Journal of the American College of Nutrition, Vol. 18, No. 6, 614-619 (1999)
Published by the American College of Nutrition
Altered Copper Status in Adult Men with Cystic Fibrosis
Susan S. Percival, PhD,
Gail P.A. Kauwell, PhD, RD,
Ellen Bowser, MS, RD, RN and
Mary Wagner, MD
Food Science and Human Nutrition Department (S.S.P., G.P.A.K.),University of Florida, Gainesville, Florida
Division of Pulmonary Diseases/Cystic Fibrosis, Department of Pediatrics (E.B., M.W.), University of Florida, Gainesville, Florida
Address reprint requests to: Susan S. Percival, Ph.D., Food Science and Human Nutrition Department, P.O. Box 110370, University of Florida, Gainesville, FL 32611.
 |
ABSTRACT
|
|---|
Objectives: To measure indices of copper status in adult men with cystic fibrosis (CF). A previous study in children showed changes in copper homeostasis compared to controls. This study was designed to investigate whether this observation persisted into adulthood.
Methods: This was a case-control age-matched study using seven men with CF and six healthy men. Blood samples were drawn into metal free tubes and fractionated into plasma, polymorphonuclear cells, mononuclear cells and erythrocytes. Cell fractions were assayed for copper and CuZn-superoxide dismutase; plasma was assayed for ceruloplasmin.
Results: The men with cystic fibrosis had significantly greater plasma copper and ceruloplasmin activity, yet had significantly lower copper-zinc superoxide dismutase activity in mononuclear and polymorphonuclear cells. Furthermore, the mononuclear cells of the cystic fibrosis subjects had about 45% percent less copper-zinc superoxide dismutase protein. Cellular copper levels were not statistically different between the two groups. A significant correlation was found between lung function and copper-zinc superoxide dismutase activity in the polymorphonuclear cells. Iron status was normal.
Conclusions: The results indicate that individuals with cystic fibrosis have altered copper distribution compared to control individuals. Some aspects are characteristic of an inflammatory response; however, other measures suggest that copper homeostasis may be abnormal. It is not known whether the deviation in copper homeostasis in these individuals is a result of poor copper absorption, inadequate dietary intake, a result of their chronic inflammation or a direct effect due to the defect in ion transport caused by the disease. However, this research suggests that the severity of the disease and the activity of a copper dependent enzyme may be related. Further work will be necessary to determine the cause of the abnormal copper homeostasis and whether correcting it has any bearing on the course of the disease.
Key words: cystic fibrosis, copper, CuZn-superoxide dismutase, copper status
|
INTRODUCTION
|
|---|
Cystic fibrosis (CF) is the most common lethal genetic disorder of Caucasians. The disease is caused by a mutation in the cystic fibrosis transmembrane conductance regulator that is responsible for the transport of chloride and sodium ions. Alterations in influx and efflux of these ions result in changes in the osmolarity of mucus and other secretions. The pancreatic duct is blocked by thick and sticky mucus that results in an inability to digest foods and a subsequent impairment in absorption of nutrients. The bronchial airways of the lungs are also blocked with mucus that impairs breathing and harbors bacteria and other infectious agents. Thus, CF patients have greater nutritional needs in fighting infections and providing energy for respiration because their absorption of nutrients is impaired. Maldigestion and malabsorption of protein, fat and fat-soluble vitamins have been fairly well characterized [1] and have decreased with the development of enzyme replacement therapy. The nutritional status of CF patients is directly correlated with their mortality and morbidity [2].
Copper is an essential trace element that has not been well studied in CF patients. Case studies of copper deficient humans document anemia and neutropenia [3,4]. Animal experiments have shown copper deficiency to result in heart, muscle, skin and bone disorders due to connective tissue impairment [5,6], possible cellular damage due to free radical toxicity [7–9], pancreatic atrophy [10,11] and impairment of the immune system [12,13]. A marginal copper deficiency in rodents has been shown to impair immunity even when standard parameters of copper status have not been affected [14]. The American population, in general, has been estimated to consume less than the estimated safe and adequate daily dietary copper intake [15,16]. CF patients may be at greater risk than the general population because their needs may be higher.
