Applications of capillary electrophoresis in nephrology.

Capillary electrophoresis has recently emerged as a powerful technique for separating components in biological samples. A family of separation methods capable of handling a diverse range of samples has been developed, the sample volumes required are very small and a wide range of specialised detectors can be employed. This review examines some methods with particular application to the analysis of urine and tubular fluid samples and references relevant applications.

A reappraisal of the carcinogenicity of surface modified asbestos fibres.

A previous study using intrapleural administration of surface-modified amosite asbestos showed a difference in the number of pleural mesotheliomas induced with C18-hydrocarbon derivatised fibres compared to native amosite asbestos. The study has been repeated with larger groups of animals (30) under specific pathogen free conditions, resulting in an increase in the mean animal survival time for both fibre-treated groups. Under these conditions there was no significant difference between the numbers of pleural mesotheliomas induced by C18 hydrocarbon-modified amosite asbestos and native amosite asbestos. The major difference between the two studies was the mean time to death from tumour of rats exposed to fibres. The C18 amosite treated rats in the first study may not have had a mean survival time long enough to allow mesotheliomas to develop.

Surface modification can affect the carcinogenicity of asbestos.

A sample of amosite asbestos was modified by effectively adding C8 and C18 hydrocarbon chains to the fibre surfaces. The altered fibres interacted less readily with cells in vitro and were less cytotoxic. In whole animals the number of mesotheliomas produced by the C8 material was the same as that with the parent material but the tumours occurred earlier. The C18 derivatized fibre was markedly less active in the production of tumours. This is the first report demonstrating that similar size fibres with differing surfaces have different pathogenic properties.

A gamma-ray detector for capillary zone electrophoresis and its use in the analysis of some radiopharmaceuticals.

The construction and evaluation of a gamma-ray detector for capillary zone electrophoresis is described. The detector was shown to have a linear response from the limit of detection 10 to 500 Bqcm-3 corresponding to 5.1 x 10(-17) to 2.55 x 10(-15) gcm-3 43Tc99m. The application of the detector for the analysis of some radiopharmaceuticals is presented.

Interaction of amosite and surface-modified amosite with a V79-4 (Chinese hamster lung) cell line.

We have been examining a number of chemically modified mineral fibers, derived from amosite asbestos, by in vitro methods to clarify the role of the fiber surface in determining biological activity. The various fibers have identical size distributions but differ in their affinities for components of the cell membrane. They were treated with boiling toluene or chemically modified by treatment with alkyldimethylchlorosilanes (R = C8, C18) that react with free-surface hydroxyl groups to form the corresponding siloxanes. Fibers in MEM supplemented with 15% fetal calf serum were added to a suspension of V79-4 cells labeled with tritiated thymidine and the mixture was incubated. Aliquots of this mixture were spun down on a density gradient to determine the degree of cell-fiber interaction. At 37 degrees C native amosite (UICC standard) stuck to cells within 15 min of incubation, and the amount of sticking was maximum within 70 min. Decreasing the temperature decreased the amount of sticking, and at 20 degrees C no sticking was observable. The chemically modified amosite and the amosite treated with boiling toluene did not stick to the cells even after 70 min. Soaking the toluene-treated amosite with aqueous solutions at room temperature for 48 hr produced a material that had the same sticking properties as the original untreated fiber. These results indicate that the silanol content, and possibly the degree of hydration of the fiber surface, is important for a fiber to stick to a cell surface.

Protein conformation changes as the result of binding to reversed-phase chromatography column materials.

The conformational changes of the protein alpha-chymotrypsinogen which may take place on reversed-phase chromatographic material of differing hydrocarbon chain lengths e.g. C4, C6, C8, C10 and phenyl, have been studied by circular dichroism as a function of 1-propanol concentration and pH of the solvent before and after binding to the reversed-phase material.

Analysis of some pharmaceuticals by high voltage capillary zone electrophoresis.

High voltage capillary zone electrophoresis (HVCZE) has been used to determine quinine, proflavine and other drugs. The technique may offer a useful alternative to chromatography in the analysis of pharmaceuticals.

Neurotoxic and antihypertensive effects of phenytoin in turkeys.

