Journal of Pediatric Gastroenterology and Nutrition
42:419Y 426 Ó April 2006 Lippincott Williams & Wilkins, Philadelphia
Fractional Calcium Absorption Is Increased in Girls with
Rett Syndrome
*†§Kathleen J. Motil, †§Rebecca J. Schultz, *†§Steven Abrams,
*†Kenneth J. Ellis, and †‡§Daniel G. Glaze
*USDA/ARS Children’s Nutrition Research Center, †Department of Pediatrics, ‡Department of Neurology,
Baylor College of Medicine, and §Texas Children’s Hospital, Houston, Texas
Results: Fractional Ca absorption was significantly higher in
RTT than in control girls (mean T SDp, 52 vs. 33 T 13%).
Dietary Ca intake (mean T SDp, 1,100 vs. 1,446 T 440 g/d) and
net Ca absorption (mean T SDp, 513 vs. 362 T 306 mg/d) did not
differ significantly between RTT and controls, respectively.
Although urinary Ca excretion did not differ between groups,
the increased urinary Ca:creatinine ratio (mean T SDp, 0.39 vs.
0.23 T 0.38) was consistent with clinical hypercalcuria and
paralleled the significantly increased urinary cortisol excretion
(mean T SDp, 3.1 vs. 1.7 T 1.1 mg/kg lean body mass per day)
in the RTT girls. BMC was significantly lower in RTT than in
controls (mean T SDp, 527 vs. 860 T 275 g). Serum Ca, P,
alkaline phosphatase, vitamin D metabolites, PTH, and osteocalcin concentrations did not differ between the groups.
Conclusion: Fractional Ca absorption showed a compensatory
increase in the presence of adequate dietary Ca intakes, mild
hypercalcuria, and pronounced bone mineral deficits in RTT
girls. Whether supplemental dietary Ca could enhance fractional
Ca absorption and improve bone mineralization in RTT girls is
unknown. JPGN 42:419Y426, 2006. Key Words: Osteopenia VNeurologic disorders VBone mineral contentVDietary
calciumVVitamin D VAnticonvulsant therapy VParathyroid
hormone VCortisol. Ó 2006 by Lippincott Williams & Wilkins
ABSTRACT
Background: Rett syndrome (RTT), an X-linked neurodevelopmental disorder primarilyaffecting girls, is characterized in
part by osteopenia and increased risk of skeletal fractures. We
hypothesized that decreased intestinal calcium (Ca) absorption
relative to dietary Ca intake and increased renal Ca excretion
might cause these problems in RTT.
Objective: We measured fractional Ca absorption, urinary Ca
loss, dietary Ca intake, and the hormonal factors regulating Ca
metabolism to determine whether abnormalities in Ca balance
might relate to poor bone mineralization in RTT girls and to
evaluate the contribution of these factors to the overall dietary
Ca needs of RTT girls.
Study Design: Ten RTT girls and 10 controls, matched for age,
sex, and pubertal status, were given a 3 day constant Ca diet that
mimicked their habitual intakes. At the end of each dietary
period, girls received single doses of 42Ca (intravenous) and 46Ca
(oral). Fractional urinary excretion of 42Ca, 46Ca, 24 hour
urinary Ca, and urinary cortisol excretion were determined.
Serum Ca, phosphorous, alkaline phosphatase, vitamin D
metabolites, parathyroid hormone (PTH), and osteocalcin were
measured in the postabsorptive state. Bone mineral content
(BMC) was measured by dual-energy x-ray absorptiometry.
INTRODUCTION
Received November 2, 2005; accepted December 28, 2005.
Address correspondence and reprint requests to Dr. Kathleen J. Motil,
Children’s Nutrition Research Center, 1100 Bates Street, Houston, TX
77030 (e-mail: [email protected]).
This work is a publication of the USDA/ARS Children’s Nutrition
Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, and has been funded in part with federal funds from
the US Department of Agriculture, Agricultural Research Service under
Cooperative Agreement Number 58-6250-1-003; the National Institutes
of Health Program Project Grant P01 HD 24234; and the National
Institutes of Health General Clinical Research Centers Grant M01 RR00188; and with funds provided by the Blue Bird Circle and International Rett Syndrome Association. The content of this publication does
not necessarily reflect the views or policies of the US Department of
Agriculture, nor does mention of trade names commercial products, or
organizations imply endorsement by these agencies.
