Isolation and purification of uremic middle molecules by multi-step liquid chromatography

Isolation and comparison of uremic sera and urine and normal sera and urine were performed by gel permeation chromatography, anion exchange chromatography and re-versed-phase high performance liquid chromatography. Two uremic middle molecular fractions (A and B) were obtained from uremic sera and urine and normal urine by gel permeation chromatography, but not from normal sera. The anion exchange chromatographic results of fraction A from different origins demonstrate that subfraction A-3 could be excreted in urine by healthy subject, but accumulated in uremic serum for renal failure of patient with uremia. After desalinization subfraction A-3 was analyzed by MALDI-TOF-MS. The results show that subfraction A-3 consists of six compounds with molecular weight 839, 873, 1007.94, 1106, 1680 and 2015 respectively. Finally, by reversed-phase high performance liquid chromatography, subfraction A-3 was further resolved into six independent fractions. Thus, the isolation and purification of six middle molecular c     

尿毒症中分子毒物的分离及飞行时间质谱分析

尿毒症被认为是因为患者肾衰而毒素在体内滞留所致^[1]。1972年Babb等^[2]提出“中分子假说”,认为分子量在300－2000范围内的中等分子量的物质是尿毒症的主要毒性物质。从此,人们作了大量的努力去分离和鉴定尿毒症中分子毒物。然而尿毒症中分子毒物的成分极其复杂^[3],从设定的中分子组分中分离得到的大都是些分子量小于800的小分子物质^[4]。因而对中分子假说一直存在争议^[5].我们对尿毒症患者及正常人的血清和尿液进行凝胶色谱分离,从尿毒症血清和尿液及正常人尿液中得到两个中分子峰A,B。将不同来源的A峰中的分子毒物进行离子交换色谱的分离和比较,得到了仅存在于尿毒症血清和正常人尿液的A－3亚峰,经脱盐和飞行时间质谱分析,确定了该组分内含有分子分别为839．69,1007．94,2015．16,16,873．69,1106．67和1680．28的6种化合物。     

尿毒症中分子毒物的分离鉴定

自从1972年Babb等提出中分子假说以来,人们做了很多努力去分离鉴定尿毒症中分子毒性物质．Chu等通过凝胶色谱和离子交换色谱发现来自尿毒症患者血清和正常人尿液的A-3亚峰物质均含有6种成分,分子量分别为839．69,873．69,1007．94,1106．67,1680．28和2015．16．该组分在病人尿液中明显缺失,说明A-3亚峰物质可能是正常人通过尿液排出,肾衰病人难以通过尿液排出,导致在体内积累的物质;     

尿毒症患者血清中中分子物质的提取及性质的初步表征

从尿毒症患者血清及腹透液中得到的中分子量物质,具有相同的凝胶色谱行为及紫外、红外吸收行为,它们在206和235nm处,有特征紫外吸收峰;在波数为3246,1516,1122和613cm~(-1)处,有特征红外吸收峰。中分子物经离子交换色谱进一步分离,发现起决定紫外及红外吸收行为的成分集中于第3子峰;其自由氨基酸和肽类物质含量均低于3％。     

尿毒症中分子毒物的分离清除及其毒性作用

尿毒症综述病症涉及人体的各个系统,本文综述了尿毒症患者可能出现的各种临床症状。这些症状被认为是由于代谢废物在患者体内滞留而造成的。由于这些代谢废物成分和生理作用的复杂性,使得分离或寻找导致某一症状的毒物极为困难。本文对见诸报道的尿毒症可能性毒物以及某些中分子物的生理或生化功能进行了综述。并且通过比较不同疗法对尿毒症中分子物的清除效果,我们认为研制选择性吸附剂,对尿毒症中分子物进行吸附清除的方法或许是一个最有前途的发展方向。     

