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Free amino acids in the caiman and rat
Comp. Biochem. Physiol., 1966, Vol. 17, pp. 583 to 598. Pergamon Press Ltd. Printed in Great Britain
FREE AMINO ACIDS IN T H E CAIMAN AND RAT* J. D. HERBERT, R. A. C O U L S O N and T. H E R N A N D E Z Departments of Biochemistry and Pharmacology, Louisiana State University School of Medicine, New Orleans, La.
(Received 19 July 1965) A b s t r a c t - - 1 . Free amino acids and total ninhydrin reactive compounds were
determined in alcoholic extracts of the tissues of fasting and fed caimans and fasting rats. 2. Total ninhydrin reactants exceeded the sum of amino acids on the chromatogram several-fold. 3. Generally only three or four amino acids, most commonly glycine, alanine, glutamine and glutamic acid, were significantly concentrated above plasma levels. No essential amino acids were concentrated in the tissues of the fasting animal 4. Species comparison showed a marked similarity in most tissues; differences were apparent in the erythrocyte, liver and lung. INTRODUCTION In vivo studies of amino acid metabolism in the caiman (Caiman latirostris) required a knowledge of free amino acids in the tissues of the fasting animal. Very little information has appeared in the literature on reptiles, and virtually no quantitative data on the Crocodilia are available. The present study was undertaken to determine the distribution and concentration of free amino acids in the caiman. The rat (Rattus norvegicus) served as a convenient subject for species comparison in parallel studies on a mammal. A wide variety of methods have been used to determine free amino acids in tissues, including non-specific (Van Slyke, 1912, 1914) and specific chemical procedures (Alexander et aL, 1945; Hamilton, 1945a, b; Christensen & Streicher, 1948), microbiological methods (Solomon et al., 1951), paper chromatographic procedures (Roberts & Frankel, 1945; Awapara et al., 1950a; Dubreuil & Tirniras, 1953 ; Roberts et al., 1957, 1958), and ion-exchange chromatographic determinations (Moore & Stein, 1954a, b; Tallan et al., 1954a; Spackrnan et aL, 1958; Nakagawa et al, 1964). Data obtained from such determinations indicated intracellular free amino nitrogen exceeded extracellular concentrations (Van Slyke & Meyer, 1914), individual tissues had characteristic free amino acid patterns (Roberts & Frankel, 1945), many species had similar free amino acid patterns for a given tissue (Roberts et al., 1957, 1958), and glycine, glutamine, alanine and glutamic acid were the amino acids most often present in high concentration in mammalian tissues * Supported in part by a grant from the National Institutes of Health, H-02062. 583
584
J.D. HERBERT,R. A. COULSONAND T. HERNANDEZ
(Christensen et al., 1948a, b; Awapara et al., 1950a; Wu, 1954; Tallan et al., 1954a). A comparison of the amino acid content of different tissues may lead to erroneous conclusions unless the nutritional state is considered. Nutritional state is difficult to control, depending not only on the length of fast before sacrifice, but also upon the nutritional history of the animal, i.e. previous diet, quantity of food intake, appetite, rate of growth, etc. Consequently, when the liver of one animal is compared with the muscle of another, the results may be misleading. In the experiments reported here, an attempt was made to compensate for this factor by analyzing about twelve tissues from each of a series of animals. Using this procedure we observed that the ratio between one tissue and another, as between one amino acid and another, was a relatively constant feature. However, differences in molar concentrations from one individual to another were considerable. MATERIALS AND METHODS Sprague-Dawley rats of both sexes, weighing 200-300 g, were fasted 24 hr and anesthetized with pentobarbital. Blood samples were taken by cardiac puncture or from the tail, and various tissues removed from the living animal were quickfrozen by pressing between lead blocks at - 20°C. The procedure, from excision to freezing, required about 5 sec maximum for each tissue. Half gramme samples of the frozen tissue were weighed and immersed in 9 vol of cold 95% alcohol, minced with scissors while immersed, homogenized in a Ser-Val high-speed micro homogenizer and centrifuged. The supernatant was stored in a freezer at - 2 0 ° C . Caimans, weighing 40-80 g, were fasted at least 4 days, and blood and tissues were prepared as for the rats. Aliquots of the alcoholic supernatants were evaporated in an air stream at about 20°C, but not to dryness. The samples were dissolved in a few drops of 0.1 N HCI and applied to a modified Moore-Stein column of an automatic analyzer. This apparatus was a modification developed by Coulson et al. (1965) in this laboratory, who added a second column to the system obtained from Technicon. Increasing the rate of flow of the buffer through the column permitted analysis of two samples in about 9 hr. The subsequent addition of a third column to the system facilitated analysis of large numbers of samples. A note about the method of evaporation is necessary. Evaporation in a water bath at temperatures ranging from 50 to 80°C resulted in partial destruction of glutamine, glycine and some basic amino acids. Evaporation at lower temperatures (20°C) in a nitrogen stream gave good recovery, but compressed air served equally well and was considerably cheaper. Calculation of per cent recovery indicated no apparent loss from air oxidation. Recovery seemed slightly better when the sample was not taken to dryness, so, in practice, a residue of 0.1-0.2 ml of fluid was left in the sample tube. Total ninhydrin reactants were determined for each sample by a modified photometric method (Troll & Cannan, 1953).
FREEAMINOACIDSIN THE CAIMANANDRAT
585
Free amino acids were determined on the whole carcass of a single caiman and a single rat by procedures similar to those used for individual tissues. RESULTS Tables 1 and 2 give the molar concentrations of twenty-two amino acids found in various tissues of the caiman and rat. Fig. 1 illustrates values obtained from determination of total ninhydrin reactants. The sum of amino acids from the column chromatographic analysis (Tables 1 and 2) has also been plotted in Fig. 1. z ¢r tu O9 09
1--
60050"0-
0
D-TOT- NIN I~I-AM AC E~-AM AC-- TOP 4
z n.,
>w tuz F-c= z
Lu
E I.o
,~
300-
e~
,.-, 20"0-
)"r"
z z
I0-0 0.0 CAIMAN
RAT
FIG. 1. T h e c r o s s - h a t c h e d areas r e p r e s e n t all t h e free a m i n o acids e x c e p t t h e four p r e s e n t i n greatest c o n c e n t r a t i o n , t h e l i n e d areas i n d i c a t e t h e a m o u n t c o n t r i b u t e d b y t h e four m a j o r a m i n o acids, a n d t h e e n t i r e b a r r e p r e s e n t s t h e total n i n h y d r i n reactants.
These summations are uniformly lower than total ninhydrin or paper chromatography values reported for the rat (Christensen et al., 1948a; Awapara et al., 1950a, b; Cravioto et al., 1951; AnseU & Richter, 1954; Wu, 1954). However, the values obtained for total ninhydrin reactants appear to be of the same order or somewhat greater than the total amino acid values previously reported. Although differences in procedure, such as choice of protein precipitant, may be a factor, it seems possible that some of the earlier values may have been influenced by contributions from ninhydrin reactive compounds which were not amino acids. The occurrence of taurine, glutathione, phospho derivatives and various small peptides has been reported in tissues (Tallan et al., 1954a) and, while we did not attempt an identification, we noted large quantities of ninhydrin reactive compounds on the chromatogram that were eluted very early by the acidic portion of the buffer. A comparison of amino acid sums in plasma and in various tissues (Fig. 1) will show that the intracellular concentrations were considerably higher than
0'082 0"03-0"16 0"056 0'03-0"10 0"284 0-19-0-46 0"119 0"07-0'23 0-158 0.11-0.25 0"157 0-14--O.22 0-163 0'11-0"26 0"135 0'07-0-27 0"278 0-12-0"39 0'151 0"09-0"25 0"183 0-10-0-34 0.144 0"08-0"33
0'011 0"01-0"02 0"056 0'03-0"10 0-227 0'11-0-37 0"385 0"16-0"91 0'341 0"27-0-61 0"071 0-05-0"09 0"262 0-17-0'37 0'171 0"10-0-28 0"203 0-10-0.28 0'438 0-31-0.56 0.354 0"20-0"51 0"229 0"16-0.36
0"090 0"05-0"14 0'089 0-06-0"11 0"252 0'13-0'50 0'168 0"11-0"26 0-275 0"18-0"40 0"134 0.08-0-18 0"152 0-10-0-17 0"118 0,05-0-20 0"224 0.13-0.28 0'171 0-10-0.28 0"213 0-13-0-26 0.203 0.12-0.45
Ser 0"362 0'13-0"88 0"195 0"07-0'43 0-205 0'05-1"08 0"054 0-0.38 0'421 0"12-1'2 0'135 0-82-2-4 0"736 0.42-1.2 1"47 0"60-3-7 2'18 0"84-3"0 4'04 2'2-7'4 4-17 2'4-6'8 0"665 0"51-1,2
Glu N H , 0"106 0'04-0.20 0"542 0"27-0"97 3"75 1-4-6.7 1"71 1"0-3-5 2"11 1"6-3.4 0-604 0'54-0.80 2"04 1'1-3'5 2-09 1'3-3'3 0"975 0'35-1"4 2"41 2.1-4.1 5-68 2'6-6.8 2"22 1.4-3.2
Glu
* Mean and ranges given; number in parentheses is number of animals,
Lung (9)
Brain (9)
Heart (9)
Muscle (9)
Stomach (7)
Gut (9)
Eye (9)
Spleen (9)
Liver (9)
Kidney (9)
Eryth. (6)
Plasma (10)
Thr
Asp
Aab~
Val
0-322 0'011 0"170 0'16-0"51 0"005-0'02 0"11-0"22 0"403 0'008 0"117 0'28-0.61 0"003-0-02 0'10-0-16 0"857 0"043 0'327 0-54-1-2 0'005-0.07 0.12-0.41 0'434 0-009 0.157 0"34-0.60 0"006-0-013 0-12-0.25 0'487 0"010 0'254 0-27-0-62 0'004-0.02 0'20-0"36 0"541 0"123 0-130 0"38-0.59 0"07-0.23 0"07-0.16 0.587 0"013 0-160 0.40-0.77 0'01-0.02 0"09-0.25 0.470 0"017 0"129 0"32-0"72 0"01-0"05 0.08-0.21 1-36 0"025 0'167 0'79-1.9 0"01-0.04 0"13-0-26 0-519 0"021 0-152 0-29-0-70 0.01-0.03 0'08-0-21 0-402 0.025 0'040 0"20-0.78 0"01-0'04 0"02-0.05 0'581 0.018 0.163 0'38-1.0 0'005-0'04 0-08-0.30
Ala
t a-Aminobutyric acid.
