Is our chronological age really the best indicator for understanding the risk of age-related diseases?
A new research study has taken their previous findings in animal testing and applied a theory to a population-based group using human urine samples in order to analyse biological age as a potentially better biomarker. Through their research, a simple urine test could soon be used as a more accurate measurement for how our bodies are aging and help to determine personalised risk profiles for disease development.
Age is typically defined as a length of time that a living entity has existed and is thus measured chronologically. Many disease risk factors highlight chronological age when it comes to determining one’s susceptibility for a particular medical condition.
Some researchers are inclined to define the natural aging process as ‘a disease’. In this sense, aging is a condition in itself which can be physically handled (i.e. manipulated), treated and even somewhat delayed, much like many other health ailments.
Age-related diseases that are associated with chronological age include the likes of:
- Cardiovascular conditions
- Hypertension (high blood pressure)
- Type 2 diabetes
- Alzheimer’s disease
This newly published research asks questions about the true nature of the aging process and disease risk. Is chronological age really the best indicator when it comes to identifying an individual’s risk of developing age-related diseases? And can accurate biomarkers be identified to determine the exact biological / physiological state of aging? The team behind the research believe so. In their view, chronological age is not entirely the same as biological age and thus may be an imprecise mode of measurement.
Analysing the state of aging
It is known that an individual’s state of aging (i.e. the rate at which the physical body ages) may be influenced by numerous factors other than merely the length of time he or she has existed since birth.
Cells in the body accumulate damage over time until the very end of a person’s life. This is referred to as oxidative damage. With an accumulation of cellular damage comes increased levels of oxidative biomarkers in the body. The rate at which cellular damage accumulates varies from one person to the next and is thought to be influenced by genetic, lifestyle and environmental factors. This means that one person’s biological age can differ significantly from another who has existed for a similar period of time (i.e. someone of a similar chronological age).
How we age biologically has an influence on our individual lifespan. Earlier biological aging could help to explain why some people develop age-related health conditions at an earlier chronological age than others.
This new study aimed to assess whether cellular damage / oxidative biomarkers could potentially serve as more accurate indicators of physiological aging than the number of years a person has been in existence.
The group of Chinese researchers recently published their analysis findings in Frontiers in Aging Neuroscience, an open-access journal, showing what they believe to be an accurate measurement of a person’s biological age which can be used to better treat the onset of age-related diseases and determine risk. (1)
So, what mode of measurement did they use to determine biological aging (or the rate of physiological aging)? The team made use of a simple urine test which analyses the amount of oxidative damage affecting human cells. Urine samples were analysed to determine the relationship between age and oxidation markers.
A liquid by-product of human metabolism, urine better reflects the oxidative state of the entire body rather than just one specific portion (i.e. an organ), and thus the biological age of a person’s physical being.
An overview of aging biomarkers
In order to identify an accurate and reliable biomarker, the research team needed to take into consideration some of the major factors associated with aging:
- Oxidative stress (damage) – This is the imbalance between reactive oxygen species and the body’s biological ability to detoxify harmful compounds and repair itself, thus resulting in cell / tissue damage.
- Protein glycation – This involves the bonding process of sugar molecules (like fructose or glucose) to proteins or lipid molecules. An accumulation can lead to the formation of plaque, a thickening of the basement membrane (a thin membrane of proteins separating the epithelium / upper skin layer from underlying tissues) and a reduction of vascular elasticity.
- DNA methylation – A mechanism arising from non-genetic influences that is used by cells to regulate gene expression. Methyl groups are added to DNA molecules, changing activity (but not the sequence), inhibiting gene transcription, and thus contributing to age-related disease biomarkers.
- Inflammation (chronic) – A localised physical reaction due to injury or infection whereby portions of the body become swollen, hot, reddened and sometimes painful. Chronic low-grade inflammation is associated with age-related diseases which develop as a result of underlying inflammatory causes.
- Cellular senescence – This refers to hindered growth of individual mitotic cells (i.e. cells undergoing cell division) which leads to impaired tissue function, and tissue accumulation that predisposes a person to disease development. A loss of tissue repair capability and the production of pro-inflammatory molecules is considered a driver of aging and the onset of age-related disease.
- Hormonal de-regulation – This involves a failure in hormone receptors within the body. Disruptions in the regulation of growth, metabolism, reproduction and responses to stress factors impacting the body have been linked to influencing the process of aging and longevity (lifespan).
Currently, it is believed that the process of aging is induced by the gradual accumulation of various molecular faults in the body’s cells and tissues over time. Small biomolecules or biopolymers, known as nucleic acids, are the key genetic material which plays a pivotal role in the synthesis of protein. By-products of normal metabolism can result in oxidative damage to biomolecules, like DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
Oxidative damage accumulates as antioxidant defence mechanisms degenerate throughout a person’s lifetime. The accumulation of oxidative markers causes errors to occur in biomolecules, which then results in protein abnormalities.
