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The present work deals with the extraction and purification of chondroitin sulfate/dermatan sulfate from skin (CSG) and bone (CBG) of corb (Sciaena umbra). Electrophoresis of these polymers in barium acetate buffer on cellulose acetate revealed two fractions similar to dermatan sulfate and chondroitin sulfate. The in vivo anticoagulant activity of both chondroitin sulfate/dermatan sulfate (CS/DS) were evaluated, at 25 and 75 mg kg−1 of body weight (b.w), using activated partial thromboplastin time (aPTT), prothrombine time (TT) and thrombin time (PT) tests. Results showed that aPTT of CSG and CBG at 75 mg kg−1 of b.w were prolonged by 1.59 and 1.48-fold respectively, compared with the control. Further, toxicity studies on liver performed by the catalytic activity of transaminases in plasma, oxidative stress markers and hepatic morphological changes demonstrated that CSG and CBG at both doses are not toxics. In summary, the higher activity and lower toxicity of both CS/DS, especially at 25 mg kg−1 of b.w, recommended these compounds as a better drug candidate.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (

Although serum protein electrophoresis is a diagnostic tool available through many veterinary laboratories, there currently are no reference intervals for protein fractions in healthy common mynahs (

Among different proteins of blood, albumin is considered a unique protein due to having special properties. Now, various protocols are used for the albumin purification worldwide, each of them has its own advantages and disadvantages. Meanwhile, a common method which is often used for the production of albumin is a combination of Cohn along with different types of chromatography. The aim of the present study was to create a concise and cost-effective albumin purification method by employing a conventional method with some modifications.In this research, the albumin was purified from human serum using chilled ethanol, followed by chromatographic methods. The purity of harvested albumin was evaluated by cellulose acetate membrane electrophoresis (CAME) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting (WB) analysis and thermostability were used for functional and stability measurement assessment, respectively.SDS-PAGE and CAME showed that the purity of purified human albumin was about 99%. Purified human albumin showed a single band with a molecular weight of 66 kDa. The results were validated by WB analysis .Also, the thermostability of purified albumin was same as the commercial albumin.This method can be a robust technique for purification of albumin in order to use clinical and research approaches.

Wound treatment and healing is complex and is comprised of an elaborate set of processes including cellular, spectroscopic and biochemical ones as well as the "reaction" of local tissue to thermal injury. Vitamin C as l-ascorbic acid (LA) prevents injurious effects of oxidants because it reduces reactive oxygen species to stable molecules, it becomes oxidized to the short-lived ascorbyl radical. As a result, antioxidant treatment may contribute to minimizing injury in burn patients. The aim of this study is to assess changes in molecular structure of collagen extracted from human epidermis burn wound scab during incubation of the epidermis in l-ascorbic acid solution. The study will be performed using FTIR and FT Raman spectroscopies. During this research it was observed that the intensity of Raman peaks increased where healing was being modified by LA. The intensity of the amide III band at 1247cm-1 relative to the intensity at 1326cm-1 was used to test tissue repair degree at the incision site. FTIR spectra were recorded from frozen specimens of serum modified by LA; an analysis of shifts in the amide I band position was conducted. The appearance of a new band for frozen samples modified by LA was observed around 1149-1220cm-1. The above conclusions confirmed the creation of hydrogen bonds between NH stretch and CO. Samples being incubated in solutions of l-ascorbic acid demonstrated the absence of electrophoretic bands of albumin. Alterations in the surface of the skin incubated in l-ascorbic acid were investigated with the use of Scanning Electron Microscopy (SEM). A decrease in external symptoms of burn injury was noted in the damaged epidermis incubated in l-ascorbic acid.

application/pdf, The aim of this study was to show the usefulness of a commercial agarose gel electrophoresis (AGE) kit (QuickGel SP) for separating bovine serum protein fractions in comparison with conventional cellulose acetate electrophoresis (CAE). Serum protein bands were verified using five reference reagents corresponding to albumin and α1-, β1-, β2-, and γ-globulins. AGE clearly revealed six separated fractions of albumin and α1-, α2-, β1-, β2-, and γ-globulin fractions in 100% and 77.8% in serum samples of dairy cows from the healthy (n=27) and diseased groups (n=27), respectively. The α1- and α2-globulins were not separated by CAE in 14.8% and 96.3% of the samples from the healthy and diseased groups, respectively, whereas β2- and γ-globulin were not separated by CAE in 96.3% and 100% of the samples from the healthy and diseased groups, respectively. More than 94% of the points for the α-globulin fractions (α1- and α2-globulins), the β-γ-globulin fractions (β1-, β2-, and γ-globulins), and the albumin/globulin ratio between AGE and CAE were within agreement on the Bland-Altman plots. However, the mean biases were not near zero in the albumin and β-γ-globulin fractions. These results suggest that the high-resolution commercial AGE kit can be utilized to separate bovine serum protein fractions. (c) Polish Academy of Sciences, Committee of Veterinary Sciences &University of Warmia and Mazury in Olsztyn 2017.

