Br. J. exp. Path. (1976) 57, 733

INHIBITION OF THE LIVER AND PLASMA PROTEIN ACUTE-PHASE RESPONSE IN MICE BY D-GALACTOSAMINE A. KOJ* AND A. DUBIN From the Department of Animal Biochemi8try, Institute of Molecular Biology, Jagiellonian Univer8ity, Grodzka 53, 31-001 Krakow, Poland Received for publication July 14, 1976

Summary.-Local inflammation evoked in Swiss albino mice by subcutaneous in-

jection of Celite resulted in a rise of liver tyrosine aminotransferase activity and plasma level of fibrinogen and seromucoid, while liver alanine aminotransferase activity and the plasma level of albumin and total protein remained unaltered. By measuring the incorporation of [14C] leucine, stimulation of liver and plasma protein synthesis by Celite injection was demonstrated. Administration of D-galactosamine (2.5 mg/10 g body weight) inhibited the enhanced synthesis of liver proteins, and especially of trauma-induced synthesis of plasma fibrinogen and seromucoid. The inhibitory effect of galactosamine was most pronounced when the amino sugar was injected simultaneously with Celite and then protein synthesis was measured 6 h later. The results obtained support the idea that high doses of galactosamine inhibit transcription of trauma-inducible mRNA in the liver and thus block the acute-phase response.

HIGH DOSES of D-galactosamine (GalN) are known to inhibit liver protein synthesis (Anukarahanonta, Shinozuka and Farber, 1973; Bauer, Lukashek and Reutter, 1974), and especially the synthesis of inducible liver enzymes, such as tyrosine aminotransferase EC 2.6.1.5 (Reynolds and Reutter, 1973). The mechanism of this phenomenon depends primarily on the deficit of UTP in the liver cell due to trapping of uridine phosphates by amino sugars (Keppler and Decker, 1969; Keppler et al., 1970; Keppler, Pausch and Decker, 1974). This deficit leads to inhibition of transcription, although some other events in glycoprotein synthesis, such as glycosylation and intracellular transport of secretory proteins, may also be involved (Bauer et al., 1974). Fibrinogen and some other plasma glycoproteins known as acute-phase reactants (AP-reactants) are synthesized in the liver in increased amounts after

various injuries (Koj, 1970, 1974). The mechanism of induced synthesis of APreactants is not fully understood but several data indicate that it includes enhanced formation of specific mRNAs. In the light of these data we attempted to investigate the effect of high doses of GalN in mice with local injury on the synthesis of albumin and typical APreactants. Simultaneously, the activity of 2 liver enzymes and total liver protein synthesis were determined. The results obtained prove specific inhibitory action of GaiN on inducible synthesis of liver and plasma proteins. MATERIALS AND METHODS Anima8.-Swiss albino approximately 25 g body libitum on a pelleted diet milk and were kept in a lighting.

virgin female mice, wt., were fed ad supplemented with room with natural

* Present address: Department of Pathology, Medical Centre-4N67, McMaster University, Hamilton, Ontario, Canada L85 4J9.

