Transient severe metastatic calcification in acute renal failure Metastatic calcification , a known complication of prolonged end-stage renal disease , is herein described for the first time in a 10-month-old boy with acute renal failure , manifesting as a painful and swollen arm . Imaging revealed diffuse calcification and technetium-99 methylene diphosphonate -LRB- 99Tc-MDP -RRB- uptake around the humerus and axilla . Calcium and vitamin D restriction , followed by intravenous administration of sodium thiosulfate caused a full symptomatic , radio - and scintigraphic improvement . Introduction Vascular -LRB- `` metastatic '' -RRB- calcification -LRB- MC -RRB- -LRB- or calcific uremic arteriolopathy -RRB- is the most common type of extra-osseous calcification in end stage renal disease , manifesting as both medial and intimal calcification of large arteries -LSB- 1 -RSB- . It usually results from a deposition of calcified products in otherwise normal tissues as a result of hyperphosphatemia with or without hypercalcemia . It may affect the visceral organs , such as kidneys , lungs , and gastric mucosa , as well as joints , eyes , and skin -LSB- 2 -RSB- . The predisposing conditions of hyperphosphatemia and high -LRB- calcium × phosphorus -RRB- concentration products need to be persistent over weeks and months , since MC has been not been described in the setup of acute renal failure -LRB- even though a similar biochemical profile is seen there too -RRB- . We describe a case of MC in a 10-month-old child presenting with hemolytic uremic syndrome -LRB- HUS -RRB- and acute renal failure -LRB- ARF -RRB- . Case report A 10-month-old male infant was recovering from his first episode of atypical HUS . He belonged to a Bedouin tribe previously diagnosed by us -LSB- 3 -RSB- with recessive factor H deficiency . His clinical course was remarkable for microangiopathic hemolytic anemia , oliguric renal failure and hypertension with a depressed myocardial function , associated with resistant hypocalcemia -LRB- total calcium as low as 5.4 mg/dl -RRB- and hyperphosphatemia -LRB- as high as 13 mg/dl -RRB- . The latter was eventually controlled with phosphor binders -LRB- aluminum hydroxide at first for several days and , later , calcium gluconate -RRB- , intravenous administration of calcium -LRB- mainly due to an episode of cardiac arrest without hyperkalemia , as previously described for HUS -LSB- 4 -RSB- -RRB- , low phosphorus diet and alphacalcidol , 0.25 μg q.d. -LRB- the latter only after serum phosphorus had been normalized -RRB- . Medications were given through a right femoral vein catheter and peripheral veins in the right arm . Peritoneal dialysis was started , using regular calcium -LRB- 3.5 meq/l -RRB- dialysate . Fresh frozen plasma -LRB- 20 ml/kg -RRB- was given daily until there was evidence of HUS remission , and was then switched to 20 ml/kg twice a week , as previously done in homozygous factor H-deficient children -LSB- 5 -RSB- . Ten days after hospitalization , he was found to have a painful and mildly swollen left arm , mainly in the elbow area , with flexion limitation . There was no intravenous device in this arm . There were no high fever , local redness or swollen joints . The plain X-ray did not reveal any lesions or fractures . A week later the left shoulder and humeral area were more swollen and tender , with no specific nodule or bone deformity . On physical examination , the swelling was deeper than the dermis -LRB- mainly in the humeral area -RRB- and painful . The superficial skin did not show signs of ischemia , necrosis , ulceration or livedo reticularis . This time the plain X ray revealed diffuse calcification , involving mostly the arm muscles and the axilla -LRB- Fig. 1a -RRB- . Calcium and phosphorus levels were 9.9 mg/dl and 6.6 mg/dl , respectively . Renal function remained stable but impaired -LRB- serum creatinine and urea concentrations were as low as 1.05 mg/dl and 90 mg/dl , respectively -RRB- . Parathyroid hormone -LRB- PTH -RRB- levels were normal -LRB- 25.7 pg/ml -RRB- . Renal sonogram showed no evidence of nephrocalcinosis . A bone scan revealed significant technetium-99 methylene diphosphonate -LRB- 99Tc-MDP -RRB- uptake along the areas that had seemed to be affected on the X-rays and physical examination -LRB- Fig. 1b -RRB- . Treatment with peritoneal dialysis , alphacalcidol and calcium salts was stopped . Ketoconazole 1 mg/kg per day was given for 3 days , in addition to prednisolone 2 mg/kg per day for a week . Renal function improved , but the -LRB- Pi × Ca -RRB- products remained above 60 mg2/dl2 . No improvement in the arm lesion was seen . On day 25 of hospitalization , intravenously administered sodium thiosulfate , 100 mg/kg q.d. , was started for 12 days . A gradual symptomatic improvement in arm range of movement and resolution of local pain was seen . At follow-up 8 weeks afterwards , X - ray and bone scan revealed significant resolution of MC -LRB- Fig. 2 -RRB- . At long-term -LRB- > 12 months -RRB- follow-up , no recurrence of MC was seen , in spite of recurrent events of HUS , eventually needing periods of peritoneal and hemodialysis . Fig. 1Chest X-ray on day 18 -LRB- a -RRB- black arrow calcification on the axilla , arrowheads calcification lines along the humerus . Tc-99m MDP scintigraphy -LRB- b -RRB- shows increased abnormal uptake in the soft tissue of the left shoulder , humerus and left scapula . Kidneys are enlarged , with increased diffuse abnormal radiotracer activityFig . 