Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase.

Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods.     

Biochemical properties of tartrate-resistant acid phosphatase in serum of adults and children.

Spectrophotometry of total acid phosphatase activity in children's sera showed an average value of 22.4 +/- 2.9 and 7.4 +/- 0.8 U/liter, for the hydrolysis of p-nitrophenyl phosphate and alpha-naphthyl phosphate, respectively. Analyses of "band 5b", after electrophoresis on acrylamide gel, gave even higher values. The values for children's sera were much higher than those for sera from adults. The multiplicity of acid phosphatases in sera of children and adults was studied by electrophoresis on acrylamide gel and by chromatography on CM-Sepharose. Both methods showed the major acid phosphatase in children's sera to be an acid pyrophosphatase, band 5b. Its catalytic properties are indistinguishable from the enzyme previously isolated from the spleen of leukemic reticuloendotheliosis.     

Lipoxygenic micromethod for specific determination of lipase activity in serum and duodenal fluid.

We propose a rapid enzymatic micromethod for the specific determination of lipase (EC 3.1.1.3) activity in serum and duodenal fluid. Free linoleic acid produced during 10-min incubation of 10 mul of sample with 1 ml of substrate (trillinolein emulsion) at 30 degrees C is converted by lipoxygenase (EC 1.99.2.1), in a coupled reaction, to its hydroperoxide, which is measured photometrically after solubilizing the reaction mixture in ethanol. Lipase activity is calculated from the rate of hydroperoxide formation, with linoleic acid as primary standard. The velocity of the reaction is greatest at pH 8.8, 35-37 degrees C, and a deoxycholate concentration of 3.6 mmol/liter. The energy of activation is 6.7 kcal/mol. The differing "apparent" Km values obtained for lipase in undiluted serum (4 X 10(-5) mol/liter) and in albumin-based diluents (1 X 10(-5) mol/liter) indicate the presence of a competitive inhibitor in the serum matrix. We detected no lipase activity in urine. Results by the proposed method correlate well with those by a copper soap extraction method (r = 0.95), but values are significantly higher for pancreatitis patients' sera (slope 1.6). The linear dynamic range extends to 1000 U/liter. Hemolysis, lipemia, and hyperbilirubinemia do not interfere. The normal range is 40-60 U/liter. Lipase activity of pancreatitis patients generally exceed 1000 U/liter during the acute phase and 250 U/liter for as long as 10 days after it.     

Simple, highly selective screening method for barbiturates in urine.

I present a new, simple colorimetric method for detecting and estimating barbiturates in urine. After the barbiturates are extracted with ether, an aliquot of the washed ether phase is added to the color reagent (a bivalent mercury/dithizone chelate in chloroform). On addition of diluted pyridine and shaking, a pinkish-violet color appears if a barbiturate is present. The overall sensitivity of the above method was evaluated by probit analysis in the case of sodium phenobarbital. The concentration of sodium phenobarbital in urine detectable at least 99% of the time was 6.72 mg/liter, with 95% confidence limits of 5.37 to 10.36 mg/liter. Sodium phenobarbital, 10 mg/liter, can be detected in the presence of phenytoin (50 mg/liter), glutethimide (100 mg/liter), or bemegride (100 mg/liter). The whole procedure requires less than 10 min. An excretion study illustrates application of the procedure.     

Fluorometric assay of serum acid or alkaline phosphatase, either in solution or on a semisolid surface.

