Calcium protein-bound

Jaynes, H. 0. and Whitney, R. McL. 1982. Resin-contact time for the determination of protein bound calcium in milk and model systems. J. Dairy Sci. 65, 1074-1083. 

In seven healthy controls, seven patients with duodenal ulcer, seven with primary hyperparathyroidism, and one with an excluded gastric antrum, intravenous secretin 3 U/ kg/hour for 90 minutes increased total protein and the protein-bound calcium fraction but did not alter the ionized calcium fraction (6). 

With prolonged bed rest, fluid retention occurs and plasma protein and albumin concentrations may be decreased by an average of 0.5 and 0.3g/dL, respectively. The concentrations of protein-bound constituents are also reduced, although mobilization of calcium from bones with an increased free ionized fraction compensates for the reduced protein-bound calcium, so serum total calcium is less affected. Serum aspartate aminotransferase activity is usually slightly less in individuals confined to bed than in those undertaking normal physical activity. Initially and paradoxically, creatine kinase (CK) activity is increased as a result of its release from skeletal muscles, but ultimately, CK activity may be less than in active, healthy individuals. Serum potassium may be reduced by up to 0.5mmol/L because of reduction of skeletal muscle mass. 

In renal failure, organic acids, phosphate, and sulfate are increased because of retention. Chronic metabolic acidosis enhances mobilixation of calcium from bone the decrease in plasma pH increases dissociation of plasma protein-bound calcium so that more Ca is filtered through the glomerulus and less is reabsorbed in the tubule (see also Chapter 45). 

Figure 49-2 Equilibria and determinations of calcium in serum. Calcium can move among three physiochemical pools (1) free calcium, (2) protein-bound calcium, and (3) calcium complexed with inorganic and organic anions. Methods for determining total calcium measure all three pools, whereas methods for determining free calcium measure only that pool.

The binding of calcium by protein and small anions is influenced by pH in vitro and in vivo. Albumin, with up to 30 binding sites for calcium,accounts for approximately 80% of the protein-bound calcium. Increasing the pH of a specimen in vitro increases the ionization and negative charge on albumin and other proteins, leading to an increase... 

Figure 49-5 Effect of pH on free and protein-bound calcium.

A common and important source of preanalytical error in the measurement of calcium is the increase in total, but not free, calcium concentration associated with tourniquet use and venous occlusion during sampling. Errors of 0.5 to 1.0 mg/dL (0.12 to 0.25 mmol/L) in total calcium may result because of the increase in protein-bound calcium caused by the efflux of water from the vascular compartment during stasis. Only small and clinically insignificant increases in free calcium have been reported with venous stasis. If a tourniquet is required, it should be applied just before sampling and released as soon as possible. 

Although ionized and protein-bound calcium are in equilibrium, release from the protein-bound fraction is slow and changes in plasma protein (especially albumin) concentration result in parallel changes in total plasma calcium. A decrease in serum albumin of 1 g/dL results in a decrease of about 0.8 mg/dL in total serum calcium. The equilibrium among the three forms of serum calcium is affected by changes in blood pH. Thus, at pH 6.8 (acidosis), about 54% of serum calcium is in the ionized form, whereas at pH 7.8 (alkalosis), only 38% is ionized. 

Tourniquets do not appear to cause artefactual elevations of serum alkaline phosphatase activities provided the period of venous occlusion does not exceed 30 seconds (S62). Longer periods of occlusion may cause tourniquet effects similar to those seen with protein-bound calcium (R3) and other circulating proteins. This factor may well be responsible for some of the variability between specimens obtained from the same individual at different times (LIO). 

In the blood, free Ca + ions are in equilibrium with calcium ions bound to proteins. As the free ions are taken up by the shell gland, more are provided by the dissociation of the protein-bound calcium. 

It is g erally assumed that it is the concentration of ionic calcium in the extracellular fluid which the parathyroid glands control by this action on the skeleton and that the variations in total plasma calcium are strictly proportional. However, Freeman and Breen (Fll) have shown that parathyroidectomy is followed by a rise in the proportion of protein-boimd calcium and that this can be reduced by injection of the hormone. This accords with the observations of Lloyd and Rose in hyperparathyroidism (L6 see Section 4.2) and with the clinical observation that hypo-parathyroid patients may develop tetany at surprisingly high levels of total plasma calcium. The effects of Ae hormone on add-base balance may be concerned in this phenomenon since acidemia reduces the proportion of protein-bound calcium. 

Another advantage (or disadvantage, depending on the problem at hand) of many electrochemical methods is that they respond to the activity of a chemical species rather than to the concentration. An example where this may be of importance is the calcium level in serum. Ion-selective electrodes respond to free, aquated Ca ions, whereas the usual clinical method for serum calcium is flame photometry, which measures the total calcium present including a large amount tied up as protein-bound calcium. The more important physiological parameter, the measure of the effective level of calcium actually available for participation in various enzymatic... 

UV-visible spectrophotometry Calcium ions form a violet-colored complex with o-cresolphthalein. Its absorbance is proportional to the calcium concentration of the sample and can be measured at 546 nm. Protein-bound calcium is released by hydrochloric acid. The interference of magnesium ions is excluded by the addition of 8-hydroxyquinoline. The method is in widespread use on mechanized analysers for clinical chemistry. 

In clinical chemistry the determination of sodium, potassium, and calcium is well standardized. In the past decades flame photometry has been reputed to be the mediod of choice in the analysis of biological samples. The advantages of this procedure are a short requirement of time and materials for sample preparation, short duration of the analytical procedure, and the possibility of automation. The procedure became improved due to the application of lithium as internal standard. Excellent precision and accuracy could be obtained in sodium and potassium determination. In calcium determination inaccuracy occurs due to the matrix. It is of disadvantage that only the determination of total calcium and not the differentiation between free and protein bound calcium is possible. Furthermore special equipment (flame photometer) is necessary. 

Calcium that is protein-bound and inorganically complexed is detectable by FAAS only after dissociation fixim these complexes. Acid is used for the dissociation of protein-bound calcium and lanthanum or strontium ions are added, usually in the sample diluent, to displace calcium from phosphate, oxalate, citrate, and other complexes (10). Interference from magnesium and other elements is reduced by using a narrow bandpass, diffraction-grating spectrophotometer set at the specific absorption line of calcium (422.7 nm). Sodium and potassium interference is eliminated by the addition of physiological concentrations of sodium and potassium to the calcium standards (111-... 

Protein-bound calcium. This is non-diffusible and accounts for a further 40% of the total. This fraction is distributed between the plasma albumin and the globulins in a ratio of about 3 1. The calcium is electrostatically bound and the amount held increases with pH because the net negative charge on the plasma proteins increases. 

Calcium is absorbed directly into plasma where a concentration of 9 to 11 mg. per 100 ml. is maintained. Calcium is present in blood in both ionized (50 to 60%) and non-ionized form, the latter bound to protein (Chapter 21). The concentration of protein-bound calcium, and hence of total blood calcium, varies with the concentration of serum proteins. The ionized calcium fraction is the one responsible for prevention of tetany. 

---