The purpose of this study was to evaluate the effect of pyridoxine deficiency on lipogenesis in the rat. It is important in studies of this type to standardize not only the food intake but also the feeding pattern of the experimental and control animals. Pair-feeding of the control rats with the deficient ones imposes on the former animals a feeding pattern similar to meal-feeding. The latter pattern elicits several adaptive changes related to energy utilization by the rat. Therefore, an attempt was made to minimize the difference in the feeding frequency between the deprived and control groups by meal-feeding of the former group. The data were compared with those obtained when only food intake was controlled. Male weanling Wistar rats were used in the present studies. The deprived rats were allowed food either ad libitum (nibbling) or for 2 hours each day (meal-feeding). The appropriate controls were given a complete diet in quantities isocaloric with the consumption of the deprived groups. Decreased fat storage as well as feed efficiency in pyridoxine deficient rats were obvious at that time, regardless of the mode of feeding employed. The fatty acid content of the epididymal adipose tissue was affected in the same manner as body fat. Pyridoxine deprivation also suppressed total body fatty acid synthesis in vivo from glucose-U-¹⁴ C, whether the animals were meal-fed or nibbling. However, the rates of fatty acid synthesis in the epididymal adipose tissue of the meal-fed deprived rats tended to exceed those observed in the control. The lipogenic capacity of liver slices from fed nibbling deprived rats exceeded that of the controls, as evidenced by increased fatty acid labelling in the presence of glucose-U-¹⁴C or acetate-1-¹⁴ C. However, when the nibbling deprived rats were fasted and refed prior to sacrifice, the incorporation of acetate-1-¹⁴ C into fatty acids was lower than in the controls. No differences in the labelling of liver fatty acids and glyceride glycerol were observed when the meal-fed deprived rats were compared with their controls. However, pyridoxine deficiency in meal-fed rats was associated with a decrease in the capacity of the liver to oxidize glucose, as comparison with the controls revealed. Epididymal adipose tissue segments from nibbling deprived rats showed less incorporation of ¹⁴C from labelled glucose into CO₂, fatty acids and glyceride glycerol than those from the corresponding controls (expressed on the basis of the deoxyribonucleic acid content of the tissue). In contrast, increased lipogenic potential of adipose tissue preparation from the deprived meal-fed rats in the presence of insulin was observed. In these rats, a decrease in adipocyte size was suggested by lipid/DNA ratio lower than that of the controls. Thus, the increased lipogenic capacity observed was possibly due to a decrease in adipocyte size, itself associated with increased sensitivity to insulin. The activities of glucose-6-phosphate dehydrogenase and malic enzyme were depressed in both the liver and the adipose tissue of the pyridoxine-deprived rats. Since these enzymes are concerned with the production of the NADPH needed for reductive fatty acid synthesis, the results were consistent with the in vivo finding. However, the activities of these enzymes did not appear to limit the in vitro lipogenic potential of the tissues investigated, since NADPH produced from glucose-6-phosphate dehydrogenase and malic enzyme alone seemed to be sufficient to support the rates of lipogenesis seen. The alterations in lipogenesis in pyridoxine deprivation observed in the present and other investigation could not be explained on the basis of the known functions of the pyridoxine-dependent enzymes. / Land and Food Systems, Faculty of / Graduate

Pyridoxine-deficient rats are known to exhibit little, if any, weight gain; they also have decreased fat stores in comparison with their pair-fed controls. The defect in energy metabolism responsible for this phenomenon is not well understood at present. This study was undertaken to investigate some aspects of glycogenesis and glycogenolysis in order to add to the present information on energy metabolism in the pyridoxine deficiency state. Meal-fed animals were used, in order to eliminate differences due to the mode of feeding between the experimental and the pair-fed control animals. Male weanling rats were fed a pyridoxine-deficient diet in one 2-hour daily meal, while the controls were pair-fed. This eliminated differences due to feeding frequency when these groups were compared with each other. Aspartate amino-transferase and alanine aminotransferase activities were assayed in liver and erythrocytes in order to verify the presence of a pyridoxine deficiency state under the conditions used in this laboratory. The activities of glycogen phosphorylase, the rate-limiting enxyme in glycogenolysis, and glycogen UDP-glucosyltransferase were assayed in liver and muscle. Glycogen