You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Ammonium bicarbonate buffers combined with hybrid surface technology columns improve the peak shape of strongly tailing lipids.

BACKGROUND: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SIGNIFICANCE: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

Salivary buffering capacity is correlated with umami but not sour taste sensitivity in healthy adult Japanese subjects.

OBJECTIVE: Saliva serves multiple important functions crucial for maintaining a healthy oral and systemic environment. Among them, the pH buffering effect, which is primarily mediated by bicarbonate ions, helps maintain oral homeostasis by neutralizing acidity from ingested foods. Therefore, higher buffering capacity, reflecting the ability to neutralize oral acidity, may influence taste sensitivity, especially for sour taste since it involves sensing H+ ions. This study aims to explore the relationship between salivary buffering capacity and taste sensitivities to the five basic tastes in healthy adult humans. DESIGN: Eighty seven healthy adult students participated in this study. Resting saliva volume was measured using the spitting method. The liquid colorimetric test was used to assess salivary buffering capacity. The whole-mouth taste testing method was employed to determine the recognition threshold for each tastant (NaCl, sucrose, citric acid, quinine-HCl, monosodium glutamate). RESULTS: Taste recognition thresholds for sour taste as well as sweet, salty, and bitter tastes showed no correlation with salivary buffering capacity. Interestingly, a negative relationship was observed between recognition threshold for umami taste and salivary buffering capacity. Furthermore, a positive correlation between salivary buffering capacity and resting saliva volume was observed. CONCLUSIONS: Salivary buffering capacity primarily influences sensitivity to umami taste, but not sour and other tastes.

Nobody's Perfect: Choice of the Buffer and the Rate of Cu2+ Ion-Peptide Interaction.

The choice of correct pH buffer is crucial in chemical studies modeling biological processes involving Cu2+ ions. Popular buffers for physiological pH are known to form Cu(II) complexes, but their impact on kinetics of Cu(II) complexation has not been considered. We performed a stopped-flow kinetic study of Cu2+ ion interactions with four popular buffers (phosphate, Tris, HEPES, and MOPS) and two buffers considered as nonbinding (MES and PIPPS). Next, we studied their effects on the rate of Cu2+ reaction with Gly-Gly-His (GGH), a tripeptide modeling physiological Cu(II) sites, which we studied previously at conditions presumably excluding the buffer interference [Kotuniak, R.; Angew. Chem., Int. Ed. 2020, 59, 11234-11239]. We observed that (i) all tested pH 7.4 buffers formed Cu(II) complexes within the stopped-flow instrument dead time; (ii) Cu(II)-peptide complexes were formed via ternary complexes with the buffers; (iii) nevertheless, Good buffers affected the observed rate of Cu(II)-GGH complex formation only slightly; (iv) Tris was a competitive inhibitor of Cu(II)-GGH complexation; while (v) phosphate was a reaction catalyst. This is particularly important as phosphate is a biological buffer.

Sucrose rinse modulates the salivary behavior of carbonic anhydrase VI and its buffering capacity: a longitudinal study in 4 to 6.5-year-old children.

Background: Carbonic anhydrase VI (CA VI) is crucial in regulating oral pH and predicting susceptibility to dental caries. The hypothesis posits that caries activity may alter the CA VI function, diminishing its capacity to regulate pH effectively and potentially exacerbating cariogenic challenges. This 1-year cohort study sought to investigate the enzymatic activity of salivary CA VI and buffering capacity following a 20% sucrose rinse in 4 to 6.5-year-old children. Method: This research involved 46 volunteers categorized into three groups based on their caries status after follow-up: caries-free (CFee), arrested caries (CArrested), and caries active (CActive). Children underwent visible biofilm examination and saliva collection for salivary flow rate, buffering capacity, and CA VI analyses before and after a 20% sucrose rinse. Results: A reduction in the buffering capacity was observed after sucrose rinse in all groups. The CA VI activity decreased significantly in CFee and CArrested groups after sucrose rinse, although it did not change in the CActive group. An improvement in the buffering capacity and salivary flow rate was found at follow-up when compared with the baseline. After 1-year follow-up, buffering capacity and salivary flow rate increased in all groups, whilst the CA VI activity reduced only in CFree and CArrested children. Conclusion: Sucrose rinse universally reduces the salivary buffering capacity, while caries activity may disrupt CA VI activity response during a cariogenic challenge. After a year, increased salivary flow enhances buffering capacity but not CA VI activity in caries-active children.

Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

Addressing the interaction of stem bromelain with different anionic surfactants, below, at and above the critical micelle concentration (cmc) in phosphate buffer at pH 7: Physicochemical, spectroscopic, & molecular docking study.

The structural stability and therapeutic activity of Stem Bromelain (BM) have been explored by unravelling the interaction of stem BM in presence of two different types of anionic surfactants namely, bile salts, NaC and NaDC and the conventional anionic surfactants, SDDS and SDBS, below, at and above the critical micelle concentration (cmc) in aqueous phosphate buffer of pH 7. Different physicochemical parameters like, surface excess (Γcmc), minimum area of surfactants at air water interface (Amin) etc. are calculated from tensiometry both in absence and presence of BM. Several inflection points (C1, C2 and C3) have been found in tensiometry profile of surfactants in presence of BM due to the conformational change of BM assisted by surfactants. Similar observation also found in isothermal titration calorimetry (ITC) profiles where the enthalpy of micellization (ΔH0obs) of surfactants in absence and presence of BM have calculated. Further, steady state absorption and fluorescence spectra monitoring the tryptophan (Trp) emission of free BM and in presence of all the surfactants at three different temperatures (288.15 K, 298.15 K, and 308.15 K) reveal the nature of fluorescence quenching of BM in presence of bile salts/surfactants. Time resolved fluorescence studies at room temperature also support to determine the several quenching parameters. The binding constant (Kb) of BM with all the surfactants and free energy of binding (∆G0 of bile salts/surfactants with BM at different temperatures have been calculated exploiting steady state fluorescence technique. It is observed that, the binding of NaC with BM is greater as compared to other surfactants while Stern-Volmer quenching constant (KSV) is found greater in presence of SDBS as compared with others which supports the surface tension and ITC data with the fact that surface activity of surfactant(s) is decreasing with the binding of the surfactants at the core or binding pocket of BM. Circular Dichroism (CD) study shows the stability of secondary structure of BM in presence of NaC and NaDC below C3, while BM lost its structural stability even at very low surfactant concentration of SDDS and SDBS which also supports the more involvement of bile salts in binding rather than surfactants. The molecular docking studies have also been substantiated for better understanding the several experimental investigations interaction of BM with the bile salts/surfactants.

Mechanisms of whole body, respiratory, acid-base buffering: a first computer-model test of three physicochemical, acid-base theories.

Acid-base disorders are currently analyzed and treated using a bicarbonate-centered approach derived from blood studies prior to the advent of digital computers, which could solve computer models capable of quantifying the complex physicochemical nature governing distribution of water and ions between fluid compartments. An alternative is the Stewart approach, which can predict the pH of a simple mixture of ions and electrically charged proteins; hence, the role of extravascular fluids has been largely ignored. The present study uses a new, comprehensive computer model of four major fluid compartments, based on a recent blood model, which included ion binding to proteins, electroneutrality constraints, and other essential physicochemical laws. The present model predicts quantitative respiratory acid-base buffering behavior in the whole body, as well as determining roles of each compartment and their species, particularly compartmental electrically charged proteins, largely responsible for buffering. The model tested an early theory that H+ was conserved in the body fluids; hence, when changing Pco2 states, intracellular buffering could be predicted by net changes in bicarbonate and protein electrical charge in the remaining fluids. Even though H+ is not conserved in the model, the theory held in simulated respiratory disorders. Model results also agreed with a second part of the theory, that ion movements between cells and interstitial fluid were linked with H+ buffering, but by electroneutrality constraints, not necessarily by some membrane-related mechanisms, and that the strong ion difference (SID), an amalgamation of ionic electrical charges, was approximately conserved when going between equilibrium states caused by Pco2 changes in the body-fluid system.NEW & NOTEWORTHY For the first time, a physicochemically based, whole body, four-compartment, computer model was used to study respiratory whole body acid-base buffering. An improved approach to quantify acid-base buffering, previously used by this author, was able to determine contributions of the various compartmental fluids to whole body buffering. The model was used to test, for the first time, three fundamental theories of whole body acid-base homeostasis, namely, H+-conservation, its linkage to ion transport, and strong ion difference conservation.

