Serum Protein Binding of the Aminoglycoside Antibiotics

The binding of four aminoglycoside antibiotics by human serum was investigated under controlled conditions of physiological pH and temperature, by means of an ultrafiltration technique. No serum binding was demonstrable for gentamicin, tobramycin, or kanamycin, whereas streptomycin was 35% bound. Previous conflicting studies are discussed, and some of the pharmacological implications are considered.     

苯丙酮尿症患者血清S-100B蛋白测定的临床意义

目的探讨苯丙酮尿症患者血清中S—100B蛋白的水平及与苯丙氨酸的关系。方法用ELISA方法测定27例初诊苯丙酮尿症患儿、5例复诊患儿及42例正常儿童血清内的S-100B蛋白水平，用高效液相色谱荧光法测定血清内的苯丙氨酸浓度并对两者进行分析比较。结果苯丙酮尿症初诊患者血清S-100B水平（0．92±0．15μg／L）显著高于复诊者（0．32±O．11μg／L）与健康对照组（0．22±0．09μ g／L），差异有统计学显著性意义（P〈0．01）；复诊者高于健康对照组，差异有统计学意义（P〈0．05）。初诊患者血清S-100B的浓度与患者体内苯丙氨酸浓度呈正相关（相关系数0．62，P〈0．01）。结论血清S-100B蛋白的水平可作为苯丙酮尿症患者监测脑损伤的指标。     

Assessment of Oritavancin Serum Protein Binding across Species

Biophysical methods to study the binding of oritavancin, a lipoglycopeptide, to serum protein are confounded by nonspecific drug adsorption to labware surfaces. We assessed oritavancin binding to serum from mouse, rat, dog, and human by a microbiological growth-based method under conditions that allow near-quantitative drug recovery. Protein binding was similar across species, ranging from 81.9% in human serum to 87.1% in dog serum. These estimates support the translation of oritavancin exposure from nonclinical studies to humans.Estimates of serum protein binding are essential to translate drug exposure from nonclinical species to humans during assessments of toxicology, pharmacokinetics, and pharmacodynamics since the free fraction dictates drug activity (3, 7, 17, 18). Recent evidence supports the concept of an “active fraction” that offers insight into the pharmacodynamic behavior of highly protein-bound drugs, such as daptomycin (20).Oritavancin is a late-stage investigational lipoglycopeptide under study for treatment of serious Gram-positive infections (6). Nonspecific binding of oritavancin to labware surfaces (1, 2) and to dialysis membranes has called into question the accuracy of previous oritavancin human serum binding estimates (85.7% to 89.9% [16]). We therefore used conditions that minimize nonspecific oritavancin binding (4, 5) to estimate its binding to serum by a single in vitro methodology for three nonclinical species (mouse, rat, and dog) and humans. Protein binding estimates were derived from serum-induced increases in oritavancin MICs (9, 10, 21). To control for any impact of serum components on bacterial growth and antibiotic activity, oritavancin activity in serum was compared to its activity in serum ultrafiltrate, which is devoid of albumin, the protein responsible for the majority of oritavancin serum binding (23). The method was benchmarked using daptomycin and ceftriaxone (8, 18, 22). (Part of this work was previously presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases as a poster [12].)Pooled serum from humans, mice, and rats was from Equitech-Bio (Kerrville, TX); pooled serum from beagle dogs was from Bioreclamation (Liverpool, NY). Serum ultrafiltrate was prepared using Centricon Plus-50 ultrafilters (Millipore, Billerica, MA), whose molecular mass cutoff (50 kDa) excludes albumin. MICs against Staphylococcus aureus ATCC 29213 were determined by broth microdilution (4) using arithmetic drug dilutions in 95% serum or 95% serum ultrafiltrate, each supplemented with 5% cation-adjusted Mueller-Hinton broth (CAMHB). Serum protein binding for each drug was calculated using the following formula: % bound = (1 − [mean MIC in serum ultrafiltrate/mean MIC in serum]) × 100.The MICs for each condition, serum source, and test agent were precise (Table ​(Table1),1), with a mean coefficient of variation of 17%. MICs as determined under CLSI M7-A8 conditions (Table ​(Table1)1) (4) were within the quality control ranges (5).

TABLE 1.

