Chuanbao Zhang

Association of branched-chain amino acids with carotid intima-media thickness and coronary artery disease risk factors.

Background Recent studies have determined that branched-chain (BCAAs) and aromatic (AAAs) amino acids are strongly correlated with obesity and atherogenic dyslipidemia and are strong predictors of diabetes. However, it is not clear if these amino acids are capable of identifying subjects with coronary artery disease (CAD), particularly with subclinical atherosclerosis who are at risk of developing CAD. Methods Four hundred and seventy two Chinese subjects (272 males and 200 females, 42–97 y of age) undergoing physical exams were recruited at random for participation in the cross-sectional study. Serum BCAAs and AAAs were measured using our previously reported isotope dilution liquid chromatography tandem mass spectrometry method. Bilateral B-mode carotid artery images for carotid intima-media thickness (cIMT) were acquired at end diastole and cIMT values more than 0.9 mm were categorized as increased. Correlations of BCAAs with cIMT and other CAD risk factors were analyzed. Results BCAAs and AAAs were significantly and positively associated with risk factors of CAD, e.g., cIMT, BMI, waist circumference, blood pressure, fasting blood glucose, TG, apoB, apoB/apoAI ratio, apoCII, apoCIII and hsCRP, and were significantly and negatively associated with HDL-C and apoAI. Stepwise multiple linear regression analysis revealed that age (β = 0.175, P<0.001), log BCAA (β = 0.147, P<0.001) and systolic blood pressure (β = 0.141, P = 0.012) were positively and independently associated with cIMT. In the logistic regression model, the most and only powerful laboratory factor correlated with increased cIMT was BCAA (the odds ratio of the fourth quartile compared to the first quartile was 2.679; P = 0.009). Conclusion BCAAs are independently correlated with increased cIMT. This correlation would open a new field of research in the mechanistic understanding and risk assessment of CAD.

Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum

Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5min. Analytical recoveries were in the range of 92.8-106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2-3.8% and 1.5-7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC-MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.

Commutability of Possible External Quality Assessment Materials for Cardiac Troponin Measurement

Background The measurement of cardiac troponin is crucial in the diagnosis of myocardial infarction. The performance of troponin measurement is most conveniently monitored by external quality assessment (EQA) programs. The commutability of EQA samples is often unknown and the effectiveness of EQA programs is limited. Methods Commutability of possible EQA materials was evaluated. Commercial control materials used in an EQA program, human serum pools prepared from patient samples, purified analyte preparations, swine sera from model animals and a set of patient samples were measured for cTnI with 4 assays including Abbott Architect, Beckman Access, Ortho Vitros and Siemens Centaur. The measurement results were logarithm-transformed, and the transformed data for patient samples were pairwise analyzed with Deming regression and 95% prediction intervals were calculated for each pair of assays. The commutability of the materials was evaluated by comparing the logarithmic results of the materials with the limits of the intervals. Matrix-related biases were estimated for noncommutable materials. The impact of matrix-related bias on EQA was analyzed and a possible correction for the bias was proposed. Results Human serum pools were commutable for all assays; purified analyte preparations were commutable for 2 of the 6 assay pairs; commercial control materials and swine sera were all noncommutable; swine sera showed no reactivity to Vitros assay. The matrix-related biases for noncommutable materials ranged from −83% to 944%. Matrix-related biases of the EQA materials caused major abnormal between-assay variations in the EQA program and correction of the biases normalized the variations. Conclusion Commutability of materials has major impact on the effectiveness of EQA programs for cTnI measurement. Human serum pools prepared from patient samples are commutable and other materials are mostly noncommutable. EQA programs should include at least one human serum pool to allow proper interpretation of EQA results.

A rapid and precise method for quantification of fatty acids in human serum cholesteryl esters by liquid chromatography and tandem mass spectrometry

We described a rapid and precise method for simultaneous quantification of eleven fatty acids in human serum cholesteryl esters (CEFAs) by liquid chromatography and tandem mass spectrometry (LC-MS/MS). After extraction of serum lipids with isopropanol, CEFAs were separated on reversed phase liquid chromatography and detected by mass spectrometry in positive ion mode with multiple reaction monitor. Individual CEFA was quantified by peak area normalization method and expressed as molar percent of total CEFAs. The run time was less than 5 min and detection limits were from 0.31 to 14.50 × 10(-5)mmol/L. Recoveries of the CEFAs ranged from 91.85% to 104.83% with a mean of 99.12%. The intra and total CVs for the measurement of CEFAs were 0.87-7.70% and 1.02-7.65%, respectively. This LC-MS/MS method required no internal standards, eliminated natural isotope interferences, and provided reproducible and reliable results for 11 major CEFAs in human serum. This method can be used in monitoring and evaluating dietary fatty acid intake. Additional studies are needed to evaluate the associations between serum CEFAs and cardiovascular disease risk factors.

