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Electrophoretic Studies of Serum Proteins of Nonpregnant and Pregnant Hamsters
Electrophoretic Studies of Serum Proteins of Nonpregnant and Pregnant Hamsters ROY P. PETERSON, M.A.," CHARLES L. TURBYFILL, M.A.,f and A. L. SODERWALL, Ph.D.
A concentration during pregnancy in humans Extraction and electrophoretic has been reported by several authors. DECREASE IN SERUM PROTEIN
4 • 9 • 10
procedures indicated a decrease in total concentration due to a decline in the albumin fraction, although an increase occurred in the alpha-!, alpha-2, and beta globulins.9 Antionades, in a review of the literature concerned with circulating hormones, indicated that these substances were loosely attached or bound to the serum proteins and migrate with them in an electric field. The rate of fetal growth varies throughout pregnancy and maternal protein metabolism can be expected to show concomitant variations. This paper reports changes that occur in the levels of the serum protein fractions of pregnant hamsters. MAl'ERIALS AND METHODS
Sera from 10 female hamsters ( 5-10 months old) were used in each group. Blood was removed during diestrus in nonpregnant females and on Days 8, 12, and 15 of the 16-day gestation period, by cardiac puncture with the animals under ether anesthesia. Smith's modification of Owen's Veronal buffer (pH 8.6 and ionic strength of 0.078) was used throughout the investigaUon.11 Electrophoretic separation was carried out in an LKB electrophoresist apparatus using cellulose acetate strips,§ 5 X 8 em. Each strip received two 8-lambda samples of serum, 3 em. from the right edge. All electrophoretic runs were of 12 hours' duration at 225 v and at room temperature. Fast green-yellowish stain ( FG) 11 and periodic acid-Schiff (PAS) stain as modified by Bodman were used to localize the total protein From the Department of Biology, University of Oregon, Eugene, Ore. This study was aided by USPHS Grant RG 6156 (C1-2). *Present address: Department of Biology, Southern University in New Orleans, La. tPresent address: Department of Zoology, University of Georgia, Athens, Ga. ;t:LKB-Produkter, Stockholm 12, Sweden. §Oxoid Consolidated Laboratories, Chicago Heights, Ill.
486
VoL. 13, No.5, 1962
SERUM PROTEIN CoNcENTRATION
487
and glycoprotein fractions, respectively. The strips were then blotted, dried, and cleared in Whitemore oil No.120. These were read on a photovolt densitometer with the forward edge of each strip serving as its zeroing point with graphs prepared according to McDonald. 7 The resulting data were subjected to analysis by the variance test. RESULTS Nonpregnant Animals
With the FG stain, 5 electrophoretically distinct bands were demonstrated. By convention these are named in order of decreasing mobility as albumin and alpha-1, alpha-2, beta, and gamma globulins. The pattern obtained is similar to that reported for the hamster by Moore. Analysis of 10 strips showed an AjG ratio of 1.61 with albumin representing 61.9 per cent and globulins, 38.1 per cent. The major globulin in all strips was the alpha-1 fraction, which included 12.9 per cent of the total protein. Figure 1 is an example of the normal electrophoretic patterns and graphs. The PAS stain revealed 4 glycoprotein-containing fractions within the globulin area. Glycoproteins were not demonstrable in the albumin fraction. The alpha-1 globulins comprised 60.7 per cent of the total glycoproteins. Table 1 and Fig. 1 indicate the means of these data and an electrophoretic pattern of PAS-stained proteins. Pregnant Animals
Eighth Day. The FG technic showed the albumin diminishing to 58 per cent with globulins being augmented to 42 per cent. Alpha-1 and gamma TABLE 1. Protein Levels (Percentage) in Serum of Nonpregnant and Pregnant Hamsters 0 Globulins Albumin Group l
f 1
Notpregnant Pregnant 8 days Pregnant 12 days Pregnant 15 days
Staint
Mean
S.D.
FG
61.92
8.99
PAS
FG
58.08
PAS
FG
53.61
PAS
FG PAS
49.48
Alpha-1 Mean
12.92 60.73 6.01 16.44 61.48 5.52 20.01 56.95 3.61 28.42 74.02
S.D.
