Defining 'full-length' recombinant factor VIII: a comparative structural analysis.

Coagulation factor VIII (FVIII) is an important glycoprotein co-factor involved in haemostasis, functioning to accelerate activation of factor X by activated factor IX. Insertion of expression vectors containing the full-length cDNA sequence of human FVIII into mammalian cell lines results in the production of recombinant factor VIII (rFVIII), typically referred to as 'full-length' rFVIII (FLrFVIII). Both FLrFVIII and plasma-derived FVIII exist primarily as heterodimeric proteins, consisting of a heterogenous light and heavy chain. The objectives of this study were to compare the structural heterogeneity of high-purity FVIII preparations and further define the term 'full length' as it refers to rFVIII protein structure. Five commercially available FVIII concentrates were characterized based on SDS-PAGE, N-terminal sequencing, and peptide and domain mapping coupled to mass spectrometry. The major heavy chain species identified in FLrFVIII included various B-domain-truncated forms of FVIII, with the predominant species terminating at Arg(1313). This study demonstrates that the use of full-sequence FVIII cDNA for the production of rFVIII does not result in a homogeneous FLrFVIII protein product. Rather, commercially available FLrFVIII represents a heterogenous mixture of various B-domain-truncated forms of the molecule, with no evidence of a contiguous, intact B-domain.

Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII:C activity in hemophilic plasma using one-stage clotting assays.

Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing one-stage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a B-domain deleted recombinant FVIII (BDDrFVIII, ReFacto). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the one-stage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL(-1). FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in one-stage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the one-stage clotting and the chromogenic substrate assay for ReFacto.

Effect of ramp slope on ventilation thresholds and VO2peak in male cyclists.

This study investigated the effect of 10 W*min(-1) (Slow ramp, SR), 30 W*min(-1) (Medium ramp, MR) and 50 W*min(-1) (Fast ramp, FR) exercise protocols on assessments of the first (VT1) and second (VT2) ventilation thresholds and peak oxygen uptake (VO(2)peak) in 12 highly-trained male cyclists (mean +/- SD age = 26 +/- 6 yr). Expired gas sampled from a mixing chamber was analyzed on-line and VT1 and VT2 were defined as two break-points in 20-s-average plots of pulmonary ventilation (V(E)), ventilatory equivalents for O(2) (V(E)/VO(2)) and CO(2) (V(E)/VCO(2)), and fractions of expired O(2) (F(E)O(2)) and CO(2) (F(E)CO(2)). Arterialized-venous blood samples were analyzed for blood-gas and acid-base status. VO(2)peak was significantly lower (p < 0.05) for SR (4.65 +/- 0.53 l small middle dot min(-1)) compared to MR (4.89 +/- 0.56 l *min(-1)) and FR (4.88 +/- 0.57 l *min(-1)) protocols. CO(2) output and blood PCO(2) were lower (p < 0.05), and V(E)/VCO(2) was higher (p < 0.05), above VT1 for SR compared to MR and FR protocols. No significant differences were observed among the protocols for VO(2), % VO(2)peak, V(E), plasma lactate ([La(-)]) and blood hydrogen ion concentration ([H(+)]), and heart rate (HR) values at VT1 or VT2. The work rate (WR) measured at VT1, VT2 and VO(2)peak increased (p < 0.05) with steeper ramp slopes. It was concluded that, in highly-trained cyclists, assessments of VT1 and VT2 are independent of ramp rate (10, 30, 50 W*min(-1)) when expressed as VO(2), % VO(2)peak, V(E), plasma [La(-)], blood [H(+)] and HR values, whereas VO(2)peak is lower during 10 W*min(-1) compared to 30 and 50 W*min(-1) ramp protocols. In addition, the WR measured at VT1, VT2 and VO(2)peak varies with the ramp slope and should be utilized cautiously when prescribing training or evaluating performance.

Reproducibility of ventilation of thresholds in trained cyclists during ramp cycle exercise.