We describe enzymatic fluorometric methods for determining activities of serum alkaline phosphatase and of serum acid phosphatase in solution and on silicone rubber pads. 4-Methylumbelliferone phosphate is used as substrate, in either tris(hydroxymethyl)aminomethane or citrate buffer. In solution, the reaction is measured at 37 degrees C in a 3-ml Pyrex cuvette. Measurements on the pads are also made at 37 degrees C, after establishing a stable substrate film by lyophilizing all reagents on the surface of the pads. Only 20 to 30 mul of substrate solution, 50 mul of buffer solution, and 1 to 10 mul of blood are necessary, making a total volume of 51 to 60 mul for each assay. The rate of appearance of the fluorescent 4-methylumbelliferone liberated from 4-methylumbelliferone phosphate by the enzymatic action is measured and equated to enzyme activity. Calibration plots of the change in fluorescence per minute vs. enzyme activity for measurements in solution and on pads show a good proportionality in the range of 30.8 to 633 U/liter for alkaline phosphatase and in the range of 0.265 to 5.3 King-Armstrong units for acid phosphatase, indicating the usefulness of these methods in the clinical laboratory.     

In vitro activities of daptomycin against 2,789 clinical isolates from 11 North American medical centers

The in vitro activity of daptomycin is affected by the concentration of calcium cations in the test medium. Mueller-Hinton broth is currently adjusted to contain 10 to 12.5 mg of magnesium per liter and 20 to 25 mg of calcium per liter, but for testing of daptomycin, greater concentrations of calcium (50 mg/liter) are recommended to better resemble the normal concentration of ionized calcium in human serum. Two levels of calcium were used for broth microdilution tests of 2,789 recent clinical isolates of gram-positive bacterial pathogens. MICs of daptomycin were two- to fourfold lower when the broth contained additional calcium. For most species, however, the percentages of strains that were inhibited by 2.0 microg of daptomycin per ml were essentially identical with the two broth media. Enterococci were the important exception; i.e., 92% were inhibited when tested in calcium-supplemented broth but only 35% were inhibited by 2.0 microg/ml without the additional calcium. This type of information should be considered when selecting criteria for defining in vitro susceptibility to daptomycin.     

Comparative stability studies of antipseudomonal beta-lactams for potential administration through portable elastomeric pumps (home therapy for cystic fibrosis patients) and motor-operated syringes (intensive care units)

The stability of antipseudomonal beta-lactams in concentrated solutions was examined in view of their potential administration by continuous infusion with external pumps (for intensive care patients) or with portable pumps carried under clothing (for cystic fibrosis patients). Aztreonam (100 g/liter), piperacillin (128 g/liter, with tazobactam), and azlocillin (128 g/liter) remained 90% stable for up to more than 24 h at 37 degrees C (mezlocillin [128 g/liter] was stable at 25 degrees C but not at 37 degrees C). Ceftazidime (120 g/liter), cefpirome (32 g/liter), and cefepime (50 g/liter) remained 90% stable for up to 24, 23.7, and 20.5 h at 25 degrees C but only for 8, 7.25, and 13 h at 37 degrees C, respectively. The control of temperature therefore appears to be critical for all three cephalosporins that cannot be recommended for use in portable pumps carried under clothes for prolonged periods for reasons of stability. Cefpirome and cefepime solutions developed an important color change (from light yellow to dark red) upon exposure when stored at 30 degrees C or higher. Degradation of ceftazidime was accompanied by the liberation of pyridine which, at 37 degrees C, was in excess of what is allowed by the U.S. Pharmacopeia, i.e., 1.1 mg/liter, after 8 and 12 h for drug concentrations of 12 and 8.3%, respectively. Imipenem and meropenem are too unstable (10% degradation at 25 degrees C after 3.5 and 5.15 h, respectively) to be recommended for use by continuous infusion. Faropenem, examined in comparison with imipenem and meropenem, proved as stable as aztreonam or piperacillin.     

