Clinical Chemistry 1 PDF

[TRANS] LESSON VIII, VIIII, X: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS AMINO ACIDS AND PROTEINS OTHER PLASMA PROTEINS PROTEINS Antitrypsin...

[TRANS] LESSON VIII, VIIII, X: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS AMINO ACIDS AND PROTEINS OTHER PLASMA PROTEINS PROTEINS Antitrypsin – alpha 1 antitrypsin (AAP) Amino acid sequence connected by PEPTIDE o key plasma protein primarily produced by the BONDS liver Amphoteric o serinproteaese inhibitor Produced in the liver except IMMUNOGLOBULINS o involved in the bodies defense mechanism Composed of C, H, O and N (16%) o it inhibits enzymes like the neutrophils Two terminals: N-terminal (NH2 - ) and C-terminal ( - elastase which is release during inflammation COOH) and can damage our connective tissue especially the lung and other organs. o So by controlling the activity of this proteaese PROTEIN STRUCTURE yung APP natin ito yung tumutulong to protect yung tissue natin particularly sa lung during inflammation o Clinical significance: pag deficient we have the genetic deficiency in AAP which will lead to the condition such as emphysema, liver disease due to unchecked activity of proteaese resulting in tissue damage pagka deficient yung AAP o Pagka naman elevated yung AAP, nagyayari ito in response pag may inflammation making it an Acute phase reactant, they are proteins whose plasma concentrations change significantly in response to inflammation, infection, or even in tissue injury PLASMA PROTEINS o Function of acute phase reactant- immune ALBUMIN AND PREALBUMIN response, pag naka detect and body natin ng pathogens yung body natin automatic na nag ALBUMIN re-release ng acute phase reactants na Highest concentration/fraction nagiging defense system natin, sila din nag General transport protein mo-modulate ng inflammation and tissue 17 days half life repair process. o Common example ng antitrypsin is the CRP PREALBUMIN or the c reactive protein – well known acute Major protein from CSF phase reactant that increase rapidly during Involve in transporting thyroxine to the brain inflammation and infection. Another example Aka transthyretin is the fibrinogen – they are involve in blood Faster than albumin clotting yung level din nito ay tumataas during Binds with RBP inflammation siya yung nag ke-create ng 2 days half life blood clot to stop the bleeding. Serum amyloid a – another marker that increases Cross easily into the CSF significantly during acute inflammatory states o Major component of alpha globulin Macroglobulin o Alpha two macroglobulin is the largest non immunoglobulin plasma protein. It is a glycoprotein synthesized, primarily in the liver. o Largest non-immunoglobulin o Elevated in nephrotic syndrome which is a kidney disorder excessive protein loss in urine. Leading to low level of albumin and other plasma proteins CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 1  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS Haptoglobin to evaluate conditions such as iron deficiency o Stress, infection, inflammation, necrosis anemia and iron overload disorders like o 4 days half life hemochromatosis. o Glycoprotein o Functions of Transferrin - Iron Transport: o Produce in the liver Transferrin binds to iron in the bloodstream o Involve in body’s response to hemolysis and delivers it to cells by interacting with o Acts as an acute phase reactant transferrin receptors on the cell surface. the o Function: binds the free hemoglobin iron-transferrin complex is internalized by o Prevents loss of iron and protects kidney cells, and iron is released for use, particularly from potential damage caused by in erythropoiesis (red blood cell production). hemoglobinuria o Recycling of Iron: Transferrin picks up iron o Levels increase in response to stress, from senescent red blood cells after they are infection, and inflammation, and even in broken down by macrophages in the liver and tissue necrosis spleen, recycling it for new red blood cell o Free hemoglobin (Hb) in the bloodstream can formation. cause oxidative damage to tissues. o Regulation of Iron Absorption: Transferrin o Haptoglobin binds free hemoglobin, forming regulates iron absorption in the intestines by a stable complex that is then cleared by controlling how much iron is delivered to the macrophages in the liver and spleen. bone marrow and other tissues. When iron o Haptoglobin levels are used to diagnose and stores are low, transferrin levels increase to monitor hemolytic anemia. enhance iron uptake and transport. o In hemolytic anemia, haptoglobin levels o Iron Overload (Hemochromatosis): In iron decrease because it binds the free overload conditions, transferrin saturation is hemoglobin released from the ruptured red high because there is an excess of iron blood cells, depleting the haptoglobin relative to transferrin. available in the blood. o Chronic Inflammation (Anemia of Chronic o Prevents Kidney Damage: Free Disease): During inflammation, transferrin hemoglobin is harmful to the kidneys; binding levels decrease, as iron is sequestered to by haptoglobin prevents hemoglobin from reduce availability to pathogens. This results filtering through the kidneys and causing in low transferrin levels, even if total body iron damage. is normal or elevated. o Cleared by the Reticuloendothelial o Pregnancy: Transferrin levels tend to System: The haptoglobin-hemoglobin increase during pregnancy to meet the complex is removed from circulation by elevated iron demands of both the mother macrophages, primarily in the liver and and developing fetus. spleen, preventing free hemoglobin from Fibrinogen persisting in the bloodstream. o Forms fibrin clot o Hemolytic Anemia: In hemolytic conditions, o A soluble plasma glycoprotein produced in red blood cells are destroyed, releasing the liver. hemoglobin into the bloodstream. This o It plays a central role in blood clotting increases the demand for haptoglobin, (hemostasis) by converting into fibrin, an leading to