Placental Metabolomics for Assessment of Sex-specific Differences in Fetal Development During Normal Gestation.

The placenta is a metabolically active interfacial organ that plays crucial roles in fetal nutrient delivery, gas exchange and waste removal reflecting dynamic maternal and fetal interactions during gestation. There is growing evidence that the sex of the placenta influences fetal responses to external stimuli in utero, such as changes in maternal nutrition and exposure to environmental stressors. However, the exact biochemical mechanisms associated with sex-specific metabolic adaptations during pregnancy and its link to placental function and fetal development remain poorly understood. Herein, multisegment injection-capillary electrophoresis-mass spectrometry is used as a high throughput metabolomics platform to characterize lyophilized placental tissue (~2 mg dried weight) from C57BL/6J mice fed a standardized diet. Over 130 authentic metabolites were consistently measured from placental extracts when using a nontargeted metabolomics workflow with stringent quality control and robust batch correction. Our work revealed distinct metabolic phenotype differences that exist between male (n = 14) and female (n = 14) placentae collected at embryonic day E18.5. Intracellular metabolites associated with fatty acid oxidation and purine degradation were found to be elevated in females as compared to male placentae (p < 0.05, effect size >0.40), including uric acid, valerylcarnitine, hexanoylcarnitine, and 3-hydroxyhexanolycarnitine. This murine model sheds new insights into sex-specific differences in placental mitochondrial function and protective mechanisms against deleterious oxidative stress that may impact fetal growth and birth outcomes later in life.

A miniaturized electrochemical toxicity biosensor based on graphene oxide quantum dots/carboxylated carbon nanotubes for assessment of priority pollutants.

The study presented a sensitive and miniaturized cell-based electrochemical biosensor to assess the toxicity of priority pollutants in the aquatic environment. Human hepatoma (HepG2) cells were used as the biological recognition agent to measure the changes of electrochemical signals and reflect the cell viability. The graphene oxide quantum dots/carboxylated carbon nanotubes hybrid was developed in a facile and green way. Based on the hybrid composite modified pencil graphite electrode, the cell culture and detection vessel was miniaturized to a 96-well plate instead of the traditional culture dish. In addition, three sensitive electrochemical signals attributed to guanine/xanthine, adenine, and hypoxanthine were detected simultaneously. The biosensor was used to evaluate the toxicity of six priority pollutants, including Cd, Hg, Pb, 2,4-dinitrophenol, 2,4,6-trichlorophenol, and pentachlorophenol. The 24h IC50 values obtained by the electrochemical biosensor were lower than those of conventional MTT assay, suggesting the enhanced sensitivity of the electrochemical assay towards heavy metals and phenols. This platform enables the label-free and sensitive detection of cell physiological status with multi-parameters and constitutes a promising approach for toxicity detection of pollutants. It makes possible for automatical and high-throughput analysis on nucleotide catabolism, which may be critical for life science and toxicology.

Simultaneous quantification of IMPDH activity and purine bases in lymphocytes using LC-MS/MS: assessment of biomarker responses to mycophenolic acid.

