Dynapenia is highly prevalent in older patients with advanced idiopathic pulmonary fibrosis

Body composition and muscle strength are emerging aspects in idiopathic pulmonary fibrosis (IPF) clinical assessment. We aimed to study the relationships of handgrip strength (HGS) with anthropometric variables, body composition, and disease staging, and to evaluate the prevalence of dynapenia in 102 clinically stable IPF patients (70 M; mean age: 69.4 years). Fat-free mass (FFM), skeletal muscle (SM) were estimated with bioimpedance analysis. HGS was measured with a digital handle dynamometer for both dominant and non-dominant body sides. Dynapenia was identified according to six recognized criteria sets. Mean body mass index (BMI) was 28.2 ± 4.7 kg/m2, with a prevalence of overweight (BMI > 25 and < 30 kg/m2) and obesity (BMI ≥ 30 kg/m2) of 35% and 37%, respectively. FFM and SM were greater in males, whereas percentage body fat was higher in women. HGS was higher and declined with age slightly more rapidly in men, showing a stronger correlation with FFM and SM. Dynapenia prevalence ranged from 20.6 to 56.9%, depending on the criteria used, and was more frequent in older patients and advanced disease. Dynapenia is highly prevalent in IPF. HGS is a promising proxy marker of muscle function to be used in clinical evaluation and follow-up programs.


Results
The main demographic and clinical characteristics of the study population are reported in Table 1. There were no differences between male and female patients in terms of age. Males were heavier (+ 16.7%) and taller (+ 9.7%), while the mean BMI was slightly higher in females (+ 3.2%). Prevalence of underweight patients (BMI < 21 kg/ m 2 ) was 7.8%, while overweight (BMI > 25 and < 30 kg/m 2 ) and obese patients (BMI ≥ 30 kg/m 2 ) were 35.3% and 37.3% of the whole study group, respectively. The 70% and 5% of patients were former or current smokers, respectively, with a median value of 25 packs/year smoked (IR . Systemic arterial hypertension (54%), gastro-esophageal reflux (27%), and type II diabetes (22%) were the most prevalent comorbidities in the group as a whole with no gender differences. Ischemic cardiovascular disease was more prevalent in males (23 vs. 6%, p < 0.05) and thyroid disease in females (21 vs. 3%, p < 0.01), whereas pulmonary hypertension (14% of cases) was only slightly more frequent in males (16 vs. 9%; p = NS). The median disease duration was 14 months (IR 6-32), with most patients under anti-fibrotic treatment with either pirfenidone (n = 44) or nintedanib (n = 49). As shown in Table 1, most patients were in GAP stages II and III (mild to moderate disease) and in TORVAN stage III, in both cases with no significant gender differences.
Lung function parameters are reported for all patients and by gender in Table 2. A mild to moderate restrictive ventilatory pattern with a similar single-breath diffusing lung capacity of the carbon monoxide (DLCO sb ) deficit was detected with no clinically significant differences between males and females. The six-minute walk distance was available in 92 patients with an overall median distance walked of 363 m (IR 233-528). Male patients (63) walked more meters than females (29) (Table 3), while percentage body fat was higher in females (+ 41.6%). All these variables did not significantly vary by the GAP or TORVAN stage.  Handgrip strength.  Table 4. Maximum-HGS, as well as D-HGS and ND-HGS, directly correlated with weight and BMI and more strongly with FFM and SM. As far as pulmonary function is concerned, maximum-HGS correlated with total lung capacity (r = 0.355, p < 0.001), but not with the forced vital capacity (FVC), the DLCO sb and the 6 min walk test (6MWT) distance. Also, neither the dyspnea nor the muscular fatigue, estimated with the Borg scale at the beginning and at the end of the 6MWT, correlated with the HGS (data not shown).
When compared to the other patients with IPF, maximum-HGS, expressed as mean ± standard error (SE), was lower in underweight patients (24.8 ± 3.0 kg vs. 29.5 ± 1.0 kg, p < 0.05) and also in those with low FFMI (23.5 ± 3.0 kg vs. 29.7 ± 1.0 kg, p < 0.05). Furthermore, after adjusting for gender, maximum-HGS (mean ± SE) was higher in patients in the GAP I stage compared to the other ones (30.8 ± 1.1 vs. 27.9 ± 1.0 kg, p < 0.05), and the same was true when TORVAN I was compared to TORVAN II-III stages (32.3 ± 1.4 vs. 28.2 ± 0.8 kg, p < 0.05); these differences disappeared when data were adjused also for age. On the contrary, no differences came out by comparing patients treated with nintedanib to those taking pirfenidone.
