BACKGROUND: Reactive oxygen species (ROS), including superoxide anion (O2) and hydrogen peroxide (H2O2), have been recognized as specific second messengers in signaling cascades involved in the growth and differentiation of cells.The generation of excessive ROS initiates cardiomyocyte apoptosis. This paper is aimed to corroborate the hypothesis that excessive amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4-dependent ROS can suppress cardiomyocyte differentiation from embryonic stem (ES) cells by induction of apoptosis.OBJECTIVE: To investigate the role of NOX4 ROS on cardiomyocyte differentiation.DESIGN: Randomized and controlled trial observation.SETTING: Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health.MATERIALS: ES cells were preserved by the laboratory of Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health, while other reagents were purchased from Sigma Company without specific notification.METHODS: The experiment was carried out in the Beijing Institute of Geriatrics, Beijing Hospital, Key Laboratory of Geriatrics, Ministry of Health from October 2003 to August 2005. ①Mouse ES cells were differentiated into embryoid bodies (Ebs). At day 4, Ebs were managed for one hour with different concentrations (1, 10, 100, 1 000 nmol/L) of H2O2, and generation of cardiomyocytes was observed at day 8 to analyze the effect of ROS on cardiomyocyte differentiation.② CGB8 cells transfected with pcDNA3.1 and pcDNA3.1-NOX4 respectively were adopted, while those untransfected CGB8 cells were taken as controls. A quantitative NBT (nitro blue tetrazolium) test was used to measure NOX4 ROS generation. The levels of NOX4 mRNA and MLC2v protein were assayed by RT-PCR and western blotting, respectively.③ROS scavenger N-acetylcysteine (5 mmol/L) and catalase (200 U/mL) managed for 2 hours before transcription were taken as controls to observe the apoptosis of CGB8 cell after NOX4 overexpressed and the expressions of p21, p53 and Bcl-2 of NOX4-transfected CGB8 cells simultaneously. And the apoptosis of ES cells was detected by Hoechst staining, TUNEL assay and DNA fragmentation.MAIN OUTCOME MEASURES: ①the role of ROS on cardiomyocyte differentiation.②the expression of NOX in ES cells.③ the production of ROS, differentiation of cardiomyocyte and apoptosis of ES cells after NOX4 overexpressed.④the feasibility that p53, p21 and Bcl-2 are involved in the apoptosis induced by the overexpression of NOX4.RESULTS: ①Different concentrations of ROS play different roles on cardiomyocyte differentiation. The exposure of Ebs to 1-100 nmol/L H2O2 for 2 hours at day 4 of culture leaded to an enhanced beating activity (P ＜ 0.01 or 0.001), whereas 1 μmol/L H2O2 depressed cardiomyocyte differentiation as compared with control conditions (P ＜ 0.001). ②NOX4 was highly expressed in ES cells, while NOX1 and NOX2 were absent and only a weak band of NOX3 was detected. The results from RT-PCR revealed that NOX4 overexpressed in CGR8 transfected with pcDNA3.1-NOX4 as compared to the control.③NBT result proved that highly-expressed NOX4 produced the excessive amounts of ROS (P ＜ 0.05). The beating activity was remarkably reduced in NOX4-overexpressing Ebs as compared to wild-type or mock-transfected Ebs (P ＜0.01). Moreover, a severe reduction of MLC2v protein in NOX4-overexpressing Ebs was also observed by western blot analysis.④p21 and p53 may be responsible for the NOX4-induced apoptosis. P53-/- cells R72D27 transfected by NOX4 did not induce apoptosis. NOX4 overexpression induced ES cell apoptosis could be prevented by overexpression of Bcl-2.CONCLUSION: The findings of this paper highlight the role of NADPH oxidase NOX4 in cardiomyocyte differentiation from ES cells. P53, p21 and Bcl-2 could be involved in the apoptotic pathway.

