Q J Med 2001; 94: 417-422
© 2001 Association of Physicians
Increased oxysterols associated with iron accumulation in the brains and visceral organs of acaeruloplasminaemia patients
From the First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu, 1 Department of Legal Medicine, Kobe University School of Medicine, Kobe, and 2 Kobe Pharmaceutical University, Kobe, Japan
Received 19 September 2000 and in revised form 23 May 2001
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Summary |
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Acaeruloplasminaemia is characterized by excessive neurovisceral iron accumulation due to mutation of the caeruloplasmin gene. Excess iron functions as a potent catalyst of biological oxidation, and increased iron concentration is associated with the products of lipid peroxidation in the serum and cerebrospinal fluid. We investigated whether the amount of iron accumulated paralleled lipid peroxidation levels in acaeruloplasminaemia tissues, examining brains and visceral organs of two affected patients at autopsy for iron and copper content, and oxysterols, including 7-hydroxycholesterol and 7-ketocholesterol, which are directly produced from cholesterol by active oxygen species. The amount of iron accumulated in various tissues was correlated with the levels of the oxysterols. These findings suggest that lipid peroxidation produced by the intracellular accumulation of iron is involved in the pathogenesis of acaeruloplasminaemia.
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Introduction |
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Caeruloplasmin, a multi-copper ferroxidase that affects the distribution of tissue iron, has antioxidant effects through the oxidation of ferrous iron to ferric iron.1,2 Acaeruloplasminaemia is characterized by iron accumulation in the retina, basal ganglia, and cerebral cortex, as well as in parenchymal tissues, due to the absence of caeruloplasmin ferroxidase activity. Clinically, the disease consists of the triad of neurological disease, retinal degeneration, and diabetes mellitus (DM).3–5 The neurological symptoms, which include involuntary movements, ataxia, and dementia, reflect the sites of iron deposition detected by MRI, as well as the regions of neurodegeneration detected at autopsy.6 Previously, we showed that an increased iron concentration was associated with the products of lipid peroxidation in the serum7 and cerebrospinal fluid (CSF).8 In those studies, monitoring was based on the formation of thiobarbituric acid-reactive substances (TBARS), but this method lacks specificity.9 Whether the amount of iron accumulated parallels lipid peroxidation levels in acaeruloplasminaemia tissues has yet to be shown. We hypothesized that oxidative stress caused by the accumulation of iron causes cell damage through lipid peroxidation in acaeruloplasminaemia, and examined brains and visceral organs of affected patients at autopsy for both iron and copper content, and oxysterols. Oxysterols, which are produced directly through free radical-mediated cholestrol peroxidation, can serve as molecular indicators of chain peroxidative damage in the cell membrane.10–12 A possible production route of oxysterol from cholesterol is shown in Figure 1
. Cholesterol (1) reacts with singlet oxygen producing 7
-hydroperoxycholest-5-en-3ß-ol (7-OOH) (2). Epimerization of 7-OOH gives 7ß-hydroperoxycholest-5-en-3ß-ol (7ß-OOH) (2). The 7-hydroperoxy-cholest-5-en-3ß-ol (7-OOH) may be reduced to 7-hydroxycholesterols (7-OH) (3), including cholest-5-en-3ß,7-diol (7-OH) and cholest-5-ene-3ß,7ß-diol (7ß-OH), or oxidized to 3ß-hydroxycholest-5-en-7-one (7-keto) (4).
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Materials
Cholesterol was obtained from Sigma. 7-Keto,7ß-OH, and 7ß-OH were purchased from Steraloids. ß-sitosterol acetate, the internal standard, also was from Steraloids.
Patients
Patient 1 was a 60-year-old man who had had diabetes mellitus and insulin therapy for 25 years, and scanning speech and forgetfulness from age 56.13 He had retinal degeneration and bilateral hearing disturbance. Facial grimacing and choreic involuntary movements of the upper extremities occurred during speech or voluntary movement of the extremities. Both his trunk and extremities showed cerebellar ataxia, and his gait was ataxic. His full-scale IQ was 68; verbal IQ was 70, and performance IQ 62 on the Wechsler Adult Intelligence Scale (WAIS). Mini-mental state examination (MMSE) score was 15. Serum caeruloplasmin was absent because of mutation of the caeruloplasmin gene, the insertion of adenine in exon 3 producing a premature stop codon.13 He had had liver dysfunction from age 57 and died from cardiac failure at age 60.
