Protein Analysis Methods Quiz

Total Proteins

• Proteins are crucial building blocks for all cells and tissues.
• They are essential for growth, development, and overall health.
• Proteins form the structural components of most organs.
• Proteins include enzymes and hormones which regulate body functions.
• A total protein test measures the amount of protein in the blood.

Kjeldahl Method

• This method quantifies protein through a three-step process: digestion, distillation, and titration.
• Digestion uses concentrated sulfuric acid (H₂SO₄), heat, and a catalyst (like selenium) to convert organic nitrogen in the sample to ammonium sulfate.
• Neutralization with sodium hydroxide (NaOH) converts ammonium sulfate into ammonia, which is then distilled and collected.
• The collected ammonia is absorbed in boric acid to form ammonium borate.
• Titration with a standard acid, using a suitable indicator, determines the total nitrogen content, from which protein content is calculated.

Kjeldahl Method for Dairy Products

• The Kjeldahl method is the standard for determining protein in dairy products.
• A factor of 6.38 is used to calculate the protein percentage from the nitrogen content.
• The calculation assumes that all nitrogen is present as protein, and that dairy protein contains 15.67% nitrogen.

Biuret Reaction

• This reaction is used to measure protein concentration.
• Peptide bonds in proteins react with copper ions (Cu²⁺) in an alkaline solution.
• The intensity of the resulting color (measured at 540nm) is directly proportional to protein concentration.
• This method is efficient for whole tissue samples and other high protein sources due to its insensitivity to free amino acids.

Biuret Test Results

• A blue solution indicates a negative result (lack of protein).
• A deep purple solution signifies a positive result (protein is present).

Folin-Ciocalteau or Lowry Method

• This method is more sensitive than the biuret method (measuring 5-150 µg of protein).
• It involves the reaction of proteins with copper ions in an alkaline solution, followed by the reduction of phosphomolybdic-tungstic acid, which produces a characteristic blue color intensity.
• This color intensity is proportional to the amount of protein present.
• The presence of interfering substances like ammonium sulfate, thiol reagents, sucrose, EDTA, and detergents is a major disadvantage.

Serum Protein Electrophoresis (SPEP)

• SPEP measures specific proteins in blood serum to diagnose diseases.
• Proteins have positive or negative electrical charges.
• In an electrical field, the proteins move depending on their charges and sizes.
• SPEP separates serum proteins into groups based on similar properties.

Blood Serum Protein Fractions

• Blood serum has two major protein groups: albumin and globulin.
• Albumin (60%): produced by the liver, maintains fluid balance, nourishes tissues, and transports various substances.
• Globulins (40%): diverse group of proteins, some produced by the liver, some by the immune system for fighting infections and transporting nutrients.
  • Alpha-1, Alpha-2, Beta, Gamma globulins are subtypes with different functions. For example, alpha-2 globulin includes haptoglobulin, which is involved in binding with hemoglobin. Alpha-1 globulin includes high-density lipoproteins (HDL) a type of cholesterol.

Albumin and Total Protein Test

• A total protein test measures all proteins in the blood.
• It compares the amount of albumin relative to globulin to calculate an albumin-to-globulin (A/G) ratio.
• Changes in the A/G ratio may indicate underlying health issues.

Total Protein Levels

• Total protein levels may increase (due to conditions involving high protein production or reduced loss of protein) or decrease (result of conditions interfering with protein production or causing increased protein loss).
• Possible increased conditions include:
  • Liver damage, kidney disease(nephrotic syndrome), dehydration, and unusually high protein production such as in certain inflammatory diseases/cancer.
• Possible decreased conditions include:
  • Malnutrition, severe liver disease, and kidney disease
  • conditions that cause the body’s immune system to attack healthy cells

Diagnostic Significance of Enzymes

• Enzymes can act as diagnostic markers for diseases.
• Enzymes also serve as reagents for biochemical estimations and detections.
• This is because tissue damage leads to release of enzymes into circulation. The amount of a particular enzyme in blood can indicate the severity or location of certain disease.

Enzyme Classification

• Enzymes are classified into six main classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases) based on the type of reaction they catalyze. Each class is further divided into subclasses.
• Each enzyme is assigned a unique number (EC number, Enzyme Commission) based on its catalytic activity.
• The EC number has six digits representing class, subclass and sub-subclass, and finally identifying number for enzyme.

Enzyme Activity Units

• International Units (IU): activity that transforms one micromol of substrate per minute
• Katal (kat): number of moles of substrate undergoing transformation per second, and unit of catalytic activity
• U, mkat, nkat are units of measurement for enzyme activity

Clinical Significance of Enzyme Estimation

• The activity of specific enzymes is helpful in clinical diagnosis, prognosis and disease monitoring.
• Single or repeated enzyme assays can contribute to a diagnosis, detecting diseases early on, assessing treatment affect, and gaining insights into the progression of a disease.

Enzymes as Diagnostic Markers in Diseases

• Enzyme estimations help diagnose myocardial infarction, liver diseases, muscle diseases, cancers, and GI tract diseases.

Enzymes in Liver Diseases

• Specific enzymes (SGOT, SGPT, ALP, GGT, 5’ nucleotidase) have characteristic patterns in blood during liver disease.
• Levels of SGPT, SGOT, etc.) may rise in diseases of liver, heart, or kidneys.
• Elevation and trends in patterns can give valuable information in these conditions.

Pseudocholinesterase

• A non-specific enzyme that hydrolyzes acyl esters.
• It is part of the functional plasma enzymes.
• Activity usually decreases in liver disease or poisoning.

Amino Acids in Blood and Urine

• Increased amino acids in blood or urine can indicate liver problems or genetic disorders.
• Crystals of certain amino acids (tyrosine, leucine) can form under specific conditions, sometimes observed in urine samples.

Urinary Crystals

• Specific types of crystals can be associated with certain diseases.

Alkaline Phosphatase in Obstructive Liver Disease

• Elevated alkaline phosphatase (ALP) in blood strongly suggests obstructive liver disease.
• Bile duct obstructions induce the production of ALP by cells lining the bile ducts.
• Progressive elevation of ALP is a significant characteristic of malignancy.
• Isoenzymes of ALP, like Regan isoenzyme, can indicate certain types of cancer.

Gamma-Glutamyl Transferase (GGT) in Liver Diseases

• GGT is an enzyme involved in various metabolic pathways, especially in the synthesis of glutathione.
• Raised GGT levels are associated with several liver conditions, cholestasis, chronic alcoholism.
• Increased GGT levels are not specific to any single condition.

5' Nucleotidase in Liver Diseases

• Elevated level of 5' nucleotidase in blood may be associated with obstructions in the flow of bile from the liver.
• 5’ nucleotidase is a specific enzyme in the liver and its level may suggest obstructive liver disease.
• Its level is also not specific to any single condition, but may give clues.

Other aspects

• The principle of different tests are briefly described
• Test methods' interpretation and normal values are mentioned
• A/G ratio, its characteristics and interpretation are addressed.