Relation Between Axial and Appendicular Skeletal Calcium and Body Weight in the Rat '

PAUL D. SAVILLE AND PATRICIA M. SMITH The Hospital for Special Surgery affiliated with The New York Hospital, Cornell University Medical Center, New York, New York

ABSTRACT The total calcium content as well as the calcium content of individual long bones has been measured in male and female rats from weaning to maturity and were found to be linear functions of body weight in both sexes. The calcium content of the total skeleton was a constant percentage of body weight in males but was a continuously increasing percentage of body weight in females. The calcium content of the tibia and femur expressed as a percentage of total body weight changed ten-fold between weaning and maturity in females but hardly changed at all in males. In mature animals above 250 gm, calcium is distributed in the different bones of the skeleton in the same proportions in both sexes though females contain much more calcium at any given body weight than males.

The total skeletal mass is of great clin- ical interest since a decrease in skeletal mass and a rising incidence of fractures occur with increasing frequency in old age. It is not possible to measure or estimate the skeletal mass with an accuracy of better than 25% in the living subject while the data on cadavers is meager and con- flicting (Mitchell et al., '45). Estimates of skeletal mass through measurement of osseous parts of extremities such as the sum of areas of compact bone in the mid- dle half of the femur (Trotter, '54) and ratios of the area of cortical layer of finger bones over total bone area (Virtama and Mahonen, '60) have been offered in the belief that they reflect skeletal mass as a whole. Forbes et al. ('53) found the skel- etal calcium content in the human body to be 99% of the total calcium. Sherman and MacLeod ( '25) showed that of the total calcium in the rat's body about 99% is in the skeleton.

The purpose of this investigation is to document the relationship between cal- cium content of the skeleton and body weight and further to clarify the relative contributions of the component parts of the skeleton to total skeletal calcium both in the male and female rat from weaning to maturity.

MATERIALS AND METHODS

Sixty male rats and 39 virgin female rats of the Charles River CD strain were

ANAT. kc., 156: 455460.

fed a standard commercial diet' and tap water and were sacrificed at predetermined ages from the time of weaning until 15 weeks of age.

Body weights of the rats were deter- mined immediately upon sacrifice. Car- casses were then eviscerated. Forelimbs and hindlimbs were disarticulated from the axial skeleton at the shoulder and hip respectively. One humerus, femur, tibia, and the remaining axial skeleton including skull were ashed separately in a muffle furnace at 500" C for 24 to 48 hours. The ash was dissolved in 2 N HC1 and brought up to predetermined volumes. Calcium content of the ashed bones and axial skele- tons was determined by a semi-automatic EDTA titration method (Mc Pherson, '65).

Individual bone calcium content was plotted as a function of body weight. Total skeletal calcium was plotted as a function of age. All regressions were calculated by the method of least squares. Values of bone calcium as percentages of total body weight and total body calcium were de- rived by substituting known values into the slope-intercept formula for a line.

1 This investigation was supported in part by Pub- lic Health Service Research grant .5. SO1 Fr-05495 from the Genexal Support Branch Diwsion of Research Facilities and Resources; U.S.P.H.S. Graduate Train- ing grant TIAM-5414 of the National Institutes of Arthritis and Metabolic Diseases; the Whitehall Foundation and Easter Seal.

2 Purina Laboratory Chow; Ralston Purina Com- pany, St. Louis, Mo.

455

456 PAUL D. SAVILLE AND PATRICIA M. SMITH

4.m-

3.500

3,000

- E 5 3 9 & 2,000-

2,500- -

0 m A

3 1,500

1.m

RESULTS Total body calcium of both male and

female rats was a sigmoid function of age (fig. 1 ) and a linear function of body weight (fig. 2). Moreover, the calcium content of individual bones was also linear functions of body weight. Figures 3 and 4 show the positive linear regression obtained when axal skeletal calcium and femur cal- cium respectively are plotted as a function of body weight. Table 1 shows the regres- sions of calcium content of whole body, axial skeleton, and individual bones as functions of body weight within each sex.

