Non-albumin Proteins Research Articles

Albumin-globulin ratio and mortality in patients on peritoneal dialysis: a retrospective study

BackgroundAlbumin-globulin ratio (AGR), a variable based on serum albumin and non-albumin proteins, has been demonstrated as a predictor of mortality in patients with malignant neoplasm. The aim of this study was to evaluate the prognostic value of AGR on peritoneal dialysis (PD) patients.MethodsWe retrospectively analyzed 602 incident PD patients from January 1st, 2008, to December 31st, 2017, at our center and followed them until December 31st, 2018. Kaplan-Meier curves and multivariate Cox regression models were applied to analyze the association between AGR and all-cause of mortality and cardiovascular mortality.ResultsThe median follow-up time was 32.17 (interquartile range = 32.80) months. During follow-up, 131 (21.8%) patients died, including 57 patients (43.5%) who died due to cardiovascular diseases. Kaplan-Meier curves showed that patients with AGR > 1.26 had better rates of survival than those with AGR ≤ 1.25 (p < 0.001). After adjusting for potential confounders, the lower AGR level was significantly associated with an increased all-cause and cardiovascular mortality [hazard ratio (HR): 1.57, 95% confidence interval (CI): 1.07–2.32, p = 0.022 and HR: 2.01, 95% CI: 1.10–3.69, p = 0.023 respectively].ConclusionsPatients with a low AGR level had an increased all-cause and cardiovascular mortality. AGR may be a useful index in identifying patients on PD at risk for CVD and all-cause of mortality.

Comparison of accompanying proteins in different therapeutic human serum albumin preparations

Pharmaceutical human serum albumin products are manufactured from donated human plasma and may contain up to 5% accompanying non-albumin proteins. It has been reported that albumin preparations manufactured by different pharmaceutical companies differed in the degree of posttranslational modifications, the redox state as well as antioxidant properties of albumin, whereas the composition of the accompanying proteins has never been comparatively analyzed. In this study, a non-targeted mass spectrometric approach was used for label-free quantification and comparison of different pharmaceutical albumin preparations. Haptoglobin and a few other proteins accounted for approximately 80% of the accompanying proteins in all products tested. Low abundance proteins were enriched by means of a combinatorial peptide ligand library (ProteoMiner, Bio-Rad). Significant differences between the amounts of several mainly low abundance proteins, such as complement factors, were observed indicating differences in the manufacturing processes of the pharmaceutical companies. The removal of the stabilizers octanoate and N-acetyltryptophan from albumin solutions using the charcoal-based Hepalbin adsorbent simultaneously reduced the accompanying proteins. For therapy evaluation of albumin preparations, the variable composition of the accompanying proteins in different albumin products should be taken into account in addition to the known heterogeneity of the albumin protein itself.

Development of a Novel Polyclonal Antibody-Based Immunoassay for the Quantitation of Non-Albumin Urinary Proteins

Background: Better diagnostic methods to detect urinary proteinuria or albuminuria, important indicators of diabetic kidney disease, are needed. Here, we describe the development and performance qualification of a new immunoassay for the quantitation of non-albumin urinary proteins of 20 to 90 kilodaltons. Methods: Urinary proteins, purified from pooled, 24-hour, diabetic urines, were immunoabsorbed to remove albumin, electrophoretically characterized, and identified by mass spectrometry. Sheep anti-urinary protein polyclonal antibodies were immunoabsorbed to remove albumin reactivity. Major immunoreactive specificities of the polyclonal antibody were identified by Western blot. A polyclonal antibody-based competitive immunoassay was developed and performanceevaluated. An unpaired t-test (α = 0.05) and a receiver-operating characteristic curve were used to evaluate the measurement of 24-hour urinary protein excretion rates in distinguishing between normal, proteinuric, and albuminuric samples. Results: Approximately 380 mg of urinary protein was purified from 6.0 g of total urinary proteins. Mass spectrometry identified more than 36 different proteins in the purified preparation. The anti-urinary protein polyclonal antibody possessed significant immunoreactivity towards transferrin, IgG chains, alpha-1-acid glycoprotein, zinc-alpha-2-glycoprotein, prostaglandin-H2-d-isomerase, and alpha-1-microglobulin. The competitive immunoassay exhibited excellent analytical and clinical performance. Measurement of urinary protein excretion rates could distinguish between normoproteinuric and proteinuric samples (p &lt; 0.0001; area under the curve = 0.6900) and between normoalbuminuric and albuminuric samples (p &lt; 0.0001; area under the curve = 0.8782). Conclusion: Measurement of urinary protein excretion rates using the urinary protein immunoassay is clinically equivalent to laboratory methods of quantitating total urinary protein or albumin in identifying proteinuria and albuminuria.

