Erythrocyte membrane polyunsaturated fatty acid profiles are associated with systemic inflammation and fish consumption in systemic lupus erythematosus: a cross-sectional study

Abstract

Objective

This study aimed to assess polyunsaturated fatty acid (PUFA) status and association with systemic inflammation and fish consumption in systemic lupus erythematosus (SLE).

Methods

Parameters of PUFA status including the proportion of omega-6 and -3 fatty acids in highly unsaturated fatty acids (omega-6%, omega-3%), essential fatty acids linoleic acid (LA) and alpha-linolenic acid (ALA), the omega-6 PUFA arachidonic acid (ARA) and the sum of key omega-3 PUFA eicosapentaenoic acid+docosahexaenoic acid (omega-3 status) were measured by gas chromatography in 68 SLE patients (88.2% female, aged 45.7±12.5 years). Associations with serum CRP, disease activity, damage and fish consumption were assessed by linear regression modelling adjusted for age, sex and body mass index. Associations are reported in terms of regression coefficients (β).

Results

Omega-6 PUFA were associated with higher CRP: omega-6% (β = 0.052, p = 0.02), the ratio of LA/ALA (β = 0.007, p = 0.02) and ARA (β = 0.308, p = 0.001). Conversely, omega-3% was associated with lower CRP (β = −0.051, p = 0.02). Increased dietary PUFA consumption from fish (g/day) was linked to a higher omega-3 status (β = 2.21, p = 0.02) and lower self-reported damage (Brief Index of Lupus Damage; β = −3.22, p = 0.02).

Conclusions

Omega-3 and omega-6 fatty acid status differentially reflect systemic inflammation in SLE and are linked to fish consumption.

Keywords

Cardiovascular disease haematological changes systemic lupus erythematosus

Introduction

Dietary intake of the essential fatty acids linoleic acid (LA; C18:2n6) and alpha-linolenic acid (ALA; C18:3n3) is a prerequisite for production of downstream long-chain polyunsaturated omega-6 and omega-3 fatty acids (omega-6 and omega-3 PUFA), respectively, within human metabolism. Both metabolic pathways require a set of enzymes such as delta-5 and delta-6 desaturase (D5D, D6D), elongases or saturases. In general, products of omega-6 PUFA metabolism result in predominantly proinflammatory molecules derived from arachidonic acid (AA; C20:4n6) such as prostaglandin E2, thromboxanes and pro-inflammatory leucotrienes, while omega-3 PUFA metabolism of eicosapentaenoic acid (EPA; C20:5n3) or docosahexaenoic acid (DHA; C22:6n3) leads to the formation of products with less pro-inflammatory, anti-inflammatory or even inflammation-resolving properties such as prostaglandin E3, leucotriene B5, protectins and resolvins.¹ In accordance with these data, beneficial health effects are associated with an increased intake of dietary omega-3 PUFA in a number of inflammatory disorders.² Measurement of PUFA status by assessing erythrocyte membrane composition represents fatty acid consumption, that is, the nutrition status of the previous 120 days,^3,4 and is advantageous as opposed to plasma sampling which may be influenced by short fasting episodes,⁵ or food frequency questionnaires (FFQ) which may be prone to bias introduced by social desirability and recall.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder characterized by autoantibody formation. The role of environmental factors such as nutrition as drivers of disease activity and flares are increasingly recognized. Indeed, evidence exists that supplementation of omega-3 PUFA may ameliorate disease activity or systemic inflammation in SLE.⁶ The majority of SLE patients seek self-empowerment and request counselling on dietary measures fit to reduce disease burden and damage.⁷ We therefore conducted a cross-sectional study in order to substantiate associations of PUFA status and metabolism with inflammation or disease activity, and to assess which dietary habits are associated with a beneficial PUFA profile.

