Total Adenylate Research Articles

Ethoxyquin attenuates enteric oxidative stress and inflammation by promoting cytokine expressions and symbiotic microbiota in heat-stressed broilers

Intestinal oxidative stress in broilers is produced by chronic heat stress (HS) and has a negative impact on poultry performance as it induces intestinal inflammation and promotes the invasion of gram-negative bacteria, such as bacterial lipopolysaccharide (LPS). Therefore, dietary inclusion of the antioxidant compound, ethoxyquin (EQ), could improve enteric antioxidant capacity, immune responses, and the epithelial barrier, and maintain the symbiotic gut microbiota community. To investigate the effects of EQ supplementation on alleviating enteric oxidative stress in heat-stressed broilers, 200 one-day-old male Ross 308 broilers were randomly assigned to 4 groups (n = 50 chicks/group; n = 10 chicks/replicate) and fed a basal diet supplemented with 0 (CT), 50 (EQ-50), 100 (EQ-100), and 200 (EQ-200) mg EQ/ kg-1 for 5 wk. The chicks were raised in floor pens inside the broiler farm at a temperature and humidity index (THI) of 29 from d 21 to d 35. Growth performance traits, relative organ index, hepatic antioxidant enzymes, serum immunity, total adenylate, and cytokine activities were improved in the EQ-50 group (linear or quadratic P < 0.05), promoting the relative mRNA expression of cytokine gene-related anti-inflammatory and growth factors. A distinct microbial community colonised the gut microbiota in the EQ-50 group, with a high relative abundance of Lactobacillus, Ligilactobacillus, Limosilactobacillus, Pediococcus, Blautia, and Faecalibacterium compared to the other groups. Dietary supplementation with 50 mg EQ/ kg-1 for 5 wk attenuates enteric oxidative stress and intestinal inflammation by enhancing serum immune and cytokine content (IgG, IL-6, and TGF-β,) and symbiotic microbiota in heat-stressed broilers. EQ promotes the expression of Hsp70, SOD2, GPx 4, IL-6, and IGF-1 cytokine gene-related anti-inflammatory and growth factors in heat-stressed hepatic broilers. Collectively, EQ-50 could be a suitable feed supplement for attenuating enteric oxidative stress and intestinal inflammation, thereby promoting the productivity of heat-stressed broilers.

A rapid method for measuring ATP + ADP + AMP in marine sediment.

In this report, I describe a method for rapid measurement of total adenylate (ATP + ADP + AMP) in marine sediment samples for estimating microbial biomass. A simple 'boil and dilute' method is described here, whereby adding boiled MilliQ water to sediments increases the detection limit for ATP + ADP + AMP up to 100-fold. The lowered detection limit of this method enabled the detection ATP + ADP + AMP in relatively low-biomass sub-seafloor sediment cores with 104 16S rRNA gene copies per gram. Concentrations of ATP + ADP + AMP correlated with 16S rRNA gene concentrations from bacteria and archaea across six different sites that range in water depth from 1 to 6000 m indicating that the ATP + ADP + AMP method can be used as an additional biomass proxy. In deep sea microbial communities, the ratio of ATP + ADP + AMP concentrations to 16S rRNA genes >1m below seafloor was significantly lower compared to communities in the upper 30 cm of sediment, which may be due to reduced cell sizes and or lower ATP + ADP + AMP concentrations per cell in the deep sea sub-seafloor biosphere. The boil and dilute method for ATP + ADP + AMP is demonstrated here to have a detection limit sufficient for measuring low biomass communities from deep sea sub-seafloor cores. The method can be applied to frozen samples, enabling measurements of ATP + ADP + AMP from frozen sediment cores stored in core repositories from past and future international drilling campaigns.

Effect of acute hypoxia on the brain energy metabolism of the scorpionfish Scorpaena porcus Linnaeus, 1758: the pattern of oxidoreductase activity and adenylate system.