storage in these tissues was also measured. Finally, the incorporation of labelled carbon atoms into blood glucose and liver glycogen following intraperitoneal injection of L-alanine-¹⁴C was assayed. Glycogen phosphorylase activity was reduced in pyridoxine-deficient animals. This defect was not accompanied by a concomitant increase in the deposition of glycogen. There was, therefore, the possibility of a decreased ability to form glycogen. Glycogen UDP-glucosyltransferase activity was normal in muscle and elevated in liver indicating, if anything, an unimpaired ability to synethesize glycogen from UDPG. A trend towards a lesser incorporation of labelled carbon atoms into the blood glucose by the pyridoxine-deficient group appeared when the results were expressed as a percent of administered dose per ml. This became statistically significant when the data was expressed in terms of the circulating glucose pool. Although not at a statistically significant level, there was a greater incorporation of labelled carbon atoms into the liver glycogen of the pyridoxine-deficient group. It appeared from these findings that the defect in energy metabolism in pyridoxine deficiency may be the result of a reduced availability of carbon skeletons and occurred prior to the formation of glycogen. Further study in this area is necessary to reveal the exact point at which energy loss occurred. / Land and Food Systems, Faculty of / Graduate

Although the importance of tricarboxylic acid cycle activity in the metabolism of aerobic bacteria is well established, detailed studies on the utilization of intermediates of the cycle designed to assess the nature, importance and control of the enzymes concerned have not been performed with pseudomonads. Results of this investigation have shown that Pseudomonas aeruginosa ATCC 9027 lacks NAD or NADP linked L-malic dehydrogenase. Studies with cell fractions have shown that an NAD and NADP independent, particulate L-malic dehydrogenase catalyses the oxidation of L-malic acid to oxalacetic acid. The labelling patterns of citrate obtained from succinate-1,4-¹⁴ C and succinate-2,3-¹⁴ C have demonstrated the involvement of the particulate malic dehydrogenase and have excluded any other possibility. Phosphofructokinase could not be detected in the cell-free extract preparations and thus accounting for the non-functional Embden-Meyerhof pathway in this organism. Cells grown in succinate medium either do not have or have extremely low levels of glucose metabolizing enzymes. The glucose effect on tricarboxylic acid cycle enzymes was not observed. Further, the addition of α-keto- glutarate and glutamate to the medium did not repress these enzymes. These observations suggest that tricarboxylic acid cycle activity is of special importance for growth and metabolism in pseudomonads. The data have also indicated that during growth in a succinate medium, pentose synthesis must occur by the action of transketolase upon compounds derived from tricarboxylic acid cycle intermediates. It has been shown that both the glucose permease and the glucose metabolizing enzymes were induced simultaneously on shift from succinate to glucose medium. The glucose permease was found to be very specific since glucose uptake was not inhibited even in the presence of 100-fold excess of α-CH glucoside, 2-deoxyglucose, galactose, fructose or mannose. Particulate malic dehydrogenase was inhibited by adenine nucleotides while it was activated by GTP and GDP. The mechanism of this regulation is not clear, however, it is obviously important in the control of tricarboxylic acid cycle activity. Addition of glutamate to the medium repressed the synthesis of glutamic dehydrogenase. High levels of malic enzyme were maintained on growth in glucose and succinate media, whereas the levels were low in acetate medium. These observations demonstrated the capacity of the organism to regulate the synthesis of enzymes in response to the particular environment. Tricarboxylic acid cycle intermediates and glyoxylate have been found to exert "fine control" over the activities of isocitrate dehydrogenase and isocitrate lyase in such a way that flow of isocitrate through the tricarboxylic acid cycle and the glyoxylate cycle is precisely regulated to suit the needs of the cell. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The metabolites of the basic tertiary arylaliphatic amine N,N,α-trimethyl-7-phenylbenzenepropanamine (RecipavrinR) from male Wistar rats were characterized by gas chromatography-mass spectrometry (GCMS). The work was undertaken in an attempt to determine the source of a novel metabolite, N-(1-methyl-3,3-diphenylpropyl) formamide. The formamide metabolite was isolated from the bile of recipavrin dosed rats only after hydrolysis with the enzyme β-glucuronidase, suggesting that it arose from a glucuronide conjugated precursor. Recipavrin was chosen for the study based on structural similarity to the narcotic analgesic methadone which was shown to give rise to a similar metabolite, 6-formamido-4,4-diphenyl-3-heptanone. The secondary formamide was not a plausible candidate for a β-glucuronidase liberated metabolite of recipavrin, suggesting that a labile aglycone was responsible for the GCMS observation of the formamide metabolite. Labile isomeric compounds, α-methyl-(N-methylene)-7-phenylbenzenepropanamine N-oxide, N-(α-methyl-7-phenylbenzenpropylidene) methylamine N-oxide, and 2-(4',4'-diphenyl-but-2'-yl) oxaziridine were synthesized as possible precursors of the formamide. N-hydroxy-a-methyl-7-phenylbenzenepropanamine, and N-hydroxy-N,α-dimethyl-7-phenylbenzenepropanamine were synthesized as candidates for labile β-glucuronidase liberated aglycone precursors of the nitrones. The biliary nonconjugated and conjugated metabolites of recipavrin were characterized in detail. In addition to the formamide, 15 different metabolites representing the N- dealkylation, oxidative deamination, N-oxidation and phenyl ring oxidation pathways were identified by GCMS. To determine if thermal decomposition of the methylene nitrone in the GC inlet was responsible for the GCMS observation of the formamide metabolite, liquid chromatography-mass spectrometry (LCMS) was used to show that the formamide and not the isomeric methylene nitrone was present in bile prior to GCMS analysis. Although the synthetic methylene nitrone was shown to degrade in the GC inlet to the formamide, the LCMS experiment ruled out the thermal generation of the biliary formamide from a nitrone precursor. The nonconjugated and conjugated metabolites of the recipavrin metabolite, norrecipavrin were characterized in detail by GCMS. Since the secondary formamide metabolite was observed in the β-glucuronidase hydrolyzed bile extract, norrecipavrin was implicated as an intermediate in the biotransformation of recipavrin to the formamide. The possibility of solvent mediated formylation or free radical oxidation of desalkyl metabolites to afford the formamides was ruled out. The methylene nitrone was shown to afford the formamide metabolite under simulated workup conditions. An alkali catalyzed Beckmann rearrangement of nitrone to amide was used to account for this transformation. The secondary hydroxylamine was shown to give rise to the methylene nitrone under simulated workup conditions. It was concluded that the oxidation of a β-glucuronidase liberated secondary hydroxylamine metabolite to the methylene nitrone followed by Beckmann rearrangement of the nitrone to the formamide was the probable source of the formamide observed by GCMS in extracts of bile from recipavrin dosed rats. The metabolism of N-methyl-N-(1-methyl-3,3- diphenylpropyl) formamide was investigated in detail to determine whether the carbinolamide, N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl) formamide was involved in the genesis of the formamide metabolite of recipavrin. The above carbinolamide and N-(1-hydroxy-1-methyl-3,3-diphenylpropyl) formamide were identified by GCMS along with 16 other metabolites representing the metabolic pathways N-deformylation, N-dealkylation, N-oxidation and phenyl ring oxidation. The carbinolamides were not found in bile from recipavrin dosed rats, ruling out the possibility of a carbinolamide glucuronide precursor of the recipavrin formamide metabolite. This was the first report of the isolation of stable dealkylation intermediates of a high molecular weight formamide. The hepatotoxicity of the anticancer agent N-methyl formamide and the solvent dimethylformamide, suggests that the recipavrin formamides could also be metabolized to toxic carbinolamide or glutathione related metabolites. / Pharmaceutical Sciences, Faculty of / Graduate

The regulation of calcium (Ca²⁺) was examined during long-term changes of neuronal excitability in the mammalian CNS. The preparations under investigation included the kindling model of epilepsy, a genetic form of epilepsy and long-term potentiation (LTP) of neuronal activity. The study also includes a discussion of the possible roles of a neuron-specific calcium-binding protein (CaBP). The findings are summarized as follows: 1) The distribution of CaBP was determined in cortical areas of the rat using a specific radioimmunoassay. The protein was found to have an unequal distribution in various cortical areas with preponderence in ventral structures. 2) Extending previous studies on the role of CaBP in kindling-induced epilepsy, its decline was correlated to the number of evoked afterdischarges (AD's) during the process of kindling. 3) Marked changes in CaBP levels were also found in the brains of the epileptic strain of mice (El). The hippocampal formation and the dorsal occipital cortex contained significantly lower CaBP than the control (CF-1) strain. The induction of seizures further decreased the levels of CaBP in the El mice. These findings are indicative of a possible genetic impairment of neuronal Ca²⁺ homeostasis in the El strain. 