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CLIN. CHEM. 36/1, 142-144 (1990)
Albumin-Adjusted Calcium Concentration in Serum Increases during Normal Pregnancy
R. B. Payne, & J. LIttle, and R. 1. Evans
Concentrations of total calcium and albumin were measured
in serum specimens from 41 women at intervals before,
during, and after 42 pregnancies. The albumin concentration
decreased but the calcium decreased more slowly, so that
the albumin-adjusted calcium concentration increased from
conception to term. These findings, taken in conjunction with
published observations of hypercalciuria, increased concentrations of 1,25-dihydroxycholecalciferol
and calcitonin in
serum, and decreased concentrations of intact parathyrin in
serum, strongly suggest that maternal ionized calcium increases throughout normal pregnancy.
Striking changes in maternal
calcium metabolism take
place during pregnancy. The plasma concentrations
of
1,25-dihydroxycholecalciferol
(1,25-DHCC) (1-3) and calcitonin (4) increase, the gastrointestinal
absorption of calcium increases (2), and there is a marked increase in
urinary calcium (2,5,6). Despite this, and despite evidence
of some reversible bone resorption in early pregnancy (7),
calcium balance is positive (5) and maternal bone structure
Department of Chemical Pathology, St. James’s University
Hospital, Leeds LS9 7TF, U.K.
Received August 18, 1989; accepted September 25, 1989.
142 CLINICAL CHEMISTRY, Vol. 36, No. 1, 1990
(7) and bone mineral content (8) remain unchanged at
term. Studies of changes in plasma concentrations of parathyrin (parathyroid hormone, PTH) during pregnancy
have given conflicting results. Workers using older one-site
radioimmunoassays
that measure fragments as well as
intact PTH have variously reported increased, normal, or
decreased concentrations
of the hormone. However, the
first study involving a specific two-site radiometric assay
for the intact PTH molecule has clearly shown lowered
concentrations
(9).
changes suggest that 1,25-DHCC synthesis
increases first, stimulating
intestinal absorption of calcium
and leading to hypercalcemia, increased calcitonin
secretion, secondary hypoparathyroidism,
and hypercalciuria.
One link in this chain has not been confirmedby measurement: hypercalcemia.
Because the albumin concentration
in plasma decreases substantially
by about 10 g/L during
pregnancy (10, 11), total calcium also decreases. Ionized
calcium
concentration measured in anaerobic specimens
has, with one exception (12), been reported as unchanged or
lowered (2,9,13-17).
However, the instruments
commonly
used to measure ionized calcium report lower values when
the albumin concentration is decreased (18-21), so that an
increase in ionized calcium during pregnancy might be
obscured by the decreasing albumin. We have therefore
examined the relationship between total calcium and albuThese
mm in serum samples collected before, during, and after
pregnancy, to seek evidence for hypercalcemia.
and Methods
Subjects
Women who were attending family-planning
clinics in
and around Leeds during 1983-1986 and who expressed
their intention to stop using contraception so as to become
pregnant were invited by one of us (R.T.E.) to take part in
a longitudinal
study of changes in serum cholinesterase
activity.
Blood specimens were collected at four-week intervals before and during pregnancy. The volunteers came
to the laboratory for venipuncture before conception and in
the early stages of pregnancy, but were visited at home
later and after delivery. Blood was obtained within 24 h of
delivery and at varying
intervals for 12 weeks subsequently. Further details of the subjects and the results of
this study have been published (22).
We took the opportunity to analyze the serum samples
for some common analytes on a Monitor Parallel discretionary analyzer (American Monitor UK Ltd., Burgess Hill,
Sussex, U.K.). Total calcium was measured by using cresolphthalein complexone, and albumin
by dye-binding with
bromcresol green after a standard reaction time of 1.5 mm.
Analytical CVs at normal concentrations in serum, calculated from duplicate measurements, were 1.1% for calcium
and 1.7% for albumin.
Serum
samples were stored at
-15 #{176}C
before analysis, and all samples from one individual were thawed and analyzed in the same batch. Results
were available for 42 normal pregnancies in 41 women.
The measured
calcium concentration was adjusted
for
albumin in two stages. First, to estimate the concentration
of non-protein-bound calcium in the sample, we subtracted
from the total calcium the product of albumin concentration
and the slope of the regression of calcium on albumin as
determined in patients with a wide range of albumin concentrations but no obvious disturbance of calcium homeostasis. Then we added to that value a constant, the average
concentration of protein-bound calcium in health (the difference between the intercept of the regression equation
and
the mean of the normal range). The equation we used was as
follows: Adjusted calcium = total calcium
(0.025 x albumin) + 1.0, with calcium in minol/L and albumin in gIL. The
slope (0.025 mmollg) and constant (1.0 mmol/L) have not
-
110,-
0
a
E
C
0
a.
a
U
951
C
90
0
C
a
.
1001
8
a.
.
