Circulating osteocalcin in primary biliary cirrhosis following liver transplantation and during treatment with ciclosporin
R.G.P.
watsot-,‘, L. Coulton’,
J.A. Kanis’, M. Lombard’,
R. William?,
J. Nettberger’
and E. Elias’
Circulating osreocakin. a noccoilagen bone protein is considered a useful indixct index of turnover. Ostecaakin levels were measured by radioimmunoassay in 57 female patients with primary biliary cirrhosis (PBC) from two cenbes, 52 normal female controls, 11 female patients following liver transpkntation for PBC and seven female patients with PfiC treated with Ciclospotin. Serum levels were sigtdficatttly less in the PBC group (median 10 @ml) compared to normals (median 15 ttglmkp < 0.001). Patients with PBC treated with Ciclospxin hadsignificantly higbeerlevektbatt untreated patients (median 23 @m/ml:p < 0.001). Levels also increased significantly following transplantation when. ~11patients were recziving Ciclosporbt (median 40 ng/ml: p c 0.001). The results suggest that the abnormal bmm metabolism of PBC is @dficantly altered by Ciclosporin and liver transplantation.
Osteoporosis is a major complication of chronic cbc4estatic liver disease (1). The aetiology has not yet been established and attempts at treatment have been disapp<ing. Bone turnover has been reported to be reduced in ptimaty biliary cirrhosis (PBC) (2) and cimulatiag
levels of
ostwcakin, a non-collagen bone matrix protein, are significantly decreased (3.4). Osteocalcitt is a small protein (approx. M&OW) which is found in bone tissues of all vertebrates. In most species it makes up to 15% (w/w) of the non-collagenow protein content, but in man it constitutes about 1% (5). Stucturally it is characterized by the presence of the amino acid pcarbnxyglutamic acid (Gk), and hence it is also known as bone Gla-protein (BGP), though its function remains untcnovm. It k present in osteobksts, osteucytes and calcified bone matrix (6). Generally blood levels are elevated in conditions charatierised by increased turnover such as Paget’s disease (7) mtd in postmenopausal osteoporosis semm levels correlate poritive-
ly with bone formation (8). Hence the reduced levels seen in PBC are considered to be a reflection of decreased OSteoblastic activity and turnover (3.4). FoUowing liver tnttsplantation for PBC it that there would be a aormaltsatioa of mnditians which produce reduced bone tumover. Tlte aim of this
pected
might beex-
study was to seek evidence for this by measuringosteomltin levels in patients with PBC before and after liver tramplantation. FoUowittg liver tranrplatttatiwt, patients at Bimtbtgham undergo att initial intensive period of immunosuppressant treatment with corticostemids and Cicl~ potin A. After a short time the mrticatemids are stopped and Ciclospotin A is continued indefinitely as the only immuao8uppressiveagent. It is apparent fmm in vitro and in viva animal experiments that Ciclosporin A k associated with changes in bone resorption (9-13) and a0 increase in osteocalcinhas been reported in one (13). Ciclosp~tin probably interferes with the production of immunologically derived sobstances,s-xl: as &mphokines,
355 which have been shown to be important in the control of bone gmwtb (14,lS). Therefore in order to examine this factor, a further aim of the study was to investigate 0s. tecakin levels in patients with PBC, who were being treated with Ciclosporin.
