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Balaban and co-workers have shown that low molecular weight compounds with slowly exchanging –NH or –OH protons may be used to alter tissue contrast via chemical exchange dependent saturation transfer (CEST) of pre-saturated spins to bulk water (1,2). Van Zijl and coworkers have demonstrated that the CEST effect can be amplified considerably by using macromolecules with a large number of chemically equivalent, exchangeable NH groups and that the amide protons of intracellular proteins may be used in a CEST experiment to image tissue pH (3) or brain tumors (4-6). In all of the early reports (endogenous CEST agents or intrinsic protein exchanging sites), the molecules used to demonstrate the CEST effect are all diamagnetic and had proton chemical shifts not much different from that of bulk water (-NH and –OH groups are typically well within 5 ppm of bulk water). A complicating and potentially competing pathway for reducing the magnetization of bulk water is the magnetization transfer effect which was demonstrated by Balaban and coworkers (7) when they used a pre-saturation pulse to saturate the broad proton signal that lies beneath the sharper bulk water signal in many tissues. This broad resonance is thought to reflect water tightly associated with tissue proteins, lipids, and/or sub-cellular structures and appears to change with progressive diseases, such as multiple sclerosis, ALS, etc. Upon saturation, exchange occurs between the bound water pool and the larger bulk water pool resulting in a decrease in bulk water signal intensity. This technique is now commonly known as magnetization transfer (MT) imaging (8). Paramagnetic analogs of diamagnetic CEST compounds were not known until the discovery of lanthanide complexes having unusually slow water exchange kinetics (9,10). As shown below, such slow water exchange paramagnetic complexes (paramagnetic CEST agents or PARACEST agents) may offer several significant advantages over diamagnetic CEST agents. One of the major advantages of these PARACEST agents is the fact that the resonance of the bound water is shifted far away from the free water and that for some lanthanides (see Figure in the preliminary results section) is outside of the range where there any appreciable effects due to MT (+/- 100 ppm at 3T).


Physical Description of the PARACEST effect. Consider two exchanging water pools, A and B, with different NMR chemical shifts. Here pool B will mean a water molecule bound to the agent, while pool A will mean bulk water. If spin B is saturated by using a continuous, selective RF saturation pulse and if A and B undergo chemical exchange during this saturation period, then the intensity of A will decrease to a new level. At steady-state, this new level is given by MA°/MA0 = 1/ (1 + kAT1A). This indicates that chemical exchange saturation transfer (CEST) effect will be observed if the T1 of pool A (bulk water) is long compared to the lifetime (1/kA) of water at this site. The model also indicates that the water at site B must have a different chemical shift than the water at site A. For maximum efficiency, the chemical system must be in the slow exchange on the NMR chemical shift time-scale. A major advantage of paramagnetic systems is that bound water molecule’s resonance is shifted much further away from the bulk water and thus the bound water lifetime can be much shorter (faster water exchange) without approaching the fast exchange limit. As we have shown, the effects of PARACEST agents can be detected at concentrations that may be well below those currently used in clinical studies. A second advantage of PARACEST agents over typical Gd3+-based agents is that they can be turned on-and-off at some specified irradiation frequency. Thus, the contrast effect can be modulated during an imaging experiment, and this could further lower the detection limit of these agents in tissue. Furthermore, these agents can be used to map micro-environment properties, such as pH, that the conventional, relaxation based agents cannot do.



 LL_Cest_fig1 LL_Cest_fig2





In the simplest CEST experiment, one applies frequency-selective RF irradiation to the agent bound spins until all nuclear magnetizations are at steady-state (i.e., the time derivatives are zero) and then samples any remaining Z magnetization of the bulk water protons (pool A) by applying a non-selective sampling RF pulse (usually a 90o pulse) to the water. Experimentally, the CEST effect can be determined from a plot of residual bulk water proton Z magnetization versus the frequency offset of the saturation pulse varied over a range of frequencies that includes the Larmor frequencies of both pools of protons. The resulting plot is referred to as a Z-spectrum (11) or a CEST spectrum (1). To obtain the optimal PARACEST effect, it would be desirable to know how the chemical properties of a PARACEST agent (concentration and exchange rates) and NMR parameters (chemical shift, T1, and T2, offset frequency, strength of RF irradiation) alter the Z-spectrum in a quantitative manner using a sound theoretical framework. One appropriate framework to describe this liquid system is the set of fundamental Bloch equations (12) modified to include the effects of chemical exchange (13,14). Van Zijl and co-workers (4) have derived expressions for the diamagnetic CEST effect. We have derived a numerical solution for the PARACEST effect for a three pool model (see (15)). In the most desirable situation, the selective RF irradiation of the exchangeable protons in a CEST agent does not directly affect the bulk water protons. The maximum PARACEST effect occurs when the RF completely saturates the protons of the agent so that their Z magnetization is zero.