4) The levels of total hippocampal Ca²⁺ and Zn²⁺ were measured by atomic absorption spectrophotometry in control and commissural-kindled animals. While no change was found in the total Ca²⁺ content of the region, hippocampal Zn²⁺ of kindled preparations was found to be significantly elevated. 5) To measure Ca²⁺ -homeostasis, the kinetic analysis of ⁴⁵Ca uptake curves was undertaken in the in vitro hippocampus. This technique was found to be a valid method for assessment of Ca²⁺-regulation in the CNS under both physiological and pathophysiological conditions. The effect of various extracellular Ca²⁺ concentrations, 2,3-dinitrophenol (DNP), calcitonin, nifedipine and 3-isobutyl-1-methylxanthine (IBMX) on ⁴⁵Ca uptake curves was examined in order to identify the two exchangeable Ca²⁺ pools derived through kinetic analysis. 6) The kinetic analysis of ⁴⁵Ca uptake curves revealed that Ca²⁺-regulation of the hippocampus is impaired following amygdala- and commissural kindling. The changes reflect an enhancement of a Ca²⁺ pool that includes free cytosolic Ca²⁺ and a concomitant decrease in the amount of buffered calcium probably as a result in the decrease of hippocampal CaBP levels. 7) A novel form of long-term potentiation (LTP) of neuronal activity in the CA1 region of the hippocampus is described. Perfusion of 100 uM of IBMX in the hippocampal slice preparation induced a long lasting increase in the amplitude of the stratum radiatum evoked population spike and EPSP responses with changes in synaptic efficacy as indicated by the altered input/output relationships. Intracellular correlates of IBMX-induced LTP included lowering of synaptic threshold and enhancement of the rate of rise of the EPSP with no alterations in the passive membrane characteristics of CA1 pyramidal neurons. The fact that IBMX was able to exert its effect even in the presence of the calcium-blocker cation Co²⁺, taken together with the drug's action on hippocampal exchangeable Ca²⁺, raises the possibility that the Ca²⁺ necessary for induction of LTP may be derived from an intraneuronal storage site. These studies indicate the significance of intracellular Ca²⁺ -regulatory mechanisms in long-term changes of neuronal excitability which occur in experimental models of epilepsy and long-term potentiation. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The effect of intramuscular corticosterone injection on post mortem muscle metabolism and shear resistance of Pectoralis Major muscle was studied. Short term studies, conducted over an 8 hour post-injection period, showed no significant effect of injection until 6 hours post injection at which time shear values increased from those of the controls. Blood glucose levels rose significantly from 2 to 8 hours post injection. A comparison of shear values of P. Major muscle from 6 week-old broilers and 8 week-old broilers after both had received corticosterone injections showed that the only difference was due to the difference of age not to any alteration in the physiological handling of the injected corticosterone. The greatest difference in treatment effect was found between birds of different stress level. Stress level was subjectively evaluated with respect to the birds reaction to a person entering the range house and to the birds response to handling. Long term studies conducted over 5 and 15 days illustrated homeostatic adjustments to daily corticosterone injections and recovery after cessation of injections. Shear testing was performed on P. Major muscle which was chilled on the carcass and on muscle excised at death. Excised muscle displayed a greater reaction to injection and cessation of injection than did intact muscle due to a lack of skeletal restriction. Shear values decreased on initial injection and increased upon cessation of injection. Homeostatic adjustments returned values to near-normal between these times. Chemical tests were found to display a much more definite reaction to corticosterone injection than did shear testing. Blood glucose and tissue glycogen increased steadily on injection of corticosterone and decreased rapidly at cessation. As expected ultimate muscle pH decreased with muscle glycogen increase. Blood cholesterol increased during injection period as endogenous corticosterone production, for which it is a precursor, was not required. / Land and Food Systems, Faculty of / Graduate

The present study may be considered endocrinological and radiobiological in nature. The endocrinology phase was concerned with studying the changes in endocrine function following the application of a stress agent. X-irradiation was chosen as the stressor in order to determine any difference in effect of this stressor from others which have been studied, e. g. heat, cold, metabolic poisons. Liver slices taken from rats at various time intervals following whole body X-irradiation were tested for their ability to metabolize hydrocortisone from a Krebs-Ringer solution.

