Download Calcium absorption measured by stable calcium isotopes (42Ca

Journal of Orthopaedic Surgery 2002, 10(1): 61–66
Calcium absorption measured by stable
calcium isotopes (42Ca & 44Ca) among northern
Chinese adolescents with low vitamin D status
Warren T K Lee and Jack C.Y. Cheng
Department of Orthopaedics & Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong
Ji Jiang, Pei Hu, and Xiaopeng Hu
Clinical Pharmacological Research Center Laboratory, Peking Union Medical College Hospital, Beijing, China
David C K Roberts
Discipline of Nutrition & Dietetics, Faculty of Medicine & Health Sciences, The University of Newcastle, Australia
INTRODUCTION
ABSTRACT
An adequate calcium intake and vitamin-D status is
important for bone mineralization in adolescents. In
Northern China, calcium intake and plasma vitaminD level of adolescents is low due to low consumption
of dairy foods and inadequate sunshine exposure. True
fractional calcium absorption (TFCA) in Chinese
adolescents has never been performed.
This study aims to evaluate nutritional adaptation
namely, TFCA and urinary calcium excretion among
Chinese adolescents in northern China.
Key words: calcium absorption, calcium intake, 25hydroxyvitamin D, puberty, Chinese
In China, calcium intakes of children are low due to
their non-dairy based diets. Calcium intakes of school
children are usually below 500 mg/d (Lee et al. 1993,
Institute of Nutrition & Food Hygiene, 1992). Results
of both cross-sectional and follow-up studies (Lee et
al. 1993, Johnson et al. 1992, Lee et al. 1994a, Lee et al.
1995a, Lee et al., 1996) have together suggested that a
persistently higher calcium intake in childhood and
adolescence may help to promote higher bone mass
in adulthood. Maximization of peak bone mass by lifelong adequate calcium intake is recognized as the best
protection against the development of osteoporosis in
later life (Institute of Medicine 1997). Until recently,
there have been limited studies to examine nutritional
adaptation among children accustomed to low calcium
diets coupled with a low vitamin D status. Whether
children with low calcium intake and low vitamin D
status are able to adapt to optimise skeletal growth is
largely unknown.
Address correspondence and reprint requests to: Dr. Warren T K LEE, Department of Orthopaedics & Traumatology, Faculty of
Medicine, The Chinese University of Hong Kong, 5/F Prince of Wales Hospital, Shatin N.T. Hong Kong. E-mail: [email protected]
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Warren T K Lee et al.
Our previous study has indicated that growing
children with normal plasma vitamin D status
accustomed to low calcium diets were able to enhance
calcium absorption and reduce urinary calcium
excretion (Lee et al. 1994b). The figure for true
fractional calcium absorption (TFCA) (54 – 63 %) was
found significantly higher then those of US children
(25-34%) consuming a calcium diet of 925 mg/d
(Abrams & Stuff 1994). This result implies that there is
a difference in calcium handling between the Chinese
and American children (Lee et al. 1995b). In fact, our
previous study was conducted in Southern China
where there is plenty of sunshine throughout the year.
Children could obtain most of their vitamin D by
regular exposure to the sun. Adequate circulating
vitamin D seems to allow nutritional adaptation to
occur (Lee et al. 1994b, 1995b). Nonetheless, the climate
in Northern China is totally different from that in the
South, where children experience long cold winters
and a lack of sunlight exposure leads to sub-optimal
vitamin D status. Among Beijing school children, the
average plasma 25(OH)-vitamin D3 (25-OHD) level in
winter is alarmingly low (13.4 nmol/L) and nearly 50%
of school children have plasma 25-OHD level below
the lower normal limit of 12.5 nmol/L (Du 1998).
Plasma 25-OHD level and calcium intakes were found
to be strong predictors on bone mass in Beijing children
(Du 1998). The prevalence of nutritional rickets in
North China is still high among children under 3 years
of age (6–44%) and adolescents (7-24%), which is
attributable to low sunshine exposure and poor source
of vitamin D in foods (Wu et al. 1991, Du 2001). It is
not known whether these children with poor vitamin
D status and subsisting on low calcium diets are able
to absorb adequate calcium for bone mineralization.
Objective
The objective of the present study was to determine
whether adolescents living in northern China with
lower vitamin D status are still capable of enhancing
calcium absorptive efficiency.
MATERIALS AND METHODS
Sixteen students (12 girls, 4 boys) aged 9 to 16 years
were recruited from schools in Beijing during
December, 1999. All the students were healthy, free
from any metabolic diseases that may interfere with
calcium and vitamin D metabolism, and without any
history of bone fractures in the previous 12 months.
