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Colorimeter : Principle, Instrumentation and Uses

Dhurba Giri — Fri, 24 Jun 2022 13:35:30 +0000

Colorimeter is a form of photometer which deals with the measurement of light transmitting power of a colored solution in order to determine the concentration of light absorbing substances present within it. It was invented by Louis J Dubosca in 1870. The concentration of colored solute in a solution is estimated by comparing its color intensity with that of standard solution containing a known concentration of solute.

Principle of Colorimeter :

When a beam of incident light of intensity I₀ passes through a solution, following events occur:

A part of incident light is reflected. It is denoted by I_r
A part of incident light is absorbed. It is denoted by I_a
Remaining incident light is transmitted. It is denoted by I_t

As I_r is kept constant by using cells with identical properties, The light that is not absorbed is transmitted through the solution and gives the solution its color. Note that color of the incident light should be complementary to that of color of the solution.

The ratio of the intensity of transmitted light (I_t) to the intensity of incident light (I₀) is called transmittance (T). Photometric instruments measure transmittance. In mathematical terms,
T = I_t÷I₀

The absorbance (A) of the solution (at a given wavelength) is defined as equal to the logarithm (base 10) of 1÷T. That is,
A = log (1÷T)

These measurements are dependent on two important laws:

Beer’s law:

When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the concentration (C) of solute in the solution.
Lambert’s law:

When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the length (L) or thickness of the solution.

When combining Beer-Lambert’s law,
Absorbance (A) α CL
Or, A= KCL
where K is a constant known as absorption coefficient.

As the path length is same (as same cuvette is used), Concentration of an unknown solution can be determined by using equation:

Instrumentation of Colorimeter :

1. Light Source:

The light source should produce energy at sufficient intensity throughout the whole visible spectrum (380-780nm). Tungsten lamp is frequently used.

2. Slit:

It allows a beam of light to path and minimize unwanted light.

3. Condensing lens:

Give parallel beam of light.

4. Monochromator:

It is used to produce monochromatic radiation (one wavelength band) from polychromatic radiation (white light) produced from light source. It allows required wavelength to pass through it. Prism, gelatin fibers, grating monochromators or interference filters can be used.

5. Sample Holder (Cuvette):

Must be transparent. Glass or clear plastic cuvettes are preferred.

6. Photo detectors:

Detector of colorimeter basically receives the resultant light beam once it has passed through the sample and converts it into electrical signal. Selenium photocell, silicon photocell, phototube, photomultiplier tube etc are used.

7. Display:

It detects and measures the electric signal and makes visible output.

Uses and Applications

In clinical laboratory, colorimeter is used for the estimation of various biochemical compounds in variety of biological samples like blood, plasma, serum, CSF, urine and other body fluids. All those methods which involve the formation of colored product with specific analyte, the analyte can be estimated quantitatively. Colorimeters are also widely used for monitoring the growth of bacterial or yeast cells in liquid cultures.

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GOD-POD Method For The Estimation Of Blood Glucose

Dhurba Giri — Tue, 22 Mar 2022 15:02:54 +0000

Glucose is the major carbohydrate present in the blood. It serves as a principal fuel for all the body tissues including the brain. It undergoes a series of chemical reactions to produce energy. Accurate and precise measurement of blood glucose level is of great importance in the diagnosis and management of diabetes and other disorders of carbohydrate metabolism, hyperglycemia, and hypoglycemia.

Different methods based on different properties of glucose are described for blood glucose estimation. They are:

1. Reduction Methods:

Ferric reduction methods

Hagedorn-Jensen ferric reduction method
Hoffman’s method

Cupric reduction methods

Somogyi-Nelsen method
Neocuproine method
Shaffer-Hartmann method
Folin-Wu method
Benedict’s method

2. Aromatic amine condensation methods:

O-toluidine method

3. Enzymatic Methods:

Glucose-oxidase Peroxidase (GOD POD) method (Trinder method)
Hexokinase method
Glucose dehydrogenase (GDH) method
Kinetic method
Polarographic method

4. Electrochemical methods:

Glucometer

GOD-POD Method for Glucose Estimation

Being more specific, easier, and more accurate, enzymatic methods are preferred these days. Among them, the GOD-POD method is the most common method of glucose estimation.

Principle:

In the presence of atmospheric oxygen, glucose present in the specimen is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid and hydrogen peroxide (H₂O₂).