Solubility vs Dissolution in Physiological Bicarbonate Buffer.

BACKGROUND: Phosphate buffer is often used as a replacement for the physiological bicarbonate buffer in pharmaceutical dissolution testing, although there are some discrepancies in their properties making it complicated to extrapolate dissolution results in phosphate to the in vivo situation. This study aims to characterize these discrepancies regarding solubility and dissolution behavior of ionizable compounds. METHODS: The dissolution of an ibuprofen powder with a known particle size distribution was simulated in silico and verified experimentally in vitro at two different doses and in two different buffers (5 mM pH 6.8 bicarbonate and phosphate). RESULTS: The results showed that there is a solubility vs. dissolution mismatch in the two buffers. This was accurately predicted by the in-house simulations based on the reversible non-equilibrium (RNE) and the Mooney models. CONCLUSIONS: The results can be explained by the existence of a relatively large gap between the initial surface pH of the drug and the bulk pH at saturation in bicarbonate but not in phosphate, which is caused by not all the interfacial reactions reaching equilibrium in bicarbonate prior to bulk saturation. This means that slurry pH measurements, while providing surface pH estimates for buffers like phosphate, are poor indicators of surface pH in the intestinal bicarbonate buffer. In addition, it showcases the importance of accounting for the H2CO3-CO2 interconversion kinetics to achieve good predictions of intestinal drug dissolution.

Drug Crystal Precipitation in Biorelevant Bicarbonate Buffer: A Well-Controlled Comparative Study with Phosphate Buffer.

The purpose of the present study was to clarify whether the precipitation profile of a drug in bicarbonate buffer (BCB) may differ from that in phosphate buffer (PPB) by a well-controlled comparative study. The precipitation profiles of structurally diverse poorly soluble drugs in BCB and PPB were evaluated by a pH-shift precipitation test or a solvent-shift precipitation test (seven weak acid drugs (pKa: 4.2 to 7.5), six weak base drugs (pKa: 4.8 to 8.4), one unionizable drug, and one zwitterionic drug). To focus on crystal precipitation processes, each ionizable drug was first completely dissolved in an HCl (pH 3.0) or NaOH (pH 11.0) aqueous solution (450 mL, 50 rpm, 37 °C). A 10-fold concentrated buffer solution (50 mL) was then added to shift the pH value to 6.5 to initiate precipitation (final volume: 500 mL, buffer capacity (ß): 4.4 mM/ΔpH (BCB: 10 mM or PPB: 8 mM), ionic strength (I): 0.14 M (adjusted by NaCl)). The pH, ß, and I values were set to be relevant to the physiology of the small intestine. For an unionizable drug, a solvent-shift method was used (1/100 dilution). To maintain the pH value of BCB, a floating lid was used to avoid the loss of CO2. The floating lid was applied also to PPB to precisely align the experimental conditions between BCB and PPB. The solid form of the precipitants was identified by powder X-ray diffraction and differential scanning microscopy. The precipitation of weak acids (pKa ≤ 5.1) and weak bases (pKa ≥ 7.3) was found to be slower in BCB than in PPB. In contrast, the precipitation profiles in BCB and PPB were similar for less ionizable or nonionizable drugs at pH 6.5. The final pH values of the bulk phase were pH 6.5 ± 0.1 after the precipitation tests in all cases. All precipitates were in their respective free forms. The precipitation of ionizable weak acids and bases was slower in BCB than in PPB. The surface pH of precipitating particles may have differed between BCB and PPB due to the slow hydration process of CO2 specific to BCB. Since BCB is a physiological buffer in the small intestine, it should be considered as an option for precipitation studies of ionizable weak acids and bases.

Dissolution Profiles of Immediate Release Products of Various Drugs in Biorelevant Bicarbonate Buffer: Comparison with Compendial Phosphate Buffer.

