Sodium Dodecyl Sulfate-agarose Gel Electrophoresis Research Articles

Description of serum symmetric dimethylarginine concentration and of urinary SDS-AGE pattern in dogs with ACTH dependent hyperadrenocorticism

Serum symmetric dimethylarginine (SDMA) and patterns of urinary protein separated by sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE) have not been investigated as biomarkers in dogs with ACTH-dependent hyperadrenocorticism (ADHAC). This exploratory prospective study aimed to evaluate SDMA, serum creatinine (sCR), and SDS-AGE in dogs with ADHAC with and without proteinuria (ADHAC-P and ADHAC-nP, respectively). Thirty-five pet dogs classified as ADHAC-P (n=16), ADHAC-nP (n=6) and healthy (n=13) were included. Renal biomarkers were evaluated in all dogs at diagnosis. Baseline concentration of SDMA was not significantly different between the three groups (P = 0.15) whereas sCr was significantly lower in dogs in ADHAC dogs compared to healthy dogs (88.0 µmol/L [70.4–132.6; 79.2–114.4]) whether they had proteinuria or not (P = 0.014 and 0.002, respectively). However, baseline concentrations of sCr and SDMA were not significantly different between dogs with ADHAC-P dogs (SDMA, 8 µg/dL [5–12; 7–9]; sCr, 57.2 µmol/L [35.2–212.2; 52.8–92.4]) and ADHAC-nP dogs (SDMA, 8.5 µg/dL [7–13; 8–10]; sCr, 70.4 µmol/L [61.6–79.2; 61.6–70.4]) (P = 0.35 and P = 0.41, respectively). Proteinuria in dogs with ADHAC-P was mainly of glomerular origin (SDS-AGE pattern: glomerular in 10/16 dogs; mixed glomerular/tubular in four dogs). In our study, SDMA was neither significantly different in dogs with ADHAC whether they were proteinuric or not, nor between ADHAC and healthy dogs. Urinary electrophoresis provides additional information to the UPC and further investigations are needed to determine whether it may help identify dogs with ADHAC-P requiring specific antiproteinuric treatment.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

The Value of Electrophoresis and Chemical Detection in the Diagnosis of Hypertensive Nephropathy.

ObjectiveTo investigate the clinical value of sodium dodecyl sulfate–agarose gel electrophoresis (SDS-AGE) of urinary proteins combined with several biochemical indices in the detection of hypertensive nephropathy.Material and MethodsIn this study, 210 patients with hypertensive nephropathy were recruited as the kidney disease group, 100 patients with simple hypertension were recruited as the hypertension group, and another 100 healthy participants were recruited as the control group. We conducted SDS-AGE of urinary proteins and urinary microalbumin (mAlb), β2-microglobulin (β2-MG), retinol-binding protein (RBP), serum cystatin C (CysC), blood urea nitrogen (BUN), and creatinine tests at the same time.ResultsThe results showed that the urinary mAlb levels in the hypertension group and hypertensive nephropathy group were higher than in the control group (P<0.05). The biochemical index detection results showed that β2-MG, RBP, serum CysC, BUN, and creatinine were higher in the hypertensive nephropathy group than in the control group and hypertension group (P<0.05). The total positive rates of urinary protein SDS-AGE and urinary mAlb, urinary β2-MG, blood CysC, blood RBP, BUN, and creatinine in 210 patients with essential hypertension nephropathy were 100.0%, 98.6%, 32.8%, 98.6%, 21.0%, 2.0%, and 20%, respectively. The total positive rate of SDS-AGE urinary protein was higher than that of the other six biochemical indices.ConclusionSDS-AGE of urinary proteins, combined with urinary mAlb, β2-MG, serum CysC, RBP, BUN, and creatinine, could improve the detection sensitivity, which would be helpful for the early and accurate diagnosis of kidney damage.