2Repeated X-ray -LRB- a -RRB- and bone scan -LRB- b -RRB- after 1 month show no abnormal findings Discussion The unique MC features in our patient were its appearance early after the diagnosis of renal failure and its transient nature . Evidence for such early appearance of this complication comes only from an animal model of ARF -LRB- adenine-induced renal failure in rats -RRB- , where microscopic metastatic calcification in aorta , coronary artery and other soft tissues were found after 4 weeks of uremia -LSB- 6 -RSB- . This was associated with hyperparathyroidism , hypocalcemia and hyperphosphatemia . In addition , a high index of suspicion may detect the MC early in the renal failure course , before it becomes ulcerated -LSB- 7 -RSB- . Current sophisticated imaging techniques suggest an incidence of subclinical or asymptomatic MC in 20 -- 100 % of patient with end-stage renal disease -LRB- ESRD -RRB- -LSB- 8 -RSB- . MC with soft tissue involvement in acute renal failure , to the best of our knowledge , has not been reported in children : using the PubMed and MESH search systems , and the terms `` calcinosis '' and `` kidney failure , acute '' -LRB- the term `` calciphylaxis '' is not an official indexing term -RRB- , we found only three case reports of adults with such combination . There was a combination of risk factors for MC in our patient , including high dose parenteral calcium administration via both intravenous and peritoneal dialysis ; high serum phosphorus levels due to renal failure and the hemolytic state ; and the use of alphacalcidol -LSB- 9 -RSB- . These may have led to MC so early in the course of his disease . The possible correlation between renal failure due to HUS , a state where the basic damage is to the endothelium in small vessels , and MC is not supported by the literature search . Uremic calcification was thought to be a largely physico-chemical process facilitated by elevated Ca × P -LRB- i.e. , `` metastatic '' calcification -RRB- . Evidence now shows that vascular smooth muscle cells actively take up phosphate to form bio-apatite . This process is associated with a phenotypic transformation of vascular smooth muscle cells during which they express osteoblast markers -LSB- 10 -RSB- . Soft tissue accumulation of Tc99m MDP may be due to passive localization of tracer in slow fluid spaces -LRB- ascites , pleural effusion -RRB- when the blood concentration of tracer is high and these spaces do not clear as rapidly as the blood pool . Tracer may also bind in necrotic tissues -LRB- myositis , myonecrosis -RRB- . Other causes of metastatic calcification include hypercalcemia from widespread destruction of bone -LRB- from metastatic cancer -RRB- , parathyroid neoplasm and hypervitaminosis D . When the solubility product of calcium and phosphate is exceeded , there is precipitation of calcium in the extracellular space . Soft tissue calcification is , thus , another cause of soft tissue uptake of Tc99m MDP -LSB- 11 -RSB- . Reported therapeutic strategies for treating and preventing MC include : increasing dialysis dose , lowering serum calcium phosphorous and Ca × P solubility products , and avoiding calcium-based Pi binders and vitamin D analogs . Unfortunately , these methods have been ineffective in some patients with MC . Intravenously administered sodium thiosulfate -LRB- ST -RRB- increases the solubility of calcium deposits -LSB- 12 -RSB- . ST was shown to be successful in the treatment of both nephrolithiasis and tumoral calcinosis . ST has antioxidant effects on endothelial cells , but its exact mechanism of effect is unclear . It undergoes mainly renal clearance . Its half-life is increased from 15 min in the normal glomerular filtration rate -LRB- GFR -RRB- to 478 min in dialysis-dependent patients . The recommended adult dose is 12.5 -- 25 g at the end of dialysis . The duration of this treatment is unclear . In our patient , a short ST course plus the withdrawal of enteral calcium and vitamin D were sufficient to resolve MC , which did not recur in the following events of HUS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hemolytic uremic syndrome
calcific uremic arteriolopathy
sodium thiosulfate
peritoneal dialysis


Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women Summary Vitamin K mediates the synthesis of proteins regulating bone metabolism . We have tested whether high vitamin K2 intake promotes bone mineral density and bone strength . Results showed that K2 improved BMC and femoral neck width , but not DXA-BMD . Hence high vitamin K2 intake may contribute to preventing postmenopausal bone loss . Introduction Since the discovery of osteocalcin , the vitamin K-dependent protein synthesized by bone tissue , attempts have been made to correlate bone vitamin K status with bone strength . The way in which these variables were measured varied in different studies . Obviously , the best way to assess bone strength is monitoring fracture incidence in a large study cohort over a long period of time . Unfortunately , this method is expensive and time consuming . Therefore , most authors have used bone mineral density as measured by DXA -LRB- DXA-BMD -RRB- as a surrogate marker for bone strength . A disadvantage of this technique is that it gives the amount of calcium per area , but does not take into account the three dimensions of the bone -LSB- 1 -RSB- . Findingreliable markers for bone vitamin K status turned out to be even more difficult . Dietary vitamin K may occur in different forms : K1 -LRB- also known as phylloquinone -RRB- and K2 , which is a group name for a series of related compounds also known as menaquinones -LSB- 2 -RSB- . The various menaquinones differ structurally in the length of their isoprenyl side chain , which may contain four isoprenoid residues in menaquinones-4 -LRB- MK-4 -RRB- up to nine residues in menaquinones-9 -LRB- MK-9 -RRB- . Still higher menaquinones have been described , but are increasingly rare in the diet . Dietary