Determination of the Dissociation Constant (Ka) of Plasma and Whole Blood in Septic Patients
- Conditions
- SepsisCritical IllnessSeptic ShockRespiratory AcidosisRespiratory AlkalosisAcid-Base Imbalance
- Interventions
- Diagnostic Test: In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in isolated plasma.Diagnostic Test: In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in whole bloodDiagnostic Test: Biomolecular analysis of plasma proteins.
- Registration Number
- NCT03966664
- Brief Summary
Alterations of acid-base equilibrium are very common in critically ill patients and understanding their pathophysiology can be important to improve clinical treatment.
- Detailed Description
Acid-base equilibrium has been object of study for more than 100 years in medicine because of its relevance in patients' management and in determining their prognosis, especially in the ICU.
A concept closely related to acid-base equilibrium is that of "buffer", term used to define any substance able to limit the changes in pH caused by the addition or loss of alkali or acid.
Depending on its physiochemical features, every buffer has one or more pH (negative logarithm of hydrogen ion concentration) values where its ability to keep pH stable is maximal. These values are defined as Ka or semi equivalence points, i.e. the pH values where the buffer dissolved in solution is half in its associated form (AH) and half in its dissociated form (A-).
Several studies tried to determine the normal values of both concentration and Ka of ATOT. However, they did not lead to univocal results. Moreover, many of these values come from studies of veterinary medicine or are the result of theoretical estimates on human plasma.
Staempfli and Constable performed a single experimental study on human plasma in 2003. These authors, however, analyzed only isolated plasma, neglecting whole blood, and computed ATOT and Ka values of healthy volunteers, while Ka and ATOT values for critically ill patients with sepsis are still unknown.
Primary aim of the present study is to quantify the acidic dissociation constant (Ka) of isolated plasma of critically ill patients with sepsis, and compare these data with normal values, i.e. obtained from healthy controls. The investigators hypothesize that plasma of critically ill septic patients has a lower Ka and that, consequently, it undergoes higher pH variations for a given perturbation of the system (variation in carbon dioxide).
Secondary aim is to quantify the Ka of whole blood of critically ill patients with sepsis and compare these data with normal values, i.e. obtained from healthy controls. The investigators hypothesize that blood of critically ill septic patients has a lower Ka and that, consequently, it undergoes higher pH variations for a given perturbation of the system (variation in carbon dioxide).
Other aims of the study are:
* quantify the Ka of plasma and whole blood of non-septic patients admitted to the ICU and compare these results with the values of septic patients and healthy volunteers.
* define the normal concentration of weak non-carbonic acids (ATOT) in plasma of septic patients and compare it with data obtained in healthy volunteers and non-septic patients.
Finally, possible structural alteration of plasma proteins will be evaluated:
* Identification of differentially modified proteoforms of serum albumin and major plasma proteins by two-dimensional electrophoresis;
* High Performance Liquid Chromatography (HPLC) to identify different Redox-forms of albumin
* Spectrophotometric evaluation of modifications of ligand binding properties of serum albumin.
Recruitment & Eligibility
- Status
- UNKNOWN
- Sex
- All
- Target Recruitment
- 90
Not provided
Not provided
Study & Design
- Study Type
- OBSERVATIONAL
- Study Design
- Not specified
- Arm && Interventions
Group Intervention Description Septic patients In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in isolated plasma. Critically ill patients of both sexes admitted to the general ICU of the participating centers with the diagnosis of sepsis will be included. Septic patients Biomolecular analysis of plasma proteins. Critically ill patients of both sexes admitted to the general ICU of the participating centers with the diagnosis of sepsis will be included. Healthy controls In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in whole blood Age and sex matched healthy volunteers. Septic patients In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in whole blood Critically ill patients of both sexes admitted to the general ICU of the participating centers with the diagnosis of sepsis will be included. Non septic patients Biomolecular analysis of plasma proteins. Non-septic patients admitted to the general ICU of the participating centers after elective non-cardiac surgery. Healthy controls Biomolecular analysis of plasma proteins. Age and sex matched healthy volunteers. Non septic patients In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in isolated plasma. Non-septic patients admitted to the general ICU of the participating centers after elective non-cardiac surgery. Healthy controls In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in isolated plasma. Age and sex matched healthy volunteers. Non septic patients In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in whole blood Non-septic patients admitted to the general ICU of the participating centers after elective non-cardiac surgery.
- Primary Outcome Measures
Name Time Method Effective dissociation constant of plasma weak acids (Ka) [dimensionless] 1 day Difference in plasma Ka between study groups.
- Secondary Outcome Measures
Name Time Method Total concentration of whole blood non-volatile buffers (Atot) [mmol/L] 1 day Difference in whole blood Atot between study groups.
Non-carbonic buffer power of whole blood due to electrolyte shifts [milliequivalents/L] 1 day Difference in Non-carbonic buffer power of whole blood due to electrolyte shifts between study groups.
Characterization of altered ligand binding properties 1 day HSA will be fractionated and dissociation constants for warfarin-SA and diazepam-SA complexes will be obtained spectrophotometrically to evaluate modifications in its ligand binding properties
Effective dissociation constant of whole blood weak acids (Ka) [dimensionless] 1 day Difference in whole blood Ka between study groups.
Non-carbonic buffer power of isolated plasma due to electrolyte shifts [milliequivalents/L] 1 day Difference in Non-carbonic buffer power of isolated plasma due to electrolyte shifts between study groups.
Total concentration of plasma non-volatile buffers (Atot) [mmol/L] 1 day Difference in plasma Atot between study groups.
Oxidized albumin [%] 1 day Difference in the percentage in oxidized albumin between groups.
Identification of differentially modified proteoforms of human serum albumin (HSA) and major plasma proteins. 1 day Samples will be analyzed by two-dimensional electrophoresis.8 After fluorescent staining and image acquisition, proteoform patterns corresponding to HSA and other major plasma proteins will be aligned and compared
Trial Locations
- Locations (2)
Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
🇮🇹Milan, Italy
Third faculty of Medicine, Charles University of Prague
🇨🇿Prague, Czechia