htklltSmdMCth&
Four groups of subjects were studied (Table 1). 1. Group 1 consisted of 57 females with PBC; thirty-three who attended the Birmingham cedre and twenty-four from the London centre. The diagnosis was confirmed histologically in each case and serum bilk&in and creatinine levels were measured at the same time as osteacalcin assay. The median bilimbic Izxk atzd ranges for the Birmingham and King’s College Hospital patients were 235 /rmoUl (raoge 26-475) and 26 pm&l (range 9-X-0). respectively. The median creatinine level and range in the Bimdngham patients was 75/rmolil (range 52-137) and in the King’s CeUege Hospital patientr 68 fimalil (range 3%US). 2. Group 2 contained 11 female patients successti~Uy transplanted for PBC at Biingbam. The interval atIer tmnsplantation when they were studied ranged from 1 month to4yearsc)mmubs, tbemeanwas 1 year3months. Nine of these subjects were included in Gmup 1 prior to transplantation. They were all receiving Ciclosparin as the only immunosuppressant drug, at a dose adjusted to give a trough whole blcad level in the range, as measured by radioimmunoassay, between 300~&Xl @ml. The mean bilirubin level and range was 20 pmalil (range 6-60) and median creatinine and range 134 Irmol/l (range 72-200). 3. Seven females with PBC at the London ware who were mated with Ciclosporbt as part of a double-blind placebo patients
eontml study to assess Ciclosporin treatment in with PBC ma& up the thii group. The dose
given was 3 n&g per day and had been continuous for 2-15 months (mean 6 months). The median bilimbin level and range was 30~mabl (range l-54) and median creatinine and range 74,umolil (range 53-145). 4. The fourth studygmoupwas made up of 52 age-matched normal female eontmk. They were part of an extensive survey of asteocakin levek in normal subjects. The sera were obtained horn the Trent Regional B&d Transfusion Service in Sheftield and all subjects signed a declaration that they were healthy and not taking any drugs apart from the contraceptive pill. Serum creatinine and bilimbin levels were not obtained. Plasma samples from each subject were stored at -70 T and the osteocalcin level subsequently measured in triplicate by a radioimm unoa.%ay me&d based on that described by Price and Nisbimoto (16) using bovine BGP as a standard and for radioicdittated tracer. Further de tails have previously been described (17). For subjects who were takb~g Cidc+nb~ A, levels were measured at their ovm cemres by radiobmn”“0. assay. Tbe results represent parent “mpound levels as well as metabolitn. Statistical comparisons between groups were made using the Mann-Whitney 11 test and paired mmparkonr of osteocakin levek in the same subjects b&xc and after transplantation were ma& wing the Wtixon Sign Rank test.
CkteDcalcin levels were lower in patients with PBC compared to normal contmk (Fe. 1) (PBC median 10 @ml; controls 15 @ml: p < O.UlJl). Levels were similar in patients from tbe two centres (Birmingham median 10 &ml, Kings 6.9 @ml). Serum bilimbin kvels were signifieantly higher in the Birmingham the King’s patients
(medians
patients compared
235 and 26 pmolil,
to
respec-
356
.
.
. . .
.“,
tively, p =z O&X) (Table 1). Following liver transplantation, oatecakin levels increased compared to pre-tramplantation levels (median 40 q/ml) (Fig. 2) and were sig nifkantly higher than controls @ < 0.001). In the patients with PBC treated with Ciclospmin, osteacalcin levels ?;&xz higher tbz!t i!! the controls (median 23 ne/ml. p < 0.05) and were higher than those wirh untreated PBC @ C 0.001). Levels in the transplanted patients tended to be
higher than in patients with PBC treated with Cyclcspo. tis. though not sigfnificantly @ = 0.09). Fig. 3 shows the osteocalcin/creatinine ratio in nine patients before and af-
OSEOCALCIN
357
IN PBC
ter liver transplantation. This was increased in eight, although there was a” increase in creatinine in seven of the same patients.
