Protein synthesis in the whole body, liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis

Infernational Journalfor Panrsifolgy Printed in Greaf Brifoin.

Vol. 12. No. 4, pp. 295-301, 1982.

ml20-7519/82/040295-07$03.00/0 Pergomon Press Lfd. Society for Parasitology

0 1982 Australian

PROTEIN SYNTHESIS IN THE WHOLE BODY, LIVER, SKELETAL MUSCLE AND KIDNEY CORTEX OF LAMBS INFECTED BY THE NEMATODE TRICHOSTRONGYL US COLUBRIFORMIS W. 0. JONES and L. E. A. SYMONS CSIRO,

Division

of Animal

Health,

McMaster Laboratory, 2037, Australia

(Received

Abstract-JONES W. 0. and muscle and kidney cortex

Private

Bag No. 1, P.O.

Glebe,

N.S.W.

13 Ju@ 1981)

L. E. A. 1982. Protein synthesis in the whole body, liver, skeletal lambs infected by the nematode Trichostrongylus colubriformis. International Journal for Parasitology 12: 295-301. Tyrosine flux and the synthesis of protein in the whole body, liver, skeletal muscle and kidney cortex and of albumin in lambs infected with Trichostrongylus colubriformis and uninfected lambs fed ad libitum or pair-fed with the infected group, were measured by constant infusion of 14C-L-tyrosine. Live weight gain was lower in the infected than in pairfed lambs, but rates of whole body protein synthesis were similar in both groups. On the other hand, compared with control lambs, there was a faster rate of protein synthesis per unit of protein consumed in infected but not in pair-fed lambs. Rates of protein synthesis per unit of body weight in infected were higher than in pair-fed lambs, but similar to the rate in control lambs. The fractional synthetic rates (FSR) of albumin and liver proteins and the amount of liver protein synthesized per day were increased by infection. The FSR and amount of protein synthesized per day were depressed in skeletal muscle and kidney cortex. Anorexia did not explain any of these changes. Infection caused a loss of protein from each of these tissues, but this loss was due to anorexia in only the liver. There was generally good correlation between concentration of RNA per g fresh weight or per mg nitrogen and the FSR of protein. However, although the RNA/DNA ratio correlated well with synthesis in skeletal muscle, it was poorly correlated for liver proteins. The relationship between the rate of growth and protein synthesis in infected lambs is discussed. INDEX

KEY WORDS:

SYMONS

Protein

of

synthesis;

whole body; sheep; liver; muscle; kidney;

albumin;

nematode;

Trichostrongyluscolubriformis.

INTRODUCTION INCORPORATION of 1%~L-leucine into protein by mice infected with Nematospiroides dubius and by guinea pigs and sheep infected with Trichostrongylus colubriformis indicated slower rates of protein synthesis in skeletal muscle and faster rates in liver (Symons & Jones, 1971, 1972, 1975, 1978; Symons, Jones & Steel, 1974). The validity of this technique for assessing protein synthesis can be questioned, since precursor specific radioactivities (S.R.) were not measured and different precursor pool sizes may affect the amount of radioactivity incorporated. Nonetheless, in some instances, concomitant changes supported the interpretation of the incorporation results. For instance, there were relevant changes in the incorporation of labelled leucine by isolated ribosomes or microsomes, the expected proportions of heavy to light ribosomes and in the case of muscle, falls in RNA concentrations. Whole body protein turnover was shown to be 295

increased in young mice infected with N. dubius (Symons & Jones, 1972). Infection of guinea pigs with T. colubriformis increased the percentage in the whole body of 14C-L-leucine in the liver and intestine but decreased that in the carcase and skin (Symons & Jones, 1981). Since these are the only data on whole body protein metabolism and the protein synthesis data referred to above was only comparative, more detailed and precise measurements of synthesis in the individual organs and the whole body are required to assess quantitatively the synthetic changes already recorded. A method for the measurement of protein synthesis in the whole body and individual tissues by constant infusion of tracer amino acid has been developed by Garlick, Millward & James (1973). Results in a number of other species using constant infusion methods have been reviewed by Reeds & Lobley (1980). This method was used to determine whole body, skeletal muscle, liver, kidney cortical protein and albumin synthesis in lambs infected with T.

