Gary D. Fullerton

NMR relaxation of protons in tissues and other macromolecular water solutions

Nuclear magnetic resonance (NMR) longitudinal (T1) and transverse (T2) relaxation parameters have been evaluated for protein solutions, cellular suspensions and tissues using both data from our laboratory and the extensive literature. It is found that this data can be generalized and explained in terms of three water phases: free water, hydration water, and crystalline water. The proposed model which we refer to as the FPD model differs from similar models in that it assumes that free and hydration water are two phases with distinct relaxation times but that T1 = T2 in each phase. In addition there is a single correlation time for each rather than a distribution as assumed in most other models. Longitudinal decay is predicted to be single exponent in character resulting from a fast exchange between the free and hydration compartments. Transverse decay is predicted to be multiphasic with crystalline (T2 approximately 10 microseconds), hydration (T2 approximately 10 msec) and free (T2 approximately 100 msec) water normally visible. The observed or effective transverse relaxation times for both the hydration and free water phases are greatly affected by the crystalline phase and are much shorter than the inherent relaxation times.

Calculation of effective dose from measurements of secondary neutron spectra and scattered photon dose from dynamic MLC IMRT for 6 MV, 15 MV, and 18 MV beam energies

Effective doses were calculated from the delivery of 6 MV, 15 MV, and 18 MV conventional and intensity-modulated radiation therapy (IMRT) prostate treatment plans. ICRP-60 tissue weighting factors were used for the calculations. Photon doses were measured in phantom for all beam energies. Neutron spectra were measured for 15 MV and 18 MV and ICRP-74 quality conversion factors used to calculate ambient dose equivalents. The ambient dose equivalents were corrected for each tissue using neutron depth dose data from the literature. The depth corrected neutron doses were then used as a measure of the neutron component of the ICRP protection quantity, organ equivalent dose. IMRT resulted in an increased photon dose to many organs. However, the IMRT treatments resulted in an overall decrease in effective dose compared to conventional radiotherapy. This decrease correlates to the ability of an intensity-modulated field to minimize dose to critical normal structures in close proximity to the treatment volume. In a comparison of the three beam energies used for the IMRT treatments, 6 MV resulted in the lowest effective dose, while 18 MV resulted in the highest effective dose. This is attributed to the large neutron contribution for 18 MV compared to no neutron contribution for 6 MV.

Characterization of proton NMR relaxation times in normal and pathological tissues by correlation with other tissue parameters

To help understand which tissue parameters best account for the water proton NMR relaxation times, the longitudinal relaxation time (T1), the transverse relaxation time (T2), and the water content of 16 tissues from normal adult rats were measured at 10.7 MHz and 29 degrees C. Regression analyses between the above and other tissue parameters were performed. These other tissue parameters included: the amounts of various organic and inorganic components, protein synthetic rate, oxygen consumption rate, and morphological composition. In addition, the differences in T1, T2, and water content values between normal liver and malignant tumor (Morris #7777 a transplantable hepatoma) were studied to help understand how a disease state can be detected and characterized by NMR spectroscopy. The results of this study and information from the literature allow the following generalizations to be made about tissue T1 and T2 values: (1) Each normal tissue has rather consistent and characteristic T1 and T2 relaxation times which are always shorter than the T1 and T2 of bulk water; (2) tissues with higher water content tend to have longer T1 relaxation times; (3) tissue T2 values are not, however, as well correlated with water content as T1 values; (4) tissues with shorter T1 values have higher calculated hydration fractions, greater amounts of rough endoplasmic reticulum, and a greater rate of protein synthetic activity; (5) tissues with higher lipid content, associated with intracellular non-membrane bounded lipid droplets, tend to have longer T2 values; (6) tissues with greater overall surface area, whether in the form of cellular membranes or intracellular or extracellular fibrillar macromolecules, tend to have shorter T2 values; (7) the differences between T1 and T2 values between tumor and normal tissues correlated with differences in the volume fraction (amounts) of extracellular fluid volumes and in the amounts of membrane and fibrillar surface area in the cells. The above generalizations should be useful in predicting T1 and T2 changes associated with specific tissue pathologies.

Collagen structure: the molecular source of the tendon magic angle effect.

