trans fatty acids ( tfas ) are created from vegetable oils through hydrogenation . in the trans configuration ,
the carbon chain extends from opposite sides of the double bond , rendering a straight molecule .
elaidic acid ( ea ; c18:1 ) is the principal tfa often found in partially hydrogenated vegetable oils , margarine , and shortening [ 1 , 2 ] .
young people in japan have come to prefer a western - style diet , and now more than 29% of the calories consumed by japanese people are provided by fat .
the consumption of trans fats increases the risk of coronary heart disease ( chd ) by raising the levels of ldl cholesterol and lowering the levels of hdl cholesterol [ 2 , 4 , 5 ] .
it has been reported that an elevated tfa blood cell content increased the risk of acute coronary syndromes ( acs ) [ 6 , 7 ] .
infants are exposed to tfa before and after birth by the transfer of fatty acids originating from the maternal diet . on average ,
mature human milk provides 3.7  g  fat/100  ml , representing about 50% of the dietary energy intake of the young infant [ 10 , 11 ] .
infants receiving human milk ingest levels of tfas and essential fatty acids that reflect short - and long - term maternal diets [ 1214 ] .
recent studies have indicated that the ea content of the maternal diet may be associated with both maternal and infant body composition in the early postpartum period .  
we previously reported that dietary fish oil intake during pregnancy and lactation provides more n-3 polyunsaturated fatty acids ( pufa ) to pups and alters their brain fatty acid composition .
however , it has been reported that prenatal essential fatty acid deficiency may result in myelin with an abnormal composition and structure , at least during the critical period of brain development .
trans - enriched isomers of -linolenic acid ( ala ) were fed to female adult rats during gestation and lactation .
the trans ala can be desaturated and elongated in vivo and thus provides trans isomers of the n-3 fatty acids eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , even in pup brain structures .  
animal studies have demonstrated that brain dha concentrations reduced after relatively high intakes of ea .
thus , a dam 's dietary ea intake during pregnancy and lactation may affect the development of the brain of her pups .
although trans ala was reportedly absorbed in the rat brain , there are no reports on the effects of dietary ea on the brain fatty acid composition of pups , to the best of our knowledge .
the aim of the present study was to determine whether a dam 's dietary ea intake during pregnancy and lactation would affect the plasma , erythrocyte , and brain fatty acid composition in pups .
we investigated the effects of the maternal ingestion of a shortening diet ( shd ) and a soybean oil diet ( sod ) on 82-day - old offspring .
body and fat tissue weights as well as plasma lipid concentrations were alsomeasured , as was the growth of the pups .
ten sperm - positive pregnant rats of the sprague - dawley strain on day 3 of pregnancy ( 10 weeks old ) were commercially obtained from clea japan ( tokyo ) .
they were housed individually in plastic cages with paper chip ( alpha - dritm , shepherd specialty papers , chicago , il ) bedding .
they were maintained in a room kept at a constant temperature ( 22  1c ) and 50% relative humidity with a 12  h dark / light cycle ( 19:007:00 ) .
they were weighed , and their food intake was measured every day between 16:00 and 18:00 during the experimental period .
the diets were prepared by adding soybean oil ( 10% ) and shortening ( 10% ) , respectively , to the ain-93 g , substituting for cornstarch .
soybean oil , the triglyceride e - test wako , the cholesterol e - test wako , and other chemicals were purchased from wako pure chemical industries ( osaka ) .
ten pregnant rats ( on day 3 of pregnancy ) were divided into two equal groups .
one group was fed the sod and the other group the shd during the experimental period ( pregnancy and lactation ) . within 24  h of birth ,
each litter was culled to 10 pups ( 5 males and 5 females ) and nursed by their dams for 21  d. on days 8 and 21 after birth , two pups ( a male and a female ) from each litter were killed by decapitation and their blood was collected .
whole blood was centrifuged for 15  min at 1400  g , and plasma was aliquoted into microcentrifuge tubes .
the buffer layer of white blood cells was removed using a pasture pipette , and then the erythrocytemembrane was aliquoted into separate microcentrifuge tubes .
perirenal fat tissue and liver were removed and weighed . after weaning ( at 21  d of age )
the pups were fed the same diet regardless of group . on days 47 , 68 , and 82 after birth ,
the pups were fasted for 16  h and sacrificed without affliction under ether anesthesia for collection of the liver , perirenal fat tissue , and brain .
