Relationships among inflammation nutrition and physiologic mechanisms establishing albumin levels in hemodialysis patients

BACKGROUND: Serum albumin concentration is a balance among its synthesis rate, fractional catabolic rate (FCR), distribution, dilution in the plasma pool and external loss. The physiologic bases for establishing the level of serum albumin in hemodialysis patients have not been defined despite the association of hypoalbuminemia with excess mortality. Albumin concentration is associated with the levels of several acute phase proteins (APPs), C-reactive protein (CRP), alpha1 acid glycoprotein (alpha1 AG), or ceruloplasmin, and with nutritional markers, such as normalized protein catabolic rate (nPCR). METHODS: To establish the relationship among parameters that regulate albumin levels and markers of nutrition and inflammation, we injected [125I]-albumin, into 64 hemodialysis patients enrolled in the HEMO study to measure albumin distribution, synthesis and FCR. These variables were related to the levels of acute phase proteins (APPs), nPCR, body mass index (BMI), external albumin loss as well as demographic variables. Albumin distribution, synthesis and FCR were calculated from kinetic modeling, as was the initial plasma volume (PV). Serum albumin, transferrin, CRP, ceruloplasmin and alpha1 AG were measured weekly. Dialysate was collected during one dialysis each week to measure albumin loss. Results were analyzed by multiple linear regression. RESULTS: Albumin concentration correlated with its synthesis rate and FCR, but not with PV or its distribution between the vascular and extravascular pools. Albumin concentration also correlated with nPCR and alpha1 AG. However, albumin synthesis was directly related most strongly to PV and BMI (or nPCR), but not to levels of APPs. By contrast, albumin FCR correlated positively with both alpha1 AG and ceruloplasmin. CONCLUSION: Albumin concentration in dialysis patients changes with inflammation and nutritional status through their effects on albumin catabolism and synthesis, respectively. Within the range of albumin levels in these patients, nutritional variables primarily affected albumin synthesis while inflammation caused hypoalbuminemia by increasing albumin FCR. Albumin synthesis also increased in proportion to PV. The result of this is that PV expansion does not contribute to hypoalbuminemia.     

In vitro and in vivo evaluation of albumin synthesis rate of porcine hepatocytes in a flat-plate bioreactor

Several configurations of extracorporeal bioartificial liver devices have been developed for the potential treatment of fulminant hepatic failure or as a bridge to liver transplantation. Recently, we developed a microchannel flat-plate bioreactor with an internal membrane oxygenator in which porcine hepatocytes are cultured as a monolayer on the bottom glass surface. In the present study, we investigated synthetic function of porcine hepatocytes in the bioreactor in both in vitro and in vivo flow circuit models. In vitro, albumin synthesis was stable in the bioreactor for up to 4 days of perfusion. In vivo, with the extracorporeal connection of the bioreactor to rat vasculature, porcine albumin was detectable for 24 h in the rat plasma. We also developed a simple mathematical model to predict the in vivo porcine albumin concentration in rat plasma. These results indicate that this configuration of a microchannel flat-plate bioreactor has potential as a liver support device and warrants further investigation.     

胎牛血清在体外培养肝细胞极性形成的作用

目的观察胎牛血清在体外培养肝细胞极性形成的作用。方法分别利用含胎牛血清和不含胎牛血清培养液培养成鼠肝细胞,并观察血清对肝细胞形态、特异性膜区域蛋白分布以及蛋白分泌的影响,以确定胎牛血清在体外肝细胞极性形成的作用。结果不含血清组,肝细胞形成肝板样结构,肝细胞膜区域蛋白特异性分布,肝细胞保持稳定的蛋白分泌并持续增长达2周之久。而含血清组肝细胞培养3d后,分化良好的小管样结构消失,肝细胞膜区域蛋白失去特异性分布,蛋白分泌逐渐减少。结论胎牛血清可阻止肝细胞极性的形成,对于肝细胞移植及生物人工肝支持具有重要意义。     

