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Original Studies |
CNRS URA 583, Université Paris V, Hôpital St. Vincent de Paul (M.Z, M.G.), 75014 Paris; France Laboratoire d’Explorations Fonctionnelles, Hôpital Necker-Enfants Malades (J.C.S., C.K.), 75015 Paris; and Centre des Bilans de Santé de l’Enfant de Paris (C.R.), 75011 Paris, France
Address all correspondence and requests for reprints to: Dr. Michèle Garabédian, CNRS URA 583, Hôpital Saint Vincent de Paul, 82 avenue Denfert Rochereau, 75014 Paris, France.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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On the other hand, the fragments formed by the breakdown of type I collagen have provided several indicators of bone resorption (1). Newly synthesized collagen fibrils are stabilized by intermolecular cross-links. The serum cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) varies in much the same way as serum PICP in growing children (4). However, serum ICTP is not considered to be a fully sensitive marker for assessing bone resorption in adults (5). The mature cross-links can also be measured directly in urine. These cross-links are trivalent cyclic pyridinium structures: pyridinoline (Pyr), also known as hydroxylysyl pyridinoline, and deoxypyridinoline (DPyr), also known as lysyl pyridinoline (6). The total excretion of Pyr (T Pyr) and DPyr (T DPyr) is commonly measured (after hydrolysis) by high performance liquid chromatography (HPLC). In healthy children (7, 8) their concentrations are expressed relative to urinary creatinine (T Pyr/Cr and T DPyr/Cr), and these ratios vary in the same way as serum PICP and ICTP. T Pyr and T DPyr levels are low in malnourished children and increase after recovery, with a significant correlation between the subsequent concentration and height gain (9). As pyridinoline cross-links in the urine are in both free and oligopeptide-bound forms, with one kind of peptide at each end of the collagen molecule (10), immunoassays have been performed to measure both the collagen type I cross-linked N-telopeptides (NTx) (11), and C-telopeptides (CrossLaps) (12). Urinary NTx levels decrease with age between infancy and adulthood (11, 13), but no information is available concerning CrossLaps during childhood.
We have studied 183 healthy infants divided into two groups according to age to 1) provide reference values for several available urinary markers of collagen degradation, including CrossLaps for infants according to age and sex; 2) examine the relationships among the various indicators of bone collagen degradation used [T Pyr and T DPyr measured by HPLC, NTx, and CrossLaps; free DPyr (F DPyr) and T DPyr measured by a recently developed RIA]; and 3) investigate the relationships between bone collagen formation and degradation in early childhood and assess the age-related and gender-related variations in these processes.
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Subjects and Methods |
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The subjects were seen in June and July 1995 at the Centre de Bilans de Santé de l’Enfant of Paris. A total of 183 healthy Caucasian children who were having an annual checkup were included. The present study was carried out on the remaining urine and serum samples. The recorded parameters were gender, age, weight, height, and dietary intake. Breast-fed children were excluded to eliminate interferences linked to the effect of breast-feeding on calcium and phosphorus metabolism. The body size parameters were measured by the nursing staff with standard procedures, including weight (kilograms) and height (centimeters), which were used to calculate body mass index (BMI) or Quetelet index as weight over the square of height. Body surface area (square meters) was obtained from the equation validated in children (14). To avoid circadian variations, all samples were taken at the same time of days, between 0800–1100 h in the morning. The children were assigned to one of three groups: 1) 10-month-old children seen after breakfast (mean age ± 1 SD, 6.3 ± 0.78 months; range, 8.5–13 months; n = 66); 2) 24-month-old children seen after breakfast (mean age ± 1 SD, 22.9 ± 1.33 months; range, 21–27.5 months; n = 49); and 3) 24-month-old children seen before breakfast (fasting group; mean age ± 1 SD, 23.3 ± 1.12 months; range, 21–27.5 months; n = 68).
Urine samples were assayed for Cr, calcium, and phosphorus. The samples were then stored at -20 C until the collagen parameters were measured.
The remainders of the serum samples were assayed for protein and total calcium before being stored at -20 C for PICP measurement.
Biochemical analysis
Serum assays. Serum protein and total calcium were measured by routine laboratory methods, and calcium was corrected for protein according to the formula of Parfitt. The values are expressed as millimoles per L. Serum concentrations of PICP were measured using an equilibrium RIA (15), and results are expressed as micrograms per L.
