Preparation of Rainbow Trout Bone Oil by Aqueous Enzymatic Methodand Identification by Gas Chromatography-Mass Spectrometry / 分析化学

Fish oil is an important nutrient component in rainbow trout bone, and the optimization of extraction by enzymatic hydrolytic method is of great significance.This study selected the alkaline protease as the hydrolytic enzyme, and optimized the process conditions of enzymatic hydrolysis of rainbow trout fish using single factor analysis method.Effects of several factors on the extraction of fish oil were studied, including the ratio of material to liquid, pH, enzymatic hydrolysis time, enzymatic hydrolysis temperature and amount of enzyme.Fatty acids were identified by gas chromatography-mass spectrometry (GC-MS).Results showed that the optimum extraction parameters of enzymatic hydrolysis were as follows: 2000 U/g alkaline protease, ratio of material to liquid of 1∶1 (w/w), pH 7.5, and extraction at 55℃ for 3h.It was found that the main composition of rainbow trout bone oil was unsaturated fatty acid with the content of 80.4% (w/w).The relative content of monounsaturated fatty acid and polyunsaturated fatty acid was about 76.9% (w/w) and 23.1% (w/w), respectively.The total content of EPA and DHA was 3.4% (w/w).This study optimized the extraction method of rainbow trout fish oil, analyzed and identified the main volatile compounds, and identified the main substances contributing to fish oil flavor.The method thus was of significance for the analysis and identification of fish oil products.

Evaluation of Hcy assay performance by circulating enzymatic method / 国际检验医学杂志

Objective To evaluate analytical performance of homocysteine (Hcy) by circulating enzymatic method .Methods Re‐ferring to CLSI evaluation project and pertinent literature ,and by combining our actual works .we designed a verification procedure and experimental method .By using these above ,the precision ,accuracy ,analytical measurement range ,clinical reportable range of Hcy by circulating enzymatic method were evaluated .Results would be compared with the declaration of the manufacturer (NingBo Medical System Biotechnology Co .,Ltd) or desirable specifications derived from biologic variation .Results The results showed that the within‐run CV were 1 .26% and 0 .84% ,and the total CV were 1 .36% and 1 .32% ,less than 10% of the manufacturer′s statement .The relative bias between the results measured for calibrator at tow levels and target value was 3 .69% and 0 .69% ,less 10% .AMR was 3 .38-51 .81 μmol/L ,and the most suitable dilution rate was 1∶3 ,so the CRR was 3 .38-155 .43 μmol/L .Con‐clusion The analytical performance of Hcy analyzed by circulating enzymatic method is consistent with the standards which manu‐facturers has proclaimed ,so it is conform to the requirements of clinical .

Development of improved enzymatic creatinine reagents / 国际检验医学杂志

Objective To develop improved enzymatic creatinine(Cr)assay reagents (self-R&D),and to investigate their appli-cation on serum detection by comparing with imported commercial Cr reagents(enzymatic Cr reagents from Toyobo)Methods En-zymatic method was used to evaluate the effect of every component and different concentrations of reagents on Cr assay by detecting the alteration of absorbance of Cr before or after the reaction.Meanwhile,the blank absorbance and analysis sensitivity of self-R&D and imported reagents,the technical indicators of precision,linearity,as well as method comparison of self-R&D reagents,were de-tected on the same automatic biochemical analyzer.Results The blank absorbance of self-R&D reagents was 0.009,and the detec-tion sensitivity was 0.13,better than that of imported Cr reagents.The coefficient of variation (CV)of high and low values of ser-um of self-R&D reagents were 1.5%,and 1.1%,respectively.The linear range was 0-2 850 μmol/L and the method comparison result was Y =0.98X +1.15 (r =0.999).The expected bias was less than the allowable error region.Using relative deviation≥10% as an index to evaluate the existence of significant interference,it shows that 35 mmol/L of creatine,3.42 mmol/L of biliru-bin,0.03 g/L of vitamin C,5 g/L of hemoglobin and 1450 FTU chyle in both low and high concentration serum samples did not interfere with the test result.Conclusion The quality of self-R&D reagents was good,and there was a good relativity between self-R&D reagents and imported Cr reagents with excellent quality.This indicates the self-R&D reagents could satisfy the application requirements of the clinics.

