ABSTRACT
BACKGROUND AND AIM: Our objective was to use metabolomics in a toxicological-relevant target tissue to gain insight into the biological processes that may underlie the negative association between air pollution exposure and oocyte quality. METHODS: Our study included 125 women undergoing in vitro fertilization at an academic fertility center in Massachusetts, US (2005-2015). A follicular fluid sample was collected during oocyte retrieval and untargeted metabolic profiling was conducted using liquid chromatography with ultra-high-resolution mass spectrometry and two chromatography columns (C18 and HILIC). Daily exposure to nitrogen dioxide (NO2), ozone, fine particulate matter, and black carbon was estimated at the women's residence using spatiotemporal models and averaged over the period of ovarian stimulation (2-weeks). Multivariable linear regression models were used to evaluate the associations between the air pollutants, number of mature oocytes, and metabolic feature intensities. A meet-in-the-middle approach was used to identify overlapping features and metabolic pathways. RESULTS: Of the air pollutants, NO2 exposure had the largest number of overlapping metabolites (C18: 105; HILIC: 91) and biological pathways (C18: 3; HILIC: 6) with number of mature oocytes. Key pathways of overlap included vitamin D3 metabolism (both columns), bile acid biosynthesis (both columns), C21-steroid hormone metabolism (HILIC), androgen and estrogen metabolism (HILIC), vitamin A metabolism (HILIC), carnitine shuttle (HILIC), and prostaglandin formation (C18). Three overlapping metabolites were confirmed with level-1 or level-2 evidence. For example, hypoxanthine, a metabolite that protects against oxidant-induced cell injury, was positively associated with NO2 exposure and negatively associated with number of mature oocytes. Minimal overlap was observed between the other pollutants and the number of mature oocytes. CONCLUSIONS: Higher exposure to NO2 during ovarian stimulation was associated with many metabolites and biologic pathways involved in endogenous vitamin metabolism, hormone synthesis, and oxidative stress that may mediate the observed associations with lower oocyte quality.
Subject(s)
Air Pollutants , Air Pollution , Biological Products , Ozone , Air Pollutants/analysis , Air Pollutants/toxicity , Air Pollution/adverse effects , Air Pollution/analysis , Androgens/analysis , Animals , Bile Acids and Salts/analysis , Biological Products/analysis , Carbon/analysis , Carnitine , Cholecalciferol/analysis , Estrogens/analysis , Female , Follicular Fluid , Hypoxanthines/analysis , Imidazoles , Metabolomics , Nitrogen Dioxide/analysis , Oocytes , Oxidants , Ozone/analysis , Particulate Matter/adverse effects , Particulate Matter/analysis , Prostaglandins/analysis , Steroids , Sulfonamides , Thiophenes , Vitamin A/analysis , Vitamins/analysisABSTRACT
In this study, the influences of organic selenium (Se, 0.002 mg/L) on the muscle flavor and texture properties of Micropterus salmonides under fasting temporary rearing (8 weeks) was investigated. Electronic nose and headspace solid-phase microextraction-gas chromatography-mass spectrometry analysis suggested that organic Se regulated the types and contents of volatile compounds, especially aldehydes and ketones, which were increased in the early temporary rearing but decreased in the late stage. Organic Se significantly increased the content of 5'-inosine monophosphate by approximately 15 % (p < 0.05), and decreased the content of hypoxanthine and hypoxanthine ribonucleoside by more than 20 % (p < 0.05). After the 8th temporary rearing week, muscle hardness and springiness increased by at least 10 % (p < 0.01), resilience and gumminess improved by at least 18 % (p < 0.05) and 5.9 % (p < 0.05), respectively. In conclusion, organic Se ameliorates the flesh quality of M. salmonides during long-term temporary rearing.
