Analytical performance of a monoclonal digoxin assay with increased specificity on the ACS:180.

Digoxin metabolites cross-react in the Ciba Corning ACS digoxin assay in proportion to their bioactivity, but have greater (near 100%) cross-reactivity in the Abbott TDx, Baxter Stratus, and Ciba Corning Magic RIA digoxin assays. We studied the analytical performance of the ACS digoxin assay and compared it with these other assays. Coefficients of variation ranged from 5.5% at 3.11 ng/ml to 8.8% at 0.57 ng/ml. Mean analytical recovery was 96.4%. Results on dilutions were linear in the range of 0.6-5.0 ng/ml. We observed no interference by hemoglobin, bilirubin, or triglycerides. Dihydrodigoxin and digitoxin had lower cross-reactivity in the ACS and Stratus assays than in the TDx and Magic assays. Digoxin-like immunoreactive factor (DLIF) in patients' sera was not detected in the ACS assay but was in the TDx, Stratus, and Magic assays. Digibind therapy seemingly did not affect digoxin results by ACS or Stratus, but did for up to 10 days after therapy for TDx and Magic. We compared digoxin results for 121 sera from 49 patients. Deming regression analysis was performed on the first specimen from each patient: ACS = 1.08(TDx)-0.17 ng/ml (r = 0.961, Sy,x = 0.164); ACS = 1.16(Stratus)-0.46 ng/ml (r = 0.973, Sy,x = 0.123); ACS = 1.00(Magic)-0.20 ng/ml (r = 0.982, Sy,x = 0.110). Discrepant results (> 2Sy,x from the regression line) were usually lower by the ACS assay (87%). Nine of 11 patients with discrepant results had renal insufficiency or hepatic disease, conditions commonly associated with increased DLIF. These observations may be explained by the improved specificity of the ACS digoxin assay.

Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity.

Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.

Phosphate transport adaptation in rat jejunum and plasma level of 1,25-dihydroxyvitamin D3.

Intestinal absorption of inorganic phosphate (Pi) increases in response to a reduction in the dietary supply of Pi. In this work this adaptive response has been characterized in jejunal brush border membrane vesicles and studied in temporal relationship with the change in the plasma level of 1,25(OH)2D3. The results indicate that in rat jejunal brush border membrane vesicles the activity of the sodium-dependent Pi transport system is stimulated by a low Pi diet. This adaptive response was the result of an increase in the Vmax and a reduction in the Km of the cotransport system. This change in Pi transport was correlated with an increase in the circulating level of 1,25(OH)2D3 in a time-related fashion. In conclusion, these results are consistent with the notion that Pi restriction leads to an increase in Pi transport activity in the luminal membrane of the intestine. A time course study suggests that the elevation in plasma 1,25(OH)2D3 might be involved in the adaptation of the intestinal Pi transport system to Pi restriction.

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers.

Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of 45Ca2+ efflux profiles. The 45Ca2+ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min-1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min-1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm-2 sec-1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.

Adaptation of phosphate transport to low phosphate diet in renal and intestinal brush border membrane vesicles: influence of sodium and pH.

The possible role of changes in the sodium (Na) affinity of the carrier for inorganic phosphate (Pi) in the adaptation of Pi transport to low Pi diet was examined in both renal and intestinal brush border membranes vesicles (BBMV) obtained from the same animal. This role was assessed by measuring the Na concentration resulting in half maximal activation of Pi transport (K0.5 Na) in renal and intestinal BBMV prepared from animals adapted to either low (LPD) or high (HPD) phosphorus diet for 7 days. The K0.5 Na was not modified by dietary Pi, in both renal and intestinal BBMV. LPD increased maximal Pi transport from 1794.8 +/- 198.0 to 2964.0 +/- 362.0 in renal and from 28.2 +/- 3.4 to 80.5 +/- 7.2 pmol/mg 10 s in intestinal BBMV. For both LPD and HPD lowering pH from 7.4 to 6 dramatically increased K0.5 Na in renal and intestinal BBMV. As compared to pH 7.4, it was enhanced by approximately 200% in both renal and intestinal membranes. This change of Na affinity with acidic pH prevented the expression of Pi transport adaptation at 100 mM Na concentration. However, at saturating Na concentrations (500 mM for renal, 300 mM for intestinal membranes), Pi transport adaptation was equally expressed at pH 6 and 7.4 in both types of membranes. Hill coefficient analysis indicates a 2:1 stoichiometry of Na to Pi in renal and intestinal membranes isolated from high or low Pi diet animals. This ratio was not modified by changes of the medium pH.

