pufa protocol

A Randomized, Masked, Controlled Study of Omega-3 Polyunsaturated Fatty Acid vs Monounsaturated Fatty

Acid Dietary Supplementation for the Treatment of Nonalcoholic Fatty Liver Disease

NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES

Grant#: U01 DK061728-03S2

CONFIDENTIAL

Final Version 04-SEP-2005

ii PUFA P&F Protocol Study Personnel

U:\KVK Research\NASH CRN\PUFA P& F\Protocol\PUFA PROTOCOL final.doc 04-SEP-2005 Last saved by jamesn STATUS: Final

STUDY PERSONNEL

Prinicipal investigator: Kris V. Kowdley, MD Professor of Medicine Division of Gastroenterology University of Washington Co-Investigators: Todd Richards, PhD Professor Department of Radiology 206-598-6725 Robert Carithers Jr., MD Professor Department of Medicine/GI 206-598-4956 David S. Weigle, MD Professor Medicine/ Metabolism 206-341-4621 Anne Larson, MD Assistant Professor Department of Medicine/GI 206-598-6700 Bruce Tung, MD Assistant Professor Department of Medicine/GI 206-685-7174 Jim Nelson, PhD Research Coordinator Department of Medicine/GI 206-221-4537 Virginia Mugford Research Coordinator Department of Medicine/GI 206-221-4538 Mario Kratz, Ph.D. Research Fellow Medicine/ Metabolism 206-685-9376 Colleen Matthys, RD Nutrition Manager Clinical Research Center 206-598-4529 Holly Callahan, RD Research Dietician Clinical Research Center 206-598-4529 Julie Bares Research Coordinator Department of Medicine/GI 206-221-4138 Samantha Stephan Research Assistant Department of Medicine/GI 206-221-3497 Narendra Siddaiah, MD Senior Fellow Department of Medicine 206-616-8475 Jacob Alexander, MD Senior Fellow Department of Medicine 206-616-8475 Study Coordinator: Jim Nelson, PhD Division of Gastroenterology

University of Washington Statistician: TBD Data Manager: Virginia Mugford Division of Gastroenterology

University of Washington Clinical Monitors: Narendra Siddaiah, MD Jacob Alexander, MD Safety Officer: Kris V. Kowdley MD

iii PUFA P&F Protocol Design Synopsis


DESIGN SYNOPSIS

Title A Randomized, Masked, Controlled Study of Omega-3 Polyunsaturated Fatty Acid vs Monounsaturated Fatty Acid Diet Supplementation for the Treatment of Nonalcoholic Fatty Liver Disease

Sponsor NIDDK

Objective To determine the effect of dietary supplementation with ω-3 polyunsaturated vs

monounsaturated fatty acids on intrahepatic fat content in patients with non-alcoholic fatty liver disease as determined by magnetic resonance spectroscopy

Study design Randomized, Masked, Controlled Treatment groups

Group A: 4g per day omega-3 polyunsaturated fatty acids (18 - 725 mg fish oil capsules containing: 18% eichosapentaenoic acid [EPA] and 12% docosahexaenoic acid [DHA])

Group B: 10g per day monounsaturated fatty acids (18 - 725 mg safflower oil

capsules containing: 80% oleic acid) Study duration

• 8-week treatment period

Sample size 30 (15 per group) Inclusion criteria

• Acceptance into NASH CRN Database Study • Exclusion from or unwillingness to participate in the NASH CRN PIVENS Study • Histological diagnosis or imaging study suggesting NAFLD with at least 20%

steatosis • Willingness to maintain study diet for duration of the study • At least 18 years of age • No contraindication for MRI scanning (i.e., pacemaker, shunts etc)

Exclusion criteria

• Use of lipid lowering drugs (i.e., statins and fibrate drugs) • Use of insulin or thiazolidinediones • Use of drugs possibly associated with NAFLD (amiodarone, methotrexate,

systemic glucocorticoids, tetracycline, tamoxifen, estrogens at doses greater than those used for hormone replacement, anabolic steroids, valproic acid, or other known hepatotoxins) for more than 2 consecutive weeks in the 2 years prior to screening

iv PUFA P&F Protocol Design Synopsis


• Initiation of anti-diabetic drugs (insulin, biguanides, sulfonylureas, metformin, thiazolidinediones) in the 3 months prior to randomization

• Initiation of anti-NASH drugs (thiazolidinediones, vitamin E, metformin, UDCA, SAM-e, betaine, milk thistle, gemfibrozil, anti-TNF therapies, probiotics) in the 3 months prior to randomization

Primary outcome • Reduction of intrahepatic fat content as determined by magnetic resonance

spectroscopy Secondary outcomes

• Change in serum aminotransferase levels • Change in lipid profile • Change in levels of proinflammatory cytokines • Change in insulin resistance determined by HOMA

v PUFA P&F Protocol Background


TABLE OF CONTENTS

STUDY PERSONNEL ...................................................................................................... II

DESIGN SYNOPSIS........................................................................................................ III

TABLE OF CONTENTS...................................................................................................V

1 BACKGROUND ........................................................................................................ 1

1.1 NONALCOHOLIC FATTY LIVER DISEASE ....................................................................................................... 1 1.1.1 Natural History and Pathogeneis......................................................................................................... 1 1.1.2 Epidemiology....................................................................................................................................... 1 1.1.3 Potential Treatment Options for NAFLD ............................................................................................ 2

1.2 LIPID METABOLISM AND NAFLD ................................................................................................................. 2 1.2.1 The Role of the Liver ........................................................................................................................... 2 1.2.2 The Role of Omega-3 Polyunsaturated Fatty Acids............................................................................ 4

1.3 PREVIOUS STUDIES OF OMEGA-3 POLYUNSATURATED FATTY ACIDS............................................................ 5 1.3.1 Human Clinical Trials .......................................................................................................................... 5 1.3.2 Animal Models of NAFLD ................................................................................................................... 6

2 NASH CLINICAL RESEARCH NETWORK.............................................................. 7

3 STUDY DESIGN........................................................................................................ 8

3.1 PRIMARY HYPOTHESIS AND STUDY OBJECTIVES ......................................................................................... 8 Hypothesis....................................................................................................................................................... 8 Study objective ................................................................................................................................................ 8 Primary outcome ............................................................................................................................................. 8 Secondary outcomes....................................................................................................................................... 8

3.2 OVERVIEW................................................................................................................................................. 8 3.3 BLINDING AND RANDOMIZATION .................................................................................................................. 9 3.4 NUTRITION COUNSELING AND ENERGY ASSESSMENT .................................................................................. 9 3.5 STUDY TREATMENT ................................................................................................................................... 9

3.5.1 Omega-3 Polyunsaturated Fatty Acid Supplements ........................................................................... 9 3.5.2 Monounsaturated Fatty Acid Supplements ....................................................................................... 10 3.5.3 Dosage .............................................................................................................................................. 10 3.5.4 Storage and Dispensing Protocol...................................................................................................... 10 3.5.5 Quality Control................................................................................................................................... 10

4 STUDY POPULATION............................................................................................ 11

4.1 SAMPLE SIZE........................................................................................................................................... 11 4.2 INCLUSION CRITERIA................................................................................................................................ 11 4.3 EXCLUSION CRITERIA............................................................................................................................... 11

5 SCHEDULE OF VISITS AND PROCEDURES ....................................................... 13

5.1 STUDY VISITS .......................................................................................................................................... 13 5.1.1 Screening Visit................................................................................................................................... 13 5.1.2 Baseline Visit ..................................................................................................................................... 13 5.1.3 Interim Visit........................................................................................................................................ 13

vi PUFA P&F Protocol Background


5.1.4 Final Visit ........................................................................................................................................... 13 5.2 PROCEDURES.......................................................................................................................................... 14

5.2.1 Measurement of Liver Fat ................................................................................................................. 14 5.2.2 Body Anthropometrics ....................................................................................................................... 15 5.2.3 Measurement of Body Fat ................................................................................................................ 15 5.2.4 Fatty Acid Oxidation .......................................................................................................................... 15 5.2.5 Lipoproteins ....................................................................................................................................... 15 5.2.6 Oral Glucose Tolerance Tests........................................................................................................... 16 5.2.7 Blood Pressure.................................................................................................................................. 16 5.2.8 Hepatic Necroinflammation and Cytokine Analysis........................................................................... 16 5.2.9 Diet, Physical Activity and Quality Of Life Questionnaires................................................................ 16

6 DATA MANAGEMENT ........................................................................................... 17

6.1 SOURCE DOCUMENTATION AND CASE REPORT FORMS ............................................................................. 17 6.2 DATA QUALITY ASSURANCE ..................................................................................................................... 17 6.3 STATISTICAL ANALYSIS ............................................................................................................................ 17

7 HUMAN SUBJECTS ISSUES................................................................................. 18

7.1 GENERAL GUIDELINES ............................................................................................................................. 18 7.2 INFORMED CONSENT................................................................................................................................ 18 7.3 HIPAA AUTHORIZATION........................................................................................................................... 18 7.4 SUBJECT CONFIDENTIALITY ...................................................................................................................... 18 7.5 REVIEW OF ORIGINAL SUBJECT RECORDS ................................................................................................ 19 7.6 AMENDMENTS TO THE PROTOCOL............................................................................................................. 19

8 DATA SAFETY MONITORING PLAN .................................................................... 20

8.1 ADVERSE EVENT GRADING........................................................................................................................ 20 8.2 STUDY MONITORING AND SAFETY REPORTS ............................................................................................. 20 8.3 ADVERSE EVENT (AE) REPORTING........................................................................................................... 20 8.4 SERIOUS AES (SAES) REPORTING........................................................................................................... 21 8.5 WITHDRAWAL OF SUBJECTS FROM STUDY ............................................................................................... 21

9 REFERENCES........................................................................................................ 22

1 PUFA P&F Protocol Background

1 BACKGROUND

1.1 NONALCOHOLIC FATTY LIVER DISE ASE

1.1.1 Natural History and Pathogeneis Nonalcoholic Fatty Liver Disease (NAFLD) encompasses the spectrum of liver disorders which, in the absence of significant alcohol consumption (<20 gm per day in women, <30 gm per day in men), range from “simple steatosis” or “fatty liver” to steatosis with necroinflammation and varying degrees of fibrosis termed Nonalcoholic steatohepatitis or NASH for short (1). NASH is a more serious form of the disease, which can ultimately lead to cirrhosis, hepatocellular carcinoma and end-stage liver disease (2-5). As shown in figure 1, the current model for the pathogenesis of NAFLD is the so-called “two-hit” hypothesis (6, 7). According to this model, the first “hit” in the pathogenesis of NASH may be any process leading to accumulation of hepatic free fatty acids resulting in a histologic picture of hepatic steatosis. If the process were to stop at that point, only “simple” steatosis would result and a balance between pro and anti-inflammatory as well as apoptotic and necrotic processes in the liver would be maintained. In the face of a second or third “hit”, this balance would be disrupted in favor of processes leading to hepatocellular injury, subsequent development of fibrosis, and progression to cirrhosis.

