Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects

Abstract Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p <0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p < 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.


Introduction
Environmental pollution causes a significant global problem for human health. Over the years, our environment has been filled with pollutants. Consequently, people are frequently exposed to different kinds of environmental contaminants which are linked to deleterious effects on public health worldwide. Among toxic metals, cadmium (Cd) and lead (Pb) are considered as chemical pollutants that require close monitoring (Mol, 2011). They are widely distributed in the environment (Madeddu et al., 2011), and contaminating the air, water, food sources and soil. Furthermore, they are also present in cigarette smoke. In addition to their harmful effect on the central nervous system, these minerals are carcinogenic, mutagenic and embryotoxic (Arguelles-Velazquez et al., 2013). The sources of exposure to Pb include cosmetics, cookware (Ragab et al., 2014), printing presses, wrapping paper, ceramics, constructing materials, textiles and even toothpaste. In fact, the main sources of Pb are water, paint, Abstract Serum toxic metals have been implicated in development of many diseases. This study investigated the association between blood levels of lead and cadmium with abnormal bone mineral density (BMD) and incidence of osteoporosis. Sixty Saudi male adults age matching were assigned into two groups: A healthy control group (n = 30) and osteoporosis patients diagnosed according to T-score (n = 30). Serum calcium, vitamin D, osteocalcin, lead, cadmium were measured. Osteoporotic group showed a highly significant elevation of blood lead and cadmium levels compared to the control group (p <0.001). BMD was negatively correlated with serum osteocalcin level compared with control. There was a significant negative correlation between the cadmium and lead levels (r=-0.465 and p-value = 0.01) and calcium (p < 0.004). Our findings suggested that high cadmium and lead were negative correlated to BMD and increased the risk factor for osteoporosis.

Association of blood heavy metal levels with osteocalcin abnormality and incidence of osteoporosis in Saudi subjects
Associação de anormalidades de osteocalcina nos níveis de metais pesados no sangue e incidência de osteoporose em indivíduos sauditas A. A. Banjabi a , K. Kannan b , T. A. Kumosani a,c,d , J. M. Yousef e , K. O. Abulnaja a,c  and S. S. Moselhy f *  30 healthy participants from the general population with no reported symptoms of osteoporosis (control), following bone mineral density (BMD) measurements using dual energy X-ray absorptiometry (DEXA). Informed verbal consent was obtained from every subject after informing the purpose of the study. Body mass index was calculated as BMI (kg/m 2 ). All of the participants underwent physical examination before enrollment in the study. The exclusion criteria included pregnancy, lactating females, the presence of organ dysfunction and terminal illness. BMD of all individuals was measured by DEXA scan (Jeddah, Saudi Arabia). Subjects were assigned to one of the study groups according to their T-score: a normal group (T-score ≥ -1) and osteoporosis group (T-score ≤ -2.5).
From each subject, 5 mL of whole blood was drawn into heparin coated tubes. Then, blood samples were immediately refrigerated and transported in cold storage to the laboratory and kept at −40 °C until they were used.

Determination of serum osteocalcin
Serum osteocalcin was determined by ELISA kit purchased from BIOMEDICA, England with sensitivity of 0.35 ng/mL.

Determination of serum calcium and vitamin D
Serum calcium was determined by calorimetric kit while Vitamin D by ELISA technique using kits from Biomedica, England.

Microwave digestion of the samples
Each investigated blood sample (1 mL) was transferred into a Teflon container, and then 5 mL of HNO 3 was added and left overnight. Next day, 0.5 mL of H 2 O 2 were added. Then, they were placed in a microwave and heated to digest.

Heavy metals measurement using inductive coupled plasma-mass spectrometry (ICP-MS)
This method was described previously, but with some modifications.

Statistical analysis
Data analyses were performed using SPSS-version 20 software. Medians were used to describe the studied samples. Univariate and multivariate regression analyses were also applied. Finally, Pearson's correlation was applied to find correlation between heavy metals and osteoporosis. P-Values < 0.05 was considered as statistically significant. Table 1 showed that, the median age of the osteoporosis patients was 65 (55.75-76) years, which cross matched the median age of the control group 62.5 (41.75-60.5). Non significant changes in the level of calcium were noted between the two groups (p = 0.882). Contrarily, vitamin D showed higher significant levels in control than that in osteoproteic group, p = 0.009. The concentration level ranges and averages of Cd and gasoline, food, soil and dust (Bosch et al., 2016). Regarding Cd, it is found in high concentrations in industrial areas and used in electroplating and galvanizing, as a cathode material for nickel-cadmium batteries, plastics and paints (Bosch et al., 2016). In the general population, food is the major source of Cd. It is also found in tobacco smoke and water (Lavado-Garcia et al., 2017). Cd and Pb are easily transported across the cell membrane in the organism and accumulated in tissues as well (Macholz, 1978). The half-life of Cd in soft tissues is estimated to be between 5 and 30 years and about 30 days for Pb. Pb mainly accumulates in the bones which comprise approximately 90% of the total amount of body Papanikolaou et al. (2005). On the other hand, Cd accumulates in the kidneys; as a result kidneys have been considered to be a critical target of Cd toxicity (Klaassen, 2003).

