Administration of edaravone and riluzole can slow the progression of the disease at the early stages of disease; thus, rapid diagnosis is vital for ALS. On the other hand, symptoms of ALS are not highly specific and can mimic the symptoms of other neurological disorders; therefore, developing prognostic and diagnostic biomarkers for ALS is immediately needed for accurate and rapid diagnosis of the disease 11 12.
Weight loss (WL) has been categorized as a clinical feature and predictive value for ALS and observed 56% to 62% in all cases 13, associated with morbidity and mortality respiratory and functional loss in ALS. Thus, bodyweight management enables the improvement of the prognosis of the patients 14. Our data showed that SOD1G93A rats had significantly lost their weights than SOD1WT rats in groups C and D, indicating WL most probably started before the first symptoms showed up (Figure 1). WL can result from different factors, including hypermetabolic state, food intake, metabolic or hormonal levels, and physical activity status 15. ROW is used to evaluate the toxicity of a substance and possible tissue damage in the organism 16. Our data showed ROW of all SOD1G93A rat tissues significantly elevated compared to the SOD1WT in groups C and D except spleen (Table 1). Thus, we can propose that ALS disease progression in the early and late stages can induce tissue impair in the liver, kidney, lung, brain, testis, heart, and spinal cord tissues (Table 1).
Biomarkers in the blood and serum can be used for early and accurate diagnosis of the disease since riluzole and edaravone, the only two approved drugs, are only effective at the early stages of ALS 2. Since ALS causes a hypermetabolic state leading to malnutrition, weight, and muscle loss in patients, various biomarkers can be used to evaluate the prognosis and progression of the ALS disease 17. We have found that albumin, TP, and Ca levels slightly increased in the SOD1G93A rats compared to the SOD1WT ones in the groups B, C, and D groups meaning increases have started at the pre-symptomatic stages as well (Table 2). An increase in serum albumin and TP correlates with the body's inflammatory status, and albumin levels positively correlate with ALS progression and survival. Additionally, albumin has antioxidant effects on metabolism 18,19 On the other hand, Ca homeostasis plays a vital role in ALS pathogenesis and disease progression, especially in SOD1-linked ALS; for instance, Ca buffering and metabolism are impaired in the motor neurons, mitochondria, and CNS even at the pre-symptomatic stages according to the various studies. Increased Ca levels in ALS patients have been found, a however possible mechanism behind Ca homeostasis should be further investigated 20,21.
Creatinine has been characterized as an independent prognostic biomarker for ALS and elevated in the earlier phases of the disease as muscle mass destruction and decreased at the late stages of the disease as a hallmark of malnutrition. Additionally, reduced creatinine levels are associated with reduced survival time and poor prognosis in male and female patients 22. Creatinine levels increased in the SOD1G93A rats of group 0 and decreased compared to the SOD1WT rays by aging and disease progression in groups A, B, C, and D (Figure 2). That indicated that creatinine could be used as a biomarker at the pre-symptomatic stages of ALS. Cholesterol is being discussed as another possible biomarker for ALS since neuron death occurs during the disease progression resulting in elevated levels of cholesterol in the CSF 23.
Interestingly increased oxidative stress in the SOD1G93A mice results in the formation of oxidized-cholesterol products existing in the serum of the rats 24. Elevated levels of cholesterol have been observed in ALS patients. Increased cholesterol levels are correlated with risk for ALS and poor survival, resulting from oxidized cholesterol products and their harmful effects on the metabolic pathways 25. We have found that cholesterol levels in the mutated rats increased compared to the wild-type ones and this increase was higher in groups C and D (Figure 2).
ALKP is a liver enzyme responsible for breaking down proteins and transporting phosphate groups, and lower levels of ALKP are associated with malnutrition in humans 26. We have shown that ALKP levels started to decrease in the SOD1G93A rats compared to the wild-type ones in group B, and the decrease reached significant levels in groups C and D (Fig. 2). There are no studies that showed the impact of the ALKP as a biomarker in the pre-symptomatic and symptomatic stages of ALS; the first time in the literature, we hypotheses that ALKP could be used as a biomarker (Fig. 2). On the other hand, bilirubin is associated with the inflammatory biomarker in inflammation-linked diseases such as ALS, multiple sclerosis (MS), Alzheimer's disease, and diabetes 27. Since we found that TBIL levels dramatically increased in the mutated rats compared to the wild-type ones at the symptomatic stages, that can be used as a possible biomarker for the ALS diagnosis (Fig. 2). Phosphate is vital for cell signaling, energy, and mineral metabolism 28. We have found that serum phosphate levels increased in the SOD1G93A rats of groups 0, A, B, and C compared to the SOD1WT rats; however, they significantly decreased in group D (Fig. 2). No published data evaluate the impact of phosphate as a potential biomarker; thus, further studies can be conducted. On the other hand, BUN levels increased in the mutated rats compared to the wild type ones according to our data (Figure 2); however, the importance of the BUN as a potential biomarker has not been studied until now, and thus a study can be conducted with the human cohort in the future.