Methodological evaluation on enzymatic method for detecting HbA1c and influence of sample pre-processing on detection result / 重庆医学

Objective To evaluate the methodological performance of the new enzymatic method for detecting glycated hemo-globin(HbAIc)and its influencing factors.Methods HbAIc was detected by the enzymatic method.The precision,anti-interfer-ence,recovery rate,accuracy and the influence of pre-processing(anti-coagulation,preservation,centrifugation)on the detection re-sults were evaluated,its correlation with HPLC and the bias degree were analyzed.Results The within-run coefficients of variation (CVs)for high,middle and low value QC samples in the enzymatic assay were 1.04%,1.26% and 1.37% respectively and the be-tween-run CVs were 1.83%,2.24% and 2.64%,respectively;the enzymatic method showed the linear correlation with HPLC(r=0.996,P 0.05);the sample was centrifuged at 500,1 000 r/min(R=15 cm)for different time(1,2,5,10 min)and at 2 000 r/min for 1 min,their detection results had statistical differences compared with the sample centrifuged=3 000 r/min for 5 min (P <0.05).Conclusion The precision,anti-interference,accuracy and linearity range of the enzymatic method all conform to the clinical requirement.Compared with the conventional method,its correlation is good with small deviation,which can entirely satisfy the demand of the HbAIc detection in clinic.

Research progress in molecular modification and pharmacological activity of Chinese materia medica polysaccharides / 中草药

Molecular modification of Chinese materia medica polysaccharides (CMMP) is considered as the technology of modification on some special structure or functional group in the main chain or the side chain of polysaccharides on the purpose of changing certain physicochemical properties and the spatial structure of polysaccharides by using physical, chemical and biological methods which can enhance the biological activity. Physical methods mainly include ultrasonic method and irradiation technology. Chemical methods refer to sulfation, phosphorylation, acetylation, carboxymethylation, alkylation, sulfonylation, selenylation, and so on. Biological method is also called the enzymatic modification containing enzyme degradation and enzymatic synthesis. In recent years, it has been shown that the physicochemical properties and spatial structure of CMMP could be changed after modification, which could make their immunopharmacology activity, such as immune adjustment, antivirus, antitumor, and antioxidant, enhanced obviously. The main modification methods of CMMP and the related pharmacological activity of its products after modification are summarized in this paper.

Study on preparation of butyryl galactose ester-modified coix component microemulsions and their anticancer activity in vitro / 中草药

Objective: To synthesize butyryl galactose ester (But-Gal) and prepare butyryl galactose ester-modified coix component microemulsions (But-Gal-CMEs) and to evaluate its physicochemical properties and anticancer activity in vitro. Methods: But-Gal was synthesized by enzyme-catalyzed reaction and the structure of the product was confirmed by 1H-NMR and FT-IR. The CMEs and But-Gal-CMEs were prepared by aqueous titration method using coix seed oil, Cremophor RH40, PEG400, But-Gal, and coixan solution as oil phase, surfactant, cosurfactant, target ligand, and aqueous phase, respectively. The average particle size, polydispersity index (PDI), and Zeta potential were detected by dynamic light scattering (DLS). The cytotoxicity of CMEs and But-Gal-CMEs aganist HepG2 cells was determined by MTT assay. The cellular uptake of CMEs and But-Gal-CMEs was detected by fluorescence microscopy. Results: The structure of But-Gal was confirmed by 1H-NMR and FT-IR. The But-Gal-CMEs displayed the spherical surface with mean droplet size of (57.68 ± 6.65) nm, PDI of 0.070 ± 0.006, and Zeta potential of (-2.95 ± 0.23) mV, respectively. MTT experiments showed that the half of HepG2 cell proliferation inhibition concentration (IC50) of But-Gal-CMEs and CMEs was 62.55 and 71.23 μg/mL. The HepG2 cell uptake results suggested that the fluorescence intensity of But-Gal-CMEs group was higher than that of CMEs group. Conclusion: The But-Gal-CMEs presents small particle size, good roundness, and good stability. In addition, But-Gal could increase the uptake rate of CMEs in HepG2 cells and enhance the inhibition of HepG2 cell proliferation.

