Vitamin D is essential for bone health (1). The main and. Thresholds for Serum 25(OH)D Concentrations With Respect to Different Outcomes

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1 ORIGINAL ARTICLE Thresholds for Serum 25(OH)D Concentrtions With Respect to Different Outcomes E. Sohl, R. T. de Jongh, M. W. Heymns, N. M. vn Schoor, nd P. Lips Deprtment of Epidemiology nd Biosttistics (E.S., M.W.H., N.M.S.), EMGO Institute for Helth nd Cre Reserch, nd Deprtment of Internl Medicine (R.T.d.J., P.L.), Endocrine Section, VU University Medicl Center, 1081 BT Amsterdm, The Netherlnds Context: Vitmin D is essentil for bone helth. In ddition, vitmin D hs recently been proposed to ply role in the pthophysiology of mny chronic diseses. Despite the lrge number of studies published on vitmin D, the threshold for sufficient serum 25-hydroxyvitmin D [25(OH)D] concentrtion is still debted nd my differ ccording to outcomes nd subgroups. Objective: The objective of the study ws to estimte the thresholds for serum 25(OH)D concentrtion with respect to the different outcomes nd for different subgroups. Design, Setting, nd Prticipnts: Observtionl dt from the Longitudinl Aging Study Amsterdm, n ongoing popultion-bsed Dutch cohort study [n 1164, men (SD) ge 75.2 (6.5) y], were used. Min Outcome Mesures: Flling, frctures, hypertension, crdiovsculr disese, blood pressure, PTH, grip strength, physicl performnce, functionl limittions, body mss index (BMI), nd mortlity were mesured. To determine thresholds, spline curves were used. Visul inspection nd the sttisticl best fit of the spline regression models were used together to estimte the best estimte of the thresholds. Results: Thresholds for serum 25(OH)D concentrtions in the whole smple rnged from 46 nmol/l (PTH) to 68 nmol/l (hypertension). On verge, women, the oldest old ( 75 y), nd individuls with high BMI ( 25 kg/m 2 ) hd lower thresholds compred with men, the youngest old (65 75 y), nd individuls with low to norml BMI ( 25 kg/m 2 ). Conclusion: The results indicte tht thresholds for serum 25(OH)D my vry ccording to different outcomes nd subgroups. This study does not support the high thresholds ( 75 nmol/l) s dvised by some experts, nd the higher requirements in women, older persons, nd those with high BMI. (J Clin Endocrinol Metb 100: , 2015) Vitmin D is essentil for bone helth (1). The min nd best known function of vitmin D is to increse the clcium bsorption from the gut to fcilitte bone minerliztion (1). In ddition, vitmin D hs recently been proposed to ply role in the function of mny other systems, such s the immune nd crdiovsculr system (2, 3), lthough cuslity hs not yet been proven for most nonclssicl outcomes (4). Despite the lrge number of studies published on vitmin D, there is still discussion on the threshold of desired ISSN Print X ISSN Online Printed in USA Copyright 2015 by the Endocrine Society Received Februry 6, Accepted April 6, First Published Online April 9, hydroxyvitmin D [25(OH)D] concentrtion. The Endocrine Society dvoctes levels of t lest 75 nmol/l (5), wheres the Institute of Medicine dvises miniml levels between 30 nd 50 nmol/l (6). Different ntionl guidelines lso dvise vrious levels. Usully the thresholds re bsed on optiml bone helth nd the level t which PTH concentrtions stop declining (7). Severl yers go, review on the evidence on thresholds of serum 25(OH)D ws published (8). The uthors stted tht levels greter thn 100 nmol/l were necessry to rech mximl bone min- Abbrevitions: BMI, body mss index; BP, blood pressure; LASA, Longitudinl Aging Study Amsterdm; MAP, men rteril pressure; 25(OH)D, 25-hydroxyvitmin D press.endocrine.org/journl/jcem J Clin Endocrinol Metb, June 2015, 100(6): doi: /jc