Method for Approximating Colloid Osmotic Pressure in Long‐Term Hemodialysis Patients

Colloid osmotic pressure (COP) is important in calculating vascular permeability during hemodialysis (HD). However, few reports have directly measured COP in HD patients. Therefore, the currently published formulas may not be clinically optimal for predicting COP for HD patients. Here, the study aims were (i) to directly measure COP in HD patients and compare the measured and predicted COP values using four previously reported formulas, and (ii) to develop a formula for approximating COP using clinical parameters. We obtained 212 measured COP values using an osmometer; the average value was 22.0 ± 0.2 mm Hg. The predicted COP based on the four different formulas was positively correlated with the measured COP (0.87 < r < 0.89), but was significantly overestimated compared to it (P < 0.001). We also performed a stepwise analysis using serum albumin and non-albumin protein concentrations and obtained the following simple formula for COP approximation: COP (mm Hg) = -7.91 + 5.64 × albumin (g/dL) + 3.00 × non-albumin proteins (g/dL). A positive linear correlation was observed between the measured COP and approximated COP using this formula (r = 0.90, P < 0.001). We calculated the mean Kr (plasma-refilling coefficient) as a marker for determining dry weight in HD patients using the measured COP and approximated COP. No differences were observed between the mean Kr derived from the measured and approximated COP. We report here significant differences between measured and predicted COP values, and have devised a simple formula for COP approximation in HD patients.

ORIGINAL RESEARCH: Comparison of methods for depletion of albumin and IgG from equine serum

Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. One-dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. For pooled serum, depletion rates varied from 35-90% for albumin and 0-94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.

Chromatography of carbon nanotubes separated albumin from other serum proteins: a method for direct analysis of their interactions

Chromatography technology was employed to clarify the mechanism of interaction between multi-wall carbon nanotubes (MWCNT) and proteins. A column (16x100 mm) was packed with purified MWCNT, and various proteins were eluted with phosphate buffered saline (PBS) with and without gradient systems. It was found that albumin in bovine serum was eluted immediately from the column without any adsorption to MWCNT. Conversely, the non-albumin proteins, including a protein of 85 kDa molecular mass and a group of proteins with molecular masses higher than 115 kDa, exhibited considerably high affinity towards MWCNT. A sample of pure bovine serum albumin was also eluted immediately from the column, while lysozyme did not elute as a peak with PBS, but eluted with 0.6 M NaCl. Fundamentally, carbon nanotubes are devoid of any electrical charge. Therefore, other forces including the hydrogen bonds, hydrophilic interactions, and van der Waals forces were most probably responsible for the differential elution behaviors. In conclusion, this chromatographic method provided a simple and direct analysis of the interactions between carbon nanotubes and the various proteins.

Respective Predictive Role of Urinary Albumin Excretion and Nonalbumin Proteinuria on Graft Loss and Death in Renal Transplant Recipients

Proteinuria is constituted by urinary albumin (UAE) and nonalbumin proteins (NAP). UAE was shown to predict ESRD and death. Whether NAP predicts graft or patient outcome is unknown in renal transplantation. We retrospectively analyzed the impact of UAE and NAP respectively on end-stage renal disease (ESRD) and death in 616 renal transplant recipients. In subjects with proteinuria <0.25 g/day, 76% of urine proteins were NAP; in those with >1 g/day, 44% of the urine proteins were NAP. Determinants of UAE and NAP were partly different: fasting glucose, body weight, donor cause of death and cyclosporine were significantly associated with NAP (but not UAE); panel reactive antibodies (PRA) and rapamycine were significantly associated with UAE (but not with NAP). NAP expressed as a continuous (HR: per g/day: 4.00 [2.85-5.63], p < 0.0001) or a categorical (presence vs. absence, HR = 29.09[8.80-96.20], p < 0.0001) parameter and UAE (per g/day, HR = 1.86 [1.24-2.78], p < 0.0001) were risk factors for graft loss in univariate analyses. NAP remained significant even after adjustment on UAE. The presence of NAP (HR: 5.37 [2.55-11.34], p < 0.0001) and macroalbuminuria (HR: 4.12 [1.65-10.29], p = 0.0024) were risk factors for death. Proteinuria is made of various proportions of UAE and NAP in renal transplantation; these two parameters provide different information on graft/patient survival.

