Biomedical subjects
B D Ross
Publications and source records attributed to B D Ross.
Gas chromatographic-mass spectrometric analysis of hexose monophosphate shunt activity in cultured cells.
A GC/MS method is described for monitoring the relative amount of glucose degraded to lactate via the hexose monophosphate shunt (HMPS) in neoplastic cells. C6 glioma cells were incubated in medium supplemented with [1-13 C]glucose and medium containing [1-13C]glucose with 0.001 mM phenazine methosulfate (PMS). The ratio of the [13C]lactate to [12C]lactate determined from the measurement of the m/z 219/220 and 117/118 ions of the trimethyl silyl derivative, was used to calculate HMPS activity. PMS increased HMPS activity in C6 glioma cells by 3.2 and 4.8 fold at 2 and 12 hours of incubation respectively. GC/MS results were compared with 1H NMR measurements of the [3-13C]lactate/[3-12C]lactate ratio. The GC/MS method was found to require less sample size and yielded better sensitivity than the NMR method.
Magnetic resonance imaging with adiabatic pulses using a single surface coil for RF transmission and signal detection.
In order to overcome the problems that arise from nonuniform B1 fields, there has been interest in developing pulses that are insensitive to large variations in RF power. Pulses derived from adiabatic passage principles that can execute spin inversion, excitation, and 90 degrees and 180 degrees plane rotations in the presence of B1 inhomogeneities have recently been described. When driven with optimized modulation functions, these pulses can execute uniform excitation, refocusing, and slice-selective inversion over a 10-fold or greater variation in B1 magnitude. This insensitivity to B1 strength enables the execution of T1- and/or T2-weighted spin-echo imaging experiments using coils, such as the surface coil, with extremely inhomogeneous B1 profiles. We have successfully acquired images with these pulses at 200 MHz using a single surface coil as the transmitter and receiver. Images of the slice definition, the region over which the excitation and refocusing pulses operate with a surface coil, and brain images obtained with slice planes perpendicular to the plane of the surface coil are presented. Results demonstrate that these pulses can be transmitted with a surface coil to yield high-quality T1- and/or T2-weighted images without B1 artifacts.
In vivo 31P and 2H NMR [corrected] studies of rat brain tumor pH and blood flow during acute hyperglycemia: differential effects between subcutaneous and intracerebral locations.
Surface coil NMR spectroscopy was used to monitor the hyperglycemia-induced alterations in pH and blood flow in vivo in C6 gliomas implanted both subcutaneously and intracerebrally in rats. Tumor pH was calculated from the chemical shift difference between PCr and Pi in the 31P NMR spectra. Subcutaneous glioma pH decreased 0.8 units by 1 h after intraperitoneal administration of an aqueous 50% glucose solution (6 g glucose per kg body weight). In contrast, hyperglycemia failed to significantly alter the pH of intracerebral gliomas which were monitored for 90 min following administration of glucose. Tumor blood flow (TBF) was determined both pre- and post-glucose administration using deuterium NMR by monitoring the time course of D2O washout following intratumoral injection of saline D2O. Subcutaneous and intracerebral TBF were found to have an average change of -78.1% (range -47.4 to -93.3%, n = 5) and -21.1% (range +6.0 to -37.8%, n = 9), respectively. In addition, laser Doppler blood flow measurements of rat skin and subcutaneous glioma revealed a dramatic reduction in blood flow in both tissues following glucose administration. These results indicate that the effects of acute hyperglycemia are site dependent and that hyperglycemia alone is not beneficial for inducing intracellular acidosis in intracerebral tumors.
31P NMR spectroscopy of the in vivo metabolism of an intracerebral glioma in the rat.
The in vivo high-energy phosphorus metabolic profile and pH of an experimental intracerebral C6 glioma in rats was examined using surface coil 31P NMR spectroscopy. Initially, phosphorus-containing metabolites of the glioma were characterized by in vivo 31P surface coil spectroscopy of subcutaneously implanted tumors and by high-resolution NMR studies of perchloric acid (PCA) extracts of both freeze-clamped subcutaneous tumor tissue and cultured cells. These studies demonstrated that the C6 glioma has reduced levels of phosphocreatine (PCr) compared to the levels found in normal rat brain. Thus, reduced spectral PCr levels were useful as a metabolic indicator for monitoring the spatial selectivity of tumor metabolism distinct from that of adjacent normal brain tissue. To maximize 31P NMR signals from intracerebral tumors, tumor cells were stereotaxically placed superficially in the brain. Proton magnetic resonance imaging (1H MRI) was used to determine the size and location of the resultant brain tumors in order to preselect rats with large superficial tumors for spectroscopic study. 31P NMR spectra of the glioma tumors revealed a consistent reduction in the PCr/ATP ratio, an increase in the Pi/ATP ratio, and a slightly increased tissue pH. No correlation was found between levels of Pi/ATP and tumor pH in subcutaneous or intracerebral gliomas and the amount of necrosis as determined histologically. This study demonstrates that phosphorus metabolites of an experimental brain tumor in the rat can be monitored in vivo with minimal contributions from adjacent normal brain tissue metabolites using surface coil 31P NMR spectroscopy.