Subject(s)
Bass , Selenium , Volatile Organic Compounds , Animals , Hypoxanthines/analysis , Muscles/chemistry , Odorants/analysis , Solid Phase Microextraction/methods , Volatile Organic Compounds/analysisABSTRACT
BACKGROUND: Improved understanding of the pathways associated with airway pathophysiologic features in COPD will identify new predictive biomarkers and novel therapeutic targets. RESEARCH QUESTION: Which physiologic pathways are altered in the airways of patients with COPD and will predict exacerbations? STUDY DESIGN AND METHODS: We applied a mass spectrometric panel of metabolomic biomarkers related to mucus hydration and inflammation to sputa from the multicenter Subpopulations and Intermediate Outcome Measures in COPD Study. Biomarkers elevated in sputa from patients with COPD were evaluated for relationships to measures of COPD disease severity and their ability to predict future exacerbations. RESULTS: Sputum supernatants from 980 patients were analyzed: 77 healthy nonsmokers, 341 smokers with preserved spirometry, and 562 patients with COPD (178 with Global Initiative on Chronic Obstructive Lung Disease [GOLD] stage 1 disease, 303 with GOLD stage 2 disease, and 81 with GOLD stage 3 disease) were analyzed. Biomarkers from multiple pathways were elevated in COPD and correlated with sputum neutrophil counts. Among the most significant analytes (false discovery rate, 0.1) were sialic acid, hypoxanthine, xanthine, methylthioadenosine, adenine, and glutathione. Sialic acid and hypoxanthine were associated strongly with measures of disease severity, and elevation of these biomarkers was associated with shorter time to exacerbation and improved prediction models of future exacerbations. INTERPRETATION: Biomarker evaluation implicated pathways involved in mucus hydration, adenosine metabolism, methionine salvage, and oxidative stress in COPD airway pathophysiologic characteristics. Therapies that target these pathways may be of benefit in COPD, and a simple model adding sputum-soluble phase biomarkers improves prediction of pulmonary exacerbations. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01969344; URL: www. CLINICALTRIALS: gov.
Subject(s)
Pulmonary Disease, Chronic Obstructive , Sputum , Biomarkers/analysis , Humans , Hypoxanthines/analysis , N-Acetylneuraminic Acid/analysis , Pulmonary Disease, Chronic Obstructive/diagnosis , Sputum/chemistryABSTRACT
We have developed a new technique to determine the concentration of hypoxanthine [Hx] in a reverse phase column using a modified high-performance liquid chromatography (HPLC) method that is faster and more reliable than those previously described. In this paper we present a formula for estimating the post mortem interval (PMI) based on this HPLC method by applying the inverse prediction method. The regression line obtained by changing the variables gives PMI = 0.183 [Hx] + 0.599 (PMI in hours, [Hx] in micromol/L, R2 = 0.531, P < 0.05).
Subject(s)
Hypoxanthines/analysis , Vitreous Body/chemistry , Chromatography, High Pressure Liquid/methods , Forensic Medicine , Humans , Postmortem Changes , Reproducibility of ResultsABSTRACT
Three radical species were detected in an EPR/ENDOR study of X-irradiated hypoxanthine.HCl.H2O single crystals at room temperature: RI was identified as the product of net H addition to C8, RII was identified as the product of net H addition to C2, and RIII was identified as the product of OH addition to C8. The observed set of radicals was the same for room-temperature irradiation as for irradiation at 10 K followed by warming the crystals to room temperature; however, the C2 H-addition and C8 OH-addition radicals were not detectable after storage of the crystals for about 2 months at room temperature. Use of selectively deuterated crystals permitted unique assignment of the observed hyperfine couplings, and results of density functional theory calculations on each of the radical structures were consistent with the experimental results. Comparison of these experimental results with others from previous crystal-based systems and model system computations provides insight into the mechanisms by which the biologically important purine C8 hydroxyl addition products are formed. The evidence from solid systems supports the mechanism of net water addition to one-electron oxidized purine bases and demonstrates the importance of a facial approach between the reactants.
Subject(s)
Electron Spin Resonance Spectroscopy/methods , Hypoxanthines/chemistry , Hypoxanthines/radiation effects , Purines/chemistry , Purines/radiation effects , Crystallography/methods , Dose-Response Relationship, Radiation , Hydroxyl Radical/chemistry , Hydroxyl Radical/radiation effects , Hypoxanthines/analysis , Molecular Conformation/radiation effects , Molecular Structure , Oxidation-Reduction/radiation effects , Radiation DosageABSTRACT
Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.