Rett syndrome (RTT) is an X-linked dominant
neurodevelopmental disorder caused by a mutation of
the MECP2 gene (1). The hallmark of the syndrome is
apparently normal development until 6 to 18 months of
life, followed by a period of rapid developmental
regression. During the period of regression, RTT girls
lose acquired speech; they replace purposeful hand use
with hand stereotypies; their cranial growth slows; and
they may experience seizures, autistic features, ataxia,
gait apraxia, and breathing abnormalities when awake
(2,3). The classic features of RTT are found primarily in
419
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420
MOTIL ET AL.
girls because the syndrome is phenotypically different
and may be lethal in males (4). Although there is no
cure for RTT, the clinical complications of the disorder
require aggressive intervention to maximize the quality
of life of affected girls.
Osteopenia is a frequent complication of RTT syndrome (5Y8). Although the cause is unknown, impaired
ambulatory ability and use of anticonvulsant medications
have both been implicated (6). Osteopenia is present in
RTT girls at an early age despite adequate dietary
calcium (Ca) intake (5Y7) and is characterized by more
profound bone demineralization than that found in
children with other neurologic disabilities or chronic
diseases (5,9). As a consequence of bone demineralization, RTT girls are at increased risk for skeletal fractures
(5Y7). In our clinic, we estimate that 25% of RTT girls
have fractures at some time in their lives.
We designed this study to extend our understanding
of osteopenia in RTT girls and provide a rationale for
dietary Ca recommendations. We characterized intestinal Ca absorption, urinary Ca loss, and dietary Ca
intake, as well as the clinical markers of bone mineral
status and hormones related to bone metabolism in RTT
girls and unaffected controls. We hypothesized that
decreased fractional Ca absorption relative to dietary Ca
intake and increased urinary Ca loss produce a net
negative Ca balance that may produce osteopenia. We
anticipated that abnormalities in hormonal markers of
Ca metabolism, such as vitamin D metabolites, parathyroid hormone (PTH), or cortisol might be associated
with osteopenia in these girls.
SUBJECTS AND METHODS
Subjects
Ten RTT girls and 10 unaffected female controls were
enrolled. We calculated that enrollment of 10 girls per group
would be sufficient to detect a 30% difference in fractional Ca
absorption (main outcome variable), assuming a standard
deviation of 6%, on the basis of previous studies in children
with bone mineral loss (10). RTT girls were matched as a
group for age, sex, and pubertal status. Efforts were made to
match heights and weights of the RTT and control girls;
however, the progressive linear and ponderal growth delays in
RTT precluded comparability in these variables between
groups (11). All RTT girls met the classic clinical diagnostic
criteria for RTT, including loss of developmental milestones,
speech, and purposeful hand movements after a period of
normal development. They also had deceleration of head
growth and development of hand stereotypies and gait apraxia
(3). MECP2 mutations were identified in all 10 girls. The RTT
girls were classified as RTT stage III or IV on the basis of the
progression of their disease (12). Although eight RTT girls
either crawled or walked in early childhood, only four could
walk, with or without assistance, at the time of this study.
Eight RTT girls had seizures that required anticonvulsant
medications including depakene, lamotrigine, carbamazepine,
or topiramate. One RTT girl received diazepam for muscle
spasms.
Each control girl gave assent for study participation; assent
was waived for RTT girls because of their cognitive impairment. All parents gave permission for the participation of their
daughter in the research study. The study protocol was
approved by the Institutional Review Board for Human
Subject Research at Baylor College of Medicine and Affiliated
Hospitals.
Study Design
All RTT and control girls were admitted for 3 days to
the Texas Children’s Hospital General Clinical Research
Center (GCRC). During admission, all girls received
controlled diets that mimicked their usual predetermined
dietary Ca intake. On day 1 of the admission, bone
mineral content (BMC) was determined by dual-energy
x-ray absorptiometry (DXA). On day 3 of admission,
dual tracer Ca (42Ca:46Ca) isotopic studies were performed. Three 8 hour urine collections were obtained
sequentially to determine the fractional excretion of
42
Ca:46Ca and 24 hour urinary Ca and cortisol excretion.
