Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429

Attainment of peak bone mass and bone turnover rate in relation to estrouscycle, pregnancy and lactation in colony-bred Sprague–Dawley rats:Suitability for studies on pathophysiology of bone and therapeutic

measures for its management

S. Senguptaa, M. Arshada, S. Sharmaa, Manoj Dubeyb, M.M. Singha,∗a Division of Endocrinology, Central Drug Research Institute, Lucknow, India

b Department of Endocrinology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India

Received 7 October 2004; accepted 7 December 2004

Abstract

Alteration in biochemical markers of bone turnover and bone mineral density (BMD) of whole body and isolated femur and tibia inf bone and, 39.3

and urinese in bodyine ratio.m calciumproestrusphatase andnificantlytersted femurrus stages ofn proximalever, clearaft)over,over duringe resorptionrate during

rs; BMD of

relation to age, estrous cycle, pregnancy and lactation and suitability of use of rat as model for studies on pathophysiology otherapeutic measures for its management were investigated. Immature rats (1, 1.5 and 2 month of age; weighing, respectively± 1.0,67.8± 2.4 and 87.2± 5.2 g) exhibited high rate of bone turnover, as evidenced by high serum osteocalcin and alkaline phosphatasecalcium/creatinine ratio. However, their BMD (whole body or of isolated long bones) was below measurable levels. Marked increaweight at 3 months (185.5± 5.2 g) was associated with low serum osteocalcin and alkaline phosphatase and urine calcium/creatinBiochemical markers and BMD attained at puberty at 3 months were maintained until 36 month of age. No significant change in seruwas observed with increasing age or on any of the biomarkers during estrous cycle, and BMD of femur and tibia isolated duringand diestrus stages was almost similar. Onset of pregnancy was associated with significant increase in serum total alkaline phososteocalcin levels, but serum calcium, urine calcium/creatinine ratio or BMD of whole body or isolated long bones were not sigdifferent from that at proestrus stage. No marked change, except increase in body weight (P< 0.05), was also evident in these paramebetween days 5 and 19 of pregnancy, irrespective of number of implantations in the uterus. A significant decrease in BMD of isola(neck and mid-shaft regions) was observed on days 5 and 21 of lactation as compared to that during pregnancy or diestrus/proestestrous cycle; the decrease being almost similar in females lactating two or six young ones. BMD of isolated tibia (global and regioto tibio-fibular separation point), though generally lower than that during cycle and pregnancy, was statistically non-significant. Howevidence of occurrence of osteoporosis during lactation, with decrease in BMD of >2.5× S.D. in isolated femur (global, neck and mid-shas well as tibia (global) was observed only when BMD data was analysed onT-/Z-score basis. Serum biochemical markers of bone turntoo, were significantly increased in comparison to cyclic rats. Findings demonstrate marked increase in body weight and bone turnfirst 3 months of age, direct correlation between peak bone mass and onset of puberty at 3 months of age and increase in bonrate during lactation. Finding of the study while might suggests possible use of rat as useful model for studies on bone turnoverlactation and post-weaning periods and extrapolation of the result to the human situation, but not in relation to ageing.© 2005 Published by Elsevier Ltd.

Keywords: Bone turnover; Peak bone mass; Ageing; Puberty; Estrous cycle; Pregnancy; Lactation; Sprague–Dawley rats; Biochemical markeisolated bones

sorosising

∗ Corresponding author. Tel.: +91 522 2613894(O)/2310757(R);fax: +91 522 2623405.

E-mail address:singhmm@rediffmail.com (M.M. Singh).

1. Introduction

With continuing demographic shift in population towarda more aged society, age-related diseases such as osteophave emerged as major public health problem. Normal age

0960-0760/$ – see front matter © 2005 Published by Elsevier Ltd.doi:10.1016/j.jsbmb.2004.12.039

422 S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429

is associated with loss of bone mineral density (BMD) anddisruption in balance of bone turnover rate and changes incrystalline properties of bone mineral deposits[1]. In human,hypogonadism in both sexes is one of the major determinantsof rapid bone loss. Marked age-related changes in bone mass,bone strength and trabecular structure may begin in womenafter menopause and this loss in men may also occur after 60years of age[2–4].

