LC-MS/MS技术检测新生儿干血斑甲基丙二酸、同型半胱氨酸cut-off值的建立

目的探讨建立高效液相色谱质谱-串联质谱联合检测(liquid chromatography-tandem mass spectrometry,LC-MS/MS)技术检测新生儿干血斑甲基丙二酸(methylmalonic acid,MMA)和同型半胱氨酸(homocysteine,Hcy)cut-off值的方法。方法收集本院分娩的新生儿MS/MS筛查中C3、C3/C2和C3/C0任一指标偏高的正常干血斑样本作为阴性样本,同时收集通过尿气相色谱质谱结合循环酶法确诊为甲基丙二酸血症合并同型半胱氨酸血症患儿的初筛干血斑样本作为阳性样本,采用LC-MS/MS技术分别检测该两类样本中MMA和Hcy的浓度。应用百分位数法并结合ROC曲线分析确定使用LC-MS/MS技术检测MMA和Hcy的cut-off值。结果共收集1 015例阴性样本和16例MMA患儿阳性样本,LC-MS/MS技术检测阳性样本的MMA和Hcy浓度均明显高于阴性样本,差异具有统计学意义(U_(MMA)=8.5,U_(Hcy)=23.5,P均0.05);MMA和Hcy的ROC曲线下面积(AUC~(ROC))均为0.999,cut-off值分别为3.145μmol/L和7.295μmol/L,诊断特异性分别为99.5%和99.3%。结论百分位数法结合ROC曲线法可用于确定本地区MMA患儿干血斑中MMA和Hcy的cut-off值,且本地区人群的MMA和Hcy cut-off值明显低于梅奥诊所提供的cut-off值。     

液相色谱-串联质谱方法检测血清中极长链脂肪酸含量

目的 建立一种检测血清中极长链脂肪酸的LC-MS/MS方法.方法 收集2009年4-6月35份疑似ALD患者血清样本,采用LC-MS/MS方法检测血清中极长链脂肪酸含量.分析加样回收率、精密度及准确度,研究在常温放置和反复冻融条件下血清样本中极长链脂肪酸含量的稳定性.同时,用该方法测定101份健康人血清中极长链脂肪酸含量,统计测定值并进行分析.随机抽取35份血清,测定结果与德国柏林医学诊断检验中心(MDI)实验室测定数值进行比对.结果 血清样本中的极长链脂肪酸在梯度洗脱条件下分离良好,杂质干扰程度小.山萮酸(behenate,C22:0)的线性范围为2～64 mg/L,加样回收率为99.92%～102.05%,日内RSD≤6%,日间RSD≤9%;木焦油酸(tetracosanoicacid,C24:0)线性范围为2～64 mg/L,加样回收率为95.12%～100.44%,日内RSD≤6%,日间RSD≤7%;蜡酸(hexacosanoic acid,C26:0)线性范围为0～8 mg/L,加样回收率为92.21%～103.71%,日内RSD≤7%,日间RSD≤8%.山萮酸、木焦油酸和蜡酸的准确度均在85%～115%之间.样本在常温条件下放置12 h、反复冻融10次可以保持稳定.检测101份健康人血清中极长链脂肪酸含量服从正态分布,山萮酸含量为(19.43±4.43)mg/L,木焦油酸含量为(19.10±4.58)mg/L,蜡酸含量为(0.21±0.11)mg/L,计算木焦油酸/山萮酸和蜡酸/山箭酸比值分别为(0.99±0.13)和(0.01±0.01).统计结果显示,成年人血清中蜡酸(0.18±0.10)mg/L和木焦油酸/山萮酸比值(1.01±0.10)和未成年人血清中蜡酸(0.21±0.08)mg/L和木焦油酸/山萮酸比值(0.99±0.14)差异无统计学意义(t分别为1.439、0.806,P均＞0.05);男性健康人血清中木焦油酸/山萮酸比值(1.05±0.10)与女性健康人血清中木焦油酸/山萮酸比值(0.97±0.10)差异有统计学意义(t=3.394,P=0.001).与德国MDI实验室比对结果发现,本研究测定的山萮酸含量(16.93±4.30)mg/L和木焦油酸含量(19.57±6.40)mg/L与德国MDI实验室测定的山萮酸含量(13.85±3.17)mg/L和木焦油酸含量(16.10±5.84)mg/L差异有统计学意义(t分别为8.401和9.914,P均=0.000),而本研究测定的蜡酸含量(0.68±0.48)mg/L、木焦油酸/山萮酸比值(1.20±0.40)和蜡酸/山萮酸比值(0.04±0.04)与德国MDI实验室测定的蜡酸含量(0.65±0.67)mg/L、木焦油酸/山萮酸比值(1.19±0.43)和蜡酸/山萮酸比值(0.05±0.05)差异无统计学意义(t分别为0.372、0.317、0.945,P均＞0.05).结论 应用LC-MS/MS方法检测血清中极长链脂肪酸,具有较好的准确度和灵敏性,特异性强,稳定性好,能为临床诊断提供重要的生化依据.

