Abstract: Osteosarcoma is the most common primary tumor of bone, occur mainly during childhood and adolescence. No reports are available in literature where serum folate and vitamin B12 have been assessed in osteosarcoma patients. Hence, the present study was planned to analyze status of folate and vitamin B12 in thirty osteosarcoma patients and compare it with thirty controls (subjects with musculoskeletal pain). Serum calcium, alkaline phosphatase levels were higher and phosphorus levels were comparable in osteosarcoma patients as compared to controls. Serum folate levels were significantly raised in osteosarcoma patients as compared to controls (p<0.001). Serum vitamin B12 levels were significantly lowered in osteosarcoma patients as compared to controls (p<0.001). Folate-mediated one-carbon metabolism (FOCM) is unequivocally linked to multiple health outcomes, including birth defects, several types of cancer and possibly cardiovascular disease and cognitive function. Tumors are known to up regulate FRα modulating the folate uptake in serum. Vitamin B12 diminishes osteoblastic activity and lowered serum vitamin B12 levels are possibly due to increased metabolic demand of the tumor. The present study suggests that these parameters can serve as useful markers for diagnosis and follow up of disease.
INTRODUCTION
Osteosarcoma is the most common primary tumor of bone, occur mainly during childhood and adolescence. Osteosarcoma develops at the sites of greatest bone growth (Rosier, 1999). Several markers for diagnosis and prognosis have been proposed in osteosarcoma namely, VEGF, bone alkaline phosphatase, osteocalcin, survivin, Erb B2 (Palmieri et al., 2009).
Alterations in one carbon metabolism related nutrients (folate, vitamin B12) in body leads to insufficient methyl group for DNA synthesis, methylation and repair, thus promoting carcinogenesis (Stover, 2009; Bailey and Gregory, 1999; Eto and Krumdieck, 1986). Several epidemiological studies have reported protective role of folate in preventing development of colorectal, breast and lung cancers (Johansson et al., 2010; Zhang et al., 1999; Heimburger et al., 1988). Some studies have reported that diet deficient in vitamin B12 enhance tumor growth (Bailey and Gregory, 1999; Eto and Krumdieck, 1986; Zhang et al., 1999; Heimburger et al., 1988).
There are conflicting results in several studies in relation to folate or vitamin B12 status and neoplasia. Epidemiological studies have demonstrated association between folic acid supplementation and increased risk of colorectal cancer (Mason et al., 2007). Also, high vitamin B12 intake has been reported to increase risk of prostate, esophageal and gastric cancer (Hultdin et al., 2005; Mayne et al., 2001).
However, no reports are available in literature where serum folate and vitamin B12 have been assessed in osteosarcoma patients. Though, deficiencies in vitamin B, along with the consequent elevated homocysteine level have been reported to be associated with bone loss, decreased bone strength and increased risk of fracture (Ahmadieh and Arabi, 2011).
Hence, the present study was planned to analyze status of folate and vitamin B12 in osteosarcoma patients.
MATERIALS AND METHODS
Thirty histopathologically confirmed cases of osteosarcoma (localized, without metastasis) were selected for the study and 30 age-matched subjects with musculoskeletal pain served as control. The study was carried out between July 2010 to June 2011.
Five milliliter blood was collected aseptically and serum was separated by centrifugation. Serum folic acid and vitamin B12 were analyzed by competitive immunoassay using direct chemiluminescent assay (Brewster, 1989; Chen et al., 1987).
Folate in the patient sample competes with acridinium ester labelled folate in the Lite Reagent for a limited amount of biotin-labelled folate binding protein. Biotin-labelled folate binding protein binds to avidin that is covalently coupled to paramagnetic particles in the Solid Phase. The sample is pre-treated to release the folate from endogenous binding proteins in the sample. The system automatically performs these steps.
Vitamin B12 from the patient sample competes with vitamin B12 labeled with acridinium ester in the lite reagent, for a limited amount of purified intrinsic factor, which is covalently coupled to paramagnetic particles in the solid phase. The assay uses releasing agent (sodium hydroxide) and DTT to release the vitamin B12 from the endogenous binding proteins in the sample and cobinamide to prevent rebinding after the solid phase is added to the sample. The system automatically performs these steps.
Statistical analysis: SPSS ver.18 was applied for various statistical analysis and student’s t-test and regression analysis was carried out. Level of significance was 0.05.
RESULTS AND DISCUSSION
Table 1 analysed various parameters in both the groups. Serum calcium levels were higher in osteosarcoma patients as compared to controls (p<0.001). Serum phosphorus levels were comparable among both the groups. Serum alkaline phosphatase were raised in osteosarcoma as compared to controls (p<0.05).
In the present study, serum folate levels were significantly increased in osteosarcoma patients as compared to controls (p<0.001, Table 2). To the best of our knowledge, no study is available where folate levels have been reported in osteosarcoma. Though low levels of folate have been reported in Paget’s disease (Polyzos et al., 2010). Rapidly dividing cells have an increased requirement for folate to maintain DNA synthesis and findings of raised serum folate levels in the present study supports the wide-spread use of antifolate in chemotherapy.
