UOIT Chemistry 2
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| 2-2s 4-1s 10x 2 of 3-2s 3-1s 12x 7T | Suggests Systemic Error
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| 1-2s 1-3s R4s | Random Error
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| Basic lipid panel: Chol, Trig, HDL, LDL |
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| Statins (example Lipitor): stimulate production of LDL receptors , removes excess LDL in bloodstream |
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| Lipid tests are not reported with routine reference intervals. They are reported with levels indicative to a patient’s risk of CHD |
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| 12h fast required for trigs/LDL (not req for chol/HDL) Cholesterol samples are to be transported at 4oC |
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| Nonfasting lipid panel -measures hdl and tc Fasting lipid panel -Measures HDL, TC and trig -LDL calculated | Checking Lipids
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| -Interferences due to peroxidase step: ascorbic acid, bilirubin, bleach -Reaction with endogenous free glycerol | Enzymatic Triglyceride Assays
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| Abell Kendall; three step method Principle of analysis: Cholesterol extracted with zeolite, esters chemically hydrolyzed (saponification); total cholesterol measured by Liebermann-Burchard reaction | Measurement of Cholesterol
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| Reference Measurement: ultracentrifugation electrophoretic mobility: (-)LDL, VLDL, HDL (+) | Lipoproteins
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| By DENSITY: particles have different sizes and densities (Vary in diameter from 5 -1000nm). increased protein content = higher density increased triglyceride = less dense | Lipoprotein Classifications
chylomicrons, VLDL, LDL, HDL
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| The sample is then examined the following day: -creamy, thick layer at the top of the sample: chylomicrons -uniform turbidity: VLDL -appearance of an orange color: LDL -clear: may be negative or may contain HDL | Standing Plasma Test
Used as a SCREENING test only.Any positive results require additional specific lipid analyses.
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| TC = HDL + LDL + VLDL VLDL = (TG/2.2) | mmol/L
Equation not used when:
when chylomicrons are present
when plasma TG concentration exceeds 4.52 mmol/L
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| QMPLS states the precision goal for cholesterol , HDL and triglyceride is 3.0% |
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| Genetic defect of the HDL receptor that predisposes individuals to high cholesterol levels (exhibit high LDL levels at birth) | Familial Hypercholesterolemia
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| Inherited genetic condition caused by a gene mutation that is passed on in an autosomal dominant fashion | Familial Hypertriglyceridemia
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| Enzyme assays use zero-order kinetics | [S]>[E]
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| Enzymatic assays use 1st-order kinetics | [S]<[E]
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| Enzyme Assay Michaelis Constant (Km) =Substrate concentration at which the reaction rate (velocity) is one-half the maximum velocity | Reflects affinity of Substrate for Enzyme
-if enzyme has small Km, it achieves maximum catalytic efficiency at low substrate concentrations (high Km means ES binding is weak!)
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| -pH -Temperature -Cofactors -Inhibitors | Factors Affecting Enzyme Activity
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| Can be competitive, non-competitive, or uncompetitive | Enzyme Inhibition
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| Liver: ALT, AST, GGT, LD Heart: CK, LD, AST Bone: ALP Muscle: CK Pancreas: AMY, LIP Prostate: ACP | ENZYME ‘PANELS’
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| Isoenzymes: two different proteins from different tissues encoded by different genes that act on the same substrate |
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| Isoforms: enzymes that differ from each other due to post-translational modifications that cause a change to the basic enzyme structure |
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| -Present in ALL tissues of the body -highest concentrations in brain, red cells, kidney, white cells, liver, lung, and myocardium (in that order) -NON-SPECIFIC marker of tissue damage - | Lactate Dehydrogenase (LD)
Catalyzes the interconversion of pyruvate and lactate
reverse reaction: P-->L favored in body
*AMI pattern: LD rises 8-12h after chest pain, peaks at 48-72h, remains elevated for 10-14 days
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| CK is the most sensitive marker for muscle tissue damage Following AMI: rises 4- 6h after chest pain, peaks at 24 – 36h, returns to normal at around 60h | Creatine Kinase (CK)
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| Typical Reference Range: Male: 55-170 U/L Female: 35-135 U/L | CK
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| INCREASE in Abs is measured at 340nm –WHY? NADPH | Creatinine Kinase (CK)
muscle, heart, brain
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| DECREASE in ABS at 340nm is proportional to the enzyme concentration; NADH becoming NAD+ | Aspartase Aminotransferase (AST)
liver and rbc
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| ELEVATED in hepatocellular diseases: Toxic hepatitis, viral hepatitis, alcoholic liver disease, metastatic cancer of liver, hepatic cellular necrosis All causes of tissue damage: right heart failure, hemolytic anemia, pre-eclampsia | alt
liver and skeletal muscle
DECREASE in ABS at 340nm is proportional to the enzyme concentration
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| Both are ↑ in acute hepatocellular damage but ALT is higher than AST (ratio is < 1) Both are ↑ in chronic liver disease (cirrhosis) but AST is much higher than ALT (ratio > 2) Both are ↑ in metastatic liver cancer but AST is higher than ALT (ratio > 2) | In healthy population AST/ALT ratio is (1 – 2)
Both AST and ALT commonly measured to evaluate liver disease.
