significant bone loss is a major feature of anorexia nervosa [ 1 , 2 ] . bone mineral density ( bmd ) has been reported to be reduced by more than 2.5  sd at either the hip or spine in 38% of women with anorexia nervosa and by more than 1.0  sd in 92% . bone loss predisposes to an increased risk of fractures [ 2 , 4 , 5 ] . the possible mechanisms of bone loss include nutritionally mediated changes in gonadal steroid concentrations   as well as alterations in the gh - igf - i system , with markedly low serum insulin - like growth factor - i ( sigf - i ) concentrations [ 711 ] . igf - i stimulates bone formation through its interaction with the type i igf - i receptor on the osteoblast [ 1216 ] . a number of studies have shown an imbalanced state of bone turnover in such patients , with decreased markers of bone formation and increased or normal markers of bone resorption . the latter observation appears to be primarily driven by estrogen deficiency , while decreased bone formation has been attributed to low sigf - i levels [ 7 , 17 ] . indeed , several studies of anorexia patients found lower levels of sigf - i and a direct correlation of this hormone with weight or body mass index ( bmi ) . however , data demonstrating that reduced serum concentrations of sigf - i contribute to low bone mineral density and whether different skeletal sites are affected in a similar way are scarce . the relative impact of estrogen deficiency versus low sigf - i on volumetric density or size is not well established . in a cohort of patients with anorexia nervosa , we studied skeletal site specificity of the association between bone density or size , and sigf - i level or sex hormone deficiency . the study is a retrospective analysis in a selected group of patients with anorexia nervosa which were consecutively referred to our consultation with data prospectively collected . the participants with anorexia nervosa met at a given time dsm - iv criteria , including weight less than 85% of expected , intense fear of being fat , disturbed body image , and amenorrhea for three or more consecutive menstrual cycles . women who had regular uterine withdrawal bleeding while receiving estrogen therapy and women with patterns of concomitant binge eating and purging type were also included . we also included 11 patients who had resumed menses , but still with an active disease . percentage of ideal body weight   and body mass index ( with weight and height measured in kilograms / height in meters ) were calculated . age at menarche , time since last menstrual period for patients with amenorrhea at time of evaluation , and total duration of amenorrhea were determined , as well as previous and current estrogen use and fracture history during the course of anorexia nervosa . calcium and protein intakes were recorded by a validated food frequency questionnaire   in a subset of 56 patients . fifty - two subjects completed a validated questionnaire for a detailed assessment of their specific weekly sports activities , including physical education classes , organized sports , recreational activities , walking , and cycling . sixty - six normal women of the same age were used as a control population . areal bmd of the lumbar spine ( ls ) in anteroposterior view , femoral neck ( fn ) and proximal femur ( pf ) were measured by dual - energy x - ray absorptiometry ( dxa ) using hologic qdr 2000 or 4500 instruments ( hologic , inc . , the coefficient of variation of repeated bmd measurements was 1%-2% [ 21 , 22 ] . the bmd ( in g / cm ) of the ls ( l2l4 ) , fn , and pf was also expressed as a t - score which compares individual bmd values to those of a young normal population of the same gender ( number of standard deviations from mean ) . according to criteria defined by the world health organization , t - scores between 1 and 2.49 indicate osteopenia , whereas t - scores below 2.5 standard deviations ( 2.5 ) indicate osteoporosis . bmd ( g / cm ) of the ls , and those of fn were also expressed in terms of standard deviations from mean gender- and age - matched controls ( z - scores ) . skeletal size was estimated from the scanned bone projected area of ls and pf . at the spine level , the height and the width at three levels ( high , middle , and lower level ) of vertebral body l2 were measured . structural geometry parameters of the hip were measured from conventional dxa outputs of the femoral neck region of interest . we measured the internal and external cortical thickness , the diameter of the femoral shaft at a fixed level of the femoral diaphysis ( 10 lines from the lower edge of the trochanter minor ) , neck width ( area divided by the mean diameter of the femoral neck ) , and hip length ( measuring the central line from the femoral head to the trochanteric region ) . the number of measured pixels was multiplied by a conversion factor of 1 pixel = 0.43  mm for the hologic 2000 device and 1 pixel = 0.45  mm for the hologic 4500 device . 