celiac disease, pancreatic dysfunction

(fatty acids combine with calcium and are precipitated

and excreted in the feces).

5. Acute pancreatitis

6. Alkalosis (calcium ions become bound to protein)

7. Osteomalacia

8. Diarrhea

9. Rickets.

Increased Ionized Calcium

1. Primary hyperparathyroidism

2. Ectopic parathyroid hormone producing tumors

3. Excess intake of vitamin D

4. Various malignancies.

Decreased Ionized Calcium

Primary hypoparathyroidism is associated with low

ionized calcium level and low total calcium level.

Be Careful

1. Thiazide diuretics may lead to impairment of urinary

calcium excretions and consequent hypercalcemia.

Clinical Chemistry 497

2. Patients with renal insufficiency who are undergoing

dialysis, a calcium-ion exchange resin is sometimes

used for hyperkalemia. The use of this resin may lead

to increased calcium levels.

3. Increased intake of magnesium and phosphates and

the excessive use of laxatives may lower the blood

calcium level. This occurs because of the increased

intestinal loss of calcium produced by these elements.

4. When decreased calcium levels are due to magnesium

deficiency, as in poor absorption from the bowel, the

administration of magnesium will correct the calcium

deficiency.

5. If a patient is known to have or suspected of having a

pH abnormality, a concurrent pH should be requested

with ionized calcium.

Interfering Factors

Many drugs can alter blood calcium levels for the list of

drugs that may alter blood calcium level.

PHOSPHORUS

Molybdate UV Method

(Courtesy: Tulip Group of Companies)

For the determination of inorganic phosphorus in serum,

plasma and urine (for in vitro diagnostic use only).

Summary

Phosphorus is mainly combined with calcium and is found in

the bones. Approximately 15% exists as inorganic phosphorus

or phosphate esters. It is involved in the carbohydrate metabolism and is a component of many other substances. Increased

levels are found in hypoparathyroidism, renal failure, bone

metastatis and liver diseases. Decreased levels are found in

hyperparathyroidism, rickets and vitamin D deficiency.

Principle

Phosphate ions in an acidic medium react with ammonium

molybdate to form a phosphomolybdate complex. This

complex has an absorbance in the ultraviolet range and

is measured at 340 nm. Intensity of the complex formed

is directly proportional to the amount of inorganic phosphorus present in the sample.

Phosphorus + Ammonium Phosphomolymolybdate bdate complex

Normal Reference Values

Serum (Adults) : 2.5–5.0 mg/dL

(Children) : 4.0–6.5 mg/dL

Urine : 0.3–1.0 g/24 hours

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 75 mL 2 × 75 mL

L1: Acid ragent 60 mL 2 × 60 mL

L2: Molybdate reagent 15 mL 2 × 15 mL

S: Phosphorus standard 5 mg/dL 5 mL 5 mL

Storage/Stability

Reagents are stable at RT (25–30°C) till the expiry

mentioned on the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent: Pour the contents of 1 bottle of L2

(molybdate reagent) into 1 bottle of L1 (Acid reagent).

This working reagent is stable for at least 6 months when

stored at 2–8°C. Upon storage the working reagent may

develop a slight blue color, however this does not affect

the performance of the reagent.

Alternatively for flexibility as much of working reagent

may be made as and when desired by mixing together

4 parts of L1 (acid reagent ) and 1 part of L2 (molybdate

reagent 2). Alternatively 0.8 mL of L1 and 0.2 mL of L2

may also be used instead of 1 mL of the working reagent

directly during the assay.

Sample Material

Serum heparinized/EDTA plasma or urine. Acidify the

urine with a few drops of conc. Hydrochloric acid and

dilute 1 + 19 before the assay, (results × 20). Inorganic

phosphorus is reported to be stable in serum for 7 days at

2–8°C.

Procedure

Wavelength/filter : 340 nm (Hg 365 nm)

Temperature : RT

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Working reagent 1.0 1.0 1.0

Distilled water 0.01

Phosphorus standard(s) - 0.01

Sample - - 0.01

498 Concise Book of Medical Laboratory Technology: Methods and Interpretations Mix well and incubate at RT for 5 minutes. Measure

the absorbance of the standard (Abs S), and test sample

(Abs T) against the Blank, within 60 minutes.

Calculations

 Abs T

Phosphorus in mg/dL = _______ × 5 Abs S

Linearity

This procedure is linear upto 20 mg/dL. If values exceed this

limit, dilute the sample with distilled water and repeat the

assay. Calculate the value using the proper dilution factor.

Note

Hemolysis interferes with the test.

Use clean glassware washed with N/10 HCI as detergents

may contain phosphate ions.

System Parameters

Reaction : UV end point Interval :

Wavelength : 340 nm Sample

volume

: 0.01 mL

Zero setting :  Reagent

blank

Reagent

volume

: 1.00 mL

Incubation

tempeerature

: RT Standard : 5 mg/dL

Incubated time : 5 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 20 mg/dL

No. of read : — Units : mg/dL

Phosphorus

Mod Gomorri’s Method

(Courtesy: Tulip Group of Companies)

For the determination of inorganic phosphorus in serum,

plasma and urine (for in vitro diagnostic use only).

Summary

Phosphorus is mainly combined with calcium and is found in

the bones. Approximately, 15% exists as inorganic phosphorus

or phosphate esters. It is involved in the carbohydrate

metabolism and is a component of many other substances.

Increased levels are found in hypoparathyroidism, renal

failure, bone metastasis and liver diseases. Decreased levels

are found in hyperparathyroidism, rickets and vitamin D

deficiency.