Adacolumn is a therapeutic mode for ulcerative colitis that achieves therapeutic efficacy through the adhesion of leukocytes to cellulose acetate beads. We used scanning electron microscopy and observed leukocyte adsorption on Adacolumn beads after granulocytapheresis/granulocyte and monocyte adsorption apheresis. We then compared results between two patients with a low and high C-reactive protein (CRP) levels to determine whether adhesion is affected by a difference in leukocyte activity depending on the level of inflammation. We found that the surface layers of the beads from both patients were covered by a clay-like layer, and spherical granulocytes were adsorbed here and there on top of it. In cross-section the adsorbed granulocytes were visible in the clay-like layer and the surface layer alike. The clay-like layer had a maximum thickness of approximately 12 μm in the low CRP patient and approximately 50 μm in the high CRP patient, so in the high CRP patient the clay-like adsorption layer was thicker. Taken together, adsorption onto beads is considered to involve an immunological mechanism. Our findings suggest that granulocytes contact and adhere to each other at the surface layer after adsorption, and that granulocyte-granulocyte adhesion is enhanced by a higher inflammatory response.

The electrophoresis on cellulose acetate membrane is most widely used because of its simplicity, and is without the use of any sophisticated instrument other than electrophoresis apparatus and the cellulose acetate strip. Here we describe a modified version of cellulose acetate membrane electrophoresis for hemoglobin separation from blood sample. Sharp, clear bands without tailing effects can be obtained with this method. The method and apparatus described here would be appropriate to separate protein fractions under 1 h at voltages up to 60 V/cm measured between the electrodes.

Nicaragua is a low-income country located in Central America. The country has a multiethnic population, with Mestizos, American Indian, and black being the most predominant ethnic groups. Nicaragua lacks a national newborn screening program and facilities dedicated to the study of hemoglobinopathies

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

Sickle cell disease affects about 150,000 births annually in Nigeria. Early diagnosis is hampered by factors such as centralized and urban localization of laboratories, high cost of diagnostic equipment and inadequate skilled manpower to operate them. The need for a low-cost, portable, easy-to-use diagnostic test for sickle cell disease is critical, especially in resource-poor countries. In this study, we evaluated the performance characteristics of a novel point-of-care testing device (SickleSCAN™), and its acceptability and feasibility, as a possible screening tool for sickle cell disease. In the first phase, we assessed the performance characteristics of SickleSCAN™ by evaluating 57 subjects comprising both children and adults attending a primary health center, for Hb SS (βS/βS; HBB: c.20A>T), Hb SC (βS/βC; HBB: c.19G>A) and Hb AS (βA/βS) using SickleSCAN™, cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC). Performance characteristics such as diagnostic sensitivity and specificity were compared to HPLC as a standard method. We subsequently undertook a second phase wherein the acceptability and feasibility of the device for sickle cell disease screening, was evaluated using semi-structured and structured questionnaires among 197 healthcare personnel and 221 subjects, respectively. Sickle cell disease was carried by 3.4% of the subjects. The diagnostic sensitivity, specificity and test efficiency of SickleSCAN™ for sickle cell disease (Hb SS and Hb SC), were 100.0, 98.2 and 98.2%, respectively. Findings from this study showed SickleSCAN™ to be a viable screening tool that can easily be applied in community-based screening for early diagnosis of sickle cell disease with little expertise and low cost.

Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis.

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties they exhibited clear differences with human Hb. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. UV-vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb-oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity of fish hemoglobins and environmental partial pressure of oxygen.

The Mekong River is known to act as a boundary between a number of terrestrial and freshwater species, including various parasites and their intermediate hosts as well as endangered mammal species. Little information is available, however, on the genetic differentiation between terrestrial invertebrates to the east and the west of this wide river. The genetic diversity among eight natural populations ofBrachytrupes portentosus(Lichtenstein, 1796) (Orthoptera: Gryllidae) collected from Thailand and the Lao People's Democratic Republic (PDR) were analyzed by multilocus enzyme electrophoresis. The allelic profiles of 20 enzymes encoding 23 loci were analyzed. An average of 41% fixed differences was detected between the populations from Thailand and Lao PDR, which are separated by the Mekong River. The percent fixed differences ranged between 4% and 26% within the populations from Thailand and between 4% and 22% within the populations from Lao PDR. A phenogram shows that the eight populations fell into two major clusters based on the Thai and Lao sampling sites. The genetic distance between the samples within Thailand and within Lao PDR was related to the distances between sampling areas. The genetic variability between populations of this cricket indicates that genetic relationships are influenced by a natural barrier as well as by the geographical distance between these allopatric populations.

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grunwald–Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species. J. Exp. Zool. 315:416–423, 2011. © 2011 Wiley-Liss, Inc.