PURPOSE: The purpose of this study was to clarify the extent to which the dissolution profiles of immediate release (IR) products of various drugs differ between biorelevant bicarbonate buffer (BCB) and compendial phosphate buffer (PPB). METHODS: The dissolution profiles of the IR products of fifteen poorly soluble ionizable drugs were measured in BCB and PPB. BCB was set to be relevant to the small intestine (pH 6.8, 10 mM). The pH was maintained using the floating lid method. The Japanese pharmacopeia second fluid (JP2, 25 mM phosphate buffer, nominal pH 6.8) was used as compendial PPB. The compendial paddle apparatus was used for the dissolution tests (500 mL, 50 rpm, 37°C). RESULTS: In 11/15 cases, a difference in dissolved% (< 0.8 or > 1.25-fold) was observed at a time point. In 4/15 cases, the ratio of the area under the dissolution curve was not equivalent (< 0.8 or > 1.25-fold). In the cases of free-form drugs, the dissolution rate tended to be slower in BCB than in JP2. In the case of salt-form drugs, a marked difference was observed for the cases that showed supersaturation. However, no trend was observed in the differences. CONCLUSIONS: Many IR products showed differences in the dissolution profiles between biorelevant BCB and compendial PPB. With the floating lid method, BCB is as simple and easy to use as PPB. Biorelevant BCB is recommended for dissolution testing.

Association between sleep pattern, salivary cariogenic bacteria and fungi populations, pH and buffering capacity in children: A comparative study.

BACKGROUND: Sleep quality has a significant impact on a child's health and is linked to oral and systemic diseases. It affects the circadian rhythm, which plays a crucial role in regulating the balance of the endocrine and hormonal systems. Current research has focused on exploring its role in the development of caries, which is influenced by inherent oral factors such as the composition of the oral microbiome and pH levels. OBJECTIVES: This study aimed to investigate the relationship between bacterial population, pH, and buffering properties of saliva and sleep patterns in 8- to 12-year-old children. MATERIAL AND METHODS: This cross-sectional study was conducted on 85 elementary school children aged 8-12 years. After obtaining written consent, non-stimulating saliva samples were collected using the spitting method. The participants' sleep pattern information was obtained with the use of the Persian version of the Children's Sleep Habits Questionnaire (CSHQ). Based on the results of the CSHQ, the participants were divided into 2 groups: those with appropriate sleep patterns; and those with inappropriate sleep patterns. The study compared the bacterial population of Streptococcus mutans, Lactobacillus spp. and Candida albicans, as well as the buffering capacity and pH of the saliva between the 2 groups. The statistical analysis employed the χ2 test, the independent samples t-test and Spearman's correlation. RESULTS: The group with inappropriate sleep patterns had significantly lower pH and buffering capacity (p < 0.001) and significantly higher colony counts of Lactobacillus and S. mutans (p < 0.001 and p = 0.012, respectively). There was no association between C. albicans and sleep patterns (p = 0.121). CONCLUSIONS: Inappropriate sleep patterns increase the population of caries-causing bacteria and reduce salivary pH and buffering capacity. This can be a significant factor in the development of dental caries in children aged 8-12 years.

Exploiting Principal Component Analysis (PCA) to reveal temperature, buffer and metal ions' role in neuromelanin (NM) synthesis by dopamine (DA) oxidative polymerization.

Neuromelanin (NM) plays a well-established role in neurological disorders pathogenesis; the mechanism of action is still discussed and the investigations in this field are limited by NM's complex and heterogeneous composition, insolubility, and low availability from human brains. An alternative can be offered by synthetic NM obtained from dopamine (DA) oxidative polymerization; however, a deep knowledge of the influence of both physicochemical parameters (T, pH, ionic strength) and other compounds in the reaction media (buffer, metal ions, other catecholamines) on DA oxidation process and, consequently, on synthetic NM features is mandatory to develop reliable NM preparation methodologies. To partially fulfill this aim, the present work focuses on defining the role of temperature, buffer and metal ions on both DA oxidation rate and DA oligomer size. DA oxidation in the specific conditions is monitored by UV-Vis spectroscopy and Principal Component Analysis (PCA) is run either on the raw spectra to model the background absorption increase, related to small DA oligomers formation, or on their first derivative to rationalize DA consumption. After having studied three case studies, 3-Way PCA is applied to directly evaluate the effect of temperature and buffer type on DA oxidation in the presence of different metal ions. Despite the proof-of-concept nature of the work and the number of compounds still to be included in the investigation, the preliminary results and the possibility to further expand the chemometric approach represent an interesting contribution to the field of in vitro simulation of NM synthesis.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

PURPOSE/STUDY QUESTION: Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? METHODS: Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). RESULTS: Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). CONCLUSION: The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing. Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Assessing the Efficacy of Buffered Versus Nonbuffered Lidocaine in Dental Extractions: A Double-Blinded Randomized Controlled Trial.

