维持性血液透析患者营养状态评估及横断面研究

发布时间：2018-05-20 21:57

本文选题：血液透析 + 营养不良　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景终末期肾脏病(end-stage renal disease, ESRD)的流行病学研究(美国肾脏病数据库系统及欧洲等资料)显示全球ESRD人数仍在持续增加,每年新增的人数超过了人口的年增长率。维持性血液透析是ESRD的一种常规治疗,也是目前应用最多的肾脏替代治疗,至今已有50余年的历史。由于肾脏功能的衰竭,大量的毒素蓄积在体内难以清除。在中国,鉴于目前的国情,绝大部分ESRD患者选择了血液透析治疗。血液透析将血液引出体外后,借助透析膜分隔血液和透析液,主要通过扩散、对流、超滤三种方式达到部分替代正常肾脏排泄水分、代谢废物、调节水电解质平衡、纠正酸中毒等功能。一般血液透析治疗仅能替代正常肾脏功能的十分之一左右。血液透析是一种体外的血液净化手段,维持性血液透析患者(maintenance hemodialysis patients, MHD)经常接触到体外循环系统如穿刺针、透析器、透析管路等。另外,一般每周2-4次往返医院进行血液透析、源源不断的经济支出以及接踵而至的各种并发症等,种种因素导致绝大部分MHD面临病痛、经济、精神等多重负担。随着透析时间的延长,往往频发营养不良、免疫紊乱、心血管疾病、不宁腿综合征(restless legs syndrome、抑郁、焦虑、睡眠障碍、钙磷代谢紊乱、贫血等。营养不良和免疫失调是MHD的显著特征。目前已有大量的数据表明营养不良是透析病人死亡的最大原因之一。营养不良可导致血液透析患者的免疫功能和体力活动功能下降,与各种感染和非感染的并发症相关,是尿毒症死亡率升高的独立危险因素。另外,尿毒症患者固有免疫和适应性免疫功能双重受损,这种免疫系统的改变与高发的心血管疾病和死亡率与有关。炎症-免疫紊乱也是导致尿毒症患者发生心血管疾病的重要机制之一,免疫活化可导致微炎症状态,引起冠状动脉粥样硬化和心血管事件的发生。目前认为免疫紊乱也是导致MHD其他多种并发症的重要原因。因此研究并干预MHD的营养状态和免疫功能对降低感染和心血管事件发生率,改善MHD的预后有重要的科研和临床意义。免疫功能障碍是导致血液透析患者感染和肿瘤高发的主要原因。色氨酸(Tryptophan,Trp)是人体的一种必需氨基酸,参与蛋白质的合成及多种组织代谢,犬尿氨酸(kynurenine,Kyn)是Trp的代谢终产物,吲哚胺2,3-双加氧酶(indloeamine-2,3-dioxygenase,IDO)是Trp代谢为Kyn的关键酶,IDO可通过抑制T细胞的增值来诱导免疫耐受。免疫耐受是尿毒症患者免疫紊乱的重要机制,树突状细胞群可通过诱导调节性T细胞介导免疫耐受。目前认为,表达IDO的树突状细胞是发挥免疫耐受作用的关键分子,使用IDO抑制剂能快速诱导T细胞介导的排斥反应,因此IDO被认为是调控免疫平衡的重要机制。既往研究也发现Trp及其代谢产物Kyn参与免疫调控,尿毒症患者Trp/Kyn的水平与低免疫反应及心血管疾病相关,但目前MHD的IDO的水平及引起免疫紊乱的机制尚未完全清楚。定量分析MHD血浆中Trp和Kyn可为了解MHD的营养状态及免疫功能提供理论依据,具有一定的科研和临床意义。高压液相色谱法(High Performance Liquid Chromatography,HPLC)是目前常见的用以检测血浆中Trp和Kyn的方法,当检测样品流经色谱柱时,检测样品中各组分因吸附力大小不同而分离,当通过检测器时,可得到相应的信号-时间曲线,从而定量各种组分。既往有研究者使用HPLC检测了慢性肾功能不全患者血浆中的Trp和Kyn,但我们在借鉴该方法检测MHD血浆中这两种氨基酸时,可能因MHD血浆中尿毒症毒素多,彼此干扰大、难以分离。为了简化操作流程,在同一色谱条件下同时检测到MHD血浆中的Trp和Kyn,本研究对此进行了探讨。研究目的 1、评估2012年12月在广东省人民医院血液净化中心行维持性血液透析的患者的营养状态,了解营养不良的发生率,分析营养不良的相关影响因素； 2、改良HPLC同时定量分析MHD血浆中的Kyn和Trp,为评估透析患者的营养状态、免疫功能,了解IDO的活性提供一定的理论依据。研究方法本研究主要采用横断面分析的研究方法,通过问卷调查、临床身体指标测量、各种实验室指标、透析数据的收集等方法搜集资料以了解MHD营养不良的现状,基本入组标准为：1)同意参与本项调查并签署知情同意书,2)维持性血液透析时间≥3个月,3)能清楚自主表达自己的意愿；采用HPLC方法检测MHD血浆中的Trp和Kyn。关于MHD营养状态的评估,本研究不仅关注了传统的血浆白蛋白及营养不良相关的主观评估,同时联合了目前国际上常用的营养不良-炎症评分(Malnutrition-Inflammation Score,MIS)及透析营养的客观评分表(Objective Score of Nutrition on Dialysis,OSND)。其中MIS评估内容涉及到MHD过去3到6个月干体重的变化、饮食摄入、胃肠道症状、营养相关的功能障碍、透析合并症、透析时间、脂肪储备实测值、肌肉消耗、身高体重指数(Body Mass Index,BMI)、血浆白蛋白、总铁结合力、转铁蛋白；OSND的评估包括过去3到6个月干体重的变化、BMI、三头肌皮褶厚度、上臂中围周径、转铁蛋白、血清白蛋白、胆固醇等。这两个量表的各项指标根据不同的等级划分不同的分数,汇总后得到最终的评估结果,其中MIS的评分标准为：8分,轻度营养不良；9-18分,中度营养不良；18分,重度营养不良,MIS正常值为0分,最高30分。