Clinical Science and Molecular Medicine (1976) 51, 399-402.
A study of changes in wholebody calcium, phosphorus, sodium
and nitrogen by neutron activation analysis in vivo in rats
on a calciumdeficient diet
K. BODDY,' R. LINDSAY,? I. HOLLOWAY,* D. A. S. SMITH,? A. ELLIOTT,*
I. ROBERTSON* A N D D. GLAROS*
Nuclear Medicine Unit, Scottish Universities Research and Reactor Centre, East Kilbride, and
? Department of Medicine, Bone Metabolism Research Unit, Western Infirmary, Glasgow, Scotland
(Received 29 April 1976)
such changes in rats subjected to variations in the
mineral constituents of their diet.
S-arY
1. A method of measuring changes in the total
body content of calcium, phosphorus, nitrogen and
sodium in rats by activation analysis in vivo is
described.
2. The change in the body content of the elements
has been measured in rats on a calcium-deficient
diet and in control animals, the body nitrogen being
used to represent lean body mass for normalization.
3. There were significant differences in Ca/N and
P/N but not in Ca/P ratios between the animals on a
deficient diet and control animals at the end of the
dietary period.
Materials and methods
The total body content of phosphorus and nitrogen
of twelve female Wistar rats was measured by total
body neutron activation analysis in vivo, a facility
developed for clinical studies (Boddy, Holloway,
Elliott, Glaros, Robertson & East, 1972; Boddy,
Holloway & Elliott, 1973) being used. A measurement of the natural body radioactivity of each animal
was made by counting in a fixed position with a
high-sensitivity shadow-shield wholebody counter
(Boddy et al., 1972; Boddy, Holloway, Elliott &
Robertson, 1975). The animals were then irradiated
unilaterally with 14 MeV neutrons for 60 s at 12 cm
SSD with a Philips type 18602 sealed tube neutron
generator. Sixty seconds later, the radioactivity
induced in the body, 28A1(from phosphorus) and
'N, was measured by wholebody counting for 600 s
live time. On the following day, when the 28A1and
3N radioactivity had decayed to negligible levels,
the total body content of calcium and sodium was
measured by a similar procedure but using neutrons
from a nuclear reactor as developed for clinial
studies (Boddy, 1966; Boddy & Alexander, 1967).
The irradiation time was 300 s and counting of the
induced radioactive isotopes 49Caand 24Na began
90 s later. The body weight of each animal was
noted.
The animals were divided randomly into two
groups of six. For 44 days, the control group r e
Key words: activation analysis in vivo, calcium,
nitrogen, phosphorus, sodium.
Introduction
In the investigation of animals subject to changes in
diet or to metabolic experiments, the measurement
of changes in the total body content of calcium,
phosphorus and sodium may be important. However,
conventional balance techniques are tedious and
inevitably are subject to cumulative errors. An
alternative method is to measure the wholebody
constituents in vivo at the beginning of the study and
at intervals thereafter by the use of total body
neutron activation analysis in vivo. The present
paper reports the use of this technique to measure
Correspondence: Dr Keith Boddy, Nuclear Medicine Unit,
Scottish Universities Research and Reactor Centre, East
Kilbride G75 OQU, Scotland.
399
K . Boddy et at.
400
ceived a normal laboratory diet containing 794 mg
of Ca, 539 mg of P and 95 mg of Mg/100 g. Over
the same period, the second group (the 'diet group')
received a standard calcium-deficient diet (Nutritional Biochemicals, U.S.A.), containing 17.3 mg of
Ca, 197 mg of P and 1012 mg of Mg/100 g. The high
magnesium content of the experimental diet tended
to induce diarrhoea and 2 days after commencement
methylcellulose (10 g/100 g of food) was added,
which effectively stopped the diarrhoea in these
animals.
The total body content of phosphorus, nitrogen,
calcium and sodium was then remeasured as before.
Paired results were compared, by using Wilcoxon's
signed ranks test, and mean values with Wilcoxon's
sum of ranks test.
