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ORIGINAL ARTICLE
Ahead of print publication  

Elevated serum leptin levels are associated with low muscle strength and muscle quality in male patients undergoing chronic hemodialysis


1 Division of Nephrology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation; School of Medicine, Tzu Chi University, Hualien, Taiwan
2 Division of Nephrology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan
3 Division of Nephrology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation; School of Post-Baccalaureate Chinese Medicine,Tzu Chi University, Hualien, Taiwan

Date of Submission03-Feb-2020
Date of Acceptance08-Apr-2020
Date of Web Publication13-Jun-2020

Correspondence Address:
Yu-Li Lin,
Division of Nephrology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, 707, Section 3, Chung-Yang Road, Hualien
Taiwan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tcmj.tcmj_20_20

  Abstract 

Objectives: Low muscle strength and poor muscle quality are highly prevalent in patients with chronic hemodialysis (HD), which lead to an increased risk of poor clinical outcomes. Leptin dysregulation is common in HD patients. Given that leptin receptors are abundant in skeletal muscle, there may be a link between leptin and muscle strength. The cross-sectional study aimed to explore the correlation of serum leptin levels with muscle strength and muscle quality in patients with chronic HD. Materials and Methods: A total of 118 chronic HD patients were included in this study. Basic characteristics, handgrip strength, body composition were assessed, and blood samples for serum leptin levels and other biochemical test were obtained. We defined skeletal muscle index (SMI) as skeletal muscle mass/height2 (kg/m2) and muscle quality as handgrip strength divided by mid-arm muscle circumference (MAMC). Patients were classified into tertile groups, according to sex-specific leptin levels. Results: We observed that patients in the higher leptin tertile tend to have a higher body weight, body mass index (BMI), body fat mass, MAMC, and SMI, while the handgrip strength and muscle quality were significantly lower. Bodyweight (r = 0.30; P = 0.001), BMI (r = 0.45; P = 0.001), body fat mass (r = 0.57;P < 0.001), and SMI (r = 0.22; P = 0.018) were positively and handgrip strength (r = −0.27; P = 0.003) and muscle quality (r = −0.35;P < 0.001) were negatively correlated with serum leptin levels, respectively. After adjusting multiple confounding factors, logarithmically transformed serum leptin levels were independently associated with handgrip strength (β = −3.29, P = 0.005) and muscle quality (β = −0.14, P = 0.009). However, gender-stratified models showed the associations were observed only in male, but not in female. Conclusion: We concluded that higher serum leptin levels are associated with low handgrip strength and poor muscle quality in male patients on chronic HD. Further studies are needed to clarify the gender differences and to evaluate the casual relationship between circulating leptin levels and muscle strength.

Keywords: Hemodialysis, Leptin, Muscle quality, Muscle strength



How to cite this URL:
Hsu BG, Wang CH, Lai YH, Kuo CH, Lin YL. Elevated serum leptin levels are associated with low muscle strength and muscle quality in male patients undergoing chronic hemodialysis. Tzu Chi Med J [Epub ahead of print] [cited 2020 Nov 28]. Available from: https://www.tcmjmed.com/preprintarticle.asp?id=286592

Bang-Gee Hsu & Chih-Hsien Wang contributed equally to this work.



  Introduction Top


Protein energy-wasting, defined as decreased body stores of protein and energy fuels, is highly prevalent in patients with end-stage renal disease (ESRD)[1],[2], which leads to adverse outcomes, as well as sarcopenia, a progressive loss of skeletal muscle mass and strength[3]. As two major criteria of sarcopenia, however, skeletal muscle strength and mass have different clinical relevance in chronic hemodialysis (HD) patients. In recent observational studies, muscle strength was more closely associated with the risk of mortality than muscle mass did[4],[5]. Moreover, poor muscle quality, defined as the ratio of muscle strength to muscle mass, was also regarded as an important predictor for mortality in chronic HD patients[6]. Accordingly, to elucidate the potential mechanisms of muscle weakness and poor muscle quality in chronic HD patients is crucial.