Toxicity and Nephrogenic Systemic Fibrosis (NSF). The association between the administration of gadolinium based contrast agents and nephrogenic systemic fibrosis (NSF) has received a great deal of recent attention. It has been known for many years that free Gd3+ is toxic (16). The fact that free Gd3+ is toxic is reported almost universally in all of the discussion sections of articles describing NSF. The authors of most of these articles then proceed to discuss the likelihood that the approved contrast agent dissociates to deposit free Gd3+ in terms of thermodynamic stability constants, conditional stability constants, and/or selective stability constants. While these parameters are important in that they reflect the thermodynamic stability of these gadolinium chelates, which in turn allows an estimate of free noncomplexed Gd3+ at equilibrium under a variety of conditions (both in vitro and in vivo), they do not provide the full story as to why some organic ligands are more likely than others to release more Gd3+. The missing component in most of the discussions published to date is the question of how fast dissociation approaches equilibrium in vivo. This question is based on chemical kinetics—not thermodynamics—a topic largely overlooked in current discussions of the physicochemical properties of these chelates. The importance of these considerations is that the stability constant governs the behavior of the agent when it reaches equilibrium or steady state, while the kinetics govern how rapidly each agent approaches steady state in vivo.

Lanthanide chemists have long recognized that in addition to having favorable thermodynamic stabilities, these chelates must also be kinetically inert—that is, their rates of formation and dissociation must be slow. It is critical to emphasize that the rates of formation—that is, how quickly these chelates form and how rapidly they reach equilibrium—are determined by characteristics that are independent of the thermodynamic stability. The rate of dissociation serves as an important parameter for comparing contrast agents in the context of the free gadolinium hypothesis—that the deposition of free Gd3+ in tissue induces NSF. The thermodynamic stability constant determines the concentrations of gadolinium chelate, free chelate, and free gadolinium at equilibrium, while the rates of formation and dissociation determine how rapidly these compounds reach equilibrium. In principle, it is possible for a chelate to have a relatively low stability constant and a slow rates of formation and dissociation; this phenomenon results in a “kinetically trapped” chelate that does not dissociate on any relevant time scale. Most lanthanide chelates have very slow rates of formation and dissociation (17).

On the basis of these considerations, the rates of transmetallation by endogenous ions should reflect only the rate-limiting step—that is, the rate of dissociation of the Gd3+ ion from each chelate. Thus, measured transmetallation rates should be surrogates for rates of dissociation of Gd3+ from these chelates. Laurent et al (18,19) determined the rates of transmetallation by zinc(II) for six approved gadolinium-based contrast agents at a neutral pH. After 5000 minutes (3.5 days), no appreciable transmetallation was observed for the DOTA based macrocyclic chelates (gadoterate meglumine, gadoteridol, and gadobutrol), whereas the linear chelates (gadopentetate dimeglumine, gadodiamide, and gadobenate dimeglumine) showed much more rapid transmetallation.

The biologic relevance of the kinetic properties of some of these contrast agents has also been studied. In 1992, Wedeking et al (20) examined the chemical properties of a number of gadolinium chelates, including their stability constants, ionic charge, lipophilicity, and size. They found that the thermodynamic and conditional stability constants could give only partial indications of which chelate was likely to induce residual Gd3+ deposition in mice. These authors found a very strong correlation between the dissociation rates of chelates in acid and the long-term deposition of Gd3+ in rat tissues, such as liver and bone (femur). The transmetallation study findings directly reflect the different kinetic stabilities of the chelates at a neutral pH and support the results of Wedeking et al (20), who found no detectable free Gd3+ in the livers and femurs of mice after intravenous administration of a macrocyclic chelate. More recently, it was shown that levels of Gd3+ in bone were markedly lower (2.5–4.0 times, depending on the analytic method used) (21,22) in patients who had received gadoteridol (a macrocyclic agent) than in those who had received gadodiamide (a linear agent).

Given these chemical and biologic factors, we suggest that discussions of the relative safety of lanthanide-based contrast agents include strong consideration of their kinetic inertness. Although pertinent related data are relatively sparse, chemical and physical considerations clearly indicate that if other factors are equal, macrocyclic agents are far less likely to dissociate and hence release free Ln3+ in vivo. If the free gadolinium hypothesis is correct—that is, if the deposition of free Gd3+ in tissue induces NSF—then the risk of NSF should be minimized with use of macrocyclic agents and the risk of these PARACEST agents inducing NSF is minimized.

