Effect of light and gamma irradiation on pyridinolines in urine

326

Abstracts

Bone Vol. 17, No. 3 September 1995~31%331

P24. Tiludronatc tablet (Sk&d@) 4OOmgld products lengthy clinical nmiasion of Paget’s disease: follow up of a dose ranging study TC Stamp, WD Frase?, Jp Sawy&*, RA Creek**, C Picot’ Royal National Orthopnrdic Hospital, Stanmore, Middlesex, ‘Royal Liverpool Hospital, Liverpool, ?? *Ssnofi Winthrop,1 Onslow Street, Guildford and +Snnofi Recherche, 9 Rue du President,

Allendc,

Gcntilly,

France

A point follow up survey took place in May 1994 of patients previously random&d to a double blind dose ranging placebo controlled study of a 12 week course of tiludronate (2OOmg, 4OOmg, 600mg or placebo) in Paget’s disease, between April 1992 and March 1993. 98 patients were eligible having received at least 11 weeks of study drug and completed the study. Complete information was collected on 85 patients and partial information derived from records on the remaining 13 patients. The median duration of follow up was least for the placebo group (671.5 days) and most for the 4OOmg tiludronate group (782 days). A survival analysis was performed on the time in days from final tiludronate treatment to date of any retreatment for Paget’s disease. The median retreatment time was 314 days for placebo and 756 days for 2OOmg but had not been reached for the 4OOmg and 6OOmg groups, only 7/27 and 6/25 respectively having required retreatment. The treatment free time was increased in all active groups compated to placebo, log rank test of the distributions p=O.O008. All active treatments showed a strong difference from placebo (p=O.O43, p=O.O@2and p=O.OOl) for 2M)mg, 4oomg and 600mg tiludronate respectively. No patients had experienced any new fractures, nor were any new adverse events related to tiludronate reported at follow up.

P25. Metabolism of the antiproliferative vitamin D analogue, EBlO89, in a cultured human keratinocytc modal VN Shankar, HLJ Makin*, NJ Schroeder*, DJH Trafford’, A-M Kissmeyef., MJ Calverley**, E Binderup’*, G Jones Department of Biochemistry, Queen’s University, Kingston, ON, Canada K7L 3N6, ‘Department of Chemical Pathology, the London Hospital Medical College, London El 2AD, UK and “Leo Pharmaceutical Products, DK-27.50; Ballerup, Denmark

EB1089 is a synthetic analogue of ls-(OH)2D3 with potential for cancer. It has an altered side chain structure featuring 26,27-dimethyl groups, insertion of an extra carbon atom (24a) at C-24 and two double bonds at C-22,23 and C-24,24a. In our studies, EB1089 (IO&I) was incubated with a human keratinocyte cell line, HF’KlA-ras, for 72hrs and four metabolites were observed, all of which possessed the same UV chromophore as EB1089, indicating retention of the side-chain unsaturation. Two metabolites were formed in sufficient quantities to permit identification by GC-MS. Though both metabolites had the same M+=758 indicating a hydroxylated version of EB1089, the two metabolites exhibited distinctively different fragmentation patterns. The mapr product showed a significant fragment at m/z 641 due to loss of 117 mass units. The minor product showed a fragment at m/z 103 and other fragments consistent with a more distal hydroxylation in the side chain. We conclude that these products are side chain monohydroxylated metabolites, the position of the minor hydroxylation site being distal to the position of the major hydroxylation site. Furthermore, the major product was oxidised by sodium m-periodate giving a cleavage product with modified UV properties. Synthetic standards are being prepared in order to confirm the identities of these metabolites. Other studies intended to pinpoint the structural feature of EB1089 which is responsible for directing this unusual target-cell side chain hydroxylation are being pursued. We conclude that EB1089 shows a unique in vitro metabolic profile which may explain its relative stability in viva. use in breast

P26. Gender differences

The restoration of a London church led to the recovery of skeletal material buried between 1729 and 1852. This gave us the opportunity to compare the rates of bone loss in the femora of these ancient samples with that of present-day individuals. We have previously shown that the females from this sample may have lost less bone density (BD) in the proximal femur than females of today [l]. The aim of this current study was to compare the rates of bone loss in the femora of the male skeletons from this sample with those of present-day males. The ancient sample comprised 78 well preserved left femora from male skeletons (age range 18-91 years; height range 152-183cm). BD was measured using dual energy X-ray absorptiometry (Lunar DPX). There was significant loss of BD in the proximJ1 femur with age (% change in femoral neck BD = -3.67% per decade of age, r=O.413, p
673-675.