The evaluation analysis of false estimates of elevated serum creatinine in patients with Waldenstrom′s macroglobulinemia through wet chemical enzymatic method / 国际检验医学杂志

Objective To explore causes and solutions of false estimates of elevated serum creatinine in patients with Walden‐strom′s macroglobulinemia throug wet chemical enzymatic method .Methods 5 cases of patients hospitalized in the Bethune Inter‐national Peace Hospital were enrolled as subjects from 2010 to 2012 .The large molecular proteins were removal from serum sam‐ples collected from patients with Waldenstrom′s macroglobulinemia by using centrifugal ultrafiltration tube .The serum creatinine levels were detected through using the wet chemical enzymatic method ,wet chemical picric acid method and dry chemical enzymatic method before and after ultrafiltration ,and data were compared .Results Before ultrafiltration ,the levels of serum creatinine of 2 cases of patients with Waldenstrom′s macroglobulinemia detected by using wet chemical enzymatic method differed with those de‐tected by using wet chemical picric acid method and dry chemical enzymatic method .While there were no obvious differences be‐tween levels of serum creatinine detected by wet chemical picric acid method and dry chemical enzymatic method .While ,after ultra‐filtration ,no obvious differences were founded in levels of serum creatinine detected by the thress methods .Conclusion The large molecular proteins should be eliminated when using the wet chemical enzymatic method in the detection of serum creatinine levels , in order to avoid abnormal increase .And the wet chemical picric acid method and dry chemical enzymatic method could also be uti‐lized to determine the accuracy ,and provide reliable determination results .

Research progress on enzymatic conversion for preparing individual ginsenosides / 中草药

Ginsenosides have antitumor, antiviral, anti-aging, immune enhancement, and other pharmacological activities, but the contents of different individual ginsenosides in herbs and their extracts are different and their pharmacological activities are different. A lot of individual ginsenosides with low contents and strong pharmacological activities are required and prepared to meet the medicinal and scientific needs. To directly improve the contents of individual ginsenosides, enzymatic preparation is a key technology. The extensive research was conducted to obtain the effective individual ginsenosides directly by enzymatic conversion. In this paper, the enzymatic conversion for preparing individual ginsenosides is reviewed to provide a theoretical basis and reference for the further development and utilization of individual ginsenosides.

Medida del colesterol de lipoproteínas de baja densidad utilizando tres metodologías / Measurement of cholesterol low-density lipoprotein using three methodologies / Medida do colesterol de lipoproteínas de baixa densidade utilizando tres metodologias

Tomando en cuenta que aún no existe una metodología estándar de rutina para la determinación del colesterol de lipoproteínas de baja densidad (LDL-c) se decidió evaluar su determinación analítica utilizando tres técnicas: determinación enzimática homogénea, precipitación con sulfato de polivinilo y fórmula de Friedewald. Fueron procesadas 98 muestras de suero a las cuales se les determinó triglicéridos (TG), colesterol total (CT), colesterol de lipoproteínas de alta densidad (HDL-c) y colesterol de lipoproteínas de baja densidad (LDL-c). Los valores promedio de CT fueron 194,46 ± 43,54 mg/dL, HDL-c 51,12 ± 12,36 mg/dL y TG 132,88 ± 76,93 mg/dL. Aun cuando el análisis de regresión mostró una buena correlación entre los valores de LDL-c, los resultados indicaron una diferencia estadísticamente significativa en los mismos cuando los niveles de TG superaron los 200 mg/dL. La misma se observó principalmente entre el método de precipitación y la fórmula de Friedewald, siendo los valores significativamente más bajos en esta última (LDL-c por precipitación: 141,3 ± 26,2 mg/dL; LDL-c por fórmula de Friedewald: 110,1 ± 35,4 mg/dL). De la misma manera se vio afectada la proporción de individuos clasificados según su riesgo coronario. Es necesario comparar las técnicas aplicadas en este estudio con la cuantificación beta para evaluar cuál tiene un mayor nivel de exactitud.

Considering that there is still no standard methodology for routine determination of low density lipoprotein (LDL-c) it was decided to evaluate their analytical determination using three techniques: homogeneous enzymatic determination, polyvinyl sulphate precipitation and Friedewald formula. Ninety-eight serum samples were processed; triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-c) and LDL-c were determined. Mean total cholesterol was 194.46 ± 43.54 mg/dL, HDL-C was 51.12 ± 12.36 mg/dL and TG was 132.88 ± 76.93 mg/dL. Although regression analysis showed a good correlation between LDL-c, the results showed a statistically significative difference in them when TG levels exceeded 200 mg/dL. It was mainly observed in the precipitation method and the Friedewald formula, the latter values being significantly lower (LDL-C by precipitation: 141.3 ± 26.2 mg/dL, LDL-C by the Friedewald formula: 110, 1 ± 35.4 mg/dL). Moreover, this difference affected the proportion of individuals classified according to their coronary risk. It is necessary to compare the techniques applied in this study with beta quantification to assess which has a higher level of accuracy.

