改良微柱法检测17-OH，17-KS和VMA及其临床应用

Clinical Application of Detecting 17-OH, 17-KS and VMA by a Modified Micro Column Method

目的完善并改良微柱分光光度检测技术，探讨其在肾上腺肿瘤、心血管等疾病中的应用价值。方法选取2010年4月～2013年3月济南军区总医院住院肾上腺肿瘤患者181例，高血压和冠心病患者168例作为实验组，同时以30例健康人作为对照组。所有研究对象均采用微柱分光光度比色法，进行24h尿液17羟皮质类固醇（17Hydroxycorticosteroids，17-OH）、17酮皮质类固醇（17-Ketosteroids，17KS）和香草扁桃酸（VanilmandelicAcid，VMA）的水平检测。在常规检测方法基础上，17—0H采用活性高岭土作为吸附剂，过柱后采用70ml／dl的无水乙醇作为洗脱液收集待测成分；17=KS检测在加热水解后，以3000r／min离心5min后再行过柱，Zimmermann显色过程稳定在4℃，60min；VMA检测则将样本PH值调至6．5～7．0时再行过柱，比色时在做样品测定的同时做试剂空白、样品空白。以蒸馏水调零测标准及其空白、样本及其空白，最后计算时将各自空白扣除，并进行回收率、精密度等方法学评价。结果①应用此方法检测17—0H，17-KS及VMA的平均回收率分别为76％，98．8％和90．8％；平均批内变异系数为9．5％，5．9％和3．8％；平均批间变异系数为10．0％，5．6％和8．4％。②17—0H在肾上腺良性肿瘤组、肾上腺恶性肿瘤组、嗜铬细胞瘤组、腹膜后肿瘤组、其他恶性肿瘤组、高血压组、冠心病组和对照组中的水平（μmol／24h）分别为30．49±18．61，34．51±18．59，32．08±10．89，30．74±25．53，67．25±27．06，44．83±32．63，39．59q-24．93和18．90±3．98，差异均有统计学意义（t=3．08，P〈0．05；t=3．19，P〈0．05；t=3．11，P〈0．05；t=2．93，P〈0．05，t=10．248，P〈0．01；t=5．19，P〈0．01；t=4．01，P〈0．01）；17=KS在肾上腺良性肿瘤组、嗜铬细胞瘤组、肾上腺恶性肿瘤组、其他恶性肿瘤组、高血压组、冠心病组和对照组中的水平（μmol／24h）分别为50．66±17．28，57．62±31．51，60．78±24．78，89．20±33．29，79．21±32．45，68．57±36．22和39．46±3．74，差异均有统计学意义（t=2．37，P〈0．05；t=3．43，P〈0．05；t=8．19，P〈0．01；t=9．99，P〈0．01，t=9．78，P〈0．01；t=7．47，P〈0．01）；VMA在肾上腺恶性肿瘤组、其他恶性肿瘤组、嗜铬细胞瘤组和对照组中的水平（μmol／24h）分别为49．34±42．29，48．97±28．92，114．63±82．94和30．85±6．09，差异均有统计学意义（t=3．01，P〈0．05；t=2．5，P〈0．05；t=7．2，P〈0．01）。③以大于17—0H，17-KS和VMA正常高值为阳性，17-OH，17-KS和VMA在肾上腺良性肿瘤组、肾上腺恶性肿瘤组、嗜铬细胞瘤组、腹膜后肿瘤组、其他恶性肿瘤组、高血压组和冠心病组的阳性栓出率分别为25％（22／89），33％（29／89）和12％（11／89）；25％（4／16），31％（5／16）和19％（3／16）；21％（7／34），44％（15／34）和79％（27／34）；20％（1／5），20oA（1／5）和20％（1／5）；63％（10／16），50％（8／16）和38％（6／16）；32％（53／168），45％（75／168）和15％（25／168）；43％（9／21），48％（10／21）和14％（3／21）。结论微柱法17—OH，17-KS及VMA的检测方法经改良后其稳定性、重复性和精密度较好，对临床肾上腺肿瘤、心血管等疾病具有=定的诊断和鉴别诊断价值。

