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Diminished Prolinemia in Chronic Chagasic Patients: A New Clue for Disease Pathology?

Trypanosoma cruzi, the etiological agent of Chagas disease, is dependent on proline for a variety of processes, such as energy metabolism, host cell invasion, differentiation, and resistance to osmotic, metabolic, and oxidative stress. On this basis, we investigated a possible relationship between prolinemia and severity of T. cruzi infection in chronic patients, as reported here. The study population consisted of 112 subjects, separated into 83 chronically T. cruzi-infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). Chagasic patients were separated into three groups: chronic asymptomatic, mild/moderate, and severe chronic chagasic cardiomyopathy (CCC) subjects. We observed a significant decrease of 11.7% in prolinemia in chagasic patients when compared to controls. Further analysis within the three groups of chagasic patients also revealed a statistically significant decrease of prolinemia in severe CCC patients compared to controls, showing a relative difference of 13.6% in proline concentrations. These data point to the possibility that collagen—which participates in the healing process of cardiac tissue—and proline metabolism in the myocardium could constitute new factors affecting the evolution of Chagas disease.


Introduction
Chagas disease is a chronic and systemic infection caused by the hemoflagellate protozoan Trypanosoma cruzi that is usually transmitted to humans by triatomine insects. The disease is endemic in the Americas, with approximately 6-7 million people chronically infected and approximately 10,000 deaths per year [1,2]. Most chronically infected individuals present a chronic asymptomatic (CA) form of the disease without demonstrable pathology, which is characterized by the lack of clinical signs and symptoms of the chronic form of the disease. The CA form can evolve with a variable degree of organ involvement, mostly cardiomyopathy and gastrointestinal disorders. Chronic chagasic cardiomyopathy (CCC) is characterized by diffuse myocarditis with inflammatory infiltrate foci, cardiac fiber damage, and prominent fibrosis. This chronic phase of the disease is also characterized by a low but persistent parasite load, which may be responsible for chronic inflammatory reactivity at the myocardial level. Depending on its clinical severity, CCC can be classified as mild, moderate, or severe [2]. Moreover, factors accounting for such diverse disease outcomes involve a complex series of interactions between the host and parasite biology. In this regard, l-proline was shown to be essential in several aspects of T. cruzi biology, such as the development of its intracellular stage [3] Molecules 2019, 24, 3167 2 of 6 and resistance to different stress conditions [4,5]. As reported in our previous studies, l-proline is also involved in energy supply and parasite survival in different environments [5,6]. Increased prolinemia (hyperprolinemia) was discovered as the cause of a metabolic syndrome in humans more than half a century ago, [7] and was classified as being type I or II depending on which enzyme is responsible for the deficiency (proline dehydrogenase causes type I hyperprolinemia, while P5C dehydrogenase causes type II hyperprolinemia) (reviewed by [8]). In addition, since hyperprolinemia started to be diagnosed, it was observed that values of prolinemia considered as physiological may vary in the range from 0.13 to 0.63 mM [9]. On this basis, we hypothesized a possible relationship between prolinemia and the severity of the T. cruzi infection. Accordingly, we have assessed the circulating levels of proline in sera from a series of chronic chagasic subjects lacking symptoms or coursing the infection with different degrees of severity of CCC.

Results
To compare proline contents among samples from chronic chagasic patients and uninfected individuals, participants were separated into four groups. Two of them consisted of T. cruzi-infected patients with mild/moderate severity (M/M, n = 26) and severe (Sev, n = 30) CCC, a third group was composed of asymptomatic infected subjects (CA, n = 27), and the fourth group consisted of healthy individuals (control group-Co, n = 29). Remarkably, among all the clinical parameters measured in those patients, glycemia was the only one that was increased when the Sev group was compared to the Co group (major group characteristics are shown in Table 1). Subjects from the CA group had normal electrocardiograms (ECGs), chest X-rays, and routine laboratory test results. The M/M group was composed of individuals with mild or moderate cardiac compromise and no congestive heart failure, but pathological ECG tracings such as complete or incomplete right bundle branch block or ventricular arrhythmia and a chest X-ray cardiothoracic ratio of <0.55. The Sev group consisted of patients presenting congestive heart failure, pathological ECG tracings, and a chest X-ray cardiothoracic ratio of >0.55 with normal CPK levels. Noteworthily, glycemia was slightly enhanced in these patients. Samples from the control group had a mean prolinemia value of 0.454 ± 0.014 mmol/L (means ± SE, n = 29), whereas the overall group of chagasic patients showed values of 0.401 ± 0.01 mmol/L (means ± SE, n = 83). Therefore, prolinemia was reduced by 11.7% in infected patients, which was statistically significant when compared to controls (p = 0.0054) (for individual values of prolinemia, see Table S1). Considering each group individually, differences reached the level of statistical significance when the prolinemia values from the Co group were compared with those of Sev patients, which presented a 13.6% decrease in proline concentrations (0.392 ± 0.018 mmol/L, p < 0.05, Figure 1). Further analysis within the groups of chagasic patients revealed no significant differences among them (for descriptive statistics of the prolinemia data, see Table S2).