Vitamin D status of Beijing adolescents is lowest during
winter due to low sunshine exposure (Du 1998).
Journal of Orthopaedic Surgery
In vivo TFCA was determined by the dual stable
isotope technique which is non-radioactive and safe
to be applied on children and adolescents (Yergey et
al. 1987, Hillman et al. 1988). The use of dual stable
isotopes technique is based on the validated method
developed by DeGrazia et al (1965) and Hillman et al.
(1988), and modified by the author (WTKL) (Lee et al.
1994b, 1995b). After an overnight fast, 44Ca (0.5 mg/
kg) was given orally by mixing with 120 ml cow’s milk
24-h in advance of the study. The milk isotope was
sub-divided into three small doses and were
administrated with the three main meals of the day.
Sub-dividing up the oral tracer into three small doses
to be given with each of the three meals in a day was
found to best represent the overall absorption of
calcium (Eastell et al. 1989). 42Ca solution for injection
was prepared, sterilised and dispensed aseptically into
a vial in the Clinical Pharmacological Research Center
Laboratory, Peking Union Medical College Hospital.
42
Ca (0.05 mg/kg) was given intravenously via the
antecubital vein by a nurse immediately after the first
oral dose. 5 ml of venous blood was drawn for the
measurement of 25-OHD and serum calcium
concentrations immediately before the i.v. injection of
42
Ca solution. Details of dosing the subjects can be
found elsewhere (Lee et al. 1994b, 1995b). 24-h urine
collection started exactly 24-h after the isotopes were
administered in order to determine the recovery of the
two isotopes in the urine.
Calcium intake was assessed by a quantitative food
frequency questionnaire specifically designed for use
among Chinese children and adolescents (Lee et al.
1996), weight and height were measured by standard
methods as described elsewhere (Lee et al. 1994b).
Plasma 25-OHD was measured by a competitive
protein assay (Amersham, Bucks., U.K.) (Lee et al.
1994b). Serum calcium and 24-h urinary calcium
concentrations were analysed by automated analysis
(Beckman Synchron CX3 Clinical System, CA, USA.).
Informed consent was obtained from the parent. The
research protocol was approved by the Human
Research Ethics Committee, The University of
Newcastle.
Mass spectrometric analysis for isotopic recovery
in urine
The organic matters of the urine were removed by
digestion upon heat with concentrated nitric acid.
Calcium was precipitated from the digested sample
by using ammonium oxalate (Fairweather-Tait 1995).
All the samples were analysed by Thermal Ionization
Mass Spectrometry using a Finnegan MAT 261 Magnet
Sector Mass Spectrometer (Finnegan-Mat GmbH,
Vol. 10 No. 1, June 2002
Calcium absorption measured by stable calcium isotopes
63
Bremen, Germany). The calculation of TFCA was based
on the fact that both intravenously and orally
administered calcium isotopes are metabolised at the
same rate once achieving equilibrium. TFCA was
determined according to the equation developed by
Yergey et al. (1987).
Statistical analysis
Descriptive statistics (mean, SD and range) were
calculated for all variables. Linear regression analysed
was employed to study the relation between different
variables and TFCA. The level of significance was set
to P ≤ 0.05, 2-tailed). Statistical analysis was performed
by using SPSS 8.0 for Windows (SPSS Inc. Chicago, IL,
USA).
RESULTS
Characteristics of the 16 subjects are shown in Table 1.
Mean age of the subjects was 12.2 ± 2.1 years. Mean
calcium intake of the subjects was 603 ± 158 mg/d.
Milk and dairy products contributed to 54% of the
daily total calcium intake (317 ± 133 mg/d), whereas
vegetables, beans and bean products contributed to
9% and 5% of the total daily calcium intake of these
adolescents.
Table 1
Characteristics of the 16 subjects
Age (y)
Weight (kg)
Height (cm)
Calcium Intake (mg/d)
Mean ± SD
Range
12.2 ± 2.1
41.2 ± 9.8
1.51 ± 0.09
603 ± 158
9–17
27.5–55.0
1.33–1.63
402–847
Mean true fractional calcium absorption (TFCA),
serum 25-OHD, serum calcium and 24-h urinary
calcium of the 16 subjects are depicted in Table 2. There
were no association between TFCA and age (r = 0.05,
p=0.86), weight (r = 0.38, p = 0.15), height (r = 0.31,
p=0.24), calcium (r = –0.16, p = 0.62) or 24-h urinary
calcium excretion (r = –0.115, p = 0.67). However, there
was a significant negative correlation between TFCA
and plasma 25-OHD (r = –0.79, p < 0.001) (Fig. 1).
