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We have investigated the modifications of the levels of intracellular markers of the oxidative stress in hepatocytes, after single or repeated injections of poly(isobutyl cyanoacrylate) (PIBCA) and polystyrene (PS) nanoparticles. Nanoparticles were administered intravenously at single doses of 20 and 100 mg kg 1 for 14 days. Levels of reduced (GSH) and oxidized (GSSG) glutathione, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CT) and the peroxidation of membrane lipids were measured. Single and repeated administration of PIBCA and PS nanoparticles induced a transient depletion of GSH and GSSG levels, a transient inhibition of SOD activity and a slight increase in CT activity. However, GPx activity was not modified and lipid peroxidation was not observed, suggesting that hepatocytes are not strongly affected by these modifications. Since nanoparticles do not distribute in hepatocytes, oxidative species could proceed from hepatic macrophages, activated after nanoparticle phagocytosis. It is unlikely that poly(alkyl cyanoacrylate) degradation products might be responsible for the oxidative attack because non-biodegradable PS nanoparticles induced the same effect. Uptake of polymeric nanoparticles by Kupffer cells in the liver induce modifications in hepatocyte antioxidant systems, probably due to the production of radical oxygen species. However, the depletion in glutathione was not great enough to initiate hepatocyte damage, since no changes in lipid peroxidation and reversible alterations were observed. This is an important factor to be considered in the use of polymeric nanoparticles as drug carriers.

We investigated the consequences of acute and subacute administration of mice with polyisobutylcyanoacrylate (PIBCA), polyisohexylcyanoacrylate (PIHCA), poly(D,L-lactic) acid (PLA), and polystyrene (PS) nanoparticles on the mononuclear phagocyte system phagocytic function. This was done by measuring the clearance rate of colloidal carbon. Single administration of PIBCA and PIHCA (but not PLA and PS) nanoparticles reduced carbon clearance in both a time- and dose-dependent fashion. Since clearance of preopsonized carbon was normal, it was assumed that PIBCA and PIHCA nanoparticles deplete opsonins specific for carbon recognition. A decrease in plasma fibronectin levels resulting from nanoparticle administration suggested its implication in their removal from blood. However, fibronectin does not seem to be responsible for PIBCA and PIHCA blockade. Phagocytic function was preserved after repeated treatment with nanoparticles, probably as a result of increased Kupffer cell phagocytic activity and the contribution of spleen macrophages. Neither toxicity nor effects due to nanoparticle hepatic accumulation were observed.

We have studied the effects of two modulations--streptozotocin-induced diabetes in vivo, and the presence of the carboxylic proton ionophore monensin in vitro--on the degradation of 3H-asialoorosomucoid ligand in isolated rat hepatocytes. The ligand was internalized by means of a synchronous wave procedure. Diabetes was associated with a marked decrease in the amount of total degraded radioactive ligand compared to that in normal cells (3.6% and 37.3% of internalized ligand respectively, at 60 min), together with increased secretion of degradation products into the incubation medium (87% and 46.3% of the total degraded ligand was secreted by diabetic and normal cells, respectively). Monensin induced similar effects in normal cells, but had no apparent effect in diabetic cells.

The hepatic asialoglycoprotein receptor is a membrane glycoprotein used as a model to study receptormediated endocytosis. In order to examine the ability of second messengers to modulate intracellular trafficking, we performed a comparative study on normal and diabetic rat hepatocytes exploring the effects of an in vivo modulation, streptozotocin-diabetes, and an in vitro modulator, vasopressin, which transduces signals via the phosphoinositide pathway. We studied three main experimental aspects: (1) constitutive endocytosis, (2) continuous ligand flux, and (3) a synchronous wave of ligand. In normal cells, vasopressin decreased ligand-binding capacity by 20%, without altering the mechanism of internalization, and decreased the level of degradation, without affecting the distribution of degradation products. Diabetic cells were characterized by a 50% decrease in cell-surface and intracellular receptor ligand-binding capacity, slowed internalization of a synchronous wave of ligand, and markedly reduced degradation with an altered distribution of degraded products. Vasopressin had no additive effect on the modification induced by diabetes. These results suggest that second messengers generated by hormones play a role in the regulation of receptor-mediated endocytosis. They also confirm that receptors are subdivided into those susceptible to modulation of any kind and those insensitive to modulation, although the boundary between the two subsets is variable.