Continuous measurement of calcium influx in mammalian nonmyelinated nerve fibers: effects of Nao, Cao, and electrical activity.

A new technique for continuous monitoring of the cellular calcium was developed and used for studying the effects of external and internal Na (Nao and Nai), external Ca (Cao), Ca ionophore A23187, and electrical activity on membrane-bound and intracellular Ca in mammalian nonmyelinated nerve fibers. Increasing Cao increased both the membrane-bound and the intracellular Ca. Lowering Nao increased the membrane-bound fraction of Ca indicating that lack of Nao enhanced the capacity of the plasma membrane to bind Ca, and produced an increase of the internal Ca pool. Increasing Nai by treatment with ouabain enhanced the Ca inflow in both, the presence and absence of Nao, presumably by stimulating the Cao/Nai exchange. The Ca ionophore A23187 produced a large and irreversible increase in the intracellular Ca without affecting the membrane-bound fraction. On the other hand, electrical activity, which is known to produce a large increase of the total Ca in squid axon, had no measurable effect on the total calcium content in our preparation. It is concluded that in mammalian nerve fibers a Ca load by exposition to Na-free solution or to A23187 produces an accumulation of Ca into the intracellular Ca stores, whereas during electrical activity the membrane-associated extrusion mechanisms are able to maintain the intracellular Ca2+ below the threshold for intracellular sequestration. Furthermore, the results indicate that the intracellular sequestration mechanisms are dependent on the internal concentration of Na.

Release of adenosine, inosine and hypoxanthine from rabbit non-myelinated nerve fibres at rest and during activity.

The composition of the efflux from desheathed rabbit vagus nerve, loaded with radioactivity by incubation in [3H]adenosine, was studied at rest and during electrical activity and after application of inhibitors of ecto-enzymes and modifications of intermediary metabolism. In addition, the degradation of externally applied ATP and adenosine was examined. [3H]ATP applied to the incubation medium was degraded to ADP, AMP, adenosine and inosine. The hydrolysis to nucleosides was inhibited by alpha, beta-methylene ADP; the appearance of AMP and nucleosides was slowed by beta, gamma-methylene ATP. Deamination of [3H]adenosine was blocked by 2-deoxycoformycin. The effluent from resting and stimulated preparations showed the presence of large amounts of inosine and hypoxanthine, smaller amounts of adenosine and adenine and traces of nucleotides. The composition of the effluent was not significantly altered by addition of alpha, beta-methylene ADP; beta, gamma-methylene ATP or 2-deoxycoformycin. Application of glucose-free solutions caused a large release of adenosine instead of inosine and hypoxanthine and a small increase in resting and stimulated efflux of 3H. Addition of 2-deoxyglucose produced a large increase in resting efflux and increased liberation of adenosine. Cyanide, 2,4-dinitrophenol, arsenate or salicylate increased the resting efflux of adenosine, inosine and hypoxanthine, and the effect of activity. It is concluded that electrical activity leads to release of adenosine, inosine and hypoxanthine, in various proportions depending on metabolic state, and that there is practically no liberation of nucleotides from nerve axons.

Effect of pH on phosphate transport into intestinal brush-border membrane vesicles.

