all animals ( charles river , yokohama , japan ) were housed under controlled temperature ( 25  
 1c ) and 12 hr : 12 hr light : dark conditions ( lights on at 07:00 hr ) with food and water 
 available ad libitum .
all of the procedures were performed in accordance 
 with the guidelines for animal care stipulated by the japanese physiological society and 
 approved by the ethics committees of miyazaki university .
all efforts were made to minimize 
 animal pain and suffering , and the number of animals used . in the first set of experiments , adult female wistar rats ( approximately 10 weeks old ) were 
 used . from 1 week
prior to sample collection , vaginal smears were taken daily from the rats 
 for determination of the estrous cycle , and accordingly , the animals were subdivided into 4 
 groups : diestrus ( n=9 ) , proestrus ( n=7 ) , estrus ( n=9 ) and metestrus ( n=16 ) .
we showed the plasma amino acid 
 concentration with supplemental table , and the percentage concentration ratios for the 
 various amino acids were also presented as circular profiles relative to the amino acid 
 concentrations of diestrus . in the next experiment
, adult female rats were mated on the day 
 of proestrus , and the day after mating was counted as day 1 of pregnancy ( p1 ) .
blood samples 
 were taken every 5 days ( p5 , 10 , 15 and 20 ; n=4 ) .
after delivery and during lactation , blood 
 samples were taken from the dams in the same way ( l1 , 5 , 10 , 15 and 20 ; n=4 ) , and we take 
 the samples from the female pups just after weaning ( n=6 ) . the percentage concentration 
 ratios for the various amino acids were also presented as circular profiles relative to the 
 concentrations determined just before pregnancy ( proestrus ) .
all blood samples were collected into heparinized capillary tubes by the tail tip incision 
 method , and these were centrifuged immediately at 4c and 14,000 rpm for 4 min .
we think 
 that the possibility of contamination of amino acids derived from erythrocyte by hemolysis 
 is almost none , since we paid scrupulous attention not to hemolyze at the blood sampling and 
 hemolyzed sample was excluded from the assay .
the resulting plasma samples were mixed with 2 
 volumes of 5% ( w / w ) trichloroacetic acid and centrifuged immediately at 4c and 10,000 rpm 
 for 20 min to remove the precipitated protein .
ufc5010bk , millipore , billerica , ma , u.s.a . ) , and the plasma 
 amino acid concentrations were measured using an automatic amino acid analyzer ( l-8800a ; 
 hitachi , tokyo , japan ) .
we focused on the 20 amino acids that are the components of 
 proteins : valine ( val ) , leucine ( leu ) , isoleucine ( ile ) , alanine ( ala ) , methionine ( met ) , 
 proline ( pro ) , tryptophan ( trp ) , phenylalanine ( phe ) , tyrosine ( tyr ) , threonine ( thr ) , 
 glutamine ( gln ) , asparagine ( asn ) , serine ( ser ) , glycine ( gly ) , cysteine ( cys ) , lysine 
 ( lys ) , arginine ( arg ) , histidine ( his ) , aspartic acid ( asp ) and glutamic acid ( glu ) .
briefly , amino acids were separated by cation - exchange chromatography and detected 
 spectrophotometrically after post - column reaction with ninhydrin reagent .
all sample 
 collections were performed at the same time ( 11:3012:30 hr ) to minimize any influence of 
 circadian rhythm .
all data were expressed as the mean  standard error of the mean ( sem ) . in the first 
 experiment ,
differences between groups at the various stages of the estrous cycle were 
 analyzed by one - way anova and post hoc tukey s test . in the other 
 experiment ,
data for amino acid levels were analyzed by one - way repeated anova , and changes 
 between time points during the experimental period were analyzed by tukey s test . the sexual 
 difference ( fig .
