Department of Food and Nutrition, Umeå University, SE-901 87 Umeå, Sweden
Department of Clinical Chemistry, Kalmar County Hospital, SE-391 85 Kalmar, Sweden
Department of Medicine, Visby Hospital, SE-621 84 Visby, Sweden
Abstract
Background
We have previously reported that rheumatoid arthritis patients, who adopted a modified Cretan Mediterranean diet, obtained a reduction in disease activity and an improvement in physical function and vitality. This shift in diet is likely to result in an altered intake of fatty acids. Therefore, the objective of the present study was to examine the dietary intake of fatty acids, as well as the fatty acid profile in serum phospholipids, during the dietary intervention study presented earlier.
Results
From baseline to the end of the study, changes in the reported consumption of various food groups were observed in the Mediterranean diet group. The change in diet resulted in a number of differences between the Mediterranean diet group and the control diet group regarding the fatty acid intake. For instance, a lower ratio of n-6 to n-3 fatty acids was observed in the Mediterranean diet group, both assessed by diet history interviews (dietary intake) and measured in serum phospholipids. Moreover, the patients in the Mediterranean diet group that showed a moderate or better clinical improvement during the study (diet responders), had a higher reported intake of n-3 fatty acids and a lower ratio of n-6 to n-3 fatty acids compared to the patients with minor or no improvement. Also the fatty acid profile in serum phospholipids differed in part between the diet responders and the diet non-responders.
Conclusion
The changes in the fatty acid profile, indicated both by dietary assessments and through fatty acids in s-phospholipids may, at least in part, explain the beneficial effects of the Cretan Mediterranean diet that we have presented earlier.
Background
We have previously reported that a shift to a modified Cretan Mediterranean diet decreased the disease activity and improved the physical function and vitality of Swedish rheumatoid arthritis (RA) patients
In supplementation studies, olive oil has sometimes been used as a placebo oil. In some of these studies improvements were reported also in the group of RA patients treated with olive oil
The relationship between n-3 fatty acids/fish intake, olive oil and RA, has also been observed in case control studies. In these studies a high consumption of fish
In view of the results from earlier studies, the beneficial effects observed in the randomized controlled trial in which the Cretan Mediterranean diet was tested
Results
Reported dietary intake
To estimate the reported consumption of foods with relevance to the fatty acid intake, a self-administrated questionnaire was used. At baseline the Cretan Mediterranean diet (MD) group (n = 26) and the control diet (CD) group (n = 25) reported a similar intake frequency of all the food groups investigated (Table
