Ice friction during speed skating.

During speed skating, the external power output delivered by the athlete is predominantly used to overcome the air and ice frictional forces. Special skates were developed and used to measure the ice frictional forces during actual speed skating. The mean coefficients of friction for the straights and curves were, respectively, 0.0046 and 0.0059. The minimum value of the coefficient of ice friction was measured at an ice surface temperature of about -7 degrees C. It was found that the coefficient of friction increases with increasing speed. In the literature, it is suggested that the relatively low friction in skating results from a thin film of liquid water on the ice surface. Theories about the presence of water between the rubbing surfaces are focused on the formation of water by pressure-melting, melting due to frictional heating and on the 'liquid-like' properties of the ice surface. From our measurements and calculations, it is concluded that the liquid-like surface properties of ice seem to be a reasonable explanation for the low friction during speed skating.


A power equation for the sprint in speed skating.

An analysis of the start of the 500 m speed skating races during the 1988 Olympic Winter Games showed a remarkably high correlation between the acceleration of the skater in the first second of the sprint and the final time (r = -0.75). In this study a power equation is used to explain this high coefficient of correlation. The performance in speed skating is determined by the capability of external power production by the speed skater. This power is necessary to overcome the air and ice friction and to increase the kinetic energy of the skater. Numerical values of the power dissipated to air and ice friction, both dependent on speed, are obtained from ice friction and wind tunnel experiments. Using aerobic and anaerobic power production as measured during supra maximal bicycle tests of international-level speed skaters, a model of the kinetics of power production is obtained. Simulation of power production and power dissipation yields values of speed and acceleration and, finally, the performance time of the sprint during speed skating. The mean split time at 100 m and the final time at 500 m in these races, derived from simulation, were 10.57 s (+/- 0.31) and 37.82 s (+/- 0.96), respectively. The coefficient of correlation between the simulated 500 m times and the actual 500 m times was 0.90. From the results of this study it can be concluded that the distribution of the available anaerobic energy is an important factor in the short lasting events. For the same amount of anaerobic energy the better sprinters appear to be able to liberate considerably more energy at the onset of the race than skaters of lower performance level.


A theoretical model for the noninvasive assessment of the transmitral pressure-flow relation.

The purpose of this paper is to formulate from the equations of fluid mechanics an equation which describes the transmitral pressure-flow relationship. According to the linear momentum equation applied to the atrioventricular coupling, the left-atrium-left-ventricle pressure difference (Pa-Pv) can be written as Pa-P v = A delta v/delta t + B v 2 + C v, where v is the transmitral blood velocity and A, B, and C are variables related to the geometry of the atrium, ventricle and mitral orifice, respectively. Based on this theory, Pa-Pv is calculated noninvasively in a patient with a nonobstructive mitral valve. Mitral flow and cardiac dimensions recorded by Doppler echocardiography are digitized and analyzed. Calculation shows that Pa-Pv reaches its peak value at the time of flow peak acceleration and has already considerably decreased at the time of peak velocity. The time course of calculated Pa-Pv is in close agreement with the published experimental catherization data. Numerical computation of early diastolic left atrium and left ventricle pressure curves based on the experimental data of others for the time constant of left ventricular relaxation, left atrial and ventricular chambers stiffness constants, combined with sine-waveform-simulated mitral flow, verifies the time course and the magnitude of Pa-Pv as predicted from flow equations. This paper provides a theoretical method for the noninvasive assessment of the transmitral pressure-flow relationship using ultrasound technique and might help to achieve a better understanding of the diastolic function as assessed by Doppler echocardiography.


Curvature characteristics and congruence of the thumb carpometacarpal joint: differences between female and male joints.

Three-dimensional geometric models of the articular surfaces of the thumb carpometacarpal (CMC) joint were constructed using precise data obtained from stereophotogrammetry (SPG). It was demonstrated that by using a least-squares surface-fitting technique, the SPG data on the surface can accurately be described by a single parametric biquintic spline function. From this mathematical description, curvature maps of the surfaces were calculated for 13 CMC joints (eight females, average 64 yr old, five males, average 70 yr old). The surface geometry of each joint was analyzed, comparisons were made between trapezial and metacarpal surfaces of the joint and differences determined between males and females. With regard to joint surface areas, the female trapezium is significantly smaller than that of the metacarpal. The shape of the female trapezial surface is also fundamentally different from that of males. No gender-related difference exists regarding the shape of the metacarpal surface. Congruence of the two opposing articular surfaces was defined by their relative principal curvatures. From these definitions, congruence in the radioulnar and dorsovolar anatomic directions, as well as the global congruence of the joint, were calculated. Most CMC joints were found to be more congruent along the radioulnar direction than the dorsovolar direction and, globally, female joints were found to be less congruent than male joints. The concept of joint congruence has played a central role in a number of hypotheses relating to the etiology of CMC joint osteoarthritis (OA), although conflicting hypotheses do exist. The precise quantitative findings of this study may lead to an improved understanding of CMC joint OA, and perhaps explain its prevalence in the female population over 55.


Prediction of the loading along the leg during snow skiing.

The complete force and moment of each cross section of the leg between the ski boot top and the knee during normal skiing were predicted from measurements of the force and moment under the toe and heel of the boot and the flexion of the ankle. The force and moment components predicted at the base of the boot were significantly different from those predicted at sites of potential injury at the boot top and the knee. The maximum torsional and maximum varus-valgus moments predicted at the knee over all subjects tested were 70 Nm and 149 Nm, which are within the estimated range of the ultimate strength of the knee without support from contracted muscles crossing the knee. Regression analyses were used to find the force components at the base of the boot that best predict the bending and torsional moments at the boot top and knee. The torsional moments at the boot top and knee are best predicted by the medial-lateral force at the toe. The varus-valgus moment at the boot top and knee are best predicted by the resultant medial-lateral force component at the base of the boot. The set of best predictors of the anterior-posterior bending moments at the boot top and knee includes the vertical force at the toe, the vertical force at the heel and the component of the total vertical force directed perpendicular to the leg.


A miniature piezoelectric polymer transducer for in vitro measurement of the dynamic contact stress distribution.

Previous studies of contact pressure measurement between articular surfaces have been mostly limited to static techniques. The purpose of our study was to develop a new dynamic technique for a direct measurement of the local contact stresses, and to apply the new method to an in vitro cadaver study of the patellofemoral joint pressures. The miniature transducer consists of a 2 mm diameter and 28 microns thick piece of piezoelectric polymer film sandwiched between two stainless steel electrodes of similar diameter. A water-resistant capsule consisting of Teflon film and Hysol epoxy was applied around the transducer. The transducer was 3 mm in diameter and 0.7 mm in thickness. A 3 mm well was made at six locations in the patella, corresponding to superior, middle, and inferior regions of both facets. Six transducers were cemented within each well, flush with the articular cartilage. The transducers were calibrated in situ before and after the experiment. The femur was rigidly fixed to the loading apparatus and the tibia was allowed to flex and extend through a 90 degrees range of motion using an Instron and a pulley system connected to the quadriceps tendon. Q angles of 0, 5, 10 and 15 degrees were established by adjusting the direction of the quadriceps tendon. Stresses ranging from 0.1-1.3 MPa were recorded at various locations. These values varied in flexion and extension. An overall decrease in these stresses was noted after tuberosity elevation up to 1.5 cm, following which increased values up to 1.8 MPa were recorded mostly in the superior section.(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo tracking of the human patella.

The purpose of this study was to describe the dynamic, in vivo, three-dimensional tracking pattern of the patella for one normal male subject. Intracortical pins were inserted into the patella, tibia, and femur. The subject performed seated and squatting knee flexion/extension, and maximum voluntary quadriceps contractions. In addition, the vastus medialis oblique was subjected to maximal electrical stimulation. Motions of the markers attached to the intracortical pins were analyzed using an automated video system. Patellar and tibial motions were determined relative to a femoral reference system. While the tibia flexed 50 degrees from full extension (seated condition), the patella flexed 30.3 degrees, tilted laterally 10.3 degrees, and shifted laterally 8.6 mm. In general, these results show qualitative agreement with the data collected from cadaveric specimens [van Kampen and Huiskes, J. orthop. Res. 8, 372-382 (1990)]. The differences present may reflect different passive constraints to patellar motions, and different relative loading of the individual quadriceps components, in our study compared to the cadaveric study. Only small differences were found between patellar motions in the seated and squatting conditions. Differences in patellar displacements produced by (1) maximal electrical stimulation of the vastus medialis oblique, and (2) maximum voluntary quadriceps contraction, at 30 degrees knee flexion and full extension, may reflect the dominant influence of passive constraints, and the vastus lateralis, on normal patellar motions. Further in vivo study of patellar tracking seems warranted to evaluate surgical and conservative interventions for patellofemoral disorders.


Periosteal and endosteal control of bone remodeling under torsional loading.

