Acoustic properties of NC100100 and their relation with the microbubble size distribution.

RATIONALE AND OBJECTIVES: NC100100 is a contrast agent for medical imaging with ultrasonography consisting of stabilized gas microbubbles in an aqueous suspension. The objective of this article is to explore the acoustic properties of NC100100 and their relation with the microbubble size distribution. The results are used to motivate the choice of a suitable assay/dosage parameter for precise control of product efficacy. METHODS: The concentration and size distribution of microbubbles in > 50 preparations of NC100100 were determined by Coulter counting, and the acoustic attenuation and backscatter efficacy were determined for all samples. The in vivo efficacy of the product was investigated by harmonic imaging of the heart in a dog model. RESULTS: The results demonstrated that the attenuation and backscatter efficacy per microbubble volume vary strongly with size, showing distinct maxima with respect to microbubble diameter. Sizes for optimal attenuation per volume ranged from 2.6 to 5.8 microns, depending on ultrasound frequency. The contribution of the smaller end tail of the microbubble distribution was shown to be negligible. From the observed size dependency for the acoustic properties, the volume concentration of microbubbles was chosen as the assay/dosage parameter for NC100100. The accuracy of this parameter as a descriptor of product efficacy was demonstrated by precise, linear relations between volume, concentration, and attenuation/backscatter. In comparison, the correlation between the microbubble number and acoustic properties was not significant. Results from the in vivo study showed a precise, linear relation between injected microbubble volume and the observed in vivo efficacy. CONCLUSIONS: The acoustic properties of NC100100 are dependent on microbubble size. The observed batch-to-batch variance in the acoustic properties of the product may be fully explained by variation in concentration and size. Microbubble volume is a more precise predictor of in vitro/in vivo efficacy than microbubble number and consequently was chosen as the assay/dosage parameter for NC100100.

Atrial natriuretic factor and renal sodium excretion during ventilation with PEEP in hypervolemic dogs.

Controlled mandatory ventilation with positive end-expiratory pressure (PEEP) reduces renal sodium excretion. To examine whether atrial natriuretic factor (ANF) is involved in the renal response to alterations in end-expiratory pressure in hypervolemic dogs, experiments were performed on anesthetized dogs with increased blood volume. Changing from PEEP to zero end-expiratory pressure (ZEEP) increased sodium excretion by 145 +/- 61 from 310 +/- 61 mumol/min and increased plasma immunoreactive (ir) ANF by 104 +/- 27 from 136 +/- 21 pg/ml. Changing from ZEEP to PEEP reduced sodium excretion by 136 +/- 36 mumol/min and reduced plasma irANF by 98 +/- 22 pg/ml. To examine a possible causal relationship, ANF (6 ng.min-1.kg body wt-1) was infused intravenously during PEEP to raise plasma irANF to the same level as during ZEEP. Sodium excretion increased by 80 +/- 36 from 290 +/- 78 mumol/min as plasma irANF increased by 96 +/- 28 from 148 +/- 28 pg/ml. We conclude that alterations in end-expiratory pressure lead to great changes in plasma irANF and sodium excretion in dogs with increased blood volume. Comparison of the effects of altering end-expiratory pressure and infusing ANF indicates that a substantial part of the changes in sodium excretion during variations in end-expiratory pressure can be attributed to changes in plasma irANF.

Quantitative 3-dimensional contrast echocardiographic determination of myocardial mass at risk and residual infarct mass after reperfusion: experimental canine studies with intravenous contrast agent NC100100.

