The effect of rat parathyroid hormone (1–34) infusion on urinary 3-hydroxypyridinium cross-link excretion in the rat

and Mineral. 19 (1992) 117-125 0169-6009/92/$05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved. Bone

117

BAM 00491

The effect of rat parathyroid nium cross-link infusion on urinary 3-hydrox excretion in the rat

C.P. Jeromea, A. Colwellb, R. Eastellb, R.G.G. Russellb and U. Trechsel” “Sandm Phmna AG, Bascl, Switzerland bUnivcrsity of Shefleld Medical School. Shqffield, UK

(Received 18 February 1992) (Accepted 10 June 1992)

Summary The utility of measurement of the urinary excretion of the 3-hydroxypyridinium cross-links, pyridinoline (Pyr) and dcoxypyridinoline (Dpyr) as indices of bone resorption in rats was investigated. Total Pyr and Dpyr excretion were measured in young rats treated by S.C.infusion with rat parathyroid hormone (l-34) (PTH) at 22-30 pg/kg/day or with diluent (controls) for 14 days. During infusion, average urinary excretion of both cross-links was significantly higher in PTH rats (Pyr: Il.77 $ 0.44 nmol/day), Dpyr: 15.81 + 0.95 nmol/day) than in controls (Pyr: 10.17 f 0.35 nmol/day, Dpyr: 12.03 + 0.67 nmol/day). These results were consistent with the magnitude of the cxpcctcd increase in bone resorption rate with this dose of PTH. The method appears to provide a sensitive measure of bone resorption for in vivo bone studies in rats.

Key words: Bone resorption; Dcoxypyridinoline; Parathyroid hormone; Pyridinoline; Rat

Introduction

Measurement of bone resorption rate in animals in viva is difficult. Loss of [3H]tetracycline from banes of, or the urinary excretion of [3H]-tetracycline in, prelabeled animals have been used as indices of bone resorption [ 1,2]. Urinary hydroxyproline excretion has been widely used as an index of bone resorption, but is non-specific and not very sensitive. Bone resorption may be measured by Correspondence to: Christopher P. Jerome, Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Medical Center Boulevard, Winston-Salem, NC 27157” 1040, USA.

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radiotracer kinetics [3] in the rat and other species. Bone resorption rate can also be calculated from histomorphometric data using certain assumptions [4,5]. More recently, 3-hydroxypyridinium cross-links, which are degradation products of collagen, have been proposed as urinary markers for bone resorption. Of these, pyridinoline (Pyr) is found predominantly in bone but also in cartilage and perhaps other tissues, whereas deoxypyridinoline (Dpyr) appears to be specific to bone, Unlike hydroxyproline, changes in dietary Pyr and Dpyr do not affect urinary concentrations [6].Urinary collagen cross-links are highly correlated with radioisotopic measurements of bane resorption [7], and the value of these assays as measures of bone resorption in man has been demonstrated [8,9]. The use of these assays in animals has received less attention. Free and total pyridinium cross-link excretion was increased at a single timepoint, 7 weeks after ovariectomy in youn rats [ 101.After 6 weeks of ethanol feeding, young male rats had decreased conjugated and total deoxypyridinoline excretion [I 11. To further evaluate the utility of these measurements as indices of bone resorption in experimental animals, the urinary excretion of total 3-hydroxypyridinium cross-links was measured in rats given a treatment known to increase bone resorption rate. We have previously demonstrated by measurement of loss of [3H]-tetracyclinefrom femur of young rats that rat parathyroid hormone (l-34) (PTH) infusion at 40-50 pg/kg causes a 2-fold increase in bone resorption rate [12].

Methods

Twelve female, 2 l-day-old CD rats were obtained from Charles River, Germany. Starting at age 33 days, the rats were fed a soy/skimmed milk based diet (Nafag diet number 8917) containing 0.8% Ca and 0.6% P. The lack of collagen in the diet precludes any possible effect of dietary components on 3-hydroxypyridinium cross-link excretion, although none was expected [6]. Also starting at 33 days old, the rats were placed in metabolism cages for 24 h on alternate days (72 h at weekends). Urine was collected in dark, sterile containers, and urine volume recorded. Aliquots of urine were frozen at --20°C pending analysis. After three urine collections, the rats were implanted with 16day osmotic minipumps (Alzet 2002, Palo Alto, CA) S.C.under isoflurane (Forene, Abbott) anaesthesia. The pumps delivered 4 @g/rat/day (approximately 30 pg/kg/day falling to 22 pg/kg/day as the animals grew) of rat parathyroid hormone (l-34) (PTH, Bachem, Switzerland) or diluent (2% cysteine HCI in 0.15 M saline). This PTH dose was approximately half that administered in a previous experiment [ 121,in which PTH infusion caused a doubling of bone resorption rate and significant growth retardation. The lower dose was selected to cause a modest increase in bone resorption rate without a confounding effect on growth and bone mass. Urine collection continued throughout the experiment. The first urine collection during treatment was during the second 24-h period after pump implantation. The pumps were removed after i4 days treatment, and the animals killed by

