T-lymphocyte and glycemic status after vitamin D treatment in type 1 diabetes: A randomized controlled trial with sequential crossover.

BACKGROUND: Type 1 diabetes mellitus (T1D) is mediated by autoaggressive T effector cells with an underlying regulatory T-cell (Treg) defect. Vitamin D deficiency is highly prevalent in T1D, which can aggravate immune dysfunction. High-dose vitamin D treatment may enhance Tregs and improve metabolism in T1D patients. METHODS: In a randomized double-blind placebo-controlled trial with crossover design, patients received either for 3 months cholecalciferol 4000 IU/d followed by 3 months placebo or the sequential alternative. Thirty-nine T1D patients (19 women and 20 men) completed the trial. RESULTS: Primary outcome was a change of Tregs, secondary HbA1C, and insulin demand. Effects were evaluated based on intra-individual changes between treatment and placebo periods for outcome measures. Exploratory analyses included vitamin D system variant genotyping and C-peptide measurements. Median 25(OH)D3 increased to 38.8 ng/ml with males showing a significantly stronger increase (p = .003). T-lymphocyte profiles did not change significantly (p > 2); however, the intra-individual change of Tregs between males and females was different with a significantly stronger increase in men (p = .017), as well as between genotypes of the vitamin D receptor (Apa, Taq, and Bsm: genotypes aa, TT, and bb; p = .004-0.015). Insulin demands declined significantly (p = .003-.039) and HbA1C improved (p < .001). Random C-peptide levels were low but rising (median, 0.125 ng/ml; range, 0.02-0.3) in 6 patients. No toxicity was observed. CONCLUSION: A daily vitamin D dose of 4000 IU for 3 months was well tolerated and enhanced Tregs in males. Glucometabolic control improved in all. Subsequent larger trials need to address ß-cell function and genotyping for individualized vitamin D doses.

Efficacy and longevity of newly developed catnip oil microcapsules against stable fly oviposition and larval growth.

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), is one of the most important pests of cattle and costs U.S. cattle producers billions of dollars in losses annually. In this study, the efficacy of catnip oil encapsulated in gelatin in oviposition deterrence and larval growth inhibition in stable flies was examined under laboratory conditions. More than 98% inhibition of stable fly larval growth and female oviposition was observed in larval and oviposition media treated with encapsulated catnip oil (0.5 g). Further, dose-response tests showed that as little as 0.1 g of encapsulated catnip oil provided > 85% oviposition deterrence. The release of nepetalactones from the capsules was more rapid when the capsules were placed on a moist substrate rather than a dry substrate. Encapsulated catnip oil also exhibited antibacterial activity, supporting the hypothesis that its inhibition of larval growth may be based on its killing of the bacteria on which larvae feed. The use of encapsulated catnip oil can provide an alternative control strategy for stable fly management.

Effect of a randomised controlled vitamin D trial on insulin resistance and glucose metabolism in patients with type 2 diabetes mellitus.

The aim of our study was to investigate the influence of a 6-month vitamin D supplementation in patients with noninsulin-requiring type 2 diabetes mellitus. We included 86 patients in a placebo-controlled, randomised, double-blind study. During 6 months patients received Vigantol oil once a week corresponding to a daily dose of 1904 IU or placebo oil, followed by 6 months of follow-up. At start and at 3-month intervals 25OHD, PTH, body mass index, HbA1c, insulin, C-peptide, and homeostasis model assessment-index were measured. The primary outcome was a change in fasting blood glucose and insulin levels. After 6 months of therapy, the verum group's 25OHD had increased to a median of 35 ng/ml in comparison to the placebo group (median 20 ng/ml, p<10-6). PTH tended to decrease in the verum group (25.5 pg/ml vs. 35.0 pg/ml, p=0.08). After 6 months of therapy, 31 patients (78%) achieved a 25OHD concentration of >20 ng/ml. Their HbA1c was significantly lower at baseline (p=0.008) and after therapy (p=0.009) than in patients with 25OHD below 20 ng/ml. C-Peptide, insulin, and HOMA-index did not change significantly in the verum group but fasting insulin was positively correlated with 25OHD concentrations after 6 months of therapy in both groups. There were no significant effects of vitamin D with a daily dose of 1904 IU on metabolic parameters in type 2 diabetes. However, the correlative findings of this study suggest a link of the 25OHD status and metabolic function in type 2 diabetes. Whether vitamin D therapy with higher doses can improve glucose metabolism needs to be investigated in follow-up trials.

