Laboratory serum biochemical tests are regarded by the Liver Study Group (LSG) of the WSAVA as an essential component of any liver investigation. The LSG categorised liver disease into four groups: vascular disorders, biliary disorders; parenchymal disorders and neoplasia. The laboratory tests that evaluate the liver have three categories. The cytosolic enzymes assess hepatocellular integrity; the cholestatic or inducible enzymes assess the biliary tree, liver excretory pathways and possible enzyme induction. The third category is the liver function tests which assess overall hepatic functional mass and portovascular integrity. The liver function tests commonly used include plasma ammonia concentration, serum bile acid concentrations and various tests that evaluate the uptake and conjugation of metabolites by the liver. Uric acid was once used as a liver test in the late 1950’s early 1960’s. Physiologically, uric acid is an attractive candidate for a liver function test. In most mammalian species serum uric acid levels only increase to the levels encountered in humans when there is hepatic dysfunction. Uric acid fell out of favour as a liver function test following the publication of two studies and one case report in the late 1950’s. The differences between hepatocellular integrity tests, cholestatic tests and tests of liver function were not fully understood at that time. The authors unfairly compared uric acid, essentially a liver function test, to a test of cholestasis. In addition the authors had very vague inclusion criteria for their liver disease cases. Despite the short-comings in these studies several prominent reference texts have since perpetuated their findings and uric acid fell out of the reckoning as a test of liver function. Many tests of liver function have been used over the years. Dynamic function tests have gained popularity again. Plasma ammonia concentration is a very reliable test of liver function but has very stringent sample-handling requirements which often make its application in the average clinic setting impractical. Serum bile acid concentrations, while not as sensitive or specific for portovascular shunting as ammonia, are widely regarded as the best test of overall liver function, especially with respect to non vascular-associated liver disease. However bile acid assays are not widely available in South Africa resulting in delays in turn-around times. In today’s climate of ever increasing costs, and demand for rapid turn around times, it would be very useful to veterinarians if a simple, rapid, cheap and robust assay could be found for evaluating functional hepatic mass. Uric acid would seem to have this potential and it is performed by most medical laboratories. In this study the serum uric acid concentrations and concentration of bile acids of a control group of normal dogs was used to compare to those in three other groups of dogs. Two of these groups had liver disease, and the third was a renal disease group. The one group of liver disease was comprised of dogs with congenital vascular anomalies while the second liver disease group was made up of dogs with various parenchymal liver diseases. Serum bile acid concentrations in the four groups were compared to the serum uric acid levels to assess the utility of uric acid as a test of liver function; and to measure the affects of diminished renal function on serum uric acid concentrations. There were significant differences in the serum bile acid concentrations between the two liver disease groups and the non-liver disease groups. Uric acid concentrations between all four groups did not differ significantly however. Serum uric acid was elevated in dogs with renal impairment. Therefore the findings in this study indicate that uric acid cannot be used as a test of liver function and is not comparable to serum bile acids in this regard. Copyright / Dissertation (MMedVet)--University of Pretoria, 2009. / Companion Animal Clinical Studies / unrestricted

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The liver is a multi-function organ that plays important roles in nutrient metabolism, biochemical transformations and blood detoxification. The purpose of the current work was to optimize Blood Oxygen Level Dependent (BOLD) liver functional MR imaging and analysis to allow the distinction between healthy volunteers and subjects with chronic liver disorders known to lead to fibrosis and reduced liver function (in this case, Hepatitis-C). Liver BOLD signal can be modulated by breathing 100% 0 2 or through intake of a meal. Previous results using these stimuli have been inconclusive when comparing healthy and diseased livers. In addition, liver BOLD analysis has been traditionally carried out using general linear models (GLM). Since the liver has a dual blood supply (portal and arterial derived), its resultant haemodynamic response is complex. This makes it too difficult to employ GLM approaches, as they require the prediction and modeling of a response function. We chose a model-free, or data-driven approach, called principle component analysis (PCA) to analyze liver data. Initial optimization was done by determining the time of maximal hepatic portal vein (HPV) blood flow following ingestion of a controlled meal (235 mL of Ensure Plus®). Statistically significant increases in HPV flow resulted at all measurement intervals, with the maximal postprandial change (71% increase in comparison to the baseline flow) at thirty minutes after ingestion. Implementing acquisition and analysis optimizations with our dual liver challenge model (hyperoxia cycling in pre- and postprandial states), the PCA approach was able to detect all of the diseased livers (n=6), while missing four of the healthy subjects (n=ll). The GLM technique, on the other hand, did not detect two of the patients and two of the healthy subjects. Thus, if this liver challenge is to be used as a screening tool, a model-free data analysis approach is suggested as more appropriate since it minimizes the chances of reporting false-negative results (based on this preliminary cohort). Although more false positives were detected with this method, it is of less concern seeing as these inaccuracies can be screened using simple blood tests. Promising results were obtained in this project, however, further studies using data-driven approaches such as partial least squares (PLS) are needed. / Thesis / Master of Science (MSc)

