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Biomedical subjects

A P Koretsky

Publications and source records attributed to A P Koretsky.

At least 19 recordsLinked to original sources
PubMed1992

2H-nuclear magnetic resonance imaging of tumor blood flow: spatial and temporal heterogeneity in a tissue-isolated mammary adenocarcinoma.

2H-Nuclear magnetic resonance imaging of deuteron accumulation in tissue following an i.v. bolus of deuterium oxide provides a noninvasive means of constructing maps of tissue perfusion. With a measured arterial input function and a simple model for tissue-capillary exchange, these data can provide quantitative estimates of local flow. This technique was tested in rat brain and then applied to the study of spatial heterogeneity and temporal variation of blood flow in the tissue-isolated R3230AC mammary adenocarcinoma. Global flow from the brain averaged 0.96 ml/min.g, in good agreement with results obtained from other methods; the perfusion of brain was relatively homogeneous. Global tumor blood flow averaged 0.32 ml/min.g, ranging from 0.11 to 0.96 ml/min.g. Imaging revealed variations in perfusion both within and between the tumors that far exceeded those expected from brain flow heterogeneity and uncertainty in the flow estimates. By obtaining repeated flow images at 30-min intervals, it was possible to show that the regional blood flow shifted with time in single pixels and in multipixel regions. These experiments show that 2H-nuclear magnetic resonance may be useful in obtaining noninvasive and quantitative measurement of temporal blood flow changes in a solid tumor in vivo.

Adenocarcinoma↗
PubMed1992

Magnetic resonance imaging of perfusion using spin inversion of arterial water.

A technique has been developed for proton magnetic resonance imaging (MRI) of perfusion, using water as a freely diffusable tracer, and its application to the measurement of cerebral blood flow (CBF) in the rat is demonstrated. The method involves labeling the inflowing water proton spins in the arterial blood by inverting them continuously at the neck region and observing the effects of inversion on the intensity of brain MRI. Solution to the Bloch equations, modified to include the effects of flow, allows regional perfusion rates to be measured from an image with spin inversion, a control image, and a T1 image. Continuous spin inversion labeling the arterial blood water was accomplished, using principles of adiabatic fast passage by applying continuous-wave radiofrequency power in the presence of a magnetic field gradient in the direction of arterial flow. In the detection slice used to measure perfusion, whole brain CBF averaged 1.39 +/- 0.19 ml.g-1.min-1 (mean +/- SEM, n = 5). The technique's sensitivity to changes in CBF was measured by using graded hypercarbia, a condition that is known to increase brain perfusion. CBF vs. pCO2 data yield a best-fit straight line described by CBF (ml.g-1.min-1) = 0.052pCO2 (mm Hg) - 0.173, in excellent agreement with values in the literature. Finally, perfusion images of a freeze-injured rat brain have been obtained, demonstrating the technique's ability to detect regional abnormalities in perfusion.

Animals↗
PubMed1992

Perfusion imaging.

Measurement of tissue perfusion is important for the functional assessment of organs in vivo. Here we report the use of 1H NMR imaging to generate perfusion maps in the rat brain at 4.7 T. Blood water flowing to the brain is saturated in the neck region with a slice-selective saturation imaging sequence, creating an endogenous tracer in the form of proximally saturated spins. Because proton T1 times are relatively long, particularly at high field strengths, saturated spins exchange with bulk water in the brain and a steady state is created where the regional concentration of saturated spins is determined by the regional blood flow and regional T1. Distal saturation applied equidistantly outside the brain serves as a control for effects of the saturation pulses. Average cerebral blood flow in normocapnic rat brain under halothane anesthesia was determined to be 105 +/- 16 cc.100 g-1.min-1 (mean +/- SEM, n = 3), in good agreement with values reported in the literature, and was sensitive to increases in arterial pCO2. This technique allows regional perfusion maps to be measured noninvasively, with the resolution of 1H MRI, and should be readily applicable to human studies.