Methods

Patients and clinical data

In this cross-sectional study, consecutive patients were recruited from the rheumatology outpatient department of Heinrich-Heine-University Düsseldorf, Germany, who fulfilled the 1997 American College of Rheumatology (ACR) SLE criteria and were >18 years of age. All patients met 2010 Systemic Lupus Erythematosus International Collaborating Clinics (SLICC)/ACR classification criteria. Patients were administered an FFQ previously validated in the German population.⁸ Answers to the items were digitalized in duplicate and controlled by EpiData (www.epidata.dk) for completeness and correctness. The amount of dietary PUFA derived from total fish consumption was calculated based on the indicated amount of fish eaten multiplied by the appropriate PUFA content indicated in the United States Department of Agriculture Food Composition Database (https://ndb.nal.usda.gov/nutrients/index). Serum high sensitive CRP was assessed as a measure of systemic inflammation. Disease activity was assessed by the Systemic Lupus Disease Activity Index-2000 (SLEDAI) and the patient-reported Systemic Lupus Activity Questionnaire (SLAQ). Damage was assessed by the SLICC damage index and the patient-reported Brief Index of Lupus Damage (BILD). Written informed consent was obtained from all patients. The study was approved by the ethics committee of the Medical Faculty of Heinrich-Heine-University Düsseldorf, and adhered to the Declaration of Helsinki.

Erythrocyte membrane fatty acid profile and desaturase activity

Blood cells were separated by density gradient centrifugation. The fatty acid composition was analysed in re-suspended blood cells (99% red blood cells) as fatty acid methyl esters (FAME) using gas chromatography with flame ionization detection, as described, with slight modifications.⁹ Briefly, 10 µL FAME C25:0 (750 µM) as internal standard and 10 µL butylated hydroxytoluene (0.02 mg/mL in methanol) were added to the samples (100 µL), and lipids were extracted with methanol/methyl tert-butyl ether. Trans-esterification of the lipid extract to FAMEs was carried out using methanolic hydrogen chloride. Calculation of the relative fatty acid pattern was based on peak areas and theoretical response factors.

The proportion of omega-3 and omega-6 PUFA (omega-3% and omega-6%) in highly unsaturated fatty acids (HUFA) was calculated, as described in Ostermann et al.¹⁰ and modified from Lands¹¹: omega-6% in HUFA = 100×(C20:3n6 + C20:4n6 +C22:4n6 + C22:5n6)/(C20:3n6 + C20:4n6 + C22:4n6 +C22:5n6 + C20:3n9 + C20:5n3 + C22:5n3 + C22:6n3); omega-3% in HUFA = 100 × (C20:5n3 + C22:5n3 +C22:6n3)/(C20:3n6 + C20:4n6 + C22:4n6 + C22:5n6 +C20:3n9 + C20:5n3 + C22:5n3 + C22:6n3).

D5D and D6D activity was assessed based on the product/substrate ratio of respective fatty acids (D5D activity: C20:4n6/C20:3n6; D6D activity: C20:3n6/C18:2n6).¹²

Statistical analysis

Associations of selected parameters of PUFA metabolism with clinical measures of inflammation or disease activity were assessed by multivariable linear regression, adjusted for age, sex and body mass index. Associations are reported in terms of regression coefficients (β). R v3.5.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for all analyses. p-Values <0.05 were considered significant. Adjusted p-values (adj. p) according to Benjamini and Hochberg are additionally reported.

Results

Patient characteristics

A total of 68 patients (88.2% female) aged 45.7 ± 12.5 years were included. Disease duration was 17 ± 8.7 years (range 2–38 years). Immunosuppressive treatment consisted mainly of antimalarials (75.7%), glucocorticoids (67.6% at a mean daily dose of 5.9 ± 6 mg) and azathioprine (18.9%). Details are outlined in Table 1. The demographics of patients are comparable to a larger German sample of SLE patients.¹³

Table 1.

Patient characteristics (N = 68).

