Marine n-3 polyunsaturated fatty acids in renal transplantation

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Marine n-3 polyunsaturated fatty acids in renal transplantation

Ivar Anders Eide

Faculty of Medicine UNIVERSITY OF OSLO

Section of Nephrology Department of Transplant Medicine

OSLO UNIVERSITY HOSPITAL RIKSHOSPITALET

2015

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© Ivar Anders Eide, 2016

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8333-191-2

Cover: Hanne Baadsgaard Utigard

Printed in Norway: 07 Media AS – www.07.no

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1 Acknowledgements

During 2010, My Svensson, Anders Hartmann and Trond Jenssen launched the idea that a strong renal transplantation research community in Oslo should join forces with a strong marine fatty acids research community in Aalborg. They came up with a project plan; an observational cohort study (presented in this thesis) and an interventional study (ORENTRA) to investigate whether marine n-3 polyunsaturated fatty acids (marine n-3 PUFAs) might be of benefit to renal transplant recipients. I was fortunate to be asked to join in. This study would not have been possible without the support of a number of special people:

Trond Jenssen. I am honored and grateful that you took on the task of being my main supervisor. You have taught me, both consciously and unconsciously, how good research should be done; the accuracy in your interpretation, your wit, your sense of humor, your enthusiasm, your pragmatism, your friendliness. I have been taking notes! Thank you for your constant support, for your loyalty, for your leadership and for giving me the opportunity to join in on your research in posttransplantation diabetes.

My Svensson. Through a wide range of contributions, from study design, via administrative and logistic support to revision of manuscripts, you have been essential for this project. I am truly grateful for your efforts, your vast knowledge of marine fatty acids and the fireworks of ideas that you are. You think faster, talk faster, walk faster than … everyone really. This and more, to me, you are first of all a warm and caring supervisor and a good friend.

Anders Hartmann. The Grand Man in Norwegian Nephrology and Transplant Medicine – I skipped “old”. It has been an honor and a privilege to be part of your research group. Insights that to me appeared beyond attainment always seemed close at hand for you. Thank you for your constant support and for multiple contributions to this project, including such

unfulfilling tasks as to help me obtain missing data on smoking habits a late Friday afternoon.

I wish to express my warm thanks for your kindness and your humble wisdom, and for creating a good working environment at The Renal Physiology Laboratory!

Anders Åsberg, Kirsten Lund, May Ellen Lauritsen, Els Breistein and Sebastian Müller / The Renal Physiology Laboratory. A sincere gratitude goes out to Kirsten, May Ellen and Els for obtaining data in the ORENTRA trial and for maintaining the biobank. What luck that Anders and Sebastian also came to work with us. With them a wave of positive energy swept over the laboratory. Thanks for scientific insight, contagious good mood and for good advice.

Finn Reinholt, Linda Dorg and co-workers at The Department of Pathology. With great patience, massive support and strong coffee you have trained me step by step to make sense of renal graft biopsies as part of a study in diabetic transplant recipients and in the ORENTRA trial. I think of Finn and Linda as my Jedi masters, full of wisdom and calm, hearing their voices in my head: Appreciate the beauty of the tissue first you must! I would also like to thank you for interesting discussions. You have convinced me that counting always counts.

Torbjørn Leivestad / The Norwegian Renal Registry, Jeppe Hagstrup Christensen, Erik Berg Schmidt and co-workers Rikke Bülow Eschen, Annette Andreassen, Birthe H. Thomsen and Inge Aardestrup at The Lipid Research Laboratory in Aalborg. Traditions are not dead weight which we drag along with us. They are the roots of the tree we cultivate. This is the 27^th PhD thesis based on data from The Norwegian Renal Registry. I would like to thank Torbjørn for

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contributing high quality data throughout the years and for interesting anecdotes. Numerous publications and PhD theses on marine n-3 PUFAs have come out The Lipid Research Laboratory in Aalborg and paved the path before me. A special thanks to Erik and Jeppe for good advice and for taking good care of me during my visits to Aalborg.

Anna Reisæter: Thank you for your support, interesting discussions and good leadership. Dag Olav Dahle: Thank you for sharing your deep understanding of numbers with me, for teaching me ultrasound techniques and for kindness. Lien Diep: Thank you for statistical help and many interesting discussions about food and science. Ida Robertsen: Thank you for teaching me pharmacokinetics, for performing the AdvaOmega study and for sharing your insight in an amusing way. Thea Halden: Thank you for collaboration on diabetes projects, for good talks and cracking jokes. Thanks to Hanne Jørgensen, Hege Pihlstrøm, Jørn Petter Lindahl, Marit Elisabeth von Düring, Jesper Fleischer and Christina Dörje for collaboration in various studies and for good company. I would like to extend my gratitude to Elisabeth, Stein, Bartek, Yulia, Kjersti, Thea, Marthe, Kristin O., Knut and Hilde S., Hallvard, Karsten, Geir, Linda, Hilde W. and fellow PhD students at “Forvalterboligen and co-workers at the observational unit and the outpatient clinic. Thanks to my roommates Espen and Harald for long sessions of pure nonsense. Thanks to Erlend for that impressive beard. Thanks to Gerd and Egil for introducing me to Nephrology and for your support. Thanks to Joe for inviting me to join his project group. Thanks to the Clinical Research Support Unit for assistance with study protocols. Thanks to “Forskningsposten”, The Research Institute of Internal Medicine and to The Norwegian Hip Fracture Register for their collaboration. Thanks to The

Department of Radiology, to the transplant surgeons at OUS and to Jens, Kristin G. and other colleagues at The Department of Endocrinology for their contributions to various studies and for good company.

I am grateful to the University of Oslo for accepting me on the PhD program and for

interesting courses. I wish also to thank Aarhus University for accepting me as a PhD student under cotutelle agreement. I would like to express a sincere thanks to the funding sources:

South-Eastern Norway Regional Health Authority, Gidske and Peter Sørensen Research Fund, The Norwegian National Association for Kidney Patients and Transplant Recipients Research Fund, Gertrude and Jack Nelson Research Fund, Freia Corporation Medical Fund, Signe and Albert Bergsmarken Research Fund, The Raagholt Foundation and Nathalia and Knut Juul Christensen Research Fund. Thanks to Pronova Biopharma for provision of study medication.

The personal pronoun in research is “we”, which I find quite relieving. Study participants are, in my opinion, part of that “we”. The ORENTRA participants also helped me to a better understanding of dietary habits, life-style factors, drug adherence and science. Thank you!