Animals↗
PubMed1992

Measurement of brain perfusion by volume-localized NMR spectroscopy using inversion of arterial water spins: accounting for transit time and cross-relaxation.

The theoretical model for perfusion measurement by NMR using arterial labeling of endogenous water is extended to include the effects of transit time and cross-relaxation of tissue water with macromolecules. Water magnetization in rat brain is monitored using the STEAM method to simultaneously determine the transit time, magnetization transfer rate constant, and perfusion. The results show that the transit time in rat brain is quite short, and thus its effect on perfusion measurement is small. It is also demonstrated both theoretically and experimentally that the steady-state effects of cross-relaxation with macromolecules on perfusion measurement are accounted for by a proper control experiment.

Animals↗
PubMed1992

Investigation of cell physiology in the animal using transgenic technology.

Over the past 10 years significant progress has been made in techniques for manipulating the genome of the animal. Production of transgenic mice has led to important insights into the regulation of gene expression, the molecular basis of cancer, immunology, and developmental biology. The tools necessary to generate transgenic mice are becoming widely available, making it possible to study a variety of problems. In this review a description of the strategies being used to address problems of interest in cell physiology using transgenic mice is given. Elucidation of the rules governing the regulation of gene expression now permits the targeted expression of a protein to a particular organ or cell type within an organ. Overexpression of proteins, expression of foreign or mutant proteins, mislocalization of proteins, and directed elimination of proteins are all procedures that can now be used to generate interesting animal models for physiological studies. The applications of these techniques to a variety of problems in normal and abnormal physiology are discussed in this review.

Animals↗
PubMed1992

The role of magnesium in postischemic cardiac dysfunction.

BACKGROUND: The biochemical basis for postischemic myocardial stunning is not fully elucidated. Magnesium is an important regulator of cellular energetic processes and excitation-contraction coupling. We hypothesized that the decrease in function in the postischemic period may be the result of an alteration in magnesium regulation. METHODS: In a Langendorf perfused rabbit heart model, we used 31P nuclear magnetic resonance spectroscopy to noninvasively determine intracellular Mg2+ and high-energy phosphate levels in the preischemic period and after a 30-minute period of normothermic ischemia. We measured adenosine triphosphate (ATP), phosphocreatine, and the phosphocreatine/inorganic phosphate ratio and calculated the free energy of ATP hydrolysis (delta GATP). On reperfusion, hearts were divided into three groups (n = 7 per group)--those receiving unmodified Krebs-Henseleit (control), 192 ng/ml dobutamine, or 5 mmol/L pyruvate. RESULTS: Function (expressed as the rate-pressure product) was approximately 77% of preischemic values in the control group, whereas in both dobutamine and pyruvate groups it returned to preischemic levels. ATP was decreased similarly in all groups in the postischemic period. Phosphocreatine/inorganic phosphate ratio and delta GATP were higher in the pyruvate group compared with the other groups. Intracellular Mg2+ was elevated significantly in the unmodified control postischemic group compared with preischemic, postischemic dobutamine, and pyruvate groups (1.0 +/- 0.12 vs 0.80 +/- 0.08, 0.64 +/- 0.08, and 0.70 +/- 0.05 mmol/L, respectively; p less than 0.05). CONCLUSIONS: We conclude that (1) postischemic "stunned" hearts have elevated Mg2+ levels in association with impaired contractile function, (2) inotropic agents improve contractile function in association with a decline in Mg2+ to preischemic levels despite differing effects on intracellular energetics, and (3) Mg2+ may play an important regulatory role in the heart after ischemia.

Animals↗
PubMed1991

Phosphocreatine protects ATP from a fructose load in transgenic mouse liver expressing creatine kinase.

