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Levels of Lipoproteins in Chicken Plasma
608
R. M. SMITH, J. KAN AND T. R. C. ROKEBY
housing problems in relation to climate. 1960 Winter Meeting, American Soc. Agr. Engineers, Memphis, Tennessee. Prince, R. P., L. M. Potter and W. W. Irish, 1961. Response of chickens to temperature and ventilation environments. Poultry Sci. 40: 102-108. Scarborough, E. N., 1957. Environmental considerations in the design of broiler houses. Summer Meeting of the American Soc. of Agricultural Engineers, June, 1957.
Siegel, P. B., and R. H. Coles, 1958. The effects of early humidity on broiler production. 55th Annual Proc. Southern Agr. Workers, pp. 240-241, 1958. Stewart, R. E., and C. N. Hinkle, 1959. Environmental requirements for poultry shelter design. Agricultural Engineering, 40: 532-535. Yeates, N. T. M., D. H. K. Lee and H. J. G. Hines, 1941. Reactions of domestic fowls to hot atmospheres. Proc. Roy. Soc. Queensland, 53: 105-128.
KAZUTAKA HOMMA AND SHUNZO KATO Department of Animal Physiology, Faculty of Agriculture, Nagoya University, Anzyo, Japan (Received for publication July 31, 1961)
B
Y ULTRACENTRIFUGAL techniques, Schjeide (1954) demonstrated that no significant amount of free lipid is present in the chicken plasma at any developmental stage, and that the concentration of plasma lipoproteins of high lipid class of the laying hen is seven times; as high as that of the rooster. This means that changes of lipid content in hen\ plasma during the egg laying period must; be considered as changes in the patterns; of plasma lipoproteins. However, routine: methods for the analysis of plasma lipoproteins require special devices and are: time consuming. In recent articles, Oncley et al. (1957),, Bernfeld et al. (1957), Bernfeld (1958)i have reported a simple and rapid methodI for the isolation of human serum lipoglobulins. The new method is based uponi the specificity of interaction betweenl beta-lipoglobulin and macromolecular sulfated polysaccharides. Florsheim and1 Gonzales (1960) have indicated that recovery of serum beta-lipoprotein by the; polyanion precipitation method is aboutt 90 percent in human and chicken seruml when Mepesulfate (sodium salt of sul--
fated polygalacturonic acid methyl ester methyl glucosides) is used as a precipitating agent. This paper reports the results of some experiments on the plasma lipoproteins of hens and roosters using the polyanion precipitation method as a tool. MATERIALS AND METHODS Experimental animals. Fourteen laying hens, 14 roosters, 7 molting hens and 5 non-laying hens of a White Leghorn X Rhode Island Red cross were used. They were kept in individual cages and fed ad libitum with the same commercial layer mash throughout the experimental periods. Refractometry of plasma proteins. In all birds, blood samples of 2 ml. each were taken by brachial vein puncture at 1 p.m. using heparin sodium as an anticoagulant. One ml. of plasma was separated by centrifugation, and 2 mg. of sodium salt of dextran sulfate, having molecular weight of approximately 80,000 (referred to as MDS) were added. While mixing MDS and the plasma, a yellowish-white precipitate was produced which deposited
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
Levels of Lipoproteins in Chicken Plasma
609
LIPOPROTEIN IN PLASMA
TABLE 1.—Electrophoretic distribution of albumin and globulin in untreated and MDS-treated plasma of the laying hen Plasma protein* (g.%) Bird No.
Untreated plasma
Supernatant of MDS-treated plasma
Albumin Globulin Albumin Globulin 1 2 3 4 5 6 7 8 9 10
2.32 1.40 2.42 1.91 1.88 1.89 1.74 2.14 2.38 2.60
4.15 4.59 3.75 3.58 4.44 3.14 3.83 4.69 5.43 4.61
2.38 1.46 2.45 1.95 1.88 1.86 1.88 2.14 2.43 2.24
2.22 3.54 2.34 2.83 2.54 2.75 2.92 3.54 2.32 3.55
Average S.D.
2.07 0.36
4.22 0.64
2.07 0.32
2.86 0.28
* Calculated from the area under scanning curve at 560 m^t. Papers were stained with Brom Phenol Blue.