Leptin, a 16-kDa hormone primarily secreted from adipose tissue, is well-established to regulate appetite and energy expenditure, glucose homeostasis, and insulin sensitivity[7]. Furthermore, leptin had been shown to trigger fatty acid oxidation in skeletal muscle[8],[9], and its receptors are abundant in skeletal muscle[10],[11]. These findings indicated that leptin signaling may play a key role in skeletal muscle homeostasis. Recently, high serum leptin levels were observed to be associated with impaired physical function in older adults, midlife women, and patients with knee osteoarthritis [12-14]. In patients with chronic kidney disease and ESRD, leptin is regarded as uremic toxin[15], which was not dialyzable by conventional HD. Thus, compared with healthy controls, serum leptin levels were markedly elevated in patients with ESRD[16]. Despite the high prevalence of both muscle weakness and leptin dysregulation in chronic HD patients, evidence regarding the association between elevated serum leptin levels and muscle weakness is limited.

Therefore, this cross-sectional study aimed to explore the correlation of serum leptin levels with both muscle strength and quality in chronic HD patients.


  Materials and Methods Top


Study participants

From January 2015 to December 2015, 160 patients who were older than 20 years and were maintained on HD using standard bicarbonate dialysate (FX class dialyzer, Fresenius Medical Care, Bad Homburg, Germany) thrice a week for more than 3 months at a medical center in Eastern Taiwan were evaluated as potential study participants. The Research Ethics Committee, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, approved the study, which was conducted under the tenets of the Helsinki Declaration (IRB104-84-B). Exclusion criteria included patients who refused to participate or who had amputated limbs, acute infection, active malignancy, and bed-ridden status. Finally, 118 patients were included in this study. The basic characteristics, HD duration, and medical history included diabetes mellitus, hypertension, hyperlipidemia, and coronary artery disease (CAD) were collected through chart review.

Anthropometric analysis

After measuring height and post-HD body weight, body mass index (BMI) was calculated as post-HD body weight (kg) divided by height squared (m). We measured triceps skinfold (TSF) and mid-arm circumference (MAC) at the level of the midpoint between acromion and olecranon from both arms. Mid-arm muscle circumference (MAMC) was calculated as MAC-π × TSF, and the average values of both arms were used for further analysis.

Total skeletal muscle mass and body fat were assessed in standing position before HD, using a portable whole-body bioelectrical impedance device (Tinita BC 706DB, Tanita Corporation, Tokyo, Japan). Skeletal muscle index (SMI) was defined as total skeletal muscle mass/height2 (kg/m2).

Muscle strength and quality

Handgrip strength was measured at the arm without vascular access, using a Jamar Plus Digital Hand Dynamometer (SI Instruments Pty Ltd., Hilton, Australia). The patients held the dynamometer in the hand to be tested, with the arm at right angles and elbow at the side of the body. Each measurement was performed with a rest of 1 min, and the average value was recorded. Muscle quality was then defined as handgrip strength (kg) divided by MAMC (cm)[17].

Biochemical investigations

About 5 mL blood sample was obtained before HD in each patient. After determining the blood cell counts (Sysmex SP-1000i, Sysmex American, Mundelein, IL, USA), the remaining blood sample was centrifuged for biochemical analyses. We measured serum levels of blood urea nitrogen, creatinine, albumin, total cholesterol, phosphorus, and C-reactive protein using an autoanalyzer (SiemensAdvia 1800, Siemens Healthcare GmbH, Henkestr, Germany). The fractional clearance index for urea (Kt/V) was calculated using a single-compartment urea kinetic model. Serum intact parathyroid hormone (PTH) levels were measured using enzyme-linked immunosorbent assays (ELISA; Diagnostic Systems Laboratories, Webster, Texas, USA), and serum leptin levels by a commercially available enzyme immunoassay (SPI-BIO, Montigny le Bretonneux, France).

