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Effect of concentration on the intestinal absorption of l-lysine in ageing rats
Exp. Geront. Vol. 4. pp. 223-230. Pergamon Press 1969. Printed in Great Britain
EFFECT OF CONCENTRATION ON THE INTESTINAL ABSORPTION OF /-LYSINE IN AGEING RATS L. PI~NZES Research Institute of Gerontology, University of Medicine, Budapest, Hungary (Received 28 May 1969)
THE STUDIESof Womack et al. (1964) suggest that "the requirement of one or more amino acids (i.e. lysine) for maintenance of body protein is higher in the old than in the young adult rat". The authors have shown that older (400-500 days) animals lose considerable weight when fed low (8 per cent) protein diet. On the other hand, Mitchell and Beadles (1950) state that in the case of wheat gluten, deficient in lysine, the biological value for the adult rat is significantly higher than for the growing rat or for the adult human. From these relationships it was concluded that the lysine requirement must have been much less in the adult rat. It is also generally believed that the rate of change with age in the requirement for this amino acid is highest in the young and continues to decrease as the organism becomes adult (Schwartz et al., 1958). Experiments by Tuttle et al. (1957, 1959) suggest that the quantitative requirements for one or more essential amino acids in men over 50 is greater when the total amount of nitrogen fed is increased. Since information about amino acid metabolism and utilization generally in senescence is still very limited and the results are often inconsistent (Tuttle et al., 1959; Watts et al., 1964), the present work was designed to study not only the intestinal absorption but the effects on the utilization of this "basic" amino acid by rats of different ages. M E T H O D S AND PROCEDURE Young, 2½-month-old, adult animals of 8 and 27½ months old Wistar female rats weighing 127.0 4- 9.5 g, 243.8 q- 18-0 g and 265.0 i 18.8 g; were kept ad lib. on standard laboratory chow, containing 20 per cent of protein. The experiment was performed under the same conditions as before and the operative procedure used has been presented in detail in a previous paper (P~nzes et al., 1968). As test-substance,/-lysine 14C/U/(sp. act. : 84 mCi/mM, radiochemical purity 99 per cent) was used. Chromatograms and spectrum analytical data indicated that this compound was in fact pure, i.e. it was considered that all the results of radioactivity were a direct indication of the derived amount of lysine present. One ml of 2,86 × 105 cpm/ml plus inactive l-lysine per 100 g body weight, as shown in the tables, was introduced into the small intestine. The test-period throughout the experiment was 20 min. Blood samples were taken from the tail every 5 min. At the end of the experiment the livers and intestines were weighed and dried. Samples, defatted by the Soxhlet method, were converted into COs by combustion and precipitated as barium carbonate (Lindenbaum et al., 1948). Radioactivity of all samples was assayed with a 1.1 mg/cm2 thin-window Geiger tube. All of the counts were corrected for self-absorption by an empirically obtained curve. B
223
224
L. PENZE8 RESULTS
Table 1 shows the percentile absorption values of lolysine from the gut of animals of different ages. It can be concluded that the rate of intestinal transport is not proportional to the concentration level and a saturation phenomenon exists at the higher concentration scales, particularly in the case of the young animals. As will also be seen in the tables, there is a general rise in the relative values of standard deviations parallel with that of the concentration levels. T h e relatively large standard deviations reflect consistent and wide variations at the highest (fifth) concentration level applied. TABLE 1.
I N T E S T I N A L ABSORPTION OF / - L Y S I N E AS PER CENT OF THE TOTAL DOSE ADMINISTERED
Initial concentration present in the small intestine (t~g substance per 100 g body weight) Young animals Adult animals Old animals
7.30 x 10 ~
2.92×103
11.67×103
4.66×104
18.60×104
64"94-10"9 ~ 45"14-10"2 47"54-11-8 35"24-2"0 54"3 4-6"4 43"1 4-7'5
29'613-6 30"64-5"6 29"5 4-3-3
25-34-5-8 28.54-7"7 25"44-6"0
9"34-6'2 22"44-13"1 15"6+4"9
Notes: 1 : Mean 4- standard deviation. Each group was of 5 animals.
TABLE 2. KINETICPARAMETERSOF THE INTESTINALABSORPTIONOF I-LYSINE# Group
Young animals
Adult animals
Old animals
a4-6a
0"9293 4- 0"1179
1"2820 4- 0"1577
1"1071 4- 0.1327
b 4- 6b/10~M1/
0"4396 4- 0.1188
0"1852 4- 0"1054
0.1616 4- 0"0108
-0"4730 4- 0.1412
-0"1444 4- 0"0840
--0.1460 4- 0-0356
-- -ab 4- 6 [ab-] /104L/ NI Estimation of K M/~M/
211"4
692"5
684.8
* For theoretical explanation and meaning of symbols see Jervis and Smyth's paper and our previous report. Absorbed amount of l-lysine (#M) is based on 1 g dried intestinal tissue. Kinetic data (estimation of the K M values) are shown in Table 2. T h e values were calculated by weighted regression analysis based on least squares weighted by the variances obtained. (Theoretical discussion and the meaning of symbols a, b and K M has already been described, P6nzes et al., 1968; but the formulae used in this present paper were modified by the variances.) Greater than 95 per cent confidence limits of K M values were obtained in the case of young animals, i.e. 132.4-524-6 and old rats: 460-1-1338.0; 95 per cent confidence limits in adults were, i.e. 354.0-1492-0 and in old rats: 490.4-1137.0; respectively. Eighty-five per cent confidence limits were: young individuals, 180-5-311-8: adult rats,
EFFECT OF CONCENTRATION O N T H E INTESTINAL ABSORPTION OF I-LYSINE
225
514.7-2260.0. It can be seen that the K u value of the y o u n g animals significantly differed from that of the adult and old animals at all confidence levels tested, whilst such differences between the adult and old rats could not be observed. Nevertheless, it seems clear that the K M value for the adults is little higher and more "indistinct" than that of the old rats. T h e specific activities of the y o u n g and aged intestines are surprisingly similar to each other at the lowest concentration level (Table 3), but the greatest rate of incorporation was found at the highest concentration used in the gut of the adult animals. Consistent decrease was observed in the radioactivities of the liver carbon content (Table 4). T h e specific activities of the young and old samples show a high degree of congruence. Considerably lesser amounts of radioactivity were detected in the adult livers, suggesting that the utilization of the test substance is greater in old than in adult rats in spite of the similarity of their K M values.