Figure 1. The “two hit” model of NAFLD pathogenesis. NAFLD is a multi-step, multi-factorial disease resulting from a variety of aberrant cellular processes within the liver.

1.1.2 Epidemiology

Insulin Resistance Oxidative Stress

FFA, Iron, TNFα

Inflammatory Cytokines

Normal

NAFL NASH

Cirrhosis

ROS Lipid peroxidation

1st Hit 2nd Hit

NF-ĸB



The prevalence of NAFLD in the U.S has been estimated to range from 7.9-39% (8-10). Recently, a large-scale study using magnetic resonance spectroscopy (Dallas Heart Study, n=2349) estimated the prevalence of NAFLD to be 33.6% (11). NAFLD is now considered to be the most common cause of elevated serum aminotransferase levels with up to 70% of these patients diagnosed with NAFLD (10). The most common risk factors for NAFLD are obesity, diabetes mellitus and hyperlipidemia. The prevalence of NAFLD rises dramatically in individuals with these co-morbid conditions with prevalence estimates ranging from 39-93% in the obese (5, 12); 21-78% in diabetics (12, 13); and 4-92% in individuals with hyperlipidemia (1, 14). Moreover, given the current epidemic of obesity and diabetes in the United States, NAFLD is expected to increase in prevalence as this segment of the population continues to grow (15, 16).

1.1.3 Potential Treatment Options for NAFLD Currently there are neither clinically proven nor widely accepted treatment therapies for NAFLD. A general recommendation of diet control, coupled with an exercise regimen to achieve weight loss, is perhaps the most often prescribed management strategy. However, the effectiveness of diet and exercise has been insufficiently evaluated in controlled clinical studies. In the last few years a number of small-scale drug therapy studies have examined the effectiveness of insulin sensitizing agents (17-25, 36), antioxidants (26-36) or lipid-lowering drugs (37-44). Many of these agents have shown promise but will need to be evaluated in long term, large-scale clinical trials. Ursodexycholic acid, (UDCA) a natural bile salt with known hepatoprotective properties has also been extensively studied (37, 42, 45-50). However, UDCA showed no beneficial effects in a large, long term, multi-center study in comparison to placebo (50). A summary of the published NAFLD treatment studies are shown in Table 1.

1.2 LIPID METABOLISM AND NAFLD

1.2.1 The Role of the Liver Lipids are the body’s primary energy reserve and yield more then twice as much energy per gram than do carbohydrates or proteins (55). The liver has a central role in the metabolism of lipids for energy production, storage and transport (see figure 2; 56). Plasma free fatty acids (FFA), released from adipose tissue, are taken up by the liver and are either oxidized in the mitochondria for ATP production or are esterified to form triglycerides (TG). The hepatic TG storage pool is continually utilized for energy production via lipolysis to FFA or incorporation into very low density lipoproteins (VLDL) for transport to meet the energy requirements of extrahepatic tissues. Steatosis occurs when the TG storage pool exceeds the oxidative and VLDL secretory capacity of the liver. Multiple mechanisms contribute to hepatic steatosis via increased circulating free fatty acid concentrations in patients with obesity and insulin resistance, two common comorbid conditions of NAFLD. These include increased visceral fat stores leading to greater hepatic FFA flux via the portal vein, decreased insulin suppression of the key lipolytic enzyme hormone-sensitive lipase (HSL) in adipocytes, increased hepatic triglyceride synthesis and impaired mitochondrial β-oxidation (57). Lipogenesis, the de novo production of lipids from carbohydrates within the liver, is another major pathway contributing to hepatic TG accumulation. As shown in figure 2, this process is independently positively regulated by both glucose and insulin (56). In both instances this effect is transcriptionally mediated through the binding of transcription factors of the basic helix-loop-helix/leucine zipper class;



Table 1 Study (Reference) Drug Design Daily dose Duration N Primary endpoint Cytoprotective agents

Laurin et al, 1996 (37) UDCA Open-label 13-15 mg/kg 12 mo 24 Improved histology Holoman et al, 2000 (47) UDCA Open-label 750 mg 6-12 mo 24 Improved LFT’s Kiyici et al, 2003 (42) UDCA Open-label 13-15 mg/kg 6 mo 17 Improved LFT’s Bagci et al, 2003 (49) UDCA Open-label 250 mg 9-12 mo 28 Improved histology Lindor et al, 2004 (50) UDCA Blind,

randomized, controlled

13-15 mg/kg 24 mo 166 No improvement

Antidiabetic Coyle et al, 1999 (17) Metformin Open-label 500 mg 4-11 mo 2 Improved histology Caldwell et al, 2001 (18) Troglitazone Open-label 400 mg 4-6 mo 10 Improved histology Acosta et al, 2001 (19) Pioglitazone Case series NR 2-12 mo 8 Improved histology Marchesini et al, 2001(20) Metformin Open-label 1500 mg 4 mo 20 Improved LFT’s Azuma et al, 2002 (21) Pioglitazone Open-label 15 mg 3 mo 7 Improved LFT’s Tetri et al, 2003 (22) Rosiglitizone Open-label 8 mg 48 wk 30 Improved histology Oruc et al, 2003 (24) Diet vs

Metformin vs Rosiglitizone

Randomized Met-1000 mg Rosi- 4 mg

3 mo 64 Improved LFT’s

Pomrat et al, 2004 (25) Pioglitozone Open-label 30 mg 48 wk 18 Improved histology Antioxidant

Fu et al, 1998 (26) LAB * Open-label NR 12 wk 4 Variable Lavine 2000 (27) # Vitamin E Open-label 400-1200 IU 4-10 mo 11 Improved LFT’s Gulbahar et al, 2000 (28) N-Acetylcysteine Open-label 1000 mg 3 mo 11 Improved LFT’s Miglio et al, 2000 (29) Betaine Blind,


300 mg 8 wk 191 Improved LFT’s

Abdelmalek et al, 2001 (30) Betaine Open-label 20 g 12 mo 8 Improved histology Yakaryilmaz et al, 2003(31) Vitamin E Open-label 400-800 mg 6 mo 16 Improved histology Kawanaka et al, 2004 (35) Vitamin E Open-label 300 mg 6 mo 10 Improved LFT’s

Antihyperlipidemic Laurin et al, 1996 (37) Clofibrate Open-label 2 g 12 mo 16 No improvement Basaranoglu et al,1999 (38) Gemfibrizol Blind,


600 mg 4 wk 46 Improved LFT’s

Saibara et al, 1999 (39) † Bezafibrate Open-label 400 mg NR 2 Improved histology Horlander et al, 2001 (40) Atorvastatin Open-label 10-30 mg 21 mo 7 Improved histology Nair et al, 2002 (41) HMG-CoA RI Case control NR ≥6 mo 13 No improvement Kiyici M et al, 2003 (42) Atorvastatin Open-label 10 mg 6 mo 27 Improved LFT’s Merat et al, 2003 (43) Probucol Open-label 500 mg 6 mo 17 Improved LFT’s Rallidis et al, 2004 (44) Pravastatin Open-label 20 mg 6 mo 5 Improved histology

Antiobesity Harrison et al, 2004 (51) Orlistat Open-label 360 mg 6 mo 10 Improved LFT’s

Other Popescu, 2003 (52) ET-RA Random 50 mg 12 wk 22 Improved LFT’s Adams et al, 2004 (53) Pentoxyflline Open-label 1600 mg 12 mo 20 Improved LFT’s Yokohama et al, 2004 (54) Losartan Open-label 50 mg 48 wk 7 Improved histology

Combinations Guma et al, 1997 (45) Diet vs. diet+ UCDA Open-label 10 mg/kg 6 mo 24 Improved LFT’s Ceriani et al, 1998 (46) Diet vs. diet+ UCDA Open-label 10 mg/kg 6 mo 31 Improved LFT’s Hasegawa et al, 2001 (32) Diet vs

diet + vitamin E Open-label, cross-over

300 mg 6 mo diet 1 yr comb.

22 Variable LFT’s & histology

Mendez-Sanchez et al, 2002 (48)

Diet vs. diet+ UCDA Blind, randomized, controlled

1200 mg 6 wk 23 LFT’s improved equally in both groups

Kugelmas et al, 2003 (33) Diet & excercise ±vitamin E

Randomized 800 IU 12 wk 16 No improvement (vit. E) Improved LFT’s (diet)

Harrison et al, 2003 (34) Vitamin E + Vitamin C

Blind, randomized, controlled

1000 IU vit.E 1000 mg vit.C

6 mo 45 No improvement LFT’s Improved histology

Sanyal et al, 2004 (36) Vitamin E alone vs vit E +pioglitozone

Randomized 400 IU vit.E 30 mg pio

6 mo 20 Combination showed Improved histology

Uygun et al, 2004 (23) Diet vs diet +metformin

Randomized 850 mg 6 mo 36 Variable LFt’s & histology

*LAB = Lecithin + Antioxidants (vit. C & E, beta caroteine, selenium) + vit. B complex ; † Drug induced NASH; # pediatric population



Figure 2. The Role of the Liver in Lipid Metabolism. The liver plays a vital role in many aspects of lipid metabolism including: the production of lipids from carbohydrates termed lipogenesis; the production of lipoproteins for storage and transport and; ATP synthesis via oxidation of free fatty acids. (Reproduced from ref. 56: Browning and Horton, JCI 2004;114:147) insulin has been shown to induce the expression (58, 59) and activity (60) of SREBP-1c (sterol regulatory binding protein-1c), while glucose stimulates the nuclear transport and activation of ChREBP (carbohydrate response element binding protein) (61). Moreover, glucose also directly induces the transcription of SREBP-1c (59). SREBP-1c is a major regulator of genes involved in lipogenesis and cholesterolgenesis including fatty acid synthase (FAS) (62), glucokinase (63), acetyl-CoA carboxylase (ACC) (64), acetyl-CoA synthetase (65) and stearoyl-CoA desaturase (SCD) (66) among others. Similarly, ChREBP has been shown to transcriptionally activate numerous lipogenic genes as well as a key mediator of glycolysis, liver type pyruvate kinase (L-PK) (67, 68). Interestingly, both ChREBP and SREBP-1c are required for the normal expression of all lipogenic genes (67, 68). In summary, the metabolic pathways responsible for the production of lipids during periods of hyperglycemia are independently but coordinately regulated by both insulin and glucose largely at the transcriptional level.

