for this cross - sectional study , we recruited men with type 2 diabetes ( with and without cna ) , and attempted to age - match them with healthy control subjects .
inclusion criteria for the diabetic participants ( with and without cna ) included ambulatory men aged between 40 and 80 years with established type 2 diabetes for more than 5 years and diabetes - derived nerve damage .
exclusion criteria included a documented history of vitamin d deficiency , thyroid disease , renal disease , or other metabolic diseases known to affect bone , as well as prior oral or intravenous antiresorptive drug use .
charcot patients had to be ambulatory and in the nonacute , convalescence stage ( stage 2 ) of the disease , which is different from acute stage ( stage 1 ) that is characterized by osseous resorption , soft - tissue swelling , and joint effusions ( 16 ) .
this study was approved by the eastern virginia medical school institutional review board , and all subjects provided written consent before participating in this study .
the inclusion criteria for the study were met by 80 men , comprising 30 healthy control subjects , 30 type 2 diabetic patients without charcot , and 20 type 2 diabetic patients with stage 2 cna .
many of the cna patients had , however , already undergone stabilization of the fractured or dislocated joints with beaming screws and other means of metallic fixation .
subjects evaluation consisted of an analysis of fasting blood chemistries ; a complete neurologic examination , including quantitative sensory , autonomic nerve function , and nerve conduction tests ; a bone density assessment ; and quantitative ultrasound imaging of bilateral calcanei .
continuous data are expressed as mean  sd ( range ) , and categoric data as n. * p < 0.05 vs. control subjects .
lower limb nerve conduction parameters were measured bilaterally by surface stimulation of the sural , peroneal , and tibial nerves .
measurements included peroneal and tibial motor nerve amplitude , conduction velocity , distal onset latency , and f wave .
all motor amplitudes were measured baseline to peak , and latencies were taken as onset latency .
motor conduction velocity was calculated as total conduction velocity ( not segmental ) using onset latencies throughout .
sensory amplitude was taken as baseline to peak , latency was reported as onset latency , and conduction velocity was calculated across the single segment using onset latency ( neuromax , excel - tech ltd . ,
quantitative sensory testing was performed using the medoc device according to previously published methods ( 17 ) .
warm and cold thermal thresholds and heat and cold pain were tested using the tsa2001/vsa3001 computer - driven sensory measuring device ( medoc , durham , nc ) .
vibration perception threshold was quantified using the medoc device and the  method of limits  protocol , where repeated , increasing vibratory stimulation is applied to the plantar aspect of the dominant great toe .
autonomic function was assessed by three tests using the anx-3.0 autonomic monitor : heart rate variability during deep breathing at six breaths per min ( e / i ratio ) and r / r variation in response to the valsalva maneuver and postural change ( ansar medical technologies , inc . ,
bmd scans of the whole body , bilateral proximal femurs , and bilateral feet were obtained using dual - energy x - ray absorptiometry ( ge lunar prodigy advance , ge healthcare , waukesha , wi ) .
scans were performed according to the manufacturer s protocols , with the exception of the feet , for which no standardized protocol exists .
mode , where a subject sat on the edge of the scan bed with the knee bent at 90 and the foot flat on the bed .
the laser marker was positioned at the midline of the ankle , at the level of the lateral malleolus . during analysis ,
the region of interest was selected manually and included only the metatarsals and phalanges to exclude any metal fixators .
test - retest reliability for this measure is high ( r = 0.96 , n = 31 ) , and the coefficient of variation is < 1% for our laboratory .
this corresponds well to other published studies of reproducibility of bmd using the hand mode ( 20 ) , and is also comparable with other regional analyses of bone , where coefficients of variation typically range from 0.5 to 1.2% . because no reference database currently exists for scans of the feet , diabetic patients were compared with sex- and age - matched control subjects .
calcaneal stiffness was determined using quantitative ultrasound imaging ( ge achilles express , waukesha , wi ) .
bone stiffness is automatically computed using an algorithm supplied by the manufacturer : stiffness index = 0.67  bua  0.28  sos  420 , where bua is broadband ultrasound attenuation and sos is the speed of sound through the tissues ( ge healthcare ) . because bone stiffness predicts fracture independent of bmd
, it is considered to be a measure of bone quality that also accounts for factors related to fracture that might not be detected by a measure of bmd alone .
fasting blood samples were obtained by antecubital venipuncture into serum separator tubes and separated immediately by refrigerated centrifugation .
the serum was stored at 80c according to instructions required by the manufacturers of the enzyme - linked immunosorbent assay ( elisa ) kit .