In addition, most MRI centers have adjusted their clinical practice to avoid injecting patients with contrast agents if there is any suspicion of renal insufficiency. Practice guidelines have been published by the American College of Radiology (ACR) (23), and if approved for human use. We suggest that the PARACEST agents will be subject to similar guidelines for their use. It is important to note that since the widespread adoption of the ACR guidelines there were no new cases of NSF reported to the FDA in 2008 and very few since then.


Responsive PARACEST Agents. This topic was covered in a more general review of responsive MR contrast agents written by Yoo and Pagel (24). In general, for gadolinium based responsive contrast agents, the chelates are designed to have alterations in relaxivity in response to specific changes in their micro-environment. For example, Vasovist, an intravascular agent which recently received clearance for human studies by the FDA, binds to human serum albumin (HSA), leading to higher relaxivity through an increased tumbling time of the adduct upon binding to this larger protein. Another example of a responsive gadolinium based contrast agent was the seminal report by Meade’s group (25) who reported on a caged chelate with no inner sphere water of hydration. This compound had increased relaxivity because of increased water accessibility to the inner sphere after beta-galactosidase enzymatically cleaved a galactopyranose ligand from the contrast agent. This agent was used to monitor the expression of beta-galactosidase in vivo.

The design of responsive PARACEST agents is based on synthesizing chelates that exhibit changes in either the chemical shift of the bound water or other exchangeable protons on the chelate and/or their lifetime(s) of exchange in response to specific alterations in their micro-environment. Examples of responsive PARACEST agents include agents that respond to temperature 26,27), redox (28), pH (29), glucose (30), lactate (31), nitric oxide (32), and enzyme activity (33). At UTSW we are focusing on an intravascular PARACEST agent and pH sensitive agents.



Literature Cited

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  2. Ward K, Balaban RS. Determination of pH using water protons and chemical exchange-dependent saturation transfer (CEST). Magn Reson Med 2000;44:799-802.
  3. Goffeney N, Bulte JW, Duyn J, Bryant LHJ, van Zijl PCM. Sensitive NMR detection of cationic-polymer-based gene delivery systems using saturation transfer via proton exchange. Journal of the American Chemical Society 2001;123:8628-8629.
  4. Snoussi K, Bulte JW, Gueron M, van Zijl PCM. Sensitive CEST Agents Based on Nucleic Acid Imino Proton Exchange: Detection of Poly(rU) and a Dendrimer-Poly(rU) Model for Nucleic Acid Delivery and Pharmacology. Magn Reson Med 2003;49:998-1005.
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  10. Zhang S, Wu K, Sherry AD. Unusually sharp dependence of water exchange rate versus Lanthanide ionic radii for a series of tetraamide complexes. Journal of the American Chemical Society 2002;124:4226-4227.
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  15. Woessner DE, Zhang S, Merritt ME, Sherry AD. Numerical solution of the bloch equations provides insights into the optimum design of PARACEST agents for MRI. Magn Reson Med 2005;53(4):790-799.
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  17. Brucher E. Kinetic stabilities of gadolinium(III) chelates used as MRI contrast agents. Krause W, editor. Berlin: Springer -Verlag; 2002. 103-122 p.
  18. Laurent S, Elst LV, Copoix F, Muller RN. Stability of MRI paramagnetic contrast media: a proton relaxometric protocol for transmetallation assessment. Invest Radiol 2001;36(2):115-122.
  19. Laurent S, Elst LV, Muller RN. Comparative study of the physicochemical properties of six clinical low molecular weight gadolinium contrast agents. Contrast Media Mol Imaging 2006;1(3):128-137.
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  21. Gibby WA, Gibby KA, Gibby WA. Comparison of Gd DTPA-BMA (Omniscan) versus Gd HP-DO3A (ProHance) retention in human bone tissue by inductively coupled plasma atomic emission spectroscopy. Invest Radiol 2004;39(3):138-142.
  22. White GW, Gibby WA, Tweedle MF. Comparison of Gd(DTPA-BMA) (Omniscan) versus Gd(HP-DO3A) (ProHance) relative to gadolinium retention in human bone tissue by inductively coupled plasma mass spectroscopy. Invest Radiol 2006;41(3):272-278.
  23. Kanal E, Barkovich AJ, Bell C, Borgstede JP, Bradley WG, Froelich JW, Gilk T, Gimbel JR, Gosbee J, Kuhni-Kaminski E, Lester JW, Nyenhuis J, Parag Y, Schaefer DJ, Sebek-Scoumis EA, Weinreb J, Zaremba LA, Wilcox P, Lucey L, Sass N, Safety ACRBRPM. ACR Guidance Document for Safe MR Practices: 2007. American Journal of Roentgenology 2007;188(6):1447-1474.
  24. Yoo B, Pagel MD. An overview of responsive MRI contrast agents for molecular imaging. Front Biosci 2008;13:1733-1752.
  25. Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Fraser SE, Meade TJ. In vivo visualization of gene expression using magnetic resonance imaging. Nature Biotechnology 2000;18(3):321-325.
  26. Li AX, Wojciechowski F, Suchy W, Jones CK, Hudson RHE, Menon RS, Bartha R. A sensitive PARACEST contrast agent for temperature MRI: Eu3+-DOTAM-glycine (Gly)-phenylalanine (Phe). Magn Reson Med 2008;59(2):374-381.
  27. Zhang S, Malloy CR, Sherry AD. MRI Thermometry Based on PARACEST Agents. Journal of the American Chemical Society 2005;127(50):17572-17573.
  28. Ratnakar SJ, Woods M, Lubag AJM, Kovacs Z, Sherry AD. Modulation of Water Exchange in Europium(III) DOTA-Tetraamide Complexes via Electronic Substituent Effects. Journal of the American Chemical Society 2008;130(1):6-7.
  29. Aime S, Castelli DD, Terreno E. Novel pH-reporter MRI contrast agents. Angew Chem Int Ed 2002;41(22):4334-4336.
  30. Zhang S, Trokowski R, Sherry AD. A Paramagnetic CEST Agent for Imaging Glucose by MRI. Journal of the American Chemical Society 2003;125(50):15288-15289.
  31. Aime S, Castelli DD, Fedeli F, Terreno E. A Paramagnetic MRI-CEST Agent Responsive to Lactate Concentration. Journal of the American Chemical Society 2002;124(32):9364-9365.
  32. Liu G, Li Y, Pagel MD. Design and Characterization of a New Irreversible Responsive PARACEST MRI Contrast Agent that Detects Nitric Oxide. Magn Reson Med 2007;58:1249-1256.
  33. Byunghee Y, Raam MS, Rosenblum RM, Pagel MD. Enzyme-responsive PARACEST MRI contrast agents: a new biomedical imaging approach for studies of the proteasome. Contrast Media & Molecular Imaging 2007;2(4):189-198.