1.2.2 The Role of Omega-3 Polyunsaturated Fatty Acids

A diet rich in omega (ω)-3 polyunsaturated fatty acids (PUFA) leads to decreased VLDL cholesterol and hypotriglyceridemia (69-76). These beneficial effects of ω-3 PUFA result from their novel ability as transcription regulators to simultaneously suppress hepatic lipogenesis while upregulating fatty acid oxidation in liver and skeletal muscle. Interestingly, ω-3 PUFA mediates these effects through multiple mechanisms on a variety of transcription factors. For example, in contrast to the upregulation of lipogenesis by insulin and glucose during states of carbohydrate excess, ω-3 PUFA function to suppress lipogenic genes by reducing the protein level, mRNA stability and transcription rate of SREBP-1c (77, 78). Omega-3 PUFA also mitigate the DNA binding affinity of at least two other transcription factors involved in regulation of lipogenic genes, NF-Y and Sp1 (79, 80).



Concomitant with the ω-3 PUFA mediated suppression of lipogenesis, is a “repartitioning” of fatty acids from storage toward oxidation and increased thermogenesis. One of the earliest detectable metabolic changes in response to dietary ω-3 PUFA intake is the reduction of the key enzyme malonyl-CoA (80, 81). Malonyl-CoA functions to regulate fatty acid flux into the mitochondria by inhibiting the activity of carnitine palmitoyltransferase-1 (CPT-1), the protein responsible for fatty acid transport into the mitochondria (82, 83).This lack of inhibition of carnitine palmitoyltransferase-1 mediated transport of FFA into the mitochondria by malonyl-CoA, results in a net increase of fatty acid β-oxidation. Omega-3 PUFA also function to increase expression of several proteins important in fatty acid oxidation within the peroxisome and mitochondria, including carnitine palmitoyltransferase-1 (84) in addition to acyl-CoA oxidase (85), mitochondrial uncoupling protein-3 (85) and mitochondrial HMG-CoA synthase (86). Upregulation of these genes by ω-3 PUFA is mediated by the transcription factor peroxisome proliferator-activated receptor (PPAR)-α. PPAR-α is a member of the steroid receptor superfamily and possesses a hydrophobic ligand binding domain and a zinc finger DNA binding motif. A conformational change occurs upon binding of PUFA to the PPAR-α ligand binding domain which results in the activation of PPAR-α. Examples of other ligands that also bind the PPAR family of transcription factors include the hypotriglyceridemic fibrate drugs and the insulin sensitizing drugs thiazolidinediones (87).

1.3 PREVIOUS STUDIES OF OMEGA-3 POLYUNSATURATED FATTY ACIDS

1.3.1 Human Clinical Trials

For the past several decades a wide range of health benefits have been attributed to the consumption of marine oils and specifically the omega-3 fatty acids. In fact, the Agency for Healthcare Research and Quality [AHRQ, a DHHS sister agency to the National Institutes of Health, the Centers for Disease Control and Prevention, and the Food and Drug Administration] has reviewed some 25,000 published studies to create ”evidence reports” of the health benefits of omega-3 polyunsaturated fatty acids on eight different health topics (88). Moreover, although dietary supplements are not regulated by the FDA, the FDA recently issued a rare “qualified health claim” for EPA and DHA omega-3 fatty acids to reduce the risk of coronary heart disease (89).

Although there are no published clinical studies evaluating the effect of omega-3 polyunsaturated fatty acids on hepatic steatosis, there are numerous clinical studies on the effect of ω-3 PUFA on hypertriglyceridemia. Meta analyses of these studies clearly show a 25-30% reduction of serum triglycerides following dietary supplementation with ω-3 polyunsaturated fatty acids (69-76). As shown in figure 3, this effect appears to be dose-dependent. It is less clear if continued treatment would also bolster these effects. The duration of studies showing a significant reduction in the level of serum triglycerides, range from two weeks to over two years (74-76). There are no data on sustainment of effects after the end of the treatment period. The effect of ω-3 PUFA on serum levels of other lipids such as total cholesterol, HDL and LDL is less clear. Most studies have shown a small non-significant increase in the level of these lipids (69-76). Many studies of ω-3 PUFA in different patient populations have reported a small yet significant reduction in blood pressure (69-76, 88). Data on the effect of ω-3 PUFA upon glucose tolerance is varied and inconclusive (70, 73, 75).



Figure 3. Meta-regression of dose of EPA + DHA intake versus net change in triglycerides (Tg.). Each point represents an individual study or study arm. Separate simple regressions were performed for each oil source type (except for the individual study arm of combined fish and fish oil). Marine oils includes non-fish animal sources including Minke whale and seal. Regression not adjusted for baseline Tg or study size. (Reproduced from reference 74: Balk E, et al. Evidence Report/Technology Assessment: Number 93. March 2004. Agency for Healthcare Research and Quality, Rockville, MD) http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hstat1a.chapter.35161

1.3.2 Animal Models of NAFLD

Several recent studies in ob/ob mice have characterized the effect of supplementation with ω-3 PUFA in this mouse model of obesity and hepatic steatosis (90-92). Seikiya et.al., have shown that administration of a diet enriched for ω-3 polyunsaturated fatty acids to ob/ob mice for as little as one week, results in marked reduction of hepatic triglyceride content and aminotransferase levels and improved insulin sensitivity (90). This study demonstrated that compared to the control diet, ω-3 PUFAs mediated these effects by reducing the mature form of the SREBP-1 protein, which in turn reduced the expression of a number of lipogenic genes. As expected, PPAR-α target genes were also induced in mice fed the ω-3 PUFA enriched diet. Similar results were obtained by Alwayn et al, using ob/ob mice given ω-3 PUFA supplementation IV (91). In a second study by this group, omega-3 fatty acid supplementation given via orogastric gavage reversed induced hepatic steatosis in C57/Bl6 mice fed a high-carbohydrate, fat-free diet and converted macrovesicular to microvesicular steatosis in B6.V-Lep(ob) obese mice (92).

7 PUFA P&F Protocol NASH CRN


2 NASH CLINICAL RESEARCH NETWORK In 2002, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), through the mechanism of RFA-DK-01-025, established a Clinical Research Network (CRN), the goal of which is to facilitate and perform clinical, scientific, epidemiological and therapeutic research in NASH. The NASH Clinical Research Network (NASH CRN) is a cooperative network of eight clinical centers and one Data Coordinating Center (DCC) (93). Clinical centers are responsible for proposing protocols, participating in their overall development, recruiting patients, conducting the research, and disseminating research findings. The individual clinical centers participate in a cooperative and interactive manner with one another and with the DCC in all aspects of the NASH CRN. The DCC supports protocol development; provides sample size calculations, statistical expertise, forms, and data analysis; supports manuscript preparation; and provides overall study coordination and quality assurance, including coordination of the activities of the Data and Safety Monitoring Board, the Steering Committee and other standing committees. The DCC also collaborates with the NIDDK Biosample (plasma, serum, and liver tissue) and Genetics (DNA) Repositories. A Steering Committee composed of the principal investigators of each clinical center in the Network, the principal investigator of the DCC, and the NIDDK Project Scientist is the main governing body of the NASH CRN. The Steering Committee has primary responsibility for the general organization of the NASH CRN, finalizing common clinical protocols and facilitating the development of a standardized nomenclature, diagnostic criteria, histological definitions, and the necessary components to the common database on patients. The Steering Committee is responsible for the conduct and monitoring of studies and reporting study results. In response to the NIDDK pilot and feasibility (P&F) program to encourage innovative, multidisciplinary research to study the pathogenesis and treatment of obesity, Dr Kowdley has received research support to perform this clinical study. This P&F study will be conducted as an ancillary study to the ongoing NASH CRN. Dr. Kris V. Kowdley MD is the Principal Investigator of both this proposal and the parent grant U01 DK61728-3. The study proposal has been reviewed and approved by the Pilot and Feasibility and Steering Committees of the NASH CRN in accordance with the NASH CRN pilot and feasibility studies policies. A pre-requisite for participation in this study is enrollment into the NASH CRN Database Study. Study participants will be recruited from the NASH CRN Database study that also do not qualify or are unwilling to participate in the PIVENS Study.

8 PUFA P&F Protocol Study Design


3 STUDY DESIGN

3.1 PRIMARY HYPOTHESIS AND STUDY OBJECTIVES

Hypothesis Dietary supplementation with ω-3 polyunsaturated fatty acids will result in a reduction of

intrahepatic fat content and decreased levels of serum triglycerides, serum aminotransferases and proinflammatory cytokines when compared to monounsaturated fatty acids.

Study objective To determine the effect of dietary supplementation with ω-3 polyunsaturated fatty acids and

monounsaturated fatty acids on intrahepatic fat content, serum aminotransferases, fasting lipids, proinflammatory cytokines and insulin resistance in patients with non-alcoholic fatty liver disease.

Primary outcome

• Reduction of intrahepatic fat content as determined by magnetic resonance spectroscopy

Secondary outcomes

• Change in serum aminotransferase levels • Change in lipid profile • Change in levels of proinflammatory cytokines • Change in insulin resistance determined by HOMA

3.2 OVERVIEW This 8 week parallel, randomized, pilot and feasibility study will determine if supplementation with ω-3 polyunsaturated fatty acids will reduce intrahepatic fat content in patients with nonalcoholic fatty liver disease in comparison to monounsaturated fatty acid supplements. Study duration is based on published clinical trials studying the effect of fish oil supplements on lipid levels (69-76) We will recruit a total of 30 participants who meet the stated inclusion criteria (see section 5).

