Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques.

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.

Orientation of water in striated frog muscle.

Proton and deuterium nuclear magnetic resonance spectra of striated frog gastrocnemius muscle exhibit angular dependence, indicating partial orientation of water in the muscle. Nonzero static dipolar and quadrupolar interactions resulting from the anisotropic motion of the water molecules modulate the spin echo decays, contributing to their nonxponential behavior.

Lung water quantitation by nuclear magnetic resonance imaging.

Nuclear magnetic resonance imaging was used to determine quantitatively the water distribution of saline-filled and normal rat lungs in both isolated lung and in situ preparations. Regional lung edema was easily detected. Studies of an isolated lung fragment indicate an accuracy of better than 1 percent and images of H2O/D2O phantoms indicate an average error of 2.7 percent.

High-resolution proton magnetic resonance spectra of a rabbit sciatic nerve.

Proton magnetic resonance spectra (220-megahertz field) of an isolated rabbit sciatic nerve in its native state have been observed and assigned to the extracellular water, intracellular water, and phospholipids of the nerve. This study indicates that the nerve fibers contain fluid-like hydrophobic regions, in agreement with the results of recent electron spin resonance spin-labeled studies of excitable membranes of nerve and muscle.

Proton relaxation rates of water in brain and brain tumors.

The distribution of relaxation rates of water in "normal" (autopsy) samples of canine and human brain shows considerable overlap with that for brain tumor samples. The following ranges of values for the spin-spin relaxation rates were observed: for normal brain gray matter, 8.6 to 11.3 sec(-1) (mean, 9.5 sec(-1)); for normal brain white matter, 13.3 to 15.7 sec(-1) (mean, 15.5 sec(-1)); for six types of malignant tumor, 4.8 to 13.4 sec(-1) (mean, 9.3 sec(-1)); for five types of benign tumor, 7.1 to 16.4 sec(-1) (mean, 11.5 sec(-1)). Spin-lattice relaxation rates showed a similar pattern. At least two nonexchanging water components with different relaxation rates were indicated by the analysis of the spin-spin relaxation measurements for the white matter samples.

Lung fluid dynamics in awake newborn lambs.

We measured steady-state lung lymph flow, lymph protein flow, and simultaneous pulmonary vascular pressures in 12 1-wk-old unanesthetized lambs and compared these measurements to those of previous studies, performed under similar conditions, on nine awake adult sheep. The purpose of these experiments was to compare newborn and adult sheep with respect to transvascular filtration of fluid and microvascular permeability to plasma proteins. We prepared the lambs surgically to isolate and collect lung lymph and measure average pulmonary arterial and left atrial pressures, allowing at least 2 days for the lambs to recover from surgery before studies began. Lambs had higher pulmonary arterial and left atrial pressures, lower lymph and plasma protein concentrations, and 57% more lymph flow per gram of dry bloodless lung than sheep; the difference in protein flow between lambs and sheep was not significant. Protein concentration in lymph relative to that in plasma was significantly lower in lambs than in sheep; but the ratio of albumin concentration to globulin concentration in both lymph and plasma was almost identical in the two groups of animals. Extravascular lung water per gram of dry bloodless lung was greater in lambs (4.82+/-0.11 g) than in sheep (4.45+/-0.08 g), but there was no histologic evidence of pulmonary edema in either group of animals. These findings suggest that lambs have more transvascular filtration of fluid per unit lung mass than sheep, but that microvascular sites for protein exchange do not differ appreciably in lambs and sheep. To test this conclusion, we measured steady-state lymph flow in three lambs before and after raising pulmonary microvascular pressure by rapid intravenous infusion of saline. Lymph flow increased as a function of the net transvascular driving pressure (hydraulic pressure gradient-protein osmotic pressure gradient). This response was almost identical to that of four sheep with pulmonary microvascular pressure augmented by inflation of a balloon in the left atrium. In eight lambs we measured the time for intravenously injected (125)I-albumin to equilibrate in lymph at half the specific activity of plasma: the protein tag equilibrated faster than in sheep. This difference could be explained partly by the higher pulmonary arterial and left atrial pressures of lambs than sheep, and possibly by the presence of more microvascular sites for protein exchange relative to the volume of distribution of protein in the lung of the younger animals.

Accumulation of hyaluronan (hyaluronic acid) in myocardial interstitial tissue parallels development of transplantation edema in heart allografts in rats.