low haptoglobin levels. insoluble protein, during the coagulation Transferrin process. o Major beta globulin o A key component of the acute-phase o Elevated in pregnancy response, meaning its levels increase in o A glycoprotein synthesized mainly in the liver. response to inflammation or tissue injury. o Acts as the primary iron transport protein in o Essential for Blood Clot Formation, fibrinogen the blood. is converted to fibrin by the enzyme thrombin o Binds and transports iron (Fe³⁺) from during blood coagulation, forming a fibrin absorption sites in the intestines, and from mesh that stabilizes blood clots. storage sites like the liver, to tissues where o As an acute-phase reactant, fibrinogen levels iron is needed, such as the bone marrow for rise during inflammation, infection, or trauma, red blood cell production. making it a useful marker for assessing o It ensures that iron is available for essential systemic inflammation. processes like hemoglobin synthesis, which o Functions of Fibrinogen - Coagulation: is necessary for oxygen transport in the Fibrinogen is cleaved by thrombin to form blood. Free iron can catalyze the production fibrin, which forms a mesh-like structure that of harmful free radicals. Transferrin binds to stabilizes the initial platelet plug during blood iron, preventing it from generating oxidative clotting. damage in tissues. o Wound Healing - The fibrin clot formed from o Marker for Iron Metabolism Disorders: fibrinogen provides a scaffold for tissue repair Transferrin levels and saturation (the amount and healing following injury. of transferrin that is bound to iron) are used CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 2  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS o Hypofibrinogenemia - Low fibrinogen levels ▪ Transports heme to the liver, where can lead to excessive bleeding due to it is degraded, and the iron is impaired clot formation. Causes may include recycled. liver disease (where fibrinogen is produced), ▪ Low hemopexin levels are seen in disseminated intravascular coagulation hemolytic anemia due to the (DIC), or inherited fibrinogen disorders. depletion of hemopexin as it binds o Hyperfibrinogenemia - High fibrinogen levels free heme. are often associated with inflammatory ▪ It serves as a marker for conditions, such as rheumatoid arthritis, or intravascular hemolysis, helping in chronic infections. Elevated fibrinogen can the diagnosis of conditions where also increase the risk of thrombosis excessive red blood cell destruction (formation of abnormal blood clots), as it occurs. promotes excessive clot formation. o Acid glycoprotein o Measured to assess clotting function, ▪ glycoprotein synthesized primarily in especially in patients with bleeding disorders the liver. or suspected disseminated intravascular ▪ It is an acute-phase reactant, coagulation (DIC). meaning its levels increase during o Useful in evaluating patients with liver inflammation, infection, and trauma. disease, as fibrinogen is produced in the ▪ Modulates the immune response by liver. binding and transporting various o High levels are linked to increased blood substances, including drugs, viscosity and thrombus formation, leading to hormones, and cytokines. a higher risk of cardiovascular events. ▪ Involved in anti-inflammatory o In disseminated intravascular coagulation responses. (DIC), fibrinogen levels can become depleted ▪ Acts as a carrier protein for lipophilic due to excessive clotting and subsequent drugs, especially in cases of consumption of clotting factors, contributing inflammation or disease. to bleeding complications. ▪ Increased AGP levels are seen in Minor proteins acute inflammation, autoimmune o Ceruloplasmin diseases, and cancer. ▪ A copper-carrying protein primarily ▪ Monitoring AGP levels is useful in synthesized in the liver. evaluating the inflammatory ▪ Functions as a ferroxidase, response in chronic conditions such facilitating the conversion of ferrous as rheumatoid arthritis and Crohn’s iron to ferric iron, allowing iron to bind disease. to transferrin for transport. o CRP ▪ Transports copper throughout the ▪ C-Reactive Protein body. ▪ A protein produced by the liver in ▪ Plays a role in iron metabolism by response to inflammation. preventing iron overload in tissues. ▪ One of the most commonly used ▪ Acts as an antioxidant, reducing biomarkers for detecting and oxidative stress by neutralizing free monitoring inflammation. radicals. ▪ Binds to phosphocholine expressed ▪ Low ceruloplasmin levels are seen in on the surface of dead or dying cells Wilson’s disease, a genetic disorder and some bacteria, marking them for where copper accumulates in organs phagocytosis (immune system like the liver, brain, and eyes. clearance). ▪ Elevated levels can occur in acute ▪ Activates the complement system, inflammation or pregnancy, as enhancing the body’s immune ceruloplasmin is an acute-phase response to infection or injury. reactant. ▪ High CRP levels are indicative of o Hemopexin acute inflammation and are ▪ A plasma glycoprotein that binds to commonly elevated in conditions free heme, which is released during such as bacterial infections, red blood cell destruction inflammatory diseases (e.g., (hemolysis). rheumatoid arthritis), and ▪ Hemopexin prevents heme- cardiovascular disease. mediated oxidative damage and iron ▪ Used as a marker to assess the risk loss. of heart attacks and strokes due to its ▪ Binds free heme with high affinity, association with atherosclerosis and protecting tissues from the toxic vascular inflammation. effects of free heme and iron. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 3  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS o AFP Hormones ▪ Alpha-Fetoprotein o Chemical messengers ▪ A protein produced by the fetal liver, o Plasma proteins serve as carriers for various yolk sac, and gastrointestinal tract hormones in the bloodstream, enhancing during pregnancy. their solubility, extending their half-life, and ▪ In adults, elevated AFP is often preventing premature degradation or associated with certain cancers. excretion. ▪ In the fetus, AFP acts as a transport o Plasma proteins control the bioavailability of protein, carrying various substances hormones, hormones bound to plasma such as bilirubin. proteins are in an inactive form, meaning they ▪ In adults, AFP levels are usually low cannot interact with receptors. but can rise in the presence of liver o Free hormones (not bound to proteins) are diseases and tumors. active and can bind to their receptors to exert ▪ Elevated AFP levels in non-pregnant their effects on target tissues. adults are associated with o This balance between free and bound hepatocellular carcinoma (a type of hormones helps maintain appropriate liver cancer) and germ cell tumors hormone levels and prevents excessive (e.g., testicular cancer). activity. ▪ In pregnancy, AFP testing is part of o Plasma proteins ensure that hormones are the maternal screening for neural delivered efficiently to their target tissues, tube defects and Down syndrome where they can bind to receptors and trigger (low AFP levels in maternal serum a biological response. can suggest Down syndrome, while o Insulin-like Growth Factor (IGF): Bound by high levels may indicate neural tube IGF-binding proteins, which regulate IGF defects like spina bifida). bioavailability and protect it from degradation. o Growth Hormone (GH): Transported by GH- FUNCTIONS binding proteins, which modulate its stability and activity. Enzymes o Low levels of SHBG can lead to increased o Biological catalysts levels of free testosterone or estrogen, o catalyze biochemical reactions in the body. potentially contributing to hormonal o enzymes speed up the rate of chemical imbalances such as polycystic ovary reactions without being consumed in the syndrome (PCOS) or androgen excess. process, ensuring various biological o High levels of TBG can result in more bound processes occur efficiently. (inactive) thyroid hormone, causing o Plasma proteins like prothrombin and symptoms of hypothyroidism even if total fibrinogen are crucial in blood coagulation. hormone levels are normal. o Prothrombin: Precursor to thrombin, an enzyme that converts fibrinogen into fibrin Transport proteins during clot formation. o Facilitate movement of ions, trace metals, o Complement proteins (C3, C4) are plasma lipids, etc. proteins that act as enzymes to activate a o Transport proteins enable the movement of cascade of immune responses. The substances throughout the circulatory complement cascade involves proteolytic system, ensuring that nutrients, hormones, cleavage of complement proteins, which and waste products reach their target organs enhances immune functions like pathogen and tissues. destruction, inflammation, and phagocytosis. o By binding to their respective substances, o Plasma proteins that act as enzymes are transport proteins regulate the free (active) often used as biomarkers for diagnosing versus bound (inactive) concentrations of diseases: Liver enzymes such as aspartate these molecules, impacting their biological aminotransferase (AST) and alanine effects. For example, only free fatty acids can aminotransferase (ALT) are measured in enter cells for energy production; those plasma to assess liver function and detect bound to albumin are not readily available for liver injury. Amylase and lipase are measured metabolism. for diagnosing pancreatitis. o Plasma proteins can act as buffers, helping o Plasma proteins can exist as zymogens, maintain pH levels in the blood by binding to inactive precursors that are activated into and releasing hydrogen ions as needed. enzymes during physiological processes. o They also stabilize the structure of hydrophobic molecules (like fatty acids) in the aqueous environment of the bloodstream. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 4  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS Immunoglobulins o The osmotic pressure generated by plasma o Produced by lymphocytes proteins allows for the effective transport of o IgG, IgA, IgM, IgD, IgE nutrients and waste products between blood o Immunoglobulins (Ig) are specialized and tissues, facilitating nutrient delivery and glycoproteins in response to antigens waste removal. (foreign substances like bacteria and Structural proteins viruses). o Provide shape and structure to organs o Immunoglobulins bind to pathogens (such as o They form the framework for tissues, bacteria and viruses), marking them for contributing to overall body structure and destruction by other immune cells. mechanical integrity. o Some immunoglobulins (especially IgM and o Collagen - The most abundant structural IgG) can activate the complement system, protein in the body, found in connective leading to lysis of pathogens. tissues, skin, bones, and cartilage. Provides o Immunoglobulins enhance phagocytosis by tensile strength and support to tissues. coating pathogens, making them easier for o Elastin - Found in connective tissues, elastin phagocytes (like macrophages) to engulf and provides elasticity and resilience, allowing destroy. tissues to stretch and return to their original o IgG: The most abundant antibody in the shape. Important in organs that require blood; provides long-term immunity and can flexibility, such as the lungs and blood cross the placenta. vessels. o IgA: Found in mucosal areas (such as the gut o Fibronectin and Laminin - Glycoproteins that and respiratory tract) and in secretions (like are components of the extracellular matrix saliva, tears, and breast milk); plays a crucial (ECM), providing structural support and role in mucosal immunity. facilitating cell adhesion, growth, and o IgE: Involved in allergic reactions and migration. responses to parasitic infections. o Structural proteins maintain the shape and Storage proteins integrity of cells and tissues, providing o Reservoirs mechanical support against forces and o Storage proteins are proteins that store