International Journal of Rheumatic Diseases 2014; 17: 321–326
ORIGINAL ARTICLE
Decreased vitamin D levels in children with familial Mediterranean fever 3 € Ahmet ANIK,1 G€ on€ ul C ß ATLI,1 Balahan MAKAY,2 Ayhan ABACI,1 Tuncay KUME, 2 1 € € Erbil UNSAL, and Ece BOBER 1
Department of Pediatric Endocrinology, Dokuz Eylul University Hospital, 2Department of Pediatric Rheumatology, Dokuz Eylul University Hospital, and 3Department of Medical Biochemistry, Dokuz Eylul University Hospital, Izmir, Turkey
Abstract Objectives: To determine the frequency of vitamin D deficiency in children with familial Mediterranean fever (FMF) and to investigate the factors associated with low vitamin D status. Design and methods: Forty-four patients with FMF and 39 age- and sex-matched healthy controls were enrolled in this study. Demographic data, FMF symptoms, disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, MEFV gene mutation, cumulative colchicine dose, compliance to treatment and serum C-reactive protein levels were recorded for each patient. Serum 25-hydroxyvitamin D levels were measured by an original commercial kit based on chemiluminescent microparticle immunoassay (CMIA). Results: The serum 25-hydroxyvitamin D levels were significantly lower in FMF patients than the healthy controls (12.9 3.6 and 16.3 5.5 ng/mL, respectively, P = 0.001). Vitamin D levels were similar in patients homozygous for M694V and other genotypes (11.8 3.7 and 13.2 3.6 ng/mL, respectively, P = 0.21). Stepwise multiple linear regression analysis confirmed that the cumulative colchicine dose was the strongest independent variable correlating with vitamin D levels (r2 = 0.194, P = 0.001). Conclusion: Our results suggest that serum 25-hydroxyvitamin D levels are decreased in children with FMF. Cumulative colchicine dose appears to negatively affect vitamin D levels. The role of colchicine on vitamin D metabolism needs to be elicited. Key words: 25-hydroxyvitamin D, children, colchicine, familial Mediterranean fever.
INTRODUCTION Familial Mediterranean fever (FMF) is a hereditary auto inflammatory disease characterized by episodic fever and combination of severe abdominal/chest pain, arthritis and erysipelas-like erythema.1 Autosomal recessively inherited mutations in the MEFV gene, which encodes pyrin or marenostrin, are the genetic causes of this disease. The mutations of the MEFV gene cause an increase in interleukin (IL)-1b production and
Correspondence: Balahan Makay, Associate Professor, Dokuz Eylul University Hospital, Department of Pediatrics, 35340 Balçova, İzmir, Turkey. Email: [email protected]
an enhanced innate immune response.2–4 In many patients, the levels of acute-phase reactants, cytokines and inflammation-induced proteins remain elevated between episodes, which suggest persistent inflammation. As a result of chronic subclinical inflammation, complications such as anemia, splenomegaly, decreased bone mineral density and amyloidosis may occur.5 Vitamin D deficiency is an important public health problem affecting children and adolescents with a reported worldwide prevalence of 30–80% in both developed and developing countries.6–8 The role of vitamin D in bone mineralization is well established. Furthermore, several recent studies have reported a link between vitamin D deficiency and certain chronic
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
A. Anık et al.
inflammatory disorders such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Behcßet’s disease.9–11 Further, an inverse correlation between vitamin D levels and disease activity in RA and SLE was also demonstrated.9,10 Only recently, two studies reported that vitamin D levels were lower than healthy controls in adult FMF patients as well.12,13 These recent findings have led to greater emphasis on treatment of vitamin D deficiency and vitamin D supplementation in rheumatological diseases. To our knowledge, vitamin D levels have not been previously investigated in children with FMF disease. The aim of this study was to determine the frequency of vitamin D deficiency in children with FMF and to investigate the factors associated with low vitamin D status.
PATIENTS AND METHODS Study population The patients who were diagnosed as FMF according to the Tel-Hashomer criteria,14 and being followed at Department of Pediatrics, Division of Rheumatology in Dokuz Eylul University Hospital, Turkey, were consecutively enrolled in the study. FMF patients who had concomitant chronic diseases such as juvenile idiopathic arthritis were not included. Further, none of the patients had chronic renal or hepatic failure, metabolic bone disease, malnutrition or drug use, which are known to interact with vitamin D levels. The patients were asked if they experienced an increase in stool frequency in the last 3 months not due to infection, in order to exclude colchicine-induced diarrhea. Healthy children who were matched for age and sex constituted the control group. This cross-sectional study was conducted between February 2012 and March 2012 in the winter season. Demographic data, FMF symptoms, MEFV mutation, disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, cumulative colchicine dose, compliance to treatment and serum C-reactive protein (CRP) levels were recorded for each patient. The disease severity score was calculated according to the scoring system suggested by Pras et al.15 All of the patients were on colchicine treatment. None of the patients in the study were in an acute attack period at the time of evaluation. The attack-free period was defined as ‘at least 2 weeks after an attack’. The starting dose of colchicine was 0.5 mg/day for children ≤ 5 years of age, and 1.0 mg/day for children > 6 years of age. Colchicine dose was increased in a stepwise fashion (0.25 or 0.5 mg/step) up to a maximum of 2.0 mg/day to control the disease activity
322
where necessary. Patients were asked if they regularly used colchicine as prescribed and defined as ‘compliant’ if they took the recommended doses. The patients who did not use colchicine regularly were defined as ‘noncompliant’. The response to colchicine was evaluated according to previously suggested principles by Ben€ Chetrit and Ozdo gan depending on the decreasing rate of annual FMF attacks.16 If colchicine reduced the attack rate by 50%, it was accepted as ‘FMF-50’ response.
