Bone resorption markers in vitamin D insufficiency

Clinica Chimica Acta 368 (2006) 48 – 52 www.elsevier.com/locate/clinchim

Invited critical review

Bone resorption markers in vitamin D insufficiency Allan G. Need T Department of Clinical Biochemistry, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia Received 16 November 2005; received in revised form 23 December 2005; accepted 23 December 2005 Available online 9 February 2006

Abstract Severe vitamin D deficiency (serum 25 hydroxyvitamin D (25(OH)D) below 12.5 nmol/L) causes rickets and osteomalacia, but there is good evidence that lesser degrees of hypovitaminosis D (vitamin D insufficiency) have deleterious effects on bone and other organs. Evidence of impaired mineralization, suggestive of vitamin D insufficiency, has been found in bone biopsies of hip fracture patients in the UK, and several studies around the world have shown a rise in serum parathyroid hormone (PTH) as 25(OH)D levels fall below 50 nmol/L. Fifty-seven percent of hospital inpatients in a Boston study had vitamin D insufficiency and their serum 25(OH)D showed an inverse relationship to their serum alkaline phosphatase (ALP) levels. Thirty-five percent of outpatients had vitamin D insufficiency in an Adelaide study, where ALP and urine hydroxyproline and pyridinium cross-links were all inversely related to serum 25(OH)D. The increased bone resorption of vitamin D insufficiency is important on two counts. Firstly, increased bone resorption may lead to increased bone loss and osteoporosis and, secondly, increased turnover appears to increase fracture risk in its own right. A consensus is developing that serum 25(OH)D levels should be maintained at 50 nmol/L or greater in the elderly to minimize the occurrence of fractures. In addition, it appears that optimal levels of bone resorption markers in this population are at or just below the mean level for premenopausal women. D 2006 Elsevier B.V. All rights reserved. Keywords: Vitamin D; Hydroxyproline; Cross-links; Alkaline phosphatase; Bone resorption

Contents 1. Vitamin D deficiency . . . . . . . . . . . . . . . . . . . . 2. Vitamin D insufficiency . . . . . . . . . . . . . . . . . . 3. Secondary hyperparathyroidism in vitamin D insufficiency 4. Prevalence of vitamin D insufficiency . . . . . . . . . . . 5. Vitamin D and bone resorption . . . . . . . . . . . . . . . 6. Bone turnover markers and fracture risk . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Evidence is building that aging populations from many parts of the world are lacking in vitamin D and that this may lead to a variety of disorders, not least of which is accelerated bone loss [1]. The excess bone loss is associated with increased parathyroid hormone levels [2] T Tel.: +61 8 8222 3391; fax: +61 8 8222 3518. E-mail address: [email protected]. 0009-8981/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.12.031

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and increased bone resorption [3]. Randomized controlled trials in elderly men and women in France and the UK suggest that vitamin D, with [4] or without [5] calcium, can correct the hyperparathyroidism and reduce fractures. This review examines the link between low vitamin D levels and increased bone resorption and the role that increased bone resorption may play in increasing the risk of fractures.

A.G. Need / Clinica Chimica Acta 368 (2006) 48 – 52

1. Vitamin D deficiency Severe vitamin D deficiency causes rickets in children and osteomalacia in adults, is associated with hypocalcaemia, impaired mineralization of bone, muscle weakness and deformity (in children), and produces the characteristic radiographic findings of flaring of the epiphyses (in children), Looser’s zones (fissure fractures) and ill-defined trabeculae [6]. Bone biopsies show increased amounts of unmineralized bone matrix (osteoid) and decreased mineralization lag time (measured with double tetracycline labelling). These changes are associated with serum 25 hydroxyvitamin D levels below 12.5 nmol/L [7]. This degree of vitamin D deficiency became rare after the recognition that sunlight generated vitamin D from 7 dehydrocholesterol in the skin and the subsequent fortification of food with vitamin D in areas where sunlight was not plentiful. However, it now appears that significant bone disease occurs with 25(OH)D levels above 12.5 nmol/L and inside the previous reference interval of about 40 – 160 nmol/L. The reference interval, of course, varied from country to country depending on the available sunlight. Patients with levels of 25(OH)D that are within previously recognized reference intervals, but which are now known to be deleterious to bone, are said to have vitamin D insufficiency.

