Blood levels, clearance rates and effects of epinephrine and norepinephrine on insulin and metabolites during alpha- and beta-adrenergic blockade in cattle in vivo, and in vitro degradation of dopamine in bovine blood

To study possible modifications of norepinephrine and epinephrine kinetics (metabolic clearance rate and half-times) by inhibition of binding to alpha- and/or beta-adrenergic receptors, norepinephrine and epinephrine were iv infused in the absence or presence of the alpha-adrenergic blocking agent phentolamine, the beta-adrenergic blocking agent propranolol, and during the combined administration of phentolamine and propranolol. In addition, recovery experiments were performed to investigate the in vitro degradation of dopamine, norepinephrine and epinephrine. Blood levels and kinetics of epinephrine were not modified by alpha- and beta-adrenergic blockade, whereas norepinephrine clearance was reduced by alpha-adrenergic blockade, and was greater than epinephrine clearance. alpha- and beta-adrenergic blockade markedly modified insulin, glucose and non-esterified fatty acid responses to epinephrine and norepinephrine. Norepinephrine and epinephrine could fully be recovered when added to bovine blood plasma. In contrast, dopamine obviously was immediately destructed by bovine, but not by human blood plasma, and should therefore barely be detectable in blood of cattle in vivo.     

Hemodialysis-associated induction of cytokines

The interleukin hypothesis relates chronic pathology in long-term end-stage renal disease (ESRD) patients to repeated stimulation of mononuclear cell cytokine production during hemodialysis. In vitro experiments demonstrated different possible mechanisms involved in hemodialysis-associated cytokine induction: adherence of mononuclear cells to the dialyzer membrane; complement activation, and the passage of cytokine-inducing bacterial fragments from contaminated dialysate through the dialyzer membrane into the blood. Studies investigating mononuclear cells from ESRD patients ex vivo suggest that these cells become activated during hemodialysis with complement-activating membranes and that the type of dialyzer membrane may influence mononuclear cell cytokine production in response to endotoxin. According to biological assays, plasma levels of interleukin-1 but not interleukin-6 activity seem to be elevated in ESRD patients compared to normal subjects and may increase further during treatment depending on the choice of the dialyzer membrane. However, to date, partly due to insufficient assay sensitivity and circulating inhibitors, measurements of interleukin-1, interleukin-6 and tumor necrosis factor in plasma by specific immunoassays could not finally prove elevated plasma cytokine levels in ESRD patients. Since circulating mononuclear cells are a major source of cytokines, studying the activation of these cells ex vivo seems to be the best approach to study hemodialysis-associated cytokine induction.     

Pharmacokinetics, central nervous system uptake, and lipid solubility of propranolol, acebutolol, and sotalol

The relation of in vitro lipophilicity, based on octanol:buffer partition ratio and on reverse-phase liquid chromatographic retention, to in vivo pharmacokinetics and central nervous system entry was evaluated for the beta-blockers propranolol, acebutolol, and sotalol. Anesthetized cats received single intravenous doses, following which plasma kinetics, cerebrospinal fluid (CSF) kinetics and brain tissue uptake were determined over the next 4 h. Propranolol, by far the most lipophilic beta-blocker in vitro, had the highest in vivo metabolic clearance and volume of distribution (Vd), the most rapid entry into CSF, and the highest brain:plasma uptake ratio (38.0). Sotalol, the most hydrophilic drug in vitro, had the lowest in vivo clearance and Vd, the slowest CSF entry, and the lowest brain:plasma ratio (0.52). Acebutolol had slightly greater in vitro lipophilicity than sotalol, intermediate values of in vivo plasma kinetics and CSF entry rate, and a slightly greater brain:plasma uptake ratio (0.71). Thus, differences among beta-blockers in lipid solubility are associated with predictable differences in plasma kinetics, and rate of entry into CSF. Furthermore, the relative extent of entry into brain is lower for hydrophilic as opposed to lipophilic beta-blockers.     

