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Conditions Affecting Plasma Amino Acid Patterns in Chickens Fed Practical and Purified Diets
Conditions Affecting Plasma Amino Acid Patterns in Chickens Fed Practical and Purified Diets K.
MARUYAMA, M.
L.
SUNDE AND A .
E.
HARPER
Departments of Poultry Science and Nutritional Sciences, University of Wisconsin, Wisconsin 53706
Madison,
(Received for publication April 28, 1975)
POULTRY SCIENCE 55: 1615-1626, 1976
INTRODUCTION
T
H E relationship between plasma amino acid concentrations and protein quality has been studied extensively in the chick. In 1953, C h a r k e y et al. c o m p a r e d plasma amino acid patterns of chicks fed s o y b e a n protein or peanut c a k e diets and also investigated the effect of various sampling procedures on the estimation of plasma amino acid c o n c e n t r a t i o n s . T h e concentration of lysine was found to increase considerably at the end of a 48 hour fasting period and to return to t h e " n o r m a l " level in four hours after refeeding. T h e y concluded that plasma lysine a n d arginine concentrations were r e s p o n s i v e t o changes in dietary supplies w h e n chicks
Research supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and the National Institute of Arthritis, Metabolism and Digestive Diseases, project number AM 10748.
were fed ad libitum. Immediately following this r e p o r t , Richardson et al. (1953) d e m o n strated reasonably good correlation of plasma amino acid concentrations with dietary a m i n o acid profiles in a study of the effects of deficiency or excessive supplementation of methionine, lysine, or valine. This relationship was also examined in t h e turkey. Tonkinson et al. (1961) observed a sharp elevation of plasma lysine, a n d a decline in p l a s m a arginine, methionine, glycine, and serine as dietary lysine was being increased t o an optimal level. A method for t h e a s s e s s m e n t of protein quality b y m e a n s of p l a s m a amino acid concentrations was devised by L o n g e n e c k e r and H a u s e (1959). This w a s based on use of t h e constant plasma amino acid concentrations in dogs during starvation as a reference stand a r d a n d determining t h e concentrations of t h e limiting amino acids post-feeding. Direct
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ABSTRACT Experiments were conducted to investigate plasma free amino acid concentrations in the chick. After one hour of fasting, total plasma amino acid concentration decreased to approximately half of the full-fed value. Within three to six hours, most amino acids had returned toward the full-fed level but did not exceed it throughout a 48 hour period of starvation. However, after 48 hours fasting lysine, threonine, and isoleucine accumulated three-fold, two-fold and two-fold of the full-fed level, respectively. Serine and glutamic acid exceeded the full-fed level at three hours and then declined. Alanine reached its highest level after six hours of fasting and then declined. In full-fed chicks diurnal variations of plasma free amino acid concentrations were observed. The lowest and highest concentrations were observed at 11 a.m. and 8 to 11 p.m., respectively under a 24-hr lighting. Reference plasma amino acid patterns are reported for chicks fed a practical diet ad libitum. In day-old chicks, concentrations of total amino acids, methionine plus one half cystine, lysine, and arginine were high. Alanine and glutamic acid concentrations were low. Most amino acid concentrations declined gradually during the first four weeks of life, but methionine plus one half cystine, phenylalanine, threonine and serine concentrations decreased sharply between two and four weeks. Lysine concentration continued to decrease in chicks fed the starter diet. At 20 weeks, plasma amino acid concentrations had decreased considerably except for methionine plus one half cystine and basic amino acids. The plasma amino acid pattern for chicks fed an isolated soybean protein diet was similar to that of chicks fed the practical diet.
1616
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
Diurnal variations in the total plasma free amino acid concentration have been reported (Squibb, 1966). Also the plasma free lysine concentration decreases with the age of the chick (Askelson and Balloun, 1963). These experiments were designed to investigate the effect of feed deprivation on plasma free amino acid concentrations and diurnal variations with ad libitum feeding and to obtain reference plasma free amino acid patterns with various diets and ages of chicks. MATERIALS AND METHODS Chickens. The experiments were conducted using chicks from the mating of new Hampshire males to Single Comb White Leghorn females. Straight-run chicks, day-old, were wing-banded, weighed, and placed in a conventional electrically heated starting battery located in a room with 24-hour lighting. Effect of Feed Deprivation and Diurnal Variation. Fifty chicks were fed a practical starter diet (Table 1) until two weeks of age.
TABLE 1.—Composition of practical type diets
Ingredient Ground yellow corn Wheat middlings Alfalfa meal Meat scrap Soybean meal Bone meal Iodized salt Fish meal Oyster shell Grit (chick size) CaC03 Vitamin-Antibiotics-Mineral mix1
Starter
Grower
(0-12 weeks)
(12-20 weeks)
%
%
52.0 10.0 5.0 5.0 20.0
63.0 10.0 3.0 2.0 18.0 1.0 0.5
— 0.5 5.0 1.0 1.0
— — —
—
2.0
0.5
0.5
1
Vitamin-Antibiotics-Mineral mix for the starter provided the following in Kg. diet: vitamin A, 2,200 I.U.; vitamin D 3 , 880 I.C.U.; riboflavin, 10 mg.; procaine penicillin, 4.5 mg.; vitamin B, 2 , 10.5 mg.; MnO, 140 mg. Vitamin-Antibiotics-Mineral mix for the grower provided the following in Kg. diet: vitamin A, 2,200 I.U.; vitamin D 3 , 700 I.C.U.; riboflavin, 20 mg.; vitamin B, 2 , 130 mg.; MnO, 110 mg. When they were weighed the number of chicks of average weight was reduced to 40. On the following day, starting at 8 a.m., 25 chicks were fasted and blood samples from three chicks were taken by heart puncture after 0-, 1-, 2-, 3-, 6-, 12-, 24-, and 48-hour periods. Similarly, 15 chicks were fed ad libitum and blood samples from three chicks were taken by heart puncture at three hour intervals starting at 8 a.m. and continuing until 8 p.m. Two trials were conducted for this experiment. In addition, two trials were conducted to examine variations of plasma free amino acid concentrations throughout a day (8 a.m. to 8 a.m.) at three hour intervals. Effect of Age on Plasma Amino Acid Pattern. A group of 30 chicks was reared on the practical starter diet for 12 weeks and a practical grower diet (Table 1) until 20 weeks of age and another group of 15 chicks was
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application of this method failed to predict limiting amino acids when the chick was used as a test animal (Smith and Scott, 1965a, b; Dean and Scott, 1966). Difficulty in application of this method with the chick resulted mainly from the considerable elevation of concentrations of lysine and threonine that occur during starvation (Gray et al., 1960). The extent of the elevation in the concentrations of amino acids varies among species (Shao and Hill, 1967; Boomgaardt and McDonald, 1969). Feeding of a non-protein diet instead of fasting helped to predict limiting amino acids for the chick to a certain extent (Hill and Olsen, 1963). An appreciable increase in plasma non-protein nitrogen during starvation (Hazelwood and Lorenz, 1959) also suggested that the supply of energyyielding nutrients is one of the major factors influencing plasma amino acid concentrations.
