Depressive symptoms are not associated with long-term integrated testosterone concentrations in hair.

OBJECTIVES: The association between depressive symptomatology and endogenous testosterone levels is inconclusive. Large inter- and intra-individual testosterone differences suggest point measurements from saliva or serum to be inadequate to map basal testosterone concentrations highlighting the potential for long-term integrated testosterone levels from hair. METHODS: Using data from a prospective cohort study, a total of 578 participants (74% female) provided complete data on depressive symptomatology, clinical features, and hair samples for quantification of testosterone concentrations at baseline. Available data of three annual follow-up examinations were used for longitudinal analyses. RESULTS: Correlation analysis showed in both, men and women, hair testosterone across all the four time points not to be significantly related to depressive symptoms. Examined clinical features were not associated with testosterone levels, except for having a current diagnosis of a psychological disorder, which was associated with reduced testosterone levels in men, but not in women. Acceptable model fit for an autoregressive cross-lagged panel analysis emerged only for the female subsample suggesting inverse cross-relations for the prediction of testosterone by depressive symptomatology and vice versa. CONCLUSIONS: Findings from this study add to the literature by showing no association between long-term integrated testosterone in hair and depressive symptomatology in men and women.

Mapping of pain circuitry in early post-natal development using manganese-enhanced MRI in rats.

Premature or ill full-term infants are subject to a number of noxious procedures as part of their necessary medical care. Although we know that human infants show neural changes in response to such procedures, we know little of the sensory or affective brain circuitry activated by pain. In rodent models, the focus has been on spinal cord and, more recently, midbrain and medulla. The present study assesses activation of brain circuits using manganese-enhanced magnetic resonance imaging (MEMRI). Uptake of manganese, a paramagnetic contrast agent that is transported across active synapses and along axons, was measured in response to a hindpaw injection of dilute formalin in 12-day-old rat pups, the age at which rats begin to show aversion learning and which is roughly the equivalent of full-term human infants. Formalin induced the oft-reported biphasic response at this age and induced a conditioned aversion to cues associated with its injection, thus demonstrating the aversiveness of the stimulation. Morphometric analyses, structural equation modeling and co-expression analysis showed that limbic and sensory paths were activated, the most prominent of which were the prefrontal and anterior cingulate cortices, nucleus accumbens, amygdala, hypothalamus, several brainstem structures, and the cerebellum. Therefore, both sensory and affective circuits, which are activated by pain in the adult, can also be activated by noxious stimulation in 12-day-old rat pups.

Ultra-short echo-time MRI detects changes in bone mineralization and water content in OVX rat bone in response to alendronate treatment.

In this work we hypothesize that bisphosphonate treatment following ovariectomy manifests in increased phosphorus and decreased water concentration, both quantifiable nondestructively with ultra-short echo-time (UTE) (31)P and (1)H-MRI techniques. We evaluated this hypothesis in ovariectomized (OVX) rats undergoing treatment with two regimens of alendronate. Sixty female four-month-old rats were divided into four groups of 15 animals each: ovariectomized (OVX), OVX treatment groups ALN1 and ALN2, receiving 5 microg/kg/day and 25 microg/kg/day of alendronate, and a sham-operated group (NO) serving as control. Treatment, starting 1 week post-surgery, lasted for 50 days at which time animals were sacrificed. Whole bones from the left and right femora were extracted from all the animals. (31)P and (1)H water concentration were measured by UTE MRI at 162 and 400 MHz in the femoral shaft and the results compared with other measures of mineral and matrix properties obtained by (31)P solution NMR, CT density, ash weight, and water measured by dehydration. Mechanical parameters (elastic modulus, EM, and ultimate strength, US) were obtained by three-point bending. The following quantities were lower in OVX relative to NO: phosphorus concentration measured by (31)P-MRI (-8%; 11.4+/-0.9 vs. 12.4+/-0.8%, p<0.005), (31)P-NMR (-4%; 12.8+/-0.4 vs. 13.3+/-0.8 %, p<0.05) and micro-CT density (-2.5%; 1316+/-34 vs. 1349+/-32 mg/cm(3), p=0.005). In contrast, water concentration by (1)H-MRI was elevated in OVX relative to NO (+6%; 15.5+/-1.7 vs. 14.6+/-1.4 %, p<0.05). Alendronate treatment increased phosphorus concentration and decreased water concentration in a dose-dependent manner, the higher dose yielding significant changes relative to values found in OVX animals: (31)P-MRI (+14%; p<0.0001), (31)P-NMR (+9%; p<0.0001), ash content (+1.5%; p<0.005), micro-CT mineralization density (+2.8%; p<0.05), and (1)H-MRI, (-19%, p<0.0001). The higher dose raised phosphorus concentration above and water concentration below NO levels: (31)P-MRI (+6%; p<0.05), (31)P-NMR (+5%; p=0.01), ash content (+1.5%; p=0.005), (1)H-MRI (-14%; p<0.0001), and drying water (-10%; p<0.0005). Finally, the group means of phosphorus concentration were positively correlated with EM and US (R(2)> or =0.98, p<0.001 to p<0.05) even though the pooled data from individual animals were not. The results highlight the implications of estrogen depletion and bisphosphonate treatment on mineral composition and mechanical properties and the potential of solid-state MR imaging to detect these changes in situ in an animal model of rat ovariectomy.