734

A. KOJ AND A. DUBIN

Chemicals.-D-galactosamine hydrochloride pure was obtained from Koch-Light (Colnbrook, Bucks., England), Celite from POCH (Gliwice, Poland), [14C] D,L-leucine (sp. act. 8-2 mCi/mM) and 131I (Na131I carrier-free, 10 mCi/ml) from IBJ (Swierk, Poland). All other compounds and reagents were high-purity preparations obtained from commercial sources. Experimental procedure.-In pilot experiments turpentine oil injected in 0-1 -ml doses per animal was employed as the irritant. However, in distinction to rabbits and rats, this procedure was toxic to mice, so in further studies local inflammation was evoked by s.c. injection of 0 3 ml of sterile Celite suspension (30 mg/rnl saline) in the scapular region. Galactosamine was freshly dissolved in pyrogen-free saline and injected i.p. in 0 3 ml volumes and dosage of 2-5 mg/10 g b.w. The dose was based on experiments of Anukarahanonta et al. (1973) and Bauer et al. (1974). The same volume of [14C] leucine solution in sterile saline was administered i.p. at a dose of 1 yCi/mouse at 90 min before killing, unless otherwise stated. In order to reduce the effect of diurnal variations in enzyme activity and plasma protein concentration the blood and liver samples were always taken at 9 p.m. Under ether anaesthesia blood was withdrawn from the inferior vena cava into a syringe moistened with 5% EDTA-Na2 solution. Pooled blood samples from 3 animals were centrifuged for 4 min at 10,000 g and plasma was collected. Immediately after blood sampling the liver was excised and chilled in 0-9% NaCl solution. Pooled livers from 3 mice were cut with scissors and a 2-g sample was homogenized with 8 ml cold saline in an all-glass homogenizer, then immediately frozen at - 200. After 2-4 days the homogenate was thawed, well shaken and centrifuged for 4 min at 10,000 g. The supernatant was used for further analysis. Isolation of plasma proteins.-A sample of 0-1 ml of EDTA-plasma was diluted 50-fold with saline and samples were used for determination of total protein. To the remaining sample 5 ml of 10% trichloracetic acid (TCA) containing carrier cold leucine was added, the protein precipitate was washed in 5% TCA, dissolved in 1 ml 0-2M NaOH and 0-05-0-2 ml samples were used in duplicates for measuring protein content and 14C radioactivity. Since during blood sampling a dilution of plasma with EDTA solution occurred, a suitable correction was applied as based on independent measurement of the total protein content in serum. In a series of determinations it was found that the total protein content of mouse serum was not affected by the expeiimental procedure and amounted on the average to 64-9 mg/ml (s.e. mean ± 0 93, N = 5). The mean value of 64-9 mg/ml was used to calculate the correction factor for plasma dilution in each examined

sample. The correction factors ranged from 1-13 to 1-29. Fibrinogen was isolated from 0-2 ml of EDTA-plasma as fibrin according to the procedure described by Koj and McFarlane (1968). The clot collected on a small glass rod was thoroughly washed in saline and dissolved in 1 ml of 0-2 M NaOH. Seromucoid fraction was isolated from 0-3 nil of EDTA-plasma by the procedure of Winzler (1955). The final protein precipitate was washed with 1% phosphotungstic acid, then with acetone and, after drying, dissolved in 1 ml 0-2 M NaOH. Duplicate 0-2-ml samples of fibrin or seromucoid solutions were used for measuring protein content and 14C radioactivity. The concentration of fibrinogen and seromucoid fraction in the blood was calculated after taking into account the correction factor for plasma dilution. Albumin was isolated from 0-2 ml of EDTAplasma by the TCA-ethanol method as described by Koj (1968). Final precipitate was dissolved in 1 ml 0-2 M NaOH. Duplicate 0-05-0-2 ml aliquots were used for determination of protein content and 14C radioactivity. Albumin concentration in mouse serum was in some experiments determined independently by the isotopic dilution method in which trace amounts of 131I-labelled mouse albumin were added to 0-2 ml of serum and albumin was isolated by TCA-ethanol procedure. The labelling of mouse albumin with 131J was carried out by the ICI method of McFarlane (1964). Measurements of albumin conbentration in the serum of control mice or those injected with Celite gave the following values (mean of 3 determinations): control mice- 32-9 mg/ml, 6 h after Celite-32-1 mg/ml, 12 h after Celite30 5 mg/ml, 24 h after Celite 29-8 mg/ml. Administration of GaiN had no appreciable effect on the albumin concentration in serum but the problem was not investigated in detail. Analysis of liver proteins and liver enzymes.The supernatant of liver homogenate was diluted 50-fold with physiological saline and used for measuring protein content, alanine aminotransferase (EC 2.6.1.2-AIAT) activity (Wr6blewski and Cabaud, 1957) and tyrosine aminotransferase activity (TAT) (Diamondstone, 1966). AIAT activity was expressed in /imol of pyruvate formed during 1 h incubation at 370 per 1 mg of protein, and TAT activity in ,umoles of p-hydroxyphenylpyruvate formed during 10 min at 37° per 1 mg protein. Another sample of 0 5 ml supernatant of liver homogenate was diluted with 0-5 ml of saline containing carrier cold leucine and protein was precipitated with 1 ml of 10% TCA. The sample was centrifuged and the resulting supernatant was additionally filtered. 0-1-0-2 ml aliquots of the filtrate were used to measure non-protein 14C radioactivities. The sediment

