Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling.

The "two-step" low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L(pg) = 1.76 microm/min-atm and E(L(p)) = 75.5 kcal/mol for control liver cells and L(pg)[cpa] = 1.18 microm/min-atm and E(L(p))[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5 degrees C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (>/=5 degrees C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5 degrees C/min. A modified Krogh model (Model 2) was constructed to account for the cell-cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1-2 degrees C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (>/=-8 degrees C).     

Measurement of water transport during freezing in mammalian liver tissue: Part II--The use of differential scanning calorimetry.

There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.     

Liver monoamine oxidase in the obese-hyperglycaemic (ob/ob) mouse.

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.     

Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. II. Mathematical Modeling

The “two-step” low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L_pg = 1.76 μm/min-atm and E_Lp = 75.5 kcal/mol for control liver cells and L_pg[cpa] = 1.18 μm/min-atm and E_Lp[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5°C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (≥5°C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5°C/min. A modified Krogh model (Model 2) was constructed to account for the cell–cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1–2°C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (≥−8°C).     

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. I. Experimental measures.

In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a "two-step" low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the "equilibrium" (infinitely slow) cooling behavior and the osmotically inactive cell volume (V(b)) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5 degrees C/min. Stereological analysis of the slam frozen (>1000 degrees C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DeltaX = 64 microm; original sinusoid (vascular) radius, r(vo) = 18.4 microm; and length of the Krogh cylinder, L = 0.71 microm (based on an isolated frog hepatocyte cell diameter of 16 microm). A significant observation was that approximately 24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r(2) = 28.7 microm, was defined (in addition to the original sinusoid radius, r(vo) = 18.4 microm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle-van't Hoff plot, the osmotically inactive cell volume, V(b) was obtained as 0.4. V(o) (where V(o) is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min.     

Mercury inhibition of fatty acid synthesis in chicks.

Male chicks were fed a commercial ration and were given drinking water which contained 0, 50, 100, 150, 200 or 300 mug of mercury/ml as mercuric chloride from hatching to 3 weeks of age. In one experiment, the mercuric chloride was administered by injection into the abdominal cavity rather than in the drinking water. At 3 weeks the chicks were killed, and the livers were removed and weighed. The activity of fatty acid synthetase in the 800 X gav supernatant fractions of the liver homogenates and in vivo incorporation of [14C]acetate into liver and carcass fatty acids and respiratory 14CO2 was determined as indicated. Administration of mercury at a treatment level of 300 mug/ml of drinking water depressed growth, feed and water consumption, liver weight, hepatic fatty acid synthetase activity, and in vivo incorporation of [14C]acetate into liver and carcass fatty acids, and increased the production of respiratory 14CO2 as compared with controls. In experiments in which graded doses of mercury were administered, body weights, liver weights, and feed and water intakes of the chicks receiving 0, 50 and 100 mug of mercury/ml of drinking water were similar to each other, but these parameters were severely depressed by 200 mug of mercury/ml of drinking water. Mercury caused a dose-related decrease of fatty acid synthetase activity. Incorporation of [14C]acetate into carcass fatty acid was depressed by 50 and 200 mug of mercury/ml of drinking water; incorporation into liver fatty acids and production of respiratory 14CO2 was not affected by mercury. Intra-abdominal injection of 6 mg of mercury/100 g body weight (as mercuric chloride) into well alimented chicks depressed hepatic fatty acid synthetase activity at 1 h post-injection. The data are consistent with the hypothesis that a portion of the effects of mercury on fatty acid synthesis are direct rather than a secondary effect of inanition.     

The accumulation of cadmium by the plaice, Pleuronectes platessa L. and the thornback ray, Raja clavata L.

The accumulation of cadmium (^115mCd) from both food and sea water by the plaice, Pleuronectes platessa L., and thornback ray, Raja clavata L., has been studied in relation to their measured cadmium concentrations. Plaice accumulated cadmium from sea water at a faster rate than rays, although the concentrations factors attained by both species as a result of such direct accumulation were very low. Both species retained cadmium from food and accumulated it in the liver, the rays relatively more than the plaice. From analyses of North Sea fish, some evidence was found for a positive linear relation between the concentrations of cadmium in plaice liver and age, but not weight, of the fish.     

Effect of sodium loading (3% NaCl) on arachidonic acid biosynthesis in rat liver microsomes.

