Loss of appetite in patients with amyotrophic lateral sclerosis is associated with weight loss and anxiety/depression

Weight loss is common in patients with Amyotrophic lateral sclerosis (ALS), and associated with disease progression. Loss of appetite has been shown to be a contributor to weight loss in patients with amyotrophic lateral sclerosis (ALS). However, the reason of loss of appetite is not clear. The Council on Nutrition appetite questionnaire (CNAQ) and the simplified nutritional appetite questionnaire (SNAQ) are short and simple appetite assessment tools, which were using in ALS patients. In our study, the CNAQ and SNAQ were translated into Chinese, and their reliability and validity were tested. The Chinese version of the CNAQ (CNAQ-C) presented more appropriate reliability and validity than the SNAQ. Among the 94 ALS patients, 50 patients (53.2%) had loss of appetite, and we found that anxiety and/or depression contributed to the loss of appetite in the ALS patients. We reconfirmed that loss of appetite was associated with greater weight loss but not with clinical features of ALS. The loss of appetite caused by emotional problems in ALS patients should be taken seriously, and early intervention should be implemented to reduce weight loss.

Comparison between ALS patients with intact appetite and loss of appetite. Among the 94 ALS patients, 44 patients (46.8%) had intact appetite, and 50 patients (53.2%) had loss of appetite. Age (p = 0.958), sex (p = 0.671) and the percentage of smoking and alcohol abuse (p = 0.893; p = 0.190) were no significant differences between ALS patients with intact appetite and loss of appetite. The weight and BMI at screening of ALS patients with intact appetite was higher than that of ALS patients with loss of appetite (p = 0.015; p = 0.005). There was no difference in other clinical measurements, including ALSFRS-R scores (p = 0.227), bulbar subscore (p = 0.690), respiratory subscore (p = 0.712), duration of disease (p = 0.943), diagnostic delay (p = 0.570) and the percentage of bulbar onset (p = 0.254) between the two groups (Table 3).
Among the 94 ALS patients, 52% of patients had anxiety and/or depression. In our results, the HADS-A and HADS-D scores of the ALS patients with intact appetite were lower than those of the ALS patients with loss of appetite (p = 0.01; p < 0.001) ( Table 3), indicating that the emotional state of patients in the normal appetite group was better than that in the loss of appetite group. Moreover, according to the HADS-A and HADS-D scores, we separated the patients into normal, borderline and abnormal groups; the CNAQ-C scores of the borderline and www.nature.com/scientificreports/ abnormal patients were lower than those of the normal patients (p < 0.001) ( Fig. 1), indicating that anxiety and depression might be potential factors associated with the loss of appetite.
To assess the cognitive and behavioural status of ALS patients, the ECAS was used in our research. Eightyone of 94 participants completed the ECAS, and the scores of 15 patients less than 81. For these patients, 40 patients (49.4%) had intact appetite, and 41 patients (50.6%) had loss of appetite. Surprisingly, there were no significant differences in the ECAS scores (p = 0.688), the ALS-special function and ALS-non-special function scores (p = 0.839; p = 0.917) between the two groups (Supplementary Table S1).