OSND的评分标准为：28-32分为正常,23-27分中度营养不良,≤22分重度营养不良,OSND正常值为5分,最高32分。调查表由研究者在MHD透析期间进行临床问询并记录结果,其中皮褶厚度、肌肉储备、中臂围周径等临床测量也在透析期间进行。所有测量使用同一个皮褶厚度测量仪和同一个标尺,每次测量前校正,每个指标测量3次,取平均值,对测量数据进行记录。同期收集入组患者的基本资料、实验室、透析数据及问卷调查内容,如年龄、基础病、维持性血液透析的时间、残余尿量、血红蛋白、血清铁、甲状旁腺素、透析器的通透性、血流量、透析模式等。各评估量表身体指数的测量、诊断及严重程度评估、实验室资料的录入由同一名研究者负责,汇总相关数据后进行统计分析。既往研究者使用HPLC检测了慢性肾功能不全患者血浆中的Trp和Kyn,本研究在借鉴该方法用于检测MHD血浆中的Trp和Kyn时,发现在色谱图上,Trp和Kyn峰与周围色谱峰融合,谱带拖尾,无法分离,难以定量分析。本研究通过调整HPLC的2个关键因素,色谱柱和流动相,拟摸索到一个比较合适的检测窗。采用的色谱柱为Sinochrom ODS-BP C18柱(4.6mm x150mm,id,4.5μm),流动相15mmol/L乙酸钠-乙酸(含5%乙腈,PH4.8),流动相流速1.0mL/min,紫外检测波长225mm。取MHD上机前全血3-4ml,置于4mlEDTA管内,采血后1小时内于常温下以2500r/min的速率,离心5分钟分离血浆,血浆经5%高氯酸溶液去除蛋白质后沉淀,取上清液20μ l直接进样分析测定。统计学分析对符合正态分布的数据用x±S表示,不符合正态分布的数据用中位数表示,采用logistic回归分析方法进行相关因素分析,P0.05认为差异有统计学意义,应用SPSS17.0软件对数据进行统计分析。研究结果 1、2012年12月广东省人民医院血液净化中心MHD营养状况及相关影响因素： 1)共217名MHD符合入选要求纳入本项调查,MIS评分为9±2分,其中轻度营养不良81人,中度营养不良135人,重度营养不良1人； 2) OSND评分为12±3分,均为重度营养不良； 3)84.4%患者血浆白蛋白未达标,30.4%患者BMI低于正常值,三头肌皮褶厚度14.5±7.0mm,中臂围24.6±3.5cm,42.9%的患者存在饮食摄入障碍,37.1%的患者有胃肠道功能不适,25%的患者存在营养相关的损伤。 4)MIS与透析时间、性别、Kt/V. Hb相关；CRP与白蛋白、血尿酸、MIS、 OSND相关。 2、在既往研究的基础上,通过调整色谱柱的填料和孔径、流动相的条件,改变了Trp和Kyn的解离度和峰保留时间,得到一个比较合适的检测窗。将原方法色谱柱SymmetryaShieldRP-18column(150mmX3.9mm,id)换成色谱柱为Sinochrom ODS-BP C18柱(4.6mmX150mm,id,4.5μm);加大了流动相的酸碱度和乙腈的含量,最终流动相为15mmol/L乙酸钠-乙酸(含5%乙腈,PH4.8),流速1.0mL/min,紫外检测波长225mm,血浆标本经5%高氯酸溶液去除蛋白质后取上清液20μl直接进样分析测定。Kyn的保留时间为6.766min,线性范围为0.08-50μmol/L,最低检测浓度0.02μmol/L, Trp的保留时间为13.072min,线性范围为0.8-500μmol/L,最低检测浓度为0.2μmol/L, Kyn和Trp日内、日间测定的相对标准偏差均小于4%。结论 1、营养不良是尿毒症患者预后的独立危险因素,MIS更适合中国人,营养不良在维持性血液患者普遍存在,需要定期评估,综合管理,及时干预； 2、血浆中Kyn和Trp与MHD的营养状态及免疫功能均相关,而营养不良可导致免疫紊乱,本研究采用HPLC的方法,在借鉴既往研究的基础上,通过调整色谱柱的直径、流动相的PH值等方法,成功地在同一条件下同时检测到了MHD血浆内的Kyn和Trp的含量,该方法简便、快速、稳定,可应用于临床和科研工作。
[Abstract]:Research background
The epidemiological study of end-stage renal disease (ESRD) (data from the American kidney disease database system and Europe) shows that the number of ESRD in the world is still increasing, and the annual increase of the number of people is more than the annual growth rate of the population. Maintenance hemodialysis is a routine treatment of ESRD and is the most used renal replacement. It has been over 50 years in the history of treatment.
