Palmitate Breath Test to Assess Fatty Acid Oxidation in Non Alcoholic Fatty Liver Disease (NAFLD)

NAFLD is the most common cause for liver enzyme abnormalities in the western world. It is
estimated to affect approximately 30% of the US population and is closely associated with the
metabolic syndrome and insulin resistance. Dysregulation of fatty acid metabolism and
abnormal function of the adipose tissue is thought to be a major feature of insulin
resistance and NAFLD. Whether fatty acid oxidation is also dysregulated in patients with
NAFLD is unclear. In this study we aim to measure total-body fatty acid oxidation of an oral
fat load using the BreathID breath test device and a meal containing 13C-labeled palmitate.
Results will be compared between patients with NAFLD, healthy controls and patients with
metabolic syndrome but not NAFLD, as well as within NAFLD patients before and after
therapeutic interventions.

- INCLUSION CRITERIA:

A. General Inclusion Criteria for all subjects:

1. Age above 21 years, male or female.

2. Estimated average alcohol consumption < 30 g/d for men or < 20 g/d for women in the 6
months prior to enrollment and no binge-drinking behavior.

B. Inclusion Criteria for NAFLD:

1. Clinical suspicion of NAFLD, defined by a liver biopsy result consistent with NAFLD or
the presence of at least two of the following criteria:

1. Suggestion of liver fat by an imaging study (ultrasound, CT scan, MRI or MR
spectroscopy) performed in the 6 months prior to enrollment.

2. Elevated aminotransferase levels (ALT > 31 U/L for men or > 19 U/L for women, or
AST > 30 U/L) on at least two occasions in the 6 months preceding enrollment.

3. Presence of the metabolic syndrome, defined according to the modified AHA/NCEP
criteria30 as the presence of at least three of:

- Abdominal obesity, defined as waist circumference > 102 cm for men or > 88
cm for women

- Elevated triglycerides (> 150 mg/dL) or the use of medication to lower
triglycerides

- Reduced HDL cholesterol (< 40 mg/DL for men or < 50 mg/dL for women)

- Elevated blood pressure (> 135/80 mmHg) or use of medication for
hypertension

- Elevated fasting glucose levels (> 100 mg/dL) or use of antidiabetic
medication

For the purpose of inclusion, the presence of overt diabetes mellitus type 2 will be
considered equivalent to the presence of the metabolic syndrome, even if the other
criteria are absent.

For the longitudinal analysis, inclusion criteria:

2. Enrolled in 13-DK-0002

or

3. Non-alcoholic liver disease subjects treated off-protocol with lifestyle intervention
or vitamin E treatment.

C. Inclusion Criteria for non-NAFLD metabolic syndrome:

1. Evidence of metabolic syndrome as defined in NAFLD criterion 1.c.

2. Normal transaminases (ALT less than or equal to 31 U/L for men or less than or equal
to19 U/L for women, or AST less than or equal to 30 U/L) at screening

3. Absence of liver fat by imaging or liver biopsy within 6 months of screening with no
(or trace) liver fat

D. Inclusion criteria for healthy volunteers

1. No history of known liver disease

2. Not on any regular systemic medications (with the exception of oral contraceptives)

3. BMI less than or equal to 25 kg/m2

4. Non diabetic

5. Normal ALT and fasting glucose

EXCLUSION CRITERIA:

1. Disorders leading to CO2 retention or significant respiratory abnormalities such as
severe congestive heart failure, severe pulmonary hypertension or severe chronic
obstructive lung disease.

2. Disorders interfering with substrate absorption such as gastric bypass surgery,
malabsorption disorders, use of orlistat or bile acid sequestrants, or extensive small
bowel resection.

3. Pregnancy or lactation

4. Inability to remain seated for the duration of the test.

5. Inability to understand and give informed consent

6. Chronic infection with hepatitis C virus (HCV) or hepatitis B virus (HBV). Patients
who were treated successfully for HCV and achieved sustained virological response can
be eligible for enrollment > 18 months after treatment cessation. Patients who are
inactive carriers of HBV (HBV DNA < 1000 copies/mL, HBeAg negative, Anti HDV negative)
for at least 12 months prior to enrollment are also eligible. Patients receiving
antiviral therapy are ineligible.

7. Concomitant liver disease such as autoimmune hepatitis, primary biliary cirrhosis,
primary sclerosing cholangitis, Wilson s disease, alpha-1 antitrypsin deficiency.

8. Presence of definite or probable drug-induced liver injury. In the case of
lipid-lowering, anti-hypertensive or anti-diabetic medications that are suspected to
cause elevation of aminotransferases, patients will be eligible if treatment is
associated with stable enzyme levels for at least 6 months and inclusion criteria 2a.
and 2c. are both present.

9. Treatment with medications known to cause fatty liver disease such as atypical
neuroleptics, tetracycline, methotrexate or tamoxifen

10. Diabetic patients requiring insulin treatment

11. Milk or soy protein allergy

12. Uncontrolled hypo/hyperthyroidism