Developed Pulse Sequence Research Articles

T1rho and T2rho based myocardial tissue characterisation in small animals at high-field MRI

Abstract Introduction Cardiac tissue characterisation using T2-based MRI promises non-invasive diagnosis or monitoring of inflammatory heart disease, toxic heart damage after chemotherapy and myocardial infarction or post-infarction patients. T2 relaxation is also an indicator for the detection of myocardial oedema or intramyocardial haemorrhage [1]. However, accurate T2 quantification is challenging due to motion, blood flow and field inhomogeneities, especially in high-field MRI [2]. In this work, we present an alternative to T2 imaging that uses spin-locking (SL) to provide a robust measurement of T2ρ relaxation. It is known from relaxation theory that T2ρ has a sensitivity almost identical to T2 [3]. In the small animal study presented here, cardiac T2ρ quantification is tested for the first time using a newly developed pulse sequence and compared with established methods. Methods All measurements were performed on a preclinical 7T MRI. T2 contrast was prepared using a CPMG (Carr-Purcell-Meiboom-Gill) sequence. T2ρ contrast was conventionally generated by a balanced-SL (BSL) preparation [4]. Furthermore, a new approach for T2ρ was developed based on a MLEV sequence (Malcolm-Levitt, [2]). In contrast to [2], all pulse delays were minimised here, producing pure T2ρ contrast (Fig. 1). The new method was first validated in phantom experiments and then tested in three mice with radial image acquisition accelerated for cardio-MRI. Results The results of the phantom study are shown in figure 2. No significant differences were found between BSL and MLEV for T2ρ quantification (mean deviation 0.74%). In vivo, significant artefacts were observed for T2 (Fig. 3), while both T2ρ methods showed improved image quality. For the evaluation of relaxation times in the left ventricle, the mean across all animals was: T2=22.9±2.6ms (R2=0.979), BSL-T2ρ=51.4±5.4ms (R2=0.983) and MLEV-T2ρ=60.0±3.4ms (R2>0.99). Thus, the MLEV-T2ρ method achieves the most robust quantification with a mean R2>0.99 and provides relaxation time maps with diagnostic image quality. Discussion: In the present study, myocardial T2ρ mapping was tested at 7T. While no significant differences between BSL and MLEV appeared in phantom experiments, significant difference was found in vivo. Here, only the MLEV technique could achieve robust quantification. One reason for the improved performance is the continuous refocusing and minimisation of free dephasing of the transverse magnetisation. The results also show that MLEV-T2ρ has improved image quality and more robust quantification compared to CPMG-T2. Conclusion Cardiac T2ρ quantification is a robust alternative to established T2 imaging and can be applied under high-field conditions. A combined measurement of T1ρ and T2ρ relaxation time could be used for native detection of myocardial fibrosis and oedema.Figure 1)Comparison of image qualityFigure 2)Results in a knockout model

Selectively Probing the Magnetic Resonance Signals of γ-Aminobutyric Acid in Human Brains In Vivo.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in human brains, playing a role in the pathogenesis of various psychiatric disorders. Current methods have some non-neglectable shortcomings and noninvasive and accurate detection of GABA in human brains is long-term challenge. To develop a pulse sequence capable of selectively detecting and quantifying the 1 H signal of GABA in human brains based on optimal controlled spin singlet order. Prospective. A phantom of GABA (pH = 7.3 ± 0.1) and 11 healthy subjects (5 females and 6 males, body mass index: 21 ± 3 kg/m2 , age: 25 ± 4 years). 7 Tesla, 3 Tesla, GABA-targeted magnetic resonance spectroscopy (GABA-MRS-7 T, GABA-MRS-3 T), magnetization prepared two rapid acquisition gradient echoes sequence. By using the developed pulse sequences applied on the phantom and healthy subjects, the signals of GABA were successfully selectively probed. Quantification of the signals yields the concentration of GABA in the dorsal anterior cingulate cortex (dACC) in human brains. Frequency. The 1 H signals of GABA in the phantom and in the human brains of healthy subjects were successfully detected. The concentration of GABA in the dACC of human brains was 3.3 ± 1.5 mM. The developed pulse sequences can be used to selectively probe the 1 H MR signals of GABA in human brains in vivo. 1 TECHNICAL EFFICACY STAGE: 1.