The metabolism of lignocaine to monoethylglycinexylidide has been found useful as an indicator of hepatic function in association with liver transplantation. It has been postulated that this might be due to the common effect of hypoxic damage on liver function and lignocaine metabolism. The aim of this work was to establish whether hepatic lignocaine elimination was impaired by hypoxia and whether lignocaine extraction ratio and clearance could be used as an indicator of hepatic function. This was studied using the isolated pig liver perfused via the hepatic artery and portal vein. To establish whether the pig liver could be used as a possible human model for this investigation and whether lignocaine had any detrimental effects on liver function and blood flow in vivo, hepatic lignocaine elimination and the effects of lignocaine administration on hepatic function and blood flow were studied in the anaesthetized pig, surgically prepared to allow sampling across the liver and direct hepatic blood flow measurement. Hepatic lignocaine elimination was then studied in the isolated perfused liver to determine whether this was similar to that found in vivo. The definitive studies required preliminary investigations not available from the literature to determine the feasibility of comparing in vivo and ex vivo hepatic function using the same liver. In addition, by studying the decay of lignocaine after bolus dose administration the necessary pharmacokinetic parameters to achieve similar constant hepatic affluent lignocaine concentrations in vivo and in the isolated preparation could be determined. The preliminary investigations showed that a sequential experiment using the same liver to compare in vivo and ex vivo function was inappropriate as the energy state of isolated perfused livers previously studied in vivo was significantly different from that in livers perfused immediately. The decay of lignocaine after a bolus dose in vivo and ex vivo could be described by a two-compartment open model and in both preparations the derived pharmacokinetic parameters from this analysis were used to achieve similar constant hepatic affluent concentrations over the study period used to determine hepatic lignocaine elimination. Lignocaine extraction ratio by the in situ pig liver was similar to that reported in man and together with hepatic clearance and intrinsic clearance was similar to that determined in the isolated state when different livers were used for this comparison. There was no detrimental effect of lignocaine administration on hepatic function and blood flow In vivo. Lignocaine extraction ratio and clearance and monoethylglycinexylidide formation were significantly impaired in livers subjected to hypoxia. Lignocaine elimination correlated strongly with hepatic cellular ATP, energy charge and ATP/ ADP ratio as well as with hepatic potassium release but less strongly with aspartate aminotransferase release when this relationship was tested using the combined data from hypoxic and normoxic livers ex vivo. These correlations were positive for hepatic adenine nucleotide status and negative for hepatic potassium and aspartate aminotransferase release. Neither hepatic alanine aminotransferase release nor lactate utilization were significantly affected by hypoxia. Lignocaine extraction ratio, hepatic oxygen consumption, ATP content, bile flow and potassium release were shown to be equivalent, more highly sensitive, and earlier indicators of hypoxic hepatic injury than hepatic aspartate aminotransferase release in the isolated perfused pig liver.