2 doi: /jc press.endocrine.org/journl/jcem 2481 erl density or the lowest risk for frctures, wheres for muscle strength serum level bove 40 nmol/l ws desirble (8). In contrst, study in Koren individuls reveled tht levels of pproximtely 50 nmol/l ws sufficient to mintin bone helth (9). In previous studies, low vitmin D sttus ws ssocited with higher blood pressure (BP) (10), higher mortlity (11), higher frcture nd flls rtes (12), higher BMI (13), nd lower physicl performnce (14, 15), but thresholds were not determined systemticlly for every outcome. Furthermore, it is known tht men serum 25(OH)D concentrtions differ between men nd women nd low nd high ge nd for individuls with different body mss index (BMI) (1, 12, 16). However, it is not cler whether the sme thresholds re pplicble to these subgroups. Therefore, this study imed to explore the thresholds of serum 25(OH)D with respect to different outcomes. In the current study, threshold is not the point t which n outcome does not occur nymore, but it is defined s the serum 25(OH)D concentrtion from which the risk for the specific outcome or outcome vlue strts to level off. Although cuslity hs not yet been proven for most nonskeletl outcomes, the results of lrge clinicl trils on these outcomes will be vilble in the next few yers. Mny observtionl studies re pointing towrd possible cusl reltionship. Therefore, we decided to include these outcomes in the current study. In ddition, we exmined whether these thresholds differ between severl subgroups, ie, men nd women nd different ge nd BMI groups. We hypothesized tht women nd older individuls would hve higher thresholds becuse women nd older subjects hve higher frcture risk (17). This higher frcture risk my be cused by lower clcium intke nd thereby requiring higher serum 25(OH)D to stimulte sufficient bsorption of clcium from the gut. Furthermore, vitmin D is ft soluble, nd therefore, 25(OH)D my be more stored in ft in persons with high BMI, lso possibly leding to higher thresholds. Mterils nd Methods Study prticipnts Dt for this study were collected within the Longitudinl Aging Study Amsterdm (LASA). LASA is n ongoing cohort study in representtive smple of the Dutch older individuls. The smpling nd dt collection procedures re described elsewhere in detil (18). Briefly, rndom ge- nd sex-strtified smple ws drwn from popultion registries of 11 municiplities in three regions in The Netherlnds. The study strted in 1992 nd 3107 individuls ged yers were included. In 2002 nd 2012, two dditionl cohorts were strted. For this study, dt from the second cycle of the first cohort, which took plce in , were used. Prticipnts who were 65 yers old nd older on Jnury 1, 1996, were invited for medicl interview in (n 1509). In 1320 prticipnts, serum 25(OH)D ws mesured. We were not ble to use the dt of 156 prticipnts due to missing vlues for the outcome vribles. Therefore, the study smple consisted of 1164 prticipnts. LASA ws pproved by the Medicl Ethics Committee of the VU University Medicl Center nd ll prticipnts gve informed consent. Outcome mesures The following vribles were considered s outcome mesures: flls, frctures, PTH, BP, crdiovsculr disese, BMI, grip strength, physicl performnce, functionl limittions, nd mortlity. These vribles were chosen becuse mny, mostly crosssectionl, studies, showed n ssocition between vitmin D sttus nd these outcomes (3, 10 15, 19, 20). Flls were prospectively recorded by using fll nd frcture clendr for 3 yers. Every 3 months prticipnts hd to send bck their clendr to the study center. A fll ws determined s n unintentionl chnge in position resulting in coming to rest t lower level or t the ground (21). A recurrent fller ws defined s n individul who fell t lest two times within 6 months (22). Times to the first nd second fll were clculted; for prticipnts who were lost to follow-up or decesed, the time until dropout ws clculted. Frctures were recorded by using two different methods. For the first 3 