CEST and PARACEST Publications


  1. Zhang S, Merritt M, Woessner DE, Lenkinski RE, Sherry AD. PARACEST Agents: Modulating MRI Contrast via Water Proton Exchange. Accounts of Chemical Research. 2003;36(10):783-90.
  2. Vinogradov E, Zhang S, Lubag A, Balschi JA, Sherry AD, Lenkinski RE. On-resonance low B1 pulses for imaging of the effects of PARACEST agents. Journal of Magnetic Resonance. 2005;176(1):54-63.
  3. Vinogradov E, He H, Lubag A, Baischi JA, Sherry AD, Lenkinski RE. MRI detection of paramagnetic chemical exchange effects in mice kidneys in vivo. Magnetic Resonance in Medicine. 2007;58(4):650-5.
  4. Dixon WT, Hancu I, James Ratnakar S, Dean Sherry A, Lenkinski RE, Alsop DC. A multislice gradient echo pulse sequence for CEST imaging. Magnetic Resonance in Medicine. 2010;63(1):253-6.
  5. Dixon WT, Ren J, Lubag AJM, Ratnakar J, Vinogradov E, Hancu I, Lenkinski RE, Sherry AD. A concentration-independent method to measure exchange rates in PARACEST agents. Magnetic Resonance in Medicine. 2010;63(3):625-32.
  6. Hancu I, Dixon WT, Woods M, Vinogradov E, Sherry AD, Lenkinski RE. CEST and PARACEST MR contrast agents. Acta Radiologica. 2010;51(8):910-23.
  7. Vinogradov E, Soesbe TC, Balschi JA, Dean Sherry A, Lenkinski RE. PCEST: Positive contrast using Chemical Exchange Saturation Transfer. Journal of Magnetic Resonance. 2011;215:64-73.
  8. Varma G, Lenkinski RE, Vinogradov E. Keyhole chemical exchange saturation transfer. Magnetic Resonance in Medicine. 2012;68(4):1228-33.
  9. Vinogradov E, Sherry AD, Lenkinski RE. CEST: From basic principles to applications, challenges and opportunities. Journal of Magnetic Resonance. 2013;229:155-72.