Binding and detoxification efficiency of albumin decline after haemodialysis.

BACKGROUND: Albumin, as the most abundant plasma protein, represents a target structure for both drug and physicochemical therapeutic approaches to eliminate uraemic toxins more efficiently. Potentially, this approach could reduce mortality of haemodialysis patients. However, little is known about albumin functional properties in these patients and its alteration by haemodialysis treatment. METHODS: The binding and detoxification efficiency of albumin were assessed by electron paramagnetic resonance spectroscopy using a spin-labelled fatty acid. Binding efficiency (BE) reflects strength and amount of bound fatty acids under certain ethanol concentration. Detoxification efficiency (DTE) reflects the molecular flexibility of the patient's albumin molecule, thus the ability to change the conformation depending on ethanol concentration. Percentage of BE and DTE are depicted in relation to healthy individuals (100%). RESULTS: Fifty-eight patients (59% male, median age 68 years, median time on haemodialysis 32 months) were included in the study. Before haemodialysis treatment, albumin binding and detoxification efficiency were substantially below healthy individuals [median BE 52% (interquartile range, IQR, 45%-59%); median DTE 38% (IQR 32-49%)]. After haemodialysis treatment, median BE and DTE significantly decreased [BE 28% (IQR 20-41%); DTE 11% (IQR 7%-27%; P < .001)]. BE and DTE decline after haemodialysis was not dependent on age, sex or treatment modalities, but was to a certain extent on the level of non-esterified fatty acids. CONCLUSION: Albumin binding and detoxification efficiency of fatty acids in maintenance haemodialysis patients were substantially below those in healthy individuals and even declined after dialysis treatment. These findings might be helpful when considering new therapeutic approaches in maintenance haemodialysis patients.

Albumin redox state of maintenance haemodialysis patients is positively altered after treatment.

BACKGROUND AND AIM: Maintenance haemodialysis patients have increased morbidity and mortality which is mainly driven by an elevated inflammation level due to the uraemic milieu. A major part of this increased inflammation level is the degree of oxidative stress which can be assessed by albumin redox state (ARS). Aim of this study was to evaluate how the ARS is affected by a haemodialysis treatment and different dialyzer properties. METHODS: ARS was determined before and after haemodialysis treatment by fractionating it into reduced human mercaptalbumin (HMA), reversibly oxidized human non-mercaptalbumin 1 (HNA-1), and irreversibly oxidized human non-mercaptalbumin 2 (HNA-2) by high-performance liquid chromatography. In healthy individuals, albumin circulates in the following proportions: HMA 70-80%, HNA-1 20-30% and HNA-2 2-5%. High flux (FX 100 [Fresenius Medical Care], BG 1.8 [Toray], Xevonta Hi 18 [B. Braun], CTA-2000 [Kawasumi]) and low flux FX10 [Fresenius Medical Care] dialyzers were used. RESULTS: 58 patients (59% male, median age 68 years, median time on haemodialysis 32 month) were included in the study. Before haemodialysis treatment, HMA (median 55.9%, IQR 50.1-61.2%) was substantially lower than in healthy individuals. Accordingly, oxidized albumin fractions were above the level of healthy individuals (median HNA-1 38.5%, IQR 33.3-43.2%; median HNA-2 5.8%, IQR 5.1-6.7%). Before haemodialysis treatment HMA was significantly higher in patients usually treated with high flux membranes (p < 0.01). After haemodialysis treatment there was a significant increase of HMA and a decrease of HNA-1 and HNA-2 (p < 0.01). These effects were more pronounced in patients treated with high flux dialyzers (p < 0.01). There were no differences of ARS alteration with regard to the dialyzer´s sterilization mode or the presence of diabetes. CONCLUSION: The study confirms that the ARS is positively altered by haemodialysis and shows for the first time that this effect depends on dialyzer properties.

Exploring Albumin Functionality Assays: A Pilot Study on Sepsis Evaluation in Intensive Care Medicine.

Human serum albumin (HSA) as the most abundant plasma protein carries multifunctional properties. A major determinant of the efficacy of albumin relies on its potent binding capacity for toxins and pharmaceutical agents. Albumin binding is impaired in pathological conditions, affecting its function as a molecular scavenger. Limited knowledge is available on the functional properties of albumin in critically ill patients with sepsis or septic shock. A prospective, non-interventional clinical trial assessed blood samples from 26 intensive care patients. Albumin-binding capacity (ABiC) was determined by quantifying the unbound fraction of the fluorescent marker, dansyl sarcosine. Electron paramagnetic resonance fatty acid spin-probe evaluated albumin's binding and detoxification efficiencies. Binding efficiency (BE) reflects the strength and amount of bound fatty acids, and detoxification efficiency (DTE) indicates the molecular flexibility of patient albumin. ABiC, BE, and DTE effectively differentiated control patients from those with sepsis or septic shock (AUROC > 0.8). The diagnostic performance of BE showed similarities to procalcitonin. Albumin functionality correlates with parameters for inflammation, hepatic, or renal insufficiency. Albumin-binding function was significantly reduced in critically ill patients with sepsis or septic shock. These findings may help develop patient-specific algorithms for new diagnostic and therapeutic approaches.