I wish to express my sincere gratitude to my father-in-law and co-author Kristian Bjerve, who introduced me to the world of marine n-3 PUFAs, constantly supported my work and

discussed major and minor issues of relevance to this project. Thanks to my brother-in-law James Eide Macpherson and my sister Magnhild for their support, revision of manuscripts and for pointing out the perks of being a PhD student, to my sister Gudrid and Karl Henrik, my mother in-law Eva, my sister-in-law Yngvil and Fredrik and to my parents Målfrid and Anders for their constant support. Finally, I wish to express a special thanks to my daughters Oda and Lea and my wife Torunn, for their immense patience and understanding and for bringing so much light and meaning to my life. A warm thanks also goes out to the rest of my family and to my friends – to me, you are a bunch of legends.

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2 List of papers

Paper I

Eide IA, Jenssen T, Hartmann A, Diep LM, Dahle DO, Reisæter AV, Bjerve KS, Christensen JH, Schmidt EB, Svensson M.

The associations between marine n-3 polyunsaturated fatty acid levels and survival after renal transplantation

Clin J Am Soc Nephrol 2015; Jul 7;10(7):1246-56.

Paper II

Eide IA, Dahle DO, Svensson M, Hartmann A, Åsberg A, Bjerve KS, Christensen JH, Schmidt EB, Lauritsen ME, Lund K, Jenssen T.

Plasma levels of marine n-3 fatty acids and cardiovascular risk markers in renal transplant recipients

Manuscript accepted for publication in European Journal of Clinical Nutrition.

Paper III

Eide IA, Jenssen T, Hartmann A, Diep LM, Dahle DO, Reisæter AV, Bjerve KS, Christensen JH, Schmidt EB, Svensson M.

Plasma levels of marine n-3 polyunsaturated fatty acid levels and renal allograft survival Nephrol Dial Transplant 2015; Sep 25: Electronic publication ahead of print.

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3 Abbreviations

AA: Arachidonic acid

β-coeff.: Regression coefficient

CI: Confidence interval

DART: Diet and Reinfarction Trial DHA: Docosahexaenoic acid DPA: Docosapentaenoic acid

DOIT: Diet and Omega-3 Interventional Trial eGFR: Estimated glomerular filtration rate EPA: Eicosapentaenoic acid

EPIC: European Prospective Investigation into Cancer and Nutrition ESRD: End-stage renal disease

GISSI-Prevenzione: Gruppo Italiano per lo Studio della Soprawivenza nell’Infarcto Miocardio Prevenzione Trial

HDL: High-density lipoprotein

HR: Hazard ratio

JELIS: Japan Eicosapentaenoic acid Lipid Intervention Study LDL: Low-density lipoprotein

PUFA: Polyunsaturated fatty acid

R²: Explained variance

RCT: Randomized controlled trial RTR: Renal transplant recipient SCD: Sudden cardiac death

Std.: Standardized

TGF: Transforming growth factor Unstd.: Unstandardized

wt%: Weight percentage of total plasma phospholipid fatty acids

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4 Summary

Background. Cardiovascular disease is the leading cause of death following renal transplantation. Marine n-3 polyunsaturated fatty acids (PUFAs) exert cardio-protective metabolic effects that may lower mortality risk after renal transplantation. Anti-inflammatory effects of marine n-3 PUFAs and potential beneficial effects on fibrosis may preserve renal graft function and prevent graft loss.

Materials and methods. This retrospective observational cohort study included 1990 Norwegian renal transplant recipients transplanted between 1999 and 2011. Associations between plasma marine n-3 PUFA levels and patient and renal graft survival were assessed by multivariable Cox proportional hazard regression analysis. Associations between plasma marine n-3 PUFA levels and cardiovascular risk markers at ten weeks post-transplant as well as change in renal graft function during the first five years after transplantation were

evaluated by linear regression analysis. Plasma phospholipids fatty acids were determined by gas chromatography. Plasma marine n-3 PUFA levels were defined as the sum of the three marine n-3 fatty acids eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) levels in weight percentage (wt%) of total plasma phospholipid fatty acids.

Results. During a median follow-up time of 6.8 years, 569 (28.6%) renal grafts were lost, either due to death of the renal transplant recipient (n=340) or graft failure in surviving patients (death censored graft loss, n=229). There were 406 deaths (20.4%) during follow-up, including 164 deaths due to cardiovascular disease (40.4% of deaths), 95 deaths due to cancer (23.4%) and 101 deaths due to infectious disease (24.9%).

Plasma marine n-3 PUFA levels ranged from 1.35 to 23.87 wt%, with a median level of 7.95 wt% (interquartile range 6.20 to 10.03 wt%). Mortality rates were lower in patients with high plasma marine n-3 PUFA levels (≥ 7.95 wt%) compared with low levels (< 7.95 wt%) across age groups, with a pooled mortality rate ratio estimate of 0.69 (confidence interval [CI] 0.57 to 0.85). When grouped according to plasma marine n-3 PUFA levels, patients belonging to the upper quartile (≥ 10.03 wt%) compared with the lower (≤ 6.20 wt%) had a 56% lower mortality risk (multivariable adjusted hazard ratio [HR] 0.44; 95% CI 0.26 to 0.75). Similar

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results were found for plasma EPA and DHA levels, when analyzed separately. In contrast, no association was found between plasma DPA levels and mortality. Patients belonging to the upper marine n-3 PUFA quartile compared with the lower had a significantly lower cardiovascular mortality risk (multivariable adjusted HR 0.18; 95% CI 0.08 to 0.42),

primarily due to strong negative associations between plasma marine n-3 PUFA levels and the risk of lethal stroke or sudden cardiac death. We found no significant association between plasma marine n-3 PUFA levels and deaths due to infectious disease. However, patients belonging to the upper plasma EPA quartile compared with the lower had a significant lower risk of death due to infectious disease. Plasma marine n-3 PUFA levels were not associated with death due to cancer.

For every 1.0 wt% increase in plasma marine n-3 PUFA level, there was an 11% reduced risk of overall graft loss (multivariable adjusted HR 0.89; 95% CI 0.84 to 0.93) and a 10%

reduced risk of death censored graft loss (multivariable adjusted HR 0.90; 95% CI 0.84 to 0.97). Higher plasma marine n-3 PUFA levels were associated with lower decline in renal graft function during the first five years after transplantation. Acute rejection episodes within the first three months after renal transplantation did not differ across marine n-3 PUFA quartiles. However, beyond three months, patients belonging to the lower marine n-3 PUFA quartile suffered more acute rejection episodes than patients in the three upper quartiles.

Plasma marine n-3 PUFA levels were associated with lower resting heart rate, lower fasting plasma glucose levels, lower plasma triglyceride levels and higher plasma high-density lipoprotein cholesterol levels, while no significant association were found with blood pressure, pulse wave velocity or plasma low-density lipoprotein cholesterol levels.

