Swine Liver Research Articles

High Performance Liquid Chromatography Determination Following Microwave Assisted Extraction of 3-Nitro-4-Hydroxyphenylarsonic Acid from Swine Liver, Kidney, and Muscle

Abstract A simple and rapid analytical method is described for the determination of 3-nitro-4-hydroxyphenylarsonic acid (roxarsone, 3-nitro) in tissues. It involves extraction of 3-nitro from swine tissues by microwave-assisted process (MAP™) followed by high performance liquid chromatography using a PRP-1 column. The compound is detected in visible region at 410 nm after post-column reaction with 0.15 N sodium hydroxide. The detection limit is estimated to be 0.25 μg g−1 for 3-nitro, equivalent to about 0.2 μg g−1 of arsenic. Official tolerances for arsenic in liver and muscle are set at 2.0 μg g−1 and 0.5 μg g−1, respectively. The method was employed to detect 3-nitro residues in incurred tissue samples from pigs fed 150 mg kg−1 of 3-nitro for a 28-d period.

The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells.

Bone marrow transplantation has been shown to induce donor-specific tolerance in rodent models. This approach could potentially be applied to xenotransplantation across discordant species barriers. To evaluate host factors resisting hematopoietic cell engraftment, we have developed two model systems utilizing the combination of swine into severe combined immunodeficient (SCID) mice. SCID mice lack functional B and T lymphocytes, and can therefore be used to evaluate nonimmune factors resisting marrow engraftment, and for adoptive transfer studies to test the role of immune cells and antibodies. First we transplanted swine bone marrow cells into SCID mice conditioned with whole-body irradiation (4 Gy). For nine weeks following the intravenous administration of 10(8) swine bone marrow cells, up to 3.8% of peripheral blood leukocytes were of swine origin, as determined by flow cytometry (FCM). These cells were all of the myeloid lineage. Swine IgG was also detectable in the serum for up to 14 weeks. The bone marrow of the reconstituted mice contained low percentages of swine myeloid cells, and swine myeloid progenitors could be detected for up to 20 weeks after bone marrow transplantation. In a second model, we grafted thymus and liver tissue from 45-69-day-old swine fetuses under the kidney capsule of 4 Gy-irradiated SCID mice. A suspension containing 10(8) swine fetal liver cells (FLC) was also administered i.p. Long-term repopulation with swine T cells was observed, with up to 1.5% swine T cells detected in the WBC, peritoneum, and spleen for at least 5.5 months postgrafting. These T cells expressed either CD4 or CD8, whereas up to 17.6% of cells in the thymic grafts expressed both CD4 and CD8. The i.p. FLC suspension was required for optimal long-term graft maintenance. Our studies show that (1) low level myeloid and B lymphocyte reconstitution can be achieved by transferring adult swine BMC to irradiated SCID recipients; (2) swine myeloid progenitors were detectable long-term in BMC of these mice, suggesting that stem cell engraftment was achieved; and (3) T cell reconstitution of SCID mice by swine progenitors requires cotransplantation of a swine stromal environment, as is provided by fetal swine thymus/liver grafts. We conclude that nonimmune factors such as those provided by species-specific stromal environments are important for reconstitution of some lineages by discordant hematopoietic stem cells.

High Performance Liquid Chromatographic Determination of Sulfisomidine and N4-Acetylsulfisomidine in Swine Tissues

Abstract A method is described for the determination of the residual sulfisomidine and its metabolite, N4-acetylsulfisomidine, in swine muscle, kidney, liver and plasma by high performance liquid chromatography. Sulfisomidine and N4-acetylsulfisomidine were extracted from the swine tissues with acetonitrile, followed by alumina column clean-up procedure, and analyzed using a reversed-phase C18 column (250×4.6mm id), with UV detection at 270nm and a mobile phase of 10mM phosphate buffer (pH 5.6)-acetonitrile (92:8; v/v). The average recoveries of sulfisomidine and N4-acetylsulfisomidine from swine tissues fortified with 0.lμg/g were ranged from 83.2 to 91.3%, 88.6 to 99.5%, respectively. The detection limits were 0.01μg/g for each drug. The applicability of the method was demonstrated by determining concentration of sulfisomidine and metabolite in tissues from swine administrated with sulfisomidine.

