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Serum CrossLaps compared with other markers of bone turnover in severely malnourished children before and after refeeding.

In growing children, bone turnover is generally high. There are several biochemical markers of bone formation and resorption that have been validated in adults by bone histomorphometry and calcium kinetics. Markers of bone formation are all measured in plasma and include bone-specific alkaline phosphatase (ALP), osteocalcin, and the C-terminal propeptide of type I collagen (PICP). Their use as markers of growth and bone formation in children is now well established (1, 2). However, most markers of bone resorption are measured in urine, including total pyridinoline (Pyd) and the more bone-specific deoxypyridinoline (Dpd). The main problems associated with the urinary markers in pediatric practice are high within-individual biological variation and the need for either timed collections (frequently impossible in children) or, for random urine samples, expressing results as a ratio to creatinine. Creatinine is itself subject to biological variation and changes with muscle mass. Dpd:creatinine excretion in children is highly variable (3) and hence relatively insensitive to therapeutic interventions.

Until recently, only one commercially available marker of bone resorption could be measured in plasma, the cross-linked telopeptide of type I collagen (ICTP). Plasma ICTP appears to be less sensitive than some of the other markers to changes in bone resorption in certain clinical situations (e.g., bisphosphonate treatment), perhaps because of metabolism by osteoclastic cathepsin K (4). There is now a new marker for bone resorption that can be measured in serum or plasma, serum CrossLaps[TM] (5), for which we have recently reported pediatric reference data (6). The CrossLaps assay is specific for the amino acid sequence EKAHD-[beta]-GGR where the aspartic acid residue (D) is [beta]-isomerized. It detects only cross-linked degradation products of C-terminal telopeptides of type I collagen and is therefore specific for bone resorption. It has been clinically evaluated in adults in whom changes in CrossLaps were inversely correlated with changes in bone mineral density in postmenopausal women treated with bisphosphonates and hormone replacement therapy (7). CrossLaps has not yet been clinically evaluated in children.

Markers of bone formation and resorption usually change in parallel, for example, during childhood growth (longitudinal bone growth and modeling) (1, 2) and during bone remodeling when the processes of bone resorption and formation are usually tightly coupled. However, it has been reported that in malnourished adolescents and adults with anorexia nervosa, there is uncoupling of bone remodeling, with a relative excess of resorption over formation (8, 9). In a recent study of severely malnourished children, we have similarly reported a marked discrepancy between low markers of bone formation and high concentrations of ICTP at presentation, consistent with such uncoupling (10). The latter study provides an appropriate clinical context in which to evaluate serum CrossLaps by examining its response to severe malnourishment and refeeding in comparison with that of other markers of bone resorption and bone formation.

We measured serum CrossLaps in a representative subgroup of 10 patients (7 boys) from an earlier larger study of 141 severely malnourished children undergoing refeeding in Dhaka, Bangladesh (10). Full details of the patients and the refeeding program are given elsewhere (10). The median age of our subgroup was 13.0 months (range, 7-36 months). For the original study, consent was obtained in Bengali from the primary caregiver of each child, and the study was approved by the Paediatrics and Reproductive Medicine Research Ethics SubCommittee of Lothian Health (Edinburgh), the Ethics Committee of the International Centre for Diarrhoeal Disease Research (Bangladesh), and the Dhaka Shishu Children's Hospital.

We analyzed plasma and urine (stored at -20 [degrees]C until analysis) from paired blood and urine samples collected at presentation (day 1) and after 30 days of refeeding. Samples were assayed in duplicate where possible. We measured serum CrossLaps with a sandwich ELISA assay (Osteometer Biotech), as described previously (5, 6). Within- and between-run CVs were 9% and 12% at 223 ng/L (manufacturer's control), 9% and 10% at 463 ng/L (pooled plasma from children with normal physiologic bone turnover), and 8% and 14% at 786 ng/L (pooled plasma from children with high physiologic bone turnover), respectively. ICTP, bone ALP, and PICP had already been measured on the same samples, as described previously (10). ICTP and PICP had been measured by RIA (Orion Diagnostica). Between-run CVs were 6% and 8% at 13.2 and 27.0 [micro]g/L for ICTP and 5%, 5%, and 5% at 117, 203, and 374 [micro]g/L for PICP. Bone ALP had been measured by an ELISA (Alkphase-B; Metra Biosystems Europe). Between-run CVs were 7%, 5%, and 7% at 15.1, 70.5, and 84.2 U/L, respectively. We measured urinary Pyd and Dpd by HPLC, using a modification (11) of the method of Pratt et al. (12). Results were expressed as a ratio to creatinine measured on the same sample. Between-run CVs were 6% at 315 [micro]mol/mol creatinine for Pyd and 9% at 65.8 [micro]mol/mol creatinine for Dpd.

