The role of ceruloplasmin in iron metabolism.

The importance of ceruloplasmin in iron metabolism was studied in swine made hypoceruloplasminemic by copper deprivation. When the plasma ceruloplasmin level fell below 1% of normal, cell-to-plasma iron flow became sufficiently impaired to cause hypoferremia, even though total body iron stores were normal. When ceruloplasmin was administered to such animals, plasma iron increased immediately and continued to rise at a rate proportional to the logarithm of the ceruloplasmin dose. The administration of inorganic copper induced increases in plasma iron only after ceruloplasmin appeared in the circulation. Thus, ceruloplasmin appeared to be essential to the normal movement of iron from cells to plasma. Studies designed to define the mechanism of action of ceruloplasmin were based on the in vitro observation that ceruloplasmin behaves as an enzyme (ferroxidase) that catalyzes oxidation of ferrous iron. Retention of injected ferrous iron in the plasma of ceruloplasmin-deficient swine was significantly less than that of ferric iron, reflecting impaired transferrin iron binding. Rat ceruloplasmin, which has little ferroxidase activity, was much less effective than porcine or human ceruloplasmin in inducing increases in plasma iron. These observations suggest that ceruloplasmin acts by virtue of its ferroxidase activity. Eight patients with Wilson's disease were evaluated in order to investigate iron metabolism in a disorder characterized by reduced ceruloplasmin levels. Evidence of iron deficiency was found in six of these, and in five of the six, plasma ceruloplasmin was less than 5% of normal. In comparison, the two patients without evidence of iron deficiency had ceruloplasmin levels of 11 and 18% of normal. It is suggested that iron deficiency tends to occur in those patients with Wilson's disease who have the severest degrees of hypoceruloplasminemia, possibly because of defective transfer of iron from intestinal mucosal cells to plasma.

A comparison of the antifertility effects of alkylating agents and vinca alkaloids in male rats.

1 The anti-fertility effects of cyclophosphamide, nitrogen mustard, vincristine and vinblastine were studied and compared in male rats. 2 The effects of the drugs on body weight and haematological values were used to monitor the pharmacological actions of the drugs. 3 All four drugs impaired fertility, the severity of the impairment depending on dose and duration of treatment. 4 Testicular size and histological appearances remained mostly normal, even in infertile animals, but seminiferous tubules were fewer in number and maturation arrest at the spermatid level was evident in some sections. 5 Recovery of drug-induced infertility occurred in 64% of treated animals, 9 to 40 weeks after cessation of treatment. 6 Morbidity and mortality were much higher with alkylating agents than with vinca alkaloids for approximately similar degrees of impairment in fertility.

Toxicodynamics of tumour promoters of mouse skin. III. Specific binding of the tumour promoter thapsigargin as measured by the cold-acetone filter assay.

A method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant, Kd, and the maximal amount of binding Bmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such a Kd for thapsigargin is similar to (a) its IC50 value for inhibiting Ca2+ uptake in the microsomal fraction from rat brain and (b) its EC50 values for inducing a rise in the cytoplasmic Ca2+ concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12-O-tetradecanoylphorbol 13-acetate.

Contamination of the environment by the current disposal methods of mercury-containing lamps in the state of Minas Gerais, Brazil.

This survey describes the degree of environmental contamination resulting from the current disposal methods of mercury-containing lamps. The territory studied for this purpose is the federal state of Minas Gerais, one of the most populated areas in the Brazilian federation of states. The results of this survey derive in part from answers received to a questionnaire mailed out to industrial firms, commercial business enterprises, hospitals and departments of public work. The sampling technique used was a nonprobabilistic (purposive sampling). Three types of disposal were found to prevail: (1) as an addition to corporate waste disposal (garbage), (2) recycling and (3) disposal by other methods. Overall, our study shows that the majority of mercury lamps are being disposed in the metropolitan area of Belo Horizonte, primarily by the public sector, followed by industrial, commercial and hospital sectors. Although recycling constitutes a relatively high fraction of the disposal methods, we find Federal regulations in Brazil regarding the disposal of hazardous, mercury-containing lamps to be far behind the state of technological achievement. This gap has permitted the adoption of disposal measures that are obsolete, incorrect, and a primary cause for the extensive contamination of the ecosystems with harmful effects to human health.