Above 100 gm weight, female rats had more total body calcium as well as a higher calcium content of individual bones at any given body weight when compared to males. The increments of bone calcium with increments of body weight in females is significantly greater than the correspond- ing increments of bone calcium with body weight in males (P < 0.001).

- -

-

-

-

Table 2 shows total skeletal calcium as a per cent of body weight. In the female rat the calcium content of the whole skele- ton is a steadily rising proportion of body weight but in the male rat, the tibia and humerus calcium is a relatively fixed per- centage of body weight and beyond the 300 gm range, the axial skeleton and femur also reach a constant proportion of body weight. Table 2 also shows that in the female rat, the femur calcium and tibia calcium are only 0.004% of body weight at 50 gm but this rises about ten-fold so that at 300 gm the femur is 0.045% of body weight while the tibia calcium is 0.036% of body weight. The male femur and tibia calcium at 50 gm is five and seven times greater percentage of total body weight than it is in females but rises more slowly than it does in the female and never attains the high values found in the female sex. Quite clearly, the calcium con- tent of female rats represents a greater

T O T A L B O D Y C A L C I U M O F N O R M A L M A L E A N D F E M A L E R A T S A S A F U N C T I O N OF A G E

500t * A ' 40 ' ' 50 ' ' 60 ' 70 ' ' 80 I ' 90 ' ' 100 " 110 ' I

AGE IN DAYS

Fig. 1 Each point represents the average calcium content of 6 or 7 animals except for the 91 day groups which represents 13 animals. The vertical bars represent plus and minus two standard errors.

RAT SKELETAL CALCIUM

5,000-

- 4,000 - E" - 5 2 3 , 0 0 0 -

!2 22,000-

0 z

2

X I a

1JQO-

0

457

- MALE, ~*6.2412x-28.9524

- -----Q EMALE, y-8.9664~-318.3784

I I I I I I I I 100 200 300 400 500 0

T O T A L B O D Y C A L C I U M v s B O D Y W E I G H T

5,000- - MALE, y=7.8919w -64.4763

---o FEMALE, y=11 .3790~-435 .9631 - 4,000 -

2 3,000 - 5

2 --J 2,Ooo - s

1,000-

I 1 I I J 0 100 200 300 400 500 0

BODY WEIGHT (gml Fig. 2 Each point represents total body calcium of 6 or 7 animals. The 400 grn male

group consisted of 13 animals. The regressions were calculated by the method of least squares. There is a significant slope difference. P < 0.001.

FEMUR C A L C I U M v s T O T A L B O D Y W E I G H T

200- MALE, y = . 3 8 2 4 ~ -8.9433

---0 FEMALE, y ,5285 x -24.1999 160 -

5 120 - Y 4 - 2 80-

40 -

I I I J 100 300 400 500

BODY WEIGHT @m, Fig. 4 Each point represents the average femur calcium of 6 or 7 animals. The 400 gm

group of males consisted of 13 animals. The regressions were calculated by the method of least squares. There is a sigmcant slope difference, P < 0.001.

TABLE 1 T h e regressions o f total body calcium, axial skeleton calcium, femur calcium, tibia calcium

were calculated by the method o f least squares and humerus' calcium respectively on body weight are listed. These regressions

Male Female

Total body calcium vs. body weight zz 11.3790~ - 435.9631 Femur calcium vs. body weight y = 0.3824~ - 8.9433 y = 0.5285~ - 24.1909 Tibia calcium vs. body weight y = 0.2847~ - 0.8468 y = 0.4218~ - 19.3159 Humerus calcium vs. body weight y = 0.1624~ - 0.5224 y = 0.2354~ - 6.9238

y = 7.8919~ - 64.4762 Axial skeletal calcium vs. body weight y = 6.241% - 28.9524 y = 8.9664~ - 318.3784

y = k x + b .

