3
INTRODUCTION
The meno pause is asso ciated with an increase in bo ne reso rptio n rate which may co ntribute to o s-teo po ro sis, fo r which the main co nsequence is an in-c re ase d risk o f frain-c ture s.1
Ho rm o ne re p lac e m e nt therapy (HRT) has been sho wn to be the mo st effi-cient preventio n and treatment o f o steo po ro sis.2
Ac-celerated po stmeno pausal bo ne lo ss is attributed to an increase in bo ne turno ver, co nsidering bo th bo ne fo rmatio n and reso rptio n due to o varian failure, with the predo minance o f reso rptio n.3,4
Bo ne fo rmatio n may be evaluated thro ugh dif-ferent bio chemical markers, such as serum co ncentra-tio n o f o steo calcine, alkaline pho sphatase and the car-bo xyl terminal pro -peptide o f type I pro co llagen.5
Ho w-ever, it has been difficult to establish a sensitive reso rp-tio n marker. Mo st studies o n the po stmeno pause pe-rio d are based o n the determinatio n o f urinary hydro x-ypro line, which is no t specific fo r bo ne co llagen but is a fractio n o riginating fro m skin co llagen, which also de-creases in the po stmeno pausal perio d.6
Urinary pyridino line (pyr) and deo xypyridino line (d-pyr) excretio n has been used as a new bo ne reso rp-tio n marker. Pyr and d-pyr are no n-reducible interlinking mo lecules o f bo ne co llagen, no t subject to significant metabo lism in the bo dy and are released during their degradatio n. Altho ugh co llagen is the majo r co mpo -nent o f several co nnective tissues, pyr is abundant in bo ne and cartilage, and d-pyr seems to be present in significant amo unts o nly in bo ne.7,8
Therefo re, urinary excretio n o f pyr and d-pyr may reflect the bo ne matrix degradatio n pro cess in a sensitive way.
Original Article
REVISTA PAULISTA DE MEDICIN AEffe ct of hormone re place me nt
the rapy on the bone mass and urinary
e xcre tion of pyridinium cross-links
Menopause Health Center, Universidade Federal de São Paulo/
Escola Paulista de Medicina, São Paulo, Brazil
a b s t r a c t
CON TEX T: The meno pause accelerates bo ne lo ss and is asso ciated with an increased bo ne turno ver. Bo ne fo rmatio n may be evaluated by several bio chemical markers. Ho wever, the establishment o f an a c c ura te ma rker fo r b o ne reso rptio n ha s b een mo re diffic ult to achieve.
OBJECTIVE: To study the effect o f ho rmo ne replacement therapy (HRT) o n bo ne mass and o n the markers o f bo ne reso rptio n: urinary excre-tio n o f pyridino line and deo xypyridino line.
DESIGN : Co ho rt co rrelatio nal study.
SETTIN G: Academic referral center.
SAM PLE: 5 3 po st-meno pausal wo men, ag ed 4 8 -5 8 years.
M AIN M EASUREM EN TS: Urinary pyr and d-pyr were measured in fasting urine samples by spectro fluo ro metry after hig h perfo rmance liquid chro mato g raphy and co rrected fo r creatinine excretio n mea-sured befo re treatment and after 1 , 2 , 4 and 1 2 mo nths. Bo ne min-e ra l d min-e nsity (BM D) w a s mmin-e a surmin-e d b y d ua l min-e nmin-e rg y X-ra y abso rptio metry (DEXA) befo re treatment and after 1 2 mo nths o f HRT.
RESULTS: The BMD after HRT was abo ut 4 .7 % (P < 0 .0 0 0 4 ); 2 % (P < 0 .0 0 2 ); and 3 % (P < 0 .0 1 ) hig her than the basal values in lumbar spine, neck and tro chanter respectively. There were no sig nificant co rrelatio ns between pyridinium cro sslinks and ag e, weig ht, meno pause duratio n and BMD. The decrease in pyr and dpyr was pro -g ressive after HRT, reachin-g 2 8 .9 % (P < 0 .0 0 0 2 ), and 4 2 % (P < 0 .0 0 0 2 ) respectively after 1 year.