2%

W. 0. JONESand I.. E. A. SYMONS

colubriformis and in uninfected animals pair-fed with them or allowed to eat ad libitum. Pair-fed controls were used since anorexia contributed to changes in incorporation of labelled leucine by tissues of infected sheep (Symons & Jones, 1975). Protein synthetic rates and RNA concentrations in the above tissues were compared to help clarify the uncertainty regarding results previously found in liver (see e.g. Symons & Jones, 1975). MATERIALS AND METHODS

Animals and infec!ion. Twenty-four worm-free ewe lambs, in one batch of 12 Merino x Border Leicester crosses and another of 12 Merinos, all aged from 15 to 23 weeks, were divided into three treatment groups of similar mean live weight. One group was infected with T. colubriformis (infected), the second group uninfected but pair-fed with sheep in the infected group (pair-fed), and the third group uninfected but fed ad libitum (control). The infected sheep were dosed orally throughout the experiment with 18,000 infective larvae per week, the dose being divided into three equal parts which were administered on Mondays, Wednesdays and Fridays. All sheep were housed in metabolism cages and given pellets containing 50% lucerne and 50% wheaten hay (11% crude protein) and water ad libitum. Experjmen~ai procedures. Food intake was recorded daily and body weight weekly. Blood was taken from the jugular vein immediately before the first dose of larvae and fortnightly thereafter, and the plasma stored at -20°C until analysed. Worm counts in infected sheep were made at the conclusion of the experiment. Between weeks 8 and 11 of infection, food intake of the infected lambs had fallen to about 50% of their corresponding controls, their plasma albumin concentrations had fallen appreciably below preinfection level, they were generally losing weight and had soft and unformed faeces. At this time all the lambs were constantly perfused by the method described for pigs by Garlick, Burk & Swick (1976). An infected lamb, its corresponding pair-fed and control were perfused concurrently on any one day. A nylon can&a was inserted into the jugular vein and taped into position the night before infusion. The following morning the cannula was connected to the syringe of a constant infusion pump and 2 @i/ml of L-[“Y(U)]-tyrosine (499 mCi/m mol) in 0.9% saline infused at the rate of 5 ml/h for 6 h. At the end of the infusion period about 30 ml of plasma was taken from the jugular vein and stored at -20°C. The animals were killed by cervical dislocation and exsanguination and the whole liver, both semitendonosus muscles and the cortices of the kidneys were removed and weighed. About 30-50 g of liver, both muscles and the kidney cortices were stored in liquid nitrogen.

