Ana María Stella

A simple method for measuring erythrocyte porphobilinogenase, and its use in the diagnosis of acute intermittent porphyria

Abstract 1. l. A simple method for measuring erythrocyte porphobilinogenase activity has been devised. 2. 2. Red blood cells are hemolysed by three freeze-thaws, the hemolysate is diluted 15 times and only 0.5 ml of this preparation is used for determining porphobilinogenase. in an incubation system also containing porphobilinogen, NaCl. MgCl 2 . Tris-HCl buffer pH 8.2; which is incubated aerobically, in the dark. at 45°. for 1 hr with shaking. 3. 3. The enzymic activity is quenched by adding TCA. 4. 4. Porphyrinogens are oxidized, protein precipitated centrifuged off and uroporphyrins formed are determined spectrophotometrically in the supernatant. 5. 5. The enzyme is stable up to 1 month if stored at —20°.

Lead toxicity in cyanobacterial porphyrin metabolism

The effect of Pb2+ on growth, tetrapyrrole photosynthetic pigment content, total free porphyrin, and 5‐aminolevulinate dehydratase (ALA‐D) activity of a cyanobacterium, Microchaete tenera, and its ability to sequester Pb2+ from the culture medium were studied. Pb2+ was assayed by graphite furnace atomic absorption spectrophotometry. M. tenera growth and chlorophyll a content were not affected by 0.5, 1.0, and 6.0 ppm of Pb2+. These treatments doubled the protein content and increased the phycobiliprotein content by four times after 7 days. The ALA‐D activity decreased in all concentrations by 63% at day 7 and by 34% at day 14. As a consequence of ALA‐D inhibition, total free porphyrin also decreased by 64% at day 7 and by 40% at day 14. The highest biomass lead uptake (7454±565 μg Pb2+/g dry weight) was observed at day 3 with 6.0 ppm of Pb2+ in the culture medium. Uptake coefficient was highest (3723±279 μg Pb2+ g−1 dry weight/ppm of applied Pb2+) with 1.0 ppm after 3 days. The increase in protein and antenna pigments on day 7 was probably a response to stress conditions and could explain why the toxic metal did not affect growth. ALA‐D inhibition and high lead biomass content confirm the importance of this enzyme as a biological indicator for stress. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 61–67, 2001

Photodynamic inactivation of red cell uroporphyrinogen decarboxylase by porphyrins

The effects of light and porphyrins on the activity of red cell uroporphyrinogen decarboxylase were studied. Photoinactivation of uroporphyrinogen decarboxylase was dependent on uroporphyrin concentration, irradiation time and temperature. Using 40 W/m2 of UV light intensity, 40-45% decreased activity was produced with 200 microM uroporphyrin I, at 37 degrees C and after 2 hr of illumination. It has been demonstrated that porphyrins photoinactivate uroporphyrinogen decarboxylase and a mechanism for this action in relation to skin lesions is proposed.

Acute intermittent porphyria--another approach to therapy.

Abstract 1. 1. Acute Intermittent Porphyria patients in early or acute attacks were treated with folic acid (30 mg/day) administered orally for not longer than 10 days. 2. 2. Both clinical and biochemical improvement immediately followed folic acid administration. 3. 3. This is a simple and short-term therapy, which can be used either for treatment or prophylaxis.

Delta aminolaevulinate dehydratase: its mechanism of action

Abstract 1. 1. Taking into account available experimental data. a mechanism of action for aminolaevulinate dehydratase is proposed. 2. 2. Besides the formation of a Schiff base between the substrate and the enzyme, the existence of a minimal functional dimer necessary for activity is also considered, as well as certain characteristics of the type of interactions involved in the inter-subunit contact sites and the role of histidine residues in the transport of protons from and to the active center.

Two cases of infantile porphyria cutanea tarda: successful treatment with oral S-adenosyl-L-methionine and low-dose oral chloroquine

In a 7‐year‐old girl and a 12‐year‐old boy, with photosensirivity and hypertrichosis, the diagnosis of familial porphyria cutanea tarda was confirmed by the characteristic pattern of urinary porphyrin excretion, diminished erythrocyte uroporphyrinogen decarboxylase and elevated plasma porphyrin index with emision maxima at 617‐618 nm. The patients were treated with a combination of low‐dose oral chloroquine and oral S‐adcnosyl‐L‐methionine (SAM); in one case alkalinization of urine was also applied. Complete clinical and biochemical recovery was achieved within 3 months. No adverse ophthalmological or other side‐effects were observed. We propose that the treatment of choice should be oral SAM (12 mg/kg/day) for 3 weeks and oral chloroquine (2 × 100 mg weekly) for about 120–150 days or until improvement of clinical and biochemical abnormalities is attained. So far no relapses have occurred. This combined therapy appears to be safe, simple, effective and very convenient for both patients and physicians.

