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Biomedical subjects

G Delfino

Publications and source records attributed to G Delfino.

At least 37 records · Page 2Linked to original sources
PubMed1986

Isoelectric focusing in low-denaturing media: visualization in renal disease of variation of the isoelectric point of albumin not related to a remarkable conformational variation.

The isoelectric properties of serum and urinary albumin from normal subjects and patients with nephrotic syndrome have been investigated in various conditions of denaturation, obtained by using urea (0-8 M) as a support in isoelectric focusing. In normal human serum, albumin is rather acidic (pI = 4.7) when focused in glycerol while the denatured form obtained by exposing the protein to 8 M urea has a much higher pI (6.1). Albumin from nephrotic patients is acidic in glycerol but at very low levels of urea (2M) it shifts from pI 4.7 to pI 6.1; the same effect has been induced by treating albumin with activated charcoal at low pH. In order to obtain more information on urea-induced changes, we have recorded the circular dichroic spectra of albumin when exposed to the concentration of urea used in gels, and we found that no conformational transition occurs for urea concentrations less than 5 M. Taken together, these observations reveal that variation of the pI of albumin in nephrotic syndrome occurs mainly due to a dissociating effect of urea on charged substances bound to this protein.

Albumins↗
PubMed1986

Conformational mediated renal selectivity towards albumin in diabetes mellitus.

In an attempt to define the nature of renal selectivity in diabetes mellitus, we have determined the free sulfhydryl (SH) groups of serum and urinary albumin in 9 normal subjects and 24 diabetic patients with various grades of renal involvement, as defined by their urinary excretion rates of albumin (alb. UER): 8 with alb. UER less than 10 micrograms/min (Group A), 6 with alb. UER between 10 and 30 micrograms/min (Group B), 5 with alb. UER between 30 and 200 micrograms/min (Group C) and 5 with alb. UER greater than 200 micrograms/min (Group D). The free SH group content of urinary albumin was three to four fold increased in comparison with its serum homologue in normal subjects and in diabetics with normal or slightly increased alb. UERs (Groups A and B). Diabetics in Group C showed a two-fold increase in free SH groups of urinary albumin compared to serum albumin and diabetics with clinical nephropathy (Group D) showed no increase at all. The SH group content of urinary albumin correlated in all diabetics with the concentration of glycosyl albumin and the of urinary/serum albumin SH groups ratio was inversely correlated with alb. UER. From these observations concerning the selectivity properties of the renal filter in normal and diabetic subjects, it is concluded that the mechanism for progression of diabetic nephropathy may be the hyperfiltration of albumin with an altered conformational state.

Adolescent↗
PubMed1985

Reaction of human serum albumin with aldoses.

The reaction of human serum albumin (HSA) with aldoses (C3-C6) and acetaldehyde has been studied. U.v. and fluorescent spectra of the HSA-glyceraldehyde and HSA-GlcN adducts reveal yellow chromophores absorbing at 300-350 nm and emitting at 435 nm. However, even limited reaction of HSA with acetaldehyde induced perturbation in the Trp microenvironment. C.d. spectra of the adducts show an average 20% decrement in mean residual ellipticity [theta], which is independent of the extent of the reaction and the aldose used. It is concluded that most of the reactions with aldoses occur at the surface of the HSA molecule. With the exception of the GlcN adduct, the HSA adducts rearrange to produce pyrrole rings on the protein surface. I.e.f. analysis shows that the pI values of the modified HSA are almost linearly correlated with the chain length of the reacting aldose: from pI 4.2 for HSA-glyceraldehyde up to pI 5.0 for HSA-GlcN.

Acetaldehyde↗
PubMed1985

Electrical charge of serum and urinary albumin in normal and diabetic humans.

