Calcium accounts for about 1.5-2.9% of body weight and is an important element in the formation of the bones and soft tissues.

In the body, 90% of calcium is present in bones as hydroxyapatite, 1% in cells, and 0.1% in the blood. Physiological functions include osmotic pressure regulation in cells, blood coagulation, muscle atrophy, nerve stimulation, and enzyme activation. In blood, 45% of calcium is bound to albumin and globulin, 10% is phosphate, and 45% is free ions.

When serum calcium levels decrease, parathyroid hormone is secreted, which promotes bone resorption, reabsorption in the kidney, and calcium absorption from the small intestine associated with the production of active vitamin D, thereby increasing the serum calcium concentration. Through these mechanisms, serum calcium concentration is strictly regulated, and normal values ​​remain within a narrow range.

Hypocalcemia is known to cause rickets, osteomalacia, tetany, osteoporosis, and arteriosclerosis, while hypercalcemia is known to be involved in depression, disorientation, fatigue, hypersensitivity, arrhythmia, and hypertension.

This method determines the calcium concentration by observing the color change in the visible region by the formation of a chelate complex between Chlorophosphonazo-Ⅲ and calcium.

Chlorophosphonazo-Ⅲ forms complexes with calcium in the neutral region. The calcium concentration can be determined by observing the absorbance at 690 nm as the main wavelength and 740-810 nm as the secondary wavelength.

◯ Samples that can be directly quantified with this kit

Examples: serum, plasma

The denaturant in the kit dissociates calcium from the protein and develops color after reducing agent reduces it. No specific pretreatment is required.

※ Do not use blood collection tubes containing EDTA which may affect the assay value.

◯ Samples that require dilution

Example: Urine

There is a possibility that the measurement range will be exceeded so dilute samples to 5x for measurement.

※ If using urine as a sample, preserve it by adding hydrochloric acid. When measuring, adjust the pH to 2~3.

※ The components of urine change from time to time, so the dilution ratio may vary. Be careful especially with the dilution ratio for early morning urine.

※ If you dilute a sample, be careful not to forget to multiply the dilution ratio when calculating the concentration.

◯ Samples that require acid extraction

Examples: cell lysates, tissue homogenates, and other aqueous solution samples

The pH of the samples was adjusted to low level to dissociates the calcium from the protein, and the supernatant was used as the assay sample. However, only the following chemicals can be measured.

・Free calcium

・Protein-bound (coordinated) calcium (Protein-Ca2+, e.g. albumin, metallothionein)

・Other coordination-bound calcium

※ Do not use lysis buffers containing EDTA as they may affect the measured values.

※ Acid digestion should be performed in the case of organic calcium.

◯ Samples that require microwave method and organic matter mixed acid decomposition

Example: Samples containing strong chelating agents such as EDTA, organic calcium (-C-Ca-C- single bonded), or calcium encapsulated in a cyclic ligand.

Acid extraction is necessary when measuring chemical species that cannot be dissociated or samples containing interfering substances such as strong calcium chelating agents.

※ After decomposition of organic matter, adjust the pH to 2-3, and use the sample as the assay sample.

(1) ) Add 2 µl of DIW, standard solution, and assay sample to wells of a 96-well plate.

(2) Add 240 µl of RA solution to each well

(3) Incubate the plate at room temperature for 10 minutes.

(4) Measure absorbance at main wavelength 690 nm (secondary wavelength 740-800 nm.)

(5) Calculate the calcium concentration using the following formula:

(1) Measurement of calcium concentration in control serums using this kit

Figure 1: Results of measurement of control serum with known calcium concentration.

(2) Measurement of serial dilution of calcium standard samples

Figure 2: Measurement results of calcium standard sample

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