477
ClinicalScience (1981) 61,477481
A comparison of radioactive calcium absorption tests with net
calcium absorption
D . H . M A R S H A L L A N D B . E. C . N O R D I N
MRC Mineral Metabolism Unit, The General Injrmary, Leeds, U.K.
(Received 29 December 1980; accepted 8 April 1981)
Summary
1. Four different methods of calculating calcium absorption by radioactive calcium procedures have been compared with each other and
with net calcium absorption in calcium-balance
studies in 100 consecutive studies on 7 1 patients.
2. All four isotope procedures yielded highly
significant correlations with net calcium absorption derived from the balance studies, but
there was little to choose between the validity of
the double-isotope and single-isotope procedure
judged by these criteria.
3. The rate of calcium absorption calculated
from one or other isotope procedure correlated
better with net calcium absorption than did the
fraction of the radioactive calcium absorbed.
4. The measurement of plasma radioactivity 1
h after single-isotope administration, corrected
for body weight, proved almost as useful as the
more complex procedures but would be expected
to underestimate calcium absorption in states of
very high bone turnover.
Key words: absorption, calcium.
a double isotope [3]. Variants of the latter have
included total-body retention of radioactive calcium after oral administration 141 and forearm
counting [51. In attempting to assess the relative
validities of these very different procedures, one is
faced with the problem that there is no ultimate
reference standard. However, the most direct
procedure, which makes the fewest assumptions,
is the calcium balance, in which calcium absorption is defined as the difference between
dietary intake and faecal output. Although
subject to considerable technical error, this is a
conventional and well-established procedure
which most workers would probably accept as a
reasonable reference standard if the balance is
performed with sufficient care and one with
which other more indirect methods can reasonably be compared.
We have therefore compared various measurements of calcium absorption using radioactive
calcium with the results obtained by balance
studies performed on the same subjects at the
same time. The results are presented in this paper.
Materials and methods
Patients
Introduction
There is no standard way of measuring calcium
absorption. A great many different methods exist
ranging from intubation with a triple lumen tube
[ l ] to calcium balance procedures and procedures involving the administration of radioactive calcium either as a single isotope [21 or as
Correspondence: Dr D. H. Marshall, MRC Mineral
Metabolism Unit, The General Infirmary, Great
George Street, Leeds LSl 3EX, U.K.
0143-5221/81/100477-05$01.50/1
The study was based on 100 consecutive sets
of observations in 71 patients (nine men, 62
women). Of these studies 36 were performed in
untreated patients and 64 in patients on a variety
of treatments. The details of the diagnostic
categories and treatments are shown in Table 1.
Clinical procedure
The balance procedure used has been
described in detail elsewhere [61. It was a 2-week
@ 1981 The Biochemical Society and the Medical Research Society
D . H . Marshall and B . E . C. Nordin
478
TABLE
1. Distribution of diagnoses and treatments of the 100 studies in 71 patients
~~
~
Diagnosis
Normal
Vertebral fractures
Femoral neck fractures
Osteomalacia
Hyperparathyroid
Renal stone
Hypoparathyroid
Hypothyroid
~~
Vitamin D or
I-u-hydroxy-vitamin D
Untreated
Other
treatment
M
F
M
F
5
3
17
2
17
15
1
3
3
5
1
2
4
I
1
7
6
Calculations
The following calculations were performed. (1)
Net calcium absorption, defined as the difference
between dietary calcium intake and faecal calcium output expressed in mmol/day. (2) The
initial fractional rate of calcium absorption after
radioactive calcium administration (A). This
measurement makes use of the two calcium
isotopes administered on days 8 and 15 and is
calculated by a standard deconvolution procedure, which has been described in detail
elsewhere 171, with 10 min intervals. After
calculating the cumulative absorbed radioactive
calcium as a function of time, the exponential fall
in unabsorbed dose in the first hour after the
initial delay is expressed as a fractional rate
constant (A). (3) The fraction of the dose of
radioactive calcium absorbed in 2 h (#); this is
derived from the deconvolution procedure and
represents the integrated rates of radioactive
calcium absorption for the first 2 h after oral
administration. (4) The mean fractional rate of
radiocalcium absorption (a) is based on single-
2
I
I
1
study with polyethylene glycol as a nonabsorbable faecal marker. Patients were equilibrated on a standard diet for the first week and
were then given intravenous radioactive calcium
(10 pCi of 47Ca as calcium chloride) on day 8,
after which daily faecal and urinary collections
are performed for a further week. The oral
radioactive calcium test was performed on day
15. The oral dose of radioactive calcium consisted of 5 pCi of 45Caor 47Cain a solution (2.0
mmol/l) of 20 mg of calcium carrier as the
chloride in 250 ml of distilled water administered
after an overnight fast at about 09.00 hours.