Abstract：

Objective To establish a method for very long chain fatty acids( VLCFA )with liquid chromatography-tandem mass spectrometry( LC-MS/MS ). Methods One hundred and one healthy cases and 35 suspected ALD patients collected from April to June in 2009 were enrolled into this study. Quantitative analyzed the concentrations of VLCFA in serum was performed using liquid chromatography-tandem mass spectrometry. The precision, accuracy and recovery were analyzed, and the stability of VLCFA concentration of sample under room temperature and repeated freeze-thawing were also investigated. Serum levels of VLCFA in 101 normal cases were determined and analyzed statistically. The results for the 35 randomly chosen serum samples were compared with those from MDI in Germany. Results Serum VLCFA were separated well under these gradient condition with small interference. The linear range of C22:0 was from 2 mg/L to 64 mg/L, the recovery was 99. 92% -102. 05%, and the relative standard deviation ( RSD ) of intra-day and inter-day was less than 6% and 9% respectively. For C24:0 they were 2-64 mg/L. 95. 12%-100. 44%. ≤6%, ≤7%,respectively. For C26:0, they were 0-8 mg/L, 92.21%-103.71%, ≤7%, ≤8%, respectively. The accuracy of C22: 0,C24:0 and C26:0 were among 85% to 115%. The samples could be stable within 12 h at room temperature and repeated 10 times freeze-thawing. The values of VLCFA in 101 normal cases followed a normal distribution and the measured values were C22:0 =( 19. 43 ±4.43 ) mg/L,C24:0 =( 19. 10 ±4. 58 )mg/L, C26:0 = ( 0. 21 ± 0. 11 ) mg/L, the ratio of C24: 0/C22:0 and C26:0/C22: 0 were ( 0. 99 ± 0. 13 )and ( 0. 01 ±0. 01 ) respectively. The statistical analysis showed the concentration of C26:0 in adults ( 0. 18±0. 10 ) mg/L and children ( 0. 21 ± 0. 08 ) mg/L, C24: 0/C22:0 in adults ( 1.01 ± 0. 10 ) and children ( 0. 99 ±0. 14 ) has no significant( t values were 1. 439,0. 806, respectively, all P ＞ 0. 05 ); the ratio of C24:0/C22:0 in male (1.05 ± 0. 10 ) and female (0.97 ± 0. 10 ) has significant difference ( t =3. 394,P =0. 001 ). Compared the values determined by MDI laboratory, the results of C22: 0( 16. 93 ±4. 30 ) mg/L,C24: 0( 19. 57 ± 6. 40 ) mg/L by this method and C22:0 ( 13.85 ± 3. 17 ) mg/L, C24:0( 16. 10 ±5.84 ) mg/L by MDI have significant differences( t = 8. 401 ,P =0. 000;t =9. 914,P =0. 000 ),but C26:0( 0.68 ±0.48 ) mg/L, C24:0/C22:0( 1.20 ±0.40 ), C26: 0/C22:0 ( 0.04 ±0.04 )by this method and C26: 0( 0. 65 ± 0. 67 ) mg/L, C24:0/C22: 0( 1.19 ± 0. 43 ), C26:0/C22: 0 ( 0. 05 ± 0. 05 )by MDI have no differences( t values were 0. 372,0. 317,0. 945 ,respectively ,all P ＞0. 05 ). Conclusions The quantitative analysis method for serum very long chain fatty acid using LC-MS/MS is accurate, sensitive,specific and stable. It could provide important biochemistry information for diagnosis in clinic.     

