The association between serum folate and osteoporosis in adults: based on the National Health and Nutrition Examination Survey

DOI: https://doi.org/10.21203/rs.3.rs-1574819/v2

Abstract

Background and Aim:

Folate, a water-soluble vitamin B, plays a vital role in the human body, osteoporosis is a multifactorial disease that has attracted extensive attention. Previous studies have found that folate can change the way of bone growth. We speculated that folate has a certain connection with osteoporosis. Meanwhile, we will further explore the relationship between folate concentration and the occurrence of osteoporosis.

Methods:

We examined data collected by the National Health and Nutrition Examination Survey over the last decade. We included 8,429 (female: 4,030; male: 4,399) participants in our final analysis and divided them into osteoporosis and non-osteoporosis groups. Logistic regression and restricted cubic spline model analyses were used to evaluate the association between folate concentration and osteoporosis using R software.

 

Results:

Folate concentrations were higher in the osteoporosis group than the non-osteoporosis group ( 57.9 ± 1.78 vs 44.15 ± 0.58 , P < 0.001). Men with higher folate concentrations exhibited higher osteoporosis risks than those with low folate concentrations (OR: 4.26, 95% CI: 1.81, 9.95). In patients aged 50–70 years, high folate levels were associated with increased incidence of osteoporosis (OR: 1.75, 95% CI: 1.14, 2.67). In the restricted cubic spline analysis, osteoporosis incidence displayed a downward trend with increasing folate concentrations. However, when folate concentration approached 50 nmol/L, osteoporosis incidence began to increase.

 

Conclusion:

Folate concentration can affect the occurrence of osteoporosis. Low folate levels can exert protective roles, whereas high levels can increase the risk of osteoporosis. The specific cut-off concentration of folate requires further investigation.

Introduction

The National Health and Nutrition Examination Survey (NHANES) is an investigative project dating back to 1959. This project selects different populations from various regions in the United States to conduct statistical investigations. By collecting personal information, it provides a population database for relevant scholars to study.

 

Osteoporosis is multifactorial disease that occurs because of decreases in bone mass,destruction of bone tissue microstructure,and increased bone fragility. [1] It primarily occurs in postmenopausal women and older individuals exerting a far more detrimental effect on the health of the latter owing to increased age-related fragility. [2-3] An ageing society has resulted in increased attention on osteoporosis, thereby making its prevention and treatment focal areas of interest. Existing research reveals that the following prevention strategies can be implemented: reasonable eating habits, moderate physical activity, and healthy lifestyles [4]

 

Serum folate, a water-soluble vitamin, is primarily involved in the metabolism of genetic materials and proteins, affects the growth and development of animals, and improves the body’s immunity. Possible causes of folate deficiency include insufficient intake or increased metabolic requirements, intestinal malabsorption, vitamin deficiency, and liver diseases [5-6]. Previous studies revealed that folate deficiency is related to neonatal neural tube defects and megaloblastic anaemia [7-8]. It was later discovered that folate deficiency can affect the occurrence of cardiovascular disease, Alzheimer’s disease, and depression [9-11]. However, folate has been shown to act on homocysteine thereby causing changes in bone mineral density (BMD) [12]. Additionally, in mice, osteoporosis induced by a high-fat diet can be inhibited by folate [13]. Existing studies have found a possible link between folate and osteoporosis in animals experiments, and there is not abundant representative population data to prove the relationship between the two. In this study, we analyzed the recent ten years of population data in the United States, and provided a large sample cohort to explore the effect of folate concentration on osteoporosis in different populations.

Materials And Methods

Study population

Data used in our study was collected over the last decade (2007 to 2018) by the NHANES, one of the largest and most comprehensive databases in the United States. Due to the COVID-19 outbreak, data from 2019 to 2020 was not collected, as it was considered incomplete and not nationally representative. Finally, data over four periods (2007–2008, 2009–2010, 2013–2014, and 2017–2018) were selected. Two periods (2011­–2012 and 2015–2016), without the osteoporosis project, were excluded. The data of 59842 participants, over the age of 20 were assessed. The NHANES database selects the representative population of each region. Different weights are matched for each person, to better estimate the characteristics of the overall population data. We extracted the following demographic data for each participant: body mass index (BMI), physical activity information, sedentary time per day (min), smoking, alcohol drinking, hypertension, diabetes, serum folate, and osteoporosis. Participants with missing information were excluded. Finally, participants (n=8,429) were selected for our study and included in the final analysis. Both red blood cell (RBC) folate and serum folate can be used as biomarkers, we chose to evaluate serum folate in consideration of its wider clinical application. [14] The survey protocol was approved by the National Centre for Health Statistics (NCHS) Research Ethics Review Board, and all participants provided written informed consent.

