Acute inflammation, acute phase serum amyloid A and cholesterol metabolism in the mouse

We describe a patient, RM, who suddenly became amnesic for premorbid autobiographic events in the absence of any known precipitating event. Learning abilities as well as semantic knowledge were normal. Knowledge of famous facts and persons was good, although not perfect. Whether RM suffered from organic or psychogenic isolated retrograde amnesia (IRA) could not be established on the basis of available clinical and neuropsychological elements. Regardless of its aetiology, RM's case respects the boundaries between semantic and episodic memory and so gives further support to the distinction between these two memory systems.     

In vivo aspects of progesterone distribution and metabolism

The end-organ response of any hormone is the result of many factors which precede the event, including biosynthesis, secretion, transport, distribution, and metabolism. These factors vary among different species. The metabolic clearance rate (MCR) of progesterone varies between 40 and 180 L./day/Kg. in man (60 to 70), monkey (40 to 50), rabbit (55 to 60), sheep (110), rat (120), and guinea pig (180). Major sites of clearance include liver, brain, and uterus. Specific metabolites of progesterone include 20 alpha-hydroxypregn-4-en-3-one (20 alphaOHP) and alpha-pregnan-3,20-dione (5 alpha-DPH). Liver, brain, and uterine clearances, extractions, and conversions of progesterone to these metabolites have been studied in various species under apparent steady-state conditions. A specific hormone action of progesterone, the appearance of uteroglobin in the rabbit uterus, has also been studied in varying horomonal states (estrogen, estrogen plus progesterone, and progesterone alone). These have all been used as examples of progesterone distribution and metabolism.     

Erythropoiesis and erythropoietin synthesis during aseptic acute inflammation

Erythropoietin (Epo) production during acute inflammation induced by s. c. turpentine administration in experimental Long-Evans rats increased in response to reduced erythropoiesis. A close correlation was found between decreased haematocrit (Hct) and increased levels of tumour necrosis factor-alpha (TNF alpha) in this experimental system. The Epo response was not different between rats with acute inflammation and anaemia and control animals with a comparable degree of anaemia. It is concluded that Epo is not an acute phase reactant, and that the Epo response in acute experimental inflammation in rats is explained by the associated development of anaemia.     

Local hemodynamics, permeability, and oxygen metabolism during acute inflammation of innervated or denervated isolated equine joints

OBJECTIVES: To determine oxygen metabolism, permeability, and blood flow in isolated joints in response to interleukin 1beta (IL-1beta) and contribution of innervation. SAMPLE POPULATION: One metacarpophalangeal (MCP) joint of 24 adult horses. PROCEDURE: The MCP joint was isolated for 6 hours in a pump-perfused, auto-oxygenated, innervated or denervated preparation. Isolated joints were assigned to the following 4 groups: control, control-denervated, inflamed, and inflamed-denervated, and inflammation was induced by intra-articular injection of IL-1beta. Circuit arterial and venous pressures, flows, and blood gas tensions, synovial fluid production, and intra-articular pressure were measured. Total vascular resistance; oxygen delivery, consumption, and extraction ratio (ER); and permeability surface area product were calculated. Synovial membrane blood flow was determined at 0, 60, and 330 minutes. Synovial membrane wet-to-dry ratio was obtained, and permeability to macromolecules was determined by intra-articular injection of Evans blue albumin and fluorescein isothiocyanate-conjugated dextran. RESULTS: Oxygen delivery and synovial membrane blood flow progressively increased but were not different among groups. Oxygen consumption and ER significantly increased in inflamed joints, as did intraarticular pressure and synovial fluid production. Inflamed joints had greater wet-to-dry ratio. Albumin permeability significantly increased in the villous synovial membrane of the inflamed groups, and dextran permeability was increased in the innervated groups, with a trend toward increased permeability in inflamed groups. CONCLUSION: Inflammation significantly increased oxygen demand, which was initially met by increased ER. Permeability to small molecules was increased with inflammation; innervation increased permeability to large molecules. Use of an isolated joint model enabled documentation of the physiologic responses of the joint to acute inflammation.     

