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PubMed2004

Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC(50) values, ranging between 2.7 and 14.3 microM) followed by robinetin, myricetin and quercetin (IC(50) values ranging between 13.6 and 21.8 microM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC(50) values of 15.0 and 22.2 microM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent K(m) for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 microM for MRP1 and 8.5 microM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1.

Animals↗
PubMed2003

Green tea catechins as a BACE1 (beta-secretase) inhibitor.

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.

Amyloid Precursor Protein Secretases↗
PubMed2006

Potential applications of immobilized bitter gourd (Momordica charantia) peroxidase in the removal of phenols from polluted water.

The potential applications of immobilized bitter gourd peroxidase in the treatment of model wastewater contaminated with phenols have been investigated. The synthetic water was treated with soluble and immobilized enzyme preparations under various experimental conditions. Maximum removal of phenols was found in the buffers of pH values 5.0-6.0 and at 40 degrees C in the presence of 0.75 mM H(2)O(2). Fourteen different phenols were independently treated with soluble and immobilized bitter gourd peroxidase in the buffer of pH 5.6 at 37 degrees C. Chlorinated phenols and native phenol were significantly removed while other substituted phenols were marginally removed by the treatment. Phloroglucinol and pyrogallol were recalcitrant to the action of bitter gourd peroxidase. Immobilized bitter gourd peroxidase preparation was capable of removing remarkably high percentage of phenols from the phenolic mixtures. Significantly higher level of total organic carbon was removed from the model wastewater containing individual phenol or complex mixture of phenols by immobilized bitter gourd peroxidase as compared to the soluble enzyme. 2,4-dichlorophenol and a phenolic mixture were also treated in a stirred batch reactor with fixed quantity of enzyme for longer duration. The soluble bitter gourd peroxidase ceased to function after 3h while the immobilized enzyme was active even after 6h of incubation with phenolic solutions.

Carbon↗
PubMed2004

Micellar electrokinetic chromatography at low pH with polyelectrolyte-coated capillaries.

The performance of capillaries coated with a poly(diallyldimethylammonium) (PDADMA) monolayer or poly(diallyldimethylammonium)-poly(styrenesulfonate) bilayer was investigated and compared under micellar electrokinetic chromatographic (MEKC) conditions. Both monolayer (positively charged) and bilayer (negatively charged) coatings with micellar (sodium dodecyl sulfate) electrolyte generated very stable and pH-independent cathodal electroosmotic flow (EOF). From the results obtained, it can be concluded that in a doubly coated capillary the second poly(styrenesulfonate) layer is replaced by sodium dodecyl sulfate micelles during flushing with micellar electrolyte. Consequently, in order to obtain a stable and pH-independent cathodal electroosmotic flow for the MEKC separations, the capillary coating with the second polyanion layer is not necessary. The importance of the PDADMA coating was illustrated by comparing MEKC separations of the common developing agents (hydroquinone, phenidone, pyrocatechol, pyrogallol and quinone) on a bare uncoated capillary with the coated capillary. The coating provides reproducible MEKC separations at low pH (pH 3.0) with relative standard deviation (R.S.D.) values for migration times and peak areas lower than 0.45 and 3.3%, respectively. Good linearities in the range from 5 x 10(-5) to 2 x 10(-3) mol l(-1) were obtained for all five compounds, with correlation coefficients higher than 0.998. The detection limits were in the range from 5 x 10(-6) mol l(-1) for pyrocatechol to 2 x 10(-5) mol l(-1) for quinone. The proposed MEKC system was applied to the determination of hydroquinone and phenidone in X-ray photographic developer solutions.

Calibration↗
PubMed2005

Problems with the estimation of urine protein by automated assays.

OBJECTIVES: Most clinical laboratories replaced their manual precipitation techniques for the determination of urinary protein with automated dye binding assays or benzethonium chloride-turbidimetric assays. Few studies have validated these assays for the measurement of urinary proteins in the normal range. DESIGN AND METHODS: This study compares four automated assays for the measurement of urinary protein to a manual Ponceau S/TCA precipitation assay. We evaluated the linearity, the precision, the analytical sensitivity, the accuracy and the recovery of different proteins for each assay. RESULTS: All assays showed good linearity with the theoretical concentration of albumin present in the sample. The coefficient of variation was below 10% at a concentration of 0.142 g/L. However, the manual Ponceau S/TCA assay demonstrated superior analytical sensitivity. Accuracy determinations showed a variable positive bias and poor correlations at concentrations below 0.1 g/L when compared to the Ponceau S/TCA assay. Small molecular weight peptides particularly affected the pyrogallol red assays but other urinary components also interfered with the automated assays. CONCLUSIONS: Most automated assays show high imprecision and poor accuracy for the measurement of urinary protein in the normal range. The Ponceau S/TCA offers a precise and accurate manual alternative to these automated assays.