The isoelectric points of albumin purified by pseudo-ligand chromatography on Affi-Gel Blue were determined simultaneously in serum and urine of 11 normal subjects and 25 diabetic patients, subdivided in groups according to their urinary excretion rates of albumin. Serum albumin was constituted by a single homogeneous peak at 4.7 (pI) in normal subjects, whereas the levels for diabetic patients covered this band and some other microheterogeneous levels, ranging from 3.5 to 7 pI. By affinity chromatography with Concanavalin A-Sepharose and immunoelectrophoretic techniques, all these micro-heterogeneous bands were characterized as glycosyl albumin. In normal subjects and diabetic patients whose urinary excretion rate of albumin was normal or increased only slightly (10 to 100 micrograms/min), the pattern of urinary albumin included a main band with normal pI (4.7) and some remarkable amounts of more anionic bands (pI between 4.0 and 4.7) if compared to the native protein, which was characterized as glycosyl albumin. Such a difference was not detected in urines of diabetic patients with clinical nephropathy. These results indicate that the non-enzymatic glycosylation of albumin is a main determinant of the excretion of this protein into urine, in spite of the anionic electrical charge. We describe also the renal selectivity properties in humans that may be viewed as a model for the study of renal disease, but the role of such a mechanism in early diabetic nephropathy remains unknown.

Adolescent↗
PubMed1985

Glycosylation of serum albumin in diabetic humans with osmolar and acid-base disorders.

The rate of nonenzymatic glycosylation of serum albumin was determined in 7 diabetic patients at the onset and during the recovery of an acute metabolic derangement as defined by hyperglycemia, hyperosmolality and metabolic acidosis of various degrees. Serum glycosyl albumin concentration (chemically determined) was decreased after 1 day in 5 patients (mean -6%) and increased in the remaining 2 (+11% and +22% respectively). The same variance was decreased in the whole group after 7 days of therapy (-18.6%). A negative statistical correlation was found by plotting initial values of serum glycosyl albumin against arterial H+ concentration and the same relationship was confirmed by multiple regression analysis which gave the following curve: t (glycosyl albumin) = 1.57 - 0.004 X (H+) + 0.000225 y (Mean blood glucose concentration) - 0.00285 z (serum osmolality) (the regression coefficients were 0.24 for x, 0.18 for y and 0.23 for z). Finally, a number of albumin isoforms with a cationic charge up to 7 pH have been detected in all patients by isoelectric focusing. Taken together all the data presented herein indicate that beside serum glucose concentration other factors (such as H+ concentration and serum osmolality) influence the rate of glycosylation of albumin, their effects being prominent and opposite to hyperglycemia.

Acid-Base Imbalance↗
PubMed1985

Urinary excretion of albumin with an altered three-dimensional conformation in diabetic functional nephropathy. Evidence for a pathogenetic role.

The free sulfhydryl group content of serum and urinary albumin has been evaluated in eight normal and 23 diabetic patients with various grades of urinary albumin excretion rates. While in normal subjects and in diabetics with either normal albuminuria or functional nephropathy, urinary albumin showed a statistically higher content of free sulfhydryl groups compared to homologous serum, diabetic patients with clinical nephropathy showed no difference. These results indicate that an increased urinary excretion of albumin altered in its conformational status is the main feature of diabetic functional nephropathy and suggest that a molecular mechanism determines the glomerular accumulation of albumin.

Adolescent↗
PubMed1985

Tubular function and renal selectivity towards glycosyl-albumin in diabetic hyperosmolar states.

The type of proteinuria and renal selectivity of glycosyl albumin has been evaluated in seven diabetic patients at the onset of plasma hyperosmolar state and after complete fluid replacement. The main feature of all patients was a marked increase in urinary excretion of beta 2-microglobulin, which promptly returned to normal after the correction of the fluid disequilibrium. This indicates that a reversible tubular injury is the first symptom of the dehydration process in diabetic patients with plasma hyperosmolality.

Adolescent↗
PubMed1985

Microheterogeneity of serum glycosyl albumin in diabetes mellitus.