Effects of hepatic artery embolization with Lipiodol and gelatin sponge particles on normal swine liver.

In order to evaluate the effects of hepatic artery embolization (HAE) with Lipiodol (Lp) and gelatin sponge particles (GSP) on swine liver tissue, we embolized the hepatic arteries of 3 pigs with GSP, 9 with Lp, and 9 with Lp + GSP. None of the pigs embolized with GSP died spontaneously during the 4-week experimental period. One pig embolized with 1 ml/kg Lp and three pigs with 2 ml/kg Lp died within 2 h. Two pigs embolized with 0.5 ml/kg Lp + GSP died the following day. The portal vein pressure (PVP) and wedge hepatic vein pressure (WHVP) values before HAE, immediately after, 1 h after, and 4 weeks after HAE showed no remarkable change in the GSP group. Remarkable and temporary elevation was observed in the more than 0.2 ml/kg Lp group. Remarkable and continuous elevation was observed in the 0.2 ml/kg Lp + GSP and 0.5 ml/kg Lp + GSP groups. Gross and histological studies demonstrated no liver damage in the GSP and Lp group. Liver infarction, circular coagulation necrosis with pseudocapsule, and liver atrophy were found in the Lp + GSP group 4 weeks after HAE. The incidence of infarction, circular coagulation necrosis, and liver atrophy in the livers embolized with 0.2 ml/kg Lp + GSP and 0.5 ml/kg Lp + GSP were 67%, 100%, and 75%, respectively. In conclusion, when the hepatic artery is embolized with Lp + GSP, the volume of Lp should be limited to less than 0.1 ml/kg.

Insulin-like growth-factor binding protein (IGFBP) serum levels and hepatic IGFBP-2 and -3 mRNA expression in diabetic and insulin-treated swine ( Sus scrofa)

1. 1. Diabetes had no significant effect on IGFBP-3 message and serum levels however, subsequent insulin treatment caused more than a two-fold increase in both hepatic IGFBP-3 mRNA and serum levels above controls ( P < 0.05). 2. 2. The induction of diabetes in pigs significantly increased the steady state levels of IGFBP-2 mRNA in the liver of young swine ( P < 0.05). 3. 3. Both liver message and serum IGFBP-2 were reduced to control levels with insulin therapy. 4. 4. We report here that in addition to its affects on IGFBP-2, insulin is involved in the regulation of IGFBP-3 expression.

Modified Method for the Determination of Ivermectin Residues in Animal Tissues

A simple, reliable method for determining residues of ivermectin in animal tissues is described combining existing sample cleanup procedures for muscle and for feed. Sample extracts are purified on alumina, C18, and silica solid-phase extraction cartridges, and ivermectin is determined by liquid chromatography with fluorescence detection. The average recovery of ivermectin from cattle, swine, and sheep muscle and liver was 87%, in the range of 5-25 ppb. Recovery from fish muscle was 77%, in the range of 5-30 ppb. The detection limit in tissue is 2 ppb.

Some toxic and essential trace metals in swine from Northern Poland

The concentrations of mercury, cadmium, lead, copper, manganese, zinc and iron have been determined in muscle (meat), liver and kidneys of swine slaughtered in the northern part of Poland between 1987 and 1991. The method of measurement was cold vapour flameless AAS for mercury and flame ASS for other trace metals. The concentrations of such toxic metals as mercury, lead and cadmium were relatively low or very low. The arithmetic mean weighted concentrations obtained related to fresh weight for muscle, liver and kidneys of swine were 1.9, 3.7 and 7.0 μg kg−1 for mercury, 5, 41 and 240 μg kg−1 for cadmium, 20, 94 and 120 μg kg−1 for lead, 1.1, 8.5 and 8.4 mg kg−1 for copper, 0.11, 1.3 and 1.3 mg kg−1 for manganese, 26, 50 and 30 mg kg−1 for zinc and 13, 54 and 63 mg kg−1 for iron, respectively. No definite time trends of mercury, cadmium and lead concentrations in swine muscle and edible organ meats during the period studied were observed, while for an essential trace metals analysed the values obtained seem to be typical. The results noted are reviewed and discussed with the data reported for muscle, liver and kidneys of swine in Poland from other studies.

