YOUNG AE KANG-LEE - ANDALFRED E. HARPER Departments of Biochemistry and Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 ABSTRACT The metabolic fate of threonine was investigated in young male rats fed 15% amino acid diets containing from 0.15% to 0.85% of L-threonine. Liver serine-threonine dehydratase (S-TDH) activity did not increase with increasing dietary threonine content. The level of threonine required for maximum weight gain was not greater than 0.55% of the diet (or about 600 ^moles/day). Tissue free threonine content of rats fed the diets with 0.15% or 0.3% of threonine was very low but increased sharply with increasing dietary threonine content above 0.3%. During ad libitum feeding of these diets containing L-[U-14C]threonine, rate of oxidation of threonine was low when intake was in the range of the requirement for maximum growth, but increased, thereafter as threonine intake increased. A 30-fold induction of liver S-TDH, by prior feeding of an 80% casein diet, did not result in increased oxidation of threonine when dietary threonine content was 0.15%. When dietary threonine content was increased to 0.5%, oxidation of threonine increased slightly but significantly. With 3% of threonine in the diet, rats previously fed a 15% casein diet had extremely high tissue threonine concentrations whereas those with high S-TDH activ ity, due to the previous feeding of the 80% casein diet, oxidized threonine rapidly and tissue threonine concentrations were elevated much less. J. Nutr. 108: 163-175, 1978. INDEXING KEY WORDS amino acid metabolism •threonine intake •serine-threonine dehydratase •threonine oxidation •tissue threonine concentration •threonine requirement (rat) Many enzymes in metabolic pathways for amino acid catabolism undergo adaptation in animals in response to changes in protein and amino acid intake ( 1-4 ). As amino acids cannot be stored in the animal body (1, 2, 5, 6), and as amino acids which are indispenable cannot be synthesized in the body (7), it is important for survival that amino acids be conserved when the supply is low and that they be

• j. j En v jJ i i OXldlZed rapidly When the supply exceeds the rermirpmpnr 19 T> WP VIQI/P r>r«> me requir UK in (¿, o;. we nave previOUsly shown that histidine is Oxidized liu.1 4.-1 J.U J- i. i e i ••

very little until the dietary supply of histi-

dine is adequate for maximum growth and that oxidation increases rapidly with increasing intake of histidine beyond the requirement. Thus, measurements of the rate of histidine oxidation in rats fed graded levels of histidine have potential as a procedure for estimating the requirement for histidine (8). Similar observations have Receivedfor,PublicationJanuary si im.

! Supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and bJT «rant AM 10748 from the National Institutes of HeaUhi Bethesda, Maryland. 2 Present address: Home Economics Building, Keimyung University 2139 Nam-Gu, Dae Myung Dong,

Daegù, Korea.

163

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Threonine Metabolism in Vivo: Effect of Threonine Intake and Prior Induction of Threonine Dehydratase in Rats1

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MATERIALS

AND METHODS

Animals. Male Holtzman rats weighing 70 to 80 g were used in all experiments. They were kept in individual suspended wire mesh cages at 23°in a room that was lighted from 21:30 to 09:30. Food and water were available ad libitum. Diets. All diets contained in percentage by weight, salt mix (30), 5; corn oil, 5; vitamin mix (30), 0.5; choline chloride, 0.2. Equal amounts of glucose monohydrate3 and cornstarch4 were added to make up 100%. All diets for the study of threonine contained 14.3% of a mixture of