0"380 0-24--0"70 0'615 0"28-1"0 1"89 0'70-4.0 0"919 0"58-1"5 1"35 0'97-2-5 0'403 0"32-0-68 1"03 0"73-1-6 0'783 0.39-1-2 5"20 2"0-7.0 0"562 0'38-1-0 0"839 0,42-1.1 0.657 0'51-0.96
Gly
TABLE 1--FREE AMINO ACID CONTENT OF TISSUES ( m M / k g WET TISSUE) IN FASTING CAIMANS*
0.047 0"03-0'06 0"040 0'03-0.07 0"050 0'03-0.14 0-052 0"03-0.11 0"089 0-05-0.14 0.104 0-03-0-39 0-068 0"05-0.09 0-074 0.04-0.12 0.192 0"07-0.28 0.047 003-0-08 0-033 0.02-0-07 0"057 0"04-0.13
Met
~q
.q
.~
~'~
OO O',
Leu
Tyr
Phe
Gab~
Om
0.192 0.052 0.091 0.071 0"14-0-32 0.03--0.11 0-07-0-16 0"03-0"15 0.155 0.049 0.087 0.024 0.019 0"11-0"22 0.03-0.08 0.07-0.12 0.005-0-05 0.01-0.06 0.232 0-071 0-118 0-093 0.086 0"13-0"36 0.04--0.11 0.08-0.17 0-01-0.32 0.04-0.21 0.240 0.077 0.109 0.027 0.027 0.16-0.36 0.03-0-13 0.04-0.20 0-02-0.07 0.02-0.05 0.305 0.078 0.146 0.116 0.025 0"26-0-43 0"05-0-13 0"08-0"33 0-01-0'46 0.02-0'08 0"160 0'077 0.147 0"138 0"014 0-13-0"21 0'02-0-23 0"07-0"26 0.02-0'17 0"003-0'04 0"023 0.216 0.056 0.093 0.005-0.06 0-11-0.31 0-05-0-07 0'06-0"16 0.077 0.203 0.058 0.110 0.02-0"14 0-12-0.27 0.04---0.09 0.07-0.19 0.060 0.242 0.061 0.121 0-05-0"09 0-16-0.42 0.04--0.14 0"05-0"24 0.036 0-201 0.057 0-084 0-01-0.08 0-11-0.32 0.04-0.11 0'05-0"13 1.99 0.012 0.066 0.030 0-048 0,04-0,08 0.02-0-07 0"03-0"08 1-3-3.6 0-003-0.03 0.216 0.059 0-110 0.030 0.043 0"13-0"32 0-05-0-08 0"08-0-14 0.004--0.10 0-01-0,14
y-Aminobutyric acid.
Plasma (10)
0'079 0.05-0.14 0.044 Eryth. (6) 0.03-0.08 0'098 Kidney (9) 0.05-0.13 0"139 Liver (9) 0.07-0.34 0"118 Spleen (9) 0"07-0"22 0"053 Eye (9) 0"03-0-07 0.086 Gut (9) 0"05-0.12 Stomach (7) 0-072 0"05-0"10 Muscle (9) 0-091 0.05-0-18 Heart (9) 0-082 0-03--0,12 Brain (9) 0.023 0.01-0.03 0.080 Lung (9) 0.03-0.13
lieu
TABLE 1--continued Try
0.193 0.004 0.11)-0.39 0.002-0-01 0.056 0.007 0.04---0.07 0.003-0-01 0.320 0-15-0"65 0.165 0-015 0-12-0"30 0--0.05 0.159 0.018 0.10-0"32 0.01-0"04 0"095 0"133 0-03-0"26 0.02-0"42 0"210 0.11-0"57 0"266 0.008 0.16-0"60 0.004-0"02 0"464 0.404 0-10-0.64 0.27-0'76 0'165 0'110 0-04--O.31 0.08-0,13 0.101 0.003 0.05-0.20 0-0.01 0.299 0-014 0-11-0-97 0.01-0-03
Lys 0-189 0.12-0-24 0.273 0.20-0-38 0.352 0.16-0-69 0.276 0.17-0-44 0.322 0'21-0-59 0.154 0.13-0-22 0.200 0"11-0-35 0.243 0.13-0"43 0"407 0.25-0"52 0-316 0.16-0.48 0.068 0.04--0.11 0.485 0.20-1.5
His
3.72 2.7-5.1 0-101 0.06-0-16 0-062 0.03-0-11 1.07 0"13-1"9 0"192 0.16-0-31 0-095 0.03-0"24 0-249 0.14-0-50 0'172 0.01-0"31 0.233 0-08-0,44 0.752 0.46-1"4 0.391 0.26-0-66
3-Me* His
0.110 0.07-0-28 0-022 0.01-0-04 0"091 0.07-0.13 0.073 0.04-4)-16 0-062 0"04-0"12 0-029 0-02-0"05 0"059 0-02-0"13 0-118 0.09-0"15 0-273 0'06-0"45 0-094 0.04-O.15 0-061 0.03-0.08 0-080 0.06-0.14
Arg
t#l O0
0
,.,
.~ ,a
o
0.237 0.20-0.26 0.129 0-11-0.14 0-380 0.25-0,82 0.300 0.25-0.36 0-487 0.39-0.58 0"292 0.22-0.36 0'677 0.44--1.2 0"260 0-03-0.47 0.428 0.09-0-48 0.310 0.22-0.37 0.411 0.33-0.45 0-349 0.30-4).41 0.212 0.18-0.23
0.012 0.01-0.02 0,007 0.005-0.01 0.584 0.51-0.83 0.277 0.16-0-40 1"68 1"3-2"0 0'154 0"11-0"20 0"550 0.20--0"77 0.246 0.23-0"26 0.120 0.05-0.22 0.560 0.47-0.65 1.62 1.3-1-9 0.611 0.49-0.82 0.584 0.43-0.71
0.231 0-17-0.30 0.188 0.18-0.19 0.650 0.49-1.1 0-243 0.11-0.44 0,479 0-14-0.98 0.197 0.08-0'41 1-11 0"39-1"9 0.338 0.18-0.52 0.412 0.15-0.73 0.311 0.17-0.51 0.372 0-17-0.64 0.277 0.14-0.45 0.284 0.12-0.46
Ser 0.545 0.44-0.62 0.256 0.20--0.32 0.323 0.09-0.46 2-68 1.5-3.5 1.27 0.98-1'7 0.726 0.63-0'83 1"82 0.54--3'9 1.58 1.2-2.0 2.34 2.1-2.7 5-99 2.5-10.0 2-59 2.2-3.1 1.12 0.65-1.5 0.894 0.69-0.99
Glu NH2 0-152 0.09-0.29 0.203 0.15-0.25 4-09 3.8--4-3 1 '64 1.4-2.2 4.87 4.4-5.5 1-19 0"96-1"4 3"54 2.5-5.0 2"46 2.4-2.6 1.10 0-57-1-4 4"45 3-7-4.8 7-89 7.4-8.3 1-84 1.3-2.2 4-38 3-9-4.8
Glu
Cit 0.447 0.37-0.50 0"438 0.37-0-53 4.07 3.6-4.9 3-05 2.6-3.3 2"80 2.0-3'7 0"839 0"75-1"1 3'04 1"7-4-8 2.17 1-8-2.4 4"87 3.0-6.0 0.765 0.55-1.2 1"12 0.86-1"4 6.93 6.0-8.3 3'55 3.5-3-7
Gly 0-323 0.24-0.38 0-243 0.21-0.26 0.902 0.56-1.8 1.45 0-97-1.9 0-957 0'64-1.3 0"624 0'54-0'86 2-25 1"2-3.8 1 "36 1"3-1"4 1 "71 1-4-2,0 1 "92 1"3-2"8 0"663 0.38-0.93 1.21 0-57-2.5 0-941 0.83-1.1
Ala
"j"c~-Aminobutyric acid.
0.012 0-01-0.02
0.059 0"02-0-09 0'017 0-01-0"03 0.141 0'10-0"18 0.045 0.04-0.05 0.094 0.06-0.12 0.112 0.08-0.13
0.053 0.03-0.08 0-024 0.02-0.03 0.014 0.01-0"02
* Mean and ranges given ; n u m b e r in parentheses is n u m b e r of animals.
Testis (7)
Lung (8)
Brain (8)
Heart (9)
Muscle (8)
Stomach (8)
Gut (13)
Eye (9)
Spleen (8)
Liver (9)
Kidney (10)
Eryth. (7)
Plasma (10)
Thr
Asp 0.012 0.01-0.02 0.011 0-005-0.02 0.068 0.01-0-12 0.049 0.02-0.09 0.041 0.01-0'07 0-053 0'01-0"10 0"062 0.02-0"13 0.024 0.01-0.05 0.016 0.01-0-03 0.022 0.004-43.05 0.033 0.03-0.04 0.031 0.02-0.05 0.013 0.01-0-02
Aab'~
TABLE 2--FREE AMINO ACID CONTENT OF TISSUES ( m M / k g WET TISSUE) IN FASTING RATS*
0.177 0.09-0.22 0"090 0.08-0.09 0.519 0-42-0.86 0,217 0,14-0.28 0-304 0-16-0-39 0"162 0-12-0"19 0"828 0.20-1-7 0-247 0.15-0"39 0.144 0-11-0-18 0"146 0.12-0-16 0"127 0.12-0.14 0.165 0.07-0.22 0-123 0.10-0.15
Val
0.091 0.06-0.11 0,054 0.05-0-06 0.142 0,07-0.36 0.086 0,04--0,14 0.177 0.10-0-24 0.117 0.08-0"13 0"537 0"15-1-0 0.160 0.10-0.27 0.076 0'05-0-10 0"077 0.06-0-11 0-086 0.07-0.10 0.073 0.05-0-10 0-074 0.06-0.10
Met
N
t~
,~O
.~ .~
t/1 OO OO
0.080 0.07-0.09 0"075 0.06--0.10 0"135 0.08-0.31 0.114 0.07-0.16 0.224 0.09-0.34 0.097 0'07-0.11 0.596 0.12-1.2 0"137 0.10--0.23 0.058 0.04-0-08 0.075 0.07-0-08 0.044 0.03-0.05 0.077 0'07---0.09 0.061 0.04--0-10
•r-Aminobutyric acid.