the plasma , liver , and brain were stored at 80c until they were analyzed .
all procedures were performed in accordance with the animal experimentation guidelines of the laboratory animal care committee of seitoku university .
triacylglycerol ( tg ) and total cholesterol ( t - cho ) concentrations in plasma as well as liver extracts were measured using test kits ( triglyceride e - test wako , cholesterol e - test wako ) .
packed erythrocytemembranes were diluted in 500  l of water and thoroughly mixed by vortex , and the mixture was kept on ice for 15  min . to initiate the lipid extraction , isopropanol ( 4  ml )
was added , and the samples were vigorously mixed by vortex and kept for 1  h with frequent mixing .
then hexane ( 6  ml ) was added , followed by frequent mixing for another hour .
the samples were subjected to centrifugation at 1200  g for 10  min , and the lipid - containing organic fraction was removed .  
fatty acid in the plasma , erythrocytemembrane , and brain extract was methylated according to the procedure of morrison and smith .
first , 1.5  ml of 0.5  mol naoh dissolved in methanol was added to 0.2  ml of plasma , and the solution was subjected to vortex mixing and then boiled in a water bath at 100c for 9  min .
next , 2  ml of boron trifluoride methanol complex methanol solution was added to the mixture , followed by vortex mixing and incubation in a water bath at 100c for 7  min . after cooling ,
n - hexane ( 3  ml ) was added to the mixture , which was shaken well . the saturated nacl solution ( 5  ml )
was added to the mixture during vortex mixing , followed by centrifugation at 1500  g for 10  min .
the n - hexane phase containing the fatty acids methyl esters ( fames ) was extracted .
the fames were analyzed by a hitachi g-3500 gas chromatograph ( gc ) fitted with a split injector ( 250c ) and a flame ionization detector ( 260c ) coupled with a chromatointegrator ( hitachi d-2500 ) .
we used a tc-70 column ( 0.25  mm i d  60  m length , 0.2  m film thickness ; gl sciences , tokyo ) , with a helium ( he ) carrier gas flow rate of 1  ml / min .
the column was isothermally kept at 190c for 15  min , heated from 190c to 200c at a heating rate of 5c / min , kept at 200c for 10  min , heated from 200c to 210c at a heating rate of 5c / min , and kept at 210c for 4  min .
each fame peak was identified with a standard fame ( funakoshi co. , tokyo ) .
during pregnancy and lactation , no significant difference in the total food intake of the dams was observed between the sod and shd groups .
no significant difference in body , liver , or perirenal fat tissue weights was observed between the two groups ( table 3 ) .
no significant difference was found in total litter size between the groups ( 13.2  2.49 and 11.0  4.64 ) .
the male and female litter sizes in the sod group were 7.00  2.24 and 6.20  1.10 , respectively , whereas those in the shd group were 6.20  1.10 and 4.60  2.19 , respectively .
therefore , the two maternal diets did not affect the litter size or the ratio of female versus male pups . within 24  h after birth ,
the pups nursed by dams fed the sod and shd weighed 7.40  0.18  g and 7.27  0.34  g , respectively , indicating no significant difference between the groups . no significant differences in plasma tg or t - cho concentrations in the dams
table 4 shows the fatty acid composition of stomach content in 8-day - old pups nursed by dams fed sod and shd .
the level of saturated fatty acids in the stomach content was the highest in both groups , and no significant difference was observed between the groups .
the monounsaturated fatty acid in the stomach content of the shd group was significantly higher than that of the sod group ( p < 0.05 ) .
the n-6 and n-3 fatty acids in the stomach content of the sod group were significantly higher than those of the shd group ( p < 0.01 ) .
the ea in the stomach contents of the shd group was 11.6  1.03 , but ea was not detected in the stomach contents of the sod group .
the ratios of n-6/n-3 of the sod and shd groups were 15.1 and 54.7 , respectively . on days 8 , 21 , 47 , 68 , and 82 after birth ,
no significant differences in body , liver , or perirenal fat tissue weights were observed between the groups ( table 5 ) .
