Discovery of Hypoxanthine and Inosine as Robust Biomarkers for Predicting the Preanalytical Quality of Human Plasma and Serum for Metabolomics.

In cold human blood, the anomalous dynamics of adenosine triphosphate (ATP) result in the progressive accumulation of adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine, and hypoxanthine. While the ATP, ADP, AMP, and IMP are confined to red blood cells (RBCs), inosine and hypoxanthine are excreted into plasma/serum. The plasma/serum levels of inosine and hypoxanthine depend on the temperature of blood and the plasma/serum contact time with the RBCs, and hence they represent robust biomarkers for evaluating the preanalytical quality of plasma/serum. These biomarkers are highly specific since they are generally absent or at very low levels in fresh plasma/serum and are highly sensitive since they are derived from ATP, one of the most abundant metabolites in blood. Further, whether blood was kept at room temperature or on ice could be predicted based on inosine levels. An analysis of >2000 plasma/serum samples processed for metabolomics-centric analyses showed alarmingly high levels of inosine and hypoxanthine. The results highlight the gravity of sample quality challenges with high risk of grossly inaccurate measurements and incorrect study outcomes. The discovery of these robust biomarkers provides new ways to address the longstanding and underappreciated preanalytical sample quality challenges in the blood metabolomics field.

Elevated blood purine levels as a biomarker of seizures and epilepsy.

OBJECTIVE: There is a major unmet need for a molecular biomarker of seizures or epilepsy that lends itself to fast, affordable detection in an easy-to-use point-of-care device. Purines such as adenosine triphosphate and adenosine are potent neuromodulators released during excessive neuronal activity that are also present in biofluids. Their biomarker potential for seizures and epilepsy in peripheral blood has, however, not yet been investigated. The aim of the present study was to determine whether blood purine nucleoside measurements can serve as a biomarker for the recent occurrence of seizures and to support the diagnosis of epilepsy. METHODS: Blood purine concentrations were measured via a point-of-care diagnostic technology based on the summated electrochemical detection of adenosine and adenosine breakdown products (inosine, hypoxanthine, and xanthine; SMARTChip). Measurements of blood purine concentrations were carried out using samples from mice subjected to intra-amygdala kainic acid-induced status epilepticus and in video-electroencephalogram (EEG)-monitored adult patients with epilepsy. RESULTS: In mice, blood purine concentrations were rapidly increased approximately two- to threefold after status epilepticus (2.32 ± .40 µmol·L-1 [control] vs. 8.93 ± 1.03 µmol·L-1 [after status epilepticus]), and levels correlated with seizure burden and postseizure neurodegeneration in the hippocampus. Blood purine concentrations were also elevated in patients with video-EEG-diagnosed epilepsy (2.39 ± .34 µmol·L-1 [control, n = 13] vs. 4.35 ± .38 µmol·L-1 [epilepsy, n = 26]). SIGNIFICANCE: Our data provide proof of concept that the measurement of blood purine concentrations may offer a rapid, low-volume bedside test to support the diagnosis of seizures and epilepsy.

Improved ultra-high performance liquid chromatographic method for simultaneous determination of five gout-related metabolites in human serum.

Creatinine and purines are gout-related metabolites commonly quantified by liquid chromatography coupled with ultraviolet and mass spectrometry. However, the high cost of liquid chromatography coupled with mass spectrometry hindered its extensive use in ordinary hospitals and clinical laboratories. Using the traditional liquid chromatography method, the full separation of these metabolites in complex biological samples is still not achieved. In this study, an improved ultra-high-performance liquid chromatography with ultraviolet spectroscopy method was reported for quantitative determination of five gout-related metabolites (i.e., creatinine, uric acid, hypoxanthine, xanthine, and inosine) in human serum within 10 min. A UHPLC system equipped with a hydrophilic C18 column was used to improve separation, shorten analysis time, and increase analysis throughput. The performance of the method was validated by evaluating linearity (squared correlation coefficient > 0.9991), recovery (92.8-100.0%, with relative standard deviation < 4.7%), accuracy (relative errors < 14.6%), precision (0.2-4.1% for intraday and 2.1-7.3% for interday) and stability (-14.1 to 8.3% in autosampler for 12 h and -13.3 to 2.2% for freeze-thaw cycles). This method was successfully applied to quantify gout-related metabolites in serum samples of healthy controls and gout patients, which was expected to be used in the clinical investigation of gout at different stages.