Comparison of reported food consumption frequencies (servings per month) between the Mediterranean Diet (MD) group and the Control Diet (CD) group.
MD group (n = 26)
CD group (n = 25)
P-value*
Time (weeks after dietary shift)
Time (weeks after start of study)
0
3
12
0
3
12
Fish
6 (6-6)
16 (6–16)
16 (16-16)§
6 (2–6)
6 (6–16)
6 (2–6)
<0.001
Shellfish
2 (0–2)
2 (0–6)
4 (2–6)‡
2 (0–2)
2 (0–2)
2 (0–2)
<0.001
Meat, minced meat or offal from pig, cattle or sheep
16 (6–16)
0 (0-0)
0 (0-0)§
16 (6–16)
6 (6–16)
6 (6–16)†
<0.001
Poultry
2 (2–6)
16 (6–16)
11 (6–16)§
2 (2–6)
2 (2–6)
2 (2–6)
<0.001
Processed meat¶(not on bread)
6 (2–6)
0 (0-0)
0 (0-0)§
6 (2–6)
6 (2–6)
2 (2–6)
<0.001
Processed meat¶on bread
11 (6–26)
0 (0-0)
0 (0-0)§
16 (2–16)
6 (4–16)
6 (2–16)
<0.001
Cheese with more than 17% fat
16 (14–26)
0 (0-0)
0 (0-0)§
26 (11–70)
16 (4–26)
16 (4–26)
0.002
Cheese with less than 17% fat
6 (0–9)
16 (0–26)
16 (6–26)‡
0 (0–4)
0 (0–6)
0 (0–11)
0.005
Dairy products with 27% fat or more
2 (0–6)
0 (0-0)
0 (0-0)‡
2 (0–6)
2 (0–6)
6 (2–6)
<0.001
Dairy products with 10–15% fat
2 (0–6)
0 (0-0)
0 (0–2)‡
2 (0–6)
0 (0–6)
0 (0–6)
0.013
Ice cream
2 (0–6)
0 (0–0.5)
0 (0–2)‡
2 (0–4)
2 (0–2)
2 (0–2)
0.015
Sweets (including chocolate)
6 (2–16)
0 (0–2)
0 (0–2)§
6 (1–6)
6 (0–6)
6 (2–6)
<0.001
Buns, cookies or cakes
6 (5–16)
0 (0–2)
0 (0–2)§
6 (2–16)
6 (2–16)
6 (2–16)
<0.001
Nuts
0 (0–2)
0 (0–2)
0 (0–3)
0 (0–2)
0 (0–2)
0 (0–2)
0.704
Seeds
0 (0-0)
0 (0–2)
0 (0–2)
0 (0-0)
0 (0-0)
0 (0-0)
0.016
Data are presented as medians (25th–75th percentiles). *The p-values refer to differences between the MD- and the CD group concerning the change from baseline to week 12. Differences between the groups were analyzed with the Mann-Whitney U test. Statistically significant change from baseline to week 12: †p < 0.05; ‡p < 0.01; §p < 0.001. Within-group differences in week 12 compared to baseline were evaluated by the Wilcoxon signed ranks test. ¶Including cured meat, sausage, pâté or the like.
Regarding the reported food choices, significantly more MD subjects than CD subjects changed their choice of milk and fermented milk to milk and fermented milk with a lower fat content, or excluded these foods totally (p = 0.003 and p = 0.038 respectively). In addition, more MD subjects than CD subjects used olive oil and canola oil, as well as the liquid margarine and half-fat margarine supplied during the study period (p < 0.001 for all comparisons).
The nutrient intake during the second half of the study was estimated by means of diet history interviews (conducted with 17 individuals in each group). Two persons in the CD group and one in the MD group were identified as under-reporters, based on the individual Goldberg cut-off
Comparison of average daily intake (excluding supplements) of energy, fat and specific fatty acids between the Mediterranean Diet (MD) group and the Control Diet (CD) group. The dietary intake is based on the diet history interviews performed between study weeks seven and twelve.
