Nuclear magnetic resonance patterns of intracellular water as a function of HeLa cell cycle.

Nuclear magnetic resonance relaxation time (T1) of the intracellular water protons and water content were measured in synchronized HeLa cells. The T1 was maximum (1020 milliseconds) in mitotic and minimum (534 milliseconds) in S phase cells. The cyclic pattern of T1 values correlated well with the chromosome condensation cycle. By treating cells with spermine, it was possible to alter T1 without a significant change in the water content. The results of this study suggest that an additional variable, namely, the conformational state of macromolecules, should be incluced in any expression explaining the shortened relaxation times of water protons in biological systems.

Distinction of normal, preneoplastic, and neoplastic mouse mammary primary cell cultures by water nuclear magnetic resonance relaxation times.

Normal, preneoplastic, and neoplastic primary cultures of mouse mammary epithelial cells were distinguishable on the basis of water proton nuclear magnetic resonance (NMR) relaxation times--i.e., spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). T1 values were 916 +/- 24 msec for normal cells, 1,029 +/- 24 msec for preneoplastic cells, and 1,155 +/- 42 msec for neoplastic cells. This method of distinction between normal and neoplastic cells (P less than 0.001) and normal and preneoplastic cells (P less than 0.005) supported previous findings in whole tissues. NMR relaxation times resulted in better distinction between these cell populations than any other technique except direct histology. The T1 and T2 values of water protons in cells grown in primary culture were higher than those of established mouse mammary cancer cell lines. The differences in T1 and T2 did not correlate with cellular hydration. The data suggested a basic difference in water-macromolecular surface interactions among normal, preneoplastic, and neoplastic cells.

Nuclear magnetic resonance studies on human breast dysplasias and neoplasms.

The ability of nuclear magnetic resonance (NMR) spectroscopy to distinguish normal, diseased non-neoplastic, and neoplastic human breast tissues was investigated with T1 and T2 relaxation times used. The results indicated that NMR relaxation times could distinguish between the mean values of breast neoplasms and other diseased or normal tissues, with P values less than 0.001. Given a single sample, the probability of classifying it nonneoplastic or carcinoma could be accomplished with 85% confidence. For human breast tissues, the relaxation time T2 may be more discriminating that T1. These results support the view that the use of NMR spectroscopy in cancer detection may be of significant value and warrants considerable more interest and effort to determine the beneficial applications and limits of this technology.

Systemic effect of benign and malignant mammary tumors on the spin-lattice relaxation time of water protons in mouse serum.

The spin-lattice relaxation time (T1) for water protons in sera was significantly (P less than 0.001) elevated above that for normal sera in mice bearing benign ductal papilloma and malignant mammary carcinoma. Serum T1 values did not differ significantly in mice with ductal hyperplasia or preneoplastic alveolar nodules. Elevated serum T1's could not be explained on the basis of serum iron levels or serum protein concentrations. This was the first report of a "systemic effect" of serum T1 elevation by a benign tumor.

Microtubule complexes correlated with growth rate and water proton relaxation times in human breast cancer cells.

Ten established human breast cancer cell lines display patterns of microtubule organization which are characterized by growth rate of the cell populations and the freedom of mobility of cellular water molecules measured by nuclear magnetic resonance spectroscopy. Cell lines with population-doubling times of 1 to 2 days demonstrate rapid mobility of water molecules by proton spin-lattice and spin-spin relaxation times (T1 greater than 750 msec, T2 greater than 120 msec) and have diffuse patterns of tubulin immunofluorescent antibody staining. Moderately fast dividing cells (population-doubling times of 3 to 7 days) have T1 values of 600 to 750 msec and show approximately 50% organized complexes of polymerized microtubules in the cytoplasm. Slow-growing cell lines demonstrate more restricted mobility of water molecules (T1 values of 500 to 600 msec) and contain abundant networks of polymerized microtubules. The three-way correlation of the physical parameter of water proton relaxation times, the structural parameter of microtubule organization, and the physiological parameter of growth suggest a close interaction of water molecules with the cytoplasmic macromolecular network in the performance of physiological function.