Defining the functions of adenosine-to-inosine RNA editing through hematology.

PURPOSE OF REVIEW: The direct modification of RNA is now understood to be widespread, evolutionarily conserved and of consequence to cellular and organismal homeostasis. adenosine-to-inosine (A-to-I) RNA editing is one of the most common mammalian RNA modifications. Transcriptome-wide maps of the A-to-I editing exist, yet functions for the majority of editing sites remain opaque. Herein we discuss how hematology has been applied to determine physiological and malignant functions of A-to-I editing. RECENT FINDINGS: Functional studies have established that A-to-I editing and ADAR1, responsible for the majority of editing in blood cells, are essential for normal blood cell homeostasis. ADAR1 edits endogenous RNA and reshapes its secondary structure, preventing MDA5 from perceiving the cells own RNA as pathogenic. Roles for ADAR1 in human leukaemia, and most recently, cancer cell intrinsic and extrinsic functions of ADAR1 have been identified that highlight ADAR1 as a therapeutic target in cancer. SUMMARY: The studies reviewed have identified the key physiological function of ADAR1 and mechanistic basis for A-to-I editing in normal physiology and have now been extended to cancer. As our understanding of the biology and consequences of A-to-I editing evolve, it may be possible to target ADAR1 function advantageously in a number of settings.

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells.

PURPOSE: To develop and validate a method for the simultaneous measurement of adenosine, guanosine, and inosine derived from mono (MP) and triphosphate (TP) forms in peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs) and dried blood spots (DBS). METHODS: Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification. RESULTS: The assay was linear for each of the three quantification ranges: 10-2000, 1.0-200 and 0.25-50 pmol/sample for adenosine, guanosine, and inosine, respectively. Intraassay (n = 6) and interassay (n = 18) precision (%CV) were within 1.7 to 16% while accuracy (%deviation) was within -11.5 to 14.7% for all three analytes. Nucleotide monophosphates were less concentrated than triphosphates (except for inosine) and levels in PBMCs were higher than RBCs for all three nucleotides (10, 55, and 5.6 fold for ATP, GTP and ITP, respectively). DBS samples had an average (SD) of -26% (22.6%) lower TP and 184% (173%) higher MP levels compared to paired RBC lysates, suggesting hydrolysis of the TP in DBS. CONCLUSION: This method was accurate and precise for physiologically relevant concentrations of adenosine, guanosine and inosine nucleotides in mono- and triphosphate forms, providing a bioanalytical tool for quantitation of nucleotides for clinical studies.

Metabolic alterations in the sera of Chinese patients with mild persistent asthma: a GC-MS-based metabolomics analysis.

AIM: To character the specific metabolomics profiles in the sera of Chinese patients with mild persistent asthma and to explore potential metabolic biomarkers. METHODS: Seventeen Chinese patients with mild persistent asthma and age- and sex-matched healthy controls were enrolled. Serum samples were collected, and serum metabolites were analyzed using GC-MS coupled with a series of multivariate statistical analyses. RESULTS: Clear intergroup separations existed between the asthmatic patients and control subjects. A list of differential metabolites and several top altered metabolic pathways were identified. The levels of succinate (an intermediate in tricarboxylic acid cycle) and inosine were highly upregulated in the asthmatic patients, suggesting a greater effort to breathe during exacerbation and hypoxic stress due to asthma. Other differential metabolites, such as 3,4-dihydroxybenzoic acid and phenylalanine, were also identified. Furthermore, the differential metabolites possessed higher values of area under the ROC curve (AUC), suggesting an excellent clinical ability for the prediction of asthma. CONCLUSION: Metabolic activity is significantly altered in the sera of Chinese patients with mild persistent asthma. The data might be helpful for identifying novel biomarkers and therapeutic targets for asthma.

Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine.

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Tubuloglomerular feedback and renal function in mice with targeted deletion of the type 1 equilibrative nucleoside transporter.

A(1) adenosine receptors (A1AR) are required for the modulation of afferent arteriolar tone by changes in luminal NaCl concentration implying that extracellular adenosine concentrations need to change in synchrony with NaCl. The present experiments were performed in mice with a null mutation in the gene for the major equilibrative nucleoside transporter ENT1 to test whether interference with adenosine disposition by cellular uptake of adenosine may modify TGF characteristics. Responses of stop flow pressure (P(SF)) to maximum flow stimulation were measured in mice with either C57Bl/6 or SWR/J genetic backgrounds. Maximum flow stimulation reduced P(SF) in ENT1(-/-) compared with wild-type (WT) mice by 1.6 ± 0.4 mmHg (n = 28) and 5.8 ± 1.1 mmHg (n = 17; P < 0.001) in C57Bl/6 and by 1.4 ± 0.4 mmHg (n = 15) and 9 ± 1.5 mmHg (n = 9; P < 0.001) in SWR/J. Plasma concentrations of adenosine and inosine were markedly higher in ENT1(-/-) than WT mice (ado: 1,179 ± 78 and 225 ± 48 pmol/ml; ino: 179 ± 24 and 47.5 ± 9 pmol/ml). Renal mRNA expressions of the four adenosine receptors, ENT2, and adenosine deaminase were not significantly different between WT and ENT1(-/-) mice. No significant differences of glomerular filtration rate or mean arterial blood pressure were found while plasma renin concentration, and heart rates were significantly lower in ENT1(-/-) animals. In conclusion, TGF responsiveness is significantly attenuated in the absence of ENT1, pointing to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels in the juxtaglomerular apparatus interstitium.

Selective determination of inosine in the presence of uric acid and hypoxanthine using modified electrode.

This article describes the selective determination of inosine (INO) in the presence of important physiological interferents, uric acid (UA) and hypoxanthine (HXN), by differential pulse voltammetry at physiological pH (7.2) using the electropolymerized film of 3-amino-5-mercapto-1,2,4-triazole (p-AMTa) modified glassy carbon (GC) electrode. The electropolymerization of AMTa was carried out by the potentiodynamic method in 0.1M H(2)SO(4). An atomic force microscopy image shows that the p-AMTa film contains a spherical-like structure. Bare GC electrode fails to resolve the voltammetric signal of INO in the presence of UA and HXN due to the surface fouling caused by the oxidized products of UA and HXN. However, p-AMTa film modified GC electrode (p-AMTa electrode) not only separates the voltammetric signals of UA, HXN, and INO, with potential differences of 730 mV between UA and HXN and 310 mV between HXN and INO, but also shows enhanced oxidation current for them. The selective determination of INO in the presence of UA and HXN at physiological pH was achieved for the first time. Using the amperometric method, we achieved the lowest detection of 50 nM for INO. The practical application of the current modified electrode was demonstrated by determining the concentration of INO in human blood serum and urine samples.

Dialysis modality-dependent changes in serum metabolites: accumulation of inosine and hypoxanthine in patients on haemodialysis.

