osteoporosis is one of the most serious public health problems for elderly people and also a major cause of the bedridden state .
this condition / disease has threatened the quality of life in old age and increased medical costs . in particular , the potential for developing osteoporosis increases dramatically after menopause in females , and estrogen deficiency causes rapid bone loss of trabecular regions in hip or lumbar spine .
estrogen deficiency is also associated with decrease in intestinal ca absorption which results in further acceleration of bone loss .
although it is well known that decrease in intestinal ca absorption is attributable to estrogen deficiency , it remains unclear what actions other gonadal hormones have for the association between bone mass and intestinal ca absorption in the estrogen deficiency state .
dehydroepiandrosterone ( dhea ) , secreted mainly from the adrenal gland and ovary , plays a critical physiological role for maintaining steroidogenesis by being used as the available precursor converted to testosterone and estrogens in various peripheral tissues such as bones , liver , brain , and skeletal muscles .
dhea concentration in the blood decreases the following ovariectomy in animals . on the other hand , dhea replacement improves bone mineral density ( bmd ) , especially a trabecular site , in the ovariectomized ( ovx ) animals [ 6 - 8 ] .
we previously observed that dhea replacement increased e2 ( estradiol ) centration in the blood .
the presence of estrogen receptors in the intestinal mucosa and estrogen stimulates intestinal calcium absorption via an estrogen receptor .
however , to our knowledge , the effect of dhea administration on intestinal ca absorption in estrogen deficiency state has not been studied yet .
accordingly , we hypothesized that dhea administration would increase intestinal ca absorption via increasing e2 concentration in the blood and prevent trabecular bone loss caused by estrogen deficiency . in the present study
, we examined bone mass in a trabecular abundant site of lumbar spine and ca balance such as intestinal ca absorption and ca accumulation in ovx rats after 8 weeks of dhea administration .
seventeen female sprague - dawley rats , 6 weeks old , were surgically ovariectomized and randomized into two groups : ovx control rats ( oc , n = 8) and ovx rats with dhea treatment ( od , n = 9 ) .
briefly , all rats were fed a diet with 1.05% calcium and 1.01% phosphate purchased from clea japan ( ce-2 , clea japan , inc . ,
dhea dissolved in sesame oil was administered to the od group intraperitoneally at 20 mg dhea / kg body weight for 8 weeks beginning one week after ovariectomy while the oc group was treated with vehicle only ( sesame oil , 0.5 ml ) .
rats were not treated with dhea or vehicle every fourth day ( i.e. , they were treated for 3 consecutive days ) .
the rats were kept in individual cages ( 15  25  19.5 cm ) and allowed access to food and distilled water ad libitum . food consumption and body weight gain
the room temperature was maintained at 24  1c , and the humidity at 50  5% .
fluorescent lights were turned on from 8:00 a.m. to 8:00 p.m. animal care and experimental procedures were approved by the animal experimental committee of the university of tsukuba .
the lumbar spine , left , and right tibiae of each rat were isolated by dissection , and all the muscle and connective tissue was carefully removed .
thereafter , bmc and bmd value for the l3-l6 lumbar spine were measured by dual - energy x - ray absorptiometry ( dxa ; aloka dcs-600r instrument ) . in the present study
, we used young ovx rats whose growth of bone is considerably influenced by body mass .
therefore , we normalized body weight for bmc to evaluate the effect of dhea on bone mass . at the each dissection
after the adhering connective tissues had been trimmed off , the femoral wet weight was measured .
thereafter , the femoral length , long width , and short width were measured with a caliper .
length was measured from the proximal tip of the femur head to the distal tip of the medial condyle .
the mediolateral ( long width ) and anteroposterior ( short width ) dimensions were measured at the midpoint of the femur diaphysis .
the bone strength of the middle diaphysis of the femur was then tested by measuring the mechanical strength , breaking force with an iio dyn-1255 instruments .
the force necessary to produce a break at the center of the femur was measured under the following conditions ; the sample space was 1.0 cm , the plunger speed was 100.0 mm / min , the load range was 50.0 kg , and the chart speed was 120.0 cm / min . in this study
, two balance studies were carried out to determine the rate of intestinal ca absorption and ca accumulation .
animals were placed in individual metabolic cages ( 24  20  18 cm ) .
the first phase was carried out on the 4th and 5th day after starting the experimental diets period ( metabolic cage phase 1 : mc 1 ) . the next phase ( mc 2 ) was carried out at the end of the experimental period . at each phase ,
urine was collected under acidic conditions by using 2ml 2n hydrochloric acid , thus preventing ca precipitate and putrefaction .
all urine was centrifuged at 2,500 rpm for 15 min to eliminate refuse . in the fecal determination ,
all daily feces were burnt to ash at 550 - 600 for approximately 18 hours , and the resulting ash was dissolved in 1n nitric acid .
the level of serum ca was measured by the inductively coupled plasma atomic emission spectroscopy ( icap - aes - 575 v nippon jarrell - ash ) . intestinal ca absorption and ca
accumulation were calculated using the amount of ca intake , the fecal ca excretion and the urinary ca excretion .
