Phosphomonoesters Research Articles

20 PHOSPHORUS METABOLITES AND INTRACELLULAR pH IN THE BRAINS OF NORMAL AND GROWTH RETARDED INFANTS

Studies by magnetic resonance spectroscopy were performed at a median age of 5 days on 18 appropriately grown (AGA) and 9 growth retarded (SGA) newborn infants with birthweights below the 3rd centile, to investigate: 1. Maturational changes in cerebral intracellular high-energy phosphorus (P) metabolite concentrations and pHi during normal gestation, and: 2. Whether any abnormalities were detectable in the SGA infants. The methods used have been described previously (Hope PL et al, Lancet 1984, ii: 366). Linear regressions were calculated for P-metabolite concentration ratios and pH. on gestational plus postnatal age for the AGA infants. Phosphocreatine (PCr)/inorganic orthophosphate (Pi) increased from 0.77±0.24 (95% confidence limits) at 28w to 1.09±0.24 at 42w (p<0.001). No significant changes were found in adenosine triphosphate (ATPl/total P, PCr/ATP, Pi/ATP, phosphomonoester (PME)/ATP, phosphodiester (PDE)/ATP, or pH., though PCr/ATP tended to rise, and Pi/ATP and PME/ATP tended to fall. Values from the SGA infants at 30-38w of gestational plus postnatal age were distributed evenly across the data from the AGA infants. We conclude: 1. PCr/Pi increased with advancing maturation of the brain, and: 2. Values for P-metabolite ratios and pHi in the SGA infants did not differ from those in the AGA infants.

Dissolved Organic Phosphorus Compounds: Do They Satisfy Planktonic Phosphate Demand in Summer?

The rate of release of phosphate from dissolved organic phosphorus (DOP) compounds generally accounted for &lt;1% of the phosphate uptake rate by seston in the open water of five diverse freshwater systems in summer. Surface water samples were taken during July and August 1984 from a eutrophic lake (East Twin Lake, OH), an acid bog lake (Triangle Bog Lake, OH), a freshwater estuarine marsh that empties into the western basin of Lake Erie (Old Woman Creek, OH), and two large mainstem reservoirs on the Savannah River (R. B. Russel Reservoir and Clarks Hill Lake, GA). In each of these, phosphatase hydrolysable phosphomonoesters (PME) often were the major fraction of DOP; phosphate release from photosensitive DOP was not detected in any of these systems at this time. The rate of release of phosphate from PME was calculated from Michaelis–Menten kinetics, and phosphatase activity was estimated spectrophotometrically using p-nitrophenyl phosphate as a model substrate. Radiometric analysis of the rate of phosphate uptake by seston showed that phosphate was sorbed to seston by at least two different processes. The total uptake rate by all uptake processes exhibited an apparent first-order dependence on the concentration of available phosphate. Typically, the velocity of uptake was 1–10 nmol∙L−1∙min−1, and the velocity of release from PME was 0.01–0.06 nmol∙L−1∙min−1.

The effects of pregnancy and parturition on phosphorus metabolites in rat uterus studied by 31P nuclear magnetic resonance.

Concentrations of phosphorus metabolites and intracellular pH have been measured in non-pregnant, late-pregnant and post-partum rat uterus using 31P nuclear magnetic resonance (31P n.m.r.). Intact uterine tissue was superfused with oxygenated de-Jalon solution at 4, 20 or 37 degrees C while inside the n.m.r. spectrometer. The phosphocreatine concentration [PCr], was higher and the inorganic phosphate concentration [Pi], lower than values determined by chemical analysis of extracts from both pregnant and non-pregnant rat uterus. [PCr] was 1.4-fold greater in late-pregnant than in non-pregnant rat uterus. Following parturition, large changes were observed in [PCr], [Pi] and in an unidentified metabolite in the phosphomonoester (PME) region of the n.m.r. spectrum. The time course of the recovery of these metabolites to prepregnant values was determined. The [PCr] remained below the non-pregnant value for at least 1 week post-partum and the [Pi] was elevated, compared to the non-pregnant value, during this period. More rapid changes were seen in the [PME], which doubled on day 0 post-partum but almost returned to its non-pregnant value on day 1 post-partum. No significant difference was observed between intracellular pH values in late-pregnant and non-pregnant rat uterus; however, there was a large acid shift following parturition. Intracellular pH depended upon the temperature at which the tissue was maintained. The effect of muscular work during parturition was investigated by comparing Caesarian-sectioned uteri with uteri which had undergone normal parturition. Uteri examined 1 day after Caesarian operation showed no differences in metabolite levels from normal, 1 day post-partum uteri. We conclude that concentrations of phosphorus metabolites depend upon the physiological state of the uterus. We suggest that the changes following parturition are not a consequence of the mechanical work performed by the uterus, but must be caused by some other event associated with parturition such as hormonal changes.