Resume Etude de l'origine et de la nature des proteines du liquide seminal par electrophorese sur acetate de cellulose L'etude du liquide seminal par electrophorese sur acetate de cellulose a permis de definir 19 bandes proteiques dont 12 ont ete caracterisees quant a leur composition et 10 quant a leur origine: quatre bandes renferment des composes prostatiques, trois renferment des composes vesiculaires et six des composes seriques; en notant le fait que certaines bandes sont constituees de plusieurs secretions specifiques. Au cours de ces etudes l'activite phosphatase acide, la presence de glycoproteines ainsi que de proteines liees au zinc ont pu etre mise en evidence. La degradation du materiel proteique au cours du vieillissement (incubation a 37°C) fait apparaitre l'obligation d'effecteur ces etudes sur du materiel frais ou congele immediatement apres la liquefaction. Les resultats obtenus sur du sperme entier et sur les differentes fractions de l'ejaculation fractionnee permettent d'envisager une utilisation clinique de cette methode, en particulier dans les cas d'azoospermie excretoire ainsi que pour les infections du tractus genital. Zusammenfassung Untersuchungen uber Ursprung und Natur der Spermaproteine mittels Cellulose-Acetat-Elektrophorese Die Elektrophorese des Spermas mittels Cellulose-Acetat erlaubt die Lokalisation von 19 Proteinbanden. 1 2 von ihnen wurden analysiert hinsichtlich ihrer Zusammensetzung und 12 von ihnen hinsichtlich ihrer Gewebsherkunft. 4 Banden waren aus Produkten der Prostata zusammengesetzt, 3 enthielten Sekrete aus den Blaschendrusen, in 6 anderen waren verschiedene Serumprodukte enthalten. Daruberhinaus wurden Untersuchungen vorgenommen auf saure Phosphataseaktivitat, Glykoproteine und Proteinbindung an Zink. Es stellte sich heraus, das ausschlieslich frische Spermaproben bzw. unmittelbar nach der Verflussigung eingefrorene Ejakulate fur eine entsprechende Analyse geeignet sind. Es wird die Auffassung vertreten, das die Spermaelektrophorese eine durchaus anwendbare Untersuchungs-methode in der klinischen Andrologie sein kann; das gilt insbesondere fur Falle mit exkretorischer Azoospermie und Infektionen im Genitaltrakt.

The detection of a γ-heavy chain disease protein in the serum and urine of a patient with a history of Hodgkin's disease in complete remission is reported. The protein has been characterized as a γ1 dimer, MW 66000, beginning at Asp 221 residue in the hinge region. No clinical or laboratory findings of malignancy have been observed in the patient since the abnormal protein was detected. The pathogenesis of this association, unreported to date, is discussed on the basis of previously administered high-dose anti-neoplastic treatments and of immunological impairment.

Iron (3 mg) was added as ferrous sulphate to 2 dl red wine, white wine and 7% alcohol and its absorption was then measured in 38 fasting male subjects. (The original concentrations of iron in the two wines were low, being 1.01-1.08 mg/l (red wine) and 0.13-0.20 (white wine]. The geometric mean absorption from red wine was only 20% of that from the alcohol solution whilst more than 4 times as much was absorbed from white wine as from the alcohol. Direct comparison showed greater absorption from white wine (10.4%) than from red wine (4.4%). Removal of about 80% of the polyphenols in red wine increased the geometric mean iron absorption from 1.9% to 3.6%. In vitro experiments indicated that iron was less soluble and less dialysable in red wines than in white wines. This was possibly due to the binding of iron to polyphenols in red wines. Electrophoretic studies suggested that the iron in white wines was complexed to hydroxycarboxylic acids.

Isoenzymatic study of leucocytic acid phosphatase under normal conditions identifies 3 isoenzymatic bands, which exhibit a noticeable cell specificity. Band 2 is granulocytic, band 3 lymphocytic and band 4 monocytic in origin. Pathologic deviations in the isoenzymatic pattern are both qualitative and quantitative. For some diseases such as chronic lymphocytic leukaemia there is a well-defined, differential pattern according to immunological B- or T-cell origin. The more significant qualitative aspects are related to the appearance of abnormal bands, especially band 3b, indicating blastic cellularity, and 5, corresponding to hairy cells. The isoenzymatic analysis of acid phosphatase activity is a simple haematologic complementary test, particularly useful in the differential diagnosis of lymphoproliferative disorders with peripheral blood manifestations.

Glycosaminoglycans and proteoglycans are macromolecules of the bone and are involved in the assembly, maturation, mineralization, and maintenance of the extracellular matrix. Heterotopic ossification is the rapid development of calcified bone tissue at ectopic sites of the body, mainly in soft tissues that normally do not ossify. The aim of this study was to characterize the molecular profiles of glycosaminoglycans and proteoglycans in normal and heterotopic bone samples to assess whether differences exist between orthotopic and heterotopic bone. Heterotopic bone tissues contained lower amounts of glycosaminoglycans compared to normal femoral bone. Structural analysis of chondroitin sulfate (CS) revealed that both heterotopic and normal femoral bones were composed mainly of 6-sulfated disaccharides. Quantitative differences in the disaccharide composition of CS, such as the decrease of 6-sulfated disaccharides in heterotopic bone with a concurrent increase of 4-sulfated and nonsulfated disaccharides, were found between ectopic bone and normal femoral bone. The proteoglycans decorin and aggrecan were both detected in all bone samples using specific antibodies. The detection of minor amounts of aggrecan in mature human bone, such as femoral bone, as well as in ectopic bone is described for the first time. These results may elucidate the phenomenon of ectopic bone formation and assist in early detection.

This study presents a comparative drug-protein, in vitro, binding profile of sodium aurothiomalate and auranofin. It was found that about 40% of total protein-bound gold is attached to albumin after incubation of aurothiomalate with whole blood for 24 h and about 29% of it was with alpha(1)-globulin and the least amount was found with gamma-globulin (6.1%). On the other hand, approximately 84% of the protein-bound auranofin gold attached to globulins of which 51% was found with beta-globulin band. It was almost equally distributed among albumin, alpha(2)-globulin and gamma-globulin, and showed least affinity for alpha(1)-globulin. The gold analyses were performed by standardless instrumental neutron activation method duly validated by use of an established atomic absorption method. The results of this study explain to some extent the difference in, in vivo, pharmacokinetics and pharmacodynamics of the two drugs.

