Combined assessment of lysine and N-acetyl cadaverine levels assist as a potential biomarker of the smoker periodontitis.

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers.

Electrified liquid-liquid interface as an electrochemical tool for the sensing of putrescine and cadaverine.

Putrescine and cadaverine are biogenic amines that serve as potential biomarkers for several types of cancers and monitoring food quality. Electrochemical sensing of putrescine and cadaverine by non-enzymatic routes remains a challenge because of their inertness at unmodified electrode surfaces and hence a liquid-liquid interface strategy has been employed for their detection. In the present study, electrochemical sensing of cadaverine and putrescine has been demonstrated by simple and facilitated ion-transfer processes using a liquid-liquid microinterface supported by a microcapillary. A microinterface was constructed in different configurations by varying the aqueous phase composition in the absence and presence of dibenzo-18-crown-6, and the ion-transfer ability of putrescine and cadaverine was studied in these configurations. A peak shaped voltammogram was observed in the backward scan, due to the linear diffusion of putrescine and cadaverine from the organic to the aqueous phase. The detection ability in the presence of dibenzo-18-crown-6 was observed in the concentration ranges of 0.25-25 µM and 0.25-40 µM for putrescine and cadaverine with detection limits of 0.11 and 0.17 µM respectively. In the presence of dibenzo-18-crown-6, the electrochemical sensing of putrescine and cadaverine was more pronounced compared to the simple ion-transfer process.

Exclusive enteral nutrition mediates gut microbial and metabolic changes that are associated with remission in children with Crohn's disease.

A nutritional intervention, exclusive enteral nutrition (EEN) can induce remission in patients with pediatric Crohn's disease (CD). We characterized changes in the fecal microbiota and metabolome to identify the mechanism of EEN. Feces of 43 children were collected prior, during and after EEN. Microbiota and metabolites were analyzed by 16S rRNA gene amplicon sequencing and NMR. Selected metabolites were evaluated in relevant model systems. Microbiota and metabolome of patients with CD and controls were different at all time points. Amino acids, primary bile salts, trimethylamine and cadaverine were elevated in patients with CD. Microbiota and metabolome differed between responders and non-responders prior to EEN. EEN decreased microbiota diversity and reduced amino acids, trimethylamine and cadaverine towards control levels. Patients with CD had reduced microbial metabolism of bile acids that partially normalized during EEN. Trimethylamine and cadaverine inhibited intestinal cell growth. TMA and cadaverine inhibited LPS-stimulated TNF-alpha and IL-6 secretion by primary human monocytes. A diet rich in free amino acids worsened inflammation in the DSS model of intestinal inflammation. Trimethylamine, cadaverine, bile salts and amino acids could play a role in the mechanism by which EEN induces remission. Prior to EEN, microbiota and metabolome are different between responders and non-responders.

Biogenic amine content during the production and ripening of Sremski kulen, Serbian traditional dry fermented sausage.

Sremski kulen is a wide diameter dry fermented sausage, produced from pork, seasoned with red spicy paprika, stuffed into pork cecum, and preserved by smoking, fermentation and drying. Due to specific ripening process, Sremski kulen is suitable for the accumulation of biogenic amines. Therefore, the aminogenesis was studied in traditionally produced Sremski kulen, taking into account the physicochemical parameters and microbial counts. The content of six biogenic amines (tryptamine, phenylethylamine, tyramine, histamine, putrescine, and cadaverine) was analyzed using high-performance liquid chromatography. The ripening process of Sremski kulen was slow followed by changes in aw and pH value as well as expressed proteolysis. The autochthonous microbiota showed pronounced decarboxylase activity. Tryptamine and phenylethylamine were detected at each examined ripening stage while histamine was not detected until the end of ripening (16.55 ± 2.33 mg/kg). Tyramine, cadaverine, and putrescine content significantly increased during the ripening period (p < .05). In the final product, cadaverine was the dominant biogenic amine (132.40 ± 5.05 mg/kg), followed by tyramine (115.80 ± 15.46 mg/kg) and putrescine (68.55 ± 2.39 mg/kg). Although the long ripening period greatly contributed to the accumulation of biogenic amines in final product, their content are not of concern from product safety aspects, but requires improvement in hygiene of production process.

Ultrasensitive, Selective, and Highly Stable Bioelectronic Nose That Detects the Liquid and Gaseous Cadaverine.

Field-effect transistor (FET) devices based on conductive nanomaterials have been used to develop biosensors. However, development of FET-based biosensors that allow efficient stability, especially in the gas phase, for obtaining reliable and reproducible responses remains a challenge. In this study, we developed a nanodisc (ND)-functionalized bioelectronic nose (NBN) based on a nickel (Ni)-decorated carboxylated polypyrrole nanoparticle (cPPyNP)-FET that offers the detection of liquid and gaseous cadaverine (CV). The TAAR13c, specifically binding to CV, which is an indicator of food spoilage, was successfully constructed in NDs. The NBN was fabricated by the oriented assembly of TAAR13c-embedded NDs (T13NDs) onto the transistor with Ni/cPPyNPs. The NBN showed high performance in selectivity and sensitivity for the detection of CV, with excellent stability in both aqueous and gas phases. Moreover, the NBN allowed efficient measurement of corrupted real-food samples. It demonstrates the ND-based device can allow the practical biosensor that provides high stability in the gas phase.

Identification and characterization of L-lysine decarboxylase from Huperzia serrata and its role in the metabolic pathway of lycopodium alkaloid.

Lysine decarboxylation is the first biosynthetic step of Huperzine A (HupA). Six cDNAs encoding lysine decarboxylases (LDCs) were cloned from Huperzia serrata by degenerate PCR and rapid amplification of cDNA ends (RACE). One HsLDC isoform was functionally characterized as lysine decarboxylase. The HsLDC exhibited greatest catalytic efficiency (kcat/Km, 2.11 s-1 mM-1) toward L-lysine in vitro among all reported plant-LDCs. Moreover, transient expression of the HsLDC in tobacco leaves specifically increased cadaverine content from zero to 0.75 mg per gram of dry mass. Additionally, a convenient and reliable method used to detect the two catalytic products was developed. With the novel method, the enzymatic products of HsLDC and HsCAO, namely cadaverine and 5-aminopentanal, respectively, were detected simultaneously both in assay with purified enzymes and in transgenic tobacco leaves. This work not only provides direct evidence of the first two-step in biosynthetic pathway of HupA in Huperzia serrata and paves the way for further elucidation of the pathway, but also enables engineering heterologous production of HupA.

Low Biofilm Lysine Content in Refractory Chronic Periodontitis.

BACKGROUND: Chronic periodontitis is controlled without antibiotics by scaling and root planing (SRP) to remove dental biofilm. It has been previously reported that the epithelial barrier to bacterial proinflammatory products is impaired when biofilm lysine falls below the minimal content of normal blood plasma. Aims were to examine whether being refractory and requiring antibiotics to supplement SRP were associated with low biofilm lysine contents. METHODS: Sixteen patients with periodontitis and six periodontally healthy volunteers (HVs) (respective mean ages: 57 ± 6 and 36 ± 8 years) were examined. Patients with periodontitis received SRP and surgery, and HVs received prophylaxis. At quarterly maintenance or prophylaxis visits during the subsequent year, therapeutic response was good (GR, n = 9) or poor (PR, n = 7; including five cigarette smokers). Biofilm cadaverine, lysine, and other amino acid (AA) contents were determined by liquid chromatography. Cadaverine mole fraction of lysine plus cadaverine (CF) indicated biofilm lysine decarboxylase activity. RESULTS: Biofilm lysine was 0.19 ± 0.10 and 0.20 ± 0.09 µmol/mg in GRs and HVs, but 0.07 ± 0.03 µmol/mg in PRs (Kruskal-Wallis: P <0.01). All AAs were depleted in biofilm from smokers, but only lysine was depleted in biofilm from non-smokers. CF was inversely associated with clinical attachment level (CAL) at baseline before therapy in all patients (R2 = 0.28, P <0.01) and with CAL change after therapy in GR (R2 = 0.49, P <0.05). Lysine and cadaverine contents discriminated PRs from GRs and HVs (Wilks' λ = 0.499, P <0.012). CONCLUSIONS: Refractory responses requiring antibiotic therapy result from smoking and/or microbial infections that starve the biofilm and epithelial attachment of lysine. Biofilm CF is associated with periodontitis severity pretherapy and extent of therapeutic response post-therapy.

Changes in Biogenic Amines and ATP-Related Compounds and Their Relation to Other Quality Changes in Common Carp (Cyprinus carpio var. Jian) Stored at 20 and 0°C.

Biogenic amines, ATP-related compounds, sensory attributes, total volatile basic nitrogen, microbial flora (total viable bacteria, Pseudomonas, Aeromonas, and H2S-producing bacteria), and free amino acids were determined in common carp (Cyprinus carpio var. Jian) stored at 20 and 0°C. Pseudomonas and H2S-producing bacteria became the dominant bacteria in carp stored at 20 and 0°C, whereas Aeromonas rapidly increased only in carp stored at 0°C. Inosine monophosphate, which is responsible for flavor and freshness, increased to a maximum of 2.37 l mol/g after 12 h at 20°C and to 4.72 l mol/g after 3 days at 0°C. Putrescine and cadaverine were the dominant amines in carp and their concentrations were significantly correlated (P < 0.05) with total volatile basic nitrogen and sensory scores in all samples during the storage. Significant correlations also were observed between histamine and total volatile basic nitrogen and sensory scores only in samples stored at 20°C. Arginine decreased while putrescine increased in all samples. A significant decrease (P < 0.05) in histidine was observed after 24 h of storage, which coincided with an increase in histamine after 36 h in samples stored at 20°C. Hypoxanthine concentrations were significantly correlated with the microbial species (P < 0.01) and sensory scores (P < 0.05) and seems to be a reliable marker for quality of carp fillets stored at 20 and 0°C.

Polyamines in conventional and organic vegetables exposed to exogenous ethylene.

Relationships between endogenous levels of polyamines by thin layer chromatography (TLC) and gas chromatography (GC), nitrate and response to the application of ethylene were established between organic and conventional vegetables (broccoli, collard greens, carrots and beets), both raw and cooked. Responses to ethylene showed that organic plants were less responsive to the growth regulator. The levels of free polyamines obtained by TLC were higher in organic vegetables. Organic broccoli showed higher levels of putrescine (Put), and cooking resulted in lowering the overall content of these amines. Conventional collard green showed the highest level of putrescine in the leaves compared with organic. Tubers of carrots and beets contain the highest levels of Put. These plants also contain high levels of spermine. GC analysis showed the highest polyamines contents compared with those obtained by TLC. Cooking process decreased putrescine and cadaverine content, both in conventionally and organically grown vegetables. Organic beets contain lower NO3(-) compared with its conventional counterpart.

Determination of trans- and cis-urocanic acid in relation to histamine, putrescine, and cadaverine contents in tuna (Auxis Thazard) at different storage temperatures.

Scombroid fish poisoning is usually associated with consumption of fish containing high levels of histamine. However, reports indicate that some cases have responded to antihistamine therapy while ingested histamine levels in these cases were low. Potentiation of histamine toxicity by some biogenic amines, and release of endogenous histamine by other compounds such as cis-urocanic acid (UCA) are some hypotheses that have been put forth to explain this anomaly. Very little is known about the effects of storage conditions on the production of both UCA isomers and biogenic amines in tuna. Thus, the production of trans- and cis-UCA, histamine, putrescine, and cadaverine in tuna during 15 d of storage at 0, 3, and 10 °C and 2 d storage at ambient temperature were monitored. The initial trans- and cis-UCA contents in fresh tuna were 2.90 and 1.47 mg/kg, respectively, whereas the levels of putrescine and cadaverine were less than 2 mg/kg, and histamine was not detected. The highest levels of trans- and cis-UCA were obtained during 15 d storage at 3 °C (23.74 and 21.79 mg/kg, respectively) while the highest concentrations of histamine (2796 mg/kg), putrescine (220.32 mg/kg) and cadaverine (1045.20 mg/kg) were obtained during storage at room temperature, 10 and 10 °C, respectively. Histamine content increased considerably during storage at 10 °C whereas trans- and cis-UCA contents changed slightly. The initial trans-UCA content decreased during storage at ambient temperature. Thus, unlike histamine, concentrations of trans- and cis-UCA did not result in elevated levels during storage of tuna.

Determination of pork spoilage by colorimetric gas sensor array based on natural pigments.

A new colorimetric gas-sensor array based on four natural pigments, that were extracted from spinach (Spinacia oleracea), red radish (Raphanus sativus L.), winter jasmine (Jasminum nudiflorum), and black rice (Oryza sativa L. indica), was developed for pork freshness evaluation. A colour change profile for each sample was obtained by differentiating the images of the sensor array before and after exposure to the odour of sample. The total viable count (TVC) per gram of pork was obtained by classical microbiological plating methods, and the biogenic amines were measured by HPLC. Biogenic amine index (BAI) for the determination of meat freshness was developed from the sum of putrescine and cadaverine. The colour change profiles were analysed using principal component analysis and correlated with conventional methods (BAI, TVC). A partial least squares (PLS) prediction model was obtained with r=0.854 and 0.933 for BAI and TVC, respectively.

The occurrence of N-nitrosamines, residual nitrite and biogenic amines in commercial dry fermented sausages and evaluation of their occasional relation.

Regarding food borne intoxications, the accumulation of biogenic amines must be avoided in all kinds of food products. Moreover, biogenic amines can function as precursors for the formation of carcinogenic N-nitrosamines when nitrite is present. To estimate the food safety of the dry fermented sausages available on the Belgian market, a screening of the residual sodium nitrite and nitrate contents, biogenic amines and volatile N-nitrosamine concentrations was performed on 101 samples. The median concentrations of residual NaNO2 and NaNO3 were each individually lower than 20mg/kg. In general, the biogenic amine accumulation remained low at the end of shelf life. Only in one product the amounts of cadaverine and putrescine reached intoxicating levels. Concerning the occurrence of N-nitrosamines, only N-nitrosopiperidine and N-nitrosomorpholine were detected in a high number of samples (resp. 22% and 28%). No correlation between the presence of N-nitrosamines and the biogenic amines content was observed. Although the N-nitrosamines could not been linked to specific product categories, the occurrence of N-nitrosopiperidine could probably be attributed to the use of pepper.

[Halitosis concomitant with chronic ENT pathology in children].

The objective of the present study was to estimate the potential of gas chromatography as a tool for diagnostics of chronic ENT pathology in the children. A total of 37 patients aged between 5 and 18 years with chronic ENT diseases and complaints of foul breath were available for the examination. Thirty one children presented with chronic tonsillitis, 3 with chronic adenoiditis, 2 with chronic rhinosinusitis, and 1 with subatrophic rhinitis. It was shown with the use of gas chromatography that the exhaled air from 34 (91.9%) patients contained cadaverin at a concentration of 0.004-0.0009 mg/ml. Three (8.1%) children exhaled methyl mercaptan (0.01-0.025 mg/m3) and hydrogen sulfide (0.002-0.006 mg/m3), and 2 (5.4%) patients exhaled the air containing dimethyl disulfide (0.009-0.002 mg/m3). The volatile sulfur-containing compounds were detected only in the children aged above 14 years. It is concluded that chronic tonsillitis is the leading etiological factor responsible for the development of the extraoral form of halitosis in the children. The foul breath in the children unlike that in the adults is most frequently due to the enhanced cadaverin concentration in the exhaled air rather than the production of volatile sulfur-containing compounds.

Analysis of synthetic canine training aids by comprehensive two-dimensional gas chromatography-time of flight mass spectrometry.

Cadaver dogs are trained on a variety of materials, including artificial or pseudo scents. The chemical components of commercially available pseudo scents are not known, so their accuracy as a decomposition odour mimic and their effectiveness as a canine training aid have not been evaluated. Two pseudo scents that are commercially available and used for training cadaver dogs were analysed using comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOFMS). The two formulations were determined to be simplistic in their composition, compared to real cadaveric volatile organic compound (VOC) mixtures, with only a few major components. The enhanced GC×GC-TOFMS peak capacity was nevertheless useful to discriminate less intense peaks from large overloaded peaks. The availability of both dimension retention times combined with the peak finding and deconvolution algorithm, enabled the chemical characterization of the two formulations. Additionally, high resolution (HR) TOFMS was used to extract molecular formulae and confirm identities of analytes. The seven compounds identified by this work have not been reported previously as volatile products of decomposition, indicating that these pseudo scents are not to be considered as an accurate representation of cadaveric decomposition odour. Further research on the olfaction of scent detection canines and the chemical composition of their target odourants needs to be conducted to develop improved canine training aids.

Measuring autophagy in macrophages.

Macroautophagy is a conserved intracellular homeostatic mechanism for the degradation of cytosolic constituents. Autophagy can promote cell survival by providing essential amino acids from the breakdown of macromolecules during periods of nutrient deprivation, and can remove damaged or excess organelles, such as mitochondria and peroxisomes. More recently, autophagy has been shown to play an important role in innate and adaptive immune responses to pathogenic bacteria in macrophages and dendritic cells. This unit presents protocols for the measurement of autophagy in macrophages.

Acceleration of purine degradation by periodontal diseases.

Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression.

Optimising cell temperature and dispersion field strength for the screening for putrescine and cadaverine with thermal desorption-gas chromatography-differential mobility spectrometry.

