nonalcoholic fatty liver disease ( nafld ) is a pathological condition that encompasses a wide spectrum of liver damage , ranging from simple steatosis to nonalcoholic steatohepatitis ( nash ) , cirrhosis , and hepatocellular carcinoma .
the pathogenesis of nafld is multifactorial and is characterized by overweight and insulin resistance ( ir ) .
recently , some studies reveal that bone mineral density ( bmd ) is also influenced by nafld .
reported a significant association between mean lumbar bmd and nafld in postmenopausal subjects after adjusting for age , body mass index ( bmi ) , alanine aminotransferase ( alt ) , smoking status , and alcohol consumption , and they concluded that postmenopausal women with nafld had an increased risk of osteoporosis .
pardee et al . reported that obese children with nafld had significantly lower bmd z - scores than obese children without nafld ; among children with nafld , those with nonalcoholic steatohepatitis ( nash ) had a significantly lower bmd z - scores than nafld children without nash .
however , when this phenomenon is explained by overweight and insulin resistance ( ir ) , there is a paradox . in obese subjects
, it has been argued that there is a positive relationship between body weight and bone mass especially in adults .
however , some recent studies reveal that severe ir may negatively affect bmd [ 4 , 5 ] .
the role of cytokines and other bone - influencing molecules released from the inflammatory liver is still unclear .
osteocalcin ( oc ) , which is a specific marker of bone formation , has been found to be decreased in liver with nafld [ 6 , 7 ] . besides its role in the calcium and bone metabolism
nafld patients present with a marked reduction in serum 25(oh ) vitamin d when compared with controls .
however , although these cytokines are associated with both nafld and bmd , none of them have been confirmed to be involved in the process whereby nafld influences bone metabolism . considering that little is known about chinese patients and that the mechanism underlying the relationship between nafld and bone defect is poorly understood , this cross - sectional study was performed in china to explore the role of overweight , ir , osteocalcin , and vitamin d in the interaction between nafld and bmd
patients were recruited from the shanghai tenth people 's hospital of tongji university from october 2011 to april 2012 .
all subjects were screened using a questionnaire on their medical history , symptoms , and findings in physical examination .
inclusion criteria were as follows : ( 1 ) subjects who were 4070 years old and ( 2 ) males or postmenopausal females with or without nafld .
( 1 ) there were other causes of chronic liver disease ( hepatitis b and c , wilson disease , -1-antitrypsin deficiency , autoimmune hepatitis , and cystic fibrosis ) , severe alcohol abuse ( > 10  g of alcohol per day ) , and drug induced fatty liver disease , especially related to amiodarone , estrogens , and vitamin a. ( 2 ) subjects were current or former smokers who quitted smoking less than 6 months ago .
( 3 ) there were conditions known to affect bone metabolism , such as kidney , thyroid or parathyroid diseases , hyperprolactinaemia , ovary excision , rheumatoid arthritis , ankylosing spondylitis , malignant tumors , and medication with agents influencing bone metabolism ( such as calcium , vitamin d , and anti - osteoporosis drugs ) .
a total of 99 males and 125 postmenopausal females were enrolled and divided into 4 groups according to the gender and the presence of nafld : males without nalfd , males with nafld , females without nafld , and females with nafld .
this study was conducted according to the principles in the declaration of helsinki , and written informed consent was collected before study .
the whole protocol was approved by the ethics committee of shanghai tenth people 's hospital of tongji university .
weight was measured with a plethysmography scale , where patients wore minimal clothing , and height was measured with a stadiometer . body mass index ( bmi ) was calculated by dividing the weight by height squared ( kg / m ) .
nafld was diagnosed based on the ultrasonographic evidence of hepatic steatosis , such as a bright hepatic echo pattern , increased echo attenuation , and loss of intrahepatic architecture .
liver ultrasonography was conducted with a 4  mhz probe ( eub-6500 , hitachi , tokyo , japan ) and evaluated by an experienced radiologist .
bmd was measured by dual - energy x - ray absorptiometry ( dexa ) ( hologic ( qdr-4000 ) , usa ) .
the bmd of lumbar spine ( l ) , right hip ( rh ) , and femoral neck ( fn ) was evaluated , and the means of l , rh , and fn served as the mean bmd ( table 4 ) .
biochemical markers , including total cholesterol ( tc ) , triglyceride ( tg ) , high density lipoprotein ( hdl ) , low density lipoprotein ( ldl ) , fasting plasma glucose ( fpg ) , fasting insulin level ( fins ) , alt , alkaline phosphatase ( akp ) , aspartate aminotransferase ( ast ) , and serum creatinine ( scr ) were measured with standard biochemical methods .