Comparison of lipoprotein (a) serum concentrations measured by six commercially available immunoassays

Background and aimsLipoprotein (a) [Lp(a)] is an established causal risk factor for cardiovascular disease (CVD), independently of low-density lipoproteins (LDL) and other risk factors. The recognition of Lp(a) as an atherogenic molecule has raised the demand for reliable quantification methods in the clinical laboratory. The aim of this work is to compare commercial immunochemical assays. MethodsWe measured Lp(a) serum concentrations using six different assays, providing Lp(a) in mg/dl (Denka Seiken, Abbott Quantia, Beckman, Diasys 21FS, and Siemens N Latex) or in nmol/l (Roche TinaQuant, Diasys 21 FS) in 144 serum samples covering the clinically relevant range of Lp(a) concentrations. All assays relied on five-point calibrations using calibrators provided by the manufacturers. Apolipoprotein(a) phenotyping was performed by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-agarose) followed by immunoblotting. ResultsMost bivariate correlation coefficients were greater than 0.90. Compared to an established IFCC-proposed reference material, the results of the different assays diverged from the target values (43.3 mg/dl or 96.6 nmol/l) by −8% (Siemens N Latex) and +22% (Abbott Quantia). Stratification of the samples into five groups with increasing Lp(a) concentrations and difference plots showed that the differences among assays were concentration-dependent. Some assays overestimated Lp(a) at high concentrations compared to the Denka Seiken assay. ConclusionsCurrent commercial immunological assays for measuring Lp(a) concentrations are differently calibrated. Their biases differ significantly across the clinically relevant concentration range in a non-linear manner. This is not conclusively explained by apolipoprotein (a) phenotypes. Further international efforts to harmonize assays for Lp(a) are needed.

Electrophoretic patterns of proteinuria in feline spontaneous chronic kidney disease.

The purpose of this study was to describe the electrophoretic patterns of proteinuria in cats at risk of and cats with chronic kidney disease (CKD), and to investigate whether the presence of high-molecular-weight (HMW) and low-molecular-weight (LMW) proteins were associated with CKD, proteinuria and/or disease progression. Healthy cats at risk of developing renal disease (n = 17) and cats affected with CKD at different stages (n = 22) were prospectively enrolled and sampled over time. Seventy urine samples were included and assayed with a commercially available sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) method. Each sample (gel lane) was inspected to identify albumin, HMW and LMW proteins, and an electrophoretic pattern (albuminuria, glomerular, tubular, mixed or negative) was assigned accordingly. Fisher's exact test was used to assess the distribution of HMW and LMW proteins in cats grouped according to International Renal Interest Society stage and to the magnitude of proteinuria, and to assess if HMW and LMW proteins at the time of inclusion were associated with the development and progression of CKD. In samples of cats at risk, the most common pattern was glomerular (84.6%); glomerular pattern was also common in cats with CKD (54.2%), although mixed proteinuria and tubular proteinuria were also present (29.5% and 11.4%, respectively). The presence of LMW proteins was associated with CKD (P <0.0001) and to a urine protein:creatinine ratio >0.2 (P = 0.025). Both HMW and LMW proteins were not associated with progression of CKD within 6 months (n = 14). Our results showed that HMW proteinuria is common in healthy cats at risk of developing CKD, although the pathological significance needs to be confirmed. The detection of LMW proteins in urine of cats suspected to be affected by CKD, especially in non-azotaemic, non-proteinuric or borderline proteinuric cats, suggests the presence of kidney damage.

Characterization of Proteinuria in Dogue de Bordeaux Dogs, a Breed Predisposed to a Familial Glomerulonephropathy: A Retrospective Study

