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Cited by 1,180Mitochondrial Function and PathologyCardiovascular and exercise physiologyAdipose Tissue and Metabolism

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

Open access
Larsen, Steen, Nielsen, Joachim, Hansen, Christina Neigaard, Nielsen, Lars Bo, Wibrand, Flemming, Stride, Nis, Schroder, Henrik Daa, Boushel, Robert +3 more·2012·The Journal of Physiology
DOI 10.1113/jphysiol.2012.230185PMC PMC3459047
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Abstract

Key points: Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects. The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein, and complex I–IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres). Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content. mtDNA was found to be a poor biomarker of mitochondrial content. Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity. Abstract Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake ( ) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein content, and complex I–IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range of (range 29.9–71.6 ml min −1 kg −1 ) and mitochondrial content (4–15% of cell volume). Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity. We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

AI snapshot

Written from the paper’s full text.

TL;DR

In 16 healthy young men, cardiolipin content, citrate synthase activity, and complex I activity were the biomarkers most strongly associated with TEM-measured mitochondrial content, while mtDNA was not. Complex IV activity was most strongly associated with muscle oxidative phosphorylation capacity.

Key findings

  • Mitochondrial fractional area ranged from 4–15% of cell volume, and VO₂max ranged from 29.9–71.6 ml min⁻¹ kg⁻¹. Cardiolipin had the strongest association with mitochondrial content (r = 0.86, P < 0.001; Lin’s Rc = 0.85), followed by CS activity (r = 0.84, P < 0.001; Rc = 0.80) and complex I activity (r = 0.78, P = 0.002; Rc = 0.77).
  • mtDNA was not related to mitochondrial content (r = 0.35, P = 0.23, n.s.).
  • For OXPHOS, defined as maximal coupled respiration with glutamate + malate + succinate (GMS3), complex IV activity had the strongest concordance (Rc = 0.64), followed by complex II activity (Rc = 0.63).
  • TMPD + ascorbate respiration correlated strongly with mitochondrial fractional area (r = 0.94, P < 0.001; Rc = 0.74), and cristae surface area correlated with mitochondrial fractional area (r = 0.93, P < 0.001; Rc = 0.91).

Methods

  • Sixteen healthy non-smoking male subjects were recruited (age 24.3 ± 0.9 years; range 20–32.6), with a large range of physical training levels; they completed a graded exercise test for VO₂max and provided vastus lateralis muscle biopsies.
  • Mitochondrial content was measured by transmission electron microscopy stereology as mitochondrial fractional area and cristae surface area; OXPHOS was measured as maximal coupled respiration in saponin-permeabilized fibres using high-resolution respirometry. Biomarkers included citrate synthase activity, cardiolipin content, mtDNA content, complex I–V protein content, and complex I–IV activity.
  • Pearson correlation coefficients and Lin’s concordance coefficient (Rc) were used; only biomarkers with significant Pearson correlations were included in Rc analysis, and Rc > 0.61 was considered a valid biomarker.

Limitations

  • Stated: the relatively low number of subjects and variability in measures meant the small difference between cardiolipin and CS activity was not enough to claim cardiolipin superior; mtDNA copy number can vary considerably (4–6 copies per cell) between individuals; cristae surface area was estimated in only 12–63 mitochondria per biopsy; three subjects failed TEM quality control, so mitochondrial content and cristae surface area were determined in only 13 subjects; TMPD + ascorbate respiration does not reach steady state and can be difficult to interpret.
  • Evident from the text: all participants were healthy young males, so findings may not generalize to females, older adults, or clinical populations.
  • Evident from the text: TEM is a two-dimensional morphological measure, and subsarcolemmal mitochondria volume recalculation assumed a cylindrical fibre radius of 40 μm.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1113/jphysiol.2012.230185

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 14 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Abstract

    Complex I activity is among the biomarkers showing the strongest association with mitochondrial content.

    “followed by CS and complex I activities”
    • Also stated in: § Abstract · plain
      “Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    The concordance between biomarkers and cristae surface area was generally lower than between biomarkers and mitochondrial surface area.

    “As can be seen in Table 2, the concordance was generally lower between the biomarkers and the cristae surface area when compared with mitochondrial surface area.”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Discussion

    CS activity has a concordance correlation coefficient (Rc) of 0.80 with mitochondrial fractional area.