The influence of experimental conditions was investigated in pulse and pulse-chase experiments involving l -[ 35 s]methionine incorporation by isolated rat hepatocytes. The incorporation of the labelled amino acid must be linear as a function of dose and time, and similar hepatocyte densities should be used to compare radioactive uptake. High hepatocyte densities give greater precision. In pulse-chase experiments, it was shown that a pool of free radioactivity moves progressively during the chase phase from the intracellular compartment. Protein-associated radioactivity must therefore be expressed as a function of both total radioactive uptake and total labelled protein.

The hepatic asialoglycoprotein receptor is the first studied mammalian lectin. Modulations in vivo by diabetes and in vitro by the carboxylic ionophore monensin gave rise to similar apparent alterations on its biosynthesis, structure and ligand binding capacity. In normal rats, the receptor (whether purified by ligand or antibody-affinity chromatography) presented a similar pattern in SDS-PAGE analysis, with a major 42-kDa band and twop minor ones (49 and 52–54 kDa). In diabetic rats, a new 38-kDa band appeared, but only after antibody-affinity purification. In vitro biosynthesis of the receptor by normal hepatocytes in the presence of 35 S-methionine showed that this 38-kDa band was present at the end of a 30-min pulse but decreased during a 180-min chase, in association with an increase in the major 42-kDa band. In diabetic cells, this evolution was retarded. Using a 30-min pulse followed by a 120-min chase in the presence of 100 μM monensin, we showed that this carboxylic ionophore had similar effects on diabetes, leading to a delay in the maturation process of the 42-kDa band and the persistent emergence of the 38-kDa species. Allowing incubation in the presence of 25 or 100 μM monensin, we observed a decrease in the number of ligand binding sites both at the surface (40%) and within the cell (28%). In hepatocytes from diabetic rats, monensin showed no additional effect on the partial diabetes-induced activation.

The ability of second messengers to modulate receptor-mediated endocytosis was studied on isolated rat hepatocytes. A 20-min preincubation with vasopressin was used as a modulation. We observed a 20% inactivation of both surface and intracellular receptors, with no change in the affinity of those remaining active. The internalization and dissociation of a synchronous wave of ligand was not affected, but its degradation was partially inhibited. Our observations suggest that second messengers such as intracellular calcium and diacylglycerol play a complex role in the intracellular trafficking associated with endocytosis.

After exposure to a concanavalin A (Con A)-unreactive variant of alpha 1-acid glycoprotein (AGP), macrophages released an inhibitor of interleukin-1 (IL-1) proliferative activity in the thymocyte comitogenic assay. This effect was observed with AGP concentrations above 100 micrograms/ml in the macrophage supernatant and would appear to be mediated by the macrophages, since native AGP had no activity on thymocyte proliferation. Preliminary physicochemical characterization showed that the factor was partially resistant to heating, undialyzable, and eluted with an apparent molecular mass of 50-100 kDa when subjected to Sephacryl S-200 chromatography. Murine IL-1 and human (h) recombinant (r) IL-1 were affected by this factor to the same extent. IL-1 and IL-2 co-induced thymocyte proliferation, which is mitogen-independent, was also inhibited, whereas hrIL-2 activity was not suppressed when assayed in thymocytes with PHA at a submitogenic concentration or in CTLL cells. The factor did not interfere with TNF alpha or hrIL-6 activity when tested against their specific cell line. These data indicate that the inhibitor may act specifically against IL-1 activity and further elucidate the possible role of AGP in the modulation of IL-1 activity via the secretion of an inhibitor.