the liver and perirenal fat tissue weights of the pups of both groups increased with growth .   on days 8 and 21 after birth ,
the plasma tg and t - cho concentrations in the pups of both groups were high but decreased after weaning ( table 6 ) .
no significant difference in plasma tg concentration was observed between the groups . on day 21 after birth ,
the plasma t - cho concentration of the shd group was significantly lower than that of the sod group ( p < 0.05 ) .
however , on day 47 after birth , no significant difference in the plasma t - cho concentration was observed between the groups .
on days 8 and 21 after birth , the saturated , n-6 , and n-3 fatty acids were significantly higher in the plasma of the sod group than in that of the shd group ( p < 0.05 ) ( table 7 ) .
on the other hand , the monounsaturated fatty acid in plasma was significantly higher in the shd group than in the sod group ( p < 0.05 ) .
ea was detected in the plasma of the shd group but not in that of the sod group .
the ea in the plasma of the shd group on day 21 after birth was significantly higher than that on day 8 after birth ( p < 0.05 ) . on day 8 after birth , compared with the fatty acid composition of the stomach contents and the plasma , the saturated fatty acid in plasma was decreased in both groups , whereas the n-6 and n-3 fatty acids were significantly increased in the plasma of both groups ( p < 0.05 ) .
although no significant difference in the saturated fatty acid in the erythrocyte membrane of the pups on days 8 and 21 after birth was observed between the groups , the n-6 and n-3 fatty acids in the erythrocyte membrane were significantly higher in pups of the sod group than in those of the shd group ( p < 0.05 ) ( table 7 ) . on days 8 and 21 after birth ,
the monounsaturated fatty acid in plasma and blood cell membrane was higher in the shd group than in the sod group ( p < 0.05 ) ( table 7 ) .
the ea in the erythrocyte membrane of the shd group was significantly higher on day 21 after birth than on day 8 after birth ( p < 0.05 ) .
in the shd group on days 8 and 21 after birth , the ea was significantly higher in plasma than in erythrocyte membrane , and n-3 fatty acid levels were significantly higher in erythrocyte membrane than in plasma ( p < 0.05 ) . the brain weight ( g/100  g  bw ) of pups of both groups decreased significantly with growth ( p < 0.05 ) ( table 8) . the total fat ( mg / g brain ) in the pup brains of both groups increased significantly with age ( p < 0.05 ) .
however , on days 8 , 21 , and 82 after birth , no significant differences in the brain weight and total fat were observed between the groups .
although no significant difference was found between the groups in the level of saturated fatty acid in the brains of pups on day 8 after birth , on day 21 the shd group had significantly higher levels than the sod group ( p < 0.05 ) .
although the level of monounsaturated fatty acid in the brains on day 8 after birth was higher in the shd group than in the sod group ( p < 0.05 ) , no significant differences were observed between the groups at 21 and 82 days after birth .
the monounsaturated fatty acid levels in the brains of pups of both groups decreased with growth ( p < 0.05 ) ( table 8) . although no significant difference between the groups was observed in the n-6 fatty acid in brains of pups on day 8 or day 21 after birth , on day 82 the shd group had higher levels than the sod group ( p < 0.05 ) .
although on days 8 and 21 after birth n-3 fatty acid levels were lower in the brains of the shd pups than in those of the sod pups ( p < 0.05 ) , no significant difference was observed between the groups at 82 days .
although the ea of the brains of pups of the shd group on day 8 after birth was 0.56  0.24% , ea was not detected on day 21 or day 82 .
on days 8 , 21 , and 82 after birth , the n-6/n-3 ratio in pup brains was higher in the shd group .
on day 8 after birth , the saturated fatty acid composition of the stomach contents of pups of both groups was higher than that of the dams fed either diet .
this indicated that a large amount of saturated fatty acid was synthesized in the dam body in order to produce high - fat milk .
reported that the specific activity of lipogenic enzyme was higher in the mammary gland and liver of lactating rats than in those of virgin animals . on the other hand ,
level of monounsaturated fatty acid , as well as of n-6 and n-3 fatty acids , was lower in the stomachs of pups in both groups than the levels of the same fatty acid composition of either diet .
we considered that this phenomenon happened because of the increased ratio of saturated fatty acid in the stomachs of the pups .
