Melatonin, calcium and vitamin D levels in adolescent idiopathic scoliosis

Introduction. Several causes have been proposed to underlie the onset and development of adolescent idiopathic scoliosis, but limited data have been published to date on the relationship between 25-OH-vitamin D, calcium, melatonin and adolescent idiopathic scoliosis. Objective. The present study evaluates the serum level of 25-OH-vitamin D, calcium and melatonin in relation to the age and sex of the patient, the Cobb angle, the seasonal variation of vitamin D. Materials and methods. A retrospective study was conducted during 2017-2019 in which patients suffering from adolescent idiopathic scoliosis, with a Cobb angle above 10 degrees, were analyzed. Statistical significance was determined using a P value of less than 0.05 in a 95% confidence interval. Outcomes. A total of 101 patients were included in the study, with a mean age of 11.61 +/2.33, 76 females and 25 males. The Cobb angle was on average 26.21o +/12.37o. Serum levels of vitamin D, calcium and melatonin were 24 ng/ml +/9.64, 9.82 mg/dl +/0.42, 8.82 pg/ml +/6.75. 55.44% had a sub-normal level of melatonin, 40.59% had insufficient levels of vitamin D. Calcium levels were within normal parameters. Melatonin level was positively correlated with vitamin D level (p = .017654, r = 0.2357). Vitamin D level correlated positively with calcium level (p = 0.01, r = 0.973), but also with the patient's age (p < 0.001, r = 0.158). Cobb angle was negatively correlated with serum vitamin D level (p < 0.01, r = −0.472). Using a 30 degree Cobb angle as a cut off point, it was observed that vitamin D levels did not differ significantly (p = 0.06), but a positive correlation was observed between melatonin level and the two groups (p < 0.00001). Conclusions. In the studied population, the calcium level remained constant within normal limits, but vitamin D and melatonin had a low level. Males had lower levels of vitamin D. As the Cobb angle increased, the serum levels of vitamin D and calcium decreased. Patients with idiopathic scoliosis should be followed from the beginning for these tests and their changes. Vitamin D and melatonin may be more important factors than have been considered so far in the onset and aggravation of idiopathic scoliosis. Larger studies, in larger populations, should be done to see if the administration of melatonin, vitamin D and calcium can have an effect on the evolution of idiopathic scoliosis of the adolescent.


INTRODUCTION
Adolescent idiopathic scoliosis represents the three-dimensional structural deformity of the spine, which usually develops after the age of 9 years [1,2]. Scoliosis affects between 2 and 4% of adolescents and about 10% of them will need a treatment method [2].
Although it is titled as idiopathic in nature, two factors remain unanimously accepted: the genetic factor and the hormonal factor [3,4].
In patients with idiopathic scoliosis, osteopenia and low bone density are often encountered, which can influence the evolution and prognosis of the disease. [12][13][14]. For a good bone density an optimal level of vitamin D and calcium is needed, elements that contribute to lower the incidence of fractures but also to improving the neuromuscular function [15,16].
Since the last century, scoliosis has been studied in laboratory animals to try to establish the etiology of this disease [17,18]. Studies conducted by Machida M et al. on pinealectomized chicken have long been a subject of controversy [19]. Following the ablation of the pineal gland, the melatonin level decreases, leading to the appearance of scoliosis to the chicken [20,21].

OBJECTIVES
Given the important role that vitamin D, calcium and melatonin can play in the etiology and development of idiopathic scoliosis of the adolescent, the present study proposes to verify the association between 25-OH-vitamin D, calcium, melatonin and the following variables: gender, age, Cobb angle. The seasonal variation for vitamin D will also be taken into account.

