尿毒症中分子毒物吸附剂的研究(Ⅰ)——戊二醛交联壳聚糖吸附剂

长期以来 ,血液净化疗法一直是临床上处理各种血液中毒的基本手段[1～ 3] .对尿毒症患者 ,目前普遍采用的治疗措施是对其进行定期的血液或腹膜透析 [1] 缓解病情 ;然而单纯血液透析疗法难以清除患者体内的中分子毒物 ,以致血液透析的患者体内 ,中分子毒物的积累会达到很高的程度 .因此 ,通过研制高效的中分子吸附剂 ,以血液灌流的方式清除中分子毒物 ,对于控制和治疗尿毒症具有重要意义 .据文献报道 ,体内蓄积的中分子毒物中肽类物质占了一定的比例[4～ 9] ,患者的许多顽固临床症状与这些毒物的体内蓄积密切相关 [1,10 ] .本课题组的研究结…     

尿毒症毒素多肽二级结构的测定和预测

尿毒症患者由于肾脏损伤,使某些代谢物质积蓄在体内而引起中毒,最终导致死亡,由于其分离纯化过程复杂,至今人们对尿毒症中分子积累物的认识仍很有限。目前,已确定的中分子毒物以蛋白质和多肽为主,在前期的工作中,我们从尿毒症患者血清中分离出分子量为1007的中分子物质,实验证明该物质可加速肾衰兔的死亡。     

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%.     

尿毒症毒素及其清除方法——兼论临床上的血液灌流治疗

长其以来,慢性肾功能衰竭一直是一种严重的疾患。本文介绍了用以解释慢性肾功能衰竭病因的几个相关假说,并对其中的尿毒症毒素学说予以了特别的关注。依据这个假说,尿毒症毒素被分成了几大类;其中,分子量位于300-5000道耳顿的中分子毒素的作用尤其引人注目。通过对现有的向种治疗慢性肾功能衰竭方法进行比较,本文认为采用血液灌流的方法,以吸附剂来清除尿毒症患者体内滞留的中分子毒素有着其独到的优势。     

尿毒症患者体液内中分子组分的多级色谱分离比较及质谱分析

采用凝胶色谱、离子交换色谱以及反相高效液相色谱法, 将尿毒症患者的尿样和血样以及正常人的尿样和血样进行了多级分离和比较. 通过凝胶色谱法, 从正常人的尿液以及尿毒症血清和尿液中分离出相对于正常人血清具有异常高浓度的A, B 2个中分子组分. 不同试样来源的A峰中分子物的离子交换色谱分离结果证明, A-3亚峰中分子物, 是尿毒症患者因肾衰难以通过尿液将其排除故而滞留在血清内的重要成分, 正常人却可以通过尿液将其排于体外. 将来自尿毒症血清及正常人尿液的A-3亚峰中分子物经脱盐处理后, 进行了基质辅助激光解吸电离-飞行时间质谱分析, 证明A-3组分由分子量分别为839, 873, 1007, 1106, 1680, 2015的6种化合物组成. 最后, 采用反相高压液相色谱法对A-3亚峰中分子组分进行了进一步分离, 得到了6个只含单一中分子化合物成分的组分. 至此, A-3组分内的中分子物得到了彻底的分离和纯化, 为进一步确定其结构以及病理生理作用奠定了基础.     

尿毒症患者血清及正常人尿液内中分子物质的分离分析

采用凝胶色谱法，以２０６ｎｍ处的紫外吸收为检测手段，分别从尿毒症患者血 清及正常人尿液内分离出Ａ，Ｂ两个中分子峰，而从正常人血清中分离出来的Ａ峰远低 于尿毒症患者血清的Ａ峰；在Ｂ峰的位置正常人血清未出现明显的吸收．运用该方法 不仅提高了凝胶色谱的分离效果，且能方便地得到中分子级分的纯品．对清除了淋洗 液组分的中分子级分进行紫外和红外扫描，结果发现来源于尿毒症患者血清及正常人 尿液的Ａ峰物质具有相同的紫外吸收且红外吸收光谱极其相似，而不同来源的Ｂ峰物 质虽然紫外吸收相同，但它们的红外吸收却存在一定的差异．采用离子交换色谱法对Ｂ 峰物质进行进一步的分离，以２３０ｎｍ处的紫外吸收为检测手段，正常人尿液的Ｂ峰物 质被分离成１７个亚峰，尿毒症血清的Ｂ峰物质被分离成１３个亚峰．其中绝大部分亚峰 的出峰位置（洗脱体积）相同．     

含氟生理活性化合物的化学基础

含氟生理活性化合物愈来愈受到人们的注目,因为分子中引进一个或数个氟原子后常常能得到活性更强、选择性更高的药物、农药及其他生理活性物质。本文试图从化学的角度对氟原子被引进分子后如何影响化合物的化学、物理性质,改变其代谢过程,从而导致生理活性的变化简要地作一综述,以期对设计具有生理活性的氟化合物时有所帮助。     