Zinc metabolism in adrenal cortical insufficiency: effects of carbohydrate-active steroids

Detailed studies of zinc kinetics were performed in two patients with adrenal cortical insufficiency to investigate the effects of carbohydrate-active steroids (CAS) on zinc metabolism. Zinc- 69m was administered intravenously to each patient under two conditions: (1) treated with CAS replacement therapy and (2) untreated, ie, without hormone treatment for five to six days. Radioactivity was measured in blood plasma, red blood cells, urine, and stool and by means of external probes placed over liver and thigh. Data were analyzed using a previously developed multicompartmental model, which describes the early phase of zinc metabolism. The results of these studies suggest that CAS promotes the internalization of zinc into red blood cells and liver cells. These results are consistent with previous in vitro and in vivo studies in which CAS was shown to induce the synthesis of metallothionein in liver cells.     

Effect of exchange transfusion of reticulocytes on in vitro and in vivo intestinal iron (Fe3+) absorption in mice

The effect of acute changes in erythron demands and in plasma-iron turnover (PIT) on the in vitro and in vivo absorption of 59Fe3+ was studied in the mouse. Hypoxic exposure for 20 h (a time at which intestinal iron absorption is markedly stimulated) induced reticulocytosis with a marked elevation in PIT. More prolonged exposure (3 d) further enhanced the plasma-iron clearance, even though the absorption of 59Fe was not further increased. Recipient mice, exchange transfused with whole blood from phenylhydrazine-treated animals, had a marked reticulocytosis and elevated PIT. However, in vivo studies exhibited only a small enhancement in intestinal 59Fe absorption. In vitro studies, in contrast, showed no changes in the kinetic parameters for duodenal Fe3+ uptake in similarly-transfused mice. Blood cells, rather than plasma, were responsible for the enhanced in vivo absorption in the transfused animals. These data indicate that acute changes in body iron demands and in PIT have only a small effect on iron absorption via a process independent of the adaptive increase in carrier-mediated uptake following chronic (3 d) hypoxia. This regulatory process, however, is inadequate to explain the adaptive changes seen during acute (20 h) hypoxic exposure.     

Principles of dialysis and dialysis of drugs

Consideration of the interactions of drugs and dialysis must include an understanding of the mechanisms of transport during dialysis, i.e., diffusion, ultrafiltration and membrane-protein binding effects. Clearance is a function of molecular size, blood and dialysate flow, membrane area and permeability, and dialyzer support geometry. Protein binding and hematocrit decrease the in vivo clearances in comparison to those measured in vitro with aqueous solutions. The effect on the serum half-life is also determined by the distribution space and clearance by other routes. Other factors such as metabolic alterations of dialysis can affect pharmacologic activity, and the clinical response is the end product of many determinants. Numerous drugs are effectively removed by hemodialysis or at a slower rate by peritoneal dialysis, which occasionally allows considerable influx. The influence of intestinal contents on elimination rates by peritoneal dialysis is unknown. Peritoneal dialysis can be influenced considerably by vasoactive drugs.     

The effect of ACTH and cortisol on aldosterone and cortisol clearance and distribution in plasma and whole blood.

The mechanisms of increased aldosterone and cortisol metabolic clearance rates (MCR) following ACTH or cortisol administration were studied in 13 subjects undergoing cardiac catheterization and in 9 healthy controls. In control subjects, the MCR (plasma) of both steroids increased by 29% (aldosterone: from 936 +/- 57 to 1204 +/- 55 l/day/m2, cortisol: from 205 +/- 12 to 264 +/- 17 l/day/m2 +/- SE) after ACTH (12 units/h) for 1 to 4 h, and by 20 and 32%, respectively, after cortisol (12 mg/h) for 1 to 2 h. In contrast, aldosterone MCR (whole blood) did not change with ACTH or cortisol administration (from 1276 +/- 57 to 1330 +/- 59 l/day/m2), indicating that the plasma MCR increase results from a redistribution of aldosterone from plasma to red cells. Aldosterone splanchnic extraction was 92 +/- 1% (n = 12) with normal morning cortisol levels, and extraction was unchanged after ACTH administration. For cortisol, however, the splanchnic extraction increased from 8 +/- 0.8% to 17.8 +/- 5.0%, and the MCR (whole blood) likewise increased by 15 to 31% (from 295 +/- 23 to 357 +/- 30 l/day/m2), after ACTH or cortisol administration. In vivo and in vitro measurements (at 37 C) of tracer aldosterone concentration in plasma and in red cells showed an increase in distribution to red cells with increasing cortisol concentrations. The results suggest that a fraction of aldosterone is bound in plasma and displaced by cortisol into red cells. There is an increased aldosterone plasma MCR, but unaltered whole blood MCR, since the liver extracts aldosterone almost completely from both plasma and red cells. The increase in cortisol MCR (plasma) results from both an increased splanchnic extraction as plasma binding sites approach saturation and a redistribution into red cells.     

