FACTORS INFLUENCING THE PROTEIN BINDING OF BUMETANIDE USING AN EQUILIBRIUM DIALYSIS TECHNIQUE

Factors that influence the plasma protein binding of bumetanide were evaluated using equilibrium dialysis. It took approximately 12 h of incubation to reach an equilibrium between plasma and isotonic phosphate buffer of pH 7.4 containing 3% dextran using a Spectrapor 2 membrane (mol. wt cut-off = 12,000-14,000) in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. Bumetanide was fairly stable in both 4% human serum albumin (HSA) and in the isotonic phosphate buffer of pH 7.4 for up to 24 h. The binding of bumetanide to 4% HSA was constant (87.5 +/- 1.73%) at bumetanide concentrations ranging from 0.1 to 100 micrograms/ml. The extents of binding were 72.0, 83.3, 88.5, 90.2, 91.3 and 91.4% at albumin concentrations of 0.5, 1, 2, 3, 4 and 5 g/100 ml, respectively, and increased with a decrease in incubation temperature; the values bound were 94.6, 90.3 and 89.3% when incubated at 4, 22 and 37 degrees C, respectively. The binding of bumetanide was independent of the buffer composition used, the quantities of AAG (up to 0.32%), heparin (up to 40 units/ml), sodium azide (up to 0.5%) and anticoagulants (EDTA, heparin and citrate). The free fraction of bumetanide in rabbit plasma (2.91%) was significantly higher than in humans (1.98%) or rats (1.85%).     

Differential effects of in vitro and in vivo hyperthermia on the production of interleukin-10

OBJECTIVE: To determine whether hyperthermia activates an anti-inflammatory response. DESIGN: A prospective study. SETTING: Heatstroke Center, Makkah, and King Faisal Specialist Hospital, Riyadh, Saudi Arabia. PATIENTS: Twenty-five heatstroke patients pre-cooling (rectal temperature 42.4 +/- 0.8 degrees C) (group 1) and 13 normothermic heat-stressed subjects were studied (group 2). Twelve of the 25 heatstroke patients were also studied post-cooling (group 3). Mononuclear cells from six healthy blood donors resting at 24 degrees C were used for in vitro study. INTERVENTIONS: Mononuclear cells were cultured at a concentration of 1 x 10(6)/ml without and with lipopolysaccharide (LPS) added at concentration of 10, 100, and 1000 ng/ml. The cells were incubated for 24 h at 37, 39, 41, and 43 degrees C. ELISA was used to measure IL-10 in the supernatant and plasma from heatstroke and heat-stressed subjects. RESULTS: All patients in group 1, 40% of group 2, and 37% of group 3, showed elevation of IL-10 (1289 +/- 2519, 248 +/- 393, and 172 +/- 226 pg/ml, respectively) compared with normal control levels, (< 100 pg/ml) P < 0.05. IL-10 level on admission did not correlate with degree of hyperthermia. During 24 h incubation at 37 degrees C without LPS, no IL-10 was detected, whereas with 10 ng/ml LPS, monocytes released 658 +/- 291 pg IL-10/10(6) cells. At 39 degrees C and 41 degrees C IL-10 release was decreased to 225 +/- 114, and 245 +/- 90 pg/10(6) cells, respectively; and was completely inhibited at 43 degrees C (67 +/- 10 pg/10(6) cells), P < 0.0001. CONCLUSION: Heat-stress with and without hyperthermia is associated with anti-inflammatory response in vivo. However, it does not seem to be the direct effect of heat on monocytes, suggesting that other environmental or genetic factors may be involved.     

3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2,D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.     

Subcellular location and properties of bactericidal factors from human neutrophils

We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins.     

Triple combination of retinoic acid, low concentration of cytarabine and dimethylformamide induces differentiation of human acute myeloid leukaemic blasts

Differentiation induction therapy provides an alternative for treatment of patients with acute myeloid leukaemia (AML) who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + low concentration of cytarabine (Ara-C) + dimethylformamide (DMF) on the differentiation of blasts from 24 AML patients was studied. Nonadherent mononuclear cells were cultured at a concentration of 5 x 10(5) cells/ml in 24-well tissue culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M RA, 10(-6) M Ara-C and/or 100 mM DMF alone and in combination with each other for 6 days in primary culture at 37 degrees C in a humidified incubator under 5% CO2. The triple combination of 10(-6) M RA + 10(-6) M Ara-C + 100 mM DMF induced 90% of blasts from 22 out of 24 AML patients to differentiate. These highly effective results justify a clinical trial of this triple combination for AML patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy.     

