In the present study, we performed untargeted profiling of circulating metabolites in a large, population-based prospective human cohort study, followed by validation in two further prospective cohorts and one longitudinal mouse study. We report the novel discovery that circulating glucuronic acid is a robust, cross-cohort and cross-species predictor of all-cause mortality in healthy individuals, as well as a predictor in humans of chronological age and healthspan-related outcomes.

The factors influencing circulating levels of glucuronic acid are poorly characterized. However, orally ingested glucuronic acid has been shown to raise serum glucuronic acid levels within an hour, suggesting that it is readily absorbed into the bloodstream [ 22 ]. The ingestion of glucuronide conjugates also increases glucuronic acid levels, likely through the absorption of glucuronic acid liberated in the gastrointestinal tract by β-glucuronidases [ 23 ]. Elevated serum levels of glucuronic acid have been reported in human studies of diabetes, hepatocellular carcinoma, hepatitis, cirrhosis, and obstructive jaundice [ 24 – 28 ]. These findings are not unambiguous, as subsequent work reported contradictory associations with hyperglycemia and hepatitis [ 25 , 29 , 30 ]. Finally, a recent metabolomic study of patients with cirrhosis identified glucuronic acid as a biomarker of disease severity and future mortality [ 31 ].

Glucuronic acid is a key metabolite of glucose involved in the detoxification of xenobiotic compounds [ 1 – 6 ]. Many of these exogenous compounds, which include pollutants and drug metabolites, undergo hepatic glucuronidation, in which they are conjugated to glucuronic acid via the enzymatic action of UDP-glucuronosyltransferases [ 7 , 8 ]. This chemical modification increases solubility in bile, facilitates urinary excretion, and is a key step in the phase II metabolism of these compounds required for their effective clearance from the body [ 9 ]. However, enzymes known as β-glucuronidases cleave these conjugates, thereby counteracting this detoxification process [ 10 ], as well as steroid hormone conjugates, thereby altering steroid metabolism [ 11 ]. Around a quarter of resident bacterial species in the human gut produce β-glucuronidase [ 12 – 14 ], which has been directly linked to increased xenobiotic-induced toxicity rescuable by inhibition of the enzyme [ 15 – 17 ]. Another vital role of glucuronic acid is as a constituent of proteoglycans, a diverse class of glycosylated proteins known primarily as components of the mammalian extracellular matrix [ 18 , 19 ], where glucuronic acid may confer increased rigidity [ 20 ]. These proteoglycans are degraded as a part of tissue remodeling by endogenous lysosomal β-glucuronidase, which, like its bacterial analogue, cleaves glucuronic acid moieties through hydrolysis [ 21 ].

We sought to further elucidate the predictive ability of glucuronic acid for age-related decline in health. In the Framingham Offspring metabolomics cohort, elevated levels of glucuronic acid were associated with poorer healthspan-related outcomes measured more than a decade in the future, including reduced overall self-rated health (p=0.11), reduced grip strength (p=0.027), reduced self-rated ability to perform heavy housework (p=0.023), higher measured times on tests of normal walking speed (p=0.018) and quick walking speed (p=9.4×10 -4 ), as well as reduced pulmonary forced expiratory volume in one second (FEV1; p=0.046), a measure of lung function ( Table 2 ). In general, an increase of glucuronic acid levels by one standard deviation corresponded to approximately the same functional decline and mortality risk expected from an additional year of age.

Figure 2. Variation of glucuronic acid levels with age. The relationship between chronological age and glucuronic acid levels in ( A ) the Estonian Biobank secondary cohort, ( B ) the Estonian Biobank primary (discovery) cohort, and ( C ) the Framingham Offspring cohort. In each case, the best-fit regression line through the data is shown, with the 95% confidence interval for this line shaded in grey. Glucuronic acid concentrations are represented as standard deviations of normalized concentrations within each dataset.

We found a significant positive correlation between glucuronic acid levels and age in the secondary Estonian Biobank cohort (N=100; r=0.41, p=2.7×10 -5 , Figure 2A ), which was selected to span a wide range of ages. This relationship also validated in the main Estonian Biobank cohort (Pearson r=0.12, p=3.7×10 -3 , Figure 2B ) and the Framingham Offspring cohort (r=0.16, p=1.3×10 -9 , Figure 2C ). Multivariate models adjusted for standard clinical covariates also identified age as a significant correlate of glucuronic acid levels in all three cohorts (p=4.3×10 -3 , 2.0×10 -8 , and 2.2×10 -5 , respectively).