Iron overload associated with congenital pyruvate kinase deficiency and high dose ascorbic acid ingestion.

Iron overload is an uncommon complication of untransfused chronic hemolytic anemias. This paper describes only the fourth reported case of erythrocyte pyruvate kinase deficiency and iron overload. Important aspects of this case are the presence of the HLA genotype commonly associated with genetic (idiopathic) hemochromatosis and a history of ingestion of large doses of ascorbic acid. Their roles in the development of iron loading and toxicity are discussed. A beneficial response to treatment with desferrioxamine was observed before significant iron removal. A mechanism for this action of desferrioxamine is proposed.

Toxicokinetics of tumour promoters of mouse skin. II. Metabolism of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin and biological activities of metabolites.

The metabolism of the phorbol diester [20-3H]12-O-tetradecanoylphorbol-13-acetate [( 3H]TPA) was studied in the back skin of NMRI mice after topical administration of a single tumour-promoting dose, dp. Up to 72 h after administration most of the radioactivity recovered from the skin surface, and from the epidermis and dermis of the treated skin area was unchanged TPA, as determined by silica gel HPLC of extracts obtained from these skin fractions. The major TPA metabolite was less polar than TPA and chromatographed in the range of long-chain TPA-20-acylates. At 72 h, it accounted for about 25, 5 and 30% of total radio-activity extracted from skin surface, epidermis and dermis respectively. Of metabolites more polar than TPA, phorbol-13-acetate (PA) by far predominated over 12-O-tetradecanoylphorbol (TP) in both the epidermis and dermis, and by 72 h its relative amount was 3.9 and 2.4% in these skin fractions. Both phorbol monoesters, PA and TP, were not detected in skin surface extracts. In addition to metabolites, various autoxidation products of TPA were present in small amounts in the extracts from each of the skin fractions. The TPA-20-acylate fraction of metabolites isolated from the extracts of skin fractions at 24 h was separated further into the individual metabolites by the combined use of argentation and reversed-phase HPLC. These individual metabolites were identified by co-chromatography with authentic reference compounds. They were TPA-20-acylates carrying saturated fatty acids (16-26 carbon atoms), cis-mono-unsaturated fatty acids (16-24 carbon atoms), linoleic acid and arachidonic acid. The conjugation of TPA with long-chain fatty acids is the first example of a new route of xenobiotic metabolism in skin. When tested for irritant activity on the mouse ear, TPA-20-acylates were about one to two orders of magnitude less active than TPA. Similarly, when TPA-20-tetradecanoate was tested for tumour-promoting activity on the back skin of NMRI mice over a dose range of dp = 50-200 nmol applied twice weekly according to the computer-assisted standard protocol 16, it was found to be of only intermediate potency as compared to the highly potent tumour promoter TPA. The results of the present investigation indicate that metabolic conjugation of TPA with long-chain fatty acids to yield TPA-20-acylates is another pathway of metabolic deactivation of TPA, thus supporting the hypothesis that TPA itself is the ultimate tumour promoter in mouse skin.

Post-marketing surveillance of drugs. The spontaneous reporting scheme: role of the Adverse Drug Reactions Advisory Committee.

Post-marketing surveillance of drugs in Australia operates predominantly through the spontaneous reporting of suspected adverse drug reactions (ADRs). Approximately 50% of reports are submitted by hospitals and the rest by individual doctors, pharmacists and dentists. Some 4500 reports ("blue cards") are now reviewed annually by the Adverse Drug Reactions Advisory Committee (ADRAC) and its Secretariat. The register of ADRs has now accumulated more than 65,000 reports. Collations and analyses of data derived from the review process are published to increase awareness by health professionals of drug associated morbidity. Continued educational efforts by professional bodies and regulatory agencies will play a key role in rationalising drug use and reducing drug induced disease.