Discussion
We translated and back-translated the CNAQ and its simplified version the SNAQ into Chinese (the CNAQ-C and SNAQ-C, respectively), and reliability and validity were tested. We found that the Chinese version of the CNAQ had sufficient reliability and validity. We showed that approximately half of the ALS patients had loss of appetite, which was partly due to anxiety and depression. Our data supported the notion that weight loss was associated with the loss of appetite and reconfirm the view that loss of appetite is a potential contributor to weight loss in ALS patients. Epidemiological evidence shows that ALS patients begin to lose weight preceding motor symptoms by several years 19 . Furthermore, patients with greater weight loss tend to have worse ALSFRS-R scores and shorter survival times 4,20 . There are many factors that contribute to weight loss with ALS, and loss of appetite has proven to be a potential contributor 8,10,11 . The CNAQ and SNAQ were developed to predict weight loss in communitydwelling adults and long-term care residents and have been used to detect the appetite of ALS patients 8,10,11 . In our research, we developed the Chinese versions of the CNAQ and SNAQ. The Cronbach's α coefficients of the CNAQ-C and SNAQ-C were 0.667 and 0.662, respectively, which were slightly lower than 0.7. In the study by Wilson et al. 12 , Cronbach's α coefficients for the CNAQ and SNAQ were 0.470 and 0.510 in the long-term care group and 0.72 and 0.70 in the community-dwelling group, respectively. This may be due to the different subjects assessed in the two studies. In addition, the CNAQ-C presented good fit in the confirmatory factor analysis as assessed by multiple indices, while some indices with the SNAQ-C were not up to standard, indicating that the CNAQ-C is more suitable for our research.
Approximately half of the participants (53.2%) demonstrated severe loss of appetite in our study, which was similar to the percentage in Holm et al. (47%) 12 and was higher than that in Ngo et al. (29%) 10  Likewise, our study also found no significant differences in bulbar and respiratory scores between ALS patients with intact appetite vs. ALS patients with loss of appetite. However, the respiratory scores of ALSFRS-R provided limited information about the actual respiratory status. In future research, more sensitive tests, as forced vital capacity, nocturnal oximetry, supine spirometry, etc. should be conducted.
Anxiety and depression are common in ALS patients. Nimish J Thakore et al. reported that 33% of patients had at least moderate depression 21 . The prevalence of anxiety in ALS patients ranged from 0 to 30% 22 . Patients with depression showed significant heterogeneity in appetite, with approximately 48% of adults with depression showing depression-related loss of appetite and approximately 35% showing depression-related increases in appetite 23 . Anxiety and depression have been confirmed to be associated with changes in appetite in a variety of diseases 24,25 . In our study, anxiety and depression showed significant differences between ALS patients with intact appetite vs. ALS patients with loss of appetite, and the CNAQ-C score in the anxiety and depression group Table 4. Correlations between CNAQ scores and baseline disease characteristics. ALS amyotrophic lateral sclerosis, ALSFRS-R ALS Functional Rating Scale-Revised, BMI body mass index, HADS-A Hospital Anxiety and Depression Scale-Axiety, HADS-D Hospital Anxiety and Depression Scale-Depression. *p < 0.05; **p < 0.01; ***p < 0.001. www.nature.com/scientificreports/ was significantly lower than that in the normal group, which had not been mentioned in previous studies, as no professional psychological scales were used. Multivariate stepwise regression analysis showed that HADS-D was independently associated with CNAQ-C, indicating that depression might be an independent correlative factor for the loss of appetite in ALS patients. Although there is not enough evidence to show that ALS patients with loss of appetite could be improved by psychological regulation, consider emotional problems and give appropriate treatment should be reasonable. The view that the motivation to eat depends on cognitive regulation of reward processes is gaining support, with control of appetite thought to involve cognitive processes such as learning, attention and memory 26 . These cognitive processes may be engaged during various aspects of appetite control, including before, during and between meals 27 . Among a sample of Chinese ALS patients, 35.71% showed cognitive impairment, and 27.38% showed behavioural abnormalities 28 . In our study, however, no association was found between ECAS and CNAQ scores. which might be due to few patients had an ECAS score below 81 in our research (only 15 patients). Interestingly, previous study has shown that, compared with ALS patients, patients with behavioural variant frontotemporal dementia (bvFTD) were more likely to show an increase in appetite 29 , which was verified that cognitive impairment had an impact on the appetite of ALS patients. Therefore, we believe that ECAS should be used as a screening indicator to evaluate cognitive and behavioral changes of ALS patients in subsequent appetite studies.