The reproducibility of peak cardiopulmonary exercise responses and the first (VT1) and second [VT2) ventilation thresholds was studied in sixteen endurance-trained male cyclists (mean +/- SD peak oxygen uptake [VO2 peak] = 63.3 +/- 7.1 ml x kg(-1) x min(-1)) during duplicate 30 W x min(-1) ramp cycling protocols. Expired gas sampled from a mixing chamber was analysed on-line and VT1 and VT2 were determined by computerised V-slope analysis and visually by two evaluators (test-retest reliability) and again by one of the evaluators 12 months later (intra-evaluator reliability) from 20-s-average respiratory data. The results demonstrated high intra-evaluator reliability (r = 0.91-0.97, P < 0.0001) for repeat determinations of VO2, work rate (WR) and heart rate (HR) at VT1 and VT2. No significant differences were observed between Tests 1 and 2 for any of the measured variables (P > 0.05). Test-retest intraclass reliability coefficients ranged from 0.86 to 0.98 (P < 0.0001) for VO2 peak, peak pulmonary ventilation (VE), carbon dioxide output (VCO2), HR and WR values, and measurements of VO2 and WR at VT2, and from 0.67 to 0.80 (P < 0.01) for measurements of VO2 and WR at VT1. The reliability of VT1 and VT2 was reduced when the thresholds were expressed as relative (%VO2 peak) (r = 0.67-0.70, P<0.01) rather than absolute (l x min(-1)) (r = 0.77-0.93, P<0.001) VO2 values. It was concluded that VO2 peak, peak VE, VCO2. HR and WR values, and VT2 are highly reproducible in trained cyclists using a 30 W x min(-1) ramp exercise function. However, determinations of VT1 are less reliable. Additionally, ventilation thresholds are more reliably described using absolute rather than relative VO2 values.

Cardiovascular regulation during head-up tilt in healthy 20-30-year-old and 70-75-year-old men.

This study compared the heart rate, finger arterial pressure (AP) and electromyographic (EMG) activity of selected anti-gravity muscles during the initial and prolonged phases of orthostatic stress in healthy young and older men. Beat-by-beat recordings of heart rate, finger systolic pressure, diastolic pressure and mean AP were made during supine rest and 5 min of 90 degrees head-up tilt (HUT) in 18 young (23+/-1 years) and 15 older (73+/-1 years) men. The EMG activity of the soleus, tibialis anterior and vastus medialis muscles was recorded. During the first 30 s following 90 degrees HUT (immediate response), the young men exhibited significant (P<0.05) decreases in finger systolic pressure, diastolic pressure and mean AP, followed by a sustained increase in finger AP during the 5 min following 90 degrees HUT (prolonged response). The immediate and prolonged finger AP and diastolic pressure responses were not significantly different (P>0.05) from the values at supine rest for the older men. The mean root mean square EMG activity of the soleus, tibialis anterior and vastus medialis muscles during 90 degrees HUT was not significantly different (P>0.05) from that at supine rest for either group. These results demonstrate that, when compared with healthy older men, young men show larger reductions in finger AP during the initial phase of orthostatic stress. However, during the prolonged phase of orthostatic stress, older men maintain resting finger AP, whereas young men demonstrate a reflex overshoot in finger AP. Finally, differences in lower-limb anti-gravity muscle activation do not account for the contrasting finger AP responses of healthy young and older men.

Does exogenous coenzyme Q10 affect aerobic capacity in endurance athletes?

