Copper Availability Contributes to Iron Perturbations in Human Nonalcoholic Fatty Liver Disease
2008; Elsevier BV; Volume: 135; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2008.04.007
ISSN1528-0012
AutoresElmar Aigner, Igor Theurl, Heike Haufe, Markus Seifert, Florian Hohla, Ludwig Scharinger, Felix Stickel, Frédéric Mourlane, Günter Weiß, Christian Datz,
Tópico(s)Folate and B Vitamins Research
ResumoBackground & Aims: Iron perturbations are frequently observed in nonalcoholic fatty liver disease (NAFLD). We aimed to investigate a potential association of copper status with disturbances of iron homeostasis in NAFLD. Methods: We retrospectively studied 140 NAFLD patients and 25 control subjects. Biochemical and hepatic iron and copper parameters were analyzed. Hepatic expression of iron regulatory molecules was investigated in liver biopsy specimens by reverse-transcription polymerase chain reaction and Western blot analysis. Results: NAFLD patients had lower hepatic copper concentrations than control subjects (21.9 ± 9.8 vs 29.6 ± 5.1 μg/g; P = .002). NAFLD patients with low serum and liver copper concentrations presented with higher serum ferritin levels (606.7 ± 265.8 vs 224.2 ± 176.0 mg/L; P < .001), increased prevalence of siderosis in liver biopsy specimens (36/46 vs 10/47 patients; P < .001), and with elevated hepatic iron concentrations (1184.4 ± 842.7 vs 319.9 ± 451.3 μg/g; P = .020). Lower serum concentrations of the copper-dependent ferroxidase ceruloplasmin (21.7 ± 4.1 vs 30.4 ± 6.4 mg/dL; P < .001) and decreased liver ferroportin (FP-1; P = .009) messenger RNA expression were found in these patients compared with NAFLD patients with high liver or serum copper concentrations. Accordingly, in rats, a reduced dietary copper intake was paralleled by a decreased hepatic FP-1 protein expression. Conclusions: A significant proportion of NAFLD patients should be considered copper deficient. Our results indicate that copper status is linked to iron homeostasis in NAFLD, suggesting that low copper bioavailability causes increased hepatic iron stores via decreased FP-1 expression and ceruloplasmin ferroxidase activity thus blocking liver iron export in copper-deficient subjects. Background & Aims: Iron perturbations are frequently observed in nonalcoholic fatty liver disease (NAFLD). We aimed to investigate a potential association of copper status with disturbances of iron homeostasis in NAFLD. Methods: We retrospectively studied 140 NAFLD patients and 25 control subjects. Biochemical and hepatic iron and copper parameters were analyzed. Hepatic expression of iron regulatory molecules was investigated in liver biopsy specimens by reverse-transcription polymerase chain reaction and Western blot analysis. Results: NAFLD patients had lower hepatic copper concentrations than control subjects (21.9 ± 9.8 vs 29.6 ± 5.1 μg/g; P = .002). NAFLD patients with low serum and liver copper concentrations presented with higher serum ferritin levels (606.7 ± 265.8 vs 224.2 ± 176.0 mg/L; P < .001), increased prevalence of siderosis in liver biopsy specimens (36/46 vs 10/47 patients; P < .001), and with elevated hepatic iron concentrations (1184.4 ± 842.7 vs 319.9 ± 451.3 μg/g; P = .020). Lower serum concentrations of the copper-dependent ferroxidase ceruloplasmin (21.7 ± 4.1 vs 30.4 ± 6.4 mg/dL; P < .001) and decreased liver ferroportin (FP-1; P = .009) messenger RNA expression were found in these patients compared with NAFLD patients with high liver or serum copper concentrations. Accordingly, in rats, a reduced dietary copper intake was paralleled by a decreased hepatic FP-1 protein expression. Conclusions: A significant proportion of NAFLD patients should be considered copper deficient. Our results indicate that copper status is linked to iron homeostasis in NAFLD, suggesting that low copper bioavailability causes increased hepatic iron stores via decreased FP-1 expression and ceruloplasmin ferroxidase activity thus blocking liver iron export in copper-deficient subjects. See Acton RT et al on page 934 in CGH. See Acton RT et al on page 934 in CGH. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome.1Neuschwander-Tetri B.A. Fatty liver and the metabolic syndrome.Curr Opin Gastroenterol. 2007; 23: 193-198Crossref PubMed Scopus (93) Google Scholar The term insulin resistance-associated hepatic iron overload (IR-HIO) syndrome describes the frequent association between hepatic steatosis and iron accumulation, as reflected by increased serum ferritin along with normal or only slightly elevated transferrin saturation.2Mendler M.H. Turlin B. 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Balancing acts: molecular control of mammalian iron metabolism.Cell. 2004; 117 (285–197)Abstract Full Text Full Text PDF PubMed Scopus (1460) Google Scholar Whereas cells acquire iron via different pathways including the uptake of transferrin bound iron via transferrin receptors and of ferrous iron via the transmembrane protein divalent metal transporter-1,4Hentze M.W. Muckenthaler M.U. Andrews N.C. Balancing acts: molecular control of mammalian iron metabolism.Cell. 2004; 117 (285–197)Abstract Full Text Full Text PDF PubMed Scopus (1460) Google Scholar respectively, so far only 1 iron exporter has been characterized, comprising a transmembrane protein termed ferroportin (FP-1) or IREG-1.5Donovan A. Brownlie A. Zhou Y. et al.Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.Nature. 2000; 403: 776-781Crossref PubMed Scopus (1403) Google Scholar, 6Abboud S. Haile D.J. A novel mammalian iron-regulated protein involved in intracellular iron metabolism.J Biol Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar Hepcidin is a master iron regulatory peptide,7Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation.Blood. 2003; 102: 783-788Crossref PubMed Scopus (1227) Google Scholar secreted mainly by hepatocytes in response to iron perturbations, inflammation, anemia, and hypoxia.8Nicolas G. Chauvet C. Viatte L. et al.The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.J Clin Invest. 2002; 110: 1037-1044Crossref PubMed Scopus (1396) Google Scholar Hepcidin exerts its regulatory functions on iron homeostasis via binding to FP-1, causing phosphorylation, internalization, and degradation of FP-1 and thus leads to the sequestration of iron by blocking its cellular export.9Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3860) Google Scholar It is well acknowledged that iron and copper metabolism are closely linked10Sharp P. The molecular basis of copper and iron interactions.Proc Nutr Soc. 2004; 63: 563-569Crossref PubMed Scopus (98) Google Scholar: whereas the enterocyte brush border enzyme cytochrome B reductase is involved in both ferric and cupric reduction,11Knopfel M. Solioz M. Characterization of a cytochrome b(558) ferric/cupric reductase from rabbit duodenal brush border membranes.Biochem Biophys Res Commun. 2002; 291: 220-225Crossref PubMed Scopus (48) Google Scholar the multicopper ferroxidase hepaestin, located at the basolateral membrane of duodenal enterocytes, is essential for cellular iron export via FP-1.12Frazer D.M. Vulpe C.D. McKie A.T. et al.Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G931-G939Crossref PubMed Google Scholar, 13McKie A.T. Barrow D. Latunde-Dada G.O. et al.An iron-regulated ferric reductase associated with the absorption of dietary iron.Science. 2001; 291: 1755-1759Crossref PubMed Scopus (873) Google Scholar Mice deficient in hephaestin (sla-mice) present with normal apical iron uptake but defective iron transfer to serum transferrin thus resulting in anemia.14Anderson G.J. Murphy T.L. Cowley L. et al.Mapping the gene for sex-linked anemia: an inherited defect of intestinal iron absorption in the mouse.Genomics. 1998; 48: 34-39Crossref PubMed Scopus (24) Google Scholar Ceruloplasmin ferroxidase activity is crucial for mobilization of iron from storage sites for incorporation into transferrin and metabolic utilization.15Osaki S. Johnson D.A. Mobilization of liver iron by ferroxidase (ceruloplasmin).J Biol Chem. 1969; 244: 5757-5758Abstract Full Text PDF PubMed Google Scholar, 16Hellman N.E. Gitlin J.D. Ceruloplasmin metabolism and function.Annu Rev Nutr. 2002; 22: 439-458Crossref PubMed Scopus (739) Google Scholar Hence, aceruloplasminemia results in increased tissue iron with high ferritin levels and mild anemia,17Harris Z.L. Klomp L.W. Gitlin J.D. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis.Am J Clin Nutr. 1998; 67: S972-S977Abstract Full Text PDF PubMed Scopus (213) Google Scholar and ceruloplasmin knockout mice accumulate iron in the liver and the reticuloendothelial system.18Harris Z.L. Durley A.P. Man T.K. et al.Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux.Proc Natl Acad Sci U S A. 1999; 96: 10812-10817Crossref PubMed Scopus (511) Google Scholar Likewise, dietary copper deficiency induces characteristic changes in expression of iron metabolism genes and impairs iron transport across the basolateral enterocyte membrane.19Auclair S. Feillet-Coudray C. Coudray C. et al.Mild copper deficiency alters gene expression of proteins involved in iron metabolism.Blood Cells Mol Dis. 2006; 36: 15-20Crossref PubMed Scopus (24) Google Scholar In this respect, copper has been shown to affect the expression of FP-1 in J774 macrophages20Chung J. Haile D.J. Wessling-Resnick M. Copper-induced ferroportin-1 expression in J774 macrophages is associated with increased iron efflux.Proc Natl Acad Sci U S A. 2004; 101: 2700-2705Crossref PubMed Scopus (52) Google Scholar and Caco-2 cells.21Tennant J. Stansfield M. Yamaji S. et al.Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells.FEBS Lett. 2002; 527: 239-244Crossref PubMed Scopus (68) Google Scholar Moreover, membrane-bound ceruloplasmin is required for the stability of FP-1, possibly explaining cerebral iron accumulation in patients with aceruloplasminemia.22De Domenico I. Ward D.M. di Patti M.C. et al.Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin.EMBO J. 2007; 26: 2823-2831Crossref PubMed Scopus (295) Google Scholar Finally, ceruloplasmin availability modifies iron loading in HFE knockout mice.23Gouya L. Muzeau F. Robreau A.M. et al.Genetic study of variation in normal mouse iron homeostasis reveals ceruloplasmin as an HFE-hemochromatosis modifier gene.Gastroenterology. 2007; 132: 679-686Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The present study aimed to investigate a possible contribution of variations of copper status and expression of the multicopper ferroxidase ceruloplasmin to iron perturbations in human NAFLD. One hundred forty consecutive patients with a diagnosis of NAFLD identified from our liver biopsy database were studied retrospectively. These patients had diagnostic liver biopsies performed at 2 different academic institutions between January of 2000 and December of 2006. Subjects with a history of relevant alcohol intake (>20 g/day), viral hepatitis, autoimmune hepatitis, primary biliary cirrhosis, Wilson's disease, or α-1-antitrypsin deficiency or patients on known steatogenic medication were excluded. None of the study patients had signs of cardiac or renal insufficiency or suffered from cancer, autoimmune diseases, or systemic infections. Patient charts were reviewed with regard to cigarette smoking and use of oral contraceptive medication because these factors are known to affect ceruloplasmin levels and functional properties.16Hellman N.E. Gitlin J.D. Ceruloplasmin metabolism and function.Annu Rev Nutr. 2002; 22: 439-458Crossref PubMed Scopus (739) Google Scholar, 24Galdston M. Levytska V. Schwartz M.S. et al.Ceruloplasmin Increased serum concentration and impaired antioxidant activity in cigarette smokers, and ability to prevent suppression of elastase inhibitory capacity of α 1-proteinase inhibitor.Am Rev Respir Dis. 1984; 129: 258-263PubMed Google Scholar To evaluate the contribution of copper to changes in NAFLD iron homeostasis, NAFLD patients were divided into tertiles according to their copper status. In 76 of 140 NAFLD patients, intrahepatic copper concentration was available. Patients were categorized into 3 groups according to low hepatic