Concentration Of Furosemide Research Articles

Furosemide-Induced Hyperglycaemia: The Implication of Glycolytic Kinases

Hyperglycaemia is a well known adverse effect of therapy with diuretics. In adipose tissue, hydrochlorothiazide and furosemide inhibit the rate of glucose transport. In skeletal muscle, furosemide decreases the rate of glucose phosphorylation and glycolysis. However, whether furosemide has any direct effect on the activities of any of the glycolytic enzymes is not known. In the present study, the effects of furosemide on the activities of the hexokinase, phosphofructokinase and pyruvate kinase were examined. Pieces of skeletal muscle (quadriceps) and liver were obtained from 10 non-diabetic subjects during surgery. Tissues were homogenized and the activities of the enzymes were measured in the presence or absence of furosemide (0-1.5 mM). Furosemide inhibited the activity of all three key glycolytic enzymes. The concentration of furosemide required to inhibit phosphofructokinase in muscle was lower than that required to inhibit the activity of this enzyme in the liver or to inhibit the activities of hexokinase and pyruvate kinase in both muscle and liver. These direct effects of furosemide may contribute to the decrease in glucose utilisation following therapy with this and similar agents in man.

External‐Standard High‐Performance Liquid Chromatographic Method for Quantitative Determination of Furosemide in Plasma by Using Solid‐Phase Extraction and On‐Line Elution

AbstractA rapid and simple external‐standard high‐performance liquid chromatographic (HPLC) method has been developed for the determination of the concentration of furosemide in plasma. The analyte is extracted with a C‐2 ethyl sorbent. On‐line elution of the analyte into the HPLC system is accomplished with an advanced automated sample processor (Varian). Furosemide is quantified by fluorescence detection within a linear range of 25 to 1000 ng/mL (average correlation coefficient, 0.9998), with a limit of detection of 1.8 ng/mL. Both internal‐ and external‐standard procedures were evaluated, and the external‐standard procedure demonstrated superior characteristics. The external‐standard procedure was precise to within a relative standard deviation of 8% and accurate with <3% error throughout the concentration range studied. The external‐standard HPLC method was used to analyze the concentration of furosemide in >1000 plasma samples obtained from patients with either normal kidney function or renal failure who had received furosemide either orally or intravenously in an experimental setting.

Activation by N-Ethylmaleimide of a Cl− -Dependent K+ Flux in Isolated Trout Hepatocytes

Isolated trout hepatocytes when swollen in hypotonic medium undergo a regulatory volume decrease (RVD), which occurs via KCl loss. The system shows characteristics similar to those of the transporter described in red cells. This led us to investigate, in trout hepatocytes, the effect of another signal known to activate this flux in red cells, i.e. treatment with the sulphhydryl-group reagent N-ethylmaleimide (NEM). NEM treatment resulted in a striking increase in ouabain-resistant K+ uptake measured by an isotope pulse uptake technique. The time course of the response to NEM was similar to that obtained with a hypotonic shock, indicating that the effect of NEM was immediate and transient. The NEM-stimulated K+ influx demonstrated the same anion sensitivity as the volume-induced K+ influx, i.e. a specific requirement for Br- or Cl-. Efflux experiments showed that NEM treatment produced a stimulation of both K+ and Cl- effluxes leading to a substantial net loss (10%) of cellular KCl, as confirmed by analysis of ionic contents. This KCl loss is consistent with the rapid cell shrinkage observed after addition of NEM. The Cl--dependent K+ influx was found to be independent of external Na+; in addition, NEM had no effect on Na+ content, indicating that Na+ is not implicated in this process. The effect of loop diuretics was tested on the NEM-stimulated K+ influx. As observed for the volume-induced K+ flux, a high concentration of furosemide (10(-3) mol l-1) is required for full inhibition of this flux; no effect was obtained with bumetanide (10(-4) mol l-1). Consequently, NEM appears to activate a KCl cotransport similar to the one induced in hypotonically swollen cells. Finally, the combination of the two treatments, NEM and hypotonic shock, was found to increase the K+ fluxes even further, suggesting additivity of the two stimuli by mutual positive interaction.