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3. Secondary hyperparathyroidism in vitamin D insufficiency The increase in serum parathyroid hormone (PTH), which occurs as serum 25(OH)D levels fall, is generally attributed to the decreased calcium absorption, which accompanies low 25(OH)D levels [7,19]. There is an inverse relation between PTH and serum ionized calcium, indicating secondary hyperparathyroidism, in postmenopausal women in Australia [20], but the relation between serum 25(OH)D and calcium absorption is weak and the change in calcium absorption noted when 25(OH)D levels rise in summer appears to be very small [21]. Also there appears to be no inverse relationship between serum PTH and calcium absorption in postmenopausal women; the relation is slightly positive (although not significantly so) [22]. It is more likely that low 25(OH)D levels cause a reduction in the calcaemic action of vitamin D on bone as demonstrated by Carlsson and Lindquist [23]. This causes a decrease in plasma ionized calcium in the fasting state, which, in turn, causes a rise in PTH. It is not yet known how serum 25(OH)D directly influences bone, since the vitamin D receptors in bone cells show much less affinity for 25(OH)D than for 1,25 dihydroxyvitamin D (1,25(OH)2D) (the ‘‘active’’ metabolite), but it is known that bone cells contain the enzyme CYP27B1, which synthesizes 1,25(OH)2D from 25(OH)D [24] suggesting that bone cells make this hormone locally and that local levels may be related to levels of circulating 25(OH)D.

2. Vitamin D insufficiency There is general agreement that low vitamin D levels, above those causing overt osteomalacia, lead to increased bone loss and an increased risk of fracture. Levels of 25(OH)D less than 12.5 nmol/L are associated with osteomalacia, while levels between 13 and 50 nmol/L indicate vitamin D insufficiency [8]. First hints of this condition came from studies of bone biopsies in hip fracture patients in the UK [9,10]. Hip fracture patients (102 women and 23 men) had more unmineralized osteoid in their biopsies than age matched subjects without hip fracture. In addition, there was more osteoid in the biopsies collected in winter, when vitamin levels are low, than in summer [11]. When 25(OH)D levels were subsequently measured in hip fracture patients (male and female) in Australia, they were found to be low (mean 39 nmol/L) [12], but not in the range found in patient with osteomalacia (< 12.5 nmol/L). Since then, several studies from the US, UK and Australia have found that parathyroid hormone levels are raised in patients with 25(OH)D levels in the range of vitamin D insufficiency [13 – 16] and there is general agreement that levels of at least 50 nmol/L are required for bone health [17], although some authors recommend levels as high as 122 nmol/L [18].

4. Prevalence of vitamin D insufficiency Thomas et al. reported in 1998 that hospitalized male and female US patients have low levels of 25(OH)D [25]. It is perhaps more surprising that ambulant postmenopausal women in sunny South Australia had low levels [20], or even that randomly selected members of the US community in the NHANES III study [26] and normal adolescents in Tasmania [27] were at risk of vitamin D insufficiency (Table 1).

Table 1 Prevalence of vitamin D insufficiency in various populations Population Community (NHANES III) White Black Adolescent males Hospital outpatients Hospital inpatients

Prevalence (%)

Age (range or mean, S.D.)

Reference

11 50 68 35 57

20 – 80 20 – 80 18 (0.8) 64 (9.3) 62 (19)