Carbohydrate metabolism of the rat C6 glioma. An in vivo 13C and in vitro 1H magnetic resonance spectroscopy study.
Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.
Acid-base regulation: has 31P NMR any answers?
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Dietary fat modulation of murine mammary tumor metabolism studied by in vivo 31P-nuclear magnetic resonance spectroscopy.
The effect of dietary fat concentration and saturation on high energy phosphate metabolites and phospholipid turnover in transplanted line 168 murine mammary tumors was studied using surface coil 31P-nuclear magnetic resonance spectroscopy. Female BALB/c mice were fed one of five diets each containing at least the minimum of essential fatty acids (EFA). Four diets contained additional safflower or palm oil for a total fat concentration of 5 or 20% by weight. The growth rate of tumors from mice fed the high safflower oil diet was significantly greater than the growth rate of tumors for mice fed all other diets including the one which contained the minimal EFA. 31P-nuclear magnetic resonance-observable phosphate metabolite ratios. ATP/Pi, ATP/phosphomonoester (ATP/PME), and PME/Pi, and tumor pH of line 168 tumors decreased with increasing tumor volume, indicating a shift from active to inactive tumor metabolism. The rates of those decreases with progressive tumor growth differed significantly among tumors of mice fed the different diets. Decreases in ATP/Pi, ATP/PME, and pH were the most rapid in the tumors of mice fed the high safflower oil diet and significantly faster than tumors of mice fed the diet containing minimum EFA. In addition, the decrease in the PME/Pi ratio of tumors was significantly greater in mice fed the high fat (high palm oil and high safflower oil) diets than mice fed the diet containing the minimum of EFA. The rate of decline of ATP/Pi and ATP/PME with progressive tumor growth was directly correlated with levels of linoleic acid as well as total unsaturated fat. High levels of a polyunsaturated fat had a significant effect on mammary tumor metabolism particularly during early stages of tumor growth. Differences in high energy phosphate metabolite dynamics relative to dietary fat were present in tumors of equal volume. Thus, dietary fat influences on mammary tumorigenesis may be related to high energy phosphate metabolites.
31P NMR spectroscopy of an experimentally induced intracerebral tumor in mice.
31P surface coil NMR spectroscopy was used to evaluate in vivo high-energy phosphorus metabolism in the brains of mice with experimentally induced primary intracranial and subcutaneous KHT sarcomas. 31P spectra of subcutaneous KHT tumors revealed a lack of detectable phosphocreatine (PCr) levels in the tumor as compared to the relatively high endogenous levels of PCr in normal mouse brain. As the intracerebral tumor size increased, there was a reduction in spectral PCr levels over a 23-day postinoculation period in situ in the brain. No histologic or spectroscopic evidence of tumor-associated necrosis or hypoxia was found. This study demonstrates that surface coil 31P NMR spectroscopy can be used to monitor changes in high-energy phosphate metabolism associated with progressive growth of an experimentally induced mouse brain tumor in situ.
Spectral resolution in clinical magnetic resonance spectroscopy.
We observe linewidths of 0.1-0.4 ppm in 31P spectra of human muscle, liver, and brain. T2 measurements of muscle phosphocreatine, together with previous brain studies, indicate that further improvements in linewidth to 0.02-0.05 ppm might often be achieved, but in some lesions magnetic susceptibility variations may limit spectral resolution.
Magnetic resonance spectroscopy for the determination of renal metabolic rate in vivo.
The method of magnetic resonance spectroscopy has been validated and applied to the determination of renal metabolic rate in vivo. Using an indwelling detector coil, 31P NMR spectra from one kidney of anesthetized rats were quantified. The concentration of ATP was the same as that determined enzymatically, but both ADP and Pi were substantially lower. Only 25% of renal Pi and virtually none of the ADP were detected by NMR. The remainder is assumed to be bound to proteins. These concentrations of metabolites contributed to a significantly increased phosphorylation potential, which in turn should increase the free energy of hydrolysis of ATP. Saturation transfer, a non-destructive magnetic technique for the measurement of chemical exchange, was readily able to detect synthesis of ATP from ADP and inorganic phosphate. The rate of ATP synthesis determined was comparable to that determined in parallel studies of renal oxygen consumption. An ATP:O ratio of approximately 2 was found, indicating that fatty acid is the preferred fuel of respiration of the rat kidney in vivo.
Glucose metabolism in renal tubular function.