Levando em consideragao que ainda nao existe uma metodologia padrao de rotina para a determinagao do colesterol de lipoproteínas de baixa densidade (LDL-c) se decidiu avaliar sua determinagao analítica utilizando tres técnicas: determinagao enzimática homogénea, precipitagao com sulfato de polivinil e fórmula de Friedewald. Foram processadas 98 amostras de soro as quais lhes foi determinado triglicerídeos (TG), colesterol total (CT), colesterol de lipoproteínas de alta densidade (HDL-c) e colesterol de lipoproteínas de baixa densidade (LDL-c). Os valores médios de CT foram 194,46 ± 43,54 mg/dL, HDL-c 51,12 ± 12,36 mg/dL e TG 132,88 ± 76,93 mg/dL. Inclusive quando a análise de regressao mostrou uma boa correlagao entre os valores de LDL-c, os resultados indicaram uma diferenga estatisticamente significativa nos mesmos quando os niveis de TG superaram os 200 mg/dL. A mesma se observou principalmente entre o método de precipitagao e a fórmula de Friedewald, sendo os valores significativamente mais baixos nesta última (LDL-c por precipitagao: 141,3 ± 26,2 mg/dL; LDL-c por fórmula de Friedewald: 110,1 ± 35,4 mg/dL). Da mesma maneira se viu afetada a proporgao de indivíduos classificados conforme seu risco coronariano. É necessário comparar as técnicas aplicadas neste estudo com a quantificagao beta para avaliar qual é que tem maior nível de exatidao.

Dietary Choline Intake of Korean Young Aldults

This study was conducted to investigate the choline intake of Korean adults for the purpose of preparing a basal data required for the establishment of choline adequate intake (AI). The subjects of 56 Korean young adults were recruited from college students of 20 to 30 years old in Daejeon city. The aliquots of foods that the subjects ate for one day were collected with use of duplicate food collection method and choline content of one day meal directly was analyzed with the use of enzymatic method. Choline intakes of male subjects were in the range of 353.5 ~ 1222.5 mg and those of female subjects were in the range of 213.1 ~ 722.3 mg. Mean intakes of choline were 658.2 +/- 243.9 mg/day in male subjects and 423.3 +/- 133.6 mg/day in female, therefore choline intake of men was about 200mg higher than that of women. Median value in total subjects was 496 mg, male's median was 608.8 mg, female's median was 419.9 mg. When the subjects were devided into 4 groups by choline intake, as less than 75%, 75 ~ 100%, 100 ~ 125% and over 125% based on choline AI of USA (males: 550 mg, females: 425 mg), there was no significant difference between men (64.3%) and wemen (67.9%) in the distribution of the subjects whose choline intake is under the range of 75 ~ 125% AI of USA. However, 10.7% of men and 21.4% of female had choline intake less than 75% AI of USA while the cases of choline intake higher than 125% AI were 25% in male and 10.7% in female. Thus, it is assumed that female case in choline-deficient state would be two times more than male. When adjusted by body weight, choline intake was 9.5 +/- 3.4 mg/kg in men, 8.1 +/- 3.1 mg/kg in women and 8.8 +/- 3.3 mg/kg in total subjects. And choline intake per 1,000 kcal of men, women and total subjects were 277.1 +/- 78.4 mg, 275.9 +/- 62.1 mg and 276.5 +/- 70.1 mg respectively. From these results, it is suggested that these levels of 276.5 +/- 70.1 mg/ 1,000 kcal or 8.8 +/- 3.3 mg/kg B.W. can be used as a reference value for the establishment of AI of choline for Korean, because overall choline intake of these subjects was not in lower state compared to other nutrients intakes obtained from calculation of the food the subjects had taken.

Measurement of HDL-cholesterol in serum by a new enzymatic method.