BACKGROUND: The body metabolism of patients with end-stage renal disease may be altered in response to long-term dialysis treatment. Moreover, the pattern of serum metabolites could change depending on the type of dialysis modality used. However, dialysis modality-dependent changes in serum metabolites are poorly understood. Our aim was to profile comprehensively serum metabolites by exploiting a novel method of (1)H-NMR-based metabonomics and identify the differences in metabolite patterns in subjects receiving haemodialysis (HD) and peritoneal dialysis (PD). METHODS: Anuric and non-diabetic HD patients were matched to PD patients for age, sex and dialysis duration. Accurate concentrations of serum metabolites were determined using the target-profiling procedure, and differences in the levels of metabolites were compared using multivariate analysis. RESULTS: Principal Components Analysis score plots showed that the metabolic patterns could be discriminated by dialysis modalities. Hypoxanthine and inosine were present only with HD, whereas serum xanthine oxidase activity and uric acid levels were not different. In contrast, PD was associated with higher levels of lactate, glucose, maltose, pyruvate, succinate, alanine, and glutamate linked to glucose metabolism and the tri-carboxylic acid cycle. Maltose appeared only in patients using icodextrin solution for PD. Known uraemic retention solutes such as urea, creatinine, myo-inositol and trimethylamine-N-oxide were increased in both dialysis groups. CONCLUSIONS: Metabonomics shows apparent differences in the profiles of serum metabolites between HD and PD, which were influenced by dialysis-related processes. Inosine and hypoxanthine are present only in HD patients, which is likely to represent more hypoxic and oxidative stress.

A rapid and simple chemiluminescence method for screening levels of inosine and hypoxanthine in non-traumatic chest pain patients.

A rapid and simple chemiluminescence method was developed for detection of inosine and hypoxanthine in human plasma. The method utilized a microplate luminometer with direct injectors to automatically dispense reagents during sample analysis. Enzymatic conversions of inosine to hypoxanthine, followed by hypoxanthine to xanthine to uric acid, generated superoxide anion radicals as a useful metabolic by-product. The free radicals react with Pholasin(®) , a sensitive photoprotein used for chemiluminescence detection, to produce measurable blue-green light. The use of Pholasin(®) and a chemiluminescence signal enhancer, Adjuvant-K™, eliminated the need for plasma clean-up steps prior to analysis. The method used 20 µL of heparinized plasma, with complete analysis of total hypoxanthine levels (inosine is metabolized to hypoxanthine using purine nucleoside phosphorylase) in approximately 3.7 min. The rapid chemiluminescence method demonstrated the capability of differentiating total hypoxanthine levels between healthy individuals, and patients presenting with non-traumatic chest pain and potential acute cardiac ischemia. The results support the potential use of chemiluminescence methodology as a diagnostic tool to rapidly screen for elevated levels of inosine and hypoxanthine in human plasma, potential biomarkers of acute cardiac ischemia.

Metabolomic Detection Between Pancreatic Cancer and Liver Metastasis Nude Mouse Models Constructed by Using the PANC1-KAI1/CD82 Cell Line.

Background: Pancreatic cancer (PC) has a poor prognosis and is prone to liver metastasis. The KAI1/CD82 gene inhibits PC metastasis. This study aimed to explore differential metabolites and enrich the pathways in serum samples between PC and liver metastasis nude mouse models stably expressing KAI1/CD82. Methods: KAI1/CD82-PLV-EF1α-MCS-IRES-Puro vector and PANC1 cell line stably expressing KAI1/CD82 were constructed for the first time. This cell line was used to construct 3 PC nude mouse models and 3 liver metastasis nude mouse models. The different metabolites and Kyoto encyclopedia of genes and genomes (KEGG) and human metabolome database (HMDB) enrichment pathways were analyzed using the serum samples of the 2 groups of nude mouse models on the basis of untargeted ultra-performance liquid chromatography-tandem mass spectrometry platform. Results: KAI1/CD82-PLV-EF1α-MCS-IRES-Puro vector and PANC1 cell line stably expressing KAI1/CD82 were constructed successfully, and all nude mouse models survived and developed cancers. Among the 1233 metabolites detected, 18 metabolites (9 upregulated and 9 downregulated) showed differences. In agreement with the literature data, the most significant differences between both groups were found in the levels of bile acids (taurocholic acid, chenodeoxycholic acid), glycine, prostaglandin E2, vitamin D, guanosine monophosphate, and inosine. Bile recreation, primary bile acid biosynthesis, and purine metabolism KEGG pathways and a series of HMDB pathways (P < .05) contained differential metabolites that may be associated with liver metastasis from PC. However, the importance of these metabolites on PC liver metastases remains to be elucidated. Conclusions: Our findings suggested that the metabolomic approach may be a useful method to detect potential biomarkers in PC.