crystalline amino acids (30) devoid of threonine. To this, threonine was added at the expense of carbohydrate to provide 0.15, 0.3, 0.4, 0.5, 0.55, 0.7, 0.85 or 3.0% in the diet. Diets used to induce different levels of liver S-TDH contained 15 or 80% of casein,5 which was added at the expense of carbohydrate. A complete amino acid mixture (30) replaced part of the casein on an equal weight basis in the diets fed during the latter part of the induction pe riod to acclimate rats to the amino acid diets fed subsequently. Diets were pre pared as gels by mixing dry diets with an equal amount of an agar solution that had been heated to boiling temperature. The agar solution was added little by little to the diet so cooling occurred rapidly. Radio active diets used in the oxidation experi ments were prepared by mixing dry diets with equal amounts of boiling 3% agar solution, as for the feeding experiment, and a solution of L-[U-14C]-threonine °in dis posable syringes as described previously (31). Each rat received 2 ¿tCiof radio activity. Tissue preparation. Plasma was prepared by centrifuging 7 the blood at 10,000 X g for 15 minutes. Liver was homogenized with four volumes of ice-cold 0.25 M su crose in a glass tissue homogenizer with a teflon pestle. Supernatant fraction for S-TDH assay was prepared by centrifug ing 7 liver homogenate at 42,000 X g for 50 minutes. Muscle (lower leg) was homoge nized 8 with nine volumes of distilled water. All tissue preparations were stored at —20° until assayed. S-TDH assay. In view of the evidence that in rats, serine dehydratase and threo nine dehydratase appear to be a single protein (22, 32), S-TDH activity was mea sured with serine as substrate by the method of Freedland and Avery (22) with a few modifications (33). Threonine assay. Threonine concentra tions in plasma, liver and muscle were de3 CPC International, Inc., Englewood Cliffs, N.J. »B.A. Raillon Co., Chicago, 111. 5 Vitamin-free casein, General Biochemicals, Inc., Chagrin Falls, Ohio. 6 New England Nuclear Corp., Boston. Mass. Spe cific radioactivity = 186 mCi/mmole. 7 Servali RC2-B centrifuge, Ivan Sorvall, Inc., Newtown, Conn. 8 Polytron homogenizer, Kinematica Gmbh., Luzern, Switzerland.

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been made with lysine by Brookes et al. (9, 10) and with methionine by Aguilar et al. (11). We have also shown that when the in take of histidine is low or barely adequate, induction of liver histidase (L-histidine ammonia-lyase EC 4.3.1.3) to high levels by previous feeding of a high protein diet does not result in increased oxidation of histidine. On the other hand, when the intake of histidine is greatly increased, the high liver histidase activity is important for rapid catabolism of excess histidine and the maintenance of homeostasis of tissue histidine concentrations (8). Weight losses of rats fed diets lacking one of the indispensable amino acids are markedly different depending on the amino acid which has been omitted. Weight loss due to threonine deficiency is more severe than weight losses due to deficiencies of several other amino acids ( 12-18 ). Also, the net protein utilization (NPU) of most amino acid mixtures devoid of one in dispensable amino acid is not zero ( 19, 20 ), but a mixture devoid of threonine gave a NPU of zero (20). These observations suggest that threonine may not be con served as well as other amino acids when intake is low. Therefore, we have studied the metabolism of threonine in rats fed diets containing graded levels of threonine. Also, since serine-threonine dehydratase (S-TDH, EC 4.2.1.16) can be induced by a high protein diet (21-29), we have also investigated the effect of prior induction of S-TDH by a high protein intake on the metabolism of threonine in rats fed differ ent levels of dietary threonine.

E. HARPER

165

METABOLISM OF THREONINE IN VIVO

(34).

EFFECT OF THREONINE INTAKE ON WT GAIN S. GAIN/FOOD ¿ Ã-

RESULTS

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Effect of dietary threonine content on threonine metabolism Growth and food efficiency. Table 1 shows average food intake, weight gain and gain:food of rats fed diets containing graded levels of threonine. Each of these variables increased as the threonine con tent of the diet was increased from 0.15% to 0.55%. Increasing dietary threonine con tent beyond 0.55% did not result in any further increases. In figure 1, weight gains of individual rats are plotted against average daily threo nine intakes. Minimum threonine intake required for maximum weight gain esti mated from this plot was about 600 ^moles/day. A plot of weight gain against

• ¿

200 400 600 800 1000 1200 THREONINE INTAKE C^iMOLE/DAY)