laparotomy. Blood samples of the molting hens were taken within 30 minutes after the time of inspection. RESULTS Composition of the precipitates. Distribution of albumin and globulin in the plasma of laying hens before and after the MDS treatment are shown in Table 1 and Figure 1. Approximately 1 g.% of plasma globulin was precipitated with MDS, whereas the albumin fraction was practically unaltered. Paper electropherograms stained with Oil Red 0 indicated that the precipitated globulins accounted for 85.63 percent (averaged from 24 determinations) of sudanophilic lipoproteins of the plasma. The precipitates showed almost identical nitrogen/phosphorus ratios (wt./wt.) of around 4 and lipid content of about 70 percent (Table 2). From the average of 5 determinations, the lipid part of the precipitated lipoproteins contained 4 percent of cholesterol and 20 percent of phospholipids. Besides,
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readily upon centrifugation at l,700Xg. for 30 minutes. Determinations of the plasma protein were made by a refractometer assuming that the specific refractive index increment of chicken plasma protein is the same as that of human serum albumin (0.186) when protein concentration is expressed in terms of g. per ml. (Doty and Edsall, 1951). The amount of the precipitated proteins was calculated as the difference between the values of the untreated plasma and the MDStreated plasma. At the dose of MDS used, the accuracy of this method was within the range of ±0.1 g. per 100 g. of the plasma. Paper electrophoresis. Paper electrophoresis of the hen plasma before and after the MDS treatment was performed in the manner described by Dessauer and Fox (1959). Drying, staining and the quantitative analysis were performed according to the description of Durrum el al. (1952). Chemical analysis. Phosphorus and nitrogen were determined as in the previous paper (Homma and Tuchida, 1959). Total lipids and alkali-labile phosphorus were determined by the method of Mclndoe (1959 a). Total cholesterol was determined by the method of Abell el al. (1952). Determination of number of ovarian follicles. Hens were injected with 0.5 ml. of 1 percent Evans Blue in saline, or with 3 mg. of Sudan III suspended in 5 percent glucose, via the left brachial vein. The number of ovarian follicles in the rapid growth phase at the time of dye injection was estimated by counting eggs with pink or blue colored yolk which were laid during the experimental period. In these hens, blood samples were taken one hour after the dye injection. When hens were in the molting state, the size and number of the ovarian follicles were inspected by
610
K . HOMMA AND S. KATO
PRECIPITATED PROTEINS
TABLE 2.—Composition of the lipoprotein-M DS complex
ALBUMIN
(%)
N / P ratio (wt./wt.)
Levels of precipitable lipoproteins in plasma (g-%)
66.1 + 5.7* (8)** 75.0 + 5.0 (3) 70.6 + 8.5 (5) 75.3 + 3.2 (3)
3.94 + 0.52(11) 4.10 + 0.52(9) 3.82 + 0.15(7) 4.12 + 0.02(4)
0.5-1.9 2.0-2.9 3.0-3.9 4.0-5.2
Lipid
it was noted that 50 to 80 percent of alkali-labile phosphorus of the lipid-free proteins of the plasma was brought into the precipitates. In the following, plasma proteins which react with MDS to form
4 --
0 o
2 39.2
a precipitate will be designated as precipitable lipoproteins. Levels of precipitable lipoproteins in chicken plasma. Concentrations of the precipitable lipoproteins in the plasma of roosters, molting hens and laying hens, are shown in Figure 2. Higher values of the precipitable lipoproteins were found only in the plasma of laying hens. Furthermore, values of total plasma pro-
o LAYING HEN o MOLTING HEN • ROOSTER
ox oo
*Mean±S.D. ** Number of determinations.
o o o0 o o o o o ° o o 0o
-
o
> ° o°o
§ a.
y
DC
a.
*yoog° °o
o
1
o c
0-
••••
mmmm
1
1
3
4
• 1
°n o
• 1
1
1
5 6 7 8 TOTAL PLASMA PROTEIN
1
9
FIG. 2. Interrelations between total plasma protein and precipitable lipoproteins. In the laying hen, three determinations were made in each bird and values were plotted separately. In the molting hen and rooster, each dot represents a different bird.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
FIG. 1. Scanning curves of paper electrophoresis of plasma proteins of the laying hen. Proteins were stained by Brom Phenol Blue. Upper curve represents the untreated plasma, and the lower one represents the supernatant of the MDS treated plasma. Precipitated lipoproteins are shown by the barred area between the two curves.
611
L I P O P R O T E I N I N PLASMA
Subsequent experiments in the winter season were designed to examine the role of ovarian follicles in regulating levels of the precipitable lipoproteins of hen plasma. Estimation of the number of ovarian follicles in the rapid growth phase was performed by the method described in the foregoing section. Fourteen laying hens were selected from the same flock of birds used in the experiment in the summer season. I t was found that, in the laying hen, increasing number of ovarian follicles caused a parallel increase of the precipitable lipoproteins in the plasma. Coefficient of correlation between the two parameters was statistically highly significant (r = 0.88; p < 0 . 0 1 ) . T h e results are shown in Figure 4. Values of molting hens obtained from a separate experiment are also included in Figure 4. Effect of estrogen on the plasma of nonlaying hens. Changes in plasma lipoproteins characteristic of the laying hen could be reproduced in the non-laying hen b y a single subcutaneous injection of
EGG LAY 0000
0000000
0000
0000
o TOTAL • PRECIPITABLE
0
5 10 15 TIME IN DAYS
FIG. 3. Time of oviposition and fluctuations in plasma proteins. 15 mg./kg. of diethylstilbestrol. Before the hormone injection, the average value of the precipitable lipoproteins of the non-laying hen was only 0.14 g.%. Fortyeight hours after the injection, it was increased to 2.96 g.%. Simultaneously, an increase in plasma globulins of nonprecipitable fraction was observed, which accounted for less t h a n 27 percent of the 0
2,5
o LAYING HEN O MOLTING HEN
_
2.0-1,5
o
_. o
1,0-0.50-
0
o
0 0
o
8
o o o o
8
o
o o 0 0
o
0
8 o o o
o o
o
8
o o o
8
8 0
s
8
o o
0
o
1 >
I
1
_L
1
1
1
1
0
1
2
3
4
5
6
7
NUMBER OF FOLLICLES FIG. 4. Interrelations between number of ovarian follicles in the rapid growth phase and levels of precipitable lipoproteins. In the laying hen, each bird was subjected to dye injection at least four times, and values obtained were plotted separately. In the molting hen, each dot represents a different bird.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
teins of the laying hen were linearly proportional to those of the precipitable lipoproteins. These lipoproteins can account for over 80 percent of the increment of plasma protein associated with egg laying. Interrelations between plasma levels of the precipitable lipoproteins and egg laying. I n order to examine whether oviposition would cause a change in the levels of plasma proteins, an experiment was conducted in the summer season of 1960. I n t h a t experiment, total protein and the precipitable lipoproteins of the plasma of 12 laying hens were determined on altern a t e days for one month. T h e results showed t h a t neither clutch size nor the time of oviposition were correlated directly to the r h y t h m of fluctuation of the plasma proteins. One typical example is shown in Figure 3.