This review of tendon/collagen structure shows that the orientational variation in MRI signals from tendon, which is referred to as the “magic angle” (MA) effect, is caused by irreducible separation of charges on the main chain of the collagen molecule. These charges are held apart in a vacuum by stereotactic restriction of protein folding due in large part to a high concentration of hydroxyproline ring residues in the amino acids of mammalian collagen. The elevated protein electrostatic energy is reduced in water by the large dielectric constant of the highly polar solvent (κ ∼ 80). The water molecules serve as dielectric molecules that are bound by an energy that is nearly equivalent to the electrostatic energy between the neighboring positive and negative charge pairs in a vacuum. These highly immobilized water molecules and secondary molecules in the hydrogen‐bonded water network are confined to the transverse plane of the tendon. Orientational restriction causes residual dipole coupling, which is directly responsible for the frequency and phase shifts observed in orientational MRI (OMRI) described by the MA effect. Reference to a wide range of biophysical measurements shows that native hydration is a monolayer on collagen hm = 1.6 g/g, which divides into two components consisting of primary hydration on polar surfaces hpp = 0.8 g/g and secondary hydration hs = 0.8 g/g bridging over hydrophobic surface regions. Primary hydration further divides into side‐chain hydration hpsc = 0.54 g/g and main‐chain hydration hpmc = 0.263 g/g. The main‐chain fraction consists of water that bridges between charges on the main chain and is responsible for almost all of the enthalpy of melting ΔH = 70 J/g‐dry mass. Main‐chain water bridges consist of one extremely immobilized Ramachandran water bridge per tripeptide hRa = 0.0658 g/g and one double water bridge per tripeptide hdwb = 0.1974 g/g, with three water molecules that are sufficiently slowed to act as the spin‐lattice relaxation sink for the entire tendon. J. Magn. Reson. Imaging 2007.

Investigation of secondary neutron dose for 18 MV dynamic MLC IMRT delivery

Secondary neutron doses from the delivery of 18 MV conventional and intensity modulated radiation therapy (IMRT) treatment plans were compared. IMRT was delivered using dynamic multileaf collimation (MLC). Additional measurements were made with static MLC using a primary collimated field size of 10 x 10 cm2 and MLC field sizes of 0 x 0, 5 x 5, and 10 x 10 cm2. Neutron spectra were measured and effective doses calculated. The IMRT treatment resulted in a higher neutron fluence and higher dose equivalent. These increases were approximately the ratio of the monitor units. The static MLC measurements were compared to Monte Carlo calculations. The actual component dimensions and materials for the Varian Clinac 2100/2300C including the MLC were modeled with MCNPX to compute the neutron fluence due to neutron production in and around the treatment head. There is excellent agreement between the calculated and measured neutron fluence for the collimated field size of 10 x 10 cm2 with the 0 x 0 cm2 MLC field. Most of the neutrons at the detector location for this geometry are directly from the accelerator head with a small contribution from room scatter. Future studies are needed to investigate the effect of different beam energies used in IMRT incorporating the effects of scattered photon dose as well as secondary neutron dose.

A MECHANISTIC VIEW OF THE NON-IDEAL OSMOTIC AND MOTIONAL BEHAVIOR OF INTRACELLULAR WATER

It is commonly assumed that essentially all of the water in cells has the same ideal motional and colligative properties as does water in bulk liquid state. This assumption is used in studies of volume regulation, transmembrane movement of solutes and electrical potentials, solute and solution motion, solute solubility and other phenomena. To get at the extent and the source of non‐ideally behaved water (an operational term dependent on the measurement method), we studied the motional and colligative properties of water in cells, in solutions of amino acids and glycine peptides whose surface characteristics are known, and in solution of bovine serum albumin, hemoglobin and some synthetic polypeptides. Solutions of individual amino acids with progressively larger hydrophobic side chains showed one perturbed water molecule (structured‐slowed in motion) per nine square angstroms of hydrophobic surface area. Water molecules adjacent to hydrophobic surfaces form pentagonal structural arrays, as shown by X‐ray diffraction studies, that are reported to be disrupted by heat, electric field, hydrostatic pressure and phosphorylation state. Hydrophilic amino acids demonstrated water destructuring (increased motion) that was attributed to dielectric realignment of dipolar water molecules in the electric field between charge groups. In solutions of proteins, several methods indicate the equivalent of 2–8 layers of structured water molecules extending beyond the protein surface, and we have recently demonstrated that induced protein conformational change modifies the extent of non‐ideally behaved water. Water self‐diffusion rate as measured in three different cell types was about half that of bulk water, indicating that most of the water in these cells was slower in motion than bulk water. In different cell types the extent of osmotically perturbed water ranged from less than half to almost all of the intracellular water. The assumption that essentially all intracellular water has ideal osmotic and motional behavior is not supported by the experimental findings. The non‐ideality of cell water is an operational term. Therefore, the amount of non‐ideally behaving water is dependent on the characteristics of water targeted, i.e. the measurement method, and a large fraction of it is explainable in mechanistic terms at a molecular level based on solute—solvent interactions.