Pseudospecific magnetic affinity beads for immunoglobulin‐G depletion from human serum

AbstractMagnetic poly(ethylene glycol dimethacrylate‐N‐methacryloyl‐(L)‐histidine methyl ester) [m‐poly(EGDMA‐MAH)] beads were prepared by suspension polymerization for the affinity depletion of immunoglobulin‐G (IgG) from human serum in a batch system. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 μmol MAH/g polymer. IgG adsorption onto the m‐poly(EGDMA) was negligible. Higher adsorption value (up to 46.8 mg/g) was obtained in which the m‐poly(EGDMA‐MAH) beads were used. IgG adsorption capacity of the magnetic beads increased with an increase in the concentration of IgG. The maximum IgG adsorption was observed at pH 6.5 for MOPS buffer. IgG molecules could be repeatedly adsorbed and eluted with these adsorbents, without noticeable loss in their IgG adsorption capacity. Adsorption capacity decreased for both increasing salt concentration and temperature. In this study, we show that m‐poly(EGDMA‐MAH) beads (wherein IgG molecules bind directly with the matrix) can be used directly for affinity depletion without further modification. Higher adsorption value was obtained from human serum (up to 85.7 mg/g). The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. The depletion efficiencies for IgG were above 85% for all studied concentrations. Eluted portion was analyzed for testing the IgG removal efficiency by two dimensional gel electrophoresis. Eluted proteins include mainly IgG, and a small number of nonalbumin proteins such as apolipoprotein A1, serotransferrin, haptoglobulin, and α1‐antitrypsin. IgA was not identified in eluted fraction. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

26 Alpha-1-antitrypsin deficiency: can it be treated and how?

Magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(l)-histidine methyl ester) [mag-poly(EGDMA-MAH) beads, 50–100 μm in diameter, were produced by suspension polymerization for affinity depletion of immunoglobulin G (IgG) from human serum. Cu2+ ions were complexed directly via MAH groups (Cu2+ loading: 4.1 μmol/g). IgG depletion studies were performed by magnetically stabilized fluidized bed column. Acetate, Tris–HCl, MES and phosphate buffers all allow adsorption of similar quantities of IgG (27.3–45.6 mg/g). MOPS and HEPES allow higher adsorption quantities (79.6 mg/g and 74.1 mg/g, respectively). Maximum adsorption capacities in MOPS buffer were 46.8 mg/g for mag-poly(EGDMA-MAH) and 102.1 mg/g for Cu2+ chelated mag-poly(EGDMA-MAH) beads. The adsorption capacity decreased drastically from 102.1 mg/g to 30.7 mg/g with the increase of the flow rate from 0.2 ml/min to 3.5 ml/min. The elution studies were performed by 1.0 M NaCl. The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 99.4%. Eluted proteins include mainly IgG, and a small number of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and α1-antitrypsin. When anti-HSA-sepharose adsorbent is used together with our metal-chelated mag-beads, IgG and HSA can be depleted in a single step.

Chip Electrophoresis as a Method for Quantifying Total Microalbuminuria

Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins. We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer's sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents. With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R2=0.9997; concentration range, 5-300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%-13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in all urine samples. The method also clearly resolved the albumin peak from interfering proteins. Unlike immunoassay, chip electrophoresis can detect both immunoreactive and nonimmunoreactive forms of albumin. This system is a simple, robust method to quantify microalbuminuria with good sensitivity, precision, and accuracy.

Pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid (AS1404), a novel vascular disrupting agent, in phase I clinical trial

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) (AS1404) is a novel antitumour agent that selectively disrupts tumour vasculature and induces cytokines. The purpose of this study was to determine the pharmacokinetics (PK) of DMXAA in cancer patients enrolled in a phase I clinical trial. DMXAA was administered as a 20-min i.v. infusion every 3 weeks and doses were escalated in cohorts of patients according to a predefined schema. PK samples were taken over the first 24 h of at least the first cycle. DMXAA was administered to 63 patients at 19 dose levels from 6 to 4,900 mg m(-2), and 3,700 mg m(-2) was established as the maximum tolerated dose. The PK observed over the dose range showed a non-linear fall in clearance from 16.1 to 1.42 l h(-1) m(-2) and resultant increase in the area under the concentration-time curve (AUC) from 1.29 to 12,400 microM h. In contrast, the increase in peak plasma concentrations from 2.17 to 1,910 microM approximated linearity. DMXAA was highly protein-bound to albumin (>99%) until saturation occurred at higher doses, leading to a rapid increase in the free fraction (up to 20%) and greater concentrations of DMXAA bound to non-albumin proteins. However, the main determinant of the non-linearity of the PK appeared to be sequential saturation of elimination mechanisms, which include hydroxylation, glucuronidation and perhaps hepatic transport proteins. This resulted in an exaggerated non-linear increase in free DMXAA plasma concentrations and AUC compared to total drug. The PK of DMXAA are well-defined, with a consistent degree of non-linearity across a very large dose range.