's approximation method . assuming a minimal intraclass correlation coefficient ( icc ) of 0.5 ( p0 ) against a desired of 0.8 ( p1 ) , based on  = 0.05 and  = 0.20 , at least 14 participants were required , if measured 3 times . cv ( % ) was computed as the 100 times the standard deviation divided by the average of 3 measures repeated in 14 patients . the mean cv of the morphological dxa parameters ranged from 0.12%    0.06% to 1.09%    0.74% . bone mineral apparent density ( bmad ) was calculated as previously described for ls and fn [ 26 , 27 ] . at the lumbar spine and femoral neck level , bmad was computed by using the equation bmad = bmc / projected area and bmad = bmc / projected area , respectively . prolactin , fsh , and tsh were determined using standard methods , for patients with amenorrhea and not receiving any form of estrogen . pth was determined by immunoradiometric assay with an intrassay coefficient of variation of 1.8%3.4% ( nichols institute diagnostics ) . calcifediol ( 25-oh - d3 ) was determined by chemiluminescence assay ( nichols advantage , san clemente , ca ) . the first 19 sigf - i determinations , that is , before march 2002 , were performed by ria after acid - ethanol extraction ( nichols institute diagnostics ) . intra- and interassay coefficients of variation were 6.4% and 10% for ria measurement , and 4.8% and 6.7% for chemiluminescence assay . the two assays were highly correlated ( r = 0.99 ) , with a regression analysis giving the following results in our laboratory : nichols advantage igf - i assay ( y ) versus nichols igf - i by extraction ( ria ) assay ( x ) ;  y = 0.84x  2.15 . igfbp-3 was measured by an enzyme - labeled chemiluminescent immunometric assay ( immulite , dpc ) . plasma and urinary calcium , phosphate , and creatinine were measured using an automatic analyser . plasma calcium was adjusted for protein levels ( adjusted ca = ca/[(protein/160 ) + 0.55 ] ) . the following parameters were calculated : ( a ) fasting urinary ca - to - creatinine ratio was taken as a reflection of net bone resorption ( bone resorption index , bri ) ; ( b ) renal tubular reabsorption of phosphate ( tmpi / gfr ) and of calcium ( trcai ) was calculated as described elsewhere [ 28 , 29 ] . the marker of bone formation osteocalcin was determined with an immunoradiometric assay ( cis - bio , gif - sur - yvette , france ) . the specific urine marker of bone resorption deoxypyridinoline was measured by fluorescence emission after acid hydrolysis and high - performance liquid chromatography separation ( bio - rad system , munich , germany ) and expressed as a creatinine ratio . when the data are not normally distributed , the summary descriptive statistics are the median and interquartile range . all non - gaussian variables were successfully normalized using simple mathematical transformations . comparisons between patients and age - matched controls for continuous variables were performed with a wilcoxon matched - pairs signed - ranks test to take into account the matched design . comparisons between two or more groups for continuous variables were performed with student 's t - test or anova for unmatched groups . correlations between bone mineral density and recorded variables were determined by simple or multiple ( backward stepwise ) linear regression analyses . standard multiple regression models were constructed for each skeletal site by using duration of anorexia nervosa , total duration of amenorrhea , age at the time of evaluation , bmi , and sigf - i as covariates . the data were analyzed using the stata statistical software package ( version 9.2 ; stata corporation , college station , tx ) . the subjects were in the early twenties ( the age range and median age and interquartile range were 15 to 46 years , median : 20 , and interquartile range : 1633 ) , had a low weight ( weight range : 26 to 60  kg , median : 43 , and interquartile range : 3055 ) , and a low bmd ( table 1 ) . the median height was 165 , with a range of 146 to 180  cm . their mean t - scores ( in patients aged more than 18 years ) were 1.52  1.23  sd for the anterior - posterior view of ls , 0.93  0.92  sd for the fn , and 1.34  0.92 for the pf , significantly lower as compared with the normal population ( table 1 ) . osteopenia was found in at least one skeletal site in 63% of patients and osteoporosis in 17% of patients . no differences in bmd expressed as t - scores were observed between patients with anorexia nervosa alone and those with the binge - purge subtype of anorexia nervosa at any site measured ( data not shown ) . eight patients reported a history of low trauma fracture ( elbow , wrist or hand ( n = 4 ) , foot or ankle ( n = 2 ) , rib ( n = 1 ) , vertebrae ( n = 1 ) ) . median time to the first fracture was 6 years ( iqr : 5 ) after the onset of the disease . their bmd tended to be lower , but not significantly , than that observed in patients ( n = 54 ) without a history of low traumatic fracture ( data not shown ) . the age of the fractured and the non - fractured was 24.5  7.7 versus 22.1  6.4 years ( ns ) , duration of amenorrhea was 50.4  60.5 versus 21.4  31.0 months ( p < 0.14 ) , and disease evolution was 7.3  4.5 versus 4.6  4.5 years ( ns ) . fifty - five patients reported at least one hospitalization during the course of the disease . girls usually complete puberty by ages 1517 , and menarche occurs generally at tanner stage 4 , at the age of 13 . in order to clarify this aspect , we determined the number of patients with a disease onset before menarche . six patients had a disease onset before the first periods . for 3 of them , three others had a delayed menarche ( after 16 ) , 4 to 5 year after disease onset , and thus possibly before pubertal skeletal growth . twenty patients ( 30% ) were current estrogen users ( median duration : 27 months , iqr : 61 ) . six patients ( 9% ) were previous estrogen users ( median duration : 8 months , iqr : 29 ) . age , age at menarche , age at the onset of anorexia nervosa , total duration of amenorrhea , protein and calcium intake , sigf - i , and physical activity did