Pmstaglandins 0 Longman
Leukotriencs and Essential Group UK Ltd 1991
Fatty
Acids
(1991) 42. 245-249
The Effects of Evening Primrose Oil, Safflower Oil and Paraffin on Plasma Fatty Acid Levels in Humans: Choice of an Appropriate Placebo for Clinical Studies on Primrose Oil D. F. Horrobin,
K. M. Ells, N. Morse-Fisher
and M. S. Manku
Efamol Research Institute, POB 818, Kentville, Nova Scotia, B4N 4H8, Canada (Reprint requests to DFH) ABSTRACT.
In a number of diseases, plasma levels of iinoleic acid are normal or elevated while those of gamma-linolenic acid (18:3n-6, GLA) and further metabolites are below normal. Evening primrose oil (EPO), similar to safflower oil (SFO) except that it contains S-9% of 18:3n-6, has been proposed as a therapeutic agent in these diseases, such as atopic eczema. There is argument as to whether an appropriate placebo for clinical studies on EPO should be an inert material such as paraffin, or a linoleic acid - containing oil such as SFO. We have therefore compared in normal humans the effects on plasma fatty acids of administering EPO, SF0 and paraffm for 10 days. Paraffin had no effect on any fatty acid in any fraction. EPO raised the level of 20:3n-6 (dihomo-gamma-linolenic acid, DGLA) the immediate metabolite of GLA but had no significant effect on arachidonic acid. In surprising contrast, SF0 raised the levels of linoleic and of arachidonic acids, without raising those of DGLA. This suggests that linoleic acid may be rapidly converted to arachidonic acid by a tightly linked enzyme sequence: GLA, in contrast, may be rapidly converted to DGLA but then only slowly on to arachidonic acid. These results are consistent with recent in vitro observations by others on rat hepatocytes and human fibroblasts.
ma linoleic acid levels are normal or above. In contrast it may be possible to correct both the biochemical abnormality and the clinical illness by providing GLA directly in the form of oils containing GLA, such as evening primrose oil (EPO). One such oil, Epogam, derived from strains of evening primrose selected and bred to give material of constant composition has recently been approved by the UK Department of Health for use in the UK National Health Service for the treatment of atopic eczema. In almost all diseases, including even those which are life threatening, it is now recognised that a substantial placebo response is likely to occur. As a result of this placebo response, a patient may experience substantial subjective and objective improvement in clinical status, even though the material administered is completely pharmacologically inert. The proof that GLA-containing oils are effective in any disease therefore requires randomised, double-blind, placebo-controlled trials to be carried out in several hundred patients in several centers. In most countries, before a claim can be made that a product is effective in treating a particular disease, organisations such as the FDA in the
INTRODUCTION In order to be fully effective as an essential fatty acid, linoleic acid must be metabolised to gammalinolenic acid (18:3n-6, GLA) and to further metabolites. In several diseases, notably atopic eczema and premenstrual syndrome, plasma phospholipid linoleic acid levels are somewhat above normal, while concentrations of GLA and further metabolites are below normal (l-3). There are a number of possible explanations for these observations, but perhaps the most likely one is that conversion of linoleic acid to GLA is slower than normal. There is also evidence that the diabetes and in association with excess alcohol intake there is impaired conversion of linoleic acid to GLA (4-6). If GLA formation is impaired, then there may be a functional deficiency of derived essential fatty acids #in spite of a normal intake of linoleic acid. Administration of linoleic acid in this situation would seem to have little merit especially when plas-
Date received 13 July 1990 Date accepted 16 November 1990 24.5
246
ProstaglandinsLeukotrienesand EssentialFatty Acids