The role of monovalent cation transport in Sindbis virus maturation and release

Alterations in intracellular monovalent cation concentrations in Sindbis virus-infected avian cells result, in part, from a reduction in Na +/K + ATPase (Na + pump) activity. Inhibition of Na + pump activity was shown previously to temporally correlate with the appearance of viral envelope proteins on the cell surface and the release of virus particles. Cells infected with envelope-defective temperature-sensitive mutants exhibited reduced Na + pump activity at the nonpermissive temperature, where viral particles are not released. By contrast, Na + pump activity was not inhibited in Sindbis virus-infected cells treated with tunicamycin or with antiviral serum, which block virus maturation and release. Diuretic-sensitive transport of 86Rb +, a K + tracer, was stimulated in cells which express virus envelope proteins, but fail to release virus particles. In these cells, the furosemide-sensitive 86Rb + influx exhibited an increase in Vmax and was responsive to changes in the extracellular concentration of NaCL Furosemide inhibited the rapid release of virus from low salt-inhibited cells after shift to isotonic conditions. Alterations in ion transport during alphavirus infection may, therefore, facilitate the efficient release of progeny virus particles.

Cumulative increase in serum furosemide concentration following repeated doses in the newborn.

In order to test whether repeat doses of the diuretic furosemide result in plasma accumulation of this drug, eight newborns (mean birthweight, 1252 gm; mean gestational age, 27.5 weeks; mean postnatal age, 44.7 days; and normal renal function) were each given a 1-mg/kg iv bolus every 12 hr for four doses and maintained normally hydrated throughout the study period. Serum furosemide peak concentrations (1 hr after administration) were measured in eight newborns, and trough levels in four newborns, by HPLC. The mean 1-hr serum furosemide level increased with repeated doses of the drug, from 3.18 +/- 0.52 to 7.95 +/- 2.36 mg/liter (mean +/- SE) (p less than .05). In contrast, the mean serum trough concentration of furosemide remained at a plateau following the first dose (1.8 +/- 0.82 to 1.98 +/- 1.81 mg/liter), suggesting attainment of a steady state. This accumulation of furosemide following repeated doses of this diuretic, therefore, reflects the decreased plasma clearance of the drug, possibly due to contraction of its volume of distribution.

Serum protein binding of furosemide in newborn infants and children.

The protein binding of furosemide was studied in the serum from 8 umbilical cords, in 51 children (aged between 2 weeks and 13.5 years) and in the plasma of 10 volunteers (aged between 28 and 42 years). The drug was added to the buffer to give a final concentration of 2 micrograms/ml. The unbound fraction of furosemide was 2.5 +/- 0.1% (cord serum) and 1.7 +/- 0.7% (adult plasma). These figures are different at a level of 0.001. The unbound fraction of furosemide reached the adult values during the 1st year of life. A correlation (level of significance greater than 0.01) was found between the unbound fraction and the age during the first 6 months of life. The furosemide binding kinetics were studied in 3 cord serum and in 3 adult plasma samples. The concentration of the drug in the buffer ranged between 1 and 16 micrograms/ml. The kinetic constants (mean +/- SEM) were: association constant (K = 10(5) M-1) 2.4 +/- 0.3 (cord serum), 2.0 +/- 0.2 (adult plasma); the number of binding sites per gram protein (n = 10(-6] was 3.2 +/- 0.5 (cord serum) and 3.9 +/- 0.7 (adult plasma). When the concentration of furosemide was increased up to 200 micrograms/ml buffer, the free fraction of the drug increased up to 4.8 +/- 0.2% (cord serum) and 2.9 +/- 0.4% (adult plasma).

Plasma protein binding of furosemide in the elderly.

