Identification of biological markers for better characterization of older subjects with physical frailty and sarcopenia.

Population aging is rapidly accelerating worldwide; however, longer life expectancy is not the only public health goal. Indeed, extended lifetime involves maintaining function and the capacity of living independently. Sarcopenia and physical frailty are both highly relevant entities with regards to functionality and autonomy of older adults. The concepts and definitions of frailty and sarcopenia have largely been revised over the years. Sarcopenia is an age-related progressive and generalized loss of skeletal muscle mass and strength. On the other hand, frailty is a state of increased vulnerability to stressors, responsible for exposing the older person to enhanced risk of adverse outcomes. Physical frailty and sarcopenia substantially overlap and several adverse outcomes of frailty are likely mediated by sarcopenia. Indeed, the concepts of sarcopenia and physical frailty can be perceived as related to the same target organ (i.e., skeletal muscle) and it may be possible to combine them into a unique definition. The biological background of such a close relationship needs to be explored and clarified as it can potentially provide novel and pivotal insights for the assessment and treatment of these conditions in old age. The aim of this paper is to indicate and discuss possible biological markers to be considered in the framing of physical frailty and sarcopenia.

Background

A healthy musculoskeletal system is necessary for physical functioning. A decrease of skeletal muscle mass is a universal consequence of aging with a broad range of functional and metabolic consequences [1]. Skeletal muscle affects a wide spectrum of vital processes that are often inadequately appreciated [2]. Clearly, skeletal muscle is responsible for movement and loss of muscle mass and quality may result in weakness and reduced mobility; however, skeletal muscle is also the largest reserve of proteins in the body. During periods of stress, under-nutrition, or starvation, it provides a continuous supply of amino acids in order to support the protein synthesis for vital organs. Skeletal muscle represents the primary site of glucose disposal, as well. A reduction of muscle mass may cause metabolic dysregulation, especially in patients with insulin resistance and type 2 diabetes. In addition, skeletal muscle is the major energy consumer and contributor to basal metabolic rate in the body. Loss of muscle represents the primary cause of age-associated reduced basal metabolic rate and decreased energy needs [3].

The age-associated loss of skeletal muscle mass, function, and quality is commonly known as “sarcopenia” [4-7]. Sarcopenia (derived from Greek sarx for flesh and penia for loss) is a term coined by Rosenberg to describe one of the most noticeable changes occurring with aging [8]. It has been defined as the “progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life and death” by the Special Interest Group of the European Sarcopenia Working Group in 2010 [9]. The term is used specifically to denote loss of muscle mass and strength associated with aging and distinguishes muscle loss due to aging from other causes, such as immobility or neurological damage. Sarcopenia is recognized as a geriatric syndrome and a key public health issue. Starting at the age of 30 years, individuals lose 1–2% of muscle per year, and by the age of 80 years, 30% of muscle mass is lost [10, 11]. The prevalence of low muscle mass is estimated to be between 10 and 25% depending on the studied population and methods applied. In octogenarians the prevalence increases up to 50% [9]. Reduced muscle function is independently associated with increased risk of functional impairment, falls, disability and mortality in older subjects [12].

Under normal circumstances, muscle homeostasis is maintained as a delicate balance between new muscle cell formation, hypertrophy, and protein loss. This balance is coordinated by the central nervous, endocrine, and immune systems. Behavioral factors (i.e., nutrition and physical activity) may also substantially modify these interactions. Every endogenous and exogenous stressor disrupting the homeostatic balance of older persons may trigger an acceleration of the sarcopenia phenomenon.

Physical frailty is strongly linked to muscle mass and function. Frailty is a multi-system impairment associated with increased vulnerability to stressors and describes individuals who are at increased risk of adverse health outcomes [13]. All experts unanimously agree on the theory of frailty and the need to push forward its study, thus promoting the implementation of the syndrome in clinics and research. However, frailty cannot be limited to a physical domain; psychological, cognitive, emotional, social and spiritual factors contribute to frailty and need to be taken into account in its definition. Physical frailty characterizes the unique core condition between sarcopenia and frailty [1]. Research on physical frailty is far more advanced than research on other aspects of frailty. A phenotypic approach to physical frailty has been introduced in clinical practice [14]. An alternative model of accumulation of deficits has also been used for measuring frailty in elderly people [15, 16]. None of these approaches seems to yield similar results in clinical practice [17]. Nevertheless, there is an overall agreement about the key role that physical function plays in the determination of the status of extreme vulnerability [18-20].