Sodium loading increases arachidonic acid (AA) metabolism by way of the prostaglandins(PGs) from series 2. Its effect on AA biosynthesis remains unknown. The purpose of the present study was to investigate the influence of sodium loading on the fatty acid composition of liver and liver microsomes, and the liver microsomal delta-6 and delta-5 desaturations of linoleic acid (LA) into AA. We found a decrease of LA and dihomo-gamma-linolenic acid (DGLA) levels in liver total lipids of Wistar rats receiving hypernatriuretic drinking water (NaCl 3%) for 60 days. At the same time AA increased. DGLA decreased and AA increased in liver microsomal total lipids. 1(14) C-LA delta-6 desaturase and 2(14) C-DGLA delta-5 desaturase activities increased in liver microsomes. These results show that, in addition to its influence on the regulation of glomerular filtration, sodium loading is involved in the regulation of liver AA biosynthesis.     

Phosphorylation status of liver by 31P-n.m.r. spectroscopy, and its implications for metabolic control. A comparison of 31P-n.m.r. spectroscopy (in vivo and in vitro) with chemical and enzymic determinations of ATP, ADP and Pi.

An investigation into the measurement of Pi and ADP in rat liver in vivo and in freeze-clamped extracts by 31P-n.m.r. spectroscopy was carried out. The concentration of Pi estimated in vivo is less than 25% [1 mM (mumol/ml of cell water)] of the value obtained from freeze-clamped liver (4 mM), whereas ADP in vivo is undetectable (1.4 mM in vitro). At 5 min after infusion of 750 mg of fructose/kg, the Pi content of liver extracts fell to 1.3 mM, whereas Pi is undetectable in vivo under these conditions [Griffiths, Stevens, Gadian, Iles & Porteous (1980) Biochem. Soc. Trans. 8, 641]. The results indicate that the lower Pi and ADP concentrations found in vivo may be due to compartmentation or binding rather than to degradation of labile organic phosphates during extraction. The results are discussed with reference to previous measurements of liver phosphates and investigations of compartmentation in the liver, as are some of the possible consequences for metabolic control in the liver of low ADP and Pi concentrations.     

Total and structured water in cancer: an NMR experimental study of serum and tissues in DMBA-induced OF1 mice.

Total water and structured water (fraction of total water which remains unfrozen below the transition point from the semisolid to solid state) were characterized by 1H NMR relaxometry in the sera and tissues of 3 groups of 30 female mice (C, H and L) receiving a single administration of DMBA and different diets. Mice given the diet H, containing the highest proportion of saturated fatty acids and processed starch, and the lowest phytochemicals content, presented the highest tumor incidence (lymphoma). This allowed 3 subgroups to be defined: subnormal (SN), small (T+) and large tumor (T++). Spin-lattice relaxation times of total water (Tlobs) in the sera and tissues did not significantly differ between C, H and L groups, and SN, T+ and T++ subgroups. In T+ mice, a decrease in the relative amount of structured water was noticed in the serum, liver and heart, while changes in the temperature dependence of the Tl of structured water (Tlsw) were observed between -21 degrees C and -42 degrees C. These results suggest a moderate increase in the rotational mobility of structured water molecules in the serum and the heart, and a pronounced decrease in the liver. Likewise, the modification of the Tlsvv temperature dependence curve's shape tends to confirm the existence of important conformational changes in the macromolecular assemblies, which markedly affect the properties of structured water, especially in the earliest stage of cancer development.     

Cadmium toxicity and liver mitochondria. I. Different effects of cadmium administered in vivo to adult, young, and ethionine-fed rats

The changes in liver mitochondrial respiratory activities and cytochrome concentrations were investigated when cadmium chloride was administered orally to adult, young, and ethionine-fed rats. Following a seven-day administration of 30 ppm cadmium in drinking water, adult rats showed no change, while young rats and ethionine-fed rats exhibited a marked increase in mitochondrial respiration with concomitant decrease of respiratory control index and P/O ratio. The concentrations of cytochromes aa3, b, and c + c1 in liver mitochondria were unchanged in adult rats, but increased significantly in ethionine-fed rats. In young rats receiving cadmium the liver mitochondrial protein increased with a slight change in the cytochrome concentration in mitochondria. It was further found that in adult rats a higher concentration (300 ppm) of cadmium in drinking water was toxic to the liver mitochondrial functions. Thus, the effect of oral administration of cadmium on the liver mitochondrial function depends on the condition of the animals.     

Effect of nutritional factors and cortisol on tyrosine aminotransferase activity in liver of brook trout, Salvelinus fontinalis Mitchill.

1. Starvation for over 50 days increased the activity of Tyrosine aminotransferase (TAT) from brook trout liver and decreased liver glycogen. 2. Cortisol (6-40 mg/100 g body wt) increased TAT activity, optimally 72 hr post-injection; liver glycogen response to cortisol was variable while tissue water either increased or remained constant. 3. Fish fed high-protein/low-carbohydrate had greater TAT activity than fish fed low-protein/high carbohydrate. There was food-induced peak of activity indicating a "diurnal rhythm" analogous to that of rat TAT. 4. TAT activity in brook trout appears to be correlated with protein catabolism.     

