Glycolysis inhibitors negatively bias blood glucose measurements: potential impact on the reported prevalence of diabetes mellitus.

BACKGROUND: Blood glucose concentrations are essential in defining diabetes mellitus. Recent guidelines advocate either of two discrete methods for sample collection and processing. One of these involves addition of glycolysis inhibitors, such as sodium fluoride-potassium oxalate (NaF-KOx) to sample collection tubes, whereas the other requires immediate refrigeration and sample separation. AIMS: To examine whether the choice of the preanalytical process has any impact on subsequent glucose determinations. METHODS: 62 healthy men participated in the study during screening for diabetes. Paired venous blood samples were collected in a serum-gel tube and a tube containing NaF-KOx (both Sarstedt, Leicester, UK). Serum was promptly separated from gel tube samples and refrigerated, whereas NaF-KOx samples were not separated until immediately before analysis. Glucose concentrations were determined using an Olympus AU 2700 analyser incorporating an automated hexokinase method. RESULTS: Mean (95% CI) glucose concentration in serum-gel tube samples was 5.2 mmol/l (5.0 to 5.4 mmol/l), whereas the concentration in tubes containing NaF-KOx was 4.9 mmol/l (4.8 to 5.1 mmol/l). A negative bias of 0.23 mmol/l (0.16 to 0.30 mmol/l) and relative negative bias of 4.7 % (3.2% to 6.3%) were observed for samples collected in NaF-KOx tubes, consistent with the combined effects of glycolysis and dilution. CONCLUSIONS: Bias associated with the use of NaF-KOx tubes may have a significant impact on the prevalence of fasting hyperglycaemia, according to current diagnostic criteria. The small but significant difference between preanalytical processes should be considered when screening for the presence of diabetes mellitus.

Excitatory and inhibitory effects of histamine on molluscan neurons.

Histamine elicited depolarization (excitation) in some neurons and hyperpolarization (inhibition) in other neurons of the central nervous system of the marine mollusc, Onchidium verruculatum. The histamine sensitive region was along the axon at some distance from the soma. H1-receptor blockers (SA-97 and mepyramine) suppressed the excitatory (H1) response without affecting the inhibitory (H2) response, while H2-receptor blockers (burimamide and metiamide) suppressed the H2-response without affecting the H1-response. The H1-response was associated with a marked increase in membrane conductance and was blocked by removal of the external Na. The H2-response consisted of a hyperpolarization without much change in conductance, compared with the hyperpolarization of same amplitude produced by glutamate in the same neuron. Passive polarization of the membrane and reduction of Cl concentrations to 1/5-1/25 caused no significant change in H2-response. The H2-response was slightly suppressed in K-free saline. Thus, it seems difficult to account for the hyperpolarization only by an increase in K or Cl conductance. Complete removal of Na and addition of ouabain blocked the H2-response, suggesting a contribution of an electrogenic Na-pump to the hyperpolarization. However, in 20 mM Na saline with or without K, histamine still caused clear hyperpolarization. In this solution, the histamine response was not affected by ouabain. Although it is difficult to exclude the possibility that an increase in K conductance may be responsible for the hyperpolarization, it is tentatively proposed as a hypothesis that the H2-response involved some active transport mechanism, different from a ouabain-sensitive electrogenic Na-pump.

An analysis of histamine-induced inhibitory response in molluscan neurons.

Mechanisms of the histamine-induced inhibitory response (the H2-response) in neurons of the marine mollusc Onchidium, were further investigated following the preceding paper. The H2-response in normal saline was blocked by ouabain, but the response recovered after a short exposure to Na-free solution containing ouabain. The recovery was only transient in the continuous presence of ouabain. When external Na was reduced to about 1/8 normal concentration (60 mM), the H2-response became sensitive to removal of external Ca, but insensitive to ouabain. The suppressing effect of Ca removal and the recovery by Ca readmission appeared very slowly. However, in about 1/3 normal Na concentration (150 mM) the H2-response was suppressed by removal of the Ca, only in the presence of ouabain. The Na-gradient may be regulated by the ouabain-insensitive transportk, such as a Na-Ca exchange in addition to the ouabain-sensitive Na-pump. The Na-Ca exchange probably dominates over the ouabain-sensitive Na-pump only when passive Na-influx is reduced in a low external Na concentration. The H2-response was markedly inhibited by DNP (5 X 10(-4)M) and cyanide (2 X 10(-3)M), while the hyperpolarization produced by glutamate, which was accompanied by a large reduction of membrane resistance, was not affected by these metabolic inhibitors. Over a wide range of external Na concentrations, the membrane potential was lower in presence than in the absence of external Ca. This may be explained by the hypothesis that there is an electrogenic Na-Ca exchange in which Ca-influx is coupled with Na-efflux. According to a similar hypothesis, the H2-response is produced by the transport system in which Ca-efflux is coupled with Na-influx and the system is controlled by the transmembrane Na gradient.

