Physiological roles of animal succinate thiokinases Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.     

Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.     

Supraphysiological concentrations of 5-aminolevulinic acid dimerize in solution to produce superoxide radical anions via a protonated dihydropyrazine intermediate

5-aminolevulinic acid (ALA) is the committed biological precursor to porphyrins. At supraphysiological concentrations ALA can dimerize to form 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY), which transfers electrons to XTT in a reaction that does not require metal ions and is specifically inhibited by superoxide dismutase. The formation of DHPY from ALA follows dimerization kinetics with a pK of 7.8+/-0.1. At pH 11.2, DHPY is relatively stable, but when the pH is dropped to 6.0 rapid conversion to 2,5-(beta-carboxyethyl)pyrazine occurs via an intermediate with an absorption maximum of 370 nm. Formation of this intermediate is pH-dependent with a pK of 6.0+/-0.1. These data indicate that ALA dimerizes to produce superoxide from a protonated form of DHPY. The significance of these results with respect to the concentrations of ALA used in photodynamic therapy, and the increased incidence of liver cancer in acute intermittent porphyria, is discussed.     

Enzymes of heme biosynthesis

 Heme is a necessary component in a variety of oxygen-binding proteins and electron-transfer proteins, and as such it occupies a central role in cellular and organismal metabolism. With only rare exceptions, organisms that utilize heme possess the entire biosynthetic pathway to produce this tetrapyrrole compound. The enzymes involved catalyze a variety of interesting reactions and utilize both common and unique cofactors and metals. Aminolevulinate dehydratase from all organisms and ferrochelatase from higher animals are both metalloenzymes, while 5-aminolevulinate synthase contains pyridoxal phosphate, and porphobilinogen deaminase possesses a unique dipyrrole cofactor. Two pathway enzymes catalyze multiple decarboxylations and yet have no cofactors, and one enzyme catalyzes a six-electron oxidation with a single FAD. To add additional scientific interest there exist biochemically and clinically distinct human genetic diseases for every step in this pathway. Received: 12 March 1997 / Accepted: 8 May 1997     

Absolute requirement for iron in the development of chemically induced uroporphyria in mice treated with 3-methylcholanthrene and 5-aminolevulinate

Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(−/−)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(−/−) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.     

Interaction of porphyrins with human organic anion transporting polypeptide 1B1

The existence of a porphyrin uptake transporter in hepatocytes has been hypothesized in recent years, but to date it has not been identified. While the linear tetrapyrrole bilirubin has been shown to be a substrate for the organic anion transporting polypeptide 1B1 (OATP1B1), similar studies have not been conducted for the cyclic tetrapyrroles (porphyrins). The aim of this study was to determine the structural features of linear and cyclic tetrapyroles necessary for interaction with OATP1B1. The interaction was quantified using HEK cells stably expressing OATP1B1 and measuring the inhibition of OATP1B1-mediated uptake of estradiol 17β-d-glucuronide in the presence or absence of various linear and cyclic tetrapyrroles. Ditaurine-conjugated bilirubin was the most potent inhibitor of uptake, with an IC50 of 5 nM, while the substitution of the taurine side chains with methyl ester eliminated the inhibition of estradiol 17β-d-glucuronide uptake. Hematoporphyrin, a cyclic tetrapyrrole with carboxyalcohol side chains at positions C-3 and C-8 and carboxyethyl side chains at positions 13 and 17 had an IC50 of 60 nM, while porphyrins lacking charged side chains such as etioporphyrin I and phthalocyanine did not inhibit OATP1B1. Chlorin e6 and hematoporphyrin were shown to be competitive inhibitors of OATP1B1-mediated uptake of bromosulfophthalein with Kis of 5.8 ± 0.3 and 1.6 ± 0.3 μM, respectively. While these studies do not provide direct evidence, they do support the assumption that tetrapyrroles are transported by OATP1B1. Additionally, these findings offer a possible explanation for the clinical observation that patients suffering from certain porphyrietic diseases have a reduced ability to excrete organic anions.     

