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

Determination of Ka of Isolated Plasma and Whole Blood of Critically Ill Patients With Sepsis, Non-septic Patients and Healthy Volunteers: an International, In-vitro Acid-base Study.

Alterations of acid-base equilibrium are very common in critically ill patients and understanding their pathophysiology can be important to improve clinical treatment.

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.

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