    “This includes cardiolipin content (Rc= 0.85), CS activity (Rc= 0.80), complex I activity (Rc= 0.77), complex II protein content (Rc= 0.72), complex IV activity (Rc= 0.71), complex V protein content (Rc= 0.69) and complex II activity (Rc= 0.67).”
    • Also stated in: § Conclusion · boosted
      “CS activity is also strongly associated with the mitochondrial content.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.
  • Findingown data
    § Abstract

    Complex II activity showed the second strongest association with muscle oxidative capacity (OXPHOS).

    “followed by complex II activity”
    • Also stated in: § Results · plain
      “GMS3 was significantly correlated to several biomarkers showing the highest concordance to complex IV activity (Rc= 0.64), and complex II activity (Rc= 0.63) both defined as a ‘substantial concordance’.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Results

    The Rc for fibre respiration using TMPD + Asc and mitochondrial fractional area was 0.74, defined as substantial concordance.

    “The Rc was 0.74 which is defined as a ‘substantial concordance’ (Rc= 0.74).”
    Quote in the paperSubstantial claim
  • May combine two claims. 17
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Findingown data
    § Results

    GMS3 was significantly correlated to several biomarkers, showing the highest concordance to complex IV activity (Rc=0.64) and complex II activity (Rc=0.63), both defined as substantial concordance.

    “GMS3 was significantly correlated to several biomarkers showing the highest concordance to complex IV activity (Rc= 0.64), and complex II activity (Rc= 0.63) both defined as a ‘substantial concordance’.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Complex V protein content, cardiolipin content, CS activity, and complex IV activity followed in concordance with cristae surface area (Rc values 0.70, 0.67, 0.67, and 0.64, respectively).

    “The biomarker that showed the strongest concordance with cristae surface area was complex I activity (Rc= 0.71) and complex II protein content (Rc= 0.71), followed by complex V protein content (Rc= 0.70), cardiolipin content (Rc= 0.67), CS activity (Rc= 0.67) and complex IV activity (Rc= 0.64).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Mitochondrial fractional area and cristae surface area were very strongly correlated and concordant.

    “we found a very strong correlation and concordance between mitochondrial fractional area and cristae surface area (Table 2)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

  • May combine two claims.

    Findingown data
    § Results

    CS activity, complex I, II and IV activity and complex II and V protein content all had Rc values defined as substantial concordance with mitochondrial fractional area.

    “CS activity, complex I, II and IV activity and complex II and V protein content all had Rc values defined as a ‘substantial concordance’.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Cardiolipin content had the strongest correlation and highest relative concordance with mitochondrial fractional area, followed by CS and complex I activities.

    “The biomarker with the strongest correlation and highest relative concordance was cardiolipin content, followed by CS and complex I activities.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

  • May combine two claims.

    Findingreasoning
    § Discussion

    Biomarkers and respiratory measures showed stronger correlation and higher concordance with mitochondrial fractional area than with cristae surface area.

    “in contrast to our hypothesis we found that the biomarkers and respiratory measures showed a stronger correlation and higher concordances with mitochondrial fractional area than cristae surface area (Table 2)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

  • May combine two claims.

    Findingown data
    § Results

    PGMSOct3 had a significant correlation with mitochondrial fractional area (r=0.65) but not as strong as GMS3.

    “The correlation between PGMSOct3 and mitochondrial fractional area was also significant (r= 0.65) but not as strong as with GMS3.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Abstract

    VO2max ranged from 29.9 to 71.6 ml min−1 kg−1.

    “range 29.9–71.6 ml min−1 kg−1”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    Respiration activated with TMPD + Asc showed the highest respiratory rate, followed by uncoupled respiration using both NADH- and FADH2-generating substrates (GMS3u).

    “As expected, respiration activated with TMPD + Asc (direct electron donor to complex IV) showed the highest respiratory rate followed by uncoupled respiration using both reduced form of nicotinamide-adenine dinucleotide (NADH)- and the reduced form of flavin adenine dinucleotide (FADH2)-generating substrates (GMS3u).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    Only complex II protein content (Rc=0.49) and complex V protein content (Rc=0.48) were significantly correlated to PGMSOct3, both defined as moderate concordance.