Subject(s)
Guanine/analysis , Hypoxanthines/analysis , Inclusion Bodies/chemistry , Paramecium tetraurelia/chemistry , Animals , Chromatography, High Pressure Liquid , Microscopy, Electron, Scanning , Mutation , Paramecium tetraurelia/growth & development , Paramecium tetraurelia/physiology , X-Ray DiffractionABSTRACT
OBJECTIVE: High concentrations of hypoxanthine and urate have been found in the blood of rats who died suddenly during induced respiratory alkalosis as well as in cases of sudden death in malignant hyperthermia-susceptible pigs challenged with halothane. The origin of these metabolites is the excessive hydrolysis of adenine nucleotides, which is associated with the production of free radicals. We wished to establish whether high levels of these compounds were also to be found in sudden infant death syndrome (SIDS) victims, compared with other causes of death. DESIGN: Vitreous humor samples were analysed for hypoxanthine and urate by high-performance liquid chromatography. SETTING: Forensic Laboratories, Salt River, Cape Town. PARTICIPANTS: Vitreous humor samples were collected from 91 infants presented for postmortem examination. MAIN OUTCOME AND RESULTS: From autopsy reports, cause of death was classified as: (i) SIDS (N = 50); (ii) acute sudden death (N = 5); and (iii) all other causes of death (N = 36). There were no differences in the hypoxanthine or urate levels of groups (i) and (iii) over the first 5 days of the postmortem period. Group (ii) levels were lower than those of both (i) and (iii). CONCLUSION: Adenine nucleotide hydrolysis is not only a feature of SIDS, and possibly results from antemortem hypoxia in most deaths. The lower concentrations found in cases of acute sudden death probably resulted only from postmortem hydrolysis of the nucleotides.
Subject(s)
Hypoxanthines/analysis , Sudden Infant Death , Uric Acid/analysis , Vitreous Body/chemistry , Adenosine Triphosphate/metabolism , Chromatography, High Pressure Liquid/methods , Free Radicals , Humans , Hydrolysis , Infant , Infant, NewbornABSTRACT
In the myocardial interstitial space, adenosine and its metabolites are important markers of ischemia, regulators of blood flow, and may produce cardioprotection against ischemia. A fast and sensitive method to assess the concentrations of adenosine and its metabolites is necessary to determine their involvement in mediating these effects. A method for the simultaneous determination of adenosine, inosine, hypoxanthine, xanthine, and uric acid in the interstitial fluid of the canine myocardium was developed using microdialysis, microbore column high-performance liquid chromatography, and a photo diode array detector (DAD). The microdialysis samples were injected directly onto a microbore C18 reverse-phase column without any prior sample preparation. Use of a DAD in this method provided many advantages. First, a DAD allowed the simultaneous detection of UV absorbance at multiple wavelengths, allowing the detection of each compound at their maximal UV absorbance. Further, the full UV absorption spectrum was recorded for each detected peak, confirming peak purity and identity. Using a microbore HPLC column and detection of UV absorbance at the maximal absorbance for each compound improve the sensitivity for all compounds. The detection limit of these compounds is 50 fmol (signal-to-noise ratio, S/N = 3). This method is useful in analyzing the temporal effect of a prolonged period of myocardial ischemia and reperfusion upon interstitial adenosine, inosine, hypoxanthine, xanthine, and uric acid concentrations in an in vivo canine model.
Subject(s)
Adenosine/metabolism , Chromatography, High Pressure Liquid/methods , Myocardial Reperfusion Injury/metabolism , Animals , Calibration , Chromatography, High Pressure Liquid/instrumentation , Dogs , Extracellular Space/metabolism , Hypoxanthine , Hypoxanthines/analysis , Inosine/analysis , Microdialysis , Sensitivity and Specificity , Time Factors , Uric Acid/analysis , Xanthine , Xanthines/analysisABSTRACT
Treatment of human respiratory tract tracheobronchial epithelial cells with gas-phase cigarette smoke led to dose-dependent DNA strand breakage that was highly correlated with multiple chemical modifications of all four DNA bases. The pattern of base damage suggests attack by hydroxyl radicals (OH.). However, by far the most important base damage in quantitative terms was formation of xanthine and hypoxanthine, presumably resulting from deamination of guanine and adenine respectively. Hence, DNA damage by cigarette smoke may involve reactive nitrogen species as well as reactive oxygen species.