Alpha-2
Beta
Gamma
Mean
S.D.
Mean
S.D.
Mean
S.D.
3.89 8.81 4.25 18.67 2.85 8.41 6.38 17.00 2.93 8.42 3.51 18:98 4.66 7.47 7.60 13.83
2.86 5.28 2.99 3.26 2.15 2.23 3.52 3.72
6.34 7.69 4.67 6.55 3.64 6.81 3.14 5.53
3.17 5.28 1.36 2.26 0.98 1.47 0.87 2.12
9.95 12.87 12.35 17.93 14.39 15.91 11.46 11.91
2.20 4.80 2.71 5.19 1.31 4.63 3.87 6.84
*Means and S.D.'s are of 10 hamsters per group. tFG: Fast green-yellowish; PAS: Periodic acid Schiff.
488
PETERSON ET AL,
FERTILITY
& STERILITY
globulins increased while alpha-2 and beta globulins decreased. The AJG ratio dropped to 1.38. All changes were significant, with respect to nonpregnant females, at the 5 per cent level, as determined by an analysis of the variance.
Fig. 1. Electrophoretic strips from nonpregnant hamster serum proteins stained with fastgreen (top) and PAS (bottom). Graph is of O.D. readings, with dashed lines indicating PAS readings.
The PAS stain disclosed that alpha-! and gamma globulin glycopi'oteins had increased appreciably. Changes in the alpha-2 and beta globulin glycoproteins occurred, but these were not statistically significant.
Twelfth Day. The FG stain indicated a further diminution in the albumin fraction to 53.6 per cent while globulins rose to 46.4 per cent. The decrease in alpha-2 and beta globulins observed at this time was offset by greater increa~s in the alpha-! and gamma globulins. The AJG ratio continued to fall (tol.l5). Although alpha-! globulin glycoproteins had decreased to 56.9 per cent, this was offset by a greater percentage increase of gamma globulin glycoproteins. Alpha-2 and beta globulin glycoproteins showed no significant variations from those of nonpregnant or 8-day pregnant animals. Fifteenth Day. The albumin fraction continued decreasing and by Day 15 was 49.4 per cent. The relative proportion of globulins represented 50.6 per cent of the total. The AJG ratio was 0.97, indicating an inversion of that for the nonpregnant. The major protein changes were in the alpha-! globulin fraction, which had increased to nearly 125 per cent above normal values. Glycoproteins in the alpha-! globulin fraction increased after Days 12 and by Day 15 had attained a new high of 75 per cent. Beta-globulin glycoproteins decreased progressively during gestation and were 5.5 per cent of the total glycoproteins on Day 15. The means and standard deviations of all groups are shown in Table 1.
VoL. 13,No.5,1962
489
SERUM PROTEIN CONCENTRATION
70
Fig. 2. Serwn protein changes during gestation.
s at
·-
30
10
0
s
4
8 DAYS
12
15
OF GESTATION
1 .1.7
1
.,
.-
1.5
Fig. 3. Albumin-globulin ratios during gestation.
1.4 0
~1.3 a:
5
:c"' 1.2
r
r
tl
1.1
'·· 1.0
0.9
e OB0~--------4----------8---------~-2--------,5- DAYS
OF
GESTATION
490
PETERSON ETAL.