Broad-Breasted White male turkeys were fed a control diet until 4 weeks of age, at which time they were randomized into a control group and two treatment groups which received 0.06% (38.7 mg/kg) and 0.09% (58.2 mg/kg) phenytoin, respectively, until termination of the experiment at 12 weeks of age. Plasma concentrations of phenytoin on the two dosages were 8.0 +/- 0.8 and 15.7 +/- 1.7 micrograms/ml. Systemic arterial blood pressure in the control turkeys was 214 +/- 5/171 +/- mm Hg and was reduced in a dose-related fashion to 185 +/- 8/143 +/- 11 and 156 +/- 5/125 +/- 4 mm Hg in the two treatment groups; likewise, the rate of systolic ejection (dp/dt maximum) was less in the phenytoin-treated turkeys. Heart rate also dropped significantly with drug administration but the difference between the two treatment groups was not significant. Evidence of neurotoxicity developed in 25% of the turkeys on the lower drug schedule; these birds had mean plasma phenytoin levels of 12.8 micrograms/ml as contrasted to the concentration of 8.0 micrograms/ml for the entire group. On 0.09% phenytoin 50% of the birds had abnormal signs and a mean phenytoin concentration of 20.5 micrograms/ml, whereas the mean drug level for the entire group on this drug level was 15.7 micrograms/ml. Early signs of neurotoxicity developed within 2 to 3 days of initiation of phenytoin and consisted of extensor rigidity of the neck and hyperactivity; at the higher drug concentrations, back pedaling and somersaulting appeared. General health and weight gain were not affected. No qualitative or quantitative changes were found in the Purkinje cells in the cerebellum of the affected turkeys.

Ultrastructure of the intraerythrocytic stage of Cytauxzoon felis.

The erythrocytes of 2 cats experimentally infected with Cytauxzoon felis were examined by light and electron microscopy. In stained blood smears, parasitized erythrocytes usually contained a single, roundish organism, but occasionally up to 4 were present in a cell. Chains of these roundish organisms also were seen. Elongated parasites, sometimes with ear-like projections, were present in a few erythrocytes. By electron microscopy, the parasite contained a poorly defined nucleus, rough endoplasmic reticulum, ribosomes, nonplicated mitochondria, food vacuoles, and a cytostome on its limiting membrane. Usually, the parasite was oval, but budding forms also were evident. Crystalloid inclusions were present in parasitized and nonparasitized erythrocytes.

Ultrastructure of schizonts in the liver of cats with experimentally induced cytauxzoonosis.

Schizonts in the liver of 2 cats with cytauxzoonosis were studied by both light and electron microscopies. By light microscopy, the cytoplasm of macrophages in the sinusoids and small vascular channels contained schizonts with cytomeres or both cytomeres and mature merozoites. By electron microscopy, it was determined that schizogony occurred in 4 stages. The earliest stage was the presence of a multilobed structure containing finely granular protoplasm in the cytoplasm of the macrophage. The 2nd stage was an increase in height and number of the lobulations on the surface of the schizont. The 3rd stage involved the development of cytomeres and the appearance of a polar ring and rhoptries in everted sacculations on the cytomere membrane. Nuclei and mitochondria were incorporated into the sacculations before the release of mature merozoites into the host cell cytoplasm. In the last stage of schizogony, following massive merozoite formation and reduction in size of the schizont, residual nuclei divided by multiple fission. Each nuclear division became incorporated into a developing merozoite having preformed rhoptries, mitochondria, and a polar ring.

Lesions in the liver and kidney of Dirofilaria immitis-infected dogs following treatment with ivermectin.

Six dogs with spontaneous heartworm disease were injected with a single dose of ivermectin. After 48 h of treatment, microfilariae counts were reduced by 92%-98% of pretreatment counts. In pretreatment biopsies examined by light and electron microscopy, microfilariae were unaltered in the sinusoids of the liver and also in the glomerular capillaries and interstitial blood vessels of the kidney. However, there was irregular thickening and dense deposits in the basement membranes of glomerular capillaries, along with a modest increase in mesangial cells and matrix. In post-treatment liver biopsies examined by light microscopy, there were numerous granulomas in the sinusoids which contained degenerated microfilariae. In post-treatment kidney biopsies there was moderate thickening of glomerular basement membranes along with pronounced proliferation of mesangial cells and matrix. Glomerular capillaries were partially or completely occluded by degenerated microfilariae. In addition, there were interstitial granulomas in the kidney. It was observed with the aid of electron microscopy that highly vacuolated and degenerated microfilariae were incorporated into granulomas in the liver sinusoids of post-treatment biopsies. In post-treatment kidney biopsies glomerular capillaries were usually occluded by degenerated microfilariae. Basement membranes were thickened and contained dense deposits. Mesangial cells and matrix were extensively increased. Interstitial granulomas in the kidney contained dead microfilariae.