Subject(s)
Adenine/analogs & derivatives , DNA Damage , DNA/chemistry , Guanine/analogs & derivatives , Hydroxyl Radical , Hypoxanthines/analysis , Smoke/adverse effects , Smoking , Xanthines/analysis , Bronchi , Cell Line , Epithelium , Humans , Hypoxanthine , XanthineABSTRACT
An assay is described for measurement of purine nucleoside phosphorylase (PNP) in plasma by high-performance liquid chromatography (HPLC). A plasma sample was incubated with hypoxanthine and ribose-1-phosphate in phosphate-free medium at pH 7.4 to catalyse the production of inosine by plasmatic PNP. The reaction was stopped by addition of perchloric acid to inactivate the enzyme and to precipitate plasma proteins. After centrifugation and neutralization of the supernatant with NaOH the increase in the substrate inosine was determined by HPLC. Plasma activities of PNP averaged 5.0 mU/ml before and 12.3 mU/ml (p < 0.001), 5 min after porcine liver transplantation. At the same time points, the plasma activities of the frequently used liver enzymes lactate dehydrogenase or alanine aminotransferase remained virtually unchanged. Thus, plasmatic activities of PNP may be a suitable and early indicator of ischemic alterations to the graft in vivo.
Subject(s)
Liver Transplantation/physiology , Liver/enzymology , Purine-Nucleoside Phosphorylase/blood , Alanine Transaminase/blood , Animals , Chromatography, High Pressure Liquid , Hypoxanthines/analysis , Indicators and Reagents , Inosine/analysis , L-Lactate Dehydrogenase/blood , Liver/physiopathology , Liver Function Tests , Phosphates/analysis , SwineABSTRACT
Previous studies in very young rats have shown that dietary nucleotides improve small intestine repair after injury or malnutrition. To investigate the potential effect of nucleotides in old rats, which have a diminished capability for intestinal repair, 17-mo-old rats were deprived of food for 5 d and then fed a nucleotide-free diet or a nucleotide-supplemented diet for 3 or 6 d. Intestinal jejunal and ileal mucosal weight, protein and DNA were evaluated as intestinal growth markers, and brush-border maltase, sucrase, lactase and aminopeptidase activities were evaluated as intestinal differentiation markers. The adenine nucleotide pool and the adenylate energy charge were also evaluated as indices of nucleotide availability. Food deprivation significantly decreased mucosal growth markers as well as differentiation markers in both jejunum and ileum. The ATP pool was also significantly depressed, but the adenylate energy charge was not significantly altered. To a certain extent, refeeding restored the losses, but in the rats that were fed the nucleotide-free diet, the restoration of the jejunum was significantly slower and the restoration of the ileum differentiation markers was incomplete compared with the rats fed the nucleotide-supplemented diet. The results suggest that dietary nucleotide intake in the elderly may accelerate the normal physiological intestinal response to refeeding after food deprivation.
Subject(s)
Aging/physiology , Food Deprivation/physiology , Ileum/drug effects , Jejunum/drug effects , Nucleotides/pharmacology , Adenine Nucleotides/analysis , Adenosine Triphosphate/analysis , Animals , Body Weight/physiology , DNA/analysis , Eating/physiology , Food, Fortified , Hypoxanthines/analysis , Ileum/growth & development , Ileum/physiology , Intestinal Mucosa/chemistry , Intestinal Mucosa/enzymology , Jejunum/growth & development , Jejunum/physiology , Lactase , Male , Microvilli/enzymology , Proteins/analysis , Random Allocation , Rats , Rats, Wistar , Sucrase/analysis , alpha-Glucosidases/analysis , beta-Galactosidase/analysisABSTRACT
A high-performance liquid chromatographic method was developed for the determination of plasma purine nucleoside phosphorylase activity. In this method, the reaction mixture consisted of 15 microliters of plasma and 285 microliters of 50 mM phosphate buffer (pH 7.4) containing 3.8 mM inosine and 0.15 mM 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid (strong xanthine oxidase inhibitor). After the reaction, the hypoxanthine produced was monitored to express plasma purine nucleoside phosphorylase activity. By this method, the activity of purine nucleoside phosphorylase was easily determined even with a small-volume plasma sample and despite its low activity in plasma. In addition, plasma purine nucleoside phosphorylase activity can be accurately determined even if the plasma is turbid. As a result, we were able to measure plasma purine nucleoside phosphorylase activity in patients with gout or asthma and healthy subjects, whereby it was demonstrated that plasma purine nucleoside phosphorylase activity was higher in patients with asthma than in either healthy subjects or patients with gout.