Methods Subjects’ heights were measured using a Harpenden stadiometer (Holtain Lmt., Crymych, Dyfed, UK) with a sensitivity of 0.1 cm and weight was measured using a SECA scale (SECA GMBH & Co. KG, Hamburg, Germany) with a sensitivity of 0.1 kg. The weight was measured with all clothing removed except underwear. After an overnight fasting, morning venous blood samples from patients with FMF and healthy subjects were drawn and collected in plain tubes. The plane tubes were centrifuged at 1200 9 g for 10 min and serum samples were removed from clots into clean Eppendorf tubes using plastic Pasteur pipettes. They were stored at 80°C until analysis. Serum 25-hydroxyvitamin D levels were measured by an original commercial kit based on chemiluminescent microparticle immunoassay (CMIA) principles (Cat no: 3L52; Abbott Diagnostics, Santa Clara, CA, USA) on an Architect i2000 auto analyzer (Abbott Diagnostics). Samples and pre-treatment reagents were combined. An aliquot of the pre-treated samples was combined with assay diluent and paramagnetic anti-vitamin D-coated microparticles to create a reaction mixture. Vitamin D in the sample bound to anti-vitamin D-coated microparticles. After incubation, a biotinylated vitamin D anti-biotin acridinium-labeled conjugate complex was added to the reaction mixture and bound to unoccupied bindings and trigger solutions were added to the reaction mixture. The resulting chemiluminescent reaction was measured as relative light units (RLUs). An indirect relationship between the amount of vitamin D in the sample and the RLUs were detected by instrument optics. The intra-assay and inter-assay coefficients of variation for determining 25-hydroxyvitamin D at 19.5 and 20.8 ng/mL were 2.4%, 3.3% (n = 10) and 2.9%, 3.1% (n = 10), respectively. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D < 15 ng/mL and severe deficiency as serum 25-hydroxyvitamin D < 5 ng/mL.17 In subjects who had low vitamin D levels, serum calcium, phosphorus, alkaline phosphatase and
International Journal of Rheumatic Diseases 2014; 17: 321–326
Vitamin D in children with FMF
parathormone levels were further evaluated by routine laboratory methods. The study protocol was approved by the ethics committee of the Dokuz Eylul University, Faculty of Medicine. Parents and children were informed about the procedure and written informed consent was obtained from the participants prior to the study.
Statistical analysis Data was evaluated using the Statistical Package for Social Sciences 11.0 program for Windows (SPSS Inc., Chicago, IL, USA) and by analyzing descriptive statistics (means, standard deviation) and by comparing dual groups using Student’s t-test and Mann–Whitney U-test when appropriate. Chi-square test was used to evaluate the differences in proportions. P-values ≤ 0.05 were considered as significant. Intercorrelations between parameters were computed through Pearson’s correlation analysis. Correlation coefficients indicated low correlation at 0.10–0.29, medium correlation at 0.30–0.49, and high correlation at ≥ 0.50. Variables with a P-value of < 0.05 in univariate correlation analysis were included in a multivariate (stepwise) linear regression analysis model to assess the independent determinants of vitamin D levels.
RESULTS Forty-four patients (20 male) with FMF and 39 healthy controls (22 male) were enrolled in the study. Ten patients (23%) were homozygous for M694V mutation and other genotypes were present in the remaining 34 patients. The clinical and laboratory characteristics of the patients are given in Table 1. Forty-four percent of the patients had a family history for FMF. Twelve patients (28%) had mild, 26 (60%) had moderate, and five (12%) had severe disease. Six patients (14%) were non-compliant by their statements. All of the patients had a good response to colchicine according to FMF-50 response. None of the patients had amyloidosis. The mean serum vitamin D level was significantly lower in patients with FMF than healthy subjects (12.9 3.6 and 16.3 5.5, respectively, P = 0.001) (Table 2). The mean vitamin D level was similar in patients homozygous for M694V and other genotypes (11.8 3.7 and 13.2 3.6, respectively, P = 0.21). Vitamin D levels were inversely correlated with cumulative doses of colchicine (r = 443, P = 0.004). Vitamin D levels were not significantly correlated with disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, serum CRP or age of the
International Journal of Rheumatic Diseases 2014; 17: 321–326
Table 1 Demographic data of the patients with familial Mediterranean fever (FMF) Parameter
Value
Age (years)* Disease duration (months)* Delay for diagnosis (months)* Duration of follow-up (months)* Dose of colchicine (mg/day)* Cumulative colchicine dose (mg) CRP (mg/L) (normal 0.1–8.2) FMF symptoms (%) Fever Peritonitis Arthritis Pleuritis Rash Disease severity score (%) Mild Moderate Severe MEFV mutation (%) M694V/M694V Other mutations
10.7 4 54 39.3 26.3 24.7 23.5 19.8 0.94 0.32 700 683 4.7 8.6 80 80 35 9 6 28 60 12 23 77
*Mean standard deviation; CRP, C-reactive protein; WBC, white blood cell.
