Swine Liver Research Articles

Changes in Iron Quantity and Taste Variance Resulting from Swine Liver Cooking

Changes in Iron Quantity and Taste Variance Resulting from Swine Liver Cooking

Hepatic Apoptotic Activity Following Transient Normothermic Inflow Occlusion and Reperfusion in the Swine Model

Accelerated hepatic apoptosis was first described in portal vein-ligated livers but has since been reported in a variety of liver injuries. Because porto-prival states can induce apoptosis we sought to determine whether transient ischemic periods followed by reperfusion would trigger such cell death. The cytokines TNF-α and TGF-β are known to facilitate apoptosis and are released in response to a number of stimuli including ischemia. We also investigated alterations in plasma and tissue levels of these cytokines which might lend support to their role in increased apoptotic activity following ischemia/reperfusion. Female pigs were used as the experimental model. Inflow occlusion of portal and hepatic arterial blood was performed to a portion of the swine liver directing the entire splanchnic flow to the remaining hepatic lobes for a period of 2 h. The livers were then reperfused and plasma and tissue samples taken for determination of apoptotic activity utilizing cell death immunoperoxidase staining of 3′-OH DNA ends generated by fragmentation and ELISA assay of histone-associated DNA fragments. Plasma and tissue levels of TNF-α and plasma levels of TGF-β were determined by ELISA assay. An increase in apoptotic activity following reperfusion was seen at Day 2 and Day 4 compared to preischemic values by the cell death stain. The ELISA cell death assay showed an increase in apoptotic activity at 60 min, Day 2, and Day 4. Moreover, the ELISA cell death assay showed enhanced apoptotic activity in “hyperperfused” hepatic lobes compared to preischemic, or resting, liver. This was also observed when compared to sham-operated animals. Surprisingly, there was no detectable increase in plasma TNF-α or TGF-β levels following ischemia/reperfusion, although homogenized liver TNF-α levels were increased at 60 min and Day 2 following reperfusion. We conclude that transient hepatic inflow occlusion followed by reperfusion can induce increased apoptotic activity in the swine model. Furthermore, increased apoptotic activity also occurs in the hyperperfused liver raising the possibility of a locally active factor or global hepatic expression of receptor activity in response to ischemia/reperfusion. This period of ischemia/reperfusion did not produce a detectable increase in circulating cytokine levels, and accelerated apoptosis could not be linked to heightened TNF-α or TGF-β plasma activity. Higher tissue levels of TNF-α could reflect enhanced binding to TNF cell surface receptors or amplified receptor expression.

Confirmation of Azaperone and Its Metabolically Reduced Form, Azaperol, in Swine Liver by Gas Chromatography/Mass Spectrometry

The method described confirms the use of the tranquilizer azaperone by detecting the parent compound and the metabolically reduced form, azaperol. Both are confirmed in swine liver at a target concentration of 10 ppb by gas chromatography/mass spectrometry (GC/MS) with electron ionization in the selected-ion-monitoring mode. Swine liver tissue is ground with dry ice. Acetonitrile is added to extract the drug from the tissue. Sodium chloride buffer is added to the extract in preparation for solid-phase extraction (SPE). The aqueous extract is loaded onto an SPE cartridge designed to extract acidic and neutral drug residues from biological matrixes. The cartridge is washed with methanol and conditioned with sodium phosphate buffer. Azaperone and azaperol residues are eluted with a 2% ammonium hydroxide in ethyl acetate. The extracts are evaporated to dryness under a stream of nitrogen and reconstituted in ethyl acetate for GC/MS analysis. A DB-1 analytical column is used to separate the compounds prior to electron ionization. The parent ion, the base peak ion, and one diagnostic fragment ion are monitored for both compounds. The method was validated with fortified tissue samples containing both azaperone and azaperol. Azaperone-incurred tissues also were analyzed, and the presence of the parent drug and the metabolically reduced form, azaperol, was confirmed.

