Assessment of tibial stiffness by vibration testing in situ--I. Identification of mode shapes in different supporting conditions

Vibrational methods to monitor fracture healing, the BRA and the IFR, are compared under different supporting conditions, excitation technique and signal processing. Mode shapes are identified by modal analysis. A wet excised human tibia and an amputation specimen are investigated. Excitation technique and signal processing caused only minor differences in the resonance frequencies. The supporting conditions had an important influence on the single bending modes changing both mode-shapes and frequencies. Thus the BRA-splint imposed a node at the malleolus. Modal analysis revealed the following modes in the two supporting conditions: BRA-splint: A 'rigid body' mode of 165 Hz in the sagittal plane. A single bending mode of 315 Hz close to the sagittal plane. IFR-hanging leg: A 'rigid-body' mode of 167 Hz close to the sagittal plane. Two single bending modes ('free-free'), a mode of 303 Hz close to the frontal plane and a mode of 470 Hz in the sagittal plane.     

Assessment of tibial stiffness by vibration testing in situ--II. Influence of soft tissues, joints and fibula

The influence of soft tissues and joints on the vibration of the human tibia was examined by modal analysis on amputated lower limbs, where the soft tissues and the fibula were dissected gradually. Measurements were made in two different set ups, IFR and BRA, which were both designed to monitor fracture healing. In IFR, vibrations are generated by hammer impact on a relaxed hanging lower leg, with the knee flexed. Resonant frequencies are determined by a computer Fourier transform procedure. In BRA, a steady state vibration is induced in a lower leg, supported near the ankle and the tibial tuberosity, using an electromagnetic shaker. Resonant frequencies are determined from the maxima in vibration amplitudes. In both set ups the soft tissues have a similar influence on the vibration of the tibia: the skin hardly influences the determined modal parameter. The mass of the muscles influences both the resonant frequency and the damping. The fibula has a stiffening effect on the tibia. The influence of the joints is small in the IFR-set up: the tibia vibrates in conditions close to those for the free-free vibration. In the BRA-set up, the supports determine the boundary conditions.     

Electric field generated by axial longitudinal vibration modes of microtubule

Microtubules are electrically polar structures fulfilling prerequisites for generation of oscillatory electric field in the kHz to GHz region. Energy supply for excitation of elasto-electrical vibrations in microtubules may be provided from GTP-hydrolysis; motor protein–microtubule interactions; and energy efflux from mitochondria. We calculated electric field generated by axial longitudinal vibration modes of microtubules for random, and coherent excitation. In case of coherent excitation of vibrations, the electric field intensity is highest at the end of microtubule. The dielectrophoretic force exerted by electric field on the surrounding molecules will influence the kinetics of microtubule polymerization via change in the probability of the transport of charge and mass particles. The electric field generated by vibrations of electrically polar cellular structures is expected to play an important role in biological self-organization.     

Total and high-density lipoprotein cholesterol measurements. Hazards in clinical interpretation.

Duplicate plasma specimens from 24 persons were sent to a community hospital, a commercial laboratory, and a university lipid research laboratory at two separate times to assess the intralaboratory and interlaboratory variations of total and high-density cholesterol measurements. For all three laboratories, the 95% confidence limits for the reproducibility of total cholesterol levels are about +/- 5.5%. For high-density lipoprotein (HDL) values, they are +/- 7%, +/- 14.45%, and +/- 9%. Interlaboratory differences for total cholesterol at the 95% confidence level are +/- 7.7% and for HDL +/- 18.4%. Construction of a "cardiac risk ratio" of total to HDL cholesterol levels is subject to confusion because small errors in HDL cholesterol levels produce large errors in the ratio.     

Sensitivity of different E. coli and Salmonella strains in mutagenicity testing calculated on the basis of selected literature

Two microbial screening test systems for gene (point) mutations, the Salmonella typhimurium assay (TA1535, TA1537, TA1538, TA98 and TA100) and the Escherichia coli WP2 reverse-mutation system (WP2, WP2uvrA, WP2pKM101 and WP2uvrApKM101), were compared with regard to sensitivity toward a broad spectrum of compounds that cause base-pair or frameshift mutations and that have known carcinogenic qualities. Based on available published literature we found that all 44 carcinogens and 9 non-carcinogens examined in both test systems also met with criteria for data acceptance drawn up by us. The results obtained are: firstly, that the Salmonella assay is decidedly better validated than the E. coli WP2 test; and secondly, that the E. coli test system sensitivity (91%) is fully on a par with the sensitivity of the Salmonella assay (72%). This last is in divergence from earlier reports, e.g. Brusick et al. (1980), and this difference must be ascribed to the new plasmid-containing strains. The many compounds not tested in the E. coli department result in fewer false negatives in the E. coli test system and their omission constitutes a bias in favour of the E. coli assay. By eliminating compounds that are negative in Salmonella and dropped from the WP2 analysis owing to insufficient data, the sensitivity of the Salmonella system is raised to 84% as compared with 91% for the WP2 assay. The results further indicate that some of the tester strains are superfluous, and show an exceedingly sensitive test can be performed by combining the best tester strains from the two test systems.     