1673 IN VIVO VARIATION OF BRAIN OXIDATIVE metabolism with therapeutic lumbar PUNCTURE (LP)

Sequential cerebral spectra of phosphorus-containing compounds, ATP, phosphocreatine (Pcr), inorganic phosphate (Pi), phosphodiester (PD), and phosphomonoester (PME), by 31-P nuclear magnetic resonance (NMR) were obtained in a severely asphyxiated premature infant with hydrocephalus and seizures. Between days 2-20, 10 therapeutic LP's were done and NMR cerebral spectra were obtained pre (a) and post (p) LP. Pcr/Pi (normal x = 1.01), a measure of bioenergetic reserve, was low at 48h; Left (L) = 0.50, Right (R) = 0.60. After the initial LP, Pcr/Pi rose to L=1.4 and R=0.6. After subsequent LP's, a sharp decrease in Pcr/Pi was followed by a rebound (a LP, L=1.50, R=1.03; p LP, L=0.87, R=0.62; 1½ hrs p LP, L=1.52, R=1.66). Pcr/Pi increased by day 8 (L=1.52, R=1.60) then stabilized during the next month (L=1.0, R=0.8). Tissue pH did not change significantly (day 5: a LP=7.07, p LP=7.02, 1½ hrs p LP=7.08; day 8: a LP=7.12, p LP=7.02, 1½ hrs p LP=1.12). PME/β-ATP, a precursor in membrane biosynthesis, declined (day 2, L=3.15, R=2.53; day 46, L=1.46, R=1.17) but changed inconsistently post LP. Acute changes in Pcr/Pi and PME/β-ATP represent changes in local cerebral metabolism. Removal of 5 cc of CSF may result in acute intracerebral tissue shifts, acute hydrostatic changes with probable effects on CSF and cerebral blood flow, and hence provision of metabolic substrate to brain tissue. These data suggest that acute transient changes in Pcr/Pi and PME/β-ATP, representing changes in local cerebral metabolism, reflect general changes in cerebral metabolic or seizure activity.

Observations of energy metabolism in neuroectodermal tumors using in vivo 31P-NMR

The energy metabolism of living tumors in rats and hamsters were investigated by obtaining in vivo 31P-NMR spectra, and the effects of chemotherapy on tumors were evaluated by observing the changes of these spectra. Tumor cells of rat glioma, human glioblastoma and human neuroblastoma were inoculated subcutaneously in the lumbar region of the animals. After the tumor grew to over 1.5 cm in diameter, in vivo 31P-NMR spectrum data was obtained selectively from the tumor with a TMR-32 spectrometer (Oxford Research Systems, U.K.). Several peaks (ATP, inorganic phosphate (Pi), phosphodiesters and phosphomonoesters (PME)) were observed in the tumors. The heights of these peaks varied widely corresponding to the tumor growth. However, the spectrum pattern of each tumor in an active stage was found to be essentially the same regardless of histological type or tumor origin. The phosphocreatine (PCr) peak was small, ATP and PME peaks were large and tissue pH calculated from the chemical shift of Pi was low in each tumor group. After intravenous injection of a large dose of a chemotherapeutic agent, ATP peaks decreased and the Pi peak increased gradually, resulting in a dominant Pi peak pattern after several hours in all groups. With lower drug doses, spectrum changes were temporarily seen in the tumors. These findings indicated that drugs with a high dose have a selective and a direct action on the energy metabolism of tumor tissues. In vivo 31P-NMR spectra measurement is very valuable not only to investigate the energy metabolism in tumor tissue but also to evaluate the effects of chemotherapy on the tumor.

Relationships Between Phosphorus Functional Classes and Alkaline Phosphatase Activity in Reservoir Lakes

ABSTRACT Dissolved phosphorus fractions present in the surface waters of a eutrophic reservoir lake in northcentral Oklahoma were characterized by chemical, enzymatic, and chromatographic techniques. Simultaneous measurements of alkaline phosphatase activity (APA) were also conducted on each lakewater sample. An inverse biphasic linear relationship was found between soluble reactive phosphorus concentrations (SRP) and APA on a seasonal basis. Phosphomonoesters (PME) comprised from 0–65 percent of the soluble unreactive phosphorus (SUP) pool and an inverse linear relationship was found between PME concentrations and APA on a seasonal basis. Gel filtration chromatography of filtered lake water from the above system and a mesotrophic lake exhibiting high clay turbidity supported the view that the majority of complex phosphorus in these systems existed as low molecular weight phosphate esters.