Venous blood samples were obtained in the postabsorptive state to measure serum Ca, phosphorus (P), vitamin
D metabolites, PTH, and osteocalcin concentrations.
Dietary Intakes
Before admission to the GCRC, the usual dietary Ca
intake and food preferences of each RTT and control
girl were estimated by the research dietitian using
24 hour dietary recall. During admission, all girls
received a constant controlled diet that mimicked their
usual dietary intake. In five RTT girls, diets were
comprised of a commercially available liquid formula
(Pediasure, Ross Laboratories, Columbus, OH) administered in boluses through a gastrostomy tube. In the other
five RTT girls and all control girls, diets consisted of
commercially prepared frozen and canned foods supplemented with dairy products or Ca-fortified orange juice.
Diets were divided into three meals and one bedtime
snack. The amount of food and beverage consumed
daily was determined by test weighing food portions
before and after eating and drinking. Daily dietary Ca,
P, protein, and energy intakes were estimated from the
amount of food consumed and the nutrient content of
each food as reported in a nutrient database (The
Minnesota Nutrition Data System, Version 4.02, Minneapolis, MN).
Bone Mineral Content
BMC was measured by DXA using a Hologic QDR
2000 instrument (Hologic, Inc., Waltham, MA) (13,14).
One hour before the test, all RTT girls received chloral
hydrate, 50 mg/kg per rectum, to minimize involuntary
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CALCIUM ABSORPTION IN RETT SYNDROME
movement. None of the control girls received sedation.
Subsequently, the whole body was scanned in the singlebeam mode while the girls reclined comfortably. The
results were analyzed with body composition software
(Hologic, version 5.56, Hologic, Inc., Waltham, MA),
assuming a fixed hydration constant (0.732 mL/g) for
lean tissue mass. The data from 10 body regions were
summed to provide values for total body BMC, nonbone
lean tissue, and total body fat mass. The in vivo
coefficient of variation using this technique for total
body BMC was less than 1%.
The BMC of RTT and control girls were converted to
z-scores based values measured in a reference pediatric
population (13). The reference pediatric database
includes whole-body DXA scans for more than 2,200
healthy children, ages 4 to 18 years, whose racial, ethnic
(white, African-American, Hispanic), and sex distributions are approximately equal. These data were used to
develop a predictive algorithm for Bnormal[ total body
BMC based on age, sex, race, ethnicity, and height (9).
Ca Isotope Studies
The dual tracer Ca isotope technique used in this
study has been described previously (10,15,16). 42Ca
and 46Ca (Oak Ridge National Laboratory, Oak Ridge,
TN) were assayed by thermal ionization mass spectroscopy and were found to contain 92% to 94% and
30% to 40% isotopic enrichment, respectively. The
isotopically labeled Ca was dissolved aseptically in
normal saline to approximate concentrations of 1.8 mg/
mL (42Ca) and 15 ug/mL (46Ca). The isotope solutions
were verified to be sterile and pyrogen free by standard
culture plate technique and Limulus amoebocyte lysate
assay (Pyrogen, Mallinckrodt, St. Louis, MO).
Sixteen hours before beginning the Ca isotope studies,
0.5 Hg/kg of 46Ca was mixed with 1 oz of whole milk
and refrigerated at j4-C overnight. After fasting overnight for 10 hours, all girls were given 0.03 mg/kg of
42
Ca intravenously over 1 to 2 minutes. Subsequently,
the premixed milk solution with the added 46Ca was
administered orally or through the gastrostomy button.
The dose of 42Ca administered intravenously and of
46
Ca mixed in whole milk was determined from the
difference in the pre- and postweights of the syringes
containing the isotopes. The bottle containing the 46Camilk mixture was rinsed thoroughly with sterile water
until the milk residue was no longer visible. All rinse
water was consumed by the girls. Care was taken to avoid
spillage of the 46Ca-milk mixture. The administration of
the breakfast meal was delayed for 30 minutes after the
consumption of the 46Ca-milk mixture.
Because of urinary incontinence in all RTT girls, a
Foley catheter was inserted using sterile technique into
the urinary bladder immediately before the 42Ca
infusion. All healthy girls voided into pre- and postweighed plastic containers fitted to the toilet. Urine
421
collected before the intravenous infusion of 42Ca was
discarded. After administration of the isotopes, urine
was collected for 24 hours in three 8 hour samples.