Serum cholesterol measured by isotope dilution liquid chromatography tandem mass spectrometry.

Abstract Background: Accurate cholesterol measurements are essential for the prevention and management of cardiovascular diseases. Quality assurance of cholesterol measurements requires reference methods. Methods: An isotope dilution liquid chromatography mass spectrometric (ID/LC/MS) method was developed. Serum samples were sampled volumetrically using automated dilutors, treated with potassium hydroxide and equilibrated with 3,4-13C2 cholesterol. The natural cholesterol and the internal standard were extracted with hexane and oxidized to cholest-4-en-3,6-diones with chromic acid. The oxidation products were separated on reversed phase LC and detected by tandem MS. The method was calibrated using aqueous cholesterol calibrators and the calibration function was established with a polynomial regression. Results: The correlation coefficients of the calibration curves were always >0.9999. The coefficients of variation (CV) of the volumetric sampling and the LC/MS analysis averaged 0.22% and 0.50%, respectively, and the total measurement CV was 0.60%. Other sources of measurement uncertainty were minor. Results on certified reference materials agreed within 1% of the certified values. Conclusions: An ID/LC/MS method for serum cholesterol has been developed. The method is simple and accurate and may be used as a candidate reference method.

Performance of electrolyte measurements assessed by a trueness verification program

Abstract Background: In this study, we analyzed frozen sera with known commutabilities for standardization of serum electrolyte measurements in China. Methods: Fresh frozen sera were sent to 187 clinical laboratories in China for measurement of four electrolytes (sodium, potassium, calcium, and magnesium). Target values were assigned by two reference laboratories. Precision (CV), trueness (bias), and accuracy [total error (TEa)] were used to evaluate measurement performance, and the tolerance limit derived from the biological variation was used as the evaluation criterion. Results: About half of the laboratories used a homogeneous system (same manufacturer for instrument, reagent and calibrator) for calcium and magnesium measurement, and more than 80% of laboratories used a homogeneous system for sodium and potassium measurement. More laboratories met the tolerance limit of imprecision (coefficient of variation [CVa]) than the tolerance limits of trueness (biasa) and TEa. For sodium, calcium, and magnesium, the minimal performance criterion derived from biological variation was used, and the pass rates for total error were approximately equal to the bias (<50%). For potassium, the pass rates for CV and TE were more than 90%. Compared with the non homogeneous system, the homogeneous system was superior for all three quality specifications. Conclusions: The use of commutable proficiency testing/external quality assessment (PT/EQA) samples with values assigned by reference methods can monitor performance and provide reliable data for improving the performance of laboratory electrolyte measurement. The homogeneous systems were superior to the non homogeneous systems, whereas accuracy of assigned values of calibrators and assay stability remained challenges.

Quantification of hemoglobin A1c by off-line HPLC separation and liquid chromatography-tandem mass spectrometry: a modification of the IFCC reference measurement procedure.

Abstract Background: The quality of hemoglobin A1c measurement is very important in the management of diabetes. A reference system has been established by the IFCC Working Group on HbA1c Standardization. We did some modification of the IFCC Reference Measurement Procedure with MS detection which significantly decreased the analysis time and improved the precision of the analytes by off-line HPLC separation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. Methods: The samples were prepared and enzymatically cleaved according to the IFCC HbA1c reference measurement procedure. Then the digest solution was injected to a reversed phase HPLC for purification and a clean fraction was collected. The fraction was analyzed by liquid chromatography-tandem mass spectrometry with an isocratic elution on a C18 column. Liner regression was used to determine the concentration of HbA1c. Results: The total analysis time which includes the off-line HPLC separation and the LC/MS/MS analysis was reduced by at least 65% compared to the existing IFCC method. The transitions of m/z 348.4→237.2 and m/z 429.4→245.2 were selected for quantification of non-glycated and glycated hexapeptide. Fifty seven hemolysate samples used in recent 3-years’ IFCC HbA1c inter-laboratory studies (2012–2014) were analyzed by the LC/MS/MS procedure for method validation. The CVS were between 0.16% and 1.87% for 4 measurements per sample in the concentration range for HbA1c between 30.4 and 145.8 mmol/mol. The relative bias of the LC/MS/MS method was varied from -3.21% to 2.47% compared to the IFCC network assigned values. Conclusions: This method is an efficient and reliable procedure for the determination of HbA1c. After thorough evaluation within the IFCC Network this modification may be implemented in the IFCC RMP for HbA1c with MS detection.