Dogue de Bordeaux dog has been reported to be predisposed to a familial glomerulonephropathy that displays some morphological modifications reported in focal and segmental glomerulosclerosis. Prevalence of quantitatively abnormal renal proteinuria was recently reported to be 33% in this breed. The nature of the proteinuria was assessed by sodium dodecyl sulfate-agarose gel electrophoresis and determinations of urinary markers (urinary retinol-binding protein, urinary N-acetyl-β-glucosaminidase, urinary albumin and urinary immunoglobulin G) on stored specimens. Diagnostic performances of sodium dodecyl sulfate-agarose gel electrophoresis to identify dogs with elevated urinary biomarkers were assessed. Samples from 102 adult Dogue de Bordeaux dogs (47 non-proteinuric [urine protein-to-creatinine ratio≤0.2], 20 borderline-proteinuric [0.2< urine protein-to-creatinine ratio ≤0.5] and 35 proteinuric dogs [urine protein-to-creatinine ratio >0.5]) were used, of which 2 were suffering from familial glomerulonephropathy. The electrophoretic protein patterns, for all but one proteinuric dog, were indicative of a glomerular origin and, in all dogs, the urinary albumin concentration related to creatinine concentration and the urinary immunoglobulin G concentration related to creatinine concentration were above the upper limit of the reference interval established for the breed. Sensitivity and specificity of sodium dodecyl sulfate-agarose gel electrophoresis identifying dogs with elevated urinary albumin concentration were 94% and 92%, respectively, while diagnostic performance of sodium dodecyl sulfate-agarose gel electrophoresis in detecting dogs with elevated urinary immunoglobulin G concentration yielded sensitivity and specificity of 90% and 74%, respectively. These results suggest that all proteinuric and some borderline-proteinuric Dogue de Bordeaux dogs likely have underlying glomerular lesions and that sodium dodecyl sulfate-agarose gel electrophoresis and urinary markers might be useful to screen dogs with borderline-proteinuria. Additional investigations are warranted to assess if these findings are related to the familial glomerulonephropathy.

Changes in serum and urine SAA concentrations and qualitative and quantitative proteinuria in Abyssinian cats with familial amyloidosis: a five-year longitudinal study (2009-2014).

BackgroundDiagnosis of familial amyloidosis (FA) in Abyssinian cats usually is made on postmortem examination.Hypothesis/ObjectivesSequential analysis of serum SAA (sSAA), urinary SAA (uSAA), urinary protein:creatinine (UPC) ratio, or sodium‐dodecylsulfate agarose gel electrophoresis (SDS‐AGE) may facilitate early identification of cats with FA.AnimalsTwenty‐three Abyssinian cats belonging to cattery A or B (low and high prevalence of FA, respectively).MethodsProspective longitudinal study using 109 blood and 100 urine samples collected over 4‐year period every 4 months, if possible, or more frequently in case of illness. Cats that died during study were necropsied. Health status of live cats was checked 5 years after enrollment. Serum amyloid A (sSAA) and urinary SAA (uSAA) were measured using ELISA kit. The UPC ratio and SDS‐AGE also was performed.ResultsFamilial amyloidosis was not identified in cattery A, whereas 7/14 cats from cattery B had FA. Serum amyloid A concentrations were not significantly different between cats in catteries A and B or between cats with or without FA, despite frequent peaks in cats from cattery B. Conversely, uSAA was significantly higher in cattery B, especially in the terminal phases of FA. Proteinuria occasionally was found in cats from both catteries, especially in those with FA. Urine protein electrophoresis identified mixed proteinuria only in cats with FA.Conclusions and Clinical ImportanceSerum amyloid A and UPC ratio are not helpful for early identification of Abyssinian cats with FA. Conversely, increases in uSAA with or without mixed proteinuria may be found before onset of clinical signs in cats with FA.

A common LPA null allele associates with lower lipoprotein(a) levels and coronary artery disease risk.