Based on the affinity for concanavalin A (Con A), human alpha 1-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the mannosyl-fucosyl receptor.

The metabolic turnover of the Hepatic Binding Protein (HBP) was investigated in streptozotocin-diabetic rats. We have already shown that diabetes induced a decreased ligand binding capacity while the immunoreactive HBP was normal. To explore the eventual modifications due to diabetic state upon the turnover of HBP, we followed the in vivo degradation of HBP and its biosynthesis in vitro . After in vivo labelling with l -[ 3 H] leucine and purification of HBP from rat livers, we found a 20% decrease in diabetic HBP half-life. By in vitro incubations of freshly isolated hepatocytes and a 2 h-pulse in the presence of l -[ 35 S] methionine, we showed that diabetes provokes an increased uptake of l -[ 35 S] methionine in hepatocytes allowing an augmented synthesis of HBP although the l -[ 35 S] methionine incorporation into total proteins was less efficient.

Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.

We have characterized a binding site for galactosyl terminal glycoproteins in hepatocytes isolated from human biopsies. The binding of asialoorosomucoid on hepatocytes previously treated by Triton X-100 was saturable, calcium-dependent and highly affine ( K a = 1.11 ± 0.87 · 10 9 M −1 ) thus corresponding to a ligand-receptor binding. The total number of receptors in the normal human liver was 140 000 ± 65 000 sites per cell. This corresponded to the value obtained in the human hepatoma cell line HepG2, but was significantly lower than for isolated rat hepatocytes. Furthermore, in hepatocytes isolated from livers with histological features of either fibrosis, cirrhosis, hepatocarcinoma with cirrhosis or nodular regenerative hyperplasia, the number of asialoglycoprotein receptors per cell was increased, while the binding affinity was unchanged.

The intermediary metabolism in hepatocytes isolated from diabetic rats has been studied. The incubation medium is a Krebs-Henseleit buffer containing albumin-bound oleate (1 mmol/l). The high rates of gluconeogenesis, ureogenesis and ketogenesis were consistent with the diabetic state of the rats. Insulin (8 X 10(-7) mol/l) decreased glucose and urea productions from alanine (10 mmol/l) by 30%; beta-hydroxybutyrate production, oleate utilization and malate efflux (calculated) from mitochondria were also decreased. No effect of insulin was found with lactate (10 mmol/l) as gluconeogenic substrate. The glycogen content of cells was constant during the time of incubation (4h). These data suggest that the oxidation-reduction state of mitochondria and the cytoplasmic oxaloacetate concentration could be important factors in the action of insulin.

We have recently shown that the administration of phenobarbital to rats leads t an increased serum α 1 -acid glycoprotein content with alterations in the relative proportion of the sugar moiety. Therefore, α 1 -acid glycoprotein was purified from normal ( α 1 -acid glycoprotein N ) and phenobarbital-treated rats ( α 1 -acid glycoprotein PB ). Glycans were separated by AX-10 chromatography and analysed by gas chromatography. It appears that, compared to α 1 -acid glycoprotein N , α 1 -acid glycoprotein PB had a higher carbohydrate content (31.7% compared to 26%) and a non-negligible amount of neutral oligosaccharide (12.2% compared to 1.3%). No tetrasialyl oligosaccharides in α 1 -acid glycoprotein PB were detected, whereas their relative proportion in α 1 -acid glycoprotein N was 27%.

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.

The initial velocity of asialoorosomucoid internalization is determined for normal and diabetic rat hepatocytes. The analysis of results according to Woolf-Hofstee's method, showed no modification of the endocytosis constant. In contrast, the maximum velocity of asialoorosomucoid internalization is decreased by threefold in diabetic rat hepatocytes as compared to normal rat hepatocytes. No modification of internalization constant is observed between the two groups of rats. This suggests that the decrease of asialoorosomucoid total uptake, previously reported for diabetic rat hepatocytes is directly related to a decrease of total surface receptor number.