1.144and 1.212counts/mgofCa(mean 1.178 k0.034
countslmg of Ca) and 33.757 and 34.023 counts/mg
of P (mean 33.89k0.13 countslmg of P). These
values are in good agreement.
Effect of diet
Results
Precision of the technique
Repeated measurements of 13N on a rat 'phantom', comprising a polyethylene bottle containing
250 ml of ammonium nitrate solution, gave a
coefficient of variation of 5 % . The difference in
radioactivity counting rate per gram of element
between a 'phantom' containing 150 ml of solution
and that containing 250 ml was 3%. This 'weight
range' essentially spanned that of the animals used.
As a more direct check on the precision of the
technique, at the end of the experimental period the
carcasses of two of the animals were analysed
chemically for calcium and phosphorus. The radioactivity counting-rates for these elements in uiuo,
which are activated respectively by thermal and fast
neutrons, were compared in each carcass with the
body contents. The weights of the carcasses were
202 g and 247 g and gave corresponding values of
The results, expressed as the mean values and
statistical significances, are summarized in Table 1.
The mean body weights of the control and diet
groups were not significantly different at the beginning of the study. By the end of the experiment there
was a significant change in both groups although the
mean weight of the group on the deficient diet was
less than that of the control group. Consequently,
the calcium, phosphorus and sodium results were
normalized to the corresponding 3N result in each
animal. As discussed below, this is equivalent to
normalizing to lean body mass (Pace & Rathbun,
1945). As only relative changes were of interest, all
of the results are expressed in arbitrary units representing corrected net counts normalized for neutron
dose and are not molar quantities. It should be
noted, particularly, that the Ca/P ratio values are
also normalized arbitrary values and d o not represent molar ratios.
In the initial measurements, there was no significant difference between the diet and control groups
in the mean values for body weight, body nitrogen
nor in the Ca/N, P/N, Na/N and Ca/P ratios. These
findings were to be expected.
In five of the six animals on the deficient diet there
was a decrease in the Ca/N ratio over the dietary
period whereas in five of the six control animals this
ratio increased. However, in neither group did the
change achieve statistical significance (P> 0.05).
When the mean values in the two groups were
TABLE
1, Changes in body composition
Mean values are shown. NS = not significant; S = significant.
~
~
Pre-diet
~
~
~
~~~
Post-diet
Group
Wt.
Na/N
Ca/N
P/N
Ca/P
(€9
Diet
Control
189.0
186.2
NS
Wt.
Na/N
CalN
PIN
CalP
3.035"'
2.776'"
NS
2.240
2.584
3.345'"
3.866")
S
S
6.688
6,68O(l)
NS
(9)
1.833
1.686
NS
2.490
2.413
NS
(l)
4442
4.156
NS
6.161
5.813
NS
200.5"'
235.1'l)
S
Significant difference post-diet and pre-diet.
Changes in dietary-deficient rats
compared, the Ca/N ratio was significantly less in
the dietary-deficient group than in the control
animals (P< 0.05).
The P/N ratio decreased significantly (P< 0.05)
in both groups of animals and the mean value in the
dietary-deficient group was significantly less (P<
0.05)than in the control animals.
In both groups of animals, the Na/N ratio increased significantly (P< 0.05) over the dietary
period, the mean increase in both cases being close
to 65%.
Five of the six animals in each group showed an
increase in the Ca/P ratio but the increase attained
statistical significance (P‘0.05) only in the control
animals. The mean values of the Ca/P ratio in the
two groups were not significantly different (P> 0.05).