612
K . HOMMA AND S. KATO TABLE 3.—Plasma proteins of non-laying hens after estrogen treatment Plasma proteins* (g.%) Hours after estrogen injection
Bird No.
Body wt. (kg.)
24
i0
48
P
T
P
T
P
8.2 10.8 9.4 9.0 10.0
2.3 4.4 3.8 1.8 2.5
1 2 3 4 5
1.45 1.60 1.20 1.23 1.38
6.5 4.5 7.0 4.8 6.0
0.26 0.12 0.13 0.04 0.16
6.4 7.0 7.8 7.0 7.4
1.4 2.2 1.4 2.2 2.8
Average
1.37
5.76
0.14
7.12
2.00
:
9.48
2.96
T, Total plasma protein. P, Precipitable lipoproteins.
increment in protein concentration induced by the hormone treatment. Results of the experiment with non-laying hens are shown in Table 3. DISCUSSION Several authors have pointed out that plasma of laying hens and roosters treated with estrogen contains larger amounts of beta-lipoproteins than that of non-laying hens and roosters (Brandt et al., 1951; Clegg et al., 1951, 1960; Caldwell and Suydam, 1959; Urist et al., 1958; Urist and Schjeide, 1961). Hillyard elal. (1956) have shown that the concentration of the lipoproteins of lower density class (1.006 or less) in the sera of cockerels is too low to measure, while lipoproteins of this class in the plasma of the heavily estrogenized roosters amount to 11 g. per 100 ml. of plasma. According to Hanahan (1960), anionic polysaccharides are the specific reagents for precipitation of low density lipoproteins of human serum (1.035 or less). These findings suggest that the polyanion precipitation method has special merit for the study of plasma lipoproteins in relation to egg laying. Results of the present experiments in the hen showed that over 80% of sudanophilic lipoproteins of high lipid content were specifically precipitated with MDS.
The chemical composition of the precipitated lipoproteins was similar to that of the so called precipitable lipophosphoprotein complex reported by Mclnode (1959 a). In a preliminary experiment of our laboratory, a preparation of low density lipoproteins was made from hen plasma by the method of Mclndoe (1959 b). It was then dissolved in cock plasma to give a concentration of 1 or 2 g.%. Recovery of the added lipoproteins from the cock plasma by the MDS precipitation method was almost complete. Rochilina (1934) has shown by refractometry, that plasma protein of the laying hen shows a cyclic decrease of approximagely 1 g.% on the day of oviposition, whereas Sturkie and Newman (1951) have shown by biuret method, that it remains fairly constant and is not influenced by the time of oviposition. Data of the present experiment show that fluctuations in the levels of plasma protein during egg laying period are due largely to the fluctuations of lipoproteins of high lipid content. These lipoproteins may be underestimated, compared to the actual, when they are calculated from protein nitrogen. Therefore, the difference in the method of determination of plasma protein used by the previous authors may be the cause of variations found in their reports.
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T
L I P O P R O T E I N I N PLASMA
Results of our experiments showed t h a t t h e levels of t h e precipitable lipoproteins of the hen plasma reflect the n u m b e r of ovarian follicles in the rapid growth phase. As estrogen administration to t h e nonlaying hen caused a rise in plasma lipoproteins to a level which is found in the laying hen, it m a y be s t a t e d t h a t
the
a m o u n t of estrogenic substances secreted the o v a r y is proportional
to
the
n u m b e r of m a t u r i n g follicles. SUMMARY D e t e r m i n a t i o n s of plasma lipoproteins of hens a n d roosters were made
using
macromolecular
as
dextran
sulfate
a
specific precipitating agent for lipoproteins of high lipid content. T h e precipitated lipoproteins did not contain a n y albumin fraction a n d showed a fairly c o n s t a n t composition. T h e concentrations of the precipitable lipoproteins in plasma were very low in nonlaying hens a n d roosters, less t h a n 1 g . % in molting hens, a n d 1 to 4 g.% in most of the laying hens. T h e degree of ovarian activity, as expressed b y t h e n u m b e r of ovarian follicles in the rapid growth phase, exhibited a positive correlation to the levels of the precipitable lipoproteins of the plasma. Increasing
amounts
of
estrogenic
sub-
stances secreted from the follicles in the rapid growth phase m a y be responsible for the rise in the precipitable lipoproteins in hen plasma t h a t occurs in association with egg laying. ACKNOWLEDGEMENT The
macromolecular
was kindly forwarded
dextran
sulfate
to us by D r .
E.