P27. Gas chromatography-mass spectromctry as a means of determining the structures of metabolites formed in oitro by cultured call lines incubated with chemical analogues of vitamin D HLJ Makin, DJH Trafford, MJ Calverley’, G Jones” 7’he London Hospital Medical College, London El 2AD, ‘Leo BaIlerup, Denmark Phnrmaceu,ical Producls, DK-2750 **Queen’s University, Kingston, Onfario, Canada K7L 3N6

and

Metabolites of vitamin D analogues produced in vitro are extracted and then separated using both straight- and reversephase HPLC systems and can usually be recognised by their characteristic UV absorption spectra using a photodiode array detector. Using the small amounts of material usually available (clrg), CC-MS of per-trimethylsilyl ethers can often provide information about the structure. It is for example, usually possible to determine the molecular ion and losses of silanol groups may also indicate the number of hydroxyls present. Further information can be obtained from the fragmentation patterns observed. For example, per-TMSi ethers of 25hydroxylated compounds give intense peaks at m/z 131 and in cases where the C?6 and 27 have been extended (as in EB1089 and KH1060), the m/z 131 peak is replaced with m/z 159. Substitution in the side chain can usually be identified by the presence of fragments which arise by cleavage of C-C bonds adjacent to the substitution. This has been particularly useful in studying the metabolism of analogues with homologated side

chains. In conjunction with other fragmentations which occur in the steroid nucleus (e.g. m/z 217 and loss of 131), metabolite structure, which can be confirmed by comparison with the synthetic compound or in particular cases by NMR, can be assigned. GC-MS can also be used after formation of cyclic boronate esters or after periodate and/or borohydride treatment. 0x0 groups can be demonstrated after formation of o-methyl oximes. 22-Oxa-steroids such as OCT and KH1060 have characteristic spectra as a result of cleavage on either side of the oxygen. GC-MS has proved to be invaluable as a means of identifying the structures of in vi&o metabolitn.

P28. Effect of light and gamma irradiation urine

on pyridinolines

in

A Blumsohn, A Colwell, K Naylor, R Eastell Department of Humon Metabolism and Clinical Biochemistry, University- of Sheffield, Sheffield

in bone loss over two centuries

B Lees, T Molleson*, TR Am&t”, C Walton, JC Stevenson Wynn InsHtute for MeUolic Research, 21 Wellingron Road, ‘Department of Palaeontology, Natural London, NW8 9SQ, History Museum, London SW7 SDB and *‘Department of Anatomy and Developmental Biology, University College London, London WClE 6BT

Background: Recent reports suggest that ambient light results in extensive degradation of urinary pyridinolines. We therefore examined the effect of light and 7 irradiation on pyridinolines and telopeptides of type I collagen in urine. Methods: Fresh undiluted urine from healthy subjects was exposed to: ijhighdose 1 irradiation (6Mrad, ‘%o source), n=lO;

Bone Vol. September

17, No. 3 1995:31%331

Abstracts

lighting x ii)-UV light 240 - 320nm x 3 days, n=6; iii) laboratory sunlight x 72 h. We measured total 24h, n=lO; iv) direct pvridinoliies by HPLC (tPyd, tDpd), free pyridinoliies by HPLC ;mmunoreactive free pyridinolines by ELISA i&d , ,FDpd), (iFPyd, iFDpd; Metra), the N-terminal tclopeptide (NTx; Osteomark, Ostex), and C-terminal telopeptide (CT’x; Crceslaps, Osteometer) of type I collagen by ELISA. Unexposed controls were measured in the same run. Results: The effect of i) gamma irradiation and ii) ultraviolet light differed for different analytes (Table). Percentresidualamlyte; tDpd y irrad. UVx72h

tPyd

Paired1-t& vs. cm~l Fl’yd

FDpd

iFPyd

<5** <5** <5** <5** 15.2” 37.4** 39.4” 17.5” 18.7” 27.6’.

?? r
?? r
iFDpd

NTx

15.5** 29.7’*

116.9. 73.6 129.6* 81.8’

CTx

UV instability was greater for free than total pyridinolines measured bv HPLC. NTx showed a slight but consistent increase afterUV 7 and irradiation, iii) Exposure to laboratory lighting x 24-hrs had no effect. iv) Exposure to direct sunlight x 3 days decreased iFDpd by 30%. Conclusions: I’yridinolines in urine are substantially more UV stable than pure aqueous standards. This is likely to be due to colour quenching or other matrix effects. Routine exposure of undiluted urine samples to laboratory lighting has no important effect on the level of these analytes.

Pm (l-84)

Basal Secretion (pmol/L/min) Pulse interval (min) pulse secretory Area (pmol/L)

1

WY 2

3

0.61

0.79

5.41 1.03

4

5

0.83

0.73

1.05

5.94

6.44

5.64

5.37

0.81

1.86

1.59

1.41

Fasting increased PTH (l-84) basal secretion, the pulse secretory area and transiently increased the pulse interval. The changes result in a “noisier” secretory pattern. Free Pyr and Dpyr secretion increased on successive days but the circadian pattern of cross link excretion persisted and in this particular subject overnight (23.00-08.00) secretion of cross links was increased over th; 5 days.