Dkcusshm This study has clearly confirmed that circulating (15teocakin levels are significantly lower than normal in Primay biliary cirrhosis. Tix number of patients with PBC in the present study is larger than in earlier studies (3.4), and the WnttVl population m”re appropriate: it is similar in size to the patient group, consists entirely of females and is closely matched for age. Patients entered into the study horn Bi~ringham had higher b&ubi” levels than the patients from King’s College Hospital, and are therefore likely to have more advanced disease (l&,9). Despite this, Onecalci” levels were similar in both groups. Ostaralci” kvek rose signifkanUy after liver trwsplantation and the indication from Pig. 2 is that levels are sustained at least up to 5 years followi”g transplantation. AU patients were taking Ciclospa’k A. Since osteaalti is eliminated by renal clearance. and Ciclosporio A tends to impair rellal fwction. astecakk levels may have increased following treatment because of a” asscziated reduced renal clearance. Hawever, the data of osteocalcin/ creatinke ratios (Fig. 3) indicates that this factor alone is unlike$ icIaca”“t for the rise in “stew&n. CiCl”sp”ri” may poteotiauy have a dire0 effect 0” “stew&” kvek. It has bee” reported to reduce bone resapticminvitro (g-1O)attdi”vivo (ll)p&hly byinhibitioo of immune ccl, &rived factors such as interkuki” and csteockst activatkg factor which have been show” to be poteat bone resorbing agents (13). The levels of osteocalck in the seven patkots with PBC treated with Cyclospoxi” A v/e= si&tiantly higher than in untreated patients, while Jcntm creatinine levels were similar in the tw”
groups. By contrast patients with PBC treated with cakiurn and vitamin D continue to have significantly lower “s. teocalci” levels than normal controls (4). Cickspork A may therefore be the principal factor in causiiip a rise in osteocakin follotig liver transplantation. The question arises as to what a” increase in tnteocalci” signifies at a cellular level. It has bee” asstmted that low levels are associated with the demonstrably low bone turnover in PBC (3). By inference it is possibk that increased levels indicate increased twmver and improved bane density and structure. In PBC lymphocytes are activated and there is an increase in serum kvels of lyntphokines (Ref. 20 and Neuberger and Adams, perwnal communication). It may he that these lymphokioa inhibit ateobkstic activity and that irhibition is removed by treatment with Ciclorpori” A. Several in vita, expetimena have demonstrated a decrease in bone resorption with Cickspmi” (9-H), and in one case there was also wide”‘% of increased bone formation (12). However, one in viva animal experiment indicates that treatment -wit,, Cictospmi” and i:; increase in 0ste”caki” may ““t necessarily be be”eticial(13). I” rats treated with Cickspmin there was sig&ica”t bone krs associated with a” increase in asterakin. There is clearly a need to imestigate bone structure in a longitudinal way in patients with PBC treated with Cyclospoti” and fouowing liver tranrpkntatio”. The early reports of the “se of Ckhnpmi” for the treatment of PBC are encouraging (2i,2).). If it catt abo ‘be shorrn to have a beneficial effect 011bone structue io patients with PBC and following liver traospk”tati”n this would be an impatant
aspect of the ma”aget”eat
of this condition.
ACkItWkdg.5E.d
This work was supported ~amme
Grant.
in part by an MRC
Prw
358
R.G.P.
watsot-,‘, L. Coulton’,
J.A. Kanis’, M. Lombard’,
R. William?,
J. Nettberger’
and E. Elias’
Circulating osreocakin. a noccoilagen bone protein is considered a useful indixct index of turnover. Ostecaakin levels were measured by radioimmunoassay in 57 female patients with primary biliary cirrhosis (PBC) from two cenbes, 52 normal female controls, 11 female patients following liver transpkntation for PBC and seven female patients with PfiC treated with Ciclospotin. Serum levels were sigtdficatttly less in the PBC group (median 10 @ml) compared to normals (median 15 ttglmkp < 0.001). Patients with PBC treated with Ciclospxin hadsignificantly higbeerlevektbatt untreated patients (median 23 @m/ml:p < 0.001). Levels also increased significantly following transplantation when. ~11patients were recziving Ciclosporbt (median 40 ng/ml: p c 0.001). The results suggest that the abnormal bmm metabolism of PBC is @dficantly altered by Ciclosporin and liver transplantation.