kavanagh et al . fed female c57/bl6 mice a trans diet containing hydrogenated vegetable oil during pregnancy and lactation and investigated the fatty acid composition in their breast milk .
the ea ( 18:1 n-9 ) in the trans diet and breast milk was 26.9% and 11.9% , respectively . in the present study ,
ea in the shd was 25.3% , and in the stomach content of the shd group on day 8 after birth it was 11.6  1.03% ( table 4 ) ; these data are mostly consistent with the results of kavanagh et al .
moreover , those authors reported that although the n-3 fatty acid was lower in the ea diet than in the control diet , the n-3 fatty acid levels in breast milk were almost the same between the groups . in our previous report
, pregnant rats were fed a corn oil diet or a perilla oil diet , and their fatty acid levels in breast milk were compared .
we reported that n-3 fatty acid was preferentially provided to breast milk when the dams were fed the n-3 fatty acid - deficient diet ; the ratio of n-6/n-3 in the breast milk in that group was lower than that in dams fed the other diet .
in the present study , the ratios of n-6/n-3 in shd and in the stomach content of the shd group were 64 and 54.7 , respectively .
the ratio of n-6/n-3 in stomach content of the shd group was lower than the ratio of n-6/n-3 in the shd group , similar to the results of the report of kavanagh et al .
there are several reports on the effects of tfa intake during pregnancy and lactation on fetuses and infants .
innis et al . indicated that a low intake of essential fatty acids during pregnancy lowed fetal development and that it was possible that tfa accumulated in the plasma of fetuses [ 10 , 26 ] . in the present study ,
tfa intake during pregnancy and lactation showed no effect on birth weight or growth after birth ( table 5 ) .
although no significant differences in plasma tg and t - cho concentrations were observed between the groups , the plasma t - cho concentration in pups on day 82 after birth tended to be higher in the shd group ( p = 0.062 ) ( table 6 ) . it was reported that tfa intake during pregnancy and lactation increased ldl - cholesterol levels in plasma and induced changes in maternal and infant body composition and the development of obesity later in childhood [ 5 , 15 ] .
it is possible that the plasma concentration in pups of the shd group was changed after 82 days of age .
compared with the plasma fatty acid composition of pups between 8 and 21 days old , the ea in plasma of the shd group on day 21 after birth was approximately twice that on day 8 after birth ( table 7 ) .
the pups were fed only breast milk until day 8 after birth . although the pups were weaned on day 21 after birth , they were fed breast milk and shd .
therefore , the pups of the shd group on day 21 after birth were fed a large quantity of ea in the shd , and this caused the ea to increase in the plasma of pups in that group .
the ea in the erythrocyte membrane of pups of the shd group on day 21 after birth was higher than that of pups at 8 days old , the same pattern that was found in plasma ( table 7 ) .
although the n-6 and n-3 fatty acids in the erythrocyte membrane were lower in the shd group than in the sod group , the ratio of n-6/n-3 in erythrocyte membrane of the shd group was higher on days 8 and 21 .
reported that , compared to controls , the blood cell membrane content of n-3 pufa was lower in acs cases ; levels of tfa and arachidonic acid ( aa ; 20:4 n-6 ) were higher .
aa is found in cell membrane phospholipids and is important in infant growth and central nervous system development .
in a recent study , significant positive relationships were found between tfa level in erythrocyte membrane and c - reactive protein ( crp ) , suggesting that the tfa intake might be a predictor of increased risk for metabolic syndrome . during pregnancy and lactation in the present study ,
tfa was provided in breast milk and increased the tfa levels in erythrocyte membrane of shd group pups .
these results indicated that tfa intake during pregnancy and lactation threatened the health of pups .   from days 8 to 21 after birth ,
the rates of increase in the total fat in pup brain were 2.6 times and 2.3 times in the sod and shd groups , respectively .
previously , we reported on the effects of a deficiency of n-3 fatty acid during pregnancy and lactation on the composition of fatty acid in the brains of rat pups . whereas fish oil contains high levels of n-3 fatty acids , such as epa and dha , there are no fatty acids in lard .
although levels of n-3 fatty acids in the brains of pups nursed by dams fed fish oil during pregnancy and lactation were higher than the levels in a group whose dams were fed lard , there was no significant difference in the ratio of n-3/n-6 in the brains of pups between the groups . in this study ,
the ratios of n-6/n-3 in the brains of pups of the shd group on days 8 , 21 , and 82 were 1.6 , 1.3 , and 1.1 times that of the sod group , respectively , and the fatty acid composition of the brains of pups remained constant ( table 7 ) .