Study design and population
The study was done in an urban area children's emergency hospital in the Pediatric Orthopedic outpatient clinic between June 2017 and October 2019.
We chose to conduct a prospective study, unblinded, non-interventional for patients diagnosed with adolescent idiopathic scoliosis, with a Cobb angle greater than 10 o . The following were exclusion criterias: scoliosis other than idiopathic in nature (lower limb ine-quality, congenital malformations, hemivertebras, muscle distrophy), postural scoliosis, known metabolic or endocrine diseases, patients wich previously had a fracture history.
A local ethical committee approved this study. A written informed consent was obtained from the parents of all the participants.
All procedures were in accordance with the ethical standards of the institutional research committee (Centre of Postgraduate Medical Education) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethical Committee of "Grigore Alexandrescu" Children's Emergency Hospital. Informed consent, as stated above, was obtained from all individual participants included in the study.

Study protocol
Patients who met the above criterias were invited to do the following blood analysis: 25-OH-vitamin D, Total calcium level and melatonin. 25-OH-vitamin D was considered normal with values between 30 ng/ml and 100 ng/ml, deficient between 20 ng/ml and 29ng/ ml, insuficient with values below 20 ng/ml. Total calcium was considered normal with values between 8.80 mg/dl and 10.6 mg/dl. Melatonin level was considered normal between 8 and 16 pg/ml if it was assessed at 8:00 a.m. and 3-8 pg/ml if it was assessed at 9:00 a.m. In order to have the smallest variations in the study group, all participants were invited to do blood tests in the morning at 8 o'clock, in the same laboratory, under the same conditions. Patients were excluded if they had positive history for recent intake of vitamin D, calcium or melatonin supplements.

Collection of data and statistical analysis
Demographic data, vitamin D, calcium, melatonin, Cobb angle were analyzed statistically. Sex was analyzed using the χ2 test. Seasonal effects of vitamin D serum level were calculated using one-way ANOVA. Pearson's product-moment correlation was used to see the correlation between vitamin D level, melatonin and age, Cobb angle. Statistical significance was determined using a P value of less than 0.05 in a 95% confidence interval. Analyses were performed with the Statistical Package for the Social Sciences (SPSS) 18.0 software.

OUTCOMES
In our study, 101 patients met the selection criteria, aged between 8 and 16 years, 76 females and 25 males, evaluated and followed within our institution, between June 2017 and October 2019, diagnosed with idiopathic scoliosis. The average age of the patients was 11.61 +/-2.33, with 95% CI between 11.15 and 12.06, as shown in table 1.
Melatonin level was on average 8.82 pg/ml +/-6.75, with a 95% CI between [7.5, 10.1]. The level of vitamin D in the group of patients was on average 24 ng/ml +/-9.64, with a 95% CI between [22.12, 25.88]. Calcium values were within the normal range, all patients were within a normal range, with an average of 9.82 mg/dl +/-0.42. The average Cobb angle in the patient group was 26.21 o +/-12.37 o , with a 95% CI between [23.79, 28.62], the maximum being 60 degrees and the minimum 11 degrees.  Analysis of the vitamin D level intable 4a in relation to gender showed a predominantly lower level for males (on average 19.6 ng/ml) compared to females (on average 25.45 ng/ml) being statistically significant (p = 0.02). Melatonin levels were slightly lower for females, but overall it was not very different compared to males, the average falling within the normal range. However, the values of the female population were more homogeneous with a standard deviation lower than that of the male. The differences were not statistically significant between the genders for melatonin values.  Comparison of the vitamin D level with the season in which the analysis was performed is shown in table 5, where significant differences are noticed (p = 0.0001). In our study, spring recorded the lowest average level of vitamin D (16.6 ng/ml on average). Statistical correlations between 25-OH-vitamin D level and melatonin, calcium, patient age, Cobb angle were made in table 6. Melatonin level was positively correlated with vitamin D level (p = .017654, r = 0.2357). Vitamin D level correlated positively with calcium level (p = 0.01, r = 0.973), but also with the patient's age (p < 0.001, r = 0.158). Cobb angle was negatively correlated with serum vitamin D level (p < 0.01, r = -0.472). Following the results shown in table 7, calculations were made regarding the statistical association between melatonin level, age and Cobb angle. Although age is positively correlated with melatonin levels, it is not statistically significant (r = 0.0715, p = 0.477). Cobb angle correlated positively with melatonin serum level (r = 0.2607, p = 0.008461). It can be seen in table 8 the division of patients taking the Cobb angle limit of 30 o . No significant statistical correlation could be observed between vitamin D level and the two groups below 30 o and over 30 0 Cobb angle (p = 0.06). The results shown in table 9 show a positive correlation between melatonin level and the two groups below 30 o and over 30 0 Cobb angle (p < 0.00001).