Disruptive forces for swine heart, liver, and spleen: their breaking stresses.

The mechanical strengths of the swine heart, liver, and spleen were determined by measuring the force that initiated the rupture of the organs. The force was loaded by the compression of a detection bar onto the organ, and the breaking stress was calculated by dividing the force by the bar's contact area. The forces were measured with five measurements in five of each type of organ with and without the serous membrane (SM). The breaking stresses of the heart were 14.1+/-0.7 kg/cm2 (mean+/-SEM) with intact epicardium and 8.1+/-0.6 without it; those of the spleen were 9.0+/-0.9 with its capsule and 1.8+/-0.1 without the capsule, and those of the liver were 4.6+/-0.3 with and 3.6+/-0.3 without the SM. A three-way layout analysis of variance showed that the organs, their SM and individual swine were independent determinants of the breaking stresses. With the intact SM, the heart was strongest and the liver was weakest. When the SM was not present, the spleen lost 80% of its strength, the liver 22% of its strength, and the heart lost 43% of its strength.

Immunoaffinity column cleanup procedure for analysis of ivermectin in swine liver

A simple and sensitive method for the analysis of ivermectin (22,23-dihydroavermectin B 1) in swine liver based on immunoaffinity column cleanup is described. The immunosorbent was prepared by coupling polyclonal anti-ivermectin antibodies to carbonyl diimidazole-activated Sepharose CL-4B. After extraction with methanol, ivermectin was cleaned up on an immunoaffinity column, and determined by reversed-phase liquid chromatography with UV absorbance detection at 245 nm. Recoveries of ivermectin from fortified samples of 5–100 μg kg −1 levels ranged 85–102%, with coefficients of variation of 6–12%. The limit of detection was 2 μg kg −1 in a 5-g sample.

Ex VivoExperiment on Radiofrequency Liver Ablation with Saline Infusion through a Screw-Tip Cannulated Electrode

Purpose: To investigate whether radiofrequency (RF) therapy with hypertonic saline infusion through a hollow screw-tip electrode can cause a lesion size suitable for liver tumor ablation. Materials and methods: RF tissue ablation of 180 sites was performed by using a hollow screw-tip electrode in 40 freshly excised swine livers. Under both power and temperature control modes, the ablation effects with and without various regimes of 5% hypertonic saline (1 ml/min) prior to and/or during the procedure were compared by measuring the size of lesions at dissection and confirmed by T1 and T2 weighted magnetic resonance (MR) imaging. Results: The maximal lesion diameter of 5.5 cm was reached at 30 W with saline infusion 1 min prior to and during 12 min of ablation. The smaller sizes (P< 0.01) between 0.3 and 2.5 cm in diameter were met with noninfusion or preinfusion-only groups. The RF ablation lesions appeared as hyper- and hypointense areas on T1 and T2 MR images, respectively. Conclusions: RF ablation in combination with present hollow screw-tip electrode and saline infusion allows for necrotic development of suitable size for liver tumor ablation. Such ablated lesions can be visualized with MR imaging.

