Age and Ageing (1975), 4, 73
INFLUENCE OF AGE AND SEX ON THE CALCIFICATION OF RAT AORTA IN RELATION TO BONE*MINERALIZATION A. J. MORGAN AND D. BELLAMY Department of Zoology, University College, Cardiff Summary
Thoracic aortas of pvg rats accumulated Ca (analysed by flame spectrophotometry) throughout mature life, with females having slightly higher levels than males at each age interval studied. Femur Ca concentration was similarly elevated in female rats, and was maintained fairly constant in both sexes after 19 weeks of age. Femur Mg and K levels showed an over-all decrease with age; whilst the Na concentration fell up to 19 weeks, and then decreased steadily. It was concluded that a simple shift of Ca from the ageing skeleton into soft tissues cannot adequately account for aortic physiosclerosis. INTRODUCTION
The rat aorta accumulates calcium with age (Freydberg-Lucas & Verzar, 1957; Urist & Adams, 1967; Hruza, 1969; Little, 1971; Oeriu et al., 1972) without accompanying pathological developments. This is the process described by Junge-Hulsing & Wagner (1968) as 'physiosclerosis' to distinguish it from the localized, massive deposition of calcium leading to the development of atherosclerotic plaques. Although it is known that sex-differences exist in the incidence of human cardiovascular disease (Furman, 1968; Walton, 1969), the investigation of possible sex-differences in the rate of arterial physiosclerosis has been limited to a few studies on human subjects (Blumenthal, Lansing & Wheeler, 1944; Blankenhorn, 1964; Kanabrocki, Fels & Kaplan, 1960), with the inherent handicaps of large individual variability and the possible contamination of tissue samples by pathological deposits. Age changes in the elemental composition of mammalian bones, especially in their calcium composition, have received much attention (Wildt, 1872; Hammett, 1925&; Weidman & Rogers, 1958; Dickerson, 1962a,6; Weiss, McBroom & Cornelison, 1969; Saville, 1969), particularly with regard to the aetiology of osteoporosis. Bone loss in the ageing human (Garn, Rohmann & Nolan, 1964; Newton-John & Morgan, 1968; Nordin et al., 1970) and dog (Detenbeck & Jowsey, 1969) is a general phenomenon, resulting in the thinning of both cortical and trabecular bone. Human bones of both sexes gradually become less dense; although the rate of decrease in density is three times greater in females than in males and is easily demonstrable in the immediately postmenopausal period in females, whereas in males it is detected in the 55 to 65-year-old group (Sobel, 1967). The study of possible sex-differences in the calcification of the rat skeleton has hitherto been largely neglected. The present study was undertaken to see whether sex-differences occur in the pattern of calcification of the thoracic aorta, and in the calcification of the femur, of ageing rats.
74
A. J. Morgan and D. Bellamy
Furthermore, this investigation considered the possibility of synchronous age-changes (controlled by a single biochemical process) occurring in these two essentially fibrous tissues. For example, calcium lost from the ageing skeleton may be linked with the process of binding within the aortic wall. Age-changes in the concentrations of other femur electrolytes were also monitored, with emphasis on their possible relationship to the processes of skeletal mineralization and soft-tissue calcification. Material and Methods Rats of the pvg strain were maintained on a standard diet (Pilsbury, Birmingham, Modified Breeding Diet) containing 14 g/kg Ca, 11 g/kg P, 0.46 mg/kg Mg, 6 g/kg Na (data provided by the manufacturer), and housed in galvanized wire cages. Twenty-five per cent of the rats of this particular strain survive to 112 weeks (Phillips: personal communication). Groups of rats of both sexes were killed by chloroform at various intervals from 8 to 113 weeks after birth: Age (weeks)
No. of males
No. of females
8 19 50 65 80 90 97 103 113
6 6 5 5 5 6 4 6 4
6 6 6 5 5 _ — 4
The thoracic aorta was dissected from its junction with the heart down to the level of the diaphragm and cleaned free of extraneous tissue with stainless steel instruments. Each aorta was dried overnight in pre-weighed silica crucibles at 105°C and weighed before ashing at 600°C in a muffle furnace. Right femurs were dissected free and immediately cleaned of flesh and connective tissue attachments. Each bone was individually dried overnight in pre-weighed glass tubes at 105 °C and weighed. The pelvic end of each dry bone was further cleaned free of tendon insertions and broken at about the level of the distal edge of the lesser trochanter. The proximal portion of each femur was subsequently dried overnight in pre-weighed silica crucibles at 105 "C, weighed, and ashed at 600°C overnight. Bone ash was weighed before being dissolved in 5 ml O.5M-HNO3 + 0.5 ml cone. 