This study was to explore the relationships between personal exposure to 10 volatile organic compounds (VOCs) and biochemical liver tests with the application of canonical correlation analysis. Data from a subsample of the 1999-2000 National Health and Nutrition Examination Survey were used. Serum albumin, total bilirubin (TB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT) served as the outcome variables. Personal exposures to benzene, chloroform, ethylbenzene, tetrachloroethene, toluene, trichloroethene, o-xylene, m-,p-xylene, 1,4-dichlorobenzene, and methyl tert-butyl ether (MTBE) were assessed through the use of passive exposure monitors worn by study participants. The first two canonical correlations were 0.3218 and 0.2575, suggesting a positive correlation mainly between the six VOCs (benzene, ethylbenzene, toluene, o-xylene, m-,p-xylene, and MTBE) and the three biochemical liver tests (albumin, ALP, and GGT) and a positive correlation mainly between the two VOCs (1,4-dichlorobenzene and tetrachloroethene) and the two biochemical liver tests (LDH and TB). Subsequent multiple linear regressions show that exposure to benzene, toluene, or MTBE was associated with serum albumin, while exposure to tetrachloroethene was associated with LDH and total bilirubin. In conclusion, exposure to certain VOCs as a group or individually may influence certain biochemical liver test results in the general population.

Weanling rats, divided into 3 groups were maintained for 15 days on a diet containing either 5 per cent casein fed ad libitum (Group 1), 20 per cent casein pair-fed to Group 1 (Group 2), or 20 per cent casein fed ad libitum (Group 3). Animals were injected with either 0.9 percent saline or phenobarbital. Phenobarbital increased microsomal protein, cytochrome P-450, and phosphatidylcholine in all dietary groups; however, in all groups the increase in P-450 was greater than for phosphatidylcholine. Protein deficiency (Groups 1 vs. 2) decreased P-450 and microsomal protein but had no effect on phosphatidylcholine contents. The fraction of phosphatidylcholine-associated sites relative to the total sites was greater during protein deficiency and was in agreement with a greater Δ A_max per nmole P-450 for ethylmorphine. Phenobarbital induction decreases the proposed fraction of phosphatidylcholine-associated P-450 sites relative to the total P-450 sites and results in a decrease in the Δ A_max per nmole P-450 for ethylmorphine. Phenobarbital increased the Δ A_max per mg of microsomal protein for aniline, which paralleled the increase in total P-450 indicating that the Type II site is independent of any association Of cytochrome P-450 with phosphatidylcholine. The V_max per mg of microsomal protein was 64-66 per cent lower in the protein deficient group. Equivalent reductions of cytochrome P-450 and activities of cytochrome P-450 and c reductases were observed. Phenobarbital induction increased enzyme activities (V_max per mg of microsomal protein) in all groups with greater percentage increases in the protein deficient animals. The data suggested that phosphatidylcholine and cytochrome P-450 play important roles in the kinetics of metabolism and binding determined after protein deficiency and/or phenobarbital induction. The dietary study was repeated using 3-methylcholanthrene as inducer. 3-methylcholanthrene produced results similar to phenobarbital in most cases; however, this inducer illicits the production of a form of P-450 termed P-448. Induction of the various parameters appears more dependent on P-448 production than phosphatidylcholine-P-450 interactions. In contrast to phenobarbital treatment, 3-methylcholanthrene treatment does not increase the specific activity for ethylmorphine metabolism and the activity of cytochrome P-450 and c reductase. Other studies indicated the K_m and V_max for ethylmorphine N-demethylation is dependent on the substrate concentration range used to determine them. The oxidase system appears to have two different activities, one involving low substrate concentrations; the other, high substrate concentrations. During induction by both phenobarbital and 3-methylcholanthrene, there is a depression in both K_m and V_max during the first few hours after injection of the inducer which corresponds to the time period when the inducer is in the greatest concentration in the cell. After this initial depression, there was an increase in both K_m and V_max , indicating induction had taken place. / Ph. D.