Parameter	n (%) or M ± SD
Female	60 (88.2%)
Disease duration	17 ± 8.7 years
BMI (kg/m²)	25.3 ± 4.6
SLAQ	6.1 ± 5.0
BILD	0.8 ± 1.4
CRP (mg/dL)	0.36 ± 0.66
C3c (mg/dL)	91.6 ± 16.5
Antimalarials	28 (75.7%)
Glucocorticoids	25 (67.6%)
Azathioprine	7 (18.9%)
Belimumab	3 (8.1%)
Rituximab	3 (8.1%)
Ciclosporine	3 (8.1%)
Mycophenolate	2 (5.4%)
Methotrexate	1 (2.7%)

SD: standard deviation; BMI: body mass index; SLAQ: Systemic Lupus Activity Questionnaire; BILD: Brief Index of Lupus Damage.

PUFA metabolites, systemic inflammation and disease activity

We hypothesized that a prevailing omega-6 PUFA status and metabolism is positively associated with inflammation, while omega-3 PUFA status favours inflammation resolution. The distribution of measured PUFA metabolites within the cohort is summarized in Table 2. The values are in the broad range of reported values for Germany albeit with comparatively low LA and high ARA values.¹⁴ The proportion of omega-6 to omega-3 essential fatty acids LA to ALA correlated to the systemic inflammation marker CRP (mg/dL; estimate (β) = 0.007 (95% confidence interval (CI) 0.00095–0.013), p = 0.024, adj. p = 0.042). Likewise, omega-6% was associated with higher CRP values (β = 0.052 (95% CI 0.0085–0.095), p = 0.02, adj. p = 0.042; Figure 1(a)), and omega-3% with lower CRP values (β = –0.051 (95% CI –0.094 to –0.0081), p = 0.021, adj. p = 0.042; Figure 1(b)). Concerning selected individual fatty acids, the omega-6 PUFA AA was linked to higher CRP values (β = 0.308 (95% CI 0.127–0.489), p = 0.001, adj. p = 0.008). There was no significant correlation of any PUFA metabolite to disease activity as measured by SLEDAI or SLAQ.

Table 2.

Distribution of measured polyunsaturated fatty acid profiles.

PUFA	Unit	M	SD	Min	Max
Arachidonic acid	% FA	13.91	0.86	10.18	16.10
Linoleic acid	% FA	9.32	1.12	6.30	13,24
Eicosapentaenoic acid	% FA	0.62	0.33	0.19	1.99
Docosahexaenoic acid	% FA	4.03	0.87	2.57	7.32
Alpha-linolenic acid	% FA	0.13	0.07	0.04	0.61
Omega-6 %	% HUFA	72.97	4.17	54.07	80.02
Omega-3 %	% HUFA	26.72	4.17	19.70	45.64
Delta-5 desaturase index	^a	9.07	1.84	5.31	13.53
Delta-6 desaturase index	^a	0.17	0.04	0.08	0.26

Distribution of individual polyunsaturated fatty acid (PUFA) profiles in the cohort of 68 systemic lupus erythematosus patients. Values represent percentage of indicated PUFA in total fatty acids (FA) or highly unsaturated FA (HUFA) as indicated.

Delta-5- and delta-6-desaturase index are calculated as outlined in the methods section.

Fish consumption, PUFA and disease activity/damage

The availability of essential fatty acid LA and ALA for omega-6 and omega-3 PUFA metabolism, respectively, is dependent upon diet, with fish consumption being of paramount importance for increased levels of long-chain omega-3 PUFA EPA and DHA, which can be combined into the omega-3 index (EPA+DHA).¹⁵ We thus hypothesized that fish consumption favours omega-3 PUFA metabolism. The omega-3 status (EPA+DHA) was higher in patients with increased dietary consumption of PUFA from fish (g/day; β = 2.21 (95% CI 0.034–4.39), p = 0.047; Figure 1(c)). No significant associations with disease activity measures were observed. However, PUFA consumption from fish was linked to lower self-reported damage (BILD; β = –3.22 (95% CI –6.1 to –0.35), p = 0.029; Figure 1(d)). Of note, the significance was lost after correction for multiple testing (adj. p = 0.1).

