This study recruited healthy children in order to study the relationship between plasma NTproCNP levels and height velocity across all phases of linear growth, as well as to define the reference range of CNP and NTproCNP as measured by RIA. The subject population accurately reflected the racial/ethnic distribution of Northeast Florida, including a low representation of people of Asian descent. Since we found no differences between racial/ethnic groups, this distribution should be comparable to other populations, with the exception of Asians, for whom we did not have enough data. Compared to the US population in general, our subjects were somewhat taller and heavier. However, within our population, we found no differences in CNP or NTproCNP levels based on height, weight or BMI, again suggesting the results should be comparable to the general population.
The results for both CNP and NTproCNP show a clear variation based on age. Levels from birth to 2 months of age have been previously reported 21
and showed very high levels in newborns that rise during the first week of life, then trend downward after six weeks of age. We show here that levels fall rapidly in the first year of life, leveling off in prepubertal children. At these ages, there is no difference between boys and girls. During the pubertal years, levels rise again. This occurs at an earlier age in girls than boys. Later in puberty, the levels drop again, eventually reaching the low adult levels. In adults, levels slowly decline during the third decade of life (manuscript in preparation). This pattern parallels that of height velocity, with the exception that, unlike height velocity, CNP and NTproCNP levels do not drop to zero at the completion of puberty. Notably, the age of the median peak NTproCNP (14.1 years for boys, 11.9 years for girls) is almost identical to the age of peak height velocity [13.6±1.1 (mean±SD) in boys and 11.5±1.2 in girls] as reported by Berkey, et al.
, for children in the US. These strong temporal links are consistent with a dominant contribution to circulating levels of NTproCNP from tissues participating in linear growth.
During puberty, CNP and NTproCNP levels similarly varied by genitalia Tanner stage, peaking in boys at Tanner stage III and IV and in girls at Tanner stage II and III. This agrees with the data by Tanner and Davies 17
for the pubertal stage at the time of peak height velocity in North American children.
A direct comparison of NTproCNP and height velocity demonstrated a strong, positive linear relationship. We had previously identified this relationship in a smaller, heterogeneous population (n
. In this larger study population studied prospectively, the correlation is even stronger (r
=0.711). Based on this result, we conclude that height velocity explains 51% of the variability of plasma NTproCNP levels in healthy children.
We have previously reported that NTproCNP levels increase in children starting growth-promoting therapy, either rhGH in children with growth hormone deficiency or idiopathic short stature, or testosterone in boys with constitutional delay of growth and puberty 11
. Using pre- and post-treatment height velocities, we identified a similar significant correlation with NTproCNP levels.
Secondary analysis identified a correlation between NTproCNP and height velocity SDS (i.e. at any given age, children growing faster than the population had higher NTproCNP levels), but no relationship between NTproCNP SDS and height SDS (i.e. children taller than the population did not have higher NTproCNP levels). This confirms the clinical dogma that tall children are not necessarily fast growing children at a given point in time. We also identified a negative relationship between NTproCNP SDS and upper-to-lower segment ratio SDS. Information from the genetically altered mice (CNP and NPR-B knockout mice and CNP transgeneic mice) as well as from clinical genetic studies (acromesomelic dysplasia, Maroteaux type, 2q37 translocations, and NPR-B activating mutations) demonstrate that CNP plays a larger role in the regulation of growth of the appendicular skeleton than the axial skeleton. Primary CNP growth disorders result in skeletal disproportion; disruption results in shortened limbs, with the distal elements being most affected, while CNP overexpression results in disproportionately long limbs, again with the distal elements being most affected. Our data suggest a similar effect is operational in healthy children. Those children with relatively high CNP production (as demonstrated by higher NTproCNP levels) have relatively longer limbs and a lower upper-to-lower segment ratio. This finding would require a prospective study for confirmation.
