A study of some constituents in human female saliva

Changes in the concentration of some constituents in women's saliva during the menstrual cycle were studied. Saliva was used because it is easier to collect than other body fluids and is continuously available for analysis. Glucose, the enzyme 17-Acetyl-D-glucosaminidase (NAG) and Calcium which are saliva constituents and belong to three different chemical groups were selected for the study. Several analytical techniques were investigated. The fluorometric assay procedure was found to be the best because of its specificity and sensitivity for the estimation of these constituents. resides the fluorametric method a spectrophotometric method was used in the NAG determination and an atomic absorption method in the calcium estimation. Glucose was estimated by an enzymatic method. This is based on the reaction of glucose with the enzymes glucose oxidase and peroxidase to yield hydrogen peroxide, which in turn oxidises a non-fluorescent substrate, p-hydroxyphenylacetic acid, to a highly fluorescent product. The saliva samples in this determination had to be centrifuged at high speed, heated in a boiling water bath, centrifuged again and then treated with a mixture of cation and anion resins to remove the substances that inhibited the enzyme system. In the determination of the NAG activity the saliva samples were diluted with citric acid/phosphate buffer, and then centrifuged at high speed. The assay was based on the enzymic hydrolysis of the non-fluorescent substrate 4-Methyl-umbelli1eryl-p-D-glucosaminide to the highly fluorescent 4-Methyl-umbelliferone• Calcium was estimated by a fluorometric procedure based upon the measurement of the fluorescence produced by the complex formed between calcein blue and calcium, at pH 9 - 13. From the results obtained from the analysis of saliva samples of several women it was found that glucose showed a significant increase in its level around the expected time of ovulation. This was found in seven cycles out of ten. Similar results were found with the enzyme NAG. No significant change in the calcium levels was observe& at any particular time of the cycle. The levels of the glucose, the activity of the enzyme NAG and the concentration of the calcium were found to change daily, and to differ from one subject to another and in the same subject from cycle to cycle. The increase observed it salivary glucose levels and the enzyme NAG activity could be monitored to predict the time of ovulation.

The release of nicotine from chewing gum formulations

The first clinically proven nicotine replacement product to obtain regulatory approval was Nicorette® gum. It provides a convenient way of delivering nicotine directly to the buccal cavity, thus, circumventing 'first-pass' elimination following gastrointestinal absorption. Since launch, Nicorette® gum has been investigated in numerous studies (clinical) which are often difficult to compare due to large variations in study design and degree of sophistication. In order to standardise testing, in 2000 the European Pharmacopoeia introduced an apparatus to investigate the in vitro release of drug substances from medical chewing gum. With use of the chewing machine, the main aims of this project were to determine factors that could affect release from Nicorette® gum, to develop an in vitro in vivo correlation and to investigate formulation variables on release of nicotine from gums. A standard in vitro test method was developed. The gum was placed in the chewing chamber with 40 mL of artificial saliva at 37'C and chewed at 60 chews per minute. The chew rate, the type of dissolution medium used, pH, volume, temperature and the ionic strength of the dissolution medium were altered to investigate the effects on release in vitro. It was found that increasing the temperature of the dissolution media and the rate at which the gums were chewed resulted in a greater release of nicotine, whilst increasing the ionic strength of the dissolution medium to 80 mM resulted in a lower release. The addition of 0.1 % sodium Jauryl sulphate to the artificial saliva was found to double the release of nicotine compared to the use of artificial saliva and water alone. Although altering the dissolution volume and the starting pH did not affect the release. The increase in pH may be insufficient to provide optimal conditions for nicotine absorption (since the rate at which nicotine is transported through the buccal membrane was found to be higher at pH values greater than 8.6 where nicotine is predominately unionised). Using a time mapping function, it was also possible to establish a level A in vitro in vivo correlation. 4 mg Nicorette® gum was chewed at various chew rates in vitro and correlated to an in vivo chew-out study. All chew rates used in vitro could be successfully used for IVIVC purposes, however statistically, chew rates of 10 and 20 chews per minute performed better than all other chew rates. Finally a series of nicotine gums was made to investigate the effect of formulation variables on release of nicotine from the gum. Using a directly compressible gum base, in comparison to Nicorette® the gums crumbled when chewed in vitro, resulting in a faster release of nicotine. To investigate the effect of altering the gum base, the concentration of sodium salts, sugar syrup, the form of the active drug, the addition sequence and the incorporation of surfactant into the gum, the traditional manufacturing method was used to make a series of gum formulations. Results showed that the time of addition of the active drug, the incorporation of surfactants and using different gum base all increased the release of nicotine from the gum. In contrast, reducing the concentration of sodium carbonate resulted in a lower release. Using a stronger nicotine ion-exchange resin delayed the release of nicotine from the gum, whilst altering the concentration of sugar syrup had little effect on the release but altered the texture of the gum.