Alteration in calcium metabolism with ageing has beenattributed to a variety of different and highly interdependentmechanisms including inherent functional changes in bonecells[5] and, at least in part, to a combination of hormonal andnutritional factors that impair regulation of calcium home-ostasis[6]. Apart from estrogen, several other hormones,including 1,25-dihydroxy vitamin D, parathyroid hormone,thyroid hormone, growth hormone, insulin like growth factor-I, other growth factors and cytokines and possibly calcitoninregulate calcium homeostasis and bone turnover rate. Nutri-tional factors, including deficiencies of Vitamin D, calcium,Vitamin K and minerals such as boron, zinc, magnesium andcopper, can interfere with normal operation of this regulatorysystem[7,8].

Onset of puberty and achievement of peak bone massare very important parameters. In case of humans, age of20–30 is known as age of peak bone mass and levels at-tained remain almost static until the age of 50 years inw n inm l sta-t ringt ofc andm sta-s doesn ins sites[

fore poro-s hileo ulatep finedw tudyi eing,e maleS imalm euticm hichi

2

2

rats,m

ternate 12-h light:12-h dark periods and free access to regu-lar pellet diet (Lipton India Ltd., Bangalore) and tap water,were used in this study. For age-related study, 21-day-oldfemale rats were randomized into nine groups of eight ratseach and kept in batches of four in plastic cages containingdry rice husk. Body weight, whole body densitometry andblood and urine samples of each rat were taken when theyattained 1, 1.5, 2, 3, 6, 9, 18, 24 and 36 months of age andwere autopsied at each time interval for collection of longbones (femur and tibia) for densitometry. For studies relatedto estrous cycle, pregnancy and lactation, 3–4-month-old vir-gin female rats (180–220 g body weight) were used. Femalesshowing diestrus (leucocytes) and proestrus (rounded epithe-lial cells with large central nucleus and rare cornified cells)type of vaginal smear picture were isolated. For studies dur-ing pregnancy and lactation, female rats were mated to co-eval males of proven fertility (day 1: day of sperm positivevaginal smear), randomized and kept individually in plasticcages. In case of rats autopsied on day 19 of pregnancy, num-ber and status of implantations in their uteri were recorded.At parturition, number of suckling young ones was adjustedrandomly to two or six per lactating female. Body weight,whole body scans and blood and urine samples of cyclic,pregnant and lactating rats were taken. Urine samples of day19 pregnant and day 5 lactating rats were not taken to avoidstress.

2

gesfi inga r first2 ivedo ines d at2a

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unc-t erumw sw teruso otteda dis-si tym

2

per-f me-t po-m soft-

omen and 60 years in men. Pregnancy and lactatioammals induce considerable changes in hormona

us as well as mineral and skeletal metabolism. Duhese physiological states[9], there is increased demandalcium and phosphate for fetal bone mineralizationilk production. In humans, although calcium homeo

is is altered during pregnancy, substantial bone lossot typically occur[10–13]. In contrast, lactation resultsubstantial bone loss, particularly at cancellous bone13].

Development of a suitable laboratory animal modelvaluation of potential agents for management of osteois is a prime need of drug research and development. Wvariectomized rat model has been demonstrated to simost-menopausal state in women, it has remained ill deith respect to ageing, pregnancy and lactation. This s

s aimed to characterize bone turnover rate during agstrous cycle, pregnancy and lactation in colony-bred feprague–Dawley rats and evaluate suitability of rat as anodel for studies on pathophysiology of bone and therapeasures for its management, no clear correlation for w

s so far available.

. Materials and methods

.1. Animals and experimental design

Colony-bred female and male Sprague–Dawleyaintained under standard conditions (22± 1◦C) with al-

.2. Urine collection

Rats were caged individually in all-glass metabolic catted with steel mesh for a total period of 48 h precedutopsy and had free access to pellet diet and tap water fo4 h of acclimatization. During next 24 h, animals recenly tap water ad libitum. Twenty-four hour fasting uramples were collected in fresh containers, centrifuge000 rpm at room temperature and stored at−20◦C untilnalyzed.

.3. Autopsy and collection of tissue

About 5 ml blood samples were collected by cardiac pure from each rat under light ether anesthesia and sas isolated and stored at−20◦C until analyzed. Animalere then autopsied by excessive ether inhalation. Uf each rat in age-related study was excised, gently blnd weighed. Femur and tibia of each rat were thenected free of adhering tissue, fixed in 70% ethanol[14]n saline and stored at−20◦C until bone mineral densi

easurement.

