Homocystinuria

Hyperhomocysteinemia (elevated plasma homocysteine levels) has been linked to increased risk of neural tube defects, cardiovascular disease, Alzheimer's dementia, pregnancy complications, and inflammatory bowel disease. Evidence for a role of hyperhomocysteinemia in the etiology of osteoporosis has recently been strengthened by the findings of two separate studies, which both reported that high homocysteine levels significantly increased risk of osteoporotic fracture. While the etiology of hyperhomocysteinemia is considered to be multifactorial (including genetic, nutritional, and lifestyle factors), a deficiency of one or more B vitamins certainly has a role. These vitamins are involved in the metabolism and clearance of homocysteine, and thus may have a protective effect against osteoporotic fracture risk.

© 2005 International Life Sciences Institute

doi: 10.1301/nr.2004.janr.29-36

Key words: homocysteine, osteoporosis, osteoporotic fracture, B vitamins, MTHFR genotype

Introduction

Homocysteine is an amino acid intermediate formed during the metabolism of methionine. There is ample evidence that high circulating homocysteine levels (also referred to as hyperhomocysteinemia) are associated with an increased risk of cardiovascular disease.1,2 Hyperhomocysteinemia has also been linked to increased risk of neural tube defects, Alzheimer's dementia, pregnancy complications, and inflammatory bowel disease.3 Very recently, two studies reported that high homocysteine levels significantly increase the risk of osteoporotic fracture.4,5 Prior to these studies, evidence for a role of homocysteine in the etiology of osteoporosis had been limited to data from association studies linking a common allelic polymorphism in the gene that encodes the methylenetetrahydrofolate reductase (MTHFR) enzyme (which is important in the clearance of homocysteine) with low bone mineral density (BMD) and/or fracture risk, together with evidence of generalized osteoporosis in patients with homocystinuria, a condition in which there is dramatically elevated plasma homocysteine levels.

Osteoporosis is a global health problem that will take on increasing significance as people live longer and the world's population continues to increase.6 Thus, prevention of osteoporosis and its complications is an essential socioeconomic priority. There is an urgent need to develop and implement nutritional approaches and policies for the prevention and treatment of osteoporosis that could, with time, offer a foundation for population-based preventive strategies. However, to develop efficient and precocious strategies in the prevention of osteoporosis, it is important to determine which modifiable factors, especially nutritional factors, influence bone health throughout life. While the etiology of hyperhomocysteinemia is considered to be multifactorial (including genetic, nutritional, and lifestyle factors), a deficiency of one or more B vitamins certainly has a role to play.3 Furthermore, some of the genetic factors underpinning hyperhomocysteinemia, with the MTHFR genotype the most extensively studied, can interact with folate status and possibly the status of other B vitamins. Therefore, provision of adequate amounts of such nutrients must now clearly be considered among the dietary strategies for osteoporosis prevention. This review will first outline the evidence for a role of homocysteine as a risk factor for osteoporosis, and will then focus on the potential protective effect of certain B vitamins due to their involvement in homocysteine metabolism and clearance.

Homocysteine and Bone

Homocystinuria, a rare autosomal recessive disease characterized by markedly elevated levels of plasma homo cysteine (>100 µmol/L), has been associated with generalized osteoporosis.7,8 The underlying pathophysiological mechanism for the occurrence of early osteoporosis in patients with homocystinuria is not completely understood.4 However, this finding has been attributed to a competitive inhibition of lysyl oxidase, an enzyme that is involved in the synthesis of cross-links that stabilize the collagen fibrils in bone,9 by high homocysteine levels.10 However, while this has been shown clearly in vitro,11 in vivo evidence of the link between homocysteine and generalized osteoporosis has been limited.12

In the general population, a mildly elevated plasma homocysteine is a common condition. Plasma homocysteine levels are influenced by genetic factors, physiological determinants (e.g., age, gender, pregnancy, postmenopausal status, and renal function), dietary and lifestyle factors (e.g., vitamins B12, B6, and B2 intake, smoking, coffee, alcohol, and exercise), and clinical conditions (e.g., deficiencies of folate, vitamins B12 and B6, as well as certain drug use).3 As mentioned previously, in addition to the condition of homocystinuria, the 677C[arrow right]T polymorphism in the MTHFR gene is probably the most extensively studied of the genetic factors underpinning hyperhomocysteinemia.