copper ( 28 μg/g, 25 patients), respectively. Because there was a highly significant correlation between hepatic and serum copper concentrations in these 76 patients (R = 0.451; P = .005), the remaining 64 of 140 NAFLD patients without available hepatic copper concentrations were also stratified into tertiles (see above) based on serum copper levels (normal range 70-130 μg/L; low serum copper levels [ 122 μg/L, 21 patients]). In total, 47 patients were studied as NAFLD with low serum and liver copper levels, 47 as NAFLD with intermediate liver and serum copper levels, and 46 patients as NAFLD with high liver and serum copper levels. Because liver biopsy specimens from truly healthy subjects could not be obtained, 25 subjects (16 females, 9 males) who underwent liver biopsy for unexplained elevation of liver enzymes were studied as "control" subjects. These patients had no evidence of liver disease and normal liver histology and were considered an acceptable control cohort for our analysis. All control subjects had normal biochemical iron and copper parameters. In none of these individuals were iron deposits detected histologically. Hepatic iron and copper concentrations were available in 18 of 25 control subjects. To screen for hereditary hemochromatosis associated, genetic testing for C282Y and H63D mutations of the HFE gene was performed in all patients as described.25Datz C. Lalloz M.R. Vogel W. et al.Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis.J Hepatol. 1997; 27: 773-779Abstract Full Text PDF PubMed Scopus (86) Google Scholar Among NAFLD patients and control subjects included in the analysis, neither homozygosity for the C282Y and H63D mutation nor compound heterozygosity for these mutations was detected. A diagnosis of diabetes was made when fasting glucose levels were above 110 mg/dL, glycosylated hemoglobin (HbA1c) was 6.0% or higher, or patients were on oral antidiabetic medication. Because insulin is known to influence ceruloplasmin expression, diabetic individuals on insulin therapy were excluded from the analysis.26Seshadri V. Fox P.L. Mukhopadhyay C.K. Dual role of insulin in transcriptional regulation of the acute phase reactant ceruloplasmin.J Biol Chem. 2002; 277: 27903-27911Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar To assess the prevalence of the metabolic syndrome (MS) in the study groups, we aimed to identify subjects fulfilling World Health Organization (WHO) clinical criteria for MS.27World Health OrganizationDefinition, diagnosis and classification of diabetes mellitus and its complications Technical Report 99.2. WHO, Geneva, Switzerland1999Google Scholar Because the waist/hip ratio and urinary albumin excretion were not available in our study population, patients were diagnosed as having MS when they suffered from insulin resistance defined as type 2 diabetes, impaired fasting glucose (100–126 mg/dL), or impaired glucose tolerance (pathologic glucose tolerance test) plus any 2 of the following: (1) arterial hypertension (use of antihypertensive medication and/or high blood pressure >140 mm Hg systolic or >90 mm Hg diastolic), (2) serum triglycerides >150 mg/dL, (3) low high-density lipoprotein (HDL) cholesterol <35 mg/dL in men or 30 (kg/m2) were present. Written informed consent was obtained from all study participants to use clinical data and obtain biopsy specimen for scientific purposes, and the study was performed in accordance with the ethical standards set forth by the Helsinki Declaration of 1975 and revised in 1983. Liver biopsy specimens were fixed in buffered formalin and embedded in paraffin. Sections were stained with H&E and Mallory trichrome for morphologic evaluation and Perl's stain to determine liver iron deposition. All liver biopsy specimens were first assessed independently by 2 pathologists unaware of clinical data and the study objective. In cases of discrepant results of the histologic examination, samples were jointly reevaluated by the 2 pathologists, and the result agreed on was used for analysis. Histologic examination of liver biopsy specimens was performed according to criteria proposed by Brunt et al.28Brunt E.M. Janney C.G. Di Bisceglie A.M. et al.Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.Am J Gastroenterol. 1999; 94: 2467-2474Crossref PubMed Scopus (3295) Google Scholar Biopsy specimen were evaluated for the degree of macrovesicular steatosis 0–3 (0, 66% of hepatocytes affected) and hepatocellular ballooning (0, no ballooning; 1, mild; and 2, marked ballooning). Inflammation was graded 0–3 (mild, moderate, and severe) according to criteria proposed, and portal inflammation was graded 0–3 as described. Fibrosis was scored as fibrosis stage 1, zone 3 pericellular fibrosis; stage 2, pericellular and portal fibrosis; stage 3, bridging fibrosis; stage 4, cirrhosis. The diagnosis of NAFLD was established in liver biopsy specimens showing steatosis with or without evidence of steatohepatitis (inflammation and hepatocyte ballooning, with or without Mallory's hyaline or fibrosis) associated with an increased BMI and alcohol consumption of less than 20 g per day. Furthermore, biopsy specimens with steatosis and mild lobular inflammation but without ballooning or perisinusoidal fibrosis were grouped with steatosis and were not classified as nonalcoholic steatohepatitis (NASH). Biopsy specimens with steatosis and any grade of fibrosis other than above were classified as NASH. In addition, biopsy specimens with steatosis and lobular inflammation (1–3) and hepatocellular ballooning (1–2) were considered as NASH in the absence of fibrosis. Siderosis was determined semiquantitatively upon histopathologic examination of Perl's-stained liver biopsy specimens: score 0, granules absent or barely discernible at a magnification of 400-fold (400×); 1, barely discernible at a magnification of 200× but easily confirmed at 400×; 2, discrete granules resolved at 100× magnification; 3, discrete granules resolved at a magnification of 25×; 4, massive granules visible even upon 10× magnification.29Searle J.L.B. Crawford D.H.G. Powell L.W. Iron storage disease.in: MacSween R.N.M. Burt A.D.P.B. 4th ed. Pathology of the liver, Churchill Livingstone2002Google Scholar Venous blood was drawn following an overnight fast for determination of liver function tests; a full blood count; serum iron status including ferritin, transferrin, transferrin saturation and serum iron; copper; ceruloplasmin; C-reactive protein; fasting glucose; and lipids and erythrocyte sedimentation rate by standardized automated laboratory methods. Hepatic iron concentration and hepatic copper concentration were determined by automated mass spectroscopy analysis in patients and control subjects and calculated as micrograms/grams of dry weight. Insulin was measured by standard laboratory techniques, and insulin resistance was calculated using homeostasis model assessment (HOMA-IR; fasting insulin (μmol/L)*fasting glucose (mmol/dL)/22.5). Serum for determination of fasting insulin was available from 12 control subjects and 75 NAFLD patients (29 patients with low, 26 with intermediate, and 20 with high serum or liver copper concentrations). Details of RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) as well as protein extraction and Western blot analysis are provided as online supporting material (see Supplementary Material online at www.gastrojournal.org). Eighteen Sprague Dawley rats were kept on Kliba Petfood Purified Diet (CH-4303; Kliba Petfood, Kaiseraugst, Switzerland), 15% casein, for 8 weeks. Thereafter, these rats were grouped randomly to receive either copper-depleted or normal diet. Copper-depleted diet contained 2 ppm copper and resulted in a daily copper intake of 0.05 mg. Normal diet contained 100 ppm of copper resulting in a daily copper intake of 2.47 mg. After 4 weeks of feeding specific diets, rats were killed. Liver protein was extracted and FP-1 Western blot analysis performed as described.30Theurl I. Ludwiczek S. Eller P. et al.Pathways for the regulation of body iron homeostasis in response to experimental iron overload.J Hepatol. 