Simultaneous liquid-chromatographic determination of vitamin K1 and vitamin E in serum.

We describe a high-performance liquid chromatographic procedure for the simultaneous measurement of vitamins K1 and E in human serum. Delipidated human serum (free of vitamins K1 and E) was used to make standard solutions of these vitamins, and cetyl naphthoate and alpha-tocopheryl acetate were the internal standards for vitamin K1 and vitamin E, respectively. A simple, novel separation method utilizing liquid-liquid partition chromatography was used as a preparative "clean-up" procedure. Cetyl naphthoate and vitamin K1 (after post-column reduction) were detected by fluorescence, alpha-tocopheryl acetate and vitamin E by ultraviolet absorption. Sensitivity (detection limit) of the assay was 30 pg for vitamin K1 and 5 ng for vitamin E per injection. The method is specific, precise, and more rapid than previously described procedures. Within- and between-assay CVs were 8.1% and 12.9%, respectively, for vitamin K1; 3.5% and 6.0%, respectively, for vitamin E. Analytical recoveries of vitamins K1 and E were 80% and 93%, respectively, from serum and from delipidated serum (standards). The average neonatal serum concentration of vitamin K1 was 83 ng/L, 2.5 mg/L for vitamin E; for normolipidemic adults, the values were 343 ng/L and 7.9 mg/L, respectively, and for hyperlipidemic adults, 541 ng/L and 11.1 mg/L, respectively.

Testicular damage due to cytotoxic drugs and recovery after cessation of therapy.

Testicular function was assessed in 32 patients who received standard chemotherapy regimens for disseminated lymphomas. Thirty-one had evidence of germ cell damage, as assessed by the finding of azoospermia and/or high plasma levels of follicle-stimulating hormone (FSH). In addition, five patients had persistently low plasma testosterone levels associated in three with elevated plasma luteinising hormone (LH) levels. Reproductive function did not recover in any patient while chemotherapy continued. Cessation of therapy was possible in 16 patients with prolonged remissions of disease. Among these, recovery of germinal epithelium differed greatly between the cyclophosphamide / vincristine / prednisone treated group and the mustine/procarbazine/vincristine/prednisone treated group. Seventy per cent of the former patients had evidence of recovery after 34 months of follow-up while only one (17%) of the latter had begun to recover at 52 months post-therapy. Serial measurement of plasma FSH levels proved useful in predicting likely recovery of spermatogenesis.

Ascorbic acid deficiency affects the metabolism of cytosolic ferritin but not lipid-associated ferritin in livers of guinea pigs.

Two physicochemically and metabolically separate pools of ferritin, namely cytosolic ferritin and lipid-associated ferritin, are present in the livers of guinea pigs. In this paper we establish that the iron content of cytosolic ferritin is dependent on and linearly related to ascorbic acid concentration, whereas changes in concentration of this vitamin do not affect the iron content of lipid-associated ferritin. In livers of ascorbic acid-deficient guinea pigs both synthesis and degradation of cytosolic ferritin are diminished equally. Consequently cytosolic ferritin is metabolized more slowly without changes in its pool size. In contrast with cytosolic ferritin, the metabolism of lipid-associated ferritin is unaffected by ascorbic acid deficiency. The differential effects of ascorbic acid deficiency on the physicochemical characteristics as well as on the metabolism of cytosolic ferritin and lipid-associated ferritin suggest that the two forms of ferritin have different functional roles.

Cytosolic ferritin and lipid-associated ferritin are metabolically different in guinea-pig livers.

