Roles of Uremic Toxins in Uremic Sacropenia
Condition: Sarcopenia
MeSH terms: Sarcopenia
Study type: Observational

Summary: 
      Background. In advanced chronic kidney disease (CKD), multiple metabolic and nutritional
      abnormalities may contribute to the impairment of skeletal muscle mass and function thus
      predisposing patients to the condition of sarcopenia. Herein, we aim to investigate the
      association of uremic toxins and sacropenia. In addition, the prevalence and mortality
      predictive power of sarcopenia, defined by different methods, in a cohort of hemodialysis
      patients.

      Methods. We plan to evaluate 300 HD patients. Sarcopenia was defined as reduced muscle
      function assessed by handgrip strength (HGS <30th percentile of a population-based reference
      adjusted for sex and age) plus diminished muscle mass assessed by different methods: (i)
      midarm muscle circumference (MAMC) <90% of reference value (A), (ii) muscle wasting by DEXA
      (B) and (iii) reduced skeletal muscle mass index (<10.76 kg/m² men; <6.76 kg/m² women)
      estimated by bioelectrical impedance analysis (BIA) (C). Serum levels of 3 established uremic
      toxins such as indoxyl sulfate, p-cresol and hippuric acid will be measured. Besides, various
      relevant inflammatory markers will also be assessed. Patients will be followed for up to 3
      years for all-cause mortality.
    

Detailed description: 
      Patients and Methods

      This study will included 300 consecutive patients receiving maintenance hemodialysis at the
      outpatient dialysis center of the Tungs Taichung Metroharbour Hospital, Taichung Taiwan.
      Recruitment will start after approval of the Ethics Committee of our hospital. Exclusion
      criteria were age of <20 and >90 years, clinical signs of acute infection during the month
      preceding the inclusion, active cancer or liver disease at the time of evaluation, previous
      diagnosis of immunological diseases and unwillingness to participate in the study. Physician
      will perform a complete chart review and interviewed each patient regarding their clinical
      history.

      All MHD patients enrolled in our study will be tested with DEXA and bioelectrical impedance
      analysis (BIA) and grip strength. Demographic data was collected and anthropometric
      measurement such as mid-arm muscle circumference (MAMC) calf circumference, and laboratory
      examination are conducted.

      Handgrip strength

      Muscle strength is assessed in the dominant hand using a Jamar hand dynamometer (Lafayette
      Instrument Company, USA). Patients are ﬁrst familiarized with the device and were then
      examined standing with both arms extended sideways from the body with the dynamometer facing
      away from the body. Patients were instructed to grip the dynamometer with the maximum
      strength in response to a voice command, and the highest value of three measurements was
      considered for the study. Handgrip strength (HGS) values under the 30th percentile from a
      speciﬁc-population reference value adjusted for age and sex were considered as reduced.

      Anthropometry

      Body mass index (BMI) is calculated as weight in kilograms divided by height in squared
      meters. MAMC is calculated according to the following equation, based on mid-arm
      circumference (measured at mid-point from the acromion to olecranon) and the triceps skinfold
      (using caliper Lange®, Cambridge Scientiﬁc Industries, Inc.).

      Values of MAMC were compared with the 50th percentile of NHANES II and standard adequacy of
      <90% was considered as reduced muscle mass .

      Bioelectrical impedance analysis

      Body composition was measured using an 8-contact electrode bioelectrical impedance analysis
      (BIA) device (Tanita BC-418, Tanita, Tokyo, Japan), and followed the standard procedure and
      the manufacturer's instructions. This BIA device was used to measure the whole body and
      segmental impedance (± 1Ω) at a frequency of 50 kHz, and it provided valid muscle mass
      estimates (kg) of each of the four extremities.[2 Appendicular muscle mass (ASM) was
      calculated as the sum of the estimated muscle mass for the arms and legs. A relative skeletal
      muscle mass index (ASM/ht2) normalized for height was defined as the ratio of ASM (kg) and
      the height squared(m2). For this analysis, we defined muscle mass cut-off points according to
      the distribution of ASM/ht2 of a young population comprising 998 healthy adults (aged 20-40
      years) or the study population. A participant was considered to have low muscle mass if his
      or her ASM/ht2 was below -2 standard deviations of the reference young adult values defined
      in previous studies (6.76 kg/m2 for men and 5.28 kg/m2 for women).

      Sarcopenia diagnosis

      The diagnosis of sarcopenia was based on the presence of derangements in both muscle function
      and muscle mass. For the purpose of the study, we considered one muscle function indicator
      (reduced HGS) associated with one of three indicators of muscle mass (MAMC, DEXA or BIA).
      Therefore, reduced HGS in association with an MAMC of <90% of the standard adequacy (Method
      A), presence of muscle wasting by DEXA (MethodB) or reduced SMMI by BIA(MethodC) were
      considered as diagnosis of sarcopenia.

