Analytical performance evaluation of the scanning capillary tube viscometer for measurement of whole blood viscosity.

BACKGROUND: Whole blood viscosity (WBV) is the resistance of blood flow in blood vessels. Increased WBV may be a cardiovascular risk factor. The proper screening of WBV can help the early detection of cardiovascular disease. We investigated the performance of a new scanning capillary tube viscometer (SCTV) for the measurement of WBV. METHODS: We evaluated the total precision of the SCTV for 20 days using three control viscosity materials, and the within-day precision with the whole blood samples of three different individuals. For the linearity evaluation, serial dilutions of a high concentration standard material were used. For the method comparison, the results of the SCTV method were compared to those of Brookfield rotating viscometer on 227 subjects. RESULTS: The SCTV had good within-run and total-run coefficient of variant (CV)s at low-, medium-, and high-concentration samples, at shear rates of 1 and 300 s(-1). The within-day CVs with the three human blood samples were 6.3%, 3.7% and 3.8% at a shear rate of 1s(-1), and 3.2%, 3.0% and 4.1% at a shear rate of 300 s(-1). The SCTV method showed an excellent linearity in the range of 84.9 to 558.2 milliPoise (mP) and 28.8 to 71.0 mP at shear rates of 1 and 300 s(-1), respectively. For the comparison study, the SCTV and the rotating viscometer showed comparable results. CONCLUSIONS: The SCTV showed a stable analytical performance, and was comparable with the rotational viscometer. This new SCTV method can be used in the clinical laboratory for various needs.

Serum lysophosphatidylcholine level is not altered in coronary artery disease.

OBJECTIVES: Lysophosphatidylcholine (LPC) is a promising biomarker for atherosclerosis and phospholipase activity. Serum LPC level in patients with coronary artery disease (CAD) was compared with controls. DESIGN AND METHODS: Eighty five CAD patients and 105 controls were enrolled. For sera from both groups of patients, twelve molecular species of LPC and lipid profile were measured. Associations with CAD were investigated and factors affecting serum LPC level were analyzed. RESULTS: Individual LPC species, inter-species ratio, and the ratio to serum lipids were not associated with CAD. Diabetes was associated with decreased level of LPC 16:1. The ratios of LPC 16:0 to LPC 18:1, LPC 16:0 to 18:2, LPC 18:0 to LPC 18:1, and LPC 18:0 to LPC 18:2 were significantly affected by sex. Current smokers had lower LPC 18:0 to LPC 18:1 ratio. CONCLUSION: Serum LPC level is not altered in patients with CAD proven by coronary angiography.

Comparison of four current 25-hydroxyvitamin D assays.

OBJECTIVES: The performance of recently developed vitamin D total assays (ADVIA Centaur and Elecsys) was compared to that of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LIASON 25-OH Vitamin D total assays. DESIGN AND METHODS: A total of 157 clinical samples and standard reference material (SRM) 972 were analyzed. RESULTS: The correlations of LC-MS/MS with the three immunoassays were acceptable. However, compared to LC-MS/MS, LIAISON and ADVIA Centaur showed negative bias, and Elecsys showed positive bias. There was a lack of agreement among the four methods with only LC-MS/MS results close to the certified values of SRM 972. The prevalence of vitamin D insufficiency (<50 nmol/L) was higher with ADVIA Centaur (51.6%) and LIAISON (52.2%) and lower with Elecsys (37.6%), compared with that of LC-MS/MS (44.6%). CONCLUSION: The new, automated total vitamin D assays show acceptable correlation with LC-MS/MS, and could be used in routine laboratories. However, standardization of vitamin D assays and consideration of assay-specific decision limits should be addressed.

The effect of CYP2C9, VKORC1 genotypes and old age on warfarin pharmacologic sensitivity in korean patients with thromboembolic disease.