Identification of in-vivo vibration modes of human tibiae by modal analysis

When attempting to evaluate the mechanical properties of human bones in vivo by mechanical vibration analysis, some essential requirements must be met. A quantitative relation between measured vibration parameters (e.g., natural frequency) and mechanical bone properties must be available, in-vivo vibration modes should correctly be identified and the associated natural frequencies reproducibly and accurately measured, the influence of joints and soft tissues must be known. These problems were addressed by modal analysis (i.e., experimental determination of natural frequencies, mode shapes and damping ratios) of human tibiae in the following situations: 1) dry excised tibiae, 2) fresh excised tibiae, 3) in-vivo tibiae, 4) tibiae in an amputated leg, in different steps of dissection. In the in-vivo measuring conditions used by the authors, the tibia vibration is practically free-free. Two single bending modes (at +/- 270 Hz and +/- 340 Hz, respectively), each of them corresponding with one principal direction for bending, were identified. The difference between the natural frequencies observed in vivo and those of fresh excised tibiae is almost completely caused by the effect of muscles (added mass and damping), whereas joints and skin play only a minor role. Frequency differences between fresh and dry excised tibiae are largely accounted for by the absence of bone marrow in the latter.     

Statistical interpretation of fluorescence energy transfer measurements in macromolecular systems.

A statistical method is presented for the interpretation of intramolecular distance measurements by the fluorescence energy transfer technique in systems for which the detailed geometries of the donor-acceptor pairs are unknown. This method enables calculation of the probability that a specified distance range corresponds to the actual distance to be measured. It makes use of the numerically calculated probability density function for the distance of interest. The two general systems considered are the single donor-acceptor pair and the multi-donor-single-acceptor transfer. In both systems, the statistical method incorporates the uncertainty in the orientation of the donor and acceptor dipoles. In addition, it can take into account the rotational mobility of the donor dipoles determined by time-dependent emission anisotropy measurements. When more than one donor is involved in the transfer process, the uncertainties associated with the number and location of individual donors and the size and shape of the donor distribution are also incorporated in calculating the distance ranges. Application of the method was demonstrated for a wide range of transfer efficiency and Ro values for the single donor-acceptor system. Specific examples are also presented for interpretation of both single donor-acceptor and multi-donor-single-acceptor energy transfer measurements performed in order to reveal the spatial relationship of the sigma subunit and the rifampicin binding site in the Escherichia coli RNA polymerase (see Wu, C.-W., Yarbrough, L. R., Wu, F. Y.-H., and Hillel, Z. (1976), Biochemistry, preceding paper in this issue). Analysis of these energy transfer data by methods which use average values of the unknown geometrical parameters of the system yielded results similar to those obtained by the statistical method. However, the statistical method represents a more realistic approach to the interpretation of energy transfer measurements since it provides information concerning the entire range of possible distances and their relative likelihood.     

In vivo tibial stiffness is maintained by whole bone morphology and cross-sectional geometry in growing female mice

Whole bone morphology, cortical geometry, and tissue material properties modulate skeletal stresses and strains that in turn influence skeletal physiology and remodeling. Understanding how bone stiffness, the relationship between applied load and tissue strain, is regulated by developmental changes in bone structure and tissue material properties is important in implementing biophysical strategies for promoting healthy bone growth and preventing bone loss. The goal of this study was to relate developmental patterns of in vivo whole bone stiffness to whole bone morphology, cross-sectional geometry, and tissue properties using a mouse axial loading model. We measured in vivo tibial stiffness in three age groups (6, 10, 16 wk old) of female C57Bl/6 mice during cyclic tibial compression. Tibial stiffness was then related to cortical geometry, longitudinal bone curvature, and tissue mineral density using microcomputed tomography (microCT). Tibial stiffness and the stresses induced by axial compression were generally maintained from 6 to 16 wks of age. Growth-related increases in cortical cross-sectional geometry and longitudinal bone curvature had counteracting effects on induced bone stresses and, therefore, maintained tibial stiffness similarly with growth. Tissue mineral density increased slightly from 6 to 16 wks of age, and although the effects of this increase on tibial stiffness were not directly measured, its role in the modulation of whole bone stiffness was likely minor over the age range examined. Thus, whole bone morphology, as characterized by longitudinal curvature, along with cortical geometry, plays an important role in modulating bone stiffness during development and should be considered when evaluating and designing in vivo loading studies and biophysical skeletal therapies.     