CON CLUSION S: Urinary pyridino line and deo xypyridino line excre-tio n decreases early in ho rmo ne replacement therapy, reflecting a decrease in the bo ne reso rptio n rate, and no co rrelatio n was o b-served with the bo ne mass evaluated by densito metry.
KEY-W ORDS: Pyridinium cro ss-links. Ho rmo ne replacement therapy. Bo ne remo deling
• Do lo res Pero vano Pardini •Anibal Tag liaferri Sabino • Ana Maria Meneses • Teresa Kasamatsu •Jo sé G ilberto Henriques Vieira
4
The purpo se o f this study was to determine the validity o f these markers in the early evaluatio n o f the efficacy o f ho rmo ne replacement therapy in relatio n to bo ne reso rptio n in meno pausal wo men.
METHODS
The pro cedures that fo llo w were in acco rdance with the ethical standards o f the co mittee respo nsible fo r human experimentatio n and with the Helsinki dec-laratio n o f 1975, as revised in 1983.
Design
Co ho rt co rrelatio nal study.
Setting
Academic referral center.
Sample
The study co nsisted o f 53 wo men with meno pause o f 3.6 years (SD 5) befo re the study, and ages o f 53 years (SD 5 median 50 years) and bo dy mass index (BMI) o f 26 kg/m2 (SD 4). They were healthy wo men witho ut
previ-o us use previ-o f any hprevi-o rmprevi-o nal previ-o r previ-o ther medicatiprevi-o n that cprevi-o uld affect bo ne metabo lism. Calcium intake, as assessed by questio ning abo ut fo o d, was between 500 and 1000 mg/ day in 26% and belo w 500 mg/day in 74% o f the patients. Meno pause was defined as ameno rrhea fo r mo re than 6 mo nths and/o r FSH serum levels o ver o r equal to 40 mIU/ L. In 96% o f the wo men the meno pause was spo ntane-o us and mean FSH levels were 65.9 mIU/L (SD 27.2).
Study Procedures
The patients were submitted to standard questio n-ing, physical and gyneco lo gical examinatio n and, when included in the study, bo ne mineral density (BMD) o f the co lumn and femur, serum go nado tro phin (LH, FSH), estradio l and urinary pyridino line and deo xypyridino line determinatio ns were perfo rmed. Then the patients started ho rmo ne replacement therapy with 100 mcg/week transdermal estradio l (Estraderm) and 5 mg/day/12days/ mo nth o ral medro xypro gestero ne (Farlutal), except fo r two patients who had hysterecto mies and used o nly es-tradio l.
Main Outcomes
Pyr and d-pyr were assayed in urine after 2 h fasting. Urine samples were co llected after 1, 2, 4 and
12 mo nths o f HRT fo r pyr and d-pyr evaluatio n and BD was repeated after 12 mo nths o f HRT. The urine was hydro lyzed and submitted to CF1 cellulo se chro ma-to graphy. The extracted pro duct was separated b y HPLC and identified using fluo rescein. Pyr and d-pyr results were expressed as pmo l/µmo l creatinine.9 The
inter-assay variatio n co efficient was 12.4% fo r pyr and 10.8% fo r d-pyr and the intra-assay was 0.6% fo r pyr and 6.7% fo r d-pyr.
Bo ne densito metry was do ne using a do uble en-ergy X-ray abso rptio n apparatus (Lunar Radiatio n DPX, Madiso n, WI) with a variatio n co efficient o f appro xi-mately 1.5% fo r bo th the co lumn and the femo ral neck. Estradio l and go nado tro phin levels were deter-mined by radio immuno assay.