The therapeutic dose of warfarin is dependent upon intrinsic patient characteristics that are highly variable. We assessed the effects of CYP2C9, VKORC1 1173 C/T polymorphisms, and old age on warfarin dosing and sensitivity by measuring plasma S-/R-warfarin levels in Korean patients. INR and the plasma S-/R-warfarin concentrations were determined in 58 patients who had the VKORC1 1173C/T CYP2C9 genotypes, were on a long-term anticoagulation regimen with warfarin, and took a daily dose of warfarin. The pharmacokinetic sensitivity of warfarin was significantly higher in the CYP2C9 *1/*3 genotypes than in the CYP2C9 *1/*1 genotypes [ratio of S-warfarin concentration/dose, 0.53 vs. 0.21; p=0.01]. Pharmacodynamic sensitivity in older patients (≥ 75 years) with the CYP2C9 *1/*1 and VKORC1 1173 TT genotypes was significantly higher as compared to younger patients (<75 years) [Ratio of INR/S-warfarin concentration, 4.88 vs. 3.41; p = 0.026]. The CYP2C9*3 allele and old age (≥ 75 years) with the VKORC1 1173 T allele were also associated with increased risk of over-anticoagulation. The increase of over-anticoagulation risk and warfarin sensitivity is related to the CYP2C9*3 allele and old age with the VKORC1 1173 T allele in Korean patients with thromboembolic disease. These findings suggest that a lower initial and maintenance dose should be considered for the patients with CYP2C9 *3 allele and advanced age in this patient population. However, due to the limited number of patients in the study population, our finding needs to be confirmed by a larger, well-controlled study.

Use of tandem mass spectrometry for newborn screening of 6 lysosomal storage disorders in a Korean population.

BACKGROUND: We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. METHODS: We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. RESULTS: The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. CONCLUSIONS: The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.

Reference interval and determinants of the serum homocysteine level in a Korean population.

In this study, we estimated the reference intervals of the serum homocysteine (Hcy) level using two automated immunoassays, and we demonstrated the effects of various factors on the Hcy level in a Korean population. We calculated the gender- and assay-specific reference intervals using the data from 809 healthy Koreans, and we assessed the effects of physiologic and lifestyle factors on the Hcy level. The upper limit was higher in males (19.21 and 19.76 µmol/l) than that in females (14.99 µmol/l and 15.16 µmol/l, AxSym and ADVIA centaur, respectively); the upper limits were comparable between the two assays. Smokers, vitamin nonusers, and persons without regular exercise showed a lower folate level and a higher Hcy level. The risk of hyperhomocysteinemia was significantly associated with the male gender (adjusted OR: 5.705, P-value: 0.008) and with the low folate level group (adjusted OR: 10.412, P-value: 0.002) on the multivariate analysis. The Hcy level was significantly different according to various factors, especially in the gender and folate level. The reference interval should be determined for each ethnic population and for each assay. The appropriate cutoff for assessing the risk for cardiovascular disease or stroke should also be validated in each population.

Newborn screening for galactosemia by a second-tier multiplex enzyme assay using UPLC-MS/MS in dried blood spots.

Galactosemia is one of the most important inherited metabolic disorders detected by newborn screening tests. Abnormal results during screening should be confirmed by enzyme activity assays. Recently, we developed a multiplex enzyme assay for galactosemia in erythrocytes using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, we proposed a second-tier multiplex enzyme assay for galactosemia that can be directly applied to dried blood spots (DBSs). Supernatants from two rehydrated-punched 3.2-mm DBSs were incubated with a reaction mixture containing [¹³C6]galactose, [¹³C2]galactose-1-phosphate, and UDP-glucose as substrates for three galactose-metabolizing enzymes. After a 4-hour incubation, the end products from the combined reaction mixture, [¹³C6]galactose-1-phosphate, UDP-[¹³C2]galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Substrates, products, and internal standards from the mixture of the three enzyme reactions were clearly separated in the UPLC-MS/MS system, with an injection cycle time of 10 min. Intra- and inter-assay imprecisions of the UPLC-MS/MS were 8.4-14.8% and 13.2-15.7% CV, respectively. Enzyme activities in DBSs from 37 normal individuals and 10 patients with enzyme deficiencies were analyzed. DBSs from galactosemia patients showed consistently lower enzyme activities as compared to those of normal individuals. In conclusion, multiplex enzyme assays using UPLC-MS/MS can be successfully applied to DBS analysis. This method allows a fast and effective second-tier test for newborns showing abnormal screening results.