Blood uridine concentration may be an indicator of the degradation of pyrimidine nucleotides during physical exercise with increasing intensity.

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.

Comparison of elimination and cardiovascular effects of adenine nucleosides administered intrapericardially or intravenously in anesthetized dog.

Intrapericardial (IP) administration of certain cardioactive agents allows investigation of local pharmacological actions on the heart and may carry potential benefit to influence myocardial function. The cardioprotective adenosine (ADO) and inosine (INO) may be the most representative candidates. Elimination and cardiovascular effects of IP and intravenously (IV) applied ADO and INO were compared on anesthetized dogs. Their pericardial and systemic concentrations were measured after consecutive administration of increasing ADO and INO doses. In the case of IP administration at the end of the incubation period, pericardial concentrations of adenine nucleosides significantly exceeded the control values. However, the IV applied ADO and INO were rapidly metabolized in the systemic plasma. As characteristic hemodynamic effects, small but sustained decrease in heart rate (IP ADO) and increase in myocardial contractility (IP INO) were observed. During IV administration, ADO and INO exerted remarkable effects on all hemodynamic variables, which then gradually disappeared in 15 minutes. In summary, the elimination of ADO and INO was significantly slower in the pericardial fluid than in the plasma. Considering the balanced cardiac actions and lack of strong systemic hemodynamic effects, IP administration of adenine nucleosides may suggest a promising approach in the local treatment of the diseased heart.

Disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics.

BACKGROUND: In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. METHODS: This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. RESULTS: The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. CONCLUSIONS: The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.

Metabolomic analysis of serum and myocardium in compensated heart failure after myocardial infarction.

AIMS: To determine the metabolic adaptations to compensated heart failure using a reproducible model of myocardial infarction and an unbiased metabolic screen. To address the limitations in sample availability and model variability observed in preclinical and clinical metabolic investigations of heart failure. MAIN METHODS: Metabolomic analysis was performed on serum and myocardial tissue from rabbits after myocardial infarction (MI) was induced by cryo-injury of the left ventricular free wall. Rabbits followed for 12 weeks after MI exhibited left ventricular dilation and depressed systolic function as determined by echocardiography. Serum and tissue from the viable left ventricular free wall, interventricular septum and right ventricle were analyzed using a gas chromatography time of flight mass spectrometry-based untargeted metabolomics assay for primary metabolites. KEY FINDINGS: Unique results included: a two- three-fold increase in taurine levels in all three ventricular regions of MI rabbits and similarly, the three regions had increased inosine levels compared to sham controls. Reduced myocardial levels of myo-inositol in the myocardium of MI animals point to altered phospholipid metabolism and membrane receptor function in heart failure. Metabolite profiles also provide evidence for responses to oxidative stress and an impairment in TCA cycle energy production in the failing heart. SIGNIFICANCE: Our results revealed metabolic changes during compensated cardiac dysfunction and suggest potential targets for altering the progression of heart failure.

Ethanol-induced activation of adenine nucleotide turnover. Evidence for a role of acetate.

Consumption of alcohol causes hyperuricemia by decreasing urate excretion and increasing its production. Our previous studies indicate that ethanol administration increases uric acid production by increasing ATP degradation to uric acid precursors. To test the hypothesis that ethanol-induced increased urate production results from acetate metabolism and enhanced adenosine triphosphate turnover, we gave intravenous sodium acetate, sodium chloride and ethanol (0.1 mmol/kg per min for 1 h) to five normal subjects. Acetate plasma levels increased from 0.04 +/- 0.01 mM (mean +/- SE) to peak values of 0.35 +/- 0.07 mM and to 0.08 +/- 0.01 mM during acetate and ethanol infusions, respectively. Urinary oxypurines increased to 223 +/- 13% and 316 +/- 44% of the base-line values during acetate and ethanol infusions, respectively. Urinary radioactivity from the adenine nucleotide pool labeled with [8-14C] adenine increased to 171 +/- 27% and to 128 +/- 8% of the base-line values after acetate and ethanol infusions. These data indicate that both ethanol and acetate increase purine nucleotide degradation by enhancing the turnover of the adenine nucleotide pool. They support the hypothesis that acetate metabolism contributes to the increased production of urate associated with ethanol intake.