bone metabolic markers including type i collagen ( ctx ) , parathyroid hormone ( pth ) , oc , calcitonin ( ct ) , bone alkaline phosphatase ( bap ) , and 25-hydroxyl - vitamin d ( 25(oh)d ) were measured by elisa ( roche ) according to the manufacturer 's instructions .
insulin sensitivity and ir were evaluated by homeostasis model assessment 2 ( homa2 ) , including homa2%b , homa2%s , and homa2ir , calculated by homa2 v2.2 ( http://www.dtu.ox.ac.uk/homa ) .
analysis of variance ( anova ) was used to compare the baseline morphometric measurements ( table 1 ) , bmd , and biomarkers in the control group and the nafld group for both genders ( tables 2 and 3 ) .
analysis of covariance was used to adjust for weight , bmi , wc , lipid profiles , and alt ( table 2 , p value ( a ) ) .
multiple linear regression analysis was performed to analyze the variables associated with mean bmd and nafld ( table 4 ) .
statistical product and service solutions version 17.0 ( spss 17.0 ) software was used for statistical analysis .
at baseline , the age , height , fpg , fins , akp , ast , and scr were comparable between the control group and the nafld group , whereas the weight , bmi , wc , lipid profiles , and alt in subjects of the nafld group were markedly different from those in the control group ( table 1 ) .
subjects in the nafld group had significantly lower bmd in rh and fn of males and in rh of females . after adjustment for parameters ( weight , bmi , wc , lipid profiles , and alt ) at baseline ,
serum bone metabolic markers including ctx , pth , oc , ct , bap , and 25(oh)d were comparable between the nafld group and the control group in both genders .
although calcium was significantly different , we did not think this difference was clinically significant . in the nafld group ,
homa2ir was higher in both genders , but homa2%s was lower in females ( table 3 ) .
three models were used to analyze the correlation between bmd and nafld taking into account all the parameters including age , bmi , waist , homa2ir , and nafld that would affect the bmd . in all the models , nafld played a very important role in the process of the influence on bone .
in our study , subjects in the nafld group had significantly lower bmd than those in the control group for both genders , and the difference was still present after adjustment for variables .
however , most clinical trials [ 1 , 9 ] showed a negative association between nafld and lumbar bmd . in the present study
, there was no significant difference in the lumbar bmd between the nalfd group and the control group , which might be attributed to the differences in the ethic and age .
since the bmd of femur neck and hip was not detected in previous studies , the bmd of femur neck and hip could not be compared among studies . at baseline ,
subjects in the nafld group were characterized by higher weight , bmi , and wc , which was in accordance with other studies in which overweight or obesity was a predictor for nafld and other serious medical conditions [ 10 , 11 ] . there is a significant relationship between bmi and relative body fat mass .
consequently , bmi is a widely accepted index of obesity , correcting for gender and ethnicity .
furthermore , there is a direct association of bmi with hepatic steatosis , nash , and advanced liver fibrosis [ 12 , 13 ] .
wc is highly correlated with visceral adipose tissue ( vat ) and a marker for abdominal obesity .
excess vat , or abdominal obesity , is a risk factor of ms as compared to individuals with fat located predominantly in the lower body and subcutaneously .
overweight , bmi and wc are related to bone mass to a certain extent because any increase in body mass , fat , or lean will expose the skeleton to greater forces during locomotion .
bone is sensitive to , but not limited to , a variety of mechanical parameters .
found that the obesity failed to protect against bone mineral loss in postmenopausal women . in our study , correlation analysis showed that there was no significant relation of bmd with weight , bmi , and wc .
hoy et al . speculated that the bone quality of overweight adolescents adapted to lean mass but not to greater fat mass , because the fat composition ratio was not significantly different between the control group and the nafld group .
ir was negatively associated with bmd in obese or t2 dm patients [ 17 , 18 ] . even having higher bone density , as in the ir state , diabetic women have lower femoral neck strength relative to load . in ir obese women
circulating concentrations of inflammatory cytokines are reckoned to be the most important factor in causing and maintaining ir .
it has been found that ir induced by 12-week high fat diet ( hfd ) could impair the osteoblastic insulin signaling , osteoblast proliferation , and osteoblast survival and result in osteoporosis of the jawbone .