Distribution of tritiated dihydromicrocystin in swine

The distribution of tritiated dihydromicrocystin i [ 3H]2H-MCLR was studied in anesthetized specific-pathogen-free pig. Two doses were administered i.m. and one dose was given via an isolated ileal loop. At 4 hr after i.v. administration of the toxin at 25 μg/kg, 64.6% of the total dose (% TD) was located in the liver, with small amounts distributed to the kidneys (1.2% TD), lungs (1.75% TD), heart (0.22% TD), ileum (0.13% TD) and spleen (0.04% TD). A similar distribution was found at 4 hr postdosing in pigs given 75 μg/kg, although the liver contained a lower fraction of the total dose, at 46.99% TD, and the kidneys had somewhat more, at 2.19% TD, than the low dose. At the high dose, the fractions of the amount given accounted for by the lungs (0.55% TD), heart (0.23% TD), ileum (0.20% TD) and spleen (0.07% TD) were similar to those at the low dose. The livers of the pigs given 75 μg/kg via the ileal loop, at 5 hr postdosing, contained 49.5% TD and the ileum had 33.94% TD. Smaller amounts were distributed to kidneys (1.04% TD), lungs (0.65% TD), heart (0.81% TD) and spleen (0.16% TD). The livers of both groups dose at 75 μg/kg contained higher concentrations of toxin, but lower percentages of the total dose, than the livers of pigs dosed at 25 μg/kg. Larger increases in serum arginase in the two 75 μg/kg groups were associated with histological evidence of more severe liver damage than at the 25 μg/kg dose. Analysis of radiolabelled compounds from hepatic tissue using fast atom bombardment mass spectrometry determined that the primary constituent was [ 3H]2H-MCLR, but two minor radioactive components were also isolated. These findings indicate that [ 3H]2H-MCLR is rapidly concentrated in the liver of swine, whether given i.v. or via an isolated ileal loop, that at extremely toxic doses uptake is slowed, and that it is as toxicologically active as the parent compound.

Hemodynamic analysis of post-reperfusion syndrom and the effect of prevention this syndrome using thromboxane A 2 synthetase inhibitor (OKY-046) in swine liver transplantation

Hemodynamic analysis of post-reperfusion syndrom and the effect of prevention this syndrome using thromboxane A 2 synthetase inhibitor (OKY-046) in swine liver transplantation

Quinoxaline-2-carboxylic acid (QCA) in swine liver and muscle.

The concentration of quinoxaline-2-carboxylic acid (QCA) determined by HPLC after alkaline hydrolysis of liver and muscle of swine, ranged from < 3 ng/g to 45.3 ng/g in liver, and from < 3 ng/g to 10.8 ng/g in muscle samples. After the 77th day of therapy QCA was found in samples of liver (9.7 ng/g). Recoveries obtained for both liver and muscle were 70% at 5 ng/g, 77% and 75% respectively at 10 ng/g, and 90% for both liver and muscle at 30 ng/g. This experiment was performed within the frame of the National Monitoring Programme of Residues in Animal Tissues in the Republic of Croatia.

THE CARDIOPULMONARY, EICOSANOID, AND TISSUE MICROANATOMIC EFFECTS OF FLUCONAZOLE DURING GRADED BACTEREMIA

Imidazole compounds have been shown to be beneficial in systemic sepsis and inflammation. The purpose of this study was to delineate the effects of fluconazole on systemic hemodynamics and on microanatomy of the heart, lung, liver, and kidney parenchyma of swine during graded bacteremia. Eighteen adult swine were studied in three groups: 1), anesthesia control; 2), septic control (Aeromonas hydrophila, 10(9)/mL, infused i.v. for 4 h); 3) fluconazole (fluconazole, 30 mg/kg i.v., followed by A. hydrophila infusion). After 4 h of graded bacteremia, autopsy was performed. Compared with the septic control group, cardiac index, oxygen delivery, and oxygen consumption were reduced significantly after fluconazole pretreatment, and mixed venous hemoglobin oxygen saturation (SVO2) and oxygen extraction were increased. Plasma thromboxane A2 and leukotriene levels were not affected by fluconazole. Computerized digital image analysis of the liver, heart, and kidney specimens revealed no statistically significant differences between the septic control group and fluconazole-pretreated animals. In the lung specimens, preinfusion of fluconazole decreased alveolar wall thickness in septic swine (anesthesia control group: 8.15 x 10(-3) +/- 1.3 x 10(-3)mm versus septic control group: 9.9 x 10(-3) +/- 1.3 x 10(-4) versus fluconazole group: 6.8 x 10(-3) +/- 1.6 x 10(-3); p < or = .05). Fluconazole pretreatment before graded bacteremia has no beneficial effect on cardiopulmonary performance or septic tissue edema of the heart, kidney, or liver. Tissue oxygen metabolism might be down-regulated by fluconazole. However, preinfusion of fluconazole appears to normalize the sepsis-induced increase in pulmonary alveolar wall thickness. The net significance of these changes on clinical outcome is not clear from these data.