МР–РЕЛАКСОМЕТРИЯ В ДИАГНОСТИКЕ РАБДОМИОЛИЗА

Purpose. Assessing the possibilities of quantitative MRI in the diagnosis and monitoring of the course of rhabdomyolysis of various etiologies. Materials and methods. The results of MRI performed by 80 patients with clinical manifestations of rhabdomyolysis were analyzed. The diagnosis of rhabdomyolysis was confirmed by measuring the level of myoglobin (more than 70 μg/l) and CPK (more than 1000 IU/l) in the absence of laboratory and instrumental signs of myocardial damage. Scanning was carried out using T1, T2, STIR protocols, as well as the developed pulse sequences for calculating the relaxation times T1, T2 and T2*. Patients underwent MR examination at least 3 times during the course of the disease. Results. In 48 cases was detected a hyperintensive signal from muscle tissue on the T2 and STIR protocols. In 25 cases were identified MR signs of rhabdomyolysis, 16 of which were confirmed by laboratory methods and or biopsy of the affected muscle. One patient with a blood myoglobin level (577 ng/ml) showed no pathological changes in MR images. Thus, in the analyzed group, the sensitivity and specificity were 94% and 71%. Quantification protocols obtained relaxation times for the affected muscle tissue. The ROC analysis method determined cutoff thresholds for rhabdomyolysis: T1 greater than 1152 ms, T2 greater than 63 ms and T2* greater than 48 ms with sensitivity and specificity, respectively: 94% and 73%, 94% and 84%, 88% and 85%. Dynamic changes in the relaxation time during the recovery of patients had correlation relationships of high and medium strength with the main laboratory markers of rhabdomyolysis. The highest values of the correlation coefficient were found between T2 and the activity of CPK, ALS, and AST (rs = 0.805, 0.813, and 0.803; p <0.05). Conclusion. An improved technique for muscle MRI with a quantitative assessment of T2 relaxation time has high sensitivity and specificity (94% and 82%) in the diagnosis of rhabdomyolysis. Recovery of

Detecting resting-state brain activity using OEF-weighted imaging

Traditional resting-state functional magnetic resonance imaging (fMRI) is mainly based on the blood oxygenation level-dependent (BOLD) contrast. The oxygen extraction fraction (OEF) represents an important parameter of brain metabolism and is a key biomarker of tissue viability, detecting the ratio of oxygen utilization to oxygen delivery. Investigating spontaneous fluctuations in the OEF-weighted signal is crucial for understanding the underlying mechanism of brain activity because of the immense energy budget during the resting state. However, due to the poor temporal resolution of OEF mapping, no studies have reported using OEF contrast to assess resting-state brain activity. In this fMRI study, we recorded brain OEF-weighted fluctuations for 10 min in healthy volunteers across two scanning visits, using our recently developed pulse sequence that can acquire whole-brain voxel-wise OEF-weighted signals with a temporal resolution of 3 s. Using both group-independent component analysis and seed-based functional connectivity analysis, we robustly identified intrinsic brain networks, including the medial visual, lateral visual, auditory, default mode and bilateral executive control networks, using OEF contrast. Furthermore, we investigated the resting-state local characteristics of brain activity based on OEF-weighted signals using regional homogeneity (ReHo) and fractional amplitude of low-frequency fluctuations (fALFF). We demonstrated that the gray matter regions of the brain, especially those in the default mode network, showed higher ReHo and fALFF values with the OEF contrast. Moreover, voxel-wise test-retest reliability comparisons across the whole brain demonstrated that the reliability of resting-state brain activity based on the OEF contrast was moderate for the network indices and high for the local activity indices, especially for ReHo. Although the reliabilities of the OEF-based indices were generally lower than those based on BOLD, the reliability of OEF-ReHo was slightly higher than that of BOLD-ReHo, with a small effect size, which indicated that OEF-ReHo could be used as a reliable index for characterizing resting-state local brain activity as a complement to BOLD. In conclusion, OEF can be used as an effective contrast to study resting-state brain activity with a medium to high test-retest reliability.

CoreMRI: A high-performance, publicly available MR simulation platform on the cloud.