Thus formed H₂O₂ oxidatively couples with 4-aminoantipyrine and phenol in presence of peroxidase (POD) to form red-colored quinoneimine dye, which is measured colorimetrically at 540nm. The intensity of the color is directly proportional to the concentration of glucose present in the specimen.

Requirements:

Specimen:

Serum, or plasma free of hemolysis. Sodium fluoride is preferred as an anticoagulant due to its antiglycolytic activity.

Reagents:

Glucose standard (100 mg/dl)
GOD-POD reagent: Enzyme reagent mixture containing glucose oxidase (GOD), peroxidase (POD), 4-aminoantipyrine, phenol, and phosphate buffer (pH≈7.0), some stabilizers and activators.

Instruments:

Test tubes
Pipettes, disposable tips, rack
Water bath
Colorimeter

Procedure:

Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
Pipette as follows:

	Blank	Standard	Test
GOD-POD Reagent	1 ml	1 ml	1 ml
Distilled water	10 µl	–	–
Glucose standard	–	10 µl	–
Sample	–	–	10 µl

Mix well and incubate at 37⁰C for 10 minutes. Or, at room temperature (25⁰C) for 30 minutes.
Measure the absorbance of the standard and test sample at 540nm (green filter) against blank within 60 minutes.

Calculation:

Calculate the concentration of blood glucose in the specimen using the following formula:

References:

Burrin, J. M., & Price, C. P. (1985). Measurement of blood glucose. Annals of clinical biochemistry.
Dandekar, S. P., Rane, S. A. (2004) Practical and Viva in Medical Biochemistry, New Delhi, Elsevier/Reed Elsevier. India PVT LTD.

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Folin-Wu Method For Estimation of Blood Glucose

Dhurba Giri — Sun, 20 Feb 2022 09:26:04 +0000

Folin-Wu method is one of the oldest methods for the estimation of blood sugar. However, it is almost obsolete for now but is in use in countries where enzyme preparations are not easy to obtain. This method is old and not specific for glucose determination since other substances (e.g. fructose, lactose, and glutathione) also bring about a reduction. The blood glucose level when estimated by the Folin-Wu method is higher than true glucose.

Principle

Proteins from the blood are removed by 10% sodium tungstate and 2/3N sulphuric acid. The glucose present in the protein-free filtrate on boiling in an alkaline medium will be changed to enediol form. This enediol will reduce cupric ions to the precipitate of cuprous oxide. This oxide is dissolved and reacted by phosphomolybdic acid to form phosphomolybdenum blue which is blue in color. Constricted tubes (Folin-Wu tubes) are used to avoid reoxidation of cuprous oxide by atmospheric oxygen. The final blue color is measured at 680 nm which is proportional to the amount of glucose present in the specimen.

Requirements

Folin Wu tubes
Colorimeter
Reagents:
1. 2/3 N H2SO4: Add 2ml H2SO4 to about 50ml of D/W and dilute up to 100ml.
2. 10% Sodium Tungstate: Dissolve 10 gm in 100ml of D/W.
3. Alkaline Copper tartarate:
  A) Dissolve 40gm sodium carbonate and 7.5 gm tartaric acid in about 400ml of D/W.
  B) Dissolve 4.5 gm copper sulphate in about 100ml D/W.
  Mix A and B and make volume up to 1000ml with D/W.
4. Phosphomolybdic acid: Dissolve 35 gm molybdic acid and 5 gm sodium tungstate in 200 ml 10% NaOH. Add it to 200ml D/W and boil for 45 minutes to remove ammonia. Cool and add slowly 125 ml of 89% phosphoric acid. Make up the volume to 500ml with D/W.
5. Distilled water
6. Glucose standard
  Stock (1g/dl): Dissolve 1 gm of glucose in 100 ml saturated benzoic acid (0.3%).
  Working standard (10mg/dl): Dilute stock 1:100 with saturated benzoic acid.

Procedure

Step 1: Preparation of protein-free filtrate:

Add 1 ml of blood to 7 ml of distilled water and mix.
Add 1 ml of 10% sodium tungstate.
Add 1 ml of 2/3N H2SO4 and mix. Allow standing for 5 minutes.
Centrifuge or filter using Whatmann number 1 filter paper.

Step 2: Testing

Set up 3 Folin-Wu tubes as follows:

Blank Standard Test

Distilled water 1 ml – –

Working glucose standard – 1 ml –

Protein-free filtrate – – 1 ml

Alkaline copper tartarate 1 ml 1 ml 1 ml
Place the tubes in a boiling water bath for 10 minutes.
Cool and add 1ml phosphomolybdic acid reagent to each tube.
Shake the tubes to get rid of air bubbles. Add distilled water up to 12.5 ml mark.
Mix and read the absorbance at 680 or red filter. Set the zero using the blank.

