Convenient purification of tritylated and detritylated oligonucleotides up to 100-mer.

Oligomers from crude phosphoramidite synthesis mixtures have been purified by reversed-phased high-performance liquid chromatography by exploiting the chromatographic variables of stationary phase pore size, chain length, and gradient shape. Chromatography was performed on oligomers up to 100-mer with mobile phases containing triethylammonium acetate/acetonitrile mixtures. Convenient guidelines are offered to enrich or purify synthetic oligomers. Tritylated oligomers up to 25 bases in length are best purified on C8 or C18, 80 A columns with moderate strength mobile phases using a combination of isocratic delays and shallow gradients. For oligomers longer than 25-mer, C3, 300 A columns provide adequate fast purification in as little as 5 min, while 300 A, C8 columns with long, slow gradients gave substantially increased purity. Chromatography of detritylated oligomers requires a modified approach. Up to 25-mer they are best purified on 80 A, C18 columns with much lower organic concentrations and shallower gradients than those used for tritylated oligomers. Detrytilated oligomers greater than 25-mer can be enriched on both C3 and C8, 300 A columns using the same conditions described for shorter detritylated oligomers.

Outcome following antimicrobial therapy for asymptomatic bacteriuria in elderly women resident in an institution.

Twenty-six elderly (mean age 83.3 +/- 8.7 years) institutionalized women with asymptomatic bacteriuria were treated with antibiotic therapy, including initial single-dose and subsequent 2 weeks' therapy, then 6 weeks' therapy if relapse occurred. Forty-seven courses of single-dose, 30 of 2 weeks', and 10 of 6 weeks' therapy were given during a 1-year period. At 8 weeks of follow-up, 57% of single-dose courses, 52% of 2-week, and 29% of 6-week had been followed by relapse, and 32%, 24%, and 29%, respectively, by reinfection. Outcome with single-dose therapy did not correlate with infecting organism, antimicrobial therapy, or presence of pyuria with the infection. However, residents who persistently relapsed following single-dose therapy appeared to be a less-well population, as evidenced by a significantly greater age, number of chronic disease diagnoses and medications, as compared to those who were cured by single-dose therapy. Thus, recurrent infection, including both relapse and reinfection is the usual short-term outcome following therapy for asymptomatic bacteriuria in this population. Differences in patient characteristics may prove useful in predicting which individuals may respond to minimal therapy.

An isolated perfused model for the study of colonic metabolism and transport.

The murine colonic perfusion model allows for the examination of absorption, metabolism, and portal transfer by the colon under physiologic conditions. This model was characterized by the use of four radiolabeled compounds: estradiol and Vitamin D3, both physiologically active circulating steroid compounds, and benzo[a]pyrene and N-acetylaminofluorene, xenobiotic carcinogens of the aromatic hydrocarbon and aromatic amide classes, respectively. Hemodynamic parameters and oxygen consumption of the preparation were stable throughout perfusion. Estradiol and N-acetylaminofluorene entered the portal vein at a rate of 2% of the lumenal dose per hour. Benzo[a]pyrene crossed at 0.4% of the lumenal dose per hour. The rate of transfer of Vitamin D3 was negligible. Analysis of the lumenal label revealed only substrate. In all experiments less than 0.02% of the applied substrate remained in the tissue compartment. Analysis of the vascular perfusate demonstrated evidence for sulfates of estradiol and N-acetylaminofluorene. Three conjugate classes were found associated with benzo[a]pyrene, constituting 42% of the portal label. Hydrolysis data suggests the presence of double conjugates of benzo[a]pyrene involving glutathione. In the case of aromatic hydrocarbons, conjugation, particularly thioether formation, implies hydroxylation and epoxide formation. For sulfation an N-acetylamino-fluorene ring or N-hydroxylation is required. The latter process could allow for the delivery of highly carcinogenic N-O sulfates to the liver.

Cefoxitin inactivation by Bacteroides fragilis.

We have surveyed the susceptibility of 1,575 clinical isolates of the Bacteroides fragilis group of organisms to cefoxitin and eight other antimicrobial agents. Eleven isolates, 0.7% of the total, were highly cefoxitin resistant and had minimum inhibitory concentrations of greater than or equal to 64 micrograms/ml. These isolates were also resistant to other beta-lactam antibiotics. Of 11 isolates, 4 were able to inactivate cefoxitin in broth cultures, as measured by microbiological and high-pressure liquid chromatography assays. Two distinct patterns of cefoxitin breakdown products were detected by high-pressure liquid chromatography analysis. The beta-lactamase inhibitors clavulanic acid and sulbactam failed to show synergism with cefoxitin. These data demonstrate that members of the B. fragilis group have acquired a novel resistance mechanism enabling them to inactivate cefoxitin.

Increased oxidation of a chemical carcinogen, benzo(a)pyrene, by colon tissue biopsy specimens from patients with ulcerative colitis.