The MTHFR enzyme, which supplies folate needed for the metabolism of homocysteine and is thus important in the clearing of this amino acid intermediate from the circulation, has a common polymorphism located to nucleotide 677 in the MTHFR gene and is caused by a single base change (C[arrow right]T) leading to an amino acid replacement of alanine with valine at position 222. This point mutation gives rise to a thermolabile variant of the MTHFR enzyme (defined by the TT MTHFR genotype), which is less effective and is associated with an increase in plasma total homocysteine levels and reduced folate levels13 (Figure 1).

The MTHFR genotype has been studied in relation to plasma homocysteine levels and risk of coronary heart disease, because raised homocysteine is an independent risk factor for coronary disease, as well as risk of Alzheimer's disease.1-3 Recently, a number of studies have investigated the association between the MTHFR genotype and bone health indices (Table 1). For example, Miyao et al.14 demonstrated that this allelic polymorphism in the MTHFR gene was associated with reduced BMD in postmenopausal Japanese women. Abrahamsen et al.15 reported that early postmenopausal Danish women with the TT MTHFR genotype had significantly lower BMD at the hip and lumbar spine and increased fracture incidence compared with those with the wild-type CC MTHFR genotype. However, MTHFR genotype did not influence bone turnover, as assessed by biochemical markers in this population; it was associated with higher plasma homocysteine levels.15

Bathum et al.16 studied the relationship between MTHFR genotype and fracture incidence in a population-based sample of Danish twins age 73 and older. After adjusting for age, gender, and body mass index, the odds-ratio of fracture risk was 1.5 per number of T alleles. Fracture risk was 1.5 times higher in the CT genotype group compared with the CC group, and 1.5 times higher in the TT group compared with the CT groups. In contrast, Jorgensen et al.17 reported an association between the C677T polymorphism (TT) in the MTHFR gene with a reduced risk of osteoporotic fracture of the forearm and hip in a case-control study in Danish postmenopausal women relative to those with the wild-type CC genotype, even though BMD at the forearm and ultrasound parameters measured at the calcaneus were similar in both genotype groups.

More recently, Villadsen et al.18 found that the TT genotype was associated with an increased risk of osteoporotic fractures in women. However, there were no significant differences in BMD of the lumbar spine, femoral neck, or total hip, or in levels of biochemical markers of bone turnover among MTHFR genotype groups.18 McLean et al.19 found that adjusted mean BMD of the hip and spine and quantitative ultrasound (which predicts fracture as well as and independent of BMD) of the calcaneus were unaffected by MTHFR genotype in 1632 male and female members of the Framingham Offspring Study (1996-2001). There was no effect of the MTHFR genotype on BMD of the lumbar spine or femoral neck or on loss of BMD from these sites in peri- and early postmenopausal Scottish women (n = 1241) participating in the Aberdeen-based Prospective Osteoporosis Screening Study.20 The reason for the discordant findings of studies investigating the relationship between MTHFR genotype and BMD/fracture risk is unclear, but it may be related to B vitamin status, because homocysteine metabolism and clearance requires folate and vitamins B2, B6, and B12.