Statistical Methods
Paired and no n-paired t tests were used to co m-pare variables within the same gro up at the beginning and after the treatment and to co mpare variables be-tween the gro ups, respectively. To evaluate values re-peated thro ugho ut the experiment, variance analysis was used and co mplemented with Tukey’s co ntrast test. In o rder to co rrelate the two variables we used Pearso n’s co rrelatio n test. The level o f significance was set at P < 0.05 (5%). Results sho wn in the text and tables are expressed as mean and standard deviatio n.
RESULTS
After 1 mo nth o f HRT d-pyr values already pre-sented a decrease o f 34% when co mpared with the baseline values (P < 0.001), and at 2 mo nths, the pyr decrease was 14% (P < 0.05). The decrease pro gressed up to 1 year o f HRT reaching 28.9% (P < 0.0002) fo r pyr and 42% (P < 0.0002) fo r d-pyr (Table 1).
Regarding bo dy mass, an increase in that o f the co lumn, femo ral neck and tro chanter was o bserved after 1 year o f HRT. BMD values were 4.7% (P < 0.0004); 2% (P < 0.002); 3% (P < 0.01) higher than the baseline values, respectively (Table 2).
On co mparing pyr and d-pyr baseline values and tho se after HRT with BMD values fo r all analyzed sites, no co rrelatio n co uld be fo und with pyr. D-pyr co rre-lated with co lumn BMD in the first mo nth o f
treat-Table I - Pyridinium cross-links be fore and afte r hormone re place me nt the rapy
After Trea tment
Va ria ble ba sa l 1 month 2 months 4 months 1 yea r
pyr pmol/ umol 4 1 .4 (1 7 ) 3 5 .6 (2 0 ) 3 5 .2 (2 1 )* 3 2 .3 (1 5 )* * 3 0 .7 (1 7 )* * *
d-pyr pmol/ umol 5 .0 (3 .8 ) 3 .3 (3 .9 )* * 3 .4 (4 .4 ) * 4 .7 (1 1 .8 ) 2 .9 (5 .2 )* * *
* * * P < 0 .0 0 0 2 ; * * P < 0 .0 0 1 ; * P < 0 .0 5 basal vs. after treatment; values in mean and standard deviatio n.
5
ment (P < 0.02; r = -0.42).
Befo re starting HRT we fo und 21 o steo po ro tic wo men (39.6%) (Z sco re = -2.50 standard deviatio ns as co mpared with yo ung adults); 8 o steo penic wo men (15%) (Z sco re between -1.00 and 2.50 standard devia-tio ns as co mpared with yo ung adults). No co rreladevia-tio n between BMD, fo r either o steo po ro tic o r o steo penic wo men with baseline pyr and d-pyr levels, was fo und in these patients.
Baseline pyr and d-pyr values did no t co rrelate with bo dy mass index (BMI), age and time o f meno -pause o f the patients.
Baseline BMD was co rrelated with BMI (co lumn P < 0.02, r = 0.49; femo ral neck P < 0.03, r = 0.46; tro -chanter P < 0.0001, r = 0.58); age (co lumn P < 0.01, r = -0.31; femo ral neck P < 0.04, r = -0.30; tro chanter P < 0.01, r = -0.38); time o f meno pause (co lumn P < 0.01, r = -0.36; femo ral neck P < 0.03; r = -0.31; tro chanter P < 0.04, r = -0.31).
DISCUSSION
Estro genic deficiency o f meno pausal wo men leads to an increase in bo ne reso rptio n, while increase in fo rmatio n is seco ndarily increased due to pheno m-ena o ccurring in parallel.10 The rate o f bo ne lo ss is due
to an imbalance between the two pro cesses and it may be expected that a sensitive reso rptio n marker wo uld be able to reflect these changes in bo ne turno ver.