Effects of albumin supplementation during cardioplegia administration: an in vitro analysis

Increasing, the colloid osmotic pressure (COP) of blood cardioplegia (BCP) may reduce myocardial edema and preserve cardiac function following cardiopulmonary bypass (CPB). The purpose of this study was to quantify the effects of albumin (ALB) supplementation on cardioplegia COP through an in vitro analysis. A self-contained cardioplegia delivery system administered supplemental ALB to four BCP ratios (1:1, 4:1, 8:1, and 20:1). In Group A, 25% ALB was combined with BCP at four delivery rates (0, 13, 25, and 50 mL ALB/L BCP), with a delivery rate of 0 mL ALB/L BCP serving as the control for all groups. Twenty-five percent ALB was added to crystalloid to create carrier solutions containing 12.5, 25, or 50 g ALB/L in Group B, while Group C combined an ALB delivery rate of 50 mL ALB/L BCP with each of the three carrier solutions. End-points included initial and post-supplementation hematocrit, total serum protein (TSP), and COP. Without supplemental ALB, TSP was less affected with increasing blood to crystalloid ratios (1:1-81.7 +/- 6.2%, 4:1-40.6 +/- 5.1%, 8:1-20.6 +/- 4.1%, 20:1-6.0 +/- 5.7%). The TSP of 1:1 and 4:1 BCP increased (p < .0003 and p < .02) across all methods of supplementation, while 8:1 BCP was similarly increased (p < .008), except with 12.5 and 25 g ALB/L carrier solutions. The greatest change from baseline COP was seen with the lower blood to crystalloid ratios (1:1-64.3 +/- 5.0% and 4:1-39.5 +/- 10.5%). In higher ratios, the effects of dilution were less profound (14.6 +/- 4.2 +/- 4.2% and 20:1-6.0 +/- 1.9%). COP of 1:1 BCP increased (p < .008) whenever ALB was added. In conclusion, TSP and COP of blood cardioplegic solutions is increased by supplemental albumin administration with quantitative enhancement dependent upon the dilutional effects of the blood to crystalloid ratio.     

粉防己碱对离体新西兰白兔阴茎海绵体cAMP和cGMP水平的影响

目的探讨粉防己碱(Tet)对离体新西兰白兔阴茎海绵体组织中环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)水平的影响,以进一步阐明其舒张阴茎海绵体组织的作用机制。方法采用~(125)I放射免疫测定法,观察Tet对离体新西兰白兔阴茎海绵体组织中cAMP和cGMP水平的影响。结果家兔阴茎海绵体组织中cAMP基础含量为(5.67±0.97)pmol/mg。Tet能直接浓度依赖性地升高阴茎海绵体组织中cAMP的含量(P＜0.05),而腺苷酸环化酶抑制剂(MDL-12,330A)对Tet升高的cAMP水平没有抑制作用(P＞0.05)。对cAMP激发剂前列腺素E_1(PGE_1)诱导产生的阴茎海绵体组织中cAMP水平,Tet也具有浓度依赖性升高作用(P＜0.05)。家兔阴茎海绵体组织中cGMP基础含量为(0.44±0.09)pmol/mg。无论是否存在鸟苷酸环化酶抑制剂(ODQ),Tet对阴茎海绵体组织中cGMP的含量均没有影响(P＞0.05)。对cGMP激发剂硝普钠(SNP)诱导产生的阴茎海绵体组织中cGMP水平,Tet也没有任何影响(P＞0.05)。结论Tet可能是通过抑制磷酸二酯酶活性而增加阴茎海绵体组织中cAMP的含量,这是Tet松弛阴茎海绵体组织的作用机制之一。     

Lipopolysaccharide suppresses albumin expression by activating NF-kappaB in rat hepatocytes