R. Scragg et al. [26] R. Scragg et al. [26] G. Jones et al. [27] A.G. Need et al. [20] M.K. Thomas et al. [25]

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5. Vitamin D and bone resorption It is well known that vitamin D deficiency (osteomalacia) causes an increase in bone resorption (presumably from the rise in PTH caused by hypocalcaemia) and a rise in serum ALP is considered a cardinal feature of the syndrome. However, bone markers are also raised in patients with vitamin D insufficiency. In the study of hospital inpatients already mentioned [25], there was an inverse relationship between serum 25(OH)D and serum alkaline phosphatase (ALP). Sahota et al. reported that UK women with vitamin D insufficiency had higher serum bone specific alkaline phosphatase and osteocalcin and higher urine hydroxyproline and deoxypyridinoline than patients with normal 25(OH)D levels [28]. Lips et al. reported in an international study of 7546 osteoporotic women that serum ALP was higher in those with lower 25(OH) levels [29]. In our Australian study of 486 postmenopausal women [3], serum PTH rose above the normal range as 25(OH)D fell below 50 nmol/L, and the urine markers of bone resorption, hydroxyproline (OHPr), pyridinoline (PYD) and deoxypyridinoline (DPD), similarly showed the biggest rise as 25(OH)D values fall below about 50 nmol/L. OHPr is the major breakdown product from collagen, the main protein of the bone matrix which makes up about 50% of the bony tissue. When bone is resorbed, calcium and phosphate are released as well, but these are re-utilized since bone formation is closely coupled to bone resorption. Hydroxyproline cannot be re-utilized because collagen formation requires proline; the proline is converted to hydroxyproline after incorporation into the collagen molecule (post-translational modification). As bone breaks down fragments of collagen of different sizes are produced with varying amounts of hydroxyproline. These fragments need to be hydrolyzed before assay of a urine sample in order to measure total hydroxyproline. The pyridinium cross-links, PYD and DPD, are also made after the collagen chain is formed and help stabilize the molecule. They are more specific for bone than OHPr, although some PYD is present in cartilage. Hydrolysis of the urine sample is again required before measurement to release PYD and DPD from collagen remnants. The ‘‘free’’ cross-links content, from non-hydrolyzed samples, may vary with different treatments for osteoporosis and is not so useful for monitoring therapy. One advantage of the pyridinium cross-link assays over hydroxyproline is that food does not contain cross-links, so 24-h urine collections can be used to monitor bone active therapy. All urine markers can be measured on a morning fasting urine but the result must be divided by the creatinine concentration to allow for urine dilution. A serious problem with the urinary markers is their high day to day variation which is largely biological. This may be overcome to some extent by the use of the new serum markers, e.g. NTX and CTX, which are metabolites of the terminal telopeptides of collagen I which nearly all come

from bone [30]. It is assumed that the observed increase in ALP in vitamin D insufficiency occurs because bone formation rises to keep up with resorption, although it is possible that some of the increase may be a result of disrupted bone formation as exhibited by the increase in osteoid and the raised mineralization lag time found in bone biopsies. It is of some concern that vitamin D insufficiency may be common in adolescent boys. Low serum 25(OH)D was found in 68% of them in one study [27] and there was an inverse association between 25(OH)D and both PYD excretion and serum bone specific alkaline phosphatase (BALP). BALP rose as 25(OH)D levels fell below 55 nmol/L and urine PYD as 25(OH)D levels fell below 43 nmol/L [27]. In our study, as already mentioned, both total serum ALP and bone resorption markers rose significantly as 25(OH)D fell below about 50 nmol/L. Vitamin D and calcium lower both PTH and ALP in elderly male and female nursing home residents [4] and it is assumed that bone resorption markers fall in the same way although data on this effect seems to be lacking. Although bone markers are increased in patients with low 25(OH)D levels the markers cannot, of course, be used in individual patients to infer their levels of 25(OH)D and this must usually be measured, although the prevalence of low levels in nursing home patients is so high it may be reasonable to treat them all as being vitamin D deficient.

6. Bone turnover markers and fracture risk Several prospective studies indicate that increased bone turnover predicts increased fracture risk. Garnero et al. [31] reported in 1996 that markers of bone resorption predicted hip fractures in elderly women in the EPIDOS study, and Ross et al. showed later that bone ALP predicted both vertebral and non-vertebral fractures in postmenopausal community-dwelling women [32]. Riggs et al. have shown that vertebral fracture reduction in a randomized trial of estrogen in postmenopausal women was due not so much to an increase in bone density as to a reduction in bone turnover [33]. This protection from fracture follows the time course of the reduction in bone turnover. In an analysis of a large randomized study of raloxifene in postmenopausal women, Sarkar et al. showed that the gradient relating vertebral fracture risk to bone density was shifted downward in those on the active drug, so that with no change in bone density the fracture risk fell by up to 40% [34]. Eastell et al. have more recently demonstrated a relationship between the fall in the newer bone markers, NTX and CTX, on risedronate therapy and reduction in vertebral fracture risk [35]. It has been postulated that increased bone remodelling may be detrimental because it causes perforation of plates or loss of trabeculae in trabecular bone [36], but it is also known that newly formed bone is less well mineralized [37] and remodelling