The shape changes that occur in the mid-diaphysis of a long bone due to adaptive remodeling induced by increasing or decreasing the axial and/or torsional loading of the bone are investigated using a simple model. In this model the mid-diaphysis of a long bone is represented as a hollow thick-walled right-circular cylinder, and different optimal strategies for bone remodeling are considered. It is shown that if such a thick-walled right-circular cylinder capable of surface remodeling is subjected to an axial compressive load and a twisting torque, then the remodeling patterns depend on whether the periosteal surface or the endosteal surface controls the limits of the remodeling process. It is shown that the effect of increasing the torque is always opposite to the effect of increasing the compressive load. Thus, similar remodeling patterns are obtained by increasing one type of loading and decreasing the other. Aside from the restriction of idealized cylindrical geometry, the only assumptions made are that the bone tissue is linearly elastic and that there exists a finite range of remodeling equilibrium stresses. Only those loading situations which maintain the bone in remodeling equilibrium are considered in this work. It follows that the results presented are independent of the specific type of rule governing the temporal evolution of the bone shape, since any such rule applies only in situations where there is active remodeling and, hence, no remodeling equilibrium.


Hemiplegic gait: a kinematic analysis using walking speed as a basis.

The kinematics of treadmill ambulation of stroke patients (N = 9) and healthy subjects (N = 4) was studied at a wide range of different velocities (i.e. 0.25-1.5 m s-1), with a focus on the transverse rotations of the trunk. Video recordings revealed, for both stroke patients and healthy subjects, similar relations between walking speed and stride length as well as stride frequency. The phase difference between pelvic and thoracic rotations (i.e. trunk rotation) and the total range of trunk rotation were almost linearly related to the walking speed. Healthy subjects showed a marked increase in pelvic rotation from 1 to 1.5 m s-1. Using dimensional analysis in a comparison between stroke patients and healthy subjects, invariances in the coordination of gait were found for stride length, stride frequency, pelvic rotation, and trunk rotation. Constant relations were obtained between, on the one hand, dimensionless velocity and, on the other, dimensionless stride length as well as stride frequency. Transitions were found between the velocities 0.75 and 1 m s-1 for dimensionless pelvic rotation and trunk rotation, indicating that, from this velocity range onwards, pelvic swing lengthens the stride: rotations of pelvis, thorax and trunk become tightly coordinated. On the basis of the dimensionless stride length, stride frequency, pelvic rotation and trunk rotation, deficits in the gait of stroke patients could be quantified. It is concluded that walking speed is an important control parameter, which should be used as a basic variable in the evaluation of the gait of stroke patients.


Influence of muscle geometry on shortening speed of fibre, aponeurosis and muscle.

The influence of muscle geometry on muscle shortening of the gastrocnemius medialis muscle (GM) of the rat was studied. Using cinematography, GM geometry was studied during isokinetic concentric activity at muscle lengths ranging from 85 to 105% of the optimum muscle length. The shortening speed of the distal fibre, the proximal aponeurosis and the muscle were determined, as well as the effect of rotation of the distal fibre and the proximal aponeurosis on the muscle speed of shortening. The results show that, due to the geometrical configuration, muscle shortening speed is not only determined by the speed of the fibre, but also to a large extent by the aponeurosis shortening speed. At optimum muscle length, the fibre and aponeurosis shortening speeds expressed relative to the muscle shortening speed amounted to 84% and 6%, respectively. At shorter muscle length, fibre speed relative to muscle speed decreased to values as low as 35%, whereas that of aponeurosis increased to values as high as 31%. Angular effects on the muscle speed of shortening can explain 10% of the muscle shortening speed at optimum muscle length and up to 34% of the muscle speed at shorter muscle length. In addition, a model was formulated to simulate the geometrical effects on muscle speed. This model, incorporating both fibre and aponeurosis length changes, contains a transfer function relating the shortening speeds of fibre and aponeurosis to muscle speed. The muscle shortening speed calculated using this transfer function demonstrated no significant differences with the speed measured experimentally.


A transversely isotropic biphasic finite element model of the meniscus.

A finite element model of the meniscus is presented, based on an axisymmetric geometric approximation of the menisci and a biphasic description of the tissue as a mixture of solid and fluid components. The highly fibrous nature of the meniscal tissue is accounted for by using a fiber-reinforced, transversely isotropic description of the solid phase. This model is used to study the response of a meniscus resting on a perfectly lubricated tibial surface and subjected to distributed loads applied to the femoral surface, and to examine the effects of changes in loading conditions at the femoral and tibial interfaces. Quantities of interest include the stress, pressure and strain distributions at discrete times early in the meniscal response, and the flow of the fluid phase relative to the solid phase. Of particular interest are regions of large tensile strains which could lead to meniscal failures such as the bucket-handle tear. We show that all components of strain are positive in regions of the outer third of the meniscus, and that the maximum tensile strain perpendicular to the circumferentially arranged fibers (largest principal strain in the axisymmetric cross section) is positive throughout most of the cross section. Changing the partition of the load on the femoral surface and the permeability at the tibial surface changes the time-dependent response, but has little effect on the strain distributions at times of the order of 5 s considered in this study. The inclusion of a transversely isotropic, fibrous representation of the solid phases is shown to be essential to proper meniscal simulation. The results demonstrate the importance of the biphasic representation since the fluid phase is shown to carry a significant part of the applied load.


Taper: an important feature of Y-bifurcations in porcine renal arteries and human cerebral arteries.

The geometry of arterial bifurcations has been shown to alter fluid flow and the propagation of both pressure and flow waves. Here we provide a more complete description of the renal artery bifurcation geometry and show that the geometry of the bifurcation is more complex than was believed previously. The objective of this study was to quantify changes in cross-sectional luminal area in systemic arterial bifurcations using the method developed by Macfarlane [Ph.D. thesis, University of Western Ontario, Ontario (1985)] to study the geometry of human cerebral bifurcations. Porcine renal arterial bifurcations from seven young (6-14 weeks) and six old (greater than 52 weeks) animals were pressure-fixed (P = 140 mmHg) for 3 h with 10% formalin. Bifurcations were embedded in a block of frozen latex paint. Serial sections were cut at 20 microns (+/- 0.01 microns) using a sledge microtome while the block face was scanned with a video camera and the images were stored on a videotape. The luminal area was measured digitally upon playback. Bifurcations from the two age groups changed in cross-sectional area not only at the flow divider but also along the so-called straight regions. Proximal linear increases in cross-sectional area were observed proximal to the apex of the bifurcation in both young and old vessels, while linear luminal area decreases were measured in the daughter branches of both young and old porcine renals. Taper was defined as the change in luminal area per unit length of parent and daughter branches, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Accuracy assessment and control point configuration when using the DLT for photogrammetry.

The direct linear transformation (DLT) is a common technique used to calibrate cameras and subsequently reconstruct points filmed with two or more cameras in a three-dimensional object space. The assessment of the accuracy of this technique, and of the influence of the distribution of control points on accuracy were examined. It was concluded that to obtain a true estimation of reconstruction accuracy, an independent assessment criterion is required, and that the use of control points distributed around the outside, rather than within the space to be calibrated, is preferred.


A simplified radiographic method for measuring bone-component motion in total knees.

Measurement of the relative motion between an implant and the surrounding bone over different time periods is valuable for assessing and comparing the component stability and predicting the potential future outcome. The RSA method, where small beads are implanted in the bone adjacent to the component, can measure implant-bone position to an accuracy of about 0.1 mm. However, the method involves special radiographic views and analytical software, not readily available. For purposes of component assessment on a more routine basis and for multi-centre trials, a method was developed where standard A-P and M-L radiographs were used. Computer software was written which estimated the out-of-plane rotations of the component relative to the plane of the film, and then carried out corrections to enhance the accuracy of calculation of the bead heights relative to the component. The theoretical errors were shown to be less than 0.04 mm for the expected range of out-of-plane rotations. When radiographs of components in simulated bones were taken at a range of rotations, the 95% confidence limits for axial displacement were found to be less than +0.3 mm, and for rotation in the plane to be 0.6 degrees. This indicated that the method was useful for studying knee components where the sinkage could reach a range of 0.5-2 mm in a 2 year period.


Velocity of ultrasound in active and passive cat medial gastrocnemius muscle.

The lengths and pinnation angles of muscle fibers in the medial gastrocnemius (MG) muscle have recently been measured in freely moving cats [Hoffer et al., Progr. Brain Res. 80, 75-85 (1989); Muscle Afferents and Spinal Control of Movement (1992)] using an ultrasound transit-time (USTT) technique. This method assumed that the velocity of ultrasound through intact muscles was constant, independent of fiber orientation, muscle activity, load, belly shape, or fiber movement. However, the velocity of ultrasound along and across the fibers has been reported to depend on the state of muscle activation in frog muscle experiments in vitro [Hatta et al., J. Physiol. 403, 193-209 (1988)]. In the present study, the assumption of constant velocity of ultrasound in the cat MG muscle was evaluated. In acute experiments, done in situ with intact blood supply, the USTT was measured along and across cat MG muscle fibers in the passive, reflexly activated and tetanically activated states, with and without changes in muscle fiber length, for situations that reproduced the length and force ranges normally used by cats during locomotion. The velocity of ultrasound was found to be independent of the state of activation or motion of the muscle, and independent of the direction of the measurement with respect to the fiber orientation, within a measurement uncertainty less than or equal to 0.2%. These results validate the use of the USTT technique for the measurement of intramuscular dimensions in freely moving animals.


A noninvasive technique for in vivo measurement of joint torques of biarticular muscles.