Microtensile measurements of single trabeculae stiffness in human femur

In this paper, the authors perform microtensile tests of single trabeculae excised from a human femur head. One of the main issues of this work is to establish some experimental procedures for preparing and testing the specimens. The use of a well-characterized microtensile apparatus allows for a low intraspecimen dispersion of the measured stiffness. Tensile/compressive tests were chosen because they appear less sensitive to errors in the cross-sectional area measurements with respect to bending tests. By these considerations, some tensile/compressive tests of plate-like trabecular specimens have been carried out. Typical stiffness values are 74.2+/-0.7Nmm(-1) for tensile tests, and 58.9+/-0.6Nmm(-1) for compressive test. Another compressive test performed on a shorter specimen yielded a stiffness value of 148.3+/-5.3Nmm(-1). The maximum applied load was about 0.5N. Rough measurements of specimens sizes yielded a Young's modulus value ranging from 1.41 to 1.89GPa.     

Interpretation of DNA vibration modes. II--The adenosine and thymidine residues involved in oligonucleotides and polynucleotides

Normal coordinate analysis of the adenosine and thymidine residues involved in the right- and left-handed conformations of oligonucleotides and polynucleotides has been performed. The valence force field, employed in this work, allowed recently to reproduce the vibrational spectra of 2'-deoxythymidine and 2'-deoxyadenosine. The calculated wavenumbers based on a non-redundant set of internal coordinates have been compared to the Raman and infrared peak positions arising from A, B, C, D and Z conformations, in the 1550-1250 cm-1 and 800-600 cm-1 spectral regions: i.e. characteristic of adenosine and thymidine residues. Moreover, a systematic study has been performed on the evolution of the vibrational wavenumbers as a function of the glycosidic angle (chi) and the sugar pucker conformation.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4–6 h.     

Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas, however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4-6 h.     

Accuracy and reproducibility of bending stiffness measurements by mechanical response tissue analysis in artificial human ulnas

Osteoporosis is characterized by reduced bone strength, but no FDA-approved medical device measures bone strength. Bone strength is strongly associated with bone stiffness, but no FDA-approved medical device measures bone stiffness either. Mechanical Response Tissue Analysis (MRTA) is a non-significant risk, non-invasive, radiation-free, vibration analysis technique for making immediate, direct functional measurements of the bending stiffness of long bones in humans in vivo. MRTA has been used for research purposes for more than 20 years, but little has been published about its accuracy. To begin to investigate its accuracy, we compared MRTA measurements of bending stiffness in 39 artificial human ulna bones to measurements made by Quasistatic Mechanical Testing (QMT). In the process, we also quantified the reproducibility (i.e., precision and repeatability) of both methods. MRTA precision (1.0±1.0%) and repeatability (3.1±3.1%) were not as high as those of QMT (0.2±0.2% and 1.3+1.7%, respectively; both p<10⁻⁴). The relationship between MRTA and QMT measurements of ulna bending stiffness was indistinguishable from the identity line (p=0.44) and paired measurements by the two methods agreed within a 95% confidence interval of ±5%. If such accuracy can be achieved on real human ulnas in situ, and if the ulna is representative of the appendicular skeleton, MRTA may prove clinically useful.     

Estimation of the relative stiffness of the molecular chain in polyelectrolytes from measurements of viscosity at different ionic strengths

A method was developed that allowed comparison of the stiffness of the chain in different polyelectrolyte from measurements of the intrinsic viscosity at different concentrations of added monovalent (sodium) salt. The response to salt was quantitatively expressed as the slope of straight lines relating the intrinsic viscosity to the reciprocal of the square-root of the ionic strength. This slope increased considerably with increasing molecular weight of the polyelectrolyte, and could serve to characterize the response to salt of different substances only when comparison was made at a constant molecular weight. An empirical parameter, B, which is the slope corresponding to an intrinsic viscosity of 1.0 at an ionic strength of 0.1 M could be correlated to the unperturbed dimensions of the molecules. A method of extrapolation, enabling the determination of B from measurements of viscosity on only one sample of unknown molecular weight, was evaluated. The empirically found correlation between Band some well established parameters of stiffness did not contrast predictions from the “fuzzy-sphere model” of Fixman, provided that reasonable assumptions regarding ion-binding and the interaction between polymer and solvent were made.     

