Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.     

Immobilized enzyme cellulose hollow fibers: I. Immobilization of heparinase

The immobilization of heparinase to tresyl-chloride-activated cellulose hollow fibers for the removal of heparin from the bloodstream was examined. Whole blood can be circulated through cellulose hollow fibers without hemolysis and the tresyl chloride chemistry provides a strong linkage which limits the release of the enzyme from the support. The tresylation and immobilization methods were modified and optimized to improve the heparinase activity retained by cellulose. Pretreatment of the hollow fibers with 0.05/V sodium hydroxide increased the degree of tresylation and the immobilization yield by a factor of five. The use of triethylamine as the organic base in the tresyl chloride activation resulted in threefold greater activity retention by the support than when pyridine was used. Together, sodium hydroxide pretreatment and triethylamine enhanced the activity retained by cellulose to 26.2 +/- 7.0% of that bound to the support. The activity retention was also a function of the technique used for immobilization. The best results were achieved when the enzyme was applied to the activated fibers once every 12 to 24 h for a total of four times. The active enzyme loading on the fibers was 0.3 mg heparin degraded/h cm(2) when 4.5 mug protein/cm(2) was bound to the fibers.     

Investigation of a whole blood fluidized bed Taylor-Couette flow device for enzymatic heparin neutralization

The use of clinical bioreactors will increase as more therapeutic proteins are being cloned, expressed, and produced at a reduced cost. The proposed use of an immobilized heparinase I reactor to make heparin anticoagulation a safer therapy is an example of how the specificity and high activity of an enzyme could be incorporated into a system to ultimately benefit a patient. However, the development of a safe and efficient bioreactor is important for the use of immobilized heparinase I and other therapeutic proteins designed for use in medical extracorporeal procedures. This study examined the possibility of using Taylor-Couette flow and "flow-induced" recirculation of the agarose beads as a way to fluidize agarose-bound heparinase in whole blood. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. A reactor based on Taylor-Couette flow was designed and modified with a tangential recirculation line. The reactor was tested for efficacy and safety in vitro in human blood. Visualization studies in water and 42% glycerol were used to determine the minimum rotation rate for efficient fluidization. The strategic placement of the recirculation line allowed recirculation of the agarose without the use of an external pump. The device removed 90% of the heparin activity within 2 min from 450 cc of human blood at a blood flow rate of 100 mL/min. Furthermore, the device maintained inlet and outlet clotting times of 269 +/- 10 and 235 +/- 6 s, respectively, demonstrating the potential for regional heparinization. Blood damage was a function of gel volume fraction and rotation rate of the inner cylinder. Hemolysis of the red cells is an important issue when Taylor vortices are combined with macroscopic solid particles such as agarose beads. A modified Taylor-Couette flow device was developed to treat whole blood and operational criteria were established to minimize hemolysis.     

Regional heparinization via simultaneous separation and reaction in a novel Taylor-Couette flow device.

The development of a safe and efficient bioreactor design has remained a challenge for the clinical application of immobilized enzymes. Specifically, the use of immobilized heparinase I has been the target of many studies to make heparin anticoagulation therapy safer for the critically ill patient with kidney failure or heart disease. We have investigated the use of Taylor-Couette flow for a novel type of bioreactor. In a previous study, we showed that the fluidization of agarose immobilized heparinase within Taylor vortices in whole blood can lead to extensive blood damage in the form of cell depletion and hemolysis. Based on these findings, we designed and developed a reactor, referred to as vortex-flow plasmapheretic reactor (VFPR), that incorporated plasmapheresis and fluidization of the agarose in the reactive compartment, separate from the whole-blood path. In the present study, immobilized heparinase I was tested as a means of achieving regional heparinization of a closed circuit. This is a method in which heparin is infused into the extracorporeal circuit predialyzer and neutralized postdialyzer. Saline studies were performed with an immobilized heparinase I-packed bed and with the VFPR. An in vitro feasibility study was performed with the VFPR using human blood. The VFPR achieved heparin conversions of 44 +/- 0.5% and 34 +/- 2% in saline and blood, respectively. In addition, the VFPR caused no blood damage. We report a novel method to achieve fluidization which depended on secondary, circumferencial flow, and was independent of the primary flow through the device.     