A higher prevalence of dynapenia was observed in the patients with low BMI only using the TESSIER criteria. Those with low FFMI only used the TESSIER or LAURETANI criteria (data not shown). On the contrary, in all cases (except ALLEY-2) dynapenia was much more prevalent in IPF patients aged ≥ 75 years (Fig. 2 4.0%) criteria. Finally, no differences emerged in the prevalence of dynapenia by comparing patients treated with nintedanib to those taking pirfenidone, regardless of the small percentage of cases with mild GI side-effects (14%). Also, no patients reported > 5% weight loss in the three months preceding the study visit.

Discussion
To the best of our knowledge this is the first report addressing from a clinical point of view the prevalence distribution of dynapenia (i.e., low muscle strength) in IPF patients. It systematically evaluated HGS in this target population, showing lower values in underweight patients or with low FFM. Dynapenia was highly prevalent, even more in patients aged > 75 years. However, the estimated proportion varied depending on the criteria set used. IPF is a chronic, progressive interstitial lung disease of unknown etiology 3,18 , characterized by a gradual decline of respiratory function up to death. Management and therapy of patients are still complex and not fully defined 27,28 . Disease progression may widely vary among patients: some rapidly deteriorate soon after diagnosis, others maintain a stable condition for years, and still, others show rapid decline following a period of stability 29 .
Alterations of nutritional status and/or body composition, which may negatively affect skeletal muscles, have seldom been described among the systemic manifestations of IPF 30 . To date, BMI has been evaluated in IPF patients with preliminary and sometimes contradictory results 7 ; the findings of this study are consistent with previous data from European countries and the United States indicating that the mean value for BMI in patients with IPF was > 25 kg/m 27 . In effect, most of the patients studied were overweight or obese (no one with a BMI > 40 kg/m 2 ), while the underweight ones were only 7.8% of the whole study sample. On the contrary, the estimated prevalence of low FFMI, which was around 8%, was less than reported in a recent paper by Jouneau et al. 11 ; those authors did not give information about the bioimpedance analysis (BIA) equation used to predict FFM, whereas in the present study a well-validated equation for COPD patients was chosen 31 .
As partly surprising and additional information, there was no relationship between FFM and SM with clinical staging systems such as GAP or TORVAN.
Although it is well known that muscle function is related to morbidity, mortality, hospitalization, quality of life, etc. in the elderly and various diseases, i.e., COPD 16,17 , only a few studies have evaluated HGS in IPF patients [18][19][20] , showing significant correlations of HGS with age (inverse) and weight (direct) 18 . Besides, mean HGS value was found to be lower in more severe patients 19,20 . Very recently, low HGS has been observed on a preliminary basis (in comparison to predicted values) in a small group of patients with IPF 21 . Of note, HGS values largely varied between studies, while the association with body composition has never been taken into consideration, nor dynapenia has been diagnosed. Unfortunately, we do not have direct information of dynapenia in our local population. Indeed, it is interesting to compare our results on HGS with those given in the very relevant paper by Dodds et al. 32 regarding normative values for grip strength in the general population. Twentyeight per cent of male patients and 25% of female patients with IPF had HGS below the 10th percentiles (for age and gender) reported by Dodds et al. and 53% and 63% below the 25th percentiles, thus strongly suggesting low muscle strength. Interestingly, we have derived percentiles for HGS in our local population for adults aged 20-60 years that are very similar to the Dodds ones. In addition, the prevalence of dynapenia in IPF patients is similar to that we have observed in COPD (unpublished results), a chronic lung disease characterized by wellknown impairments in the musculoskeletal system.
The findings of our study make it possible to assess the relationships of HGS with various potential predictors. In line with that has been observed in the general population 32 and COPD patients 33 HGS showed an inverse correlation with age, as already reported 18 , a direct correlation with weight, FM, and (slightly stronger) with FFM and SM. These observations were confirmed by multiple regression analysis. Models including gender, age explained around 70% of the total variance, and weight or (as an alternative) gender, age and FFM; in other words, no increase in the prediction power was observed substituting FFM for weight. It is also significant to point out that HGS was lower in underweight IPF patients or those with low FFM or low SM. We have also appraised whether muscle strength could be affected by disease severity; quite surprisingly, in line with findings on body composition (see above), we found that HGS did not vary depending on GAP or TORVAN stages, with this finding further confirmed by the weak association found between HGS and lung function.
