EFP TEST FOR CALCiUM

Calcium, being the most abundant mineral in the human body, is the major material used in bones and shells mineralization, wherein it helps build strong bones and teeth. It is important for heart function, and helps with muscle contraction, nerve signaling and blood clotting as well. Aside from that, it is also one of the major extra – cellular fluids that functions as a signal for many cellular processes in which all cells need in order to work properly.

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Normal values of calcium vary as to the amount of serum albumin, a protein wherein calcium is bound. Rather than the total calcium, biologic effect of calcium is determined by the amount of ionized calcium. It does not vary with the level of albumin, thus, it is useful to be measured when that protein is not in normal ranges or when a calcium disorder is suspected despite normal level of total calcium.

Determination of Calcium through Flame Photometry

 

Blood Calcium determination by Emission Flame Photometry presents peculiar difficulties. If precipitation and other pretreatment of the sample are to be avoided, the serum must be diluted at least tenfold, so that the final concentration of calcium is 1mg/100ml. this diluted calcium solution emits very little light from a flame. The use of an interference filter permits the partial isolation of the calcium oxide red bands, provided that the didymeium glass is used to reduce the excess sodium light also emitted. In an acetylene -  air flame, maximum output of energy in the calcium band is ensured, and the intensity of this  light, although very low, can be measured either by the use of a PHOTO - CONDUCTIVE cell

 

by R. W. R. Baker

 

Measurement of calcium concentrations is not achieved by using samples of diluted serum used for sodium and potassium determination, since calcium’s lines of emission are much less intense than those of sodium and potassium. Reduced viscosity by precipitation of proteins with trichloroacetic acid are adequately used. A correction is necessary for the effect of sodium and potassium upon calcium emission upon flame background at the wave-length used for calcium measurements.

METHOD

SERUM PREPARATION

Pipette l.00 cc. of serum and 5.00 cc. of distilled water into 15 cc. conical centrifuge tubes. 4.00 cc. of 10 per cent trichloroacetic acid is added, mix by inverting ten times, stand for ten minutes in order to precipitate the proteins. Centrifuge at 2000 r.p.m. for 10 minutes. Supernatant fluid is decanted and transferred into 5cc beakers.

STOCK SOLUTION PREPARATION

By using 1 liter of distilled water as diluent,  0.5004g Calcium Carbonate is dissolved and added to 10cc of concentrated hydrochloric acid, forming 10 mmeq stock solution of calcium. For recovery experiments, a standard with 1.0 m.eq. of calcium per liter was prepared. The reading for the calcium blank was determined with a solution containing 14.0 m.eq. of sodium and 0.5 m.eq. of potassium per liter of 4 per cent trichloroacetic acid.

The instrument used was a Beckman quartz spectrophotometer with a flame attachment. Light from the flame was measured on the transmission scale of the instrument, since the units on this scale are related linearly to the intensity of the light. The settings of the instrument are somewhat arbitrary, the following serving only as a guide. Calibration curves may vary somewhat with different settings and different instruments and should be checked for each instrument. 

• Oxygen-22 inches of water pressure.
• Gas---Adjusted to give medium small cones of blue flame, about 0.3 to 0.4 cm. in height. Shorter cones tended to cause an unstable reading. The propane pressure gage read about 1.2 cm. but was not used in flame adjustment.
• Air-20 pounds per sq. in.
• Wave-Length-The 556 rnp calcium oxide line is the most satisfactory for calcium measurement.
• Sensitivity-Adjusted to give readings on the transmission scale of about 70; about four turns of the sensitivity knob from the counter-clockwise limit with switch position 0.1. 
• Phototube-The ultraviolet tube was used at 556 mp; it is more sensitive than the red phototube.

MEASUREMENT

The reading on the transmission scale was recorded for distilled water, for the sodium- and potassium-containing calcium blank, or the working standard, and for the unknown, followed by repeated between the two readings. After three pairs of agreeing readings were obtained, the calcium blank and distilled water readings were checked. by reading the transmission at 589 rnp and at 767 rnp of the standard and unknown solutions. For this purpose the slit,was decreased to about 8.2 mm., the sensitivity was increased if necessary to 1.0, and the red photocell was used. Readings of the standard and unknown to determine the average difference When necessary, data for a sodium and potassium correction were obtained.

references:

R.W.R.Baker, Clinical Chemistry, 2004

HENRY GRIFFITH, CLINICAL LABORATORY CHEMISTRY, 2006