Alpha-foetoprotein and oestrogen metabolism. I. -Influence of alpha-foetoprotein on the metabolism of steroids by rat liver microsomes in vitro.

Rat alpha-foetoprotein (AFP) was shown to inhibit the formation of water soluble metabolities of oestrone and oestradiol by incubation with microsomes from rat liver in the presence of NADPH. The results support the proposal that in young animals the low activity of enzymes responsible of oestradiol metabolism may be due in part to the presence of AFP and not only to the low level of these enzymes.     

Assessment of heavy metals around Abakaliki metropolis and potential bioaccumulation and biochemical effects on the liver,kidney, and erythrocyte of rats

This study investigated changes in kidney, liver and erythrocyte of male rats after exposure to ground water samples collected in the studied areas and treated water sample. Results reveal high levels of heavy metals in water from studied areas and a concomitant accumulation of metals in liver, kidney and blood of rats. Heavy metal contaminated underground water (HMCUW) caused a significant increase in levels of malondialdehyde (MDA) and decreased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), including glutathione (GSH) levels. Principal component analysis implicated geogenic and anthropogenic factors for underground water contamination by heavy metals in the areas. However, activities of gamma-glutamyl transferase (γ-GT), 51 nucleotidase (51NT), glutathione peroxidase (GSH-Px), and myeloperoxidase (MPO) significantly increased in kidney, liver and erythrocytes of rats compared with control group (p < 0.001). We observed significant (p < 0.001) increase in serum electrolytes, urea and creatinine levels compared to control. Heavy metals in HMCUW are above ambient levels and possess significant decrease in triiodothyronine (T3) and thyroxine (T4) levels. Histology revealed alterations in architecture of the kidney and liver. Prolonged exposure to HMCUW could induce oxidative damage in kidney, liver and erythrocyte which could elicit liver and kidney dysfunctions.     

The state of water in biological systems as studied by proton and deuterium relaxation.

Careful experiments on the measurement of the intensity of the deuterium NMR signal for 2-H2 O in muscle and in its distillate were performed, and they showed that all 2-H2 O muscle is "NMR visible". The spin-lattice relaxation time (T1) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to --70 degrees C. T1 values of deuterons in 2H2 O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to --20 degrees C. Calculations on T1 for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T1 values compared to pure water and the frequency dependence of T1 are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

Nuclear magnetic resonance studies of substrate interaction with cobalt substituted alcohol dehydrogenase from liver.

The role of zinc in liver alcohol dehydrogenase has been studied by replacement of 1.3 and 3.5 of the four Zn(II) ions with Co(II) and measuring the effects of the paramagnetic Co(II) on the relaxation rates of the protons of water, ethanol, and isobutyramide. Water relaxation studies at 8, 24, 100, and 220 MHz indicate two classes of bound Co(II). The similar to 2 readily replaced Co(II) ions retain one fast exchanging water proton in their inner coordination spheres, while the similar to 2 slowly exchanging Co(II) ions coordinate no detectable water protons, indicating that the former replaced Zn(II) at the "catalytic sites" and the latter replaced Zn(II) at the "structural sites" detected crystallographically. Ethanol, acetaldehyde, and isobutyramide bind with appropriate affinities to the Co(II) substituted alcohol dehydrogenases decreasing the number of fast exchanging protons at the catalytic Co(II) site by greater than or equal to 54 percent. Coenzyme binding causes smaller changes in the water relaxation rate which may be due to local conformation changes. The paramagnetic effects of Co(II) at the catalytic site on the relaxation rates of the methyl protons of isobutyramide at 100 and 220 MHz indicate that this analog binds at a site 9.1 A from the catalytic Co(II). This distance decreases to 6.9 A when NADH is bound, and a Co(II) to methyne proton distance of 6.6 A is determined indicating a conformation change leading to the formation of a second sphere enzyme-Co(II)-isobutyramide complex in which a hydroxyl or water ligand intervenes between the metal and the substrate analog. Similar behavior is observed in the enzyme-ethanol complexes. The paramagnetic effects of Co(II), at the catalytic site, on the relaxation rates of the protons of ethanol at 100 and 220 MHz, indicate that this substrate bind at a site 12-14 A distant from the catalytic Co(II) but that this distancedecreases to 6.3 A in the abortive enzyme-NADH-ethanol complex. The role of the catalytic Co(II) thus appears to be the activation of a hydroxyl or water ligand which polarizes the aldehyde carbonyl group by hydrogen bonding. The role of the structural Co(II), which is more distant from isobutyramide (9-11 A), may be that of a template for protein conformation changes. By combining the present distances with those from previous magnetic resonance studies on the liver enzyme, the arrangement of coenzyme, metal, and substrate at the active site in solution can be constructed. This arrangement is consistent with that of ADP-ribose and zinc in the crystalline complex of liver alcohol dehydrogenase as determined by X-ray crystallography (Branden et al., (1973), Proc. Natl. Acad. Sci. U.S.A.70, 2439).     