Testis (7)
Lung (8)
Brain (8)
Heart (9)
Muscle (8)
Stomach (8)
G u t (13)
Eye (9)
Spleen (8)
Liver (9)
Kidney (10)
Eryth. (7)
Plasma (10)
Ileu
0.155 0"15-0-16 0'097 0"07-0-14 0.376 0.19-0.98 0.292 0-17-0-43 0-566 0.20---0-86 0"187 0.16--0.24 1"74 0-27-3.8 0.398 0-24.-0.64 0.168 0.16-0.18 0.177 0.16-0.19 0.106 0.05-0.16 0"146 0.13-0.17 0.216 0.12-0.32
Leu 0.092 0.09-0.10 0.064 0.06-0.08 0.136 0.09-0.27 0.102 0.05-0.15 0.196 0.12-0.25 0.168 0.14-0.18 0-790 0"22-1"5 0.169 0.11-0.30 0.099 0.08-0-12 0.091 0"06-0.16 0-047 0.04-0-05 0.072 0.04--0-09 0.123 0"09-0-16
Tyr 0.086 0-08-0.09 0.062 0-06-0-08 0-162 0.08-0-41 0-115 0-05--0-19 0.259 0-10--0.35 0.146 0-10-0.20 0.970 0-20-2-0 0.212 0.12-0.35 0.077 0.07-0.08 0.067 0.06-0.07 0.058 0.04--0.07 0.089 0.07-0.10 0.117 0"06-0.16
Phe
Orn
0.046 0.03-0.08 0"033 0.02-0.05 0"024 0.004--0.06 0.074 0.03-0.11 0.241 0"031 0.01-0.47 0.01-0.06 0.252 0.037 0.22-0.36 0.02-0"06 0.232 0.128 0.04--0"42 0.02-0"34 0.060 0-042 0.01-0"13 0.03-0.06 0.026 0.02-0-03 0.017 0.004-0-04 2.15 0.022 1.0-3.3 0.01-0-04 0.061 0.051 0-02-0.13 0.03-0-06 0-103 0.013 0.01-0.18 0.01-0.02
G a b ;~
TABLE 2~continued Try
0.406 0.065 0-32-0.47 0.04-0.09 0.215 0-20-0-23 0-343 0-18-0-57 0-321 0.18-0.48 0.368 0-13--0.65 0.488 0-28-1.0 1.03 0.031 0-16-2.4 0.01-0.07 0.321 0-18-0.49 0'810 0.642 0.37-1.3 0-12-1.4 0'833 0.35-2.4 0"226 0.20-4).26 0"285 0.17-0.49 0"349 0-012 0.23-0.54 0-01-0.02
Lys 0-111 0'07-0.17 0-066 0.05-0"08 0.238 0.19-0.39 0.412 0-35-0.46 0.436 0.15-0-67 0.126 0.12q)'14 0-682 0.15-1"5 0-258 0.15-0"38 0-302 0.24-0.38 0.169 0.10-0.36 0.077 0.07-0-08 0-114 0.10~-13 0.114 0.08-0.16
His
0.020 0.01-0-03
0.547 0.27-0.73
0-025 0.02-0"03
3-Me His
0.161 0-11-0"21 0.113 0-11-0.12 0.077 0.04--0-17 0.002 0-0.01 0.061 0.02-0.09 0.106 0.1ff-4).12 0.450 0.07-1.0 0.128 0.10-0.17 0.096 0.04--0"18 0.134 0.07-0"28 0.042 0.02-0"06 0.046 0.03-0"06 0.090 0.06-0.11
Agr
Ln O0
O
590
J.D. HERBERT,R. A. COULSONANDT. HERNANDEZ
extracellular values. But when the values of the top four amino acids were subtracted from the sum, as in Fig. 1, it was apparent that the remainder were not concentrated significantly above plasma levels. The top four constituted 50-80 per cent of the intracellular amino acids and the remainder were roughly equivalent to their plasma values. One exception was rat gut, in which the remainder varied over a wide range of concentrations (see Table 2). These animals were fasting, and the excised gut segments appeared empty, so, to minimize autolysis and avoid inadvertent extraction of cellular amino acids, the segments were not washed. Thus some variation may have been due to residual dietary amino acids or partially catabolized epithelial sheddings in the lumen. Figure 2 illustrates the four amino acids that were concentrated in each tissue. A cursory examination will indicate the general importance of glycine, alanine, glutamic acid and glutamine. Aspartic acid appeared in the top four in only three tissues: rat brain and spleen and caiman liver; no other amino acid was represented in the top four more than twice. These select few occurred in characteristic ratios in the various tissues. A general similarity was seen between the two species, notable exceptions being the erythrocyte, liver and lung.
Erythrocyte The erythrocyte of the caiman contained a compound, appearing in the position of 3-methyl histidine, in quantities equal to the entire remainder of amino acids. This compound, first reported in the urine of man and cat (Tallan et al., 1954b), did not appear in the erythrocyte of the rat, dog or turtle, but did occur in the alligator erythrocyte in quantities comparable to those in the caiman. Its metabolic function is unknown; it does not appear to be related to the presence of a nucleus in the red blood cell, since the compound was absent in the turtle erythrocyte. Quantities of this compound in caiman stomach, heart, brain and lung were relatively high, possibly above the amounts which could be contributed by blood contained in these tissues. The erythrocyte of the rat showed little concentrating ability, although glycine and glutamic acid appeared to achieve a slight concentration gradient when corrections for cell water were applied.
Kidney Amino acid concentrations in the two species were similar, and total values were comparable with other tissue totals in contrast to some reports that kidney contains unusually high concentrations of amino acids (Solomon et al., 1951).
Liver Species differences were readily apparent in this tissue. Whereas the glycine and glutamine content of rat liver remained consistently high, glutamine, particularly, showed great variation in the caiman liver. In the fasting caiman, glutamine is often immeasurably small, but may be present in significant quantities. It rises greatly after eating and falls slowly as the exogenous amino acids not needed
FREE AMINO ACIDS I N THE CAIMAN AND RAT
591
for protein synthesis are catabolized (Coulson & Hernandez, 1965). Values for total liver amino acids in the rat were consistently lower, relative to other tissues, than other values reported in the literature (Christensen et al., 1948a; Wu, 1954) for reasons which are not clear. Spleen Some similarity is evident, though caiman spleen had a rather high content of 3-methyl histidine, a reflection, perhaps, of the high content of erythrocytes in this organ.
Eye The values obtained for this tissue were, in effect, an average of the amino acid concentrations of its various structural components: sclera, retina, lens, aqueous and vitreous humors. The small size of the animals used made it impractical to attempt analyses of its components. However, we analyzed three components of dog eye separately and found that the aqueous humor resembled plasma except for a rather low glycine level, vitreous humor concentrated glycine and glutamic acid to some degree, and the lens showed high values for glycine and glutamic acid. The caiman eye was characterized by large amounts of a peptide emerging before aspartic acid, which, upon hydrolysis, yielded equimolar quantities of glycine and glutamic acid as two of its components. Further identification of this compound was not attempted. Gut and stomach Again the major constituents were the same in both species. In rat gut, however, the remainder of the amino acids, particularly the essentials, varied greatly. Skeletal muscle The prominence of glycine in skeletal muscle of both species was a very constant feature of this tissue, as was the presence of tryptophane, a compound found only in trace amounts in most tissues. Rat muscle contained rather large amounts of 3-methyl histidine, which was not found in appreciable quantity in other rat tissues. Heart and brain High quantities of glutamine and glutamic acid in heart and the prominence of glutamic acid, glutamine and y-aminobutyric acid in brain verified numerous observations on various species reported in the literature (Hamilton, 1945b; Awapara et al., 1950a, b; Frankel, 1950a; Cravioto et al., 1951; Ansell & Richter, 1954; Tallan et al., 1954a; Roberts et al., 1957, 1958; Okumara et al., 1959). Lung Rat lung contained large quantities of glycine, while caiman lung yielded glutamic acid in greatest quantity, an apparent species difference.
v I-
Z ~
'-1
"
GUT
PLASMA
KIDNEY
LIVER
I
STOMACH
HEART
Npi
MUSCLE
-'- N N
~ Y T E S
~-
BRAIN
SPLEEN
LUNO
EYE
I
TESTIS
FIG. 2. A graphic representation of the data of Tables 1 and 2. Four compounds constitute 50-80 per cent of the total free amino acids of the tissues. Column R represents the remaining amino acids, plotted according to their value, but without identification.
0'0.-
6O-
@10-
b~
t~
~q
©
>
t~
bo
593
FREE A M I N O ACIDS I N THE CAIMAN AND RAT
The prominence of the "big four" amino acids indicated in Fig. 2 is further illustrated by examination of free amino acids extracted from the whole carcass. Table 3 gives molar concentrations of carcass amino acids in a single caiman and a single rat. In both animals the amino acids present in greatest concentration were glycine, glutamine, glutamic acid and alanine. TABLE 3 - - C ~ c A s s From AMINO ACIDS ( m M / k g WET TISSUE) Asp Caiman Rat
Caiman Rat
Thr
Glu NH2 0"131 0"024 0.090 1.30 0.188 0"200 0.204 2.23
0"680 0.003 1-32 0.082
Lcu
Orn
Tyr
Ser
Phe
Gabt
Glu
Cit
Lys
Gly
Ala
Aab*
Val
I
~W
Ileu
1.37 0-655 0.014 0.060 0"013 0"022 3.23 1-00 0.006 0.115 0'057 0"099
Try
His
3-Me His
Arg
0.072 0"024 0.066 0-022 0.013 0.207 0-264 0-220 0.241 0.112 0.142 0"077 0.092 0"023 0"033 0.150 0"018 0"242 0.120 0.066
* =-Aminobutyric acid.
Met
Sum 5.603 9"694
t ~,-Aminobutyric acid.
2=01
>,-
o
:t 4'0~0-
~ ~
CELL/PLAS o o RAT 0 ,.'~ 12"2 23":5 6"1 44"1
2"4
26"0q4"1
3 . 7 10'2 58"6
1"6
CAIMAN RAT FIO. 3. T h e relationship between certain intracellular and extracellular amino
acids in the entire carcasses of one 50 g rat and one 50 g caiman. It is evident that only a few amino acids are highly concentrated inside the cells. To illustrate just how much these compounds were concentrated, corrections were made for cell solid content and extracellular fluid dilution, yielding values in mM/kg cell water. These values are compared with plasma values in Fig. 3 and their ratios calculated. Aspartic acid was added because its plasma value was very low and intracellular concentration was obvious, even though it did not
-0"02 -0'05 -0"04 -0-06 -0"05 -0"06 0 -0-03 -0.05 --0-04 --0'03 -0.05
Phe
Tyr
-0"01 -0'04 -0"03 -0-04 -0'04 -0-05 +0"03 -0.02 -0.04 --0'04 --0.02 -0-02
+0.05 -0.01 -0.06 +0-31 -0"09 -0-11 -0.08 -0.06 +0-16 -0.08 -0.10 --0'04
+0.04 -0.03 +0.09 -0'23 +0"04 +0"05 +0-18 +0.04 -0-02 +0'22 +0.14 +0-07
Thr
+0.27
+0.04 +0'03 +0"34 -0"02
Gab ~
+0.10 +0.03 +0.07 +0"01 -0"14 +0'06 +0.01 +0.05 +0'28 -0-04 -0.10 --0.06
Ser
-0'01 -0-01 -0'01 -tr. +0-01 +0'01 +0-02 -0.02 +0-17 +0.01 +0.01 +0-02
Om
+1.22 -0.12 +2.52 +1'51 +0"03 +0'03 +1.33 -0-05 +4'70 +2-76 +0.39 +0.56
Glu NH2
+0"02 -0"04 -0'14 +0"02 -0"06 -0"04 -0-10 --0.06 +0.76 --0.04 --0.02 -0.17
Lys
+0.32 +0.01 +5.05 +0'79 +2"42 +0"06 +2"09 --0.04 -0'19 +1-11 +0'70 +0.62
Glu
Gly
-0-12 --0.01
Tyr +0"14 -0-12 +0'09 -0-01 -0-01 +0'07 +0'08 -0.03 +0-16 --0.16 +0.01 -0.07
His
+0.98 +0-33 +0.64 +1'14 +0-26 +0.38 +0.94 +0-58 +2"19 +0-64 +0.12 +0.64
Ala
(rnM/kg WET
+1-72 +0'26 +0"99 +0'48 +0-25 +0'36 +1.89 +0.72 +3"73 +0-84 +0"30 +0.01
C H A N G E IN AMINO ACIDS
+tr. -0-01 -0'05 +0"04 -0"03 -tr. -0.02 -0'06 +0.55 --0.02 --0.04 -0-02
Arg
+0.03 0 -0-12 +0'12 -0.04 -0.02 +0.03 +0.06 +0"02 -0.02 -0.02 +0.02
Val
+4"88 +0"33 +9"08 +4"17 +2"42 +0'52 +6"66 +1.14 +12.27 +5.12 +1.29 +1.52
Ileu
+0-12 -0.07 +0.04 -~ 0"03 -0'03 -tr -i 0.18 +0"12 +0"01 -0.01 -0.01 -i 0.08
Leu
,* 7-Aminobu~yric
289 105 194 173 130 111 204 117 192 151 108 122
% of control
+0.03 +0.01 -0.01 -0.03 -0-01 -0-02 +0.04 -0.02 -0'02 -0.02 -0.01 +tr. Total change
+0.13 -0.01 +0.06 +0"07 +0.06 +0'02 +0.07 +0"08 +0"01 +0.16 +0.01 +0.14
~Iet
? ~-Aminobutyric acid.
