0021-972X/90/7002-0467$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 70, No. 2 Printed in U.S.A.

Multiple Osteocalcin Fragments in Human Urine and Serum as Detected by a Midmolecule Osteocalcin Radioimmunoassay* ARCH K. TAYLOR, SUSAN LINKHART, SUBBURAMAN MOHAN, ROBERT A. CHRISTENSON, FREDERICK R. SINGER, AND DAVID J. BAYLINK Jerry Pettis Veterans Administration Hospital (A.K.T., S.L., D.J.B.), Departments of Biochemistry (S.M., D.J.B.), Physiology (S.M.), Pediatrics (R.A.C), and Medicine (S.M., D.J.B.), Loma Linda University, Loma Linda, California 92357; and University of Southern California School of Medicine (F.R.S.), Los Angeles, California 90033

ABSTRACT. Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating levels of human osteocalcin (OC) correlate with the skeletal isoenzyme of alkaline phosphatase and can be used as an index of bone formation. A disadvantage of using serum OC as a marker of formation is its diurnal variation. To address this problem we carried out our studies to determine the usefulness of urine in the assessment of bone turnover. Using a midmolecule specific human OC RIA, we were able to detect OC in urine of normal adults (42 MgeQ/g creatinine), normal children (849 Mgeq/g creatinine), and Paget's disease patients (613 Mgeq/g creatinine). Immunoreactive fragments of OC in human urine and human serum were separated by high pressure liquid chromatography. Multiple fragments were found in normal adult urine that were not detected in

normal adult serum. Uremic and Paget's disease sera contain several immunoreactive forms of OC, other than the intact molecule, not found in normal adult serum. Additionally, both Paget's disease sera and urine contained a specific peak of immunoreactive material, eluting at 25% acetonitrile, that was not found in any other serum or urine tested. Urinary OC (uOC) correlated with both skeletal alkaline phosphatase (r = 0.91) and serum OC (r = 0.83), indices of skeletal formation. While uOC has a diurnal variation similar to that of serum OC, determinations of 24-h uOC give integrated values of daily bone turnover rates. Z-Score analysis indicates that uOC (z = 14.04) is better able to distinguish between normal children with high bone turnover and normal adults than either skeletal alkaline phosphatase (z = 8.87) or serum OC (z = 9.01). {J Clin Endocrinol Metab 70: 467, 1990)

O

dropping during the late morning and early afternoon (10), it is important that serum samples be collected at the same time each day when various patient samples are to be compared. This is especially critical if serial measurements are made to follow bone formation within a single patient. The serum half-life of OC is very short due to its small size and rapid clearance from circulation by glomerular filtration in the kidneys (4). Although the presence of GLA can be detected in the urine, its measurements cannot be used as a specific marker for bone formation because the amino acid is also found in other proteins (5). It has been estimated that 80% of the total urinary level of GLA is from turnover of the vitamin K-dependent blood-clotting factors and not from OC clearance. On the other hand, a determination of the 24-h clearance of OC in the urine would allow an integrated assessment of OC formation during the day and would eliminate the potential problems evolving from its diurnal variation. Our aims in this study were to examine the possibility of measuring OC in urine (uOC) using our midmolecule

STEOCALCIN (OC; also called bone gla protein or BGP) is a 49-amino acid vitamin K-dependent calcium-binding protein that is found in bone matrix and circulating in serum (1-4). The presence of 7-carboxyglutamic acid (GLA) residues confers a high affinity for hydroxyapatite to the protein (5, 6). It is produced by osteoblasts and can constitute up to 20% of the noncollagenous proteins found in bovine bone matrix (2) and 0.03% of those found in human bone matrix (7, 8). Evidence indicates that circulating levels of OC correlate with skeletal alkaline phosphatase and can be used as an index of bone formation (7, 9). However, because serum OC (sOC) undergoes a large diurnal variation, peaking during the late night and early morning and Received April 24, 1989. Address all correspondence and requests for reprints to: Arch K. Taylor, Ph.D., Research Service (151), Jerry L. Pettis Veterans Administration Hospital, 11201 Benton Street, Loma Linda, California 92357. * This work was supported by NIH Grant AR-31062 (to S.M.), V.A. Research Support (to A.K.T. and S.L.), V.A. MI Award (to D.J.B.), NIH General Clinical Research Center Grant RR-43 (to F.S.), and the National Institute of Biogerontology (Madison, WI). 467

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 September 2015. at 00:12 For personal use only. No other uses without permission. . All rights reserved.

JCE & M • 1990 Vol70-No2

TAYLOR ET AL.

468

human OC RIA (7) and a commercially available bovine assay and to evaluate the usefulness of urine in the assessment of OC in bone metabolism.

Materials and Methods Subjects studied Urine and sera were collected between 0800-1100 h from 30 normal adults, 30 Paget's disease patients, and 6 normal children between the ages of 4-10 yr.

been shown to react with a midmolecule epitope of the OC molecule and is capable of detecting fragments of hOC. The bovine OC RIA was performed as described by the manufacturer (BTI). This assay is a rabbit antibovine OC assay that shows cross-reactivity with human OC. The assay is described by the manufacturer as a whole molecule assay. Urinary creatinine was determined using a commercially available creatinine kit (Sigma, St. Louis, MO).