Altered 5-aminolevulinic acid metabolimn leading to pseudoporphyria in hemodialysed patients

Hemodialysed patients with no history of porphyria may present neurological symptoms similar to those seen in acute porphyrias. Porphyria has been associated with an increase in plasma levels of 5-aminolevulinic acid and porphobilinogen. Our aim was to evaluate these parameters and the activities of the enzymes involved in the first steps of heme metabolism in non-porphyric hemodialysed patients. The activities of 5-aminolevulinate dehydratase and deaminase were determined in red blood cells (RBC) from 78 hemodialysed patients, before and after dialysis. Plasma levels of 5-aminolevulinic acid, porphobilinogen and zinc were also measured. These parameters were also measured in 40 volunteers to obtain controls levels. The levels of 5-aminolevulinic acid (0.98 +/- 0.09 microgram/ml) and porphobilinogen (1.32 +/- 0.13 micrograms/ml) were raised in non-porphyric patients prior to hemodialysis (P < 0.001) compared with controls (5-aminolevulinic acid 0.13 +/- 0.02 microgram/ml; porphobilinogen 0.90 +/- 0.09 microgram/ml). After dialysis there was a decrease in both 5-aminolevulinic acid (to 0.61 +/- 0.05 microgram/ml) and porphobilinogen (to 1.10 +/- 0.16 micrograms/ml) although both parameters remained higher than controls (P < 0.001). The activities of both 5-aminolevulinate dehydratase (0.550 +/- 0.095 U/ml RBC), and deaminase (54.13 +/- 9.13 U/ml RBC) were diminished in blood samples of patients before dialysis (P < 0.001) compared to controls (dehydratase 0.975 +/- 0.115 U/ml RBC; deaminase 77.32 +/- 10.00 U/ml RBC). After dialysis 5-aminolevulinate dehydratase activity was partially recovered (to 0.666 +/- 0.100 U/ml RBC) while deaminase returned to normal values (73.45 +/- 9.46 U/ml RBC). The plasma zinc concentration in hemodialysed patients (44 +/- 12 micrograms/100 ml) was significantly lower than controls (105 +/- 30 micrograms/100 ml, P < 0.001). Addition of 22.5 mM zinc to the dehydratase reaction mixture raised the activity of 5-aminolevulinate dehydratase in blood samples of hemodialysed patients taken before and after dialysis. The study reports a partial loss of activity of 5-aminolevulinate dehydratase and deaminase activities in red blood cells from non-porphyric patients undergoing hemodialysis. Since plasma zinc levels were below normal in hemodialysed patients, and the activity of 5-aminolevulinate dehydratase could be restored by the addition of zinc, it is suggested that these abnormalities in heme metabolism may be explained by altered zinc and associated antioxidant status following dialysis.

Pyrrylmethane intermediates in the synthesis of uroporphirigonen III by soybean callus porphobilinogenase

Abstract 1. 1. Further evidence for the formation of pyrrolic intermediates resulting from action of soybean callus porphobilinogenase on porphobilinogen in the absence or presence of ammonia, under anaerobic conditions, is reported. 2. 2. It was found that the activity of porphobilinogenase was affected by the presence of air; the yield of uroporphyrinogens was very low, although consumption of substrate was not greatly modified. These results suggested that some intermediate in the reaction was still being formed, but that this compound must be in the reduced form to be further converted into uroporphyrinogens. It was observed that ammonia probably produced as accumulation of pyrryl intermediates, but that they synthesize uroporphyrinogen III at a slower rate. 3. 3. Attempts were made to detect the formation of an enzyme-substrate complex, but they all failed.

Studies on erythrocyte aminolaevulinate dehydratase I. Its purification and possible therapeutic applications

Abstract 1. 1. A method for purifying human erythrocytes ALA-D, using a mixture of n -butanol and chloroform, which denature hemoglobin, followed by ammonium sulphate fractionation and affinity chromatography yielding a 1600-fold purified enzyme, is described. 2. 2. By oxidation of Sephadex G-25 with NaIO 4 , a polyaldehyde, is obtained which can be covalently bound to the ALA-D; however the immobilized enzyme is inactive, because essential ϵ-amino groups at the active site were involved in the coupling. Similar experiments with another enzyme, Rhodanese, resulted in an active insolubilized preparation. 3. 3. By suspending the carrier-enzyme in buffer, slow solubilization with simultaneous release of protein occurs, indicating that this approach might find important therapeutical applications in the treatment of enzyme deficiencies.

Porphyrin biosynthesis—immobilized enzymes and ligands VIII. Studies on the purification of δ-aminolaevulinate dehydratase from Euglena gracilis

Highly stable selective adsorbents for δ-aminolaevulinate dehydratase (ALA-D) were prepared by attaching the substrate to agarose beads, either directly or through an extension arm. Columns containing these biospecific adsorbents can completely bind the enzyme present in extracts of Euglena gracilis Z strain. Elution is obtained by changing the ionic strength of the buffer. Due to their ease of preparation and high stability these adsorbents may be of general value for purification of ALA-D from different sources.