A cellulose acetate plate electrophoresis method for analysis of pharmaceutical heparin and its potential glycosaminoglycan impurities, e.g. dermatan sulfate, chondroitin sulfate and oversulfated chondroitin sulfate, is presented. Heparin is chemically degraded by application of nitrous acid and residual glycosaminoglycans are electrophoretically separated thereafter. After staining using Alcian blue 8GS, these glycosaminoglycan impurities can be quantified by means of comparison to a dermatan sulfate standard. Results of a validation study of this analytical method are shown, demonstrating its feasibility for routine use in analytical quality control labs under GMP conditions.

Background: Although rainbow trout (Oncorhynchus mykiss, Walbaum) are one of the most-studied fish, electrophoretic techniques and classification of serum protein fractions have not been standardized, such that clinically useful values are lacking. Objective: The aim of the present study was to evaluate preliminarily the serum protein fractions of rainbow trout using automated cellulose acetate electrophoresis and densitometry. Methods: Serum samples from 25 rainbow trout (Oncorhynchus mykiss, Walbaum) were electrophoresed on cellulose acetate plates and quantified using densitometry. Results: A maximum of 6 fractions were identified and numbered, in order of decreasing mobility, as I, II, III, IV, V, and VI. In 3 of 25 (12%) samples, 6 fractions were identified; in 18 (72%) samples, 5 fractions were identified; and in 4 (16%) samples, 4 fractions were identified. Fractions I, V, and VI were always clearly identifiable, whereas fractions II and IV were frequently fused and indistinguishable from fraction III. The pattern with 5 fractions was the most probable type (χ2, P

Hyaluronic acid (HA) samples showing inhibition effect on digestion with testicular hyaluronidase (HAase) were found from 16 commercially available HA products, which were supplied from 11 different manufacturers. Most of these HA samples (six samples) were derived from the rooster comb, and one sample was derived from the human umbilical cord. HA oligosaccharides produced by exhaustive digestion of these HA samples with testicular HAases were monitored by capillary electrophoresis, and we found that a few HA samples gave no oligosaccharide products. Detailed analysis of HA samples by cellulose acetate membrane electrophoresis revealed that the HA samples were not digested with HAase because of the presence of a small amount of dermatan sulfate (DS). Analysis of disaccharide units of these HA samples produced by digestion with chondroitinase ABC supported the observations. And the content of DS in the sample was estimated to be ca. 8%. In contrast, these HA samples were easily digested with bacterial hyaluronate lyases from Streptomyces hyalurolyticus and Streptococcus dysgalactiae and gave endproducts of unsaturated disaccharide or unsaturated tetra- or hexasaccharides. The results suggested that the inhibitory effect of DS on HAase is specific to endo-type hydrolase (i.e. testicular HAase). In addition, pharmaceutical preparations of HA derived from rooster comb were easily digested with testicular HAase. These findings will be useful information for clinical or cosmetic use of HA preparations in terms of their half-life.

Summary The performances of two commercial screening tests for glucoses-phosphate dehydrogenase deficiency (Sigma fluorescent spot test and Sigma colorimetric method) were assessed in order to determine their usefulness in a routine haematology laboratory. As a first step, three ranges for enzyme activity were determined as follows: a ‘normal’ range determined from the 95% confidence limit of assays carried out on 114 normal adult males, a deficient range calculated as 25% of the upper limit of normal or less, and an intermediate range between the lower and upper values of these ranges. These values were 4.9–11.8 u/g Hb, 0–2.9 u/g Hb and 3.0–4.8 u/g Hb respectively. A separate normal range was also determined for females and was 5.5–12.8 u/g Hb. The two screening tests were then assessed against these values and the kits were found to be equally reliable at predicting normals and deficients but less reliable at detecting all female heterozygotes. The criteria for using the different procedures were evaluated. It is concluded that screening procedures are useful only when a large number of tests are routinely performed or in the absence of facilities for assays, as they offer no advantage in cost or time over the assay procedure. The colorimetric test (single vials) is easier and cheaper to use if isolated tests are performed, whereas the fluorescent spot test (with some modifications) is more useful for carrying out numerous tests simultaneously.

Urine from 348 patients of a hospital for the mentally handicapped in Wessex was screened for abnormalities of aminoacid and mucopolysaccharide excretion. Persistent aminoacid abnormalities were found in 16 patients: two had phenylketonuria and one a severe variant form of hyperphenylalaninaemia (dihydropteridine reductase deficiency). In at least nine of the other patients, the observed abnormality was probably a secondary phenomenon. Two patients excreted excessive amounts of a glycosaminoglycan identified tentatively as hyaluronic acid. The study also uncovered one undiagnosed diabetic and three patients with urinary tract infection.