3d ) were analyzed by student s t - test . to visualize the results , the concentrations of amino acids among the experimental groups 

for the 2-dimensional circular plane figures , 
 we plotted the percentage concentration of each amino acid relative to the control level ( at 
 diestrus or proestrus ) . in the three - dimensional figures , principal components analysis 
 ( pca )
was performed to reduce the dimensionality of the data set and to identify new 
 meaningful underlying variables . to obtain a 
 more accurate result in the latter analysis
, we used 40 kinds of amino acids and their 
 metabolic intermediates , including taurine , sarcosine , citrulline , ornithine and 
 hydroxyproline , for pca analysis .
the actual concentrations of the various amino acids in plasma are shown in tables 1table 1alterations in plasma amino acid concentrations during the estrous cycle in 
 female rats and age - matched male ratsamino acids(mol
/ l)diestrus(n=9)proestrus(n=7)estrus(n=9)metestrus(n=16)age - matched male(n=4)valine175.8  6.2182.0  12.1168.9  5.6189.6  4.3157.8  4.8leucine124.4  3.8129.3  7.5126.0  4.0135.2  4.6110.5  4.2isoleucine72.8  2.077.7  5.174.7  2.378.9  1.465.9  3.4alanine408.9  10.7459.5  27.3453.2  24.7444.0  13.4349.1  31.2*methionine66.6  3.363.4  4.964.1  1.774.5  2.956.3  3.4proline223.7  8.9217.6  17.0215.1  9.1238.4  5.9170.5  6.5*tryptophan141.9  6.8129.9  7.0130.1  6.3145.3  3.6106.1  7.4*phenylalanine67.0  2.171.4  4.170.9  2.073.0  1.866.7  1.8tyrosine67.6  2.574.4  5.669.3  2.874.6  3.091.2  4.7*threonine320.4  18.0357.3  40.1332.4  25.5363.9  15.4265.5  5.0glutamine650.5  30.4637.5  25.6619.1  20.5689.8  18.4556.3  16.6asparagine51.0  3.248.2  4.244.4  2.153.1  2.244.5  2.8serine261.6  11.2275.4  19.4267.8  9.8283.9  9.8197.5  7.9*glycine281.1  13.1283.1  17.5263.8  7.9282.3  8.8255.2  3.8cysteine1.5  0.41.4  0.41.7  0.71.4  0.3ndlysine491.2  19.2479.6  30.4461.9  21.9521.9  20.7336.8  21.5*arginine178.5  12.6176.5  4.8196.5  9.0251.7  28.1125.8  14.0*histidine58.3  2.359.8  3.157.0  1.662.3  1.851.3  0.6aspartate10.8  1.711.8  1.312.7  1.714.6  1.67.0  1.0glutamate91.3  8.286.4  7.894.3  6.999.7  7.681.6  13.4the data represent the mean  standard error of the mean ( sem , n=716 ) . * ; 
 p<0.05 vs. diestrus by student s t - test . , 2table 2alterations in plasma amino acid concentrations during pregnancyamino acids ( mol / l)proestrus ( p0)p5p10p15p20valine172.3  17.9247.3  83.1305.8  85.3211.1  61.1201.1  64.6leucine123.6  11.0124.5  6.0119.6  4.0107.5  2.6103.1  9.6isoleucine73.3  7.674.9  3.671.6  2.266.3  1.068.5  7.4alanine414.3  26.3361.6  14.6348.2  15.7373.8  49.7510.6  33.6methionine59.3  4.360.3  4.167.5  4.457.8  5.654.0  3.2proline191.6  12.3186.3  13.0171.7  8.8175.2  12.6184.1  9.1tryptophan119.3  6.2100.6  1.2106.2  6.5101.8  4.362.4  5.5*phenylalanine67.8  3.661.1  2.364.5  3.258.8  3.159.8  4.9tyrosine74.8  5.264.0  3.274.4  6.751.9  5.8 * 50.3  4.9*threonine276.0  16.1235.1  13.9240.6  21.0268.7  13.8292.1  23.9glutamine601.1  11.4614.6  20.7623.2  36.4631.1  39.5598.8  24.3asparagine43.1  5.040.1  1.637.4  1.637.7  2.550.9  3.2serine246.4  17.6230.7  6.2207.5  5.3 * 202.8  8.8 * 221.8  6.3glycine272.2  21.2259.5  9.3232.2  14.0158.1  7.9 * 127.6  3.6*cysteinendndndndndlysine435.8  19.8422.1  48.3418.6  31.2527.4  30.9623.4  60.3*arginine160.8  20.0174.6  9.4166.2  4.4162.1  24.8140.4  8.7histidine59.3  4.658.9  1.557.8  3.144.8  3.2 * 36.6  2.1*aspartate11.9  2.310.3  2.18.3  0.89.2  1.19.0  0.6glutamate87.6  12.582.8  15.478.1  10.480.2  10.183.3  10.6the data represent the mean  standard error of the mean ( sem , n=4 ) . * ; 