Safety of heart-lung machines during prolonged standby.

BACKGROUND: Coronary artery bypass surgery is increasingly performed without an extra-corporeal circuit. A heart-lung machine (HLM) is kept on standby for safety reasons, but rarely used. The tubes of the machines are currently discarded after every operation. Costs and waste would be avoided if HLMs could stay on standby for longer periods of time. We therefore investigated the sterility of intra-tube fluid over time. MATERIALS AND METHODS: Four machines were tested. The tube system was fixed and filled with priming solution. Samples of intra-tube fluid and fluid from the fluid bags were taken after 0, 12, 24, 36, 48 and 72 h. Microbiological cultures were performed by direct inoculation and membrane filtration and incubated up to 14 days. Endotoxin levels were also determined. RESULTS: Bacteria were grown from two samples only and were considered as contaminants. Endotoxin concentrations never exceeded acceptable levels. CONCLUSION: To keep HLMs on standby for 72 h is probably safe. The prolonged use of HLMs will contribute to cost and waste reduction in open heart surgery.

The value of heparin concentration monitoring in off-pump coronary bypass surgery.

BACKGROUND/OBJECTIVE: In recent years, a remarcable increase in off-pump coronary bypass surgery (OPCAB) was observed. The identical anticoagulation treatment in cardiac surgery with cardiopulmonary bypass (CPB) and OPCAB makes the hemodynamic management in OPCAB procedures essential, since a perioperative compensation of blood loss is difficult and the hemodynamic stability has to be maintained by infusions. The aim of this study was to evaluate the circulating heparin concentration measurement in OPCAB patients by using the heparin/protamine titration method with Hepcon/HMSplus (HMS). PATIENTS AND METHOD: In 8 patients (3F/5M) undergoing elective OPCAB, the calculated heparin dose was administered and heparin concentration was registered together with activated clotting time (ACT) 5 min after administration. Measurements were carried out in 45 min periods and additional heparin was administered if the measured heparin concentration was lower than the calculated. The protamin dose was also automatically calculated by HMS. RESULTS: The mean operation time was 155 +/- 36 min (80-210) with 2.3 bypass grafts per patient. There was a significant difference between the demand for heparin as determined by HMS and the conventionally calculated value (p<0.05). Similarly, the calculated and measured concentration of heparin diverged significantly from each other (p<0.05), independent of ACT. In spite of an initially higher administration of heparin as calculated by HMS, the concentration of circulating heparin was below the estimated value in 7 patients which made a further administration of heparin necessary. The 24 h postoperative blood loss was 550 +/- 176 mL (300-850). No rethoracotomy for bleeding was necessary. CONCLUSIONS: HMS could be a useful method for appropriate anticoagulative treatment in OPCAB procedures, if there is a sufficient hemodynamic management with restrictive administration of infusions guaranted during surgery.

New surface biopolymers for oxygenators: an in vitro hemocompatibility test of poly(2-methoxyethylacrylate).