After thoroughly mixing each sample, a 50 mL aliquot
of urine was obtained and stored at j70-C for analysis
of the isotopic enrichment of Ca. A 2% aliquot of each
8 hour urine sample was pooled and stored at j70-C for
analysis of Ca, cortisol, and creatinine concentrations.
Serum Mineral and Hormone Profiles
On the morning of the Ca isotope studies, a venous
blood sample was obtained in the postabsorptive state
for the determination of serum Ca, P, alkaline phosphatase, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D,
PTH, and osteocalcin concentrations.
Analytical Techniques
The isotopic enrichment of Ca in urine was determined using a Finnigan MAT Thermoquad mass
spectrometer (Bremen, Federal Republic of Germany).
After precipitation of the urinary Ca with ammonium
oxalate, each sample was analyzed for its ratio of 42Ca/
48
Ca and 46Ca/48Ca. Each sample ratio was compared
with the naturally occurring ratio, and the result was
expressed as a percent of excess (14,17). The precision
for the enriched samples was less than 1%.
Total Ca concentration was measured on all pooled
urine samples by flame atomic absorption spectrophotometry (Flame Atomic Absorption Spectrophotometer,
Model 3030B, Norwalk, CT). Urinary cortisol concentrations were measured by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA). Serum Ca, P,
and alkaline phosphatase and urinary creatinine were
measured by an automated system (Vitros, Johnson &
Johnson Clinical Diagnostics, Rochester, NY). Vitamin
D metabolites were measured by radioimmunoassay
(Endocrine Sciences Laboratory, Calabasas Hills, CA).
PTH concentrations were measured by a two-site
chemiluminescent assay (Endocrine Sciences Laboratory, Calabasas Hills, CA). Osteocalcin was measured
by an ELISA technique (Novacalcin, Metra Biosystems,
Mountainview, CA).
Calculations
The fractional absorption of Ca (%) was calculated as
the ratio of the accumulated oral versus intravenous
tracer in urine during the 24 hours after tracer administration (10,15,16). Total Ca absorption (mg/d) was
calculated as the multiple of the fractional absorption of
Ca and dietary Ca intake. Net Ca balance (mg/kg per
day) was calculated as the difference between total
dietary Ca absorbed and the sum of urinary, endogenous
fecal, and miscellaneous Ca losses. Endogenous fecal
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MOTIL ET AL.
and miscellaneous Ca losses were estimated to be
50 mg/d (17).
Statistical Analysis
Descriptive statistics (mean T SD) were calculated
with MiniTab statistical software (Version 11.0, MiniTab, Inc., State Park, PA). Two-sample t-tests were
applied to detect differences in growth and body
composition measures between RTT and control girls.
Because of significant differences in height z-scores,
general linear modeling was used to detect differences
between RTT and control girls in the following outcome
variables: fractional Ca absorption; total dietary Ca
absorption; urinary Ca and cortisol excretion; dietary Ca
intake; and BMC, lean body mass, and body fat. Two
sample t-tests were applied to detect differences in
serum Ca, P, alkaline phosphatase, 25-hydroxy- and
1,25-dihydroxyvitamin D, PTH, and osteocalcin concentrations between both groups of girls. Analysis of
variance was applied to detect differences in fractional
Ca absorption and BMC in RTT girls on the basis of
their ambulatory status and their use of anticonvulsants.
Linear regression was applied to determine relationships
among the variables dietary Ca intake, intestinal Ca
absorption, urinary Ca and cortisol excretion, BMC,
vitamin D metabolites, PTH, and age. Significance was
determined at P G 0.05.
RESULTS
Characteristic of Subjects
The characteristics of the RTT and control girls
obtained included age, race, ethnicity, Tanner stage,
Rett stage, height and weight expressed as absolute and
z-score values, and body mass index (Table 1). Mean
age, height, body weight, weight z-score, and body mass
index were not significantly different in RTT and
control girls. Height z-scores and head circumference
measurements were significantly lower in RTT than
control girls (10). The stage of pubertal development in
the majority of RTT and control girls was Tanner I or II.
The MECP2 mutations identified in RTT girls included
R306C, Q208X, R106W, R168X/1188-1189insA, 1308
del TC, exon 1 deletion (1 of each); T158M, deletions in
exons 3 and 4 (2 of each). The X-inactivation status of
the RTT girls was unknown.