The protein binding of furosemide was investigated in plasma from 22 old and 11 young subjects by equilibrium dialysis. The unbound fraction of furosemide was 3.16% in plasma from the elderly and 1.7% in plasma from the young. A significant correlation was found between the unbound fraction of furosemide and the plasma concentration of albumin. The average number of binding sites was 3.8 (elderly) and 2.7 (young) 10(-6) mol/g albumin. The average association constant (K) was 4.3 (elderly) and 4.2 (young) 10(5) M-1. By increasing the concentration of furosemide up to 200 micrograms/ml buffer the unbound fraction of the drug rose to 5.2% (elderly) and 3.5% (young).

Effect of chloral hydrate on renal excretion of furosemide in rabbits

The mechanism of interaction of furosemide (FS) with chloral hydrate (CH) was investigated in rabbits. The co-administration of CH markedly enhanced the plasma concentration of FS, and reduced the amount of cumulative urinary excretion of FS after intravenous bolus injection. Furthermore, the co-administration of CH was found to significantly decrease the renal clearance (Clr) of FS, but to have no effect on its extrarenal clearance (Clnr). These clearly indicate that the interaction of FS with CH is generated during renal excretion. Interestingly, the co-administration of CH reduced the amount of cumulative urinary excretion of phenolsulfonphthalein, which is excreted exclusively by tubular secretion, whereas it did not affect the amount of cumulative urinary excretion of inulin, which is excreted exclusively by glomerular filtration. The co-administration of CH caused no change in urinary pH after intravenous bolus injection of FS. These findings suggest that the co-administration of CH may depress the renal excretion of FS by inhibiting the tubular secretion.

Furosemide inhibits glucose transport in isolated rat adipocytes via direct inactivation of carrier proteins.

Furosemide inhibits 3-O-methyl-D-glucose equilibrium flux in isolated adipocytes. The inhibition is saturable with an increasing concentration of furosemide and shows a noncompetitive type of kinetics. Both basal and insulin-stimulated fluxes are equally affected by the inhibition. Hydrochlorothiazide and piretanide also inhibit the flux with a similar potency, whereas bumetanide, a more potent diuretic, is much less potent. To understand the molecular basis of this inhibition, effects of furosemide on the glucose-sensitive cytochaslasin B binding activities of adipocytes were studied. Furosemide inhibits the glucose-sensitive cytochalasin B binding of both microsomal and plasma membrane preparations. For both preparations, the inhibition is time dependent and only slowly reversible, is saturable with an increasing concentration of furosemide, shows a noncompetitive type of kinetics with apparent Ki (the inhibitor concentration that gives the half-maximum effect) of 3.5 and 0.7 mM after 2 and 18 h incubation, respectively, and is essentially identical between the basal and insulin-stimulated adipocytes. The inhibition develops with a first-order rate constant of approximately 0.12/h at 4 degrees C. These results indicate that furosemide inhibits glucose transport in adipocytes by directly inactivating transport carriers of both plasma membranes and microsomal reserve pool. This inactivation of glucose carrier may play a part in the diuretic-induced glucose intolerance frequently observed during diuretic therapy.

High concentrations of furosemide inhibit serum binding of thyroxine.

Serum samples taken from four patients who had low serum T4 concentrations (less than 2 micrograms/dl) during severe non-thyroidal illness were found to contain a heat-stable, dialyzable inhibitor of 125I T4 binding to plasma proteins. Inhibitory activity coincided with high dose furosemide treatment for oliguric renal failure. Inhibition was proportional to the serum furosemide concentration and the effect was reproduced in vitro by addition of furosemide to normal serum. The inhibitory effect diminished with serum dilution while maintaining the same relative concentration of furosemide. A time-course study in one patient demonstrated a close temporal relationship between high serum concentrations of furosemide and subnormal T4, associated with T3 resin uptake values compatible with increased occupancy of T4-binding globulin by a competitor. These findings demonstrate that furosemide in high concentrations can inhibit T4 binding in plasma and may be a factor contributing to the development of the low T4 state in critical illness.

