同位素稀释液相色谱串联质谱法测定血清总甘油

目的 建立同位素稀释液相色谱串联质谱测定血清总甘油的方法.方法 以[13C3]-甘油作内标,用氢氧化钾异丙醇溶液水解血清甘油酯为游离甘油,将游离甘油转化为苯甲酸酯,用同位素稀释液相色谱串联质谱(LC/MS/MS)分离检测,用标准曲线法定量.结果 甘油/内标峰面积比与甘油浓度(0.565～4.517 mmol/L)线性相关系数大于0.999 9;测定不同浓度血清总甘油批内变异系数(CV)平均为0.52%(范围0.21%～2.62%),总CV平均为1.15%(范围0.62%～2.00%);分析国际和国家标准物质,测定值与认证值的偏倚小于1%(-0.20%～1.06%).结论 建立同位素稀释液相色谱串联质谱测定血清总甘油方法,方法特异、精密、准确,可望用作血清总甘油测定参考方法.     

高效液相色谱测定血清甘油三酯的研究——一种参考方法的建议

目的　建立特异、精密的血清总甘油和游离甘油测定方法 ,以便甘油三酯测定标准化。方法　以 1,2 ,4 丁三醇作内标 ,用柱前衍生高效液相色谱技术分析血清皂化前后的甘油含量 (两者分别代表血清游离甘油和总甘油浓度 )。结果　测定总甘油相对不精密度小于 2 % ,游离甘油小于 4% ;总甘油平均回收率 10 0 0 % ,游离甘油 99 7% ,与同位素稀释 /质谱法的相对偏差不大于± 2 %。结论方法特异、精密、简单 ,可望用作血清甘油三酯测定参考方法。     

甘油内空白法与GPO-POD法检测血清三酰甘油

目的 探讨甘油内空白法与甘油磷酸氧化酶(GPO)-过氧化物酶(POD)法测定生化质控血清中三酰甘油(TG)的差异.方法 采用两种方法测定7个不同品牌共17个批号生化室内质控血清和卫生部2006年常规化学室间质评样本三酰甘油,比较两法测定结果的异同.结果 甘油内空白法与GPO-POD法测定不同品牌不同批次生化室内质控血清三酰甘油的结果无明显相关,Roche与Leadman的TG结果可相差1倍以上;而Aalto和Bio Rad的结果非常相近.不同批次的卫生部室间质评样本结果与此类似.结论 部分品牌的生化质控血清含大量的游离甘油,两法测定质控血清的结果可能出现明显差异.     

Automated procedure for kinetic measurement of total triglycerides (as glycerol) in serum with the Gilford System 3500

An automated procedure is used on the Gilford 3500 Computer-Directed Analyzer to measure serum triglycerides indirectly by using aqueous glycerol standards. Most enzymatic methods require long hydrolysis or awkward saponification. The method of Bucolo and David [clin. Chem. 19, 476 (1973)], in which lipase and glycerol kinase are used, is modified. The kinetic procedure described eliminates the need for a serum blank. It uses "Eskalab" bulk reagents and reduces both time and cost per test by measuring the decreasing NADH concentration from the glycerol kinase reaction at 340 nm after enzymatic hydrolysis at room temperature. The change in absorbance of the standard during a 14-s measuring time is used in the ratiometric calculation of the unknowns. The use of a stable aqueous standard that can be reliably and accurately prepared makes this method ideal for the traceability desired by many organizations. Reagent blank drift did not affect the results. A correlation coefficient of 0.991 for comparison with the manual endpoint method and a typical CV of 2.25% show this method to be accurate and reliable.     

Liquid-chromatographic measurement of creatinine in serum and urine

We describe the adaptation of a "high-performance" liquid chromatographic method for determination of creatinine in serum and urine. The proposed method is simple, rapid, precise, and accurate. The retention time for creatinine can be varied simply by changing the KH2PO4 concentration in the mobile phase: acetonitrile/aqueous KH2PO4 (1/4 by vol). Within-day precision (CV) was 1.2-3.6% in serum chromatographed with an internal standard, and 2.3-2.8% in serum when an external standard was used. Between-day precision (CV) was 1.3-2.1% in serum and 1.3-2.7% in urine (with an external standard). Analytical recoveries of creatinine added to serum were 94-100% for the method with an internal standard, 95-103% with an external standard.     

