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J Pediatr 1983;102:947-50. Osteocalcin Concentrations in Plasma Prepared with Different Anticoagulants. MIchael J. Power,1 Bernadette ..... CW, Callahan PX.
chromatography/radioimmunoassay

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CLIN. CHEM. 37/2, 281-284 (1991)

Osteocalcin Concentrations in Plasma Prepared with Different Anticoagulants MIchael J. Power,1 Bernadette

O’Dwyer’

EugeneBreen,2 and PatrickF. Fottreil”3

We investigated the effects on plasma osteocalcin concentrations of different anticoagulants used to collect the blood samples. Plasma osteocalcin concentrations measured by enzyme immunoassay and radioimmunoassay are influenced by the nature of the anticoagulants used. The most significant difference between concentrations found in plasma and serum was seen with oxalate/fluonde anticoagulant, which reduced osteocalcin concentrations to 37.3% of serum values. This is probably related to increased hemolysis with this anticoagulant compared

with osteocalcin concentrations in plasma prepared with other anticoagulants. Samples prepared with sodium cit-

rate (0.105 mol/L) or lithium hepann gave values 92.4% and 83.6% of those obtained with matched serum samples. Osteocalcin concentrations were relatively stable in plasma and serum at -20 #{176}C for two freeze/thaw cycles. In blood from 100 patients there was a good correlation between osteocalcin concentrations in serum and plasma (lithium heparin) (r2 = 0.831); the slope and intercept (±SE) were 0.924 ± 0.04 and 4.92 ± 1.25 pg/L, respectively. However, in 10 patients, serum osteocalcin concentrations were two- to threefold higher than those in matched plasma samples. Additional Keyphrasee: calcium-binding protein bone gla protein metabolic bone disease assay enzyme immunoassay .

osteoporosis radioimmuno-

Osteocalcin is a bone-specific protein consisting of 49 amino acids, three of which are ‘y.carboxyglutamic acid (1). Synthesized by osteoblasts, osteocalcin enters the blood (2-4), where its concentration in plasma reflects the rate of bone formation rather than the rate of bone resorption (5). Measurements of plasma osteocalcin are used in various clinical situations, including metabolic bone diseases, hyperthyroidism, liver cirrhosis, renal disorders, and diseases related to excess glucocorticoids (6-12). Among the published immunoassays of osteocalcm (13-17) is one from this laboratory, a relatively rapid enzymoimmunoassay (EIA) involving use of a monocloDepartments of’ Biochemistry, icine, University College Hospital,

University College, and2 MedGalway, Ireland. 3Addresacorrespondence to this author. Received September 7, 1990; accepted December 12, 1990.

nal antibody (18). Published ranges of plasma osteocalcin concentrations in controls’ and patients’ samples vary widely (19). One reason for this is the use of various polyclonal antisera, which differ in the extent to which they may recognize some of the multiple forms of osteocalcin in the circulation. In addition to intact osteocalcin, other immunoreactive forms in plasma are a high-molecularmass form (100 kDa) (19, 20) and peptides of lower molecular mass (1000-1500 Da) (21-23). Here we show that anticoagulants used to prepare plasma samples influence the immunoreactivity of osteocalcin and may represent another reason for the wide range of published osteocalcin values.

MaterIals and Methods Subjects: Plasma and serum were prepared from the same blood samples from two groups of patients (A and B), selected without conscious bias in the outpatient department of University College Hospital. Group A consisted of 20 women, ages 30-60 years, who had no clinical history or symptoms of bone-related disorders or conditions known to influence bone metabolism; one patient with anorexia nervosa was under investigation for osteoporosis. Samples from these subjectswere used for the studies described in Tables 1-3. Group B consisted of 100 subjects, both men and women: 40, ages 18-38 years, had no clinical history or symptoms of bone-related disorders; 60, ages 50-90 years, included

patients with hyperparathyroidism, Paget’s disease, Osteoporosis,and renal disease. We used 36 samples from these 120 subjects, Groups A and B, selected without consciousbias, for the studies of the stability of osteocalcin. Serum and plasma: Immediately after collection, portions of each blood sample were put into tubes that contained various anticoagulants or no anticoagulant (for serum preparation). Samples were kept at 4#{176}C until serum and plasma were separated-usually after -24 h, but some samples were separated