      Laboratorial parameters

      All blood samples are collected during the midweek dialysis from the AV fistula, immediately
      after the insertion of the dialysis cannula but before the administration of heparin. Blood
      is sampled in 4 c.c. Venoject II tubes, centrifuged (10 min, 3000 rpm) and immediately store
      at -70°C until assayed. Serum albumin, urea, creatinine, and total protein concentrations in
      serum are determined according to standard methods. The serum levels of hsCRP are measured
      using a Behring Nephelometer II (Dade Behring, Tokyo, Japan). Serum concentrations of total
      p-cresol sulfate, hippuric acid and IS (i.e., combined free and protein bound fractions) are
      analyzed with High-performance liquid chromatography (HPLC). Briefly, for binding
      competition, 200μl serum to which we added 20μl 0.50mM 1-naphthalenesulfonic acid (internal
      standard) was vortex-mixed with 250μl 0.24M sodium octanoate (binding competitor).After
      incubation at room temperature for 5min, we added 2ml cold acetone to precipitate proteins.
      Following vortex-mixing and centrifuging at 4 ◦C, 1860×g for 20 min, the supernatant was
      transferred to 12mm×100mm, GL 14 glass test tubes and 2ml dichloromethane was added. After
      vortex-mixing and centrifuging at 4 ◦C, 1860×g for 10min, 200μl of the upper layer was
      transferred to glass autosampler vials, followed by addition of 20μl 1M HCl and 15μl was
      injected onto the HPLC. The HPLC analysis was performed on an Agilent 1100 series LC (Santa
      Clara, CA),and Agilent ChemStations software were used for the chromatographic analysis. The
      separation was carried out on a ZORBAX SB-C18 Solv Saver Plus HPLC column (5 μm, 3.0 mm×150
      mm).at a flow rate of 0.6 ml/min. Mobile phase A is 0.2% trifluoroacetic acid in Milli-Q
      water and mobile phase B is 0.2% trifluoroacetic acid in acetonitrile. The analytical method
      consists of an isocratic run with 92% mobile phase A for 23 min.. Each analytical run was
      followed by a 1.3 min washout gradient to 100% B. Column temperature was 25 ◦C, and
      autosampler tray temperature was 6 ◦C. We quantified the analytes by using the analyte to
      standard peak area ratio on a Agilent 1100 High Performance Fluorescence detector G1321A and
      Agilent 1100 Series UV-Visible detectors G1314A. Detector settings were λex 260 nm/λem288nm
      for p-cresyl sulfate and λex 280 nm/λem 390nm for indoxyl sulfate and internal standard.
      Hippuric acid was monitored by UV-Vis detector at 254 nm. Quantitative results are obtained
      and calculated in terms of their concentrations (mg/L).

      Blood samples are also collected before dialysis for measurement of oxidative stress (CML,
      Nε-(carboxymethyl)lysine ) and inflammatory markers (IL-6). Serum Nε-(carboxymethyl)lysine
      (CML) was determined using enzyme-linked immunosorbent assay (R&D system, Minneapolis, MN,
      USA). Similarly, the concentration of IL-6 is also assessed using Quantikine Human
      Interleukin Immunoassay (R&D Systems Minneapolis, MN, USA) with a detection limit of 15 pg/L
      and 0.12 pg/mL, respectively. In addition, serum irisin, urotensin II, follistatin and alpha
      1 antichymotrypsin concentrations are measured by using the human enzyme-linked immunosorbent
      assay (ELISA) kits in accordance with the manufacturer's instructions. The All determinations
      are performed in duplicate, and the mean value is used.

      Statistical analyses

      The variables are expressed in mean±SD, median (interquartile range) or proportions. Variable
      distributions are tested by e.g. Shapiro Wilk test, and those not normally distributed are
      standardized by using z-score.Studentt-test or Chi-square test is employed for the
      comparisons between sarcopenic and non-sarcopenic patients. Kappa test is used to evaluate
      the agreement among the methods. Survival analyses are made with the Kaplan-Meier survival
      curve and the Cox proportional hazard model. The univariate and multivariate Cox-regression
      analyses are presented as hazard ratio [HR; 95% conﬁdence intervals (CI)]. Statistical
      signiﬁcance is set at the level of P<0.05, and the analyses are performed by using the SPSS
      software version 18 (SPSS, Inc., Chicago, IL, USA)
    

Observational model: Case-Crossover
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        hemodialysis (HD) patients
      
Keywords: sarcopenia, uremic toxins