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causes focal areas of weakness so there may be a number of reasons for this phenomenon. This is not to say that bone turnover has no benefit; it allows the bones to adapt to changes in the way they are used and allows for removal of fatigue damage. Nevertheless, it appears that the prevailing level of bone remodelling in postmenopausal women with osteoporosis is too high for bone health and that any trend for an increase, such as that caused by vitamin D insufficiency, will increase the fracture risk. It has been suggested that an optimal level of bone resorption is achieved when bone marker values are half a standard deviation below the premenopausal mean [35]. A consensus is developing that serum 25(OH)D should be maintained at 50 nmol/L or above to minimize the occurrence of fractures.

References [1] von Muhlen DG, Greendale GA, Garland CF, Wan L, Barrett-Connor E. Vitamin D, parathyroid hormone levels and bone mineral density in community-dwelling older women: the Rancho Bernardo Study. Osteoporos Int 2005;16:1721 – 6. [2] Souberbielle J-C, Cormier C, Kindermans C, et al. Vitamin D status and redefining serum parathyroid hormone reference range in the elderly. J Clin Endocrinol Metab 2001;86:3086 – 90. [3] Jesudason D, Need AG, Horowitz M, O’Loughlin PD, Morris HA, Nordin BEC. Relationship between serum 25-hydroxvitamin D and bone resorption markers in vitamin D insufficiency. Bone 2002;31: 626 – 30. [4] Chapuy MC, Arlot ME, Duboeuf F, et al. Vitamin D3 and calcium to prevent hip fractures in elderly women. N Engl J Med 1992;327: 1137 – 42. [5] Trivedi DP, Doll R, Khaw KT. Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial. Br Med J 2003;326:469 – 74. [6] Leonard MB, Shore RM. Radiological evaluation of bone mineral in children. In: Favus MJ, editor. Primer on the metabolic bone diseases and disorders of mineral metabolism. 5th edition. American Society for Bone and Mineral Research; 2003, p. 173. [7] Parfitt AM, Qiu S, Rao DS. The mineralization index—a new approach to the histomorphometric appraisal of osteomalacia. Bone 2004;35:320 – 5. [8] Chapuy M-C, Preziosi P, Maamer M, et al. Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int 1997; 7:439 – 43. [9] Chalmers J, Conacher WDH, Gardner DL, Scott PJ. Osteomalacia—a common disease in elderly women. J Bone Jt Surg 1967;49B:403 – 23. [10] Aaron J, Gallagher JC, Anderson J, et al. Frequency of osteomalacia and osteoporosis in fractures of the proximal femur. Lancet 1974;1: 229 – 33. [11] Aaron J, Gallagher JC, Nordin BEC. Seasonal variation of histological osteomalacia in femoral neck fractures. Lancet 1974;2:84 – 5. [12] Morris HA, Morrison GW, Burr M, Thomas DW, Nordin BEC. Vitamin D and femoral neck fractures in elderly South Australian women. Med J Aust 1984;140:519 – 21. [13] Parfitt AM, Gallagher JC, Heaney RP, Johnston CC, Neer R, Whedon GG. Vitamin D and bone health. Am J Clin Nutr 1982; 36:1014 – 31. [14] Krall EA, Sahyoun N, Tannenbaum S, Dallal GE, Dawson-Hughes B. Effect of vitamin D intake on seasonal variation in parathyroid

[15]

[16]

[17] [18]

[19] [20]