Osteoporosis is a major complication of chronic cbc4estatic liver disease (1). The aetiology has not yet been established and attempts at treatment have been disapp<ing. Bone turnover has been reported to be reduced in ptimaty biliary cirrhosis (PBC) (2) and cimulatiag
levels of
ostwcakin, a non-collagen bone matrix protein, are significantly decreased (3.4). Osteocalcitt is a small protein (approx. M&OW) which is found in bone tissues of all vertebrates. In most species it makes up to 15% (w/w) of the non-collagenow protein content, but in man it constitutes about 1% (5). Stucturally it is characterized by the presence of the amino acid pcarbnxyglutamic acid (Gk), and hence it is also known as bone Gla-protein (BGP), though its function remains untcnovm. It k present in osteobksts, osteucytes and calcified bone matrix (6). Generally blood levels are elevated in conditions charatierised by increased turnover such as Paget’s disease (7) mtd in postmenopausal osteoporosis semm levels correlate poritive-
ly with bone formation (8). Hence the reduced levels seen in PBC are considered to be a reflection of decreased OSteoblastic activity and turnover (3.4). FoUowing liver tnttsplantation for PBC it that there would be a aormaltsatioa of mnditians which produce reduced bone tumover. Tlte aim of this
pected
might beex-
study was to seek evidence for this by measuringosteomltin levels in patients with PBC before and after liver tramplantation. FoUowittg liver tranrplatttatiwt, patients at Bimtbtgham undergo att initial intensive period of immunosuppressant treatment with corticostemids and Cicl~ potin A. After a short time the mrticatemids are stopped and Ciclospotin A is continued indefinitely as the only immuao8uppressiveagent. It is apparent fmm in vitro and in viva animal experiments that Ciclosporin A k associated with changes in bone resorption (9-13) and a0 increase in osteocalcinhas been reported in one (13). Ciclosp~tin probably interferes with the production of immunologically derived sobstances,s-xl: as &mphokines,
355 which have been shown to be important in the control of bone gmwtb (14,lS). Therefore in order to examine this factor, a further aim of the study was to investigate 0s. tecakin levels in patients with PBC, who were being treated with Ciclosporin.
htklltSmdMCth&
Four groups of subjects were studied (Table 1). 1. Group 1 consisted of 57 females with PBC; thirty-three who attended the Birmingham cedre and twenty-four from the London centre. The diagnosis was confirmed histologically in each case and serum bilk&in and creatinine levels were measured at the same time as osteacalcin assay. The median bilimbic Izxk atzd ranges for the Birmingham and King’s College Hospital patients were 235 /rmoUl (raoge 26-475) and 26 pm&l (range 9-X-0). respectively. The median creatinine level and range in the Bimdngham patients was 75/rmolil (range 52-137) and in the King’s CeUege Hospital patientr 68 fimalil (range 3%US). 2. Group 2 contained 11 female patients successti~Uy transplanted for PBC at Biingbam. The interval atIer tmnsplantation when they were studied ranged from 1 month to4yearsc)mmubs, tbemeanwas 1 year3months. Nine of these subjects were included in Gmup 1 prior to transplantation. They were all receiving Ciclosparin as the only immunosuppressant drug, at a dose adjusted to give a trough whole blcad level in the range, as measured by radioimmunoassay, between 300~&Xl @ml. The mean bilirubin level and range was 20 pmalil (range 6-60) and median creatinine and range 134 Irmol/l (range 72-200). 3. Seven females with PBC at the London ware who were mated with Ciclosporbt as part of a double-blind placebo patients
eontml study to assess Ciclosporin treatment in with PBC ma& up the thii group. The dose
given was 3 n&g per day and had been continuous for 2-15 months (mean 6 months). The median bilimbin level and range was 30~mabl (range l-54) and median creatinine and range 74,umolil (range 53-145). 4. The fourth studygmoupwas made up of 52 age-matched normal female eontmk. They were part of an extensive survey of asteocakin levek in normal subjects. The sera were obtained horn the Trent Regional B&d Transfusion Service in Sheftield and all subjects signed a declaration that they were healthy and not taking any drugs apart from the contraceptive pill. Serum creatinine and bilimbin levels were not obtained. Plasma samples from each subject were stored at -70 T and the osteocalcin level subsequently measured in triplicate by a radioimm unoa.%ay me&d based on that described by Price and Nisbimoto (16) using bovine BGP as a standard and for radioicdittated tracer. Further de tails have previously been described (17). For subjects who were takb~g Cidc+nb~ A, levels were measured at their ovm cemres by radiobmn”“0. assay. Tbe results represent parent “mpound levels as well as metabolitn. Statistical comparisons between groups were made using the Mann-Whitney 11 test and paired mmparkonr of osteocakin levek in the same subjects b&xc and after transplantation were ma& wing the Wtixon Sign Rank test.
CkteDcalcin levels were lower in patients with PBC compared to normal contmk (Fe. 1) (PBC median 10 @ml; controls 15 @ml: p < O.UlJl). Levels were similar in patients from tbe two centres (Birmingham median 10 &ml, Kings 6.9 @ml). Serum bilimbin kvels were signifieantly higher in the Birmingham the King’s patients
(medians
patients compared
235 and 26 pmolil,
to
respec-
356
.