+0"20 -0"06 -0"02 -0"54 -0"13 -0-04 -0.12 -0-07 --0'15 --0.21 --0.23
3-Me His
+0.02 0 +0-01 +0'01 +0"05 -0.07 +0.01 -tr. +0"04 +0.01 -tr. +0.02
Aabt
TISSUE) AFTER FEEDING*
* Average of five caimans killed 24 hr after feeding compared to control values in Table 1. acid.
Plasma Eryth. Kidney Liver Spleen Eye Gut Stomach Muscle Heart Brain Lung
Plasma Eryth. Kidney Liver Spleen Eye Gut Stomach Muscle Heart Brain Lung
Asp
TABLE 4
N
>
> ~J
.~ ¢) O
",ID 4~
FREE A M I N O ACIDS I N T H E C A I M A N AND RAT
595
achieve large quantitative values in any tissue tested. The remaining amino acids are plotted as well to show that these are not generally concentrated by the cell. Highest ratios are seen for aspartic and glutamic acids, two compounds which attain primarily an extracellular fluid distribution when administered exogenously. Glycine, alanine and glutamine all attain a body water distribution when injected. Table 4 presents the changes in free amino acids in the tissues occurring when ten small caimans (100 g) were permitted to eat all of the meat they desired. One day was allowed for digestion, absorption and distribution; the animals were then killed and the tissues were analyzed in the usual way. The results were informative in several respects. The majority of the amino acids did not rise appreciably in any tissue, and although the total free amino acid concentrations were about twice those of the control group, it is evident that three or four of the "non-essential" ones were responsible for most of the increase. Striated muscle, the largest tissue in the body, showed the greatest millimolar increase in total amino acids after feeding, and over 80 per cent of the gain was due to glutamine, glycine and alanine. Although glutamic acid was also increased considerably in several organs, the total rise of this compound in the entire body was not very great since the organs sharing in the increase had a total weight considerably less than the muscle. The eye, which is neither very active metabolically nor subject to rapid growth, was affected very little. It is significant that the three amino acids which increased the most were also the ones present in the fasting plasma in great concentration and, in addition, were also the ones responsible for most of the synthesis of excretion products of nitrogen metabolism. It is difficult not to conclude that these amino acids represent one stage in the degradation of those amino acids present in excess of that required for protein synthesis. DISCUSSION It is evident that only a few amino acids are maintained at high concentrations in the tissues of fasting caimans and fasting rats. The significance of such selective concentration remains to be explored. These amino acids are almost certainly neither direct products of protein degradation nor source materials for protein synthesis, since essential amino acids were not concentrated to any extent. Three of the amino acids most often concentrated, glycine, alanine and glutamine, are rapidly metabolized and serve as precursors of ammonia and uric acid when injected into an intact alligator (Coulson & Hernandez, 1964). Thus these compounds may serve as vehicles for the disposal of nitrogen produced in catabolism of protein. However, this theory does not explain why the cells maintain high intracellular gradients of these amino acids, or why these three should occur in characteristic ratios in each tissue. It fails also to explain the presence of high concentrations of glutamic acid, a compound which is slowly metabolized when administered exogenously. The characteristic amino acid patterns in different tissues suggest a difference in the primary metabolic reactions which occur in these tissues. It is not necessary
596
J.D. HERBERT,R. A. COULSONANDT. HERNANDEZ
to postulate mutually exclusive reactions; a difference in relative frequency could as easily account for the dissimilarity. Roberts & Frankel (1950a, b, 1951) have demonstrated a catabolic pathway for glutamic acid in brain via the intermediates, 7-aminobutyric acid and succinic semialdehyde. The high levels of glutamine, glutamic acid and 7-aminobutyric acid in brain suggest that this pathway may be important in the in vivo metabolism of this tissue. In kidney the low levels of glutamine may be significant in light of the kidney's role in ammonia formation, reputedly from the amide nitrogen of glutamine (Nash & Benedict, 1921; Van Slyke et al., 1943). However, glycine and alanine, two compounds equalling glutamine as ammonia precursors in the alligator (Coulson & Hernandez, 1964), are maintained at high concentrations in the kidney. The general prominence of glutamic acid may reflect its postulated role in transamination, though some doubt has been expressed as to the importance of transamination in the live animal (Coulson & Hernandez, 1965). SUMMARY 1. Alcoholic extracts of various tissues of the fasting caiman and fasting rat were analyzed for free amino acids by column chromatography and for total ninhydrin reactants by a colorimetric method. 2. Total ninhydrin reactive compounds exceeded the sum of amino acids on the chromatogram several-fold, indicating a high content of ninhydrin reactants which were not amino acids. 3. Each tissue contained high concentrations of three or four amino acids in characteristic ratios. Glycine, glutamine, glutamic acid and alanine were the most plentiful. 4. Comparison of the two species showed a marked similarity in most tissues; species differences were apparent in the erythrocyte, liver and lung. 5. Feeding increased glutamine, glycine, glutamic acid and alanine in most tissues of the caiman while affecting the essential amino acids very little. Acknowledgement--We are grateful to Mr. Jacob Watson for the preparation of the figures.
REFERENCES ALEXANDERB., LANDWEHRG. • SELIGMANA. M. (1945) A specific micro-method for the colorimetric determination of glycine in blood and urine. 3t. biol. Chem. 160, 51-59. ANSELLG. B. & RICHTERD. (1954) A note on free amino acid content of rat brain. Biochem. ft. 57, 70-73. AWAPA~ J., LANDUAA. J. & FtmmT R. (1950a) Distribution of free amino acids and related substances in organs of the rat. Biochim. biophys. Acta 5, 457-462. AWAPARAJ., LANDUAA. J., FImRSTR. & SmI~ B. (1950b) Free 7-aminobutyric acid in brain, ft. biol. Chem. 187, 35-39. CI~ISTENSEN H. N., ROTHW~LLJ. T., SEAm R. A. & STm~ICI~RJ. (1948a) Association between rapid growth and elevated cell concentrations of amino acids: in regenerating liver after partial hepatectomy in the rat. 3t. biol. Chem. 175, 101-105.
A M I N O ACIDS I N T H E C A I M A N A N D RAT
597
CHI~ISTENS~ H. N. & ST~SICHER J. (1948) Association between rapid growth and elevated cell concentrations of amino acids: in fetal tissues..7, biol. Chem. 175, 95-100. CHmSTENSEN H. N., STmzlcmm J. & ELBING~ R. L. (1948b) Effects of feeding individuM amino acids upon the distribution of other amino acids between cells and extracellular fluid..7, biol. Chem. 172, 515-524. COtJLSON R. A. & H~m'~ANDEZT . (1964) Biochemistry of the Alligator, p. 78. Louisiana State University Press, Baton Rouge. COULSON R. A. & H ~ A N D E Z T . (1965) Amino acid metabolism in the alligator. Fed. Proc. Symp. 24, 927-940. COULSON R. A., HERNANDEZ T. & BYERS V. (1965) A simple two-column system for the rapid analysis of plasma amino acids. Analyt. Biochem. 10, 159-162. C~vlOTO R. O., MASSmU G. & IzQt:IEmJO J. J. (1951) Free amino acids in rat brain during insulin shock. Proc. Soc. exp. Biol. Med. 78, 856-858. DUBREUIL R. & TIMn~S P. S. (1953) Effect of cortisone on free amino acids in serum and organs of the rabbit. Am..7. Physiol. 174, 20-26. HAMILTON P. B. (1945a) Gasometric determination of glutamine amino acid carboxyl nitrogen in plasma and tissue filtrates by the ninhydrin-carbon dioxide method..7, biol. Chem. 158, 375-396. HAMILTON P. B. (1945b) Glutamine: a major constituent of free ~t-amino acids in animal tissues and blood plasma..7, biol. Chem. 158, 397-409. MOORE S. & STEIN W. (1954a) Procedures for the chromatographic determination of amino acids on 4 per cent cross-linked sulfonated polystyrene resins..7, biol. Chem. 211, 893-906. MOORE S. & STEIN W. (1954b) A modified ninhydrin reagent for the photometric deterruination of amino acids and related compounds..7, biol. Chem. 211, 907-914. NAKAGAWAH., LINDSAY R. H. & COHEN P. P. (1964) Composition and labeling patterns of "free" and protein amino acids in Rana catesbeiana tadpoles and frogs. Arch. Biochem. Biophys. 106, 299-306. NASH T . P. & BENEDICT S. R. (1921) T h e ammonia content of the blood, and its bearing on the mechanism of acid neutralization in the animal organism..7, biol. Chem. 48, 463-488. OKUMARA N., OTSUKI S. & AOYAMAT . (1959) Studies on the free amino acids and related compounds in the brains of fish, amphibia, reptile, ayes, and m a m m a l . . 7 . Biochem. 46, 207-212. ROBERTS E. & FRANKEL S. (1945) Free amino acids in normal and neoplastic tissues of mice as studied by paper chromatography. Cancer Res. 9, 645-648. ROBERTS E. & FRANKEL S. (1950a) 7-Aminobutyric acid in brain: its formation from glutamic acid..7, biol. Chem. 187, 55-63. ROBERTS E. & FRANKEL S. (1950b) Glutamic acid decarboxylase in b r a i n . . 7 , biol. Chem. 188, 789-795. ROBERTS E. & FRANKEL S. (1951) Further studies of glutamic acid decarboxylase in brain. .7. biol. Chem. 190, 505-512. ROBERTS E., LOWE I. P., CHANIN M. & JELIN~K B. (1957) Free or easily extractable amino acids of the heart muscle of various species..7, exp. Zool. 135, 239-254. ROBERTS E., LOWE I. P., GUTH L. & JELINEK B. (1958) Distribution of ~,-aminobutyric acid and other amino acids in nervous tissue of various species..7, exp. Zool. 138, 313-328. SOLOMON J. D., JOI~SON C. A., S h ~ r ~ a A. L. & BERGEIM O. (1951) T h e determination of free and total amino acids in rat tissues..7, biol. Chem. 189, 629-635. SPACKMAN D. H., STEIN W . & M O O R E S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. TALLAN H. H., M O O l ~ S. & STEIN W . (1954a) Studies on the free amino acids and related compounds in the tissues of the cat..7, biol.Chem. 211, 927-939.
598
J. D. HERBERT,R. A. COULSONAND T. HERNANDEZ
TALLAN H. H., STEIN W. • MOORE S. (1954b) 3-Methylhistidine, a new amino acid from human urine. J. biol. Chem. 206, 825-834. TROLL W. & CANNAN R. K. (1953) A modified photometric ninhydrin method for the analysis of amino and imino acids. J. biol. Chem. 200, 803-811. VAN SLYKE D. D. (1912) The quantitative determination of aliphatic amino groups. J. biol. Chem. 12, 275-284. VAN SLYKED. D. (1914) The fate of protein digestion products in the b o d y - - I I . Determination of amino nitrogen in the tissues. J. biol. Chem. 16, 187-195. VAN SLYKE D. D. & MEYER G. M. (1914) T h e fate of protein digestion products in the b o d y - - I I I . T h e absorption of amino acids from the blood by the tissues. J. biol. Chem. 16, 197-212. VAN SLYKE D. D., PHILLIPS R. A., HAMILTON P. B., ARCHIBALDR. M., FUTCHERP. H. & HILLER A. (1943) Glutamine as source material of urinary ammonia. J. biol. Chem. 150, 481-482. W u C. (1954) Metabolism of free amino acids in fasted and zein-fed rats. J. biol. Chem. 207, 775-786.