DISCUSSIONS
Discussions on the link between melatonin and the etiology of scoliosis have aroused interest among researchers in recent decades [22], starting with the studies of Machida et al., who concluded that following removal of the pineal gland chickens will develop a scoliosis similar to idiopathic scoliosis of the adolescent [17,18,21]. The same group studied melatonin levels in a group of 30 patients and found that 35% of them had lower levels at night compared to the control group. Bagnall KM et al. restored the study, but on a much smaller group of patients, 7 in the study group and 7 in the control group, regarding the level of melatonin during the day and at night, without finding different results. Fagan AB et al. measured the level of urinary excreted melatonin for 24 hours, following a batch of 19 patients with scoliosis and 23 patients in the control group without proving a statistically significant difference [24]. Brodner et al. followed the circadian secretion of melatonin and the urinary excretion of 6-hydroxy-melatonin-sulfate in a batch of 9 patients with scoliosis who were to be operated on a batch of age-matched and gender-matched controls, but without finding statistically significant differences [25].
In our study group, 101 patients were monitored for melatonin level, and the results showed that more than half of the patients (56 -55.44%) had a low melatonin level. Melatonin level was positively correlated with 25-OH-vitamin D level, but had a weak positive correlation with Cobb angle when patients were divided into two groups, with Cobb angle below 30 and above 30 degrees, but statistically significant. One of the limitations of this study is the lack of comparison with a lot of healthy patients, of the same age and sex, regarding the level of melatonin.
Children may suffer from insufficient levels of vitamin D for various reasons. In the cold season (winter) there is less sun exposure, but also insufficient dietary intake of vitamin D [25,26]. Dietary intake of vitamin D, sun exposure and exercise level were not quantified in the present study. All patients were asked if they were using vitamin D, calcium or other supplements, and the answer was negative for all.
The optimal vitamin D level, measured by 25-OH-vitamin D, was proposed for a value of over 30 ng/ml, with an ideal value between 40 and 60 ng/ml. Insufficiency is considered when the level is below 20 ng/ml, and between 20 and 29 ng/ml the patient is considered to have an deficiency level [28,29].
A number of authors [30,31] have shown that patients with idiopathic scoliosis of the adolescent have a lower level of vitamin D compared to a control group. One of the limitations of this study is the lack of comparison with an age-mached control group. However, a low level of vitamin D will have an impact on bone mineral density and the development of deformities, such as idiopathic scoliosis of the adolescent [32,33].
Calcium level was within the limits of normal for all patients. Goździalska A et al. followed in his study calcium level in a group of 200 patients, both in the control group and in the study group, where although he found significant differences between groups, the values were within the normal limits [34].

CONCLUSIONS
Patients included in this study had low levels of vitamin D, significant differences being observed between boys and girls, boys being more affected. Calcium level was within the limits of normal, with small variations. Melatonin level was below the normal limit, with more than half of the patients below the normal limit.
The positive correlation between vitamin D and calcium, together with the negative correlation with Cobb angle, is yet another proof that patients with idiopathic scoliosis should be investigated regularly for these analyzes. The level of melatonin remained constant, regardless of the value of the Cobb angle, being slightly higher in patients whose Cobb angle exceeds 30 degrees.
Vitamin D and melatonin may be more important factors than have been considered so far in the onset and aggravation of idiopathic scoliosis. Larger studies, in larger populations, should be done to see if the administration of melatonin, vitamin D and calcium can have an effect on the evolution of idiopathic scoliosis of the adolescent.