Estimation of hemoglobin content
- 1. Estimation of hemoglobin content Dr. Nitin Kumar Sen Assistant professor Department of Pharmacology SJIPR, Palghar
- 2. Theory- Hemoglobin is the protein molecule in red blood cells that carries oxygen from the lungs to the tissues and returns carbon dioxide from the tissues back to the lungs. It is abbreviated as Hb, or Hgb. Hemoglobin is made up of four protein molecules (globulin chains) that are connected together. The normal adult Hb molecule contains two alpha-chains and two beta-globulin chains. Each globulin chain contains iron containing porphyrin compound termed heme. Iron atom is located within this heme. Each heme binds to one oxygen molecule (HbO2-oxyhemoglobin). Hence Hb molecule can bind four oxygen molecules (HbO8). The heme part of the Hb is also responsible for the red color of the blood. Normal value- The normal range for hemoglobin is: in male-13 to 18 gm/dl, in female-12 to 16 gm/dl.
- 3. Formation of hemoglobin- Formation of hemoglobin begins in the proerythroblast stage during the synthesis of erythrocytes. The following are the stages of hemoglobin synthesis. 1. 1.The succinyl CoA formed in the Krebs metabolic stage, binds with glycine to form a pyrrole molecule. 2. 2. Four pyrroles combine to form protoporphyrin IX. 3. 3. Protoporphyrin IX combines with ferrous iron (Fe++) to form the heme molecule. 4. 4. Each heme molecule combines with a long polypeptide chain, a globin synthesized by ribosomes, forming a subunit of hemoglobin called a hemoglobin chain. 5. 5.Each chain has a molecular weight of 16,000, four of these chains bind together to form whole 6. hemoglobin molecule.
- 4. Functions of hemoglobin- The hemoglobin present in the blood carries oxygen molecules in the form of oxy-hemoglobin and delivers the oxygen to the tissue cells and carrying the carbon dioxide (metabolic waste) to the alveoli of the lung. The carbon dioxide eliminated from the lung through expiration.
- 5. Principle- When blood is mixed with 0.1N HCl and kept for 5-7 minutes, the hemoglobin is converts into the yellowish brown coloured acid hematin. The acid-hematin is then diluted with distilled water till the color of the acid hematin matches that of the yellowish brown solution present in the calibration tube. The hemoglobin is estimated by reading the value directly from the scale. (Please note that 100 percent on the scale corresponds to 14.5 to 15 gm %). Fetal hemoglobin is not converted to acid hematin and therefore this technique is not used for small infants.
- 6. Apparatus and accessories. ● Sahli’s hemoglobinometer (comparator)- This consists of yellowish brown standard tubes. Opaque white glass is present at the back to provide uniform illumination. ● Sahli’s hemoglobin tube which is marked in grams percent g% (2-24) and percentage % (10-140) ● Hemoglobin pipette or Sahlis’ pipette (marked at 0.02 or 20μl) attached to a flexible rubber tube with white sucker, no bulb. ● Stirrer- Thin glass rod. ● N/10 HCl or 0.1N HCl. ● Distilled water ● Spirit swabs.
- 8. Procedure- ● N/10 HCl (0.1NHCl) was placed in to the Sahli’s graduated tube the mark 2 grams. ● With the help of Sahli’s pipette blood was pipette out exactly up to 20μl mark. ● The blood adhering to the outer part of the pipette was wiped off with the help of cotton. ● The collected blood sample was added to the N/10 HCl present in the Sahli’s tube. ● The contents mixed with the help of stirrer, and allowed the tube for 10 minutes. ● Then distilled water added drop in to the tube till the color of the solution matches with the standard yellowish brown color. ● The reading of the lower meniscus was noted in grams and percentage.
- 10. Observations and report- 1. The hemoglobin content of my blood is ------------- gm% ----------- %. 2. The oxygen carrying capacity of my blood = ---------- gm x 1.34 ml = ml% 3. The iron content of my blood is ------------- gm % x 3.35 mg = ------------- mg iron %.