INHIBITION OF ACUTE PHASE RESPONSE was suspended in 5 ml of 5% TCA, heated for 15 min at 90° and washed in cold 5% TCA. Finally it was dissolved in 1 ml 0-2 M NaOH and duplicate samples were used for measuring 14C activity and protein content. Determination of protein content and of

radioactivities.-Protein content in plasma and liver preparations was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard. 14C radioactivities were determined using a dioxane-based Bray scintillation mixture in a Packard liquid scintillation spectrometer model 2111 with an efficiency of 75%. 1311 radioactivities were determined in a well-type scintillation counter (USB-2, Swierk) with an efficiency of 42%. Calculations.-Specific activities of plasma proteins cannot be regarded as the best indicators of the acute-phase response since they neglect important changes in the pool size of AP-reactants (Koj, 1968, 1974). For this reason total activities incorporated into individual proteins (fibrinogen, seromucoid, albumin) present in 1 ml of plasma were calculated

taking into account the actual protein concentration corrected for plasma dilution. For statistical analysis the mean values and standard error of the mean (s.e.) were calculated and the significance of differences between experimental groups was evaluated by Student's t test. RESULTS

Systemic response to injury is known to include several phenomena, among them increased synthesis of inducible liver enzymes and liver-produced plasma glycoproteins (for references see Koj, 1974). Fig. 1 shows changes in the concentration of some plasma proteins and activities of 2 liver enzymes in mice injected with Celite. The level of albumin slightly decreased after injury in agreement with previous observations in rabbits

0

HS3

735

200

a, 0.1

-H

04

12 Hours after Celite FiG. 1.-Changes in the concentration of some plasma proteins and in the activity of 2 liver enzymes in mice following subcutaneous injection of Celite at time 0. Relative values were calculated in respect of control intact mice. The results are the mean of 5 experiments (except 24 h mean of 2 determinations); * liver tyrosine aminotransferase activity; A liver alanine aminotransferase activity; E plasma albumin level; A plasma fibrinogen level; 0 plasma seromucoid fraction level.

736

A. KOJ AND A. DUBIN

(Koj, 1968). On the other hand, the level of fibrinogen and seromucoid rose continuously up to 24 h after injury suggesting enhanced synthesis of these proteins. Specific activities of liver AlAT ranged from 19 to 34 ,umol of pyruvate per mg protein but the mean values deviated by less than 10 % from those of the control group. Hence AlAT activity was not significantly affected by injection of Celite. In the case of TAT a sharp rise in enzyme activity occurred with a maximum at approximately 6 h after injury. Tsukada and co-workers (1968) and Geller, Yuviler and Schapiro (1969) reported the maximum of adaptive response of rat liver TAT at 4 h after laparotomy in rats. Reynolds and Reutter (1973) demonstrated that dexamethasone-induced TAT increase in rat liver can be totally inhibited by simultaneous administration of galactosamine (3.75 mg/10 g body wt.). Fig. 2 shows that, when galactosamine was injected to mice i.p. at a dosage of 2-5 mg/10 g simultaneously with Celite, the rise of enzyme activity was conspicuously reduced (the difference between Groups 5 and 6 being statistically signi-

ficant at P < 0-01). This may indicate similarity in the mechanism of induction of liver TAT by hormones and injury. However, at later periods after administration of Celite, this dose of GaIN had little effect on TAT activity (no statistical differences were found between Groups 7-9 and the saline-injected controls). It should be added that administration of galactosamine did not alter AlAT activity. Direct evidence on the inhibition of protein synthesis by galactosamine was obtained in isotopic experiments. In order to find out the period of maximum labelling of plasma and liver proteins in mice following a single injection of [14C] leucine, the experiment depicted in Fig. 3 was carried out. It is evident that liver proteins show a broad plateau between 60 and 90 min, while plasma proteins exhibit a definite maximum at 90 min. For this reason the animals were usually killed 90 min after injection of [14C] leucine. Preliminary observations indicated that administration of Celite or GalN (2.5 mg/10 g body wt.) had no effect on the curve of appearance of labelled proteins in plasma. Bauer et al. (1974) observed a delay in plasma glycoprotein

8

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4

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NaCl

6h

NaCl 12h

GalN 6 h

GalN 12 h

Celite Cel 6h 6 h GalN 6h

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FIG. 2. Changes in liver tyrosine aminotransferase activity following various treatments of mice. Celite was injected s.c. and saline (NaCl) and galactosamine (GalN) i.p. (2-5 mg GalN per 10 g body wt.) 6 or 12 h before sacrifice. The enzyme activities are given in ,umol of product formed per mg liver protein in 10 min and are the mean + s.e. of 5 to 11 experiments.