Theoretical analysis of the effect of convective flow on solute transport and insulin release in a hollow fiber bioartificial pancreas

The bioartificial pancreas, in which transplanted pancreatic tissue or isolated cells are cultured on a hollow fiber membrane, is an attractive approach to restore physiologic insulin delivery in the treatment of diabetes. Insulin response in prototype devices has been unacceptable due to the large mass transport limitations associated with the membrane and the surrounding shell region. Although available theoretical analyses provide some insight into the combined effects of transport and reaction in the bioartificial pancreas, they cannot quantitatively account for the effects of convective recirculation flow, complex intrinsic insulin secretory kinetics, and non-uniform distribution of pancreatic cells. We have developed a detailed model for glucose and insulin transport and insulin secretion in the hollow fiber bioartificial pancreas based on the solution of the mass and momentum conservation equations describing flow and transport in the lumen, matrix, and shell. Model predictions are in good agreement with literature data obtained in a hollow fiber device with minimal radial convective flow. Although no quantitative data are available for a device with significant radial convection, model simulations demonstrate that convective recirculation flow can dramatically improve insulin response, allowing the device to accurately capture the bi-phasic insulin secretion characteristic of the normal physiologic response. Results provide fundamental insights into the coupling between kinetics and transport in the hollow fiber system and a rational basis for the design of clinical devices.     

Apparent reduction of endogenous creatinine clearance by salicylate treatment.

A prospective study of nine patients with rheumatoid arthritis and 11 healthy volunteers who started salicylate treatment showed that in 18 of the 20 subjects creatinine clearance values fell the average being 25%. Serum creatinine concentration was increased by an average of 38%, although blood urea levels were unchanged. Since 51Cr-edetic acid clearance tests performed at the same time in 12 of the subjects were not affected, the fall in creatinine clearance was probably not due to impaired glomerular function, and salicylate ingestion may therefore invalidate the creatinine clearance test as an index of glomerular filtration rate. It is clearly important to inquire into the patient''s analgesic comsumption when using the creatinine clearance test to assess glomerular function.     

Carvedilol: a beta blocker with antioxidant property protects against gentamicin-induced nephrotoxicity in rats

Gentamicin is an antibiotic effective against gram negative infections, whose clinical use is limited by its nephrotoxicity. Since the pathogenesis of gentamicin-induced nephrotoxicity involves oxygen free radicals, the antioxidant carvedilol may protect against gentamicin-induced renal toxicity. We therefore tested this hypothesis using a rat model of gentamicin nephrotoxicity. Carvedilol (2 mg/kg) was administered intraperitoneally 3 days before and 8 days concurrently with gentamicin (80 mg/kg BW). Estimations of urine creatinine, glucose, blood urea, serum creatinine, plasma and kidney tissue malondialdehyde (MDA) were carried out, after the last dose of gentamicin. Kidneys were also examined for morphological changes. Gentamicin caused marked nephrotoxicity as evidenced by increase in blood urea, serum creatinine and decreased in creatinine clearance. Blood urea and serum creatinine was increased by 883% and 480% respectively with gentamicin compared to control. Carvedilol protected the rats from gentamicin induced nephrotoxicity. Rise in blood urea, serum creatinine and decrease in creatinine clearance was significantly prevented by carvedilol. There was 190% and 377% rise in plasma and kidney tissue MDA with gentamicin. Carvedilol prevented the gentamicin induced rise in both plasma and kidney tissue MDA. Kidney from gentamicin treated rats, histologically showed necrosis and desquamation of tubular epithelial cells in renal cortex, whereas it was very much comparable to control with carvedilol. In conclusion, carvedilol with its antioxidant property protected the rats from gentamicin-induced nephrotoxicity.     

