Über die Ausscheidung der Koproporphyrin-Isomere I und III bei Erkrankungen der Leber und des Blutes

Abstract

54Gotten u. Doyen: Ausscheidung der Koproporphyrin-Isomere I und HI buffer pH 4.2 add 4.5 mg Na-alginate and heat until the alginate is dissolved. After cooling to room temperature add 50 mg o-tolidine 2HC1. Standard curve for Hgb: lyze freshly obtained erythrocytes with destilled water, centrifuge and determine Hgb-concentration in the supernatant with the cyanide method (1). Prepare dilutions from 1-10 yWg Hgb/m/ with saline and measure the peroxidase activity with the standard method. A straight line should be obtained by plotting max. absorbance over the amount of Hgb (compare fig. 6a). Standard curve for Hgb-Hp in the presence of Na-alginate : Prepare Hpsolution with 8 /ig pure Hp/1 m/ dcst. water (the actual amount of the Hp-preparation depends on its purity). Mix equal volumes of the Hp-solution and a standardized Hgb-solution, containing 8 g Hgb/m/. 1 ml of the mixture contains 4 g Hgb as Hgb-Hp^com-plex. Prepare dilutions 0.5-4 & Hgb/m/ and measure the peroxidase activity as described, but substitute the plain o-tolidinetartrate-buffer with the alginate containing solution. In plotting max. absorbance over the amount of Hgb a curve as shown in figure 6b will be obtained. Unknown urine: Dilute urine 1:500 with saline and use 1 m/in the standard method. If the max. absorbance is at least .1, read Hgbconcentration from standard curve and multiply by 500. If the Hgb-concentration in the urine is too low to permit a 1:500 solution (no measurable reaction), the urine has to be treated with sephadex. Prepare a Sephadex-G-50 column (0.8 X 20 cm) with 1% saline. To 0.5 ra/ urine add 2.5 mg Hp (calculated as pure Hp) and mix well. Put the 5.0 m/ on the Sephadex column, elute with 1% saline and collect 2.0 m/ fractions (approx. 10). Take an appropriate aliquot (up to 1.0 m/) of each fraction and measure the peroxidase activity using the reagent-buffer with Na-alginate. With aliquots less than 1.0 m/ make up the volume difference with saline. For each fraction the amount of Hgb per 2.0m/ fraction is calculated (standard curve c). The sum is the amount of Hgb per 5.0 m/ of urine. For each Sephadex column the percentage of recovery of Hgb-peroxidase is determined. Prepare Hgb-solution with 4 //g Hgb/m/ saline and add an equal volume of a Hp-solution with 4 § pure Hp/m/. From this mixture put 5.0 m/ (= 10 g Hgb as Hp-complex) on column and procede as described for urine. The percent recovery obtained from this determination is used to correct the value obtained for urines with this particular column. The columns can be washed with saline and used repeatedly. The excretion of coproporphyrin in normal health, in liver ailments of various origin, and in a series of blood ailments was studied by extraction, high voltage electrophoresis and paper chromatography, and isomers I and III were determined. In normal persons the urinary level was 42-181 (88.6 ±30.2) ^g/24 hr. (males) and 43-135 (70.8 j-28.1) ^g/24hr. total coproporphyrin. Coproporphyrin III was predominant in healthy men and women (64.8 and 62.2% resp.). In ailm...