Plasma protein buffer system

Plasma Protein Buffer System and Plasma Base Excess The buffer value (p) of the nonbicarbonate buffers of plasma is about 7.7 mmol/L at pH 7.40 and a normal plasma protein concentration of 72 g/L. Proteins, especially albumin, account for the greatest portion (95%) of the nonbicarbonate buffer value of the plasma. The most important buffer groups of proteins in the physiological pH range are the imidazole groups of histidines (pimolecule contains 16 histidines. 

An average rate of metabolic activity produces roughly 22,000 mEq acid per day. If all of this acid were dissolved at one time in unbuffered body fluids, their pH would be less than 1. However, the pH of the blood is normally maintained between 7.36 and 7.44, and intracellular pH at approximately 7.1 (between 6.9 and 7.4). The widest range of extracellular pH over which the metabolic functions of the liver, the beating of the heart, and conduction of neural impulses can be maintained is 6.8 to 7.8. Thus, until the acid produced from metabolism can be excreted as CO2 in expired air and as ions in the urine, it needs to be buffered in the body fluids. The major buffer systems in the body are the bicarbonate-carbonic acid buffer system, which operates principally in extracellular fluid the hemoglobin buffer system in red blood cells the phosphate buffer system in all types of cells and the protein buffer system of cells and plasma. 

To test this hypothesis, very low density lipoprotein (VLDL, d<1.0 gm/ml), low density lipoprotein (LDL, d=l.02-1.063) and high density lipoprotein (HDL, d=l.09-1.21) were isolated from outdated human plasma by ultracentrifugation according to established procedures (27,28), using potassium bromide for density adjustments and stored at -20° C in the presence of 20% sucrose before use. The purity of individual lipoprotein fractions thus obtained was established by polyacrylamide gel electrophoresis in sodium dodecyl buffer system (2 ) and filtration through a Sepha-rose 6B column, equilibrated with 0.2 M potassium bromide in 0.1 M sodium phosphate buffer, pH 7.2. Protein (30) and cholesterol... 

An important factor in the interaction of foreign surfaces with blood is the rapid adsorption of plasma proteins onto such surfaces when they are exposed to blood (4). For this reason the adsorption of radioactively tagged blood components on heparinized and unheparinized surfaces was measured. Proteins were dissolved in approximate physiological concentrations in a buffered (pH 7.35) physiological saline solution and the solutions were exposed to the test surfaces for 2 hours at 37 °C. in a static system. After the exposure, the surfaces were rinsed with physiological saline and distilled water and then dried. The amount of protein on the surfaces was determined in a 27r-gas flow proportional counter (7). As shown in Table III, although both heparinized surfaces were nonthrombogenic, there is no consistent pattern of either increased or decreased adsorption of the proteins caused by the heparinization. In-... 

Many proteins, and notably albumin, contain weak acidic and basic groups within their structure. Therefore, plasma and other proteins form important buffering systems. Intracellular proteins limit pH changes within cells, whilst the protein matrix of bone can buffer large amounts of hydrogen ions in patients with chronic acidosis. 

As discussed in Chapter 1, at a blood pH of 7.4, the ratio of [HCO3 ] to [H2CO3] is 20 1 and the system s buffering capacity can neutralize a large amount of acid. The system is independently regulated by the kidneys, which control the plasma HCOj level, and by the respiratory rate, which regulates the Pco2- Protein and phosphate buffer systems also operate in plasma and erythrocytes. Proteins are especially important buffers in the intracellular fluid. The hydroxyapatite of bone also acts as a buffer. 

The pH of blood plasma is maintained at about 7.40 by several buffer systems, the most important of which is the HCO3/H2CO3 system. In the erythrocyte, where the pH is 7.25, the principal buffer systems are HCO3/H2CO3 and hemoglobin. The hemoglobin molecule is a complex protein molecule (molar mass 65,000 g) that contains a number of ionizable protons. As a very rough approximation, we can treat it as a monoprotic acid of the form HHb ... 

The study of membrane and nucleic acid systems presents greater complexities. The study of lipids and insoluble membrane proteins in association with them can be achieved in solution scattering [75,76] by the use of lipid vesicles to solubilize the system of interest. Membrane proteins can alternatively be studied by solubilization using detergent micelles. The exception to this generalization is the group of plasma hpoproteins which are readily soluble in aqueous buffers. Systems with nucleic acids (i.e. protein-nucleic acid complexes, viruses, ribosomes and chromatin) [24-27,77,78] are not affected by these solubihty problems. However, lipid and nucleic acid systems are both further complicated in their analyses by the polyionic character of these macromolecules. Particular care is required concerning the partial specific volumes of the individual components to be used within the system of interest. 

A buffer system prevents marked changes in the pH of a solution when an acid or base is added. Three major buffer systems of the blood are the bicarbonate buffer, the phosphate buffer, and the plasma proteins. The most important of these is the bicarbonate buffer system, consisting of a mixture of bicarbonate ions (HCO3 ) and carbonic acid (H2CO3). 

Although the bicarbonate system is not the strongest buffer system in the body (the most powerful and plentiful one utilizes the proteins of plasma and cells), it is very important because the concentrations of both bicarbonate and carbonic acid are regulated by the kidneys and by the respiratory system. Without the respiratory and urinary processes, the capacity of buffers would eventually be exceeded. 

The blood contains three major buffer systems the bicarbonate system, the phosphate system, and the plasma protein system. The most important is the bicarbonate system, which consists of a mixture of bicarbonate ions and carbonic acid. 

In order to achieve high concentrations of HCOj" from dissolved CO2, the resulting protons must be removed, i.e. accommodated by a buffer system. The principal buffers serving this function are plasma proteins (accounting for about 10% of the protons), erythrocyte phosphate (about 20%) and erythrocyte hemoglobin (60-70%). For the role of hemoglobin, see Bohr effect. Hemoglobin. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

Blood is far more complicated than phosphate as a buffer system. There are many different chemicals contributing to buffering. Each haemoglobin molecule and each plasma protein molecule has many groups which donate and accept hydrogen ions and which have pK values in the physiological range. The result... 

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