Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

The overall goal of this In Vivo magnetic resonance technique and exercise protocol is to establish a robust and standardized method for the noninvasive measurement of mitochondrial oxidative phosphorylation capacity. Skeletal muscle mitochondrial oxidative phosphorylation capacity can be measured noninvasively with In Vivo phosphorous 31 magnetic resonance spectroscopy. This standardized technique will facilitate the development of targeted therapies to improve oxidative phosphorylation capacity.

The main advantages of this technique are that it is noninvasive allowing for serial investigation, and that it is well tolerated by individuals spanning a wide range of functional capabilities. Generally, individuals new to this method will struggle because the equipment set up and subject positioning are technically intensive and crucially important to obtain good results. Prior to imaging, first obtain written and informed consent from the subject and thoroughly screen them for MRI safety.

Then, set up for the experiment. This protocol is demonstrated using a Siemens Avanto 1.5 Tesla system. Begin prepping for imaging by first plugging the phosphorous 31 coil into the table coil connector closest to the bore.

Then, place a large triangle foam cushion near the other end of the scanner exam table, but not directly on the coil. Also, place a head pillow at the of the MR exam table farthest from the bore, then instruct the subject to lie supine with their feet first on the MR table. Place a foam cushion under their knees to support the leg in a partially flexed position.

Be sure to position the subject close to the right side of the table in order to center the left thigh as closely to the magnet isocenter as possible to ensure optimal B0 homogeneity. Then, provide the subject with ear plugs or headphones for hearing protection. Position the phosphorous 31 coil on the subject’s left quadriceps at approximately the mid point between the patella and the femoral head.

Then, secure it to the leg using straps. The coil should be over the lateral portion of the leg, above the vastus lateralis. Secure a baby oil bottle to the medial aspect of the thigh with the same straps used to secure the coil to the leg, then bind the subject’s legs together with a strap placed below the coil and above the knee.

Secure the subject’s legs to the MR table with additional straps, one above the knee, and one midway between the knee and the ankle. Allow the subject to practice the isometric exercise to ensure that the straps provide sufficient resistance. Then, once the subject is comfortable, use the laser light guide to mark the center of the coil and move the table to the magnet isocenter using the centering landmark.

For the baseline phase of the spectroscopy acquisition, instruct the subject to lie still and relax their leg muscles to minimize motion artifacts. Begin by acquiring a triplane localizer to verify positioning and identify the location of the coil. Also, acquire a second localizer, but open the slice view on the first triplane localizer.

Next, set up the phosphorous 31 spectroscopy sequence with non-localized pulse acquired sequence parameters as seen here. Center and rotate the slice orientation by left clicking and holding the slice group, and match the final orientation of the slices to the position of the oil bottle. Now, place the shim box by dragging the second triplane localizer into the viewing window, then drag the spectroscopy sequence into the protocol window and double click to open.

Use the position tool bar to visualize the shim voxel which will appear on the localizer images. The voxel can be moved by left clicking and holding it in the center of the box, and it’s size and rotation can be changed by left clicking and holding it at the corner of the box. Place the shim box directly below the coil, and parallel to the plane of the quadriceps to ensure B0 field homogeneity.

Use the localizer images to identify the sensitive region of the coil, and adjust the shim box to encompass this region within the muscle. Open the acquisition viewer window and select the head icon in the tool bar to allow viewing of the spectroscopy acquisition in real time, then run the sequence to obtain a single test spectrum. Next, observe the resulting spectrum and examine the quality of the B0 shimming.

Look for a prominent phosphocreatine peak centered at zero parts per million without significant noise. If the spectrum appears noisy, reposition the shim box and reacquire. Copy the test spectroscopy sequence with the best spectral quality and use it for all subsequent measurements.

Increase the number of measurements from one to 10, then acquire while the subject is at rest. Next, for exercise acquisition, apply the shim settings from the previous scan and set the sequence to acquire 20 measurements. Instruct the subject to first remain at rest for two measurements, then provide the subject with a count down to indicate when to start the exercise.

At this point, the subject should initiate knee extension flexion as forcefully and as rapidly as possible against the resistance of the straps for approximately 30 seconds. After a 30%drop in the PCR peak height is observed, instruct the subject to terminate the exercise and rest. Then, acquire 20 additional post exercise measurements immediately without pause or shimming.

After the acquisitions are complete, ensure exercise quality by comparing the phosphocreatine peak heights at the beginning and end of exercise. This graph shows the recovery of the phosphocreatine concentration after it’s depletion with the rapid quasi-static knee extension exercise. The line represent the fit of the exponential recovery function.

A Bland-Altman analysis of phosphocreatine recovery time demonstrates a mean difference and standard deviation of 1.03 plus or minus 4.83 seconds, and a between trials coefficient of variation of 4.66. Shown here is a representative phosphocreatine recovery curve from the examination of a nonambulatory subject. Note that a phosphocreatine depletion of 64%was attained with this exercise protocol.

Finally, this comparison of phosphocreatine recovery times demonstrates sequentially poorer mitochondrial oxidative capacity in control, non-diabetic, and diabetic subjects. Once mastered, this technique can be done in less than 20 minutes if it’s performed properly. While attempting this procedure, it’s important to remember to both verify that the spectrum has sufficient signal prior to exercise and to ensure that exercise results in a phosphocreatine depletion of at least 30%Following this procedure, other methods like fat muscle quantification imaging can be performed in order to calculate muscle mass, and fat content, which are also important to overall skeletal muscle function.

This technique can be used to investigate many diseases including Friedreich’s ataxia, where it allowed researchers to correlate genetic markers of disease severity in mitochondrial oxidative capacity. After watching this video you should have a good understanding of how to perform an In Vivo phosphorous 31 magnetic resonance spectroscopy scan and exercise protocol. Don’t forget that working with magnetic resonance scanners requires stringent safety measures, and that all MR experiments should be performed by adequately trained personnel.