IntroductionA Cloud-ORiented Engine for advanced MRI simulations (coreMRI) is presented in this study. The aim was to develop the first advanced MR simulation platform delivered as a web service through an on-demand, scalable cloud-based and GPU-based infrastructure. We hypothesized that such an online MR simulation platform could be utilized as a virtual MRI scanner but also as a cloud-based, high-performance engine for advanced MR simulations in simulation-based quantitative MR (qMR) methods.Methods and resultsThe simulation framework of coreMRI was based on the solution of the Bloch equations and utilized a ground-up-approach design based on the principles already published in the literature. The development of a front-end environment allowed the connection of the end-users to the GPU-equipped instances on the cloud. The coreMRI simulation platform was based on a modular design where individual modules (such as the Gadgetron reconstruction framework and a newly developed Pulse Sequence Designer) could be inserted in the main simulation framework. Different types and sources of pulse sequences and anatomical models were utilized in this study revealing the flexibility that the coreMRI simulation platform offers to the users. The performance and scalability of coreMRI were also examined on multi-GPU configurations on the cloud, showing that a multi-GPU computer on the cloud equipped with a newer generation of GPU cards could significantly mitigate the prolonged execution times that accompany more realistic MRI and qMR simulations.ConclusionscoreMRI is available to the entire MR community, whereas its high performance and scalability allow its users to configure advanced MRI experiments without the constraints imposed by experimentation in a true MRI scanner (such as time constraint and limited availability of MR scanners), without upfront investment for purchasing advanced computer systems and without any user expertise on computer programming or MR physics. coreMRI is available to the users through the webpage https://www.coreMRI.org.

Measuring the signs of the methyl 1H chemical shift differences between major and 'invisible' minor protein conformational states using methyl 1H multi-quantum spectroscopy.

Carr-Purcell-Meiboom-Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and 'invisible' minor states. The exchange rate(s) ([Formula: see text]), population(s) of the minor state(s) and the absolute value of the chemical shift difference [Formula: see text] (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of [Formula: see text] that is required to reconstruct the spectrum of the 'invisible' minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl [Formula: see text] CPMG experiment (Yuwen in Angew Chem 55:11490-11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of [Formula: see text] is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~18kDa cavity mutant of T4 Lysozyme ([Formula: see text]) and (2) the [Formula: see text]kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus ([Formula: see text]).

Measurement of 15N longitudinal relaxation rates in 15NH4+ spin systems to characterise rotational correlation times and chemical exchange

Many chemical and biological processes rely on the movement of monovalent cations and an understanding of such processes can therefore only be achieved by characterising the dynamics of the involved ions. It has recently been shown that 15N-ammonium can be used as a proxy for potassium to probe potassium binding in bio-molecules such as DNA quadruplexes and enzymes. Moreover, equations have been derived to describe the time-evolution of 15N-based spin density operator elements of 15NH4+ spin systems. Herein NMR pulse sequences are derived to select specific spin density matrix elements of the 15NH4+ spin system and to measure their longitudinal relaxation in order to characterise the rotational correlation time of the 15NH4+ ion as well as report on chemical exchange events of the 15NH4+ ion. Applications to 15NH4+ in acidic aqueous solutions are used to cross-validate the developed pulse sequence while measurements of spin-relaxation rates of 15NH4+ bound to a 41kDa domain of the bacterial Hsp70 homologue DnaK are presented to show the general applicability of the derived pulse sequence. The rotational correlation time obtained for 15N-ammonium bound to DnaK is similar to the correlation time that describes the rotation about the threefold axis of a methyl group. The methodology presented here provides, together with the previous theoretical framework, an important step towards characterising the motional properties of cations in macromolecular systems.

Reduction of Scan Time and Echo Time in 3D Time-of-Flight (TOF) MR Angiography, Using Reverse Elliptical Centric Dual-Echo Acquisition

In the conventional single-echo three dimension (3D) time-of-flight (TOF) magnetic resonance angiography (MRA), it always takes long scan time to obtain the good images. In addition, high-resolution MRA with flow compensation requires long echo time (TE). Here, a reverse elliptical centric dual-echo 3D TOF MRA pulse sequence was developed to reduce the scan time and minimize the effective TE. Dual-echo and reverse elliptical centric acquisition in the phase encoding and slice phase encoding direction were used to reduce the scan time by 61%. Furthermore, the effective echo time (TE) was also minimized from 4.8ms to 4.1ms. The developed pulse sequence was tested on clinical MRI systems and the contrast to noise ratio (CNR) was measured to evaluate the results. The results show that the CNR only decrease by 2.5% with the scan time reduced to 39% when compared with the conventional single-echo 3D TOF. Compared with the conventional dual-echo 3D TOF acquisition, the developed pulse sequence brings advantages in three aspects: (1) reduce scan time by 22%; (2) increase CNR by 10%; (3) reduce the artifacts induced by dual-echo effects. In addition, while keeping the similar scan time as the conventional single-echo 3D TOF acquisition, the results show that reverse elliptical centric dual-echo 3D TOF acquisition increase CNR by 54.3%.

