Simultaneous measurement of whole blood vitamin B1 and vitamin B6 using LC-ESI-MS/MS.

Vitamin B1 and B6 have recently been included in the Dutch clinical guidelines for the general practitioner in the differential diagnosis of dementia. To keep up with the sharp rise in the number of requests, an LC-MS/MS method using stable isotopes as internal standards was developed. The active vitamers thiamine pyrophosphate (TPP) and pyridoxal-5'-phosphate (PLP) in whole blood are simultaneously measured with a short run time of 2min. Whole blood is mixed with internal standard solution containing both TPP-d3 and PLP-d3, followed by deproteinization with a trichloroacetic acid (TCA) solution. A UPLC-MS/MS system from Waters™ was used for chromatographic separation and subsequent detection by electrospray ionization in the positive mode with mass transitions of 425.1>121.85 for TPP and 247.9>149.9 for PLP. The method is linear across the range of 12-4870 nmol/L for TPP and 6-4850 nmol/L for PLP. The mean intra-assay and inter-assay precision are 3.5% and 7.6% respectively for TPP and 3.4% and 6.1% for PLP. The relative matrix effect (TPP 97%, PLP 93%), recovery (TPP 99%, PLP 94%) and lower limit of quantification (TPP 12 nmol/L, PLP 6 nmol/L) meet the applied acceptance criteria. The comparison of the new LC-ESI-MS/MS method for TPP with our current HPLC-Fluorescence method for total thiamine yields the following equation: TPP LC-MS/MS=0.97×total thiamine HPLC - 10.61 (r2=0.94). The comparison of the new LC-ESI-MS/MS method for PLP with our current LC-ESI-MS/MS method results in PLP LC-MS/MS new=1.01×PLP LC-MS/MS old - 1.58 (r2=0.99). In conclusion, this LC-MS/MS based assay is characterized by simple sample processing with a short run time and comparison with the current methods is excellent. The new LC-MS/MS method is a convenient method to determine TPP and PLP in whole blood for both clinical routine and research applications.

Modelling asthma in macaques: longitudinal changes in cellular and molecular markers.

The aim of the present study was to determine whether systemic sensitisation and chronic aeroallergen challenge in macaques replicate the classical and emerging immunology and molecular pathology of human asthma. Macaques were immunised and periodically challenged over 2 yrs with house dust mite allergen. At key time-points, serum, bronchoalveolar lavage (BAL) and bronchial biopsies were assayed for genes, proteins and lymphocyte subpopulations relevant to clinical asthma. Immunisation and periodic airway challenge induced changes in immunoglobulin E, airway physiology and eosinophilia consistent with chronic, dual-phase asthma. Sensitisation increased interleukin (IL)-1ß and -6 concentrations in serum, and IL-13 expression in BAL cells. Airway challenge increased: early expression of IL-5, -6, -13 and -19, and eotaxin; and variable late-phase expression of IL-4, -5 and -13, and thymus- and activation-regulated chemokine in BAL cells. CD4+ lymphocytes comprised 30% of the CD3+ cells in BAL, increasing to 50% in the late phase. Natural killer T-cells represented <3% of the CD3+ cells. Corticosteroid treatment reduced serum histamine levels, percentage of CD4+ cells and monocyte-derived chemokine expression, while increasing CD3+ and CD8+ cells in BAL. Sensitisation and periodic aeroallergen challenge of cynomolgus macaques results in physiological, cellular, molecular and protein phenotypes, and therapeutic responses observed in human asthma, providing a model system useful in target and biomarker discovery, and translational asthma research.

Selected contribution: HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone.

Cyclic nucleotide-induced relaxation of maximally activated arterial smooth muscle has two phases. 1) The initial relaxation transient is typically characterized by a rapid reduction in force associated with brief reductions in myoplasmic Ca(2+) concentration ([Ca(2+)](i)) and myosin regulatory light chain (MRLC) phosphorylation on serine (Ser)-19 (Ser(19)). 2) The sustained inhibitory response is typically associated with Ser(16) phosphorylation of heat shock protein 20 (HSP20) without sustained reductions in [Ca(2+)](i) or MRLC phosphorylation. We investigated whether the extent of Ser(16)-HSP20 phosphorylation quantitatively correlated with the sustained inhibitory response. With addition of nitroglycerin to histamine-stimulated swine carotid media, the initial relaxation transient was associated with a decrease in MRLC phosphorylation without an increase in Ser(16)-HSP20 phosphorylation. During the sustained phase of nitroglycerin-induced relaxation and during force redevelopment induced by washout of nitroglycerin in the continued presence of histamine, the level of Ser(16)-HSP20 phosphorylation, but not MRLC phosphorylation, correlated with inhibition of force. Forskolin, which increases cAMP concentration, also induced a sustained inhibitory response that was associated with increases in Ser(16)-HSP20 phosphorylation without reductions in MRLC phosphorylation levels. Forskolin increased Ser(16)-HSP20 phosphorylation to a greater extent and inhibited force more completely than that observed with nitroglycerin. Increases in Ser(16)-HSP20 phosphorylation correlated with the degree of force inhibition regardless of whether the relaxation was induced by nitroglycerin or forskolin. These data are consistent with the hypothesis that Ser(16)-HSP20 phosphorylation may be a cyclic nucleotide-dependent, yet MRLC phosphorylation-independent, inhibitor of smooth muscle contractile force.

Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3',5'-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMP-mediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.

Minor role of a Ca2+-depleted sarcoplasmic reticulum in heterologous desensitization of smooth muscle to K+.

Exposure of porcine carotid artery smooth muscle (PCASM) to histamine was followed by a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of a steady-state myoplasmic Ca2+ concentration ([Ca2+]i). We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized PCASM cells is the same before and after a desensitizing histamine treatment, then the transient attenuation of the increase in [Ca2+]i may be due to accelerated uptake of Ca2+ by a partially depleted sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier. We tested this hypothesis by eliciting responses of "desensitized PCASM" to 40 mM KCl in the presence of cyclopiazonic acid (CPA), an SR Ca2+-ATPase inhibitor. Contractions of CPA-treated tissues were attenuated less than those of tissues not treated with CPA, but they were not abolished. CPA-insensitive mechanism(s) dominated the desensitization. We conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into PCASM in response to 40 mM KCl.

Heterologous desensitization of smooth muscle to K+ depolarization: retarded stimulus-[Ca2+]i coupling.

To understand the phenomenon of postreceptor heterologous desensitization, we exposed porcine carotid media to 40 mM KCl physiological saline solution both before and after intervening treatment with histamine. Increasing histamine concentration or duration of exposure or decreasing the interval between histamine exposure and KCl progressively slowed the contractile responses to K+ depolarization. A delay in initiation and a slower rate of rise of KCl-induced stress in histamine-pretreated muscle were preceded by a slower rate of rise of aequorin-estimated myoplasmic Ca2+ concentration ([Ca2+]i), myosin regulatory light chain (MRLC) phosphorylation, and tissue stiffness, with no detectable change in the Ca2+ sensitivity of MRLC phosphorylation. This heterologous desensitization was not a diminished steady-state force but instead a profound slowing of contraction rates. This slowing was a manifestation of retardation of the rate at which [Ca2+]i rises to the level appropriate for the stimulus. The lack of rapid initial [Ca2+]i and cross-bridge phosphorylation transients as a consequence of histamine pretreatment resulted in very slow cross-bridge cycling rates and rates of force development (latch).

Water compartmentalization and extracellular tortuosity after osmotic changes in cerebellum of Trachemys scripta.

1. Water compartmentalization in the turtle cerebellum subject to media of different osmolalities was quantified by combining extracellular diffusion analysis with wet weight and dry weight measurements. The diffusion analysis also determined the tortuosity of the extracellular space. 2. Isolated cerebella were immersed in normal, oxygenated physiological saline (302 mosmol kg-1), hypotonic saline (238 mosmol kg-1) and a series of hypertonic salines (up to 668 mosmol kg-1). The osmolality was varied by altering the NaCl content. 3. Extracellular volume fraction and tortuosity of the granular layer of the cerebellum were determined from measurements of ionophoretically induced diffusion profiles of tetramethylammonium, using ion-selective microelectrodes. The volume fraction was 0.22 in normal saline, 0.12 in hypotonic medium and 0.60 in the most hypertonic medium. Tortuosity was 1.70 in the normal saline, 1.79 in the hypotonic and 1.50 in the most hypertonic saline. 4. The water content, defined as (wet weight-dry weight)/wet weight, of a typical isolated cerebellum (including granular, Purkinje cell and molecular layers) was 82.9%. It increased to 85.2% in hypotonic saline and decreased to 80.1% in the most hypertonic saline. 5. Measurements of extracellular volume fraction and water content were combined to show that hypotonic solutions caused water to move from the extracellular to the intracellular compartment while hypertonic solutions caused water to move from the intracellular to extracellular compartment, with only a relatively small changes in total water in both cases. 6. These results suggest the use of the isolated turtle cerebellum as a model system for studying light scattering or diffusion-weighted magnetic resonance imaging.

Pleural pressure measured with a rib capsule in anesthetized pneumonectomized rabbits.

Pleural pressure was measured at end expiration in anesthetized rabbits using a rib capsule placed in the right fifth rib. Three groups of rabbits were studied in right and left lateral recumbency at least 8 weeks after surgery; rabbits that had undergone left pneumonectomy (Px, n = 8), rabbits that had undergone left pneumonectomy with wax plombage (Px + W, n = 7), and sham-operated control rabbits (S, n = 6). In S, Px, and Px + W rabbits in the left lateral position (lung and capsule nondependent), pleural pressure was -2.11 +/- 0.88 (mean +/- SD), -2.65 +/- 0.23, and -1.96 +/- 0.55 cmH2O respectively. In S, Px and Px + W rabbits in the right lateral position (lung and capsule dependent), pleural pressure was 0.64 +/- 0.22, 0.85 +/- 1.42, and 0.48 +/- 1.77 cmH2O respectively. In each position, pleural pressure did not differ among groups. This suggests that the compensatory increase in lung volume and reduced lung compliance following pneumonectomy in rabbits (Olson, J. Appl. Physiol. 74: 415-422, 1993) was not simply due to hyperinflation of the remaining lung.

