Basic studies on the equivalent cross-relaxation rate imaging (equivalent CRI)--phantom studies.

We have studied saturation transfer in hydrophilic, cross-linked copolymer gels from irradiated polymer protons to observed water protons, using f2 (ppm) profiles of [1 - (I(infinity)/I(0))], [(I(0)/I(infinity)) - 1] or 1/T(IS)(H2O), where I(0) and I(infinity) are the longitudinal magnetization of the observed water protons before and after long-time-f2-irradiation on polymer protons, respectively, and 1/T(IS)(H2O) is the cross-relaxation rate. (A) [1 - (I(infinity)/I(0))] (magnetization transfer ratio, MTR) was used in magnetic resonance imaging (MRI) as the MTR imaging. 1/T(IS)(H2O) (cross-relaxation rate) was used in the imaging of the magnetization transfer rate constant. This method was quite time-consuming compared with MTR imaging. However, f2 (ppm) profiles of [(I(0)/I(infinity)) - 1] correlated well with corresponding profiles of 1/T(IS)(H2O), because [(I(0)/I(infinity)) - 1] is equal to 1/[T(IS)(H2O)/T1(H2O)]. These results lead us to the conclusion that [(I(0)/I(infinity)) - 1] might be applicable to cross-relaxation rate (CR)-like imaging, i.e. equivalent CRI. (B) W (%) (dry weight) profiles of [(I(0)/I(infinity)) - 1] and 1/T(IS)(H2O), obtained by near-resonance f2-irradiation, seem to indicate participation of molecular rigidity and an amount of bound water. However, those values, monitored with off-resonance f2-irradiation, seem to be independent of monomer composition and to indicate mainly participation of rigidity, i.e. W (%) of copolymer gels.

Application of the transition state theory to water transport across cell membranes.

We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.

Effect of diabetic retinopathy on redox state of aqueous humor and serum albumin in patients with senile cataract.

PURPOSE: To investigate the oxidative status of albumin in the aqueous humor and serum of senile cataract patients with diabetes in order to clarify the pathogenesis of this condition. METHODS: High-performance liquid chromatography (HPLC) was employed to measure the reduced form of albumin (mercaptalbumin) and the oxidized form of albumin (nonmercaptalbumin) in serum and aqueous humor. The mercaptalbumin, nonmercaptalbumin-1, and nonmercaptalbumin-2 fractions in aqueous humor obtained at the start of cataract surgery and in serum obtained intraoperatively were analyzed by HPLC in 7 senile cataract patients with diabetic retinopathy (2 men and 5 women aged 70+/-9.8 years). RESULTS: The mean content (%) of mercaptalbumin, nonmercaptalbumin-1, and nonmercaptalbumin-2 in serum albumin from the diabetic patients was 60.3+/-7.8, 36.9+/-6.6, and 2.7+/-1.7%, respectively, while the corresponding values for aqueous humor albumin were 40.0+/-14.1, 52.4+/-12.6, and 7.5+/-3.6%. When the mercaptalbumin content (%) of aqueous humor albumin was compared between patients with active and inactive diabetic retinopathy, the respective values were 47.0+/-9.1% and 22.8+/-5.7%. A significant correlation mercaptalbumin content (%) of aqueous humor albumin did not show with the HbA1c level, but there was still a relationship (Y = 5.0 x - 2.7, r = 0.80, and p < 0.052). CONCLUSION: The increase of mercaptalbumin (the reduced form of albumin) in the aqueous humor of patients with diabetic retinopathy probably resulted from an increase of retinal vascular permeability.

Saturation transfer ratio imaging in invasive ductal carcinomas of the breast.

A prospective study was performed to investigate the correlations between saturation transfer ratio (STR) and histologic parameters of invasive ductal carcinomas in human breast. The histologic parameters investigated were the extent of fibrosis in the intercellular matrix, dysplastic changes of nuclei, and mitotic index. Twenty-seven patients with breast carcinoma were examined using an off-resonance saturation pulse in conjunction with conventional field-echo T(1)-weighted imaging at frequency offsets of 448 Hz and 1200 Hz from water resonance. The values of STR at frequency offset of 1200 Hz (STR(1200)) increased from non-scirrhous carcinoma to scirrhous carcinoma. Although STR(1200) showed correlation with the extent of fibrosis in the intercellular matrix (p<0.01, n = 27), they did not correlate with the dysplastic changes of nuclei or mitotic index. On the other hand, the values of STR at frequency offset of 448 Hz (STR(448)) demonstrated close correlation to dysplastic changes of nuclei and mitotic index (p<0.01, n = 27). STR(1200) correlates with the structural characteristics and STR(448) correlates with the nature of malignant cells with regard to nuclear dysplasia and mitotic potential.