Conclusion. Higher plasma marine n-3 PUFA levels were independently associated with better patient and graft survival after renal transplantation. We also found significant beneficial associations between plasma marine n-3 PUFA levels and plasma triglyceride levels, resting heart rate, fasting plasma glucose levels, plasma high-density lipoprotein cholesterol levels, acute rejection rate and renal graft function, which might explain a lower mortality and graft loss risk in patients with high plasma marine n-3 PUFA levels.

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5 Introduction

In 1929, Dr. George Burr described the first essential fatty acids (1). This changed the perception of fatty acids from just a source of calories to potent dietary factors (2). Some polyunsaturated fatty acids (PUFAs) are essential – they cannot be synthesized by humans.

They are necessary for survival and need to be consumed (3). There are two types of essential fatty acids, n-6 PUFAs and n-3 PUFAs named according to the position of the first double bond between carbon atoms, counted from the methyl end of the fatty acid (Figure 1).

Consumption of seafood provides the essential marine fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (4).

Cell membrane phospholipids incorporate fatty acids (4). The length of the fatty acid, as well as number and position of double bonds, determine their biological properties and affect cell membrane structure and function. Unsaturated double bonds make it harder for phospholipids to pack together by creating twists and turns to an otherwise straight hydrocarbon chain (Figure 1), increasing the viscosity of cell membranes. Fatty acid composition also affects the activity of cell membrane bound proteins like receptors, enzymes and ion channels (4).

In the late 1960s, Dr. Hans Olaf Bang and Dr. Jørn Dyerberg measured plasma lipid levels and fatty acid composition in Greenland Eskimos. Despite a high fat diet, Greenland Eskimos had low plasma triglyceride and total cholesterol levels (5). Compared with a Danish

population, they had a lower incidence of cardiovascular and infectious diseases (6). The diet rich in marine fatty acids was a plausible explanation (6). Although these pioneer studies have received some critique for choice of methods (7), similar associations have been found in other populations with a high marine n-3 PUFA intake (8, 9).

Carboxyl end

Double bonds

n-3 position from the methyl end Methyl end

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Dr. Dyerberg later became a chief physician at Aalborg University Hospital, where he set up a lipid research laboratory. This laboratory, which performed the fatty acid analysis in the current study (Figure 2), has been the cornerstone of an extensive marine n-3 PUFA research activity in Denmark. During the last four decades several beneficial effects of marine n-3 PUFAs have been described, some of which might be of particular importance for renal transplant recipients (RTRs) (Table 1).

The concept of organ transplantation is old. In India, skin transplantations were performed already in the second century BC, inspired by the transplanted elephant head of Ganesha, the Hindu God of Wisdom (10). In Europe, the history of organ transplantation dates back to the fourth Century AD. In 348, the twin brothers Cosmas and Damian, both trained physicians, transplanted a black lower right limb of a recently deceased Ethiopian Gladiator to a white Roman to replace a cancerous leg. Surprisingly, the transplanted organ (graft) survived several days (10). Around 1850, European surgeons started to perform skin transplantations and systematically record observations. It became evident that skin transplants harvested from the recipient, as opposed to donor grafts, survived longer. They also found that a second donor graft was rejected earlier than the first, suggesting that immunological barriers might be the cause of organ transplant (graft) rejection (11).

In 1954, Dr. Joseph Murray performed a live donor renal transplantation in genetically identical twins. The recipient died eight years later with a functioning graft (12). This case showed that long-term patient and graft survival was possible after organ transplantation. This would not have been possible without insights in blood groups, the human leukocyte antigen

Column retention time (min) 40 45 50 EPA

DPA DHA

Column Automatic

chart recorder Detector

Temperature regulator Helium (carrier gas)

Flow regulator Pressure

regulator

Injection port Sample

injector

4) Fatty acid quantification by gas chromatography

Fatty acid analysis

≥ 500 ml plasma 1) Extraction of lipids

2) Isolation of phospholipid fraction 3) Transmethylation of fatty acids

Step 4 start with injection of transmethylated fatty acids

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system and concepts of immunosuppression and rejection (13-15). The discovery of

immunosuppressant properties of some substances, especially prednisolone in the late 1940’s and cyclosporine A in 1969, greatly improved organ transplant survival (16, 17).

The first renal transplantation in Norway was performed by Dr. Leif Efskind in 1956 (18).

The same year, hemodialysis was offered for the first time in Norway (19). In other Nordic countries, organ transplantations were performed from 1964 and in 1969 a Scandinavian transplant program was initiated. The shared deceased donor pool for the Scandinavian countries and improved life expectancy for uremic patients due to dialysis therapy made it possible to offer a renal transplant to an increasing number of patients during the 1970’s and 1980’s (19). The introduction of cyclosporine A at our center in 1983 dramatically improved short-term graft survival (20).

In Norway, all organ transplantations are performed at Oslo University Hospital

Rikshospitalet, which serves a population of about 5.2 million inhabitants. In recent years, 264 to 302 renal transplantations have been performed annually at our center (20). Although the supply of renal grafts is still less than the demand, median time on wait-list for renal transplantation has gradually been reduced and is now only about eight months (20).

Patient outcome after renal transplantations performed at Oslo University Hospital have been recorded since 1963 (20). Around 1980 Dr. Torbjørn Leivestad started to systematically register all Norwegian patients on dialysis therapy and renal transplant recipients (RTRs), in order to plan the transplant activity at our center (20). The registry expanded to become The Norwegian Renal Registry some years later. It is now based upon annual reports from all nephrologists working in Norway, includes all Norwegian patients on renal replacement therapy (dialysis therapy or renal transplantation) and is continuously updated.

During the last few decades, improved surgical procedures and immunosuppressive regimens have resulted in excellent short-term renal graft survival (21). However, progressive fibrosis continues to be an obstacle to long-term graft survival, which has changed very little in recent years (21). Compared with dialysis therapy, renal transplantation reduces the risk of

cardiovascular mortality in uremic patients (22). Still, RTRs suffer an annual cardiovascular mortality rate twice that of the general population (22). This is partly due to a high prevalence of traditional cardiovascular risk factors in patients with end-stage renal disease (ESRD) and the uremic phase prior to transplantation (22). Dyslipidemic and hyperglycemic side-effects

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from immunosuppressive drugs and impaired renal graft function also pose a threat to patient and graft survival (22). New therapeutic agents that might lower mortality risk and / or improve long-term renal graft survival are sought.