BACKGROUND: The severity of hypoalbuminemia has been shown to be related to morbidity and mortality in critical illness, illustrating the need for better understanding of the molecular mechanism of hypoalbuminemia. Lipopolysaccharide(LPS) is a key mediator which induces hypoalbuminemia in sepsis and septic shock. The present studies were performed to identify whether the reduction of albumin expression induced by LPS was mediated by activating nuclear factor kappa B(NF-kappaB) in cultured rat hepatocytes. MATERIALS AND METHODS: Primary rat hepatocytes were divided into five groups treated with normal saline or 1 ng/ml, 0.01 microg/ml, 0.1 microg/ml, or 1 microg/ml of LPS for 24 h. The albumin level in the supernatant and NF-kappaB activity in hepatocytes were measured. Hepatocytes were pretreated for 30 min with SN50 (a highly selected inhibitor of NF-kappaB) at different concentrations (10, 30, and 50 microg/ml). After 24 h of treatment with 1 microg/ml of LPS, the culture medium was measured for albumin level. Meanwhile, NF-kappaB activity in hepatocytes was assayed. RESULTS: LPS dramatically decreased albumin expression and enhanced NF-kappaB activity in rat hepatocytes, especially in the 1 microg/ml LPS group. This reduction in albumin expression induced by LPS can be completely inhibited by SN50 in different concentrations, and the maximal increase in albumin was observed at a SN50 dosage of 30 microg/ml. CONCLUSIONS: The results suggest that LPS inhibits albumin expression by activating NF-kappaB signaling. NF-kappaB is a critical signaling pathway in LPS-induced hypoalbuminemia which it is worthwhile to understand in studying the molecular mechanism of hypoalbuminemia in sepsis and septic shock.     

体外培养肝细胞胆汁分泌功能的研究

目的：使体外培养肝细胞获得胆汁分泌功能.方法：利用三明治构型培养成SD大鼠肝细胞并观察其胆小管结构及其胆汁分泌功能的形成,并以单层胶原培养肝细胞作对照。结果：免疫细胞化学显示三明治构型肝细胞培养24h后,胆小管结构初步形成,随着培养时间延长,胆小管结构更加清晰,120h后形成胆小管网络,而在对照组肝细胞,胆小管结构形成后,随着培养时间延长逐渐模糊,120h后胆小管结构消失,没有形成胆小管网络。其次,荧光二乙酯的肝细胞代谢显示,三明治构型肝细胞培养96h后即有胆汁分泌功能,而在对照组,肝细胞没有胆汁分泌功能。结论：三明治构型培养肝细胞在体外重建体内胆小管结构,保持了体内肝细胞胆汁分泌功能的特点。     

Isolated hepatocytes versus hepatocyte spheroids: in vitro culture of rat hepatocytes

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 +/- 4.4%), while in G2 it was 72.1 +/- 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.     

体外肝细胞极性的重建

目的 使培养肝细胞在体外重建细胞极性。方法 利用三明治构型培养成鼠肝细胞并观察其形态、特异性膜区域蛋白分布并以单层胶原培养肝细胞作对照。结果 三明治构型肝细胞呈单层生长，形成肝板样结构，伴胆小管网络形成，免疫组织化学证明肝细胞膜区域蛋白呈特异性分布。而在对照组，肝细胞基本没有形成肝板样结构，肝细胞膜区域蛋白没有形成特异性分布。结论 三明治构型培养肝细胞在体外重建细胞极性，保留了体内肝细胞的特点。     

Effects of serum from patients with acute liver failure due to paracetamol overdose on human hepatocytes in vitro