Pharmacokinetic and pharmacodynamic study of the combination of furosemide retard and triamterene

The pharmacodynamics and pharmacokinetics of the combination of furosemide retard (30 mg)/triamterene (50 mg) were compared with furosemide (30 mg) in 18 healthy male volunteers aged 39.3 +/- 6.3 years. After the administration of furosemide the onset of its effect was very rapid, reaching a maximum between 1.5 to 3 h, and followed by rebound after 9 to 10.5 h. In contrast the combination furosemide retard/triamterene showed a protracted course with a duration of effect up to 12 h. The general effect over 12h of the two preparations was equivalent with respect to the excretion of urine, sodium, chloride and calcium, but the combination caused significantly less excretion of potassium (p less than or equal to 0.05) than furosemide. After a lag-phase of 33.9 +/- 5.4 min the maximum plasma concentration of furosemide was reached after 3.47 +/- 0.66 h, and the elimination half-life was approximately 2 h. After a lag-phase of 33.0 +/- 17.8 min the maximum plasma concentration of the main metabolite of triamterene, the OH-TA sulphuric acid ester, was reached after 1.7 +/- 0.59 h, and its elimination half-life amounted to 1.25 +/- 0.37 h. Because of the sustained release of furosemide from the retard-formulation, its principal pharmacokinetic parameters were better adapted to those of triamterene. The consequences were not only a protracted effect but also an improved electrolyte profile, especially with regard to reduced loss of potassium. In the case of renal insufficiency, however, the potassium level in serum might be increased to an undesirable extent.

Sodium-lithium exchange and sodium-potassium cotransport in human erythrocytes. Part 1: Evaluation of a simple uptake test to assess the activity of the two transport systems.

A reliable and simple uptake test is proposed to assess the activity of the sodium lithium (Na+-Li+) countertransport and the sodium-potassium (Na+-K+) cotransport system of human erythrocytes. The test consists in measurement of phloretin-sensitive Li+ uptake from magnesium (Mg2+) media containing 2 mM Li+ and of furosemide-sensitive rubidium (Rb+) uptake from Na+ media containing 5 mM Rb+. Both Li+ and Rb+ are determined by atomic absorption spectrophotometry. The particular experimental conditions of the uptake assay are analyzed with respect to parameters that potentially affect the results, such as pH, cell volume, internal Na+, external Na+ and Rb+, presence of other ions, and concentration and protein-binding of furosemide, respectively. It is found that the activity of the two transport systems varies more than threefold among normotensive individuals, the activities in erythrocytes of each individual donor remaining stable within +/- 10% over more than two red cell life spans. The Li+ and Rb+ uptake rates are highly correlated to Li+ and K+ or Na+ release rates as determined by the methods of Canessa et al. and Garay et al. The test can thus be applied for routine screening in the search of "abnormalities" of red cell cation transport in hypertensive patients.

The use of HPLC to elucidate the metabolism and urinary excretion of furosemide and its metabolic products.

Three principles for the use of HPLC with spectrophotometric detection to determine the concentration of furosemide and of 4-chloro-5-sulfamoyl anthranilic acid (CSA) were studied. A reversed phase microbondapack C18 column was used for the separation of either unchanged furosemide (I), CSA (II) or the diazo product of CSA (III). The sensitivity of the methods for the determination of furosemide added to serum in vitro were for I 0.05, for II 0.020 and for III 0.01 microgram/ml. The methods I and II were used to study the excretion pattern in the urine of furosemide and CSA after intravenous injection of 80 mg to 10 normal subjects. An average of 48.1 +/- 9.8% (S.D.) of the furosemide was excreted during the first hour as unchanged furosemide. Simultaneously 1.9 +/- 0.8% of the dose was excreted as CSA. Only very low concentrations of CSA were found in serum and that metabolite apparently was excreted with a higher renal clearance than furosemide during the period 30-60 min. after the administration (30.3 +/- 145 ml/min. for CSA and 88 +/- 23 ml/min. for furosemide). During the 24 hour period following the administration 52.6 +/- 8.1 mg was excreted as unchanged furosemide and 11.4 +/- 5.2 mg as a glucuronidated product.