Intrahepatic fat will be measured by proton magnetic resonance spectroscopy (1H MRS). This technique has been widely used to measure liver fat and is significantly correlated with histologic fat measurements (94-99). The percentage of liver fat will be calculated as the ratio of the fat resonance signal divided by the sum of the water and fat resonance signals.

Secondary outcome measurements for this study include: changes in serum aminotransferase levels; change in lipid profile; change in insulin resistance and change in levels of proinflammatory cytokines.



This study will provide preliminary data on the efficacy of fish oil supplementation as a simple and widely available treatment of fatty liver disease.

3.3 BLINDING AND RANDOMIZATION Study investigators will be blinded to the treatment assignment of all 30 subjects. Subjects will be stratified according to whether or not they have diabetes mellitus. Subjects will then be evenly randomized into either group A or B shown below. Subjects will not be informed as to which group they will be in, however subjects receiving the fish oil capsules may notice a slight “fishy” taste or smell.

Group A: omega-3 polyunsaturated fatty acid supplementation (fish oil capsule containing: 18% eichosapentaenoic acid [EPA] and 12% docosahexaenoic acid [DHA])

Group B : monounsaturated fatty acid supplement (safflower oil capsule containing: 80% oleic

acid)

3.4 NUTRITION COUNSELING AND ENERGY ASSESSMENT During the screening visit participants will meet with UW GCRC research dieticians and will be asked to provide a 24-hour diet recall. Subjects will then be counseled on preparation and maintenance of a proper well balanced diet (16% protein, 50% carbohydrates, 34% fat) based on a modified Therapeutic Lifestyle Changes diet of the National Cholesterol Education Program (100). Compliance to the recommended diet will be reassessed at the 4 week interim visit.

3.5 STUDY TREATMENT The ω-3 PUFA and MUFA supplements will be purchased from Capsugel Liquids Group, Greenwood SC (A subsidiary of Pfizer Inc.). Both supplements will be supplied as 725 mg hard shell capsules.

3.5.1 Omega-3 Polyunsaturated Fatty Acid Supplements Patients in Group A will receive omega-3 polyunsaturated fatty acid supplements. Each 725 mg fish oil capsule contains 30% ω-3 PUFA (18% eichosapentaenoic acid [EPA] and 12% docosahexaenoic acid [DHA]). Each capsule also contains 1 mg (1.3 IU) of vitamin E (d-alpha tocopherol) as a stabilizing agent. Vitamin E has also been shown to have beneficial effects on NAFLD, albeit at much higher doses (ie 400-1200 IU per day, see table 1). We have chosen fish oil supplements containing the highest ratio of EPA:DHA shown to maximally decrease de novo lipogenesis (101).



3.5.2 Monounsaturated Fatty Acid Supplements Patients in Group B will receive the monounsaturated fatty acid supplement containing 80% oleic acid safflower oil. Each MUFA capsule will also contain an equivalent amount of vitamin E to the ω-3 PUFA supplements.

3.5.3 Dosage Subjects in both groups will receive 18 – 725 mg capsules per day. We will instruct the subjects to take the capsules with each meal so that the number of capsules ingested is equally divided between breakfast, lunch and dinner. Group A patients will receive a dose of 4 g of omega 3 PUFA per day, while group B patients will receive 10 g of oleic acid MUFA per day.

3.5.4 Storage and Dispensing Protocol

Study treatment supplies will be stored at 15-25°C (59-77°F) away from light, by the University of Washington IDS Pharmacy. The supplements will be dispensed by the IDS Pharmacy at the baseline and 4 week visit (see schedule of events). The bottle numbers, dispensing date, stop date, and any missed doses of the study treatment will be recorded on the study Treatment Dispensation Log kept at IDS and the Treatment Dosing Log case report form and maintained in the Study Database. The subject will be instructed to return all unused study treatment in the original package at each visit. Research staff will tally capsules remaining in each bottle as an additional measure of compliance. The capsule count, together with the start and stop dates must be recorded on the drug accountability page for each study visit.

3.5.5 Quality Control The formulation documentation provided by Capsugel Liquids Group is attached as appendix A. We have also independently assayed the purity and composition of each lot of supplements and this information is included in the appendix. Both the MUFA and PUFA capsules have been tested for heavy metal contamination and passed as shown below. Capsugel Analytical Test Specification Results Arsenic ≤ 0.1 ppm Pass Lead ≤ 0.1 ppm Pass Cadmium ≤ 0.1 ppm Pass Mercury ≤ 0.025 ppm Pass

11 PUFA P&F Protocol Study Population


4 STUDY POPULATION

4.1 SAMPLE SIZE We will use the well established effect of omega-3 PUFA on lowering serum triglycerides to estimate our sample size. There have been numerous studies in similar patient populations (ie., hyperlipidemic, diabetic and or obese subjects) which have consistently shown a 10-40 % reduction in serum triglycerides (73, 74). We anticipate that omega-3 PUFA should have a similar effect in lowering hepatic triglycerides that it does to lower serum triglycerides.

Therefore we have chosen a study done in patients with visceral obesity (a common co-morbid condition associated with NAFLD) using the same dose 4g and a similar time course (6 wks vs 8 wks in our study)(29). Data on serum triglyceride levels before and after 6 weeks of fish oil supplementation showed estimates (mean +/- sd) baseline 2.00 +/- 1.21 mmol/L and 8 weeks 1.50 +/- 0.73 mmol/L with correlation between baseline level and eight-week change rho = -0.89. These values lead to an estimate of the sd of the change in triglyceride levels of 0.60 mmol/L or 30% of baseline levels. From the same study, twelve patients on placebo had values: baseline 1.69 +/- .62 mmol/L and 6 weeks 1.57 +/- .52. Assuming the a more conservative correlation within patient (.8) than above yields an estimate of the standard deviation of the change from baseline of 24% in the placebo group. Assuming the standard deviation of the change in inter-hepatic fat content will be similar in percent change to those witnessed in serum triglyceride levels, a sample size of 15 per group will have 80% and 90% powers to detect differences of 29% and 34% in change between groups, respectively We anticipate an attrition rate of 10% therefore we will plan to enroll a total of 33 subjects to replace dropouts.

4.2 INCLUSION CRITERI A • Acceptance into NASH CRN Database Study • Exclusion from or unwillingness to participate in the NASH CRN PIVENS Study • Histological diagnosis or imaging study suggesting NAFLD with at least 20% steatosis • Willingness to maintain study diet for duration of the study • At least 18 years of age

4.3 EXCLUSION CRITERIA • Use of lipid lowering drugs (i.e., statins and fibrate drugs) • Use of insulin or thiazolidinediones • Hemoglobin HbA1c level >8 %

12 PUFA P&F Protocol Study Population


• Use of drugs possibly associated with NAFLD (amiodarone, methotrexate, systemic glucocorticoids, tetracycline, tamoxifen, estrogens at doses greater than those used for hormone replacement, anabolic steroids, valproic acid, or other known hepatotoxins) for more than 2 consecutive weeks in the 2 years prior to screening

• Initiation of anti-diabetic drugs (insulin, biguanides, sulfonylureas, metformin, thiazolidinediones) in the 3 months prior to randomization

• Initiation of anti-NASH drugs (thiazolidinediones, vitamin E, metformin, UDCA, SAM-e, betaine, milk thistle, gemfibrozil, anti-TNF therapies, probiotics) in the 3 months prior to randomization

13 PUFA P&F Protocol Schedule/Procedures


5 SCHEDULE OF VISITS AND PROCEDURES

5.1 STUDY VISITS

5.1.1 Screening Visit After obtaining informed consent, a screening office visit is conducted by the research coordinator to insure compliance with the inclusion and exclusion criteria. We will normally coincide this visit with the subject’s NASH CRN Database Study follow-up visit. During this visit, subjects are asked to complete questionnaires about eating and physical activity habits and their general quality of life. The subjects also are given a physical examination and anthropomorphic measurements. Those subjects who meet eligibility criteria will meet with the CRC nutrition research coordinators and will be asked for a 24 hour dietary recall. Subjects will be counseled on preparing and maintaining a well balanced diet. Subjects will be asked to complete a 3-day food record and mail it back to the nutrition department.

5.1.2 Baseline Visit Subjects will be admitted into the UWMC GCRC on the morning of the visit, following a 12 hr overnight fast. Approximately 50 ml (3.4 tablespoons) of blood will be drawn for measurement of fasting lipids, serum aminotransferases, inflammatory cytokines, fatty acid oxidation, highly sensitive CRP and adiponectin. We will also draw 25 ml (1.7 tablespoons) of blood for storage of serum and plasma sample for future research purposes. We will also collect 4 ml of urine for future research purposes. Subjects will then undergo a 120 min oral glucose tolerance test to determine their calculated insulin sensitivity. During this period the subject will be weighed and measured for body composition. Blood pressure measurements will be performed. DEXA scanning will be performed to determine each subject’s percentage and distribution of body fat. Following the OGTT the subject will be given lunch then taken to the Radiology Department to assess intra-hepatic fat distribution as determined by proton magnetic resonance spectroscopy (1H MRS).

5.1.3 Interim Visit Three weeks into the study patients will again meet with the research nutritionists to reassess their compliance with dietary recommendations. Subjects will receive an additional supply of study treatment supplements at this time. Subjects are also asked to complete questionnaires about their physical activity habits and general quality of life at this visit.