1H-NMR and raman studies on perforating trauma-induced cataract formation in a mouse lens.

In order to provide new insight into the molecular mechanism of perforating trauma-induced cataract formation in an 8-week-old ddY mouse lens, we performed an in situ investigation into changes in the water-protein and/or protein-protein interactions by using 500 MHz (1)H-NMR spectroscopy, and into structural alterations in lens proteins by using Raman spectroscopy. Cross-relaxation times of water protons in the perforated opaque lens were considerably shorter than those in the intact transparent lens, whereas there was no significant difference in water content, suggesting a drastic change in water-protein and protein-protein interactions in the perforated lens. In addition, there was no significant difference in the intensity ratios of several key Raman bands between intact and perforated lenses, indicating that no significant local and overall conformational changes in lens protein itself occur in the perforated lens. The present (1)H-NMR and Raman results lead us to the conclusion that changes leading to lens opacification in the perforating trauma-induced cataract appear to involve the rapid formation of immobile large lens protein aggregates without formation of intra- and intermolecular disulfide linkages, and rapid increase in a fraction of bound water associated with large protein aggregates.

H-NMR and CD studies on the structural transition of serum albumin in the acidic region--the N-->F transition.

Helical content (f(alpha)) of bovine mercaptalbumin (BMA) showed the characteristic two-step decrease in the acidic region, one corresponding to the N-->F transition (pH 4.40-->3.75; f(alpha), 0.68-->0.58) and the other to the F-->E transition (the acid-expansion) (pH 3.60-->2.90; falpha, 0.58-->0.48). However, falpha of human serum albumin (HSA) mainly decreased in the N-->F transition (N-->F, pH 4.6-->3.4; falpha, 0.70-->-0.55 and F-->E, below pH 3.0; falpha, 0.55-->0.52). The difference in pH-profile of f(alpha) between BMA and HSA might be due to the microheterogeneity. The 1H-NMR spectra and cross-relaxation times (T(IS)) from irradiated to observed protein protons, which reflect the structural fluctuation and/or mobilability in proteins, were measured on the N-, F-, E-forms of HSA and BMA, and the N*-form (8.23 M urea, neutral pD) of iodoacetamide-blocked HSA (IA-HSA) and bovine serum albumin (IA-BSA). The 1H-NMR spectra and elongations of T(IS) values for the F- and E-forms of HSA and the E-form of BMA were quite similar to those for the N*-form of IA-HSA and IA-BSA, indicating the liberation of the intramolecular motion in the F- and E-forms. Those for the F-form of BMA were intermediate between the N- and E-form. The present results together with the reported data on hydrodynamic radii and D-H exchange reaction, indicate that the F-form of HSA and presumably BMA has a native-like globule form with a highly helical state and fluctuating tertiary structure. Thus, all of the present findings on the F-form of serum albumin seem to be in accord with the structural features for the F-form suggested by Foster's group (1-3, 19, 20, 22, 23) and the molten globule state demonstrated by Dolgikh et al. (40), and Ohgushi and Wada (36, 37).

Conformational changes in seventeen cystine disulfide bridges of bovine serum albumin proved by Raman spectroscopy.

Conformational changes in cystine disulfide bridges of bovine serum albumin during acid-induced isomerization (N --> F and F --> E transitions) have been studied with Raman spectroscopy. In an X-ray crystallographic study of human serum albumin, Carter and Ho reported that all disulfide bridges of the albumin molecule are in the gauche-gauche-gauche conformation [1]. On the other hand, the solution structure of bovine serum albumin examined by Raman spectroscopy differs from its crystal structure in the conformation of some of the disulfide bridges. Two Raman bands were detected at 520 and 505 cm(-1) in the disulfide stretching mode region, suggesting that the 17 disulfide bridges in the N-form of bovine serum albumin solution take both the gauche-gauche-gauche and gauche-gauche-trans conformations. The ratio of the peak intensities at 520 and 505 cm(-1) (I505/I520) is increased from 1.6 to 2.1 and from 2.1 to 6.3 on going from the N- to the F-form and from the F- to the E-form, respectively, indicating that the gauche-gauche-trans conformation of the disulfide bridges is converted to a gauche-gauche-gauche one which is the most energetically stable form during the acid-induced isomerization. However, small amounts of gauche-gauche-trans conformation still remain even in the E-form.