BACKGROUND: The development of biomarkers describing the individual responses to the immunosuppressant mycophenolic acid (MPA) has focused on the target enzyme activity [inosine 5'-monophosphate dehydrogenase (IMPDH)]. An extended strategy is to quantify the metabolic consequences of IMPDH inhibition. The aim of this study was to develop an assay for quantification of IMPDH activity and related purine bases and to provide preliminary data on the behavior of these biomarkers during clinical exposure to MPA. METHODS: Liquid chromatography-mass spectrometry was used to determine xanthine (IMPDH activity in incubated cell lysate), hypoxanthine, guanine, and adenine derived from free nucleotides in lymphocytes. Analytical performance was assessed, and the biomarkers were examined in CD4⁺ cells from 2 groups: Healthy individuals in a single-dose MPA study (n = 5) and liver transplant recipients on MPA therapy (n = 15). RESULTS: Coefficients of variation between series were below 10% and 15% for measurement of the purines and IMPDH activity, respectively. Although IMPDH was inhibited, the purine levels increased in response to MPA in 3 of the 5 healthy individuals, and this positive response seemed to be associated with IMPDH1 c.579 + 119 G/G and c.580 - 106 G/G. In the liver transplant study, guanine was not reduced in response to the transient drop in IMPDH activity after MPA dosing. However, there were trends toward decrease in guanine and elevation of hypoxanthine during prolonged MPA therapy. The guanine/hypoxanthine ratio (median) was 37% lower and the adenine level was 21% lower at day 17 compared with day 4 after transplantation. CONCLUSIONS: The assay allows precise quantification of IMPDH activity, hypoxanthine, guanine, and adenine in lymphocytes. Some individuals may possess a counteracting purine response to the MPA-mediated inhibition of IMPDH. Reduction of the guanine/hypoxanthine ratio may be related to prolonged inhibition of IMPDH and seems as an intriguing pharmacodynamic biomarker for MPA.

Fuzzy intervention in biological phenomena.

An important objective of modeling biological phenomena is to develop therapeutic intervention strategies to move an undesirable state of a diseased network toward a more desirable one. Such transitions can be achieved by the use of drugs to act on some genes/metabolites that affect the undesirable behavior. Due to the fact that biological phenomena are complex processes with nonlinear dynamics that are impossible to perfectly represent with a mathematical model, the need for model-free nonlinear intervention strategies that are capable of guiding the target variables to their desired values often arises. In many applications, fuzzy systems have been found to be very useful for parameter estimation, model development and control design of nonlinear processes. In this paper, a model-free fuzzy intervention strategy (that does not require a mathematical model of the biological phenomenon) is proposed to guide the target variables of biological systems to their desired values. The proposed fuzzy intervention strategy is applied to three different biological models: a glycolytic-glycogenolytic pathway model, a purine metabolism pathway model, and a generic pathway model. The simulation results for all models demonstrate the effectiveness of the proposed scheme.

Assessment of dietary ratios of red clover and grass silages on milk production and milk quality in dairy cows.

Twenty-four multiparous Holstein-Friesian dairy cows were used in a replicated 4 x 4 Latin square changeover design experiment to test the effects of changing from ryegrass (Lolium perenne) silage to red clover (Trifolium pratense) silage in graded proportions on feed intakes, milk production, milk organoleptic qualities, and whole-body nitrogen partitioning. Four dietary treatments, comprising ad libitum access to 1 of 4 forage mixtures plus a standard allowance of 4 kg/d dairy concentrates, were offered. The 4 forage mixtures were, on a dry matter (DM) basis: 1) 100% grass silage, 2) 66% grass silage: 34% red clover silage, 3) 34% grass silage: 66% red clover silage, and 4) 100% red clover silage. In each of 4 experimental periods, there were 21 d for adaptation to diets and 7 d for measurements. There was an increase in both DM intakes and milk yields as the proportion of red clover in the diet increased. However, the increase in milk yield was not as great as the increase in DM intake, so that the efficiency of milk production, in terms of yield (kg) of milk per kg of DM intake, decreased. The concentrations of protein, milk fat, and the shorter chain saturated fatty acids decreased, whereas C18 polyunsaturated fatty acids (PUFA) and long-chain PUFA (C20+) increased as the proportion of red clover in the diet increased. There was little effect of dietary treatment on the organoleptic qualities of milk as assessed by taste panel analysis. There were no effects on the aroma of milk, on aftertaste, or overall liking of the milk. Milk was thicker and creamier in color when cows were fed grass silage compared with red clover silage. The flavor of milk was largely unaffected by dietary treatment. In conclusion, increasing the proportion of red clover in the diet of dairy cows increased feed intakes and milk yields, decreased the concentration of fat and protein in milk, increased PUFA for healthiness, and had little effect on milk organoleptic characteristics.

Assessment of nerve stimulation-induced release of purines from mouse kidneys by tandem mass spectrometry.