Discussion

Despite a great reported eagerness to receive and follow dietary counselling, few SLE patients receive such advice.⁷ This seems especially concerning, since SLE patients tend to follow an unhealthy diet.^16,17 In the present study, we focused on PUFA and assessed PUFA status by both FFQ and erythrocyte membrane PUFA composition, which arguably more accurately reflects nutritional status than FFQ due to recall errors and social desirability bias.⁵ PUFA represent an interesting class of macronutrients in this regard, as their intake is highly dependent upon nutrition and may differentially influence health and inflammation. In particular, omega-3 PUFA status and metabolism results in the formation of metabolites associated with inflammation resolving properties, while omega-6 PUFA metabolism favours production of inflammatory mediators.² In this regard, we recently demonstrated that erythrocyte PUFA patterns correlate linearly with the level of their corresponding eicosanoids and other oxylipins.¹⁸

In the present study, we found a consistent association of omega-6 PUFA metabolites and markers with increased systemic inflammation, and lower systemic inflammation in instances of a high proportion of omega-3 PUFA in SLE patients. In our study, CRP levels were in the normal range. However, subclinical inflammation assessed by variations of serum CRP levels in predominantly ‘normal’ ranges have previously been linked to relevant clinical parameters such as disease activity and surrogate markers for atherosclerosis in SLE patients.^17,19 Previous interventional studies which administered omega-3 PUFA containing fish oil to SLE patients produced conflicting results in this regard: amelioration of systemic inflammatory markers was seen in some^20,21 but not all studies.^22,23 Of note, individual nutrition status omega-3 PUFA content in the blood cells was not assessed in these studies. However, based on our previous studies, we hypothesize that the background diet is a critically important factor. In particular, a common Western diet may not be overcome by administration of essential omega-3 PUFA at a fixed dose, and more complex nutritional interventions may be necessary (e.g. contemporaneously reducing LA intake).^24,25 Complex dietary interventions have previously been shown to be effective in maintaining a diet associated with better health, including higher fish consumption in SLE patients.²⁶ Therefore, assessment of the nutritional status omega-3 PUFA content may be important to dose macronutrients in clinical studies correctly. In line with this notion, the present study suggests that monitoring PUFA status is feasible for monitoring patients on diverse diets.

Concerning disease activity measures, we did not find a direct association of omega-3 PUFA levels, including the EPA and DHA levels in erythrocytes, with lower disease activity, as has been reported previously.²⁷ However, in the latter study, FFQ were used to estimate omega-3 PUFA levels, while we employed erythrocyte membrane PUFA composition, which is more accurate, since individual differences in absorption and metabolism are thereby accounted for.⁵ Indeed, when total PUFA consumption from fish was calculated from our FFQ, increased levels were associated with lower self-reported damage scores in the present study. Trials employing fixed doses of omega-3 PUFA supplementation reported conflicting results with no effect^21,23 versus amelioration of SLE disease activity.²² Of note, all studies were carried out in different populations (Brazil, the USA and Northern Ireland, respectively) and at fixed doses of omega-3 PUFA supplementation. Again, background diet may constitute an important factor.^24,25 EPA and DHA intestinal uptake, for instance, is particularly dependent upon the total fat content of a meal.²⁸ We therefore assessed the diet of SLE patients by an FFQ. There was a significant association of the omega-3 status with increased fish consumption. This is in accordance with fish and fish oil being major sources of nutritional long-chain omega-3 PUFA.¹⁵ Obviously, besides fatty acids, other food ingredients may play important roles in modifying health of SLE patients, such as vitamins,²⁹ particularly vitamin D,⁶ salt intake,³⁰ extra-virgin olive oil or phenolic fractions of olive oil.²⁹ Adjustments for this complex interplay were not possible in the present study. However, a tailored approach to individualized dietary counselling is likely necessary for optimal clinical results. Furthermore, the effect of immunosuppressive treatments on PUFA metabolism is largely unknown. Interdependence between glucocorticoid intake and diet composition has been reported.³¹ In the current study, we did not note any associations with a specific immunosuppressive drug, but treatments were diverse, and the study was not powered to recognize such differences.

