Bone is a dynamic organ that serves mechanical and homeostatic functions. It undergoes a continual self-regeneration process called remodeling. Remodeling removes old bone and replaces it with new bone. This regenerative process occurs in distinct areas of bone known as bone metabolic units (BMU). Within each BMU, bone formation by osteoblasts and bone resorption by osteoclasts are coupled tightly and in a delicate balance to maintain bone mass and strength. Adolescence is a critical period for peak bone mass accrual (1). It is well-documented that male adolescents were seen to have achieved a higher peak bone mass than females due to differences in body size, muscle mass, and puberty (2). Evidence has shown that any interference with bone mass metabolism (either bone formation or resorption) during adolescence has been linked to the risks of fracture and increases the risk of fracture and skeletal disease later in life (3–5).

Serum cross-linked C-telopeptide of type I collagen (CTX) is one of the bone biochemical markers of bone turnover, a biomarker of osteoclast activity whereby it is used to assess bone resorption (6). In regard to biochemical markers of bone turnover, it can be presented in two: (i) markers of bone formation, which reflect osteoblast activity and are byproducts of collagen synthesis, matrix proteins or osteoblastic enzymes, and (ii) markers of bone resorption, which reflect osteoclast activity and are for the most part degradation products of type I collagen (6, 7). Vitamin D has been demonstrated to play an essential role in bone health both indirectly and directly (8–10). Indirectly, the function of vitamin D in bone is to maintain calcium homeostasis by increasing intestinal absorption of calcium (9, 10) and directly via the vitamin D receptor system (8).

Vitamin D is essential for optimal bone health, as it plays a crucial role in regulating calcium and phosphorus metabolism, which are vital for bone formation and maintenance. Adequate levels of vitamin D facilitate the absorption of calcium from the intestine, ensuring that the body has sufficient calcium to support bone mineralization. Furthermore, vitamin D directly influences osteoblast function, promoting bone formation while also inhibiting excessive bone resorption by osteoclasts. This balance is critical, especially during adolescence, a key period for peak bone mass development. Insufficient vitamin D levels can lead to decreased bone density, increasing the risk of fractures and long-term skeletal disorders. Thus, maintaining adequate vitamin D status is fundamental for the development of strong, healthy bones during the crucial growth phases of adolescence.

Even though there is substantial data on biochemical markers of bone turnover, there is limited evidence of the C-terminal telopeptide (CTX) of bone turnover in adolescent populations with low calcium and vitamin D concentrations. The importance of vitamin D together with dietary calcium intake for bone health has been confirmed by several meta-analyses (9, 10). It is known that the primary role of calcium is to help build and maintain the bones, whereas vitamin D functions to regulate intestinal calcium absorption by stimulating bone resorption to maintain the serum calcium concentration (11).

Hence, a lack of vitamin D may affect bone growth and impair the acquisition of peak bone mass, particularly during adolescence. Furthermore, the concentration of circulating 25(OH)D required for optimal bone health in adolescents is uncertain. Several observational studies have reported a negative association between serum 25(OH)D and biomarkers of bone resorption among children and adolescents with vitamin D deficiency or insufficiency (12, 13), though some studies revealed no significant association between serum 25(OH)D and bone biomarkers (14, 15). Pludowski et al. (5) have described in the vitamin D supplement guidelines that the vitamin D concentration (25(OH)D < 50 nmol/L) has clinical implications for bone health disease. Vitamin D (25(OH)D < 50 nmol/L) influences 20 to 40% of peak bone mass, which leads to high bone resorption and defects in bone formation (4, 5, 16, 17).

Evidence from Malaysia and Southeast Asia region remains limited, despite the high prevalence of both low calcium intake and vitamin D deficiency among adolescents. Presently, most Malaysian adolescents (91.7%) fail to meet the Recommended Nutrient intake for dietary calcium, which is set at 1000 mg/day, and have insufficient levels of vitamin D (25(OH)D < 50 nmol/L) (18). Suriawati et al. (18) reported that the average calcium intake among 13-year-old Malaysians is only 370 ± 12 mg/day. Additionally, Quah et al. (20) found that 15-year-old Malaysians have serum 25(OH)D below 50 nmol/L (92.0%). Various factors, including sex, ethnicity, living area, obesity, skin type, clothes style, and vitamin D dietary intake, have been identified as influences on vitamin D concentrations among Malaysian adolescents (19, 20). Based on these findings, we hypothesized that among 15-year-old Malaysian adolescents, lower dietary calcium intake and insufficient serum concentrations of 25-hydroxyvitamin D (25(OH)D) are associated with increased levels of C-terminal telopeptide (CTX), indicating higher bone resorption and impaired bone turnover, ultimately affecting overall bone health. Therefore, this study aimed to investigate the association of bone turnover (CTX) with low calcium intake and 25(OH)D concentrations among 15-year-old Malaysian adolescents. This study is important as it examined these associations within a large, multi-ethnic Malaysian adolescent cohort, providing region-specific insights including behavior, participants characteristics and clothing attire which may explore further serum vitamin D concentrations that may influence CTX level. This will complement data from European and other Asian studies.