Researched DNA oxidative products include 8-Oxo-7,8-dihydro-2′-deoxyguanine (also referred to as 8-oxodGsn) and 8-oxo-7,8-dihydroguanosine (known as 8-oxoGsn), with studies having been focussed on their susceptibility to ROS (reactive oxygen species) and ability to induce and increase the mutation frequency of organisms. Oxidised DNA can be repaired by enabling excision products (those which are extracted) to be transported across cell membranes and expelled into plasma, cerebrospinal fluid and urine without the need to be metabolised. Left alone, levels of 8-oxodGsn and 8-oxoGsn gradually increase until the end of a person’s life, elevating risk for disease onset.
8-oxodGsn and 8-oxoGsn products can reflect the oxidative state of the entire body, and thus urine testing is a viable assessment option for quantification. To determine accurate quantification of 8-oxodGsn and 8-oxoGsn products derived from bodily fluids, the research team used a liquid chromatography with tandem mass spectrometry (LC-MS/MS) based system which is less affected by cross-contamination of the two DNA products. The team felt that their selected chromatographic method could enable them to separate sample impurities during a high-performance liquid chromatography-electrochemical detection (HPLC) phase of the process and better distinguish between types of guanosines (a purine nucleoside / enzyme that is encoded by NP genes).
This team previously used this chromatographic method in a study with mice (2) and found that 8-oxoGsn in urine could be accurately measured and age-dependant increases in biomarkers could be successfully identified. Thus, showing potential for effectively evaluating the biological aging process.
This prompted the team to think a step ahead and apply the same research process to human urine samples to test whether they could determine accuracy in biological age in the same way. The team believe that this assessment method is better suited for routine measurement due to its ability to analyse large quantities of urine samples. This makes the method capable of being accurately applied to large-scale epidemiological studies when looking at how certain diseases affect certain populations and why in order to develop better strategies of treatment down the line.
How was the study conducted?
The research study was approved by the Ethics Committee of West China Hospital at the Sichuan University (Chengdu, China).
The research team randomly selected 1 228 Chinese individuals (615 of which were female and 613 were male). The age range of these individuals was between 2 and 90. Adult participants were recruited from the West China Hospital (Sichuan University) while undergoing routine health checks between January and April 2016. Children were recruited from the West China kindergarten.
Consent (obtained through consent forms by adult individuals or parents of children) was given for the collection of spot urine samples from a predominantly Sichuanese population. The samples were then filtered through a study inclusion criteria assessment.
To meet the criteria, recruited participants had to have…
- No history of psychiatric or somatic (i.e. physical body) abnormalities
- No history of medication use, smoking or consumption of alcoholic substances 2 weeks prior to sample collection
Eligible participants were then sub-divided into age classes:
- 1 to 10: 102 (51 male and 51 female)
- 11 to 20: 70 (52 male and 18 female)
- 21 to 30: 167 (79 male and 88 female)
- 31 to 40: 186 (89 male and 97 female)
- 41 to 50: 202 (102 male and 100 female)
- 51 to 60: 205 (102 male and 103 female)
- 61 to 70: 115 (48 male and 67 female)
- 71 to 80: 149 (68 male and 81 female)
- 81 to 90: 32 (22 male and 10 female)
Urine samples were frozen and thawed when ready for use and were then separated into two for analysis. One split sample was analysed to determine concentrations of 8-oxodGsn and 8-oxoGsn using ultra-high-performance LC-MS/MS with a UPLC-MS/MS (a triple quadrupole mass spectrometer).
The team measured creatinine levels (compounds of creatine which are metabolised and then excreted in urine) in the other split sample by means of the Cobas P800 system (designed for high throughput applications). These levels could then be determined as a fixed concentration which was subtracted from each participant’s sample in order to provide a correct result that was free of non-specific reactions.
Samples were validated using three different method assessments, all of which were performed in triplicate:
- Linearity and sensitivity: This entailed analysing standard solutions of 8-oxodGsn and 8-oxoGsn measured in ng/ml (nanogram/millilitres).
- Accuracy (recovery study): This involved the identification of low-level urine, medium-level urine and high-level urine. Low levels of creatinine in urine could indicate abnormalities with kidney function and therefore kidney problems or disease. To assess accuracy, the team performed a recovery study of the samples as a way to measure proportional errors. The 8-oxodGsn and 8-oxoGsn split samples were spiked with varying amounts of urine from the others in order to determine low, medium or high concentrations.
- ‘Within-day’ versus ‘between-day’ precision study: The low, medium and high urine concentrations were then assessed according to ‘within-day’ and ‘between-day’ evaluations. For ‘within-day’, samples were analysed 20 times (consecutively) in a single day. For ‘between-day’ the same urine samples were assessed for 20 consecutive days.
What were the study findings?
The research team found no significant differences in 8-oxodGsn and 8-oxoGsn content values in the urinary samples of males and premenopausal females under the age of 60. From the age of 61, however, these levels appeared to be considerably higher in postmenopausal females than in males.