In light of these reported-albeit somewhat inconsistent-associations between MTHFR genotype and BMD/fracture risk, together with evidence for the involvement of markedly elevated homocysteine levels in skeletal disease, it is possible, if not likely, that slightly elevated levels of plasma homocysteine have a role in the etiology of osteoporosis in the normal population. Although an earlier report in elderly subjects (age >65 years; n = 46) failed to find a relationship between homocysteine levels and BMD,21 two recent relatively large studies have provided further evidence of a link between homocysteinemia and risk of osteoporotic fracture.

van Meurs et al.4 showed in a group of Dutch adult males and females (n = 2406; age >55) participating in two prospective, population-based studies (two independent cohorts from the Rotterdam study and the Longitudinal Aging Study Amsterdam), that those in the highest quartile of homocysteine levels had twice the risk of a nonvertebral osteoporotic fracture (50% of which were hip and wrist fractures) relative to those in the other three quartiles (homocysteine 75th percentile cutoff values differed by age and cohort, but ranged between 11.4 and 38.6 µmol/L). Interestingly, similar risk estimates appeared to be present in the two cohorts of the Rotterdam study and in the Amsterdam study, suggesting a consistent association within this Dutch population. Homocysteine levels had no effect on BMD at the femoral neck or lumbar spine, which led the investigators to speculate that homocysteine interferes with the development of the microarchitecture of bone independently of the amount of mineral in the bone.

In the second study, published in the same issue of the New England Journal of Medicine, McLean et al.5 reported that the risk of hip fracture in men and women from the Framingham Study (n = 1999; age 59) was increased by nearly a factor of 4 in men and a factor of 2 in women for the highest quartile of (non-fasting) plasma homocysteine (20.8 and 18.6 µmol/L for men and women, respectively) compared with the lowest quartile (8.5 and 7.6 µmol/L for men and women, respectively). The investigators suggest, however, that the apparent differences in risk according to sex may be explained by the lower background incidence of hip fracture in men. This notion was supported by their findings that the difference in absolute risk between the highest and lowest quartiles for men and women were similar. BMD was not assessed in this study. Interestingly, the high homocysteine level appeared to have an effect of a size similar to that of established risk factors for fracture (low BMD, cognitive impairment, and recent falls) and for cardiovascular disease (hypercholesterolemia and hypertension).5

The high homocysteine levels in these studies may be reflective of poor nutritional status. However, when the data on fracture risk from one cohort in the Netherlands study were adjusted for dietary intake (calories, protein, calcium, vitamins B6 and B12, folate, and 25-hydroxyvitamin D), the risk estimates were not altered.4

Role for B Vitamins in Protection against Homocysteine-Related Risk of Osteoporosis

As mentioned previously, the common allelic polymorphism (C677T) in the gene that encodes the MTHFR enzyme has been variably associated with BMD in postmenopausal women. Some of the discordant findings on its effect on bone may arise from a possible gene-nutrient interaction between one or more of the B vitamins and MTHFR genotype. The MTHFR and associated enzymes, together with a number of the B complex vitamins, are required for clearing homocysteine from the circulation (Figure 2). In support of this notion, McLean et al.19 reported that, despite a lack of MTHFR genotypic effects on quantitative ultrasound or BMD in the entire study population (n = 1632), when subjects were stratified on the basis of plasma folate levels (above and below 4 ng/mL), then trends for associations between MTHFR genotype and bone phenotypes were evident. In subjects with plasma folate less than 4 ng/mL, those with the TT genotype had lower mean quantitative ultrasound of the heel (P = 0.06) and BMD of Ward's area (P = 0.08) than those subjects with at least one wild-type allele (CC + CT genotype group). On the other hand, somewhat surprisingly, in subjects with plasma folate ≥4 ng/mL, those with the TT genotype had significantly (P