Physiological correction of hereditary mild hypofibrinogenemia during pregnancy

Introduction

Hereditary hypofibrinogenemia is a rare fibrinogen disorder characterised by decreased levels of fibrinogen. Pregnant women with hypofibrinogenemia are at risk of adverse obstetrical outcomes, depending on the fibrinogen level.

Aim

We investigated how the physiological changes of hemostasis throughout the pregnancy impact the hemostatic balance in a woman with hereditary mild hypofibrinogenemia.

Methods

Fibrin clot properties were analyzed by turbidimetry and scanning electron microscopy, clot weight and red blood cells retention were measured by whole clot contraction, and in vitro thrombin generation was assessed by calibrated automated thrombogram and ex vivo by TAT.

Results

Throughout the pregnancy, the fibrinogen levels increased reaching normal values in the third trimester (activity 3.1 g/L, antigen 3.2 g/L). In parallel, the fibrin polymerisation increased, the fibrinolysis decreased, the fibrin clot network became denser with thicker fibrin fibers, and the fibrin clot weight and red blood cells retention increased, reaching control's value at the third trimester. Similarly, in vitro and ex vitro thrombin generation increased, reaching maximum values at the delivery.

Conclusion

In this case of hereditary mild hypofibrinogenemia we observed a physiological increase of fibrinogen and thrombin generation. Future studies should focus on moderate and severe hypofibrinogenemia, to assess fibrinogen variation and the overall impact of increased TG on the hemostasis balance.     

Measurement of backfiltration rates during hemodialysis with highly permeable membranes.

A method for determining local transmembrane fluid movement in a commercial hemodialyzer at low dialysate flow rates by measuring changes along the dialyzer length in the local concentration of a marker macromolecule added to the dialysis solution has been developed. The method was evaluated in vitro at zero net ultrafiltration using dialyzers containing polysulfone (n = 4) and cuprophane (n = 3) membranes. The local concentration of the marker macromolecule along the dialyzer length was higher than the input dialysate concentration only during experiments with dialyzers containing polysulfone membranes. These observations provide direct empirical evidence that fluid movement in the dialysate to blood direction, i.e., backfiltration, occurs during hemodialysis with this highly permeable membrane. Net rates of backfiltration for the dialyzer containing polysulfone membrane were also calculated from changes in the local concentration of the marker macromolecule and mass balance considerations. The calculated backfiltration rates increased with increasing blood flow rate and trended upward with increasing dialysate flow rate. The described methodology provides a novel approach for the further characterization of fluid and solute transport during hemodialysis with highly permeable membranes.     

Does monitoring of pre-/post-dialyzer pressure difference improve efficiency in intermittent hemodialysis?

