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Hemoglobin and Porphyrins Objectives

Increase in ala porphobilinogen or both causes

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increase in ALA, porphobilinogen, or both causes neuropsychiatric symptoms including abdominal pain, vomiting constipation, tachycardia, hypertension, psychiatric symptoms, fever, leukocytosis, and paresthesia 2 most common screening tests for urinary PBG are the Watson-Schwartz and the Hoesch tests
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Hemoglobin and Porphyrins – Objectives PROTOPORPHYRIN--- =PROTO UROPORPHYRIN--- =URO COPROPORPHYRIN--- =COPRO 2. Explain the difference between an acquired and inherited porphyria—also list what tests could be performed to differentiate between these two conditions. An acquired porphyria is a disorder that are not the result of an inherited biochemical defect in heme synthesis but a result of another disorder, toxin, or drug interfering with heme synthesis. Secondary porphyrias can be distinguished from true porphyrias by measuring levels of urinary ALA and PBG. In secondary porphyrias ALA levels are increased in the urine whereas PBG excretion usually remains normal. 3. Explain the basic principles for the following methods for measuring porphyrins: Watson-Schwaltz, Hoesch, Fluorometric measurements for porphyrins (LAB STUDENTS ONLY). Watson-Schwaltz--- screening test for PBG; PBG forms a red-orange color when mixed w/ Ehrlich’s reagent; an extraction w/ chloroform or butanol is performed to differentiate PBG from interfering substances such as urobilinogen or indole; if a cherry red color remains in the aqueous phase after the addition of chloroform or butanol, this indicates a positive test for PBG. Hoesch--- screening test for PBG; the reagent in this test does not react w/ urobilinogen therefore it is sometimes used to confirm the results of the Watson-Schwaltz test; porphobilinogen and ALA are determined quantitatively by successively separation in 2 ion-exchange columns; an aliquot of urine is loaded on an anion-exchange or alumina column that retains PBG while ALA passes thru; following washes to remove interfering substances, PBG is eluded w/ acetic acid and measured spectrophotometrically using Ehrlich’s reagent; the eluate from the 1 st column is loaded on a cation-exchange column to retain ALA and then eluted w/ sodium acetate; the eluted ALA is reacted w/ Ehrlich’s reagent after 1 st being condensed w/ acetylacetone to form a pyrrole and then measured spectrophotometrically Fluorometric measurements--- a quantitative procedure; based on enhanced fluorescence of porphyrins when in acidic solution; usually at an excitation wavelength of 400-405 nm and an emission wavelength of 594-598 nm; standard solutions of coproporphyrin, uroporphyrin, and possibly, protoporphyrin are used to calculate the porphyrin concentrations in the smaples; each porphyrin exhibits different different excitation and emission wavelengths (solvent dependent) and some procedures used average wavelengths while other procedures include multiple measurements and calculation of levels of each porphyrin
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increase in ALA porphobilinogen or both causes...

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