although the ea in shd dams taken during pregnancy and lactation was 25.3% , the ratio of ea decreased as the ea moved into breast milk , plasma , and blood cells and brains of pups on day 8 after birth .
n-3 fatty acids are not consumed as a source of energy because of their poor intake ; these acids are used in the composition of the body . on the other hand
, it is supposed that the ea is used as a source of energy with oleic acid ( c18 - 1 ) , which is contained abundantly in shd .
ea was not detected in pup brains of the shd group , although the pups of the shd group on day 21 after birth showed 25.3% ea in shd and the erythrocyte membrane was 8.6% ea .
it was supposed that very small amounts of ea in the brains of pups of the shd group were detected because the blood brain barrier is not yet formed by day 8 after birth .
lipids in the maternal diet were reported to affect the myelination of the rat brain .
moreover , dha was essential for myelination of the rat brain , and dha deficiency during infancy delays brain development , while in aging a dha deficiency accelerates deterioration of brain functions [ 30 , 31 ] .
while the ala in shd was only 0.1% , the synthesized n-3 pufa from ala moved from the dams into the pups via breast milk , and the n-3 pufa was synthesized in body of pups . on day 82 after birth , no significant difference in the n-3 fatty acid levels in the brains of pups was observed between the groups ( table 8) .
therefore , it was considered that dha was synthesized in the bodies of the pups . in one study
, pregnant mice were fed a diet containing 21.5% tfa and 0.1% ala , and the relationship between the fatty acid composition of the brain and behavioral development in 7-week - old mice was investigated .
the brain dha level of the tfa group was lower than that of the control group .
reversal learning in the t - water maze was significantly slower in the tfa group compared with the control group .
the authors of that study referred that future studies of the long - term effects of dietary tfa during the pre- and postnatal periods should measure behavioral development and neural function of brain .
fed pregnant and lactating rats a diet containing 14.1% tfa and investigated the relationship between relative fatty acid composition in total lipids of the hippocampi of offspring and the spatial memories of young rats .
although tfa was detected in the brains of pups immediately after birth , the effects of dietary tfa on brain development and function remain unclear . 
the fao / who joint meeting of fat and fatty acid in human nutrition ,  held in japan in 2008 , set out the target to intake of tfa less than 1% of total energy intake .
kawabata et al . pointed out that daily tfa intake could become higher than the predicted value if processed foods containing high levels of tfa were consumed , while the average intake of tfa in young women was lower than the who - recommended energy ratio ( < 1% ) .
it is advisable to avoid especially high levels of tfa - containing foods during pregnancy and lactation . in summary
, we have shown that ea intake during pregnancy and lactation supplies ea to plasma , remains in the erythrocyte membrane of pups , and moves into the brain in early infancy .
we have also shown that the plasma t - cho concentration in pups on day 82 after birth tended to be higher in the shd group ( p = 0.062 ) .
these data highlight the need for further studies which should examine in more detail the long - term effects of dietary tfa during the pre- and postnatal periods and measure behavioral development and neural function of brain .
 to investigate the effects of a dam 's dietary elaidic acid ( ea ) intake during pregnancy and lactation on the fatty acid composition of plasma , erythrocyte membrane , and brain in rat pups , we fed two groups of dams either a soybean oil diet ( sod ) or a shortening diet ( shd ) containing soybean oil ( 10% ) or shortening ( 10% ) , respectively . although ea was not detected in the sod , ea accounted for 25.3% of all fatty acid content in the shd . on day 8 after birth 
 , the ea levels in the stomach , plasma , and erythrocyte membrane of pups nursed by the dams fed the shd were 11.6  1.03% , 7.18  1.20% , and 5.82  1.00% , respectively . 
 although on day 8 after birth the ea level of the brains of pups nursed by shd - fed dams was 0.56  0.24% , ea was not detected on day 21 or day 82 after birth . 
 these results suggest that ea intake during pregnancy and lactation supplies ea to plasma , remains in the erythrocyte membrane of pups , and moves into the brain in early infancy . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 