Determination of the Dissociation Constant (Ka) of Plasma and Whole Blood in Septic Patients

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.

• In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in isolated plasma. (Diagnostic test): Collection of a venous blood sample, centrifugation in order to harvest isolated plasma and performance of in-vitro tonometry in order to assess Ka and Atot.
• In vitro determination the dissociation constant (Ka) and total amount of non-volatile buffers (Atot) in whole blood (Diagnostic test): Collection of a venous blood sample and performance of in-vitro tonometry in order to assess Ka and Atot.
• Biomolecular analysis of plasma proteins. (Diagnostic test): Bidimensional electrophoresis, determination of oxidized albumin fraction, characterization of altered ligand binding properties of plasma albumin.

Primary outcomes

• Effective dissociation constant of plasma weak acids (Ka) [dimensionless] [1 day]: Difference in plasma Ka between study groups.

Secondary outcomes

• Effective dissociation constant of whole blood weak acids (Ka) [dimensionless] [1 day]: Difference in whole blood Ka between study groups.
• Total concentration of plasma non-volatile buffers (Atot) [mmol/L] [1 day]: Difference in plasma Atot between study groups.
• 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.
• 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.
• Oxidized albumin [%] [1 day]: Difference in the percentage in oxidized albumin between 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
• 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

Sex: All, ages: 18 Years to any, accepts healthy volunteers

Group 1: Septic patients

Inclusion Criteria for Group 1:

* Diagnosis of Sepsis
* Age > 18 years
* Informed or deferred informed consent

Exclusion Criteria for Group 1:

* Pregnancy
* Bilirubin > 4 mg/dL
* Minor or major thalassemia
* Transfusion of more than 4 Units of packed red blood cells and/or 1 L of plasma during the 24 hours prior to enrollment

Group 2: Healthy volunteers

Inclusion criteria for Group 2:

* informed consent
* Age > 18 years

Exclusion criteria for Group 2:

• Pregnancy

Group 3: Non-septic patients

Inclusion criteria for Group 3:

* Informed consent
* Age >18 years
* Planned ICU admission after elective surgery

Exclusion criteria for Group 3:

* Diagnosis of sepsis
* Pregnancy
* Bilirubin >4 mg/dL
* Liver cirrhosis
* Onco-hematological diseases
* Minor or major thalassemia
* Transfusion of more than 4 Units of packed red blood cells and/or 1 L of plasma during the 24 hours prior to enrollment

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• Thomas Langer, MD, Principal investigator, Fondazione IRCCS Ca' Granda Hospital, Milano, Italy
• Antonio M Pesenti, MD, Study chair, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
• Frantisek Duska, MD, Principal investigator, Third faculty of Medicine, Charles University, Prague
• Giacomo Grasselli, MD, Study chair, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
• Paul Elbers, MD, Study chair, Amsterdam UMC, Department of Intensive Care Medicine