119

decapitation under isoflurane anaesthesia 1 week later. One PTH-treated rat died before the end of treatment; data for this animal are included up to experimental day 7. Quantitation of total 3-hydroxypyridinium cross-links in urine [13 J Sample preparation.

Pyridinoline and deoxypyridinoline standards were prepared from canine femur using the method of Black et al. [14]. Analyses were performed in duplicate. 50 ~1 of urine, 200 ~1 of distilled water, and 250 ~1 of concentrated HCl were placed in 14 x 100 screw-capped tubes (Northern Media), and hydrolysed overnight at 110°C. Five ml aliquots of a stirred solution of 5% CFI cellulose in n-butanol:glacial acetic acid:water (4:1:1) were added to disposable plastic columns (Polyprep, Biorad Labs). 500 ~1 glacial acetic acid, 500 ~1 of the stirred mixture, and 2.0 ml of n-butanol were added to each hydrolysate. The mixture was vortexed and the samples applied to the columns. The tubes were washed with 2.0 ml of the 4:l: 1 solvent mixture, and applied to the columns, which were then washed with 2 x 10 ml of the 4: 1:1 solvent mixture. The 3-hydroxypyridinium compounds were then eluted from the columns with 5 ml water into S-ml plastic centrifuge tubes. The top (butanolic) phase was aspirated off and the samples evaporated to dryness at 40°C using a Lfntrifugal evaporator (Uniscience Ltd.). The dried samples were reconstituted in 250 ~1 1% heptafluorobutyric acid and injected directly into the HPLC. Instrumentation. The HPLC instrument consisted of two Jasco 880-PU pumps

with high pressure mixing, a Waters 7 12 autosampler, a Jasco 821-FP fluorescence detector, and a Waters 820 data acquisition system. 20 11 was injected onto a 33 x 4.6-mm, 3-pm octadecyldimethylsilane (ODS) column (Supelchem (UK) Ltd.). The separation was achieved isocratically within 4 min at 1.0 ml/min, using two solvents A:B (90:10). Solvent A was 20 mM ammonium chloride, 10 mM heptafluorobutyric acid, pH 2.25, and solvent B was 75% acetonitrile, 25% solvent A. After every 10 injections a column wash and reequilibration was performed to maintain the column efficiency.. The temperature of the column effluent was maintained at 12°C by passing through a 15cm stainless steel coil (0.009 in ID) immersed in a jacket of coolant from an LKB 2219 Multitemp II thermostatic circulator, prior to being monitored by the fluorescence detector. The fluorescence was monitored with the detector excitation at 295 nm and emission at 395 nm, a.nd quantitated using external standardisation. Recovery of pyridinoline and deoxypyridinoline from the cellulose columns was calculated using aqueous standards and urines with these compounds added and put through the entire sample preparation. The intra- and interassay variation were less than 11%. The recovery was 92.1 + 5.2% (n = 15) for pyridinoline and 91.7 +, 7.1% (n = 15)for deoxypyridinoline. The response of the fluorescence detector was linear to at least ZOO0 nmol/l pyridinoline and 2500 nmol/l deoxypyridinoline under the assay conditions.

120 Other measwemen ts

Urine creatinines were measured, after appropriate dilution with water, using a Co’sasBio Centrifugal Analyser (Roche) and a commercially available kit (Unikit II Roche). Serum osteocalcin was measured by radioimmunoassay using rat osteocalcin >99.9% pure, goat anti-rat osteocalcin, and [12’1]rat osteocalcin (Biomedical Technologies, Inc, Stoughton, MA, USA). Serum calcium was measured by a calorimetric method using mcthylthymol blue as an indicator. , Left tibias and femurs were dehydrated and defatted in graded alcohols and acetone, and dried to constant weight (dry, defatted weight) in a vacuum dessicator.