K1 originates mainly from green , leafy vegetables , whereas menaquinones are produced by bacteria and are mostly found in fermented foods such as cheese and the Japanese food natto -LRB- fermented soy beans -RRB- -LSB- 3 , 4 -RSB- . Synthetic forms of vitamin K available in food supplements or pharmaceutical preparations are K1 and MK-4 , recently also the natural MK-7 has become available . The recommended daily intake of vitamin K -LRB- all forms -RRB- is between 100 and 120 μg/day , but this value is based on the hepatic vitamin K requirement for blood clotting factor synthesis . The requirements of other tissues have not yet been defined , but are probably higher . In 1984 , Hart and coworkers described a case-control study in which patients with femur neck fractures had lower circulating vitamin K1 concentrations than age - and sex-matched controls -LSB- 5 -RSB- . In a second study from the same authors it was found that subjects with low serum vitamin K1 concentrations had a lower DXA-BMD than those with normal or high serum vitamin K1 -LSB- 6 -RSB- . A potential bias in these studies is that low circulating vitamin K may be a marker for general poor nutritional status and that low DXA-BMD and increased fracture risk are associated with the combined deficiency of essential minerals , vitamins and proteins rather than with vitamin K alone . Others have published population-based studies in which an inverse correlation was found between fracture risk and vitamin K1 intake -LSB- 7 , 8 -RSB- . It was found that the association between vitamin K1 intake and fracture risk was much stronger than that between vitamin K1 intake and DXA-BMD -LSB- 9 -RSB- . A drawback of these studies is that no attempts were made to also monitor vitamin K2 intake . Although in the western society K2 forms only 10 -- 20 % of the total vitamin K intake -LSB- 3 , 4 -RSB- , it is absorbed from the food matrix much better than K1 and may cover 50 % of the total vitamin K absorbed . Moreover , it has been demonstrated that vitamin K2 rather than K1 is preferentially taken up by extrahepatic tissues such as bone and arteries -LSB- 10 -RSB- . Hence also vitamin K1 intake seems to be a questionable marker for bone vitamin K status . Possibly the best marker available today is circulating undercarboxylated osteocalcin -LRB- ucOC -RRB- . Either the absolute concentration , the ratio between ucOC and total osteocalcin , or the ratio between ucOC and carboxylated osteocalcin -LRB- cOC -RRB- are used in the literature . The group of Delmas -LSB- 11 -- 13 -RSB- and Luukinen et al. -LSB- 14 -RSB- reported a number of independent studies suggesting a strongly increased fracture risk in populations with elevated serum ucOC ; later studies by the French group -LSB- 15 -RSB- and others -LSB- 9 , 16 -RSB- showed that ucOC was inversely associated with DXA-BMD . It is at least remarkable that in all approaches , associations between vitamin K status and fracture risk were demonstrated more readily than those between K status and DXA-BMD . In a supplementation study with vitamin K1 alone no effect on DXA-BMD was observed during a 2-year treatment with 10 mg/day -LSB- 17 -RSB- . In another study from the same group , however , a synergistic effect of 1 mg/day of K1 with minerals and vitamin D -LSB- 18 -RSB- was found . In this study the rate of bone loss in postmenopausal women was reported to be 35 % lower in the presence of minerals and vitamins K1 and D as compared to the group receiving minerals and vitamin D without vitamin K . A number of Japanese studies reported variable effects of vitamin K2 alone , but substantial beneficial effects if combined with vitamin D -LSB- 19 -RSB- . It has been reported by many different authors that in the healthy population serum osteocalcin is incompletely carboxylated , which is indicative for poor vitamin K status of bone -LSB- 11 , 14 , 16 , 20 -RSB- . Based on our present knowledge of vitamin K-dependent proteins , it must be expected that non-carboxylated osteocalcin is not functional . Since the molecular function of osteocalcin has remained unclear thus far , we do not know whether it contributes to bone strength at all . From transgenic osteocalcin-deficient mice , we know that osteocalcin contributes to regulating the form and the dimensions of bone -LSB- 21 -RSB- . This may also be important for humans . The importance of bone geometry for bone strength is rapidly gaining interest -LSB- 22 -- 26 -RSB- . Although DXA-BMD is still the determinant most generally used in the clinical evaluation of hip fracture risk , it has been stipulated that its uncritical use may lead to size-related artifacts in the estimation of bone strength and in the identification of fracture risk -LSB- 27 -RSB- . As was mentioned by Heaney -LSB- 28 -RSB- , the ultimate concern in studying bone status is bone strength . Holding other variables constant , strength will increase both as bone mass increases and as bone size increases . When estimating bone strength , two strategies to also compensate for the dimensions of bone have been proposed . First , it is encouraged that densitometric comparisons between groups are based on bone mineral content -LRB- BMC -RRB- rather than on DXA-BMD -LSB- 28 -RSB- . Second , bone dimensions are used as independent determinants for bone strength . Important geometric parameters are the hip axis length -LRB- HAL -RRB- and the femoral neck width -LRB- FNW -RRB- . Patients with low DXA-BMD or after experiencing a hip fracture had an increased FNW , suggesting an attempt to compensate for the increased fracture risk at this critical site -LSB- 26 -RSB- . On the other hand , it seems obvious that at comparable DXA-BMD a larger FNW will positively contribute