yers of follow-up, frctures were reported with fll nd frcture clendr ( through ), which hd to be sent bck to the study center every 3 months. Second, questions on the occurrence of frctures in the lst 3 yers were sked during the interviews of the next mesurement cycles ( through ). So in totl, 10-yer frcture follow-up period is vilble. The dte nd type of frcture were verified by generl prctitioners or in hospitls. When prticipnt ws lost to follow-up or decesed, informtion on the occurrence of frctures ws sked from the generl prctitioners. Frctures of the hnd, hed, finger, foot, nd toe were excluded becuse these re not likely to be osteoporotic. Time to first frcture ws clculted or, when no frcture occurred, the time to end of follow-up, deth, or to loss of follow up. Prticipnts were thus in the nlyses only for the time they were t risk. PTH ws mesured by using n immunordiometric ssy (Incstr Corp). The interssy coefficient of vrition ws 12%. BP (millimeters of mercury) ws mesured using n utomtic device (Omron model HEM-706; Omron Corp) fter t lest 5 minutes of rest t the upper left rm in sitting position. Men rteril pressure (MAP) ws clculted using the following formul: distolic pressure 1/3 (systolic pressure distolic pressure). Hypertension ws defined s systolic pressure greter thn 160 mm Hg nd/or distolic pressure greter thn 90 mm Hg, nd/or the use of ntihypertensive mediction ccording to the guidelines for older individuls of the Dutch generl prctitioners (23). Crdiovsculr disese ws bsed on self-report. During the medicl interview, prticipnts were sked questions on whether they hd chronic crdiovsculr disese. BMI (kilogrms per squre meter) ws clculted from the mesured weight nd the mesured height in squre meters. Body weight (kilogrms) ws mesured without upper clothes nd

3 2482 Sohl et l Thresholds for Serum 25(OH)D J Clin Endocrinol Metb, June 2015, 100(6): shoes using clibrted blnce bem scle. Body height (meters) ws mesured using stdiometer. Grip strength (kilogrms) ws mesured using clibrted strin-guged dynmometer (Tkei TKK 5001; Tkei Scientific Instruments Co Ltd). Prticipnts hd to perform two mximum efforts with ech hnd, when stnding with their hnd longside their body. The men grip strength ws clculted s the men of the mximum scores of the right nd left hnds. Physicl performnce ws determined using three different tests, the wlking test in which prticipnts hd to wlk 3 m, turn round, nd wlk bck s quickly s possible; the chir stnd test, in which prticipnts hd to stnd up from nd sit down on chir without using their hnds for five times s quickly s possible; nd the tndem stnd test, in which they hd to stnd in tndem position (one foot directly in front of the toes of the other foot) for mximum of 10 seconds. The scores of the individul test were summed nd the totl scores rnged from 0 to 12, with 12 points indicting the best performnce. Functionl limittions were ssessed by sking questions on the bility to perform ctivities of dily life: using their own or public trnsporttion, cutting their own toenils, dressing nd undressing themselves, wlking outside for 5 minutes without stopping, sitting down nd stnding up from chir, nd wlking up nd down stircse of 15 steps without resting. A dichotomiztion ws mde between hving limittions in ny of these ctivities vs no limittions in ny these ctivities. The exct dte of deth ws collected from the municiplity registries in which prticipnts were living. Time until deth from to ws clculted. The nlyses on frctures, flling, recurrent flling, nd mortlity were bsed on longitudinl dt, wheres the other outcomes were cross-sectionl. Serum 25(OH)D mesurements Morning blood smples were tken from the prticipnts, who were llowed to et te or tost but no diry products. Smples were centrifuged nd stored t 20 C until determintion in A competitive binding protein ssy