Results Recovery of human OC during extraction

Sample preparation Urine samples were extracted using Baker-10 SPE C-18 columns. The columns were primed following the manufacturers instructions. One milliliter of urine was loaded onto each column, and the columns were washed with 5 mL 0.1% trifluoroacetic acid (TFA) in water to desalt the urine, removing various materials that interfere with the RIA for human OC. The OC was eluted from the columns with 5 mL 50% acetonitrile (AcN) in 0.1% TFA-water. The samples were evaporated to dry ness using a Speed-Vac concentrator (Savant Instruments, Inc., Hicksville, NY). The dried samples were redissolved in 1 mL of 0.1 M NH4HCO3 and assayed using our midmolecule human OC RIA (7). Serum samples from the same patients were assayed at the same time as urine samples. Recovery of OC by this method was determined by two methods. First, known amounts of iodinated human OC were added to urine before extraction, and the isolated labeled human OC was counted. Second, known amounts of intact human OC were added to urine before extraction and recovery of immunoreactive hOC was determined by RIA. Additionally, intact OC in 0.1 M NH4HCO3 buffer was extracted in a manner similar to that used to extract urine to determine if the existence of fragmented OC was an artifact of the extraction method. OC fragments

uOC fragments were separated on a high pressure liquid chromatography (HPLC) C-4 reverse phase column (Bio-Rad, Richmond, CA) using a 15-45% AcN gradient in 150 min. Twominute fractions were collected, evaporated to dryness, reconstituted with 1 mL 0.1 M NH4HCO3, and assayed using our assay and a commercially available bovine OC RIA (BTI, Stoughton, MA). Serum samples were subjected to Sephacryl S-200 gel filtration in 0.1 M NH4HCO3 to remove large mol wt proteins. Fractions containing immunoreactive OC, as measured by either our human OC RIA or the commercial bovine OC RIA, were pooled, lyophilized, reconstituted with 0.1% TFA, subjected to HPLC reverse phase separation, and assayed as described above. Serum and urine assays The skeletal isoenzyme of alkaline phosphatase (sAlP) was determined as previously described (11). The human OC RIA was performed as previously described (7). This assay is a guinea pig antihuman OC assay that has

Extraction of urine spiked with known amounts of iodinated human OC recovered greater than 95% of the labeled material (data not shown), while extraction of urine spiked with intact human OC recovered greater than 94% of the immunoreactive human OC (see Table 1). Although urine contains large amounts of immunoreactive OC and does not need to be concentrated, this extraction is necessary to desalt the urine to remove material from the urine that interfered with our human OC RIA. Intact OC that had been extracted with the C18 columns and subjected to HPLC chromatography remained intact (data not shown), indicating that the existence of immunoreactive fragments in urine and the lack of intact OC in urine were not artifacts caused by the desalting step. HPLC fractionation of normal urine and serum All normal adults urine samples (n = 5) contained immunoreactive fragments of OC, none of which eluted after 80 min (31% AcN; Fig. 1). A similar pattern was found in the urine of normal children (data not shown), even though the total amount of OC in this urine was significantly higher than that in normal adults (Table 2). Although the majority of sOC eluted as intact OC (35.4% AcN; Fig. 1, arrow), other fragments were also detected with the human OC assay. However, all of these serum fragments eluted after 80 min, and none of them corresponded with those in urine fractions. The commercial bovine RIA, which is described as an intact OC TABLE 1. Recovery of OC from urine during extraction Sample no. 1 1 1 1 2 2 2 2

Added human OC

Expected

Actual

(flg/h)

(Mg/L)

(Mg/L)

5.89° 6.89 10.89 15.89 16.60° 17.60 21.60 26.60

5.89 6.92 10.57 15.02 16.60 17.52 20.98 25.93

1.0 5.0 10.0

1.0 5.0 10.0

% Recovered 100.0 100.4 97.1 94.5 100.0 99.5 97.1 97.5

" Expected values for unspiked samples are the mean of five determinations.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 September 2015. at 00:12 For personal use only. No other uses without permission. . All rights reserved.

OC FRAGMENTS IN URINE AND SERUM

20

40

60

80

100

120

140

160

MINUTES

20

40

469

60

80

100

120

140

160

MINUTES

FlG. 2. Immunoreactive human OC fragments in renal failure serum. Serum was fractionated by reverse phase chromatography, and fractions were tested for immunoreactive material as described in the text. The arrow indicates the location of the intact human OC.

immunoreactive fragments not detected in normal adult human serum. Some of these extra peaks correspond to those in the urine of normal adults, giving further evidence that the immunoreactive material found in urine is the result of renal clearance of serum OC. 40

60

80

100

120

140

160

MINUTES

FlG. 1. Immunoreactive human OC fragments in normal adult urine (a) and normal adult serum (b). Urine and serum were subjected to reverse phase HPLC fractionation, and fractions were measured for immunoreactive material as described in the text. The arrow indicates where intact human OC elutes. TABLE 2. Urine OC in various metabolic conditions

Adults (n = 30)

uOC

sOC

(jugeq/g creat)

(Mg/L)

Skeletal A1P (/ukat/L)

41.99 33.15

7.12 2.56

0.48 0.16

Children (n = 6)

848.7 139.1

29.8 4.81

P (vs. adults) z (us. adults)

Multiple osteocalcin fragments in human urine and serum as detected by a midmolecule osteocalcin radioimmunoassay.

Reliable markers of bone formation are essential to the investigation of metabolic bone disorders. In this regard, evidence indicates that circulating...
664KB Sizes 0 Downloads 0 Views