TABLE 3. SPECIFICACTIVITYOF THE SMALLINTESTINE# (cpm per 10 mg carbon) Initial concentration present in the small intestine (/~g substance per 100 g body weight) Young animals'[" Adult animals~ Old animals§
7"30 × l0 s 2.92 × 10a
1359 4- 126 948 ± 136 1234 4- 105
848 4- 267 952 ± 292 936 4- 270
11"67 × 10a 4"66 x 104
18.60 × 104
710 4- 137 680 + 84 579 4- 211
469 ± 209 668 4- 190 433 4- 267
780 ± 418 630 4- 69 573 4- 173
* All samples were dried and defatted. t Percentile carbon content of the dried and defatted samples: 14.25 4- 1" 54. 1: Percentile carbon content of the dried and defatted samples : 13- 80 4- 2" 03. § Percentile carbon content of the dried and defatted samples: 13" 50 ± 1" 94.
TABLE 4.
SPECIFIC ACTIVITY OF THE LIVER #
(cpm per 10 mg carbon) Initial concentration present in the small intestine (/~g substance per 100 body weight) Young animalst Adult animals$ Old animals§
7"30 × 102 2"92 × 10a
1252 4- 363 716 4- 120 1164 4- 109
663 ± 244 415 ± 78 703 4- 118
11"67 x 103 4.66 × 104 18"60 × 104
435 4- 94 319 + 94 237 4- 25
318 4- 119 159 4- 57 209 ± 106
* All samples were dried and defatted. t Percentile carbon content of the dried and defatted samples : 21" 0 ± 3" 2. Percentile carbon content of the dried and defatted samples : 19- 0 ± 3" 0. § Percentile carbon content of the dried and defatted samples : 20" 6 ± 3" 2.
147 ± 78 ± 132 ±
88 31 32
0.074 O-006 f 0.006
0.074 & 0.052 0.016 & O-013
f 0.018 f 0.011
0.015 0.036
0.035 f 0.008 0.036 + O-012
+ 0.029
15’ 0.039 20’ 0.064 f 0.037
0.029
O-032 zt 0.017 O-046 f 0.025
o-040 f 0.017 0.041 f 0.024
15’ O-103 & 0.026 20’ 0.096 f 0.046
i 0.019
0.014
0.012 f 0.008 O-025 f O-012
5’ 0.032 f 0.015 10’ O-072 f O-038
f 0.010
f 0.011
0.022
f 0.016
0.030
* 0.007 * 0.032
f 0.043
10’ 0.044
O-188 * 0.034
5’ 0.022
20’
f 0.021 f O-049
0.040 0.062
&
x 103
f 0.011
0.029
11.67
rt 0.021
x 103
INTHEBLOOD~
zk 0.036
0.036 0.043
10’ 0.105 f 0.031 15’ 0.124 f 0.023
2.92
0.034
x 102
5’2 0.084 & 0.066
7.30
5. PERCENTILERADIOACTIVITYFOUND
* 0.012 0.040
& 0.033
0.015 0.041 0.055 0.092
0.047 f o-014 0.075 f 0.037
f 0.048
zk 0.036
* 0.031
* 0.008
0.073 f 0.046 f 0.017
0.020
0.013
0.027 f 0.022
o-022 f 0.013
f 0.014
+ 0.009 f 0.037
f 0.006
0.016 0.016 0.034
f 0.023
0.078
0.039
0.066 f 0.014 + 0.009
0.103 f 0.083
0.011
0.042 f 0.022
f
5
x 104
0.052
0.044
f 0.018
0.024
18.60
x 104
4.66
* Cpm/l ml of blood in o/0 of the total dose (Total dose administered into the small intestine regarded as 100 per cent). t Minutes after introduction of the radioactive substance into the intestine.
Old animals
Adult animals
Young animals
Initial concentration present in the small intestine (rg substance per 100 g body wt.)