737

INHIBITION OF ACUTE PHASE RESPONSE

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80 C)

60 4.,

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40

_-

20

30

60 90 Time after injection of

120min

14C-leuciZie

FIc. 3.--14C radioactivities in plasma and liver of mice following i.p. injection of 1 ,Ci [14C] D, Lleucine. Relative values were calculated after assuming the maximum as equal to 100%; 0 trichloroacetic acid soluble 14C activity of liver homogenate; 0 protein-bound 14C activity of liver homogenate; LI] plasma albumin 14C activity; A plasma fibrinogen 14C activity.

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FIc. 4.-Changes in liver protein-bound 14C activity following various treatments of mice. Celite was injected s.c. and saline (NaCl) or galactosamine (GalN) i.p. (2-5 mg GalN per 10 g body wt.) 6 or 12 h before sacrifice. 1 ,uCi of [14C] D,L-leucine in 0 3 ml saline was given i.p. in all cases at 90 min before sacrifice. The results are the mean of 5 to 11 experiments and are compared with the control group 1 (injected with saline 6 h before sacrifice): (a) significantly different from Group 1 at P < 0.05; (b) significantly different from Group 5 at P < 0-01; (c) significantly different from Group 1 at P < 0-01.

738

A. KOJ AND A. DUBIN

secretion by rat liver following injection of GaiN but they used higher doses of this amino sugar while the proteins were labelled with [14C] galactose or [3H] fucose. Fig. 4 shows changes in labelling of liver proteins with [14C] leucine following various treatment of mice. For the sake of clarity the diagram represents the relative values calculated in respect of control group (1.). The mean liver protein specific activity in this group was found to be 210 dpm/mg (s.e. + 10-7). When the mean absolute values of experimental groups were subjected to statistical analysis it was found that injection of Celite stimulated liver protein synthesis (Groups 5, 7 and 8) while GalN administered simultaneously with Celite at 6 h before sacrifice inhibited this response (Group 6). In other experimental groups the observed differences were statistically not significant. It should be mentioned that, when in a single experiment the time of exposure to [14C] leucine was shortened to 30 min, the inhibitory effect of GalN was slightly more pronounced but the overall pattern remained unchanged. Both Anukarahanonta et al. (1973) and Bauer et al. (1974) observed much stronger inhibition of liver protein synthesis in rats by GalN but they employed either pulse-labelling with [14C] amino acids or injected higher doses of this amino sugar. On the other hand, our observations confirm earlier suggestions of Reutter (cited after Bauer

et al., 1974) that GalN has no effect on the accumulation of a labelled amino acid in the liver since we found the non-protein 14C radioactivity in TCA supernatant of liver homogenate unchanged by treatment of mice with galactosamine or Celite. The Table shows that injection of galactosamine had only a small effect on the radioactivity incorporated into total plasma protein or albumin but it reduced dramatically the labelling of fibrinogen in Celite-injected mice (Group 3 vs Group 4). It is clear, moreover, that the ratio of total activities incorporated into fibrinogen and albumin is particularly useful in estimating the degree of acute-phase response and its inhibition by D-galactosamine. As demonstrated previously (Koj, 1968; Koj and Dubin, 1974) such procedure decreases individual variability resulting from different external factors and from changes in the intracellular activity of the labelled precursor. Assuming a similar content of leucine in mouse fibrinogen and albumin, this ratio corresponds to the relative synthesis rate of fibrinogen. Hence the data presented in the Table indicate that the relative synthesis of fibrinogen was increased almost 3 times at 6 h after injection of Celite, but the simultaneous administration of galactosamine inhibited this reaction. It should be also noted that the inflammation evoked in mice by Celite had only a small effect on the total proteinto-albumin activity ratio in distinction to the perfused rat liver (Koj and Dubin,

TABLE.-Comparison of [14C] Leucine Activities Incorporated into Plasma Proteins of Mice Injected with Celite, Galactosamine or Saline 6 h before Sacrifice Total protein Fibrinogen Total protein to albumin Fibrinogen to albumin activity activity ratio activity activity ratio arroup Treatment dpm/ml x 100 dpm/ml x 100 1 Control 30500 218 830 5 93 2 Galactosamine 27950 193 746 5-15 3 Celite 16340 37910 232 2353 14-40 4 Celite + galactosamine 13560 26980 199 1202 8-86 Celite was injected s.c., saline and galactosamine i.p. (2-5 mg GaIN per 10 g body wt.). At 90 min before sacrifice each mouse received 1 uCi [14C] D,L-leucine i.p. Pooled plasmas from 3 mice in each experimental group were analysed. Radioactivities in total protein, albumin and fibrinogen are given as dpm/ml plasma (recalculated from specific activities of isolated proteins and their content in plasma). Results represent Albumin activity dpm/ml 13990 14480

a single experiment.