Inhibition of hepatic triglyceride formation by clofibrate

The effect of clofibrate (CPIB) on hepatic glycerolipid formation has been studied in vivo and in vitro in the rat. Feeding 0.25% CPIB in laboratory chow significantly reduced serum triglyceride levels by 6 hr and concomitantly decreased the rate of glycerol-(14)C incorporation into hepatic and serum glycerides, in vivo. These changes persisted for at least 14 days. A similar decrease in serum triglyceride and glycerol incorporation into hepatic glycerides was observed in rats fed high glucose diets containing 0.25% CPIB. Serum glycerol was reduced by feeding CPIB for 14 days. The formation of diglyceride and triglyceride from (14)C-sn-glycerol-3-P by rat liver homogenates was inhibited by addition of 1-40 mM CPIB to the reaction mixture. These results suggest that CPIB reduces hepatic glycerolipid synthesis, possibly by inhibition of one or more reactions in the esterification of sn-glycerol-3-P. This change may account for the early fall in serum triglyceride. At later time periods, serum glycerol levels fall and in some experiments, hepatic triglyceride content increases. Therefore, it is likely that additional metabolic alterations may contribute to the sustained hypotriglyceridemic effects of CPIB.     

Effects of nicotinic acid treatment on glyceride formation and lipolysis in adipose tissue of hyperlipidemic patients

Summary Thirty-one weight-stable patients with different types of hyperlipoproteinemia were treated daily with 4g nicotinic acid for 6 weeks. Effects of this therapy on adipose tissue metabolism were evaluated. By using biopsy specimens of subcutaneous adipose tissue, fatty acid and glucose incorporation into adipose tissue glycerides were measured in vitro as well as glycerol and fatty acid release, which allowed us to estimate adipose tissue lipolysis. The amount of fatty acids produced by lipolysis and thereafter utilized within adipose tissue without being released (fatty acid retention) was estimated. Fatty acid and glucose incorporation into adipose tissue, glycerol release and fatty acid retention values increased, but serum triglyceride levels decreased (allP<0.001) after nicotinic acid treatment. The change in fatty acid incorporation was positively correlated with changes in glucose incorporation into adipose tissue (r=0.53,P<0.01) and fatty acid retention (r=0.76,P<0.001). Although adipose tissue lipolysis, measured as glycerol release, increased, the lipolyzed fatty acids were retained in adipose tissue, suggesting an enhanced synthesis of glycerides both from exogenous and endogenous sources. The increase in fatty acid incorporation into adipose tissue indicates that the decrease in serum triglyceride levels produced by nicotinic acid treatment may partly be due to the fact that this drug promotes incorporation of fatty acids, derived from lipoprotein-carried triglycerides in the blood, into adipose tissue glycerides.     

Quantitation of serum bile acids by isotope dilution with 13C-labelled homologs

Quantitation of individual bile acids in serum can be carried out with radioimmunoassays or with gas chromatography. The most specific measurements are obtained with combined gas chromatography/mass spectrometry employing stable isotope labelled bile acids as internal standards. So far only the use of deuterated internal standards has been described for this purpose. 24-13C-labelled bile acids have not been used since correction for the natural abundance of the 13C contribution has to be made. Furthermore, the maximal degree of labelling of 13C-labelled bile acids is about 90%. This requires additional correction for the percentage of unlabelled molecules. Using 13C-labelled bile acids as internal standards and adequate corrections for isotope interferences we have measured individual serum bile acids in healthy volunteers by inverse isotope dilution with coefficient of variation (CV) values of 5.4-6.2% determined for the total procedure of sample preparation and analytical technique. In fasting serum of 15 healthy volunteers chenodeoxycholic acid averaged 0.98 +/- SD 0.77 mumol/l, cholic acid 0.49 +/- 0.39 mumol/l and deoxycholic acid 1.07 +/- 0.68 mumol/l. Two hours postprandially these values increased to 2.42 +/- 1.46 mumol/l for chenodeoxycholic acid, 0.81 +/- 0.45 mumol/l for cholic acid and 1.66 +/- 1.02 mumol/l for deoxycholic acid. These data agree well with those described in the literature obtained with deuterated internal standards. It may be concluded that 24-13C-labelled bile acids are suitable as internal standards for quantitation of serum bile acids, if corrections for isotope interferences are made.     

