The analyses presented in this report represent a new approach to the characterization of age-related differences in VPA effects on a biological system. In the past, attention has focused on a terminal olefin metabolite of VPA, 4-ene-VPA, due to its structural similarity to known hepatotoxins, such as 4-pentenoic acid. Kondo et al.
reported that 4-ene-VPA formation is increased in younger children and declines with increasing age (23
), but these age-related differences have not been replicated by others (20
). Evidence for increased bioactivation of VPA in younger children has been presented by Gopaul et al.
on the basis of increased urinary concentrations of N-acetylcysteine conjugates of (E)
-2,4-diene VPA, reflecting detoxication of reactive VPA metabolites by glutathione conjugation (26
In urinary metabolite studies it is common practice to apply a correction factor using urinary creatinine concentrations to adjust for the effects of hydration status on observed drug and metabolite concentrations. However, creatinine production increases with age between birth and adolescence with the acquisition of muscle mass, and is affected by gestational age in newborns and anorexia in adolescents (summarized in (33
)). Thus, in a comprehensive analysis of VPA metabolite profiles in 91 children receiving VPA both as monotherapy and polytherapy, we were unable to replicate any age-related changes in urinary VPA metabolite concentrations when developmental changes in urinary creatinine concentration were taken into consideration (manuscript submitted for review). We therefore considered that changes in body composition characteristic of growth and development in children are likely associated with increased metabolic demands. Given that impairment of mitochondrial β-oxidation has been implicated in VPA hepatotoxicity, and developmental changes in mitochondrial function may be implied by the age-dependent changes in normal ranges for urinary organic acid concentrations used in pediatric settings to diagnosis inborn errors of metabolism, we hypothesized that the developmental context in which VPA is administered may be an important determinant for age-related differences in the risk of serious forms of toxicity. The data presented in this study represent an initial exploration of this alternative approach.
There are few, if any, studies using comprehensive profiling of urinary organic acids or other biomarkers to assess alterations to mitochondrial metabolism by VPA on a population basis in children. The data presented in this study provide a unique opportunity to examine the variability in endogenous metabolic consequences of VPA in children, and to formulate hypotheses for further prospective studies. Data models revealed that a systematic metabolic change occurred in response to VPA treatment in children without overt hepatic damage, although significant interindividual variability in metabolic responses to VPA was observed. A final key finding was an age-dependent metabolic response to VPA, thus giving a new perspective on the unexplained VPA toxicity in young children to guide future investigation. Because of the retrospective analysis of residual urine samples, interpretation is limited to the identification and description of potentially interesting metabolic changes and issues related to pediatric metabolic profiling, subject to validation in future prospective investigations.
Age trends are a significant issue in the metabolic profiling of pediatric populations. As discussed above creatinine excretion changes with age, but is commonly used to account for fluctuations in urine concentration between samples. Thus, correction for urinary creatinine concentration typically introduces an age trend into the data (if one was not already present). Furthermore, those involved in the medical treatment of young children are well aware that the range of normal values for individual organic acids changes with increasing postnatal age, despite these values routinely being reported relative to urinary creatinine. The challenge in studies of this type is to differentiate changes due to the intervention from the background noise generated by increasing age, particularly if the factor of interest has its own age dependency. An important finding in this study is that organic acid profiles changed with time, and this change was independent of drug administration. To the extent that individual organic acids can serve as surrogate biochemical markers of mitochondrial dysfunction, the data raise the possibility of a mitochondrial “phenotype”, and that this phenotype changes with age. A major challenge will be to refine assessment of mitochondrial phenotype to determine the phenotype (i.e., at a younger age) most susceptible to toxicity, and thus provide testable hypotheses for the mechanisms of toxicity.
A differential effect of VPA on organic acids was noted in the PCA model, as illustrated from the varying distance of VPA subjects to the control/CBZ cluster. These metabolic changes are drug-specific, but not disease-specific, as CBZ subjects serving as the disease control do not differ from the healthy controls. Furthermore, an age-dependent metabolic response to VPA was observed. Both PCA and discriminant analysis showed deviation of the VPA metabolic profiles from the control and CBZ groups. Clustering revealed groupings of VPA subjects according to age, and identified two subsets of young subjects within the VPA group. Finally, it is notable that there is considerable inter-individual variability in the extent to which individual patients deviate from the control/CBZ cluster, with some VPA patients indistinguishable from the control and CBZ groups while others deviate considerably. Taken together, one can view VPA administration in children as perturbation of a dynamic system with a medium chain fatty acid load, and that consequences in terms of mitochondrial function may change significantly throughout childhood. In this context, it would not be unreasonable to observe inter-individual variability in the ability to respond to that perturbation across a treated population with, perhaps, more limited ability to adapt at discrete ages/developmental stages.
A major limitation of this study is its design. The goal of the original investigation was to characterize population variability in the patterns of VPA biotransformation and the effect of age on those profiles. This report represents a change in perspective from “what children’s developing systems do to the drug” to “what the drug does to children’s developing systems”, and necessarily involved secondary analysis of the samples to address that change in perspective. All subjects in this study were deemed healthy by standard laboratory measures and so it was not possible to address specifically the relationship between organic acid profiles and hepatotoxicity. Also, the study was designed to collect urine under steady state conditions after patients had been stabilized on their doses of VPA and CBZ. Thus, it was not possible to assess the extent to which organic acid profiles changed following VPA treatment. Nevertheless, the insights gained provide valuable insights and testable hypotheses for future studies.
An additional limitation is the scope of analytes used to assess metabolic effects of VPA. Urinary organic acid profiles are routinely available in tertiary care pediatric settings, and were used in this investigation as a surrogate measure of metabolic function and, potentially, mitochondrial function. Expanding the repertoire of analytes to interrogate the response of a broader complement of biological pathways to VPA administration, analogous to the metabolomic approaches that have been applied in both animal (34
) and human (35
) studies to identify endogenous profiles predictive of acetaminophen-induced hepatotoxicity, has the potential to elucidate biological mechanisms and identify sensitive and specific biomarkers for toxicity, especially those predictive of children at highest risk for serious toxicity.
Overall, this study indicates that human pediatric metabolic profiling of individual response to VPA provides a new perspective for investigating the mechanistic basis of age-related susceptibility to VPA hepatotoxicity. Clearly, prospective investigation is needed to more precisely identify the factors contributing to variability in VPA metabolic response. Longitudinal studies designed to better identify individual toxicity risk by examining VPA metabolic response over time are needed.