PLASMA AMINO ACID PATTERNS
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TABLE 2.—Pre-fasting and 48-hours fasting plasma free amino acid concentrations'-2 Pre-fasting3
Total
(jtmoles/100 ml. |7**4 61** 49* 81* 65* 61** 11* 22* 22* 10* 24* 34* 14* 30* 501
Molar % 4 12 10 16 13 12 2 4 4 2 5 7 3 6 100
(xmoles/100 ml. 7*4
Molar %
58* 49** 188* 74** 53* 10* 21* 37* 25** 30* 115* j j** 32**
1 8 7 26 10 7 2 3 5 4 4 16 2 5
710
100
'Averages of two trials. 2 In each trial, plasma from three chicks within a group were pooled for amino acid analysis. 3 Samples were taken from the chicks fed a practical starter diet for two weeks at day 15, 0800 hr. and at day 17, 0800 hr. following a 48 hr. fasting. 4 * and ** indicate that concentrations fell in the range of less and more than ±15% of the average, respectively.
reared on an isolated soybean protein diet (Al-Ubaidi and Bird, 1964) for two weeks. Three trials were conducted for this experiment. Blood samples were taken from five chicks by heart puncture at the ages of day-old, one, two, four, and six weeks and from the wing veins of three chicks at the ages of 12 and 20 weeks. Sample Preparation for Amino Acid Analysis. One to three ml. of whole blood in a heparinized syringe from each chick was immediately transferred into a centrifuge tube jacketed with ice and then centrifuged at 28,700 x g for 20 minutes. Equal quantities of the plasma obtained within a treatment were pooled and deproteinized with 15% sulfosalicylic acid to make the final concentration of the protein-free supernatant solution 3% in sulfosalicylic acid. The sam-
1. Technicon Instruments Co., Ardsley, NY.
pies were kept frozen until analysis with an amino acid analyzer. 1 RESULTS AND DISCUSSION The Effect of Feed Deprivation on Plasma Free Amino Acid Concentrations. After two weeks on the practical diet, the average weights of the chicks were 141 g. and 138 g. in two trials. The pre-fasting and the 48-hr. fasting plasma free amino acid concentrations are shown in Table 2. At the end of a 48-hr. fast, considerable elevations in the concentration of threonine, valine, isoleucine and lysine and a depression in the concentration of aspartic acid were noted. The concentrations of other amino acids were virtually unchanged. Consequently, molar ratios of plasma free amino acids were appreciably altered. The molar percentage of threonine, valine, isoleucine, and lysine collectively was increased from 29% to 51%. The pre-fasting concentrations were set at 100% and the
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Aspartic acid Glutamic acid Alanine Threonine Serine Glycine Methionine + 1 / 2 Cystine Phenylalanine + Tyrosine Valine Isoleucine Leucine Lysine Histidine Arginine
48-hr. fasting3
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
300
fe
total amino
3
M
thr
O
Ki lys
>
acid
O) c 200' D
1 00
&?
s
i 4
50
4 8 hr
FIG. 1. Effect of fasting on total concentration of free amino acids and concentrations of threonine and lysine in the plasma of chicks. Data are a percent of pre-fasting concentration.
relative concentrations of plasma free amino acids measured during fasting are plotted in Fig. 1, 2 and 3. Plasma total amino acid concentration (Fig. 1) decreased to approximately half of the pre-fasting concentration in one hr. after removal of the feed. After this initial drop, it then returned to approximately the pre-fasting concentration at the end of three hr. fasting. During prolonged starvation, concentration of plasma total amino acids did not change greatly through 48 hr. except for the low value at 12 hr. Plasma lysine concentration was elevated after six hr. of fasting and reached concentrations of two- to three-fold the prefasting concentration at 24 to 48 hr. Somewhat
similar changes were observed in plasma threonine (Fig. 1). These observations are in general agreement with earlier observations (Gray et al., 1960). Marked reduction in plasma free amino acid concentrations was observed at the 12-hr. period of fasting. Plasma total amino acid concentration was reduced as low as with one-hr. fasting. Yet the concentrations of lysine, threonine (Fig. 1) and isoleucine (Fig. 2) were not reduced. A similar trend in fluctuation of fasting amino acid concentrations was observed in swine except that the reduction was found to be non-significant (Richardson et al.. 1965). Concentrations of plasma branched-chain
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I
1619
PLASMA AMINO ACID PATTERNS
3 fasting
6
12
4 8
hr
period
FIG. 2. Effect of fasting on concentrations of valine, leucine and isoleucine in the plasma of chicks. Data are a percent of pre-fasting concentration.
amino acids (Fig. 2) were reduced moderately during the first 6 to 12 hr. but progressively increased after 24 to 48 hr. of fasting. Plasma isoleucine concentration was elevated considerably (2.2-fold) more than the concentrations of the other branched-chain amino acids. Hill and Olsen (1963) observed that fasting concentrations of plasma lysine, threonine, and branched-chain amino acids in four week-old chicks reached a plateau after 24 hr. of starvation. However, in the present experiment using two week-old chicks, the concentrations of lysine, threonine, isoleucine and valine continued to increase for 48 hr. of fasting. It is possible that difference in the responses of plasma free amino acid concentrations to fasting is accounted for by the path-
ways of protein metabolism which undergo change in the course of development. Since the embryo contains a considerable amount of fat, but only a trace of carbohydrate, the gluconeogenic pathway is highly active and its activity lessens after hatching (Nelson et al., 1966; Wallace and Newsholme, 1967). Accumulations of lysine, threonine and branched-chain amino acids during starvation would result from the breakdown of tissue proteins and slow-oxidation of these amino acids. It is probable that the lower fasting plasma lysine concentration observed in two week-old chicks may be accounted for by higher availability of NADPH which is required for lysine degradation (Wang and Nesheim, 1972) compared to that in older chicks, since the pentose phosphate shunt diminishes
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2
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
10O O)
fasting
period
FIG. 3. Effect of fasting on concentrations of aspartic acid, glutamic acid, alanine, serine and glycine in the plasma of chicks. Data are a percent of pre-fasting concentration.
in the early developmental period (Wenger, 1967). Another possibility is that reutilization of amino acids for tissue protein synthesis is more efficient in younger chicks. The moderate increases observed in plasma branched-chain amino acid concentrations are surprising in view of the widespread occurrence of leucine-a-keto-glutarate transaminase along with moderately inducible branched-chain keto-acid dehydrogenase in kidney and muscle (Featherston and Horn. 1973). Hazelwood and Lorenz (1959) demonstrated that during starvation gluconeogencsis was highly active. Changes in concentrations of some glucogenic amino acids during fasting are shown in Fig. 3. Concentrations of these
amino acids were generally reduced by fasting with the 12-hr. fasting concentrations being 50% or less of the pre-fasting concentrations. Plasma glycine and aspartic acid concentrations were reduced during the entire fasting period. The reduced concentration of plasma glycine could occur because of the demand for glycine for uric acid synthesis in conjunction with gluconeogenesis. Plasma alanine concentration remained near the pre-fasting level until after six hr. of fasting and then declined sharply at 12 hr., while plasma aspartic acid, glutamic acid and serine concentrations rose for three hr. after fasting began, and then began to fall. This may be related to the role of alanine as a carrier of nitrogen formed in extrahepatic tissues
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I 0)
1621
PLASMA AMINO ACID PATTERNS
aspartic acid for gluconeogenesis. Hence, it appeared that the highest fasting plasma alanine concentrations and the rapid decline in concentrations of plasma aspartic acid and glutamic acid at the six-hour period were a likely indication of the onset of gluconeogenesis in the course of fasting. Decline in plasma free amino acid concentrations at 12 hours of fasting appeared somewhat similar to that at one hour. However, the first decline was probably due to rapid uptake of amino acids for protein synthesis following ingestion of feed and the second decline was probably due to increased catabolism of the available amino acids for gluconeogenesis. Between 12 and 48 hr., amino acids were evidently released from labile tissue proteins as rapidly as they were used for gluconeogenesis. Otherwise, plasma concentrations could not have been maintained. Considerable elevations of lysine, threonine and isoleucine concentrations dur-
TABLE 3.—Concentrations of plasma free amino acids over a 24-hour period with three-hour intervals' Sampling time Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Methionine + 1/2 Cystine Valine Leucine Isoleucine Phenylalanine Tyrosine Total
A.M.
P.M.
A.M. 8
11
2
58 18 36 12 110 88 47 74 57 12 29 29 14 12 13 585
29 17 31 8 97 74 34 64 54 11 24 28 10 11 10 502
47 18 44 11 112 96 50 84 60 12 31 32 14 15 13 639
5
8
ll2
22
jxmoles/100 ml. plasma 60 77 56 43 26 18 20 21 38 43 53 43 5 10 13 13 110 147 146 124 82 98 106 84 35 27 51 53 67 89 89 75 62 74 66 62 15 14 14 15 27 27 34 29 20 33 29 33 14 13 12 14 11 13 14 14 12 15 13 14 579 696 625 735
52
Pooled SE3
54 11 28 5 104 82 31 71 44 11 23 23 11 9 10 517
7 3 7 5 13 10 8 12 7 1 4 4 2 2 2 68
'Concentrations were averages of four trials unless indicated and plasma samples were taken from three chicks and pooled within a group in each trial. 'Average of two trials. 3 Pooled SE was computed from concentrations at 8 a.m., 11 a.m., 2 p.m., 5 p.m. and 8 p.m. in four trials.