5.1.4 Final Visit The final visit will be a repeat of all procedures which were done at the baseline visit.



Table 2. Data Collection Schedule

5.2 PROCEDURES

5.2.1 Measurement of Liver Fat To assess intra-hepatic fat we will utilize proton magnetic resonance spectroscopy (1H MRS). This technique utilizes the differential resonance frequencies of lipid and water protons placed in a magnetic field. A 3-4 ppm difference can be detected in the proton resonance frequencies of methyl and methylene groups on lipid molecules relative to resonance frequency of water. We will utilize an image-guided localized double spin echo sequence method (95-97). This technique has been widely used to measure liver fat and is significantly correlated to computed tomography and histologic measurements (63,71,84,85,94-99). The percentage of liver fat will be calculated as the ratio fat signal divided by the sum of the water and fat signals. Measurements will be obtained with subjects on a 1.5 Tesla Signa Scanner whole body system (General Electric Medical Systems, Version 5.8 hardware and software) which is located in the Department of Radiology at the University of Washington Medical Center. Sagittal, coronal, and axial MR slices through the right lobe of the liver will be acquired, and a 27-cm3 spectroscopic volume of interest will be positioned, avoiding major blood vessels, intrahepatic bile ducts, and the lateral margin of the liver. The voxel size and position will be optimized to prevent contamination of signal from liver by signal from abdominal adipose fat. We will use a relatively large voxel to be able to collect good quality data in a short time and to minimize patient time in the magnet. After the system is tuned and shimmed, spectra will be collected using the GE -body coil for radio frequency transmission and signal reception. A PRESS sequence will be used for spatial localization and signal acquisition with the parameters interpulse delay Tr _ 3 s, spin echo time Te _ 25 ms, 16 acquisitions,

Procedure Screening Visit Enrollment visit Intrerim Visit @ 4 wks

Final Visit @ 8 wks

Informed Consent X

Nutrition Counseling and Diet Assessment

X X

Questionnaires X X X

Dispense Supplements X X

MRS Liver Fat Quantification X X

Anthropometric Measurements

X X

DEXA Scan X X

Fasting Lipids X X

OGTT X X

Inflammatory Cytokines X X

Aminotransferase Levels (AST, ALT)

X X

Fatty Acid Oxidation X X

Indirect Calorimetry X X

Blood Pressure X X

Specimen Banking X X



and 1,024 data points over a 1,000-Hz spectral width. Only signals from a selected volume element will be collected. Areas of resonances from protons of water and methylene groups in fatty acid chains of the hepatic triglyceride will be evaluated with a line-fit procedure and software developed at the University of Washington. The subject will be inside the scanner for about 45 minutes.

5.2.2 Body Anthropometrics All measurements will be taken in duplicate at the beginning of each visit.

• Weight will be measured without shoes using an electronic scale to the nearest tenth of a kg. in the morning after voiding and before breakfast.

• Height will be measured without shoes to the nearest 0.5 cm using a stadiometer.

From the weight and height measurements, BMI (kg/m2) will be calculated.

Waist and hip circumference will be measured using a Gulick II Tape Measure (this tape measure applies a known amount of tension (4 ounces) to the measuring tape, thus increasing reproducibility and accuracy)

• Waist circumference will be measured to the nearest 0.5 cm in the standing position. The measure should be taken around the abdomen horizontally at the midpoint between the highest point of the iliac crest and lowest part of the costal margin in the mid-axillary line.

• Hip circumference will be measured to the nearest 0.5 cm in the standing position at the levels

of the trochanter and through the symphysis pubis.

• Tricep skinfold measurement will be measured on the right tricep to the nearest 0.5 mm using a Harpenden skinfold caliper.

5.2.3 Measurement of Body Fat To determine total body fat content/mass, dual-energy X-ray absorptiometry (DEXA) will be performed on the GCRC. A whole body scan is performed to evaluate body fat (102). The coefficient of variation (CV) for total fat mass is 2.6-2.9% (102).

5.2.4 Fatty Acid Oxidation An indication of β oxidation levels of free fatty acids will be determined by assaying levels of the serum marker β-OH butyrate using a β-hydroxybutyrate kit (Sigma Chemical Co., St. Louis, MO). Oxidized LDL plasma levels will be determined by ELISA. Indirect calorimetry will be utilized to estimate differential glucose and lipid oxidation (103).

5.2.5 Lipoproteins Fasting blood samples will be obtained at the time of the first basal sample of the OGTT. Plasma total cholesterol, triglycerides (TG), HDL-C, HDL2 and HDL3 will be measured at the Northwest Lipid Research Laboratory as previously described (104). LDL will be calculated by the Friedwald formula: LDL-C=total cholesterol – HDL-c – (TG/5). The relative flotation rate (Rf) as a measure of LDL peak density will be determined by density gradient ultracentrifugation (105). FFAs will be collected in orlistat to inhibit lipolysis of triglyceride and will be measured by enzymatic calorimetric quantification using a kit (Wako).



5.2.6 Oral Glucose Tolerance Tests Following a 12 hour overnight fast, an intravenous catheter will be placed in a peripheral arm vein for blood sampling. Twenty minutes later, three basal samples at 0, 5 and 10 minutes will be drawn. Then the subjects will drink a standard 75 gram beverage and blood samples will be obtained at 30, 60, 90 and 120 minutes for determination of plasma glucose, insulin and C-peptide. Glucose tolerance will be categorized based on the 2 hour glucose level according to published guidelines (normal <140mg/dl, IGT 140-200mg/dl, diabetes >200mg/dl). The efficiency of glucose disposal will be determined as the incremental area under the curve (AUC) for glucose. The insulin and C-peptide responses will also be quantified as the incremental response at 30 minutes and as the incremental AUC for 120 minutes.

5.2.7 Blood Pressure Seated blood pressure will be measured at the brachial artery using an automated sphygmomanometer after the subject has been at rest for five minutes. The systolic and diastolic fifth phase pressures will be recorded on three separate occasions separated by 30 seconds. The average of the three measurements will be used as the systolic and diastolic pressures. Subjects on blood pressure medication will continue to take their blood pressure medication as prescribed.

5.2.8 Hepatic Necroinflammation and Cytokine Analysis Serum levels of ALT, AST, IL-6, TNF-αR2 and will be determined using a standard ELISA assay. Highly sensitive CRP will be measured by nephelometry (Bade-Behring). Adiponectin will be assayed from fasting plasma by standard ELISA. In total approximately 60 ml (4 tablespoons) of blood will be drawn for these assays.

5.2.9 Diet, Physical Activity and Quality Of Life Questionnaires Research participants will be asked to fill out the block food questionnaire as part of their participation within the NAFLD Database study. Participants will be asked to fill out 24 hour diet recall, brief physical activity and quality of life questionnaires at screening, 4 and 8 weeks.

17 PUFA P&F Protocol Data Management


6 DATA MANAGEMENT

6.1 SOURCE DOCUMENTATION AND CASE REPORT FORMS The principal investigator and his research staff will be responsible for preparing and maintaining adequate and accurate case histories designed to record all observations and other data pertinent to the study for each study participant, using the case report forms (CRFs) created for the study. All information recorded on the CRFs for this study must be consistent with the patient’s source documentation. The data collected during the study should be entered in the study charts in black ink using a ballpoint pen. If an error is made, then the error is to be crossed out with a single horizontal line, the new information clearly recorded next to the error, and the correction initialed and dated. Correction fluid is not to be used. Study staff will enter information from the CRFs into an electronic confidential database created using Microsoft Access. A record of patient screen failures will be maintained for patients who do not qualify for enrollment. This record will be maintained until manuscript submission. A record of all procedures performed on the GCRC will be added to the patients official medical records.

6.2 DAT A QUALITY ASSURANCE During the course of the study, the Study Coordinator will review protocol compliance, compare CRFs and individual patient’s medical records, assess drug accountability, and ensure that the study is being conducted according to pertinent regulatory requirements. CRFs will be verified with source documentation. The review of medical records will be performed in a manner to ensure that patient confidentiality is maintained. The Data Manager will compare the original source document for each patient to data entered into the Database. Instances of missing or uninterpretable data will be discussed with the principal investigator and research coordinator for resolution. After CRFs are verified, the Data Manager will perform a quality assurance audit.

6.3 STATISTICAL ANALYSIS Values for intrahepatic fat content, serum liver enzymes, BMI, lipid profile, cytokines and fatty acid oxidation will be expressed as mean ±SEM. Baseline and post treatment changes in intrahepatic fat content will be compared between placebo and treatment groups using a two-sample t test. Spearman rank correlation will be used to examine the relationship between serum liver enzymes and grades of hepatic fat determined by MRS. Statistical analysis will be performed using STATA statistical software. A final review of the statistical analysis will be performed by statisticians at UW GCRC.

18 PUFA P&F Protocol Human Subjects


7 HUMAN SUBJECTS ISSUES

7.1 GENERAL GUIDELINES The trial will be carried out in accordance with the Declaration of Helsinki (October 2000) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) and the Health Insurance Portability and Accountability Act (HIPAA) regulations. The University of Washington IRB must approve the trial before the commencement of the study and all the guidelines provided by the Office of Human Research Projects (OHRP) and HIPAA must be followed. Scientific activities of the proposed study are subject to scientific misconduct policies of University of Washington.

7.2 INFORMED CONSENT The investigators are responsible for development of the informed consent document. The informed consent document will be used to explain, in simple terms, the procedures, risks and benefits of the trial to the subject and family member, if applicable. The Informed Consent will also state that the subject is free to withdraw from the study at any time for any reason without penalty or loss to benefits s/he otherwise entitled. If amendments to the protocol occur after admitting subjects to the study that affect the subject’s participation, study participants will be notified in writing of the changes as an addendum to the previously signed Informed Consent document. Subjects will be asked to sign the revised consent form. The subject will be presented with the Informed Consent form at the screening visit. The subject will be given all the necessary time to read and sign the Informed Consent. The Study Clinician and the Study Coordinator will be available to answer any questions. No further procedures will occur until the subject has signed the Informed Consent. Informed consent is an ongoing process between the investigator and the study participant and does not stop once the potential subject signs the form.

7.3 HIPAA AUTHORIZ ATION All consented potential subjects will be asked to sign a separate authorization for study personnel to review their clinical records and enter their Personal Health Information (PHI) in the study related documents in accordance with the Health Insurance Portability and Accountability Act (HIPAA) to be implemented as of April 14, 2003. Additionally, all the investigators will have signed a confidentiality agreement for the trial. This agreement supersedes the HIPAA federal law in the state of Washington and allows investigators to access records indefinitely or until expiration of the confidentiality agreement.

