Raimo Tenhunen

Haem arginate: a new stable haem compound

Intravenous administration of haem in acute hepatic porphyrias inhibits the induction of β‐aminolaevulinic acid synthase, reduces the formation of potentially harmful metabolites of porphyrin synthesis and corrects the haem deficiency. Typically, haem therapy has been given in the form of haematin—haem dissolved in alkali. Such haematin solutions are, however, extremely unstable. Thus, the rapid decomposition of this therapeutic agent may have been responsible for the ineffectiveness of treatment in some clinical states and adverse reactions may have been caused by haematin degradation products. There is, therefore, a need for a stable, effective and well‐tolerated haem preparation. We have prepared certain highly soluble haem compounds of which haem arginate has proved to be the most promising. Pure haemin was isolated from HIV and hepatitis B negative human blood. The haem derivatives prepared were screened as substrates for haem oxygenase. Haem arginate and haem lysinate were found to be as good substrates as methaemalbumin. Stock solutions of haem arginate were stable for 2 years at +6 °C. After dilution with sterile isotonic saline the haem arginate infusion was clearly more stable than haematin solutions made in the laboratory or prepared by dissolving commercial lyophilized haematin. The antiporphyrogenic effect of haem arginate (even after storage for two years) in 2‐allyl‐2‐isopropylacet‐amide‐induced experimental porphyria of rats was equal to that of freshly prepared haematin. The acute oral toxicity of haem arginate was low compared with the parenterally administered drug, indicating poor oral bioavailability. The acute toxic effects after high intravenous or intraperitoneal doses were directed to the liver. Conventional haematin caused thrombophlebitic reactions at the site of infusion in all rabbits, but haem arginate did not cause any thrombophlebitis even after repeated infusions. The dose of haem arginate used in clinical trials, 0ṁ2 mg of haem kg−1 min−1 during 15 min (= total dose 3 mg kg−1 diluted in 100 mL isotonic saline), did not cause any clinically significant changes in the numerous haemostatic parameters measured in patients, and ten times higher infusion rates or total doses were needed to cause a decrease in the blood pressure of rats. Haem arginate has proven to have many advantages over conventional haematin solutions.

Purification and characterization of an acid glutathione S-transferase from human lung

An acid glutathione S-transferase (EC: 2.5.1.18) from human lung was purified and characterized. The purification procedure included two isoelectric focusing runs, Sephadex G-100 gel filtration, glutathione-affinity chromatography, and Sephadex G-75 gel filtration. With respect to the properties studied the acid lung transferase differed from human liver transferases alpha-epsilon, but it bore a close resemblance to the other human low pI transferases. Bilirubin affected the kinetics of the lung enzyme markedly differently as compared with transferase p, suggesting possible nonidentity between these enzymes. The acid lung transferase represented about 97% of the total glutathione transferase activity of the lung 100,000 g supernatant used in this work.

Heme arginate treatment for myelodysplastic syndromes

Heme arginate was given to 26 patients with a myelodysplastic syndrome (MDS) as infusions of 2-3 mg/kg body weight weekly for 8-12 weeks. Most of the patients first received a loading dose on four consecutive days. Six of the patients showed improvement in cytopenias during the therapy. In three of the responders severely depressed blood cell counts recovered to normal or close to normal. So far the maximum duration of a response after the cessation of the treatment is 25 months, and the two ongoing responses have lasted for 11 and 12 months, respectively. In two responders of the eight patients with more than 15% ring sideroblasts the number of ring sideroblasts decreased during the treatment but remained unchanged in six non-responders. The responders were characterized by a low or low normal heme synthase activity which increased during the treatment, whereas the non-responders showed a higher mean heme synthase activity which decreased during the treatment. In general, the responders had significantly fewer defects in heme synthetic enzyme activities than the non-responders. FAB type, karyotype or growth pattern in in vitro cultures of hematopoietic progenitors did not predict the response. Apart from one case of mild venous irritation, no other adverse effects were seen. The present study shows that heme arginate induces beneficial effects on cytopenia in some MDS patients and has very few side-effects.

Homozygous variegate porphyria. A severe skin disease of infancy.

A boy exhibited severe bullous skin disease a few days after birth, followed by increased fragility of the exposed skin in spring and summer. Examination at 2 1/2 years of age led to characteristic biochemical findings: increased excretion of fecal porphyrins (coproporphyrin 121 to 131 and protoporphyrin 467 to 576 nmol/g dry weight), and increased erythrocyte protoporphyrin concentration (3643 to 4840 nmol/I). Lymphocyte protoporphyrinogen oxidase activity was very low in the patient (0.4 nmol/mg protein/h) and half‐normal (2.7 and 2.3 nmol/mg protein/h) in the parents, suggesting that the patient had homozygous variegate porphyria. Severe skin symptoms and a high concentration of red cell protoporphyrin concentration in an infant should prompt suspicion of homozygous acute hepatic porphyria.

