Undercarboxylated osteocalcin and bone mass in 8 12 year old children with cystic fibrosis

Transcription

1 Journal of Cystic Fibrosis 7 (2008) Undercarboxylated osteocalcin and bone mass in 8 12 year old children with cystic fibrosis M.S. Fewtrell a,, C. Benden b, J.E. Williams a, S. Chomtho a, F. Ginty b, S.V. Nigdikar c, A. Jaffe b a MRC Childhood Nutrition Research Centre, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK b Portex Respiratory Medicine Unit, Great Ormond Street Hospital for Children National Health Service Trust, London WC1N 1EH, UK c MRC Human Nutrition Research, Elsie Widdowson Laboratory, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK Received 9 October 2007; received in revised form 23 November 2007; accepted 28 November 2007 Available online 21 February 2008 Abstract Young adults with cystic fibrosis (CF) frequently develop bone disease. One suggested aetiological factor is suboptimal vitamin K status with impaired carboxylation of osteocalcin and abnormal bone formation. Methods: We measured bone mineralization and turnover in thirty-two 8 12 year old CF patients (14 boys) using Dual Energy X-ray absorptiometry (whole body (WB) and lumbar spine (LS)), 25-OH Vitamin D, PTH and markers of bone formation (plasma osteocalcin, N-terminal pro-peptide of type 1 collagen (P1NP)), plus an indirect measure of vitamin K status, undercarboxylated osteocalcin (uc-oc). Results: LS bone mineral density (BMD) standard deviation (SD) scores were b 1.0 in 20% of subjects. Size-adjusted LS and WB bone mass was normal. Compared to reference data, % uc-oc was high and P1NP low. LS bone mass was predicted by % uc-oc but not other markers (0.4% decrease in size-adjusted LSBMC (p=0.05); 0.04 SD decrease in LSBMAD (p=0.04) per 1% increase in uc-oc). Conclusion: Markers suggestive of sub-optimal vitamin K status and low bone formation were present despite normal size-adjusted bone mass. The association between LSBMC and % uc-oc is consistent with the hypothesis that sub-optimal vitamin K status is a risk factor for CF bone disease. This should ideally be investigated in an intervention trial European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Bone mineralization; Bone turnover; Vitamin K; Undercarboxylated osteocalcin 1. Introduction With earlier diagnosis and improved treatment, the life expectancy of patients with cystic fibrosis (CF) has steadily improved over the last few decades, and is likely to improve Abbreviations: CF, Cystic fibrosis; DXA, Dual energy X-ray absorptiometry; BMD, bone mineral density; BMC, bone mineral content; BMAD, bone mineral apparent density; OC, Osteocalcin; uc-oc, undercarboxylated osteocalcin. This work was supported by funding from the Medical Research Council, UK. Some of the data included in this manuscript were presented at the 2nd International Meeting on Children's Bone Health in Sorrento, May Abstract: Bone mineralization and turnover in children with cystic fibrosis. Fewtrell MS, Benden C, Williams JE, Biassoni L, Ginty F, Jaffe A. Bone 2005;36:S33(O24). Corresponding author. Tel.: ; fax: address: m.fewtrell@ich.ucl.ac.uk (M.S. Fewtrell). further in the future. However, a number of longer-term health problems have become apparent, including CF bone disease. There are now many reports of bone pain and spontaneous fractures in young adults with CF [1]. Histomorphometry in individuals with low bone mass measured using Dual Energy X- ray Absorptiometry (DXA) shows significantly reduced cancellous bone volume with low bone formation at both tissue and cellular level [2]. A recent study reported normal bone mass in 5 10 year old children with CF but significantly reduced whole body and wrist bone mass in older children and adolescents aged years [3], consistent with the hypothesis that CF bone disease develops or becomes apparent during adolescence and young adulthood. As highlighted and discussed in detail in recent consensus documents [1,4], the aetiology of CF bone disease is likely to be multifactorial, with poor nutritional intake, malabsorption, chronic inflammation and infection, reduced levels of physical /$ - see front matter 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi: /j.jcf