ws used (Nichols Dignostics). The interssy coefficient of vrition ws 10%. Subgroups Age nd sex were derived from popultion registries. BMI ws clculted s described bove. To define the two subgroups of BMI, the common cutoff point of 25 kg/m 2, which is ner the medin BMI, ws used. Age ws dichotomized round the medin ge, nmely 75 yers. Sttisticl nlysis Serum 25(OH)D ws used s continuous independent vrible in ll nlyses. Thresholds were estimted in four steps. First, spline plots were mde to determine the reltionship between serum 25(OH)D nd the outcomes. For continuous vribles, the splines curves plotted the outcome vrible ginst serum 25(OH)D, wheres the outcome ws log odds or log hzrds for dichotomous nd longitudinl outcomes, respectively. Subsequently it ws determined whether there ws cliniclly relevnt difference in outcome between high nd low serum 25(OH)D concentrtions in our smple. The mgnitude of the cliniclly relevnt difference ws bsed on the literture if possible. For BP (distolic, systolic, nd MAP), difference of 6 mm Hg ws considered relevnt (24), nd for grip strength this ws 6 kg (25). A 5% weight loss is often considered s cliniclly relevnt in the literture (26); therefore, chnge in the BMI of pproximtely 1 point ws used in this study. For physicl performnce, difference of 1 point in totl score ws considered s cliniclly relevnt (27). For differences in odds nd hzrd rtios, miniml difference of 0.5 ws considered s relevnt. When cliniclly relevnt difference in outcomes (risks or vlues) ws not observed between high nd low serum 25(OH)D, no further steps were performed. Second, on the bsis of the spline curves, nonliner reltionship hd to be confirmed becuse no threshold will exist in liner ssocition. This ws done by using the nonlinerity test within spline regression nlysis. A vlue of P.2 ws considered s nonliner. Third, the threshold of serum 25(OH)D ws estimted visully nd ws defined s the point t which the outcome vlue strted to increse or decrese less rpidly (28) or the lowest vlue on the spline curve ws chosen in cse of U-shped reltionship. Fourth, these visully determined thresholds were used in further nlyses to determine the definite thresholds by the following procedure. Spline regression nlyses were performed using three knots. The first nd third knots remined the fixed stndrd knots bsed on the distribution of vlues (the 10th nd 90th percentile). A rnge of possible vlues round the visully determined threshold vlue ( 10 nmol/l) ws explored for the second knot. The vlue used s the second knot, which resulted in the model with the best fit, ws defined s the definite threshold vlue (28). The best model fit ws ssessed by the highest C-index for dichotomous outcome vribles nd for survivl nlyses nd by the R 2 for continuous outcome vribles. To evlute the performnce of the models, we chose mesures tht reflect discrimintion becuse these re considered more importnt for clinicins. Furthermore, the C-index for discrimintion is closely relted to the R 2. With these mesures we were ble to use comprble performnce mesures independent of the type of model. Tble 1 provides summry of this procedure. An djustment for confounders ws not mde to represent the true vlues of the generl popultion. Dichotomous outcomes, ie, the risk of flling, being recurrent fller, functionl limittions, hypertension, Tble 1. Summry of the Procedure to Determine the Thresholds Step Gol 1 Spline curves Confirmtion of cliniclly relevnt difference 2 Spline regression, test of nonlinerity Confirmtion of nonliner ssocition (P.2) 3 Spline curves, visul inspection Visul determintion of the threshold 4 Spline regression, best model fit Determintion of the definite threshold

