The Influence of a Personalized Intervention Program—AGA@4life—in the Cardiovascular Diseases: A Biochemical Approach

Abstract

Aging is a complex process inherent to and inevitable in humans. With life expectancy rising, there are concerns about the senior population’s wellbeing, and a hope of preventing certain diseases such as cardiovascular diseases. To achieve it, this study resorts to the implementation of an interventional program based on the comprehensive geriatric assessment model [AGA@4life]. The aim is to evaluate the effect of a new nutritional and exercise regime and evaluate possible changes in nitric oxide (NO) metabolites and endothelin 1 (ET-1). An intervention study was developed with 17 participants with ages of 65 and above. They were evaluated in the beginning [T0] and after eight weeks [T1], where NO metabolites and ET-1 levels were determined by enzymatic assays and the slot blot technique, respectively. There was a significant decrease in ET-1 levels in both the control (p < 0.001) and intervention (p = 0.04) groups from T0 to T1, but there was only a tendency for a decrease in the NO metabolite’s levels in the same conditions [p > 0.05]. Even though the NO metabolite levels did not increase as expected, possibly because of an increase in oxidative stress, the ET-1 levels decreased as expected and the overall results are promising, proving this program could have a beneficial effect on the geriatric population.

1. Introduction

Aging is a complex process that will eventually happen to every living being. In humans, it is influenced by numerous factors including genetic variables as well as environmental factors, and it is characterized by progressive changes in body mass composition that will eventually lead to a functional decline at both the cellular and organ levels with the passing of time [1,2]. Over the last 20+ years, life expectancy has risen by at least 6 years, and, in Europe, the average age is between 75.8 and 85.7 years old [3]. One reason for the improvement in life expectancy is the fast advances in technology, but, probably, the major reason is the improvement in health care and nutrition as well as a more efficient infrastructure and greater access to basic supplies [4]. With this increase and the decline in birth rates, there are a few challenges that are surfacing concerning the health and wellbeing of the senior population that are of extreme priority to be addressed.

With the age advancement, it is almost certain that some sort of chronic disease is bound to develop; among these, the most common ones are cardiovascular diseases (CDs), cancer, stroke, chronic obstructive pulmonary disease, chronic kidney disease, type 2 diabetes, and Alzheimer’s disease [1,4,5]. The advancement in age also contributes to the exacerbation of chronic systemic inflammation, oxidative stress, DNA damage, the decline in mitochondrial function, and cellular senescence, thus meeting all the necessary conditions for the onset of metabolic disorders [4,5].

One of the dramatic changes that are characteristic of human aging is the general increase in the total fat mass, that plays an important role when it comes to certain diseases [6,7]. The excess or dysfunctional fat tissue appears to accelerate these diseases and, in consequence, reduce the life span of an individual, while interventions that delay or limit the fat tissue are associated with an enhanced life span. Two of the most sought-after interventions for this specific problem are caloric restriction and the implementation of a physical exercise regime [7,8]. The cardiovascular system is one of the most affected systems in a high-fat body, which can lead to certain CDs such as hypertension, heart failure, coronary heart disease, and atherosclerosis, paving the way to heart attacks and strokes [9].

CDs are one of the most common causes of death in the world especially in developed countries, and, even though there have been some improvements in the prevention of this type of diseases, their prevalence continues to rise over the years [10,11,12]. These are chronic diseases that gradually evolve throughout life and are usually asymptomatic for a long time. They can be caused by multiple factors, some of them invariable, such as age and gender among others, and others variable, such as smoking, physical inactivity, poor eating habits, etc. [13]. Due to the increase in CDs over the years, there has been an increase in awareness in the population and some prevention tactics have been encouraged within the population, one of them being the change in poor lifestyle habits [13,14]. There have been some studies proving that the key to maintaining a healthy lifestyle and, therefore, preventing some of these cardiac complications is the adoption of a more balanced diet and a regular exercise regime in order to stay active.

When it comes to nutrition, it has been shown that a diet that consists of an intake of foods with less cholesterol has helped to lessen the development of certain complications, such as atherosclerosis, which can ultimately lead to the development of more serious CDs [15]. We have had proof with the help of several studies that a healthy change in the diet has many benefits in the overall health of a patient, coupled with a healthy exercise regime. As well as a poor diet, the American Heart Association has also established that a sedentary lifestyle is a major modifiable risk factor for cardiovascular diseases and the embracing of a more active lifestyle has had a major effect in the prevention and treatment of such diseases. By being active, not only do the muscles strengthen, one of them being the heart, but body fat is also kept at a lower level, preventing the development of cardiac complications [16,17].