Depletion and bioavailability of 14C-oxibendazole residues in swine tissues

Groups of male swine were administered a single oral dose of 14C-oxibendazole in a gelatin capsule at a level of 15 mg kg −1 bodyweight and killed after 10 h, 24h, and 7 days withdrawal. Combustion analysis indicated that liver was the only tissue which contained significant residues. Owing to the insolubility of oxibendazole, absorption was variable, especially at the early time points. Total residues were highest after 24h withdrawal (4 ppm) and depleted relatively slowly to approximately 1.8 ppm after 7 days. Extractable residues decreased from 35% to 11% over this same period. When 14C-oxibendazole was fortified into control, lyophilized, pelleted swine liver and fed to rats utilizing the Gallo-Torres model, bioavailability was greater than 94%. In contrast, bioavailability was substantially reduced to approximately 40% when liver containing 14C-oxibendazole residues from dosed swine was fed. This percentage remained constant for liver samples from the 24h or 7 day withdrawal swine. The data were consistent with the rapid metabolism of oxibendazole and the early formation of a high percentage of bound tissue residues. The significant differences in residue bioavailability and extraction efficiency between the fortified control and dosed tissues indicate that little, if any, parent oxibendazole is present in the dosed tissues, even at early withdrawal times. As non-bioavailable residues are of no toxicological concern, the 60% reduction in bioavailability is significant from a regulatory perspective. The wider safety margin, relative to the established acceptable daily intake (ADI) for oxibendazole, is beneficial to the consumer in terms of human food safety.

Determination of Carbadox and Its Metabolite, Quinoxaline-2-carboxylic Acid, in Swine Tissues by HPLC

A simple and rapid method for the simultaneous determination of carbadox (CDX) and quinoxaline-2-carboxylic acid (QCA), the most persistent metabolite, in swine tissues has been developed by HPLC with photodiode array detection. The drugs were extracted with 0.3% metaphosphoric acid-MeOH (7:3, v/v), and the extracts were cleaned up with a Bond Elut C18 cartridge. The HPLC separation was performed on a Wakosil-II 5C18 RS column (150×4.6mm i. d.) with a gradient system of 0.01M phosphate buffer (pH 2.5)-MeCN as the mobile phase, at the flow-rate of 0.6ml/min. The calibration graphs were rectilinear from 0.5 to 50ng for CDX and QCA. The recoveries of CDX and QCA from swine muscle and liver fortified at the level of 0.1μg/g were 85.0-94.3%, with high precision. The limits of detection were 0.005-0.01μg/g for CDX and QCA. Further, a preliminary investigation was carried out in order to determine the toxic metabolite desoxy-carbadox.

A facilitated technique for hepatectomy of porcine liver.

The swine liver has been used for experimental transplantation and as an ex vivo support system for patients with fulminant hepatic failure. Preservation of liver function after the cold storage period can be accomplished by complying with several key steps during the procurement: Keep the procedure short, provide extensive exposure, allow only minimal dissection, irrigate the portal system through the splenic vein and thereby obtain regular blood flow through the organ up to the clamping point, and ice the liver abruptly in situ. This retrieval technique was used and evaluated in 10 pigs. The method allowed a short and successful organ procurement while preserving normal function of the liver ex vivo.

