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CCHM 2 Prelim LEC - Lecture notes
Clinical Chemistry 2 (MDT 3122L)
Our Lady of Fatima University
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Clinical Chemistry 2
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science
PRELIM
ENZYMOLOGY
Enzymes ● Have very small amount in the body ● Measured based on their activity, not by absolute quantity ● Essential to Physiologic Functioning: ○ Hydration of CO 2 ○ Nerve conduction ○ Muscle contraction ○ Nutrient degradation ○ Energy use ● Biological protein that catalyze a reaction ● Enzymes can be found inside the cells ● When the cells have been damaged the enzyme will be released, and that is the time the concentration of enzyme in the blood vessels increases ● Each organ has a specific enzyme ● It catalyzes single or specific reaction only ● Reactants: ○ Enzyme and substrate, the combination of these reactants called “ES Complex” ● Products: ○ Fast chemical reaction ● Parts of the enzyme Catalytic Mechanism of Enzymes ● A chemical reaction may occur spontaneously if the free energy or available kinetic energy is higher for the reactant than the products (lower energy). ● Activation Energy ○ Chemical reaction may occur when reactants have energy to break their bond and collide (enzymes and substrate) to form new bond Enzymes are highly Specific: Absolute Specificity ○ Strictest model ○ Enzymes combine only into a one substrate and catalyze only a single reaction. Group Specificity ○ Combines all the substrates with a particular chemical group. Bond Specificity ○ Combines all substrates with a particular chemical bond. ■ EX: Ionic bond, covalent bond, hydrogen bond and metallic bond Stereoisomeric Specificity ○ Combines all substrates with a specific optical isomers. ■ Optical isomers - mirror image of chemical groups, or that have the same numbers for example hydrogen, oxygen, etc. but have a different positioning. Factors that influence Enzymatic Reaction
Substrate concentration/enzyme concentration ● Follows the hypothesis of Michaelis & Menten “even in low substrate concentration, the substrate can readily bind with free enzyme” ● Enzymatic Reaction can be: ○ First-Order Kinetic ■ The reaction rate of enzymatic reaction is directly proportional to substrate concentration. ■ Control or increase the substrate concentration to increase enzymatic reaction ○ Zero-order Kinetic ■ enzymatic reaction depends only on the enzyme concentration. ■ Control or increase the enzyme concentration to increase enzymatic reaction ○ Saturation Kinetic ■ once the reaction reaches its maximum rate of chemical reaction it will no longer produce fast chemical reaction.
Ph ● Enzymes are protein that carry a net molecular charge ● Normal range: 7-8 pH (neutral) ● Usually enzymes can be denatured by extreme pH ● But there are also enzyme that requires extreme pH
Temperature ● Temperature coefficient ○ in every 10°C increase with temperature it will result to two folds increase in activity of enzyme (Activity/reaction increases 2x) ● increase reaction = increase reaction ● High temperature = rapid/faster reaction ● 37°C (25 or 30*C) - most reactions occur with this temp ● 40-50°C- denaturation ● 60-65°C- inactivation ● Low temp - reversible inactivation activation of an enzyme ○ Repeated thawing can cause denaturation of enzymes
Co-factors ➔ Non-protein entities that must bind to an enzyme before a reaction occurs. Activators ○ inorganic, metallic/non-metallic ○ Metallic: Ca, Fe, Mg, Mn, Zn, K ○ Non-metallic: Br, Cl ○ Function: Alter spatial configuration of enzyme Coenzyme ○ organic ○ Ex. NAD, nucleotide phosphatase and vitamins ○ Function: Serve as a second substrate for enzymatic reaction.
Inhibitors ● Interferes chemical reaction Types of Inhibition ○ Competitive Inhibition- targets active site ○ Non-Competitive Inhibition- targets allosteric site ○ Uncompetitive Inhibition- disables ES complex
Storage -20°C ● Long term preservation of enzyme ● Freezing temp 2-8°C ● General temperature for preservation of enzyme ● Refrigerator temp 15 to 25-30°C ● Room Temp ● For cold labile enzymes, ex. LDH (LD 4 &LD 5 )
Hemolysis ● Lysis/destruction of rbc
Lactescence/Milky specimen ● Decrease with enzyme measurement/concentration. Enzymatic Reactions ● 2 Methods in measuring the enzymatic reactions: ○ Fixed-time (end point) ➔ Reactants are combined → it allows to react on a designated time → stopped → measured ○ Continuous-Monitoring/Kinetic Assay ➔ Multiple measurement of reaction with designated specific time interval ➔ Used to measure enzyme activity Enzymes is a protein (Amino Acid) ● Primary Structure ➔ Simplest ➔ made up of only specific amino acid sequence ● Secondary Structure ➔ Polypeptide chain twisting ● Tertiary structure ➔ Foldings of polypeptide chain ● Quaternary structure ➔ Complex and functional ➔ Spatial relationship between subunits of tertiary structure Isoenzymes ● Same catalytic reaction but different physical properties Example ○ CK -> 3 ○ LD -> 5 Differentiated by: ○ Electrophoretic mobility ○ Solubility ○ Resistance to inactivation Nomenclature ● Naming of enzymes ● Developed by the E. (Enzyme Commission) ● Enzyme are grouped into 6:
Oxido-reductase ➔ Catalyze oxidation reduction reaction between substrate
Transferase ➔ Catalyze a transfer of a group other than hydrogen between one substrate to another
Hydrolase ➔ Catalyze hydrolysis of various bonds
Lyase ➔ Catalyze the removal of groups without hydrolysis ➔ Product contains double bond
Isomerase ➔ Catalyze the interconversion of geometric optical and positional isomers
Ligases ➔ Catalyze the joining of two substrate molecules
CREATINE KINASE
○ Common Abbr: CPK (Creatine Phosphokinase) ○ Standard Abbr: CK ● Associated with ATP generation in contractile system (muscle) ● Function: in the muscle cells it stores Creatine phosphate that is important in ATP production ● reaction catalyzes ○ Creatine phosphate + ADP ⇔ Creatine +ATP ● It may vary depends on muscle mass, gender, race, and age Tissue sources:
- Skeletal muscle
- Heart muscle
- Brain tissue Isoenzymes: ○ Dimer with 2 subunits and 2 possible forms (B-brain or M-muscle) CK-1 <1% CK-BB Brain type CK-2 <6% CK-MB Hybrid type CK-3 94-100% CK-MM Muscle type ISOENZYMES CK-MM (CK-3) ● Major fraction in serum ● Elevated also in muscle activity, IM injection ● Increased in cardiac and skeletal muscle disorders ● Elevated in hypothyroidism because of increased membrane permeability of the cell and decreased clearance of creatine kinase CK-BB ● Seldomly found in the plasma ● Have a high molecular size ● Have a short half life (1-5 HRS) CK-MB ● 20% of cardiac tissue contains ck-mb, and very little to other tissues ● Myocardium is the only tissue from which CK-MB enters the serum, meaning it is quite specific to the heart muscle ● Indicator of myocardial damage (AMI) Rise: 4-8 hrs Peak: 12-24 hrs Normalize: 48-72 hrs ● Other cardiac markers ○ LDH, and AST ○ Troponin, and Myoglobin (non enzyme) ● Other diagnostic significance: ○ AMI ○ Muscular Dystrophy Duchenne Type ■ degenerative form of muscular disorder, loss of muscle ■ CK increased 50-100x ○ Cerebrovascular Accident (CVA) & other brain conditions ○ Hypothyroidism ○ malignant hyperpyrexia ○ Reye’s syndrome ○ Vibrio Vulnificus
ATYPICAL FORM OF CK
○ Abnormal forms Macro CK ● Found midway between MM and MB ● Theories: ○ Made up of CK-BB that is bound to IgG ○ Made up of lipoprotein (LPP) bound with CK-MM ● No specific disorder is associated ● Mostly seen in female more than 50 years old ○ Related to sex and age of the pX Mitochondrial CK (CK-Mi) ● Found before the CK-MM in the electrophoresis ● Bound to the exterior surface of the mitochondrial membrane of the Muscle, brain, & liver. ● Found in severe illness, malignant tumor and cardiac abnormalities METHODS USED FOR THE MEASUREMENT OF ISOENZYMES OF CK Electrophoresis ● Reference method Ion Exchange Chromatography ● More sensitive but pricey ● Problem with the bad column: ○ CK-MM merge with CK-MB ○ CK-BB eluted with CK-MB Antibodies ● used specifically for CK-MB determination for dx of AMI ● Anti-M antibody ○ inhibits all the activity of M subunit. ■ CK-BB (no effect) ■ CK-MB (multiply the remaining B subunit into 2) ■ CK-MM (eliminate M subunits) ○ Do not have effect on Macro CK and CK-MI Immunoassay ● Detects MB reliably with minimal reactivity ● Detects enzyme protein rather than activity METHODS
- Tanzer-Gilbarg Assay (pH9 @ 340 nm) ● Forward/Direct reaction ○ Creatine + ATP ⇔ Creatine Phosphate + ADP CK ○ ADP+Phosphoenol ⇔ Pyruvate pyruvate + ATP Pyruvate kinase (PK) ○ Pyruvate+NADH+H ⇔ lactate+NAD LDH
- Oliver-Rosalki Assay (pH6 @ 340 nm) ● Reverse/Indirect reaction ○ Creatine Phosphate + ADP ⇔ Creatine + ATP CK ○ ATP + glucose ⇔ ADP + G6P Hexokinase (HK)
○ G6P + NADPH ⇔ 6-phosphogluconate + NADPH G6PD REFERENCE VALUE/RANGE ● Male: 15-160 U/L @37°C ● Female: 15-130 U/L @ 37°C ● CK-MB: <6% SAMPLE ● Avoid Hemolysis of RBC that will release AK that will give a falsely elevated CK ● Storage: (must be in dark place) ○ 4 °C - 7 days ○ -20°C - 1 month LACTATE DEHYDROGENASE (LDH) ● Catalyzes the interconversion of lactic acid and pyruvic acid ○ Lactate + NAD ⇔ Pyruvate + NADH ● Coenzyme: NAD Isoenzymes: ○ Tetrametric molecules containing 4 subunits of two possible forms LD1 14-26% HHHH HEART & RBC LD2 29-39% HHHM LD3 20-26% HHMM
LUNGS,
LYMPHOCYTE,
SPLEEN,
PANCREAS
LD4 8-16% HMMM LIVER
LD5 6-16% MMMM SKELETAL MUSCLE
ISOENZYMES
LD 1 & LD 2