In a previous study, we examined copper status in blood and blood cells of children and adolescents with CF compared to age and gender matched controls [17]. We showed that there was no difference in plasma copper or ceruloplasmin (Cp) specific activity. Other nutritional parameters, like zinc and iron, were also not different. However, we found that the two copper dependent enzymes, cytochrome c oxidase and CuZn-superoxide dismutase (CuZn-SOD), were significantly lower in the blood cells of CF patients than those of controls. The study in children had limitations because the patients were from a wide age range: from prepubescent children aged 10 years through young adults aged 22 years. Both genders were studied, and the control groups had several overweight individuals. The copper parameters that were examined might have been variable due to body weight or hormonal factors, thus masking any significant differences. The current study was undertaken in adult males to minimize these variables and to determine if these changes persisted into adulthood.
|
METHODS
|
|---|
Subject Selection
Seven adult males diagnosed with CF were recruited from the Cystic Fibrosis Center at the Pediatric Pulmonary Division of the University of Florida. They were diagnosed on the basis of duplicate sweat tests with chloride and sodium concentration >60 mmol/L; all but one subject were pancreatic insufficient and taking pancreatic enzyme replacement therapy. CF subjects took a supplement (Scandipharm, Inc, Birmingham, AL) containing vitamins A, D, E, K, beta-carotene, water-soluble vitamins and zinc (15 mg zinc per day). Although zinc is known to interfere with copper absorption, this level is not high enough to have an impact. Subjects were limited to those who were not acutely ill nor had had a recent exacerbation of their CF. They were recruited at a CF Education Day program.
Six healthy adult males were recruited as control subjects from students enrolled in the University of Florida’s College of Agriculture. Selection criteria for both groups included the following: 1) 18 to 32 years of age, 2) no acute illness and 3) no anti-inflammatory medication. Weight was determined, height and, in the case of CF patients only, the percent of Forced Vital Capacity (FVC) and the percent of Forced Expiratory Volume (FEV-1); white blood cell counts and percent polymorphonuclear cells were obtained from the patient charts. FVC and FEV-1 are measures of pulmonary function. Informed written consent was obtained from all subjects, and the Institutional Review Board approved the protocol for the Protection of Human Subjects at the J. Hillis Miller Health Sciences Center.
Blood Collection
Fifteen mL of venous blood was drawn from each subject into metal-free collection tubes containing EDTA. Subjects had fasted overnight. Whole blood hemoglobin concentration was determined spectrophotometrically as cyanmethemoglobin (Drabkin’s Reagent, Sigma Diagnostics, St. Louis, MO). Hematocrit was determined by drawing duplicate samples of whole blood into capillary tubes and centrifuging for five minutes at 12,000 rpm. A micro hematocrit tube reading device was used to determine hematocrit levels. The remaining whole blood was layered on Ficoll-Hypaque, density 1.114, (Mono-Poly Resolving Medium®, ICN Biomedical, Costa Mesa, CA) and centrifuged at 25 C, 1000 x g for 60 minutes to separate whole blood into plasma, mononuclear cells (MN), polymorphonuclear cells (PMN) and erythrocytes. All processing and storage tubes were washed in 30 mmol/L nitric acid followed by thorough rinsing with distilled, deionized MilliQ water. Plasma was removed and stored at -85 C. The MN cells fraction is approximately 85% lymphocytes and 15% monocytes. The MN cells and PMN were washed twice with a balanced salt solution devoid of Ca2+ or Mg2+. The cell pellets were re-suspended in buffer containing 0.155 mol/L NH4Cl, 0.012 mol/L NaHCO3 and 0.105 mmol/L EDTA for 60 seconds to lyse contaminating erythrocytes. The cells then underwent a final washing in the balanced salt solution. MN and PMN cell pellets were re-suspended in a volume of distilled deionized H2O containing 10 µmol/L phenyl methyl sulfonyl fluoride to achieve a cell protein concentration of approximately 2.0 g/L. All isolated cell samples were sonicated, then stored at -85 C.
Biochemical Assays
Copper levels in the plasma and in sonicated homogenates of the PMN and MN cell fractions were determined by graphite furnace atomic absorption spectrophotometry. SeronormTM (Accurate Chemical Scientific Co., Westbury, NY) was used to validate the copper concentration. Variation from the known Seronorm copper concentration was less than 3.1%. The daily coefficient of variation was less than 8%. Matrix effects were evaluated and the slope of the standard additions varied less than 0.8% from the slope of the standard curve.
Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity was measured by inhibiting pyrogallol autoxidation as previously described [18,19]. Cu/Zn-SOD activity was measured after extraction with 0.4 vol of chloroform:ethanol (15:25) to eliminate dismutase activity derived from the mangano form of superoxide dismutase. One unit of activity is that which will inhibit the rate of pyrogallol autoxidation by 50%. ELISA methodology was used to estimate the amount of Cu/Zn-SOD protein in the MN cell fraction using a rabbit anti-human polyclonal antibody produced in our lab.
Plasma ceruloplasmin (Cp) oxidase was determined spectrophotometrically at 540 nm using o-dianisidine as the substrate [20]. The Cp ELISA was performed with antibodies purchased from Dako Corp (Carpenteria, CA), and the procedure of DiSilvestro et al. was followed [21]. The protein content in cell fractions was determined by the Coomassie dye-binding assay as described by the manufacturer (BioRad Laboratories, Richmond, CA).
Statistical Analyses
Statistical evaluation of the data was by Student’s unpaired t test and regression analysis using the software program SigmaStat (version 1.01) produced by Jandel Scientific (San Rafael, CA). Significantly different means and regressions were established by p values <0.05.
An F-statistic was use to test the hypothesis of equality of variance among subjects within each group. If the equality of variances was not rejected, then the hypothesis of equality of the means of the groups was tested using a t statistic. If the equality of variances was rejected, then the hypothesis of equality of means of the groups was tested using an approximate t statistic.
|
RESULTS
|
|---|
Demographics of the subject population are shown in Table 1. There were no significant differences between the mean values for age, height, weight and percent ideal body weight (IBW) of the CF men compared to the control men. Hemoglobin and hematocrit were not different between the groups.
In the CF group, there were significantly more Cp oxidase activity and Cp protein concentration in the plasma (Table 2). There were 38% more Cp protein in the CF group and 46% more Cp activity. The specific activity of Cp was not different between the two groups.
Plasma copper was significantly greater by 50% in the CF group than in the controls (Table 3). Copper values in the MN and the polymorphonuclear cell fractions were not statistically different.
The activity of CuZn-SOD was significantly lower in the PMN and the MN cells of the CF men (Table 4). The CF group had 46% less CuZn-SOD activity in the PMN fraction and 70% less activity in the MN fraction. The analysis for CuZn-SOD protein levels in the MN fraction showed that there was also significantly less CuZn-SOD protein in the CF group. The CF group had 43% less CuZn-SOD protein relative to 70% less activity resulting in significantly less specific activity in the MN cells of the CF men than in those of controls.
The clinical parameters of lung function and white blood cell counts were correlated with measures of copper status, which included copper levels, Cp measures and CuZn-SOD activity. The activity of CuZn-SOD in the PMN cell fraction was significantly and strongly correlated with FEV-1 (Fig. 1), FVC (data not shown) and percent of PMN (Fig. 2), having p values equal to 0.006, 0.019 and 0.030, respectively. Other indices did not correlate with one another, nor did they correlate with lung function or white cell counts.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1. Correlation between lung function and the activity of CuZn-SOD in polymorphonuclear cells in adult men with cystic fibrosis. FEV-1 values were obtained from the patient charts. CuZn-SOD was determined as described in the methods.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2. Correlation between the percentage of peripheral polymorphonuclear leukocytes with the activity of CuZn-SOD in the same cells. No data on the percent PMNs were available for two CF men.
|
|
|
DISCUSSION
|
|---|
The purpose of this study was to extend our observations of copper homeostasis in adult individuals with cystic fibrosis. The previous study focused on children and adolescents of ages before, during, and after puberty as well as both genders. This cohort was much more homogeneous than the previous study.
The two groups of men who volunteered for this study were very closely matched in age and height. CF individuals are usually smaller than their healthy controls. The fact that the average weight is similar between the two groups is due to one CF individual who was 170% of his ideal body weight. If this individual is not included in the calculation for mean weight, the difference in weight between healthy and CF men becomes significant: control men=111.9±9.2% IBW versus CF men=89.1±5.0% IBW, p=0.0002.
The indices of copper homeostasis that we measured were different between the two groups of men. The adult CF group had significantly higher Cp activity and Cp protein levels in the blood compared to the controls, although the values were within the normal published clinical range. The specific activity of Cp, however, was not different between the two groups. High Cp is characteristic of an inflammatory response [22]. We hypothesized that if the CF men were copper deficient, the specific activity of Cp would be lower in the CF group due to the fact there is not enough cofactor to provide the total activity, regardless of the level of Cp. In the previous study with children, the specific activity of Cp was 40% lower in the subjects than in the controls, yet this did not achieve significance at the p<0.05 level.