4 doi: /jc press.endocrine.org/journl/jcem 2483 Tble 2. Smple Chrcteristics LASA Study Smple Sex Age BMI Men Women <75 y >75 y <25 kg/m 2 >25 kg/m 2 N Age, y 75.2 (6.5) 75.3 (6.5) 75.2 (6.5) 69.8 (2.8) 81.0 (3.7) 75.5 (6.7) 75.1 (6.4) 75 y 596 (51.2) 291 (49.9) 305 (52.5) 202 (50.5) 394 (51.6) 75 y 568 (48.8) 292 (50.1) 276 (47.5) 198 (49.5) 370 (48.4) Gender Men, n, % 583 (50.1) 291 (48.8) 292 (51.4) 211 (52.8) 372 (48.7) Women, n, % 581 (49.9) 305 (51.2) 276 (48.6) 189 (47.3) 392 (51.3) Serum 25(OH)D, nmol/l 54.5 (24.1) 59.0 (24.5) 49.9 (22.9) 61.7 (23.5) 46.9 (22.8) 57.1 (24.9) 53.1 (23.6) PTH, pmol/l 3.2 [ ] 3.1 [ ] 3.2 [ ] 2.9 [ ] 3.5 [ ] 3.0 [ ] 3.2 [ ] Any frcture, n, % 134 (11.5) 45 (7.7) 89 (15.3) 59 (9.9) 75 (13.2) 41 (10.3) 93 (12.2) Time to ny frcture, d 1341 [ ] 1134 [ ] 1541 [ ] 1694 [ ] 1095 [ ] 1968 [ ] 1095 [ ] Flling, n, % 635 (54.62) 286 (49.1) 349 (60.1) 308 (51.7) 327 (57.6) 223 (55.8) 412 (53.9) Time to first fll, wk 40 [16 83] 45.5 [16 83] 38 [16 84] 39 [16 81] 41 [16 83] 41 [20 79] 39 [14 83] Recurrent fller, n, % 297 (25.5) 146 (25.0) 151 (26.0) 124 (20.8) 173 (30.5) 117 (29.3) 180 (23.6) Time to second fll, wk 61 [30 99] 62 [30 98] 59 [30 102] 61 [20 89] 63 [33 108] 61 [35 92] 61 [27 102] Hypertension, n, % 743 (63.8) 370 (63.5) 373 (64.2) 348 (58.4) 395 (69.5) 205 (51.2) 538 (70.4) BP (systolic), mm Hg (25.7) (25.1) (26.3) (24.3) (26.8) (25.7) (25.4) BP (distolic), mm Hg 83.5 (13.5) 83.8 (14.1) 83.1 (12.9) 84.7 (13.1) 82.1 (13.8) 81.3 (12.7) 84.6 (13.8) MAP, mm Hg (15.9) (15.8) (16.0) (15.1) (16.6) (15.6) (15.8) Crdiovsculr disese, n, % 278 (23.9) 165 (28.3) 113 (19.4) 86 (14.4) 192 (33.8) 80 (20.0) 198 (25.9) BMI, kg/m (4.0) 26.2 (3.3) 27.4 (4.6) 26.9 (3.8) 26.8 (4.3) 22.7 (1.9) 28.9 (3.1) 25 kg/m (34.4) 211 (36.2) 189 (32.5) 202 (33.9) 198 (34.9) 25 kg/m (65.6) 372 (63.8) 392 (67.5) 394 (66.1) 370 (65.1) Decesed, n, % 469 (42.6) 300 (51.5) 196 (33.7) 143 (24.0) 353 (62.1) 177 (44.3) 319 (41.8) Time to deth, wk 279 [ ] 269 [ ] 302 [ ] 280 [ ] 278 [ ] 268 [ ] 289 [ ] Grip strength, kg 28.6 (9.9) 36.0 (7.9) 21.2 (4.8) 31.1 (10.1) 26.1 (9.0) 28.0 (9.2) 29.0 (10.2) Physicl performnce (0 12) 7.5 (3.1) 8.1 (2.8) 7.0 (3.2) 8.7 (2.5) 6.3 (3.2) 7.8 (3.1) 7.4 (3.1) Functionl limittions, n, % 630 (54.1) 277 (47.5) 353 (60.8) 241 (40.4) 389 (68.5) 175 (43.8) 455 (59.6) Vlues re men (SD), n (percentge), or medin [interqurtile rnge]. Dshes indicte no nlyses were performed for this subgroup. nd crdiovsculr disese, were nlyzed using logistic regression nlyses within the spline nlyses. Continuous outcomes, ie, grip strength, physicl performnce, PTH, systolic nd distolic pressures, MAP, nd BMI, were nlyzed using spline regression nlyses bsed on liner regression. These spline regression nlyses re bsed only on liner regression nd re not ssuming liner reltionship s liner regression itself does. The longitudinl outcomes, ie, time to first frcture, to first nd second fll, nd to deth, were nlyzed using Cox-regression nlyses. Results The chrcteristics of the study smple re shown in Tble 2. In totl, 1164 individuls were nlyzed nd their men (SD) ge ws 75.2 (6.5) yers. Men nd women were similrly distributed. Men (SD) serum 25(OH)D ws 54.5 (24.1) nmol/l. Serum 25(OH)D concentrtions less thn 25 nmol/l, nmol/l, nmol/l, nd 75 nmol/l or greter were present in 10.1%, 36.2%, 34.9%, nd 18.9% of the individuls, respectively. Tble 3 presents the results for the nlyses on the estimtion of the thresholds for serum 25(OH)D. In the nlyses in the whole smple, the optiml serum 25(OH)D concentrtions rnged from 46 (PTH) to 68 nmol/l (hypertension). Thresholds could not be determined for risk of flling, crdiovsculr disese, systolic nd distolic BPs, men rteril pressure, BMI, nd frctures becuse of lck of clinicl relevnce or nonlinerity of the ssocitions (see Tble 3 for reson per outcome). For the BP vribles, there ppered to be threshold, but the difference in BP between the optiml nd worst point ws only pproximtely 2 4 mm Hg nd thus ws considered