Data in
High output from industrial sewage and organic compounds are mainly responsible for environmental pollution. There is an inverse relationship, and increased significantly in downriver polluted areas (Schulz and Martins-Junior, 2001). It was reported that, high dietary cadmium levels increased risk of osteopenia or osteoporosis. In addition, lead exposure may increased risk of osteopenia or osteoporosis (Jalili et al., 2020).
Osteocalcin is calcium binding protein synthesized by osteocyte and osteoblasts and a sensitive biomarker for bone formation due to its role in calcification. Blood levels of osteocalcin are generally related to the rate of bone formation (Ishak et al., 2015).
Osteoporosis, literally "porous bone", is a bone disorder that weakens bones, making them more likely to break. It affects millions of people worldwide, predominantly postmenopausal women while Saudi Arabia is among the countries with highest incidence of osteoporotic fractures. It is a multi-factorial bone disorder and involves the interaction between genes, endocrine function, nutritional and environmental factors (Gartell et al., 1986). Dermience et al. (2015) identified the toxic effects of Pb and Cd on bone metabolism (Castelli et al., 2005) and higher fracture risk has also been reported from toxic exposure to Cd (Higazy et al., 2010;Castelli et al., 2005). In spite of the fact that these metals are naturally-occurring in our environment and the exposure to them is unavoidable, their osteo-toxic effects have not been extensively investigated in Saudi Arabia. Therefore, the rational of current study is to investigate the association of toxic metals (Cd and Pb) with risk of osteoporotic bones in Saudi subjects.

Subjects
Samples of the present study were collected from King Fahd Hospital, Jeddah, located in the Western province of Saudi Arabia from March 2017 to January 2018. The research protocol was approved by the National Committee (approval no. A00406). Sixty Saudi subjects were included in this study, age and sex-matched. They were equally divided into two groups, 30 osteoproteic patients according to T-score, whereas the other group was comprised of Pb in the osteoporosis group were 0.02-0.09 (0.03) and 0.1-0.9 (0.41), respectively. Osteocalcin level was significantly increased in osteoporosis group compared with control group. Pearson correlation showed a negative correlation with BMD. On the other hand, the concentration level ranges and averages of Cd and Pb in the control group were 0.01-0.03 (0.02) and 0.1-0.4 (0.26), respectively. Table 2 demonstrated that the blood levels of both Cd and Pb in the osteoporosis group were higher than those in the control (p <0.001 and 0.001), respectively.
Uni-variant logistic regression test verified that age and vitamin D have significant effects on osteoporosis, p-value = 0.001 and 0.004 and ORs = 1.083 and 0.924, respectively. Conversely, BMI and calcium did not show a significant effect (Table 3). With regard to multivariant logistic regression, the model was significant (p-value<0.0005 and Nagelkerke R Square =0.504) and revealed that age and vitamin D have significant effects on the incidence of osteoporosis, p =0.002 and 0.005, while ORs = 1.11 and 0.914, respectively. In contrast, BMI and calcium failed to show any significant effect (Table 4). Pearson's correlation analysis was also performed and proved a positive correlation between Cd and Pb, r = 0.505 and p-value = 0.004, in the osteoporosis samples. On the other hand, the control samples recorded a negative correlation (r = -0.465 and p-value = 0.01) between Cd and Pb ( Table 5).
The Dual energy X-ray absorptiometry (DEXA) refrences range for BMD indicated by the value of T score, if T-score was -1.0 or more this is normal healthy bone density. While if T-score (-1.0 to -2.5), it indicated osteopenia and if T-score lower than -2.5 it indicated osteoporosis. Our results indicated that obese women showed T score = -2.31 while overweight women showed T score of -1.76 compared with control group T score =-1.4 with p<0.001 for both.

Discussion
Bone is a target organ for toxic metals as Cd and Pb with increased risk of fragility fractures Berglund et al. (2000). These metals were correlated with lower BMD and osteoporosis. Lead was found to be associated with reduced bone calcification (Jarup and Akesson, 2009). In the present study, osteoporotic patients, showed significantly increased blood levels of Cd and Pb as compared with control group (Delmas, 2008). The mechanism of association between toxic metals and fragility was attributed to renal tissue damage, which lead to decreased calcium reabsorption. Another explanation was also regarded for Cd due to it interfere with calcium reabsorption. In addition, vitamin D deficiency contributed in bone loss, decreased BMD and increased bone resorption to maintain blood calcium (Saltman and Strause, 1993). However, Pb, can replace the calcium content in hydroxyapatite and has a higher affinity for osteocalcin than calcium (Staessen et al., 1999;Akesson et al., 2006). The current study emphasized the association between Cd and Pb in the blood and the incidence of osteoporosis in Saudi population, which is consistent with previously reported studies of populations in Japan, Sweden and Belgium (Jin et al., 2004;Wang et al., 2003;Chen et al., 2011). In opposition with our findings, no association was reported between urinary cadmium and BMD in female Japanese farmers (Lv et al., 2017). This contradictory result could be explained by the valuable effects of exercise performed by the working women on bone, since physical exercise was consistently reported to enhance muscle strength and improve bone density (Schutte et al., 2008).
The increased level of osteocalcin in osteoprosis is attributed to decrease BMD and increased bone turnover. A high bone turnover can disrupt the trabecular architecture which reduces the bone strength in osteoporosis, ultimately resulting in decreased levels of bone mineral density.
Our data revealed that there is a link between blood lead and cadmium and the risk of osteoporosis. The mechanisms are complex. It is suggested that high cadmium levels decrease the formation of calcitriol (active Vitamin D) in blood, thereby decreasing calcium absorption; increasing  bone resorption and break down the collagen matrix, inhibiting osteoblasts; and altering the expression of genes involved in bone homeostasis (Hsu et al., 2009). Cadmium can also induce reactive oxygen species production and oxidative stress (Kawakami et al., 2013), both the mentioned reasons can induce osteoporosis (Hsu et al., 2014). Also, lead is known for suppression of key genes for leptin and adiponectin, which are required to maintain healthy BMD.

Conclusion
We observed that in comparison to healthy subjects, osteoporotic patients have higher levels of lead and cadmium which are known to reduce bone mineral density and hence lead to osteoporosis.