Labyrinthine Barriers and Cochlear Homeostasis

The blood-labyrinth barrier is a concept used to explain the stability of composition of the labyrinthine fluids in spite of systemic alterations in blood composition. This blood-labyrinth barrier concept was tested by injecting various test substances into the systemic circulation of experimental animals and recovering these compounds in perilymph. The concentration of each test substance in perilymph lagged behind that of serum, and the transport of a series of test substances was found to be inversely related to the molecular weight and diameter. Among the osmotic agents injected, glycerol and urea penetrated into perilymph to a considerable degree, however, mannitol did not enter perilymph in any significant amount. This may explain the clinical differences noted with these agents in testing for Meniere's disease. Furosemide, an ototoxic diuretic, was found to penetrate into perilymph after intravenous injection into chinchillas. The concentration of furosemide, measured by high pressure liquid chromatography, was fairly constant at the time of full recovery of endocochlear potential after doses of 50-200 mg/kg. The principle of correlating drug concentration in serum and in inner ear fluids with pathophysiologic effect may provide a prediction of threshold concentration of ototoxic effect by measurement of serum concentration of the drug. More extensive studies are necessary to clarify the role of the blood-labyrinth barrier in the regulatory mechanisms which maintain the homeostasis in the inner ear and the pathology which may follow when this homeostasis is disrupted.

Renal tubular secretion and effects of furosemide

Continuous intravenous infusion of furosemide (8 mg/hr) to 6 healthy subjects induced an average diuresis at steady state of 667 +/- 144 ml/30 min (+/- SD) with a mean plasma concentration of furosemide of 623 +/- 209 ng/ml. The urinary output of Cl- was 50.4 +/- 7.5, of Na+ 47.7 +/- 8.7, and of K+ 5.4 +/- 0.6 mmole/30 min. Intravenous injection of probenecid (1 gm) raised the plasma furosemide level to a maximum of 1,584 +/- 151 ng/ml. Despite this, the urinary excretion of water, Cl-, Na+, and K+ decreased to 52%, 39%, 39%, and 52%, respectively, of control values. Probenecid greatly reduced the urinary excretion and renal clearance of furosemide. There was no or negative correlation between the plasma levels of furosemide and its diuretic and saluretic effects. The urinary excretion and renal clearance of the diuretic correlated positively with these effects. No effect of probenecid on protein binding of furosemide was detected. The findings show that the diuretic effects of furosemide depend on active tubular secretion of the drug and thus on its tubular fluid concentration.

Elimination kinetics of furosemide in perilymph and serum of the chinchilla. Neuropharmacologic correlates.

This study was done to determine the comparative elimination kinetics of furosemide from chinchilla perilymph and serum, and to correlate perilymph concentration with changes in endocochlear potential. The elimination kinetics of furosemide (FU) were determined in sera and perilymph obtained from chinchillas injected with 100 mg/kg i.v. of FU. Concentrations of FU exhibited a linear decay pattern in serum and perilymph over the initial 60 minutes. The rate of decline of furosemide levels in perilymph was about four times slower than the rate of fall in serum. Chronic treatment (25 mg/kg i.p. every 12 hours) did not appear to influence the level of drug at 60 minutes after a dose of FU (100 mg/kg IV). Chinchillas were also studied following doses of FU ranging from 25--200 mg/kg i.v. to see the effect on endocochlear potential (EP). A positive correlation was found between FU dosage, the maximum millivolt reduction of EP and the time to initiation of recovery of EP. The perilymph concentration of furosemide when the EP began to recover was 5 microgram/ml (1.5 x 10(-5) M). Knowledge of furosemide kinetics may ultimately be applied to prevent ototoxicity in patients.