'Analar' HNO3 in the crucible. This solution required further dilution before electrolyte analysis in the flame spectrophotometer: for Na and K analysis, 0.2 ml of the above solution was mixed with 5 ml deionized water in hard-glass tubes (American Instruments Co. Inc.)—solution A; for Ca and Mg analysis, 0.5 ml of solution A was mixed with 3.5 ml deionized water and 1 ml 5 per cent La3+ solution (to give a final concentration of 1 per cent La3+)—solution B. Solutions were dispensed using Eppendorf microlitre pipettes and the glass vials were sealed with Parafilm. The spectrophotometer used was a Pye/Unicam SP90A with a SP20 pen-recorder attachment. Ca and Mg were analysed in the absorption mode, Na and K were analysed in the emission mode. Instrumental settings were as described in the Pye/Unicam SP90A method sheets. All data were expressed as Mean ± S.D. The significance of the difference between means for male and female rats of a given age was determined by the Student's t-test. RESULTS
Body Weight and Femur Dry Weight Males were significantly heavier than females at each age (Fig. 1). The body weight of both sexes increased rapidly between 8 and 19 weeks, followed by a slightly slower rate
Influence of Age and Sex on the Calcification of Rat Aorta
75
450
(4)
350
(6), 250
o o m (4) 150
100 0
30
60 Age (weeks)
120
Fig. 1. Growth curves of ageing pvg rats. Numbers in parentheses above and below the lines refer to the number of observations on males and females, respectively (Q 0 = males, O O = females). Statistical evaluation (males v. females): P 00 CO 00 CO ^ "^-CN 00
nNnH
60
oo in oa Tt- i-H C N , _ CNCOt^OOCNsO t^-00
in cs ^h oo v
0.1 (n.s.) : P > 0 . 1 (n.s.)
K/Na 8 week v. 8 week 50 week v. 50 week 65 week v. 65 week 90 week v. 90 week
: P>0.05 (n.s.) : P
INFLUENCE OF AGE AND SEX ON THE CALCIFICATION OF RAT AORTA IN RELATION TO BONE*MINERALIZATION A. J. MORGAN AND D. BELLAMY Department of Zoology, University College, Cardiff Summary
Thoracic aortas of pvg rats accumulated Ca (analysed by flame spectrophotometry) throughout mature life, with females having slightly higher levels than males at each age interval studied. Femur Ca concentration was similarly elevated in female rats, and was maintained fairly constant in both sexes after 19 weeks of age. Femur Mg and K levels showed an over-all decrease with age; whilst the Na concentration fell up to 19 weeks, and then decreased steadily. It was concluded that a simple shift of Ca from the ageing skeleton into soft tissues cannot adequately account for aortic physiosclerosis. INTRODUCTION
The rat aorta accumulates calcium with age (Freydberg-Lucas & Verzar, 1957; Urist & Adams, 1967; Hruza, 1969; Little, 1971; Oeriu et al., 1972) without accompanying pathological developments. This is the process described by Junge-Hulsing & Wagner (1968) as 'physiosclerosis' to distinguish it from the localized, massive deposition of calcium leading to the development of atherosclerotic plaques. Although it is known that sex-differences exist in the incidence of human cardiovascular disease (Furman, 1968; Walton, 1969), the investigation of possible sex-differences in the rate of arterial physiosclerosis has been limited to a few studies on human subjects (Blumenthal, Lansing & Wheeler, 1944; Blankenhorn, 1964; Kanabrocki, Fels & Kaplan, 1960), with the inherent handicaps of large individual variability and the possible contamination of tissue samples by pathological deposits. Age changes in the elemental composition of mammalian bones, especially in their calcium composition, have received much attention (Wildt, 1872; Hammett, 1925&; Weidman & Rogers, 1958; Dickerson, 1962a,6; Weiss, McBroom & Cornelison, 1969; Saville, 1969), particularly with regard to the aetiology of osteoporosis. Bone loss in the ageing human (Garn, Rohmann & Nolan, 1964; Newton-John & Morgan, 1968; Nordin et al., 1970) and dog (Detenbeck & Jowsey, 1969) is a general phenomenon, resulting in the thinning of both cortical and trabecular bone. Human bones of both sexes gradually become less dense; although the rate of decrease in density is three times greater in females than in males and is easily demonstrable in the immediately postmenopausal period in females, whereas in males it is detected in the 55 to 65-year-old group (Sobel, 1967). The study of possible sex-differences in the calcification of the rat skeleton has hitherto been largely neglected. The present study was undertaken to see whether sex-differences occur in the pattern of calcification of the thoracic aorta, and in the calcification of the femur, of ageing rats.
