Estimation of tumor oxygenation and metabolic rate using 31P MRS: correlation of longitudinal relaxation with tumor growth rate and DNA synthesis.

31P MRS longitudinal relaxation times (T1) were determined for C3H murine fibrosarcomas (FSaII), and mammary carcinomas (MCaIV). Tumors were implanted in the foot dorsum, and were 100-300 mm3 in volume. T1s were repeated after the animal was allowed to breathe 100% oxygen for 30 min and then again 36-48 hr following 30 Gy. The spectrum were obtained using an 8.5 T spectrometer with a 8 cm bore and a 1.4 cm single turn antenna coil. The 31P relaxation times for untreated tumors in air breathing animals were: 3.78 sec for phosphomonoesters, 4.37 sec for inorganic phosphate (Pi), 2.73 sec for phosphocreatine, 1.37 sec for gamma ATP, 1.14 sec for alpha ATP, and 1.18 sec for beta ATP. The Pi T1s were 4.37 and 4.70 sec in control and irradiated tumors in air breathing animals. Respiration of oxygen for 30 min reduced the T1s to 3.02 and 2.62 sec in control and irradiated tumors respectively. The Pi T1 of an anoxic tumor, determined on an in situ tumor 60 min after death was 5.93 sec. The oxygen breathing induced decrease in the T1 of Pi is unlikely to have been caused by the paramagnetic properties of oxygen alone, and suggests a component of increased magnetization transfer secondary to the ATPase reaction. Oxygen breathing following 30 Gy, resulted in a decreased growth time (800 mm3 endpoint) and an increased proportion of cells in S-phase. These results support the hypothesis that the decrease in Pi T1 measured with oxygen breathing is a measure of tumor oxygen tension and metabolic rate, and suggests that T1 measurement may indirectly predict tumor growth rate and DNA synthesis.

Effects of hydralazine on in vivo tumor energy metabolism, hematopoietic radiation sensitivity, and cardiovascular parameters.

Energy metabolism of murine FSaII foot tumors was studied by in vivo 31P-MRS in C3Hf/Sed mice. Spectroscopy was performed following exposure to escalating doses of hydralazine (HYD) ip. At 0.25 mg/kg, HYD caused a 20% increase in PCr/Pi and had no significant effect on mean arterial blood pressure. HYD doses greater than or equal to 2 mg/kg lead to hypotension which was associated with a decrease in PCr, NTP, pH, and an increase in Pi (p less than 0.01 for control vs 10 mg/kg HYD). When mice were given ip injections of HYD (0.25, 1, 2 and 10 mg/kg) 10 min prior to whole body irradiation, spleen stem cell survival after 6 Gy was increased (2.19 colonies in control animals vs 6.74 colonies per spleen in animals treated with greater than or equal to 2 mg/kg HYD), as was the LD50/30 dose (6.49 Gy [control] vs 9.00 Gy [10 mg/kg HYD]). The data provide evidence that PCr/Pi is a useful indicator of perfusion efficiency (and indirectly of hypoxic cell fraction) in FSaII tumors. These observations suggest that HYD may be a useful adjuvant for hyperthermic treatment of tumors and for potentiation of agents specifically toxic to hypoxic or nutrient-deprived cancer cells. HYD should be used with care in patients receiving radiation treatments or other therapies for which hypoxia can unfavorably affect treatment outcome.

Effects of hypercapnia on brain pHi and phosphate metabolite regulation by 31P-NMR.

The ability of brain cells to regulate intracellular pH (pHi) and several phosphate metabolites was evaluated during 1 h of hypercapnia (inspiratory CO2 fraction of 0.10 and 0.05) in anesthetized rats by 31P high-field (145.6 MHz) nuclear magnetic resonance spectroscopy. Body temperature was maintained at 37 +/- 0.5 degrees C. Fully relaxed spectra were obtained for controls and 30-50 min after CO2 loading and CO2 withdrawal. Spectra were taken serially every 2.5 min after gas mixtures were changed. Brain pHi decreased 0.10 +/- 0.02 units [7.06 +/- 0.01 (SE)] to 6.96 +/- 0.01 (P less than 0.001) after 30-50 min of 10% CO2 breathing, and arterial pH decreased 0.24 +/- 0.01 units. Brain pHi decreased by 0.045 +/- 0.01 units (7.05 +/- 0.01 to 7.01 +/- 0.01, P less than 0.05) during 5% CO2 breathing. Brain pHi returned to control values after 30-50 min of CO2 washout in both groups. In three of six animals breathing 10% CO2, there was an undershoot in brain pHi by 0.07-0.09 units between 2.5 and 20 min of hypercapnia. Three animals exhibited an overshoot in pHi by 0.06-0.11 units between 7.5 and 17.5 min during CO2 washout. Phosphocreatine-to-Pi and Pi-to-beta-ATP ratios changed during hypercapnia and returned to base line after withdrawal of CO2. The findings of a smaller brain pHi change than arterial pH change and undershoots and overshoots in pHi support the view that pHi regulation involves active processes such as transmembrane ion transport.

