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PubMed1987

Lactate dehydrogenase, isoenzyme patterns and cation levels in human breast gross cyst fluid.

In order to investigate the diagnostic and/or prognostic value of total activity and isoenzymatic patterns of lactate dehydrogenase, 24 human breast gross cystic fluids were studied. A comparison of total lactate dehydrogenase activity to the serum level revealed an increased activity in about 63% of the cases examined; moreover, a significant increase in the slow-moving lactate dehydrogenase 4 and 5 isoenzymes was observed in some cyst fluids. The levels of Na+ and K+ concentrations were also analyzed and two classes of cysts were identified: one presenting Na+ and K+ levels similar to those found in extracellular compartment; the other with high K+ and low Na+ levels, characteristic of an intracellular fluid. This latter pattern could indicate an active metabolism of the epithelial cells lining the cysts. This breast cyst fluid also showed increased levels of lactate dehydrogenase 4 and 5 isoenzymes. The correlation between an increased activity of lactate dehydrogenase 4 and 5 isoenzymes and high K+ and low Na+ levels could be the expression of a high biosynthetic activity and of an anaerobic metabolism in some cysts, suggesting the evolution of the breast gross cyst lesion to malignancy. The importance of these observations is discussed.

Body Fluids↗
PubMed2004

In vivo muscle amino acid transport involves two distinct processes.

We have tested the hypothesis that transit through the interstitial fluid, rather than across cell membranes, is rate limiting for amino acid uptake from blood into muscle in human subjects. To quantify muscle transmembrane transport of naturally occurring amino acids, we developed a novel 4-pool model that distinguishes between the interstitial and intracellular fluid compartments. Transport kinetics of phenylalanine, leucine, lysine, and alanine were quantified using tracers labeled with stable isotopes. The results indicate that interstitial fluid is a functional compartment insofar as amino acid kinetics are concerned. In the case of leucine and alanine, transit between blood and interstitial fluid was potentially rate limiting for muscle amino acid uptake and release in the postabsorptive state. For example, in the case of leucine, the rate of transport between blood and interstitial fluid compared with the corresponding rate between interstitial fluid and muscle was 247 +/- 36 vs. 610 +/- 95 nmol.min(-1).100 ml leg(-1), respectively (P < 0.05). Our results are consistent with the process of diffusion governing transit from blood to interstitial fluid without selectivity, and of specific amino acid transport systems with varying degrees of efficiency governing transit from interstitial fluid to muscle. These results imply that changes in factors that affect the transit of amino acids from blood through interstitial fluid, such as muscle blood flow or edema, could play a major role in controlling the rate of muscle amino acid uptake.

Adult↗
PubMed1995

Acid-base regulation during hypothermia. a brief review.

Acid-base physiology has mainly focused on mechanisms that maintain normal, extracellular pH at a constant temperature. Usually it is the arterial blood which is considered. As reliable pH meters became generally available in the 1950s it was observed that an arterial pH of about 7.39-7.42 was maintained remarkably constant in normal man and in mammals in general. This moderate degree of blood alkalosis is maintained by chemical buffering, by appropriate adjustment of the lung ventilation and by the kidneys. To measure pH intracellularly was more difficult, but not impossible, and over some time it became apparent that intracellular fluid was close to neutrality. pH values around 6.8 was found. This is a favorable state for retention of metabolites inside the cells. From an analysis by Davis (1) of the ionization constants of several hundred watersoluble biosynthetic intermediates one may argue that the ideal intracellular pH would occur near the neutrality of water where most of these compounds are ionized and thus captured within the cells, with little tendency to escape across the cell membrane. Apparently, if cells are to defend their neutrality and also to eliminate their acid metabolites and CO2, there must be a considerable transmembrane H+ gradient: The hydrogen ion concentration at neutrality is 160 nmol/L (pH 6.8) and that of blood 40 nmol/l (pH 7.4).

Acid-Base Equilibrium↗
PubMed1980

Control of ion distribution in isolated smooth muscle cells. I. Potassium.