Many lipophilic pesticides are known to be transferred to offspring via the mother's milk. The present study was conducted to determine how much endrin was transferred from endrin resistant and susceptible dams to their suckling pups and to further characterize the effects that endrin may have on the hepatic mixed function oxidase (MFO) system in the pups. Dosing of the dams with endrin began one day after birth with either (l) oral doses of endrin in corn oil or (2) a mixture of endrin in ground feed. The total amount of endrin in the pup was determined by gas chromatography. MFO activity was determined in 2-1/2 week old pups and for adult animals using maximal activities for the demethylation of ethylmorphine and hydroxylation of aniline. No difference in the amount of endrin present in the pups was observed between strains provided both received equal amounts of endrin. MFO activity for endrin dosed mature animals and for 2-1/2 week old pups from endrin dosed dams exhibited a significant decrease from control activities. There was no difference in MFO activities between age groups. A significantly higher ethylmorphine demethylase activity was observed in comparing the resistant to the susceptible strain but there was no significant difference in aniline hydroxylase activity. / Master of Science

Hydrogen sulfide (H2S), is a gasotransmitter with several key roles in metabolism and vascular function. The effects of H2S are dependent on concentration and target organ. For example, increased H2S concentrations impair liver metabolic function but protect against vascular dysfunction and atherosclerosis. Thiosulfate sulfurtransferase (TST), a nuclear encoded mitochondrial matrix enzyme, is proposed to be a component of the sulfide oxidising unit (SOU) which metabolises H2S. Preliminary data has shown that Tst deletion in mice (Tst-/-) increases circulating H2S levels measured in whole blood. Therefore, it was hypothesised that Tst-/- mice would exhibit worsened metabolic function in the liver but also protection of vascular function under conditions of vascular stress e.g. atherosclerosis. Liver metabolism was assessed by extensive metabolic phenotyping of Tst-/-mice fed control diet and in conditions of metabolic dysfunction induced by a high fat diet (HFD). Tst deletion altered glucose metabolism in mice; gluconeogenesis was increased in liver from Tst-/-mice fed control diet. Glucose intolerance in HFD-fed Tst-/-mice was also more severe than HFDfed C57BL/6 controls. In vitro metabolic investigations in primary hepatocytes isolated from Tst-/-mice demonstrated that mitochondrial ATP-linked and leak respiration were increased compared to controls. The effect of Tst deletion on vascular function was investigated in Tst- /-mice fed control or HFD using myography. Tst deletion did not alter vessel function when mice were maintained on a normal diet. HFD feeding (20 weeks) reduced maximal vessel constriction in the presence of endothelial nitric oxide synthase and cyclooxygenase inhibitors in C57BL/6 aorta. However, in Tst-/-mice fed HFD there was no reduction in maximal constriction suggesting a protective action of Tst deletion. The effects of Tst deletion on atherosclerotic lesions was investigated by generating double knock-out (DKO) mice by deletion of the Tst gene in ApoE-/- mice and (ApoE-/-Tst-/-). Atherosclerotic lesion formation was accelerated by feeding mice a western diet. Within the brachiocephalic branch lesion volume and total vessel volume were reduced in DKO mice fed western diet for 12 weeks, indicating that Tst deletion reduced lesion formation. Plasma cholesterol was reduced in DKO mice compared to ApoE-/- controls and a trend towards reduced systolic blood pressure was also noted. Overall this work supported the hypothesis that Tst deletion engenders metabolic dysfunction but vascular protection. The findings are consistent with the reported effects of increased H2S signalling. Overall inhibition of TST represents a novel target for treatment of atherosclerosis, with the caveat that glycaemia may be worsened due to hepatic metabolic dysfunction.

Under conditions of high self-regulatory effort, peripheral organ systems have been found to slow, potentially to rearrange energetic priorities in favor of the brain. The present study tested an expansion of this model by exploring the possibility that alcohol metabolism (i.e., liver function) may slow during self-regulation. We also anticipated that high trait self-control would attenuate the effect of condition on metabolism. Twelve males aged 21-25 completed two conditions in counterbalanced order. During each session, the participant received 0.33 ml/kg of absolute alcohol for a target peak blood alcohol concentration (BAC) of 0.03 g%. Participants then performed tasks (self-regulatory tasks in the high self-regulation condition and identical tasks without a self-regulatory component in the low self-regulation condition) and BAC was measured throughout. Although there was no main effect of condition, trait self-regulation moderated the effect of condition on alcohol metabolism such that only those with lower trait self-control had slower alcohol metabolism under high self-regulatory effort. These results provide support for the hypothesis that liver function may indeed be altered by self-regulatory effort. In addition to suggesting the liver as a target organ for psychophysiological research, these data provide further support for slowing of peripheral systems during high self-regulatory demand.