Effects of short-term low-dose heparin administration on biochemical parameters of bone turnover

Bone and Mineral, 22 (1993) 27-32

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© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0169-6009/93/$06.00 BAM 00580

Effects of short-term low-dose heparin administration on biochemical parameters of bone turnover H.E. v a n d e r Wiel a, P. L i p s a, P.C. H u i j g e n s b a n d J.C. N e t e l e n b o s a aDepartment of Endocrinology, Free University Hospital Amsterdam, The Netherlands bDepartment of Hematology, Free University Hospital Amsterdam, The Netherlands (Received 31 July 1992) (Accepted 10 February 1993)

Summary Heparin therapy may cause osteoporosis. The effects of short-term low-dose heparin are not known. We have studied the effects of short-term heparin administration, twice daily 5000 IU s.c., for 10 days on the biochemical parameters of bone turnover in six healthy male volunteers. No effects were observed on the urinary excretion of hydroxyproline and calcium. Serum levels of cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), a new marker of bone resorption, did not change significantly. A slight but significant decrease in serum alkaline phosphatase was observed. TmP/GFR increased significantly during heparin administration. In all volunteers a uniform increase in serum transaminases appeared which completely reversed after discontinuation of heparin administration. We conclude that short-term low-dose heparin administration does not change biochemical parameters of bone resorption, but has a small significant suppressing effect on serum alkaline phosphatase levels. Heparin administration resulted in a significant but transient increase of serum transaminase levels. Key words: Heparin; Osteoporosis; Hydroxyproline; Transaminase

Introduction Heparin is a popular drug used for prophylaxis and therapy of thrombosis. Heparin has several adverse effects: thrombopenia, thrombosis, and a frequently overlooked hepatotoxicity, which is usually reversible [1-3]. A side effect of long-term heparin administration is osteoporosis. However, less than 50 patients have been documented in the literature [4]. From the case reports and two prospective studies [5,6] it can be deduced that dosage and treatment duration are important. Osteoporosis has not been described when dosages of less than 10 000 IU/day are given or when the treatment period is less than 10 weeks. The true incidence of osteoporosis due Correspondence to: H.E. van der Wiel, Department of Endocrinology, Free University Hospital, PO Box 7057, 1007MB Amsterdam, The Netherlands.

28 to heparin treatment is not known. In most case reports osteoporosis is not welldefined. Patients were either pregnant women who did not have another choice of anticoagulant or, in the earlier studies, older patients with cardiovascular diseases I5,7]. In this study we prospectively evaluated the effects of short-term low-dose heparin administration on biochemical parameters of bone turnover in six healthy male volunteers.

Subjects and Methods Six healthy men aged 39 ± 8 years participated in the study. After obtaining basal values for each parameter described below they started with 5000 IU heparin subcutaneously every 12 h (Liquemin Roche, Hoffmann-Laroche BV Mijdrecht, the Netherlands), for 10 days. The participants injected the heparin themselves. During administration of heparin normal daily activities were performed. The protocol was approved by the ethical committee of the hospital, and all volunteers gave informed consent. Fasting blood samples were obtained for measurement of serum calcium, phosphate, albumin, creatinine, alkaline phosphatase, osteocalcin and serum levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) on day 0, 4 and 10 of drug administration. Fasting serum PTH (1-84), serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels were determined on day 0 and day 10. Fasting 2-h urine for the determination of calcium, phosphate, creatinine and hydroxyproline was sampled on day -4 (i.e. 4 days before commencement of heparin administration), 0, 2, 4, 7 and 10. Adverse effects were monitored by determination of serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT) and platelets on day 0, 4, 7, 10, and 3 weeks after stopping heparin administration. Calcium, phosphate, albumin, creatinine, alkaline phosphatase, SGOT, SGPT and platelets were determined using standard laboratory methods. Serum calcium was corrected for serum albumin using the following equation: corrected calcium = calcium + ( 4 0 - albumin (g/l)) x 0.02 mmol/l. Hydroxyproline was measured by high-performance liquid chromatography as described previously [8]. The within run coefficient of variation of the hydroxyproline/creatinine ratio in a 2-h fasting urine is 3.2% [8]. Serum samples for analysis of vitamin D metabolites were chromatographed by a Sep-Pak C 18 cartridge, and further purification was by gradient HPLC. Serum 25-hydroxyvitamin D concentrations were measured by competitive protein binding assay using rat serum as binding protein. The intra- and interassay coefficients of variation are 5 and 6%, respectively. The detection limit is 3 nmol/l. Serum 1,25-dihydroxyvitamin D concentrations were measured by competitive protein binding assay using chicken duodenal receptor as binding protein. The intra- and interassay coefficients of variation are 6 and 15%, respectively. The detection limit is 10 pmol/l. Serum intact human PTH (1-84) was measured by a two-step immunochemical method involving aminoterminal immunoextraction followed by a sensitive midregion aminoassay. The intra- and interassay coefficients of variation are 8.3 and 10.2%. The detection limit is 0.8 pmol/l [9]. Serum osteocalcin was analyzed by an RIA method using a kit from Incstar Corporation. Serum ICTP was analyzed with a RIA kit from Orion Diagnostica, Espoo, Finland [10].