A careful examination of concepts of sarcopenia and physical frailty shows that they share many common points [1]. In fact, several adverse outcomes of frailty and sarcopenia are likely associated and sometimes one may determine the other. Sarcopenia is also associated with modifications in biological functions, including inflammation, glucose regulation, hormone production, cellular communication and protein storage. In this regard, the identification of specific biological markers that can be quantified in a reliable and cost-effective manner is important. Such biomarkers may serve in the qualitative assessment of the physical function impairment, represent potential targets for interventions, and support the clinical and research follow-up of the condition of interest.

Biological markers of physical frailty and sarcopenia

Definition of a biomarker

A biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” [21]. The optimal biomarker should be quantified in an accurate and reproducible manner and the assay feasible at reasonable cost. It should add new information that cannot be obtained by a careful clinical assessment alone. More importantly, the biomarker needs to show a strong correlation between the disease and its outcome in clinical studies. The ideal biomarker should support the clinician’s decisions in the management of the condition of interest. Last but not least, a biomarker can be used in research trials, making it more suitable for screening, baseline evaluation, and/or definition of outcomes. There are biomarkers for screening (to identify the target population), for assessment, and for follow-up. For example, biomarkers for detection and diagnosis may not be the same as those that ideally track disease progression. A biomarker is defined as any substance, structure, or process that can be measured in the body or its products, influence or predict the incidence of outcome or disease, and can be used in research. In this definition, biological markers “(blood, urine, etc.), functional tests or imaging markers are included. In this paper, we will only focus on biological markers of sarcopenia and physical frailty.

Sarcopenia may be considered a biological substrate for the development of physical frailty. In this context, several biological markers have been shown to be associated with skeletal muscle mass, strength, and function, thus representing potential markers for the effect of the studied interventions. There is not only one biological marker that perfectly matches the sarcopenia and physical frailty criteria, but there is a range of complementary biomarkers, that will together constitute the ideal panel of markers (Fig. 1).

Figure 1

Biological markers in relation to sarcopenia and physical frailty

Elevated inflammatory markers associated with lower muscle mass and strength

In older age, a low-grade inflammatory state characterized by increased concentrations of cytokines and acute phase proteins is common [22, 23]. TNF-α, IL-1β, IL-6, and IL-18, and C-reactive protein (CRP) and fibrinogen are among the cytokines and acute phase proteins that have been frequently studied in describing such chronic inflammatory states [24]. This phenomenon, also called “inflammaging,” results from an imbalance between pro- and anti-inflammatory networks [25]. Muscle mass and strength are inversely associated with plasma concentrations of IL-6 and TNF-α in well-functioning older men and women [26-28], which is only partially explained by decline in muscle strength and slowed walking speed [29]. Moreover, elevated inflammatory markers predict mobility limitation, independent of cardiovascular disease events and severe illness [30].

Products of oxidative damage contribute to sarcopenia and physical frailty

One of the factors that could play a key role in triggering sarcopenia and physical frailty is the accumulation of reactive oxygen species (ROS). ROS are generated by the addition of a single electron to the oxygen molecule. ROS are by-products found in practically all tissues and are usually generated in the mitochondrial respiratory chain. Such reactive elements are often harmful, resulting in oxidative stress that can damage other cellular components such as DNA, proteins, lipids, etc., which in turn results in subsequent damage to cells and tissues. Cells respond to oxidative stress by variations in the rate of cell growth, changes in cell cycle length, and increase of their defensive mechanisms (i.e. antioxidant defense system). Free radicals cause severe damage if they are not promptly eliminated by the action of anti-oxidant agents. The levels of these damaged macromolecules and lipids increase with age [31, 32].

Protein carbonyls are known markers of oxidative stress and accumulate with aging [33]. Protein carbonylation leads to cellular dysfunction and a decline in tissue function and is involved in the pathogenesis of sarcopenia [34]. Serum protein carbonyls are independently associated with grip strength [35].

Advanced glycation end products (AGEs), bioactive compounds that are formed by nonenzymatic glycation of proteins, lipids, and DNA, play a role in the pathogenesis of sarcopenia and physical frailty [36]. Elevated serum AGEs are associated with poor muscle strength [37].

Serum creatinine and urinary creatinine excretion as a marker of muscle mass

Creatine is a naturally occurring nitrogen-containing compound found in the diet, primarily in red meat and seafood [38]. The majority of creatine is stored in skeletal muscle as phosphocreatine (PCr), a high-energy phosphate involved in the rapid resynthesis of adenosine triphosphate (ATP) during intense muscle contraction [38]. Aging may have a negative impact on high-energy phosphate metabolism [39-41].