大鼠去卵巢后血清酶和骨元素镁的变化

目的观察大鼠去卵巢后血中3种磷酸酶和骨元素的变化,探讨雌激素水平下降引起骨质疏松的原理。方法60周龄雌性SD大鼠30只,随机分为实验组和对照组各15只,平均体质量(385±13)g,手术前取血,测定血清碱性磷酸酶,酸性磷酸酶和骨特异性碱性磷酸酶。实验组切除双侧卵巢,对照组假性切除卵巢,继续饲养6个月后,取血清测定上述3种磷酸酶活性,30只大鼠全部处死,取髂骨,测定骨的元素含量。用中子活化方法测定骨的钙、镁、钠、锰、锶和钾元素的含量。结果术前血清碱性磷酸酶、酸性磷酸酶和骨特异性碱性磷酸酶的活性分别为(21.2±7.1),(19.3±4.8)和(15.1±3.5)U/L,术后血清碱性磷酸酶、酸性磷酸酶和骨特异性碱性磷酸酶的活性分别为(15.4±5.8),(12.9±2.4)和(5.6±1.1)U/L,摘除卵巢后上述各项生化指标均显著降低(P<0.001)。大鼠切除卵巢后实验组髂骨镁元素含量为(4.32±0.2O)mg/g,对照组为(5.12±0.15)mg/g。实验组含量显著低于对照组(t=12.40,P<0.001)。钙、钠、锰、锶和钾元素含量两组比较差异无显著性意义。结论大鼠去卵巢6个月后血清碱性磷酸酶、酸性磷酸酶、骨特异性碱性磷酸酶和髂骨的镁元素均显著降低。     

Diagnosis of hydatid cyst by percutaneous aspiration: value of electrolyte determinations

A study was made of the fluid from 11 hydatid cysts (10 hepatic, one renal) and from 50 nonparasitic cysts (25 renal, 10 ovarian, 10 pancreatic and five hepatic). Our analysis indicated that the chloride (range 62-95 mEq/liter), the sodium (range 125-139 mEq/liter) and the potassium content (range 5.1-7.5 mEq/liter) of the former differed significantly (p less than 0.01) from those of the controls. Hydatid cysts may be indistinguishable from others with both CT and ultrasound and therefore the electrolyte data are of diagnostic value when a hydatid cyst is accidentally punctured percutaneously.     

Effect of calcium, magnesium, and zinc on ticarcillin and tobramycin alone and in combination against Pseudomonas aeruginosa.

Correlation between in vitro and in vivo test results for synergy between carboxypenicillins and aminoglycosides against Pseudomonas aeruginosa is poor. Although the divalent cation content of culture media is known to affect aminoglycoside susceptibility testing for P. aeruginosa, this effect of divalent cations has not been examined for synergy testing of carboxypenicillin-aminoglycoside interaction against P. aeruginosa. The minimal inhibitory concentrations (MICs) of tobramycin and ticarcillin and the interaction of these drugs in combination were studied by a microtitration method for 36 strains of P. aeruginosa in Mueller-Hinton broth with varying supplements of calcium, magnesium, and zinc. The supplementation of Mueller-Hinton broth to 50 or 100 mg of calcium per liter had a significant effect in increasing the tobramycin MIC (P less than 0.01), as well as decreasing the degree of synergy between ticarcillin and tobramycin (P less than 0.01). Supplementation to 20 mg of magnesium per liter, 1.0 mg of zinc per liter, or both did not significantly affect tobramycin MIC or the interaction of tobramycin and ticarcillin. Supplementation to 50 or 100 mg of calcium per liter rendered any additional effect of magnesium and zinc on aminoglycoside MIC and aminoglycoside-carboxypenicillin interaction negligible. If these results for ticarcillin and tobramycin are confirmed for other carboxypenicillins and aminoglycosides, then the Mueller-Hinton broth used for P. aeruginosa aminoglycoside susceptibility and synergy testing may need to be supplemented only with calcium at a concentration of 50 mg/liter.     

Alkaline phosphatase. II. Conditions affecting determination of total activity in serum.

