Rapid Centrifugation Technique Research Articles

Comparison of Rapid Centrifugation Technique with Conventional Centrifugation for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) Testing.

Prothrombin Time (PT) and activated partial thromboplastin time (APTT) are frequently performed coagulation tests in patients with coagulation disorders especially in critical care areas and in monitoring patients on anticoagulation therapy. In coagulation testing, sample processing especially centrifugation is one of the most critical steps that affect turnaround time(TAT). This study was carried out over a period of 1year. Three hundred paired samples from patients sent for PT and APTT estimation were included. One sample was centrifuged in a regular bench top centrifuge at 1500g for 20min. The other sample was divided into two polypropylene aliquots and centrifuged in a microcentrifuge at 13000g for 3min. The plasma obtained from both methods was tested for PT and APTT using the automated method on STA Compact coagulometer (Stago) using commercial thromboplastin STAR-NeoplastineR C1 Plus and phospholipid (cephalin), STAR-C K PRESTR 5 respectively. Data were analyzed using descriptive statistics, Student t test, correlation coefficient and Bland-Altman plots. Mean PT, INR and APTT forboth centrifugation methods was comparable with no statistically significant difference (p > 0.05). PT, INR and APTT also showed good correlation (r > 0.98) when compared between the two methods of centrifugation. Bland-Altman comparison between rapid and conventional methods of centrifugation for PT, INR and APTT also showed acceptable agreement. Rapid centrifugation technique for routine coagulation testing can be used safely with a significant reduction in the TAT. This can benefit patients in critical care settings and those on outpatient oral anticoagulant therapy.

Regulation of ion content and cell volume in isolated rat renal IMCD cells under hypertonic conditions.

The adaptive responses of cell volume and intracellular inorganic electrolytes to hypertonicity were investigated in isolated inner medullary collecting duct (IMCD) cells in vitro. Intracellular water and element content were determined simultaneously by a rapid centrifugation technique and electron-probe microanalysis, respectively. In the absence of adenosine 3',5'-cyclic monophosphate (cAMP), cells isolated at 600 mosM (268 mM NaCl) shrank in 900 mosM buffer (418 mM NaCl) within 100 s to 79 +/- 2% of the control volume and showed no regulatory volume increase (RVI). K+ content decreased steadily during the first 5 min to 18% below control. K+ concentration initially increased 25 +/- 2% above control and was normalized after 5 min to 144 +/- 10 mM (n = 6). Na+ and Cl- content increased slowly during the 60-min incubation period; at this point intracellular Na+ and Cl- concentrations were 97 +/- 6 and 59 +/- 7% (n = 6) above control levels (75 +/- 12 mM Na+ and 143 +/- 29 mM Cl-), respectively. In the presence of 10(-4) M 8-bromo-cAMP (8-BrcAMP; used as a substitute for antidiuretic hormone), IMCD cells showed RVI within 5 min. K+ content initially dropped 18 +/- 2% below control and recovered during the next 4 min but was 12 +/- 4% below control after 60 min. K+ concentration showed similar changes. Na+ and Cl- content increased to a larger extent than in the absence of 8-BrcAMP to 95 +/- 10% (Na+) and 47 +/- 6% (Cl-) above control after 60 min, whereas Na+ and Cl- concentrations were the same as without 8-BrcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Florida red tide brevetoxins and binding in fish brain synaptosomes

Equilibrium binding of the lipid-soluble neurotoxins isolated from the marine red tide dinoflagellate Ptychodiscus brevis to Tilapia brain synaptosomes is described. Tritiated brevetoxin PbTx-3 binding studies were carried out at 23°C and specific binding was measured using a rapid centrifugation technique. Rosenthal analysis of specific toxin binding yields a K D of 6.1 nM and a B max of 1.4 pmol/mg protein and suggests a single class of site. Tritiated PbTx-3 can be displaced from its specific binding site by PbTx-1, PbTx-2, and PbTx-3, the three most potent brevetoxins. In vivo ichthyotoxicity correlates in a positive fashion with displacing ability in the assay. Binding of brevetoxins to specific receptor sites in fish excitable tissues is proposed as an initial event in ichthyotoxicity observed during Florida red tides.