The objective of this work was to develop a noninvasive method to measure the joint torques produced by biarticular muscles at two joints simultaneously. During intramuscular stimulation of the cat medial gastrocnemius (MG) muscle, torques at the ankle and knee joints were calculated from forces measured in two dimensions at the end point of the cat paw under isometric conditions. The method was verified by the known anatomical properties of cat MG muscle and the tibialis anterior (TA) muscle. The MG muscle was shown to produce a significant flexion torque at the knee, besides an extension torque at the ankle. This was in agreement with its anatomical arrangement. The TA muscle produced primarily an ankle flexion torque. The small knee torque, due to measurement errors, yielded an estimate of measurement accuracy of 3.0 +/- 2.1% (n = 52). The coupling ratio of the MG muscle, defined as T(ankle)/T(knee), varied significantly with both knee and ankle angles. The profile of MG mechanical coupling agreed qualitatively with changes in limb configuration. The method can be used to measure recruitment properties of electrically stimulated biarticular muscles, and may potentially be used to study the biomechanics of biarticular coupling.


Characterizing bone strain distributions in vivo using three triple rosette strain gages.

Three triple-element rosette strain gages were attached to the equine third metacarpal midshaft to record site-specific strains engendered by locomotion. The distribution of strains acting upon the midshaft cross section were characterized using a combined beam theory and finite element model analysis that did not presume the manner by which the bone was inertially loaded. A medium-speed trot (3.6 ms-1) was chosen as a representative speed and gait, with normal and shear strains, and strain energy density (SED) distributions determined throughout the stance and subsequent swing phase. Importantly, the sites of maximum compression (-2400 mu epsilon), tension (810 mu epsilon), shear (1500 mu epsilon), and SED (54 kPa) were not located at any of the gage attachment sites, emphasizing that a minimum of three rosette gages are necessary to resolve the peaks and locations of functionally induced normal and shear strains. Considering the nonuniform strain distributions across the cortex, we conclude that the third metacarpal is subject to a complex loading milieu comprised of bending, axial compression, end shear, and torsion. As this complex manner of loading was consistent through the entire stance phase, it would appear that, at least during the trot, specific sites within the same cross section are subject to vastly different magnitudes of strain stimulus.


Different methods to estimate total power and its components during lifting.

A more fundamental understanding about the act of manual lifting can be provided by the assessment of the total production of power and the power generated in joints. The present study is concerned with the validity of the estimations of these parameters. Four subjects lifted an 18.8 kg load while they were filmed and ground reaction forces were measured. The total generated power was calculated in three ways: (1) by summation of joint powers, (2) on the basis of the rate of change of the summed energy contents of human body segments, and (3) on the basis of the rate of change of the body energy estimated from ground reaction forces. The results were compared. Furthermore, at a segmental level the power supplied to or absorbed from a segment was compared to the rate of change of its energy content. The resulting instantaneous power curves from the three different methods showed a high level of agreement, which supports their validity. However, some minor discrepancies were observed. The major cause of the observed difference between the rate of change of the summed segmental energy contents and the summed joint powers was found at a segmental level. It was observed that segmental link lengths (i.e. distances between proximal and distal markers) changed during movement, which yielded discrepancies between the power flow to or from a segment and the rate of change of its energy content.


Intermuscular coordination in a sprint push-off.

This study was designed to investigate the patterns of intermuscular coordination during a sprinting event. In previous research it was found that despite the indeterminacy problem of movement control, movements like vertical jumping, speed skating and cycling are performed in a stereotyped manner. It was hypothesized that this might be due to constraints associated with the transformation of joint rotations into the desired translation. The objective of the present study was to determine the extent to which the intermuscular coordination patterns during other movements also are performed in a stereotyped manner and, if that is true, whether this can be understood on the basis of such constraints. Seven elite sprint runners were instructed to execute an explosive sprinting dash. Ground reaction forces and cinematographic data were recorded for the second stance phase of the sprint. Simultaneously, electromyographic activity of nine leg muscles was recorded telemetrically. Linked-segment modeling was used to obtain net joint moments and net joint powers. Different athletes appeared to perform the sprint in a stereotyped manner. The muscle coordination pattern is characterized by a proximo to distal sequence in timing of the monoarticular muscles. When compared to the sequential pattern found in jumping, the biarticular hamstrings and rectus femoris muscles behave differently; in the sprint a more pronounced reciprocal activity between these muscles exists. The resulting movement pattern is characterized by a sequence of upper leg extension and plantar flexion. The observed sequence in timing of muscle activation patterns is aimed at solving the problems associated with the earlier identified geometrical and anatomical constraint. However, the coordination pattern cannot be fully understood on the basis of these constraints. A specific constraint is identified with respect to the direction of the ground reaction force, which explains the pronounced reciprocal activity of the biarticular hamstring and rectus femoris muscles. The intermuscular coordination pattern in the sprint can be seen as a compromise between the specific requirement of the sprint and the advantageous effect of a proximo to distal sequence as found previously for jumping.


Predicted and observed shapes of human mandibular condyles.

A mathematical model based on linear programming was used to study the directions of the joint forces used to maintain the human jaw in three-dimensional static equilibrium when producing bite forces of 100 N to a maximum of 1000 N down the long axis of a central incisor, first premolar, first molar and third molar. Seven different versions of the model were studied. The two simplest versions minimized the total muscle tension and the total joint load, respectively. Assuming that the joint force direction must be normal to some part of the articular surface of the condyle, neither version produced directions consistent with the observed shapes of human condyles. The other five versions minimized different combinations of muscle tensions and joint loads. Two of these versions produced joint force directions compatible with the shapes of condyles. Both minimized total muscle tension plus the (vertical) joint load on the back of the condyle. The results suggest that joint mechanoreceptors (probably non-directional) as well as muscle receptors contribute to the neuromuscular control of bite forces. Our results are consistent with some recent observations [Marshall and Tatton, Exp. Brain Res. 83, 137-150 (1990)] of the cat knee joint.


The role of the shaft in the golf swing.

Current marketing of golf clubs places great emphasis on the importance of the correct choice of shaft in relation to the golfer. The design of shafts is based on a body of received wisdom for which there appears to be little in the way of hard evidence, either of a theoretical or experimental nature. In this paper the behaviour of the shaft in the golf swing is investigated using a suitable dynamic computer simulation and by making direct strain gauge measurements on the shaft during actual golf swings. The conclusion is, contrary to popular belief, that shaft bending flexibility plays a minor dynamic role in the golf swing and that the conventional tests associated with shaft specification are peculiarly inappropriate to the swing dynamics; other tests are proposed. A concomitant conclusion is that it should be difficult for the golfer to actually identify shaft flexibility. It is found that if golfers are asked to hit golf balls with sets of clubs having different shafts but identical swingweights the success rate in identifying the shaft is surprisingly low.


Biomechanical consequences of callus development in Hoffmann, Wagner, Orthofix and Ilizarov external fixators.

A theoretical analysis by a finite elements model (FEM) of some external fixators (Hoffmann, Wagner, Orthofix and Ilizarov) was carried out. This study considered a logarithmic progress of callus elastic characteristics. A standard configuration of each fixator was defined where design and application characteristics were modified. A comparison among standard configurations and influence of every variation was made with regard to displacement and load transmission at the fracture site. An experimental evaluation of standard configurations was performed with a testing machine. After experimental validation of the theoretical model was achieved, an application of physiological loads which act on a fractured limb during normal gait was analysed. A minimal contribution from an external fixator to the total rigidity of the bone-callus-fixator system was assessed when a callus showing minimum elastic characteristics had just been established. Insufficient rigidity from the fixation devices to assure an adequate immobilization during the early stages of fracture healing was verified. However, regardless of the external fixator, callus development was the overriding element for the rigidity of the fixator-bone system.


Complete disappearance of metastatic abdominal tumors from gastric cancer after treatment with irsogladine maleate.

A 60-year-old woman with gastric cancer had undergone partial gastrectomy in September 1989. Pathological examination revealed a poorly differentiated adenocarcinoma of pT3pN3pM0 (not resected for cure), stage IV. Postoperative adjuvant therapy comprised 1-(tetrahydro-2-furanyl)-5-fluorouracil plus uracil and OK-432. On 11 August 1990, two forefinger-tip-sized tumors were palpated beneath the operation scar. They increase in size, the superior tumor reaching 4x3 cm, the inferior tumor 5x3 cm on 5 September. Then, on 17 September, the inferior tumor was resected but the superior tumor remained; the histological type was poorly differentiated adenocarcinoma. After the operation, from 20 September, she was given 4 mg irsogladine maleate orally every day. On 8 October, there was no increase in the size of the superior tumor. By 29 October, the superior tumor had disappeared and no further tumor appeared thereafter; the patient showed no sign of relapse.


Effect of chemotherapy combined with caffeine for osteosarcoma.