The effect of pH on the rate of phosphate (Pi) uptake was studied in rabbit duodenal brush-border membrane vesicles. Pi uptake was found to be sodium dependent at all pH values tested (5.7-8.1). Further, the rate of Pi uptake depended on pH; for instance, with 100 mM external sodium, reducing the pH from 8.1 to 6.8 or 5.7 doubled the rate of Pi influx. At 100 mM external sodium, experiments under initial rate conditions, carried out with varying Pi concentrations and at pH values of 6, 6.8, or 7.6, showed that sodium-dependent Pi uptake was saturable at the three pH values tested; the apparent Km expressed in function of total Pi was not dependent on pH. Vmax was not affected between pH 6 and 6.8 but was significantly reduced at pH 7.6. Lowering external sodium lowered Vmax at all pH values investigated. At acid and alkaline pH the rate of Pi uptake was a sigmoidal function of the external sodium concentration. Hill coefficients, calculated from these experiments, exceeded unity and were unaffected by pH. At saturating sodium concentrations, the rate of Pi uptake was higher at pH 6 than at pH 7.6. The [Na]0.5 was lower at pH 7.6 than at pH 6. Further, sodium-dependent Pi uptake appeared to be electrogenic at acid and alkaline pH. It is concluded that the pH dependence of intestinal Pi transport is not an expression of preferential transport of monovalent or divalent phosphate. The pH dependence appears to reflect properties of the sodium-phosphate cotransport mechanism and is in part related to changes in the affinity of the transport system for sodium.

Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers.

Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate . The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using 45Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2 mM calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.

Uptake of adenosine and release of adenine derivatives in mammalian non-myelinated nerve fibres at rest and during activity.

1. Influx of adenosine into rabbit non-myelinated nerve fibres was measured using [2-(3)H]adenosine. The uptake of radioactivity increased linearly with duration of incubation for up to 60 min and adenosine concentration up to 200 mum. The uptake at different adenosine concentrations showed a saturable component with a half-maximal activation at 17.1 mum and a linear part.2. The radioactivity taken up was rapidly incorporated into AMP, ADP and ATP. Isotopic equilibrium between the nucleotides was achieved within 15 min.3. The uptake of (3)H from 0.2 mum-adenosine was almost completely inhibited by addition of 200 mum-adenosine and to a similar extent by 200 mum-tubercidin and AMP; a 70% inhibition was found with ATP and ADP; alpha, beta methylene-ADP had no effect.4. ATP, ADP and AMP added to the extracellular medium of a desheathed vagus were slowly hydrolysed.5. In preparations loaded with [2-(3)H]adenosine and then washed with adenosine and label-free solution there was a steady efflux of radioactivity amounting to 0.18 x 10(-3)/min. Addition of adenosine or tubercidin transiently increased the efflux.6. Electrical stimulation caused an extra release of radioactivity. The extra fractional loss was 21.8 x 10(-6)/impulse in preparations that had rested for several hours; it decreased to 2.3 x 10(-6)/impulse when stimulation was applied after a 30 min rest.7. The radioactivity of the resting efflux and of the extra efflux after stimulation was found mostly in inosine and hypoxanthine; adenosine and adenine accounted for only 3%, and the nucleotides for less than 1% of the efflux.8. Adenosine added to the external medium of a desheathed nerve was slowly deaminated.9. It is concluded that inosine and hypoxanthine found in the effluent from desheathed vagus nerve trunk result from release of these compounds from nerve fibres and not from extracellular breakdown of released ATP or adenosine.10. Electrical activity in non-myelinated nerve fibres of the nerve trunk thus causes the release of metabolites (inosine and hypoxanthine) together with small amounts of adenosine and adenine, while release of ATP and other nucleotides is almost completely absent.

Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers.

Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9 mM, calcium and without phosphate, the fractional rate of loss was 1.62 X 10(-3) min-1 at 120 min after the beginning of the washing period and fell slowly (9% hr-1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50 mM calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 micro M lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that a least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.

Phosphate ion transport in rabbit brain synaptosomes.

Synaptosomes (vesicles of nerve endings) isolated from rabbit brain were studied as a model system for the uptake of inorganic phosphate. The phosphate uptake showed a sodium-dependent, saturable component with a Kt of 0.29 mM. The sodium-dependent component was larger at pH 6 than at pH 7.4 or 8. Application of potassium salts, ouabain, monensin, nigericin or FCCP decreased the uptake. The results indicate that the sodium-sensitive phosphate influx is dependent on the Na+ gradient and on the membrane potential, which might act, preferentially, on the transport of the monovalent phosphate ion.

