Kinetics of cyclocreatine and Na(+) cotransport in human breast cancer cells: mechanism of activity.

The growth-inhibitory effect of cyclocreatine (CCr) and the kinetics of CCr and Na(+) cotransport were investigated in MCF7 human breast cancer cells and its adriamycin-resistant subline with use of (31)P- and (23)Na-NMR spectroscopy. The growth-inhibitory effect in the resistant line occurred at a lower CCr concentration and was more pronounced than in the wild-type line. This correlated with an approximately 10-fold higher affinity of CCr to the transporter in the resistant line. The passive diffusion coefficient of CCr was also higher in the resistant line by three- to fourfold. The transport of CCr was accompanied by a rapid increase in intracellular Na(+). This increase was found to depend on the rate of CCr transport and varied differently with CCr concentration in the two cell lines. It is proposed that the cotransport of CCr and Na(+) followed by increased Na(+) concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.

Multiple bond 13C-13C spin-spin coupling provides complementary information in a 13C NMR isotopomer analysis of glutamate.

Most 13C nuclear magnetic resonance (NMR) isotopomer analyses relate a metabolic index of interest to populations of 13C isotopomers as reported by one-bond 13C-13C spin-spin couplings. Metabolic conditions that produce highly enriched citric acid cycle intermediates often lead to 13C NMR spectra of metabolites such as glutamate that show extra multiplets due to long-range couplings. It can be demonstrated from 13C NMR spectra of hearts perfused with mixtures of acetate plus propionate that multiplets in glutamate C2 arising from 3J25 coupling provide a direct readout of acetyl-CoA fractional enrichment (FC1 and FC3), while multiplets in glutamate C5 arising from 2J35 and 3J25 couplings quantitatively reflect enrichment of the anaplerotic substrate.

NMR temperature measurements using a paramagnetic lanthanide complex.

NMR thermometry has previously suffered from poor thermal resolution owing to the relatively weak dependence of chemical shift on temperature in diamagnetic molecules. In contrast, the shifts of nuclear spins near a paramagnetic center exhibit strong temperature dependencies. The chemical shifts of the thulium 1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) complex (TmDOTP5-) have been studied as a function of temperature, pH, and Ca2+ concentration over ranges which may be encountered in vivo. The results demonstrate that the 1H and 31P shifts in TmDOTP5- are highly sensitive to temperature and may be used for NMR thermometry with excellent accuracy and resolution. A new technique is also described which permits simultaneous measurements of temperature and pH changes from the shifts of multiple TmDOTP5- spectral lines.

The determination of magnesium in human blood plasma by 31P magnetic resonance spectroscopy using a macrocyclic reporter ligand.

The ligand 1,4,7-triazacyclononane-1,4,7-tris(methylene methylphosphinic acid), NOTMP, was used to measure free MgII levels in blood plasma by 31P MRS. Separate resonances were observed for the free ligand and the MgII complex and the ratio of their resonance areas was used to evaluate the free, ionized MgII concentration, [Mg]free. The CaII and the ZnII complexes gave rise to separate resonances in the 31P spectrum in an aqueous sample. In human blood plasma samples, however, these resonances were never observed thus excluding the interference of these metal ions. Heparin, up to 150 units/ml, had no influence on the Mg-NOTMP equilibrium. The 31P MRS methodology was applied to twenty human blood plasma samples. Total MgII ([Mg]total), as measured by atomic absorption spectroscopy, averaged 0.85 +/- 0.12 mM while free ionized MgII ([Mg]free) measured by 31P MRS was 0.66 +/- 0.09 mM. The 31P MRS method gave inherently larger values for free ionized MgII than that reported by ion-selective electrodes (ISE). This was traced to a redistribution of existing plasma MgII species after the addition of about 2 mM of NOTMP. Calculations using existing thermodynamic data show that the ionized MgII concentration (iMg) and the concentration of MgII weakly complexed to small anions (Mg(comp)) both drop after the addition of NOTMP, with Mg(comp) dropping to negligible levels. Thus, the 31P MRS method appears to be less sensitive to variations in the concentration of weakly binding anions (bicarbonate, carbonate, chloride, lactate, phosphate, etc.) than the ISE method. Our data indicates that the difference between Mg(total), as measured by atomic absorption spectroscopy, and Mg(free), as measured by 31P MRS, provides an direct estimate of the protein bound MgII fraction.