Nine patients with osteosarcoma were treated by chemotherapy combined with caffeine and surgery. All primary tumors showed complete histological response to preoperative chemotherapy consisting of three intraarterial infusions of cisplatin and caffeine without/with doxorubicin and two systemic high-dose methotrexate combined with vincristine. Limb-salvage surgery was performed in eight patients with marginal procedure, which led to the preservation of good limb function. Below-knee amputation was done in one patient with calcaneal osteosarcoma. There has been neither local recurrence nor lung metastasis in seven patients with conventional osteosarcoma during a median follow-up period of 28 months. Lung metastases leading to death were observed in one patient with small-cell osteosarcoma despite complete destruction of the primary tumor by preoperative chemotherapy. Chemotherapy combined with caffeine administration deserves further extensive and large-scale study in osteosarcoma.


Is bile or are pancreaticoduodenal secretions related to gastric carcinogenesis in rats with reflux through the pylorus?

Male Wistar rats were subjected to one of three types of operative reflux procedure that allowed part or all of the duodenal contents to flow back into the stomach through the pylorus, thus producing models of bile reflux alone, pancreaticoduodenal reflux alone, and combined reflux. All surviving animals were killed 50 weeks after surgery and the development of gastric cancer was assessed. No cancer was seen in 16 animals with pancreaticoduodenal reflux or in 32 control animals with gastrotomy, whereas 2/8 animals with bile reflux and 11/29 animals with combined reflux had gastric carcinoma. Compared with the control group, the incidence of carcinoma in animals with bile or combined reflux was significantly higher (P less than 0.05 and P less than 0.01 respectively). All carcinomas developed in the antral area near the pylorus. Adenomas were observed only in the groups of animals developing carcinoma and occurred in the same region of the stomach. These results suggest that bile, and not pancreaticoduodenal secretions, is the component of the duodenal contents responsible for the development of gastric carcinoma.


Molecular-genetic analysis of ocular adnexal benign lymphoid hyperplasias by a two-step polymerase-chain-reaction.

Twelve biopsied ocular adnexal benign lymphoid hyperplasias (OABLH) satisfying benign histological criteria were investigated for clonal immunoglobulin (Ig) heavy-chain gene rearrangement by means of a two-step polymerase chain reaction (PCR) method using formalin-fixed and paraffin-embedded tissue. Of the 12, 4 (33%) demonstrated clear single bands of the rearranged gene for the Ig heavy-chain, of between 100 and 150 base pairs. The selected cases were all free of malignant lymphoma and all of the lesions were small (2 x 2-22 x 6 mm; median 4.5 x 3 mm). Histopathological and cytological features were not essentially different between monoclonal and non-monoclonal examples. Immunohistochemistry was of little benefit in separating the two. It is concluded that OABLH demonstrating a benign clinical course frequently contain monoclonal B cell populations suggesting a continuous progressive spectrum of lesions in B cell neoplasia. In addition, the significance of molecular-genetic analysis for OABLH and the utility of the two-step PCR method should be emphasized.


Induction/repair of strand breakage in mature and nascent DNA of cultured Chinese hamster ovary cells exposed to hydrogen peroxide.

Treatment of cultured mammalian cells with hydrogen peroxide results in the production of extensive DNA damage. Strand breakage was produced at the level of either nascent or mature DNA and the former target appeared slightly more resistant than the latter. Although inhibitors of the enzyme poly(ADP-ribose) polymerase similarly retarded the repair of such lesions, removal of DNA strand breaks was much slower for the newly synthesized DNA as compared to mature DNA.


Hyaluronidase enhances the activity of adriamycin in breast cancer models in vitro and in vivo.

The effect of hyaluronidase and a combination of hyaluronidase with Adriamycin was investigated on several breast cancer models in vitro and in vivo. In vitro enzyme treatment (using concentrations up to 80,000 IU/1) of murine (MXT-, MXT +/-, and MXT+) and human (MCF-7, ZR-75-1 and T-47-D) breast cancer cell lines did not inhibit tumour cell proliferation (measured by a kinetic crystal violet assay) in either case. Although high-dose hyaluronidase (1.2 x 10(6) IU/kg) was ineffective, when administered peritumourally to the MXT M3.2 mammary carcinoma of the B6D2F1 mouse, it is remarkable that five "megadoses" were excellently tolerated. However, the antineoplastic activity of Adriamycin against the oestrogen-receptor-positive variant of the MXT tumour was significantly enhanced by combination with concentrations of hyaluronidase that were inactive per se, both in vitro and in vivo. Interestingly, the enhancement of the in vivo antitumour activity was not compromised by toxic side-effects.


Prognostic factors in WHO grade 2 transitional-cell bladder cancer (TCC); a novel two-grade classification system for TCC based on mitotic index.

A retrospective histological analysis has been carried out on 537 cases of transitional-cell bladder carcinoma, followed-up over a period of 9 years. In the first part of the study WHO grade 2 tumours were analysed and a number of independent factors predictive for survival identified. In a multivariate analysis the T category and M/V index (number of mitotic figures/mm2 neoplastic epithelium) were the most important prognostic factors. In a subsequent analysis of the whole series of 537 cases, overall the M/V index was not as important in predicting survival as the stage of the tumour. However, in superficial tumours (Ta-T1) subsequent analysis showed that the M/V index alone could be used to predict survival.


Tobacco-related cancer in relation to prevalence of drinking and smoking in eastern Austria.

Eastern Austrian regional mortality patterns of oral cancer (oral cavity, pharynx and larynx) and oesophageal, lung and urinary bladder cancer were compared to smoker rates and to liver cirrhosis mortality by type of residence: Vienna (1.7 x 10(6) inhabitants), middle towns (50,000-100,000 and 10,000-50,000 inhabitants), small towns (2000-10,000 inhabitants) and rural areas categorized by agrarian quota less than or equal to 10%, 10%-20% and greater than 20%. The study area (Vienna, Lower Austria and Burgenland) covers 23,600 km2 with 3.23 x 10(6) inhabitants. In men, liver cirrhosis correlated negatively with smoker rates (r = 0.74, P = 0.1). Deaths from oral cancer and oesophageal cancer correlated significantly with deaths from liver cirrhosis (r = 0.81, P = 0.03; r = 0.78, P = 0.04, respectively) but not with smoker rates; lung cancer and bladder cancer correlated significantly with smoker rates (r = 0.91, P = 0.01; r0.83, P = 0.04, respectively), but not with liver cirrhosis. In women, similar urban-rural gradients for all parameters resulted in a positive correlation between liver cirrhosis and smoker rates (r = 0.59, P = 0.22) and a significant correlation of lung cancer with liver cirrhosis (r = 0.75, P = 0.05). Oral cancer correlated significantly with liver cirrhosis (r = 0.83, P = 0.02), but not with smoker rates; lung cancer correlated more significantly with smoker rates (r = 0.92, P = 0.01) than with liver cirrhosis; bladder cancer correlated positively with smoker rates (r = 0.70, P = 0.12). Geographical distribution of oral and oesophageal cancer in Eastern Austria seems thus to be highly subject to the prevalence of heavy drinking. Sociocultural influences upon the occurrence of these cancers seem to be mediated through drinking habits rather than through smoking habits alone.


Evaluation of tissue polypeptide antigen serum levels for monitoring disease activity during chemotherapy in patients with transitional carcinoma of the urinary tract.

In 28 patients with transitional carcinoma of the urinary tract, all treated with chemotherapy, serial measurements of serum tissue polypeptide antigen (TPA) were performed and correlated to clinical evaluations of response. At the start of chemotherapy elevated levels of TPA were found in 4 out of 14 patients with T2-4NO-2MO tumours and in 7 out of 14 patients with distant metastases. In most patients with elevated TPA levels who responded to chemotherapy, TPA levels rapidly returned to normal. False positive elevations of TPA were observed in 2 patients. It is concluded that serial measurement of TPA for monitoring disease activity has limited value because of the low sensitivity of TPA, especially for patients with early-stage cancer, and because of the occurrence of false positive results.


Determination of phospholipid fatty acid and triacylglycerol composition of rat caecal mucosa.

The lipid composition of rat caecal mucosa, including the fatty acid composition of phospholipids and triacylglycerols, has been examined by capillary gas chromatography. Thirty-seven peaks were resolved, ranging in chain length from 12 to 24 carbon atoms. Preliminary identification of fatty acids by comparison with authentic standards was confirmed by gas chromatography-mass spectrometry using electron-impact ionization. The neutral and polar components were examined. Fatty acid methyl esters were quantified in absolute amounts with respect to the percentage of total phospholipid and triacylglycerols. The results show significantly higher levels of 16:0, 18:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n-6) in phospholipids, and higher levels of 16:0, 18:1(n-9) and 18:2(n-6) in triacylglycerols. On the other hand, analysis of caecal triacylglycerols revealed sn-glycerol-palmitate-oleate-palmitate, sn-glycerol-palmitate-linoleate-palmitate and sn-glycerol-palmitate-linoleate-oleate as major components.


Determination of pentoxifylline and its metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction.

A simple and reliable method for the determination of pentoxifylline and its main metabolites in human plasma has been developed using high-performance liquid chromatography. After selective solid-phase extraction, pentoxifylline, its metabolites and an internal standard, 7-(2'-chloroethyl)theophylline, were separated on a 5-micron LiChrospher 100 RP-18 column using water-dioxan-acetonitrile (87:6.5:6.5, v/v/v) acidified with acetic acid (0.5%, v/v) as the mobile phase. The analytes were detected at 275 nm. The lowest detectable concentration for all analytes was 25 ng/ml; the recovery was 85%. The assay has been successfully applied to analysis of these compounds in human plasma after administration of an oral dose of 400 mg of pentoxifylline to healthy volunteers.