2.30 (0.09) 1.12 (0.08) 52.2 (13) 2.85 (0.8) 4.5 (1.5) 104 (27) 60.5 (18) 3.0 15.3(6.5) 0.12(0.07) 1.02(0.12) 12 (4) 13 (9) 198 (9)

0.93 0.58 0.03 0.10 0.07 ND ND 0.52 0.70 0.47 0.011 0.0 0.0 0.69

ANOVA P value 2.3 (0.08) I. 15 (0.12) 48.4 (14) 2.58 (0.49) ND ND ND 2.9 16(4.9) 0.15(0.08) 1.19(0.17) 25 (1 l) 31 (24) 189 (9)

Day 4 mean (SD) -0.11,0.10 -0.08,0.16 -9.1,-0.07" -0.7,0.17 ND ND ND -0.5,0.3 -3,4 -0.04,0.1 0.08,0.24 @ 4,24* 0.8,36* -33,13

95% CI

2.29 (0.07) 1.12(0.13) 49.7 (13.6) 2.53 (0.7) 3.3 (1.1) 110.1 (17) 56.7 (14.5) 2.8 15.3(5.1) 0. ! 3(0.06) 1.17(0.21) 36 [9] 80 (22) 195 (13)

Day 10 mean (SD)

-0.07,0.04 -0.12,0.12 -4.2,-0.8" -0.65,0.02 -2.6,0.17 -34,46 -9.3,1.7 -0.5,0.10 -3.6,3.6 -0.05.0.06 -0.02,0.31 12,37 @ 45,89 @ -24,18

95% CI

Serum calcium is corrected for serum albumin. ND denotes not determined. Hp/Cr denotes hydroxyproline/creatinine ratio in fasting 2-h urine. Ca/Cr denotes calcium/creatinine ratio in fasting 2-h urine. *P < 0.05, @P < 0.01. ICTP is the carboxy-terminal telopeptide region of type I collagen, cross-linked by pyridinoline and is a serum marker for bone resorption. 95% CI denotes 95% confidence interval for the difference between value measured and basal value (t = 0). ANOVA denotes Analysis of Variance for repeated measurements.

Calcium (retool/I) Phosphate (mmol/l) A Ph. (units/l) Osteocalcin (ng/ml) PTH (pmoFl) 1,25(OH)2vit D (pmolA) 25(OH)vit D (nmol/l) ICTP O~g/1) Hp/Cr (#mol/mmol) Ca/Cr (mmol/mmol) TmP/GFR (mmol/I) SGOT (units/l) SGPT (units/l) Platelets (109/I)

Basal value mean(SD)

Serum and urinary values during short-term heparin administration

Table 1

30 The intra- and interassay coefficients of variation were 5 and 6%. ICTP is the carboxyterminal telopeptide region of type I collagen, cross-linked by pyridinoline and liberated during degradation of collagen type I. Type I collagen is the main collagen type found in mineralized bone. TmP/GFR was calculated using the nomogram of Bijvoet et al. [11]. Heparin levels were measured before initiation of administration at day 0 and at day 7, when peak levels 1.5 h after administration and nadir levels just before the next administration were measured. For statistical analysis, an analysis of variance for repeated measurements was done. If this yielded significant results, 95% confidence intervals (95% CI) were calculated for the difference between the time points and t = 0.