Bioequivalence of sustained release theophylline formulations

The bioequivalence of sustained release theophylline formulations, marketed in the United Kingdom, has been investigated in relation to the co-administration of food in both single dose and steady state volunteer studies. The effect of food on pharmacokinetic parameters and their clinical relevance was researched. Experimentation using drug induced modification of gastric motility to ascertain the component influences of the rate of gastric emptying on the absorption of theophylline from sustained release formulations was conducted. Prolongation of time to maximum plasma theophylline concentration by food reported in the literature and its clinical importance was investigated in once daily compared with twice daily administration of sustained release theophylline formulations and smoking habit. The correlation between saliva and plasma theophylline concentrations as a means of developing a non-invasive sampling techniques was examined. Data obtained from in vitro dissolution studies was compared with in vivo results. This thesis has shown no significant differences occurred in the pharmacokinetic parameters measured between sustained release formulations available in the United Kingdom. The investigations into the influence of food on prolongation of time to maximum plasma theophylline concentration and other measured pharmacokinetic parameters demonstrated no important pharmacokinetic or clinical effects. Smoking adults taking sustained release theophylline formulations had similar drug clearances to those reported in the literature for smokers taking plain uncoated theophylline formulations. KEY WORDS Bioequivalence Theophylline Sustained Release Food Pharmacokinetics RONALD PURKISS SUBMITTED FOR

A structural investigation of the alkali metal site distribution within bioactive glass using neutron diffraction and multinuclear solid state NMR

The atomic-scale structure of Bioglass and the effect of substituting lithium for sodium within these glasses have been investigated using neutron diffraction and solid state magic angle spinning (MAS) NMR. Applying an effective isomorphic substitution difference function to the neutron diffraction data has enabled the Na-O and Li-O nearest-neighbour correlations to be isolated from the overlapping Ca-O, O-(P)-O and O-(Si)-O correlations. These results reveal that Na and Li behave in a similar manner within the glassy matrix and do not disrupt the short range order of the network former. Residual differences are attributed solely to the variation in ionic radius between the two species. Successful simplification of the 2 saliva, and hence to the bioactivity of the material. Detailed structural knowledge is therefore a prerequisite for optimizing material design.

An examination of the calcium and strontium site distribution in bioactive glasses through isomorphic neutron diffraction, X-ray diffraction, EXAFS and multinuclear solid state NMR.

Strontium has been substituted for calcium in the glass series (SiO2)49.46(Na2O)26.38(P2O5)1.07(CaO)23.08x(SrO)x (where x = 0, 11.54, 23.08) to elucidate their underlying atomic-scale structural characteristics as a basis for understanding features related to the bioactivity. These bioactive glasses have been investigated using isomorphic neutron and X-ray diffraction, Sr K-edge EXAFS and solid state 17O, 23Na, 29Si, 31P and 43Ca magic-angle-spinning (MAS) NMR. An effective isomorphic substitution first-order difference function has been applied to the neutron diffraction data, confirming that Ca and Sr behave in a similar manner within the glass network, with residual differences attributed to solely the variation in ionic radius between the two species. The diffraction data provides the first direct experimental evidence of split Ca–O nearest-neighbour correlations in these melt quench bioactive glasses, together with an analogous splitting of the Sr–O correlations; the correlations are attributed to the metal ions correlated either to bridging or to non-bridging oxygen atoms. Triple quantum (3Q) 43Ca MAS NMR corroborates the split Ca–O correlations. Successful simplification of the 2 < r (A) < 3 region via the difference method has also revealed two distinct Na environments. These environments are attributed to sodium correlated either to bridging or to nonbridging oxygen atoms. Complementary multinuclear MAS NMR, Sr K-edge EXAFS and X-ray diffraction data supports the structural model presented. The structural sites present will be intimately related to their release properties in physiological fluids such as plasma and saliva, and hence the bioactivity of the material. Detailed structural knowledge is therefore a prerequisite for optimising material design.