Increased levels of lipoprotein(a) are a highly heritable risk factor for coronary artery disease (CAD). The genetic determinants of lipoprotein(a) levels are mainly because of genetic variation in the apolipoprotein(a) gene (LPA). We have tested the association of a relatively common null allele of LPA with lipoprotein(a) levels and CAD risk in a large case-control cohort. We have also examined how null allele genotyping complements apolipoprotein(a) isoform typing to refine the relationship between LPA isoform size and circulating lipoprotein(a) levels. The LPA null allele (rs41272114) was genotyped in the PROCARDIS (Precocious Coronary Artery Disease) case-control cohort (4073 CAD cases and 4225 controls). Lipoprotein(a) levels were measured in 909 CAD cases and 922 controls; apolipoprotein(a) isoform size was estimated using sodium dodecyl sulfate-agarose gel electrophoresis and a high-throughput quantitative polymerase chain reaction-based method. Null carriers are common (null allele frequency, 3%) and have significantly lower circulating lipoprotein(a) levels (P=2.1×10(-10)) and reduced CAD risk (odds ratio, 0.79 [0.66-0.97]; P=0.023) compared with noncarriers. An additive allelic model of apolipoprotein(a) isoform size, refined by null allele genotype and quantitative polymerase chain reaction values, showed a sigmoid relationship with lipoprotein(a) levels, with baseline levels for longer isoform alleles and progressively higher levels of lipoprotein(a) for shorter isoform alleles. The LPA null allele (rs41272114) is associated with decreased circulating lipoprotein(a) levels and decreased CAD risk. Incorporating rs41272114 refined apolipoprotein(a) isoform size typing obtained by immunoblotting and quantitative polymerase chain reaction. A joint genomic and isoform analysis revealed details of the relationship between apolipoprotein(a) isoform size and circulating lipoprotein(a) level consistent with a threshold effect on lipoprotein secretion.

Clinicopathological findings in non‐human primate recipients of porcine renal xenografts: quantitative and qualitative evaluation of proteinuria

Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ. Eight bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio (UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE), respectively. The main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine samples, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS-AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all samples collected post-transplantation. The study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS-AGE technique in urine samples of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight (LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function.

Low-molecular-weight lipoprotein (a) and low relative lymphocyte concentration are significant and independent risk factors for coronary heart disease in patients with type 2 diabetes mellitus: Lp(a) phenotype, lymphocyte, and coronary heart disease

BackgroundThe aim of the present prospective study was to examine whether lipoprotein (a) [Lp(a)] phenotypes and/or low relative lymphocyte concentration (LRLC) are independently associated with coronary heart disease (CHD) in patients with type 2 diabetes mellitus (T2DM).MethodsSerum Lp(a) concentration, Lp(a) phenotypes, and RLC were analyzed in 214 subjects. Lp(a) phenotypes were classified into 7 subtypes according to sodium dodecyl sulfate-agarose gel electrophoresis by Western blotting. Subjects were assigned to the low-molecular-weight (LMW (number of KIV repeats: 11–22) ) and high-molecular-weight (HMW( number of KIV repeats: >22 )) Lp(a) groups according to Lp(a) phenotype and to the LRLC (RLC: <20.3%) and normal RLC (NRLC; RLC: ≥20.3%) groups according to RLC. A CHD event was defined as the occurrence of angina pectoris or myocardial infarction during the follow-up period.ResultsDuring the follow-up period, 30 cases of CHD events were verified. Neutrophil count showed no correlation with CHD, while relative neutrophil concentration and RLC showed positive and negative correlations, respectively, with CHD. The Cox proportional hazard model analysis revealed the following hazard ratios adjusted for LMW Lp(a), LRLC, and LMW Lp(a) + LRLC: (4.31; 95% confidence interval [CI], 1.99-9.32; P < 0.01, 3.621; 95% CI, 1.50-8.75; P < 0.05, and 7.15; 95% CI, 2.17-23.56; P < 0.01, respectively).ConclusionsOur results suggest that both LMW Lp(a) and LRLC are significant and independent risk factors for CHD and that the combination thereof more strongly predicts CHD in patients with T2DM.

Sodium dodecyl sulfate–agarose gel electrophoresis for the detection and isolation of amyloid curli fibers

Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)–agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS–agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS–agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS–agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.

High Serum and Urine Lysozyme Levels in a Dog with Acute Myeloid Leukemia

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine proteincreatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate-agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5-8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.