Table 2 Comparison of patients and healthy controls Parameter
FMF patients (n = 44)
Age (years) Gender (F/M) Vitamin D level (ng/mL)
10.7 4 24/20 12.9 3.6
Healthy controls (n = 39)
P-value
9.2 4.2 17/22 16.3 5.5
0.10* 0.32† 0.001*
Data are given as means standard deviation. *Student’s t-test. †Chisquare test.
patient. Stepwise multiple linear regression analysis confirmed that the cumulative colchicine dose was the only strongest independent variable correlated with vitamin D levels (r2 = 0.194, P = 0.001) (Table 3). Vitamin D levels were similar in females and males in the whole study population (14 5 and 15 4.8, respectively, P = 0.29). Thirty-two patients (72%) and 18 healthy controls (46%) had vitamin D deficiency (P = 0.014). Severe vitamin D deficiency was observed in only one FMF patient. Further evaluation showed normal serum calcium, phosphorus and alkaline phosphatase and parathormone levels in patients with low vitamin D (data not shown).
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Table 3 Multivariate stepwise linear regression analysis (dependent variable: vitamin D level) Variable Cumulative colchicine dose
B 0.002
SRC (ß) 0.440
t 3.024
P-value 0.001
r2
P-value
0.194
B, coefficient of regression; SRC, standardized regression coefficient (Durbin-Watson: 1.7).
None of the patients or controls had growth retardation or clinical signs of rickets. Further, none of the patients declared a history of diarrhea in the last 3 months.
DISCUSSION The results of this study demonstrated that serum 25-hydroxyvitamin D levels were significantly lower in children with FMF than their healthy peers. Additionally, vitamin D levels were inversely correlated with cumulative doses of colchicine; however, there was no significant correlation between vitamin D levels and acute-phase reactants, disease duration, disease severity score or patient age. To date, several studies have revealed a link between vitamin D deficiency and various chronic autoinflammatory diseases such as inflammatory bowel disease,18 systemic lupus erythematosus,10,19 rheumatoid arthtiris,9,20 multiple sclerosis21 and type 1 diabetes mellitus.22 Two recent studies also showed lower serum vitamin D levels in adult FMF patients than healthy controls.11,12 Similarly, we found lower serum vitamin D levels among children with FMF compared with healthy children. Erten et al.12 found a significant negative correlation between low serum vitamin D levels and acute-phase reactants in their study. They suggested that decreased vitamin D levels in FMF patients could be another indirect sign of ongoing inflammation. We could not show a significant correlation between vitamin D levels and acute-phase reactants in our study. In the previous study they enrolled 99 FMF patients; however, we had a smaller number of patients, which might contribute to the lack of correlation between vitamin D and acute-phase response. Further, we included attackfree patients for at least 2 weeks and the mean CRP level of our patients was remarkably lower than their patients (4.7 8.6 vs. 14.3 34.3). We found a negative correlation between cumulative colchicine doses and serum vitamin D levels. Similarly, Karatay et al.11 found low 25-hydroxyvitamin D levels
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in patients with Behcet’s disease and they also demonstrated a strong relation between colchicine use and low serum vitamin D levels. They mentioned that the largest impact was due to colchicine therapy, although decreased vitamin D levels were also associated with smoking and alcohol intake. They did not report the dose and duration of colchicine therapy. Therefore, our study raised the question whether cumulative colchicine dose had an impact on vitamin D metabolism. Although the exact mechanism of colchicine in FMF is unknown, colchicine inhibits the intracellular microtubuli, interfering with intracellular granular transport and secretion of mediators.23 In normal monocytes, microtubules mediate intracellular transport of 25 (OH)D3 to mitochondria and the translocation of the 1,25(OH)D3-receptor complex to the nucleus. Previously, Kamimura et al.24 demonstrated that integrity of the microtubule network was critical for a normal genomic response to 1,25(OH)2 vitamin D3. They also showed that colchicine caused a dose-dependent reduction of 1,25(OH)2 vitamin D3 production by monocytes in vitro. Additionally, they found that disruption of microtubule integrity totally blocked the ability of exogenous 1,25(OH)2 vitamin D3 to inhibit its own production and to induce 24-hydroxylase activity in human monocytes. Despite the fact that we could not measure the serum levels of 1,25(OH)2 vitamin D3 and 24,25(OH)2 vitamin D3 in this study, the presence of low serum 25(OH) vitamin D3 levels suggests that disrupted microtubule integrity due to colchicine use may result in increased production of 1,25 (OH)2 vitamin D3 and 24,25(OH)2 vitamin D3, which might have led to this apparently low vitamin D status. An alternative hypothesis about the negative correlation of vitamin D and cumulative colchicine dose might be gastrointestinal malabsorption and gastrointestinal adverse effects related to colchicine. Indeed, in some patients, colchicine may cause gastrointestinal side effects, such as diarrhea. Padeh et al.25 reported that 14% of their patients developed diarrhea while using colchicine. Our patients did not declare an increase in the stool frequency in this study. Previous studies showed that colchicine might induce reversible vitamin B12 malabsorption by altering the function of ileal mucosa, but not by intestinal hypermotility.26,27 However, to date, no study has investigated whether colchicine causes intestinal malabsorption of vitamin D. Another remarkable result of this study is the high rate of vitamin D deficiency in healthy controls (46%) as well as in FMF patients (72%). Erten et al.12 also