Blood urea content changes in man under hypokinesia

It has been demonstrated that hypokinesia (diminished muscular activity) leads to an increase in blood urea content in man. Against this background the objective of this investigation was to determine blood urea content under hypokinesia (HK) on 17 physically healthy men aged 19-23 yr. They were divided into three groups: the 1st group (5 men) was examined under HK, the 2nd group (4 men) was studied during the background period (BGP) as well as in the readaptation period (RTP), and the 3rd group (8 men) was placed under ordinary conditions and served as control. For the simulation of the hypokinetic effect the men were kept under a rigorous bed rest regime for 16 days. Blood urea, blood creatinine, urine urea, and urine creatinine were measured. The results were processed statistically. The most pronounced increased urea content was observed in the men with an initial low concentration (3.3-4.2 mmole/liter). Variations in the urea concentration were analogous and manifested a reduction during the initial days and an elevation thereafter. Creatinine excretion and clearance were reduced uniformly and significantly during the initial 10 days of HK. It was concluded that diminished muscular activity induced an increase in urea content and a decrease in creatinine clearance in man.     

Optimization of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of in vitro matured bovine oocytes

The aim of the current work was to evaluate applicability of triacetate cellulose hollow fiber vitrification (HFV) method for cryopreservation of groups of in vitro matured bovine oocytes (12–17 oocytes per device). We also attempted to optimize HFV protocol by altering concentration of non-permeating cryoprotectant (sucrose) in vitrification solution and concentration of extracellular Ca²⁺ by using a calcium-free base medium for preparation of vitrification/rewarming solutions with ethylene glycol (EG) as a single permeating cryoprotectant. Neither of modifications of HFV protocol significantly affected survival or fertilization rates of the vitrified bovine oocytes. Embryo development rates in the vitrification groups were lower than those in the control (31.2% of blastocysts at Day 8 post IVF). Use of vitrification/rewarming solutions with lower Ca²⁺ concentration and EG did not significantly improve embryo development rates. An increase of sucrose concentration in vitrification solution from 0.5 to 1.0 M significantly improved blastocyst yield on Day 8 post IVF (21.1–23.4% vs 3.1–3.5%; p < 0.05). Obtained results indicated that sufficient dehydration of the oocytes and/or the solution surrounding them in hollow fiber before immersion into liquid nitrogen is an important factor for successful vitrification. Use of HFV method allowed simplification and standardization of vitrification/rewarming procedures. Triacetate cellulose hollow fibers can be used successfully for cryopeservation of groups of in vitro matured bovine oocytes.     

Fiber-optic fluorescence imaging

Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.     

Ginkgo biloba extract ameliorates gentamicin-induced nephrotoxicity in rats.

The effect of Ginkgo biloba (EGb), a plant extract with an antioxidant effect, has been studied on gentamicin-induced nephrotoxicity in male wistar rats. Ginkgo biloba extract (300 mg/kg BW) was administered orally 2 days before and 8 days concurrently with gentamicin (80 mg/kg BW). Saline treated animals served as control. Estimations of urine creatinine, glucose, blood urea, serum creatinine, plasma and kidney tissue MDA were carried out after 8 days of gentamicin treatment. Kidneys were examined using histological techniques. Blood urea and serum creatinine were increased by 896% and 461% respectively, with gentamicin, compared to saline treated group. Creatinine clearance was significantly decreased with gentamicin. Ginkgo biloba extract protected rats from gentamicin-induced nephrotoxicity. Changes in blood urea, serum creatinine and creatinine clearance induced by gentamicin were significantly prevented by Ginkgo biloba extract. There was a 177% and 374% rise in plasma and kidney tissue MDA with gentamicin, which were significantly reduced to normal with Ginkgo biloba extract. Histomorphology showed necrosis and desquamation of tubular epithelial cells in renal cortex with gentamicin, while it was normal and comparable to control with Ginkgo biloba extract. These data suggest that supplementation of Ginkgo biloba extract may be helpful to reduce gentamicin nephrotoxicity.     

Effects of Renal Function on Plasma Digoxin Levels in Elderly Ambulant Patients in Domiciliary Practice

An investigation into the relations between the daily dose of digoxin, drug regimen, serum digoxin concentration, and creatinine and digoxin clearance was carried out in a group of elderly ambulant patients in domiciliary practice. Moderate to severe impairment of renal function was found both in patients taking digoxin and in elderly control subjects. Plasma digoxin levels were not related to blood urea concentrations or creatinine clearance. Digoxin clearance was less than creatinine clearance. Now that plasma digoxin levels can be measured relatively easily their estimation should become part of clinical practice.     