Stoichiometry-Dependent Formation of Quantum Dot−Antibody Bioconjugates: A Complementary Atomic Force Microscopy and Agarose Gel Electrophoresis Study

The unique advantages of quantum dot (QD) bioconjugates have motivated their application in biological assays. However, physical characterization of bioconjugated QDs after surface modification has often been overlooked. Here, biotinylated antibodies (biotin-IgG) were attached to commercial streptavidin-conjugated quantum dots (strep-QDs) at different stoichiometric ratios, and these QD bioconjugates were characterized with atomic force microscopy and discontinuous sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE). The results from these complementary analytical techniques showed that the molar ratio determined the relative sizes, molecular weights and morphologies of the QD bioconjugates. Additionally, the novel discontinuous SDS-AGE analysis confirmed specific binding between biotin-IgG and strep-QDs. Researchers who design QD bioconjugates for cell-based assays should consider stoichiometry-dependent differences in the physical properties of their QD bioconjugates.

Sodium dodecyl sulfate agarose gel electropheresis and electroelution of high molecular weight human salivary mucin

The aim of this study was to test the applicability of sodium dodecyl sulfate (SDS) agarose gel electrophoresis, electroelution and electrophoretic filtration as methods of separation, detection, and purification of high molecular weight human salivary mucin. SDS agarose gel electrophoresis of whole saliva and mucin prepared by density gradient ultracentrifugation revealed bands with molecular weights in excess of 450 kDa and between 1.6x10(6) and 2x10(6) Da. Electroelution of material from gel and subsequent electrophoresis resulted in highly purified high molecular weight material. Electrophoretic filtration, a method of collecting the material remaining from whole saliva in the slot after penetration of low molecular weight constituents into SDS polyacrylamide gel, failed to produce pure high molecular weight material. It is concluded that SDS agarose gel electrophoresis and electroelution are suitable methods for studying high molecular weight salivary mucin glycoproteins.

Free Light‐Chain Proteinuria and Normal Renal Histopathology and Function in 11 Dogs Exposed to Leishmania infantum, Ehrlichia canis, and Babesia canis

The purpose of this retrospective study was to investigate the relationship among proteinuria consisting of immunoglobulin free light chains (FLCs), renal histopathologic findings, and routine markers of renal function in 11 dogs exposed to Leishmania infantum (n = 8), Ehrlichia canis (n = 2), and Babesia canis (n = 1). FLC proteinuria was suspected based on identification of a 22‐ to 27‐kDa band by sodium dodecyl sulfate‐agarose gel electrophoresis (SDS‐AGE) and later confirmed by immunofixation electrophoresis. SDS‐AGE identified an isolated band of 22–27 kDa in 8 dogs, whereas the remaining 3 had a 22‐ to 27‐kDa band and an additional band of 67–72 kDa. The median urine protein‐to‐urine creatinine ratio was 0.37 (range, 0.11‐2.24) and increased ratios were found in 6 dogs (54.5%) (reference value, &lt;0.7). All dogs underwent histologic examination of renal percutaneous biopsy specimens and determination of serum creatinine and urea concentrations. Tissue samples for light microscopy were stained with hematoxylin‐eosin, periodic acid‐Schiff, Goldners trichrome, and methenamine silver. In the study group, the glomerular tufts, mesangium, tubulointerstitium, and vessels appeared unaffected. The median serum creatinine concentration in these 11 dogs was 1.3 mg/dL (range, 0.8‐1.5 mg/dL; reference range, 0.6‐1.5 mg/dL), whereas the concentration for urea was 28 mg/dL (range, 22–52 mg/dL; reference range, 20–50 mg/dL). All dogs had normal renal morphology and had normal serum creatinine and urea concentrations, suggesting that immunoglobulin FLC may be detected in the urine of dogs exposed to L infantum, E canis, and B canis without any apparent structural or functional renal derangement.

Free Light-Chain Proteinuria and Normal Renal Histopathology and Function in 11 Dogs Exposed to Leishmania infantum, Ehrlichia canis, and Babesia canis