International Journal of Rheumatic Diseases 2014; 17: 321–326
Vitamin D in children with FMF
found similar results in their study. The main source of vitamin D is solar UV radiation falling on the skin. The reasons for decreased serum 25-hydroxyvitamin D levels in children with FMF may be secondary to venous sampling time which overlapped with the winter season. We did not evaluate the dietary vitamin D intake, physical activity status and sunlight exposure, which might contribute to lower vitamin D levels in this study population. Low socioeconomic status is another factor inversely effecting vitamin D levels.28 However, we did not consider the socioeconomic status of our study population by objective instruments. Furthermore, increased skin pigmentation reduces the capacity of skin to synthesise vitamin D.29 A genetic predisposition to vitamin D deficiency and altered vitamin D level has also been reported among Asian people.30 Genetic polymorphisms of vitamin D receptors in Turkish people require further investigation. An alternative explanation for the lower vitamin D levels in FMF patients may be the possibility that patients with FMF may be going outside less and getting less sun exposure than their healthy peers because of their sickness. Those on the highest dose of colchicine may be the sickest patients, going outside even less than those with milder FMF symptoms who also require less colchicine. However, to date, there is no data about the possible restriction of spending time outside regarding FMF patients in the literature. This study had some limitations. First, the number of patients and control groups were small. Second, it would be optimal to measure 25(OH)-D concentrations prior to colchicine use; however, in the present study all of the patients were already on colchicine therapy. The other limitations are the lack of evaluating dietary vitamin D intake, physical activity status and sunlight exposure of the subjects. In conclusion, serum 25-hydroxyvitamin D vitamin levels were lower in children with FMF than healthy controls. It is not clear whether vitamin D levels observed in FMF patients are a triggering factor or a consequence of the disease. Further, the role of colchicine on vitamin D metabolism needs to be elicited.
REFERENCES 1 Ben-Chetrit E, Levy M (1998) Familial Mediterranean fever. Lancet 351, 659–64. 2 Chae JJ, Aksentijevich I, Kastner DL (2009) Advances in the understanding of familial Mediterranean fever and possibilities for targeted therapy. Br J Haematol 146, 467–68.
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3 Guz G, Kanbay M, Ozturk MA (2009) Current perspectives on familial Mediterranean fever. Curr Opin Infect Dis 22, 309–15. 4 Ozen S (2009) Mutations/polymorphisms in a monogenetic autoinflammatory disease may be susceptibility markers for certain rheumatic diseases: lessons from the bedside for the benchside. Clin Exp Rheumatol 27 (2 Suppl. 53), 29–31. 5 Ben-Zvi I, Livneh A (2011) Chronic inflammation in FMF: markers, risk factors, outcomes and therapy. Nat Rev Rheumatol 7, 105–12. 6 Whiting SJ, Barton CN (2005) Vitamin D intake: a global perspective of current status. J Nutr 135, 310–16. 7 Holick MF (2007) Vitamin D deficiency. N Engl J Med 357, 266–81. 8 Andıran N, Celik N, Akca H, Dogan G (2012) Vitamin D deficiency in children and adolescents. J Clin Res Pediatr Endocrinol 4, 25–9. 9 Cutolo M, Otsa K, Uprus M, Paolino S, Seriolo B (2007) Vitamin D in rheumatoid arthritis. Autoimmun Rev 7, 59–64. 10 Bogaczewicz J, Sysa-Jedrzejowska A, Arkuszewska C et al. (2012) Vitamin D status in systemic lupus erythematosus patients and its association with selected clinical and laboratory parameters. Lupus 21, 477–84. 11 Karatay S, Yildirim K, Karakuzu A et al. (2011) Vitamin D status in patients with Behcet’s Disease. Clinics (Sao Paulo) 66, 721–23. 12 Erten S, Altunoglu A, Ceylan GG, Maras Y, Koca C, Yuksel A (2011) Low plasma vitamin D levels in patients with familial Mediterranean fever. Rheumatol Int 32, 3845–49. 13 Kisacik B, Kaya SU, Pehlivan Y, Tasliyurt T, Sayarlioglu M, Onat AM (2011) Decreased vitamin D levels in patients with familial Mediterranean fever. Rheumatol Int 33, 1355–57. 14 Livneh A, Langevitz P, Zemer D et al. (1997) Criteria for the diagnosis of familial Mediterranean fever. Arthritis Rheum 40, 1879–85. 15 Pras E, Livneh A, Balow JE et al. (1998) Clinical differences between North African and Iraqi Jews with familial Mediterranean fever. Am J Med Genet 75, 216–9. 16 Ben-Chetrit E, Ozdogan H (2008) Non-response to colchicine in FMF–definition, causes and suggested solutions. Clin Exp Rheumatol 26 (4 Suppl. 50), 49–51. 17 Misra M, Pacaud D, Petryk A, Collett-Solberg PF, Kappy M (2008) Drug and therapeutics committee of the Lawson Wilkins pediatric Endocrine society. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics 122, 398–417. 18 Jahnsen J, Falch JA, Mowinckel P, Aadland E (2002) Vitamin D status, parathyroid hormone and bone mineral density in patients with inflammatory bowel disease. Scand J Gastroenterol 37, 192–9.