Relationship between energetic, ionic, and functional status in the perfused rat heart following thermal injury: a 31P and 23Na NMR study.

To test the hypothesis that energy deficits and intracellular ion derangements may be the cellular basis for intrinsic myocardial dysfunction in rats after burn trauma, we examined ATP metabolism, intracellular pH, sodium, and mechanical performance simultaneously in perfused beating hearts from sham burn or burned rats (43% TBSA, 3 degrees scald burn, resuscitated for 24 hr with lactated Ringer's solution, Parkland formula). Intracellular calcium was also measured in myocytes harvested from parallel groups of sham burn and burn resuscitated rats. Burn trauma caused a 46% decrease in left ventricular developed pressure, a 69% decrease in +dP/dtmax, and a 72% decrease in -dP/dtmax. Intracellular to external standard sodium ratio increased (+58%) from 0.318 +/- 0.027 to 0.500 +/- 0.048 (P < 0.05), and intracellular calcium increased (+67%) from 206 +/- 13 to 445 +/- 37 nM (P < 0.01). Burn hearts exhibited decreased functional response to isoproterenol challenge compared to sham burn controls, but energy metabolism was similar in all hearts, regardless of burn injury. Our data suggest that burn trauma alters intracellular cardiomyocyte calcium and sodium homeostasis, and ionic derangements are not related to either altered intracellular pH or high energy phosphate deficits.

Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts.

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.

The design of macrocyclic ligands for monitoring magnesium in tissue by 31P NMR.

A series of ligands based upon 1,4,7-triazacyclononane, containing variable numbers of acetate vs methylenephosphonate ester or methylenephosphonate sidechains, has been synthesized and characterized as possible 31P NMR probes for monitoring [Mg2+]free. The diacetate monophosphonate and diacetate monophosphonate ester mixed ligands proved to have conditional (dissociation) constants at pH 7.4 in an appropriate range for measuring typical levels of intracellular [Mg2+]free, and binding selectivities for Mg2+ over Ca2+ that ranged from 1.4 to 6.8. The 31P resonances of these ligands were well downfield of typical tissue phosphate metabolite resonances, and addition of Mg2+ to any one of these ligands resulted in well-resolved resonances for HL and MgL (where L = ligand), in slow chemical exchange. The chemical shift differences between the HL and MgL species varied from 2.1 to 2.6 ppm for three different ligands. The conditional Mg(2+)-L binding constants were sensitive to pH in the physiological range, due to protonation of a single macrocyclic nitrogen at high pH (log K1 values ranged from 10 to 12). However, given conditions that allow an independent assessment of pH (i.e., from the 31P chemical shift of Pi), we show that accurate KD values can be estimated from the known thermodynamic stability constants (KMgL) and ligand protonation constants (Ki). This makes these ligands potentially useful for monitoring [Mg2+]free by 31P NMR over a variety of solution conditions, even during conditions when the pH may be changing.

Effects of oxidant exposure on substrate utilization and high-energy phosphates in isolated rat hearts.