Abnormal striatal resting-state functional connectivity in adolescents with obsessive–compulsive disorder

Neuroimaging research has implicated abnormalities in cortico-striatal-thalamic-cortical (CSTC) circuitry in pediatric obsessive–compulsive disorder (OCD). In this study, resting-state functional magnetic resonance imaging (R-fMRI) was used to investigate functional connectivity in the CSTC circuitry in adolescents with OCD. Imaging was obtained with the Human Connectome Project (HCP) scanner using newly developed pulse sequences which allow for higher spatial and temporal resolution. Fifteen adolescents with OCD and 13 age- and gender-matched healthy controls (ages 12–19) underwent R-fMRI on the 3T HCP scanner. Twenty-four minutes of resting-state scans (two consecutive 12-min scans) were acquired. We investigated functional connectivity of the striatum using a seed-based, whole brain approach with anatomically-defined seeds placed in the bilateral caudate, putamen, and nucleus accumbens. Adolescents with OCD compared with controls exhibited significantly lower functional connectivity between the left putamen and a single cluster of right-sided cortical areas including parts of the orbitofrontal cortex, inferior frontal gyrus, insula, and operculum. Preliminary findings suggest that impaired striatal connectivity in adolescents with OCD in part falls within the predicted CSTC network, and also involves impaired connections between a key CSTC network region (i.e., putamen) and key regions in the salience network (i.e., insula/operculum). The relevance of impaired putamen-insula/operculum connectivity in OCD is discussed.

Multi-spin echo spatial encoding provides three-fold improvement of temperature precision during intermolecular zero quantum thermometry.

Intermolecular multiple quantum coherences (iMQCs) are a source of MR contrast with applications including temperature imaging, anisotropy mapping, and brown fat imaging. Because all applications are limited by signal-to-noise ratio (SNR), we developed a pulse sequence that detects intermolecular zero quantum coherences with improved SNR. A previously developed pulse sequence that detects iMQCs, HOMOGENIZED with off resonance transfer (HOT), was modified with a multi-spin echo spatial encoding scheme (MSE-HOT). MSE-HOT uses a series of refocusing pulses to generate a stack of images that are averaged in postprocessing for higher SNR. MSE-HOT performance was quantified by measuring its temperature accuracy and precision during hyperthermia of ex vivo red bone marrow samples. MSE-HOT yielded a three-fold improvement in temperature precision relative to previous pulse sequences. Sources of improved precision were 1) echo averaging and 2) suppression of J-coupling in the methylene protons of fat. MSE-HOT measured temperature change with an accuracy of 0.6°C. MSE-HOT improved the temperature accuracy and precision of HOT to a level that is sufficient for hyperthermia of bone marrow.

Composite-180° pulse-based symmetry sequences to recouple proton chemical shift anisotropy tensors under ultrafast MAS solid-state NMR spectroscopy

There is considerable interest in the measurement of proton (1H) chemical shift anisotropy (CSA) tensors to obtain deeper insights into H-bonding interactions which find numerous applications in chemical and biological systems. However, the presence of strong 1H/1H dipolar interaction makes it difficult to determine small size 1H CSAs from the homogeneously broadened NMR spectra. Previously reported pulse sequences for 1H CSA recoupling are prone to the effects of radio frequency field (B1) inhomogeneity. In the present work we have carried out a systematic study using both numerical and experimental approaches to evaluate γ-encoded radio frequency (RF) pulse sequences based on R-symmetries that recouple 1H CSA in the indirect dimension of a 2D 1H/1H anisotropic/isotropic chemical shift correlation experiment under ultrafast magic angle spinning (MAS) frequencies. The spectral resolution and sensitivity can be significantly improved in both frequency dimensions of the 2D 1H/1H correlation spectrum without decoupling 1H/1H dipolar couplings but by using ultrafast MAS rates up to 70kHz. We successfully demonstrate that with a reasonable RF field requirement (<200kHz) a set of symmetry-based recoupling sequences, with a series of phase-alternating 270°0–90°180 composite-180° pulses, are more robust in combating B1 inhomogeneity effects. In addition, our results show that the new pulse sequences render remarkable 1H CSA recoupling efficiency and undistorted CSA lineshapes. Experimental results on citric acid and malonic acid comparing the efficiencies of these newly developed pulse sequences with that of previously reported CSA recoupling pulse sequences are also reported under ultrafast MAS conditions.