TABLE
EFFECT OF CONCENTRATION
ON THE INTESTINAL ABSORPTION OF I-LYSINE
227
Table 5 shows the changes in blood radioactivity. As mentioned before, the relatively large values of standard deviation occur mainly at higher concentrations, particularly in the case of adults. This observation clearly reflects the wide variations in the percentile absorption as well. On the date derived in Table 5, it seems clear that the specific activities of young animals were greater than those of adult and old rats. The animals of the adult group showed the lowest value of radioactivity and in old rats there was a tendency for the specific activity at the 20 min period to rise more sharply than in the adult, and still more than in the young, animals. DISCUSSION
The lysine absorbed from the small intestine is considered as "luminal loss" and based on dried tissue weight unit. The percentile absorption values demonstrated in Table 1, and the kinetic parameters presented in Table 2, support the concept that energy-dependent systems are of significance in transporting this amino acid (DiBella, 1960; Fearon and Bird, 1967; Finch and Hird, 1960; Hagihira et al., 1961; Robinson, 1966), and are of the same order as in our previous in vivo study (P6nzes et al., 1968) lasting similarly for 20 min and using dl-methionine. In his in vivo experiments with anesthetized 150-200 g male rats, DiBella found that the absorption of l-lysine from the rat's small intestine was 86.0-83.5 per cent. The applied amount of l-lysine was 520 ~M in Ringer-bicarbonate buffer and the duration of the absorption test was 60 min. The data show that there is a perceptible difference in the l-lysine utilization of the young and adult rats. Although the intestinal utilization in'the old animals does not differ significantly from that of the adult rats, the radioactivity incorporated in the liver tissue is similar to that found in young rats. The high relative rates of intestinal absorption found in the young animals seem to support Mitchell's classical concept (1947) that lysine is primarily needed for rapid protein synthesis as found in growing rats, while it is practically dispensable in the adult. The Michaelis constant of the young animals (211.4 ~M) proved to be less than that found by Finch and Hird (1960). In their in vitro experiment the K u value calculated from the rates of uptake at 1 mM and 10 mM for starved, young female rats was as great as 0.55 mM. The reason for this must be sought not only in the difference between in vivo and in vitro conditions but in the difference of body weight of animals (the authors used much heavier and older rats than we did), and in the different starving periods before experiment. The concentration levels were also entirely different. The non-essential difference observed between the KM value of the young adult and old animals can evidently be explained by the relative high values of standard deviations at the fifth and greatest concentration level. It is worth mentioning here the finding of Hagihira et al. (1961) in their in vitro study that the maximal rates of transport for l-lysine, /-arginine and dl-ornithine at 1.0 m M initial concentration were only 1/10 to 1/20 of the rates for the transport of some of the neutral amino acids, i.e. glycine, l-alanine and l-proline. Hagihira et al. (1961) emphasize that the small capacity for the intestinal transport of the dibasic amino acids explains the difficulty in obtaining significant net transport with relative high initial concentrations. As Robinson (1966) has pointed out, the dibasic amino acids are readily saturated because of their relatively small capacity, since active transfer can not be distinguished at a substrate concentrations of 5 mM or 10 mM. Thus, the uptake of l-phenylalanine will be saturated at a substrate level of
228
L. P~NZES
15 mM, whilst that of l-arginine is almost saturated at a substrate concentratxon of 3 mM. Robinson showed that both dibasic acids, l-arginine and l-lysine, are among those amino acids which proved to be the most efficiently absorbed when the intestinal mucosa is presented with an equimolar mixture of 20 amino acids, each at concentration of 1 mM. The reason for this he explains by the level of physiological need, since these are only two basic amino acids, generally found in protein hydrolysates, as compared to other 18, neutral, amino acids. Decrease of the specific activity of the intestine samples towards the highest concentration scales was found to be less definite than the liver samples. It is apparent that considerably greater specific activity was measured in the adult intestine at the highest concentration used; this corresponds to the lowest value of the specific activity of liver samples of the adult animals. In vitro studies on the distribution of amino acids between intestinal mucosa and external (bathing) fluid may give us some information about exceptional role of l-lysine in mucosal transport. On the basis of "everted sac" experiments on the inhibition of normal gut mucosal uptake of l-lysine-14C and l-cystine-35S by a second amino acid (Thief et al., 1964), it appeared that lysine, arginine and cystine very likely form one family; i.e. the three amino acids compete for the same intestinal uptake mechanism. Similar observations are also described by Finch and Hird who have indicated that /-lysine and probably l-ornithine and l-arginine may not compete for a common transfer mechanism with the other/-amino acids. However, this mucosal uptake equally covers both accumulation within, and metabolic transformation in the intestinal tissue. Finch and Hird have shown that l-lysine is not metabolised by the tissue in vitro, but is almost fully recoverable and shows a more continuous time progress of uptake than any other /-amino acids over the range 4 min to 50 min. The relative high values of standard deviations in the specific activity of the blood samples interested us particularly. It is known that the method of drawing blood from the rat's tail could give rise to several sources of error, although, there are many references in the literature based on