The results reported here for NTproCNP are from an in-house RIA. An ELISA kit is commercially available. When the two assays are compared, the RIA gave values roughly five times those of the ELISA. The values have a significant degree of correlation, however. Cross-platform analysis showed the standards used in both assays are comparable and hence not the source of the difference. High performance liquid chromatography analysis of plasma shows that the NTproCNP peak is broad and contains degraded, lower molecular weight forms 13
. We also have evidence that the carboxy-terminal amino acid of the predicted peptide is not present in circulating NTproCNP, suggesting that carboxy-terminal degradation occurs in vivo
. In addition, the first two residues from the amino-terminus of circulating NTproCNP are removed by endogenous dipeptidyl peptidase-4 activity. The ELISA utilizes antibodies raised against amino acids 1–19 and 30–50. Any amount of terminal degradation would likely reduce the amount of product being measured by the ELISA. The RIA utilizes a polyclonal antiserum raised to the synthetic peptide proCNP1–15
which is the amino-terminus of the propeptide and is therefore not affected by carboxy-terminal degradation. The epitope of the antiserum used by the RIA requires residues 3–15 of proCNP; loss of the two aminoterminal amino acids would not affect binding. We believe this is the source of the difference between the assays. Given the differences in the two platforms, it would not be appropriate to use the reference range we present here for results obtained with the commercial ELISA.
Many peptides have been identified as potential biomarkers correlating with linear growth, most of them products of bone metabolism. They include markers of bone synthesis 23,24
and bone resorption 25
, all of which increase during puberty 25,26
. Among these, bone specific alkaline phosphatase (BsALP)23
, urinary deoxypyridinoline cross-links (DPD)27
and the cartilage specific matrix protein (COMP)28
appeared to show the best correlation with linear growth when assessed during periods characterized by rapid growth. However longitudinal studies show little or no relation to height velocity in healthy children 29,30
. In contrast to products of bone metabolism, CNP is synthesized by chondrocytes and acts to promote expansion within all zones of the growth plate 31
. While it is rapidly degraded at its source, the bio-inactive metabolite of the prohormone (NTproCNP) enters the circulation and is therefore a logical candidate as a marker of skeletal growth. However, like classical bone turnover markers, CNP synthesis is not limited to the skeleton; it is also expressed in a range of mammalian tissues including osteoblasts 32
, brain, reproductive tissues and vascular endothelium 33
. Contributions made by such tissues to circulating levels are difficult to assess although in the adult, where plasma levels of NTproCNP are relatively invariant 10
, it is likely that constitutive production from the vasculature, bone, heart and liver 34
all contribute to some degree. The much higher levels in children, the evidence of an arterio–venous CNP gradient across bone dense tissues in growing lambs 35
and the marked and concordant changes in NTproCNP accompanying changes in height velocity 10,11,21,36
indicate that the growing skeleton is a dominant source in children. This view is further supported by our finding in the present study showing that in the context of normal growth throughout childhood, 51% of the variance in NTproCNP concentration is determined by variation in height velocity.
To our knowledge, NTproCNP is the first peptide described whose blood levels correlate with height velocity in healthy children of all ages. As such, there is potential clinical utility in assessing NTproCNP levels. In the evaluation of children with short stature, NTproCNP levels might differentiate those with short stature, but normal height velocity from those with poor current growth. Levels before and during growth-promoting therapy may provide information about the efficacy of the therapy. And finally, levels may be useful in identifying when growth is completed. The first step in evaluating these potential uses is the development of a reference range for NTproCNP, as presented here. Future work should establish the impact of renal function or other potentially confounding effects of drugs or illness on plasma concentrations of NTproCNP, and their interpretation in children with growth disorders.
In summary, CNP is expressed and acts in the epiphyseal growth plate, plays a key regulatory role of the growth plate and hence in linear growth. NTproCNP, a marker of CNP biosynthesis, strongly correlates with height velocity in healthy children and in children receiving growth promoting therapies. This is strong evidence for NTproCNP being a biomarker for linear growth.