Pulse sequences for uniform perfluorocarbon droplet vaporization and ultrasound imaging

Phase-change contrast agents (PCCAs) consist of liquid perfluorocarbon droplets that can be vaporized into gas-filled microbubbles by pulsed ultrasound waves at diagnostic pressures and frequencies. These activatable contrast agents provide benefits of longer circulating times and smaller sizes relative to conventional microbubble contrast agents. However, optimizing ultrasound-induced activation of these agents requires coordinated pulse sequences not found on current clinical systems, in order to both initiate droplet vaporization and image the resulting microbubble population. Specifically, the activation process must provide a spatially uniform distribution of microbubbles and needs to occur quickly enough to image the vaporized agents before they migrate out of the imaging field of view. The development and evaluation of protocols for PCCA-enhanced ultrasound imaging using a commercial array transducer are described. The developed pulse sequences consist of three states: (1) initial imaging at sub-activation pressures, (2) activating droplets within a selected region of interest, and (3) imaging the resulting microbubbles. Bubble clouds produced by the vaporization of decafluorobutane and octafluoropropane droplets were characterized as a function of focused pulse parameters and acoustic field location. Pulse sequences were designed to manipulate the geometries of discrete microbubble clouds using electronic steering, and cloud spacing was tailored to build a uniform vaporization field. The complete pulse sequence was demonstrated in the water bath and then in vivo in a rodent kidney. The resulting contrast provided a significant increase (>15dB) in signal intensity.

RES-TOCSY: A facile approach for accurate determination of magnitudes, and relative signs of nJHF

The RES-TOCSY experiment for accurate determination of heteronuclear nJHF is reported. The main feature of the proposed technique is the accurate measurement of magnitudes of heteronuclear couplings from the displacement of cross sections of the 2D spectrum and their relative signs from the slopes of their displacement vectors. The experiment is highly advantageous as the couplings of smaller magnitudes hidden within line widths could also be accurately determined, and also in situations when the spectrum does not display any coupling fine structures. The efficient utility of the developed pulse sequence is unambiguously established on fluorine containing aromatic and aliphatic molecules.

15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide (15)N is used as a probe of solvent exchange. The experiment is performed in 50% H(2)O-50% D(2)O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, (15)N(D), into protonated species, (15)N(H), as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of (15)N(H/D)-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5-2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.

Reduction of flow-related signal loss in flow-compensated 3D TOF MR angiography, using variable echo time (3D TOF-VTE).

High-resolution MRA with phase/frequency flow compensation may require very long echo times (TEs). Variable TE (VTE) was implemented into flow-compensated 3D TOF to minimize the effective TE and reduce the flow-related signal void. The k-space of the 3D TOF was divided into segment groups ranging from two to 32 segments with different TEs. The TEs were minimized and the flow-compensation gradient lobes were calculated to null the total first moment at the peak of the echo for each segment. Possible artifacts and off-resonance effects were evaluated, with respect to the number of TE segments, using the point spread function (PSF) and corresponding experiments. The optimal number of TE segments for the least artifact was determined to be one-half of the number of slices. Two types of artifacts caused by VTE were predicted and subsequently observed. The developed pulse sequence 3D TOF-VTE was tested on clinical MRI systems, by performing scans of the cervical carotid artery and intracranial carotid artery at the carotid siphon. The signal distribution near the bifurcation and the siphon was much more uniform with VTE, and the flow-related signal loss was greatly reduced. The resultant MR angiograms provided improved vessel detail. The results show that VTE improved the quality of flow-compensated 3D TOF MRA.

Direct NMR evidence that prolidase is specific for the trans isomer of imidodipeptide substrates

The in vitro hydrolysis by porcine kidney prolidase of the imidodipeptide L-alanyl-L-proline was monitored by using 1H high-resolution NMR spectroscopy. The dipeptide exists as an equilibrium mixture of isomers with cis or trans conformation about the peptide bond. The 13C and 1H NMR spectra of the dipeptide displayed well-resolved resonances for each isomer. Inversion-transfer NMR spectroscopy, with a recently developed pulse sequence, was used with a range of temperatures to calculate the unitary rate constants for the exchange between isomers. A new analytical procedure was introduced for directly obtaining estimates of the unitary rate constants from inversion-transfer data. Arrhenius analysis yielded an activation energy for the isomerization of 87.0 +/- 4.1 kJ mol-1. 1H NMR time courses of the prolidase-catalyzed hydrolysis of L-alanyl-L-proline showed a faster removal of the trans isomer as the [enzyme]/[substrate] ratio was increased. The transient-kinetic information coupled with the steady-state kinetic parameters of the enzyme was used to develop two possible models of the overall hydrolytic reaction. Numerical integration of the relevant differential equations using the experimentally determined rate constants gave simulated progress curves that enabled selection of one of the proposed schemes as being the most likely; this proposal entailed absolute specificity of prolidase for the trans isomer of L-alanyl-L-proline. Finally, on the basis of the present work, and information from the literature, we have proposed a new model of the active site of the enzyme.