31P magnetic resonance spectroscopy

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The 31P magnetic resonance spectroscopy ( 31P MRS ) is a special form of magnetic resonance imaging in which the relative concentrations of specific substances, such as phosphates of energy metabolism can be determined in the fabric. It is mainly used in scientific studies on muscle physiology and sports medicine .

Basics

The 31-phosphorus magnetic resonance spectroscopy allows non-invasive insight into the mitochondrial function of the muscle cells . It was derived from NMR spectroscopy , whereby the naturally occurring isotope of phosphorus is excited with a specific frequency of 25.8 MHz in a magnetic field of 1.5 Tesla . When using a clinical magnetic resonance tomography (MRT) system with the ability to perform spectroscopy at a magnetic field between 1.5 and 3 Tesla, quantitative changes in phosphocreatine (PCr), inorganic phosphate, phosphomonoester and phosphodiester as well as α-, β- and γ-phosphates can be observed of adenosine triphosphate (ATP) can be detected. However, at the α and γ positions there is an overlay with the phosphates of ADP and nicotinamide adenine dinucleotide (NAD + / NADH). The β position of ATP is not overlaid by other phosphates and can therefore be used for a quantification of ATP.

In order to measure metabolite changes in the muscle under stress, a high temporal resolution is sought, which is in a range between 4 and 20 s. This allows the changes to be recorded well, especially at the beginning of an exercise.

The comparison between the two spectra, which come from the same muscle, shows the importance of the repetition time (TR) on the signal level. The spectrum with the TR of 1s has been amplified by the Nucleus Overhauser Effect (NOE).

Quantification of the metabolites

There are various methods of quantifying the metabolites measured in muscle using the 31P-MRS . One possibility is to use an external standard that is introduced into the measuring arrangement. Phenylphosphonic acid [10mM] is an example of such an external standard, which is measured along with the metabolites in the muscle. Since the concentration of the external standard is known, the concentrations of the other metabolites can be measured. One problem with this method, however, is the fact that the external standard is located outside the living body and is therefore only partially comparable with metabolites within the body.

Another method is to use ATP as a so-called internal standard. The resting ATP concentration of the muscle shows little variability between different people and is usually given as 8.2 mmol / l. The advantage is that the ATP is intracellular and is therefore subject to the same conditions as the metabolites to be quantified. This method of quantification is mainly used.

In order to be able to quantify metabolites, the partial saturations that result from the different relaxation times of the metabolites must be balanced. In the case of a fully saturated spectrum, which is usually measured with a repetition time of 30 s, the different relaxation times are irrelevant. However, since it is necessary to record the dynamic changes in the muscle under load with a higher temporal resolution than 30 s, a much shorter repetition time is used for this. Since the metabolites relax with different times during the measurement, the ratio of the integrals between the peaks changes. This ratio no longer corresponds to the actual concentrations and must be corrected. It is possible to obtain a fully relaxed spectrum with the help of a repetition time of 30 s in the resting state before the exercise protocol, which is then used to correct the ratios of the metabolites to one another. However, the concentrations of the metabolites can also be corrected mathematically with a constant repetition time using the known relaxation times.

Changes in the energy-rich phosphates at the beginning of muscle work

At the beginning of a muscle work, the contractions increasingly split adenosine triphosphate on the myofibrils . As a result, more adenosine diphosphate (ADP) and inorganic phosphate (P) as well as [H +] ions are produced. The creatine kinase in the myofibrils now transfers a phosphate of the PCr to the ADP, which is rephosphorylated to ATP. The remaining creatine is rephosphorylated at the mitochondria in the cytosol to PCr with consumption of ATP. This reaction is mediated by a creatine kinase on the outer membrane of the mitochondria. PCr is thus a shuttle for the transport of energy-rich phosphates from the mitochondria to the myofibrils.

The ATP production in the mitochondria is initially too low to completely rephosphorylate the accumulating creatine . This leads to a progressive decrease in PCr in the cytosol. This is also referred to as the anaerobic phase, as the muscle cell tries to meet the ATP requirement through lactic acid fermentation . This does not succeed, so that the progressive decrease in PCr cannot be stopped. But this creates lactate , which serves as a buffer for the [H + ] ions produced during ATP hydrolysis .

State of equilibrium of changes in the energy-rich phosphates

The time course of the spectra is shown for 3 consecutive load levels and the recovery phase.

In the further course, the mitochondrial capacity is increased in accordance with the metabolic requirements by enzyme activation and by improving the oxygen supply by means of increased tissue perfusion. As a result, the phase of progressive PCr decay changes into an equilibrium of PCr hydrolysis and rephosphorylation. This equilibrium is usually established in a mono-exponential course. The time constants , which indicate the point in time at which the progressive decrease in PCr falls to a state of equilibrium, are between 30 and 60 seconds in healthy people and also correlate with the increase in blood flow in the supplying arteries.

Recent studies have also shown that with an incremental muscle load, the blood flow in the supplying artery increases as the work intensity increases. In each increment there is an equilibrium of the PCr decay. The PCr levels at the end of an increment show a linear correlation with the work intensity of the muscle.

