Clinical Chem 2 Day 2 1st Periodical PDF

TOTAL PROTEINS are important building blocks of all cells and tissues. They are important for body growth, development, and health. They form the structural part of most organs and make up enzymes and hormones that regulate body functions. This test measures the amount of protein in your blood. 1....

TOTAL PROTEINS are important building blocks of all cells and tissues. They are important for body growth, development, and health. They form the structural part of most organs and make up enzymes and hormones that regulate body functions. This test measures the amount of protein in your blood. 1. The Kjeldahl method involves a three-step approach to the quantification of protein: digestion, distillation, and titration. Digestion of organic material is achieved using concentrated H2SO4, heat, K2SO4 (to raise the boiling point), and a catalyst (e.g., selenium) to speed up the reaction. This process converts any nitrogen in the sample to ammonium sulfate. The digestate is neutralized by the addition of NaOH, which converts the ammonium sulfate to ammonia, which is distilled off and collected in a receiving flask of excess boric acid, forming ammonium borate. The residual boric acid is then titrated with a standard acid with the use of a suitable end-point indicator to estimate the total nitrogen content of the sample.  Kjeldahl method The Kjeldahl method, published in 1883, has traditionally been the accepted reference method for the determination of protein in dairy products. The percentage of protein present is calculated from the nitrogen content using a factor of 6.38 for dairy products (calculated assuming that dairy protein contains 15.67% nitrogen, 100 ÷ 15.67 = 6.38) and assumes that all the nitrogen in the product is present as protein. 2. The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer–Lambert law. In this assay, the copper(II) binds with nitrogen present in the peptides of proteins. In a secondary reaction, the copper(II) is reduced to copper(I). Due to its insensitivity and little interference by free amino acids, this assay is most useful for whole tissue samples and other sources with high protein concentration. 3. Folin–Ciocalteau or Lowry method While the biuret method is sensitive in the range 0.5 to 2.5 mg protein per assay, the Lowry method is 1 to 2 orders of magnitude more sensitive (5 to 150 μg). The main disadvantage of the Lowry method is the number of interfering substances; these include ammonium sulfate, thiol reagents, sucrose, EDTA, Tris, and Triton X-100. The final color in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phosphomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue color. 4. The serum protein electrophoresis (SPEP) Test measures specific proteins in the blood to help identify some diseases. Proteins are substances made up of smaller building blocks called amino acids. Proteins carry a positive or a negative electrical charge, and they move in fluid when placed in an electrical field. Serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups of similar size, shape, and charge. Blood serum contains two major protein groups: albumin and globulin. Both albumin and globulin carry substances through the bloodstream. Using protein electrophoresis, these two groups can be separated into five smaller groups (fractions): Albumin. Albumin proteins keep the blood from leaking out of blood vessels. Albumin also helps carry some medicines and other substances through the blood and is important for tissue growth and healing. More than half of the protein in blood serum is albumin. Alpha-1 globulin. High-density lipoprotein (HDL), the "good" type of cholesterol, is included in this fraction. Alpha-2 globulin. A protein called haptoglobin, which binds with hemoglobin, is included in the alpha-2 globulin fraction. Beta globulin. Beta globulin proteins help carry substances, such as iron, through the bloodstream and help fight infection. Gamma globulin. These proteins are also called antibodies. They help prevent and fight infection. Gamma globulins bind to foreign substances, such as bacteria or viruses, causing them to be destroyed by the immune system.  Albumin is made by the liver and makes up about 60% of the total protein. Albumin keeps fluid from leaking out of blood vessels, nourishes tissues, and transports hormones, vitamins, drugs, and substances like calcium throughout the body. Globulins make up the remaining 40% of proteins in the blood. The globulins are a varied group of proteins, some produced by the liver and some by the immune system. They help fight infection and transport nutrients. Normal Values Total protein: 6.4 to 8.3 g/dL Albumin: 3.5 to 5.0 g/dL Alpha-1 globulin: 0.1 to 0.3 g/dL Alpha-2 globulin: 0.6 to 1.0 g/dL Beta globulin: 0.7 to 1.2 g/dL Gamma globulin: 0.7 to 1.6 g/dL Note: g/dL = grams per deciliter  5. Refractometry is the analytical method of measuring substances' refractive index (one of their fundamental physical properties) in order to, for example, assess their composition or purity. A refractometer is the instrument used to measure refractive index ("RI"). 6. Turbidimetry is based on the measurement of the loss of intensity of transmitted light in an emulsion (or solution containing fine particles) due to the scattering effect of particles suspended in it. Nephelometry is based on the measurement of scattered light by a solution containing fine particles. Light scattering can also be used for the measurement of concentration in polymer solutions 7. SALT FRACTIONATION The process of "salting out" is a purification method that relies on the basis of protein solubility. It relies on the principle that most proteins are less soluble in solutions of high salt concentrations because the addition of salt ions shield proteins with multi-ion charges. Those charges help protein molecules interact, aggregate, and precipitate. Proteins contain various sequences and compositions of amino acids. Therefore, their solubility to water differs depending on the level of hydrophobic or hydrophilic properties of the surface. Proteins with surfaces that have greater hydrophobic properties will readily precipitate. What is an albumin and total protein test? The TOTAL PROTEIN compares the amount of albumin with globulin and calculates what is called the A/G ratio. A change in this ratio can provide your healthcare practitioner with a clue as to the cause of the change in protein levels. Proteins circulate throughout your blood to help your body maintain fluid balance. Albumin is a type of protein the liver makes. It’s one of the most abundant proteins in your blood. Your liver makes two main proteins: albumin and globulin. Low levels can mean damage or disease. Keep in mind that the immune system also produces globulin TESTNG FOR ALBUMIN TESTING FOR ALBUMIN 1. PRINCIPLE The method is based on the specific binding of bromocresol green (BCG), an anionic dye, and the protein at acid pH produce a color change of the indicator from yellow – green to green –blue with the resulting shift in the absorption wavelength of the complex. The intensity of the color formed is proportional to the concentration of albumin in the sample. BCG + Albumin PH 4.3 BCG-albumin complex IMPORTANT TO NOTE The AGR was calculated using the equation AGR=albumin/ (total protein-albumin) OR : A/G RATIO= ALBUMIN/GLOBULIN ( TOTAL PROTEIN-ALBUMIN) Total protein levels in the blood may increase or decrease, to a greater or lesser degree, with various conditions. Total protein levels may decrease in conditions that: Interfere with production of albumin or globulin proteins, such as malnutrition or severe liver disease Increase the breakdown or loss of protein, such as kidney disease (nephrotic syndrome) Increase or expand the volume of plasma, the liquid part of blood (diluting the blood), such as congestive heart failure Total protein levels may increase with conditions that cause: Abnormally high production of protein (e.g., inflammatory disorders, multiple myeloma) Dehydration Low total protein: You could have a liver or kidney disorder, or a digestive disorder like celiac disease (your body can’t absorb protein the way it should). High total protein: Too much protein in your blood can be a sign of chronic infection or inflammation (like HIV/AIDS or viral hepatitis). It can also be an early sign of a bone marrow disorder.  Low A/G ratio: This might be the sign an autoimmune disorder, where your body’s immune system attacks healthy cells. It can also point to kidney disease or cirrhosis, which is inflammation and scarring of the liver. In some cases, a low A/G ratio can be a sign of a tumor in your bone marrow. High A/G ratio: This can be a sign of disease in your liver, (FATTY LIVER) kidney, or intestines. It’s also linked to low thyroid activity and leukemia.