The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes.

An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes.

Excess purine degradation in exercising muscles of patients with glycogen storage disease types V and VII.

To investigate purine catabolism in exercising muscles of patients with muscle glycogen storage disease, we performed ischemic forearm exercise tests and quantitated metabolites appearing in cubital venous blood. Two patients with glycogen storage disease type V and three with glycogen storage disease type VII participated in this study. Basal lactate concentrations lowered in every patient with glycogen storage disease type V or type VII. Two patients with glycogen storage disease type VII, who had markedly elevated concentrations of serum uric acid (14.3 and 11.9 mg/dl, respectively), showed high basal concentrations of ammonia (118 and 79 mumol/liter, respectively; 23 +/- 4 mumol/liter in healthy controls) and of hypoxanthine (23.4 and 20.4 mumol/liter, respectively; 2.0 +/- 0.4 mumol/liter in healthy controls). Other patients showed near normal measurements of these metabolites. After forearm exercise, ammonia, inosine, and hypoxanthine levels increased greatly in every patient studied, in contrast with the lack of increase in lactate levels. The incremental area under the concentration curves for venous ammonia was 13-fold greater in the glycogen storage disease group than in controls (1,120 +/- 182 vs. 83 +/- 26 mumol X min/liter). The incremental areas of inosine and hypoxanthine were also greater in the glycogen storage disease group (29.2 +/- 7.2 vs. 0.4 +/- 0.1 and 134.6 +/- 23.1 vs. 14.9 +/- 3.2 mumol X min/liter, respectively). The incremental areas of ammonia in controls and in glycogen storage disease patients strongly correlated with those of hypoxanthine (r = 0.984, n = 11, P less than 0.005). These findings indicated that excess purine degradation occurred in the exercising muscles of patients with glycogen storage disease types V and VII, and suggested that the ATP pool in the exercising muscles may be deranged because of defective glycogenolysis or glycolysis.

Release of adenosine from human hearts during angina induced by rapid atrial pacing.

This study was designed to determine whether human hearts release adenosine, a possible regulator of coronary flow, during temporary myocardial ischemia and, if so, to examine the mechanisms involved. Release of adenosine from canine hearts had been reported during reactive hyperemia following brief coronary occlusion, and we initially confirmed this observation in six dogs hearts. Angina was then produced in 15 patients with anginal syndrome and severe coronary atherosclerosis by rapid atrial pacing during diagnostic studies. In 13 of these patients, adenosine appeared in coronary sinus blood, at a mean level of 40 nmol/100 ml blood (SE = +/-9). In 11 of these 13, adenosine was not detectable in control or recovery samples; when measured, there was concomitant production of lactate and minimal leakage of K(+), but no significant release of creatine phosphokinase, lactic acid dehydrogenase, creatine, or Na(+). THERE WAS NO DETECTABLE RELEASE OF ADENOSINE BY HEARTS DURING PACING OR EXERCISE IN THREE CONTROL GROUPS OF PATIENTS: nine with anginal syndrome and severe coronary atherosclerosis who did not develop angina or produce lactate during rapid pacing, five with normal coronaries and no myocardial disease, and three with normal coronaries but with left ventricular failure. The results indicate that human hearts release significant amounts of adenosine during severe regional myocardial ischemia and anaerobic metabolism. Adenosine release might provide a useful supplementary index of the early effects of ischemia on myocardial metabolism, and might influence regional coronary flow during or after angina pectoris.