    “Only complex II protein content (Rc= 0.49) and complex V protein content (Rc= 0.48) were significantly correlated to PGMSOct3, both defined as having a ‘moderate concordance’.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Cristae surface area was strongly correlated and showed strong concordance with mitochondrial fractional area.

    “The cristae surface area was strongly correlated and showed a strong concordance with mitochondrial fractional area”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

  • May combine two claims.

    Findingown data
    § Discussion

    Cardiolipin content had a stronger correlation (r=0.86 vs. CS activity r=0.84) and higher relative concordance (Rc=0.85 vs. Rc=0.80) with mitochondrial content than CS activity.

    “Even though our data suggest that cardiolipin had a stronger correlation (cardiolipin content r = 0.86 vs. CS activity r = 0.84) and higher relative concordance (cardiolipin content Rc= 0.85 vs. CS activity Rc= 0.80) with mitochondrial content”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § Methods

    Samples from three subjects failed quality control, so mitochondrial content and cristae surface area were determined in only thirteen subjects.

    “Samples from three subjects failed quality control. Mitochondrial content and cristae surface area was therefore only determined in thirteen subjects.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Introduction

    Studies on isolated mitochondria or permeabilized muscle fibres have shown that acute exercise, high fat diet, type 2 diabetes, chronic obstructive pulmonary disease and obesity may change the intrinsic properties of the mitochondria.

    “Studies on isolated mitochondria or permeabilized muscle fibres have shown that acute exercise (Fernström et al. 2004), high fat diet (Anderson et al. 2009), type 2 diabetes (Phielix et al. 2008), chronic obstructive pulmonary disease (Naimi et al. 2011) and obesity (Anderson et al. 2008) may change the intrinsic properties of the mitochondria.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Fernström et al. 2004

      Not checked in the free run.

    • cites Anderson et al. 2009

      Not checked in the free run.

    • cites Phielix et al. 2008

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Introduction

    Studies have also shown that intrinsic mitochondrial functionality is not altered with acute exercise, type 2 diabetes and obesity.

    “Hence, studies have also shown that the intrinsic mitochondrial functionality is not altered with acute exercise (Molnar et al. 2006), type 2 diabetes (Boushel et al. 2007; Hey-Mogensen et al. 2010) and obesity (Ara et al. 2011).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Molnar et al. 2006

      Not checked in the free run.

    • cites Boushel et al. 2007

      Not checked in the free run.

    • cites Hey-Mogensen et al. 2010

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § Results

    The integrity of the outer mitochondrial membrane was assessed by addition of cytochrome c in protocols 1-3 and 5, and no effect of this addition was seen.

    “The integrity of the outer mitochondrial membrane was assessed by addition of cytochrome c in protocols 1–3 and 5. No effect of this addition was seen (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Methods

    All respiratory protocols were made in duplicates.

    “All respiratory protocols were made in duplicates.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning
    § Introduction

    The array of diverse biomarkers currently used for normalization may explain some of the controversies that exist in some of the above-mentioned areas.

    “The array of diverse biomarkers currently used for normalization may explain some of the controversies that exist in some of the above-mentioned areas.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    The mitochondrial population exists in a three-dimensional network.

    “Even though it is well established that the mitochondrial population exists in a three-dimensional network (Ogata & Yamasaki, 1997), two-dimensional imaging using transmission electron microscopy (TEM) is still regarded as the golden standard for measuring mitochondrial fractional area (mitochondrial content).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ogata & Yamasaki, 1997

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    CS activity can be acutely regulated by exercise.

    “In contrast to cardiolipin, CS activity can be acutely regulated by exercise (Tonkonogi et al. 1997).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tonkonogi et al. 1997

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § Discussion

    When using TMPD + ascorbate as substrate the respiratory rate does not reach a steady state but displays a spiked response, which can be difficult to interpret.

    “Furthermore, when using TMPD + ascorbate as substrate the respiratory rate does not reach a steady state but rather it displays a spiked response, which can be difficult to interpret.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Conclusion

    Cardiolipin has the strongest association with mitochondrial content and is the most valid biomarker for investigating changes in mitochondrial content and for calculating intrinsic mitochondrial functionality.