Impaired plasma protein binding of phenytoin in uremia and displacement effect of salicylic acid.

The plasma protein binding of phenytoin (DPH) was studied by equilibrium dialysis at 37 degrees C in plasma from uremic patients and healthy subjects. Scatchard plot analyses demonstrated a decreased association constant Ka for the DPH-albumin interaction in the uremic plasma (mean 1.76 - 10(3) M-1 +/-SD 0.12 and a mean 4.10 - 10(3) M-1 +/- SD 0.24 in normal plasma). Studies on separated fractions of serum did not indicate any significant binding of DPH to proteins other than albumin. The nonlinear mathematical relationship between bound DPH and serum albumin could be linearized at low drug concentrations by plotting the ratio of bound/unbound DPH against albumin concentration. The displacement effect of salicylic acid at a concentration of 276 mug/ml (2mM) and DPH was considerable in plasma from normal subjects. In uremic plasma the effect was of much smaller magnitude.     

Effect of protein binding of daptomycin on MIC and antibacterial activity.

A higher rate of clinical failures in patients treated with daptomycin (2 mg/kg of body weight, given once daily) compared with rates in patients treated with conventional regimens caused early termination of this comparative clinical trial. One explanation for these failures could be that daptomycin is highly protein bound and that the concentration of the unbound active drug is too low for antibacterial activity. To assess this explanation, we studied the binding of daptomycin to proteins by using an ultrafiltration method. pH (7.0 to 7.4), temperature (25 or 37 degrees C), or daily freezing and thawing over 2 months had no effect on binding of daptomycin to proteins. We found that daptomycin was bound to albumin (90%) at 4 g/100 ml. Binding of daptomycin was not concentration dependent (2.5 to 80 micrograms/ml). In human serum samples spiked with daptomycin, average binding was 94% +/- 2.4%. In 6 subjects given an intravenous infusion of daptomycin (3 mg/kg), average binding was 90% +/- 2.1%. Susceptibility studies showed that a concentration in serum 20 times the unbound concentration was needed to equal the MIC of the total drug. These results indicate that daptomycin is highly bound (90 to 94%) to albumin and that clinical failure to daptomycin can in part be explained by the low concentration of the unbound drug.     

Immunoglobulin G independent activation of the classical complement pathway by monosodium urate crystals.

10 mg of monosodium urate crystals reduced the CH50 of 1 ml of human serum by 57% after 30 min at 37 degrees C. C1, C4, and C3 depletion of 52, 68, and 46% were typical of classical pathway activation. C1 binding and activation occurred when urate crystals were incubated with isolated precursor C1, and required the intact macromolecule, C1qrs. Activation of isolated C1 by urate crystals was not diminished by F(ab')2 anti-Fc under conditions in which C1 activation by aggregated immunoglobulin (G) was blocked by the F(ab')2 antibody.     

Apparent kinetics of angiotensin converting enzyme: hydrolysis of [3H]benzoyl-phenylalanyl-alanyl-proline in the isolated perfused lung

Isolated perfused rabbit lungs were used to study the hydrolysis of [3H]benzoyl-phenylalanyl-alanyl-proline [( 3H]BPAP), a synthetic substrate for angiotensin converting enzyme (ACE). Lungs were perfused, at constant flow rates, with physiologic medium containing added BPAP and the concentration of its metabolite, [3H]benzoyl-phenylalaline, was measured in lung effluent. Hydrolysis of BPAP (4.2 microM) was 64.1 +/- 3.3% at 37 degrees C and was significantly decreased (P less than .01) to 10.1 +/- 8.7% by the addition of the ACE inhibitor, MK422 (10(-6) M). Disappearance (i.e., hydrolysis) of immunoreactive angiotensin I was also inhibited by MK422. Hydrolysis of BPAP was saturable and calculated apparent kinetic constants were Km = 13 microM and Vmax = 50 nmol/sec/lung. When the perfusion medium temperature was 10 degrees C, apparent Km was unchanged, whereas Vmax was significantly (P less than .05) decreased. At BPAP concentrations sufficient to depress metabolism to less than 20%, perfusion pressure was unchanged. Hydrolysis of BPAP under first-order conditions was independent of flow over the range 10 to 50 ml/min. However, increase in flow rate to 100 ml/min/lung was associated with decreased BPAP metabolism. These data indicate that BPAP is a substrate for pulmonary ACE in vitro and substantiate its use in intact animals because: 1) it is without physiologic effect in high doses; and 2) calculated apparent kinetics in isolated lungs under these conditions of steady-state concentrations were similar to those obtained from earlier studies that utilized bolus injection techniques in intact animals.     