74
A. J. Morgan and D. Bellamy
Furthermore, this investigation considered the possibility of synchronous age-changes (controlled by a single biochemical process) occurring in these two essentially fibrous tissues. For example, calcium lost from the ageing skeleton may be linked with the process of binding within the aortic wall. Age-changes in the concentrations of other femur electrolytes were also monitored, with emphasis on their possible relationship to the processes of skeletal mineralization and soft-tissue calcification. Material and Methods Rats of the pvg strain were maintained on a standard diet (Pilsbury, Birmingham, Modified Breeding Diet) containing 14 g/kg Ca, 11 g/kg P, 0.46 mg/kg Mg, 6 g/kg Na (data provided by the manufacturer), and housed in galvanized wire cages. Twenty-five per cent of the rats of this particular strain survive to 112 weeks (Phillips: personal communication). Groups of rats of both sexes were killed by chloroform at various intervals from 8 to 113 weeks after birth: Age (weeks)
No. of males
No. of females
8 19 50 65 80 90 97 103 113
6 6 5 5 5 6 4 6 4
6 6 6 5 5 _ — 4
The thoracic aorta was dissected from its junction with the heart down to the level of the diaphragm and cleaned free of extraneous tissue with stainless steel instruments. Each aorta was dried overnight in pre-weighed silica crucibles at 105°C and weighed before ashing at 600°C in a muffle furnace. Right femurs were dissected free and immediately cleaned of flesh and connective tissue attachments. Each bone was individually dried overnight in pre-weighed glass tubes at 105 °C and weighed. The pelvic end of each dry bone was further cleaned free of tendon insertions and broken at about the level of the distal edge of the lesser trochanter. The proximal portion of each femur was subsequently dried overnight in pre-weighed silica crucibles at 105 "C, weighed, and ashed at 600°C overnight. Bone ash was weighed before being dissolved in 5 ml O.5M-HNO3 + 0.5 ml cone. 'Analar' HNO3 in the crucible. This solution required further dilution before electrolyte analysis in the flame spectrophotometer: for Na and K analysis, 0.2 ml of the above solution was mixed with 5 ml deionized water in hard-glass tubes (American Instruments Co. Inc.)—solution A; for Ca and Mg analysis, 0.5 ml of solution A was mixed with 3.5 ml deionized water and 1 ml 5 per cent La3+ solution (to give a final concentration of 1 per cent La3+)—solution B. Solutions were dispensed using Eppendorf microlitre pipettes and the glass vials were sealed with Parafilm. The spectrophotometer used was a Pye/Unicam SP90A with a SP20 pen-recorder attachment. Ca and Mg were analysed in the absorption mode, Na and K were analysed in the emission mode. Instrumental settings were as described in the Pye/Unicam SP90A method sheets. All data were expressed as Mean ± S.D. The significance of the difference between means for male and female rats of a given age was determined by the Student's t-test. RESULTS
Body Weight and Femur Dry Weight Males were significantly heavier than females at each age (Fig. 1). The body weight of both sexes increased rapidly between 8 and 19 weeks, followed by a slightly slower rate
Influence of Age and Sex on the Calcification of Rat Aorta
75
450
(4)
350
(6), 250
o o m (4) 150
100 0
30
60 Age (weeks)
120
Fig. 1. Growth curves of ageing pvg rats. Numbers in parentheses above and below the lines refer to the number of observations on males and females, respectively (Q 0 = males, O O = females). Statistical evaluation (males v. females): P 00 CO 00 CO ^ "^-CN 00
nNnH
60
oo in oa Tt- i-H C N , _ CNCOt^OOCNsO t^-00
in cs ^h oo v
0.1 (n.s.) : P > 0 . 1 (n.s.)
K/Na 8 week v. 8 week 50 week v. 50 week 65 week v. 65 week 90 week v. 90 week
: P>0.05 (n.s.) : P