Table 1. Established and proposed (*) effects of marine n-3 polyunsaturated fatty acids, mortality and graft failure risk factors in patients who have received a renal transplant

Marine n-3 PUFAs effects Mortality risk factors (22) Graft failure risk factors (22, 23) Reduce triglycerides (5, 24) Recipient age Recipient age

Increase high-density lipoprotein cholesterol

(5, 24) Dyslipidemia Human leukocyte antigen match between donor and recipient Increase heart rate

variability

(25) Diabetes mellitus Drug non-adherence, chronic and / or acute rejections

Anti-hypertensive effect (26) Hypertension Hypertension

Anti-thrombotic effect (27) Cardiovascular disease Ischemia / reperfusion injury Improve endothelial function (28) Delayed and / or impaired

graft function

Delayed and / or impaired graft function

Less arterial calcification (9) Time in dialysis therapy Recurrent or de novo glomerular disease

Atherosclerotic plaque stabilization

(29) Donor age and type (living / deceased donor)

Donor age and type (living / deceased donor)

Anti-inflammatory effect (30) Viral and bacterial infections Viral and bacterial infections Anti-fibrotic effect * (31) Cancer Interstitial fibrosis and tubular

atrophy

Renoprotective effect * (32) Smoking Calcineurin inhibitor

nephrotoxicity

Increase immunotolerance * (33) Obesity Nephron mass

Other proposed or established effects of marine n-3 polyunsaturated fatty acids, that might influence health after renal transplantation, include increased cell membrane fluidity, cytoprotection against oxidative stress by autophagy, reduced angiogenesis, induction of apoptosis of cancer cells, detoxification of pollutant and reactive oxygen species, increased gastrointestinal transit time and absorption of calcineurin inhibitors, improved hemodialysis graft patency, lowering of homocysteine levels, lower remnant lipoprotein levels, lower fibroblast growth factor 23 levels, improved bone metabolism, reduced lipid peroxidation, modulation of brain monoamine pathways, modulation of endocannabinoid signal pathways and modulation of sodium and calcium transport (4, 30, 34-52).

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5.1 Marine n-3 polyunsaturated fatty acids in renal transplantation

To date, no observational studies and only small randomized controlled trials (RCTs) (42, 53- 72) have studied the effects of marine n-3 PUFAs in RTRs (Table 2). Meta-analyses of these studies reported a significant reduction in plasma triglyceride levels, a minor reduction of diastolic blood pressure and slightly increased plasma high-density lipoprotein (HDL) cholesterol levels in patients who received marine n-3 PUFA supplements compared with controls (73-75). The effect of marine n-3 PUFA supplementation on renal graft function is unclear (73, 74). Due to a low number of events, meta-analyses of RCTs could not evaluate effects of marine n-3 PUFAs on mortality and graft loss incidence (7 deaths and 45 graft losses out of 846 patients) and acute rejection rates were not significantly different in the interventional group compared with the control group (73, 74).

Marine n-3 PUFAs might possibly improve patient survival in RTRs through various cardio- protective effects (Table 1). Uremic dyslipidemia is characterized by high triglyceride and low HDL cholesterol levels (76). Although renal transplantation improve lipid profiles (76), dyslipidemia is common in RTRs (22). Statin therapy, which primarily lower low-density lipoprotein (LDL) cholesterol, has been reported to lower the risk of major cardiovascular events in RTRs (77). At our center, three out of four RTRs receive statin therapy at one year post transplantation (20). A triglyceride-lowering effect of marine n-3 PUFAs has been repeatedly shown in various patient populations (37), also in patients on statin therapy (78).

Only a small cross-over study has investigated the additional effect of marine n-3 PUFA supplementation to statin therapy in RTRs and found that the combined treatment lowered triglyceride levels more efficiently than statins alone (72).

Patients with ESRD suffer a particularly high risk of sudden cardiac death (SCD) (79), and SCD is still prevalent after renal transplantation (22). Marine n-3 PUFAs lower resting heart rate and increase heart rate variability, which might reduce the risk of arrhythmias and SCD (44). To our knowledge, no previous study has investigated the relationship between marine n-3 PUFAs and heart rate in renal transplantation.

Marine n-3 PUFAs may possibly exert beneficial effects on renal grafts. EPA competes with n-6 PUFA arachidonic acid (AA) as substrate in the cyclooxygenase and lipoxygenase pathways. The cyclooxygenase pathway produces the prostaglandin hormones that initiate the inflammation process (30). Prostaglandins synthesized from EPA, as opposed to AA, are not pro-inflammatory (30). Specialized pro-resolving lipid mediators (protectins and resolvins),

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synthesized from marine n-3 PUFAs in the cyclooxygenase pathway, reduce lymphocyte infiltration and inflammatory cytokine production (80). Another branch of the

cyclooxygenase pathway leads to synthesis of the vasoactive hormone thromboxane. AA derived thromboxane cause vasoconstriction, as opposed to thromboxane synthesized from EPA (30). Calcineurin inhibitors reduce blood flow into the glomeruli, which might reduce renal graft function (81). This side-effect might be counteracted by EPA (30, 42).

In the lipoxygenase pathway, EPA derived leukotrienes not only reduce vascular and bronchial constriction, but also the ability of leukocytes to detect cytokines, thereby reducing mitigation of leukocytes towards inflamed areas (30).

Inflammation causes release of both inflammatory and pro-fibrotic cytokines (31). Pro- inflammatory cytokines stimulate fibroblasts to synthesize and deposit proteins that intrude between cells (31). This process, called fibrosis, obliterates normal tissue structure and change the environment surrounding the cells, therefore also their function and increase the risk of cell death (31). Progressive fibrosis in the renal graft will eventually lead to graft failure (82). Marine n-3 PUFAs have been found to reduce fibroblast proliferation and collagen synthesis (31), which are essential steps in development and progression of fibrosis (83). Finally, EPA may exert immunomodulatory effects through upregulation of regulatory T-lymphocytes (33). These cells modulate the immune system to maintain tolerance to antigens and thereby not only reduce the tendency to reject a renal graft, but also reduce chronic low-grade inflammation (33). Anti-inflammatory effects of marine n-3 PUFAs are scarcely studied in RTRs (64).

Hypertension may cause renal damage (84). Marine n-3 PUFA supplementation has been found to lower blood pressure in both non-transplant and transplant populations (26, 55, 85).

Since the endothelium regulates vascular tone and permeability, endothelium dysfunction will impair renal function (86). Marine n-3 PUFAs may improve endothelial function (87). In renal transplantation, no interventional study has investigated the effects of marine n-3 PUFAs on arterial stiffness or endothelial function to date.

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Table 2. Marine n-3 polyunsaturated fatty acids in renal transplantation. Studies performed to date. Panel A: Study characteristics.