Statistical analysis

Continuous variables are expressed as mean ± standard deviation or as median and interquartile range based on data distribution. Categorical variables are expressed as absolute (n) and relative frequency (%). The differences between males and females were compared using independent t-test or Mann–Whitney U-test for continuous variables and the Chi-square test or Fisher's exact test for categorical variables. Patients were then classified into three groups according to sex-specific leptin levels. These variables among groups were analyzed by one-way analysis of variance or the Cochran–Armitage test for trend. Clinical variables that correlated with serum leptin levels were evaluated through Pearson's correlation analysis. Serum leptin levels, HD duration, albumin, intact PTH, and C-reactive protein showed skewed distribution and were log-transformed before analysis. Finally, the association of serum leptin levels with handgrip strength and muscle quality were examined through univariate and multivariate linear regression. Statistical analysis was performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA). A P < 0.05 was considered statistically significant.


  Results Top


[Table 1] shows the clinical variables of the 118 HD patients. The mean age was 63.2 ± 13.2 years. Among them, 43 (36.4%) had diabetes mellitus, 56 (47.5%) had hypertension, 32 (27.1%) had hyperlipidemia, and 78 (66.1%) had CAD. Compared with males, female patients had longer HD duration, lower height, body weight, BMI, MAMC, SMI, handgrip strength, muscle quality, and serum creatinine levels, while body fat mass, total cholesterol, Kt/V and serum leptin levels were significantly higher.
Table 1: Clinical variables of the 118 hemodialysis patients

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Clinical characteristics of the study population stratified by sex-specific tertiles of serum leptin levels were exhibited in [Table 2]. Increasing tertiles of serum leptin levels were significantly associated with greater weight (P < 0.001), BMI (P < 0.001), body fat mass (P < 0.001), MAMC (P = 0.028), SMI (P = 0.001), and lower handgrip strength (P = 0.045) and muscle quality (P = 0.001).
Table 2: Clinical characteristics of the study population stratified by sex-specific tertiles of serum leptin levels

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The continuous variables correlated with logarithmically transformed serum leptin levels are shown in [Table 3]. The leptin had a positive correlation with body weight (r = 0.30; P = 0.001), BMI (r = 0.45; P = 0.001), body fat mass (r = 0.57; P < 0.001), and SMI (r = 0.22; P = 0.018), while had an inverse correlation with handgrip strength (r = −0.27; P = 0.003) and muscle quality (r = −0.35; P < 0.001).
Table 3: Correlation between serum leptin levels and clinical variables among 118 hemodialysis patients

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[Table 4] shows the unadjusted and adjusted linear regression analysis of serum leptin levels with handgrip strength and muscle quality. After fully adjustment of age, sex, HD duration, diabetes mellitus, hypertension, CAD, SMI, body fat mass, albumin, hemoglobin, and C-reactive protein, we demonstrated that logarithmically transformed serum leptin levels were negatively associated with both handgrip strength (β = −3.29, P = 0.005) and muscle quality (β = −0.14, P = 0.009). Sensitivity analyses were performed among 82 patients who had handgrip strength measurement in the dominant arm, which showed similar results [Supplementary Table 1].
Table 4: Multivariate linear regression analysis of serum leptin levels correlated with handgrip strength and muscle quality among 118 hemodialysis patients

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Given the significant differences of serum leptin levels and muscle function between males and females, the associations between serum leptin levels, handgrip strength, and muscle quality were further stratified by gender [Table 5]. The negative associations of serum leptin levels with handgrip strength (β = −5.47, P = 0.011 for males; β = −0.57, P = 0.642 for females) and muscle quality (β = −0.22, P = 0.021 for males; β = −0.04, P = 0.534 for males) were only observed in males, not in females.
Table 5: Multivariate linear regression analysis of serum leptin levels correlated with handgrip strength and muscle quality, stratified by gender

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  Discussion Top


In this cross-sectional study of chronic HD adults, higher-serum leptin concentration predicted handgrip strength weakness and poor muscle quality. These associations were independent of body fat mass, skeletal muscle mass, and serum C-reactive protein levels, which suggests the independent role of leptin on handgrip strength and muscle quality. However, these associations were only observed in males but not in females.

