Tuesday, February 18, 2014

Hyperpolarization without persistent radicals

The paper for this week is

"Hyperpolarization without persistent radical for in vivo real-time metabolic imaging"


Eichhorn, Takado, Salameh, Capozzi, Cheng, Hyacinthe, Mishkovsky, Roussel and Comment

PNAS 2013 110 18064



There is a lot going on in this short and well-written paper.  I highly recommend that you read it yourself, because I don't quite know where to start.  I guess I have to start somewhere, so let's jump into metabolic imaging.

For most organic chemists busy dissolving their precious compounds into 500 uL of CDCl3 and transferring to 5 mm NMR tubes, it might be a shock to learn that using high end MRI scanners, it is possible to take spatially- and temporally-resolved spectra on mice (or humans).  Yes.  You can lay in a (high end) MRI scanner and someone can take an NMR spectrum of a specific anatomical region, say the brain or the heart.  Some organic chemist should ask the question - why doesn't my department buy an MRI scanner and let me (and all my lab mates) set up a reaction flask inside and take NMR spectra of little regions of my flask as the reaction proceeds?  Frankly, the spectra are crummy relative to conventional NMR.  One trick used to improve spectral quality is to use 13C-labeled material and record 13C NMR spectra.  Going back to biological samples, it is possible to measure the real-time, spatially-resolved conversion of substrates into metabolites in this technique.  This technique is called metabolic imaging. 

Sensitivity is one big problem with metabolic imaging, leading various groups (and companies, etc.) to borrow any available tools to increase signal-to-noise.  One powerful tool that is all the rage in solid state NMR of biological macromolecules these days is dynamic nuclear polarization or DNP. 

One of the best explanations of DNP is on the website of Bridge12, a company that sells hardware to do this type of experiment.


By the way, I'll mention that Bridge12 maintains a nice literature blog at http://blog.bridge12.com/

To summarize briefly, a sample is mixed with persistent radicals.  Because the unpaired electron has a much larger gyromagnetic ratio, the population difference between spin-down and spin-up states is larger, which explains why EPR is so much more sensitive than NMR.  The trick behind DNP is to use microwave irradiation to saturate the EPR signal of the persistent radicals and transfer the polarization to the nuclei in the sample, much like the classic steady state NOE experiment.  This transfer leads to an enhancement of signal.

For any clinical application, though, DNP is hard to pull off.  Because you cannot inject radicals into a patient, you have to filter them out after transfer.  Meanwhile everything is relaxing and some of the signal enhancement that you have fought so hard (and paid so much) to get is lost.  You get about a minute.  But in this minute, impressive results have been collected, allowing real-time detection of metabolic intermediates in vivo.  The authors assert that "most preclinical developments have focused on samples of neat pyruvic acid (PA) to which suitable persistent radicals are added."  PA is important because "this molecule has a central position in the glycolytic pathyway" and "is a powerful marker for cancer metabolism."   

As the title of the publication suggest the authors of this paper describe a way to take advantage of the sensitivity gains of DNP without persistent radicals.  They make tiny beads of neat PA and extract an electron by UV irradiation to make a radical.  They do the DNP experiment to enhance the 13C signal of PA.  When these beads are dissolved in water, all you get is hyperpolarized pyruvate, pyruvate hydrate and acetate (and CO2 gas).  All this extra signal make it possible to record spatially and temporally resolved 13C spectra inside a mouse and watch the formation of lactate and breakdown of pyruvate!

What do the authors actually do?

They devise a clever experimental setup so that droplets of 2 +/- 0.5 uL of pyruvic acid are dripped one-by-one into a 3 mm EPR tube (http://www.wilmad-labglass.com/Products/705-PQ-6-25/) sitting inside a EPR dewar (http://www.wilmad-labglass.com/Products/WG-850-B-Q/) filled with liquid nitrogen.  Each drop is flash frozen into a little bead ~1.5 mm in diameter.  Approximately a dozen beads are collect in each EPR tube.  I picture bubble tea in my head.  Then each EPR tube filled with frozen beads and liquid nitrogen is irradiated for 1 hour using a high-power 365-nm LED array.

What happens in this setup?  First, radical are created.  Figure 1B and C show the X-band EPR spectrum of natural abundance PA beads and [1-13C] PA beads at 77 K after 1 hour of UV irradiation.

For readers unfamiliar with EPR, it helps to think of EPR in terms of NMR, except EPR focuses on unpaired electrons.  The x-axis is magnetic field instead of frequency (or ppm) because in EPR the magnetic field is swept during the experiment.  The y-axis is the first derivative of absorbance instead of absorbance.  At the point on the x-axis where the signal switches from positive to negative is the maximum of the absorbance.  If you study Fig 1B, you will see four such crossing.  If you study the intensity of the bands you can convince yourself that the absorbance spectrum is a quartet.  The reason is that a delocalized unpaired electron is coupled to three equivalent protons (the methyl group of PA).  As a control, the authors also make beads with 13C enrichment at the 1 position.  In this case, the unpaired electron is coupled to one 13C nuclei and 3 equivalent protons.  The absorbance spectrum is a doublet of quartets.  If you study the EPR spectrum in Fig 1C you can convince yourself that you see a doublet of quartets. 

What is actually going on in the beads upon UV irradiation in not consequential for this publication.  To quote the authors "in aqueous solution, PA undergoes efficient photodecarboxylation. ... The radicals produced by the low-temperature UV irradiation of the pure acid  ... are most likely related to intermediary products postulated for this photodecarboxylation mechanism."  In summary it does not matter why, but the authors can produce PA radical at concentrations of 15 mM.  They get the concentration from quantitative EPR.

Approximately seven frozen beads of [U-13C] PA are dissolved in ~500 uL of D2O to make a 900 mM solution, which is transferred to 5 mm NMR tube.  The authors record a 1D 13C NMR spectrum with no 1H decoupling on a 400 MHz NMR.  The interscan delay equals 180 s and the number of scans equal 512 for a total acquisiton time ~17 hours.  The result is shown in Fig. 2.

The authors assign several 13C resonances to pyruvate, pyruvate hydrate, acetate and CO2.  The authors interpretation of this result is that upon dissolution, PA radical decomposes to CO2 and acetate!  There is no EPR signal upon dissolution and the 13C T1 are the same as non-UV irradiated PA.  Together these data indicate that there is no radicals and these samples can be injected or perfused into live animals.

To get hyperpolarization, though, you still have to do the microwave saturation experiment.  The authors state that using their system, 13C polarization of 10% can be achieved in 2.5 h at 5 T and 1.2 K using 50 mW microwave power.  I have no idea if that is impressive or not.  It sounds very expensive, though.  By way of comparison, using traditional persistent radicals, 13C polarization of 60% can be achieved in the same amount of time.

To demonstrate the potential of their method, the authors collect in vivo metabolic images on a mouse.  80 mM hyperpolarized pyruvate solutions are prepared from UV-irradiated PA beads.  A 300 uL bolus was injected into mouse femoral vein.  A 13C spectrum localized in the mouse head was measured every 3 s for 75 s.  Holy crap!  A 13C spectrum every three seconds!  Figure 4A shows the
spectra.  The inset spectrum is a sum projection (I assume).    

What is more is that the authors can collect metabolic images 5 mm^3 spatial resolution, 3 s time resolution.  Fig. 4B and C show 13C images using interleaved selective excitations of pyruvate (B) and lactate (C) superimposed on 1H anatomical images of the mouse head.  

Figure 4 is very impressive and it is clear why the authors think they are onto something.  Endogenous DNP is clearly a huge step towards routine clinical applications.  I look forward to following this field. 


researchblogging.org citation code

Eichhorn TR, Takado Y, Salameh N, Capozzi A, Cheng T, Hyacinthe JN, Mishkovsky M, Roussel C, & Comment A (2013). Hyperpolarization without persistent radicals for in vivo real-time metabolic imaging. Proceedings of the National Academy of Sciences of the United States of America, 110 (45), 18064-9 PMID: 24145405


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