A distinct pool of liver ferritin has been described in man, guinea pigs and rats [Cham, Roeser, Nikles & Ridgway (1986) Clin. Chim. Acta 158, 71-79]. This ferritin accounts for approx. 30% of total intracellular ferritin. It differs from previously described cytosolic and 'microsomal-fraction' ferritin by its firm association with lipid and by the absence of heat-stability at 75 degrees C. The present study demonstrates that cytosolic ferritin and lipid-associated ferritin in guinea-pig livers have distinctly different rates of turnover. Cytosolic ferritin has a rate of turnover approx. 3.5 times as high as lipid-associated ferritin. The apparent metabolic heterogeneity suggests that the two forms of ferritin may have different functional roles.

Quantitation of reticuloendothelial iron kinetics in humans.

Reticuloendothelial iron kinetics were investigated in a simultaneous dual-isotope study in 10 healthy adult subjects in whom 55Fe-ferric hydroxide phosphate colloid was used to label the reticuloendothelial iron pools, and 59Fe-transferrin was used to define plasma iron kinetics. The simultaneous clearance of 55Fe and 59Fe from plasma and the uptake of each into red blood cells were measured over 14 days. The 55Fe-colloid was cleared almost immediately, and its iron was rapidly released to bind to plasma transferrin. Red cell incorporation of 55Fe was, however, much slower than that of 59Fe bound to transferrin in vitro. The data were analyzed by a new model of reticuloendothelial iron metabolism that contained two reticuloendothelial iron pools; one had a rapid turnover and donated iron to transferrin, and the other, a storage pool, had a slower turnover. The transit pool contained a mean of 164 mumol iron with little variation between subjects, whereas the storage pool was somewhat larger (mean 873 mumol iron) and showed more marked variation between subjects. In general an equal proportion of the iron leaving the transit pool went to transferrin and to the storage pool. The distribution between the two routes did not appear to be related either to plasma iron concentration, latent iron-binding capacity, or transferrin saturation.

A procedure for the purification of ferritin from human liver by heating a methanol-treated homogenate.

A simple, rapid technique for purification of ferritin from human liver tissue is described. Methanol, at a final concentration of 40% (v/v) in liver homogenate, precipitates the majority of proteins but does not affect ferritin. Subsequent heating of this homogenate at 75 degrees C for 10 min results in a purified ferritin preparation as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis. The resultant purified ferritin contained the same amount of iron as the original endogenous ferritin. There were no significant differences (paired t tests) in the amount of protein in the purified ferritin preparation when measured by rocket immunoelectrophoresis and by the Lowry procedure, suggesting that the antigenecity of ferritin was unaffected by the methanol and heat treatment. Both endogenous liver ferritin and radiolabeled human liver ferritin added to liver homogenates were recovered after methanol and heat treatment with similar yields (77 +/- 7% and 70 +/- 2%, respectively) when compared with the standard treatment of heating a homogenate at 75 degrees C. The overall ferritin yield with this rapid procedure was 40%.

Burkitt's lymphoma in Australia.

An Australian case of Burkitt's lymphoma is reported. The clinical features of large ovarian masses and subsequent bone marrow invasion, as well as the results of investigations related to the Epstein-Barr virus, were more consistent with the American than the African type of Burkitt's lymphoma. After a good initial response to cyclophosphamide and vincristine, the tumour rapidly became resistant to these and other therapeutic measures and the patient died 10 months after diagnosis.

Hodgkin's disease: results of treatment with combination chemotherapy.

Combination chemotherapy, which consisted of nitrogen mustard, a vinca alkaloid, procarbazine, and prednisone, was given to 60 patients with disseminated or recurrent Hodgkin's disease. A complete remission of disease was observed in 41 patients, with a median duration of 35+ months. The longest continuing remission was 89 months, and 52% of patients who achieved a complete remission were alive five years from the start of treatment. In contrast, patients who only had a partial remission (11 patients), or failed to respond to therapy (8 patients) fared badly, with a less than 50% survival rate at one year. Response to drug therapy could not be predicted on the basis of sex, age or tumour histology, but patients who had previously received both radiotherapy and chemotherapy had a poor prognosis. Drug toxicity was substantial, but was considered acceptable in view of the clear benefit of treatment to most patients. Further improvement in the outlook of patients with advanced Hodgkin's disease is likely to result from modifications of current drug regimens, possibly combined with non-radical radiotherapy.