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery.

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt<-->nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical_ patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA+HNA)]+/-standard deviation, fHMA+/-sigma], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were 0.636+/-0.050 (n = 83) and 0.615+/-0.062 (n = 14), respectively. fHMA values were markedly lower than those for healthy male adults of 0.750+/-0.028 (n = 28). fHMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in cardiac surgical patients. Although fHMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients never recovered even on the 21st postoperative day.

Cross-relaxation times of normal and biochemically induced osteoarthritic rabbit knee cartilages.

We measured the spin-lattice relaxation times (T1) of water protons and intermolecular cross-relaxation times (T(IS)) from irradiated protein protons (f2-irradiation at 1.95 or -4.00 ppm) of rabbit normal and monoiodoacetate-induced degenerated knee articular cartilages to observed water protons. The mean values of T1 (T1) for control and degenerated rabbit knee cartilages were 1.87+/-0.15 (mean+/-SD, n=29) and 1.82+/-0.13 s (n=34), respectively. The mean values of water content (W(H2O)) for control and degenerated rabbit knee cartilages were 82.9+/-2.09 (n=26) and 83.1+/-2.57% (n=28), respectively. These values were not statistically different from each other. However, the mean values of T(IS) (T(IS)) for normal knee articular cartilage were significantly different from those for degenerated cartilage: (normal), T(IS) (f2=1.95 ppm)=2.46+/-0.62 s (n=28), T(IS) (f2=-4.00 ppm)=4.25+/-1.26 s (n=26); (degenerated), T(IS) (f2=1.95 ppm)=1.99+/-0.76s (n=34), T(IS) (f2=-4.00 ppm)=3.33+/-0.76 s (n=31). Obtained results may be attributed to the reported switchover from type II to type I collagen syntheses in osteoarthritic cartilage, resulting in broad collagen fibers. This specificity of cross-relaxation effect may prove useful in the noninvasive and pathophysiological evaluation of cartilage tissues in vivo.

Structural characterization of the molten globule and native states of ovalbumin: a 1H NMR study.

Molecular characteristics of ovalbumin (OVA) in the acidic (pD 3.08, the E-form) and neutral [pD 7.29, the N-form (native form)] regions were studied by measuring effective radii, 1H NMR spectra, spin-echo 1H NMR spectra and cross-relaxation times (TIS) from irradiated to observed protein protons which are particularly sensitive for detection of the mobile segments and/or structural looseness in proteins. 1H NMR spectra did not show significant differences between the N- and E-forms except for the spectral lines in the CH3, epsilon CH2 and aromatic regions. Effective radii and TIS values for main- and side-chains showed 1.08 and 1.5- to 2.0-fold increases on going from the N- to E-forms, respectively. The elongation of TIS values might indicate the appearance of the fluctuating tertiary structure in the E-form. Molecular characteristics of the E-form, inferred from reported far ultraviolet-circular dichroism (UV-CD) spectra in the peptide region, near UV-CD spectra in the aromatic region [Koseki et al. (1988) J. Biochem. 103, 425-430], effective radii and especially elongation of TIS values might indicate that the E-form could be in the molten globule state. The onset of denaturation of OVA using TIS measurements was also studied.

Comparative 1H-NMR studies on the physical state of water in soft contact lens and mouse lens.

The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin-lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%.

Spin-lattice relaxation times of water in polarized and depolarized rabbit vagus nerves.

Spin-lattice relaxation times (T1) of intracellular water of the nonmyelinated fibers of rabbit cervical vagus nerve were measured using a paramagnetic shift reagent, s-FDF. Spin-lattice relaxation decay curves were composed of the fast (T1,F) and slow (T1,S) relaxation components. The mean values of T1,F and T1,S in the polarized nerve fibers at 25 degrees C were 0.12 +/- 0.04 and 0.61 +/- 0.13 sec., respectively, and their fractions were 0.71 +/- 0.07 and 0.29 +/- 0.07, respectively (n = 21). On the other hand, those values in the depolarized nerve fibers were 0.16 +/- 0.01 sec., 0.77 +/- 0.17 sec., 0.77 +/- 0.10 and 0.23 +/- 0.10, respectively (n = 15). T1,F and T1,S for the depolarized nerve fibers were significantly elongated (P < 0.01).