 

Serum folate

More than a decade ago, serum folate levels were measured using the Quantaphase II Folate radio assay kit (Bio-Rad Laboratories, Hercules, CA). Subsequently, the microbiologic assay (MBA) was developed to test folate content, which is considered more accurate [15]. Since 2011, total serum folate has been calculated as the sum of 5 forms of folate, namely 5-methyltetrahydrofolate, folic acid, 5-formyltetrahydrofolate, tetrahydrofolate, and 5, 10-methenyltetrahydrofolate by isotope-dilution high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [16]. We classified folate concentration into three grades (according to the interquartile range): low, medium, and high. Q1 was considered low grade; Q2–Q3, intermediate grade; and Q4, high grade. 

 

Osteoporosis 

Information about osteoporosis was screened from the questionnaire data and the dual-energy X-ray absorptiometry (DXA) test. Responses of ‘Yes’ to family questions such as: ‘Have you ever been diagnosed with osteoporosis/brittle bones?’ was accepted as osteoporosis. Additionally, DXA is the most commonly used method for BMD measurement in a clinic, and has the advantages of high speed and low radiation. According to the World Health Organization criteria, osteoporosis should be considered if bone mineral density of the lumbar spine or femoral neck is < 2.5 standard deviation (SD) of healthy person of the same sex. [17]

 

Covariates

In the NHANES database, we selected the following as covariates: age, race, sex, BMI, physical activity, sedentary time, smoking, alcohol consumption, hypertension, and diabetes. BMI uses international units (kg/m2). Physical activity was defined as: ‘How much time do you spend in a typical day doing moderate-intensity activity?’ Sedentary time was defined as ‘How much time do you usually spend in a typical day sitting?’. Three levels distinguished the degree of smoking: never (the participant has smoked <100 cigarettes during their entire life), former smoker (the participant smoked in the past but has completely stopped), and current smoker (the participant has smoked >100 cigarettes during their life and currently smoke, either on some days or daily). Three levels distinguished the degree of drinking: never (the participant has hardly consumed alcohol in their life), light (the participant consumes alcohol occasionally throughout the year), and moderate to heavy (weekly or daily consumption). The diagnostic criteria for hypertension were: ‘Have you ever been diagnosed with hypertension by a doctor?’ or ‘Are you currently using any antihypertensive medication?’. The diagnostic criteria for diabetes were: ‘Have you ever been diagnosed with diabetes by a doctor?’ or ‘Are you currently using insulin, or any other anti-diabetic medication?’ The above information can be found in the questionnaire data or examination data.

 

Statistical analysis

R software (version 4.1.2) was used for data statistics and analysis. R is a language and environment for statistical computing and graphics, developed at Bell Laboratories (formerly AT&T, now Lucent Technologies). The Student t-test was used to compare normally distributed measurement data, and the χ2 test was used to compare the count data. Serum folate concentration was divided into three grades according to the quartile (low grade: Q1, middle grade: Q2–Q3, high grade: Q4). Taking the lower grade as reference, logistic regression analysis was performed to analyse the association between folate and osteoporosis. Stratified analysis was conducted for age, sex, race, and the results were expressed as odds ratios (ORs) and corresponding 95% confidence intervals (CIs). The restricted cubic spline (RCS) model was used to analyse the nonlinear association between folate and osteoporosis. Due to the large sample size, four points (5th, 35th, 65th, and 95th) of folate concentration were used as knots, and the mean was used as the reference point [18-19]. The NHANES adopted complex multi-stage sampling, and our data was analysed after matching with appropriate weights. Unless otherwise specified, all data were considered statistically significant with a two-tailed P value < 0.05.

