A rapid, reproducible method for coating Rotorod Sampler collector rods with silicone grease.

BACKGROUND: The Rotorod Sampler (Sampling Technologies, St. Louis Park, MN) is a rotating-arm impactor that recovers airborne particles on two rapidly moving plastic collector rods. For decades, the standard method for applying silicone grease to collector rods has been with one's finger. Although this method can yield excellent results when performed by practiced investigators, a relatively high skill level is required, and significant intrapreparer variability has been reported in the medical and technical literature. OBJECTIVE: The purpose of this investigation was to develop and evaluate a new method for coating Rotorod collector rods with silicone grease. METHODS: Collector rods were coated with silicone grease by dipping them into a solution consisting of silicone grease and hexane. Pollen recovery by these dipped collector rods was compared with pollen counts obtained with hand-greased collector rods. RESULTS: Twenty-three paired samples were obtained during five sampling periods. Pollen recovery by the hand-greased and dipped collector rods was similar (P = 0.410). Dipped collector rods generally offered a lower standard deviation than hand-greased collector rods, however, differences were not statistically significant (F = 1.782, P = 0.087). Dipped collector rods were also superior to hand-greased collector rods in several qualitative categories such as grease uniformity, time required for microscopic analysis, and visual quality. CONCLUSIONS: Dipped collector rods offered a time-efficient means to obtain atmospheric samples with excellent visual quality. The resulting pollen counts were similar to data obtained via the standard, manual method. Allergists are encouraged to consider using this new method in their office practices and for drug studies.

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.

Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria.

Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase.

Comparison of bile porphyrin concentrations in cattle and human beings with protoporphyria.

Blood and bile porphyrin concentrations were measured in cattle with protoporphyria and compared with those in human beings with the disease. Whereas the mean RBC porphyrin concentration in cattle was 18-fold greater than in human beings, the mean bile porphyrin concentration was only 78% greater. Sequential measurements over a 30-hour period in 1 animal with a bile fistula indicated that the ratio of total porphyrin to total bile acid in bile varied minimally. When the animal was given an IV infusion of taurocholate, the biliary excretion rate of porphyrin increased in parallel with that of bile acid, because of enhancement of bile flow. Thus, in cattle with protophorphyria, the concentration of porphyrin in bile is low compared with that of porphyrin in RBC, in contrast with findings in human beings, and adequate amounts of bile acids are secreted to maintain efficient protoporphyrin excretion. This explains, in part, why hepatobiliary disease has not been observed in cattle with protoporphyria, but has been seen in human beings with the disease.

Porphyria and porphyrin metabolism.

Porphyrins, their reduced congeners (porphyrinogens), and their precursors are accumulated and excreted in excessive amounts in the porphyrias because of defects in the enzymes of heme biosynthesis. The nature of these defects is being defined using biochemical and molecular biological techniques. The principal clinical manifestations in the porphyrias, photocutaneous lesions and neurological dysfunction, are linked to the biochemical abnormalities, and appropriate therapeutic interventions have accordingly been developed. The exogenous administration of metalloporphyrins and porphyrin derivatives, unlike the harmful effects of porphyrins in the porphyrias, may be of use in some clinical conditions, such as the treatment of hyperbilirubinemic states and the detection and therapy of certain cancers.

Modulation of hepatic ferrochelatase activity by dietary manipulation of mitochondrial phospholipid fatty acyl groups.

Ferrochelatase is an enzyme bound to the inner mitochondrial membrane, which is important in heme biosynthesis. Activity of purified ferrochelatase is affected by the presence of certain fatty acids. In the present study, we examined whether the activity of ferrochelatase is altered by dietary manipulation of the composition of mitochondrial membrane phospholipid fatty acyl groups. Rats were fed diets containing triolein, safflower or menhaden oil as 5% (w/w) of the diet. After 3 weeks, the animals were killed and liver mitochondria were isolated. Phospholipid fatty acid composition and ferrochelatase activity were assayed in the isolated mitochondria. Marked differences were seen. The proportion of oleic acid was highest in the triolein oil-fed group, that of linoleic and arachidonic acid was highest in the safflower oil-fed group and the proportion of eicosapentaenoic acid was highest in the menhaden oil-fed group. Ferrochelatase activity was greatest in the triolein oil-fed group and lowest in the menhaden oil-fed group regardless of whether the mitochondria were intact, sonicated or sonicated and treated with Tween 20. Mixing of mitochondria from menhaden oil-fed rats with triolein oil resulted in a significant increase in ferrochelatase activity. Membrane fluidity and activities of the mitochondrial membrane enzymes succinic dehydrogenase and cytochrome oxidase did not differ among the groups. We conclude that dietary manipulation of mitochondrial membrane phospholipid fatty acyl group composition can directly modulate hepatic ferrochelatase activity. This has potential application in the treatment of protoporphyria, the genetic disorder in which ferrochelatase activity is deficient.