Moryi, Director of t h e Research Laboratory of Meito Co. L t d . , N a g o y a . REFERENCES Abell, L. L., B. B. Levy, B. B. Brodie and F. E. Kendall, 1952. A simplified method for the estimation of total cholesterol in serum and demon-
stration of its specificity. J. Biol. Chem. 195: 357-366. Bernfeld, P., V. M. Donahue and M. E. Berkowitz, 1957. Interaction of human serum beta-lipoglobulin with polyanions. J. Biol. Chem. 226: 51-64. Bernfeld, P., 1958. The Lipoproteins, Methods and Clinical Significance. 24-36. S. Karger Pub., New York and Basel. Brandt, L. W., R. E. Clegg and A. C. Andrews, 1951. The effect of age and degree of maturity on the serum proteins of the chicken. J. Biol. Chem. 191: 105-112. Caldwell, C. T., and D. E. Suydam, 1959. Quantitative study of estrogen-induced atherosclerosis in cockerels. Proc. Soc. Exp. Biol. Med. 101: 299302. Clegg, R. E., P. E. Sanford, R. E. Hein, A. C. Andrews, J. S. Hughes and C. D. Mueller, 1951. Electrophoretic comparison of the serum proteins of normal and diethylstilbestrol treated cockerels. Science, 114: 437-438. Clegg, R. E., A. T. Ericson and U. K. Misra, 1960. Effect of high levels of dietary cholesterol on the serum proteins of the chicken. Poultry Sci. 39: 35-39. Dessauer, H. C , and W. Fox, 1959. Changes in ovarian follicle composition with plasma levels of snakes during estrus. Amer. J. Physiol. 197: 360-366. Doty, P., and J. T. Edsall, 1951. Advances in Protein Chemistry. VI. 55. Academic Press Inc., New York. Durrum, E. L., M. H. Paul and E. R. B. Smith, 1952. Lipid detection in paper electrophoresis. Science, 116: 428-430. Florsheim, W. H., and C. Gonzales, 1960. Comparison of ultracentrifuge and polyanion precipitation methods for serum beta-lipoproteins. Proc. Soc. Exp. Biol. Med. 104: 618-620. Hanahan, D. J., 1960. Lipide Chemistry. John Wiley and Sons, New York. Hillyard, L. A., C. Entenman and I. L. Chaikoff, 1956. Concentration and composition of serum lipoproteins of cholesterol fed and stilbestrolinjected birds. J. Biol. Chem. 223:359-368. Homma, K., and K. Tuchida, 1959. NaCl-acetate turbidity test. Poultry Sci. 38:1426-1430. Mclndoe, W. M., 1959 a. A lipophosphoprotein complex in hen plasma associated with yolk production. Biochem. J. 72: 153-159. Mclndoe, W. M., 1959 b. Lipoproteins of high lipid content from egg yolk. Biochem. J. 73: Proc. 45-16. Oncley, J. L., K. W. Walton and D. G. Cornwell, 1957. A rapid method for the bulk isolation of
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
from
613
614
K . HOMMA AND S. KATO ing, ovulation, number of blood samples taken and plasma volume. Poultry Sci. 30: 240-248. Urist, M. R., A. 0 . Schjeide and F. C. McLean, 1958. The partition and binding of calcium in the serum of the laying hen and of the estrogenized rooster. Endocrinology, 63: 570-585. Urist, M. R., and A. O. Schjeide, 1961. The partition of calcium and protein in the blood of oviparous vertebrates during estrus. J. Gen. Physiol. 44:743-755.
Some Criteria for the Evaluation of Protein Supplements in Chick Growth Trials A . A . WOODHAM AND IAN McDONALD Rowett Research Institute, Bucksbum, Aberdeen (Received for publication July 31,1961)
V
ARIOUS criteria may be used to assess the performance of chicks in tests intended to compare the nutritive value of different sources of supplementary protein. They extend from the simple comparison of weight gain over a given age range to the use of relatively complicated expressions such as the "gross protein value" (GPV), put forward by Heiman et al. (1939). The GPV technique has been used in this laboratory over a period of twelve years and various modifications have been made from time to time. Between 1957 and 1959 over 600 individual GPV determinations were carried out using a modified method (Duckworth et al., 1961). This formed part of a collaborative investigation into the variable quality of commercial protein concentrates used in the United Kingdom for pig and poultry feeding. It was felt that the considerable body of data which had been accumulated on growth rates, feed consumptions, etc., might be used to check whether the GPV calculation could be improved by slight modification and whether it was in fact
more useful than some simpler criterion obtainable from the same results. A suggested alternative method of calculation (Anwar, 1960) has also been investigated. MATERIALS AND METHODS
In the project described above, tests were made on a wide range of protein concentrates. The meat meals and cottonseed meals have been arbitrarily selected from among these to represent animal and vegetable proteins respectively. Tests were made on 29 different samples of meat and meat/bone meal, and 17 different samples of cottonseed meal. Each sample was tested in at least two separate experiments, the total number of individual results being 78 for the 29 meat meals and 39 for the 17 cottonseed meals. Tables 1 and 2 show the mean values of a number of possible criteria by which the meals might be assessed. To clarify the relationships between them, algebraic definitions of each criterion are given below. Certain chemical tests were also made on each sample of meal. The "available lysine value" (ALV) (Carpen-
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
beta-lipoproteins from human plasma. J. Amer. Chem. Soc. 79: 4666^671. Rochilina, M., 1934. Les proteines du sang et la. ponte des poules. Bull. Soc. de Chim. Biol. 16: 1644-1651. Schjeide, A. O., 1954. Studies on the New Hampshire chicken embryo. III. Nitrogen and lipide analyses of ultracentrifugal fractions of plasma. J. Biol. Chem. 211:355-362. Sturkie, P. D., and H. J. Newman, 1951. Plasma proteins of chickens as influenced by time of lay-
R. M. SMITH, J. KAN AND T. R. C. ROKEBY
housing problems in relation to climate. 1960 Winter Meeting, American Soc. Agr. Engineers, Memphis, Tennessee. Prince, R. P., L. M. Potter and W. W. Irish, 1961. Response of chickens to temperature and ventilation environments. Poultry Sci. 40: 102-108. Scarborough, E. N., 1957. Environmental considerations in the design of broiler houses. Summer Meeting of the American Soc. of Agricultural Engineers, June, 1957.