Extension of a Liquid Chromatographic Method for N-Methylcarbamate Pesticides in Cattle, Swine, and Poultry Liver

Liver samples of bovine, swine, and poultry were fortified with 0, 5, 10, and 20 ppb mixed standards of an additional 6 carbamates as part of a method extension study. Each of the 3 species in this study was analyzed 4 times at 0, 5, 10, and 20 ppb fortification levels. The average of 12 recoveries of 6 carbamates at all 3 fortification levels was greater than 90%, with a maximum coefficient of variation less than 18%.

A High Omega 3 Fatty Acid Diet Alters Fatty Acid Composition of Heart, Liver, Kidney, Adipose Tissue and Skeletal Muscle in Swine

The fatty acid profiles and total lipid contents of two skeletal muscles, adipose tissue, heart, liver and kidney of swine fed a diet rich in omega 3 (n-3) fatty acids (i.e., 5% fish oil) was investigated. These values were compared to those determined for animals which were fed an equal caloric diet low in n-3 fatty acids (i.e., 5% coconut oil). All supplementations were given over a 13-week period. The lipids were extracted with chloroform-methanol, trans-esterified and the relative fatty acid methyl-esters concentrations were determined using capillary gas chromatography. The fish oil diet significantly enhanced the relative amounts of n-3 fatty acids (i.e., eicosapentaenoic acid and docosahexaenoic acid) in all tissues examined. In the heart, liver and kidney, the increases in n-3 fatty acids were compensated by decreases primarily in arachidonic acid, but in the other tissues the contents of lauric and myristic acids were also reduced. In general, the n-3 fatty acid contents were 40-165% higher in the animals fed the fish oil. Supplementation of n-3 fatty acids in swine induced a significant incorporation of these fatty acids throughout the body, however the extent of this incorporation differed between tissues perhaps due to tissue-specific metabolic pathways.

Breath ethane: A specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver

A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.

Prolongation of cardiac xenograft survival by double filtration plasmapheresis and ex vivo immunoadsorption.

A double filtration plasmapheresis (DFPP) technique and ex vivo immunoadsorption were applied to remove natural antibodies and avoid hyperacute rejection in discordant xenotransplantation. A swine heart was heterotopically transplanted into a dog's neck after DFPP and ex vivo immunoadsorption using a swine spleen or liver. Mean graft survival time was prolonged to 240 +/- 141 min in the group treated by combined DFPP and splenic adsorption, and 225 +/- 62 min in the group treated by combined DFPP and hepatic adsorption, whereas it was 9 +/- 5 min in the group without any treatment (p < 0.01). Mean removal rates of IgG and IgM were 85.5% and 93.3%, respectively. Anti-swine lymphocytotoxic antibodies and hemagglutination antibodies were also effectively removed. Deposits of canine IgM and C3 on the vascular endothelium of the graft were observed on immunofluorescence, which suggested that natural antibodies in the IgM fraction played an important role in xenohyperacute rejection.

The effect of exposure to a Δcya/ Δcrp mutant of Salmonella typhimurium on the subsequent colonization of swine by the wild-type parent strain

This study characterizes the clinical response and colonization pattern of caesarean-derived, colostrum-deprived swine exposed to a Δcya/ Δcrp mutant (χ4233) of S. typhimurium and challenged with the wild-type parent strain. χ4233 was mildly virulent in swine and induced transient fever and soft stools. χ4233 colonized the ileum, cecum, liver, spleen, tonsils, and mandibular and ileocolic lymph nodes of swine in a manner similar to the parental wild-type, but the numbers of S. typhimurium (χ4233) in the ileum were 100-to 1000-fold less than those of pigs exposed to the parental wild-type. Pigs exposed to χ4233 21 days before parental wild-type challenge demonstrated a milder clinical response to challenge than did pigs that did not receive χ4233. The wild-type populations in the ilea of χ4233-exposed pigs after challenge were 100- to 10,000-fold less than those in pigs not receiving χ4233. The liver, spleen, and ileocolic lymph nodes were cleared of wild-type S. typhimurium more quickly after challenge in χ4233-exposed pigs. The populations of χ4233 in the ilea of exposed pigs after wild-type challenge were also less than would have been expected in unchallenged pigs. Thus, exposure of swine to a Δcya/ Δcrp mutant of S. typhimurium modulated the subsequent response to parental wild-type challenge and reduced carrier populations of wild-type S. typhimurium in infected swine.