Nuclear magnetic resonance study of squid giant axon.

Using a spin-echo technique, the spin-lattice and spin-spin relaxation times (T1 and T2) of water protons in a single nerve fiber (giant axon of squid) were determined. Similar measurements were also carried out on axoplasm extruded from these nerve fibers. It was found that the relaxation times of water protons of both the intact fiber and the extruded axoplasm are approximately equal (and much less than those of a free solution), suggesting that the relaxation times of cellular water are shortened mainly by water-protein interactions rather than by water-membrane interactions.

Effect of electric field on physical states of cell-associated water in germinating morning glory seeds observed by 1H-NMR.

Morning glory seeds in dry conditions (0.099 g H2O/dry wt.) were exposed to electric fields and germinated. The physical state of water in the germinating seeds of both control and exposed groups were examined using 1H-NMR spectroscopy and NMR microscopy. Three water fractions were observed which were characterized by different relaxation times (T1) and chemical shifts. The average region containing long T1 fractions was approximately 50 micrometer in diameter and consisted of half-permeable barriers. The maximum intracellular water transport rate was 2.3x10-5 cm2/s. The treatment with electric field (500 kV/m for 60 min) increased the fraction with the shortest T1 and decreased that with the longest T1. Because the total water content in the treated seeds (3.4 g H2O/dry wt.) was similar to that in the untreated seeds (3.9 g H2O/dry wt.), the treated seeds held more water in a condition in restricted motion than the untreated seeds. It is thought that the membrane systems were affected by the electric polarization which led to an unusual accumulation of water and the hydration of stored macromolecules during the imbibition process. This set of events led to excessive swelling of stored macromolecules, resulting in the disruption of membrane systems and irregular organization of tissue structures.

The spin-lattice relaxation times of water associated with early post mortem changes in skeletal muscle.

We studied the spin-echo signal of muscle water in a large time domain and found that the motion of the nuclear magnetic moment of tissue water cannot be characterized by a single spin-lattice relaxation time (T1). The relaxation time T1B, which is the T1 characterized by those protons with a slower relaxation rate, is influenced by the early post mortem changes in skeletal muscle. T1B increased with time after the tissue was taken from the animal and reached a maximum at 3 h. However, the weighted average of T1 of all water protons (T1A) did not change throughout the time course of the experiments.

Effect of insulin on potassium flux and water and electrolyte content of muscles from normal and from hypophysectomized rats.

It was reported previously that insulin hyperpolarized rat skeletal muscle and decreased K(+) flux in both directions. The observations on K(+) flux are now extended to take advantage of the greater sensitivity to insulin of hyperphysectomized rats. Insulin caused a shift of water from extracellular to intracellular space if glucose was present, but not in its absence. Insulin caused net gain of muscle fiber K(+), though not necessarily an increase in K(+) concentration in fiber water. It probably also decreased intrafiber Na(+) and Cl(-). Insulin decreased K(+) efflux. The effect was dose-dependent. Muscles from hypophysectomized rats were more sensitive to the action of insulin on K(+) flux than were those from normal rats. The effect was demonstrable within the time resolution of the system, suggesting that insulin's action is on cell surfaces. K(+) influx was also decreased by insulin. Bookkeeping suggests that some K(+) influx be called active. Insulin seemed to decrease active K(+) influx and passive K(+) efflux. It is not resolved whether insulin has a true dual effect or whether it acts only on passive fluxes in both directions (the apparent action on active K(+) influx being an artefact of incomplete definition of passive flux) or whether a single alteration in the membrane may affect both active and passive fluxes.

Magnesium supplementation in protein-calorie malnutrition.