Urinary assays. Urinary Cr (U Cr) and calcium (U Ca) were measured by an automated chemistry analyzer (Beckman Synchron CX system, Beckman Instruments, Brea, CA). Phosphorus was measured with a Technicon colorimetric method (Bayer Diagnostics, France). The results for U Ca and phosphorus are expressed as millimoles per mmol Cr excreted.
T Pyr and T DPyr measurements by HPLC
Urine samples were diluted with equal volumes of pyrogen-free water before measurement according to a previously described method (16). Briefly, the cross-links were extracted from the acid-hydrolyzed samples by cellulose chromatography and separated by HPLC on a reverse phase C18 column with fluorescence detection. The standards of Pyr and DPyr were prepared from human bone according to the method of Eyre (17). The interassay variations were 6.5% for T Pyr and 10.9% for T DPyr. Urinary concentrations are expressed as nanomoles per mmol Cr.
F DPyr and T DPyr RIA
Samples were analyzed by a new RIA (Pyrilinks-D RIA, Metra Biosystems, Mountain View, CA) for F DPyr and T DPyr. The Pyrilinks-D RIA is a competitive RIA that uses a monoclonal anti-DPyr antibody (purified murine monoclonal anti-DPyr antibody) coated on the inner surface of polystyrene tubes. This antibody is the same as that used in the previously validated enzyme-linked immunosorbent assay (ELISA) for urinary deoxypyridinoline (18). DPyr in the sample competes with 125I-labeled DPyr for the antibody. The incubated tubes were washed to remove unbound [125I]DPyr and then counted. Pyrilinks-D reacts less than 1% with free pyridinoline. The sensitivity determined as 2 SD at zero standard was 2 nmol/L. When spiking 40 nmol/L DPyr in three urine samples containing 11.5, 33.4, and 64.4 nmol/L endogenous DPyr, the recoveries ranged from 90–100%. The intraassay (n = 18) and interassay (n = 37) coefficients of variation assessed in three samples with different concentrations were less than 6% and 8%, respectively.
F DPyr assay.
Samples, standards, and controls (50 µL)
were diluted in 450 µL wash buffer; 100 µL diluted standards,
controls, and samples were dispensed in duplicate in the coated tubes,
followed by 200 µL cold (2–8 C) [125I]DPyr. Two
additional tubes were included for total counts. The tubes were gently
vortexed and left in the dark at 2–8 C for 2 h. The supernatants
were then discarded, and 4 mL cold (2–8 C) wash buffer were placed in
each tube except those for the total counts. The tubes were decanted,
and supernatants were discarded. The [125I]DPyr bound was
counted in a suitable 
-counter and quantified by comparison with the
standard curve obtained with purified bovine bone DPyr in 10 mmol/L
phosphoric acid. All values are expressed as nanomoles per mmol Cr.
T DPyr assay. Urine samples were hydrolyzed before analysis as recommended by the manufacturer. Each urine sample (200 µL) was boiled in an equal volume of 6 N HCl and heated at 110 C for 18 h in screw-capped glass tubes. Hydrolyzed samples were neutralized by adding 50 µL hydrolysate to 200 µL 1 N NaOH. The neutralized hydrolysate (50 µL) was diluted in 450 µL assay buffer. The following steps were the same as those for F DPyr measurement. Results for the hydrolyzed samples were multiplied by 10 to obtain T DPyr concentrations and expressed as nanomoles per mmol Cr.
CrossLaps.
CrossLaps was measured by ELISA (CrossLaps ELISA,
Osteometer, Rodovre, Denmark). This assay uses a polyclonal antiserum
raised against an immobilized synthetic peptide with an amino acid
sequence (EKAHDGGR) specific for part of the C-telopeptide of the
1(I) collagen chain (12). Results are expressed as
micrograms per mmol Cr.
NTx. NTx was measured by ELISA (Osteomark, Ostex International, Seattle, WA) (11). The results are expressed as nanomoles of bone collagen equivalents per mmol Cr.
Calculated parameters
The free fraction of deoxypyridinoline (F DPyr/T DPyr) and the ratio of T Pyr to T DPyr (Pyr/DPyr) in urine were calculated. The peptide-bound DPyr, (B DPyr) was estimated as T DPyr minus F DPyr and expressed as nanomoles per mmol Cr.
Statistical analysis
All results are expressed as the mean ± 1 SD, except when indicated. Comparisons between groups were assessed by Student’s t test for unpaired data. Variables not known to be Gaussian distributed (BMI, body surface area, serum PICP, and all urinary parameters) underwent logarithmic (ln) transformation before using Student’s t test. Relationships between parameters were assessed by Spearman’s rank correlation. P < 0.05 was significant.