Two-dimensional high-performance liquid chromatographic method to assay p-hydroxyphenylphenylhydantoin enantiomers in biological fluids and stereoselectivity of enzyme induction in phenytoin metabolism.

A two-dimensional high-performance liquid chromatographic method was developed to assay the enantiomers of a major phenytoin metabolite, p-hydroxyphenylphenylhydantoin (p-HPPH). Racemic p-HPPH was first separated from phenytoin and other interfering peaks by a reversed-phase column and monitored by an ultraviolet detector. At the retention time of p-HPPH, the racemic p-HPPH peak was automatically transferred to a chiral ligand-exchange column to separate R-p-HPPH and S-p-HPPH by a time-programmed column-switching valve. The ratio of enantiomers was measured by a second ultraviolet detector. The method can be used to assay R- and S-p-HPPH enantiomers with reasonable sensitivity and reproducibility. By using this method, the stereoselectivity of enzyme induction and inhibition of phenytoin metabolism was investigated. Male rats were treated with phenobarbital, 3-methylcholanthrene, acetone, Aroclor 1254, pregnenolone-16 alpha-carbonitrile, dexamethasone and isosafrole. Microsomes were prepared from the rat liver and phenytoin hydroxylation was measured. Pretreatment with phenobarbital, pregnenolone-16 alpha-carbonitrile or acetone induced phenytoin metabolism non-stereoselectively. Pretreatment with dexamethasone decreased R-p-HPPH formation without affecting the formation of S-p-HPPH. Liver microsomes from female rats showed a higher S-p-HPPH formation, whereas R-p-HPPH formation remained the same. Various inhibitors were added to inhibit phenytoin metabolism by control microsomes. Sulphaphenazole, ketoconazole, 4,4-di(p-methoxyphenyl)hydantoin, cimetidine and diazepam inhibited the formation of R- and S-p-HPPH. Quinidine, tolbutamide and mephenytoin showed no significant inhibitory activity. None of these inhibitors showed stereoselectivity.


Determination of manidipine enantiomers in human serum using chiral chromatography and column-switching liquid chromatography.

A stereoselective and highly sensitive method using chiral chromatography and successive column-switching liquid chromatography is described for the determination of manidipine enantiomers in human serum. A human serum sample obtained after ingestion of manidipine was extracted twice with a mixture of n-hexane-diethyl ether under alkaline conditions. The enantiomers in the extract were separated on a chiral stationary phase column (Chiralcel OJ), and the effluents containing the respective enantiomers were collected. Each fraction was then analysed by column-switching liquid chromatography. The proposed stereoselective method offered high sensitivity: detection limits for both isomers were 0.2 ng/ml in human serum, both at a signal-to-noise ratio of 3. The method is suitable for the pharmacokinetic studies of manidipine enantiomers.


Determination of baicalin and baicalein in rat plasma by high-performance liquid chromatography with electrochemical detection.

A rapid and sensitive method, using electrochemical detection, has been developed for the determination of baicalin and baicalein, the flavonoid of Scutellariae radix, in rat plasma. Following separation by high-performance liquid chromatography, baicalin and baicalein were oxidized at a glassy carbon electrode to permit selective electrochemical detection. Absolute detection limits were found to be 5 ng/ml from 50 microliters of plasma for baicalin and 2 ng/ml from 100 microliters of plasma for baicalein. The resulting assays were suitable for pharmacokinetic studies of baicalin and baicalein in rats.


Combined thin-layer chromatography-photography-densitometry for the quantification of ifosfamide and its principal metabolites in urine, cerebrospinal fluid and plasma.

A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.


In vivo transformation of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by human nasal mucosa.

A method for the determination of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) by gas chromatography-mass spectrometry (GC-MS) in samples obtained from healthy subjects by nasal lavage is presented. HETEs were extracted from samples obtained by nasal lavage using C18 solid-phase cartridges. The purification of 12-HETE and 5-HETE was carried out in two consecutive steps of purification by reversed-phase high-performance liquid chromatography. Methylated and trimethylsilylated fractions were separated by GC-MS with electron-impact ionization. The production of 12-HETE by human nasal mucosa was confirmed by GC-MS.


Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography.

A method for the enantiomeric analysis of amino acids of mammalian tissues is described. An excellent resolution of D- and L-enantiomers of common protein amino acids was achieved by employing a combination of thin-layer chromatography and high-performance liquid chromatography. D-Enantiomers and L-enantiomers of glutamate, aspartate, glutamine, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine and histidine, as well as glycine were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. The amino acid diastereomers were separated by two-dimensional thin-layer chromatography. Each amino acid diastereomer was then analysed by reversed-phase high-performance liquid chromatography for the resolution of D- and L-enantiomers. Very sharp peaks were obtained using a conventional octadecylsilyl-bonded column, and the possibility of analysing these amino acids (except tyrosine and histidine) in subnanomole amounts was indicated. The method was used to demonstrate the presence of D-enantiomers of alanine, proline and serine in mouse kidney.


Simultaneous determination of allantoin and oxypurines in biological fluids by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the separation and quantification of allantoin and oxypurines in plasma and urine samples. Urine was analyzed directly and plasma after acid deproteinisation with perchloric acid. Separation and quantification of purine derivatives was achieved using two Spherisorb ODS-5 column (250 mm x 4.6 mm I.D.) connected in series together with a NH4H2PO4-NH4H2PO4-acetonitrile (80:20) gradient and monitoring the effluent at 205 nm. The average recoveries of standard compounds added to urine and plasma samples were 96 and 97%, respectively, using allopurinol as internal standard. The within-day variability was less than 7% and the day-to-day coefficient of variation less than 11% indicating a good precision of the method.


Analysis of meconium for cocaine in neonates.

A solid-phase extraction method was developed for the extraction of first-day meconium samples from premature infants of cocaine-dependent mothers. Extracts were analysed by high-performance liquid chromatography and gas chromatography-mass spectrometry for cocaine and its metabolites. Control stools showed no drug. Meconium from cocaine-dependent mothers showed cocaine in the range 0.1-0.78 micrograms/g. Benzoylecgonine, ecgonine and ecognine methyl ester were not present in the samples, which suggests that the metabolism of cocaine in the premature neonate is limited.


Simple, rapid and selective method using high-performance liquid chromatography for the determination of bretylium in plasma.

A high-performance liquid chromatographic method with ultraviolet detection has been developed for the determination of bretylium in plasma. Following a single-step solid-phase extraction procedure, bretylium is selectively isolated and well recovered from plasma. The assay sensitivity is 0.156 micrograms/ml from 250-microliters plasma samples and its linearity was assessed up to 40 micrograms/ml. The method is accurate (101.0 +/- 5.4%) and precise (maximum coefficient of variation of 8%). It provides a simple and time-saving alternative to existing methods and is particularly suitable for pharmacokinetic studies.


Sensitive determination of josamycin and rokitamycin in plasma by high-performance liquid chromatography with fluorescence detection.

Rokitamycin and josamycin were successfully derivatized with dansylhydrazine in 20 min at 60 degrees C. Rokitamycin and josamycin levels were determined in plasma after ion-pair extraction into hexane-isoamyl alcohol with lauryl sulphate and precolumn derivatization. Resolution was obtained by liquid chromatography with fluorescence detection (352/537 nm) in 12 min. The limit of detection was 20 ng/ml macrolide starting from 1 ml of plasma, and linearity was demonstrated between 50 and 400 ng/ml. Inter-run coefficients of variation were 10.2% at 100 ng/ml and 9.1% at 300 ng/ml. The system was reliably used for pharmacokinetic studies in plasma.


Development and use of a stability-indicating high-performance liquid chromatographic assay for Meldrum's acid.

A high-performance liquid chromatographic method was developed that separates Meldrum's acid from its primary decomposition products, malonic acid and acetone. The method uses a reversed-phase column under isocratic conditions, with detection by ultraviolet absorption at 210 nm. Quantitation of the parent molecule and the acid decomposition product was possible over concentration ranges of 0.1-10.0 and 0.1-2.0 mg/ml, respectively. Acetone could be determined only at much higher concentrations. Using the malonic acid concentration as a measure of decomposition, this method was used to determine the hydrolytic stability of Meldrum's acid and its skin penetration properties.


Rapid determination of creatine, phosphocreatine, purine bases and nucleotides (ATP, ADP, AMP, GTP, GDP) in heart biopsies by gradient ion-pair reversed-phase liquid chromatography.

