Dysequilibrium of the PTH-FGF23-vitamin D axis in relapsing remitting multiple sclerosis; a longitudinal study

Dysequilibrium of the PTH-FGF23-vitamin D axis in relapsing remitting multiple sclerosis; a... Background: Parathyroid glands of people with relapsing remitting multiple sclerosis (RRMS) fail to respond to low serum 25-hydroxyvitamin D (25OHD) and low serum calcium, which are stimuli for parathyroid hormone (PTH) secretion. This led us to hypothesise: that there is suppression of PTH in RRMS due to higher than normal serum concentrations of fibroblast growth factor 23 (FGF23). We therefore sought evidence for dysregulation of the PTH-FGF23-vitamin D axis in RRMS. Methods: Longitudinal study (winter to summer) with fasting venepunctures. For RRMS subjects who recruited a healthy control (HC) friend, pairs analyses were performed. For each pair, the within-pair difference of the variable of interest was calculated (RRMS minus HC). Then, the median of the differences from all pairs was compared against a median of zero (Wilcoxon) and the 95% confidence interval of that median difference (CI) was calculated (Sign Test). Results: RRMS had lowerwinterPTHthan HC, P = 0.005, (CI -2.4 to 0.5 pmol/L, n = 28 pairs), and lower summer PTH, P = 0.04, (CI -1.8 to 0.5, n = 21 pairs). Lower PTH associates physiologically with lower intact FGF23 (iFGF23), yet RRMS had higher iFGF23 than HC in winter, P =0.04, (CI-3to15pg/mL, n = 28 pairs) and iFGF23 levels comparable to HC in summer, P = 0.14, (CI -5 to 13, n = 21 pairs). As PTH stimulates and FGF23 reduces, renal 1-alpha hydroxylase enzyme activity, which synthesises serum 1,25-dihyroxyvitamin D (1,25(OH) D) from serum 25OHD, we examined the ratio of serum 1,25(OH) Dto 2 2 serum 25OHD. In winter, this ratiowaslowerinRRMSversusHC, P = 0.013, (CI -1.2 to - 0.3, n =28 pairs). Conclusions: This study revealed a dysequilibrium of the PTH-FGF23-vitamin D axis in RRMS, with lower plasma PTH, higher plasma iFGF23 and a lower serum 1,25(OH) D to 25OHD ratio in RRMS compared with HC subjects. This dysequilibrium is consistent with the study hypothesis that in RRMS there is suppression of the parathyroid glands by inappropriately high plasma concentrations of iFGF23. Studying the basis of this dysequilibrium may provide insight into the pathogenesis of RRMS. Keywords: Multiple sclerosis, Vitamin D, Parathyroid hormone, Fibroblast growth factor 23 Background tubules and immune cells (macrophages, dendritic cells, VitaminDissynthesisedinskinuponexposuretoultravio- and potentially B and T lymphocytes) to a very potent form let light and converted in the liver to 25-hydroxyvitamin D of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) D), by (25OHD), the main vitamin D metabolite in serum and the 1-alpha hydroxylase (Tanaka and DeLuca 1981; Reichel clinical indicator of vitamin D nutrition (Parfitt et al. 1982; et al. 1987a; Reichel et al. 1987b; Bacchetta et al. 2013; Hollis 1996). Serum 25OHD is converted in the kidney Shimada et al. 2004; Shimada et al. 2005;Enioutina et al. 2009; Carvalho et al. 2017), which is increased by parathyroid hormone (PTH) and reduced by fibroblast * Correspondence: mark.stein@mh.org.au growth factor-23 (FGF23) (Tanaka and DeLuca 1981; The Royal Melbourne Hospital, Parkville, Australia Bacchetta et al. 2013; Shimada et al. 2004;Shimada et Department of Diabetes and Endocrinology, The Royal Melbourne Hospital, Parkville, VIC 3050, Australia al. 2005; Gattineni et al. 2011). Serum 1,25(OH) Dis Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stein et al. Molecular Medicine (2018) 24:27 Page 2 of 9 degraded by renal 24-hydroxylase, which is reduced by Methods PTH and increased by FGF23 (Tanaka and DeLuca Subjects 1981; Shimada et al. 2004; Gattineni et al. 2011). PTH People with RRMS (aged ≥18 years) were recruited from and FGF23 thus regulate serum 1,25(OH) D concentra- hospital clinics and through publicity on the MS tions through opposite effects on synthetic and de- Australia Victoria website in July–August 2011. They gradative hydroxylases. were asked to bring one or more healthy friends (aged These regulatory pathways include serum 1,25(OH) D ≥18 years) to serve as healthy controls (HC). itself, which feeds back both on the enzymes The study was approved by the Human Research Eth- 1-alpha-hydroxylase and 24-hydroxylase (Colston et al. ics Committees of Melbourne Health and Eastern 1977) and on the hormones PTH and FGF23. Health, Victoria. All subjects provided written informed 1,25(OH) D directly stimulates the synthesis of FGF23 consent. by osteoblasts and osteocytes (Shimada et al. 2004; Exclusions to filter out conditions affecting mineral Nguyen-Yamamoto et al. 2017). It can increase the absorp- metabolism were: tion of dietary calcium and phosphate, and higher serum Pregnancy, breast-feeding, fracture, bone/joint surgery, concentrations of calcium and phosphate may feedback to treatment with raloxifene, alendronate, risedronate, zole- stimulate FGF23 secretion (Shimada et al. 2005; dronate, strontium ranelate or teriparetide in the previ- Nguyen-Yamamoto et al. 2017; Quinn et al. 2013). Serum ous 6 months. 1,25(OH) D also feeds back directly on the parathyroid Except for the oral contraceptive, treatment with es- glands to reduce PTH synthesis (Silver et al. 1986)and trogen or progesterone, testosterone, phenytoin, valpro- also indirectly by its action to increase serum concen- ate or levetiracetam in the previous 3 months. trations of FGF23, which may then further reduce Treatment with oral or systemic glucocorticoid in the PTH synthesis and secretion (Ben-Dov et al. 2007; previous month. Lavi-Moshayoff et al. 2010). Completing another feed- Treatment with furosemide or thiazide diuretic in the back loop, PTH, the secretion of which is reduced by previous week. FGF23, in turn stimulates FGF23 synthesis by osteo- Inability to ambulate 300 m without assistance of an- blasts and osteocytes (Ben-Dov et al. 2007; other person. Lavi-Moshayoff et al. 2010). HC were excluded if a 1st- or 2nd-degree relative had FGF23 circulates as an intact molecule (iFGF23), a history of demyelination. which is cleaved to release a C-terminal fragment The following were permitted: oral contraceptives, asthma (cFGF23) (Razzaque and Lanske 2007; Blau and Collins inhalers, over-counter vitamin/mineral supplements. 2015). FGF23 binds a variety of FGF receptor subtypes, We sought to enrol at least 20 individuals with RRMS either directly or in conjunction with the Klotho recep- and 20 HC based on the sample size used to report that tor (Gattineni et al. 2011; Razzaque and Lanske 2007; people with RRMS failed to increase their serum PTH Blau and Collins 2015; Kurosu et al. 2006). A soluble concentrations in winter (Soilu-Hänninen et al. 2008). alpha fragment of the Klotho receptor may also circulate Between August 19th-September 28th 2011 subjects and may serve to stimulate FGF23 synthesis (Smith et al. had fasting ‘winter’ venepuncture at collection centres of 2012). Melbourne Pathology (Sonic Healthcare, Collingwood, In winter, a decrease in sunlight exposure and vitamin Victoria). Venepuncture was performed between D synthesis is associated with an increase in serum 7:30 am–10 am to control for circadian variation in PTH, which maintains the serum calcium concentration. PTH. Subjects were asked to avoid alcohol for at least In people with relapsing remitting multiple sclerosis 24 h before venepuncture. Each RRMS-HC friend pair (RRMS), Soilu-Hänninen et al. (Soilu-Hänninen et al. attended the same centre at the same time and was ran- 2008) observed that the winter rise in serum PTH was domised (via central computer) as to within-pair vene- blunted and was associated with lower serum calcium, puncture order. They completed questionnaires compared to healthy controls (HC). It appeared that regarding anthropometrics, personal and family health. the parathyroid glands of people with RRMS failed to Body mass index (BMI) was calculated as weight (kg)/ respond to two stimuli for PTH secretion, namely lower [height (m)] . concentrations of vitamin D and serum calcium. This Subjects with serum 25OHD < 50 nM were advised that led us to hypothesise that there is suppression of PTH their vitamin D nutrition was suboptimal. They were in RRMS due to higher than normal serum concentra- given their serum 25OHD result to take to their primary tions of FGF23. In the present study, we sought evi- care physician with the recommendation that they com- dence for dysregulation of the PTH-FGF23-vitamin D mence vitamin D3 (1000 IU daily). We did not mandate axis in RRMS, in a longitudinal study from winter to or monitor such supplementation as we planned to dir- summer. ectly measure serum 25OHD on repeat venepuncture. Stein et al. Molecular Medicine (2018) 24:27 Page 3 of 9 Subjects were invited to return February 2012 for of zero (Wilcoxon) and the 95% CI of that median dif- identical ‘summer’ collections, preserving within-pair ference was calculated (Sign Test). venepuncture order. Modelling (general linear modelling/multiple regres- sion/path analysis) was not performed as bi-directional Laboratory assays causality (Ben-Dov et al. 2007) renders such approaches EDTA blood tubes were centrifuged immediately, as invalid (personal communication, Professor Terry Speed, were serum separator tubes after blood had clotted. Bioinformatics Division, Walter and Eliza Hall Institute Plasma for PTH assay was transported at ambient of Medical Research, Melbourne). temperature (Glendenning et al. 2002). Remaining Statistical analyses were performed on Minitab 13.1 and plasma and sera were frozen on site then transported for 17 (http://www.minitab.com). Outliers were defined by same day general biochemistry analysis or for storage at the Minitab definition, i.e. > 1.5 x interquartile range − 80 C. Melbourne Pathology Central Laboratory (Col- (IQR) outside the IQR. P < 0.05 was considered significant. lingwood, Melbourne) measured serum biochemistry Hypotheses with an a priori direction, viz. lower PTH, (Modular c701 chemistry analyser; Roche, Mannheim, higher FGF23 (RRMS versus HC), were tested one-tailed Germany), plasma human intact PTH (Modular e602 (Armitage and Berry 1994). Other hypotheses were tested immunoassay analyser; Roche, Mannheim, Germany) two-tailed. with coefficient of variation (CV) = 4.0% (2–50 pmol/L) and serum 25OHD (Liaison analyser in winter, Liaison Results XL analyser in summer; DiaSorin, Turin, Italy), with CV 55 RRMS and 35 HC subjects were recruited in winter. = 7.5% (40–280 nmol/L). Calcium corrected for albumin Their median (IQR) ages were 46.5 (36.5–52.5) and 49.5 (Cacorr) was calculated as total calcium (mmol/L) (38.5–56.0) years, respectively (P = 0.2). RRMS and HC + (40-albumin [g/L]) × 0.02. were 71% and 43% female (F), respectively, (P = 0.007). Sullivan Nicolaides Pathology, (Sonic Healthcare, Bris- Subjects were excluded for non-return of the questionnaire bane, Australia) measured plasma intact FGF23 (iFGF23) (3 RRMS, 2 HC) and confounding medication (2 HC), by ELISA (CY4000; Kainos Laboratories, Tokyo, Japan) leaving 83 winter subjects. Of RRMS, 14 (27%) were (Yamazaki et al. 2002 and see also Imel et al. 2006 and on no therapy, 6 (12%) took glatiramer acetate, 26 Smith et al. 2013), with interassay CV = 3% (at mean (50%) interferon-beta, 2 (4%) natalizumab and 4 (8%) 71.8 pg/mL) and 4% (at mean 203.3 pg/mL), plasma fingolimod; 38% were taking vitamin D compared to C-terminal FGF23 peptide (FGF23c) by ELISA (Immu- 16% HC (P = 0.02), and 12% were taking calcium notopics, San Clemente, USA) with interassay CV = 9% compared to 6% HC (P =0.4). (at mean 32.3) and 1% (at mean 293) and plasma soluble Median (IQR) BMIs were: RRMS 23.9 (21.2–29.2) and alpha klotho (Klotho) by ELISA (IBL, Hamburg HC 26.2 (22.8–29.0) kg/m (P = 0.4). Two RRMS females Germany), with interassay CV = 4% (at mean 1130.8 pg/ with outlier BMIs (43.3, 43.0) and 1 RRMS female with mL) and 4% (at mean 1322.3 pg/mL). Serum ferritin was BMI 16.9 (not clinically consistent with normal mineral assayed with the Architect kit, (Abbott, Abbott Park, metabolism) were excluded. One female HC had a failed Illinois, USA). Serum 1,25OH D was measured by im- winter venepuncture. This left 79 subjects (49 RRMS, 30 munoassay (IDS iSYS, Tyne and Wear, UK). HC) for whom winter plasma PTH and other analytes For ELISAs, summer and winter specimens from each were studied for cohort analyses. Of these, 61 (38 RRMS, RRMS-HC pair were measured in the same assay (within 23 HC) agreed to return for summer venepuncture for the same ELISA plate). Laboratory personnel were blind measurement of plasma PTH and other analytes. After to source of specimens. the above exclusions, 28 RRMS-HC pairs remained for winter analyses and 23 RRMS-HC pairs for summer ana- Statistics lyses. We did not ascertain the reasons why some sub- The frequencies of characteristics of RRMS and HC sub- jects declined to return in summer. jects were compared by Fisher’s exact test. Cohort me- dian values for biochemical analytes of RRMS and HC 25OHD and 1,25(OH) D subjects were compared by Mann-Whitney test. The Serum 25OHD increased from winter to summer in 95% confidence intervals (CIs) for differences between both RRMS and HC (P < 0.01). However, for serum cohort medians were calculated by Moods Median Test. 1,25(OH) D a significant seasonal rise was detected only For RRMS subjects who had recruited an HC friend, in the HC (P = 0.02) (Fig. 1, Table 1). pairs analyses were performed. For each pair, the within-pair difference of the variable of interest was cal- PTH and iFGF23 culated (RRMS minus HC). Then, the median of the dif- Pairs analysis revealed that subjects with RRMS had ferences from all pairs was compared against a median lower plasma PTH than HC in winter and summer Stein et al. Molecular Medicine (2018) 24:27 Page 4 of 9 (Fig. 2, Table 2). For example, the median winter Examination of potential confounders within-pair difference (RRMS-HC) in plasma PTH was The RRMS cohort contained a higher proportion of fe- − 1.6 pmol/L (P = 0.005). To put the magnitude of this males, andplasmaPTH mayvarywithrenal functionand difference in context, it is over half the magnitude of the plasma FGF23 with serum magnesium and ferritin (Takeda HC IQR for winter plasma PTH (Table 1). et al. 2011; Braithwaite et al. 2012; Durham et al. 2007). In subjects with RRMS, plasma iFGF23 was the same Therefore, we explored post-hoc whether the lower plasma as or higher than in HC (Fig. 3, Table 2). For example, PTH and paradoxically similar or higher plasma iFGF23 in the median winter within-pair difference (RRMS-HC) in subjects with RRMS could be due to confounding by sex or plasma iFGF23 was 5 pg/mL (P = 0.04). To put the mag- serum creatinine, magnesium, ferritin or 25OHD. nitude of this difference in context, it approximates one Either for all subjects combined, or within the separate third the magnitude of the HC IQR for winter plasma RRMS and HC cohorts, plasma PTH and plasma iFGF23 iFGF23 (Table 1). did not differ by sex and in winter neither was associated with serum creatinine, which itself did not differ be- Ratio of serum 1,25(OH)2D to 25OHD tween RRMS and HC. As PTH increases and iFGF23 reduces the activity of renal The median within-pair difference (RRMS-HC) in serum 1-alpha hydroxylase, which synthesises serum 1,25(OH) D magnesium was not significantly different from zero (both from serum 25OHD, (and as PTH reduces and iFGF23 in- in winter and summer). The median within-pair difference creases the activity of renal 24-hydroxylase which de- (RRMS-HC) in serum ferritin was not significantly different grades serum 1,25(OH) D) we compared the ratio of the from zero (both in winter and summer). Furthermore, there serum concentrations of 1,25(OH) Dand 25OHD be- was no correlation between within-pair differences in tween RRMS and HC. Subjects with RRMS had a lower plasma iFGF23 and within-pair differences in serum ferritin. ratio of serum 1,25(OH) D (pmol/L) to serum 25OHD The bivariate relationship between serum 25OHD (nmol/L). The median winter within-pair difference and plasma PTH was weak (Fig. 5). Winter serum (RRMS-HC) in this ratio was − 0.7 (P = 0.013, 95% CI -1.2 25OHD correlated inversely with winter plasma PTH to − 0.3, n =28 pairs) (Fig. 4). To put the magnitude of this for all subjects combined (r = − 0.27, P = 0.02) and difference in context, it is over one third the magnitude of within the RRMS (r = − 0.32, P = 0.03) but not the HC the HC IQR for the winter ratio of serum 1,25(OH) D cohort (r = − 0.06, P = 0.7). In summer, serum 25OHD (pmol/L) to serum 25OHD (nmol/L); as the median (IQR) did not correlate with plasma PTH. Furthermore, HC winter ratio was 3.1 (2.0–3.8). serum 25OHD did not correlate with plasma iFGF23, In the smaller sample of summer pairs, the median plasma FGF23c, plasma klotho or serum ferritin. within-pair difference (RRMS-HC) of the ratio of serum In summary, lower plasma PTH and similar or higher 1,25(OH) D to serum 25OHD was − 0.3, which did not plasma iFGF23 in RRMS compared with HC subjects reach significance (P = 0.07, 95% CI -0.9 to 0.1, n =22 pairs) was not due to confounding by sex or serum creatinine, (Fig. 4), potentially because of the smaller sample size. magnesium, ferritin or 25OHD. Fig. 1 Serum 25OHD and serum 1,25(OH) D Individual values (open circles), cohort medians and interquartile ranges (boxes) are plotted for those subjects who had both winter and summer measurement of serum 1,25(OH) D. Serum 25OHD increased from winter to summer in both HC and RRMS subjects. However, only HC demonstrated a significant seasonal rise in serum 1,25(OH) D 2 Stein et al. Molecular Medicine (2018) 24:27 Page 5 of 9 Table 1 Cohort analyses Chemical RRMS HC 95% CI for difference (RRMS-HC) in medians P value median (IQR) median (IQR) For 25OHD and PTH, n = 49 for RRMS and 30 for HC. For all other analytes below, n = 38 for RRMS and n = 25 for HC Winter 25OHD 58 (40–89) 43 (36–71) -3 to 32 0.09 *** *** Summer 25OHD 87 (70–108) 81 (70–92) −6 to 18 0.4 Winter PTH 4.5 (3.7–6.0) 5.8 (4.1–6.6) −1.6 to −0.3 0.02 Summer PTH 4.7 (3.9–5.7) 5.0 (4.0–6.0) − 1.5 to 0.5 0.2 Winter iFGF 43 (36–56) 41 (35–49) −5 to 8 0.14 Summer iFGF 46 (40–60) 47 (38–52) −6 to 6 0.3 Winter FGFc 95 (76–136) 85 (73–112) − 12 to 38 0.08 Summer FGFc 93 (77–114) 90 (72–124) − 15 to 18 0.3 Winter Klotho 682 (517–817) 677 (553–964) − 182 to 134 0.6 Summer Klotho 588 (491–743) 732 (571–976) − 257 to 16 0.08 Winter Ferritin 103 (58–214) 73 (24–170) −34 to 75 0.2 Summer Ferritin 98 (44–185) 90 (35–148) −32 to 67 0.5 Winter 125(OH) D 148 (116–181) 139 (121–155) −19 to 25 0.4 + ** Summer 125(OH) D 168 (133–201) 176 (147–204) −27 to 27 0.7 Restricted to subjects who provided both a winter and summer specimen, For all analytes below n = 38 for RRMS and n = 23 for HC Winter 25OHD 59 (38–91) 47 (37–79) −17 to 32 0.4 ** ** Summer 25OHD 87 (70–108) 81 (70–92) −6 to 18 0.4 Winter PTH 4.5 (3.8–6.1) 5.8 (4.0–6.6) −1.7 to 0.3 0.09 Summer PTH 4.7 (3.9–5.7) 5.0 (4.0–6.0) −1.5 to 0.5 0.3 Winter iFGF 45 (36–59) 45 (35–50) −6 to 9 0.3 Summer iFGF 46 (40–60) 49 (40–52) −6 to 5 0.4 Winter FGFc 93 (77–134) 96 (76–123) − 23 to 24 0.4 Summer FGFc 93 (77–114) 90 (75–134) − 15 to 17 0.5 Winter Klotho 681 (523–749) 677 (569–1034) − 154 to 104 0.5 Summer Klotho 588 (491–743) 725 (563–984) − 231 to 27 0.1 Winter Ferritin 108 (60–224) 62 (21–153) −4 to 87 0.06 Summer Ferritin 98 (44–188) 89 (32–134) −25 to 67 0.4 Winter 125(OH) D 150 (114–193) 148 (126–160) −16 to 31 0.5 ++ * Summer 125(OH) D 168 (133–201) 177 (145–204) −27 to 27 0.7 Abbreviations: IQR interquartile range * ** *** + ++ P: < 0.05, < 0.005, < 0.0005, 0.07, 0.23 for within-cohort, winter versus summer values for 25OHD and 1,25(OH) D tabulated above Discussion We present evidence for dysequilibrium of the PTH-FGF23-vitamin D axis in RRMS. Subjects with RRMS had lower plasma PTH concentrations than HC, yet their plasma iFGF was the same as or higher than HC. In winter, when vitamin D nutrition was lowest, they had a lower serum 1,25(OH) D to 25OHD ratio and in summer they failed to demonstrate a rise in serum 1,25(OH) D, as observed in HC. These findings are consistent with our initial hypothesis that there is suppression of PTH in RRMS, associated with higher Fig. 2 Within-pair differences (RRMS-HC) in plasma PTH serum FGF23. The magnitude of this dysequilibrium was Stein et al. Molecular Medicine (2018) 24:27 Page 6 of 9 Table 2 Pairs analyses (subject with RRMS minus HC friend) Chemical Median difference (IQR) 95% CI for median difference P value Analyses of all pairs: n = 28 pairs (winter) 21–23 (summer) Winter 25OHD 17 (−8 to 48) −5 to 37 0.03 Summer 25OHD 11 (− 12 to 41) −9 to 35 0.09 Winter PTH − 1.6 (− 2.6 to 0.6) −2.4 to 0.5 0.005 Summer PTH −1.1 (−2.0 to 0.7) −1.8 to 0.5 0.04 Winter iFGF 5 (−6 to 18) −3 to 15 0.04 Summer iFGF 4 (−6 to 15) −5 to 13 0.14 Winter FGFc 7 (−25 to 49) −15 to 45 0.15 Summer FGFc −4(−34 to 29) −26 to 19 0.7 Winter Klotho −58 (− 314 to 147) − 252 to 88 0.4 Summer Klotho − 206 (− 469 to 117) − 386 to 40 0.05 Winter Ferritin 50 (−58 to 89) −45 to 63 0.3 Summer Ferritin 27 (−57 to 90) −36 to 79 0.4 Winter 125(OH)D6(−20 to 29) −9 to 18 0.4 Summer 125(OH) D −4(−50 to 18) −42 to 15 0.5 BMI −1.4 (−5.6 to 4.4) −4.5 to 2.4 0.8 Restricted to pairs who provided winter and summer specimens: n =21–23 pairs Winter 25OHD 7 (−8 to 51) −6 to 46 0.08 Summer 25OHD 11 (−12 to 41) −9 to 35 0.09 Winter PTH −1.5 (−2.6 to 0.6) −2.5 to 0.6 0.02 Summer PTH −1.1 (−2.0 to 0.7) − 1.8 to 0.5 0.04 Winter iFGF 6 (−4 to 16) −3 to 15 0.05 Summer iFGF 4 (−6 to 15) −5 to 13 0.14 Winter FGFc 3 (−36 to 47) −25 to 42 0.4 Summer FGFc −4(−34 to 29) −26 to 19 0.7 Winter Klotho −81 (− 349 to 96) − 297 to 88 0.2 Summer Klotho −206 (−469 to 117) −386 to 40 0.05 Winter Ferritin 52 (−54 to 164) −21 to 87 0.13 Summer Ferritin 27 (−57 to 90) −36 to 79 0.4 Winter 125(OH)D2(−32 to 30) −19 to 22 0.7 Summer 125(OH) D −4(−50 to 18) −42 to 15 0.5 No significant differences were detected across season Abbreviations: IQR interquartile range Fig. 4 Within-pair differences (RRMS-HC) in the serum 1,25(OH) Dto Fig. 3 Within-pair differences (RRMS-HC) in plasma iFGF23 25OHD ratio Stein et al. Molecular Medicine (2018) 24:27 Page 7 of 9 Fig. 5 Plasma PTH and Serum 25OHD Plasma PTH in winter (open circles) and summer (solid circles) plotted against serum 25OHD for subjects who provided both winter and summer venepuncture. Three outliers (serum 25OHD > 150 nM) were excluded to enlarge the scale of the X-axis large. The median winter RRMS-HC difference in their MS cohort grouped primary progressive and sec- plasma PTH exceeded half the HC IQR for winter ondary progressive MS together with RRMS (Lonergan plasma PTH and the median winter RRMS-HC differ- et al. 2011; McKenna et al. 2018). Blau and Collins (Blau ence in plasma iFGF23 approximated a third of the HC and Collins 2015) wrote “The action of FGF23 on the IQR for winter iFGF23. Similarly, the median winter parathyroid gland has been reported to suppress PTH RRMS-HC difference in the ratio of serum 1,25(OH) D secretion in vitro and in rodent models, but demonstra- to 25OHD exceeded a third of the HC IQR for the win- tion of a similar effect in humans is lacking.” Although ter value of this ratio. our findings are correlative they are consistent with a Dysequilibrium of the PTH-FGF23-vitamin D axis data physiological effect of FGF23 to suppress the parathyroid exhibited internal consistency. Thus, lower plasma PTH glands in RRMS. and higher plasma iFGF23 observed in RRMS would be We were not able to determine if dysequilibrium of expected to lead to lower renal 1-alpha hydroxylase ac- the PTH-FGF23-vitamin D axis precedes or follows the tivity in RRMS and thus decreased synthesis of serum pathophysiology of RRMS. It is interesting, however, that 1,25(OH) D from 25OHD (Tanaka and DeLuca 1981; while we find differences (RRMS vs HC) in the levels of Bacchetta et al. 2013; Shimada et al. 2004; Shimada et al. hormones (PTH and iFGF23) that regulate the activity 2005). Lower plasma PTH and higher plasma iFGF23 in of the enzymes 1-alpha hydroxylase and 24-hydroxylase, RRMS would also be expected to lead to higher renal which respectively synthesise and degrade the potent 24-hydroxylase activity in RRMS with increased degrad- vitamin D metabolite 1,25(OH) D, others find differ- ation of serum 1,25(OH) D (Tanaka and DeLuca 1981; ences between people with MS versus HC in the genes Bacchetta et al. 2013; Shimada et al. 2004; Shimada et al. coding for these same enzymes (Pierrot-Deseilligny and 2005). Accordingly, the winter serum 1,25(OH) Dto Souberbielle 2017). This certainly raises the possibility 25OHD ratio was lower in RRMS than HC and subjects that dysequilibrium of the PTH-FG23-vitamin D axis with RRMS did not demonstrate the expected summer could have a pathogenic role. In addition, because PTH rise in serum 1,25(OH) D observed in HC. Unlike the and FGF23 also regulate extra-renal 1-alpha hydroxylase Finnish study of Soilu-Hänninen et al. (Soilu-Hänninen in innate and adaptive immune cells, (for example see et al. 2008), we did not find a lower winter serum cal- Ref Bacchetta et al. 2013) this dysequilibrium has the cium concentration in RRMS, which might be explained potential to modify immune-inflammatory processes in by the fact that the winter serum 25OHD was higher in MS via autocrine as well as endocrine vitamin D metab- the Australian subjects. Irish researchers examined PTH olism, in keeping with evidence that impaired vitamin D at low levels of 25OHD (Lonergan et al. 2011; McKenna nutrition is associated with MS (Pierrot-Deseilligny and et al. 2018). In one study (Lonergan et al. 2011) a signifi- Souberbielle 2017; Munger et al. 2006; van der Mei et al. cant inverse correlation of PTH with 25OHD was 2007; Simpson Jr et al. 2010). present in controls but not in people with MS. Those It will be important to determine whether plasma studies, however, did not report plasma calcium and PTH and plasma iFGF23 could be used as biomarkers to Stein et al. Molecular Medicine (2018) 24:27 Page 8 of 9 identify individuals at risk for RRMS or predict disease basis of this dysequilibrium may provide insight into the course and response to therapy. It will also be important pathogenesis of RRMS and requires further investigation. to determine whether there is dysequilibrium in the Abbreviations PTH-FGF23-vitamin D axis in other autoimmune dis- 1,25(OH) D: 1,25-dihydroxyvitamin D; 25OHD: 25-hydroxyvitamin D; eases such as type 1 diabetes, systemic lupus erythema- CI: Confidence interval; FGF23: Fibroblast growth factor 23; FGFc: c-terminal fibroblast growth factor 23; HC: Healthy control(s); iFGF23: Intact fibroblast tosus and rheumatoid arthritis that exhibit an incidence growth factor 23; IQR: Interquartile range; Klotho: Soluble alpha klotho; or disease activity which correlates with vitamin D nutri- PTH: Parathyroid hormone; RRMS: Relapsing remitting multiple sclerosis tion (Munger et al. 2013; Watad et al. 2017). This study has several limitations. Not all of the differ- Acknowledgements Professor TJ Martin (St Vincent’s Institute of Medical Research, Melbourne) ences between RRMS and HC demonstrated by and Professor Seiji Fukumoto (University of Tokyo) gave helpful advice on within-pair analysis were seen in comparison of RRMS the iFGF23 assay. Professor Terry Speed (Walter and Eliza Hall Institute of and HC cohort medians. Furthermore, not all subjects Medical Research) and Professor Ian Gordon (Statistical Consulting Centre, University of Melbourne) provided statistical advice and review. Mary Tanner with RRMS recruited a HC friend to the study. While (Royal Melbourne Hospital), Kelly-Jane Lazarus (Box Hill Hospital) and Elizabeth cohort analyses have the advantage of larger sample size, McDonald (Medical Director, MS Australia Victoria) helped with recruitment. analysis of RRMS-HC pairs may be more sensitive as it Bob Hutton (Royal Melbourne Hospital Information Technology Department) assisted with the study website. Dr. Ken Sikaris (Pathologist, Melbourne controls for pre-analytical specimen handling measure- Pathology and Chair, International Federation Clinical Chemistry Committee on ment error (each RRMS-HC friend pair attended the Analytical Quality) advised and supervised general biochemistry. Hayley Tacon, same specimen collection centre at the same time), Eliza Ticknell and Marzi DeGaris (Melbourne Pathology) advised and assisted with specimen collection. Melissa Gresle (University of Melbourne) helped with which may contribute up to 50% of total analyte meas- sample storage. Karen Young, Michael Freemantle, Melissa Nelson, Eric Simons urement error (Plebani 2006). To reduce analytical la- (Sullivan Nicolaides Pathology) performed the iFGF, FGFc, klotho, ferritin and boratory error, winter and summer specimens from each 1,25(OH)2D assays. Advanced Professional Systems Pty Ltd. assisted with Medilink software to remind subjects of venepuncture arrangements. RRMS-HC pair were analysed within the same ELISA plate. In addition, we deliberately recruited HC from Funding friends of the people with RRMS to minimise bias from This study was funded by charitable research grants from The Myer Foundation unmeasured lifestyle, social and demographic variables Australia (Martyn Myer [President], Christine Edwards [then CEO]) and Multiple Sclerosis Research Australia (Professor Bill Carroll [then Director], Heather Cato that could potentially mask disease-specific differences. [then MS Grants Research Co-ordinator]). These funding bodies gave generous A further potential limitation is that we did not measure support and encouragement but had no role in the design of the study, in the markers of inflammation, which could confound the in- collection, analysis, and interpretation of data or in writing the manuscript. terpretation of serum ferritin concentrations, if people Availability of data and materials with RRMS had more inflammation than HC. Lower Clinical data set not available. As was standard at the year of study inception serum ferritin concentrations have been associated with and institutional human research ethics committees review, human research ethics committee permission was not sought or granted and informed higher serum concentrations of FGF23c and iFGF23 consent was not obtained for public repository deposition of patient data or (Braithwaite et al. 2012; Durham et al. 2007), but this re- sharing the data set with researchers outside our institutions’ research team. lationship appears to be assay specific (Durham et al. 2007). In particular, the Kainos iFGF23 assay used in this Authors’ contributions Study concept and design: All authors. Acquisition, analysis, or interpretation study was not affected by low concentrations of serum of data: All authors. Drafting of the manuscript: MS, LCH. Critical revision of ferritin (Durham et al. 2007). Hence, potential confound- the manuscript for important intellectual content: All authors. Statistical ing of serum ferritin by inflammation in the RRMS co- analysis: MS. Obtained funding: All authors. Administrative, technical, or material support: All authors. Study supervision: All authors. All authors read hort would not explain higher RRMS plasma and approved the final manuscript. concentrations of iFGF23 in this study. Finally, the fact that some subjects chose not to return for summer Ethics approval and consent to participate The study was approved by the Human Research Ethics Committees of venepuncture may have introduced bias into the study. Melbourne Health and Eastern Health, Victoria. All subjects provided written However, subjects were recruited both from hospital MS informed consent. clinics and from the community, supporting the generalizability of the results. Competing interests The authors declare that they have no competing interests. Conclusions This study revealed a dysequilibrium of the Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in PTH-FGF23-vitamin D axis in RRMS, with lower plasma published maps and institutional affiliations. PTH, higher plasma iFGF23 and a lower serum 1,25(OH) D to 25OHD ratio in RRMS compared with HC subjects. This Author details 1 2 The Royal Melbourne Hospital, Parkville, Australia. Department of Diabetes dysequilibrium is consistent with the study hypothesis that and Endocrinology, The Royal Melbourne Hospital, Parkville, VIC 3050, in RRMS there is suppression of the parathyroid glands by 3 Australia. Walter and Eliza Hall Institute of Medical Research, Parkville, 4 5 inappropriately high plasma concentrations of iFGF23. The Australia. Sullivan Nicolaides Pathology, Brisbane, Australia. Florey Stein et al. Molecular Medicine (2018) 24:27 Page 9 of 9 Neuroscience Institutes, Parkville, Australia. 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Dysequilibrium of the PTH-FGF23-vitamin D axis in relapsing remitting multiple sclerosis; a longitudinal study

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Biomedicine; Molecular Medicine
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Abstract

Background: Parathyroid glands of people with relapsing remitting multiple sclerosis (RRMS) fail to respond to low serum 25-hydroxyvitamin D (25OHD) and low serum calcium, which are stimuli for parathyroid hormone (PTH) secretion. This led us to hypothesise: that there is suppression of PTH in RRMS due to higher than normal serum concentrations of fibroblast growth factor 23 (FGF23). We therefore sought evidence for dysregulation of the PTH-FGF23-vitamin D axis in RRMS. Methods: Longitudinal study (winter to summer) with fasting venepunctures. For RRMS subjects who recruited a healthy control (HC) friend, pairs analyses were performed. For each pair, the within-pair difference of the variable of interest was calculated (RRMS minus HC). Then, the median of the differences from all pairs was compared against a median of zero (Wilcoxon) and the 95% confidence interval of that median difference (CI) was calculated (Sign Test). Results: RRMS had lowerwinterPTHthan HC, P = 0.005, (CI -2.4 to 0.5 pmol/L, n = 28 pairs), and lower summer PTH, P = 0.04, (CI -1.8 to 0.5, n = 21 pairs). Lower PTH associates physiologically with lower intact FGF23 (iFGF23), yet RRMS had higher iFGF23 than HC in winter, P =0.04, (CI-3to15pg/mL, n = 28 pairs) and iFGF23 levels comparable to HC in summer, P = 0.14, (CI -5 to 13, n = 21 pairs). As PTH stimulates and FGF23 reduces, renal 1-alpha hydroxylase enzyme activity, which synthesises serum 1,25-dihyroxyvitamin D (1,25(OH) D) from serum 25OHD, we examined the ratio of serum 1,25(OH) Dto 2 2 serum 25OHD. In winter, this ratiowaslowerinRRMSversusHC, P = 0.013, (CI -1.2 to - 0.3, n =28 pairs). Conclusions: This study revealed a dysequilibrium of the PTH-FGF23-vitamin D axis in RRMS, with lower plasma PTH, higher plasma iFGF23 and a lower serum 1,25(OH) D to 25OHD ratio in RRMS compared with HC subjects. This dysequilibrium is consistent with the study hypothesis that in RRMS there is suppression of the parathyroid glands by inappropriately high plasma concentrations of iFGF23. Studying the basis of this dysequilibrium may provide insight into the pathogenesis of RRMS. Keywords: Multiple sclerosis, Vitamin D, Parathyroid hormone, Fibroblast growth factor 23 Background tubules and immune cells (macrophages, dendritic cells, VitaminDissynthesisedinskinuponexposuretoultravio- and potentially B and T lymphocytes) to a very potent form let light and converted in the liver to 25-hydroxyvitamin D of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) D), by (25OHD), the main vitamin D metabolite in serum and the 1-alpha hydroxylase (Tanaka and DeLuca 1981; Reichel clinical indicator of vitamin D nutrition (Parfitt et al. 1982; et al. 1987a; Reichel et al. 1987b; Bacchetta et al. 2013; Hollis 1996). Serum 25OHD is converted in the kidney Shimada et al. 2004; Shimada et al. 2005;Enioutina et al. 2009; Carvalho et al. 2017), which is increased by parathyroid hormone (PTH) and reduced by fibroblast * Correspondence: mark.stein@mh.org.au growth factor-23 (FGF23) (Tanaka and DeLuca 1981; The Royal Melbourne Hospital, Parkville, Australia Bacchetta et al. 2013; Shimada et al. 2004;Shimada et Department of Diabetes and Endocrinology, The Royal Melbourne Hospital, Parkville, VIC 3050, Australia al. 2005; Gattineni et al. 2011). Serum 1,25(OH) Dis Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stein et al. Molecular Medicine (2018) 24:27 Page 2 of 9 degraded by renal 24-hydroxylase, which is reduced by Methods PTH and increased by FGF23 (Tanaka and DeLuca Subjects 1981; Shimada et al. 2004; Gattineni et al. 2011). PTH People with RRMS (aged ≥18 years) were recruited from and FGF23 thus regulate serum 1,25(OH) D concentra- hospital clinics and through publicity on the MS tions through opposite effects on synthetic and de- Australia Victoria website in July–August 2011. They gradative hydroxylases. were asked to bring one or more healthy friends (aged These regulatory pathways include serum 1,25(OH) D ≥18 years) to serve as healthy controls (HC). itself, which feeds back both on the enzymes The study was approved by the Human Research Eth- 1-alpha-hydroxylase and 24-hydroxylase (Colston et al. ics Committees of Melbourne Health and Eastern 1977) and on the hormones PTH and FGF23. Health, Victoria. All subjects provided written informed 1,25(OH) D directly stimulates the synthesis of FGF23 consent. by osteoblasts and osteocytes (Shimada et al. 2004; Exclusions to filter out conditions affecting mineral Nguyen-Yamamoto et al. 2017). It can increase the absorp- metabolism were: tion of dietary calcium and phosphate, and higher serum Pregnancy, breast-feeding, fracture, bone/joint surgery, concentrations of calcium and phosphate may feedback to treatment with raloxifene, alendronate, risedronate, zole- stimulate FGF23 secretion (Shimada et al. 2005; dronate, strontium ranelate or teriparetide in the previ- Nguyen-Yamamoto et al. 2017; Quinn et al. 2013). Serum ous 6 months. 1,25(OH) D also feeds back directly on the parathyroid Except for the oral contraceptive, treatment with es- glands to reduce PTH synthesis (Silver et al. 1986)and trogen or progesterone, testosterone, phenytoin, valpro- also indirectly by its action to increase serum concen- ate or levetiracetam in the previous 3 months. trations of FGF23, which may then further reduce Treatment with oral or systemic glucocorticoid in the PTH synthesis and secretion (Ben-Dov et al. 2007; previous month. Lavi-Moshayoff et al. 2010). Completing another feed- Treatment with furosemide or thiazide diuretic in the back loop, PTH, the secretion of which is reduced by previous week. FGF23, in turn stimulates FGF23 synthesis by osteo- Inability to ambulate 300 m without assistance of an- blasts and osteocytes (Ben-Dov et al. 2007; other person. Lavi-Moshayoff et al. 2010). HC were excluded if a 1st- or 2nd-degree relative had FGF23 circulates as an intact molecule (iFGF23), a history of demyelination. which is cleaved to release a C-terminal fragment The following were permitted: oral contraceptives, asthma (cFGF23) (Razzaque and Lanske 2007; Blau and Collins inhalers, over-counter vitamin/mineral supplements. 2015). FGF23 binds a variety of FGF receptor subtypes, We sought to enrol at least 20 individuals with RRMS either directly or in conjunction with the Klotho recep- and 20 HC based on the sample size used to report that tor (Gattineni et al. 2011; Razzaque and Lanske 2007; people with RRMS failed to increase their serum PTH Blau and Collins 2015; Kurosu et al. 2006). A soluble concentrations in winter (Soilu-Hänninen et al. 2008). alpha fragment of the Klotho receptor may also circulate Between August 19th-September 28th 2011 subjects and may serve to stimulate FGF23 synthesis (Smith et al. had fasting ‘winter’ venepuncture at collection centres of 2012). Melbourne Pathology (Sonic Healthcare, Collingwood, In winter, a decrease in sunlight exposure and vitamin Victoria). Venepuncture was performed between D synthesis is associated with an increase in serum 7:30 am–10 am to control for circadian variation in PTH, which maintains the serum calcium concentration. PTH. Subjects were asked to avoid alcohol for at least In people with relapsing remitting multiple sclerosis 24 h before venepuncture. Each RRMS-HC friend pair (RRMS), Soilu-Hänninen et al. (Soilu-Hänninen et al. attended the same centre at the same time and was ran- 2008) observed that the winter rise in serum PTH was domised (via central computer) as to within-pair vene- blunted and was associated with lower serum calcium, puncture order. They completed questionnaires compared to healthy controls (HC). It appeared that regarding anthropometrics, personal and family health. the parathyroid glands of people with RRMS failed to Body mass index (BMI) was calculated as weight (kg)/ respond to two stimuli for PTH secretion, namely lower [height (m)] . concentrations of vitamin D and serum calcium. This Subjects with serum 25OHD < 50 nM were advised that led us to hypothesise that there is suppression of PTH their vitamin D nutrition was suboptimal. They were in RRMS due to higher than normal serum concentra- given their serum 25OHD result to take to their primary tions of FGF23. In the present study, we sought evi- care physician with the recommendation that they com- dence for dysregulation of the PTH-FGF23-vitamin D mence vitamin D3 (1000 IU daily). We did not mandate axis in RRMS, in a longitudinal study from winter to or monitor such supplementation as we planned to dir- summer. ectly measure serum 25OHD on repeat venepuncture. Stein et al. Molecular Medicine (2018) 24:27 Page 3 of 9 Subjects were invited to return February 2012 for of zero (Wilcoxon) and the 95% CI of that median dif- identical ‘summer’ collections, preserving within-pair ference was calculated (Sign Test). venepuncture order. Modelling (general linear modelling/multiple regres- sion/path analysis) was not performed as bi-directional Laboratory assays causality (Ben-Dov et al. 2007) renders such approaches EDTA blood tubes were centrifuged immediately, as invalid (personal communication, Professor Terry Speed, were serum separator tubes after blood had clotted. Bioinformatics Division, Walter and Eliza Hall Institute Plasma for PTH assay was transported at ambient of Medical Research, Melbourne). temperature (Glendenning et al. 2002). Remaining Statistical analyses were performed on Minitab 13.1 and plasma and sera were frozen on site then transported for 17 (http://www.minitab.com). Outliers were defined by same day general biochemistry analysis or for storage at the Minitab definition, i.e. > 1.5 x interquartile range − 80 C. Melbourne Pathology Central Laboratory (Col- (IQR) outside the IQR. P < 0.05 was considered significant. lingwood, Melbourne) measured serum biochemistry Hypotheses with an a priori direction, viz. lower PTH, (Modular c701 chemistry analyser; Roche, Mannheim, higher FGF23 (RRMS versus HC), were tested one-tailed Germany), plasma human intact PTH (Modular e602 (Armitage and Berry 1994). Other hypotheses were tested immunoassay analyser; Roche, Mannheim, Germany) two-tailed. with coefficient of variation (CV) = 4.0% (2–50 pmol/L) and serum 25OHD (Liaison analyser in winter, Liaison Results XL analyser in summer; DiaSorin, Turin, Italy), with CV 55 RRMS and 35 HC subjects were recruited in winter. = 7.5% (40–280 nmol/L). Calcium corrected for albumin Their median (IQR) ages were 46.5 (36.5–52.5) and 49.5 (Cacorr) was calculated as total calcium (mmol/L) (38.5–56.0) years, respectively (P = 0.2). RRMS and HC + (40-albumin [g/L]) × 0.02. were 71% and 43% female (F), respectively, (P = 0.007). Sullivan Nicolaides Pathology, (Sonic Healthcare, Bris- Subjects were excluded for non-return of the questionnaire bane, Australia) measured plasma intact FGF23 (iFGF23) (3 RRMS, 2 HC) and confounding medication (2 HC), by ELISA (CY4000; Kainos Laboratories, Tokyo, Japan) leaving 83 winter subjects. Of RRMS, 14 (27%) were (Yamazaki et al. 2002 and see also Imel et al. 2006 and on no therapy, 6 (12%) took glatiramer acetate, 26 Smith et al. 2013), with interassay CV = 3% (at mean (50%) interferon-beta, 2 (4%) natalizumab and 4 (8%) 71.8 pg/mL) and 4% (at mean 203.3 pg/mL), plasma fingolimod; 38% were taking vitamin D compared to C-terminal FGF23 peptide (FGF23c) by ELISA (Immu- 16% HC (P = 0.02), and 12% were taking calcium notopics, San Clemente, USA) with interassay CV = 9% compared to 6% HC (P =0.4). (at mean 32.3) and 1% (at mean 293) and plasma soluble Median (IQR) BMIs were: RRMS 23.9 (21.2–29.2) and alpha klotho (Klotho) by ELISA (IBL, Hamburg HC 26.2 (22.8–29.0) kg/m (P = 0.4). Two RRMS females Germany), with interassay CV = 4% (at mean 1130.8 pg/ with outlier BMIs (43.3, 43.0) and 1 RRMS female with mL) and 4% (at mean 1322.3 pg/mL). Serum ferritin was BMI 16.9 (not clinically consistent with normal mineral assayed with the Architect kit, (Abbott, Abbott Park, metabolism) were excluded. One female HC had a failed Illinois, USA). Serum 1,25OH D was measured by im- winter venepuncture. This left 79 subjects (49 RRMS, 30 munoassay (IDS iSYS, Tyne and Wear, UK). HC) for whom winter plasma PTH and other analytes For ELISAs, summer and winter specimens from each were studied for cohort analyses. Of these, 61 (38 RRMS, RRMS-HC pair were measured in the same assay (within 23 HC) agreed to return for summer venepuncture for the same ELISA plate). Laboratory personnel were blind measurement of plasma PTH and other analytes. After to source of specimens. the above exclusions, 28 RRMS-HC pairs remained for winter analyses and 23 RRMS-HC pairs for summer ana- Statistics lyses. We did not ascertain the reasons why some sub- The frequencies of characteristics of RRMS and HC sub- jects declined to return in summer. jects were compared by Fisher’s exact test. Cohort me- dian values for biochemical analytes of RRMS and HC 25OHD and 1,25(OH) D subjects were compared by Mann-Whitney test. The Serum 25OHD increased from winter to summer in 95% confidence intervals (CIs) for differences between both RRMS and HC (P < 0.01). However, for serum cohort medians were calculated by Moods Median Test. 1,25(OH) D a significant seasonal rise was detected only For RRMS subjects who had recruited an HC friend, in the HC (P = 0.02) (Fig. 1, Table 1). pairs analyses were performed. For each pair, the within-pair difference of the variable of interest was cal- PTH and iFGF23 culated (RRMS minus HC). Then, the median of the dif- Pairs analysis revealed that subjects with RRMS had ferences from all pairs was compared against a median lower plasma PTH than HC in winter and summer Stein et al. Molecular Medicine (2018) 24:27 Page 4 of 9 (Fig. 2, Table 2). For example, the median winter Examination of potential confounders within-pair difference (RRMS-HC) in plasma PTH was The RRMS cohort contained a higher proportion of fe- − 1.6 pmol/L (P = 0.005). To put the magnitude of this males, andplasmaPTH mayvarywithrenal functionand difference in context, it is over half the magnitude of the plasma FGF23 with serum magnesium and ferritin (Takeda HC IQR for winter plasma PTH (Table 1). et al. 2011; Braithwaite et al. 2012; Durham et al. 2007). In subjects with RRMS, plasma iFGF23 was the same Therefore, we explored post-hoc whether the lower plasma as or higher than in HC (Fig. 3, Table 2). For example, PTH and paradoxically similar or higher plasma iFGF23 in the median winter within-pair difference (RRMS-HC) in subjects with RRMS could be due to confounding by sex or plasma iFGF23 was 5 pg/mL (P = 0.04). To put the mag- serum creatinine, magnesium, ferritin or 25OHD. nitude of this difference in context, it approximates one Either for all subjects combined, or within the separate third the magnitude of the HC IQR for winter plasma RRMS and HC cohorts, plasma PTH and plasma iFGF23 iFGF23 (Table 1). did not differ by sex and in winter neither was associated with serum creatinine, which itself did not differ be- Ratio of serum 1,25(OH)2D to 25OHD tween RRMS and HC. As PTH increases and iFGF23 reduces the activity of renal The median within-pair difference (RRMS-HC) in serum 1-alpha hydroxylase, which synthesises serum 1,25(OH) D magnesium was not significantly different from zero (both from serum 25OHD, (and as PTH reduces and iFGF23 in- in winter and summer). The median within-pair difference creases the activity of renal 24-hydroxylase which de- (RRMS-HC) in serum ferritin was not significantly different grades serum 1,25(OH) D) we compared the ratio of the from zero (both in winter and summer). Furthermore, there serum concentrations of 1,25(OH) Dand 25OHD be- was no correlation between within-pair differences in tween RRMS and HC. Subjects with RRMS had a lower plasma iFGF23 and within-pair differences in serum ferritin. ratio of serum 1,25(OH) D (pmol/L) to serum 25OHD The bivariate relationship between serum 25OHD (nmol/L). The median winter within-pair difference and plasma PTH was weak (Fig. 5). Winter serum (RRMS-HC) in this ratio was − 0.7 (P = 0.013, 95% CI -1.2 25OHD correlated inversely with winter plasma PTH to − 0.3, n =28 pairs) (Fig. 4). To put the magnitude of this for all subjects combined (r = − 0.27, P = 0.02) and difference in context, it is over one third the magnitude of within the RRMS (r = − 0.32, P = 0.03) but not the HC the HC IQR for the winter ratio of serum 1,25(OH) D cohort (r = − 0.06, P = 0.7). In summer, serum 25OHD (pmol/L) to serum 25OHD (nmol/L); as the median (IQR) did not correlate with plasma PTH. Furthermore, HC winter ratio was 3.1 (2.0–3.8). serum 25OHD did not correlate with plasma iFGF23, In the smaller sample of summer pairs, the median plasma FGF23c, plasma klotho or serum ferritin. within-pair difference (RRMS-HC) of the ratio of serum In summary, lower plasma PTH and similar or higher 1,25(OH) D to serum 25OHD was − 0.3, which did not plasma iFGF23 in RRMS compared with HC subjects reach significance (P = 0.07, 95% CI -0.9 to 0.1, n =22 pairs) was not due to confounding by sex or serum creatinine, (Fig. 4), potentially because of the smaller sample size. magnesium, ferritin or 25OHD. Fig. 1 Serum 25OHD and serum 1,25(OH) D Individual values (open circles), cohort medians and interquartile ranges (boxes) are plotted for those subjects who had both winter and summer measurement of serum 1,25(OH) D. Serum 25OHD increased from winter to summer in both HC and RRMS subjects. However, only HC demonstrated a significant seasonal rise in serum 1,25(OH) D 2 Stein et al. Molecular Medicine (2018) 24:27 Page 5 of 9 Table 1 Cohort analyses Chemical RRMS HC 95% CI for difference (RRMS-HC) in medians P value median (IQR) median (IQR) For 25OHD and PTH, n = 49 for RRMS and 30 for HC. For all other analytes below, n = 38 for RRMS and n = 25 for HC Winter 25OHD 58 (40–89) 43 (36–71) -3 to 32 0.09 *** *** Summer 25OHD 87 (70–108) 81 (70–92) −6 to 18 0.4 Winter PTH 4.5 (3.7–6.0) 5.8 (4.1–6.6) −1.6 to −0.3 0.02 Summer PTH 4.7 (3.9–5.7) 5.0 (4.0–6.0) − 1.5 to 0.5 0.2 Winter iFGF 43 (36–56) 41 (35–49) −5 to 8 0.14 Summer iFGF 46 (40–60) 47 (38–52) −6 to 6 0.3 Winter FGFc 95 (76–136) 85 (73–112) − 12 to 38 0.08 Summer FGFc 93 (77–114) 90 (72–124) − 15 to 18 0.3 Winter Klotho 682 (517–817) 677 (553–964) − 182 to 134 0.6 Summer Klotho 588 (491–743) 732 (571–976) − 257 to 16 0.08 Winter Ferritin 103 (58–214) 73 (24–170) −34 to 75 0.2 Summer Ferritin 98 (44–185) 90 (35–148) −32 to 67 0.5 Winter 125(OH) D 148 (116–181) 139 (121–155) −19 to 25 0.4 + ** Summer 125(OH) D 168 (133–201) 176 (147–204) −27 to 27 0.7 Restricted to subjects who provided both a winter and summer specimen, For all analytes below n = 38 for RRMS and n = 23 for HC Winter 25OHD 59 (38–91) 47 (37–79) −17 to 32 0.4 ** ** Summer 25OHD 87 (70–108) 81 (70–92) −6 to 18 0.4 Winter PTH 4.5 (3.8–6.1) 5.8 (4.0–6.6) −1.7 to 0.3 0.09 Summer PTH 4.7 (3.9–5.7) 5.0 (4.0–6.0) −1.5 to 0.5 0.3 Winter iFGF 45 (36–59) 45 (35–50) −6 to 9 0.3 Summer iFGF 46 (40–60) 49 (40–52) −6 to 5 0.4 Winter FGFc 93 (77–134) 96 (76–123) − 23 to 24 0.4 Summer FGFc 93 (77–114) 90 (75–134) − 15 to 17 0.5 Winter Klotho 681 (523–749) 677 (569–1034) − 154 to 104 0.5 Summer Klotho 588 (491–743) 725 (563–984) − 231 to 27 0.1 Winter Ferritin 108 (60–224) 62 (21–153) −4 to 87 0.06 Summer Ferritin 98 (44–188) 89 (32–134) −25 to 67 0.4 Winter 125(OH) D 150 (114–193) 148 (126–160) −16 to 31 0.5 ++ * Summer 125(OH) D 168 (133–201) 177 (145–204) −27 to 27 0.7 Abbreviations: IQR interquartile range * ** *** + ++ P: < 0.05, < 0.005, < 0.0005, 0.07, 0.23 for within-cohort, winter versus summer values for 25OHD and 1,25(OH) D tabulated above Discussion We present evidence for dysequilibrium of the PTH-FGF23-vitamin D axis in RRMS. Subjects with RRMS had lower plasma PTH concentrations than HC, yet their plasma iFGF was the same as or higher than HC. In winter, when vitamin D nutrition was lowest, they had a lower serum 1,25(OH) D to 25OHD ratio and in summer they failed to demonstrate a rise in serum 1,25(OH) D, as observed in HC. These findings are consistent with our initial hypothesis that there is suppression of PTH in RRMS, associated with higher Fig. 2 Within-pair differences (RRMS-HC) in plasma PTH serum FGF23. The magnitude of this dysequilibrium was Stein et al. Molecular Medicine (2018) 24:27 Page 6 of 9 Table 2 Pairs analyses (subject with RRMS minus HC friend) Chemical Median difference (IQR) 95% CI for median difference P value Analyses of all pairs: n = 28 pairs (winter) 21–23 (summer) Winter 25OHD 17 (−8 to 48) −5 to 37 0.03 Summer 25OHD 11 (− 12 to 41) −9 to 35 0.09 Winter PTH − 1.6 (− 2.6 to 0.6) −2.4 to 0.5 0.005 Summer PTH −1.1 (−2.0 to 0.7) −1.8 to 0.5 0.04 Winter iFGF 5 (−6 to 18) −3 to 15 0.04 Summer iFGF 4 (−6 to 15) −5 to 13 0.14 Winter FGFc 7 (−25 to 49) −15 to 45 0.15 Summer FGFc −4(−34 to 29) −26 to 19 0.7 Winter Klotho −58 (− 314 to 147) − 252 to 88 0.4 Summer Klotho − 206 (− 469 to 117) − 386 to 40 0.05 Winter Ferritin 50 (−58 to 89) −45 to 63 0.3 Summer Ferritin 27 (−57 to 90) −36 to 79 0.4 Winter 125(OH)D6(−20 to 29) −9 to 18 0.4 Summer 125(OH) D −4(−50 to 18) −42 to 15 0.5 BMI −1.4 (−5.6 to 4.4) −4.5 to 2.4 0.8 Restricted to pairs who provided winter and summer specimens: n =21–23 pairs Winter 25OHD 7 (−8 to 51) −6 to 46 0.08 Summer 25OHD 11 (−12 to 41) −9 to 35 0.09 Winter PTH −1.5 (−2.6 to 0.6) −2.5 to 0.6 0.02 Summer PTH −1.1 (−2.0 to 0.7) − 1.8 to 0.5 0.04 Winter iFGF 6 (−4 to 16) −3 to 15 0.05 Summer iFGF 4 (−6 to 15) −5 to 13 0.14 Winter FGFc 3 (−36 to 47) −25 to 42 0.4 Summer FGFc −4(−34 to 29) −26 to 19 0.7 Winter Klotho −81 (− 349 to 96) − 297 to 88 0.2 Summer Klotho −206 (−469 to 117) −386 to 40 0.05 Winter Ferritin 52 (−54 to 164) −21 to 87 0.13 Summer Ferritin 27 (−57 to 90) −36 to 79 0.4 Winter 125(OH)D2(−32 to 30) −19 to 22 0.7 Summer 125(OH) D −4(−50 to 18) −42 to 15 0.5 No significant differences were detected across season Abbreviations: IQR interquartile range Fig. 4 Within-pair differences (RRMS-HC) in the serum 1,25(OH) Dto Fig. 3 Within-pair differences (RRMS-HC) in plasma iFGF23 25OHD ratio Stein et al. Molecular Medicine (2018) 24:27 Page 7 of 9 Fig. 5 Plasma PTH and Serum 25OHD Plasma PTH in winter (open circles) and summer (solid circles) plotted against serum 25OHD for subjects who provided both winter and summer venepuncture. Three outliers (serum 25OHD > 150 nM) were excluded to enlarge the scale of the X-axis large. The median winter RRMS-HC difference in their MS cohort grouped primary progressive and sec- plasma PTH exceeded half the HC IQR for winter ondary progressive MS together with RRMS (Lonergan plasma PTH and the median winter RRMS-HC differ- et al. 2011; McKenna et al. 2018). Blau and Collins (Blau ence in plasma iFGF23 approximated a third of the HC and Collins 2015) wrote “The action of FGF23 on the IQR for winter iFGF23. Similarly, the median winter parathyroid gland has been reported to suppress PTH RRMS-HC difference in the ratio of serum 1,25(OH) D secretion in vitro and in rodent models, but demonstra- to 25OHD exceeded a third of the HC IQR for the win- tion of a similar effect in humans is lacking.” Although ter value of this ratio. our findings are correlative they are consistent with a Dysequilibrium of the PTH-FGF23-vitamin D axis data physiological effect of FGF23 to suppress the parathyroid exhibited internal consistency. Thus, lower plasma PTH glands in RRMS. and higher plasma iFGF23 observed in RRMS would be We were not able to determine if dysequilibrium of expected to lead to lower renal 1-alpha hydroxylase ac- the PTH-FGF23-vitamin D axis precedes or follows the tivity in RRMS and thus decreased synthesis of serum pathophysiology of RRMS. It is interesting, however, that 1,25(OH) D from 25OHD (Tanaka and DeLuca 1981; while we find differences (RRMS vs HC) in the levels of Bacchetta et al. 2013; Shimada et al. 2004; Shimada et al. hormones (PTH and iFGF23) that regulate the activity 2005). Lower plasma PTH and higher plasma iFGF23 in of the enzymes 1-alpha hydroxylase and 24-hydroxylase, RRMS would also be expected to lead to higher renal which respectively synthesise and degrade the potent 24-hydroxylase activity in RRMS with increased degrad- vitamin D metabolite 1,25(OH) D, others find differ- ation of serum 1,25(OH) D (Tanaka and DeLuca 1981; ences between people with MS versus HC in the genes Bacchetta et al. 2013; Shimada et al. 2004; Shimada et al. coding for these same enzymes (Pierrot-Deseilligny and 2005). Accordingly, the winter serum 1,25(OH) Dto Souberbielle 2017). This certainly raises the possibility 25OHD ratio was lower in RRMS than HC and subjects that dysequilibrium of the PTH-FG23-vitamin D axis with RRMS did not demonstrate the expected summer could have a pathogenic role. In addition, because PTH rise in serum 1,25(OH) D observed in HC. Unlike the and FGF23 also regulate extra-renal 1-alpha hydroxylase Finnish study of Soilu-Hänninen et al. (Soilu-Hänninen in innate and adaptive immune cells, (for example see et al. 2008), we did not find a lower winter serum cal- Ref Bacchetta et al. 2013) this dysequilibrium has the cium concentration in RRMS, which might be explained potential to modify immune-inflammatory processes in by the fact that the winter serum 25OHD was higher in MS via autocrine as well as endocrine vitamin D metab- the Australian subjects. Irish researchers examined PTH olism, in keeping with evidence that impaired vitamin D at low levels of 25OHD (Lonergan et al. 2011; McKenna nutrition is associated with MS (Pierrot-Deseilligny and et al. 2018). In one study (Lonergan et al. 2011) a signifi- Souberbielle 2017; Munger et al. 2006; van der Mei et al. cant inverse correlation of PTH with 25OHD was 2007; Simpson Jr et al. 2010). present in controls but not in people with MS. Those It will be important to determine whether plasma studies, however, did not report plasma calcium and PTH and plasma iFGF23 could be used as biomarkers to Stein et al. Molecular Medicine (2018) 24:27 Page 8 of 9 identify individuals at risk for RRMS or predict disease basis of this dysequilibrium may provide insight into the course and response to therapy. It will also be important pathogenesis of RRMS and requires further investigation. to determine whether there is dysequilibrium in the Abbreviations PTH-FGF23-vitamin D axis in other autoimmune dis- 1,25(OH) D: 1,25-dihydroxyvitamin D; 25OHD: 25-hydroxyvitamin D; eases such as type 1 diabetes, systemic lupus erythema- CI: Confidence interval; FGF23: Fibroblast growth factor 23; FGFc: c-terminal fibroblast growth factor 23; HC: Healthy control(s); iFGF23: Intact fibroblast tosus and rheumatoid arthritis that exhibit an incidence growth factor 23; IQR: Interquartile range; Klotho: Soluble alpha klotho; or disease activity which correlates with vitamin D nutri- PTH: Parathyroid hormone; RRMS: Relapsing remitting multiple sclerosis tion (Munger et al. 2013; Watad et al. 2017). This study has several limitations. Not all of the differ- Acknowledgements Professor TJ Martin (St Vincent’s Institute of Medical Research, Melbourne) ences between RRMS and HC demonstrated by and Professor Seiji Fukumoto (University of Tokyo) gave helpful advice on within-pair analysis were seen in comparison of RRMS the iFGF23 assay. Professor Terry Speed (Walter and Eliza Hall Institute of and HC cohort medians. Furthermore, not all subjects Medical Research) and Professor Ian Gordon (Statistical Consulting Centre, University of Melbourne) provided statistical advice and review. Mary Tanner with RRMS recruited a HC friend to the study. While (Royal Melbourne Hospital), Kelly-Jane Lazarus (Box Hill Hospital) and Elizabeth cohort analyses have the advantage of larger sample size, McDonald (Medical Director, MS Australia Victoria) helped with recruitment. analysis of RRMS-HC pairs may be more sensitive as it Bob Hutton (Royal Melbourne Hospital Information Technology Department) assisted with the study website. Dr. Ken Sikaris (Pathologist, Melbourne controls for pre-analytical specimen handling measure- Pathology and Chair, International Federation Clinical Chemistry Committee on ment error (each RRMS-HC friend pair attended the Analytical Quality) advised and supervised general biochemistry. Hayley Tacon, same specimen collection centre at the same time), Eliza Ticknell and Marzi DeGaris (Melbourne Pathology) advised and assisted with specimen collection. Melissa Gresle (University of Melbourne) helped with which may contribute up to 50% of total analyte meas- sample storage. Karen Young, Michael Freemantle, Melissa Nelson, Eric Simons urement error (Plebani 2006). To reduce analytical la- (Sullivan Nicolaides Pathology) performed the iFGF, FGFc, klotho, ferritin and boratory error, winter and summer specimens from each 1,25(OH)2D assays. Advanced Professional Systems Pty Ltd. assisted with Medilink software to remind subjects of venepuncture arrangements. RRMS-HC pair were analysed within the same ELISA plate. In addition, we deliberately recruited HC from Funding friends of the people with RRMS to minimise bias from This study was funded by charitable research grants from The Myer Foundation unmeasured lifestyle, social and demographic variables Australia (Martyn Myer [President], Christine Edwards [then CEO]) and Multiple Sclerosis Research Australia (Professor Bill Carroll [then Director], Heather Cato that could potentially mask disease-specific differences. [then MS Grants Research Co-ordinator]). These funding bodies gave generous A further potential limitation is that we did not measure support and encouragement but had no role in the design of the study, in the markers of inflammation, which could confound the in- collection, analysis, and interpretation of data or in writing the manuscript. terpretation of serum ferritin concentrations, if people Availability of data and materials with RRMS had more inflammation than HC. Lower Clinical data set not available. As was standard at the year of study inception serum ferritin concentrations have been associated with and institutional human research ethics committees review, human research ethics committee permission was not sought or granted and informed higher serum concentrations of FGF23c and iFGF23 consent was not obtained for public repository deposition of patient data or (Braithwaite et al. 2012; Durham et al. 2007), but this re- sharing the data set with researchers outside our institutions’ research team. lationship appears to be assay specific (Durham et al. 2007). In particular, the Kainos iFGF23 assay used in this Authors’ contributions Study concept and design: All authors. Acquisition, analysis, or interpretation study was not affected by low concentrations of serum of data: All authors. Drafting of the manuscript: MS, LCH. Critical revision of ferritin (Durham et al. 2007). Hence, potential confound- the manuscript for important intellectual content: All authors. Statistical ing of serum ferritin by inflammation in the RRMS co- analysis: MS. Obtained funding: All authors. Administrative, technical, or material support: All authors. Study supervision: All authors. All authors read hort would not explain higher RRMS plasma and approved the final manuscript. concentrations of iFGF23 in this study. Finally, the fact that some subjects chose not to return for summer Ethics approval and consent to participate The study was approved by the Human Research Ethics Committees of venepuncture may have introduced bias into the study. Melbourne Health and Eastern Health, Victoria. All subjects provided written However, subjects were recruited both from hospital MS informed consent. clinics and from the community, supporting the generalizability of the results. Competing interests The authors declare that they have no competing interests. Conclusions This study revealed a dysequilibrium of the Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in PTH-FGF23-vitamin D axis in RRMS, with lower plasma published maps and institutional affiliations. PTH, higher plasma iFGF23 and a lower serum 1,25(OH) D to 25OHD ratio in RRMS compared with HC subjects. This Author details 1 2 The Royal Melbourne Hospital, Parkville, Australia. Department of Diabetes dysequilibrium is consistent with the study hypothesis that and Endocrinology, The Royal Melbourne Hospital, Parkville, VIC 3050, in RRMS there is suppression of the parathyroid glands by 3 Australia. Walter and Eliza Hall Institute of Medical Research, Parkville, 4 5 inappropriately high plasma concentrations of iFGF23. The Australia. Sullivan Nicolaides Pathology, Brisbane, Australia. Florey Stein et al. Molecular Medicine (2018) 24:27 Page 9 of 9 Neuroscience Institutes, Parkville, Australia. 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Molecular MedicineSpringer Journals

Published: May 30, 2018

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