samples were subsequently evaluated in duplicate for srage by the quantikine rage enzyme - linked immunoassay ( r&d systems , minneapolis , mn ) ; cross - linked n - telopeptides of type 1 ( ntx ) collagen , as a measure of bone resorption ( osteomark ntx elisa / eia , wampole laboratories , princeton , nj ) ; and osteocalcin , as a measure of bone formation ( alpco diagnostics , salem , nh ) .
group differences on each dependent measure were evaluated using one - way anova with a least significant differences post hoc analysis .
a multiple linear stepwise regression analysis was run to help identify factors that may distinguish those who develop cna from those who do not .
all analyses were run using spss 18.0 software ( spss inc . , chicago , il ) with significance set at p < 0.05 .
lower limb nerve conduction parameters were measured bilaterally by surface stimulation of the sural , peroneal , and tibial nerves .
measurements included peroneal and tibial motor nerve amplitude , conduction velocity , distal onset latency , and f wave .
all motor amplitudes were measured baseline to peak , and latencies were taken as onset latency .
motor conduction velocity was calculated as total conduction velocity ( not segmental ) using onset latencies throughout .
sensory amplitude was taken as baseline to peak , latency was reported as onset latency , and conduction velocity was calculated across the single segment using onset latency ( neuromax , excel - tech ltd . ,
quantitative sensory testing was performed using the medoc device according to previously published methods ( 17 ) .
warm and cold thermal thresholds and heat and cold pain were tested using the tsa2001/vsa3001 computer - driven sensory measuring device ( medoc , durham , nc ) .
vibration perception threshold was quantified using the medoc device and the  method of limits  protocol , where repeated , increasing vibratory stimulation is applied to the plantar aspect of the dominant great toe .
autonomic function was assessed by three tests using the anx-3.0 autonomic monitor : heart rate variability during deep breathing at six breaths per min ( e / i ratio ) and r / r variation in response to the valsalva maneuver and postural change ( ansar medical technologies , inc . ,
bmd scans of the whole body , bilateral proximal femurs , and bilateral feet were obtained using dual - energy x - ray absorptiometry ( ge lunar prodigy advance , ge healthcare , waukesha , wi ) .
scans were performed according to the manufacturer s protocols , with the exception of the feet , for which no standardized protocol exists .
mode , where a subject sat on the edge of the scan bed with the knee bent at 90 and the foot flat on the bed .
the laser marker was positioned at the midline of the ankle , at the level of the lateral malleolus . during analysis ,
the region of interest was selected manually and included only the metatarsals and phalanges to exclude any metal fixators .
test - retest reliability for this measure is high ( r = 0.96 , n = 31 ) , and the coefficient of variation is < 1% for our laboratory .
this corresponds well to other published studies of reproducibility of bmd using the hand mode ( 20 ) , and is also comparable with other regional analyses of bone , where coefficients of variation typically range from 0.5 to 1.2% . because no reference database currently exists for scans of the feet , diabetic patients were compared with sex- and age - matched control subjects .
calcaneal stiffness was determined using quantitative ultrasound imaging ( ge achilles express , waukesha , wi ) .
bone stiffness is automatically computed using an algorithm supplied by the manufacturer : stiffness index = 0.67  bua  0.28  sos  420 , where bua is broadband ultrasound attenuation and sos is the speed of sound through the tissues ( ge healthcare ) . because bone stiffness predicts fracture independent of bmd
, it is considered to be a measure of bone quality that also accounts for factors related to fracture that might not be detected by a measure of bmd alone .
fasting blood samples were obtained by antecubital venipuncture into serum separator tubes and separated immediately by refrigerated centrifugation .
the serum was stored at 80c according to instructions required by the manufacturers of the enzyme - linked immunosorbent assay ( elisa ) kit .
samples were subsequently evaluated in duplicate for srage by the quantikine rage enzyme - linked immunoassay ( r&d systems , minneapolis , mn ) ; cross - linked n - telopeptides of type 1 ( ntx ) collagen , as a measure of bone resorption ( osteomark ntx elisa / eia , wampole laboratories , princeton , nj ) ; and osteocalcin , as a measure of bone formation ( alpco diagnostics , salem , nh ) .
group differences on each dependent measure were evaluated using one - way anova with a least significant differences post hoc analysis .
a multiple linear stepwise regression analysis was run to help identify factors that may distinguish those who develop cna from those who do not .