Immunoglobulin G depletion from human serum with metal-chelated beads under magnetic field

Magnetic poly(ethylene glycol dimethacrylate-N-methacryloyl-(L)-histidine methyl ester) [mag-poly(EGDMA-MAH) beads, 50-100 microm in diameter, were produced by suspension polymerization for affinity depletion of immunoglobulin G (IgG) from human serum. Cu2+ ions were complexed directly via MAH groups (Cu2+ loading: 4.1 micromol/g). IgG depletion studies were performed by magnetically stabilized fluidized bed column. Acetate, Tris-HCl, MES and phosphate buffers all allow adsorption of similar quantities of IgG (27.3-45.6 mg/g). MOPS and HEPES allow higher adsorption quantities (79.6 mg/g and 74.1mg/g, respectively). Maximum adsorption capacities in MOPS buffer were 46.8 mg/g for mag-poly(EGDMA-MAH) and 102.1mg/g for Cu2+ chelated mag-poly(EGDMA-MAH) beads. The adsorption capacity decreased drastically from 102.1mg/g to 30.7 mg/g with the increase of the flow rate from 0.2 ml/min to 3.5 ml/min. The elution studies were performed by 1.0M NaCl. The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 99.4%. Eluted proteins include mainly IgG, and a small number of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin. When anti-HSA-sepharose adsorbent is used together with our metal-chelated mag-beads, IgG and HSA can be depleted in a single step.

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.

The analysis and characterisation of immuno-unreactive urinary albumin in healthy volunteers

Objectives: To compare the analysis of different forms of intact albumin in urine from healthy volunteers. To determine contamination by common non-albumin proteins on HPLC analysis of urinary albumin and of purified immuno-unreactive albumin. Design and methods: Overnight urine samples collected from healthy volunteers were analysed for total albumin (immunoreactive plus immuno-unreactive) by HPLC and densitometry following native PAGE separation and for immunoreactive albumin by RIA. The contamination by non-albumin proteins of the HPLC analysis of urinary albumin and of immuno-unreactive albumin preparations was determined by ELISA. Immuno-unreactive albumin was tested for Co 2+-binding capacity. Results and conclusions: HPLC analysis of healthy urine generates higher ACR values than immunological methods due to the presence of immuno-unreactive albumin. Immuno-unreactive albumin cannot be accounted for by the non-albumin urinary proteins tested. Isolated immuno-unreactive albumin is not recognised by antibodies to common urinary proteins or by an array of anti-albumin antibodies and behaves like serum albumin in terms of HPLC elution, native PAGE migration, and cobalt ion binding.

Congenital analbuminaemia: biochemical and clinical implications. A case report and literature review

Congenital analbuminaemia was diagnosed in a small-for-gestational-age neonate presenting with placental and body oedema, an unusual presentation of this rare autosomal recessive disorder. A review of 39 reported cases in the literature shows that the clinical symptoms are always remarkably mild and that the diagnosis is rarely made in infancy. The absence of albumin appears to be partly counterbalanced by high levels of non-albumin proteins and circulatory adaptations. However, congenital analbuminemia can have important complications: lipodystrophy and hypercholesterolaemia, possibly leading to atherosclerosis. Other possible complications reported in literature are hypercoagulability, osteoporosis, respiratory tract infections, intrauterine growth retardation and intrauterine death. Moreover, albumin-binding drugs should be used with caution. Congenital analbuminaemia is a rare disorder with remarkably mild signs and symptoms at all ages. Although often thought to be innocent, this disorder may have important clinical complications.

Cellular Oxidant Stress and Advanced Glycation Endproducts of Albumin: Caveats of the Dichlorofluorescein Assay*

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO–BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO–BSA had increased affinity for Cu2+ and Ca2+, but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (HSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO–BSA is less hydrophobic than BSA. Finally, BSA also prevented H2O2 catalyzed DCF fluorescence more potently than MGO–BSA. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin.