It is also possible that vitamin B status per se could influence the relationship between homocysteine concentrations and osteoporotic fracture. For example, deficiency of folate and vitamins B6 and B12, which are major determinants of homocysteine concentrations in older persons,22,23 rather than the homocysteine concentration itself, may be responsible for the observed effect on risk of osteoporostic fracture. Pernicious anemia has been shown to increase bone loss and risk of osteoporotic fracture,24,25 while vitamin B12 status has recently been shown to be associated with bone mineral content and BMD in older women26 and in frail elderly women (but not men).27 Cagnacci et al.28 reported that in postmenopausal women, folate but not homocysteine or vitamin B12, was independently related to BMD. On the other hand, McLean et al.19 showed that adult men and women with plasma folate status ≥4 ng/mL had approximately 2% higher BMD and quantitative ultrasound measures than those with plasma folate below 4 ng/mL. However, when homocysteine levels were added to the model, folate was no longer significantly associated with any bone phenotype. Unfortunately, the potential influence of B vitamin status on the relationship between homocysteine levels and fracture risk was not assessed in either of the two recent epidemiological studies.4,5 In the study by McLean et al.,5 baseline dietary data were not available. In the study by van Meurs et al.,4 dietary intake of calories, protein, calcium, vitamins B6 and B12, folate, and 25-hydroxyvitamin D were assessed by food frequency questionnaire in only one of the Rotterdam Study cohorts, but not in the other cohort or in the Amsterdam cohort. Indeed, van Meurs et al.4 suggested that in view of the inherent limitation of measuring dietary intake by means of food frequency questionnaires, nutritional deficiency cannot be completely ruled out as a cofounder in their study.

Conclusion and Future Directions

It is clear from the available data that homocysteine can be added to the growing list of risk factors for osteoporotic fractures. This is an important development, because it has been suggested that the prevention of osteoporosis relies heavily on identification of risk factors that can be reversed readily.29 It is less clear whether a causal relationship between increased homocysteine levels and osteoporotic fracture really exists. Proof of such a causal relationship must be a high priority and this will require further research. While van Meurs et al.4 have suggested that this causal relationship could be established by intervention studies aimed at lowering serum homocysteine levels, Raisz29 has recently argued that a randomized, placebo-controlled clinical trial to determine whether reducing homocysteine levels with nutritional supplements decreases the incidence of fracture in patients with osteoporosis would not be ethical, since effective therapy is available to those patients.

In the absence of such studies, the association between elevated homocysteine and risk of fracture and BMD needs to be confirmed in other large population studies. These studies should include estimates of B vitamin intakes and measures of status indices of certain B vitamins, as well as the MTHFR genotype. This will allow for investigations of MTHFR genotype-folate interactions, but also potential interactions with other B vitamins and potential nutrient-nutrient (B vitamin) interactions, which may influence the relationship of homocysteine and osteoporotic fracture. For example, McNutly et al.30 showed that in healthy subjects (age 19-63), the high homocysteine concentration typically associated with homozygosity for 677C[arrow right]T variant of MTHFR occurs only with poor vitamin B2 status. McKinley et al.31 reported that low-dose vitamin B6 lowers plasma homocysteine in healthy subjects who are both folatc and B2 replete. Inclusion of dietary assessment in future population studies would also allow for control of other potential dietary confounders such as coffee and alcohol intake levels.3

Further exploration of the relationship between homocysteine and osteoporotic fracture could also be facilitated, as suggested by Raisz,29 by collection of data on fracture from studies of the effects of lowering homocysteine levels in cardiovascular disease. The emphasis of this research must be directed toward understanding the underlying mechanisms by which homocysteine increases fracture risk.

If homocysteine concentration is truly a causal mechanism for risk of fracture, the public health implications could be substantial. In the United States, a mandatory folic acid fortification policy introduced in 1998 has proved to be highly effective in reducing the prevalence of mild hyperhomocysteinemia (fasting homocysteine >13 µmol/L) from 18.9% to 9.8%, and of low folate status (

In conclusion, while many questions still remain at this time, nonetheless, the possibility that a dietary-based intervention aimed at reducing hyperhomocysteinemia and its associated risk of hip fractures is intriguing and worthy of research efforts. Such information would also help in the ongoing debate about whether mandatory folic acid fortification programs should be introduced in some countries.

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14. Miyao M, Morita H, Hosoi T, et al. Association of methylenetetrahydrofolate reductase (MTHFR) polymorphism with bone mineral density in postmenopausal Japanese women. Calcif Tissue Int. 2000;66:190-194.