Results

In this study, a total of 8,429 participants were selected (Table 1). The proportions of males and females were almost equivalent (52.19 and 47.81%, respectively). Females with osteoporosis accounted for 81.58% of the data, considerably exceeding males (P < 0.001). The occurrence of osteoporosis was 27.82% in participants who performed moderate physical activity daily and 72.18% in participants who performed no physical activity (P < 0.001). The proportions of hypertension and diabetes in the osteoporosis group was 58.08 and 34.59% (P < 0.001), respectively. The serum folate concentration in participants with osteoporosis was 57.9 ± 1.78 nmol/L, which was higher than that of participants without osteoporosis (P < 0.001).

 

Logistic regression analysis

We used logistic regression to analyse the association between folate and osteoporosis among different subgroups (Table 2). The occurrence of osteoporosis was significantly increased in men with middle and high levels of folate compared to those with low levels (OR: 3.51, 95% CI: 1.55, 7.91 and OR: 8.02, 95% CI: 3.63, 17.66). After adjusting for the covariates, the occurrence of osteoporosis was still significantly increased (OR: 3.14, 95% CI: 1.46, 6.73 and OR: 4.26, 95% CI: 1.81, 9.95). In women, before adjustment, 1.76 times more participants had high levels of folate compared to those with low levels (OR: 1.76, 95% CI: 1.29, 2.4). After adjustment, there was no statistically significant difference between participants with the two levels of folate. In participants aged 50–70 years, middle and high levels of folate were associated with an increased incidence of osteoporosis compared with low levels (OR: 1.71, 95% CI: 1.03, 2.82 and OR: 1.75, 95% CI: 1.14, 2.67), and no differences in folate levels were observed after adjusting for covariates. In the analysis for different races subgroups, high folate levels in some races increased the incidence of osteoporosis (all P < 0.05).

 

RCS model analysis 

There was a nonlinear association between folate concentration and osteoporosis. In the total population, initially, the occurrence of osteoporosis exhibited a downward trend as folate concentration increased, until the incidence reached the lowest point at ~25 nmol/L folate. Subsequently, increased folate levels were associated with an increase in the occurrence of osteoporosis, but remained below the normal level. After 50 nmol/L, the occurrence of osteoporosis increased significantly. In both men and women, the association between osteoporosis and folate were similar to that in the total population. In age stratification, <50 years and >70 years were insignificant, whereas between the ages of 50 and 70 years, a continuous increase in osteoporosis with increased folate concentration was observed (Figures 1, 2).

Discussion

Our study investigated the association between folate levels and osteoporosis in the US population over the last decade. Due to the unique properties and functions of folate, it plays a fundamental role in organisms. Recent studies have demonstrated that folate may regulate lipid metabolism and oxidative stress reaction, activate the AMP-activated protein kinase (AMPK) pathway, and reduce high-fat diet induced osteoporosis. Additionally, it has also been found that vitamins B and folate supplements can improve BMD [13, 20]. All these suggest that folate may affect bone formation.

 

In this study, age, sex, hypertension and physical activity were found to influence the occurrence of osteoporosis, which is consistent with findings of previous studies [21]. The proportions of diabetes in osteoporosis was higher than non-osteoporosis (P<0.001). It could be due to increased BMD in diabetic patients, and the risk of fracture eventually increases in these patients, it is related to the duration of diabetes and complications [22-23]. However, more research is required in this regard.

 

Folate concentration was significantly higher in the osteoporosis group than in the non-osteoporosis group (P < 0.001). We divided folate into low, medium, and high grades to analyse. We observed that in men, the risk of osteoporosis with moderate and high folate levels was significantly higher than that with low folate levels, and higher folate levels increased the risk of osteoporosis suggesting that folate may affect bone formation, and high folate levels may exert an inhibitory effect. In age stratification, between the ages of 50 and 70 years, medium and high levels of folate showed a higher risk of osteoporosis than lower levels. After adjusting for covariates, such as sex, no significant difference was found in the results, which may be related to female hormones. Women experience menopause at ~50 years of age, and the loss of oestrogen after menopause could lead to bone loss, thereby increasing the overall occurrence of osteoporosis [24]. No association between folate and osteoporosis was found in the group older than 70 years. It was considered that decreased secretion of hormones in older individuals can stimulate osteoclasts and inhibit osteoblasts [25]. This accompanied by decline of organ functions and physical activity, results in bone decline, which affects the occurrence of osteoporosis. In racial stratification, except for multi-ethnic minorities, other races exhibited an increased risk of osteoporosis with the increase of folate levels. This shows that folate is associated with osteoporosis; however, the association between different races requires further investigation. 