2005; 43: 711-719Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Statistical analyses were carried out using SPSS statistics package (SPSS, Inc, Chicago, IL). Calculations for statistical differences in clinical and laboratory characteristics between the various groups were carried out by ANOVA or nonparametric Kruskal–Wallis test. Proportions were compared using Fisher exact test and χ2 method. Student t test or Mann–Whitney U test in case of non-Gaussian distribution of parameters was used to calculate differences in hepatic mRNA expression of iron metabolism genes as determined by RT-PCR technique. Associations among the various parameters in the different groups were calculated using Spearman rank correlation technique and a Bonferroni correction for multiple testings. Patient groups were not different regarding age and rates of heterozygosity for carriers of HFE C282Y and H63D mutations. HFE mutation carrier rates were comparable with the prevalence of the HFE mutations of the white population studied.25Datz C. Lalloz M.R. Vogel W. et al.Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis.J Hepatol. 1997; 27: 773-779Abstract Full Text PDF PubMed Scopus (86) Google Scholar Among NAFLD patients, we detected a higher degree of hepatic steatosis, a higher BMI, an increased prevalence of diabetes, and higher triglyceride levels in patients with low as opposed to those with high serum and liver copper concentrations. No differences were found with regard to the prevalence of fibrosis on liver biopsy, histologic diagnosis of NASH, liver transaminase levels, fasting glucose, total cholesterol, and low-density lipoprotein (LDL) and HDL cholesterol levels as well as the number of patients smoking (for details, see Table 1). Only 4 women took oral contraceptive medication, which excludes a relevant effect of these drugs to ceruloplasmin concentrations in our cohort because these patients did not differ from the other patients of the respective group. In general, NAFLD subjects had lower hepatic copper concentrations (21.9 ± 9.8 μg/g) compared with control subjects (29.6 ± 5.1, P = .002).Table 1Clinical, Histologic, and Biochemical Characteristics of Control Subjects and NAFLD Patients According to Copper ConcentrationsControlLow copperIntermediateHigh copperP value (ANOVA)Number of Pts25464747—Female (%)16 (64.0)16 (35.8)21 (44.7%)19 (40.4)—Age, y43.6 (±9.3)53.7 (±8.4)aDenotes significance at P < .05 as compared with control subjects.51.8 (±12.2)aDenotes significance at P < .05 as compared with control subjects.49.4 (±9.6)aDenotes significance at P < .05 as compared with control subjects.—HFE C282Y Heteroc. (%)3 (12)4 (8.7)4 (8.5)3 (6.4)—HFE H63D Heteroc. (%)4 (16)7 (15.2)6 (16%)7 (14.9)—Pts with NASH (%)0 (0)10 (21.3)aDenotes significance at P < .05 as compared with control subjects.7 (14.9%)aDenotes significance at P < .05 as compared with control subjects.6 (12.7%)aDenotes significance at P < .05 as compared with control subjects.—Hepatic steatosis (%)0 (±0)36.5 (±19.4)32.6 (±16.5)25.8 (±16.7).007Pts with fibrosis (%)0 (0)8 (17.4)6 (12.8)7 (14.9)—Degree of siderosis, 0−400.91 (±0.62)aDenotes significance at P < .05 as compared with control subjects.0.68 (±0.63)aDenotes significance at P < .05 as compared with control subjects.0.21 (±0.41)aDenotes significance at P < .05 as compared with control subjects.<.001Hepatic copper, μg/gbData available from 76 (hepatic copper concentration) or 75 patients (HOMA-IR).29.6 (±5.1)11.9 (±3.9)22.5 (±4.0)34.4 (±8.0)<.001Serum copper, 70−130 mg/L119.7 (±25.7)93.4 (±11.8)106.3 (±13.7)128.6 (±13.1)<.001BMI, kg/m225.5 (±2.2)29.0 (±3.4)aDenotes significance at P < .05 as compared with control subjects.28.3 (±2.3)aDenotes significance at P < .05 as compared with control subjects.27.8 (±3.0)aDenotes significance at P < .05 as compared with control subjects..016BMI >301 (4%)17 (37.0%)13 (27.7%)11 (23.4%)—Fasting glucose, mg/dL89.8 (±6.6)108.8 (±14.9)aDenotes significance at P < .05 as compared with control subjects.106.1 (±18.0)aDenotes significance at P < .05 as compared