Redox Imbalance and the Development of Cystic Fibrosis Diabetes

Aim 1: Three groups of subjects will be evaluated: 1. 27 CF children with class I-III
mutations aged 1 to 9 years with normal glucose tolerance (NGT), 2. 27 age-matched controls
with NGT, and 3. 15 CF children with class IV-VI CFTR mutations ages 1 to 9 years. After
obtaining informed consent from the parents, subjects will undergo an overnight fast with
nothing to eat or drink except water for 10 hours. The children will be asked to withhold all
short-acting bronchodilators prior to the start of the study visit and to withhold all
long-acting bronchodilators for a minimum of ten hours prior to the start of the study visit.
Prior to the study visit, parents will be given a three-day diet journal in which they will
record in detail everything each child consumes during the recording period. Details include
amount of food or drink and brand names as well as pancreatic enzyme replacement therapy
(PERT) dose given with each meal or snack. The recording period will include two week days
and one weekend day. The children will have an intravenous (IV) line placed and blood drawn
for measurement of plasma glucose, insulin, and cysteine (CyS)/cystine (CySS) redox state.
Then the child will be given 1.75 gm/kg to a maximum of 75 gm of an oral glucose solution to
drink and will be coached to drink it within 10 minutes. Blood will be drawn 30 minutes after
glucose ingestion for repeat measures of glucose, insulin, and redox status. Blood will be
drawn 2 hours after glucose ingestion for repeat measures of glucose, insulin, and redox
status. Subjects who completed the protocol will be approached to reconsent and repeat the
protocol annually to track their level of glucose intolerance and monitor for conversion to
CF Diabetes.

For children who are able, exhaled breath condensate (EBC) will also be collected prior to
the OGTT and immediately following completion of the 2 hour blood draw. EBC will be analyzed
for Cys/CySS redox coupling.

Aim 2a: Forty-four CF subjects 12 years of age or older with NGT or impaired glucose will
undergo a meal challenge with half randomized to receive a test meal with a high glycemic
index and half a meal with a low index. Subjects will fast overnight for 10 hours taking
nothing to eat or drink except water. Subjects will be instructed to withhold all
short-acting bronchodilators for a minimum of four hours prior to the start of the study
visit and withhold all long-acting bronchodilators for a minimum of ten hours prior to the
start of the study visit. Following informed consent, participants will first undergo
indirect calorimetry for determination of resting metabolic rate and substrate oxidation;
this will occur only in participants 16 years old and older. An intravenous line (IV) will
then be inserted; and an iPro sensor placed for continuous glucose monitoring (CGM);
placement of the CGM will be optional as our experience with this protocol indicates that
some CF patients will not volunteer for CGM placement but will for IV placement. If the iPro
sensor is inserted; it must be in place for a minimum of 60 minutes prior to the baseline
blood draw to allow the iPro sensor device to detect interstitial glucose levels accurately.
Blood will be drawn for measurement of glucose, insulin, Cys/CySS redox status,
acylcarnitine, total carnitine, proteomics and metabolomics analysis on subjects 16 years
older and older. For the subjects 12 years old to 15 years old, blood will be drawn for
measurement of glucose, insulin, Cys/CySS redox status, and proteomics. The subject will then
be instructed to eat one of two tests meals in ten minutes. Subjects must complete the meal.
Meals will be isocaloric breakfasts with one meal having a high glycemic index and one a low.
The nutrient composition of each meal will be 10 kcal per kg, 50% kcal from carbohydrates,
20% kcal from protein, and 30% kcal from fat. Blood will be drawn for repeat measures of
glucose, insulin, CyS/CySS redox state1, 2, and 3 hours after the test meal on all subjects.
Blood will be drawn for proteomics at base line and at 3 hours on all subjects. Additional
blood will be drawn at baseline and 2 hrs after the meal for measurement of acylcarnitine,
total carnitine,and metabolomics and biomarkers related to fatty acid metabolism on subjects
16 years old and older. Following the 3 hour blood draw, the IV line and iPro sensor, if
placed, will be discontinued. A whole-body dual energy X-ray absorptiometry (DEXA) scan will
be administered at the completion of the 3 hr blood draw for analysis of body composition;
this test will be performed only on those subjects 16 years old and older. Female
participants will be required to complete a urine pregnancy test prior to the DEXA scan to
confirm a non-pregnant status. If the participant's urine pregnancy test shows a positive
pregnancy, the DEXA scan will not be completed. Following the DEXA scan, the subject will be
discharged home. Prior to their study visit, participants16 years and older will be given
instructions to complete a 3-day food record for estimation of usual dietary intake as part
of their standard CF care.