The widespread observation of magnesium depletion in edematous malnutrition has been confirmed in Guatemalan children. The magnesium requirement during initial stages of therapy has been estimated as 2.7 mEq/kg per day. This may be achieved by adding 0.5% MgSO4.7H2O to a solution containing 15% dextromaltase and 1.5% KCl which is used to dilute whole milk; two parts milk and one part dilution mixture. The replacement of magnesium deficits was not essential for recovery from edematous malnutrition, however, the present evidence suggested that the rate of recovery was accelerated by approximately 2 weeks in those children who received the supplement.

Sequential changes in body composition during infection: electron probe study IV.

Alterations occur in human muscle electrolyte and water composition in response to infection. There appear to be at least two basic mechanisms; the first is an exchange of sodium for potassium without alteration in water content of muscle. The second is an increase in cellular Na and water without a loss of K on a dry weight basis. In a series of studies in monkeys, Salmonella typhimurium sepsis was induced as an experimental model. Both patterns of muscle response to infection were detected. Electron probe microanalysis revealed that the loss of K concentration was due to an accumulation of intracellular saline which dilute the K content. The mechanism of this is unclear; however, a concomitant increase in undertermined osmoles in the serum suggests that there may be an increase in organic osmoles within the cell which leads to the dilution of intracellular K concentration.

Electrolyte metabolism in rhesus monkeys with experimental salmonella sepsis.

Prior investigations in the human indicate that alterations occur in electrolyte balance and serum concentration during infectious diseases. In order to explore these alterations in greater detail, electrolyte metabolism has been investigated in rhesus monkeys with a sublethal illness induced by intravenous inoculation with Salmonella typhimurium. The response to this illness was evaluated by a variety of measurements including serum and muscle electrolyte composition and renal function studies. In the animals with ad libitum dietary intake, a loss in muscle and serum potassium concentrations was evident within 24 h after inoculation. This was reflected in increased urinary potassium losses during the febrile phase of illness. Serum and muscle K concentrations returned to normal after 5 days of illness. Sodium and water content of muscle responded to infection in a more complex pattern. During the febrile phase, muscle sodium and water increased and sodium concentrations in serum and urine were elevated. During convalescence, renal retention of sodium was marked and overlapped the period of weight loss and the increased urine volume. This asynchrony in recovery of normal renal function appeared to be the cause of relatively large swings in plasma sodium concentrations during the early convalescent period. These investigations indicate that the altered serum concentrations in infectious diseases are the sum of renal and extrarenal factors controlling electrolyte metabolism, and that some of the most remarkable alterations occur during early convalescence as renal function returns to normal.

In vivo NMR imaging and T1 measurements of water protons in the human brain.

Nuclear magnetic resonance (NMR) proton density images of the human brain have been made by the FONAR method. Spin-lattice relaxation times, T1, of water hydrogen protons have been determined at random positions within frontal and temporal regions of the human brain. The primary purpose of this ongoing research is to accumulate a large data base of normal T1 values for water protons in normal human brain tissue. Our experience to date includes 31 measurements on 18 volunteer subjects, and the mean value +/- standard deviation is 215 +/- 42 msec. In addition, two metastatic lesions of the brain were studied and found to have T1 values longer than those for normal brain tissue.

Effect of nuclear magnetic resonance on chromosomes of mouse bone marrow cells.

Three groups of six male Balb/c mice, subjected to 30 MHz continuous wave NMR exposure in a static magnetic field of 7.05 K Gauss for one hour, were each compared to another group of ten unexposed mice with respect to chromatid and chromosomal aberrations. The exposed groups were sacrificed at two hours, 24 hours and 48 hours following NMR exposure respectively. Control mice were sacrificed 24 hours after sham-exposure. All groups had approximately 0.02 apparent aberrations per cell. These apparent aberrations were in the form of metacentric chromosomes, possibly resulting from a union of chromosomes at their centromeres or possibly simply chromosomes in association. The results are consistent with earlier in vitro findings that NMR exposure causes no adverse cytogenetic effects.

Evaluation of muscle degeneration in inherited muscular dystrophy by nuclear magnetic resonance techniques.