Oxypurines, Protein, Glucose and the Functional State of Blood Vasculature Are Markers of Renal Calcium Stone-forming Processes? Observations in Men with Idiopathic Recurrent Calcium Urolithiasis

In idiopathic calcium urolithiasis the relationships between oxypurines, accompanying proteins and glucose in urine and plasma, and the associated metabolic activity (MA) are unknown. To establish whether MA is related to these parameters and to calcium oxalate crystallization, or whether it reflects a reaction of metabolism to systemic insults was the major goal of the work. One hundred fifty one males were studied in three trials: trial 1 (n=130 patients) and trial 2 (n=24 patients) were cross-sectional; trial 3 included 11 patients and 14 controls). Mean age was 46 years (trials 1 and 2) and 29 years (trial 3). In trial 1 the stratification was based on the median urinary oxypurine excretion, in trial 2 on the median plasma oxypurine concentration (below or above: Low and High subgroups). No dietary restrictions were imposed, but standardized ambulatory laboratory testing was carried out. MA was quantitated by a score. Established analytical methods were used, except for oxypurine measurement which was done by high performance liquid chromatography. Patients with kidney stones tended to be overweight (body mass index >25 kg/(m)2) and to have fasting hyperglycemia. In trial 1 severe oxypurinuria, and especially severe xanthinuria, was associated with an increase in urinary pH, creatinine clearance, proteins, uric acid, malonedialdehyde (indicator of lipid peroxidation), systolic blood pressure, and with a decrease in plasma uric acid (synonymous with a decrease of antioxidant capacity). Tubular reabsorption of proteins and stone-forming substances was diminished but MA remained unchanged despite slightly increased calcium oxalate crystal growth. In trial 2 high adenosine and xanthine coincided with elevated systolic and diastolic blood pressure, high uric acid with high urinary malonedialdehyde, high summed oxypurines minus uric acid with an increase of diastolic blood pressure, glycemia and MA; urinary nitrate (indicator of systemic vasodilation) was unchanged. In trial 3 patients' oxypurinemia and proteinuria were normal, but body mass index, glycemia and insulinemia were increased. Urinary total protein, albumin and non-albumin proteins were positively predicted (multivariate regression analysis) by urinary xanthine, glucose and pH (trial 1); MA was positively (trial 3) or negatively (trial 2) predicted by urinary total protein. In calcium urolithiasis, a disorder of affluence, 1) oxypurinuria and proteinuria and oxypurinemia and MA appear causally linked, presumably via oxidant/antioxidant imbalance-induced renal tissue damage; 2) urinary proteins may act as inhibitors or promoters of stone-forming processes; 3) a stone-initiating role of impaired vasodilatation is conjectural; 4) overweight, obesity, mild glucosuria and hyperdynamic blood circulation are regular signs.

Hypoalbuminemia causes high blood viscosity by increasing red cell lysophosphatidylcholine