MD group (n = 17)
CD group (n = 17)
P-value*
Energy (MJ)
8.8 ± 1.6
9.8 ± 3.2
p = 0.242
Fat (g)
60.4 ± 21.9
89.3 ± 33.1
p = 0.005
Total saturated fatty acids (g)
18.3 ± 8.2
40.5 ± 18.3
p < 0.001
Total monounsaturated fatty acids (g)
25.3 ± 10.5
31.9 ± 11.1
p = 0.088
Total polyunsaturated fatty acids (g)
11.8 ± 3.9
10.6 ± 3.5
p = 0.381
Total n-6 fatty acids (g)†
7.9 ± 2.5
8.2 ± 2.7
p = 0.743
Total n-3 fatty acids (g)‡
3.1 ± 1.3
2.0 ± 0.9
p = 0.008
Ratio n-6:n-3
2.7 ± 0.6
4.4 ± 0.9
p < 0.001
Fat (E%§)
25.0 ± 5.3
33.7 ± 5.6
p < 0.001
Total saturated fatty acids (E%)
7.5 ± 2.4
15.0 ± 3.7
p < 0.001
Total monounsaturated fatty acids (E%)
10.5 ± 2.8
12.2 ± 2.2
p = 0.067
Total polyunsaturated fatty acids (E%)
5.0 ± 1.1
4.1 ± 1.1
p = 0.028¶
4:0–10:0
0.77(0.32–1.59)
3.51(1.72–4.79)
p < 0.001
12:0
0.44(0.28–0.93)
1.67(0.93–2.56)
p < 0.001
14:0
1.81(1.07–2.90)
4.68(2.79–6.19)
p < 0.001
16:0
9.46(7.29–13.29)
18.54(14.98–26.00)
p < 0.001
18:0
2.80(1.99–4.34)
6.85(5.94–9.97)
p < 0.001
20:0
0.14(0.10–0.19)
0.19(0.16–0.38)
p = 0.016
16:1n-7
0.95(0.70–1.22)
1.25(1.05–1.84)
p = 0.014
18:1n-9
20.50(14.98–28.05)
27.55(21.52–35.67)
p = 0.049
18:2n-6
7.42(5.80–9.46)
7.90(5.78–9.77)
p = 0.892
18:3n-3
1.79(1.23–2.36)
1.42(1.09–2.09)
p = 0.454
20:4n-6
0.08(0.05–0.10)
0.08(0.04–0.13)
p = 0.708
20:5n-3
0.35(0.23–0.59)
0.11(0.06–0.18)
p < 0.001
22:5, n-3 and n-6
0.07(0.05–0.11)
0.02(0.01–0.05)
p = 0.001
22:6n-3
0.73(0.44–1.03)
0.21(0.12–0.30)
p < 0.001
Data are presented as mean ± SD for normally distributed variables and as medians (25th–75th percentiles) for variables with skew distributions. *The P-values refer to the difference between diet- and control group. Differences between groups were analyzed by the Students t-test for independent samples for normally distributed variables and by the Mann-Whitney U test for variables with skew distributions; †sum of 18:2n-6 and 20:4n-6; ‡sum of 18:3n-3, 20:5n-3 and 22:6n-3; §E% = percent of total energy. ¶Difference between groups regarding E% polyunsaturated fatty acids was not significant (p = 0.101) when under- and over-reporters were excluded.
According to the results from the diet history interviews, the MD group had a lower intake of fat and saturated fatty acids (SFAs), compared to the CD group (Table
Fatty acid composition in serum phospholipids
At baseline there were no significant differences between the groups regarding the composition of fatty acids in serum phospholipids (Table
Comparison of the fatty acid composition (per cent of total fatty acids) in serum phospholipids between the Mediterranean Diet (MD) group and the Control Diet (CD) group.
MD group (n = 26)
CD group (n = 25)
P-value*
Time (weeks after dietary shift)
Time (weeks after start of study)
0
3
12
0
3
12
16:0
28.3 (23.6–31.0)
29.0 (24.6–32.2)