BACKGROUND/OBJECTIVE: The continuous interaction of blood with artificial contact surfaces under cardiopulmonary bypass can lead to a substantial damage of blood cells and plasma factors. Surface biopolymers in oxygenation systems can help increasing the hemocompatibility, often combined with anticoagulative agents such as heparin. The poly(2-methoxyethylacrylate) [PMEA] is a new heparin-free polymer. The objective of this experimental study was to evaluate the hemocompatibility of a PMEA-coated oxygenator (Terumo Capiox RX-25) (PTX) under standardized in vitro conditions compared to two ionic-bound and one covalent-bound heparin-coated models. METHOD: Each oxygenator was mounted in a separate standardized closed circulation system. Heparinized (5 IE/mL) fresh human blood from the same donor (hemodilution: Ringer's solution) was used. Circulation time: 120 - 180 min with a flow rate of 4.0 L/min. Blood samplings: at the beginning, 5. min and every 30 min of the circulation. PARAMETERS: platelets, granulocytes, plasma factors (p-selectin, alpha-granulomeres expression, and TAT(III)-complex). After the experiment, oxygenators were dismantled and examined by scanning electron microscopy. RESULTS: All of the oxygenators led to an initial reduction of platelets and granulocytes. PTX had the lowest platelet and granulocyte reduction rates. With a lower p-selectin release compared to covalent-bound heparin-coated oxygenator and higher expression of alpha-granulomeres compared to ionic-bound heparin-coated oxygenators, the results of PTX indicated that a high number of circulating platelets were intact on the PMEA surface. TAT(III)-complex showed a steady increase in all of the oxygenators during the tests, more remarkably in PTX. In contrast to ionic-bound heparin-coated oxygenators, the electron microscopy displayed virtually no cellular accumulation on hollow fiber and housing surfaces of PTX and covalent-bound heparin-coated oxygenator. CONCLUSIONS: 1. The hemocompatibility characteristics of PTX were remarkably better than ionic-bound heparin-coated oxygenators and slightly better than the covalent-bound heparin-coated model under in vitro conditions. 2. The PMEA coating can be a useful alternative for patients with heparin-associated disorders. 3. The clinical feasibility of PTX should be evaluated under in vivo conditions.

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids.

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, (13)C and (31)P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1-(13)C-glucose was almost entirely to 3-(13)C-lactate (88 +/- 12%, n = 7), even though the perfusion medium was equilibrated with 95% O(2). Labeling was also observed in other glycolytic metabolites, primarily alanine and alpha-glycerolphosphate. A low level of (13)C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1-(13)C-glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high-resolution (13)C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of < or =0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2-(13)C-acetate into other metabolites was detected either in intact perfused spheroids or in high-resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3-(13)C-propionate replaced acetate, the high-energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3-(13)C-propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B(12), a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2- and 3-labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity.

Muscarinic properties of compounds related to arecaidine propargyl ester.

A series of new analogues of the arecaidine propargyl ester (CAS 35516-99-5), APE, 1a) with alcohols consisting of 4 or 5 carbon atoms were investigated at muscarinic receptor subtypes. The muscarinic activity of the quaternary and tertiary salts of the APE-related compounds were assayed on the isolated guinea-pig ileum (M3 receptor subtype) and guinea-pig left atria (M2 receptor subtype) as well as on rabbit isolated vas deferens (M1 receptor subtype). The structural variations made in the APE molecule, replacing the triple bond in the ester side chain with structures such as double bond, an allene moiety, a single bond, a cyclopropyl group or two triple bonds should alter the selectivity and potency in favour of the M2 subtype. Enhanced, though modest, selectivity for M2 receptors was achieved with the 2-butynyl ester 2a. The other structural variations resulted in a loss of potency, but not necessarily of efficacy.

Inhibition of tumor cell proliferation by dexamethasone: 31P NMR studies of RIF-1 fibrosarcoma cells perfused in vitro.