One of the potential etiologies of CDs is increased oxidative stress, which is defined as the imbalance between reactive oxygen species (ROS) production and antioxidant defenses in a biological system [18]. It is necessary to maintain a basal level of ROS to maintain various cellular functions, and an imbalance in the production of these ROS, specifically an excess in ROS production, is associated with many CDs. To maintain the ROS levels, there are certain mechanisms that prevent an excess in their formation [19]. When in normal concentrations, ROS are known to modulate numerous pathways important for the systemic control of vascular resistance and blood pressure, including the bioavailability of nitric oxide (NO). NO is important in the cardiac system since its presence diffuses to smooth muscle cells, causing relaxation and vasodilatation. When there is an increase in ROS production, there is going to be a decrease in the NO’s bioavailability, which can lead to hypertension, and, eventually, more serious cardiac complications [20]. Another important player in the development of CDs is the enhancement of the endothelin-1 (ET-1) hormone, which regulates the cardiac system contraction through the concentration of intercellular Ca²⁺. An increase in this hormone can reduce the time of diastolic relaxation, increasing the contractility of the heart [21].

The AGA@4life program is an intervention program based on an individual and multidisciplinary assessment protocol, from which intervention strategies were implemented, adjusted to each participant’s needs, and aimed at preventing frailty and functional, cognitive, and social decline in seniors, supported by differentiated and multidisciplinary technical skills. This study aimed to evaluate the impact of the AGA@4life program by comprehending a nutritional intervention, modifying and correcting dietary errors, while implementing a balanced diet plan adjusted to the nutritional needs of each user; as well as evaluate the impact of a tailored exercise program in biochemical parameters in old adults in a day care center [22].

Therefore, the main objective of this study was to evaluate the impact of the intervention program in NO metabolites and ET-1 levels before these new dietary and exercise implementations and eight weeks after the patients have been under the new regime, to assess whether there were any improvements regarding the parameters that can lead to an improved quality of life and the possible prevention of CDs.

2. Materials and Methods

2.1. Study Design

The study was a non-randomized intervention study which involved a multidisciplinary team including a physiotherapist, a dietitian, a physiologist, psychologists, audiologists, and biomedical laboratory scientists in order to implement the AGA@4life program. The program had the purpose of evaluating the effect of a new tailored exercise program and a new dietary regime in the biochemical profile of NO metabolites, and ET-1 in senior population. This program consisted of six phases: data gathering, resorting to a diagnostic evaluation of the participants; discussion between the multidisciplinary team of each case; definition of the intervention plan; implementation of said plan with the help of the participants, family members, and caregivers; monitoring of the response to the plan; and finally, revising the intervention according to the outcome. The study included two evaluations of the different groups at different time points, one in the beginning of the study and one eight weeks after the implementation.

2.2. Population and Sample

There were 34 participants enrolled in the program, who were recruited from a day care center in Coimbra district, Portugal, and the average age of the participants was of 80.2 ± 10.6 years old. There were both female and male participants and all were physically autonomous with no prior cerebrovascular or neurological disorders [22].

A baseline diagnostic evaluation was performed on all participants (T0) which included the gathering of biochemical determinations such as NO metabolites and ET-1. For the evaluation, 10 mL of peripheral blood was collected by venous puncture for serum and plasma K₃-EDTA tubes, early in the morning, and participants fasted overnight. A Mini Nutritional Assessment (MNA) questionnaire, validated in Portugal, was used to assess nutritional risk; a Food Frequency Questionnaire, also validated in Portugal, established food intake and frequency history; while a 24 h assessment determined the food intake during that period. Anthropometric data (AD) were collected, including height, weight, body mass index, waist/hip ratio, and circumferences of the waist, arm, and leg, using a non-stretchable tape measure. Bioelectrical impedance analysis was employed to determine the percentages of fat and lean mass. Brachial blood pressure, with the participant in sitting position, with the arm supported at the level of the heart, free of any constrictive effect of clothing, and after a 5 min resting period, were evaluated in three consecutive measurements.