.4. Bone mineral density measurement

Before autopsy, whole body scan of each rat wasormed on an Hologic QDR-4500A fan-beam densitoer, calibrated daily with Hologic hydroxyapatite anthroorphic spine phantom using manufacturer provided

S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429 423

ware for small animals. Scans were performed on animalsunder light ether anesthesia positioned prone with care toavoid superimposition of bones with standard callimation ofX-ray beam and scan speed of 1.67 mm/s (2.5 lines/mm).Tail was looped around to lie almost parallel to the ani-mal and was included in scan window. In case of wholebody densitometry, measurements were performed on dayof autopsy. BMD of isolated bones was measured usingidentical regions of interest (femur: global, neck and mid-shaft; tibia: global and region about 2 mm proximal totibio-fibular separation point) and scan speed of 1 mm/s(4 lines/mm).

2.5. Biochemical markers of bone turnover

Serum total alkaline phosphatase activity was esti-mated by commercially available kits (#396494; BoehringerMannheim, Germany). The assay is based on colorimet-ric estimation of p-nitrophenol formed after breakdownof p-nitrophenylphosphate by alkaline phosphatase. Ab-sorbance was read at 405 nm using pre-programmed semi-automatic photometer (model 5010, Boehringer Mannheim,Germany). For serum osteocalcin estimation, one stepsandwich ELISA using streptavidin technology (#1822047;Boehringer Mannheim, Germany) was used. Assay quantita-tively detects most important stable metabolite, N-terminalm asec t os-t Thisa urest ure-m y isb nti-b inityo 44,w ocal-c con-t kits( d ontc s inu ratiotc rcialk sur-i inew ro-v vari-a m-i

2

ancef answ

3. Results

3.1. Body weight

There was a marked increase in body weight between 1 and3 months of age with maximum increase (113%;P< 0.01)occurring between second and third months of age coincid-ing with the period of onset of puberty. While peak bodyweight of 243.3± 12.8 g was attained at 18 months of age,the successive increase between 3 and 6, 6 and 9, 9 and 18months of age was only of the order of 12.6% (P< 0.05),6.6 and 9.1% (P< 0.05), respectively. This was followed bygradual decrease in body weight to reach almost the 3 monthlevel at 36 months of age. There was no apparent differencein body weight of rats in diestrus or proestrus stages of es-trous cycle. Body weight also remained almost unaltered un-til day 5 of pregnancy. This was followed by statisticallysignificant (P< 0.05) increase on day 19 of pregnancy dueprobably to inclusion of weight of fetuses, which decreasedto almost proestrus level following parturition (day 19 post-coitum versus days 5 or 21 post-partum,P< 0.05). There wasno difference in body weight in rats lactating two or six youngones, adjusted randomly on day of parturition, until day 21post-partum.

3.2. Uterine weight

r per1 f age,w ally,m sa

3

evelsf d at1 reasei rkedd werem ver-s t2 nthso dt ge ofc teo-c y ond cep-t r-i ays 5oA llow-i velr tage.N reg-n ef-

iddle fragment (N-mid fragment; aa 1–43), of proteleavage between aa 43 and 44, in addition to intaceocalcin, which is unstable under routine conditions.ssay, performed routinely on day of sampling, ass

hat short interim time between sampling and measents had no effect on results. Specificity of assaased on the fact that it employs two monoclonal aodies specifically directed against epitopes in the vicf N-terminal or N-side labile amino acid bridge 43–hich do not cross-react with other endogenous ostein fragments. Assay for measurement of calcium ionent in serum and urine samples using commercial#1553593; Boehringer Mannheim, Germany) was basehe principle that calcium forms a violet complex witho-resolphthalein complexone in alkaline medium. Valuerine samples were represented as calcium/creatinine

o correct variation in individual urine volumes[15]. Urinereatinine was estimated colorimetrically using commeits (#977721; Boehringer Mannheim, Germany), meang rate of formation of coloured complex by creatinith picrate in alkaline medium. All the above kits are pided with internal standards. The inter- and intra-assaytions for biomarkers and BMD were within normal li

ts.

.6. Statistical analysis

Means of groups were compared by analysis of variollowed by multiple comparison. Individual testing of meas done by Newman–Keuls test.

Maximum uterine weight (represented on absolute o0 g body weight bases), was also attained at 3 months ohich was maintained until 36 months of age. Incidentaximum increase (482%,P< 0.01) in uterine weight walso observed between 2 and 3 months of age.