The impact on tumor cell metabolism of a substantial reduction in cell proliferation rate without acute cytotoxicity was examined in cultured RIF-1 tumor cells following treatment with an antiproliferative steroid, dexamethasone (DEX). After 48 h exposure to 4 mM DEX, acute cell viability was essentially unchanged: cells were 93 +/- 2% trypan blue excluding in both control and treated cultures (all values are mean +/- SD). The fraction of actively proliferating cells in the S phase (as indicated by incorporation of 5-bromodeoxyuridine) was only 4 +/- 3%, compared with 13 +/- 3% in age-matched control cultures (n =4, paired t-test: p < 0.004) and 23 +/- 7% at the beginning of the treatment. Three days of DEX treatment resulted in a limited increase in the level of apoptosis (programmed cell death): cells did not become rounded or detached, but the fraction expressing apoptotic DNA fragmentation (susceptible to nick end labeling by terminal deoxy-nucleotidyl transferase) was 15 +/- 7%, vs 2 +/- 1% in control cultures (p < 0.02). Despite a 75% inhibition of cell proliferation, DEX caused only a modest change in the 31P NMR spectra of RIF-1 cells in vitro. The ratio of phosphocreatine to nucleoside triphosphates (NTP) was 30% higher, on average, in treated than in control cells (n = 8, paired t-test, p < 0.02), even when both treated and control cell densities were low. The level of total phosphomonoester (relative to NTP) was lower at low cell density, but this was independent of whether cells were growing rapidly (control low density) or were growth inhibited by DEX. Neither the ratio of phosphocholine to NTP nor the intracellular pH was significantly different in DEX-treated cells.

Stereoselective interaction of uncharged esters at four muscarinic receptor subtypes.

We investigated the binding and pharmacological properties of the esters of 3,3-dimethylbutan-1-ol (the carbon analogue of choline) with either diphenylglycolic acid, (R)-phenylcyclohexylglycolic acid, or (S)-phenylcyclohexylglycolic acid [BS-6181, (R)-BS-7826 and (S)-BS-7826, respectively] at muscarinic M1, M2, M3 (Hm3) and M4 receptors. The three uncharged compounds were muscarinic receptor antagonists, with pA2 or pKi values between 7.9 and 5.6. The achiral ester BS-6181 displayed highest affinity for M1, M3 (Hm3) and M4 receptors (pA2 or pKi = 7.2-7.6) and lower affinity for M2 receptors (pA2 or pKi = 6.7 and 6.8). The four muscarinic receptor subtypes were able to distinguish between the two enantiomers of the cyclohexyl derivative of BS-6181 [(R)- and (S)-BS-7826], with a preference for the (R)-isomer (up to 79-fold). Interestingly, the (S)-enantiomer of BS-7826, being the distomer, was found to be M4 selective (pKi/M4 = 6.9; pA2 or pKi/M1-M3 (Hm3) = 5.6-6.2). These results indicate that uncharged compounds may (stereo)selectively bind to muscarinic receptors via hydrophobic interactions. Thus, an ionic bond between muscarinic ligands and an anionic site of the receptor is not absolutely necessary for recognition of muscarinic receptors.

Influence of monovalent cations on the binding of a charged and an uncharged ('carbo'-)muscarinic antagonist to muscarinic receptors.