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19 Kamen DL, Cooper GS, Bouali H, Shaftman SR, Hollis BW, Gilkeson GS (2006) Vitamin D deficiency in systemic lupus erythematosus. Autoimmun Rev 5, 114–7. 20 Cutolo M, Otsa K, Laas K et al. (2006) Circannual vitamin D serum levels and disease activity in rheumatoid arthritis: Northern versus Southern Europe. Clin Exp Rheumatol 24, 702–4. 21 Correale J, Ysrraelit MC, Gaitan MI (2011) Vitamin Dmediated immune regulation in multiple sclerosis. J Neurol Sci 311, 23–31. 22 Takiishi T, Gysemans C, Bouillon R, Mathieu C (2010) Vitamin D and diabetes. Endocrinol Metab Clin North Am 39, 419–46. 23 Ben Chetrit E, Levy M (1998) Colchicine: 1998 update. Semin Arthritis Rheum 28, 48–59. 24 Kamimura S, Gallieni M, Zhong M, Beron W, Slatopolsky E, Dusso A (1995) Microtubules mediate cellular 25-hydroxyvitamin D3 trafficking and the genomic response to 1,25-dihydroxyvitamin D3 in normal human monocytes. J Biol Chem 270, 22160–66.
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25 Padeh S, Gerstein M, Berkun Y (2012) Colchicine is a safe drug in children with familial Mediterranean fever. J Pediatr 161, 1142–46. 26 Race TF, Peas IC, Faloon WW (1970) Intestinal malabsorption induced by oral colchicine: comparison with neomycin and cathartic agents. Am J Med Sci 259, 32–41. (Abstract). 27 Webb DI, Chodos RB, Mahar CQ, Faloon WW (1968) Mechanism of vitamin B12 malabsorption in patient receiving colchicine. N Engl J Med 279, 845–50. 28 Ozkan B (2010) Nutritional rickets. J Clin Res Pediatr Endocrinol 2, 137–43. 29 Clemens TL, Adams JS, Henderson SL (1982) Increased skin pigmentation reduces the capacity of skin to synthesise vitamin D3. Lancet 1, 74–76. 30 Shaw NJ, Pal BR (2002) Vitamin D deficiency in UK Asian families: activating a new concern. Arch Dis Child 86, 147–49.
International Journal of Rheumatic Diseases 2014; 17: 321–326
ORIGINAL ARTICLE
Decreased vitamin D levels in children with familial Mediterranean fever 3 € Ahmet ANIK,1 G€ on€ ul C ß ATLI,1 Balahan MAKAY,2 Ayhan ABACI,1 Tuncay KUME, 2 1 € € Erbil UNSAL, and Ece BOBER 1
Department of Pediatric Endocrinology, Dokuz Eylul University Hospital, 2Department of Pediatric Rheumatology, Dokuz Eylul University Hospital, and 3Department of Medical Biochemistry, Dokuz Eylul University Hospital, Izmir, Turkey
Abstract Objectives: To determine the frequency of vitamin D deficiency in children with familial Mediterranean fever (FMF) and to investigate the factors associated with low vitamin D status. Design and methods: Forty-four patients with FMF and 39 age- and sex-matched healthy controls were enrolled in this study. Demographic data, FMF symptoms, disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, MEFV gene mutation, cumulative colchicine dose, compliance to treatment and serum C-reactive protein levels were recorded for each patient. Serum 25-hydroxyvitamin D levels were measured by an original commercial kit based on chemiluminescent microparticle immunoassay (CMIA). Results: The serum 25-hydroxyvitamin D levels were significantly lower in FMF patients than the healthy controls (12.9 3.6 and 16.3 5.5 ng/mL, respectively, P = 0.001). Vitamin D levels were similar in patients homozygous for M694V and other genotypes (11.8 3.7 and 13.2 3.6 ng/mL, respectively, P = 0.21). Stepwise multiple linear regression analysis confirmed that the cumulative colchicine dose was the strongest independent variable correlating with vitamin D levels (r2 = 0.194, P = 0.001). Conclusion: Our results suggest that serum 25-hydroxyvitamin D levels are decreased in children with FMF. Cumulative colchicine dose appears to negatively affect vitamin D levels. The role of colchicine on vitamin D metabolism needs to be elicited. Key words: 25-hydroxyvitamin D, children, colchicine, familial Mediterranean fever.