This study examined the associations between dietary calcium intake, serum 25(OH)D concentrations, and bone turnover (CTX) among Malaysian adolescents. Current findings showed that low calcium intake was significantly associated with higher CTX levels, whereas vitamin D deficiency, although highly prevalent, was not associated with CTX. This result is consistent with the biological role of calcium as the main substrate for bone mineralization, where inadequate intake may directly increase bone resorption. In contrast, the lack of association between 25(OH)D and CTX may reflect that vitamin D concentrations were uniformly low in this population, possibly below the threshold at which variability in bone turnover could be detected. Previous studies in European and Asian cohorts (11, 15, 26) reported similar findings, emphasizing the dominant role of calcium intake in regulating bone turnover during adolescence. However, some studies acknowledged that lacking vitamin D leads to defective bone formation and mineralization, including increasing bone resorption and ultimately increasing the CTX concentrations (4, 27, 28). While, Suriawati et al. (18) conducted a study to investigate the relationship between the dietary intake, vitamin D and bone mineral content (BMC) in 13-year-old Malaysian adolescents and reported that those subjects with a higher intake of vitamin D and a higher combination of the intake of vitamin D and calcium resulted in significantly higher BMC quartiles. Current study, where the outcome was the CTX concentrations, found that higher bone turnover CTX concentrations were not significantly associated with low serum 25(OH)D among adolescents with low calcium intake. The potential explanation involves the optimal PTH concentration in serum that may be associated with vitamin D concentrations, whereby in this study, the average PTH concentration among adolescents was only 5.16 ± 3.35 pmol/L, which is borderline the normal range concentration.

Though the serum CTX concentration is a sensitive biomarker of bone resorption because it responds quickly to bone metabolism changes (26), and vitamin D is essential for bone formation and mineralization (4, 5), a study by Marwaha et al. (11) suggested that bone remodeling requires optimal concentrations of serum PTH. A low concentration of PTH may be associated with low bone formation and high concentrations of PTH with high bone turnover. Hence, suppression of PTH with vitamin D supplementation is associated with decreased bone resorption, as observed in their vitamin D supplementation study (11). On the other hand, a few studies suggested that the 25(OH)D is associated with bone turnover only when the calcium intake is above the median intake. In contrast, not when the calcium intake is lower than the median (29–31). The present study population’s calcium intake was also low, less than 50% of the recommended nutrient intake. Together with the prevalence of vitamin D deficiency (25(OH)D < 50 nmol/L) was high, 63.8% with females had a higher rate than males (91.9% vs. 45.4%), and the concentration of PTH of the study population also borderline average range concentration, thus, these conditions could be possible explanations for the absence of impact between 25(OH)D and CTX concentrations in the present study.

Nevertheless, apart from low calcium intake, the multivariate analysis revealed that the factor of urban residents having outdoor activities between 2 p.m. and 4 p.m., sunscreen avoidance, and always wearing a hijab/hat were significantly associated with a higher concentration of CTX among adolescents in this study. The current results showed that adolescents who live in urban areas had been found to have higher CTX concentrations than those living in rural areas. It is known that urbanization has changed dietary patterns and lifestyles, leading urban adolescents to a sedentary lifestyle and being less physically active, lacking exercise, improper nutrition, and tobacco and alcohol use (32). These health pattern changes have been linked to the risks of many diseases, including impacting bone turnover markers, indicating increased concentrations of bone resorption. Furthermore, low calcium intake and 25(OH)D concentrations among adolescents in this study may not suffice to provide additional calcium to control the homeostasis process (28).

Our results also found that those who had outdoor activities between 2 p.m. and 4 p.m. had higher CTX concentrations. This contrasted with previous studies (33, 34). Theoretically, exposure to sunlight between 10.00 a.m. and 4.00 p.m. would absorb ultraviolet light to ensure that vitamin D is sufficient to maintain bone health functions and calcium balance. It is essential to bone formation and mineralization (16, 17). It has been suggested that approximately 20–30 min of sun exposure between 10.00 a.m. and 4.00 p.m. at least twice a week to the face, arms, legs, or back without sunscreen will usually provide sufficient daily vitamin D requirements (33, 35). Thus, reducing bone resorption and lowering bone resorption markers (e.g., CTX).