not differ according to estrogen use and menstrual history at the time of examination ( table 2 ) . amenorrheic women had a significantly lower bmi ( 15.3  2.2 in untreated women versus 15.6  2.0 in current estrogen users , p < 0.05 ) . age- and sex - adjusted bmd values ( z - score ) were similar ( data not shown ) . serum levels of albumin and protein were lower than the normal range in 17% and 29% of patients , respectively ( table 2 ) , while sigf - i was below the lower limit of the normal level in 58% of patients . the sigf - i levels correlated with bmi ( r = 0.43 , p < 0.001 ) . nmol / l ) and one displayed a deficiency ( below 25  nmol / l ) . markers of bone turnover ( total alkaline phosphatase , but not osteocalcin , and fasting urinary calcium - to - creatinine ratio ) were lower in estrogen users or estrogen replete patients ( table 2 ) . bone area was in contrast similar or tended to be higher in anorexia nervosa patients . thus , compatible with a reduced volumetric bone density , rather than altered bone geometry , bmad was lower , as was also cortical thickness at the level of the femoral shaft . bone geometry parameters were compared to the control population according to the disease onset , that is , in those below or above the median age of disease onset , and this did not affect the results ( data not shown ) . ls bmd correlated to the duration of anorexia nervosa , total duration of amenorrhea , sigf - i , and bmi . fn bmd was associated ( p < 0.05 for all comparisons , table 4 ) with age at the time of evaluation , duration of anorexia nervosa , total duration of amenorrhea , sigf - i , and igfbp-3 . bmd at any site was not correlated with the degree of physical activity . to assess the relative contribution of the variables in determining bmd the following parameters were included : duration of anorexia nervosa , total duration of amenorrhea , age at the time of evaluation , bmi , and sigf - i . physical activity was not included in the model , as severely affected patients were unable to exert a high level of such activity . furthermore , many patients were advised to avoid sports activities in order to conserve their energy . given the high variability of the recorded calcium and protein intakes and the absence of any correlation in the univariate model , these two variables were not included . ls bmd was significantly correlated with total duration of amenorrhea ( p < 0.01 ) . the serum level of igf - i was the only significant independent predictor of fn bmd and remained significant even after adjusting for height . we performed the same analysis excluding the 20 patients receiving a hormone replacement therapy at the time of evaluation and found similar results ( data not shown ) . we examined whether the influence of the sigf - i level on bmd could be explained by an effect on true volumetric density or on bone size . the sigf - i levels correlated with fn bmad ( multiple regression model : r = 0.001 , p < 0.04 ) , but not with hip geometry variables ( neck width , hip axis length , cortical thickness , and femoral shaft diameter ) . there was a correlation between osteocalcin ( a bone - specific formation marker ) and nutritional variables such as bmi , 25-hydroxy vitamin d , sigf - i , and igf - bp3 . in a multiple stepwise backward regression model , bmi and 25-hydroxy vitamin d were independent predictors of serum osteocalcin levels . deoxypyridinoline and calcium - to - creatinine ratios were significantly higher in patients with amenorrhea at the time of evaluation as compared with those spontaneously menstruating or being treated with estrogen ( table 2 ) . deoxypyridinoline positively correlated with age at the time of evaluation , amenorrhea at the time of evaluation , 25-hydroxy vitamin d , and pth levels . in a stepwise multiple regression , the serum pth level and amenorrhea at the time of evaluation were the most significant predictors of deoxypyridinoline levels , explaining 36% of its variance . our study showed a high prevalence and profound degree of low bone mineral mass in women with anorexia nervosa . indeed , osteoporosis was present in 17% of patients and osteopenia in 63% . altogether , 12% reported a history of low trauma fracture . our results suggest that low bone mass is the consequence of a reduced bone density rather than a reduced bone size . it may result from a number of mechanisms , including estrogen deficiency and inadequate nutritional intakes . we found , as other groups , that age - adjusted bone mineral density correlates with the duration of amenorrhea [ 6 , 9 , 30 ] . in our population , estrogen deficiency seemed to equally affect the spine and the proximal femur . decreased calcium intake and excessive physical activity may also impact on the degree of bone loss , but we did not find any correlation between physical activity and ls bmd . a beneficial effect of physical activity had been suggested in an observational study published by seeman et al . . conversely , another recent study showed that excessive moderate loading exercise may put patients at higher risk of low bone mass , but high bone loading activities may provoke bone accrual during recovery . in our study , data about nutritional intakes and exercise were obtained through a questionnaire completed by the patients , with its associated limitations . since denial is common in anorexia nervosa , these data should be considered with caution . undernutrition and nutritionally dependent factors are likely to play a major role in the bone