We describe a technique for examining unidirectional ion movements in suspensions of enzymatically disaggregated smooth muscle cells derived from stomach muscle of the toad. This technique has been used to analyze the movement of 42K across these cells. This analysis was greatly simplified by the finding that the cells were in a steady state with respect to K+ distribution after isolation. The potassium contents of the isolated cells were identical to those of intact smooth muscle (131 mM/liter intracellular fluid) and stable for over 4 h; moreover, the unidirectional influx and efflux rates were equal. An additional simplification was provided by the finding that virtually all the K+ exchanges in a manner predicted for a simple two-compartment system consisting of an extracellular and an intracellular space. Transmembrane K+ flux in these cells averaged 1.2 pmol.cm-2.s-1 at room temperature. A large portion (approximately 80%) of 42K influx appeared to be mediated by a saturable transport system with an apparent Km of 0.6 mM and an apparent Vmax of 1.3 pmol.cm-2.s-1. The calculated resting membrane permeability to K+ in these isolated smooth muscle cells, assuming a membrane potential of -50 mV, was 2.9 X 10(-8) cm/s. The calculated gK+ was 2.7 mumho/cm2 constituting only a small fraction of the total membrane conductance as measured electrophysiologically. The latter finding suggests that the resting membrane potential in the isolated cells must be determined by ions in addition to K+. We propose that these methods for studying ion movements in smooth muscle should aid in unraveling the mechanisms responsible for controlling the distribution of ions both at rest, as in the present study, as well as in response to neurotransmitters.

Animals↗
PubMed1986

Volume influences on thirst and vasopressin secretion in dehydrated sheep.

The contribution of extracellular fluid volume (ECFV) to water consumption and plasma vasopressin concentration (PAVP) after water deprivation for 52 h was examined in sheep. Intravenous infusion of isotonic NaCl, equivalent to either estimated ECFV loss or total body water loss, significantly reduced water intake by 37% when water was offered 3 h after infusion but not when water was offered 1 h after infusion. Plasma osmolality (POsm) was reduced after 3 h. Infusion of 200 mM NaCl, which maintained POsm, decreased water consumption by the same degree as isotonic NaCl infusion. Thus large decreases in POsm had no effect on water intake in this experimental protocol. Lack of inhibition of drinking 1 h after infusion suggests that the decrease observed after 3 h may have been mediated by receptors in the interstitial fluid (ISF) compartment and not the intravascular compartment. PAVP was reduced 3 h after infusion of NaCl but not at 1 or 2 h after infusion. POsm was also decreased at 3 h. Thus reduction of PAVP by NaCl infusion may have been caused by either ISF or intracellular fluid volume expansion.

Animals↗
PubMed1983

Mobilised fluid volumes after induced hyperosmolality in the rat.

Hyperosmolar infusions of the inert pentose sugar, xylose (MW 150), were used to induce hyperosmolar states in non-starved and starved rats. Using 51Cr EDTA and RIHSA the extracellular fluid (ECF) and plasma fluid volumes (PV) were determined before and after infusions. The cause of weight loss after 24-30 h starvation was also examined. Equal osmolar provocation in starved and non-starved animals caused the same degree of hyperosmolality. The greater the osmolality increase the larger the volume of intracellular fluid mobilised. Despite the total ECF volume increments being large relative to PV, this fluid compartment remained hardly effected by the fluid released from the cells. No evidence could be found to support 24-30 h starvation as causing a measurable fluid balance defect, a finding of considerable importance when considering short term problems arising out of starvation. The strict control of PV in normovolemic rats has again been confirmed.

Animals↗
PubMedDate not supplied

New concepts in the pulmonary disposition of antibiotics.

Despite interpretive problems associated with studies of antibiotic concentrations in tissues, it is of major interest to correlate the presence of the drug at significant concentrations in lung tissues and fluids at a particular site with its clinical efficacy. All the models described above have their uses as well as their limitations. Measurement of antibiotic concentrations achieved in lung parenchyma, ELF, bronchial mucosa of bronchial secretions has shown variable levels for the same drug. Many respiratory infections are caused by obligate or facultative intracellular pathogens, which may be eradicated as a result of intracellular penetration and accumulation of quinolones, of macrolides, as shown in several models of phagocytic cells, and as a result of intracellular antibacterial activities. For bacterial infections located in extracellular pulmonary sites, a knowledge of achievable concentrations of beta-lactams, of aminoglycosides, of quinolones should be of value, whereas for bacteria multiplying in alveolar macrophages the high concentrations of the new macrolides or new fluoroquinolones that can be achieved in extravascular and intracellular fluids should have clinical relevance. Animal models of severe pneumonia, as well as studies in patients hospitalized in intensive care units, have provided correlation between clinical outcomes and high lung tissue and intramacrophagic concentrations of fluoroquinolones, or of macrolides in legionellosis, or of ceftazidime or imipenem in pseudomonal pneumonia.