The Monoglyceride Pathway of Fat Absorption in Man

The absorption of fat was studied in five male subjects with cannulation of the thoracic duct in the neck by the administration of doubly labeled monoglycerides, or triglyceride as well as labeled free glycerol or labeled free oleic acid, by gastric or duodenal intubation.Total recoveries of the administered glyceride radioactivity from the lymph lipids ranged from 35 to 53% for the glycerol label (tritium) and from 35 to 57% for the fatty acid label ((14)C). The recovery of administered radioactive free glycerol in lymph lipids was only 4.1%, even when given in mixture with bile salts, fatty acid, and monoglyceride.A comparison of the isotope ratios of the two components (glycerol and fatty acid) of the lymph glycerides with the ratios of these components of the original meal glyceride showed little change during the initial period of fat absorption, indicating that the doubly labeled monoglycerides passed into the lymph intact. During the later part of the period of major fat absorption, the ratios in lymph lipids changed due to loss of glycerol representation, indicating monoglyceride hydrolysis and portal venous diversion of free glycerol.Confirmation of the intact nature of 2-monoglyceride during absorption was made by analyzing the amount and position of the labeled fatty acid in the lymph triglycerides. The percentage of labeled fatty acid in the various positions of the lymph triglycerides was virtually identical with that of the meal during the initial period of fat absorption and then changed reflecting isomerization of fatty acids and subsequent complete hydrolysis of the glycerides.The 2-monoglyceride pathway appears to be the major route of fat absorption for man during normal digestion and absorption of dietary triglyceride.     

Mass fragmentographic analysis of total cholesterol in serum using a heptadeuterated internal standard

A mass fragmentographic method for the determination of total cholesterol in serum using heptadeuterated [25,26,26,26,27,27,27-²H]cholesterol as internal standard is presented. The results obtained are compared with a colorimetric and gas chromatographic method which were previously proposed as reference methods.Criteria for the development of absolute measurement by means of mass fragmentography and stable isotopically labelled internal standards are given.The conclusion is drawn that, at present, mass fragmentographic methods for the determination of total cholesterol in serum do not fulfil the criteria required for absolute methods.     

ARCHITECT C16000全自动生化分析仪检测血清胱抑素C的性能评价

目的对ARCHITECT C16000全自动生化分析仪检测血清胱抑素C(Cys C)的分析性能进行评价。方法根据相关标准,验证ARCHITECT C16000全自动生化分析仪检测血清Cys C的精密度、正确度、分析测量范围、临床可报告范围、抗干扰性能、生物参考区间。结果低水平质控品、高水平质控品及混合血清标本的批内变异系数(CV)均小于1/4室间质评允许总误差(6.25%),批间CV均小于1/3室间质评允许总误差(8.33%),也均小于厂商标准(10.00%)。5个水平校准品检测值与靶值相对偏移的绝对值均小于1/2室间质评允许总误差(12.50%)。参比系统和待评系统检测结果一致性好。实测值与预期值的线性回归方程为Y=1.000 X+0.094,r=0.9991,r≥0.975,斜率在0.97~1.03,Cys C的分析测量范围为0.14~9.91 mg/L。Cys C可接受的最大稀释倍数为20倍,临床可报告范围上限为198.20 mg/L。含不同水平游离胆红素、结合胆红素、血红蛋白及乳糜标本的检测值与对照标本检测值相对偏差的绝对值均小于厂商标准(10.00%)。生物参考区间验证中,超出参考区间的Cys C检测结果不超过10.00%,通过验证。结论ARCHITECT C16000全自动生化分析仪检测Cys C的性能符合临床要求,可应用于临床。     