Structure and properties of hydroxyapatite/cellulose nanocomposite films

The aim of this study was to develop a new inorganic-organic hybrid film. Nanohydroxyapaptite (nHAP) particles as the inorganic phase was mixed with cellulose in 7 wt.% NaOH/12 wt.% urea aqueous solution with cooling to prepare a blend solution, and then inorganic-organic hybrid films were fabricated by coagulating with Na₂SO₄ aqueous solution. The structure and properties of the hybrid films were characterized by high resolution transmitting electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM), thermo-gravimetric analysis (TGA), Fourier transform infra-red (FT-IR) spectra, wide angle X-ray diffraction (WAXD) and tensile testing. The results revealed that the HAP nanoparticles with mean diameter of about 30 nm were uniformly dispersed and well immobilized in the hybrid film as a result of the role of the nano-and micropores in the cellulose substrate. A strong interaction existed between HAP and cellulose matrix, and their thermal stability and mechanical strength were improved as a result of good miscibility. Furthermore, the results of 293T cell viability assay indicated that the HAP/cellulose films had excellent biocompatibility and safety, showing potential applications in biomaterials.     

Controlled trial of azathioprine in the nephrotic syndrome secondary to idiopathic membranous glomerulonephritis.

In a controlled double-blind trial five patients with the nephrotic syndrome due to idiopathic membranous glomerulonephritis received azathioprine, 2.5 mg/kg-d, while four others received placebo. After 1 year of treatment there was no significant difference between the two groups with regard to the changes in leukocyte count, values for hemoglobin, serum creatinine, blood urea nitrogen or serum albumin, 24-hour excretion of protein in the urine, or creatinine clearance. In this study azathiprine appeared not to be useful in the treatment of idiopathic membranous glomerulonephritis.     

Effect of pentoxifylline on cyclosporine-induced nephrotoxicity in rats

Effect of unique hemorrheologic agent pentoxifylline (PTX) was investigated on cyclosporine (CsA) induced nephrotoxicity in rats. Compared to saline control, CsA produced significant increase in blood urea and serum creatinine. Pentoxifylline treatment prevented the CsA-induced rise in blood urea and serum creatinine. Creatinine clearance (Ccr) and lithium clearance (Licr) was decreased with CsA. PTX treatment prevented the CsA-induced decrease in Ccr and Licr. Malondialdehyde (MDA) was increased with CsA compared to saline treated animals. PTX prevented the CsA-induced MDA rise. Kidney form CsA treated rat showed marked vacuolar degeneration of tubular epithelium with excess of microcalcification. Severity of the lesions was markedly reduced in rats treated with PTX plus CsA. The results indicate that PTX reduces CsA-induced renal toxicity in rats.     

前列地尔联合尿毒清颗粒对慢性肾衰竭患者血清甲状旁腺激素、同型半胱氨酸水平及临床疗效的影响

目的:探究前列地尔联合尿毒清颗粒对慢性肾衰竭患者血清甲状旁腺激素、同型半胱氨酸及临床疗效的影响。方法:收集我院收治的慢性肾衰竭患者74例,根据随机对照表分为对照组和试验组,每组37例。对照组予以前列地尔注射液治疗,试验组在对照组基础上予以尿毒清颗粒治疗。观察并比较两组患者临床疗效,治疗前后血清肌酐(SCr)、尿素氮(BUN)、血尿酸(UA)、内生肌酐清除率(Ccr)、超敏C反应蛋白(hs-CRP)、同型半胱氨酸(Hcy)、甲状旁腺素(PTH)、β2微球蛋白(β2-MG)水平以及不良反应的发生情况。结果:与对照组相比,试验组治疗后临床总有效率较高(P0.05)。两组治疗后血清肌酐、尿素氮、血尿酸水平下降(P0.05),内生肌酐清除率水平升高(P0.05);与对照组相比,试验组血清肌酐、尿素氮、血尿酸水平较低(P0.05),内生肌酐清除率水平较高(P0.05)。两组治疗后超敏C反应蛋白、同型半胱氨酸、甲状旁腺素以及β2微球蛋白水平降低(P0.05);与对照组相比,试验组超敏C反应蛋白、同型半胱氨酸、甲状旁腺素以及β2微球蛋白水平较低(P0.05)。两组不良反应发生情况相比差异无统计学意义(P0.05)。结论:前列地尔联合尿毒清颗粒对慢性肾衰竭患者的临床疗效显著,安全性较高,可能与其下调血清甲状旁腺激素以、同型半胱氨酸及β2微球蛋白水平有关。     

