ALS is a disease characterized by irreversible neuronal necrosis. The overall prognosis of the disease is poor. At present, there is no method for early diagnosis and effective treatment. Previous animal experiments have confirmed that biomarkers may be used as an indicator for early diagnosis and prognosis of ALS. In this study, we explored the difference between the serum indicators of ALS patients and the control group, hoping to find indicators that may prompt the diagnosis and prognosis of ALS, so as to provide a basis for further experiments.
4.1. Creatinine can assess the functional status and muscle strength of the patients, predict the disease progression rate and prompt prognosis.
The blood creatinine is converted from creatine. The creatinine level is a direct reflection of the creatine pool, which reflects the mitochondrial function of muscles [12]. A Japanese study showed that patients with ALS had lower serum creatinine levels compared with healthy controls. In addition, the ALS function rating scale and the rate of decrease in forced vital capacity (FVC) are negatively correlated with serum creatinine levels [13]. ALSFRS-R and FVC are effective indicators for evaluating limb function and respiratory function in patients with ALS, respectively. The results of Van [14] showed that there is a strong correlation between muscle strength and serum creatinine level. In this study, we observed a smaller variation of serum creatinine decline rate in patients than that of ALSFRS-R, suggesting that the rate of creatinine decline is a more stable predictor of ALS disease progression than ALSFRS-R, and it is more effective in patients with limb symptoms. The results of a large study in Huaxi Hospital showed that the serum creatinine of ALS patients was significantly lower than that of the healthy control group after adjusting for BMI and age, and that patients with low serum creatinine levels were more likely to develop severe motor disorders and low BMI values. But they thought that there was no significant difference in serum creatinine level among ALS patients with different disease sites, and there is no relationship between the serum creatinine level and the survival time of ALS patients[15]. Chio et al. [16] suggested that lower creatinine levels was closely associated with poorer clinical function at diagnosis (clinical function is evaluated by ALSFRS-R score and FVC). The study showed that serum creatinine is related to survival, and the predictive sensitivity of serum creatinine to mortality is similar to FVC, ALSFRS-R score and age. Patin et al [17] suggested that the creatinine level of patients with limb disease was lower than that of patients with medulla oblongata disease. This difference still exists after excluding confounding factors such as gender and weight. The above studies all support blood creatinine as an accurate measure of muscle quality, and can evaluate limb function, disease progression rate and prognosis. In this study, the creatinine level of the ALS group were significantly lower than that of the healthy control group. The results of multivariate analysis showed that: creatinine level is an independent risk factor for ALSFRS-R and DPR. Within a certain range, higher creatinine level indicate better limb function and slower disease progression. Serum creatinine is a cheap and easy-to-obtain biomarker with good reproducibility, so it can be used extensively and repeatedly in clinical practice to evaluate the limb function and prognosis of patients with ALS. Creatinine can also be used in animal experiments to evaluate the limb function of mice, and in the progress of drugs in the drug experimental development, creatinine can be used to evaluate the effect of drugs to help develop effective treatment.
4.2 Higher blood lipids indicate better limb function and slower disease progression.
Studies have shown that ALS patients have a higher probability of dyslipidemia compared with the general population. Two studies from the United Kingdom and Germany found that the prevalence of hypercholesterolemia and hypertriglyceridemia in ALS patients was significantly increased [18–19]. Some researchers believe that hyperlipidemia is a protective factor for ALS. After muscle denervation, the body's efforts to regenerate nerves lead to an increase in energy demand, and the increased demand is supplemented by lipids, which can also explain the decrease of blood lipid levels in patients with advanced disease progression. And the total body fat decreased [18]. The study by Dupuis et al. [20] concluded that blood total cholesterol, low-density lipoprotein (LDL), and ratio of low-density lipoprotein to high-density lipoprotein (LDL/HDL) in ALS patients were significantly higher than those in the control group. The study on ALS population by Okamoto et al. [21] also showed that a large amount of high-fat diet can reduce the symptoms of ALS. The study by Zinman et al. [22] showed that compared with patients without lipid-lowering drugs, ALS patients with lipid-lowering drugs had a significant increase in the rate of decrease in ALSFRS-R score and forced vital capacity. In this study, the blood total cholesterol and triglyceride levels of the ALS group were significantly higher than those of the control group. However, only the total cholesterol level was significantly correlated with the disease progression rate after correlation analysis. As the total cholesterol level increased, the disease progression rate decreased. The presence of dyslipidemia in ALS patients is increasingly recognized. The exact cause of hyperlipidemia in ALS is still uncertain: it may be a compensatory mechanism, lipids are the preferred energy source for skeletal muscle, and the regeneration of denervated muscles may lead to increased energy demand in ALS. It is currently believed that higher blood lipids may be related to better limb function and slower progression. In ALS patients, lipid-lowering drugs should be used carefully, especially to avoid statins, which may aggravate muscle damage.