Several potential mechanisms explained the association between high leptin levels and muscle weakness in our chronic HD patients. Leptin was well-recognized as a proinflammatory adipokine [18-21], and its high serum levels in patients with ESRD were associated with chronic inflammation status, as well as impaired insulin sensitivity [22-24]. Both of the above conditions have detrimental effects on skeletal muscle homeostasis[25]. Besides, there were well-established links between hyperleptinemia and increased risk of cardio-metabolic diseases[26],[27], which has a negative impact on both muscle strength and quality. Despite this, the association between high leptin levels and poor muscle strength and quality remained significant after our full adjustment, included age, sex, HD duration, DM, HTN, CAD, SMI, body fat mass, Kt/V, albumin, hemoglobin, and C-reactive protein. This may implicate an independent role of leptin on skeletal muscle homeostasis.

Reduced skeletal muscle mass was observed in leptin-deficient Ob/Ob mice, while leptin administration showed beneficial effect on the growth of skeletal muscle mass[28]. Recently, leptin signaling was also shown to stimulate vascular endothelial growth factor A production by skeletal myocytes, which suggests its regulatory role in skeletal muscle angiogenesis[29]. Hyperleptinemia in ESRD patients may actually implicate leptin resistance, in which normal leptin signaling was blunted. This may explain our observation between high serum leptin levels and low muscle strength. Moreover, leptin resistance impairs fatty acid oxidation in muscle[9] and leads to increase in intramuscular fat infiltration, which contributed to the decrease in muscle quality[8],[30]. However, these intramuscular changes may not result in a reduction in either MAMC or total skeletal muscle mass volume.

Several observational studies showed a close link between serum leptin levels and physical function in different populations. In premenopausal mid-life women, high serum leptin levels were independently associated with poor physical performance, included stair climb, sit-to-stand, 2-pound lift times, and forward reach distance[13]. In the seniors-ENRICA cohort, which evaluated community-dwelling older men and women, higher leptin levels were associated with poor self-reported physical performance and lower extremity function, assessed by Short Physical Performance Battery[12]. In patients with knee osteoarthritis, serum leptin levels were inversely related to muscle strength and physical performance[14]. These observations were in line with our findings in chronic HD patients.

Interestingly, the negative associations between serum leptin levels and handgrip strength were only observed in males, but not in females in our study. This finding indicated the potentially different impacts of serum leptin levels on handgrip strength between males and females. The discrepancies may be attributed to gender differences in the leptin biological activity, as well as the differences in the expression of skeletal muscle leptin receptors, which is partly mediated by sex hormones[31],[32]. Nevertheless, further studies are needed to clarify this critical issue.

We acknowledged several limitations to our study. First, handgrip strength was measured at the arm without vascular access, not the dominant hand. However, our sensitivity analyses, which included the patients assessed in the dominant arm, showed similar trends. Second, we did not evaluate muscle strength of lower extremities and physical performance, such as 6-min walk test, repeated sit-to-stand, Short Physical Performance Battery test. Third, inflammatory cytokines other C-reactive protein, such as interleukin-6 and TNF-α, were not measured. Finally, this is a cross-sectional study, and the physiologic role of leptin on muscle strength, and quality could not be elucidated in the current study.


  Conclusion Top


We showed that higher serum leptin levels were associated with handgrip strength weakness and poor muscle quality in male patients undergoing chronic HD. The molecular mechanisms by which leptin exerts its effects in skeletal muscle homeostasis in chronic HD patients, as well as the causes of gender differences in the associations between leptin and muscle strength, should be further investigated.