High density lipoprotein (HDL) are responsible for the reverse transport of cholesterol from the peripheral cells to the liver. Here, cholesterol is metabolised. Monitoring of HDL-C in serum is of clinical importance since an inverse correlation exists between serum HDL-C concentrations and the risk of atheroselerotic disease. Elevated HDL-C concentrations are protective against coronary heart disease. In this study, an automated enzymatic method for the direct determination of HDL-C in serum was found to have good precision and accuracy. Reproducibility was determined daily using control precinorm? L in an internal protocol (n=20), within-run CV = 1.3% and between-day CV = 2.013%. The comparison of the HDL-C enzymatic assay with HDL-C precipitating agent was good with a correlation coefficient r = 0.965, Y = 1.981+ 0.959X, n =172. The normal range of HDL-C concentration in serum by this enzymatic method was 36.16 -86.08 mg./dl. Thus, a HDL-C level of over 35 mg/dl., shows the risk is not high and is protective against coronary heart disease.

Measurement of LDL-cholesterol in serum by new enzymatic method.

Low density Lipoprotein (LDL) plays a key role in causing and influencing the progression of atherosclerosis and coronary sclerosis in particular. The LDLs are derived from Very Low Density Lipoproteins (VLDLs). The elimination of LDL from plasma takes place mainly by the liver parenchymal cells via specific LDL receptors. The LDL-cholesterol value is the most powerful clinical predictor among all of the individual parameters with respect to coronary atherosclerosis., Therefore, therapies focusing on lipid reduction primarily target the reduction of LDL-cholesterol which is then expressed in prevention of atherosclerosis. This new enzymatic automated method for direct determinaton of LDL-cholesterol in serum had good precision and accuracy. Reproducibility was determined daily using control precinorm? L in an internal protocol (n = 20), within-run CV = 0.856%, and between-run CV = 2.6%. The comparison of the LDL-C level by enzymatic assayed with the calculated LDL-C using the HDL-C determination by enzymatic assayed and by precipitation, showed a good correlation ( r = 0.989, Y = 0.966X - 7.255, n = 188 and r = 0.977, Y = 0.957X - 7.049, n = 188). The normal range of LDL-C concentration in serum by this enzymatic method was 57.47 - 221.39 mg./dl. Thus,a normal level of LDL-C should be < 200 mg./dl. for good warning. A level > 200 mg/dl warns the doctors that the patient has a high risk of coronary atherosclerosis.

A novel enzymatic method for measuring serum conjugated bilirubin / 中华检验医学杂志

Objective To establish a new enzymatic method for the determination of serum conjugated bilirubin (CB) by the use of bilirubin oxidase (BOD). Method Only the CB was oxidized by BOD under the conditions of pH 5.5 in the presence of sodium fluoride NaF and N-acetylcysteine (NAC) which could prevent the unconjugated and delta bilirubin oxidized. CB was quantitatively determined from a decrease in the absorbance at 450 nm caused by CB oxidization. Results The intra-assay CVs were 6.52% and 0.33% (n=20) at CB concentrations of 17.65 and 301.49 ?mol/L, respectively. The inter-assay CVs were 9.90% and 2.72% at 31.50 and 184.12 ?mol/L, respectively. The linearity was up to 320 ?mol /L. The optimum concentrations of selective inhibitors were 2.5 mmol/L for NaF and 2.5 mmol/L for NAC. The results of serum CB (Y) determined by this method correlated well with those determined by diazo-dye method (Y=0.839X-7.965, r=0.969). Conclusion The proposed reaction conditions could effectively inhibit the oxidation of unconjugated bilirubin by BOD, and thus increase the specificity of CB assay.

Comparative analysis of venous blood lactate levels by automatic lactate strip kit (accusport) and by standard marbach’s enzymatic method.

Lactic acidosis is an emerging life-threatening condition that needs to be diagnosed and treated as early as possible. The complete analysis of blood lactate levels by standard method takes at least a few hours and is not available at all times. The automatic lactate strip kit (Accusport) will be more practical for diagnosis and treatment of lactic acidosis patients. This study showed the results of blood lactate determined by standard enzymatic method of Marbach compared to the lactate strip. The results showed a strong correlation between the two methods (r2=0.966). The correlation increased in the case of high blood lactate levels (r2=0.978) and decreased in normal blood lactate levels (r2=0.943). Overall lactate values measured from the strip method were lower than those from the enzymatic method. From this study we can calculate a constant factor of 0.981 which when multiplied with the value of blood lactate analysed by lactate strip then added with 0.532, the result will be equal to that from Marbach’s enzymatic method.