The interpretation of CO2 equilibration data to obtain plaque fluid buffer capacities, and comparison with results obtained by titration.

Titration measurements of pooled plaque fluid buffering capacity (Shellis and Dibdin, 1988), which showed a broadly defined minimum at pH 7, were compared with recent curves published by Carey et al. (1988a), which they obtained by an ultra-micro CO2-equilibration technique and which suggested a quite different profile, peaking sharply at pH 7.1. When analyzed in a different, more conventional way, the raw measurements in the latter study become more consistent with our own results and with earlier findings of Tatevossian (1977). In particular, we conclude that the peak at pH 7.1 is an artifact, and that Carey et al. underestimated buffer capacities below pH 6.4 and above pH 7.4. Rationales for the two modes of analysis are compared, and possible reasons for the remaining differences between the re-analyzed CO2-equilibration results and the titration results are discussed. Suggestions for the improvement of the accuracy of the CO2-equilibration technique are put forward.

Separation characteristics of alkylated guanines in high-performance liquid chromatography.

The retention behavior of thirteen alkylated guanines on normal-phase silica gel and amino columns and on reversed-phase ODS and phenyl columns was studied. The larger the alkyl substituent at the same position of guanine the weaker was the retention in the normal-phase chromatographic system and the greater the retention during reversed-phase chromatography. O6-Derivatives possess the lowest polarity in each set of isomers. An amino column was found to be of highest efficiency in terms of separation of the set of ethylguanine isomers and of benzylguanines studied. A phenyl column provided the best resolution of methylated guanines.

Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin.

A competitive ELISA assay has been developed that permits reproducible quantitation of the anticoagulant hirudin in buffer and urine. Coupling of peroxidase and hirudin was performed with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. In both solvents the lower limit of sensitivity was 8 ng hirudin/ml (0.08 AT-U) while the upper limit was 7.7 micrograms/ml (78.45 AT-U).

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing.

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Stabilized normal-phase high-performance liquid chromatographic analysis of aspirin and salicylic acid in solid pharmaceutical dosage forms.

A simultaneous analysis of aspirin and nonaspirin salicylates in solid pharmaceutical dosage forms is described. Two separate extraction procedures are employed, one for plain aspirin tablets and one for tablets containing aspirin plus buffers or antacids. The analyses of the extracted samples are accomplished by a stabilized normal-phase high-performance liquid chromatographic (HPLC) procedure. Prepared samples and standards are stable for up to 24 h, and the methodology is suitable for an automated HPLC system.

Visualization of carrier ampholyte patterns in granular gel slabs. 1. Applications to analytical electrofocusing.

1. The isoelectric banding patterns of three commercial carrier ampholytes (pH range 2-11) were visualized in both Sephadex gel slabs and on paper prints, by means of ultraviolet light. 2. The fluorescence pattern of Servalyt is described in detail. This pattern, which has sharply delineated bands, is characteristic, and is reproducible from run to run with different batches of Servalyt. A total of 56 fluorescent bands were identified on gel slabs, and 62 on prints. 3. Methods are described for identifying individual fluorescent bands on prints. A classification scheme for organizing the banding pattern of Servalyt and naming individual bands is presented. 4. On gel slabs and on prints, the fluorescent pattern of a carrier ampholyte may be used analytically as a map for the location and identification of focused protein bands. The utility of the fluorescent pattern for characterising commercial ampholyte mixtures is discussed. 5. Individual fluorescent bands on the gel slab have characteristic pH values, which are constant from run to run and remain constant in the presence of focused sample proteins. On prints, these bands serve as permanent internal markers for assigning isoelectric points to protein bands.

An analysis of the buffer system in the rumen of dairy cattle.

A method is presented for the analysis of buffer systems in the rumen using the first derivation of titration curves. Bicarbonate and volatile fatty acids (VFA) are the main components of the buffering system in the rumen fluid of dairy cattle under widely different feeding conditions. Phosphate from saliva is of little importance as a buffer, but neutralizes acids produced in the rumen. After studying five cows during the peripartal period a spontaneous and transient increase in the concentrations of VFA and a soluble marker (PEG) as well as a drop in pH and in the bicarbonate concentrations not related to feeding was observed in two animals that were sampled several hours before parturition. The potential risk of provoking rumen disturbances upon feeding animals close to the time of parturition, when buffering capacity may be minimal, is stressed.

Gastric acid secretion rate and buffer content of the stomach after eating. Results in normal subjects and in patients with duodenal ulcer.

New methods are described by which the buffer content and the rate and pattern of net gastric acid secretion in human subjects fed normal meals can be measured by use of sodium bicarbonate infusion to control intragastric pH. With these techniques, it was shown that the rate of acid secretion in response to a steak meal in seven duodenal ulcer patients was twice the rate achieved in six control subjects and that the amount of acid secreted after eating exceeded the peak histamine response in the ulcer patients but not in the controls. Meal-stimulated acid secretion, expressed as a function of the peak histamine response, was roughly correlated with the serum gastrin concentration (r = 0.45), but it was concluded that other factors must also contribute to the higher than normal secretory responses to a meal found in duodenal ulcer patients. Measurement of buffer content of the stomach revealed that the duodenal ulcer patients emptied the meal buffer at a much more rapid rate than the normal subjects. By 2 h after eating, the ulcer subjects had less than half as much buffer in their stomachs as the controls. The combination of acid hypersecretion and rapid buffer emptying leads to abnormally high gastric acidity after a meal in duodenal ulcer patients. These results suggest that, in addition to a large parietal cell mass, parietal cell responsiveness to a meal and the rate of buffer emptying may be important in the pathogenesis of duodenal ulcer.

Microbioassay of antimicrobial agents.

A previously described agar-diffusion technique for microbioassay of antimicrobial agents has been modified to increase sensitivity of the technique and to extend the range of antimicrobial agents to which it is applicable. This microtechnique requires only 0.02 ml of an unknown test sample for assay, and is capable of measuring minute concentrations of antibiotics in buffer, serum, and urine. In some cases, up to a 20-fold increase in sensitivity is gained relative to other published standardized methods and the error of this method is less than +/-5%. Buffer standard curves have been established for this technique, concurrently with serum standard curves, yielding information on antimicrobial serum-binding and demonstrating linearity of the data points compared to the estimated regression line for the microconcentration ranges covered by this technique. This microassay technique is particularly well suited for pediatric research and for other investigations where sample volumes are small and quantitative accuracy is desired. Dilution of clinical samples to attain concentrations falling with the range of this assay makes the technique readily adaptable and suitable for general clinical pharmacological studies. The microassay technique has been standardized in buffer solutions and in normal human serum pools for the following antimicrobials: ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, cephaloglycin, cephalexin, cephaloridine, cephalothin, erythromycin, rifamycin amino methyl piperazine, kanamycin, neomycin, streptomycin, colistin, polymyxin B, doxycycline, minocycline, oxytetracycline, tetracycline, and chloramphenicol.