to bone strength . In this way it is understandable that also and increase of BMC may contribute to bone strength , although it should be reminded that it is not the mass per se but the distribution of mass that is crucial for bone strength . The importance of combining bone geometry with DXA-BMD for expressing bone strength and fracture risk was realized by Karlamangla et al. , who developed mathematical equations for calculating indices for compression strength , bending strength , and impact strength of the femoral neck -LSB- 29 -RSB- . In these equations , DXA-BMD is combined with HAL , FNW , height and body weight . We have used the same equations to analyse the data from a placebo-controlled intervention trial in which we compared the effects of vitamin K2 on DXA-BMD and bone strength indices in postmenopausal women , who were recruited from the apparently healthy population and thus represent the large group of free living elderly subjects . Additional or synergistic effects of calcium and vitamin D were not included in the design of our study . Subjects and methods Subjects The study was designed for apparently healthy , non-osteoporotic women . Participants were recruited by local newspapers . Inclusion criteria were female gender , age between 55 and 75 years at intake , Caucasian race , apparently healthy , at least 2 years postmenopausal and willingness to sign informed consent . Exclusion criteria were : a history of metabolic bone disease -LRB- s -RRB- or recent bone fractures -LRB- less than one year -RRB- , low bone density -LRB- T-score < − 2.5 -RRB- , ovariectomy , hysterectomy , oral anticoagulant treatment , hormone replacement therapy , treatment with bisphosphonates , calcitonin , prednisone , heparin , or vitamin K-containing vitamin concentrates and food supplements . Also subjects who had received an investigational new drug within the last 12 months were excluded from the trial . In total 325 women met the criteria and were randomized into our study . The participants were pre-stratified according to age : 105 in the age of 55 -- 65 years , and 220 in the age of 65 -- 75 years . The study protocol was approved by the University Hospital medical ethics committee . Masking Participants were identified by a single randomization number according to the randomization schedule generated by the University Hospital Pharmacy using a computer-generated random permutation procedure in the software package SPSS . Participants were randomly assigned to treatment with either 45 mg/day of MK-4 -LRB- menatetrenone , EISAI Co , Tokyo , Japan -RRB- or placebo , and compliance with treatment was decided from pill counts during home visits . The daily dose of MK-4 was given in three capsules of 15 mg each , which had to be taken at three time points spread over the day , preferably after the meal . The participant randomization codes were allocated sequentially in the order in which the participants were enrolled . After completion of all analyses the randomization code was disclosed to the investigator . Measurements The clinical end points chosen to evaluate the effect of the study treatment on bone were bone mineral density -LRB- DXA-BMD -RRB- , bone mineral content -LRB- BMC -RRB- and bone strength indices of the femoral neck . DXA-BMD and BMC were measured at baseline and after 1 , 2 and 3 years of treatment by dual energy X-ray absorptiometry -LRB- DXA -RRB- at the site of the left total hip and femoral neck , as well as the vertebrae -LRB- L2-L4 -RRB- using a Hologic QDR 4500-A -LRB- Waltham MA , USA -RRB- . The in vivo precision -LRB- CV -RRB- after repeated measurements in 10 healthy adults -LRB- 30 -- 40 years old -RRB- was 0.85 % for the spine and 0.78 % for the femur neck in short term -LRB- one day interval -RRB- and 1.03 % and 0.98 % in long term -LRB- one-year interval -RRB- precision measurements . The long-term precision of FNW assessment was 1.53 % . At each time point , standing height and body weight were determined with standardized equipment and the body mass index -LRB- BMI -RRB- was calculated as the weight in kilograms divided by the square of the height in meters . The daily calcium intake was assessed using a validated food frequency questionnaire . Indices for compression strength -LRB- CSI -RRB- , bending strength -LRB- BSI -RRB- and impact strength -LRB- ISI -RRB- at the site of the femoral neck were calculated from the mean femoral neck width -LRB- FNW -RRB- and hip axis length -LRB- HAL -RRB- , together with height , weight and femoral neck DXA-BMD as described by Karlamangla et al. -LSB- 29 -RSB- . The mean FNW was obtained from the 1.5 cm wide femoral neck region of interest -LRB- area -LRB- cm2 -RRB- / 1.5 -LRB- cm -RRB- -RRB- . The HAL reflects the distance along the femoral neck axis from the lateral margin at the base of the greater trochanter to the inner pelvic brim . The equations used were : Sample collection Fasting blood was taken and 2-h fasting urine was collected at baseline and after 3 , 6 , 12 and 36 months of treatment . After blood was taken , it was allowed to clot at room temperature for half an hour . Serum was prepared after centrifugation at 3,000 rpm , during 15 minutes . It was subdivided in small aliquots and kept at − 80 °C until use . Urine was collected as the second morning void after an overnight fast . Biochemical measurements In serum the following markers were tested , all based on enzyme immuno assays -LRB- EIA -RRB- : total osteocalcin -LRB- t-OC , BioSource Europe SA -RRB- ; under-carboxylated osteocalcin -LRB- uc-OC -RRB- and carboxylated osteocalcin -LRB- c-OC -RRB- both obtained from Takara Shuzo Co Ltd. , Shiga , Japan ; bone-specific alkaline phosphatase -LRB- BAP , Alkphase B , Quidel/Metra