after

3 h. The

following anticoagulants were used in Vacutainer Tubes (Becton-Dickinson, Meylan Cedex, France): EDTA (75 gIL), lithium heparin (14 300 USP units/L), potassium oxalate/sodium fluoride (10 and 12.5 mg, respectively, per 5-mL tube, i.e., 2.0 and 2.5 g/L), and CLINICALCHEMISTRY, Vol. 37, No. 2, 1991 281

sodium citrate (0.105 and 0.129 mol/L). Plasma and serum were separated by centriftigation at 1000 x g for 15 mm at 4#{176}C. Samples were stored at -20 #{176}C and assayed for osteocalcin within two weeks. Osteocalcin assay: Osteocalcin was measured by radioimmunoassay (19) and solid-phase EIA on microtiter plates with use of an osteocalcin-horseradish peroxidase (EC 1.11.1.7) conjugate and a monoclonal antibody raised against bovine osteocalcin (18). Osteocalcin concentrations in serum or plasma (lithium heparin, or EDTA) as measured with these assays agreed well with published values. Working standard solutions were prepared from purified bovine osteocalcin; human plasma samples containing, per liter, 1, 11, or 21 g of endogenous human osteocalcin were used as controls. Results Effect of Different Anticoagulants Osteocalcin concentrations were lower in plasma than in serum and varied with the anticoagulant used in samples from six patients in Group A (Table 1). Plasma prepared in the presence of sodium citrate (0.105 molIL)

had the highest concentration of immunoreactive osteocalcin, whereas samples with potassium oxalate/sodium fluoride had the lowest (Table 1). Blood samples from these patients were kept at 4#{176}C for 24 h before plasma and serum were separated. A similar trend was obtained with serum and plasma from five other patients in Group A when blood was stored at room temperature, -15 #{176}C, for 24 h (Table 2, tube set 5). Osteocalcin concentrations in serum correlated well with those in plasma prepared with potassium oxalate/ sodium fluoride Er2 = 0.78; slope and intercept (±SE), 0.526 (0.10) and -1.86 (1.92) g/L, respectively] and between the latter and plasma prepared with lithium heparin [r2 = 0.84; slope and intercept, 1.79 (0.28) and 3.84 (2.42) g/L, respectively]. None of the anticoagulants interfered with the EIA of control samples of osteocalcin. The average (± SE) analytical recovery of purified bovine osteocalcin added to serum and plasma samples prepared with the different anticoagulants was as follows: lithium heparin, 102 (6.6)%; EDTA, 116 Table 1. Influence of Antlcoagulants on Osteocalcin ConcentratIons In Plasma Osteocalcin concn, Anticoagulant

Serum(none) Oxalate/fluoride Sodiumcitrate 0.129 mol/L 0.105 mol/L EDTA, 75 gIL

Lithiumhepann

Mean

SEN

8.95 3.3

0.4 0.3

6.3 8.4 5.8 7.5

0.2 0.3 0.1 0.1

% of serum value 100

37.3 70.4 94.2

65.1 83.6

Blood samples from six patients in Group A were stored at 4#{176}C for 24 h without aritlcoagulants (serum) and with the above anticoagulants (plasma). Osteocalcin was measured by EIA after storage at -20#{176}C for about two weeks.

282 CLINICAL CHEMISTRY, Vol. 37, No. 2, 1991

(18.5)%; sodium citrate (0.105 mol/L), 86 (10.4)%; and oxalate/fluoride, 93.0 (18)% (n = 4). The stability of the osteocalcin, as measured by EIA, was similar in the presence of the various anticoagulants in the six di!ferent samples. The recovery of osteocalcin in the different samples after 24 h at 4#{176}C was higher (by 12%) in the lithium heparin samples than in plasma samples prepared with the other anticoagulants, but this was not statistically significant. When a hemolyzed plasma sample was added to purified osteocalcin and analyzed for osteocalcinby EIA

