Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia.

The fluorescent porphyrin in the erythrocytes of patients with lead intoxication or with iron deficiency anemia is zinc protoporphyrin that is bound to globin moieties, probably at heme binding sites.

Solution photochemistry of thymine and uracil.

With dienes as specific triplet quenchers, it has been shown that the photodimerization of thymine in acetonitrile proceeds entirely through the triplet state and the photodimerization of uracil in acetonitrile or in water proceeds in part through the triplet state. The photohydration of uracil probably does not involve the triplet state. Efficiencies of intersystem crossing of thymine and uracil in acetonitrile were determined.

Cholesterol hydroperoxide formation in red cell membranes and photohemolysis in erythropoietic protoporphyria.

3beta-Hydroxy-5alpha-hydroperoxy-Delta(6)-cholestene is produced in protoporphyrin-containing red blood cell ghosts irradiated with approximately 400-nanometer light in the presence of oxygen. Incorporation of this cholesterol photooxidation product into normal red blood cells leads to increased osmotic fragility and eventual hemolysis. These results may be relevant to photohemolysis associated with erythropoietic protoporphyria.

Comparative study of protoporphyrins in erythropoietic protoporphyria and griseofulvin-induced murine protoporphyria. Binding affinities, distribution, and fluorescence spectra in various blood fractions.

Excess erythrocyte protoporphyrins of human congenital erythropoietic protoporphyria and of griseofulvin-induced murine hepatic protoporphyria were found to be associated with hemoglobin and stroma fractions in similar relationships. More than 99.5% of total erythrocyte protoporphyrin was bound to hemoglobin in each case. However, profound differences were found when protoporphyrin concentration was measured in erythrocytes that had been segregated into populations of progressive age on discontinuous density gradients. In erythropoietic protoporphyria, porphyrin content diminished rapidly with age; in murine protoporphyria, the aging erythrocyte populations became progressively more porphyrin rich. In vitro diffusion of protoporphyrin from plasma across the intact erythrocyte membrane was demonstrated. The equimolar binding affinity of protoporphyrin to hemoglobin was shown to be 40 times that of protoporphyrin to serum albumin. This strong affinity provides the driving force for the observed transmembrane diffusion, and explains the high erythrocyte/plasma porphyrin ratio in murine hepatic protoporphyria. The opposite rapid efflux of intra-erythrocytic protoporphyrin into plasma previously shown in uncomplicated erythropoietic protoporphyria occurs despite this strong hemoglobin affinity, implying continuous efficient clearance of protoporphyrin from plasma by the liver. Furthermore, these and other data suggest that a hepatic synthetic source for any significant fraction of the blood protoporphyrin in erythropoietic protoporphyria is highly improbable.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes, both in vivo and in vitro.

In lead intoxication photosensitivity is usually absent, despite concentrations of protoporphyrin in the erythrocytes equal to or greater than in erythropoietic protoporphyria. Profound differences in the distribution of protoporphyrin in aging erythrocytes were demonstrated by age-dependent fractionation of cells on discontinuous density gradients. In erythropoietic protoporphyria the concentration of protoporphyrin declined extremely rapidly with erythrocyte age; the bulk of the protoporphyrin was lost in less than 3 days and the concentration of fluorescent erythrocytes in the gradient paralleled the decline of protoporphyrin. In lead intoxication the protoporphyrin concentration declined only slightly with cell aging and erythrocytes of all ages fluoresced. In the bone marrow from a patient with erythropoietic protoporphyria all reticulocytes, but only occasional late normoblasts, fluoresced, suggesting a single population. Sterile incubation in plasma (pH 7.5) demonstrated rapid diffusion of protoporphyrin from the erythrocytes in erythropoietic protoporphyria, but not in lead intoxication. Plasma protoporphyrin was elevated in erythropoietic protoporphyria, but not in lead intoxication. Estimates of the daily loss of protoporphyrin from erythropoietic tissue in erythropoietic proporphyria suggested an order of magnitude similar to the total blood protoporphyrin. Therefore, it is not necessary to postulate a preponderant extraerythropoietic source to explain the amount of fecal excretion. A significant amount of the diffused protoporphyrin probably reaches the skin with resulting photosensitivity. In contrast, in lead intoxication protoporphyrin remains within the erythrocyte throughout its life span ; there is no diffusion into the plasma and hence no photosensitivity.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. II. Different binding of erythrocyte protoporphyrin to hemoglobin.