Two-dimensional contrast echocardiography has been shown to enable the evaluation of myocardial perfusion abnormalities. However, its ability to quantify a regional myocardial mass is limited. The goal of this study was to examine the quantitative value of 3-dimensional echocardiography (3DE) in the estimation of myocardial mass at risk, salvaged mass, and residual infarct mass after intravenous injection of contrast. We created acute coronary occlusion, followed by reperfusion in 10 dogs. Three-dimensional echocardiographic data were acquired at the end of each stage, and the perfusion defect mass and dysfunctional mass were measured. The true mass at risk and infarct mass were determined by anatomic methods. The anatomic mass at risk (x) (27.1+/-14.6 g or 23.8%+/-9.7% of the left ventricle [%LV]) correlated well with the 3DE-determined perfusion defect mass (y) during coronary occlusion (y = 0.5x+8.9; r = 0.90; P<.001; mean difference -4.8+/-8.1 g; or y = 0.7x + 6.5; r = 0.83, P<.01; mean difference -0.1+/-5.4 %LV). Good correlation was also found between the anatomic infarct mass (x) (9.3+/-8.1 g or 9.1+/-8.8 %LV) and the 3DE perfusion defect mass after reperfusion (y) (y = 1.2x+1.2; r = 0.93; P<.001; mean difference 2.3+/-4.0 g; or y = 1. 3x, r = 0.98, P <.0001; mean difference 2.7+/-3.7 %LV). The salvaged mass was 13.6 +/-11.0 %LV from anatomic methods and 14.2+/-13.0 %LV by 3DE. To conclude, with the use of intravenous contrast, 3DE could quantify the actual mass at risk during acute ischemia, and in the setting of reperfusion, the residual infarct mass and salvaged mass.

Destruction of contrast microbubbles and the association with inertial cavitation.

The destruction of insonified Sonazoid microbubbles and its association with inertial cavitation in vitro utilizing an active acoustic detector was investigated. The experimental observation indicated that contrast microbubbles could be damaged at moderate acoustic pressures of 0.6-1.6 MPa (0.4-1.0 in mechanical index, MI). A damaged bubble could be dissolved into the medium on the order of 1 ms, implying that the destruction at moderate pressures is a relatively slow (relative to inertial bubble collapse), nonviolent dissolution process following the disruption of encapsulating surface materials. Inertial cavitation events in the presence of contrast microbubbles were observed using multiple highly intense ultrasound (US) pulses (>1.6 MPa). This observation suggested that intense US might disintegrate contrast microbubbles, and fragments of disintegrated microbubbles could be activated by an upcoming highly intense imaging pulse. The above results imply that inertial cavitation is unlikely to take place in the presence of Sonazoid contrast microbubbles when exposed to diagnostic US with an MI <1.

4-Methylpyrazole (4-MP) is effectively removed by haemodialysis in the pig model.

The safety and pharmacokinetics of the alcohol dehydrogenase inhibitor 4-MP have recently been evaluated and its clinical use in ethylene glycol poisoning in France is promising. The dialysability of 4-MP is not known. This is important as hemodialysis is one of the cornerstones in the treatment of (late) ethylene glycol and methanol poisonings. We therefore studied the dialysability of 4-MP in the pig model. Anesthesized pigs (maintained on respirator) given 4-MP, 10 mg/kg, served as controls (n = 3) to the pigs (n = 3) given 15 mg/kg and hemodialyzed for 4 h. 4-MP plasma elimination curves for both groups were compared for 12 h and dialysance data calculated. 4-MP (MW 82) was removed by the same rate as urea (MW 60) by the 0.3 m2 dialysator, thus indicating no significant protein binding of 4-MP. The mean dialysance of 4-MP (and urea) in the three pigs were 61 (63), 51 (53) and 56 (48) ml/min, at a blood flow of 75 ml/min. The amount of 4-MP removed by hemodialysis in 4 h was 57-76 mg compared to a urinary excretion of 1-3 mg over 12 h. We conclude that the dialysability of 4-MP is significant and this must be taken into account when these two treatment procedures are combined.

Analysis of reabsorbate concentrations in the proximal tubules of dog kidneys during osmotic diuresis.

To examine to what extent the reabsorbate concentrations, calculated as the flux ratios between solutes and water, represent the fluid composition in the lateral intercellular space (LIS) in the proximal tubules, reabsorption was stimulated by elevating PCO2 from 5 to 13 kPa before and during infusion of mannitol to a plasma concentration of 70 mM in volume-expanded dogs receiving ethacrynic acid. The reabsorbate concentration of NaHCO3 increased by 50 mM during mannitol infusion. The real concentration of NaHCO3 in LIS could not, however, be elevated by this amount, since the driving forces for fluid reabsorption then would have increased during osmotic diuresis due to diffusion of mannitol into LIS from plasma. A model analysis of diffusion in LIS showed that transcellular transport can only lead to trivial increases of LIS concentrations compared to plasma, whereas diffusion across tight junctions can increase LIS concentrations by several mM. NaCl diffusion and coupled transcellular water transport may therefore represent a significant contribution to total bicarbonate-dependent NaCl and water reabsorption in the proximal tubules.