Oritavancin, ceftriaxone, and daptomycin MICs against S. aureus ATCC 29213 in cation-adjusted Mueller-Hinton broth and 95% serum ultrafiltrate and 95% serum from human, mouse, rat, and dog

Action of Polymyxin B on Bacterial Membranes: Phosphatidylglycerol- and Cardiolipin-Induced Susceptibility to Polymyxin B in Acholeplasma laidlawii B

To identify the polymyxin receptor molecules in the membranes of living microorganisms, fusion of intact Acholeplasma laidlawii B with lipid vesicles was investigated according to the procedure of Grant and McConnell (1973). The naturally polymyxin-resistant A. laidlawii B was treated with phospholipid vesicles prepared from purified phospholipids of the polymyxin-susceptible Salmonella typhimurium G30. A. laidlawii B absorbed between 15 and 45% of its own lipid content of the added tritium-labeled phospholipids without loss of viability. Association with the acidic components phosphatidylglycerol and cardiolipin produced a 10- to 30-fold increase in polymyxin susceptibility, which was not obtained with egg-phosphatidylcholine and mixed phosphatidylcholine-phosphatidylethanolamine vesicles. The polymyxin-sensitized cells bound 12 times more radioactive antibiotic than resistant cells. The phosphatidylglycerol-induced susceptibility was abolished by serum fraction V (Cohn) proteins.     

Rate of Binding of Antibiotics to Canine Serum Protein

The time rates of binding of three antibiotics of similar chemical structure, each with differing degrees of protein binding, were determined. Cephaloridine, which is 10% bound by serum proteins, was bound at a more rapid rate than cephalothin, which is 40% bound by serum protein. Cefazolin, bound 80%, required for longest time period for maximum binding to occur. The rate of protein binding appears directly related to the total percentage bound. The data from this study indicate that prolonged rates of binding of highly protein-bound drugs may influence pharmacological studies.     

血清视黄醇结合蛋白4 及骨桥蛋白与绝经后骨质疏松症的相关性研究

目的　探讨绝经后妇女血清视黄醇结合蛋白4（retinol binding protein 4,RBP4）及骨桥蛋白（osteopontin,OPN） 水平与骨密度（bone mineral density,BMD） 的关系, 分析其在绝经后骨质疏松症（postmenopousalosteoporosis,PMOP）中的应用价值。方法　对262 例绝经后妇女进行研究双能X 线骨密度仪测量腰椎1-4（L1-4）和左侧股骨颈（FN）,测定血中RBP4,OPN,I 型原胶原N- 端前肽（PINP）,β- 胶原降解产物（β-CTX）,甲状旁腺素（PTH）,骨钙素（OC）,血钙（Ca）及血磷（P）水平。ROC 曲线分析RBP4 及OPN 诊断PMOP 的价值。结果　骨质疏松组血清 RBP4(μg/ml)（25.13±4.74 , 18.46±4.25 和15.30±3.87）及OPN(ng/ml)（13.58±4.42 , 10.47±3.46 和8.24±3.13）水平均明显高于骨量减少组和正常组,差异有统计学意义（均P ＜ 0.05）。相关分析显示,骨质疏松症组血清RBP4及OPN 水平与β-CTX 呈正相关（r=0.291,0.284,均P ＜ 0.05）,与Ca,L1-4 及左FN 骨密度呈负相关（r = - 0.247,-0.513,-0.486,-0.265,-0.556,-0.574,均P ＜ 0.05）。ROC 曲线显示,血清RBP4 及OPN 水平为21.53μg/ml 和12.27 ng/ml 是诊断PMOP 的最佳截值。结论　血清RBP4 及OPN 水平升高与低BMD 相关,是影响PMOP 发生的关键因子。     

Inactivation of Endotoxin by Polymyxin B

The limulus gelation assay was utilized to investigate endotoxin inactivation by a number of antibiotics in vitro. Endotoxin activity was sharply reduced by polymyxin B and sodium colistimethate. The effect of the polymyxin was not significantly inhibited by 0.001 M calcium or 90% serum. Crude endotoxins from a variety of aerobic gram-negative bacteria, including several not previously studied, could be inactivated 1 or more logs by as little as 1 mug of polymyxin B per ml, whereas Bacteroides fragilis endotoxin was poorly detoxified. A 10,000-fold range in the relative susceptibility of different endotoxins to inactivation by polymyxin B was found. The endotoxin most susceptible to polymyxin B was derived from an organism resistant to polymyxin B by disk sensitivity testing, suggesting that the bacteriocidal and endotoxin detoxifying properties of polymyxin need not be directly related.     