 p<0.05 vs. non - pregnant female rats group . , 3table 3alterations in plasma amino acid concentrations during lactation in dams and 
 the pups just after weaningamino acids(mol / l)proestrus ( p0)l1l5l10l15l20pupsvaline172.3  17.9130.5  32.2154.6  12.0182.8  1.4168.7  9.8129.2  24.4206.8  10.2leucine123.6  11.097.5  10.9115.6  8.4133.6  3.5129.6  6.5117.7  18.2146.5  8.8isoleucine73.3  7.666.1  4.273.6  5.985.5  1.679.8  4.074.2  11.297.0  3.8alanine414.3  26.3739.3  50.2 * 621.8  41.0585.1  35.2608.9  61.9572.1  44.2738.9  33.5methionine59.3  4.370.0  6.682.5  3.7 * 98.7  4.2 * 99.3  5.3 * 90.5  6.4 * 86.1  2.7proline191.6  12.3288.6  27.7 * 268.7  18.7 * 259.0  8.9 * 268.7  14.9 * 239.4  15.3358.2  39.7tryptophan119.3  6.2106.1  12.8113.3  2.5118.1  7.2115.4  9.9113.7  8.999.5  4.9phenylalanine67.8  3.666.3  4.867.7  4.770.9  2.171.8  6.465.0  6.476.4  5.6tyrosine74.8  5.285.0  8.387.4  10.681.9  6.384.4  7.569.0  9.8148.1  17.3threonine276.0  16.1386.4  60.6390.9  21.2377.4  20.5347.2  17.1316.8  23.4219.5  12.7glutamine601.1  11.4818.9  40.7 * 704.7  43.1711.7  37.1752.6  35.3 * 688.8  25.6626.0  46.6asparagine43.1  5.090.5  9 * 71.4  2 * 84.3  3.1 * 91.7  1.9 * 76.5  2.2 * 75.7  12.9serine246.4  17.6422.5  29.7 * 372.0  30.4 * 366.3  12.4 * 353.0  12.1 * 321.4  15.1300.6  24.0glycine272.2  21.2400.6  34 * 421.6  41.6 * 465.5  20.6 * 461.7  22.7 * 381.3  14.7613.2  25.6cysteinendndndndndndndlysine435.8  19.8686.5  100.8 * 521.1  34.9550.1  30.8489.6  2.6459.8  32.2232.9  16arginine160.8  20213.8  20.8196.5  17.3194.9  8.6182.5  5.5178.7  13.9240.2  26.6histidine59.3  4.656.3  4.566.2  3.378.6  3.4 * 72.3  3.263.2  6.7119.4  7.7aspartate11.9  2.311.4  2.013.4  5.85.5 
1.47.2  0.76.8  1.216.9  2.2glutamate87.6  12.576.8  1.793.1  16.766.1  5.865.7  4.871.1  6.6119.3  7.8the data represent the mean  standard error of the mean ( sem , n=4 , 6 ) . * ; 
 p<0.05 vs. non - pregnant female rat group , ; 
 p<0.05 vs. dams on day 20 of lactation group .. to compare the types of amino acid changes occurring during the estrous 
 cycle , pregnancy or lactation , we displayed the ratios of the respective amino acids at each 
 stage in the form of circular profiles relative to the corresponding values for diestrus or 
 proestrus .
the data represent the mean  standard error of the mean ( sem , n=716 ) . * ; 
 p<0.05 vs. diestrus by student s t - test .
the data represent the mean  standard error of the mean ( sem , n=4 ) . * ; 
 p<0.05 vs. non - pregnant female rats group .
the data represent the mean  standard error of the mean ( sem , n=4 , 6 ) . * ; 
 p<0.05 vs. non - pregnant female rat group , ; 
 p<0.05 vs. dams on day 20 of lactation group .
the levels of the individual amino acids in plasma were almost the same among proestrus , 
 estrus , metestrus and diestrus , and their circular profiles showed no significant 
 differences ( fig .