尿毒症中分子毒物吸附剂的研究——Ⅱ. 交联剂链长对壳聚糖树脂吸附性能的影响

用甲醛、乙二醛和戊二醛作为交联剂,以反向悬浮的方法制备了3种不同的交联壳聚糖树脂吸附剂,并对其中的后两种用NaBH₄进行了还原处理.经红外光谱表征和扫描电子显微镜观察,氢化还原处理有效地提高了吸附剂的化学稳定性;而交联剂的不同则对吸附剂的表面形态有着明显的影响.通过对尿毒症中分子毒物的模拟物和牛血清白蛋白的吸附研究发现,3种壳聚糖树脂吸附剂都在符合临床血液灌流要求的时间里达到了吸附平衡,在对牛血清白蛋白几乎不发生吸附作用的同时,其对模拟的中分子毒物则都产生了一定的吸附作用,但吸附量有所不同;随着交联剂脂肪链的增长,吸附剂对模拟的中分子毒物的吸附能力逐渐增强;以戊二醛为交联剂时,吸附剂的吸附效果最好.     

壳聚糖与尿素的相互作用及其应用研究进展

尿素能够显著破坏甲壳素/壳聚糖分子氢键结构和疏水相互作用,增加其临界胶束浓度,促进多糖大分子的溶解,并能减少其在溶液中的自聚集现象.碱-尿素水溶液可以作为一种新型的甲壳素/壳聚糖绿色溶剂,有望用于对刺激性要求较为苛刻的食品、生物医学等领域.壳聚糖衍生物特别是其与过渡金属离子的配合物具有良好的尿素吸附功能,可用于尿毒症患者血液中小分子毒物的吸附,对机体刺激性小且不吸附血清蛋白等生物大分子.有望成为血液灌流治疗法中清除尿素等小分子毒性物质的良好吸附剂.壳聚糖还可以作为包膜材料,制备壳聚糖包膜尿素,与普通的包膜尿素相比性能更为优越.     

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％。     

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     

大孔吸附树脂对尿毒症和烧伤毒血症血浆中分子物质体外吸附的研究

研究了大孔吸附树脂对尿毒症和烧伤毒血症患者血浆中分子物质的体外吸附,用Sephadex G15柱层析法对血浆中分子物质进行吸附前、后的组分分析,并进行了血浆模拟灌流实验,观察中分子物质的清除率,初步探讨了大孔吸附树脂孔结构对中分子物质的吸附机理。     

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,     

Study by means of high-performance liquid chromatography of solutes that decrease theophylline/protein binding in the serum of uremic patients.

Substantial changes in protein binding of drugs occur during the progression of renal insufficiency. Protein-bound uremic solutes play a role in the inhibition of drug protein binding. We previously demonstrated that hippuric acid in uremic ultrafiltrate was an inhibitor of the theophylline protein binding. The present study was undertaken to extend the yield of protein-bound uremic solutes by displacing ligands in uremic serum from their binding sites by five deproteinization methods. The inhibitory effect on theophylline protein binding of the deproteinized uremic serum was higher than with ultrafiltrate (p < 0.05). The influence of 30 semi-preparative HPLC fractions from deproteinized uremic serum on the theophylline protein binding was evaluated to identify the responsible compounds and to compare their relative individual impact. The theophylline protein binding was calculated as a percentage (bound versus total). The most important decrease of the protein binding was observed in HPLC fractions 6, 10 to 13, 15 and 28 with protein binding of: 61.5 +/- 10.8, 64.5 +/- 7.6, 60.9 +/- 10.1, 47.5 +/- 3.3, 60.0 +/- 6.7, 60.7 +/- 6.3 and 61.3 +/- 6.9%, respectively versus 69.1 +/- 2.4% for control serum (p < 0.05). The responsible compounds were characterized in the fractions by co-elution: 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), indole-3-acetic acid, indoxyl sulfate, hippuric acid, p-hydroxyhippuric acid and tryptophan. Their concentration was determined by analytical HPLC and a solution containing these compounds at the same concentration as in deproteinized uremic serum was composed. This solution was added to control serum and decreased the theophylline protein binding from 69.0 +/- 4.4% to 61.3 +/- 1.3%, which was less important than in genuine uremic serum (44.4 +/- 3.8%, p < 0.05). Dose-response curves with the characterized compounds revealed that the most important role in binding inhibition could be attributed to hippuric acid and CMPF. Our data suggests that the yield of protein binding inhibiting compounds is more important with deproteinized uremic serum than with uremic ultrafiltrate. The identified uremic compounds are not entirely representative for the decreased protein binding of theophylline, indicating that additional factors than those identified in this study affect the protein binding as well.     