Immunogenicity and reactogenicity of Avaxim (160 AU) as compared with Havrix (1440 EL.U) as a booster following primary immunization with Havrix (1440 EL.U) against hepatitis A.

BACKGROUND: Hepatitis A vaccination is recommended for travelers from the UK to areas of moderate or high endemicity. Two licensed hepatitis A vaccines are now available in the UK, and this trial was undertaken to determine whether Avaxim can be used as a booster following a primary course of Havrix. METHODS: One hundred and eighty-five subjects were randomized to receive a booster dose of either Avaxim (n=92) or Havrix (n=93), 6 to 7 months after a primary dose of Havrix. Subjects were observed for 30 minutes for immediate reactions and subsequently completed a diary card for a further 2 weeks. Serology samples for HAV antibody titers were taken at 28 6 7 days later. RESULTS: One month following the booster dose, all subjects in both treatment groups achieved HAV antibody titers >= 20 mIU/mL. In the Avaxim group, geometric mean titer (GMT) values increased from 642 mIU/mL (97.5% CI 330-1250 mIU/mL) to 6669 mIU/mL (4566-9740 miu/mL), compared with 739 mIU/mL (379-1443 mIU/mL) at baseline to 4460 mIU/mL (2880-6908 mIU/mL) following the administration of Havrix. The increase in GMT following the administration of Avaxim was significantly greater than that following Havrix (p=.02). Eight percent of subjects reported pain at the injection site following a booster dose of Havrix, compared with none following Avaxim. This difference in reactogenicity was statistically significant (p=.01). In all other respects, both preparations were safe and equally well tolerated. CONCLUSION: Either Avaxim or Havrix may be given as a booster dose of hepatitis A vaccine when Havrix has been administered as the primary dose.

Smooth muscle from the respiratory tract: Effects of altered gas tensions.

STRIPS of bovine tracheal smooth muscle were subjected to changes in the gas tensions of the Krebs solution bathing them. Contractions were produced by 50 Hz sine-wave electrical field stimulation, either in single bursts, or repetitively throughout the experiment.Hypoxia (Po2 99, 55 or 22 mm Hg, pH 7.4) decreased the response to single bursts of stimuli by 22-56 per cent (P<0.01). Hypoxia also reduced the resting tension by 72 per cent (P<0.005). All changes due to altered gas tensions were, at least partially, reversible by returning the tissue to standard conditions.Hypercapnic acidosis (Pco2 140-150 mm Hg, pH 6.7-7.0) reduced the response to single bursts of stimuli by 6 per cent (P<0.01) and reduced the response to repetitive bursts of stimuli by 16 per cent (P<0.01). Acapnic alkalosis (Pco2 0 mm Hg, pH 7.9) did not significantly alter the response to repetitive stimulation.It is concluded that hypoxia, and to a lesser extent hypercapnia, can directly modify tone in the airways, and may contribute to regulation of airflow in the tracheobronchial tree.

Excitation and contraction in bovine tracheal smooth muscle.

1. The smooth muscle layer of the bovine trachea was studied in vitro with the micro-electrode and sucrose-gap techniques. The membrane potential was stable at--47-6 plus or minus 0-98 (S.E. of mean) mV, and there was no spontaneous electrical or mechanical activity. 2. The cell membrane had strong rectifying properties, making it impossible to elicit action potentials by electrical stimulation in normal Krebs Solution. The rectification was abolished by TEA (30 mmol/l), which depolarized the membrane and produced plateau-type action potentials. 3. The spontaneous repetitive action potentials produced by TEA were associated with rhythmic oscillatory contractions of the muscle strips. 4. Histamine caused an increased tone, with superimposed rhythmic fluctuations in tension. The electrical response consisted of depolarization, with rhythmic slow oscillations in potential (slow waves) which were synchronous with the fluctuations in tension. 5. Acetylcholine produced smooth, tonic contractures of tracheal muscle strips, and caused simple depolarization of the membrane. No action potentials were recorded. 6. In calcium-free solutions containing EGTA, the mechanical response to TEA was completely abolished; the response to histamine was greatly reduced; the response to acetylcholine was reduced to a lesser extent. All responses reverted to normal when normal concentrations of extracellular calcium were restored. 7. Lanthanum added to the bathing solution abolished the contraction due to TEA even though the solution contained calcium. It reduced the histamine-induced contraction to 26% of control, and reduced the acetylcholine-induced contraction to 58% of control; extracellular calcium was present throughout. 8. It is suggested that TEA produces contraction by promoting influx of calcium ions into the cytoplasm. Acetylcholine, and to a smaller extent histamine, are less dependent upon the presence of extracellular calcium, and may be capable of releasing calcium sequestered within the cell; acetylcholine appears to be more effective in releasing sequestered calcium.