Brain amino acid concentrations during diving and acid-base stress in turtles.

To assess the role of brain amino acid neurotransmitters in the breath hold of diving animals, concentrations of free amino acids present in the brains of turtles immediately after 2 h of apneic diving (at 20 degrees C) were measured. Additionally, the same measurements were performed on four other groups of animals subjected to 2 h of hypercapnia (8% CO2 in air), anoxia (N2 breathing), anoxia plus hypercapnia (8% CO2-92% N2), or air breathing (control). Significant changes in the concentrations of the inhibitory amino acid neurotransmitters known to affect respiration [gamma-aminobutyric acid (GABA) and taurine] were seen. GABA increased significantly in those animals subjected to anoxia, whereas taurine decreased significantly in the diving animals and increased significantly in those subjected to anoxia plus hypercapnia. These results suggest that the attenuated central ventilatory drive during diving in these animals may be related to alterations in brain concentrations of GABA and taurine.

Viability of the neonatal rat isolated brainstem preparation by 31P MRS.

The isolated brainstem-spinal axis from the neonatal rat is an established model for studying neuronal responses of the ventilatory control system, however, its viability has not been clearly established. We studied the brainstem-spinal axis from newborn rats at 8.5 T with 31P NMR spectroscopy. The relative pattern of high energy phosphates (HEPs) was similar to that reported for the in vivo neonatal brain. The average pHi was 0.2 to 0.4 units less than the pHi for the in vivo neonatal brain. The HEPs and pHi were stable for 6 h, suggesting extended in vitro viability.

Temperature-induced changes in turtle CSF pH and central control of ventilation.

Cerebrospinal fluid (CSF) and arterial blood acid-base variables, as well as respiratory minute volume (RMV), were measured at three different body temperatures in unanesthetized turtles. RMV remained constant while CSF pH decreased 0.015 U/degrees C with increasing body temperature. These results show that ventilation is not tracking CSF pH when body temperature is the independent variable; however, perfusion of the brain ventricular system of turtles with mock CSF, in which ion-dependent pH decreases were produced, caused large increases in ventilation which were independent of body temperature. Comparisons between turtles and goats reveal that central chemical control of ventilation is functioning similarly in the two animals and appears to fit the alphastat model of ventilatory control proposed by Reeves. These data strongly suggest that both animals are maintaining the fractional dissociation of imidazole constant in central receptive structures, indicating that this system is the model for central chemical control of ventilation in air-breathing vertebrates.

Acid-base stress and central chemical control of ventilation in turtles.

Central chemical control of ventilation in turtles, Chrysemys (Pseudemys) scripta, has been characterized by alterations in breathing frequency (f). To assess the effects of acid-base stress on the central ventilatory response, three groups of animals were subjected to 2 h of anoxia (98% N2-2% CO2), 2 h of hypercapnia (8% CO2 in air), or 2 h of anoxia plus hypercapnia (92% N2-8% CO2). Ventilatory responses were measured continuously, and arterial blood and cerebrospinal fluid (CSF) acid-base variables were determined after 2 h. Results show that central ventilatory responses (f) were significantly elevated in CSF acidosis caused by CO2 over those caused by the accumulation of CSF fixed acid alone (anoxia). The increases in f did not correlate with decreases in estimated CSF pH, since it was much lower in anoxia (7.19) than in hypercapnia (7.37). Our results strongly suggest that the central nervous system acidosis caused by anoxia produces centrally mediated ventilatory change in a fundamentally different way from hypercapnia.