Pathologic sideroblasts and siderocytes associated with chloramphenicol therapy in a dog.

Siderotic granules were recognized in blood erythrocytes from a male Boxer dog with suppurative prostatitis, cystitis and pyelonephritis that was being given high dosage chloramphenicol therapy. Siderotic inclusions were recognized in the cytoplasm of 96% of the rubricytes and metarubricytes in a bone marrow aspirate. Siderotic inclusions were numerous and in some cases formed a ring around the nucleus. This perinuclear location suggested that pathologic mitochondrial iron accumulation had occurred, resulting in the formation of "ringed" sideroblasts. The occurrence of pathologic sideroblasts was confirmed by electron microscopy. Blood siderocytes and bone marrow sideroblasts disappeared after cessation of chloramphenicol therapy.

Cartilaginous metaplasia of the thoracic aorta of control turkeys and exacerbation by beta-aminopropionitrile.

Seventeen of sixty distal extremities of the thoracic aortas of 12-week-old control male turkeys and 37 of 40 distal extremities of the aortas of turkeys fed 0.07% beta-aminopropionitrile (BAPN) from 4 to 12 weeks of age contained areas of cartilaginous metaplasia when examined by light microscopy. The cartilaginous areas were generally elongated and located in the subendothelium of control turkeys, but a roundish area of cartilage was occasionally evident in the deep media. The magnitude of chondroplasia was enhanced by feeding BAPN; the extensive lesion usually extended from the subendothelium to deep in the media. Regardless of treatment, chondrocytes were pleomorphic, contained vacuoles, and had cytoplasmic processes. The cells were separated by pools of proteoglycans and connective tissue. The ultrastructure of chondrocytes in the aortas of both treatment groups was typical of this cell type. They had undulations or projections of the cell membranes. The cisternae of endoplasmic reticulum were dilated and contained electron-translucent material which was similar to extracellular proteoglycans. Golgi apparatus, free ribosomes, mitochondria, glycogen granules, filaments, and a centriole also were present in the cytoplasm. The extracellular matrix, which included collagenous and elastic fibers and also delicate fibrils and interconnecting matrix granules, separated adjacent chondrocytes by spaces of varying size.

Tetrameres columbicola (Nematoda: Spiruridae) infection of pigeons: ultrastructure of the gravid female in glands of the proventriculus.

The light and electron microscopies of Tetrameres columbicola gravid females in sections of the parasitized proventriculus of pigeons were studied. By light microscopy, the most conspicuous structures in the sectioned parasite were the intestine, ovary, and especially the uterus that contained numerous eggs. By electron microscopy, there was a thick mat of pigment-coated microvilli on the surfaces of the intestinal epithelial cells. The germinal zone of the ovary contained nonmembrane-bound oocytes, but oocytes were confined by a membrane in the growth zone of the ovary. The core of the spermatheca contained oocytes and the periphery harbored sperm. In this location, the unfertilized oocyte had pseudopods; sperm had invaginations of the plasma membrane. After fertilization, there was proliferation of ribosomes within the oocyte. Embryonating eggs in the uterus had thick shells and were partially enveloped by elongations of the uterine epithelial cells. Surfaces of the epithelial cells were pleated and they had electron opaque areas at the points of the pleats. Larvae in eggs had a well developed annulated cuticle and muscular layer. Somatic muscle cells had tailed appendages that protruded into the pseudocoelom. The single layer of cells beneath the hypodermis had lateral processes at the base of the cells that interdigitated with similarly elongated processes of adjacent muscle cells. Striated fibers were present in the central portion of the cells.

A unique connective tissue structure in the aortic media of the chicken.