Siegel, P. B., and R. H. Coles, 1958. The effects of early humidity on broiler production. 55th Annual Proc. Southern Agr. Workers, pp. 240-241, 1958. Stewart, R. E., and C. N. Hinkle, 1959. Environmental requirements for poultry shelter design. Agricultural Engineering, 40: 532-535. Yeates, N. T. M., D. H. K. Lee and H. J. G. Hines, 1941. Reactions of domestic fowls to hot atmospheres. Proc. Roy. Soc. Queensland, 53: 105-128.
KAZUTAKA HOMMA AND SHUNZO KATO Department of Animal Physiology, Faculty of Agriculture, Nagoya University, Anzyo, Japan (Received for publication July 31, 1961)
B
Y ULTRACENTRIFUGAL techniques, Schjeide (1954) demonstrated that no significant amount of free lipid is present in the chicken plasma at any developmental stage, and that the concentration of plasma lipoproteins of high lipid class of the laying hen is seven times; as high as that of the rooster. This means that changes of lipid content in hen\ plasma during the egg laying period must; be considered as changes in the patterns; of plasma lipoproteins. However, routine: methods for the analysis of plasma lipoproteins require special devices and are: time consuming. In recent articles, Oncley et al. (1957),, Bernfeld et al. (1957), Bernfeld (1958)i have reported a simple and rapid methodI for the isolation of human serum lipoglobulins. The new method is based uponi the specificity of interaction betweenl beta-lipoglobulin and macromolecular sulfated polysaccharides. Florsheim and1 Gonzales (1960) have indicated that recovery of serum beta-lipoprotein by the; polyanion precipitation method is aboutt 90 percent in human and chicken seruml when Mepesulfate (sodium salt of sul--
fated polygalacturonic acid methyl ester methyl glucosides) is used as a precipitating agent. This paper reports the results of some experiments on the plasma lipoproteins of hens and roosters using the polyanion precipitation method as a tool. MATERIALS AND METHODS Experimental animals. Fourteen laying hens, 14 roosters, 7 molting hens and 5 non-laying hens of a White Leghorn X Rhode Island Red cross were used. They were kept in individual cages and fed ad libitum with the same commercial layer mash throughout the experimental periods. Refractometry of plasma proteins. In all birds, blood samples of 2 ml. each were taken by brachial vein puncture at 1 p.m. using heparin sodium as an anticoagulant. One ml. of plasma was separated by centrifugation, and 2 mg. of sodium salt of dextran sulfate, having molecular weight of approximately 80,000 (referred to as MDS) were added. While mixing MDS and the plasma, a yellowish-white precipitate was produced which deposited
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
Levels of Lipoproteins in Chicken Plasma
609
LIPOPROTEIN IN PLASMA
TABLE 1.—Electrophoretic distribution of albumin and globulin in untreated and MDS-treated plasma of the laying hen Plasma protein* (g.%) Bird No.
Untreated plasma
Supernatant of MDS-treated plasma
Albumin Globulin Albumin Globulin 1 2 3 4 5 6 7 8 9 10
2.32 1.40 2.42 1.91 1.88 1.89 1.74 2.14 2.38 2.60
4.15 4.59 3.75 3.58 4.44 3.14 3.83 4.69 5.43 4.61
2.38 1.46 2.45 1.95 1.88 1.86 1.88 2.14 2.43 2.24
2.22 3.54 2.34 2.83 2.54 2.75 2.92 3.54 2.32 3.55
Average S.D.
2.07 0.36
4.22 0.64
2.07 0.32
2.86 0.28
* Calculated from the area under scanning curve at 560 m^t. Papers were stained with Brom Phenol Blue.
