Conversion of thyroxine into tri-iodothyronine by rat liver homogenate.

By using a highly specific radioimmunoassay the formation of tri-iodothyronine by the deiodination of thyroxine was studied in rat liver homogenate. Several observations suggest that the reaction observed is enzymic in nature. Pre-heating the homogenate for 30 min at 56 degrees C completely abolished conversion of thyroxine into tri-iodothyronine; the component of rat liver homogenate responsible could be saturated with substrate; iodotyrosines displayed competitive activity. Between 0 degrees and 37 degrees C, the tri-iodothyronine-production rate was positively correlated with incubation temperature. The addition of NAD+ enhanced conversion into tri-iodothyronine, which suggests that an oxidative mechanism is involved. 5-Propyl-2-thiouracil and 6-propyl-2-thiouracil, both known to prevent deiodination in vivo, greatly decreased the deiodiantion activity of rat liver homogenate.     

The localization of phenolic and tyrosyl ring thyroxine deiodination in rat and mouse retina

To elucidate T₄ metabolism in various cell types of rat retina, 5-monodeiodinating and 5′-monodeiodinating activities were studied in retinal cell layers obtained by selective cytotoxic action of monosodium glutamate on bipolar and ganglion cell layers and by iodoacetate effect on photoreceptor cells. Concomitantly these enzyme activities were studied in C3H/HeN mouse retina genetically deprived of photoreceptor cells. Deiodinase activities were low in rat and mouse retina deprived of photoreceptors. The 5′-monodeiodination rate of T₄ was higher than T₄ tyrosyl ring deiodination in cell layers examined and the highest values were found in the photoreceptor cells. Data support the hypothesis that phenolic and tyrosyl ring deiodinase activities are present in the photoreceptor cells. Their reciprocal changes may regulate the nuclear function which in turn controls the rhythmical renewal of rod outer segments.     

Effect of ethanol and linoleic acid on changes in biliary excretion of iodothyronines possibly related to thyroxine deiodination in rat liver

Groups of male rats weighing about 350 g were inserted polyethylene tubings into bile duct and femoral vein under pentobarbital anesthesia. Several iodothyronines (i.e. T4, T3, rT3, 3,5-T2, 3,3'-T2 and 3',5'-T2) were estimated in 2-hr portions of bile with the aid of specific radioimmunoassay. After the infusion of ethanol (0.3 ml/hr/rat for 4 hr) an increase of biliary excretion of rT3 and a decrease of 3,5-T2 was found as compared to controls. When 5 mg linoleic acid was added to 1.2 ml ethanol, the increase of rT3 was significantly higher than that after ethanol only and, in addition, significant increase of 3',5'-T2 excretion was found. It was concluded that both ethanol and unsaturated fatty acids may inhibit 5'-monodeiodination in the liver and that unsaturated nonesterified fatty acids may exert such effect even when administered intravenously without underlying metabolic disorders.     

Demonstration of collagenase activity in rat liver homogenate

Collagenase activity became detectable in rat liver homogenate by washing liver tissue repeatedly with buffered saline before homogenization. This enzyme activity was inhibited by adding minute quantities of serum. These data suggest that collagenase is active $\text{in situ}$ in the liver, but is made inactive during the homogenization by forming a complex with contaminating serum factors.     

Inhibition of hepatic deiodination of thyroxine is caused by selenium deficiency in rats.

Selenium (Se) deficiency produced up to a 14-fold decrease in hepatic tri-iodothyronine (T3) production from thyroxine (T4) in vitro. The T3 production rate could not be restored by the addition of a variety of cofactors, nor by the addition of control homogenate. The impairment in hepatic T3 production observed in Se deficiency was reflected in the concentrations of thyroid hormones circulating in plasma, T4 being increased approx. 40% and T3 being decreased by 30%. However, the fall in plasma T3 concentrations was smaller than might be expected in view of the marked decreased in T3 production. Se deficiency had no measurable effect on plasma reverse-tri-iodothyronine concentrations. The data suggest that Se deficiency produces an inhibition of both 5- and 5'-deiodination, consistent with the widely held view that these reactions are catalysed by the same enzyme complex. The mechanism of inhibition appears not be mediated by changes in thiol levels, but a direct role of Se in the activity of the deiodinase complex cannot be excluded.     

Preservation of native properties of mitochondria in rat liver homogenate

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.     

Effect of diet and L-thyroxine on deiodination of L-thyroxine in rat liver

Male Wistar rats on 3 or 50% fat diet for 14 weeks were treated during 6 weeks with L-thyroxine (5 or 25 or 50 microgram 100 g body weight/2 times weekly, sc.) and the activity of L-thyroxine-deiodinase was determined in supernatans of liver homogenates. With increasing thyroxine lading the deiodinating activity increases statistically significantly within each diet group. The liver of animals on 50% fat diet deiodinates increasing thyroxine doses to a lesser extent than livers of animals on 3% fat diet. It is discussed that high fat diet influences thyroid function and that thyroxine shares effectiveness and undergoes deiodination also in other tissues, probably in fat tissue, to a higher extent.     

Reduction of various lipid hydroperoxides by rat liver homogenate

Rates of disappearance of hydroperoxy groups of various lipid peroxides added to rat liver homogenate differed from each other. The hydroperoxy group of linoleic acid disappeared rapidly, while those of L-3-phosphatidyl choline dilinoleoyl and trilinolenin disappeared slowly. The hydroperoxy groups of cholesterol linoleate were stable in the homogenate. Most of the conjugated dienes of these lipid peroxides remained. The hydroperoxy groups of the unsaturated fatty acids of the phosphatidyl choline were found to be changed to hydroxy groups as analyzed by high-performance liquid chromatography.     

Polyamines effect on subcellular fractionation of rat liver homogenate

1) Spermidine and spermine added to the homogenizing medium are able to increase the sedimentation velocity of mitochondria, smooth microsomes, lysosomes, Golgi apparatus and plasma membranes. Spermine at 0.5 mM enhances the sedimentation and at 3 mM is able to sediment, at 600 g for 10 min, almost the totality of membranous components of the cell. 2) Smooth microsomes were used as a model to study the nature of spermine effect. The amount of spermine bound to 1 g of smooth microsomes would increase their density of about 0.02%. In the presence of 1 mM spermine the great majority of smooth microsomes are unable to pass through a 10 μ filter indicating an extensive aggregation of the particles. 3) Spermine induced aggregation of smooth microsomes can be reversed by either heparin or poly-D-glutamic acid.     

Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.