statistical significance ( p < 0.05 ) was determined using an unpaired student s t - test between groups . two - way analysis of variance ( anova ) was used for determining the effects of time and group on intestinal ca absorption and accumulation .
statistical analysis was carried out by one - way anova followed by fisher`s f - test for multiple comparisons among four points in intestinal ca absorption and accumulation .
statistical analysis was performed using spss for windows ( version 20.0 j ; spss inc . ,
seventeen female sprague - dawley rats , 6 weeks old , were surgically ovariectomized and randomized into two groups : ovx control rats ( oc , n = 8) and ovx rats with dhea treatment ( od , n = 9 ) .
briefly , all rats were fed a diet with 1.05% calcium and 1.01% phosphate purchased from clea japan ( ce-2 , clea japan , inc . ,
dhea dissolved in sesame oil was administered to the od group intraperitoneally at 20 mg dhea / kg body weight for 8 weeks beginning one week after ovariectomy while the oc group was treated with vehicle only ( sesame oil , 0.5 ml ) .
rats were not treated with dhea or vehicle every fourth day ( i.e. , they were treated for 3 consecutive days ) .
the rats were kept in individual cages ( 15  25  19.5 cm ) and allowed access to food and distilled water ad libitum . food consumption and body weight gain
the room temperature was maintained at 24  1c , and the humidity at 50  5% .
fluorescent lights were turned on from 8:00 a.m. to 8:00 p.m. animal care and experimental procedures were approved by the animal experimental committee of the university of tsukuba .
the lumbar spine , left , and right tibiae of each rat were isolated by dissection , and all the muscle and connective tissue was carefully removed .
thereafter , bmc and bmd value for the l3-l6 lumbar spine were measured by dual - energy x - ray absorptiometry ( dxa ; aloka dcs-600r instrument ) . in the present study
, we used young ovx rats whose growth of bone is considerably influenced by body mass .
therefore , we normalized body weight for bmc to evaluate the effect of dhea on bone mass .
after the adhering connective tissues had been trimmed off , the femoral wet weight was measured .
thereafter , the femoral length , long width , and short width were measured with a caliper .
length was measured from the proximal tip of the femur head to the distal tip of the medial condyle .
the mediolateral ( long width ) and anteroposterior ( short width ) dimensions were measured at the midpoint of the femur diaphysis .
the bone strength of the middle diaphysis of the femur was then tested by measuring the mechanical strength , breaking force with an iio dyn-1255 instruments .
the force necessary to produce a break at the center of the femur was measured under the following conditions ; the sample space was 1.0 cm , the plunger speed was 100.0 mm / min , the load range was 50.0 kg , and the chart speed was 120.0 cm / min .
in this study , two balance studies were carried out to determine the rate of intestinal ca absorption and ca accumulation .
animals were placed in individual metabolic cages ( 24  20  18 cm ) .
the first phase was carried out on the 4th and 5th day after starting the experimental diets period ( metabolic cage phase 1 : mc 1 ) . the next phase ( mc 2 ) was carried out at the end of the experimental period . at each phase ,
urine was collected under acidic conditions by using 2ml 2n hydrochloric acid , thus preventing ca precipitate and putrefaction .
all urine was centrifuged at 2,500 rpm for 15 min to eliminate refuse . in the fecal determination ,
all daily feces were burnt to ash at 550 - 600 for approximately 18 hours , and the resulting ash was dissolved in 1n nitric acid .
the level of serum ca was measured by the inductively coupled plasma atomic emission spectroscopy ( icap - aes - 575 v nippon jarrell - ash ) .
intestinal ca absorption and ca accumulation were calculated using the amount of ca intake , the fecal ca excretion and the urinary ca excretion .
all the data are expressed as mean  se . statistical significance ( p < 0.05 ) was determined using an unpaired student s t - test between groups .
two - way analysis of variance ( anova ) was used for determining the effects of time and group on intestinal ca absorption and accumulation .
statistical analysis was carried out by one - way anova followed by fisher`s f - test for multiple comparisons among four points in intestinal ca absorption and accumulation .
statistical analysis was performed using spss for windows ( version 20.0 j ; spss inc . , chicago , il , usa ) .
the body weight gain , food intake , and food efficiency are presented in table 1 .
the initial body weight did not differ between the groups . the final body weight and the body weight gain were significantly lower in the od group than in the oc group .
the bmc normalized by body weight of the lumbar spine ( trabecular - abundant region ) in the od group was found to be significantly higher compared to that in the oc group . to eliminate the effect of body weight on growing bone
, we normalized body weight for bmc for evaluating the effect of dhea on bone mass .
the bmd of the lumbar spine in the od group tended to be higher compared to that in the oc group , but it did reach statistically significant levels .
the femoral wet weight normalized by body weight in the od group was found to be significantly higher compared to that in the oc group , but the long and short width and length of femur did not differ between the groups ( table 2 ) .