Patient 2 was a 66-year-old woman who had had blepharospasm and retinal degeneration for 15 years.3 From age 60, she developed grimacing, rigidity, and diabetes. Neurological examination at age 65 found bilateral blepharospasm synchronized with perioral spasm and neck dystonia. Her speech was scanning, and her gait slightly ataxic. Her full-scale IQ was 80, verbal IQ 82, and performance IQ 76 on the WAIS. MMSE score was 25. Her laboratory findings were less severe than those of patient 1 (Table 1). The mutation in her ceruloplasmin gene was a 5-bp insertion in exon 7.14 She died from pancreatic cancer and cardiac failure at age 66. There was no pathological evidence of myocardial infarction in either patient.
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Laboratory methods
Brains and visceral organs were obtained at autopsy within 6 h of death from the two patients with acaeruloplasminaemia and from four control subjects, mean age 63 years (two men and two women, 60–66 years old). The control subjects had arteriosclerosis and asymptomatic cerebral lacunae infarctions, and the cause of their deaths was acute myocardial infarction. The basal ganglia (putamen and globus pallidus), cerebral cortex (hippocampus and angular gyrus), and cerebellar cortex were separated from the brain less than 1 h after autopsy. Samples of each brain area, liver, pancreas, heart and kidney were cut immediately into small sections that then were used for biochemical examination.
Metal concentrations
Fresh tissue was dried in a microwave digestion unit (MLS-1200 MEGA, Milestone General) until a constant weight (10–15 mg) was obtained. The dried powder was weighed, and 0.7 ml samples of the ashed solutions were prepared with 0.1 M HCl. Sample solutions were analysed directly in an atomic absorption spectrophotometer (Hitachi Z 6100). Data were calculated on a dry-weight basis, because tissue density was not uniform in all the brain areas.
Oxysterol determination
Total lipid was extracted by the method of Folch et al.15 The cholesterol-rich fraction was isolated from the total lipid by solid-phase extraction, as reported previously.11 Oxysterols were determined with a Hitachi L-7000 series liquid chromatography system, comprising an L-7100 pump (Hitachi), L-7400 UV detector (Hitachi) set at 210 nm, and a Chromatopac C-R6A integrator (Shimadzu). A TSK gel ODS-80Ts column (Tosoh) was used (250x4.6 mm internal diameter), and methanol (100%) was delivered as the mobile phase at a flow rate of 0.7 ml/min. Standard curves were prepared by analysing 50, 100, 250, and 500 ng of 7-OH; 100, 200, 500, and 1000 ng of 7-keto; and 400, 800, 2000, and 4000 ng of cholesterol with 625 ng of the internal standard. Individual peak areas were calculated with an integrator. Ratios of the oxysterols to the internal standard also were calculated for the standard compounds and the lipid extracts. Regression lines of the oxysterol ratios to the internal standard vs. the standard concentrations were linear.
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Results |
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Iron concentrations in the acaeruloplasminaemia patients were markedly increased in the brain and visceral organs, compared with controls (Table 2
). The distribution in order of iron level in both the acaeruloplasminaemia and control brains was globus pallidus>putamen>cerebral cortex, cerebellar cortex. Iron contents were markedly elevated in the basal ganglia of patients with acaeruloplasminaemia. The content in the livers was greater than in the brain. The amounts of iron accumulated in all the regions examined were greater in patient 1 than in patient 2. Copper levels, unlike iron, were evenly elevated in all the regions examined in the patients with acaeruloplasminaemia.
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Concentrations of 7-OH, as well as 7-keto, were much higher in the brain tissues than in the peripheral organs in both the acaeruloplasminaemia patients and controls (Table 3
), being highest in the angular gyrus. Levels in basal ganglia tissue obtained from the acaeruloplasminaemia patients were >3–5 times the control values, and the cerebellar and cerebral cortexes showed an approximately 2- to 3-fold increase. Ratios of the 7-OH+7-keto concentrations per cholesterol concentration (7-OH+7-keto/Cho), an index of oxysterol production from cholesterol, also were elevated to about 0.30–0.45 in the patients’ brains, whereas in the controls they were 0.06–0.11. These ratios were higher in the basal ganglia of the affected patients than in the hippocampus and cerebellar cortex. 7-OH was markedly increased in the patients' liver, pancreas and kidney, whereas none was detected in the controls. 7-Keto also was >4 times the control values in those organs. The ratios of 7-OH+7-keto/Cho in the patients therefore were >15 times the control values. Concentrations of 7-OH, as well as 7-keto, in the patients' hearts were about twice the control values, but the oxysterol index was much higher in the heart than in the other organs in the aceruloplasminemia patients. Values for all the regions examined were larger in patient 1 than in patient 2.