laparotomy. Blood samples of the molting hens were taken within 30 minutes after the time of inspection. RESULTS Composition of the precipitates. Distribution of albumin and globulin in the plasma of laying hens before and after the MDS treatment are shown in Table 1 and Figure 1. Approximately 1 g.% of plasma globulin was precipitated with MDS, whereas the albumin fraction was practically unaltered. Paper electropherograms stained with Oil Red 0 indicated that the precipitated globulins accounted for 85.63 percent (averaged from 24 determinations) of sudanophilic lipoproteins of the plasma. The precipitates showed almost identical nitrogen/phosphorus ratios (wt./wt.) of around 4 and lipid content of about 70 percent (Table 2). From the average of 5 determinations, the lipid part of the precipitated lipoproteins contained 4 percent of cholesterol and 20 percent of phospholipids. Besides,
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on May 31, 2015
readily upon centrifugation at l,700Xg. for 30 minutes. Determinations of the plasma protein were made by a refractometer assuming that the specific refractive index increment of chicken plasma protein is the same as that of human serum albumin (0.186) when protein concentration is expressed in terms of g. per ml. (Doty and Edsall, 1951). The amount of the precipitated proteins was calculated as the difference between the values of the untreated plasma and the MDStreated plasma. At the dose of MDS used, the accuracy of this method was within the range of ±0.1 g. per 100 g. of the plasma. Paper electrophoresis. Paper electrophoresis of the hen plasma before and after the MDS treatment was performed in the manner described by Dessauer and Fox (1959). Drying, staining and the quantitative analysis were performed according to the description of Durrum el al. (1952). Chemical analysis. Phosphorus and nitrogen were determined as in the previous paper (Homma and Tuchida, 1959). Total lipids and alkali-labile phosphorus were determined by the method of Mclndoe (1959 a). Total cholesterol was determined by the method of Abell el al. (1952). Determination of number of ovarian follicles. Hens were injected with 0.5 ml. of 1 percent Evans Blue in saline, or with 3 mg. of Sudan III suspended in 5 percent glucose, via the left brachial vein. The number of ovarian follicles in the rapid growth phase at the time of dye injection was estimated by counting eggs with pink or blue colored yolk which were laid during the experimental period. In these hens, blood samples were taken one hour after the dye injection. When hens were in the molting state, the size and number of the ovarian follicles were inspected by
610
K . HOMMA AND S. KATO
PRECIPITATED PROTEINS
TABLE 2.—Composition of the lipoprotein-M DS complex
ALBUMIN
(%)
N / P ratio (wt./wt.)
Levels of precipitable lipoproteins in plasma (g-%)
66.1 + 5.7* (8)** 75.0 + 5.0 (3) 70.6 + 8.5 (5) 75.3 + 3.2 (3)
3.94 + 0.52(11) 4.10 + 0.52(9) 3.82 + 0.15(7) 4.12 + 0.02(4)
0.5-1.9 2.0-2.9 3.0-3.9 4.0-5.2
Lipid
it was noted that 50 to 80 percent of alkali-labile phosphorus of the lipid-free proteins of the plasma was brought into the precipitates. In the following, plasma proteins which react with MDS to form
4 --
0 o
2 39.2
a precipitate will be designated as precipitable lipoproteins. Levels of precipitable lipoproteins in chicken plasma. Concentrations of the precipitable lipoproteins in the plasma of roosters, molting hens and laying hens, are shown in Figure 2. Higher values of the precipitable lipoproteins were found only in the plasma of laying hens. Furthermore, values of total plasma pro-
o LAYING HEN o MOLTING HEN • ROOSTER
ox oo
*Mean±S.D. ** Number of determinations.
o o o0 o o o o o ° o o 0o
-
o
> ° o°o
§ a.
y
DC
a.
*yoog° °o
o
1
o c
0-
••••
mmmm
1
1
3
4
• 1
°n o
• 1
1
1
5 6 7 8 TOTAL PLASMA PROTEIN
1
9
FIG. 2. Interrelations between total plasma protein and precipitable lipoproteins. In the laying hen, three determinations were made in each bird and values were plotted separately. In the molting hen and rooster, each dot represents a different bird.
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FIG. 1. Scanning curves of paper electrophoresis of plasma proteins of the laying hen. Proteins were stained by Brom Phenol Blue. Upper curve represents the untreated plasma, and the lower one represents the supernatant of the MDS treated plasma. Precipitated lipoproteins are shown by the barred area between the two curves.
611
L I P O P R O T E I N I N PLASMA
Subsequent experiments in the winter season were designed to examine the role of ovarian follicles in regulating levels of the precipitable lipoproteins of hen plasma. Estimation of the number of ovarian follicles in the rapid growth phase was performed by the method described in the foregoing section. Fourteen laying hens were selected from the same flock of birds used in the experiment in the summer season. I t was found that, in the laying hen, increasing number of ovarian follicles caused a parallel increase of the precipitable lipoproteins in the plasma. Coefficient of correlation between the two parameters was statistically highly significant (r = 0.88; p < 0 . 0 1 ) . T h e results are shown in Figure 4. Values of molting hens obtained from a separate experiment are also included in Figure 4. Effect of estrogen on the plasma of nonlaying hens. Changes in plasma lipoproteins characteristic of the laying hen could be reproduced in the non-laying hen b y a single subcutaneous injection of
EGG LAY 0000
0000000
0000
0000
o TOTAL • PRECIPITABLE
0
5 10 15 TIME IN DAYS
FIG. 3. Time of oviposition and fluctuations in plasma proteins. 15 mg./kg. of diethylstilbestrol. Before the hormone injection, the average value of the precipitable lipoproteins of the non-laying hen was only 0.14 g.%. Fortyeight hours after the injection, it was increased to 2.96 g.%. Simultaneously, an increase in plasma globulins of nonprecipitable fraction was observed, which accounted for less t h a n 27 percent of the 0
2,5
o LAYING HEN O MOLTING HEN
_
2.0-1,5
o
_. o
1,0-0.50-
0
o
0 0
o
8
o o o o
8
o
o o 0 0
o
0
8 o o o
o o
o
8
o o o
8
8 0
s
8
o o
0
o
1 >
I
1
_L
1
1
1
1
0
1
2
3
4
5
6
7
NUMBER OF FOLLICLES FIG. 4. Interrelations between number of ovarian follicles in the rapid growth phase and levels of precipitable lipoproteins. In the laying hen, each bird was subjected to dye injection at least four times, and values obtained were plotted separately. In the molting hen, each dot represents a different bird.