The effects of anti-insulin antibodies and cross-reactivity with human recombinant insulin analogues in the E170 insulin immunometric assay.

BACKGROUND: Insulin assays are affected by varying degrees of interference from anti-insulin antibodies (IAs) and by cross-reactivity with recombinant insulin analogues. We evaluated the usefulness of the E170 insulin assay by assessing IA effects and cross-reactivity with 2 analogues. METHODS: Sera were obtained from 59 type 2 diabetes patients receiving continuous subcutaneous insulin infusion and 18 healthy controls. Insulin levels were determined using an E170 analyzer. To investigate the effects of IAs, we performed IA radioimmunoassays, and analyzed the differences between directly measured insulin (direct insulin) and polyethylene glycol (PEG)-treated insulins (free, IA-unbound; total, IA-bound and unbound insulin). We performed in-vitro cross-reactivity tests with insulin aspart and insulin glulisine. RESULTS: In IA-positive patients, E170 free insulin levels measured using the E170 analyzer were significantly lower than the direct insulin levels. The mean value of the direct/free insulin ratio and IA-bound insulin, which were calculated as the difference between total and free insulin, increased significantly as endogenous IA levels increased. The E170 insulin assay showed low cross-reactivities with both analogues (< 0.7%). CONCLUSIONS: IAs interfered with E170 insulin assay, and the extent of interference correlated with the IA levels, which may be attributable to the increase in IA-bound insulin, and not to an error in the assay. The E170 insulin assay may measure only endogenous insulin since cross-reactivity is low. Our results suggest that the measurement of free insulin after PEG pre-treatment could be useful for beta cell function assessment in diabetic patients undergoing insulin therapy.

Determination of carbohydrate-deficient transferrin levels by using capillary electrophoresis in a Korean population.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). METHODS: We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. RESULTS: The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (κ coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and γ-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). CONCLUSIONS: The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.

Molecular and biochemical characterization of the GALT gene in Korean patients with galactose-1-phosphate uridyltransferase deficiency.

BACKGROUND: Three different types of galactosemia have been described, and the most common form occurs due to a deficiency in the galactose-1-phosphate uridyltransferase (GALT) enzyme activity. METHODS: To investigate the molecular defects of the GALT gene, PCR-direct sequencing was performed with genomic DNA from 18 Korean patients with reduced GALT activity. RESULTS: Of the 18 patients tested, 13 (72.2%) had previously reported variants: Duarte variant (12 patients), p.R201H (1 patient), and g.A1962G. In addition, we identified six novel sequence variations by PCR-direct sequencing: five sequence variations in coding regions (p.H31R, p.L116I, p.Q169H, p.H186P and p.R333R), and one in an intron (g.2621A>G). Of 100 normal individuals tested, 4 were heterozygous for the Duarte variant, which indicates a Duarte allele frequency of 2%. Biochemical characteristics of the novel genetic alterations were determined: enzyme activity for exonic alterations and splicing for intron. CONCLUSION: The genetic constitution of the GALT gene is responsible for galactosemia in the Korean population.

Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed. RESULTS: Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells. CONCLUSIONS: This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

The relationship between Lewis/Secretor genotypes and serum carbohydrate antigen 19-9 levels in a Korean population.