Structural characterisation of hypoxia-mimicking bioactive glasses

Nickel and cobalt are both known to stimulate the hypoxia-inducible factor-1 (HIF-1a), thus significantly improving blood vessel formation in tissue engineering applications. We have manufactured nickel and cobalt doped bioactive glasses to act as a controlled delivery mechanism of these ions. The resultant structural consequences have been investigated using the methods of isotopic and isomorphic substitution applied to neutron diffraction. The structural sites present will be intimately related to their release properties in physiological fluids such as plasma and saliva, and hence the bioactivity of the material. Detailed structural knowledge is therefore a prerequisite for optimising material design. Results show that nickel and cobalt adopt a mixed structural role within these bioactive glasses occupying both network-forming (tetrahedral) and network-modifying (5-fold) geometries. Two thirds of the Ni (or Co) occupies a five-fold geometry with the remaining third in a tetrahedral environment. A direct comparison of the primary structural correlations (e.g. Si-O, Ca-O, Na-O and O-Si-O) between the archetypal 45S5 Bioglass® and the Ni and Co glasses studied here reveal no significant differences. This indicates that the addition of Ni (or Co) will have no adverse effects on the existing structure, and thus on in vitro/in vivo dissolution rates and therefore bioactivity of these glasses.

Cortisol - ein geeigneter physiologischer Indikator für Belastungen am Arbeitsplatz?

Die vorliegende Studie prüft Zusammenhänge zwischen Arbeitsintensität, Tätigkeitsspielraum, sozialer Arbeitsumgebung (Kooperation/Kommunikation, soziale Unterstützung, soziale Stressoren) und Stresserleben am Arbeitsplatz mit der basalen Cortisolsekretion im Speichel (Tagesprofil, Aufwachreaktion und Variation über den Tag). Insgesamt 46 Erwerbstätige aus dem Bankwesen sammelten an zwei aufeinander folgenden Arbeitstagen je vier Speichelproben (beim Aufwachen, 30 min nach dem Aufwachen, 14 Uhr und unmittelbar vor dem Zubettgehen), aus denen die individuelle Cortisolkonzentration (Mittelwert aus den jeweils zugehörigen Proben) bestimmt wurde. Die Tätigkeitsmerkmale wurden sowohl mittels Fragebögen als auch objektiv, d.?h. unabhängig vom Arbeitsplatzinhaber, erhoben. Alter, Geschlecht, Rauchen, Body-Mass-Index, gesundheitliche Beeinträchtigungen sowie eventuelle Abweichungen bei der Probensammlung wurden als mögliche Drittvariablen berücksichtigt. Im Ergebnis zeigte sich, dass subjektiv erlebte, geringe soziale Unterstützung und hohe soziale Stressoren mit einer erhöhten Aufwachreaktion bzw. mit einer erhöhten Variation über den Tag assoziiert waren. Für die Arbeitsintensität, den Tätigkeitsspielraum sowie für die objektiv erhobene Kooperation/Kommunikation waren keine Effekte nachweisbar. Die Ergebnisse lassen vermuten, dass sowohl die Belastungs- als auch deren Erhebungsart für den Nachweis von Effekten im Hinblick auf die Cortisolsekretion bei Erwerbstätigen von Bedeutung sind. The present study examines associations between job demands, job control, social work environment (cooperation/communication, social support, social stressors), and strain at work with basal salivary cortisol (day profiles, cortisol awakening reaction, diurnal variation). Forty-six employees collected four saliva samples (immediately after waking up, 30 min after waking up, at 2 p.m. and immediately before going to bed) each on two consecutive working days. We computed the mean across the two days for each of the four saliva samples per employee. Job characteristics were assessed by self-reports as well as by objective job analysis. Analyses were controlled for possible confounding effects of age, gender, smoking, body-mass index, health impairments, and non compliance with the cortisol protocol. Results show that subjectively experienced low social support and high social stressors at work were associated with elevated cortisol awakening reaction and elevated diurnal variation. We found no effects for job demands, job control or objectively assessed cooperation/communication. Our results suggest that both the type of job characteristic as well as the type of measurement of job characteristics have to be taken into account when relating them to employees’ cortisol secretion.

