Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.     

Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy

The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.     

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+].     

31P-NMR studies of metabolite compartmentation in Fasciola hepatica

Fasciola hepatica, the common liver fluke, is an anaerobic parasitic worm. Possible compartmentation of metabolites between different cell types, metabolic compartments, and free and macromolecule-bound species was investigated using 31P-NMR. A spectrum of the intact worm shows unusual metabolic features, among which are large amounts of glycerolphosphorylcholine, phospholipids mobile on the NMR time-scale, and free cytosolic ADP. Spectra from cells as different as those in oral sucker tissue and eggs showed similar features. Acidosis after serotonin administration was associated with parallel changes in chemical shifts of intracellular Pi and glucose 6-phosphate, suggesting that they are in the same metabolic compartment. Although 13.4 +/- 1.1 mumol/g wet wt. (n = 3) Mg2+ is present in fluke tissue, a considerable fraction is sequestered out of the cytosol. The intracellular free [Mg2+] was independently estimated from the chemical shifts of ATP and ADP as 1.6 +/- 0.5 mM and 2.9 +/- 0.7 mM, respectively. Quantitation of observable phosphate-containing metabolites in whole tissue and in perchlorate extracts demonstrated that 60% of the total ADP and 50% of the total Pi are 'NMR-invisible' in the intact fluke and therefore probably bound to macromolecules in the cells. The apparent ATP/ADP X Pi free concentration ratio is much lower in this anaerobic tissue than in mammalian oxidative tissues.     

Further studies on alterations in magnesium binding during cold storage of erythrocytes

Free intracellular Mg2+ concentration ([Mg2+]i) was measured in cold-stored human erythrocytes by the method of null-point titration with ionophore A23187. [Mg2+]i was 311 +/- 41 microM (mean +/- S.D.) for cells stored 0-10 days, increasing to 458 +/- 64 microM for cells stored 22-48 days. The values for stored cells were higher than those previously determined by a 31P-NMR method (Bock et al. (1985) Blood 65, 1526-1530); however, the null-point method requires extensive washing of the cells, which we have found to increase NMR-measured [Mg2+]i. The null-point values still represent a small fraction of total cell Mg2+, and confirm that binding of Mg2+ to ligands other than ATP and 2,3-bisphosphoglycerate must increase during storage. As an initial test of whether this may imply suboptimal availability of Mg2+ for cell preservation, we used A23187 to prepare erythrocytes with altered Mg2+ content, then removed ionophore and stored the cells in plasma-free medium for up to 2 weeks. Higher Mg2+ content had a very significant positive correlation (P less than 0.0001) with higher cell ATP concentrations. Storage did not significantly affect basal or Na+-stimulated efflux of Mg2+ from Mg2+-loaded red cells.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Calcium affects phosphoinositide turnover in human erythrocytes

Changes in extracellular Ca2+ concentration ([Ca2+]) were observed to affect 32Pi incorporation into polyphosphoinositides (PPI) and phosphatidic acid (PA) of human erythrocytes. A decrease of extracellular [Ca2+] from 1.5 mmol/l to 0.04 mumol/l increased the specific radioactivity (S.A.) of phosphatidylinositol 4,5-bisphosphate to 182% and that of phosphatidylinositol 4-phosphate to 120% of controls. Simultaneously S.A. and concentration of PA decreased. Further decrease of the extracellular [Ca2+] from 0.04 mumol/l to lower values as well as depletion of intracellular Ca2+ using ionophore A 23187 in Ca2(+)-free medium did not accelerate the PPI turnover rates any more. None of the above changes in extracellular [Ca2+] had any effect on the phosphorylation pattern of erythrocyte membrane proteins. Isolated erythrocyte membranes were incubated in the presence of [gamma-32P]ATP in media with various [Ca2+]. The decrease of [Ca2+] from 0.04 mumol/l (physiological concentration inside the cell) to lower values did not influence the turnover of PPI and PA monoester phosphates. Only after [Ca2+] was increased to 1-5 mumol/l an increase of PPI and PA turnover was observed. Our data suggest that the changes in extracellular [Ca2+] affect the metabolism of PPI and PA (despite the intracellular location of the latter) and may thus influence the properties of red cell plasma membrane.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Intracellular-free magnesium in the smooth muscle of guinea pig taenia caeci: a concomitant analysis for magnesium and pH upon sodium removal