BACKGROUND: Injections using buffered lidocaine may decrease discomfort, have a quicker onset, and be a more efficacious local anesthetic. Previous studies have been inconclusive in the oral context. PURPOSE: To address if bicarbonate buffered 2% lidocaine can decrease pain from the use of local anesthesia, has a quicker onset time, and is more efficacious. STUDY DESIGN: The design was a single-center double-blinded randomized control trial, set in an outpatient oral and maxillofacial clinic housed in the University of Cincinnati Medical Center. Inclusion criteria for the study were patients requiring a single tooth extraction due either to caries or periodontal disease. PREDICTOR VARIABLE: The predictor variable was the local anesthetic used either nonbuffered 2% lidocaine with 1:100,000 epinephrine (control) or bicarbonate buffered 2% lidocaine with 1:100,000 epinephrine (study) was randomly assigned. MAIN OUTCOME VARIABLES: Primary outcome variables were injection pain score, and postoperative pain, time to anesthetic onset, and the number of rounds of injections required to achieve adequate anesthesia. COVARIATES: The covariates were jaw involved, age, sex, and race, American Society of Anesthesiologists score, body mass index, current tobacco use, history of psychiatric illness, chronic pain, and preoperative pain score. ANALYSES: Test statistics were calculated using Wilcoxon rank-sum test, Kruskal-Wallis test, Spearman rank correlation test, χ2 test for bivariate analyses, and Fisher's exact test. P values ≤ .05 were considered statistically significant. RESULTS: The final sample was 114 subjects. The mean age of the sample was 42.97 years, standard deviation ±13.43 years. The sample was 39.47% male. The racial demographics were Caucasian (62.28%) and African American (33.33%). Buffered lidocaine did not have a statistically significant relationship with any of the outcomes. The jaw involved had a statistically significant association to the injection pain score (P value = .006), and the number of rounds of anesthetic required (P value = .047). Age showed a statistically significant association to injection pain score (P value = .032), and the number of rounds of anesthetic required (P value = .027). Finally, preoperative pain had a statistically significant relationship with injection pain score (P value = < .001). CONCLUSION AND RELEVANCE: In this study, bicarbonate buffered lidocaine did not exhibit any discernible advantages over nonbuffered lidocaine for any study outcomes.

Evaluation of acute intravenous toxicity of HEPES: Is Good's buffer good and safe enough for clinical utilization in nuclear medicine?

OBJECTIVE: Good's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation. EXPERIMENTAL: A pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats. RESULTS: No mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4-5 days of dosing. CONCLUSION: The amount of HEPES to be administered during clinical intervention is usually much lower (typically 1-2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation.

Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Evaluation of the efficacy of buffered local anesthetic in extraction of infected teeth-randomized double-blind study.

PURPOSE: In the presence of infection, acidic pH of a lignocaine local anesthetic causes undesirable effects such as burning on injection, relatively slow onset, and lack of numbness. Buffered lignocaine will increase the pH of the solution and may resolve above problems. Thus, the objective of this study is to compare the efficacy of buffered lignocaine with that of commercial lignocaine. METHOD: Seventy patients with infected teeth were randomly divided into two equal groups. The study group received buffered lignocaine (8.4% sodium bicarbonate added to 2% lignocaine mixture) while the control received commercial lignocaine preparation (2% lignocaine with 1:80,000 adrenaline). Burning while injection, pain using VAS scale and onset of action with EPT (electric pulp tester) were recorded. RESULTS: In the study group, the VAS score after injection was 1.20 ± 0.68 and the control group was 2.57 ± 0.92 (p = 0.001). There was a statistically significant reduction in pain reduction in the study group. The time of onset was 3.97 ± 0.71 and 5.67 ± 1.15 min, respectively, and the difference was statically significant. Only one-third of the study group experienced burning on injection as compared to two-thirds in the control group. CONCLUSION: Buffered lignocaine is more effective as compared to commercial lignocaine in the extraction of infected teeth. CTRI NUMBER: CTRI/2022/01/039476.