The activity of oxidoreductases, malate dehydrogenase and lactate dehydrogenase (MDH, 1.1.1.37; LDH, 1.1.1.27), as well as parameters of adenylate system-[ATP], [ADP], [AMP], total adenylate pool (AP), and adenylate energy charge (AEC) in medulla oblongata (MB) and forebrain, midbrain, and diencephalon (FDMB)-were studied in the scorpionfish under acute hypoxia (0.9-1.2mgO2·L-1, 90min). A higher MDH activity level was observed in MB and FDMB, as compared to LDH (p < 0.05). At the same time, MB showed a higher adenylate content and increased AP (p < 0.05). AEC did not exceed ~ 0.7 (vs. the maximum of this index ~ 0.9-1.0) in the brain of the scorpionfish indicating adaptation of the tissue energy status to hypoxia. A rapid decrease in MDH activity (p < 0.05) was observed in MB under acute hypoxia. These changes were accompanied by insignificant LDH activation. A pronounced LDH activation (p < 0.05), a decrease in MDH activity, and the highest AP raise (p < 0.05) were observed in FDMB, suggesting activation of glycolysis and simultaneous decrease in the rate of ATP consumption. MB and FDMB demonstrated the ability to a relative retention of AEC during hypoxia. The unidirectional metabolic adaptation was based on the intensification of glycolysis, a decrease of ATP consumption, and a subsequent increase in adenylate concentration that allowed the scorpionfish brain structures to maintain the energy status under acute hypoxia.

A Comprehensive Analysis of ATP Tests: Practical Use and Recent Progress in the Total Adenylate Test for the Effective Monitoring of Hygiene

Rapid hygiene monitoring tests based on the presence of ATP have been widely used in the food industry to ensure that adequate cleanliness is maintained. In this study, the practical applications and limitations of these tests and recent technological progress for facilitating more accurate control were evaluated. The presence of ATP on a surface indicates improper cleaning and the presence of contaminants, including organic debris and bacteria. Food residues are indicators of insufficient cleaning and are direct hazards because they may provide safe harbors for bacteria, provide sources of nutrients for bacterial growth, interfere with the antimicrobial activity of disinfectants, and support the formation of biofilms. Residues of allergenic foods on a surface may increase the risk of allergen cross-contact. However, ATP tests cannot detect bacteria or allergenic proteins directly. To ensure efficient use of commercially available ATP tests, in-depth knowledge is needed regarding their practical applications, methods for determining pass-fail limits, and differences in performance. Conventional ATP tests have limitations due to possible hydrolysis of ATP to ADP and AMP, which further hinders the identification of food residues. To overcome this problem, a total adenylate test was developed that could detect ATP+ADP+AMP (A3 test). The A3 test is suitable for the detection of adenylates from food residues and useful for verification of hygiene levels. The A3 test in conjunction with other methods, such as microorganism culture and food allergen tests, may be a useful strategy for identifying contamination sources and facilitating effective hygiene management.

Gap Junction–Mediated Transport of Metabolites Between Stem Cells and Vascular Endothelial Cells

We have previously demonstrated that small molecular transfer, such as glucose, between hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) and vascular endothelial cells via gap junctions constitutes an important mechanism of stem cell therapy. Cell metabolites are high-potential small-molecule candidates that can be transferred to small molecules between stem cells and vascular endothelial cells. Here, we investigated the differences in metabolite levels between stem cells (HSCs and MSCs), vascular endothelial cells, and the levels of circulating non-hematopoietic white blood cells (WBCs). The results showed remarkable differences in metabolite concentrations between cells. Significantly higher concentrations of adenosine triphosphate (ATP), guanosine triphosphate (GTP), total adenylate or guanylate levels, glycolytic intermediates, and amino acids were found in HSCs compared with vascular endothelial cells. In contrast, there was no significant difference in the metabolism of MSCs and vascular endothelial cells. From the results of this study, it became clear that HSCs and MSCs differ in their metabolites. That is, metabolites that transfer between stem cells and vascular endothelial cells differ between HSCs and MSCs. HSCs may donate various metabolites, several glycolytic and tricarboxylic acid cycle metabolites, and amino acids to damaged vascular endothelial cells as energy sources and activate the energy metabolism of vascular endothelial cells. In contrast, MSCs and vascular endothelial cells regulate each other under normal conditions. As the existing MSCs cannot ameliorate the dysregulation during insult, exogenous MSCs administered by cell therapy may help restore normal metabolic function in the vascular endothelial cells by taking up excess energy sources from the lumens of blood vessels. Results of this study suggested that the appropriate timing of cell therapy is different between HSCs and MSCs.

