Our results suggest that an inverse association between age and BDR in asthmatics aged 5–25 years may be enhanced with higher levels of 2-hydroxyglutarate, while increased levels of cholesterol esters, GABA and ribothymidine may attenuate the age-associated BDR decline. BDR, the change in airway constriction before and after the administration of a short-acting β 2 -agonist, is strongly correlated with asthma control [5
], and has been shown to decrease with age [6
]. Although most studies demonstrating age-related effects have been conducted in middle-aged individuals, there is some evidence to suggest decline may begin as early as adolescence [4
2-hydroxyglutarate is an alpha-hydroxy acid form of glutaric acid. Although 2-hydroxyglutarate has not previously been associated with lung function or asthma, it has been associated with hypoxia in primary cultures of lung cells [9
]. 2-hydroxyglutarate is involved in the butanoate metabolism pathway, which regulates the GAD (Glutamic Acid Decarboxylase)-mediated decarboxylation of L-glutamate into GABA. GABA, an inhibitory neurotransmitter, is known to participate in regulation of contractility of airway smooth muscle [10
]. GABA has a number of positive effects on lung function, including relaxing airway smooth muscle, inhibiting muscle conduction, decreasing resistance in airway breathing channels, decreasing contractility of trachea muscle, reducing neurogenic extravasation, reducing anticholinergic bronchoconstriction, and regulating mucus hypersecretion [10
]. Thus, increased levels of GABA metabolites may help to mitigate the decreases in BDR shown to accompanying aging. Further work is required to understand the role of 2-hydroxybutarate in this relationship.
Four highly correlated cholesterol esters were also among the replicated metabolites, based on a p
-value of <0.1. Cholesterol esters are dietary lipids, which play an important role in the mediation of inflammation and immune function [12
]. A dysregulated immune system is one of the key facets of asthma and asthmatic lung function, and immune function is known to decline with age [14
]. Increased levels of some cholesterol esters may thus lessen age-associated immune and lung function changes.
In CAMP, GABA and the cholesterol esters were shown to decline with age, but this only reached significance amongst the older participants. Similarly, most associations were inverse but did not reach significance in the GACRS. There is scant literature on the association between age and blood GABA levels, but plasma levels of total cholesterol and cholesterol esters have been reported to increase with age in some studies [15
], in contrast to our findings. However, these metabolites are highly influenced by diet, which is itself influenced by age, among multiple other factors. Our findings suggest a possible interaction between age and GABA and cholesterol esters, and individuals with high levels of these metabolites may be able to reduce the normal process of age-associated BDR decline. In particular, GABA and the GABAergic system have previously been proposed as a compelling new therapeutic avenue for asthma [11
]. These findings suggest GABA may be particularly useful to help slow or reverse age-related decline in lung function.
Given that sex differences in asthma phenotypes may emerge in the window from childhood to adulthood, we examined the replicated age*metabolite interactions in sex-stratified models. There was some evidence for sex-specific variation, which may relate to observed differences in the metabolome that accompany puberty [16
]. Intriguingly, the interaction of 2-hydroxyglutarate with age appeared to be shifted towards an earlier age in females, mirroring the age of pubertal onset which typically occurs earlier in females. The differences by sex in the cholesterol esters are also of note, given their observed associations with sex in the literature [17
]. However, these stratified analyses were limited by small numbers and further studies are needed to determine whether age-related changes in metabolite profiles influence sex-specific lung function and asthma treatment responses.
One of the key strengths of this study is that we were able to identify metabolites that may help to understand the biology of age-related differences in therapeutic response in asthma. The key to precision medicine is a complete understanding of disease mechanisms; how these differ between individuals with the same apparent disease phenotypes and influence therapeutic response. Age has consistently been shown to be one of the key factors underlying these pathogenic and therapeutic differences among individuals with asthma. Although the number of metabolomic studies of asthma is increasing, most asthma studies are in either distinct adult or child populations, and studies in adolescent populations or that consider the influence of ageing are lacking [18
]. Furthermore, few have considered the metabolomics of BDR or treatment response. This current study is unique in its utilization of metabolomic profiling to explore BDR among a population of asthmatics over multiple time-points spanning both childhood, adolescence, and early adulthood. An additional strength of this study was the ability to replicate our findings in an independent cohort.
Despite these strengths, a few caveats deserve mention. First, there were some notable differences between the studies, which may explain the limited replication. CAMP is a longitudinal study which encompassed a much wider age range than GACRS, which is cross-sectional in design with only a single time-point. This difference in age range may be of particular importance, as the greatest age related differences in metabolite levels in CAMP were shown to occur in late adolescence/early adulthood, an age range not captured by the GACRS population. The longitudinal nature of CAMP may also introduce bias as all samples from the three time-points were processed and sent for metabolomics profiling at the same time, meaning that the storage time that the samples experienced differed by as many as 16 years. It has been shown that increased storage time, as well as repeated freeze thaw samples can influence the quality and concentration of metabolites [19
]. However, we note our samples were stored at -80C following best practice guidelines, and that this effect is metabolite class dependent and our metabolites of interest are not among those shown to be most affected [19
Second, there were differences in the racial structure of the two cohorts; CAMP included multiple races, while all participants in GACRS were Hispanic. We adjusted for race in CAMP and we found that these results were largely unchanged when we additionally explored adjustment by ancestry score. however we were underpowered to stratify by race and therefore our race stratified results were largely non-significant, particularly among the Black and Hispanic populations. Nevertheless, we note that the directions of effects were largely consistent across the populations. We also have abundant data demonstrating that the asthma-relevant genetic findings in CAMP are generalizable to GACRS [21
], including over 15 studies of validated susceptibility loci with similar effect sizes; and we have previously replicated metabolomics findings between the two populations [25
].Consequently despite the differences in study characteristics we consider GACRS a very strong replication population for CAMP.
In CAMP, metabolomic profiling was performed on serum, while in Costa Rica plasma was used. Nevertheless, metabolomics studies comparing results from plasma and serum show that although the specific metabolites may differ, the overall biological conclusions are likely to be the same [26
]. It is therefore notable that we saw multiple cholesterol esters among the significantly interacting metabolites in both populations, although the actual metabolites were not always identical. It should also be noted that our conceptual model assumes circulating blood is a representative tissue for lung function. While other tissues such as airways brushing and bronchoalveolar lavage fluid, may be closer to the lung, metabolomics profiling of such biospecimens has been shown to be limited by issues such as contamination, dilution, lack of standardization and their invasive nature [27
]. Mounting research demonstrates the suitability and success of blood omic-based lung disease studies [18
]. Furthermore, blood is clinically relevant and readily accessible; vital for clinical translation which is the ultimate aim of many metabolomic studies of complex diseases.
Finally, Most of the metabolites reported as significant were not robust to correction for multiple testing according the Benjamini-Hochberg procedure [33
]. However, there are currently no consensus standards for multiple testing correction in metabolomics; most commonly applied correction procedures are considered too stringent. This is due to the existence of metabolites within regulated connected biological pathways; the metabolites comprising these pathways, particularly those involved in the same biochemical reactions, are highly correlated and therefore cannot be considered truly independent. Thus, we considered a liberal p
-value threshold whilst also reporting the FDR-corrected results. Finally, the beta coefficients for the change in BDR were somewhat modest, and further work is needed to consider potential clinical utility.