AbstractCystic fibrosis is a serious illness that affects thousands of individuals in this country. Often seen solely as a respiratory disease, people sometimes neglect the other dangers that stem from CF, including malnutrition. Teenage girls suffering from cystic fibrosis are especially at risk due to their increasing nutritional needs during the course of pubertal development. Many minerals, such as calcium, zinc, and iron, have been studied in relation to cystic fibrosis, but there is still not much information available on another essential element: copper. This study assesses the copper levels in adolescent females with CF by measuring copper activity and copper concentration in the patients’ blood using western blotting and substrate assays. Results show normal copper activity, around 0.25 g/L for the p-phenylenediamine oxidase activity and around 70 U/L for o-Dianisidine, but abnormal and puzzling copper concentration levels of 1000 to 1200 µg/L, which is somewhat higher than normal. Further studies need to be conducted to evaluate the long term effects of CF on copper status in this population. Concrete information on copper status and nutrition in CF patients can lead to a better quality of life for these patients.

Angel Li:

Introduction• Cystic fibrosis affects about 30,000 people in the United States

and is the most common inherited disease leading to a shortened life span among white people.

• CF inhibits a patient’s ability to digest and absorb necessary dietary antioxidants, which is a possible cause of disease progression.

• Nutrients and antioxidants are especially important in female adolescence, where the most growth and development occurs.

• Little data exists examining components of copper homeostasis in persons with CF.

• Due to a new “aging” CF patient population, it is essential to monitor nutritional status in order to improve patients’ quality of life

CYSTIC FIBROSIS & COPPER• Cystic fibrosis is an autosomal recessive disease

that causes the body to produce an abnormally thick, sticky mucus that clogs the small tubes and ducts of the lungs and digestive system.

• In addition to lung infections, pancreatic insufficiency leads to maldigestion, malabsorption, and, ultimately, malnutrition.

• Copper is an essential trace element that also functions as a cofactor in numerous enzymes that catalyze oxidation-reduction reactions.

• Copper deficiency is common among people with CF or other diseases that result in malabsorption of nutrients.

SUBJECTS & METHODS• Serum samples obtained from larger calcium kinetic study

conducted by Schulze et al.• 23 girls aged 7 to 18 were recruited from The Johns

Hopkins Hospital Cystic Fibrosis Center as well as two other regional CF centers (in Harrisburg, PA and Wilmington, DE).

• Laboratory procedures:– Polyacrylamide gel electrophoresis– Western blotting– Atomic absorption spectrophotometry– p-Phenylenediamine substrate assay– o-Dianisidine dihydrochloride substrate assay

Demographics Baseline Follow-Up n 23 17 Age (years) 12.8 ± 3.3 13.9 ± 3.5 Age Range 7 – 17 8 – 19 Height (cm) 147.8 ± 14.9 150.9 ± 13.0 Weight (kg) 40.9 ± 13.5 45.4 ± 13.3 Tanner Stage Range 1 – 5 1 – 5 Cp Activity (PPD g/L)

0.25 ± 0.08 0.27 ± 0.10 Cp Activity (o-D Units/L) 73.1 ± 27.0 67.3 ± 29.4 Plasma Cp (ratio*) 0.61 ± 0.23 0.90 ± 0.17 Plasma Copper (µg/L) 1002.0 ± 188.4 1197.8 ± 363.2

Table 1. Demographics and Copper/Ceruloplasmin Status in Adolescent

Females with Cystic Fibrosis

*In relation to positive control (when PC=1)**Values represent the means±S.D.

Baseline vs. follow-up

Table 2. Average Change per Subject in Baseline vs. Follow-Up Data (with S.D.)

Analysis Performed Average % Change per Subject Cp Activity (PPD)

4.6 ± 40.0 Cp Activity (o-D) -11.1 ± 33.8 Plasma Cp 45.1 ± 50.1 Plasma Copper 20.5 ± 30.0 Values represent the percentage±S.D.

Western Blot (Baseline)

Fig 3. Western Blot Results for Baseline Samples

Western blot (follow-up)

Fig 4. Western Blot Results for Follow-Up Samples

discussion• Copper status in adolescent females with cystic fibrosis is

comparable to that of healthy individuals.– All values fell within normal range when compared to already published data of

normal adolescents as well as those suffering from CF.– Little change between both ceruloplasmin activity and copper

concentration at baseline versus follow-up.• Serum ceruloplasmin concentration was higher in the follow-

up data than that of the baseline.– Since ceruloplasmin is an acute phase reactant, the rise in

concentration could be attributed to the protein’s response to inflammation, disease, and trauma, possibly correlating to disease progression in CF.

• It is extremely difficult to analyze longitudinal data; further studies need to be conducted to evaluate CF and copper status.

AcknowledgementsI would like to thank my mentor, Dr. Kimberly O’Brien, for guiding me throughout this project and helping me with the writing of this paper. I am also indebted to Dr. Z. Leah Harris as well as Dr. Xuying Xu and Sokhon Pin for allowing me to conduct my research in their lab and helping me in every step of my laboratory work. Thank you to Dr. Kerry Schulze and Jung Park for teaching me the many useful data analysis tools that I needed to write this paper. And last but not least, I would like to acknowledge my mentor teacher, Mrs. Michelle Bagley, for providing me with the opportunity to have such a wonderful high school research experience.

CF LUNGS and PANCREAS

a

b

cFig 1. (a) mucus-filled lungs (b) normal pancreas

(c) pancreas from a cystic fibrosis patient

http://www.hipusa.com/eTools/webmd/A-Z_Encyclopedia/CysticFibrosis.jpg http://medgen.genetics.utah.edu/photographs/pages/cystic_fibrosis.htm

PAGE & Western Blotting

a

b

Fig 2. (a)

polyacrylamide gel

electrophoresis (b) gel transfer

(c) protein-antibody binding

http://www.biology.arizona.edu/immunology/activities/western_blot/graphics/electro.gif

http://www.nationaldiagnostics.com/ http://www1.amershambiosciences.com/