pressure. essential nutrients and provide a reservoir of o They play a crucial role in wound healing and energy and amino acids for the body. tissue repair by forming a scaffold for new o Storage proteins provide a readily available tissue formation. source of essential nutrients (like amino acids and iron) that the body can utilize during periods of increased demand (growth, injury, or illness). o Buffering Function - Some storage proteins can help maintain osmotic pressure and stabilize the blood’s pH. Energy source o ATP source for tissues and muscles o plasma proteins can also serve as energy sources under certain conditions (starvation, prolonged exercise). Osmotic force o Prevents cell lysis by facilitating the entry and exit of water in the cells o Osmotic force refers to the pressure exerted by solutes (like plasma proteins) in a solution that drives the movement of water across semi-permeable membranes. o Albumin - The primary contributor to colloid osmotic pressure (also known as oncotic pressure) in the blood. By attracting and holding water in the blood vessels, albumin helps to maintain blood volume and pressure. o Prevent excessive leakage of fluid from the bloodstream into surrounding tissues, thus helping to maintain normal blood volume and tissue hydration. o This is critical in preventing edema (swelling due to fluid accumulation) and ensuring adequate circulation. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 5  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS METHODS OF ANALYSIS During digestion, oxidation occurs, producing gases Kjeldahl (Nitrogen component of proteins) such as carbon monoxide o widely used analytical technique for (CO), carbon dioxide determining the nitrogen content of organic (CO₂), water (H₂O), and compounds, particularly proteins. Since sulfur dioxide (SO₂). proteins are made up of amino acids, which This step ensures that all contain nitrogen, this method allows for the organic matter is converted estimation of protein content in a sample. into inorganic components. o sequential steps: ▪ NITROGEN CONVERSION ▪ PRECIPITATION – TCA or Tungstic Addition of ammonium Acid bisulfite then distill with boric To remove non-protein acid nitrogenous compounds and To prepare the ammonium concentrate the protein ions for quantification. nitrogen. After digestion, an addition Add trichloroacetic acid of ammonium bisulfite is (TCA) or tungstic acid to made to the solution. This the sample solution. These compound reacts with the acids precipitate proteins by ammonium ions to form a denaturing them and stable ammonium salt. causing them to aggregate. The solution is then distilled The precipitated proteins with boric acid. The can then be separated from ammonia released during the solution by centrifugation this distillation reacts with or filtration. boric acid to form ▪ DIGESTION – Sulfuric acid H2SO4 ammonium borate, which Ginagamit to digest the can be titrated. sample ▪ TITRATION – titrate with HCl To convert organic nitrogen To quantify the amount of into inorganic forms nitrogen in the sample. (ammonium ions). The distillate containing The precipitate (if TCA was ammonium borate is titrated used) is digested using with a standard solution of concentrated sulfuric acid hydrochloric acid (HCl) until (H₂SO₄). a color change occurs During digestion, the sulfuric (indicating the endpoint). acid hydrolyzes the proteins, The volume of HCl used for breaking them down into titration is directly their constituent amino acids proportional to the amount of and releasing nitrogen as nitrogen in the original ammonium sulfate. sample, allowing for the ▪ BOILING – 340 to 360 degrees calculation of protein content To facilitate the digestion using the nitrogen-to-protein process. conversion factor The mixture is boiled at (commonly 6.25 for many temperatures between 340 proteins). to 360 degrees Celsius. Kjeldahl Modifications: to enhance the sensitivity This high temperature and specificity of nitrogen determination in various helps in the complete samples. digestion of the organic o Nesslerization (ammonium dimercuric iodide) material, converting all ▪ Uses potassium, mercury and iodine nitrogen into ammonium ▪ Product: ammonium mercuric ions. chloride (yellow solution) ▪ OXIDATION – produce CO, CO2, ▪ To detect and quantify ammonium H2O, SO2 ions in a sample. To further break down the ▪ Ammonium ions react with the organic matter and convert reagent to form ammonium nitrogen into a measurable mercuric chloride. form. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 6  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS ▪ A yellow solution indicating the ▪ The Lowry method is widely used in presence of ammonium ions, which biochemical research for determining can be quantified protein concentrations due to its spectrophotometrically. sensitivity. ▪ Nesslerization is particularly useful in UV absorption (280nm) detecting low concentrations of Salt fractionation ammonium in environmental and Sodium sulfate, Sodium sulfite, ammonium sulfate, clinical samples. methanol o Berthelot (indophenol blue) ▪ Uses sodium nitroprusside and alkaline hypochlorite ▪ Product: indophenol blue solution ▪ To measure ammonium ions in a sample through colorimetric analysis. ▪ Ammonium ions react with sodium nitroprusside in an alkaline solution to produce indophenol blue. ▪ A blue-colored solution, with intensity proportional to the concentration of ammonium ions. ▪ This method is widely used in clinical PROTEIN ELECTROPHORESIS laboratories for determining ammonia levels in blood and urine Protein electrophoresis is a laboratory technique samples. used to separate proteins based on their size and o Biuret charge. This method is crucial for analyzing protein ▪ Cupric sulfate, Sodium tartrate, composition in biological samples and diagnosing NaOH and Kl various medical conditions. ▪ To estimate the total protein content Cellulose Acetate - A medium that allows for the based on the presence of