INHIBITION OF ACUTE PHASE RESPONSE

739

0 H: H r 0 xs

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GalN

GalN

Celite

6 h

12 h

6 h

Cel.6h GalN 6h

Celite Cel.12h Cel.12h 12 h GalN 12h Ga 2MN Ch

FIG. 5. The effect of galactosamine and Celite on the relative rates of incorporation of [14C] leucine into plasma fibrinogen in comparison with albumin. Relative synthesis of fibrinogen was calculated as shown in the Table. The results are the mean of 5 to 11 experiments + s.e. (a) significantly different from Group 1 at P < 0 001; (b) significantly different from Group 5 at P < 0.01. For other experimental details see Fig. 4.

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b

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5

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6 I

I

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6 h

I -,--

I

Cel.6h Celite Cel.12h Cel.12h 12 h GalN 12h GalN 6h GalN 6h

FIG. 6. The effect of galactosamine and Celite on the relative rates of incorporation of [14C] leucine into plasma seromucoid fraction in comparison with albumin. For explanation see Fig. 5. (a) Significantly different from Group 1 at P < 0 01; (b) significantly different from Group 5 at P < 0-05.

740

A. KOJ AND A. DUBIN

high doses of GaIN act primarily by blocking transcription. At a later time after injury GaIN is less effective because some trauma-inducible mRNA might already have been produced. It should be mentioned here that high doses of galactosamine failed to inhibit increased synthesis of fibrinogen and seromucoid by the perfused rat liver which was isolated 20 h after injection of turpentine oil into liver donors (Koj and Dubin, unpublished). The acute-phase response is a complicated sequence of events including an early inflammatory response and formation of intermediate humoral factors which are transported by blood to the liver and stimulate hepatocytes (Koj, 1974). The action of galactosamine on these events is not known and it cannot be overlooked. However, most of the metabolic effects of galactosamine are limited to liver, and this amino sugar is probably less cytotoxic than actinomycin D. Hence our experiments indicate that at suitable doses galactosamine can be DISCUSSION used as a rather specific inhibitor of the Several authors have demonstrated liver and plasma protein acute-phase that trauma-induced synthesis of some response. liver enzymes and liver-produced plasma glycoproteins is inhibited by actinomycin This study was carried out within the D administered shortly after injury (for research project co-ordinated by the references see Koj, 1974). On the other Institute of Biochemistry and Biophysics, hand, synthesis of albumin was not signi- Polish Academy of Sciences. We are ficantly suppressed by this antibiotic grateful to Professor M. Sarnecka-Keller (Neuhaus, Balegno and Chandler, 1966; for helpful discussions and critical reading Balegno and Neuhaus, 1970). When of the manuscript. The skilful technical actinomycin D was administered at a assistance of Mrs M. Weigt-Wadas is later time after injury, it failed to inhibit gratefully acknowledged. the augmented formation of seromucoid (Neuhaus et al., 1966) or fibrinogen (Sarcione, 1970). This pattern greatly REFERENCES resembles our results obtained in mice ANUKARAHANONTA, T., SHINOZUKA, H. & FARBER, injected with Celite and galactosamine. E. (1973) Inhibition of Protein Synthesis in Rat Liver by D-galactosamine. Res. Com. Chem. The acute-phase reaction of liver TAT and Pathol. Pharmacol., 5, 481. plasma fibrinogen and seromucoid was BALEGNO, H. W. & NEUHAUS, 0. W. (1970) Effect blocked only when GaIN was administered of Insulin on the Injury-stimulated Synthesis of Serum Albumin in the Rat. Life Sci. II, 9, 1039. simultaneously with Celite and the response BAITER, C. H., LUKASHEK, R. & REUTTER, W. G. was assayed 6 h later. This remains in (1974) Studies on the Golgi Apparatus. Cumulative Inhibition of Protein and Glycoprotein agreement with the generally accepted Secretion by D-galactosamine. Biochem. J., 142, notion that transcription is the essential 221. step in the acute-phase response, and that DIAMONDSTONE, T. I. (1966) Assay of Tyrosine

1974). It must be remembered, however, that apart from other experimental differences between perfused system and in vivo conditions, in the intact animal [14C] amino acid is incorporated also into plasma proteins of extrahepatic origin, such as immunoglobulins, which do not belong to the acute-phase reactants. The ratios of fibrinogen to albumin activities were calculated in all experiments and the mean values are shown in Fig. 5. It can be stated that injection of Celite resulted in a conspicuous rise of fibrinogen synthesis rate. Galactosamine blocked this response when administered simultaneously with the inflammatory stimulus, but at later periods was ineffective. Similar results were obtained with the seromucoid fraction (Fig. 6), although the effects of both Celite and galactosamine on the acute-phase response were less pronounced and the scatter of experimental results was greater.