To further validate the observed relationship between glucuronic acid levels and mortality, we performed metabolomic profiling on the sera of 196 27-month-old female mice from a genetically heterogenous background followed from birth through 33 months of age. In this murine cohort, glucuronic acid levels were also a significant predictor of all-cause mortality (HR=1.29, p=0.018), and a weakly significant difference in survival was observed between the top and bottom quartiles of glucuronic acid levels in this cohort (p=0.1, Figure 1C ).

We investigated whether the predictive ability of glucuronic acid for mortality would be attenuated by including other known mortality biomarkers in the regression model. The Framingham Offspring cohort allowed us to correct for seven of the most commonly studied biomarkers of mortality, including fasting glucose, HDL cholesterol, LDL cholesterol, triglycerides, creatinine, HbA1c, and albumin. In a multivariate Cox regression model adding these to our baseline clinical covariates, glucuronic acid levels remained a significant predictor of mortality (HR=1.12, p=0.044).

We evaluated this association in two replication cohorts, the Framingham Offspring cohort and TwinsUK study cohort, by Cox regression corrected for clinical covariates. Demographic characteristics of these cohorts are shown in Table 1A . As in the Estonian cohort, we found that glucuronic acid levels predicted all-cause mortality in both the Framingham Offspring (N=1,479; HR=1.13, p=0.032; Kaplan-Meier curve in Figure 1B) and in the TwinsUK (N=1,761; HR=1.25, p=0.017) cohorts.

We performed untargeted metabolomics on the Estonian Biobank cohort and found 69 of 569 identified metabolites to be predictive at FDR < 0.05 of all-cause mortality by Cox regression corrected for clinical covariates. Glucuronic acid ranked 9 th by p-value and was highly significant after correcting for multiple hypothesis testing (HR=1.44, p=2.9×10 -6 , FDR=5.0×10 -4 ), with higher levels leading to shorter lives on average. A Kaplan-Meier survival curve comparing the top and bottom quartiles of glucuronic acid levels in the Estonian Biobank cohort is shown in Figure 1A and demonstrated a significant difference in survival between the curves (p=1.7×10 -6 ).

Discussion

We have demonstrated that glucuronic acid levels are robust predictors of all-cause mortality and correlate with future healthspan-related outcomes. The effect size of the relationship with mortality (hazard ratios between 1.1 and 1.4 in our cohorts) is comparable to that of existing, clinically-important biomarkers of mortality such as cholesterol (HR=1.12 per mmol/L increase) and systolic blood pressure (HR=1.13 per 10 mm Hg increase) [32, 33]. Importantly, the predictive utility of glucuronic acid persists after adjustment for standard clinical covariates and other accepted predictors of mortality, including factors such as demographics, BMI, smoking status, blood lipids, HbA1c, creatinine, and albumin, indicating that the predictive ability of glucuronic acid for mortality is independent of these existing markers and their related biological mechanisms [34]. Moreover, we have demonstrated novel associations between glucuronic acid levels and future healthspan-related outcomes, including physical abilities, functional capabilities, and self-rated health, suggesting that the risk of mortality associated with elevated glucuronic acid levels is accompanied by a general decline in healthspan. Finally, we found glucuronic acid to be strongly positively correlated with age in three human cohorts with mean age ranging from 40 to 70 years (Table 1), an association that remained statistically significant following adjustment for clinical covariates. Notably, the simultaneous association of glucuronic acid levels with age, lifespan (as determined by all-cause mortality), and healthspan-related outcomes strongly argues that glucuronic acid is a biomarker of biological aging. Depending on the specific biology that underlies this relationship, glucuronic acid may also relate to the pathogenesis of these outcomes, and hence also of biological aging.