.3. Biochemical markers of bone turnover

Serum total alkaline phosphatase and osteocalcin lollowed almost similar pattern, with high levels observe, 1.5 and 2 months of age. There was a significant dec

n these parameters at 2 months of age, followed by maecrease to reach low levels at 3 months of age, whichaintained until 36 months of age (1, 1.5 or 2 months

us 3, 6, 9, 18, 24 or 36 months,P< 0.01;Fig. 1). Levels amonths of age, though lower than that at 1 or 1.5 mo

f age, were significantly higher (P< 0.01) when compareo those at 3 months of age. There was no effect of staycle on serum total alkaline phosphatase activity or osalcin concentration. Their levels increased significantlay 5 of pregnancy (i.e., day of maximal endometrial re

ivity to blastocyst signals[16]) and were maintained dung entire pregnancy and lactation (proestrus versus dr 19 post-coitum or days 5 or 21 post-partum,P< 0.05).pparent decrease in alkaline phosphatase activity fo

ng parturition was statistically non-significant and the leemained significantly higher than that at proestrus sumber of implantations in the uterus on day 19 of pancy or number of suckling young (two or six) had no

424 S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429

Fig. 1. Age-related changes in serum and urine biochemical markers of bone turnover in colony-bred female Sprague–Dawley rats. Note high levels of serumtotal alkaline phosphatase (A) and osteocalcin (B) exhibiting high bone turnover rate at 1, 1.5 and 2 months of age followed by a significant decrease toreachlow levels at 3 months of age, which was then maintained until 36 months. Serum calcium concentration (C) remained almost unaltered throughout 1–36months of age, but high rate of excretion of calcium in urine, based on high calcium/creatinine ratio (D) was observed until 2 month of age. The low levelofcalcium excretion attained at 3 months of age was maintained until 36 months of age. (a)P< 0.05 and (b)P< 0.01, vs. corresponding preceding age group; (c)P< 0.05, vs. corresponding 1 month age group; (d)P< 0.01, vs. corresponding 1.5 and 2 month age groups; (f)P< 0.01, vs. corresponding 3 month age group.All other relevant comparisons were statistically non-significant.

fect on serum total alkaline phosphatase or osteocalcin levels(Fig. 2).

Serum calcium concentration remained almost unalteredthroughout 1–36 months of age, different stages of estrouscycle, pregnancy and lactation in rats, irrespective of dif-ferent number of implantations during pregnancy and num-ber of suckling young ones during lactation (Figs. 1 and 2).High rate of excretion of calcium in urine, based on high cal-cium/creatinine ratio, in rats was observed until 2 month ofage. Low level of calcium excretion attained at 3 months ofage was maintained until 36 months of age (1, 1.5, 2 monthsversus 3, 6, 9, 18, 24 or 36 months of age,P< 0.05 toP< 0.01;Fig. 1). There was also no apparent effect of stage of es-trous cycle, pregnancy or lactation on calcium/creatinine ra-tio (Fig. 2).

3.4. Bone mineral density

Whole body (global) BMD remained very low until 2months of age. This was followed by a marked (P< 0.01)increase to reach peak levels at 3 months of age, which wasthen maintained until 36 months of age (1, 1.5 or 2 monthsversus 3, 6, 18, 24 or 36 month of age,P< 0.01;Fig. 3). In-terestingly, BMD (global) of long bones (femur and tibia) at1, 1.5 and 2 months of age was below measurable limits of

densitometer even when they were taken together, in situ orafter isolation, in the same window. Stage of estrous cycle,pregnancy or lactation had no effect on whole body BMD inrats (Table 1). As in case of whole body scans, peak BMDlevels of isolated femur and tibia (global as well as all regionsof interest, viz. neck and mid-shaft of femur and region prox-imal to tibio-fibular separation point of tibia) was observed at3 months of age and was maintained until 36 months of age,with BMD of femur being generally more than that of tibia atall time intervals (Fig. 3). There was, however, no significantdifference in BMD between neck and mid-shaft of isolatedfemur or TFSP region of isolated tibia. BMD of isolated fe-mur and tibia as well as their regions of interest evaluatedwas almost similar during diestrus and proestrus stages ofestrous cycle and remained unaltered until day 19 of preg-nancy. A significant decrease in BMD of isolated femur (neckand mid-shaft) was observed on days 5 and 21 of lactationas compared to that during pregnancy or at diestrus/proestrusstages of estrous cycle; the decrease being almost similarin females lactating two or six young ones (Table 1). BMD(global) of isolated femur on day 21 of lactation, thoughlower than that on days 5 (7.7 and 9.0% in females lactat-ing two or six young ones, respectively) and 19 (12.7 and13.9%) of pregnancy or day 5 of lactation (4.4%) was signif-icant (P< 0.05) only in comparison to cyclic (proestrus: 13.3