all analyses were run using spss 18.0 software ( spss inc . , chicago , il ) with significance set at p < 0.05 .
we evaluated 80 adult men , aged 55.3  9.0 years , consisting of 30 healthy control subjects , 30 type 2 diabetic patients , and 20 type 2 diabetic patients diagnosed with stage 2 cna . according to the classification scheme of sanders and frykberg ( 16 ) to describe neuropathic osteoarthropathy patterns of joint involvement ,
28% of cna patients displayed pattern 2 ( tarsometatarsal / lisfranc s joint involvement ) , 45% displayed pattern 3 ( midtarsal and naviculocuneiform joint involvement ) , and 27% displayed pattern 4 ( ankle and subtalar joint involvement ) .
no patients were classified as pattern 1 ( forefoot ) or pattern 5 ( calcaneus ) , which might have otherwise interfered with the measurement of bmd or calcaneal stiffness , or both .
cna patients had similar duration of diabetes but higher hba1c levels than their counterparts with diabetes ( 8.3  1.6 vs. 7.0  2.9% , table 1 ) . fasting serum blood urea nitrogen and creatinine values for all diabetic and cna patients were within the normal reference ranges .
cna and diabetic patients were slightly older , had a greater bmi , and a higher waist - to - hip ratio than control subjects .
diabetic and cna patients showed more peripheral nerve damage than control subjects , and cna subjects exhibited the highest degree of sympathetic and parasympathetic dysfunction and small and large fiber neuropathy compared with the other study groups ( table 2 ) .
peripheral and autonomic neuropathy tests continuous data are expressed as mean  sd ; categoric data as n. * p < 0.05 vs. control subjects . p < 0.05 vs. diabetic and control subjects .
bmd of the proximal femur was slightly higher at the femoral neck and greater trochanter for diabetic patients than for cna or control individuals , but bmd of the feet was not significantly different across groups ( table 3 ) .
calcaneal stiffness was similar for diabetic and control subjects but was markedly reduced in the cna patients ( p < 0.01 ) .
these results indicate that ultrasound imaging may detect features that are not detectable by bmd scans using dual - energy x - ray absorptiometry .
blood biomarkers , bone density , and stiffness data are presented as mean  sd . * p < 0.05 vs. diabetic and control subjects . p < 0.05 vs. control subjects .
cna patients displayed srage values more than seven times lower than control subjects and more than three times lower than diabetic patients ( p < 0.05 ) . across all subjects
, there was a significant positive correlation between the calcaneal bone stiffness index and srage ( r = 0.35 , p < 0.01 ) and a negative correlation between stiffness index and osteocalcin ( r = 0.39 , p < 0.001 ) .
these results demonstrate a cross - sectional relationship between an impaired age defense , increased bone turnover , and reduced bone stiffness . in multiple linear stepwise regression analysis ,
when measures of large and small fiber neuropathy , autonomic neuropathy , blood measures , bmd , and bone stiffness were entered into the model , 74.5% of group membership was predicted by vibration perception , pressure perception , and srage ( r = 0.745 , p < 0.001 ) . similarly , vibration perception and sympathetic valsalva response accounted for 50.3% of the variability in bone stiffness ( r = 0.503 , p < 0.001 ) , indicating a possible relationship of both large fiber and autonomic neuropathies in the progression to cna .
the two most important findings from this study were 1 ) an indication that charcot patients have an impaired rage defense mechanism with an accompanying increase in serum markers of bone turnover , and 2 ) reduced bone stiffness in the calcaneus , not accompanied by reductions in bone mineral density . in this study ,
srage values in cna patients were reduced by 86% , but the diabetic patients had a 50% reduction compared with control subjects .
hyperglycemia generates higher levels of age , but without adequate srage to bind age , it and other ligands tend to accumulate in tissues with slow turnover , such as tendon , skin , bone , amyloid plaques , and cartilage ( 21 ) .
accumulation of age has been shown to reduce osteoblastic activity via an increase in rage that increases rankl activation that leads to enhanced osteoclastogenesis and impaired matrix mineralization ( 10,11,22 ) .
although our biomarker for bone resorption ( ntx ) was not different between groups , this does not preclude the possibility that bone may be inadequately mineralized and not contributing to bone strength .
studies have demonstrated that bone toughness and stiffness are affected by age cross - link formation , and the elastic modulus is independent of other determinants of bone strength such as bone density and microarchitecture ( 23,24 ) .
our finding that bone stiffness detected by quantitative ultrasound imaging was markedly lower in cna subjects , despite bmd values in the feet and proximal femur within normal reference ranges , supports the notion that bone stiffness is not necessarily correlated with bmd in certain disease states .