Biogenic amines, and putrescine and cadaverine in particular, have significant importance in the area of food quality monitoring, and are also potentially important markers of infection, for cancer, diabetes, arthritis and cystic fibrosis. A thermal desorption-gas chromatograph-heated differential mobility spectrometer was constructed and the significant effect of interactions between cell temperature and dispersion field strength on the observed responses studied. The experiment design was a Box-Wilson central composite design (CCD) over the levels of 10-24 kVcm(-1) for dispersion field strength and 100-130 degrees C for cell temperature. The optimum values were estimated to be 16.22 kVcm(-1) and 116 degrees C for putrescine and 14.78 kVcm(-1) and 112 degrees C for cadaverine, respectively with an ammonia dopant at 19 mgm(-3). An amine test atmosphere generator was constructed and produced stable concentrations of putrescine (7 mgm(-3)) and cadaverine (4 mgm(-3)) vapours at 50+/-0.5 degrees C. Tenax TA-Carbotrap adsorbent tubes were used to sample putrescine and cadaverine vapour standards and a linear response function over the range of sample masses 5-20 ng was obtained at 15.0 kVcm(-1) 115 degrees C, with a R(2) of 0.99 for both putrescine and cadaverine. The sample mass at the limit of detection was estimated to be 3 ng for putrescine and cadaverine. Preliminary data from sampling the headspace of chicken meat revealed a 62% increase in the recovered masses of putrescine from 0.84 to 1.36 ng in the sampled air.

Determination of rat hepatic polyamines by electron-capture gas chromatography.

INTRODUCTION: An efficient and reproducible electron-capture gas chromatographic protocol that allows the simultaneous detection and quantification of the polyamines putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), and spermidine (N-[3-aminopropyl]-1,4-diaminobutane) was developed. METHODS: Hepatic tissue from male Sprague-Dawley rats was used for analysis. The polyamines and the internal standard (sertraline) were extracted and derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column (narrow-bore fused silica column; 25 mm x 0.32 mm) and an electron-capture detector. RESULTS: PFBC reacts with the amine functions of the polyamines examined here to produce PFB derivatives with high sensitivity on electron-capture detection. The method permitted the quantitative analyses of all three amines in rat hepatic tissue; the concentration of putrescine, but not spermidine, was increased significantly following a 14-day administration of the diamine oxidase (DAO) inhibitor aminoguanidine. Cadaverine was also present at increased concentrations in hepatic homogenates from aminoguanidine-treated rats. DISCUSSION: Extractive derivatization with PFBC followed by gas chromatographic analysis using electron-capture detection results in a rapid and reproducible assay that permits the simultaneous detection and quantification of putrescine, cadaverine, and spermidine in biological tissue.

Gas chromatographic method for putrescine and cadaverine in shrimp.

A gas-liquid chromatographic method developed for the determination of putrescine and cadaverine in fishery products was modified for application to the determination of diamines in shrimp. Addition of potassium chloride and hydrochloric acid to the methanol-water extraction solvent resulted in increased recovery of the diamines and minimized gel formation. The recovery of putrescine increased on average from 64 to 98%, and the recovery of cadaverine increased from 85 to 93%. The chromatographic separation of the derivatized diamines was significantly improved with a change from an OV-225 column (cyanopropyl methyl phenyl methyl silicone) to a more polar HP-Innowax column (crosslinked polyethylene glycol). Background levels of putrescine and cadaverine in known high-quality shrimp ranged from 0 to 0.7 ppm. Shrimp that failed sensory examination generally contained putrescine at levels >4.8 ppm and cadaverine at levels >1.3 ppm.

Gas chromatographic method for putrescine and cadaverine in canned tuna and mahimahi and fluorometric method for histamine (minor modification of AOAC Official Method 977.13): collaborative study.

A collaborative study was conducted to test a modification to the AOAC fluorometric method for histamine (AOAC Official Method 977.13) that substitutes 75% methanol as the extracting solvent. All other steps remain unchanged. The extracts prepared with 75% methanol were also used to collaboratively test a gas chromatographic (GC) method for determination of putrescine and cadaverine in seafood. In the GC method, the extracted diamines are converted to fluorinated derivatives, the reaction mixtures are passed through solid-phase extraction columns, and the derivatives are quantitated by electron capture GC after separation on an OV-225 column. Fourteen laboratories using the GC method for putrescine and cadaverine and 16 laboratories using the fluorometric method for histamine analyzed 14 canned tuna and raw mahimahi (including blind duplicates and a spike) containing 0.2-2.6 ppm putrescine, 0.6-9.1 ppm cadaverine, and 0.6-154 ppm histamine. At the 5 ppm level, recoveries ranged from 71 to 102% for putrescine and 77 to 112% for cadaverine; the respective repeatability relative standard deviations (RSDr) were 5.2 and 15%, and the respective reproducibility relative standard deviations (RSDR) were 8.8 and 18%. At the 50 ppm level, histamine recoveries ranged from 84 to 125%, RSDr was 3.6%, and RSDR was 9.4%. The GC method for determination of putrescine in canned tuna and cadaverine in canned tuna and mahimahi has been adopted first action by AOAC INTERNATIONAL, and the AOAC Official Method 977.13, Histamine in Seafood, Fluorometric Method, has been modified.