Fig. 1 Effect of threonine intake on weight gain. Values are for rats described in table 1. Each point represents an individual rat. Threonine in take was estimated from food intake and dietary threonine content. The regression line for the three lower levels of dietary threonine was drawn using least squares method. The horizontal part of the curve is based on the average weight gain with the three higher levels of dietary threonine.

dietary threonine content indicated that the threonine requirement for maximum weight gain and gain:food was not greater than 0.55% of the diet. Tissue threonine concentrations. Tissue threonine concentrations of rats fed diets graded in the level of threonine are shown in figure 2. Plasma threonine concentration remained low until dietary threonine con tent exceeded 0.3%, then increased gradu ally until dietary threonine content reached TABLE 1 0.55%, whereupon it increased sharply Effect of threonine content of the diet on food intake with each further increment of threonine. and weight gain1 The pattern of change in liver threonine concentration followed the same trend as Dietary intake2g/day5.8±0.1310.9±0.413.6±0.414.9±0.414.5±0.514.6±0.4Weight gain2g/day-0.92±0.062.1 for plasma threonine concentration. Muscle threonine%0.150.30.40.550.70.85Food food-0.16±0.010.19±0.010.33±0.010.44±0.010.45±0.010.47±0.01 threonine concentration decreased slightly as the dietary threonine content was in creased from 0.15% to 0.3% and then in ±0.194.5 creased with increasing increments of ±0.256.6 threonine. These results differ somewhat ±0.246.5 from those obtained with histidine (8) in ±0.36.9 that tissue histidine concentration pla±0.1Gain: teaued when dietary histidine content was 1All rats were fed a diet containing 7.5% of casein slightly above the requirement. and 7.5% of a complete amino acid mixture for 2 In vivo threonine oxidation. After the days and then a diet containing 15% of the complete amino acid mixture for the next 2 days before various diets containing graded levels of experimental diets were fed. Experimental diets were threonine had been fed for 13 to 15 days, fed for 13 to 15 days. 2Food intake and weight were measured every other day. 3Mean±sBMof oxidation experiments were carried out on eight rats. Initial body weights were 70 to 80 g.

"Technicon Instruments, Tarrytown, N.Y.

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termined using an amino acid analyzer.8 Tissue samples were deproteinized with 15% sulfosalicylic acid. Supernatates were gassed with N2 in ampules, sealed and then heated at 75°for 2.5 hours to hydrolyze asparagine and glutamine. In vivo oxidation experiments. In vivo oxidation experiments were carried out in open-circuit glass metabolism cages as de scribed previously (8). Statistical analysis. The significance of the difference between two arithmetic means was evaluated by Student's i-test

166

YOUNG AE KANG-LEE

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Fig. 2 Effect of dietary threonine content on plasma and tissue threonine concentrations. Values are for samples taken at the end of the oxidation experiment described in table 2. Each point repre sents the average of four rats. Vertical lines indi cate SEM.

rats fed their respective diets containing L-[U-I4C]-threonine. Recoveries of radio activity at the end of the 12-hour oxidation

E. HARPER

experiment are shown in table 2. The per centage of absorbed radioactivity recovered in expired CO2 was fairly high with the lowest dietary level of threonine, but de creased with increasing dietary level of threonine up to 0.4%, then increased with further increases in dietary threonine con tent. The probability of threonine recycling through coprophagy is low as the experi ment was of only 12-hour duration and the design of the cages was such as to keep coprophagy to a minimum. The percentage of radioactivity recovered in urine was small for all groups but showed a pattern of change similar to that for CO2. The per centage of absorbed dose retained in the body tended to show a change in the op posite direction. About 90% of the ab sorbed dose was retained in the body in all groups. A considerable proportion of the absorbed dose (9% to 12%) was found in liver, but very little was recovered in brain (2did not increase as rapidly with in creasing level of dietary threonine above the requirement as was previously observed with histidine ( 8 ). The sum of the amounts of threonine utilized for weight gain, oxi dized to CO2 and excreted into urine should account for most of the threonine absorbed. Despite the assumptions made in the calculations, the amounts of threo nine accounted for agree reasonably well with the amounts absorbed for the two lower levels of dietary threonine but were considerably lower than the amounts ab sorbed for the four higher levels. This dis crepancy may be explained partly by the substantial increases in the size of free threonine pools with increasing content of threonine in the diet. The total amounts of free threonine in plasma,11 liver and mus cle,12which account for about 50% of body weight, were 8.4, 6.4, 24.3, 68.2, 161.8 and 231.4 Amólesfor 0.15, 0.3, 0.4, 0.55, 0.7 and