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teins of the laying hen were linearly proportional to those of the precipitable lipoproteins. These lipoproteins can account for over 80 percent of the increment of plasma protein associated with egg laying. Interrelations between plasma levels of the precipitable lipoproteins and egg laying. I n order to examine whether oviposition would cause a change in the levels of plasma proteins, an experiment was conducted in the summer season of 1960. I n t h a t experiment, total protein and the precipitable lipoproteins of the plasma of 12 laying hens were determined on altern a t e days for one month. T h e results showed t h a t neither clutch size nor the time of oviposition were correlated directly to the r h y t h m of fluctuation of the plasma proteins. One typical example is shown in Figure 3.
612
K . HOMMA AND S. KATO TABLE 3.—Plasma proteins of non-laying hens after estrogen treatment Plasma proteins* (g.%) Hours after estrogen injection
Bird No.
Body wt. (kg.)
24
i0
48
P
T
P
T
P
8.2 10.8 9.4 9.0 10.0
2.3 4.4 3.8 1.8 2.5
1 2 3 4 5
1.45 1.60 1.20 1.23 1.38
6.5 4.5 7.0 4.8 6.0
0.26 0.12 0.13 0.04 0.16
6.4 7.0 7.8 7.0 7.4
1.4 2.2 1.4 2.2 2.8
Average
1.37
5.76
0.14
7.12
2.00
:
9.48
2.96
T, Total plasma protein. P, Precipitable lipoproteins.
increment in protein concentration induced by the hormone treatment. Results of the experiment with non-laying hens are shown in Table 3. DISCUSSION Several authors have pointed out that plasma of laying hens and roosters treated with estrogen contains larger amounts of beta-lipoproteins than that of non-laying hens and roosters (Brandt et al., 1951; Clegg et al., 1951, 1960; Caldwell and Suydam, 1959; Urist et al., 1958; Urist and Schjeide, 1961). Hillyard elal. (1956) have shown that the concentration of the lipoproteins of lower density class (1.006 or less) in the sera of cockerels is too low to measure, while lipoproteins of this class in the plasma of the heavily estrogenized roosters amount to 11 g. per 100 ml. of plasma. According to Hanahan (1960), anionic polysaccharides are the specific reagents for precipitation of low density lipoproteins of human serum (1.035 or less). These findings suggest that the polyanion precipitation method has special merit for the study of plasma lipoproteins in relation to egg laying. Results of the present experiments in the hen showed that over 80% of sudanophilic lipoproteins of high lipid content were specifically precipitated with MDS.
The chemical composition of the precipitated lipoproteins was similar to that of the so called precipitable lipophosphoprotein complex reported by Mclnode (1959 a). In a preliminary experiment of our laboratory, a preparation of low density lipoproteins was made from hen plasma by the method of Mclndoe (1959 b). It was then dissolved in cock plasma to give a concentration of 1 or 2 g.%. Recovery of the added lipoproteins from the cock plasma by the MDS precipitation method was almost complete. Rochilina (1934) has shown by refractometry, that plasma protein of the laying hen shows a cyclic decrease of approximagely 1 g.% on the day of oviposition, whereas Sturkie and Newman (1951) have shown by biuret method, that it remains fairly constant and is not influenced by the time of oviposition. Data of the present experiment show that fluctuations in the levels of plasma protein during egg laying period are due largely to the fluctuations of lipoproteins of high lipid content. These lipoproteins may be underestimated, compared to the actual, when they are calculated from protein nitrogen. Therefore, the difference in the method of determination of plasma protein used by the previous authors may be the cause of variations found in their reports.
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T
L I P O P R O T E I N I N PLASMA
Results of our experiments showed t h a t t h e levels of t h e precipitable lipoproteins of the hen plasma reflect the n u m b e r of ovarian follicles in the rapid growth phase. As estrogen administration to t h e nonlaying hen caused a rise in plasma lipoproteins to a level which is found in the laying hen, it m a y be s t a t e d t h a t
the
a m o u n t of estrogenic substances secreted the o v a r y is proportional
to
the
n u m b e r of m a t u r i n g follicles. SUMMARY D e t e r m i n a t i o n s of plasma lipoproteins of hens a n d roosters were made
using
macromolecular
as
dextran
sulfate
a
specific precipitating agent for lipoproteins of high lipid content. T h e precipitated lipoproteins did not contain a n y albumin fraction a n d showed a fairly c o n s t a n t composition. T h e concentrations of the precipitable lipoproteins in plasma were very low in nonlaying hens a n d roosters, less t h a n 1 g . % in molting hens, a n d 1 to 4 g.% in most of the laying hens. T h e degree of ovarian activity, as expressed b y t h e n u m b e r of ovarian follicles in the rapid growth phase, exhibited a positive correlation to the levels of the precipitable lipoproteins of the plasma. Increasing
amounts
of
estrogenic
sub-
stances secreted from the follicles in the rapid growth phase m a y be responsible for the rise in the precipitable lipoproteins in hen plasma t h a t occurs in association with egg laying. ACKNOWLEDGEMENT The
macromolecular
was kindly forwarded
dextran
sulfate
to us by D r .
E.