Ref Yr First author Country

Base- line*

Dur- ation

Dose

EPA+DHA Placebo

Sample size

Lost#

%

(53) 89 Urakaze Japan 2yr 26w 2.2 g/day None 30 0

(54) 90 van der Heide Netherlands 3d 4w 3.0 g/day Coconut oil 31 0 (55) 90 van der Heide Netherlands 36w 12w 3.0 g/day Corn oil 21 14 (56) 92 van der Heide Netherlands 3d 4w 3.0 g/day Coconut oil 88 0 (57) 93 van der Heide Netherlands 3d 52w 3.0 g/day Coconut oil 66 9

(58) 92 Berthoux France 3d 52w 2.7 g/day None 32 9

(59) 94 Corda France 12w 27w 1.2 g/day Olive oil 23 0

(60) 95 Bennett US 16w 26w 2.7 g/day† Corn oil 90 32

(61) 95 Maachi France 3d 52w 2.4 g/day None 80 2

(62) 96

Kooijmans-

Coutinho Netherlands 3d 12w 3.0 g/day Coconut oil 50 9

(42) 98 Busnach Italy 1d 52w 2.6 g/day‡ Olive oil 42 0

(63) 00 Santos Portugal 2d 52w 3.0 g/day Unknown 60 0

(64) 02 Hernandez Spain 2d 12w 1.9 g/day Soy oil 91 0

(65) 89 Sweny UK 14 pts with chronic rejection. Marine n-3 PUFA, no placebo.

(66) 93 Schut Netherlands Cross-over, 29 pts. Marine n-3 PUFA 16w, corn oil 16w.

(67) 95 Hansen Denmark 18 pts + 9 healthy controls, both received marine n-3 PUFA 10w.

(68) 04 Singer Israel Parenteral marine n-3 PUFA to 8 donors and their recipients.

(69) 13 Sabbatini Italy 26 pts received diet rich in fatty fish vs no dietary advice Three studies have compared the effects of marine n-3 PUFAs vs statins after renal transplantation:

(70) 97 Castro Portugal 1yr 12w 3.0 g/day Simvastatin 43 0

(71) 97 Rodriguez Spain 26w 26w 3.0 g/day Lovastatin 34 6

(72) 01 Grekas Greece Cross-over, 24 pts. Pravastatin vs pravastatin + marine n-3 PUFA.

Abbreviations: yr: year, d: days, w: weeks, pts: patients, vs: versus, EPA: eicosapentaenoic acid, DHA:

docosahexaenoic acid. *: After time of transplantation. #: Lost to follow-up. †: Some patients received 5.4 g EPA+DHA / day. ‡: Some patients received 5.1 g EPA+DHA / day.

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Table 2 continued. Panel B: Results.

Ref

Deaths (I / P)

Graft failure

(I / P)

Acute rejection

(I / P)

Effects (interventional group compared with placebo group) on lipids, blood pressure, cyclosporine A trough levels and renal graft function (53) 0 / 0 0 / 0 0 / 0 No effect on triglyceride level, BP or graft function

(54) 0 / 0 0 / 0 3 / 6 No effect on BP, graft function or CyA level

(55) Lower BP, better graft function, no effect on CyA level

(56) 0 / 0 1 / 1 15 / 12 No effect on BP, graft function or CyA level (57) 0 / 1 1 / 4 8 / 20 No effect on BP, graft function or CyA level (58) 0 / 0 3 / 4 15 / 13 Better graft function, no effect on lipids or CyA level (59) 0 / 0 0 / 0 No effect on lipids or graft function

(60) 0 / 0 0 / 0 8 / 5 Lower diastolic BP, no effect on systolic BP, graft function or lipids (61) 0 / 0 5 / 5 29 / 32

Better graft function, lower triglyceride levels, no effect on CyA or cholesterol levels

(62) 1 / 3 4 / 3 24 / 14 No effect on lipids, BP, graft function or CyA levels

(42) 2 / 0 2 / 2 3 / 3*

Better graft function, lower triglyceride and higher HDL cholesterol levels, no effect on total cholesterol or CyA levels

(63) 0 / 0 0 / 0 5 / 8 No effect on lipids, BP, graft function or CyA levels

(64) 0 / 0 6 / 4 20 / 19

Lower total cholesterol levels, no effects on triglycerides, BP, graft function or CyA levels. Effects on cytokines presented in section 9.6 (65)

Better graft function, lower triglyceride levels, no effect on cholesterol levels

(66) No effect on BP or graft function

(67) Lower BP, no effect on graft function or CyA levels

(68) No effect on graft function

(69) Lower triglyceride and cholesterol levels, no effect on graft function (70) 0 / 0 0 / 0 No difference in lipid levels between the groups

(71) 0 / 0 0 / 0

Lower triglyceride levels in the marine n-3 PUFA group and lower cholesterol levels in the statin group

(72) Lower triglyceride levels in the combined treatment group

I: Interventional group. P: Placebo group. BP: Blood pressure. CyA: Cyclosporine A. *: Patients, not episodes.

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5.2 Marine n-3 polyunsaturated fatty acids in patients with chronic kidney disease and other patient populations at high risk of cardiovascular events

Although large epidemiological studies report inconsistent results (Table 3), most of the studies suggest an overall negative association between fish consumption and cardiovascular morbidity and mortality (9, 88-102). For decades, concordant evidence from interventional studies also suggested cardio-protective effects of marine n-3 PUFAs (4). This view has been challenged in recent meta-analyses of RCTs (103-105), indicating no significant effects of marine n-3 PUFA supplementation on cardiovascular morbidity or mortality. These meta- analyses were hampered by substantial heterogeneity, received critique for their selection of studies (106) and other meta-analyses of RCTs reached the opposite conclusion (107, 108).

Nonetheless, the evidence for additional cardioprotective effects of marine n-3 PUFAs to optimal anti-thrombotic, anti-hypertensive and statin therapy is unclear, as most RCTs performed after the year 2000 have failed to show significant beneficial effects of marine n-3 PUFA supplementation on cardiovascular morbidity and mortality (78, 109-121). These drugs share many of the beneficial effects of marine n-3 PUFAs (4, 30, 37). However, some patient populations might benefit from a high marine n-3 PUFA intake, including patients with dyslipidemia (78), chronic kidney disease (32, 79, 122) and transplant recipients (123-129).

In patients on hemodialysis therapy, plasma marine n-3 PUFA levels were negatively associated with the risk of SCD (79) and marine n-3 PUFA supplementation reduced the incidence of myocardial infarction (MI) (122). Marine n-3 PUFA supplementation have been found to slow down the decline in renal function in patients with IgA nephritis (32). Improved renal function after marine n-3 PUFA supplementation has also been reported in patients without renal disease (117, 124, 127). Marine n-3 PUFA supplementation has also been shown to improve arteriovenous graft patency (41, 130, 131) and most studies in ESRD patients report a modest improvement of lipid profile (38, 130, 132-137).

A few interventional studies have investigated the effects of marine n-3 PUFAs after cardiac transplantation, and report improved lipid profile, lower blood pressure and lower resting heart rate (123-126). Studies on the effects of marine n-3 PUFAs in liver and bone marrow transplant recipients are scarce, but promising (127-129). To our knowledge, no study has investigated the effects of marine n-3 PUFAs in skin or lung transplantation. Due to possible gastrointestinal side-effects (74), marine n-3 PUFAs are probably not the drug of choice in intestinal or pancreas transplant recipients.

