Percentage Ash Content of Nonhuman Primate Long Limb B o n e d DAVID B. BURR Department of Anutomy, University of Kunsas Medical Center, Kansas City, Kansas 66103
KEY WORDS Percentage ash weight cal fractions
. Methodology
. Macaque . Bone chemi-
ABSTRACT
Trotter and Hixon ('76) and Vose and Roach ('72) presented conflicting data about percentage ash content of the long limb bones of M. mulatta and M. nemestrina. The suggestion of Trotter and Hixon ('76) that this discrepancy was due to methods of preparing the bone prior to ashing, rather than to species differences, was tested using data collected by Gong ('72) on the volumetric composition of nonhuman primate bone. Masses of the various chemical fractions of bone reported by Gong ('72) were used to compute ash content as a percentage of total dry weight and ash content as a percentage of fat-free dry weight. Good agreement was found between ash content as a percentage of total dry weight and the data of Vose and Roach ('72); good agreement was also found between ash content as a percentage of fat-free dry weight and the data of Trotter and Hixon ('76). This suggests that the data of Vose and Roach ('72) conflict with those of Trotter and Hixon ('76) because Vose and Roach ('72) failed to defat the bones prior to ashing. No actual species differences exist in the ash content of bones of M. mulatta and M.nemestrina. The data of Trotter and Hixon ('76) are most likely a more adequate reflection of both the magnitude and the regional similarity of ash content in nonhuman primate bone.
The percentage ash content of various components of the skeleton has long been used a s a measure of relative differences in mineralization (Trotter, '73; Trotter and Hixon, '74; Trotter and Peterson, '55, '621, and t h e strength and behavior of bone as a mechanical member has been predicted based upon these data (Vose and Kubala, '59; Mather, '68; Wilson, '77). However, differences in preparation of material prior to ashing has often produced conflicting results when these techniques are applied to nonhuman primate bone Nose and Roach, '72; Trotter and Hixon, '76). It is essential to have accurate basic data about regional differences in ash content of nonhuman primate bone in order to make accurate interpretations about skeletal adaptation to specific environmental niches and behavioral modes. Furthermore, since nonhuman primates have been used as models for mineral metabolism in humans (Kazarian and AM. J. PHYS. ANTHROP. (19791 51; 361-364.
Von Gierke, '69; Mack, '71; Morey and Baylink, '781, it is necessary that accurate basic data be available to those researchers wishing to make comparisons between nonhuman primates and humans. The purpose of this short communication is to clarify one basic disagreement concerning regional differences in ash content in the long bones of t h e extremities so t h a t basic data on the strength of various components of the skeletal system can be used in comparative research. Vose and Roach ('72) ashed the dried bones of four adult male M. nemestrina. Although the bones were dissected and cleaned manually prior to ashing, none of the bones was defatted and cartilage was left intact. They found t h a t the fibula had the greatest mean ash content as a percentage of bone weight (58.1%)of any of the long bones of the extremities. This
' Supported by KUMC Committee on Research Grant 1514
361
362
DAVID B. BURR TABLE 1
Masses ofchemical fractions and percentage ash content in the non-fat-free long bones of Macaca Fat
Femur Tibia Fibula Ulna Radius Humerus
2.86 2.77 0.30 0.76 0.94 1.43
Volatile Jnorgamc fraction
Nonfatty organic fraction
Ash
0.65 0.46 0.11 0.24 0.23 0.45
5.68 3.92 0.78 1.84 1.71 3.78
9.45 6.78 1.58 3.51 3.28 6.60
% ash in the fatty skeleton
50.70 48.67 57.04 55.28 53.25 53.83
% ash in the fatty qkeleton
48.80 48.40 58.10
-
53.00 54.40
Masses (grams) computed from volume tractions reported by Gong ('721. ash Computed from Gong ('721 as total dry weight 'O0' ' From Vose and Roach 1'72).