Subject(s)
Chromatography, High Pressure Liquid/methods , Hypoxanthines/analysis , Inosine/metabolism , Purine-Nucleoside Phosphorylase/blood , Adult , Aged , Asthma/enzymology , Calibration , Febuxostat , Female , Gout/enzymology , Gout Suppressants/metabolism , Humans , Hypoxanthine , Hypoxanthines/metabolism , Male , Middle Aged , Purine-Nucleoside Phosphorylase/metabolism , Thiazoles/metabolism , Uric Acid/analysis , Uric Acid/metabolism , Xanthine Oxidase/antagonists & inhibitors , Xanthine Oxidase/metabolismABSTRACT
Hypoglycemia increases the vulnerability of the perinatal brain to asphyxia, but it is not known if hypoglycemia-induced changes in cerebral hemodynamics and vascular reactivity underlie this vulnerability. This study tested the hypothesis that hypoglycemia exacerbates postischemic hypoperfusion, and impairs postischemic CO2 reactivity. The authors also examined the hypothesis that postischemic hypoperfusion is associated with a reduction in the interstitial concentration of the vasodilator metabolite adenosine. Global cerebral ischemia of 10 minutes duration was induced in newborn pigs anesthetized with isoflurane by occlusion of subclavian and brachiocephalic arteries; cortical cerebral blood flow (CBF) and interstitial adenosine concentration were evaluated simultaneously using the combined hydrogen clearance/microdialysis technique. Hypoglycemia (blood glucose < 25 mg/dl) was induced by regular insulin (25 IU/kg) administered intravenously 2 hours prior to induction of ischemia. In the eight normoglycemic animals, baseline CBF was 38 +/- 4 ml/min/100 gm and baseline adenosine concentration was 1.2 +/- 0.1 microM; in the eight hypoglycemic animals, these values were 39% (p < 0.05) and 62% (p < 0.05) greater, respectively, under baseline conditions. At 1 hour of postischemic reperfusion in normoglycemic animals, CBF was reduced 39% relative to the preischemic baseline (p < 0.01), concomitant with a 27% reduction (p < 0.05) in adenosine concentration, suggesting that this lowered concentration may underlie delayed hypoperfusion. These postischemic reductions in CBF and interstitial adenosine concentration were significantly greater in hypoglycemic animals, with CBF and adenosine concentration reduced 70% (p < 0.001) and 71% (p < 0.01), respectively, relative to baseline. In nine animals preischemic reactivity to hypercapnia was unaffected by hypoglycemia. Postischemic hypercapnic reactivity was retained in the eight normoglycemic animals, but was attenuated 73% (p < 0.05) in hypoglycemic animals. Thus, in the newborn pig, hypoglycemia exacerbates postischemic cortical hypoperfusion and impairs postischemic cerebrovascular reactivity to hypercapnia.
Subject(s)
Adenosine/metabolism , Brain Ischemia/physiopathology , Cerebral Cortex/metabolism , Cerebrovascular Circulation/physiology , Hypercapnia/physiopathology , Hypoglycemia/physiopathology , Adenosine/analysis , Animals , Animals, Newborn , Blood Pressure/physiology , Carbon Dioxide/blood , Extracellular Space/chemistry , Extracellular Space/metabolism , Hypercapnia/blood , Hypoxanthine , Hypoxanthines/analysis , Hypoxanthines/metabolism , Inosine/analysis , Inosine/metabolism , Swine , Xanthine , Xanthines/analysis , Xanthines/metabolismABSTRACT
The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction's role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.