Due to renal failure, a large number of toxins are hard to be removed in the body. In China, in view of the current national conditions, most ESRD patients have chosen hemodialysis treatment. After hemodialysis, the blood is extracted from the body, and the hemodialysis membrane is used to separate the blood and dialysate with the aid of the dialysis membrane, which can be partially replaced by three ways of spreading, convection and ultrafiltration. The kidney Excrets water, metabolize waste, regulates the balance of water and electrolyte, and corrects the function of acidosis. General hemodialysis can only replace about 1/10 of normal kidney function. Hemodialysis is an in vitro blood purification method, and maintenance hemodialysis patients (maintenance hemodialysis patients, MHD) often come into contact with the body. The ring system such as puncture needle, dialyzer, dialysis pipeline, etc., in addition, 2-4 times a week to and from the hospital to carry out hemodialysis, the continuous economic expenditure and the following complications, a variety of factors lead to the overwhelming majority of MHD face the pain, economic, mental and multiple burdens. With the extension of dialysis time, often frequent malnutrition, Immune disorders, cardiovascular diseases, restless legs syndrome (restless legs syndrome), depression, anxiety, sleep disorders, calcium and phosphorus metabolism disorders, anemia and so on.
Malnutrition and immune disorders are the prominent features of MHD. There is a large number of data showing that malnutrition is one of the biggest causes of death in dialysis patients. Malnutrition can lead to a decline in immune function and physical activity in hemodialysis patients, associated with various infections and non infectious complications, and is the only increase in the mortality of uremia. In addition, the inherent immunity and adaptive immune function of uremia patients are double damaged, and the changes of this immune system are associated with high incidence of cardiovascular disease and mortality. Inflammation - immune disorders are also one of the important mechanisms of cardiovascular disease in patients with uremia. Immune activation can lead to the state of micro inflammation and cause the crown. The occurrence of atherosclerosis and cardiovascular events is considered as an important cause of many other complications of MHD. Therefore, the study and intervention of the nutritional status and immune function of MHD have important scientific and clinical significance in reducing the incidence of infection and cardiovascular events and improving the prognosis of MHD.