7.4 SUBJECT CONFIDENTIALITY All laboratory specimens, evaluation forms, reports, and other records will be identified by a coded number to maintain subject confidentiality. All records that mention the subject’s name (medical history, screening form, informed consent) will be kept in the patient’s chart in a locked file cabinet separate from the case report forms. All computer entry and networking programs will be done with coded numbers only. Clinical information will not be released without written permission of the subject. Research staff will sign a confidentiality agreement before they are authorized to have direct contact with the subject or the subject’s name. In signing the confidentiality agreement, all study

19 PUFA P&F Protocol Human Subjects


personnel will be committed to follow procedures already in practice at University of Washington Medical Center to protect the privacy of study subjects.

7.5 REVIEW OF ORIGINAL SUBJECT RECORDS Once subjects have signed the HIPAA authorization, records that identify the subject and the Informed consent forms signed by the subject may be viewed by members for the Research Team who have signed a confidentiality agreement, the University of Washington IRB members. Auditing by outside parties may be carried out on the CRFs that identify subjects by coded numbers only.

7.6 AMENDMENTS TO THE PROTOCOL Changes to the protocol and the informed consent form by the investigators are not permitted once the study documentation has been approved by the University of Washington IRB. Should an amendment be necessary, the principal investigator will submit an official request to the IRB coordinator and enclose the amendment as an appendix to the study protocol. IRB approval will be obtained before changes to the protocol can be implemented.

20 PUFA P&F Protocol DSMP


8 Data Safety Monitoring Plan

8.1 ADVERSE EVENT GRADING An adverse event (AE) is any untoward medical occurrence in a subject, not necessarily having a causal relationship with the study. AE information will be collected from the time the subject signs the informed consent until study termination. A serious adverse event (SAE) is any untoward medical occurrence that:

• results in death, • is life-threatening, • requires inpatient hospitalization or prolongation of existing hospitalization, • results in persistent or significant disability/incapacity,

AE's are graded as Mild (no limitation of usual activities), Moderate (some limitation) or Severe (inability to carry out usual activities) and attributed according to the relationship to the study procedures as Not related, Unlikely, Possible, Probable, or Definite

8.2 STUDY MONITORING AND SAFETY REPORTS The study coordinator will review source documents and Case Report Forms to verify accuracy of AE reports. Once CRFs are validated by the Study Coordinator, the Data Manager will verify and code the dataset and will generate periodic Interim Safety Reports for the PI’s review. These reports will contain AE data, study conduct, enrollment and drop-out data. Annual Reports will be submitted to the UW IRB and GCRC and will contain:

• The number of adverse events and an explanation of how each event was handled • The number of complaints and how each complaint was handled • The number of subject withdrawals and an explanation of why the subject withdrew or was

withdrawn • The number of protocol violations and how each was handled

Safety Officer: The PI will perform the duties of the Safety Officer in compliance with NIH regulations.

8.3 ADVERSE EVENT (AE) REPORTING Adverse events will be reported to the IRB, GCRC and NIDDK, in accordance with current NIH and local or state regulations. Timely and complete reporting of all AEs will assist the principal investigator in identifying any untoward medical occurrence, thereby allowing: 1) protection of safety of study subjects; 2) appropriate modification of study protocols; and 3) improvements in study design or procedures. AEs may be spontaneously reported by a subject or elicited during questioning and examination of a subject. All identified AEs will be recorded and described on the appropriate Non-



serious or Serious AE page of the CRF. If known, the diagnosis underlying a constellation of AEs should be recorded, rather than its individual symptoms. Any unexpected adverse events will be reported to the UW HSD within 24 hours. An Adverse Event Report form will be obtained from HSD and returned to the UW HSD and GCRC within 5 working days.

Subjects experiencing AEs that cause interruption or discontinuation of treatment or those experiencing AEs that are present at the end of their participation in the study should receive follow-up as medically appropriate. The principal investigator will report to the University of Washington HSD and GCRC the outcome of any AE that caused permanent discontinuation or that was present at the end of the study particularly if the AE was considered by the principal investigator to be related in any way to the study procedures.

8.4 SERIOUS AES (SAES) REPORTING SAEs should be reported following the subjects’ written consent to participate in the study until the end of follow-up. Adverse events classified as “serious” (SAEs) require expeditious handling and reporting to the University of Washington IRB, GCRC and comply with regulatory requirements. All SAEs will be followed up to resolution or until condition of patient is stable and no change is expected. SAEs will be reported to the GCRC in real time, and phoned in to the University of Washington HSD @ 543-0098 within 24 hours of learning its occurrence. The completed form will be mailed to HSD as well.

8.5 WITHDRAWAL OF SUBJECTS FROM STUDY A subject may withdraw from the study at any time if:

• He/she decides to withdraw (nevertheless, if a subject withdraws, the Principal Investigator will make a reasonable effort to determine the reason for the subject’s withdrawal from the study)

The Principal Investigator may decide to withdraw the subject from the study if:

• He/she experiences a serious AE (grade 4) or a non-serious severe AE (grade 3) related or unrelated to treatment

• He/she is instructed by a health provider to take prescription drugs, over-the-counter drugs or supplements that are excluded from the study

• Any other reason for which the investigator believes is in the subject’s best interest to withdraw.

22 PUFA P&F Protocol References

9 REFERENCES 1. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty

liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999; 116(6):1413-1419.

2. Charlton MR, Kasparova P, Weston S, Lindor KD, Maorkendler Y, Wiesner RH et al. The

frequency of non-alcoholic steatohepatitis as a cause of advanced liver disease. Liver Transpl 2001; 7(7):608-14.

3. Teli MR, James OF, Burt AD, Bennett MK, Day CP. The natural history of nonalcoholic fatty liver:

a follow-up study. Hepatology 1995; 22(6):1714-1719. 4. Propst A, Propst T, Judmaier G, Vogel W. Prognosis in nonalcoholic steatohepatitis [letter;

comment]. Gastroenterology 1995; 108(5):1607. 5. Powell EE, Cooksley WG, Hanson R, Searle J, Halliday JW, Powell LW. The natural history of

nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years. Hepatology 1990; 11(1):74-80

6. Day CP, James OF. Steatohepatitis: a tale of two "hits"? [editorial]. Gastroenterology 1998;

114(4):842-845. 7. Day CP. Pathogenesis of steatohepatitis. Best Pract Res Clin Gastroenterol. 2002 Oct;16(5):663-

78. 8. McCullough AJ. Update on nonalcoholic fatty liver disease. J Clin Gastroenterol 2002;

34(3):255-262. 9. Falck-Ytter Y, Younossi ZM, Marchesini G, McCullough AJ. Clinical features and natural history of

nonalcoholic steatosis syndromes. Semin Liver Dis 2001; 21(1):17-26. 10. Szczepaniak LS, Nurenberg P, Leonard D, Browning JD, Reingold JS, Grundy S, Hobbs HH,

Dobbins RL. Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population. Am J Physiol Endocrinol Metab. 2005 Feb;288(2):E462-8.

11. Clark JM, Brancati FL, Diehl AM. The prevalence and etiology of elevated aminotransferase levels

in the United States. Am J Gastroenterol 2003; 98(5):960-967. 12. Bacon BR, Farahvash MJ, Janney CG, Neuschwander-Tetri BA. Nonalcoholic steatohepatitis: an

expanded clinical entity. Gastroenterology 1994; 107:1103-9. 13. Creutzfeldt W, Frerichs H, Sickinger K. Liver diseases and diabetes mellitus. Prog Liver Dis

1970;3:371-407. 14. Lee RG. Nonalcoholic steatohepatitis: a study of 49 patients. Hum Pathol 1989; 20(6):594-598.



15. Youssef WI, McCullough AJ. Steatohepatitis in obese individuals. Best Pract Res Clin Gastroenterol. 2002 Oct;16(5):733-47

16. Scheen AJ, Luyckx FH. Obesity and liver disease. Best Pract Res Clin Endocrinol Metab. 2002

Dec;16(4):703-16. 17. Coyle WJ, Delaney N, Yoshihashi A, Lawson P. Metformin treatment in patients with nonalcoholic

steatohepatitis normalizes LFTs and improves histology [Abstract]. Gastroenterology 1999;116:A1198.

18. Caldwell SH, Hespenheide EE, Redick JA, Iezzoni JC, Battle EH, Sheppard BL. A pilot study of a

thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis. Am J Gastroenterol 2001;96:519-525.

19. Acosta RC, Molina EG, O’Brien CB, Cobo MC, Amaro R, Neff GW, Schiff ER. The use of

pioglitazone in nonalcoholic steatohepatitis [Abstract]. Gastroenterology 2001;120:A546. 20. Marchesini G, Brizi M, Bianchi G, Tomassetti S, Zoli M, Melchionda N. Metformin in non-alcoholic

steatohepatitis [Letter]. Lancet 2001;358:893-894. 21. Azuma T, Tomita K, Kato S, Adachi M, Inokuchi N, Kitamura N,Nishimura T, et al. A pilot study of

a thiazolidinediones, pioglitazone, in nonalcoholic steatohepatitis [Abstract]. Hepatology 2002;36:406A.

22. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, Oliver D, Bacon BR. Improved nonalcoholic

steatohepatitis after 48 weeks of treatment with the PPAR-gamma ligand rosiglitazone. Hepatology 2003; 38(4):1008-1017.

23. Uygun A, Kadayifci A, Isik At, et al. Metformin in the treatment of patients with non-alcoholic

steatohepatitis. Aliment Pharmacol Ther 2004;19:537– 44. 24. Oruc N, Dereli D, Yilmaz C, Yuce G, Gunsar F, Akarca US. Beneficial effects of insulin sensitizing

agents on fatty liver disease;preliminary results. [Abstract] J Hepatol 2003;38(Suppl. 2):198. 25. Promrat K, Lutchman G, Uwaifo GI, Freedman RJ, Soza A, Heller T et al. A pilot study of

pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 2004; 39(1):188-196. 26. Fu CS, Esrason K, Alshak NS, Conteas CN, Simmons VJ. Dietary lecithin, anti-oxidant and

vitamin B complex (LAB) decrease hepatic steatosis in patients with NASH [Abstract]. Gastroenterology 1998;114:A1243.

27. Lavine JE. Vitamin E treatment of nonalcoholic steatohepatitis in children: a pilot study. J Pediatr

2000;136:734-738. 28. Gulbahar O, Karasu ZA, Ersoz G, Akarca US, Musoglu A. Treatment of nonalcoholic

steatohepatitis with N-acetylcysteine [Abstract]. Gastroenterology 2000;118:A1444. 29. Miglio F, Rovati LC, Santoro A, Setnikar I. Efficacy and safety of oral betaine glucuronate in non-

alcoholic steatohepatitis. A doubleblind, randomized, parallel-group, placebo-controlled prospective clinical study. ArzneimittelForschung 2000;50(8):722– 7.



