively) but not in the cerebellar cortex. Approximately half of the MA users had levels of 4-hydroxynonenal falling above the upper limit of the control range in caudate and frontal cortex. A subgroup of MA users with high brain drug levels had higher concentrations of the aldehydes. Our data suggest that MA exposure in human causes, as in experimental animals, above-normal formation of potentially toxic lipid peroxidation products in brain. This provides evidence for involvement of oxygen-based free radicals in the action of MA in both dopamine-rich (caudate) and -poor (cerebral cortex) areas of human brain.

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. RESULTS: Significant association of the PTPN22 1858T allele with RA was detected in both the Toronto-based RA cohort (P = 1.6 x 10(-6), odds ratio [OR] 1.8) and the Halifax-based RA cohort (P = 9.4 x 10(-4), OR 1.94). Association of the risk allele with RA was not affected by sex, age at disease onset, or the presence of either rheumatoid factor or rheumatoid nodules. No association between the PTPN22 risk allele and Crohn's disease was detected. CONCLUSION: These observations confirm the association of RA susceptibility with the PTPN22 1858T allele. However, the data also reveal a lack of association between this variant and Crohn's disease, suggesting that the PTPN22 1858T allele is a risk allele for multiple, but not all, autoimmune diseases.

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. RESULTS: The SLC22A -TC haplotype was strongly associated ( P < .0001) with CD in the non-Jewish subgroup of this cohort, and the combination of SLC22A -TC homozygosity and one or more of the common CARD15 disease susceptibility alleles engendered a 7.5-fold increase in risk for CD ( P = 9 x 10 -8 ) and a 4.5-fold increase in risk for ileal disease ( P = .001). The risk haplotype showed only a suggestive association with CD in the Jewish subgroup and no association with UC in the cohort or in subgroups stratified by CARD15 genotypes. CONCLUSIONS: The SLC22A -TC haplotype acts together with CARD15 disease susceptibility alleles to increase risk for CD and ileal disease among CD patients but does not contribute to risk for UC in this Canadian cohort. The association of the SLC22A -TC haplotype and CARD15 alleles with ileal disease suggests that these variants have biologically intertwined effects in the pathogenesis of CD.

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. METHODS: Nine hundred eighteen unrelated patients with RA, 507 patients with Crohn's disease, and 623 healthy controls were genotyped for the putatively RA-associated slc2F1 and slc2F2 variants and the Crohn's disease-associated SLC22A4 1672T variant. RESULTS: Neither slc2F1 nor slc2F2 showed evidence for association with RA, the allele frequencies of these variants being significantly different in the Canadian population compared with those reported in the Japanese population, but not significantly different between patients with RA and controls. In addition, associations between the 1672T Crohn's disease risk allele and RA or between the slc2F1-A and slc2F2-T risk alleles and Crohn's disease were not detected in this study cohort, and the latter 2 alleles were not in linkage disequilibrium with the 1672T variant. CONCLUSION: These observations do not support roles for any of the previously identified SLC22A4 disease risk alleles in RA susceptibility in the Canadian population. The slc2F1/slc2F2 risk alleles were not associated with Crohn's disease nor in linkage disequilibrium with the Crohn's disease-associated 1672T variant, and accordingly, also appear to be irrelevant to Crohn's disease susceptibility in the population under study.

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). In a clinical reference set, 37% of individuals with serum B12 less than 200 pmol/L had plasma MMA concentrations within the reference interval (75-378 nmol/L), rendering cobalamin deficiency unlikely. The observation illustrates that MMA assay may be a useful adjunct test in assessing patients with low serum B12.