Calculation of the time constants of the phosphocreatine changes

Screenshot of a non-linear regression analysis in R of PCr changes under a constant muscle load

The time course of PCr is examined with the aid of a non-linear regression analysis or curve fitting, a mono-exponential model being used predominantly. As a result, the time constant of the PCr curve can be estimated, which indicates the point in time at which the progressive portion of the PCr drop changes into the plateau that corresponds to the aerobic phase. The PCr time constants also correlate with the mitochondrial oxygen turnover and are therefore also an indication of the mitochondrial function. In particular, the PCr time constant during the regeneration period depends almost exclusively on mitochondrial function. The PCr time constant can be calculated in the SPSS statistics program. Other suitable programs are also Origin or GNU R .

Phosphocreatine kinetics and mitochondrial function

PCr is a shuttle molecule that transports high- energy phosphates from the mitochondrion to the myofibril so that the ADP that is produced during muscle contraction can be regenerated into ATP. This allows the ATP at the myofibril to be kept constant under aerobic conditions. At the myofibril, the ATP is split by an ATPase when it contracts. When the energy-rich phosphate group is transferred from the PCr to the ADP, creatine remains . The creatine is rephosphorylated again at the mitochondrion, splitting ATP. Since creatine is only rephosphorylated to PCr if the ATP synthesis in the mitochondrion is sufficient, conclusions can be drawn about mitochondrial function and mitochondrial oxygen turnover via the PCr kinetics.

It is also recognized that post-exercise PCr regeneration, in particular, is a mirror of mitochondrial function. If the time constant of the PCr regeneration is extended, there is a mitochondrial dysfunction.

Ergometer

For this examination, an MR-compatible ergometer is required to generate an energy release in the muscle, which then leads to the metabolic shifts in the muscle described above. The ergometer should enable constant energy output over a period of several minutes. Since this method is mainly used for scientific questions, most working groups use self-made variants that manage the energy output via air pressure, cable pulls with weights or rubber bands. Since the calf muscles are relatively easy to use in an MRI system, they are often examined. That is why there are relatively many variants of pedal ergometers.

Applications

The 31P-MRS for studying the metabolism of high-energy phosphates in muscles is mainly used in muscle physiology and sports medicine. Other areas of application are metabolic diseases and storage diseases such as McArdle's disease . These diseases usually lead to an impairment of the mitochondrial function, which can be measured and quantified using the 31P-MRS of the skeletal muscle under stress. Other congenital and acquired mitochondrial disorders can also be examined with it. In recent years this method has been increasingly used for the scientific investigation of the energy metabolism in the calf muscles in patients with peripheral arterial occlusive disease ( PAD ).

Individual evidence

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  2. ↑ Stubbs et al. 31P-magnetic resonance spectroscopy studies of nucleated and non-nucleated erythrocytes; time domain data analysis (VARPRO) incorporating prior knowledge can give information on the binding of ADP. Biochim Biophys Acta 1291, 1996, pp 143-148.
  3. ↑ a b Schocke et al. High-energy phosphate metabolism in the exercising muscle of patients with peripheral arterial disease. VASA 37, 2008, pp. 199-210.
  4. ↑ Hitchins et al. 31P NMR quantitation of phosphorus metabolites in rat heart and skeletal muscle in vivo. Am J Physiol-Heart Circul Physiol 281, 2001, pp. H882-H887.
  5. ↑ Kemp et al. Absolute quantification of phosphorus metabolite concentrations in human muscle in vivo by 31 P MRS: a quantitative review. NMR Biomed 20, 2007, pp. 555-565.
  6. ↑ Meyerspeer et al. Relaxation times of 31 P-metabolites in human calf muscle at 3 T. Magn Reson Med. 49, 2003, pp. 620-625.
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  10. ↑ Iotti et al. Inorganic phosphate is transported into mitochondria in the absence of ATP biosynthesis: an in vivo 31P NMR study in the human skeletal muscle. Biochem Biophys Res Commun . 225, 1996, pp. 191-194.
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  12. ↑ Iotti et al. The mono-exponential pattern of phosphocreatine recovery after muscle exercise is a particular case of a more complex behavior. Biochim Biophys Acta. 1608, 2004, pp. 131-139.
  13. ↑ Greiner et al. High-energy phosphate metabolism in the calf muscle of healthy humans during incremental calf exercise with and without moderate cuff stenosis. Eur J Appl Physiol. 99 (5), 2007, pp. 519-531.
  14. ↑ Nevill et al. A model for phosphocreatine resynthesis. J Appl Physiol. 82, 1997, pp. 329-335.
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  16. ↑ Forbes et al. Short-term high-intensity interval training improves phosphocreatine recovery kinetics following moderate-intensity exercise in humans. Appl Physiol Nutr Metab. 33 (6), 2008, pp. 1124-31.
  17. ↑ Quistorff et al. A simple calf muscle ergometer for use in a standard whole-body MR scanner. Magn Reson Med. 13, 1990, pp. 444-449.
  18. ↑ Francescato et al. Two-pedal ergometer for in vivo MRS studies of human calf muscles. Magn Reson Med 46, 2001, pp. 1000-1005.
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  22. ↑ Vorgerd et al. Treatment of glycogenosys type V (McArdle disease) with creatine and ketogenic diet with clinical scores and with 31P-MRS on working leg muscle. Acta Myol. 26 (1), 2007, pp. 61-3.
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