| Epidemiological studies (United States Renal Data System, European data, et al.) about end-stage renal disease(ESRD) show that the number of ESRD patients continues to grow around the world, and the annual number of new patients exceeds the population growth. Maintenance hemodialysis, with a history of around50years, is a conventional treatment for ESRD and is the most widely technology for renal replacement.Large amounts of toxins are difficult to remove on account of renal failure. In view of the actual conditions of China, most of ESRD patients choose hemodialysis. After drawing the blood out of body, hemodialysis separates the blood and dialysate dialysis by dialysis membrane, then through diffusion, convection and ultrafiltration to excrete water and metabolic wastes, finally achieve the goal of keeping the balance of water and electrolyte,correcting acidosis and so on. However, only about one-tenth of normal renal function can be replaced by hemodialysis. In addition, as an blood purification methods in vitro, hemodialysis make patients exposure to cardiopulmonary bypass system such as needle, dialyzers, dialysis tubing, etc. Furthermore, patients have to shoulder the burdens of economy, spirit, illnesses by reason of the frequent dialysis times (2-4times per week), a steady stream of economic costs and various of complications. a variety of complications will ensue with time in hemodialysis, which is often prone to malnutrition, immune disorders, restless legs syndrome (RLS), depression, anxiety, sleep disorders, calcium and phosphorus metabolism disorders, anemia, etc.Malnutrition and immune disorders are the characteristic features of maintenance hemodialysis patients. At present, a large number of study indicate that malnutrition is one of the biggest causes of death in dialysis patients. Mainly as it can make the patient’s immune function and physical activity decline, which is associated with various infections and non-infectious complications and is a independent risk factor for elevated mortality in uremia. In addition, the high rate of mortality of cardiovascular disease in uremic patients is related to change of immune system, whose innate and adaptive immune function is double damaged. Inflammation-immune disorders is also one of the important mechanisms of cardiovascular disease in patients with uremia, immune activation can lead to micro-inflammation, causing the occurrence of coronary atherosclerosis and cardiovascular events. The immune disorder is a major cause of most other complications, too. Therefore, study and intervention nutritional status and immune function of MHD have research and clinical significance for reducing the incidence of infections, cardiovascular events, and improving the prognosis of MHD.What’ more, immune dysfunction is the chief cause of the high incidence of infection and tumors in dialysis patients. Tryptophan (Trp) is an essential amino acids for human, which take part in the synthesis and metabolism of proteins. Kynurenine(Kyn) is the final product of tryptophan metabolism, indoleamine2,3dioxygenase (IDO) is the key enzyme when tryptophan is metabolized to kynurenine. immune tolerance is an important mechanism of immune disorders for uremic patients. Dendritic cell population can be mediated the immune tolerance by induced regulatory T cells. Now that the IDO-expressing dendritic cells are key molecules of the immune tolerance, using IDO inhibitors can induce T-cell mediated rejection rapidly, so IDO is considered to be an important mechanism for regulating the immune balance. Previous studies have also found that Trp and its metabolites Kyn involved in immune regulation, the Trp/Kyn levels of uremic patients is associated with low immune response and cardiovascular disease, However, there is no clear answer for the current level of IDO and immune disorders mechanism in MHD.Quantitative analysis Kyn and Trp in plasma can provide a theoretical basis for understanding nutritional status and immune function of MHD, which inhabit some research and clinical significance. High Performance Liquid Chromatography(HPLC) is a commonly method for detecting Trp and Kyn. When the test sample flow through the column, due to different adsorption of components. the test sample size separation, The detector were obtained the corresponding signal-time curves of the various components once fow through it, then quantitative them. When using the previous HPLC to detect Trp and Kyn for MHD, we learn that both amino acids were seperated hardly, probably due to the plasma uremic toxins more and complex, they may interfere with each other and difficult to separate. To simplify the process, detected Trp and Kyn in plasma successfully under the same chromatographic conditions for MHD, is the aim to this study.Objection1. To assess the nutritional status in maintenance hemodialysis patients in December2012of blood purification center in Guangdong general Hospital, learn the prevalence of malnutrition and analysis related factors;2. To improved a HPLC to quantitative analysis Kyn and Trp in plasma of MHD simultaneously, then provide a theoretical basis on assess the nutritional status、 immune function and to understand the activity of IDO in dialysis patients.MethodsCross-sectional analysis is the main research method for this study. Through collecting questionnaires, clinical physical measurement, various laboratories and dialysis data to learn the current situation of malnutrition.The basic