4.3 Uric acid reduced the damage to neurons caused by oxidative stress.
Uric acid (UA) is the main end product of human purine metabolism. As a natural antioxidant, it can remove superoxide and reduce neuronal death caused by oxidative stress. The study of Bakshi et al. showed that urate significantly reduced the death of motor neuron cells induced by hydrogen peroxide in an astrocyte-dependent manner [23]. A study in Japan showed that the serum UA level of ALS patients was significantly higher than that of the healthy control group, and that the serum UA level was positively correlated with BMI, ALSFRS-R score and creatinine level, and negatively correlated with DPR [24]. Paganoni et al. [25] reported that when creatinine and BMI were controlled, serum uric acid level was an important predictor of ALS survival rate and dysfunction. A prospective study observed that people with higher serum uric acid levels than their age- and sex-matched control group have a slightly lower risk of ALS in the future [26]. The meta-analysis results of Zhang et al. [27] showed that the serum UA level of ALS patients was significantly lower than that of the control group, and the serum UA level of ALS patients was negatively correlated with the risk of death. Nicholson et al. [28] increased the blood UA level by intravenous infusion of inosine in 25 ALS patients. The results showed that the biomarkers of oxidative were significantly reduced, and ALSFRS-R was improved compared to before treatment. Although the results of various studies are partially contradictory, most of the current experimental results believe that uric acid as an antioxidant can reduce the damage of oxidative stress to neurons, which is a protective factor for ALS. In this study, the level of uric acid in the ALS group was significantly lower than that in the control group, but there was no correlation with ALSFRS-R and DPR in the multivariate correlation analysis. Further clinical trials are needed to determine whether uric acid has a protective effect in the onset of ALS, increasing blood uric acid levels, and whether it can reduce neuronal loss, improve patient limb function and extend life span.
4.4 Creatine kinase, homocysteine, and cystatin C are future research directions
Creatine kinase (CK) is an enzyme that catalyzes the reversible conversion of creatine and provides energy for muscle contraction. Linkhart et al. [29] believe that the degeneration of innervated motor neurons leads to increased muscle cell membrane permeability and CK release. CK levels may be related to the severity of lower motor neuron loss and muscle atrophy in patients with ALS. Tai et al. [30] found that the serum CK level was associated with the persistent low F wave of the median nerve in the EMG, which confirmed that the CK level in ALS patients was related to the severity of lower motor nerve loss. Their other study showed that the survival time of patients with elevated CK levels was significantly longer than that of patients with normal CK levels [31], and every 10 U/L increase in CK reduced the risk of death by 54.3% (95% CI 0.221–0.947). However, the results of Gibson et al. [32] showed that low CK levels (< 200U/L) were associated with longer survival (P < 0.01) after excluding factors such as onset location, age, and gender. In this study, 31 of 103 ALS patients had elevated serum CK, accounting for 30%, all of which were mild to moderately elevated, and none exceeded 1000 U/L. The CK level of the ALS group was significantly higher than that of the control group. However, no correlation was observed between the CK level and the patient's physical function score and disease progression rate. It may be related to the small sample size and the lack of long-term follow-up of patients. At present, the mechanism of the increase in CK levels in ALS patients is not yet clear. There are different research results on the significance of serum CK levels in ALS, and larger sample studies and more animal experiments are needed to further clarify.