Essentials of glomerulotubular balance.

In the proximal tubules, fractional reabsorption remains essentially unchanged during variations in glomerular filtration rate (GFR). Glomerulotubular balance (GTB), defined as the linear relationship between proximal tubular reabsorption and GFR, is quantitatively the most important regulator of tubular reabsorption, which may be stopped by inhibiting Na, K-ATPase activity completely. However, ouabain in doses inhibiting 80% of the Na, K-ATPases, exerts no effect on proximal reabsorption of water, NaCl and NaHCO3. At constant plasma pH, the same relationship between filtered and reabsorbed bicarbonate is obtained whether bicarbonate reabsorption is altered by varying GFR or plasma concentration of bicarbonate. In contrast, a selective rise in plasma NaCl concentration at constant plasma pH (hypernatremia) reduces NaHCO3 reabsorption and fails to stimulate NaCl reabsorption. Other characteristics of proximal tubular reabsorption are that nonreabsorbable solutes, such as mannitol, inhibit water and NaCl reabsorption with little or no change in NaHCO3 reabsorption and renal oxygen consumption. Mannitol reduces the slope of the GTB curve for NaCl but not for NaHCO3. Hypertonic NaHCO3 exerts an osmotic effect on proximal water and NaCl reabsorption comparable to that of mannitol, whereas hypertonic NaCl is without osmotic effect. By reducing plasma pH (hypercapnia at high plasma bicarbonate concentration), the slope of the GTB curves for NaCl and NaHCO3 can be greatly increased. By raising plasma pH either by hypocapnia or bicarbonate loading, proximal reabsorption of NaHCO3 and NaCl is greatly depressed and remains almost unaltered during variations of GFR (abolished GTB). Similarly, carbonic anhydrase inhibitors, such as acetazolamide, reduce the reabsorption of NaCl and NaHCO3 in the same proportion as a rise in plasma pH, and abolish GTB. Examinations of proximal tubular oxygen consumption indicate that the energy requirement for NaHCO3 reabsorption is as expected for transcellular transport by Na, K-ATPases, whereas proximal NaCl reabsorption requires no additional energy. These data indicate that transcellular energy-requiring NaHCO3 reabsorption provides the main osmotic force across the tight junction for paracellular reabsorption of proximal tubular fluid containing NaCl and other solutes of low reflection coefficient. The main factors influencing GTB are the filtered load of bicarbonate, plasma pH and nonreabsorbable solutes in the proximal tubular fluid.

How bicarbonate loading inhibits tubular reabsorption of NaCl in dog kidneys.

During continuous infusion of ethacrynic acid in dogs, changes in glomerular filtration rate (GFR) and PCO2 at constant plasma bicarbonate concentration (PHCO3) alter bicarbonate and chloride reabsorption in a ratio of 1:2. This ratio did not apply when PHCO3 was raised by bicarbonate loading in 11 anaesthetized volume-expanded dogs. A rise in PHCO3 from 30 to 54 mM at constant PCO2 and GFR reduced sodium reabsorption during ethacrynic acid infusion from 3586 +/- 725 to 2449 +/- 403 mumol min-1. Bicarbonate and chloride reabsorption were reduced in a ratio of 1:10. When plasma pH was restored from 7.8 to 7.5 by raising PCO2, the inhibitory effect on chloride reabsorption was halved. At constant plasma pH 7.5 a rise in PHCO3 from 20 to 30 mM reduced chloride reabsorption by 20%. A further 30% inhibition was caused by raising PHCO3 from 30 to 54 mM. Bicarbonate reabsorption was highest at PHCO3 54 mM, suggesting a large capacity for bicarbonate reabsorption if PHCO3 is raised at constant plasma pH 7.5. Water and NaCl reabsorption remaining during ethacrynic acid infusion is almost equally inhibited by alkalosis and by an osmotic effect of unreabsorbed NaHCO3.