Two measures of iron status were not different between the two groups. Therefore, we conclude that any aberrations of copper are not due to a general malabsorption of minerals.
CuZn-SOD is a copper dependent enzyme that has potential to be a functional indicator of copper status. The MN and the PMN cell fractions of the CF individuals contained significantly less CuZn-SOD activity, suggesting an abnormality in copper homeostasis in this group. Rodent studies have shown that inflammation reduced the activity of CuZn-SOD in the livers of copper-adequate, -marginal and -deficient rats [23]; however, more research is needed to determine how this enzyme is influenced by chronic inflammation in other cell types. Also, it remains to be tested whether the CuZn-SOD activity in the CF individuals is lower due to a nutritional deficiency of copper, chronic inflammation or a defect caused by the CF disease.
It is noteworthy that the amount of CuZn-SOD protein in the MN cell fraction was also lower. Reduced CuZn-SOD protein has not been described in any dietary copper deficient animal study, and, as such, this enzyme has been described as a constitutively expressed protein [24]. An explanation for this is lacking.
Alda and Garay observed that copper transport into erythrocytes required the chloride anion exchanger [25]. From these results, we speculate that copper transport into cells may possibly be influenced by the defect in chloride transport in cystic fibrosis. Defective transport of copper into the cell would result in lower CuZn-SOD activity.
Copper levels in the leukocytes of the adults were not significantly different. Although the utility of this measurement of copper status has not been shown, it does suggest that the adult men with CF were not overtly copper deficient. It also suggests that circulating white blood cells do not acquire more copper from plasma that has high copper and Cp levels because correlations between Cp or plasma copper levels and leukocyte copper content were not significant. Furthermore, the activity of intracellular CuZn-SOD was not influenced by high serum copper levels as the activity of this enzyme was lower in cystic fibrosis patients.
The correlation between clinical tests and copper status parameters was determined as a potential marker in evaluating the progression or the severity of the disease. The activity of CuZn-SOD in the PMN cell fractions was highly correlated with both measures of lung function as well as the number of PMN cells in the blood; the lower the activity, the worse the lung impairment. Whether low CuZn-SOD activity is a cause of the symptoms or an effect of the disease is not known. CuZn-SOD functions to dismutate the superoxide anion, which is an important antioxidant mechanism for the cell. Hirase et al. showed that the life span of red blood cells was reduced in a woman diagnosed with overt copper deficiency [26]. Her erythrocyte CuZn-SOD activity was 20% of normal; they suggested this as the reason for the shortened life span of the cell. Accordingly, low activity of CuZn-SOD in the neutrophil might reduce the life span of this cell type in individuals with CF.
In summary, this research suggests that copper homeostasis in CF subjects is different from that in healthy controls. The changes in copper homeostasis are not a result of a general mineral abnormality because iron status was not affected in the men with CF. Further research will now be necessary to determine if this aberrant copper distribution is due to a defect in copper transport as a result of the chloride transport defect, a redistribution of copper because of the chronic inflammation or because of difficulties in copper absorption or dietary intake.
|
FOOTNOTES
|
|---|
Supported by NIH Grants MCJ-129612 and DDK R29-43518.
Florida Agricultural Experiment Station Journal Series Number R-07135.
Received April 1, 1999.
Accepted August 1, 1999.
|
REFERENCES
|
|---|
- Pencharz PB, Durie PR: Nutritional management of cystic fibrosis.
Ann Rev Nutr
13:
111–136,
1993.[Medline]
- Corey M, McLaughlin FJ, Williams M, Levinson H: A comparison of survival, growth, and pulmonary function in patients with cystic fibrosis in Boston and Toronto.
J Clin Epidemiol
6:
583–591,
1988.
- Danks DM: Copper deficiency in humans.
Ann Rev Nutr
8:
235–257,
1988.[Medline]
- Graham GG, Cordano A:
Copper deficiency in human subjects. In Prasad AS, Oberleas D (eds): "Trace Elements in Human Health and Disease. Volume 1 Zinc and Copper." New York: Academic Press, pp.
363–372,
1976.
- Medeiros DM, Davidson J, Jenkins JE: A unified prospective on copper deficiency and cardiomyopathy.
Proc Soc Exp Biol Med
203:
262–273,
1993.[Abstract]
- Romero-Chapman N, Lee J, Tinker D, Uriu-Hare JY, Keen CL, Rucker RR: Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin.