this method. But the observed fluctuations in specific activities could equally be real (Waterlow and Stephen, 1967), since the amount of the testsubstance, i.e. free amino acid in the blood at any moment, is low compared to the proportion entering and leaving the amino acid pool. The constant rise in the radioactivity values at higher concentration levels in all groups may be related to the rate of their removal from the blood by the different body tissues; i.e. the greater the blood radioactivity level the higher the initial concentration of l-lysine introduced into the intestinal lumen. In addition, Guggenheim et al. (1960) have estimated that the level of an amino acid is dependent upon the rate of its absorption from the intestine. This was found in the present study. Two characteristics were observed: on one hand, a permanent increase within the same initial concentration used and on the other hand, an elevation at successive higher concentration levels. The former is conceivably time-dependent, the second might be based on tissue uptake. It is concluded from the data that at higher concentration rates the tissue uptake of radioactivity was lower in comparison to the amounts detected at the first three concentration levels. A relatively high blood specific activity measured in the young animals might be expected from an intensive tissue uptake, but against this a more pronounced increase was noted in the case of the young adult and old animals. Nevertheless, it must be pointed out that the 20 min period, which is adequate to establish the rate of amino acid
EFFECT OF CONCENTRATIONON THE INTESTINALABSORPTIONOF I-LYSINE
229
absorption, was too short to determine the main characteristics of radioactive lysine turnover in the blood. In Waterl6w and Stephen's study, in which rats were infused intravenously for periods up to 7 hr with/-radiolysine, it was found that after 4 hr the specific activity of free lysine in the blood reached a plateau-level. From this the authors calculated the total lysine flux into or out of the blood stream. In male rats there was a tendency for the flux per 100 g body weight to decrease with increasing weight and age of the rats. Waterlow and Stephen added that the overall rate of lysine efflux in young rats corresponds to the faster breakdown of plasma albumin in infants as against adults. It seems, therefore, logical to accept that protein metabolism behaves in very much the same way as energy metabolism--its intensity is the greatest in the young organism. In sum, the findings give no unambiguous indication that lysine absorption from the gut increases with ageing. The literature is conflicting. Swendseid and Tuttle (cit. Berry, 1968) on the basis of their balance studies state that " I n men over 50 years of age •.. there is an indication of an increased requirements for essential nitrogen as compared with the needs for young men. The requirements for methionine and lysine appear to be at least double those of younger men". On the other hand, Watts et al. (1964) support the view that old men do not require larger amounts of certain amino acids than do young men. The conclusion of our present findings is that the lysine requirement is the greatest in the 2½-month-old rats, and the demand of the aged animals is not less, and may be greater than that of the young adults. REFERENCES
BERRY, W. T. C. (1968) Proc. Nutrit. Soc. 27, 191. DI BELLA,S. (1960) Experientia 16/8, 367. FEARON, J. R. and Bran, F. H. (1967)ft. Nutrit. 93, 198. FINCH, L. R. and Him), F. J. R. (1960) Biochim. biophys. Acta 43, 268, 278. GUGOENHEIM, K., HALEVY, S. and FRIEDMANN,N. (1960) Arch. Biochem. Biophys. 91, 6. HAOIHIRA,H., LIN, E. C., SAMIY,A. H. and WILSON,T. H. (1961) Biochem. biophys. Res. Comm. 4, 478. JERVlS, L. and SMYTH,D. H. (1959) J. Physiol. 149, 433. LINDENBAUM,A., SCHUBERT,J. and ARMSTRONG,W. D. (1948) Anal. Chem. 20, 1120. MITCHELL,H. H. (1947) Arch. Biochem. 12, 293. MITCHELL, U. H. and BEADLES,J. R. (1950) y. Nutrit. 40, 25. PI~NZES, L., SIMON, G. and WINTER, M. (11968) Exp. Geront. 3, 257. ROBINSON, J. W. L. (1966) Certain Aspects of Intestinal Amino Acid Absorption. Universit6 de Lausanne, Darmstadt, Verlag der Odenwald-H. Frotseher KG. SCHWARTZ,H. G., TAYLOR,M. W. and FISHER,H. (1958)ff. Nutrit. 65, 25. THIER, S., Fox, M. and SECAL, S. (1964) Science 143, 482. TUTTLE, S. G., SWENDSEID,M. E., MULCARE, D., GRIFFITH, W. H. and BASSETT, S. H. (1957) Metabolism 6, 564. TUTTLE, S. G., SWENDSEIO,M. E., MULCARE,D., GRIFFITH,W. H. and BASSETT, S. H. (1959) Metabolism 8, 61. WATERLOW,J. C. and STEPHEN,J. M. L. (1967) Clin. Sci. 33, 489• WATTS, J. H., MANN,A. N., BRADLEY,L. and THOMPSON, D. J. (1964)ff. Geront. 19, 370. WOMACK, M., MARSHALL,M. W. and HXLDEBRANI),H. E. (1964) ft. Geront. 19, 45.
Summary--In vivo studies on rats of different ages show that the greatest rate of absorption and utilization of l-lysine-14C was found in 2½-month-old rats whilst the demand of the 27½-month-old animals is not conspicuously less, and may be greater than, that of young adults.
230
L. PI~NZF_,S R ~ s u m 6 - - L ' o b s e r v a t i o n in vivo de rats de diff4rents figes r6v~le que la proportion d'absorption et d'utilisation la plus 41ev4e de l-lysine-t4C a 4t4 constat4e chez des rats fig4s de 2½ tools, alors que les besoins des anlmaux fig6s de 27½ mois ne sont pas singuli6rement moindres et p e u v e n t m ~ m e ~tre sup4rieurs ~ ceux de jeunes adultes. Zusammenfassung--fn ~ ' v o - U n t e r s u c h u n g e n an Ratten unterschiedlichen Alters ergaben, dab die grfl3te A u f n a h m e - u n d Einbaurate y o n / - 1 4 C - L y s i n bei 2½ M o n a t e alten Rat-ten g e f u n d e n wird, w~_rend der Bedarf bei 27½ M o n a t e alten T i e r e n nicht ausgesprochen geringer u n d mfglicherweise gr6Ber als bei j u n g e n Adulten ist. Pe31oMe--HCcIIe~OBaHH~ in vivo Ha rpbIcax pa3mPmoro Bo3pacTa nora3am4, tITO HaH6om, maa cropocrb nornomcIma H yrzaaaamm l-aHamaa-14C 05aapyxmnacl, y rpblc no3pacTe 2½ MP..CffI~eB,Tor~a KaIC nOTpe6HOCTb y )IKtIBOTHblXB Bo3pacre 27½ MeCfllIen B HO3Ha~IHTeJIbHOi~CTeIIeHMHIDK0H MOXOT6MTb BblKI0qOM y MOHO~blXB3pOCJIbIX.