There are multiple mechanisms that might link levels of circulating glucuronic acid to age, mortality, and healthspan. One of the most compelling and best-understood possibilities relates to the cleavage of glucuronic acid from glucuronidated xenobiotics and steroid hormones by intestinal bacteria, a process that releases glucuronic acid as well as the toxin or steroid, making both available for reabsorption into the bloodstream in a process called enterohepatic recirculation [10]. Since activity of the responsible enzyme, β-glucuronidase, varies greatly among microbial species [12, 35], the composition of a person’s intestinal microbiome has a direct influence on this process. A microbiome that rapidly cleaves glucuronide conjugates may thus produce elevated glucuronic acid and also interfere with xenobiotic elimination and steroid metabolism. The substrates of glucuronide conjugation include xenobiotics such as environmental toxins and drug metabolites with pro-inflammatory and immunosuppressive effects [36–38] as well as endogenous steroids with tumorigenic effects at high concentrations [39, 40], and impairment of glucuronidation by intestinal β-glucuronidase can cause organ toxicity, inflammatory disorders, and carcinogenesis [16, 41–44]. Consequently, changes in microbiome composition could easily underlie the observed correlation between higher glucuronic acid levels, age, healthspan-related outcomes, and mortality [45].

This hypothesis is supported by other evidence from the literature. Among older people, bacterial β-glucuronidase activity levels are increased relative to young people (Mroczyńska and Libudzisz, 2010), and microbiome differences can distinguish healthy, independent older people from those who tend to be frail, sick, and require long-term residential care [46, 47]. Microbiome composition has also been linked to the onset of numerous age-related diseases, including atherosclerosis [48], type 2 diabetes [49], Alzheimer’s disease [50], chronic kidney disease [51], and nonalcoholic steatohepatitis [52], all of which contribute to reduced healthspan and increased mortality. Furthermore, bacterial β-glucuronidase activity changes in the appropriate direction with dietary modifications. For example, red meat consumption alters the gut microbiome [53, 54], elevates fecal β-glucuronidase activity [55], and correlates with increased future diabetes, cardiovascular disease, and mortality [56]. In contrast, dietary fiber consumption increases microbial diversity [57], reduces fecal β-glucuronidase activity [58–60], and correlates with lower all-cause and cause-specific mortality [61–63].

While this hypothesis is compelling, many other possibilities exist. For example, bacterial β-glucuronidase activity may be affected by gastrointestinal pH [64, 65] and hepatic glucuronidation may be impaired by chronic renal failure [66], both of which are independent of microbiome composition. Another potential mechanism linking glucuronic acid levels to disease states involves endogenous human β-glucuronidase, which localizes primarily to the lysosome and degrades glycosaminoglycans during normal and pathologic remodeling of the extracellular matrix (ECM) via hydrolytic liberation of glucuronic acid [21]. ECM remodeling is increased in aging and age-related diseases [67–69], with ECM degradation fragments in serum even being employed as disease biomarkers in some cases [70–72]. Moreover, lysosomal membrane permeabilization, an observation to inflammation and cell death [73–79] can cause the release of endogenous β-glucuronidase into the bloodstream [80], where it cleaves glucuronidated conjugates and may contribute to circulating glucuronic acid levels [81, 82]. In these scenarios, elevated glucuronic acid levels could be a result of ECM remodeling, inflammation, or cell death caused by concurrent disease. This seems less likely in our discovery cohort, where participants were free of major diseases at sample collection, but could still be consistent with subclinical disease. Finally, in addition to the above mechanisms related to glucuronidation, glucuronic acid may directly elicit an inflammatory response through an interaction with toll-like receptor 4 (TLR4) [83], which has been implicated in the pathophysiology of age-related diseases [84–86]. Of course, substantial further research is warranted to distinguish the relative contributions of these various hypotheses to the link between circulating glucuronic acid levels and aging, healthspan, and mortality.

The present study has a number of limitations. First, it is observational and retrospective in nature; however, it derives strength from the persistence of association across three high-quality, prospective human cohort studies in different countries, as well as in a lifespan study of genetically heterogenous mice. Second, despite the geographic diversity of our cohorts (Estonia, USA, and the United Kingdom), study participants were still predominantly of European descent, and the generalizability of these results to other demographic groups is uncertain, although the positive cross-species replication is encouraging. Third, the human cohorts were under-powered to evaluate relationships of glucuronic acid with the incidence of most individual diseases.

In summary, circulating glucuronic acid levels predict mortality in humans and mice, and in humans also associate with chronological age and predict healthspan-related outcomes. These simultaneous associations with age and with factors defining both lifespan and healthspan provide strong evidence that glucuronic acid is a biomarker of longevity and healthspan, as well as underlying biological age.