Colonic biopsy specimens from patients with ulcerative colitis and normal subjects were studied for the ability to metabolize an environmental carcinogen, benzo(a)pyrene. Approximately 73% of 30 colonic biopsy specimens from 7 ulcerative colitis patients could metabolize benzo(a)pyrene to oxidized products, with an average production of 11.6 nmol/mg biopsy protein. In contrast, 39% of 23 biopsy specimens from 5 normal persons showed metabolic activity, with an average of 2.79 nmol benzo(a)pyrene metabolites/mg biopsy protein. Thus, benzo(a)pyrene oxidation activity in colonic tissue from colitis patients was, on the average, fourfold greater than that in normal subjects. This elevated metabolic activity appeared to be unrelated to the state of inflammation in the biopsy section. There was a tendency toward increased metabolic activity in the distal colon. Although there is no evidence that benzo(a)pyrene itself is "the colon carcinogen," this chemical belongs to a broad class of environmental carcinogens, the polycyclic aromatic hydrocarbons. Our findings suggest that the colonic mucosa of patients with ulcerative colitis has a greater ability than that of normal subjects to oxidize such chemicals possibly to electrophiles with higher mutagenic potential.

An internally-standardized assay for amphotericin B in tissues and plasma.

A high-performance liquid chromatographic (HPLC) method with p-nitrophenol as internal standard is described for the rapid analysis of amphotericin B recovered by methanolic extraction from tissues and plasma. Programmed, gradient elution of the ODS column was used with detection by tungsten light at 388 nm. Standard curves were derived based on the peak height ratios. The lowest reproducible limit of the assay was 0.04 micrograms/ml with plasma. The extraction and chromatographic procedures recovered 53-71% of the amphotericin B from each of these sources. The coefficient of variation of the recovery ratios was less than 18% from plasma over a range of concentrations of amphotericin B from 0.08 to 10.0 micrograms/ml. Recovery from tissues, studied over a narrower concentration range, showed a similar degree of precision. Variations in precolumns apparently resulting in selective binding of the amphotericin B were found to have a systematic but important influence on recovery efficiency. No substances were detected which interfered with the assay procedures as described. By incorporating an internal standard we have enhanced the reliability and flexibility of the HPLC assay for amphotericin B especially for assay of tissues.

Enzymatic methylation of microsomal metabolites of benzo(a)pyrene.

These studies suggest that the microsomal metabolism of benzo(a)pyrene (BP) produces metabolites which can be methylated by the catechol-o-methyltransferase (COMT)/S-adenosylmethionine (SAM) enzyme/donor combination. Induced microsomes converted 12 to 15% of substrate BP to polar products. Approximately 0.06% of substrate BP was recovered as COMT/SAM-reactive substances. In tests for specificity, COMT/SAM was found to react with catechols, but not with dihydrodiols, quinones, a phenol, an epoxide, or 1,4-hydroquinone. Organic extracts of COMT/[14C]SAM incubations with BP were fractionated by high-performance liquid chromatography. The appearance of radiolabeled chromatographic bands required the presence of substrate BP, microsomes, and COMT/[14C]SAM. When the Ames mutagenesis assay was supplemented with COMT/SAM, a 36% reduction was observed in the number of revertant colonies induced by the microsomal oxidation of BP. In contrast, the mutagenic properties of 2-aminofluorene were not affected by COMT/SAM. These observations indicate that COMT/SAM does not generally inhibit mixed-function oxidase activity but rather reacts with substances which are activated by ring oxygenations.

Assay of gentamicin and tobramycin in sera of patients by gas-liquid chromatography.

Therapeutic levels of gentamicin and tobramycin in the sera of patients were measured by gas-liquid chromatography. Concentration-response curves for both drugs were linear over an expected therapeutic range of 1.3 to 12.5 mug/ml (coefficient of determination was >0.97). Coefficients of variation for chromatographic response to gentamicin varied from 6.3 to 9.6%, and to tobramycin from 3.8 to 13.5%. Paired gas-liquid chromatography and microbiological assays for patient serum aminoglycoside levels were performed on 106 gentamicin and 40 tobramycin sera. At levels <2.0 mug/ml, the average difference of estimates between the two assay techniques for gentamicin and tobramycin were, respectively, 38 and 29%. At drug concentrations >2.0 mug/ml, the mean difference between paired estimates was near 20% for both aminoglycosides. The speed, precision, and accuracy of the gas-liquid chromatography assay indicate that it can be a useful alternative to the microbiological procedure for the determination of gentamicin and tobramycin levels in human serum.

Gas-liquid chromatographic method for the assay of aminoglycoside antibiotics in serum.