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Results |
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The values for body size parameters are given in Table 1
. Boys had greater weight, height, and
body surface area than girls at 10 and 24 months of age. Before further
biological analysis, the effect of fasting was tested on parameters of
calcium metabolism and on CrossLaps excretion in 117 children seen at
24 months of age. Fasting had no effect on the serum and urine
parameters (Table 2). The data obtained
for all 24-month-old children were, therefore, pooled before analysis
according to gender and comparison to 10-month-old children.
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). Analysis of their spot urine samples
showed higher creatinine concentration in both sexes, lower
phosphorus/Cr ratios in boys, and higher U Ca/Cr ratios in girls
compared to corresponding values in the 10-month-old children. For the
same age, gender had no clear effect on these parameters.
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Urine samples from 38 infants, aged 10 months, were assayed by
HPLC for T Pyr and T DPyr. Expressed as ratios of Cr, the T Pyr and T
DPyr values measured by HPLC were highly correlated (r = 0.91;
P = 0.0001; Fig. 1a).
Results were compared to those obtained by assaying the same samples
for CrossLaps by ELISA, for NTx by ELISA, and for F DPyr and T DPyr
using a recently developed RIA. As ratios of Cr, T DPyr measured by the
reference HPLC method was closely correlated (P =
0.0001) with F DPyr (r = 0.82) and T DPyr (r = 0.80) measured
by RIA as well as with CrossLaps (r = 0.72; Fig. 1, b, c, and d).
It was less tightly correlated with NTx (r = 0.53;
P = 0.004; Fig. 1e).
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The serum PICP concentrations of both boys and girls were
significantly lower at 24 months than at 10 months of age (Table 4). When each of the age groups was
considered separately, there was an inverse correlation between age and
serum PICP concentration in the boys aged 10 months (r = -0.53;
P = 0.02), but not in the older boys or in the girls.
Like serum PICP, urinary F DPyr/Cr, T DPyr/Cr, and NTx/Cr levels were
significantly lower at 24 months than at 10 months of age in boys. In
contrast, CrossLaps/Cr did not change in boys with increasing age, and
none of the tested urinary markers of collagen degradation showed
age-related changes in girls.
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None of the parameters analyzed in children aged 10 months
differed between boys and girls (Table 4
). The serum PICP levels and
the urinary F DPyr/T DPyr ratios of boys and girls aged 24 months were
also the same, but the 24-month-old girls had significantly higher
values than boys for urinary F DPyr/Cr, T DPyr/Cr, B DPyr/Cr,
CrossLaps/Cr, and NTx/Cr (Table 4).
Relationships between markers of collagen formation and degradation
Serum PICP did not correlate with T Pyr/Cr, T DPyr/Cr measured by HPLC or RIA, F DPyr/Cr, or B DPyr/Cr in any group. In contrast, correlations were found between serum PICP and CrossLaps/Cr levels in the 10-month-old boys (r = 0.70; P = 0.048) and between serum PICP and NTx/Cr ratios in the 24-month-old girls (r = 0.57; P = 0.021).
Relationships between collagen markers and body size parameters
For all subjects, the serum PICP concentration showed a
significant negative correlation with weight, height, body surface
area, and BMI, but this correlation was not found within the groups of
infants separated according to age (Fig. 2). Only the girls aged 24 months showed
a significant positive correlation between BMI and serum PICP (r =
0.51; P = 0.041).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Similar to serum PICP, several urinary indicators of bone resorption have been shown to be very high during infancy and to decrease with age. This was shown for T Pyr/Cr and T DPyr/Cr (7, 8, 20, 21, 22) and for NTx/Cr (11, 13) in mixed populations of boys and girls. The results of the present survey also show high values of T Pyr/Cr, T DPyr/Cr, F DPyr/Cr, and NTx/Cr in infants. The proportion of F DPyr was similar to that in healthy children (23) and adults (24), but lower than that in adolescents (24). High values for T DPyr were found with both the HPLC assay and the newly developed RIA assay that uses the monoclonal antibody developed for F DPyr measurement by ELISA (18). Close correlations between the RIA values for F DPyr and T DPyr and the HPLC values for T DPyr suggest that this RIA is a suitable method to assay DPyr in urine. The present results also show an age-related decrease in the urinary excretion of these markers in boys between 10–24 months of age, but there was no age-related decrease in girls. Another marker of type I collagen degradation, urinary CrossLaps/Cr, was analyzed. No information is yet available on this marker during childhood. Like the other markers, high levels of CrossLaps/Cr were found in infants; they were 5- to 7-fold higher than those in healthy premenopausal women (25, 26). However, this marker differed from NTx and other cross-links as it showed no age-related decrease during the first 2 yr of life.