血清总同型半胱氨酸候选参考测量程序(液相色谱串联质谱法)的建立及性能评估

目的建立一种基于液相色谱串联质谱(LC-MS/MS)技术的血清总同型半胱氨酸(Hcy)候选参考测量程序并对其性能进行评价。方法采用一种简单的蛋白沉淀方法对血清样本进行前处理,然后采用LC-MS/MS定量检测总Hcy,参照美国临床实验室标准化协会(CLSI) C62-A文件和C50-A文件对建立的候选参考方法进行线性、检测限与定量限、基质效应、精密度、正确度等基本分析性能验证。结果 LC-MS/MS检测总Hcy的线性范围为0.5～200.0μmol/L。定量限和检测限分别为0.31 nmol/g、0.06 nmol/g。3种不同比例(1∶1、80∶20、20∶80)的血清与溶液混合物的相对基质效应分别为1.94%、1.91%、1.78%。批内、批间变异系数(CV)分别为2%和1%。3种浓度(30.58、49.21、65.42 nmol/g)的加标样本平均加标回收率分别为99.8%、100.2%、100.8%。测定NIST SRM 1950标准物质的结果偏移1%。样本处理后分别在室温[(23±2)℃]和自动进样器(温度为10℃)中放置24 h,检测结果均非常稳定。结论成功建立了基于LC-MS/MS技术的血清总Hcy候选参考测量程序。该参考测量程序准确度高、精密度好,能够用于常规临床检验方法的量值溯源,保证测定结果的准确性。     

视黄醇结合蛋白4在妊娠期糖尿病患者中的表达及其临床意义

目的：分析视黄醇结合蛋白4（retinol-binding protein4,RBP4）在妊娠期糖尿病患者和健康孕妇血清中的浓度差异及其与临床、病理特征的关系。方法：检测18例妊娠期糖尿病（GDM）和212例健康孕妇血清中RBP4的表达,分别于孕18周、孕20周、孕28周及产后8周空腹收集血清。利用酶联免疫吸附实验（ELISA）检测血清RBP4的表达。用HOMA-IR（homeosta-sis model assessment）模型评价胰岛素抵抗程度。结果：所有孕妇血清RBP4水平在产前各时间点呈时间依赖性升高。产后8周（中位数,15.35μg/mL;四分位数,11.32～27.85μg/mL）孕妇血清RBP4水平均下降。孕20周（中位数,45.72μg/mL;四分位数,33.34～58.69μg/mL）、孕28周（中位数,52.34μg/mL;四分位数,42.65～73.54μg/mL）时,GDM患者血清RBP4水平高于健康孕妇（孕20周：中位数,19.13μg/mL;四分位数,15.23～22.65μg/mL;孕28周：中位数,42.54μg/mL;四分位数,24.56～55.21μg/mL）。孕18周（中位数,16.80μg/mL;四分位数,14.58～28.67μg/mL）和产后8周（中位数,15.35μg/mL;四分位数,11.32～27.85μg/mL）,GDM患者血清RBP4水平和健康孕妇无显著差异（孕18周：中位数,15.78μg/mL;四分位数,10.23～19.35μg/mL;产后8周：中位数,13.54μg/mL;四分位数,9.21～18.35μg/mL）。孕20周时血清RBP4水平和HOMA-IR（相关系数r=0.872;P=0.002）、体质量指数、血清三酰甘油和低密度脂蛋白呈正相关,和高密度脂蛋白呈负相关。结论：孕20周血清RBP4水平可能成为GDM的早期诊断指标。     

尿素、尿酸和总蛋白复合冰冻人血清国家标准品的研制

目的研制尿素、尿酸和总蛋白复合冰冻人血清国家标准品,用于校准和评价常规方法,促进尿素、尿酸和总蛋白测定的标准化。方法收集含不同浓度尿素、尿酸和总蛋白的血清(无溶血、脂血和黄疸)样本,过滤除菌后分装,于-70℃保存。采用单因素方差分析评价候选品的均匀性。通过线性回归方差分析进行稳定性研究。采用参考方法或经验证的方法定值,计算不确定度。对候选品及25份新鲜血清样本在参考方法(或经验证的方法)及3个常规检测系统间的互通性进行评估。结果候选品中尿素、尿酸和总蛋白均匀性检验F值分别为1.071 4、1.339 9和1.275 0,均F0.05。在-20℃条件下,候选品中尿素、尿酸和总蛋白均至少可稳定37 d;在2~8℃条件下,尿素可稳定27 d,尿酸及总蛋白至少可稳定37 d;在20~25℃条件下,尿素仅能稳定3 d,尿酸可稳定8 d,总蛋白可稳定27 d。定值结果分别为:尿素(5.68±0.17)mmol/L(k=2)、尿酸(288.68±3.96)μmol/L(k=2)、总蛋白(67.03±3.46)g/L(k=2)。尿素、尿酸和总蛋白浓度全部在25份血清样本的回归直线95%可信区间范围内。结论研制的尿素、尿酸和总蛋白复合冰冻人血清国家标准品候选品均匀性、稳定性、互通性良好,定值准确、可靠。     