Anti-Bacterial Agents↗
PubMed1981

Body fluid composition in chronic renal failure.

Studies were performed to assess body fluid composition alterations occurring in 10 patients undergoing chronic hemodialysis. Red cell mass (RCM), plasma volume (PV), extracellular fluid volume (ECF), and total body water (TBW) were assessed 24 hours following hemodialysis to dry weight, following infusion of normal saline to increase body weight by approximately 2 kg, and following hemodialysis to decrease body weight by approximately 2 kg. Results demonstrate that TBW constituted from 48.5 to 51.0% of body weight. The ECF constituted from 41.8 to 46.3% of the total body water. The ratio of PV to interstitial fluid (ISF) volume (ISF = ECF - PV) approximated 1.1 to 1.2:3. Salt and water alterations (isotonic saline infusion and ultrafiltration) were restricted to the ECF component of the TBW. In the ECF, it was principally the ISF that buffered salt and water expansion or depletion. There was no evidence for volume alterations in the intracellular fluid (ICF = TBW - ECF). We conclude that the ISF is the buffer zone which maintains the proper balance and relationship between vascular capacity and volume.

Adult↗
PubMed1983

Body fluid distribution in the maintenance of DOCA-salt hypertension in rats.

Body fluid volumes and their relation to mean arterial pressure and plasma renin activity (PRA) were examined in heminephrectomized rats after 4 wk of treatment with deoxy-corticosterone acetate (DOCA) and placed on one of three levels of salt intake, either high (D-HS), normal (D-NS), or low (D-LS); sham-operated rats, which received heminephrectomy and no DOCA treatment, also received high (S-HS), normal (S-NS), or low (S-LS) intakes of salt. Body fluid volumes were measured as the distribution volumes of radioiodinated serum albumin, 35SO4, and tritiated water for plasma volume (PV), extracellular fluid volume (EFV), and total body water (TBW), respectively. Approximately the same degrees of hypertension occurred in the D-HS and D-NS rats, but the D-LS rats were normotensive. PV and EFV were increased only in the D-HS rats, with no prominent changes occurring in the D-NS rats. Intracellular fluid volume (ICF) was not changed in the D-NS rats when compared with the S-NS rats. The ratios of PV/EFV and EFV/TBW in the DOCA-treated groups on high or normal salt were not different from their controls. PRA was greatly suppressed in the D-HS and D-NS rats when compared with all other groups. In another group of D-HS rats, sodium was restricted for 2 wk; in this group the mean arterial pressure fell to control levels without significant changes in PV, but interstitial fluid volume was reduced to normal levels. These results demonstrated that 1) in DOCA-salt hypertensive rats there is expansion of body fluid volumes that are proportionally distributed among the PV, EFV, and ICF; 2) increases in body fluid volumes are not necessary for DOCA to maintain hypertension; 3) a certain minimal amount of dietary sodium is necessary for the development and maintenance of hypertension; and 4) following DOCA treatment the suppression of PRA is not due solely to expansion of body fluid volumes.

Animals↗
PubMed1988

Direct measurement of CAT activity by incubation of CAT-expressing cells in medium containing chloramphenicol.

We describe a simple, flexible, rapid, sensitive and accurate in vivo assay of the bacterial chloramphenicol acetyltransferase (CAT) enzyme expressed in mammalian cells. The assay is based on the ability of the substrate and products of this enzyme reaction (viz. chloramphenicol and its acetylated derivatives) to equilibrate rapidly between the cells and the surrounding tissue culture medium. We find that chloramphenicol added to the culture medium readily enters the cells and becomes acetylated by the intracellular CAT enzyme. The acetyl derivatives leave the cell and appear rapidly in the culture medium. Due to the large excess of the extracellular compared to the intracellular fluid and due to rapid equilibration of chloramphenicol and its derivatives between them, we find that the bulk of the chloramphenicol and its acetyl derivatives are present in the culture medium at any given time point. Chloramphenicol and its acetylated products are extracted from the medium with ethyl acetate and resolved by thin layer chromatography giving an accurate measurement of the intracellular CAT activity. Sensitive and accurate quantitation of CAT activity in this assay is made possible by the addition of trace amounts of 14C-labeled chloramphenicol to the medium.