I
TABLE 2
fatty organic constituent within the bone increased the initial weight of t h e bone t o which the inorganic constituent is compared by percentage after ashing. This reduces the percenX ash in the 5% ash in the tage values for the inorganic component, parfat-free fat-free skeleton ' skeleton : ticularly for those large bones in which the organic component will be greatest. Male Female The suggestion of Trotter and Hixon ('76) 68.60 68.90 62.46 Femur t h a t this discrepancy was due to methods of 68.10 68.40 Tibia 63.36 preparation, rather than to individual or spe69.10 66.95 69.00 Fibula 68.00 68.60 cies differences, was tested by using indepen65.61 Ulna 68.60 68.70 65.73 Radius dent data collected by Gong ('72) on the 68.80 68.80 Humerus 63.58 volumetric composition of nonhuman primate bone. Gong ('72) cleaned and fixed the skele' Computed from Gong ("721 as ash +ash x 100 nonfatty tons of five adult maleM. mulatta. All soft tisorganic fraction sue including cartilage was removed from the From Trotter and Hixon 1'76) bones. Water, fat, nonfatty organic, volatile inorganic and ash fractions of the bone were was followed by the humerus (54.4%),radius extracted using techniques summarized in (53.0%),femur (48.8%) and tibia (48.4%). Gong et al. ('64). Masses of the various fracUlnae were not included in this study. They tions were collected and volumes computed by found that the mean ash percentage of the dividing the mass of the fraction by its specific combined bones of the upper extremity was gravity. These volumes were then normalized 5.4%greater than the mean ash percentage of to a skeletal volume of 140 cm3. the femur and tibia combined. The volumes of the various fractions reTrotter and Hixon ('76) ashed the dried ported by Gong ('72) were converted to their bones of six male and seven female adult M. original masses by multiplying by the respecmulatta. All the bones were defatted and the = 1.00 g/ cartilage removed. In contrast to Vose and tive specific gravity of each (water cm3; fat = 0.912 g/cm"; volatile inorganic = Roach ('721, they found differences between the long bones of less than 1.0%ash content. 1.43 g/cm3; nonfatty organic = 1.745 g/cm3; Furthermore, as Trotter and Hixon ('76) ash = 3.188 g/cm3). The ash fraction of themselves point out, for "comparable parts of various long bones of the extremities was comthe skeleton each percentage ash weight from pared as a percentage of that bone's total dry the nemestrina monkey is lower than ours weight. Results similar to those of Vose and . . .for the Macaca mulatta" (p. 231). They Roach ('72) were obtained (table 1): the fibula suggested t h a t the discrepancy in these data had t h e largest percentage ash content might be explained by differences in method. (57.04%),followed by t h e ulna (55.28%), Vose and Roach ('72) failed to defat t h e bone humerus (53.83%), radius (53.25561, femur prior to ashing. The increased weight of the (50.70%)and tibia (48.67%).In all cases these Percentage ash content in the fat-free long bones of Macaca
ASH CONTENT IN PRIMATE BONE
percentage values differed from those of Vose and Roach ('72) for each bone by less than 2.0. They are not only similar in magnitude, but topographic distinctions are also maintained: the fibula provided the greatest percentage of ash of any of the long bones, while the femur and tibia provided the least. Using the recomputed data from Gong ('721, the mean percentage of ash weight of the combined humerus and radius was 3.81%greater than the percentage ash weight of the combined femur and tibia. This also compares favorably with Vose and Roach's ('72) computed value of 5.40% greater. If methodological differences between the work of Trotter and Hixon ('76) and that of Vose and Roach ('72) are in fact responsible for the differences between their results, it follows that results similar to Trotter and Hixon's ('76) would be expected if percentage ash weights were calculated as a function of only the organic and ash fractions. Assuming that the drying and defatting procedures employed by Trotter and Hixon ('76) eliminated all fat as well as the volatile inorganic component of the bone, the ash weight of each of the various long bones was compared as a percentage of each bone's dried and defatted weight (ash fraction nonfatty organic fraction). Examination of table 2 indicates that this procedure produces results that are indeed very similar in magnitude