2 308 M.S. Fewtrell et al. / Journal of Cystic Fibrosis 7 (2008) activity, corticosteroid use and pubertal delay all contributing to varying degrees. Recently, the role of sub-optimal vitamin K status has also been debated. This fat-soluble vitamin is vital as a cofactor for the post-translational conversion of glutamyl residues to γ-carboxyglutamyl residues in vitamin K-dependent proteins such as prothrombin and osteocalcin (OC). OC is produced by osteoblasts and is the main non-collagenous protein in bone. In its carboxylated form it binds to hydroxyapatite in bone and is believed to play a regulatory role in bone formation, mineralization and resorption, positively affecting bone quality [5,6]. In contrast, undercarboxylated OC (uc-oc) binds less effectively to hydroxyapatite and a significant association has been found between fracture incidence and uc-oc in elderly subjects [7,8]. The liver is more efficient than bone in using available vitamin K. Hence uc-oc is the first functional marker to appear in vitamin K deficiency and the last to respond to supplements. Furthermore, it is regarded as a better marker of vitamin K status than serum vitamin K concentrations which vary markedly according to recent dietary intake [9 11]. Although routinely supplemented with other fat-soluble vitamins (A, D and E), in many UK centres CF patients do not routinely receive vitamin K supplements [12]. Some degree of uncarboxylation of OC is present in healthy individuals [9,10], but several studies have reported high proportions of uc-oc as well as other indicators of sub-optimal vitamin K status in patients with CF [13 17]. Moreover, supplementation with vitamin K has been shown to reduce the concentration of Proteins Induced by Vitamin K Absence (PIVKA-II) [15], the proportion of uc-oc [14] and the absolute concentration of uc- OC [17]. However, there are few data from populations with simultaneous measurements of vitamin K status and bone mass, hence a relationship between sub-optimal vitamin K status and CF bone disease has not been established. The aim of our study was to measure bone mass and markers of bone turnover in a population of 8 12 year old children with CF, and specifically to investigate the relationship between these parameters and the proportion of uc-oc as a measure of sub-optimal vitamin K status. 2. Methods Children aged 8 to 12 years attending the CF clinic at Great Ormond Street Hospital for Children (London, UK) for annual review were recruited and studied between October 1st 2002 and September 30th The complete study protocol included measurement of body composition (not reported here) using whole body plethysmography, which requires the subject to remain still with a regular quiet breathing pattern for a few minutes at a time. Children who were considered to be too unwell to comply with this measurement due to their respiratory status were excluded from the study. Parents or guardians gave informed consent and assent was obtained from the child. The study was approved by the Great Ormond Street Hospital for Children National Health Service Trust and Institute of Child Health Research Ethics Committee. All children had a DXA scan (Lunar Prodigy, GE Medical Systems, US; Encore 2002 software) of whole body (WB) and lumbar spine (LS) performed whilst wearing light indoor clothing. The radiation exposure was approximately 2.2 μsv. Weight was recorded using digital scales and height measured using a stadiometer. Pubertal stage was self-assessed using line drawings showing the different Tanner stages for breast or genital development and for pubic hair. Children were classified as pre-pubertal if they rated themselves stage 1 for both parameters, and as pubertal if any parameter was rated as 2 or above. Blood was obtained for measurement of markers of bone formation (plasma osteocalcin (N-Mid osteocalcin, Nordic Bioscience Diagnostics A/S, Herlev, Denmark) and N-terminal pro-peptide of type 1 collagen (P1NP; Orion Diagnostica, Finland) and for PTH and 25-OH vitamin D (DiaSorin LTD, Wokingham, UK). Samples were obtained in a non-fasting state at the time of routine sampling for clinical purposes. Plasma or serum were separated as soon as possible and stored at 80 C prior to analysis. Samples were run in duplicate and repeated if the %CV was N 10. With the exception of osteocalcin and undercarboxylated osteocalcin, all samples were analysed in one run. 25-OH vitamin D measurements were monitored by the vitamin D external quality assurance scheme (DEQUAS). Plasma total OC (t-oc) and uc-oc were analysed by onestep ELISA. This assay measures intact osteocalcin and the large N-terminal-mid molecule fragment. Uc-OC was measured using the same assay following a hydroxyapatite binding stage in a modification of the method described by Gundberg [10]. Intra-assay CVs were 3.3% for t-oc and 5.1% for uc-oc. The percentage of uc-oc (% uc-oc) was calculated as (uncarboxylated/total) 100. Demographic data (sex, age, and genotype) were collected. Laboratory spirometry (percentage forced expiratory volume in 1 s, FEV 1 ; percentage forced vital capacity, FVC) was performed according