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(Felig et al., 1970). In the chick, hepatic gluconeogenesis is progressively decreased after hatching and renal gluconeogenesis becomes considerably more important than in mammals (Evans and Scholz, 1973). In addition to branched-chain amino acids, serine is also catabolized to a considerable extent in the kidney of the chick since L-serine dehydratase is distributed mainly in the kidney (Ascarelli and Bruckental, 1969) and absent from the liver where serine is catabolized in an alternate pathway (Yoshida and Kikuchi, 1971). Another possibility is that the delayed reduction in plasma alanine concentration is accounted for by a considerably lesser operational capacity of glutamic-pyruvic transaminase compared with that of glutamicoxalo acetic transaminase in the liver (Maruyama et al., 1976). The rapid reduction in plasma aspartic acid concentration during this period is a reflection of enhanced utilization of
1622
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
150
o z o o
/
ill
TVs
*' / .
-i Ul Q:
>Ly«
50
V/ i TIME
$
r~
II
PM OF DAY
FIG. 4. Within a day in total concentration of free amino acids and concentrations of valine, lysine and arginine in the plasma of chicks fed ad libitum under a 24-hour lighting regimen. Data are a percent of concentration at 8 a.m. ing this period would seem to indicate that these amino acids are not degraded as rapidly as the others. Diurnal Variations of Plasma Free Amino Acid Concentrations. Changes in the concentrations of plasma free amino acids within a day under ad libitum feeding conditions are shown in Table 3. The values shown are average plasma free amino acid concentrations in two or four trials. Plasma amino acid concentrations in the late evening were generally higher than in the morning. When concentrations at 11 a.m. and at 8 p.m. were compared, a large increase in relative value (71 to 93% increase) was observed in lysine and arginine and the total plasma free amino acid was increased by 46%.
Fluctuations within a day were observed in the concentrations of most plasma free amino acids. The diurnal pattern observed was similar for most of individual amino acids but with different magnitudes. The pattern was characterized by oscillation with peaks at 2 p.m. and at 8 to 11 p.m. This pattern was also shown in the total concentration of plasma free amino acids. Figure 4 illustrates the diurnal pattern with total amino acids, valine, arginine and lysine. Valine and arginine concentrations demonstrated a fall at 11 a.m. and a gradual fall starting at 8 p.m. until 5 a.m. The plasma free lysine concentration showed an extensive fall at 11 a.m. and it exhibited a delayed rise which reached a peak at 11 p.m. Concentrations of branched-chain amino
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p 100 o <
PLASMA AMINO ACID PATTERNS
The Effect of Age on Plasma Amino Acid Pattern. Few observations have been made on the effect of age on plasma free amino acid concentrations in chicks (Askelsen and Balloun, 1963). Hence, the study was undertaken to determine changes in the plasma amino acid pattern with age. Experiments were carried out with ad libitum feeding of practical type diets (Table 1) and by sampling at 3 p.m. The plasma amino acid pattern of chicks fed the isolated soybean protein diet for one or two weeks was also studied. The isolated soybean protein diet provided a similar amino acid profile and performance of chicks for up to two-weeks of age when compared with the chick starter diet. Three trials were conducted and average plasma amino acid concentrations are shown in Table 4. It was generally observed that concentrations of dispensable amino acids, glycine and threonine had larger SEM values. Plasma amino acid concentrations of chicks fed the isolated soybean protein diet differed little from those of chicks fed the practical
starter diet with possible exceptions of leucine, phenylalanine, and arginine at one week and lysine at two weeks. With increasing age of chicks, total plasma amino acid concentration decreased gradually. The lowest total plasma amino acid concentration was observed at 4 weeks when the starter diet was fed from 0 to 12 weeks of age. A large decline in concentrations of individual amino acids from either day old or one week of age was observed at this time in threonine, serine, valine, methionine plus one half cystine, isoleucine, phenylalanine lysine and arginine. The decline in the plasma free amino acid concentrations was associated with a high rate of gain in weight during the first four weeks. The concentration of lysine decreased consistently throughout the feeding of the practical starter diet. Askelsen and Balloun (1963) also reported a decrease in lysine with age (from two weeks to eight weeks). This decrease continued until 12 weeks in the present study. Plasma amino acid concentrations of day-old chicks were generally higher than those of older chicks. It was particularly noted in concentrations of methionine plus one half cystine, lysine and arginine. On the other hand, concentrations of alanine and glutamic acid were lower at one day of age. High plasma lysine concentration at one day of age with large variation was probably attributable to the difference in individual hatching times. The difference between 12 week plasma levels and 20 week plasma levels were probably due, at least partly, to the difference in the protein content of the diet (20% protein versus 15% protein in the diet). The amount of protein in the diet of the 12 week old chicks was excessive and this may account for elevation of plasma serine, glycine, alanine and branched-chain amino acids concentrations. Considerable variation in plasma concentrations of dispensable amino acids may also be the result of excessive protein
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acids fluctuated in a similar manner regardless of the concentration. Amino acids with relatively small concentration also showed about the same degree of fluctuation. An exception was the concentration of threonine which progressively increased during the period of 11 a.m. through 11 p.m. A similar trend was observed in the concentration of methionine plus a half cystine (Table 3). From the diurnal variations observed, it appeared that the 11 a.m. fall was somewhat similar to the observation made after onehour of fasting when a rapid uptake of amino acids for protein synthesis probably occurred. The 2 p.m. rise was probably the consequence of the condition in which a massive flow of dietary amino acids saturated the plasma free amino acid pool. Whatever the metabolic basis may be, diurnal fluctuations of plasma amino acid concentrations obviously occur in the chick under a 24-hour lighting.
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5 86 66 24 55 70 32
10 14 25 14 15 45 12 27 500
4a 5 lObc 16a 3bc 2a led 3a
± 2a ± la ±2a ± 0.3a ± 0.6b ±7a ± 0.2bc ± 4a ±41a
± ± ± ± ± ± ±
0.2ab 3ab labc 2abc 11a 7a 2a ± 2b ± 0.9de ± 2b ±2a ± 2c ± 3d ± 0.5bc ± lbc ± 18bc
11 10 17 10 10 35 10 24 410
10 16 16 13 23 39 10 20 466
lbc lb 2b la 0.7a 2cd lbc led 8ab
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ±
ISPD
11 12 16 15 13 56 11 22 478
lb 2cd 2b 0.7a 0.8b lb lab 3bc 3ab
g121
± ± ± ± ± ± ± ± ±
5 8 15 14 6 37 8 16 331 310
± 0.2 ± 0.1 ± 0.4 ± 4 ± 0.2 ±2 ±0.4 ± 0.5 ± 16cd
±0.2 ± 3 ± 3 ± 0.4 ± 2 ± 1 ± 0.6
PD
4 Week
Practical diet. 'Isolated soybean protein diet. "•Average plasma free amino acid concentration ± SEM in three trials. 'Values within the same amino acid without a common letter are significantly different (p < 0.05).
2
2 Week (j.moles/100 ml. plasma 4 5 5 ± lab 0.3b 59 86 ± 2ab la 49 76 ± 3a 7abc 14 17 ± 2bc 3bc 40 47 ± 4 a 5a 37 69 ± 5a 4cd 18 22 ± 3cde lde
PD 3 96 66 18 41 41 18
ISPD 3 5 ± 0.9ab 66 ± 8cd 72 ± 9ab 31 ± 6a 42 ± 3a 77 ± 4a 26±2c
1 Week
± 0.4bc ± O.lbc ± la ± 2a ±2b ±0.2c ± 0.9a ± 0.6b ± 18a
± ± ± ± ± ± ±
PD 2
67 125 71 38 Body weight 1 Blood samples were taken from Chicks fed ad libitum at 3 p.m.