peptide separation of proteins based on their size and charge. bonds. Used for simple, rapid protein separations and can ▪ Proteins react with cupric ions in an provide high resolution for serum proteins. alkaline medium to form a violet- Agarose - A gel matrix made from agarose colored complex (the Biuret polysaccharides, suitable for larger proteins and reaction). nucleic acids. ▪ A colored solution whose intensity Polyacrylamide Gel - The most commonly used can be measured medium for protein electrophoresis due to its spectrophotometrically, with versatility and resolution. Allows for precise control absorbance correlating with protein over pore size, making it ideal for separating proteins concentration. of varying sizes. ▪ The Biuret method is a commonly Buffer at pH 8.6 - A pH of 8.6 is typically optimal for used technique for determining total separating most proteins, as it ensures that proteins protein content in biological samples, are negatively charged and migrate towards the including serum and urine. anode during electrophoresis. o Lowry Migration is from ▪ Phosphotungstic and CATHODE TO ANODE phosphomolybdic acid Dye used: ▪ Oxidation of tyrosine, trptophan and o CBB (Coomassie histidine Brilliant Blue) – ▪ To quantify proteins by measuring Ponceau S the reduction of specific reagents in (reversible stain the presence of amino acids. used to visualize ▪ The method involves the oxidation of proteins on aromatic amino acids (tyrosine, nitrocellulose tryptophan, and histidine) in the membranes after protein, resulting in a blue-colored transfer from gels. It's commonly used for complex. quick checks during the Western blotting ▪ The resultant color can be quantified process.) and Amido black (A dye that binds using spectrophotometry, with strongly to proteins, providing a dark blue intensity related to protein color for visualization on the gel.) concentration. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 7  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS o Lipoproteins – ORO (Oil Red O - Used INFLAMMATION WITH ELEVATED ACUTE PHASE specifically for visualizing lipoproteins. This REACTANTS (Α1, Α2 AND Β) dye stains lipids within the proteins, helping to differentiate lipoprotein classes in electrophoresis.) and Sudan Black (Another lipid-specific stain that provides visualization of lipids and lipoproteins in gels.) o Glycoprotein – PAS (Periodic Acid-Schiff - Used for staining glycoproteins, which contain carbohydrate groups. The PAS stain reacts with the sugar moieties, producing a pink or red color that indicates the presence of glycoproteins.) NON-PROTEIN NITROGENOUS COMPOUNDS / BIOCHEMISTRY, METHODS OF ANALYSIS AND CLINICAL NORMAL ELECTROPHORESIS GRAPH AFTER DENSITOMETRY SIGNIFICANCE UREA Most abundant NPN in the blood (45-50%) PROTEIN METABOLISM (amino groups and ammonia) significant non-protein nitrogenous compound (NPN) in the blood and plays a crucial role in the body's nitrogen metabolism. Urea is primarily produced in the liver as a result of protein metabolism. Urea serves as a primary vehicle for the excretion of BETA-GAMMA BRIDGE IN CIRRHOSIS excess nitrogen from the body. Urea contributes to the osmotic pressure of blood, which helps regulate the movement of fluids between compartments in the body ( blood and tissues). Urea levels in the blood reflect the body’s nitrogen balance, indicating whether there is an excess or deficiency of protein intake. ALBUMIN “SPIKED” PATTERN IN NEPHROTIC SYNDROME LABORATORY ANALYSIS Avoid hemolysis!!! can lead to the release of intracellular components into the plasma, including potassium, lactate dehydrogenase, and hemoglobin. Hemolysis can artificially elevate urea levels and other test results, leading to misinterpretation. MONOCLONAL “SPIKE” PATTERN Avoid using SODIUM CITRATE (It can interfere with urea and other nitrogenous compound measurements due to dilution effects.) and SODIUM FLUORIDE (Acts as a glycolytic inhibitor and can affect the accuracy of certain biochemical assays by preventing glucose consumption, leading to altered metabolite levels.) additives for plasma Timed specimen (24 hour collection) for urine samples, provides a comprehensive assessment of kidney function and the body’s nitrogen balance by measuring total urea excretion over a complete day. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 8  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS Slightly affected by high protein diet but NOT TO A Fearon reaction (Diacetyl Monoxime) SIGNIFICANT LEVEL. While protein intake can o chemical method measures urea by forming affect urea levels, normal dietary habits should be a colored complex when urea reacts with maintained for reliable results. diacetyl monoxime in an acidic medium. o Requires boiling (100 degrees ) o PFF (Proteins Free Filter): Typically METHODS OF ANALYSIS requires the removal of proteins from the sample. Coupled Enzymatic (340 nm, KINETIC) o Obsolete method (Chemical method) o use of specific enzymes to catalyze reactions o Urea reacts with diacetyl in the presence of that result in measurable products. an acid (often sulfuric acid). o The typical wavelength used for detection is o The product of the reaction is a pink 340 nm, corresponding to the absorbance of chromogen, detectable at 520 nm. NADH, a cofactor that is reduced in the reaction. Diacetyl + acid + urea -> pink chromogen (520nm) o Urea is converted to ammonia by urease Color enhancers: Thiosernicarbazide and ferric ions enzyme, which then undergoes a series of enzymatic reactions (often involving glutamate dehydrogenase and other enzymes). o The reaction produces NADH, and the change in absorbance at 340 nm is monitored kinetically. o provides a sensitive and accurate measurement of urea levels and is widely used in clinical laboratories. Conductimetric and/or pH determination o Measures NH4 + and CO3 2- o measures changes in electrical conductivity or pH during chemical reactions involving ammonium ions (NH₄⁺) and carbonate