Proteins purified by affinity chromatography on Blue-Sepharose CL-6B from serum of 10 normal controls and 19 Type I diabetic patients were studied by means of combined ultrathin isoelectric focusing and photochemical silver stain. While only a single band of protein (characterized as albumin by crossed immunoelectrophoresis) with a pI of 4.7 was found in serum of normal subjects, 10 out of the diabetic group showed some bands of proteins with a pI greater than 4.7 and a single one with a pI less than 4.7. Concanavalin A-Sepharose removed all these bands with altered pIs which were further characterized as albumin by fused rocket immunoelectrophoresis of the eluates from Concanavalin A-Sepharose, direct immunofixation after isoelectric focusing of proteins with high purified anti-albumin antibodies, SDS-polyacrylamide gradient pore electrophoresis, aminoacidic analysis. The gas-chromatographic analysis of carbohydrates released from both the albumin bound to Concanavalin A-Sepharose and that not bound, revealed in addition to two unidentified peaks, the presence of glucose, galactose and mannose whose contents were greatly increased in albumin with affinity for the lectin. Serum glycosyl albumin concentration was not statistically different in serum of diabetic patients displaying cationic glycosyl albumin in comparison to patients without these proteins (0.2261 +/- 0.0186 versus 0.1874 +/- 0.015 nmole HMF/nmole albumin), whereas the first group showed statistically higher urinary excretion rates of albumin (28.6 +/- 1.2 micrograms/min versus 4.6 +/- 0.2 micrograms/min).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1984

Glycosyl albumin and diabetic microalbuminuria: demonstration of an altered renal handling.

In attempt to elucidate the link between the nonenzymatic glycosylation of proteins and the diabetic functional nephropathy, renal handling of glycosyl albumin has been evaluated in 15 normal subjects and 29 insulin-dependent diabetic patients divided in three groups according to their urinary excretion rates of albumin (Ualb): (group A) ten diabetic patients with Ualb less than 10 micrograms/m', (group B) 12 patients with Ualb between 10 and 100 micrograms/m', and (group C) seven patients with Ualb greater than 100 micrograms/m'. Albumin was purified with Blue-Sepharose CL-6B. The carbohydrate bound to albumin was determined chemically with thiobarbituric acid after the acid hydrolysis of the protein. Serum glycosyl albumin concentration in normal subjects was 0.1256 +/- 0.009 nmoles of hydroxymethylfurfural per nanomole of albumin, in group A, 0.1900 +/- 0.0124; in group B, 0.2199 +/- 0.0177; and in group C, 0.2224 +/- 0.02732. Urinary glycosyl albumin concentration was 1.8467 + 0.2132 in normal subjects, 1.4369 +/- 0.3355 in group A, 1.008 +/- 0.1584 in group B, and 0.2614 + 0.0295 in group C. In normal subjects and patients without apparent nephropathy (groups A and B), the clearance of albumin correlated with the serum concentration of glycosyl albumin. In all patients (groups A, B, and C) the urinary-serum glycosyl albumin concentration ratio was correlated inversely with albumin clearance. These data show that in normal subjects and diabetic patients with normal excretion rates of albumin and microalbuminuric diabetic patients the passage of glycosyl albumin through the glomerular wall is facilitated in contrast to normal albumin and that glycosyl albumin plays an important role in the pathogenesis of diabetic functional nephropathy.

Adolescent↗
PubMed1983

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate.

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.

Concanavalin A↗
PubMed1983

Determination of a glycosyl subunit of human serum albumin by concanavalin A-sepharose.

A new procedure has been devised for the isolation of a glycosyl subunit of albumin from human serum. After the selective removal from serum proteins with affinity chromatography on Blue-Sepharose CL-6B, albumin was applied to a column of concanavalin-A Sepharose which resolved the protein in two subunits with different specific colour activity for carbohydrates, as tested with thiobarbituric acid assay. The glycosyl albumin bound to concanavalin-A Sepharose was homogeneous when examined by immunoelectrophoresis and sodium dodecyl-sulphate polyacrylamide electrophoresis, whereas it showed a microheterogeneity when tested by isoelectric focusing. The procedure was applied to a model system as well as to serum from normal and diabetic patients.

Carbohydrates↗