to those reported by Trotter and Hixon ('76). Percentage ash weights of each individual bone differ from those measured by Trotter and Hixon ('76). The differences range from 2.05%to 6.14%acceptable levels of difference for data collected on separate random samples. The data of Trotter and Hixon ('76) differed from those of Vose and Roach ('72) by more than simply magnitude. Vose and Roach ('72) indicated substantial regional differences between the different long bones. It would seem difficult to represent such differences in range as indicative of methodological differences if the methodology employed was standard procedure for all bones within each separate study. However, inspection of tables 1 and 2 indicates that percentage ash weights computed with and without the fatty organic component will produce such results even when computed from the same sample. Percentage values computed from Gong's ('72) data show a much greater range of values for the various long bones when calculated with the f a t t y organic component included than when calcu-
+
363
lated without the fat fraction. When the fatty organic component is included in the calculation (table 11, percentage ash content ranges from 48.67 to 55.28%(8.37%);when this component and volatile inorganic component are excluded in the calculation (table 21,percentage ash content ranges from 62.46 to 66.95% (4.49%).The most reasonable explanation for this is that there is greater regional variation in the fat content of the long bones than there is in the mineral content. Consequently, the regional differences that Vose and Roach ('72) measured are most likely reflections of regional differences in bone fat content rather than an accurate indication of differences in mineral. The data of Trotter and Hixon ('76) are most likely a more accurate reflection of both the magnitude and the regional similarity of ash content in nonhuman primate bones. These results confirm that Trotter and Hixon ('76) were correct in asserting that methodological differences between their study and that of Vose and Roach ('72) were responsible for differences in magnitude and range of their final results. If researchers are to make accurate interpretations o f ' relative bone strength based on its inorganic component, the fatty organic component should be eliminated prior to ashing. The data of Trotter and Hixon ("76)are therefore a more adequate reflection of inorganic content in nonhuman primate bone than are the data of Vose and Roach ('72) and future comparisons with other nonhuman primates or with humans should rely on these more accurate data. LITERATURE CITED Gong, J. K. 1972 Volumetric composition of the monkey skeleton. Anat. Rec., 172: 543-549. Gong, J. K., J. S. Arnold and S. H. Cohn 1964 Composition of trabecular and cortical bone. Anat. Rec., 149: 325-331. Kazarian, L.E., and H. E. Von Gierke 1969 Bone loss as a result of immobilization and chelation. Preliminary results in Mncaca mulatta. Clin. Orthop., 65: 67-75. Mack, P. B. 1971 Bone density changes in a MUCQCU nemestrina monkey during the biosatellite 3 project. Aerospace Med., 42: 828-833. Mather, B. S. 1968 The effect of variation in specific gravity and ash content on the mechanical properties of human compact bone. J. Biomech., I: 207-210. Morey, E . R., and D.J. Baylink 1978 Inhibition of bone formation during space flight. Science, 201: 1138-1141. Trotter, M. 1973 Percentage ash weight of young human skeletons. Growth, 37: 153-163. Trotter, M., and B. B. Hixon 1974 Sequential changes in weight, density and percentage ash weight of human skeletons from an early fetal period through old age. Anat. Rec., 179: 1-18.
364
DAVID B. BURR
1976 The density of long limb bones and the percentage ash weight of the skeleton of Macaca mulatta. Am. J. Phys. Anthrop., 44: 223-232. Trotter, M., and R. R. Peterson 1955 Ash weight of human skeletons in percent of their dry, fat-free weight. Anat. Rec., 123: 341-358. 1962 The relationship of ash weight and organic weight of human skeletons. J. Bone J t . Surg., 44A: 669-681.
Vose, G. P., and A. L. Kubala 1959 Bone strength-its relationship to X-ray determined ash content. Human Biol., 31: 262-270. Vose, G. P., and T. L. Roach 1972 Ash content of bones in the pigtail monkey, Macaca nemestrina. Aerospace Med., 43: 291-292. Wilson, C. R. 1977 Bone-mineral content of the femoral neck and spine versus the radius or ulna. J. Bone Jt. Surg., 59A: 665-669.