Young and old human erythrocytes, separated in vitro according to age, were labeled at the surface-sialic acid residues by sodium periodate and borotritide treatment. No qualitative difference was observed between the sialic acid derivatives of young- and old-erythrocyte membranes. The number of labeled residues was significantly decreased in the in vivo aged erythrocytes (12 +/- 2.8 X 10(6); n = 8) when compared to the young ones (19.4 +/- 2.8 X 10(6); n = 8). Analysis of the labeled glycoproteins by sodium dodecyl sulfate gel-electrophoresis showed that this decrease affects the PAS-1, PAS-4, PAS-2, and PAS-3 bands (glycophorins A and B) of the old-erythrocyte membranes.

The concanavalin A-unreactive variants of alpha 1-acid glycoprotein introduced in culture medium of monocytes/macrophages induces the inhibition of thymocyte proliferative activity of interleukin 1. LPS or LPS receptors were not involved in the activity of alpha 1-acid glycoprotein on macrophages. alpha 1-acid glycoprotein did not show any direct effect on thymocytes whereas the monocyte/macrophage-supernatant inhibited the interleukin 1 proliferative effect. The activity of tumor necrosis factor and interleukin 2 was not altered by the alpha 1-acid glycoprotein-macrophage supernatant.

The calibration curves for evaluating the degree of desialylation of C1-inactivator (sialic acid content 12.5%) and haemopexin (sialic acid content 4%) have been plotted. No desialylation of either glycoprotein occurs in normal subjects. In the patients (liver damage) studied, C1-inactivator is often desialylated, whereas haemopexin is not. In a previous report, we had shown that alpha1-acid glycoprotein is more often desialylated than alpha1-antitrypsin. Thus, it appears that the degree of desialylation of the sialic acid-rich glycoproteins is a more sensitive index of the severity of hepatic injury than that of the sialic acid-poor glycoproteins. This could be due to a defect in the sialylation process during synthesis.

The in vivo kinetic of removal of 3H asialo-orosomucoid from plasma was investigated in control and streptozotocin-diabetic rats after intravenous injection of 1 mg of asialo-orosomucoid/100 g body wt. Michaelis-Menten kinetics of disappearance were observed. In diabetic rats the maximal rate (Vmax) of disappearance of 3H asialo-orosomucoid was decreased by 30% with no modification of Michaelis constant. Since no accumulation of desialylated orosomucoid in the circulation was observed, the slower rate of removal of 3H asialo-orosomucoid was attributed to a decrease in the number of hepatic asialoglycoprotein receptors which are largely involved in the catabolism of asialoglycoproteins. Our estimate on in vivo maximal rates was 10- to 20-fold greater than our previous in vitro estimate of the maximal rate of endocytosis. In contrast, the values of the Michaelis constant obtained in vivo and in vitro were very similar.

A diet rich in proteins is given to rats for at least 20 days; this regimen causes metabolic modifications. They can be measured in vitro by the gluconeogenesis of hepatocytes from fasted animals. The presence of fatty acids in the incubation medium is necessary to reveal an increase in gluconeogenesis, which varies according to the substrate. There is a concomitant increase in fatty acid catabolism; this is in good correlation with ATP needs.