Objective To examine the changes of serum total and high density lipoprotein cholesterol during whole blood incubation and to investigate the possible mechanisms responsible for the instability.Methods A method for the measurement of serum total cholesterol (TC), total free cholesterol (TFC), high density lipoprotein cholesterol (HDLC) and high density lipoprotein free cholesterol (HDLFC) by high performance liquid chromatography (HPLC) was developed. Whole blood specimens were incubated at 25℃ and 4℃ for various period of time and serum TC, TFC, HDLC and HDLFC were measured.Results The new HPLC method showed a mean within-run CV of 0.22%~0.51%. The averaged changes during the incubations ranged 0.6%~2.0% for TC and 2.0%~4.1% for HDLC.Conclusion An HPLC method has been established that is highly precise and can be used for detecting subtle cholesterol changes in biological samples. Different cholesterol exchanges or transfers between blood cells and lipoproteins exist during various incubations. Prolonged whole blood storage that causes serum TC and HDLC changes should be avoided in clinical lipid measurements.

Objective To establish a method for measuring serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and provide an assay that can be applied to theevaluation of the level of smoke exposure and to the risk analysis of smoking related diseases.Methods Blood samples were collected from 94 apparently healthy subjects from October to December in 2010 and centrifuged,and the sera were separated.Serum samples were mixed with [ D3 ] -cotinine ( as the internal standard) and treated with acetonitriles to precipitate protein.After centrifugation,the supernatants were transferred and evaporated under a stream of nitrogen until dryness and reconstituted with mobile phase.Then the residuals were analyzed by LC/MS/MS system with multiple reaction monitor model; the concentration of cotinine were quantified by the isotope internal standard method and the stand curve was employed with a series of calibration.To estimate the precision of the method,five frozen serum pools were repeatedly analyzed in five runs,and every pool was analyzed in triplicate.In addition,the recovery rates were analyzed with the serum sample added with different levels of standard.The stability of cotinine in serum preserved at room temperature,4 ℃ and - 80 ℃,respectively.Finally,the levels of cotinine of 94 healthy subjects were measured to evaluate the distribution of cotinine with different smoke statuses.Results Serum cotinine measured by ID-LC/MS/MS was separated well with few interferences.The correlation coefficients between the peak area ratios and cotinine concentrations were higher than 0.9993.The values of within-run coefficients of variation (CV) of five frozen serum pools (0.68,48.42,94.34,250.95 and 287.04 μg/L) were 2.19％,0.78％,0.75％,0.65％ and 0.67％,respectively.The values of total CV were 4.71％,1.40％,1.98％,1.10％ and 1.03％,respectively.The limit of detection (LOD) and limit of quantitation ( LOQ ) were 0.013 and 0.050 μg/L,respectively.The analytical recoveries ranged from 99.22％ to 102.67％.The samples could maintain stability within 2 d at room temperature,7 d at 4 ℃ and 3 months at -80 ℃ resulting the accuracy of measurements from 99.28％ to 100.87％ and the CV＜5％.The levels of cotinine of 94 healthy subjects were measured and shown skewed and leptokurtic distribution.The concentrations of twenty smokers,fourteen former smokers and sixty non-smokers were 116.40 (63.17 -241.12),0.67 (0.15 - 0.95 ) and 0.22 (0.15 - 0.42 ) μg/L,respectively.Furthermore,the level of cotinine of former smokers (Z =-2.12,P ＜0.05) and smokers (Z =-6.67,P ＜0.001) were statistically higher than non-smokers.Conclusions An ID-LC/MS/MS method for serum cotinine detection has been established.It is hoped that the method will be applied to the assessment of smoke exposure and its association with the risks of smoking related diseases since it is simple,specific,precise,sensitive and accurate.