This study is concerned with the regulation of intracellular-free Mg2+ concentration ([Mg2+]i) in the smooth muscle of guinea pig taenia caeci. To assess an interaction of Ca2+ on the Na(+)-dependent Mg(2+)- extrusion mechanism (Na(+)-Mg2+ exchange), effects of Na+ removal (N- methyl-D-glucamine substitution) were examined in Ca(2+)-containing solutions. As changes in pHi in Na(+)-free solutions perturb estimation of [Mg2+]i using the single chemical shift only of the beta-ATP peak in 31P NMR (nuclear magnetic resonance) spectra, [Mg2+]i and pHi were concomitantly estimated from the chemical shifts of the gamma- and beta- peaks. When extracellular Na+ was substituted with N-methyl-D- glucamine, [Mg2+]i was reversibly increased. This increase in [Mg2+]i was eliminated in Mg(2+)-free solutions and enhanced in excess Mg2+ solutions. ATP content fluctuated little during removal and readmission of Na+, indicating that [Mg2+]i changes were not induced by Mg2+ release from ATP, and that Mg(2+)-extruding system would not be inhibited by fuel restriction. A slow acidification in Na(+)-free solutions and transient alkalosis by a readmission of Na+ were observed regardless of the extracellular Mg2+ concentration. When the extracellular Ca2+ concentration was increased from normal (2.4 mM) to 12 mM, only a marginal increase in [Mg2+]i was caused by Na+ removal, whereas a similar slow acidosis was observed, indicating that extracellular Ca2+ inhibits Mg2+ entry, and that the increase in [Mg2+]i is negligible through competition between Mg2+ and Ca2+ in intracellular sites. These results imply that Na(+)-Mg2+ exchange is the main mechanism to maintain low [Mg2+]i even under physiological conditions.     

An Na+-stimulated Mg2+-transport system in human red blood cells

The initial rate of net Mg2+ efflux was measured in human red blood cells by atomic absorption. In fresh erythrocytes incubated in Na+,K+-Ringer's medium this rate was 7.3 +/- 2.8 mumol/l cells per h (mean +/- S.D. of 14 subjects) with an energy of activation of 13 200 cal/mol. Cells with total Mg2+ contents ([ Mg]i) ranging from 1.8 to 24 mmol/l cells were prepared by using a modified p-chloromercuribenzenesulphonate method. Mg2+ efflux was strongly stimulated by increases in [Mg]i and in external Na+ concentrations ([ Na]o). A kinetic analysis of Mg2+ efflux as a function of [Mg]i and [Na]o revealed the existence of two components: an Na+-stimulated Mg2+ efflux, which exhibited a Michaelian-like dependence of free internal Mg2+ content (apparent dissociation constant = 2.6 +/- 1.4 mmol/l cells; mean +/- S.D. of six subjects) and on external Na+ concentration (apparent dissociation constant = 20.5 +/- 1.9 mM; mean +/- S.D. of four subjects) and a variable maximal rate ranging from 35 to 370 mumol/l cells per h, and an Na+-independent Mg2+ efflux, which showed a linear dependence on internal Mg2+ content with a rate constant of (6.6 +/- 0.7) X 10(-3) h-1. Fluxes catalyzed by the Na+-stimulated Mg2+ carrier were partially dependent on the ATP content of the cells and completely inhibited by quinidine (IC50 = 50 microM) and by Mn2+ (IC50 = 0.5-1.0 mM).     

Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes

1. Erythrocytes in whole blood samples from dogs with phosphofructokinase (PFK) deficiency had lower 2,3-diphosphoglycerate (2,3-DPG) concentrations, higher ATP concentrations, and were more alkaline fragile than normal canine erythrocytes. 2. Reticulocytes from a PFK-deficient dog contained nearly three times the ATP concentration of normal canine erythrocytes, and had 2,3-DPG concentrations similar to normal canine erythrocytes. 3. PFK-deficient reticulocytes are not alkaline fragile. 4. The erythrocyte 2,3-DPG concentration in whole blood samples from PFK-deficient dogs was increased to normal by in vitro incubation with dihydroxyacetone, pyruvate and phosphate. This incubation resulted in only a slight increase in ATP concentration. 5. The alkaline fragility of these 2,3-DPG replenished PFK-deficient erythrocytes was normal. 6. Findings in this study indicate that the increased alkaline fragility of canine PFK-deficient erythrocytes is the result of decreased intracellular 2,3-DPG concentration.     