The correction factors for sucrose gap measurements and their practical applications.

The distribution of extracellular and intracellular potential in the sucrose gap apparatus, previously established for a single fiber using the cable equations for a core conductor model (Jirounek and Straub, Biophys. J., 11:1, 1971), is obtained for a multifiber preparation. The exact equation is derived relating the true membrane potential change to the measured potential differences across the sucrose gap, the junction potentials between sucrose and physiological solution, the membrane potential in the sucrose region, and the electrical parameters of the preparation in each region of the sucrose gap. The extracellular potential distribution has been measured using a modified sucrose gap apparatus for the frog sciatic nerve and the rabbit vagus nerve. The results indicate a hyperpolarization of the preparations in the sucrose region, of 60--75 mV. The hyperpolarization is independent of the presence of junction potentials. The calculation of the correction terms in the equation relating the actual to the measured potential change is illustrated for the case of complete depolarization by KC1 on one side of the sucrose gap. The correction terms in the equation are given for various experimental conditions, and a number of nomographic charts are presented, by means of which the correction factors can be rapidly evaluated.

Regulation of Na-dependent phosphate influx across the mucosal border of duodenum by 1,25-dihydroxycholecalciferol.

Animals teated with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), at doses which decrease the renal production and/or the plasma levels of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], display a reduced net absorption of phosphate. In this study we investigated whether EHDP-treatment and administration of 1,25(OH)2D3 to EHDP-treated animals affected the phosphate influx across the mucosal border of rabbit duodenum. The initial rate of phosphate influx into mucosal cells was measured in isolated intestine. In control, untreated rabbits, the phosphate influx shows a saturable, Na-dependent component and a diffusional, Na-independent uptake. In tissue from rabbits treated for 3 days with EHDP, the phosphate influx was found to be strongly reduced. EHDP-treatment decreased the Na-dependent, carrier mediated phosphate influx in duodenum. Administration of 1,25(OH)2D3 to EHDP-treated animals reversed the reduced phosphate influx. These effects were mainly apparent through changes in the J(mc)(max) of the phosphate influx, which was decreased from 211 +/- 38.7 nmol/cm2h in controls to 42.1 +/- 18.1 nmole/cm2 h in the EHDP-treated group and increased to 413 +/- 43.6 nmole/cm2 h by 1,25(OH)2D3. The treatment did not appear to affect the diffusional, Na-independent phosphate influx. EHDP-treatment did not affect the influx of alanine in this segment suggesting that EHDP-treatment affects only 1,25(OH)2D3-dependent transport mechanisms. The results suggest that 1,25(OH)2D3 modulates the number of carrier sites available at the mucosal membrane for Na-dependent phosphate entry.

Oxygen consumption and phosphate efflux in mammalian non-myelinated nerve fibres.

1. A comparison has been made between the efflux of labelled phosphate from the non-myelinated fibres of the desheathed rabbit vagus nerve at 37 degrees C and the corresponding O2 consumption at rest and during activity, and during a variety of experimental interventions. 2. The resting rate constant of phosphate efflux was 2.61 X 10(-3) min-1: electrical stimulation (10 sec-1, 3 min) produced an extra fractional loss of 6.75 X 10(-6) impulse-1. 3. The corresponding resting O2 consumption was 0.484 m-mole x kg-1 impulse-1. 4. Ouabain (100 microM) produced a sustained depression (of about 40%) of the resting O2 consumption, accompanied by a transient fall (of about 14%) in the rate constant of phosphate efflux. 5. Na salicylate (10 mM) or Na arsenate (1 mM) produced a much larger increase in phosphate efflux than in resting O2 consumption. 6. Changing the external phosphate concentration (between 0.02 and 2 mM), addition of acetylcholine (1.7 mM), and addition of lanthanum (20 microM)--all of which are known to affect markedly the phosphate efflux in rabbit non-myelinated fibres--had little or no effect on the resting O2 consumption or, where tested, on the extra O2 consumption with electrical stimulation. 7. Changing the external Ca concentration (between 0.09 and 9 mM) had only minor effects on the O2 consumption (resting and stimulated) and on the rate constant of resting phosphate efflux. 8. It is concluded that although changes in metabolism of the nerve produce changes in the phosphate efflux expected on the basis of the concomitant changes in the internal concentration of inorganic phosphate, the converse is not true; and increases and decreases in the rate constant of phosphate efflux do not necessarily signal the corresponding metabolic changes.