ObjectiveTo investigate the biological variations ( CVI,CVG ) of serum high-density (HDL2-C,HDL3-C ) and low-density (LDLa-C,LDLb-C ) lipoprotein subfractions and high-density lipoprotein cholesterol esterification rates (FERHDL and MERHDL ).MethodsTwenty healthy volunteers,10 males and 10 females,were recruited for this study from September to October,2010.Blood was collected four times from each individual with a 2-week interval between each sampling.Serum lipoprotein subfraction cholesterol levels were measured by ultracentrifugation/HPLC,FERHDL and MERHDL were measured by HPLC.Within-subject ( CVI) and between-subject ( CVG.) biological variations and quality specifications for precision,bias and total error were calculated.Results The average CVI of this group were 5.5％ and 7.2％ for HDL3-C and HDL2-C,11.2％ and 18.7％ for LDLa-C and LDLb-C,11.95％ and 12.3％ for FERHDL and MERHDL,respectively.The CVG for HDL2-C was 45.5％,much higher than that of HDL3-C (8.7％),and FERHDL(49.5％ ) had a higher CVG than MERHDL (30.6％ ).For each analyte,there was a considerable variation of CVIamongindividuals.ConclusionsBiologicalvariationsof lipoprotein subfractions,FERHDI and MERHDL have been estimated.These rsults will play an important role in quality specifications and cardiovascular disease risk assessment.

Objective To develop an HPLC method for the measurement of n-3 fatty acid index of serum cholesteryl esters.Methods Serum triglycerides were hydrolyzed with ethanolic sodium hydroxide and cholesteryl esters (CEs) were extracted with hexane.The extracted CEs were analyzed by reversed phase HPLC with a UV detection at 205 nm.Cholesteryl eicosapentaenoate and docosahexaenoate ( major n-3 fatty acid cholesteryl esters) were identified by liquid chromatography-tandem mass spectrometry and cholesterol in each CE fraction was measured.Peak areas of CEs were corrected for cholesterol and CE n-3 index was calculated using the corrected peak area and expressed as the percentage of n-3 fatty acid CEs in total CEs.Results The HPLC analysis can be finished in 6 minutes.Triglycerides which interfere with the determination of n-3 fatty acid index, were hydrolyzed with ethanolic sodium hydroxide (4 mol/L) in 30 seconds.The within-run and total CVs for CE n-3 index averaged 0.66% and 0.90%, respectively.CE n-3 indexes of 70 volunteers and 36 coronary heart disease patients apparently healthy subjects and patients with coronary heart disease in Beijing Hospital appeared to be positively skewed and leptokurtic distribution ( skewness = 1.25, kurtosis = 1.70 ).The median of n-3 indices were 0.98% ( 0.37% - 2.40% ).The logarithm of n-3 index appeared to be normal distribution and the average is 0.003 7% with standard deviations of 0.15.The distribution of n-3 indices of gender groups was similar with the total.The medians of females and males were 1.08% (0.60% -2.40%) and 0.95% (0.37% -2.11%) respectively, and the former were significantly higher than the latter( t = - 3.021, P = 0.003 ).Conclusion A new method for the measurement of n-3 index of serum cholesteryl esters by HPLC has been established.It is simple and precise and can be used in predicting cardiovascular diseases risks and monitoring dietary intake of n-3 fatty acids.

Objective To evaluate the analytical performance of 7 homogeneous HDL-cholesterol reagents. Methods An altracentrifugation-HPLC method was used as the comparison method. Fourty fresh patient samples were analyzed by homogeneous methods and the comparison method. The homogeneous methods were all performed on a Hitachi 7170A chemistry analyzer according to the manufacturer's instructions, Precision, accuracy and total errors were analyzed. Results The homogeneous assays typically demonstrated within run coefficient variance(CV) of < 1%, and total CV of < 3%. Methods A, B and D showed average bias, bias at the medical decision points and total errors all within the NCEP performance criteria and method C and F unacceptable biases (-19.74% and 11.46%, respectively) and total errors according to the NCEP criteria. However, all the homogenous methods (A-F) had total errors of < 30%, as required by the US Clinical Laboratory Improvement Amendment (CLIA). Conclusions Homogeneous HDL-C assays have been shown to be reasonably precise, but discrepant results have been observed with some of the assays. Clinical laboratories should pay more attention on selecting and validating homogeneous HDL-C reagents.