A study of excitatory neuromuscular transmission in the bovine trachea.

1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.

Relaxation following contraction in tonically contracted smooth muscle from the bovine trachea.

Isometric tension was recorded from strips of bovine tracheal smooth muscle in which the tone had been artificially raised by agonist drugs such as histamine and carbachol. Application of exogenous acetylcholine produced a biphasic response consisting of an initial contraction followed by a more prolonged relaxation before tone was restored to normal. Atropine blocked both components of the biphasic response to exogenous acetylcholine. Tetrodotoxin blocked neither phase of the response to exogenous acetylcholine even though a similar biphasic response to electrical stimulation was severely disrupted. Application of exogenous substance P produced a biphasic response of similar magnitude and form to that produced by acetylcholine. Application of exogenous histamine (tone raised by carbachol) also produced a biphasic response although higher concentrations were required to produce a relaxation of equal magnitude to that produced by acetylcholine. It is concluded that the inhibitory component of the biphasic response to exogenous acetylcholine occurs as a non-specific sequel to contraction.

Contractures produced by carbamate anticholinesterases in bovine tracheal smooth muscle.

1. In isolated strips of bovine tracheal muscle the carbamate anticholinesterases, neostigmine and eserine caused similar, slowly-developing, sustained spasms which were concentration-related in the range 10(-7)-10(-4) mol/l; these spasms could be abolished either by withdrawing the anticholinesterase or by addition of atropine (5 X 10(-7) mol/l). 2. Depletion of tissue stores of acetylcholine using hemicholinium-3 with or without electrical stimulation rendered the muscle unresponsive to neostigmine (10(-6) mol/l). Responses to acetylcholine itself were not impaired. 3. A low concentration of neostigmine (10(-8) mol/l) did not cause spasm but enhanced the contractile response of bovine trachealis to acetylcholine, carbachol and histamine. This concentration of neostigmine also increased the muscle's contraction upon exposure to a high-potassium solution, even in the presence of atropine (5 X 10(-7) mol/l). 4. It is concluded that neostigmine and eserine cause spasm not only by preventing breakdown of endogenously released acetylcholine but also by stimulating release of acetylcholine from nerve terminals and by a non-specific enhancement of muscle contraction.

The contractile response of smooth muscle to immersion in hypertonic solutions.

1. Strips of bovine tracheal muscle and rabbit aorta produced sustained contractions on perfusion with Krebs solution made twice normal strength by addition of sucrose. The contractures were relaxed on return to normal Krebs solution. 2. Similar contractures were produced by tracheal muscle strips in Krebs solutions made twice normal strength by addition of galactose, glucose or NaCl whereas urea caused only a transient contraction. 3. In twice normal strength Krebs solution (sucrose added) the basal tension of rat portal vein and guinea-pig taenia coli was increased. Spontaneous mechanical activity was maintained, but the frequency of contractions was reduced. 4. The hypertonic contracture of bovine trachea in twice normal strength Krebs solution (sucrose added) was reduced by 15% by omission of Ca from the bathing fluid (0.1 mmol/l EGTA added). Severe Ca depletion, by prolonged washing in Ca-free Krebs with 12.5 mmol/l EGTA and Carbachol added, resulted in a 77% reduction in the hypertonic contracture. 5. In twice normal Krebs solution (sucrose added), the hypertonic contracture was partially relaxed by isoprenaline (4 x 10(-6) mol/l); the contractile response to carbachol was reduced; the contractile response to high-K Krebs solution was maintained. 6. Atropine (5 x 10(-7) mol/l) abolished the contractile response to carbachol, but had no effect on the hypertonic contracture. 7. It is suggested that the contraction of bovine tracheal strips in hypertonic solutions is mainly due to activation of the contractile myofilaments rather than simple cell shrinkage. Hypertonic solutions may also interfere with some steps in the excitation-contraction coupling sequence.