Creatine is the precursor of creatinine. In the steady-state and with stable kidney function, creatinine is usually produced at a relatively constant rate by the body depending on the absolute amount of muscle mass [42]. Creatinine is filtered out of the blood by the glomeruli (and is excreted to a smaller extent in the proximal tubules of the kidney). Since there is little to no tubular reabsorption of creatinine, its renal clearance is often used to estimate glomerular filtration rate. Under stable kidney function, the serum or plasma concentration of serum creatinine can also reflect skeletal muscle mass, if its non-muscle-mass-dependent variations (such as due to renal filtration or meat intake) can be accurately accounted for [42].

A new biological technique to estimate muscle mass has been developed recently by using a dose of creatine labelled with a non-radioactive tracer (deuterium). The isotope, enclosed in a gel capsule is ingested, and a urine sample collected several days later is used to estimate deuterated creatine by mass spectroscopy. The measured dilution space is strongly correlated with total body skeletal muscle mass measured with MRI [43].

To the best of our knowledge, the relationship between serum or urinary creatinine excretion and muscle strength has not been demonstrated. However, several studies provided evidence for the effects of creatine supplementation on muscle strength [44, 45].

Endocrine function

Decline in muscle mass and parallel decline of muscle function are attributed to a progressive shift from anabolic to catabolic metabolism with a reduced capacity for synthesizing new proteins and repairing muscle damage [46]. The defect in muscle protein homeostasis may be related to changes in circulating levels of hormones.

The age-associated decline in the production of dehydroepiandrosterone sulfate (DHEAS) is an important determinant of reduced muscle mass and strength in older persons [47]. There is evidence that sex hormones (testosterone, estrogens, and DHEAS), whose levels decrease with age, exert an important role in the age-related onset of sarcopenia [48]. DHEAS may affect muscle performance. The skeletal muscle is able to convert DHEA into active androgens and estrogens, and to stimulate insulin-like growth factor-1 (IGF-1), which is important for muscle growth and recovery [49]. The maintenance of adult muscle depends on satellite cell activation, proliferation, survival, and differentiation, all of which can also be stimulated by testosterone [50, 51]. The effects of testosterone on muscle can be categorized as anabolic, anti-catabolic, and potentially anti-inflammatory [52, 53].

It has been proposed that testosterone stimulates skeletal muscle protein synthesis, improve recycling of intracellular amino acids, and promote the activity of motor neurons [52]. However, the proposed effects of testosterone on muscle protein degradation are not straightforward. It appears that short-term testosterone administration does not change the breakdown rate of muscle proteins, whereas treatment for several months decreases muscle protein breakdown [52]. Testosterone promotes commitment of pluripotent stem cells to myogenic lineage but inhibits their differentiation into adipocytes via an androgen receptor-mediated pathway, suggesting the rationale for its well-known effects on the reduction in body fat mass and the increase in fat-free mass and insulin sensitivity [54].

The link between vitamin D and skeletal muscle health has been well-described in clinical studies [55]. There is a broad range of muscle deficits associated with varying degrees of vitamin D insufficiency, whereas supplementation with various forms of vitamin D has mostly beneficial effects. The identification of the vitamin D receptor (VDR) in skeletal muscle tissue provides solid evidence for its direct effect on physical frailty and sarcopenia [56, 57]. Some studies have identified genomic effects of vitamin D, leading to the synthesis of new proteins that affect muscle cell contractility, proliferation, and differentiation [58, 59].

Loss of skeletal muscle mass has also been associated with insulin resistance and high glycated hemoglobin HbA1C concentrations. Skeletal muscle is a primary tissue responsible for insulin-mediated glucose disposal; thus, low muscle mass may cause reduced insulin-mediated glucose disposal. However, type II muscle fibers, which are less responsive to the metabolic actions of insulin [60], are lost to a greater extent than type I fibers in age-related muscle atrophy [61]. Moreover, some studies have shown that insulin resistance precedes the development of frailty [62-65].

Urine proteomics panel of muscle protein breakdown: measure of muscle catabolism

Urinary proteins should be regarded as a potential source of biomarkers for several disorders of muscle catabolism. Over the past few years, great technological advances have occurred in proteomics, and a large number of proteins in the urinary proteome of healthy people have been identified [65-69]. Thus far, protein–protein interaction data (interactome) has been widely used for the identification of biomarkers, with the assumption that the interactions of proteins may well reflect the health status. More than a biomarker, measurement of urine proteomics panel of muscle protein breakdown is a technique for simultaneously assessing multiple biomarkers and seeing how they interact.