0.85% threonine groups, respectively. Since recoveries of radioactivity, as shown in table 2, were over 95% it would appear that a substantial proportion of the ab sorbed threonine was retained as free thre onine or threonine metabolites during the 12 hours of the oxidation experiments. Liver S-TDH activity. S-TDH activity of livers taken at the end of the oxidation ex periment are shown in table 5. In agree ment with earlier reports (22, 24), threo nine did not induce hepatic S-TDH activ ity. In fact, S-TDH activity tended to decrease with increasing dietary threonine content but the differences observed were not significant. Effect of threonine intake and prior induction of S-TDH on threonine metabolism Rats were allowed to adjust for 6 days to diets containing either 15% or 80% of casein to induce different levels of liver S-TDH; then animals from each treatment group were fed diets containing 15% of amino acids and 0.15, 0.5 or 3.0% of threo nine. Oxidation experiments were done on the second day after the rats had been changed to the amino acid diet regimen. All rats were fed ad libitum their respec tive diets containing L-[U-14C]-threonine during the 12-hour dark period and expired 11Plasma volumes were estimated according to Fernandez et al. (78). 12Total muscle weights were estimated according to Miller (79).

169

METABOLISM OF THREONINE IN VIVO TABLE 5 Effect of the dietary threonine content on liver serine-threonine dehydratase activity1

activityunit/liver1.38±0.282.74

liver0.44±0.11"0.45±0.180.35

wt93.3±0.36.0±0.17.6±0.38.4±0.38.7±0.59.

wt.1.62 100 g body

±0.332.20±0.571.69±0.521.54±0.391.34 ±0.732.58±0.762.73±0.732.31±0.402.33 ±0.120.33±0.100.28±0.060.25±0.05S-TDH ±0.51unit/

±0.221.31±0.29Liver

1Values are for livers taken at the end of the oxidation experiment described in table 2. of NADH disappeared per minute at 30°,pH 8.5. 3Mean ±SEMof four rats.

CC>2was collected for the entire 12-hour feeding period. Liver S-TDH activity and plasma and liver threonine concentrations of samples taken at the end of the oxidation experi ment and percentage of absorbed radio activity expired as 14CO2 for the 12-hour period are shown in table 6. Liver S-TDH activity was induced greatly in groups pre treated with the 80% casein diet. The average increase in S-TDH activity was 36.8-, 42.6- or 51.4-fold depending on whether the enzyme activity was expressed per g liver, per liver or per 100 g body weight. S-TDH activity decreased with in creasing threonine content of the amino acid diet regardless of whether the rats had previously been fed 15% or 80% casein diets and the difference between the ex treme values was significant. With 0.15% or 0.5% of threonine in the diet, the proportions of the absorbed dose oxidized by rats previously fed the 15% casein diet were similar to those observed in the initial experiment (table 2). The percent oxidized increased only slightly as the threonine content of the diet was in creased from 0.85% (10.9% of the ab sorbed dose, table 2) to 3.0% (12.6% of the absorbed dose). Rats previously fed the 80% casein diet oxidized only slightly more of the absorbed threonine than those pretreated with 15% casein when the threonine content of the diet was 0.15% or 0.5%. The increase with 0.5% threonine was, nevertheless, significant. However, when rats in which S-TDH had been in duced by prior feeding of the 80% casein diet were subsequently fed the diet con