EFFECT OF CONCENTRATION ON THE INTESTINAL ABSORPTION OF /-LYSINE IN AGEING RATS L. PI~NZES Research Institute of Gerontology, University of Medicine, Budapest, Hungary (Received 28 May 1969)
THE STUDIESof Womack et al. (1964) suggest that "the requirement of one or more amino acids (i.e. lysine) for maintenance of body protein is higher in the old than in the young adult rat". The authors have shown that older (400-500 days) animals lose considerable weight when fed low (8 per cent) protein diet. On the other hand, Mitchell and Beadles (1950) state that in the case of wheat gluten, deficient in lysine, the biological value for the adult rat is significantly higher than for the growing rat or for the adult human. From these relationships it was concluded that the lysine requirement must have been much less in the adult rat. It is also generally believed that the rate of change with age in the requirement for this amino acid is highest in the young and continues to decrease as the organism becomes adult (Schwartz et al., 1958). Experiments by Tuttle et al. (1957, 1959) suggest that the quantitative requirements for one or more essential amino acids in men over 50 is greater when the total amount of nitrogen fed is increased. Since information about amino acid metabolism and utilization generally in senescence is still very limited and the results are often inconsistent (Tuttle et al., 1959; Watts et al., 1964), the present work was designed to study not only the intestinal absorption but the effects on the utilization of this "basic" amino acid by rats of different ages. M E T H O D S AND PROCEDURE Young, 2½-month-old, adult animals of 8 and 27½ months old Wistar female rats weighing 127.0 4- 9.5 g, 243.8 q- 18-0 g and 265.0 i 18.8 g; were kept ad lib. on standard laboratory chow, containing 20 per cent of protein. The experiment was performed under the same conditions as before and the operative procedure used has been presented in detail in a previous paper (P~nzes et al., 1968). As test-substance,/-lysine 14C/U/(sp. act. : 84 mCi/mM, radiochemical purity 99 per cent) was used. Chromatograms and spectrum analytical data indicated that this compound was in fact pure, i.e. it was considered that all the results of radioactivity were a direct indication of the derived amount of lysine present. One ml of 2,86 × 105 cpm/ml plus inactive l-lysine per 100 g body weight, as shown in the tables, was introduced into the small intestine. The test-period throughout the experiment was 20 min. Blood samples were taken from the tail every 5 min. At the end of the experiment the livers and intestines were weighed and dried. Samples, defatted by the Soxhlet method, were converted into COs by combustion and precipitated as barium carbonate (Lindenbaum et al., 1948). Radioactivity of all samples was assayed with a 1.1 mg/cm2 thin-window Geiger tube. All of the counts were corrected for self-absorption by an empirically obtained curve. B
223
224
L. PENZE8 RESULTS
Table 1 shows the percentile absorption values of lolysine from the gut of animals of different ages. It can be concluded that the rate of intestinal transport is not proportional to the concentration level and a saturation phenomenon exists at the higher concentration scales, particularly in the case of the young animals. As will also be seen in the tables, there is a general rise in the relative values of standard deviations parallel with that of the concentration levels. T h e relatively large standard deviations reflect consistent and wide variations at the highest (fifth) concentration level applied. TABLE 1.
I N T E S T I N A L ABSORPTION OF / - L Y S I N E AS PER CENT OF THE TOTAL DOSE ADMINISTERED
Initial concentration present in the small intestine (t~g substance per 100 g body weight) Young animals Adult animals Old animals
7.30 x 10 ~
2.92×103
11.67×103
4.66×104
18.60×104
64"94-10"9 ~ 45"14-10"2 47"54-11-8 35"24-2"0 54"3 4-6"4 43"1 4-7'5
29'613-6 30"64-5"6 29"5 4-3-3
25-34-5-8 28.54-7"7 25"44-6"0
9"34-6'2 22"44-13"1 15"6+4"9
Notes: 1 : Mean 4- standard deviation. Each group was of 5 animals.
TABLE 2. KINETICPARAMETERSOF THE INTESTINALABSORPTIONOF I-LYSINE# Group
Young animals
Adult animals
Old animals
a4-6a
0"9293 4- 0"1179
1"2820 4- 0"1577
1"1071 4- 0.1327
b 4- 6b/10~M1/
0"4396 4- 0.1188
0"1852 4- 0"1054
0.1616 4- 0"0108
-0"4730 4- 0.1412
-0"1444 4- 0"0840
--0.1460 4- 0-0356
-- -ab 4- 6 [ab-] /104L/ NI Estimation of K M/~M/
211"4
692"5
684.8
* For theoretical explanation and meaning of symbols see Jervis and Smyth's paper and our previous report. Absorbed amount of l-lysine (#M) is based on 1 g dried intestinal tissue. Kinetic data (estimation of the K M values) are shown in Table 2. T h e values were calculated by weighted regression analysis based on least squares weighted by the variances obtained. (Theoretical discussion and the meaning of symbols a, b and K M has already been described, P6nzes et al., 1968; but the formulae used in this present paper were modified by the variances.) Greater than 95 per cent confidence limits of K M values were obtained in the case of young animals, i.e. 132.4-524-6 and old rats: 460-1-1338.0; 95 per cent confidence limits in adults were, i.e. 354.0-1492-0 and in old rats: 490.4-1137.0; respectively. Eighty-five per cent confidence limits were: young individuals, 180-5-311-8: adult rats,
EFFECT OF CONCENTRATION O N T H E INTESTINAL ABSORPTION OF I-LYSINE
225
514.7-2260.0. It can be seen that the K u value of the y o u n g animals significantly differed from that of the adult and old animals at all confidence levels tested, whilst such differences between the adult and old rats could not be observed. Nevertheless, it seems clear that the K M value for the adults is little higher and more "indistinct" than that of the old rats. T h e specific activities of the y o u n g and aged intestines are surprisingly similar to each other at the lowest concentration level (Table 3), but the greatest rate of incorporation was found at the highest concentration used in the gut of the adult animals. Consistent decrease was observed in the radioactivities of the liver carbon content (Table 4). T h e specific activities of the young and old samples show a high degree of congruence. Considerably lesser amounts of radioactivity were detected in the adult livers, suggesting that the utilization of the test substance is greater in old than in adult rats in spite of the similarity of their K M values.