Dynapenia is defined as the loss of muscle strength associated with aging (or even nutrition-related diseases) not caused by neurological or muscular disorders 14 ; the reduction of muscle strength is not necessarily linked to or caused by a decrease in skeletal muscle mass 34 . From a clinical point of view, dynapenia (usually recognized as low HGS values) is associated with reduced ability to perform daily living activities, increased morbidity and mortality, and worse quality of life 15,16 . According to the recent EWGSOP-2 consensus 15 , dynapenia is the first criterion to consider for the diagnosis of sarcopenia. Considering chronic lung diseases, there are few reports on dynapenia in COPD patients 35,36 and none in patients with IPF. Facing this background, one of the main objectives of the present study was to evaluate the prevalence of dynapenia according to various cutoffs proposed by the literature; in all cases, a subject/patient is dynapenic if his/her HGS falls below a defined threshold value. The cutoffs have been derived in different ways: for example, the recent 2019 EWGSOP consensus 20 set threshold values based on data published by Dodds et al. 32 as mean HGS of young adults minus 2.5 standard deviations (SD), while Lauretani et al. 37 referred to the ability of HGS to discriminate elderly individuals with reduced mobility. Consequently, although there is a certain similarity, the selected cutoffs vary from 26 to 33.1 kg for men and from 16 to 21 kg for women (see "Methods" section).
At first glance, mean HGS values of patients with IPF appear to be relatively low; in effect, the prevalence of dynapenia was high, but indeed varied considerably according to the selected criteria both in male(from 20.0 to 51.4%) and female patients (from 25.0 to 68.8%), with a tendency towards higher values in the latter. There was a clear effect of age as dynapenia was significantly more often detected in patients aged 75 years or more; for instance, the prevalence increased from 15.9 to 46.4% with the EWGSOP-2 criteria and from 48.6 to 78.6% with the TESSIER criteria. These findings are not unexpected since a decline of HGS with aging is a well-known phenomenon 15,38 ,while the cutoffs are the same for all subjects. On the other hand, it is worth noting that in most instances, the prevalence of dynapenia did not differ in underweight patients or those with low FFM. Finally, www.nature.com/scientificreports/ the comparison concerning the severity of disease, dynapenia was more prevalent in the more advanced GAP or TORVAN stages. Overall, as recently stated 21 , and based on what is known for COPD, the impairment of muscle function in patients with IPF is likely to be multifactorial and due to muscle disuse, hypoxaemia, inflammation, oxidative stress, etc. Dynapenia seems to be a relevant clinical problem especially in older patients. Limitations of our study include the single-center setting along with the cross-sectional and retrospective design. The study population included patients intercepted at different time points of the disease, and most of them were already on anti-fibrotic therapy. Unfortunately, the sudden occurrence of the COVID-19 pandemic prevented any effort of recruitment and follow-up of new cases. Longitudinal studies will help understand the impact of dynapenia and altered body composition on disease presentation and progression/survival, maybe in combination with the quality of life evaluation. An additional limitation of our study is the lack of data on physical activities. Certainly, future efforts will help address this issue as well. Any inter-relation with anti-fibrotic therapies also merits to be further investigated. With respect to this topic, although we found no associations of either body composition, HGS, and dynapenia with the currently used drugs, nintedanib and pirfenidone, to the best of our knowledge, the present study represents the first attempt in exploring this area. Recently, Jouneau et al. have shown, by retrospectively evaluating the pooled data of the two Inpulsis trials, that the rate of FVC decline was greater in patients with low baseline BMI and those with > 5% weight loss over 52 weeks. Interestingly, nintedanib reduced lung function decline independently of BMI and had a greater effect in patients with > 5% weight loss 8 . In addition, Suzuki et al. have shown in two retrospective case series that IPF patients under antifibrotic therapy had skeletal muscle loss and that sarcopenia was a prognostic factor of reduced survival 39 .
In conclusion, the present study systematically evaluated HGS in IPF, showing lower values in underweight patients or with low FFM. HGS varied, at least in part, depending on weight, body composition, and especially age. The age-related decline in muscle strength persisted even after correction for body composition. From a clinical point of view, dynapenia (i.e., low muscle strength) was found to be highly prevalent, even more in patients aged > 75 years, although the estimated proportion varied depending on the criteria set used. Overall, the point prevalence estimate of dynapenia tended to be higher in GAP stages II-III than GAP stage I and in TORVAN stages II-III than TORVAN stage I. Finally, no differences emerged in HGS or prevalence of dynapenia by comparing patients treated with nintedanib to those taking pirfenidone.
Based on the evidence gathered, the measurement of HGS appears to be a promising proxy index of muscle function of IPF patients. Further studies are needed to better understand how this variable can best be used in the disease's clinical management and identify that part of patients that requires special attention in terms of applied nutrition and motor rehabilitation. Endpoints of particular interest would be, along with survival, also physical activity, quality of life, disease progression and anti-fibrotic therapies.

