PubMed Health⌕ Search

Biomedical subjects

L J Neuringer

Publications and source records attributed to L J Neuringer.

At least 19 recordsLinked to original sources
PubMed1993

Quantitative differential effects of rhodamine 123 on normal cells and human colon cancer cells by magnetic resonance spectroscopy.

Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.

Adenosine Triphosphate↗
PubMed1993

13C- and 31P-NMR studies of human colon cancer in-vitro and in-vivo.

We report comparative 31P-NMR studies in-vivo and in-vitro of the human adenocarcinoma cell line HCT-116 in a high-density, perfused microcarrier culture and as a tumour from the same cell line grown in three different immune-suppressed animal models (NIH triple deficient, Nude, SCID). The phosphate metabolite ratios, pHNMR and intracellular free magnesium, derived from the 31P-NMR spectra, were compared for the in-vivo and in-vitro systems. Results obtained with HCT-116 cells on microcarrier beads are quantitatively similar to that of small (122 mm3), tumours in-vivo derived from the same cell line in any of the immune-suppressed animal systems studied. This suggests that in-vitro microcarrier cell culture serves as a useful model system for deriving information about metabolism of small, tumours in-vivo. It offers the additional advantages of allowing for precise control of substrate milieu, perfusion and oxygenation. The microcarrier system was also used to measure flux through glycolysis and the pentose cycle. In particular, we measured glucose utilization and the production of lactate, alanine, glutamine and glycogen in proton-decoupled 13C-NMR experiments following administration of [1-13C]glucose. We found that (63% +/- 6%) of the glucose utilized was released as [3-13C] lactate in the presence of oxygen, indicating that the HCT-116 cells have a high level of aerobic glycolysis. Serial labelling experiments with [1-13C] glucose and [6-13C] glucose reveal that at least (11.6% +/- 1.3%) of the glucose utilized enters the pentose cycle. We determined that (6.9% +/- 1.2%) of the glucose utilized is recycled to glucose via the pentose cycle while (4.7% +/- 1.4%) of the glucose utilized enters the pentose cycle to form lactate. The high rate of recycling via the pentose cycle suggests that a significant fraction of cellular NADPH is generated by the pentose cycle as opposed to generation by the malate-pyruvate shuttle.

Adenocarcinoma↗
PubMed1993

Creatine kinase-catalyzed reaction rate in the cyanide-poisoned mouse brain.

Brain creatine kinase (CK)-catalyzed phosphorus flux from phosphocreatine (PC) to ATP was measured in vivo in young adult mice made reversibly hypoxic by injection of cyanide. Phosphorus spectra and saturation transfer measurements of CK-catalyzed flux were acquired using a high-field (8.45 T) nuclear magnetic resonance (NMR) spectrometer. After low cyanide doses (1-3 mg/kg of body weight), there were no measurable changes in brain pH or in concentrations of PC, the nucleoside triphosphates (including ATP), and Pi. The CK-catalyzed phosphorus flux increased about 75% after the low cyanide dose. Higher doses (4-6 mg/kg) produced a transient 30-40% decrease in PC concentration, doubling of Pi, and a 0.2 unit decrease in pH. The CK-catalyzed phosphorus flux decreased 50-80% after the higher cyanide doses. This decrease in phosphorus flux was present long after reactant concentrations returned to precyanide values. It is proposed that the increase in brain CK-catalyzed phosphorus flux with the lower cyanide doses is due to an increase in ADP concentration. The large, prolonged decrease in CK-catalyzed reaction rate in the moderately poisoned brain may be due to loss of activity of the mitochondrial CK isoform.

Adenosine Triphosphate↗
PubMed1991

Angiogenesis determines blood flow, metabolism, growth rate, and ATPase kinetics of tumors growing in an irradiated bed: 31P and 2H nuclear magnetic resonance studies.