.
. . .
.“,
tively, p =z O&X) (Table 1). Following liver transplantation, oatecakin levels increased compared to pre-tramplantation levels (median 40 q/ml) (Fig. 2) and were sig nifkantly higher than controls @ < 0.001). In the patients with PBC treated with Ciclospmin, osteacalcin levels ?;&xz higher tbz!t i!! the controls (median 23 ne/ml. p < 0.05) and were higher than those wirh untreated PBC @ C 0.001). Levels in the transplanted patients tended to be
higher than in patients with PBC treated with Cyclcspo. tis. though not sigfnificantly @ = 0.09). Fig. 3 shows the osteocalcin/creatinine ratio in nine patients before and af-
OSEOCALCIN
357
IN PBC
ter liver transplantation. This was increased in eight, although there was a” increase in creatinine in seven of the same patients.
Dkcusshm This study has clearly confirmed that circulating (15teocakin levels are significantly lower than normal in Primay biliary cirrhosis. Tix number of patients with PBC in the present study is larger than in earlier studies (3.4), and the WnttVl population m”re appropriate: it is similar in size to the patient group, consists entirely of females and is closely matched for age. Patients entered into the study horn Bi~ringham had higher b&ubi” levels than the patients from King’s College Hospital, and are therefore likely to have more advanced disease (l&,9). Despite this, Onecalci” levels were similar in both groups. Ostaralci” kvek rose signifkanUy after liver trwsplantation and the indication from Pig. 2 is that levels are sustained at least up to 5 years followi”g transplantation. AU patients were taking Ciclospa’k A. Since osteaalti is eliminated by renal clearance. and Ciclosporio A tends to impair rellal fwction. astecakk levels may have increased following treatment because of a” asscziated reduced renal clearance. Hawever, the data of osteocalcin/ creatinke ratios (Fig. 3) indicates that this factor alone is unlike$ icIaca”“t for the rise in “stew&n. CiCl”sp”ri” may poteotiauy have a dire0 effect 0” “stew&” kvek. It has bee” reported to reduce bone resapticminvitro (g-1O)attdi”vivo (ll)p&hly byinhibitioo of immune ccl, &rived factors such as interkuki” and csteockst activatkg factor which have been show” to be poteat bone resorbing agents (13). The levels of osteocalck in the seven patkots with PBC treated with Cyclospoxi” A v/e= si&tiantly higher than in untreated patients, while Jcntm creatinine levels were similar in the tw”
groups. By contrast patients with PBC treated with cakiurn and vitamin D continue to have significantly lower “s. teocalci” levels than normal controls (4). Cickspork A may therefore be the principal factor in causiiip a rise in osteocakin follotig liver transplantation. The question arises as to what a” increase in tnteocalci” signifies at a cellular level. It has bee” asstmted that low levels are associated with the demonstrably low bone turnover in PBC (3). By inference it is possibk that increased levels indicate increased twmver and improved bane density and structure. In PBC lymphocytes are activated and there is an increase in serum kvels of lyntphokines (Ref. 20 and Neuberger and Adams, perwnal communication). It may he that these lymphokioa inhibit ateobkstic activity and that irhibition is removed by treatment with Ciclorpori” A. Several in vita, expetimena have demonstrated a decrease in bone resorption with Cickspmi” (9-H), and in one case there was also wide”‘% of increased bone formation (12). However, one in viva animal experiment indicates that treatment -wit,, Cictospmi” and i:; increase in 0ste”caki” may ““t necessarily be be”eticial(13). I” rats treated with Cickspmin there was sig&ica”t bone krs associated with a” increase in asterakin. There is clearly a need to imestigate bone structure in a longitudinal way in patients with PBC treated with Cyclospoti” and fouowing liver tranrpkntatio”. The early reports of the “se of Ckhnpmi” for the treatment of PBC are encouraging (2i,2).). If it catt abo ‘be shorrn to have a beneficial effect 011bone structue io patients with PBC and following liver traospk”tati”n this would be an impatant
aspect of the ma”aget”eat
of this condition.
ACkItWkdg.5E.d
This work was supported ~amme
Grant.
in part by an MRC
Prw
358