27.8 (25.3–31.3)
28.9 (27.2–31.7)
29.4 (27.1–32.3)
29.8 (26.6–32.1)
0.797
18:0
14.5 (13.1–15.2)
13.7 (12.2–15.3)
13.7 (12.4–15.7)
15.0 (13.2–17.1)
14.6 (13.3–16.5)
15.0 (13.3–17.0)
0.584
20:0
0.0 (0.0–0.1)
0.0 (0.0–0.2)
0.0 (0.0–0.3)†
0.0 (0.0–0.2)
0.0 (0.0–0.2)
0.0 (0.0–0.2)
0.100
24:0
1.4 (1.0–2.2)
1.4 (1.0–1.9)
1.6 (1.1–2.0)
1.4 (1.0–1.6)
1.1 (0.8–1.7)
1.3 (0.8–1.8)
0.959
Total saturated fatty acids
44.0 (39.1–46.8)
44.7 (37.8–48.3)
43.9 (39.6–49.4)
45.6 (41.4–49.9)
45.6 (41.1–49.1)
46.0 (42.7–49.9)
0.705
16:1n-7
0.8 (0.6–0.9)
0.7 (0.6–0.8)
0.8 (0.6–0.9)
0.7 (0.6–1.1)
0.8 (0.6–0.9)
0.8 (0.6–1.0)
0.339
18:1n-7
1.7 (1.3–1.8)
2.0 (1.4–2.2)
1.8 (1.6–2.0)
1.7 (1.3–1.9)
1.7 (1.5–2.1)
1.8 (1.5–2.0)
0.557
18:1n-9
9.9 (8.2–10.9)
9.7 (8.3–11.2)
9.4 (7.3–10.6)
10.2 (8.3–11.1)
10.6 (8.9–11.5)
10.9 (8.3–11.5)
0.087
22:1n-11
0.6 (0.2–1.6)
0.6 (0.2–1.5)
0.8 (0.8–1.8)
0.6 (0.0–1.3)
0.3 (0.0–1.0)
0.6 (0.0–1.1)
0.356
Total monounsaturated fatty acids
13.1 (11.4–13.8)
12.8 (11.2–14.6)
12.6 (11.3–13.8)
13.3 (11.8–14.4)
13.6 (11.6–14.7)
13.9 (11.4–15.0)
0.207
18:2n-6
16.7 (11.5–19.9)
16.5 (10.7–18.6)
14.4 (11.0–16.9)
16.0 (12.5–19.2)
16.7 (12.8–18.1)
17.0 (13.5–19.5)
0.044
20:2n-6
0.4 (0.2–0.7)
0.3 (0.2–0.5)
0.4 (0.2–0.6)
0.3 (0.2–0.5)
0.3 (0.2–0.5)
0.4 (0.3–0.6)
0.458
20:3n-6
2.6 (1.8–3.2)
2.5 (1.7–3.0)
2.2 (1.6–2.6)‡
2.1 (1.5–3.1)
2.2 (1.7–2.8)
2.4 (1.8–3.1)
0.003
20:4n-6
6.9 (5.0–7.7)
6.8 (4.8–8.2)
6.6 (4.1–8.0)
5.9 (4.1–9.8)
6.6 (4.2–8.5)
6.5 (4.8–8.7)
0.144
Total n-6 fatty acids
25.2 (18.9–30.7)
26.8 (17.5–30.0)
23.8 (17.6–28.3)†
24.4 (18.4–32.2)
25.1 (19.3–29.9)
25.2 (21.5–30.5)
0.008
18:3n-3
0.3 (0.0–0.4)
0.2 (0.0–0.4)
0.4 (0.0–0.4)
0.3 (0.2–0.4)
0.3 (0.1–0.5)
0.3 (0.0–0.5)
0.395
20:5n-3
1.4 (1.1–1.8)
1.8 (1.1–2.8)
2.1 (1.4–3.3)§
1.1 (1.0–1.5)
1.1 (1.0–1.7)
1.1 (0.9–1.4)
<0.001
22:5n-3
0.7 (0.2–1.7)
0.6 (0.2–1.4)
0.8 (0.2–1.7)
0.5 (0.2–1.3)
0.6 (0.2–1.0)
0.6 (0.3–1.0)
0.395
22:6n-3
3.9 (2.2–4.8)
4.2 (3.1–6.6)
5.0 (2.5–7.2)§
3.0 (1.5–4.3)
3.5 (1.7–4.2)
3.0 (2.2–4.5)
<0.001
Total n-3 fatty acids
6.1 (5.2–7.4)
7.3 (5.3–10.0)
8.6 (6.0–11.7)§
5.2 (4.3–7.0)
4.9 (3.7–6.4)
5.2 (3.8–7.0)
0.001
n-6:n-3 ratio
4.2 (3.0–5.6)
3.1 (2.7–3.7)
2.5 (2.1–3.5)‡
4.6 (3.6–5.6)
4.4 (3.4–5.6)
4.9 (4.1–5.6)
0.002
Total polyunsaturated fatty acids
32.7 (25.9–38.4)
33.5 (24.4–40.1)
32.4 (22.7–39.0)
29.3 (22.3–37.5)
31.0 (24.0–36.6)
30.9 (25.7–37.4)
0.072
Data are presented as medians (25th–75th percentiles). *The p-values refer to differences between the MD- and the CD group concerning the change from baseline to week 12. Differences between the groups were analyzed with the Mann-Whitney U test. Statistically significant change from baseline to week 12: †p < 0.05, ‡p < 0.01, §p < 0.001. Within-group differences in week 12 compared to baseline were evaluated by the Wilcoxon signed ranks test.
Spearman's correlation coefficients between the reported intake of fatty acids or foods and fatty acids in serum phospholipids.