An NMR method to characterize multiple water compartments on mammalian collagen

A molecular model is proposed to explain water 1H NMR spin‐lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well‐separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h 1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h 2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h 3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.

Evidence that collagen and tendon have monolayer water coverage in the native state

This paper investigates an alternative explanation for widely reported paradoxical intracellular water properties. The most frequent biological explanation assumes water structure extending multiple layers from surfaces of compactly folded macromolecules to explain large amounts of perturbed water. Long range water structuring, however, contradicts molecular models widely accepted by the scientific majority. This study questions whether the paradoxical cell water could result from larger than expected amounts of first layer interfacial water on internal protein surfaces rather than structured multilayers. Native mammalian tendon is selected for the study because (1) the organ consists of highly compact structures of a single macromolecular protein—collagen, (2) molecular structure and geometry of collagen is well characterized by X‐ray diffraction, (3) molecular structure extends to the macroscopic tendon level and (4) perturbed water behavior similar to cellular water is reported on tendon. Native tendon holds 1.6 g water/g dry mass. The 62% native water content simulates the water content of many cell types. MicroCT studies of tendon dilatometry as a function of hydration are measured and correlated to X‐ray diffraction measurements of interaxial separation. Correlations show that native tendon has sufficient water for only a single monolayer of interfacial water. Thus the paradoxical properties of water in native tendon are first‐layer interfacial water properties. Similar water behavior on globular proteins suggests that paradoxical cell water behavior could be caused by larger than expected amounts of first layer interfacial water on internal and external macromolecular surfaces of cell components.

Water in tendon: Orientational analysis of the free induction decay

The orientation dependence of the free induction decay (FID) of 1H NMR water signal in ex vivo bovine digital flexor tendon at the native level of hydration is reported. Residual dipolar coupling due to the overall tissue anisotropy produces a 6:1 change in the signal intensity as an angle between the long axis of a specimen and the external magnetic field is changed from the “magic angle” of 54.7° to 0°. The strength of residual dipolar interactions between water protons was estimated by orientational analysis of the signal intensity to be equal to 780 Hz. Apparent signal maxima are observed at orientations 8–13° away from 54.7° due to an inhomogeneous contribution to the decay. A small fraction of total water in tendon is detectable at all orientations and exhibits a shift in the precession frequency. It is hypothesized that this water fraction resides in the interconnecting gaps at the ends of collagen molecules. The gaps have a disordered environment that allows for a zero time average of dipolar interactions. Measured frequency and phase shifts are interpreted as signatures of the bulk magnetic susceptibility effect due to geometry of the cavity formed by adjacent gaps at the ends of the collagen molecules. The multiexponentiality of the FID decay is hypothesized to be due to the exchange between orientationally restricted water structured along the length of the collagen molecule and disordered water in the cavity. Magn Reson Med 54:280–288, 2005.

Prospective tissue-mimicking materials for use in NMR imaging phantoms

Water-based proteinaceous gels, which--with appropriate additives--are stable with time and possess a high melting point, have been used as base materials in ultrasonically tissue-mimicking materials. In the present work, versions of these gels having various concentrations of glycerol and graphite particles were studied regarding their NMR T1 and T2 dependencies at a proton Larmor frequency of 10.7 MHz. It has been found that T1 depends primarily on the concentration of glycerol and T2 depends primarily on the graphite particle concentration. Also, the ranges of T1 and T2 likely span those which exist for soft tissue parenchymae. Thus, these materials are good candidates for use as NMR tissue-mimicking materials. T1 and T2 also vary with gelatin concentration. The latter fact, together with the strong dependence of T2 on graphite concentration, mean that effective contrast-resolution phantoms and anthropomorphic phantoms with stable T1 and T2 distributions can be produced.