are presented as mean f SEM in tables and graphs. Placebo and PTHtreated animals were simultaneously compared at all timepoints using a multiple comparison prwxicre [ 151.Daily averages were calculated for all urinary parameters for sack animal for days -6 to -1 (before infusion) and days 2 to I8 (during nd are shown in Table 2. They were compared between groups by Student’s c-test. Paired comparisons over time were not done due to the con. founding e%ct of growth. Data

Rl!SUltS

During inf;lsion, PTH-treated rats had slightly lower body weight than placebotreated rats, but after removal of the pumps, weight returned towards control values (Table 1). Bone dry defiltted weights were slightly, but not significantly, lower in PTH-treated animals at the end of the experiment (approximately 1 week Table 1

of PTH infusion’ on body weight, serum calcium and osteocalcin, and bone mass (mean + SEM) Effct

Placebo

PTH-infused

P

Day -9

I 17.68 f 3.20

llR.IO

f

3.44

NS

Day 12

195.18 k 4.71

173.35 f

8.05

< 0.05

Day 21

214.08 + 6.02

192.62 k 10.50

Body weight (g)

< 0.1

Serum (day 21) Calcium

(mmoI/I)

2.18 & 0.03

2.22 & 0.06

NS

CMeocalcin

(@ml)

4.75 f

5.44 1_ 8.36

NS

0.29

Dry defatted weight (day 21) Tibia Femur

(mg)

349.27 _t ! i .02

325.74 + 8.:c

NS

(mg)

417.57 + 16.23

380.74 + 10.50

NS

’ Rat PTH (l-34) subcutaneous infusion started day I and ended day 15.

-

121

URINE I‘ VBLUMIS ml/day 14..

10

,I' ,'

CREATININE mmolfday

il i I)’

--

.-

--_.-__-

Placebo or PTB Infusion I-

10

I.)-- Placebo Urine Volume 0

2

PTH Urine Volume ~- Placebo Creatinine @. PTH Creatinine

0 I-I--- )__-___-I1_...(.-_(_---_I- _,~----l--l16 2 4 8 10 13 -G -4 -1

0

18

Fig.1. Effectsof placeboand rPTH (l-34) (PTH) infusion on daily urine and creatinineoutput in young rats. Error bars indicate the mean f SEM of four to six animals. Statistically significant differences bebveenplacebo-and PTH-infused rats indicated (“P
after removal of the pumps), and serum calcium and osteocalcin were not significantly different. These data indicate that the selected dose of PTH caused minimal effects on growth to confound the interpretation of results. PTH infusion tended to increase urine output during the first week of treatment (Fig. 1). Urine volume actually exceeded the volume of the collection flasks for three of six PTH-infused animals on day 3, so this point is missing in graphs of data depending on urine volume. Daily creatinine output was not different between groups (Fig. 1) throughout the experiment, again suggesting that growth and metabolic effects of PTH infusion were minimal. Whether the data were expressed as daily output or with creatinine as a referent, there were consistent trends towards increased excretion of both pyridinoline and deoxypyridinoline commencing 3-7 days after infusion started, with significant differences between group means at later time points (Fig. 2 and 3). Daily output of both cross-links declined towards control levels 4 days after the pumps were removed. Average cross-link excretion was significantly increased during PTH infusion

122 24

18

16

14

12

10 PYR tlmQl]ciay

DPYR 12 nmolfday

8

--_ Placebo or PTR Infusion

6

4 q--Placebo PYR ...O-., PTN PYR --&-- Placebe DPYR ....@.,. PTH DPYR

2,

0

_,7,~--__-~_~_~_!.

-6

-4

---------,_-

-1

2

4

, _____,_.+--f-

8

10

13

16

10

EXPERIMENT DAY Fig. 2. Effects of placebo and rPTH (l-34) (PTH) infusion on daily urinary pyridinoline (PYR) and deoxypyridinoline(DPYR) output in young rats. Error bars indicate the mean f SEM of four to six animals. Statistically significant differencesbetweenplacebo-and PTH-infused rats for both PYR and DPYR indicated (aP<0.05, bP
(Table 2), but urine volume, creatinine output and creatinine concentration were not different between groups.