The effect of orally supplemented coenzyme Q10 (CoQ10) on plasma CoQ10 concentration and aerobic capacity in endurance athletes was evaluated. Eighteen volunteer male road cyclists and triathletes, 8 in a CoQ10 supplementation group (QG) and 10 in a placebo group (PG), successfully completed the experimental protocol. Subjects were evaluated during and following graded cycling exercise tests, which were performed before and after 28 days of supplementation with 1 mg.kg-1.day-1 of CoQ10 or placebo. The presupplementation plasma CoQ10 concentration was significantly increased from 0.91 +/- 0.13 microgram.ml-1 to 1.97 +/- 0.27 microgram.ml-1 in QG following supplementation (p < .05). However, the CoQ10 supplementation regime had no consistently significant effect on oxygen uptake, anaerobic and respiratory compensation thresholds, blood lactate, glucose and triglyceride kinetics, heart rate, and blood pressure during and after graded cycling to exhaustion.

Sampling arterialised-venous blood from a dorsal-hand vein during exercise without compromising a competitive cycling position.

The arterialisation of dorsal-hand venous blood, using a new hand-heating sleeve that did not disrupt a normal competitive cycling position, was evaluated in 10 male cyclists. Hand-heating was achieved by pumping heated water through a rubberised panel which rested against the dorsal and ventral surfaces of the right hand. Subjects were cannulated near the dorsal venous arch in both hands and blood samples were collected simultaneously at rest under normothermic conditions for 9 subjects, and at rest and after 10, 20 and 30 min of exercise at 50% of the anaerobic threshold for 10 subjects, when only the right hand was heated. No bilateral differences for PO2, PCO2 or pH were observed between normothermic hands at rest (p > 0.05). The heating sleeve increased the hand temperature (p < 0.05) and produced a significant (p < 0.05) arterialisation of blood samples collected from a dorsal-hand vein, compared to values observed in the non-heated hand, at rest and during 30 min of exercise. PO2 and pH were about 10 mm Hg and 0.01 units higher, and PCO2 was about 1.5 mm Hg lower, for samples collected from the heated compared to the non-heated hand. This new method of hand-heating was effective in arterialising venous blood and minimised the need for variation to a normal, competitive cycling position.

Reliability of using the D-max method to define physiological responses to incremental exercise testing.

The purpose of this study was to examine the reliability of using a mathematical method, D-max, to define blood lactate kinetics in response to an incremental exercise test, and to compare the physiological responses corresponding to the workload at D-max with those at the traditional 4 mmol l-1 lactate threshold and ventilatory thresholds. Ten male endurance trained athletes, with an average (+/- SD) age of 25.6 +/- 8.2 years and maximal oxygen consumption of 64.0 +/- 1.7 ml kg-1 min-1, performed an incremental cycling test on two occasions separated by four weeks. The expired gas was analysed on-line and plasma lactate concentration was analysed for each workload and at exhaustion. The lactate response to exercise was represented by a third-order polynomial regression curve. The D-max was defined as the point on the regression curve that yields the maximal distance to the straight line formed by the two end points of the curve. The results demonstrated a high test-retest reliability (intraclass correlation coefficients 0.77-0.93, p < 0.01) in oxygen consumption, heart rate and exercise intensity at both D-max point and exhaustion. No significant differences were found in the mean values of the variables between the two tests. It is concluded that the D-max appears to be a reliable method for defining the individual physiological responses to exercise tests, with the advantage of objectivity. However, there is no evidence to support the theory that the exercise intensity defined by the D-max method is superior to that defined by other methods to prescribe training intensity or predict aerobic performance for athletes. Further investigations are warranted to examine the validity of using this method in exercise prescription.

Changes in cortical amino acids during electrical kindling in rats.

The effect of kindling rats by electrical stimulation of the frontal cortex for approx. 3 months on the concentrations of amino acids (taurine, aspartate, glutamate, glutamine and GABA) in the cerebral cortex has been studied, as well as the release of endogenous amino acids from kindled slices of brain in vitro. In these kindled rats, killed 1 week after the last shock, there were no changes in any concentrations of amino acids. However, when cortical slices, prepared from either the control or kindled rats, were stimulated in vitro by exposure to veratrine, more endogenous glutamate and aspartate were released from the kindled tissue than from the control. The neurotransmitter amino acids, glutamate and aspartate, may be involved in the permanent effects of electrical kindling.