高效液相色谱-蒸发光散射法测定人肠黏膜通透性的方法学及其临床应用研究

目的进行高效液相色谱-蒸发光散射法测定人肠黏膜通通性的方法学研究，同时应用此方法研究重度融性乙型病毒性肝炎患者肠道屏障功能的变化并探讨其治疗方法。方法（1）采用高效液相色谱-蒸发光散射法检测30例健康人（正常组）和90例重度慢性乙型病毒性肝炎患者尿乳果糖、甘露醇排泄率比值（L／M）的变化，同时测定血清二胺氧化酶（DAO）的变化，以此两项指标来反映肠道屏障功能的变化并将两组加以比较。（2）将90例重度慢性乙型病毒性肝炎患者按随机数字表法分为对照组、乳果糖组，谷氨酰胺组3组，每组30例。对照组采用常规治疗；乳果糖组在常规治疗的基础上加用乳果糖10g，3：L／d；谷氨酰胺组在常规治疗的基础上加用谷氨酰胺颗粒10g，3：L／d。疗程均为2周。于治疗前后分别测定3组患者的肝功能、内毒素（ET）、肿瘤坏死因子（TNF-α）、DAO、白介素-2（IL-2）、白介素-8（IL-8）、T淋巴细胞亚群、L／M等，并采用协方差分析法加以比较。结果采用高效液相色谱-蒸发光散射法测定L／M的变化，甘露醇和乳果糖能得到很好地分离，乳果糖的线性范围为0．65—13．0μg/L（r=0．9980），甘露醇的线性范围为0．74～14．7／μg／L（r=0．9983）。乳果糖和甘露醇日内精密度的相对标准偏差（RSD）分别为1．56％、0．65％（n=6），日间精密度的RSD分别为1．77％、0．99％（n=6）。乳果糖的加样回收率为98．68％～100．4％，平均值为99．51％；甘露醇的加样回收率为99．52％-100．5％，平均值为99．94％。乳果糖在尿中的最低检测浓度为94．2-L，甘露醇在尿中的最低检测浓度为29．9μg/L。重度慢性乙型病毒性肝炎患者L／M、DAO水平明显升高（P〈0．01）。治疗2周后，与对照组相比，乳果糖组TBIL、ET、TNF-α的变化差异有统计学意义（P〈0．05）；与对照?

Clinical Study and Technology of High Performance Liquid Chromatography with Evaporative Light Scattering Detector in Human Intestinal Permeability Evaluation

Objective To establish a new method that can detect the lactulose（L）and mannitol（M）in urine by high performance liquid chromatography with evaporative light scattering detector （HPLC-ELSD）, and study the change of intestinal barrier in patients with heavy degree chronic hepatitis B and clinical therapy simultaneously. Methods （ 1 ） 30 healthy people （ normal group） and 90 cases of heavy degree chronic hepatitis B （liver disease group）were measured the change of intestinal permeability by urine lactulose/mannitol ratio （L/M） with HPLC-ELSD. Meanwhile ,the serum diamine oxidase （DAO）was measured respectively and compared each other. （2）90 cases heavy degree chronic hepatitis B patients were randomly divided into three groups：the control group, the lactulose group and the glntamine group,each group had 30 cases. In the control group,the patients were treated as usual,in the lactulose group,the patients were added lactulose 10 g,thrice a day besides usual treatment. In the glutamine group, the patients were added glutamine 10 g, thrice a day, besides usual treatment. The total treatment period was two weeks. Before and after treatment ,parameters such as liver function,endotoxin（ET） ,tumor necrosis factor-α （TNF-α）, DAO, interleukin-2 （ IL-2 ）, interleukin-8 （ IL-8 ）, T lymphocyte subgroup and L/M in the three groups were measured respectively, which were comparsed after two weeks＇treatment. Results （ 1 ） Lactulose and mannitol could be well separated by this method. The linear range of laetulose was 0. 65 - 13.0 μg/L（ r = 0. 9980） ,the linear range of mannitol was 0. 74 - 14.7 μg/L（ r = 0. 9983 ）. The within-day precision relative standard deviation （RSD） was 0. 65% for lactulose and 1.56% for mannitol. The recovery rate ranged from 98.68% - 100.5% for lactulose and 99.52% - 100.4% for mannitol. The lowest detection limit of lactulose and rnannitol were 94.2 μg/L and 29.9 μg/L,respeetively. （2）Compared with healthy people,in heavy degr