1.alterations in plasma amino acids concentrations ( a  c , f ) , body weight ( d ) and daily 
 food intake ( e ) at various stages of the estrous cycle in female rats and also in 
 age - matched male rats .
the concentrations of individual plasma amino acids are shown 
 as a percentage ratio relative to the concentrations in diestrus females . a : results 
 for proestrus ( red line ) , b : estrus ( yellow line ) , c : metestrus ( green line ) and f : 
 age - matched males ( blue line ) . the data for body weight and food intake represent the 
 mean  standard error of the mean ( sem ) .
d , diestrus ( white bar ) ; p , proestrus ( black 
 bar ) ; e , estrus ( gray bar ) ; m , metestrus ( diagonal bar ) .
amino acids indicated by red 
 shading and asterisks showed a significant difference vs. diestrus females 
 ( p<0.05 ) . , table 1 ) . on the other hand , the shapes of the circular profiles 
 changed during the course of pregnancy ( fig .
2.temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight 
 ( b ) and food intake ( c ) during pregnancy in female rats ( n=4/group ) .
the 
 concentrations of individual plasma amino acids are shown as a percent ratio relative 
 to the concentrations in non - pregnant female rats ( proestrus ; n=4 , black line ) .
amino acids indicated by red shading and asterisks 
 showed a significant difference vs. non - pregnant females 
 ( p<0.05 ) . , 
 table 2 ) .
the plasma ser level decreased significantly in mid and late ( p10 and 15 ) 
 pregnancy , and tyr , gly and his decreased significantly at the late and end stages ( p15 and 
 20 ) of pregnancy , whereas trp and lys significantly decreased and increased at the end of 
 pregnancy , respectively .
alterations in plasma amino acids concentrations ( a  c , f ) , body weight ( d ) and daily 
 food intake ( e ) at various stages of the estrous cycle in female rats and also in 
 age - matched male rats .
the concentrations of individual plasma amino acids are shown 
 as a percentage ratio relative to the concentrations in diestrus females .
a : results 
 for proestrus ( red line ) , b : estrus ( yellow line ) , c : metestrus ( green line ) and f : 
 age - matched males ( blue line ) . the data for body weight and food intake represent the 
 mean  standard error of the mean ( sem ) .
d , diestrus ( white bar ) ; p , proestrus ( black 
 bar ) ; e , estrus ( gray bar ) ; m , metestrus ( diagonal bar ) .
amino acids indicated by red 
 shading and asterisks showed a significant difference vs. diestrus females 
 ( p<0.05 ) .
temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight 
 ( b ) and food intake ( c ) during pregnancy in female rats ( n=4/group ) .
the 
 concentrations of individual plasma amino acids are shown as a percent ratio relative 
 to the concentrations in non - pregnant female rats ( proestrus ; n=4 , black line ) .
amino acids indicated by red shading and asterisks 
 showed a significant difference vs. non - pregnant females 
 ( p<0.05 ) .
much larger changes in the circular profiles of plasma amino acids were observed during the 
 lactation period ( fig . 

3.temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight 
 ( b ) and food intake ( c ) during lactation in female rats ( n=4/group ) .
the 
 concentrations of individual plasma amino acids are shown as a percentage ratio 
 relative to the concentrations in non - pregnant females ( proestrus ; n=4 , black line ) .
d 
 shows the results of the pups just after weaning ( n=6 , blue line ) compared with the 
 dams on the day 20 of lactation ( n=4 , red line ) .
amino acids indicated by red shading and asterisks showed a significant difference vs. 
 each control group ( p<0.05 ) . , table 3 ) : many amino acids showed significant increases , and none showed a significant 
 decrease .
plasma pro , ser and gly levels increased continuously from day 1 until day 15 of 
 lactation .
the levels of asn and met increased significantly from days 1 and 5 respectively 
 until the end of lactation , ala and lys were increased significantly only on day 1 of 
 lactation , his was increased on day 10 , and gln was increased on days 1 and 15 .