ions (CO₃²⁻). o The addition of urea to a solution containing URIC ACID suitable reagents can result in the formation of ammonium ions, which will change the Second most abundant NPN in the blood (10%) conductivity and/or pH of the solution. PURINE and NUCLEIC ACID METABOLISM o measurement can be used to infer the Insoluble at high concentrations (>6.8 mg/dL) or in concentration of urea based on the changes acidic pH which can lead to crystal formation. observed. Uric acid is primarily produced from the metabolism Isotope Dilution Mass Spectrometry (reference of purines, which are nitrogen-containing method) compounds found in many foods and are also o IDMS is a highly precise method that uses released during the breakdown of nucleic acids (DNA stable or radioactive isotopes to quantify the and RNA). amount of urea in a sample. The breakdown of purines leads to the formation of o A known amount of an isotopically labeled hypoxanthine, which is further converted to urea standard is added to the sample. xanthine and then to uric acid by the enzyme o The ratio of the isotopes in the sample is xanthine oxidase. measured using mass spectrometry, allowing Purines → Hypoxanthine → Xanthine → Uric Acid for the determination of the concentration of urea based on the dilution principle. o IDMS is considered a reference method for urea analysis due to its high accuracy and specificity. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 9  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS LABORATORY ANALYSIS o The intensity of the colored complex can be measured spectrophotometrically, allowing Avoid icteric samples (PEROXIDASE) for quantification of uric acid levels based on o Icteric samples, which contain elevated the absorbance. levels of bilirubin, can interfere with o enzymatic method uses the enzyme uricase laboratory assays, particularly those that rely to catalyze the conversion of uric acid to on enzymatic reactions involving allantoin, which is then quantified indirectly peroxidase. through the production of hydrogen peroxide o High bilirubin levels can lead to falsely (H₂O₂). elevated or inaccurate uric acid readings. o method is highly specific and sensitive, AVOID LIPEMIC (Lipemia can cause turbidity in making it a popular choice for clinical samples, leading to interference in photometric laboratories to measure uric acid levels measurements and affecting accuracy.) AND accurately. HEMOLYZED SAMPLE! (Hemolysis releases Isotope Dilution Mass Spectrometry (reference hemoglobin and other intracellular components into method) the plasma, which can affect uric acid levels and other biochemical parameters) !! Do not use EDTA or Sodium Fluoride anticoagulants/additives Use HEPARIN for plasma samples Affected by diet (food high in uric acid) but a recent meal WON’T AFFECT THE OVERALL CONCENTRATION Bilirubin and ascorbic acid destroys peroxidase enzyme o High levels of bilirubin can interfere with uric acid assays, especially those that use peroxidase-based methods. o Ascorbic acid (Vitamin C) can also interfere with some colorimetric assays, leading to falsely low uric acid readings. METHODS OF ANALYSIS CREATININE/CREATINE Approximately 5% (Creatinine) and 2% (Creatine) of Caraway method (requires PFF) the total NPNs o Uric Acid + Phosphotungstic acid -> allantoin o Both compounds are closely related to + Tungsten blue (600 nm) muscle metabolism and energy production. o involves the reaction of uric acid with Important compounds related sa muscle metabolism phosphotungstic acid, leading to the and play a significant role in assessing kidney formation of allantoin and a color complex function. known as tungsten blue. Product of muscle contraction and energy (ATP) o Requires Proteins Free Filter (PFF) to usage remove proteins that could interfere with the Majority is excreted in the URINE assay. o Uric acid reacts with phosphotungstic acid One of the best organic determinants of the to produce allantoin and tungsten blue. glomerular filtration rate o The resultant tungsten blue complex is Creatine - Synthesized from the amino acids measured photometrically at 600 nm. arginine, glycine, and methionine primarily in the Enzymatic method liver, but also in the kidneys and pancreas. Stored o Uric acid + H2O -> Allantoin + H2O2 in muscle tissue, where it plays a crucial role in (URICASE) energy production. ▪ Uric acid reacts with water in the Creatinine - Creatine undergoes a non-enzymatic presence of uricase conversion to creatinine, which occurs at a relatively o H2O2 + Indicator dye -> colored complex constant rate, conversion happens as creatine is (PEROXIDASE) used during muscle contraction and energy (ATP) ▪ The generated hydrogen peroxide utilization, making creatinine a byproduct of muscle reacts with an indicator dye in the metabolism. presence of peroxidase to form a colored complex. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 10  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS LABORATORY ANALYSIS ENZYMATIC REACTIONS Avoid hemolysis and icteric samples (Jaffe method) Creatininase-Hydrogen peroxide - method uses the High protein ingestion might have TRANSIENT effect sequential action of several enzymes to convert Timed specimen for urine samples (Creatinine creatinine into a measurable colored compound Clearance) through a series of reactions. Some methods are affected by lipemic samples o Creatinine -> Creatine (Creatininase) o Creatine -> Sarcosine + Urea (Creatine Ascorbic acid, glucose and alpha ketoacids may amidinohydrolase) affect Jaffe reaction o Sarcosine -> Glycine + Formaldehyde + Hydrogen Peroxide (Sarcosine oxidase) METHODS OF ANALYSIS o Hydrogen peroxide -> Colored dye (Peroxidase reaction) Jaffe Method (Chemical Method) Creatininase-Creatine Kinase - method also o Creatinine + Picric acid -> red-orange involves the initial conversion of creatinine to creatine complex (red tautomer) but focuses on the subsequent phosphorylation o Jaffe method is based on the reaction