Determination of Ractopamine Hydrochloride in Swine and Turkey Tissues by Liquid Chromatography with Coulometric Detection

A liquid chromatographic (LC) method is described for determination of ractopamine hydrochloride (LY031537) in swine and turkey tissues. Liver, kidney, muscle, and fat samples were homogenized in methanol. An aliquot of the extract was evaporated, diluted with water, and buffered to pH 10.5 +/- 0.5 with sodium carbonate to convert ractopamine hydrochloride to a free base. The free base was extracted from the buffered sample with ethyl acetate. The extract was further purified on a Bond Elut acid-washed silica solid-phase extraction cartridge. After converting the free base back to the salt form with pH 4.5 buffer, analytical separation and quantitation of ractopamine hydrochloride was performed on an IBM C18 column with coulometric detection at +600 mV. The limit of detection of the method was approximately 0.5 ppb as determined in swine liver. Overall recovery levels ranged between 75 and 100% for samples of liver, kidney, muscle, and fat fortified at 1 to 100 ppb. The coefficients of variation ranged from 2 to 18% for samples fortified at 1 to 100 ppb.

Determination of ceftiofur and its desfuroylceftiofur-related metabolites in swine tissues by high-performance liquid chromatography

An HPLC method was developed and validated for the determination of ceftiofur-related metabolites that have the potential to be microbiologically active in swine muscle, kidney, liver and fat. Its performance was evaluated against incurred-residue swine tissues. This method is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their conjugate sulfur containing moiety using dithioerythritol to yield desfuroylceftiofur, and further stabilization to desfuroylceftiofur acetamide. The limit of quantitation was 0.1 μg ceftiofur equivalents/g tissue. The assay is specific for ceftiofur-related metabolites when evaluated against commercially available antibiotics for swine.

Production and metabolism of dopamine and norepinephrine in mesenteric organs and liver of swine

Concentrations of catecholamines and their metabolites in plasma entering and exiting mesenteric organs, liver, and kidneys were examined to assess the regional production and metabolism of catecholamines in anesthetized swine. Higher portal venous than arterial plasma concentrations of norepinephrine and its metabolites, 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), and of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), indicated substantial production of both catecholamines by mesenteric organs, representing 45-50% of total body production. Most dopamine was not metabolized to norepinephrine, suggesting a large nonnoradrenergic contribution to total body dopamine production. Concentrations of norepinephrine, DHPG, normetanephrine, and MHPG were lower in plasma exiting than entering the liver, whereas concentrations of the end-product of norepinephrine metabolism, vanillylmandelic acid (VMA), were higher in outflowing than inflowing plasma. Over 94% of the VMA removed by the kidneys was produced in the liver, consistent with the hepatic source of this metabolite. Removal of DOPAC and HVA by the liver exceeded removal by the kidneys or production by mesenteric organs, indicating greater hepatic than renal elimination of these metabolites from the body and underestimation of mesenteric organ dopamine production based on summed spillovers of dopamine metabolites from the liver into the systemic circulation.

Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts

Preservation injury to bile ducts is a serious problem in liver transplantation, especially when preservation exceeds 12 hours. The authors hypothesized that the injury was caused by contact of bile ducts with bile salts during cold preservation and might be preventable by infusion of more hydrophilic bile salts. Swine livers were harvested after intraportal infusions of saline (control), of the hydrophobic bile salt taurodeoxycholate, or of the hydrophilic bile salts tauroursodeoxycholate or dehydrocholate. The effect of infusing a combination of hydrophilic and hydrophobic bile acids was also studied. Bile samples were taken before and during the infusions. Then livers were perfused with UW solution, ducts were flushed retrograde with UW, and livers were stored at 0 to 1°C for 20 hours. Bile ducts were harvested after preservation, and coded microscopic slides of the specimens were examined by light microscopy. There was large variability in baseline bile salt concentration. Injury after preservation consisted of sloughing and pyknosis of surface and glandular epithelium. The histologic injury score determined after preservation was directly related to bile salt concentration in bile ducts at the time of flushing. During bile salt infusions, the infused bile salt replaced most or all of the other bile salts present in bile. Severe postpreservation injury of intrahepatic ducts occurred after taurodeoxycholate infusions, but injury was minimal when either of the two hydrophilic bile salts was infused. The mixture of bile acids produced intermediate results. Retrograde flushing with UW does not prevent injury to intrahepatic ducts. The authors conclude that the injury is caused by contact with bile salts, is dependent on bile salt concentration and composition, and is preventable.

Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts

Preservation injury to bile ducts is a serious problem in liver transplantation, especially when preservation exceeds 12 hours. The authors hypothesized that the injury was caused by contact of bile ducts with bile salts during cold preservation and might be preventable by infusion of more hydrophilic bile salts. Swine livers were harvested after intraportal infusions of saline (control), of the hydrophobic bile salt taurodeoxycholate, or of the hydrophilic bile salts tauroursodeoxycholate or dehydrocholate. The effect of infusing a combination of hydrophilic and hydrophobic bile acids was also studied. Bile samples were taken before and during the infusions. Then livers were perfused with UW solution, ducts were flushed retrograde with UW, and livers were stored at 0 to 1 degree C for 20 hours. Bile ducts were harvested after preservation, and coded microscopic slides of the specimens were examined by light microscopy. There was large variability in baseline bile salt concentration. Injury after preservation consisted of sloughing and pyknosis of surface and glandular epithelium. The histologic injury score determined after preservation was directly related to bile salt concentration in bile ducts at the time of flushing. During bile salt infusions, the infused bile salt replaced most or all of the other bile salts present in bile. Severe postpreservation injury of intrahepatic ducts occurred after taurodeoxycholate infusions, but injury was minimal when either of the two hydrophilic bile salts was infused. The mixture of bile acids produced intermediate results. Retrograde flushing with UW does not prevent injury to intrahepatic ducts. The authors conclude that the injury is caused by contact with bile salts, is dependent on bile salt concentration and composition, and is preventable.

Analysis of tilmicosin in swine liver extracts by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

Tilmicosin is a semisynthetic macrolide antibiotic used in the treatment of respiratory disease in cattle and swine. The technique of liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) was employed in an on-line capacity for the analysis of tilmicosin in extracts from spiked swine liver. Increasing the potential in the cone/skimmer region of the ion source resulted in an increased abundance of unique structurally indicating fragment ions of tilmicosin. Three ions, [M+H]+ and two diagnostic fragment ions were chosen for confirmation of the presence of tilmicosin in swine liver tissue extracts using the mass spectrometer in selected-ion monitoring mode. The ion abundance ratios arising from any given combination of ions in data acquired from extracts of swine liver tissue spiked with tilmicosin at 5 and 10 ppm were within +/- 10% of the corresponding mean standard abundance ratio, and duplicate sample analyses exhibited < 10% relative standard deviation. These results suggest the potential for the application of LC/APCI-MS as a confirmatory technique for tilmicosin in swine liver.

Simultaneous Determination of Carbadox and Oxytetracycline in Swine Tissues by High Performance Liquid Chromatography

A simple, rapid and reliable high performance liquid chromatographic (HPLC) method for the simultaneous determination of carbadox and oxytetracycline in swine muscle and liver has been developed. The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on a Wakosil-II 5C18 RS column (150×4.6mm i. d.) with 0.05M sodium dihydrogenphosphate (pH 4.5)-acetonitrile (87:13) as the mobile phase at the flow-rate of 1.0ml/min. The drugs were detected by UV absorbance measurement at 360nm. The calibration graphs were rectilinear from 0.1 to 50ng for carbadox, and from 1 to 50ng for oxytetracycline. The recoveries of the drugs from swine muscle and liver fortified at the level of 0.1μg/g were 83.7-85.6%, with high precision. The limits of detection were 0.002μg/g for carbadox and 0.05μg/g for oxytetracycline in swine muscle. The accuracy and reproducibility of the present method were sufficient for residue analysis.