Isotope Studies
Dietary Ca, P, protein, energy intake, measures of Ca
absorption, and urinary Ca loss in RTT and controls are
shown in Table 2. Dietary Ca, P, protein, and energy
intakes were not significantly different between the
groups when adjusted for differences in height z-scores.
The average dietary Ca intake of RTT girls approximated the Dietary Reference Intake (DRI) for this
nutrient (116 T 46%); however, three RTT girls
consumed Ca in amounts less than 90% of the DRI.
Fractional Ca absorption was significantly greater in
RTT than controls when adjusted for differences in
height z-scores; however, net daily Ca absorption was
not significantly different between the groups. Fractional
Ca absorption was not associated with serum 25hydroxyvitamin D, 1,25-dihydroxyvitamin D, PTH, or
osteocalcin concentrations (data not shown). Neither
fractional nor net Ca absorption changed significantly
with increasing age in either group (data not shown).
Fractional Ca absorption was not significantly different
in ambulatory and nonambulatory RTT girls (53 T 9 vs.
38 T 9%, respectively); between RTT girls who ever
walked and those who never walked (46 T 7 vs. 40 T
19%, respectively); and between RTT girls who received
anticonvulsant medication and those who did not (49 T
7 vs. 36 T 13%, respectively).
Urinary Ca excretion, expressed as absolute amounts
or in relation to body weight, lean body mass, and
TABLE 1. Characteristics of girls affected with Rett syndrome and unaffected age-matched,
unaffected controls
Variable*
Rett syndrome (n = 10)
Control (n = 10)
P value†
Age (y)
Tanner stage (I:II:III:IV:V)‡
Rett stage (III:IV)
Racial distribution (C:A:H)§
Head circumference (cm)
Height (cm)
Height-for-age (z-score)
Weight (kg)
Weight-for-age (z-score)
Body mass index (kg/m2)
8.5 T 2.9
5:3:1:1:0
5:5
6:1:3
49.1 T 1.4
118.8 T 13.0
j1.6 T 1.4
25.4 T 8.4
j0.6 T 1.8
17.6 T 4.4
8.4 T 2.1
8:0:2:0:0
Y
3:2:5
52.5 T 1.7
126.8 T 12.6
j0.4 T 0.8
28.2 T 7.9
+0.1 T 0.7
17.2 T 1.7
NS
Y
Y
Y
G0.01
NS
G0.05
NS
NS
NS
*Variables are shown as mean + SD.
†Two-Sample t-test.
‡I = Prepubertal, V = Pubertal.
§C = Caucasian, A = African-American, H = Hispanic.
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CALCIUM ABSORPTION IN RETT SYNDROME
423
TABLE 2. Dietary intakes, intestinal absorption, and urinary excretion of calcium (Ca), and net Ca balance (corrected for
differences in height z-scores) in girls affected with Rett syndrome and unaffected age- and sex-matched controls
Variable
Dietary intake
Energy (kcal/d)
Protein (g/d)
Calcium (mg/d)
Phosphorus (mg/d)
Intestinal Ca absorption
Fractional (%)
Total (mg/d)
Urinary Ca excretion
Total (mg/d)
Total (mg/kg per day)
Total (mg/kg lean body mass per day)
Ca (mg):creatinine (mg)
Urinary cortisol excretion
Total (mcg/d)
Total (mcg/kg per day)
Total (mcg/kg lean body mass per day)
Cortisol (mcg):creatinine (mg)
Ca balance (mg/kg per day)
Rett Syndrome (n = 10)
Control (n = 10)
Pooled SD
P value
1345
43
1100
934
1635
52
1446
988
423
15
440
350
NS
NS
NS
NS
52
660
33
491
13
286
G0.01
NS
100
4.8
6.9
0.39
78
2.9
4.9
0.22
91
4.1
5.6
0.36
NS
NS
NS
NS
51
2.0
3.2
0.16
513
33
1.2
1.7
0.10
362
24
0.9
1.1
0.08
306
NS
NS
G0.05
NS
NS
urinary creatinine excretion, was not significantly
different in RTT and controls when adjusted for differences in height z-scores. However, in RTT girls, urinary
Ca excretion, expressed either as the Ca:creatinine ratio
or related to body weight, exceeded the upper limit of
clinical reference values (0.25 and 4 mg/kg daily,
respectively). Urinary Ca excretion was not associated
with dietary Ca intake in RTT or control girls (data not
shown). Urinary cortisol excretion, expressed in relation
to lean body mass but not in relation to body weight,
absolute amounts, or as the cortisol:creatinine ratio, was
significantly higher in RTT than in control girls when
adjusted for differences in height z-scores (Table 2).