SUMMARY: Aim: The diagnostic approach for renal diseases with the electrophoretic pattern of urinary protein on cellulose acetate (CA) membrane differentiates the causes of proteinuria. However, this method has not been used routinely because of its difficulty in obtaining a clear image. This study was performed in order to re-evaluate this method with an improved system. Methods: Using the newly developed system of CA membrane electrophoresis and its visualization, we examined fresh urine from patients (n = 100) who subsequently underwent renal biopsy and compared the results with the histological findings. Results: The improved method of urine electrophoresis with CA membrane provided clear images and was sensitive enough for urine samples to be applied without concentration. The profiles of proteinuria were clearly classified into three patterns: glomerular, tubular or mixed. The profiles exhibited a good agreement with the histological findings of renal biopsy. Conclusion: The recognition of damaged portions in kidney through the profiles of proteinuria by this system could be practically effective for understanding the kidney disease at bedside.

Background Glycosaminoglycans are found in human tissues including plasma. They encompass chondroitin sulphates, heparan sulphate/heparin, hyaluronic acid, and keratan sulphate. Glycosaminoglycans, in particular heparan sulphate and heparin, are strongly associated with plasma proteins, so that their purification results quite difficult. Methods In order to study the distribution of glycosaminoglycans in plasma subfractions, we developed a novel method that allows their identification even if they were still associated with proteins or peptides. Plasma was fractionated following the procedure of Cohn–Oncley, and each fraction was treated with proteases. After centrifugation, glycosaminoglycan/protein complexes in the supernatant were analysed using a modified cellulose acetate electrophoresis which allowed identification of glycosaminoglycans in mixtures of glycosaminoglycans/proteins. Results Chondroitin sulphate was recovered in cryoprecipitate and in all Cohn–Oncley fractions. Glycosaminoglycans belonging to the class of heparan sulphate/heparin, however, were recovered in the cryoprecipitate and in fractions I and IV-1, and, in smaller amount, in fraction II + III. Conclusions Since the largest amount of plasma proteins is partitioned in Factions II + III and V, these results demonstrate that heparan sulphate/heparin are not randomly distributed in Cohn–Oncley fractions and are associated with certain plasma proteins. This association might play a role in the physiological function of heparan sulphate/heparin, regulating hemostasis and atherogenesis.

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1-5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14-97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan-hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.

A new diffusive gradients in thin films (DGT) device, using Pb(II) ion-imprinted silica (IIS) as the binding agents and commercial cellulose acetate dialysis (CAD) membrane as the diffusion layer (CAD/IIS-DGT), has been developed and evaluated for sampling and measurement of free Pb(II) species. The CAD/IIS-DGT devices were successfully applied to the measurement of free Pb(II) species in synthetic solutions, in natural freshwaters and in industrial wastewaters. The CAD/IIS-DGT provides reliable results over pH range of 4.5-6.5 and a wide range of ionic strength from 1.0×10(-3) to 0.7 mol L(-1). The concentrations of the free Pb(II) species in synthetic solution containing different concentrations of ligands measured by CAD/IIS-DGT showed a good agreement with the value measured by Pb-ion selective electrode. Field deployments of the CAD/IIS-DGT devices allowed accurate measurements of the concentrations of free Pb(II) species.

Alcian blue staining has been widely used to visualize acidic mucins and mucopolysaccharides in supported molecular matrix electrophoresis (SMME) and on membrane transferred from electrophoresis gels. Mucins with low acidic glycan content, however, cannot be stained with Alcian blue, which is one of the major drawbacks of this staining method. On the other hand, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content; however, periodic acid-Schiff staining decomposes glycans. Here, we introduce succinylation-Alcian blue staining as an alternative staining method to visualize mucins, regardless of the acidic residue content, and without glycan decomposition.

Due to the diminished stocks of the 2(nd) batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for human immunoglobulin for electrophoresis, in 2013, the European Directorate for the Quality of Medicines and HealthCare (EDQM) initiated an international collaborative study for the establishment of a replacement batch. The study was run under the aegis of the Biological Standardisation Programme (BSP). Seventeen laboratories participated in the collaborative study to verify the suitability of the candidate reference preparation according to the Ph. Eur. monographs 0338 and 0918 using the zone electrophoresis (ZE) method with either cellulose acetate and/or agarose as the testing medium. The candidate preparation was found suitable for the intended purpose and was subsequently adopted by the Ph. Eur. Commission as the human immunoglobulin for electrophoresis BRP batch 3 with an assigned range for immunoglobulin of 79.8 % to 86.4 % of the total protein content.

Context.—Apparent hemoglobinopathies caused by blood transfusions rarely have been reported in the scientific literature. Objective.—To interpret the abnormal hemoglobins appearing as small peaks on hemoglobin chromatograms or electrophoresis membranes. Design.—In the clinical laboratories of a university hospital and a metropolitan hospital affiliated with a medical school, we interpreted hemoglobin chromatograms and electrophoresis membranes; correlated them with patients' medical, laboratory, and transfusion records; and when possible, identified the abnormal hemoglobin in the donors' transfusion segments. Results.—We detected 52 incidences of apparent hemoglobinopathies in 32 recipients caused by blood transfusion, of which 46 were hemoglobin C, 4 were hemoglobin S, and 2 were hemoglobin O-Arab. When first detected, the abnormal hemoglobins in recipients ranged from 0.8% to 14% (median, 5.6%). Multiple transfusions with abnormal hemoglobins occurred in 11 patients with 2 patients receiving hemoglobin C blood 5 separate times. One patient received hemoglobin C and later S, and another patient received C and later O-Arab. Conclusions.—Apparent hemoglobinopathies caused by blood transfusions are far more common than previously reported and represent diagnostic challenges. Misdiagnosis could lead to unnecessary testing, treatment, and counseling. If a hemoglobinopathy from a unit of transfused blood is identified in a recipient, we recommend notifying the donor of that abnormality.

Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorphism among ten clinical and 11 non-clinical isolates of Trichoderma. Initial testing of 13 enzyme systems for activity and resolution of bands showed that seven were appropriate for identifying the different species. Each of the enzyme systems investigated (glucose-6-phosphate dehydrogenase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, peptidases A, B and D, and phosphoglucomutase) was diagnostic for at least one species. On the basis of the results of isoenzyme analysis, several isolates identified originally as Trichoderma pseudokoningii, T. koningii or T. citrinoviride were re-identified as T. longibrachiatum, in agreement with sequence analysis data for the internal transcribed spacer region of the isolates. The availability of a quick, inexpensive and reliable diagnostic tool for the identification of T. longibrachiatum isolates is important, as most clinical Trichoderma isolates belong to T. longibrachiatum. Furthermore, as many different enzyme systems are available, the method may also be suitable for the identification of other clinically relevant fungal species.

Diet is now one of the well established means in the management of diabetes. Bitter gourd and spent turmeric at 10% level were tested for their efficacy on glycosaminoglycan metabolism in various tissues viz., liver, spleen, lungs, heart and testis in control, diabetic and treated rats. The glycosaminoglycans (GAGs) were isolated from defatted and dried tissues. The contents of sulfated GAGs decreased in all the tissues and the decrease was more prominent in heart and testis. In the isolated GAGs, contents of total sugar, amino sugar, uronic acid and sulfate were studied. Decrease in total sugar content was maximum in testis. Amino sugar content decreased considerably in testis (38%) and lungs (15%). The content of uronic acid also decreased in testis (33%) besides heart (29%) and liver (25%). Sulfate groups in GAGs perform pivotal functions in many biological events and decrease in sulfate content was significant in heart (40%), testis (37%) and liver (37%). GAGs profile on the cellulose acetate electrophoresis revealed that heparan sulfate (HS), hyaluronic acid (HA) and chondroitin sulfate/dermatan sulfate (CS/DS) were present in liver, spleen and lungs. HS, CS were present in heart, DS/CS was observed in testis. The observed beneficial effects in GAGs metabolism during diabetes may be due to the presence of high amounts of dietary fibres present in bitter gourd and spent turmeric, besides, possible presence of bioactive compounds in one or both of them.

Hemicelluloses (A and B) were isolated from an Indo-African hybrid variety of finger millet (ragi, Eleusine coracana) by extracting the starch-free residue with 10% sodium hydroxide under a continuous stream of nitrogen, and changes in their sugar composition during malting for 96 h were studied. Hemicellulose B, obtained in higher yield from both native (N) and malted (M) flours, was found to be completely soluble in water, richer in uronic acid, and more viscogenic than hemicelullose A. Fractional precipitation of hemicellulose B by ammonium sulfate resulted in four precipitable fractions (F-60, F-70, F-80, and F-100) and a nonprecipitable (NP) fraction varying in their yield and arabinose, xylose, galactose, and glucose contents. A progressive increase in the pentose-to-hexose ratio (P:H) from 0.42:1.0 in F-60 to 1.94:1.0 in NP was observed in native hemicellulose B fractions; however, in malted hemicellulose B the P:H ratio increased from 0.43:1.0 in F-60 to 1.56:1.0 in F-80 and then decreased to 1.13:1.0 in NP. The major fraction, F-70 (N, 44.5%; M, 38.5%), was separated into eight subfractions on DEAE-cellulose by successive elution with water, ammonium carbonate (AC) (0.1, 0.2, and 0.3 M AC), and sodium hydroxide (0.1 and 0.2 M) differing in their yield and neutral sugar composition. The purity of the major glucuronoarabinoxylan fraction (0.1 M AC eluted) was ascertained by Sepharose CL-4B, HPSEC, cellulose acetate, and capillary electrophoresis methods. A significant decrease in the molecular mass of arabinoxylan from 1200 to 1120 kDa upon malting for 96 h is an indication of cell wall degradation by the inducible cell wall degrading enzymes.

Background Information regarding leishmaniasis recidiva cutis (LRC), a clinical variant of cutaneous leishmaniasis, in the New World is scarce. LRC is characterized by slowly progressing lesion(s) that appear after a variable period of time, from months to years, in or around the scar of an apparently clinically healed sore. Patients and methods Six patients are reported who presented with crusted, papular lesions located on the edge of a healed scar, with a mean of 18.2 months of slowly progressive evolution. The isolated strains of Leishmania parasites were characterized by enzyme electrophoresis. Eleven enzyme systems were assayed. Skin biopsies from the active border of the lesions were taken for histopathology. Results Tissue sections showed a granulomatous, lymphohistiocytic, dermal infiltrate containing Langhans’ giant cells. The anamnestic data, together with the clinical and histopathologic findings, support the diagnosis of LRC. The isoenzyme profile of Leishmania parasites isolated from five of the six patients identified them as Leishmania (Viannia) panamensis. Conclusions These findings are the first reported evidence of LRC within the clinical spectrum of American tegumentary leishmaniasis in Ecuador, and of its causative agent. The existence of LRC has future implications for both disease treatment and vaccine development.