Adenosine, formed from AMP and metabolized to inosine, modulates renal sympathetic neurotransmission. The present study had two goals: 1) to develop ultrasensitive and specific mass spectrometry-based assays for cAMP, AMP, adenosine, inosine, and, for comparison, guanosine using state-of-the-art tandem liquid chromatography-mass spectrometry (LC-MS-MS); and 2) to quantify the effects of renal sympathetic nerve stimulation on the release of cAMP, AMP, adenosine, inosine, and guanosine from the isolated, perfused mouse kidney. Using LC-MS-MS, we developed highly sensitive (detection limit of 0.02-0.05 pg/microl) and accurate (r(2) > 0.99) assays for all the aforementioned compounds. In the perfused mouse kidney (n = 9), periarterial (renal sympathetic) nerve stimulation elicited a frequency-dependent (0, 3, 5, 7, and 9 Hz) and significant (p = 0.0148, repeated measures analysis of variance) increase in the concentration of inosine in the renal venous perfusate (29 +/- 8, 51 +/- 8, 54 +/- 11, 65 +/- 15, and 80 +/- 20 pg/microl, respectively), yet concomitantly decreased (p = 0.0239, repeated measures analysis of variance) the concentration of AMP in the renal venous perfusate (3.8 +/- 1.3, 3.2 +/- 1.7, 2.4 +/- 1.5, 2.0 +/- 1.1, and 1.1 +/- 0.4 pg/microl, respectively). No significant changes were observed in the levels of adenosine, cAMP, or guanosine in the renal venous perfusate. These results indicate that using state-of-the-art mass spectrometric methods, it is possible to investigate trace amounts of purines released from mouse organs in perfusion and that renal sympathetic nerve stimulation is associated with a robust increase in the main metabolite of adenosine (inosine), while concomitantly decreasing AMP. This suggests that renal sympathetic nerve stimulation influences the efficiency of AMP conversion to adenosine and hence to inosine.

Purines: from premise to promise.

Geoff Burnstock's remarkable insight and tenacity has established the area of purinergic research as a bona fide target for drug discovery. While efforts in P1 receptor-based medicinal chemistry and biology efforts over the past 25 years have not reached the level of success that the pharmaceutical industry investment may have anticipated, the P2 area, with knowledge of the selective localization of members of the P2X and P2Y family members and data from transgenic knockouts, has identified several potential therapeutic areas of major promise including cystic fibrosis, chronic bronchitis, male contraception and neurodegeneration. In addition, interest in the potential of purinergic therapeutics has extended outside the major pharmaceutical companies to the 'biotech industry' resulting in an environment where the inherent risks of 'first in field' in a therapeutic area may be more appropriately nurtured.

In vitro assessment of salvage pathways for pyrimidine bases in rat liver and brain.

In this paper we extend our previous observation on the mobilization of the ribose moiety from guanosine to xanthine catalyzed by rat liver extracts (Giorgelli et al., Biochim. Biophys. Acta 1335 (1997) 16-22). The data show that in rat liver and brain extracts the activated ribose, stemming from inosine and guanosine phosphorolysis as ribose 1-phosphate, can be used to salvage uracil to uracil nucleotides. Uridine is an intermediate. The salvage process occurs even in the presence of excess inorganic phosphate suggesting that uridine phosphorylase may function in vivo as an anabolic enzyme. Ribose 5-phosphate cannot substitute for inosine, guanosine or ribose 1-phosphate as ribose donor. When inorganic phosphate was substituted with arsenate, hindering the formation of ribose 1-phosphate, no ribose transfer could be observed. A similar pathway occurs at the deoxy level. The deoxyribose moiety of deoxyinosine can be used to salvage thymine to thymine nucleotides, again in the presence of excess inorganic phosphate. Our results introduce a novel aspect of the salvage pathway, in which ribose 1-phosphate seems to play a pivotal role.

Assessment of purine metabolism in human renal transplantation.