BACKGROUND: Continuous monitoring of pre-/post-dialyzer pressure difference (DeltaP) is widely used in continuous renal replacement therapies to monitor extracorporeal circuit function. The aim of this study was to verify whether DeltaP may help to identify chronic subclinical worsening of dialysis quality due to incomplete dialyzer clotting in intermittent hemodialysis. METHODS: Nine chronic hemodialysis patients were enrolled in the study and dialyzed twice (high-flux polysulfone dialyzer) with DeltaP and urea-clearance monitoring: the first session with a standard anticoagulation and the second without. To verify whether a visible clotting of the dialyzer precedes or follows a significant DeltaP increase, we checked the dialyzers for the presence of red clots after a saline flush performed when a 50% increase in DeltaP was registered. RESULTS: In the second dialysis session after a 50% increase in DeltaP (documented in 7/9 patients), all dialyzers, after saline flush, showed a visible fiber clotting but not a significant reduction (>15%) in urea clearance. In the majority of the patients (6/7), until a few minutes before complete occlusion of the extracorporeal circuit, the urea clearance did not change significantly (-8.9 +/- 12.7%). CONCLUSIONS: The usual check of the presence or absence of red clots in the dialyzer at the end of the dialysis session is enough, in the absence of red clots, to ensure that dialyzer efficiency is maintained during the whole treatment. Contrary to what is applied in CRRT, a continuous monitoring of DeltaP during intermittent hemodialysis would not significantly help to unmask unnoticed inefficient hemodialysis sessions.     

Dual role of diffusion in tissue gas exchange: blood-tissue equilibration and diffusion shunt

The role of diffusion in tissue gas exchange is investigated using a simple mathematical model which incorporates both tissue-blood equilibration and gas transfer between arterial and venous vessels with counter-current flow, leading to 'diffusion shunt'. Both increasing the diffusion coefficient of the gas considered or decreasing the blood flow results in two antagonistic effects: (i) improvement of blood/tissue equilibration, (ii) increase in extent of diffusion shunt. The diffusion shunt retards inert gas wash-out (local tissue clearance) and leads thus, if not taken into account, to an underestimation of capillary blood flow calculated from the wash-out rate constant. For O2 (and CO2) the diffusion shunt reduces the efficacy of blood/tissue transfer, but its extent is expected to be only moderate because of the chemical combination of these gases in blood.     

Equilibration of intravascular albumin with lung lymph in unanesthetized sheep

In 16 unanesthetized sheep with chronic lung lymph fistulas we measured pulmonary vascular pressures, lymph flow, lymph and plasma total protein and albumin concentration. We determined the rate of equilibration of radioiodinated albumin between plasma and lung interstitial fluid (lung lymph) in three steady-state conditions; baseline (n = 14), increased pulmonary microvascular pressure (n = 9) and increased microvascular permeability (n = 4). The tracer protein equilibration proceeded according to single compartment wash-in kinetics in all experiments. Lung lymph flow averaged 5.3 +/- 2.8 (S.D.)ml/h under baseline conditions, 16.1 +/- 10.6 ml/h during increased pressure and 37.3 +/- 29.4 ml/h during increased permeability. The half time of equilibration averaged 2.9 +/- 1.0 h, 2.2 +/- 1.0 h and 0.7 +/- 0.2 h, respectively. Lung interstitial fluid equilibrates with plasma proteins more rapidly than most other organs. The marked difference between increased permeability and the other conditions demonstrates the sensitivity of this method. No evidence was obtained that any tracer protein entered lung lymph within the caudal mediastinal lymph node.     

Busulfan clearance in renal failure and hemodialysis

The impact of hemodialysis on the clearance of busulfan was determined in a patient with chronic renal failure undergoing autologous peripheral stem cell transplantation for non-Hodgkin's lymphoma. The extraction ratio for busulfan across the dialyzer was 0.530 +/- 0.026 at a blood flow of 400 ml/min, which corresponds to a hemodialysis clearance of 2.23 +/- 0.11 ml/min/kg body weight. Apparent oral clearance of busulfan without hemodialysis was 3.38 +/- 0.56 ml/min/kg. Thus, a 4 h hemodialysis session enhanced the apparent oral clearance of busulfan by 65%. We conclude that hemodialysis effectively removes busulfan from circulating blood, but a standard hemodialysis period (ie, 4 h) does not significantly alter busulfan exposure. Bone Marrow Transplantation(2000) 25, 201-203.     