At T0, using the MNA and AD, the nutritional needs were estimated, and an individualized diet plan was created, considering the clinical history, anamnesis, and biochemical parameters of each participant. The food plan was then explained and provided to each day center user. This intervention included auditing meal preparations, and, based on the findings, training was provided to those responsible for daily meal preparation. The training covered topics such as healthy eating and the specific nutritional needs of the participants. Recommendations were also given to correct the main dietary errors observed. A diet manual was developed, offering a variety of diet types, each programmed and categorized, with indications of forbidden and permitted foods and dishes based on the nutritional needs of the different center users. They were all then submitted to a new individually made dietary plan that contemplated all of the needs of each individual.

The group was subdivided into two groups according to their willingness to integrate the exercise program. The first group was the control group (CG) where 7 of the 17 participants did not follow an exercise regime; the intervention group (IG) was composed of the remaining 10 participants to whom were attributed the new exercise plan.

After 8 weeks (T1), only 17 participants had concluded the program, with the dropouts being justified by hospital internments and exchange of day center, in which all 17 (11 female and 6 male) underwent a new evaluation. The biochemical evaluation consisted of the evaluation of the same parameters to determine whether or not there had been a change in the results [22].

2.3. Enzymatic Assays

To determine the NO metabolite concentration, a Total Nitric Oxide and Nitrate/Nitrite kit (R&D Systems, Minneapolis, MN, USA) was used, which determines NO metabolite concentrations based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The reaction is followed by colorimetric detection of nitrite as an azo dye product of the Griess Reaction. This is based on the two-step diazotization reaction in which acidified NO₂⁻ produces a nitrosating agent, reacting with sulfanilic acid and, consequently, producing the diazonium ion that is coupled to N-ethylenediamine to form the chromophoric azo-derivative which absorbs light at 540–570 nm [23].

2.4. Slot Blot

ET-1 was determined using the antibody clone (1:1000) 2E1 (R&D Systems, Abinddon, UK) by slot blot, which is a variation of the standard enzyme immunoassay; this technique was performed as described in Caseiro et al. [24]. The results are captured in film and the images are treated in the Image Lab program (Bio-Rad, Hercules, CA, USA). The results are expressed in arbitrary units (AU).

2.5. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6.01 software. For this prospective study (two time points), considering the endothelin levels in serum and an effect size d ≈ 1.4, a sample of 16 participants was estimated and given a test power of 0.85 (1 − β), a confidence level of 95%, and a predicted sampling error of 5% (α < 0.05). After careful evaluation of the type of study, the statistic study that fits with all the variables was a two-way ANOVA, followed by multiple comparisons by post hoc Tukey’s test (alpha = 0.05). The effect size was estimated, based on Cohen’s standardized value.

2.6. Ethics

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Polytechnic Institute of Coimbra (approval 8/2018). The anonymity and confidentiality of the collected data were guaranteed, and all participants signed an informed consent form prior to the study. There is no conflict of interest to be declared.

3. Results

3.1. Blood Pressure

The evaluation of systolic blood pressure levels showed a tendency of a decrease in T1, in the intervention group, but without statistical significance (Figure 1).

3.2. NO Metabolite Levels

When evaluating the NO metabolite levels in both groups, we can see that there is a lot of variability in the results between the participants; therefore, we see no statistically significant changes in the results (Figure 2).

3.3. ET-1 Levels

After evaluating the different groups at both timepoints, we can see a statistically significant decrease in the ET-1 levels between T0 and T1 in the CG (p < 0.001) (Figure 2) going from a mean of 8.45 (SD = 1.98) in T0 to a mean of 5.23 (SD = 0.80) in T1. There was also a statistically significant decrease in the IG also between T0 and T1 (p = 0.04) (Figure 2) going from a mean of 5.73 (SD = 1.11) in T0 to a mean of 4.13 (SD = 1.01) in T1. When compared to the CG, the IG tends to have lower ET-1 levels even though it is not statistically significant (p = 0.31) (Figure 3). The effect size was estimated, based on Cohen’s standardized value, with a value of 1.51 for GC and 1.06 for CG.

4. Discussion

The biological, psychological, and social context of the senior is very specific, so aging and the physiological processes associated with it are determinant factors of the effectiveness of a program which aims to promote a healthy lifestyle for an individual [25]. As the elder population is a fragile and vulnerable group associated with multiple chronic conditions, or comorbidity and disability, the finding of a program to promote their health needs to be carefully evaluated [26]. Considering the specific traits of this target population, it seems clear that any plan or action to confront it must rely on an individualized, multidimensional, and interdisciplinary approach, which is the basis of the AGA@4life program [22].