Calcium as a per cent of body weight for the whole skeleton, the axial skeleton, the femur, the tibia and the humerus in males and females between 50 and 500 gm are listed. The data was derived f r o m the regressions in table 1 by substituting into the formula y = kx+b, the slope ( k ] and the intercept (b ) from table 1 and body weights (x ) listed thereby deriving the calcium content o f the various parts of the skeleton (y).

TABLE 2

Total ca. Axial skeletal ca. Femur ca' Tibia ca. Humerus ca.

M F M F ~ Wt (1 bone) (1 bone) ( 1 bone )

M F M F M F

50 60 70 75 90 100 125 150 200 250 300 350 400 450 500

- 0.66 0.68 0.70 0.70 0.72 0.72 0.74 0.75 0.76 0.76 0.77 0.77 0.77 0.77 0.78

0.27 0.41 0.52 0.56 0.65 0.70 0.79 0.85 0.92 0.96 0.99

0.57 0.26 0.58 0.37 0.58 0.44 0.59 0.47 0.59 0.54 0.60 0.59 0.60 0.64 0.61 0.68 0.61 0.74 0.61 0.77 0.61 0.79 0.62 0.62 0.62 0.62

0.020 0.023 0.026 0.026 0.028 0.029 0,031 0.032 0.034 0,035 0.035 0.036 0.036 0.036 0.036

0.004 0.013 0.018 0.021 0.026 0.029 0,034 0.037 0.041 0.043 0.045

0.030 0.030 0.030 0.030 0.029 0.029 0.029 0.029 0.029 0.029 0.029 0.029 0.029 0.029 0.029

0.004 0.010 0.015 0.016 0.020 0.023 0.027 0.029 0.033 0.034 0.036

~~

0.015 0.015 0.015 0.016 0.016 0.016 0,016 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.016

0.010 0.012 0.014 0.014 0.016 0.017 0.018 0.019 0.020 0.021 0.021

y = kx+ b.

RAT SKELETAL CALCIUM 459

C A L C I U M A S A PER CENT OF BODY W E I G H T

- &-----Q

--------------------- ---~Total body calcium, female ---Axial skeleton calcium, female

-Total body calcium, male --+ Axial skeleton calcium, male

Fig. 5 Total body calcium and axial skeleton calcium of male and female rats was de- rived from the least square regressions listed in table 1, By substituting these regressions and known weights in the formula y = kx + b thus deriving y, calcium content of the body or axial skeleton, at the respective weights and then expressing this as a per cent of body weight.

TABLE 3

The calcium content of the axial sekleton, femur, tibia and humerus as a per cent of total skeletal calcium for males and females between 50 and 500 gm is listed. Calcium content of the various parts o f the skeleton was derived from the least squares regressions. in table 1 before being expressed as a percentage o f the total skeletal calcium.

Axial skeletal ca. Femur ca. Tibia ca. Humerus ca.

M F M F M F M F Wt.

50 85.8 97.7 3.1 1.7 4.6 1.3 2.3 3.6 60 84.5 89.0 3.4 3.0 4.4 2.4 2.2 2.9 70 83.6 85.8 3.6 3.5 4.3 2.8 2.2 2.7 75 83.2 84.8 3.7 3.7 4.2 2.9 2.2 2.6 90 82.5 83.1 3.9 4.0 4.1 3.2 2.2 2.4

82.1 82.4 4.1 4.1 4.0 3.3 2.2 2.4 81.5 81.3 4.2 4.2 3.9 3.4 2.1 2.3

100 125 150 81.1 80.8 4.3 4.3 3.9 3.5 2.1 2.2 200 80.5 80.2 4.5 4.4 3.8 3.5 2.1 2.2

4.5 4.5 3.8 3.6 2.1 2.2 300 80.0 79.6 4.6 4.5 3.7 3.6 2.1 2.1 350 79.9 4.6 3.7 2.1 400 79.8 4.7 3.7 2.1 450 79.7 4.7 3.7 2.1 so0 79.7 4.7 3.7 2.1

250 80.2 79.8

y = k x + b .