The lack of an effect of furosemide on uterine prostaglandin metabolism [formula omitted

We determined the effect of 2 mg/kg intravenous furosemide on the production and metabolism of prostaglandin E 2 in the utero-placental unit of pregnant dogs. Uterine venous prostaglandins E 2 and 15-keto-13,14-dihydro E 2 were measured by gas chromatography-mass spectrometry. Even though the dose of furosemide was adequate to effect a good diuresis, neither the production nor the metabolism of prostaglandin E 2 by the uterus was altered by that dose of the drug. Using radioactive microspheres to measure hemodynamic parameters, we observed no change in uterine vascular resistance while renal vascular resistance decreased. Although the renal concentration of furosemide may be higher than the uteroplacental concentration, there is so far no evidence in vivo that usual doses of furosemide enhance the production or inhibit the metabolism of prostaglandin E 2.

Distribution, elimination and natriuretic effect of furosemide in patients with severe arterial hypertension

Furosemide 40 mg was injected intravenously in 7 patients with severe hypertension and vascular complications. A two compartment, open model was used to describe the disappearance of the drug from serum. The mean serum clearance (Cls=1.83 ml/min · kg) was significantly reduced compared to the mean Cls-value of a group of normals (2.96 ml/min · kg). A significant correlation was found between Cls and mean blood pressure, as well as between Cls and renal clearance (mean Clr=0.83 ml/min · kg); extrapolation of the regression line yielded a Cls-value of 50 ml/min for Clr=0. The Clr was also significantly negatively correlated with mean blood pressure. Protein binding of furosemide was normal, except in one patient, who had considerable impairment of renal function. Apparently more than 90% of unchanged furosemide passed in urine was excreted by tubular transport. A highly significant negative correlation was found between Cls and the fraction of furosemide excreted as a glucuronide. During the first two hours, significantly less sodium was excreted by the patients than by a comparable group of normal subjects. The correlation between serum concentration of furosemide and the amount excreted of sodium was not significant, but highly significant correlations were found between the amounts of furosemide and sodium excreted by the kidney in 0–30 and 0–60 min. In all the individual patients an approximately linear relationship with wide variation in the slope was found between the cumulative excretion of furosemide and sodium from 0–30 min to 0–60 and to 0–120 min. After 120 min deviations were observed in the curves from 4 of the patients, which indicated that smaller and smaller additional amounts of sodium were excreted with constant additional amounts of furosemide.

Diuretic effect of furosemide in acute glomerulonephritis

Twenty-five children with the diagnosis of acute glomerulonephritis were treated with oral or intravenous doses of furosemide. The intravenous administration of 1 mg/kg or greater resulted in an increase in urine volume in all patients. Oral doses of less than 2 mg/kg were not as effective, but there was wide variation in diuretic response to the drug. In 13 patients, plasma concentrations of furosemide were measured. The plasma half-life varied from 2.3 to 4.4 hours after intravenous administration of the drug. The plasma concentration of furosemide did not correlate with diuretic response.

Furosemide accumulation by renal tissue

Furosemide accumulation by rabbit kidney cortical slices increased with incubation time, reaching a plateau by 60–90 min. Furosemide uptake by kidney slices from rabbits, dogs, mice and rats was inhibited by dinitrophenol, ouabain and probenecid, and was decreased by incubation at 0°, anoxia or the use of sodium-free medium. Furosemide slice/medium ratios decreased with increasing concentration of furosemide in the incubation medium. Addition of furosemide to medium containing rat kidney slices and either p-aminohippurate (PAH) or N-methylnicotinamide (NMN) produced a dose-related inhibition of PAH, but not NMN, accumulation. It is concluded that furosemide is actively transported by the renal organic anion transport system.