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased and growth rate is slowed, these tumors are assumed to be metabolically deprived with hypoperfusion. In this study, we directly measured the effect of tumor bed irradiation on blood flow, growth rate, rate of nucleoside triphosphate (NTP) turnover, and metabolic state using 31P and 2H nuclear magnetic resonance, and an intradermal assay for angiogenesis. (NTP turnover refers to ATP-synthetase mediated NTP turnover that is visible to 31P nuclear magnetic resonance using the technique of saturation transfer.) A decrease in the number of small blood vessels perfusing tumors in a preirradiated bed was found. Most of the decrease was due to a loss of vessels with diameters less than 0.04 mm. When tumors growing in preirradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed in most tumors. Consistent with these observations, the phosphocreatine/inorganic phosphate and nucleoside triphosphate/inorganic phosphate ratios were significantly lower in the tumors growing in a preirradiated bed compared with tumors in a nonirradiated bed. The blood flow rate was similar to control for tumors less than 100 mm3 (45.8 versus 40.5 ml/100 g/min, P = not significant), but was significantly lower than control for tumors greater than 100 mm3 (40.4 versus 12.2 ml/100 g/min, P less than 0.01). The NTP turnover rates correlated (P less than 0.005, r = 0.66) with the volume doubling rate (1/tumor volume doubling time), but for tumors approximately 100 mm3 in size neither the volume doubling rate nor the NTP turnover rate of tumors growing in an irradiated bed was statistically lower than control [NTP turnover: 14 +/- 3%/s versus 9 +/- 2%/s; volume doubling rate: 0.47 +/- 0.07/day versus 0.33 +/- 0.04/day (mean +/- SE)]. A large intertumor variability of all metabolic parameters was observed.

Adenosine Triphosphatases↗
PubMed1991

Maturational increase in mouse brain creatine kinase reaction rates shown by phosphorus magnetic resonance.

In-vivo phosphorus fluxes in the reaction catalyzed by creatine kinase (CK) were measured in brains of mice from 3 to 40 days of age using high-field (8.45 T) phosphorus magnetic resonance and the saturation transfer technique. This technique gives the ratio of chemical flux to reactant concentration directly and allows the calculation of pseudo-rate constants for the forward direction from PC to ATP (kf) and for the reverse direction (kr). The spin-lattice relaxation times (T1) for phosphocreatine (PC) and for the nucleoside triphosphate (NTP) nuclei, estimated by the progressive saturation technique, did not change during this age period. The PC concentration doubled relative to the NTP concentration over the first month of life. The kf and the flux of phosphorus nuclei in the forward direction increased 2- to 3-fold in the very narrow time period from 12 to 15 days of age. Brain phosphorus flux from PC to ATP thus increased 4- to 6-fold in the first month of life. An increase at least that large occurred in the reverse direction, but the kr could not be measured consistently in the younger animals using the saturation transfer technique. Phosphorus fluxes were equal in the forward and reverse directions in the mature brain. The capacity to increase rates of glycolysis and tissue respiration in response to increased energy demand appears in the same narrow age period as the increase in CK-catalyzed reaction rates in the developing rodent brain. We propose that these coincident changes in brain energy metabolism reflect the maturation of mechanisms for coupling cell energy production to rapid changes in energy requirements.

Adenosine Triphosphate↗
PubMed1991

MR angiography without subtraction.

A new NMR method for producing angiograms with strong suppression of the static spin signal and without the need for subtraction is described and demonstrated. A velocity-selective pulse sequence was implemented whereby the magnetization of all stationary spins is driven to the -z axis and is not detected, while maximizing the signal intensity of the moving spins. A theory of the method is presented and gives good agreement with experimental results obtained on a flow phantom. It is shown theoretically and experimentally that high-quality velocity-independent angiograms of the head and neck can be obtained with strong suppression of static spin signal when TR approximately T1. The method can be extended to produce three-dimensional angiograms.

Angiography↗
PubMed1991

Multiecho, spin-echo sequence to eliminate unwanted echoes.

Two types of artifacts--a mirror-reversed image about the phase-encode direction and a wave-like intensity variation across the image--may appear when using a conventional multiecho spin-echo sequence. We describe and demonstrate a new method, which eliminates such artifacts, using only one excitation per phase-encoding step and without the need for spoiler gradients.