Results

Results are summarized in Table 1 and Fig. 1. As can be seen, low-dose heparin administration did not influence the urinary parameters of bone resorption. Serum carboxy-terminal telopeptide (ICTP), a new marker for bone resorption, did not change significantly. As for the bone formation parameters, serum alkaline phosphatase decreased slightly but significantly during administration (P = 0.03). The serum level of osteocalcin did not decrease significantly (P = 0.10). A change in serum 1,25dihydroxyvitamin D was not observed. TmP/GFR increased significantly during heparin administration. All volunteers had small hematomas at the place of injection, but this did not interfere with daily activities. No decrease in platelet count was observed. A significant increase in SGOT and SGPT was observed in all participating volunteers. The increase in SGPT was more pronounced than the increase in SGOT. No clinical symptoms accompanied the rise of liver enzymes and values decreased to baseline values within three weeks after cessation of heparin administration. Peak heparin levels at day 7 were 0.29 4- 0.23 IU/ml and nadir levels were 0.13 4- 0.05 IU/ml.

-z~-

Hp/Cr

- - 0 - - Ca/Cr 0.36

30 I

heparin so.

I

24

0.30 0.24

18

\,,\

A ~ I / -

-

A

0.18

12 0.12 6

0

~ o

0.06 i -4

I

I

I

I

I

0

2

4

7

10

0.(2,0

clays

Fig. 1. Urinary hydroxyproline/creatinineand calcium/creatinineratio before(-4 days)and during low-

dose heparin treatment. Values are expressedas mean ± SD.

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Discussion We were unable to demonstrate significant effects of low-dose heparin administration on the biochemical parameters of bone resorption, i.e. the urinary parameters, as well as serum ICTP. ICTP is a sensitive and specific marker of bone resorption [10]. This might imply that either heparin in this dosage and treatment duration does not have measurable effects, or the sample size was too small for detecting any effects. The latter is improbable because, as is evident from the confidence intervals, only very small changes were found in this homogenous population. Several mechanisms have been suggested for the effects of heparin on bone turnover [12-14]: an increased effect of parathyroid hormone on osteoclasts; increased bone resorption due to heparin-related collagenase activity; decreased osteoblastic function due to a direct effect of heparin on osteoblasts; and alterations in vitamin D metabolism which result in decreased levels of 1,25-dihydroxyvitamin D [13]. In vitro it was found that heparin inhibits collagen synthesis [14]. We found a small but significant decrease in serum alkaline phosphatase, possibly indicating a decreased bone formation. However serum osteocalcin did not decrease significantly. The increase in TmP/GFR may be caused by a direct effect of heparin on the renal phosphate handling or by a decrease of serum PTH, but the latter was only borderline significant (P = 0.07). The heparin induced osteoporosis found in pregnant women and patients with cardiovascular disease may be a combination of the effects of a higher dose of heparin and the effects of relative immobility on bone resorption and formation. As can be seen from our data, short-term administration in healthy mobile adults does not result in pronounced effects and it remains to be established whether a higher dose would have resulted in a greater effect. In the literature, only two patients with heparin-proven osteoporosis have undergone a bone biopsy [15,16], one of them showing increased resorption and decreased formation [15]. If low-dose heparin had effects on bone resorption we would have seen these effects reflected in biochemical parameters of bone resorption, analogous to the effect of immobilization on biochemical and histomorphometric parameters of bone resorption [17-19]. In this study we found a uniform reversible increase in serum transaminase levels in all subjects, consistent with earlier reports [2,3], suggesting a direct hepatotoxic effect. This suggests that heparin should be added to the differential diagnosis of liver enzyme disturbances in patients requiring this therapy. In summary, low-dose heparin, which is frequently used for prophylaxis of thrombosis in clinical patients, did not result in changes in biochemical parameters of bone resorption in healthy mobile volunteers. A slight but significant decrease was observed in serum alkaline phosphatase. All volunteers showed an asymptomatic increase in serum transaminase levels. No other side effects were observed.

Acknowledgements We want to express our gratitude to our colleagues R. Jonkhof and K.G. van der Hem for cooperating in this study, to Dr C. Popp for measuring serum ICTP and to Mr R. Barto and Dr. W.J.F. van der Vijgh for measurement of vitamin D metabolites.

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