When standardized as the creatinine ratio, urinary Ca
excretion had a positive association with urinary cortisol
excretion in RTT and control girls (P G 0.05, r = 0.66).
Urinary Ca excretion was not associated with 1, 25dihydroxyvitamin D or PTH concentrations.
Hormone/Mineral Profiles
The hormone and mineral profiles related to body Ca
metabolism are shown in Table 3. Serum Ca, P, alkaline
phosphatase, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin
D, PTH, and osteocalcin concentrations were not
significantly different in RTT and control girls.
Bone Mineral Content and Body Composition
BMC, lean body mass, and body fat are shown in
Table 4. BMC (grams and z-score) and lean tissue mass
were significantly lower, and body fat was significantly
higher in RTT than in controls when adjusted for height
z-scores. The proportion of body weight comprised by
lean body mass was significantly lower, whereas the
proportion of body weight comprised by fat was
significantly higher in RTT than in controls. BMC was
not associated with dietary Ca intake when adjusted for
age or with fractional and net Ca absorption (data not
shown). A significant interaction between BMC and age
was detected in RTT and controls (Fig. 1). BMC
increased significantly with increasing age in both
groups (P G 0.001, r = 0.85), but the rate of BMC
accretion over time was significantly lower in RTT than
controls (57 T 23 vs. 118 T 16 g/y, respectively). BMC
was not associated with urinary cortisol excretion or
serum vitamin D metabolite, PTH, and osteocalcin
concentrations in RTT or controls (data not shown).
TABLE 3. Serum hormone and mineral profile of girls affected with Rett syndrome and unaffected
age- and sex-matched controls
Variable
Rett syndrome (n = 10)
Control (n = 10)
P value
Calcium (mmol/L)
Phosphorus (mmol/L)
Alkaline phosphatase (U/L)
Osteocalcin (nmol/L)
25-Hydroxyvitamin D (nmol/L)
1,25-Dihydroxyvitamin D (pmol/L)
Parathyroid hormone (ng/L)
2.42 T 0.12
1.61 T 0.29
234 T 109
3.93 T 1.54
57 T 22
166 T 36
26 T 11
2.52 T 0.15
1.71 T 0.10
246 T 54
4.27 T 1.54
65 T 17
169 T 42
26 T 15
NS
NS
NS
NS
NS
NS
NS
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424
MOTIL ET AL.
TABLE 4. Body composition, including bone mineral content, lean body mass, and body fat (corrected
for differences in height z-scores) of girls affected with Rett syndrome and unaffected age- and
sex-matched controls
Variable
Body composition
Bone mineral content (g)
z-score
Lean body mass (kg)
Percent body weight
Body fat (kg)
Percent body weight
Rett Syndrome (n = 10)
527
j1.47
15.0
61
10.1
37
BMC, when adjusted for age, did not differ significantly
in RTT girls who were currently ambulatory and those
who were not (mean T SDp, 531 vs. 514 T 197 g,
respectively), between RTT girls who used anticonvulsant medications and those who did not (mean T SDp,
559 vs. 392 T 177 g, respectively), and between RTT
girls who ever walked and those who never walked
(mean T SDp, 593 vs. 379 T 262 g, respectively).
DISCUSSION
Osteopenia frequently complicates the clinical course
of RTT (5Y7). We hypothesized that decreased intestinal
Ca absorption relative to dietary Ca intake accompanied
by increased renal Ca excretion might account for the
osteopenia in RTT. We found that, in the presence of
adequate dietary Ca intake, fractional Ca absorption
showed a significant compensatory increase in RTT
girls, resulting in rates of net Ca absorption that did not
differ significantly from those of unaffected controls.