1051-
85
80
L
L
L
Results
The mean concentrations
of total calcium and albumin
in
serum were determined
in specimens collected within each
four-week period of pregnancy and at intervals afterwards,
and expressed as a percentage of the mean values before
conception (Figure
1). The total calcium concentration
decreased less steeply than would be predicted
from the
relationship between calcium and albumin if ionized cal-
cium were unchanged. In the last trimester of pregnancy,
the total calcium concentration began to increase while the
mean albumin concentration was still declining. However,
adjusted calcium increased linearly from conception to
term (r = 0.962; P <0.001). Quantitative
differences between concentrations in the specimens taken shortly before
conception and before delivery are summarized
in Table 1.
Mean bicarbonate concentration decreased
by 3.1 mmol/L
from conception to delivery (P <0.001). The anion gap did
not change significantly.
Immediately
after delivery there was an abrupt decrease
in both total calcium and albumin concentration, presumably because of redistribution of protein and fluid. However, adjusted calcium remained unchanged. Total calcium
and albumin values then increased and the adjusted
calcium values decreased exponentially
to reach preconception values by eight weeks post partum.
Discussion
The gradual increase
in albumin-adjusted
calcium we
observed during pregnancy could be due to (a) an increasing underestimation of albumin concentration
as pregnancy progressed, (b) a gradual increase in protein-bound
or complexed calcium with no change in ionized calcium, so
that the slope of the regression we used to adjust calcium
for albumin became increasingly
inappropriate, or (c) a
true increase in ionized calcium.
Albumin can be underestimated
in the presence of
metabolites that interfere with the chemistry of the method
used for its measurement.
For example, substances retained during renal failure result in spuriously low albumin values when measured by a dye-binding method involving bromcresol purple (26, 27). However, there have
been no reports of metabolite interference with the bromcresol green method we used. Protein-bound
calcium increases in pregnancy as a consequence of the mild compensated respiratory alkalosis, with an increase of pH of about
Table 1. Change In Serum Concentrations
in 42 Normal
Pregnancies from Shortly before Conception to Shortly
before Delivery
Calcium, mmol/L
75L
0
differed significantly
in assessments made on patients in
this hospital over a period of 15 years (23-25).
Student’s paired t-test was used to assess changes in
concentration
from shortly before conception to shortly
before delivery.
I
40
I
80
I
120
I
160
200
Duration of gestation (days)
240
280
I
0
40
Albumin,
g/L
80
Days after delivery
FIg. 1. Changes in concentrationsin serum during pregnancy and
the puerperlum in 42 pregnancies in 41 women
Mean serum albumin(U), total
calcIum(0),and albumin-adjustedcalcium (S)
concentrations,
expressed as a percentageof the meanconcentrations
inthe
specimensshortly preceding conception (0 days)
Mean change
Standarddeviation
Lower95% limit
Upper95% limit
2.79
-15.3
-4.1
Measured
-O.08
0.092
-0.26
+0.11
Adjusted
+0.16*
0.103
-0.04
+0.38
*signifndy (P <0.001) different from zero.
CLINICALCHEMISTRY, Vol. 36, No. 1,
1990
143
0.02 unit and a decrease in bicarbonate of about 3 mmol/L.
However, such binding can account,at most, for a change of
only 1% compared with the 7% we observed(Figure1).An
increase in complexed calcium is improbable because the
concentration of bicarbonate, the principal complexing anion, decreased and the anion gap did not change. Thus, the
largest part of the increase in adjusted calcium is most
probably due to an increase in ionized calcium.
Why has the concentration of ionized calcium been reported to be unchanged or lowered during pregnancy (2,9,
12-17)? The commonly used ionized calcium analyzers
show a positive relationship between the calcium result
and the albumin concentration in a sample (18-21), almost
certainly an effect of reference electrode design and composition (28). Because the apparent ionized calcium decreases
as albumin does (20), a true increase during pregnancy
could be obscured.
The gradual linear increase in adjusted calcium during
pregnancy accompanies a similar linear increase in serum
1,25-DHCC (1). It seems likely that the sequence of events
leading to hypercalcemia
and hypercalciuria is initiated by
hormonal changes that increase renal (29) or placental (30,
31) synthesis of 1,25-DHCC. Fetal FF11-related
protein
may be responsible, animal experiments
having shown this
protein to be present in placenta as well as fetal parathyroid; this protein is also thought to control calcium transport from mother to fetus (32). It may well be that the
activity of placental 1-a-hydroxylase
is controlled by FFHrelated protein, by analogy with the control of the renal
enzyme by FFH.
The present coherent interpretation
of changes in calcium homeostasis during pregnancy has depended on recognizing the nonspecificity of two clinical laboratory methods: ionized calcium measurement
and single-antibody
radioimmunoassay
for FF11.
Clinically,
it is worth remembering
that an increase in
albumin-adjusted
calcium in serum by up to about 0.40
mmol/L is normal in pregnancy (Table 1), as is urinary
calciumexcretion as high as 15 mmol/day (6); neither is an
indication for intensive investigation of calcium homeostasis.
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