血清脂肪酶速率比浊法

本文报道了适合国内条件的血清脂肪酶(LPA)速率比浊法。用纯化橄榄油作底物,浓度为273.6μmol/L,缓冲液pH9.8,去氧胆酸钠浓度为1g/L.两份血清作重复测定,CV 为4.68～5.2%,精密度优于国内报道的快速比浊法.在不加昂贵的辅脂肪酶的条件下,基本解决了正常血清用固定时间的快速比浊法几乎测不出LPA 活性的问题。测定103例(男52例,女51例)献血员血清,LPA 活性全距为0～39.7U/L;百分位数法求得95%上限30.75U/L;男女之间无显著差别(P>0.9)。本法简单快速,反应在5分钟内完成,能满足急诊需要。     

胰岛素样生长因子及生长激素与胎儿体重的关系

目的:了解胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)及生长激素与胎儿出生时体重的关系.方法:对妊娠足月健康产妇71例所分娩的胎儿按其体重不同分为3组,用放射免疫方法测定胎儿脐静脉血清IGF-1与生长激素浓度.结果:脐静脉血清IGF-1浓度分别为大体重组195～637(中位数416)μg/L,中体重组169～285(中位数227)μg/L,小体重组159～209(中位数184)μg/L.大体重组脐静脉血清IGF-1浓度高于中体重组和小体重组(分别为P＜0.05,P＜0.01),中体重组的哜静脉血清IGF-1又高于小体重组(P＜0.05).脐静脉血清生长激素浓度:大体重组(36±13)μg/L,中体重纽(27±6)μg/L,小体重纽(20±10)μg/L.大体重纽脐静脉血清生长激素浓度高于中体重组和小体重组(分别为P＜0.05,P＜0.01),中体重组的脐静脉血清生长激素又高于小体重组(P＜0.05).结论:胎儿出生时的体重与IGF-1和生长激素有一定关系,IGF-1与生长激素是胎儿在母亲子宫体内生长发育中的重要激素.     

健康人群胰岛素测定稳定性及参考范围调查

目的 建立本实验室人血清胰岛素(Ins)参考范围并分析测定稳定性.方法 对Architect I2000化学发光免疫分析系统进行方法学评价;对413例健康成年人进行血清Ins检测,分析生理和实验室指标对Ins的影响,建立Ins参考范围;对Ins在不同温度下的稳定性进行探讨.结果 Ins检测批内和天间不精密度分别为1.67%和2.60%.各年龄组Ins水平无统计学差异(P>0.05).女性Ins水平(中位数5.7 mU/L)高于男性(中位数5.0 mU/L)(z＝3.696,P＜0.05).以第97.5%位数为参考值上限,第2.5%位数为参考值下限,女性Ins参考范围为2.6～11.8 mU/L,男性为2.3～11.6 mU/L.胰岛素与体质量指数、三酰甘油呈正相关,与高密度脂蛋白呈负相关.Ins水平在25、4、-20 ℃至少可稳定4 h、24 h、7 d.结论 性别是影响人血清Ins水平的重要因素,应建立不同性别Ins参考范围;胰岛素水平在各种温度条件下并非很稳定.     