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS. CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in combination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived from CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use method for detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CS by CAME and that exhibit CPC titration behavior similar to CS.

A new electrophoretic migration type of alkaline phosphatase 1 (Akp1) was found on the cellulose acetate electrophoresis for kidney and liver homogenates of KWHM mouse, a newly established inbred strain derived from the Korean wild mouse (Mus musculus molossinus). This new type of alkaline phosphatase 1 was distinguished from previously reported AKP1A and AKP1B types in the mouse, and tentatively named AKP1C. In genetic analysis by mating experiments between KWHM and C57BL/6J (AKP1A) or BALB/cA (AKP1B), the phenotypic segregation ratios of AKP1A : AKP1AC : AKP1C or AKP1B : AKP1BC : AKP1C were 1 : 2 : 1 in both groups of F2 generations. It was therefore concluded that AKP1C type is controlled by Akp1c allele which is codominant with Akp1a and Akp1b alleles.

The year 2006 marks the 50th anniversary of the discovery of cellulose acetate (CA) electrophoresis by Joachim Kohn, a pathologist at Queen Mary’s Hospital in Roehampton, London. During a career in pathology that began in 1950 and spanned 37 years, Kohn published more than 50 papers in clinical laboratory medicine. He was the first to report the use of CA microbiology filters as solid supports for zone electrophoresis and the separation of hemoglobin phenotypes on CA membranes. Kohn also invented a new electrophoresis chamber and an 8-position stamp applicator especially for use with CA membranes. Beginning in 1957, Kohn pioneered the development of CA techniques for immunoelectrophoresis, counter immunoelectrophoresis, radial immunodiffusion, protein blotting, and immunofixation. He also designed a transport dressing for burn patients and was the first person to describe the use of an enzyme-based dipstick for measuring fingerstick blood glucose concentrations. This short review highlights Kohn’s discovery of CA electrophoresis and his contributions to the development of this procedure.

Background The aim of the present work was to characterize the molecular defects of a slow-migrating (albumin Zagreb) and a fast-migrating (albumin Krapina) genetic variant of human serum albumin detected in heterozygous persons living in Croatia and to elucidate the fatty acid-binding properties of the two alloalbumins. Methods Purification and structural identification of the variants were performed by conventional protein chemistry methods, whereas types and amounts of albumin-bound, endogenous fatty acids were determined by gas chromatography. Results Protein sequencing established that albumin Zagreb is a proalbumin variant (−1Arg→Gln), and that albumin Krapina is due to a mutation within the mature polypeptide chain (573Lys→Glu). The gas chromatographic results showed that the fatty acid-binding properties of the proalbumin variant are normal, while the amino acid substitution in position 573 resulted in a general decrease of fatty acid binding. Conclusions The structural defects of the first alloalbumins, detected by routine clinical electrophoresis among the Croatian population, were characterized. Albumin Zagreb is caused by a hot-spot mutation occurring in a CpG sequence in the albumin gene. It is commonly assumed that bisalbuminaemia has no direct clinical relevance. However, the present study suggests that naturally occurring mutations can affect the ligand-binding properties of human serum albumin.

Multilocus enzyme electrophoresis was used to compare specimens of the parasitic nematodeCloacina obtusafrom the stomach of the eastern grey kangaroo,Macropus giganteusand the western grey kangaroo,M. fuliginosus. Allelic variation among nematodes was detected at 17 (85%) of 20 loci, but there was only a single fixed genetic difference (at the locus for isocitrate dehydrogenase, IDH) betweenC. obtusafromM. fuliginosusand those fromM. giganteusin areas where each host occurred in allopatry. However, this fixed difference was not apparent within the zone of host sympatry. Although electrophoretic data indicate genetic divergence among allopatric populations ofC. obtusain the two host species, the magnitude of the electrophoretic difference (5%) between these populations does not refute the hypothesis thatC. obtusarepresents a single species. The ’usual’ situation for parasitic helminths of grey kangaroos is that pairs of parasite species occur in the two host species. This situation differs forC. obtusa, where there has been a lack of speciation following a speciation event in its macropodid marsupial hosts. This finding suggests that a speciation event in the host does not necessarily lead to a speciation event for all its parasites and further highlights our lack of understanding of which processes drive speciation in parasites.

Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T. cruzi IIb and T. cruzi IId, respectively. Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media. We tried to reproduce this observation, but used a different cloning method of individual organisms. Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only. The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes. The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.

In order to improve our knowledge about the taxonomic status and the population structure of the causative agent of Human African Trypanosomiasis in the Central African subregion, 169 newly isolated stocks, of which 16 came from pigs, and 5 reference stocks, were characterized by multilocus enzyme electrophoresis, for 17 genetic loci. We identified 22 different isoenzyme profiles or zymodemes, many of which showed limited differences between them. These zymodemes were equated to multilocus genotypes. UPGMA dendrograms revealed one main group:Trypanosoma brucei gambiensegroup I and 3T. brucei‘non-gambiense’ stocks.T. b. gambiensegroup I zymodemes were very homogenous, grouping all the human stocks and 31% of the pig stocks. Two main zymodemes (Z1 and Z3) grouping 74% of the stocks were found in different remote countries. The genetic distances were relatively high inT. brucei‘non-gambiense’ zymodemes, regrouping 69% of pig stocks. The analysis of linkage disequilibrium was in favour of a predominantly clonal population structure. This was supported by the ubiquitous occurrence of the main zymodemes, suggesting genetic stability in time and space of this parasite's natural clones. However, in some cases an epidemic population structure could not be ruled out. Our study also suggested that the domestic pig was a probable reservoir host forT. b. gambiensegroup I in Cameroon.