Methods
Study population. The study population was composed of 102 (70 males and 32 females) consecutive patients with clinically stable IPF referring to our outpatient clinic from February 1st, 2019, to March 1st, 2020. They included nine treatment naïve IPF patients and 93 patients already on anti-fibrotic therapy with pirfenidone (n = 44) or nintedanib (n = 49). No patients were previously treated or were taking inhaled or systemic corticosteroids. IPF diagnosis was revised in all patients according to the 2018 official diagnostic criteria 3 . Coexistance of paraseptal/centrolobular emphysema was detected in a small percentage of cases (8%). Exclusion criteria were related to the diagnosis of respiratory diseases other than IPF, acute exacerbation in the four weeks before the study visit, and lung cancer coexistence. Additional exclusion criteria were related to osteo-muscular and neurological disorders or presence of pace-maker/implantable cardioverter defibrillator. Hospitalization in the three months preceding the study visit was reported in no cases. The study was retrospectively conducted in accordance with the amended Declaration of Helsinki and was approved by the local Ethics committee (Federico II University. Registration number: 120/2020). Enrolled patients gave their written informed consent to participate in the study, and all data of interest were anonymously collected into a dedicated database. Spirometry, lung volumes measurement, and determination of the hemoglobin (Hb)-adjusted DLCO sb were performed using a computer-assisted spirometer (Quark PFT 2008 Suite Version Cosmed Ltd, Rome, Italy) according to international standards [40][41][42] . The 6MWT was performed by trained hospital staff according to guidelines in those patients with a basal peripheral oxygen saturation > 90% in ambient air 43 . The Borg scale was used to assess the level of dyspnea and muscular fatigue at the beginning and at the end of the test. The GAP (stages I, II and III) was recorded as previously described. Accordingly, the TORVAN (stages I, II, III and IV) score, which is a disease complexity index accounting for comorbidities that impact on IPF prognosis, was evaluated as well 44,45 . Anthropometry and body composition. Body weight and stature were measured to the nearest 0.1 kg and 0.5 cm with a mechanical column scale and a stadiometer, respectively (SECA 711 and SECA 220, Hamburg, Germany); BMI was then calculated as body weight (in kg) divided by stature squared (in m 2 ). Body composition was assessed by BIA. Measurements were carried out with a Human Im-Touch analyzer (© DS Medica S.r.l., Milan, Italy) in standardized conditions (i.e., ambient temperature 23-25 °C, fast > 4 h, empty bladder, supine position for at least 10 min before testing). After cleaning the skin surface, the patients were asked to lie down with their legs and arms slightly abducted to avoid any contact between the limbs and the trunk. A standard tetra-polar technique was used: measuring electrodes were placed on wrist and ankle dorsal surface, while injecting electrodes were on the dorsal surface of the hand and the foot, respectively. Impedance (Z) was measured for both the D and ND body side with an electrical current of 800 mA. Concerning BIA-based estimates of body composition, FFM and SM were determined using BIA equations proposed by Rutten et al. 31 for patients with COPD and by Jenssen et al. 46  www.nature.com/scientificreports/ Muscle strength. HGS was measured by the same operator following standard procedures using a Dynex dynamometer (MD systems Inc. Ohio USA) to assess the isometric strength of the D and ND arm. Patients were instructed to stand upright with their shoulder adducted and neutrally rotated, elbow fully extended, and forearm and wrist neutrally positioned during the study. A pre-test was done, allowing the patient to become familiar with the instrument. Three measurements were performed for each hand, one minute apart, alternating between the dominant and non-dominant sides 47,48 . Maximum values were derived for each arm (D-HGS and ND-HGS), and maximum HGS was finally derived as the highest value of six attempts.
Dynapenia. Dynapenia was defined according to six different criteria sets, five of which derived from consensus documents on the diagnosis of sarcopenia: the FRIED 49 and LAURETANI 37 criteria were proposed by the 2010 EWGSOP consensus 50 , the ALLEY-1 and ALLEY-2 criteria 38 by the FNIH Sarcopenia Project 51 , and the EWGSOP-2 criteria by the corresponding 2019 consensus 15 , based on data by Dodds et al. 32 . In addition, the TESSIER criteria 52 , which were recently established in a large sample of Canadian population, were also selected. Whatever the criteria used, a subject/patient is dynapenic if his/her HGS falls below a defined threshold value.
The cutoff values for various criteria sets were as follows. Statistical analysis. Results are expressed as mean ± SD or SE, median value (and interquartile range = IR), and frequency, where appropriate. Statistical significance was pre-determined as p < 0.05. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS Inc, Chicago, IL, USA) version 24. ANOVA with the post-hoc Tukey test and the general linear model (GLM) were used to compare groups and assess the effects of factors on a single dependent variable (in the case, even after adjusting for covariates). Partial correlation and multiple regression analysis were utilized to identify predictors of a given dependent variable.
Ethics approval and consent for publication. The Ethics committee approved the study of the Federico II University of Naples, Italy (Registration number: 120/2020; 22/05/2020). All patients signed informed consent.

Data availability
Data are available upon reasonable request.