elevated bone formation activity in charcot patients is likely the result of an accumulation of microfractures , and an excessive attempt at bone repair to maintain bone integrity of the foot .
so taken together , our finding that cna patients display higher levels of bone formation markers and reduced srage may suggest that cna patients are susceptible to age - mediated osteoblast apoptosis , thereby limiting their ability to repair bone .
this represents another possible pathway linking cna to bone loss besides the rankl / osteoprotegerin pathway
. it would be valuable to investigate whether certain drugs that raise rage levels ( e.g. , ace inhibitors , statins , and glitazones ) ( 25 ) , may also have a role in the prevention of charcot progression in patients with severe neuropathy .
we have shown , along with others , that charcot patients display significantly worse autonomic neuropathy ( both sympathetic and parasympathetic ) than diabetic or control subjects .
this is an important finding , because it has recently been suggested that the presence of cna may coincide with a loss of sympathetic activity that allows shunt vessels to open , which increases perfusion of the feet and may speed bone resorption ( 12 ) .
simple autonomic function tests may indeed be useful tools to detect an increased predisposition to developing cna in those with diabetes .
a limitation of our study was the slightly older age of the diabetic and cna patients compared with control subjects , despite an attempt to age - match subjects .
in addition , bone stiffness and rage levels were still different between diabetic and cna individuals , despite the presence of diabetes for a similar duration .
therefore , we were not concerned that these comparisons were confounded by the 5-year difference in average age between the diabetic and cna subjects and the control group .
although all of our cna patients were in the chronic stage 2 of the disease , we did include patients who had recently undergone operative stabilization .
this type of stabilization , however , does not represent a  cure  or even improvement of the underlying disease process .
it is possible , however , that postsurgical cna patients may possess a different biochemical profile than presurgical patients .
none of our cna patients had involvement of the calcaneus or metatarsals , so we were confident that our measures of bone stiffness and bmd were accurate .
lastly , we did not evaluate classic inflammation or oxidative stress markers , which would have added significantly to the argument that reductions in srage promote inflammation or oxidative stress , or both . in summary
, we show that there is a spectrum of srage deficiency wherein diabetes reduces srage levels by 50% , suggesting a role in the complications of diabetes .
furthermore , cna patients who have progressed to stage 2 have an 86% reduction in srage and extremely advanced somatic and autonomic neuropathy that may prevent a normal defense against oxidative stress and may predispose them to reductions in bone integrity and strength . to further elucidate the deleterious effects of cna on collagen and bone quality ,
future studies could evaluate components of bone quality such as bone microarchitecture and mineralization from calcaneal biopsy specimens of cna patients .
in addition , it would be very valuable to fully explore the protective potential of medications that elevate rage on bone formation pathways in this vulnerable population .
 objectivethis study investigated the relationship between circulating soluble receptor for advanced glycation end products ( srage ) and parameters of bone health in patients with charcot neuroarthropathy ( cna).research design and methodseighty men ( aged 55.3  9.0 years ) , including 30 healthy control subjects , 30 type 2 diabetic patients without charcot , and 20 type 2 diabetic patients with stage 2 ( nonacute ) cna , underwent evaluations of peripheral and autonomic neuropathy , nerve conduction , markers of bone turnover , bone mineral density , and bone stiffness of the calcaneus.resultscna patients had worse peripheral and autonomic neuropathy and a lower bone stiffness index than diabetic or control individuals ( 77.1 , 103.3 , and 105.1 , respectively ; p < 0.05 ) , but no difference in bone mineral density ( p > 0.05 ) . 
 cna subjects also had lower srage levels than control ( 162 vs. 1,140 pg / ml ; p < 0.01 ) and diabetic ( 162 vs. 522 pg / ml ; p < 0.05 ) subjects , and higher circulating osteocalcin levels.conclusionscna patients had significantly lower circulating srage , with an accompanying increase in serum markers of bone turnover , and reduced bone stiffness in the calcaneus not accompanied by reductions in bone mineral density . 
 these data suggest a failure of rage defense mechanisms against oxidative stress in diabetes . 
 future studies should determine if medications that increase srage activity could be useful in mitigating progression to cna . 
RESEARCH DESIGN AND METHODS
Nerve conduction studies
Quantitative sensory tests
Autonomic function tests
BMD
Calcaneal stiffness
Blood measures
Statistical analysis
RESULTS
CONCLUSIONS