Summary A plasmatic glycoprotein is submitted to a mild periodate oxydation and its pharmacological activity is studied. This glycoprotein contains much N acetyl Neuraminic Acid (NANA = 15 p. cent), and it reduces the biological activity of histamine on smooth muscle such as guinea pig ileum. With mild oxydation, oxydation ratio consumed m-periodate/NANA = 1.10, 1.55, 1.90, we establish that one mole of HCHO is liberated for one mole of NANA. We also identify the 8 NANA and 7 NANA derivatives. The only 8 carbon derivative is obtained when about one mole of m-periodate is consumed for one mole of NANA. The 7 carbon derivative appears as soon as the consumption of a second mole leads to a second cleavage. These results prove that the oxydation is limited to the sole N acetyl neuraminic acid and more precisely to the lateral polyhydroxylic chain. Under these conditions, pharmacological activity gradually decreases, it disappears as soon as the lateral polyhydroxylic chain is completely cut off.

en Electroimmunodiffusion methods of Laurell and radial immunodiffusion method of Mancini are compared for the qualitative and quantitative analysis of native and desialylated α 1 -acid glycoprotein. Samples are incubated under different conditions at decreasing pH (3.5 to 0.5 pH units), with increasing ionic strength and with neuraminidase during different time intervals. Results show a pronounced decrease in electrophoretic mobility of α 1 -acid glycoprotein treated either with acidic reagents or with neuraminidase (ionic strength has no effect). Such a procedure might involve chemical or enzymatic hydrolysis by which sialyl residues are removed. This hydrolysis implicates lower results in the estimation of the desialylated glycoprotein by electroimmunodiffusion. On the other hand, the amounts of α 1 -acid glycoprotein evaluated by radial immunodiffusion are not modified after incubation. This is expected since diffusion and antigenic properties are not related to the sialic acid content. The data suggest that radial immunodiffusion, less accurate and sensitive than electroimmunodiffusion, is nevertheless more adequate for estimating native and desialylated α 1 -acid glycoprotein.

Lipogenesis from U(14C) lactate was studied in hepatocytes isolated from obese Zucker rats (fa/fa) their lean littermates (Fa/?) and Sprague Dawley rats. The distribution of radioactive carbon between the glycerol and the fatty acid moieties of the acylglycerols were studied. Radioactive lactate was better utilized for glycerol formation than it was for fatty acid formation in the obese rats. However, when oleate was added to the hepatocytic incubation medium, radioactive lactate was preferentially incorporated into the fatty acid moiety of the acylglycerols. Zucker obesity classified as a "metabolic obesity" by Meyer (1) depends upon abnormalities in carbohydrate metabolism associated with increased lipogenesis. This might be explained by biochemical shifts in the utilization of nutrients (2). Among the nutrients, lactate seems to be a better source of carbon than glucose for lipid synthesis (3). It has been shown that there is an increased hepatic portal blood concentration of lactate several hours after eating: about 4 mM in Wistar rats (4) and 10-15 mM in obese Zucker rats (3). We are interested in determinating the incorporation of carbon from lactate either into glycerol or into fatty acyl moieties of hepatic acylglycerols, and in determining the influence of exogenous fatty acids on acylglycerol synthesis, since a high level of circulating fatty acids in Zucker obese rats has been reported (5). Our purpose was to determine the incorporation of lactate into glycerol and fatty acyl moieties of acylglycerols, under the influence of oleate. Hepatocytes were isolated from ad libitum fed obese Zucker rats (fa/fa), their lean littermates (Fa/?) and Sprague-Dawley rats (SD). Incorporation of lactate was studied for three hours, in order to exclude short-term regulation effects and to allow oleate to be distributed into all cellular compartments.

This report concerns the plasma factor responsible for the decrease of the biological activity of histamine on the isolated guinea-pig ileum.1 The results presented prove that its plasma factor combines with histamine.2 The combination between histamine and our protein causes a decrease in its inhibitory effect: the histamine bound to the plasma factor cannot be determined either by a fluorimetric or biological method, and acid hydrolysis is necessary to release the histamine from its complex.

The authors determine the titre of two monospecific immunosera by the radial immunodiffusion method. Because of the precision and rapidity of this technique, it is most suitable for:--periodic quality control of an immunoserum--the quantitative estimation of specific immunoglobulins adsorbed to the supporting medium in affinity chromatography.