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Table 3. Effects of marine n-3 polyunsaturated fatty acids in patient populations other than renal transplant recipients.

Ref Yr First author Country Study population, design, sample size, follow-up time and findings Epidemiological studies in the general population

(88) 86 Norell Sweden Twin registry, n=10,966, DQ, 14yr follow-up. Lower cardiovascular mortality.

(89) 95 Ascherio US Health Professionals Study. Men 45-85yr, n=44,895, DQ, up to 11yr follow-up. No association with coronary artery disease incidence.

(90) 98 Albert US Physicians Health Stud. Men 40-85yr, DQ, n=20,551. Lower overall mortality, lower SCD incidence, but no effect on cardiovascular mortality.

(91) 02 Albert US Physicians Health Study, nested case control study, n=278. Less SCD with increasing plasma marine n-3 PUFA levels.

(102) 97 Daviglus US Chicago Western Electric Study. Men 40-55yr, n=1,822, dietary interview, mean follow-up 26yr. Lower cardiovascular mortality.

(92) 97 Pietinen Finland Men 50-70yr who were current smokers, n=21,930, DQ, 6yr follow-up.

Non-significant tendency towards increased mortality.

(93) 01 Yuan China Men 45-65yr, n=18,244, DQ, 12yr follow-up. Lower overall mortality, but no association with cardiovascular mortality.

(100) 01 Iso US Nurses’ Health Study. Women 35-60yr, n=79,839, DQ, 14yr follow- up. Higher fish consumption; lower stroke risk in non-aspirin users.

(98) 02 Hu US Nurses’ Health Study. Lower risk of coronary artery disease and cardiovascular mortality.

(99) 08 Sun US Nurses’ Health Study. Nested case control study, n=434. Lower risk of MI, lower triglyceride and higher HDL cholesterol levels with higher EPA and DHA, but not with DPA levels

(95) 05 Mozaffarian US Cardiovascular Health Study. Age ≥65yr, n=45,722, DQ, 14yr follow- up. Lower incidence of coronary artery disease.

(96) 05 Mozaffarian US Cardiovascular Health Study. Lower incidence of heart failure.

(94) 06 Iso Japan JPHC. Age 40-60yr, n=41,578, DQ, 11yr follow-up. Lower risk of coronary artery disease.

(9) 08 Sekikawa Japan Japanese in Japan, Japanese in US, Whites in US, cross-sectional study, n=868. The “Japanese” factor is related to a diet rich in marine fatty acids; lower carotid artery thickness and coronary artery calcification.

(97) 10 Bjerregaard Denmark Age 50-65yr, n=57,053, DQ, 7.5yr follow-up. Lower risk of coronary artery disease in men, but not in women.

(101) 10 Pottala US Heart and Soul Study. Adult patients, n=956, fatty acid analysis, 5.9yr follow-up. Higher marine n-3 PUFA levels were associated with lower mortality risk.

Interventional studies in the general population

(121) 10 Einvik Norway DOIT. RCT, men 65-75yr (28% established CVD), n=563, 2.1 g EPA+DHA vs corn oil for 3yr. Non-significant tendency towards lower overall and cardiovascular moratlity.

(120) 13 Roncaglioni Italy RCT, patients at high risk of CVD, n=12,513, 0.8 g EPA+DHA vs olive oil for 5yr. No effect on cardiovascular morbidity and mortality.

Diabetes

(119) 13 Bosch Netherl. ORIGIN. RCT, patients at high risk of CVD, n=12,513, 0.8 g EPA+DHA vs olive oil for 5yr. No effect on cardiovascular morbidity and mortality.

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Table 3 continued.

Ref Yr First author Country Study population, design, sample size, follow-up time and findings Cardiovascular disease

(110) 89 Burr UK DART 1. RCT, MI survivors, n=2,033, dietary advice or supplements (0.2-0.9 g EPA+DHA) vs no dietary advice, 2yr follow-up. Lower overall mortality, but no effect on MI incidence or cardiovascular mortality.

(109) 03 Ness UK DART 2. No long-term benefits in participants from the first study.

(138) 96 Eritsland Norway RCT, patients who underwent coronary artery bypass grafts, n=610, 1yr follow-up. Lower incidence of vein graft occlusion.

(139) 99 Johansen Norway RCT, patients who underwent percutaneous transluminal coronary angioplasty, n=529, 26w follow-up. No effect on restenosis incidence.

(111) 99 Marchioli Italy GISSI-Prevenzione. RCT, MI survivors, n=11,324, 0.9 g EPA+DHA vs vitamin E vs placebo for 3.5yr. Lower incidence of composite endpoint; myocardial reinfarction, stroke or mortality.

(112) 08 Tavazzi Italy GISSI-Heart Failure. RCT, patients with heart failure, n=6,920, 0.9 g EPA+DHA vs vitamin E vs placebo for 4yr. Lower overall and cardiovascular mortality.

(140) 01 Nilsen Norway RCT, MI survivors, n=300, 3.6 g EPA+DHA vs corn oil for a median of 1.5yr. Higher HDL cholesterol, lower triglycerides, but no effect on composite major cardiovascular event and mortality endpoint.

(141) 03 Erkkilä Finland Observational cohort study, established coronary artery disease, n=415, fatty acid analysis, 5yr follow-up. Lower overall mortality with higher EPA and DHA, EPA also associated with cardiovascular mortality.

(113) 06 Brouwer Netherl. RCT, patients with ICD after documented ventricular arrhythmia, n=546, 0.8 g EPA+DHA for 1yr. Non-significant tendency towards less ICD intervention due to ventricular arrhythmia.

(114) 10 Galan France SU.FOL.OM3. RCT, patients with history of ischemic heart disease or stroke, n=2,501, 0.6 g EPA+DHA vs vitamin B vs placebo for 5yr. No effect on composite endpoint (MI, stroke or cardiovascular mortality).

(118) 10 Rauch Germany OMEGA. RCT, MI survivors, n=3,851, 0.8 g EPA+DHA vs olive oil for 1yr. No effect on major cardiovascular events or mortality.

(115) 10 Kromhout Netherl. Alpha Omega. RCT, MI survivors, n=4,837, 0.4 g EPA+DHA vs placebo for 3.5yr. No effect on major cardiovascular events.

(116) 12 Eussen Netherl. Alpha Omega. Reduced major cardiovascular events in non-statin users.

(117) 14 Hoogeveen Netherl. Alpha Omega. Improved renal function.

Renal disease

(122) 06 Svensson Denmark RCT, ESRD, n=206, 1.7 g EPA+DHA vs olive oil for 2yr. Lower incidence of MI and lower triglyceride levels.

(79) 13 Friedman US Matched case control study, ESRD, n=400 (out of 10,044). Lower odds of SCD with higher levels of marine n-3 PUFAs.

