Possibility of using salivary ultra-weak chemiluminescence as a biomarker for feelings of anxiety in hospital settings.

The aim of this study was to assess whether a particular value of noninvasive salivary ultra-weak chemiluminescence (UCL) could be used as a biomarker of psychological stress. Our study covered two groups. Group 1 comprised six healthy volunteers who stayed in a hospital for one night and group 2 comprised 15 patients with lung cancer and 24 patients with respiratory diseases other than lung cancer who were in hospital for an extended stay. First, we evaluated the UCL of saliva from six healthy volunteers before and after one night in hospital. Immunoglobulin A (IgA) concentrations were also measured. The integrated intensity value of UCL was correlated with the IgA concentration (correlation coefficient 0.90). Second, in the case of a long hospital stay, we found that the maximum salivary UCL intensities were higher in patients with lung cancer than in those with respiratory diseases other than lung cancer or in 28 healthy controls. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of drug crystallinity in aqueous suspension using terahertz time-domain attenuated total reflection spectroscopy.

Terahertz pulsed spectroscopy has recently been demonstrated to be a novel technique for the investigation of the solid-state properties of pharmaceutical materials. In this study, we directly measured the crystallinity of a drug suspended in water, using a terahertz pulsed attenuated total reflection (ATR) method. The dihydropyridine calcium channel blocker nifedipine is classified as a poorly soluble drug; its most stable crystalline form is known as form I. Transmission spectra, collected from 0.2 to 2.0 THz (6.6 to 66 cm(-1) ), of nifedipine crystals had a strong absorption peak at 1.2 THz (40 cm(-1) ) at room temperature. When the nifedipine crystals were mixed with poloxamer 188 and suspended in water, the resulting spectra measured using the ATR method had a peak at the same frequency as in the spectra obtained in transmission mode. Furthermore, the peak area was proportional to the amount of crystals. The upward sloping baseline in the spectra, corresponding to water absorption, decreased stepwise with increasing amounts of crystalline particles. We confirmed that the spectra gave excellent quantitative results, using partial least-squares regression analysis. The results suggest the possibility of using this method for qualitative and quantitative assessments of crystalline drugs in suspension.

Development of a saliva collection device for use in the field.

BACKGROUND: For monitoring biomarkers, saliva has several distinct advantages over other biological fluids. Saliva sampling is relatively non-invasive and enables the collection from either adults or infants under many different circumstances. However, there is no collection device that can be speedily used for analysis in the field. The aim of the present study was to evaluate the suitability of a new device, termed Muddler, compared with commercially available collection devices. METHODS: Saliva was collected from healthy volunteers. The collection devices such as Muddler, eye sponge, Salivette® Cotton (SC) and Salivette® Synthetic (SS) were evaluated in terms of the volume and/or composition of the collected saliva. The amounts of immunoglobulin A (IgA) and lactofferin in saliva were assessed by the enzyme-linked immunosorbent assays with the corresponding antibodies. Amylase activity was measured using a commercially available kit, and high molecular weight complexes including mucin were assessed by SDS-PAGE staining. RESULTS: A newly developed Muddler, which was made of plastic plate, was the best device for collecting a constant volume of saliva among all the devices examined in the present study. Furthermore, Muddler can collect without change in composition of salivary proteins such as IgA, lactoferrin, amylase, and mucin complex, whereas the levels of the salivary proteins obtained with all the commercial devices used were clearly different from those in original saliva. CONCLUSIONS: The newly developed Muddler was the best collection device in terms of the accuracy of collection and the reliability of measurements among all the devices examined in the present study.

Simultaneous monitoring of superoxides and intracellular calcium ions in neutrophils by chemiluminescence and fluorescence: evaluation of action mechanisms of bioactive compounds in foods.

We have developed a measuring system for simultaneous monitoring of chemiluminescence and fluorescence, which indicate respectively, (i) generation of superoxide anion radicals (O2(-•)) and (ii) change in the intracellular calcium ion concentration ([Ca(2+)]i) of neutrophils triggered by the mechanism of innate immune response. We applied this measuring system for establishing a method to distinguish between anti-inflammatory actions and antioxidant actions caused by bioactive compounds. We evaluated anti-inflammatory agents (zinc ion [Zn(2+)] and ibuprofen) and antioxidants (superoxide dismutase [SOD] and ascorbic acid). It was shown that ibuprofen and Zn(2+) were anti-inflammatory while SOD and ascorbic acid were anti-oxidative. We conclude that it is possible to determine the mechanism of action of bioactive compounds using this method.

Preparation of polymeric nanoparticles of cyclosporin A using infrared pulsed laser.

Nanoparticle formation of poorly water-soluble drugs is a means of providing much benefit for improving solubility and bioavailability. We showed that laser irradiation of drugs can be a novel tool for dispersing drug nanoparticles in water. Using our method, we were able to produce nanoparticles containing immunosuppressant drug, cyclosporin A, which shows poor solubility toward water, with high levels of the drug using polyvinyl pyrrolidone and sodium dodecyl sulfate as stabilizing agents. The absence of degradation products was confirmed and the loss of pharmaceutical activity with an inhibitory effect on the interleukin-2 production of Jurkat T cells did not occur. Cyclosporin A nanoparticles showed a spherical shape and their particle size was distributed uniformly around 200 nm. Powder X-ray diffraction analysis suggested that cyclosporin A in the nanoparticles was in an amorphous state. In the measurement of solubility rate, the nanoparticle formulation showed a higher rate than that which had not been processed. At present, although this laser irradiation technology has low productivity, it is expected as a new technology for drug nanoparticle manufacturing together with the development of a new laser device.

Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils--a promising candidate formulation for amnioinfusion fluid.

Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress.

Optical diagnosis of gastric cancer using near-infrared multichannel Raman spectroscopy with a 1064-nm excitation wavelength.

BACKGROUND: Gastric cancer is one of the most common cancers in Japan. The use of endoscopy is increasing, along with the number of histological examinations of specimens obtained by endoscopy. However, it takes several days to reach a diagnosis, which increases the medical expense. Raman spectroscopy is one of the available optical techniques, and the Raman spectrum for each molecule and tissue is characteristic and specific. The present study investigated whether Raman spectroscopy can be used to diagnose gastric cancer. METHODS: A total of 251 fresh biopsy specimens of gastric carcinoma and non-neoplastic mucosa were obtained from 49 gastric cancer patients at endoscopy. Without any pretreatment, the fresh specimens were measured with a near-infrared multichannel Raman spectroscopic system with an excitation wavelength of 1064 nm, and Raman spectra specific for the specimens were obtained. A principal component analysis (PCA) was performed to distinguish gastric cancer and non-neoplastic tissue, and a discriminant analysis was used to evaluate the accuracy of the gastric cancer diagnosis. RESULTS: The Raman spectra for cancer specimens differed from those for non-neoplastic specimens, especially at around 1644 cm(-1). Sensitivity was 66%, specificity was 73%, and accuracy was 70%. The accuracy of diagnosis using the single Raman scattering intensity at 1644 cm(-1) was 70%, consistent with the PCA result. CONCLUSIONS: The present results indicate that near-infrared multichannel Raman spectroscopy with a 1064-nm excitation wavelength is useful for gastric cancer diagnosis. Establishment of a Raman diagnostic system for gastric cancer may improve the clinical diagnosis of gastric cancer and be beneficial for patients.

Comparison of peroxidase response to mental arithmetic stress in saliva of smokers and non-smokers.

Saliva is the first body fluid to encounter exogenous materials or gases such as cigarette smoke (CS). The aim of this study was to examine whether smoking affects oral peroxidase (OPO) reactivity to mental stress. The subjects were 39 non-smokers and 10 smokers. In the experiment, the Kraepelin psychodiagnostic test as a psychological stressor and saliva was sampled 30 min before, just before, immediately after, and 30 min after the beginning of the test. OPO reactivity to the test between smokers and non-smokers was measured in addition to uric acid concentration, flow rate, IgA, thiocyanate (SCN-) concentration, amylase activity as a salivary stress marker, and ultra-weak chemiluminescence (UCL) level, which is indicative of salivary antioxidative and antibacterial abilities. Moreover, we studied the effect of smoking on the response of salivary peroxidase (SPO) and myeloperoxidase (MPO) activity to mental stress, respectively. The results showed that the IgA concentration, amylase activity, SCN(- concentration, and UCL level are higher in the non-smoking group than smoking group and the IgA concentration and UCL level increased in the non-smokers significantly just after the Kraepelin test. The levels of SCN-) were higher in smokers than in non-smokers and OPO activity was greater in the non-smoking group in all sessions. Furthermore, only the non-smokers had significantly increased MPO activity just after the test. MPO may play a crucial role in the response to acute psychological stress besides inflammation, and CS suppresses this response significantly.

Simultaneous measurement of superoxide generation and intracellular Ca2+ concentration reveals the effect of extracellular Ca2+ on rapid and transient contents of superoxide generation in differentiated THP-1 cells.

We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca2+ concentration ([Ca2+]i) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated superoxide response occurred without change in [Ca2+]i. The cells treated with TMB-8, an intracellular Ca2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca2+]i elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca2+]i elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca2+ influx from the extracellular domain. Verapamil, voltage-dependent Ca2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca2+ release or extracellular Ca2+ influx, respectively. Taking these results together, the characteristic extracellular Ca2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca2+]i.

Simultaneous measurement of superoxide generation and intracellular calcium ion of neutrophil-like culture cells.

We have developed a novel method for simultaneously measuring fluorescence and chemiluminescence. The generation of superoxide anion and the intracellular Ca(2+) ion concentration of neutrophil-like cells stimulated by agonists were measured in real time by our method. Our results were in agreement with the intracellular signalling in the neutrophils. We also found that the presence of Zn(2+) ion inhibited both the generation of superoxide anion and the influx of Ca(2+) ions.

Ultra-weak photon emission from human hand: influence of temperature and oxygen concentration on emission.

We have studied ultra-weak photon emission (UPE) from living organisms. We report here some features of the UPE from human hand by means of photon counting techniques. The intensity of the UPE depended on the position of human hand; nail>finger>palm. As the temperature declined, the intensity of the UPE from the palm decreased. Further, as oxygen concentration around the palm was lowered, the intensity of the UPE from the palm decreased. These results show the UPE from the palm partly contains emissions based on oxidation reaction on skin surface as a potential. When we used mineral oil between the photomultiplier tube and the palm, the intensity of the UPE increased twice as much, which indicates the UPE from the inside of the skin certainly exists. The fact may be explained by refractive index matching. As mentioned above, we considered the generation mechanism of photons emitted from the human hand.