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Dietary threonine0.150.30.40.550.70.85unif/g

2Unit = ¿»moles

taining 3% of L-threonine, they oxidized 31.4% of the absorbed dose, a 2.5-fold in crease over the value for those previously fed the 15% casein diet. Plasma threonine concentration of rats previously fed the 15% casein diet in creased twofold as dietary threonine con tent was increased from 0.15% to 0.5%. Increasing the dietary threonine content from 0.5% to 3.0% resulted in a 45-fold increase in plasma threonine concentration. Plasma threonine concentration of rats previously fed the 80% casein diet also in creased with increasing threonine content of the diet, but much less than in rats previously fed the 15% casein diet. There was no difference between the plasma threonine concentrations of the groups previously fed 15% or 80% of casein when the threonine content of the diet was 0.15%. These results are consistent with the higher rate of oxidation observed for rats pretreated with 80% casein. Patterns of change in liver threonine con centration followed the same trends as for plasma threonine concentration. DISCUSSION The amount of threonine oxidized (table 4) remained relatively constant until threo nine intake approached the requirement, then increased with increasing intake of threonine without any increase in total measurable S-TDH activity. Bloxam (35) found that rates of glucose and urea pro duction by the perfused rat liver did not increase in proportion to changes in perfusate threonine concentration but showed

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AND ALFRED

METABOLISM

OF THREONINE

171

and lysine (43, 44) in vivo and with his tidine (46) in perfused liver. These results are, in general, similar to those of the previous study in histidine metabolism in vivo (8) except that threo nine oxidation was less responsive than histidine oxidation to changes in substrate intake by the rat. Also, concentrations of free threonine in plasma and tissues in creased more dramatically in response to increasing threonine intake (fig. 3) than did those of histidine to increasing histidine intake (8). Yamashita and Ashida (51) have shown that the catabolic breakdown of [U-14C]-threonine to CO2 in rats fed a high threonine diet did not increase as much as that of [U-14C]-lysine in rats fed a high lysine diet. Relative changes in the concentrations of free threonine in blood and muscle in relation to threonine intake are reported to be greater than those of free lysine in these tissues in relation to lysine intake ( 51, 52 ) and a large intake of threonine results in greatly elevated tissue threonine concentrations (53-57). Several studies indicate that threonine may not be conserved as efficiently as other amino acids ( 12-18 ). Weight loss due to a complete amino acid deficiency is least when lysine is deleted and greatest when threonine is deleted. Also, values for NPU of amino acid mixtures devoid of a single amino acid are not always zero, as would be expected on the basis of chemical score (19, 20). Only with deficiencies of threenine, isoleucine or total sulfur-containing amino acids were the expected NPU values of zero observed (20). Yamashita and Ashida (18) observed that oxidation of lysine by rats fed a lysine-free diet de creased considerably more than did oxida tion of threonine by rats fed a threoninefree diet and that the reduction of free lysine content of tissues was much less than that of threonine after the respective treat ments. Chu and Hegsted " (58) found that lysine-a-ketoglutarate reducÃ-ase activity falls in response to low lysine intake whereas S-TDH activity is not influenced by threonine intake and suggested that the different responses of the two catabolic en zymes to their substrates might explain the "Chu, S. H. W. & Hegsted, D. M. (1976) Dietary regulation of lysine-ketoglutarate reducÃ-aseactivity in the rat. Federation Proc. 35, 257 (Abstr.).