Heterogeneity of metabolic activity along the nephron points to a very varied relationship between glucose metabolism and ion transport. Glycolysis is linked closely to free-water clearance and possibly to sodium, potassium, and hydrogen ion transport. Glucose oxidation, while not the major source of renal energy, is crucial in sodium, potassium, and phosphate reabsorption. Gluconeogenesis recovers carbon compounds generated during the process of renal ammoniagenesis. Glucose synthesis and active sodium transport appear to compete for renal ATP, although no regulatory function for this competition has been identified. Glucose formed in the proximal tubule may support free-water clearance in adjacent distal tubule, but is not thought to contribute to any medullary function. The complex network of biosynthetic and catabolic pathways of glucose metabolism may have evolved in the kidney to protect the organism against wide variations in glucose demand which would otherwise be unavoidable during the course of rapidly fluctuating renal electrolyte loads.
Glutamine synthesis in the perfused rat kidney and in isolated rat cortical tubules: regulation by pH.
The possible involvement of renal glutamine synthesis in the regulation of ammoniagenesis during metabolic acidosis has been investigated. Using an isotope dilution technique glutamine synthesis has been demonstrated in the intact perfused rat kidney, despite zero net balance for glutamine in this preparation. Inhibition of glutamine synthesis resulted in increased ammonia production in isolated cortical tubules. The rates of these processes were comparable. We conclude that recycling of nitrogen through glutamine synthesis and glutamine hydrolysis could play a quantitatively significant role in the control of ammonia excretion by the kidney, particularly in acute acidosis where the fall in pH seems to inhibit glutamine synthesis.
Formation of n.m.r.-invisible ADP during renal ischaemia in rats.
Measurement of the adenine nucleotide and inorganic phosphate content of normoxic and ischaemic kidney in vivo has been made, comparing enzymic assay (after freeze-clamping and acid extraction) with quantification by 31P-n.m.r. Both methods give similar results for ATP, and n.m.r. quantification of Pi gives a value 25-50% of that obtained by enzymic assay. ADP, which is largely invisible to n.m.r. in the normoxic kidney, remains invisible during ischaemia despite a 2-3 fold rise in enzymically assayed ADP. N.m.r. and enzymic assay of the acid extracts give similar values for all metabolites measured. The question of ADP binding in the kidney is discussed, as are the implications for the metabolic regulation of ADP-dependent reactions.
The influence of renal function on lactate and glucose metabolism.
The relationship of lactate metabolism to renal function was studied in the isolated perfused rat kidney. A new radioisotopic method has been developed that enables the simultaneous measurement of lactate production and consumption in the presence of physiological concentrations of both lactate and glucose. In kidneys from fed rats, when glucose was absent, lactate production was only 12 mumol/h per g dry wt, and in kidneys from starved rats there was no lactate production, indicating that neither the phosphoenolpyruvate/pyruvate substrate cycle nor other analogous cycles for the recycling of lactate carbon are operating in the intact kidney cortex. Lactate production from glucose occurred at a high rate, at the same time as lactate consumption, demonstrating that lactate recycling between renal cortex and medulla can occur in the intact kidney. Lactate production from glucose correlated with glomerular filtration rate (P less than 0.001), urine flow rate (P less than 0.01) and sodium reabsorption (P less than 0.05). There was significant basal lactate production at zero glomerular filtration rate. Lactate consumption was not correlated with any renal function. When Na+ reabsorption was inhibited with the diuretic frusemide, or when filtration was entirely prevented (the 'non'-filtering kidney'), lactate production was decreased by 39% and 50% respectively. Basal lactate production determined in this way was the same as that calculated above by linear regression. Prevention of filtration, but not the addition of frusemide, significantly inhibited lactate consumption. It is concluded that glycolysis is required for medullary Na+ transport, and that some different transport function(s) require lactate oxidation.
Phosphorus metabolites by NMR.
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Effect of hibernation on liver and kidney metabolism in 13-lined ground squirrels.
Metabolic rates and adenine nucleotide content of liver and kidney from hibernating ground squirrels were measured and compared to rats to study the biochemical adaptation to hibernation. High rates of renal and hepatic gluconeogenesis were observed in squirrels, particularly from propionate and glycerol compared to rat. During hibernation and starvation soluble phosphoenolpyruvate carboxykinase activity was increased in both liver and kidney. Although metabolic rates are decreased during hibernation the results suggest that the enzymic complement is maintained at high activity even during torpor.
Carnitine as a possible adjunct in parenteral feeding.
Carnitine is necessary for the transport of fatty acids across the inner mitochondrial membrane, and depletion in response to Intralipid infusion has previously been demonstrated. This study investigates whether orally administered L-carnitine increases tolerance to a lipid load given intravenously. Eight patients with active inflammatory bowel disease, being treated with intravenous prednisolone, were studied. Intralipid was infused on two occasions. Triglycerides and ketone bodies rose in a reproducible manner. Carnitine did not influence these changes. Carnitine excretion rose after an oral dose indicating that carnitine was absorbed, but carnitine excretion was increased in the steroid-treated individuals and rose after oral prednisolone in two healthy subjects. It is concluded that under the conditions of this study oral carnitine is without demonstrable effect on the handling of an intravenous lipid load.