This study aimed to investigate lipoprotein metabolism in male hamsters fed diets considered to be atherogenic in humans. Livers from adult male hamsters were selected to study aspects of apolipoprotein B metabolism. Isolated hepatocytes in suspension were compared with those maintained under tissue culture conditions. Liver slices were also prepared and compared with isolated suspended hepatocytes. Freshly prepared hepatocytes from the animals were incubated with radiolabelled precursors in suspension, or they were maintained under tissue culture conditions; liver slices were also investigated. The rates of total protein synthesis were of the same order in each of these systems, but protein secretion was impaired in liver slices, probably as a result of diffusion problems associated with the altered architecture of the sliced tissue. Albumin constituted 40 - 50% of the secreted proteins in each system. The rates of VLDL synthesis were increased in cells and slices prepared from animals previously fed sucrose- or fat-rich diets, but the secretion of VLDL was inhibited when diets contained unsaturated fat. The overall synthesis of apolipoprotein B was enhanced by fat-feeding; in the case of suspended hepatocytes, secretion of this protein was decreased when the preceding diet contained fats that were unsaturated; while in the case of liver slices, secretion was paradoxically enhanced. Apolipoprotein B was not degraded at significant rates in hepatocytes prepared from either control or fat-fed hamsters.

Hypothesis: Glucose is known to be protective in moderate low flow ischaemia due to the production of glycolytic ATP. However, it is questioned whether glucose would still be protective in ultra-low flow ischaemia. Firstly, glycolysis is thought to be inhibited, and secondly, deleterious glycolytic metabolites accumulate. Our hypothesis was that in ultra-low flow ischaemia, glucose utilisation is not inhibited at the level of glycolysis, but by delivery. Increased delivery of glucose should result in increased production of protective glycolytic ATP, but the rate of metabolite accumulation would also increase. Using ultra low flow rates, I wished to investigate how to achieve optimal rates of glycolysis, and how such rates would be balanced by any detrimental component of metabolite accumulation. Methods: The isolated Langendorff-perfused rat heart, with a left ventricular balloon to record ischaemic contracture and reperfusion stunning, was used, with severe flow restriction. Glucose concentrations were changed and pre-ischaemic glycogen contents were altered by perfusion with different substrates (acetate - depletion~ glucose + insulin - loading) or by preconditioning, with 5 min ischaemia and 5 min reperfusion prior to sustained ischaemia. Results: Analysis of glucose uptake relative to delivery showed that in severe low flow ischaemia, the extraction of glucose was increased, and glycolysis was thus limited more by substrate supply than by enzyme inhibition. Analysis of metabolites confirmed this concept. The optimal glucose concentration during severe low flow ischaemia was 11 mM, giving maximal recovery on reperfusion. Both lower and higher glucose concentrations increased ischaemic contracture. Changes in pre-ischaemic glycogen levels correlated with the time to onset of contracture, such that a reduction in glycogen accelerated contracture. Prior glycogen depletion or loading did not improve functional recovery. The benefits of preconditioning on reperfusion function following sustained total global ischaemia could not be related to glycogen depletion. If preconditioning were followed by sustained low flow ischaemia, glucose uptake was increased, but no benefit was found, possibly because a low residual flow abolished the effects of preconditioning. Many of the above results are consistent with the hypothesis that too low a rate of glycolysis results. in insufficient ATP production for protection, while excess glycolytic rates lead to excess metabolite accumulation with detrimental effects. Conclusions: Provision of glucose at the correct concentration, when the benefit associated with glycolytic ATP outweighs the detriment associated with moderate metabolite accumulation, is protective to the low-flow ischaemic myocardium, which can upregulate its ability to extract glucose. Improved residual flow enhances this benefit. Prior glycogen depletion is not beneficial, despite a reduced metabolite accumulation. This mechanism cannot be related to the protective effect of preconditioning.

Presented for the degree of Doctor of Science of University of the Uiituatersrand. October, 1964. / ffiy interest in iron metabolism was initially aroused in 1948 by a young patient with idiopathic haemochromatosis who was admitted to Professor Elliott*© ward while I was serving my medical internship, With the support of the Council for Scientific and Industrial Research it was possible to carry out radioisotopic studies on this patient and over the next four years a number of other subjects with the same disease ware investigated. As the study continued, attention was also directed to the siderosis which is so common in adult Bantu, and to the Iron overload which results from the administration of repeated blood transfusions to subjects with refractory anaemias. / IT2018