血清中HBsAg浓度差异在乙型肝炎标志物模式分析中价值

目的分析不同浓度HBsAg血清中乙型肝炎标志物表现模式,以揭示其在人群中的分布特征。方法采用ELISA法与微粒子酶免疫分析技术(microparticle enzyme immunoassay,MEIA)测定5987例非肝炎流行区住院及门诊患者血清中HBsAg及其表面抗体(抗HBs)、乙肝e抗原及e抗体(HBeAg、抗HBe)和乙肝病毒核心抗体(抗HBc);根据定值参比血清和样本HBsAg荧光速率值/阴性对照荧光速率值之比(S/N值),再结合中和确证试验结果来确定HBsAg浓度,同时分析乙肝病毒血清学标志物模式;对低浓度(HBsAg≤1μg/L)再用PCR-ELISA法定量测定HBV DNA。结果:共检出HBsAg阳性784例,HBsAg浓度在5μg/L以上有636例,占HBsAg阳性81.1%;HBsAg浓度在2~5μg/L的有47例(5.99%);1~2μg/L的有69例(8.80%);1μg/L以下的有32例(4.08%)。尤其高浓度(HBsAg>5μg/L)和低浓度(HBsAg≤1μg/L)在人群中分布率分别为10.62%和0.53%;而中等浓度(1μg/L相似文献   

液相色谱串联质谱法和循环酶法测定血清中同型半胱氨酸的相关性

目的分析液相色谱串联质谱(LC-MS/MS)法与循环酶法测定人血清中同型半胱氨酸(homocysteine,HCY)浓度的相关性。方法收集63例患者的血清样本,用LC-MS/MS法和循环酶法分别测定相同样本的HCY浓度,并评价2种方法的相关性。结果 LC-MS/MS法与循环酶法对HCY浓度测定的结果分别为(19.11±15.69)μmol/L和(16.95±14.41)μmol/L,差异有统计学意义(t=6.25,P0.05)。2种方法的线性回归方程为YLC-MS/MS法=1.074X循环酶法+0.892,相关系数(R)=0.987,相关性较好。结论 LC-MS/MS法与循环酶法测定血清中HCY浓度的相关性较好,LC-MS/MS法适用于临床对HCY的检测。     

Holotranscobalamin in laboratory diagnosis of cobalamin deficiency compared to total cobalamin and methylmalonic acid.

BACKGROUND: Cobalamin-saturated transcobalamin, also called holotranscobalamin (holoTC), constitutes only between 6% and 20% of total plasma B(12). Serum concentration of holoTC is a new marker in laboratory diagnosis of cobalamin deficiency. We tested the utility of holoTC in assessing vitamin B(12) status. METHODS: We measured concentrations of holoTC and methylmalonic acid (MMA) in 1018 serum samples that were referred to our laboratory for total cobalamin testing. RESULTS: Concentrations of MMA were lower in females compared to males and this difference was no more significant after adjusting for serum creatinine. Moreover, age was associated with higher concentrations of serum MMA, higher holoTC and slightly higher concentrations of total cobalamin. Higher concentrations of serum creatinine were associated with higher concentrations of MMA and holoTC. However, no association between serum creatinine and total cobalamin was observed. Only subjects with normal serum creatinine showed a negative correlation between serum holoTC and MMA (r= -0.36, p<0.001). In subjects with MMA > or =300 nmol/L and holoTC < or =35 pmol/L, concentrations of total cobalamin were well within the normal range (median; 25th/75th percentiles=212; 171/272 pmol/L). Receiver operating characteristic (ROC) curve analysis displayed a higher sensitivity and specificity for holoTC compared with vitamin B(12) for detecting concentrations of MMA > or =300 nmol/L in individuals with normal renal function. CONCLUSIONS: Compared to total cobalamin, we observed a better performance of holoTC assay in detecting elevated concentrations of MMA in subjects with normal renal function. The majority of subjects with combined low holoTC and elevated MMA had normal concentrations of total cobalamin. HoloTC can be used as a first line parameter in detecting cobalamin deficiency.     