TABLE 3. SPECIFICACTIVITYOF THE SMALLINTESTINE# (cpm per 10 mg carbon) Initial concentration present in the small intestine (/~g substance per 100 g body weight) Young animals'[" Adult animals~ Old animals§
7"30 × l0 s 2.92 × 10a
1359 4- 126 948 ± 136 1234 4- 105
848 4- 267 952 ± 292 936 4- 270
11"67 × 10a 4"66 x 104
18.60 × 104
710 4- 137 680 + 84 579 4- 211
469 ± 209 668 4- 190 433 4- 267
780 ± 418 630 4- 69 573 4- 173
* All samples were dried and defatted. t Percentile carbon content of the dried and defatted samples: 14.25 4- 1" 54. 1: Percentile carbon content of the dried and defatted samples : 13- 80 4- 2" 03. § Percentile carbon content of the dried and defatted samples: 13" 50 ± 1" 94.
TABLE 4.
SPECIFIC ACTIVITY OF THE LIVER #
(cpm per 10 mg carbon) Initial concentration present in the small intestine (/~g substance per 100 body weight) Young animalst Adult animals$ Old animals§
7"30 × 102 2"92 × 10a
1252 4- 363 716 4- 120 1164 4- 109
663 ± 244 415 ± 78 703 4- 118
11"67 x 103 4.66 × 104 18"60 × 104
435 4- 94 319 + 94 237 4- 25
318 4- 119 159 4- 57 209 ± 106
* All samples were dried and defatted. t Percentile carbon content of the dried and defatted samples : 21" 0 ± 3" 2. Percentile carbon content of the dried and defatted samples : 19- 0 ± 3" 0. § Percentile carbon content of the dried and defatted samples : 20" 6 ± 3" 2.
147 ± 78 ± 132 ±
88 31 32
0.074 O-006 f 0.006
0.074 & 0.052 0.016 & O-013
f 0.018 f 0.011
0.015 0.036
0.035 f 0.008 0.036 + O-012
+ 0.029
15’ 0.039 20’ 0.064 f 0.037
0.029
O-032 zt 0.017 O-046 f 0.025
o-040 f 0.017 0.041 f 0.024
15’ O-103 & 0.026 20’ 0.096 f 0.046
i 0.019
0.014
0.012 f 0.008 O-025 f O-012
5’ 0.032 f 0.015 10’ O-072 f O-038
f 0.010
f 0.011
0.022
f 0.016
0.030
* 0.007 * 0.032
f 0.043
10’ 0.044
O-188 * 0.034
5’ 0.022
20’
f 0.021 f O-049
0.040 0.062
&
x 103
f 0.011
0.029
11.67
rt 0.021
x 103
INTHEBLOOD~
zk 0.036
0.036 0.043
10’ 0.105 f 0.031 15’ 0.124 f 0.023
2.92
0.034
x 102
5’2 0.084 & 0.066
7.30
5. PERCENTILERADIOACTIVITYFOUND
* 0.012 0.040
& 0.033
0.015 0.041 0.055 0.092
0.047 f o-014 0.075 f 0.037
f 0.048
zk 0.036
* 0.031
* 0.008
0.073 f 0.046 f 0.017
0.020
0.013
0.027 f 0.022
o-022 f 0.013
f 0.014
+ 0.009 f 0.037
f 0.006
0.016 0.016 0.034
f 0.023
0.078
0.039
0.066 f 0.014 + 0.009
0.103 f 0.083
0.011
0.042 f 0.022
f
5
x 104
0.052
0.044
f 0.018
0.024
18.60
x 104
4.66
* Cpm/l ml of blood in o/0 of the total dose (Total dose administered into the small intestine regarded as 100 per cent). t Minutes after introduction of the radioactive substance into the intestine.
Old animals
Adult animals
Young animals
Initial concentration present in the small intestine (rg substance per 100 g body wt.)