Dacryolithiasis is one of the causes of acquired nasolacrimal duct obstruction, although its origin is unclear. The purpose of this study was to investigate the differences in tear constituents between patients with nasolacrimal duct obstruction with dacryoliths and those without dacryoliths. In a prospective case-control study, undiluted tears were collected from 30 eyes from 30 patients with partial acquired nasolacrimal duct obstruction (PANDO) consisting of 17 PANDO patients with dacryoliths and 13 PANDO patients without dacryoliths. The pH, and the Na+, K+, Cl–, total calcium and total protein concentrations were determined. The composition of the tear proteins was assessed by cellulose acetate electrophoresis. In the tear samples from 17 eyes with PANDO/dacryoliths, the mean ± SD K+ level was 15.6 ± 3.1 mEq/l. In the tear samples from 13 PANDO controls, the mean ± SD K+ level was 19.8 ± 4.9 mEq/l. The difference between the two groups was statistically significant (p = 0.02). In the tear samples from PANDO/dacryolith patients, the mean ± SD total protein level was 129.0 ± 72.9 mg/dl. In the tear samples from the PANDO controls the mean ± SD total protein level was 261.6 ± 132.5 mg/dl. The difference between the two groups was statistically significant (p = 0.00). In the tear protein fractions from electrophoresis, a low level of lysozyme was observed in the PANDO/dacryolith samples compared with the PANDO control samples (p = 0.03). The tears from patients with PANDO due to dacryoliths showed a change in the concentrations of electrolytes and protein, particularly lysozyme, compared with that of the patients with PANDO without dacryoliths, which may be related to the pathogenesis of dacryoliths.

The aim of this study was to clarify the relationship between the relative mobility of M protein in various disease states using cellulose acetate membrane electrophoresis. To examine the carbohydrate chain of the M protein, sera from patients with multiple myeloma (MM), various cancers, and benign disease were treated with neuraminidase. The relative mobility in benign disease and MM patient sera following neuraminidase treatment varied among individuals, while that of IgG M protein in sera from cancer patients was from 0.2 to 0.3. Thus, the relative mobility in cancer patients was narrower than in those with MM or benign disease. However, after neuraminidase treatment, there was no significant difference between relative mobility in cancer patient's sera and those in other disease patients. J. Clin. Lab. Anal. 18:182–186, 2004. © 2004 Wiley-Liss, Inc.

A hybrid zone involving the deep-sea mussels, Bathymodiolus azoricus and B. puteoserpentis , was recently discovered at Broken Spur hydrothermal vent field (29 °° °° 10 ′′ N, 43 °° °° 10 ′′ W) along an intermediate segment of the Mid-Atlantic Ridge axis. Examination of nuclear (allozymes) and cytoplasmic (mitochondrial DNA) gene markers in a new sample from Broken Spur revealed significant cytonuclear disequilibrium caused by an excess of the parental types (coupling phase) and a deficiency of recombinants (repulsion phase). An assignment test of individual multilocus genotypes also revealed an excess of parental genotypes in the admixed population. These results support the hypothesis that the Broken Spur mussel population comprises a nonequilibrium mixture of parental immigrants and hybrid individuals.

Thirty-one stocks of Trypanosoma cruzi, the agent of Chagas disease, representative of the genetic variability of the 2 principal lineages, that subdivide T. cruzi, were selected on the basis of previous multilocus enzyme electrophoresis analysis using 21 loci. Analyses were performed with lower numbers of loci to explore the impact of the number of loci on the robustness of the phylogenies obtained, and to identify the loci that have more impact on the phylogeny. Analyses were performed with numerical (UPGMA) and cladistical (Wagner parsimony analysis) methods for all sets of loci. Robustness of the phylogenies obtained was estimated by bootstrap analysis. Low numbers of randomly selected loci (6) were sufficient to demonstrate genetic heterogeneity among the stocks studied. However, they were unable to give reliable phylogenetic information. A higher number of randomly selected loci (15 and more) were required to reach this goal. All loci did not convey equivalent information. The more variable loci detected a greater genetic heterogeneity among the stocks, whereas the least variable loci were better for robust clustering. Finally, analysis was performed with only 5 and 9 loci bearing synapomorphic allozyme characters previously identified among larger samples of stocks. A set of 9 such loci was able to uncover both genetic heterogeneity among the stocks and to build robust phylogenies. It can therefore be recommended as a minimum set of isoenzyme loci that bring maximal information for all studies aiming to explore the phylogenetic diversity of a new set of T. cruzi stocks and for any preliminary genetic typing. Moreover, our results show that bootstrap analysis, like any statistics, is highly dependent upon the information available and that absolute bootstrap figures should be cautiously interpreted.