● Indicator of AMI and Intravascular hemolysis ● Flipped pattern: when LD1>LD ○ Indication of AMI and hemolyzed sample LD 3 ● pulmonary involvement ● Carcinoma (CA) LD 4 ● intrahepatic disorder LD 5 ● Muscular dystrophies LD 6- Alcohol Dehydrogenase ● 6TH Band to the electrophoresis ● Arteriosclerotic cardiovascular failure ● Presence of LD6 indicates Great prognosis and impending death MEASUREMENT OF ISOENZYMES: ● Electrophoresis ● Immunoinhibition or chemical inhibition ● Substrate affinity ➔ There is a chemical assay that uses Alpha Hydroxybutyrate dehydrogenase, an enzyme that has great affinity to H subunit for measurement of LD DIAGNOSTIC SIGNIFICANCE: ● Elevated level: ○ Renal, ○ Hepatic ○ Cardiac, ○ Skeletal ● Highest level ○ pernicious and hemolytic disorder ○ Hematologic ○ neoplastic disorder AMI: ● Rise: 12-24 hrs ● Peak: 48-72 hrs ● Normalize: after 10 days METHODS
- Wacker Method (8.3-8 ph @ 340 nm) ● Forward/Direct Reaction (lactate is converted to pyruvate)
- Wrobleuski and La Due (7.1-7 @340nm) ● 3 times faster, small sample is needed ● Susceptible to substrate exhaustion & loss of linearity SAMPLE ● Avoid hemolysis ● Content RBC of LDH is 100-150x compared to outside the RBC ● Storage: room temperature ● Must be read within 24hrs ● Avoid cold temperature as it may destroy LD4 & LD5 (most labile) REFERENCE VALUE/RANGE
● 100-225 U/L
ASPARTATE AMINOTRANSFERASE (AST)
● Under Transaminase/Transferase ● Aka SGOT (Serum Glutamic Oxaloacetic Transaminase) ● involved in the transfer of amino group between aspartate and alpha keto acids (very important for the synthesis and degradation of amino acid as a source of energy) ● Gluconeogenesis is the production of glucose from noncarbohydrate sources including the amino acid ● Coenzyme: Pyridoxal Phosphate (2nd substrate) ○ Aspartate + a-ketoglutarate ⇔ oxaloacetate + glutamate ISOENZYMES: ● Cytoplasmic Isoenzyme ● Mitochondrial Isoenzyme TISSUE SOURCES:
- Cardiac tissue
- Liver ➔ Highest elevation of AST is seen in Acute Hepatocellular Disorder ➔ In Viral Hepatitis its concentration will increase 100X ➔ Cirrhosis: level may increase up to 4x
- Skeletal muscle ➔ Muscular dystrophy: concentration increase up tp 4-8x AMI: ● Rise: 6-8 hrs ● Peak: 24-48 hrs ● Normalize: 15th after AMI METHOD: Karmen Method ● Aspartate + a-ketoglutarate ⇔ oxaloacetate + glutamate ast ● Oxaloacetate + NADH + H ⇔ malate + NAD Mdh SAMPLE: ● AVOID HEMOLYSIS OF SAMPLE ● Storage of sample: In refrigerator can last up to 3-4 days
ACID PHOSPHATASE (ACP)
● Major source: Prostate ● Minor sources: RBC, platelets, bone USES: ● forensic chemistry ○ Activity of ACP can last up to 4 days into vaginal washing ○ Presence of >50 iu/L is indicative of presence of prostatic secretion ● detection of cancer (Prostate cancer, much better if combined with tumor marker PSA) METHOD: Shinowara Method ● pH 5 ● Immunochemical technique (RIA) ● Immunoprecipitation ● Immunoenzymatic assay SPECIAL CONSIDERATION ON ENZYME ACTIVITY ● Prostatic ACP - inhibited by L-tartrate ● Red cell ACP - inhibited by cupric ion and formaldehyde ● Prostatic CA - better determine by combination of PSA SAMPLE ● Avoid hemolysis ● At room temp for 1-2 hrs, level of ACP may decrease ● Read the sample immediately ● Storage: 4°C LAST UP TO 2 DAYS REFERENCE VALUE/RANGE ● Total ACP: 2 -11 U/L ● Prostatic ACP: 0-3 ng/Ml AMYLASE (AMS/AMY) ● Pancreatic markers ● It catalyzes the breakdown of starch and glycogen. ● Present in mouth already ● Smallest enzymes in terms of size ● Can pass through glomerulus of kidney ● Normal in urine ● Earliest pancreatic marker ISOENZYMES: S-TYPE ○ Ptyalin type ○ Salivary gland ○ Anodal P-TYPE ○ Amylopsin ○ Pancreas ○ Cathodal TISSUE SOURCES: ● Major tissue sources: acinar cells of salivary gland and pancreas ● Other tissue sources: adipose tissue, fallopian tube, small intestine, skeletal muscle SIGNIFICANCE: ● Acute pancreatitis - level of amylase will increase in blood and urine) AMS LPS Rise: 2-12 hrs 6 hrs Peak: 24 hrs 24 hrs Normalise: 3-5 days 8-14 days ● Parotitis - inflammation of parotid gland ● Renal failure- increase of ams to blood because of failure of it to be excreted MACROAMYLASE ● Abnormal form of AMS ● AMS bound to Immunoglobulin REFERENCE VALUE/RANGE ● 60-180 SU/dL ● 95-290U/L METHOD: ● Inhibitor: wheat germ lectin,& TAG ● Substrate: starch
- Saccharogenic ● Measures the amount of reducing sugar produced by the hydrolysis of starch by the usual glucose methods. ● Classic reference method expressed in SU.
- Amyloclastic ● Measures AMS activity by following the decreases in substrate concentration (degradation of the starch).
- Chromogenic ● Measures AMS activity by the increase in color intensity of the soluble ● dye-substrate solution produced in the reaction.