N-terminal procollagen peptides: measure of muscle fibrosis

Type III collagen in soft connective tissues, such as muscle and skin, is synthesized from larger procollagen III molecules carrying peptide extensions at both N- and C-terminal ends [70-72]. The N- and C-terminal extensions of procollagen III are removed by specific proteinases during the final stages of collagen synthesis, and released into the circulation in stoichiometric amounts [72]. Procollagen type III N-terminal peptide (P3NP), a product of this proteolytic cleavage during collagen synthesis in connective tissue, can be measured in the human serum, and its circulating concentrations have been described in children, healthy adults, acromegalic subjects, and athletes [73-77]. P3NP levels vary in response to exercise, testosterone, and growth hormone (GH) [75, 78–82], and could represent useful markers of GH doping in sports [75, 76, 80–85]. Plasma concentrations of P3NP represent an interesting marker of skeletal muscle remodeling. Indeed, serum P3NP concentrations reflect lean body mass and appendicular skeletal muscle mass [86].

Overexpression of myostatin leads to muscle atrophy

Myostatin is a member of the transforming growth factor-β superfamily and is known to be a negative regulator of skeletal muscle myogenesis and functions as an inhibitor of muscle growth [87-89]. Myostatin-deficient mice have increased muscle mass, whereas overexpression of myostatin leads to muscle atrophy [88, 90]. Although alterations in myostatin expression and activity in the context of aging are not fully understood, aging is associated with upregulated myostatin expression in humans [91]. Thus, older myostatin null mice exhibit resistance to the sarcopenic phenotype [92-94] and neutralize antibodies to myostatin, leading to an increase in muscle mass and improved measures of muscle performance, including grip strength [95-97].

Agrin fragment: measure of neuromuscular junction function

Agrin, an extracellular proteoglycan, is synthesized in motor neurons, transported along the axons and finally released into the synaptic basal lamina, where it induces postsynaptic differentiation (including acetylcholine receptor clustering). Agrin is therefore essential for the formation and stabilization of neuromuscular junctions [98]. Agrin is inactivated after cleavage by neurotrypsin, a synaptic protease, which frees a soluble 22 kDa C-terminal agrin fragment (CAF) that can be detected in human serum [99-101]. Experiments with transgenic mice overexpressing neurotrypsin in spinal motor neurons have shown the full sarcopenia phenotype, including a reduced number of muscle fibers, increased heterogeneity of fiber thickness, more centralized nuclei, fiber-type grouping and an increased proportion of type I fibers [102]. Thus, elevated levels of CAF cause degeneration of neuromuscular junctions and indicate that sarcopenia contributes to physical frailty.

Which biomarker is more reflective of sarcopenia and physical frailty?

Given the syndromic nature of sarcopenia and physical frailty, no unique biomarker has all the features to reflect sarcopenia and physical frailty, but a panel of complementary biomarkers (likely belonging to multiple classes: imaging, biological markers, and functional tests) would be most useful. Together they constitute the ideal panel of markers. The first objective is to evaluate current biomarkers (described above) and the thresholds for correlation with clinical outcome, and perhaps with therapeutic intervention in clinical trials. The results of these trials will tell us whether biomarkers and thresholds can be upheld in accordance to the above-mentioned criteria for good biomarkers. It is likely that some of the suggested thresholds will have to be adjusted. The second objective is to identify novel biomarkers of sarcopenia. Biomarkers derived from blood or urine can easily be measured in a standardized and low-cost way and are therefore very attractive. Finally, further studies are necessary to understand how sarcopenia and physical frailty intersect with muscle tissue and to define specific biomarkers according to their relevance (diagnosis, follow-up, research, etc.).

Conclusion

The recognition of sarcopenia as a major component of physical frailty implies that interventions that target the skeletal muscle may provide therapeutic and preventive advantages against frailty and its clinical correlates. Observational studies and some randomized clinical trials have suggested a positive effect of regular physical activity and nutritional interventions on improving physical function and/or reducing symptoms of disability in healthy older individuals and those at risk for mobility disability [103]. In this context, one research priority is to investigate and define novel biomarkers allowing an improved assessment, characterization and follow-up of elderly people with physical frailty and sarcopenia. It is necessary to identify a segment of the aging population at risk for adverse outcomes whose medical needs are presently unmet, partly due to the current unclear definition of frailty. We have to investigate the possibility of translating the model of physical frailty and sarcopenia into a clinical intervention (e.g. multidomain intervention) with potentially positive effects aimed at preventing mobility disability. The results generated by these studies will have relevant clinical and public health implications, filling an important gap in knowledge for practicing evidence-based geriatric medicine.