Modulation of polymorphonuclear leukocyte microbicidal activity and oxidative metabolism by fibrinogen degradation products D and E.

Fibrinogen degradation products (FDP) D and E are typically present in blood of patients with disseminated intravascular coagulation and related conditions in which granulocyte (PMN) defense against bacterial infection may be compromised. This study was intended to determine whether FDP modify PMN functions critical to their bactericidal activity. Incubation of human PMN and Escherichia coli with 50-100 micrograms/ml FDP did not affect phagocytosis, but reduced by greater than 90% the cells' ability to inhibit bacterial colony growth compared with control PMN incubated with albumin or fibrinogen. FDP (10-100 micrograms/ml) inhibited PMN O2- release and chemotaxis stimulated by FMLP by 17-50% (P less than 0.005) and 41% (P less than 0.01), respectively. Fragment E3, and not fragment D1, was primarily responsible for inhibition of FMLP-induced PMN O2- release. Phorbol myristate acetate (10 ng/ml), 1-oleoyl-2-acetylglycerol (10(-6) M), AA (4.2 x 10(-5) M), and zymosan-activated serum-stimulated PMN O2- release were also decreased 37-63% by FDP compared with control protein. There are at least two mechanisms by which FDP may impair PMN responses. With respect to FMLP, FDP (16-100 micrograms/ml) inhibited specific binding to the cell surface over a ligand concentration range of 1.4-85 nM [3H]FMLP. In contrast, FDP did not effect the extent of phorbol ester binding to PMN but blocked activation of protein kinase C. These data suggest that elevated plasma FDP inhibit several PMN functions critical to the bactericidal role of these inflammatory cells.     

Antiproliferative synergism of the allylamine SF 86-327 and ketoconazole on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi.

We have investigated the growth-inhibitory effects of two ergosterol biosynthesis inhibitors, the dioxolane imidazole ketoconazole and the allylamine SF 86-327, alone and in combination, on the proliferative stages of Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease. Proliferation of epimastigotes in liver infusion-tryptose medium at 28 degrees C was immediately arrested by any of these drugs at greater than or equal to 3 x 10(-5) M; cell lysis occurred 24 h later. Below that concentration, SF 86-327 at concentrations down to 1 x 10(-6) M stopped growth after 48 h. In contrast, ketoconazole slowed cell growth only moderately, but proliferation finally stopped and cell lysis occurred after 120 h at 3 x 10(-6) M. Synergistic effects could be observed when the two drugs were used in combination: the concentration of SF 86-327 required to reduce the cell growth to 25% of controls in 144 h was reduced 33-fold in the presence of 1 x 10(-6) M ketoconazole, which by itself reduced growth only by 30%. Amastigotes, proliferating in Vero cells at 37 degrees C, were much more susceptible to both drugs, but ketoconazole was definitely a more potent antiparasitic agent than the allylamine in this system: whereas the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 1 x 10(-7) M and that required to completely eradicate the parasite was 3 x 10(-6) M, for ketoconazole these concentrations were 1 x 10(-10) M and 1 x 10(-8) M, respectively. Again, strong synergistic effects were observed when the drugs were used in combination: the concentration of SF 86-327 required to reduce the number of infected cells to 50% of controls was 100-fold lower in the presence of 10(-11) M ketoconazole, which by itself had no effects on amastigote proliferation. The parasite was completely eradicated when the drugs were used in combination at concentrations as low as 10(-9) M. Synergy of the antiproliferative effects of the drugs on both froms of the parasite was further demonstrated by concave isobolograms. On the other hand, SF 86-327 at 10(-5) M had no effects on the proliferation of Vero cells, whereas ketoconazole at 10(-7) M reduced the proliferation of these cells by 50%.     