Multi-modality study of the compositional and mechanical implications of hypomineralization in a rabbit model of osteomalacia.

Osteomalacia is characterized by hypomineralization of the bone associated with increased water content. In this work we evaluate the hypotheses that 1) 3D solid-state magnetic resonance imaging (MRI) of (31)P (SSI-PH) and (1)H (SSI-WATER) of cortical bone can quantify the key characteristics of osteomalacia induced by low-phosphate diet; and 2) return to normophosphatemic diet (NO) results in recovery of these indices to normal levels. Twenty female five-week old rabbits were divided into four groups. Five animals were fed a normal diet for 8 weeks (NOI); five a hypophosphatemic diet (0.09%) for the same period to induce osteomalacia (HYI). To examine the effect of recovery from hypophosphatemia an additional five animals received a hypophosphatemic diet for 8 weeks, after which they were returned to a normal diet for 6 weeks (HYII). Finally, five animals received a normal diet for the entire 14 weeks (NOII). The NOI and HYI animals were sacrificed after 8 weeks, the NOII and HYII groups after 14 weeks. Cortical bone was extracted from the left and right tibiae of all the animals. Water content was measured by SSI-WATER and by a previously reported spectroscopic proton-deuteron nuclear magnetic resonance (NMR) exchange technique (NMR-WATER), phosphorus content by SSI-PH. All MRI and NMR experiments were performed on a 9.4 T spectroscopy/micro-imaging system. Degree of mineralization of bone (DMB) was measured by micro-CT and elastic modulus and ultimate strength by 3-point bending. The following parameters were lower in the hypophosphatemic group: phosphorus content measured by SSI-PH (9.5+/-0.4 versus 11.1+/-0.3 wt.%, p<0.0001), ash content (63.9+/-1.7 versus 65.4+/-1.1 wt.%, p=0.05), ultimate strength, (96.3+/-16.0 versus 130.7+/-6.4 N/mm(2), p=0.001), and DMB (1115+/-28 versus 1176+/-24 mg/cm(3), p=0.003); SSI-WATER: 16.1+/-1.5 versus 14.4+/-1.1 wt.%, p=0.04; NMR-WATER: 19.0+/-0.6 versus 17.4+/-1.2 wt.%, p=0.01. Return to a normophosphatemic diet reduced or eliminated these differences (SSI-PH: 9.5+/-0.9 versus 10.6+/-0.8 wt.%, p=0.04; DMB: 1124+/-31 versus 1137+/-10 mg/cm(3), p=0.2; US: 95.6+/-18.6 versus 103.9+/-7.5 N/mm(2), p=0.2; SSI-WATER: 12.4+/-0.6 versus 12.2+/-0.3 wt.%, p=0.3) indicating recovery of the mineral density close to normal levels. Phosphorus content measured by SSI-PH was significantly correlated with DMB measured by micro-CT (r(2)=0.47, p=0.001) as well as with ultimate strength (r(2)=0.54, p=0.0004). The results show that the methods presented have potential for in situ assessment of mineralization and water, both critical to the bone's mechanical behavior.