A method for measuring proteins in low concentrations applying the zone immunoelectrophoresis assay is reported. The low detection limit makes it possible to measure alpha 1-acid glycoprotein in rat serum and also to quantify the secretion of this protein after concentration of the incubation media containing less than 10(7) isolated rat hepatocytes. The method is simple and consumes very small quantities of antiserum.

The number of cell surface and total asialoglycoprotein receptors was investigated in normal and diabetic rat hepatocytes using 2 methods: ligand and polyclonal antibody binding. An identical number of immunoreactive receptors was found in both types of cells, while the ligand binding activity of cell surface receptors was reduced by 58% in diabetic rats compared with normal ones, or by 33% for total cell receptors.

The total capacity of hepatocytes to bind asialoorosomucoid was measured on normal and streptozctocin diabetic rats. 4 days after the streptozotocin injection, a slight decrease of total receptor concentration was observed while a more marked reduction of cell surface receptor occurred. In animals sacrified 11 days after the streptozotocin injection, the total capacity of hepatocytes to bind asialoorosomucoid was about 70 % of the normal level.

Rats with Jejunoileal bypass were used to study the biological activity of the hepatic binding protein. Hepatocytes were prepared 11 weeks after surgical procedure, and presence of asialoo-rosomucoid in serum has been determined. Compared to control rat hepatocytes, endocytosis of [3H]asialoorosomucoid by bypassed rat hepatocytes was decreased by about 60%. This was due to a decreased number of total and surface receptors. In most sera, an accumulation of asialoorosomucoid was found. A reduction of protein synthesis or turnover could be considered.

The binding of asialoglycoproteins by hepatic binding protein was studied in freshly isolated hepatocytes from genetically diabetic BB Wistar rats. The number of cell surface asialoglycoprotein receptors was dramatically decreased (58,000 +/- 38,000 for diabetic rats compared to 267,000 +/- 70,000 for normal rats), while the association equilibrium constant was not changed. These results parallel those obtained with streptozotocin-diabetic rats and support the hypothesis that insulin deprivation is responsible for the decrease in the receptor number.

The inhibitory effect of sodium arsenite and azide on asialoorosomucoid endocytosis was tested using isolated rat hepatocytes. Under either continuous flux conditions or a single synchronous wave of ligand endocytosis we confirm that azide inhibits the recycling of the receptors and we provide evidence for the involvement of thiol groups in the internalization step. In addition pretreatment of hepatocytes with azide allows us to demonstrate that receptor endocytosis proceeds independently of the presence of any specific ligand.

Oxidation by galactose oxidase followed by reduction with tritiated sodium borohydride appears to be an excellent tool to label a protein. By this technique, we have labeled an histamine blocking protein. We try now to modify the experimental conditions in order to have a clear cut between specific and non specific binding of this protein to guinea-pig ileum.

Normal mouse spleen cells treated with periodate were stimulated to undergo blastogenesis. In contrast spleen cells from nude mice did not respond to periodate. Such a treatment of normal and nude spleen cells led to the oxidation of the cell surface sialic acid residues with formation of N-AN8. Prior treatment with neuraminidase of normal spleen cells greatly impaired their capacity to respond to periodate activation with a decrease in the amount of N-AN8 formed.

When the two immunological methods, radial immunodiffusion (RID) and electroimmunodiffusion (EID), were used for the determination of alpha 1-acid glycoprotein, a significant discrepancy in the results was encountered depending on the degree of sialylation. It appeared that with desialylated alpha 1-acid glycoprotein, amounts estimated by EID were much lower than those actually present as assayed by the RID method. Determination of glycoprotein samples treated with neuraminidase for varying periods of time revealed an increasing underestimation by the EID method with decreasing sialic acid content. Partial resialylation of asialo-alpha 1-acid glycoprotein by bovine colostrum beta-galactoside alpha (2 leads to 6) sialyltransferase on the other hand resulted in less underestimation. Differential determination of alpha 1-acid glycoprotein by the two immunological methods thus offers a method for the estimation of the degree of sialylation of alpha 1-acid glycoprotein and other sialoglycoproteins in serum and body fluids.