Animals↗
PubMed1987

Amino acid supply and protein metabolism in Ehrlich ascites tumour cells. 2. Incorporation of 14C-tyrosine.

The incorporation of 14C-tyrosine into protein of incubated Ehrlich ascites tumour cells was measured over a 10-fold range of external amino acid concentrations, the composition of which simulated that of mouse intraperitoneal fluid. Incorporation was linear with time and the rate of incorporation increased with increasing external amino acid concentration up to at least six times intraperitoneal concentrations. Further increases in external amino acid supply did not yield a concomitant increase in the rate of tyrosine incorporation, a maximum fractional protein synthetic rate of approximately 100% d-1 being achieved. The specific activity of tyrosine in intracellular fluid was approximately 95% of that of extracellular fluid, irrespective of the external amino acid concentration.

Amino Acids↗
PubMed1988

Aspiration and characterization of predentin fluid in developing rat teeth by means of a micropuncture and micro-analytical technique.

A fluid phase was aspirated in vivo and in vitro from predentin or pulp of developing rat teeth by means of a micropuncture technique. Pooled aspirates (approx. 2 nL) were analyzed for P, Na, K, Ca, Mg, and S by electron probe microtechniques (Lechene and Warner, 1979). Compared with pulp fluid, currently and previously studied cartilage fluids, as well as serum, predentin fluid showed elevated K, depressed Na, Cl, and Ca, as well as increased P. Statistical analysis was possible for only a few groups of comparisons among the elemental profiles. Ultrastructural examination of the aspiration site and of the aspirates showed no evidence of contamination with cell organelles or other formed elements. The micropuncture technique used was a critically precise and laborious procedure; possible contamination with intracellular fluid could not be avoided. The consistently low Mg concentration found in the aspirates, however, supports our view that the samples were primarily extracellular.

Animals↗
PubMed1982

Induced hyperosmolality effects on extracellular fluid volume, lymph flow and osmolar equilibrium: studies in the rat.

The effects of experimentally induced hyperosmolality has been studied in rats. Renal vascular pedicle ligation was performed on all animals to prevent renal clearance of the infused solute, xylose, and allow accurate ECF space determinations with 51Cr EDTA. Small volume hyperosmolar xylose infusions caused parallel elevation of the osmolality of thoracic duct lymph and arterial plasma. A positive correlation was obtained between lymph osmolality and flow alterations. The maximal osmolality elevation of 22 mosm X kg-1 H2O was associated with an intracellular fluid mobilisation which was equal to 7.1% of the ECF space. The infused and mobilised fluid volumes were distributed unequally between the intra and extra vascular compartments of the ECF.

Animals↗
PubMed1981

Fluid shifts from myofibrillar to sarcoplasmic space in the myocardium of exhausted rats.

Integrity of the myocardium following a swim to exhaustion (STE) was assessed by quantitative light and electron microscopy in young adult female rats. Myocardial fiber diameter and mitochondrial (MITO) number, size, and volume fraction (VF) were unaffected by the STE, Also, the STE did not alter ventricular water content. In contrast, myofibrillar (MYO) VF was reduced and sarcoplasmic (SARCO) VF elevated (both P less than 0.05) at exhaustion. As both cell and MITO absolute volume were apparently unchanged at exhaustion, an intracellular fluid shift from MYO to SARCO space must have occurred, without affecting MITO volume. Neither swim training nor low-protein diet modified these exhaustion-induced changes. These data provide a possible explanation at the ultrastructural level for the documented reduction in performance of the heart in the exhausted state.

Animals↗
PubMed1992

Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection.