Acid-base status and electrolytes in red blood cells and plasma of turtles submerged at 3 degrees C.

Two groups of adult Chrysemys picta bellii were studied; control turtles were maintained at 24 degrees C with free access to air, and diving animals were continuously submerged for 8.5-9.5 wk in aerated water at 3 degrees C. Cold submergence elicited a substantial lactic acidosis (blood pH 7.70; plasma [lactate-] 42.5 meq/l H2O). This acid load was largely balanced by changes in plasma strong ions; [Cl-] decreased, and [Ca2+] and [Mg2+] increased. Phosphorus nuclear magnetic resonance showed a significant reduction in the transerythrocyte pH gradient of diving animals (control, delta pH 0.66-0.90; diving, 0.00-0.11). This relative alkalosis of submerged turtle red cells was related to changes in blood cell (RBC) volume and composition; mean corpuscular concentration of hemoglobin [( Hb]) and RBC H2O content measurements revealed a 39% increase in RBC volume. Cell volume change was effected by a proportionally greater influx of K+ than Cl-, providing ionic compensation for the lactic acidosis. The obligatory H2O influx, which decreased the effective concentration of protein anion, and a 77% reduction of RBC [ATP] further contributed to the relative alkalosis. The combined effects of RBC alkalization and reduced [ATP] account for the observed Hb-O2 binding properties previously reported for submerged turtles at 3 degrees C (15).

Central chemical control of ventilation and response of turtles to inspired CO2.

The role of central chemosensors in the overall ventilatory response of freshwater turtles (Chrysemys scripta elegans) to the addition of CO2 in inspired gas was measured. Centrally mediated ventilatory responses were isolated in the unanesthetized animal by combining CO2 breathing and brain ventricular perfusion with mock cerebrospinal fluid (CSF) of varying acid-base status. Breathing 4.5% CO2 resulted in increases in both ventilatory frequency (f) and tidal volume (VT), with increases in VT providing most of the overall ventilatory change. Alterations in the acid-base status of the perfusate produced highly significant changes in f. VT changes were divorced from the acid-base status of the mock CSF perfusate. We therefore conclude that ventilatory changes in turtles, mediated by central chemosensors, are primarily affected by alterations in f. VT changes, associated with acid-base homeostatic mechanisms, are mediated by receptors outside the blood-brain barrier in these animals. On the basis of these data, we hypothesize that the increase in f observed when turtles breathe 4.5% CO2 is primarily mediated by the central chemosensors.

Differences between CSF and plasma Na+ and K+ activities and concentrations.

[Na+] and [K+] determined by ion-selective electrodes (ISE) by direct potentiometric determination were compared with those obtained by flame photometry in plasma and in cisternal cerebrospinal fluid (CSF) obtained from pentobarbital-anesthetized dogs. Comparisons of pre- and postanesthesia values showed no change in plasma [Na+] but a 5% fall in plasma [K+]. CSF and plasma Na+ activities were identical, indicating that Na+ does not contribute to the potential difference between them. CSF K+ activity was 74% of that in plasma. The CSF-to-plasma molar and molal ratios differed from the activity (ISE) ratios. Concentrations of Na+ and K+ were also determined in an electrolyte solution and in the same solution with albumin added. Flame values differed from ISE values in plasma, CSF, and solutions. Albumin lowered flame values to a greater extent than ISE values. Bicarbonate lowered ISE values. ISE ratios, which equal activity ratios, rather than molar or molal ratios should be used in thermodynamic equilibrium equations, whereas molar concentrations should be used in electroneutrality equations or in determination of the strong ion difference.

1H-NMR measurement of fractional dissociation of imidazole in intact animals.

The alphastat hypothesis states that intracellular acid-base status is regulated to maintain constancy of the fractional dissociation of intracellular protein and enzyme imidazole-histidine (alpha-imidazole). A major drawback of this theory has been the lack of a means to directly measure alpha-imidazole in intact animals. We developed a method for directly measuring alpha-imidazole in intact unanesthetized animals using 1H-nuclear magnetic resonance spectroscopy (NMR). We measured carnosine alpha-imidazole of white skeletal muscle from intact unanesthetized newts at three body temperatures (10, 20, and 30 degrees C). alpha-Imidazole remained constant, approximately 0.56, with alterations in body temperature, whereas intracellular pH (pHi) changed significantly (-0.015 U/degrees C), affirming the validity of the imidazole alphastat hypothesis for this tissue. This method was also used to determine the pK values of the imidazole moiety of carnosine and the imidazole moiety alone over a temperature (T) range 4-40 degrees C. The pK values of carnosine differed from those of imidazole, but the delta pK/delta T was the same. pHi was also determined using 31P-NMR and found to be the same as that calculated from carnosine alpha-imidazole values. Therefore Pi and carnosine share a similar pHi environment. We describe a novel technique to directly measure alpha-imidazole in intact tissue.

