Phosphorus-31 NMR spectroscopy

31P magnetic resonance spectroscopy ( 31P -MRS) is a special form of magnetic resonance imaging, in which the relative concentrations of certain substances, such as phosphates of energy metabolism, are determined in the tissue. It is mainly used in scientific studies of muscle physiology and sports medicine.

Basics

The 31 -phosphorus magnetic resonance spectroscopy allows non-invasive insight into the mitochondrial function of muscle cells. It is derived from the NMR spectroscopy, wherein the naturally occurring isotope of phosphorus having a specific frequency of 25.8 MHz excited at a magnetic field of 1.5 Tesla. When using a clinical magnetic resonance imaging (MRI ) system with the capability of spectroscopy in a magnetic field between 1.5 and 3 Tesla can be quantitative changes in phosphocreatine ( PCr ), inorganic phosphate, phosphomonoester and phosphodiester and the α -, β - and γ - phosphates of adenosine triphosphate (ATP ) can be detected. However, in the α - and γ - position takes place 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 thus be used for quantification of ATP.

To measure changes in muscle metabolites under load, a high temporal resolution is desired, the S is in a range between 4 and 20. Thus, the changes especially at the beginning of a load can be well captured.

Quantification of metabolites

There are several methods to quantify the metabolites, which are measured by means of the 31P -MRS muscle. One possibility is to use an external standard that is introduced in the measuring arrangement. Phenylphosphonic acid [ 10 mM ] is an example of such an external standard that is measured together with the metabolite 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 is, however, the fact that the external standard is located outside the living body and thus with metabolites within the body is only partially comparable.

Another method is to use ATP as a so-called internal standard. The ATP concentration in the resting state of the muscle has only a low variability between different people and is usually given as 8.2 mmol / l. The advantage is that the ATP intracellularly located and is thus subject to the same conditions to quantify the metabolites. This method of quantification is mainly used.

In order to quantify metabolites must Partialsaturierungen that come through different relaxation times of the metabolites into existence, be compensated. In a fully saturated spectrum, which is usually measured with a repetition time of 30 s, the different relaxation times do not matter. Since it is necessary, however, under load with a higher temporal resolution than recorded for 30 s, the dynamic changes in the muscle, this has a much lower repetition is used. Since the metabolites to relax to a different time during the measurement, the ratio of the integrals under the peaks varied from one another. This ratio is out of sync with the actual concentrations and must be corrected. It is possible in the idle state before the loading protocol fully nonrelaxed spectrum using a repetition time of 30 s to obtain, which is then used to correct the ratios of the metabolites with each other. The concentrations of the metabolites can also be corrected via the well-known relaxation times also calculated at a constant repetition.

Changes in high-energy phosphates at the beginning of muscular work

At the beginning of muscle adenosine triphosphate work is increasingly cleaved at the myofibrils through the contractions. This increasingly fall adenosine diphosphate ( ADP) and inorganic phosphate (P) and [H ] ions at. The creatine kinase in the myofibrils now transfers a phosphate of PCr to ADP, which is so rephosphorylated to ATP. The remaining creatine is rephosphorylated at the mitochondria to the cytosol PCr with consumption of ATP. This reaction is mediated by creatine kinase in the outer membrane of the mitochondria. PCr is thus a shuttle for the transport of high-energy phosphates from the mitochondria to the myofibrils.

ATP production in the mitochondria is too low at the start to complete the accumulation rephosphorylieren creatine. This leads to a progressive decrease in PCr cytosol. One speaks here of the anaerobic phase, since the muscle cell tries to meet the ATP requirement by the lactic acid fermentation. This is not possible, so that the progressive PCr waste can not be stopped. However, this creates lactate, which serves as a buffer for the ATP produced in the hydrolysis of [H ] ions.

Changes in the equilibrium state of the energy-rich phosphate

Subsequently, the mitochondrial capacity is increased according to the metabolic requirements by enzyme activation and by improving the oxygen supply through enhanced tissue perfusion. Thus, the phase of progressive PCr decay turns into a balance of PCr hydrolysis and rephosphorylation. This equilibrium is usually one in a mono- exponential. The time constants that specify the time of the envelope of progressive PCr decrease the equilibrium state, be in healthy people between 30 to 60 seconds and also correlate with the increase of blood flow in the feeding arteries.

Recent studies have also shown that when an incremental load on the muscle blood flow in the feeding artery increases with increasing work intensity. This leads to an increment in each equilibrium PCR decay. The PCr level at the end of an increment show a linear correlation to the labor intensity of the muscle.

Calculation of the time constant of the changes phosphocreatine

The time run of PCR is analyzed using a non-linear regression analysis and curve fitting, being mainly used is a mono- exponential model. Characterized the time constant of the PCR curve can be estimated which indicates the time at which transitions of the progressive portion of the PCR drop in the plateau, which corresponds to the aerobic phase. The PCr time constants correlate well with the mitochondrial oxygen consumption and thus also an indication of mitochondrial function. Specifically, the PCR time constant during the regeneration time depends almost exclusively on the mitochondrial function. The calculation of the PCr time constants can be made in the statistics program SPSS. Other suitable programs are also Origin or GNU R.

Phosphocreatine kinetics and mitochondrial function

PCR is a shuttle molecule, the energy-rich phosphates transported from the mitochondrion to the myofibril, so that the ADP which is produced during muscle contraction, it can be regenerated into ATP. Thereby can be kept constant at the myofibril under aerobic conditions, the ATP. The myofibril, the ATP is cleaved by an ATPase in their contraction. During the transfer of high-energy phosphate group from PCr to ADP creatine remains. The creatine is rephosphorylated on the mitochondria under ATP splitting again. Since creatine is only rephosphorylated to PCr when ATP synthesis in the mitochondrion sufficient for conclusions can be drawn about the PCr kinetics on mitochondrial function and mitochondrial oxygen consumption.

It is also recognized that in particular the regeneration of PCr after a load is a reflection of mitochondrial function. Is the time constant of the PCR regeneration extended, there is a mitochondrial disorder.

Ergometer

For this study, an MR -compatible ergometer is needed to generate muscle energy output, which then leads to the above-described metabolic shifts in muscle. The ergometer should allow a period of several minutes, a constant energy output. Since this method is mainly used for scientific issues, most use workgroup homemade versions, effect the energy output over air pressure, cables with weights or elastic bands. Since the calf muscle is relatively easy to load in an MRI system, this is often studied. Therefore, there are also quite a lot of variants of pedal ergometers.

Applications

The 31P -MRS for the study of the metabolism of energy-rich phosphates in the muscle is mainly used in muscle physiology and sports medicine. Other application areas include metabolic disorders and memory disorders such as the Crohn's McArdle. Such diseases often leads to a disturbance of mitochondrial function which can be measured and quantified by means of the 31P -MRS of the skeletal muscle during exercise. Other congenital and acquired mitochondropathies can thus be investigated. In recent years this method has been increasingly used for the scientific investigation of the energy metabolism in the calf muscle in patients with peripheral arterial disease (PAD ).