7 Aspartic acid 78 Threonine 85 Serine 17 Glutamic acid 41 Glycine 38 Alanine 49 Valine Methionine + 1 / 2 Cystine 18 19 Isoleucine Leucine 24 10 Tyrosine 13 Phenylalanine Lysine 65 10 Histidine 41 Arginine Total 515
Day-old
TABLE 4.—Change in plasma free amino acid concentrations during the gr
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PLASMA AMINO ACID PATTERNS
T h e information in this report indicates clearly that it is necessary to state in the experimental p r o c e d u r e t h e time of t h e day t h e blood samples were taken. It is also necessary to indicate any fasting period involved. T h e possibility exists that differences could be s h o w n b e t w e e n a 24 h o u r lighting period as presented in this report and one involving a natural day length or a standard 14 hour day as used in many laboratories. T h e d e c r e a s e in plasma amino acids observed as the birds b e c a m e older is also an important factor that must b e considered when comparing results from different laboratories or e x p e r i m e n t s within a laboratory. ACKNOWLEDGEMENT The authors wish t o acknowledge the gift of the Dextrin from t h e C o r n P r o d u c t s C o . , Argo, Illinois, through the courtesy of Dr. J. D . Shroder.
REFERENCES Al-Ubaidi, Y. Y., and H. R. Bird, 1964. Assay for the unidentified growth factor in dried whey. Poultry Sci. 43: 1484-1488. Ascarelli, I., and I. Bruckental, 1969. Location and properties of serine dehydrase in the chick. Comp. Biochem. Physiol. 29: 1277-1279. Askelson, C. E., and S. L. Balloun, 1963. Influence of age and dietary protein on certain free amino acids in chick blood plasma. Poultry Sci. 42: 140-146. Boomgaardt, J., and B. E. McDonald, 1969. Compari-
son of fasting plasma amino acid patterns in the pig, rat and chicken. Can. J. Physiol. Pharmacol. 47: 392-395. Charkey, L. W., W. K. Manning, A. K. Kano, F. X. Gassner, M. L. Hopwood and I. L. Madsen, 1953. A further study of vitamin B, 2 in relation to amino acid metabolism in the chick. Poultry Sci. 32: 630-642. Dean, W. F., and H. M. Scott, 1966. Use of free amino acids concentrations in blood plasma of chick to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88: 75-83. Evans, R. M., and R. W. Scholz, 1973. Development of renal gluconeogenesis in chicks fed high fat and high protein "carbohydrate-free" diets. J. Nutr. 103: 242-250. Featherston, W. R., and G. W. Horn, 1973. Dietary influences on the activities of enzymes involved in branched-chain amino acid catabolism in the chick. J. Nutr. 103: 757-765. Felig, P., T. Pozefsky, E. Marliss and G. F. Cahill, Jr., 1970. Alanine: key role in gluconeogenesis. Science, 167: 1003-1004. Gray, J. A., E. M. Olsen, D. C. Hill and H. D. Branion, 1960. Effect of a dietary lysine deficiency on the concentration of amino acids in the deproteinized blood plasma. Can. J. Biochem. Physiol. 38: 435441. Hazelwood, R. L., and F. W. Lorenz, 1959. Effects of fasting and insulin on carbohydrate metabolism of the domestic fowl. Am. J. Physiol. 197: 47-51. Hill, D. C , and E. M. Olsen, 1963. Effect of starvation and a non protein diet on blood plasma amino acids, and observations on the detection of amino acids limiting growth of chicks fed purified diets. J. Nutr. 79: 303-310. Longenecker, J. B., and N. L. Hause, 1959. Relationship between plasma amino acids and composition of the ingested protein. Arch. Biochem. Biophys. 84: 46-59. Maruyama, K., M. L. Sunde and A. E. Harper, 1976. Is L-glutamic acid nutritionally a dispensable amino acid for the chick? Poultry Sci. 55: 45-60. National Research Council, 1971. Nutrient Requirements of Poultry. N.A.S., Washington, D.C. Nelson, P., G. Yarnell and S. R. Wagle, 1966. Biochemical studies of the developing embryo. II. Studies on C0 2 fixation enzymes. Arch. Biochem. Biophys. 114: 543-546. Richardson, L. R., L. G. Blaylock and C. M. Lyman, 1953. Influence of dietary amino acid supplements on the free amino acids in the blood plasma of chicks. J. Nutr. 51: 515-522. Richardson, L. R., F. Hale and S. J. Ritchey, 1965. Effect of fasting and level of dietary protein on
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intake. After switching birds from t h e starter diet to the grower diet, concentrations of most of amino acids decreased considerably, with exceptions of methionine plus one half cystine and basic a m i n o acids. T h e trend of decreasing concentrations of methionine plus a half cystine and lysine was somewhat surprising, since the birds were kept on the starter diet until 12 w e e k s . T h e requirements of both methionine plus cystine and lysine d e c r e a s e from the period of 0 to 6 w e e k s and during t h e period of 6 to 14 w e e k s ( N . R . C . , 1971).
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
Tonkinson, L. V., K. E. Dunkelgod, E. W. Gleaves, R. H. Thayer and R. J. Sirny, 1961. Effect of dietary lysine on free amino acid concentrations in blood plasma of growing turkeys. Poultry Sci. 40: 11061117. Wallace, J. C , and E. A. Newsholme, 1967. A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl. Biochem. J. 104: 378-384. Wang, S. H., and M. C. Nesheim, 1972. Degradation of lysine in chicks. J. Nutr. 102: 583-596. Wenger, E., B. S. Wenger and P. A. Kitos, 1967. Pentose phosphate pathway activity of the chick embryo in ovo. J. Exp. Zool. 166: 263-270. Yoshida, T., and G. Kikuchi, 1971. Significance of the glycine cleavage system in glycine and serine catabolism in avian liver. Arch. Biochem. Biophys. 145: 658-668.
Immunological and Compositional Patterns of Lipoproteins in Chicken (Gallus domesticus) Plasma J. Y-L. Yu, L. D. CAMPBELL AND R. R. MARQUARDT
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Received for publication June 23, 1975)
ABSTRACT Immunological and compositional patterns of plasma lipoproteins in the laying and nonlaying hen (Gallus domesticus) and rooster plasmas were compared. Hen plasma contained three immunologically distinct lipoproteins: very low density (VLDL) and low density (LDL); high density (HDL); and lipovitellin (LV). Rooster plasma contained VLDL and LDL; and HDL. Laying hen plasma in comparison to the other two groups of birds contained a high content of VLDL and LV. The plasma lipoprotein pattern of nonlaying hens and roosters were similar except for the relatively high content of LDL and the presence of low levels of LV in nonlaying hen plasma. In general, triglyceride, phospholipid and cholesterol contents were similar for each lipoprotein class in laying hen, nonlaying hen and rooster plasma. POULTRY SCIENCE 55: 1626-1631, 1976
INTRODUCTION
L
of lay have been characterized via analytical
IPOPROTEINS are the transport media
ultracentrifugation
for lipids in the blood of mammals and
(1956), Schjeide et al. (1963) and Schjeide
birds (Frederickson etal., 1967). Lipoproteins
and Wilkens (1964). Hillyard etal. (1956) have
also serve as precursors of egg yolk lipid
compared the composition and concentra-
in the laying hen (Schjeide
tions of plasma lipoproteins in normal and
et al.,
1963;
by Schjeide
and
Urist
1972; Yu
stilbestrol injected roosters. Characterization
and Marquardt, 1973; Gornall and Kuksis,
of plasma lipoproteins in the laying hen by
1973).
the use of preparative ultracentrifugation was
Husbands, 1971; Hillyard et ai,
Plasma lipoproteins in the hen at the onset
recently undertaken by Husbands (1970) and
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free amino acids in pig plasma. J. Anim. Sci. 24: 368-372. Shao, T. C , and D. C. Hill, 1967. A comparison of the effect of dietary fat and carbohydrate on free amino acids in the blood of chicks. Can. J. Physiol. Pharmacol. 45: 225-234. Smith, R. E., and H. M. Scott, 1965a. Use of free amino acid concentrations in blood plasma in evaluating the amino acid adequacy of intact proteins for chick growth. I. Free amino acid patterns of blood plasma of chicks fed unheated and heated fishmeal proteins. J. Nutr. 86: 37-44. Smith, R. E., and H. M. Scott, 1965b. Use of free amino acid concentrations in blood plasma in evaluating the amino acid adequacy of intact proteins for chick growth. II. Free amino acid patterns of blood plasma of chicks fed sesame and raw, heated and overheated soybean meals. J. Nutr. 86: 45-50. Squibb, R. L., 1966. Nature of the free amino-acid pool in avian tissues. Nature, 209: 710-711.