BACKGROUND: The Lewis histo-blood group system consists of 2 major antigens-Lea and Leb-and a sialyl Lewis antigen-carbohydrate antigen (CA) 19-9. We investigated the distribution of Lewis genotypes and evaluated the relationship between the Lewis/Secretor genotypes and the serum level of CA 19-9 in a Korean population to identify whether the serum CA 19-9 levels are influenced by the Lewis/Secretor genotypes. METHODS: The study included 242 individuals who had no malignancies. Lewis genotyping was performed for the 59T>G, 508G>A and 1067T>A polymorphic sites. The Secretor genotype was determined through analysis of the 357C>T and 385A>T polymorphic sites and the fusion gene. Serum CA 19-9 level was analyzed using an electrochemiluminescence immunoassay. RESULTS: Individuals carrying the 3 common genotypes-Le/Le, Le/le(59,508), and Le/le(59,1067)-accounted for 95% of the study population. In the Korean population, the allelic frequencies of Le, Le(59), le(59,508), and le(59,1067) were 0.731, 0.010, 0.223, and 0.035, respectively. We found a significant difference in serum CA 19-9 concentrations among the 9 Lewis/Secretor genotype groups (P<0.001). The serum CA 19-9 levels in subjects with genotype groups 1 and 2 (Le/- and se/se) were higher than those with genotype groups 3-6 (Le/- and Se/-; 15.63 vs 6.64 kU/L, P<0.001). CONCLUSIONS: Le/Le, Le/le(59,508), and Le/le(59,1067) are frequent Lewis genotypes in Koreans. Because serum CA 19-9 levels are significantly influenced by the Lewis/Secretor genotypes, caution is suggested when interpreting the serum CA 19-9 levels.

Estimating the biases of the Korean national cholesterol proficiency test.

It is recommended that clinical laboratories keep the bias of serum total cholesterol analysis at < or =3.0% compared to a reference method. In Korea, national cholesterol proficiency testing has long been available, but there has been little information about the magnitude of analytical bias. The authors calculated the bias of the peer group mean for Korea's national cholesterol proficiency test through an indirect approach that overcomes the potential matrix effect of proficiency test materials. One laboratory was selected among the proficiency test participants to represent Korean laboratories. Total cholesterol levels of six fresh serums spanning a wide range of concentrations were measured by the representative laboratory and three reference laboratories. The relationship between the proficiency test mean and the reference method mean was established by linear regression analysis. The peer group mean of the proficiency test was calculated to have a bias of +2.4 to +2.5% at the medical decision levels. When grouped by instrument and reagent, 29 to 66% of the laboratories showed biases <3.0%. Thus it was determined that the peer group mean of the Korean cholesterol proficiency test has an acceptable level of positive bias. The indirect approach used in this study provides a practical model for estimating cholesterol analytical bias for proficiency testing.

Performance analysis of the ARCHITECT anti-cyclic citrullinated peptide antibody in the diagnosis of rheumatoid arthritis.

BACKGROUND: Anti-cyclic citrullinated peptide (CCP) antibody is emerging as an important diagnostic marker for rheumatoid arthritis (RA). We evaluated the analytical and diagnostic performance of the ARCHITECT anti-CCP (Abbott Diagnostics), a new fully automated chemiluminescent microparticle immunoassay. METHODS: Serum samples from 69 patients with RA and 86 non-RA patients were used to evaluate the performance of the ARCHITECT anti-CCP assay, and the results were compared with those of EliA CCP (Phadia). The optimal cut-off value was calculated using receiver operating characteristic (ROC) curve analysis. RESULTS: Within-run and total imprecision (%CV) of the ARCHITECT anti-CCP were <6% and good linearity was observed over the claimed range. The areas under the ROC curves for the ARCHITECT anti-CCP and EliA CCP were 0.90 and 0.89, respectively. The sensitivity and specificity were 76.8% and 95.3% for the ARCHITECT anti-CCP and 78.3% and 95.3% for EliA CCP using the manufacturer's cut-off thresholds. Both assays showed sensitivity of 84.1% and specificity of 94.2% using the optimal cut-off values. CONCLUSIONS: The analytical performance of the ARCHITECT anti-CCP was satisfactory and diagnostic performance was comparable to that of EliA CCP. The use of optimal cut-off thresholds can yield higher sensitivity with minimal loss of specificity.

[Performance evaluation of the Piccolo xpress Point-of-care Chemistry Analyzer].