Determination and developmental changes in carbonic anhydrase III in swine liver

1. 1. CA-III was measured by enzyme-immunoassay in the livers of male and female swine aged from the fetus to 5 years old. 2. 2. Non sexual dimorphism in porcine liver could be detected at 6 months, but stag showed twice as much as swine of the same age. 3. 3. The concentration of Ca-III in the liver increased during development up to 6 months of age, followed by decline due to senescence.

Clostridium botulinum Type C in Healthy Swine in Japan

Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver. Liver specimens were cultivated in chopped meat-glucose medium and the cultures were examined for botulinum toxin. In cattle, none of the cultures of 100 liver specimens yielded the toxin. In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms. One of the five farms where the carrier-state swine were present was surveyed for about 2 years to determine whether the carrier-state was transient or resident. C. botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture. These data suggest that it is not uncommon for healthy swine to carry C. botulinum type C in the liver and that there is a close relationship between C. botulinum carrier-state in swine and the presence of this organism in their raising environments. In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C. botulinum type C was obtained.

An Analytical Survey of Aflatoxins in Tissues from Swine Grown in Regions Reporting 1988 Aflatoxin-Contaminated Corn

A joint project was undertaken by the Food Safety and Inspection Service (FSIS) and the Agriculture Research Service branches of the U.S. Department of Agriculture to determine the presence of aflatoxins in the U.S. meat supply during a drought year. In 1988, high incidences of aflatoxins occurred in corn grown in regions of the Midwest, Southeast, and South. Six states were identified as having serious aflatoxin contamination in their corn crop: Virginia, North and South Carolina, Texas, Iowa, and Illinois. Swine liver and pillars of diaphragm (muscle) tissues were sampled by federal FSIS Inspectors in plants located in these states. A worstcase sampling plan was conducted. Samples were taken in January 1989 from hogs fed corn soon after harvest and in April 1989 from hogs fed corn originally stored and then fed in the spring. A modification of the official AOAC method for the thin-layer chromatography (TLC) determination of aflatoxins in animal tissue was used to permit quantitation by LC with fluorescence detection. The official AOAC TLC confirmation of identity method was used to confirm all positive samples with B1 concentrations greater than 0.04 ppb and M1 concentrations greater than 0.1 ppb. Sixty samples in the January group and 100 samples in the April group were assayed. Concentrations of aflatoxins B1 and M1 in the first group of pig livers ranged from 0.04 to 0.06 ppb. The identity of aflatoxin B1 was confirmed in all positive samples. Aflatoxin M1 could not be confirmed in any of the positive liver samples because the method was insufficiently sensitive for this aflatoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppressive mechanism of 15-deoxyspergualin on sinusoidal lining cells in swine liver transplantation: Suppression of MHC class II antigens and interleukin-1 production

To elucidate the precise mechanism of action of 15-deoxyspergualin (DSG) in swine liver transplantation, the expression of MHC class II antigens (Ia) on hepatic sinusoidal lining cells (SLC) and their production of interleukin-1 (IL-1) were examined. In our previous study, we isolated sinusoidal endothelial cells (SEC) and Kupffer cells (KC) by enzymatic digestion and centrifugal elutriation, and demonstrated that both SEC and KC present alloantigens effectively and generated IL-1 in response to allogenic or lipopolysaccharide stimulation. Animals were divided into three groups: group 1, nontransplanted normal controls ( n = 3); group 2, no immunosuppressive treatment following liver transplantation ( n = 5); group 3, DSG (0.8 mg/kg/day) intravenously for 7 days following liver transplantation ( n = 5). At 1 week after transplantation, the three liver grafts in groups 2 and 3 were processed for the study of Ia expression and IL-1 production on SEC and KC. The expression of Ia was detected in 21.5 ± 4.7% of SEC and 24.3 ± 11.1% of KC in group 1. In group 3, Ia expression was suppressed compared with group 2, being 3.6 ± 2.8% versus 22.0 ± 2.8% for SEC ( P < 0.02) and 15.5 ± 11.3% versus 24.3 ± 7.1% for KC. IL-1 production by SEC and KC was respectively 11,483 ± 3311 cpm and 9077 ± 2161 cpm in group 1. In group 3, IL-1 production was inhibited compared with that in group 2, being 7190 ± 883 cpm versus 19.297 ± 5182 cpm for SEC ( P < 0.05) and 16,130 ± 3769 cpm versus 25,857 ± 3963 cpm for KC. In addition, we demonstrated histologically that DSG suppressed liver allograft rejection.