NO is a potent vasodilator and is extremely important in the control of the normal vascular functions of the endothelium [27]. It is synthesized in vascular endothelial cells from the L-arginine amino acid by the enzyme endothelial nitric oxide synthase (eNOS). This enzyme is constitutively expressed and is, therefore, always present in endothelial cells. However, its activity is tightly controlled by intracellular calcium levels (Ca²⁺) [28]. Endothelium-dependent vasodilators such as acetylcholine act by increasing intracellular calcium, therefore increasing eNOS activity causing NO release. The NO produced diffuses to the smooth muscle layer and stimulates the enzyme soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP), thus causing vasorelaxation [29]. It has been studied that NO has a significant role in the cardiovascular system, specifically in hypertension [30,31]. One thing that can help with hypertension is the improvement in the NO bioavailability which, according to some research, physical exercise can enhance, improving the endothelial function [32]. Even though it was expected that, after the new program, there would be an increase in the NO metabolite concentrations, that did not happen. One possible justification for the decrease in NO metabolite concentration in both groups after the implementation of the new regime is that this change in the participants’ normal routine could have triggered an increase in oxidative stress, especially in the IG that also had a new workout regime, which may have led to NO consumption, forming peroxynitrite.

In healthy individuals, ET-1 and NO metabolite production are balanced to maintain a normal vascular tone but, sometimes, some aberrant ET-1-mediated effects can lead to the development of cardiac diseases. ET-1 is a hormone responsible for the maintenance of the vascular tone. ETᴀ receptors are expressed on vascular smooth muscle cells and ETᴃ receptors on both endothelial and vascular smooth muscle cells. ET-1 is produced by endothelial cells (ECs), which then diffuses through the basal side in the vascular wall and binds to both ETᴀ and ETᴃ receptors on smooth muscle cells, which leads to a vasoconstriction of the vessels [21,33,34]. It is therefore important to also evaluate the ET-1 values to determine if the levels of these parameters are balanced or not. As expected, there was a decrease in ET-1 levels between T0 and T1 in both groups [35,36], which agrees with the previous studies where it was shown that a regular exercise regime reduces the values of ET-1 in the plasma [37]. We were expecting a higher decrease in the IG group when compared to the CG, but, since ET-1 is also highly influenced by oxidative stress, it is not unusual that there was a smaller decrease in those values since there was a possible raise in oxidative stress due to the physical exercise [38].

As this is a senior population, the adjustment to a new routine takes a longer period of time, and, by changing their dietary regime, the adaptation can be a bit difficult. The same goes for the workout regime since the senior population tends to be more sedentary than a younger population, so starting a workout regime after a while of being less active can put a strain on the body while it is not fully adjusted. With this in mind, a better approach in a future study would be to extend the program duration so the participants could fully adjust to the program and the benefits could really be evident, as well as increasing the number of participants. This expansion would not only be good in terms of increasing the sample size in order to consolidate the results but would also be beneficial to the population under study by having them live a healthier life and reap the benefits that come with that. With an extension of the program, it would also be interesting to understand the differences between male and female participants since the results could also be influenced by genders. It would also be interesting to consider another control group in which no changes in lifestyle would be applied so we could understand the true effect of the new diet program. With this in mind, it is still safe to say that, looking at these as well as the previously published results [22], they are very promising and the program really has a positive impact on the lives of the participants, not only when it comes to CD markers such as NO metabolites and ET-1, as well as triglycerides and both high- and low-density lipoprotein cholesterol (c-HDL and c-LDL, respectively), but also in several different aspects, making them healthier overall.

5. Conclusions

The AGA@4life program, a multidisciplinary intervention program for seniors, has several benefits when it comes to the overall health of the participants. In the senior group, an improvement in their health is also an improvement in other factors such as autonomy, independence, mobility, and social adaptation. As CDs are the focal point of this work, we can say that, in the studied cardiovascular markers, namely, the NO metabolites and ET-1, we can see an improvement, especially in the ET-1 levels, between the two timepoints of the study, potentially improving cardiac function as well as possibly decreasing the risk of developing said diseases. Overall, we conclude that both a healthy and diverse diet as well as a personalized exercise program have a great impact on the overall health of the participants, especially the diet program. This program is another proof that living a healthy lifestyle with a good diet and a regular workout regime plays an important part in preventing and treating a lot of different health problems no matter the age.