Observations on the mechanism for the active extrusion of lithium in mammalian non-myelinated nerve fibres.

1. A study has been made of the O2 consumption, and the corresponding efflux of labelled phosphate, from the non-myelinated fibres of the desheathed rabbit vagus nerve at 37 degrees C in Locke solutions in which various ions were substituted for Na, and also in the presence of ouabain. 2. Switching from Na-Locke solution to Li-Locke solution produced a small transient decrease in the resting O2 consumption (of 14%), which rapidly recovered to its original value. This was accompanied by an initial brief rise followed by a maintained fall in the resting phosphate efflux. 3. In Li-Locke solution, ouabain (100 microM) produced a fall in the resting O2 consumption of 40%, i.e. similar to that produced in Na-Locke solution. Any depression of the resting phosphate efflux was absent or small. 4. In choline-Locke solution, in Tris-Locke solution, in K-Locke solution or in sucrose-Locke solution the resting O2 consumption, which fell by 30-40%, was insensitive to the addition of ouabain (100 microM). 5. Addition of either Na ions or of Li ions partially restored respiration in choline-Locke solution, Li being an order of magnitude less effective than Na. 6. In choline-Locke solution the internal K content was not affected by ouabain. However, if Li (77 mM) was present in the bathing solution ouabain (100 microM) produced a 30% fall in the internal K content. 7. It is concluded that these effects of Li, and their alteration by ouabain, reflect the activity of a mechanism for the active extrusion of Li ions. It is suggested that the mechanism for the active extrusion of Li is the same as that for Na. 8. There also seems to be a site for Li that controls the phosphate efflux and which is half-maximally activated with external Li concentrations of about 2-4 mM.

Release of inorganic phosphate during activity in mammalian non-myelinated nerve fibres.

1. The efflux of labelled phosphate was measured in desheathed rabbit vagus nerve at rest and during activity.2. In solutions with 2 mM-phosphate and 1 mM-K the rate constant of the resting efflux was 2.7 x 10(-3) min(-1); stimulation caused an extra fractional loss of 2.8 x 10(-6) impulse(-1).3. Lowering the phosphate concentration decreased the resting and the stimulated efflux; with 0.2 mM-phosphate the corresponding values were 1.9 x 10(-3) min(-1) and 1.8 x 10(-6) impulse(-1), respectively.4. Increasing the K to 5.6 mM decreased both resting and stimulated efflux.5. Lowering the temperature decreased the resting efflux with a Q(10) of 2.9 and the stimulated efflux with a Q(10) of 8.1.6. Chromatography of the effluent showed that at rest and during activity at least 96% of the radiophosphate was in the orthophosphate fraction.7. Replacing the Na of the solution by Li lowered the rate constant of the resting efflux to 0.8 x 10(-3) min(-1) and abolished the extra release during activity, without reduction of the action potential.8. The presence of ouabain did not affect the resting efflux, except at 100 muM, when a transient reduction was found. The extra fractional loss was not affected with 0.001 muM; with 0.01-0.5 muM, it was reduced without much change in the action potential, and abolished at higher concentrations.9. The results agree with the hypothesis that the extra release results from an increase in internal inorganic phosphate caused by increased break-down of ATP during recovery.10. Comparison with the O(2) consumption shows that about 1% of the inorganic phosphate liberated at the inside of the axons escapes to the outside.