Uptake of N-nitrosobis(2-oxopropyl)amine by isolated rat and hamster hepatocytes: species differences and evidence for an active carrier-mediated transport process.

The rates of uptake of the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) by hepatocytes isolated from Fischer rats and Syrian hamsters were determined in order to investigate species differences in cellular transport of the carcinogen. Initial rates of uptake of [1-14C]BOP by hepatocytes were measured using a rapid centrifugation technique. At cell densities from 1.5 to 6 x 10(6) cells/ml, initial rates of uptake were as much as 4-fold more rapid in hamster hepatocytes than in those of the rat. The cell/medium distribution ratio for hamster hepatocytes reached a value of 9.0 after a 20-min incubation with an extracellular BOP concentration of 20 microM. Under the same conditions, the cell/medium distribution ratio for rat hepatocytes was only 2.4. These results indicated that BOP uptake proceeded against a concentration gradient and was more rapid in hamster hepatocytes. In both species, the rates of uptake were saturable with increasing concentration (2-685 microM) and displayed biphasic kinetics characteristic of high-affinity (Km less than 20 microM) and low-affinity (Km greater than 30 microM) processes for the uptake of BOP. Evidence for the involvement of an ATP-dependent active carrier-mediated transport process was obtained from experiments in which hepatocytes were preincubated with metabolic inhibitors. Significant inhibition of uptake was observed in the presence of KCN, carbonyl cyanide-3-chlorophenylhydrazone, antimycin A, oligomycin and other agents which interfere with electron transport or ATP generation. Based on the reduction in uptake rates, rat hepatocytes were more sensitive to the effects of these inhibitors. These results suggest that the entry of BOP into hepatocytes is under cellular regulation and that the more rapid rate of uptake in liver cells of the hamster may be one factor responsible for the observation that BOP is a more potent hepatotoxin and carcinogen in this species.

Characteristics and specificity of the glucocorticoid "carrier" of rat liver plasma membrane.

The uptake of corticosterone by highly purified plasma membrane vesicles of rat liver was studied by a rapid-centrifugation technique which allows uptake measurements within 5 s. The vesicles are free of soluble cytoplasmic constituents. Therefore, association of hormone with the vesicle is attributed entirely to components of the vesicle-membrane. Half maximal uptake is reached at 8 s at 21 degrees C. At 15 degrees C transition of the lipid state in the membrane leads to a decrease of uptake, a characteristic property common to membrane mediated processes. The uptake of corticosterone is saturable and reversible but does not follow normal saturation kinetics. The apparent dissociation constants of three uptake systems bear direct relation to the concentration of free corticosterone in rat plasma (4-16 nM) supporting a physiological role for the system. Uptake of corticosterone decreases with decreases in vesicular volume; about 50% of the hormone is bound specifically and 50% is transported to the lumen of the vesicle. Since outflow of intravesicular hormone also occurs readily, the uptake and transport is proposed to be mediated by putative "carriers". The "carrier" preferentially transports glucocorticoids; dexamethasone is not taken up by this putative molecule. Steroids with 5 alpha conformation are more potent inhibitors of the "carrier" for corticosterone than 5 beta-steroids. Androgens and estrogens are weak competitors of corticosterone. The affinity of the "carrier" for several hormones differs considerably from that of the cytoplasmic receptor. Morris hepatoma cells (MH 3924) do not take up corticosterone. Our results prompt us to propose the hypothesis that the transport function of the "carrier" and the binding of the hormone by the cytoplasmic receptor are two different entities; perturbation of the "carrier" may lead to steroid unresponsiveness. Normal expression of steroid hormone activity is manifested in the concerted action of the functionally sound cell membrane "carrier" and the intracellular receptor.