There are conflicting reports in relation to folate and cancer development. Some studies support the concept that diminished folate status predisposes to development of several cancers such as colon, breast, lung, pancreas and cervix (Glynn et al., 1996; Almadori et al., 2002; Kato et al., 1999).
| Table 1: | Various parameters in both osteosarcoma and control groups
(Mean±SD) |
ns: Not significant, **p<0.01, ***p<0.01, ***p<0.01,
significant when compared to control |
|
| Table 2: | Serum folic acid and vitamin B12 levels in both osteosarcoma and control groups (Mean±SD) |
*p<0.001, significant when compared to control |
|
| Table 3: | Correlation between various parameters (r-values) |
ns: Not significant, **p<0.01, significant when compared
to control |
|
Increased risk of colorectal cancer has been reported with folate supplementation and high serum folate levels (Weinstein et al., 2008), Reduced Folate Carrier (RFC), a folate carrier protein is expressed in almost all tissues and its primary physiological function is tissue redistribution of folate (Whetstine et al., 2002). Decreased RFC expression and transport mediated defects of folate have been reported in osteosarcoma (Yang et al., 2008).
Tumors are known to up regulate folate receptors and folate receptor (FRα) has been reported to over expressed in osteosarcoma tissue samples (Yang et al., 2007). It has been reported that FRα is found in normal tissues and in malignant cells it loses its polarity and become contactable to blood stream, thus modulating cell folate uptake (Yang et al., 2007).
In the present study, serum vitamin B12 levels were significantly decreased in osteosarcoma patients as compared to controls (Table 2, p<0.001). Status of vitamin B12 in osteosarcoma is not known and no report of vitamin B12 status in osteosarcoma is available in literature. However, high serum cobalamin has been reported in chronic myeloid leukemia, multiple myeloma (Ermens et al., 1993). Several in vitro and clinical studies have indicated that cobalamin may have effect (direct or indirect) on osteoclastic activity (Whetstine et al., 2002; Yang et al., 2008; Yang et al., 2007; Ermens et al., 1993).
Serum alkaline phosphatase was inversely correlated with vitamin B12 in osteosarcoma and positively correlated with controls and was not statistically significant (Table 3). Effect of vitamin B12 on proliferation and cellular alkaline phosphatase activity in human bone marrow stromal osteoprogenitor cells (hbMSC) and osteoblastic cells (UMR106 cells) has been reported (Kim et al., 1996). They suggested that vitamin B12 deficiency suppresses osteoblastic activity that may contribute to osteoporosis and fractures.
A significant negative correlation was observed between serum calcium and vitamin B12 in both osteosarcoma and controls and values were higher in osteosarcoma (Table 3). Low vitamin B12 levels suppress osteoblastic activity and release calcium from bone (Kim et al., 1996). Finding of inverse correlation between vitamin B12 and serum calcium in the present study lend support to this statement.
In the present study a significant inverse correlation was observed between serum folate and vitamin B12 levels (Table 3). It is not clearly documented in literature whether imbalance between folate and vitamin B12 associated with any other adverse effects, low serum folate and vitamin B6 concentrations but not low serum vitamin B12 concentrations, have been reported to be associated with an altered morphology of human bone (Holstein et al., 2009).
There is compelling evidence that habitual intake of adequate quantities of folate helps to reduce the risk of developing colorectal cancer in general population (Ratan et al., 2008). In addition, there is little question that folate may act in a paradoxical, cancer promoting fashion under certain experimental conditions. Ample evidence now exists to indicate that increasing folic acid intake of women in the periconception period substantial diminishes the risk of pregnancies complicated by a neural tube defect (Ratan et al., 2008). Evidence from animal studies suggests a possible association between high intakes of folic acid and promotion of cancer development and progression. There is also a time trend study from the USA and Canada that suggests colorectal cancer incidence increased at around the same time mandatory fortification with folic acid was introduced (Wald and Oakley, 2007).
Vitamin B12 has been shown to stimulate osteoblast proliferation and alkaline phosphatase activity and vitamin B12 deficiency has been associated with defective functional maturation of osteoblasts. Recent publications indicate a shift to more evidence of osteoclast stimulation by high homocysteine and low vitamin B12 concentrations (Van Wijngaarden et al., 2011). Kim et al. (1996) suggested that a suppressed activity of osteoblasts may contribute to osteoporosis and fractures in patients with vitamin B12 deficiency. Herrmann et al. (2007) demonstrated a strong stimulatory effect of low concentrations of folate, vitamin B12 and B6 on osteoclasts activity, suggesting a mechanistic role of low B-vitamin concentrations for bone degradation.
Folate has a dual effect on cancer, protecting against cancer initiation but facilitating progression and growth of preneoplastic cells and subclinical cancers, which are common in the population. Thus, a high folic acid intake may be harmful for some people. At present, no information concerning the effect of maternal folic acid supplementation on bone cancer risk in the offspring exists in humans. Epigenetic and metabolic programming takes places during embryogenesis and hence the embryonic stage is highly susceptible to changes in the intrauterine environment, which may influence the risk of developing cancer in adulthood (Ly et al., 2011).
Findings of raised folate and vitamin B12 in young subjects with osteosarcoma in the present study raise the possibility of supplementation of folate and occurrence of osteosarcoma in these subjects. It may be added that in India, periconception maternal folic acid supplementation is in practice owing to high risk of neural tube defects. Further studies are required to explore dual effect of folate on cancer, especially bone cancer.
Tumors are known to up regulate FRα modulating the folate uptake in serum. Vitamin B12 diminishes osteoblastic activity and lowered serum vitamin B12 levels are possibly due to increased metabolic demand of the tumor. Thus, vitamin B12 supplementation, along with newer antifolate derivative and drugs targeting FRα proteins may be of value in treatment of osteosarcoma in future.
CONCLUSION
Serum folate levels were significantly raised and vitamin B12 levels were significantly lowered in osteosarcoma patients as compared to controls. The present study suggests that these parameters can serve as useful markers for diagnosis and follow up of disease and raise the possibility of supplementation of folate, vitamin B12 and occurrence of osteosarcoma in these subjects.