Fate of haem after parenteral administration of haem arginate to rabbits

Rabbits were injected either intravenously or intramuscularly with [14C]haem arginate and [59Fe]haem arginate (haem 5 mg kg−1). The main part (80%) of AUCINF of labelled haem was associated with the β‐phase, T½ being about 6 h. Only 1% of the haem dose had been taken up by the red blood cells. In contrast, the iron moiety from the haem molecule was effectively utilized. Thirty days post‐injection of [59Fe]haem arginate, 40% of the dose after intravenous injection and 60% after intramuscular injection was circulating with the red cells. Radioactivity was shown to concentrate in the liver, where haem is mainly metabolized and eliminated. An accumulation of haem in the adrenals was also evident. Haem itself did not concentrate in the bone marrow, and a negligible amount of radioactivity was recovered from brain, implying a poor penetration of the blood brain barrier.

Meme biosynthesis in sideroblastic anemia

Abstract The activities of δ-aminolevulinic acid synthase (ALA-S), δ-aminolevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthase (Uro-I-S), uroporphyrinogen decarboxylase (Uro-DC) and heme synthase have been studied in peripheral red blood cells of 9 patients with hereditary sideroblastic anemia (HSA) and 8 patients with refractory idiopathic sideroblastic anemia (RISA). In one family with HSA the ALA-S activity was decreased, but was normalized during therapy by different pyridoxine preparations. In the other family the ALA-S activity was increased. In RISA a decreased ALA-S activity and an increased Uro-I-S activity were constant findings.

Haem arginate as a treatment for myelodysplastic syndromes.

Summary. We report on two patients with a myelodysplastic syndrome in whom the blood cell counts markedly improved during treatment with haem arginate. One patient received haem arginate only, the other haem arginate in combination with low dose androgen. The drug was given as weekly infusions of 2‐3 mg/kg body weight for 8 ‐ 12 weeks. In one patient the percentage of ringed and other abnormal sideroblasts in the bone marrow was clearly reduced as a result of the treatment. In both patients the effect on blood cell counts lasted for several months after the cessation of the haem arginate treatment. Eleven other patients showed no clear response. No adverse effects of the infusions were observed. Further studies on the possible therapeutic role of haem arginate are indicated.

Skin changes in variegate porphyria

SummaryThe skin of 20 patients with variegate porphyria (VP) was studied using light, fluorescent, and electron microscopy. Twelve patients had skin symptoms and markedly increased fecal protoporphyrin excretion. Their sun-exposed skin was characterized by homogeneous PAS-positive thickening and IgG deposition in the vessel walls. The basic ultrastructural change was thickening of the vascular walls caused by reduplication of the basal lamina and perivascular deposition of amorphous material. Qualitatively similar but less prominent histopathological changes occurred in sun-protected skin in some of the patients. Six patients had no skin symptoms but an increased porphyrin excretion. The light microscopical changes were comparable to those in the patients with skin symptoms, but the ultrastructural changes were less severe. No abnormal histopathological changes occurred in two symptomless patients who had low lymphocyte protoporphyrinogen oxidase activity but normal fecal porphyrin excretion. These results show that the primary site of skin damage in VP is the vessel wall, and that histopathological changes of the skin also occur in porphyric patients who have never had skin symptoms. Factors determining the occurrence of skin symptoms in VP are discussed.

Rapid normalization of antipyrine oxidation by heme in variegate porphyria.

Effects of heme on hepatic xenobiotic drug metabolism were investigated in eight subjects with variegate porphyria. A single infusion of heme arginate (3 mg/kg heme) reversed rapidly the prolonged mean elimination half‐life of antipyrine from 27.2 to 12.7 hours (p < 0.001) and increased total clearance from 0.23 to 0.44 ml/min/kg (p < 0.001). Excretion of 6β‐hydroxycortisol and d‐glucaric acid increased significantly during heme infusion. Excretion of urinary porphyrin precursors increased during the antipyrine test but was normalized by heme. It is concluded that in variegate porphyria a partial block in heme biosynthesis results in subnormal capacity of P450‐associated monooxygenases, but this is easily normalized by exogenous heme.

Optical and EPR spectroscopy studies on haem arginate, a new compound used for treatment of porphyria

A protohaem compound, used for treatment of porphyrias, has been studied to elucidate its state of aggregation. EPR and absorption spectroscopy measurements reveal that 38.3 mM protohaem, dissolved in 40% 1,2-propanediol/10% ethanol/water solution, also containing 153 mM arginine, is partly EPR silent. It exists as high molecular weight aggregates and probably also as mu-oxo-dimers. Dilution in the aqueous alcohol solution dissolves the aggregates first to oligomers and dimers, and finally to monomers (Kdiss = 24 X 10(-6)M). When haem is diluted in 0.9% sodium chloride, a fully monomeric state is not reached even at 1 microM concentration. At 3.5 microM concentration, that used for infusion in patients, the haem is still totally aggregated.