MARUYAMA, M.
L.
SUNDE AND A .
E.
HARPER
Departments of Poultry Science and Nutritional Sciences, University of Wisconsin, Wisconsin 53706
Madison,
(Received for publication April 28, 1975)
POULTRY SCIENCE 55: 1615-1626, 1976
INTRODUCTION
T
H E relationship between plasma amino acid concentrations and protein quality has been studied extensively in the chick. In 1953, C h a r k e y et al. c o m p a r e d plasma amino acid patterns of chicks fed s o y b e a n protein or peanut c a k e diets and also investigated the effect of various sampling procedures on the estimation of plasma amino acid c o n c e n t r a t i o n s . T h e concentration of lysine was found to increase considerably at the end of a 48 hour fasting period and to return to t h e " n o r m a l " level in four hours after refeeding. T h e y concluded that plasma lysine a n d arginine concentrations were r e s p o n s i v e t o changes in dietary supplies w h e n chicks
Research supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, and the National Institute of Arthritis, Metabolism and Digestive Diseases, project number AM 10748.
were fed ad libitum. Immediately following this r e p o r t , Richardson et al. (1953) d e m o n strated reasonably good correlation of plasma amino acid concentrations with dietary a m i n o acid profiles in a study of the effects of deficiency or excessive supplementation of methionine, lysine, or valine. This relationship was also examined in t h e turkey. Tonkinson et al. (1961) observed a sharp elevation of plasma lysine, a n d a decline in p l a s m a arginine, methionine, glycine, and serine as dietary lysine was being increased t o an optimal level. A method for t h e a s s e s s m e n t of protein quality b y m e a n s of p l a s m a amino acid concentrations was devised by L o n g e n e c k e r and H a u s e (1959). This w a s based on use of t h e constant plasma amino acid concentrations in dogs during starvation as a reference stand a r d a n d determining t h e concentrations of t h e limiting amino acids post-feeding. Direct
1615
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ABSTRACT Experiments were conducted to investigate plasma free amino acid concentrations in the chick. After one hour of fasting, total plasma amino acid concentration decreased to approximately half of the full-fed value. Within three to six hours, most amino acids had returned toward the full-fed level but did not exceed it throughout a 48 hour period of starvation. However, after 48 hours fasting lysine, threonine, and isoleucine accumulated three-fold, two-fold and two-fold of the full-fed level, respectively. Serine and glutamic acid exceeded the full-fed level at three hours and then declined. Alanine reached its highest level after six hours of fasting and then declined. In full-fed chicks diurnal variations of plasma free amino acid concentrations were observed. The lowest and highest concentrations were observed at 11 a.m. and 8 to 11 p.m., respectively under a 24-hr lighting. Reference plasma amino acid patterns are reported for chicks fed a practical diet ad libitum. In day-old chicks, concentrations of total amino acids, methionine plus one half cystine, lysine, and arginine were high. Alanine and glutamic acid concentrations were low. Most amino acid concentrations declined gradually during the first four weeks of life, but methionine plus one half cystine, phenylalanine, threonine and serine concentrations decreased sharply between two and four weeks. Lysine concentration continued to decrease in chicks fed the starter diet. At 20 weeks, plasma amino acid concentrations had decreased considerably except for methionine plus one half cystine and basic amino acids. The plasma amino acid pattern for chicks fed an isolated soybean protein diet was similar to that of chicks fed the practical diet.
1616
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
Diurnal variations in the total plasma free amino acid concentration have been reported (Squibb, 1966). Also the plasma free lysine concentration decreases with the age of the chick (Askelson and Balloun, 1963). These experiments were designed to investigate the effect of feed deprivation on plasma free amino acid concentrations and diurnal variations with ad libitum feeding and to obtain reference plasma free amino acid patterns with various diets and ages of chicks. MATERIALS AND METHODS Chickens. The experiments were conducted using chicks from the mating of new Hampshire males to Single Comb White Leghorn females. Straight-run chicks, day-old, were wing-banded, weighed, and placed in a conventional electrically heated starting battery located in a room with 24-hour lighting. Effect of Feed Deprivation and Diurnal Variation. Fifty chicks were fed a practical starter diet (Table 1) until two weeks of age.
TABLE 1.—Composition of practical type diets
Ingredient Ground yellow corn Wheat middlings Alfalfa meal Meat scrap Soybean meal Bone meal Iodized salt Fish meal Oyster shell Grit (chick size) CaC03 Vitamin-Antibiotics-Mineral mix1
Starter
Grower
(0-12 weeks)
(12-20 weeks)
%
%
52.0 10.0 5.0 5.0 20.0
63.0 10.0 3.0 2.0 18.0 1.0 0.5
— 0.5 5.0 1.0 1.0
— — —
—
2.0
0.5
0.5
1
Vitamin-Antibiotics-Mineral mix for the starter provided the following in Kg. diet: vitamin A, 2,200 I.U.; vitamin D 3 , 880 I.C.U.; riboflavin, 10 mg.; procaine penicillin, 4.5 mg.; vitamin B, 2 , 10.5 mg.; MnO, 140 mg. Vitamin-Antibiotics-Mineral mix for the grower provided the following in Kg. diet: vitamin A, 2,200 I.U.; vitamin D 3 , 700 I.C.U.; riboflavin, 20 mg.; vitamin B, 2 , 130 mg.; MnO, 110 mg. When they were weighed the number of chicks of average weight was reduced to 40. On the following day, starting at 8 a.m., 25 chicks were fasted and blood samples from three chicks were taken by heart puncture after 0-, 1-, 2-, 3-, 6-, 12-, 24-, and 48-hour periods. Similarly, 15 chicks were fed ad libitum and blood samples from three chicks were taken by heart puncture at three hour intervals starting at 8 a.m. and continuing until 8 p.m. Two trials were conducted for this experiment. In addition, two trials were conducted to examine variations of plasma free amino acid concentrations throughout a day (8 a.m. to 8 a.m.) at three hour intervals. Effect of Age on Plasma Amino Acid Pattern. A group of 30 chicks was reared on the practical starter diet for 12 weeks and a practical grower diet (Table 1) until 20 weeks of age and another group of 15 chicks was
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application of this method failed to predict limiting amino acids when the chick was used as a test animal (Smith and Scott, 1965a, b; Dean and Scott, 1966). Difficulty in application of this method with the chick resulted mainly from the considerable elevation of concentrations of lysine and threonine that occur during starvation (Gray et al., 1960). The extent of the elevation in the concentrations of amino acids varies among species (Shao and Hill, 1967; Boomgaardt and McDonald, 1969). Feeding of a non-protein diet instead of fasting helped to predict limiting amino acids for the chick to a certain extent (Hill and Olsen, 1963). An appreciable increase in plasma non-protein nitrogen during starvation (Hazelwood and Lorenz, 1959) also suggested that the supply of energyyielding nutrients is one of the major factors influencing plasma amino acid concentrations.
PLASMA AMINO ACID PATTERNS
1617
TABLE 2.—Pre-fasting and 48-hours fasting plasma free amino acid concentrations'-2 Pre-fasting3
Total
(jtmoles/100 ml. |7**4 61** 49* 81* 65* 61** 11* 22* 22* 10* 24* 34* 14* 30* 501
Molar % 4 12 10 16 13 12 2 4 4 2 5 7 3 6 100
(xmoles/100 ml. 7*4
Molar %
58* 49** 188* 74** 53* 10* 21* 37* 25** 30* 115* j j** 32**
1 8 7 26 10 7 2 3 5 4 4 16 2 5
710
100
'Averages of two trials. 2 In each trial, plasma from three chicks within a group were pooled for amino acid analysis. 3 Samples were taken from the chicks fed a practical starter diet for two weeks at day 15, 0800 hr. and at day 17, 0800 hr. following a 48 hr. fasting. 4 * and ** indicate that concentrations fell in the range of less and more than ±15% of the average, respectively.