Ascitic Fluid Cephalosporin Concentrations: Influence of Protein Binding and Serum Pharmacokinetics

Mongrel dogs with ascites created by inferior vena cava ligation were given cephalothin, cephaloridine, cefazolin, and cefamandole to evaluate the effect of protein binding and serum pharmacokinetics on the distribution of cephalosporins into ascitic fluid. Antibiotics were given intramuscularly (15 mg/kg) every 4 h for a total of eight doses. Antibiotic binding to dog serum and ascitic fluid was measured by ultracentrifugation. Binding of the cephalosporins to dog serum ranged from 31% for cephaloridine to 46% for cephalothin, considerably lower than human serum binding for cefazolin, cephalothin, and cefamandole. Antibiotic binding to ascitic fluid was only slightly lower than that to serum. Ascitic fluid antibiotic concentrations, which approached equilibrium at 16 to 28 h, were significantly higher for cefazolin and cephaloridine than for cephalothin and cefamandole. However, serum concentrations were also higher for cefazolin and cephaloridine, and percent penetration (ratio of serum peak to ascites peak × 100) was not statistically different among the four drugs. Binding of these cephalosporins to extravascular fluid protein was an important factor that determined the total ascitic fluid antibiotic level achieved. A formula utilizing the log mean serum level and binding to serum and extravascular fluid protein was used to accurately predict ascitic fluid drug levels at equilibrium.     

Polymyxin B induces lysis of marine pseudoalteromonads

Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that "what is true for Escherichia coli is true for all bacteria" does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.     

Treatment of Murine Coccidioidomycosis with Polymyxin B

An agar dilution method was employed to test the susceptibility of 12 strains of Coccidioides immitis to polymyxin B (PB). After 3 days of incubation, eight strains were markedly inhibited by 5.0 mug of PB per ml and four strains did not grow. PB at 10 mug/ml inhibited the growth of all strains through 20 days of incubation. To determine whether PB has anticoccidioidal activity in vivo, mice in groups of 30 were infected intraperitoneally with a mean lethal dose (LD(50)) or 20 LD(50) of arthrospores of C. immitis ATCC 28868 (Silveira). Treatment by intraperitoneal injection of PB (2.5 mg/kg) was begun 2 or 5 days after challenge. By 40 days after infection, 47 and 7% of the untreated mice challenged with an LD(50), respectively, were alive. Of mice infected with an LD(50) or 20 LD(50) and treated with PB beginning 2 days after the challenge, 90% of each group were alive by day 40. Initiation of PB therapy 5 days after infection permitted survival of 84% of the mice infected with an LD(50); however, only 27% of the mice infected with 20 LD(50) survived by day 40. In this latter group there was evidence that PB prolonged life since 56% of the treated mice were alive by day 15 as compared with 30% of the controls. PB in vivo was fungistatic since the majority of treated mice had C. immitis in the liver, lungs, and spleen.     

Uptake of Polymyxin B into Renal Cells

Polymyxin B is increasingly used as a treatment of last resort against multidrug-resistant Gram-negative infections. Using a mammalian kidney cell line, we demonstrated that polymyxin B uptake into proximal tubular epithelial cells was saturable and occurred primarily through the apical membrane, suggesting the involvement of transporters in the renal uptake of polymyxin B. Megalin might play a role in the uptake and accumulation of polymyxin B into renal cells.     

ELISA法测定血清S100B蛋白

目的建立ELISA测定血清S100B蛋白的方法。方法制备S100B单克隆和多克隆抗体,以双抗体夹心法测定血清S100B蛋白。结果方法的平均回收率为93.7%-104%,批内及批间平均变异分别为3.7%和5.9%,方法的线性为0-12.5μg/l,参考值范围为0.14-0.38μg/l。结论该法简便、快速、特异、敏感,适于临床常规应用。     

Developmental Pattern of a Serum Binding Protein for Multiplication Stimulating Activity in the Rat

The concentration of multiplication stimulating activity (MSA), an insulinlike growth factor (IGF), is high in fetal rat serum. We now report that MSA is exclusively associated wth an albumin-size binding protein in fetal rat serum; the growth hormone-dependent, gamma globulin-size binding protein, which predominates in the older animal, is absent from fetal rat serum. When ¹²⁵I-MSA was incubated with fetal rat serum and then gel filtered on Sephadex G-200, specific radioactivity eluted in the void volume (peak I) and the albumin region (peak III); by contrast, specific radioactivity eluted mainly in the gamma globulin region (peak II) in adult rat serum. Pools of the Sephadex G-200 fractions were chromatographed on Sephadex G-50, in 1 M acetic acid, to separate the binding protein from IGF activity. Analysis of IGF activity by chick embryo fibroblast bioassay, competitive protein binding assay, and MSA by radioimmunoassay revealed that all the IGF activity and MSA in fetal rat serum resided in peak III. Measurement of MSA binding capacity of the stripped binding protein by Scatchard analysis demonstrated that the majority of binding capacity also was found in peak III in fetal rat serum; most of MSA binding capacity was in peak II in adult rat serum. In fetal rat sera, in addition to the peak III binding protein, which is the major carrier of endogenous MSA, there is a component in peak I capable of specifically binding ¹²⁵I-MSA. This component elutes as a single species from a Sepharose-6B column. As MSA associated with peak III gradually declined in early neonatal life, peak II-associated IGF activity measured by chick embryo fibroblast bioassay showed a rise of activity with a peak at 5 d of neonatal life, a nadir at 20 d, with an increase again to attain adult levels. These studies demonstrate that the MSA binding protein in the fetus is different from the growth hormone-dependent binding protein in adult life.     