A gas-liquid chromatographic (GLC) method is presented for the rapid analysis of gentamicin, tobramycin, netilmicin, and amikacin from human serum. This procedure may have application to all aminoglycoside drugs. The three isomers of gentamicin are resolved as two bands, while tobramycin, netilmicin, and amikacin appear in this system as single bands. Normal serum constituents do not interfere with chromatograms. Thus far, no assay interference has been found in cases where other drugs and antibiotics were administered concurrently with aminoglycoside therapy. Dose-response data demonstrating linear recovery are included for all four aminoglycosides as well as a comparison of the GLC method with the microbiological method for the assay of gentamicin and amikacin. Quantitation is based upon the relative response of the antibiotics to a fixed amount of the internal standards, either kanamycin A or paromomycin B. These standards are clearly resolved as symmetrical peaks from the antibiotics of assay interest. Isothermal chromatographic analysis time is less than 8 min, while total assay time per single serum specimen is approximately 50 min. Preparation of serum includes: precipitation, evaporative drying of the supernatant, a two-stage derivatization (N-trimethylsilylimidazole, N-heptafluorobutyrylimidazole), and a single hexane extraction with a water wash. The methodology described may be applied to the analysis of other compounds (e.g., saccharides, amino-saccharides, amino acids, etc.) which do not rapidly partition into an organic phase.

Internal standards for gas chromatographic analysis of metabolic end products from anaerobic bacteria.

2-Methylpentanoic acid and benzoic acid are suggested for use as routine internal standards for gas chromatographic analysis of microbial end products.

Effect of dissolved oxygen and Eh and Bacteroides fragilis during continuous culture.

Bacteroides fragilis subsp. fragilis was maintained in a chemostat modified for anaerobic conditions to test the effects of dissolved oxygen and Eh on growth. Using a defined medium containing glucose and a dilution rate of 0.16 h -1, a stable population of 3 X 10(9) colony-forming units/ml was present. At this steady state, the pH was 5.6, the Eh was -50 mV, and the dissolved oxygen concentration was 0% atmospheric saturation. The Eh was then adjusted to +300 mV by adding potassium ferricyanide while oxygen was excluded; in this system there were no demonstrable changes from the steady state in viable cells, pH, glucose concentration, or volatile fatty acid production. In other experiments oxygen was introduced into the original steady state at a dissolved oxygen concentration of 10% atmospheric saturation for a period of 6 to 8 h. During O2 exposure, the viable cell count decreased at a rate comparable to the theoretical washout rate for a static bacterial culture. Similar results were obtained with a dissolved oxygen concentration of 25 and 100%. Other effects of O2 exposure included an increase in Eh from -50 to +250 mV, a decrease in glucose consumption, and a decrease in volatile fatty acid production. These results suggest that dissolved oxygen has a bacteriostatic effect on B. fragilis in continuous culture, which may be independent of changes in Eh alone.

Rapid diagnosis of anaerobic infections by direct gas-liquid chromatography of clinical speciments.

Current methods to isolate and identify anaerobic bacteria are laborious and time consuming. It was postulated that the short-chain fatty acids (SCFA) produced by these organisms might serve as microbial markers in clinical material. 98 specimens of pus or serous fluid were analyzed by gas-liquid chromatography, and findings were compared with culture results. Good correlations were found for the recovery of anaerobic Gram-negative bacilli and the presence of isobutyric, butyric, and succinic acids. 19 of 20 specimens with significant amounts of these acids (greater than 0.01 mumol/ml) yielded bacteroides or fusobacteria. Culture of the single "false-positive" specimen failed to grow anaerobic Gram-negative bacilli, although clinical data and Gram-stain suggested their presence. 77 of 78 specimens which has insignificant concentrations of the marker acids failed to yield anaerobic, Gram-negative bacilli in culture. The single "false-negative" specimen yielded Bacteroides pneumosintes, an organism which does not ferment carbohydrates. It is concluded that direct gas-liquid chromatographic analysis of clinical specimens provides a rapid presumptive test for the presence of anaerobic, Gram-negative bacilli.

Effects of time and growth media on short-chain fatty acid production by Bacteroides fragilis.

Gas-liquid chromatography was used to monitor the evolution of short chain fatty acids by Bacteroides fragilis in five media. Acetic and succinic acids, the prominent end products encountered, were readily detected within 24 h. Propionic, isobutyric, isovaleric, and lactic acids were usually recorded in more limited quantities. Maximum rates of bacterial multiplication, glucose catabolism, and end-production coincided with the first 24 h in carbohydrate-supplemented media. Extended incubation (672 h) favored substantial succinate increases in three of five media. These observations suggest that incubation time and composition of the medium are important determinants in short chain fatty acid production by B. fragilis.

Rapid gas chromatographic technique for presumptive detection of Clostridium botulinum in contaminated food.

A simple gas-liquid chromatography end-product assay is reported for butyric and other short-chain fatty acids as presumptive indicators of Clostridium botulinum contamination in food.