30. Abdelmalek MF, Angulo P, Jorgensen RA, Sylvestre PB, Lindor KD. Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study. Am J Gastroenterol 2001;96:2711-2717.

31. Yakaryilmaz F, Guliter S, Ozenirler O, Akyol G. Vitamin E treatment for patients with nonalcoholic

steatohepatitis: results of a pilot study. [Abstract] J Hepatol 2003;38(Suppl. 2):203. 32. Hasegawa T, Yoneda M, Nakamura K, et al. Plasma transforming factor-b1 level and efficacy of a-

tocopherol in patients with nonalcoholic steatohepatitis: a pilot study. Aliment Pharmacol Ther 2001;15:1667–72.

33. Kugelmas M, Hill DB, Vivian B, Marsano L, McClain CJ. Cytokines and NASH: a pilot study of the

effects of lifestyle modification and vitamin E. Hepatology. 2003 Aug;38(2):413-9. 34. Harrison SA, Torgerson S, Hayashi P,Ward J, Schenker S. Vitamin E and vitamin C treatment

improves fibrosis in patients with nonalcoholic steatohepatitis. Am Gastroenterol 98(11):2485–2490, 2003

35. Kawanaka M, Mahmood S, Niiyama G, Izumi A, et al. Hepatol Res. 2004 May;29(1):39-41. 36. Sanyal AJ, Mofrad PS, Contos MJ, Sargeant C, et al. A pilot study of vitamin E versus vitamin E

and pioglitazone for the treatment of nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol. 2004 Dec;2(12):1107-15.

37. Laurin J, Lindor K, Crippin JS, Gossard A, Gores GJ, Ludwig J, et al. Ursodeoxycholic acid or

clofibrate in the treatment of non-alcoholinduced steatohepatitis: a pilot study. Hepatology 1996;23(6): 1464–7.

38. Basaranoglu M, Acbay O, Sonsuz A. A controlled trial of gemfibrozil in the treatment of patients

with nonalcoholic steatohepatitis [Letter; Comment]. J Hepatol 1999;31:384. 39. Saibara T, Onishi S, Ogawa Y, Yoshida S, Enzan H. Bezafibrate for tamoxifen-induced non-

alcoholic steatohepatitis [Letter]. Lancet 1999; 353:1802. 40. Horlander JC, Kwo PY, Cummings OW, Koukoulis G. Atorvastatin for the treatment of NASH

[Abstract]. Gastroenterology 2001;120:A544. 41. Nair S, Wiseman M. HMG-CoA reductase inhibitors in no alcoholic fatty liver disease: is their

potential hepatotoxicity an issue in these patients? A case control study based on histology [Abstract]. Hepatology 2002;36:409A.

42. Kiyici M, Gulten M, Gurel S, Nak SG, et al. Ursodeoxycholic acid and atorvastatin in the treatment

of nonalcoholic steatohepatitis. Can J Gastroenterol. 2003 Dec;17(12):713-8.

43. Merat S, Malekzadeh R, Sohrabi MR, Hormazdi M, Naserimoghadam S, Mikaeli J, et al. Probucol in the treatment of nonalcoholic steatohepatitis: an open-labeled study. J Clin Gastroenterol 2003; 36(3):266–8.

44. Rallidis LS, Drakoulis CK, Parasi AS. Pravastatin in patients with nonalcoholic steatohepatitis:

results of a pilot study. Atherosclerosis 174 (2004) 193–196



45. Guma G, Viola L, Thome´ M, Galdame O, Alverez E. Ursodeoxycholic acid in the treatment of nonalcoholic steatohepatitis: results of a prospective clinical controlled trial [Abstract]. Hepatology 1997;26:387A.

46. Ceriani R, Bunati S, Morini L, Sacchi E, Colombo G. Effect of ursodeoxycholic acid plus diet in

patients with nonalcoholic steatohepatitis (abstract). Hepatology 1998;28:386A. 47. Holoman J, Glasa J, Kasar J et al. Serum markers of liver fibrosis in patients with non-alcoholic

steatohepatitis (NASH) (abstract). J Hepatology 2000; 32:A210. 48. Mendez-Sanchez N, Gonzalez V, Pichardo-Bahena R, Uribe M. Weight reduction and

ursodeoxycholic acid in subjects with nonalcoholic fatty liver disease: a randomized, double-blind, placebo-controlled trial [Abstract]. Hepatology 2002;36:412A.

49. Bagci S, Uygun A, Kadayifci A, Bektas A, Ates Y, Erdil A, et al. Ursodeoxycholic acid in the

treatment of nonalcoholic steatohepatitis. [Abstract] J Hepatol 2003;38(Suppl. 2):195. 50. Lindor KD, Kowdley KV, Heathcote EJ, et al. Ursodeoxycholic acid for treatment of nonalcoholic

steatohepatitis: results of a randomized trial. Hepatology 39(3):770–778, 2004 51. Harrison SA, Fincke C, Helinski D, Torgerson S, HayashI P. A pilot study of orlistat treatment in

obese, non-alcoholic steatohepatitis patients. Aliment Pharmacol Ther 2004; 20: 623–628. 52. Popescu A. Preliminary results of the treatment with an endothelin receptor antagonist (ET-RA) in

patients with nonalcoholic steatohepatitis (NASH) associating metabolic disorders. [Abstract] J Hepatol 2003;38(Suppl. 2):199.

53. Adams LA, Angulo P. Vitamins E and C for the treatment of NASH: duplication of results but lack

of demonstration of efficacy. Am J Gastroenterol 98(11):2348–2350, 2003 54. Yokohama S, Yoneda M, Haneda M, Okamoto S, et al. Therapeutic efficacy of an angiotensin II

receptor antagonist in patients with nonalcoholic steatohepatitis. Hepatology. 2004 Nov;40(5):1222-5.

55. Ophardt C. Virtual Chembook. http://www.elmhurst.edu/~chm/vchembook/620fattyacid.html. 2003 56. Browning JD, Horton JD. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest.

2004 Jul;114(2):147-52. 57. Lewis GF, Carpentier A, Adeli K, Giacca A. Disordered fat storage and mobilization in the

pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev. 2002 Apr;23(2):201-29. 58. Foretz, M., Guichard, C., Ferre, P., and Foufelle, F. 1999. Sterol regulatory element binding

protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc. Natl. Acad. Sci. U. S. A. 96:12737–12742.

59. Hasty AH, Shimano H, Yahagi N, Amemiya-Kudo M, Perrey S et al. Sterol regulatory element-

binding protein-1 is regulated by glucose at the transcriptional level. J Biol Chem. 2000 Oct 6;275(40):31069-77.



60. Kotzka J, Muller-Wieland D, Koponen A, Njamen D, Kremer L, Roth G, Munck M, Knebel B, Krone W. ADD1/SREBP-1c mediates insulin-induced gene expression linked to the MAP kinase pathway. Biochem Biophys Res Commun. 1998 Aug 19;249(2):375-9.

61. Yamashita, H., et al. 2001. A glucose-responsive transcription factor that regulates carbohydrate

metabolism in the liver. Proc. Natl. Acad. Sci. U. S. A. 98:9116–9121. 62. Clarke SD, Armstrong MK, Jump DB. Dietary polyunsaturated fats uniquely suppress rat liver fatty

acid synthase and S14 mRNA content. J Nutr 1990; 120(2):225-231.

63. Jump DB, Clarke SD, Thelen A, Liimatta M. Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. J Lipid Res 1994; 35(6):1076-1084.

64. Katsurada A, Iritani N, Fukuda H, Matsumura Y, Nishimoto N, Noguchi T et al. Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-CoA carboxylase in rat liver. Eur J Biochem 1990; 190(2):435-441.

65. Martin G, Schoonjans K, Lefebvre AM, Staels B, Auwerx J. Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthetase genes by PPARalpha and PPARgamma activators. J Biol Chem 1997; 272(45):28210-28217.

66. Bene H, Lasky D, Ntambi JM. Cloning and characterization of the human stearoyl-CoA desaturase gene promoter: transcriptional activation by sterol regulatory element binding protein and repression by polyunsaturated fatty acids and cholesterol. Biochem Biophys Res Commun 2001; 284(5):1194-1198.

67. Ishii S, Iizuka K, Miller BC, Uyeda K. Carbohydrate response element binding protein directly

promotes lipogenic enzyme gene transcription. Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15597-602.

68. Iizuka K, Bruick RK, Liang G, Horton JD, Uyeda K. Deficiency of carbohydrate response element-

binding protein (ChREBP) reduces lipogenesis as well as glycolysis. Proc Natl Acad Sci U S A. 2004 May 11;101(19):7281-6.

69. Carroll DN, Roth MT. Evidence for the cardioprotective effects of omega-3 Fatty acids. Ann

Pharmacother 2002; 36(12):1950-1956.

70. Friedberg CE, Janssen MJ, Heine RJ, Grobbee DE. Fish oil and glycemic control in diabetes. A meta-analysis. Diabetes Care 1998; 21(4):494-500.

71. Mensink RP, Zock PL, Kester AD, Katan MB. Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials. Am J Clin Nutr 2003; 77(5):1146-1155.

72. Harris WS. n-3 fatty acids and serum lipoproteins: human studies. Am J Clin Nutr 1997; 65(5 Suppl):1645S-1654S.

73. MacLean CH, Mojica WA, Morton SC, et al. Effects of Omega-3 Fatty Acids on Lipids and

Glycemic Control in Type II Diabetes and the Metabolic Syndrome and on Inflammatory Bowel Disease, Rheumatoid Arthritis, Renal Disease, Systemic Lupus Erythematosus, and Osteoporosis.