to laboratory protocols, based on adult American Thoracic Society (ATS: [18]) and European Respiratory Society (ERS) standards for spirometry [19]. Computerised frontal chest radiographs were assessed for radiological features of CF lung disease using the modified Chrispin Norman chest radiograph scoring system (CNS), which applies a maximum score of 38 for radiological signs of severe lung pathology [20,21] Statistics Weight, height and Body Mass Index (BMI SD; weight/ height 2 ) scores were calculated using British reference data. WB and LS (L2-4) BMDSD scores for age and sex were obtained from the manufacturer's reference database. To adjust for the effects of bone size, bone mineral apparent density (BMAD) was calculated for the lumbar spine as BMC/BA 1.5 and BMAD SD scores were calculated using our local reference data for children aged 5 19 years [22]. These data were collected by our research group in a project establishing reference bone and body composition data using the same DXA machine over the same period. The power relationship between BMC and BA determined by log log regression for our subjects was in fact 1.3 and did not differ significantly between patients

3 M.S. Fewtrell et al. / Journal of Cystic Fibrosis 7 (2008) and controls. Analyses were repeated with BMAD calculated as BMC/BA 1.3 and results were unchanged. Results are therefore presented here with BMAD calculated as BMC/BA 1.5 for comparison with other studies. We also calculated SD scores for whole body BMC adjusted for bone area and height, again using our local reference data. Associations between variables were assessed using Spearman rank correlation. Multiple regression analysis was used to examine relationships between bone biochemistry parameters and bone mass after adjusting for potential confounding factors. Because % uc-oc may not fully adjust uc-oc for total OC in some populations, t-oc was also added to regression models. 3. Results 32 children (14 boys) aged years were studied and 28 provided a blood sample. Their characteristics are shown in Table 1. All children were pancreatic insufficient and were taking pancreatic enzyme supplements. None had CF-related liver disease. Children were routinely supplemented with vitamin A (5000 IU/day), vitamin E (150 IU/day) and 25-OH vitamin D (600 IU/day). Two children were receiving oral prednisolone at the time of measurement in both cases a short-term 3-week course to treat allergic bronchopulmonary aspergillosis. Six children were taking inhaled steroids (beclomethasone or budesonide). The majority of children were ΔF508 homozygous (n=23). CF lung disease in the study population was mild (FEV 1 N80%) in 58%, moderate (FEV %) in 36% and severe (FEV 1 b40%) in 6%. Modified Chrispin Norman scores ranged between 2 and 22 (median 9). Five children reported a previous fracture. In only one case was this considered to have occurred with minimal trauma Bone mass (Table 2) The mean WBBMD SD score was normal in both prepubertal and pubertal children. Only one child had a SD score below 1.0. Similarly, the mean WBBMC SD score adjusted Table 1 Descriptive data for participants, according to pubertal status (mean (SD)) Pre-pubertal Pubertal n=27 n=5 Age (years) 9.71 (0.99) (0.93) Weight SDS 0.55 (0.95) 0.05 (1.77) Height SDS 0.67 (1.064) (1.30) BMI SDS 0.25 (1.0) (1.77) Boys n (%) 13 (48) 1 (20) CF genotype ΔF508/ ΔF ΔF508 heterozygote 4 2 Other 4 FEV1 (% predicted) a 81 (74,91) 81 (50,96) Chrispin Norman score a 9 (5,15) 6 (4,12) Calcium intake (g/day) 1772 (1219) 1557 (573) pb0.005 compared to 0 (1-sample t-test). a Median (25th, 75th centiles). Table 2 Bone densitometry and bone turnover markers, according to pubertal status Pre-pubertal Pubertal DXA N 27 5 Whole body BMD SD (0.68) 0.09 (0.73) N (%) b (4) 0 Whole body BMC SD 0.99 (1.27) 0.15 (0.89) Lumbar spine BMD SD 0.40 (0.68) 0.62 (1.17) n (%) b (22) 1 LS BMC SD 0.65 (0.97) 0.44 (1.47) LS BA SD 0.66 (1.42) 0.07 (1.34) LS BMAD SD 0.10 (0.88) 0.62 (0.69) n (%) b (19) 1 Biochemistry N 24 4 Plasma osteocalcin (ng/ml) 98.6 (29.7) (40.7) % uc-oc 35 (8.7) 45 (11) P1NP mcg/l 401 (148) a 449 (54) a PTH (pg/ml) (14.62) (8.95) 25OH-vitamin D (nmol/l) 70.2 (17.0) 57.5 (12.0) min, max 35.7, , 72.5 a Mean (SD) values from healthy Swiss children: pre-pubertal 760 (219); early pubertal girls 1043 (440), boys 938 (324) mcg/l [22]; Mean values from healthy Dutch children: pre-pubertal girls and boys approximately 600, stage 2 boys 600, stage 2 girls 800 mcg/l, taken from Fig. 2 in [24]. for bone area and height was also normal, and only one pubertal child had a score b 1.0. In contrast, the mean LSBMD SD was less than zero in both pre-pubertal and pubertal children, reaching significance for the larger pre-pubertal group. 