Sodium balance in renal failure. A comparison of patients with normal subjects under extremes of sodium intake

To gain insight into the factors involved in the maintenance of sodium balance in patients with chronic renal failure, we studied 10 patients with a creatinine clearance of 11.5 +/- 4.0 ml/min after equilibrium on 20 and 120 mEq of sodium per day. The measurements included blood pressure, plasma volume, blood volume, extracellular fluid volume, plasma renin activity, plasma aldosterone, and plasma norepinephrine. For comparison, eight normal volunteers were studied after equilibration on 20, 200, and 1128 mEq of sodium per day. The latter intake was chosen to match the high sodium intake per residual renal function in the patients. In the patients, equilibrium after raised sodium intake was accompanied by a marked increase in blood pressure and blood volume, a moderate fall in plasma renin activity and levels of aldosterone and norepinephrine, and only little expansion of the interstitial space. The 24-hour creatinine clearance rose by 21.2 +/- 7.2%. Fractional sodium excretion (X 100%) was 5.3 +/- 0.8% during the 120 mEq sodium diet. In the normal volunteers, increasing the sodium intake from 20 to 1128 mEq/day evoked no consistent change in blood pressure but caused a comparable rise in blood volume, considerable suppression of plasma renin activity, aldosterone, and norepinephrine, and a much larger increase in interstitial volume. Their creatinine clearance had risen by 22.4 +/- 6.5%, and their fractional sodium excretion during the 1128 mEq sodium intake was 3.9 +/- 0.2%.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic model of calcium mass balance during dialysis therapy

A kinetic model of Ca mass balance during dialysis has been developed. It is a single-compartment, variable-volume model to compute Ca mass balance during dialysis in its volume of distribution, the extracellular fluid. The model was used to analyze literature data which were suitable for the assessment of Ca mass balance over the course of dialysis. The modeled analyses predicted the serial plasma Ca concentrations very well. The mass balance analyses revealed a pool of rapidly diffusible Ca beyond the extracellular fluid distribution volume where Ca could be mobilized (M+(Ca)) or sequestered (M-(Ca)) very rapidly at rate equal but opposite in sign to dialyzer flux and thus effectively maintain near constant plasma Ca in the face of dialyzer Ca concentration gradients. This pool is likely the large pool of diffusible (miscible) Ca in connective tissue and on bone surfaces. Analysis of net Ca flux during dialysis with Cdi(Ca) = 2.50 mEq/l suggests that 80% of patients are in positive Ca balance during dialysis. Further studies are required to verify the model and to develop a model of interdialytic Ca mass balance.     

Half-life of plasma growth hormone in young and old conscious female rats

The kinetics of disappearance of plasma GH was studied in young (3-4 months) and old (24-27 months) Sprague-Dawley female rats. Conscious, free moving animals carrying indwelling atrial and carotid cannulas received a single injection of 125I-rGH via the carotid cannula. Sequential blood samples were removed at intervals during the following hour, and total (TR) and immunoprecipitable radioactivity (IPR) were determined in the corresponding plasmas. Both TR and IPR displayed biexponential kinetics in vivo which did not differ significantly, for each variable, between young and old animals. The volumes of distribution of GH were also similar in both age-groups. The IPR/TR ratio, an estimate of GH inactivation within the plasma space, showed a decreasing sigmoid-shaped kinetics in vivo with a time of semi-inactivation (ti1/2) of 23.8 +/- 1.2 and 29.0 +/- 1.0 min (mean +/- SE) for young and old rats, respectively (P less than 0.02). The estrous status did not significantly affect ti1/2 values in vivo. The in vitro t1/2 was estimated by incubating plasma from the young and old animals at 37 degrees C with 125I-rGH for several hours. The IPR/TR ratio displayed a linear kinetics in vitro with t1/2 values of 23.7 +/- 1.7 and 25.8 +/- 1.9 h (NS) for young and old animals, respectively. The above results show that GH catabolism decreases slightly with age in the female rat, although it is unlikely that this change has a significant effect on plasma levels of GH. The data also suggest that GH is physiologically inactivated in the extravascular space.     