obesity status - associated ir may induce high circulating intact ( ifgf-23 ) and c - terminal fgf-23 ( ctfgf-23 ) , which is positively associated with pth and negatively associated with vitamin d , resulting in low bone mass .
elevated alt and nafld are associated with ir , and thus , alt has been proposed as a useful serum marker for early ir , which reflected a balance between oxidative stress and antioxidant response [ 24 , 25 ] .
failed to find low bmd in nafld patients , but elevated serum alt was found to be associated with lower bmd . in our study , negative association was noted between alt and bmd in nafld group , which was consistent with previously reported studies .
alt , an indicator of inflammation state in either the liver or system , will affect bone metabolism .
osteoblasts can secrete oc , but recent work indicates that , besides acting as a specific bone marker for bone formation , oc is also associated with insulin secretion and sensitivity .
after administration of recombinant oc , insulin secretion increases , blood glucose decreases , and experimental obesity is attenuated .
consequently , oc has been considered to link bone to energy metabolism , although this was not evident in our study .
possible explanations for this discrepancy may be that only total serum oc was tested , whereas undercarboxylated osteocalcin ( ucoc ) affects energy metabolism . secondly , the association between oc and steatohepatitis is not confirmed by multivariate analysis .
thirdly , decreased serum oc in chinese nafld patients as compared to subjects in western countries suggests the potential role of ethnicity .
vitamin d receptors are present in several cell types and play an important role in both energy metabolism and bone metabolism .
25(oh)d is a major circulating form of vitamin d and constitutes the best clinical indicator of vitamin d stores .
reported that nafld patients had a marked reduction in serum 25(oh)d as compared to controls , and this decrease was closely associated with the histopathological features of nafld .
however , a report on 102 nafld patients and 54 healthy controls showed no difference in the serum vitamin d , which was consistent with our results . in our study , we did not take the seasonal variation of vitamin d ; since it was a cross - sectional study , we could not balance every impact factor .
moreover , vitamin d was not the only one of the factors that affect the bone metabolism ; there were still other markers including type i collagen ( ctx ) , parathyroid hormone ( pth ) , oc , calcitonin ( ct ) , and bone alkaline phosphatase ( bap ) .
future studies are needed to clarify the correlation between vitamin d status and nafld . this retrospective study still had some limitations .
firstly , being a cross - sectional study , it failed to completely exclude that some correlations were incidental .
secondly , bmd could be influenced by many factors such as physical activity , calcium / vitamin d intake , and peak bone mass , which were not evaluated in this study .
thirdly , only total oc , instead of carboxylated and uncarboxylated forms of oc , was measured .
moreover , we did not perform hepatic biopsy to evaluate the severity of nafld . that was because hepatic biopsy which was considered as the golden standard to diagnose nafld was invasive .
liver ultrasonography was also considered an effective way to investigate whether the fat existed in the liver .
further studies are needed to evaluate the effect of nafld on bmd in different extent .
for example , we could conduct magnetic resonance spectroscopy ( mrs ) on the subjects or a hepatic biopsy on the animal model of nafld . in conclusion
, bmd is significantly decreased in elderly male and female chinese nafld patients as compared to controls .
further studies are needed to identify the causative factors , especially the role of ir .
 
 aim . to investigate bone metabolic characteristics in chinese adults with nonalcoholic fatty liver disease ( nafld ) . methods . 
 a total of 224 patients ( 99 males and 125 postmenopausal females ) were recruited and divided into 4 groups : males without nafld , males with nafld , females without nafld , and females with nafld . bone mineral density ( bmd ) was evaluated according to body mass index ( bmi ) , waist circumference ( wc ) , and serum biomarkers . 
  cell function was evaluated by homa2%b , homa2%s , and homa2ir . 
 results . males in the nafld group had lower bmd of the right hip and the femoral neck ( 0.852  0.117 versus 0.930  0.123 , p = 0.002 ; 0.736  0.119 versus 0.812  0.132 , p = 0.004 ) , and females had lower bmd of the right hip ( 0.725  0.141 versus 0.805  0.145 , p = 0.002 ) even after adjusted for weight , bmi , waist , hdl , and alt . 
 there was no significant difference in bone metabolic markers between patients with and without nafld . 
 nafld was an important factor that affected the bone ; moreover , the effect attenuated when homa2ir entered into the model ( r2 = 0.160 ,  = 0.172 , and p = 0.008 ) . conclusions . 
 nafld exerts a detrimental effect on bmd in both males and females 
 . insulin resistance may play an important role in this pathophysiological process . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion