Wednesday, October 29, 2014

Electron Paramagnetic Resonance of hair!

I wanted to switch gears a bit and do a paper on Electron Paramagnetic Resonance (EPR), also know as Electron Spin Resonance (ESR).  The paper for this weeks is

Electron spin resonance (ESR/EPR) of free radicals observed in human red hair: a new, simple empirical method of determination of pheomelanin/eumelanin ratio in hair.


Chikvaidze EN, Partskhaladze TM and Gogoladze TV;jsessionid=39B010DF34D055C5F27880DD49E628F1.f01t04

I discussed EPR briefly in an earlier post (  To review, one can think about EPR much like you think about NMR.  In a simple 1D 1H NMR spectrum parameters like the resonance frequency (the chemical shift plus any scalar couplings) and peak integrals can be interpreted to understand properties of the molecule, for instance the molecular structure of a small drug-like organic molecule.  There are similar parameters for EPR.  In EPR the resonance frequency is reported as the "g-factor."  Instead of depending on the shielding of nuclei by electrons like the chemical shift in NMR, the g-factor depends on the coupling of the spin motion of the electron to the orbital motion (spin-orbit coupling).  These days every organic chemistry textbook contains a table of chemical shifts classified by functional group.  With EPR, on the other hand, I am not aware of any standard tables of g-factors.  I don't want to suggest that the spin-orbit coupling is not sensitive to electronic structure.  Of course it is!  Spin-orbit coupling depends on which orbital the unpaired electron resides.  For metals, the g-factor is crucial.  For organic (oxygen, carbon and nitrogen) radicals, though, it seems like all g-factors are ~ 2.  Although the g-factor is not diagnostic in these cases, the coupling to magnetic nuclei are!  The unpaired electron will experience "hyperfine" coupling to nuclei, such as 14N (nuclear spin I = 1).  The electron is split into 2I+1 lines.  Hence if the nuclei is 15N (I = 1/2) the EPR signal is a doublet.  If the nuclei is 14N, the EPR signal is a triplet, but all three legs have identical height.  (As an aside, I'll mention to organic chemists that they should look at the CDCl3 signal in a 1D 13C spectrum to see an equivalent effect).  EPR spectra differ in two key ways from NMR spectra.  First, the x-axis is magnetic field (in gauss or telsa) not frequency.  Second, the y-axis is the first derivative of the absorbance.

Now that we are all experts on EPR, what is it that Chikvaidze and co-workers are measuring?  There is a branched polymer in skin and hair called melanin that controls pigmentation color.  This polymer is made of varying amounts of two monomers, called eumelanin and pheomelanin.  The former is associated with dark (brown or black) colors, the latter with red.  Eumelanin contains a O-C-C-O semiquinone and gives an EPR signal consistent with an oxygen radical (a singlet).  Pheomelanin contains a O-C-C-N semiquinonimine and gives and EPR signal consistent with a nitrogen radical (triplet).  According to the authors the measurement of the concentration of pheomelanin in skin samples is "an issue of great interest in the world" because UV-mediated breakdown of this molecule produces reactive oxygen species which "might help to explain the relatively high incidence of skin cancer among red-haired individuals."  There are assays available to determine the ratio of pheomelanin/eumelanin in hair samples that involve chemical treatments, etc.  Because these molecules are paramagnetic, it is possible to use EPR as a non-invasive assay to determine the amount of pheomelanin (ug/mg) in hair samples.

What did the authors do?  They collected 113 hair samples from their students (42 black, 28 dark brown, 27 red and 16 blond - I assume each sample is from a different student.  It is not clear if all samples are used in this study.) and divided the samples in bundles of equal mass (40 mg) and length (1.5 cm).  Because they need to make very accurate measurements of the g-factor, the authors use a standard of Mn2+ (in MgO powder).  I assume the standard is in a sealed capillary which is placed in a 4 mm EPR tube along with the hair bundle.  EPR are measured using an X-band EPR spectrometer at room temperature.

The EPR spectrum of black hair is a "slightly asymmetric singlet" with g-factor = 2.0035-2.0036.  The spectrum of black hair looks identical for each donor (data not shown).  For red hair, on the other hand, the spectrum vary depending on the donor.  The authors classify two distinct types of spectra (type I and type II) with g-factor = 2.0038-2.0047 shown in Figure 2:

The interpretation of this data is that the EPR spectrum of black hair is essentially the spectrum of eumelanin, whereas the spectrum of red hair is a superposition of eumelanin and pheomelanin.

The authors used microwave saturation to filter one of the components of red hair.  Let me try to explain saturation succinctly.  If the rate of spin flips between the spin-down and spin-up state is faster than the rate of relaxation back to the ground (spin-down) state, then the intensity of the signal is attenuated by saturation.  The rate of spin flips depends on microwave power.  The relaxation rate depends on R1 (= 1/T1).  Let's say there are two signals in the EPR spectrum.  One has a small R1 (aka large T1, aka slow relaxation) and the other has a large R1 (aka small T1, aka fast relaxation).  We can then choose a microwave power such that the rate of spin flips is larger than the small R1, but smaller than the large R1.  So the slow relaxing signal is saturated and maybe even disappears.  One can play this game with a 10 uM solution of TEMPOL radical under an atmosphere of air (which includes paramagnetic oxygen that will increase R1) or nitrogen.  At high microwave power the slow relaxing (large T1) nitrogen atmosphere sample will saturate and give no signal, but the fast relaxing (small T1) air atmosphere sample will not.

Figure 3 shows the EPR spectrum of red hair as a function of microwave power:

The authors interpretation is that "the triplet (hyperfine coupling = 0.372 mT, g-factor = 2.0055) .. evident after saturation of the singlet at maximum microwave power, corresponds to pure pheomelanin in the .. spectrum."

Now the authors are prepared to address their main goal: determine the ratio of pheomelanin/eumelanin in hair.  Given that pheomelanin has a g-factor of 2.0055 and eumelanin has a g-factor of ~2.0036, the authors hope to use the experimental g-factor of hair to determine the ratio.  Normally, one would make a calibration curve with different ratios to test the fitness of this method. Not so simple in this case.  One can mix different ratios of red and black hair, if you assume black hair is pure eumelanin.  Figure 4 shows the calibration curve.

Using data from Ito et al. (Pigm. Cell Melanoma Res. 2011 24, 605) that correlates hair color to concentration of pheomelanin in ug/mg, the authors make another calibration curve to convert the measured g-factor into pheomelanin concentration, shown in Figure 7.


My only beef with this paper is that the accuracy and precision of this method to measure pheomelanin and the ratio of pheomelanin:eumelanin is not addressed.  Let me state it another way.  Let's assume you can distinguish hair that contains 0.8 ug/mg pheomelanin from hair that contains 4.8 ug/mg.  Can you distinguish 2.4 ug/mg from 2.5 ug/mg?  To answer this question I did some simulations in MatLab using EasySpin ( a supercool simulation program.  My script is at the end of the blog.

The first plot shows the simulated EPR spectra of blond (in blue) vs red (in green) hair using data from Table 2 in the paper. 

Using the zero crossing to estimate the g-factor for the blond hair equals 2.0035 and for the red hair equals 2.0037, which is somewhat smaller than the 2.0046 from the Table 2 of the paper.  OK fair enough.  We can tell blond from red hair using the g-factor.

Two different red hair samples with pheo/eu% equal to 59 (blue) and 54 (green) look identical to me (see below).


Maybe I'm screwing something up with my simulations, but based on these results I am skeptical that X-band EPR can pick up subtle difference is pheomelanin:eumelanin.  Q-band (35 GHz) is a different story, though.  Signals at different g-factors no longer overlap and using double integrals, it may be possible to estimate pheomelanin concentration.  Below is the Q-band simulation of pheo/eu% equal to 59 (blue) and 54 (green).  I also assumed smaller lwpp to exaggerate the transitions.

Overall, I like this paper because it made me think about EPR and play with EasySpin.  It also got me worried about hair in my EPR cavity!  I'll have to keep the gingers away from my instrument.


EasySpin MatLab script

% Mixture of pheomelanin and eumelanin
% based on data from Chikvaidze et al. MRC 2014


% Experimental parameters
Xp.mwFreq = 9.43;
Xp.Range = [334 338];

% Component 1
Eu.g = 2.0035;
Eu.lwpp = 0.5;

% Component 2
Pheo.g = 2.0055;
A = 0.372;
Pheo.A = mt2mhz(A);
Pheo.Nucs = 'N';
Pheo.lwpp = 0.5;

% Relative abundances
Eu.weight = 0.46;
Pheo.weight = 0.54;

% One call to pepper
[B1,spc1] = pepper({Eu,Pheo},Xp);

% Relative abundances
Eu.weight = 0.41;
Pheo.weight = 0.59;

% One call to pepper
[B2,spc2] = pepper({Eu,Pheo},Xp);

plot(B1,spc1,B2,spc2); Chikvaidze, E., Partskhaladze, T., & Gogoladze, T. (2014). Electron spin resonance (ESR/EPR) of free radicals observed in human red hair: a new, simple empirical method of determination of pheomelanin/eumelanin ratio in hair Magnetic Resonance in Chemistry, 52 (7), 377-382 DOI: 10.1002/mrc.4075

Monday, October 13, 2014

You don't need that big expensive magnet to do NMR!!!

As chemists we often focus on parameters like chemical shift, scalar coupling and integrals that can be measured in an NMR spectrum and interpreted to understand qualitative and quantitative information about molecules.  There are applications where these parameters are less important and the longitudinal (T1) and transverse (T2) relaxation time constants and/or the self-diffusion coefficient (D) are critical.  A lot of these applications do not involve the types of samples that organic chemists or biochemists prepare (~600 uL in a 5 mm NMR tube).  In fact, sometimes T1, T2 and D need to be measured in extreme environmental conditions.  You can't drag your 11.7 T magnet to the south pole!  Even in more benign environments, a big magnet is not necessary for T1, T2 and D measurements needed for tasks like food characterization and oil-well logging.

Today I am going to discuss two papers that explore NMR without a big and expensive magnet!

The first paper is

"Ultra-low-field NMR relaxation and diffusion measurements using an optical magnetometer"


Ganssle PJ, Shin HD, Seltzer SJ, Bajaj VS, Ledbetter MP, Budker D, Knappe S, Kitching J and Pines A.

Angew Chem Int Ed Engl. 2014 53 9766-70
doi: 10.1002/anie.201403416

The authors design and demonstrate an ultra low field (ULF) NMR capable of performing industrially relevant measurement (T1, T2 and D) for the characterization of mixtures of hydrocarbons and water.  The authors claim that their instrument is the first step towards a compact, inexpensive and robust NMR sensor which operates at the Earth's magnetic field. 

How does this work?

Conventional NMR detectors use a coil to detect transverse magnetization.  The ULF NMR uses a magnetometer like what is in your cell phone as a compass.  The specific magnetometer is optically detected (the author's do not really explain the detector in this paper).  I don't pretend to understand all the details of this detector, which is called a spin exchange relaxation-free (SERF) configuration magnetometer.  The important thing to understand is that the detector measures longitudinal magnetization (along the z direction), in contrast with traditional NMR coils.  In fact, during acquisition, a series of 180 degree pulses are applied to sample to flip the spins between the +z and -z direction.  The authors convert the "average magnitude of change in magnetometer signal in response to a pi pulse" into a sensible signal.   

The authors want to design "a NMR sensor which operates at the Earth's magnetic field", but for now they have to make a few compromises to get a prototype.  First, there isn't much longitudinal magnetization in samples polarized by the Earth's magnetic field, so the authors apply a "pre-polarizing" 2 T field.  Second, the sample chamber is not really at the Earth's magnetic field, which (according to Wikipedia is at 25–65 uT (microtesla).  By the way, a strong refrigerator magnet has a field of about 10 mT.  At any rate, the chamber is designed to have no magnetic fields - there is some sort of special shielding to remove the Earth's magnetic field.  Then a weak "bias" magnetic field (50 uT in this case) is applied to the chamber.  This field is designed to be turned off during detection (the SERF detector has optimal response at zero field) and during pulses.  The reason for turning off the field during pulses is so that "the device does not need to be re-tuned when the bias field is changed."   Overall, their set up looks like the following:

The pulse sequences used to measure T1, T2 and D are the following:

If you study these pulse sequences, they will make a lot of sense.  The T1 measurement does not use the inversion recovery pulse sequence, instead longitudinal relaxation time constant is measured "as an exponential decay of the spin magnetization as a function of an increasing delay time between the sample pre-polarization and measurement."  You also see the unusual detections strategy.  Longitudinal magnetization is flipped between -z and z by a pi pulse.  The T2 measurement is a standard CPMG train.  "The inter-pulse spacing (tau) is held constant and the number of pulses (n) is varied."  Diffusion is CPMG with a gradient.

One thing I find pretty cool about this set up is that it uses conventional 5 mm NMR tubes!

What do the authors measure?

They record the relaxation time constants (T1 and T2) of some common solvents: water, methanol, ethanol and hydrocarbons at 0.5 G and 37 C.  They also make mixtures of hydrocarbons and water.  How did they do that?  These liquids are immiscible!  In fact, they used "a coaxial insert which separated liquid in a smaller, 3.3 mm NMR tube from the liquid in a standard thin walled 5 mm NMR tube."  I'm not sure I would call that a mixture, but why split hairs!  The data looks like the following:

Finally the authors can make 2D plots of T2 v T1 or T2 v D to demonstrate how readily these solvents can be distinguished based on these relaxation properties:

The authors conclude that this device is an "important first step towards the development of compact, inexpensive devices which can take advantage of the Earth's high homogeneous ambient magnetic field."  They acknowledge "the limitations present in these experiment ... are artifacts of the design of the device" but feel that they make a "compelling case for future research".

I think the device described in this publication is interesting and important.  I have a few questions, though.  How long does it take to make each T1, T2 and D measurement shown in figure 3 (each dot on the curve)?  Can T1, T2 and D distinguish miscible liquids, such as water-methanol or water-ethanol?  I work at a University and the proof of the vodka is a major concern on football days!  Finally, how big is the instrument, really?  It is hard for me to get a feel for the size based on Figure 1.  It looks almost as big as a superconducting NMR magnet.

The second paper is

"Scalable NMR spectroscopy with semiconductor chips"


Dongwan Ha, Jeffrey Paulsen, Nan Sun, Yi-Qiao Song and Donhee Ham

In contrast with the first paper, which described an unconventional detection scheme, this paper describes a miniaturized NMR system that is ~12 cm^3, weighs 7.3 kg and can perform 1D 1H, multi-pulse and heteronuclear experiments.  By contrast, a commercially available system like the Picospin45 ( is 20.3 cm x 14.6 x 29.2 cm, weighs 4.7 kg and can only do 1D 1H.  The major innovation by the authors is the miniaturization of the electronics into a 4 mm^2 chip.  The technical details are best understood by an electrical engineer, so I won't explain too much.  The net result is a system that looks like the following:

Obviously there is quite a bit missing from the picture, but the penny on the left gives a general idea of the size of the system.  The Larmor frequency is 21.8 MHz.  Samples are 1 mm capillaries with ~0.8 uL of sample.  What can the system do?  Figure 3 shows 1D 1H for 7 small molecules (acquisition times on the order of seconds to minutes).

Frankly, this data looks like 21.8 MHz NMR spectra.  The magnet is shimmed to 0.13 ppm resolution (~2 Hz).  So you can make out 7 Hz couplings, but small couplings or lines closer than ~0.1 ppm blend together.  The spectra of aspirin, serine and glucose are not useful for chemical characterization.

One of the advantages of the design is pulse programming.  The ability to control pulses and delays enables the authors to go beyond traditional 1Ds and do multidimensional NMR.  Figure 4 shows the JRES and 2D phase sensitive COSY on neat ethanol and 1.5 M alanine dissolved in D2O (acquisition times are 15 and 73 minutes, respectively).

The authors can also collect HSQC and HMQC on 13C enriched methanol in 17 and 34 minutes, respectively.  Note that there is no decoupling during acquisition (t2), meaning the peaks are split by the 1JCH.

The authors round the paper out with a relaxometry experiment on a crude oil sample.  They also introduce a clever processing hack to handle temperature fluctuations, which present a "significant obstacle towards portable NMR."  Their solution deserves a longer explanation in this blog post, but lets face it, it is getting way too long.


To wrap up this post, I'll note that I am not convinced that either system discussed above or a commercial benchtop NMR (from PicoSpin, Magritek or Nanalysis) can ever replace a trusty 400-600 MHz NMR for resonance assignment in organic chemistry or biochemistry.  I will concede that not all chemical characterization requires a 400-600 MHz NMR.  To paraphrase John Edwards from Process NMR associates at the MestreNova Users meeting prior to the ENC this year "if your spectrum looks like crap at 400 MHz, it won't look too much worse at 90 MHz, so why waste time on a superconducting magnet."  The authors of the papers I reviewed today present two novel NMResque instruments capable of making measurements nearly identical to a high field superconducting system.  The trick will be to continue to develop these systems and find applications where these systems outperform conventional systems.  


Ganssle, P., Shin, H., Seltzer, S., Bajaj, V., Ledbetter, M., Budker, D., Knappe, S., Kitching, J., & Pines, A. (2014). Ultra-Low-Field NMR Relaxation and Diffusion Measurements Using an Optical Magnetometer Angewandte Chemie International Edition, 53 (37), 9766-9770 DOI: 10.1002/anie.201403416  

Ha, D., Paulsen, J., Sun, N., Song, Y., & Ham, D. (2014). Scalable NMR spectroscopy with semiconductor chips Proceedings of the National Academy of Sciences, 111 (33), 11955-11960 DOI: 10.1073/pnas.1402015111

Friday, April 18, 2014

ENC 2014 Post 1

SPECIAL NOTE:  I wrote this post at the ENC, but for some reason it never got posted.  (I blame the crappy wi-fi in my hotel!).  Sorry ...

Day 1 of the 2014 ENC

The first session was the Laukien Prize session.

The prestigious Laukien prize is given to recognize excellence in experimental nuclear magnetic resonance published within the last three years.  The 2014 Laukien prize is awarded to a group of six leading solid-state NMR spectroscopists for their development and application of magic-angle spinning (MAS) ssNMR experiments for the determination of 3D protein structures and the study of associated molecular dynamics processes.

The winners are:

Marc Baldus - Utrecht University
Mei Hong - Iowa State University
Ann McDermott - Columbia University
Beat Meier - ETH Zurich
Hartmut Oschkinat - Leibniz-Institute (FMP) Berlin
Robert Tycho - NIH

Each winner was given 15 minutes to give a research overview, which was very exciting!

The second session was called "Electron Meet Nuclei", which was a cute title for a DNP session.  The organizers pointed out that 2014 is the 70th anniversary of the discovery of magnetic resonance (MR).  MR was discovered in 1944 by Zovoisky at Kazan University, USSR, who observed the EPR signal of CuSO4 and CuCl2.  The session featured a great talk by Bob Griffin from MIT and I really enjoyed a whirlwind of a talk by Mei Hong about ssNMR to probe plant cell wall structure.

After lunch there was a poster session.  I always love the poster session.  I learn so much.  I really enjoyed a plant metabolomics study of watermelon.  (Poster 167) The presenter (Iqbal Mahmud from Claflin University in South Carolina) looks for biomarkers that correlate with resistance to Powdery Mildrew (PM), a nasty fungus that ruins crops and costs growers a lot of money.  He found several and made a hypothesis regarding the pathways upregulated in PM resistant varieties.  Now collaborators at the Department of Agriculture are treating watermelon to engage this pathway to see if it confers resistance.  What I really liked about his poster was the experimental design.  The presenter and collaborators made grafts to assess the translocation of biomarkers, instead of just hunting through watermelon juice with no hypothesis.  I guess you could call this work "untargeted metabolomics", but, by good experimental design, the authors certainly increased their odds of finding a relevant target!

Day 2

The AM session began with the presentation of the JMR awards.  2014 is the inaugural year of this award, which is picked from abstracts submitted from graduate students and post-docs and includes a one year subscription to JMR and $350.  The winners are

Joseph Courtney UIUC
Michael Loretz ETH Zurich
Moritz Zaiss German Cancer Research, Heidelberg


The first session was "Biomolecular Structure and Function".  All talks were outstanding.  I was especially impressed with Jim Prestegard's talk "Sparse-Labeling and Long-Range Constraints: Structure and Function of Glycoproteins".  Professor Prestegard discussed work in his lab using selective labeling and PRE to explore how glycan structure and dynamics impacts function of glycoproteins, in particular, Immunoglobin G (IgG).  Glycoproteins are notoriously challenging to study because the heterogeneity, but the CCRC in Georgia has developed a number of clever tricks to hijack enzymes to selectively label glycans.  The examples discussed in this project all involve labeling galactose on a long branched glycan chain.  There are two galactoses on this glycan and there is evidence of chemical exchange broadening.  Rex measurements result in kex and some structural information on two major species.  It seems the the glycan flips between a "free" and "bound" structure.  The bound structure agrees with crystallography and the sugars are buried inside the IgG.  In the free structures, the glycan hangs out of the bottom of IgG.  A very long (microsecond) MD simulation suggests that the glycan does shuffle between the inside and outside.  Very cool work.

Next, there were parallel session.  I was torn because I wanted to hear some talks from both sessions, but I opted to stay in one room and listen to all the "Drug Design Ligand Interactions" session.  Once again, the talks were excellent.  Kevin Gardner gave a great talk, as always, and two student talks (by Quentin Chappuis from EPFL Lausanne and Johannes Bjornmeras from Stockholm U) were both riveting.  In the afternoon session, there were tutorials, including a lecture by one of the great NMR teachers in the world, Professor James Keeler from Cambridge.  He wrote a great book (which I own and read every chance I get) and posted many lectures on YouTube.  

Overall a great day!  THere is a special workshop tonight regarding the National Research Council Report on High Field Spectrometers in the US, which discusses NSF policy on mechanisms for funding and siting new high field NMRs.  A representative from NSF will be on hand.

Thursday, March 27, 2014

ENC 2014 Post 2

Another great day at the ENC.

The early AM session "Dynamics" included a very interesting talk by Cyril Charlier from CNRS, Paris.  The title is "Nanosecond Time Scale Motions in Proteins Revealed by High-Resolution NMR Rexalometry."  The concept is that it would be helpful to protein NMR people to measure T2 (and T1 and NOE) at many different fields, especially really low fields, to characterize fast time scale dynamics.  Of course, low fields means less polarization and resolution.  So you end up with lots of overlap and low signal-to-noise.  The authors realized that inside a NMR magnet -  it is only at the nominal field for a small area, then the field drops off.  So if you can design a "shuttle" system to move the sample inside the magnet you can turn a NMR at one field (say 800 MHz) to a magnet that has all fields from 800 MHz to 45 MHz.  The limitation (for the time being, at least) is that the probe sits at one spot, so you have to pulse and record at 800 MHz.  How can you use this trick.  Relaxometry.  They developed a variant of the HSQC in which you shuttle the sample up to low field for a "relaxation period" then shuttle back to acquire.  For every scan it has to jump up to low field then back down.  They also developed some theory and software to handle the combined effects of relaxation at high field (during pulses and acquisition), at low field (while it sits high in the magnet) and shuttling.  At this stage they've only reported applications to ubiquitin, but this idea is interesting and I want to see where it goes.

There were parallel sessions in the late AM.  I couldn't decide if I wanted to go to Biomolecular Structure and Function II or Hyperpolarization.  I decided to go the the Hyperpolarization, because this session featured non-DNP hyperpolarization (optical polarization, LLS and photo-cidnp).  I heard Silvia Cavagnero from UW-Madison discuss progress with her photo-cidnp system.  There were posters in the afternoon and parallel sessions on in vivo biomedical MRI/MRS and Solids. I probably should have attended the in vivo session, but by this time I was ready for a short break, so I took this opportunity to see the city and visit an old friend in Boston. 

Overall, a great day.  One more full day left and back home on Friday.  I'll probably make one more post on Friday.


PS - I'll mention that I saw the news reports on the huge fire in the Back Bay yesterday.  I just read on Twitter that two fire fighters were killed in the line of duty!  As a family member of a first responder, I am very saddened to hear about the loss of life of a fire fighter and I extend my heartfelt sympathy to the families, friends and co-workers.

Saturday, March 22, 2014

The 55th Experimental Nuclear Magnetic Resonance Conference

Hello all.

This week I will be blogging about the 55th Experimental Nuclear Magnetic Resonance Conference (ENC) in Boston, Ma.  This conference is the granddaddy of all NMR conferences and usually contains a very wide variety of talks on all sub-disciplines of NMR.

The program can be found at

I hope to give daily updates, but I don't want to make promises that I can't keep.  The hospitality suites are very inviting at ENC.  I am looking forward to a great week.


By the way, I have been slack on blogging lately due to several huge projects which are now complete.  I hope to get back to bi-weekly blog posts after ENC.

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

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 ( sitting inside a EPR dewar ( 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. 

*** 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


Monday, February 10, 2014

Papers I am reading ...

It has been a big week at Sit and Spin.  Last Tuesday morning just after I posted my review of an Angew. Chem. paper on NMR of very large protein complexes (, I jumped over to twitter to publicize the blog.  I don't remember the exact chain of events, but eventually my blog was mentioned by @SeeArrOh, an organic chemist and blogger ( with a wide readership.  As a consequence, the blog got more hits in one day than I had in the previous three months combined!  

In parallel, I saw an interesting news article on Nature ( highlighting the correlation between citations in research blogs and citations in the scientific literature.  This article reminded me why I got into the blogging game.  I think there is a lot of great research being done by hard-working and thoughtful scientists in the field of NMR.  I am concerned that the broader community of chemists is not aware of this work, or at least as aware as they are about work in other branches of chemistry.  Perhaps, in some small way, this blog can help to remedy this problem.  

Additionally, the Nature news article alerted me to the site  I am ashamed to admit that I was unaware of and underlying the movement underway in science communications.  This site is such a great aggregate of information.  I have spent so much time digging through it that I went ahead and applied for an account.  I would be honored if the editors include this blog.  

Of course with added attention there will be added pressure to write more posts.  It takes me a while to write a complete critique.  It probably takes almost as much time as it took me to make powerpoint slides for journal club in group meetings back when I was a post-doc.  And I only had to do that a few times per year!  I am trying to get a blog post out every other week.  

In case I don't get the opportunity to discuss all of them, here is a list of papers in my "to blog" pile.  I picked these out of the most prominent journals that publish NMR papers.  The criteria I use is the following: "Does the title and abstract seem interesting to a wide audience?"  If anyone out there finds something that I am missing, please contact me.  

Here is my list.  I'll try to get to two or three of these in the next month or so.

Hyperpolarization without persistent radicals for in vivo real-time metabolic imaging.

Eichhorn et al.

PNAS 2013 110 18064

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers.

Sekhar A, Kay LE.

PNAS 2013 110 12867

Diastereotopic splitting in the 13C NMR spectra of sulfur homofullerenes and methanofullerenes with chiral fragments.

Tulyabaev AR, Tuktarov AR, Khalilov LM.
MRC 2014 52, 3

Biophysical aspects of cyclodextrin interaction with paraoxon.

Soni SD, Bhonsle JB, Garcia GE.
MRC 2014 
Solution Structure of a G-quadruplex Bound to the Bisquinolinium Compound Phen-DC3.

Chung WJ, Heddi B, Hamon F, Teulade-Fichou MP, Phan AT.

Structure elucidation and NMR assignments of two unusual xanthones from Lomatogonium carinthiacum (Wulf) Reichb.

Wang Q, Bao B, Chen Y.

Structural elucidation and NMR assignments of two new pyrrolosesquiterpenes from Streptomyces sp. Hd7-21.

Liu DZ, Liang BW.

Automatic assignment of 1H-NMR spectra of small molecules.

Cobas C, Seoane F, Vaz E, Bernstein MA, Dominguez S, Pérez M, Sýkora S.