FREE AMINO ACIDS IN T H E CAIMAN AND RAT* J. D. HERBERT, R. A. C O U L S O N and T. H E R N A N D E Z Departments of Biochemistry and Pharmacology, Louisiana State University School of Medicine, New Orleans, La.
(Received 19 July 1965) A b s t r a c t - - 1 . Free amino acids and total ninhydrin reactive compounds were
determined in alcoholic extracts of the tissues of fasting and fed caimans and fasting rats. 2. Total ninhydrin reactants exceeded the sum of amino acids on the chromatogram several-fold. 3. Generally only three or four amino acids, most commonly glycine, alanine, glutamine and glutamic acid, were significantly concentrated above plasma levels. No essential amino acids were concentrated in the tissues of the fasting animal 4. Species comparison showed a marked similarity in most tissues; differences were apparent in the erythrocyte, liver and lung. INTRODUCTION In vivo studies of amino acid metabolism in the caiman (Caiman latirostris) required a knowledge of free amino acids in the tissues of the fasting animal. Very little information has appeared in the literature on reptiles, and virtually no quantitative data on the Crocodilia are available. The present study was undertaken to determine the distribution and concentration of free amino acids in the caiman. The rat (Rattus norvegicus) served as a convenient subject for species comparison in parallel studies on a mammal. A wide variety of methods have been used to determine free amino acids in tissues, including non-specific (Van Slyke, 1912, 1914) and specific chemical procedures (Alexander et aL, 1945; Hamilton, 1945a, b; Christensen & Streicher, 1948), microbiological methods (Solomon et al., 1951), paper chromatographic procedures (Roberts & Frankel, 1945; Awapara et al., 1950a; Dubreuil & Tirniras, 1953 ; Roberts et al., 1957, 1958), and ion-exchange chromatographic determinations (Moore & Stein, 1954a, b; Tallan et al., 1954a; Spackrnan et aL, 1958; Nakagawa et al, 1964). Data obtained from such determinations indicated intracellular free amino nitrogen exceeded extracellular concentrations (Van Slyke & Meyer, 1914), individual tissues had characteristic free amino acid patterns (Roberts & Frankel, 1945), many species had similar free amino acid patterns for a given tissue (Roberts et al., 1957, 1958), and glycine, glutamine, alanine and glutamic acid were the amino acids most often present in high concentration in mammalian tissues * Supported in part by a grant from the National Institutes of Health, H-02062. 583
584
J.D. HERBERT,R. A. COULSONAND T. HERNANDEZ
(Christensen et al., 1948a, b; Awapara et al., 1950a; Wu, 1954; Tallan et al., 1954a). A comparison of the amino acid content of different tissues may lead to erroneous conclusions unless the nutritional state is considered. Nutritional state is difficult to control, depending not only on the length of fast before sacrifice, but also upon the nutritional history of the animal, i.e. previous diet, quantity of food intake, appetite, rate of growth, etc. Consequently, when the liver of one animal is compared with the muscle of another, the results may be misleading. In the experiments reported here, an attempt was made to compensate for this factor by analyzing about twelve tissues from each of a series of animals. Using this procedure we observed that the ratio between one tissue and another, as between one amino acid and another, was a relatively constant feature. However, differences in molar concentrations from one individual to another were considerable. MATERIALS AND METHODS Sprague-Dawley rats of both sexes, weighing 200-300 g, were fasted 24 hr and anesthetized with pentobarbital. Blood samples were taken by cardiac puncture or from the tail, and various tissues removed from the living animal were quickfrozen by pressing between lead blocks at - 20°C. The procedure, from excision to freezing, required about 5 sec maximum for each tissue. Half gramme samples of the frozen tissue were weighed and immersed in 9 vol of cold 95% alcohol, minced with scissors while immersed, homogenized in a Ser-Val high-speed micro homogenizer and centrifuged. The supernatant was stored in a freezer at - 2 0 ° C . Caimans, weighing 40-80 g, were fasted at least 4 days, and blood and tissues were prepared as for the rats. Aliquots of the alcoholic supernatants were evaporated in an air stream at about 20°C, but not to dryness. The samples were dissolved in a few drops of 0.1 N HCI and applied to a modified Moore-Stein column of an automatic analyzer. This apparatus was a modification developed by Coulson et al. (1965) in this laboratory, who added a second column to the system obtained from Technicon. Increasing the rate of flow of the buffer through the column permitted analysis of two samples in about 9 hr. The subsequent addition of a third column to the system facilitated analysis of large numbers of samples. A note about the method of evaporation is necessary. Evaporation in a water bath at temperatures ranging from 50 to 80°C resulted in partial destruction of glutamine, glycine and some basic amino acids. Evaporation at lower temperatures (20°C) in a nitrogen stream gave good recovery, but compressed air served equally well and was considerably cheaper. Calculation of per cent recovery indicated no apparent loss from air oxidation. Recovery seemed slightly better when the sample was not taken to dryness, so, in practice, a residue of 0.1-0.2 ml of fluid was left in the sample tube. Total ninhydrin reactants were determined for each sample by a modified photometric method (Troll & Cannan, 1953).
FREEAMINOACIDSIN THE CAIMANANDRAT
585
Free amino acids were determined on the whole carcass of a single caiman and a single rat by procedures similar to those used for individual tissues. RESULTS Tables 1 and 2 give the molar concentrations of twenty-two amino acids found in various tissues of the caiman and rat. Fig. 1 illustrates values obtained from determination of total ninhydrin reactants. The sum of amino acids from the column chromatographic analysis (Tables 1 and 2) has also been plotted in Fig. 1. z ¢r tu O9 09
1--
60050"0-
0
D-TOT- NIN I~I-AM AC E~-AM AC-- TOP 4
z n.,
>w tuz F-c= z
Lu
E I.o
,~
300-
e~
,.-, 20"0-
)"r"
z z
I0-0 0.0 CAIMAN
RAT
FIG. 1. T h e c r o s s - h a t c h e d areas r e p r e s e n t all t h e free a m i n o acids e x c e p t t h e four p r e s e n t i n greatest c o n c e n t r a t i o n , t h e l i n e d areas i n d i c a t e t h e a m o u n t c o n t r i b u t e d b y t h e four m a j o r a m i n o acids, a n d t h e e n t i r e b a r r e p r e s e n t s t h e total n i n h y d r i n reactants.
These summations are uniformly lower than total ninhydrin or paper chromatography values reported for the rat (Christensen et al., 1948a; Awapara et al., 1950a, b; Cravioto et al., 1951; AnseU & Richter, 1954; Wu, 1954). However, the values obtained for total ninhydrin reactants appear to be of the same order or somewhat greater than the total amino acid values previously reported. Although differences in procedure, such as choice of protein precipitant, may be a factor, it seems possible that some of the earlier values may have been influenced by contributions from ninhydrin reactive compounds which were not amino acids. The occurrence of taurine, glutathione, phospho derivatives and various small peptides has been reported in tissues (Tallan et al., 1954a) and, while we did not attempt an identification, we noted large quantities of ninhydrin reactive compounds on the chromatogram that were eluted very early by the acidic portion of the buffer. A comparison of amino acid sums in plasma and in various tissues (Fig. 1) will show that the intracellular concentrations were considerably higher than
0'082 0"03-0"16 0"056 0'03-0"10 0"284 0-19-0-46 0"119 0"07-0'23 0-158 0.11-0.25 0"157 0-14--O.22 0-163 0'11-0"26 0"135 0'07-0-27 0"278 0-12-0"39 0'151 0"09-0"25 0"183 0-10-0-34 0.144 0"08-0"33
0'011 0"01-0"02 0"056 0'03-0"10 0-227 0'11-0-37 0"385 0"16-0"91 0'341 0"27-0-61 0"071 0-05-0"09 0"262 0-17-0'37 0'171 0"10-0-28 0"203 0-10-0.28 0'438 0-31-0.56 0.354 0"20-0"51 0"229 0"16-0.36
0"090 0"05-0"14 0'089 0-06-0"11 0"252 0'13-0'50 0'168 0"11-0"26 0-275 0"18-0"40 0"134 0.08-0-18 0"152 0-10-0-17 0"118 0,05-0-20 0"224 0.13-0.28 0'171 0-10-0.28 0"213 0-13-0-26 0.203 0.12-0.45
Ser 0"362 0'13-0"88 0"195 0"07-0'43 0-205 0'05-1"08 0"054 0-0.38 0'421 0"12-1'2 0'135 0-82-2-4 0"736 0.42-1.2 1"47 0"60-3-7 2'18 0"84-3"0 4'04 2'2-7'4 4-17 2'4-6'8 0"665 0"51-1,2
Glu N H , 0"106 0'04-0.20 0"542 0"27-0"97 3"75 1-4-6.7 1"71 1"0-3-5 2"11 1"6-3.4 0-604 0'54-0.80 2"04 1'1-3'5 2-09 1'3-3'3 0"975 0'35-1"4 2"41 2.1-4.1 5-68 2'6-6.8 2"22 1.4-3.2
Glu
* Mean and ranges given; number in parentheses is number of animals,
Lung (9)
Brain (9)
Heart (9)
Muscle (9)
Stomach (7)
Gut (9)
Eye (9)
Spleen (9)
Liver (9)
Kidney (9)
Eryth. (6)
Plasma (10)
Thr
Asp
Aab~
Val
0-322 0'011 0"170 0'16-0"51 0"005-0'02 0"11-0"22 0"403 0'008 0"117 0'28-0.61 0"003-0-02 0'10-0-16 0"857 0"043 0'327 0-54-1-2 0'005-0.07 0.12-0.41 0'434 0-009 0.157 0"34-0.60 0"006-0-013 0-12-0.25 0'487 0"010 0'254 0-27-0-62 0'004-0.02 0'20-0"36 0"541 0"123 0-130 0"38-0.59 0"07-0.23 0"07-0.16 0.587 0"013 0-160 0.40-0.77 0'01-0.02 0"09-0.25 0.470 0"017 0"129 0"32-0"72 0"01-0"05 0.08-0.21 1-36 0"025 0'167 0'79-1.9 0"01-0.04 0"13-0-26 0-519 0"021 0-152 0-29-0-70 0.01-0.03 0'08-0-21 0-402 0.025 0'040 0"20-0.78 0"01-0'04 0"02-0.05 0'581 0.018 0.163 0'38-1.0 0'005-0'04 0-08-0.30
Ala
t a-Aminobutyric acid.