[21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

51

hormone secretion in postmenopausal women. N Engl J Med 1989; 321:1777 – 83. Gloth MF, Gundberg CM, Hollis BW, Haddad JG, Tobin JD. Vitamin D deficiency in homebound elderly persons. JAMA 1995;274:1683 – 6. Sahota O, Mundey MK, San P, Godber IM, Lawson N, Hosking DJ. The relationship between vitamin D and parathyroid hormone: calcium homeostasis, bone turnover, and bone mineral density in postmenopausal women with established osteoporosis. Bone 2004; 35:312 – 9. Malabanan A, Veronikis IE, Holick MF. Refining vitamin D insufficiency. Lancet 1998;351:805 – 6. Kinyamu HK, Gallagher JC, Rafferty KA, Balhorn KE. Dietary calcium and vitamin D intake in elderly women: effect on serum parathyroid hormone and vitamin D metabolites. Am J Clin Nutr 1998;67:342 – 8. Seeman E. Pathogenesis of bone fragility in women and men. Lancet 2002;359:1841 – 50. Need AG, O’Loughlin PD, Morris HA, Horowitz M, Nordin BEC. The effects of age and other variables on serum parathyroid hormone in postmenopausal women attending an osteoporosis center. J Clin Endocr Metab 2004;89:1646 – 9. Barger-Lux M, Heaney RP. Effects of above average summer sun exposure on serum 25-hydroxyvitamin D and calcium absorption. J Clin Endocr Metab 2002;87:4952 – 6. Need AG, Horowitz M, O’Loughlin PD, Coates PS, Nordin BEC. What is the cause of the hyperparathyroidism in vitamin D deficiency? J Bone Miner Res 2005;20(Suppl 1):S390. Carlsson A, Lindquist B. Comparison of intestinal and skeletal effects of vitamin D in relation to dosage. Acta Physiol Scand 1955; 35:53 – 5. Omdahl J, Morris HA, May BK. Hydroxylase enzymes of the vitamin D pathway: expression, function and regulation. Annu Rev Nutr 2002; 22:139 – 66. Thomas MK, Lloyd-Jones DM, Thadhani RI, et al. Hypovitaminosis D in medical inpatients. N Engl J Med 1998;338:777 – 83. Scragg R, Sowers M, Bell C. Serum 25-hydroxyvitamin D, diabetes, and ethnicity in the third national health and nutrition examination survey. Diabetes Care 2004;27:2813 – 8. Jones G, Dwyer T, Hynes KL, Parameswaran V, Greenaway TM. Vitamin D insufficiency in adolescent males in southern Tasmania: prevalence, determinants, and relationship to bone turnover markers. Osteoporos Int 2005;16:636 – 41. Sahota O, Masud T, San P, Hosking DJ. Vitamin D insufficiency increases bone turnover markers and enhances bone loss at the hip in patients with established vertebral osteoporosis. Clin Endocrinol 1999; 51:217 – 21. Lips P, Duong T, Oleksik A, et al. A global study of vitamin D status and parathyroid function in postmenopausal women with osteoporosis: baseline data from the Multiple Outcomes of Raloxifene Evaluation Clinical trial. J Clin Endcor Metab 2001; 86:1212 – 21. Seibel MJ. Biochemical markers of bone turnover: Part I. Biochemistry and variability. Clin Biochem Rev 2005;26:97 – 122. Garnero P, Hausherr E, Chapuy MC, et al. Markers of bone resorption predict hip fracture in elderly women: the EPIDOS prospective study. J Bone Miner Res 1996;11:1531 – 8. Ross PD, Kress BC, Parson RE, Wasnich RD, Armour KA, Mizrahi IA. Serum bone alkaline phosphatase and calcaneus bone density predict fractures: a prospective study. Osteoporos Int 2000; 11:76 – 82. Riggs BL, Melton III LJ, O’Fallon WM. Drug therapy for vertebral fractures in osteoporosis: evidence that decreases in bone turnover and increases in bone mass both determine antifracture efficacy. Bone 1996;18:197S – 210S. Sarkar S, Mitlak BH, Wong M, Stock JL, Black DM, Harper KD. Relationships between bone mineral density and incident

52

A.G. Need / Clinica Chimica Acta 368 (2006) 48 – 52

vertebral fractures with raloxifene therapy. J Bone Miner Res 2002;17:1 – 10. [35] Eastell R, Barton I, Hannon RA, Chines A, Garnero P, Delmas PD. Relationship of early changes in bone resorption to the reduction in fracture risk with risedronate. J Bone Miner Res 2003; 18:1051 – 6.

[36] Parfitt AM. High bone turnover is intrinsically harmful: two paths to a similar conclusion. J Bone Miner Res 2002;17:1558 – 9. [37] Boivin GY, Chevassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone density by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone 2000; 27:687 – 94.