The purpose of this retrospective study was to investigate the relationship among proteinuria consisting of immunoglobulin free light chains (FLCs), renal histopathologic findings, and routine markers of renal function in 11 dogs exposed to Leishmania infantum (n = 8), Ehrlichia canis (n = 2), and Babesia canis (n = 1). FLC proteinuria was suspected based on identification of a 22- to 27-kDa band by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) and later confirmed by immunofixation electrophoresis. SDS-AGE identified an isolated band of 22-27 kDa in 8 dogs, whereas the remaining 3 had a 22- to 27-kDa band and an additional band of 67-72 kDa. The median urine protein-to-urine creatinine ratio was 0.37 (range, 0.11-2.24) and increased ratios were found in 6 dogs (54.5%) (reference value, <0.7). All dogs underwent histologic examination of renal percutaneous biopsy specimens and determination of serum creatinine and urea concentrations. Tissue samples for light microscopy were stained with hematoxylin-eosin, periodic acid-Schiff, Goldners trichrome, and methenamine silver. In the study group, the glomerular tufts, mesangium, tubulointerstitium, and vessels appeared unaffected. The median serum creatinine concentration in these 11 dogs was 1.3 mg/dL (range, 0.8-1.5 mg/dL; reference range, 0.6-1.5 mg/dL), whereas the concentration for urea was 28 mg/dL (range, 22-52 mg/dL; reference range, 20-50 mg/dL). All dogs had normal renal morphology and had normal serum creatinine and urea concentrations, suggesting that immunoglobulin FLC may be detected in the urine of dogs exposed to L. infantum, E. canis, and B. canis without any apparent structural or functional renal derangement.

Lipoprotein(a) and prevalent cardiovascular disease in a dialysis population: the choices for healthy outcomes in caring for ESRD (CHOICE) study

Background:Levels of lipoprotein(a) [Lp(a)], an atherogenic lipoprotein, are elevated in patients with end-stage renal disease and inversely related to the size of apolipoprotein(a) [apo(a)], a glycoprotein bound to Lp(a). We studied the association of Lp(a) level and apo(a) size with prevalent atherosclerotic cardiovascular disease (ASCVD) in 871 incident dialysis patients (261 blacks, 565 whites, 45 other). Methods:Lp(a) was measured by an apo(a) size-independent enzyme-linked immunoassay; and apo(a) size was measured by sodium dodecyl sulfate-agarose gel electrophoresis. Prevalent ASCVD, derived from medical records, was defined as coronary heart disease or cerebral or peripheral vascular disease. Adjustment variables included age, sex, race, dialysis modality, diabetes, serum creatinine level, albumin level, and low-density lipoprotein cholesterol level. Results:ASCVD prevalence was 58%. Median Lp(a) levels for those with compared with those without ASCVD were 38 versus 35 nmol/L for whites (P = 0.49) and 100 versus 74 nmol/L for blacks, respectively (P = 0.35). Lp(a) level was associated with ASCVD among those younger than 60 years (odds ratio [OR] for +1 interquartile range of Lp(a), 1.5; P = 0.02), but not among those 60 years and older (OR, 1.0; P = 0.82; Pinteraction by age, 0.08). ORs were 1.3 for all whites (P = 0.03) and 1.1 for all blacks (P = 0.87; Pinteractionby race = 0.53). ORs of ASCVD were 1.7 for whites younger than 60 years (P = 0.01) and 1.2 for blacks younger than 60 years (P = 0.77; Pinteraction by race = 0.42). No association between apo(a) isoform size and ASCVD was present. Conclusion:In an incident dialysis cohort, Lp(a) level was associated with prevalent ASCVD among whites younger than 60 years, but not among blacks or those older than 60 years. Apo(a) isoform size was not associated with prevalent ASCVD. These data suggest that baseline ASCVD is unlikely to strongly confound the potential associations of Lp(a) level and prospectively ascertained ASCVD among incident dialysis patients.

Differences between genotyping and phenotyping methods for assessing apolipoprotein(a) size polymorphisms.