    “We conclude the following: (1) Cardiolipin has the strongest association with the mitochondrial content and is the most valid biomarker for investigating changes in mitochondrial content and for calculating intrinsic mitochondrial functionality.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.
22 of 22

  • The paper relies on this without support.

    There is no existing knowledge of which commonly used marker of mitochondrial content has the strongest association with a morphological measure of actual mitochondrial content.

    “However, it is not known which of these commonly used markers of mitochondrial content has the strongest association with a morphological measure of the actual mitochondrial content.”

  • The authors state this as an assumption.

    A large range of physical training levels among recruited subjects produces large variations in mitochondrial content.

    “In order to increase the sensitivity of the statistical analysis the recruited subjects represented a large range of physical training levels and accordingly were assumed to exhibit large variations in mitochondrial content.”

  • The paper relies on this without support.

    Mitochondrial fractional area measured by two-dimensional TEM represents actual mitochondrial content in skeletal muscle.

    “In the present study we determined mitochondrial fractional area and cristae surface area using two-dimensional TEM as representative measures of mitochondrial content.”

  • The paper relies on this without support.

    CS activity may contribute to the oxidation of substrates in some respiratory protocols.

    “Furthermore, CS activity may contribute to the oxidation of substrates in some respiratory protocols.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract names a different correlation test from the methods

    Minor

    The abstract says Spearman correlation tests were used, whereas the Statistics section says Pearson correlation coefficients were calculated and Table 2 labels its coefficients as Pearson correlations.

  • CS concordance with cristae area differs from Table 2

    Minor

    The results report a concordance coefficient of 0.67 for CS activity and cristae surface area, but Table 2 gives 0.64 for that pairing. The table assigns 0.67 to cardiolipin instead.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Anderson EJ, 2008, Obesity is associated with reduced glutathione content, increased mitochondrial H2O2 emitting potential and a more oxidized redox environment in human skeletal muscle, Diabetes, 57, A430
  2. Mogensen M, 2006, Mitochondrial respiratory function is decreased in skeletal muscles of patients with type 2 diabetes, Diabetologia, 49, 121

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Related

Authors
Larsen, S., Nielsen, J., Hansen, C. N., Nielsen, L. B., Wibrand, F., Stride, N., Schroder, H. D., Boushel, R., Helge, J. W., Dela, F., & Hey‐Mogensen, M.
Affiliations
  • University of Copenhagen (DK)
  • University of Southern Denmark (DK)
  • Copenhagen University Hospital (DK)
ORCID
Venue
The Journal of Physiology
Publisher
Wiley
Published
2012-07
Volume
590
Issue
14
Pages
3349-3360
ISSN
  • 0022-3751 (ISSN-L)
  • 1469-7793
DOI
10.1113/jphysiol.2012.230185
PMC
PMC3459047
OpenAlex
W2143588920
Semantic Scholar
fd65ead65b8d92d37b4a872d5ac261c288c4585d
Cited by
1,180
References
45
Citations by year
2026: 67 · 2025: 71 · 2024: 98 · 2023: 102 · 2022: 113 · 2021: 134 · 2020: 105 · 2019: 115 · 2018: 99 · 2017: 67 · 2016: 74 · 2015: 55 · 2014: 41 · 2013: 31 · 2012: 7
FWCI
17.21
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 23 d)
Locations
  • The Journal of Physiology (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion)
  • University of Southern Denmark Research Portal (University of Southern Denmark) (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Health Sciences › Medicine › Complementary and alternative medicine › Cardiovascular and exercise physiology
  • Health Sciences › Medicine › Physiology › Adipose Tissue and Metabolism
Keywords
mitochondrial content, skeletal muscle, biomarkers, muscle oxidative capacity, citrate synthase, cardiolipin, mitochondrial DNA, complex I activity, complex IV activity, oxidative phosphorylation, vastus lateralis, research participants
MeSH terms
Adenosine Triphosphatases, Adult, Cardiolipins, Carrier Proteins, Citrate (si)-Synthase, Exercise Test, Humans, Male, Membrane Proteins, Mitochondria, Muscle, Oxidative Phosphorylation, Oxygen Consumption, Biomarkers, Muscle Fibers, Skeletal, Mitochondrial Proton-Translocating ATPases, Electron Transport Complex I, Microscopy, Electron, Transmission, Quadriceps Muscle

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