	Blank	Standard	Test
Distilled water	1 ml	–	–
Working glucose standard	–	1 ml	–
Protein-free filtrate	–	–	1 ml
Alkaline copper tartarate	1 ml	1 ml	1 ml

Calculations

Calculate the concentration of glucose in the blood specimen using the following
formula:

Note: 1 ml of blood was diluted 1:10 for protein precipitation. 1 ml of the diluted blood was then used for test. Therefore, the actual volume of blood used for the test is 0.1 ml.
Also, 1 ml of the working standard (10 mg/dl) contains 0.1 mg of glucose.

References

Kolhatkar, A., Ochei, J., & McGraw, T. (2008). Medical Laboratory Science: Theory and Practice.
Naigaonkar, A.V., (2007). A Manual Of Medical Laboratory Technology.
Geetha, D. K. (2011). Practical Biochemistry. Jaypee Brothers Medical Publishers (P) Ltd, UK.

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How to Convert ‘g’ to RPM and Vice Versa in a Centrifuge?

Dhurba Giri — Sat, 11 Sep 2021 16:37:08 +0000

Centrifuagtion is a technique that helps to separate mixtures by applying centrifugal force. A centrifuge machine works by using a principle of sedimentation. Under the influence of gravitational force, substances separate according to their density.

When dealing with centrifuge machines, we come across two different units of measurement: The Revolutions per minute (RPM) and Relative centrifugal force (RCF) or g-force. In fact these units are not same. This article discusses about what they actually are and how they are realated to each other.

Revolutions per minute (RPM)

RPM (Revolutions per minute) basically describes how fast the centrifuge goes. It is a measurement of how fast the centrifuge rotor does a full rotation in one minute. The force applied to the contents varies by the size of the centrifuge rotor.

Relative centrifugal force (RCF)

RCF (Relative centrifugal force) is the amount of force exerted on the contents on the rotor, resulting from revolutions of the rotor. It depends on the rotation speed and radius of the rotor. It is relative to the force of earth’s gravity and is measured in force*gravity (or g-force).

Which one to use?

The bigger the radius the more acceleration is applied to the samples for the same RPM. Since different centrifuge machines may have different rotor sizes, using RCF or g-force will give very accurate setting for experiments. RCF remains constant irrelevant of the centrifuge you are using

Converting RPM to RCF and vice versa

To calculate RCF from RPM, use following equation:

To calculate RPM from RCF, use following equation:

Where,

RCF(g) = Relative Centrifugal Force
r = Radius of the rotor (cm). It is the distance from the rotor axis to the bottom of the tube.
RPM(n) = Revolutions Per Minute

References

https://handling-solutions.eppendorf.com/sample-handling/centrifugation/safe-use-of-centrifuges/basics-in-centrifugation/
https://www.westlab.com/blog/2019/01/29/difference-between-rcf-and-rpm-in-centrifugation
https://blog.btlabsystems.com/blog/rpm-rcf-g-force
http://www.fao.org/3/ac802e/ac802e0u.htm

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Diacetyl Monoxime (DAM) Method for Estimation of Urea

Dhurba Giri — Sun, 28 Mar 2021 15:26:32 +0000

Urea is a waste product formed in liver following the breakdown of proteins. It passes into the blood, is filtered out of the kidneys and excreted in urine. Thus determination of blood urea is the most widely used screening test for the evaluation of kidney function. There are various methods used for the estimation of urea in a laboratory. Diacetyl monoxime (DAM) method is an older method.

Principle

Proteins are first precipitated by trichloroacetic acid. The urea present in the protein-free filtrate reacts with diacetyl monoxime in a hot acidic medium in presence of ferric/cadmium ions and thiosemicarbazide to form pink or red colored complex- diazine. The intensity of the color developed is measured photometrically at 530nm, which is directly proportional to the concentration of the urea present in the fluid.

Requirements

Apparatus:
Colorimeter
Conical flasks and test tubes to hold 20ml
Pipettes: 50ul, 0.1ml, 0.5ml, 5 ml
Measuring cylinder, 50 ml
Water bath at 100°C
Reagents:
Benzoic acid
Ferric Chloride
Diacetyl monoxime
orthophosphoric acid
Thiosemicarbazide
Tricholoroacetic acid
Urea
Specimen:
Serum, heparinized plasma or fluoride plasma.