Nuclear magnetic resonance (NMR) techniques were applied to study the muscular dystrophy in chicks. The water proton spin-lattice relaxation times (T1) of fast, slow, and mixed muscles and plasma were measured. The T1 values of dystrophic pectoralis major and posterior latissimus dorsi (PLD) were significantly higher than those of the normal pectoralis and PLD muscles. The present results establish a direct relationship between the differences in T1 values and the severity of muscle degeneration. Consistent with this conclusion, it was also found that the T1 values of muscles unaffected in muscular dystrophy, namely, the gastrocnemius, and anterior latissimus dorsi (ALD), were not different between the normal and dystrophic chicks. Although the affected muscles of dystrophic chicks contained higher percent water and fat than those of normal chicks, the results show that the higher T1 values in dystrophic muscles were not solely due to variations in their water content. The increase in the T1 values is principally a result of altered interaction between cellular water and macromolecules in the diseased muscles. These data also point out the potential use of NMR imaging in evaluating muscle degeneration.

Acute ethanol decreases NMR relaxation times of water hydrogen protons in fish brain.

The traditional belief about ethanol's mechanism of action is based on ethanol's lipophilicity and capability to penetrate and disorder lipid bilayers. This traditional belief is now being supplanted by growing evidence that ethanol has relatively selective actions on certain synaptic receptors, such as those for NMDA, serotonin, and GABA. It was recently argued that these receptor specificities are secondary to a preferential ability of ethanol to displace membrane bound water in the domains of certain receptors. The data obtained in this study are consistent with the original hypothesis: any disorganization of cellular water by ethanol will be detectable by proton nuclear magnetic resonance (NMR) spectroscopy. In particular, the relaxation times of water hydrogen protons reflect how constrained water molecules are by the macromolecules within cells. The relaxation time of "bulk" water is lengthened relative to water molecules that are under the influence of electromagnetic fields of macromolecular surfaces within cells. Here, we tested this hypothesis in living fish, which dosed themselves by swimming in water that had added ethanol. Estimates of brain alcohol at 5 min after initial exposure revealed that the brain concentration was only about 1/3 that of the water in which they were swimming. The average value of the NMR relaxation time T1, but not T2, was decreased at 5 min (when brain concentrations were on the order 100 mM) and reached statistical significance at 10 and 30 min after initial exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

A hypothesis defining an objective end point for the relief of chronic pain.

A three-part hypothesis for an objective end point for pain is presented: 1) chronic pain results in a characteristic, but reproducible, pattern for the distribution of T lymphocytes in the various phases of their cell cycle; 2) Significant reduction or complete loss of chronic pain will cause a reproducible change in the distribution of T lymphocytes in their cell cycle; 3) The change in T lymphocytes cell cycle distribution will be a function of the degree of recovery from the pain experience. A preliminary test of the hypothesis is presented. The cell cycle distribution of T cell lymphocytes was determined in a group of 10 subjects (experiencing chronic pain) before and after participating in a 10-day educotherapy program given by a master teacher. Associated with a significant reduction of pain was a highly significant shift of the T cell lymphocytes into the S phase of the cell cycle. This observation is consistent with parts one and two of the hypothesis.

Evidence for structuring of water in growing oocytes: an X-ray microanalysis and nuclear magnetic resonance study.

Energy dispersive X-ray microanalysis measured the Na, K, Cl, P, Mg, S and Ca contents (mM/kg dry weight) in the nucleus and yolk-free cytoplasm of growing Xenopus laevis oocytes quick frozen in the ovary. Nuclear magnetic resonance measurements of T2, the transverse relaxation time of water protons, were obtained on small immature oocytes and on large, fully grown oocytes. Changes in the nucleus and cytoplasmic content were observed for all elements except for Ca. Nuclear/cytoplasmic macroscopic gradients of K, Cl, Na, and Mg increase with growth. The T2 times of large oocytes were found to be shorter than those of small oocytes. The data in this report further support the hypothesis that intraoocytic water and elements do not exist in an ideal dilute solution and that changes in the states of water and elements occur during oocytic growth.