Mechanism of chemical-induced toxicity: I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K + and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.

L-thyroxine enters the rat liver cell by simple diffusion.

The mode of uptake of L-[125I]thyroxine by freshly isolated rat liver parenchymal cells was studied by a rapid centrifugation technique. Using conditions for measuring initial rates of uptake, uptake by liver cells was not saturable when exposed to hormone concentrations in the incubation medium ranging from 2 pmol/l to 10 mumol/l. The Arrhenius plot was linear from 2 to 37 degrees C; the temperature coefficient was 1.4. The uptake of L-[125I]thyroxine by liver cells was 35% when compared with that of L-[125I]tri-iodothyronine. In the presence of 2.8% bovine serum albumin the rate of uptake of L-[125I]thyroxine by liver cells was reduced by 90%. These results suggest that L-[125I]thyroxine enters the rat liver parenchymal cell by simple diffusion and only the free hormone crosses the plasma membrane.

Study of fluxes at low concentrations of L-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient.

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.

A comparison of the phosphorylation potential and electrochemical proton gradient in mung bean mitochondria and phosphorylating sub-mitochondrial particles

The phosphorylation potential ( ΔG p ) and the electrochemical proton gradient (Δ \\ ̄ gmH +) normally maintained during respiration or ATP hydrolysis by mung bean hypocotyl mitochondria and phosphorylating sub-mitochondrial particles have been investigated. Phosphorylation potential experiments using safranine and oxonol-VI, as membrane potential markers for mitochondria and sub-mitochondrial particles, respectively, suggest that the ‘null point’ ΔG p (i.e., the phosphorylation potential at which no change in optical signal occurred) corresponds to a value of 15.2 ± 0.7 kcal/mol in mitochondria and 11.2 ± 0.3 kcal/mol in sub-mitochondrial particles. The value of Δ \\ ̄ gmH + generated by the hydrolysis of ATP was estimated using ion distribution techniques. In each case a rapid centrifugation technique was used to separate the organelle from the suspending medium. The total Δ \\ ̄ gmH + generated in each case was approx. 200 mV being composed of both membrane potential and pH components. A comparison of Δ \\ ̄ gmH + with ΔG p indicates that the apparent H +/ATP ratio in mung bean mitochondria is 3.4 ± 0.2 while in phosphorylating sub-mitochondrial particles it is 2.2 ± 0.1.

Mitochondrial pyruvate transport and its hormonal regulation

A quick glance at any diagram of major metabolic pathways reveals the central position that pyruvate plays in mammalian metabolism. Indeed it is true to say that pyruvate lies at the major crossroads of catabolic and anabolic pathways involved in the metabolism of sugars, fats and amino acids. Closer examination of these pathways indicates that in most cases pyruvate must enter the mitochondria before further metabolism occurs through the action of pyruvate dehydrogenase or pyruvate carboxylase. Only in the case of reduction to lactate or transamination to alanine may pyruvate be metabolised in the cytoplasm, and in the latter case a mitochondrial pathway is also present (Mendes-Mourao et al., 1975). The pKa for pyruvate is low (2.49) so that at physiological pH the concentration of the free acid will be negligible. Since free diffusion of hydrophilic ions across the inner mitochondrial membrane does not occur to an appreciable extent, it is to be expected that pyruvate enters the mitochondria by a carrier mediated mechanism. However, despite this powerful circumstantial evidence, unequivocal demonstration that such a specific transport mechanism exists had to await the discovery of a specific inhibitor of mitochondrial pyruvate transport. This was achieved in 1974 by Halestrap & Denton (1974) who showed that sc-cyano-4-hydroxycinnamate and its analogues inhibit mitochondrial pyruvate transport at PMolar concentrations. Prior to this Papa et al. (1971) had provided some evidence that pyruvate transport involves a specific carrier by using a rapid centrifugation technique to study the kinetics of pyruvate uptake into mitochondria. This work was criticized by Zahlten et al. (1972) as demonstrating binding of pyruvate to mitochondrial membranes rather than net transport, but this criticism was later shown to be false (Brouwer et nl., 1973; Papa & Paradies, 1974). A further query was raised by Bakker & Van Dam (1974) who suggested that some pyruvate transport might not be carrier mediated and could occur by a non specific exchange diffusion similar to that which could be demonstrated in phospholipid membrane vesicles. The demonstration that a-cyano-4-hydroxycinnamate specifically inhibited both pyruvate entry into and efflux from rat liver mitochondria allowed these two problems to be eliminated and enabled the properties of the transport system to be studied in some detail.

