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[Determination of the degree of base protonation in isionic solutions of DNA from the curve of conductometric titration].

Conductance of the DNA isoionic solution at different degrees of neutralization has been investigated at 25 degrees. There are regions on the conductometric titration curve corresponding to titration of: 1) primary phosphoryl groups 2) guanine 3) adenine 4) adenine and cytosine in half-protonated pairs 5) thymine. The pH intervals corresponding to protonation of bases in isoionic solutions are shifted into alkaline region compared with Na-DNA solutions. It has also been found for molar per cent of unprotonatad bases to equal to that of thymine determined as from conductometric titration curve so from chromatographic analysis of the DNA hydrolysate.

DNA↗
PubMed1985

The titration hypothesis revisited: nicotine gum reduces smoking intensity.

Supplemental nicotine gum reduced the intensity of smoking in six one-and-a-half to two-pack-a-day smokers. The study involved two 4-hour self-paced smoking sessions where nicotine and placebo chewing gum were administered in a double-blind fashion. Puff volume, puff duration, inhaled volume, inhaled duration, and interpuff interval were calculated for each puff on each cigarette. Blood was drawn for nicotine levels at regular intervals as well as before and after each cigarette. Cardiovascular measures were made at regular intervals. The nicotine gum reduced smoking frequency and intensity as predicted by the titration hypothesis. Precise titration (i.e., equal nicotine blood levels on both test days) was confounded by changes in smoked nicotine delivery produced by the gum. The gum, whether placebo or active, increased smoked nicotine absorption. However, while nicotine blood levels were slightly higher on nicotine gum day, the differences may not be biologically meaningful since heart rate and blood pressure increases were similar on both days. Difficulties testing the titration hypothesis are discussed.

Adult↗
PubMed1985

Endpoint titration and immunotherapy.

Inhalant allergy, or "atopy" as it is now termed, is the best understood form of allergy today. In some circles, it is the only recognized form of allergy. While an overall picture of its effects on the body and a reasonable approach to its treatment now exist, many problems remain to be solved and much improvement in its treatment will probably occur within the next several years. Many new approaches to treatment of aeroallergens are now available; however, all are compared with the skin test, which is and has been the baseline for testing and treatment. Endpoint titration provides a quantitative means for undertaking treatment of aeroallergen sensitivity. In no other way does it differ from the forms of skin testing that have been widely used for generations. The practitioners of endpoint titration feel that this difference is highly significant in simplifying, validating, and shortening the necessary period of therapy. While the concept of endpoint titration is not difficult, it is by definition a quantitative form of testing and requires a degree of expertise in performing it correctly. While a good understanding of the method may be gained from the literature, adequate hands-on experience should be obtained by any physician prior to instituting the technique as a treatment modality. Once mastered, it becomes a reliable baseline for all forms of inhalant allergy care.

Allergens↗
PubMed1971

Titration of diphtheria and tetanus antitoxins in sera of low titre.

Available methods for titrating diphtheria and tetanus antitoxin at low concentrations in human or animal blood are surveyed, with special attention to the amount of serum required for the test.In vivo methods, especially the rabbit or guinea-pig intradermal test for diphtheria and the mouse test for tetanus, are precise and reliable. If, however, serum levels as low as about 0.001 IU/ml have to be determined, rather large amounts of serum are required: moreover, the tests are rather time-consuming and expensive.Tissue culture methods are available only for diphtheria antitoxin titration. The titres found coincide very well with those from animal tests. The titrations are less time-consuming and more economical. These methods seem to be very promising for the replacement of animal tests. Of the real in vitro methods, the haemagglutination procedure has been investigated most thoroughly and used most frequently. Low titres can be measured using small amounts of serum, but the titres thus obtained may differ considerably from those obtained in animal tests, at least for individual sera. For mass screening, the method is very suitable.A number of other methods are briefly discussed, but they are either less suitable or have not yet been sufficiently investigated.

Animals↗
PubMed1976

Incompatibility between C3b and B of guinea-pig and man and its influence on the titration of the alternative pathway factors D and B in these two species.

The incubation of the proteins C3b, B, D and C3 induces enzymatic activation of B and of C3. This system of purified components was applied to the titration of the guinea-pig and human factors D and B in various homologous and heterologous reaction mixtures. On the basis of functional as well as of immunological tests, the D enzymes of both species were found to be related proteins. First, titrations of factor B revealed that both D enzymes activate homologous and heterologous B with comparable efficiency. Secondly, a rabbit anti-D antiserum raised against guinea-pig D was found to form a precipitation line of partial identity with guinea-pig and human D. In contrast to the compatibility between heterologous D and B, heterologous combinations of C3b and B were found to be incompatible. This incompatibility was indicated by low titres of D in the presence of heterologous C3b and B; in contrast, in the presence of homologous C3b and B, the titres of D were up to one hundred-fold higher. The reason for this effect was found to reside in inefficient complex formation between heterologous C3b and B. Therefore titrations of D and B should be only performed in the presence of homologous C3b and B.