Plasma samples were obtained at 10, 20, 40, 90
and 180 min after the intravenous radioactive
calcium and at 15, 30, 45, 60, 90 and 120 min
after the oral dose.
F
M
1
1
TABLE2. Correlation matrix between calculated parameters for calcium absorption on
100 studies
See text for definitions of symbols.
Measurement
Net
\/A
$
dd
0.740
0.630
0.913
0.764
0.872
0.773
0.700
0.851
0,819
0.927
isotope measurements only [81, where the fractional rates of absorption and clearance are obtained
by non-linear curve fitting. (5) The fraction
of the dose of radioactive calcium circulating in
the plasma and extracellular fluid 1 h after the
oral dose (Fc);this is derived from the fraction of
the dose per litre of plasma 1 h after administration multiplied by 15% of the total-body
weight to allow for the extracellular calcium [91.
Results
In the following analysis the initial fractional rate
(A) and the mean fractional rate (a)have been
transformed to their square roots to normalize
their distribution and yield linear correlation. The
correlation matrix embracing all the data is
shown in Table 2 and the relationships between
net calcium absorption by the balance method on
the one hand and the various radioactive calcium
measures on the other are illustrated in Figs. 1-4.
The relation between net calcium absorption
and the square-root of the initial rate of radioactive calcium absorption (A) is shown in Fig. 1.
There was a highly significant correlation between these two variables ( r = 0.74; P < 0.001).
The regression equation is: net = 13-5 x ,/A 6.6 and the SD about the line is 3.3 mmol/day.
Calcium absorption
=I
a
1 5 t
479
20
L
15
-
* *
a
*
h
P
3
zE
10-
v
8
b
b
5 -
P
EL
b
2
sm
0 '
Y
s
0
r=0.74
b
-10
I
I
0
0
0.1
0.5
-5L--
-10 0
L
I
1.0
2.0
FIG. 1. Relation between initial fractional rate of
calcium absorption based on double-isotope calculation (A) and net calcium absorption from balance
studies.
0.6
0.8
1.0
Fraction absorbed at 2 h
3.0
I (square-rootscale)
0.4
0.2
FIG.2. Relation between fraction of radioactive calcium absorbed at 2 h based on the double-isotope
calculation (4) and net calcium absorption from
balance studies.
e
The relation between the fraction of the dose
absorbed in 2 h derived from the double-isotope
procedure (#) is shown in Fig. 2. The correlation
coefficient was again highly significant ( r = 0.63;
P < 0.001), though not as high as in Fig. 1. The
regression equation is: net = 14.7 x # - 4.3 and
the SD about the line is 3.8 mmol/day.
The relation between net calcium absorption
and the square-root of the average fractional rate
of absorption from the single-isotope procedure
(4 is shown in Fig. 3. The correlation between
them was highly significant ( r = 07.6&
;'
<
0.001) and very similar to that between net
calcium absorption and 1 (Fig. 1). The regression
is: net = 17.0 x \/A - 7.3 and the SD about the
line is 3.2 mmol/day.
The relation between net calcium absorption
and the fraction of the single-isotope dose
circulating at 1 h corrected for body weight (Fc)
is shown in Fig. 4. The correlation coefficient was
highly significant (r = 0.70; P < 0.001). The
regression equation is: net = 32.7 x Fc - 3.2 and
the SD about the line is 3.5 mmol/day.
Since the double-isotope calculation which
gives the highest correlation with net calcium
absorption is r = 0.74, we have compared the
single-isotope calculations with 1 in Figs. 5 and 6.
It should be noted that these are not independent
measurements, since they have in common the
plasma concentrations from the oral dose.
Fig. 5 shows the relationship between J1and
-
*Or
15
h
P
P
L
P
10
.
-/
.*
v
-
.-8
EL
!2
5 -
sm
u,
z
O
------
-1ok
0
,
.
0.1
0.5
I
,
,
1.0
1.5
2.0
square-root scale)
FIG.3. Relation between fractional rate of radioactive calcium absorption after single-isotope test (u)
and net calcium absorption from balance studies.
Ja; that between Ji and Fc is shown in Fig. 6.
The correlation coefficient was very significant
( r = 0.85; P < 0-OOl), but there was slightly
more scatter than in Fig. 5.