1. The effect of the buffer concentration on binding of [3H]-N-methylscopolamine to muscarinic receptors M2 was tested in rat heart. Tracer binding was of low affinity in a 20 mM imidazole buffer (pKD 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate buffer concentration (pKD 9.92 to 9.22), slightly inhibited by an increase of the Tris/HC1 buffer concentration from 20 to 100 mM (pKD 9.70 to 9.47) and unaffected by an increase of the histidine/HC1 buffer concentration from 20 to 100 mM (pKD 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardiac M2 receptors and to transiently expressed wild-type and (Y533-->F) mutant m3 muscarinic receptors in COS-7 cells. 2. Equilibrium [3H]-N-methylscopolamine binding to cardiac M2 receptors was inhibited, apparently competitively, by monovalent salts (LiCl > or = NaCl > or = KCl). In contrast, binding of the uncharged 3,3-dimethylbutan-1-ol ester of diphenylglycolic acid (BS-6181) was facilitated by addition of monovalent salts (LiCl > or = NaCl > or = KCl) to the binding buffer. This cation binding pattern is consistent with interaction with a large, negative field strength binding site, such as, for instance, a carboxylic acid. 3. In the presence of 100 mM NaCl, [3H]-N-methylscopolamine had a similar affinity for the wild-type m3 receptor (pKD 9.85) and for a (Y533-->F) mutant m3 receptor (pKD 9.68). However, in the absence of added salts, the tracer had a significantly lower affinity for the mutated (pKD 10.19) as compared to the wild-type (pKD 10.70) m3 receptor. BS-6181 had a significantly lower affinity for the (Y533-->F) mutant m3 muscarinic receptor, as compared to the wild-type m3 receptor, both in the absence (pKD 6.19-6.72) in the presence (pKD 6.48-7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncharged ester and of [3H]-N-methylscopolamine to the m3 receptor were decreased by the mutation. 4. Taken together, these results support the hypothesis that monovalent cations from the buffer may interact with the cation binding site of the receptors (an aspartate residue in the third transmembrane helix of muscarinic receptors). Buffer cations may inhibit competitively the binding of (charged) muscarinic ligands having a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric 'carbo-analogues'. Mutation of the (Y533-->F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.

Glucose metabolism in RIF-1 tumors after reduction in blood flow: an in vivo 13C and 31P NMR study.

Low pH appears to enhance the effectiveness of therapeutic hyperthermia. 13C and 31P NMR spectroscopy have been employed to examine the possibility that elevating glucose in a solid tumor while simultaneously reducing tumor blood flow would induce a more profound acidosis than either treatment alone. When blood flow in RIF-1 tumors was acutely reduced by administration of hydralazine and additional glucose was delivered locally by intratumoral injection, tumor acidosis (as determined by 31P NMR spectroscopy) during the period of reduced blood flow was not enhanced, relative to administration of hydralazine alone. Tumor NTP/P1 ratios decreased significantly within 20 min of hydralazine administration, whether or not glucose was injected, although NTP/P1 ratios were slightly higher in tumors that received extra glucose. Tumor lactate concentrations were not significantly different in glucose-supplemented tumors, despite glucose concentrations that were 4 to 5 times higher. When the added glucose was labeled with 13C, no correlation was detected between the pH in an individual tumor and the intensity of the 3-[13C]-lactate resonance in the same tumor.

31P NMR spectroscopic studies of the effects of cyclophosphamide on perfused RIF-1 tumor cells.

To determine whether direct cellular effects of chemotherapy are responsible for 31P NMR spectral changes observed in treated tumors in vivo, RIF-1 fibrosarcoma cells were examined in vitro before, during, and after treatment with 4-hydroperoxycyclophosphamide (4-HC), an activated form of cyclophosphamide. When RIF-1 cells were treated with 4-HC in a metabolically stable but nonproliferating state, the 31P NMR spectra were identical with those of untreated cells for up to 70 h. When actively proliferating RIF-1 cells were treated with 4-HC, the intensities of the nucleotide triphosphate resonances, which increased linearly during control cell growth, remained constant for 50 h or longer. These studies demonstrate that the bioenergetic improvement observed following treatment of RIF-1 tumors in vivo [S.-J. Li, J.P. Wehrle, S.S. Rajan, R.G. Steen, J.D. Glickson, and J. Hilton, Cancer Res. 48, 4736 (1988)] does not result from direct effects of cyclophosphamide metabolites on RIF-1 cell metabolism, but rather from indirect effects of treatment on tumor or host physiology.

Gel-entrapment of perfluorocarbons: a fluorine-19 NMR spectroscopic method for monitoring oxygen concentration in cell perfusion systems.