Age-related change in redox state of human serum albumin.

Human serum albumin (HSA) is the mixture of human mercaptalbumin (HMA, reduced form) and human nonmercaptalbumin (HNA, oxidized form). We developed a rapid and concise HPLC system to obtain the clear resolution of HSA into HMA and HNA, using an Asahipak GS-520H column. The mean value of the fraction of HMA (f(HMA)) for healthy young male subjects was 0.76 +/- 0.04 (n = 54). However, the f(HMA, 60-90) value for healthy elderly subjects (where the numbers in brackets indicate the range of ages) was 0.48 +/- 0.06 (n = 183). In healthy elderly subjects, f(HMA) was significantly lower than in healthy young male subjects, indicating that HSA in the elderly becomes more oxidized than in the young subjects. Consequently, we suggest that one of the important functions of serum albumin could be to participate in the maintenance of a constant redox potential in the extracellular fluids, thus securing a certain redox buffer capacity. f(HMA) on HSA might reflect this redox buffer capacity with age.

The kinetic studies on the intramolecular SH, S-S exchange reaction of bovine mercaptalbumin.

Bovine mercaptalbumin (BMA) has 17 disulfide bonds and one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction (N-A isomerization, molecular aging) in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). The aging reaction was completely reversible and strongly affected by environmental factors, such as pH, temperature, ionic strength, Ca2+, nonbranched short-chain fatty acids, etc. Disulfide configuration (or pairing of disulfide bonds) was affected by the environmental factors. Obtained results might support the concept of Klotz (1966) that protein conformation (or three-dimensional structure) is dependent upon (i) the primary structure and (ii) constituents of the solvent.

Increased oxidized form of human serum albumin in patients with diabetes mellitus.

High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on Asahipak GS-520H columns at neutral pH (6.87) showed a clear resolution of human mercaptalbumin (HMA) and nonmercaptalbumin (HNA), which are reduced and oxidized form of HSA, respectively. We studied the conversion of HMA to HNA (mercapt-nonmercapt conversion) as an index of oxidative change of the tissues and organs in 28 normal subjects and in a total of 47 patients with non-insulin dependent diabetes mellitus (NIDDM). Mean (+/- SD) values of the HMA fraction of HSA, f(HMA), [HMA/(HMA + HNA)], was significantly lower in NIDDM patients than in normal subjects (0.63 +/- 0.067 vs 0.75 +/- 0.028, P < 0.001). It was lower in poorly controlled NIDDM patients (0.63 +/- 0.058, n = 20) than in well controlled NIDDM patients (0.67 +/- 0.032, n = 9) (P < 0.05). Plasma glucose values sampled on occasions including overnight fasting and postprandial ones (r = -0.441, n = 47, P < 0.01), but not plasma glucose values sampled on overnight fasting (r = -0.345, n = 29) or postprandial (r = -0.467, n = 18) conditions and HbA1c (r = -0.211, n = 34), negatively correlated with the f(HMA) values, indicating that mercapt-nonmercapt conversion may not be due to cumulative hyperglycemia over a month, but due to short-term alteration in blood glucose level. The presence or absence of diabetic complications including nephropathy, retinopathy and neuropathy did not affect the f(HMA) values. In conclusion, decreased f(HMA) values in the diabetic patients suggested the presence of a rapidly altered oxidative change of albumin due to hyperglycemia.

Proton NMR studies on ischemic rat brain tissue.

The spin-lattice relaxation time (T1) of water protons and the cross-relaxation time (TIS) between irradiated protein protons and observed water protons were measured in order to study water-macromolecular interactions in ischemic rat brain tissues. Tissues were obtained by bilateral common carotid artery occlusion from stroke-prone spontaneously hypertensive rats. Water, Na, and K contents were measured in ischemic brain tissue at the same time. Water and Na content increased while the TIS value and K content decreased following ischemic insults. The T1 value did not change until 180 min after ischemia had been induced. Changes in the TIS value occurred earlier than changes observed for the T1 value, water, and electrolyte contents. Results indicate that the value of TIS may be useful for detecting cerebral ischemia and that the physical structure of water-macromolecular interaction may be altered soon after ischemic onset in brain tissue.

Proton nuclear magnetic resonance studies on water structure in peritumoral edematous brain tissue.