The subjects in the study are predominantly girls;
therefore, the results are also analysed and presented
by grouping all the girls in one group. The
characteristics of the adolescent girls are shown in
Table 3. Mean calcium intake of the girls was 591 ±
164 mg/d. Mean TFCA, serum 25-OHD, serum
Figure 1 Correlation between True Fractional Calcium
Absorption (TFCA) and 25-(OH)Vitamin D3 (25-OHD) in 16
Beijing Adolescents.
Table 2
True fractional calcium absorption (TFCA),
plasma 25-OHD, serum calcium and 24-h
urinary calcium of the 16 subjects
TFCA (%)
25-OHD (nmol/L)
Serum calcium (mmol/L)
24-h urinary calcium (mg/d)
Mean ± SD
Range
57.4 ± 15.4
34.3 ± 12
2.3 ± 0.15
87.5 ± 59.2
28.6–83.0
15–60.75
1.9–2.6
16–204.8
calcium and 24-h urinary calcium of the 12 girls are
depicted in Table 4. There were no association between
TFCA and age (r = –0.11, p=0.73), weight (r = 0.10, p =
0.75), height (r = 0.16, p=0.93), dietary calcium (r = 0.13,
p = 0.69) or 24-h urinary calcium excretion (r = –0.17,
p = 0.59). Again, there was a significant negative
correlation between TFCA and plasma 25-OHD (r =
–0.73, p = 0.008).
Table 3
Characteristics of the 12 Beijing adolescent girls
Age (y)
Weight (kg)
Height (cm)
Calcium intake (mg/d)
Mean ± SD
Range
12.4 ± 2.4
42.1 ± 9.2
1.52 ± 0.09
591 ± 164
9–17
28–55
1.33–1.63
402–847
Table 4
True fractional calcium absorption (TFCA), serum 25-OHD,
serum calcium and 24-h urinary calcium
of the 12 Beijing adolescent girls
Mean ± SD
TFCA (%)
60.4 ± 14.4
25-OHD (nmol/L)
30.54 ± 9.835
Serum calcium (mmol/L)
2.29 ± 0.16
24-h urinary calcium (mg/d) 79.9 ± 49.6
Range
41.3–83.0
15–47.25
1.9–2.5
22.4–185.6
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Warren T K Lee et al.
DISCUSSION
The present study was piloted to evaluate true
fractional calcium absorption in relation to vitamin D
status and urinary calcium excretion in a group of
adolescents with poor vitamin D status living in
northern China. The number of subjects in this pilot
study was limited to 16 due to the high costs of the
isotopes (HK$3,600 per subject in 1999) and the
laboratory cost for treating urine samples (HK$400 per
subject).
Traditionally, calcium absorption has been
determined by balance studies. However, this
approach fails to differentiate the endogenous calcium
from the dietary source, and hence underestimates the
true fraction of calcium absorbed. Furthermore, the
balance technique is tedious, labour intensive, and has
inherent problems of incomplete urine and fecal
collection. Although TFCA can be determined by using
radioisotopes of calcium (DeGrazia et al. 1965), the
inherent potential hazards of ionizing radiation limit
its use in growing children. The recent development
of a dual stable isotope technique provides a safe, nonradioactive and accurate method of measuring in vivo
TFCA in infants and children (Hillman et al. 1988, Lee
et al. 1994b, Abrams et al. 1997). Most importantly, the
dual stable isotopes technique can correct for the
endogenous calcium secreted into the gut and greatly
improves the accuracy of the estimation of TFCA. By
collecting a urine sample 24 hours after the absorption
test, TFCA can be evaluated by determining the ratio
of the dual stable isotopes recovered in the urinary
sample using a thermal ionisation mass spectrometer
(Lee et al. 1994b, 1995b; Abrams et al. 1997). The dual
stable isotopes technique is less time consuming, at
one day compared to 3-4 weeks with the traditional
balance method. Thus, the new technique also
improves subject compliance (Lee et al. 1995b; Abrams
et al. 1997). In fact, the dual stable isotope technique is
currently adopted by international nutrition expert
groups (Department of Health, 1991, Institute of
Medicine, 1997) as the gold standard to determine in
vivo calcium bioavailability. The determination of in
vivo calcium bioavailability is essential to establish
calcium requirement and thus recommended dietary
allowances (RDAs) for calcium (Department of Health,
1991).