Hepatic and bile porphyrins in patients with protoporphyria and liver failure.

The livers of patients who have protoporphyria and hepatic failure contain large amounts of pigment crystals. Two such patients underwent liver transplantation, providing the opportunity to identify the pigment crystals. Portions of liver were digested enzymically, sedimented through a sucrose gradient, treated with 1% sodium dodecylsulfate, and centrifuged to purify the crystals. Spectrophotometric and high-performance liquid chromatography analysis demonstrated them to be composed of protoporphyrin. Bile samples were obtained from the 2 patients, 4 other patients who did not have liver disease, and 10 control subjects. The porphyrin concentrations in bile from the 6 patients were significantly increased above controls (range 254-7884 micrograms/dl compared with 11-109 micrograms/dl). The ratio of protoporphyrin to bile acid in bile distinguished the 2 patients with advanced liver disease (3105 and 2756 micrograms/mmol) from the 4 patients without liver disease (range 61-926 micrograms/mmol). Thus, analysis of bile from patients with protoporphyria may help in evaluating their hepatobiliary status.

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase.

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.

Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of 5-aminolevulinic acid and heme from 4,5-dioxovalerate in the rat.

We previously demonstrated an alternate pathway for the biosynthesis of 5-aminolevulinic acid (ALA) in bovine liver mitochondria and of tetrapyrroles in suspensions of rat hepatocytes (1980. J. Biol. Chem. 255: 3742; 1981. Proc. Natl. Acad. Sci. USA. 78: 5335). This pathway involves a transamination reaction that incorporates the intact 5-carbon skeleton of 4,5-dioxovaleric acid (DOVA) into ALA. We investigated this alternate pathway in vivo by the intraperitoneal injection of DOVA into rats. Incorporation of DOVA and [5-14C]DOVA into urinary ALA and hepatic and erythroid heme was quantified and compared with the incorporation of [4-14C]ALA and [2-14C]glycine into heme. Within 3 h of injection of 175 mumol of DOVA, urinary ALA excretion increased 2.4-fold over controls. After injection of [5-14C]DOVA, 0.11% of the radioactivity was recovered as urinary ALA, which quantitatively accounted for the 2.4-fold increase in ALA excretion. After the injection 175 mumol of [5-14C]DOVA, 0.14% of the radioactivity was recovered after 3 h as hepatic heme. The injection of 1.75 mmol of [2-14C]glycine or 175 mumol of [4-14C]ALA resulted in recovery of 0.2 and 3.4%, respectively, of the radioactivity as hepatic heme after 3 h. These doses of radiolabeled DOVA, glycine, and ALA were injected into rats with phenylhydrazine-induced anemia. Recovery of radioactivity after 3 h as splenic (erythroid) heme was 0.35% for DOVA, 0.072% for glycine, and 0.25% for ALA. These studies establish that the intact 5-carbon skeleton of DOVA can be incorporated into ALA and heme in vivo.

Uroporphyrinogen decarboxylase. A method for measuring enzyme activity.

A method for measuring the activity of uroporphyrinogen decarboxylase from a variety of sources is described. Porphyrinogen substrates are generated by enzymic synthesis or chemical reduction. Substrates and enzyme are incubated under standardized conditions. The reactions are then stopped and the reaction products, as well as unmetabolized substrate, are absorbed on tale, eluted and esterified. Porphyrin esters are then separated and quantified using high performance liquid chromatography.

The role of ferrichrome reductase in iron metabolism of Ustilago sphaerogena.

Ferrichrome, the ferric ionophore for Ustilago sphaerogena, can serve as a source of iron for the enzyme ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) in this organism, but only after enzymatic removal of the iron from its carrier. U. sphaerogena contains a specific ferrichrome reductase (NADH:ferrichrome oxidoreductase) which catalyzes cellular dissociation of the complex by reduction of the metal to the ferrous state. A spectrophotometric assay was developed based on trapping of the ferrous ion produced by ferrozine. There is an apparent inhibition by oxygen which is thought to be due to re-oxidation of the metal under the assay conditions. The close structural analogue, ferrichrome A, is not a substrate, nor is the ester type siderochrome ferric hexahydro-N,N',N"-triacetylfusarinine C. Aluminum desferriferrichrome is inhibitory. The importance of this enzyme for the metabolism of iron in this organism is discussed.