It is difficult to interpret urinary data from young children. Spot urine samples are usually preferred to 24-h samples because it is not easy to collect urine for long periods of time. Cross-links excretion does not appear to be directly influenced by renal function in infancy (21), and the assessment of collagen degradation products based on their concentration relative to creatinine excretion in first void urine samples is considered to be valid in healthy children (8). However, the urinary excretion of creatinine depends on protein intake and skeletal muscle mass, which is closely related to growth. Thus, identical values at 10 and 24 months of age, as is the case for T DPyr/Cr and NTx/Cr in females and for CrossLaps/Cr in both sexes, may, in fact, reflect an increase in the absolute excretion of these degradation products with increasing age, as urinary Cr increases during this period. Alternatively, part of the decrease observed in T DPyr/Cr and NTx/Cr in boys results from the increase in urinary Cr, and the absolute decrease in the degradation of collagen may thus be smaller than that in their ratio to Cr.
Nevertheless, comparative analysis of the data obtained with different markers of collagen degradation show clear differences among these markers. First, there was no correlation between serum PICP and T Pyr/Cr, T DPyr/Cr, or F DPyr/Cr in any of the four groups of infants when analyzed separated according to age and sex, unlike the situation in adolescents (27). However, serum PICP correlated with urinary CrossLaps/Cr in boys aged 10 months and with NTx/Cr in girls aged 24 months. These relationships suggest that urinary CrossLaps and NTx excretions may partly reflect the rate of collagen production during rapid growth. In agreement with this hypothesis, antibodies to NTx and CrossLaps are thought to react with all type I collagen peptides rather than only with bone-derived ones (28). Second, CrossLaps/Cr did not decrease during the first 2 yr of life, unlike the other markers of collagen degradation. This suggests differences in production of the various cross-links during early childhood, presumably linked to differences in maturation of the enzymatic systems involved.
The simultaneous analysis of the changes in collagen synthesis and degradation markers also shows sex-linked differences and a certain degree of uncoupling of the two processes during early infancy. Gender did not appear to influence collagen metabolism in the 10-month-old infants. It also did not influence PICP levels in the 24-month-old children. However, levels of all degradation markers were significantly higher in girls than in boys at this age. Moreover, the parallel decreases in serum PICP and urinary degradation markers observed in boys between 10–24 months of age did not occur in girls. Thus, unlike the boys, the girls appear to maintain a high degree of collagen degradation up to the age of 24 months despite a decrease in their rate of collagen formation. The lack of a close link between type I collagen formation and degradation is not surprising because of the different regulatory systems involved in the control of skeletal growth, bone modeling, and bone remodeling. Urinary excretion of T DPyr and, to a lesser extent, of T Pyr is considered to reflect bone resorption in adults. If one assumes that this is also the case during infancy, the present results suggest that the timing of the changes in the resorption rate of the skeleton during the first 2 yr of life differ with the sex. However, as mentioned above, urinary excretion of the cross-links may not reflect bone resorption alone in rapidly growing children and may in part be dependent on overall somatic growth. Earlier studies in healthy children found no correlation between body size parameters and pyridinoline cross-links (29), but F DPyr/Cr showed some correlation with height at 10 months of age, and CrossLaps/Cr and NTx/Cr showed some correlations with weight, BMI, and body surface area at 24 months of age. Thus, the gender-related differences in urinary degradation markers may reflect differences in the production of the collagen degradation markers by skeletal and nonskeletal cells.
In conclusion, the present cross-sectional study provides reference values for markers of collagen metabolism during infancy, including CrossLaps. They show the potential usefulness of a newly developed RIA for measuring F DPyr and T DPyr in healthy children, which is more convenient than the time-consuming HPLC method. They show differences in the excretions of the various collagen degradation indicators and suggest that some of these indicators, especially urinary CrossLaps and NTx, commonly referred to as markers of bone resorption, may, in fact, reflect the overall type I collagen turnover during early childhood. Finally, they show gender-related differences in the metabolism of type I collagen as early as 2 yr of age.
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Acknowledgments |
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Footnotes |
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Received February 26, 1997.
Revised May 19, 1997.
Accepted May 27, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) and 24 months (+).