3d shows the results of the pups just after 
 weaning compared with the dams on day 20 of lactation .
many amino acids ( val , ala , pro , tyr , 
 gly , his , asp and glu ) showed significant increases , and only 2 amino acids ( thr and lys ) 
 were decreased .
temporal alterations in plasma amino acid concentrations ( a , red line ) , body weight 
 ( b ) and food intake ( c ) during lactation in female rats ( n=4/group ) .
the 
 concentrations of individual plasma amino acids are shown as a percentage ratio 
 relative to the concentrations in non - pregnant females ( proestrus ; n=4 , black line ) .
d 
 shows the results of the pups just after weaning ( n=6 , blue line ) compared with the 
 dams on the day 20 of lactation ( n=4 , red line ) .
amino acids indicated by red shading and asterisks showed a significant difference vs. 
 each control group ( p<0.05 ) .
pca analysis revealed clear differences in the 3d plots among the groups ( fig . 4a and 4bfig .
4.principal component analysis ( pca ) plots from amino acid concentration data of 
 individuals ( a ) and median ( b ) in each experiment .
black plot ; male rats age - matched 
 with females in the estrous cycle experiment , blue plot ; estrous cycle experiment , red 
 plot ; pregnancy period and green plot ; lactation period . ) . on the pca plot , we added the plasma amino acid levels for an age - matched male 
 group ( n=4 ) to confirm whether or not a sex difference was evident .
the positions of each 
 sample were plotted against the axes of the first three components ( pc1 , pc2 and pc3 ) in a 
 3d space and were colored according to each experimental group ( black plot ; male rats 
 age - matched with the females used in the estrous cycle experiment , blue plot ; estrous cycle 
 experiment , red plot ; pregnancy period and green plot ; lactation period ) .
pca analysis 
 allowed visual identification of data patterns and highlighted similarities and differences 
 among the reproductive stages in female rats .
the dispersion of the points assembled in each group . principal component analysis ( pca ) plots from amino acid concentration data of 
 individuals ( a ) and median ( b ) in each experiment .
black plot ; male rats age - matched 
 with females in the estrous cycle experiment , blue plot ; estrous cycle experiment , red 
 plot ; pregnancy period and green plot ; lactation period .
presentation of the amino acid profiles in a 2-dimensional circular plane , using the levels 
 of amino acids during diestrus or proestrus as controls , demonstrated that the profiles 
 changed during pregnancy and lactation in dams , but not during the estrous cycle . in 
 addition , 3-dimensional pca analysis revealed a different distribution of plasma amino acid 
 levels among reproductive stages or sex , especially in late pregnancy and early lactation in 
 dams .
these observations suggest that the levels of amino acids in female rats differ from 
 those in males and that they change according to reproductive stage .
generally , it has been considered that protein metabolism differs between males and 
 females , since the sexes differ considerably in muscle mass and energy consumption . some of these differences may be correlated with sex 
 steroid hormones : testosterone and estrogen stimulate and inhibit protein synthesis , 
 respectively [ 14 , 27 ] .
also , it has been reported that ovariectomy causes an increase of bcca and a 
 decrease of ala in plasma .
changes in the circular amino acid profiles in dams during pregnancy or lactation may 
 result from transitional changes in the plasma levels of steroid .
on the other hand , in rats 
 during the estrous cycle , there were no significant differences in the circular profiles , 
 even though plasma steroid hormone levels do vary during the cycle .
although the reason for 
 this unexpected result is unknown , it may be results of short term change of steroid levels 
 in estrus cycle .
another possibility is that pregnant or lactating dams have a higher 
 nutritional demand and food intake in comparison with normal rats .
the present study also demonstrated an increase of food intake in 
 pregnant and lactating dams . in spite of the increase of food intake during pregnancy , most of the significant changes 
 in plasma amino acid levels were falls , rather than increases .
in addition , most of the 
 amino acids that showed reduction during pregnancy were glycogenic amino acids .
in pregnant 
 rats , increase of insulin and ketone body , and decrease of glucose are observed in 
 peripheral blood [ 5 , 13 ] .
therefore , decrease of glycogenic amino acid during pregnancy may be due to 
 promoting the gluconeogenesis for supply of glucose to the fetus .
another possible reason 
 for the reduction in many plasma amino acid levels may have been an increase in the plasma 
 progesterone level .