ALS is a hypermetabolic disease; however, the possible mechanisms contributing to the homeostasis of the energy metabolism is not known. Besides serum biochemistry biomarkers, we evaluated serum hormone and glucose levels in the SOD1WT and SOD1G93A rats (Fig. 2). Glucose metabolism is considered one of the possible targeting approaches to cure people with ALS since it is vital for OXPHOS, synthesis of neurotransmitters, and oxidative stress metabolism 29. Glucagon induces catabolism of the glycogen storages of the body resulting in the glucose release into the blood. On the other hand, c-peptide can be measured instead of insulin since it is more stable and has a longer half-life.
Additionally, it is produced in equal amounts with insulin, and both are responsible for lowering blood glucose 30. Interestingly we have found that serum glucagon levels increased in the SOD1G93A rats compared to the SOD1WT rats in groups B, C, and D (Fig. 3). C-peptide levels increased in the mutated rats compared to wild-type animals in groups 0, A, and B; however, they decreased in groups C and D (Figure 3). Despite increased glucagon and decreased c-peptide levels, glucose levels in the SOD1G93A rats increased in groups A and B. Still, they significantly reduced in groups C and D (Figıre 3).
On the other hand, PYY is a gut hormone involving energy expenditure, appetite, and fat oxidation. Increased levels of PYY in the blood result in increased energy expenditure, elevated levels of fat oxidation, and decreased appetite 31. However, we have found that PYY levels decreased in the mutated rats compared to the wild-type ones in the pre-symptomatic stages but increased in groups C and D (Figuıre 3). MCP-1 expression is induced by insulin and plays a role in diabetes because of impaired glucose metabolism. Increased levels of MCP-1 are associated with impairment in adipocyte function decreased glucose uptake into the cells 32. We have found that MCP-1 levels increased in the SOD1G93A rats compared to SOD1WT rats in all groups, where MCP-1 levels decreased in the wild-type rats by aging (Fig. 3). All these data indicate that increased catabolic activity and impairment in glucose metabolism have started at the pre-symptomatic stages and worsened at the late stages of ALS. Thus, PYY, MCP-1, glucagon, c-peptide, and glucose levels can be evaluated as possible biomarkers for ALS prognosis and progression as one of the hallmarks of impaired glucose metabolism 29.
We have also evaluated blood biomarkers as potential biomarkers in the SOD1WT and SOD1G93A rats that have become a hot topic in a couple of years. RBC and MCH levels were suggested as possible early biomarkers of early stages of ALS by an in-silico study 33. Additionally, decreased levels of RBC count have been reported as an early biomarker in ALS 34. Additionally, increased HGB levels are associated with prognosis and decreased survival rate in ALS 35. We have found that RBC, HGB, and MCH levels decreased in the SOD1G93A rats in groups 0, A, C, and D; however, they increased in group D (Table 3). We suggest that MCH, HGB, and RBC count can be considered possible biomarkers in ALS and give insight into disease progression.
Blood biomarkers indicating the impairment in the peripheral immune system and inflammation are vital for ALS prognosis and diagnosis since neuroinflammation contributes to ALS pathogenesis 36. Increased levels of monocyte, granulocyte, and neutrophil and decreased lymphocyte counts have previously been reported in ALS patients36–38. We found that monocyte and granulocyte counts have started to increase in the mutated rats in the pre-symptomatic (B) and continued in the symptomatic stages (C and D) (Figure 4). In groups B, C, and D, lymphocyte levels decreased in the SOD1G93A rats (Figure 4). Therefore, granulocyte, lymphocyte, and monocyte levels can be used as potential biomarkers in the ALS prognosis and progression, indicating the impaired inflammation and immune response, which is one of the hallmarks of the disease 1,36. In this study, we have reported that MCH, RDW-SD, MCV, RDW-CV, PCT, and HCT values significantly decreased in the SOD1G93A rats in comparison with SOD1WT rats at the early stage (C) and late-stage (D) of ALS first time in the literature (Fig. 4). We have found that WBC levels decreased in the mutated rats compared to the wild-type ones in all groups and PLT levels decreased in the mutated rats in the pre-symptomatic (B) and symptomatic groups (C and D) first time literature as well (Fig. 4). All the indicated parameters can be used as hallmarks of the impaired blood homeostasis and immune system since WBC, PLT, MCH, RDW-SD, HCT, MCV, PCT, and RDW-CV levels are impaired in the alteration of the indicated metabolisms 39. Furthermore, these parameters can be evaluated in the human cohort to validate the accuracy of our data.
Serum mineral and trace element levels are vital for the various metabolisms such as antioxidant homeostasis, blood metabolism, neuron integrity, and nutrition balance, for instance, Fe plays a crucial role in the energy, blood, and oxidative stress metabolisms and elevated levels of Fe indicate higher levels of oxidative stress in the organism. Additionally, elevated levels of iron can disturb Ca, Cu, and Zn levels in the body. We have found that Zn, Fe, Ca, Na, Mg, and K levels were impaired in the SOD1G93A rats compared to the SOD1WT rats that may indicate impaired oxidative stress metabolism, neuronal homeostasis, and mineral metabolism 2,10,40–44 (Fig. 5). Thus, these minerals and trace elements can be evaluated as potential biomarkers for ALS prognosis and progression. In conclusion, first time in the literature, we have investigated possible biomarkers in the blood and serum by eliminating the effects of aging and indicated promising biomarkers could be used in the ALS disease prognosis and progression.