 

Considering that folate levels do not display a simple linear association with osteoporosis, we used RCS to describe it. In the total population, as folate concentration increased, the risk of osteoporosis gradually decreased, reaching the lowest point at 25 nmol/L, and subsequently there was a J-shaped association between the two. In the stratified analysis, similar results were observed among the subgroups of men and women, aged 50–70 years old. Folate levels below 50 nmol/L appear to have a protective effect against osteoporosis, whereas levels exceeding 50 nmol/L appear to be a risk factor, which provides a reference range for the possible prevention of osteoporosis. These results are in agreement with those of a similar study conducted by Stanley et al. who found that both high and low folate levels increase cardiovascular mortality in hypertensive individuals [26]

 

Since the 1990s, the government of the United States has implemented adding folate to flour and grain to improve  folate levels of the population [27]. Implementation of the folate policy has also played a significant role in reducing the occurrence of several diseases. In 2018, the United States discontinued the policy of adding folate, causing some speculation. Some studies reported that excessive folate is not beneficial and can increase cardiovascular events and cause mortality [28-29]. Professor Evans, in a 15-year study, highlighted that high serum folate is associated with increased risk of death in adults with diabetes [30]. The current explanation is that excessive folate may increase the level of unbound folate and increase the degradation of folate, and also control biological methylation and nucleotide synthesis, thereby damaging DNA integrity [31,32]. Moreover, excessive intake of natural folate might not cause poisoning; however, long-term high dose synthetic folate intake can produce a large amount of unmetabolised folate, which may reduce the cytotoxicity of natural killer cells [33]. Excessively high levels may also affect the absorption of other nutrients or mask the symptoms of vitamin B12 deficiency [34]

 

To our knowledge, it is first of its kind and uses a database with abundant representative population data to reveal the association between folate concentration and osteoporosis. We have also determined the folate concentration cut-off value to prevent osteoporosis, it could be used to guide folate supplement regulations. At the same time, this study had some limitations. Dietary intake is an important factor affecting folate levels. However, there are currently insufficient data available regarding this. Additionally, since this was a cross-sectional study, it could not reveal a prognostic association between folate and osteoporosis. 

 

In conclusion, the concentration of folate can affect the occurrence of osteoporosis; low levels of folate can play a protective role, whereas high levels can be a risk factor. Further studies are required to ascertain the specific folate cut-off value.

Abbreviations

NHANES

National Health and Nutrition Examination Survey

NTDs

Neonatal neural tube defects

MA

Megaloblastic anemia

BMD

Bone mineral density

COVID-19

Corona Virus Disease 2019

BMI

Body mass index

NCHS

National Center for Health Statistics

DXA

Dual-energy X-ray absorptiometry

WTO

World Health Organization

ORs

Odds Ratios

CIs

Corresponding 95% confidence intervals

RCS

Restricted cubic spline

Declarations

Data Availability Statement: The datasets used and/or analysed during the current study are available at https://wwwn.cdc.gov/nchs/nhanes/nhanes3/DataFiles.aspx

 

Acknowledgments: We thank Professor Jia for guidance on the content of the article and Doctor Yang for his support in data analysis.

 

Author Contributions: Senjie Li: Conceptualisation, Data curation, Formal analysis, Investigation, Writing- original draft, Writing-review & editing. Dongqing Lv: Conceptualisation, Investigation, Methodology, Project administration, Writing-original draft, Writing-review & editing. Hong Yang: Conceptualisation, Investigation, Methodology, Project administration, Software. Shao Yi Yan: Project administration, Formal analysis, Investigation. Lei Wu: Conceptualisation, Methodology, Data curation, Supervision, Writing-review & editing. Yongping Jia: Conceptualisation, Methodology, Project administration, Supervision, Writing-review & editing.