In vivo inter-organ protein metabolism of the splanchnic region and muscle during trauma, cancer and enteral nutrition

The study of protein kinetics has entered a new era by the recognition that whole body protein turnover only poorly reflects the true events occurring in several organs and with regard to the multitude of proteins present in the body. It is also increasingly recognized that the simultaneous synthesis and degradation of proteins is important in regulation and adaptation during several metabolic conditions like starvation, feeding, after trauma, and during exercise. Especially important is the recognition that the kinetics of individual proteins may change in opposite directions, thereby leading to fluxes of alpha-amino-nitrogen that serve to adapt to and survive a changing environment. At present, much emphasis is put upon molecular biological regulation. However, it is important that the metabolic processes that occur in the intact organism are still poorly defined. New technology allows the exploration of these processes, which should therefore prompt the initiation of further research in this area.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer

We have studied the proliferation of cells in two models of chemical hepatocarcinogenesis. The cells were genetically labeled in vivo using retrovirally mediated transfer of the Escherichia coli beta-galactosidase marker gene coupled to a nuclear localization signal (nls-lacZ gene). In the first carcinogenic model, rats were fed a choline-deficient diet containing 2-acetylaminofluorene, and their livers were perfused with recombinant retrovirus at the onset of oval cell proliferation. The second model was based on the administration of diethylnitrosamine coupled with a partial hepatectomy and is thought to induce cancer with no involvement of oval cells. Analysis of beta-galactosidase expression in the liver at various times after gene transfer revealed the presence of large clusters of positive cells in both models. Moreover, the beta-galactosidase-positive cells displayed morphologic, antigenic, and enzymatic profiles consistent with a hepatocyte phenotype. Our results, therefore, provide evidence for a strikingly similar clonal proliferation of apparently normal hepatocytes during the course of 2-acetylaminofluorene- as well as diethylnitrosamine-induced liver carcinogenesis.     

A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate

Heparan sulfate is a highly sulfated carbohydrate polymer that binds to and modulates the activities of numerous proteins. The formation of these protein-binding domains in heparan sulfate is dependent on a series of biosynthetic reactions that modify the polysaccharide backbone; the initiating and rate-limiting steps of this process are the N-deacetylation and N-sulfation of N-acetylglucosamine residues in the polymer. We now report that in the rat liver, biosynthesis of heparan sulfate utilizes a single protein that possesses both N-deacetylase and N-sulfotransferase activities. This was accomplished by demonstrating that both activities resided in a purified soluble fusion protein containing the Golgi-lumenal portion of the enzyme. We propose that this protein be renamed the rat liver Golgi heparan sulfate N-deacetylase/N-sulfotransferase.     

In vivo metabolism of alpha-tocopherol in lipoproteins and liver: studies on rabbits in response to acute cholesterol loading

OBJECTIVE: To investigate the transport of alpha-tocopherol in lipoproteins of rabbits under normal diet and under acute loading of cholesterol. DESIGN: Two New Zealand White rabbits were fed 14C-alpha-tocopherol acetate in a single oral dose and the recovery of radiolabel in lipoproteins and plasma was monitored. Low density lipoprotein (LDL) from these animals was obtained and labeled with [3H] cholesteryl ester. Three other rabbits were injected with this double-labeled LDL in the native form; while three other animals received this LDL in the acetylated form. RESULTS: Plasma clearance, liver uptake and levels of radiolabel in high density lipoprotein (HDL) of animals injected with 14C[3H]acetyl LDL were significantly higher than those in animals injected with 14C[3H]native LDL. Larger particles of HDL, rich in apolipoprotein E (apoE) carried significantly higher levels of both labels in rabbits injected with acetylated LDL. CONCLUSION: These results provide evidence for in vivo mechanisms of "reverse alpha-tocopherol transport", analogous to "reverse cholesterol transport".     

In vivo modification of 3'-phosphoadenosine 5'-phosphosulfate and sulfate by infusion of sodium sulfate, cysteine, and methionine

The importance of tissue sulfate concentrations in regulating 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthesis is not known. Therefore, this study was conducted to determine the influence of increased availability of inorganic sulfate on steady-state PAPS concentrations in various tissues. To increase tissue sulfate concentrations, 2-16 mmol/kg of sodium sulfate and sulfur-containing amino acids (cysteine or methionine) were infused intravenously for 2 hr into pentobarbital-anesthetized rats. Serial blood samples were taken during the infusion and analyzed for sulfate concentrations. After 2 hr of infusion, liver, kidney, and brain were removed for quantification of tissue PAPS and sulfate concentrations. Infusion of sodium sulfate, cysteine, and methionine increased serum and tissue sulfate concentrations in a dose- and time-dependent manner. Serum sulfate concentrations increased from 0.8 to 14 mM during the infusion of sodium sulfate, whereas infusions of cysteine and methionine increased serum sulfate concentrations to 4.8 and 2.0 mM, respectively. Tissue sulfate concentrations also increased during sulfate infusion. Liver sulfate concentration increased from 0.8 to 4.8 mM, kidney concentration increased from 0.6 to 31 mM, and brain concentration increased from 0.1 to 0.6 mM. Similar to the serum sulfate concentrations, sulfate infusion was the most effective in increasing tissue sulfate concentrations, cysteine was intermediate, and methionine the least effective. Although sulfate concentrations in liver, kidney, and brain increased 6-, 50-, and 6-fold by infusing sulfate, respectively; tissue PAPS levels were not altered markedly. Hepatic PAPS concentrations increased significantly (30-35%) only when infused with the higher doses (8 or 16 mmol/kg/2 hr) of sodium sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo murine studies on the biochemical mechanism of acetaminophen cataractogenicity