Electromagnetic Phenomena↗
PubMed1991

Spoiling of transverse magnetization in steady-state sequences.

A detailed analysis is presented of a method to eliminate transverse magnetization prior to each rf excitation in pulse sequences with TR less than T2. It is shown that artifact-free images with high T1 contrast can be obtained only if a phase shift that is incremented during each TR interval is applied to the transverse magnetization. Computer simulations are used to show that when this phase increment is 117 degrees, the steady-state transverse magnetization prior to each rf pulse is nulled over a wide range of T1, T2, and rf tip angles, resulting in optimal T1 contrast. Such nulling of steady-state transverse magnetization cannot be obtained by using large gradient pulses, or gradients of random or linearly incremented amplitude. Images of phantoms and human subjects confirm the theoretical predictions.

Artifacts↗
PubMed1991

Tumors growing in irradiated tissue: oxygenation, metabolic state, and pH.

Experimental tumors growing in irradiated tissue have been used to study the biological differences characteristic of locally recurrent tumors. Animal tumors were early generation isotransplants of a spontaneous fibrosarcoma in a C3Hf/Sed mouse, designated FSa-II. Since the hypoxic cell fraction of tumors growing in irradiated tissue is increased, these tumors are assumed to be metabolically deprived with hypoperfusion and acidosis. In this study we directly measured the oxygen partial pressure (pO2) distribution, metabolic state, and pH of tumors growing in an irradiated tumor bed using oxygen sensitive electrodes and 31P-NMR. The results confirmed a three-fold increase in the number of pO2 readings less than or equal to 2.5 mmHg and also showed increased acidosis with a 0.17 unit decrease in pHNMR. When tumors growing in pre-irradiated tissue reached approximately 100 mm3 in volume, a high frequency of gross and microscopic necrosis and hemorrhage was already observed. Consistent with these observations, the phosphocreatine/inorganic phosphate (PCr/Pi) and nucleoside triphosphate/inorganic phosphate (NTP/Pi) ratios were significantly lower in the tumors in a pre-irradiated bed compared to tumors in a non-irradiated bed (PCr/Pi: 0.51 vs 0.79, p less than 0.05; and NTP/Pi: 0.64 vs 0.93, p less than 0.05). The longitudinal relaxation time (T1) of Pi was numerically shorter in control tumors (consistent with the better tissue oxygenation), but this did not reach statistical significance (2.09 +/- .11 sec vs 2.25 +/- .16 sec).

Animals↗
PubMed1990

NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition.

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.

Amino Acid Sequence↗
PubMed1990

Lack of efficacy of water-suppressed proton nuclear magnetic resonance spectroscopy of plasma for the detection of malignant tumors.

Water-suppressed proton nuclear magnetic resonance (NMR) spectroscopy of plasma has been proposed by Fossel et al. (N Engl J Med 1986; 315:1369-76) as a technique for detecting malignant tumors. In their analysis, plasma samples from patients with cancer were clearly distinguished from those of normal subjects by measuring and averaging the methyl and methylene line widths of plasma lipoproteins in NMR spectrums. To evaluate this diagnostic procedure further, we collected and analyzed by NMR spectroscopy 145 samples of plasma from patients who served as controls, most of whom were undergoing orthopedic or cardiac surgery (n = 66); patients with a variety of untreated malignant tumors (n = 25) or treated malignant tumors (n = 18); and patients with hyperplastic or "premalignant" diseases, such as benign prostatic hyperplasia and ulcerative colitis (n = 36). All the samples were coded, and NMR spectroscopy was performed without knowledge of the patients' clinical status. There were no significant differences in the NMR line widths among the four study groups (P greater than 0.05 for all pairwise comparisons). The specificity and sensitivity of this method for distinguishing the control patients (mean line width [+/- SD], 44.0 +/- 7.4 Hz) from those with untreated cancer (43.8 +/- 6.9 Hz) were poor, with a false positive rate of 52 percent (34 of 66) and a false negative rate of 56 percent (14 of 25). Inverse correlations of line widths with age (P less than 0.01) and with the plasma triglyceride level (P less than 0.001) were detected. We conclude that NMR spectroscopy of plasma is not an accurate test for the detection of malignant tumors.