Effect of prostaglandin E 1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro

The effect of PGE 1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in huna, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1–6 μg protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE 1-concentrations (100 pM to 100 μM). In separate experiments the effect of PGE 1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p<0.001) lower binding capacity for 123I-LDL. PGE 1 significantly (p<0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and th binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE 1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to specific apo-B-receptors in a dose-dependent manner (p<0.01–0.001) with an ED 50 of 123±64 nM for human liver, 90±102 nM for rat liver obtained during anasthesia, 74±23 nM for rat liver obtained after decapotation and 941±121 nM for swine liver. In human liver iloprost (ED 50=876±53 nM) and PGI 2 (ED 50=52±12 nM) were less effective than PGE 1, PGE 2 had no effect on LDL-induced competition. It is concluded that PGE 1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE 1.

Determination of Carbadox-Related Residues in Swine Liver by Gas Chromatography/Mass Spectrometry with Ion Trap Detection

The ion trap detector (ITD), in combination with a capillary gas chromatograph and under chemical ionization conditions, offers sufficient sensitivity to determine carbadox-related residues as the methyl ester derivative of quinoxaline-2-carboxylic acid at 3 micrograms/kg or higher in porcine liver. A tetradeuterated internal standard of QME effectively compensates for losses incurred during sample preparation. The method produced mean levels of 3.3 (+/- 0.5), 5.5 (+/- 0.8), and 10.1 (+/- 0.9) micrograms/kg for liver fortified at 3, 5, and 10 micrograms/kg. When applied to analysis of samples containing incurred residues of 14C-carbadox at the low microgram/kg level, results were comparable to those obtained by reverse isotope dilution analysis.

Quantitation and Confirmation of Sulfamethazine Residues in Swine Muscle and Liver by LC and GC/MS

The present paper describes a liquid chromatographic (LC) method for purification of crude swine tissue extracts before gas chromatographic/mass spectrometric (GC/MS) quantitation and confirmation of sulfamethazine at low ppb levels. Fractions corresponding to sulfamethazine were collected, evaporated to dryness, N-methylated with diazomethane, concentrated, and analyzed by GC/MS. A mass spectrometer was set to selected ion monitoring (SIM) mode. Ions 233, 227, 228, and 92 m/z were detected. Ratio 227/233 m/z (sulfamethazine/internal standard, [phenyl 13C6] sulfamethazine) was used for quantitation, while ratios 228/227 and 92/227 m/z, respectively, were used for confirmation. Quantitation in spiked blank muscle tissue was tested from 100 to 1 ppb and found acceptable at all concentrations studied; coefficients of variations ranged from 4.9 to 14.4%. Similar results were obtained for liver tissue from 5 to 20 ppb; coefficients of variation ranged from 1.2 to 9.1%.

牛および豚の肝臓および腎臓におけるメチオニンスルホキシド還元酵素の性質と活性

反芻家畜のメチオニンスルホキシド(MSO)還元能は豚などの単胃動物よりも高いかもしれないとの仮説のもとに,屠場試料を用いて牛および豚の肝臓および腎臓におけるMSO還元活性を比較した.后性はMSOからのメチオニン(Met)生成量で表した.まず,各組織のMSO還元活性の存在を確認した後,それぞれのMSO還元酵素の性質を検討した.その結果,最適pH,最適温度,基質特異性は,各動物間および組織間に差が見られた,続いて,最適pHおよび39°C(生理的温度)の条件下で,また,還元剤として肝臓にはNADH,腎臓にはNADPH(各1mM)を添加し,最大活性が得られる基質濃度で,牛37頭および豚10頭の活性を測定した.その結果,nmolMet/h/g組織重で表すと,肝臓においては,牛(平均220)は豚(平均97)の2.3倍,腎臓においては,牛(平均586)は豚(平均269)の2.2倍の活性が見られ,肝臓および腎臓ともに,牛は豚よりも有意(P<0.001)に高い活性をもつことが分った.したがって,牛は,MSOをMetとして利用する能力が,豚よりも高いと考えられた.