Determination of binding characteristics as a measure for effective albumin using different methods.

BACKGROUND & AIMS: Transport functions of albumin are of clinical and pharmacological interest and are determined by albumin's properties like posttranslational modifications or bound ligands. Both are affected in pathological conditions and in therapeutic grade albumin solutions. The term effective albumin concentration was introduced as a measure of functionally intact albumin. Our aim was to evaluate the impact of ligands and modifications with different approaches as a measure of effective albumin. APPROACH & RESULTS: We used a spin labelled fatty acid and dansylsarcosine to characterize binding properties of albumin i) prepared from plasma of patients and healthy control donors, ii) measured directly out of plasma, iii) research grade albumin, iv) in vitro modified albumin, and v) therapeutic infusion solutions before and after removal of stabilizers. Bilirubin is the main determinant for binding function in patients' albumin. In in vitro prepared albumin bound fatty acids correlated with impaired binding. Human nonmercaptalbumin1, not human nonmercaptalbumin2, showed reduced binding properties. Binding and transport function of therapeutic albumin was severely impaired and restored by filtration. Glycation of research grade albumin had no effect on the binding of dansylsarcosine and only a minor effect on fatty acid binding. CONCLUSIONS: Our results suggest that effective albumin -in terms of binding properties- is primarily determined by bound ligands and only to a minor extent by posttranslational modifications. Characterizing albumin directly from plasma better reflects the physiological situation whereas in the case of therapeutic grade albumin stabilizers should be removed to make its binding properties accessible.

Spin electron paramagnetic resonance of albumin for diagnosis of oral squamous cell carcinoma (OSCC).

OBJECTIVES: Albumin has a known capability to modulate free serum concentrations of proteins produced by tumour cells. The technique of spin probe labelling of albumin followed by electron paramagnetic resonance (EPR) spectroscopy may allow identification of these structural and functional changes, which regularly occur as consequence of binding tumour metabolites as ligands. The aim of the present study was a proof of principle evaluation of EPR-analysis of peripheral blood samples as possible predictor for oral squamous cell carcinoma (OSCC). MATERIAL AND METHODS: The present study is designed as gender-matched cohort. EPR was tested after retrieval of peripheral blood samples. The study group is represented by 32 patients with OSCC, and the control group consisted of 30 healthy patients. RESULTS: Overall analysis exhibited a diagnostic sensitivity of 72% (23/32 OSCC group) and a specificity of 80% (24/30 control group). Subgroup analysis revealed ten patients with elevated leukocytes (>10,000/µl; n = 9 [OSCC group] and n = 1 [control group]). After exclusion of patients with elevated white blood cell count, sensitivity considerably increased to 87% and specificity to 83%. CONCLUSION: EPR analysis of peripheral blood samples might be appropriate to support the clinician in primary and follow-up diagnosis of potential tumours such as OSCC. Unfortunately, subgroup analysis characterises the method vulnerable to inflammation. CLINICAL RELEVANCE: Nevertheless, our preliminary results are intriguing, as diagnosis of OSCC appears possible by simple peripheral blood examination. Thus, further appraisal of this novel method with inclusion of different tumour entities, systemic conditions and inflammation in a larger study population appears highly valuable.

Analysis of albumin fatty acid binding capacity in patients with benign and malignant colorectal diseases using electron spin resonance (ESR) spectroscopy.

INTRODUCTION: In colorectal cancer (CRC), no biological marker is known that could serve both as a marker for detection and prognosis. Electron spin resonance (ESR) spectroscopy of spin-labeled fatty acid (FA) molecules binding to human serum albumin is a suitable method for the detection of conformational changes and alterations of transport function of albumin through changes in its FA binding capabilities. OBJECTIVE: The aim of this study was to examine whether the FA binding to albumin is detectably and significantly altered in CRC patients when compared with patients having benign colorectal diseases. MATERIALS AND METHODS: One hundred four patients operatively or endoscopically treated for CRC, sigmoid diverticulitis, or a colorectal adenoma were examined before procedure. Albumin was analyzed by ESR with spin-labeled FA. A determination ratio (DR) was calculated from the measured ESR spectra as ratios of the fraction of FA that is tightly bound vs. the fractions that are loosely interacting with albumin or are unbound. RESULTS AND DISCUSSIONS: Patients with CRC showed significantly lower DR values (DR, -0.09 +/- 0.98 vs. 0.61 +/- 1.43) than patients with benign colorectal diseases, consistent with a change of conformation and transport function of albumin in CRC. Within the CRC group, with advanced tumor stage, the difference in DR values increased. ESR of FA binding to albumin thus seems to be suitable for detection of patients with CRC. Furthermore, a correlation with advanced tumor stage can be established. CONCLUSIONS: These results suggest that a further evaluation of the role of ESR in patients with all stages of CRC should take place. It should also be examined whether ESR might play a role in detecting CRC in a larger panel of patients.