Aim 2b. Forty-four CF subjects 12 years of age or older with NGT or impaired glucose will
initially undergo a test beverage challenge, using a test soda that contains 60% fructose and
40% glucose or fruit juice that contains a combination fructose, glucose, and sucrose at a
dose of 1.75 grams per kilogram body weight to a maximum of 75 grams. One to four weeks later
all subjects will have an OGTT, that is, ingestion of oral glucose solution at a dose of 1.75
grams per kilogram to a maximum of 75 grams. Of the 44 subjects drinking the test beverage,
twenty-two will be administered the soda, and twenty-two will be administered fruit juice
Subjects will fast overnight for 10 hours taking nothing to eat or drink except water.
Subjects will be instructed to withhold all short-acting bronchodilators for a minimum of
four hours prior to the start of the study visit and withhold all long-acting bronchodilators
for a minimum of ten hours prior to the start of the study visit. After obtaining informed
consent participants will have an intravenous line (IV) inserted. Blood will be drawn for
measurements of glucose, insulin, and Cys/CySS redox status at baseline or zero minutes. The
subject will then be instructed to drink a test beverage, either a test soda or fruit juice,
in ten minutes that was prepared by the research bio-nutritionist. Repeat blood samples will
be drawn for measures of glucose, insulin, and CyS/CySS redox state ½ hour, 1 hour and 2
hours from the initial consumption of the test beverage drink. During the study visit a
beverage intake questionnaire will be administered to assess the patient's overall intake of
sweetened beverages. At the completion of the visit the participant will be scheduled for a
subsequent visit in one to four weeks for repeat testing using the same guidelines as above,
but the test drink to be used is a glucose solution (glucola) used for the standard OGTT. The
75 gram glucola drink will be used for testing; the dose administered will be determined by
multiplying the patients weight x 1.75 grams; the dose administered is not to exceed 75
grams. Subjects must consume all of the drink. Blood samples will be drawn at baseline (prior
to glucola intake) and then repeat blood samples will be drawn for measures of glucose,
insulin, and CyS/CySS redox state ½ hour, 1 hour and 2 hours from the initial consumption of
the glucola drink. Insertion of the continuous glucose monitor is optional during the test
beverage challenge and the redoxy OGTT.

Aim 1:

Inclusion Criteria:

For CF children -

1. CF diagnosed by pilocarpine electrophoresis sweat test;

2. CF Transmembrane Conductance Regulator (CFTR) mutation analysis showing two Class I to
III mutations;

3. Aged 1 to 9 years;

4. On a clinically stable medical regimen for at least three weeks;

5. No IV antibiotics for a respiratory exacerbation for at least three weeks; and

6. No hospitalization for at least six weeks.

For age-matched controls -

1. No acute illness for at least six weeks;

2. Never been hospitalized except at birth following a full term delivery;

3. Aged 1 to 9 years; and

4. Without any chronic illness requiring prescription medications.

Exclusion Criteria:

For CF children -

1. Current or past diagnosis of CFRD; or

2. Parents unwilling to have an IV inserted for blood draws.

For controls -

1. Parents unwilling to have an IV inserted for blood draws.

Aim 2:

Inclusion Criteria:

1. CF diagnosed by pilocarpine electrophoresis sweat test;

2. CFTR mutation analysis showing two Class I to III mutations;

3. Aged 12 years or older;

4. Normal OGTT done within 6 months prior to study visit;

5. On a clinically stable medical regimen for at least three weeks; and

6. No IV antibiotics for a respiratory exacerbation for at least three weeks.

Exclusion Criteria:

1. Current or past diagnosis of CFRD; or

2. Current or past diagnosis of CF prediabetes by OGTT.

3. Allergy or intolerance to egg or dairy products