Fatty acids in s-phospholipids
Dietary fatty acids/foods
EPA
DHA
α-LNA
total n-3
EPA g/day
0.58*
0.24
0.04
0.43†
EPA g/100 g total fat
0.58*
0.32
0.04
0.49*
DHA g/day
0.60*
0.19
-0.05
0.40†
DHA g/100 g total fat
0.57*
0.26
-0.09
0.45*
α-LNA g/day
0.13
-0.09
0.38†
0.03
α-LNA g/100 g total fat
0.24
0.11
0.29
0.20
Change in fish intake‡
0.48*
0.46*
0.14
0.51*
Change in shellfish intake‡
0.33†
0.48*
0.03
0.34†
*p < 0.01; †p < 0.05; ‡change from baseline to week 12 in relation to change in percentage of fatty acids in s-phospholipids.
Furthermore, changes in the reported consumption frequencies of fish and shellfish were positively related to changes in EPA, DHA and the total percentage of n-3 fatty acids in s-phospholipids (Table
Differences between diet responders and diet non-responders
When the MD group was divided into diet responders (n = 15) and diet non-responders (n = 11), based on their clinical improvement during the study, the diet responders had a higher reported relative intake (fatty acids expressed in relation to the total fat intake) of EPA, docosapentaenoic acid (22:5, which in the food composition database used includes both n-3 and n-6 isomers) and DHA compared to the diet non-responders (p = 0.032, p = 0.040 and p = 0.011 respectively). This resulted in a lower intake ratio of n-6 to n-3 fatty acids in the group of diet-responders. The median n-6 to n-3 ratio was 2.2 in the diet responders and 3.0 in the diet non-responders (p = 0.032). Also the intake of the MUFA palmitoleic acid (16:1n-7), in relation to the total fat intake, was higher in the diet responders (p = 0.015), while the E% of total MUFAs was higher in the diet non-responders (p = 0.048). However, when the intake of total MUFAs was expressed in relation to the total fat intake there was no significant difference between the groups (p = 0.380).
Regarding the fatty acid composition in serum phospholipids there were some differences between the diet responders and the diet non-responders at baseline. The diet responders had a higher relative amount of lignoceric acid (24:0) and lower relative amounts of arachidic acid (20:0), vaccenic acid (18:1n-7) and α-LNA (p = 0.042, p = 0.022, p = 0.007 and p = 0.024 respectively). The change in fatty acid composition from baseline to the end of the study also differed between the groups. The relative amounts of the SFAs palmitic acid (16:0) and stearic acid (18:0), as well as the total SFAs, increased in the diet non-responders, while these fatty acids decreased in the diet responders (p = 0.040, p = 0.033 and p = 0.007 respectively for differences between the groups). On the other hand, the n-3 fatty acid docosapentaenoic acid (22:5n-3) increased in the diet responders while a slight decrease was observed in the diet non-responders (p = 0.048). Regarding the other n-3 fatty acids in serum phospholipids, no significant differences were found between the groups.
No significant differences between the groups were observed concerning the reported consumption of food groups. However, from baseline to the end of the study there was a tendency towards a greater increase in the fish consumption (p = 0.059), as well as a greater decrease in the consumption of dairy products with 10–15% fat (p = 0.076), in the group of diet responders compared to the non-responders.
Discussion
Dietary intake of fat and specific fatty acids
In this study we used a questionnaire to estimate the reported consumption of foods with relevance to the fatty acid intake, and diet history interviews to assess the intake of fat and specific fatty acids. In the MD group, the changes in the reported consumption of food items were in line with the advice given to the patients, i.e. to replace meat, and processed meat with fish and poultry, to use olive oil, canola oil or margarine based on canola oil, and to choose low fat dairy products. This was also reflected in the reported intake of fat assessed by the diet history interviews. For instance, choosing low fat dairy products instead of higher fat alternatives is likely to result in a decreased intake of both total fat and of SFAs. Thus, both assessments of dietary intake indicated that the MD group changed their dietary intake in the direction aimed at.