慢性肾衰患者血清中分子物质浓度变化及其对Na,K-ATP酶的抑制作用

慢性肾功能衰竭是指在各种慢性肾脏疾病的基础上,缓慢出现肾功能减退而不可逆转的肾衰竭综合症由于患者肾功能衰竭,使得一些正常人本可以排出体外的代谢产物滞留在体内,导致肌体的一系列病变,涉及到人体的肠胃、免疫、心血管、内分泌、皮肤和骨骼等各个系统．20世纪70年代以来,中分子物质在患者体内的毒性作用开始引起众多研究者的关注．然而,由于这类物质成分复杂,对其分离鉴定工作还面临着很多困难,它们与慢性肾衰患者的临床症状之间的因果关系及致病机理仍不甚明确．在前期研究中,我们采用凝胶色谱法分离尿毒症患者血清,得到两个中分子物质峰A和B,     

尿毒症中分子毒物吸附剂的研究(Ⅲ)——酸性氨基酸修饰交联壳聚糖

以球状壳聚糖为载体,酸性氨基酸为配体,合成了尿毒症中分子毒物吸附剂,并测试了其血液相容性.为了研究吸附剂对尿毒症患者体内多肽蓄积物的吸附性能,选取分子量不同的6种多肽作为体内蓄积多肽的模拟物进行吸附实验.研究表明,吸附剂对多肽模拟物产生一定的吸附作用.在此基础上又对尿毒症患者血清超滤液进行了吸附实验,结果显示,吸附剂对血清中分子级分的清除率为10.3%.     

Analytical isotachophoresis of uremic blood samples

Uremic blood samples were analyzed for ionogenic substances using analytical isotachophoresis. Multicomponent separations proved that the uremic state shows significant differences from the normal state, especially with regard to anionic low-molecular-weight substances. As a quantitative parameter the ratio of anionic higher-molecular-weight substances to anionic low-molecular-weight substances is proposed: the HL ratio. Separation patterns and HL ratios were studied during nine weeks for one patient on chronic hemodialysis. The patient showed a low HL ratio due to excess of low-molecular-weight substances. Separation patterns before and after hemodialysis showed clear differences and the HL ratio increased. The method of analysis is neither time- nor sample-consuming and sample preparation is not needed. Experimental procedures are easily standardized and results are reliable.     

Growth impairment of primary chondrocyte cells by serum of rats with chronic renal failure

Insulin-like growth factor (IGF)/IGF binding protein (IGFBP) abnormalities may be important in the pathogenesis of growth failure in chronic renal failure (CRF). We induced experimental CRF by 5/6 nephrectomy in Sprague Dawley rats (100 g) and observed for 2 weeks comparing with sham-operated pair-fed control rats (Sham- C). CRF rats gained 30% less height than Sham- C rats (P < 0.01). Serum IGFBP profiles by Western ligand blot revealed that IGFBP4 was elevated two fold in CRF rats (P < 0.01 vs. Sham-C). However, IGFBP4 mRNA levels in liver or skeletal muscle were not different in two groups. To determine if the increase of serum IGFBP4 in CRF retarded the growth of cartilage, epiphyseal chondrocytes were isolated from CRF or control rats and cultured in the presence of control or CRF rat sera. Incubation with 10% CRF serum reduced proliferations of normal chondrocytes and L6 rat skeletal muscle cells. In contrast, 10% CRF serum did not inhibit the growth of CRF chondrocytes. Rat sera from two groups were separated into two different fractions, high (>10 kDa, containing IGFBPs) and low (<10 kDa, containing free IGF) molecular weight fractions using a gel filtration column. Both fractions obtained from CRF sera decreased the growth of control chondrocytes up to 40% compared with those from control sera. We suggest that the pathogenesis of growth failure in CRF may be involved in the increase of circulating IGFBP4 as well as the unidentified small molecular weight uremic serum factors which block the growth of chondrocytes in growth plate.     