Construction and calibration of a 50 T/m z-gradient coil for quantitative diffusion microimaging.

q-Space imaging is capable of providing quantitative geometrical information of structures at cellular resolution. However, the size of restrictions that can be probed hinges on available gradient amplitude and places very high demands on gradient performance. In this work we describe the design and construction of a small, high-amplitude (50 T/m) z-gradient coil, interfaced with a commercial 9.4 T microimaging system. We also describe a method to calibrate the coil for quantitative measurements of molecular diffusion at very high-gradient amplitudes. Calibration showed linear current response up to 50 T/m, with a gain=1.255 T/m/A. The z-gradient coil was combined with the commercial x- and y-gradients for tri-axial imaging, and its performance was demonstrated by ADC maps of free water and by q-space experiments on water sequestered around polystyrene microspheres (4.5 microm diameter), which showed the expected diffraction peak. In addition, diffusion-weighted images of a fixed mouse spinal cord illustrated the capability of this coil for quantitative imaging of tissue microstructure.

Measurement of phosphorus content in normal and osteomalacic rabbit bone by solid-state 3D radial imaging.

In osteomalacia decreased mineralization reduces the stiffness and static strength of bone. We hypothesized that hypomineralization in osteomalacic bone could be quantified by solid-state (31)P magnetic resonance imaging (SS-MRI). Hypomineralization was measured with a 3D radial imaging technique at 162 MHz (9.4T) in rabbit cortical bone of hypophosphatemic (HY) and normophosphatemic (NO) animals. The results were compared with those obtained by quantitative micro-CT (micro-CT) and (31)P solution NMR. 3D images of 277 microm isotropic voxel size were obtained in 1.7 hr with SNR approximately 9. Mineral content was lower in the HY relative to the NO group (SS-MRI: 9.48 +/- 0.4 vs. 11.15 +/- 0.31 phosphorus wet wt %, P < 0.0001; micro-CT: 1114.6 +/- 28.3 vs. 1175.7 +/- 23.5 mg mineral/cm(3); P = 0.003). T(1) was shorter in the HY group (47.2 +/- 3.5 vs. 54.1 +/- 2.7 s, P = 0.004), which suggests that relaxation occurs via a dipole-dipole (DD) mechanism involving exchangeable water protons, which are more prevalent in bone from osteomalacic animals.

[The art of horseshoeing--between empiricism and science]. / Die Kunst des Hufbeschlags--zwischen Empirie und Wissenschaft.

To correctly shoe a horse requires the farriers to have a good working knowledge of postural anomalies and movement patterns, as well as of the different concepts of horseshoeing and to be able to apply the appropriate technique to every individual horse they shoe. The correct technique for specific problem cases is frequently a subject of debate amongst specialists and many theories would benefit from objective gait analyses. The case study presented examines the influence of different shoeing conditions on selected gait analysis parameters. The measurements were conducted on a Warmblood mare: (A) shod with long toes, (B) properly trimmed without shoes, (C) conventionally shod with rolled toes and finally (D) shod using the 4-point technique. Data on force-, time- and distance parameters were recorded using an instrumented treadmill. First contact and breakover of the hooves were documented using high-speed videography. A long toe resulted in a prolongation of the breakover time and, therefore, in a prolongation of the second half of the stance phase. Additionally, the prolonged stance duration associated with an unaltered force impulse, led to decreased force peaks. It was possible to objectively record differences between the trimmed, unshod foot, the shod long-toe and the shod rolled toe configurations. The differences between the rolled toe and the 4 point shoe however, were minimal. Gait analysis is a technique well suited for objective evaluation of different shoeing techniques under standardised conditions.

Retinoic acid induced myelomeningocele in fetal rats: characterization by histopathological analysis and magnetic resonance imaging.