S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429 425

Fig. 2. Change in serum and urine biochemical markers of bone turnover during estrous cycle, pregnancy and lactation in colony-bred adult femaleSprague–Dawley rats. Note almost similar levels of serum total alkaline phosphatase activity (A) and osteocalcin concentration (B) during diestrus andproestrus stages of estrous cycle. The levels increased significantly on day 5 post-coitum (i.e., the day of maximal endometrial receptivity to blastocyst signals)and were maintained during the entire pregnancy and lactation. There was also no apparent effect of stage of estrous cycle, pregnancy or lactation on serumcalcium (C) or urine calcium/creatinine ratio (D). Number of implantations in the uterus on day 19 of pregnancy or number of suckling young [two or six,adjusted randomly at parturition] had no significant effect on their levels. Di: diestrus, Pro: proestrus; (a)P< 0.05 and (b)P< 0.01, vs. corresponding proestrusgroup. Urine samples of day 19 pregnant and day 5 lactating rats were not taken to avoid stress. All other relevant comparisons were statistically non-significant.

and 14.5%; diestrus: 12.7 and 13.9%) females. In compari-son, isolated tibia (global and region proximal to TFSP) didnot present any change/decrease in BMD during lactation.Clear evidence of occurrence of osteoporosis during lacta-tion with decrease in BMD of >2.5× S.D. in isolated femur(global, neck and mid-shaft) as well as tibia (global) wasobserved only when BMD data was analysed onT-/Z-scorebasis (Table 2).

4. Discussion

With continuing demographic shift in population towardsmore aged society, age-related diseases including osteoporo-sis have emerged as major public health problem. Normalageing in humans is known to be associated with disruptionin the balance of bone turnover rate, changes in crystallineproperties of bone mineral deposits and loss of bone mineraldensity[1,2]. Results of this study demonstrate marked in-crease in body weight and bone turnover rate during first 3months of age. There was a direct correlation between peakbone mass and onset of puberty at 3 months of age and,

as in human, increase in bone resorption rate during lacta-tion. However, lack of effect on biochemical markers of boneturnover and BMD even until 36 months of age, after attain-ment of peak bone mass, suggests unsuitability of use of ratas model for development of therapeutic measures for man-agement of age-related osteoporosis in humans. Pertinently,estrogen deficiency following ovariectomy leads to increasedbone turnover and the status 28 days post-ovariectomy in ratsis considered to be comparable to that in post-menopausalwomen[17] and is known to serve as a good laboratory an-imal model for study on pathogenesis and management ofestrogen deficiency (including post-menopausal) osteoporo-sis[18].

We have also not observed any major difference in boneturnover rate, based on serum and urine biochemical markersand BMD, between diestrus and proestrus stages of estrouscycle in rat. Reports of significantly higher levels of serumPTH [19] and 1,25-(OH)2 vitamin D at or around the timeof ovulation[20] and serum osteocalcin peak during lutealphase[21] have been reported. While proestrus stage in ratcorresponds to the time of ovulation/mid-cycle in primates,lack of effect of stage of estrous cycle on markers of bone

426 S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429

Fig. 3. Age-related changes in bone mineral density of whole body (A) and regions of interest of isolated long bones (B–F) in colony-bred femaleSprague–Dawley rats. Note very low whole body BMD (A) until 2 months of age, followed by marked increase to reach peak levels at 3 months, whichwas then maintained until 36 months of the age. BMD of femur (B–D) and tibia (E and F) at 1, 1.5 and 2 months of age was below measurable limits ofdensitometer even when they were taken together in situ or after isolation in the same window. Peak BMD levels of isolated femur and tibia (global as well asall regions of interest) were observed at 3 months of age and were maintained until 36 months of age, with BMD of femur being generally more than that oftibia at all time intervals. TFSP: window about 2 mm proximal to tibio-fibular separation point. Other conditions are the same as inFig. 1.

turnover in rat in this study might be due comparatively toshort (4–5 days) duration of estrous cycle and absence of afunctional luteal phase in each cycle in rat and other labora-tory rodents. It may be pertinent to mention that reports ofabsence of any change in serum PTH, calcitonin, osteocal-cin and 1,25-(OH)2 vitamin D levels during entire menstrualcycles in women are also available[20,21].