Biochem J
275:
657–662,
1991.
- Taylor CG, Bettger WJ, Bray TM: Effect of dietary zinc or copper deficiency on the primary free radical defense system in rats.
J Nutr
118:
613–621,
1988.
- Chao PY, Allen KGD: Glutathione production in copper-deficient isolated rat hepatocytes.
Free Radic Biol Med
12:
145–150,
1992.[Medline]
- Bartoli GM, Palozza P, Piccioni E: Enhanced sensitivity to oxidative stress in Cu,ZnSOD depleted rat erythrocytes.
Biochim Biophys Acta
1123:
291–295,
1992.[Medline]
- Weaver FC, Sorenson RL, Kobienia B: Nonenzymatic isolation and culture of adult islets from atrophic pancreata of copper deficient rats: a morphological analysis.
In Vitro Cell Dev Biol
24:
108–116,
1988.[Medline]
- Kishimoto S, Iwamoto S, Masutani S, Yamamoto R, Jo T, Saji F, Terada N, Sasaki Y, Imaoka S, Sugiyama T: Apoptosis of acinar cells in the pancreas of rats fed on a copper-depleted diet.
Exp Toxicol Pathol
45:
489–495,
1994.[Medline]
- Prohaska JR, Failla ML:
Copper and Immunity. In Klurfeld DM (ed): "Human Nutrition—A Comprehensive Treatise, Volume 8: Nutrition and Immunology." New York: Plenum Press, pp.
309–332,
1993.
- Lukasewycz OA, Prohaska JR: The immune response in copper deficiency.
Ann NY Acad Sci
587:
147–159,
1990.[Medline]
- Hopkins RG, Failla ML: Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conventional indicators of copper status.
J Nutr
125:
2658–2668,
1995.
- Klevay LM, Reck SJ, Jacob RA, Logan GM, Munoz JM, Sandstead HH: The human requirement for copper. I. Healthy men fed conventional American diets.
Am J Clin Nutr
33:
45–50,
1980.[Abstract/Free Full Text]
- Pennington JAT, Young BE, Wilson DB, Johnson RD, Vanderveen JE: Mineral content of foods and total diets: the selected minerals in foods survey, 1982–1984.
J Am Diet Assoc
86:
876–891,
1986.[Medline]
- Percival SS, Bowser EK, Wagner MH: Reduced copper enzyme activities in blood cells of children with cystic fibrosis.
Am J Clin Nutr
62:
633–638,
1995.[Abstract/Free Full Text]
- Percival SS: Cu/Zn superoxide dismutase activity does not parallel copper levels in copper supplemented HL-60 cells.
Biol Trace Elem Res
38:
63–72,
1993.[Medline]
- Percival SS, Bae B, Patrice M: Copper is required to maintain Cu/Zn superoxide dismutase during HL-60 cell differentiation.
Proc Soc Exp Biol Med
203:
78–83,
1993.[Abstract]
- Schosinsky KH, Lehmann HP, Beeler MF: Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride.
Clin Chem
20:
1556–1563,
1974.[Abstract]
- DiSilvestro RA, David EA: Enzyme immunoassay for ceruloplasmin: application to cancer patient serum.
Clin Chim Acta
158:
287–292,
1986.[Medline]
- Cousins RJ: Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin.
Physiol Rev
65:
238–309,
1985.[Free Full Text]
- DiSilvestro RA, Marten JT: Effects of inflammation and copper intake on rat liver and erythrocyte CuZn-superoxide dismutase activity levels.
J Nutr
12:
1223–1227,
1990.
- Percival SS, Harris ED: Regulation of CuZn Superoxide dismutase with copper. Ceruloplasmin maintains functional enzyme levels during differentiation of K562 cells.
Biochem J
274:
153–158,
1991.
- Alda JO, Garay R: Chloride (or bicarbonate)-dependent copper uptake through the anion exchanger in human red blood cells.
Am J Physiol (Cell Physiol)
259:
C570–C576,
1990.[Abstract/Free Full Text]
- Hirase N, Abe Y, Sadamura S, Yufu Y, Muta K, Umemura T, Nishimura J, Nawata H, Ideguchi H: Anemia and neutropenia in a case of copper deficiency: Role of copper in normal hematopoiesis.
Acta Haematol
87:
195–197,
1992.[Medline]
TOP
ABSTRACT
INTRODUCTION