- Coupled-enzyme ● Measures AMS activity by the continuous monitoring technique. ● Usually performed in lab Increased AMY ● Acute pancreatitis ● Mumps ● Alcoholism ● Ectopic Pregnancy ● Peptic Ulcer LIPASE (LPS) ● Triacylglycerol acylhydrolase ● Pancreatic markers ● An enzyme that hydrolyzes the ester linkages of fats to produce alcohol and fatty acid. ● Major tissue source: Acinar cell of Pancreas ● More pancreas specific ● Pancreatic markers: ○ Amylase ○ Lipase ○ Trypsin ○ Chymotrypsin ○ elastase - SIGNIFICANCE: ● Acute pancreatitis ○ inc of lipase ● Chronic pancreatitis ○ There is a reduction of Lipase METHOD: ● use OLIVE OIL, TRIOLEIN addition of COLIPASE (make assay more sensitive to acute pancreatitis) Cherry Crandal method ● hydrolysis of olive oil after incubation for 24 hours @ 37 *C & titration of fatty acids using NaOH
● Reference method for lps determination ○ TAG + H2O ⇔ monoglyceride and fatty acids LPS REFERENCE VALUE/RANGE ● 0-1/mL Miscellaneous enzymes ALDOLASE ● Aka fructose-1,6-diphosphate aldolase ● splits fructose-1,6-diphosphate into two triose phosphate molecules in metabolism of glucose ISOENZYMES: ● Aldolase A - in skeletal muscle ● Aldolase B -in WBC, liver and kidney ● Aldolase C - in brain tissue Increase level: ● skeletal muscle disorder ● Leukemia ● Hemolytic Anemia ● hepatic carcinomas 5’ NUCLEOTIDASE ● Liver enzyme ● marker for hepatobiliary disease REFERENCE VALUE/RANGE ● 0-1 units GAMMA GLUTAMYL TRANSFERASE (GGT) ● It catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at a gamma carboxyl group. ● Used as an indicator of liver disease ● Clinically applied mainly to evaluate liver and biliary system SOURCES: ○ Liver (in lining of canaliculi) ○ kidney, ○ prostate & ○ Pancreas CLINICAL SIGNIFICANCE: ● detection of Hepatobiliary disorder ● Highest concentration is seen in biliary tract obstruction ● Sensitive indicator of alcoholism (esp. Occult alcoholism) ● Elevated among individual taking : warfarin, phenobarbital, & phenytoin therapy ● Reaction catalyzed by GGT ○ Glutathione + Amino acid →GGT Glutamyl Peptide + cysteinyl glycine ● METHOD: Rosalki & Tarrow Method ● SUBSTRATE: gamma- glutamyl-p-nitroanilide REFERENCE VALUE/RANGE ● 5-30 U/L Female ● 6-45U/L Male Cholinesterase/Pseudocholinesterase ● Used to motor effect of muscle relaxant after surgery ● Marker for insecticide and pesticide poisonings ● METHOD: Ellman techniques or potentiometry REFERENCE VALUE/RANGE ● Plasma: 0.5-1 ph unit Angiotensin Converting Enzyme ● FUNCTION: convert angiotensin 1 to angiotensin 2 ● indicator of neuronal dysfunction specifically Alzeimher’s disease ● SOURCES: macrophage and epithelioid cells Ceruloplasmin ● copper carrying protein & an enzyme ● Liver marker ● Indicative of hepatolenticular disease (wilsons’ disease) (value is declined) Glucose-6-Phosphate Dehydrogenase ● deficiency of this enzyme can lead to drug-induced hemolytic anemia (antimalarial drug, primaquine) ● Newborn screening marker ● SPECIMEN: Red cell or serum REFERENCE VALUE/RANGE ● 10-15U/g Hb or 1200-200mU/L packed RBC Ornithine Carbamoyl Transferase ● Hepatobiliary disease REFERENCE VALUE/RANGE
● 8-
Hormones affecting sodium level: 1. Aldosterone ○ Sodium retaining hormone ○ Produced when there is decline of blood volume or increased blood pressure ○ Maalat daw siya 2. Atrial natriuretic peptide (ANP) ○ Produced from the atrium of the heart in response to increasing blood pressure and volume 3. Angiotensin II ○ Member of RAAS system ○ Promotes vasoconstriction and activates the production of aldosterone HYPONATREMIA ● Low level of sodium in to the blood
- Increased sodium loss a. Hypoadrenalism ○ Decreased aldosterone reaction b. K deficiency ○ when serum k is low, the tubules will conserve k & excrete Na in exchange c. Diuretic use ○ Some diuretics target sodium d. Ketonuria e. Salt losing nephropathy f. Vomiting/ diarrhea g. Severe burns
- Increased water retention a. renal failure b. nephrotic syndrome & hepatic cirrhosis ○ plasma protein decreases which dec. Colloid Osmotic Pressure then causes edema. c. aldosterone deficiency d. cancer e. SIADH
- Water Imbalance a. Excess water intake ○ Polydipsia b. SIADH ○ due to CNS, malignancies and Trauma c. Pseudohyponatremia ○ due to increased lipids and proteins Classification of Hyponatremia by Osmolality With Low Osmolality With Normal Osmolality (all are cations) With High Osmolality ○ Inc. sodium loss ○ inc,. Water retention ○ Inc. non sodium ions ○ Lithium excess ○ Inc. gamma globulins ○ Severe hyperkalemia, hypercalcemia, & proteinemia ○ Pseudohyponatre mia ○ Hyperglycemia ○ Mannitol infusion ○ Hyperlipidemia ○ Pseudohyperkale mia (in vitro hemolysis) Symptoms of Hyponatremia ● It depends on the serum level of sodium: ○ 125-130 mmol/L - GI ○ < 125 mmol/L - neuropsychiatric symptoms (headache, N/V, lethargy, Ataksia, muscle weakness) ○ < 120 mmol/L - medical emergnecy Treatment of hyponatremia ● treatment is directed at correction of the condition that caused either water loss or Na+ loss in excess water loss. ○ Sodium replacement ○ Fluid restriction ○ Giving hypertonic saline HYPERNATREMIA ● Increase level of sodium in the blood