The traditional Cretan Mediterranean diet is well known for its high content of MUFAs from olive oil. According to a study by Cleland at al, the incorporation of EPA into neutrophil membranes after fish-oil supplementation was higher in healthy subjects eating a diet high in OA and low in LA, compared to subjects on a diet high in LA
Among the most important characteristics of the experimental diet was the amount of n-3 fatty acids and the relation between n-6 and n-3 fatty acids. The original Cretan Mediterranean diet was rich in n-3 fatty acids from both animal sources and plant sources
A modified Cretan Mediterranean diet, similar to our experimental diet, has been tested by de Lorgeril et al
In the present study, the reported total intake of n-3 fatty acids in the MD group was 3.1 g/day and of these 1.2 g were EPA + DHA. Most studies of dietary supplementation with fish oil have used between 1 and 7.1 g EPA + DHA per day
Fatty acid composition in serum phospholipids
The dietary intake of fat is often difficult to assess by means of dietary assessment methods. Therefore, there is a need for objective markers of fatty acid intake to be used as a complement to dietary assessment methods
The use of fatty acid patterns in serum phospholipids as biomarkers of the dietary intake is, limited by the fact that not only diet affects the levels of fatty acids in biological specimens
Sometimes, the relative fatty acid levels reflect the intake of specific foods, especially when the food item is the major source of the fatty acid in question. In this study changes in the reported consumption of fish and shellfish were related to changes in the percentage of both EPA, DHA and total n-3 fatty acids, with Spearman's correlation ranging from 0.46 to 0.51 for fish and from 0.33 to 0.48 for shellfish. Regarding fish intake, this is in agreement with other studies
Can the altered intake of fatty acids explain the beneficial effects on RA disease activity?
To analyse the possible connection between the altered intake of fatty acids in the MD group and the improvement regarding disease activity, we divided the MD group into diet responders and diet non-responders, based on the individual change in disease activity during the study. It is important to remember that only 15 (diet responders) and 11 (diet non-responders) patients were compared in this analysis. Hence, the power to detect differences between diet responders and diet non-responders was fairly low. Still, when these two groups were compared differences in both the reported intake of fatty acids and the fatty acid composition in serum phospholipids were found. Overall, the results point towards a more favourable fatty acid intake in the group of diet responders, at least regarding PUFAs, which in turn suggests a better compliance to the experimental diet in this group. Thus, the differences found between diet responders and diet non-responders indicate that the fatty acid intake contributed to the positive effect of this diet on RA disease activity.
Conclusion
In conclusion, the changes in the reported consumption of food items in the MD group were in line with the advice given during the study. As a result, the total fat intake was lower in the MD group compared to the CD group, and in the MD group a lower percentage of the energy intake was derived from SFAs. The MD group also had a lower intake ratio of n-6:n-3 fatty acids. A corresponding change in the relation between n-6 and n-3 fatty acids was observed in s-phospholipids. Furthermore, the MD patients who were characterised as diet responders, had a higher reported intake of long chain n-3 fatty acids and a lower ratio of n-6 to n-3 fatty acids compared to the diet non-responders. Also the fatty acid profile in serum phospholipids differed in part between the diet responders and the diet non-responders. These findings point towards a better compliance to the experimental diet in the group of diet responders.
Altogether, the changes in the fatty acid profile indicated by three methods, a questionnaire, diet history interviews and fatty acids in s-phospholipids may, at least in part, explain the beneficial effects on the clinical measurements demonstrated earlier
Methods
Patients, study design and the experimental diet
The study was a randomised, parallel, dietary intervention study over three months. In total, 56 patients with RA were included in the study. Of these, 26 MD subjects (21 women and 5 men, mean age 58 years) and 25 CD subjects (20 women and 5 men, mean age 59 years) completed the study. The patients, study design and the dietary intervention have been described in detail elsewhere
The experimental diet used in the present study was based on the Cretan Mediterranean diet previously tested by de Lorgeril et al, in a secondary prevention study of coronary heart disease
To promote good compliance with the Mediterranean diet some food items were supplied free to the MD group, namely: frozen vegetables, tea, olive oil, canola oil and the liquid and half-fat margarine based on canola oil. Olive oil and canola oil, were supplied by Karlshamns AB, vegetables by Nestlé Sweden AB and margarine and tea by Van den Bergh Foods AB.