FERTILITY
& ·STERILITY
Figures 2 and 3 were derived from the da.;ta in Table 1 and show graphically the protein changes and AjG ratio changes during gestation. DISCUSSION
The progressive decline in the AjG ratio during pregnancy has been reported by Brown for the human; however, he did not report a change in alpha-1 globulin. Roy, after studying the effects of protein-free diets on pregnancy in the rat, concluded that paper electrophoresis indicated minor changes in serum proteins. Apparently, normal diets allow major changes in humans and hamsters. The changes seen in the alpha-1 globulins in hamsters are similar to those reported for humans in studies by Studnitz, in which alpha-1, alpha-2, and beta globulin levels were fortified. Engle and Woods reported similar results for the alpha-1 fraction. The glycoproteins in the alpha-1 globulin fraction have been reported to increase during pregnancy, neoplastic disease, and a number of other pathologic conditions in other species. 2 • 3 • 13 The significance of these variations is still a matter of conjecture. Serum protein variations during pregnancy are indicative of an increased metabolic turnover of these fractions. It has been suggested by Jaffay that such changes may result from the shunting of albumin synthesis into the synthesis of other body proteins. It was based on a study of the kinetics of albumin metabolism during growth. Albumin, according to this theory, can serve as the source of amino acids for the synthesis of body proteins either directly, without complete hydrolysis, or by contributing to the tissue aminoacid pool. Brown has advanced the idea that variations may also result from a progressive hemodilution during pregnancy. With the advent of fetal growth, quantitative and qualitative changes in the maternal proteins may reflect alterations in endocrine secretory output. It has been reported, for instance, that gonadotropins increase during pregnancy. These hormones, as well as adrenocorticotropins, thyroxine, insulin, and some steroids, are associated with the alpha globulins. 1 Bioassays of Cohn fractions prepared by cold ethanol fractionation have implicated these and other hormones with specific serum proteins. 1 • 5 • 13 It is suggested that the variations in serum proteins reported in this and other investigations may arise from greater or lesser hormonal output. SUMMARY
Serum from nonpregnant and pregnant hamsters was examined by cellulose acetate zone electrophoresis.
y
VoL.13, No.5, 1962
y
1. The albumin fraction decreases during gestation while globulins show a concurrent increase. The AjG ratio undergoes a reversal during late gestation as a result of a decrease in albumin and an increase in globulins. 2. Glycoproteins show variations during gestation, with the major change being a drop in alpha-! glycoproteins on Day 12 and a rise above nonpregnant values on Day 15 of a 16-day gestation period. 3. The relation between serum protein electrophoretic bands, endocrines, and metabolic activity was discussed. Further investigation into the correlation between these serum protein variations and pituitary secretions is anticipated.
~-
n n
•r n rs
SERUM PROTEIN CoNCENTRATION
Dept. of Biology University of Oreg011 Eugene, Ore.
h
Is ~r
1-
~r
)-
m
ln .t. 11~
n, :>f ;e le
LY
u-
491
REFERENCES I. ANTONIADEs, H. N. Circulating hormones. Plasma Proteins 2:105, 1960. 2. BERGSTRAND, C. G., and CzAR, B. Paper electrophoretic study of human fetal serum proteins with the demonstration of a new protein fraction. Scand..]. Clin. & Lab. Invest. 9:277, 1957. 3. BoDMAN, J. The separation of serum glycoproteins by continuous electrophoresis. Lab. Prac. Part 1:517, 1957. 4. BRoWN, T. Electrophoretic analysis of serum proteins in pregnancy; A preliminary study. ]. Obst. & Gynaec; Brit. Emp. 61:781, 1954. 5. ENGLE, R. L., and WooDs, K. R. Comparative biochemistry and embryology. Plasma Proteins 2:183, 1960. . 6. JAFFAY, H. The metabolism of serum proteins: Ill Kinetics of serum protein metabolism during growth. /. Biol. Chem. 285:2352, 1960. 7. McDoNALD, H. J. Area under peaks: Dropping of perpendiculars versus extending curves to baseline. In Ciba Foundati~ Symposium on Paper Electrophoresis, ed. by E. Wolstenholme, 1956, p. 149. . . . 8. MooRE, D. H. Species differences in seruni protein patterns. ]. Biol. Chem. 161:21, 1945. 9. PlLLIERO, S. J., and SToNE, M. L. Serum levels and electrophoretic patterns in pregnancy. Anat. Rec.189:311, 1961. 10. RoY, S. K., and LEATHEM, J. H. Reproduction and protein nutrition. Anat. Rec. 189: 269, 1961. 11. SMITH, I. Chromatographic and..Electrophoretic Techniques. Vol. 2: Zone Electrophoresis. Heinemann, London, 1960, p. 9. 12. STUDNITZ, W. voN Studies on serum proteins in pregnancy. Scand.]. Clin. & Lab. Invest. 7:324, 1955. · · · 13. WINZL,ER, R. J. Glycoproteins. Plasma Proteins 1:309, 1960.