- EXCESS WATER LOSS a. diabetes insipidus b. renal tubular disorder c. prolonged diarrhea d. profuse sweating e. severe burns f. vomiting
- DECREASE WATER INTAKE a. Older persons b. Infants c. Mental impairment
- INCREASED INTAKE OR RETENTION a. Hyperaldosteronism b. Sodium bicarbonate infusion c. Hyperadrenocorticism Hypernatremia Related to Urine Osmolality I. Low Urine Osmolality ● Diabetes Insipidus ○ impaired secretion of AVP or kidney do not respond to AVP that causes high urine output (hypotonic fluid). II. Normal Urine Osmolality ● Partial defect in AVP release or response ● Osmotic diuresis III. High Urine Osmolality ● Loss of thirst ● Insensible loss ● GI loss of hypotonic fluid Symptoms of Hypernatremia: ● Altered mental status, lethargy, irritability, restlessness, muscle twitching, thirst, etc. Treatment: ● Directed at underlying conditions Methods:
Ion Selective Electrode (ISE) ○ Most used routine method for protein build up
Atomic Absorption Spectrophotometry (AAS)
Flame Emission Spectrophotometry (FES)
Colorimetry
Chemical method ○ Outdated ○ Requires large sample ○ Lack of precision CHLORIDE (Cl) ● Is the major extracellular anion (outside the cell) (-) ● Cl- shift is secondary to the movement of Na and HCO 3. ● Completely absorb in GI tract ● Eliminated through sweating ● Aldosterone - nagmamaalat ● Hypernatremia = Hyperchloremia Functions: ● Maintain Osmolality ● Maintain Blood volume ● Maintain Electroneutrality ● Hyperchloremia ● Hypochloremia Loss of Cl ● Vomiting ● Diabetic ketoacidosis ● Aldosterone deficiency ● Salt losing nephritis Determination: ● Sample: serum, plasma, urine (24 H), and sweat (used for determination of cystic fibrosis-chromosomal disorder in C7, characterized by producing of thick and sticky secretion that is high in electrolyte ) ● Lithium heparin- anticoagulant of choice Method: ● Ion Selective Electrode (ISE) ● Coulometric - amperometric Titration ○ Silver bind to Cl ● Mercuric titration (Schales-Schales) chloridometer ● Digital chloridometer REFERENCE VALUE/RANGE ● Plasma, Serum: 98-107 mmol/L ● Urine (24H): 110 - 250 mmol/Day PHOSPHATE (PO 4 ) ● Predominant ICF anion ● Omnipresent in nature ○ Present everywhere ● Participates in important biochemical processes: ○ Genetic materials - DNA & RNA are complex phosphodiesters ○ Coenzymes - mostly are esters of phosphoric or pyrophosphoric acid. ○ Reservoir of biochemical energy - ATP, creatine PO4, phosphoenolpyruvate ○ Affects 2,3-BPG Regulation:
● ↑Intestine - absorb phosphate from the food
● ↓Kidney - excrete or reabsorb phosphate
● Bone - store phosphate ● Hormones
○ ↓PTH - increase renal excretion of phosphate
○ ↑Vit. D - promote absorption in intestine and reabsorption
in the kidney
○ ↑Growth Hormone - decrease renal excretion of
phosphate through urination
○ ↓Calcitonin - promotes the deposition to the bone
Distribution: ● Bone (80%) ● Soft tissue (20%) ● serum/plasma (1%) Types of PO 4 ● Total PO 4 - 12 mg/dl ● Organic - 8-9 mg/dl ● Inorganic Po 4 - 3-4 mg/dl (measure in lab) HYPOPHOSPHATEMIA ● Increased renal excretion ● Hyperparathyroidism ● Decreased intestinal absorption ● Vit. D deficiency ● Antacid use HYPERPHOSPHATEMIA ● Increased intake ● Increased release of cellular phosphate ● Neonates ( not yet have developed PTH & vit. D metabolism Determination: ● Serum, Lithium heparin, 24 H urine ● Avoid hemolysis ● It shows Circadian rhythm (inconsistent throughout the day) ○ Highest level - late morning ○ Lowest level - evening ● Fiske-Subbarow ○ PO4 + Ammonium molybdate ->colored complex REFERENCE VALUE/RANGE ● Adult: 0.78-1 mmol/L ● Urine: 13-42 mmol/day LACTATE ● It is a by-product of an emergency mechanism that produces a small amount of ATP when oxygen delivery is severely diminished. ● It is a normal product of glucose metabolism (glycolysis). ● The conversion of pyruvate to lactate is activated when a deficiency of oxygen leads to an accumulation of excess NADH. ● Pyruvate is converted to acetyl CoA which enters citric acid cycle (aerobic metabolism) or anaerobic metabolism ○ Aerobic yields 38 mol ATP ○ Anaerobic produce 2 mol ATP ● Excess lactate in blood is an early, sensitive, and quantitative indicator of the severity of oxygen deprivation. Regulation: ● Liver is the major organ for removing lactate by converting lactate back to glucose by a process called gluconeogenesis. Clinical Application: ● Useful for metabolic monitoring in critically ill Pt. ● For indicating severity of illness ● For objectively determining patient prognosis