Results of previous experiments on isolated purified isoenzymes of alkaline phosphatase from humans have been confirmed on sera containing relatively large activities of the different isoenzymes. The most remarkable finding is that activation by N-ethylaminoethanol is much more pronounced, in the case of the intestinal and placental isoenzymes, than is activation by diethanolamine. For several reasons, it is suggested that N-ethylaminoethanol is the buffer of choice, 0.1 mol/liter concentration for routine measurements and 1 mol/liter in those cases where the determination of the intestinal or placental isoenzymes is important. Mg2+ could be omitted because its addition increases the activity only marginally. Normal values for adults with use of 0.1 mol/liter N-ethylaminoethanol are 59 +/- 36 (2 SD) U/liter (n = 126).     

Low-dose dexamethasone as adjunctive therapy for disseminated Mycobacterium avium complex infections in AIDS patients.

Five human immunodeficiency virus-infected patients with disseminated Mycobacterium avium complex infection had progressive weight loss and persistent fever despite multidrug antimycobacterial therapy. These patients were given daily low-dose oral dexamethasone (typically 2 mg/day) as adjunctive therapy. All had substantial and sustained weight gain (12 to 50% of pre-steroid treatment body weight [P < 0.03]), reduction in fever, and an improved sense of well-being. The serum albumin level increased during dexamethasone therapy (from 3.06 +/- 0.59 g/dl [mean +/- standard deviation] to 3.9 +/- 0.22 g/dl [P < 0.01]), while the serum alkaline phosphatase level fell (from 368 +/- 247 U/liter to 128 +/- 43.6 U/liter [P < 0.04]). Further studies of the potential role for corticosteroids in the management of disseminated M. avium complex infections in human immunodeficiency virus-infected patients are warranted.     

Macromolecular alkaline phosphatase and an immunoglobulin G that inhibited alkaline phosphatase in a patient's serum

Macromolecular alkaline phosphatase (EC 3.1.3.1) was found in the serum of a patient suffering from myasthenia gravis (adult type II) complicated with thymoma, and was shown by immunoelectrophoresis to be bound to immunoglobulins A and G (IgG). Placental alkaline phosphatase, complexed with either the patient's serum or IgG purified from the patient's serum, remained at the origin on electrophoresis, with significant loss of activity. Intestinal alkaline phosphatase, complexed with either the patient's serum or the patient's IgG, migrated to a position similar to that of the macromolecular alkaline phosphatase in the patient's serum on electrophoresis. About 50% of the placental alkaline phosphatase activity was inhibited with 0.1-0.2 g of the patient's IgG per liter, but 6.93 g of the IgG per liter was required for about 20% inhibition of the intestinal alkaline phosphatase activity. The complex of intestinal alkaline phosphatase with the patient's IgG was fairly heat stable. From these results, we concluded that the macromolecular alkaline phosphatase in the patient's serum consisted of intestinal alkaline phosphatase and IgG that was specific for placental alkaline phosphatase.     

In vitro activity of tosufloxacin, a new quinolone, against respiratory pathogens derived from cystic fibrosis sputum.

By using broth microdilution methods, the in vitro activity of tosufloxacin (A-64730), a new quinolone, was compared with those of other agents, including five quinolones, against geographically diverse cystic fibrosis sputum isolates obtained from 26 cystic fibrosis centers in the United States. These included Pseudomonas aeruginosa, conventional as well as especially resistant (ceftazidime, aztreonam, gentamicin, and/or tobramycin) isolates: Escherichia coli; Pseudomonas cepacia; Staphylococcus aureus; and Haemophilus influenzae. Tosufloxacin MICs for 50 and 90% of isolates of standard P. aeruginosa were 0.5 and 2.0 mg/liter, for resistant P. aeruginosa they were 4.0 and greater than 16.0 mg/liter, for E. coli they were less than or equal to 0.016 mg/liter, for P. cepacia they were 4.0 and 8.0 mg/liter, for S. aureus they were 0.063 and 0.063 mg/liter, and for H. influenzae they were less than or equal to 0.016 and 0.032 mg/liter, respectively. Tosufloxacin activities against standard and resistant strains of P. aeruginosa were similar to those of comparative quinolones. Against E. coli, tosufloxacin activity was similar to those of other quinolones. Against S. aureus, tosufloxacin activity was similar to those of trimethoprim-sulfamethoxazole and cephalexin, but tosufloxacin was more active than other agents. Against H. influenzae, tosufloxacin activity was similar to those of other quinolones. There was minor diminution of activity at pH 8.2 but major diminution of activity at pH 5.2 and at inoculum sizes of greater than or equal to 10(7) CFU/ml. Activity was unaffected by sputum but was enhanced by serum and by the omission of cation supplementation. Tosufloxacin has consistent activity against common cystic fibrosis pathogens. Its high degree of activity against S. aureus with activity maintained against P. aeruginosa and other gram-negative bacteria of interest suggests that further in vitro studies and assessment of activity in in vivo models of cystic fibrosis pulmonary infections are warranted.     

