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

Sepsis Clinical Trial

— ENTERPRISE  

Official title:

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.

Clinical Trial Details — Status: Recruiting

Administrative data

• NCT number: NCT03966664
• Other study ID #: ENTERPRISE
• Status: Recruiting
• Start date: June 3, 2019
• Est. completion date: June 2021

Study information

• Verified date: June 2019
• Source: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico
• Contact: Thomas Langer, MD
• Phone: 0255033232
• Email: thomas.langer[@]unimi.it
• Is FDA regulated: No
• Study type: Observational

Clinical Trial Summary

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

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 information / eligibility

• Status: Recruiting
• Enrollment: 90
• Est. completion date: June 2021
• Est. primary completion date: June 2021
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years and older

Eligibility

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

Related Conditions & MeSH terms

• Acid-Base Imbalance
• Acidosis
• Alkalosis
• Alkalosis, Respiratory
• Critical Illness
• Respiratory Acidosis
• Respiratory Alkalosis
• Sepsis
• Septic Shock
• Shock, Septic

Intervention

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

Locations

Country	Name	City	State
Czechia	Third faculty of Medicine, Charles University of Prague	Prague
Italy	Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico	Milan

Sponsors (2)

Lead Sponsor	Collaborator
Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico	Charles University, Czech Republic

Countries where clinical trial is conducted

Czechia,  Italy, 

References & Publications (8)

Fencl V, Leith DE. Stewart's quantitative acid-base chemistry: applications in biology and medicine. Respir Physiol. 1993 Jan;91(1):1-16. Review. — View Citation

Figge J, Mydosh T, Fencl V. Serum proteins and acid-base equilibria: a follow-up. J Lab Clin Med. 1992 Nov;120(5):713-9. — View Citation

Henderson LJ. THE REGULATION OF NEUTRALITY IN THE ANIMAL BODY. Science. 1913 Mar 14;37(950):389-95. — View Citation

Langer T, Scotti E, Carlesso E, Protti A, Zani L, Chierichetti M, Caironi P, Gattinoni L. Electrolyte shifts across the artificial lung in patients on extracorporeal membrane oxygenation: interdependence between partial pressure of carbon dioxide and strong ion difference. J Crit Care. 2015 Feb;30(1):2-6. doi: 10.1016/j.jcrc.2014.09.013. Epub 2014 Sep 22. — View Citation

Lee SW, Hong YS, Park DW, Choi SH, Moon SW, Park JS, Kim JY, Baek KJ. Lactic acidosis not hyperlactatemia as a predictor of in hospital mortality in septic emergency patients. Emerg Med J. 2008 Oct;25(10):659-65. doi: 10.1136/emj.2007.055558. — View Citation

LEEUWEN AM. NET CATION EQUIVALENCY ('BASE BINDING POWER') OF THE PLASMA PROTEINS. Acta Med Scand. 1964;176:SUPPL 422: 1+. — View Citation

Staempfli HR, Constable PD. Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma. J Appl Physiol (1985). 2003 Aug;95(2):620-30. Epub 2003 Mar 28. — View Citation

Stampfli HR, Misiaszek S, Lumsden JH, Carlson GP, Heigenhauser GJ. Weak acid-concentration Atot and dissociation constant Ka of plasma proteins in racehorses. Equine Vet J Suppl. 1999 Jul;(30):438-42. — View Citation

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Effective dissociation constant of plasma weak acids (Ka) [dimensionless]	Difference in plasma Ka between study groups.	1 day
Secondary	Effective dissociation constant of whole blood weak acids (Ka) [dimensionless]	Difference in whole blood Ka between study groups.	1 day
Secondary	Total concentration of plasma non-volatile buffers (Atot) [mmol/L]	Difference in plasma Atot between study groups.	1 day
Secondary	Total concentration of whole blood non-volatile buffers (Atot) [mmol/L]	Difference in whole blood Atot between study groups.	1 day
Secondary	Non-carbonic buffer power of whole blood due to electrolyte shifts [milliequivalents/L]	Difference in Non-carbonic buffer power of whole blood due to electrolyte shifts between study groups.	1 day
Secondary	Non-carbonic buffer power of isolated plasma due to electrolyte shifts [milliequivalents/L]	Difference in Non-carbonic buffer power of isolated plasma due to electrolyte shifts between study groups.	1 day
Secondary	Oxidized albumin [%]	Difference in the percentage in oxidized albumin between groups.	1 day
Secondary	Characterization of altered ligand binding properties	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	1 day
Secondary	Identification of differentially modified proteoforms of human serum albumin (HSA) and major plasma proteins.	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	1 day