Immune dysfunction is the main cause of high incidence of infection and tumor in hemodialysis patients. Tryptophan (Trp) is an essential amino acid in the human body. It is involved in the synthesis of protein and the metabolism of various tissues. Kynurenine (Kyn) is the metabolic end product of Trp, and the indolamine 2,3- dioxygenase (indloeamine-2,3-dioxygenase, IDO) It is the key enzyme of Trp metabolism as Kyn. IDO can induce immune tolerance by inhibiting the increment of T cells. Immune tolerance is an important mechanism of immune disorder in patients with uremia. Dendritic cells can mediate immune tolerance by inducing regulatory T cells. At present, the dendritic cells expressing IDO are the key molecules of immune tolerance. IDO inhibitors can quickly induce T cell mediated rejection, so IDO is considered an important mechanism for regulating immune balance. Previous studies have also found that Trp and its metabolite Kyn are involved in immunoregulation. The level of Trp/Kyn in uremia patients is associated with low immune responses and cardiovascular disease, but the level of IDO in MHD and the cause of immune disorder at present. The disorderly mechanism is not completely clear.
The quantitative analysis of Trp and Kyn in MHD plasma can provide a theoretical basis for understanding the nutritional status and immune function of MHD. It has certain scientific and clinical significance. High pressure liquid chromatography (High Performance Liquid Chromatography, HPLC) is a common method for detecting Trp and Kyn in plasma. Each component in the sample is separated because of the different size of the adsorption force. When the detector is used, the corresponding signal time curve can be obtained to quantify various components. Previous researchers used HPLC to detect Trp and Kyn in the plasma of patients with chronic renal insufficiency, but we may use this method to detect the two kinds of amino acids in MHD plasma. In order to simplify the operation process, the Trp and Kyn in the plasma of MHD are simultaneously detected in the same chromatographic condition. This study has been discussed in this study, in order to simplify the operation process, the MHD plasma has many uremic toxins.
research objective
1, to evaluate the nutritional status of patients with maintenance hemodialysis in the blood purification center of Guangdong General Hospital in December 2012, to understand the incidence of malnutrition and to analyze the related factors of malnutrition.
2, the quantitative analysis of Kyn and Trp in MHD plasma by modified HPLC provides a theoretical basis for assessing the nutritional status, immune function and understanding of the activity of IDO in dialysis patients.
research method
This study mainly adopts the method of cross-sectional analysis. Through questionnaire survey, clinical physical indicators measurement, various laboratory indicators, dialysis data collection and other methods to collect data to understand the status of MHD malnutrition, the basic entry standard is: 1) consent to participate in this regulation and sign informed consent, 2) maintenance hemodialysis time. More than 3 months, 3) can clearly express their wishes; HPLC method was used to detect Trp and Kyn. in MHD plasma.
With regard to the assessment of the nutritional status of MHD, this study not only focused on the subjective assessment of the traditional plasma albumin and malnutrition, but also combined the current international common malnutrition score (Malnutrition-Inflammation Score, MIS) and the objective assessment of dialysate (Objective Score of Nutrition on Dialysis, OSND). The MIS assessment involves the changes in dry weight of MHD over the past 3 to 6 months, dietary intake, gastrointestinal symptoms, nutritional related dysfunction, dialysis complication, dialysis time, the measured value of fat reserve, muscle consumption, Body Mass Index, BMI, plasma albumin, total iron binding force, transferrin, and OSND assessment included The changes in body weight for 3 to 6 months, BMI, skin fold thickness of triceps, circumference of the upper arm, transferrin, serum albumin, cholesterol and so on. The indexes of the two scales were divided according to different grades, and the final evaluation results were collected, in which the scoring criteria for MIS were: 8, mild malnutrition; 9-18 points. Dystrophy, 18 points, severe dystrophy, MIS normal value of 0 points, and the highest score of 30.OSND, were normal, 23-27 moderate dystrophy, less than 22 points severe dystrophy, OSND normal value 5, and the highest 32. The questionnaire was conducted by the researchers during the MHD dialysis period and recorded the results, of which the skin fold thickness, Clinical measurements such as the muscle reserve and the circumference of the middle arm were also carried out during the dialysis. All measurements were made with the same skin fold thickness measuring instrument and the same scale, corrected before each measurement. Each measure was measured 3 times, the average value was taken, and the measured data were recorded. The basic data, laboratory, dialysis data and questionnaire survey were collected during the same period. Content, such as age, basic disease, time of maintenance hemodialysis, residual urine volume, hemoglobin, serum iron, parathyroid hormone, permeability of dialysator, blood flow, dialysis mode, etc.. Measurement of body index, diagnosis and severity assessment of each assessment scale. The entry of laboratory data is responsible for the same researcher. Statistical analysis.