BACKGROUND: Point-of-care (POC) tests are used increasingly due to fast results and simple test procedures, which enables rapid diagnosis and therapeutic monitoring. We evaluated the performance of the Piccolo xpress Chemistry Analyzer (Abaxis, USA) a POC chemistry analyzer. METHODS: Fourteen analytes, Na(+), K(+), Cl(-), Ca(2+), total carbon dioxide, AST, ALT, total bilirubin, alkaline phosphatase, blood urea nitrogen, creatinine, albumin, total protein, and glucose; were measured simultaneously with a 100 microL of whole blood sample using a Comprehensive Metabolic Reagent disk. Within-run and total precision and linearity were evaluated according to CLSI EP15-A and EP6-A guidelines, respectively. Comparison with a central laboratory chemistry analyzer was performed using 144 patient samples. RESULTS: The coefficients of variations of within-run and total precision were all within 5% for three levels except for total carbon dioxide, ALT, alkaline phosphatase, total bilirubin, and creatinine in low level, and creatinine in middle level. The results of 14 analytes were linear within a commonly encountered range in clinical samples (r(2)> or =0.98). More than 10% of samples in Na(+), AST, ALT, glucose, BUN did not satisfy CLIA analytical quality requirement. CONCLUSIONS: The Piccolo xpress Chemistry Analyzer can analyze multiple analytes with a minimal amount of whole blood in a short time. It showed an acceptable performance for precision, linearity and comparison with central laboratory analyzer. It can be useful as a screening tests modality in mobile clinics, ambulances, and field clinics for military use, and for pediatric patients from whom enough sample volume is difficult to obtain.

Tacrolimus concentrations in relation to CYP3A and ABCB1 polymorphisms among solid organ transplant recipients in Korea.

BACKGROUND: Cytochrome P450 3A (CYP3A) and the drug transporter P-glycoprotein (P-gp) affect the bioavailability of tacrolimus, the most commonly used immunosuppressive agent in organ transplant recipients. We have determined the genotypic frequencies of the CYP3A and ATP-binding cassette sub-family B member 1 (ABCB1) genes, which encode the CYP3A and P-gp proteins, respectively, in Korean organ transplant recipients and donors, and have assessed the influence of CYP3A and ABCB1 polymorphisms on tacrolimus concentrations. METHODS: Using chip-based MALDI-TOF mass spectrometry, 506 solid organ transplant recipients and 62 corresponding of liver transplant donors were genotyped for CYP3A4*6, CYP3A4*18, CYP3A5*3, CYP3A5P1*3, ABCB1 c.2677G>A/T, and ABCB1 c.3435C>T alleles, and their steady-state blood concentrations of tacrolimus were measured. RESULTS: Frequencies of variant alleles among the transplant recipients were CYP3A5*3 76.8%, CYP3A5P1*3 75.9%, ABCB1 c.2677A/T 52.8%, ABCB1 c.3435T 36.9%, CYP3A4*18 1.9%, and CYP3A4*6 0.3%. The CYP3A5P1*3 allele was strongly linked to the CYP3A5*3 allele (r=0.816). Patients with the CYP3A5*3 and CYP3A5P1*3 alleles showed higher blood tacrolimus concentrations per adjusted dose ratio than did patients with wild-type alleles, among both liver transplant donors and renal transplant recipients. CONCLUSION: The CYP3A5 genotype of the liver is considered to show the most important association with tacrolimus concentrations. Ultimately, genotyping for CYP3A5 may help optimal individualization of immunosuppressive drug therapy for patients undergoing solid organ transplantation.

A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.