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Analyses. Specific radioactivities of free tyrosine in plasma and free and protein-bound tyrosine in tissues were determined as described by Garlick et al. (1976). Albumin was isolated from plasma by the addition of 20 ml saturated ammonium sulphate to 20ml plasma. The precipitate was spun down in a refrigerated centrifuge at 50,OOOgfor 20 min, the supernatant adjusted to pH 4.4 with 5 M H,SO, and the precipitate recovered as before. This precipitate was dissolved and the pH adjusted to 7-8 with 1 M NaOH. Free SO& was removed by dialysis in distilled water at 4”Cand the material redialysed against 0.02 M phosphate buffer, pH 8, for 24 h. It was then applied to an A50 DEAE-Sephadex 25 x 4.50mm column with a gradient of 0.02-0.4 Mphosphate buffer, pH 8, to elute the albumin. The albumin was then hydrolysed and the specific activity of bound tyrosine determined as for other tissues. RNA, DNA and nitrogen of tissues were estimated as previously described (Symons & Jones, 1971, 1975). Plasma albumin and globulin concentrations were estimated as described by Steel, Symons & Jones (1980) and plasma urea by an automated method (Technicon method AAII-01 for the Autoanalyzer II). Ca~c~~atjons.For estimation of the tyrosine flux, i.e. the flow of tyrosine from the plasma free amino acid pool, and the fractional synthetic rate (FSR) Garlick ef ai. (1973, 1976) should be consulted. The amount of protein synthesized by the whole animal was calculated from the tyrosine flux on the assumption that sheep protein contains 3% tyrosine. The amount of protein synthesized in the liver, skeletal muscle and the cortex of the kidney was calculated from the FSR using the total protein (from total nitrogen determinations) of each tissue. A computer programme was used to calculate the rate constant and the plateau S.R. of plasma tyrosine. Another computer interative procedure was used to determine the FSR from the ratio of proteinbound to free tyrosine S.R. in each tissue. The liver intracellular free tyrosine S.R. was used to calculate albumin synthesis. Statistical analrs;is. Data was analyzed using analysis of variance in which times of experiments were removed as block factors. Differences between means were tested for significance at the 5% level. Muscle FSR was also examined b; the Wilcoxon’s signed rank test. RESULTS

Food intake and body weight Infected lambs harboured over 30,000 adult parasites, except for one with 5,200, and their protein intake over the final seven days was about 48% of controls. Final body weights and weekly rate of gain of the infected lambs were significantly less than those of the controls. The rate of growth of the infected group was also less than the pair-fed animals (Table 1).

TABLE ~-DAILY PROTEIN INTAKE OVER FINAL SEVEN DAYS, FINAL BODY WElCki~s AND RATES 0F CHANGE OF LAMBS INFECTED WlTH Trichostrongylus colubriformis. MEANSOF EIGHT SHEEP PER EXPERIMENTALGROUP

Mean protein intake (g d-l) Body weight (kg) Body weight gain (kg week-l)

Infected

Pair-fed

CORtrOlS

63” 23.0” -0.02c

65b 27.2b 0.38”

13@ 30.6a 0*68a

S.E.D.*

8 5.5 0.10

*~.~.~.=standard error of difference between means. Means with different superscripts differ significantfy at the 5% level.

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Whole body protein synthesis in trichostrongylosis

Tyrosine flux and protein synthesk Whole body. Contrary to the observations for pigs by Garlick et al. (1976), preliminary measurements

showed that the appearance of a plateau of the S.R. of free tyrosine in blood was not affected by feeding or fasting. Consequently, lambs were allowed to eat during infusion. The rate constants of the exponentials that described the formation of the plateau of free tyrosine S.R. in plasma were 40, 40 and 43 per day in the infected, pair-fed and control animals, respectively. In the whole body the protein synthesized per day is directly proportional to the tyrosine flux, nonetheless, tyrosine flux has been reported since this is the parameter determined experimentally. In the infected and pair-fed lambs the flux was depressed relative to the controls (Table 2). However, tyrosine flux per kg body weight was increased in infected compared to pair-fed animals, but

297

was similar to that in the controls. When allowance was made for protein ingested, tyrosine flux in infected lambs exceeded that of the controls. Liver. The FSR and daily synthesis of liver protein by the infected lambs was greater than in both the uninfected groups (Table 3). Liver RNA per g fresh weight or per mg nitrogen reflected the increased liver protein synthesis in infected lambs. However, RNA/DNA concentration ratio and total liver content of RNA were lower in both the infected and pair-fed groups than in the controls. Total liver nitrogen was reduced in both infected and pair-fed lambs. Albumin, globulin and urea. The plasma concentration of albumin in the infected lambs was depressed compared with both uninfected groups and with preinfection levels (Table 4) but the increased plasma globulin concentration was not significantly different.