Increased curvature of hollow fiber membranes could up‐regulate differential functions of renal tubular cell layers

Tissue engineering devices as in vitro cell culture systems in scaffolds has encountered the bottleneck due to their much lower cell functions than real tissues/organs in vivo. Such situation has been improved in some extent by mimicking the cell microenvironments in vivo from either chemical or physical ways. However, microenvironmental curvature, commonly seen in real tissues/organs, has never been manipulated to regulate the cell performance in vitro. In this regard, this paper fabricated polysulfone membranes with or without polyethylene glycol modification to investigate the impact of curvature on two renal tubular cells. Regardless the varying membrane curvatures among hollow fiber membranes of different diameters and flat membrane of zero curvature, both renal cells could well attach at 4 h of seeding and form similar confluent layers at 6 days on each membrane. Nevertheless, the renal cells on hollow fibers, though showing confluent morphology as those on flat membranes, expressed higher renal functions and, moreover, the renal functions significantly increased with the membrane curvature among hollow fibers. Such upregulation on functions was unassociated with mass transport barrier of hollow fibers, because the cultures on lengthwise cut hollow fibers without mass transfer barrier showed same curvature effect on renal functions as whole hollow fibers. It could be proposed that the curvature of hollow fiber membrane approaching to the large curvature in kidney tubules increased the mechanical stress in the renal cells and thus might up‐regulate the renal cell functions. In conclusion, the increase of substrate curvature could up‐regulate the cell functions without altering the confluent cell morphology and this finding will facilitate the design of functional tissue engineering devices. Biotechnol. Bioeng. 2013; 110: 2173–2183. © 2013 Wiley Periodicals, Inc.     

The Plasma Phenolsulfonphthalein Index (PSPI) of Renal Function: II. Correlation with Other Parameters of Renal Function and Indications for Use

The plasma phenolsulfonphthalein index (PSPI) was determined in 175 subjects and was compared with levels of blood urea nitrogen (BUN) and serum creatinine, 15-minute urinary excretion of PSP, and clearance of creatinine, PAH and inulin. Statistical analyses indicate that the PSPI measures the same function as the 15-minute urine test when PSP is administered in a dosage of 1 mg./kg. body weight; and that, although the index cannot be used as a precise substitute for PAH clearance, it is equally repeatable and no greater error is associated with its measurement. The PSPI has been found of most value when complete, accurately timed urine collections are unobtainable because of urological abnormality or inability of the patient to co-operate.     

Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126

A common approach to construct a bioartificial renal tubule system is to utilize renal tubular cells seeded in porous polymer membrane hollow fibers. We have reported that overgrowth of renal tubular cells was not beneficial for the transport and reabsorption functions of bioartificial tubules. Therefore, long-term maintenance of a confluent monolayer of cells in hollow fibers is essential and technically challenging. In this study, we examined whether MEK inhibitor, U0126, could maintain the monolayer of Lewis-lung cancer porcine kidney 1 (LLC-PK(1)) cells on polystyrene plates and in a dialysis module housing hollow fibers made of ethylene vinyl alcohol (EVAL). We also evaluated the leakage of urea nitrogen (UN) and creatinine (Cr) through the cell-lined hollow fibers, and reabsorption of glucose and sodium by the cells, comparing the U0126-treated cells with nontreated cells in the module. Treatment with 50micromol l(-1) U0126 prevented the overgrowth of cells cultured on polystyrene plates. Moreover, U0126-treatment reduced the leakage of UN, and increased the reabsorption of electrolytes in 65cm(2) modules. Scanning electron microscopy revealed that it also prevented the overconfluence of cells in modules. Therefore, application of U0126 is a potentially effective method to improve the performance of the device.     

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.