reared on an isolated soybean protein diet (Al-Ubaidi and Bird, 1964) for two weeks. Three trials were conducted for this experiment. Blood samples were taken from five chicks by heart puncture at the ages of day-old, one, two, four, and six weeks and from the wing veins of three chicks at the ages of 12 and 20 weeks. Sample Preparation for Amino Acid Analysis. One to three ml. of whole blood in a heparinized syringe from each chick was immediately transferred into a centrifuge tube jacketed with ice and then centrifuged at 28,700 x g for 20 minutes. Equal quantities of the plasma obtained within a treatment were pooled and deproteinized with 15% sulfosalicylic acid to make the final concentration of the protein-free supernatant solution 3% in sulfosalicylic acid. The sam-
1. Technicon Instruments Co., Ardsley, NY.
pies were kept frozen until analysis with an amino acid analyzer. 1 RESULTS AND DISCUSSION The Effect of Feed Deprivation on Plasma Free Amino Acid Concentrations. After two weeks on the practical diet, the average weights of the chicks were 141 g. and 138 g. in two trials. The pre-fasting and the 48-hr. fasting plasma free amino acid concentrations are shown in Table 2. At the end of a 48-hr. fast, considerable elevations in the concentration of threonine, valine, isoleucine and lysine and a depression in the concentration of aspartic acid were noted. The concentrations of other amino acids were virtually unchanged. Consequently, molar ratios of plasma free amino acids were appreciably altered. The molar percentage of threonine, valine, isoleucine, and lysine collectively was increased from 29% to 51%. The pre-fasting concentrations were set at 100% and the
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Aspartic acid Glutamic acid Alanine Threonine Serine Glycine Methionine + 1 / 2 Cystine Phenylalanine + Tyrosine Valine Isoleucine Leucine Lysine Histidine Arginine
48-hr. fasting3
1618
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
300
fe
total amino
3
M
thr
O
Ki lys
>
acid
O) c 200' D
1 00
&?
s
i 4
50
4 8 hr
FIG. 1. Effect of fasting on total concentration of free amino acids and concentrations of threonine and lysine in the plasma of chicks. Data are a percent of pre-fasting concentration.
relative concentrations of plasma free amino acids measured during fasting are plotted in Fig. 1, 2 and 3. Plasma total amino acid concentration (Fig. 1) decreased to approximately half of the pre-fasting concentration in one hr. after removal of the feed. After this initial drop, it then returned to approximately the pre-fasting concentration at the end of three hr. fasting. During prolonged starvation, concentration of plasma total amino acids did not change greatly through 48 hr. except for the low value at 12 hr. Plasma lysine concentration was elevated after six hr. of fasting and reached concentrations of two- to three-fold the prefasting concentration at 24 to 48 hr. Somewhat
similar changes were observed in plasma threonine (Fig. 1). These observations are in general agreement with earlier observations (Gray et al., 1960). Marked reduction in plasma free amino acid concentrations was observed at the 12-hr. period of fasting. Plasma total amino acid concentration was reduced as low as with one-hr. fasting. Yet the concentrations of lysine, threonine (Fig. 1) and isoleucine (Fig. 2) were not reduced. A similar trend in fluctuation of fasting amino acid concentrations was observed in swine except that the reduction was found to be non-significant (Richardson et al.. 1965). Concentrations of plasma branched-chain
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I
1619
PLASMA AMINO ACID PATTERNS
3 fasting
6
12
4 8
hr
period
FIG. 2. Effect of fasting on concentrations of valine, leucine and isoleucine in the plasma of chicks. Data are a percent of pre-fasting concentration.
amino acids (Fig. 2) were reduced moderately during the first 6 to 12 hr. but progressively increased after 24 to 48 hr. of fasting. Plasma isoleucine concentration was elevated considerably (2.2-fold) more than the concentrations of the other branched-chain amino acids. Hill and Olsen (1963) observed that fasting concentrations of plasma lysine, threonine, and branched-chain amino acids in four week-old chicks reached a plateau after 24 hr. of starvation. However, in the present experiment using two week-old chicks, the concentrations of lysine, threonine, isoleucine and valine continued to increase for 48 hr. of fasting. It is possible that difference in the responses of plasma free amino acid concentrations to fasting is accounted for by the path-
ways of protein metabolism which undergo change in the course of development. Since the embryo contains a considerable amount of fat, but only a trace of carbohydrate, the gluconeogenic pathway is highly active and its activity lessens after hatching (Nelson et al., 1966; Wallace and Newsholme, 1967). Accumulations of lysine, threonine and branched-chain amino acids during starvation would result from the breakdown of tissue proteins and slow-oxidation of these amino acids. It is probable that the lower fasting plasma lysine concentration observed in two week-old chicks may be accounted for by higher availability of NADPH which is required for lysine degradation (Wang and Nesheim, 1972) compared to that in older chicks, since the pentose phosphate shunt diminishes
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2
1620
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
10O O)
fasting
period
FIG. 3. Effect of fasting on concentrations of aspartic acid, glutamic acid, alanine, serine and glycine in the plasma of chicks. Data are a percent of pre-fasting concentration.
in the early developmental period (Wenger, 1967). Another possibility is that reutilization of amino acids for tissue protein synthesis is more efficient in younger chicks. The moderate increases observed in plasma branched-chain amino acid concentrations are surprising in view of the widespread occurrence of leucine-a-keto-glutarate transaminase along with moderately inducible branched-chain keto-acid dehydrogenase in kidney and muscle (Featherston and Horn. 1973). Hazelwood and Lorenz (1959) demonstrated that during starvation gluconeogencsis was highly active. Changes in concentrations of some glucogenic amino acids during fasting are shown in Fig. 3. Concentrations of these
amino acids were generally reduced by fasting with the 12-hr. fasting concentrations being 50% or less of the pre-fasting concentrations. Plasma glycine and aspartic acid concentrations were reduced during the entire fasting period. The reduced concentration of plasma glycine could occur because of the demand for glycine for uric acid synthesis in conjunction with gluconeogenesis. Plasma alanine concentration remained near the pre-fasting level until after six hr. of fasting and then declined sharply at 12 hr., while plasma aspartic acid, glutamic acid and serine concentrations rose for three hr. after fasting began, and then began to fall. This may be related to the role of alanine as a carrier of nitrogen formed in extrahepatic tissues
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I 0)
1621
PLASMA AMINO ACID PATTERNS
aspartic acid for gluconeogenesis. Hence, it appeared that the highest fasting plasma alanine concentrations and the rapid decline in concentrations of plasma aspartic acid and glutamic acid at the six-hour period were a likely indication of the onset of gluconeogenesis in the course of fasting. Decline in plasma free amino acid concentrations at 12 hours of fasting appeared somewhat similar to that at one hour. However, the first decline was probably due to rapid uptake of amino acids for protein synthesis following ingestion of feed and the second decline was probably due to increased catabolism of the available amino acids for gluconeogenesis. Between 12 and 48 hr., amino acids were evidently released from labile tissue proteins as rapidly as they were used for gluconeogenesis. Otherwise, plasma concentrations could not have been maintained. Considerable elevations of lysine, threonine and isoleucine concentrations dur-
TABLE 3.—Concentrations of plasma free amino acids over a 24-hour period with three-hour intervals' Sampling time Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Methionine + 1/2 Cystine Valine Leucine Isoleucine Phenylalanine Tyrosine Total
A.M.
P.M.
A.M. 8
11
2
58 18 36 12 110 88 47 74 57 12 29 29 14 12 13 585
29 17 31 8 97 74 34 64 54 11 24 28 10 11 10 502
47 18 44 11 112 96 50 84 60 12 31 32 14 15 13 639
5
8
ll2
22
jxmoles/100 ml. plasma 60 77 56 43 26 18 20 21 38 43 53 43 5 10 13 13 110 147 146 124 82 98 106 84 35 27 51 53 67 89 89 75 62 74 66 62 15 14 14 15 27 27 34 29 20 33 29 33 14 13 12 14 11 13 14 14 12 15 13 14 579 696 625 735
52
Pooled SE3
54 11 28 5 104 82 31 71 44 11 23 23 11 9 10 517
7 3 7 5 13 10 8 12 7 1 4 4 2 2 2 68
'Concentrations were averages of four trials unless indicated and plasma samples were taken from three chicks and pooled within a group in each trial. 'Average of two trials. 3 Pooled SE was computed from concentrations at 8 a.m., 11 a.m., 2 p.m., 5 p.m. and 8 p.m. in four trials.