A simple binary solvent method has been developed for the simultaneous determination of creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, GTP, GDP, IMP, NAD, inosine, adenosine, hypoxanthine and xanthine. This allows separation of the most important nucleotides present in myocardial biopsies as, for example, in studies using 31P NMR spectroscopy. In NMR spectra ATP and PCr are the only visible high-energy phosphates, therefore the status of other nucleotides and bases cannot be determined. The nucleotides, AMP degradation products, PCr and Cr in pig and rat heart muscle were resolved with 35 mM K2HPO4, 6 mM tetrabutylammonium hydrogensulfate buffer, pH 6.0, and a binary acetonitrile gradient on medium-bore, 250 mm or 125 mm x 3.9-4.6 mm I.D. steel octadecyl-bonded (C18) columns at a flow-rate of 1.5 or 1.0 ml/min. This method, optimized for use with older high-performance liquid chromatography pumps (100 microliters displacement heads), resolves the major porcine and rat myocardial nucleotides and degradation products within 22 min. The amounts found in normoxic porcine muscle are: Cr 9.21 +/- 0.75; hypoxanthine 1.40 +/- 0.14; PCr 7.20 +/- 1.2; IMP 1.34 +/- 0.13; beta NAD 1.82 +/- 0.23; AMP 0.10 +/- 0.04; GDP 0.05 +/- 0.02; ADP 1.23 +/- 0.09; GTP 0.19 +/- 0.01; ATP 4.45 +/- 0.32 mumol/g wet weight. The method, incorporating adenosine tetraphosphate as an internal standard, allows the documentation of changes in both the high-energy phosphates and their degradation products in a single analysis of myocardial samples as small as 200 micrograms (wet weight).


Glassy carbon pre-column for direct determination of acetylcholine and choline in biological samples using liquid chromatography with electrochemical detection.

The determination of acetylcholine and choline has been quite successfully accomplished using liquid chromatography with electrochemical detection following the original reports of Potter et al. [J. Neurochem., 41 (1984) 188]. A post-column reactor containing acetylcholinesterase and choline oxidase allows conversion of the desired species into hydrogen peroxide, an electrochemically active substance. However, the direct injection of tissue homogenates and other biological samples into such a system exhibits quite large solvent fronts and unidentified peaks. Using a pre-column packed with glassy carbon particles, we were able to dramatically decrease the size of the solvent front for such injections and tentatively identify the unknown peaks to be caused, at least in part, by common catecholamines. The glassy carbon pre-column, in addition to increasing the selectivity of the results, allowed the required chromatographic time per sample to be decreased from 20 to 10 min.


Determination of platelet monoamine oxidase activity by high-performance liquid chromatography with electrochemical detection.

A procedure for determining human platelet monoamine oxidase (MAO) with dopamine (DA) as substrate is described. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used to separate and detect components of the reaction mixture. The method for platelet preparation was also improved and only 2 ml of blood were required. Following a 10-min incubation of the platelet preparation with DA in 0.1 M Tris buffer (pH 9.0), excess DA substrate was removed by adsorption on a cation-exchange resin. The reaction product, 3,4-dihydroxyphenylacetaldehyde, was adsorbed on acid-washed alumina, eluted with 0.1 M perchloric acid and analyzed by HPLC. Simple, clean chromatograms were obtained with good reproducibility using 3,4-dihydroxybenzylamine as an internal standard. The within-sample, between-samples and between-day relative standard deviations were 0.9, 3.7 and 6.1%, respectively. The apparent Michaelis constant and maximum velocity were 0.10 mM and 0.37 nmol/min.mg protein, respectively. This HPLC-ED method offers a good alternative to methods using radioactivity.


Lipophilicity determination of some monoamine oxidase inhibitors: the effect of methanol and ammonium chloride.

The lipophilicity (RM value) of seventeen monoamine oxidase inhibitory drugs was determined by reversed-phase thin-layer chromatography, and the effect of salt concentration on the reversed-phase retention was studied by adding ammonium chloride to the eluent. Each drug exhibited regular retention behaviour, its RM value linearly decreasing with increasing concentration of methanol in the eluent. Ammonium chloride decreased the retention: the effect was higher at lower salt concentrations, which indicates that the phenomenon is of saturation character. The influence of ammonium chloride depended on the concentration of methanol (on the dielectric constant of the eluent) suggesting that methanol suppresses the dissociation of ammonium chloride resulting in a modified salting-in effect.


Analysis of 3,5,6-trichloropyridinol in human urine using negative-ion chemical ionization gas chromatography-mass spectrometry.

A sensitive gas chromatographic-negative-ion chemical ionization mass spectrometric (GC-NCI-MS) method was developed to measure levels of the chlorpyrifos metabolite 3,5,6-trichloropyridinol (3,5,6-TCP) in human urine. The metabolic 3,5,6-TCP was isolated from urine by acid hydrolysis of urine aliquots, followed by diethyl ether extraction. The residues of ether extraction were taken up in o-xylene and derivatized with N-(tert.-butyldimethylsilyl)-N-methyltrifluoroacetamide (overall five-fold concentration). The structural isomer 3,4,5-trichloropyridinol was used as an internal standard in this analysis. This method was found to be linear for the determination of 3,5,6-TCP over the range 0.8-792 ng/ml. The limit of detection for 3,5,6-TCP in human urine was estimated to be 0.5 ng/ml. Low levels of 3,5,6-TCP (1-18 ng/ml of urine) were identified in control human urine samples. Structural confirmation of the background 3,5,6-TCP was obtained via GC-NCIMS-MS analysis.


High-performance liquid chromatography-thermospray mass spectrometry of hydroxy-polyunsaturated fatty acid acetyl derivatives.

A method for the determination of hydroxy-polyenoic acids has been developed. Hydroxy groups of hydroxy-polyunsaturated fatty acid were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, fifteen kinds of hydroxy-polyunsaturated fatty acid derivative could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatograph without a gradient system. Generally, the base ion, [M + H - n(60)]+, is produced through elimination of acetic acid (n = number of the hydroxy group of hydroxy-polyunsaturated fatty acid). The detection limit for these derivatives was ca. 0.2 pmol at the levels of hydroxy-polyenoic acids prior to derivatization. They could be analysed in the range 0.5-25 pmol. The assay was successfully applied to hydroxy-polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acid was added.


Rapid and efficient purification of cimetropium bromide and mifentidine drug metabolite mixtures derived from microsomal incubates for analysis by mass spectrometry.

A comparative study of the use of organic solvent extraction versus Sep-Pak C18 cartridges in the recovery and analysis of phase I (unconjugated) drug metabolites using mass spectrometry is presented. Standard mixtures of putative metabolites of the anticholinergic drug cimetropium bromide and the H2-antagonist mifentidine were purified from inactivated liver microsomal preparations using both methods, and subsequently the recovery of each compound was quantitated. In general, the percentage recovery and degree of purification were greater when using Sep-Pak C18 cartridges compared with organic solvent extraction. Even more efficient recovery was achieved when zinc sulphate precipitation of proteins in the liver microsomal mixtures was carried out prior to analysis. Also, the HPLC-grade solvents used in this study contained a variety of ultraviolet-inactive, hydrophobic components. This leads to problems of suppression in fast atom bombardment mass spectrometric analysis. Using Sep-Pak C18 cartridges directly prior to analysis by fast atom bombardment with single or tandem mass spectrometry leads to far superior mass spectral results compared with organic solvent extraction.


Monitoring of ajmaline in plasma with high-performance liquid chromatography.

A rapid, reliable and sensitive assay for routine determination of ajmaline in plasma by high-performance liquid chromatography with fluorimetric detection is presented. A low limit of detection in plasma (less than 1 ng/ml ajmaline) could be achieved by the extraction of plasma samples and the use of fluorimetric detection. Deproteinization of the plasma sample instead of extraction, or the use of an ultraviolet detector, yielded a higher limit of detection (less than 50 ng/ml). Two different eluents were studied. Eluent 1 allowed clear separation of ajmaline from isoajmaline and sandwicine, but did not separate isoajamaline from sandwicine. With eluent 2, separation of isoajmaline and sandwicine was achieved, but separation of ajmaline from sandwicine was less optimal than with eluent 1. Therefore, eluent 1 was used for further clinical studies. No interference was observed from therapeutic doses of other commonly co-administered drugs, such as acetylsalicylic acid, digoxin, digitoxin, ranitidine, dopamine, dobutamine, furosemide, captopril or glycerol trinitrate. In addition, the chemical stability of ajmaline and a possible rearrangement of ajmaline to its stereoisomers isoajmaline and sandwicine was studied in vivo and in vitro. Ajmaline proved to be unusually stable under both in vivo and in vitro conditions.


Automated high-performance liquid chromatography of iopentol in human plasma and whole blood using on-line dialysis as sample preparation.

A fully automated high-performance liquid chromatographic method has been developed for the analysis of the radiographic contrast agent iopentol in human plasma and whole blood. This method is based on on-line sample preparation with dialysis followed by pre-column enrichment of the dialysate. The method was compared with a manual sample preparation method based on protein precipitation with tetrahydrofuran. The inter-assay and intra-assay variations and the limits of quantitation were the same for both methods. The on-line combination of dialysis and column-switching high-performance liquid chromatography was shown to be a reliable and time-saving technique for monitoring iopentol in human whole blood and plasma.


Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.


Immobilized lectin columns: useful tools for the fractionation and structural analysis of oligosaccharides.

Elucidation of the binding specificity of a concanavalin A-Sepharose column led to the possibility of the affinity chromatography of oligosaccharides and glycopeptides with the use of immobilized lectin columns. Subsequent addition of immobilized erythroagglutinating phytohaemagglutinin, Aleuria aurantia lectin, Datura stramonium agglutinin, Ricinus communis agglutinin and Allomyrina dichotoma agglutinin to the range of well characterized lectin columns has afforded a way to fractionate a mixture of N-linked oligosaccharides even to a single component.