Animals↗
PubMed1976

Human cell-mediated cytotoxicity estimated by lymphocyte titration.

Human lymphocyte immunity to tumor-derived target cells was estimated by titrating lymphocyte concentration to achieve a 50% reduction of target cell survival. This lymphocyte titration assay gave estimates of cytotoxicity that were different from those obtained with the conventional cell-mediated cytotoxicity assays but were more proportional to lymphocyte activity. Estimates of cytotoxicity obtained using the lymphocyte titration assay were reproducible upon repeated testin over the course of several months and were relatively unaffected by two- to fourfold variations in target cell concentration. Target cell-specific cytotoxicity was reproducible but often did not appear to be tumor specific.

Antibody Formation↗
PubMed1983

Charge determination of proteins with polyelectrolyte titration.

A recently developed photometric version of polyelectrolyte titration was applied for the determination of the number of charged residues on globular proteins. Based on the observation that oppositely charged polyelectrolytes form, in general, stoichiometric polyelectrolyte complexes, the protein solutions were incubated in excess with an oppositely charged polyelectrolyte, and the residual amount was back-titrated using o-toluidine blue for end point detection. It was found that within the range pH 2 to pH 9 the interaction of the polyelectrolytes, potassium polyvinylsulfate, polydiallylammonium chloride, and N-methylglycolchitosan iodide, with various proteins of known amino acid composition (ribonuclease A, trypsin, chymotrypsin A, pepsin, cytochrome c) occurs stoichiometrically through 1:1 ion pair interaction, irrespective of the spatial distribution of the interacting ionic sites. The close correspondence between the experimental data for the net charge and the calculated balance of ionized residues for the proteins at a given pH indicates that in the native structure of these proteins oppositely charged ionic functions are largely neutralized by the formation of intramolecular salt linkages. It is concluded that polyelectrolyte titration offers an easy access to the determination of the surface charge of proteins and other biopolymers. The data further support the notion of the importance of electrostatic cooperative interactions in biological systems.

Animals↗
PubMed1983

Liquid chromatography, microfluorometry, and dye-titration determination of vitamin C in fresh fruit and vegetables.

The vitamin C content of several fresh fruit and vegetables was determined by a liquid chromatographic (LC) method which gave simultaneous separate values for ascorbic acid and dehydroascorbic acid (DHA) and by the official AOAC methods of microfluorometry and dye-titration. The levels of ascorbic acid obtained by LC and dye-titration were in good agreement, except for a few colored products where it was difficult to determine the end point of the titration. The combined values for ascorbic acid and DHA obtained by LC and microfluorometry were in agreement for most produce, but for about one-third of the samples, the values obtained by microfluorometry were significantly higher.

Ascorbic Acid↗
PubMed1983

NMR titration studies of histidine 57 and the [methylene-13C]PMS group in the phenylmethanesulfonyl (PMS) derivative of Streptomyces erythraeus trypsin.

1H and 13C NMR titrations were performed on PMS-St. trypsin derived by PMSF modification of Ser 195 in Streptomyces erythraeus trypsin, which is devoid of auto-catalytic degradation activity at pH approximately 8. NMR titration of the imidazole C2 proton showed that His 57 had the pKa value of 6.9 in both native St. trypsin and PMS-St. trypsin, suggesting that the adjacent hydroxyl group of Ser 195 had no effect or a very weak effect on the acid-base properties of the imidazole ring in the catalytic triad. A small change in the chemical shift of the isotopically enriched methylene carbon in the PMS moiety of [methylene-13C]PMS-St. trypsin was observed between pH 6 and 8. The titration curve had an inflexion at pH 6.9 and the mode of transition was apparently sigmoidal, although a Hill coefficient of more than unity was suggested. It is thus likely that His 57 is responsible for this transition. Based on these results, the role of His 57 in the catalytic triad of the active site in serine protease is discussed.

Amino Acids↗
PubMed1994

Stimulus dose-titration in ECT: a 2-year clinical experience.

One hundred and thirty-four patients referred for electroconvulsive therapy (ECT) over a 2-year period underwent standardized stimulus dose-titration. No significant adverse events were associated with the dose-titration procedure. Our data demonstrate that seizure threshold increases with age (p = 0.0001) and is higher with bilateral electrode placement (p = 0.0001). Contrary to other studies, our data show the effect of gender is not statistically significant (p = 0.54). If consensus regarding optimal dosing strategies relative to seizure threshold can be achieved, dose-titration may be adopted as a clinically useful step in optimizing ECT technique.

Adult↗
PubMed1994

[The relationship between titrated acidity (mEq/l) and pH of human gastric juice: a study based on the data estimated by pH-meter].