Weight loss strategies, stress, and cognitive function:supervised versus unsupervised dieting

The early stages of dieting to lose weight have been associated with neuro-psychological impairments. Previous work has not elucidated whether these impairments are a function solely of unsupported or supported dieting. Raised cortico-steroid levels have been implicated as a possible causal mechanism. Healthy, overweight, pre-menopausal women were randomised to one of three conditions in which they dieted either as part of a commercially available weight loss group, dieted without any group support or acted as non-dieting controls for 8 weeks. Testing occurred at baseline and at 1, 4 and 8 weeks post baseline. During each session, participants completed measures of simple reaction time, motor speed, vigilance, immediate verbal recall, visuo-spatial processing and (at Week 1 only) executive function. Cortisol levels were gathered at the beginning and 30 min into each test session, via saliva samples. Also, food intake was self-recorded prior to each session and fasting body weight and percentage body fat were measured at each session. Participants in the unsupported diet condition displayed poorer vigilance performance (p=0.001) and impaired executive planning function (p=0.013) (along with a marginally significant trend for poorer visual recall (p=0.089)) after 1 week of dieting. No such impairments were observed in the other two groups. In addition, the unsupported dieters experienced a significant rise in salivary cortisol levels after 1 week of dieting (p<0.001). Both dieting groups lost roughly the same amount of body mass (p=0.011) over the course of the 8 weeks of dieting, although only the unsupported dieters experienced a significant drop in percentage body fat over the course of dieting (p=0.016). The precise causal nature of the relationship between stress, cortisol, unsupported dieting and cognitive function is, however, uncertain and should be the focus of further research. © 2005 Elsevier Ltd. All rights reserved.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

1 Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2 In the presence of indomethacin (3μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0 ± 0.1, n = 5). 3 Establishment of a rank order of potency using the EP4 > EP2 agonist 11-deoxy PGE1, and the EP2 > EP4 agonist PGE1-OH (mean pEC 50 of 7.6 ± 0.1 (n = 6) and 6.4 ± 0.1 (n = 4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations < 1 μM failed to relax the tissues. 4 Blockade of EP 4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration-response curves, exhibiting pA2 and pKB values of 5.7 ± 0.1, n = 3, and 8.4, n = 3, respectively. 5 The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3 ± 0.1 (n = 4) and 8.1 ± 0.1 (n = 9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGFα2 failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4 ± 0.3, n = 3). 6 These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