TABLE 2-TYROSINE FLL'X AND RATE OF PROTEIN SYNTHESIS IN THE WHOLE BODY OF LAMBS INFECTED WITH Tr~chosrro~g~t~~ colubriformis

Tyrosine flux (mmole h-l) Protein synthesized (g d-l) Tyrosine flux per kg body weight (mmole h-1) Tyrosine flux per g protein intake (mmole h-1) Number of sheep

infected

Pair-fed

2,54b 368b

2.14” 31Ob

3.83= 555a

0.30

0.115a

0.08 1b

0.127a

0.026

0.041b 6

@033ab 7

0*029= 8

OGO5

COIltrOk

S.E.D.

See footnotes to Table 1. TABLE~-FRACTIONALSYNTHESISRATE,PROTEINSYNTHESIZEDAND RNA AND NITROGEN CONCENTRATIONS IN THE LIVER OF LAMBS INFECTED WITH Trichostrongylus colubriformis. EIGHT LAMBS EXCEPT WHERE INDICATED IN PARENTHESES

infected FSR (d-1) Protein synthesized (g d-l) RNA/f.w.* (mg g-1) RNA/N (mg mg-I) RNA/DNA (mg mg-I) Total RNA (g) Total nitrogen (g)

Pair-fed

COIItrOk

0.724b(6) 0469a(6) 0.346=(7) 47.7b(6) 3 1.2qq 29.4a(7) 622b 5.55a 5.80a o-21 Ia 0.234” 0*199= 3.02” 2.82b 3.50a 2.31c 1.96b 2.75a IO.lb 9.4b 14.0a __

S.E.D.

0.083 6-5 0.14 o@J9 0.12 0.10 0.7

See footnotes to Table 1. *f.w. = fresh weight. TABLE d--PLASMA ALBUMIN, GLOBULIN AND UREA CONCENTRATION AND ALBUMINSYNTHESIS OFLAMBSINFECTEDWITH Trichostrongyluscolubriformis. MEANS OF EIGHT UNLESS OTHERWISE INDICATED IN PARENTHESES

Infected Albumin (g I-1) Globulin (g I-1) Urea N (mg 1-t) Albumin % pre-infection Albumin FSR (d-l) See footnotes to Table 1.

22.7b 32.1(7) 226b 7Ob O.lSlb(6)

Pair-fed 33,6= 27.6

Controls

32+9a 30.9 I soa 176a 102a 98a O.O67a(S) O.O88a(6)

S.E.D.

2-i 2.2 20 6 0.018

298

W. 0. JONESand

L. E. A.

SYMONS

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1982

TABLE ~-FRACTIONAL SYNTHESIS RATE, PROTEINSYNTHESIZED PER DAY AND RNA AND NITROGEN CONCENTRATIONS IN THE PAIRED SEMITENDONOSUS MUSCLES OF SHEEP INFECTED WITH Trichostrongylus colubriformis. EIGHT LAMBS EXCEPT WHERE INDICATED IN PARENTHESES -._

Infected

Pair-fed

COIlWOk

FSRt (d-1) 0,0347b(6) O.O363ab(7) 0.242b(6) 0.319a(7) Protein synthesized (g d-l) 0.699b 0.678b RNA/f.w. (mg g-1) 0.032b 0.038ab RNA/N (mg mg-1) 1.98b 1.7lb RNA/DNA (mg mg-1) OG47b 0.039b Total RNA (g) I .4Ja 1.06b Total nitrogen (g) _..-. See footnotes to Table 1. j-Significance tested by Wilcoxon’s signed rank test.

0.0392a 0.345= 0.817a 0.043a 2-6Oa 0.060a

S.E.D.

