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Isotopic studies of the conversion of oxophlorins and their ferrihaems into bile pigments in the rat.

ABSTRACT: 1. Tritiated oxymesoporphyrins and their ferrihaems were tested as possible intermediates in the catabolism of haemoglobin. The tritiated compounds were injected into rats with biliary fistulae and the incorporation of the isotope into bile, bile pigment, urine, faeces, liver, kidney and spleen was measured. 2. alpha-Oxymesoferrihaem was extensively converted into bile pigment and specifically to the expected mesobilirubin. 3. beta-Oxymesoferrihaem was poorly converted into bile pigment and was not converted into mesobiliverdin IXbeta. The latter was independently shown to be excreted rapidly in bile. 4. The free oxyporphyrins were also poor precursors of bile pigment, and alpha-oxymesoporphyrin competed with bilirubin for excretion by the liver. 5. By analogy with the results obtained with alpha-oxymesoferrihaem it is concluded that alpha-oxyprotoferrihaem is an intermediate in the catabolism of haemoglobin, undergoing further oxidation to bile pigment under the catalysis of an enzyme of definite specificity.

SUBMITTER: Kondo T

PROVIDER: S-EPMC1176635 | biostudies-other | 1971 Feb

REPOSITORIES: biostudies-other

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Project description:1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4-8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with (14)C by using 5-amino[4-(14)C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major (14)C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with (59)Fe by using (59)FeCl(3), norethindrone administration resulted in the detection of (59)Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H(2)SO(4). These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.

Project description:1. The half-life of [(15)N]urea was found to be significantly longer than that of [(14)C]urea injected at the same time, the differences being due to endogenous catabolism of urea, which is accompanied by little or no reutilization of (14)C but is approx. 20% for (15)N. [(15)N]Urea therefore appears to be valueless as an indicator of nitrogen metabolism unless the extents of endogenous catabolism of urea and of fractional reutilization of (15)N can be separately estimated. 2. Though measurements of the radioactivity of expired (14)CO(2) confirmed the existence of considerable urea catabolism these could not be used for quantitative assessments. 3. Alternative graphical methods based on [(14)C]urea specific activities in plasma and urine samples were used to calculate the fraction of urea production that is excreted. Values by the two methods were in good agreement and showed that some animals excrete less than half the urea that they produce. 4. Specific activity differences between simultaneous samples of urinary and plasma urea reflect the presence of a pool of urea in the kidney that is not in equilibrium with the body urea pool. Calculations indicate the presence of urea in the kidney that in some cases may represent as much as 15% of the body pool, and in two animals in which post-mortem renal analyses were performed the masses of urea found agreed closely with the calculated values. 5. A model for urea metabolism is proposed that includes this pool in the excretory pathway. The related theory is shown to be adequate to explain the shape of the specific activity curves of urinary urea from the time of injection and the constant delay of the specific activity of urinary urea, relative to that of plasma urea, that is observed after a short preliminary equilibration period. 6. The body urea pool was calculated from the activity retained at 1.5hr. by excluding renal activity and the corrected specific activity of plasma urea at the same time. The urea pool was calculated to be distributed at the plasma concentration in a substantially smaller water volume than that found by injecting tritiated water in five animals. Reasons for this are discussed. 7. Urea synthesis rates calculated from the pool values are in close agreement with rates calculated from the mass of urea recovered in the urine and the fraction of newly synthesized urea that is excreted.

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Isotopic studies of the conversion of oxophlorins and their ferrihaems into bile pigments in the rat.

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