Discussion
Total body activation analysis in viuo was first
demonstrated in animals by Mayneord, Martin &
Layne (1949)with high-energy X-rays. Subsequently,
by using neutron irradiation, the technique has been
successfully used in man by several groups (Anderson, Osborn, Tomlinson, Newton, Rundo, Salmon
& Smith, 1964; Chamberlain, Fremlin, Peters &
Philip, 1968;Palmer, Nelp, Murano & Rich, 1968;
Cohn, Dombrowski & Fairchild, 1970;Boddy et al.,
1972). The feasibility of measuring the total body
content of various elements in small animals by
neutron activation analysis in vivo has been reported
by several workers (Nagai, Fujii, Muto & Inouye,
1969; Williams, Cargol, Pailthorp & Nelp, 1970;
Biggin & Morgan, 1971 ; Weber & Andrews, 1972;
Chasteland & Comar, 1972;Shukla & Cohn, 1973;
Dombrowski, Wallach, Shukla & Cohn, 1973;
Palmer, 1973). However, although a number of
clinical investigations have been described in man,
we are not aware of any previous analogous studies
in animals applying these techniques to a specific
medical problem.
The aims of the present study were to explore the
usefulness of the technique in sequential studies of
the body composition of animals and, in particular,
to examine the influence of a calcium-deficient diet
on skeletal composition in rats. The precision of the
method was shown to be satisfactory. There seems
little doubt of the effectivenessof the technique since
it can provide a simultaneous measurement of total
body calcium, phosphorus, nitrogen and sodium in a
semi-automated fashion without killing the animals.
401
The statistical significance of sequential changes can
be evaluated and a study concluded at an appropriate time. A further important advantage of the
method is that changes in mineral content can be
related to body nitrogen, as the I3N counting rate,
and, hence, to lean body mass (Pace & Rathbun,
1945). This is especially useful in growing animals
where dietary or therapeuticregimens may cause nonspecific changes in the rate of growth without proportional changes in skeletal mass. For example, in
the present study, there was a significantly smaller
change in the body weight of the diet animals than in
the control animals and analysis of the counting
rates per se of calcium, phosphorus and sodium
could be misleading. An apparent ‘deficiency’ might
simply be a corollary of the reduced overall generalized growth of the diet animals. For this reason, we
have preferred to interpret the results relative to
nitrogen which correlated closely with lean body
mass (Pace & Rathbun, 1945).
The Ca/N and P/N ratios were reduced in a
remarkably similar proportion in the diet animals
compared with the control animals. These findings
represent therefore a true failure of animals to gain
mineral on the low-calcium and low-phosphorus and
high-magnesium diet. There was an increase in Ca/P
ratio (although these do not represent molar
quantities), which was less pronounced in the diet
animals. This could imply a change in skeletal or
soft tissue composition with age in favour of
calcium compared with phosphorus, and the effect
would be less marked in the animals on a diet relatively deficient in calcium.
There was a marked change in the Na/N ratio of
about 65% over the dietary period, which was
similar in both the diet and the control animals. The
dietary intake of sodium was similar in the two
groups. However, the method measures the total
body content and does not distinguish between
uptake into soft tissue and bone, which might have
been different in the two groups of animals.
Acknowledgments
This work was supported by grants and studentships
(A.E. and I.R.) from the Medical Research Council
and financial assistance from the .National Fund for
Research into Crippling Diseases, which are gratefully acknowledged. We thank Professor G . M.
Wilson and Professor H. W. Wilson for their interest
and encouragement.
402
K. Boddy et al.
References
ANDERSON,
J., OSBORN,
S.B., TOMLINSON,
R.W.S., NEWTON,
D., RUNDO,J., SALMON,
L. & SMITH,J.W. (1964) Neutron
activation analysis in man in vivo: a new technique in
medical investigation. Lancer, ii, 1201-1205.
BIGGIN,H.C. & MORGAN,
W.D. (1971) Fast neutron activation analysis of the major body elements. Journal of
Nuclear Medicine, 12, 808-814.
BODDY,K. (1966) In vivo activation analysis of iodine in the
thyroid gland-a preliminary study. In: Proceedings of the
7th Syniposiuni on Radioactive Isotopes in Clinical Medicine
and Research, p. 377. Urban and Schwarzenberg, Munich.