Applications of high throughput microsomal stability assay in drug discovery

High throughput in vitro microsomal stability assays are widely used in drug discovery as an indicator for in vivo stability, which affects pharmacokinetics. This is based on in-depth research involving a limited number of model drug-like compounds that are cleared predominantly by cytochrome P450 metabolism. However, drug discovery compounds are often not drug-like, are assessed with high throughput assays, and have many potential uncharacterized in vivo clearance mechanisms. Therefore, it is important to determine the correlation between high throughput in vitro microsomal stability data and abbreviated discovery in vivo pharmacokinetics study data for a set of drug discovery compounds in order to have evidence for how the in vitro assay can be reliably applied by discovery teams for making critical decisions. In this study the relationship between in vitro single time point high throughput microsomal stability and in vivo clearance from abbreviated drug discovery pharmacokinetics studies was examined using 306 real world drug discovery compounds. The results showed that in vitro Phase I microsomal stability t(1/2) is significantly correlated to in vivo clearance with a p-value<0.001. For compounds with low in vitro rat microsomal stability (t(1/2)<15 min), 87% showed high clearance in vivo (CL>25 mL/min/kg). This demonstrates that high throughput microsomal stability data are very effective in identifying compounds with significant clearance liabilities in vivo. For compounds with high in vitro rat microsomal stability (t(1/2)>15 min), no significant differentiation was observed between high and low clearance compounds. This is likely owing to other clearance pathways, in addition to cytochrome P450 metabolism that enhances in vivo clearance. This finding supports the strategy used by medicinal chemists and drug discovery teams of applying the in vitro data to triage compounds for in vivo PK and efficacy studies and guide structural modification to improve metabolic stability. When in vitro and in vivo data are both available for a compound, potential in vivo clearance pathways can be diagnosed to guide further discovery studies.     

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;     

Generation of superoxide anions during the reaction of guanidino compounds with methylglyoxal

Uremic toxins are accumulated in the blood of patients with chronic renal failure (CRF), although alteration of the toxicity by the interaction of various uremic retention products has not been precisely clarified. In this study, we found that cytochrome c added to incubation mixtures containing guanidino compounds and methylglyoxal in phosphate buffer solution (pH 7.4) resulted in reduction of cytochrome c. Superoxide anions were generated from incubation mixtures of each guanidino compound with methylglyoxal, because the reduction was inhibited by the addition of superoxide dismutase. The incubation mixture containing each guanidino compound and methylglyoxal had different rates of generation of the superoxide anion from other mixtures. A relatively higher superoxide anion formation rate was observed in the incubation mixture containing Arg and methylglyoxal (7.9 +/- 0.5nmol x m(-1) x min(-1)), or in the incubation mixture containing methylguanidine and methylglyoxal (6.3 +/- 0.6 nmol x ml(-1) min(-1)). These findings suggest that interactions of various uremic retention products which accumulate in the blood of uremic patients may generate reactive oxygen species and may be involved in the oxidative stress observed in CRF patients. The addition of aminoguanidine, which is known to inhibit the formation of advanced glycation end products, to a mixture of guanidino compounds and methylglyoxal inhibited reactions between guanidino compounds and methylglyoxal.     

Investigation of uremic analytes in hemodialysate and their structural elucidation from accurate mass maps generated by a multi‐dimensional liquid chromatography/mass spectrometry approach

Historically, structural elucidation of unknown analytes by mass spectrometry alone has involved tandem mass spectrometry experiments using electron ionization. Most target molecules for bioanalysis in the metabolome are unsuitable for detection by this previous methodology. Recent publications have used high‐resolution accurate mass analysis using an LTQ‐Orbitrap with the more modern approach of electrospray ionization to identify new metabolites of known metabolic pathways. We have investigated the use of this methodology to build accurate mass fragmentation maps for the structural elucidation of unknown compounds. This has included the development and validation of a novel multi‐dimensional LC/MS/MS methodology to identify known uremic analytes in a clinical hemodialysate sample. Good inter‐ and intra‐day reproducibility of both chromatographic stages with a high degree of mass accuracy and precision was achieved with the multi‐dimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) system. Fragmentation maps were generated most successfully using collision‐induced dissociation (CID) as, unlike high‐energy CID (HCD), ions formed by this technique could be fragmented further. Structural elucidation is more challenging for large analytes >270 Da and distinguishing between isomers where their initial fragmentation pattern is insufficiently different. For small molecules (<200 Da), where fragmentation data may be obtained without loss of signal intensity, complete structures can be proposed from just the accurate mass fragmentation data. This methodology has led to the discovery of a selection of known uremic analytes and two completely novel moieties with chemical structural assignments made. Copyright © 2009 John Wiley & Sons, Ltd.