Glycerol phosphorylcholine (GPC) and serine ethanolamine phosphodiester (SEP): evolutionary mirrored metabolites and their potential metabolic roles.

Water-soluble phosphodiesters (WSPDE) are a prominent feature of many 31P-NMR spectra; however, their role has remained somewhat of a mystery. What has been missed in almost all previous studies is the fact that two classes of WSPDE exist in vertebrates: those in mammals and those in the other (reptile-avian) line. The first is represented by glycerol phosphorylcholine and the second by serine ethanolamine phosphodiester. A further examination of the literature suggests a common role for all WSPDE as lysophospholipase inhibitors and therefore net sparers of phospholipids by decreasing phospholipid metabolic throughput.

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.

In vivo 19F NMR studies of hyperthermia: hydrophobic environments probed by halothane.

The steady-state distribution of the general anesthetic halothane in different rat tissues, including a renal adenocarcinoma with and without hyperthermia treatment, has been evaluated by in vivo 19F NMR spectroscopy. The 19F spectra of halothane (which is a hydrophobic probe) from within tissue show differences in the partitioning between normal rat tissues and adenocarcinoma. Muscle, as a control tissue, exhibits a single large resonance around 0 ppm. However, the adenocarcinoma exhibits two slow-exchanging resonances separated by 0.3 ppm with the one at the more hydrophobic chemical shift being more sensitive to hyperthermia treatment. The results from this tumor model suggest that 19F NMR spectroscopy may be useful first in detecting a change in hydrophobic environments using a lipophilic probe such as halothane, and secondly in monitoring the effects of hyperthermia, a treatment whose effectiveness may involve changes at the level of the plasma membrane. Under conditions of continuous delivery, a resonance which is not detected in the spectra of halothane in excised tissue appears 5 ppm downfield from the resonance for halothane localized in tissues. A rotating frame experiment is used to show that this resonance is derived from anesthetic absorbed on the tissue surface.

Percutaneous absorption of flurbiprofen in the hairless rat measured in vivo using 19F magnetic resonance spectroscopy.

The objective of this investigation was to develop a new methodology using 19F-magnetic resonance spectroscopy (MRS) to measure the in vivo percutaneous absorption of flurbiprofen through hairless rat skin. A 2% W/V flurbiprofen gel (Klucel HF, hydroxypropyl cellulose 1.5% to 2% W/V) containing isopropyl alcohol, water, and propylene glycol (55:35:10 v/v/v) was prepared. A 2-mg dose (100 mg of gel) was applied to the skin of the lower back of an anesthetized hairless rat, contained with a rubber o-ring, and occluded with a lexan plastic cover slip. The animal was placed on an MR surface coil (3.5-cm diameter tuned to 19F) and measurements taken continuously over approximately 3 h in 10-min intervals with a 2-tesla GE CSI nuclear magnetic resonance (NMR) spectrometer. One measures the disappearance of MR signal intensity per interval, which directly relates to the percent of drug disappearance over time, which in turn was converted to a flux value. The flux of flurbiprofen in vivo was found to be 95 +/- 22 micrograms/cm2/h. This is approximately four times greater than the flux of flurbiprofen through excised human skin reported by Akhter and Barry (22 +/- 14 micrograms/cm2/h). This new in vivo method measures drug disappearance and can be readily transferred to man. This method may be adapted to study other fluorine compounds or other nuclei with magnetic properties. It avoids exposure of a patient or animal to the radiation used in x-ray fluorescence methods or to 14C- or 3H-radiolabeled drugs.

31P NMR investigation of vanadium-treated chick muscle.

The phosphorus NMR profile of normal and vanadium-treated chick muscle was obtained in vivo. The data show that the differentiation of breast and thigh muscles in terms of pH, lipid related metabolites, and bioenergetic parameters can be readily followed. Although the vanadium-treated chicks showed substantial retardation of growth, the only NMR parameter that was significantly affected by dietary vanadium was the pH of breast muscle, which was substantially more acidic in the vanadium-treated animals.

ATP, ADP, and magnesium mixed solutions: in vitro 31P NMR characterization and in vivo application to lens.

The difference between the 31P NMR resonance position of the beta-peak of ADP and the gamma-peak of ATP at a fixed monovalent ion concentration level and pH is shown in vitro to depend only on free magnesium concentration. This difference can vary by more than 1 ppm depending on the pH of the solution and free magnesium concentration. Using 31P NMR and ion-selective electrodes we have constructed experimental curves to show how calibration of experimental results can be achieved. Theoretical calculations can be used to predict the functional dependence of the chemical shift difference on the above parameters. However, using the reported ion dissociation constants the fit was not exact and the discrepancy increased at higher pH values. To demonstrate that this technique can yield valid in vivo results and to analyze a previously unreported system where free magnesium levels vary, sample spectra from lenses and enucleated eyes are given. The data show a disagreement between the present methods and the more conventional ATP method. The reason for this difference is not known although some possible reasons are suggested.

Cytosolic water-soluble phosphodiester in vertebrate lens.

The water-soluble phosphate profiles determined by 31P nuclear magnetic resonance (NMR) of a broad range of vertebrate lenses are presented. We identify threonine ethanolamine phosphodiester (TEP) in intact lenses and lens extracts by its chemical shift and its physical and chemical characteristics. Amongst the species studied, we give the first characterization of frog lens where not only serine ethanolamine phosphodiesters (SEP) but TEP are found.