气-质联用技术测定尿有机酸方法的建立及在遗传代谢病诊断中的应用

目的 利用气-质联用技术测定尿液中有机酸种类及含量的变化,为先天性遗传代谢病,特别是有机酸代谢紊乱提供新的诊断依据.方法 收集临床高度怀疑为有机酸代谢紊乱的195例患儿的尿液标本,根据肌酐含量取相应体积的尿样和内标,加盐酸羟胺对带羟基的有机酸进行圬化反应生成酮体后,用乙酸乙酯和乙醚萃取有机酸,三甲基硅烷衍生;测定采用Agilent GC/MS 6890/5973i气相质谱仪,选择扫描模式检测,测定质荷比(m/z)范围在50～550 m/z的所有有机酸,数据处理采用Agilent GCMSD ChemStationGl701DA软件.用健康对照样本中加人的内标和阳性对照样本中加入的阳性有机酸进行分析方法的线性范围、精密度、准确性、样本回收率和残留分析.结果 该方法可测定尿液中100余种有机酸,以健康对照者尿样中加入的二甲基丙二酸(MMA)和2-苯基丁酸(2-PA)内标为参考,最低检测极限为2.5～2.8μmol/L;批内和批间变异系数均<10%,样本前处理批间变异系数为11.4%;样品回收率为95%～105%,残留分析<1%,所有参数均符合临床检测的要求.用该方法检测的临床患儿尿样中,发现诊断阳性病例12例,包括6例甲基丙二酸血症、1例丙酸血症、3例酪氨酸血症Ⅰ型、1例枫糖尿病和1例Ketosis病(酮症病).结论本研究建立了气-质联用技术分析测定尿液中有机酸的方法,可用于先天性遗传代谢性疾病的筛查诊断.     

Rapid 2nd-tier test for measurement of 3-OH-propionic and methylmalonic acids on dried blood spots: reducing the false-positive rate for propionylcarnitine during expanded newborn screening by liquid chromatography-tandem mass spectrometry

BACKGROUND: The expansion of newborn screening programs has increased the number of newborns diagnosed with inborn errors of metabolism in the presymptomatic phase, but it has also increased the number of costly, stress-producing false-positive results. Because propionylcarnitine (C3) is one of the analytes most frequently responsible for false-positive results, we aimed to develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify free methylmalonic (MMA) and 3-OH propionic (3OH-PA) acids in blood spots. METHODS: We studied newborn screening spots from 250 healthy controls; 124 from infants with abnormal C3, of whom only 5 (4%) were truly affected; 124 from infants with altered isolated methylmalonylcarnitine; and 4 from clinically diagnosed patients. Whole blood was eluted from a 3.2-mm dried blood spot by a CH(3)CN/H(2)O 7:3 and 5 mL/L formic. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray without derivatization. Total analysis time was 5 min per sample. RESULTS: The assays were linear up to 3300 nmol/L for both metabolites. Intra- and interassay imprecision data were 3.6%-8% and 3.1%-6%, respectively, for MMA and 5.2%-20% and 3.6%-17% for 3OH-PA. Limit of detection and limit of quantitation were 1.95 and 4.2 micromol/L, respectively, for MMA and 8 and 10 micromol/L for 3OH-PA. The recoveries were 92.9%-106.1%. No deterioration was noted on the columns after 500 chromatographic runs. If the new method had been used as a 2nd-tier test for the 124 samples, only the 5 true positives would have been recalled for additional samples, and the positive predictive value would have been 100%. CONCLUSIONS: This method has the potential to markedly reduce false-positive results and the associated costs and anxiety. It may also be suitable for diagnosing and routinely monitoring blood spots for methylmalonic aciduria and propionic acidemia.     

An improved assay for plasma methylmalonic acid using chemical ionization gas chromatography mass spectrometry

OBJECTIVES: To develop a precise and sensitive assay for methylmalonic acid (MMA) using positive chemical ionization gas chromatography mass spectrometry (CI GC-MS), and to illustrate its clinical utility. METHODS: Using the developed assay, reference intervals were determined with 108 ambulatory individuals, and potential clinical utility examined in 178 consecutive patients with possible cobalamin deficiency (serum B12<200 nmol/L). RESULTS AND CONCLUSIONS: Methylmalonic acid measured by CI GC-MS was precise (CV: 4-5%), and sensitive (limit of quantitation: 37 nmol/L). In a clinical reference set, 37% of individuals with serum B12 less than 200 pmol/L had plasma MMA concentrations within the reference interval (75-378 nmol/L), rendering cobalamin deficiency unlikely. The observation illustrates that MMA assay may be a useful adjunct test in assessing patients with low serum B12.     