Membrane characterization and solute diffusion in porous composite nanocellulose membranes for hemodialysis

The membrane and solute diffusion properties of Cladophora cellulose and polypyrrole (PPy) functionalized Cladophora cellulose were analyzed to investigate the feasibility of using electroactive membranes in hemodialysis. The membranes were characterized with scanning electron microscopy, ζ-potentiometry, He-pycnometry, N₂ gas adsorption, and Hg porosimetry. The diffusion properties across the studied membranes for three model uremic toxins, i.e. creatinine, vitamin B12 and bovine serum albumin, were also analyzed. The characterization work revealed that the studied membranes present an open structure of weakly negatively charged nanofibers with an average pore size of 21 and 53 nm for pristine cellulose and PPy-Cladophora cellulose, respectively. The results showed that the diffusion of uremic toxins across the PPy-Cladophora cellulose membrane was faster than through pure cellulose membrane, which was related to the higher porosity and larger average pore size of the former. Since it was found that the average pore size of the membranes was larger than the hydrodynamic radius of the studied model solutes, it was concluded that these types of membranes are favorable to expand the Mw spectrum of uremic toxins to also include conditions associated with accumulation of large pathologic proteins during hemodialysis. The large average pore size of the composite membrane could also be exploited to ensure high-fluxes of solutes through the membrane while simultaneously extracting ions by an externally applied electric current.     

Present status of hemoperfusion/hemodialysis in Italy

The use of charcoal hemoperfusion in the treatment of chronic renal failure has been proposed and applied by several authors. The availability of coating membranes of increased biocompatibility currently allows a safer and wider use of this purifying technique. It has been recently demonstrated that long-term treatment with combined hemodialysis/hemoperfusion yields an improvement of certain dialysis-resistant uremic signs in patients on regular dialysis treatment, while in selected patients it affords a marked reduction (up to one-third) in the overall time of treatment per week. The tolerance of long-term treatment is good. In line with these findings, a multicenter study has been carried out in Italy with two main aims: (1) to see whether long-term treatment with charcoal hemoperfusion is really safe and substantially free from side effects; (2) to verify in a larger and more varied population of patients whether such long-term treatment actually improves certain uremic signs persisting despite adequate dialysis treatment. A third phase of the multicentric study (reducing the weekly time of treatment) is currently being worked on. Five nephrology and dialysis departments took part in the study: in Bologna, Rome, Chieti, Ancona, and Lecce.     

Numerical analysis of density gradient centrifugation profiles from eukaryotic DNA

A numerical method for the deconvolution of superimposed Gaussian distributions with a unique solution has been proposed by Medgyessy [10]. We have tested the usefulness of this method for the analysis of density gradient centrifugation profiles from eukaryotic DNA, which are normally composed from overlapping Gaussian distributed profiles of several subcomponents with different mean buoyant densities. From the analysis of human DNA and from model calculations we conclude that major subcomponents can be identified by this method, if they differ in their buoyant density by approximatly 0.005 g/ml. Minor components can only be identified if the total DNA has been fractionated according to buoyant density and the analysis is performed on the density profiles of the subfractions. This procedure represents a quick method to determine a reliable minimum number of subcomponents of DNA.This paper was presented at the VI. Symposium on Analytical Ultracentrifugation, Marburg, FRG, February 16–17, 1989.     

Analytical aspects of hemodialysis

Clinical and biomedical analysis is one of the most significant fields of modern analytical chemistry. Uremia is a significant public-health problem. Chronic hemodialysis, a common renal replacement therapy, is life saving for more than a million uremic patients world-wide and this number is increasing every year. This therapy consumes significant parts of national budgets for health as its costs are comparable with those for intensive care. Hemodialysis treatment should, just like any other therapy, be individually prescribed, and adequately administrated and assessed.

This article reports on the analytical needs connected with biomedical assessment of this therapy. I discuss the main analytical aspects of this therapy, including target analytes, choice of samples, and analytical and bioanalytical systems dedicated to the control of therapy, as well as methods required for the quantitation of hemodialysis using analytical data.     

Profiles in chronic diseases. I. Investigations of steroid profiles in uremia (author's transl)]

Steroid profiles of hemofiltrates of uremic patients contain as main steroids the sulfates of 11beta-hydroxyetiocholanolone, 11-ketoetiocholanolone, 11beta-hydroxyandrosterone and 11-ketoandrosterone. In blood of uremic patients androstenediol is the main steroid of the sulfate fraction, while in blood of healthy persons dehydroepiandrosterone sulfate is the main steroid. The gradual decrease of the kidney function is characterized by an increase of 11-oxigenated androstane conjugates in urine.     