In vitro measurements and interpretation of total antioxidant capacity

Background

One of the strategies most commonly used to assess a free radical-antioxidant balance in chemical and biological systems is the determination of the total antioxidant capacity (TAC). A large amount of research has been published using TAC. However, it remains unclear which is the significance of these investigations for understanding the biological importance of free radical reactions.

Scope of review

This review discusses the relevance and limitations of TAC for the assessment of the antioxidant activities present in food and food derivatives, and in body tissues and fluids.

Major conclusions

TAC determinations are simple, inexpensive, and able to evaluate the capacity of known and unknown antioxidants and their additive, synergistic and/or antagonistic actions, in chemical and biological systems. However, different TAC assays correlate poorly with each other, since each TAC assay is sensitive to a particular combination of compounds, but exclude many others. The TAC values for foods cannot be translated to the in vivo (human) antioxidant defenses, and furthermore, to health effects provided by that food.

General significance

Up to date, conclusions that can be drawn from the extensive amount of research done using TAC of foods or populations should not be considered when used for making decisions affecting population health. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Alterations of lithium clearance in rats by different modes of lithium administration

This study examines the effects of acute versus dietary lithium administration on proximal tubular fluid output (Vprox) and sodium clearance in 6 groups of unrestrained, conscious rats. Vprox was estimated on the basis of the renal lithium clearance. The aim was to find the mode of lithium administration which least influences the proximal and distal reabsorption of sodium. The lithium doses used resulted in serum lithium concentrations between 0.2 and 0.3 mmol/l with no difference between the groups. Acute intravenous lithium administration increased lithium clearance by 40% and sodium clearance by 109%. Administration by gastric tube increased lithium clearance by 22% and sodium clearance by 78% in comparison to dietary administration of lithium. Potassium excretion did not change by acute lithium administration. The data presented indicate that prior to measurements of lithium clearance, lithium should be administered in the diet for 2 days, since acute lithium administration, intravenously or by gastric tube, causes great changes in renal tubular reabsorption.     

Spectral broadening of the Soret band in myoglobin: an interpretation by the full spectrum of low-frequency modes from a normal modes analysis

In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus supporting the applicability of the short-times approximation to the analysis of hemeprotein spectra; in particular, they clearly indicate the presence of spectral heterogeneity in the Soret band of carbon-monoxy myoglobin. The analyses also show that a temperature dependence of the Gaussian width parameter steeper than the hyperbolic cotangent law predicted by the Einstein harmonic oscillator and/or a temperature dependence of inhomogeneous broadening are not sufficient to obtain quantitative information on the magnitude of an-harmonic contributions to the iron-heme plane motion. However, the dependence of the previous two quantities may be used to obtain semiquantitative information on the overall coupling of the Soret transition to the low-frequency modes and therefore on the dynamic properties of the heme pocket in different states of the protein.     

Normal modes of vibration in bovine pancreatic trypsin inhibitor and its mechanical property

The normal mode analysis of conformational fluctuation is carried out for a small globular protein, bovine pancreatic trypsin inhibitor. Results are analyzed mainly to reveal the mechanical construction of the protein molecule. We take dihedral angles, including peptide omega angles, as independent variables for the normal mode analysis. There are 306 such angles in this molecule. Motions in modes with frequencies lower than 120 cm-1 are shown to involve atoms in the whole protein molecule, and spatial change of displacement vectors is continuous, i.e., those of atoms near in space are similar. To quantitate the observation of the continuity, a correlation function of direction vectors of atomic displacements is calculated. From this function we define a quantity that is interpreted as the wave length of an equivalent elastic plane wave. From this quantity we deduce effective Young's modulus for each mode. For the mode with the lowest frequency 4.4 cm-1, it turned out to be 0.8 x 10(9) dyn cm-2, the value two orders of magnitude softer than, for instance, alpha-helices. Prompted by this observation, the four lowest frequency modes and also the harmonic motions in the thermal equilibrium are analyzed further mainly to detect relatively rigid structural elements in the molecule. From this analysis emerges a mechanical picture of the protein molecule that is made up of relatively rigid elements held together by very soft parts.