within 1 h, the osteocalcin concentration decreased by 50%. However, assay of the same samples by RIA overestimated osteocalcin values by 100-400%. This effect was not due to the presenceof hemoglobin because adding pure bovine hemoglobin (Sigma Chemical Co., St. Louis, MO; 12.5 zg/L) to solutions of osteocalcin standards gave no change in osteocalcin concentrations when measured by EIA or RIA. Durationof Contactwith Anticoagulant Immunoreactive osteocalcin concentrations were compared in serum and plasma (prepared with lithium heparin and potassium oxalate/sodium fluoride) in blood samples from five patients in Group A. Samples were separated from erythrocytes 3 h after taking the blood sample, and one aliquot was frozen at -20 #{176}C. The remaining aliquots were allowed to stand at room temperature (15 #{176}C) for a further 9, 21, and 45 h before storage at -20 #{176}C. The osteocalcin concentrations in these samples (Table 2, tube sets 1-4) decreased with time, and the trend was similar to the results in Table 3, which had been incubated at 4#{176}C. Separation of serum and plasma samples from erythrocytes after 24 h rather than 3 h at room temperature (15 #{176}C) led to lower osteocalcin concentrations in five patients from Group A (Table 2, tube set 5). Osteocalcin concentrations also decreased during storage of blood samples (with lithium heparin as anticoagulant) from

nine other patients in Group A at 4#{176}C (Table 3) but not to the same extent as occurred at room temperature (Table 2, tube set 5). Stability of Osteocalcin

The immunoreactivity of osteocalcin in matched serum and plasma (lithium heparin) samples from 20 patients was measured before and after storage at -20 #{176}C about every two weeks for two months. The immunoreactivity did not decrease in either serum or plasma after one freeze/thaw cycle Er2 = 0.98; slope and intercept (±SE), 1.02 (0.03) and 0.12(0.85) g/L, respectively], and decreased only slightly after the second freeze/thaw cycle Er2 = 0.96; slope and intercept, 0.87 (0.05) and 0.149 (2.06) g/L, respectively]. But it decreased by about 40% after four freeze/thaw cycles, and the correlation lessened [r = 0.74; slope and intercept, 0.61 (0.09) and 0.77 (3.2) pg/L, respectively].

Table 2. Osteocalctn ConcentratIons In Serum and Plasma after IncubatIon wIth Cellular Material

Tubeset

AntIcoagulant

1

3

4

5

% of osteocalcln concn remaining after 3 h with cellular material

Incubation time,h

Mean

SEN

3 3

24.2 25.2

3.14 3.34

100.0

3 12 12

10.8

2.33

100.0

19.4

3.06

80.2

18.7

2.71

74.2

Serum Lithium heparin

2

Osteocalcin, iig/L

100.0

Oxalatelfluoride Serum Lithium hepann Oxalate/fluoride Serum Lithium heparin Oxalate/fluoride Serum Lithium heparin Oxalatelfluoride Serum

12

6,0

1.02

55.6

24 24 24 48 48 48 24

14.3 12.2 3.9 9.9 8.9 3.1

2.56

59.1

2.83

48.4

0.70 1.74 2.55

35.1 40.8 35.1

Lithium hepann

24

11.6 8.7

Oxalate/fluoride

24

1.8

0.64

33.3

2.27

47.9 34.6 16.6

2.62 0.43

Bloodsamples from five patients in Group A were incubated at room temperature(15 #{176}C). Tube sets 1-4 were separatedfrom the erythrocytesbefore storage at -20#{176}C. Tube set 5 was incubatedfor 24 h in the presenceof erythrocytes,separated,and stored at -20#{176}C. All sampleswere assayedfor osteocalcinby EIA after about two weeks of storage.

Table 3. Osteocalcin ConcentratIons in Plasma Separated from Blood Stored for Different TImes OsteocaIcln concn, pg/L Storage period, h

Mean

SEN

26.5 19.8 15.3

5.7 4.0 2.8

MedIan

of mean

concn after 2.5 h

22.0 100.0 15.8 74.7 13.3 57.7 a Blood from nine patients in Group A was stored at 4#{176}C in lithium hepann 2.5

24.0 48.0

for various periods before plasma was separated from blood cells.