INTRODUCTION Familial Mediterranean fever (FMF) is a hereditary auto inflammatory disease characterized by episodic fever and combination of severe abdominal/chest pain, arthritis and erysipelas-like erythema.1 Autosomal recessively inherited mutations in the MEFV gene, which encodes pyrin or marenostrin, are the genetic causes of this disease. The mutations of the MEFV gene cause an increase in interleukin (IL)-1b production and
Correspondence: Balahan Makay, Associate Professor, Dokuz Eylul University Hospital, Department of Pediatrics, 35340 Balçova, İzmir, Turkey. Email: [email protected]
an enhanced innate immune response.2–4 In many patients, the levels of acute-phase reactants, cytokines and inflammation-induced proteins remain elevated between episodes, which suggest persistent inflammation. As a result of chronic subclinical inflammation, complications such as anemia, splenomegaly, decreased bone mineral density and amyloidosis may occur.5 Vitamin D deficiency is an important public health problem affecting children and adolescents with a reported worldwide prevalence of 30–80% in both developed and developing countries.6–8 The role of vitamin D in bone mineralization is well established. Furthermore, several recent studies have reported a link between vitamin D deficiency and certain chronic
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
A. Anık et al.
inflammatory disorders such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Behcßet’s disease.9–11 Further, an inverse correlation between vitamin D levels and disease activity in RA and SLE was also demonstrated.9,10 Only recently, two studies reported that vitamin D levels were lower than healthy controls in adult FMF patients as well.12,13 These recent findings have led to greater emphasis on treatment of vitamin D deficiency and vitamin D supplementation in rheumatological diseases. To our knowledge, vitamin D levels have not been previously investigated in children with FMF disease. The aim of this study was to determine the frequency of vitamin D deficiency in children with FMF and to investigate the factors associated with low vitamin D status.
PATIENTS AND METHODS Study population The patients who were diagnosed as FMF according to the Tel-Hashomer criteria,14 and being followed at Department of Pediatrics, Division of Rheumatology in Dokuz Eylul University Hospital, Turkey, were consecutively enrolled in the study. FMF patients who had concomitant chronic diseases such as juvenile idiopathic arthritis were not included. Further, none of the patients had chronic renal or hepatic failure, metabolic bone disease, malnutrition or drug use, which are known to interact with vitamin D levels. The patients were asked if they experienced an increase in stool frequency in the last 3 months not due to infection, in order to exclude colchicine-induced diarrhea. Healthy children who were matched for age and sex constituted the control group. This cross-sectional study was conducted between February 2012 and March 2012 in the winter season. Demographic data, FMF symptoms, MEFV mutation, disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, cumulative colchicine dose, compliance to treatment and serum C-reactive protein (CRP) levels were recorded for each patient. The disease severity score was calculated according to the scoring system suggested by Pras et al.15 All of the patients were on colchicine treatment. None of the patients in the study were in an acute attack period at the time of evaluation. The attack-free period was defined as ‘at least 2 weeks after an attack’. The starting dose of colchicine was 0.5 mg/day for children ≤ 5 years of age, and 1.0 mg/day for children > 6 years of age. Colchicine dose was increased in a stepwise fashion (0.25 or 0.5 mg/step) up to a maximum of 2.0 mg/day to control the disease activity
322
where necessary. Patients were asked if they regularly used colchicine as prescribed and defined as ‘compliant’ if they took the recommended doses. The patients who did not use colchicine regularly were defined as ‘noncompliant’. The response to colchicine was evaluated according to previously suggested principles by Ben€ Chetrit and Ozdo gan depending on the decreasing rate of annual FMF attacks.16 If colchicine reduced the attack rate by 50%, it was accepted as ‘FMF-50’ response.
Methods Subjects’ heights were measured using a Harpenden stadiometer (Holtain Lmt., Crymych, Dyfed, UK) with a sensitivity of 0.1 cm and weight was measured using a SECA scale (SECA GMBH & Co. KG, Hamburg, Germany) with a sensitivity of 0.1 kg. The weight was measured with all clothing removed except underwear. After an overnight fasting, morning venous blood samples from patients with FMF and healthy subjects were drawn and collected in plain tubes. The plane tubes were centrifuged at 1200 9 g for 10 min and serum samples were removed from clots into clean Eppendorf tubes using plastic Pasteur pipettes. They were stored at 80°C until analysis. Serum 25-hydroxyvitamin D levels were measured by an original commercial kit based on chemiluminescent microparticle immunoassay (CMIA) principles (Cat no: 3L52; Abbott Diagnostics, Santa Clara, CA, USA) on an Architect i2000 auto analyzer (Abbott Diagnostics). Samples and pre-treatment reagents were combined. An aliquot of the pre-treated samples was combined with assay diluent and paramagnetic anti-vitamin D-coated microparticles to create a reaction mixture. Vitamin D in the sample bound to anti-vitamin D-coated microparticles. After incubation, a biotinylated vitamin D anti-biotin acridinium-labeled conjugate complex was added to the reaction mixture and bound to unoccupied bindings and trigger solutions were added to the reaction mixture. The resulting chemiluminescent reaction was measured as relative light units (RLUs). An indirect relationship between the amount of vitamin D in the sample and the RLUs were detected by instrument optics. The intra-assay and inter-assay coefficients of variation for determining 25-hydroxyvitamin D at 19.5 and 20.8 ng/mL were 2.4%, 3.3% (n = 10) and 2.9%, 3.1% (n = 10), respectively. Vitamin D deficiency was defined as serum 25-hydroxyvitamin D < 15 ng/mL and severe deficiency as serum 25-hydroxyvitamin D < 5 ng/mL.17 In subjects who had low vitamin D levels, serum calcium, phosphorus, alkaline phosphatase and
International Journal of Rheumatic Diseases 2014; 17: 321–326
Vitamin D in children with FMF
parathormone levels were further evaluated by routine laboratory methods. The study protocol was approved by the ethics committee of the Dokuz Eylul University, Faculty of Medicine. Parents and children were informed about the procedure and written informed consent was obtained from the participants prior to the study.