Alterations in the functional capacity of albumin in patients with decompensated cirrhosis is associated with increased mortality.

UNLABELLED: Albumin concentration is diminished in patients with liver failure. Albumin infusion improves survival of cirrhotic patients with spontaneous bacterial peritonitis, and it is hypothesized that this may be due in part to its detoxifying capabilities. The aim of this study was to perform detailed quantitative and qualitative assessment of albumin function in patients with cirrhosis. Healthy controls and patients with acute deterioration of cirrhosis requiring hospital admission (n = 34) were included. Albumin function was assessed using affinity of the fatty acid binding sites using a spin label (16 doxyl-stearate) titration and electron paramagnetic resonance spectroscopy and ischemia-modified albumin (IMA) was measured. Twenty-two patients developed acute-on-chronic liver failure. Twelve were treated with the Molecular Adsorbents Recirculating System (MARS) and 10 with standard medical therapy. For each parameter measured, the patients' albumin had reduced functional ability, which worsened with disease severity. Fifteen patients died, and IMA, expressed as an albumin ratio (IMAR), was significantly higher in nonsurvivors compared with survivors (P < 0.001; area under the receiver operating curve = 0.8). No change in the patients' albumin function was observed following MARS therapy. A significant negative correlation between IMAR and the fatty acid binding coefficients for sites 1 and 2 (P < 0.001 for both) was observed, indicating possible sites of association on the protein. CONCLUSION: The results of this study suggests marked dysfunction of albumin function in advanced cirrhosis and provide further evidence for damage to the circulating albumin, which is not reversed by MARS therapy. IMAR correlates with disease severity and may have prognostic use in acute-on-chronic liver failure.

Fatty acid binding sites of human and bovine albumins: differences observed by spin probe ESR.

Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5-8.0) and temperatures (25-50 degrees C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.

Recognition of malignant processes with neural nets from ESR spectra of serum albumin.

Cancer diseases are the focus of intense research due to their frequent occurrence. It is known from the literature that serum proteins are changed in the case of malignant processes. Changes of albumin conformation, transport efficiency, and binding characteristics can be determined by electron spin resonance spectroscopy (ESR). The present study analysed the binding/dissociation function of albumin with an ESR method using 16-doxyl stearate spin probe as reporter molecule and ethanol as modifier of hydrophobic interactions. Native and frozen plasma of healthy donors (608 samples), patients with malignant diseases (423 samples), and patients with benign conditions (221 samples) were analysed. The global specificity was 91% and the sensitivity 96%. In look-back samples of 27 donors, a malignant process could be detected up to 30 months before clinical diagnosis. To recognise different entities of malignant diseases from the ESR spectra, Artificial neural networks were implemented. For 48 female donors with breast cancer, the recognition specificity was 85%. Other carcinoma entities (22 colon, 18 prostate, 12 stomach) were recognised with specificities between 75% and 84%. Should these specificity values be reproduced in larger studies, the described method could be used as a new specific tumour marker for the early detection of malignant processes. Since transmission of cancer via blood transfusion cannot be excluded as yet, the described ESR method could also be used as a quality test for plasma products.

Inhibition of basal and interleukin-1-induced VCAM-1 expression by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells.

Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMCPHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMCLOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMCPHGPx and SMCLOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMCPHGPx to SMCLOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMCPHGPx, whereas in SMCLOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx and 75% in SMCLOX. VCAM-1-inducibility was abrogated in SMCLOX but not in SMCPHGPx. Accordingly, NFkappaB-driven reporter gene activation by IL-1 was unaffected in SMCPHGPx but abolished in SMCLOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMCLOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system.

Selenium-dependent enzymes in endothelial cell function.

Glutathione peroxidases and thioredoxin reductases are the main selenoproteins expressed by endothelial cells. These enzymes reduce hydroperoxides, their role in endothelial cell physiology, however, by far exceeds prevention of oxidative damage. Reactive oxygen and nitrogen species, especially superoxide, hydroperoxides, and nitric oxide, are crucial signaling molecules in endothelial cells. Their production is regulated by vascular NAD(P)H oxidases and the endothelial nitric oxide synthase. Their metabolism and physiological functions are coordinated by glutathione peroxidases and the thioredoxin/thioredoxin reductase system. Endothelial selenoproteins are involved in the regulation of the vascular tone by maintaining the superoxide anion/nitric oxide balance, of cell adhesion by controlling cell adhesion molecule expression, of apoptosis via inhibition/activation of apoptosis signal-regulating kinase-1, and of eicosanoid production by controlling the activity of cyclooxygenases and lipoxygenases. Accordingly, they regulate inflammatory processes and atherogenesis. The underlying mechanisms are various and differ between individual selenoproteins. Scavenging of hydroperoxides not only prevents oxidative damage, but also interferes with signaling cascades and enzymes involved. Modulation of proteins by hydroperoxide-driven thiol/disulfide exchange is a novel mechanism that needs to be further investigated. A better understanding of the complex interplay of selenoproteins in regulating endothelial cell functions will help to develop a rationale for an improvement of health by an optimum selenium supply.