Longitudinal Relaxation Rate Of Water Protons Research Articles

A relaxometric method for the assessment of intestinal permeability based on the oral administration of gadolinium-based MRI contrast agents.

Herein, a new relaxometric method for the assessment of intestinal permeability based on the oral administration of clinically approved gadolinium (Gd)-based MRI contrast agents (CAs) is proposed. The fast, easily performed and cheap measurement of the longitudinal water proton relaxation rate (R1) in urine reports the amount of paramagnetic probe that has escaped the gastrointestinal tract. The proposed method appears to be a compelling alternative to the available methods for the assessment of intestinal permeability. The method was tested on the murine model of dextran sulfate sodium (DSS)-induced colitis in comparison with healthy mice. Three CAs were tested, namely ProHance®, MultiHance® and Magnevist®. Urine was collected for 24 h after the oral ingestion of the Gd-containing CA at day 3-4 (severe damage stage) and day 8-9 (recovery stage) after treatment with DSS. The Gd content in urine measured by (1)H relaxometry was confirmed by inductively coupled plasma-mass spectrometry (ICP-MS). The extent of urinary excretion was given as a percentage of excreted Gd over the total ingested dose. The method was validated by comparing the results obtained with the established methodology based on the lactulose/mannitol and sucralose tests. For ProHance and Magnevist, the excreted amounts in the severe stage of damage were 2.5-3 times higher than in control mice. At the recovery stage, no significant differences were observed with respect to healthy mice. Overall, a very good correlation with the lactulose/mannitol and sucralose results was obtained. In the case of MultiHance, the percentage of excreted Gd complex was not significantly different from that of control mice in either the severe or recovery stages. The difference from ProHance and Magnevist was explained on the basis of the (known) partial biliary excretion of MultiHance in mice.

A Quantitative Relaxometric Version of the ELISA Test for the Measurement of Cell Surface Biomarkers

Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an in vitro diagnostic method based on the release of paramagnetic species from relaxometrically “silent” liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×105 has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.

A Quantitative Relaxometric Version of the ELISA Test for the Measurement of Cell Surface Biomarkers

Quantitative measurement of marker expression in diseased cells is still a topic of considerable interest and different methodologies are currently under intense scrutiny. This work aims at developing an in vitro diagnostic method based on the release of paramagnetic species from relaxometrically "silent" liposomes operated by the action of a phospholipase A2 (PLA2) previously targeted to the epitope of interest. The released paramagnetic species causes an increase of the longitudinal water proton relaxation rate proportional to the number of PLA2 bound to the cell outer surface. The sensitivity of the herein proposed method, named R-ELISA, was attempted in the detection of folate receptor expression on human ovarian cancer cells by functionalizing PLA2 with folic acid. Receptor/cell number of 8.3×10(5) has been measured on IGROV-1 cells. The R-ELISA assay can detect nanomolar cell suspension receptor concentrations and has been validated by well-established spectrofluorimetric procedures.

Determination of water permeability of paramagnetic liposomes of interest in MRI field

The water permeability of various liposome membranes has been determined at 298K by measuring the NMR longitudinal water proton relaxation rate of vesicles encapsulating the clinically approved Gd-HPDO3A complex (HPDO3A=10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). Two basic formulations based on DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyl-oleylphosphatidylcholine) phospholipids were selected and investigated. Furthermore, the permeability changes caused by the membrane incorporation of amphiphiles like cholesterol and/or metal complexes of interest for designing improved liposome-based MRI contrast agents, were also investigated. The incorporation of cholesterol and metal complexes bearing C18 saturated chains in POPC-based liposomes reduces the water diffusivity across the membrane bilayer. On the contrary, the incorporation of a macrocyclic metal complex bearing four C12 alkylic chains, one for each coordination arm of the ligand, considerably enhances the water permeability in DPPC-based liposomes. Finally, it is reported that the permeability of POPC-based bilayer is increased when the liposomes are subjected to an osmotic stress.

A benzene-core trinuclear GdIIIcomplex: towards the optimization of relaxivity for MRI contrast agent applications at high magnetic field

A novel ligand, H(12)L, based on a trimethylbenzene core bearing three methylenediethylenetriamine-N,N,N'',N''-tetraacetate moieties (-CH(2)DTTA(4-)) for Gd(3+) chelation has been synthesized, and its trinuclear Gd(3+) complex [Gd(3)L(H(2)O)(6)](3-) investigated with respect to MRI contrast agent applications. A multiple-field, variable-temperature (17)O NMR and proton relaxivity study on [Gd(3)L(H(2)O)(6)](3-) yielded the parameters characterizing water exchange and rotational dynamics. On the basis of the (17)O chemical shifts, bishydration of Gd(3+) could be evidenced. The water exchange rate, k(ex)(298)=9.0+/-3.0 s(-1) is around twice as high as k(ex)(298) of the commercial [Gd(DTPA)(H(2)O)](2-) and comparable to those on analogous Gd(3+)-DTTA chelates. Despite the relatively small size of the complex, the rotational dynamics had to be described with the Lipari-Szabo approach, by separating global and local motions. The difference between the local and global rotational correlation times, tau(lO)(298)=170+/-10 ps and tau(gO)(298)=540+/-100 ps respectively, shows that [Gd(3)L(H(2)O)(6)](3-) is not fully rigid; its flexibility originates from the CH(2) linker between the benzene core and the poly(amino carboxylate) moiety. As a consequence of the two inner-sphere water molecules per Gd(3+), their close to optimal exchange rate and the appropriate size and limited flexibility of the molecule, [Gd(3)L(H(2)O)(6)](3-) has remarkable proton relaxivities when compared with commercial contrast agents, particularly at high magnetic fields (r(1)=21.6, 17.0 and 10.7 mM(-1)s(-1) at 60, 200 and 400 MHz respectively, at 25 degrees C; r(1) is the paramagnetic enhancement of the longitudinal water proton relaxation rate, referred to 1 mM concentration of Gd(3+)).