 

Funding: This study was supported by the Beijing Medical Award Foundation of China (No. YXJL202103530603).

 

Informed Consent Statement: All participants provided informed consent. Consent for publication: Not applicable.

 

Conflicts of Interest: The authors declare no conflict of interest.

References

  1. E SirisR AdlerJ Bilezikian,et al. The clinical diagnosis of osteoporosis: a position statement from the National Bone Health Alliance Working Group.Osteoporos Int. 2014;25:1439-1443. doi: 10.1007/s00198-014-2655-z
  2. Wright C, Looker C, Saag G, et al. The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine. J Bone Miner Res. 2014;29:2520-6. doi: 10.1002/jbmr.2269.
  3. Burge R, Dawson-Hughes B, Solomon DH, et al. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025. J Bone Miner Res. 2007;22:465-75. doi: 10.1359/jbmr.061113.
  4. Maria Felicia F, Giuseppe L, Mariangela C. How physical activity across the life span can reduce the impact of bone ageing: A literature review. Int J Environ Res Public Health. 2020;17(6):1862. doi: 10.3390/ijerph17061862.
  5. Gropper SS, Smith JL. Advanced nutrition and human metabolism, 6th Edition [Internet]. Wadsworth Belmont, CA;2013.p.344-353. Available from: https://www.cengage.co.uk/books/9781133104056/.
  6. Bailey RL, Dodd KW, Gahche JJ, et al. Total folate and folic acid intake from foods and dietary supplements in the United States: 2003 – 2006. Am J ClinNutr. 2010;91(1):231–237. doi: 10.3945/ajcn.2009.28427231–237.
  7. Daly LE, Kirke PN, Molloy A, et al. Folate levels and neural tube defects. Implications for prevention. J Am Med Assoc. 1995;274:1698-1702. doi: 10.1001/jama.1995.03530210052030.
  8. Socha DS, DeSouza SI, Flagg A, et al. Severe megaloblastic anemia: Vitamin deficiency and other causes. Cleve Clin J Med. 2020;87(3):153-164. doi: 10.3949/ccjm.87a.19072.
  9. Obeid R, Holzgreve W, Pietrzik K, et al. Folate supplementation for prevention of congenital heart defects and low birth weight:an update. CardiovascDiagnTher. 2019; 9(Suppl2):S424-S433. doi: 10.21037/cdt.2019.02.03.
  10. Robinson N, Grabowski P, Rehman I. Alzheimer's disease pathogenesis: Is there a role for folate? Mech Ageing Dev. 2018;174:86-94. doi:10.1016/j.mad.2017.10.001.
  11. Khosravi M, Sotoudeh G, Amini M, et al. The relationship between dietary patterns and depression mediated by serum levels of Folate and vitamin B12. BMC Psychiatry. 2020;20(1):63. doi: 10.1186/s12888-020-2455-2.
  12. Herrmann M, Widmann T, Herrmann W. Homocysteine - a newly recognised risk factor for osteoporosis.ClinChem Lab Med. 2005;43(10):1111-7. doi: 10.1515/CCLM.2005.194.
  13. Haiting H, Yaxi Zhang, Yue S, et al. Folic Acid Attenuates High-Fat Diet-Induced Osteoporosis Through the AMPK Signaling Pathway. Front Cell Dev Biol. 2022;9: 791880. doi:10.3389/fcell.2021.791880. 
  14. Yetley E, Pfeiffer C, Phinney K, et al. Biomarkers of folate status in NHANES:a roundtable summary. Am J Clin Nutr. 2011;94:303S-12S. doi:https://doi.org/10.3945/ajcn.111.013011.
  15. National Center for Health Statistics. National Health and Nutrition Examination Survey: Laboratory Procedure Manual. Available at: https://wwwn.cdc.gov/nchs/data/nhanes/2009-2010/labmethods/FOLATE_F_MET.PDF. October 2011.
  16. National Center for Health Statistics. National Health and Nutrition Examination Survey: Laboratory Procedure Manual.Available at: https://wwwn.cdc.gov/nchs/data/nhanes/2017-2018/labmethods/FOLFMS-J-MET-508.pdf. July 2020.
