中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2009年
12期
1354-1359
,共6页
罗昔波%唐爱国%莫喜明%皮兰敢%敖翔
囉昔波%唐愛國%莫喜明%皮蘭敢%敖翔
라석파%당애국%막희명%피란감%오상
色谱法%高压液相%犬尿氨酸%色氨酸%关节炎%类风湿
色譜法%高壓液相%犬尿氨痠%色氨痠%關節炎%類風濕
색보법%고압액상%견뇨안산%색안산%관절염%류풍습
Chromatography%high performance liquid%Kynurenine%Tryptophan%Rheumatoid arthritis
目的 探讨高效液相色谱荧光检测法(HPLC-FLD)同时测定血清色氨酸(TRP)和犬尿氨酸(KYN)对诊断类风湿关节炎(RA)的临床意义.方法 血清标本加等量5%(V/V)高氯酸溶液去除蛋白,离心取上清液20 μl,直接进样分析.色谱柱为HpemilC_8柱(300 mm×6.0 mm i.d,10 μm);流动相为0.25 mol/L醋酸锌和50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 ml/min;0～10 min荧光检测器的激发波长和发射波长分别为365 nm和480 nm,10 min后激发波长和发射波长分别变换为254 nm和404 nm.同时,用该方法测定120名健康成人和110例RA患者血清TRP、KYN和TRP/KYN比值(K/T),并评价其诊断RA的敏感度、特异度和方法学效能.结果 血清标本的KYN和TRP保留时间分别为8.1和11.5 min,两者分离良好.KYN线性范围为0.098 ～19.600 μmol/L,最低检测浓度为0.04 μmol/L;回收率为90.8%～96.2%,日内变异系数为3.68%,日间变异率为4.97%.TRP的线性范围为4.9～196.0μmol/L.最低检测限为0.005 μmol/L,回收率为92.6～106.9%,13内变异系数为3.63%,日间变异系数为4.44%.在本试验的色谱条件下测定苯丙氨酸(Phe)、酪氨酸(Tyr)、犬尿喹啉酸(KYNA)、5-羟色胺(5-HT)和Cr均无干扰.RA组患者血清KYN含量和K/T比值[(2.06±0.38) μmol/L和(55.46±5.81)×10~(-3)]与健康对照组[(1.51±0.35)μmol/L和(32.54 ±9.00)×~(-3)]比较,均显著升高(U=3 251.0,t=10 741,P均为0.000),而RA组TRP含量[(38.24±5.27)μmol/L]与健康对照组[(47.52±5.79)μmol/L]比较,则显著降低(t=10.399,P=0.000).K/T比值诊断RA的敏感度、特异度分别为83.6%(92/110)、85.8%(103/120).结论 HPLC-FLD同时测定血清TRP和KYN的方法精密度、回收率、抗干扰能力、线性范围等均符合临床检测要求.K/T比值可作为RA的辅助性诊断指标.
目的 探討高效液相色譜熒光檢測法(HPLC-FLD)同時測定血清色氨痠(TRP)和犬尿氨痠(KYN)對診斷類風濕關節炎(RA)的臨床意義.方法 血清標本加等量5%(V/V)高氯痠溶液去除蛋白,離心取上清液20 μl,直接進樣分析.色譜柱為HpemilC_8柱(300 mm×6.0 mm i.d,10 μm);流動相為0.25 mol/L醋痠鋅和50 mmol/L醋痠溶液(含3%乙腈),流速為1.5 ml/min;0～10 min熒光檢測器的激髮波長和髮射波長分彆為365 nm和480 nm,10 min後激髮波長和髮射波長分彆變換為254 nm和404 nm.同時,用該方法測定120名健康成人和110例RA患者血清TRP、KYN和TRP/KYN比值(K/T),併評價其診斷RA的敏感度、特異度和方法學效能.結果 血清標本的KYN和TRP保留時間分彆為8.1和11.5 min,兩者分離良好.KYN線性範圍為0.098 ～19.600 μmol/L,最低檢測濃度為0.04 μmol/L;迴收率為90.8%～96.2%,日內變異繫數為3.68%,日間變異率為4.97%.TRP的線性範圍為4.9～196.0μmol/L.最低檢測限為0.005 μmol/L,迴收率為92.6～106.9%,13內變異繫數為3.63%,日間變異繫數為4.44%.在本試驗的色譜條件下測定苯丙氨痠(Phe)、酪氨痠(Tyr)、犬尿喹啉痠(KYNA)、5-羥色胺(5-HT)和Cr均無榦擾.RA組患者血清KYN含量和K/T比值[(2.06±0.38) μmol/L和(55.46±5.81)×10~(-3)]與健康對照組[(1.51±0.35)μmol/L和(32.54 ±9.00)×~(-3)]比較,均顯著升高(U=3 251.0,t=10 741,P均為0.000),而RA組TRP含量[(38.24±5.27)μmol/L]與健康對照組[(47.52±5.79)μmol/L]比較,則顯著降低(t=10.399,P=0.000).K/T比值診斷RA的敏感度、特異度分彆為83.6%(92/110)、85.8%(103/120).結論 HPLC-FLD同時測定血清TRP和KYN的方法精密度、迴收率、抗榦擾能力、線性範圍等均符閤臨床檢測要求.K/T比值可作為RA的輔助性診斷指標.
목적 탐토고효액상색보형광검측법(HPLC-FLD)동시측정혈청색안산(TRP)화견뇨안산(KYN)대진단류풍습관절염(RA)적림상의의.방법 혈청표본가등량5%(V/V)고록산용액거제단백,리심취상청액20 μl,직접진양분석.색보주위HpemilC_8주(300 mm×6.0 mm i.d,10 μm);류동상위0.25 mol/L작산자화50 mmol/L작산용액(함3%을정),류속위1.5 ml/min;0～10 min형광검측기적격발파장화발사파장분별위365 nm화480 nm,10 min후격발파장화발사파장분별변환위254 nm화404 nm.동시,용해방법측정120명건강성인화110례RA환자혈청TRP、KYN화TRP/KYN비치(K/T),병평개기진단RA적민감도、특이도화방법학효능.결과 혈청표본적KYN화TRP보류시간분별위8.1화11.5 min,량자분리량호.KYN선성범위위0.098 ～19.600 μmol/L,최저검측농도위0.04 μmol/L;회수솔위90.8%～96.2%,일내변이계수위3.68%,일간변이솔위4.97%.TRP적선성범위위4.9～196.0μmol/L.최저검측한위0.005 μmol/L,회수솔위92.6～106.9%,13내변이계수위3.63%,일간변이계수위4.44%.재본시험적색보조건하측정분병안산(Phe)、락안산(Tyr)、견뇨규람산(KYNA)、5-간색알(5-HT)화Cr균무간우.RA조환자혈청KYN함량화K/T비치[(2.06±0.38) μmol/L화(55.46±5.81)×10~(-3)]여건강대조조[(1.51±0.35)μmol/L화(32.54 ±9.00)×~(-3)]비교,균현저승고(U=3 251.0,t=10 741,P균위0.000),이RA조TRP함량[(38.24±5.27)μmol/L]여건강대조조[(47.52±5.79)μmol/L]비교,칙현저강저(t=10.399,P=0.000).K/T비치진단RA적민감도、특이도분별위83.6%(92/110)、85.8%(103/120).결론 HPLC-FLD동시측정혈청TRP화KYN적방법정밀도、회수솔、항간우능력、선성범위등균부합림상검측요구.K/T비치가작위RA적보조성진단지표.
Objective To explore the clinical significance of serum kynurenine and tryptophan by HPLC fluorescence detection in patients with rheumatoid arthritis ( RA). Methods Serum samples were deproteinized by equal volume of 5% ( V/V) perchloric acid. It employed a Hypersil C8 column and a mobile phase consisted of 0. 25 mol/L zinc acetate, 50 mmol/L acetic acid with 3% (V/V) acetonitrile at a flow rate of 1. 5 ml/min. The fluorescence excitation and emission wavelengths were operated at 365 nm and 480 nm respectively at the beginning of the run, and 10 minutes later, the excitation and emission wavelength changed to 254 nm and 404 nm. TRP and KYN of health group and RA group were determined by HPLC-FLD. Results The retention time of KYN was 8. 1 min;the linearity was from 0. 098 μmol/L to 19. 600 μmol/L with the detection limit of 0. 04 μmol/L , the recovery were 90. 8%-96. 2% , and the intra-day and inter-day variations were 3. 68% and 4. 97% respectively. The retention time of TRP was 11. 5 min, the linearity of the assay was from 4. 9 μmol/L to 196. 0 junol/L, the detection limits was 0. 005 μmol/L,the recovery of TRP was 92. 6% -106. 9% , the intraday and interday coefficients of variations were 3. 63% and 4.44% .respectively. Phenylalanine, tyrosine, serotonin, kynurenic acid and creatinine didn't interfere in the assay. Compared with healthy subjects, there were significantly increased concentration of serum KYN and K/T Ratio [(1.51 ±0.35) nol/L vs(2. 06 ±0. 38) ujnol/L,tf = 3 251. 0,P =0. 000; (32. 54 ± 9. 00) × 10~(-3) vs(55. 46 ±5. 81) × 10~(-3) ,t = 10 741 ,P =0. 000] and significantly decreased concentrations of TRP [(47.52±5.79) μmol/L vs (38.24±5.27) μmol/L; t = 10. 399,P =0. 000) ] in the patients with RA. The sensitivity, specificity of K/T ratio for diagnsis RA was 83. 6% (92/110) and 85. 8 ( 103/ 120)% respectively. Conclusions A new method is established for simultaneous determination of KYN and TRP in serum by HPLC-FLD with on-column derivatization. The method is simple and rapid, and its precision, sensitivity and specificity are satisfactory for clinical and scientific study. K/T Ratio is a supporting diagnosis biomarker of RA.