Acco rding to previo us studies, the meno pause leads to a significant increase in urinary pyridino line and deo xypyridino line excretio n. Uebelhart et al o b-served an increase o f 128% fo r pyr and 20% fo r d-pyr (in 24-ho ur urine samples) when co mpared with pre-meno pausal wo men in the age range o f 31 ± 6 years,11
sho wing that the reso rptio n pro cess is quite active. The effect o f HRT o n the bo ne mass o f meno -pausal wo men is mainly due to reductio n in reso rp-tio n, reflecting a decrease in the frequency o f activa-tio n in new bo ne remo deling units.12,13 In fact, urinary
pyr and d-pyr determinatio ns have sho wn that these substances are quite specific markers o f bo ne reso rp-tio n. Our findings were that within the first mo nth o f
HRT in the case o f pyr, and the seco nd mo nth fo r d-pyr, a significant decrease co uld be o bserved in co m-pariso n with the pretreatment values. This reinfo rces the value o f these markers, in additio n to demo nstrat-ing their early estro genic effect o n bo ne.
Uebelhart o bserved that after 6 mo nths o f HRT, pyr and d-pyr levels returned to premeno pausal val-ues.14 Ho wever, o ur findings sho wed a co ntinuo us
de-c re ase o f 28.9% and 42%, re sp e de-c tive ly, sim ilar to Hassager’s findings.1 It is pro bable that after this length
o f HRT, stabilizatio n o f the reso rptio n pro cess and co nsequently the marker levels o ccurs, in acco rdance with findings by Schlemmer, who se patients were fo l-lo wed-up fo r 10 years.15
Since the po stmeno pausal b o ne lo ss rate is determined b y the ratio o f fo rmatio n to reso rptio n pheno mena, we wo uld expect that sensitive reso rp-tio n markers such as pyr and d-pyr wo uld co rrelate with mineral b o ne density as assessed b y densito m-etry. Ho wever, except fo r d-pyr in the first mo nth o f HRT, which co rrelated with co lumn BMD, no co rrelatio n b etween markers and b o dy mass co uld b e o b -served, whether at b aseline co nditio ns o r after treat-ment. Other autho rs have o b tained the same results, reinfo rcing the idea that these markers are no t use-ful fo r the diagno sis o f patients with o steo po ro sis, b ut may help in therapeutic mo nito ring.14 In co ntrast
to Schlemmer, who fo und different pyr and d-pyr val-ues in o steo po ro tic and o steo penic patients diag-no sed b y BMD,15 o ur patients sho wed no difference
between the gro ups with o r witho ut o steo po ro sis and o steo penia. One explanatio n fo r this finding is that it may b e due to the fact that b o ne mass measure-ment, as assessed b y densito metry, is made o nly at so me sites such as the co lumn, femur o r radius and evaluatio n o f urinary markers reflects co llagen deg-radatio n o f the who le skeleto n, including different segments which may present different turno ver and b o ne lo ss rates.16
Mo st previo us studies o n urinary pyr and d-pyr excretio n refer to alteratio ns o bserved in the po stmeno pausal perio d in co mpariso n with the prestmeno -pausal co nditio n.5,14,17 There are no t many lo
ngitudi-nal studies that extend beyo nd 6 mo nths o f HRT.
CONCLUSION
We c o nc lud e that urinary p yrid ino line and deo xypyridino line excretio n decreases during ho rmo ne replacement therapy, reflecting a decrease in the bo ne reso rptio n rate. This decrease is early and is therefo re helpful in the evaluatio n o f therapeutic efficacy.
Table 2 - Bone mine ral de nsity be fore and afte r hormone re place me nt the rapy
BM D•, g/ cm2
Loca tion ba sa l 1 yea r
Lumba r spine 0 .9 7 (0 .1 8 ) 1 .0 2 (0 .1 8 )* * *
N eck 0 .8 2 (0 .1 3 ) 0 .8 4 (0 .1 2 )* *
Trocha nter 0 .7 0 (0 .1 4 ) 0 .7 2 (0 .1 3 )*
* * * P < 0 .0 0 0 4 ; * * P < 0 .0 0 2 ; * P < 0 .0 1 basal vs. after treatment; values in mean and standard de\viatio n. • Bo ne mineral density
6
1. Hassager C, Co lwell A, Assiri AMA, Eastel R, Russel RGG, Christiansen C. Effect o f meno pause and ho rmo ne replacement therapy o n urinary excretio n o f pyridinium cro ss-sectio nal study. Clinical Endo crino lo gy 1992;37:45-50.