Renal Loss ● Diuretics ○ thiazides ● Nephritis ● Renal tubular acidosis ○ as tubular excretion of H+ decreases (acidosis), K+ excretion increases to maintain electroneutrality. ● Hyperaldosteronism ● Cushing’s syndrome & mineralocorticoids ● Hypomagnesemia ○ enhance sec. of Aldosterone Cellular Shift ● Alkalosis ○ as the cell promote loss of H+ from within, then K & Na enters the cell to promote electroneutrality ● Insulin overdose Decreased Intake Symptoms of Hypokalemia ● Occurs when K level is <3 mmol/L ● weakness, fatigue & constipation. Muscle weakness/ paralysis Treatment of Hypokalemia ● KCl replacement ● IV replacement Sources: ● Dried fruits, nuts, cereals, banana and orange juice HYPERKALEMIA Causes Decreased Renal Excretion ● Acute/ chronic renal failure ● Hypoaldosteronism ● Addison’s disease (low cortisol level) ● Diuretics Cellular Shift ● Acidosis ○ as H+ moves into the cell, K+ moves out of the cell= electroneutrality ● Muscle or cellular injury ● Chemotherapy ● Leukemia ● hemolysis Increased Intake ● Oral/IV K+ replacement Artifactual ● Hemolysis ● Thrombocytosis ● Fist clenching/ tourniquet Drugs causing Hyperkalemia: ● Captopril - inhibits ACE ● NSAIDS - inhibits aldosterone ● Spironolactone - potassium sparing diuretics ● Digoxin - inhibits Na-K pumps ● Cyclosporine - immunosuppressive drug, that inhibits the renal response to the aldosterone ● Heparin - inhibit aldosterone secretions Symptoms: ● Muscle weakness, tingling, numbness, mental confusion, cardiac arrhythmias, & cardiac arrest. Specimen Consideration: ● Coagulation - promote release of K from the platelet ○ Potassium in serum - 0-0. ● Prolonged tourniquet application ● Ice ● Hemolysis: slight (↑3%) and gross hemolysis (↑30%). Determination: ● Serum, plasma, and 24 hr urine. ○ Anticoagulant of choice for plasma - HEPARIN ● Ion selective electrode (ISE) with Valinomycin Gel REFERENCE VALUE/RANGE ● Serum: 3.5-5 mmol/L ● Plasma: ○ Male: 3.5-4 mmol/L ○ Female: 3.4-4 mmol/L ● Urine (24 H); 25-125 mmol/L BICARBONATE (HCO 3 ) ● Second most abundant anion in ECF ● Total CO2 comprise of : ○ CO 2 ■ potentially toxic ■ Acid ■ Controlled by lungs ○ Bicarbonate ion (HCO 3 ) (it account 90% of CO 2 ) ■ Base for alkaline ■ Controlled by kidneys ○ Carbonic acid H 2 CO 3 ○ Dissolved CO 2 ○ Total CO 2 is indicative of HCO 3 measurement ● It is a major component of Buffering system of the Blood ○ CO 2 + H 20 ←→H 2 CO 3 ←→HCO 3 + H ○ Enzyme that regulate: Carbonic anhydrase ○ Normal value of the blood 7 to 7. ○ Kidney - it control the base and excrete excess HCO 3 ○ Lungs - it control the acid and excrete excess CO 2 ○ Buffer - substances that resist the sudden changes of pH in blood ○ Enzyme: carbonic anhydrase Regulation: ● Kidney: reabsorption of HCO 3 as CO 2 ○ Semipermeable with bicarbonate ○ PCT - reabsorb 85-90% of CO 2 ○ DCT - reabsorb 15% of CO 2 Clinical Application: ● Acid-base imbalances ○ cause changes in HCO 3 and CO 2 levels. ● Metabolic acidosis ○ Indication of kidney problem and lungs will response to it ○ a decreased HCO 3 may occur as HCO 3 combines with H+ to produce CO 2 which is exhaled by the lungs. ○ Lungs aims to eliminate CO 2 (acid) ● Metabolic alkalosis ○ Problem with the kidney ○ elevated total CO 2 concentrations occur as HCO 3 is retained, often with increased pCO 2 as a result of compensation by hypoventilation. ○ Lungs retains CO 2 ■ Typical causes: severe vomiting, hypokalemia, and
excessive alkali intake. Determination for Carbon Dioxide: ● serum or plasma ● LITHIUM HEPARIN as anticoagulant ● Arterial or whole blood could be used ● Specimen should be collected anaerobically ○ Failed to cap the sample will lead to CO 2 will decrease by 6 mmol/L 2 Common Methods are: ● Ion selective electrode (ISE) ○ for measuring total CO2 uses an acid reagent to convert all forms of CO2 to CO2 gas and is measured by a pCO electrode. ● Enzymatic Method ○ It alkalines the sample to convert all forms of CO2 to HCO3. ■ HCO3 is used to carboxylate Phosphoenolpyruvate (PEP) in the presence of PEP carboxylase (enzyme), which catalyzes the formation of oxaloacetate. ■ This is coupled in the ff. reaction, in which NADH is consumed as a result of the action of Malete dehydrogenase (MDH). Phosphoenolpyruvate + HC03 Oxaloacetate + H2PO Oxaloacetate + NADH + H Malate + NAD REFERENCE VALUE/RANGE ● Plasma/Serum: 23-29 mmol/L MAGNESIUM (Mg) ● 4th most abundant cation in the body ● 2nd most abundant cation in the ICF ● An average man that weighs 70 kg approx. 