A concentration during pregnancy in humans Extraction and electrophoretic has been reported by several authors. DECREASE IN SERUM PROTEIN
4 • 9 • 10
procedures indicated a decrease in total concentration due to a decline in the albumin fraction, although an increase occurred in the alpha-!, alpha-2, and beta globulins.9 Antionades, in a review of the literature concerned with circulating hormones, indicated that these substances were loosely attached or bound to the serum proteins and migrate with them in an electric field. The rate of fetal growth varies throughout pregnancy and maternal protein metabolism can be expected to show concomitant variations. This paper reports changes that occur in the levels of the serum protein fractions of pregnant hamsters. MAl'ERIALS AND METHODS
Sera from 10 female hamsters ( 5-10 months old) were used in each group. Blood was removed during diestrus in nonpregnant females and on Days 8, 12, and 15 of the 16-day gestation period, by cardiac puncture with the animals under ether anesthesia. Smith's modification of Owen's Veronal buffer (pH 8.6 and ionic strength of 0.078) was used throughout the investigaUon.11 Electrophoretic separation was carried out in an LKB electrophoresist apparatus using cellulose acetate strips,§ 5 X 8 em. Each strip received two 8-lambda samples of serum, 3 em. from the right edge. All electrophoretic runs were of 12 hours' duration at 225 v and at room temperature. Fast green-yellowish stain ( FG) 11 and periodic acid-Schiff (PAS) stain as modified by Bodman were used to localize the total protein From the Department of Biology, University of Oregon, Eugene, Ore. This study was aided by USPHS Grant RG 6156 (C1-2). *Present address: Department of Biology, Southern University in New Orleans, La. tPresent address: Department of Zoology, University of Georgia, Athens, Ga. ;t:LKB-Produkter, Stockholm 12, Sweden. §Oxoid Consolidated Laboratories, Chicago Heights, Ill.
486
VoL. 13, No.5, 1962
SERUM PROTEIN CoNcENTRATION
487
and glycoprotein fractions, respectively. The strips were then blotted, dried, and cleared in Whitemore oil No.120. These were read on a photovolt densitometer with the forward edge of each strip serving as its zeroing point with graphs prepared according to McDonald. 7 The resulting data were subjected to analysis by the variance test. RESULTS Nonpregnant Animals
With the FG stain, 5 electrophoretically distinct bands were demonstrated. By convention these are named in order of decreasing mobility as albumin and alpha-1, alpha-2, beta, and gamma globulins. The pattern obtained is similar to that reported for the hamster by Moore. Analysis of 10 strips showed an AjG ratio of 1.61 with albumin representing 61.9 per cent and globulins, 38.1 per cent. The major globulin in all strips was the alpha-1 fraction, which included 12.9 per cent of the total protein. Figure 1 is an example of the normal electrophoretic patterns and graphs. The PAS stain revealed 4 glycoprotein-containing fractions within the globulin area. Glycoproteins were not demonstrable in the albumin fraction. The alpha-1 globulins comprised 60.7 per cent of the total glycoproteins. Table 1 and Fig. 1 indicate the means of these data and an electrophoretic pattern of PAS-stained proteins. Pregnant Animals
Eighth Day. The FG technic showed the albumin diminishing to 58 per cent with globulins being augmented to 42 per cent. Alpha-1 and gamma TABLE 1. Protein Levels (Percentage) in Serum of Nonpregnant and Pregnant Hamsters 0 Globulins Albumin Group l
f 1
Notpregnant Pregnant 8 days Pregnant 12 days Pregnant 15 days
Staint
Mean
S.D.
FG
61.92
8.99
PAS
FG
58.08
PAS
FG
53.61
PAS
FG PAS
49.48
Alpha-1 Mean
12.92 60.73 6.01 16.44 61.48 5.52 20.01 56.95 3.61 28.42 74.02
S.D.
Alpha-2
Beta
Gamma
Mean
S.D.
Mean
S.D.