Relaxometric Determination of the Exchange Rate of the Coordinated Water Protons in a Neutral GdIII Chelate

AbstractThe exchange rate of the coordinated water molecule in the neutral complex [Gd(DTPA‐BBA)(H2O)] (DTPA‐BBA = 1,7‐bis[(N‐benzylcarbamoyl)methyl]‐1,4,7‐triazaheptane‐1,4,7‐triacetate or diethylenetriaminopentaacetate N,N′‐bis(benzylamide)) is slower than in the parent complex [Gd(DTPA)(H2O)]2‐. From the analysis of the temperature dependence of the solvent 17O NMR transverse relaxation time in an aqueous solution of the paramagnetic complex, a value of 4.5 × 105 s‐1 (at 298 K) is obtained for the exchange rate of the coordinated water molecule. This rate constant does not vary in the pH range 7–12. Conversely, over the same pH range and at 298 K and 20 MHz, the longitudinal water proton relaxivity increases from 4.8 to 6.5 s‐1 mM‐1. The analysis of the dependence of the longitudinal water proton relaxation rate on magnetic field and temperature at pH 7 and pH 12 shows that the increase in relaxivity at basic pH has to be assigned to the contribution of the prototropic exchange at the water molecule in the inner coordination sphere of the metal ion. This exchange process is catalyzed by OH‐ ions (kP = 1.7 × 109 M‐1 s‐1 at 298 K) and causes an increase in the observed relaxivity when it occurs at a rate larger than the exchange rate of the entire water molecule. At pH 12 the limiting effect of the slow exchange rate for the coordinated water molecule is removed, and the longitudinal water proton relaxivity measured at this pH then represents the maximum value attainable for this complex.

Interactions of phospho- and dephosphosuccinyl coenzyme A synthetase with manganous ion and substrates. Studies of manganese complexes by NMR relaxation rates of water protons.

The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn. Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.

Interaction of Manganous Ion, Substrates, and Anions with Arginine Kinase: MAGNETIC RELAXATION RATE STUDIES OF WATER PROTONS AND KINETIC ANION EFFECTS

Abstract The interactions of ADP and the arginine enantiomers with arginine kinase have been investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to the paramagnetic Mn(II) ion in the enzymesubstrate complexes. Anion effects on the kinetics of the forward reaction of arginine kinase, on the PRR of quaternary arginine kinase-MnADP-arginine complexes, and on the binding of l- and d-arginine in the quaternary complex also have been examined. Manganous ion binds to arginine kinase at approximately two sites with a Kd value of 0.55 mm, and the PRR enhancement of the binary complex, eb, at 24.3 MHz and 25° is 9.6. MnADP binds quite strongly to the enzyme, K2 = 7.5 µm, and the resulting ternary complex has a highly enhanced PRR, et = 18.9. Although MnATP binds more weakly to the enzyme than MnADP by an order of magnitude, K2 = 79 µm, the enhancement factor for the ATP ternary complex, et = 16.7, is within 12% of the value for the ADP ternary complex. Free ADP binds about 7 times more weakly to the enzyme than does MnADP, whereas the binding of ATP is only slightly weaker than that of MnATP. The substrate l-arginine binds to the ternary E-MnADP complex forming an abortive quaternary complex which has a PRR value that is approximately 50% of the value for the ternary complex. The dissociation constant, Kdiss, for l-arginine in the quaternary complex, 0.15 mm, was determined from titration data using PRR as the parameter. Although d-arginine binds to the ternary complex about 300 times more weakly than l-arginine, the PRR value of the quaternary complex containing d-arginine is within 10% of the value of the l-arginine-containing complex. The addition of 10 mm nitrate produces a 20-fold decrease in the dissociation constant for d- or l-arginine from the quaternary complex. The dissociation constant of nitrate from the complex with l-arginine (Kdiss = 0.06 mm) is increased 50-fold upon substitution of d-arginine, and nitrate produces a decrease of 48% in the PRR of the quaternary complex in the presence of l-arginine and of 36% in the presence of d-arginine. The planar anions, thiocyanate, nitrite, and formate, lower the PRR of the quaternary complex to a small extent. However, the tetrahedral anions, sulfate, perchlorate, and trichloroacetate, are ineffective in this respect. Nitrate both lowers the PRR to a greater extent and binds about 2 orders of magnitude more tightly than any of the other anions. In addition, nitrate inhibits the forward reaction of arginine kinase, produces a marked curvature in the reaction progress curve, and augments the inhibition by MgADP. Apparently this anion locks the enzyme into a transition-state analog complex in which all components are bound very tightly. The effects of nitrate on arginine kinase complexes are quite similar to the anion's effects on creatine kinase complexes. The results for both enzymes are consistent with the hypothesis that the nitrate anion acts as an analog of the transferable phosphoryl group in its trigonal bipyramidal configuration in the transition state. These observations lend further support to the contention that basic aspects of the catalytic mechanism are similar for both guanidino kinases.