31P NMR spectroscopy, chemical analysis, and free Mg2+ of rabbit bladder and uterine smooth muscle

31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.     

Phosphatic metabolites, intracellular pH and free [Mg2+] in single, intact porcine carotid artery segments studied by 31P-NMR

Superfused porcine carotid artery segments (approximately 7 cm lengths) were analyzed by 31P-NMR spectroscopic methods to characterize the 31P spectrum of arterial smooth muscle and to determine the influence of passive stretch (intraluminal pressurization, 95-100 mmHg) on cellular phosphatic metabolite levels, intracellular pH and free magnesium concentration ([Mg2+free]i). Equilibrated, single, intact arteries were studied under steady-state, constant flow conditions at 37 degrees C. Phosphoethanolamine, phosphocholine, inorganic phosphate (Pi), phosphocreatine (PCr) and nucleoside triphosphates (NTP), primarily ATP, were the principle metabolites detected in the 31P-NMR spectrum of intact arterial smooth muscle. The concentration of these metabolites and intracellular pH, as determined from the referenced chemical shift of Pi, were unaffected by pressurization. The PCr:Pi ratios determined for nonpressurized (flaccid) and pressurized arteries were 1.2 +/- 0.1 and 1.3 +/- 0.3, respectively. Intracellular pH averaged 7.02 +/- 0.02 (mean +/- 1 S.D.) for flaccid arteries vs. 7.03 +/- 0.05 for pressurized arteries. The upfield chemical shift of the beta-ATP peak, which has been described in other types of smooth muscle, was also observed in these experiments. Interestingly, pressurization significantly shifted the resonance position of this peak, which was interpreted to represent a change in [Mg2+free]i. The average [Mg2+free]i of flaccid artery preparations was computed to be 0.54 +/- 0.03 x 10(-3) M, as compared to 0.99 +/- 0.10 x 10(-3) M for pressurized arteries. This change in [Mg2+free]i was evident within the first hour following pressurization and persisted thereafter. These findings suggest that altering the resting length of vascular smooth muscle produces a change in [Mg2+free]i. This shift in free Mg2+ levels may act as a metabolic signal triggering a change in vascular smooth muscle metabolism, an effect which has been reported to occur in smooth muscle in response to stretch.     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Cytoplasmic pH and free Mg2+ in lymphocytes

Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly- trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced no net movement of Mg2+ or H+; (b) by Mg- sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.     

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2-3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (Pi) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of Pi. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and Pi. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 +/- 2.5 (n = 5), Krebs-Henseleit saline 42 +/- 2.7 (n = 6) ml/min, P less than 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 +/- 0.7 (n = 5), Krebs-Henseleit saline 1.7 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.01. For [ATP]: FC-43 1.8 +/- 0.7 (n = 5), Krebs-Henseleit saline 0.9 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [Pi] determined by both methods, 36% of Pi in FC-43-perfused hearts, and only 24% of Pi in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of Pi is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.     

NMR-invisible ATP in rat heart and its change in ischemia

The subcellular compartmentalization of adenosine 5'-triphosphate (ATP) in isolated perfused rat heart and its relation to energy depletion in ischemia were examined by 31P nuclear magnetic resonance (31P-NMR) spectroscopy and chemical analyses. The signal intensities of the beta-phosphate of ATP and creatine phosphate in the 31P-NMR were standardized by the intracellular volume ratio measured with 23Na-NMR to determine the actual content of each. During aerobic perfusion the ATP content determined by NMR (13.7 +/- 2.2 mumol/g dry weight) was significantly lower than that found by chemical analysis (22.4 +/- 0.7 mumol/g dry weight), while the creatine phosphate contents determined by the two methods were the same. During ischemia at 33 degrees C, the signal of the beta-phosphate of ATP in the 31P-NMR spectrum decreased progressively, disappearing completely after 16 min. But at this time 5.7 +/- 1.7 mumol/g dry weight of myocardial ATP was still detected by chemical analysis. These results indicated that there were two different compartments of intracellular ATP in the heart, only one of which is detectable by 31P-NMR spectroscopy, and that during ischemia the ATP that is detectable, which seems to be the free ATP in the cytosol, decreased more rapidly than the ATP in the other compartment.     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.