0+030 0.034 0.047 0.003 0.25 0.005 0.09

1.40a

TABLE ~-FRACTIONAL SYNTHESIS RATE, PROTEIN SYNTHESIZED PER DAY AND AND NITROGEN CONCENTRATIONS IN THE KIDNEY CORTEX OF LAMBS INFECTED WITH Trichostroffgyl~scolubr~~ormjs.EIGHT LAMBS EXCEPT WHERE INDICATED IN PARENTHESES

RNA

FSR (d-1) Protein synthesized (g d-1) RNA/f.w. (mg g-1) RNA/N (mg mg-1) RNA/DNA (mg mg-1) Total RNA (g) Total nitrogen (g)

Infected

Pair-fed

0*305a(6) lG+lb(6) 4-22 0.19 0.9sb Os2Ob 1.07b

0,542b(7) 4,63a(7) 4.06 0.17 0.96” 0.21” 1.30ab

Controls

S.E.D. -.

0.4OOab 3*91= 4.03 0.17 1.19a 0.25a

1.54a

0.07 I 0.87 0.15 0.01 0.05 0.01 0.11

See footnotes to Table f . Plasma

urea was increased

FSR of

albumin

in infected

in the infected

lambs

Iambs.

The

was 2 or

3

times higher than in uninfected lambs. Muscle. In spite of the small differences and the variation found in the FSR of muscle protein which

made the analysis of variance not significant, a consistentiy lower value in the infected group returned a significant result when tested by Wilcoxon’s signed rank test (see Table 5). The FSR of muscle protein in the infected animals was significantly lower than that in the controls, but not that in the pair-fed animals. The amount of muscle protein synthesised per day by the infected lambs was significantly lower than by either the pair-fed or control animals. On the other hand, although the concentrations and total amount of RNA in muscle of infected lambs were generally significantly lower than in controls they did not differ significantly from values in pair-fed lambs. The total amount of nitrogen in muscle was lower in the infected lambs than in either pair-fed or control animals. Kidney. In the kidney cortex, the FSR and daily protein synthesized were depressed in the infected group compared with the pair-fed group (Table 6). The daily protein synthesis was also depressed compared to the controls. Total RNA and RNA per unit DNA in the infected and pair-fed animals were de-

pressed, whereas there were no differences between experimental groups when RNA was expressed in terms of fresh weight or nitrogen. Total nitrogen was depressed in the infected lambs compared with the other two groups. ~e~af~o~shi~ betweerz FSR and RNA co~~e~fraf~ons

Correlation coefficients of mean FSR to mean RNA concentration for the three tissues, for the three treatment groups and for all values combined are recorded in Table 7. RNA expressed in terms of TABLE MEAN

DNA,

~-C~IWELATI~N

COEFFICIENTS 0~ MEAN FSR ~0 PER g FRESH WEIGHT, mg NITROGEN AND mg FOREACH'rISSUE,EXPERIMENTALGROUP,ANDFORALL TISSUESCOMBINED ._

RNA

Liver Muscle Kidney cortex

FSR V. RNA (f.w.)-’ RNA N-1 RNA DNA-’ 1.oo 0.76 -0.50 0.97 0.63 1.00 -0.66 -0.71 -0.08

Infected Pair-fed Controls

0.96 0.91 0.88

Combined tissues

0.89

-

0.91 0.94 0.95

0.72 -0.19 -0.26

0.91

0.11

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Whole body protein synthesis in trichostrongylosis