Subject(s)
Actins/physiology , Adenosine Triphosphate/deficiency , Cytoskeleton/physiology , Intercellular Junctions/physiology , Actins/ultrastructure , Adenosine Diphosphate/analysis , Adenosine Triphosphate/analysis , Animals , Cells, Cultured , Cytoskeleton/ultrastructure , Dogs , Electric Impedance , Epithelial Cells , Epithelium/physiology , Fluorescent Antibody Technique , Fluorescent Dyes , Freeze Fracturing , Hypoxanthine , Hypoxanthines/analysis , Image Processing, Computer-Assisted , Intercellular Junctions/ultrastructure , Kidney/cytology , Microscopy, Confocal , Microscopy, Electron , Microtubules/physiology , Microtubules/ultrastructure , Models, StructuralABSTRACT
Beta-endorphin may induce respiratory depression and bradycardia. Elevated levels of hypoxanthine (HX) in vitreous humour (VH) may possibly indicate hypoxia before death. Furthermore, gliosis in the brain stem may reflect a previous hypoxic/ischaemic injury in the brain. In the present study we relate beta-endorphin immunoreactivity (BENDI) in the CSF to the presence or absence of reactive astrocytosis in the nucleus olivae inferior (NOI). The relationship between the HX concentration in VH and the number of reactive astrocytes in sudden infant death (SID) cases (n = 17) and controls (n = 23) was also studied. The number of reactive astrocytes was examined in the NOI by immunohistochemical demonstration of glial fibrillary acidic protein (GFAP). The BENDI in CSF and the number of reactive astrocytes in the NOI divided the SID victims into two subpopulations (P < 0.01). One had a median of < 4 fmol/ml BENDI in CSF (range < 4) and 2 reactive astrocytes (range 0-15), and was similar to the controls that died from infections. The other subpopulation had a median of 260 fmol/ml BENDI in CSF (range 160-400) and 13 reactive astrocytes (range 7-33), similar to the control infants with previous hypoxia. In this latter SID subpopulation the number of reactive astrocytes correlated positively with BENDI in CSF (r = 0.7, P < 0.05). All the SID victims had elevated levels of HX in VH. In the SID subpopulation with high level of BENDI in CSF and increased number of activated astrocytes, the correlation factor between HX in VH and activated astrocytes was r = 0.7 (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Astrocytes , Brain Stem/chemistry , Gliosis/physiopathology , Hypoxanthines/analysis , Sudden Infant Death , Vitreous Body/chemistry , beta-Endorphin/cerebrospinal fluid , Astrocytes/pathology , Astrocytes/physiology , Brain Stem/pathology , Cell Count , Female , Glial Fibrillary Acidic Protein/analysis , Gliosis/pathology , Humans , Hypoxanthine , Infant , Male , Olivary Nucleus/pathology , Olivary Nucleus/physiology , Radioimmunoassay , Reproducibility of Results , Sudden Infant Death/cerebrospinal fluid , Sudden Infant Death/pathologyABSTRACT
A reversed-phase high-performance liquid chromatographic method has been developed for the analysis of purine and pyrimidine bases, uric acid and nucleosides largely relating to the purine synthetic and degradation metabolic pathways, with particular attention to the separation of hypoxanthine, xanthine and guanine. Complete separation and quantitation of the purines has been accomplished in the nanogram-microgram scale on conventional 4.6 mm I.D. columns with a standard gradient HPLC instrumentation as well as on 1 mm I.D. microbore columns with a dedicated isocratic micro-HPLC system using a dioxane-sodium acetate buffer. For the definite identification of components in excreta of ticks a GC-MS method has been described involving formation and GC of the trimethysilyl derivatives on a 25-m DB-5 column directly coupled with an ion trap detector. The methods are demonstrated on the analysis of the purine metabolites having an assembly pheromone effect on argasid ticks.