Preparation of Regaents

Reagent 1: Trichloroacetic acid, 50g/l (5%) solution
Trichloroacetic acid = 10g
Distilled water = upto 200ml
Reagent 2: Diacetyl monoxime (2,3-butanedione monoxime) solution
Diacetyl Monoxime = 2g
Distilled water = upto 500ml
Reagent 3: Acid reagent
Concentrated sulfuric acid = 44ml
Orthophosphoric acid (H3PO4), 85% = 66ml
Cadmium sulfate = 1.6 g
Thiosemicarbazide = 50mg
Distilled water = upto 500ml
Reagent 4: Colour reagent
Acid reagent (Reagent 3) = 50ml
Diacetyl monoxime reagent = 50ml
Reagent 5: Benzoic acid solution 1 g/l
Benzoic acid = 1 g
Distilled Water = 1000ml.
Reagent 6: Urea stock reference solution, 125mmol/l
Urea = 750mg
Benzoic acid, 1 g/l (0.1%) solution = upto 100ml
Reagent 7: Urea working reference solution, 10mmol/l
Urea stock reference solution = 8ml
Benzoic acid (C7H6O2), 1 g/l (0.1%) solution = upto 100ml

Preparation of Sample

To obtain protein free filtrate, take 50 ul of whole blood/serum/plasma in a centrifuge tube. Add 1 ml of TCA solution and mix. Centrifuge at high speed (3000 g) for 5 minutes to sediment the precipitated proteins and obtain a clear supenatent fluid. Do same for standard/control sample.

Procedure

Take three (or more if needed) large test-tubes and label as follows:
Blank tube (B)
Standard tube (S)
Test tube (T)

Pipette into each tube as follows:

	Test	Standard	Blank
Color Reagent (Reagent 4)	3 ml	3 ml	3 ml
Protein Free Filtrate	0.1 ml	–	–
Urea Standard 10 mmol/l	–	0.1 ml	–
Distilled water	–	–	0.1 ml

Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for exactly 15 minutes to allow the red color to develop.
Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes.
Measure the colour produced in a colorimeter at a wavelength of 530nm.

Calculations

Calculate the concentration of urea in the blood specimen using the following formula:

References

World Health Organization, 2003. Manual of basic techniques for a health laboratory. World Health Organization.
World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
Godkar, P.B. and Godkar, D.P., 2003. Textbook of medical laboratory technology. Bhalani.
Mukherjee, K.L., 2013. Medical Laboratory Technology Volume 3 (Vol. 3). Tata McGraw-Hill Education.

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O-Toluidine Method for Estimation of Blood Glucose

Dhurba Giri — Sat, 27 Mar 2021 17:16:22 +0000

The O-toluidine method is an older method of blood glucose estimation. This method is no longer used today because O-toluidine is believed to be a carcinogen and is replaced by enzymatic methods. This method is still popular because of its simplicity, sensitivity and accuracy.

Principle

The proteins are first precipitated by tricholoroacetic acid. The glucose present in a protein free filtrate react with O-toluidine (primary aromatic amine) in a hot acidic medium to form a stable green colored complex, namely N-glycosamine. The presence of thiourea stabilizes the o-toluidine reaction. The intensity of the color developed is measured photometrically at 630nm, which is directly proportional to the concentration of the glucose present in the fluid.

Requirements

Apparatus:
Graduated pipettes
Test tubes
Micropipettes
Heating Bath, 100C
Spectrophotometer, wavelength 630nm
Reagents:
Benzoic acid
D-glucose
Glacial acetic acid
O-toluidine
Thiourea
Trichloroacetic acid
Specimen:
Collect 2-3 ml of blood in a fluoride tube. Use plasma for testing. Serum/Whole blood can also be used. Prepare protein-free filtrate if the sample is grossly hemolyzed or icteric. Add 100 ul specimen to 900 ul of 5gm/dl TCA, mix and centrifuge to get the filtrate.

Preparation of Regaents

Benzoic acid solution 1 g/l:
Dissolve 1 gm of benzoic acid in water and make upto 1 liter. Prepare the benzoic acid at least 24 hours before use.
Stock glucose solution 100 mg/dl:
Dissolve 1 gm of glucose in 1 liter of benzoic acid solution (1 g/l).
O-toluidine reagent:
Dissolve 1.5 gm thiourea in 940 ml of glacial acetic acid. When completely dissolved, add 60ml of O-toluidine. Mix well and store in amber bottle.