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a sigmoidal response; threonine appeared to be conserved when the concentration was low but rates of gluconeogenesis and ureogenesis from threonine increased rap idly when threonine concentration in creased above the physiologic range. The Km of S-TDH for threonine is 84 to 130 mM (24, 36, 37), which is much higher than the liver free threonine concentrations of 1.0 to 4.0 mM ( after correcting for water content of liver of 70% ) of rats fed 0.85% of threonine or less (fig. 2). Under these conditions, the rate of threonine oxidation should increase if liver threonine concen tration increases (38), as has been ob served for histidine (8), methionine (39), tryptophan (40, 41), tyrosine (40), phenylalanine (42), lysine (43, 44) and arginine (45) in vivo; and with histidine (46), tryptophan (40) and phenylalanine (47) in perfused rat liver. When dietary threonine content was low, a 30-fold increase in the S-TDH activ ity (table 6) did not result in a significant increase in the oxidation of threonine; also, tissue threonine concentrations were not different despite the great difference in the level of S-TDH. The Km of threonyl t-RNA synthetase is 4.3 X IO"6 M (48), much lower than that of S-TDH, which is 8.4 x IO-2 to 13 X IO-2 M (24, 36, 37). The low Km of the threonine activating enzyme and the high rate of protein synthesis in rapidly growing animals, apparently pro vide an efficient trapping system for threo nine despite greatly elevated degrading enzyme activity (table 6). Similar obser vations have been made with respect to histidine (8) and lysine (44). However, Austic and Nesheim (45) observed a high correlation between the level of kidney arginase activity and in vivo oxidation of arginine by chicks consuming argininedeficient diets. When the dietary supply of threonine was increased greatly, rats with high S-TDH activity owing to pre vious ingestion of an 80% casein diet, oxidized absorbed 14C-threonine to COa at a significantly faster rate than those pre viously fed a 15% casein diet. Increased capacity to catabolize a load of amino acid due to induction of degrading enzymes has also been observed with histidine (8, 46), tyrosine (49), tryptophan (39, 50)

IN VIVO

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E. HARPER

relationship between plasma threonine con centrations and the dietary level of threo nine. An effect of the length of feeding period on the plasma amino acid response curve has been demonstrated by several investigators (64-66). Differences among results obtained in these studies may also be due to the differences in the experi mental conditions such as the time of sam pling after feeding (67-70) and feeding schedule (71,72). The dietary level of threonine required for maximum growth determined in the present study (0.50%) agreed closely with the threonine requirement of the growing rat of 0.51 % estimated by Rama Rao et al. (73) using the protein efficiency ratio method, but was somewhat higher than values reported by other investigators; 0.42% by McLaughlan and Illman using plasma threonine concentrations (63), and 0.43% by Pick and Meade using the growth assay and protein efficiency ratio (74). Plotting threonine oxidation against di etary threonine content or threonine intake resulted in curves with a distinct break point. The requirement for threonine esti mated from the amounts of threonine oxidized agreed quite well with the re quirement values based on the growth as say. Oxidation of lysine by rats (10) fed diets containing graded amounts of lysine, by sheep given abomasal infusions of graded levels of lysine (9), and oxidation of L-[methyl-14C]-methionine 15by rats fed diets containing graded amounts of methionine ( 11 ) increased gradually and then sharply with increasing amounts of the respective amino acid ingested or infused, showing a break point near the require ment level. Herrstrom et al.16 have made similar observations on oxidation of lysine by liver slices from rats fed diets contain ing graded levels of lysine. In contrast to these observations, in rats (11) fed diets containing graded amounts of methionine, oxidation of L-[l-14C]-me»Hall. W. K., Doty, J. E. & Eaton, A. G. (1940) Availability of D,L-threonlne and D,L-allothreonlne for the formation of carbohydrate In the rat. Am. J. Physiol. 129, 372P. "Aguilar, T. S., Benevenga, N. J. & Harper, A. E. (1971) Effect of dietary methionine level on Its metabolism. J. Anim. Sci. 33, 1145 (Abstr.). 16Herrstrom, G. A., Owens, P. N. & Garrigus, U. S. (1972) Amino acid requirements determined by in vitro oxidation. Federation Proc. 31, 731 (Abstr.).