C57BL/6 and DBA/2 mice are, respectively, susceptible and resistant both to the induction of aryl hydrocarbon hydroxylase (cytochrome P450 1A1, or CYP1A1) and to the cataractogenicity of acetaminophen, which may involve its bioactivation to a toxic reactive intermediate, catalysed by P450 and (or) prostaglandin H synthase (PHS). Following induction of P450 using beta-naphthoflavone, the cataractogenicity of acetaminophen (400 mg/kg ip) in C57BL/6 mice was reduced by pretreatment with the P450 inhibitors SKF 525A and metyrapone, the glutathione precursor N-acetylcysteine, the antioxidant vitamin E, and the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p < 0.05). Acetaminophen (200 mg/kg) cataractogenicity was enhanced by pretreatment with the glutathione depletor diethyl maleate (DEM) and the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) (p < 0.05). No significant effect on acetaminophen cataractogenicity was observed using the PHS cyclooxygenase inhibitors aspirin or naproxen, or the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Accordingly, acetaminophen cataractogenicity in C57BL/6 mice does not appear to be dependent upon bioactivation by PHS. In DBA/2 mice treated with beta-naphthoflavone, a high dose of acetaminophen (750 mg/kg ip) was not cataractogenic, even after pretreatment with DEM, BSO, or BCNU. The resistance of DBA/2 mice to acetaminophen cataractogenesis, despite concomitant pretreatments with an inducer of P450 and several agents that interfere with glutathione-dependent detoxifying pathways, suggests differences in this strain involving cytoprotective pathways subsequent to acetaminophen bioactivation and detoxification of the cataractogenic reactive intermediate. These results indicate that acetaminophen cataractogenicity in C57BL/6 mice results from P450-catalysed bioactivation of acetaminophen to a reactive intermediate, possibly a benzoquinone imine and (or) a free radical, the toxicity of which is reduced by glutathione-dependent reactions.     

Renal glomerular basement membrane. In vivo biosynthesis and turnover in normal rats

Glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive glycine and proline, and subsequently purified by osmotic lysis followed by sequential treatment with detergents. Analysis of tail tendons from these animals allowed comparison of basement membrane biosynthesis and degradation with these parameters in the newly synthesized fractions of fibrillar collagen. Peak radiolabeling with [3H]glycine occurred within 24 h, declining steadily thereafter in both basement membranes and salt-soluble tail tendon collagen. Calculated turnover times for [3H]glycine-labeled glomerular basement membrane and salt-soluble tail collagen were similar. Turnover of the collagenous portion of glomerular basement membrane was slightly longer, comparable to the acetic acid-soluble fraction of fibrillar collagen. Glomerular basement membrane is readily labeled after parenteral injection of radioactive precursors. Its biologic half-life is comparable to that of soluble fibrillar collagen, indicating a more rapid turnover than previously believed.     

In vivo evidence of enhanced guanylyl cyclase activation during the hyperdynamic circulation of acute liver failure

The different efficiencies of sucrose and trehalose in protecting isolated spinach (Spinacia oleracea L.) thylakoids against freeze-thaw damage is quantitatively related to their ability to reduce the solute loading of the vesicles during freezing. In the present paper we show that this effect is based on a reduction of the solute permeability of the membranes. Permeability was measured with 14C-labeled glucose at temperatures between 0 and 10 degrees C. Glucose permeability was reduced by both sucrose and trehalose, with trehalose effective at much lower concentrations than sucrose. An analysis of the temperature dependence of glucose permeability in the presence and absence of trehalose revealed that a 50% reduction in permeability resulted from a 10% increase in activation energy and a 30% decrease in activation entropy. Using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH), we found that the reduced permeability of the membranes in the presence of trehalose was unaccompanied by a reduction in lipid fluidity. This also excluded the possibility of a solute-induced liquid crystalline to gel phase transition. A reduced partitioning of the hydrophobicity-sensitive dye merocyanine 540 into thylakoids and into membranes containing 50% digalactosyldiacylglycerol in the presence of trehalose as compared to sucrose and glucose showed that the lipid headgroup region of these membranes became less accessible for solutes. No significant difference in merocyanine partitioning in the presence of trehalose as compared to sucrose or glucose was apparent when monogalactosyldiacylglycerol dispersions or phosphatidylcholine vesicles were investigated.     

In vivo bioavailability and metabolism of topical diclofenac lotion in human volunteers

OBJECTIVE: To characterize the long-term impact of four hormone therapy regimens on insulin and glucose concentrations measured during a standard oral glucose tolerance test. RESEARCH DESIGN AND METHODS: The Postmenopausal Estrogen/Progestin Intervention Study was a 3-year placebo-controlled randomized trial to assess effects of four hormone regimens on cardiovascular risk factors. This efficacy analysis describes glucose and insulin concentrations from 788 adherent women at baseline and at 1 and 3 years' postrandomization. RESULTS: When compared with women taking placebo, those taking conjugated equine estrogen (CEE) at 0.625 mg/day with or without a progestational agent had mean fasting insulin levels that were 16.1% lower, mean fasting glucose levels 2.2 mg/dl lower, and mean 2-h glucose levels 6.4 mg/dl higher (each nominal P < 0.05). No significant differences were apparent between women taking CEE only versus the three progestin regimens: medroxyprogesterone acetate (MPA) at 2.5 mg daily (continuous MPA), MPA at 10 mg on days 1-12 (cyclical MPA), and micronized progesterone (MP) (cyclical) at 200 mg on days 1-12. The impact of hormone therapy on insulin and glucose depended on baseline levels of fasting insulin and 1-h glucose (P < 0.05). However, the treatment effects on carbohydrate metabolism appeared to be consistent across participant subgroups formed by lifestyle, clinical, and demographic characteristics. CONCLUSIONS: Oral hormone therapy involving 0.625 mg/day of CEE may modestly decrease fasting levels of insulin and glucose. Postchallenge glucose concentrations are increased, however, which may indicate delayed glucose clearance.     

In vivo platelet and T-lymphocyte activities during pulmonary tuberculosis

Platelets have been suggested to play a role in the inflammatory response, including defence against bacteria. The aims of this study were to determine in vivo platelet activity during the clinical course of pulmonary tuberculosis and to investigate whether or not there is a correlation between the magnitude of platelet activation and the extent of the pulmonary disease. T-lymphocyte activity was also analysed in the patients. Platelet factor-4 (PF4) and soluble interleukin-2 receptor-alpha (sIL-2Ralpha) concentrations were used as markers of platelet and T-lymphocyte activation, respectively. Twenty-five patients with pulmonary tuberculosis were studied. Fifteen healthy subjects served as a control group. The levels of both sIL-2Ralpha (3,000+/-1,948 pg x mL(-1)) and PF4 (103.1+/-6.7 IU x mL(-1)) were significantly higher in the patients with tuberculosis than in the control group (984+/-360 pg x mL(-1) and 78.2+/-23.9 IU x mL(-1), respectively) (Mann-Whitney U-test, p<0.001 for both comparisons). The plasma PF4 levels were found to be well correlated with the extent of pulmonary lesions on chest radiography (the Spearman's bivariate correlation analysis, r=0.65, p<0.001). However, sIL-2Ralpha concentrations did not correlate with the extent of disease. In conclusion, it has been suggested that platelet and T-lymphocyte activation occurs during pulmonary tuberculosis. The good correlation between platelet activation and the extent of pulmonary tuberculosis might be ascribed to a pathophysiological role of platelets in pulmonary tuberculosis.     

Synergistic effect of alcohol and nicotine on glycosaminoglycan metabolism in rats

Oral administration of alcohol along with nicotine decreased all the glycosaminoglycan (GAG) fractions except hyaluronic acid in aorta and liver of rats. Decreased activity of the enzymes involved in the biosynthesis of precursors of GAG and increased activity of many of GAG hydrolysing enzymes indicate decreased biosynthesis and increased degradation of GAG. Sulphate metabolism in liver was also significantly altered by administration of both alcohol and nicotine showing considerable decrease in the concentration of sulphated GAG.     

In vivo cerebral metabolism and central benzodiazepine-receptor binding in temporal lobe epilepsy

Positron emission tomography measured interictal cerebral glucose metabolism with [18F]fluorodeoxyglucose and central benzodiazepine-receptor binding with [11C]flumazenil in 10 mesial temporal lobe epilepsy (TLE) patients and in normal subjects. Eight TLE patients had mesial temporal, lateral temporal, and thalamic hypometabolism ipsilateral to EEG ictal onsets, with additional extratemporal hypometabolism in four. One had unilateral anterior mesial temporal hypometabolism only, and one had normal metabolism. Each patient had decreased benzodiazepine-receptor binding in the ipsilateral anterior mesial temporal region, without neocortical changes. Thus, interictal metabolic dysfunction is variable and usually extensive in TLE, whereas decreased central benzodiazepine-receptor density is more restricted to mesial temporal areas. Metabolic patterns in TLE may reflect diaschisis, while benzodiazepine-receptor changes may reflect localized neuronal and synaptic loss that is specific to the epileptogenic zone. [11C]Flumazenil imaging may be useful in presurgical evaluation of refractory complex partial seizures.