Spin-lattice relaxation times (T1) of water protons and cross-relaxation times (TIS) between irradiated protein and water protons were measured to study the water structure in peritumoral edematous brain tissue of rats. Despite small changes in T1, water, and electrolyte contents, TIS values of water protons were significantly reduced in peritumoral edematous brain tissue when compared to those of the controls. Results indicate that the water structure in brain tissues may become altered at an early stage of edemic formation without causing any significant changes in cellular hydration. TIS might serve as a sensitive parameter for studying the water structure in a variety of tissues, such as in edematous brain tissue.

Quantitative and qualitative changes of serum albumin in CAPD patients.

This study was conducted to assess quantitative and qualitative changes in serum albumin in CAPD patients. For twenty-six CAPD patients as well as age-, sex- and dialysis history-matched HD patients, biochemical and physiological parameters including urea kinetics were determined. Albumin was qualitatively evaluated by HPLC. The CAPD patients showed significant decreases in serum albumin and the reduced form of albumin accompanied by a lower protein catabolic rate(PCR) compared to the HD patients. Thirty five % of CAPD patients showed mild to moderate hypoalbuminemia, associated with a higher incidence of peritonitis and longer hospital stay. Patients with hypoalbuminemia had less of the reduced form of albumin. A weak positive correlation was found between serum albumin concentration and KT/V, and PCR. Thus, in CAPD patients, there occur quantitative and qualitative changes in serum albumin. Hypoalbuminemia may be a risk factor for peritonitis and is due in part to insufficient dialysis and protein intake. Intermolecular change in albumin may possibly be due to uremia per se.

Circular dichroic and 1H-NMR studies on the aged form of bovine plasma albumin.

Bovine plasma albumin (BPA) has 17 disulfide bonds and approximately one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). 1) Fractions of alpha-helix (f alpha) and beta-form (f beta) of iodoacetamide-blocked non-aged BPA (IA-BPA) at pH 6.5 (the N-form) and 9.0 (the B-form) in the absence of added salt were 0.70, 0.12 and 0.62, 0.18, respectively (Era et al. (1990]. However, there were no changes in f alpha and f beta of the iodoacetamide-blocked A-form (IA-A-form) over the pH range from 5.5 to 9.1 in the absence of added salt or in 0.10 M KCl (f alpha approximately 0.60, f beta approximately 0.20), indicating that the secondary structure of the IA-A-form might be similar to that of non-aged IA-BPA at pH 9.0 (B-form) in the absence of added salt, that is, the frozen B-form, stabilized covalently by the repairing of disulfide bonds. 2) The rigidity of the A- and IA-A-forms, as monitored by cross-relaxation times between irradiated and observed protein protons, was similar to or slightly higher than that of non-aged IA-BPA or BMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural transition of bovine plasma albumin in the alkaline region--the N-B transition.

Bovine plasma albumin (BPA) has approximately one SH group (Cys-34) which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form. So, the N-B transition at ionic strength above 0.20 and below 0.10 was studied using BPA and iodoacetamide-blocked BPA (IA-BPA), respectively. (1) pH profiles of [theta]262 and [theta]268 of BPA in 0.20 M KCl showed the characteristic changes in the pH region 7.0-9.0, corresponding to the N-B transition. On going from pH 7.0 to 9.0 in 0.10 M KCl or NaCl, IA-BPA did not show significant changes in rotational relaxation times of tryptophyl fluorophors, CD-resolved secondary structures, spin-echo 1H-n.m.r. spectra and cross-relaxation times (TIS) between irradiated and observed protein protons, which might reflect the rigidity of the domains and/or subdomains. On the other hand, rotational relaxation times of 1-anilino-8-naphthalenesulfonate-IA-BPA complex (IA-BPA-ANS0.9, molar ratio of ANS to IA-BPA = 0.9/1) showed significant decreases from 131 to 114 ns on going from the N- to the B-forms in 0.10 M KCl. The above results and reported experimental evidence might indicate that on going from the N- to the B-forms in 0.10 M KCl or NaCl, the mutual movement of subdomains, connected with a flexible hinge region (Brown & Shockley (1982)) might increase without loss in the helicity and the rigidity of subdomains. (2) The N-B transition of IA-BPA in the absence of salt was quite different from those in 0.10 M KCl or NaCl. Decreases in the helicity and the intramolecular rigidity, as monitored by TIS-measurements, were observed on going from the N- to the B-forms.