  17. Kanis JA, Cooper C, Rizzoli R, et al. European guidance for the diagnosis and management of osteoporosis in postmenopausal women.Osteoporos Int. 2019;30(1): 3-44. doi:10.1007/s00198-018-4704-5. 
  18. F.E. Harrell, Regression Modeling Strategies: with Applications to Linear Models, Logistic Regression, and Survival Analysis (Second Edition), Springer, New York, 2015.
  19. Martin O'Donnell, Salim Y, Andrew M, et al. Urinary sodium and potassium excretion and risk of cardiovascular events. JAMA. 2011;306(20):2229-38. doi: 10.1001/jama.2011.1729.
  20. Mariangela R, Alice T, Federica F. Adequate intake and supplementation of B vitamins, in particular folic acid, can play a protective role in bone health. Curr Aging Sci. 2021 Oct 4. doi: 10.2174/1874609814666211005101730. Online ahead of print.
  21. JL Kelsey. Risk factors for osteoporosis and associated fractures. Public Health Rep Sep-Oct 1989;104 Suppl(Suppl):14-20.
  22. Irzal H, Blazenka M, Vesna C, et al. Increased bone mineral density in postmenopausal women with type 2 diabetes mellitus. Ann Saudi Med. 2008;28:102- 104. doi: 10.5144/0256-4947.2008.102.
  23. de Liefde II, van der M, de Laet CE, et al. Bone mineral density and fracture risk in type 2 diabetes melli-tus: the Rotterdam Study.Osteoprosis Int. 2005;16:1713-1720. doi: 10.1007/s00198-005-1909-1. 
  24. Bijelic R, Milicevic S, Balaban J. Risk Factors for Osteoporosis in Postmenopausal Women. Med Arch. 2017;71(1):25-28. doi: 10.5455/medarh.2017.71.25-28. 
  25. Coughlan T, Dockery F. Osteoporosis and fracture risk in older people. Clin Med (Lond). 2014;14(2):187-91. doi: 0.7861/clinmedicine.14-2-187.
  26. Stanley N, Emeka I, Logan TC, et al. Association between serum folate and cardiovascular deaths among adults with hypertension. Eur J ClinNutr. 2020;74(6):970-978. doi: 10.1038/s41430-019-0533-7.
  27. Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Final rule. 21 CFR Parts 136, 137, and 139. Fed Regist. 1996;61:8781-8789.
  28. Yang P, Bin D, Zhiqiang W. Serum folate concentrations and all-cause, cardiovascular disease and cancer mortality: A cohort study based on 1999-2010 National Health and Nutrition Examination Survey (NHANES). Int J Cardiol. 2016;219:136-42. doi: 10.1016/j.ijcard.2016.06.024. 
  29. Y Peng, Z Wang. Red blood cell folate concentrations and coronary heart disease prevalence: A cross-sectional study based on 1999-2012 National Health and Nutrition Examination Survey. NutrMetabCardiovasc Dis. 2017;27(11):1015-1020. doi: 10.1016/j.numecd.2017.07.007. 
  30. Evans A, Emeka I, Akwasi AY, et al. Serum folate levels and fatality among diabetic adults: A 15-y follow-up study of a national cohort. Nutrition. 2016;32(4):468-73. doi: 10.1016/j.nut.2015.10.021. 
  31. Natalia VO, Natalia IK, Steven NR, et al. Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation.BiochemPharmacol. 2006;72(2):256-66. doi: 10.1016/j.bcp.2006.04.005. 
  32. Julia S, Joel B, Sang-Woon C, Too much folate: a risk factor for cancer and cardiovascular disease?CurrOpinClinNutrMetab Care. 2009;12(1):30–36. doi: 10.1097/MCO.0b013e32831cec62.
  33. Aron M, Breeana M, Bess S, et al. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytotoxicity among postmenopausal women. J Nutr. 2006;136(1):189-94. doi: 10.1093/jn/136.1.189.
  34. K R Patel, A Sobczyńska-Malefora. The adverse effects of an excessive folic acid intake. Eur J Clin Nutr. 2017;71(2):159-163. doi: 10.1038/ejcn.2016.194