Procedure

Take three (or more if needed) large test-tubes and label as follows:
Blank tube (B)
Standard tube (S)
Test tube (T)

Pipette into each tube as follows:

	Test (T)	Standard (S)	Blank (B)
O-toluidine reagent	3 ml	3 ml	3 ml
Serum/Plasma	50 μl	–	–
Glucose Standard 100 mg/dl	–	50 μl	–
Distilled water	–	–	50 μl

Note: If protein free filtrate is used instead of serum/plasma, pipette 500 ul of protein free filtrate instead of 50 μl serum/plasma.

Mix the contents of each tube. Place all the tubes in the water-bath at 100°C for exactly 12 minutes.
Remove the tubes and allow them to cool in a beaker of cold water for 5 minutes.
Measure the colour produced in a colorimeter at a wavelength of 630nm.

Calculations

Calculate the concentration of glucose in the blood specimen using the following
formula:

References

World Health Organization, 2003. Manual of basic techniques for a health laboratory. World Health Organization.
World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
Mukherjee, K.L., 2013. Medical Laboratory Technology Volume 3 (Vol. 3). Tata McGraw-Hill Education.

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Sulphosalicylic Acid Test for Proteinuria: Principle and Procedure

Dhurba Giri — Sun, 21 Mar 2021 14:54:41 +0000

Most plasma proteins are too large to pass through the glomeruli of the kidney. The small amount of protein which does filter through is normally reabsorbed back into the blood by the kidney tubules. Only trace amounts of protein (less than 150 mg per 24 h) can therefore be found in normal urine. These proteins include Tamm-horsfall protein (Maximum-40%), Albumin (20%), Immunoglobulins, Hormones, Enzymes and Mucopolysaccharides.

When more than trace amounts of protein are found in urine, this is termed proteinuria. Detection of proteinuria is an important indicator of renal disease because protein has a very low maximal tubular rate of reabsorption. The following methods are used to test for proteinuria:

Qualitative Tests:
Heat and acetic acid test
Sulphosalicyclic acid test
Hellers nitric acid test
Quantitative Tests:
Esbach’s method
Aufrecht’s method
Other tests:
Protein reagent strip test
Biuret test
Urine protein electrophoresis

Sulphosalicylic Acid Test

Principle

Proteins are precipitated by 5-sulphosalicylic acid. Any resulting turbidity will give an estimation of the amount of protein present in the urine which can be subjectively quantitated visually or more precisely quantitated using photometry. Cells and casts in the urine must be removed by centrifuging before carrying out the test. The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.

Requirements

Specimen:
Random urine
Apparatus:
Centrifuge, general purpose
Graduated pipettes/micropipettes
Measuring Cylinder
Test Tubes
pH paper/ pH meter
Reagents:
5-sulphosalicylic acid solution-3%
Glacial acetic acid-10%

Procedure

Check the pH of a portion of urine, if it is alkaline or neutral, add 10% acetic acid solution, drop by drop, until it is just acidic (about pH 6).
If the urine is cloudy, filter or centrifuge the urine (5 minutes, 2000-3000 rpm).
Take 2ml clear urine in a test tube.
Add 2 ml 5-sulphosalicylic acid solution and mix. Do not shake. Examine for turbidity against a dark background.

Result and Interpretation

Grade the turbidity as follows:

Negative : No cloudiness
Trace: Faint turbidity.
1+ : definite turbidity
2+ : Heavy turbidity but no flocculation
3+ : Heavy turbidity with light flocculation.
4+ : Heavy turbidity with heavy flocculation.

Note:

Normal urine doesn’t contain detectable protein by this method. A false positive result may be obtained if the patient is receiving tolbutamide, penicillin and some other drugs. High concentration of urates in the urine may cause a false positive result due to precipitation of urate in an acidic urine.

References

World Health Organization, 1986. Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies. In Methods recommended for essential clinical chemical and haematological tests for intermediate hospital laboratories/Working Group on Assessment of Clinical Technologies.
Ridley J.W. (2018) Procedures for Complete Urinalysis/Confirmation Testing. In: Fundamentals of the Study of Urine and Body Fluids. Springer, Cham. https://doi.org/10.1007/978-3-319-78417-5_10
Cheesbrough, M., 1981. Medical laboratory manual for tropical countries (Vol. 1). M. Cheesbrough, 14 Bevills Close, Doddington, Cambridgeshire, PE15 OTT..