Urinary Ca excretion was not significantly different in
RTT and controls, but it exceeded the upper limits of
clinical reference values and was consistent with mild
hypercalcuria in the RTT girls (16). Although hormonal
abnormalities were not associated with reduced BMC in
FIG. 1. Relation between total body bone mineral content (BMC)
and age in girls with Rett syndrome (black circles) and unaffected
age-matched controls (white circles); interaction, P G 0.05; Rett
(BMC [g] = 59 + 57.2 Age [y], P G 0.05, r = 0.64); Control (BMC
[g] = j45 + 107 Age [y], P G 0.01, r = 0.88).
Control (n = 10)
860
j0.31
20.1
75
5.6
21
Pooled SD
275
0.56
4.7
7
4.4
7
P value
G0.05
G0.01
G0.05
G0.01
G0.05
G0.01
RTT girls, the positive relation between urinary cortisol
and Ca excretion raises the possibility that increased
bone resorption in RTT may be hormonally mediated.
Adequate dietary Ca intake is an important factor for
maintenance of bone mineral status. We found that the
habitual dietary Ca intakes of RTT girls as a group was
close to the DRI for this nutrient (18). However, three
individuals had Ca intakes that were less than 90% of
DRI. We did not measure directly the Ca content of the
food consumed by the girls, but we believe that our
estimates accurately represent their daily Ca intake.
Five RTT girls received a commercial formula by way
of gastrostomy as their primary food source, making the
estimate of their daily Ca intake relatively reliable. We
recognize, however, that 24 hour dietary recall may not
be representative of habitual patterns of food consumption, particularly in RTT girls in whom oral food was
the sole source of nutrient intake.
Our study demonstrates that a defect in fractional Ca
absorption does not contribute to osteopenia in RTT. To
the contrary, we found that fractional Ca absorption was
greater by a factor of 1.6 in RTT than in control girls, a
value that exceeds the fractional Ca absorption in
children with other chronic disorders (10,16). Fractional
Ca absorption did not increase with increasing age,
presumably because the majority of the RTT and control
girls were prepubertal or Tanner stage II (19). Although
height-for-age z-scores and mobility were compromised
in the RTT girls, the increase in fractional Ca absorption
suggests that the dietary Ca intake of the RTT girls was
insufficient to meet their metabolic needs for bone mineralization (20). Others have shown that, in the presence
of normal vitamin D status, the absorptive capacity of
the intestinal tract of children habitually taking a diet
low in Ca increases (20). We assume that modest
deficits in dietary Ca intake in RTT girls relative to their
metabolic needs could lead to substantial bone mineral
deficits over a prolonged period (21). Nevertheless, net
Ca absorption, calculated as the multiple of dietary Ca
intake and fractional Ca absorption and Ca balance, did
not differ significantly between RTT and control girls.
The nonsignificant increase in net Ca retention in RTT
girls reflects the inherent problems of quantifying Ca
balance because of inaccuracies in estimated dietary Ca
J Pediatr Gastroenterol Nutr, Vol. 42, No. 4, April 2006
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CALCIUM ABSORPTION IN RETT SYNDROME
intake and endogenous Ca loss. Whether fractional and net
Ca absorption would increase further in response to
supplemental dietary Ca and ultimately improve bone
mineralization in RTT girls is unknown.
Although urinary Ca losses did not differ between
RTT and control girls, we considered the RTT girls as a
group to be mildly hypercalcuric because their daily
urinary Ca losses were greater than 4 mg/kg (16,22),
and their Ca:creatinine ratio exceeded the clinical
reference value of 0.25. Our study was insufficiently
powered to detect differences in this secondary outcome
variable because of the variability of the measurement
among the girls. The hypercalcuria in RTT most likely
represents an increase in the excretion of Ca in response
to impaired bone mineralization. Although we did not
measure bone turnover, our findings suggest that
osteopenia in RTT girls is a consequence of increased
bone resorption rather than decreased bone formation.
The similarity of serum osteocalcin concentrations between
RTT and control girls, in the presence of somewhat higher
urinary Ca losses, supports this notion.
The physiologic mechanisms producing increased
bone resorption in RTT are unknown. Bone resorption
may be associated with hyperparathyroidism, but the
normal PTH concentrations in the RTT girls make this
etiology unlikely. Hypercortisolism has been associated
with decreased bone formation, increased urinary Ca
excretion, and decreased Ca absorption (10,23,24). We
found a direct association between urinary Ca and
cortisol excretion, an observation that parallels similar
findings in adolescents with anorexia nervosa (10).