Standardization of serum fructosamine assays

We have calibrated a secondary serum protein standard by use (as primary standards) of samples of albumin and polylysine glycated with [14C]glucose in vitro, the glycation of which was assessed by radioactivity measurements and by elementary analysis for C and N. Using this standard for calibration in our improved fructosamine assay, one obtains an average fructosamine value of 247 mumol/L for nondiabetic individuals (or, in terms of total serum protein, 3.2 mumol/g)--about a tenth the value we obtained when we used the fructosamine assay of Johnson et al. (Clin Chim Acta 1983;127:87-95), standardized with desoxymorpholinofructose. In contrast, results corresponded well with the value for mean glycation of serum proteins, 3 mumol/g, determined by a furosine/HPLC method. Evidently the proposed procedure, in which a standard sharing the binding characteristics of endogenous glycated proteins is used together with our modified new fructosamine assay, leads to more realistic values for the concentrations of glycated serum proteins.     

同位素稀释液相色谱串联质谱法测定血清葡萄糖

目的 建立一种使用同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)测定血清葡萄糖的候选参考方法.方法 以[~(13)C_6]葡萄糖为内标,用重量法准确地与血清混合,除去蛋白后在碱性条件下与1-苯基-3-甲基-5-吡唑酮反应,用LC/MS/MS测定葡萄糖和内标衍生产物,以包括法定量.结果 血清葡萄糖测定的批内、批间和总变异系数的平均值分别为0.36%(范围0.28%-0.42%)、0.47%(范围0.20%-0.67%)和0.61%(范围0.42%-0.76%).加样回收试验的回收率范围为99.0%-100.9%.分析参考物质SRM 965a,测定结果与认定值的平均偏差为-0.20%(范围-0.39%-+0.11%).结论 建立了ID-LC/MS/MS法测定血清葡萄糖的方法,方法准确、精密、简便,可望作为血清葡萄糖测定的参考方法.     

Isotope dilution/mass spectrometry of serum cholesterol with [3,4-13C]cholesterol: proposed definitive method

We describe a new gas-chromatographic/mass-spectrometric (GC/MS) isotope-dilution method for determination of serum cholesterol. The method has been fully optimized and documented to provide the high accuracy and precision expected for a Definitive Method. In the presence of [3,4-13C]cholesterol, cholesteryl esters in serum are hydrolyzed under optimum conditions and the entire cholesterol pool is extracted and derivatized to silyl ethers. The cholesterol derivatives are resolved from other sterols by gas-liquid chromatography on a fused silica column, and selected ions characteristic of cholesterol and the [3,4-13C]cholesterol are monitored with a GC/MS quandrupole system. We estimated the cholesterol content of samples by bracketing each sample with standards of comparable cholesterol concentration that also contained the [3,4-13C]cholesterol. The procedure was highly reproducible (CV less than 0.5%), better accuracy and precision being obtained with [3,4-13C]cholesterol than with heptadeuterated cholesterol. Mean values per gram of dry serum for one serum pool assayed by this method and that of the National Bureau of Standards differed by 0.5%. We conclude that the method satisfies the criteria for a Definitive Method.     

Fatty Acid Incorporation into Human Adipose Tissue in Hypertrigiyceridaemia*

The fatty acid and glucose incorporation into glycerides and glycerol release from adipose tissue were determined in a middle-aged population of 109 men and 41 women. 43 men and 19 women were rrormolipidaemic. The same analysis was also carried out in 13 male and 9 female normolipidaemic students. Needle biopsy specimens of adipose tissue were incubated in vitro in an albumin medium containing ³H-fatty acids and ¹⁴C-glucose. After two hours of incubation values for fatty acid and glucose incorporation were calculated from the incorporation of ³H-activity into the fatty acids and ¹⁴C-activity into the glycerol moiety of extracted glycerides. The mean values for fatty acid incorporation were lower in all types of hypertriglyceridaemic subjects (II B, III, IV and V) than in the normolipidaemic control subjects. In the male hypertriglyceridaemic population 36 % had values for fatty acid incorporation below the 5th percentile of the normolipidaemic group and 14 % had values below the lowest normal value. The rate of fatty acid incorporation was negatively correlated with the serum triglyceride concentration. This correlation remained unchanged when partial correlation was performed when the influence of body weight was eliminated. Fatty acid and glucose incorporation correlated positively. Incorporation of glucose behaved in the same way as described above for incorporation of fatty acids. Glycerol and fatty acid release was the same in the normo- and hypertriglyceridaemic groups. It is likely that the removal of plasma triglycerides from blood requires hydrolysis of triglycerides to fatty acids and the subsequent removal of the fatty acids. The hypothesis has been formulated that when the former process is normal, a defect of fatty acid removal (a low rate of fatty acid incorporation into glycerides) may be responsible for an impaired removal of plasma triglyceride-fatty acids. A low rate of fatty acid incorporation may contribute to the development of hypertrigiyceridaemia, according to this hypothesis.     