The relationship between titrated acidity and pH was studied using the 3206 samples of human gastric juice, which were collected by the intragastric tube in the basal and gastrin stimulated conditions. The pH of samples subjected to the present study ranged from 0.95 and 6.9. The results showed that a certain value of pH did not always indicate an identical value of titrated acidity, and that the coefficient of variation was greater as pH value rose. As a whole, however, the acidity became lower with the increase of pH value. Thus, the relationship between pH and titrated acidity could be expressed closely as "Y = 369.19 - 424.09X + 203.66X2 - 48.29X3 + 5.57X4 - 0.25X5), where X = pH and Y = acidity. The conversion table from pH to acidity, which was calculated by this expression, was presented for the clinical use.

Adult↗
PubMed1993

[Granule-accumulation in VERO cells used for determination of antitoxin titration by micro cell culture method: I. Influence of mouse serum].

When the titration of diphtheria antitoxin of mouse serum was carried out by micro cell culture method using VERO cells, a large number of granules were observed in the cells. In order to examine the influence of this phenomenon on the titration of antitoxin, kinetics of the granule-accumulation was investigated. The granule-accumulation occurred in the cells in the culture medium to which either immunized or normal mouse serum was added. The granule-rich cells appeared at the dilution of the serum less than 1:32 and increased in number with the concentration of the serum. After 4 days of incubation 88% of the cells showed granule-accumulation when undiluted serum was added. Besides the mouse serum, those from guinea-pigs, horses, fetal calves and humans were examined. However, intensive accumulation of granules such as shown with mouse serum was not observed. From these results it was suggested that mouse serum might have some unknown mechanism which caused the remarkable accumulation of granules in VERO cells. The nature of granule and influence of this phenomenon on the titration of diphtheria antitoxin will be presented in an accompanying paper.

Animals↗
PubMed1993

Nature of the low activity of S-methyl-coenzyme M reductase as determined by active site titrations.

Purified S-methyl-coenzyme M reductase (methylreductase) exhibits a very low fraction of its in vivo activity, suggesting either enzyme inactivation during cell lysis and chromatographic purification or the lack of an activating component in assay mixtures. Evidence that all methylreductase molecules in the purified protein can catalyze slow substrate turnover is found in a study of turnover-dependent in vitro incorporation of radiolabeled HS-CoM at the enzyme active site (Hartzell, P. L., Donnelly, M. I., and Wolfe, R. S. (1987) J. Biol. Chem. 262, 5581-5586). We have conducted active site titrations of purified methylreductase and of a highly active partially purified preparation (Rospert, S., Bocher, R., Albracht, S. P. J., and Thauer, R. K. (1991) FEBS Lett. 291, 371-375) using the reversible competitive inhibitor bromopropanesulfonate (K(i) = 0.05 microM). Curve fitting the data based on an equilibrium binding model shows that 0.1-1.4% of purified methylreductase has functional inhibitor binding sites while up to 25% of a highly active preparation binds the inhibitor. An EPR titration of highly active methylreductase with this inhibitor is consistent with this result, showing that the MCR-red1 and -red2 EPR signals (Albracht, S. P. J., Ankel-Fuchs, D., Bocher, R., Ellermann, J., Moll, J., van der Zwann, J. W., and Thauer, R. K. (1988) Biochim. Biophys. Acta 955, 86-102) are titrated in parallel with this active fraction. Attempts to observe turnover-dependent uptake of radiolabel from [thio-35S]2-methylthioethane-sulfonate by methylreductase were unsuccessful. These results suggest that the low activity of purified methylreductase is due primarily to low percentages of catalytically competent enzyme.

Alkanesulfonates↗
PubMed1995

Spectrophotometric titration of phenoxyl groups of sheep brain tubulin.

Spectrophotometric titration of phenoxyl group of sheep brain tubulin carried out in 6M guanidine hydrochloride indicated 36 tyrosine residues per dimer of tubulin. A plot of log alpha/(1-alpha) versus pH showed that 26 residues titrated with apparent pK of 10.4 and 10 residues titrated with apparent pK of 11.0. The high pK value of tyrosine could be attributed to the possibility that the molecules containing tyrosine residues were not completely utilised as these residues could have been partially shielded from the solvent. Alternatively, they could have proximal negative charges.

Animals↗
PubMed1977

Reductive titration of cytochrome P-450 in rat liver microsomes.

Microsomal cytochromes b5 and P-450 of rat liver have been titrated with standardized sodium dithionite solution with a newly developed titrating apparatus that allows spectrophotometric monitoring of the reduction process while strictly O2-free conditions are maintained throughout the procedure. Cytochrome b5 and other electron acceptors in the microsomal preparation were saturated with reducing equivalents prior to addition of carbon monoxide to the system. Continued titration in the presence of CO revealed that 1 electron equivalent was required for the formation of P-450(Fe2+)-CO. These results are in agreement with previous findings of 1 electron equivalence for cytochrome P-450 of adrenocortical mitochondria and P-450CAM.

