Urinary Osteocalcin as a Marker of Bone Metabolism Kaisa K. Ivaska, 1* Sanna-Maria Ka ¨ ko ¨ nen, 1 Paul Gerdhem, 2 Karl J. Obrant, 2 Kim Pettersson, 3 and H. Kalervo Va ¨a ¨ na ¨ nen 1 Background: Osteocalcin (OC) is produced by osteoblasts during bone formation, and circulating OC has been used in clinical investigations as a marker of bone metabolism. OC is excreted into urine by glomerular filtration and can be found in urine as midmolecule fragments. Methods: We developed and evaluated three immuno- assays (U-MidOC, U-LongOC, and U-TotalOC) for the detection of various molecular forms of urine OC (U- OC). We evaluated the association of U-OC with other markers of bone turnover and with bone mass in 1044 elderly women and studied seasonal and circadian vari- ation of U-OC. Results: U-OC correlated with other bone turnover mark- ers [Spearman correlation (r), 0.30 – 0.57; P <0.0001], dem- onstrating the association between U-OC and skeletal metabolism. There was also a significant association be- tween bone metabolism assessed by U-OC quartiles and bone mass assessed by total body bone mineral content (P <0.0001). The seasonal effects appeared to be rather small, but we observed a significant circadian rhythm similar to the one reported for serum OC with high values in the morning and low values in the afternoon. Conclusions: The three immunoassays had unique specificities toward different naturally occurring U-OC fragments. U-OC concentrations measured with any of these assays correlated with bone turnover rates as- sessed by conventional serum markers of bone metab- olism. The measurement of OC in urine samples could be used as an index of bone turnover in monitoring bone metabolism. © 2005 American Association for Clinical Chemistry Bone turnover markers provide dynamic and rapid mea- sures of total body skeletal metabolism and may be clinically useful tools, e.g., in monitoring osteoporosis therapy. At least some markers appear promising for determinations of fracture risk, rate of bone loss, or disease severity [for reviews, see Refs. (1–3 )]. Osteocalcin (OC) 4 is a small, bone-specific protein (M r 5800) pro- duced by osteoblastic cells during bone formation (4–6). A fraction of OC enters the blood, where it can be detected, and circulating OC has been widely measured to assess bone turnover (7, 8). In addition to the newly synthesized OC derived from osteoblasts, the circulating OC pool probably also includes molecules derived from the resorption process when OC embedded in the bone matrix is released (9, 10). The main route of circulating OC catabolism is renal filtration and degradation (11 ), and immunoreactive OC is present in urine (12 ). Urine OC (U-OC) is a heteroge- neous pool of various OC fragments (13–15 ) that reflect potentially diverse degradation cascades and that consist mainly of the middle portion of the molecule truncated at both the amino and carboxy termini. Intact, unfragmented OC has not been found in urine. The introduction of serum OC (S-OC) assays into routine clinical practice has been limited by the presence of multiple forms of OC in the circulation and differences among assays in the ability to detect these forms (16 ). It is unclear whether OC fragments are generated by proteol- ysis of intact OC in blood, are formed during biosynthesis, or whether some fragments are derived directly from bone resorption. OC molecules of resorptive origin would be of clinical interest, but the circulating resorptive forms may be masked in serum samples by de novo-synthesized 1 Institute of Biomedicine, Department of Anatomy, and 3 Department of Biotechnology, University of Turku, Turku, Finland. 2 Department of Orthopaedics, Malmo ¨ University Hospital, Malmo ¨, Swe- den. *Address correspondence to this author at: Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland. Fax 358-2-333-7352; e-mail kaisa.ivaska@utu.fi. Received October 13, 2004; accepted December 14, 2004. Previously published online at DOI: 10.1373/clinchem.2004.043901 4 Nonstandard abbreviations: OC, osteocalcin; U-OC, urine osteocalcin; S-OC, serum osteocalcin; Mab, monoclonal antibody; Gla, -carboxyglutamic acid; MALDI-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; BMC, bone mineral content; aBMD, areal bone mineral density; S-BoneALP, serum bone-specific alkaline phosphatase; CTX and NTx, C- and N-terminal cross-linked telopeptides of type I collagen; and TRACP5b, tartrate-resistant acid phosphatase isoenzyme 5b. Clinical Chemistry 51:3 618 – 628 (2005) Endocrinology and Metabolism 618