The effects of an intraperitoneal dose of fructose on hepatic metabolism in transgenic mice expressing creatine kinase in liver were investigated using phosphorus-31 nuclear magnetic resonance (31P-NMR). Transgenic mice were fed diets containing varying amounts of creatine (Cr; 0-12%). It has previously been shown that 31P-NMR spectra of transgenic mice have a peak due to phosphocreatine (PCr), the intensity of which was proportional to the amount of Cr in the diet. No PCr peak was detected in control mice or transgenic mice not fed Cr. In the present study NMR spectra were collected before and for a 1-h recovery period after infusion of 0.15 mmol/10 g body wt fructose. In all mice infusion of fructose resulted in a two- to threefold elevation of phosphomonoesters. In control and non-Cr-fed transgenic mice this was accompanied by a 60% reduction of the inorganic phosphate (Pi) and a 50% fall in ATP. In transgenic mice fed Cr, the extent of reduction of Pi was dependent on the level of PCr and was markedly reduced compared with controls. Falls in Pi of 46, 24, and 6% were detected 12.5 min after fructose infusion in low, intermediate, and high PCr-containing livers, respectively. The presence of PCr also protected hepatic ATP levels from a fructose load. Transgenic mice fed on high or intermediate Cr diets showed no significant loss of ATP. However, livers with low levels of PCr lost ATP during a fructose challenge. From the equilibrium established by creatine kinase, free ADP levels were calculated throughout the fructose dose. Fructose caused a 2.5-fold increase in free ADP. This rise in ADP was independent of the total Cr or whether Pi and ATP were reduced by fructose infusion. These results indicate that an increase in ADP is not sufficient to cause depletion of ATP during a fructose challenge.

Adenosine Triphosphate↗
PubMed1990

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration.

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.

Adenosine Diphosphate↗
PubMed1990

Measurement of cerebral blood flow in rat brain by 19F-NMR detection of trifluoromethane washout.

The washout of trifluoromethane (CHF3) from rat cerebral cortex was monitored by 19F NMR. After 15 min of inhalation of 67% CHF3/33% O2 the fluorine signal detected was in a steady state. The CHF3 was switched off rapidly at the endotracheal tube and the washout detected with 12-s time resolution. Two models were used to extract flow information, a simple exponential fit and a model which accounts for arterial CHF3 recirculation. In both cases, a two-compartment model fit the data significantly better than a one-compartment model. In both models, the faster time component varied with increasing pCO2, but no significant change in the slow component was detected. At control values of pCO2, there was a small difference in washout rate constants derived from the two models. At high pCO2, when tissue washout was comparable to arterial washout of CHF3, the model which accounted for arterial recirculation gave higher flows. Using this two-compartment model with correction for recirculation, a control flow (pCO2 = 35 mm Hg) of 0.73 +/- 0.04 ml/min/g was measured. Increasing plasma pCO2 increased the apparent flow six- to sevenfold with a 4.4% increase in flow per millimeter of Hg change in CO2. These results are qualitatively in agreement with results found by others using the washout of 133Xe. However, this method yields values for flow that are lower than those obtained using 133Xe washout, probably because of diffusion limitations of CHF3.

Animals↗
PubMed1990

Nuclear magnetic resonance determination of flow, lactate, and phosphate metabolites during amphetamine stimulation of the rat brain.

The effects of amphetamine on rat cerebral blood flow, lactate, and phosphate levels were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Proton NMR imaging was used to determine the region of brain being studied. Blood flow was estimated by detecting the washout of trifluoromethane using 19F NMR. A dose of 20 mg/kg D-amphetamine sulphate stimulated flow fourfold over control values for at least 1 h. Even though amphetamine stimulated a large increase in flow, there were no significant changes in levels of inorganic phosphate, ATP, phosphocreatine, pH or Mg2+ as determined by 31P NMR. This was true for doses of 10 and 20 mg/kg D-amphetamine sulphate. No significant changes occurred in 1H NMR detected levels of lactate with a 20 mg/kg dose of amphetamines. Lactate levels increased slightly 30 min after administration of 10 mg/kg D-amphetamine sulphate. The results call into question the central role of changes in levels of phosphates in coupling increased cortical activity to increases in metabolism.

Amphetamine↗
PubMed1990

Absence of pH changes during altered work in the in vivo sheep heart: a 31P-NMR investigation.