24g of Mg is present Distribution: ● Bone (53%) ● Muscle, other organs and soft tissue (46%) ● Serum & RBC (1%) ○ In Serum, it has 3 forms ■ Bound to albumin (34%) ■ Free/ ionized state (61%) ● Active form of electrolytes ■ Complexed with other ions (5%) Functions: ● Involve in myocardial rhythm and contractility of the heart ● Serves as cofactors in enzyme activity ● Regulation of ATP in ion pump ● Blood coagulation ● Neuromuscular excitability ● Production of ATP from the glucose ● Cofactors for more than 300 enzymes ● Important for glycolysis, transcellular ion transport, neuromuscular transmission, synthesis carbohydrates, Protein, lipids and nucleic acid , and release of and response to certain hormones. Sources: ● Raw nuts, dry cereal, hard drinking water (high mineral content), vegetable, meat, fish ● Deficient: processed food and instant food Regulations: ● Intestine absorbs 20-65% ● Kidneys reabsorbed when there is deficiency and excretes when overload ● Henle’s loop- major renal regulatory site ○ Ascending LH may reabsorbs 50-60% of filtered Mg ○ PCT may reabsorbs 25-30%% of filtered Mg ○ DCT may reabsorbs 2-5% of filtered Mg Hormones ● Parathyroid Hormone ○ Inc renal reabsorption of Mg from the urine, enhance intestinal absorption of Mg ● Aldosterone ○ Increase renal excretion of Mg ● Thyroxine ○ Increase renal excretion of Mg HYPOMAGNESEMIA Reduced Intake ● Poor diet/starvation ● Prolonged magnesium- ● deficient IV therapy ● Chronic Alcoholism Decreased Absorption ● Malabsorption syndrome ● Surgical resection of S. ● Nasogastric suction ● Pancreatitis- LPS binds Mg ● Vomiting ● Diarrhea ● Laxative abuse ● Neonatal - malabsorption ● Congenital- malabsorption Increased Excretion-Renal ● Tubular disorder ● Glomerulonephritis ● pyelonephritis Increased Excretion-Endoc rine ● Hyperparathyroidism & Hypercalcemia (inc. renal excretion as a result of excess Ca+) ● Hyperaldosteronism (inc. renal excretion) Increased Excretion-Drug Induced ● Diuretics ● Antibiotics ● Cyclosporine ● Digitalis Miscellaneous ● Excess lactation ● Pregnancy Drugs Causing loss of Mg through the kidney ● gentamicin, diuretics, cisplatin ○ has a nephrotoxic effect that inhibits the ability of the renal to conserve Mg), cyclosporine, furosemide. ● Furosemide ○ loop diuretics, especially effective in increasing renal loss of Mg2+. ● Thiazide ○ requires a longer period to cause hypomagnesemia. ● Cisplatin- ○ has a nephrotoxicity effect that inhibits the ability of the renal tubule to conserve Mg. ● Cyclosporine ○ an immunosuppressant, severely inhibits the renal tubular reabsorption of Mg and has many adverse effects. ● Cardiac glycosides (Digoxin & Digitalis)
○ Renal Symptoms (nephrolithiasis, nephrocalcinosis). Treatment ● In menopausal women estrogen replacement is combined with Ca+ replacement and salt & water intake to encourage Ca++ excretion. ○ Biphosphanate - main drug class used to lower Ca levels which prevents bone resorption. Determination: ● Total Calcium- serum or Lithium heparin ○ No to: venous stasis & EDTA & Citrate ● Ionized Calcium- prepared sample is heparinized whole blood ○ Anaerobically collected ○ Serum from ETS may be used if clotting & centrifugation are done quickly (<30 mins. ) at room temperature. ○ Urine (24 hrs) - should be acidified with 6 mol/L HCl ○ Collected with accurate time Methods: ● Total Ca++ analysis ● Ortho-Cresolphthalein Complexone (CPC) or Arsenzo III dye to form a complex with Ca. It uses 8-hydroxyquinolone to prevent Mg interference. ● AAS- reference method but rarely used. ● ISE- for ionized Ca++ REFERENCE VALUE/RANGE ● Total Calcium- serum/plasma ○ Child, < 12y/o: 2.20-2/L ○ Adult: 2.15-2 mmol/L ● Ionized Calcium-Serum ○ Child- 1.20-1 mmol/L ○ Adult: 1.16-1 mmol/L ● Ionized Calcium- Plasma ○ Adult: 1.03-1 mmol/L ● Ionized Calcium- Whole Blood ○ Adult: 1.15-1 mmol/L ● Total Calcium – Urine (24H) ○ Adult: 2.50-7 mmol/d
TRACE ELEMENTS
● are consist of metals, except: selenium, the halogens, fluoride and iodine ● 2 types: 1. Essential Trace Elements ○ Deficiency impairs biochemical functions 2. Non-Essential Trace Elements ○ High dose can cause toxicity ○ No deficiency ● In the body these have a very small amount ○ Trace Elements (mg/dL) - Iron, Copper, Zinc ○ Ultra Trace Elements (ug/L) - Selenium, Chromium, Manganese ARSENIC (As) ● Ubiquitous in nature ● Non-essential TE Uses: ● pesticide, poison gasses, ammunition, pigments, semiconductors, and medicinal drugs Health Effect: no known function ● Arsenic Trioxide as treatment to Acute Promyelocytic Leukemia Absorption, Transport, & Excretion: ● Ingestion (predatory fish), water, inhalation Symptoms: ● GI Symptoms (nausea, vomiting [N/V], abdominal pain, and water diarrhea), ● Bone Marrow (pancytopenia, anemia, and basophilic stippling which can also occur in lead exposure) ● Cardiovascular (electrocardiographic