By using biotin-labeled proteoglycan core protein, hyaluronan (hyaluronic acid; HA) was visualized in rat heart grafts at different times (2, 4, and 6 d) after transplantation. In normal, nontransplanted hearts HA was present in the adventitia of arteries and veins and in the myocardial interstitial tissue. An increased accumulation of HA was evident in the edematous interstitial tissue, infiltrated with lymphocytes, on day 4 after allogeneic transplantation, and was even more pronounced by day 6. No apparent increase in HA was seen in syngeneic grafts. Biochemical assay of HA in heart tissue demonstrated that the myocardial content of HA had increased 60% by day 2 after transplantation in allogeneic as well as syngeneic grafts, indicating that surgical trauma may induce some HA accumulation in heart grafts. The extractable amount of HA declined during the following days in the syngeneic grafts, but increased progressively during the development of rejection in the allogeneic grafts, and increased on average three times by day 6. The relative water content also increased progressively during rejection of allogeneic grafts and correlated with the HA accumulation. The interstitial accumulation of HA, a glycosaminoglycan with unique water-binding qualities, is presumably implicated in the development of interstitial edema during rejection of heart grafts.

Interrelation between alterations in pulmonary mechanics and hemodynamics in acute myocardial infarction.

Pulmonary mechanics were evaluated in 30 patients with acute myocardial infarction by measuring forced expiratory flow rates and total pulmonary resistance (R(T)) with the oscillometric method at the resonant frequency of the chest (6-8) cycle/s). During the first 3 days after infarction, forced expiratory volume (FEV) and forced mid-expiratory flow rate (FEF(25-75%)) were 69% and 60% of predicted values, respectively. 10 or more wk later these values were 95% and 91%. Initially, R(T) was 52% greater than predicted, but was only 4% greater 10 or more wk later. In 11 patients R(T) was measured at both resonant frequency and at 3 cycle/s. Five of these patients had no clinical signs of heart failure, but nine had abnormally high values of pulmonary artery pressure, "wedge" pressure and pulmonary extravascular water volume. All of these patients recovered. Initially, R(T) at resonance was 50% and R(T) at 3 cycle/s was 130% greater than predicted values. 2-3 wk later these figures were -3% and +6% of those predicted, respectively. At 10 wk or more, significant frequency dependence of R(T) had disappeared (R(T) at 3 cycle/s was 7% greater than R(T) at resonance). Isoproterenol inhalation in six patients caused no change in flow rates, R(T) at resonance, or R(T) at 3 cycle/s. R(T) at resonance and at 3 cycle/s, FEV, and FEF(25-75%) correlated significantly with the pulmonary vascular pressures. Patients with more marked arterial hypoxia and larger values for extravascular water volume had greater elevations of R(T) and depression of FEF(25-75%), but linear correlations were not significant. Clinical signs of congestive heart failure significantly correlated with a fall in FEV and FEF(25-75%), the development of frequency dependence of R(T), and elevation of the pulmonary wedge pressure. The initial elevation of R(T) and low flow rates indicate a modest degree of airway obstruction in acute myocardial infarction. Lack of response to isoproterenol suggests that bronchial muscular constriction is not a major factor. Frequency dependence of R(T) accompanied by elevated pulmonary vascular pressures and extravascular water volume indicates that pulmonary congestion causes the development of uneven time constants in the airways. Vascular engorgement and interstitial edema from elevated vascular pressures causing narrowing of the peripheral airways and closure of collateral airways could account for the above findings.

The relationship between right duct lymph flow and extravascular lung water in dogs given alpha-naphthylthiourea.

The relationship between right duct lymph flow and extravascular lung water was studied in 3 normal dogs and 15 dogs with pulmonary edema induced by alpha-naphthylthiourea (ANTU). Right duct lymph was collected in a pouch created by ligating jugular, subclavian, and brachiocephalic veins. Extravascular lung water was measured in vivo by double indicator dilution and post-mortem by weighting lungs before and after drying. Cardiac output, pulmonary artery and pulmonary artery wedge pressures, and the concentration of protein and electrolytes in plasma and right duct lymph were determined. Eight lungs were examined by light and electron microscopy. There was a direct relationship between right duct lymph flow (RDLF in milliters per hour per gram dry lung) and extravascular lung water (Qwl in milliliters per gram dry lung) which was best described by the equation RDLF=0.75-0.26 Qwl+0.03 (Qwl).2 Dogs with severe ANTU-induced edema had extensive lung capillary endothelial destruction but only mild interstitial swelling and no visible damage to type I alveolar epithelial cells. Cardiac output, pulmonary artery and wedge pressures, and protein and electrolyte concentrations did not correlate with either extravascular water or right duct flow. Thus, in ANTU-induced pulmonary edema right duct lymph flow was directly related to extravascular lung water with the highest flows occurring with severe edema. The absence of a rapid increase in lymph flow with small increases in extravascular water may be due to early sequestration of fluid in the alveolar space. Hemodynamic changes did not account for changes in lung water or lymph flow. The pulmonary interstitial factors relating increased extravascular water to lymph drainage remain to be determined.

Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep.

We did experiments to determine whether beta-adrenergic agonists increase lung liquid clearance in anesthetized ventilated adult sheep and, if so, whether the increase is mediated by beta receptors and what mechanism is involved. We instilled 100 ml of autologous serum either alone or with a beta-adrenergic agonist (terbutaline, 10(-5) M, or epinephrine, 5.5 X 10(-6) M) into one lower lobe. After 4 h both terbutaline and epinephrine increased lung liquid clearance. The increase in lung liquid clearance was inhibited when propranolol (a beta blocker) or amiloride (a sodium channel blocker) was added to the terbutaline. Increased clearance was not explained by changes in pulmonary hemodynamics, pulmonary blood flow, or lung lymph flow. We conclude that beta-adrenergic agonists increase lung liquid clearance in anesthetized intact adult sheep. This increase is mediated through beta receptors and probably depends on increased active transport of sodium across the alveolar barrier.

An experimental model of cachexia induced by a xenografted human tumor.

A hypernephroma removed from a male patient who had lost 30 kg in weight in the 2 months preceding surgery was established in immunosuppressed CBA/Lac mice as a nonmetastasizing transplantable xenograft. The xenografted tumors, although comprising less than 5% of the total body weight of the mice, produced considerable weight loss (greater than 25%). A slight reduction in food intake of tumor-bearing mice was noted, but some animals bearing mouse or human tumors not inducing cachexia had equally low food intake without accompanying weight losses. No obvious defects in gastrointestinal histology or absorption were observed. The precise mechanism(s) producing the severe cachexia remains to be established.

Transport of molecules in the tumor interstitium: a review.

The transport of fluid and solute molecules in the interstitium is governed by the biological and physicochemical properties of the interstitial compartment as well as the physicochemical properties of the test molecule. The composition of the interstitial compartment of neoplastic tissues is significantly different from that of most normal tissues. In general the tumor interstitial compartment is characterized by large interstitial space, high collagen concentration, low proteoglycan and hyaluronate concentrations, high interstitial fluid pressure and flow, absence of anatomically well-defined functioning lymphatic network, high effective interstitial diffusion coefficient of macromolecules, as well as large hydraulic conductivity and interstitial convection compared to most normal tissues. While these factors favor movement of macromolecules in the tumor interstitium, high interstitial pressure and low microvascular pressure may retard extravasation of molecules and cells, especially in large tumors. These differences in transport parameters have major implications in tumor growth and metastases, as well as in tumor detection and treatment.

Nutritional support of bone marrow transplant recipients: a prospective, randomized clinical trial comparing total parenteral nutrition to an enteral feeding program.

Although standard supportive care for bone marrow transplant (BMT) recipients includes total parenteral nutrition (TPN), it has not been shown that this is the most appropriate method of nutritional support. To determine whether current BMT recipients require TPN during the early recovery period, we conducted a prospective, randomized clinical trial comparing TPN and an individualized enteral feeding program (counseling, high protein snacks and/or tube feeding). Nutritional assessment included measurement of serum proteins, anthropometry, and body composition analysis. For the latter, total body water and extracellular fluid were measured by standard radioisotope dilution techniques and used to quantitate body cell mass and body fat plus extracellular solids (FAT + ECS). In 27 TPN patients, body composition 28 days after BMT, expressed as a percentage of baseline, was body cell mass, 100%, extracellular fluid, 108%, FAT + ECS, 108%, and in 30 enteral feeding program patients, was body cell mass, 93%, extracellular fluid, 104%, and FAT + ECS, 94%. Only the difference in FAT + ECS was statistically significant (p less than 0.01). Compared to the enteral feeding program, TPN was associated with more days of diuretic use, more frequent hyperglycemia, and more frequent catheter removal (prompted by catheter-related complications), but less frequent hypomagnesemia. There were no significant differences in the rate of hematopoietic recovery, length of hospitalization, or survival, but nutrition-related costs were 2.3 times greater in the TPN group. We conclude that TPN is not clearly superior to individualized enteral feeding and recommend that TPN be reserved for BMT patients who demonstrate intolerance to enteral feeding.