Saturation transfer from gamma-ATP to inorganic phosphate was used to assign the intracellular inorganic phosphate resonance of the phosphorus-31 nuclear magnetic resonance spectrum of heart obtained from adult sheep under Halothane anesthesia. The 31P chemical shift of intracellular inorganic phosphate was then used as a probe of myocardial pH. Resting myocardial pH was found to be 7.03 +/- 0.02. The effects of increasing myocardial work on myocardial pH were examined using external pacing and phenylephrine infusion alone or in combination to produce steady-state increases in the rate-pressure product. No alteration in myocardial pH was observed with up to 4-fold increases in rate-pressure product. No changes in high-energy phosphates were observed except at the highest rate-pressure products obtained, where small increases in inorganic phosphate and decreases in the phosphocreatine/ATP ratio were observed. In addition, the transition to a new steady state was studied with a 20-s time resolution after initiation of pacing. Again, no changes in pH or levels of phosphates were detected during the transition to increased work.

Adenosine Triphosphate↗
PubMed1990

NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels.

To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 mumol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (t1/2 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 +/- 0.004 mumol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.

Adenosine Diphosphate↗
PubMed1989

The B isozyme of creatine kinase is active as a fusion protein in Escherichia coli: in vivo detection by 31P NMR.

A cDNA encoding the B isozyme of creatine kinase (CKB) has been expressed in Escherichia coli from a fusion with lacZ carried by lambda gt11. Western blots indicate that a stable polypeptide with the appropriate mobility for the beta-galactosidase-creatine kinase (beta-gal-CKB) fusion protein cross-reacts with both beta-gal and CKB antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the lambda gt11-CKB construct have a CK activity of 1.54 +/- 0.07 mumol/min per mg protein. The fusion protein appears to provide this activity because immunoprecipitation of protein with beta-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli.

Animals↗
PubMed1988

Activation of dehydrogenase activity and cardiac respiration: a 31P-NMR study.

31P-NMR studies were performed to determine the tissue phosphate and oxygen consumption effects of known maneuvers on the activation of pyruvate dehydrogenase during work jumps in the perfused rat heart. In control studies of the glucose-perfused heart, work jumps, with pacing, resulted in a 32% increase in oxygen consumption (QO2) from 1.72 +/- 0.09 to 2.29 +/- 0.12 mmol O2.h-1.g dry wt-1. During this transition no significant change in the high energy phosphates were detected. In contrast, work jumps did cause changes in the phosphates when the activation of pyruvate dehydrogenase was blocked with 2.5 micrograms of ruthenium red per milliliter or maximally stimulated with 11 mM pyruvate before the increase in work. The observed increase in QO2 and inorganic phosphate and calculated increase in ADP are consistent with these phosphates controlling mitochondrial respiration under these conditions. These results suggest that the activation of pyruvate dehydrogenase and/or other dehydrogenases may be an important step in the orchestration of work and QO2.

Animals↗
PubMed1988

Potassium adaptation: 39K-NMR evidence for intracellular compartmentalization of K+.

To investigate the effects of K+ uptake on the intracellular environment, both 39K-nuclear magnetic resonance (NMR) and K+-selective electrodes were used to measure K+ activity with acute K+ loading in control and K+-adapted rats. These results were then compared with tissue K+, measured by flame photometry. There was a lower NMR K+ visibility (ratio of NMR signal to tissue content) in muscle and liver in K+-adapted rats, compared with controls before and after an acute K+ load. This lower K+ visibility in K+-adapted rats was confirmed in liver homogenate with the K+-specific electrode. In liver homogenates from control and K+-adapted rats, addition of RbCl (300 mumol/g) increased the NMR K+ signal more in K+-adapted rats (19 +/- 1.1 mumol/g) than controls (11 +/- 1.0 mumol/g, P less than 0.01). This is consistent with the displacement of K+, by Rb+, from NMR-undetected sites. These results suggest that some 10-15% of intracellular K+ may be within a compartment not detectable by NMR or electrodes and that chronic K+ loading leads to an increased capacity of this compartment.

Adaptation, Physiological↗