the breaking force at femoral diaphysis site ( cortical boneabundant ) did not differ between the groups ( table 2 ) .
the intestinal ca absorption , rate of intestinal ca absorption , ca accumulation , and rate of ca accumulation decreased from mc 1 ( the 4th and 5th of the experimental diet period ) to mc 2 ( the end of the experimental period ) , but the interaction of time and group was not observed . in both mc1 and mc2 , all parameters did not differ between the groups .
the present study demonstrated that dhea administration increased the bone mass of lumbar spine in ovariectomized rats . on the other hand
we found that the bone mass of lumbar spine ( trabecular abundant site ) was increased after 8 weeks of dhea administration .
this result is similar to the result of our previous study   and turner et al.s study   which showed increase in trabecular bone mass of tibia .
dhea particularly may have a positive effect on trabecular bone mass but not in cortical areas in the diaphysis region .
the reason for this site - specific effect remains unclear yet , but our result further supported that dhea administration may be effective for preventing the reduction of trabecular abundant bones at which fracture frequently occurs for postmenopausal women with osteoporosis . in the present study ,
femoral weight normalized by body weight was higher in rats treated with dhea than in control rats .
the reason for the higher femoral weight would be supported by martel et al. study that the total bmc of femur increased by dhea administration in ovariectomized rats . also , we measured the femoral strength . to our knowledge
, no studies have examined the effect of dhea treatment on the bone strength of diaphysis site .
however , we did not observe any effect of dhea on the bone strength at the midpoint of the femur diaphysis .
the lack of effect of dhea on the bone strength might be partly influenced by the non - effect of dhea on the bone mass at the diaphysial area .
our previous study showed that e2 concentration in the blood was higher in ovariectomized rats treated with dhea than in ovariectomized control rats .
although evidence of a direct effect of estrogen promoting intestinal ca absorption has been reported , our results revealed that deha administration may have no effect on intestinal ca absorption .
as reason for the lack of dhea effect , intestinal ca absorption might have increased by dhea administration in an earlier phase during 8 weeks of the experimental period .
ca accumulation at the end point of the present experiment did not differ between the groups , meaning that the effect of dhea on bone had already reached its ceiling peak level . as another possible reason for the lack of dhea effect
, we used 1.05% ca diet which is an enough ca amount for maintaining the bone health of ovariectomized rats .
some studies related to examining intestinal ca absorption used to use 0.3 - 0.5% ca diet condition   which can lead to bone loss and induce higher ca absorption . as the result of our previous study , the serum dhea concentration was approximately 9-fold higher in dhea - treated rats than in control rats .
thus , we cautiously assumed that ca absorption would have increased if we treated lower dose of dhea than that in the present study .
therefore , future research that focuses on the dose - dependent effect of dhea administration is necessary .
in addition , we observed that dhea administration increased bmc normalized body weight even though it did not increase bmd . during the growing phase , bmd
sometimes may not precisely reflect the alteration of bone mass since growth of bone is considerably influenced by body mass . because bmd is calculated by dividing the bmc with area ( cm )
, the bmd measurement by dxa did not provide the details of bone parameters such as cortical width and periosteal circumferences .
thus , the parameters should be examined in detail by using the methods of pqct , ct , or bone morphometry in future studies , especially when using animals in the growing phase .
the present study confirmed the positive effect of dhea on trabecular bone mass of lumbar spine in ovariectomized rats . on the other hand , dhea administration did not affect intestinal ca absorption .
we suggest that dhea administration might have limited impact on intestinal ca absorption in estrogen deficiency state .
 [ purpose]the effect of dehydroepiandrosterone ( dhea ) administration on intestinal calcium ( ca ) absorption in estrogen deficiency state has not been studied yet . 
 we examined the bone mineral content ( bmc ) of lumbar spine and ca balance such as intestinal ca absorption and ca accumulation in ovariectomized ( ovx ) rats after 8 weeks of dhea administration.[methods]seventeen female sprague - dawley rats , 6 weeks old , were randomized into two groups : ovx control rats ( oc , n = 8) and ovx rats with dhea treatment ( od , n = 9 ) . 
 dhea was administered to the od group intraperitoneally at 20 mg dhea / kg body weight for 8 weeks while the oc group was treated with vehicle only.[results]the bmc normalized by body weight of the lumbar spine ( trabecular - abundant region ) in the od group was found to be significantly higher compared to that in the oc group . 
 the femoral wet weight normalized by body weight in the od group was significantly higher compared to that in the oc group . the intestinal ca absorption , rate of intestinal ca absorption , ca accumulation , and rate of ca accumulation decreased from the 4th and 5th of the experimental diet period to the end of the experimental period , but interaction of time and group was not observed . in both periods 
 , all parameters did not differ between the groups.[conclusion]the present study confirmed the positive effect of dhea on trabecular bone mass in ovariectomized rats . on the other hand 
 , dhea administration might have limited the impact on intestinal ca absorption in estrogen deficiency state . 