0"380 0-24--0"70 0'615 0"28-1"0 1"89 0'70-4.0 0"919 0"58-1"5 1"35 0'97-2-5 0'403 0"32-0-68 1"03 0"73-1-6 0'783 0.39-1-2 5"20 2"0-7.0 0"562 0'38-1-0 0"839 0,42-1.1 0.657 0'51-0.96
Gly
TABLE 1--FREE AMINO ACID CONTENT OF TISSUES ( m M / k g WET TISSUE) IN FASTING CAIMANS*
0.047 0"03-0'06 0"040 0'03-0.07 0"050 0'03-0.14 0-052 0"03-0.11 0"089 0-05-0.14 0.104 0-03-0-39 0-068 0"05-0.09 0-074 0.04-0.12 0.192 0"07-0.28 0.047 003-0-08 0-033 0.02-0-07 0"057 0"04-0.13
Met
~q
.q
.~
~'~
OO O',
Leu
Tyr
Phe
Gab~
Om
0.192 0.052 0.091 0.071 0"14-0-32 0.03--0.11 0-07-0-16 0"03-0"15 0.155 0.049 0.087 0.024 0.019 0"11-0"22 0.03-0.08 0.07-0.12 0.005-0-05 0.01-0.06 0.232 0-071 0-118 0-093 0.086 0"13-0"36 0.04--0.11 0.08-0.17 0-01-0.32 0.04-0.21 0.240 0.077 0.109 0.027 0.027 0.16-0.36 0.03-0-13 0.04-0.20 0-02-0.07 0.02-0.05 0.305 0.078 0.146 0.116 0.025 0"26-0-43 0"05-0-13 0"08-0"33 0-01-0'46 0.02-0'08 0"160 0'077 0.147 0"138 0"014 0-13-0"21 0'02-0-23 0"07-0"26 0.02-0'17 0"003-0'04 0"023 0.216 0.056 0.093 0.005-0.06 0-11-0.31 0-05-0-07 0'06-0"16 0.077 0.203 0.058 0.110 0.02-0"14 0-12-0.27 0.04---0.09 0.07-0.19 0.060 0.242 0.061 0.121 0-05-0"09 0-16-0.42 0.04--0.14 0"05-0"24 0.036 0-201 0.057 0-084 0-01-0.08 0-11-0.32 0.04-0.11 0'05-0"13 1.99 0.012 0.066 0.030 0-048 0,04-0,08 0.02-0-07 0"03-0"08 1-3-3.6 0-003-0.03 0.216 0.059 0-110 0.030 0.043 0"13-0"32 0-05-0-08 0"08-0-14 0.004--0.10 0-01-0,14
y-Aminobutyric acid.
Plasma (10)
0'079 0.05-0.14 0.044 Eryth. (6) 0.03-0.08 0'098 Kidney (9) 0.05-0.13 0"139 Liver (9) 0.07-0.34 0"118 Spleen (9) 0"07-0"22 0"053 Eye (9) 0"03-0-07 0.086 Gut (9) 0"05-0.12 Stomach (7) 0-072 0"05-0"10 Muscle (9) 0-091 0.05-0-18 Heart (9) 0-082 0-03--0,12 Brain (9) 0.023 0.01-0.03 0.080 Lung (9) 0.03-0.13
lieu
TABLE 1--continued Try
0.193 0.004 0.11)-0.39 0.002-0-01 0.056 0.007 0.04---0.07 0.003-0-01 0.320 0-15-0"65 0.165 0-015 0-12-0"30 0--0.05 0.159 0.018 0.10-0"32 0.01-0"04 0"095 0"133 0-03-0"26 0.02-0"42 0"210 0.11-0"57 0"266 0.008 0.16-0"60 0.004-0"02 0"464 0.404 0-10-0.64 0.27-0'76 0'165 0'110 0-04--O.31 0.08-0,13 0.101 0.003 0.05-0.20 0-0.01 0.299 0-014 0-11-0-97 0.01-0-03
Lys 0-189 0.12-0-24 0.273 0.20-0-38 0.352 0.16-0-69 0.276 0.17-0-44 0.322 0'21-0-59 0.154 0.13-0-22 0.200 0"11-0-35 0.243 0.13-0"43 0"407 0.25-0"52 0-316 0.16-0.48 0.068 0.04--0.11 0.485 0.20-1.5
His
3.72 2.7-5.1 0-101 0.06-0-16 0-062 0.03-0-11 1.07 0"13-1"9 0"192 0.16-0-31 0-095 0.03-0"24 0-249 0.14-0-50 0'172 0.01-0"31 0.233 0-08-0,44 0.752 0.46-1"4 0.391 0.26-0-66
3-Me* His
0.110 0.07-0-28 0-022 0.01-0-04 0"091 0.07-0.13 0.073 0.04-4)-16 0-062 0"04-0"12 0-029 0-02-0"05 0"059 0-02-0"13 0-118 0.09-0"15 0-273 0'06-0"45 0-094 0.04-O.15 0-061 0.03-0.08 0-080 0.06-0.14
Arg
t#l O0
0
,.,
.~ ,a
o
0.237 0.20-0.26 0.129 0-11-0.14 0-380 0.25-0,82 0.300 0.25-0.36 0-487 0.39-0.58 0"292 0.22-0.36 0'677 0.44--1.2 0"260 0-03-0.47 0.428 0.09-0-48 0.310 0.22-0.37 0.411 0.33-0.45 0-349 0.30-4).41 0.212 0.18-0.23
0.012 0.01-0.02 0,007 0.005-0.01 0.584 0.51-0.83 0.277 0.16-0-40 1"68 1"3-2"0 0'154 0"11-0"20 0"550 0.20--0"77 0.246 0.23-0"26 0.120 0.05-0.22 0.560 0.47-0.65 1.62 1.3-1-9 0.611 0.49-0.82 0.584 0.43-0.71
0.231 0-17-0.30 0.188 0.18-0.19 0.650 0.49-1.1 0-243 0.11-0.44 0,479 0-14-0.98 0.197 0.08-0'41 1-11 0"39-1"9 0.338 0.18-0.52 0.412 0.15-0.73 0.311 0.17-0.51 0.372 0-17-0.64 0.277 0.14-0.45 0.284 0.12-0.46
Ser 0.545 0.44-0.62 0.256 0.20--0.32 0.323 0.09-0.46 2-68 1.5-3.5 1.27 0.98-1'7 0.726 0.63-0'83 1"82 0.54--3'9 1.58 1.2-2.0 2.34 2.1-2.7 5-99 2.5-10.0 2-59 2.2-3.1 1.12 0.65-1.5 0.894 0.69-0.99
Glu NH2 0-152 0.09-0.29 0.203 0.15-0.25 4-09 3.8--4-3 1 '64 1.4-2.2 4.87 4.4-5.5 1-19 0"96-1"4 3"54 2.5-5.0 2"46 2.4-2.6 1.10 0-57-1-4 4"45 3-7-4.8 7-89 7.4-8.3 1-84 1.3-2.2 4-38 3-9-4.8
Glu
Cit 0.447 0.37-0.50 0"438 0.37-0-53 4.07 3.6-4.9 3-05 2.6-3.3 2"80 2.0-3'7 0"839 0"75-1"1 3'04 1"7-4-8 2.17 1-8-2.4 4"87 3.0-6.0 0.765 0.55-1.2 1"12 0.86-1"4 6.93 6.0-8.3 3'55 3.5-3-7
Gly 0-323 0.24-0.38 0-243 0.21-0.26 0.902 0.56-1.8 1.45 0-97-1.9 0-957 0'64-1.3 0"624 0'54-0'86 2-25 1"2-3.8 1 "36 1"3-1"4 1 "71 1-4-2,0 1 "92 1"3-2"8 0"663 0.38-0.93 1.21 0-57-2.5 0-941 0.83-1.1
Ala
"j"c~-Aminobutyric acid.
0.012 0-01-0.02
0.059 0"02-0-09 0'017 0-01-0"03 0.141 0'10-0"18 0.045 0.04-0.05 0.094 0.06-0.12 0.112 0.08-0.13
0.053 0.03-0.08 0-024 0.02-0.03 0.014 0.01-0"02
* Mean and ranges given ; n u m b e r in parentheses is n u m b e r of animals.
Testis (7)
Lung (8)
Brain (8)
Heart (9)
Muscle (8)
Stomach (8)
Gut (13)
Eye (9)
Spleen (8)
Liver (9)
Kidney (10)
Eryth. (7)
Plasma (10)
Thr
Asp 0.012 0.01-0.02 0.011 0-005-0.02 0.068 0.01-0-12 0.049 0.02-0.09 0.041 0.01-0'07 0-053 0'01-0"10 0"062 0.02-0"13 0.024 0.01-0.05 0.016 0.01-0-03 0.022 0.004-43.05 0.033 0.03-0.04 0.031 0.02-0.05 0.013 0.01-0-02
Aab'~
TABLE 2--FREE AMINO ACID CONTENT OF TISSUES ( m M / k g WET TISSUE) IN FASTING RATS*
0.177 0.09-0.22 0"090 0.08-0.09 0.519 0-42-0.86 0,217 0,14-0.28 0-304 0-16-0-39 0"162 0-12-0"19 0"828 0.20-1-7 0-247 0.15-0"39 0.144 0-11-0-18 0"146 0.12-0-16 0"127 0.12-0.14 0.165 0.07-0.22 0-123 0.10-0.15
Val
0.091 0.06-0.11 0,054 0.05-0-06 0.142 0,07-0.36 0.086 0,04--0,14 0.177 0.10-0-24 0.117 0.08-0"13 0"537 0"15-1-0 0.160 0.10-0.27 0.076 0'05-0-10 0"077 0.06-0-11 0-086 0.07-0.10 0.073 0.05-0-10 0-074 0.06-0.10
Met
N
t~
,~O
.~ .~
t/1 OO OO
0.080 0.07-0.09 0"075 0.06--0.10 0"135 0.08-0.31 0.114 0.07-0.16 0.224 0.09-0.34 0.097 0'07-0.11 0.596 0.12-1.2 0"137 0.10--0.23 0.058 0.04-0-08 0.075 0.07-0-08 0.044 0.03-0.05 0.077 0'07---0.09 0.061 0.04--0-10
•r-Aminobutyric acid.