15. Abrahamsen B, Madsen JS, Tofteng CL, et al. A common methylenetetrahydrofolate reductase (C677T) polymorphism is associated with low bone mineral density and increased fracture incidence after menopause: longitudinal data from the Danish osteoporosis prevention study. J Bone Miner Res. 2003;18:723-729.

16. Bathum L, Von Bornemann Hjelmborg J, Christiansen L, et al. Evidence for an association of methylene tetrahydrofolate reductase polymorphism C677T and an increased risk of fractures: results from a population-based Danish twin study. Osteoporos Int. 2004;15:659-664.

17. Jorgensen HL, Madsen J S, Madsen B, et al. Association of a common allelic polymorphism (C677T) in the methylene tetrahydrofolate reductase gene with a reduced risk of osteoporotic fractures: a case control study in Danish postmenopausal women. Calcif Tissue Int. 2002;71:386-392.

18. Villadsen MM, Bunger MH, Carstens M, Stenkjaer L, Langdahl BL. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with osteoporotic vertebral fractures, but is a weak predictor of BMD. Osteoporos Int. 2004;[Epub ahead of print].

19. McLean RR, Karasik D, Selhub J, et al. Association of a common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene with bone phenotypes depends on plasma folate status. J. Bone Miner Res. 2004;19:410-418.

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21. Browner WS, Malinow MR. Homocyst(e)inaemia and bone density in elderly women. Lancet. 1991;338:1470.

22. Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 1993;270:2693-2698.

23. Johnson MA, Hawthorne NA, Brackett WR, et al. Hyperhomocysteinemia and vitamin B-12 deficiency in elderly using Title IIIc nutrition services. Am J Clin Nutr. 2003;77:211-220.

24. Eastell R, Vieira NE, Yergey AL, et al. Pernicious anaemia as a risk factor for osteoporosis. Clin Sci (Lond). 1992;82:681-685.

25. Goerss JB, Kim CH, Atkinson EJ, Eastell R, O'Fallon WM, Melton LJ 3rd. Risk of fractures in patients with pernicious anemia. J Bone Miner Res. 1992;7:573-579.

26. Stone KL, Bauer DC, Sellmeyer D, Cummings SR. Low serum vitamin B-12 levels are associated with increased hip bone loss in older women: a prospective study. J Clin Endocrinol Metab. 2004;89:1217-1221.

27. Dhonukshe-Rutten RA, Lips M, de Jong N, et al. Vitamin B-12 status is associated with bone mineral content and bone mineral density in frail elderly women but not in men. J Nutr. 2003;133:801-807.

28. Cagnacci A, Baldassari F, Rivolta G, Arangino S, Volpe A. Relation of homocysteine, folate, and vitamin B12 to bone mineral density of postmenopausal women. Bone. 2003;33:956-959.

29. Raisz LG. Homocysteine and osteoporotic fractures-culprit or bystander? N Engl J Med. 2004;350;2089-2090.

30. McNulty H, McKinley MC, Wilson B, et al. Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements. Am J Clin Nutr. 2002;76:436-441.

31. McKinley MC, McNulty H, McPartlin J, et al. Low-dose vitamin B-6 effectively lowers fasting plasma homocysteine in healthy elderly persons who are folate and riboflavin replete. Am J Clin Nutr. 2001;73:759-764.

32. Jacques PF, Selhub J, Bostom AG, Wilson PW, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. 1999;340:1449-1454.

33. Honein MA, Paulozzi LJ, Mathews TJ, Erickson JD, Wong LY. Impact of folic acid fortification of the US food supply on the occurrence of neural tube defects. JAMA. 2001;285:2981-2986.

Kevin D. Cashman, PhD

Prof. Cashman is with the Department of Food and Nutritional Sciences and the Department of Medicine, University College, Cork, Ireland.

Address for correspondence: Department of Food and Nutritional Sciences and Department of Medicine, University College, Cork, Ireland; Phone: 353-21-4901317; Fax: 353-21-4270244; E-mail: k.cashman@ucc.ie.

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