Interaction between drugs and potassium-rich solutions in producing contraction in bovine tracheal smooth muscle: studies in normal and calcium-depleted tissues.

1. The contracture normally induced in isolated bovine tracheal smooth muscle by potassium-rich solution was abolished by removal of the extracellular calcium. The contraction returned when calcium was added to the solution in a concentration greater than 0.05 mmol/l. 2. The amplitudes of the potassium-contracture, and of the contractile responses to histamine and acetylcholine in normal physiological solutions, declined at low temperatures (15-25 degrees C). If drugs were added during the plateau phase of the potassium contracture, the extra tension developed above the contracture did not change with temperature. 3. Calcium-depletion reduced the responses to drugs, and repeated application of the drugs in calcium-free solution produced progressively smaller contractions, suggesting that an intracellular store of calcium was being used up. 4. Depolarization of calcium-depleted tracheal muscle by high-K+ solution without calcium produced responses to drugs which were larger than those in sodium-based calcium-free solutions. There was no potentiation in a solution in which sodium was replaced by sucrose, suggesting that potassium was not acting simply by replacing the sodium. 5. It is suggested that depolarization of the membrane by potassium makes available a fraction of bound calcium which was not available in calcium-free sodium-based solution.

The quest for the inhibitory neurotransmitter in bovine tracheal smooth muscle.

The effect of inhibitory nerve stimulation on the mechanical, membrane potential and membrane conductance responses of isolated bovine tracheal smooth muscle has been studied. Membrane responses were measured in a sucrose-gap apparatus. In order to record inhibitory responses, it was necessary to increase tone in the preparation by applying a drug such as histamine. When tone was raised, repetitive field stimulation of intrinsic nerves caused depolarization and contraction, followed by relaxation and a suppression of histamine-induced slow waves. Hyperpolarization of the membrane was only seen following prolonged nerve stimulation, and there was no change in membrane conductance. The inhibitory effect of nerve stimulation was abolished by tetrodotoxin, but was not abolished by atropine, indomethacin, propranolol, naloxone or the purinergic blockers quinidine and theophylline. It was not satisfactorily mimicked by catecholamines, by gamma-amino-n-butyric acid (GABA) or by purines. Nerves with catecholamine fluorescence could not be found in the tracheal muscle layer. Neither adrenergic nor purinergic types of nerve terminal could be found in the tracheal muscle layer during ultrastructural examination of over one thousand nerve profiles. Vasoactive intestinal peptide (VIP) caused relaxation of the histamine-contracted tracheal muscle, suppressed the slow wave and caused slight hyperpolarization at higher concentrations, without affecting the membrane conductance. VIP was found in samples of tracheal muscle at a mean concentration of 1.95 ng/g. When the effluent solution flowing past isolated tracheal muscle strips was assayed for VIP, samples collected during inhibitory nerve stimulation had much higher concentrations of the peptide than samples collected before stimulation, after stimulation, or during stimulation in the presence of tetrodotoxin (10(-6) mol/l). The VIP content of the effluent during control periods was 73.8 pg/ml, and during stimulation was 167.5 pg/ml. It is suggested that VIP might be the non-adrenergic inhibitory neurotransmitter in bovine tracheal smooth muscle.

Effect of food on the comparative pharmacokinetics of modified-release morphine tablet formulations: Oramorph SR and MST Continus.

The relative bioavailability and pharmacokinetic profiles of Oramorph SR (OSR) and MST Continus (MST), were evaluated by a randomized, four-way cross-over study in 24 healthy, male volunteers given single oral (30 mg) doses whilst fasting or after a high-fat breakfast. Mean Cmax, tmax, AUC (0,24h), AUC and tlag were significantly greater in fed compared with fasting subjects. Overall relative bioavailability of the two formulations (log AUC), was within the acceptable 80-125% limits for bioequivalence both fed and fasting. Mean fasting Cmax for OSR was greater than MST (P < 0.05) but there was no difference between formulations in mean fed Cmax. No statistically significant difference between OSR and MST was found for other parameters nor in the incidence of adverse events. These results suggest that OSR and MST are bioequivalent and that if patients were to transfer between formulations, dosage adjustment would be unnecessary, irrespective of their meal schedules or food intake.