BioArchive自动液氮储存系统保存的胎盘脐血的冷冻生物学特征

为了解在特定降温和复温过程中于BioArchive自动液氮储存系统保存的胎盘脐血 (PCB)的冷冻生物学特征 ,采用Procount方法流式细胞术测定CD34+ 细胞含量 ,细胞集落形成单位 (CFU)检测CD34+ 细胞的增殖能力 ,台盼蓝拒染法和白细胞分类测定有核细胞活存率和白细胞分类变化。结果显示 ,冻存复温后PCB有核细胞平均活存率为 (73.3± 12 .5 ) % ;PCBCD34+ 细胞的含量在冻存前为 (0 .3± 0 .2 1) % ,冻存复温后为 (0 .4 5± 0 .36 ) %。冻存复温后PCBCFU GM / G/ GEMM形成能力为冻存前的 (97.9± 39.9) %。比较大、小冷冻袋冻存复温后PCB细胞活存率和CFU GM/ G/ GEMM的形成能力无显著差异。另外 ,冻存复温后PCB的白细胞分类明显改变 ,粒细胞百分比明显降低 ,淋巴细胞和单核细胞百分比明显升高。结论 :在本研究中所采用的降温与复温程序不适于保存粒细胞 ,但是能够很好地保存淋巴细胞和单核细胞 ,尤其是CD34+ 的幼稚造血细胞。     

Differential effect of DPI 201-106 on the sensitivity of the myofilaments to Ca2+ in intact and skinned trabeculae from control and myopathic human hearts.

Activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration ([Ca++]i) result from phosphatidylinositol biphosphate (PIP2) breakdown. We previously demonstrated that PKC activation inhibits arginine vasopressin (AVP)-induced osmotic water flow in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. To estimate the potential significance of PIP2 turnover as a modulator of water transport in this nephron segment, we examined the effect of Ca on AVP action and explored the mechanisms of action of PKC and increased [Ca++]i. In rabbit CCTs perfused at 37 degrees C, pretreatment with bath A23187 (2 x 10(-8) M, 2 x 10(-6) M), a Ca ionophore, almost totally suppressed AVP (10 microU/ml)-induced peak hydraulic conductivity (Lp). The suppression by 2 x 10(-8) M A23187 was as potent as that by 2 x 10(-6) M A23187, and significant even when it was administered 10 min after AVP. When phorbol myristate acetate (PMA, 10(-9) M), a PKC activator, and A23187 (2 x 10(-8) M) were placed in the bath simultaneously, the combined suppressive effect on peak Lp was greater than that of either inhibitor alone. However, the mechanisms of inhibition by PMA and A23187 were different. While both 10(-7) and 10(-9) M PMA suppression are primarily post-cAMP, A23187 predominantly suppressed a pre-cAMP step: 10(-4) M chlorophenylthio-cAMP-induced peak Lp was not affected by 2 x 10(-8) M A23187, and only partially inhibited by 2 x 10(-6) M A23187. The PMA (10(-7) M) suppression of AVP-induced peak Lp was totally reversed by bath staurosporine (10(-7) M), a PKC inhibitor, but not attenuated by either bath indomethacin (5 x 10(-6) M) or low Ca (1-2 x 10(-6) M) bath medium. In contrast, the A23187 (2 x 10(-8) M) suppression of the peak Lp was not affected by staurosporine, but was significantly reversed by indomethacin or low Ca bath medium. We conclude: (a) Elevation of [Ca++]i, as well as activation of PKC, suppresses the hydroosmotic effect of AVP on CCT at 37 degrees C. (b) When stimulated simultaneously these two intracellular mediators are additive in their antagonism of AVP action. These results suggest that stimulated PIP2 breakdown may be an important modulator of water transport in CCT. (c) Different mechanisms underlie PKC and Ca-mediated suppression of the AVP-induced water transport. The inhibition of AVP action by increased [Ca++]i is primarily pre-cAMP, and involves a cyclooxygenase metabolite(s) of arachidonic acid, while the inhibition by PKC is post-cAMP, and independent of cyclooxygenase products of arachidonic acid.     

Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.