Urine methylmalonic acid measurements for the assessment of cobalamin deficiency related to neuropsychiatric disorders

BACKGROUND: Detection of cobalamin deficiency is clinically important for a better understanding of neuropsychiatric diseases, and why the deficiency occurs more frequently than previously anticipated. However, serum cobalamin measurements have a limited ability to diagnose a deficiency state. OBJECTIVE: To evaluate functional cobalamin status in neuropsychiatric patients using an appropriate photometric urine methylmalonic acid (MMA) determination method that could be easily adapted to all routine clinical laboratories. METHODS: We modified the old photometric method used for determining urinary MMA concentrations. MMA measurements were made in first morning urine samples with normalizing by creatinine concentrations. The serum cobalamin, total homocysteine (tHcy), folate, red cell folate, and urinary MMA concentrations taken from 17 psychosis, 28 depression, 16 dementia patients and 47 healthy people were analyzed using the ROC, correlation and multiple regression analysis.RESULTS: The modified method was found to have better recovery (96-103%) and CV% values than the old method. Mean +/- SDs of uMMA and cobalamin concentrations were 11.49 +/- 4.93 mmol/mol creatinine, and 231 +/- 151 pg/mL in psychosis and depression group, and 6.04 +/- 1.93 mmol/mol creatinine and 308 +/- 140 pg/mL in control group, respectively. Those in the dementia group were 11.53 +/- 4.0 mmol/mol creatinine and 231 +/- 84 pg/mL, and in the control group 6.05 +/- 1.94 mmol/mol creatinine and 364 +/- 188 pg/mL. There was a good correlation between urinary MMA and serum Vitamin B(12) determinations for all groups at a confidence level (p) of 99%. The correlation between urinary MMA and red cell folate was also significant at p = 95% for depression, psychosis and control groups, and p = 99% for dementia group. In the ROC analyses, area under the curve values for uMMA, B12 and tHcy were 0.842, 0.796 and 0.728, respectively. CONCLUSIONS: A sensitive and easy photometric method has been presented. When cobalamin deficiency is suspected in neuropsychiatric patients, photometric urinary MMA determination analysis can be the first diagnostic test used. If the urinary MMA concentration is above the reference value, serum cobalamin levels can be determined for further diagnosis.     

Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry

BACKGROUND: The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography-mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. METHODS: Serum or plasma samples (100 microL) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were analyzed by GC-MS in the selected-ion monitoring mode. RESULTS: Detection limits (signal-to-noise ratio, 5:1) were between 0.03 micromol/L (MMA) and 10 micromol/L (Ser, tCys). The assay was linear to 100 micromol/L for MMA and tHcy and to 1000 micromol/L for Met, tCys, Ser, and Gly. The within-day CVs ranged from 0.7% to 3.6% (n = 20), and the between-day CVs from 2.1% to 8.1% (n = 20). The recovery was between 79% and 99% for the different analytes. CONCLUSION: This assay combines a simple and automated sample preparation with selective and sensitive GC-MS analysis and is well suited for the combined measurement of MMA, tHcy, and the related amino acids.     

Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B(12) deficiency. METHODS: We added 1 nmol of the internal standard MMA-d(3) to 500 microL of plasma or 100 microL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 degrees C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d(3,) respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. RESULTS: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 micromol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030 x -0.032 (S(y|x) = 1.03 micromol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3. 8-8.5% and 1.3-3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 micromol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 micromol/L), 96.0% (0.60 micromol/L), and 94.8% (2.02 micromol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100-150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. CONCLUSIONS: This method is well suited for large-scale MMA testing (> or =100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.     

Comparison of serum and plasma methylmalonic acid measurements in 13 laboratories: An international study

BACKGROUND: Detection of cobalamin deficiency is increasingly important, and methylmalonic acid (MMA) appears to be a useful marker. Information on interlaboratory variation and on methodological differences for MMA in serum and plasma is limited. METHODS: Using gas chromatography/mass spectrometry, 13 laboratories participated in a 2-day analysis of 8 serum and 11 EDTA-plasma specimens. Results were analyzed for imprecision, recovery, and differences among laboratories and methods. RESULTS: The mean among-laboratory imprecision (CV) was 19% and 21% for serum and plasma samples, respectively, and 9.3% and 7.8% for serum and plasma samples with added MMA, respectively. The mean within-laboratory (among-run) CV was 13% for both serum and plasma samples and 5.2% and 4.9% for serum and plasma samples with added MMA. Within-method imprecision was the same or higher than among-method imprecision. The mean among-laboratory recovery of MMA was 105% and 95% in serum and plasma, respectively. Most laboratories showed a proportional bias relative to the consensus mean of up to 15%. Two laboratories reported results that on average were almost 30% higher than the consensus mean. CONCLUSIONS: No method differences were found, but significant among-laboratory imprecision was found in the present study. Improvements are needed to reduce the analytical imprecision of most laboratories, and attention must be focused on calibration issues. Differences among laboratories can be improved by introducing high-quality reference materials and by instituting external quality assessment programs.     