Women between the ages of 51 and 60 saw a sharp increase in oxidation products. In males of the same age group, increases were more gradual. The team noted that males in the 51 to 60 age group had 8-oxodGsn / creatinine levels that were 10% higher than those noted in men aged 41 to 50. By comparison, females aged 51 to 60 saw a 30% increase compared to those in the 41 to 50 age group. The team also reported similar tendencies in the older age groupings (aged 60 and upwards).
With these findings, the team determined that oxidative products increase at a higher rate in post-menopausal women than in men of the same age. This, they believe, has a lot to do with the decreased production of oestrogen (the primary female sex hormone) in the body at this stage of life, as well as changes in iron content.
Reduced oestrogen production in post-menopausal women results in decreased anti-oxidative capacity. Iron content is also higher in post-menopausal women and thus also contributes to elevations in urinary 8-oxodGsn excretion levels. While younger women lose iron content through their monthly menstrual periods, post-menopausal females retain higher levels of iron which is known to result in increased oxidative stress and damage.
Gradual increases in 8-oxodGsn / creatinine levels occurred throughout the various age groupings from 21 to 30 onwards. Levels from each age group were compared to those of the 11 to 20 age group. The most significant increases appeared to reflect from the 51 to 60 age group and this, the team believes, can be attributed to the degeneration of anti-oxidative capability as a person reaches the senior stages of life. The levels were lowest among younger generations between the ages of 11 and 30, by comparison.
Thus, the team found that 8-oxodGsn and 8-oxoGsn in urine showed a definite correlation with age. 8-oxodGsn and 8-oxoGsn levels increased throughout the different adult age groups, which correlates with the degenerative patterns of anti-oxidant defence systems as people age.
Creatinine levels also measured differently throughout the different age groups. Children in the age group 1 to 10 displayed lower levels than those participants aged 11 to 20.
Children participating in the study, however, appeared to show a much higher level of nucleic acids than the older groupings. The team then took the decision to assess a small grouping of babies - 5 infants aged between 1 and 5 months, and 6 babies aged between 6 and 12 months.
The 8-oxodGsn and 8-oxoGsn content of babies aged 6 to 12 months had markedly higher values than the children in the 1 to 10 age group. Based on their assessment, this could indicate that there is an imbalance between the oxidative and anti-oxidative states of new-born infants. The research team believe that the higher values can be attributed to the process of childbirth – a baby (foetus) moves from the intrauterine environment that is lacking in adequate oxygen supply to another which has more oxygen content. A new-born’s antioxidant defence system is also weaker at birth, gradually maturing with age as a little one develops.
Many previous studies have focussed on assessing urinary 8-oxodGsn only. 8-oxoGsn is largely regarded as a chemical product that originates from RNA and has thus had limited focus in studies in relation to aging. The research team believes that RNA plays a key role in protein translation and should therefore be included in study along with urinary 8-oxodGsn. Any modifications to these molecules results in the accumulation of abnormal protein formations, which has a direct impact on the process of aging.
The research team feel that 8-oxoGsn is thus not only a reliable biomarker of aging, it may also be a better one than 8-oxodGsn for two reasons:
- 8-oxoGsn levels showed an approximate 2-fold increase result than 8-oxodGsn when assessing age groups.
- 8-oxoGsn levels also better correlated with a person’s state of aging – i.e. it better reflects a person’s actual physiological aging stage.
In this regard, the research team feel that they have been able to demonstrate that a simple, sensitive and relatively quick urine sample test could provide a reliable method of analysis of the urinary excretion of 8-oxoGsn and 8-oxodGsn. As such, this urine test (with 8-oxoGsn content) could prove a better evaluation method showing increases in oxidative damage (thus serving as a biomarker) more accurately for the process of biological aging in adults. Accurate measurements of this biomarker, the team believes, could then help those in the medical field to better predict the risk of age-related disease in adults… and all with a very simple urine test.
"Urinary 8-oxoGsn may reflect the real condition of our bodies better than our chronological age and may help us to predict the risk of age-related diseases," says Jian-Ping Cai, one of the researchers who was involved in the study.
The team now hopes that their rapid analysis method may prove useful in future large-scale aging research studies, which will enable greater numbers of participant samples to be analysed over a shorter period of time. In this way, assessing the impact of age-related disease can potentially be better addressed and prove helpful in improving the diagnosis and treatment of specific conditions.
1. Frontiers in Aging Neuroscience. 27 February 2018. Urinary 8-oxo-7,8-dihydroguanosine as a Potential Biomarker of Aging: https://www.frontiersin.org/articles/10.3389/fnagi.2018.00034/full [Accessed 01.03.2018]
2. US National Library of Medicine National Institutes of Health. May 2012. Age-dependent increases in the oxidative damage of DNA, RNA, and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS: urinary 8-oxoguanosine as a novel biomarker of aging: https://www.ncbi.nlm.nih.gov/pubmed/22348977 [Accessed 01.03.2018]