OBJECTIVE: Our goal was to investigate the effects of serum from patients with acute liver failure due to paracetamol (acetaminophen) overdose on the function of human hepatocytes in vitro. METHODS: Freshly isolated human hepatocytes plated on collagen-coated culture plates were, incubated (24 hours 37 degrees C) in medium containing pooled human sera (0%-80%) obtained from normal individuals or from patients with acute liver failure due to paracetamol overdose. The effects of the sera on cell function were assessed using MTT, [14C]-leucine incorporation, and cytochrome P450 (CYP1A1/2) activity assays. RESULTS: The overall cellular metabolic activity was significantly greater at all concentrations after exposure to acute liver failure serum compared to normal serum. There were no significant differences in the decreases produced by pooled acute liver failure and normal sera at concentrations up to 80% on the [14C]-leucine incorporation or CYP1A1/2 activity. CONCLUSION: The overall cell function/activity of human hepatocytes was not impaired in vitro on exposure to serum from patients with acute liver failure due to paracetamol overdose.     

地塞米松对单层培养的大鼠肝细胞分泌机能的影响

目的 研究地塞米松对大鼠肝细胞分泌机能的影响。方法 用Seglen两步灌注法从F344大鼠肝脏中灌注并以低温离心分离肝细胞，然后单层培养。在Willam's E培养基中加入地塞米松，通过位相差显微镜观察细胞形态；并通过荧光染料分泌实验及Rhodamin-Phallodin染色后荧光显微镜观察胆汁分泌机能和细胞骨架的改变。结果 培养基中加入地塞米松后，肝细胞毛细胆管样结构扩张，荧光染料在扩张的毛细胆管样结构中聚集，细胞骨架蛋白在毛细胆管样结构周围聚集。结论 地塞米松促进大鼠肝细胞胆汁分泌，导致肝细胞毛细胆管样结构扩张。     

高纯度透析浓缩液对血液透析患者血清白细胞介素6、肿瘤坏死因子α和白蛋白水平的影响

目的比较高纯度透析浓缩液和普通透析浓缩液对长期透析患者血清促炎症因子白细胞介素(6IL鄄6)、肿瘤坏死因子α(TNF鄄α)和血清白蛋白的影响。方法采用前瞻性临床对照研究,将85例维持性血液透析患者随机分为两组,分别采用普通透析浓缩液(常规组,例)和42高纯度透析浓缩液(高纯度组,例)进行常规低通量血液透析治疗并随访4312个。月比较常规组与高纯度组患者在血清IL鄄6、TNF鄄α、血清白蛋白、干体重、体重指数(BMI)、上臂中肌肉周径(MAC)、血红蛋白、红细胞压积、白细胞计数、中性粒细胞计数以及红细胞生成素应用剂量上的差异。结果高纯度组43例,常规组42例,两组患者间年龄、性别比例、透析龄、BMI、Kt/V值和血清IL鄄6、TNF鄄α水平差异无统计学意义。与基础水平比较,随访结束时高纯度组患者血清IL鄄6[(6.91±5.13)pg/ml比(3.06±2.42)pg/ml]和TNF鄄α水平[(14.78±4.61)pg/ml比(13.60±4.24)pg/ml]显著下降;血清白蛋白[(35.9±3.7)g/L比(37.6±3.4)g/L]、血红蛋白[(82.4±24.7)g/L比(88.2±22.9)g/L]及红细胞压积(0.25±0.07比0.28±0.05)均显著上升。与常规组比较,随访结束时高纯度组血清IL鄄6[(3.06±2.42)pg/ml比(4.22±3.99)pg/ml]和TNF鄄α水平[(13.60±4.24)pg/ml比(15.79±6.38)pg/ml均显著下降     

内毒素诱导大鼠肝细胞白蛋白表达下降的分子机制

目的　探讨内毒素诱导肝细胞白蛋白表达下降的分子机制。方法　 1μg/ml内毒素刺激肝细胞后 ,分别在 0 ,2 ,8,12和 2 4h时留取肝细胞及上清检测白蛋白mRNA及其蛋白水平的变化。细胞内信号蛋白p38激酶和ERK激酶的特异性阻断剂SB2 0 35 80和PD980 5 9预处理肝细胞后检测上清中白蛋白的浓度。结果　内毒素刺激后 2 4h白蛋白mRNA下降约为 30 % ,与此同时白蛋白浓度下降约 5 0 %。SB2 0 35 80和PD980 5 9可以在体外抑制内毒素诱导肝细胞白蛋白表达的下降。结论　内毒素在转录水平抑制肝细胞白蛋白mRNA的表达来抑制白蛋白的合成。这一过程与细胞内信号传导通路p38和ERK激酶密切相关 ,进一步阐明了感染时低白蛋白血症的分子机制。     