Discussion
The variability and severity of the symptoms of Chagas disease depend on many factors, such as the parasite strain [10], parasite load, infection route [2], host immunity [11], and number of reinfections [12]. In addition, the parasite persistence at specific sites of the infected host induces a local immune response, which is responsible for tissue damage, especially of myocardial cells [11]. CCC is characterized by inflammation, fibrosis, myocytolysis, vasculitis, and parasitic persistence, leading to different degrees of clinical manifestations, i.e., mild, moderate, or severe [2]. Our findings point to a possible association between prolinemia and disease pathology during T. cruzi infection. Considering that the results were different from our initial hypothesis, we reinforce the suggestion that the diminished prolinemia in Sev subjects can be related to the clinical evolution of Chagas disease. As previously mentioned, the reported serum proline concentration in healthy individuals compromise and no congestive heart failure, but pathological ECG tracings such as complete or incomplete right bundle branch block or ventricular arrhythmia and a chest X-ray cardiothoracic ratio of <0.55. The Sev group consisted of patients presenting congestive heart failure, pathological ECG tracings, and a chest X-ray cardiothoracic ratio of >0.55 with normal CPK levels. Noteworthily, glycemia was slightly enhanced in these patients. Samples from the control group had a mean prolinemia value of 0.454 ± 0.014 mmol/L (means ± SE, n = 29), whereas the overall group of chagasic patients showed values of 0.401 ± 0.01 mmol/L (means ± SE, n = 83). Therefore, prolinemia was reduced by 11.7% in infected patients, which was statistically significant when compared to controls (p = 0.0054) (for individual values of prolinemia, see Table S1). Considering each group individually, differences reached the level of statistical significance when the prolinemia values from the Co group were compared with those of Sev patients, which presented a 13.6% decrease in proline concentrations (0.392 ± 0.018 mmol/L, p < 0.05, Figure 1).
Further analysis within the groups of chagasic patients revealed no significant differences among them (for descriptive statistics of the prolinemia data, see Table S2).

Discussion
The variability and severity of the symptoms of Chagas disease depend on many factors, such as the parasite strain [10], parasite load, infection route [2], host immunity [11], and number of reinfections [12]. In addition, the parasite persistence at specific sites of the infected host induces a local immune response, which is responsible for tissue damage, especially of myocardial cells [11]. CCC is characterized by inflammation, fibrosis, myocytolysis, vasculitis, and parasitic persistence, leading to different degrees of clinical manifestations, i.e., mild, moderate, or severe [2]. Our findings point to a possible association between prolinemia and disease pathology during T. cruzi infection. Considering that the results were different from our initial hypothesis, we reinforce the suggestion that the diminished prolinemia in Sev subjects can be related to the clinical evolution of Chagas disease. As previously mentioned, the reported serum proline concentration in healthy individuals is within the range of 0.13 and 0.63 mmol/L [9]. We also observed a slight increase in glycemia in the Sev group with respect to uninfected controls. It has been previously shown that proline can be a precursor for gluconeogenesis [13] and that lactate is an allosteric regulator of proline oxidase (the first enzyme in the proline oxidation pathway). On this basis, it was suggested that lactate may influence the hepatic availability of proline, which can be used for glucose biosynthesis, resulting in increased glycemia [14]. Further investigations are necessary to confirm the existence of a link between these two metabolic parameters. The relationship between prolinemia and some types of disease also has been studied in cancer research. Decrease of serum proline has been related to renal cell carcinoma [15], oral cancer [16], colorectal cancer [17], and esophageal cancer [9]. Some of these studies suggest that the lower level of serum proline could be an indication of overutilization of amino acids in tumor tissue [9].
Prolidase (E.C.3.4.13.9) is a cytosolic exopeptidase that is widely distributed in humans and animals, which splits imidodipeptides (originated from the degradation of procollagen, collagen, and proteins) into free proline or hydroxyproline within the cytoplasm [18]. In addition to its primary biological function in the collagen degradation metabolism, prolidase also participates in the recycling of proline from dipeptides to resynthetize collagen [19]. Thus, plasma prolidase activity might be a possible indicator of collagen catabolism, as already shown in chronic liver disease [18]. Furthermore, Myara et al. [19] suggest that plasma prolidase activity might be high in the early stage of fibrosis and might subsequently drop in an advanced fibrosis stage. This way, the disturbance of the collagen metabolism could interfere with the free proline concentration. This idea is consistent with previous findings showing that fibroblasts obtained from prolidase-deficient subjects had higher collagen degradation rates and lower proline levels when compared to control cells [20]. Moreover, it was recently suggested that a decrease in prolidase activity is an indicator of advanced fibrosis [21].
Regarding Chagas disease, it is known that severe CCC patients usually have myocardial fibrosis, which is defined also by the progressive accumulation of fibrillar extracellular matrix (ECM) in this tissue. These changes occur as a repair mechanism in response to chronic cardiac damage caused by several factors, including an intense inflammatory response (reviewed by [2]). Considering this background, we propose the following: (i) Severe CCC patients have diminished prolinemia because proline is the main substrate for the synthesis of collagen destined for the reparative process of cardiac tissue. Thus, the extension of myocardial fibrosis would be inversely related to the serum proline amount. (ii) Plasma prolidase activity might decrease in advanced fibrosis compared to the early stage of fibrosis, leading to a decrease in free proline in the tissue fluids. This phenomenon probably stems from decreased collagen turnover, which in turn is secondary to decreased functional heart tissue [22]. In summary, our results point to collagen and proline metabolism in the myocardium as being new factors to be investigated to gain better insight into the physiopathology of Chagas disease.

Study Design and Participants
The study population consisted of 112 subjects, separated into 83 chronically T. cruzi-infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). None of these patients were under specific treatment (i.e., benznidazole or nifurtimox), and none had concomitant pathological disorders. Exclusion criteria comprised neuroendocrine disturbances, metabolic diseases (such as diabetes), immunological diseases, and treatment with hormones or immunomodulators. Control subjects were seronegative to T. cruzi-specific tests. The diagnosis was based on at least two positive serological findings (either by ELISA, hemagglutination, or immunofluorescence), together with clinical symptoms, chest X-rays, and 12-lead resting electrocardiograms (ECGs). Routine laboratory, glycemia, and creatine phosphokinase (CPK) parameters were also assayed.