KEY WORDS Percentage ash weight cal fractions
. Methodology
. Macaque . Bone chemi-
ABSTRACT
Trotter and Hixon ('76) and Vose and Roach ('72) presented conflicting data about percentage ash content of the long limb bones of M. mulatta and M. nemestrina. The suggestion of Trotter and Hixon ('76) that this discrepancy was due to methods of preparing the bone prior to ashing, rather than to species differences, was tested using data collected by Gong ('72) on the volumetric composition of nonhuman primate bone. Masses of the various chemical fractions of bone reported by Gong ('72) were used to compute ash content as a percentage of total dry weight and ash content as a percentage of fat-free dry weight. Good agreement was found between ash content as a percentage of total dry weight and the data of Vose and Roach ('72); good agreement was also found between ash content as a percentage of fat-free dry weight and the data of Trotter and Hixon ('76). This suggests that the data of Vose and Roach ('72) conflict with those of Trotter and Hixon ('76) because Vose and Roach ('72) failed to defat the bones prior to ashing. No actual species differences exist in the ash content of bones of M. mulatta and M.nemestrina. The data of Trotter and Hixon ('76) are most likely a more adequate reflection of both the magnitude and the regional similarity of ash content in nonhuman primate bone.
The percentage ash content of various components of the skeleton has long been used a s a measure of relative differences in mineralization (Trotter, '73; Trotter and Hixon, '74; Trotter and Peterson, '55, '621, and t h e strength and behavior of bone as a mechanical member has been predicted based upon these data (Vose and Kubala, '59; Mather, '68; Wilson, '77). However, differences in preparation of material prior to ashing has often produced conflicting results when these techniques are applied to nonhuman primate bone Nose and Roach, '72; Trotter and Hixon, '76). It is essential to have accurate basic data about regional differences in ash content of nonhuman primate bone in order to make accurate interpretations about skeletal adaptation to specific environmental niches and behavioral modes. Furthermore, since nonhuman primates have been used as models for mineral metabolism in humans (Kazarian and AM. J. PHYS. ANTHROP. (19791 51; 361-364.
Von Gierke, '69; Mack, '71; Morey and Baylink, '781, it is necessary that accurate basic data be available to those researchers wishing to make comparisons between nonhuman primates and humans. The purpose of this short communication is to clarify one basic disagreement concerning regional differences in ash content in the long bones of t h e extremities so t h a t basic data on the strength of various components of the skeletal system can be used in comparative research. Vose and Roach ('72) ashed the dried bones of four adult male M. nemestrina. Although the bones were dissected and cleaned manually prior to ashing, none of the bones was defatted and cartilage was left intact. They found t h a t the fibula had the greatest mean ash content as a percentage of bone weight (58.1%)of any of the long bones of the extremities. This
' Supported by KUMC Committee on Research Grant 1514
361
362
DAVID B. BURR TABLE 1
Masses ofchemical fractions and percentage ash content in the non-fat-free long bones of Macaca Fat
Femur Tibia Fibula Ulna Radius Humerus
2.86 2.77 0.30 0.76 0.94 1.43
Volatile Jnorgamc fraction
Nonfatty organic fraction
Ash
0.65 0.46 0.11 0.24 0.23 0.45
5.68 3.92 0.78 1.84 1.71 3.78
9.45 6.78 1.58 3.51 3.28 6.60
% ash in the fatty skeleton
50.70 48.67 57.04 55.28 53.25 53.83
% ash in the fatty qkeleton
48.80 48.40 58.10
-
53.00 54.40
Masses (grams) computed from volume tractions reported by Gong ('721. ash Computed from Gong ('721 as total dry weight 'O0' ' From Vose and Roach 1'72).