Moryi, Director of t h e Research Laboratory of Meito Co. L t d . , N a g o y a . REFERENCES Abell, L. L., B. B. Levy, B. B. Brodie and F. E. Kendall, 1952. A simplified method for the estimation of total cholesterol in serum and demon-
stration of its specificity. J. Biol. Chem. 195: 357-366. Bernfeld, P., V. M. Donahue and M. E. Berkowitz, 1957. Interaction of human serum beta-lipoglobulin with polyanions. J. Biol. Chem. 226: 51-64. Bernfeld, P., 1958. The Lipoproteins, Methods and Clinical Significance. 24-36. S. Karger Pub., New York and Basel. Brandt, L. W., R. E. Clegg and A. C. Andrews, 1951. The effect of age and degree of maturity on the serum proteins of the chicken. J. Biol. Chem. 191: 105-112. Caldwell, C. T., and D. E. Suydam, 1959. Quantitative study of estrogen-induced atherosclerosis in cockerels. Proc. Soc. Exp. Biol. Med. 101: 299302. Clegg, R. E., P. E. Sanford, R. E. Hein, A. C. Andrews, J. S. Hughes and C. D. Mueller, 1951. Electrophoretic comparison of the serum proteins of normal and diethylstilbestrol treated cockerels. Science, 114: 437-438. Clegg, R. E., A. T. Ericson and U. K. Misra, 1960. Effect of high levels of dietary cholesterol on the serum proteins of the chicken. Poultry Sci. 39: 35-39. Dessauer, H. C , and W. Fox, 1959. Changes in ovarian follicle composition with plasma levels of snakes during estrus. Amer. J. Physiol. 197: 360-366. Doty, P., and J. T. Edsall, 1951. Advances in Protein Chemistry. VI. 55. Academic Press Inc., New York. Durrum, E. L., M. H. Paul and E. R. B. Smith, 1952. Lipid detection in paper electrophoresis. Science, 116: 428-430. Florsheim, W. H., and C. Gonzales, 1960. Comparison of ultracentrifuge and polyanion precipitation methods for serum beta-lipoproteins. Proc. Soc. Exp. Biol. Med. 104: 618-620. Hanahan, D. J., 1960. Lipide Chemistry. John Wiley and Sons, New York. Hillyard, L. A., C. Entenman and I. L. Chaikoff, 1956. Concentration and composition of serum lipoproteins of cholesterol fed and stilbestrolinjected birds. J. Biol. Chem. 223:359-368. Homma, K., and K. Tuchida, 1959. NaCl-acetate turbidity test. Poultry Sci. 38:1426-1430. Mclndoe, W. M., 1959 a. A lipophosphoprotein complex in hen plasma associated with yolk production. Biochem. J. 72: 153-159. Mclndoe, W. M., 1959 b. Lipoproteins of high lipid content from egg yolk. Biochem. J. 73: Proc. 45-16. Oncley, J. L., K. W. Walton and D. G. Cornwell, 1957. A rapid method for the bulk isolation of
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from
613
614
K . HOMMA AND S. KATO ing, ovulation, number of blood samples taken and plasma volume. Poultry Sci. 30: 240-248. Urist, M. R., A. 0 . Schjeide and F. C. McLean, 1958. The partition and binding of calcium in the serum of the laying hen and of the estrogenized rooster. Endocrinology, 63: 570-585. Urist, M. R., and A. O. Schjeide, 1961. The partition of calcium and protein in the blood of oviparous vertebrates during estrus. J. Gen. Physiol. 44:743-755.
Some Criteria for the Evaluation of Protein Supplements in Chick Growth Trials A . A . WOODHAM AND IAN McDONALD Rowett Research Institute, Bucksbum, Aberdeen (Received for publication July 31,1961)
V
ARIOUS criteria may be used to assess the performance of chicks in tests intended to compare the nutritive value of different sources of supplementary protein. They extend from the simple comparison of weight gain over a given age range to the use of relatively complicated expressions such as the "gross protein value" (GPV), put forward by Heiman et al. (1939). The GPV technique has been used in this laboratory over a period of twelve years and various modifications have been made from time to time. Between 1957 and 1959 over 600 individual GPV determinations were carried out using a modified method (Duckworth et al., 1961). This formed part of a collaborative investigation into the variable quality of commercial protein concentrates used in the United Kingdom for pig and poultry feeding. It was felt that the considerable body of data which had been accumulated on growth rates, feed consumptions, etc., might be used to check whether the GPV calculation could be improved by slight modification and whether it was in fact
more useful than some simpler criterion obtainable from the same results. A suggested alternative method of calculation (Anwar, 1960) has also been investigated. MATERIALS AND METHODS
In the project described above, tests were made on a wide range of protein concentrates. The meat meals and cottonseed meals have been arbitrarily selected from among these to represent animal and vegetable proteins respectively. Tests were made on 29 different samples of meat and meat/bone meal, and 17 different samples of cottonseed meal. Each sample was tested in at least two separate experiments, the total number of individual results being 78 for the 29 meat meals and 39 for the 17 cottonseed meals. Tables 1 and 2 show the mean values of a number of possible criteria by which the meals might be assessed. To clarify the relationships between them, algebraic definitions of each criterion are given below. Certain chemical tests were also made on each sample of meal. The "available lysine value" (ALV) (Carpen-
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beta-lipoproteins from human plasma. J. Amer. Chem. Soc. 79: 4666^671. Rochilina, M., 1934. Les proteines du sang et la. ponte des poules. Bull. Soc. de Chim. Biol. 16: 1644-1651. Schjeide, A. O., 1954. Studies on the New Hampshire chicken embryo. III. Nitrogen and lipide analyses of ultracentrifugal fractions of plasma. J. Biol. Chem. 211:355-362. Sturkie, P. D., and H. J. Newman, 1951. Plasma proteins of chickens as influenced by time of lay-