2. Belchetz PE. Ho rmo nal treatment o f po stmeno pausal wo men. N Engl J Med 1994;330(15):1062-71.
3. Nilas L, Ch ris tian s e n C. Th e p ath o p h ys io lo g y o f p e ri an d po stmeno pausal bo ne lo ss.Br J Obstet Gynaeco l 1989;96:580-7.
4. No rdin BEC, Po lley KJ. Metabo lic co nsequences o f the meno pause: a cro ss-sectio nal, lo ngitudinal, and interventio n study o n 557 no rmal po stmeno pausal wo men. Calcif Tissue Int 1987;41(Suppl 1):1-58.
5. Prestwo o d KM, Pilbeam CC, Burleso n JA, et al. The sho rt term effects o f co njugated estro gen o n b o ne turno ver in o lder wo men. J Clin Endo crino l Metab 1994;79:366-71.
6. Brincat M, Mo niz CF, Kabalan S, et al. Decline in skin co llagen co ntent and metacarpal index after the meno pause and its preventio n with sex ho rmo ne replacement. Br J Obstet Gynaeco l 1987;94:126-9.
7. Delmas PD, Schlemmer A, Gineyts E, Riis BJ, Christiansen C. Urinary excretio n o f pyridino line cro ss-links co rrelates with bo ne turno ver measured in iliac crest bio psy in patients with vertebral o steo po ro sis. J Bo ne Min Res 1991;6:639-44.
8. Eyre DR. Co llag e n c ro s s - lin kin g am in o ac id s . Me th En zym o l 1987;144:115-39.
9. Black D, Duncan A, Ro bins SP. Quantitative analysis o f the pyridinium cro ss-links o f co llagen in urine using io n-paired reversed phase high-perfo rmance liquid chro mato graphy. Analytical Bio chem 1988;169:197-203.
REFERENCES
10. Parfitt AM. The co ntributio n o f bo ne histo lo gy to understanding the patho genesis and impro ving the management o f o steo po ro sis.Clin Invest 1982;5:163-7.
11. Uebelhart D, Gineyts E, Chapuy MC, Delmas PD. 1990. Urinary excretio n o f pyridinium cro ss-links: a new marker o f bo ne reso rptio n in metabo lic bo ne disease. Bo ne Mineral 1982;8:87-96.
12. Riggs BL, Jo wsey J, Go ldschmit RS, Kelly PJ, Ho ffman DL, Arnaud CD. Sho rt and lo ng term effects o f estro gen and synthetic anabo lic ho rmo ne in po stmeno pausal o steo po ro sis.J Clin Invest 1972;51:1659-63.
13. Parfitt AM. Treatment o f o steo po ro sis: theo retical po ssibilities. Clin Invest Med1982;5:181-3.
14. Uebelhart D, Schlemmer A, Jo hansen JS, Gineyts E, Christiansen C, Delmas PD. Effect o f meno pause and ho rmo ne replacement therapy o n the urinary excretio n o f pyridinium cro ss-links.J Clin Endo crino l Metab 1991;72:367-73.
15. Sc hle m m e r A, Hassage r C, De lm as PD, Christianse n C. Urinary excretio n o f pyridinium cro ss-links in healthy wo men: the lo ng term e ffe c ts o f m e no p ause and e stro ge n/p ro ge ste ro ne the rap y. Clin Endo crino l 1994;40:777-82.
16. Parfitt AM. Bo ne remo deling: relatio nship to the amo unt and structure o f bo ne, and the patho genesis and preventio n o f fractures. In: Riggs BL, Melto n LJ, edito rs. Osteo po ro sis. New Yo rk: Raven Press;1988:45-94.
