Erythrocyte, buffering

The plasma bicarbonate level is normally a reflection of both the erythrocyte buffering mechanisms and the renal acid-base homeostatic mechanisms. The former affects the actual bicarbonate level but not the standard bicarbonate. The standard bicarbonate gives a measure of the non-respiratory contribution and its measurement is therefore useful in acute respiratory disorders when a metabolic component is involved. 

Boutron, P. Peyridieu, J.-F. (1993). Absence of toxicity for erythrocytes of buffered solutions containing high coixientrations of 2,3-butanediol and sugars. Cryobiol. 30,658 (abstract). 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

Table 6.3 displays the solution pKa values of the promoters capable of facilitating the electron transfer of Cu, Zn-SOD listed in Table 6.1. The —COOH-termi-nated SAMs are mostly negatively charged in phosphate buffer (pH 7.0). Although the bovine erythrocyte Cu, Zn-SOD has a net negative charge at pH 7.0 (p/ = 4.9), an electrostatic interaction is still expected to occur between the SAMs and the positively charged amino acid moieties (typically —NI13). Besides, the hydrogen bonding between —COOH groups and the amino acid residues is believed to comprehensively...

In 1977, Kellogg and Fridovich [28] showed that superoxide produced by the XO-acetaldehyde system initiated the oxidation of liposomes and hemolysis of erythrocytes. Lipid peroxidation was inhibited by SOD and catalase but not the hydroxyl radical scavenger mannitol. Gutteridge et al. [29] showed that the superoxide-generating system (aldehyde-XO) oxidized lipid micelles and decomposed deoxyribose. Superoxide and iron ions are apparently involved in the NADPH-dependent lipid peroxidation in human placental mitochondria [30], Ohyashiki and Nunomura [31] have found that the ferric ion-dependent lipid peroxidation of phospholipid liposomes was enhanced under acidic conditions (from pH 7.4 to 5.5). This reaction was inhibited by SOD, catalase, and hydroxyl radical scavengers. Ohyashiki and Nunomura suggested that superoxide, hydrogen peroxide, and hydroxyl radicals participate in the initiation of liposome oxidation. It has also been shown [32] that SOD inhibited the chain oxidation of methyl linoleate (but not methyl oleate) in phosphate buffer. 

Blood is the transport medium of the body. Plasma, which accounts for approximately 60% of the total volume, carries a wide range of small and medium-sized metabolites some are simply dissolved in solution (93% of the plasma is water), others are carried by specific carrier proteins. The chemical composition of the plasma is complex and reflects the chemical composition inside cells, which is why blood tests are so commonly used in diagnosis to see the biochemical events occurring in tissues. The formed cellular elements of the blood perform several functions defence against blood loss from bleeding (platelets, also called thrombocytes), defence against infection and immune surveillance (white cells, leucocytes), and gas transport and pH buffering (red cells, erythrocytes). 

This occurs because deoxy-Hb forms carbamino-complexes with C02 more readily than oxy-Hb. Secondly, deoxy-Hb is a better buffer of H+ than oxy-Hb and this increases the amount of HC03 formed. Once formed, HC03 diffuses out of the erythrocyte. To maintain electrical neutrality Cl- moves in. This is known as the Cl shift or the Hamburger effect. 

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

AmB formulations were dispersed in phosphate-buffered saline (PBS) at different concentrations (0.1 lOOpg/mL) and incubated for five minutes at 37°C. Freshly isolated human erythrocytes were then added to a final hematocrit of 2% and incubated at the same temperature for 30 minutes. After centrifugation, the supernatant was removed and the RBC pellet was lysed with sterile water. The hemoglobin remaining in the pellet was estimated from its absorption at 560 nm recorded with a spectrophotometer. The percentage hemolysis was calculated from the difference between the hemoglobin remaining in the test samples and the control incubated with PBS alone. 

During anaerobic glycolysis in the muscles and erythrocytes, glucose is converted into lactate, releasing protons in the process (see p. 338). The synthesis of the ketone bodies acetoacetic acid and 3-hydroxybutyric acid in the liver (see p. 312) also releases protons. Normally, the amounts formed are small and of little influence on the proton balance. If acids are formed in large amounts, however (e. g., during starvation or in diabetes mellitus see p. 160), they strain the buffer systems and can lead to a reduction in pH (metabolic acidoses lactacidosis or ketoacidosis). 

Due to their high concentration, plasma proteins—and hemoglobin in the erythrocytes in particular—provide about one-quarter of the blood s buffering capacity. The buffering effect of proteins involves contributions from all of the ionizable side chains. At the pH value of blood, the acidic amino acids (Asp, Glu) and histidine are particularly effective. 

Erythrocyte distribution in plasma and buffer No need for mechanism to separate bound and free drug - adsorption issues minimized. For highly bound compounds, analytical precision no longer a prerequisite. Low throughput, more complex assay format. [40]... 

A 100-pi aliquot of thawed erythrocytes is mixed well with 900 pi hemolysis buffer. This diluted hemolysate is used to determine hemoglobin concentration (e.g., by a routine technique). 

The enzyme reaction is performed in duplicate. Incubation buffer (200 pi) is mixed with 50 pi diluted hemolysate and incubated at 37°C for 5 min, then 50 pi substrate solution is added and the tubes are kept at 37°C in the dark for a further 60 min. One null value per experiment with hemolysis buffer instead of erythrocytes is treated the same way. 

The Bohr effect is closely related to the major roles that hemoglobin plays in disposing of the C02 produced in tissues, and in controlling the blood pH. While oxygen is being delivered to the tissues in the venous blood, the C02 is being removed from the tissues (fig. 5.5). The C02 that diffuses into the erythrocytes is rapidly converted into carbonic acid, which in turn dissociates into H+ and HC03. The protons produced by this dissociation would lower the pH and reverse the dissociation if it were not for the buffer-... 

In the isolated perfused liver experiments, buffers containing no erythrocytes or serum proteins are used to examine the direct interaction of a gene drag with tissues and to avoid the interaction of a gene drag with blood components and possible contamination of nucleases. 

Resuspend the cell pellet in labeling buffer (about 5 mL final total volume) and proceed to count recovered cells in triplicate (see Note 7). An automated particle counter is highly recommended because it is possible to set size gates for cancer cells, erythrocytes, and leukocytes. 

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