In the present study, we tested urine samples from a large population of children and adults to characterize metabolic features in different age stages. Urine metabolism characteristics from early life to old age were profiled, which is the first systematic comparison study based on such a large and healthy population.
Gender-dependent metabolism status in children and adults
In the present study, we characterized the urine metabolomes of males and females in children and adults. Consistent with previous studies [4, 6, 7], amino acid metabolism, including tryptophan metabolism and arginine and proline metabolism, showed gender differences in both children and adults. In females/girls, tryptophan metabolites, indoleacrylic acid, indolylacryloylglycine and 5-hydroxyindoleacetic acid showed higher levels compared with males/boys. 5-Hydroxyindoleacetic acid is a breakdown product of serotonin that is excreted in the urine and is mainly involved in serotonin degradation and serotonin receptor signaling regulation. The higher level in females could be partly explained by greater precursor, such as tryptophan, availability [12]. Studies on experimental animals have revealed the effects of gonadal hormones on the indoleamine system, and in the rat brain, 5-hydroxyindoleacetic acid is higher in females than in males [13]. These results were consistent with our present urine results.
In addition, children and adults showed different gender-dependent metabolic statuses. In the child population, purine metabolites, steroid metabolites and amino acid metabolites showed specific gender dependence. For example, uric acid, an oxidation product of purine, showed a significantly higher level in boys compared with girls but showed no gender variations in adults. Renal excretion of uric acid in children differs from that in adults. It was reported that the younger the child, the greater the excretion of uric acid [14]. Altered urine uric acid level is an indispensable marker in detecting rare inborn errors of metabolism [15]. The level of uric acid in urine was higher in boys than girls, probably contributing to the higher prevalence of hyperuricemia in boys than in girls [16].
Compared to children, we identified more specific gender-dependent metabolites in adults. Most of the specific gender-dependent metabolites in adults showed higher levels in males, including fatty acids, acylcarnitines, steroid hormones and dipeptides. Higher levels of acylcarnitines in males were found in our previous study, indicating a higher activity of fatty acid oxidation and energy production in males compared to females [7]. In addition, a higher level of steroid hormone biosynthesis was found in males, consistent with previous studies [17].
Age-dependent metabolism status in children and adults
For children and adults, we found many common age-dependent pathways between boys (males) and girls (females), indicating common metabolism status variations with age in humans. For instance, pantothenate and CoA biosynthesis, fatty acid biosynthesis and tryptophan metabolism were found to be changed with age in both children and adults. These pathways correspond to energy demand changes with aging [4]. Interestingly, tryptophan metabolism was only found to be age dependent in boys and males, while no significant changes were found in girls and females. Metabolites of tryptophanol and 5-methoxytryptophan were reported to be associated with increased cellular anti-inflammatory and blood circulation properties [18], probably reflecting age-associated metabolism activity differences between genders. In addition, we found that the pathway of steroid hormone biosynthesis was changed with age in adults and children. As a whole, steroid hormone biosynthesis showed high activity during the adolescent and youth stages, corresponding to sexual development [19].
Additionally, children and adults showed different age-dependent metabolic statuses. Particularly in the children population, the fatty acid biosynthesis pathway was found to be age dependent. The results showed a positive correlation with increasing age. Fatty acids constitute a large energy source for the body. Increased fatty acid metabolism indicated high ATP generation with age in a children population [20]. In adults, pyrimidine metabolism and caffeine metabolism were found to be age dependent. Pyrimidine metabolism was found to be positively correlated with aging, showing the highest level in the old adults. Deoxyuridine, a naturally occurring nucleoside, is considered to be an antimetabolite that is converted to deoxyuridine triphosphate during DNA synthesis. Disturbance of DNA synthesis may modulate the aging process and contribute to the high incidence of cancer with aging [21].
Metabolomic characteristics during each age stage
Metabolomic characteristics during the pre- and primary school stages During early life in the preschool and primary school stages, gender differences were relatively smaller than in other age stages, as shown in Fig 4. During the preschool stage, pathways of pantothenate and CoA biosynthesis, pyrimidine metabolism, vitamin B6 and alanine metabolism showed high activity in girls and boys. These active pathways were associated with energy and nutrient supply. These metabolic characteristics correspond to the physiological characteristics during this life stage, high metabolism activity for rapid growth and development demands [4]. Research on children aged from 2-7 years old suggested that pantothenate and CoA biosynthesis, pyrimidine metabolism and vitamin B6 metabolism were significantly related to autism spectrum disorder (ASD) [22]. These results highlight the importance for these pathways to maintain homeostasis during preschool.