with control subjects.103.3 (±17.2)aDenotes significance at P < .05 as compared with control subjects..321Diabetes0 (0%)18 (39.1%)14 (29.8%)10 (21.3%).073HOMA-IRbData available from 76 (hepatic copper concentration) or 75 patients (HOMA-IR).1.69 (±0.47)5.09 (±3.71)aDenotes significance at P < .05 as compared with control subjects.4.24 (±3.44)2.89 (±1.74).009Triglycerides, mg/dL107.0 (±54.4)211.1 (±116.1)aDenotes significance at P < .05 as compared with control subjects.156.6 (±84.9)aDenotes significance at P < .05 as compared with control subjects.153.0 (±68.1)aDenotes significance at P < .05 as compared with control subjects..023Triglycerides >1504 (16%)26 (56.5%)aDenotes significance at P < .05 as compared with control subjects.22 (46.8%)22 (46.8%)—Low HDL1 (4%)9 (19.6%)6 (12.3%)5 (10.6%)—Hypertension2 (8%)18 (39.1%)aDenotes significance at P < .05 as compared with control subjects.13 (27.7%)9 (19.1%).041Metabolic syndrome0 (0%)18 (39.1%)aDenotes significance at P < .05 as compared with control subjects.9 (19.1%)6 (12.7%).005Serum iron, 59−158 mg/L103.6 (±33.8)135.8 (±37.4)aDenotes significance at P < .05 as compared with control subjects.115.4 (±39.6)103.2 (±39.6)<.001Tf Sat (%)26.9 (±10.3)36.4 (±8.1)aDenotes significance at P < .05 as compared with control subjects.33.3 (±9.7)28.4 (±6.8).013AST, 10−32 U/mL32.5 (±12.9)40.5 (±21.5)40.8 (±17.8)34.6 (±17.3).181ALT, 10−32 U/mL46.6 (±22.1)66.4 (±41.9)aDenotes significance at P < .05 as compared with control subjects.70.9 (±39.5)aDenotes significance at P < .05 as compared with control subjects.59.6 (±35.9).451AP, 40–129 U/mL96.7 (±46.3)82.0 (±23.1)aDenotes significance at P < .05 as compared with control subjects.82.5 (±26.9)aDenotes significance at P < .05 as compared with control subjects.98.9 (±42.6).029GGT, 10−71 U/mL109.8 (±52.6)61.2 (±36.9)98.1 (±77.1)90.2 (±55.7).229Cholesterol, mg/dL224.7 (±48.9)223.1 (±48.7)215.5 (±44.1)233.1 (±46.1).324LDL cholesterol, mg/dL140.3 (±48.4)141.0 (±45.6)141.1 (±37.9)149.5 (±42.0).371HDL cholesterol, mg/dL70.0 (±18.9)51.8 (±17.3)54.2 (±14.4)55.9 (±17.9).294Hemoglobin, g/dL14.6 (±1.5)15.6 (±1.2)aDenotes significance at P < .05 as compared with control subjects.15.5 (±1.2)aDenotes significance at P < .05 as compared with control subjects.15.1 (±1.5).101Smokers (%)4 (16)5 (10.8)6 (12.7)5 (10.6)—NOTE. Data are shown as means (±SD). P value (ANOVA) denotes significance level comparing NAFLD patients grouped as low serum and liver copper compared with patients grouped as high serum and liver copper as calculated by ANOVA. Proportions were compared using Fisher exact test.Pts, patients; NASH, nonalcoholic steatohepatitis; HIC, hepatic iron concentration; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; GGT, γ-glutamyl-transpeptidase; Tf Sat, transferrin saturation; (%) refers to percentage of patients in each study group.a Denotes significance at P < .05 as compared with control subjects.b Data available from 76 (hepatic copper concentration) or 75 patients (HOMA-IR). Open table in a new tab NOTE. Data are shown as means (±SD). P value (ANOVA) denotes significance level comparing NAFLD patients grouped as low serum and liver copper compared with patients grouped as high serum and liver copper as calculated by ANOVA. Proportions were compared using Fisher exact test. Pts, patients; NASH, nonalcoholic steatohepatitis; HIC, hepatic iron concentration; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; GGT, γ-glutamyl-transpeptidase; Tf Sat, transferrin saturation; (%) refers to percentage of patients in each study group. Patients with low serum or liver copper concentrations revealed a higher prevalence of isolated criteria of MS and fully established MS according to the modified WHO criteria applied and increased insulin resistance as calculated by HOMA-IR (Table 1). Moreover, HOMA-IR showed an inverse correlation with serum and liver variables of copper metabolism (Table 2). The comparison of 23 NASH patients with 117 NAFLD patients did not reveal a significant
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