s cliniclly nonrelevnt. For the other vribles without threshold, the P vlue for nonlinerity ws greter thn.2, nd therefore, nonliner reltionship ws not ssumed. Figures 1 nd 2 provide some exmples of the determintion of the thresholds. When nlyzing the different subgroups, the differences in the thresholds of serum 25(OH)D were observed (Tble 3). On verge, but not for ll outcomes, the threshold for women ws lower thn for men. Thresholds for men rnged from 48 to 68 nmol/l, wheres for women the rnge ws nmol/l. The sme trend ws observed for individuls younger thn 75 yers of ge (51 72 nmol/l) compred with n ge of 75 yers or older (38 61 nmol/l) nd for individuls with low to norml BMI (46 76 nmol/l) compred with high BMI (42 64 nmol/l). Discussion This study showed tht thresholds for serum 25(OH)D vry between different subgroups nd outcomes. It ws

5 2484 Sohl et l Thresholds for Serum 25(OH)D J Clin Endocrinol Metb, June 2015, 100(6): Tble 3. Threshold Vlues in Nnomoles per Liter With Respect to Different Outcomes nd Subgroups Sex Age Body Mss Index Totl Group Men Women <75 y >75 y <25 kg/m 2 >25 kg/m 2 Dichotomous Risk of flling b b b Risk of recurrent flling Hypertension b b Crdiovsculr disese Functionl limittions Continuous PTH Systolic BP b b b b 76 b Distolic BP b b b b b b b MAP b b b b b b b Grip strength Physicl performnce BMI 45 Survivl Mortlity Frctures 41 b b Flling b b 61 Recurrent flling Vlues re in nnomoles per liter. Dshes indicte no nlyses were performed for this subgroup. No nonliner ssocition. b No cliniclly relevnt difference. found tht thresholds were lower for women, the oldest old ( 75 y), nd higher BMI ( 25 kg/m 2 ), compred with men, younger old, nd low to norml BMI, respectively. Thresholds for serum 25(OH)D concentrtions in the whole smple rnged from 46 (PTH) to 68 (hypertension) nmol/l. In ddition, no cler thresholds could be estimted for BP indices, crdiovsculr disese, frctures, nd BMI becuse of lck of clinicl relevnce or liner reltionship. To the best of our knowledge, this is the first study to show remrkble differences in thresholds for serum 25(OH)D between different subgroups. The underlying mechnisms why women, the older old, nd individuls with high BMI hve lower thresholds hve to be studied in further reserch. A priori, we hypothesized tht these groups would hve higher thresholds. On the other hnd, it cn lso be hypothesized tht in individuls with high BMI, the serum 25(OH)D concentrtion reflects less well the totl body 25(OH)D becuse it is stored in ft cells (29). Therefore, the threshold for serum 25(OH)D cn be lower in these individuls becuse there will be 25(OH)D vilble from the dipocytes. In ddition, older individuls my hve lower thresholds compred to younger individuls, becuse it cn be imgined tht other vribles, such s chronic diseses, hve greter influence on the studied outcomes thn serum 25(OH)D. In ddition, it cnnot be excluded tht unmesured confounding fctors, such s vitmin D binding protein levels or clcium intke, ply role. Surprisingly, previous studies on ssocitions between vitmin D sttus nd severl outcomes in LASA reveled tht when n interction ws found, the ssocition ws most significnt within the subgroup with the highest thresholds found in the current study. For exmple, serum 25(OH)D ws only longitudinlly ssocited with expirtory pek flow in men (30), with the 1-yer risk of recurrent flling (31) nd 6-yer frcture risk (12) in individuls ged yers, nd with quntittive ultrsound prmeters nd bone minerl density only in individuls with low to norml BMI (32). The highest men serum 25(OH)D levels were lso found in these subgroups; this my prtly explin why the thresholds were lso higher. However, if lower threshold ws found only due to lower men vlues, then one would expect tht the line of the grph ws still incresing or decresing until the highest