Albuminuria↗
PubMed2006

Fe(III) improves antioxidant and cytoprotecting activities of mangiferin.

Iron-induced oxidative stress has been implicated in several pathological processes. In the present work we provide evidence for the formation of a mangiferin:Fe(III) complex (2:1), shown by means of either iron-induced changes in the UV/visible spectrum of mangiferin or by reduction of the anodic current peak in the voltammogram of that compound; we demonstrate, in addition, that the ferric complex is more effective than mangiferin itself in scavenging superoxide radicals generated by pyrogallol autoxidation, as well as in protecting hepatocytes from reactive oxygen species mediated hypoxia/reoxygenation injury. Moreover, we found that the mangiferin:Fe(III) complex reacts more readily with horseradish peroxidase/H(2)O(2) than does mangiferin by itself. We postulate that mangiferin could afford protection against iron/reactive oxygen species-mediated pathological processes by means of both iron chelating and iron-stimulated superoxide radical scavenging activity.

Animals↗
PubMed2003

Reaction rate constants of superoxide scavenging by plant antioxidants.

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO2) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 x 10(3) mol(-1) l s(-1) for morin to 2.9 x 10(7) mol(-1) l s(-1) for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.

Antioxidants↗
PubMed2004

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats.

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.

Administration, Oral↗
PubMed2004

Characterization of the influence of nitric oxide donors on intestinal absorption of macromolecules.

To characterize the influence of nitric oxide (NO) donors on the intestinal absorption of macromolecules, the relationship between the release rate of NO from NO donors and their absorption-enhancing effects and the effects of several scavengers and generators on the absorption-enhancing effects of NO donor were investigated. The t1/2 values of the NO release rate from 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-1-propanamine (NOC5), 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12) are 25, 5 and 100min, respectively. The absorption-enhancing effects of NO donors on the absorption of fluorescein isothiocyanate dextrans with an average molecular weight of 4400 (FD-4) are NOC5 > NOC7 > NOC12 in the colon. The lowest enhancing effect of NOC12 may be due to the slow rate of NO release. The enhancing effect of NOC7 rapidly disappeared compared with the effect of NOC5. The results raise the possibility that the difference between NOC5 and NOC7 on enhancing effect is related to the t1/2 of the NO release. The NOC7-induced enhancing effect was prevented by the co-administration of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide sodium salt (C-PTIO), an NO scavenger; tiron, an O2(-) scavenger; mannitol, an OH* scavenger, and deferoxamine, peroxynitrate scavenger. Pyrogallol, an O2(-) generator, potentiated the NOC7-induced enhancing effect. These results support a role for peroxynitrate, and possibly OH*, in the NO donor-induced intestinal enhancing effect.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2004

Venusol from Gunnera perpensa: structural and activity studies.

From the aqueous extract of the dry rhizomes of Gunnera perpensa the minor components pyrogallol, succinic acid, lactic acid, and the trimethyl ether of ellagic acid glucoside were isolated. The major constituent was identified as Z-venusol, a phenylpropanoid glucoside. Its structure was verified by X-ray diffraction. Tests on isolated uterine smooth muscle from rats showed that the whole extract stimulated a direct contractile response and induced a state of continuous contractility of the uterus once all additives had been removed from the organ bath. By contrast, venusol did not trigger the direct contractile response but induced the state of continuous contractility once the organ bath was flushed.

Animals↗
PubMed2004

Organic matter transformation in the environment investigated by quantitative electron paramagnetic resonance (EPR) spectroscopy: studies on lignins.

The lignins separated from angiosperm and gymnosperm trees, peat and xylitic brown coal were investigated by quantitative EPR. Observed free radicals in lignins are sensitive to alkaline environment. Gaseous ammonia interacting with solid lignins in resonance cavity shifts quinone-hydroquinone equilibria towards formation of semiquinone anions. Complexation of copper(II) by lignins causes drastic decrease of the semiquinones in the matrices. Formation of lignin-Pb(II) complexes yielded radicals characterised by unusually low g-value (1.9999-2.0003). Monomeric structural units of the investigated lignins were recognised by pyrolysis with in situ methylation by tetramethylammonium hydroxide. Although for the natural lignins the mixture of normal semiquinone signals at g about 2.0034 and signals at g 1.9999 were observed, some monomeric components of lignins (e.g., caffeic acid, pyrogallol) gave pure lines at g = 1.9999. The bacterial oxidative biodegradation of lignin monomeric components and their Pb(II) complexes resulted in increase of the radical signals.

Bacillus subtilis↗
PubMed2005

Complex secretory products in the oviduct of the plethodontid salamander Bolitoglossa dofleini (Amphibia, Urodela).