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| ELEVATED in: Chronic alcoholism* Abuse of antidepressants and anticonvulsants Hepatitis, cirrhosis Acute cholecystitis ↑↑Biliary obstruction (obstructive liver disease) Metastatic liver carcinoma DECREASED in hypothyroidism | Gamma Glutamyltransferase (GGT)
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| p-nitroaniline is yellow. Measure INCREASE in color at 410nm | Gamma Glutamyltransferase (GGT) assay
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| RBC Cholinesterase -measured in amniotic fluid to confirm neural tube defect when AfAfp elevated Cholinesterase -CHE used routinely outside of North America as a non-specific marker for liver disease (decreased) | Cholinesterase (CHE or CHOLIN)
Enzymes that catalyze the hydrolysis of acyl-esters to alcohols and carboxylates
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| -DECREASE seen in pesticide poisoning -Patients w/variant CHE exhibit prolonged apnea and paralysis when receiving anesthetics (succinylcholine or mivacurium) -identified by assaying both total activity and the extent of enzyme inhibition by dibucaine | Cholinesterase (CHE or CHOLIN)
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| -Paget’s disease -Obstructive biliary disease -Pregnancy (16-20wks from placental ALP) Children (rapid bone growth) -Lowered in:Cretinism, dwarfism, hypothyroidism, scurvy, severe malnutrition, gross anemia, hypophosphatasemia | ALP catalyze the hydrolysis of various phosphoesters at an optimum pH of 10
Requires Mg++ as an activator, Zn++
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| p-nitrophenol (yellow) (read at 405nm) ALP increases 3-10% upon standing at RT or at 4oC for several hours Can be 25% higher after a fatty meal | ALP
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| -Catalyzes α-1, 4-glycosidic bonds in starch, glycogen (requires Ca/Cl- cofactors) -mostly in acinar cells of pancreas and in salivary glands -ELEVATED in: Acute pancreatitis,Pancreatic obstruction,Perforated peptic ulcer,Mumps,Ingest opiate analgesics | Amylase
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| AMY is high in acute pancreatitis AMY is normal in appendicitis | STAT
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| Can NOT assay for amylase if patient has been administered opiate analgesics (common in acute pancreatitis) =falsely HIGH results | Amylase
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| Test urine AMY! If NEG then ↑ serum AMY due to macroamylase Contamination with saliva produces false positive results!!! | Amylase
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| -Present primarily in pancreas -ELEVATED in: Acute pancreatitis Penetrating duodenal ulcers Perforated peptic ulcers Intestinal obstruction | Lipase
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| ELEVATED in metastatic or invasive cancer of the prostate (returns to normal w/in 3-4 days after sugery) Paget’s disease (osteitis deformans) Metastatic breast cancer Minor elevations in thrombocytosis, Gaucher’s disease, and metastatic bone cancer | Acid Phosphatase (ACP)
Present in prostate, rbcs, platelets, spleen, liver, osteoclasts, breast
Same reaction principle as ALP but at pH 5.0
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| very little protein and few cells | SF
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| C: Frozen M: RT H: refrigerated |
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| Appearance (Gross examination): performed at the bedside and in the laboratory. Report COLOR and CLARITY: provides 1st diagnostic information Normal Sf is clear and colorless (viscosity like water) | Spinal Fluid – Gross examination
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| -Traumatic tap due to the puncture of a blood vessel during collection -Uneven distribution of blood cerebral hemorrhage: evenly distributed throughout 3 tubes -traumatic tap: heaviest blood in 1st tube, gradual diminishing amts in tubes 2 and 3. |
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| HI SFPRO (>0.60g/L): associated with meningitis Glucose can differentiate between bacterial or viral cause of meningitis LO SFGLU (<3.0mmol/L): bacterial meningitis NORMAL SFGLU: viral meningitis HI SFGLU: patient likely hyperglycemic | Meningitis
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| LACTATE: levels in Sf are unrelated to plasma levels Used mainly to differentiate source of meningitis >2.5 mmol/L: bacterial, tubercular, fungal meningitis More consistent than glucose. WHY?? <2.5 mmol/L: associated with viral meningitis. | Lactate levels fall rapidly if successful treatment
Offers sensitive method to monitor effectiveness of antibiotic treatment
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| Normal synovial fluid is clear to pale yellow and viscous | Synovial Fluid
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| Viscosity: decreased in infection -neutrophils release hyaluronidase which destroys the hyaluronic acid required for joint lubrication String Test performed at the bedside. Non-inflammatory fluids “stringout” from 3-6 cm | Synovial Fluid
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| Transudates have low protein value and form as a result of changes to hydrostatic or colloidal osmotic pressure | Exudates have high protein content and are caused by increased capillary permeability from diseases that involve direct inflammation of the surfaces of the body cavities or by the presence of tumors