Energy requirement of sodium reabsorption in the thick ascending limb of Henle's loop in the dog kidney: effects of bumetanide and ouabain.

To examine whether sodium reabsorption in the thick ascending limb of Henle's loop (TALH) in the dog kidney has a passive component, the ratios between reductions in sodium reabsorption and oxygen consumption (delta Na/delta Qo2 ratio) were measured by inhibiting tubular transport with bumetanide (30 micrograms kg-1) and ouabain (120 ng kg-1 intrarenally). Clearance studies were performed in volume expanded dogs treated with acetazolamide (100 mg kg-1) or maleate (400 mg kg-1). In five acetazolamide-treated dogs, bumetanide gave a delta Na/delta Qo2 ratio of 29.9 +/- 2.5, whereas the combination of bumetanide and ouabain gave 19.0 +/- 0.6. When ouabain was given before bumetanide, ouabain gave a delta Na/delta Qo2 ratio of 19.2 +/- 1.1 and the combination gave 19.9 +/- 1.2. In the maleate-treated dogs, bumetanide gave a delta Na/Qo2 ratio 30.3 +/- 1.7, and the combination of bumetanide and ouabain gave 27.1 +/- 1.5. To localize the metabolic effect of bumetanide and ouabain, local heat production was measured at 18 places in four kidneys with copper-constantan thermocouples. Bumetanide reduced metabolic rate in the outer medulla by 51 +/- 4%, and in the cortex by 16 +/- 6%. Subsequent infusion of ouabain reduced metabolic rate in the outer medulla by only 9 +/- 3%, whereas cortical metabolism was reduced by 33 +/- 4%. The results show that bumetanide mainly acts in the outer medullar where TALH is located, whereas the additional effect of ouabain is mainly located in cortical segment of the nephron including the proximal tubules. Bumetanide inhibits the reabsorption of 30 mol sodium for each mole oxygen consumed, which show that for each 18 mol sodium that are transported through the cells in the TALH in dog kidneys. 12 mol (40%) are transported along the paracellular route without additional requirement of energy.

Loop diuretics reduce lithium reabsorption without affecting bicarbonate and phosphate reabsorption.

The effects of the loop diuretics ethacrynic acid and bumetanide on lithium, bicarbonate and phosphate reabsorption were compared in 16 anaesthetized, normovolaemic dogs. In six dogs, ethacrynic acid (3 mg kg-1 body wt) significantly reduced absolute lithium reabsorption from 29.3 +/- 4.1 to 19.0 +/- 3.4 mumol min-1, fractional lithium reabsorption from 0.65 +/- 0.04 to 0.37 +/- 0.04 and fractional chloride reabsorption from 1.00 +/- 0.00 to 0.65 +/- 0.02. Bicarbonate and phosphate reabsorption did not decrease significantly. In six other dogs, bumetanide (30 micrograms kg-1 body wt) gave similar results. Absolute lithium reabsorption significantly decreased from 34.0 +/- 2.2 to 18.1 +/- 2.6 mumol min-1 and fractional lithium reabsorption decreased from 0.50 +/- 0.03 to 0.25 +/- 0.03. Fractional chloride reabsorption decreased from 0.98 +/- 0.00 to 0.61 +/- 0.05, whereas bicarbonate and phosphate reabsorption were not significantly altered. Thus, both loop diuretics greatly reduced lithium reabsorption. We propose that loop diuretics inhibit passive lithium reabsorption in the thick ascending limb of Henle's loop by reducing the lumen-positive electrical potential that drives passive cation transport.

Effect of maleate on tubular protein reabsorption in dog kidneys.

