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4 classifications of biochemical substances in the body |
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-proteins -carbohydrates -nucleotides -lipids |
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sources of proteins in body |
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-enzymes -hormones -building blocks of cellular substances -chromosomes |
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-some contain nitrogen, phosphorus, sulfur -R group, amino group, carboxyl group |
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-bond formed when 2 amino acids link together to make a protein -loses an H2O in the process -this is what is tested for in biuret reaction |
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-contains biuret reagent (organic compound that reacts with a cupric ion in an alkaline medium) -Colored complex formed between amino group, carboxyl group, cupric ion -tests for peptide bonds -results indicate presence of proteins in serum The intensity of the violet color is proportional to the number of peptide bonds (# of proteins) -Sensitivity Range: 1.5 – 15 g/dL -Used for large amounts of protein -dilutions decrease turbidity & pigment effects |
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enzymes are used to break peptide bonds -pepsin, trypsin, chymotrypsin (digestive tract) |
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-albumin -most globulins -hemoglobin -made in liver (except gamma globulins) -function: transport, supply amino acids (when needed), regulate water balance |
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colloidal osmotic pressure |
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-maintains blood volumes and water content between the tissues -important that rate of flow through capillaries is maintained -too much flow --> edema |
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-molecular weight is approx 65,000. -large body protein, cannot easily pass through cell membranes -Regulates plasma volume (colloidal pressure) -Soluble in water (saline) -Coagulates in heat (think egg white) -Its presence in plasma creates an osmotic force that maintains fluid volume within the vascular space. -Made in liver, can see increased level in liver -liver disease: albumin levels drop -Electrophoresis: Albumin is most negatively charged-travels farthest to + pole |
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-major waste product of mammals -comes from breakdown of amino acids -efficiently removed by kidneys -normal level of urea in blood = 8-22mg/dL -blood levels dependent on protein and fluid intake |
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-occurs in liver -converts 2 molecules of ammonia and 1 molecule of CO2 into 1 molecule of urea -ammonia must be converted because it is highly toxic to us |
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-deamination of amino acids produces ammonia -then ammonia is converted to urea -urea is the form by which wastes are excreted from body |
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-natural result of muscle function -Created from creatine phosphate in the muscle -Creatine phosphate readily converted to ATP -has 3N instead of 2N, so more easily excreted -Normal blood levels = 0.5-1.5 mg/dL -good evaluator of kidney function -high level of creatinine in serum = kidney failure |
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-A metabolic waste product from NH3 -Derived from purines in diet & synthesized in body from nucleic acid synthesis -Found normally in blood due to normal cell destruction -increased in excessive cell destruction ex. leukemia, cancer-treating drugs -Purines produced in body; also found in drugs and medicines |
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-method of quantitating proteins -8.6 pH = net negative charge -Different proteins (aa sequence) carry different charges -Separates proteins according to size and charge Larger proteins, more positive proteins stay near starting point (negative end) |
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molecular weight ranges from ~45,000 to 900,000; Mainly a transport protein Some are antibodies Has 4 fractions: α1, α2, β, ∂ (alpha-1, alpha-2, Beta, Gamma) Globulins are proteins that include gamma globulins (antibodies, not made in liver) and a variety of enzymes, and carrier and transport proteins The specific profile of globulins is determined by protein electrophoresis (SPEP) MADE in LIVER, can see increased level in liver |
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Serum Protein ElectroPhoresis (SPEP) |
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Chart shows progress of protein across gel electrophoresis plate
migration from negative to positive charge
Once the migration is complete, the strip is stained and the intensity of the color bands are read in a spectrophotometric-type instrument (densitometer). We get a tracing of curves that is then quantitated. |
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Point of Application (POA) |
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-where proteins are injected into gel plate -negatively charged end of plate |
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Serum Protein Electrophoresis (SPE) |
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shows normal electrophoresis results for different types of protein |
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-shown as broad band (“bridge”) across the Beta and gamma regions -seen in liver diseases |
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-Heat coagulates protein -Solution becomes turbid, we measure turbidity -CAUTION: Interference from other substances can affect turbidity -The main problem with this method is that heated urine tends to smell- we usually don't use this method |
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-Acid alters protein structure -Causes precipitation and measure this turbidity -Two acids generally used: Sulfosalicylic acid (SSA)-urines Trichloroacetic acid (TCA)-CSF proteins |
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-add sulfosalicylic acid to a urine specimen -observe turbidity as the acid denatures the proteins in the urine
Results: Neg: no turbidity Trace: slight turbidity against dark background but not against a light background 1+: Slight turbidity, transparent, visible against light 2+: Turbidity, not transparent, no flocculation 3+: Turbid with some flocculant particles 4+: Heavy flocculation with precipitation of particles |
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-Rapid, easy screening for protein, dipstick method -2 chemicals in the pad: -Citrate buffer – maintains pH of 3.0 -Bromphenol blue – indicator that responds to change in pH -Protein error of indicators – at constant pH of 3, changes color if protein is present -Very alkaline/very acidic pH (buffer can’t keep pH at 3)-could get false positive result for protein |
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-an increase in the blood level of total protein. (high protein in blood) -total proteins rarely go above 9 g/dL -2 causes: -dehydration -multiple myeloma |
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-a cause of hyperproteinemia -caused by either a decreased fluid intake or an excessive fluid loss from vomiting, diarrhea -Relative increase: The absolute quantity of protein is unaltered, but the concentration is increased because the water volume is decreased |
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-a cause of hyperproteinemia -plasma cell abnormality; proliferation of plasma cells in marrow -a plasma cell is a transformed B lymphocyte whose function is to produce antibodies -Antibodies are gamma globulins -These plasma cells are synthesized and secreting gamma globulins known as myeloma proteins -they are abnormal -electrophoresis shows monoclonal spike in gamma region -Symptoms: -Bone lesions – Ca leaves bone; hypercalcemia (increase in calcium) -Recurring infections – impaired Antibody synthesis -Immunodeficiency -N/N anemia; blood smear shows rouleaux due to increased protein |
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-result of electrophoresis test that indicates multiple myeloma -spike is in gamma region because body makes gamma globulins in multiple myeloma |
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-Rouleaux: RBCs stack like coins, stick together -caused by the excess of protein in blood (hyperproteinuria) |
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seen with myeloma Very small molecular weight protein (Kappa or Lambda chains) excreted into urine Bence Jones precipitates at 560 C normal protein ppts at at 700 C Chronic renal failure can develop |
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-decrease in level of total protein -Causes: -nephrotic syndrome -severe burns -excessive blood loss -malabsorption -drugs -liver disease |
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Nephrotic syndrome (hypoproteinemia) |
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-marked proteinuria -hypoalbuminemia- losing albumin through the kidneys -edema -increased serum creatinine levels -complication of glomerular disease, systemic diseases (diabetes) |
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severe burns (hypoproteinemia) |
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-large amounts of protein lost -increased H2O replacement -More water than protein, looks like hypoproteinemia |
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excessive blood loss (hypoproteinemia) |
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-large amounts of protein lost -water replacement -Hemoglobin is signficant -Dilution factor is important |
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malabsorption (hypoproteinemia) |
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-digested food is not reabsorbed into blood stream nor transported to liver for protein synthesis -Can’t make enough proteins |
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-certain therapeutic drugs impair protein synthesis -Alcohol can impair protein synthesis because of damage to the liver |
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liver disease (hypoproteinemia) |
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-most common disease of hypoproteinemia -Such disease states as hepatitis, cirrhosis, can cause liver damage -normally functioning liver cells are essential for normal protein synthesis -Albumin and some globulins are synthesized in the liver. |
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