Immobilization of protein ligands with methyl vinyl ether-maleic anhydride copolymer.

Methyl vinyl ether-maleic anhydride copolymer (MMAC) is a water-insoluble polymer with an acid anhydride group which reacts with amino groups of ligands to form stable amide bonds. MMAC was used to immobilize protein ligands on two kinds of supports, the wells of plastic microtitre plates for enzyme-linked immunosorbent assay and related methods, and gels for affinity adsorbents. The wells were first coated with MMAC and then allowed to react with proteins. The immobilization of proteins by this method was efficient and occurred in a dose-dependent manner. Shodex Et123, a gel having amino groups, was incubated with MMAC, and then the activated Shodex was used to immobilize high concentrations of proteins. Concanavalin A-Shodex thus obtained had high affinities and was successfully used for the high-performance liquid affinity chromatography of sugar derivatives on a short column.


Immobilized ferredoxins for affinity chromatography of ferredoxin-dependent enzymes.

An immobilized ferredoxin more stable than the conventional immobilized spinach ferrodoxin was prepared by reacting CNBr-Sepharose with ferredoxins isolated from barley and Synechococcus vulcanus, a thermophilic blue-green alga. The dissociation constants of immobilized ferredoxin from spinach, barley and S. vulcanus for spinach ferredoxin-NADP reductase were 0.922, 2.505 and 5.209 microM, respectively, whereas those for barley ferredoxin-NADP reductase were 1.159, 0.579 and 2.851 microM, respectively. The order of stability was S. vulcanus greater than barley greater than spinach. The immobilized ferredoxin was applied to the simultaneous detection of ferredoxin-dependent enzymes in spinach chloroplasts. Over 20 polypeptides were detected. Synechococcus ferredoxin could also be immobilized on a Toyopearl gel and repeatedly used in an automated high-performance liquid chromatographic system.


Membrane-based receptor affinity chromatography.

Membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, has been developed for the purification of human interleukin-2 and related biomolecules. The multi-purpose affinity membrane used in this study consisted of a soluble form of interleukin-2 receptor (IL-2R) chemically bonded to hollow-fiber membranes in an oriented fashion. A model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) was used to study the important factors influencing the performance of MRAC, including support morphology, mass transfer rate and adsorption kinetics. All three are shown to be highly efficient. MRAC has been successfully applied to the purification of anti-Tac-H, recombinant human interleukin-2 (rIL-2) and interleukin 2-Pseudomonas exotoxin fusion protein (IL2-PE40). Overall, MRAC was found to be a viable, scalable and extremely productive affinity purification method.


Kinetic aspects of membrane-based immunoaffinity chromatography.

With a view towards the efficient large-scale purification of recombinant proteins, factors influencing antigen-antibody adsorption kinetics were studied in a model hollow-fiber membrane-based immunosorbent. Non-diffusion-controlled, homogeneous adsorption kinetics are approached in membranes. It is shown that adsorption kinetics, rather than mass transfer, first becomes limiting in membrane-based immunoaffinity chromatography (MIC). Antigen adsorption is not kinetically limited, even at low feed-stream antigen concentrations. Binding efficients approach theoretical values when antibody coupling densities are decreased sufficiently. Antigen breakthrough during adsorption occurs near the membrane's observed binding capacity. The results of these kinetic studies were essential in the development of highly efficient and productive MIC systems for the purification of three recombinantly produced biotherapeutics, interferon-alpha 2a, interleukin-2 and interleukin-2 receptor.


Short-cut method for predicting the productivity of affinity chromatography.

A method was developed for predicting the productivity P (amount of the target protein recovered per unit volume per unit time) of affinity chromatography (AFC) and similar chromatographic operations. The constant-pattern approach was employed for describing the breakthrough curve. It was found that P increases with increasing u at low u regions and there is a maximum in the P-u curve. The u value that gives the maximum P (uM) was derived as a function of the number of transfer units, n. The experimental breakthrough curves for several different packing media systems such as 40- and 10-microns porous particles, 2.5-microns non-porous particles and surface-derivatized membrane media were measured, from which the n values were determined. The uM values were then calculated using the n values. It is suggested that the present method is useful for the rapid survey of the operating conditions in scaling-up AFC and similar chromatographic operations.


Arginine-tail method, an affinity tag procedure utilizing anhydrotrypsin agarose.

The arginine-tail method is a recently developed affinity tag procedure utilizing immobilized anhydrotrypsin for specific enrichment of a recombinant protein. Three model proteins (originally human beta-galactoside-binding lectin with a relative sub-unit molecular mass of 14,000) were prepared by mutagenesis, each of which has a tail of either Arg, Gly-Arg, or Gly-Gly-Arg at the C-terminus. All of them retained their original sugar-binding activity and antigenicity, and became recognizable by anhydrotrypsin. They were adsorbed on an anhydrotrypsin-agarose column at pH 5 after treatment with 4 M urea or 10 mM HCl to expose the C-terminal tails. The adsorbed arginine-tailed lectins were eluted either specifically with benzoylglycylarginine (Bz-Gly-Arg) or with 5 mM HCl. Added arginine was removed by carboxypeptidase B, but very slowly. When urea-denatured Escherichia coli lysate containing one of the arginine-tailed lectins was applied to the column, the lectin was adsorbed together with some host proteins, which are expected to have arginine or lysine at their C-termini. However, more than a ten-fold enrichment was attained by this procedure. The described method would be useful for purifying various recombinant proteins, even those which are inactive as a result of mutagenesis. The smallest tag arginine at the C-terminus should have a minimum effect on both the structure and function of a target protein.


Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.


Galactosylation as a tool for the stabilization and immobilization of proteins.

This paper presents a brief overview of the role that the carbohydrate moieties of biologically active glycoproteins play in the stabilization and oriented immobilization of these proteins on solid supports. The synthetic galactosylation of hydrophobic areas or their surroundings on the protein surface improves the structural stability of native proteins against inactivation by the interaction of water with hydrophobic clusters. The lowering of the degree solvation of tyrosine residues in galactosylated trypsin and the model substance N-carbobenzoxy-L-glutamyl-L-tyrosine was proved by Raman spectroscopy. D-Galactose residues can be selectively oxidized, either with periodate or enzymatically, and the aldehyde groups thus formed are used for the immobilization of glycoproteins on solid supports with hydrazide groups under mild conditions.


Improved affinity chromatographic purification of D-mannose-N-acetyl-D-glucosamine-specific lectin from the bark of Sophora japonica eliminating the loss by sugar specific self-aggregation.

A novel D-mannose-N-acetyl-D-glucosamine-specific lectin of Sophora japonica bark, B-SJA-II, which showed self-aggregation based on sugar specificity, was purified by affinity chromatography on maltamyl-Sepharose subsequent to chromatographic separation on lactamyl-Sepharose to remove a major D-galactose-N-acetyl-D-galactosamine specific lectin, B-SJA-I. However, the yield of this method was low as a result of the sugar-specific precipitation and binding to other glycoproteins. A modified method was developed to circumvent this problem. All the purification procedures, except for the final chromatographic separation, were carried out in the presence of the haptenic sugar and the sugar-specific adsorption of B-SJA-II onto the adsorbent was carried out in a dialysis bag by gradually removing the sugar. This method gave a yield eight times higher than the original method.


Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.


Isolation and characterization of isolectins from Erythrina variegata seeds.

Three isolectins were isolated from seeds of Erythrina variegata (Linn.) var. Orientalis by ion-exchange chromatography, followed by affinity chromatography on lactose-Sepharose 4B and acid-treated Sepharose 4B columns. The purified isolectins (EVLI, EVLII and EVLIII) are all specific for galactopyranosides and N-acetylgalactosamine, and their affinities for simple sugars are EVLIII greater than EVLII greater than EVLI. EVLI and EVLIII are homodimers made up of an A-subunit of molecular mass 36,000 and a B-subunit of molecular mass 33,000, whereas EVLII is a heterodimer composed of the A- and B-subunits. Upon treatment with trifluoromethansulphonic acid, the molecular masses of both subunits decreased to 31,000. Rechromatography of EVLII on the acid-treated Sepharose 4B column again produced the homodimeric lectins (EVLI and EVLIII). It is suggested that the constituent subunits of Erythrina variegata isolectins are eschangeable with each other in vitro.


Purification and characterization of carbohydrate-binding peptides from Lotus tetragonolobus and Ulex europeus seed lectins using affinity chromatography.

Carbohydrate-binding peptides of several anti-H(O) leguminous lectins were obtained from endoproteinase Asp-N or Lys-C digests of L-fucose-binding Lotus tetragonolobus lectin (LTA) and Ulex europeus lectin I (UEA-I) and from that of a di-N-acetylchitobiose-binding Ulex europeus lectin II (UEA-II) by affinity chromatography on columns of Fuc-Gel (for LTA and UEA-I) and on a column of a mixture of several oligomers of N-acetyl-D-glucosamine (GlcNAc) coupled to Sepharose 4B (GlcNAc oligomer-Sepharose 4B) (for UEA-II). These peptides were retained on the Fuc-Gel or GlcNAc oligomer-Sepharose 4B column and were presumed to have an affinity for the columns. The amino acid sequences of the retarded peptides were determined using a protein sequencer.