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Heat and Acetic Acid Test for Proteinuria: Principle and Procedure

Dhurba Giri — Sat, 13 Mar 2021 16:05:51 +0000

Most plasma proteins are too large to pass through the glomeruli of the kidney. The small amount of protein which does filter through is normally reabsorbed back into the blood by the kidney tubules. Only trace amounts of protein (less than 150 mg per 24 h) can therefore be found in normal urine. These proteins include Tamm-horsfall protein (Maximum-40%), Albumin (20%), Immunoglobulins, Hormones, Enzymes and Mucopolysaccharides.

Qualitative Tests:

Heat and acetic acid test
Sulphosalicyclic acid test
Hellers nitric acid test

Quantitative Tests:

Esbach’s method
Aufrecht’s method

Other tests:

Protein reagent strip test
Biuret test
Urine protein electrophoresis

Heat and acetic acid test

Principle:

This test is based on the principle that proteins get precipitated when boiled in an acidic medium.

Procedure:

Take 5-10ml clear urine in a test tube.
Boil the upper portion over a flame.
Compare the heated part with the lower part. Cloudiness or turbidity indicates the presence of either proteins or phosphates/carbonates.
Add 2-4 drops of 10% glacial acetic acid and boil the upper portion again.
If turbidity is still present, protein is present in urine. If turbidity disappears, that is due to phosphates or carbonates present in urine.

Result and Interpretation:

Grade the turbidity as follows:

Negative : No cloudiness
Trace: Barely visible cloudiness.
1+ : definite cloud without granular flocculation
2+ : heavy and granular cloud without granular flocculation
3+ : densed cloud with marked flocculation.
4+ : thick curdy precipitation and coagulation

References

Sood R. Concise book of Medical Laboratory Technology. Jaypee Brothers Pvt. Limited; 2015.
Cheesbrough M. Medical laboratory manual for tropical countries. M. Cheesbrough, 14 Bevills Close, Doddington, Cambridgeshire, PE15 OTT.; 1981.

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Gerhardt’s Test For Ketone Bodies: Principle, Procedure and Interpretation

Dhurba Giri — Sat, 12 Dec 2020 15:46:45 +0000

The term ketone bodies refers to three intermediate products of fat metabolism, namely acetone (2%), acetoacetic acid (20%) and beta-hydroxybutyrate (78%). Under normal conditions, metabolized fats are completely broken down to water and carbon dioxide, hence negligible amount (1mg/24 hrs) of ketone bodies are excreted in urine.

When the rate of production exceeds, excess ketone bodies are eliminated in urine and the condition is known as ketonuria. Two conditions most commonly associated are starvation and Diabetes mellitus. Ketonuria is also seen in case of prolonged vomiting, severe diarrhoea, anesthesia, liver damage, high fat intake and low carbohydrate intake.

Various methods are available for detecting ketones in urine:

Rothera’s test
Gerhardt’s test
Lang’s test
Lindeman’s test
Han’s test
Tablet test

The commonly used methods for ketonuria are based on the principle of Rothera’s nitroprusside test. However, other tests with different principle can also be used. Gerhardt’s Test is not very sensitive test because it can only detect about 25 to 50 mg/dl of acetoacetic acid.

Principle of Gerhardt’s test

Gerhardt’s test is based on the reaction of ferric chloride with acetoacetic acid to form a port wine or Bordeaux red color. This test detects acetoacetic acid only, Acetone and beta-hydroxybutyrate can’t be detected by this method. It also detects salicylates in urine.

Requirements

Urine specimen
Test tubes
10% Ferric chloride:
(10ml of ferric chloride in 100 ml of distilled water)

Procedure of Gerhardt’s test

Transfer about 3-5 ml of urine to a test tube.
Add 5ml of ferric chloride drop by drop to the urine. If phosphates are present, they get precipitated as ferric phosphates.
On addition of a slight excess of ferric chloride, if diacetic acid is present, Bordeaux red color will develop.
To confirm the presence of acetoacetic acid, boil the test solution for 5 minutes.
Observe the color change.

Observations and Results

If the color disappears, then acetoacetic acid is present. The acetoacetic acid on boiling loses carbon dioxide and is converted to acetone. Acetone doesn’t react with ferric chloride.
If the color persists, acetoacetic acid is absent. Previous color is due to salicylates.