Rapid Hygiene Assay Sensitive to Cumulative Adenylate Homologues Exhibits Equal or Higher Frequencies of Soil Contamination Detection than Assay Limited to ATP Detection

Based upon regulatory and food industry-driven food safety standards, there is a need for rapid, accurate methods for assessing sanitary conditions. A commonly used assay is based on the assessment of the biochemical molecule adenosine triphosphate (ATP). A more recent assay, the total adenylate homologue-based (AXP) assay, targets the cumulative presence of ATP and its dephosphorylated homologues, adenosine diphosphate and adenosine monophosphate. Yet there is little information that compares the practical performance of these two assays. This work examined these two assay types with a comparative study in a grade A dairy foods processing plant and a licensed and inspected meat processing facility. A total of 1,920 concomitant analyses were conducted with main variables of assay type, processing facility type, and hygiene zone category. Statistical process control methodology was used to calculate 95% confidence control limits; data beyond those limits were considered contamination events. Results demonstrated that overall, the AXP assay detected contamination events approximately two times more often than the assay based on ATP only. This increase in the rate of contamination event detection was especially prevalent in the meat processing facility, where across all hygienic zones, there were 38 versus 85 contaminations events detected for the ATP and AXP assays, respectively. Across hygiene zones, the AXP data displayed either an equal or an increased incidence of soil detection compared with data from the ATP assay. This study provides applied evidence that assays solely dependent on ATP concentrations are less able to detect soil contaminants under conditions that favor ATP dephosphorylation reactions.

An Explanation Based on Energy-Related Changes for Blue Mussel Mytilus edulis Coping With Seawater Acidification.

As ocean acidification (OA) is gradually increasing, concerns regarding its ecological impacts on marine organisms are growing. Our previous studies have shown that seawater acidification exerted adverse effects on physiological processes of the blue mussel Mytilus edulis, and the aim of the present study was to obtain energy-related evidence to verify and explain our previous findings. Thus, the same acidification system (pH: 7.7 or 7.1; acidification method: HCl addition or CO2 enrichment; experimental period: 21d) was set up, and the energy-related changes were assessed. The results showed that the energy charge (EC) and the gene expressions of cytochrome C oxidase (COX) reflecting the ATP synthesis rate increased significantly after acidification treatments. What’s more, the mussels exposed to acidification allocated more energy to gills and hemocytes. However, the total adenylate pool (TAP) and the final adenosine triphosphate (ATP) in M. edulis decreased significantly, especially in CO2 treatment group at pH 7.1. It was interesting to note that, TAP, ATP, and COXs gene expressions in CO2 treatment groups were all significantly lower than that in HCl treatment groups at the same pH, verifying that CO2-induced acidification exhibited more deleterious impacts on M. edulis, and ions besides H+ produced by CO2 dissolution were possible causes. In conclusion, energy-related changes in M. edulis responded actively to seawater acidification and varied with different acidification conditions, while the constraints they had at higher acidification levels suggest that M. edulis will have a limited tolerance to increasing OA in the future.

Assessing the Performance of a Real-Time Total Adenylate (ATP+ADP+AMP) Detection Assay for Surface Hygiene Monitoring in Food Manufacturing Plants and Commercial Kitchens

Environmental hygiene monitoring in the food processing environment has become important in current food safety programs to ensure safe food production. However, conventional monitoring of surface hygiene based on visual inspection and microbial counts is slow, tedious, and thus unable to support the current risk-based management system. Therefore, this study was conducted to assess the performance of a real-time total adenylate assay that detected ATP+ADP+AMP (A3) for food contact surface hygiene in 13 food processing plants and two commercial kitchens in Malaysia. The A3 value was compared with the microbial count (aerobic plate count [APC]) on food contact surfaces. Receiver-operating characteristic (ROC) analysis was performed to assess the reliability of the data and to determine the optimal threshold value for hygiene indication of food contact surfaces. Overall, the A3 value demonstrated a weak positive relationship with APC. However, the A3 value significantly correlated with APC for food processing environments associated with raw meat and raw food ingredients such as fruit that harbor a high microbial load. ROC analysis suggested an optimal threshold for the A3 value of 500 relative light units to balance the sensitivity and specificity at 0.728 and 0.719, respectively. The A3 assay as a hygiene indicator for food contact surfaces had an efficiency of 72.1%, indicating its reliability as a general hygiene indicator.

5'-Adenosine monophosphate deaminase regulation in ground squirrels during hibernation.

Hibernation is an important winter survival strategy for many small mammals. By sinking into a deep torpor where metabolic rate can be as low as 1-5% of the resting rate in euthermia, animals accrue huge energy savings that allow survival, typically without eating, for many months. Hibernating ground squirrels show a net reduction in the total adenylate pool of skeletal muscle during torpor, but the ATP/ADP ratio and adenylate energy charge remain stable. A key enzyme involved in managing adenylate pool size is 5'-adenosine monophosphate deaminase (AMPD). Assessing skeletal muscle AMPD from both Richardson's ground squirrels (Urocitellus richardsonii) (RGS) and 13-lined ground squirrels (Ictidomys tridecemlineatus) (TLGS), the present study shows that muscle AMPD of euthermic versus hibernating animals displays markedly different kinetic properties, differential responses to temperature and to effectors, and is regulated by reversible protein phosphorylation. AMPD activity decreased during hibernation in both TLGS and RGS skeletal muscle, by 70 and 84%, respectively. Stimulation of total protein phosphatases, total serine/threonine protein phosphatases, PP1, PP2B or PP2C, all reduced AMPD activity between 54 and 92% in extracts of euthermic RGS muscle. The same incubation did not change the activity of AMPD from muscle of hibernating animals. Oppositely, both euthermic and hibernating AMPD showed a strong increase in activity when incubated under conditions that promoted the enzyme phosphorylation by PKA, PKC or PKG. Overall, the data indicate that both low activity of AMPD and low affinity of the enzyme for AMP during torpor reduce the rate of adenylate degradation, the primary driver of these changes being covalent phosphorylation of AMPD.

Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

Magnesium Signaling in Plants.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

АКТИВНОСТЬ ФЕРМЕНТОВ ЭНЕРГЕТИЧЕСКОГО ОБМЕНА И АДЕНИЛАТНАЯ СИСТЕМА КАМЕР СЕРДЦА ЧЕРНОМОРСКОЙ СКОРПЕНЫ ( SCORPAENA PORCUS L.) ПРИ ОСТРОЙ ГИПОКСИИ

The fish heart is a unique model to compare the resistance to hypoxia of its two chambers (atrium and ventricle), which are different in the structure and functional loading. The activity of oxydoreductases malate dehydrogenase (MDH, 1.1.1.37) and lactate dehydrogenase (LDH, 1.1.1.27), as well as the parameters of the adenylate system in the heart chambers of a Black Sea scorpaena, were studied under acute hypoxia (0.9–1.2 mg O2·L–1, 90 min). Despite the leading functional role of the ventricle, MDH activity in this heart compartment tended to decrease compared to the atrium in the absence of differences in LDH activity. At the same time, the difference in the level of adenylates (ATP, ADP, AMP), total adenylate pool (AP), and adenylate energy charge (AEС) between the atrium and ventricle was statistically nonsignificant, although the absolute value of the ventricular AP was almost twice as large as the atrial AP. The AEC values of the atrium and ventricle perfused only with venous blood did not exceed ~0.7 (vs. the maximum of this parameter ~0.9–1.0), apparently reflecting the energy status of tissues initially adapted to hypoxia. Under acute hypoxia, there were found two strategies for energy metabolism transformation in the heart chambers in the form of a 2.4-fold drop in MDH activity (р < 0.05) in the atrium and a 2.2-fold increment in LDH activity (р < 0.05) in the ventricle. Probably, the decline in MDH activity in the atrial tissue was determined by a more passive function of this heart chamber in providing the blood flow. The exposure to acute hypoxia led to a decrease in the level of adenylate nucleotides and an AEC decline in the heart chambers, as pronounced most distinctly in the ventricular myocardium. When decreasing PO2, the AEC in the heart chambers shifted within quite a narrow range (from 0.7 to 0.6), indicating the retention of a certain stationary energy status achieved by inhibition of ATP consumption or demand. The putative mechanism for retaining the AEC may be based on the negative chronotropic effect of hypoxia.

Exploring the Biochemical Basis of the Meridian Tropism Theory for the Qi-Invigorating Traditional Chinese Medicine Herb Panax ginseng.

We examined the effect of the Qi-invigorating Traditional Chinese Medicines (TCM) herb Panax ginseng (P.G.) on mitochondrial functions and cellular antioxidant capacity in different organs of mice. We found that the P.G. extracts had a significant effect on tissues of mice, with the generation of total adenylate pool (TAP) enhanced in all visceral tissues, but not for the brain. The mitochondrial membrane potential (MMP) and antioxidant capacity reflected by superoxide dismutase (SOD) and glutathione (GSH) increased only for the meridian tissues that P.G. belongs to including Heart, Spleen and Lung. Reactive oxygen species (ROS), as a combined result of the increased energy metabolism and antioxidant capacity, varied in different organs. We concluded that: 1) the Qi-invigorating TCM herb P.G. had a significant effect on mice by enhancing TAP production in all of the visceral tissues examined, except for the brain; 2) for the meridional tissues of P.G. (Heart, Spleen and Lung), the P.G. extracts not only promoted the TAP production, but also boosted the antioxidant capacity demonstrated by the simultaneous increase in TAP, and SOD and GSH.

Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects

AbstractSevere congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-β result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony–stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient's neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was <25% of control values, but phox proteins appeared quantitatively normal. Extensive metabolomics analysis at steady state and upon incubation with stable isotope–labeled tracers (U-13C-glucose, 13C,15N-glutamine, and U-13C-fructose) demonstrated dramatic impairments in early glycolysis (hexose phosphate levels), hexosemonophosphate shunt (required for the generation of the NADPH), and the total adenylate pool, which could explain the dramatic cell dysfunction displayed by the patient's neutrophils. Preliminary experiments with fructose supplementation to bypass the enzyme block demonstrated that the metabolic profile could be reversed, but was not sustained long enough for functional improvement. In human deficiency of G6PC3, metabolic defects resulting from the enzyme deficiency account for diverse neutrophil functional defects and present a major risk of infection.

Sanitation Monitoring of Stainless Steel Surfaces with a Test for Total Adenylates

Sanitation Monitoring of Stainless Steel Surfaces with a Test for Total Adenylates

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Comparison of Detection Limits for Allergenic Foods between Total Adenylate (ATP+ADP+AMP) Hygiene Monitoring Test and Several Hygiene Monitoring Approaches

Validation and verification of cleaning and inspection methods are essential to prevent the spread of allergens via cross-contact. Among the hygiene monitoring tests used on-site, the ATP test is rapid and provides quantifiable results. Nevertheless, because a wide variety of foods contain significant amount of ADP and/or AMP due to the degradation of ATP, the ATP+ADP+AMP (A3) test is preferred for detecting food debris. Hence, the A3 test may be valuable in screening food debris that may contain residual allergens. In this study, the detection limits of the A3 test for 40 foods that are regulated in several countries as allergenic were compared with those of the other hygiene monitoring tests used on-site: the conventional ATP test with similar sensitivity for ATP, the protein swab test that detects as little as 50 μg of protein, and the lateral flow immunoassay (LFI). The A3 test demonstrated lower detection limits than did the ATP test. The detection sensitivity of the A3 test was greater than that of the protein swab test except for its use on gelatin (extracted protein). The cleaning validation performance using a stainless steel model in fish and meat revealed that the A3 test is efficient in verifying the levels of remaining food debris. Although LFI displayed the best sensitivities for 10 of 14 foods, it is not commercially available for some specific allergens; however, the A3 test can detect such food debris. Moreover, the detection limits of the A3 test were preferable or comparable to those of LFI for crustacean shellfish and for processed grains, with the exception of wheat flour and buckwheat. A field study in a food processing plant demonstrated that the amount of both A3 and milk protein (enzyme-linked immunosorbent assay) considerably decreased as the cleaning steps progressed. Therefore, the A3 test is effective in detecting the risk for allergen cross-contact after inadequate cleaning.

590-P: Modulation of Adenine Phosphoribosyltransferase-Mediated Purine Salvage to Accelerate Diabetic Wound Healing

Aim: Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. Our aim was to evaluate the role of APRT in diabetic wound healing. Methods: The molecular mechanism of APRT-mediated cell survival was validated by the haploid HAP1 cell. The adenylate nucleotides in NIH-3T3 fibroblast response to adenine was monitoring by HPLC-MS/MS method. The effects of cell behaviors were evaluated by mice NIH-3T3 fibroblast and human epithelial keratinocytes. Genetically diabetic mice received surgery for full-thickness of the cutaneous wound and the adenine addition as the substrate for APRT on the wound. Results: In the wild type and APRT knockout HAP1 cell, our data revealed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. The time-course of monitoring the total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. In mice NIH-3T3 fibroblasts and human NHEK keratinocytes, adenine elevated cellular ATP levels in a dose-dependent manner and facilitated cell migration. The up-regulated APRT found in wound skin was correlated with the demands of wound repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. Conclusions: Our data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. This study provided an explanation for the up-regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation. Disclosure G. Young: Employee; Self; Energenesis-Biomedical. J. Lin: Employee; Self; Energenesis-Biomedical. Y. Cheng: Employee; Self; Energenesis-Biomedical. H. Chen: Board Member; Self; Energenesis-Biomedical.

Metabolomic analysis of SMP30/GNL knockout mice treated with fermented vegetable and fruit extract (OM-X®)

Background: A dietary supplement developed in Japan, OM-X®, is the result of extended fermentation of dozens of edible vegetables, fruits, seaweeds, mushrooms, and includes 12 strains of Lactic acid bacteria (LAB) and Bifidobacterium. The supplements powerful antioxidant, anti-inflammatory properties, and regulatory effect on metabolizing liver enzymes have been reported.Senescence marker protein-30 (SMP30)/Gluconolactonase (GNL) Knockout (KO) mice, which lack the ability to biosynthesize Vitamin C (VC) in the body, were used in this study. The mice exhibited decreased antioxidant capacity in the blood, decreased liver function, and had poor hair growth due to the VC deficiency. The aim of the study was to examine the effects of oral administration of OM-X® on VC deficiency from the viewpoint of comprehensive analysis of liver metabolism.Methods: SMP30/GNL KO and C57BL/6 (WT) mice were used in this study. The KO mice were divided into the following three groups: VC non-administration (VC minus), VC (0.15 W/V %) administration (VC plus), and no VC plus 0.6% OM-X® administration (OM-X®) groups (n = 6 per group). Mice were kept under experimental conditions until they were euthanized at week 8 (age: 16 weeks old). Body weight and plasma biochemistry levels were measured.. Metabolomic analyses of harvested livers were performed.Results: In the VC minus group, a significant decrease in body weight and significant changes in plasma biochemistry parameters compared with the WT control or VC plus groups was observed. In the OM-X® group, significant suppression or a suppressive trend was observed compared with the VC minus group. Liver metabolomic analysis showed that total adenylate levels decreased in the VC minus group, whereas OM-X® administration significantly elevated total adenylate levels. In addition, the ratios of Glutathione (GSH)/ Glutathione disulfide (GSSG) and S-adenosylmethionine (SAM)/ S-adenosyl-L-homocysteine (SAH) and total glutathione levels were all significantly higher in the OM-X® group than in the VC minus group. The glycerol 3-phosphate/ Dihydroxyacetone phosphate (DHAP) and putrescine/spermidine ratios were elevated in the VC minus group, whereas it was significantly lower in the OM-X® group.Conclusion: The results showed that in SMP30/GNL KO mice, some organ damage may have occurred during VC deficiency, as indicated by weight loss, hepatic injury, and changes in triglyceride-related markers. OM-X® had an effect on energy charge maintenance by elevating total adenylate levels, inducing antioxidant capacity via enhancing glutathione levels, and promoting protein synthesis, including polyamine synthesis. Altogether, the results revealed that OM-X® prevents the adverse biological changes caused by VC deficiency in SMP30/GNL KO mice.Keywords: OM-X®, SMP30/GNL KO mice, Vitamin C, metabolomic analysis.

Validation Study of LuciPacTM A3 Surface for Hygiene Monitoring through Detection of ATP, ADP, and AMP from Stainless Steel Surfaces: AOAC Performance Tested MethodSM 051901.

The LuciPac A3 Surface Hygiene Monitoring System based on the detection of total adenylate, ATP+ADP+AMP (A3), has been developed by Kikkoman Biochemifa. This A3 swabbing assay kit was validated for Performance Tested MethodsSM (PTM) certification. The LuciPac A3 Surface Hygiene Monitoring System was evaluated for limit of detection (LOD) for each adenylate with pure analyte solutions, detection of food residues and microbial residues on stainless steel surfaces, interference by disinfectants, and selectivity of the method response. Pure analyte studies performed by the method developer and the independent laboratory showed good linearity (R2 > 0.9854) and repeatability precision (RSDr < 20% for ≥2.5 fmol/assay). The LOD values for each adenylate were around 10 relative light units or 2.5 fmol/assay. The repeatability precision in the method developer laboratory for the matrix study (raw chicken breast, sliced deli ham, orange juice, yogurt, and apple pie) and the microbial study (Cronobacter sakazakii, Lactobacillus acidophilus, and Saccharomyces cerevisiae) were 8-30% and 10-35%, respectively. The repeatability precision of independent laboratory testing was 13-27% for orange juice and 16-43% for ham. Interference by several disinfectants indicate that rinsing is recommended to be performed after the use of sanitizing agents and before testing with LuciPac A3 Surface. Selectivity testing revealed that no positive interference and no inhibition were caused by adenylate analogues. Instrument variation, lot-to-lot consistency, accelerated stability (30°C, 5 weeks) were confirmed, and the method was shown to be robust against shaking time. The LuciPac A3 Surface has been successfully validated. This A3 swabbing assay kit was qualified for PTM certification No. 051901.