Anaerobiosis↗
PubMed1998

Improved protamine-sensitive membrane electrode for monitoring heparin concentrations in whole blood via protamine titration.

An improved protamine-sensitive electrode based on a polymeric membrane doped with the charged ion exchanger dinonylnaphthalenesulfonate (DNNS) is used for monitoring heparin concentrations in whole blood. The electrode exhibits significant nonequilibrium potentiometric response to polycationic protamine over the concentration range of 0.5-20 mg/L in undiluted whole-blood samples. The sensor can serve as a simple end point detector for the determination of heparin via potentiometric titrations with protamine. Whole-blood heparin concentrations determined by the electrode method (n > or = 157) correlate well with other protamine titration-based methods, including the commercial Hepcon HMS assay (r = 0.934) and a previously reported potentiometric heparin sensor-based method (r = 0.973). Reasonable correlation was also found with a commercial chromogenic anti-Xa heparin assay (r = 0.891) with corresponding plasma samples and appropriate correction for whole-blood hematocrit levels. Whereas a significant positive bias (0.62 kU/L; P < 0.001) is observed between the anti-Xa assay and the protamine sensor methods, insignificant bias is observed between the protamine sensor and the Hepcon HMS tests (0.08 kU/L; P = 0.02). The possibility of fully automating these titrations offers a potentially simple, inexpensive, and accurate method for monitoring heparin concentrations in whole blood.

Bias↗
PubMed1978

[Titration of serum and antirabies immunoglobulins by the radial immunodiffusion method].

Titration on mice of sera and immunoglobulin rabies anibodies is expensive, tedious and long. We are proposing a cheap, easy and rapid method which could be useful during the preparation of immunoglobulin or for the final titration. The antigen is a purified inactivated vaccine cultured on human diploid cells and purified by dextran sulfate and ultracentrifugation; it is included in agar on a glass slide. The reference serum was given us by Dr Atanasiu from Pasteur Institute; its titre was compared with international reference on mice test and was given a titre of 65 IU per ml. Five specific rabies immunoglobulin and 3 sera were received from two producers for titration. For IDR test, immunoglobulin and reference serum dilutions are diposed in wells of the same agar slide and then incubated at ambiant room temperature during 48 hours. Migration of each Ig and serum dilution is measured after coloration reported on a graph and compared. Each of the samples was checked 4 times by radial immunodiffusion test and twice by seroneutralization test on mice. Statistical analysis of the results by Student's test demonstrates that both methods are comparable.

Animals↗
PubMed2000

ImmunoCAP and HY*TEC enzyme immunoassays in the detection of allergen-specific IgE compared with serial skin end-point titration by receiver operating characteristic analysis.

OBJECTIVES: In this study, we compared 2 different immunoassays, HY*TEC enzyme immunoassay (Hycor Biomedical Inc, Garden Grove, CA) and Pharmacia Upjohn ImmunoCAP (Pharmacia and Upjohn, Kalamazoo, MI) using skin end-point titration as a gold standard by receiver operating characteristic analysis. MATERIALS: One hundred patients were enrolled in this study and were tested with the above 3 modalities. The results were analyzed by receiver operating characteristic analysis, in which the curves give a decision criterion for any diagnostic test relative to its sensitivity and specificity against an accepted gold standard test. Only inhalant allergens (Dermatophagoides pteronyssinus, Dermatophagoides farina, cat dander, giant ragweed, English plantain weed, timothy grass, Alternaria tenuis, Aspergillus fumigatus, meadow fescue, oak tree, white ash tree, and lamb's-quarter weed) were studied. RESULTS: The results obtained with both ImmunoCAP and HY*TEC showed a good correlation with skin end-point titration for D pteronyssinus, D farina, cat dander, giant ragweed, timothy grass, A tenuis, and meadow fescue, with sensitivities ranging from 71% to 88%. However, the sensitivity of both tests to English plantain weed, lamb's-quarter weed, oak tree, and white ash tree was lower (66% down to 39%). Additionally, HY*TEC results were not satisfactory for detecting specific IgE to A fumigatus (30% sensitivity). The overall sensitivity for each test was 65.67% for the HY*TEC and 71.25% for the ImmunoCAP. CONCLUSION: The 2 in vitro testing systems are comparable for most of the allergens, except for A fumigatus, English plantain, lamb's-quarter, and white ash tree; ImmunoCAP and HY*TEC would benefit from improved technology for detecting these specific IgE antibodies. The HY*TEC assay did not give acceptable results for A fumigatus. The overall assay characteristics were good to excellent for each system tested.

Adolescent↗