Y

P29. The in nitro and in oivo metabolism of EB 1089 A-M Kissmeyer, VN Shank&, G Jones’, HLJ Makin” Leo Pharmaceuticnl Products, DK-2750 Ballerup, Denmnrk, ‘Queen’s University, Kingston, ON, Canada KiL 3N6 md “London Hospital Medical College, London, El 2AD, UK EB 1089 is a novel analogue of the physiologically active metabolite of vitamin D3, la,25-dihydroxycholecalciferol on (1,25(OH)2D3). EB 1089 is active, at very low concentration, ccl1 growth regulation both itI vitro and in viva. EB 1089 has hen sclcctcd for clinical trials in cancer patients. In the present study, we have investigated the metabolic stability of EB 1089 and its metabolic profile in different iti vitro and in viuo models. The serum half-life of EB 1089 in rats was 2.2 hours (calculated from 0.5-6 h after i.v. dosing), which was comparable with the half-life of 1,25(OH)zD3 determined under the same conditions. EB 1089 is therefore considered as a metabolically stable analogue and a suitable candidate for systemic use. EB 1089 was metabolised approximately twice as fast as 1,25(OH)zD3 in liver microsomes obtained from rat, minipig and man. The formation of several metabolites were observed in these incubations as well as in liver samples collected from EB 1089 dosed rats. The number of metabolites were based on the number of peaks observed from HPLC. Incubations of the H-ras transformed human keratinocyte cell line HPKlA-ras (R.Kremer, McGill Univ.) with EB 1089 also resulted in the formation of several metabolites (identified in companion abstract by Shankar et al, this meeting). Four of the major metabolites were isolated and identified as side-chain hydroxylated analogues of EB 1089. Based on the PDA spectra, the two double bonds in the side-chain appear to be intact in the major metabolites.

P30. Changes in PTH (l-84) pulsatility and urinary cross-links excretion during a 96 hr fast I’ Newlands, A Black, GH Beastall, FC Logue, WD Fraser Department of Clinical Chemistry, Royal Liwrpwl University Hospital, Liverpool L7 8XP Fasting alters the circadian rhythm of PTH (I-84) with loss of the overnight rise and increased variability of secretion which may be due to alterations in m (l-84) pulsatility. These changes in PTH (I-84) secretion could affect urinary excretion of Pyr and Dpyr. A normal male volunteer was venesected daily at 1 min intervals for 1 hour between 10.00-11.00 prior to and during a 96 h fast. Urine was collected 3 hourly from 08.00-23.00 and once overnight 23.00-08.00. PTH (l-84) was measured by IRMA and deconvolutional analysis performed.

P31. Quantitative mapping of calcium and protein in mouse bone usineya new X-ray imaging technique CJ Buckley, N Khaleque, MW Robins’, X Zhang” Departments of Physics and ‘Physiology, King’s College, The Strand, London WCZR 2LS, UK and *‘Department of Physics, SUNY at Stony Brook, NY 11794, USA A technique for quantitatively mapping calcium and protein without stains in undemineralised, embedded bone tissue sections has been developed. The technique can resolve features down to 50nm with both elemental and molecular bond sensitivity. The images below were formed by combining a number of images taken at different soft x-ray energies using a scanning transmission x-ray microscope. The technique has been developed to image and quantify the calcium and protein contents of normal and osteoporotic (ovariectomised) mice femoral necks in spatial maps for comparative studies. Bone tissue from these mice are at the embedding stage. However, we have mapped calcium and protein in undemineralised, embedded and unstained O.lrm thick femoral head sections from normal and obese mice. The results are shown below.

Quantitative maps of calcium (left) and protein (right) in normal (above) and obese (below) mouse femoral head. The bones were resected from sacrificed mice after 150 days. The O.lpm thick sections were imaged by scanning x-ray microscopy*, where images at seven different x-ray energies were combined to create those above. The concentration is indicated by a continuous brightness scale. For calcium the peak brightness is equivalent to 2 x 10-6 g/d and for protein it is 5 x @g/cm*. The scale bar is 50pm. 1. CJ Buckley et. al. Bone 13: 104 (1992).

P32. Effects of gammalinolenic acid (GLA) and eicosapentaenoic acid (EPA) on bone calcium and on urinary collagen cross link excretion in young male rats MC Kruger, N Claasen, HC Potgieter, DF Horrobin’ Department of Physiology, University of Pretoria, S Africa and ‘Scotia Pharmaceutic&, Guildford GUI IBA, UK Several decades ago essential fatty acid (EFA) deficient animals were shown to develop severe osteoporosis. The main dietary EFAS are linoleic (LA) and alpha-linolenic (ALA) acids, but these exert most of their biological effects by being converted to metabolites such as GLA from LA and EPA from ALA. We

327