Biosystems San Diego , CA , USA -RRB- ; N-telopeptides of type I collagen -LRB- sNTX , Osteomark sNTx , Ostex International , Inc. , Seattle , WA , USA -RRB- and 25-Hydroxy vitamin D -LRB- 25-OH D , OCTEIA 25-hydroxy vitamin D , IDS , UK -RRB- . In the urine we measured free deoxypyridinoline by using an enzyme immunoassay -LRB- DPD , Quidel/Metra Biosystems -RRB- . Urinary calcium -LRB- Ca -RRB- and creatinine -LRB- creat -RRB- were assessed according to standard methods . Dpd and Ca were corrected for the urinary creat concentration . Statistics The required numbers of participants in both age groups were assessed using separate power analyses -LRB- power of 90 % and a significance level of 0.05 -RRB- . In the younger age group it was assumed that in the placebo group the mean bone loss is approximately 1.5 % -LRB- 4.5 % in 3 years -RRB- . The lowest detectable effect was set at an improvement of 30 % -LRB- = 3.15 % bone loss after 3 years -RRB- . Taking into account a drop-out frequency of 12 % per year this group should consist of at least 76 subjects . In the older age group the mean bone loss was assumed to amount 1 % per year -LRB- 3 % in 3 years -RRB- . With a lowest detectable effect of 30 % -LRB- = 2.1 % bone loss in 3 years -RRB- and a drop-out frequency of 19 % per year , the minimal size of this group is 218 subjects . Statistical analysis was performed using the statistical package SPSS -LRB- SPSS Corp , Chicago , IL -RRB- . The calculations are based on the intention-to-treat principle . Drop-outs were included in the analyses , with the last data available in all subsequent measuring points . All follow-up measurements were related to the baseline values and the proportional changes were calculated for each subject . Differences between and within the different groups were assessed with the unpaired and paired Student 's t-test , respectively . All data are given as mean values ± SE . The linear regression model was used to investigate the effect of treatment after adjustment for potential confounders . Change of FNW , HAL and bone strength indices after 3 years of treatment were used as dependent variables and age and BMI as independent variables . Results Baseline measurements and follow-up Three hundred fifty-one women were recruited for the study , 26 of whom were excluded before randomization for different reasons . After randomization there were 161 women receiving MK-4 and 164 women receiving placebo . The MK-4 group consisted of 54 women younger than 65 years of age and 107 women older than 65 years of age ; in the placebo group these numbers were 51 and 113 , respectively . The baseline characteristics of all participants in the placebo and MK-4 group are given in Table 1 . No significant differences were found between both groups . However , when comparing the two age groups there was an age-related increase in weight and BMI and an age-related decrease in height -LRB- Table 2 -RRB- . DXA-BMD and BMC in the older age group were significantly lower at the site of the femoral neck , but not at the total hip and spine . From all serum markers for bone metabolism only BAP was higher in the older age group -LRB- borderline significant -RRB- . Although there was an age-related decrease in serum vitamin D concentration , all participants had levels which were within the normal range at baseline -LRB- Tables 1 and 2 -RRB- and at all subsequent measurements -LRB- data not shown -RRB- . Urinary DPD/creat was markedly lower in the older age group compared to the younger group . Table 1Baseline characteristics of all participants subdivided according to treatment Placebo n = 164MK-4 n = 161P-valueAnthropomorphic variables : Age -LRB- years -RRB- 66.0 ± 0.565.9 ± 0.40.9 Years since menopause17 .7 ± 0.717.2 ± 0.60.6 Weight -LRB- kg -RRB- 71.8 ± 1.070.3 ± 0.90.3 Height -LRB- cm -RRB- 162 ± 0.5161 ± 0.50.2 BMI -LRB- kg/m2 -RRB- 27.3 ± 0.327.1 ± 0.40.7 Calcium intake -LRB- mg/day -RRB- 811 ± 26870 ± 320.1Non-smoking -LRB- % -RRB- 87871.0 Bone density characteristics : DXA-BMD -LRB- g/cm2 -RRB- of : femoral neck0 .688 ± 0.0070.706 ± 0.0080.1 Total hip0 .842 ± 0.0090.861 ± 0.0080.1 Lumbar L2-L40.931 ± 0.0120.929 ± 0.0130.9 BMC -LRB- g -RRB- of : femoral neck3 .54 ± 0.043.65 ± 0.040.06 Total hip30 .6 ± 0.4331.4 ± 0.390.2 Lumbar L2-L443.1 ± 0.7242.4 ± 0.760.6 Hip geometry : Mean FNW -LRB- cm -RRB- 3.43 ± 0.023.45 ± 0.0170.4 HAL -LRB- cm -RRB- 11.6 ± 0.0711.7 ± 0.060.2 Bone strength indices : CSI -LRB- g/kg . m -RRB- 3.41 ± 0.0553.52 ± 0.0520.1 BSI -LRB- g/kg . m -RRB- 1.00 ± 0.0171.04 ± 0.0170.2 ISI -LRB- g/kg . m -RRB- 0.24 ± 0.0040.26 ± 0.0040.03 Serum markers for bone metabolism : c-OC -LRB- ng/mL -RRB- 6.1 ± 0.26.2 ± 0.21.0uc-OC -LRB- ng/mL -RRB- 3.3 ± 0.23.3 ± 0.20.4t-OC -LRB- ng/mL -RRB- 13.2 ± 0.413.2 ± 0.50.7 BAP -LRB- ng/mL -RRB- 23.1 ± 0.723.6 ± 0.80.2 NTX -LRB- nM -RRB- 10.8 ± 0.210.7 ± 0.30.525-OH D -LRB- nM -RRB- 67.2 ± 2.372.3 ± 2.60.09 Urine markers for bone metabolism : Creatinine -LRB- mmol/L -RRB- 3.5 ± 0.33.6 ± 0.21.0 DPD / creat -LRB- μmol/mol -RRB- 7.4 ± 0.27.5 ± 0.20.7 Calcium / creat -LRB- mol/mol -RRB- 0.29 ± 0.020.29 ± 0.010.7 Only the BMC and the impact strength index of the femoral neck in the MK-4 group were slightly higher than in the placebo one . This difference is accounted for by expressing MK-4-induced changes as a percentage of the baseline values . All data are given ± SE.Table 2Baseline characteristics of participants subdivided according to age Age 55 -- 65 n = 105Age 65 -- 75 n = 220P-valueAnthropomorphic variables : Age -LRB- years -RRB- 59.0 ± 0.369.3 ± 0.2 Years since menopause9 .7 ± 0.521.2 ± 0.4 Weight -LRB- kg -RRB- 67.4 ± 1.072.8 ± 0.80.0001 Height -LRB- cm -RRB- 163 ± 0.6161 ± 0.40.003 BMI -LRB- kg/m2 -RRB- 25.4 ± 0.428.1 ± 0.30.0001 Calcium intake -LRB- mg/day -RRB- 816 ± 34858 ± 270.4Non-smoking -LRB- % -RRB- 88870.9 Bone density characteristics : DXA-BMD -LRB- g/cm2 -RRB- of : femoral neck0 .716 ± 0.0100.689 ± 0.0060.02 Total hip0 .871 ± 0.0120.842 ± 0.0070.03 Lumbar L2-L40.940 ± 0.0150.925 ± 0.0110.4 BMC -LRB- g -RRB- of : femoral neck3 .69 ± 0.063.55 ± 0.030.02 Total hip31 .4 ± 0.630.7 ± 0.30.3 Lumbar L2-L444.0 ± 0.942.1 ± 0.60.1 Hip geometry : Mean FNW -LRB- cm -RRB- : 3.44 ± 0.0223.44 ± 0.0160.9 HAL -LRB- cm -RRB- : 11.8 ± 0.0811.6 ± 0.060.03 Bone strength indices : CSI -LRB- g/kg m -RRB- : 3.75 ± 0.0683.31 ± 0.0390.0001 BSI -LRB- g/kg m -RRB- : 1.09 ± 0.0220.98 ± 0.0130.0001 ISI -LRB- g/kg m -RRB- : 0.27 ± 0.0050.24 ± 0.0030.0001 Serum markers for bone metabolism : c-OC -LRB- ng/mL -RRB- 5.9 ± 0.26.3 ± 0.20.2uc-OC -LRB- ng/mL -RRB- 3.3 ± 0.23.2 ± 0.10.8t-OC -LRB- ng/mL -RRB- 13.9 ± 0.612.8 ± 0.40.1 BAP -LRB- ng/mL -RRB- 21.8 ± 0.824.1 ± 0.70.05 NTX -LRB- nM -RRB- 11.0 ± 0.310.6 ± 0.20.225-OH D -LRB- nM -RRB- 76.5 ± 2.766.3 ± 2.30.005 Urine markers for bone metabolism : creatinine -LRB- mmol/L -RRB- 3.5 ± 0.33.6 ± 0.20.8 DPD / creat -LRB- nmol/mmol -RRB- 8.2 ± 0.27.1 ± 0.20.0001 Calcium / creat -LRB- mmol/mmol -RRB- 0.29 ± 0.020.29 ± 0.010.9 The differences in all age-related variables were highly significant . All data are given ± SE . After 3 years 257 women had completed the study : 133 women in the MK-4 group and 124 women in the placebo group . In total 68 women stopped during the study . This was 21 % of the total . The largest group of participants -LRB- n = 30 -RRB- stopped during the first 3 months of the trial . The most important reasons for discontinuation in this time period were various health problems -LRB- n = 18 -RRB- , low DXA-BMD -LRB- n = 17 -RRB- , lack of motivation -LRB- n = 10 -RRB- and complaints about the capsules -LRB- n = 9 -RRB- . Subjects who stopped because of low DXA-BMD did so after consultation during the trial with their general practitioner ; there were two complaints about the capsules in the MK-4 group and seven in the placebo group . Two participants died during the trial : one brain tumor and one cerebrovascular accident . Both casualties were in the placebo group . In total , 36 negative or positive side effects were mentioned by the participants . In the MK-4 group 16 women experienced negative effects and four experienced positive effects of the capsules ; in the placebo group the numbers were 15 and one , respectively . The most frequent negative effects were gain of weight and gastro-intestinal complaints . However , there was no difference in total numbers of complaints between the MK-4 and the placebo group . Effect of MK-4 supplementation Effects on serum markers -LRB- if detectable -RRB- were maximal after 12 months of treatment , with no further increase of the difference from placebo at 36 months . In Fig. 1 we have plotted the changes of bone markers in serum during the first year . As was to be expected , a statistically significant effect of vitamin K treatment was observed on the serum concentrations of cOC and ucOC . The markers for bone formation -LRB- tOC and BAP -RRB- were also significantly higher in the MK-4 group as compared to the placebo one . No effect was observed on markers for bone resorption , or on DXA-BMD at any of the sites measured -LRB- Fig. 2a -RRB- . Also urinary calcium excretion was not affected by MK-4 treatment -LRB- data not shown -RRB- . It turned out that in the MK-4 group the BMC of the femoral neck decreased at a significantly lower rate than in the placebo one -LRB- Fig. 2b -RRB- , and that this effect was paralleled by a significant increase in the femoral neck width , suggesting bone accrual at this critical site -LRB- Fig. 2c -RRB- . The hip axis length -LRB- HAL -RRB- seemed to slightly increase with age in an MK-4 independent manner . From the DXA-BMD , the FNW and the HAL we have calculated the various indices for strength of the femur neck , and it resulted that MK-4 had a significant effect on all three indices -LRB- Fig. 2d -RRB- . Notably the bending strength remained almost constant after the three years of intervention . Fig.1EffectofMK-4 on markers for bone metabolism during the first year of treatment . Bone formation markers are presented in graphs a -- d -LRB- cOC , ucOC , tOC and BAP , respectively -RRB- and bone resorption markers in graph E and F -LRB- NTX and DPD/creat respectively -RRB- . Closed symbols : MK-4 ; open symbols : placebo . Error bars represent SE . Significance of differences MK-4 compared to placebo : # : p < 0.05 ; ## : p < 0.005 Fig. 2Effects of MK-4 on bone after 3 years of treatment . All data are expressed as a percentage of the respective baseline values . Graph A shows the data of DXA-BMD of the femoral neck -LRB- DXA-BMDFN -RRB- , total hip -LRB- DXA-BMDtotalhip -RRB- and lumbar spine -LRB- DXA-BMDLS -RRB- ; graph B shows the BMC of the femoral neck -LRB- BMCFN -RRB- , total hip -LRB- BMCtotalhip -RRB- and lumbar spine -LRB- BMCLS -RRB- ; graph C shows the femoral neck width -LRB- FNW -RRB- and hip axis length -LRB- HAL -RRB- and graph D shows the indices of the compression strength -LRB- CSI -RRB- , bending strength -LRB- BSI -RRB- and impact strength -LRB- ISI -RRB- . Open bars : placebo ; hatched bars : MK-4 . Error bars represent SE . Significance of change from baseline : * : p < 0.05 , ** : p < 0.005 ; significance of difference MK-4 compared to placebo : # : p < 0.05 , ## : p < 0.005 Using a linear regression model , we have tested the statistical significance of the MK-4 induced effects . As is shown in Table 3 , the differences in FNW , CSI , BSI and ISI between the MK-4 and placebo group were significant , both before and after