Financial support and sponsorship

This study was supported by a grant from Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan (TCRD105-04).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kovesdy CP, Kopple JD, Kalantar-Zadeh K. Management of protein-energy wasting in non-dialysis-dependent chronic kidney disease: Reconciling low protein intake with nutritional therapy. Am J Clin Nutr 2013;97:1163-77.  Back to cited text no. 1
    
2.
Jadeja YP, Kher V. Protein energy wasting in chronic kidney disease: An update with focus on nutritional interventions to improve outcomes. Indian J Endocrinol Metab 2012;16:246-51.  Back to cited text no. 2
    
3.
Fouque D, Kalantar-Zadeh K, Kopple J, Cano N, Chauveau P, Cuppari L, et al. A proposed nomenclature and diagnostic criteria for protein-energy wasting in acute and chronic kidney disease. Kidney Int 2008;73:391-8.  Back to cited text no. 3
    
4.
Isoyama N, Qureshi AR, Avesani CM, Lindholm B, Bàràny P, Heimbürger O, et al. Comparative associations of muscle mass and muscle strength with mortality in dialysis patients. Clin J Am Soc Nephrol 2014;9:1720-8.  Back to cited text no. 4
    
5.
Kittiskulnam P, Chertow GM, Carrero JJ, Delgado C, Kaysen GA, Johansen KL. Clinical investigation: Sarcopenia and its individual criteria are associated, in part, with mortality among patients on hemodialysis. Kidney Int 2017;92:238-47.  Back to cited text no. 5
    
6.
Yoda M, Inaba M, Okuno S, Yoda K, Yamada S, Imanishi Y, et al. Poor muscle quality as a predictor of high mortality independent of diabetes in hemodialysis patients. Biomed Pharmacother 2012;66:266-70.  Back to cited text no. 6
    
7.
Mantzoros CS, Magkos F, Brinkoetter M, Sienkiewicz E, Dardeno TA, Kim SY, et al. Leptin in human physiology and pathophysiology. Am J Physiol Endocrinol Metab 2011;301:E567-84.  Back to cited text no. 7
    
8.
Dyck DJ, Heigenhauser GJ, Bruce CR. The role of adipokines as regulators of skeletal muscle fatty acid metabolism and insulin sensitivity. Acta physiologica (Oxford, England) 2006;186:5-16.  Back to cited text no. 8
    
9.
Minokoshi Y, Toda C, Okamoto S. Regulatory role of leptin in glucose and lipid metabolism in skeletal muscle. Indian J Endocrinol Metab 2012;16 (Suppl 3):S562-8.  Back to cited text no. 9
    
10.
Guerra B, Santana A, Fuentes T, Delgado-Guerra S, Cabrera-Socorro A, Dorado C, et al. Leptin receptors in human skeletal muscle. J Appl Physiol (1985) 2007;102:1786-92.  Back to cited text no. 10
    
11.
Hamrick MW, Shi X, Ding K, Isales CM. Leptin receptor expression in skeletal muscle declines with aging: A mechanism linking altered leptin signaling with frailty and sarcopenia. Calcified Tissue Int 2008;82:S229.  Back to cited text no. 11
    
12.
Lana A, Struijk E, Guallar-Castillón P, Martín-Moreno JM, Rodríguez Artalejo F, Lopez-Garcia E. Leptin concentration and risk of impaired physical function in older adults: The Seniors-ENRICA cohort. Age Ageing 2016;45:819-26.  Back to cited text no. 12
    
13.
Karvonen-Gutierrez CA, Zheng H, Mancuso P, Harlow SD. Higher leptin and adiponectin concentrations predict poorer performance-based physical functioning in midlife women: The Michigan study of women's health across the nation. J Gerontol A Biol Sci 2016;71:508-14.  Back to cited text no. 13
    
14.
Manoy P, Anomasiri W, Yuktanandana P, Tanavalee A, Ngarmukos S, Tanpowpong T, et al. Elevated serum leptin levels are associated with low vitamin D, sarcopenic obesity, poor muscle strength, and physical performance in knee osteoarthritis. Biomarkers 2017;22:723-30.  Back to cited text no. 14
    
15.
Alix PM, Guebre-Egziabher F, Soulage CO. Leptin as an uremic toxin: Deleterious role of leptin in chronic kidney disease. Biochimie 2014;105:12-21.  Back to cited text no. 15
    
16.
Sharma K, Considine RV, Michael B, Dunn SR, Weisberg LS, Kurnik BR, et al. Plasma leptin is partly cleared by the kidney and is elevated in hemodialysis patients. Kidney Int 1997;51:1980-5.  Back to cited text no. 16
    