(32) 94 Donadio US RCT, IgA nephritis, n=106, 3.2 g EPA+DHA vs olive oil for 2yr. Less decline in renal function compared with placebo.

(38) 99 Ando Japan RCT, ESRD, n= 38, 1.6 g EPA vs placebo for 18w. Lower levels of oxidized LDL cholesterol and remnant lipoproteins as well as lower triglyceride levels.

(41) 02 Schmitz US RCT, ESRD, n=24 , 3.2 g EPA+DHA vs corn oil for 1yr. Better arteriovenous graft patency, lower systemic BP and lower venous outflow resistance.

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Table 3 continued.

Ref Yr First author Country Study population, design, sample size, follow-up time and findings Dyslipidemia

(78) 07 Yokoyama Japan JELIS. RCT, n=14,981, 1.8 g EPA+statin vs statin alone for 4.5yr.

Lower triglycerides and lower cardiovascular mortality, but no effect on SCD incidence.

Heart, liver and bone marrow transplantation

(125) 93 Ventura US RCT, heart transplantation, n=20, 3.0 g EPA+DHA vs corn oil for 52w.

Lower mean arterial BP and lower left ventricular mass.

(123) 97 Andreassen Norway RCT, heart transplantation, n=30, 3.4 g EPA+DHA vs corn oil for 27w.

Lower triglyceride levels, lower systolic and diastolic BP.

(124) 01 Holm Norway RCT, heart transplantation, n=55, 3.4 g EPA+DHA vs corn oil for 52w.

Improved renal function, lower systemic vascular recistance.

(126) 06 Harris US Clinical study, heart transplantation, n=18, 1.0 to 3.4 g EPA+DHA for 16 to 27w. Lower resting heart rate.

(127) 95 Badalamenti Italy RCT, liver transplantation, n=27, 3.5 g EPA+DHA vs corn oil for 8w.

Better renal blood flow, lower urine thromboxane A2 levels.

(128) 12 Zhu China Clinical study, liver transplantation, n=66, Parenteral EPA+DHA for 1w vs standard care. Improved patient survival and graft function.

(129) 01 Takatsuka Japan Clinical study, bone marrow transplantation, n=17, 1.8 g EPA vs no placebo for 30w. Improved patient survival, less severe GVHD.

Ongoing interventional studies

ORENTRA Renal transplant recipients, n=132, 2.7 g EPA+DHA vs olive oil for 44w. Detailed description of study design in section 10.

OMEMI MI survivors, n=1400, 1.8 g EPA+DHA vs corn oil for 2yr. Composite primary endpoint: Death, new non-fatal MI, revascularization or stroke

VITAL General population >50yr, n=25,874, 1.0 g EPA+DHA vs vitamin D vs combined treatment vs placebo, mean duration 5yr, for primary prevention of CVD and cancer ASCEND Diabetes, n=15,480, aspirin with either 1.0 g EPA+DHA or placebo for a minimum of

5yr. Primary endpoints fatal and non-fatal MI and strokes

Reduce-It Mixed dyslipidemia, high risk of / established CVD, n=8,000, statins with or without EPA, mean duration 5yr. Composite cardiovascular morbidity /mortality endpoint SuperiorSVG Coronary artery (saphenous vein) graft bypass, n=1,550, 1.1 g EPA+DHA vs placebo

for 1 yr, for prevention of graft occlusion and cardiovascular mortality

EPA: Eicosapentaenoic acid. DHA: Docosahexaenoic acid. BP: Blood pressure. MI: Myocardial infarction.

SCD: Sudden cardiac death. DQ: Dietary questionnaire. Ig: Immunoglobulin. Netherl.: Netherlands. n: number.

w: weeks. g: grams. yr: years. RCT: Randomized controlled trial. ESRD: End-stage renal disease. GVHD: Graft versus host disease. ORIGIN: Outcome reduction with initial glargine intervention. SU.FOL.OM3:

Supplementation with folate, vitamin B6, vitamin B12 and / omega-3 fatty acids trial. JELIS: Japan Eicosapentaenoic acid Lipid Intervention Study. GISSI: Gruppo Italiano per lo Studio della Soprawivenza nell’Infarcto Miocardio Prevenzione Trial. DART: Diet and Reinfarction Trial. DOIT: Diet and Omega-3 Interventional Trial. JPHC: Japan public health center-based prospective study. OMEMI: Omega-3 fatty acids in elderly patients with acute myocardial infarction. SuperiorSVG: Superior saphenous vein graft trial. VITAL:

Vitamin D and omega-3 trial. ASCEND: A study of cardiovascular events in diabetes. ORENTRA: Omega-3 fatty acids in renal transplantation.

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5.3 Marine n-3 polyunsaturated fatty acid consumption in Norway

EPA and DHA enter the human food chain through fish and marine mammals that feed on marine phytoplankton (4). The main source of marine n-3 PUFAs in a typical Nordic diet is various species of fish with a high content of EPA and DHA in their muscles (Figure 3) (142).

Some people also consume marine n-3 PUFA supplements, which in Norway are most often made from cod liver oil (Norwegian: tran). Lean fish, like cod, store marine n-3 PUFAs in the liver instead of the muscles.

Different dietary habits, income distribution and social infrastructure between and within countries probably explain why some countries with rich fishing grounds have low fish consumption and vice versa (Figure 4) (143-146). In some European countries, fish consumption is associated with higher educational level, higher socioeconomic group and various life-style factors considered to be healthy (147). In Norway, unlike many other countries, fish is not expensive (148), fish consumption is relatively high (149) and mostly influenced by regional dietary habits (150), with easy access to fresh fish along the Norwegian coastline. Fish consumption was not associated with other dietary or life-style factors in a large Norwegian cohort (150). The European Prospective Investigation into Cancer and Nutrition (EPIC) study concluded that a typical Nordic diet was characterized by a high consumption of fish, root vegetables and dark bread (149). However, dietary habits are constantly changing. Consistent with previous reports (151), recipient age was positively associated with plasma marine n-3 PUFA levels and negatively associated with plasma n-6 PUFA levels. Consistent with previous reports (152, 153), we found an inverse association between plasma levels of marine n-3 PUFAs and n-6 PUFAs, suggesting different dietary patterns in different age groups. Mean daily intake of marine n-3 fatty acids EPA and DHA is about 0.9 g in Japan (94), 0.1 g in the US (79, 154) and 0.7 g in Norway (153). The

background consumption of marine n-3 PUFAs in a Norwegian cohort is similar to the low- dose marine n-3 PUFA supplements used in some RCTs (114, 115). At our center, we recommend a high intake of fatty fish after renal transplantation in line with dietary recommendations for patients with dyslipidemia or established cardiovascular disease (combined EPA and DHA intake ≥ 1g per day) (155). Based on plasma marine n-3 PUFA levels, we assume that most of the patients in our cohort had a lower marine n-3 PUFA intake than the recommendation. There is limited evidence to suggest specific threshold values for effects of marine n-3 PUFAs (52).