Phosphorus and fluorine NMR examination of the anesthetized newt (Notopthalamus viridescens).

We have examined newts by 19F-NMR using the anesthetic halothane as a probe and in another set of experiments taken 31P-NMR spectra under similar conditions. The spectra were recorded from the animal's tail. The water soluble 31P-NMR signals point to little difference between anesthetized and unanesthetized newts except for the potential disappearance of two pools of inorganic phosphate in the anesthetized animals. The 19F spectra show two anesthetic populations in the tail which the phosphorus spectra suggest arise from populations of halothane in muscle and in lipid.

Effects of temperature on muscle pHi and phosphate metabolites in newts and lungless salamanders.

The effect of acute alterations in body temperature (BT) on intracellular pH (pHi) and phosphate metabolites was assessed in white skeletal muscle of intact newts and lungless red-backed salamanders using 31P-nuclear magnetic resonance spectroscopy. pHi decreased with increasing BT in the tail muscle of both newts and lungless red-backed salamanders. The change in pH with change in temperature from 10 to 30 degrees C was -0.018 U/degrees C in newts and -0.041 U/degrees C in red backs. The calculated alpha-imidazole for skeletal muscle cytosol did not change (0.56) in newts from 10 to 30 degrees C but fell from 0.69 to 0.43 in red-backed salamanders. Phosphocreatine (PCr)/Pi fell and Pi/beta-ATP rose with increasing temperature in both newts and red backs; however, the change was much greater in red backs. Providing the red backs with O2 at 30 degrees C led to higher pH and alpha-imidazole, comparable to that of newts, along with increased PCr/Pi and lower Pi/beta-ATP. Thus newts maintain white skeletal muscle cell cytosol alpha-imidazole constant with changes in BT, whereas red backs apparently do not. However, at the BT of preference, red backs and newts maintain similar muscle pHi and alpha-imidazole. The method of gas exchange appears to strongly influence the ability of an animal to maintain its acid-base status over a range of temperatures, and our results suggest that behavioral regulation of BT may involve alpha-imidazole regulation as well.

Unknown phosphate compounds in tail muscle of intact conscious newts by 31P NMR.

Unknown phosphate resonances at 0 and -21.6 ppm have been identified in 31P NMR spectra of tail muscle of unanesthetized newts which do not correspond to known phosphate-bearing compounds in skeletal muscle cells. The concentrations of both unknowns decrease markedly during muscular activity and severe hypoxia (conditions associated with decreased intracellular pH and increased cellular levels of inorganic phosphate). The unknown at 0 ppm increases in concentration with imposition of moderate hypoxia. Our data suggest that these unknowns may be liable storage compounds for a high energy phosphate bond, and are involved in newt skeletal muscle phosphogen metabolism.

NMR spectroscopy as an investigative technique in physiology.

Relating physiological variables on an organ system level to metabolic function within the intracellular environment has been exceedingly difficult because of a paucity of techniques. Most of the tools at our command necessitate either the removal or destruction of tissues before measurements can be made. Recently, NMR spectroscopy has been applied to several important questions relating organ system and cellular physiology. NMR has the distinct advantage of being noninvasive and nondestructive, allowing the investigator to make repetitive measurements of intracellular variables while manipulating experimental variables that are important on the organ system level. In this review we shall present several examples of such NMR investigations so that the reader will gain some appreciation of the potential of this relatively new technique. Cellular acid-base homeostatic mechanisms, high-energy phosphate metabolism, and regulation of anaerobic glycolysis will be discussed for such diverse cellular populations as mammalian brain, mammalian heart muscle, salamander skeletal muscle, amphibian skin, and invertebrate muscle. In addition, the role of phosphomonoesters and phosphodiesters in lipid metabolism for several tissues in different species will be evaluated.