Subject(s)
Chromatography, Gas/methods , Chromatography, High Pressure Liquid/methods , Guanine/analysis , Hypoxanthines/analysis , Xanthines/analysis , Animals , Feces/chemistry , Gas Chromatography-Mass Spectrometry , Hypoxanthine , Ions , Ticks , XanthineABSTRACT
Hypoxanthine concentrations in vitreous humor were determined in 107 cases of sudden infant death syndrome (SIDS) and compared with levels in 4 cases of borderline SIDS, 26 cases of infectious death and 16 cases of sudden violent death. The hypoxanthine measurements were made using a high-performance liquid chromatography method. The hypoxanthine levels were significantly (p < 0.01) higher in SIDS than in violent deaths, while no significant difference was found between SIDS and infectious deaths. The present report demonstrates a similar distribution pattern of hypoxanthine levels in vitreous humor in SIDS and infectious death. We have previously described signs of immune stimulation both in peripheral organs and in the central nervous system in these conditions. This indicates that the death mechanism in SIDS has some similarities with infectious death.
Subject(s)
Hypoxanthines/analysis , Sudden Infant Death , Vitreous Body/chemistry , Age Factors , Child , Child, Preschool , Communicable Diseases , Female , Humans , Hypoxanthine , Infant , Infant, Newborn , MaleABSTRACT
The levels of pseudouridine (psi rd), uridine (Urd), hypoxanthine (Hyp), xanthine (Xan) and uric acid (UA) in both blood and urine samples were assayed by a reversed-phase high performance liquid chromatographic (HPLC) method. The evaluation of the sampling procedures showed that the levels of plasma oxypurines were overestimated by more than 15% for Hyp and 35% for Xan when heparinized plasma separation was delayed for 5 minutes after sampling. The fact that oxypurines levels of serum were significantly higher than those of plasma was also documented. Hence, for the accurate measurement of plasma oxypurines, plasma should be immediately separated from the blood with anticoagulants after it is collected. Physiological variation under a suitable sample preparation was also studied in 6 healthy male volunteers after their usual and a low purine diet for 4 days. The mean plasma levels for 6 male volunteers on a usual diet were 2.52 +/- 0.19 mumol/l for psi rd, 290.1 +/- 88.8 for UA, 49.4 +/- 1.24 for Urd, 0.82 +/- 0.30 for Hyp and 0.49 +/- 0.12 for Xan. On a low purine diet, the declines in the plasma oxypurine concentrations and the renal excretions of UA as well as oxypurine were demonstrated. When a dietary purine intake was restricted, the reference range of the Hyp level in plasma wa 0.48 +/- 0.26, and Xan was 0.36 +/- 0.17 mumol/l. Dietary influences on purine-pyrimidine metabolites should also be considered for the accurate measurement of their concentrations in blood and urine.
Subject(s)
Diet , Purines/metabolism , Pyrimidines/metabolism , Specimen Handling , Chromatography, High Pressure Liquid/methods , Humans , Hypoxanthine , Hypoxanthines/analysis , Male , Pseudouridine/analysis , Uric Acid/analysis , Uridine/analysis , Xanthine , Xanthines/analysisABSTRACT
Hypoxanthine (Hx) is a degradation product of adenosine. Increased concentrations were reported in cases of hypoxia as well as with prolonged postmortem interval (PMI). Hx is recommended as an indicator of prolonged (cerebral) hypoxia, for example in vitamins of sudden infant death as well as a new biochemical method for estimation of postmortem time. The correlation of vitreous Hx values with the time since death was reported to be even higher than the vitreous potassium (K+) values. The authors' investigations on 92 bodies with known time since death gave a completely opposite result: a much higher correlation between vitreous K+ and time since death than vitreous Hx. The possible discrepancies between these different results will be discussed (disturbing of intra-ocular fluid dynamics by repeated sample-taking in the study of Rognum et al. The results published so far on vitreous Hx values in sudden infant death syndrome (SIDS) cases as an indicator for a prolonged cerebral hypoxia are also not convincing. When vitreous concentrations of newborn infants or infants of age < 6 months are compared to those of older infants or adults the vitreous diameter must be taken into consideration (diffusion gradient; Fick's law of diffusion). The discrepant results on vitreous Hx as a measure of vital hypoxia and PMI will be discussed. The authors' results on Hx determinations on cerebrospinal fluid in comparison to cerebrospinal spinal (CSF) potassium will also be briefly addressed.