Albumin deficiency is accompanied by a reduction in red cell deformability and blood hyperviscosity. Albumin deficiency increases plasma fibrinogen and triglyceride levels and may alter red cell membrane lipid composition. These options, which could all contribute to reduced red cell deformability (RCD) and hyperviscosity, were studied in the Nagase analbuminemic rat (NAR), a mutant Sprague Dawley rat (CON), characterized by normal total protein levels, with an absolute deficiency of albumin, but elevated levels of non-albumin proteins and hyperlipidemia. Plasma protein-binding of the polar phopholipid lysophosphatidylcholine (LPC) was markedly decreased. LPC comprised only 26 +/- 1% of total plasma phospholipids as compared to 42 +/- 2% in CON. NAR red cells in CON plasma had a viscosity that was similar to CON red cells in CON plasma. Conversely, CON red cells in NAR plasma show an increased viscosity as compared to CON red cells in CON plasma. The maximum deformation index of both NAR and CON red cells was markedly decreased in NAR plasma as compared to either NAR or CON cells in CON plasma (0.04 +/- 0.03 and 0.02 +/- 0.02 vs. 0.22 +/- 0.06 and 0.15 +/- 0.04, respectively; P < 0.05). Thus, plasma composition causes hyperviscosity and reduced RCD in NAR. Fibrinogen is not responsible since red cells in serum and red cells in plasma had a similar viscosity and differences in viscosity and RCD between NAR and CON were maintained. Plasma triglycerides are also not responsible since the viscosity of red cells in serum with a 50% reduction in triglycerides was not reduced. LPC levels in red cells were increased in NAR (8.7 +/- 0.2 vs. 5.5 +/- 0.3% of total phospholipids; P < 0.01). Adding albumin to NAR blood dose-dependently decreased whole blood viscosity, despite marked increases in plasma viscosity, and increased RCD of NAR cells (from 0.04 +/- 0.03 to 0.21 +/- 0.01; P < 0.05). There was also some effect on CON RCD of similar albumin addition to CON blood (from 0.15 +/- 0.04 to 0.29 +/- 0.03; P < 0.05). Adding albumin to NAR blood reduced red cell LPC content and increased plasma LPC content in a dose-dependent fashion, whereas there were only slight effects of adding albumin to CON blood. There was a reciprocal relation between red cell LPC and the other polar phospholipids in the red cell membrane, probably indicating exchange. The maximum deformability index of either NAR or CON cells was not affected much by adding LPC to CON plasma (NAR, from 0.22 +/- 0.06 to 0.18 +/- 0.10; CON, from 0.15 +/- 0.04 to 0.12 +/- 0.05; NS), whereas adding LPC to NAR plasma caused the red cells to become rigid. Adding LPC to CON red cells in NAR plasma caused a much stronger increase in relative LPC content (from 6.6 +/- 0.7 to 10.9 +/- 0.9%; P < 0.05) than adding LPC to CON red cells in CON plasma (from 5.6 +/- 0.4 to 6.4 +/- 0.8%; NS). Thus, in the absence of albumin, LPC in red blood cells is increased. As a consequence of the latter, RCD is decreased and whole blood viscosity increased. Alterations in red cell phospholipids are far more important than increases in plasma fibrinogen or triglycerides in determining hyperviscosity of blood and reduced RCD in NAR.

Comparison of pharmacokinetic parameters of a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate.

The alteration of the pharmacokinetic parameters of the polypeptide BBI through conjugation with palmitic acid was examined. 125I-BBI or 125I-Pal-BBI was administered iv to 6 week old CF-1 mice at a dose of 3 mg/kg. The mice were sacrificed at 5, 10, 20, 60, 120, 240, 360, and 480 min and the total radioactivity was determined for blood and each organ. The blood was analyzed on a Sephadex G-50 size-exclusion column to determine the amount of intact polypeptide present in the blood. From the amount of intact polypeptide at each time point, the pharmacokinetic parameters were determined. By conjugating three palmitic acids to each BBI molecule, the area under the curve (AUC) and mean residence time (MRT) increase by a factor of 10.8 and 2.8, respectively. There was also a difference in the organ distribution between the two treatments; while 125I-BBI was rapidly cleared from the kidneys, 125I-Pal-BBI was predominantly to the liver. Subsequent studies suggested that the binding of the conjugate to non-albumin serum proteins was most likely the cause of the altered pharmacokinetics. The residence time in the blood and the lipophilicity of BBI were increased upon conjugation with palmitic acid through a reversible disulfide linkage. Pharmacokinetic studies showed an increase in the AUC and a decrease in kidney clearance in palmitic acid conjugates, indicating a potential increase of the therapeutic efficacy of the polypeptide drug.

Blood Distribution of Mycophenolic Acid

RS-61443 (RS) a morpholinoethyl ester of mycophenolic acid (MPA), can be considered a prodrug, as immunosuppressive activity is expressed only after hydrolysis to MPA upon absorption. Little is known about the blood distribution of MPA; such information would have an impact on the medium used for analysis of the drug in clinical trials. This was investigated by spiking whole blood having an initial temperature of either 4 degrees or 22 degrees C with increasing amounts of MPA ranging from 100 to 10,000 micrograms/L. These drug concentrations span the range seen when immunosuppressive doses of the RS are administered. This was followed by incubation of the blood at 37 degrees C for 0-120 min prior to separation of the cells. The drug concentration was measured in the plasma and whole blood fractions by high-performance liquid chromatography. MPA was almost exclusively found in the plasma fraction and did not exhibit any temperature or concentration dependence. The free or unbound fraction of MPA over the same concentration range was determined by ultracentrifugation and demonstrated a concentration dependence ranging from 7.2 to 16.5% of total drug for a concentration range spanning 500-10,000 micrograms/L. The drug was found to be primarily associated with the non-albumin proteins in the plasma. Less than 10% of the drug was found to be bound to lipoproteins. The data suggest that from an analytical standpoint, plasma, rather than whole blood, would be the most suitable medium for analysis because of the higher concentrations of the drug found in this fraction.