Previous researchers used HPLC to detect Trp and Kyn in plasma of patients with chronic renal insufficiency. This study was used for reference to the detection of Trp and Kyn in MHD plasma. It was found that on the chromatogram, the peaks of Trp and Kyn were fused with the peaks of the surrounding chromatograph, and the band trailing, unable to be separated, was difficult to quantify. This study by adjusting 2 key factors of HPLC, The chromatographic column and the mobile phase are used to find a suitable detection window. The chromatographic column used is Sinochrom ODS-BP C18 column (4.6mm x150mm, ID, 4.5 u m), the mobile phase 15mmol/L acetate acetic acid (5% acetonitrile, PH4.8), the flow phase velocity 1.0mL/min, and the UV detection wavelength 225mm. take the whole blood before the MHD, and 1 hours after the blood collection. At the rate of 2500r/min at normal temperature, plasma was separated by centrifugation for 5 minutes. The plasma was precipitated after 5% perchloric acid solution was removed, and the supernatant was obtained by direct injection of 20 mu L.
Statistical analysis
The data that conform to the normal distribution are expressed in X + S, the data that do not conform to the normal distribution are expressed in the median, and the correlation factors are analyzed by the logistic regression analysis method. The difference is statistically significant by P0.05, and the data are statistically analyzed with the SPSS17.0 software.
Research results
Nutritional status and related factors of MHD in the blood purification center of Guangdong General Hospital in December 12012:
1) a total of 217 MHD were included in the survey. The MIS score was 9 + 2, of which 81 were mild dystrophy, 135 were moderate dystrophy and 1 were severe dystrophy.
2) the OSND score was 12 + 3, all of which were severe malnutrition.
3) in 84.4% patients, plasma albumin did not reach the standard, 30.4% patients were lower than normal BMI, triceps skin fold thickness 14.5 + 7.0mm, middle arm circumference 24.6 + 3.5cm, 42.9% of patients with dietary intake disorders, 37.1% of the patients with gastrointestinal dysfunction, 25% of the patients with nutritional related damage.
4) MIS was correlated with dialysis time, gender and Kt/V. Hb; CRP was associated with albumin, serum uric acid, MIS and OSND.
2, on the basis of previous studies, by adjusting the packing and pore size of the column and the conditions of the mobile phase, the dissolution and peak retention time of Trp and Kyn were changed, and a suitable detection window was obtained. The original method of chromatographic column SymmetryaShieldRP-18column (150mmX3.9mm, ID) was replaced by a Sinochrom ODS-BP C18 column (4.6mmX150mm, ID, 4.5). The acidity and alkalinity of the mobile phase and the content of acetonitrile were increased, the final flow phase was 15mmol/L acetate sodium acetate (5% acetonitrile, PH4.8), the flow velocity 1.0mL/min, the UV detection wavelength 225mm, the plasma specimen after 5% perchloric acid removal of the protein and the supernatant 20 mu l direct injection analysis and determination of.Kyn was 6.766min, and the linear range was 0.08-5 The minimum detection concentration is 0 mu mol/L, the minimum detection concentration is 0.02 mu, the retention time of Trp is 13.072min, the linear range is 0.8-500 mu mol/L, the minimum detection concentration is 0.2 u mol/L, the relative standard deviation of daytime determination is less than 4%. within Kyn and Trp days.
conclusion
1, malnutrition is an independent risk factor for the prognosis of uremic patients. MIS is more suitable for Chinese people. Malnutrition is common in the maintenance of blood patients. Regular assessment, comprehensive management and timely intervention are needed.
2, Kyn and Trp in plasma are related to the nutritional status and immune function of MHD, and malnutrition can lead to immune disorders. This study uses the method of HPLC, and on the basis of previous studies, by adjusting the diameter of the column and the pH value of the mobile phase, the content of Kyn and Trp in the MHD plasma is successfully detected in the same condition. The method is simple, rapid and stable, and can be applied to clinical and scientific research.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.5

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