TABLE
EFFECT OF CONCENTRATION
ON THE INTESTINAL ABSORPTION OF I-LYSINE
227
Table 5 shows the changes in blood radioactivity. As mentioned before, the relatively large values of standard deviation occur mainly at higher concentrations, particularly in the case of adults. This observation clearly reflects the wide variations in the percentile absorption as well. On the date derived in Table 5, it seems clear that the specific activities of young animals were greater than those of adult and old rats. The animals of the adult group showed the lowest value of radioactivity and in old rats there was a tendency for the specific activity at the 20 min period to rise more sharply than in the adult, and still more than in the young, animals. DISCUSSION
The lysine absorbed from the small intestine is considered as "luminal loss" and based on dried tissue weight unit. The percentile absorption values demonstrated in Table 1, and the kinetic parameters presented in Table 2, support the concept that energy-dependent systems are of significance in transporting this amino acid (DiBella, 1960; Fearon and Bird, 1967; Finch and Hird, 1960; Hagihira et al., 1961; Robinson, 1966), and are of the same order as in our previous in vivo study (P6nzes et al., 1968) lasting similarly for 20 min and using dl-methionine. In his in vivo experiments with anesthetized 150-200 g male rats, DiBella found that the absorption of l-lysine from the rat's small intestine was 86.0-83.5 per cent. The applied amount of l-lysine was 520 ~M in Ringer-bicarbonate buffer and the duration of the absorption test was 60 min. The data show that there is a perceptible difference in the l-lysine utilization of the young and adult rats. Although the intestinal utilization in'the old animals does not differ significantly from that of the adult rats, the radioactivity incorporated in the liver tissue is similar to that found in young rats. The high relative rates of intestinal absorption found in the young animals seem to support Mitchell's classical concept (1947) that lysine is primarily needed for rapid protein synthesis as found in growing rats, while it is practically dispensable in the adult. The Michaelis constant of the young animals (211.4 ~M) proved to be less than that found by Finch and Hird (1960). In their in vitro experiment the K u value calculated from the rates of uptake at 1 mM and 10 mM for starved, young female rats was as great as 0.55 mM. The reason for this must be sought not only in the difference between in vivo and in vitro conditions but in the difference of body weight of animals (the authors used much heavier and older rats than we did), and in the different starving periods before experiment. The concentration levels were also entirely different. The non-essential difference observed between the KM value of the young adult and old animals can evidently be explained by the relative high values of standard deviations at the fifth and greatest concentration level. It is worth mentioning here the finding of Hagihira et al. (1961) in their in vitro study that the maximal rates of transport for l-lysine, /-arginine and dl-ornithine at 1.0 m M initial concentration were only 1/10 to 1/20 of the rates for the transport of some of the neutral amino acids, i.e. glycine, l-alanine and l-proline. Hagihira et al. (1961) emphasize that the small capacity for the intestinal transport of the dibasic amino acids explains the difficulty in obtaining significant net transport with relative high initial concentrations. As Robinson (1966) has pointed out, the dibasic amino acids are readily saturated because of their relatively small capacity, since active transfer can not be distinguished at a substrate concentrations of 5 mM or 10 mM. Thus, the uptake of l-phenylalanine will be saturated at a substrate level of
228
L. P~NZES
15 mM, whilst that of l-arginine is almost saturated at a substrate concentratxon of 3 mM. Robinson showed that both dibasic acids, l-arginine and l-lysine, are among those amino acids which proved to be the most efficiently absorbed when the intestinal mucosa is presented with an equimolar mixture of 20 amino acids, each at concentration of 1 mM. The reason for this he explains by the level of physiological need, since these are only two basic amino acids, generally found in protein hydrolysates, as compared to other 18, neutral, amino acids. Decrease of the specific activity of the intestine samples towards the highest concentration scales was found to be less definite than the liver samples. It is apparent that considerably greater specific activity was measured in the adult intestine at the highest concentration used; this corresponds to the lowest value of the specific activity of liver samples of the adult animals. In vitro studies on the distribution of amino acids between intestinal mucosa and external (bathing) fluid may give us some information about exceptional role of l-lysine in mucosal transport. On the basis of "everted sac" experiments on the inhibition of normal gut mucosal uptake of l-lysine-14C and l-cystine-35S by a second amino acid (Thief et al., 1964), it appeared that lysine, arginine and cystine very likely form one family; i.e. the three amino acids compete for the same intestinal uptake mechanism. Similar observations are also described by Finch and Hird who have indicated that /-lysine and probably l-ornithine and l-arginine may not compete for a common transfer mechanism with the other/-amino acids. However, this mucosal uptake equally covers both accumulation within, and metabolic transformation in the intestinal tissue. Finch and Hird have shown that l-lysine is not metabolised by the tissue in vitro, but is almost fully recoverable and shows a more continuous time progress of uptake than any other /-amino acids over the range 4 min to 50 min. The relative high values of standard deviations in the specific activity of the blood samples interested us particularly. It is known that the method of drawing blood from the rat's tail could give rise to several sources of error, although, there are many references in the literature based on