fresh weight or nitrogen generally correlated well with FSR, except in kidney. There was poor correlation between FSR and the ratio of RNA to DNA, except for muscle, or for the infected animals as a group. DISCUSSION The weekly dose of larvae produced significant worm burdens, reduced food consumption and rate of live weight gain as expected from previous experience (Steel el al., 1980) and affected protein metabolism without endangering the life of the lamb. Measurement of protein synthesis by the constant infusion method produced results in the controls that compare satisfactorily with earlier reports. The rate constant of the exponential expression which described the rate of increase of plasma S.R. to a plateau in these lambs, weighing about 3 1 kg, can be compared with a constant of 50 in 76 kg pigs (Garlick et al., 1976). The rate of whole body protein synthesis of 555 g per day by these lambs is within the range of 600 and 400 g per day reported in sheep weighing 20 and 63 kg respectively (Buttery, Beckerton & Lubbock, 1977; Reeds & Lobley, 1980). In general, FSR is inversely related to body weight. The FSR of 0.346 per day for liver protein in the controls is within the range of 0.23-0.68 per day which has been reported in cattle, pigs, rabbits and rats (Lobley, Milne, Lovie, Reeds & Pennie, 1980; Garlick et al., 1973, 1976; Nicholas, Lobley & Harris, 1977). The FSR of 0.039 per day for skeletal muscle of the controls is also within the range of 0.02-0.048 for skeletal muscle in the above species. Measurements of tyrosine flux indicated a reduced whole body protein synthesis in infected and pair-fed animals (Table 2) which was consistent with their lower body weight and food intake. When allowance was made for these factors different trends become evident. Tyrosine flux per unit body weight of the infected lambs was significantly increased compared with the pair-fed lambs. The increase was such that the flux on a body weight basis in infected lambs was virtually the same as in uninfected lambs fed ad lib. This demonstrates the importance of using pair-fed control lambs, since a specific effect on protein metabolism due to parasitism, but not attributable to anorexia, is clearly indicated. The faster flux per unit of protein intake by the infected animals shows the extra metabolic burden on a nutritional basis. The calculation of whole body protein synthesis (Table 2) assumes that all tyrosine leaving the free plasma pool is used for protein synthesis. While Garlick et al. (1973) considered other pathways, particularly oxidation, in their calculation, it is possible that in infected animals a greater proportion of tyrosine is being catabolised. However, the tyrosine flux is still a valid indication of the replacement of this amino acid in the whole body. The concomitant increase in tyrosine flux and plasma urea in the infected lambs (Table 4) is consistent with the sugges-

299

tion of Roseby (1973) that increased urea production was due to increased amino acid catabolism in sheep with trichostrongylosis. A greater enteric loss of plasma occurs in T. colubriformis infected than in pair-fed sheep (Barker, 1973). In addition to plasma protein increased enteric loss of other proteins, such as exfoliated epithelial cells and mucoproteins is likely (see Steel & Symons, 1979). Much of this endogenous protein is probably reabsorbed from the ileum (Symons, 1976). This loss and reabsorption of amino acids would contribute to the increased amino acid flux observed in infected lambs. Furthermore, the loss of nitrogen from liver, muscle and kidney (Tables 3, 5 and 6) and possibly from other tissues, indicates further release of amino acids into the circulation. The most important component contributing to tyrosine flux is protein synthesis per se. Increased protein synthesis was found in the liver (Table 3, discussed later). The increased plasma globulin concentration in trichostrongylosis of sheep (Steel et al., 1980; Table 4) suggest increased globulin synthesis. As well, protein synthesis associated with the developing immunological response is likely to be increased. Of course, a decrease in protein synthesis, such as found in muscle and kidney cortex (Tables 5 & 6) occuring alone would cause a decreased flux rate, but in spite of this, the net flux rate was increased. The rate of liver protein synthesis in the infected lambs was markedly increased and yet there was clearly no change related to anorexia. This synthesis must have been of fixed liver proteins, as Garlick et al. (1973) point out that the method used measures only these proteins, since plasma proteins (including albumin) synthesized in the liver remain therein for only about 30 min. The faster rate of liver protein synthesis confirms the interpretation of earlier experiments (Symons & Jones, 1971, 1978). Increased liver protein synthesis in infected animals is consistent with the increased flux of tyrosine on a body weight basis. This flux presumably would reflect an increased amino acid turnover associated with increased metabolic activity in the liver, such as an increased synthesis of enzymes associated with amino acid metabolism, for example those of the urea cycle. The observed increase in plasma urea is consistent with this hypothesis. Although elevated enteric plasma protein loss has been considered responsible for increased liver synthesis of exported proteins found in guinea pigs with trichostrongylosis, (Symons et al., 1974), it must be emphasised that it is the synthesis of fixed liver proteins that has been measured in the present study. Another possible reason is the effect of higher plasma cortisol and lower plasma insulin concentrations in sheep infected by T. colubriformis reported by Prichard, Hennessy & Griffiths (1974). The relationship of RNA concentration to FSR in liver is discussed later. Anorexia apparently accounted for