22% of pre-pubertal children and 1/5 of the pubertal children had SD scores below 1.0. Both LSBMC and BA SD scores were significantly less than zero for the pre-pubertal children. These children were, however, significantly short and light for their age. LSBMAD SD scores were calculated to adjust for bone size, and were not significantly different from zero in either group. Nevertheless, 19% of pre-pubertal and 1/5 of the pubertal children had BMAD SD scores below 1.0. Measurements of bone mass did not differ significantly between children treated with inhaled steroids and those who were not treated (Table 2) Markers of bone turnover, PTH and 25-OH vitamin D(Table 2) 25-OH vitamin D concentrations ranged from 35.7 to nmol/l (within the local reference range). No child had a value in the range generally accepted as deficient in a non- CF population (b25 nmol/l). One child had a value b37.5 nmol/ l and four children had values between 37.5 and 50 nmol/l. Serum PTH concentrations ranged from 5.3 to 70.6 pg/ml, with one pre-pubertal child having a value N56 pg/ml (the upper limit of normal for this assay in adults); this child had a 25-OH vitamin D concentration of 53 nmol/l. However, 25-OH vitamin D and PTH were significantly negatively correlated (r = 0.40, p=0.04). There was also a significant seasonal effect on 25-OH vitamin D concentrations (p = 0.04) on analysis of variance

4 310 M.S. Fewtrell et al. / Journal of Cystic Fibrosis 7 (2008) (ANOVA) with the highest concentrations seen in samples taken during late summer, and the lowest concentrations during winter. % uc-oc ranged from 22 to 62%. Mean (95% CI) values previously reported using the same assay by our laboratory in elderly Chinese women [23] were 18.6 (15.3, 22.9)% whilst those from elderly British women with lower dietary vitamin K intakes and less optimal vitamin K status were 30.3 (27.7, 33.1)%. Using a similar (hydroxyapatite binding) assay with a quoted reference value for % uc-oc of 21%, Beker [16] reported a mean % uc-oc of 31 in young adults with CF who were not receiving vitamin K supplements; the mean value fell to 17% when the same individuals received supplements. More recently, Kalkwarf et al. [24] reported a median % uc-oc of 13.6, with a range of , in 245 healthy girls aged between 3 and 16 years. Unpublished data (Kalkwarf, personal communication) from this study for girls aged years (the age-range of our CF patients) showed a mean (SD) % uc-oc of 14.0 (4.1) with a range of 6.0 to 24.3, whilst the values for pre-pubertal and Tanner stage II girls were 12.2 and 15.3% respectively. The assay used in this study measured intact OC which is thought to bind hydroxyapatite equivalently to the large mid-molecular fragment. Thus, although caution must be exercised in comparing results from different assays and different populations, collectively, the available data suggest that our CF patients had a high mean % uc-oc. Concentrations of P1NP in our subjects were lower than those previously reported using the same assay in healthy Swiss [25] or Dutch [26] adolescents of the same sex and pubertal stage Correlations between bone turnover markers, PTH and 25-OH vitamin D t-oc was positively correlated with P1NP (r =0.483, p=0.009). % uc-oc and uc-oc showed no significant correlations with any other parameter measured Bone mass and bone biochemistry There were no significant correlations between WBBMD or LSBMD and any biochemical marker. However, after adjusting for age, sex, pubertal status and size, LSBMC was predicted by % uc-oc, with a 0.3% decrease in LSBMC for each 1% increase in uc-oc (p = 0.05: Table 3). This association remained when t-oc was added to the model, and t-oc was not itself a significant predictor of LSBMC. No other biochemical marker predicted WB or LSBMC in this analysis. LS BMAD SD was significantly negatively correlated with both uc-oc (r = 0.41, p = 0.03) and % uc-oc (r = 0.44, p = 0.02), but not with any other biochemical parameter, including t-oc. After adjusting for pubertal status, a 1% increase in % uc- OC was associated with a 0.04SD decrease in BMAD SD (p=0.02: Table 3). The associations between bone mass and % uc-oc remained significant after adjusting for treatment with inhaled steroids, and after excluding the two children who were receiving a short course of oral steroids at the time of the study. Table 3 Regression analyses investigating predictors of (i)ls BMC; (ii) LS BMAD SD score Variable B T p (i) % uc-oc Pubertal stage Weight Height Bone area b0.001 Adjusted R ; RSD 0.08 n=28 (ii) % uc-oc Pubertal stage Height Adjusted R ; RSD 0.72 