it has been reported that administration of progesterone to humans 
 causes a marked reduction of many amino acids in plasma . however , if progesterone is the main reason for the decrease of amino acids , 
 this does not explain why many amino acids showed increases in their levels in lactating 
 dams , whose plasma progesterone level is high .
the only amino acid that showed an increased level in plasma at the end of pregnancy was 
 lys ( a ketogenic amino acid ) .
although the reason for this is unclear , lys is utilized for 
 acetyl - coenzyme a , some of which is employed for cholesterol synthesis .
furthermore , it has 
 been reported that dietary lys and arg are related to plasma cholesterol levels and that a 
 decrease in the ratio of arg to lys is associated with an increase in the plasma cholesterol 
 level .
cholesterol may be necessary for the 
 rapid increase in the level of estrogen at the end of pregnancy for initiation of 
 lactation .
in contrast with pregnant dams , all of the amino acids that showed significant changes in 
 lactating dams increased .
all of these , except for lys and met , were non - essential and 
 glycogenic amino acids . during the lactation in comparison with pregnancy ,
therefore , these non - essential and glycogenic amino acids may be for 
 supplying sufficient glucose or amino acid to product the milk in the mammary gland .
the 
 increase in the lys level on the initial day of lactation may have been due to the increase 
 observed at the end of pregnancy .
ala was significantly increased in early lactation ( l1 ) 
 and showed a tendency to increase as lactation proceeded .
it has 
 also been reported that the expression of amino acid transporter ( aat ) and lipid 
 synthesis - related genes in the mammary gland is increased more during lactation than during 
 pregnancy in the mouse .
in addition , the amino 
 acid concentrations in secreted milk during lactation are different from those of maternal 
 plasma . in the fetus immediately before 
 delivery , amino acid levels are known to be higher than in the mother . on the basis of these observations
, we expected that the plasma 
 amino acid levels in rats just after weaning might be higher than in mature rats , and 
 measured the plasma amino acid levels in pups just after weaning . as expected , many kinds of 
 amino acid in plasma showed levels markedly higher than in the dam ( fig .
it might be generally believed that the increase of food 
 intake results in increase of amino acid levels in blood .
certainly , increase of food intake 
 brings about increase of plasma amino acid levels in hyperphagic animals like the ob / ob 
 mouse .
however , this correlation between food 
 intake and plasma amino acid levels did not apply to pregnant rats , since main change in 
 amino acid level in pregnant rats was decrease , but not increase , nevertheless , food intake 
 increased . in lactating dam , however , some amino acids concomitant with food intake 
 increased in the present study also . but , the rises of bcaa were not recognized in lactating 
 dam .
therefore , we supposed that the plasma amino acid levels in lactating dam were 
 influenced by not only food intake , but also metabolic change . in the present study , we showed that imbalance of amino acid profiles strongly reflects the 
 metabolic changes occurring in vivo and that such data would be applicable 
 to various fields , such as diagnosis , treatment with infusion to patient , nutrition for the 
 pregnant women and production of artificial milk .
 we measured the plasma levels of amino acids at various reproductive stages in female 
 rats , including the estrous cycle , pregnancy and lactation , and compared the resulting 
 amino acid profiles using two- or three - dimensional figures . 
 these figures revealed that 
 the amino acid profiles of pregnant and lactating dams differed considerably from those 
 during the estrous cycle or in male rats . 
 the plasma levels of individual amino acids were 
 almost the same between proestrus , estrus , metestrus and diestrus , and their profiles did 
 not differ significantly . 
 however , the amino acid profiles changed during pregnancy and 
 lactation in dams . 
 the plasma ser level decreased significantly in mid and late pregnancy , 
 whereas tyr , gly and his decreased significantly in the late and end stages of pregnancy , 
 and trp and lys significantly decreased and increased at the end of pregnancy , 
 respectively . 
 much larger changes in amino acid profiles were observed during lactation , 
 when the levels of many amino acids increased significantly , and none showed a significant 
 decrease . 
 plasma pro , ser and gly levels increased continuously from day 1 until day 15 of 
 lactation , whereas asn and met increased significantly from days 1 and 5 respectively 
 until the end of lactation . 
 these results suggest that the profiles of plasma amino acids 
 show characteristic changes according to reproductive stage and that it may be necessary 
 to consider such differences when performing amino acid - based diagnosis . 