I
TABLE 2
fatty organic constituent within the bone increased the initial weight of t h e bone t o which the inorganic constituent is compared by percentage after ashing. This reduces the percenX ash in the 5% ash in the tage values for the inorganic component, parfat-free fat-free skeleton ' skeleton : ticularly for those large bones in which the organic component will be greatest. Male Female The suggestion of Trotter and Hixon ('76) 68.60 68.90 62.46 Femur t h a t this discrepancy was due to methods of 68.10 68.40 Tibia 63.36 preparation, rather than to individual or spe69.10 66.95 69.00 Fibula 68.00 68.60 cies differences, was tested by using indepen65.61 Ulna 68.60 68.70 65.73 Radius dent data collected by Gong ('72) on the 68.80 68.80 Humerus 63.58 volumetric composition of nonhuman primate bone. Gong ('72) cleaned and fixed the skele' Computed from Gong ("721 as ash +ash x 100 nonfatty tons of five adult maleM. mulatta. All soft tisorganic fraction sue including cartilage was removed from the From Trotter and Hixon 1'76) bones. Water, fat, nonfatty organic, volatile inorganic and ash fractions of the bone were was followed by the humerus (54.4%),radius extracted using techniques summarized in (53.0%),femur (48.8%) and tibia (48.4%). Gong et al. ('64). Masses of the various fracUlnae were not included in this study. They tions were collected and volumes computed by found that the mean ash percentage of the dividing the mass of the fraction by its specific combined bones of the upper extremity was gravity. These volumes were then normalized 5.4%greater than the mean ash percentage of to a skeletal volume of 140 cm3. the femur and tibia combined. The volumes of the various fractions reTrotter and Hixon ('76) ashed the dried ported by Gong ('72) were converted to their bones of six male and seven female adult M. original masses by multiplying by the respecmulatta. All the bones were defatted and the = 1.00 g/ cartilage removed. In contrast to Vose and tive specific gravity of each (water cm3; fat = 0.912 g/cm"; volatile inorganic = Roach ('721, they found differences between the long bones of less than 1.0%ash content. 1.43 g/cm3; nonfatty organic = 1.745 g/cm3; Furthermore, as Trotter and Hixon ('76) ash = 3.188 g/cm3). The ash fraction of themselves point out, for "comparable parts of various long bones of the extremities was comthe skeleton each percentage ash weight from pared as a percentage of that bone's total dry the nemestrina monkey is lower than ours weight. Results similar to those of Vose and . . .for the Macaca mulatta" (p. 231). They Roach ('72) were obtained (table 1): the fibula suggested t h a t the discrepancy in these data had t h e largest percentage ash content might be explained by differences in method. (57.04%),followed by t h e ulna (55.28%), Vose and Roach ('72) failed to defat t h e bone humerus (53.83%), radius (53.25561, femur prior to ashing. The increased weight of the (50.70%)and tibia (48.67%).In all cases these Percentage ash content in the fat-free long bones of Macaca
ASH CONTENT IN PRIMATE BONE
percentage values differed from those of Vose and Roach ('72) for each bone by less than 2.0. They are not only similar in magnitude, but topographic distinctions are also maintained: the fibula provided the greatest percentage of ash of any of the long bones, while the femur and tibia provided the least. Using the recomputed data from Gong ('721, the mean percentage of ash weight of the combined humerus and radius was 3.81%greater than the percentage ash weight of the combined femur and tibia. This also compares favorably with Vose and Roach's ('72) computed value of 5.40% greater. If methodological differences between the work of Trotter and Hixon ('76) and that of Vose and Roach ('72) are in fact responsible for the differences between their results, it follows that results similar to Trotter and Hixon's ('76) would be expected if percentage ash weights were calculated as a function of only the organic and ash fractions. Assuming that the drying and defatting procedures employed by Trotter and Hixon ('76) eliminated all fat as well as the volatile inorganic component of the bone, the ash weight of each of the various long bones was compared as a percentage of each bone's dried and defatted weight (ash fraction nonfatty organic fraction). Examination of table 2 indicates that this procedure produces results that are indeed very similar in magnitude