In summary, our current study suggests that omega-6 fatty acid metabolites are associated with systemic inflammation, while omega-3 fatty acids favour reduced inflammatory markers and are associated with higher PUFA consumption from fish in SLE patients.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

ORCID iD

S Vordenbäumen

References

Fabian

Kimler

Hursting

SD.

Omega-3 fatty acids for breast cancer prevention and survivorship. Breast Cancer Res 2015; 17: 62.

Galli

Calder

PC.

Effects of fat and fatty acid intake on inflammatory and immune responses: a critical review. Ann Nutr Metab 2009; 55: 123–139.

Brown

Pang

Roberts

DC.

Persistent changes in the fatty acid composition of erythrocyte membranes after moderate intake of n-3 polyunsaturated fatty acids: study design implications. Am J Clin Nutr 1991; 54: 668–673.

Harris

Varvel

Pottala

Warnick

McConnell

JP.

Comparative effects of an acute dose of fish oil on omega-3 fatty acid levels in red blood cells versus plasma: implications for clinical utility. J Clin Lipidol 2013; 7: 433–440.

Rifkin

Shrubsole

Cai

, et al. PUFA levels in erythrocyte membrane phospholipids are differentially associated with colorectal adenoma risk. Br J Nutr 2017; 117: 1615–1622.

De Medeiros

MCS

Medeiros

JCA

De Medeiros

Leitãi

JCGC

Knackfuss

MI.

Dietary intervention and health in patients with systemic lupus erythematosus: a systematic review of the evidence. Crit Rev Food Sci Nutr 2019; 59: 2666–2673.

Robinson

Mcdonnell

Wincup

, et al. Diet and lupus: what do the patients think? Lupus 2019; 28: 755–763.

Haftenberger

Heuer

Heidemann

Kube

Krems

Mensink

GBM.

Relative validation of a food frequency questionnaire for national health and nutrition monitoring. Nutr J 2010; 9: 36.

Ostermann

Müller

Willenberg

Schnebb

NH.

Determining the fatty acid composition in plasma and tissues as fatty acid methyl esters using gas chromatography – a comparison of different derivatization and extraction procedures. Prostaglandins Leukot Essent Fatty Acids 2014; 91: 235–241.

10.

Ostermann

Reutzel

Hartung

, et al. A diet rich in omega-3 fatty acids enhances expression of soluble epoxide hydrolase in murine brain. Prostaglandins Other Lipid Mediat 2017; 133: 79–87.

11.

Lands

A critique of paradoxes in current advice on dietary lipids. Prog Lipid Res 2008; 47: 77–106.

12.

Cormier

Rudkowska

Lemieux

Couture

Julien

Vohl

MC.

Effects of FADS and ELOVL polymorphisms on indexes of desaturase and elongase activities: results from a pre–post fish oil supplementation. Genes Nutr 2014; 9: 437.

13.

Chehab

Sauer

Richter

, et al. Medical adherence in patients with systemic lupus erythematosus in Germany: predictors and reasons for non-adherence – a cross-sectional analysis of the LuLa-cohort. Lupus 2018; 27: 1652–1660.

14.

Stark

Van Elswyk

Higgins

Weatherford

Salem

Jr.

Global survey of the omega-3 fatty acids, docosahexaenoic acid and eicosapentaenoic acid in the blood stream of healthy adults. Prog Lipid Res 2016; 63: 132–152.

15.

Constantin

M-M

Nita

Olteanu

, et al. Significance and impact of dietary factors on systemic lupus erythematosus pathogenesis. Exp Ther Med 2019; 17: 1085–1090.

16.

Xie

Han

, et al. Metabolic disturbances associated with systemic lupus erythematosus. PLoS One 2012; 7:e37210.