17. Marab in i R, Sirto ri P, Ch io n n a R, Barzizza L, Ru b in ac c i A. Galacto sylhydro xylysine and pyridinium cro ss links in mo nito ring the bo ne respo nse to ho rmo ne replacement therapy. J Endo crino l Invest 1996;19:154-8.
r e s u m o
CON TEXTO: A perda ó ssea acelerada o bservada na pó s meno pausa é atribuída a um incremento do turno ver ó sseo , co m predo mínio da reabso rção em deco rrência da falência o variana. Ao co ntrário da fo rmação tem sido difícil de estabelecer um marcado r sensível de reabso rção .
OBJETIVOS: Avaliar o efeito da terapia de repo sicão ho rmo nal (TRH) na densidade e no s marcado res urinário s de reabso rção ó ssea, piridino lina (PIR) e deo xipiridino lina (D-PIR).
TIPO DE ESTUDO: Estudo co o rte de co rrelação .
LOCAL: Centro universitário de referência.
PACIEN TES: 5 3 mulheres meno pausadas há 3 ,6 ± 5 ano s, idade mediana de 5 3 ano s.
VARIÁVEIS ESTUDADAS: A excreção urinária de PIR e D-PIR na urina de jejum ,analisada po r HPLC e expressa em pmo l / mmo l de creatinina ,fo i avaliada em co ndiçõ es basais e apó s 1 ,2 ,4 e 1 2 meses de TRH. Realiz aram densito metria ó ssea (DO ) em co luna e fêmur antes e apó s 1 2 meses de TRH.
RESULTADOS: A DO apó s TRH fo i 4 .7 %(P<0 .0 0 0 4 ); 2 %(P<0 .0 0 2 ); and 3 %(P<0 .0 1 ) maio r que o s valo res basais na co luna , co lo do fêmur e tro canter respectivamente. O bservo u-se um decréscimo do s valo res de PIR e D-PIR preco ce e pro g ressivo , ating indo 2 8 ,9 % (P<0 .0 0 0 2 ) e 4 2 % (P<0 .0 0 0 2 ) apó s 1 ano de TRH respectivamente. N ão ho uve co rrelação entre o s valo res de DO e o s de PIR E D-PIR.
CON CLUSÕES: A excreção urinária de PIR e D-PIR co nstituem um marcado r sensível da reabso rção ó ssea.O decréscimo preco ce a to rna útil na avaliação da eficácia terapeutica.
PALAVRAS-CHAVE: Excreção urinária de piridino linas. Terapia de repo sição ho rmo mal. Remo delação ó ssea.
Dolore s Pe rovano Pardini, MD, PhD. Universidade Federal de São Paulo / Esco la Paulista de Medicina, São Paulo , Brazil.
Anibal Tagliafe rri Sabino, MD. Department o f Obstetrics, Universidade Federal de São Paulo /Esco la Paulista de Medicina, São Paulo , Brazil.
Ana Maria Me ne se s, MD. Universidade Federal de São Paulo /Esco la Paulista de Medicina, São Paulo , Brazil.
Te re sa Kasamatsu. Department o f Medicine, Universidade Federal de São Paulo /Esco la Paulista de Medicina, São Paulo , Brazil.
José Gilbe rto He nrique s Vie ira, MD, PhD. Universidade Federal de São Paulo /Esco la Paulista de Medicina, São Paulo , Brazil.
Source s of funding: No t declared.
Conflict of inte re st: No t declared.
Last re ce ive d: 4 August 1999.
Acce pte d: 17 August 1999.
Addre ss for corre sponde nce :
Do lo res Pero vano
Disciplina de Endo crino lo gia, Departamento de Medicina, Universidade Federal de São Paulo /Esco la Paulista de Medicina
Caixa Po stal 20.266
São Paulo /SP - Brasil - CEP 04034-970 E-mail: dpardini@ uo l.co m.br
p u b lis hin g in fo r m a t io n
