The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin

A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes.2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25–100 mM) resulted in a significant inhibition of protein synthesis.3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells.4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats.5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA.6. Acute administration of ethanol did not inhibit procollagen synthesis.7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes.8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

Albumin, fibrinogen and transferrin synthesis in isolated rat hepatocyte suspensions. A model for the study of plasma protein synthesis.

A system using hepatocyte suspensions in vitro was developed for studying the synthesis of albumin, fibrinogen and transferrin. Conditions for optimum survival of the hepatocyte and for synthesis of these plasma proteins were defined for this system. These conditions included the use of horse serum (17.5 percent, v/v, heat-inactivated), an enriched medium (Waymouth's MB 752/1), an O2 tension of between 18.7 times 10(3) and 26.7 times 10(3) Pa and constant stirring. Albumin, fibrinogen and transferrin synthesis rates were obtained of 0.32 p 0.094(10), 0.12 p 0.030(11) and 0.097 p 0.017(10) [mean p S.D. (n)]mg/h per g of hepatocytes respectively. These rates were maintained for the first 12h of study and synthesis continued at a diminished rate up to 48h. The synthesis of albumin was decreased in a medium containing less amino acids and glucose, but that of fibrinogen was substantially unaffected. ATP concentrations up to 12h and RNA/DNA ratios up to 24h were comparable with values in vivo. The ability to study cells up to 48h permitted us to find that the addition of a mixture of hormones consisting of glucagon, cortisol, tri-iodothyronine and growth hormone enhanced fibrinogen synthesis. Addition of insulin to the above mixture resulted in increased synthesis for albumin and transferrin but not for fibrinogen.     

The measurement of synthesis rates of albumin and fibrinogen in rabbits

1. A formula is proposed for calculating fractional synthesis rates of liver-produced plasma proteins that dispenses with urinary information or information about the size of the urea pool in the body or the fraction of urea that is endogenously catabolized. 2. Synthesis rates obtained for albumin and fibrinogen agreed well with corresponding catabolic rates for the ¹³¹I-labelled proteins except in two of the fibrinogen measurements. 3. Significant reutilization of ¹⁴C occurs in some animals after [¹⁴C]carbonate injections, giving rise to errors in the calculation of protein synthesis rates. These can best be avoided by using results obtained by injecting [¹³C]urea simultaneously. [¹⁵N]urea is shown not to be satisfactory for this purpose.     

A study on the intracellular transport of prothrombin, albumin and transferrin in rat

The intracellular transport of prothrombin in rat has been studied and compared with the transport of albumin and transferrin. The proteins were immunoisolated from plasma samples after pulse labelling with [3H]leucine and the secretion kinetics were determined. The half-times for secretion (t1/2) were approx. 30, 53 and 75 min for albumin, prothrombin and transferrin, respectively, whereas the minimal transit time for prothrombin was approx. 30 min, and those for albumin and transferrin 15-20 min. After injection of vitamin K-1 into warfarin-treated rats, the accumulated prothrombin precursor was gamma-carboxylated and secreted with a t1/2 of 37 min. This indicates that the gamma-carboxylation of prothrombin in rough endoplasmic reticulum cannot account for the delay in the transport of prothrombin as compared to albumin. Comparison of the incorporation of [3H]leucine and [3H]glucosamine into plasma prothrombin and transferrin suggested that transferrin is secreted randomly from an intracellular pool, whereas prothrombin is transported in a more orderly sequence. Moreover, treatment of rough microsomes with 0.05% sodium deoxycholate indicated that prothrombin is more tightly associated with the membranes of rough endoplasmic reticulum than albumin and transferrin.     

The effect of iron and ethanol on rat hepatocyte collagen synthesis.

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

The effect of hypophysectomy and growth hormone replacement on albumin synthesis and catabolism in the rat

Albumin synthesis and catabolism were respectively measured by McFarlane's (1963) sodium [(14)C]carbonate method and I-labelled albumin in hypophysectomized rats, both untreated and treated with growth hormone. Hypophysectomy resulted in a decrease in both albumin synthesis and catabolic rates. These changes as shown by pair-feeding experiments could not be ascribed to decreased food intake alone. Growth hormone was shown to partially restore both albumin synthesis and catabolic rates. It is proposed that growth hormone stimulates albumin synthesis and that its effect on albumin catabolism is secondary to changes in the mass of the intravascular albumin pool.     

The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes.

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.     

The effect of dietary protein deficiency on albumin synthesis and on the concentration of active albumin messenger ribonucleic acid in rat liver.

After severe hepatic injury induced by dimethylnitrosamine, approximately a 4-fold increase in hepatic prolyl 4-hydroxylase activity occurred within 4 days, whereas the increases in total immunoreactive prolyl 4-hydroxylase protein and in prolyl 3-hydroxylase activity were only about 1.4-fold. The different magnitudes of the increases in the prolyl 4-hydroxylase and 3-hydroxylase activities were verified after partial purification of the enzymes by gel filtration. The data support previous reports indicating differential increases in the activities of individual enzymes of collagen biosynthesis in hepatic injury. Separation of prolyl 4-hydroxylase tetramers from the monomer-size protein by gel filtration indicated that the increase in enzyme activity was similar to that in enzyme tetramers, and an increase had also occurred in the ratio of enzyme tetramers to total enzyme protein. Thus the specific activity of the tetramers had remained unchanged in liver injury. The administration of dimethylnitrosamine was also accompanied by a marked increase in the immunoreactive prolyl 4-hydroxylase protein concentration in the serum, and a similar effect was also noted after carbon tetrachloride administration, results suggesting that the increases originated in the liver.     

The acute effects of ethanol on protein synthesis in isolated rat liver cells are pH-dependent

Acute administration of ethanol to isolated rat liver cells induced a pH-dependent inhibition of protein synthesis. The effect of the alcohol was highest at pH 7.0 and nil at pH 7.8. 4-methyl-pyrazole partially reversed the action of ethanol only below pH 7.4. Time-course experiments suggested that ethanol could act preventing the initiation of new polypeptide chains stimulated by D-glucose, and that this effect is abolished at pH 7.8.     

Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

Enhanced synthesis of albumin and fibrinogen at high altitude.

The acute effects of active and passive ascent to high altitude on plasma volume (PV) and rates of synthesis of albumin and fibrinogen have been examined. Measurements were made in two groups of healthy volunteers, initially at low altitude (550 m) and again on the day after ascent to high altitude (4,559 m). One group ascended by helicopter (air group, n = 8), whereas the other group climbed (foot group, n = 9), so that the separate contribution of physical exertion to the response could be delineated. PV was measured by dilution of (125)I-labeled albumin, whereas synthesis rates of albumin and fibrinogen were determined from the incorporation of isotope into protein after injection of [ring-(2)H(5)]phenylalanine. In the air group, there was no change in PV at high altitude, whereas, in the foot group, there was a 10% increase in PV (P < 0.01). Albumin synthesis (mg. kg(-1). day(-1)) increased by 13% in the air group (P = 0.058) and by 32% in the foot group (P < 0.001). Fibrinogen synthesis (mg. kg(-1). day(-1)) increased by 40% in the air group (P = 0.068) and by 100% in the foot group (P < 0.001). Hypoxia and alkalosis at high altitude did not differ between the groups. Plasma interleukin-6 was increased modestly in both groups but C-reactive protein was not changed in either group. It is concluded that increases in PV and plasma protein synthesis at high altitude result mainly from the physical exercise associated with climbing. However, a small stimulation of albumin and fibrinogen synthesis may be attributable to hypobaric hypoxia alone.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.