300

W. 0. JONESand L. E. A. SYMONS

the loss of nitrogen in liver (Table 3) as was found earlier (Symons & Jones, 1975). The hypoalbuminaemia of the infected lambs, which is characteristic of several gastrointestinal nematode infections, including trichostrongylosis of sheep (Coop, Sykes &Angus, 1976), is attributable to enteric plasma loss and a decreased amount of albumin synthesised (Steel et al., 1980). The faster FSR of albumin in the infected lambs is consistent with the faster rate of albumin turnover in lambs with trichostrongylosis (Steel et al., 1980). Anorexia was clearly not responsible for the higher FSR of albumin in the liver. The anorexia associated with T. colubriformis infection was previously shown to account for the depressed incorporation of leucine into skeletal muscle protein (Symons & Jones, 1971, 1975, 1978). In the present experiment, reduced food intake did not significantly reduce the rate of muscle protein synthesis in uninfected lambs and, similarly, loss of muscle nitrogen was observed in the infected, but not in the pair-fed lambs. However, significantly decreased muscle RNA which would be consistent with reduced protein synthesis was observed in both infected and pair-fed lambs. The inappetance of the infected lambs was not as great as previously (Symons & Jones, 1975), and may account for the unchanged protein synthetic rates found in the pairfed animals. The kidney cortex was examined since proteins synthesized therein are not exported and its protein synthetic rate is similar to that of liver. Anorexia was not responsible for the reduced protein synthesis in the kidney cortex of infected lambs (Table 6). The lower nitrogen content of kidney cortex in infected lambs was consistent with the reduced protein synthesis. There was some uncertainty in regard to the effect of infection and the anorexia associated with it on RNA concentrations. Falls occurred only in total RNA and RNA/DNA ratio in both infected and pair-fed animals, which apparently indicated a lack of relationship of RNA and protein synthesis in the kidney cortex. Correlation coefficients showed that concentration of RNA per unit of fresh weight or nitrogen was indicative of the relative rates of protein synthesis in liver, skeletal muscle and tissues generally. This agrees with other reports (Earl, Laurent, Everett, Bonnin & Sparrow, 1978; Garlick et al., 1976). These relationships are unaffected by infection with T. colubrtformis or by limiting food consumption of uninfected animals. The poor correlation between RNA concentration and protein synthesis of the kidney cortex is possibly related to the small changes of RNA being compared. On the other hand, although RNA relative to DNA is indicative of relative protein synthesis in muscle, it is a quite unsatisfactory marker for protein synthesis of liver proteins. This finding explains the earlier apparently contradictory observation that rates of synthesis of

I.J.P. VOL. 12. 1982

liver proteins increased despite falls in RNA/DNA ratios (Symons & Jones, 1971, 1975). The more precise methods used in this work have clarified most of the doubts about previous findings and have shown particularly differences between the anorexic and other effects of infection on protein synthesis in trichostrongylosis. The results obtained indicate a diversion of amino acid away from productive processes in infected lambs e.g. synthesis of muscle protein, into non-productive e.g. synthesis of liver proteins and recycling through the gut as a consequence of enteric protein loss or into other pathways required to maintain life. This diversion does not occur, to the same extent, as a result of anorexia and so explains why pair-fed animals do not lose weight to the same extent as infected animals. Acknowledgements-The Mr. M. Stewart, and statistical analysis of acknowledged.

valued technical assistance of the advice on mathematical and Dr. D. Griffiths, are gratefully

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