changes), ● CNS (encephalopathy and polyneuropathy, ● Renal (renal insufficiency and renal failure), ● Hepatic (hepatitis or inflammation of the liver) Chronic Exposure: ● dermatologic, hepatic, cardiovascular, CNS, malignant change Methods: ● Inductively coupled plasma mass spectrometry (ICP-MS), ● Graphite furnace atomic absorption spectrometry (GFAAS) or ● Hydride generation atomic absorption spectroscopy (HGAAS) common method ● Arsenic Speciation ○ Differentiate the different factors or forms of arsenic to its determination ○ Atomic Emission Spectrophotometer (AES) is performed ○ After this conduct element analysis Sources: ● Natural sources ○ volcanoes & weathering of minerals. ● Anthropogenic sources ○ pollution, cigarette smoking, production of metals, burning of foil, fossil fuels, timber, & agriculture Forms ● Non-toxic-Organic form: ○ Arsenobetaine ○ Arsenocholine ● Toxic-Inorganic Form: ○ Pentavalent ○ Trivalent ○ Methylated form ■ White powder of Arsenic Trioxide odorless and tasteless even in 0.01-0 ■ acute exposure can result to death REFERENCE VALUE/RANGE ● Blood: <23 ug/L ● Urine: < 50 ug/L or <0 ug/day CADMIUM (Cd) ● Soft, buish-white metal, easily cut with knife ● Non-essential TE Uses: ● pigments, batteries, metal plating, & plastic industries Health Effect: no known function ● Protein-Cd Adducts ○ when protein bind, Cadmium causes its denaturation, resulting in a loss of function Absorption, Transport, & Excretion: ● Ingestion (not efficient, 5%), inhalation (effective, 10-50%) ● Cd bound to blood is 70%- use for monitoring for chronic exposure ● Baby are free from Cd ● Almost 90% is excreted in the body Toxicity ● Renal dysfunction ○ very common ○ Can cause proteinuria ● Inhalation of vapor ○ Can cause nasal epithelial damage and also in the lungs ● Bone, immune, blood, nervous system ● Chemical pneumonitis & edema ○ Caused by Fumes ● N/V, abdominal pain ○ By ingestion Methods: ● Graphite furnace atomic absorption spectrometry (GFAAS), ● Inductively coupled plasma mass spectrometry (ICP-MS), ● Inductively coupled plasma atomic emission spectroscopy (ICP-AES) Sources: ● Burning of fossil fuels and incineration of waste REFERENCE VALUE/RANGE ● Urine: < 2 ug/L ○ 24 hr urine - < 3 ug/Day ● Blood: <5 ug/L ○ Special consideration: yellow-colored plastic in particular often contains Cd.
ESSENTIAL ELEMENT FUNCTION DEFICIENCY TOXICITY
CHROMIUM (Cr) ● Derived from the greek word chroma = color ● It makes ruby red and emerald green ● Known carcinogenic agent that may cause lung carcinoma ● 2 valency of chromium is trivalent and hexavalent ● enhances insulin action; for glucose and lipid metabolism ● insulin resistance, ● impaired glucose tolerance (type 2 DM), ● hyperlipidemia ● skin ulcers, renal and hepatic necrosis COBALT (Co) ● Cobalt Hgb synthesis; components of Vit. B (cobalamin) ● anemia, ● growth retardation ● heart failure, ● hypothyroidism COPPER (Cu) ● Cellular respiration; ● collagen synthesis ● menke's kinky hair syndrome ○ Fatal and progressive brain disease ○ Characterized by kinky or stinky hair and retardation of growth, and mental retardation ○ Occur in 3mos or 5yrs of age ● muscle weakness ● interferes with absorption of iron & zinc ● Wilson’s disease ○ Can cause neurologic disorder and liver dysfunction ○ Kayser-Fleischer ring, green copper deposits around the cornea FLUORINE (F) ● prevents dental carries ● dental carries ● Fluorosis ○ Mottled teeth, opaque white due to over deposition of F IODINE (I) ● thyroid hormone synthesis ● goiter, cretinism, myxedema ● Thyrotoxicosis IRON (Fe) ● Oxygen transport, component of Hgb ● IDA ● Hemochromatosis ○ Build up of too much iodine in the body; skin, liver, hearts, and joints. MANGANESE (Mn) ● bone and connective tissue functions ● skeletal defects ● Psychiatric disorders, Parkinson's disease MOLYBDENUM (Mo) ● DNA metabolism ● growth depression, cretinism, goiter ● anemia, thyrotoxicosis SELENIUM (Se) ● Prevents oxidative, damage of lipids ● Anti dandruff shampoo ● keshan disease ○ endemic, highly lethal cardiomyopathy ○ Caused by deficiency of Se and infection with Coxsackie virus ● hair & nail loss, liver failure
● kashinbeck disease ○ Chronic osteochondropathy disease ○ Other possible cause are deficiency in Iodine and grain contaminated with mycotoxin (fungi), water pollution with organic materials and fulvic acid ZINC (Zn) ● Protein synthesis ● acrodermatitis enteropathica (inability to absorb zinc in the intestine), growth retardation, immune deficiency, infertility, delayed wound heling, osteoporosis ● gastrointestinal irritation
CCHM 2 Prelim LEC - Lecture notes
Course: Clinical Chemistry 2 (MDT 3122L)
University: Our Lady of Fatima University
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