The ultrastructure of the secretory granules in the cells of the subdivisions of the oviduct in the neotropical plethodontid salamander Bolitoglossa dofleini was studied by transmission electron microscopy. In addition, we applied the cationic dye Cuprolinic Blue (CB) at different electrolyte concentrations to demonstrate proteoglycans, and the pyrogallol red-copper (PR-C) method to stain proteins at the ultrastructural level. The entire oviduct is lined by a simple epithelium that contains ciliated and microvillous cells in the first subdivision, the aglandular pars recta; microvillous cells show a moderate secretory activity. The following pars convoluta is differentiated into five glandular subdivisions and the aglandular "uterine portion". Especially in the glandular parts, the epithelium is arranged in longitudinal folds. At their crests ciliated and microvillous cells similar to those in the pars recta occur. Gland cells are crowded with secretory granules that differ in their structural complexity (with and without electron-dense spheres or masses; elaborated, homogeneous or granular matrix; spherical; distorted) along the various subdivisions. Further, as suggested by the CB-technique, the cranial subdivisions contain large amounts of sulphated proteoglycans that decrease in the caudal direction. Carboxylated proteglycans appear to be present in all subdivisions examined. Electron-dense spheres of secretory granules are largely free of CB-precipitates, but stain more or less intensely with PR-C. The ultrastructure of the pars recta, and especially the "uterine portion" indicates transporting capability. The epithelial cells of the "uterus" have coated pits and a considerable amount of lysosome-like bodies.

Animals↗
PubMed2003

Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay.

It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less.

Chemistry Techniques, Analytical↗
PubMed2003

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation.

OBJECTIVE: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. METHODS: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. RESULTS: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O(2)(-)) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O(2)(-)-generator, abolished relaxant responses to ACh. CONCLUSION: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect.

Acetylcholine↗
PubMed2000

Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA.

Tannic acid has numerous food and pharmacological applications. It is an additive in medicinal products, and is used as a flavouring agent and as an anti-oxidant in various foods and beverages. We have previously shown that tannic acid in the presence of Cu(II) causes DNA degradation through generation of reactive oxygen species. On the other hand, it exhibits antimutagenic and anticarcinogenic activities, and induces apoptosis in animal cells. It is known that most plant-derived polyphenolic anti-oxidants also act as pro-oxidants under certain conditions. In this paper, we compare the anti-oxidant and pro-oxidant properties of tannic acid and its structural component gallic acid. It is shown that tannic acid is the most efficient generator of the hydroxyl radical in the presence of Cu(II), as compared with gallic acid and its analogues syringic acid and pyrogallol. The anti-oxidant activity of tannic acid was studied by its effect on hydroxyl radical and singlet oxygen mediated cleavage of plasmid DNA. Again, tannic acid provided the maximum protection against cleavage, while gallic acid and its structural analogues were found to be non-inhibitory or partially inhibitory. The results suggest that the structural features of tannic acid that are important for its anti-oxidant action are also those that contribute to the generation of hydroxyl radicals in the presence of Cu(II). Restriction analysis of treated phage DNA and thermal melting profiles of calf thymus DNA indicated that tannic acid strongly binds to DNA. Indirect evidence indicates that modification of DNA bases may also occur.

Animals↗
PubMed2003

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes.

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.

Animals↗
PubMed2002

Detection of low-molecular-weight proteins in urine by dipsticks.

BACKGROUND: Testing of urines with dipsticks for proteinuria, glycosuria, etc., is common practice. A deficiency with currently available dipsticks is their lack of chemical sensitivity and underestimation of low-molecular-weight proteins such as light chains. METHODS: We experimented with a number of dyes that gave an easily recognized color change on dipsticks for various low-molecular-weight proteins such as alpha-1-glycoprotein, alpha-1- and beta-2-microglobulin, and kappa and lambda light chains. We were successful in formulating a dye for impregnating dipsticks that gave a color change with low-molecular-weight proteins. RESULTS: Most dipsticks will measure proteins down to about 1 g/l. Our composite of two dyes (described here as the "TPR" dipsticks) gave reproducible results for protein concentrations of >/=300 mg/l, and detected low-molecular proteins. The TPR reagent is resistant to interferences from many compounds; also, the protein results are not altered in a given urine at a pH between 5 and 8. CONCLUSIONS: We have developed a dipstick that detects low-molecular-weight proteins. The dipsticks are easy to use and are suitable for outpatient or point-of-care testing. The precision of the dipsticks is satisfactory and is only marginally lower than quantitative spectrophotometric methods using pyrogallol red (PYR).

Coloring Agents↗
PubMed2000

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.

Albuminuria↗