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 glutamine 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 lactulose 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 mannitol were 94.2 μg/L and 29.9 μg/L,respectively.(2)Compared with healthy people,in heavy degree chronic hepatitis B patients,the level of L/M and DAO were all significantly raised(P0.01).(2)Compared with control group,in the lactulose group,TBIL,ET and TNF-α changed significantly after two weeks' treatment(P0.05).Compared with control group,ALT,TBIL,TNF-α,ET,IL-2,L/M and DAO in the glutamine group were changed significantly after treatment(P0.05).Compared with the lactulose group,ALT,TBIL,TNF-α,IL-2 and L/M in the glutamine group changed significantly after treatment (P0.05).Conclusion (1) HPLC-ELSD is a good,precise,simple and convenient method to evaluate the intestinal permeability change in clinical practice.(2)There is the intestinal barrier damage in heavy degree chronic hepatitis B patients.(3)Compared with control group,lactulose or glutamine add-on therapy can ameliorates the endotoxemia and inflammatory response,attenuates the liver function,but the glutamine's clinical effect is better,and can also reverse the intestinal barrier damage.