During primary school stage, the most active metabolism pathway is tryptophan metabolism, lipid metabolism, nicotinate and nicotinamide metabolism and histidine metabolism. These metabolic features were corresponding to the main physiological characteristic-visual development, blood circulation increasing and rapid metabolism activity during this stage. Although gender differences were small during the primary school stage, metabolites with the highest level in boys and girls showed specific features. In boys, tryptophan metabolites, such as 5-methoxytryptophan, were found to show the highest level in boys. 5-Methoxytryptophan is an endogenous tryptophan metabolite with anti-inflammatory properties [18]. In addition, 5-methoxytryptophan was reported to be involved in the cyclic metabolism of the retina [23], ventricular remodeling and maintaining liver function [24, 25]. In girls, urocanic acid, a breakdown (deamination) product of histidine, showed the highest level. Urocanic acid is one of the essential components of human skin [26]. It could accumulate in the epidermis and may be both a UV protectant and an immunoregulator. A higher level of these metabolites in girls may contribute to skin development during this age stage. It was reported that skin disorders are more common among girls than boys aged 6 to 17 years [27], which could be affected by the immunomodulatory effects of urocanic acid [28].
Metabolomic characteristics during adolescence and youth During adolescence and the youth stage, gender differences become more significant, partly due to changes in hormone and endocrine levels. The main metabolic feature during these stages was fatty acid oxidation and biosynthesis, androgen and estrogen metabolism and steroidogenesis. These metabolic characteristics correspond to pubertal development, neurodevelopmental changes and heightened stress sensitivity during adolescence and youth stages. Cortisol, androstenol, testosterone, and their glucuronide metabolites showed higher levels during this period. Cortisol is the main glucocorticoid secreted by the adrenal cortex and is involved in the stress response. Synergies between cortisol reactivity and testosterone were reported to influence antisocial behavior in young adolescence. The youth with high diurnal testosterone, combined with high or moderate cortisol reactivity, were significantly higher on antisocial behavior and attention behavior problems [29].
In addition to the common metabolic features during adolescence and the youth stage, boys and girls also showed specific metabolic characteristics. During adolescence, spermidine biosynthesis was higher in boys. One of the involved metabolites was 5-methylthioadenosine, a byproduct of polyamine synthesis in DNA turnover cycles that increases with inflammation to modulate cellular stress. It has been shown to influence the regulation of gene expression, proliferation, differentiation, and apoptosis [30]. Higher serum levels of 5-methylthioadenosine have been reported in youth boys when compared to girls [31]. A direct association between 5-methylthioadenosine and high metabolic risk was found in boys, possibly driven by proinflammatory pathways [32]. While in adolescent girls, fatty acid oxidation and biosynthesis showed high activity. Acylcarnitines showed the highest level, indicating high activity of carnitine acetyltransferase in mitochondria [33]. These results indicated the preferred metabolic fuel-from fatty acid oxidation in adolescent girls [33].
In the youth males aged 20 to 30 years, linoleic acid metabolites showed higher levels in addition to steroid metabolism. The involved metabolite eicosapentaenoic acid serves as the precursor for prostaglandin-3. It could enhance the production of the cytoprotective prostanoid 15d-PGJ2 [34], which corresponds to the elevated prostaglandin production in youth males [35]. In contrast to youth males, arachidonic acid metabolites showed higher levels in youth females. It was reported that in females, arachidonic acid metabolism could rescue anti-inflammatory and butyrate-producing microbiota, then upregulate GPR41 and GPR109A and control hypothalamic inflammation [36].
Metabolomic characteristics during the middle and old stages During the middle age stage, males and females showed the most significant gender differences. In males, the main metabolic features were vitamin B6 and purine metabolism. 8-Hydroxy-7-methylguanine, a methylated purine base, showed higher levels in males. High methylated purine bases were found in tumor-bearing patients compared to healthy controls [37]. Urine alkylated purines were partly derived from covalently bound adducts in DNA formed from exposure to carcinogenic alkylating agents [38]. Purine disorders may be associated with serious, sometimes life-threatening consequences. In females, the metabolism pathway of steroidogenesis and caffeine metabolism showed high activity. Menopausal symptom are an unavoidable problem in females during this period, which could contribute to some metabolic disorders. Caffeine metabolism was reported to be associated with menopausal symptoms, particularly vasomotor symptoms [39].
For the stage aged above 50 years, the gender difference decreased. During this period, organ metabolism activity gradually slows down. Energy-supply metabolism pathways, such as fatty acids and amino acids, showed low levels. In contrast, steroidogenesis, caffeine and pyrimidine metabolism showed high levels. Higher levels of pyrimidine metabolites in the old stage have adverse effects on health [38]. Additionally, several cognitive impairment-related metabolites, including acetylhistidine and steroid hormones, were found to be higher in the old population, partly contributing to the high incidence of cognitive impairment diseases at older ages [40].