adjustment for age and BMI . The effects on BMC , FNW and bone strength indices were observed for both age groups . As is shown in Fig. 3 the loss of bone strength in the MK-4 supplemented group was almost negligible in the group of 65 years and older . Table 3Effect of MK-4 treatment on geometrical variables Unadjusted adjusted mean difference -LRB- SE -RRB- pMean difference -LRB- SE -RRB- pFNW -LRB- % -RRB- +1.30 -LRB- 0.50 -RRB- 0.01 +1.34 -LRB- 0.50 -RRB- 0.009 HAL -LRB- % -RRB- − 0.25 -LRB- 0.26 -RRB- 0.3 +0.23 -LRB- 0.22 -RRB- 0.9 CSI -LRB- % -RRB- +2.26 -LRB- 0.77 -RRB- 0.004 +2.03 -LRB- 0.76 -RRB- 0.008 BSI -LRB- % -RRB- +3.80 -LRB- 1.2 -RRB- 0.002 +3.83 -LRB- 1.2 -RRB- 0.001 ISI -LRB- % -RRB- +1.98 -LRB- 0.82 -RRB- 0.02 +1.72 -LRB- 0.79 -RRB- 0.03 The increase/decrease of each variable was calculated as a percentage from its baseline value . The differences between the change in MK-4 group and that in the placebo group are given . A positive value reflects a more pronounced effect in the MK-4 , a negative value reflects a more pronounced effect in the placebo group . The values are given before and after adjustment for age and BMI , adjusted values are quoted at the mean values for age and BMI of the total group -LRB- age = 65.9 years ; BMI = 27.2 kg/m2 -RRB- . Fig. 3Effect of MK-4 on bone strength indices in 2 different age groups -LRB- 55 -- 65 and 65 -- 75 years of age -RRB- . All data are expressed as the mean percent change relative to the respective baseline values after 3 years of treatment with either MK-4 or placebo . Graph A shows the compact strength index -LRB- CSI -RRB- , graph B the bending strength index -LRB- BSI -RRB- and graph C the impact strength index -LRB- ISI -RRB- . Open bars : placebo ; hatched bars : MK-4 . Error bars represent SE . Significance of change from baseline : * : p < 0.05 , ** : p < 0.005 ; significance of difference MK-4 compared to placebo : # : p < 0.05 Discussion In this paper we demonstrate that the main effect of vitamin K2 on bone in the hip is an increase of the femoral neck BMC and width , resulting in maintenance of the calculated bone strength even at decreasing DXA-BMD after the menopause . This makes K2 to an interesting compound for combination therapy with other food supplements -LRB- calcium , vitamin D -RRB- or drugs -LRB- bisphosphonates -RRB- with known effects on also DXA-BMD . The potential synergism between K2 and other supplements/drugs , however , requires further investigation . In the literature there are many indications that vitamin Kcontributes to the maintenance of optimal bone strength . Besides various population-based studies and a number of clinical intervention trials investigating the effects of increased vitamin K intake , it has also been reported that subjects receiving vitamin K antagonists -LRB- oral anticoagulant therapy -RRB- have lower DXA-BMD and increased fracture risk -LSB- 30 -- 32 -RSB- . Also experimental animals developed osteopenia upon treatment with the vitamin K antagonist warfarin -LSB- 33 -RSB- . On the other hand we must conclude , however , that in intervention trials the reported effects on DXA-BMD of vitamin K alone were small ; most studies published thus far indicate that only in combination with calcium and vitamin D , vitamin K has a synergistic effect on DXA-BMD -LSB- 18 , 19 , 34 -RSB- . Osteocalcin is the most abundant vitamin K-dependent protein in bone , and the phenotype of transgenic osteocalcin-deficient mice suggests that it is primarily involved in the correct arrangement of hydroxyapatite crystals and in structuring the correct dimensions of bone -LSB- 21 -RSB- . Both factors may contribute to the total bone strength . Hence studies in which DXA-BMD is the only clinical endpoint do not give conclusive evidence on bone strength . If , for instance , the femoral neck DXA-BMD remains constant but increases in width the bone will become stronger and more resistant to fracture . Therefore , we have used mathematical procedures for calculating three indices for bone strength at the site of the femoral neck . Despite the very high dose of MK-4 used in the present trial , no effect was found on the DXA-BMD of the femoral neck , the total hip or the lumbar spine . On the other hand , significant positive effects were observed both on the femoral neck BMC and width . The relation between DXA-BMD , BMC and FNW can be seen from the formula : DXA-BMD = BMC/area , where FNW is one of the dimensions of the surface area . In case FNW is increasing , the projected area of the DXA-BMD measurement will increase too . In case BMC remains constant , the DXA-BMD values will decrease . In case FNW increases and BMC increases adequately , DXA-BMD remains constant , with a concomitant increase of bone strength . The importance of the FNW for the calculated bone strength is obvious from the equations for all three bone strength indices . Our results showed that during the entire intervention period there was no or very little decrease in bone strength in the K2 treated group , whereas there was substantial loss of bone strength in the placebo one . The favourable effect was found both in the young postmenopausal group -LRB- 55 -- 65 years old -RRB- as well as in the older group -LRB- 65 -- 75 years old -RRB- . Since together with vitamin D and calcium vitamin K was also reported to have a positive effect on DXA-BMD it might even be speculated that the combination of these supplements will result in an increase of bone strength . After we obtained these data , we re-analysed previous studies from our lab , and it turned out that vitamin K1 had only little or no effect on the FNW . It should be pointed out , however , that the doses used in these studies were 1 mg/day and 10 