Effect of propylthiouracil on liver regeneration in rats after partial hepatectomy.

The effect of propylthiouracil (PTU) on the growth activity of intact liver and liver regenerating after partial (65-70%) hepatectomy (PH) was studied in rats. PTU (Propycil, Kali-Chemie, FRG) was dissolved in drinking water (1 g PTU per litre) and this was given to the rats, as their sole source of fluids, three days before PH and then up to the end of the experiment. In rats given PTU, marked inhibition of liver DNA synthesis and the mitotic activity of hepatocytes was found after PH. This effect was potentiated to some extent by partial inanition of the experimental animals given PTU, as demonstrated in a paired feeding test in control rats. PTU inhibition of DNA synthesis in intact and regenerating liver also took effect in thyroidectomized rats, even with substitution (thyroid hormone) therapy. The experiments demonstrated that the effect of propylthiouracil on DNA synthesis in the liver is mediated primarily by way of its direct effect on the liver.     

In vivo assessment of microcystin-LR-induced hepatotoxicity in the rat using proton nuclear magnetic resonance (1H-NMR) imaging.

Microcystin-LR (MCLR)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-350 g) using magnetic resonance imaging (MRI). Following the intraperitoneal administration of MCLR (LD(50)), a region of damage, characterised by increased signal intensity on T(2)-weighted images, was seen proximal to the hepatic portal vein in the liver. Similarly, increased signal intensity was seen in the chemical-shift selective images (CSSI) of water frequency, proximal to the hepatic portal vein in the liver. This indicates that the increased signal intensity observed in the T(2)-weighted images was due to an increased amount of magnetic resonance (MR) visible protons in the tissue which represents an oedematous response. Image analysis of regions of apparent damage around the hepatic portal vein indicated a statistically significant increase in signal intensity in this region. Mitochondrial swelling and lipid inclusions were observed by transmission electron microscopy (TEM) in samples obtained from the oedematous regions of the liver using spatial coordinates from the magnetic resonance (MR) images. Massive haemorrhagic necrosis and nuclear swelling were observed by light microscopy in the centrilobular regions of the lobules.     

Increased rate of cholesterologenesis--a possible cause of hypercholesterolemia in experimental chronic renal failure in rats.

Hypercholesterolemia plays an important role in the lipid abnormalities in chronic renal failure (CRF). It is thought to contribute to both a progression of renal failure and atherosclerosis. Despite intensive research, the etiopathogenesis of hypercholesterolemia in CRF patients is still obscure. The present study was designed to evaluate the possible role of cholesterol overproduction in the development of hypercholesterolemia associated with experimental CRF. We found that plasma total cholesterol and cholesterol distributed in VLDL, LDL and HDL concentrations were significantly enhanced in CRF rats. Simultaneously, the rate of liver cholesterol biosynthesis in vivo (measured by determining the incorporation of tritium from tritiated water intraperitoneally injected into cholesterol ), liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and liver HMG-CoA reductase mRNA presence were elevated. Significant increases in activity of liver malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, NADPH-producing enzyme (required for cholesterol synthesis) have also been observed in CRF rats. In conclusion, the increased rate of liver cholesterol biosynthesis due to increase of HMG-CoA reductase and NADPH-producing enzyme gene expression could be one of the possible causes of hypercholesterolemia in CRF animals.     

Separation of two L-glutamine-hydroxylamine glutamyltransferases from rat liver.

The purification of glutamine synthetase (GS) from rat liver demonstrates that a small portion of glutamine-hydroxylamine-glutamyltransferase activity (GT) remains associated with GS activity (GT(S)). As GS is purified from the water extract, the ratio between GT(S) activity (GT(T)) is left to be extracted by KC1 from the pellet and, on further purification, appears to be independent of GS activity. Subtle differences in pH optimum, substrate requirement and reaction rates on addition of cofactors and amino acids in vitro and in responses to hormonal stimuli in vivo indicate that the glutamine transfer reaction may be catalyzed by two distinguishable proteins; only the minor component may be identical to GS.