Cortical levels of nucleotides and their degradation products from 42 transplanted human kidneys have been studied. Biopsies were performed during renal harvesting just before cooling, at the end of cold storage, and following reinstallment of renal blood circulation. ATP levels fell, and AMP and degradation products (inosine monophosphate [IMP], inosine, adenosine, and hypoxanthine) increased during cold storage and returned to near-normal values 30 min after recirculation. The major degradation product found was hypoxanthine, indicating very poor xanthine oxidase activity in human kidneys. The sum of adenine nucleotides (ATP+ADP+AMP) did not significantly decrease after cold storage, but adenylate energy charge (ATP+1/2ADP/ATP+ADP+AMP) was reduced to half, being recovered in implanted kidneys. The sum of adenine nucleotides was significantly reduced after implantation. The rate of acute tubular necrosis was higher in kidneys preserved for more than 30 hr. Kidneys with acute tubular necrosis had significantly lower levels of the total pool of adenine nucleotides at reperfusion, but there was no correlation between incidence of acute tubular necrosis and ATP or other metabolite levels in the kidneys before or during cold preservation. The success of human kidney transplantation does not seem to depend only on the pool of residual nucleotides at the end of cold storage but on other factors that determine the ability of the cell to recover a normal energy state after reperfusion.

Attenuation of warm ischemic injury of rat lung by inflation with room air--assessment of cellular components and the surfactant in the bronchoalveolar lavage fluid in relation to changes in cellular adenosine triphosphate.

Studies were made on the effects in rat lungs of aerobic and anaerobic conditions on the intracellular levels of adenosine triphosphate and its related metabolites, the releases of intracellular enzymes, and the secretion of pulmonary surfactant. After warm ischemia for 120 min, the ATP content of lungs inflated with air was significantly higher (8.0 +/- 1.2 mumol/g dry weight) than those of deflated lungs and lungs inflated with nitrogen (0.8 +/- 0.7 mumol/g dry weight and 2.0 +/- 0.7 mumol/g dry weight, respectively; P < 0.001). The amounts of intracellular enzymes, such as lactate dehydrogenase, cytosolic and mitochondrial aspartate aminotransferase, and protein in the bronchoalveolar lavage fluid (BALF) of air-inflated lungs were significantly less than those in BALFs of deflated and nitrogen-inflated lungs (P < 0.001). The BALF-contents of dipalmitoyl phosphatidylcholine (DPPC), the main component of alveolar surfactant of aerobic and anaerobic ischemic lung were, however, similar. During 120-min warm ischemia after lavage, air-inflated lungs secreted significantly more DPPC into the alveolar space than nitrogen-inflated lungs did (P < 0.001). We conclude that cell membranes in the lungs are damaged under anaerobic conditions, but that inflation of ischemic lungs with air is effective for protecting them from cell injury and for maintaining the intracellular level of ATP and the ability of the cells to secrete pulmonary surfactant.

[Environmentally-induced causes of aging-dependent DNA modifications and possible consequences: current methods for the assessment of genotoxic potential of mixed pollution]. / Umweltbedingte Ursachen alternsabhängiger DNA-Veränderungen und mögliche Konsequenzen: Neue Methoden zur Erfassung des gentoxischen Potentials von Mischpollutionen.

The deoxyribonucleic acid (DNA) is subject of a series of modifications with time, which are connected with the development of cancer and the process of aging. However, an efficient and nearly perfect repair system works against them, which eliminates most but not all of these changes. A series of tests has been developed for assessment of genotoxic potential of compounds and mixed pollution of environment. Three fast assay systems (umu test, DIT test, and Visko test) are discussed, whose results are obtained more quickly than by using conventional methods (e.g., Ames test or sister chromatid exchange).

Assessment of purine-dopamine interactions in 6-hydroxydopamine-lesioned rats: evidence for pre- and postsynaptic influences by adenosine.