serum 25(OH)D concentrtions observed in our study without reching plteu or without showing U-shped curve. Therefore, relly lower threshold for these subgroups is suggested compred with their counterprts. However, further reserch is needed to clrify the underlying mechnisms nd to estblish whether the sme thresholds exist in popultions with higher concentrtions of serum 25(OH)D. Previous studies on the estimtion of thresholds re scrce nd re minly bsed on bone outcomes nd PTH concentrtions. On the one hnd, the use of PTH s n outcome mesure cn be justified becuse PTH stimultes bone loss (33). On the other hnd, PTH concentrtions fluctute with dietry hbits, time of the dy, renl func-

6 doi: /jc press.endocrine.org/journl/jcem 2485 Figure 1. The determintion of the threshold vlues for three different outcomes, one for ech type of sttisticl nlysis. Smll dotted lines re 95% confidence intervls. The rrows indicte the visully determined thresholds. The x-xis represents serum 25(OH)D in nnomoles per liter; the y-xis represents the outcome vribles. A, Liner regression: PTH (in picomoles per liter), visully 56 nnomoles per liter, fter sttisticl nlyses 46 nnomoles per liter. B, Logistic regression: hypertension (log odds), visully 67 nnomoles per liter, fter sttisticl nlyses, 68 nnomoles per liter. C, Cox regression: mortlity (log reltive hzrd), visully 55 nnomoles per liter, fter sttisticl nlyses, 56 nnomoles per liter. tion, nd physicl ctivity (33 37). Therefore, wide rnge of thresholds hve been found ( nmol/l) (33). In our study, thresholds for serum 25(OH)D concentrtions bsed on PTH levels re more or less in the sme rnge s these for other outcomes, lthough in the whole smple, the thresholds were lower bsed on PTH thn bsed on other outcomes. In ddition, severl previous studies on thresholds re bsed on the postintervention serum 25(OH)D levels of clinicl trils supplementing vitmin D (8), nd therefore, it is difficult to compre these results with our popultion-bsed observtionl study. In the future, dose-response clinicl trils supplementing vitmin D will be necessry to estblish definite thresholds, but the results of the current study cn be considered s first step in exploring thresholds for different outcomes nd subgroups. This informtion cn be used when designing these dose-response clinicl trils. When considering the results, it is remrkble tht thresholds could be estimted for hypertension but not for ny of the other BP vribles. These vribles minly filed to pss the criterion with regrd to clinicl relevnce. This cn be explined by the fct tht individuls using ntihypertensive drugs were lso clssified s hving hypertension, wheres in the other vribles, only the mesured BP vlues were tken into ccount. In cse of well-controlled hypertension, the BP will be comprble with individuls without hypertension, nd therefore, no cler ssocition cn be found. Also, in previous reserch within

7 2486 Sohl et l Thresholds for Serum 25(OH)D J Clin Endocrinol Metb, June 2015, 100(6): Figure 2. The determintion of the threshold vlues for hypertension ccording to the different subgroups. Smll dotted lines re 95% confidence intervls. The rrows indicte the visully determined thresholds. The x-xis represents serum 25(OH)D in nnomoles per liter; the y-xis represents the log odds for hypertension. A, Totl group: visully, 67 nnomoles per liter, fter sttisticl nlyses, 68 nnomoles per liter. B, Men: visully, 67 nnomoles per liter, fter sttisticl nlyses, 68 nnomoles per liter. C, Women: visully, 49 nnomoles per liter, fter sttisticl nlyses, 49 nnomoles per liter. D, Age younger thn 75 yers: no cliniclly relevnt difference. E, Age 75 yers or older: visully, 54 nnomoles per liter, fter sttisticl nlyses, 61 nnomoles per liter. F, BMI less thn 25 kg/m 2 : visully, 67 nnomoles per liter, fter sttisticl nlyses, 58 nnomoles per liter. G, BMI 