Objective To perfect and modify micro column spectrophotometric detection technology,discusses its application value in adrenal tumors,cardi0vascular and other diseases. Methods Collected 181 cases of adrenal tumor patients, 168 cases with hypertension and coronary heart disease patients as the experimental group, 30 cases in the control group from April 2010 to March 2013 in General Hospital of Jinan Military Area. All the objects were detected for 24h urinary 17 hydroxycor- ticosteroid （ 17-ttydroxycorticosteroids, 17-OH ）, 17 ketone corticosteroids （ 17-Ketosteroids, 17-KS） and vanillylmandelic acid （Vanilmandelic, Acid,VMA）） level by micro column spectrophotometric colorimetry. On the basis of conventional de- tection methods, 17-OH used active kaolin as adsorbent,column by 70 ml/dl ethanol as eluant collection component;Detec- tion of 17-KS in heating after hydrolysis,to 3 000 r/min centrifugal 5 min after the column,Zimmermann color process was stable at 4℃ for 60 minutes. VMA detection would regulate pH to 6.5-7.0 and column,colorimetry in determination of the reagent blank, sample blank. Distilled water zero measuring standard and blank, sample and blank, the calculation would de- duct each blank subtraction,and did the recovery, precision evaluation. Results ①The application of this method in detection of 17 OH, the average recovery of 17-KS and VMA was 76 %, 98.8 % and 90.8 % respectively; the average batch coefficientof variation was 9.5%,5.9% and 3.8% respectively;average intraassay coefficient of variation was 10% ,5.6% and 8.40/00 respectively. ②17-OH in adrenal benign tumor group, adrenal malignant tumor group, pheochromocytoma group, retr0perito- neal tumor group,other malignant tumor group, hypertension, coronary heart disease group and the control group in the lev- els were 30.49±18.61 μmol/24 h,34.51±18.59 μmol/24 h,32.08±10.89 gmol/24 h,30. 74±25. 53 μmol/24 h,67.25± 27.06 μmol/24 h,44.83±32.63 μmol/24 h,39.59±24.93 μmol/24 h and 18.90±3.98 gmol/24 h respectively,and the difference was statistically significant （t = 3.08, P 〈 0.05 ; t = 3.19, P 〈 0.05 % t = 3.11, P 〈 0.05 ; t = 2.93, P 〈 0.05, t = 10.248, t = 5.19, P〈 0.01 ; P〈 0.01 ; t = 4.01, P〈 0.01 ） ; 17-KS in adrenal benign tumor group, pheochromocytoma, adrenal malignant tumor group group, other malignant tumor group, hypertension, coronary heart disease group and the control group in the levels were 50.66 ± 17.28 μmol/24 h, 57.62 4 31.51 μmol/24 h, 60.78 ± 24.78 μmol/24 h, 89.20 ± 33.29 μmol/24 h,79.21±32.45 μmol/24 h,68.57±36.22 μmol/24 h and 39.46±3.74 μmol/24 h respectively,and the difference was statistically significant （t=2.37,P〈0.05;t=3.43,P〈0.05;t=8.19,P〈0.01;t=9.99,P〈0.01,t=9.78,P〈0.01; t= 7.47,P〈0.01）;VMA in adrenal malignant tumor group, other malignant tumor group, pheochromocytoma group, and control group in the levels were 49.34±42.29 〉mol/24 h,48.97±28.92 μmol/24 h,l14. 63±82. 94 μmol/24 h and 30.85 ±6.09μmol/24 h respectively, and the difference was statistically significant （t= 3.01, P〈0.05 ; t= 2.95, P〈0.05 ; t= 7.29,P〈0.01）. ③At greater than 17 OH,17 KS,VMA normal value for positive,17-OH,17-KS and VMA in adrenal be nign tumor group, adrenal malignant tumor group, pheochromocytoma group, retroperitoneal tumor group, other malignant tumor group, hypertension and coronary heart disease group, the positive rates were 25 % （ 22/89 ）, 33 %/0 （ 29/89 ）, 12 % （ 11 / 89） ;25%（4/16） ,g1%（5/16） ,19%（3/16） %21% （7/34）,44% （15/34）,79% （27/34） ;20% （1/5）,20% （1/5）,20% （1/5） 63%（%/%6）%5%（8/%6）%38%（6/16）%32%（53/%68）%45%（75/%68）%5%（25/%68）;43%（9/2%）,48%（%/2%）,%4%（3/ 21） respectively. Conclusion Improved micro column method for deteding 17-OH, 17-KS and VMA had the stability, re- peatability and precision. It has the value of diagnosis and differential diagnosis of adrenal tumors and the clinical cardiovas- cular disease.