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Glycolysis: All Steps Explained with Diagrams, Enzymes, Products and Energy Yield

Dhurba Giri — Wed, 06 Feb 2019 14:07:49 +0000

Glycolysis is the major pathway for the utilization of glucose in the body. Glycolysis is defined as the sequence of reactions for the breakdown of Glucose (6-carbon molecule) to two molecules of pyruvic acid (3-carbon molecule) under aerobic conditions; or lactate under anaerobic conditions along with the production of ATP. This was described by Embden, Meyerhof and Parnas. Hence, it is also called as Embden Meyerhof pathway.

Site of Glycolysis

Glycolysis takes place in cytosol of all the cells of the body. It is the major pathway for ATP synthesis in tissues lacking mitochondria, e.g. erythrocytes, cornea, lens etc.

Types of Glycolysis

Glycolysis is the only pathway which can operate aerobically and anaerobically.

Aerobic Glycolysis: It occurs when oxygen is readily available. Final product is pyruvate along with the production of ATPs.

Anaerobic Glycolysis: It occurs when oxygen is scarce. Final product is lactate along with the production of ATPs.

Steps of Glycolysis

The breakdown of glucose molecule is brought about by sequential reaction of 10 enzymes which can be divided into two phases:

Phase 1: Preparatory Phase
This phase is also called glucose activation phase. In the first stage of glycolysis, glucose is phosphorylated, isomerized, phosphorylated again and cleaved to yield two triose molecules. This comprises the first five reactions, 1, 2, 3, 4 and 5 which convert Glucose to two molecules of Glyceraldehyde-3-Phosphate. These reactions consume 2ATPs per glucose.

Phase 2: Payoff Phase
This phase is also called energy extraction phase. In stage, a series of changes convert glyceraldehyde 3-phosphate to pyruvate. This stage produces 4 ATPs per glucose molecule for a net yield of 2 ATPs per glucose.

Step 1: Uptake and Phosphorylation of Glucose

Glucose is phosphorylated to glucose-6-phosphate.
This reaction is catalysed by the specific enzyme glucokinase in liver cells and by non specific enzyme hexokinase in liver and extrahepatic tissue. The enzyme splits the ATP into ADP, and the phosphate group is added onto the glucose.
This is irreversible regulatory reaction step of glycolysis.
This is the flux generating step of glycolysis.
Mg2+ acts as cofactor.

Step 2: Isomerization of Glucose-6-Phosphate

Glucose-6-phosphate is isomerised to Fructose-6-phosphate by the enzyme Phosphohexose Isomerase.
It involves Aldose-Ketose Isomerism.
It is freely reversible reaction.

Step 3: Phosphorylation of Fructose-6-Phosphate

Fructose-6-phosphate is further phosphorylated to Fructose 1,6-bisphosphate.
One ATP is utilized. The enzyme phosphofructokinase-1 catalyses the transfer of a phosphate group from ATP to fructose-6-phosphate.
This step is second irreversible step in glycolysis.
Rate limiting commited step of glycolysis.
Also called bottle neck of glycolysis.

Step 4: Cleavage of Fructose 1,6-Bisphosphate

Enzyme aldolase splits 6-carbon Fructose 1,6-bisphosphate into two 3-carbon compounds, namely, Glyceraldehyde-3-phosphate and Dihydroxy acetone phosphate (DHAP).
Aldolase is a Lyase.
This reaction is reversible.

Step 5: Isomerization of Dihydroxyacetone Phosphate

As GAP is on the direct pathway of glycolysis, whereas DHAP is not. DHAP is isomerized to Glyceraldehyde 3-phosphate by the enzyme phosphotriose isomerase.
Hence two molecules of glyceraldehyde 3-phosphate are formed from one molecule of glucose.
This inter conversion is reversible.

Step 6: Dehydrogenation of Glyceraldehyde-3-Phosphate

Glyceraldehyde-3-phosphate is oxidised to a high energy compound 1,3-bisphosphoglycerate by enzyme glyceraldehyde-3-phosphate dehydrogenase.
It is a NAD dependent reversible reaction which generates NADH. This NADH enters in mitochondria by Malate-Aspartate shuttle or Glycerophosphate shuttle under aerobic conditions. But in anaerobic conditions, NADH is utilized by Lactate Dehydrogenase, NAD+ is regenerated.

Step 7: Conversion of 1,3-Bisphosphoglycerate to 3-Phosphoglycerate

The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate.
This is only kinase reaction in glycolysis, which is reversible.
This step generates ATP at substarate level phosphorylation.