During the PTH infusion period, excretion of both cross-links was 2540% higher in treated animals than in controls (Table 2). This is consistent with the magnitude of the expected increase in bone resorption rate, since we have previously found that infusion of a Zfold higher dose of rat PTH (l-34) causes a doubling of bone resorption rate [12]. Although there are no other published data on rat PTH (l-34) infusion, it has been previously shown that continuous infusion of bovine PTH increases static histomorphometric indices of bone resorption in rats [la, 17] and dogs [ 181. Intravenous infusion of bovine PTH

3001 I

123

T

250

PYR/CR nmol/mmol

1 200-r 150 Placebo or PTH Infusion

EXPERIMENT DAY

DPYR/CR mnol/mmol

Placebo or PTH Infusion

100 t 50

I

0

+. Placebo PYR/CR ....o-.. PT# FYR/CR -=+- Placebo DPYR/CR Fig.3. Effects of placebo and rPTH (l-34) (PTH) infusion on urinary pyridinoline/creatinine (PYR/CR)

and deoxypyridinoline/creatinine (DPYR/CR) in young rats. Error bars indicated the mean + SEM of five to six animals. Statistically significant differences between placebo- and PHT-infused rats indicated (nP
(l-34) also caused an increase in urinary hydroxyproline excretion in women [19,20]. Although the animals, were growing throughout the experiment, daily output of Pyr and Dpyr were constant in controls. Expression of the results as ratios to creatinine revealed a decline with age in controls. Since creatinine production is related to muscle mass and therefore to skeletal size, this result is consistent with a progressive decline in bone turnover rate with age [5]. In PTH-infused animals, daily cross-link excretion remained high despite slight growth retardation, and relative to creatinine remained near pre-treatment levels, indicating that PTN maintained high bone turnover rates in these growing animals. These results indicate the need to take into account changes in body weight, creatinine excretion and urinary volume in interpretation of cross-link excretion data.

124 Table 2 Daily average values for urinary parameters before and during PTH infusion’ in

young rats (mean f SEM)

Beforeinfusion Urine volume @Way) Creatinine (wow) Cfeatinine output (nmol/day) (nmol/day) Pyridinoline DeoxYpyridinoline (nmoljday) (nmol/mmol) Pyr@Winine Dpyr/crcatinine (nmol/mmol) pYr/Dpyr Durin@linhsion Urine volume OWdw) Crealinine @mow Creatinine output (nmoljday) (nmol/day) Pyridinoline Deoxypyridinoline (nmol/day) Pyrjcreatinine (nmol/mmol) Dpyr/creatinine (nmol/mmol) Pyr/Dpyr

Placebo

PTH-infused

P

PTH/placebo (%)

8.94 f 1.47 5.06 f 0.59 38.89 f I.10 9.43 $ 0.50 12.18 f 0.92 255.21 f 14.28 322.30 f 26.78 0.79 f 0.05

8.71 f 5.40 f 39.28 f 8.92 f 10.63 f 229.40 f 284.02 f 0.83 f

NS NS NS NS

97.47 f 23.33 106.75 f 23.96 101.00 f II.51 94.56 f 1I.25 87.29 f 10.45 89.89 f 8.37 88.12 f 9.47 105.67 f 7.16

II.15 f 1.2.5 5.57 f 0.66 57.05 f 3.26 10.17 f 0.35 12.03 f 0.67 179.85 f 5.07 212.72 f II.65 0.86 f 0.04

12.95 f I.14 4.39 f 0.30 53.71 f 2.37 II.77 f 0.44 IS.81 f 0.95 225.84 f 10.07 300.10 f 18.81 0.77 f 0.02

1.52 1.04 4.33 0.95 0.99 17.08 19.33 0.03

NS NS

NS NS

NS

NS NS eo.05 co.01
116.12 f 78.72 f 94.15 f 115.72 f 131.41 k 125.57 f 141.07 f 89.79 f

16.50 10.73 6.80 2.87 10.78 6.62 II.74 4.46

’ Rat PTH (l-34) subcutaneousinfusion started day 1 and ended day 15.

Excretion measured as ratios to creatinine may be the optimal method of In the two previous rat studies, either free [IO] or conjugated [l l] cross-link excretion, but not both, were significantly altered by experimental treatments; however, total excretion was also significantly changed in both reports. Our data support these flndings that measurement of total 3-hydroxypyridinium crosslinks in urine provides a sensitive index of bone resorption rate in rats. As in previous studies, modest increases in cross-link excretion were statistically significant with the relatively small sample size of six. Since bone resorption rate is otherwise difftcult to quantify, this method would seem to offer a valuable additional measurement for in tivo studies of bone metabolism.

Aekaowledgements We thank Peter Ingold, Erika Ryser and Martina Frank for excellent technical assistance.

125

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