Estrogen-induced changes in high-energy phosphate metabolism in rat uterus: 31P NMR studies.

Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found to be the following: creatine phosphate (CP), 2.1 +/- 0.2; nucleoside triphosphates, mainly adenosine 5'-triphosphate (ATP), 4.6 +/- 0.4; phospho monoesters, primarily sugar phosphates (SP), 5.4 +/- 0.7; and inorganic phosphate (Pi), 0.8 +/- 0.4. Adenosine 5'-diphosphate (ADP) concentration was estimated to be approximately 40 mumol/kg wet weight from the assumed equilibrium of the creatine kinase reaction. The concentration of CP, and to lesser extent ATP and SP, declined within the first 1.5-3 h after injection of 17 beta-estradiol, returned to control values between 6 and 12 h, and then increased, reaching maximal concentrations at 24 h. From the fractions of the total soluble ATP in free and Mg2+-bound forms, [free Mg2+] in the untreated uterus was estimated to be 0.2-0.4 mmol/kg wet weight. An increase in [free Mg2+] in the uterus was detected 1.5 h after estrogen injection. A subsequent parallel increase in the ratio of ATP to CP concentrations suggests that estrogen can also affect the apparent creatine kinase equilibrium by modulating [free Mg2+].

STUDIES OF OXIDATIVE METABOLITES USING 31-P NMR SPECTROSCOPY IN NEWBORN NEUROLOGIC DISORDERS

We have previously reported that cerebral spectra from newborn babies have high phosphomonoester (PME) concentration (7 m mole/kg), low phosphocreatine (PCr) concentration (1 m mole/kg), low PCr/Pi (inorganic phosphate) ratio (2), and intracellular pH 7.1. We present 31-P NMR results in: maple syrup urine disease, congenital lactic acidosis, gluconeogenic disorder, fetal alcohol syndrome, focal seizures and stroke, severe neurogenic arthrogryposis, lobar holoprosencephaly, and Group B strep meningitis. Spectroscopy was performed after recovery from acute metabolic or infectious abnormalities on the hypothesis that there are persistent changes in oxidative metabolites. Thus, during the study, babies were not stressed or given metabolic challenges. As a group, these babies had a phosphate potential (PCr/Pi 1.0) which is characteristic of lactic acidosis. In the child with focal seizures and stroke, PCr/Pi was 0.8 in the injured hemisphere vs 1.8 in the normal hemisphere. In the baby with severe neurogenic arthrogryposis, PCr/Pi in resting muscle was 0.3 vs > 7.0 for normal resting muscle. While there were individual differences, the mean concentration of PCr and PME and mean pH did not differ significantly from control infants. These data suggest that a wide variety of neonatal neurologic syndromes may cause persistent changes in oxidative metabolites and that 31-P NMR spectroscopy may yield significant information on the minimal value of PCr/Pi that is consistent with aerobic metabolism. (NIH T35-HD-07217-1OAI and NIH-HD-15973-01)

The reactivity of phosphomono-and phosphodiester groups in oligonucleotides

The rate constants were estimated by phosphorus NMR spectroscopy for the reactions of alcohols (Tr-dT, 2-cyanoethanol) in pyridine with the main types of the reactive phosphorylating intermediates formed by treatment of pdT-Ac, pdTpdT-Ac, Tr-dTpdT-Ac, Tr-dTpdTpdT-Ac with 2, 4, 6-triisopropylbenzene-sulfonyl chloride (TPS): 1) B type derivatives with phosphomono ester (PME) group converted to a phosphoryl pyridinium residue; 2) C type derivatives with PME and phosphodiester (PDE) groups converted to trisubstituted pyrophosphate; 3) D type derivatives with PDE groups converted to tetrasubstituted pyrophosphate. The two latter types are partially present as cyclic intramolecular pyrophosphates Ci and Di. The reactivity of the intermediates decrease in the series B greater than Ci approximately Di greater than C approximately D. The Ci derivative of pdTpdT-Ac when obtained in dimethylformamide was found to be rather stable to hydrolysis and could be separated from the other dinucleotide derivatives by ion-exchange chromatography. The Arrhenius parameters of all steps of the conversion of PME group of pdT-Ac to B derivative and of the reaction of TPS with PDE group of dinucleoside phosphate Tr-dTpdT-Ac were measured.