Biochemical consequences of phosphorylcholines low pK with special relevance to human semen: passive pH regulation.

1. The significant difference in pK between phosphorylcholine (PCh) and inorganic phosphate (Pi) of over 1 pH unit results in PCh being a source of acid buffering potential and an actual sink for protons when it is hydrolyzed. Phosphorylethanolamine (PEth) can fill the same role. 2. Both semen and tissues having substantial anaerobic metabolism among which are those of fetal origin can face circumstances of non-pathological acidosis. 3. We demonstrate from the literature that all of those tissues with the two understandable exceptions of erythrocytes and striated muscle, have high levels of PEth and/or PCh. 4. Since these phosphomonoesters can serve as lipid metabolites as well as low-pK fixed-phosphate buffer it appears they can serve a dual function in these tissues. 5. Their concentration would vary depending on the physiological and biochemical conditions present and we would call this a "Swiss army knife" theory of metabolic function.

31P NMR studies of excised gray and white calf brain.

1. 31P NMR examination of isolated calf gray and white matter reveals that white matter contains higher levels of the phosphodiester glycerolphosphoryl choline (GPC) than gray. 2. It is suggested that GPC may play a role in maintaining the level of phospholipids present by inhibition of phospholipases. 3. The spectra also reveal a skewed peak whose maximum is at -11 ppm which is inferred to arise from myelin-like structures. 4. The results show that phosphorus spectra from the brain must be carefully considered whether they arise from the same type tissue or represent a mixed sample since variation in results may represent anatomy as well as physiology.

In vivo 31P nuclear magnetic resonance studies on the absorption of triphenyl phosphite and tri-o-cresyl phosphate following subcutaneous administration in hens.

Tri-o-cresyl phosphate (TOCP) and triphenyl phosphite (TPP) are known to be neurotoxic in several species. In a previous study, we found that the subcutaneous administration of the compounds may result in toxicological effects which are prolonged in comparison to administration by other routes. In order to test the hypothesis that slow absorption from the injection site could account for our results, we monitored the disappearance of either compound from the injection site using in vivo 31P nuclear magnetic resonance (NMR). In addition, the test samples and some potential metabolites were examined in vitro with NMR. The disappearance of equimolar doses of subcutaneously injected TOCP (1187 mg/kg) and TPP (1000 mg/kg) from the injection site, with time, showed a biphasic pattern. The first phase took place within a few hours, while the second phase was very slow, with a half-life of about 2 weeks for both compounds. These results may account for the prolonged neuropathy target enzyme inhibition and explain the delayed neurotoxicity produced by subcutaneous injection of TOCP and TPP. Two animals given TPP exhibited an atypical pattern, in that the TPP apparently converted to diphenyl phosphonic acid within several hours of injection. In these hens, this phenomenon was accompanied by acute lethality. The conversion to diphenyl phosphonic acid also took place when the TPP was placed in an aqueous solution in vitro. Diphenyl phosphonic acid may play a role in the unique toxicity of TPP.

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.

Fluorine-19 NMR studies of tumor-bearing rats treated with difluoromethylornithine.

Copenhagen rats with implanted metastatic prostate carcinomas have been treated with the drug alpha-difluoromethylornithine (DFMO), an inhibitor of the enzyme ornithine decarboxylase. In vivo and in vitro 19F NMR observations were then carried out on a variety of organs and tissues. The distribution of the drug strongly favored tumor over surrounding muscle. DFMO, which gives a spectrum similar to that of the pH indicator difluoromethylalanine, has potential for determination of in vivo pH. However, in contrast to the alanine analog, DFMO exhibits a considerably smaller shift dependence in response to pH changes.

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.

In vivo 31P nuclear magnetic resonance studies of arsenite induced changes in hepatic phosphate levels.

Hepatic phosphate resonances were evaluated in vivo by 31P nuclear magnetic resonance (31P-NMR) following a single intravenous dose of sodium arsenite (10 mg/kg). Acute in vivo administration of arsenite rapidly decreased intracellular pools of all ATP phosphate with concomitant increases in inorganic phosphate and phosphomonoesters. In the phosphodiester resonance region, glycerolphosphorylcholine was also increased. The data suggest that liver cannot compensate for the rapid loss of NAD-linked substrate oxidation via other metabolic pathways, such as glycolysis for the production of ATP, and also demonstrate that 31P-NMR spectroscopy can disclose time-dependent metabolic changes of the liver in vivo.

Magnetic resonance spectroscopy of the musculoskeletal system.

Magnetic resonance spectroscopy can be used to characterize the bioenergetic state of the musculoskeletal system, and several marker compounds related to the tissue's biochemistry have been found to exist. Potential new techniques involving better spatial localization, spectral editing, and examination of nuclei other than phosphorus are in the developmental stage. Their development over the next few years will determine the extent to which MRS will become a universally used medical tool. However, the results with 31P alone guarantee a continued role in the study of muscular disease, peripheral vascular disease, and hypoxia and ischemia of the neonatal brain.

31P-NMR characterization of hen egg yolk and egg white.

The avian egg is an isolated system where embryonic development can easily be studied. Furthermore, the system can be subjected to various environmental constraints. Its size is such that it can easily be accommodated in most NMR instruments. This is demonstrated by a recent 31P-NMR surface coil experiment where the increase of embryo size and development could be judged by the levels of ATP and phosphocreatine (Belton, P.S., Gordon, R.E., Jones, J.M. and Shaw, D. (1983) Br. Poult. Sci. 24, 429-433).

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.