Discussion
In the absence of an ultimate standard of calcium
absorption, we have attempted to assess the
D. H . Marshall and B. E . C. Nordin
480
..
20 L
t
3-0
I
15
**
a
2.0
/*
.
...%'
.*
1.51
u
H
r=0.851
-5t
-101
r=
.
,
0
.
0.70
.
,
,
0.4
0.2
0.6
FIG.4. Relation between the fraction of radioactive
calcium dose circulating 1 h after single-isotope
administration (F,) and net calcium absorption from
balance studies.
-
1.5
h
d
v)
u
1.0
g
0
0.5
Y
*
-
0.1
r=
0.87
~
0
0.1
0.5
1.0
0
0.2
0.4
0.6
Fraction of dose in 15% body wt.
Fraction of dose in 15% body wt.
1.5
2.0
square-root scale)
FIG.5. Relation between the mean fractional rate of
radioactive calcium absorption after a single isotope
(a)and the initial fractional rate of radioactive calcium
absorption derived from the double-isotope calculation
(a).
validity of various radioactive calcium procedures and calculations by comparing them with
each other and with net calcium absorption
measured by the balance technique in an unselected series of 100 studies in 71 patients with a
variety of diagnoses, some untreated and others
FIG.6. Relation between the fraction of dose circulating 1 h after single-isotope administration (F,) and the
initial rate of radioactive calcium absorption derived
from the double-isotope calculation (A).
treated. In this way, we have been able to perform
our comparisons over a wide range of absorptive
states.
Net absorption has been taken as the reference
standard rather than 'true' absorption which
requries, in addition to the measurement of
dietary and faecal calcium, the estimation of
endogenous faecal calcium. This endogenous
excretion is small in comparison with the total
faecal calcium at these intakes and subject to a
relatively large error in its estimation. This 'true'
absorption has not been found to be significantly
bettefi than the 'net' absorption to the radioactive
isotope tests.
Taking the net calcium absorption, measured
as accurately as we were able and knowing that it
was subject to an error of not less than 0.5
mmol/day, we have shown that there is little to
choose between the various radioactive calcium
procedures. In particular, the calculation of
radioactive calcium absorption rates from
analysis of six blood samples after a singleisotope test is at least as good, on this criterion,
as the initial rate of absorption calculated from
the double-isotope test. Again, and using the
same criterion, there is surprisingly little lost by
confining the analysis to a single blood sample
taken 1 h after administration of the dose and
corrected for body weight. However, it is of
interest that the measurement of calcium absorption rate (1 and cc) agrees better with net
calcium absorption than does the measurement of
Calcium absorption
the fraction of the dose absorbed (@,confirming
the observations of Reeve et al. [lo].
Internal comparison of the different radioactive calcium methods again shows such high
correlations between them as to cast considerable
doubt on the need for a double-isotope procedure
or even multiple blood sampling, after a single
isotope, unless high precision is required,
although it must be borne in mind that the
various procedures share a number of common
measurements which must contribute to these
very high correlations. These correlations
obtained despite the inclusion in the material of
patients with high bone turnover due to osteomalacia and hyperparathyroidism. The implication is that the six blood-sample singleisotope test is as good as the double-isotope test
in correcting for variations in bone uptake. Even
the simplest procedure involving a single blood
sample, where no correction for bone uptake is
possible, will inevitably tend to underestimate
calcium absorption in states of very high bone
turnover such as severe hyperparathyroidism and
osteomalacia, particularly perhaps after vitamin
D treatment of the latter. Examination of our
data shows that some of the scatter in Fig. 6 is
accounted for in this way; doubtless the same
would be true in Paget's disease.
Nonetheless, and these reservations notwithstanding, the single-isotope single blood-sample
procedure would appear to be adequate for most
clinical purposes. If the object of a calciumabsorption test is to establish whether the patient
is a malabsorber, normal absorber or high
absorber of calcium, then this simple procedure
would appear to be adequate and has the
enormous merit of being suitable for multiple
observations on the same subject both because of
the minimal discomfort to the patient and the
minimal technical and medical time that it
31
48 1
involves. Where it is essential to measure calcium
absorption with high precision, we do not doubt
that multiple blood sampling or even doubleisotope procedures may be required, but it is our
strong impression at the present time that the
considerable variation in calcium absorption, not
only between but also within individuals,
generally makes high-precision measurement a
somewhat uneconomic exercise.
References
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[31 DE GRAZIA,J.A., IVANOVICH,
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