Statistical analysis Data was evaluated using the Statistical Package for Social Sciences 11.0 program for Windows (SPSS Inc., Chicago, IL, USA) and by analyzing descriptive statistics (means, standard deviation) and by comparing dual groups using Student’s t-test and Mann–Whitney U-test when appropriate. Chi-square test was used to evaluate the differences in proportions. P-values ≤ 0.05 were considered as significant. Intercorrelations between parameters were computed through Pearson’s correlation analysis. Correlation coefficients indicated low correlation at 0.10–0.29, medium correlation at 0.30–0.49, and high correlation at ≥ 0.50. Variables with a P-value of < 0.05 in univariate correlation analysis were included in a multivariate (stepwise) linear regression analysis model to assess the independent determinants of vitamin D levels.
RESULTS Forty-four patients (20 male) with FMF and 39 healthy controls (22 male) were enrolled in the study. Ten patients (23%) were homozygous for M694V mutation and other genotypes were present in the remaining 34 patients. The clinical and laboratory characteristics of the patients are given in Table 1. Forty-four percent of the patients had a family history for FMF. Twelve patients (28%) had mild, 26 (60%) had moderate, and five (12%) had severe disease. Six patients (14%) were non-compliant by their statements. All of the patients had a good response to colchicine according to FMF-50 response. None of the patients had amyloidosis. The mean serum vitamin D level was significantly lower in patients with FMF than healthy subjects (12.9 3.6 and 16.3 5.5, respectively, P = 0.001) (Table 2). The mean vitamin D level was similar in patients homozygous for M694V and other genotypes (11.8 3.7 and 13.2 3.6, respectively, P = 0.21). Vitamin D levels were inversely correlated with cumulative doses of colchicine (r = 443, P = 0.004). Vitamin D levels were not significantly correlated with disease duration, time to delay for diagnosis, duration of follow-up, disease severity score, serum CRP or age of the
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Table 1 Demographic data of the patients with familial Mediterranean fever (FMF) Parameter
Value
Age (years)* Disease duration (months)* Delay for diagnosis (months)* Duration of follow-up (months)* Dose of colchicine (mg/day)* Cumulative colchicine dose (mg) CRP (mg/L) (normal 0.1–8.2) FMF symptoms (%) Fever Peritonitis Arthritis Pleuritis Rash Disease severity score (%) Mild Moderate Severe MEFV mutation (%) M694V/M694V Other mutations
10.7 4 54 39.3 26.3 24.7 23.5 19.8 0.94 0.32 700 683 4.7 8.6 80 80 35 9 6 28 60 12 23 77
*Mean standard deviation; CRP, C-reactive protein; WBC, white blood cell.
Table 2 Comparison of patients and healthy controls Parameter
FMF patients (n = 44)
Age (years) Gender (F/M) Vitamin D level (ng/mL)
10.7 4 24/20 12.9 3.6
Healthy controls (n = 39)
P-value
9.2 4.2 17/22 16.3 5.5
0.10* 0.32† 0.001*
Data are given as means standard deviation. *Student’s t-test. †Chisquare test.
patient. Stepwise multiple linear regression analysis confirmed that the cumulative colchicine dose was the only strongest independent variable correlated with vitamin D levels (r2 = 0.194, P = 0.001) (Table 3). Vitamin D levels were similar in females and males in the whole study population (14 5 and 15 4.8, respectively, P = 0.29). Thirty-two patients (72%) and 18 healthy controls (46%) had vitamin D deficiency (P = 0.014). Severe vitamin D deficiency was observed in only one FMF patient. Further evaluation showed normal serum calcium, phosphorus and alkaline phosphatase and parathormone levels in patients with low vitamin D (data not shown).
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Table 3 Multivariate stepwise linear regression analysis (dependent variable: vitamin D level) Variable Cumulative colchicine dose
B 0.002
SRC (ß) 0.440
t 3.024
P-value 0.001
r2
P-value
0.194
B, coefficient of regression; SRC, standardized regression coefficient (Durbin-Watson: 1.7).
None of the patients or controls had growth retardation or clinical signs of rickets. Further, none of the patients declared a history of diarrhea in the last 3 months.