Step 8: Inter-Molecular Shift of Phosphate Group

Phosphoglycerate mutase shifts the phosphate group from 3rd to 2nd carbon atom, converting 3-phospho glycerate to 2-phospho glycerate.
This is reversible reaction.
Mg2+ is essential for this reaction.

Step 9: Dehydration of 2-Phosphoglycerate

Enolase converts 2-phosphoglycerate to phosphenol pyruvate.
Enolase is dependent on Mn2+ and Mg2+.
This is reversible reaction.
Fluoride inhibits enolase. This property can be used when it is required to prevent glycolysis in blood prior to the estimation of blood glucose.

Step 10: Conversion of Phosphoenolpyruvate to Pyruvate

Pyruvate kinase catalyzes the conversion of phosphenol pyruvate to pyruvate.
This is the second step in glycolysis that generates ATP at substrate level phosphorylation.
This is irreversible reaction.

Additional Step in Anaerobic Condition

When animal tissues cannot be supplied with sufficient oxygen to support aerobic oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated from NADH by the reduction of pyruvate to lactate by Lactate Dehydrogenase (LDH).
Tissues that function under hypoxic conditions eg. skeletal muscle, smooth muscle, erythrocytes produce lactate. In erythrocytes, even under aerobic conditions, glycolysis terminates in lactate because of absence of mitochondria.

Net energy yield in Glycolysis

Energy Yield in Aerobic Glycolysis

Step No.	Enzyme	Source	No. of ATP
1	Hexokinase	–	-1
3	Phosphofructokinase	–	-1
6	Glyceraldehyde-3-phosphate dehydrogenase	NADH	(+2.5)x2=5
7	Phosphoglycerate Kinase	ATP	(+1)x2=2
10	Pyruvate Kinase	ATP	(+1)x2=2
	Net Energy Yield		9-2= 7 ATPs

Energy Yield in Anaerobic Glycolysis

Step No.	Enzyme	Source	No. of ATP
1	Hexokinase	–	-1
3	Phosphofructokinase	–	-1
7	Phosphoglycerate Kinase	ATP	(+1)x2=2
10	Pyruvate Kinase	ATP	(+1)x2=2
	Net Energy Yield		4-2= 2 ATPs

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Colorimeter : Principle, Instrumentation and Uses

Principle of Colorimeter :

Beer’s law:

Lambert’s law:

Instrumentation of Colorimeter :

1. Light Source:

2. Slit:

3. Condensing lens:

4. Monochromator:

5. Sample Holder (Cuvette):

6. Photo detectors:

7. Display:

Uses and Applications

GOD-POD Method For The Estimation Of Blood Glucose

1. Reduction Methods:

2. Aromatic amine condensation methods:

3. Enzymatic Methods:

4. Electrochemical methods:

GOD-POD Method for Glucose Estimation

Principle:

Requirements:

Specimen:

Reagents:

Instruments:

Procedure:

Calculation:

References:

Folin-Wu Method For Estimation of Blood Glucose

Principle

Requirements

Procedure

Step 1: Preparation of protein-free filtrate:

Step 2: Testing

Calculations

References

How to Convert ‘g’ to RPM and Vice Versa in a Centrifuge?

Revolutions per minute (RPM)

Relative centrifugal force (RCF)

Which one to use?

Converting RPM to RCF and vice versa

References

Diacetyl Monoxime (DAM) Method for Estimation of Urea

Principle

Requirements

Preparation of Regaents

Preparation of Sample

Procedure

Calculations

References

O-Toluidine Method for Estimation of Blood Glucose

Principle

Requirements

Preparation of Regaents

Procedure

Calculations

References

Sulphosalicylic Acid Test for Proteinuria: Principle and Procedure

Sulphosalicylic Acid Test

Principle

Requirements

Procedure

Result and Interpretation

Note:

References

Heat and Acetic Acid Test for Proteinuria: Principle and Procedure

Heat and acetic acid test

Principle:

Procedure:

Result and Interpretation:

References

Gerhardt’s Test For Ketone Bodies: Principle, Procedure and Interpretation

Principle of Gerhardt’s test

Requirements

Procedure of Gerhardt’s test

Observations and Results

Glycolysis: All Steps Explained with Diagrams, Enzymes, Products and Energy Yield

Site of Glycolysis

Types of Glycolysis

Steps of Glycolysis