Assessment of enzyme measurement procedures in China through a trueness verification program.

BACKGROUND Since 2003, the National Center for Clinical Laboratories (NCCL) has organized a network of reference laboratories and several survey programs to improve standardization in China. METHODS We analyzed the 2015 trueness verification program to assess the status of enzyme measurement standardization. Commutable serum-based materials were prepared and sent to 10 reference laboratories to assign target values for 2 enzymes (alanine aminotransferase-pyridoxal phosphate [ALT-pp] and γ-glutamyltransferase [GGT]) using IFCC reference measurement procedures. RESULTS Analytical performance was assessed for compliance to 3 indexes: trueness (bias), imprecision (CV), and accuracy (total error). Of the 250 participating laboratories, about half (≥124) used heterogeneous systems. More laboratories met the tolerance limit of imprecision than of trueness or accuracy. Except at the lowest concentration, the CV pass rates were >90% for the 2 enzymes. The optimal performance criterion derived from biological variation yielded pass rates for total error (ALT 77%, GGT 80%) that were higher than for bias (ALT 63%, GGT 73%). CONCLUSIONS PT/EQA results for commutable samples can be used to assess trueness against reference measurement procedures. Despite global and national standardization programs, bias remains a critical limitation of current enzyme measurement procedures in China.

Serum triglyceride measurements: the commutability of reference materials and the accuracy of results

Abstract Background We aimed to evaluate the commutability of external quality assessment (EQA) materials, aqueous solutions, and commercial reference materials (calibrators and controls), and the accuracy of routine systems for serum triglyceride measurements. Methods According to the clinical and laboratory standards institute (CLSI) EP14-A3 protocol, we analyzed 43 fresh patient specimens and 32 processed materials including lyophilized samples, human serum pools, liquid reagents, swine sera and aqueous solutions by 14 routine methods (evaluated methods) and an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC/MS/MS) (comparative method). The accuracy of the routine method was evaluated by analyzing the absolute bias, relative bias, and the bias at three medical decision levels based on CLSI EP9-A3. Results Frozen serum samples and swine sera were commutable for all of the assays. The EQA/PT materials, commercial calibrators and control materials showed matrix effects differently on routine methods. The aqueous glycerol solutions were generally noncommutable for routine method. All except one routine analytical systems met the National Cholesterol Education Program (NCEP) recommended analytical performance guideline analytical quality criteria for total error. Conclusions Matrix effects and calibration biases existed in measurements of serum triglyceride. Continued efforts are needed to improve the accuracy and comparability of routine measurements.

A candidate reference method for serum calcium measurement by inductively coupled plasma mass spectrometry.

BACKGROUND Calcium is an important serum ion which is frequently assayed in clinical laboratories. Since quality assurance requires reference methods, the establishment of a candidate reference method for serum calcium measurement is important. METHOD An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were spiked gravimetrically with aluminum internal standard, digested with 69% ultrapure nitric acid and diluted to test concentration. Then the (44)Ca/(27)Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators and the calibration function was established with bracketing method. RESULTS The correlation coefficients between the measured (44)Ca/(27)Al ratios and the analyte concentrations ratios were all >0.9999. The coefficients of variation of the measurements were 0.27% and 0.16% for two spiked serum. The analytical recovery was 100.24%. The accuracy of the measurement was also verified through measurement of certified reference materials. Comparison with recognized reference method and international inter-laboratory comparisons gave satisfied results. CONCLUSION New ICP-MS method is specific, precise, simple, and low in cost, and may be used as a candidate reference method in the standardization of serum calcium measurement.