In vitro activities of gatifloxacin, two other quinolones, and five nonquinolone antimicrobials against obligately anaerobic bacteria.

The activity of the new fluoroquinolone gatifloxacin was compared with those of other quinolones and antimicrobial agents of other classes against 294 anaerobes by the broth microdilution technique. For all strains tested, gatifloxacin MICs at which 50 and 90% of the isolates were inhibited were 0.5 and 2 mg/liter, respectively, and were 3 to 4 dilution steps lower than, e.g., ciprofloxacin.     

In vitro activities of isavuconazole and other antifungal agents against Candida bloodstream isolates

Isavuconazole is the active component of the new azole antifungal agent BAL8557, which is entering phase III clinical development. This study was conducted to compare the in vitro activities of isavuconazole and five other antifungal agents against 296 Candida isolates that were recovered consecutively from blood cultures between 1995 and 2004 at a tertiary care university hospital. Microdilution testing was done in accordance with CLSI (formerly NCCLS) guideline M27-A2 in RPMI-1640 MOPS (morpholinepropanesulfonic acid) broth. The antifungal agents tested were amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and isavuconazole. C. albicans was the most common species, representing 57.1% of all isolates. There was no trend found in favor of non-Candida albicans species over time. In terms of MIC(50)s, isavuconazole was more active (0.004 mg/liter) than amphotericin B (0.5 mg/liter), itraconazole (0.008 mg/liter), voriconazole (0.03 mg/liter), flucytosine (0.125 mg/liter), and fluconazole (8 mg/liter). For isavuconazole, MIC(50)s/MIC(90)s ranged from 000.2/0.004 mg/liter for C. albicans to 0.25/0.5 mg/liter for C. glabrata. Two percent of isolates (C. glabrata and C. krusei) were resistant to fluconazole; C. albicans strains resistant to fluconazole were not detected. There were only two isolates with MICs for isavuconazole that were >0.5 mg/liter: both were C. glabrata isolates, and the MICs were 2 and 4 mg/liter, respectively. In conclusion, isavuconazole is highly active against Candida bloodstream isolates, including fluconazole-resistant strains. It was more active than itraconazole and voriconazole against C. albicans and C. glabrata and appears to be a promising agent against systemic Candida infections.     

蜂胶与三种化学防腐剂抑菌性能的比较

目的研究蜂胶与某些化学防腐剂的抑菌性能及其稳定性。方法采用肉汤稀释法和定量抑菌试验法,对蜂胶及三种化学防腐剂抑菌性能进行比较观察。结果蜂胶对金黄色葡萄球菌、大肠埃希菌和黑曲霉菌的最低抑制浓度依次为7.8、31.25和31.25 g/L。苯甲酸钠对金黄色葡萄球菌最低抑制浓度为62.5 g/L,山梨酸和山梨酸钾对金黄色葡萄球菌、大肠埃希菌和黑曲霉菌的最低抑制浓度达到125 g/L以上。含10 g/L的蜂胶溶液对金黄色葡萄球菌作用10 min,抑菌率可达99.92%;对大肠埃希菌作用20 min,抑菌率仅为67.22%。苯甲酸钠、山梨酸、山梨酸钾对金黄色葡萄球菌和大肠埃希菌作用20 min,抑菌率均达不到50%。含量10 g/L蜂胶经55℃存放14 d,对金黄色葡萄球菌杀灭效果无明显下降。结论蜂胶对细菌繁殖体和霉菌都有较强的抑菌作用,其储存性能稳定;蜂胶抑菌性能明显优于苯甲酸钠、山梨酸、山梨酸钾等防腐剂。     