to those reported by Trotter and Hixon ('76). Percentage ash weights of each individual bone differ from those measured by Trotter and Hixon ('76). The differences range from 2.05%to 6.14%acceptable levels of difference for data collected on separate random samples. The data of Trotter and Hixon ('76) differed from those of Vose and Roach ('72) by more than simply magnitude. Vose and Roach ('72) indicated substantial regional differences between the different long bones. It would seem difficult to represent such differences in range as indicative of methodological differences if the methodology employed was standard procedure for all bones within each separate study. However, inspection of tables 1 and 2 indicates that percentage ash weights computed with and without the fatty organic component will produce such results even when computed from the same sample. Percentage values computed from Gong's ('72) data show a much greater range of values for the various long bones when calculated with the f a t t y organic component included than when calcu-
+
363
lated without the fat fraction. When the fatty organic component is included in the calculation (table 11, percentage ash content ranges from 48.67 to 55.28%(8.37%);when this component and volatile inorganic component are excluded in the calculation (table 21,percentage ash content ranges from 62.46 to 66.95% (4.49%).The most reasonable explanation for this is that there is greater regional variation in the fat content of the long bones than there is in the mineral content. Consequently, the regional differences that Vose and Roach ('72) measured are most likely reflections of regional differences in bone fat content rather than an accurate indication of differences in mineral. The data of Trotter and Hixon ('76) are most likely a more accurate reflection of both the magnitude and the regional similarity of ash content in nonhuman primate bones. These results confirm that Trotter and Hixon ('76) were correct in asserting that methodological differences between their study and that of Vose and Roach ('72) were responsible for differences in magnitude and range of their final results. If researchers are to make accurate interpretations o f ' relative bone strength based on its inorganic component, the fatty organic component should be eliminated prior to ashing. The data of Trotter and Hixon ("76)are therefore a more adequate reflection of inorganic content in nonhuman primate bone than are the data of Vose and Roach ('72) and future comparisons with other nonhuman primates or with humans should rely on these more accurate data. LITERATURE CITED Gong, J. K. 1972 Volumetric composition of the monkey skeleton. Anat. Rec., 172: 543-549. Gong, J. K., J. S. Arnold and S. H. Cohn 1964 Composition of trabecular and cortical bone. Anat. Rec., 149: 325-331. Kazarian, L.E., and H. E. Von Gierke 1969 Bone loss as a result of immobilization and chelation. Preliminary results in Mncaca mulatta. Clin. Orthop., 65: 67-75. Mack, P. B. 1971 Bone density changes in a MUCQCU nemestrina monkey during the biosatellite 3 project. Aerospace Med., 42: 828-833. Mather, B. S. 1968 The effect of variation in specific gravity and ash content on the mechanical properties of human compact bone. J. Biomech., I: 207-210. Morey, E . R., and D.J. Baylink 1978 Inhibition of bone formation during space flight. Science, 201: 1138-1141. Trotter, M. 1973 Percentage ash weight of young human skeletons. Growth, 37: 153-163. Trotter, M., and B. B. Hixon 1974 Sequential changes in weight, density and percentage ash weight of human skeletons from an early fetal period through old age. Anat. Rec., 179: 1-18.
364
DAVID B. BURR
1976 The density of long limb bones and the percentage ash weight of the skeleton of Macaca mulatta. Am. J. Phys. Anthrop., 44: 223-232. Trotter, M., and R. R. Peterson 1955 Ash weight of human skeletons in percent of their dry, fat-free weight. Anat. Rec., 123: 341-358. 1962 The relationship of ash weight and organic weight of human skeletons. J. Bone J t . Surg., 44A: 669-681.
Vose, G. P., and A. L. Kubala 1959 Bone strength-its relationship to X-ray determined ash content. Human Biol., 31: 262-270. Vose, G. P., and T. L. Roach 1972 Ash content of bones in the pigtail monkey, Macaca nemestrina. Aerospace Med., 43: 291-292. Wilson, C. R. 1977 Bone-mineral content of the femoral neck and spine versus the radius or ulna. J. Bone Jt. Surg., 59A: 665-669.