Reduced Survival of Isotope-Labelled Rh(D)-Negative Donor Red Cells in a Patient with Anti-LWab

The serum of an 85-year-old Caucasian male with no history of blood transfusion contained an IgG3 antibody with anti-LWab specificity. The antibody failed to react with dithiothreitol-treated red cells, and there was a marked reduction in titre of the antibody with pronase-treated cells, findings consistent with an antibody having this specificity. High association values were obtained in a mononuclear phagocyte assay when LW-positive red cells, sensitised in vitro with the patient's serum antibody, were incubated with peripheral blood monocytes from the patient. In vivo red cell survival studies demonstrated that 99mTc-labelled rhesus-negative (rr), LW-positive red cells had 53% survival at 1 h. The IgG subclass of the antibody, mononuclear phagocyte assay results and in vivo survival studies predicted a significant reduction in the posttransfusion survival of therapeutic volumes of rhesus-negative (rr), LW-positive red cells.     

Norepinephrine kinetics during insulin-induced hypoglycemia

Norepinephrine (NE) kinetics (plasma appearance rate, clearance, and forearm extraction) were measured during insulin-induced hypoglycemia in six healthy subjects. NE clearance did not change during hypoglycemia, indicating that the increase in plasma NE during hypoglycemia is due to an increased plasma appearance rate of NE. Forearm extraction of 3H-NE and of endogenous plasma epinephrine decreased significantly during hypoglycemia, probably due to an increase in regional blood flow.     

Determination of gastric bicarbonate secretion in the human without acid suppression

Gastric CO2/HCO3 was determined in absence of simultaneous inhibition of acid secretion by intra- and extragastric pCO2/pH measurements in 23 persons and calculated using the equation of Henderson-Hasselbalch. pCO2 was measured with use of a new electrode. The characteristics of the CO2 selective electrode membrane were tested in vitro. The CO2 selective membrane proved to be stable against 0.1 n HCl, gastric fluid with a pH of 1, and bile as well as mechanical irritations. 96.8 +/- 0.52% of given amounts of bicarbonate were detected in test fluids ranging in pH from 2.5-7.1. In gas and fluid, a linear relationship between given and measured bicarbonate of y = 0.97 x +1.03 with a correlation coefficient of 0.99 was observed. In vivo at pH less than 6, the intragastric pCO2 was significantly higher than that in venous blood. CO2 diffusion could be prevented by equilibration of intragastric pCO2 with venous pCO2. pCO2 was then measured in fluid as well as in reaspirated gas. Thus 94.6 +/- 8.8%, HCO3- that had been instilled intragastrically, could be detected. Without equilibration and resultant volume increase of the gas phase, bicarbonate secretion was 296.4 +/- 41.9 mumol/h under basal conditions and 520.8 +/- 252.8 mumol/h after acid stimulation. With equilibration, a bicarbonate secretion of 574.4 +/- 66.7 and 754.4 +/- 81.2 mumol/h was observed during basal and stimulated acid secretion. Our results indicate that bicarbonate measurements in absence of acid suppression require determination of CO2 in the fluid as well as in the gas phase.     

Kinetics of protein import into isolated Xenopus oocyte nuclei

An in vitro assay for nucleocytoplasmic transport was established in which signal-dependent protein import is reproduced faithfully by isolated purified nuclei. The assay permits the precise quantification of import kinetics and the discrimination between translocation through the nuclear envelope and intranuclear transport. Nuclei were manually isolated from Xenopus oocytes and after manual purification incubated with a medium containing a green fluorescent transport substrate, karyopherins alpha2 and beta1, a red fluorescent control substrate, an energy mix and, for keeping an osmotic balance, 20% (wt/vol) BSA. Import of transport substrates into the nucleus and exclusion of the control substrate were monitored simultaneously by two-color confocal microscopy. Two widely differing import substrates were used: the recombinant protein P4K [480 kDa, four nuclear localization sequences (NLSs) per P4K tetramer], and NLS-BSA (90 kDa, 15 NLSs). The measurements suggested that import, at the specific conditions used in this study, consisted of two consecutive processes: (i) the rapid equilibration of the concentration difference across the nuclear envelope, a process involving binding and translocation of substrate by the nuclear pore complex, and (ii) the dissipation of the intranuclear concentration difference by diffusion.