Defensin NP-1, derived from the neutrophils of rabbits, was tested for its fungistatic and fungicidal activity against strains of Cryptococcus neoformans. The MICs for the encapsulated strains tested ranged from 3.75 to 15.0 micrograms of NP-1 per ml. The minimum fungicidal concentrations for these strains were similar to the MICs. An acapsular strain, however, had a lower MIC of 0.93 and minimum fungicidal concentration of 1.88 micrograms/ml. NP-1 demonstrated time-dependent and concentration-dependent killing of C. neoformans. Killing occurred rapidly in the first 20 min of exposure to NP-1 and was maximum at 90 to 120 min. Killing of C. neoformans by NP-1 was concentration dependent with 31% +/- 9% survival at 25 micrograms/ml, 13% +/- 4% survival at 50 micrograms/ml, 9% +/- 5% survival at 75 micrograms/ml, and 5% +/- 3% survival at 100 micrograms/ml. NP-1's fungicidal effect on C. neoformans was also inoculum dependent, with increased activity observed at 10(4) versus 10(5) or 10(6) cells per ml. In addition, stationary-phase C. neoformans was less susceptible to NP-1 killing than yeast cells in the logarithmic phase. Subinhibitory concentrations of both NP-1 (0.25 x MIC) and fluconazole (0.25 x MIC) acted synergistically in inhibiting growth of C. neoformans. Similar combinations of NP-1 and amphotericin B, however, did not yield synergy.     

Comparative nuclear magnetic resonance studies of water permeability of red blood cells from maternal venous blood and newborn umbilical cord blood.

Comparative morphological and nuclear magnetic resonance (NMR) measurements of the diffusional permeability (Pd) were performed on red blood cells (RBCs) from maternal venous blood and fetal RBCs, isolated from cord blood taken at delivery. Fetal RBC had a diameter of 8.79+/-0.03 microm (mean+/-standard deviation, SD), a volume of 103 microm3 and a surface area of 157 microm2. We report here the first comparative measurements of Pd of maternal and fetal RBCs by using a Mn2+-doping NMR technique. The values of Pd were, in the case of maternal RBC, 3.7 x 10(-3) cm/s at 15 degrees C, 4.1 x 10(-3) cm/s at 10 degrees C, 4.9 x 10(-3) cm/s at 25 degrees C, 5.2 x 10(-3) cm/s at 30 degrees C and 7.2 x 10(-3) cm/s at 37 degrees C. For fetal RBC all corresponding Pd values were almost half, namely 2.0 x 10(-3) cm/s at 15 degrees C, 2.3 x 10(-3) cm/s at 20 degrees C, 2.8 x 10(-3) cm/s at 25 degrees C, 3.4 x 10(-3) cm/s at 30 degrees C and 4.4 x 10(-3) cm/s at 37 degrees C. The decreased Pd values of fetal RBCs were probably due to lower channel-mediated water permeability compared with adult RBCs. The values of the activation energy for water permeability (E(a,d)) were significantly higher for fetal RBCs (27.6+/-5.0 kJ/mol) than for adult RBCs (22.8+/-2.7 kJ/mol). A positive correlation between the Pd values of the two kinds of RBCs was found. This points to the genetic basis for the determination of RBC water permeability.     

Clearance of Immunoreactive Somatostatin by Perfused Rat Liver

Other investigators have demonstrated that concentrations of immunoreactive somatostatin (IRS) are higher in blood from the hepatic portal vein or its tributaries than in blood from the hepatic or peripheral systemic veins of man and animals. This suggests that there is hepatic extraction of IRS from the portal system in vivo. In the rat, portal vein plasma IRS is reported to be heterogeneous and to contain, in part, a 1,600 mol wt form of IRS which is immunochemically similar to synthetic somatostatin and not significantly bound to high molecular weight plasma protein. Our study was undertaken to determine directly whether unbound synthetic cyclic somatostatin was cleared by the rat liver perfused through the hepatic portal vein in vitro with a recirculating, plasma-free, erythrocyte-containing perfusate.At 37 degrees C and pH 7.40, perfusate IRS, at initial concentrations (1,728 pg/ml) within the range previously reported in rat portal venous blood, was removed by the liver at a rate commensurate with first-order kinetics. Hepatic clearance was 0.84+/-0.04 ml/min per g postperfusion wet weight (SE). Hepatic extraction was 36+/-2%, and t((1/2)) was 20.0+/-1.3 min. Recovery of IRS from the perfusate without the liver was >85%, excluding significant degradation by the medium. Clearance, extraction, and t((1/2)) of IRS were not changed by an unphysiologic IRS concentration (621,500 pg/ml), or by pharmacologic concentrations of insulin (8.2 muM) or glucagon (2.9 muM).The t((1/2)) was prolonged significantly to 28.2+/-1.9 and 45.6+/-4.7 min during perfusions at liver temperatures of 25 degrees and 16 degrees C, respectively. At 37 degrees C, the t((1/2)) was also significantly increased to 28.7+/-3.2 and 24.2+/-1.1 min at perfusate pH 7.06 and 6.78, respectively.These studies indicate that the rat liver clears unbound IRS from the perfusate by a first-order kinetic process that is (a) unsaturable at pharmacologic concentrations, (b) temperature-sensitive and, to a lesser extent, influenced by lowered pH, and (c) not affected by insulin and glucagon. The liver would appear to play an important role in the metabolism of the 1,600 mol wt form of somatostatin. Clearance of endogenous IRS by the liver should be considered in the interpretation of IRS concentrations in the peripheral systemic veins.     