To examine the effects on protein and electrolyte reabsorption of reducing the energy supply to the proximal tubules, an inhibitor of the citric acid cycle, maleate (600 mg.kg-1), was administered to anesthetized dogs during continuous ethacrynic acid infusion. One hour after infusion, maleate reduced renal oxygen consumption from 128 +/- 3 to 48 +/- 6 mumol.min-1. Comparisons at similar GFR showed that maleate reduced bicarbonate reabsorption by 65%, chloride reabsorption by 60% and phosphate reabsorption by 90%. Tubular reabsorption of lysozyme, determined by the 'trapped-label' method, was reduced by 97%. Total protein excretion in urine increased from 0.12 to 1.0 mg.min-1 and was not associated with a significant increase in brush border and lysosome marker enzymes. However, by superimposing a carbonic anhydrase inhibitor, acetazolamide (100 mg.kg-1), electrolyte reabsorption was slightly further reduced but protein excretion increased to 2.7 mg.min-1, coincidentally with a dramatic increase in enzyme excretion: approximately 20-fold in the brush border enzymes, alanine aminopeptidase and alkaline phosphatase, and 10-fold in the lysosomal enzymes, acid phosphatase and N-acetyl-beta-glucosaminidase. Our data indicate that maleate stops protein reabsorption without signs of acute tubular damage, whereas subsequent administration of acetazolamide results in tubular desquamation and albumin leakage.

Dissociation between water and lithium transport during acute changes in plasma potassium concentration in dog kidney.

Lithium clearance is often used as a marker for proximal tubular water transport. Proximal tubular transport may be modulated by changing plasma potassium concentration. The aim of the present study was to examine the effect of acute changes in plasma potassium concentration on proximal tubular fluid and lithium transport. Clearance studies were performed in seven anaesthetised, volume-expanded dogs treated with amiloride (1 mg kg-1 body weight) to block distal tubular potassium secretion, and with bumetanide (30 micrograms kg-1 body weight) to inhibit sodium reabsorption in Henle's loop. When plasma potassium concentration was raised from 2.6 +/- 0.2 to 7.9 +/- 0.2 mmol l-1, water reabsorption decreased from 23.9 +/- 2.9 to 19.8 +/- 2.2 ml min-1, whereas lithium reabsorption increased from 10.5 +/- 2.3 to 18.1 +/- 2.3 mumol min-1, at constant glomerular filtration rate. We conclude that acute elevation of plasma potassium concentration inhibits proximal tubular fluid reabsorption, but stimulates renal lithium reabsorption. Thus, lithium reabsorption cannot be used as a marker for proximal tubular transport during acute changes in plasma potassium concentration.

Hypernatremia inhibits NaHCO3 reabsorption and associated NaCl reabsorption in dogs.

To examine the effect of selective rise of plasma NaCl concentration (hypernatremia) on NaHCO3 reabsorption and associated NaCl reabsorption remaining during continuous ethacrynic acid infusion, hypertonic NaCl solution was infused in three groups of anesthetized volume-expanded dogs. In six dogs examined at constant hematocrit and plasma pH, bicarbonate and water reabsorptions were inversely related to PNa and reduced by 37% and 39% respectively by raising PNa from 140 to 200 mM. Chloride reabsorption remained essentially constant until PNa exceeded 170 to 180 mM. At PNa 200 mM, sodium reabsorption was reduced by 22 +/- 6%. In six other dogs, mechanical variations of GFR showed that the inhibitory effects of hypernatremia (PNa 199 +/- 3 mM) were less pronounced at low GFR. After subsequent administration of acetazolamide (30 mg/kg body wt), only 20% of control bicarbonate reabsorption remained and glomerulo-tubular balance was completely abolished. Both hypernatremia and acetazolamide inhibited NaHCO3 and NaCl reabsorption in a molar ratio of about 1:2, as in normonatremic dogs. Finally, experiments in six dogs showed that the inhibitory effects of hypernatremia (PNa 213 +/- 4 mM) were not altered by varying PCO2 and plasma pH. We conclude that hypernatremia inhibits paracellular water and NaCl reabsorption in the proximal tubules by reducing the osmotic force caused by transcellular NaHCO3 reabsorption. A rise in PNa does not stimulate transcellular NaCl reabsorption during distal inhibition by ethacrynic acid.

Plasma potassium concentration as a determinant of proximal tubular NaCl and NaHCO3 reabsorption in dog kidneys.