The effects of a xanthine oxidase-mediated free radical-generating system containing purine and iron-loaded transferrin or solutions containing hydrogen peroxide and iron-loaded transferrin on substrate utilization and high-energy phosphates were evaluated by nuclear magnetic resonance (NMR) spectroscopy in isolated perfused rat hearts. Hearts were supplied with lactate, acetate, and glucose, and the contribution of each substrate to acetyl coenzyme A was measured in control hearts and in the presence of a free radical-generating system. Perfused hearts were monitored by 31P NMR, and tissue extracts were analyzed by 13C NMR. Free radicals decreased the phosphocreatine and beta-ATP peak areas and reduced contractile function. Under control conditions, lactate, acetate, and endogenous sources were the major contributors of acetyl coenzyme A units, with only 5% originating from glucose. In the presence of a xanthine oxidase-mediated free radical-generating system, the glucose contribution increased to 54%, while contributions from acetate and endogenous sources were significantly reduced. Both 13C and 31P NMR analyses showed no significant accumulation of glycolytic sugar phosphates, suggesting little inhibition of glyceraldehyde-3-phosphate dehydrogenase. The increased contribution of glucose to the tricarboxylic acid cycle relative to acetate and endogenous sources is consistent with activation of pyruvate dehydrogenase. In contrast, hearts exposed to a hydrogen peroxide-based free radical-generating system showed an increase in lactate utilization, a decrease in acetate utilization, and no change in glucose utilization compared with control hearts. Glycolytic sugar phosphates were found to accumulate, suggesting possible inhibition of glyceraldehyde-3-phosphate. Thus, different radicals or their metabolites may have varying effects on myocardial metabolism.

Preferential elimination of pivalate with supplemental carnitine via formation of pivaloylcarnitine in man.

1. To evaluate the effectiveness of carnitine administration in aiding the elimination of pivalate liberated from pivampicillin, studies were undertaken on seven paediatric patients treated for 7 days with combined pivampicillin and molar excess of carnitine. 2. A 22-fold increase occurred in urinary carnitine ester excretion on the last day of treatment (2967 +/- 604 versus 134 +/- 50 mumol/day, p < 0.05); the pivaloylcarnitine was identified with 13C-n.m.r. Only pivalate was detected in the urinary carnitine ester g.l.c. profile, the amount of this ester was equal to 92% of the daily pivalate intake. 3. The renal clearance rate of carnitine esters significantly exceeded that of creatinine indicating that the carnitine ester was eliminated by active transport. 4. The plasma concentration and urinary output of free carnitine were not changed significantly by the treatment, and the free and esterified carnitine concentrations in red cells remained unchanged indicating that carnitine deficiency was prevented.

Thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) as a 23Na shift reagent for the in vivo rat liver.

The use of thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate (TmDOTP5-) as an in vivo 23Na NMR shift reagent for rat liver was evaluated by collecting interleaved 23Na and 31P spectra. Infusion of 80 mM TmDOTP5- without added Ca2+ produced baseline-resolved peaks from intra- and extracellular sodium without producing any changes in phosphate metabolite resonances or intracellular pH. Several key physiological parameters measured in parallel groups of animals confirmed that liver physiology is largely unaffected by this shift reagent. A direct comparison of TmDOTP5- versus DyTTHA3- showed that after infusion of 5-8 times more DyTTHA3-, the extracellular sodium peak shifted by the same amount as with TmDOTP5-, but the two 23Na resonances were very broad and not resolved. The baseline-resolved peaks with TmDOTP5- allowed us to measure the in vivo T1 and T2 relaxation characteristics of intra- and extracellular Na+. The measured T1, T2s, and T2f values and the relative contributions from the slow and fast T2 components for intracellular Na+ in liver did not differ significantly from the values reported for perfused frog heart. The T1 and T2 relaxation curves of the extracellular Na+ resonances fit a monoexponential function. Analysis of the relative contribution of the fast- and slow-relaxing T2 components from intracellular Na+ resulted in a calculated visibility factor of 69 +/- 4% and the intracellular Na+ concentration calculated from the NMR peak intensity ratio, the measured visibility factor, and literature values of intra- and extracellular volume was 19 mM. These results indicate that TmDOTP5- promises to be quite useful as an in vivo shift reagent for liver and other organs.