Pregnancy and lactation in mammals induce considerablechanges in hormonal status as well as in mineral and skele-tal metabolism. There is increased demand of calcium andphosphate for fetal bone mineralization and milk production.It has been shown that there is increase in intestinal absorp-tion of calcium associated with elevated levels of 1,25-(OH)2vitamin D. It is believed that increasing requirements of cal-

cium and phosphate for development of fetal rat are also metthrough increased mobilization of minerals from maternalskeleton[9]. We have observed significant increase in bodyweight on day 19 of pregnancy when compared with cyclic orday 5 post-coitum rats, due probably to inclusion of weightof the fetuses. There was also a significant increase in serumbiochemical markers of bone formation viz. serum total al-kaline phosphatase and osteocalcin in pregnant rats, but theBMD levels (whole body or of isolated long bones) or urineexcretion of calcium (calcium/creatinine ratio) were not sig-nificantly altered. While high levels of serum total alkalinephosphatase observed on day 19 of pregnancy in this studymight also be due to increased secretion of its placental iso-form, similar levels of serum alkaline phosphatase, bone Gla

S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429 427

Tabl

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Cha

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(g/c

m2)

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1381

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0.14

18±

0.00

140.

1431

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0.14

72±

0.00

240.

1329

±0.

0095

0.13

38±

0.00

420.

1368

±0.

0019

0.13

47±

0.00

82

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1896±

0.00

330.

1910

±0.

0077

0.17

94±

0.00

400.

1896

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33±

0.00

200.

1708

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1772

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.

protein, urine crosslinks and bone mass (whole body andlumbar vertebrae) have been reported in non-pregnant andpregnant Cynomolgus monkeys[22]. In women, too, whilecalcium homeostasis is altered during pregnancy, substantialbone loss does not typically occur[10–13].

In contrast to pregnancy, lactation is known to result in asubstantial loss of bone mass, particularly at cancellous bonesites. Varying degree of bone loss during lactation has beenreported to occur in humans, monkeys, dogs, sheep, pigs, etc.,cf. [13]. Histomorphometric studies in dog indicate signifi-cantly elevated bone turnover during lactation, with greaterincrease in bone resorption than bone formation[13]. In thepresent study in rat, a significant decrease in BMD when com-pared to cyclic or pregnant rats suggesting loss of bone massduring lactation has been observed. However, clear evidenceof occurrence of osteoporosis during lactation, with decreasein BMD of >2.5× S.D. in isolated femur (global, neck andmid-shaft) and tibia (global) was observed only when BMDdata was analysed onT-/Z-score basis. Interestingly, the de-crease was almost similar to that observed 30 days after bilat-eral ovariectomy in our previous study[23], with decrease inBMD of >2.5× S.D. of the corresponding sham intact[23]or proestrus stage (this study) rats in all regions of interest(viz. femur: global, neck and mid-shaft; tibia: global), exceptTFSP region of isolated tibia, which registered no changeeither 30 days after ovariectomy in adult cyclic rats[23] or2 nes( -t unga ap ldr teds -oldf BMD[ isa d tod tinctf teo-p lvingb althf thersa ealths

onet sixy s inc dyw hisi e toh de-p ling[ im-p

nel iatedw loss

1 days post-partum in rats lactating two or six young oTables 1 and 2). Pertinently,T-/Z-score[24] compares paient’s bone mass with mean bone mass of normal yodult reference population (T-score) or with mean BMD oferson of the same age (Z-score), if applicable to rats, wouefer both toT- andZ-scores, since this study was conducimultaneously on randomized colony-bred 3–4-monthemale rats, an age when they achieve almost maximum23,25]. The analysis of BMD data onT-/Z-score basis in thnd earlier[25] studies further suggests the urgent neeefine threshold boundaries for laboratory animals, dis

rom those used clinically, to ascertain incidence of osorosis, osteopenia and normal states in studies invoone turnover including development of new drugs, he

oods, diets or supplements for pregnant/lactating mond ageing population and to effectively determine the htatus of animal colonies.

We did not observe any significant difference in burnover rate or BMD levels in rats lactating two oroung ones, randomly adjusted at parturition. This ionformation with lack of significant difference in boeight or any of the biomarkers of bone turnover. T

s interesting since in women lactation anovulation duypothalamus–pituitary–ovarian (H–P–O) suppressionends primarily on frequency and intensity of suck

26,27]. In rat and mouse, number of young suckling isortant in determining delay of implantation[28].