of reactions to generate ATP, which can be quantified. creatinine with picric acid in an alkaline o Creatinine -> Creatine (Creatininase) solution to form a colored complex. o Creatine + ATP -> Creatine Phosphate + o Creatinine reacts with picric acid to form a ADP (Creatine Kinase) red-orange complex (known as the red o ADP + Phosphoenolpyruvate -> Pyruvate + tautomer ATP (Pyruvate Kinase) o Creatinine reacts with picric acid to form a o Pyruvate + NADH -> Lactate + NAD (Lactate red-orange complex (known as the red Dehydrogenase) tautomer o Jaffe method is widely used for its simplicity and cost-effectiveness, but it may have METHODS OF ANALYSIS limitations due to interference from other Conway (titration) substances in the sample. o Microdiffusion chamber to separate and Modifications of Jaffe Method measure uric acid through titration. o Use of adsorbents (Fuller’s o The sample containing uric acid is placed in earth/Aluminum magnesium silicate and a chamber that allows for the diffusion of uric Lloyd’s reagent/sodium aluminum acid into a surrounding medium containing a silicate) reagent. ▪ These adsorbents can be used to o known for its simplicity and effectiveness in reduce interference from other quantifying uric acid, although it may be less compounds in the sample, thereby commonly used in modern laboratories improving the specificity of the Jaffe compared to other methods. reaction. Enzymatic method o Kinetic measurement - involves measuring o Glutamate Dehydrogenase (urease method) the rate of formation of the red-orange o enzymatic method measures uric acid by complex rather than its final concentration. It converting it into ammonia, which can be can help enhance sensitivity and reduce quantified. interference from other substances that may o enzymatic method is highly specific and be present in the sample. sensitive, making it a preferred choice in Isotope Dilution Mass Spectrometry many clinical laboratories for uric acid Enzymatic method measurement. o Creatininase-Hydrogen peroxide Dry slide method ▪ Creatinine is converted to creatine o Bromphenol blue - The dry slide contains by the enzyme creatininase. The bromphenol blue, a pH indicator that changes reaction produces hydrogen color in response to the concentration of uric peroxide (H₂O₂), which can be acid in the sample. measured. o dry slide method is a colorimetric technique o Creatininase-Creatine Kinase that uses a dry reagent slide to analyze uric ▪ creatine formed from creatinine is acid levels in a sample. further acted upon by creatine o dry slide method offers a convenient, rapid, kinase, producing additional and easy-to-use option for measuring uric measurable products. acid levels in clinical settings. Ion Selective/Specific Electrode (potentiometry) CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 11  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS CREATININE CLEARANCE LIVER FUNCTION TESTS LIVER key clinical measurement used to assess kidney function by evaluating the ability of the kidneys to filter The liver is a vital organ responsible for numerous creatinine from the blood. metabolic functions, detoxification processes, and Creatinine is: synthesis of essential biomolecules. Liver function o Endogenous - Creatinine is produced tests (LFTs) are a group of blood tests used to assess naturally in the body, primarily from the the health and functionality of the liver. breakdown of creatine, which is found in Chief metabolite of the body muscle tissue. Its production is relatively Located at the upper quadrant of the abdominal cavity constant and proportional to muscle mass. Components o Synthesized at a constant rate - The o Functional units: hepatocytes are the synthesis of creatinine occurs at a constant primary functional cells of the liver. They rate, typically reflecting an individual’s perform most of the liver's metabolic muscle mass and activity levels. This rate can processes, including the synthesis of proteins vary slightly based on factors such as age, and bile, and the detoxification of various sex, and physical activity. metabolites. o Majority is cleared by renal filtration - The o Macrophages: Kupffer cells are specialized majority of creatinine is excreted by the macrophages located in the liver that play a kidneys through glomerular filtration. Since crucial role in the immune response. They creatinine is not significantly reabsorbed or help to filter and phagocytize pathogens, secreted by the renal tubules, its levels in the dead cells, and other debris from the blood urine can be used as a reliable indicator of that passes through the liver. kidney function. o Passageways: bile canals and hepatic duct defined as the volume of blood plasma that is ▪ Bile canals (canaliculi) are small completely cleared of creatinine by the kidneys per ducts formed between adjacent unit time, typically expressed in milliliters per minute hepatocytes where bile is secreted. (mL/min). ▪ The hepatic duct collects bile from Creatinine clearance – relationship between urine the liver and transports it to the and serum creatinine: gallbladder for storage or directly to U = urine creatinine concentration (mg/dL) the small intestine for digestion. V = total urine volume collected (mL) S = serum creatinine concentration (mg/dL) T = time of collection (usually in minutes) TUBULAR REABSORPTION TESTS Phensulfonthalein test o Secretory ability o used to evaluate the secretory ability of the renal tubules. o Phenolsulfonphthalein is a dye that is filtered at the glomerulus and normally secreted by the renal tubules. The test measures how LIVER FUNCTION TESTS effectively the kidneys can secrete this dye. Enzymes: Urine specific gravity o AST Aspartate Aminotransferase o Concentrating ability of renal tubules ▪ An enzyme involved in amino acid o Specific gravity measures the concentration metabolism; found in the liver, heart, of solutes in the urine, reflecting the kidney's muscle, and kidneys. ability to concentrate or dilute urine relative to ▪ Elevated levels may indicate liver plasma. damage, but can also arise from heart or muscle conditions. AST is often interpreted alongside ALT for liver function assessment. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 12  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS o ALT Alanine Aminotransferase oInternational Normalized Ratio (INR) ▪ Primarily found in the liver; crucial for ▪ A standardized measure of PT that amino acid metabolism. accounts for variations in testing ▪ Elevated ALT levels are more methods. specific to liver injury compared to ▪ INR is used to monitor AST. High levels may indicate anticoagulation therapy and assess hepatitis, fatty liver disease, or liver liver function; elevated levels damage from drugs or toxins. suggest impaired liver synthetic o ALP Alkaline Phosphatase capacity. ▪ An enzyme related to bile duct Macromolecules: Protein, Carbohydrates and Lipids function; also found in bone, kidney, and placenta. ▪ Elevated ALP levels can indicate BILIRUBIN ANALYSIS cholestasis (bile flow obstruction), liver disease, or bone disorders. PHOTOSENSITIVE o GGT Gamma-Glutamyl Transferase o Bilirubin is sensitive to light and can degrade ▪ An enzyme involved in the upon exposure to sunlight or artificial light, metabolism of glutathione and drug which can affect the accuracy of detoxification; primarily found in the measurements. liver. o Samples should be protected from light ▪ Elevated GGT levels can indicate during collection, transport, and storage. liver disease, bile duct obstruction, or Avoid hemolysis alcohol abuse. It is often used in Analyze TOTAL AND DIRECT BILIRUBIN only conjunction with ALP to differentiate Two Methods: between liver and bone disease. o Evelyn and Malloy (560 nm) Metabolites ▪ This colorimetric method uses a o Bilirubin reaction that converts bilirubin into a ▪ breakdown product of hemoglobin; colored complex, which can then be processed by the liver for excretion in measured spectrophotometrically. bile. Total and direct bilirubin levels ▪ The measurement is typically taken are measured. Elevated bilirubin can at 560 nm. indicate liver dysfunction, hemolysis, ▪ The total bilirubin in the sample or bile duct obstruction. reacts with specific reagents to o Bile Acids produce a measurable color, which ▪ Compounds produced by the liver to correlates with the bilirubin aid in the digestion and absorption of concentration. fats. Increased bile acids in the blood ▪ This method is widely used and may suggest cholestasis or impaired provides accurate measurements of liver function. both total and direct bilirubin levels. o Blood Ammonia o Jendrassik and Grof (600 nm) ▪ A byproduct of protein metabolism; ▪ This method is also based on a normally converted to urea by the colorimetric reaction where bilirubin liver. Elevated blood ammonia levels reacts with specific reagents to can indicate hepatic encephalopathy produce a colored compound. or severe liver failure, as the liver is ▪ The measurement is taken at 600 unable to detoxify ammonia nm. efficiently. ▪ The total bilirubin reacts with the Synthetic: reagents, and the intensity of the o Bromsulphalein color developed is measured to ▪ Assesses the liver's ability to excrete determine bilirubin concentration. the dye bromsulphalein, which is ▪ known for its high sensitivity and taken up by liver cells. specificity, particularly in detecting o Hippuric Acid test low levels of bilirubin. ▪ Evaluates the liver's ability to conjugate and excrete hippuric acid. Hematologic: o Prothrombin time ▪ Measures the time it takes for blood to clot; assesses the liver’s production of clotting factors. ▪ Prolonged PT can indicate liver dysfunction or vitamin K deficiency. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 13  TRANS: AMINO ACIDS AND PROTEINS, PLASMA PROTEINS, NONPROTEIN NITROGENS REACTIVE PRINCIPLE Enzymatic o These methods utilize specific enzymes to convert bilirubin into measurable products, typically involving reactions that yield a color change. o Enzymatic methods are generally more specific than colorimetric methods, minimizing interference from other substances in the sample. o They can provide accurate measurements of JENDRASSIK AND GROF total and direct bilirubin levels. ✓ Sensitive, REFERENCE METHOD o Considered a reference method for bilirubin analysis, it provides a standard against which other methods can be compared. ✓ Unaffected by hemoglobin up to 750 mg/dl o without significant interference, allowing for accurate bilirubin measurement even in hemolyzed samples. ✓ Minimal turbidity by protein precipitation Bilirubin + DSA -> azobilirubin + excess DSA o Bilirubin in the serum reacts with diazotized sulfanilic acid (DSA) to form azobilirubin and an excess of DSA. Excess DSA + Ascorbic acid -> Destroy excess DSA o Any excess DSA that remains after the initial reaction is reacted with ascorbic acid, which effectively destroys the excess DSA, preventing it from interfering with subsequent measurements. Azobilirubin + alkaline tartrate -> blue complex (600nm) o The azobilirubin then reacts with alkaline tartrate to produce a stable blue complex. OTHER BILIRUBIN ANALYSIS METHODS Qualitative Methods o Ehrlich’s method (Qualitative) ▪ qualitative test that relies on the reaction of bilirubin with Ehrlich's reagent, which contains p- dimethylaminobenzaldehyde (DMAB). ▪ primarily used for qualitative detection of bilirubin, particularly in urine. A positive result suggests the presence of bilirubin, which can be indicative of liver disease or hemolysis. o Van de Bergh (modified Ehrlich’s) ▪ modifies the original Ehrlich’s method to improve specificity and sensitivity for measuring bilirubin, particularly distinguishing between direct and indirect bilirubin. ▪ provides more detailed information regarding bilirubin status and can be useful in diagnosing specific types of liver disease or hemolytic conditions. CARODAN. E CORNEJO. HJ GUDIO. JA MERCADO. DA YAON. RA | 3Y-MT 14