Objective To standardize total cholesterol (TC) and HDL cholesterol (HDLC) analytical systems with the US CDC TC reference method and HDLC designated comparison method (DCM). Methods CDC TC reference method and HDLC DCM were set up and the quality was controlled by participating in the CDC Cholesterol Reference Method laboratory Network (CRMLN) bimonthly survey. The performance of 21 TC or HDLC analytical systems from 3 manufacturers were tested with the methods according to the CRMLN certification protocols. Results The coefficient variation (CV) of TC analyses with the reference method in 18 surveys averaged 0.29% and the bias versus CDC target value 0.1%. The DCM HDLC CV in 17 surveys averaged 0.010 mmol/L(0.39 mg/dl) and the averaged biases versus CDC target and group mean were - 0.019 mmol/L (-0.72 mg/dl) and - 0.006 mmol/L (-0.25 mg/dl), respectively. Most of the TC and HDLC analysis events (> 90%) satisfied the CRMLN accuracy and precision criteria for the reference method and DCM. Eighteen of the 21 tested TC or HDLC systems met the performance criteria for analytical systems and were certified for traceability by CDC. Conclusions A reference method for cholesterol and a DCM for HDLC and performed within an international reference laboratory network have been established and used for certification of TC and HDLC analytical systems, Further application of the methods to the standardization of lipid analysis are expected.

Objective To develop an HPLC method for the measurement of fractional and molar esterification rate of serum HDL(FER HDL and MERHDL).Methods Blood samples were mixed with 5,5'-dithiobis-(2-nitrobenzoic acid)(DTNB)and the sera were separated.Serum HDL fractions were prepared by precipitation with Dextran sulfate and magnesium and the fractions were incubated at 37℃for 1 h in the presence and absence of 2-mercaptoethanol(ME).Free cholesterol levels of the HDL fractions were analyzed by HPLC and FERHDL and MERHDL were calculated.Results Under the selected conditions,serum free cholesterol could be stabilized by inhibition of LCAT with DTNB and the inhibition be reversed by ME. The total CVs for FERHDL and MERHDL were 1.59%-3.74% and 1.64%-2.88%,respectively.The averages of FERHDL and MERHDL in 70 apparently healthy subjects were 18.7%/h and 42.7 μmol·L-1·h-1 with standard deviations of 7.2%/h and 11.8 μmol·L-1·h-1·respectively,and the medians were 16.1%/h and 11.8 μmol·L-1·h-1.Close correlations of FERHDL.and MERHDL with other cardiovascular disease risk factors were observed.Conclusion A new method for the measurements of FERHDL and MERHDL by HPLC has been estabished. The method is safe, precise and simple and applications in the assessment of cardiovascular diseases risks are expected.

Objective To prepare serum cholesterol and triglycerides reference materials.Methods Blood units were collected from healthy donors and the sera separated and screened for cholesterol and triglycerides levels.Four serum pools were prepared by pooling sera grouped according to cholesterol and triglyceride levels.The materials were tested for homogeneity and stability and their values for total cholesterol,total glycerol and free glycerol and triglycerides were assigned by HPLC methods.Results The materials were tested to be homogeneous and stable for at least 4 years at-20℃.The certified values (reference value±expanded uncertainty)of the 4 materials for total cholesterol were(5.110±0.065) mmol/L,(4.761±0.062)mmol/L,(3.941±0.050)mmol/L and(3.158±0.041)mmol/L,respectively;for total glycerol(2.212±0.043)mmol/L,(1.679±0.033)mmol/L,(1.275±0.027)mmol/L and(1.067±0.023)mmol/L;for free glycerol(0.142±0.005)mmol/L,(0.149±0.004)mmol/L,(0.146±0.003)mmol/L and(0.122±0.003)mmol/L;and for triglycerides(2.069±0.043)mmol/L,(1.530±0.033)mmol/L,(1.129±0.027)mmol/L and(0.945±0.023)mmol/L.Conclusion Certified reference materials for serum total cholesterol,total glycerol,free glycerol and triglycerides have been prepared.