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(Felig et al., 1970). In the chick, hepatic gluconeogenesis is progressively decreased after hatching and renal gluconeogenesis becomes considerably more important than in mammals (Evans and Scholz, 1973). In addition to branched-chain amino acids, serine is also catabolized to a considerable extent in the kidney of the chick since L-serine dehydratase is distributed mainly in the kidney (Ascarelli and Bruckental, 1969) and absent from the liver where serine is catabolized in an alternate pathway (Yoshida and Kikuchi, 1971). Another possibility is that the delayed reduction in plasma alanine concentration is accounted for by a considerably lesser operational capacity of glutamic-pyruvic transaminase compared with that of glutamicoxalo acetic transaminase in the liver (Maruyama et al., 1976). The rapid reduction in plasma aspartic acid concentration during this period is a reflection of enhanced utilization of
1622
K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
150
o z o o
/
ill
TVs
*' / .
-i Ul Q:
>Ly«
50
V/ i TIME
$
r~
II
PM OF DAY
FIG. 4. Within a day in total concentration of free amino acids and concentrations of valine, lysine and arginine in the plasma of chicks fed ad libitum under a 24-hour lighting regimen. Data are a percent of concentration at 8 a.m. ing this period would seem to indicate that these amino acids are not degraded as rapidly as the others. Diurnal Variations of Plasma Free Amino Acid Concentrations. Changes in the concentrations of plasma free amino acids within a day under ad libitum feeding conditions are shown in Table 3. The values shown are average plasma free amino acid concentrations in two or four trials. Plasma amino acid concentrations in the late evening were generally higher than in the morning. When concentrations at 11 a.m. and at 8 p.m. were compared, a large increase in relative value (71 to 93% increase) was observed in lysine and arginine and the total plasma free amino acid was increased by 46%.
Fluctuations within a day were observed in the concentrations of most plasma free amino acids. The diurnal pattern observed was similar for most of individual amino acids but with different magnitudes. The pattern was characterized by oscillation with peaks at 2 p.m. and at 8 to 11 p.m. This pattern was also shown in the total concentration of plasma free amino acids. Figure 4 illustrates the diurnal pattern with total amino acids, valine, arginine and lysine. Valine and arginine concentrations demonstrated a fall at 11 a.m. and a gradual fall starting at 8 p.m. until 5 a.m. The plasma free lysine concentration showed an extensive fall at 11 a.m. and it exhibited a delayed rise which reached a peak at 11 p.m. Concentrations of branched-chain amino
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p 100 o <
PLASMA AMINO ACID PATTERNS
The Effect of Age on Plasma Amino Acid Pattern. Few observations have been made on the effect of age on plasma free amino acid concentrations in chicks (Askelsen and Balloun, 1963). Hence, the study was undertaken to determine changes in the plasma amino acid pattern with age. Experiments were carried out with ad libitum feeding of practical type diets (Table 1) and by sampling at 3 p.m. The plasma amino acid pattern of chicks fed the isolated soybean protein diet for one or two weeks was also studied. The isolated soybean protein diet provided a similar amino acid profile and performance of chicks for up to two-weeks of age when compared with the chick starter diet. Three trials were conducted and average plasma amino acid concentrations are shown in Table 4. It was generally observed that concentrations of dispensable amino acids, glycine and threonine had larger SEM values. Plasma amino acid concentrations of chicks fed the isolated soybean protein diet differed little from those of chicks fed the practical
starter diet with possible exceptions of leucine, phenylalanine, and arginine at one week and lysine at two weeks. With increasing age of chicks, total plasma amino acid concentration decreased gradually. The lowest total plasma amino acid concentration was observed at 4 weeks when the starter diet was fed from 0 to 12 weeks of age. A large decline in concentrations of individual amino acids from either day old or one week of age was observed at this time in threonine, serine, valine, methionine plus one half cystine, isoleucine, phenylalanine lysine and arginine. The decline in the plasma free amino acid concentrations was associated with a high rate of gain in weight during the first four weeks. The concentration of lysine decreased consistently throughout the feeding of the practical starter diet. Askelsen and Balloun (1963) also reported a decrease in lysine with age (from two weeks to eight weeks). This decrease continued until 12 weeks in the present study. Plasma amino acid concentrations of day-old chicks were generally higher than those of older chicks. It was particularly noted in concentrations of methionine plus one half cystine, lysine and arginine. On the other hand, concentrations of alanine and glutamic acid were lower at one day of age. High plasma lysine concentration at one day of age with large variation was probably attributable to the difference in individual hatching times. The difference between 12 week plasma levels and 20 week plasma levels were probably due, at least partly, to the difference in the protein content of the diet (20% protein versus 15% protein in the diet). The amount of protein in the diet of the 12 week old chicks was excessive and this may account for elevation of plasma serine, glycine, alanine and branched-chain amino acids concentrations. Considerable variation in plasma concentrations of dispensable amino acids may also be the result of excessive protein
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acids fluctuated in a similar manner regardless of the concentration. Amino acids with relatively small concentration also showed about the same degree of fluctuation. An exception was the concentration of threonine which progressively increased during the period of 11 a.m. through 11 p.m. A similar trend was observed in the concentration of methionine plus a half cystine (Table 3). From the diurnal variations observed, it appeared that the 11 a.m. fall was somewhat similar to the observation made after onehour of fasting when a rapid uptake of amino acids for protein synthesis probably occurred. The 2 p.m. rise was probably the consequence of the condition in which a massive flow of dietary amino acids saturated the plasma free amino acid pool. Whatever the metabolic basis may be, diurnal fluctuations of plasma amino acid concentrations obviously occur in the chick under a 24-hour lighting.
1623
5 86 66 24 55 70 32
10 14 25 14 15 45 12 27 500
4a 5 lObc 16a 3bc 2a led 3a
± 2a ± la ±2a ± 0.3a ± 0.6b ±7a ± 0.2bc ± 4a ±41a
± ± ± ± ± ± ±
0.2ab 3ab labc 2abc 11a 7a 2a ± 2b ± 0.9de ± 2b ±2a ± 2c ± 3d ± 0.5bc ± lbc ± 18bc
11 10 17 10 10 35 10 24 410
10 16 16 13 23 39 10 20 466
lbc lb 2b la 0.7a 2cd lbc led 8ab
± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ±
ISPD
11 12 16 15 13 56 11 22 478
lb 2cd 2b 0.7a 0.8b lb lab 3bc 3ab
g121
± ± ± ± ± ± ± ± ±
5 8 15 14 6 37 8 16 331 310
± 0.2 ± 0.1 ± 0.4 ± 4 ± 0.2 ±2 ±0.4 ± 0.5 ± 16cd
±0.2 ± 3 ± 3 ± 0.4 ± 2 ± 1 ± 0.6
PD
4 Week
Practical diet. 'Isolated soybean protein diet. "•Average plasma free amino acid concentration ± SEM in three trials. 'Values within the same amino acid without a common letter are significantly different (p < 0.05).
2
2 Week (j.moles/100 ml. plasma 4 5 5 ± lab 0.3b 59 86 ± 2ab la 49 76 ± 3a 7abc 14 17 ± 2bc 3bc 40 47 ± 4 a 5a 37 69 ± 5a 4cd 18 22 ± 3cde lde
PD 3 96 66 18 41 41 18
ISPD 3 5 ± 0.9ab 66 ± 8cd 72 ± 9ab 31 ± 6a 42 ± 3a 77 ± 4a 26±2c
1 Week
± 0.4bc ± O.lbc ± la ± 2a ±2b ±0.2c ± 0.9a ± 0.6b ± 18a
± ± ± ± ± ± ±
PD 2
67 125 71 38 Body weight 1 Blood samples were taken from Chicks fed ad libitum at 3 p.m.