Determination of amylase activity and amylase isoenzymes in serum and urine using a solid phase blue starch substrate.

The alpha-amylase of serum and urine was determined in 40 healthy people using the modified "Phadebas Amylase Test". The original method was modified by decreasing the incubation volume to one millilitre and by adding to all urine speciemens a small amount of albumin in saline. The normal values obtained are slightly higher than those obtained by the original method. The amylase isoenzymes were likewise determined from the serum and urine of the same 40 healthy people. For separation were used electrophoresis on cellulose acetate and Phadebas tablets as substrate. These urine and serum isoamylases were investigated and compared. The distributions obtained deviate somewhat from ones reported earlier. The clinical usefulness of isoamylase is briefly discussed.     

Determination of serum glucose by isotope dilution mass spectrometry: candidate definitive method.

We report a rather simple method to determine glucose concentration in serum, using isotope dilution mass spectrometry and [13C6]glucose as internal standard. The procedure involves a single step of sample purification and the conversion of the analyte into its aldononitrile pentaacetate. The between-day and within-day contribution to total variance for a single measurement was determined by assaying Standard Reference Material (SRM) 909 serum. The method was then applied to measurement of glucose concentration in three lyophilized sera: SRM 909 and two other commercially available sera. In the two studies, the concentration of SRM 909 serum was found to be 0.8% above and 0.3% below the reported value (6.25 mmol/L), respectively; the overall coefficient of variation for determinations in all sera ranged from 0.37% to 0.56%. The precision and the accuracy of the method satisfy the requirements for a Definitive Method.     

Enzymatic measurement of serum glycerol using a COBAS-BIO centrifugal analyzer

An enzymatic method for the measurement of glycerol using glycerol kinase coupled to pyruvate kinase and lactate dehydrogenase was adapted to a COBAS-BIO centrifugal analyzer. Routine standardization is not required under the optimized conditions since the calculation factor derived from the measurement of pure glycerol standard was found to be identical to the theoretical value. Excellent correlation between the method and the DuPont ACA triglyceride method was obtained. The coefficients of variation for within-run and day-to-day precision were less than 4%. Only 2 microL of serum is required, making this an excellent means for monitoring therapeutic glycerol administration in premature infants with hydrocephalus. The method has the sensitivity to measure glycerol concentrations as low as 0.1 mmol/L. This, together with the rapid analysis time (28 specimens in 10 min), makes it suitable for the correction of glycerol interference in the measurement of serum triglyceride.     

Hydroxyurea interferes negatively with triglyceride measurement by a glycerol oxidase method

Measured triglyceride concentrations were extremely low (less than 100 mg/L) in the serum of some patients who were receiving hydroxyurea for myeloproliferative diseases. The assay being used to quantify triglycerides was a "cascaded" enzymatic method involving (a) lipase, to generate glycerol from triglycerides; (b) glycerol oxidase, to convert glycerol to glyceraldehyde, with generation of hydrogen peroxide; and (c) peroxidase, which acts on the hydrogen peroxide with subsequent coupled generation of a red-violet quinone (reagent system used in the Technicon RA-1000). Hydroxyurea added to serum samples appeared to inhibit the action of glycerol oxidase, with a stoichiometric relation to the concentration of substrate (a decrease of roughly 2.4 mmol/L in measured triglyceride per 1 mmol of hydroxyurea per liter). A different enzymatic assay for triglycerides, which involves glycerol kinase (Beckman Instruments) did not show this effect of hydroxyurea.