Zinc, copper and aluminium in the corpuscular components of peripheral blood in patients with pre-terminal and terminal renal failure

Summary Trace elements play an essential role in the activation and regulation of metabolic processes. By using the flameless graphite furnace-AAS, accurate results can be obtained. In order to verify blood cells as more representative than plasma to reflect the whole body content of trace elements at uremic state, two patient groups, one with preterminal renal failure (n=40) and the second with chronic renal failure undergoing hemodialysis (n=20) were examined. The concentration levels of zinc, copper and aluminium were measured in different matrices, i.e. whole blood, plasma and corpuscular blood components. The analytical technique is eligible to isolate mono- and polymorphnuclear leucocytes for the determination of intracellular contents. In agreement with other reports, zinc and copper were decreased and aluminium was increased in the plasma of both patient groups. Different results could be found for intracellular contents with mean zinc concentration of erythrocytes, thrombocytes and leucocytes between mean and higher levels, though staying within normal values. Copper was decreased in erythrocytes and increased in thrombocytes, but not detectable in leucocytes. The results of this study point out the differences between the plasmatic and intracellular alteration of trace element contents in uremic patients. However, the concentration levels of all cellular compartments show high standard deviation, which shows the necessity of measuring additional matrices, e.g. plasma. Uremic symptoms relate to the disorders of the trace element homeostasis. Regarding the increased fragility of erythrocytes with shortened life duration, decreased copper contents could play an essential role. Increased aluminium concentration levels might also be one of the interfering factors in the uremic thrombocytopathy. Yet the measurement of the plasma content is inevitable to diagnose and reflect the whole body content of trace elements at uremic state. By keeping analysis control, important information about progression and influence of therapy can be obtained.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Anion-exchange chromatography of normal and monoclonal serum immunoglobulins

Anion-exchange chromatography was used for separations of the three main immunoglobulin classes in human sera. The material included normal sera as well as sera with monoclonal immunoglobulin components. The Mono Q anion-exchange column was eluted with a stepwise gradient formed by 0.05 and 0.3 M phosphate buffers. No indications of negative effects on the sample components were observed and the protein recovery was high. A fast, easy and reproducible separation of immunoglobulins G, A and M was obtained.     

Improved phenotyping of alpha 1-antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: first demonstration of a deficient protein variant in the ACT system.

Genetic variation of human alpha 1-antichymotrypsin (ACT) was investigated in sera using thin-layer polyacrylamide gel isoelectric focusing (pH range 4.0-6.5) followed by immunoprinting with a monospecific anti-human ACT antibody. Sialidase-treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant "ACT Bochum" was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.     

High‐resolution capillary zone electrophoresis and mass spectrometry for distinction of undersialylated and hypoglycosylated transferrin glycoforms in body fluids

High‐resolution capillary zone electrophoresis is used to distinguish transferrin glycoforms present in human serum, cerebrospinal fluid, and serum treated with neuraminidase and N‐glycosidase F. The obtained data are compared to mass spectrometry data from the literature. The main focus is on the analysis of the various asialo‐transferrin, monosialo‐transferrin, and disialo‐transferrin molecules found in these samples. The features of capillary zone electrophoresis and mass spectrometry are reviewed and highlighted in the context of the analysis of undersialylated and hypoglycosylated transferrin molecules. High‐resolution capillary zone electrophoresis represents an effective tool to assess the diversity of transferrin patterns whereas mass spectrometry is the method of choice to elucidate structural identification about the glycoforms. Hypoglycosylated transferrin glycoforms present in sera of alcohol abusers and normal subjects are structurally identical to those in sera of patients with a congenital disorder of glycosylation type I. Asialo‐transferrin, monosialo‐transferrin and disialo‐transferrin observed in sera of patients with a type II congenital disorder of glycosylation or a hemolytic uremic syndrome, in cerebrospinal fluid and after treatment of serum with neuraminidase are undersialylated transferrin glycoforms with two N‐glycans of varying structure. Undersialylated disialo‐transferrin is also observed in sera with high levels of trisialo‐transferrin.