BODDY,
K. &ALEXANDER,
W.D. (1967) Clinical experience of
in vivo activation analysis of iodine in the thyroid gland: an
assessment of the problems. In: Proceedings of the Syniposiurn on Nuclear Activation Techniques in the Life Sciences,
p. 583. I.A.E.A., Vienna.
BODDY,K., HOLLOWAY,
I. & ELLIOTT,A. (1973) Preliminary
results of measuring total body calcium with a new facility
for total body in vivo activation analysis. In: Proceedings of
the Panel on in vivo Neutron Activation Analysis, p. 163.
I.A.E.A., Vienna.
BODDY,K., HOLLOWAY,
I., ELLIOTT,A., GLAROS,D.,
ROBERTSON,
I. & EAST,B.W. (1972) Low cost facilities for
partial body and total body in vivo activation analysis in the
clinical environment. In: Proceedings of the Symposium on
Niiclear Acrivation Techniques in the Life Sciences, p. 589.
I.A.E.A., Vienna.
BODDY,K., HOLLOWAY,
I., ELLIOTT,A. & ROBERTSON,
I.
(1975) A high sensitivity dual-detector shadow-shield
whole-body counter for clinical use, particularly total
body in vivo activation analysis. Physics in Medicine and
Biology, 20, 295-304.
CHAMBERLAIN,
M.J., FREMLIN,
J.H., PETERS,
D.K. & PHILIP,
H. (1968) Total body calcium by whole-body neutron
activation: new technique for study of bone disease.
British Medical Journal, ii, 581-585.
CHASTELAND,
M. & COMAR,D. (1972) Dosage par radioactivation neutronique in vivo de quelques elements con-
tenus dans I'organisme du rat. International Journal of
Applied Radiation and Isotopes, 23,209-218.
COHN,S.H., DOMBROWSKI,
C.S. & FAIRCHILD,
R.G. (1970)
In vivo neutron activation analysis of calcium content in
man. International Journal of Applied Radiation and
I.OtOpeS, 21, 127-1 37.
DOMBROWSKI,
C.S., WALLACH,
S., SHUKLA,
K.K. & COHN,
S.H. (1973) Determination of whole-body magnesium by
in vivo neutron activation. International Joiirnal of Nuclear
Medicine and Biology, 1, 15-21.
MAYNEORD,
W.V., MARTIN,J.H. & LAYNE,D.A. (1949)
Production ofradioactivity in animal tissues by high energy
X-rays. Nature (London), 164,728-730.
NAGAI,T., FUJII,I., MUTO,H. & INOUYE,
T. (1969) Totalbody nitrogen and protein determined by in vivo fastneutron activation analysis. Journal of Niiclear Medicine,
10, 192-196.
PACE,N. & RATHBUN,
E.N. (1945) Studies on body composition. The body water and chemically combined nitrogen content in relation to fat content. Journal of Biological
Chemistry, 158, 685-691.
PALMER,
H.E. (1973) Feasibility of determining total-body
calcium in animals and humans by measuring 37Ar in
expired air after neutron irradiation. Journal of Nuclear
Medicine, 14, 522-527.
PALMER,
H.E., NELP,W.B., MURANO,
R. &RICH,C.R. (1968)
The feasibility of in vivo neutron activation analysis of
total body calciumand other elements of body composition.
Physics in Medicine and Biology, 13, 269-279.
SHUKLA,
K.K. & COHN,S.H. (1973) Measurement ofcalcium
in rats by total body neutron activation analysis. International Journal of Nuclear Medicine and Biology, 1,73-18.
WEBER,
D.A. & ANDREWS,
H.L. (1972) Neutron activation
analysis of calcium in biological samples. Journal of Nuclear
Medicine, 13, 293-299.
WILLIAMS,
J.L., CARGOL,
L.H., PAILTHORP,
K.G. & NELP,
W.B. (1970) Neutron activation analysis of total-body
phosphorus in mice. Journal of Nuclear Medicine, 11.
576-579.