inclusion criteria are:1) agreed to involved in this investigation and signed an informed consent form;2) hemodialysis time≥3months;3) be able to express their wishes clearly and independently; Using HPLC method to quantitatively detect Try and Kyn in plasma of maintenance hemodialysis patients.On the investigation of the nutritional status, not only do we concerned about the traditional assessment of serum albumin, but also joint malnutrition-inflammation score (MIS) and Objective Score of Nutrition on Dialysis (OSND), which MIS assessment relates to the dry weight changes during th past3-6months, dietary intake, gastrointestinal symptoms, nutrition-related disorders, complications of dialysis and dialysis time, the measured value of fat reserves, muscle wasting, body mass index(BMI), serum albumin, total iron binding capacity, transferrin; OSND assessment including weight changes over the last3-6months, BMI, triceps skinfold thickness, upper arm circumference circumference, ferritin, serum albumin and cholesterol. Which MIS’s ratings criteria which is:<8points, mild malnutrition;9-18points, moderate malnutrition;>18points, severe malnutrition. Which the normal is0, the maximum30points. OSND rating criteria:28-32,23-27minutes into normal moderate malnutrition,≤22minutes of severe malnutrition, OSND normal for5minutes, up to32points. Survey carried out by doctors during dialysis, the same to measure skinfold thickness, arm muscle circumference reserves, the circumference and other clinical measurements. Using the same measure skinfold thickness measurements meter and with the same ruler. Calibration each before measurement. Each indicator measured three times, get the mean value of the measurement data. Collecting the basic data for the same period of dialysis patients,such as the laboratory, dialysis and survey data content, age, underlying disease, hemodialysis time, residual urine volume, hemoglobin, serum iron, parathyroid hormone, dialyzer permeability, blood flow, dialysis mode. Each assessment scale, diagnosis and assess the degreee of severity are assessed by the same researcher.Collecting the relevant data for statistical analysis.Using HPLC to detect Try and Kyn in Plasma of maintenance hemodialysis patients, the column was Sinochrom ODS-BP C18column (4.6mm×150mm, id.5μm), mobile phase15mmol/L sodium acetate-acetic acid (containing5%acetonitrile, PH4.8), mobile phase flow rate1.0mL/min, detection wavelength225mm, take2-3ml whole blood form arteriovenous fistula of maintenance hemodialysis patients before hemodialysis, placed in4ml EDTA tube, separation the plasma with2500r/min rate for5minutes at room temperature during1hour, plasma samples were dealed with5%perchloric acid solution, get20u1supernatant protein samples to analyzed directly.Statistical AnalysisFor normally distributed data ues X±S, any data does not meet the normal distribution then use the median. Using logistic regression analysis method to analyze the ralative factors, P<0.05that the difference statistically significant, apply SPSS17.0statistical software for data analysis.Results1、The nutritional status of MHD in December2012of Blood Purification Center in Guangdong General Hospital:1) A total of217MHD were enrolled, MIS score was9±2points,81people with mild malnutrition, Moderate malnutrition135people,1with severe malnutrition;2) OSND score was12±3points, all were severe malnutrition;3) The serum albumin of84.4%patients below normal,30.4%patients’BMI lower than normal, triceps skinfold thickness14.5±7.0mm, the arm circumference24.6±3.5cm,42.9%of patients with dietary intake disorders,37.1%of patients with functional gastrointestinal discomfort,25%of patients with nutrition-related injuries;4) MIS was correlate with duration of dialysis, gender, Kt/V and Hb; CRP correlated with albumin, uric acid, MIS and OSND.2、On the basis of previous studies, by adjusting the column packing and pore size, condition of the mobile phase, Changed the degree of dissociation and the retention time of Trp and Kyn. the column SymmetryaShieldRP-18column (150mmX3.9mm, id,5μm) column was replaced by Sinochrom ODS-BP C18column (4.6mmX150mm, id,4.5μm). what’s more, adjust the content and pH of the mobile phase of acetonitrile, the mobile phase was15mmol/L sodium acetate-acetic acid (containing5%acetonitrile, PH4.8), flow rate was1.0mL/min, detection wavelength225mm, plasma specimens were5%perchloric acid solution after removal of the supernatant protein samples were analyzed by direct measurement. Kynurenine retention time6.766min, the linear range of0.08-50μmol/L, the minimum detectable concentration0.02μmol/L, tryptophan retention time13.072min, the linear range of0.8-500μmol/L, the minimum detectable concentration was0.2μmol/L, kynurenine and tryptophan days, the relative standard deviation of day were less than4%.Conclusions1. Malnutrition is an independent risk factor for the prognosis of patients with uremia. MIS is more suitable for Chinese people. Malnutrition is prevalent in maintenance hemodialysis patients, It is necessary to assess regularly, manage generally and intervent timely; 2. On the basis of previous study, by adjusting the diameter of the column, PH value and other methods of mobile phase by HPLC. We detected the Kyn and Trp under the same conditions successfully in maintenance hemodialysis patients. The method is simple,fast, stable, which can be used in clinical and research work. |
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