Intracellular contents reflect the specific history of a cell including innate physiological heterogeneity as well as differing levels of exposure to environmental influences. A method capable of analyzing a variety of species from within a single human erythrocyte is demonstrated. Guided by a microscope, individual cells can be drawn into open capillaries of 10-microns i.d. On contact with a low ionic strength buffer solution, the cell lyses and releases its intracellular fluid. The ionic components are then separated by capillary electrophoresis. For glutathione, microderivatization with a fluorescent reagent can be accomplished in vitro with monobromobimane. The effects of extracellular oxidizing and reducing agents on the glutathione levels can thus be followed. For sodium and potassium, or any other ionic species, charge displacement of a fluorescent cation results in indirect fluorescence detection. The two detection modes are suitable for intracellular components present at low-attomole and sub-femtomole levels, respectively.

Electrophoresis↗
PubMed1997

Profound natriuresis, extracellular fluid volume contraction, and hypernatremia with hypertonic losses following trauma.

A young male sustained very serious head and soft tissue injuries in a motor vehicle accident (MVA). Three interesting problems developed in the sodium (Na) and water area in the second week in hospital. First, on day 11 after the MVA, his urine output increased to 3 liters per day; the urine osmolality was 1000 mOsm/kg H2O and Na and Cl were the principal urine osmoles. There appeared to be a salt wasting syndrome because he had a very large natriuresis (close to 900 mmol/24 hr) at a time when his central venous pressure was low. To help identify the nephron site responsible for a natriuresis with a high urine osmolality, additional studies were carried out in normal volunteers who took a loop or a thiazide diuretic on different occasions while ADH was acting. The pattern of natriuresis in the patient was similar to that after the thiazide but not the loop diuretic. The second problem concerned his hypernatremia (153 mM) because his urine was hypertonic and his intravenous therapy was isotonic saline. To explain hypernatremia while receiving more electrolyte-free water, we speculated that there was a water shift into cells resulting from particles generated and retained in his intracellular fluid. Given the large shift of water required, a lesion in muscle was suspected, a form of rhabdomyolysis. The third problem concerned the rate of catabolism of lean body mass. The metabolic consequences of generating these intracellular particles and the large amount of urea that was excreted could reflect a large degree of protein catabolism.

Adult↗
PubMed1989

An endogenous factor induces heterogeneity of binding sites of selective muscarinic receptor antagonists in rat heart.

According to molecular biological and pharmacological criteria, rat heart membranes normally express only one muscarinic receptor subtype. The selective antagonists pirenzepine and AF-DX 116 bind to this receptor with a single affinity: low and high, respectively. We report here that an endogenous, intracellular factor alters the affinity of selective antagonists for muscarinic receptors in the heart. Thus, when the intracellular fluid is added back to rat heart membranes, both pirenzepine and AF-DX 116 bind to two receptor sites. Approximately 30% of the receptors bind pirenzepine with high affinity and AF-DX 116 with low affinity. Thus, while cardiac muscarinic receptors are coded for by a single mRNA and are therefore genetically homogeneous, the resulting receptor protein might behave like a mixture of receptor subtypes in intact tissues due to the influence of intracellular factors on receptor conformation.

Animals↗
PubMed1989

The determination of mass of metabolites with tracers.

Application of tracers in vivo for the determination of replacement and mass of bloodborne compounds at steady state is discussed. Theory and methods to determine mass with tracers (total amount of compound-tracee-within the body) for compartmental and noncompartmental systems are presented, and their limitations examined. Methods to derive mass from the specific activity curves after bolus injection or infusion of tracer are described using graphic procedures or by equations using the parameters of exponential curves. The relationship between assumed models and the interpretation of tracer data is examined. The determination of both replacement (appearance, which equals utilization at steady state) and mass of most compounds present in both extracellular and intracellular fluids (such as lactate and amino acids) requires the application of the A-V mode for tracer administration and sampling of blood. Recycling of carbon affects the determination of mass with 14C. Estimates of true mass are provided with tritium-labeled compounds, even when tritium loss is by exchange with protons or through futile cycling. Estimates of the amount (body mass) of lactate, alanine, glutamate, and proline obtained with tritium-labeled compounds are presented. Most of these masses are intracellular. The concentration of lactate in tissues equals or is greater, and that of amino acids much greater than that in plasma. Hence, the so-called "distribution space" for these compounds, calculated conventionally by dividing mass by plasma concentration, would appear to be equal to or greater than the body water of lactate, and several liters per kilogram for amino acids.

Alanine↗