mg/day , which is considerably lower than the 45 mg/day used in the present study . The first question that may be raised is whether the very high dose of vitamin K used in our study is required to obtain the observed effect . The present recommendations for vitamin K intake vary between 90 and 120 micrograms per day , and in a dose of 45 milligrams per day the vitamin is used as a drug rather than as a food supplement . It should be realized , however , that MK-4 has a very short biological halflife time -LRB- 1 -- 2 h -RRB- , which is the reason why it was given three times daily in a dose of 15 mg per helping . Even at this regimen its short halflife will result in fluctuating K2 serum levels , which may explain the requirement of a high dose . At this time no data are available on the efficacy of MK-4 at lower doses , for instance in the range between 100 -- 500 micrograms per day . A possible alternative to MK-4 might be using MK-7 , the K2 vitamin most abundantly found in the Japanese food natto -LRB- fermented soy beans -RRB- . MK-7 has probably a comparable effect as MK-4 -LSB- 35 -RSB- , but it has a halflife in the circulation of 3 days , resulting in more constant plasma levels and accumulation in the blood and various tissues . Therefore , MK-7 is the most logical choice for use as a low-dose food supplement , because even at low intake relatively high blood and tissue levels can be obtained . Clinical trials in which the efficacy of MK-7 is tested are , however , lacking at this time . From our study it is not clear whether the high MK-4 regimen will increase vertebral bone strength in a similar way as observed for the DXA-derived femoral neck bone strength . To our knowledge no reliable formulas are available for calculating vertebral compression strength , the most important strength index for the vertebrae . However , the substantial and significant effect of MK-4 on the lumbar spine BMC warrants more elaborate studies in which the effect on vertebral fracture risk is investigated . A substantial risk reduction for vertebral fractures by long-term MK-4 treatment has also been reported by a number of Japanese studies -LSB- 36 , 37 -RSB- . Another question that needs to be answered is why K2 has a much more pronounced effect than K1 . One reason may be the different doses used : 45 mg/day for MK-4 and 1 -- 10 mg/day for K1 . Based on our present knowledge , all three doses are an excess ; it seems not unlikely , therefore , that there is a qualitative difference between K1 and K2 . The only difference between both vitamins is the aliphatic side chain , which does not influence the coenzyme function , but which does affect the distribution of vitamin K over the lipoproteins in the blood , and the transport to and absorption by the various tissues . It has been demonstrated in human volunteers that both K1 and K2 are transported to the liver via triglycerides , but that only K2 vitamins -LRB- both MK-4 and MK-7 -RRB- are incorporated into the LDL fraction for transport to extrahepatic tissues -LSB- 39 -RSB- . It is not surprising , therefore , that in animal experiments it was found that K1 mainly accumulates in the liver , whereas MK-4 is far more abundant in other tissues including pancreas , bone and vessel wall -LSB- 10 -RSB- . In that perspective , K2 seems to be the most logical choice for supplementation because notably bone and arteries -LRB- and not the liver -RRB- were reported to have a sub-optimal vitamin K status in the majority of the healthy population . A limitation of this study is that it was conducted in non-osteoporotic women , in whom we tried to decrease bone loss and to maintain bone strength . Therefore , this study does not allow conclusions regarding beneficial effects of vitamin K2 on bone quality in women who have been diagnosed with osteoporosis already . A further limitation is that the study was exclusively conducted on women , so that no conclusions can be drawn for bone loss in men . A further limitation of our study was that the DXA-BMD of the spine was determined only in the postero-anterior -LRB- PA -RRB- position . This measurement includes not only the trabecular bone of the vertebral body , but also a substantial amount of cortical bone from posterior elements . During ageing , degenerative changes in the spine such as osteoarthritis or sclerosis of the endplates may occur . In those cases , PA projection may result in incorrect interpretation of the DXA-BMD and BMC values because the observed increase is due to degenerative changes and is not based on treatment effect . Lateral DXA-BMD measurements evaluate exclusively the vertebral body , allowing more sensitive detection of postmenopausal bone loss or DXA-BMD changes during therapy -LSB- 38 -RSB- . At the start of this study lateral DXA-BMD monitoring , however , was not yet available for clinical trials in our hospital . In conclusion we have demonstrated that vitamin K2 is capable of improving both the BMC and bone geometry at critical sites , leading to maintenance of the DXA-derived femoral neck bone strength in postmenopausal women during a 3-year study period . Even in the very high doses used in our study , adverse side effects of MK-4 were minor and not different from the placebo group . Therefore , it seems desirable to evaluate the cost-benefits of supplementing low dose vitamin K2 to all postmenopausal women . The optimal dose , the preferred form -LRB- MK-4 or MK-7 -RRB- , and synergistic effects of complementary food supplements -LRB- calcium , vitamin D -RRB- or medication -LRB- bisphosphonates -RRB- , however , require further research .

O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

bone mineral content
fracture risk
osteoporosis
vitamin k