A method for evaluating the degree of desialylation of α 1 -AGP and α 1 -at has been developed. It consists in their simultaneous determination by radial immunodiffusion (RID) and electroimmunodiffusion (EID). When a desialylation exists, an underestimation by EID relative to RID is found. 1. (1) No significant desialylation of α 1 -AGP and α 1 -AT occurred in normal subjects. 2. (2) No correlation between desialylation of α 1 -AGP and α 1 -AT and their amounts existed. 3. (3) Desialylation was preferentially observed in patients with severe hepatic damage but also with inflammatory disorders.

The albumin, orosomucoid and alpha 2-macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine-induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure it.

When radial immunodiffusion (RID) and electroimmunodiffusion (EID) were used for the determination of rat alpha 1-acid glycoprotein (alpha 1-AGP) a significant discrepancy in the results was encountered depending on the degree of sialylation. When alpha 1-AGP was desialylated, the amounts estimated by EID were much lower than those actually present as assayed by the RID method. The relationship between the percentage of desialylation of alpha 1-AGP and the percentage of its underestimation by EID relative to RID was determined and a calibration curve was plotted to evaluate the degree of desialylation of rat alpha 1-AGP. When compared to other procedures (rat membrane inhibition assay and isoelectrofocusing), the proposed method was easier to perform and allowed the specific evaluation of the degree of undersialylation of the glycoprotein.

The binding and the total uptake of [3H]asialoorosomucoid was studied in normal rats, streptozotocin-diabetic rats and insulin-treated diabetic rats. 1 The binding of [3H]asialoorosomucoi'd to normal rat hepatocytes gave a curvilinear plot when analysed by the Scatchard method, suggesting either two classes of independent receptor sites with different apparent association constants or a negative cooperativity in binding. The data, plotted according to the method of De Meyts and Roth showed a decrease of the affinity constant with respect to site occupancy. 2 The Scatchard plot obtained for the [3H]asialoorosomucoid binding to streptozotocin-diabetic rat hepatocytes was curvilinear. According to the independent site model, the receptor number of each class was decreased, without any change of the apparent association constants. According to the model of De Meyts and Roth, no modification of the interaction factor and apparent affinity constants was observed, whilst the total number of receptor sites was decreased by half in streptozotocin-diabetic rats as compared with normal rats. 3 Study of the total uptake of [3H]asialoorosomucoId by hepatocytes from each group of rats showed a decrease by half of the endocytosis in streptozotocin-diabetic rats as compared with normal rats. 4 Insulin treatment of the streptozotocin-diabetic rats restored both parameters studied to normal level, whilst the withdrawal of insulin treatment decreased their level to that of streptozotocin-diabetic rats. These results are related to possible modifications of the hepatic binding protein, receptor for the plasma asialoglycoproteins, in the diabetic state.

Using high concentrations of extracellular [3H]asialoorosomucoid we obtained the steady-state level of [3]asialoorosomucoid endocytosis by isolated hepatocytes from normal and streptozotocin diabetic rats. At the steady-state of the overall reaction, the intracellular amount of [3H]asialoorosomucoid did not change with time, the apparent overall rate of [3H]asialoorosomucoid degradation was close to that of [3H]asialoorosomucoid internalization, in both normal and diabetic rat hepatocytes. Although in diabetic cells the intracellular amount of [3H]asialoorosomucoid was about three-times lower than in normal cells, the same fraction of intracellular asialoorosomucoid was degraded per time interval by both normal and diabetic cells. The apparent first-order rate constant of steady-state degradation was about 0.011 min−1 for both normal and diabetic cells. In diabetic rat hepatocytes, the decrease of the clearance of serum asialoglycoproteins was directly correlated to the variation of cell surface receptor number, without any modification of internalization and degradation steps.