Summary, Evidence Report/Technology Assessment: Number 89. AHRQ Publication Number 04-E012-1, March 2004. Agency for Healthcare Research and Quality, Rockville, MD. http://www.ahrq.gov/clinic/epcsums/o3lipidsum.htm

74. Balk E, Chung M, Lichtenstein A, et al. Effects of Omega-3 Fatty Acids on Cardiovascular Risk

Factors and Intermediate Markers of Cardiovascular Disease. Summary, Evidence Report/Technology Assessment: Number 93. AHRQ Publication Number 04-E010-1, March 2004. Agency for Healthcare Research and Quality, Rockville, MD. http://www.ahrq.gov/clinic/epcsums/o3cardrisksum.htm

75. Montori VM, Farmer A, Wollan PC, Dinneen SF. Fish oil supplementation in type 2 diabetes: a

quantitative systematic review. Diabetes Care. 2000 Sep;23(9):1407-15.

76. Lewis A, Lookinland S, Beckstrand RL, Tiedeman ME. Treatment of hypertriglyceridemia with omega-3 fatty acids: a systematic review. J Am Acad Nurse Pract. 2004 Sep;16(9):384-95.

77. Xu J, Nakamura MT, Cho HP, Clarke SD. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats. J Biol Chem 1999; 274(33):23577-23583.

78. Worgall TS, Sturley SL, Seo T, Osborne TF, Deckelbaum RJ. Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein. J Biol Chem 1998; 273(40):25537-25540.

79. Teran-Garcia M, Rufo C, Nakamura MT, Osborne TF, Clarke SD. NF-Y involvement in the polyunsaturated fat inhibition of fatty acid synthase gene transcription. Biochem Biophys Res Commun 2002; 290(4):1295-1299.

80. Clarke SD. Nonalcoholic steatosis and steatohepatitis. I. Molecular mechanism for polyunsaturated fatty acid regulation of gene transcription. Am J Physiol Gastrointest Liver Physiol 2001; 281(4):G865-G869.

81. Clarke SD. Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance. Br J Nutr 2000; 83 Suppl 1:S59-S66.

82. Brown NF, Esser V, Foster DW, McGarry JD. Expression of a cDNA for rat liver carnitine palmitoyltransferase I in yeast establishes that catalytic activity and malonyl-CoA sensitivity reside in a single polypeptide. J Biol Chem 1994; 269(42):26438-26442.

83. McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem 1997; 244(1):1-14.

84. Mascaro C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D. Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem 1998; 273(15):8560-8563.

85. Baillie RA, Takada R, Nakamura M, Clarke SD. Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition. Prostaglandins Leukot Essent Fatty Acids 1999; 60(5-6):351-356.



86. Rodriguez JC, Gil-Gomez G, Hegardt FG, Haro D. Peroxisome proliferator-activated receptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by fatty acids. J Biol Chem 1994; 269(29):18767-18772.

87. Staels B, Schoonjans K, Fruchart JC, Auwerx J. The effects of fibrates and thiazolidinediones on plasma triglyceride metabolism are mediated by distinct peroxisome proliferator activated receptors (PPARs). Biochimie 1997; 79(2-3):95-99.

88. Agency for Healthcare Research and Quality website: http://www.ahrq.gov/clinic/epcix.htm. 89. The US Food and Drug Administration News Release (P04-89) September 8, 2004

http://www.fda.gov/bbs/topics/news/2004/NEW01115.html 90. Sekiya M, Yahagi N, Matsuzaka T, Najima Y, et al. Polyunsaturated fatty acids ameliorate hepatic

steatosis in obese mice by SREBP-1 suppression. Hepatology. 2003 Dec;38(6):1529-39. 91. Alwayn IP, Gura K, Nose V, Zausche B, et al. Omega-3 fatty acid supplementation prevents

hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr Res. 2005 Mar;57(3):445-52.

92. Alwayn IP, Andersson C, Zauscher B, Gura K, Nose V, Puder M. Omega-3 fatty acids improve

hepatic steatosis in a murine model: potential implications for the marginal steatotic liver donor. Transplantation. 2005 Mar 15;79(5):606-8.

93. Anonymous. Nonalcoholic Steatohepatitis Clinical Research Network. Hepatology 2003;37:244. 94. Brechtel K, Jacob S, Machann J, Hauer B, Nielsen M, Meissner HP et al. Acquired generalized

lipoatrophy (AGL): highly selective MR lipid imaging and localized (1)H-MRS. J Magn Reson Imaging 2000; 12(2):306-310.

95. Longo R, Ricci C, Masutti F, Vidimari R, Croce LS, Bercich L et al. Fatty infiltration of the liver.

Quantification by 1H localized magnetic resonance spectroscopy and comparison with computed tomography. Invest Radiol 1993; 28(4):297-302.

96. Longo R, Pollesello P, Ricci C, Masutti F, Kvam BJ, Bercich L et al. Proton MR spectroscopy in

quantitative in vivo determination of fat content in human liver steatosis. J Magn Reson Imaging 1995; 5(3):281-285.

97. Schonfeld G, Patterson BW, Yablonskiy DA, Tanoli TS, Averna M, Elias N et al. Fatty liver in

familial hypobetalipoproteinemia: triglyceride assembly into VLDL particles is affected by the extent of hepatic steatosis. J Lipid Res 2003; 44(3):470-478.

98. Szczepaniak LS, Babcock EE, Schick F, Dobbins RL, Garg A, Burns DK et al. Measurement of intracellular triglyceride stores by H spectroscopy: validation in vivo. Am J Physiol 1999; 276(5 Pt 1):E977-E989.

99. Tarasow E, Siergiejczyk L, Panasiuk A, Kubas B, Dzienis W, Prokopowicz D et al. MR proton spectroscopy in liver examinations of healthy individuals in vivo. Med Sci Monit 2002; 8(2):MT36-MT40.



100. Therapeutic Lifestyle Changes diet of the National Cholesterol Education Program: http://nhlbisupport.com/chd1/tlc_lifestyles.htm

101. Rambjor GS, Walen AI, Windsor SL, Harris WS. Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans. Lipids 1996; 31 Suppl:S45-S49.

102. Fuller NJ, Laskey MA, Elia M. Assessment of the composition of major body regions by dual-energy X-ray absorptiometry (DEXA), with special reference to limb muscle mass. Clin Physiol 1992; 12(3):253-266.

103. Perseghin G. Pathogenesis of obesity and diabetes mellitus: insights provided by indirect

calorimetry in humans. Acta Diabetol 2001; 38(1):7-21.

104. Warnick GR. Enzymatic methods for quantification of lipoprotein lipids. Methods Enzymol 1986; 129:101-123.

105. Chung BH, Wilkinson T, Geer JC, Segrest JP. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 1980; 21(3):284-291.

30 PUFA P&F Protocol Appendix


Appendix

SUPPLEMENT FORMULATION DOCUMENTS

Nelson, James

From: Rhodes, Ronald [[email protected]]

Sent: Friday, July 22, 2005 11:42 AM

To: Nelson, James

Cc: Oliver-Smith, Glenda

Subject: RE: Customer Request

Page 1 of 3

7/22/2005

Good Afternoon I will list out the test completed during our incoming test for your review. If you have any further questions, please let me know. Regards, Ronald Rhodes Analytical Test Specification Results Arsenic ≤ 0.1 ppm Pass Lead ≤ 0.1 ppm Pass Cadmium ≤ 0.1 ppm Pass Mercury ≤ 0.025 ppm Pass

From: Nelson, James [mailto:[email protected]] Sent: Friday, July 22, 2005 1:19 PM To: Rhodes, Ronald Cc: Oliver-Smith, Glenda Subject: RE: Customer Request Dear Ronald, Thank you for the information regarding Mercury testing on the fish oil we ordered (lot LI000423). Did you also test for any other heavy metal contaminants? Just in case the committee decides to ask. Please note the specific question from our IRB.

1. The committee raised questions about the potential for heavy metal contamination, particularly mercury, in the ώ-3 fish oil capsules. Please supply documentation from Pfizer that the capsules have been tested for this contamination.

I appreciate your help. Thanks Jim Nelson FYI Please change the address you for me (shown Below) as the address you have for me is for another researcher who ordered capsules last year and is tired of getting correspondence about my order (including invoices and bills etc)

James E. Nelson, Ph.D. Research Coordinator

From: Oliver-Smith, Glenda [mailto:[email protected]] Sent: Friday, July 22, 2005 8:50 AM To: Nelson, James Subject: FW: Customer Request Hi Jim, Please see the attached document and contact me if you need anything further. Regards, Glenda L. Oliver-Smith Direct Marketing Sales Capsugel, A Division of Pfizer 864-942-3818 or 888-783-6361 Fax: 864-942-3079 Visit us at www.capsugel.com

From: Rhodes, Ronald Sent: Friday, July 22, 2005 11:48 AM To: Oliver-Smith, Glenda Subject: RE: Customer Request Let me know if you need any further assistance. Ronald

From: Brown, Melanie (Greenwood) Sent: Friday, July 22, 2005 11:04 AM To: Rhodes, Ronald Subject: Customer Request Importance: High Ronald, The lot the customer is requesting the information below is LI000423. Regards, Melanie B Brown Capsugel Liquids Group 535 N. Emerald Road

Department of Medicine Division of Gastroenterology University of Washington Medical Center 1959 N.E. Pacific Street Box 356424 Seattle, WA 98195 Phone: (206) 221-4537 Fax: (206) 616-7805 Email: [email protected]

Page 2 of 3

7/22/2005

Phone: (864) 942-3787 Fax: (864) 942-3858

From: Oliver-Smith, Glenda Sent: Thursday, July 21, 2005 4:36 PM To: Brown, Melanie (Greenwood) Subject: University of Washington Hi Melanie, Jim Nelson from University of Washington ([email protected]) – 206-221-4537 called today asking for an analysis on the mercury content for the fish oil capsule that we made for them (Order number: C0007993-1-1). The resource number is: LG6L0040580001. Please copy me on the response. Thanks, Glenda L. Oliver-Smith Direct Marketing Sales Capsugel, A Division of Pfizer 864-942-3818 or 888-783-6361 Fax: 864-942-3079 Visit us at www.capsugel.com

Privileged, confidential or patient identifiable information may be contained in this message. This information is meant only for the use of the intended recipients. If you are not the intended recipient, or if the message has been addressed to you in error, do not read, disclose, reproduce, distribute, disseminate or otherwise use this transmission. Instead, please notify the sender by reply e-mail, and then destroy all copies of the message and any attachments.

Page 3 of 3

7/22/2005

pufa protocol

Health & Medicine