DISCUSSION The results of this study demonstrated that serum 25-hydroxyvitamin D levels were significantly lower in children with FMF than their healthy peers. Additionally, vitamin D levels were inversely correlated with cumulative doses of colchicine; however, there was no significant correlation between vitamin D levels and acute-phase reactants, disease duration, disease severity score or patient age. To date, several studies have revealed a link between vitamin D deficiency and various chronic autoinflammatory diseases such as inflammatory bowel disease,18 systemic lupus erythematosus,10,19 rheumatoid arthtiris,9,20 multiple sclerosis21 and type 1 diabetes mellitus.22 Two recent studies also showed lower serum vitamin D levels in adult FMF patients than healthy controls.11,12 Similarly, we found lower serum vitamin D levels among children with FMF compared with healthy children. Erten et al.12 found a significant negative correlation between low serum vitamin D levels and acute-phase reactants in their study. They suggested that decreased vitamin D levels in FMF patients could be another indirect sign of ongoing inflammation. We could not show a significant correlation between vitamin D levels and acute-phase reactants in our study. In the previous study they enrolled 99 FMF patients; however, we had a smaller number of patients, which might contribute to the lack of correlation between vitamin D and acute-phase response. Further, we included attackfree patients for at least 2 weeks and the mean CRP level of our patients was remarkably lower than their patients (4.7 8.6 vs. 14.3 34.3). We found a negative correlation between cumulative colchicine doses and serum vitamin D levels. Similarly, Karatay et al.11 found low 25-hydroxyvitamin D levels
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in patients with Behcet’s disease and they also demonstrated a strong relation between colchicine use and low serum vitamin D levels. They mentioned that the largest impact was due to colchicine therapy, although decreased vitamin D levels were also associated with smoking and alcohol intake. They did not report the dose and duration of colchicine therapy. Therefore, our study raised the question whether cumulative colchicine dose had an impact on vitamin D metabolism. Although the exact mechanism of colchicine in FMF is unknown, colchicine inhibits the intracellular microtubuli, interfering with intracellular granular transport and secretion of mediators.23 In normal monocytes, microtubules mediate intracellular transport of 25 (OH)D3 to mitochondria and the translocation of the 1,25(OH)D3-receptor complex to the nucleus. Previously, Kamimura et al.24 demonstrated that integrity of the microtubule network was critical for a normal genomic response to 1,25(OH)2 vitamin D3. They also showed that colchicine caused a dose-dependent reduction of 1,25(OH)2 vitamin D3 production by monocytes in vitro. Additionally, they found that disruption of microtubule integrity totally blocked the ability of exogenous 1,25(OH)2 vitamin D3 to inhibit its own production and to induce 24-hydroxylase activity in human monocytes. Despite the fact that we could not measure the serum levels of 1,25(OH)2 vitamin D3 and 24,25(OH)2 vitamin D3 in this study, the presence of low serum 25(OH) vitamin D3 levels suggests that disrupted microtubule integrity due to colchicine use may result in increased production of 1,25 (OH)2 vitamin D3 and 24,25(OH)2 vitamin D3, which might have led to this apparently low vitamin D status. An alternative hypothesis about the negative correlation of vitamin D and cumulative colchicine dose might be gastrointestinal malabsorption and gastrointestinal adverse effects related to colchicine. Indeed, in some patients, colchicine may cause gastrointestinal side effects, such as diarrhea. Padeh et al.25 reported that 14% of their patients developed diarrhea while using colchicine. Our patients did not declare an increase in the stool frequency in this study. Previous studies showed that colchicine might induce reversible vitamin B12 malabsorption by altering the function of ileal mucosa, but not by intestinal hypermotility.26,27 However, to date, no study has investigated whether colchicine causes intestinal malabsorption of vitamin D. Another remarkable result of this study is the high rate of vitamin D deficiency in healthy controls (46%) as well as in FMF patients (72%). Erten et al.12 also
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Vitamin D in children with FMF
found similar results in their study. The main source of vitamin D is solar UV radiation falling on the skin. The reasons for decreased serum 25-hydroxyvitamin D levels in children with FMF may be secondary to venous sampling time which overlapped with the winter season. We did not evaluate the dietary vitamin D intake, physical activity status and sunlight exposure, which might contribute to lower vitamin D levels in this study population. Low socioeconomic status is another factor inversely effecting vitamin D levels.28 However, we did not consider the socioeconomic status of our study population by objective instruments. Furthermore, increased skin pigmentation reduces the capacity of skin to synthesise vitamin D.29 A genetic predisposition to vitamin D deficiency and altered vitamin D level has also been reported among Asian people.30 Genetic polymorphisms of vitamin D receptors in Turkish people require further investigation. An alternative explanation for the lower vitamin D levels in FMF patients may be the possibility that patients with FMF may be going outside less and getting less sun exposure than their healthy peers because of their sickness. Those on the highest dose of colchicine may be the sickest patients, going outside even less than those with milder FMF symptoms who also require less colchicine. However, to date, there is no data about the possible restriction of spending time outside regarding FMF patients in the literature. This study had some limitations. First, the number of patients and control groups were small. Second, it would be optimal to measure 25(OH)-D concentrations prior to colchicine use; however, in the present study all of the patients were already on colchicine therapy. The other limitations are the lack of evaluating dietary vitamin D intake, physical activity status and sunlight exposure of the subjects. In conclusion, serum 25-hydroxyvitamin D vitamin levels were lower in children with FMF than healthy controls. It is not clear whether vitamin D levels observed in FMF patients are a triggering factor or a consequence of the disease. Further, the role of colchicine on vitamin D metabolism needs to be elicited.
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