The prevention of human neural tube defects by folic acid administration and the potential for fetal surgical intervention for myelomeningocele (MMC) have renewed interest in the molecular pathways and pathophysiology of spina bifida. Animal models for assessment of the early developmental biology and pathophysiology of this lesion are needed. The goal of this study was to develop and characterize a non-surgical rat model of MMC. Time-dated Sprague-Dawley rats were gavage fed different doses of retinoic acid (RA) dissolved in olive oil at E10 (maternal n = 55, fetal n = 505). Control animals received olive oil alone (maternal n = 20, fetal n = 265) or were untreated (maternal n = 5, fetal n = 63). Fetuses were analyzed by detailed histopathology and MRI. Overall, isolated MMC occurred in 60.7% (307/505) of RA-exposed fetuses and no controls. Histopathology confirmed the entire spectrum of severity observed in human MMC, ranging from exposure of the cord with intact neural elements to complete cord destruction. MRI of the brain of MMC fetuses confirmed structural changes similar to humans with Arnold-Chiari malformation, including downward displacement of the cerebellum to just above the foramen magnum and compression of the developing medulla into a small posterior fossa. In conclusion, the RA-induced rat model of MMC is developmentally and anatomically analogous to human MMC. This relatively efficient and cost-effective model of MMC should facilitate investigation of the developmental biology and pathophysiology of MMC, and may be useful for the evaluation of further strategies for prenatal treatment.

Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas.

A method was developed for obtaining high signal-to-noise 13C NMR spectra of intracellular compounds in metabolically active cultured cells. The method allows TCA cycle labeling kinetics to be determined in real time without significant oxygen transport limitations. Cells were immobilized on the surface of nonporous microcarriers that were either uncoated or coated with polypeptides and used in a 12-cm3 packed bed. The methods were tested with two EMT6 mouse mammary tumor cell lines, one strongly adherent and the other moderately adherent, and a weakly adherent mouse insulinoma line (betaHC9). For both EMT6 lines, NTP and oxygen consumption measurements indicated that the number of cells in the spectrometer ranged from 6 x 10(8) to 1 x 10(9). During infusion of [1-13C]glucose, labeling in C-4 glutamate (indicative of flux into the first half of the TCA cycle) could be detected with 15-min resolution. However, labeling for C-3 and C-2 glutamate (indicative of complete TCA cycle activity) was fivefold lower and difficult to quantify. To increase TCA cycle labeling, cells were infused with medium containing [1,6-13C2]glucose. A 2.5-fold increase was observed in C-4 glutamate labeling and C-3 and C-2 glutamate labeling could be monitored with 30-min resolution. Citrate synthase activity was indirectly detected in real time, as [3,4-13C2]glutamate was formed from [2-13C]oxaloacetate and [2-13C]acetate (of acetyl-CoA). Cell mass levels observed with betaHC9 cells were somewhat lower. However, the 13C S/N was sufficient to allow real-time monitoring of the response of intracellular metabolite labeling to a step change in glucose and a combined glutamine/serum pulse.

Multipoint mapping for imaging of semi-solid materials.

Multipoint k-space mapping is a hybrid between constant-time (single-point mapping) and spin-warp imaging, involving sampling of a k-line segment of r points per TR cycle. In this work the method was implemented for NMR imaging of semi-solid materials on a 400 MHz micro-imaging system and two different k-space sampling strategies were investigated to minimize the adverse effects from relaxation-induced k-space signal modulation. Signal attenuation from T(2) decay results in artifacts whose nature depends on the k-space sampling strategy. The artifacts can be minimized by increasing the readout gradient amplitude, by PSF deconvolution or by oversampling in readout direction. Finally, implementation of a T(2) selective RF excitation demonstrates the feasibility of obtaining short-T(2) contrast even in the presence of tissues with long-T(2). The method's potential is illustrated with 3D proton images of short-T(2) materials such as synthetic polymers and bone.

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E.

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts.

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

Galactose metabolism in normal human lymphoblasts studied by (1)H, (13)C and (31)P NMR spectroscopy of extracts.

The development of tools to follow and quantitate the fate of galactose in mammalian cells is crucial to the study and understanding of the inherited disorders of galactose metabolism. In this study we incubated normal human lymphoblasts with 1- or 2-(13)C galactose for 2.5 or 5 h and prepared TCA extracts of the cells. The various galactose metabolites were identified and quantified using a combination of proton, carbon and phosphorus NMR spectra. Galactose-1-phosphate (gal-1P), uridine diphosphogalactose, uridine diphosphoglucose and galactitol were present in the extracts. Average levels for gal-1P were around 10 nmol/mg protein and for uridine diphosphoglucose, uridine diphosphogalactose and galactitol in the range of 0.5-2 nmol/mg protein. Galactonate was never found in any conditions. Percentage labeling could be estimated for gal-1P and for the ribose carbons of AMP. The labeling agrees with a conversion of galactose to glucose through the Leloir pathway.

Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE.

To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;-150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;-191) of apoE3 and apoE4 bound to the LDLR with approximately 100-fold greater affinity than the 22-kDa fragment of apoE2. The pK(a) values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pK(a) of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic alpha-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity. The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pK(a) of K146 significantly, but it increased the pK(a) of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.

Effects of lipid interaction on the lysine microenvironments in apolipoprotein E.

Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1-191) in the lipid-free and lipid-associated states. As shown by (1)H,(13)C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3.dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK(a) values above 10. In apoE3.DMPC complexes, however, all eight lysines were resolved, and the pK(a) values were 9.2-11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK(a) values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3.DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.

Chronic metabolic acidosis increases NaDC-1 mRNA and protein abundance in rat kidney.

BACKGROUND: Chronic metabolic acidosis increases, while alkali feeding inhibits, proximal tubule citrate absorption. The activity of the apical membrane Na+/citrate cotransporter is increased in metabolic acidosis, but is not altered by alkali feeding. METHODS: Renal cortical mRNA and brush border membrane protein abundances of sodium/dicarboxylate-1 (NaDC-1), the apical membrane Na+/citrate transporter, were measured. RESULTS: By immunohistochemistry, NaDC-1 was localized to the apical membrane of the proximal tubule. Chronic metabolic acidosis caused an increase in NaDC-1 protein abundance that was maximal in the S2 segment and that increased with time. Metabolic acidosis also increased NaDC-1 mRNA abundance, but this was first seen at three hours and correlated with the severity of the metabolic acidosis. Alkali feeding had no effect on NaDC-1 protein or mRNA abundance. CONCLUSIONS: Chronic metabolic acidosis increases renal cortical NaDC-1 mRNA abundance and apical membrane NaDC-1 protein abundance, while alkali feeding is without effect on NaDC-1.

Metabolic control of sodium transport in streptozotocin-induced diabetic rat hearts.

Diabetic and control cardiomyocytes encapsulated in agarose beads and superfused with modified medium 199 were studied with 23Na- and 31P-NMR. Baseline intracellular Na+ was higher in diabetic (0.076 +/- 0.01 micromoles/mg protein) than in control (0.04 +/- 0.01 micromoles/mg protein) (p < 0.05). Baseline betaATP and phosphocreatine (PCr) (peak area divided by the peak area of the standard, methylene diphosphonate) were lower in diabetic than in control, e.g., betaATP control, 0.70 +/- 0.07; betaATP diabetic, 0. 49 +/- 0.04 (p < 0.027); PCr control, 1.20 +/- 0.13; PCr diabetic, 0. 83 +/- 0.11 (p < 0.03). This suggests that diabetic cardiomyocytes have depressed bioenergetic function, which may contribute to abnormal Na,K-ATPase function, and thus, an increase in intracellular Na+. In the experiments presented herein, three interventions (2-deoxyglucose, dinitrophenol, or ouabain infusions) were used to determine whether, and the extent to which, energy deficits or abnormalities in Na,K-ATPase function contribute to the increase in intracellular Na+. In diabetic cardiomyocytes, 2-deoxyglucose and ouabain had minimal effect on intracellular Na+, suggesting baseline depression of, or resetting of both glycolytic and Na,K-ATPase function, whereas in control both agents caused significant increases in intracellular Na+after 63 min exposure: 2-deoxyglucose control, 32.9 +/- 8.1%; 2-deoxyglucose diabetic, -4.6 +/- 6% (p < 0.05); ouabain control, 50.5 +/- 8.8%; ouabain diabetic, 21.2 +/- 9.2% (p < 0.05). In both animal models, dinitrophenol was associated with large increases in intracellular Na+: control, 119.0 +/- 26.9%; diabetic, 138.2 +/- 12.6%. Except for the dinitrophenol intervention, where betaATP and PCr decreased to levels below 31P-NMR detection, the energetic metabolites were not lowered to levels that would compromise sarcolemmal function (Na,K-ATPase) in either control or diabetic cardiomyocytes. In conclusion, in diabetic cardiomyocytes, even though abnormal glycolytic and Na, K-ATPase function was associated with increases in intracellular Na+, these increases were not directly related to global energy deficit.