Transdermal self-permeation enhancement of ibuprofen.

The objective of this study was to prepare saturated solutions of ibuprofen, of different concentrations, and to investigate their effect on permeation of ibuprofen across rat epidermis. Ibuprofen saturated solutions were prepared using 0.1, 0.2, 0.3 and 0.4 M disodium hydrogen phosphate solution (DHP). The solubility of ibuprofen in DHP increased as the molarity of DHP increased. Thus the four saturated solutions of ibuprofen (0.1M-DHP-IBU, 0.2M-DHP-IBU, 0.3M-DHP-IBU and 0.4M-DHP-IBU) have different concentrations of the same drug, and showed same pH (pH 7.0+/-1). The permeability study was also carried out using human epidermis and silastic membrane. Permeation rate of ibuprofen across rat epidermis and human epidermis from 0.4M-DHP-IBU was much greater than from 0.1M-DHP-IBU. The magnitudes of increase in the drug flux were 46.4-fold with rat epidermis and 9.4-fold with human epidermis. Such a great increase in drug flux was not observed with silastic membrane, only 1.4-fold. This suggests that the increased drug flux is likely due to drug-skin interaction and not the increased concentration of ibuprofen per se. Surface tension (ST) measurements of DHP versus ibuprofen concentration showed ST reduction of DHP, from 72 to 27.9 dyn/cm. This is an indication that ibuprofen acted as ionic surfactant and the observed skin permeability enhancement is attributed to disruption of stratum corneum barrier. Results of DSC study supported this assumption. DSC of untreated rat stratum corneum samples showed lipid transitions at 41.9+/-0.0 degrees C (T1), 55.1+/-1.6 degrees C (T(x)), 70.2+/-0.1 degrees C (T2) and 77.5+/-0.1 degrees C (T3), while those pretreated with 0.4M-DHP-IBU did not show the first three lipid transitions. Also, pretreatment of rat epidermis with 0.4M-DHP-IBU enhanced permeation of diclofenac sodium greater than 1250-fold. This corroborates that ibuprofen not only enhances its own permeation but also that of other drugs, such as diclofenac sodium.     

Comparison of Netilmicin with Gentamicin in the Therapy of Experimental Escherichia coli Meningitis

Netilmicin (SCH 20569), a new broad-spectrum aminoglycoside derived from sisomicin, was compared with gentamicin in the therapy of experimental Escherichia coli meningitis in rabbits. Meningitis was produced in 48 animals by the intracisternal inoculation of 10(5)E. coli colony-forming units. The minimum bactericidal concentration was 2 mug/ml against the test strain for both gentamicin and netilmicin. The two aminoglycosides demonstrated comparable penetration into the cerebrospinal fluid (CSF). The mean percent penetration [(CSF concentration/serum concentration) x 100%] was 22.5 +/- 6.0 and 20.6 +/- 7.2 for netilmicin and gentamicin, respectively (P = 0.18). However, netilmicin achieved bactericidal activity in the CSF at lower levels than did gentamicin. When mean CSF concentrations ranged from 4 to 8 mug/ml, mean CSF bacterial titers decreased 2.98 logs in rabbits treated with netilmicin but only 0.16 log in rabbits treated with gentamicin. A 2-log decrease in CSF bacterial counts was produced by a mean CSF concentration of 1.4 mug of netilmicin per ml as compared to 14.1 mug of gentamicin per ml. Because of its reduced toxicity and greater in vivo bactericidal activity, netilmicin may offer an advantage over gentamicin in the therapy of gram-negative bacillary meningitis.