Evaluation of telavancin activity versus daptomycin and vancomycin against daptomycin-nonsusceptible Staphylococcus aureus in an in vitro pharmacokinetic/pharmacodynamic model

Daptomycin-nonsusceptible (DNS) Staphylococcus aureus strains have been reported over the last several years. Telavancin is a lipoglycopeptide with a dual mechanism of action, as it inhibits peptidoglycan polymerization/cross-linking and disrupts the membrane potential. Three clinical DNS S. aureus strains, CB1814, R6212, and SA-684, were evaluated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations (starting inoculum, 10(8.5) CFU/g) for 120 h. Simulated regimens included telavancin at 10 mg/kg every 24 h (q24h; peak, 87.5 mg/liter; t(1/2), 7.5 h), daptomycin at 6 mg/kg q24h (peak, 95.7 mg/liter; t(1/2), 8 h), and vancomycin at 1 g q12h (peak, 30 mg/liter; t(1/2), 6 h). Differences in CFU/g between regimens at 24 through 120 h were evaluated by analysis of variance with a Tukey's post hoc test. Bactericidal activity was defined as a ≥3-log(10) CFU/g decrease in colony count from the initial inoculum. MIC values were 1, 0.25, and 0.5 mg/liter (telavancin), 4, 2, and 2 mg/liter (daptomycin), and 2, 2, and 2 mg/liter (vancomycin) for CB1814, R6212, and SA-684, respectively. Telavancin displayed bactericidal activities against R6212 (32 to 120 h; -4.31 log(10) CFU/g), SA-684 (56 to 120 h; -3.06 log(10) CFU/g), and CB1814 (48 to 120 h; -4.9 log(10) CFU/g). Daptomycin displayed initial bactericidal activity followed by regrowth with all three strains. Vancomycin did not exhibit sustained bactericidal activity against any strain. At 120 h, telavancin was significantly better at reducing colony counts than vancomycin against all three tested strains and better than daptomycin against CB1814 (P < 0.05). Telavancin displayed bactericidal activity in vitro against DNS S. aureus isolates.     

Arginase in patients with breast cancer

The mean arginase activity in breast cancers (n = 80) was significantly higher than in control tissues and it accounted for 0.31 +/- 0.23 U/g wet tissue and 0.083 +/- 0.061 U/g (P < 0.05), respectively. With the cutoff value of 0.1 U/g wet tissue, raised arginase activity was observed in 74% of tumors. The preoperative arginase activity in blood serum from women with breast cancer was 11.2 +/- 7.9 U/l (n = 115), and it was significantly higher than in 70 healthy controls, where it was 5.7 +/- 2.4 U/l (P < 0.05). With the cutoff value for normal serum arginase activity above 8.0 U/l, the activity was raised in 10% of control individuals, and in 63% of women with breast cancer. The sensitivity and specificity of the arginase test in blood serum were 63% and 60%, respectively. Two isoforms immunologically identical to human kidney arginases (L-arginine amidinohydrolase) were found in both normal and cancerous breast tissues. The level of anionic form was similar in control and cancerous tissues, whereas the cationic isoform predominated in breast cancer. The cationic isoform was the only one present in serum of both ill and healthy women, and its level was higher in patients with breast cancer. Thus, it can be concluded that the cationic isoform is responsible for the increase of arginase activity in serum of patients with breast cancer.