BACKGROUND: Many genetic variations of GALK1 have been identified in the patients with galactokinase (GALK1) deficiency. However, the molecular characteristics of GALK1 in individuals with elevated GALK1 activity are relatively unknown. METHODS: We investigated the relationship between elevated GALK1 activity and the molecular GALK1 gene variations, and the molecular mechanism underlying elevated GALK1 activity. PCR products from 63 subjects, without any attenuation of galactose degradation enzymes, were sequenced to screen for nucleotide alterations in the GALK1 promoter. RESULTS: Three nucleotide substitutions were identified: c.-179A>G, c.-27A>C, and c.-22T>C. With respect to the c.-22T>C mutation, GALK1 activity in 13 subjects with the T/C or C/C genotype was significantly higher than those in 50 subjects with the T/T genotype (p < 0.001). The dual luciferase reporter assay in Hep3B cells showed that the luciferase activity with the GALK1 promoter with the c.-22C mutant allele increased approximately 2.5-fold, compared to that with the c.-22T. A specific DNA-protein complex was observed in an electrophoretic mobility shift assay, with slightly higher affinity to c.-22C than to c.-22T. CONCLUSION: The c.-22T>C mutation, which was observed frequently in individuals with elevated GALK1 activity, increased the expression of a reporter gene through enhanced binding of a currently unidentified nuclear protein. These results suggest that the elevated GALK1 activity resulted from enhanced gene expression, due to nucleotide variation within GALK1 promoter.

Electrospray ionization-tandem mass spectrometry analysis of the mycolic acid profiles for the identification of common clinical isolates of mycobacterial species.

Mycolic acids are unique and complex molecular structures found in mycobacterial species. In the present study, we investigated whether electrospray ionization-tandem mass spectrometry (ESI-MS/MS) can be used to identify mycobacterial species based on their mycolic acid profiles. Clinical isolates of Mycobacterium tuberculosis complex and 18 nontuberculosis mycobacterial (NTM) species identified by PCR-restriction fragment length polymorphism (RFLP) or real-time PCR were used for this analysis. Crude lipid extracts were prepared by saponifying 1-2 colonies of individual isolates of mycobacterial species and by chloroform and methanol (2:1, v/v) extraction. ESI-MS/MS in negative ion mode with high cone voltage and collision energy was used for mycolic acid profiling analysis. Combinatorial precursor ion scans of m/z 395, 367, and 339 in the range of m/z 1000-1400 resulted in spectra specific to individual mycobacteria. M. tuberculosis complex and M. pulveris showed major ions by performing precursor ion scans on m/z 395 and 367, while other NTM species showed major ions by performing scans on m/z 367 and 339. The different NTM species examined showed different species dependent mycolic acid profiles. In conclusion, we describe a rapid, reliable, and informative ESI-MS/MS protocol for mycolic acid profiling in mycobacterial species, which allows mycobacterial species to be easily identified in clinical laboratories.

CD36 polymorphism and its relationship with body mass index and coronary artery disease in a Korean population.

BACKGROUND: CD36 is a multifunctional membrane receptor and a cell-adhesion molecule that is expressed in platelets, monocytes/macrophages, microvascular endothelial cells, cardiac monocytes and adipocytes. In this study, we investigated whether genetic polymorphisms of the CD36 gene are associated with risk of coronary artery disease (CAD) in a Korean population. METHODS: PCR and polyacrylamide gel electrophoresis or PCR-restriction fragment length polymorphism assays were performed to analyze the following CD36 gene polymorphisms: a (TG) repeat in intron 3 and the base substitution 478C>T (Pro90Ser). A total of 219 patients with significant CAD and 236 control subjects were examined with regard to their genotypes, lipid profiles and other risk factors for CAD. RESULTS: The frequency of (TG) 11- or 12-repeat homozygotes was significantly higher in male CAD patients than in control men (28.4% vs. 15.7%, OR=2.13, p=0.018). Homozygosity for the (TG) 11- or 12-repeat allele was also significantly associated with a higher body mass index (BMI) compared to non-carriers in 134 control men after controlling for age, smoking and hypertension, and explains a 13% BMI variation observed in this study (p=0.015, analysis of covariance). For the 478C >T mutation, which has been reported to be associated with CD36 deficiency, there was no difference in the frequency of the 478T allele between CAD patients and control subjects. However, the 478T allele was found to be closely linked with a (TG) 11- or 12-repeat allele of intron 3 in the control subjects (chi2=18.88, p<0.001). CONCLUSIONS: The (TG) repeat polymorphism in intron 3 of the CD36 gene is associated with a higher BMI and cardiovascular risk for men in a Korean population.