Linear regression analyses. Model (i) with LS BMC as dependent variable and bone area, weight, height, pubertal stage (0=pre-pubertal, 1=pubertal) and % uc- OC as independent variables. Model (ii) with LS BMAD SD score as dependent variable and pubertal stage, weight (ln) and % uc-oc as independent variables Disease severity, bone mass and bone turnover Modified Chrispin Norman scores were significantly negatively correlated with t-oc (r= 0.46, p=0.014), uc-oc (r= 0.39, p=0.04) and P1NP (r= 0.63, pb0.001). FEV 1 was positively correlated with P1NP (r = 0.52, p = 0.005). These findings suggest that more severe lung disease is associated with evidence of lower bone formation. However, these markers of CF lung disease severity were not predictive of bone mass. 4. Discussion Our study showed that 8 12 year old children with CF had normal bone mass when adjusted for their reduced size. Despite this, bone turnover markers were consistent with reduced bone formation, with a higher than expected proportion of uc-oc, a sensitive marker of sub-optimal vitamin K status. The proportion of uc-oc was in turn a significant predictor of size-adjusted bone mass. In contrast, 25-OH vitamin D status did not predict bone mass. Furthermore, although markers of lung function were associated with markers of bone turnover, the severity of lung disease did not itself predict bone mass. Previous reports of bone health in older children and adolescents with CF have generally demonstrated that they have low bone mass, although in some studies bone mass becomes normal when adjusted for reduced body size. However, it has been suggested that deficits in bone mass appear or become apparent during adolescence [3], leading to clinically apparent bone disease in early adult life. Our results suggest that biochemical findings consistent with abnormal bone formation may precede measurable abnormalities in bone mass, and that strategies to improve gains in bone mass during childhood and, in particular, during the adolescent growth spurt, may need to be started earlier in childhood. Such measures would obviously include attention to improving nutrition in general, preventing deterioration of lung function and physical activity, but might also include increased vitamin D intake and vitamin K supplements. Although a causal role for vitamin K in CF bone disease has not been proven, collective data from a number of sources,

5 M.S. Fewtrell et al. / Journal of Cystic Fibrosis 7 (2008) including now the data from our own study, are fairly compelling. Thus, children and adolescents with CF have been shown to have sub-optimal vitamin K status and increased concentrations of PIVKA's and uc-oc, which are reversible to at least some degree following vitamin K supplements. CF patients also have low bone mass and frequently develop clinically apparent bone disease in early adult life. However, few studies have combined measurements of vitamin K status with assessment of bone mass. In a recent study in 106 UK children with CF, Conway et al. reported that vitamin K deficiency was common with a mean % uc-oc of 71% [14]. This result was obtained using a specific immunoassay and the figure is therefore not directly comparable to our result which was obtained using a hydroxyapatite binding step followed by measurement of uc-oc. Our study has some limitations, including the lack of agematched reference data for % uc-oc using the identical assay. However, we made inferences using available data from other groups of subjects. The N-Mid OC kit used in our study is regarded as the assay of choice for measuring uc-oc since it measures both the intact and the large mid-molecular fragment, both of which bind hydroxyapatite equivalently. Although caution must be exercised in comparing results from different assays and different populations, we consider that collectively, the available data suggest that our CF patients had a high mean % uc-oc. Osteocalcin assays are widely recognised to present problems with interpretation, as the numerous available kits detect different parts of the protein and give very different absolute values, making direct cross-comparison between kits difficult. These issues apply to all studies in this field. Moreover, measurements of uc-oc using the hydroxyapatite binding method are affected not only by vitamin K status, but also by the total amount of OC in the sample [10], which makes interpretation of results in children, in whom OC concentrations can vary considerably, more difficult. Importantly, it also casts some doubt on the value of using age and sex matched control subjects, since unless t-oc concentrations are also matched, interpreting % uc-oc