Effect of a high energy, low carbohydrate diet on serum levels of lipids and lipoproteins.

The effect of a high energy, low carbohydrate (ketogenic) diet on serum levels of lipids and lipoproteins was studied in two normal individuals. A marked rise in serum levels of cholesterol and phospholipid occurred during the diet, but serum triglyceride levels remained within the normal range. High density lipoprotein cholesterol level did not change, and the rise in serum level of cholesterol levels with its distribution in lipoproteins, these observed changes pose a potential atherogenic risk. Serum lipid and lipoprotein levels reverted to normal on discontinuation of the diet. A modest weight loss observed during the diet was regained within 24 hours of the diet's cessation.

Toxicokinetics of tumor promoters of mouse skin. I. Metabolism of phorbol and ingenol esters by mouse liver microsomes.

The metabolism of ten selected phorbol esters and of one ingenol ester was investigated in mouse liver microsomes fortified with cofactors. The major metabolites were generated by an esterolytic activity in the microsomes. The following 12,13-diesters were cleaved readily to yield the 13-monoesters: 12-O-tetradecanoylphorbol-13-acetate, phorbol-12,13-dipropionate, bis(13-O-acetylphorbol)-12, 12'-tetradecanedioate, phorbol-12,13-dibenzoate; the monoesters 12-O-tetradecanoylphorbol and 3-O-tetradecanoylingenol yielded the corresponding parent alcohols. Other 12,13-di- or 13-monoesters such as 'inverse TPA', i.e. 12-O-acetylphorbol-13-tetradecanoate, phorbol-12,13-didecanoate, 12-O-retinoylphorbol-13-acetate, phorbol-13-decanoate and 12-O-tetradecanoyl-4 alpha-phorbol-13-acetate were either comparatively or completely resistant to esterolysis. The data indicate that metabolism of diterpene esters depends on the nature and position of the acyl moiety, as well as on the structure of the diterpene moiety. The esterolytic activity in mouse liver microsomes can be inhibited by i.p. administration of bis(4-nitrophenyl)phosphate to mice. Evidence for other relevant metabolic pathways besides esterolysis was lacking.

Flucloxacillin associated cholestatic hepatitis. An Australian and Swedish epidemic?

The clinico-pathological entity of flucloxacillin-associated cholestatic hepatitis is described and the recognition and documentation of cholestasis associated with flucloxacillin and with related isoxazolyl-penicillins (cloxacillin, dicloxacillin) is examined on an international basis, with particular reference to Australia. Data were obtained from the literature, from the Australian adverse drug reaction monitoring agency and from the Collaborative Centre for International Drug Monitoring (World Health Organisation) in Sweden. Approximately 600 cases of flucloxacillin-associated cholestatic hepatitis were collected, as well as 164 cases associated with other isoxazolyl penicillins. Jaundice and pruritus may first appear several weeks after administration of the drug has ceased and typically are severe and protracted. Liver tests may be abnormal for months after symptomatic recovery. Death is uncommon. Liver pathology shows centrizonal bile stasis with portal tract inflammation and variable loss of bile ducts. Approximately 1 in 15,000 users of flucloxacillin will develop the reaction. Increasing age (> 55 years) and prolonged intake (> 14 days) are particular risk factors. Cholestasis associated with cloxacillin/dicloxacillin appears to be similar in nature but is less well defined. Recognition and reporting of the reaction have been uncommon in the United Kingdom inter alia and high in Sweden and Australia, although estimates of risk have been similar. In Australia, the remarkably high rate of reports appears to be the result of sustained publicity for the reaction. There is only a trickle of reports of cholestatic hepatitis in association with the use of cloxacillin and dicloxacillin from the USA and Canada. The high level of awareness of the reaction and consequential regulatory action so far have not resulted in a diminution of its occurrence in Australia.