Plasma/Serum Correlation Osteocalcin concentrations, measured by RIA with use of a polyclonal antibody (19), correlated well in matched serum and plasma (lithium heparin) samples from 100 patients from Group B [r2 = 0.831; slope and intercept (±SE), 0.924 (0.04) and 4.92 (1.25) g/L, respectively]. However, in 10 samples from this group, osteocalcin concentrations were two- to threefold higher in serum than in plasma. The latter samples were taken from both controls and patients with osteoporosisand renal impairment. They were of the same age range as the 90 individuals whose matched serum and plasma osteocalcin concentrations were similar. A similar result was obtained when the same samples were reassayed by EIA with use of a monoclonal antibody. The differences between serum and plasma persisted after one freeze/thaw cycle. DIscussion These studies showed that plasma osteocalcin values measured by EIA or RIA are influenced by the type of anticoagulants. Osteocalcin concentrations in plasma prepared with potassium oxalate/sodium fluoride were considerably lower than those in matched serum values

and decreased further when erythrocytes were allowed in contact with plasma and this anticoagulant for 24 h at 4#{176}C or at room temperature (Tables 1 and 2). This might be due to the hydrolysis of osteocalcin by peptide hydrolases released from erythrocytes in the presence of the anticoagulant, as suggested by recent studies (21). Further evidence for this is also the increased hemolysis seen with potassium oxalate/sodium fluoride compared with other anticoagulants. Peptide hydrolase activity apparently remains active after separation of erythrocytes because osteocalcin concentrations continue to decrease with storage at room temperature (Table 2). Concentrations decreased faster in the oxalate/fluoride

samples, which were also the most hemolyzed. Osteocalcin values were stable in plasma and serum stored at -20 #{176}C for at least one freeze/thaw cycle but decreased after two cycles. This is probably caused by peptide hycirolase or proteinase activity because pure osteocalcin standards withstand repeated freeze/thaw cycles and are unaffected by heating to 70#{176}C for 15 miii (24). Here no loss of immunoreactivity occurred with storage at 4 #{176}C for seven days. In contrast, fresh serum

and plasma samples containing either endogenous human osteocalcin or added bovine osteocalcin decreased by 25% after 24 h at 4#{176}C. After 24 h at 4#{176}C, serum and plasma samples, previously subjected to two freeze/thaw cycles within two months, lost only 10% of the osteocalcin that had been added to them. This is probably attributable to reduction in proteolytic activity as a

result of freezing and thawing. There was a good correlation between osteocalcin concentrations in 100 matched serum and plasma (lithium heparin) samples, in agreement with results from other studies (24, 25). However, in 10 patients, serum osteocalcin concentrations were two- to threefold those of plasma. This difference persisted when samples were CLINICAL CHEMISTRY, Vol. 37, No. 2, 1991 283

assayed by EIA and RIA and when a polyclonal or a monoclonal antibody was used. We have no explanation for this difference, but it indicates that one should interpret high serum osteocalcin values with caution. We previously noticed that when some serum samples are subjected to gel filtration on a Sephadex G-100 column, an immunoreactive osteocalcinpeak appears at the void volume as well as at the anticipated location (19). This peak was not present in all samples and may be a contributing factor to this difference between serum and plasma. This peak at the void volume has been reported by another group (20) but not by others (26). In contrast with a recent report (21), we detected no immunoreactive osteocalcin peaks at the molecular mass region of 1000-1500 Da. This indicates that our antibodies, both monoclonal and polyclonal, do not detect osteocalcin peptides that may be released as a result of the bone resorption process. We recommend that, for the measurement of osteocalcm concentration in serum or plasma, one should be mindful of the anticoagulant used and should not use oxalate/fluoride. Plasma or serum samples should be stored as soon as possible at -20 or -70 #{176}C, in small volumes. Samples should not be thawed more than twice before osteocalcin is measured. We are grateful

to Bioresearch

Ireland for support.

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