will remain difficult. In order to address the potential effect of t-oc on % uc-oc, we adjusted for t-oc in our regression analysis, and found that the addition of t-oc to the model did not affect the findings. Reported associations between markers of bone turnover and bone mass in healthy children and adolescents have been inconsistent [26,27]. Kalkwarf et al. specifically examined associations between indicators of vitamin K status and current BMC or BMC gain over a 4 year period in healthy girls, and found no consistent relationship [24]. Nevertheless, it is plausible that any relationship might become more apparent in conditions where bone turnover is abnormal. Conway et al. reported that % uc-oc was significantly correlated with bone formation markers, themselves negatively correlated with measurements of BMD and BMC in children with CF. However, % uc-oc was not itself correlated with bone density in this cohort. These findings contrast with our own data where % uc-oc but not other bone turnover markers were significant predictors of bone mass. The reason for this discrepancy remains unclear, but could relate to the different assays used, or to other characteristics of the study populations, including, for example, vitamin D status. The majority of our subjects had 25-OH vitamin D concentrations well within the local reference range, whilst vitamin D concentrations in the study by Conway et al. were lower (mean 43.8 nmol/l, 95% CI ). Recent US [1] and UK [4] consensus statements on bone disease in CF recommended that concentrations of 25-OH vitamin D should be maintained between 75 and 150 nmol/l, although it was recognised that data to support this recommendation were limited. According to these criteria, 18/28 (64 %) of our patients, the majority of the patients studied by Conway et al, as well as most of the healthy local children and adolescents previously measured by our laboratory with the same assay have sub-optimal vitamin D status [28]. However, as recently reported [29], the different 25- OH Vitamin D assays available plus the large inter-laboratory variation that exists for the same assay presents a problem making comparisons across different studies. Further work is clearly required to establish the ideal physiologic 25-OH vitamin D concentrations for maximising bone health in CF patients. Achieving the higher concentrations suggested by the US consensus document may itself be problematic. A recent study of 134 adults with CF found that in many cases, vitamin D status failed to improve following vitamin D doses recommended by the US and UK consensus documents [30]. This suggests that higher doses or alternative strategies may be required. Nevertheless, it is important to appreciate that, as is the case for vitamin K, a causal link between vitamin D status and CF bone disease has not been established. Moreover, whilst our study found an association between sub-optimal vitamin K status and bone mass, we found no such association for vitamin D status. Although both sub-optimal vitamin K status and low bone mass appear common in patients with CF, it remains to be determined whether supplementing with vitamin K actually improves bone mass and prevents bone disease. In normal elderly populations, low vitamin K concentrations and increased % uc-oc are associated with increased risk of hip fracture [31] and both parameters predict the risk of hip fracture independently of bone density [32]. A Japanese study showed significant reduction in osteoporotic bone loss after high supplemental doses of vitamin K2 [33], whilst a single randomised trial of vitamin K1 supplementation (1mg/day) suggested that supplementation resulted in reduced bone loss at the femoral neck over a 3 year period [34]. Data from animal models are consistent with these findings, and show that vitamin K stimulates bone formation and reduces resorption [35]. Furthermore, vitamin K may act synergistically with vitamin D [36,37]. In a recent survey of Paediatric Specialist CF Centres in the UK, only 18% reported using routine vitamin K supplementation [12], with doses ranging from 0.3 mg to 10 mg/day, perhaps reflecting inconsistencies in recommendations from different groups in the US, Europe and the UK [1,4,38,39]. All expert groups have stressed the need for further research in this area before an optimal dose can be stated confidently. Ideally, the potential role of vitamin K for preventing or reducing the incidence of CF bone disease should be investigated in a randomised trial, assessing the effects of vitamin K supplementation and dose on both bone turnover and bone mass, and, in the long-term, on the development of bone disease.