Nevertheless, sun-protection behavior and clothing styles (such as long sleeves, long pants, and wearing a hijab/hat) could eliminate ultraviolet absorption and lower vitamin D concentrations. This was in line with the present findings. The present study found that those who had sunscreen avoidance behaviors and never wore hijab/hat had lower CTX concentrations than those who applied for sunscreen protection. These explanations could be why our study found that those who had outdoor activities between 2 p.m. and 4 p.m. had higher CTX concentrations. Additionally, as most of the participants in this study were females and Malays, these factors (sunscreen avoidance and clothing styles) were synonyms. Malaysian women, like some Asian populations, prefer to have fairer skin (34). Therefore, although they had outdoor activities between 10.00 a.m. and 4.00 p.m., they might apply sunscreen protective behavior, concealing their skin with long sleeves and long pants and wearing a hat.

The present study found that the calcium intake among adolescents was lower than the recommended Malaysian adolescent nutrient intake, which is in line with previous studies that reported low calcium intake among Malaysian adolescents due to a low intake of milk (18). A few studies have shown milk consumption was positively correlated with decreasing the serum CTX concentration in adolescents (36–39). This is comparable to the present results that a low calcium intake is associated with higher CTX concentrations. The relatively low calcium intake observed in this study may account for the lack of significant association between higher vitamin D levels and increased CTX concentration (p = 0.127). This could be attributed to the dual role of vitamin D. When there is an adequate supply of calcium, vitamin D and its metabolites can enhance the calcium balance and promote mineral deposition in the bone matrix. Conversely, in the presence of calcium deficiency, 1,25(OH)2D may stimulate bone resorption while concurrently inhibiting bone mineralization to maintain serum calcium homeostasis, potentially at the expense of bone mass (31, 40). This emphasizes the importance of a sufficient calcium intake. In addition, in the Malaysia National Health and Morbidity Survey 2022, only 23.2% of adolescents consumed milk / milk products at least two times daily (41).

This is the first study investigating the association of bone turnover (CTX) with low calcium intake and 25(OH)D concentrations among adolescents in Malaysia. In addition, to our knowledge, this study had a larger sample of Southeast Asia investigating the CTX bone turnover marker among adolescents. Current study supports inadequacy of both calcium and vitamin D insufficiency are highly prevalent. This large, multi-ethnic adolescent sample from middle income country will complement the existing literature from Western and Northern Asian populations. The inclusion of PTH as a covariate in our analysis further strengthens the robustness of our findings. PTH is a central regulator of calcium and vitamin D metabolism, and its inclusion confirmed that inadequate calcium intake remained significantly associated with higher CTX levels independent of PTH concentrations. These data are essential for future intervention studies which aim to decrease bone resorption. However, several limitations should be considered in the interpretation of the present study. First, the cross-sectional design precludes determinations of cause and effect. Second, dietary intake was based on self-reported 7-day diet history interviews, which may be subject to recall bias (20, 42). However, trained dietitians conducted interviews where audit checks were in place, and the method has been validated in Malaysian adolescents. However, vitamin D supplementation data were not collected, although this is unlikely to have significantly affected results for this age group as current Malaysia Ministry of Health policy is focusing on food first dietary-based approach. Despite these limitations, our study has important public health implications. The finding that calcium intake, rather than vitamin D deficiency, is associated with elevated bone turnover highlights the urgent need for interventions to increase calcium intake among Malaysian adolescents. Strategies such as school-based nutrition programs, expansion of food fortification program, and ensuring the affordability of calcium-rich foods may help to improve bone health outcomes in this vulnerable age group. Lastly, it should be noted that the recruitment of students involved only those attending national schools because more than 85% of Malaysian students go to national schools. Therefore, these findings cannot be generalized to all Malaysian adolescents as it does not include participants from east Malaysia.

In conclusion, this study found that low dietary calcium intake, rather than vitamin D deficiency, was significantly associated with elevated CTX levels, a marker of bone resorption, among Malaysian adolescents. This is one of the first large-scale studies in Southeast Asia to explore these associations, providing empirical evidence from a multi-ethnic Malaysian adolescent cohort. This study also highlights the urgent need for public health strategies that promote calcium intake through improved dietary habits, food fortification initiatives, and affordable access to calcium-rich foods. Longitudinal and interventional studies are warranted to confirm these associations and to explore the combined role of calcium and vitamin D in optimizing adolescent bone health. Further research is warranted to ascertain the determinants of CTX concentrations.