Oxygenation is a major determinant of the physiological state of cultured cells. 19F NMR can be used to determine the oxygen concentration available to cells immobilized in a gel matrix by measuring the relaxation rate (1/T1) of perfluorocarbons (PFC) incorporated into the gel matrix. In calcium alginate gel beads without cells the relaxation rate (1/T1) of the trifluoromethyl group of perfluorotripropylamine (FTPA) varies linearly with oxygen concentration, with a slope of 1.26 +/- 0.15 x 10(-3) s-1 microM-1 and an intercept of 0.50 +/- 0.04 s-1. During perfusion with medium equilibrated with 95%/5% O2/CO2, changes in PFC T1s indicate that the average oxygen concentration was reduced from 894 +/- 102 microM in the absence of cells to 476 +/- 65 microM and 475 +/- 50 microM in the presence of 0.7 x 10(8) EMT6/Ro and RIF-1 murine tumor cells per milliliter of gel, respectively. The presence of 0.2 microliters of FTPA/ml of gel had no effect on the energy status of the cells as indicated by 31P NMR spectra. To calculate oxygen gradients within the beads from the average PFC T1 of the sample, a mathematical model was used assuming that oxygen is the limiting nutrient for cell metabolism and that the cellular oxygen consumption rate is independent of oxygen concentration. Data for EMT6/Ro cells were fit using experimentally determined perfusion parameters together with literature values for cell volume and oxygen consumption rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of absolute phosphate metabolite concentrations in RIF-1 tumors in vivo by 31P-1H-2H NMR spectroscopy using water as an internal intensity reference.

The absolute metabolite quantification method of Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] in which the tissue water proton signal is used as an internal intensity standard and its more recent variation in which NMR peak intensities are referenced to that of the natural abundance deuterium signal of water [Li et al., SMRM Abstr. 2, 825 (1988); Song et al., Magn. Reson. Med. 25, 45 (1992) have been implemented to obtain absolute phosphate metabolite concentrations in subcutaneous RIF-1 tumors during untreated growth and following treatment with 5-fluorouracil. The equivalence of these two hydrogen isotopes as intensity standards and the validity of their use in the determination of absolute metabolite concentrations in vivo by NMR has been demonstrated. On matched in vivo and extract tumor samples (n = 5), excellent agreement has been obtained between nucleoside triphosphate concentrations determined by NMR and those derived by HPLC analysis for the control tumors. Following 3 days of untreated growth, absolute concentrations of phosphate metabolites in RIF-1 tumors (n = 10) decreased significantly, except for the Pi concentration which did not vary. For the treated tumors (n = 10) there were no changes in metabolite concentrations except for a decrease in the PCr and, possibly, Pi concentrations. The PCr/Pi ratio in the latter tumors did not change. These observations suggest that changes in absolute metabolite concentrations may be more sensitive indices of response to therapy than changes in metabolite peak amplitude ratios, a parameter commonly used to express in vivo NMR data.

31P NMR spectroscopic study of the effects of gamma-irradiation on RIF-1 tumor cells perfused in vitro.

In order to examine the mechanisms underlying radiation-induced changes in phosphorus metabolite levels observed in RIF-1 tumors in vivo, RIF-1 cells in culture were perfused for up to 70 h following gamma-irradiation with 0-25 Gy and monitored continuously by 31P NMR spectroscopy at 8.5 T. Cells immobilized in the sample volume by incorporation into calcium alginate beads were bioenergetically stable, but did not replicate at the cell density used. Following an initial increase in PCr and NTP, which occurred in both control and irradiated cells, a dramatic decline in high-energy phosphates was detected beginning 24-30 h after irradiation with 15 or 25 Gy. In contrast, unirradiated cells or cells treated with 10 Gy remained metabolically stable for up to 72 h. The metabolic changes induced by irradiation of the cultured cells, which reflected cell death and lysis, were distinctly different from those observed in RIF-1 tumors in vivo during the same postirradiation time interval--an increase in high-energy relative to low-energy phosphates. This suggests that the spectral changes in vivo do not result from direct modification of cellular energy metabolism by radiation injury.