Mean
S.D.
3.89 8.81 4.25 18.67 2.85 8.41 6.38 17.00 2.93 8.42 3.51 18:98 4.66 7.47 7.60 13.83
2.86 5.28 2.99 3.26 2.15 2.23 3.52 3.72
6.34 7.69 4.67 6.55 3.64 6.81 3.14 5.53
3.17 5.28 1.36 2.26 0.98 1.47 0.87 2.12
9.95 12.87 12.35 17.93 14.39 15.91 11.46 11.91
2.20 4.80 2.71 5.19 1.31 4.63 3.87 6.84
*Means and S.D.'s are of 10 hamsters per group. tFG: Fast green-yellowish; PAS: Periodic acid Schiff.
488
PETERSON ET AL,
FERTILITY
& STERILITY
globulins increased while alpha-2 and beta globulins decreased. The AJG ratio dropped to 1.38. All changes were significant, with respect to nonpregnant females, at the 5 per cent level, as determined by an analysis of the variance.
Fig. 1. Electrophoretic strips from nonpregnant hamster serum proteins stained with fastgreen (top) and PAS (bottom). Graph is of O.D. readings, with dashed lines indicating PAS readings.
The PAS stain disclosed that alpha-! and gamma globulin glycopi'oteins had increased appreciably. Changes in the alpha-2 and beta globulin glycoproteins occurred, but these were not statistically significant.
Twelfth Day. The FG stain indicated a further diminution in the albumin fraction to 53.6 per cent while globulins rose to 46.4 per cent. The decrease in alpha-2 and beta globulins observed at this time was offset by greater increa~s in the alpha-! and gamma globulins. The AJG ratio continued to fall (tol.l5). Although alpha-! globulin glycoproteins had decreased to 56.9 per cent, this was offset by a greater percentage increase of gamma globulin glycoproteins. Alpha-2 and beta globulin glycoproteins showed no significant variations from those of nonpregnant or 8-day pregnant animals. Fifteenth Day. The albumin fraction continued decreasing and by Day 15 was 49.4 per cent. The relative proportion of globulins represented 50.6 per cent of the total. The AJG ratio was 0.97, indicating an inversion of that for the nonpregnant. The major protein changes were in the alpha-! globulin fraction, which had increased to nearly 125 per cent above normal values. Glycoproteins in the alpha-! globulin fraction increased after Days 12 and by Day 15 had attained a new high of 75 per cent. Beta-globulin glycoproteins decreased progressively during gestation and were 5.5 per cent of the total glycoproteins on Day 15. The means and standard deviations of all groups are shown in Table 1.
VoL. 13,No.5,1962
489
SERUM PROTEIN CONCENTRATION
70
Fig. 2. Serwn protein changes during gestation.
s at
·-
30
10
0
s
4
8 DAYS
12
15
OF GESTATION
1 .1.7
1
.,
.-
1.5
Fig. 3. Albumin-globulin ratios during gestation.
1.4 0
~1.3 a:
5
:c"' 1.2
r
r
tl
1.1
'·· 1.0
0.9
e OB0~--------4----------8---------~-2--------,5- DAYS
OF
GESTATION
490
PETERSON ETAL.