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Discussion |
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The mechanisms of tissue injury in acaeruloplasminaemia have yet to be clarified, but increased oxidative stress has attracted much attention. This is because iron promotes the formation of a variety of reactive oxygen species which can induce cellular damage and facilitate the decomposition of lipid peroxides, and caeruloplasmin inhibits the auto-oxidation of lipids induced by the action of inorganic iron.1,2 Elsewhere, we reported marked excess of lipid peroxidation in the serum7 and CSF8 of patients with acaeruloplasminaemia. Malonaldehyde (MDA) formation also was increased in the acaeruloplasminaemia brains.16 Lipid peroxidation usually has been monitored by the formation of TBARS or MDA, but these methods lack specificity and tend to be influenced to some extent by assay conditions.9 The recent development of HPLC with chemiluminescence detection has made the direct measurement with high sensitivity and specificity of lipid peroxides possible. The finding that oxysterols were elevated in various tissue homogenates from acaeruloplasminaemia patients is direct evidence that increased lipid peroxidation occurred.
The brain, by virtue of its high lipid and oxygen consumption, as well as elevated metal concentrations (e.g. iron), is an ideal site for degeneration by oxidative stress. It is relatively poor in many antioxidants, such as catalase.17 In acaeruloplasminaemia, the increase in the oxysterol concentration in the basal ganglia was relatively small, compared with increases in the other brain regions, but cholesterol concentrations differed in the various brain regions. As compared with the oxysterol index, which reflects the cholesterol peroxidation rate, this index was markedly elevated in the basal ganglia of acaeruloplasminaemia brain. Involuntary movement may indicate sites at which lipid peroxidation was increased. These regions may not be in a burned-out state, but extensive loss of neurons was observed.6 The accumulated iron is not evenly distributed within the acaeruloplasminaemia brain: the basal ganglia have relatively large concentrations, whereas the cerebral cortex and cerebellum have fairly low ones. The amounts of iron accumulated paralleled the oxysterol levels in the acaeruloplasminaemia brains. The iron accumulation may result in the increased lipid peroxidation that induces neuronal cell damage. The brain iron content and oxysterol index for our patient 1, who showed progressive involuntary movement, ataxia, and dementia, were higher than those in patient 2, whose symptoms were mild. Increased susceptibility to lipid peroxidation secondary to iron accumulation may be closely related to disease severity. The iron concentrations in the visceral tissues in the present patients were increased more than 10-fold over the control values. Oxysterols in the peripheral organs of the patients also were much higher than the control subjects. Beta cells in the pancreas are particularly vulnerable to the cytotoxic effects of oxidative stress. Free radicals generated in the presence of iron react together, initiating lipid peroxidation and cell damage.18 Iron-mediated lipid peroxidation may have a role in the development of diabetes in the acaeruloplasminaemia patients. The heart also is susceptible to free radical attack, because of a highly oxygenated structure. The oxysterol index in the heart in the control subjects was relatively high as compared with the other visceral organs, probably because of increased lipid peroxidation caused by ischaemic heart injury. However, it was greater in the patients than in the controls, since accumulated iron has the ability to promote membrane lipid peroxidation. Since hepatic fibrosis or cirrhosis occurs with hepatic iron concentrations above a threshold of approximately 22.3 mg/g dry weight,19 hepatic injury in the present patients might be slight, to judge from the laboratory findings. In patients with acaeruloplasminaemia, the balance between pro- and antioxidant components may shift to a higher pro oxidant status. Once such oxidative stress becomes chronic, it may be difficult to return to normal by compensative mechanisms.
An inverse relationship has been shown between the tissue contents of iron and copper in the liver,20 but copper levels tended to increase in the brain as well as other organs of the patients with acaeruloplasminaemia despite the larger increase in iron content. The significance of these findings is unknown. At the least, copper-induced, increased pro-oxidant capabilities may be involved in the pathophysiology in this disease. Caeruloplasmin does not have an essential function in copper transport from the liver to the brain, even though it donates copper ions to tissues.
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Notes |
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Address correspondence to Dr H. Miyajima, The First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. e-mail: miyajima{at}hama\|[hyphen]\|med.ac.jp
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