According to Krebs et al.[29], lactation associated booss and its recovery post-weaning is negatively associth parity. While a suggestion of attenuation of bone

428 S. Sengupta et al. / Journal of Steroid Biochemistry & Molecular Biology 94 (2005) 421–429

Table 2Evaluation of change in BMD in relation to estrous cycle, pregnancy and lactation based onT-/Z-score in female Sprague–Dawley rats

Parameter Femur Tibia

Global Neck Mid-shaft Global TFSPa

Mean± S.D. of rats in proestrus stage 0.1910± 0.0077 0.1943± 0.0063 0.1805± 0.0033 0.1768± 0.0036 0.1523± 0.00672.5× S.D. of rats in proestrus stage 0.0193 0.0158 0.0083 0.0090 0.0168

Proestrus vs. diestrusDifference in mean BMD −0.0014 −0.0042 +0.0011f −0.0062 +0.0017f

Proestrus vs. day 5 of pregnancyDifference in mean BMD −0.0116e −0.0023 −0.0033 −0.0117d −0.0012

Proestrus vs. day 19 of pregnancyDifference in mean BMD −0.0014 −0.0024 −0.0001 −0.0104d −0.0017

Proestrus vs. day 5 of lactationDifference in mean BMD −0.0177b,e −0.0288b,d −0.0176b,d −0.0232b,d −0.0123b,e

−0.0202c,d −0.0399c,d −0.0210c,d −0.0197c,d −0.0080c,e

Proestrus vs. day 21 of lactationDifference in mean BMD −0.0254b,d −0.0307b,d −0.0192b,d −0.0255b,d −0.0006b

−0.0277c,d −0.0301c,d −0.0187c,d −0.0173c,d −0.0041c

a Window about 2 mm proximal to tibio-fibular separation point (TFSP).b Females suckling two young ones, randomly adjusted on day 1 post-partum.c Females suckling six young ones, randomly adjusted on day 1 post-partum.d Decrease in BMD by >2.5× S.D. of corresponding control value is a condition commonly referred to as osteoporosis.e Decrease in BMD by >1.0× S.D., but <2.5× S.D., of the control value is a condition commonly referred to as osteopenia[24].f Increase in BMD over corresponding control value.

by generous dietary ratio of calcium to protein has been made[29], several studies demonstrating lack of preventive effectof calcium supplementation on bone loss during lactation orpost-weaning are available[30–33]. According to a study infemale Cynomolgus monkeys, having close similarities towomen in reproductive physiology and skeletal structure, re-covery in bone mass even after 6 months post-weaning wasnot large enough to offset loss that had occurred during first 4months of lactation[22]. Considering this and the increasedrisk of loss of bone mass particularly in women with closelyspaced pregnancies, women nursing more than one child orlactating adolescents who might still not have attained peakbone mass[32], need for development of an agent that caneffectively prevent increased bone resorption during lacta-tion and/or augment bone mineralization during immediatelypost-weaning period in women can hardly be overempha-sized. However, since most chemicals/hormones ingested bynursing mother are excreted in milk and find their way intobody/system of suckling infant and their potential adverseeffects on newborn and infant health[34], need for develop-ment of alternative effective drug regimens with safer profilesin breast feeding or timing of drug dosing to minimize accu-mulation in breast milk has been suggested[34]. Ability ofcertain SERMs[24,25,35–37]to prevent estrogen deficiencyosteoporosis[38] suggests their potential to prevent lacta-tion associated bone loss, which is also a hypo-estrogenics ce ana itht r-i ghts bone

turnover rate during lactation and post-weaning periods andextrapolation of result to human situation, but not in relationto ageing.

Acknowledgements

Authors thank Dr. C.M. Gupta, Director, for interest in thestudy, Dr. Vijaylakshmi Bhatia, Department of Endocrinol-ogy, Sanjay Gandhi Post-Graduate Institute of Medical Sci-ences, Lucknow, for BMD measurements, Dr. Mukesh Sri-vastava for statistical analysis of the data and Mr. B.P. Mishraand Mr. Jagdish Prasad for efficient handling of animals. Thisstudy received financial support from the Ministry of Healthand Family Welfare, Government of India. CDRI communi-cation no. 6367.

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