The CD subjects were instructed to adhere to their ordinary diet. If the subjects of the study used any dietary supplementation (e.g. fish oils, vitamins, minerals, etc.) prior to the study this was recorded. All such supplementation had to be kept unchanged during the study.
Dietary assessments
To assess the dietary intake both a self-administered questionnaire and diet history interviews were used. The questionnaire was completed by the patients of both groups at baseline, in week three and twelve. This questionnaire included both open and closed questions, mainly concerning food choices, and it was designed to investigate compliance with the Mediterranean diet. Regarding questions on frequencies of food intake, the subjects should state their average intake of various food items by marking one of six alternatives ranging from "rarely or never" to "two or more times per day". To enable comparisons of the consumption between the two groups, as well as consumption at different points in time, the food frequencies were converted to average consumption per month. For example, if a consumption of 3–5 times per week was marked this would be converted to 16 times per month.
Diet history interviews, covering the dietary intake during the past month, were carried out to obtain more detailed data on the energy and nutrient intake during the intervention period. The interviews were performed between study weeks seven and twelve and were conducted with 34 patients from both the MD group and the CD group (15 women and 2 men from each group). The only selection criterion for taking part in the diet history interviews was that the subjects were included in the study on February 15th 1999, or later. Fore more details regarding the questionnaire and the diet history interviews see reference 34.
The nutritional analysis package MATs 4_03e was used to calculate the estimated intake of energy and nutrients based on the diet history interviews. This program is based on the Swedish National Food Administration's food composition database, PC-kost (version 2_97). If composite food items and supplements not listed in the database were reported, the nutrient content was entered manually. The energy and nutrient intake was calculated both including and excluding dietary supplements. Still, when dietary supplements were included, the total intake of n-3 fatty acids increased marginally in the MD group and not at all in the CD group. Therefore, only the dietary intake without supplements will be presented. However, when the correlation between the reported dietary intake of fatty acids and their relative content in s-phospholipids were analyzed, the results from the diet history interviews
Validation of the diet history interviews
A validation of the diet history interviews by means of the doubly labelled water method (performed for nine subjects) and biological markers of the intake of protein, sodium and potassium has been presented elsewhere
Categorization of diet responders and diet non-responders
In this intervention study the disease activity was assessed by means of a combined index called disease activity score from 28 joints (DAS28)
Determination of the composition of fatty acids in serum phospholipids
Sampling
Blood samples for the analysis of fatty acids in serum phospholipids were taken at baseline and in weeks three and twelve. After a one-night fast, samples were collected in tubes without additives. The blood was cooled in ice water for 30 min before centrifugation at 1500
Folch extraction
Serum (1 ml) was extracted using methanol (2 ml) and chloroform (4 ml). Potassium chloride, 0,88%; (2 ml with the water content of the sample excluded) was added and the mixture was shaken thoroughly for 30 seconds before being allowed to settle. The aqueous layer was removed, one fourth of the volume of the organic phase of methanol-saline (1:1, v/v) was added and the washing procedure was repeated. The bottom layer containing the purified lipids was collected and the solvent was evaporated in a gentle stream of nitrogen at +30°C
Separation of lipid classes using thin layer chromatography (TLC)
The purified lipids were solved in 40 μl chloroform containing 0,05% (v:v) of antioxidant butylated hydroxytoluene (BHT, 10% in ethanol (w:v)) and separated on silica gel plates 60, F254 (MERCK) by TLC, using petroleumether: diethyl ether: acetic acid (81:18:1). The different lipid classes were visualized using UV-light at 254 nm. The standards used were L-α-Phosphatidylcholine and L-α-Phosphatidylethanolamine, Larodan Fine Chemicals. The phospholipid spots were transferred into screw-capped glass tubes for preparation of fatty acid methyl esters
Transesterification using sodium methoxide
The silica gel containing the lipids was dissolved in sodium-dried diethyl ether (0,5 ml) and methyl acetate (20 μl). Sodium methoxide, 0,5 M, in dry methanol (20 μl) was added. After 15 minutes at room temperature the reaction was stopped by the addition of acetic acid (2 μl). The solvent was evaporated in a gentle stream of nitrogen at +30°C. Iso-hexane with BHT (0,05%; 1 ml) was added and the mixture was centrifuged at 1500
GC analysis
The composition of the fatty acids was determined using gas chromatography. Of the purified methylated sample 1 μl was injected into a HP 5890 series II gas chromatograph suited with a FID and an HP-FFAP capillary column (30 m × 0,25 mm × 0,25 μm). The He carrier gas flow was 13 ml/min. The detector temperature was 260°C. The injector split 1:50 at 220°C. A temperature programme was used with an initial temperature of 160°C held for 5 minutes, raised from 160 to 220°C at a rate of 2°C/min and 220°C held for 30 minutes.