this method. But the observed fluctuations in specific activities could equally be real (Waterlow and Stephen, 1967), since the amount of the testsubstance, i.e. free amino acid in the blood at any moment, is low compared to the proportion entering and leaving the amino acid pool. The constant rise in the radioactivity values at higher concentration levels in all groups may be related to the rate of their removal from the blood by the different body tissues; i.e. the greater the blood radioactivity level the higher the initial concentration of l-lysine introduced into the intestinal lumen. In addition, Guggenheim et al. (1960) have estimated that the level of an amino acid is dependent upon the rate of its absorption from the intestine. This was found in the present study. Two characteristics were observed: on one hand, a permanent increase within the same initial concentration used and on the other hand, an elevation at successive higher concentration levels. The former is conceivably time-dependent, the second might be based on tissue uptake. It is concluded from the data that at higher concentration rates the tissue uptake of radioactivity was lower in comparison to the amounts detected at the first three concentration levels. A relatively high blood specific activity measured in the young animals might be expected from an intensive tissue uptake, but against this a more pronounced increase was noted in the case of the young adult and old animals. Nevertheless, it must be pointed out that the 20 min period, which is adequate to establish the rate of amino acid
EFFECT OF CONCENTRATIONON THE INTESTINALABSORPTIONOF I-LYSINE
229
absorption, was too short to determine the main characteristics of radioactive lysine turnover in the blood. In Waterl6w and Stephen's study, in which rats were infused intravenously for periods up to 7 hr with/-radiolysine, it was found that after 4 hr the specific activity of free lysine in the blood reached a plateau-level. From this the authors calculated the total lysine flux into or out of the blood stream. In male rats there was a tendency for the flux per 100 g body weight to decrease with increasing weight and age of the rats. Waterlow and Stephen added that the overall rate of lysine efflux in young rats corresponds to the faster breakdown of plasma albumin in infants as against adults. It seems, therefore, logical to accept that protein metabolism behaves in very much the same way as energy metabolism--its intensity is the greatest in the young organism. In sum, the findings give no unambiguous indication that lysine absorption from the gut increases with ageing. The literature is conflicting. Swendseid and Tuttle (cit. Berry, 1968) on the basis of their balance studies state that " I n men over 50 years of age •.. there is an indication of an increased requirements for essential nitrogen as compared with the needs for young men. The requirements for methionine and lysine appear to be at least double those of younger men". On the other hand, Watts et al. (1964) support the view that old men do not require larger amounts of certain amino acids than do young men. The conclusion of our present findings is that the lysine requirement is the greatest in the 2½-month-old rats, and the demand of the aged animals is not less, and may be greater than that of the young adults. REFERENCES
BERRY, W. T. C. (1968) Proc. Nutrit. Soc. 27, 191. DI BELLA,S. (1960) Experientia 16/8, 367. FEARON, J. R. and Bran, F. H. (1967)ft. Nutrit. 93, 198. FINCH, L. R. and Him), F. J. R. (1960) Biochim. biophys. Acta 43, 268, 278. GUGOENHEIM, K., HALEVY, S. and FRIEDMANN,N. (1960) Arch. Biochem. Biophys. 91, 6. HAOIHIRA,H., LIN, E. C., SAMIY,A. H. and WILSON,T. H. (1961) Biochem. biophys. Res. Comm. 4, 478. JERVlS, L. and SMYTH,D. H. (1959) J. Physiol. 149, 433. LINDENBAUM,A., SCHUBERT,J. and ARMSTRONG,W. D. (1948) Anal. Chem. 20, 1120. MITCHELL,H. H. (1947) Arch. Biochem. 12, 293. MITCHELL, U. H. and BEADLES,J. R. (1950) y. Nutrit. 40, 25. PI~NZES, L., SIMON, G. and WINTER, M. (11968) Exp. Geront. 3, 257. ROBINSON, J. W. L. (1966) Certain Aspects of Intestinal Amino Acid Absorption. Universit6 de Lausanne, Darmstadt, Verlag der Odenwald-H. Frotseher KG. SCHWARTZ,H. G., TAYLOR,M. W. and FISHER,H. (1958)ff. Nutrit. 65, 25. THIER, S., Fox, M. and SECAL, S. (1964) Science 143, 482. TUTTLE, S. G., SWENDSEID,M. E., MULCARE, D., GRIFFITH, W. H. and BASSETT, S. H. (1957) Metabolism 6, 564. TUTTLE, S. G., SWENDSEIO,M. E., MULCARE,D., GRIFFITH,W. H. and BASSETT, S. H. (1959) Metabolism 8, 61. WATERLOW,J. C. and STEPHEN,J. M. L. (1967) Clin. Sci. 33, 489• WATTS, J. H., MANN,A. N., BRADLEY,L. and THOMPSON, D. J. (1964)ff. Geront. 19, 370. WOMACK, M., MARSHALL,M. W. and HXLDEBRANI),H. E. (1964) ft. Geront. 19, 45.
Summary--In vivo studies on rats of different ages show that the greatest rate of absorption and utilization of l-lysine-14C was found in 2½-month-old rats whilst the demand of the 27½-month-old animals is not conspicuously less, and may be greater than, that of young adults.
230
L. PI~NZF_,S R ~ s u m 6 - - L ' o b s e r v a t i o n in vivo de rats de diff4rents figes r6v~le que la proportion d'absorption et d'utilisation la plus 41ev4e de l-lysine-t4C a 4t4 constat4e chez des rats fig4s de 2½ tools, alors que les besoins des anlmaux fig6s de 27½ mois ne sont pas singuli6rement moindres et p e u v e n t m ~ m e ~tre sup4rieurs ~ ceux de jeunes adultes. Zusammenfassung--fn ~ ' v o - U n t e r s u c h u n g e n an Ratten unterschiedlichen Alters ergaben, dab die grfl3te A u f n a h m e - u n d Einbaurate y o n / - 1 4 C - L y s i n bei 2½ M o n a t e alten Rat-ten g e f u n d e n wird, w~_rend der Bedarf bei 27½ M o n a t e alten T i e r e n nicht ausgesprochen geringer u n d mfglicherweise gr6Ber als bei j u n g e n Adulten ist. Pe31oMe--HCcIIe~OBaHH~ in vivo Ha rpbIcax pa3mPmoro Bo3pacTa nora3am4, tITO HaH6om, maa cropocrb nornomcIma H yrzaaaamm l-aHamaa-14C 05aapyxmnacl, y rpblc no3pacTe 2½ MP..CffI~eB,Tor~a KaIC nOTpe6HOCTb y )IKtIBOTHblXB Bo3pacre 27½ MeCfllIen B HO3Ha~IHTeJIbHOi~CTeIIeHMHIDK0H MOXOT6MTb BblKI0qOM y MOHO~blXB3pOCJIbIX.