International session at the BCLA conference

Full text: It seems a long time ago now since we were at the BCLA conference. The excellent FIFA World Cup in Brazil kept us occupied over the summer as well as Formula 1, Wimbledon, Tour de France, Commonwealth Games and of course exam paper marking! The BCLA conference this year was held in Birmingham at the International Convention Centre which again proved to be a great venue. The number of attendees overall was up on previous years, and at a record high of 1500 people. Amongst the highlights at this year's annual conference was the live surgery link where Professor Sunil Shah demonstrated the differences in technique between traditional phacoemulsification cataract surgery and femtosecond assisted phacoemulsification cataract surgery. Dr. Raquel Gil Cazorla, a research optometrist at Aston University, assisted in the procedure including calibrating the femtosecond laser. Another highlight for me was the session that I chaired, which was the international session organised by IACLE (International Association of CL Educators). There was a talk by Mirjam van Tilborg about dry eye prevalence in the Netherlands and how it was managed by medical general practitioners (GPs) or optometrists. It was interesting to know that there are only 2 schools of optometry there and currently under 1000 registered optometrists there. It also seems that GPs were more likely to blame CL as the cause for dry eye whereas optometrists who had a fuller range of tests were better at solving the issue. The next part of the session included the presentation of 5 selected posters from around the world. The posters were also displayed in the main poster area but were selected to be presented here as they had international relevance. The posters were: 1. Motivators and Barriers for Contact Lens Recommendation and Wear by Nilesh Thite (India) 2. Contact lens hygiene among Saudi wearers by Dr. Ali Masmaly (Saudi) 3. Trends of contact lens prescribing and patterns of contact lens practice in Jordan by Dr. Mera Haddad (Jordan) 4. Contact Lens Behaviour in Greece by Dr. Dimitra Makrynioti (Greece) 5. How practitioners inform ametropes about the benefits of contact lenses and overcome the potential barriers: an Italian survey, by Dr. Fabrizio Zeri (Italy) It was interesting to learn about CL practice in different parts, for example the CL wearing population ration in Saudi Arabia is around 1:2 Male:Female (similar to other parts of the world) and although the sale of CL is restricted to registered practitioners there are many unregistered outlets, like clothing stores, that flout the rules. In Jordan some older practitioners will still advise patients to use tap water or even saliva! But thankfully the newer generation of practitioners have been educated not to advise this. In Greece one of the concerns was that some practitioners may advise patients to use disposable lenses for longer than they should and again it seems to be the practitioners with inadequate education that would do this. In India it was found that cost was one barrier to using contact lenses but also some practitioners felt that they shouldn’t offer CLs due to cost too. In Italy sensitive eyes and CL care and maintenance were the barriers to CL wear but the motivators were vision and comfort and aesthetics. Finally the international session ended with the IACLE travel award and educator awards presented by IACLE president Shehzad Naroo and BCLA President Andrew Yorke. The travel award went to Wang Ling, Jinling Institute of Technology, Nanjing, China. There were 3 regional Contact Lens Educator of the Year Awards sponsored by Coopervision and presented by Dr. J.C. Aragorn of Coopervision. 1. Asia Pacific Region – Dr. Rajeswari Mahadevan of Sankara Nethralaya Medical Research Foundation, Chennai, India 2. Americas Region – Dr. Sergio Garcia of University of La Salle, Bogotá and the University Santo Tomás, Bucaramanga, Colombia 3. Europe/Africa – Middle East Region: Dr. Eef van der Worp, affiliated with the University of Maastricht, the Netherlands The posters above were just a small selection of those displayed at this year's BCLA conference. If you missed the BCLA conference then you can see the abstracts for all posters and talks in a virtual issue of CLAE very soon. The poster competition was kindly sponsored by Elsevier. The poster winner this year was: Joan Gispets – Corneal and Anterior Chamber Parameters in Keratoconus The 3 runners up were: Debby Yeung – Scleral Lens Central Corneal Clearance Assessment with Biomicroscopy Sarah L. Smith – Subjective Grading of Lid Margin Staining Heiko Pult – Impact of Soft Contact Lenses on Lid Parallel Conjunctival Folds My final two highlights are a little more personal. Firstly, I was awarded Honorary Life Fellowship of the BCLA for my work with the Journal, and I would like to thank the BCLA, Elsevier, the editorial board of CLAE, the reviewers and the authors for their support of my role. My final highlight from the BCLA conference this year was the final presentation of the conference – the BCLA Gold Medal award. The recipient this year was Professor Philip Morgan with his talk ‘Changing the world with contact lenses’. Phil was the person who advised me to go to my first BCLA conference in 1994 (funnily he didn’t attend himself as he was busy getting married!) and now 20 years later he was being honoured with the accolade of being the BCLA Gold Medallist. The date of his BCLA medal addressed was shared with his father's birthday so a double celebration for Phil. Well done to outgoing BCLA President Andy Yorke and his team at the BCLA (including Nick Rumney, Cheryl Donnelly, Sarah Greenwood and Amir Khan) on an excellent conference. And finally welcome to new President Susan Bowers. Copyright © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Plasma membrane abundance of human aquaporin 5 is dynamically regulated by multiple pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.