FERTILITY
& ·STERILITY
Figures 2 and 3 were derived from the da.;ta in Table 1 and show graphically the protein changes and AjG ratio changes during gestation. DISCUSSION
The progressive decline in the AjG ratio during pregnancy has been reported by Brown for the human; however, he did not report a change in alpha-1 globulin. Roy, after studying the effects of protein-free diets on pregnancy in the rat, concluded that paper electrophoresis indicated minor changes in serum proteins. Apparently, normal diets allow major changes in humans and hamsters. The changes seen in the alpha-1 globulins in hamsters are similar to those reported for humans in studies by Studnitz, in which alpha-1, alpha-2, and beta globulin levels were fortified. Engle and Woods reported similar results for the alpha-1 fraction. The glycoproteins in the alpha-1 globulin fraction have been reported to increase during pregnancy, neoplastic disease, and a number of other pathologic conditions in other species. 2 • 3 • 13 The significance of these variations is still a matter of conjecture. Serum protein variations during pregnancy are indicative of an increased metabolic turnover of these fractions. It has been suggested by Jaffay that such changes may result from the shunting of albumin synthesis into the synthesis of other body proteins. It was based on a study of the kinetics of albumin metabolism during growth. Albumin, according to this theory, can serve as the source of amino acids for the synthesis of body proteins either directly, without complete hydrolysis, or by contributing to the tissue aminoacid pool. Brown has advanced the idea that variations may also result from a progressive hemodilution during pregnancy. With the advent of fetal growth, quantitative and qualitative changes in the maternal proteins may reflect alterations in endocrine secretory output. It has been reported, for instance, that gonadotropins increase during pregnancy. These hormones, as well as adrenocorticotropins, thyroxine, insulin, and some steroids, are associated with the alpha globulins. 1 Bioassays of Cohn fractions prepared by cold ethanol fractionation have implicated these and other hormones with specific serum proteins. 1 • 5 • 13 It is suggested that the variations in serum proteins reported in this and other investigations may arise from greater or lesser hormonal output. SUMMARY
Serum from nonpregnant and pregnant hamsters was examined by cellulose acetate zone electrophoresis.
y
VoL.13, No.5, 1962
y
1. The albumin fraction decreases during gestation while globulins show a concurrent increase. The AjG ratio undergoes a reversal during late gestation as a result of a decrease in albumin and an increase in globulins. 2. Glycoproteins show variations during gestation, with the major change being a drop in alpha-! glycoproteins on Day 12 and a rise above nonpregnant values on Day 15 of a 16-day gestation period. 3. The relation between serum protein electrophoretic bands, endocrines, and metabolic activity was discussed. Further investigation into the correlation between these serum protein variations and pituitary secretions is anticipated.
~-
n n
•r n rs
SERUM PROTEIN CoNCENTRATION
Dept. of Biology University of Oreg011 Eugene, Ore.
h
Is ~r
1-
~r
)-
m
ln .t. 11~
n, :>f ;e le
LY
u-
491
REFERENCES I. ANTONIADEs, H. N. Circulating hormones. Plasma Proteins 2:105, 1960. 2. BERGSTRAND, C. G., and CzAR, B. Paper electrophoretic study of human fetal serum proteins with the demonstration of a new protein fraction. Scand..]. Clin. & Lab. Invest. 9:277, 1957. 3. BoDMAN, J. The separation of serum glycoproteins by continuous electrophoresis. Lab. Prac. Part 1:517, 1957. 4. BRoWN, T. Electrophoretic analysis of serum proteins in pregnancy; A preliminary study. ]. Obst. & Gynaec; Brit. Emp. 61:781, 1954. 5. ENGLE, R. L., and WooDs, K. R. Comparative biochemistry and embryology. Plasma Proteins 2:183, 1960. . 6. JAFFAY, H. The metabolism of serum proteins: Ill Kinetics of serum protein metabolism during growth. /. Biol. Chem. 285:2352, 1960. 7. McDoNALD, H. J. Area under peaks: Dropping of perpendiculars versus extending curves to baseline. In Ciba Foundati~ Symposium on Paper Electrophoresis, ed. by E. Wolstenholme, 1956, p. 149. . . . 8. MooRE, D. H. Species differences in seruni protein patterns. ]. Biol. Chem. 161:21, 1945. 9. PlLLIERO, S. J., and SToNE, M. L. Serum levels and electrophoretic patterns in pregnancy. Anat. Rec.189:311, 1961. 10. RoY, S. K., and LEATHEM, J. H. Reproduction and protein nutrition. Anat. Rec. 189: 269, 1961. 11. SMITH, I. Chromatographic and..Electrophoretic Techniques. Vol. 2: Zone Electrophoresis. Heinemann, London, 1960, p. 9. 12. STUDNITZ, W. voN Studies on serum proteins in pregnancy. Scand.]. Clin. & Lab. Invest. 7:324, 1955. · · · 13. WINZL,ER, R. J. Glycoproteins. Plasma Proteins 1:309, 1960.