25 kg/m 2 or greter: no cliniclly relevnt difference. LASA, no significnt ssocition between vitmin D sttus nd BP ws observed (38). Some remrks hve to be mde on the method of the estimtion of the thresholds. A spline regression curve is good method to show the shpe of the ssocition; it uses more informtion of the dt, s compred with ctegoriztionofthedt(39). However, itisquestionble whether the thresholds found in this study rely on the distribution of serum25(oh)dinourdtset(40). Becuse the serum 25(OH)D concentrtions re reltively low, high threshold could priori not be found due to the lck of prticipnts with these concentrtions. But on the other hnd, if the threshold,ingenerl,isinthehigherconcentrtions of serum 25(OH)D, one would expect n ongoing incresing or decresing line of the grph until the highest serum 25(OH)D levels in our study smple. For most of the outcomes, this ws not the cse becuse the line of the grph levels off t reltively low serum 25(OH)D concentrtions. Only for crdiovsculr disese nd BMI, there ws n ongoing decresing line with higher serum 25(OH)D levels, nd high threshold is therefore still possible. Becuseonly19%oftheindividulshd serum 25(OH)D concentrtions of 75 nmol/l or greter, reduced sttisticl power in the highest rnge of serum 25(OH)D concentrtions could lso hve cused more imprecision, nd the reduced sttisticl power could ply role in not finding nonliner reltionship. Therefore, to find out whether there re thresholds for these outcomes, further (longitudinl) studies in popultions with higher serum 25(OH)D concentrtion should be performed to vlidte our results. Previous studies on thresholds of serum 25(OH)D usully only rely on the sttisticl best rtio of sensitivity nd specificity, without tking the clinicl relevnce into ccount. We used four-step pproch, in which we first, systemticlly determined whether there ws cliniclly relevnt

8 doi: /jc press.endocrine.org/journl/jcem 2487 difference nd, second, whether it ws possible to determine threshold by using test for nonlinerity. By using this pproch, we were ble to show thresholds tht were of clinicl relevnce nd tht were true thresholds in nonliner reltionship. This study hs some further limittions nd strengths. First, most of the nlyses in this study re bsed on crosssectionl dt. Second, some of the outcomes used re outcomes of which the cuslity with regrd to vitmin D is still being questioned (4). Therefore, before using the results of these nlyses in updting guidelines on optiml vitmin D concentrtions nd on supplementtion, the cusl reltionship with vitmin D hs first to be proven. Third, dt on crdiovsculr disese ws bsed on selfreport. Although previous reserch reveled tht the greement between self-reported chronic diseses nd dt from generl prctitioners is firly ccurte (41), this could hve cused some imprecision. The min strengths of this study re its lrge popultion-bsed study smple, the determintion of mny outcome vribles within the sme study smple, nd, for severl outcomes, long follow-up time ws vilble. Furthermore, the thresholds were determined systemticlly for ll of the potentilly relevnt outcomes. In conclusion, the min finding of this study is tht thresholds for serum 25(OH)D differ ccording to outcome nd subgroup. This indictes tht future guidelines my need to consider more subgroups. In ddition, this study does not support reltively high required levels of serum 25(OH)D, especilly not for older persons, women, nd persons with high BMI. However, the thresholds first hve to be vlidted in other popultions with other distributions of serum 25(OH)D concentrtions nd in dose-response clinicl trils. Acknowledgments We cknowledge the tem nd the prticipnts of the Longitudinl Aging Study Amsterdm. Address ll correspondence nd requests for reprints to: N. 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