The interstitium between smooth muscle cells in the media of the abdominal aorta of the chicken contains basement membranes, glycosaminoglycan, stout elastic fibers, extensive bundles of collagenous fibers, and a unique striated structure. In cross section, this striated, hexagonal structure resembles a honeycomb, each hexagon consisting of 6 isosceles triangles. Microtubule-like structures are present at each corner and center of a hexagon, and 3 delicate filaments are located equidistantly between putative microtubules. The periodicity evident in longitudinal section is the result of a constant repetition of microtubule-like elements. From staining with phosphotungstic acid it appears that the striated connective tissue structures are proteinacous and might serve as a reinforcing structure where smooth muscle cells are separated by dilated extracellular spaces.

Acute experimentally induced aflatoxicosis in the weanling pony.

Nineteen weanling ponies and 1 adult pony were given a single oral dose of aflatoxin B1 (AFB1). Dosages were: 0, 0.5, 1, 2, 4, 5, 6, and 7.4 mg of AFB1/kg of body weight. Vital signs were monitored, and whole blood and serum collected for analysis of serum enzymes, prothrombin time, blood cell counts, and serum urea nitrogen. Ponies that died were examined for gross lesions, and tissues were collected for histopathologic examination and analysis of AFB1 and AFM1 residues. Two of the 4 ponies given the 2 mg/kg dose and all ponies given the larger dosages died within 76 hours. Clinical signs included increased rectal temperature, faster heart and respiratory rates, abdominal straining, bloody feces, and tetanic convulsions. At necropsy, ponies that died of acute aflatoxicosis showed visceral petechiae and hepatic focal lesions. Histopathologic changes included severe hepatic necrosis, vacuolation, and bile duct hyperplasia. Aflatoxins B1 and M1 were recovered from liver, kidney, skeletal muscle, and gastrointestinal contents. One other pony given the 2 mg/kg dose died 32 days after dosing, and 1 control pony died after 70 days. Continuous elevations in prothrombin time and serum aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase levels were observed in ponies dosed at 4 mg/kg or more. Significant (P less than 0.05) elevations in these values, which peaked 2 to 3 days after dosing, were seen in ponies given the 2 mg/kg dose. This group also had significant increases over controls in PCV and hemoglobin concentration 5 days after dosing.

Modification of the effects of aflatoxin B1 and warfarin in young pigs given selenium.

Selenium may be related to the hepatic metabolism of the coumarin compounds aflatoxin B1 and warfarin. Selenium evidently increased the pharmacologic activity of warfarin, probably due to a displacement of warfarin from albumin by selenium, the close relationship among selenium, vitamin E, and sulphur-containing groups (eg, glutathione), or the antioxidant effect of selenium. A diet containing selenium in a concentration of 2.5 mg/kg of feed was protective against the toxic effects of both coumarins in pigs given 4 daily oral doses of 0.2 mg/kg of body weight. Selenium, as glutathione peroxidase, at least in part, protects the hepatic cells against the toxic effects of aflatoxin B1 and warfarin. The protection was demonstrated by alteration of clinical responses and hematologic (prothrombin times), electrophoretic, and clinical chemistry values. It also was demonstrated that selenium at 2.5 mg/kg of feed does not produce toxic effects; however, dietary selenium at a concentration of 5 mg/kg (and in the presence of both toxic agents) was toxic for young pigs within the 3-week experimental period. Warfarin was more active as an anticoagulant than aflatoxin B1.

Effect of high levels of Larvadex on reproduction in Leghorn breeders.

The insect growth inhibitor, Larvadex, was fed to egg type breeder hens for 8 weeks at levels of 0, 50, 100, 500, 1000, and 2000 ppm and to the same strain of males at 0 and 2000 ppm. All birds were kept in individual cages. Fertility was determined following artificial insemination. Egg production was significantly increased in Experiment 1 but numerically decreased in Experiment 2 by feeding 1000 ppm Larvadex. Feeding 2000 ppm significantly decreased egg production in both experiments. Egg weight was highest at 100 ppm and decreased with higher treatment levels. Specific gravity of eggs was improved with all Larvadex treatments. Fertility was not affected by treatment of females or males. Hatchability was reduced by the 1000 and 2000 ppm levels in the hen's diet. Semen quality was not significantly affected by feeding Larvadex at 2000 ppm.