Analysis of methylmalonic acid in plasma by liquid chromatography-tandem mass spectrometry

BACKGROUND: Methylmalonic acid (MMA) is a biochemical marker for cobalamin deficiency, particularly in cases where the cobalamin concentration is moderately decreased or in the low-normal range. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is a rapid, robust method that has been used in MMA analysis. We developed a simple method combining solid-phase extraction (SPE) and derivatization to prepare serum or plasma for LC-MS/MS analysis of MMA. METHODS: Deuterated internal standard d(3)-MMA was added to serum or plasma before SPE on strong anion-exchange (SAX) columns. After elution with HCl-butanol (10:90 by volume) and addition of 1 g/L formic acid, the samples were simultaneously derivatized and evaporated by heating to 70 degrees C for 15 min followed by 54 degrees C overnight in uncapped vials. Acetonitrile and 1 g/L formic acid were added to the samples before injection into the LC-MS/MS system. MMA and d(3)-MMA were quantified in the multiple-reaction monitoring mode. Calibrators were prepared in serum by the standard addition method. RESULTS: The MMA assay was linear up to 200 micromol/L. Interassay CVs were 6.7%, 5.0%, and 5.0% for mean concentrations of 0.15, 0.36, and 0.65 micromol/L, respectively. CONCLUSIONS: Our simplified sample preparation and derivatization method is suitable for use in MMA analyses. MMA elutes with the derivatization reagent, and derivatization and evaporation are performed simply by leaving the uncapped vials in a heating block overnight. The method shows good linearity and precision.     

高效液相色谱-荧光检测法在慢性肾功能不全患者血清芳香族氨基酸检测中的临床价值

目的 建立同时测定血清AAA含量的HPLC-FLD法,探讨CRI患者血清AAA含量变化及其临床应用价值。方法血清标本来自于100名健康体检者和80例CRI患者。将CRI患者按2002年美国肾脏基金会(NKF)诊断分期标准进行分期：CKD 2期4例、CKD 3期12例、CKD 4期12例和CKD 5期52例;按CRI不同病因分组：慢性肾炎型32例、糖尿病型36例和高血压型12例。血清经高氯酸去蛋白后,离心取上清液测定,外标法定量。采用Megres C18色谱柱,流动相为乙腈：水（体积比为1∶9）,流速为1.0 ml/min,荧光检测器在不同时间段设定特定波长对血清AAA进行测定。对健康对照组和CRI患者组血清中AAA总量、Tyr、Phe和Trp含量及不同分期和不同病因CRI患者血清Tyr、Phe和Trp含量进行比较,同时评价血清AAA总量诊断CRI的敏感度与特异度。结果Tyr、Phe和Trp线性范围分别为0.550～275.000、3.050 ～1 220.000和0.049～49.000 μmol/L,最低检测限分别为0.014、0.500和0.005 μmol/L,平均回收率分别为100.9％、101.3％和98.5％,日内精密度为2.32％～3.92％（平均为3.13％）,日间精密度为3.18％ ～4.20％（平均为3.58％）。CRI患者组血清AAA总量、Tyr、Trp含量及Tyr/Phe比值分别为(135.74±12.23)、(52.27±8.25)、(21.49±4.25) μmoL/L和[0.87（0.68 ～1.05）],低于健康对照组的(174.47±11.57)、（63.53±4.68）、(44.22±3.67) μmol/L和[0.97（0.94～1.00）],差异均有统计学意义（t=- 14.709、4.452、22.100,U=266.000,P均＜0.05）。不同分期CRI患者Tyr、Phe和Trp含量差异无统计学意义;Tyr含量在慢性肾炎组、高血压组和糖尿病组间差异无统计学意义,Phe在慢性肾炎组与高血压组、慢性肾炎组与糖尿病组间差异有统计学意义（U= 114.00、395.00,P均＜0.05）,Trp在慢性肾炎组与糖尿病组间差异有统计学意义(U=349.00,P＜0.05)。血清AAA总量诊断CRI的敏感度、特异度分别为90％ (72/80)和100％ (100/100)。结论HPLC-FLD法测定血清AAA简便、快速,敏感度高及特异度好,同时测定血清AAA含量对CRI患者的诊断和评价有一定价值。