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| LD above 0.45 of upper limit of normal (ULN) serum value cholesterol greater than 1.16 mmol/L Total protein greater than 30 g/L if none is present, the fluid is virtually always a transudate | Modern Criteria: If at least one of the following three criteria is present, the fluid is virtually always an exudate
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| pH: differentiates between benign and complicated parapneumonic effusions in the pleural cavity. pH >7.3 (benign) effusion resolves spontaneously pH <7.2 patient requires tube drainage Fibrinogen: transudates don’t clot, exudates clot | Additional Markers for Serous Fluids:
Glucose: decreased in Exudates, normal in Transudates
Amylase: Elevated pleural fluid amylase is seen with pancreatitis and esophageal rupture.
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| Steatorrhea: increased fat in stool |
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| ->2.5ml/150g of blood in stool is pathologically significant but not always visible -mass screening for colorectal cancer Pos FOB = HI predictive value for colorectal cancer | FOB
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| FOBT: detects pseudoperoxidase activity of Hb |
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| Ration of 2:1 (lecithin:sphingomyelin) or greater indicates that the lungs are mature enough for delivery. | Amniotic Fluid
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| NPNs clinically: are closely associated with kidney function are often associated with liver function | Non Protein Nitrogenous (NPN) Substances: compounds that contain nitrogen that aren’t proteins (do NOT contain peptide bonds)
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| Pre-Renal: loss of appropriate blood supply Renal: loss of functional kidney tissue Post-Renal: blockage of urinary tract preventing urine outflow |
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| urea=bun*2.14 | urea ri: 3-7mmol/L
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| Decreased Urea -Advanced liver disease (decreased synthesis by the liver) Low protein diet Pregnancy (due to increased filtration rate) |
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| Fearon Reaction: direct estimation of urea Condensation of urea with diacetyl to form a colored diazine derivative which is measured spectrophotometrically at 540nm. |
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| In the presence of nitroprusside and in an alkaline medium, Ammonium is reacted w/phenol and hypochlorite to produce colored indophenol | Berthelot Method
Sodium nitroprusside acts as a catalyst
Indophenol is a blue chromophore that absorbs at 560nm.Interference from ascorbic acid
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| AMMONIA contamination!! detergents, smoke, urine Na Fluoride inhibits urease and can’t be used Sodium citrate also inhibits urease and can’t be used Ammonium heparin causes false POS and can’t be used | Testing for Urea
Pre-Analytical Factors
Urine samples must be refrigerated to avoid bacterial decomposition of urea to ammonia
Add thymol as a preservative and growth inhibitor
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| all creatinine is excreted in the urine evaluate kidney function | Creatinine
Nitrogenous waste product produced in the liver from slow but spontaneous degradation of creatine phosphate in muscle
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| Male: 50-130 umol/ Female: 45-100 umol/L | Creatinine RI
Unaffected by diet
Increases with age
In “normal”, healthy individuals creatinine levels in the blood are low and constant
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| Creatinine is oxidized by saturated picric acid in an alkaline solution to form creatinine picrate, a red-orange chromogen read at 510-520nm | Methods of Creatinine Analysis
The Jaffe Reaction
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| w/ normal SCr: -HI Urea due to PRE-renal causes such as prerenal azotemia, high protein intake w/ HI SCr: -HI Urea due to POST-renal causes such as an obstruction | Urea/Creatinine Ratio
Due to LO urea associated with decreased urea production (low protein intake, severe liver disease, tubular necrosis)
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| Need a substance that is freely filtered through the glomerulus and not bound to protein not reabsorbed by the tubules is physiologically inert | Clearance Tests
Examples:
INULIN is the best substance for this but must be administered and we don’t have a reliable, easy inulin assay
CREATININE almost as good but is ENDOGENOUS (produced by the body) and we can easily measure it
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| CC=[U*V]/[P*86400] V=volume of 24hrs urine in mL/d for corrected: *[1.73/A] A= patient body surface area in square meters | Creatinine Clearance
Typical Corrected Creatinine Clearance RI:
1.25 – 2.10 mL/s
CrCl <1.00 mL/s indicates loss of about 50% of functional nephron capacity and is classified as moderate kidney disease
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| What is another common use for urine creatinine analysis?? To measure completeness of 24h urine collections! | Adults excrete 1.2-1.5g of creatinine/day
An excretion rate of <0.8g/d is indicative that some of the 24h urine was discarded by the patient!!