Effects of amino acids and albumin on erythropoietin carbamoylation

Background

Anemia in chronic renal failure results from inadequate production of erythropoietin and decrease in its biological activity, and the reduced activity of erythropoietin is caused by the presence of plasma inhibitors of erythropoietin. It is reported that one of the inhibitors of erythropoietin is cyanate, a potential uremic toxin formed spontaneously from increased urea due to decreased renal function, and erythropoietin loses its biological activity due to negatively charged cyanate. The purpose of this study is to investigate the protective effects of amino acids, positively charged amino groups, and albumin binding of several toxins on erythropoietin carbamoylation.

Methods

The degree of change in erythropoietin structure by cyanate was measured by the trinitrobenzenesulphonate reaction and Western blotting. The loss of biological activity of erythropoietin caused by cyanate was measured by injecting erythropoietin into rats with chronic renal failure.

Results

The free amino groups in erythropoietin decreased under cyanate treatment in a time- and concentration-dependent manner. In the cyanate treatment group, of the twenty amino acids, phenylalanine, valine, tryptophan, threonine, and lysine prevented the structural modification of erythropoietin, according to Western blot analysis. In addition, of the three proteins, albumin prevented the structural modification of erythropoietin. As for the cyanate with erythropoietin treatment group, only lysine and albumin prevented the loss of biological activity of erythropoietin in the rats.

Conclusion

The results of this study suggest that lysine and albumin may play a protective role against renal anemia by erythropoietin carbamoylation in chronic renal failure.     

Effects of anesthetic agents and physiologic changes on intraoperative motor evoked potentials

Motor evoked potentials (MEPs) have shown promise as a valuable tool for monitoring intraoperative motor tract function and reducing postoperative plegia. MEP monitoring has been reported to contribute to deficit prevention during resection of tumors adjacent to motor structures in the cerebral cortex and spine, and in detecting spinal ischemia during thoracic aortic reconstruction. Many commonly used anesthetic agents have long been known to depress MEP responses and reduce MEP specificity for motor injury detection. Although new stimulation techniques have broadened the spectrum of anesthetics that can be used during MEP monitoring, certain agents continue to have dose-dependent effects on MEP reliability. Understanding the effects of anesthetic agents and physiologic alterations on MEPs is imperative to increasing the acceptance and application of this technique in the prevention of intraoperative motor tract injury. This review is intended as an overview of the effects of anesthetics and physiology on the reproducibility of intraoperative myogenic MEP responses, rather than an analysis of the sensitivity and specificity of this monitoring method in the prevention of motor injury.     

微囊化猪肝细胞培养的研究

目的　微囊包裹猪原代肝细胞并进行普通培养 ,观察肝细胞形态变化。方法　在不含小牛血清的RPMI 164 0培养基中培养 ,光镜下观察培养过程中肝细胞形态变化 ,检测不同时期培养上清中白蛋白及尿素含量。结果　微囊肝细胞可较长期存活 ,微囊肝细胞组与游离肝细胞组上清蛋白分泌在前 3d差异无显著性 (P >0 .0 5 ) ,4d后差异有显著性 (P <0 .0 5 ) ,而上清尿素合成在前 2d差异无显著性 (P >0 .0 5 ) ,3d后差异有显著性 (P <0 .0 5 )。结论　微囊材料可制备微囊化猪原代肝细胞 ,并进行体外培养 ,与游离肝细胞比较具有更好分泌合成功能。