Homocysteine (HCY) is a sulfhydryl-containing amino acid produced by the demethylation of methionine. HCY has a variety of potential neuropathological mechanisms of action that may be related to ALS, including free radical damage and cytoplasmic calcium overload, mitochondrial dysfunction, activation of the apoptotic pathway, and excitotoxic amino acid-mediated damage [33–35]. The study of Zoccolella et al. [36] showed that compared with healthy controls, the serum HCY level of ALS patients was significantly increased, and higher HCY was associated with faster disease progression. Zhang et al. [37] divided SOD1 transgenic mice into early group (30d), pre-symptomatic group (60d), symptom group (90d) and terminal group (120d). They found that the blood HCY level of each stage of the disease is different And HCY was significantly increased in the symptom group, suggesting that elevated HCY may aggravate neuronal damage. Their subsequent study showed that folic acid alone or in combination with folic acid and vitamin B12 can significantly reduce the blood HCY level of SOD1 transgenic mice, delay the occurrence of ALS, prolong the survival time of mice, and increase the anterior horn of the spinal cord of mice. The number of motor neurons is significant [38]. A Japanese small sample double-blind clinical trial of 24 ALS patients found that short-term (4 weeks) high-dose (0.5mg/d) administration of vitamin B12 effectively improved the compound motor action potential on the electromyogram of ALS patients, suggesting that the loss of spinal cord motor neurons is improved [39]. In our study, the serum HCY level of the ALS group was significantly higher than that of the control group, and there was no significant correlation between ALSFSR and DPR and HCY levels in the correlation analysis. If you want to get a more precise conclusion, it is more necessary to conduct a cohort study on patients in the ALS group. Use "vitamin B12 and folic acid" to reduce the patient's HCY, and perform longitudinal comparisons to observe whether lowering the blood HCY level can improve the patient's muscles. Features. We believe that HCY may be one of the factors that cause neuronal damage in ALS. Even if ALS cannot be cured, the treatment of reducing HCY still has a positive meaning and it is an important part of the treatment of ALS.
Cystatin C is an endogenous cysteine protease inhibitor, which plays an important role in the repair of the nervous system after injury and disease. The neuroprotective activity of CysC includes inducing autophagy in nerve cells, promoting the degradation of misfolded or unfolded proteins, and preventing the accumulation of abnormal mutant proteins [40]. Currently, most studies on CysC in ALS focus on cerebrospinal fluid, and there are few blood-based studies. Studies have shown that compared with healthy controls, CysC levels are significantly reduced in the cerebrospinal fluid of patients with ALS [41]. A longitudinal study analysis showed that the level of CysC in the cerebrospinal fluid of fast-progressors was lower, while those with slower progress showed an increasing trend of CysC levels, and higher cerebrospinal fluid CysC levels suggested slower progress [42]. In a study, purified and recombinant CysC from Escherichia coli was continuously pumped into the lateral ventricle of SOD1 mice, and CysC was labeled with serum lectin. The results showed that CysC was successfully delivered to the lumbar spinal cord and mainly distributed in the lumbar spinal cord after 1 week of administration. In the anterior horn neurons, continuous infusion of CysC prolonged the survival time of SOD1 mice by an average of 9.6 days (6.48% of the total survival time) [43]. Tetsuka [44]et al.believed that CysC levels in cerebrospinal fluid and blood were independently regulated, and there was no difference in blood CysC in ALS patients with varying degrees of severity. Based on numerous studies, it can be considered that CysC in cerebrospinal fluid is a reliable protective agent, but CysC in serum has not shown the potential as a diagnostic marker or a prognostic marker. In this study, the blood CysC level of the ALS group was significantly higher than that of the control group, and the correlation analysis had nothing to do with the functional score and disease progression rate. Further researches were needed to determine whether there is a correlation between cerebrospinal fluid and CysC in the blood, and whether increasing the level of CysC in the blood can improve the function, delay the progression, and improve the prognosis of ALS patients.