INHIBITION OF ACUTE PHASE RESPONSE Transaminase Activity by Conversion of pHydroxyphenylpyruvate to p-Hydroxybenzaldehyde. Analyt. Biochem., 16, 395. GELLER, E., YUVILER, A. & SCHAPIRO, S. (1969) Tyrosine Aminotransferase: Activation or Repression by a Stress. Proc. Soc. exp. Biol. Med., 130, 458. KEPPLER, D. & DECKER, K. (1969) Studies on the Mechanism of Galactosamine Hepatitis: Accumulation of Galactosamine- 1-phosphate and its Inhibition of UDP-glucose Pyrophosphorylase. Eur. J. Biochem., 10, 219. KEPPLER, D., RUDIGIER, J., BISCHOFF, E. & DECKER, K. (1970) The Trapping of Uridine Phosphates by D-galactosamine, D-glucosamine and 2-deoxy-D-galactose. Eur. J. Biochem., 17, 246. KEPPLER, D., PAUSCH, J. & DECKER, K. (1974) Selective Uridine Triphosphate Deficiency Induced by D-galactosamine in Liver and Reversed by Pyrimidine Nucleotide Precursors. Effect on Ribonucleic Acid Synthesis. J. Biol. Chem., 249, 211. KoJ, A. (1968) The Measurement of Absolute and Relative Synthesis Rates of Fibrinogen in Vivo. Biochim. biophys. Acta., 165, 97. KoJ, A. (1970) Synthesis and Turnover of Acutephase Reactants. In Ciba Foundation Symposium on Energy Metabolism in Trauma. Ed. R. Porter and J. Knight. London: Churchill. p. 79. KoJ, A. (1974) Acute-phase Reactants. In Structure and Function of Plasma Proteins. Ed. A. C. Allison. London and New York: Plenum Press, Vol. 1, p. 73. KoJ, A. & McFARLANE, A. S. (1968) The Effect of Endotoxin on Plasma Albumin and Fibrinogen Synthesis Rates as Measured by the 14C Carbonate Method. Biochem. J., 108, 137.

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KoJ, A. & DUBIN, A. (1974) On the Hormonal Modulation of Acute-Phase Plasma Protein Synthesis in Perfused Rat Liver. Acta biochim. polon., 21, 159. LowRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265. McFARLANE, A. S. (1964) Metabolism of Plasma Proteins. In Mammalian Protein Metabolism. Ed. H. N. Munro and J. B. Allison. New York and London: Acad. Press. Vol. 1, p. 297. NEUHAIJS, 0. W., BALEGNo, H. F. & CHANDLER, A. M. (1966) Induction of Plasma Protein Synthesis in Response to Trauma. Am. J. Physiol., 211, 151. REYNOLDS, R. D. & REUTTER, W. (1973) Inhibition of Induction of Rat Liver Tyrosine Aminotransferase by D-Galactosamine. J. Biol. Chem., 248, 1562. SARCIONE, R. J. (1970) Regulation of Plasma a2 Acute-Phase Globulin Synthesis in Rat Liver. In Plasma Protein Metabolism. Ed. M. A. Rothschild and T. Waldmann. New York and London: Acad. Press. p. 427. TSUKADA, K., OURA, H., NAEKASHIMA, S. & HAYASAKT, N. (1968) Adaptive Response of Tryptophan Pyrrolase and Tyrosine Transaminase in Rat Liver after Partial Hepatectomy and Laparatomy. Biochim. biophys. Acta, 165, 218. WINZLER, R. J. (1955) Determination of Serum Glycoproteins. In Methods of Biochemical Analysis. Ed. D. Glick. New York: Acad. Press. Vol. 2. p. 279. WROBLEwSKI, F.J& CABAUD, P. (1957) Colorimetric Measurement of Serum Glutamic'Pyruvic Transanimase. Am. J. clin. Path., 27, 235.