Relationship between cancellous bone induced magnetic field and ultrastructure in a rat ovariectomy model.

The site-dependent variations in trabecular bone morphology were studied in the rat tibia by magnitude and phase difference three-dimensional nuclear magnetic resonance microscopy and image processing, and the implications of ovariectomy were evaluated. Specimens excised from the proximal tibial metaphysis in ovariectomized (n = 7) and intact control (n = 4) rats were imaged at 9.4T with their anatomic axes parallel to the direction of the magnetic field. An echo-offset 3D rapid spin-echo excitation pulse sequence was used to generate phase difference maps, from which the standard deviation of the phase difference, sigma(delta psi), was calculated. In addition, a fictitious rate constant, R2', was calculated from the slope of the exponential portion of the Fourier transform of the phase difference histogram. Trabecular bone volume fraction was also determined in the same volume of interest. The results show strong correlations between bone volume fraction and both sigma(delta psi) and R2', suggesting that these parameters could be useful for nondestructive assessment of trabecular bone volume.

Covalent modifications to 2'-deoxyguanosine by 4-oxo-2-nonenal, a novel product of lipid peroxidation.

Two major products (adducts A and B) from the reaction of 2-deoxyguanosine (dGuo) with 13-hydroperoxylinoleic acid were detected by liquid chromatography/mass spectrometry (LC/MS). Adducts A and B were also the major products formed enzymatically when dGuo was incubated in the presence of linoleic acid and lipoxygenase. The mass spectral fragmentation patterns of adducts A and B suggested that unique modifications to the nucleoside had been introduced. This resulted in the characterization of a novel bifunctional electrophile, 4-oxo-2-nonenal, as the principal breakdown product of linoleic acid hydroperoxide. In subsequent studies, adduct A was found to be a substituted ethano dGuo adduct that was a mixture of three isomers (A(1)-A(3)) that all decomposed to form adduct B. Adduct A(1) was the hemiacetal form of 3-(2-deoxy-beta-D-erythropentafuranosyl)-3,5,6, 7-tetrahydro-6-hydroxy-7-(heptane-2-one)-9H-imidazo[1, 2-alpha]purine-9-one. Adducts A(2) and A(3) were the diastereomers of the open chain ketone form. Adduct B was the substituted etheno dGuo adduct, 3-(2-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(heptane-2 -one)-9-hydroxy[1,2-alpha]purine, the dehydration product of adducts A(1)-A(3). Identical covalent modifications to dGuo were observed when calf-thymus DNA was treated with 4-oxo-2-nonenal. These data illustrate the diversity of reactive electrophiles produced from the peroxidative decomposition of lipids and have implications in fully assessing the role of lipid peroxidation in mutagenesis and carcinogenesis.

The diagnostic accuracy of ex vivo MRI for human atherosclerotic plaque characterization.

Recent evidence indicates that the type of atherosclerotic plaque, rather than the degree of obstruction to flow, is an important determinant of the risk of cardiovascular complications. In previous work, the feasibility of using MRI for the characterization of plaque components was shown. This study extends the previous work to all the plaque components and shows the accuracy of this method. Twenty-two human carotid endarterectomy specimens underwent ex vivo MRI and histopathological examination. Sixty-six cross sections were matched between MRI and histopathology. In each cross section, the presence or absence of plaque components were prospectively identified on the MRI images. The overall sensitivity and specificity for each tissue component were very high. Calcification and fibrocellular tissue were readily identified. Lipid core was also identifiable. However, thrombus was the plaque component for which MRI had the lowest sensitivity. A semiautomated algorithm was created to identify all major atherosclerotic plaque components. MRI can characterize carotid artery plaques with a high level of sensitivity and specificity. Application of these results in the clinical setting may be feasible in the near future.