Association of Uremic Sarcopenia and Mitochondrial Copy Number and Its Clinical Correlates
Condition: Sarcopenia; Mitochondrial DNA
MeSH terms: Sarcopenia
Study type: Observational

Summary: 
      Sarcopenia is the decline of muscle mass and strength with age. Evidence suggests that
      oxidative stress and molecular inflammation play important roles in age-related muscle
      atrophy. The two factors may interfere with the balance between protein synthesis and
      breakdown, cause mitochondrial dysfunction, and induce apoptosis. Sarcopenia, inflammation
      and oxidative stress is highly prevalent in hemodialysis patients and may contribute to
      mortality. The copy number of mitochondrial DNA (mtDNA) is affected by oxidative stress in
      blood circulation. This study aimed to test whether mtDNA copy number correlates with
      oxidative stress and some uremic toxins in nondiabetic hemodialysis(HD) patients. 200
      nondiabetic hemodialysis patients and 50 healthy subjects will be enrolled. This study will
      be performed to investigate quantitative changes in mtDNA occur in HD patients with and
      without sarcopenia. Copy number of mtDNA in leukocyte DNA is determined by real-time
      polymerase chain reaction in HD patients and 50 age- and sex-matched control subjects. In
      addition, correlation of the alterations of albumin redox status, 8-isoprostane, plasma IL-6
      ,LBP and TNF-a and as well as various uremic toxins will be performed.
    

Detailed description: 
      Research design and methods Subjects In this study, we shall recruit 200 patients on
      maintenance hemodialysis. In addition, 50 age- and sex-matched subjects act as controls. The
      HD patients have been on stable dialysis for at least 3 months HD patients are being treated
      three times weekly with standard bicarbonate dialysis (Na 138 mmol/L, HCO3 35 mmol/L, K 1.5
      mmol/L, Ca 1.25 mmol/L, Mg 0.75 mmol/L) and by high-flux HD with synthetic membranes
      (dialysis filters surface area: 1.7 to 2.1 m2). All HD patients enroll in our study will be
      tested with DEXA and handgrip strength. Demographic data will be collected and laboratory
      examination are conducted as listed below.

      Biochemical Determinations Blood samples for laboratory testing were drawn from the venous
      end of a vascular access at the beginning of the hemodialysis session and then stored at
      -80°C until time of analysis. The high sensitive C-reactive protein (hsCRP) levels were
      determined by a commercial immunoturbidimetric assay using a Hitachi autoanalyzer (model
      7170). The detection limit and interval for CRP was 0.1 mg/L and 0.1-500.0 mg/L,
      respectively. The baseline serum albumin was measured by the bromocresol green method on a
      Hitachi autoanalyzer (model 7170). Serum levels of cholesterol, triglyceride, and low- and
      high-density lipoprotein cholesterol were determined by standard laboratory methods.

      Determination of relative leucocyte mtDNA copy number The real-time PCR reaction was
      performed in triplicate for each reaction. The 10ul PCR reaction contains 1X TaqMan Universal
      PCR Master Mix buffer, 500nM of each primer, 200nM of TaqMan probe, and 0.2-2ng of total
      genomic DNA extract, PCR conditions are 2 min at 50℃, 10 min at 95℃, followed by 40 cycles of
      15 s of denaturation at 95℃ and 60 s of annealing/extension at 60℃. Real-time quantitative
      analysis was performed on StepOne Real-Time PCR system(ABI). The primers and probes for
      real-time PCR are listed in Table 1. The PCR products that involve mitochondria DNA(MtDNA)
      and nuclear DNA.

      Standard Curve:

      Standard DNA solutions for the mitochondrial genome and the nuclear 18S rRNA dene (nDNA) were
      generated from PCR products cloned in a vector of pCR2.1-TOPO. Serial dilutions were made and
      RT-QPCR reactions were performed to construct the standard curve from Ct values and the
      number of copies of the standard plasmid DNA.