Testis (7)
Lung (8)
Brain (8)
Heart (9)
Muscle (8)
Stomach (8)
G u t (13)
Eye (9)
Spleen (8)
Liver (9)
Kidney (10)
Eryth. (7)
Plasma (10)
Ileu
0.155 0"15-0-16 0'097 0"07-0-14 0.376 0.19-0.98 0.292 0-17-0-43 0-566 0.20---0-86 0"187 0.16--0.24 1"74 0-27-3.8 0.398 0-24.-0.64 0.168 0.16-0.18 0.177 0.16-0.19 0.106 0.05-0.16 0"146 0.13-0.17 0.216 0.12-0.32
Leu 0.092 0.09-0.10 0.064 0.06-0.08 0.136 0.09-0.27 0.102 0.05-0.15 0.196 0.12-0.25 0.168 0.14-0.18 0-790 0"22-1"5 0.169 0.11-0.30 0.099 0.08-0-12 0.091 0"06-0.16 0-047 0.04-0-05 0.072 0.04--0-09 0.123 0"09-0-16
Tyr 0.086 0-08-0.09 0.062 0-06-0-08 0-162 0.08-0-41 0-115 0-05--0-19 0.259 0-10--0.35 0.146 0-10-0.20 0.970 0-20-2-0 0.212 0.12-0.35 0.077 0.07-0.08 0.067 0.06-0.07 0.058 0.04--0.07 0.089 0.07-0.10 0.117 0"06-0.16
Phe
Orn
0.046 0.03-0.08 0"033 0.02-0.05 0"024 0.004--0.06 0.074 0.03-0.11 0.241 0"031 0.01-0.47 0.01-0.06 0.252 0.037 0.22-0.36 0.02-0"06 0.232 0.128 0.04--0"42 0.02-0"34 0.060 0-042 0.01-0"13 0.03-0.06 0.026 0.02-0-03 0.017 0.004-0-04 2.15 0.022 1.0-3.3 0.01-0-04 0.061 0.051 0-02-0.13 0.03-0-06 0-103 0.013 0.01-0.18 0.01-0.02
G a b ;~
TABLE 2~continued Try
0.406 0.065 0-32-0.47 0.04-0.09 0.215 0-20-0-23 0-343 0-18-0-57 0-321 0.18-0.48 0.368 0-13--0.65 0.488 0-28-1.0 1.03 0.031 0-16-2.4 0.01-0.07 0.321 0-18-0.49 0'810 0.642 0.37-1.3 0-12-1.4 0'833 0.35-2.4 0"226 0.20-4).26 0"285 0.17-0.49 0"349 0-012 0.23-0.54 0-01-0.02
Lys 0-111 0'07-0.17 0-066 0.05-0"08 0.238 0.19-0.39 0.412 0-35-0.46 0.436 0.15-0-67 0.126 0.12q)'14 0-682 0.15-1"5 0-258 0.15-0"38 0-302 0.24-0.38 0.169 0.10-0.36 0.077 0.07-0-08 0-114 0.10~-13 0.114 0.08-0.16
His
0.020 0.01-0-03
0.547 0.27-0.73
0-025 0.02-0"03
3-Me His
0.161 0-11-0"21 0.113 0-11-0.12 0.077 0.04--0-17 0.002 0-0.01 0.061 0.02-0.09 0.106 0.1ff-4).12 0.450 0.07-1.0 0.128 0.10-0.17 0.096 0.04--0"18 0.134 0.07-0"28 0.042 0.02-0"06 0.046 0.03-0"06 0.090 0.06-0.11
Agr
Ln O0
O
590
J.D. HERBERT,R. A. COULSONANDT. HERNANDEZ
extracellular values. But when the values of the top four amino acids were subtracted from the sum, as in Fig. 1, it was apparent that the remainder were not concentrated significantly above plasma levels. The top four constituted 50-80 per cent of the intracellular amino acids and the remainder were roughly equivalent to their plasma values. One exception was rat gut, in which the remainder varied over a wide range of concentrations (see Table 2). These animals were fasting, and the excised gut segments appeared empty, so, to minimize autolysis and avoid inadvertent extraction of cellular amino acids, the segments were not washed. Thus some variation may have been due to residual dietary amino acids or partially catabolized epithelial sheddings in the lumen. Figure 2 illustrates the four amino acids that were concentrated in each tissue. A cursory examination will indicate the general importance of glycine, alanine, glutamic acid and glutamine. Aspartic acid appeared in the top four in only three tissues: rat brain and spleen and caiman liver; no other amino acid was represented in the top four more than twice. These select few occurred in characteristic ratios in the various tissues. A general similarity was seen between the two species, notable exceptions being the erythrocyte, liver and lung.
Erythrocyte The erythrocyte of the caiman contained a compound, appearing in the position of 3-methyl histidine, in quantities equal to the entire remainder of amino acids. This compound, first reported in the urine of man and cat (Tallan et al., 1954b), did not appear in the erythrocyte of the rat, dog or turtle, but did occur in the alligator erythrocyte in quantities comparable to those in the caiman. Its metabolic function is unknown; it does not appear to be related to the presence of a nucleus in the red blood cell, since the compound was absent in the turtle erythrocyte. Quantities of this compound in caiman stomach, heart, brain and lung were relatively high, possibly above the amounts which could be contributed by blood contained in these tissues. The erythrocyte of the rat showed little concentrating ability, although glycine and glutamic acid appeared to achieve a slight concentration gradient when corrections for cell water were applied.
Kidney Amino acid concentrations in the two species were similar, and total values were comparable with other tissue totals in contrast to some reports that kidney contains unusually high concentrations of amino acids (Solomon et al., 1951).
Liver Species differences were readily apparent in this tissue. Whereas the glycine and glutamine content of rat liver remained consistently high, glutamine, particularly, showed great variation in the caiman liver. In the fasting caiman, glutamine is often immeasurably small, but may be present in significant quantities. It rises greatly after eating and falls slowly as the exogenous amino acids not needed
FREE AMINO ACIDS I N THE CAIMAN AND RAT
591
for protein synthesis are catabolized (Coulson & Hernandez, 1965). Values for total liver amino acids in the rat were consistently lower, relative to other tissues, than other values reported in the literature (Christensen et al., 1948a; Wu, 1954) for reasons which are not clear. Spleen Some similarity is evident, though caiman spleen had a rather high content of 3-methyl histidine, a reflection, perhaps, of the high content of erythrocytes in this organ.
Eye The values obtained for this tissue were, in effect, an average of the amino acid concentrations of its various structural components: sclera, retina, lens, aqueous and vitreous humors. The small size of the animals used made it impractical to attempt analyses of its components. However, we analyzed three components of dog eye separately and found that the aqueous humor resembled plasma except for a rather low glycine level, vitreous humor concentrated glycine and glutamic acid to some degree, and the lens showed high values for glycine and glutamic acid. The caiman eye was characterized by large amounts of a peptide emerging before aspartic acid, which, upon hydrolysis, yielded equimolar quantities of glycine and glutamic acid as two of its components. Further identification of this compound was not attempted. Gut and stomach Again the major constituents were the same in both species. In rat gut, however, the remainder of the amino acids, particularly the essentials, varied greatly. Skeletal muscle The prominence of glycine in skeletal muscle of both species was a very constant feature of this tissue, as was the presence of tryptophane, a compound found only in trace amounts in most tissues. Rat muscle contained rather large amounts of 3-methyl histidine, which was not found in appreciable quantity in other rat tissues. Heart and brain High quantities of glutamine and glutamic acid in heart and the prominence of glutamic acid, glutamine and y-aminobutyric acid in brain verified numerous observations on various species reported in the literature (Hamilton, 1945b; Awapara et al., 1950a, b; Frankel, 1950a; Cravioto et al., 1951; Ansell & Richter, 1954; Tallan et al., 1954a; Roberts et al., 1957, 1958; Okumara et al., 1959). Lung Rat lung contained large quantities of glycine, while caiman lung yielded glutamic acid in greatest quantity, an apparent species difference.
v I-
Z ~
'-1
"
GUT
PLASMA
KIDNEY
LIVER
I
STOMACH
HEART
Npi
MUSCLE
-'- N N
~ Y T E S
~-
BRAIN
SPLEEN
LUNO
EYE
I
TESTIS
FIG. 2. A graphic representation of the data of Tables 1 and 2. Four compounds constitute 50-80 per cent of the total free amino acids of the tissues. Column R represents the remaining amino acids, plotted according to their value, but without identification.
0'0.-
6O-
@10-
b~
t~
~q
©
>
t~
bo
593
FREE A M I N O ACIDS I N THE CAIMAN AND RAT
The prominence of the "big four" amino acids indicated in Fig. 2 is further illustrated by examination of free amino acids extracted from the whole carcass. Table 3 gives molar concentrations of carcass amino acids in a single caiman and a single rat. In both animals the amino acids present in greatest concentration were glycine, glutamine, glutamic acid and alanine. TABLE 3 - - C ~ c A s s From AMINO ACIDS ( m M / k g WET TISSUE) Asp Caiman Rat
Caiman Rat
Thr
Glu NH2 0"131 0"024 0.090 1.30 0.188 0"200 0.204 2.23
0"680 0.003 1-32 0.082
Lcu
Orn
Tyr
Ser
Phe
Gabt
Glu
Cit
Lys
Gly
Ala
Aab*
Val
I
~W
Ileu
1.37 0-655 0.014 0.060 0"013 0"022 3.23 1-00 0.006 0.115 0'057 0"099
Try
His
3-Me His
Arg
0.072 0"024 0.066 0-022 0.013 0.207 0-264 0-220 0.241 0.112 0.142 0"077 0.092 0"023 0"033 0.150 0"018 0"242 0.120 0.066
* =-Aminobutyric acid.
Met
Sum 5.603 9"694
t ~,-Aminobutyric acid.
2=01
>,-
o
:t 4'0~0-
~ ~
CELL/PLAS o o RAT 0 ,.'~ 12"2 23":5 6"1 44"1
2"4
26"0q4"1
3 . 7 10'2 58"6
1"6
CAIMAN RAT FIO. 3. T h e relationship between certain intracellular and extracellular amino
acids in the entire carcasses of one 50 g rat and one 50 g caiman. It is evident that only a few amino acids are highly concentrated inside the cells. To illustrate just how much these compounds were concentrated, corrections were made for cell solid content and extracellular fluid dilution, yielding values in mM/kg cell water. These values are compared with plasma values in Fig. 3 and their ratios calculated. Aspartic acid was added because its plasma value was very low and intracellular concentration was obvious, even though it did not
-0"02 -0'05 -0"04 -0-06 -0"05 -0"06 0 -0-03 -0.05 --0-04 --0'03 -0.05
Phe
Tyr
-0"01 -0'04 -0"03 -0-04 -0'04 -0-05 +0"03 -0.02 -0.04 --0'04 --0.02 -0-02
+0.05 -0.01 -0.06 +0-31 -0"09 -0-11 -0.08 -0.06 +0-16 -0.08 -0.10 --0'04
+0.04 -0.03 +0.09 -0'23 +0"04 +0"05 +0-18 +0.04 -0-02 +0'22 +0.14 +0-07
Thr
+0.27
+0.04 +0'03 +0"34 -0"02
Gab ~
+0.10 +0.03 +0.07 +0"01 -0"14 +0'06 +0.01 +0.05 +0'28 -0-04 -0.10 --0.06
Ser
-0'01 -0-01 -0'01 -tr. +0-01 +0'01 +0-02 -0.02 +0-17 +0.01 +0.01 +0-02
Om
+1.22 -0.12 +2.52 +1'51 +0"03 +0'03 +1.33 -0-05 +4'70 +2-76 +0.39 +0.56
Glu NH2
+0"02 -0"04 -0'14 +0"02 -0"06 -0"04 -0-10 --0.06 +0.76 --0.04 --0.02 -0.17
Lys
+0.32 +0.01 +5.05 +0'79 +2"42 +0"06 +2"09 --0.04 -0'19 +1-11 +0'70 +0.62
Glu
Gly
-0-12 --0.01
Tyr +0"14 -0-12 +0'09 -0-01 -0-01 +0'07 +0'08 -0.03 +0-16 --0.16 +0.01 -0.07
His
+0.98 +0-33 +0.64 +1'14 +0-26 +0.38 +0.94 +0-58 +2"19 +0-64 +0.12 +0.64
Ala
(rnM/kg WET
+1-72 +0'26 +0"99 +0'48 +0-25 +0'36 +1.89 +0.72 +3"73 +0-84 +0"30 +0.01
C H A N G E IN AMINO ACIDS
+tr. -0-01 -0'05 +0"04 -0"03 -tr. -0.02 -0'06 +0.55 --0.02 --0.04 -0-02
Arg
+0.03 0 -0-12 +0'12 -0.04 -0.02 +0.03 +0.06 +0"02 -0.02 -0.02 +0.02
Val
+4"88 +0"33 +9"08 +4"17 +2"42 +0'52 +6"66 +1.14 +12.27 +5.12 +1.29 +1.52
Ileu
+0-12 -0.07 +0.04 -~ 0"03 -0'03 -tr -i 0.18 +0"12 +0"01 -0.01 -0.01 -i 0.08
Leu
,* 7-Aminobu~yric
289 105 194 173 130 111 204 117 192 151 108 122
% of control
+0.03 +0.01 -0.01 -0.03 -0-01 -0-02 +0.04 -0.02 -0'02 -0.02 -0.01 +tr. Total change
+0.13 -0.01 +0.06 +0"07 +0.06 +0'02 +0.07 +0"08 +0"01 +0.16 +0.01 +0.14
~Iet
? ~-Aminobutyric acid.