Lesch-Nyhan syndrome involves disorders of both purine and dopamine metabolism. Neonatal lesioning of dopaminergic neurons with 6-hydroxydopamine (6-OHDA) has been proposed as a rodent model of the dopamine deficiency in this childhood disorder. In the present studies, the functional interaction between purines and dopamine was examined in adult rats which received 6-OHDA lesions either as neonates or as adults. Even though dopamine levels were decreased by at least 92%, both neonatal- and adult-6-OHDA-lesioned rats had normal hypoxanthine-guanine phosphoribosyltransferase function and purine nucleotide levels (adenosine, ADP, ATP and AMP), indicating that hypoxanthine-guanine phosphoribosyltransferase is not localized only to dopaminergic neurons in striatum. However, the 6-OHDA-lesioned animals were supersensitive to the locomotor activating effects of the adenosine antagonist, theophylline, with the response being greater in adult-6-OHDA-lesioned rats. This effect was presynaptic to dopaminergic neurons as indicated by alpha-methyltyrosine blockade of the theophylline response and its reinstatement by L-dopa. The presynaptic nature of this action of theophylline was supported further by a lack of interaction between theophylline and the direct acting D1- and D2-dopamine agonists, SKF-38393 and LY-171555, respectively. After systemic administration of SKF-38393 or L-dopa, central microinjection of the adenosine agonists, 2-chloroadenosine or 5'-N-ethylcarboxamide adenosine, were effective in preventing self mutilation induced by these dopamine agonists in neonatally lesioned rats. Relative potencies of the adenosine agonists for A1 and A2-adenosine receptors suggested involvement of an A2-adenosine receptor in this action.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium falciparum: assessment of in vitro growth by [3H]hypoxanthine incorporation.

To evaluate rapidly Plasmodium falciparum growth in Vitro, [3H]hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, [3H]hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting [3H]hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.

Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase.

The conjugate of L-alanosine [L-2-amino-3-(N-hydroxyN-nitrosamino)propionic acid] and 5-amino-4-imidazolecarboxylic acid ribonucleotide has been synthesized in good yield by enzymatic means, using partially purified chicken liver 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (EC 6.3.2.6). The chromatographic behavior of this molecule was characterized, as was its ability to inhibit adenylosuccinate synthetase, an enzyme long considered to be the locus of action of the drug. The Ki of-L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide versus a partially purified adenylosuccinate synthetase frm the L5178y/AR leukemia of C57BL X DBA/2 F1 (hereafter called BD2F1) mice was 0.228 microM, whereas the Ki of L-alanosine was 57.23 mM. Administration of 50 microCi of DL-[1-14C]alanosine along with unlabeled L-alanosine (500 mg/kg) to BD2F1 mice bearing s.c. nodules of Leukemia L5178Y/AR resulted in the accumulation in tumors of a material with properties compatible with those of L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide. It coeluted with L-alanosyl-5-amino-r-imidazolecarboxylic acid ribonucleotide in the high-resolution chromatographic system used, was Bratton-Marshall positive, and inhibited adenylosuccinate synthetase strongly. In tumor nodules 2 hr after dosage, the concentration of this compound approximated 70 microM. Under the same circumstances, the intratumoral concentration of L-alanosine was found to be 440 microM. At this concentration, the antibiotic itself exerts only a marginal inhibition of leukemic adenylosuccinate synthetase. In ancillary studies, it was shown for the first time in vivo that the parenteral administration of L-alanosine reduces the specific activity of intratumoral adenylosuccinate synthetase by 70% and depresses the synthesis of DNA to an equivalent or greater extent; adenine but not hypoxanthine (both at 250 mg/kg) was able to reverse the latter inhibition. No effect on purine salvage enzymes was exerted by L-alanosine. Viewed in concert, these experiments establish that the adduct of L-alanosine with 5-amino-4-imidazolecarboxylic acid is formed by neoplastic cells in vivo and that this anabolite is most probably responsible for the inhibition of adneylosuccinate synthetase and, in turn, for the diminished synthesis of DNA seen after a therapeutic dose of L-alanosine.