17.
Landi F, Calvani R, Tosato M, Martone AM, Fusco D, Sisto A, et al. Age-related variations of muscle mass, strength, and physical performance in community-dwellers: Results from the Milan EXPO Survey. J Am Med Dir Assoc 2017;18:88.e17-25.  Back to cited text no. 17
    
18.
Kwon H, Pessin JE. Adipokines mediate inflammation and insulin resistance. Front Endocrinol (Lausanne) 2013;4:71.  Back to cited text no. 18
    
19.
Tazawa R, Uchida K, Fujimaki H, Miyagi M, Inoue G, Sekiguchi H, et al. Elevated leptin levels induce inflammation through IL-6 in skeletal muscle of aged female rats. BMC Musculoskelet Disord 2019;20:199.  Back to cited text no. 19
    
20.
Iikuni N, Lam QL, Lu L, Matarese G, La Cava A. Leptin and Inflammation. Curr Immunol Rev 2008;4:70-9.  Back to cited text no. 20
    
21.
Agrawal S, Gollapudi S, Su H, Gupta S. Leptin activates human B cells to secrete TNF-α, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathway. J Clin Immunol 2011;31:472-8.  Back to cited text no. 21
    
22.
Mak RH, Cheung W, Cone RD, Marks DL. Leptin and inflammation-associated cachexia in chronic kidney disease. Kidney Int 2006;69:794-7.  Back to cited text no. 22
    
23.
Zanetti M, Barazzoni R, Guarnieri G. Inflammation and insulin resistance in uremia. J Ren Nutr 2008;18:70-5.  Back to cited text no. 23
    
24.
Stenvinkel P, Heimbürger O, Lönnqvist F. Serum leptin concentrations correlate to plasma insulin concentrations independent of body fat content in chronic renal failure. Nephrol Dial Transplant 1997;12:1321-5.  Back to cited text no. 24
    
25.
Fahal IH. Uraemic sarcopenia: Aetiology and implications. Nephrol Dial Transplant 2014;29:1655-65.  Back to cited text no. 25
    
26.
Korolczuk A, Dudka J. Increased risk of cardiovascular complications in chronic kidney disease: A possible role of leptin. Curr Pharm Des 2014;20:666-74.  Back to cited text no. 26
    
27.
Kuo CH, Lin YL, Lee CJ, Wang CH, Lai YH, Liou HH, et al. Hyperleptinemia positively associated with central arterial stiffness in hemodialysis patients. PLoS One 2018;13:e0190694.  Back to cited text no. 27
    
28.
Sáinz N, Rodríguez A, Catalán V, Becerril S, Ramírez B, Gómez-Ambrosi J, et al. Leptin administration favors muscle mass accretion by decreasing FoxO3a and increasing PGC-1alpha in ob/ob mice. PLoS One 2009;4:e6808.  Back to cited text no. 28
    
29.
Nwadozi E, Ng A, Strömberg A, Liu HY, Olsson K, Gustafsson T, et al. Leptin is a physiological regulator of skeletal muscle angiogenesis and is locally produced by PDGFRα and PDGFRβ expressing perivascular cells. Angiogenesis 2019;22:103-15.  Back to cited text no. 29
    
30.
Hamrick MW, McGee-Lawrence ME, Frechette DM. Fatty Infiltration of Skeletal Muscle: Mechanisms and Comparisons with Bone Marrow Adiposity. Front Endocrinol (Lausanne) 2016;7:69.  Back to cited text no. 30
    
31.
Valencak TG, Osterrieder A, Schulz TJ. Sex matters: The effects of biological sex on adipose tissue biology and energy metabolism. Redox Biol 2017;12:806-13.  Back to cited text no. 31
    
32.
Guerra B, Fuentes T, Delgado-Guerra S, Guadalupe-Grau A, Olmedillas H, Santana A, et al. Gender dimorphism in skeletal muscle leptin receptors, serum leptin and insulin sensitivity. PLoS One 2008;3:e3466.  Back to cited text no. 32
    



 
 
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