loss associated with anorexia nervosa . there were marked effects of nutritional status on bone formation markers which correlated with bmi , 25-hydroxy vitamin d , serum igf - i , and igfbp-3 , in our study as in others [ 7 , 8 , 34 ] . caloric and protein deprivation in anorexia nervosa leads to increased gh and decreased igf - i . this could be explained by a downregulation of the gh receptor or its postreceptor mechanisms , inducing a fall in the gh - dependent serum proteins igf - i and igfbp-3 levels . the rise in gh is presumably due to decreased feedback inhibition by sigf - i . fibroblast growth factor-21 may be a putative factor of gh resistance in anorexia nervosa . our study confirms the low sigf - i levels previously reported in low - weight patients with anorexia nervosa [ 9 , 10 ] . sixty percent of the patients had sigf - i values below the age - adjusted reference range ; yet , only 17% of the patients showed lower than normal serum albumin concentration , a usual indicator of nutritional status . sigf - i is closely related to nutritional status as suggested by the highly significant correlation with bmi ( p < 0.001 ) . several studies showed that sigf - i concentration is a more reliable index of nutritional status as compared with others markers [ 3739 ] . the most intriguing finding was the high and independent association of sigf - i and igfbp-3 levels with age - adjusted fn bmd . we examined whether differences in bone size may explain the association . in a first step this adjustment did not affect the relation between fn bmd and sigf - i . in a second step , parameters of structural geometry of the hip were measured using conventional dxa outputs obtained from the fn region of interest . we measured medial and lateral cortical thickness at a fixed level of the femoral diaphysis , neck width , hip lengths , and femoral shaft diameter . , we examined the impact of another analytic strategy , that of bmad , a volumetric bone density estimate . we found a positive correlation which suggests that the relation between sigf - i and fn bmd might not be related to differences in bone size . note that part of the femoral neck region of interest is intracapsular and thus devoid of periosteum . a recent study ( hip structural analysis ) found an increase in the outer and inner diameter of the hip and a decrease in the cortical thickness at the femoral shaft . anorexia nervosa affects patients at a time of acquisition of peak bone mass , and one possible consequence is growth retardation and an effect on bone modelling . short stature may be present in those starting in the infancy or early childhood . for those beginning their eating disorders later , that is , during or after puberty , a study found that majority of patients with anorexia reached their expected height . in our population , the median age of onset of anorexia was 16 years . a small number of patient had a disease onset before menarche , and had probably reached their adult stature and peak bone mass . this study has several limitations , particularly the relative heterogeneity of the study population , with three groups of patients at different stages of severity ( active disease , and spontaneously menstruated ) and taking or not estrogens . it may be advocated that the inclusion of patients who were taking estrogen confounds the interpretation of the results that spine bone mineral density was related to hypogonadism . although the benefits of estrogen in the bone density issue of patient with anorexia nervosa were refuted in the last decade , a recent publication suggested that physiologic estrogen replacement given transdermally may be beneficial to bone . excluding those patients taking estrogen male patients were not included in the study even if they display similar hormonal issues , given the aim of study in analyzing bone size . 
 in conclusion , in a large cohort of patients with anorexia nervosa , spine and hip bmd values were related to hypogonadism , whereas sigf - i was the most significant predictor of bone mass at the fn level . our morphometric analysis suggests that the site - specific effect of sigf - i could be related to reduced volumetric bmd rather than to modified hip geometry .
we investigated the relative effect of amenorrhea and insulin - like growth factor - i ( sigf - i ) levels on cancellous and cortical bone density and size . 
 we investigated 66 adult women with anorexia nervosa . 
 lumbar spine and proximal femur bone mineral density was measured by dxa . 
 we calculated bone mineral apparent density . 
 structural geometry of the spine and the hip was determined from dxa images . weight and bmi , but not height , as well as bone mineral content and density , but not area and geometry parameters , were lower in patients with anorexia nervosa as compared with the control group . 
 amenorrhea , disease duration , and sigf - i were significantly associated with lumbar spine and proximal femur bmd . in a multiple regression model 
 , we found that sigf - i was the only significant independent predictor of proximal femur bmd , while duration of amenorrhea was the only factor associated with lumbar spine bmd . 
 finally , femoral neck bone mineral apparent density , but not hip geometry variables , was correlated with sigf - i . in anorexia 
 nervosa , spine bmd was related to hypogonadism , whereas sigf - i predicted proximal femur bmd . 
 the site - specific effect of sigf - i could be related to reduced volumetric bmd rather than to modified hip geometry .