17.

Pocovi-Gerardino

Correa-Rodríguez

Rubio

J-LC

Fernández

Ortego-Centeno

Rueda-Medina

Diet quality and high-sensitivity C-reactive protein in patients with systemic lupus erythematosus. Biol Res Nurs 2019; 21: 107–113.

18.

Ostermann

West

Schoenfeld

, et al. Plasma oxylipins respond in a linear dose-response manner with increased intake of EPA and DHA: results from a randomized controlled trial in healthy humans. Am J Clin Nutr 2019; 109: 1251–1263.

19.

Mercurio

Lobasso

Barbieri

, et al. Inflammatory, serological and vascular determinants of cardiovascular disease in systemic lupus erythematosus patients. Int J Mol Sci 2019; 20: 2154.

20.

Curado Borges

De Miranda Moura Dos Santos

Weiss Telles

Melo De Andrade

Toulson Davisson Correia

Lanna

CCD.

Omega-3 fatty acids, inflammatory status and biochemical markers of patients with systemic lupus erythematosus: a pilot study. Rev Bras Reumatol Engl Ed 2017; 57: 526–534.

21.

Arriens

Hynan

Lerman

Karp

Mohan

Placebo-controlled randomized clinical trial of fish oil’s impact on fatigue, quality of life, and disease activity in systemic lupus erythematosus. Nutr J 2015; 14: 82.

22.

Wright

O’Prey

McHenry

, et al. A randomised interventional trial of omega-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus. Ann Rheum Dis 2008; 67: 841–848.

23.

Bello

Fang

Fazeli

, et al. Omega-3 in SLE: a double-blind, placebo-controlled randomized clinical trial of endothelial dysfunction and disease activity in systemic lupus erythematosus. Rheumatol Int 2013; 33: 2789–2796.

24.

Greupner

Kutzner

Pagenkopf

, et al. Effects of a low and a high dietary LA/ALA ratio on long-chain PUFA concentrations in red blood cells. Food Funct 2018; 9: 4742–4754.

25.

Greupner

Kutzner

Nolte

, et al. Effects of a 12-week high-α-linolenic acid intervention on EPA and DHA concentrations in red blood cells and plasma oxylipin pattern in subjects with a low EPA and DHA status. Food Funct 2018; 9: 1587–1600.

26.

Everett

Wolf

Contento

Haiduc

Richey

Erkan

Short-term patient-centered nutrition counseling impacts weight and nutrient intake in patients with systemic lupus erythematosus. Lupus 2015; 24: 1321–1326.

27.

Charoenwoodhipong

Harlow

Marder

, et al. Dietary omega polyunsaturated fatty acid intake and patient-reported outcomes in systemic lupus erythematosus: the Michigan Lupus Epidemiology & Surveillance (MILES) Program. Arthritis Care Res. Epub ahead of print 10 May 2019. DOI: 10.1002/acr.23925.

28.

Davidson

Johnson

Rooney

Kyle

Kling

DF.

A novel omega-3 free fatty acid formulation has dramatically improved bioavailability during a low-fat diet compared with omega-3-acid ethyl esters: the ECLIPSE (Epanova(®) compared to Lovaza(®) in a pharmacokinetic single-dose evaluation) study. J Clin Lipidol 2012; 6: 573–584.

29.

Aparicio-Soto

Sánchez-Hidalgo

, Alarcón-de-la-Lastra C. An update on diet and nutritional factors in systemic lupus erythematosus management. Nutr Res Rev 2017; 30: 118–137.

30.

Scrivo

Perricone

Altobelli

, et al. Dietary habits bursting into the complex pathogenesis of autoimmune diseases: the emerging role of salt from experimental and clinical studies. Nutrients 2019; 11.

31.

Lourdudoss

Hafström

Frostegård

Van Vollenhoven

The association between diet and glucocorticoid treatment in patients with SLE. Lupus Sci Med 2016; 3: e000135.