Chemical structures and descriptions

TCA cycle metabolites

What is measured?

Citrate (Cit), isocitrate (iCit), α-ketoglutarate (aKG), fumarate (Fum), malate (Mal), pyruvate (Pyr), lactate (Lac), and succinate (Suc; under development).
Method(s): GC-MS/MS.

The tricarboxylic acid cycle and related metabolites

The tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or Krebs cycle) is a series of reactions forming a loop that ultimately generate energy, mainly by oxidation of acetyl-CoA generated from glycolysis, and catabolism of fatty acids and amino acids. The energy is stored in guanosine triphosphate (GTP), NADH and FADH2 (Figure 1). The citric acid cycle also provides precursors for many compounds, including certain amino acids.
Recent basic research have demonstrated roles of Krebs cycle intermediates in intra- and intercellular signalling affecting physiology and pathogenesis (1). Krebs cycle intermediates have been linked to DNA and histone methylation (α-ketoglutarate and α-hydroxyglutarate), immune modulation (acetyl-CoA, itaconate, fumarate and succinate), thermogenesis (succinate), inhibition of hypoxia state and HIF-α stabilization (succinate, fumarate and α-hydroxyglutarate), and some are considered as “oncometabolites” defined as conventional metabolites that, when aberrantly accumulated, have pro-oncogenic functions (α-hydroxyglutarate, succinate (1), fumarate (2) and lactate (3)).
Studies on metabolomics involving Krebs cycle intermediates in relation to human health and disease usually include few patients and have been performed only recently. These metabolites have been related to BMI, cardiovascular disease (pyruvate, citrate, succinate), diabetes (pyruvate, isocitrate, succinate), NAFLD (isocitrate and citrate), longevity (isocitrate), asthma (succinate), disease activity in rheumatoid arthritis patients (itaconate), and worsening of clinical outcome in cancer patients (succinate, fumarate and α-hydroxyglutarate).

Indication(s)

To investigate the metabolomic signature of human diseases.

Specimen, collection and processing

Matrix: EDTA plasma (preferred) or serum.
Volume: Minimum volume is 60 µL, but 200 µL is optimal and allows reanalysis.
Preparation and stability: Krebs cycle metabolites are probably stable in serum/plasma, but there are concern over oxaloacetate. Samples should be put on ice immediately after collection and stored at -80 °C.

Transportation

Frozen, on dry ice. (for general instruction on transportation, click here)

Reported values, interpretation

Reported values: Cit: 90-200 µmol/L; cAco: 0.9-1.6 µmol/L; iCit: 2-22 µmol/L; aKG: 2-40 µmol/L; Suc: 2-12 µmol/L; Fum: 0.5-4 µmol/L; Mal: 2-21 µmol/L; Oaa: – ; Oxa: 6-12 µmol/L; Pyr: 80-160 µmol/L; Lac: 500-1600 µmol/L; Ita: 0.2-2.3 µmol/L.
Intraclass correlation coefficient (ICC): na.

Literature

1. Martínez-Reyes, I., & Chandel, N. S. (2020). Mitochondrial TCA cycle metabolites control physiology and disease. Nat Commun, 11, 102.
2. Yong, C., Stewart, G. D., & Frezza, C. (2020). Oncometabolites in renal cancer. Nat Rev Nephrol, 16, 156-172.
3. Hayes, C., Donohoe, C. L., Davern, M., & Donlon, N. E. (2021). The oncogenic and clinical implications of lactate induced immunosuppression in the tumour microenvironment. Cancer Lett, 500, 75-86.

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Statistical power is the probability that a statistical test will correctly reject a false null hypothesis (H0​) when a specific alternative hypothesis (H1​) is true. H0​ is the null hypothesis, which states there is no effect or no difference. H1​ is the alternative hypothesis, which states there is a real effect or difference. Alpha (α) is the probability of a Type I error (a false positive), which is the risk of incorrectly rejecting the H0​ when it is actually true. You set this value before the experiment, commonly at 0.05. Beta (β) is the probability of a Type II error (a false negative), which is the risk of failing to reject the H0​ when it is actually false.

Power is calculated as 1−β. Increasing power means you are decreasing the probability of making a Type II error.

Several factors can be adjusted to increase the power of a statistical test:

  • Effect Size: This is the magnitude of the difference you are trying to detect. A larger effect size is easier to detect, thus increasing power. 

  • Sample Size: The number of observations in a study. A larger sample size provides more information about the population, reducing the margin of error and increasing the power to detect a true effect.

  • Variation: Refers to the spread or standard deviation of the data within the population. Less variation makes it easier to distinguish a real effect from random noise, thereby increasing power.

  • Alpha (): Increasing the alpha level (e.g., from 0.05 to 0.10) also increases power, but at the cost of a higher risk of a Type I error. This trade-off is often undesirable.

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