      Determination of mtDNA/nDNA Ratio as a measure of mtDNA Content:

      The copy number of the mtDNA and the nDNA is calculated using the threshold cycle number (Ct)
      and intrapolating from the standard curve. The ratio of the copy number of mtDNA to the copy
      number of nDNA is measurement of mtDNA content.[30] Measurement of the Albumin Redox State
      Measurement of the albumin redox state was performed using the high-performance liquid
      chromatography (HPLC) method reported previously [31]. The HPLC-fluorescence detection
      (HPLC-FD) system consisted of an AS-8010 autosampler (injection volume, 2 mL per specimen;
      Tosoh, Tokyo, Japan) and a Model FS-8000 fluorescence detector (excitation wavelength, 280
      nm; emission wavelength, 340 nm) with a CCPM double-plunger pump (Tosoh) in conjunction with
      a SC-8020 system controller (Tosoh). A Shodex-Asahipak ES-502N 7C column [Showa Denko, Tokyo,
      Japan; 10×0.76 cm (inner diameter), dimethylaminoethyl-form for ion-exchange HPLC, column
      temperature, 35°C±0.5°C] or in some instances two Asahipak GS-520H columns [Asahi Chemical
      Industry; Kawasaki, Japan; 25×0.75 cm (inner diameter) maintained at 32°C] were used. Linear
      gradient elution was carried out with an ethanol level increasing from 0% to 5% in 0.05 M
      sodium acetate and 0.40 M sodium sulfate buffer (pH 4.85; acetate-sulfate buffer) at a flow
      rate of 1.0 mL/min. Deaeration of the buffer solution was performed by helium bubbling. Based
      on the HPLC profiles of HSA obtained from these procedures, the values for each fraction were
      subjected to numerical curve fitting, and the fractions of HMA, HNA-1, and HNA-2 to total HSA
      were calculated.

      Measurements of Liposaccharide-binding protein (LBP) The LBP was determined from serum
      samples and controls using standardized enzymelinked immunosorbent assay (ELISA) methods, and
      serum from normal control subjects was used for interassay variation.

      Total of 8-iso-PGF2-alpha Analysis Total 8-iso-PGF2-alpha concentrations in plasma were
      measured by a specific enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, Mich).
      Samples were centrifuged and the supernatants were collected, purified as stated previously
      and stored at -80°C until assayed. Samples were assayed at 50 μl after a 1:10 or 1:20
      dilution and read at 420 nm in a 96 well microplate reader. The assay has been validated to
      obtain a high correlation (0.95) between added known amounts of 8-isoprostane and the
      concentration measured by EIA and has been directly validated by gas chromatography/mass
      spectrometry. The antiserum used in this assay has a 100%cross reactivity with
      8-iso-PGF2-alpha 0.2% each with PGF2-alpha, PGF3, PGE1, PGE2, 0.1% each with
      6-keto-PGF1-alpha. The detection limit of the assay is 4 pg/ml and the range of standard
      curve was from 3.9 to 500 pg/ml.

      Measurements of Serum total p-cresol and Indoxyl sulfate Plasma concentrations of total
      p-cresol sulfate(PCS) and indoxyl sulfate( IS) 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. Detector settings were λex 260 nm/λem288nm for p-cresyl sulfate
      and λex 280 nm/λem 390nm for indoxyl sulfate and internal standard. Quantitative results are
      obtained and calculated in terms of their concentrations (mg/L).

      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 (Table 2) from a speciﬁc-population reference value adjusted for age and sex were
      considered as reduced [32].

      Dual-Energy X-ray Absorptiometry (DEXA) DEXA is a widely used reference method for body FFM
      and FM measurements.10 DEXA was performed using a Lunar Prodigy (GE Medical Systems, Madison,
      WI). Whole-body scans were performed according to manufacturer's instructions, and body FM
      (FMDEXA), LTM (LTMDEXA) and bone mineral content are analyzed using the manufacturer's
      software. The DEXA method uses an X-ray tube with a filter to generate low-energy (40 kV).
      and high-energy (70 or 100 kV) photons. When photonsat different energy levels pass through
      tissue, their absorptions can be expressed as a ratio of attenuation at lower or higher
      energy levels. DEXA estimate of FFM was calculated as a sum of LTM and bone mineral content
      estimates.

      All patients will be examined by the same observer. Overall, AWGS recommends using 2 standard
      deviations below the mean muscle mass of young reference group or the lower quintile as the
      cutoff value determination. Moreover, AWGS recommends using height-adjusted skeletal muscle
      mass instead of weight-adjusted skeletal muscle mass, and the suggested cutoff values were
      7.0 kg/m2 in men and 5.4 kg/m2 in women by using DEXA.
    

Observational model: Case-Crossover
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        patients have to be at least 20-years-old and on outpatient hemodialysis (HD) for at least
        3 months.
      
Keywords: oxidative stress; Sarcopenia; copy number of mitochondrial DNA (mtDNA)