+0"20 -0"06 -0"02 -0"54 -0"13 -0-04 -0.12 -0-07 --0'15 --0.21 --0.23
3-Me His
+0.02 0 +0-01 +0'01 +0"05 -0.07 +0.01 -tr. +0"04 +0.01 -tr. +0.02
Aabt
TISSUE) AFTER FEEDING*
* Average of five caimans killed 24 hr after feeding compared to control values in Table 1. acid.
Plasma Eryth. Kidney Liver Spleen Eye Gut Stomach Muscle Heart Brain Lung
Plasma Eryth. Kidney Liver Spleen Eye Gut Stomach Muscle Heart Brain Lung
Asp
TABLE 4
N
>
> ~J
.~ ¢) O
",ID 4~
FREE A M I N O ACIDS I N T H E C A I M A N AND RAT
595
achieve large quantitative values in any tissue tested. The remaining amino acids are plotted as well to show that these are not generally concentrated by the cell. Highest ratios are seen for aspartic and glutamic acids, two compounds which attain primarily an extracellular fluid distribution when administered exogenously. Glycine, alanine and glutamine all attain a body water distribution when injected. Table 4 presents the changes in free amino acids in the tissues occurring when ten small caimans (100 g) were permitted to eat all of the meat they desired. One day was allowed for digestion, absorption and distribution; the animals were then killed and the tissues were analyzed in the usual way. The results were informative in several respects. The majority of the amino acids did not rise appreciably in any tissue, and although the total free amino acid concentrations were about twice those of the control group, it is evident that three or four of the "non-essential" ones were responsible for most of the increase. Striated muscle, the largest tissue in the body, showed the greatest millimolar increase in total amino acids after feeding, and over 80 per cent of the gain was due to glutamine, glycine and alanine. Although glutamic acid was also increased considerably in several organs, the total rise of this compound in the entire body was not very great since the organs sharing in the increase had a total weight considerably less than the muscle. The eye, which is neither very active metabolically nor subject to rapid growth, was affected very little. It is significant that the three amino acids which increased the most were also the ones present in the fasting plasma in great concentration and, in addition, were also the ones responsible for most of the synthesis of excretion products of nitrogen metabolism. It is difficult not to conclude that these amino acids represent one stage in the degradation of those amino acids present in excess of that required for protein synthesis. DISCUSSION It is evident that only a few amino acids are maintained at high concentrations in the tissues of fasting caimans and fasting rats. The significance of such selective concentration remains to be explored. These amino acids are almost certainly neither direct products of protein degradation nor source materials for protein synthesis, since essential amino acids were not concentrated to any extent. Three of the amino acids most often concentrated, glycine, alanine and glutamine, are rapidly metabolized and serve as precursors of ammonia and uric acid when injected into an intact alligator (Coulson & Hernandez, 1964). Thus these compounds may serve as vehicles for the disposal of nitrogen produced in catabolism of protein. However, this theory does not explain why the cells maintain high intracellular gradients of these amino acids, or why these three should occur in characteristic ratios in each tissue. It fails also to explain the presence of high concentrations of glutamic acid, a compound which is slowly metabolized when administered exogenously. The characteristic amino acid patterns in different tissues suggest a difference in the primary metabolic reactions which occur in these tissues. It is not necessary
596
J.D. HERBERT,R. A. COULSONANDT. HERNANDEZ
to postulate mutually exclusive reactions; a difference in relative frequency could as easily account for the dissimilarity. Roberts & Frankel (1950a, b, 1951) have demonstrated a catabolic pathway for glutamic acid in brain via the intermediates, 7-aminobutyric acid and succinic semialdehyde. The high levels of glutamine, glutamic acid and 7-aminobutyric acid in brain suggest that this pathway may be important in the in vivo metabolism of this tissue. In kidney the low levels of glutamine may be significant in light of the kidney's role in ammonia formation, reputedly from the amide nitrogen of glutamine (Nash & Benedict, 1921; Van Slyke et al., 1943). However, glycine and alanine, two compounds equalling glutamine as ammonia precursors in the alligator (Coulson & Hernandez, 1964), are maintained at high concentrations in the kidney. The general prominence of glutamic acid may reflect its postulated role in transamination, though some doubt has been expressed as to the importance of transamination in the live animal (Coulson & Hernandez, 1965). SUMMARY 1. Alcoholic extracts of various tissues of the fasting caiman and fasting rat were analyzed for free amino acids by column chromatography and for total ninhydrin reactants by a colorimetric method. 2. Total ninhydrin reactive compounds exceeded the sum of amino acids on the chromatogram several-fold, indicating a high content of ninhydrin reactants which were not amino acids. 3. Each tissue contained high concentrations of three or four amino acids in characteristic ratios. Glycine, glutamine, glutamic acid and alanine were the most plentiful. 4. Comparison of the two species showed a marked similarity in most tissues; species differences were apparent in the erythrocyte, liver and lung. 5. Feeding increased glutamine, glycine, glutamic acid and alanine in most tissues of the caiman while affecting the essential amino acids very little. Acknowledgement--We are grateful to Mr. Jacob Watson for the preparation of the figures.
REFERENCES ALEXANDERB., LANDWEHRG. • SELIGMANA. M. (1945) A specific micro-method for the colorimetric determination of glycine in blood and urine. 3t. biol. Chem. 160, 51-59. ANSELLG. B. & RICHTERD. (1954) A note on free amino acid content of rat brain. Biochem. ft. 57, 70-73. AWAPA~ J., LANDUAA. J. & FtmmT R. (1950a) Distribution of free amino acids and related substances in organs of the rat. Biochim. biophys. Acta 5, 457-462. AWAPARAJ., LANDUAA. J., FImRSTR. & SmI~ B. (1950b) Free 7-aminobutyric acid in brain, ft. biol. Chem. 187, 35-39. CI~ISTENSEN H. N., ROTHW~LLJ. T., SEAm R. A. & STm~ICI~RJ. (1948a) Association between rapid growth and elevated cell concentrations of amino acids: in regenerating liver after partial hepatectomy in the rat. 3t. biol. Chem. 175, 101-105.
A M I N O ACIDS I N T H E C A I M A N A N D RAT
597
CHI~ISTENS~ H. N. & ST~SICHER J. (1948) Association between rapid growth and elevated cell concentrations of amino acids: in fetal tissues..7, biol. Chem. 175, 95-100. CHmSTENSEN H. N., STmzlcmm J. & ELBING~ R. L. (1948b) Effects of feeding individuM amino acids upon the distribution of other amino acids between cells and extracellular fluid..7, biol. Chem. 172, 515-524. COtJLSON R. A. & H~m'~ANDEZT . (1964) Biochemistry of the Alligator, p. 78. Louisiana State University Press, Baton Rouge. COULSON R. A. & H ~ A N D E Z T . (1965) Amino acid metabolism in the alligator. Fed. Proc. Symp. 24, 927-940. COULSON R. A., HERNANDEZ T. & BYERS V. (1965) A simple two-column system for the rapid analysis of plasma amino acids. Analyt. Biochem. 10, 159-162. C~vlOTO R. O., MASSmU G. & IzQt:IEmJO J. J. (1951) Free amino acids in rat brain during insulin shock. Proc. Soc. exp. Biol. Med. 78, 856-858. DUBREUIL R. & TIMn~S P. S. (1953) Effect of cortisone on free amino acids in serum and organs of the rabbit. Am..7. Physiol. 174, 20-26. HAMILTON P. B. (1945a) Gasometric determination of glutamine amino acid carboxyl nitrogen in plasma and tissue filtrates by the ninhydrin-carbon dioxide method..7, biol. Chem. 158, 375-396. HAMILTON P. B. (1945b) Glutamine: a major constituent of free ~t-amino acids in animal tissues and blood plasma..7, biol. Chem. 158, 397-409. MOORE S. & STEIN W. (1954a) Procedures for the chromatographic determination of amino acids on 4 per cent cross-linked sulfonated polystyrene resins..7, biol. Chem. 211, 893-906. MOORE S. & STEIN W. (1954b) A modified ninhydrin reagent for the photometric deterruination of amino acids and related compounds..7, biol. Chem. 211, 907-914. NAKAGAWAH., LINDSAY R. H. & COHEN P. P. (1964) Composition and labeling patterns of "free" and protein amino acids in Rana catesbeiana tadpoles and frogs. Arch. Biochem. Biophys. 106, 299-306. NASH T . P. & BENEDICT S. R. (1921) T h e ammonia content of the blood, and its bearing on the mechanism of acid neutralization in the animal organism..7, biol. Chem. 48, 463-488. OKUMARA N., OTSUKI S. & AOYAMAT . (1959) Studies on the free amino acids and related compounds in the brains of fish, amphibia, reptile, ayes, and m a m m a l . . 7 . Biochem. 46, 207-212. ROBERTS E. & FRANKEL S. (1945) Free amino acids in normal and neoplastic tissues of mice as studied by paper chromatography. Cancer Res. 9, 645-648. ROBERTS E. & FRANKEL S. (1950a) 7-Aminobutyric acid in brain: its formation from glutamic acid..7, biol. Chem. 187, 55-63. ROBERTS E. & FRANKEL S. (1950b) Glutamic acid decarboxylase in b r a i n . . 7 , biol. Chem. 188, 789-795. ROBERTS E. & FRANKEL S. (1951) Further studies of glutamic acid decarboxylase in brain. .7. biol. Chem. 190, 505-512. ROBERTS E., LOWE I. P., CHANIN M. & JELIN~K B. (1957) Free or easily extractable amino acids of the heart muscle of various species..7, exp. Zool. 135, 239-254. ROBERTS E., LOWE I. P., GUTH L. & JELINEK B. (1958) Distribution of ~,-aminobutyric acid and other amino acids in nervous tissue of various species..7, exp. Zool. 138, 313-328. SOLOMON J. D., JOI~SON C. A., S h ~ r ~ a A. L. & BERGEIM O. (1951) T h e determination of free and total amino acids in rat tissues..7, biol. Chem. 189, 629-635. SPACKMAN D. H., STEIN W . & M O O R E S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. TALLAN H. H., M O O l ~ S. & STEIN W . (1954a) Studies on the free amino acids and related compounds in the tissues of the cat..7, biol.Chem. 211, 927-939.
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J. D. HERBERT,R. A. COULSONAND T. HERNANDEZ
TALLAN H. H., STEIN W. • MOORE S. (1954b) 3-Methylhistidine, a new amino acid from human urine. J. biol. Chem. 206, 825-834. TROLL W. & CANNAN R. K. (1953) A modified photometric ninhydrin method for the analysis of amino and imino acids. J. biol. Chem. 200, 803-811. VAN SLYKE D. D. (1912) The quantitative determination of aliphatic amino groups. J. biol. Chem. 12, 275-284. VAN SLYKED. D. (1914) The fate of protein digestion products in the b o d y - - I I . Determination of amino nitrogen in the tissues. J. biol. Chem. 16, 187-195. VAN SLYKE D. D. & MEYER G. M. (1914) T h e fate of protein digestion products in the b o d y - - I I I . T h e absorption of amino acids from the blood by the tissues. J. biol. Chem. 16, 197-212. VAN SLYKE D. D., PHILLIPS R. A., HAMILTON P. B., ARCHIBALDR. M., FUTCHERP. H. & HILLER A. (1943) Glutamine as source material of urinary ammonia. J. biol. Chem. 150, 481-482. W u C. (1954) Metabolism of free amino acids in fasted and zein-fed rats. J. biol. Chem. 207, 775-786.