To examine whether an acute increase in plasma potassium concentration ([K]p) may inhibit proximal tubular transport, clearance studies were performed in seven anaesthetized, volume expanded dogs treated with amiloride (1 mg kg-1 body wt) to block tubular potassium secretion, and with bumetanide (30 micrograms kg-1 body wt) to inhibit NaCl reabsorption in Henle's loop. As [K]p was raised in steps from 2.6 +/- 0.2 to 7.9 +/- 0.2 mM, bicarbonate, chloride, and sodium reabsorption decreased by 232 +/- 56, 520 +/- 59 and 958 +/- 112 mumol min-1, respectively, at constant glomerular filtration rate (GFR). On average, the molar ratio between the inhibitory effects on bicarbonate and chloride reabsorption were 1:2.2-2.4. Reabsorption was calculated at GFR 100 ml min-1: (reabsorption 100/GFR (mmol min-1). It was inversely correlated to ln [K]p with r = -0.82 for bicarbonate and with r = -0.89 for chloride. Fractional potassium reabsorption remained constant at 0.31 +/- 0.03. Administration of acetazolamide (100 mg kg-1 body wt) in eight dogs at [K]p 8 mM reduced fractional reabsorption of bicarbonate, chloride and sodium as much as in previous studies on normokalaemic dogs. We conclude that acute elevation of [K]p inhibits NaHCO3 transport and passive proximal tubular NaCl reabsorption. This inhibition is not related to changes in potassium secretion and carbonic anhydrase activity, but may be secondary to depolarization of the basolateral membrane.

Low oxygen cost of carbonic anhydrase-dependent sodium reabsorption in the dog kidney.

To examine the oxygen requirement of carbonic anhydrase-dependent sodium reabsorption in the proximal tubule, 18 anaesthetized dogs were studied under conditions of saturated distal NaCl reabsorption; the latter was accomplished by volume expansion (all groups) combined with infusion of loop diuretics (groups 1 and 3). Acetazolamide reduced HCO3- reabsorption by 602 +/- 32 mumol min-1 (55%, group 1) and by 777 +/- 103 mumol min-1 (66%, group 2). This was accompanied with a reduction in sodium reabsorption and oxygen consumption in a molar delta Na/delta O2 ratio of about 45 in both groups of dogs. The delta HCO3/delta O2 ratio averaged 16 +/- 1, which was not significantly different from the theoretical value of 18 expected for transcellular sodium transport by Na+, K+-ATPase. Mannitol (group 3) reduced NaCl reabsorption by 37 +/- 2% without affecting NaHCO3 reabsorption or oxygen consumption significantly. We conclude that carbonic anhydrase-dependent NaCl reabsorption in the proximal tubules is passive, and that NaHCO3 reabsorption is the only important active sodium transport which is sensitive to inhibition of carbonic anhydrase.

Difference between hypertonic NaCl and NaHCO3 as osmotic diuretics in dog kidneys.

To compare the osmotic inhibitory effects of NaCl and NaHCO3 on proximal tubular fluid reabsorption, plasma osmolality was raised by 40 mosmol kg-1 H2O by infusing hypertonic NaCl and NaHCO3 in volume-expanded dogs receiving ethacrynic acid. In five dogs studied at constant plasma pH 7.5, both NaCl and NaHCO3 reduced water reabsorption by 29 +/- 2%. However, NaCl infusion reduced bicarbonate reabsorption by 31 +/- 2%, whereas bicarbonate reabsorption remained unchanged during NaHCO3 infusion. In six dogs, bicarbonate reabsorption was kept constant during NaCl and NaHCO3 infusion by adjustments of plasma pH. At similar glomerular filtration rates (42.4 +/- 2.9 ml min-1), water reabsorption was 28.7 +/- 1.7 ml min-1 in the control period, 29.4 +/- 2.5 ml min-1 during hypertonic NaCl infusion and 20.6 +/- 1.2 ml min-1 during hypertonic NaHCO3 infusion. Therefore, NaCl did not reduce proximal tubular water reabsorption by a direct osmotic effect. By calculating the regression coefficient for the relationship between measured chloride reabsorption and maximal convective chloride flux, the effective reflection coefficient for NaCl averaged 0.11 +/- 0.01. The combination of a low reflection coefficient and high permeability may explain why hypertonic NaCl is not an osmotic diuretic.