Effect of a regional increase in alveolar pressure on pulmonary blood flow.

We examined whether wedging a catheter (0.5 cm OD) into a subsegmental airway in dog (n = 6) or pig lungs (n = 5) and increasing pressure in the distal lung segment affected pulmonary blood flow. Dogs and pigs were anesthetized and studied in the prone position. Pulmonary blood flow was measured by injecting radiolabeled microspheres (15 microns diam) into the right atrium when airway pressure (Pao) was 0 cmH2O and pressure in the segment distal to the wedged catheter (Ps) was 0, 5, or 15 cmH2O and when Pao = Ps = 15 cmH2O. The lungs were excised, air-dried, and sectioned. Blood flow per gram dry weight normalized to cardiac output to the right or left lung, as appropriate, was calculated for the test segment, a control segment in the opposite lung corresponding anatomically to the test segment, the remainder of the lung containing the test segment (test lung), and the remainder of the lung containing the control segment (control lung). The presence of the catheter reduced blood flow in the test segment compared with that in the control segment and in the test lung. Blood flow was not affected by increasing pressure in the test segment. We conclude that, in studies designed to measure collateral ventilation in dog lungs, the presence of the wedged catheter is likely to have a greater effect on blood flow than the increase in pressure associated with measuring collateral airway resistance.

Pleural pressure as a function of body position in rabbits.

Pleural pressure was measured at end expiration in spontaneously breathing anesthetized rabbits. A liquid-filled capsule was implanted into a rib to measure pleural liquid pressure with minimal distortion of the pleural space. Capsule position relative to lung height was measured from thoracic radiographs. Measurements were made when the rabbits were in the prone, supine, right lateral, and left lateral positions. Average lung heights in the prone and supine positions were 4.21 +/- 0.58 and 4.42 +/- 0.51 (SD) cm, respectively (n = 7). Pleural pressure was -2.60 +/- 1.87 (SD) cmH2O at 50.2 +/- 7.75% lung height in the prone position and -3.10 +/- 1.22 cmH2O at 51.4 +/- 6.75% lung height in the supine position. There was no difference between the values recorded in the prone and supine positions. Placement of the capsule into the right or left chest had no effect on the magnitude of the pleural pressure recorded in rabbits in right and left lateral recumbency (n = 12). Measurements over the nondependent lung were repeatable when rabbits were turned between the right and left lateral positions. Lung height in laterally recumbent rabbits averaged 4.55 +/- 0.52 (SD) cm.

Detection and prevalence of hearing loss in a cohort of children following serogroup B, meningococcal infection 1983-1987.

A study of hearing loss in 50 surviving children aged 2 months to 17 years, hospitalised with serogroup beta meningococcal infection between 1983-1987, indicated that 35 (70%) had a hearing assessment. The majority were admitted under the care of the paediatricians. Older children tended not to have a follow-up hearing assessment. Impairment of hearing was found in 3, (8.6% of those tested, or 6% of the whole group). Two similar studies in the early and late 1970s indicate comparable prevalence estimates. There is little indication that over the past 17 years the prevalence of hearing loss has declined.

Effect of temperature upon carbon dioxide stores in the snake Coluber constrictor and the turtle Chrysemys scripta.

The effect of temperature upon respiratory exchange ratio (R) was measured in snakes (Coluber constrictor) and turtles (Chrysemys scripta). Increasing body temperature produced a transient elevation of R, and lowering body temperature transiently depressed R. These thermal effects resulted from an 'excess' and a 'deficit' CO2 elimination, respectively. Steady-state blood CO2 content (CCO2) in C. constrictor decreased linearly with rising temperature. Plasma bicarbonate concentration, calculated from in vivo arterial PCO2 and pH, followed the same pattern. Also, time courses of blood CCO2 were consistent with the metabolic studies. Less than half of the change in blood CCO2 could be explained by shifts of the in vitro CO2 dissociation curve; the remainder was contributed by other tissues. Blood lactate levels changed little with temperature. Based upon the blood studies, the predicted quantity of CO2 eliminated from the extracellular space when temperature increases is about 29% of the excess CO2 eliminated from the snakes. Thus, CCO2 in other tissues also decreases with rising temperature. It is concluded that reptiles function as open systems with respect to CCO2, which does not agree with alphastat control. Systemic arterial PO2 and PCO2 increased with rising body temperature in C. constrictor. The mechanisms producing these increases are discussed.