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Figure 3. The content of eicosapentaenoic and docosahexaenoic acid (grams) in the flesh of various species of fish and in other seafoods.

The figure is based on data from the US Department of Agriculture Dietary Guidelines for Americans 2015 Report (142).

Figure 4. Fish consumption per capita per year in selected countries – having a coastline helps, but is not the only determinant of fish consumption.

The figure is based on data from the UN Food and Agriculture Globefish State of the World Fisheries and Aquaculture Report 2014 (143).

Country Argentina Spain Germany Greece Iran Afghanistan Japan

Fish consumption (kg/yr) 6.0 43.0 14.2 19.6 9.1 0.1 51.7

% fish of total animal protein 2.9 19.9 7.3 8.6 11.6 0.2 37.3

Country USA UK

Fish consumption (kg/yr) 21.7 19.0

% fish of total animal protein 7.4 9.2

Norway Sweden Denmark Russia

53.4 31.1 23.0 22.3

23.4 11.8 13.5 14.0

The figure is based on data from the UN Food and Agriculture Globefish State of the World Fischeries and Aquaculture Report 2014 Shrimp: EPA 0.17g / 100g.

DHA 0.14g / 100g.

Cod: EPA 0.01g / 100g. DHA 0.15g / 100g.

Pollock: EPA 0.09g / 100g. DHA 0.45g / 100g.

Trout: EPA 0.47g / 100g. DHA 0.52g / 100g.

Plaice: EPA 0.24g / 100g. DHA 0.26g / 100g.

Crab: EPA 0.12g / 100g.

DHA 0.30g / 100g.

Herring: EPA 0.91g / 100g.

DHA 1.11g / 100g.

Salmon: EPA 0.41g / 100g. DHA 1.43g / 100g.

Mussels: EPA 0.14g / 100g. DHA 0.15g / 100g.

Mackerel: EPA 0.50g / 100g.

DHA 0.70g / 100g.

Abbrevations: EPA: Eicosapentaenoic acid. DHA: Docosahexaenoic acid

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5.4 Epidemiological and statistical considerations

We used two types of observational study designs in the present study: Cohort (paper I and paper III) and cross-sectional study design (paper II).

Cohort study design: We followed patients from baseline ten weeks after renal transplantation to the onset of an event (death or graft loss) or to the end of the observational period (1^st of February 2014) for patients who did not suffer an event (censoring). We were particularly interested in associations between the primary exposure variable plasma marine n-3 PUFA levels (the sum of plasma EPA, DHA and docosapentaenoic acid [DPA] levels measured at ten weeks post-transplant) and risk of certain events, which we assessed by Cox proportional hazard regression analysis (156). Cox regression estimates the relative hazard of an event for explanatory variables included in the Cox model. In this study, we may interpret relative hazard as relative risk. Consequently, hazard ratio (HR) = 1.0 indicate a neutral risk, HR > 1.0 indicate an increased risk and HR < 1.0 a decreased risk of the event in question. We assumed that plasma marine n-3 PUFA levels had a linear association with events and that associations were stable throughout the observational period (proportional hazard). These assumptions were tested as they had to be met in order to accept the Cox regression model (156).

In addition to plasma marine n-3 PUFA levels, other explanatory variables were added to the Cox models (covariates). A variable that influence the association between the primary exposure variable and outcome (usually an event) or dependent variable (e.g. cardiovascular risk markers) is called a confounder and might either hide, exaggerate or distort the true estimates of the association between the primary exposure variable and outcome / dependent variable (e.g. between plasma marine n-3 PUFAs and cardiovascular mortality). As an example, current smoking was negatively associated with plasma marine n-3 PUFA levels.

Without adjustment for smoking status in the Cox regression analysis, associations between plasma marine n-3 PUFA levels and cardiovascular mortality would be influenced by the association between smoking and cardiovascular mortality. We used pre-defined traditional and transplant-specific risk factors for death and graft loss as covariates in the Cox models.

Competing risk in survival analysis occurs when an event (e.g. death due to cardiovascular disease) modifies the chance for another event to occur (e.g. death due to cancer). Some risk factors (e.g. current smoking) increase the risk of both events, but one event may be more likely to occur before the other (e.g. death due to cardiovascular disease occured before the

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patient would have died of cancer). Under these circumstances standard Cox regression will not be optimal for assessment of associations between exposure and certain events (in this example smoking status and death due to cancer, but theoretically a similar scenario could be the case for marine n-3 PUFAs) (157). Renal graft dysfunction increases the risk of both graft failure and death (22), and therefore death competes with the risk of death censored graft loss (158). In contrast, overall graft loss comprises both grafts lost to recipient death and death censored graft loss and would therefore not be influenced by competing risk due to recipient death. In the setting of possible competing risk, standard Cox regression could be used when the research question investigates the etiological relationship between risk factors and a given outcome (e.g. plasma marine n-3 PUFA levels and cardiovascular mortality) (157). This approach requires the assumption that competing risks are independent (which would not be the case for smoking and death due to cancer, whereas it is more likely to be the case with plasma marine n-3 PUFA levels). Subdistribution hazard method is an alternative approach which was investigated for cause-specific mortality in this study and would especially be appropriate if we were interested in predicting the probability of a given outcome at a given time for a patient population with certain characteristics (e.g. current smokers and risk of death due to cancer) (157). Some argue that results obtained by both approaches should be reported when competing risks are suspected, and statistical packages have been updated in recent years to perform these analyses (159). Since Kaplan-Meier patient and graft survival curves for events susceptible to competing risk might be misleading (160), we chose to use the Kaplan-Meier method only for all-cause mortality and overall graft loss (161).

Cross-sectional study design: We collected data on explanatory variables (including plasma marine n-3 PUFA levels) and parameters of interest (various cardiovascular risk markers) at ten weeks post-transplant. Linear regression was used to study correlations between plasma marine n-3 PUFA levels and cardiovascular risk markers, with and without adjustment for other explanatory variables (162). Unstandardized (Unstd.) regression coefficients (β-coeff.) indicate the slope of regression lines, whereas standardized regression coefficients (Std. β- coeff.) scale the explanatory variable to its standard deviation. Std. β-coeff. make it possible to compare relative associations of different explanatory variables with the same dependent variable. Linear regression may also be used to assess how much of the variance (R²) in the dependent variable (e.g. fasting plasma glucose) is explained by the explanatory variables included in the final regression models. In age and gender adjusted models, recipient age and gender, as well as plasma marine n-3 PUFA levels were forced into the final model. In

Marine n-3 polyunsaturated fatty acids in renal transplantation