The mean TFCA of the 16 subjects was higher than
the US counterparts (25–34%) with calcium intake at
925 mg/d (Abrams & Stuff, 1994). In addition, the
mean TFCA from the present study was comparable
to the author’s previous calcium absorption study
among 7-year-old children in Hong Kong (TFCA: 54–
63%, dietary calcium intake: 862 mg/d and plasma 25-
Journal of Orthopaedic Surgery
OHD 83.25 nmol/L). The findings from the present
study and our previous studies (Lee et al. 1994b, 1995b)
confirmed that there is a difference in TFCA between
the Chinese and Caucasian children and adolescents.
An adequate vitamin D status is important for
enhancing calcium absorption (Lee et al. 1993), the
mean serum level of 25-OHD of the subjects in the
present study was close to the lower normal limit of
27.5 nmol/L.
It is interesting to find that there was a strong
inverse correlation between TFCA and plasma 25OHD, the explanation for this phenomenon is
uncertain and it has never been reported before. It is
speculated that adolescents with escalated demand for
bone mineralization during pubertal bone growth may
have adapted to a habitually lower vitamin D status
by increasing the rate of conversion of 25-OHD in the
kidney to the active metabolite of vitamin D 3
(1,25(OH)D3). Further study is necessary to look into
the profile of calcium homeostasis in this group of
adolescents in order to understand the mechanism of
nutritional adaptation. Mean 24-hour urinary calcium
excretion was lower than 100 mg/d implied that there
was higher renal conservation of calcium in this group
of adolescents in order to retain sufficient calcium for
bone growth.
Results of the current study will provide some new
scientific data to revise the Recommended Dietary
Allowances (RDAs) for children and adolescents in
China. Furthermore, countries in South East Asia are
in a joint effort to establish local RDAs figures based
on research studies on local populations (Tee, 1998).
Findings from this study will provide some reference
data for the formulation of RDAs in Asian children
and adolescents subsisting on non-dairy based diets.
Standard deviations of the outcomes given in Table
2 were wide among the 16 subjects with inter-subject
coefficient of variations (CV = SD/Mean x 100%) of
TFCA, 25-OHD and 24-h urinary calcium at 26.8%, 35%
and 67% respectively. These discrepancies may be due
to a wide age range (9 to 17 years old), individual
variation in calcium intake (402–847 mg/d) and
calcium handling. In our previous TFCA study, the
studied children (n=34) were all aged 7, and the CV of
TFCA was 13–17%. Furthermore, subjects in our
previous study were stratified into two groups based
on calcium intake (< 500 mg/d and > 500 mg/d) prior
to group mean comparison (Lee et al. 1994b). On the
other hand, inter-subject CV of TFCA was also wide
in other studies conducted in USA (Abrams & Stuff
1994; Abrams et al. 1995; O’Brien et al. 1996) (21–39%),
and the age range of the subjects in those studies were
also large (5 to 17 years or 8 to 16 years).
The inter-subject CV of plasma 25-OHD in the
Vol. 10 No. 1, June 2002
present study was 35% which might be attributable to
the extent of sunshine exposure and oral vitamin D
intake. Such a wide inter-subject CV was also observed
in previous studies among Black and White girls aged
5–17 years in USA (17–39%) (Abrams & Stuff 1994;
Abrams et al. 1995; O”Brien et al. 1996). On the other
hand, the inter-subject CV of 24-h urinary calcium
excretion was even greater in the present study (67.7%)
which is related to dietary intake of sodium and protein
and most importantly the stage of pubertal growth —
urinary calcium concentration markedly reduces in
peripubertal period but not in post-pubertal period
(Abrams & Stuff 1994; Abrams et al. 1995). Such a large
inter-subject CV of 24-h urinary calcium was also
observed in other studies (53–78%) (Abrams & Stuff
1994; Abrams et al. 1995).
In conclusion, the study demonstrated that
Calcium absorption measured by stable calcium isotopes
65
growing individuals with sub-optimal vitamin D
status are still capable of enhancing TFCA and
reducing urinary calcium absorption to allow for
adequate calcium for bone growth and mineralization.
The research findings from this study will provide
scientific data as regard to nutritional adaptation in
growing children and adolescents, in particular
missing data on intestinal absorption, urinary calcium
excretion among children and adolescents accustomed
to non milk based diet and poor vitamin D status.
ACKNOWLEDGEMENTS
The study was supported in part by a research grant
from the Research Management Committee, The
University of Newcastle, NSW, Australia.
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