6 312 M.S. Fewtrell et al. / Journal of Cystic Fibrosis 7 (2008) Conflict of interest statement There are no conflicting interests. Acknowledgments The authors thank Catherine Wilson for performing DXA scans and the assistance with data collection; Charlotte Dawson and Denise Sheehan (CF Nurse Specialists); Dr. Ann Prentice (MRC-Human Nutrition Research, Cambridge) for facilitating the work and for the helpful comments on the manuscript; Caren Gundberg, Heidi Kalkwarf and Inez Schoenmakers for the helpful comments on interpretation on bone turnover assays; and Ann Laidlaw, Janet Bennett and Louise McKenna at MRC-Human Nutrition Research, Cambridge, for performing the assays. References [1] Aris RM, Merkel PA, Bachrach LK, Borowitz DS, Boyle MP, Elkin SL, et al. Guide to bone health and disease in cystic fibrosis. J Clin Endocrinol Metab 2005;90: [2] Elkin SL, Vedi S, Bord S, Garrahan NJ, Hodson ME, Compston JE. Histomorphometric analysis of bone biopsies from the iliac crest of adults with cystic fibrosis. Am J Respir Crit Care Med 2002;166: [3] Buntain HM, Greer RM, Schluter PJ, Wong JC, Batch JA, Potter JM, et al. Bone mineral density in Australian children, adolescents and adults with cystic fibrosis: a controlled cross-sectional study. Thorax 2004;59: [4] Bone mineralisation in Cystic Fibrosis. Report of the UK Cystic Fibrosis Trust Bone Mineralisation Working Group Cystic Fibrosis Trust ISBN [5] Hauschka PV, Lian JB, Cole DE, Gunberg CM. Osteocalcin and matrix Gla protein: vitamin K dependent proteins in bone. Physiol Rev 1989;69: [6] Liu G, Peacock M. Age-related changes in serum undercarboxylated osteocalcin and its relationships with bone density, bone quality, and hip fracture. Calcif Tissue Int 1998;62(4): [7] Luukinen H, Kakonen SM, Pettersson K, Koski K, Laippala P, Lovgren T, et al. Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin. J Bone Mineral Res 2000;15: [8] Vergnaud P, Garnero P, Meunier PJ, Breart G, Kamihagi K, Delmas PD. Undercarboxylated osteocalcin measured with a specific immunoassay predicts hip fracture in elderly women: the EPIDOS Study. J Clin Endocrinol Metab 1997;82: [9] Vermeer C, Jie KS, Knapen MH. Role of vitamin K in bone metabolism. Annu Rev Nutr 1995;15:1 22. [10] Gundberg CM, Nieman SD, Abrams S, Rosen H. Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin. J Clin Endocrinol metab 1998;83: [11] Shearer MJ. Vitamin K. Lancet 1995;345: [12] Urquhart DS, Fitzpatrick M, Cope J, Jaffe A. Vitamin k prescribing patterns and bone health surveillance in UK children with cystic fibrosis. J Hum Nutr Diet 2007;20(6): [13] Rashid R, Durie P, Andrew M, Kalnins D, Shin J, Corey M, et al. Prevalence of vitamin K deficiency in cystic fibrosis. Am J Clin Nutr 1999;70: [14] Conway SP, Wolfe SP, Brownlee KG, White H, Oldroyd B, Truscott JG, et al. Vitamin K status among children with cystic fibrosis and its relationship to bone mineral density and bone turnover. Pediatrics 2005;115: [15] Wilson DC, Rashid M, Durie PR, Tsang A, Kalnins D, Andrew M, et al. Treatment of vitamin K deficiency in cystic fibrosis: effectiveness of a daily fat-soluble vitamin combination. J Pediatr 2001;138: [16] Beker LT, Ahrens RA, Fink RJ, O'Brien ME, Davidson KW, Sokoll LJ, et al. Effect of vitamin K1 supplementation on vitamin K status in cystic fibrosis patients. JPGN 1997;24: [17] Nicolaidou P, Stavrinadis I, Loukou I, Papadopoulou A, Georgouli H, Douros K, et al. The effect of vitamin K supplementation on biochemical markers of bone formation in children and adolescents with cystic fibrosis. Eur J Pediatr : online first 2006, doi: /s [18] Standardization of Spirometry. Update. American