Whether cortisol secretion is involved in the pathogenesis of bone mineral loss in RTT girls through
neuroendocrine mechanisms is speculative (25). Finally,
the regulatory effect of the MECP2 gene on bone
mineral metabolism is unknown. Deregulation in the
control of neuronal activity, particularly in the context
of Ca-dependent transcription pathways, may underlie
the pathology of bone mineral loss and osteopenia in
RTT (26).
Osteopenia may affect girls with RTT more severely
than those with other neurologic conditions such as
cerebral palsy, cystic fibrosis, juvenile dermatomyositis,
and chronic liver disease (5,9). Hass et al. (5) reported
BMC values of RTT girls that were 62% lower than
those of girls with cerebral palsy. The BMC of the RTT
girls in our study was 39% lower than that of agematched controls of similar pubertal status. Their BMC
z-score approximated that of other children with
cerebral palsy (27). The degree of osteopenia in the
RTT girls in our study was similar to that reported by
Haas et al. (5), although the latter group had a broader
range of age and pubertal status. Our study and that of
Hass et al. (5) demonstrated that osteopenia occurs early
in RTT girls. In our study, the interaction between BMC
and age in RTT and control girls suggests that the BMC
of RTT girls may deviate from normal as early as age
425
3 years. Unlike the data from Haas et al. (5), the RTT
girls in our study showed increasing BMC with
increasing age, albeit at a twofold lower rate than that
of the controls. These observations underscore the need
to better understand the mechanisms producing osteopenia in Rett girls to develop effective interventions to
reduce the risk of fractures.
Immobility and the use of anticonvulsants have been
thought to contribute to osteopenia in RTT girls (28,29).
In our study, neither the ambulatory status nor the use of
anticonvulsants affected BMC. Although fractional Ca
absorption tended to be higher in RTT girls who were
ambulatory or received anticonvulsants, the small
number of girls in each group precluded confirming a
significant difference. Ca homeostasis is regulated in
part by the action of 25-hydroxyvitamin D on intestinal
Ca absorption and by 1,25-dihydroxyvitamin D and
PTH on bone and kidney. In our study, fractional Ca
absorption was not associated with 25-hydroxyvitamin
D. Furthermore, urinary Ca excretion and BMC were
not associated with 1,25-dihydroxyvitamin D or PTH
concentrations, all of which were within normal range
for age in RTT and control girls. We did not characterize the vitamin D receptor gene and cannot comment
on the relation between a restriction fragment length
polymorphism and osteopenia in RTT girls (30).
Our study was not designed to evaluate dietary
strategies to enhance bone mineralization in RTT girls.
Thus, we did not find an association between dietary
intake and fractional Ca absorption or BMC in RTT
girls, possibly because of the narrow range of their
dietary Ca intake (31). However, classic Ca balance
studies in adolescent females demonstrate increased Ca
retention with higher Ca intake (32). Bone mass also
tends to increase with higher Ca intake. Indeed,
placebo-controlled trials show that an increase in dietary
Ca is associated with increased bone mineral density in
children and adolescents (33Y41). Others have suggested that supplemental vitamin D, especially early in
life, may enhance bone mineral mass in later childhood
(42). However, the efficacy of these dietary interventions in RTT remains to be determined.
In conclusion, we have shown that a defect in
intestinal Ca absorption does not account for the
profound osteopenia of RTT girls. Rather, fractional
Ca absorption showed a compensatory increase in the
presence of adequate dietary Ca intakes, mild hypercalcuria, and bone mineral deficits in the RTT girls.
Whether supplemental dietary Ca further enhances
fractional Ca absorption and ultimately improves bone
mineralization in RTT girls is unknown.
Acknowledgments: The authors thank the girls and their
families for their participation in this study; the nursing and
dietary staff of the General Clinical Research Center, Texas
Children’s Hospital, for study support; R. Shypailo and J. Posada
for technical support; S. Vaidya, M. Thotathuchery, and Lucinda
J Pediatr Gastroenterol Nutr, Vol. 42, No. 4, April 2006
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426
MOTIL ET AL.
Clarke for laboratory analyses; and K. Fraley and E.O. Smith,
PhD, for statistical support.
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