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difference in lysine and threonine conser vation in rats subjected to deficiencies of these amino acids. When overall histidine utilization was estimated from values for weight gain, oxi dation and excretion (8), the totals agreed reasonably well with the amounts of histi dine absorbed during the same period. Similar calculations for threonine in the Eresent study, however, showed that, with igh levels of dietary threonine, the amounts of threonine accounted for were considerably less than those absorbed (table 4). Part of this discrepancy can be accounted for by the great enlargement of free threonine pools with high threonine intake. It is also possible that some threo nine was converted to carbohydrate or fat. Yamashita and Ashida (51) have shown that while catabolic breakdown of [U-14C]threonine to CO2 in rats fed excess threo nine did not increase as much as that of [U-14C]lysine in rats fed a diet containing excess lysine, more threonine than lysine was converted to non-protein substances by rats fed excesses of these amino acids. Hall et al.14 (59) have also observed a high rate of synthesis of liver glycogen in rats starved and then fed large amounts of threonine. In the present study, free threonine con centration of plasma, liver and muscle were very low when the dietary content of threo nine was low and increased sharply as the dietary content of threonine exceeded 0.3%, a level considerably lower than that required for maximum weight gain. Kihlberg (60) has observed similar results in rats fed amino acid diets containing graded amounts of threonine. Pion and coworkers (52, 56) observed that, in rats fed diets containing graded levels of threonine for 2 weeks, free threonine concentrations in blood and muscle increased only slowly until dietary threonine content was ade quate and then increased sharply with higher levels of dietary threonine. They (61, 62) have also observed a similar re lationship between threonine intake and the concentration of free threonine in blood in preruminant calves. In contrast to these observations, in a short-term study with rats, McLaughlan and Illman (63) observed an almost linear

AND ALFRED

METABOLISM

OF THREONINE

LITERATURE

6. 7. 8.

9.

10.

11.

12.

13.

14.

15.

CITED

1. Harper, A. E. (1971) Adaptability and amino acid requirements. In: Metabolic Adap tation and Nutrition. Proc. of the special ses sion held during the 9th meeting of the Pan American Health Organization Advisory Com mittee on Medical Research, 1970. Scientific Publications No. 222, pp. 8-20, Washington, D.C. 2. Harper, A. E. (1974) Control mechanisms in amino acid metabolism. In: The Control of Metabolism (Sink, J. D., éd.),pp. 49-74, Pennsylvania State University Press, Univer sity Park, Pa. 3. Harper, A. E. (1975) Metabolic adapta tion to adequate and inadequate amino acid supply. In: Nutrition (Chavez, A., Bourges, H. & Basta, S., eds.), Vol. 1, pp. 1-8. Proc. 9th Intl. Congr. Nutrition, Mexico, 1972, Karger, Basel. 4. Munro, H. N. (1972) A general survey of mechanisms regulating protein metabolism in mammals. In: Mammalian Protein Metabolism (Munro, H. N., ed.), Vol. 4, pp. 3-130, Aca demic Press, New York. 5. Elman, R. (1939) Time factor in retention

16.

17.

18. 19.

20.

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thionine increased steadily with increasing dietary methionine content over the entire range of intake as might be expected for an amino acid that is a precursor of another and is degraded during the conversion. In a study on piglets, in which oxidation rate increased initially then tended to plateau with the higher methionine intakes (75), a single infusion of labeled methionine was given 1 hour after one of two daily meals and data were expressed as percentage of administered dose recovered in CO2. Adamson and Fisher (76) have reported similar results with rabbits adapted to diets con taining graded amounts of arginine and then given an intravenous injection of [U-14C]arginine. Percentage of adminis tered dose recovered in expired CO2 in 5 hours, during which time food was pro vided ad libitum, increased with increasing level of dietary arginine initially, then plateaued. In both of these studies the labeled amino acids would have been diluted to differing degrees by the influx of amino acids from the gut as well as by the en dogenous amino acids from protein catabolism. Differences in the specific radio activity of free methionine or arginine at the site of oxidation with the different di etary levels of the amino acid might have been partly responsible for the oxidation patterns they observed.

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