Acidic solvents extract the same porphyrin-protoporphyrin-from the erythrocytes of patients with either erythropoietic protoporphyria or lead intoxication. However, extractable protoporphyrin disappears rapidly, both in vivo and in vitro, from erythrocytes in erythropoietic protoporphyria but slowly, if at all, in lead intoxication. Consistent with these observations, fluorescence spectroscopy revealed that the intracellular state of the erythrocyte protoporphyrin is different in the two diseases. Spectrofluorometric measurements coupled with fractionations and biochemical syntheses showed that in erythropoietic protoporphyria the protoporphyrin is bound as the free base to hemoglobin molecules at sites other than the heme binding sites. In lead intoxication the fluorescent porphyrin is also bound to hemoglobin but is present as zinc protoporphyrin. The data suggest that the zinc protoporphyrin is bound at heme binding sites. Acidic extraction solvents remove the chelated zinc, but zinc protoporphyrin may be extracted intact from erythrocytes with acetone, ethanol, or the detergent Ammonyx-LO.

Interaction of rabbit hemopexin with bilirubin.

The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.

Fluorescence methods in the diagnosis and management of diseases of tetrapyrrole metabolism.

Under appropriate conditions fluorescent porphyrins and bilirubin present in blood and other body fluids can be examined spectrofluorometrically without prior extraction. Uses of such direct fluorescence spectroscopy of porphyrins and bilirubin in studies and diagnoses of diseases associated with abnormal or impaired heme synthesis and metabolism are reviewed. The method of "front-face" fluorometry which allows quantitative assays of fluorescent porphyrins and bilirubin in small undiluted blood specimens is described.

Rapid fluorometric assay of bilirubin and bilirubin binding capacity in blood of jaundiced neonates: comparisons with other methods.

The concentrations of total blood bilirubin, albumin-bound bilirubin, and the reserve and total bilirubin binding capacities of 35 neonatal blood samples (28 patients) were determined by automated front-face fluorometry ((hematofluorometer). These values were compared to results of diazo determinations, Sephadex gel filtration, and peroxidase-oxidation methods. Total blood bilirubin level by fluorometry agreed well with the total plasma bilirubin level by diazotization (r = .96, sigma = 1.7 mg/100 ml). Albumin-bound bilirubin concentrations by fluorometry also correlated well with diazo values (r = .95, sigma = 1.9 mg/100 ml) and were slightly lower than the total blood bilirubin concentrations. Values for total bilirubin binding capacity determined by fluorometry agreed well with results obtained for the same specimens by Sephadex gel filtration (n = 28, r = .97, sigma = 1.8 mg/100 ml) and by peroxidase-catalyzed oxidation (n = 25, r = .97, sigma = 1.7 mg/100 ml). The agreement among the results obtained by the three methods indicates a well-defined in vitro end point at which available primary or "tight" binding sites on albumin are saturated with bilirubin. In this clinical experience the coefficient of variation of results with the hematofluorometer was 8.4% for total blood bilirubin and 6.5% for total binding capacity. A comparison of "sick" with "well" infants revealed that the fraction of bilirubin not bound to albumin was significantly different for these two groups. The assays made with the hematofluorometer are quick (10 to 15 minutes) and require only a small quantity (approximately 150 microliters) of blood.

A rapid fluorometric method for determining bilirubin levels and binding in the blood of neonates: comparisons with a diazo method and with 2-(4'-hydroxybenzene)azobenzoic acid dye binding.

A simple, rapid fluorometric method for determining the albumin-bound bilirubin concentration, total blood bilirubin concentration, and the bilirubin reserve-binding capacity of albumin was clinically evaluated using blood specimens from 79 neonates. This study showed that these bilirubin determinations, made by means of the Bell Laboratories hematofluorometer, correlated well with plasma bilirubin levels obtained by a diazotization (Jendrassik-Grof) method. Hematofluorometer reserve-binding capacities correlated very well with 2-(4'-hydroxybenzene)azobenzoic acid (HABA) dye reserve-binding capacities for specimens of artificially jaundiced adult blood. For specimens of neonatal blood the HABA dye reserve capacity was, on the average, higher than the hematofluorometer reserve-binding capacity, particularly for specimens from low-birth-weight babies (less than 2,000 gm). Comparison of HABA reserve capacity and hematofluorometer reserve capacity for high-birth-weight babies (greater than 2,000 gm) gave data very similar to those for adult blood specimens. The specific bilirubin-binding capacity of albumin was found to be greater for infants whose birth weight exceeded 2,000 gm than for the lower birth weight group. The total blood bilirubin concentration obtained by the hematofluorometer is shown to be significantly higher than the concentration of bilirubin bound to albumin, an indication of other important compartments of bilirubin in blood.

Fluorescence studies of protoporphyrin. Transport and clearance.

By means of spectrofluorometry porphyrins can be assayed directly in body fluids without extraction. Rapid diagnosis of various primary and secondary porphyrinemias can be made based upon such fluorometric measurements. Fluorometric methods can also provide information about porphyrin binding sites that is useful in understanding porphyrin transport and clearance. Free erythrocyte protoporphyrin produced in the circulating red cells of lead intoxicated Mallard ducks rapidly diffuses from those cells.