Extraction of Soil Solution Using a Simple Centrifugation Method for Pesticide Adsorption‐Desorption Studies

AbstractA simple and rapid centrifugation technique has been devised to extract small samples of soil solution from moist pesticide‐treated soils to allow measurement of the pesticide equilibrium concentration. Energy status of the soil water extracted was characterized by correlating the amount of centrifugal force applied, soil moisture content, and matric potential. By varying the size of soil samples and the centrifugal force applied, a sizable volume of soil solution (40 to 53% of the moisture content at or below 0.3 bar suction) was obtained from the soil. The pesticide equilibrium concentration of the soil solution remained relatively constant throughout the centrifugation process.

Hormone action at the membrane level VI. Binding of (—)-[ 3H]dihydroalprenolol and (±)-[ 3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[ 3H]isoproterenol and (—)-[ 3H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K d of 195 nM and has 2200 sites/cell. The binding of (—)-[ 3H]dihydroalprenolol shows one type of site with a K d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[ 3H]isoproterenol as compared to the (—)-[ 3H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.

Hormone action at the membrane level. V. Binding of ( [formula omitted])-[ 3H]isoproterenol to intact chicken erythrocytes and erythrocyte ghosts

The binding of ( ±)-[7- 3H]isoproterenol to intac chicken erythrocytes has been investigated by a rapid centrifugation technique. The binding is displaceable by a one thousand-fold excess of cold isoproterenol and consists of two fractions, only one of which is inhibitable by the beta antagonist (−)-propranolol. The total displaceable binding to intact cells amounts to 80 or 127 molecules per cell at a( ±)-isoproterenol concentration of 0.4 μM depending on the method employed to analyze the binding. Under similar conditions, the total displaceable binding to isolated membrane ghosts is 12 600 molecules per cell. The propranolol-inhibitable binding to intact cell reaches saturation within 5 min at 4 °C and gives by Scatchard analysis a maximum binding of 108 molecules per cell and with a K D of 0.4 μM. 50% inhibition of binding is obtained with 0.3 μM unlabeled (−)-isoproterenol as compared to 20 μM unlabeled (−)-isoproterenol. The binding of isoproterenol thus shows a marked stereospecific preference for the (−)-isomer.

Avian mitochondrial glutamine metabolism.

Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.

Light-dependent anion transport in isolated spinach chloroplasts

A rapid centrifugation technique is described which provides direct measurements of transient anion movements in illuminated chloroplasts. The results obtained by the technique are consistent with proposed mechanisms of chloroplast anion transport in which light-induced proton uptake determines the movement of other ions in chloroplasts. 1. 1. Direct measurements of weak acid anion transport (succinate, phosphate) during illumination support the hypothesis that proton association with weak acid anions within the chloroplasts produces concentration gradients of the uncharged weak acid species and results in net anion efflux. 2. 2. Influx of dissociated anion (Cl −) occurs in the presence of dissociated cations (Na +, K +) and weak base cations (NH 4 +). In both instances Cl − influx is presumably necessary to maintain charge balance across the membrane during proton uptake. Both the direction and quantity of anion transport in illuminated chloroplasts are predictable from the principles of charge balance and pH-dependent shifts in weak acid concentrations. Sufficient quantities of osmotically active ions are transported to account for the observed volume changes in grana by osmotic mechanisms.