The fatty acid methyl esters were identified by comparison with the retention times of the standards: GLC-68D and GLC-68A, Nu-Chek-Prep and Qualimix Fish 89 – 5540, Larodan Fine Chemicals.
The individual fatty acids were expressed as the percentage of total fatty acids (relative amount). The relative amounts were quantified by integrating the area under the peak and dividing the results by the total area of all fatty acids.
Statistical methods
The statistical analyses were performed using SPSS for Windows version 11.0.1. The differences in fat intake between groups were analyzed using the Student's t-test for independent samples, when the variables were normally distributed. For fatty acids with skewed distributions the Mann-Whitney U test was used. As regards the reported consumption of food items and the composition of fatty acids in serum phospholipids, the Mann-Whitney U-test was performed to test differences between groups at baseline. For these variables, within group differences from baseline to week 12 were evaluated by means of the Wilcoxon signed ranks test.
The Spearman's rank correlation was used to evaluate the association between the reported dietary intake of fatty acids (assessed by diet history interviews including supplements) and their relative content in s-phospholipids. When calculating the correlation, the mean value of the results from week six and twelve were used regarding fatty acids in s-phospholipids, since the diet history interviews were performed between study weeks seven and twelve.
Spearman's rank correlation was also used to evaluate the association between the reported consumption of fish (estimated by means of the questionnaire) and the long chain n-3 fatty acids in s-phospholipids. For these assessments the change from baseline to week 12 was used.
All results were considered statistically significant at a two-tailed p-value of <0.05.
List of abbreviations
AA, arachidonic acid; BMI, body mass index; CD, control diet; DAS 28, disease activity score from 28 joints; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid, E%, percentage of the energy intake; FIL, food intake level; LA, linoleic acid; α-LNA, α-Linolenic acid; MD, Mediterranean diet; MUFA, monounsaturated fatty acids; OA, oleic acid; PUFA, polyunsaturated fatty acids; RA, rheumatoid arthritis; SFA, saturated fatty acids
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
LH participated in the conception and design, acquisition of data, data analysis, interpretation of data and the writing of the manuscript. IN participated in the data analysis, the critical revision of the manuscript and the writing of parts of the paper. LS participated in the conception and design, acquisition of data, interpretation of data and the critical revision of the manuscript. GJ participated in the conception and design, data analysis, interpretation of data, and the critical revision of the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
We would like to express our gratitude to our collaborators, Lena Martinsson, Eva Wolke, Lena Henningsson, Marianne Olsson, Ann-Louise Karlsson, Mona Bäckström and Gunnel Gustavsson for administrative assistance, information, advice and support to the patients concerning their diet, help with the clinical examinations, and in the handling of blood samples for chemical analyses.
We would like to thank Maria Bengtsson and Jenny Wannstedt for performing diet history interviews, Petra Rydén for her help with the processing of food questionnaires, and Christel Larsson, Ulla Johansson, Magdalena Rosell, Agneta Hörnell, Anette Jonsäll and Ylva Mattsson-Sydner for valuable discussions.
We also wish to express our gratitude to all the participants.
The study was supported by grants from the Faculty of Social Sciences of Umeå University, the Swedish Foundation for Health Care Sciences and Allergy Research, the Health Research Council in the Southeast of Sweden, the Swedish Rheumatism Association, the Swedish Nutrition Foundation, the JC Kempe Memorial Scholarship Fund, the "Borgerskapet i Umeå" Fund and the "Uppsala Hemsysterskola" Fund.