460 PAUL D. SAVILLE AND PATRICIA M. SMITH

DISCUSSION

Sherman and MacLeod ('25) showed that although male albino rats have more gross weight of calcium than female rats at any given age, female rats have more calcium at any given body weight. Our data confirms this finding and further shows quantitative differences in calcium content between various parts of the rat's skeleton and between individual bones of male and female rats.

The syndrome of osteoporosis in humans is very much more common in females than in males (Albright et al., '40) so that the finding of more skeletal calcium in females compared with male rats of the same weight at first suggests a species dif- ference between humans and rats. How- ever, Garn et al. ('63) measuring the corti- cal thickness of the second metacarpal in nearly 500 individuals, found that al- though men have thicker cortices at any given age compared with women, women have smaller bones than men, so that they have a greater amount of cortical bone in terms of cross sectional area or total bone volume than men. Furthermore, this greater bone density in women was main- tained until the sixth decade when there was a dramatic decrease of cortical bone per unit of cross sectional area so that older men had denser bone than older women. Comparison of total skeletal cal- cium and of long bone calcium in male and female rats corresponds rather closely to comparisons of bone mass per unit volume or cross sectional area of human meta- carpal before the sixth decade of life.

From this study it is apparent that body weight is a good predictor of total skeletal mass in the male rat but not in the female. Similarly, the humerus calcium content is also a constant proportion of total skeletal calcium while the femur and tibia calcium are only slightly less constant in the male. In the female, humerus calcium is fairly

constant but tibia and femur are less con- stant proportions of the skeletal calcium. Clearly, in the human adult, body weight which can vary greatly over a short time will not be a reliable predictor of skeletal calcium content. From this study it would seem that in normal rats the calcium con- tent of long bones is variably reliable as a predictor of skeletal mass. On the other hand, the fact that these relations change with age and sex and from bone to bone, suggests the possibility that in skeletal dis- ease one bone may not change at the same rate or in the same direction as the rest of the skeleton. Therefore, in keeping with the views of Forbes and associates ('53), caution is advocated in accepting densit- ometry of osseous extremities as accurately reflecting total skeletal changes in skeletal diseases.

LITERATURE CITED 1940

Postmenopausal osteoporosis. Tran. Assn. Am.

Forbes, R. M., A. R. Cooper and H. H. Mitchell 1953 Composition of adult human body as determined by chemical analysis. J. Biochem.,

1963 The developmental nature of bone changes during aging. In: Relations of Development and Aging. Ed. by J. E. Birren. Charles C Thomas, Springfield, Illinois. Chap. IV: 41-62.

Mc Pherson, G . E. 1965 Stable calcium isotopes as tracers in studies of mineral metabolism. Acta Orthop. Scan., Supp., 78: 4-86.

Mitchell, H. H., T. S. Hamilton, F. R. Steggerda and H. W. Bean 1945 The chemical composi- tion of the adult human body and its bearing on the biochemistry of growth. J. Biochem.,

Sherman, H. C., and F. L. MacLeod 1925 The calcium content of the body in relation to age, growth, and food. J. Biochem., 64: 429-459.

Trotter, M. 1954 A preliminary study of esti- mation of weight of the skeleton. Am. J. Phys. Anthrop., 12: 537-551.

Virtama, P., and H. Mahonen 1960 Thickness of the cortical layer as an estimation of min. era1 content of human finger bones. Brit. J. Radiol., 33: 60-62.

Albright, F., E. Bloomberg and P. A. Smith

Phys., 55: 298-305.

203: 359-366. Garn, S. M., C. G. Rohmann and P. Nolan

158: 625-637.