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| Relatively insoluble: At pH>5.6 prominent form is the uric acid ion monosodium urate which is water soluble At pH <5.6 exists as uric acid which is not soluble | Uric Acid
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| Overproduction of urate: Gout Lesch-Nyhan syndrome* High protein diet Excess tissue destruction (excessive nucleic acid turnover) due to radiation, chemotherapy Myeloproliferative disorders: LEUKEMIA Severe starvation (cell destruction) | Uric Acid
Na Fluoride and EDTA inhibits uricase!!
DO NOT USE!!!!!!
Hemolyzed samples should be avoided
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| Measurement can be accomplished by: Decreased UV absorbance (282-292nm) as urate is consumed Coupling H2O2 to an indicator system using peroxidase | Uricase Method: enzyme uricase catalyzes oxidation of uric acid to allantoin
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| Plasma Ammonia used to monitor progression of Hepatic COMA Hemolyzed samples may NOT be used | Ammonia RI:10-40 umol/L
Used by liver to produce UREA
At physiological pH, most is ionized as ammonium ion
NH4+ is TOXIC to CNS cells (especially brain)
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| TBIL RI: <17umol/L | Prehepatic: problem occurs before liver due to increased bilirubin synthesis
Hepatic: problem occurs in liver due to hepatocellular or cholestatic disease
Posthepatic: problem occurs after liver due to biliary obstruction
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| ↑TBIL due to ↑ uBIL ↑Unconj bili now excreted into bile = ↑urobilinogen produced = ↑urobilinogen in urine, ↑urobilin (and dark brown stools) NO bilirubin in urine!!! WHY? –not water soluble | pre-hepatic jaundice
Rate of hemolysis exceeds the liver’s ability to conjugate the bilirubin for normal excretion in urine/feces
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| Failure to conjugate bilirubin:inc conc of free unconjugated/albumin-bound bilirubins Fail to transport cBIL into bile canaliculi:increased cBIL back up into blood/urine Fail to re-excrete re-circulated urobilinogen: inc urobilinogen in blood and urine | Hepatic Jaundice
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| cBIL backs up, ruptures bile canaliculi and enters blood ↑ cBIL and POS bilirubin in the urine (cBil is water soluble) Blockage prevents bilirubin entry into intestines ↓ urobilinogen produced, ↓ urobilin produced = white chalky pale looking stools | Post-hepatic Jaundice
↑TBIL, ↑cBIL, and normal uBIL
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| Reacts directly w/ CONJUGATED bilirubin (and ∆ bilirubin) Referred to as DIRECT bilirubin (DBIL) Requires an accelerator to solubilize unconjugated bilirubin in order to have it react with the reagent Unconjugated bilirubin = INDIRECT bilirubin (IBIL) | Diazo method
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| 1)Boiling Point Elevation 2)Freezing Point Depression 3)Vapor Pressure Depression 4)Osmotic Pressure | 4 colligative properties
properties of a solution that are influenced by the number of molecules (solute particles) in the solution but NOT their individual composition
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| Normal osmolality in plasma is 280 - 300 mosm/kg |
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| ICF: K, PO4- ECF: Na, Cl, HCO3- |
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| reference range = 50 – 1400 mosm/kg H2O urine osmo | Urine Osmolality: also used to determine renal concentrating ability (reflects renal function)
Random urine osm >600 mosm/kg suggests normal renal function
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| Calculation based upon substances in plasma that contribute significantly to the osmolality: Calculation for S.I. Units: Calc OSMO = (2 x Na+) + Glucose + Urea | Osmolar gap=measured osmo-calc osmo
A “normal” osmolal gap should be between +/-2 mmol/L (mosm/kg)
ie. Agreement between the measured and calculated values
What is the clinical significance of an elevated osmolar gap???? Volatile materials
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| Osmolal Gap MOST OFTEN used as a screen for Volatiles: Only applicable to FPD (NOT VPD) |
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