In our study, 52% of patients had anxiety and/or depression. Interestingly, we found that after removing the patients with anxiety and/or depression, still had sixteen patients (approximately 33%) with loss of appetite, which shows that emotion dysfunction was not the only reason for the loss of appetite in ALS patients. Unexpectedly, similar to previous results, the weight and BMI at the screening of ALS patients with intact appetite were higher than those of ALS patients with loss of appetite, and there were no differences in other demographic data and clinical measurements between the two groups (Supplementary Table S3). Likewise, no significant differences were found in the mean ECAS, ALS-special function and ALS-non-special function scores between ALS patients with intact appetite and ALS patients with loss of appetite (Supplementary Table S4). Similarly, the CNAQ score has no correlation with other clinical indicators except the weight loss and BMI decrease since diagnosis (Supplementary Table S5). The disruption of central energy homeostasis may play an important role. The hypothalamus, the main central organ that regulates appetite, has been shown to atrophy in ALS patients, even in the premorbid stage, and the degree of atrophy was correlated with BMI 30 . Moreover, changes in appetite-regulating AgRP (increased) and POMC (decreased) neurons have also been demonstrated in ALS mouse model 31 . The volume of multiple brain regions involved in appetite regulation has also been reported to be reduced in ALS patients 32 . In future studies, the effect of central organ alterations on the appetite of ALS patients should be considered.
Our research has the following limitations: (1) We did not collect CNAQ-C information in a control group to compare with the appetite of ALS patients, and we lacked an assessment of energy intake. (2) This study did not have follow-up data. Although the study confirmed the relationship between weight loss and appetite loss, it did not confirm that the CNAQ-C can predict weight loss in the next 6 months. In addition to confirming the functional predictions of the CNAQ, it is necessary to clarify whether there is a relationship between the patient's appetite and disease progression and survival. In conclusion, we identified a new risk factor for loss of appetite in ALS patients, and we emphasized the possibility of other mechanisms. Identification of the mechanisms underlying for the loss of appetite in ALS patients, such as changes in the central nervous system, hormones, and mood, can lead to implementing targeted treatments or using appetite-enhancing drugs based on the mechanisms to prevent patients from losing weight.

Methods
Participants. Patients with possible, probable, or definite ALS were included in the study, and all patients met the revised E1 Escorial criteria for ALS from Peking University Third Hospital. The exclusion criteria included digestive system diseases, thyroid diseases, diabetes and other wasting diseases; gastrostomy or nasal feeding and other patients unable to eat; and/or a history of other neurological disorders. A total of 94 patients were enrolled. All participants underwent clinical testing and epidemiological investigations at screening, such as age, weight, BMI, ALS Functional Rating Scale-Revised (ALSFRS-R) scores, duration of disease, diagnosis delay. Smoking was defined as having smoked at least 1 cigarette a day for at least 1 year or more than 360 cigarettes in total for a year. Drinking was defined as having an average of 2 or more drinks per week for more than 1 year. This study was approved by the Research Ethics Committee of Peking University Third Hospital. Written informed consent was provided by all participants. All methods were performed in accordance with relevant guidelines and regulations.
Translation of the CNAQ and SNAQ. The CNAQ is an 8-item questionnaire about participants' appetite, hunger frequency, satiety, taste, eating behaviour, and mood. The score for each question is 1-5 points, the total score range is 8-40 points, and scores ≤ 28 points are considered indicative of loss of appetite 12 . The SNAQ is a simplified version of the questionnaire composed of 4 questions from the CNAQ. Similarly, each item is rated on a 5-point scale, the total score range is 4-20 points, and scores ≤ 14 points are considered indicative of loss of appetite 12 . We obtained permission from the original article's author John E Morley to develop the Chinese versions of the CNAQ and SNAQ. Using standardized translation and back-translation methods, the Chinese version (CNAQ-C) was developed by a nutrition researcher, a physician, and a neurobiology researcher, repeated translation and back-translation occurred until equivalent English expressions were attained, and they were approved by the original author (Supplementary Table S6). All participants needed to recall their feelings or behaviours from the past month to answer each question.