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.

Laser Raman investigation of intact single muscle fibers. On the state of water in muscle tissue.

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nubilus). The spectra in the region of the O-H (or O-2H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of "structured" intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 +/- 0.01 for H2O and 1.05 +/- 0.03 for 2H2O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state of -5 degrees C, which corresponds to 1 g of water per g of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.

Correlation of relaxation time with water content in muscle and brain tissues.

Proton T1 of water in muscle and brain tissues was studied as a function of water content at 25 degrees C and 5, 30, and 100 MHz. Muscle samples were bathed in modified Krebs solutions that have the same ionic strength but different osmolarity in order to change the water content. At all three frequencies their 1/T1 extrapolated to the same value of 0.6 S-1 for zero dry weight, indicating that T1 for the majority of water molecules in normal muscle and brain is frequency-independent. Dehydrated muscle, homogenized brain tissue suspended in modified Krebs solution and dehydrated brain tissue provide samples with wide ranges of water content. Their T1 values at all three frequencies can be well accounted for by a simple equation derived from the two-state model.

A proton magnetic resonance study of water in human stratum corneum.

Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum mounted at a specific orientation to the applied magnetic fild, three distinct absorptions may be seen having different chemical shifts and spin-lattice relaxation times (T1). All T1 values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.

NMR measurement of intracellular water volume in rat kidney proximal tubules.

Knowledge of cell water volume is essential for the measurement of concentrations of intracellular ions and metabolites in kidney proximal tubules. We have developed a method which utilizes 35Cl-NMR as a measure of extracellular volume and 2H-NMR, in combination with a membrane-impermeable shift-reagent [Dy-DTPA]2-, as a measure of the ratio of intra- and extracellular water volumes. Measurement of extracellular volume by 35Cl-NMR is possible, since the resonance of intracellular 35Cl is too broad to be detectable in kidney cells. The 2H-NMR measurement exploits the fact that only extracellular water is in direct contact with [Dy-DTPA]2-. However, rapid exchange of water across the cell membrane results in only a single 2H2O resonance at a chemical shift which is a weighted average of the shifted extra- and unshifted intracellular water resonances. Expression of the extracellular volume as a fraction of the total volume by fCl and as a fraction of the total water-volume by fD, permits the calculation of the fractional cell-water content fw = [(1/fD)-1]/[(1/fCl)-1]. This approach was applied to proximal tubular suspensions prepared from the rat kidney. The water content was found to be 76.9 +/- 1.8% (n = 6) at 37 degrees C. Increasing extracellular osmolality from 295 to 390 mOsm/kg H2O, by addition of mannitol, decreased the water content by 21%. Our results are in good agreement with those obtained by the gravimetric method.

The state of water in biological systems as studied by proton and deuterium relaxation.

Careful experiments on the measurement of the intensity of the deuterium NMR signal for 2-H2 O in muscle and in its distillate were performed, and they showed that all 2-H2 O muscle is "NMR visible". The spin-lattice relaxation time (T1) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to --70 degrees C. T1 values of deuterons in 2H2 O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to --20 degrees C. Calculations on T1 for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T1 values compared to pure water and the frequency dependence of T1 are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.

The density and free water of cholinergic synaptic vesicles as a function of osmotic pressure.

Synaptic vesicles from the cholinergic electromotor nerve terminals of Torpedo marmorata are among the most uniform subcellular organelles known and are osmotically sensitive. Changes in density accompanying osmotic perturbation have enabled changes in water content to be calculated; when referred to a standard state of known volume and water content, fractional and absolute water contents could be calculated for the perturbed states and compared with the fractional free water content as measured by the glycerol space. Under hyperosmotic conditions, discrepancies were found between these two estimates, the glycerol space falling more rapidly than the water space predicted from the density change. This is attributed to a failure of glycerol to displace water imbibed by the membrane as it collapses round an aqueous core of decreasing volume. 'Reserve' vesicles obeyed a relationship between density, osmotic load and osmolality derived for a perfect osmometer, and independent estimates of fractional free water content under standard conditions and osmotic load were made. The former of these agreed well with the glycerol space under standard conditions and the latter agreed with previous estimates of the osmotic load using morphological and analytical data and an assumed activity coefficient of 0.65. Finally, it was possible to model the interconversion of reserve and recycling vesicles more accurately than in previous work.