It was deemed impractical to try to compare biochemical data in the study group of malnourished Bangladeshi children with reference values from well-nourished Bangladeshi children both for ethical reasons and because such children may be difficult to identify. Instead, we compared results in the study group with our own median and interquartile reference values derived from well-nourished, age-matched (6-36 months) Scottish children who presented mainly to the casualty and outpatient departments of our hospital with various minor conditions that were considered not to have an effect on bone turnover or growth (6, 13, 14).

Data were expressed as medians and interquartile intervals. Day 1 and 30 results were compared by Wilcoxon signed-ranks tests. Spearman rank correlations with correction for ties were used to compare variables at each time point. P <0.05 was regarded as significant.

At presentation, the median weight-for-height SD score was -3.6 (range, -4.3 to -2.3) and the median height-for-age SD score was -3.6 (range, -6.2 to -1.9). After 90 days of refeeding, the median weight-for-height SD score improved to -1.3 (range, -2.0 to 0.0) and the median height-for-age SD score increased slightly to -3.2 (range, -6.1 to -1.3). Markers of bone formation were low (bone ALP) or low-normal (PICP) at presentation compared with the Scottish reference group and increased markedly in all patients in response to refeeding (P = 0.002; Table 1). Both plasma markers of bone resorption, CrossLaps and ICTP, were very high in all patients at presentation and decreased on refeeding in all patients (P = 0.002; Table 1 and Fig. 1, A and B). The urinary markers of bone resorption, Pyd and Dpd, were very variable (Table 1 and Fig. 1, C and D). Pyd largely overlapped the Scottish reference group and increased further on refeeding in 8 of 10 patients (P = 0.03). Dpd also largely overlapped the Scottish reference group and showed no consistent change on refeeding.

The only significant correlations among markers were between serum CrossLaps and ICTP on day 1 ([r.sub.s] = 0.75; P = 0.01) and day 30 ([r.sub.s] = 0.94; P <0.0001), between Pyd and Dpd on day 30 ([r.sub.s] = 0.65; P = 0.04), and between bone ALP and PICP on day 30 ([r.sub.s] = 0.71; P = 0.02).

In this subgroup of malnourished children, therefore, as in the main study (10), markers of bone formation (bone ALP and PICP) were low or low-normal at presentation and increased after refeeding. Conversely, as in the main study (10), ICTP was initially high compared with age matched Scottish children and decreased on refeeding, Like ICTP, serum CrossLaps was high in all 10 malnourished children and decreased on refeeding in every child. In contrast, urinary excretion of Pyd and Dpd overlapped Scottish reference values at presentation and showed variable responses to refeeding. This discrepancy may have been partly attributable to changes in renal handling of the urinary markers. Concomitant changes in muscle turnover might also have contributed to altered creatinine production and excretion, thereby affecting results expressed as a ratio to creatinine. Timed collections in such young children are not a viable alternative. We conclude that Pyd and Dpd are poor markers of bone resorption in this clinical situation.


Serum CrossLaps was highly correlated with ICTP at presentation and, to an even greater extent, after refeeding. By contrast, CrossLaps and ICTP showed no significant correlations with either (a) the urinary markers of bone resorption, Pyd and Dpd, or (b) markers of bone formation, even during catch-up weight gain when collagen synthesis, as reflected in PICP, was apparently supranormal. This suggests that CrossLaps and ICTP are not merely markers of overall bone collagen turnover. Our contrasting observations that both ICTP and CrossLaps were high at presentation and decreased during refeeding, whereas markers of bone formation were low at presentation and increased during refeeding, confirms that bone formation and resorption are uncoupled in severely malnourished children, as described for adolescents and young adults with anorexia nervosa (8, 9). We conclude that serum CrossLaps is a valid marker of bone resorption in children in this clinical situation.

This study was funded by a Small Project Grant from Lothian University Hospitals NHS Trust. We are grateful to Dr. Conor Doherty for permission to use the stored samples from a previous collaborative study for this evaluation (10).


(1.) Crofton PM, Kelnar CJH. Bone and collagen markers in paediatric practice [Review]. Int J Clin Pract 1998;52:557-65.

(2.) Crofton PM. New aspects of biochemical markers of bone turnover in paediatrics [Review]. In: Schonau E, Matkovic V, eds. Prevention of osteoporosis-a paediatric task? Proceedings of the 2nd International Workshop on Paediatric Osteology, Cologne, 1997. Amsterdam: Elsevier, 1998:3-14.

(3.) Shaw NJ, Dutton J, Fraser WD, Smith CS. Urinary pyridinoline and deoxypyridinoline excretion in children. Clin Endocrinol 1995;42:607-12.