Uroporphyria and hepatic carcinogenesis induced by polychlorinated biphenyls-iron interaction: absence in the Cyp1a2(-/-) knockout mouse

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.     

Detection of DNA variations in the polymorphic hydroxymethylbilane synthase gene by high-resolution melting analysis

Acute intermittent porphyria (AIP) represents the most frequent type of acute porphyria. The underlying cause is a defect in the hydroxymethylbilane synthase (HMBS) gene. Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. We established the diagnostic tool, high-resolution melting (HRM), for diagnosing AIP. Of 13 amplicons amplified by PCR in the presence of the LCGreen Plus dye, 4 showed polymorphic backgrounds. The ability of the HRM method to detect DNA variations in the HMBS gene was tested on a DNA sample with 10 known mutations by a curve shape scan using the LightScanner instrument. Furthermore, genomic DNA (gDNA) samples from 97 individuals with suspected hepatic porphyria were tested. All possible genotypes from each of four polymorphic amplicons were detected. Each of the 10 mutations tested had an altered melting profile compared with the melting profile of the controls. Screening the group of subjects with suspected hepatic porphyria revealed nine different DNA variations, four of which were novel. In conclusion, HRM is a fast, cost-effective prescreening method for detecting DNA variations in the HMBS gene. Therefore, the screening can be easily applied to a porphyria family if misdiagnosis or rare dual porphyria is suspected.     

Major and trace elements in whole blood of phlebotomized patients with porphyria cutanea tarda

Porphyria cutanea tarda (PCT) is a disorder of hem biosynthesis resulting from a decreased activity of the uroporphyrinogen decarboxylase enzyme. Hem precursors are accumulated in the blood, liver and skin. Inherited and acquired factors also contribute to the pathogenesis of PCT. Hem precursors and porphyrins are excreted with urine and faeces. Whole blood of 8 PCT patients and 6 volunteers of Caucasian origin were analysed. In addition to routine laboratory measurements, 19 elements (Al, B, Ba, Ca, Cd, Co, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, S, V, Zn) were determined by means of inductively coupled plasma optical emission spectrometry (ICP-OES). Mg, P and S concentrations in whole blood were decreased significantly (p<0.05), whereas Ba was increased in PCT patients compared to controls. Metabolic alterations are reflected in the correlation of parameters. Positive correlations were found between the element pairs of Zn-Al, Zn-Mg, Zn-Mn, B-S, Fe-Mg, K-P, Mg-Mn for PCT patients, whereas in the control group Al-Mn, Ca-Cu, Ca-Na, Cu-Mg, Fe-K, Mg-Na, Zn-P showed positive correlations.     

Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyria-independent

Hexachlorobenzene (HCB) alters phospholipid and heme metabolisms in the liver and Harderian gland. The effects of HCB on phospholipid metabolism, in an organ considered to be non-responsive to its porphyrinogenic effects, remain to be studied. Therefore, as the brain is an organ with this feature, this paper analyzes the effects of HCB on brain phospholipid composition in order to investigate if there is any relationship between HCB-induced porphyrin metabolism disruption and phospholipid alterations. For this purpose, a time-course study of HCB effects on brain phospholipids was performed in two strains of rats differing in their susceptibility to acquire hepatic porphyria: Chbb THOM (low); and Wistar (high). This paper shows for the first time that rat brain phospholipids are affected by HCB exposure. Comparative studies show that HCB-induced disturbances in brain phospholipid patterns are time and strain-dependent. Thus, whereas major phospholipids, phosphatidylcholine and phosphatidylethanolamine were more altered in Wistar rats, minor phospholipids, phosphatidylinositol and phosphatidylserine were more affected in Chbb THOM rats. HCB intoxication led to a sphingomyelin/phosphatidylcholine molar ratio lower than the normal, in both strains. As was expected, brain porphyrin content was not altered by HCB intoxication in either strain. It can be concluded that HCB is able to alter brain phospholipid metabolism in a strain-dependent fashion, and in the absence of alterations in brain heme metabolism. In addition, HCB-induced disturbances in brain phospholipids were not related to the degree of hepatic porphyria achieved by the rats.