血清维生素D结合蛋白水平检测对大鼠肝毒性诊断的评价研究

目的 评价血清维生素D结合蛋白(vitamin D binding protein,VDBP)用于大鼠肝毒性诊断的价值。方法 用酮康唑(ketoconazole,KTZ)、四氯化碳(carbon tetrachloride,CCl₄)和对乙酰氨基酚(acetaminophen,APAP)在雄性大鼠中构建3种肝毒性模型,在不同时间点从腹腔大静脉采集外周血,3 000 r/min离心制备血清。用ELISA法测定血清中VDBP含量,用全自动生化分析仪测定血清中的丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、碱性磷酸酶(alkaline phosphatase,ALP)、总胆红素(total bilirubin,TBIL)、总胆汁酸(total biliary acid,TBA)、胆碱酯酶(cholinesterase,CHE)、谷氨酸脱氢酶(glutamate dehydrogenase,GLDH)、5'核苷酸酶(5'-nucleoticlase,5'-NT)、单胺氧化酶(monoamine oxidase,MAO)和苹果酸脱氢酶(malate dehydrogenase,MDH)含量。实验组与对照组进行t检验分析,检查指标值的差异是否具有统计学意义。结果 在CCl₄肝毒性模型中,与对照组相比,ALT,AST,TBIL和CHE在CCl₄给药后4 h起升高,差异均具有统计学意义(均P<0.05)。GLDH,MAO和MDH在给药后6 h起升高,差异均具有统计学意义(均P<0.05)。在KTZ肝毒性模型中,与对照组相比,ALT在KTZ给药6天后升高,差异具有统计学意义(P<0.05)。AST和TBIL在给药后8天升高,差异具有统计学意义(P<0.05)。在APAP肝毒性模型中,与对照组相比,ALP在APAP给药后4 h和8 h点升高,差异具有统计学意义(P<0.05)。CHE在给药后8 h点升高,差异具有统计学意义(P<0.05)。ALT和TBIL在给药后24 h点升高,差异具有统计学意义(P<0.05)。在3种肝毒性模型中,VDBP含量在给药后的所有时间点都降低,差异均具有统计学意义(均P<0.05)。结论 VDBP比其它肝功能指标能更灵敏地预测肝毒性的发生。     

2型糖尿病患者血清视黄醇结合蛋白4与血清超敏C反应蛋白、胰岛素抵抗的相关性研究

目的探讨2型糖尿病（type 2 diabetes mellitus,T2DM）患者血清视黄醇蛋白4（RBP4）与血清超敏C反应蛋白（hs-CRP）、胰岛素抵抗（IR）的关系。方法选取61例首次诊断的T2DM患者作为T2DM组,25例健康体检者作为对照组,检测2组血清RBP4、空腹血糖（FBG）、餐后2 h血糖（2 h PBG）、胰岛素（FINS）、hs-CRP水平。同时,测定身高、体质量,计算体质指数（BMI）。采用稳态模型法计算胰岛素抵抗指数（Homa-IR）。结果 T2DM组BMI、血清FBG、2 h PBG、FINS、hs-CRP、RBP4及Homa-IR均显著高于对照组（均P〈0.05）。Pearson相关分析结果显示,血清RBP4水平与BMI、血清FINS及Homa-IR、血清hs-CRP水平呈正相关（r=0.230、0.244、0.393、0.511,P〈0.05或P〈0.01）。多元逐步回归分析结果显示,Homa-IR、血清hs-CRP为RBP4的独立相关因素。结论 T2DM患者血清RBP4、hs-CRP水平显著升高,RBP4可能参与了IR与T2DM的发生、发展,RBP4可能为一新的炎症标志物。     

Polymyxin B and polymyxin B nonapeptide alter cytoplasmic membrane permeability in Escherichia coli

The effects of polymyxin B and polymyxin B nonapeptide (PMBN) on the permeability of the Escherichia coli cytoplasmic membrane were investigated. Both compounds caused loss of free amino acids, uracil and K+ from E. coli. The rates of loss promoted by polymyxin B were one and a half to two-fold greater than those caused by PMBN. Although PMBN mediated loss of low molecular weight substances from E. coli, it was not bactericidal. In contrast, polymyxin B treated E. coli lysed and rapidly lost viability. We suggest that the bactericidal activity of polymyxin B may be related to its previously reported ability to release cytoplasmic proteins from bacteria.