(4.) Sassi M-L, Eriksen H, Risteli L, Niemi S, Mansell J, Gowen M, et al. Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 2000;26:367-73.

(5.) Rosenquist C, Fledelius C, Christgau S, Pedersen BJ, Bonde M, Qvist P, et al. Serum CrossLaps one step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from C-terminal telopeptides of type I collagen. Clin Chem 1998;44:2281-9.

(6.) Crofton PM, Evans N, Taylor MR, Holland CV. Serum CrossLaps: pediatric reference intervals from birth to 19 years of age. Clin Chem 2002;48: 671-3.

(7.) Christgau S, Rosenquist C, Alexandersen P, Hannover Bjarnason N, Ravn P, Fledelius C, et al. Clinical evaluation of the serum CrossLaps one step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen C-telopeptides. Clin Chem 1998;44: 2290-300.

(8.) Stefanis N, Mackintosh C, Abraha HD, Treasure J, Moniz C. Dissociation of bone turnover in anorexia nervosa. Ann Clin Biochem 1998;35:709-19.

(9.) Audi L, Vargas DM, Gussinye M, Yeste D, Marti G, Carrascosa A. Clinical and biochemical determinants of bone metabolism and bone mass in adolescent female patients with anorexia nervosa. Pediatr Res 2002;51:497-504.

(10.) Doherty CP, Crofton PM, Sarkar MAK, Shakur MS, Wade JC, Kelnar CJH, et al. Malnutrition, zinc supplementation and catch-up growth: changes in insulin-like growth factor I, its binding proteins, bone formation and collagen turnover. Clin Endocrinol 2002;57:391-9.

(11.) Crofton PM, Ahmed SF, Wade JC, Stephen R, Elmlinger MW, Ranke MB, et al. Effects of intensive chemotherapy on bone and collagen turnover and the growth hormone axis in children with acute lymphoblastic leukemia. J Clin Endocrinol Metab 1998;83:3121-9.

(12.) Pratt DA, Daniloff Y, Duncan A, Robins SP. Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography. Anal Biochem 1992;207:168-75.

(13.) Crofton PM. Wheat-germ lectin affinity electrophoresis for alkaline phosphatase isoforms in children: age-dependent reference ranges and changes in liver and bone disease. Clin Chem 1992;38:663-70.

(14.) Crofton PM, Ahmed SF, Wade JC, Elmlinger MW, Ranke MB, Kelnar CJH, et al. Bone turnover and growth during and after continuing chemotherapy in children with acute lymphoblastic leukaemia. Pediatr Res 2000;48:490-6.

Patricia M. Crofton, [1,2] * Nancy Evans, [2] and Rhona Stephen [2] ([1] Department of Paediatric Biochemistry, Royal Hospital for Sick Children, Sciennes Rd., Edinburgh EH9 1LF, United Kingdom, [2] Section of Child Life and Health, Department of Reproductive and Developmental Sciences, University of Edinburgh, Sylvan Place, Edinburgh EH9 1UW, United Kingdom; * address correspondence to this author at: Department of Paediatric Biochemistry, Royal Hospital for Sick Children, Sciennes Rd., Edinburgh EH9 1LF, United Kingdom; fax 44-131-536-0410, e-mail
Table 1. Changes in bone markers during the first month of
refeeding in 10 severely malnourished infants.

 Median (interquartile range)

Marker (a) Day 1 Day 30 group (b)

Bone resorption markers
 CrossLaps, ng/L 2085 841 (c) 274
 (1675-2899) (594-1042) (244-374)
 ICTP, [micro]g/L 59 19 (c) 15
 (49-83) (15-24) (13-18)
 Pyd, [micro]mol/mol 508 843 (d) 310
 creatinine (292-662) (612-953) (213-407)
 Dpd, [micro]mol/mol 70 93 65
 creatinine (45-84) (64-128) (42-87)
Bone formation markers
 Bone ALP, U/L 34 66 (c) 64
 (21-40) (48-72) (54-76)
 PICP, [micro]g/L 388 961 (c) 488
 (309-472) (877-1093) (382-609)

(a) CrossLaps, ICTP, bone ALP, and PICP were measured in plasma,
and Pyd and Dpd were measured in urine.

(b) Age-matched (6-36 months) Scottish children with no evidence
of disorders likely to affect bone turnover or growth: n = 11 for
CrossLaps; n = 14 for Pyd/Dpd; n = 30 for ICTP and PICP; n = 93
for bone ALP.

(c,d) Compared with day 1 values (Wilcoxon signed-ranks):
(c) P <0.01; (d) P <0.05.
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Title Annotation:Technical Briefs
Author:Crofton, Patricia M.; Evans, Nancy; Stephen, Rhona
Publication:Clinical Chemistry
Date:Jan 1, 2003
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