Thoracic Society. Am J Respir Crit Care Med 1995;152: [19] Roca J, Burgos F, Sunyer J, Saez M, Chinn S, Anto JM, et al. Reference values for forced spirometry. Group of the European Community Respiratory Health Survey Eur Respir J 1998;11: [20] Chrispin AR, Norman AP. The systematic evaluation of the chest radiograph in cystic fibrosis. Pediatr Radiol 1974;2(2): [21] Benden C, Wallis C, Owens CM, Ridout DA, Dinwiddie R. The Chrispin Norman score in cystic fibrosis: doing away with the lateral view. Eur Respir J 2005;26: [22] Fewtrell MS, Gordon I, Biassoni L, Cole TJ. Dual X-ray absorptiometry (DXA) of the lumbar spine in a clinical paediatric setting: does the method of size-adjustment matter? Bone 2005;37: [23] Yan L, Zhou B, Greenberg D, Wang L, Nigdikar S, Prynne C, et al. Vitamin K status of older individuals in northern China is superior to that of older individuals in the UK. Br J Nutr 2004;92: [24] Kalkwarf HJ, Khoury JC, Bean J, Elliot JG. Vitamin K, bone turnover and bone mass in girls. Am J Clin Nutr 2004;80: [25] Ginty F, Cavadini C, Michaud PA, et al. Effects of usual nutrient intake and vitamin D status on markers of bone turnover in Swiss adolescents. Eur J Clin Nutr 2004;58: [26] van Coeverden SCCM, Netelenbos JC, de Ridder CM, Roos JC, Popp- Snijders C, Delemarre-van de Waal HR. Bone metabolism markers and bone mass in healthy pubertal boys and girls. Clinical Endocrinology 2002;57: [27] Cadogan J, Blumsohn A, Barker ME, Eastell R. A longitudinal study of bone gain in pubertal girls:anthropometric and biochemical correlates. J Bone Miner Res 1998;13: [28] Fewtrell MS, Prentice A, Jones SC, Bishop NJ, Stirling D, Buffenstein R, et al. Bone mineralization and turnover in preterm infants at 8 12 years of age: the effects of early diet. J Bone Miner Res 1999;14: [29] Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, Hansen KE, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab 2004;89: [30] Boyle MP, Noschese ML, Watts SL, Davis ME, Stenner SE, Lechtzin N. Failure of high-dose ergocalciferol to correct vitamin D deficiency in adults with cystic fibrosis. Am J Respir Crit Care Med 2005;172: [31] Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz GA. Vitamin K intake and hip fractures in women: a prospective study. Am J Clin Nutr 1999;69:74 9. [32] Booth SL, Tucker KL, Chen H, Hannan MT, Gagnon DR, Cupples LA, et al. Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women. Am J Clin Nutr 2000;71: [33] Shiraki M, Shiraki Y, Aoki C, Miura M. Vitamin K2 (menatetrenone) effectively prevents fractures and sustains lumbar spine bone mineral density in osteoporosis. J Bone Min Res 2000;15: [34] Braam LA, Knapen MH, Geusens P, Brouns F, Hamulyak K, Gerichhausen MJ, et al. Vitamin K1 supplementation retards bone loss in postmenopausal women between 50 and 60 years of age. Calcif Tiss Int 2003;73:21 6. [35] Vermeer C, Jie KS, Knappen MH. Role of vitamin K in bone metabolism. Annu Rev Nutr 1995;15:1 22. [36] Matsunaga S, Ito H, Sakou T. The effect of vitamin K and D supplementation on ovariectomy-induced bone loss. Calcif Tiss Int 1999;65: [37] Bolton-Smith C, Mole PA, McMurdo MET, Paterson CR, Shearer MJ. Two-year intervention with phylloquinone (vitamin K1), vitamin D and calcium: effect on bone mineral content of older women. Ann Nutr Metab 2001;45(suppl 1):46. [38] Sinaasappel M, Stern M, Littlewood J, Wolfe S, Steinkamp G, Heijerman HG, et al. Nutrition in patients with cystic fibrosis: a European Consensus. J Cyst Fibros 2002;1: [39] Borowitz D, Baker R, Stallings V. Consensus report on nutrition for pediatric patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2002;35: