Fibroblast growth factor 23 contributes to diminished bone mineral density in childhood inflammatory bowel disease

We hypothesize that FGF23 is increased in patients with IBD and, being a phosphaturic hormone, may contribute to the diminished BMD noticed in this patient population. So the aim was to assess relation of serum FGF-23 to BMD among children with IBD.

1- Diagnosis of IBD based on the Porto criteria [19].

2- Disease flare that was assessed using Pediatric Ulcerative Colitis Activity Index (PUCAI) for UC [20] and modified Pediatric Crohn’s disease Activity Index (PCDAI) for CD [21].

3- No steroid therapy for at least three months prior to enrollment in this study.

1- Critically ill patients who cannot be transferred for DXA procedure.

2- Concomitant endocrinal, renal or genetic bone diseases.

After approval of the study by the local ethics committee, informed consents were taken from the guardians of all patients, as well as assents from older children.

A full medical history and general examination, including anthropometric assessment of height for age with its Z score as well as body mass index (BMI), were done [22].

Laboratory assessment included complete blood count [Coulter 1660], C-reactive protein [AVITEX- CRP latex test [23]], ESR [Westergren method [24]], serum calcium [colorimetric method [25]], serum alkaline phosphatase [autoanalyzer], serum phosphorus [direct UV method without reduction [26]] and urinary phosphorus [colorimetric methods using an autoanalyzer (Multistat III Plus, Instrumentation Laboratory, Inc, Lexington, MA)]. Colonoscopy and esophago-gastrodudenoscopy [Pentax videosopes, PENYAX GmbH CO. Juulius Vosseler Strasse 104, 22527 Hamburg, Germany] with histopathologic assessment of multiple biopsies were done for all patients. Serum creatinine was measured using Synchron cx7 (Beckman Inc.).

Measurement of 25 (OH) D₃ was done using radioiodine (¹²⁵I)-based RIA kits (DiaSorin, Stillwater, MN) [27] with unit of measurement being ng/ml. Values below 15 ng/ml were considered indicative of vitamin D deficiency [28] and values below 8 ng/ml were considered indicative of severe vitamin D deficiency [29]. Measurement of 1, 25 (OH)₂ D₃ was done using Human 1, 25-Dihydroxy-Vitamin D RIA Kit (IBL International GmbH) with unit of measurement being pg/ml [30]. PTH level was measured by the Immulite 2000 Intact PTH assay.

Serum FGF23 was measured using a C-terminal human FGF23 ELISA (Immunotopics, San Clemente, CA) [31]. The plasma was stored at –20 C till assessment at end of study. It was expressed as a concentration of pg/ml with a sensitivity of 4 pg/ml.

BMD was determined by DXA Lunar scan [DPX-MD 2001, GE medical systems, USA]. Device was calibrated daily, and the technical error calculated to be less than 1%. Z-scores were calculated from BMD values using the reference data for bone density for age and sex [32] and corrected to bone age which was assessed from X Rays of the left hand [33]. The values of total body BMD were used for analysis.Values between −1.0 and −2.5 denoted a mild decrease in BMD and < −2.5 were diagnostic of severe decrease [34].

The computer program SPSS for Windows, release 10.0 (SPSS, inc, Chicago, IL, USA) was used for data entry and analysis. All quantitative data were analyzed as mean and standard deviation (SD). Student’s t test was used to compare quantitative data between different groups. Chi square test was used to compare qualitative variables between different groups. Multiple regression analysis was performed.

The diminished BMD during flare with significant improvement in remission agrees with previous similar results in IBD patients using either total body or lumbar spine bone mineral density [38‐40]. Herzog et al. [41] and Ahmed et al. [42] reported significant values of 26% and 22% respectively. Schmidt et al. [40] showed no difference between UC and CD regarding the frequency of low BMD.

This degree of improvement was achieved in a period of 7.12 +/− 2.8 months between the flare and remission assessment. Improvement of BMD in such a relatively short period is consistent with Viapiana et al. [43] who found that a 3 months period of intensive nutritional therapy in anorexia nervosa was sufficient to show the significant improvement in BMD. Similarly, Bhambri et al. [44] found that BMD measured by DXA was significantly improved but not to base line after therapy for 2.8+/−1.4 months.

The residual diminished BMD in a significant proportion of IBD children, during remission, suggests that nutritional therapy, IBD specific therapy and/or their duration are not sufficient to restore BMD to complete normality.

The mean serum level of 25 (OH) vitamin D₃, as a measure of vitamin D status [45], showed significantly lower values during flare of UC and CD compared to controls. Deficiency (< 15 ng/ml) or severe deficiency (< 8 ng/ml) were uncommon among patients with IBD during flare and markedly improved after remission. The vitamin D nutritional state returned to normal after achieving disease remission, although the intake of vitamin D and calcium had not changed. This may indicate that the state of hypovitaminosis D is a reflection of disordered bioavailability with disease activity rather than isolated nutritional deficiency. The normalization of vitamin D nutritional status with residual low bone mineral density in IBD patients implies that VD is not the sole player in the development of IBD osteopathy. Sentongo et al. [28] found that there was no association between hypovitaminosis D and bone mineral density (P = 0.10) and Hessov et al. [6] found reduction in trabecular bone mass in the presence of normal mean serum levels of the three vitamin D metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and 1,25-dihydroxyvitamin D₃) after ileal resection in CD. Higher values of 1,25- dihydroxyvitamin D₃ during flare of UC and CD may be due to inflammation-induced intestinal over-expression of activating enzymes. Abreu et al. [8] also demonstrated high levels of 1,25- dihydroxyvitamin D₃ in IBD patients, more so in CD patients. They explained this by demonstrating an increased expression of intestinal 1 alpha hydroxylase in CD patients which would contribute to increased activation of vitamin D. This may be reinforced by our finding that levels of active form were dramatically reduced to control values or even less after achieving disease remission.

FGF23 was significantly higher during disease flare in both UC and CD groups, and while significantly decreasing during remission, did not return back to normal control values. Although phosphatonins including FGF23 inhibit 1 α hydroxylase enzyme in the kidney [10] yet, the high levels of active vitamin D in this study minimizes this role in our context

This finding was not reported in previous clinical studies on IBD patients. It would be related to the high values of TNF alpha reported in such patients [17]. Uno et al. [18] demonstrated that in experimental animals, TNF-alpha decreases Phex mRNA and protein expression via a transcriptional mechanism and this is at least in part responsible for inhibition of osteoblast mineralization. The Phex mRNA and protein is known to be involved in degradation of FGF23 [46]. So we can propose that higher values of FGF23 with its phosphaturic action [10] and the resultant hypophosphatemia may share, among other factors, in the process of diminished BMD in these particular patients. The high levels of FGF23 cannot be attributed to impaired renal functions as serum creatinine was within normal for all patients. 1,25-dihydroxyvitamin D₃ may exert its largest effect on FGF23 expression/production when exposed to high levels of extracellular Pi in osteoblasts/osteocytes [47].

Parathyroid hormone was found to be significantly higher during disease flare than in remission. Moreover, remission values are still significantly higher than in controls. The relation of PTH to bone metabolism is established. This establishes the role of altered PTH in the cascade of osteopathy seen in these cases. The FGF23 polypeptide also suppresses the expression of vitamin D 1-hydroxylase and PTH, resulting in a reduction in serum 1,25-dihydroxyvitamin D₃[48] and PTH [49] levels. So our results, showing high 1, 25(OH)₂ VD₃ and PTH, suggest that FGF23 elevation is a secondary event rather than a primary incident. The TNF alpha role may augment this elevation through decrease of FGF23 degradation.

The etiology of IBD related bone disease seems to be multi-factorial with inflammation itself being the most important factor [4]. In our present work, bone mineral density was low during disease flare despite absence of steroid therapy for at least 3 months prior to enrolment. It dramatically increased after remission even with the use of steroids in all patients. This may suggest a lesser role of steroids on the bone mineral density in IBD patients. Walther et al. [50] found a similar proportion of osteoporosis in steroid-naive (12%) and steroid-treated (11%) pediatric IBD patients. Paganelli et al. [4] reported that the correlation between the lifetime cumulative dose of steroids or duration of treatment and BMD was not statistically significant.

Regression analysis for different clinical and laboratory variables in the ulcerative colitis showed that the only significant determining factors were FGF23 followed by serum calcium. On the other hand in CD, it showed many significant determining factors affecting BMD. On top comes 1,25-dihydroxyvitamin D₃, followed by urinary phosphorus and FGF23. The significant role of vitamin D in CD rather than in UC group may reflect a problem related to inflammation or absorption in the former group.

Previous studies have found that BMI correlated with BMD in IBD both in children [39]. But our analysis did not show an impact of BMI on the BMD in view of other variables.

The marked improvement noted in our CD group might be related to use of infliximab, a TNF inhibitor, in some of them. Franchimont et al. [51] and Ryan et al. [52] found that Infliximab rapidly improves bone formation in adults with IBD.

Controversy exists about the relation between decreased BMD and fracture risk among children with IBD [53]. However, low BMD during childhood is associated with increased fracture risk later in life [54]. Bone metabolism in children is characterized by predominant bone modeling with simultaneous activity of both osteoblasts and osteoclasts on different parts of the bone. Therefore, the principles of bone loss observed in adult patients with chronic inflammation are not directly applicable to children [55]. Understanding the specific underlying mechanisms for decreased BMD among children with IBD may help to implement more effective therapies for this complication.