The aim of the present study was to analyze, on a double-blind basis, the relationships between the apolipoprotein(a) (apo(a)) gene and protein size polymorphisms in healthy volunteers (n = 99) and patients with premature myocardial infarction (n = 91). Apo(a) genotypes were determined by pulse-field electrophoresis and phenotypes were separated by sodium dodecyl sulfate-agarose gel electrophoresis. Results showed that phenotyping overestimated apo(a) size with respect to genotyping (mean (SD) = 3.7 (3.4) kringle units; p < 0.001) in subjects with a double-band genotype, although both measurements were highly correlated (r = 0.83; p < 0.001). We also observed that the protein band in subjects with a single-band phenotype was related more closely to the smallest allele than to the largest allele band. The correlation of plasma lipoprotein(a) (Lp(a)) concentration was stronger with the phenotype than with the genotype. We hypothesize that post-translational modifications in the apo(a) molecule may be the most plausible explanation for the discrepancies observed. In conclusion, the present study highlights the dissimilarities between phenotyping and genotyping methods for the measurement of apo(a) size and suggests that laboratories should carefully consider these relationships and the transfer of results between such methodologies.

Sensitive Method for Detection and Semiquantification of Bence Jones Protein by Cellulose Acetate Membrane Electrophoresis Using Colloidal Silver Staining

The monoclonal free light chain of immunoglobulin, Bence Jones protein (BJP), is associated with malignant monoclonal gammopathies, in particular with multiple myeloma, lymphoproliferative diseases such as Waldenstrom macroglobulinemia and malignant lymphoma, amyloidosis associated with light chain, and light chain deposition disease (1)(2). The detection of urinary BJP is useful for diagnosing and evaluating the prognosis for monoclonal gammopathies (3)(4)(5)(6)(7). BJP can be detected as a sharp band by urinary protein electrophoresis (UPE). UPE on cellulose acetate, cellulose nitrate, and agarose has been reported. More recently, sodium dodecyl sulfate-agarose gel electrophoresis and capillary electrophoresis of urinary proteins have been reported (8)(9)(10)(11)(12)(13). Electrophoresis on cellulose acetate membranes is carried out for serum protein fractions in many clinical laboratories, and it is a simple and easily reproducible technique. After UPE on cellulose acetate, membranes are stained with solutions containing Acid-violet 17 and Coomassie Brilliant Blue; however, extensive preconcentration of urine before electrophoresis generally is recommended because of the low concentrations of urinary proteins. This procedure for concentrating urine is time-consuming and has problems such as protein loss, aggregation, and degradation. Although highly sensitive staining methods using colloidal gold solution without preconcentration of urine have been reported (14)(15)(16), these methods require 2–3 h for protein staining. We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for cellulose acetate membranes (17). In this study, we further modified the staining method and developed a more rapid and sensitive colloidal silver staining method to detect small amounts of BJP that does not require preconcentration of urine. We used urine samples obtained from inpatients with multiple …

Idiopathic immune‐mediated acquired von Willebrand's disease in a patient with angiodysplasia: Demonstration of an unusual inhibitor causing a functional defect and rapid clearance of von Willebrand factor

A case of idiopathic immune-mediated von Willebrand's disease (AvWD) associated angiodysplasia and recurrent lower gastrointestinal bleeding is reported. Coagulation parameters at presentation were activated partial thromboplastin time of 41 sec, bleeding time >15 min, factor VIII procoagulant activity, 5%; von Willebrand factor antigen (WF:Ag) 5%, and vWF:ristocetirn cofactor activity 11% sodium dodecyl sulfate-agarose gel electrophoresis pattern of plasma vWF showed a pattern similar to type II vWD. An in vitro inhibitor against vWF in the immunoglobulin (Ig)G fraction of the patient's plasma was demonstrated vWF parameters showed a short-lived increase after 1-deamino-8-D-arginine vasopressin (DDAVP) administration. The patient's bleeding episodes were initially managed adequately with cryoprecipitate replacement therapy and DDAVP, to which she became refractory. No significant improvement was achieved following the institution of immunosuppressive therapy in the form of high-dose steroids and cyclophosphamide. She was then treated with intravenous immunoglobulin (IvIg) to which she showed an adequate response in terms of her clinical situation and her hemostatic parameters. The patient is on maintenance treatment with repeated courses of IvIg based on vWF parameter monitoring. To our knowledge, this is the third reported association between idiopathic immune-mediated AvWD and angiodysplasia. Am. J. Hematol. 60:151–157, 1999. © 1999 Wiley-Liss, Inc.