7 Aspartic acid 78 Threonine 85 Serine 17 Glutamic acid 41 Glycine 38 Alanine 49 Valine Methionine + 1 / 2 Cystine 18 19 Isoleucine Leucine 24 10 Tyrosine 13 Phenylalanine Lysine 65 10 Histidine 41 Arginine Total 515
Day-old
TABLE 4.—Change in plasma free amino acid concentrations during the gr
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PLASMA AMINO ACID PATTERNS
T h e information in this report indicates clearly that it is necessary to state in the experimental p r o c e d u r e t h e time of t h e day t h e blood samples were taken. It is also necessary to indicate any fasting period involved. T h e possibility exists that differences could be s h o w n b e t w e e n a 24 h o u r lighting period as presented in this report and one involving a natural day length or a standard 14 hour day as used in many laboratories. T h e d e c r e a s e in plasma amino acids observed as the birds b e c a m e older is also an important factor that must b e considered when comparing results from different laboratories or e x p e r i m e n t s within a laboratory. ACKNOWLEDGEMENT The authors wish t o acknowledge the gift of the Dextrin from t h e C o r n P r o d u c t s C o . , Argo, Illinois, through the courtesy of Dr. J. D . Shroder.
REFERENCES Al-Ubaidi, Y. Y., and H. R. Bird, 1964. Assay for the unidentified growth factor in dried whey. Poultry Sci. 43: 1484-1488. Ascarelli, I., and I. Bruckental, 1969. Location and properties of serine dehydrase in the chick. Comp. Biochem. Physiol. 29: 1277-1279. Askelson, C. E., and S. L. Balloun, 1963. Influence of age and dietary protein on certain free amino acids in chick blood plasma. Poultry Sci. 42: 140-146. Boomgaardt, J., and B. E. McDonald, 1969. Compari-
son of fasting plasma amino acid patterns in the pig, rat and chicken. Can. J. Physiol. Pharmacol. 47: 392-395. Charkey, L. W., W. K. Manning, A. K. Kano, F. X. Gassner, M. L. Hopwood and I. L. Madsen, 1953. A further study of vitamin B, 2 in relation to amino acid metabolism in the chick. Poultry Sci. 32: 630-642. Dean, W. F., and H. M. Scott, 1966. Use of free amino acids concentrations in blood plasma of chick to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88: 75-83. Evans, R. M., and R. W. Scholz, 1973. Development of renal gluconeogenesis in chicks fed high fat and high protein "carbohydrate-free" diets. J. Nutr. 103: 242-250. Featherston, W. R., and G. W. Horn, 1973. Dietary influences on the activities of enzymes involved in branched-chain amino acid catabolism in the chick. J. Nutr. 103: 757-765. Felig, P., T. Pozefsky, E. Marliss and G. F. Cahill, Jr., 1970. Alanine: key role in gluconeogenesis. Science, 167: 1003-1004. Gray, J. A., E. M. Olsen, D. C. Hill and H. D. Branion, 1960. Effect of a dietary lysine deficiency on the concentration of amino acids in the deproteinized blood plasma. Can. J. Biochem. Physiol. 38: 435441. Hazelwood, R. L., and F. W. Lorenz, 1959. Effects of fasting and insulin on carbohydrate metabolism of the domestic fowl. Am. J. Physiol. 197: 47-51. Hill, D. C , and E. M. Olsen, 1963. Effect of starvation and a non protein diet on blood plasma amino acids, and observations on the detection of amino acids limiting growth of chicks fed purified diets. J. Nutr. 79: 303-310. Longenecker, J. B., and N. L. Hause, 1959. Relationship between plasma amino acids and composition of the ingested protein. Arch. Biochem. Biophys. 84: 46-59. Maruyama, K., M. L. Sunde and A. E. Harper, 1976. Is L-glutamic acid nutritionally a dispensable amino acid for the chick? Poultry Sci. 55: 45-60. National Research Council, 1971. Nutrient Requirements of Poultry. N.A.S., Washington, D.C. Nelson, P., G. Yarnell and S. R. Wagle, 1966. Biochemical studies of the developing embryo. II. Studies on C0 2 fixation enzymes. Arch. Biochem. Biophys. 114: 543-546. Richardson, L. R., L. G. Blaylock and C. M. Lyman, 1953. Influence of dietary amino acid supplements on the free amino acids in the blood plasma of chicks. J. Nutr. 51: 515-522. Richardson, L. R., F. Hale and S. J. Ritchey, 1965. Effect of fasting and level of dietary protein on
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intake. After switching birds from t h e starter diet to the grower diet, concentrations of most of amino acids decreased considerably, with exceptions of methionine plus one half cystine and basic a m i n o acids. T h e trend of decreasing concentrations of methionine plus a half cystine and lysine was somewhat surprising, since the birds were kept on the starter diet until 12 w e e k s . T h e requirements of both methionine plus cystine and lysine d e c r e a s e from the period of 0 to 6 w e e k s and during t h e period of 6 to 14 w e e k s ( N . R . C . , 1971).
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K. MARUYAMA, M. L. SUNDE AND A. E. HARPER
Tonkinson, L. V., K. E. Dunkelgod, E. W. Gleaves, R. H. Thayer and R. J. Sirny, 1961. Effect of dietary lysine on free amino acid concentrations in blood plasma of growing turkeys. Poultry Sci. 40: 11061117. Wallace, J. C , and E. A. Newsholme, 1967. A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl. Biochem. J. 104: 378-384. Wang, S. H., and M. C. Nesheim, 1972. Degradation of lysine in chicks. J. Nutr. 102: 583-596. Wenger, E., B. S. Wenger and P. A. Kitos, 1967. Pentose phosphate pathway activity of the chick embryo in ovo. J. Exp. Zool. 166: 263-270. Yoshida, T., and G. Kikuchi, 1971. Significance of the glycine cleavage system in glycine and serine catabolism in avian liver. Arch. Biochem. Biophys. 145: 658-668.
Immunological and Compositional Patterns of Lipoproteins in Chicken (Gallus domesticus) Plasma J. Y-L. Yu, L. D. CAMPBELL AND R. R. MARQUARDT
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Received for publication June 23, 1975)
ABSTRACT Immunological and compositional patterns of plasma lipoproteins in the laying and nonlaying hen (Gallus domesticus) and rooster plasmas were compared. Hen plasma contained three immunologically distinct lipoproteins: very low density (VLDL) and low density (LDL); high density (HDL); and lipovitellin (LV). Rooster plasma contained VLDL and LDL; and HDL. Laying hen plasma in comparison to the other two groups of birds contained a high content of VLDL and LV. The plasma lipoprotein pattern of nonlaying hens and roosters were similar except for the relatively high content of LDL and the presence of low levels of LV in nonlaying hen plasma. In general, triglyceride, phospholipid and cholesterol contents were similar for each lipoprotein class in laying hen, nonlaying hen and rooster plasma. POULTRY SCIENCE 55: 1626-1631, 1976
INTRODUCTION
L
of lay have been characterized via analytical
IPOPROTEINS are the transport media
ultracentrifugation
for lipids in the blood of mammals and
(1956), Schjeide et al. (1963) and Schjeide
birds (Frederickson etal., 1967). Lipoproteins
and Wilkens (1964). Hillyard etal. (1956) have
also serve as precursors of egg yolk lipid
compared the composition and concentra-
in the laying hen (Schjeide
tions of plasma lipoproteins in normal and
et al.,
1963;
by Schjeide
and
Urist
1972; Yu
stilbestrol injected roosters. Characterization
and Marquardt, 1973; Gornall and Kuksis,
of plasma lipoproteins in the laying hen by
1973).
the use of preparative ultracentrifugation was
Husbands, 1971; Hillyard et ai,
Plasma lipoproteins in the hen at the onset
recently undertaken by Husbands (1970) and
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free amino acids in pig plasma. J. Anim. Sci. 24: 368-372. Shao, T. C , and D. C. Hill, 1967. A comparison of the effect of dietary fat and carbohydrate on free amino acids in the blood of chicks. Can. J. Physiol. Pharmacol. 45: 225-234. Smith, R. E., and H. M. Scott, 1965a. Use of free amino acid concentrations in blood plasma in evaluating the amino acid adequacy of intact proteins for chick growth. I. Free amino acid patterns of blood plasma of chicks fed unheated and heated fishmeal proteins. J. Nutr. 86: 37-44. Smith, R. E., and H. M. Scott, 1965b. Use of free amino acid concentrations in blood plasma in evaluating the amino acid adequacy of intact proteins for chick growth. II. Free amino acid patterns of blood plasma of chicks fed sesame and raw, heated and overheated soybean meals. J. Nutr. 86: 45-50. Squibb, R. L., 1966. Nature of the free amino-acid pool in avian tissues. Nature, 209: 710-711.