False Negative Reactions↗
PubMed1990

Motion-insensitive, steady-state free precession imaging.

Steady-state free precession (SSFP) pulse sequences employing gradient reversal echoes and short repetition time (TR) between successive rf excitation pulses offer high signal-to-noise ratio per unit time. However, SSFP sequences are very sensitive to motion. A new SSFP method is presented which avoids the image artifacts and loss of signal intensity due to motion. The pulse sequence is designed so that the time integral of each of the three gradients is zero over each TR time interval. The signal then consists of numerous echoes which are superimposed. These echoes are isolated by combining the data from N different scans. In each scan a specific phase shift is added during every TR interval. Each of these N isolated echoes produces a motion-insensitive, artifact-free image. Because all the echoes are sampled simultaneously, the signal-to-noise ratio per unit time in this SSFP method is higher than in existing SSFP techniques which sample only one echo at a time. The new method was implemented and used to produce both two- and three-dimensional images of the head and cervical spin of a human patient. In these images the high signal intensity of cerebrospinal fluid is preserved regardless of its motion. Further work is required to evaluate the imaging parameters (TR, TE, rf tip angle) so as to give optimal tissue contrast for the various echoes.

Humans↗
PubMed1990

Failure of 1H nuclear magnetic resonance spectroscopy of blood plasma to detect malignancy.

Water-suppressed proton nuclear magnetic resonance (NMR) of plasma was proposed as a technique for detecting malignant tumors. In that analysis, bloods drawn from cancer patients at the Beth Israel Hospital (BIH; Boston, MA), were easily distinguished from normal subjects by measuring and averaging the proton NMR methyl and methylene line widths of plasma lipoproteins. We collected blood at the Massachusetts General Hospital (MGH), including from normal controls, patients with untreated and treated malignant tumors, and patients with nontumor diseases. The plasma NMR analyses were carried out blind. The code was not broken until all patient charts and pathology records were reviewed, plasma analyses were completed, and patients had been divided into appropriate clinical groups. Analysis of these data showed no differences between the means of the study groups (false-positive and false-negative frequencies 46% and 57%, respectively). An inverse correlation of methyl/methylene line widths with age (P less than .01), and a correlation with nitrate-requiring cardiovascular disease (P less than .05) was, however, evident. This test cannot be validly used to detect malignancy.

Adult↗
PubMed1989

Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics.

The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25----Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.

Circular Dichroism↗
PubMed1989

Brain creatine phosphate and creatine kinase in mice fed an analogue of creatine.

Brain phosphocreatine (PCr) concentration and creatine kinase (CK) activity have been studied by 31P nuclear magnetic resonance (NMR) spectroscopy in mice fed an analogue of creatine, beta-guanidinopropionic acid (GPA). The phosphorylated analogue (GPAP), which almost completely replaces PCr in skeletal muscle, is a poor substrate for CK. Mice, which received GPA in food (2%) and water (0.5%) for up to 9 months beginning at 35 days of age, were normal in appearance and activity. Maximal brain GPAP concentration, reached after two weeks of feedings, was approximately equal to the concentration of PCr. The concentration of PCr decreased at least 20% relative to that of the nucleoside triphosphates. When GPA feedings were stopped, GPAP disappeared in about 20 days from skeletal muscle, but only after 40-50 days from brain. Steady-state NMR saturation transfer studies showed a markedly reduced chemical exchange rate from PCr to ATP in brains of GPA-fed mice. These results suggest a compartmentation of brain PCr. The GPA-accessible PCr compartment has a slow rate of PCr turnover compared to skeletal muscle. The slow reaction rate of the GPA-inaccessible PCr as a CK substrate is consistent with the hypothesis that this residual PCr is the same compartment which is stable in hypoxic or seizing animals.

Adenosine Triphosphate↗