Determination of the carbohydrate-binding site of Bauhinia purpurea lectin by affinity chromatography.

To determine the carbohydrate-binding site of Bauhinia purpurea lectin (BPA), a D-galactose- and lactose-binding lectin, a peptide which interacts with lactose was purified from endoproteinase Asp-N digests of BPA by chromatography on a lactose-Sepharose column. It consists of nine amino acids and its amino acid sequence is Asp-Thr-Trp-Pro-Asn-Thr-Glu-Trp-Ser. A tryptic fragment with the ability to interact with lactose was also purified and found to contain this sequence, consisting of nine amino acids. This nonapeptide was aligned in a part of the metal-binding region conserved in all legume lectins. The chemical synthesis of the nonapeptide was carried out by a solid-phase method and the synthetic peptide showed a lactose-specific binding activity in the presence of calcium. A chimeric lectin gene was constructed using a cDNA coding BPA in which the nonapeptide sequence was replaced by the corresponding region of the alpha-D-mannose binding Lens culinaris lectins. Although BPA is specific for beta-D-galactose, the chimeric lectin expressed in Escherichia coli was found to bind alpha-D-mannosyl-bovine serum albumin and this binding was inhibited by D-mannose.


Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.


Use of antibody fragments in immunoaffinity chromatography. Comparison of FV fragments, VH fragments and paralog peptides.

Some new antibody fragments have recently been described: FV fragments (Mr 25,000), VH fragments or "dAbs" (12,500) and paralog peptides (1000-2000). FV fragments, VH fragments and a paralog peptide that had been derived from a parent antibody with a specificity for hen lysozyme were produced. All three reagents were immobilized on Sepharose and evaluated for their ability to recover hen lysozyme from "spiked" serum and to separate hen lysozyme from turkey lysozyme. The FV column had excellent specificity for hen lysozyme, the VH column had significantly reduced specificity and the paralog peptide column did not bind lysozyme at all.


Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein.

A method is described for the rapid and efficient affinity chromatographic purification of murine monoclonal immunoglobulin M (IgM) which utilizes immobilized rabbit mannan binding protein (MBP). This solid-phase matrix is shown to bind IgM-class antibodies from a variety of species. Conditions reported show a binding capacity of IgM from murine ascites of nearly 1 mg/ml of immobilized MBP support. The prepared gel is shown to possess an ability to bind not only mouse IgM, but also human and bovine IgM, although with a lesser affinity. The matrix can be regenerated and reused at least ten times without any apparent loss of binding capacity or specificity. Mouse monoclonal IgM purified from ascites fluid using this method is greater than 95% pure as shown by high-performance liquid chromatography analysis.


Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.


Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.


High-performance affinity chromatography system for the rapid, efficient assay of glycated albumin.

A high-performance affinity chromatographic system was constructed and shown to permit highly reproducible, rapid, automatic assays of serum glycated albumin (GA) by separation of albumin (Alb) on an anion-exchange column (Asahipak ES-502N) packed with a vinyl alcohol copolymer bearing diethylamino groups and consecutive separation of GA on a column packed with a vinyl alcohol copolymer bearing boronate groups. The first column selectively retained Alb free of other serum proteins and permitted at least a 95% recovery of sample Alb. The purity of the Alb peak was confirmed by two-dimensional electrophoresis. Chromatographic analyses of human serum Alb incubated with glucose on the second column showed that the peak area for GA increased in accordance with the incubation time and suggested selective adsorption of GA on the second column. Optimization of the conditions for the two-column system reduced the analysis time to 10 min. Analyses of human sera with the present system showed GA to be 16.1 +/- 1.1% (mean +/- S.D.) of total Alb in non-diabetic children and 39.9 +/- 9.1% (mean +/- S.D.) in diabetic children (0-17 years old).


Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.


Purification of sufficiently gamma-carboxylated recombinant protein C and its derivatives. Calcium-dependent affinity shift in immunoaffinity and ion-exchange chromatography.

Protein C, which is an important anti-thrombotic factor in the blood coagulation cascade, undergoes several post-translational modifications. gamma-Carboxylation on nine glutamic acid residues at the N-terminal region of the light chain [gamma-carboxylated glutamic acid (Gla) domain] is considered to be critical for full anti-clotting activity. It is also known that when recombinant protein C is expressed in animal cells this particular modification is often lost. We were successful in preparing a monoclonal antibody (PC01) which distinguishes the sufficiently gamma-carboxylated protein from the rest by its specific affinity for the Ca(2+)-induced conformational change of the former, and thereby developed a simple process of purifying sufficiently gamma-carboxylated protein C. Culture supernatant of Chinese hamster ovary cell transformants was first applied to Q-Sepharose and recombinant protein C was partially purified. It was then loaded onto a PC01 affinity column in the presence of 5 mM calcium chloride. Sufficiently gamma-carboxylated protein C was retained while insufficient-carboxylated protein C quickly passed through. The former was eluted with 5 mM EDTA efficiently and with high purity, contained eight Gla units per molecule, and had similar anti-clotting activity. The flow-through was relatively impure protein C which contained five Gla units per molecule and showed limited anti-clotting activity. We extended the application of the Ca(2+)-induced conformational change to conventional ion-exchange chromatography. The sufficiently gamma-carboxylated protein C was found to elute earlier in the salt gradient from an anion-exchange column in the presence of 5 mM calcium chloride being fully separated from the insufficiently carboxylated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)


Interactions and uses of antisense peptides in affinity technology.

Antisense peptides, amino acid sequences encoded in the antisense strand of DNA, can interact with significant affinity and selectivity with their corresponding sensepeptides. Experimentally, sense-antisense peptide recognition has been observed repeatedly. However, skepticism about the biological relevance of this phenomenon has persisted. This is due in part to the unexpected and somewhat couterintutive nature of the interaction as well as to its non-universality as an empirical observation. Nonetheless, antisense peptides in several cases investigated so far have been used as immobilized ligands for the successful affinity chromatographic separation of native (sense) peptides and proteins. For example, immobilized antisense peptides corresponding to Arg8-vasopressin (AVP) have been used to separate vasopressin from oxytocin chromatographically as well as to affinity capture AVP-receptor complex. These results, together with improved understanding of the general features of amino acid sequence which drive antisense-sense peptide interactions as well as new ideas for making antisense peptides chimeras, are beginning to suggest improved ways to make antisense-related peptides as affinity agents for separation as well as for other biotechnology applications.


Interaction of amphipathic model lipopeptides with phospholipid bilayers.

In order to investigate the conformation and localization of lipopeptides in lipid bilayers, a basic model peptide with a long alkyl chain, Ac-Ser-Val-Lys-Amy-Ser-Trp-Lys-Val-NHCH3 Amy-1; Amy = alpha-aminomyristic acid) was synthesized. Its interaction with neutral and acidic phospholipid bilayers was studied by circular dichroism (CD) spectroscopy, dye leakage and fluorescence measurements. Another peptide, Ac-Leu-Ala-Arg-Leu-Trp-Amy-Arg-Leu-Leu-Ala-Arg-Leu-NHCH3 (Amy-2), which was prepared previously, was used for comparison. The CD data indicated that Amy-1 took a beta-turn and/or a beta-structure in the absence and presence of liposomes. Amy-2 formed a beta-structure in aqueous solution and an alpha-helical structure in liposomes. The dye leakage ability of Amy-1 was much weaker than that of Amy-2. Fluorescence spectroscopic data suggest that the peptides are immersed in lipid bilayers. Based on these results, discussion is made in terms of localization of the peptides in lipid bilayers.


Purification and partial characterization of phospholipase A2 isoforms from human placenta.

Five isoforms of the human placental phospholipase A2 were identified and purified to near homogeneity. The purification of these enzymes involved gel permeation, ion-exchange and affinity chromatography. The apparent relative molecular mass of these proteins is 70,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These enzymes have pH optima of 7 and 8. Two-dimensional gel electrophoresis of these enzymes revealed distinct pH optima for each of the isoforms with values ranging from 4.0 to 6.5. Three of the isoforms require calcium for activity whereas the other two forms exhibit 50% of their maximum activity without the presence of calcium.


Trypsin and affinity chromatography.

Affinity adsorbents for trypsin which were prepared by immobilizing product-type ligands, that is, peptides having C-terminal arginine, proved to be effective not only for preparative purposes but also for basic research on molecular recognition. The properties of the binding site of trypsin were revealed by chromatographic experiments. Quantitative analysis based on the theory of frontal affinity chromatography proved to be extremely effective. As an extension of the product-type ligands, peptide argininals were also used and information on the mechanism of action of these inhibitors was obtained. Anhydrotrypsin, which lost the hydroxyl group of Ser183, was found to gain increased binding ability for product-type compounds. This inactivated enzyme was also used as an immobilized ligand and the unique affinity adsorbent thus prepared proved to be extremely effective for the separation of peptides and recombinant proteins based on their C-terminal structures. High-performance affinity chromatography of trypsin and related enzymes using a polymer-based support was also developed.


Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.