USA, the Dept. of Health in the UK, or Health and Welfare in Canada, must be satisfied that the trials have been well conducted, that the results are reliable and that an appropriate placebo has been used. We have been involved in organising clinical trials on EPO and have encountered disagreements as to what should be used as the placebo. Most of these trials have involved the administration of about 4 g/day of EPO whose fatty acid composition is shown in Table 1. Four g per day of EPO contains about 2.5 g of linoleic acid and 320 mg of GLA. Possible placebos include other linoleic acid-co@aining oils which contain no GLA such as safflower oil, or inert materials which contain no essential fatty acids such as liquid paraffin. Many, but not all,, non-medical scientists in the lipid field have strongly advocated the use of linoleic acid-containing bils as placebos in trials of EPO in conditions such as atopic eczema. They have been interested, not so much in the clinical result of whether or not patients improve over their baseline state, but in the difference between effects of linoleic acid and GLA. Such scientists have therefore urged us to use safflower oil as placebo. This is in spite of the fact that linoleic acid is unlikely to be of clinical benefit since the patients concerned clearly have no dietary deficiency of linoleic acid and have plasma levels of linoleic acid which may be sqmewhat above normal. In contrast, many, but, not all, physicians and regulatory officials have expressed different concerns. They are not interested in the details of essential fatty acid metabolism but in whether patients given EPO improve or not as compared with what happens with a truly inert placebo. They have pointed out that there is no evidence that safflower oil, or any other linoleic acid-containing oil is an inert placebo. Linoleic acid is known to be a precursor of many substances, which could theoretically have pro-inflammatory actions. Some physicians therefore argued that linoleic. acid might make atopic eczema worse. In this situation, if EPO had no effect at all, making patients neiiher worse nor better, it would appear to be better than the linoleic acid placebo because it was doing less harm. This might be a result which tiould interest lipid biochemists but it would be of no interest to physicians whose concern is with clinical improvement over baseline. The physicians and some regulatory officials therefore argued for the use of a truly inert substance as placebo. Liquid paraffin was suggested. At a dose of 4 g/day this has no pharmacological actions. Fifteen to 30 g per day are required to produce a laxative effect. We took the view that these theoretical arguments could be resolved only by an experimental study. We have therefore compared the effects of
administering 4 g/day of Epogam EPO, safflower oil and liquid paraffin for 10 days to normal individuals. In some respects the results were surprising and indicated that the regulators and physicians were correct to be suspicious of possible pharmacological effects of safflower oil.
SUBJECTS AND METHODS Subjects were university students and laboratory personnel who appeared to be in good health. A blood sample was withdrawn from each subject prior to the start of the study. Each subject then took 4 x 500 mg capsules each morning and evening for 10 days (4 g/day in total) and another blood sample was taken on the 11th day. Forty-two subjects took Epogam EPO, 24 subjects took safflower oil (SFO), and 10 subjects took liquid paraffin. Each group contained both males and females but separate analysis showed no sex differences in the results: the two sexes were therefore grouped together. The lipids were extracted from the plasma, the lipid fractions separated by thin layer chromatography, the separate fractions transmethylated and analysed by gas chromatography according to methods previously described (7). Table 1 Compositionof the fatty acids in the safflower oil and evening primrose oil used in the studies described in this paper Safflower Linoleic (18:2n-6) Gamma-linolenic (18:3n-6) Oleic (18: ln-9) Stearic (18:0) Palmitic (16:0) Alpha-linolenic (18:3n-3) Others
79.0 10.7 2.1 6.9 0.6 0.7
Evening Primrose 70.2 8.7 12.7 1.8 5.8 0.8
RESULTS The before and after treatment concentrations of fatty acids in the phospholipid, triglyceride and cholesterol ester fractions are shown in Tables 24. There were no changes in the total amounts of phospholipid, cholesterol esters or triglycerides. There were no changes in response to paraffin in any fatty acid in any lipid fraction, indicating that paraffin was a truly inert placebo with regard to fatty acid levels. EPO, in contrast, produced significant elevations of dihomo-gamma-linolenic acid (DGLA, 20:3n-6), the immediate elongated metabolite of GLA, in the plasma phospholipid fraction. Oleic acid concentrations fell significantly in triglyceride and cholesterol
Choice of an Aooronriate
Placebo for Clinical Studies on Primrose Oils
Table 2 Concentrations of fatty acids (in mg/lOtl mg total lipid present) in the total phospholipid fractions in the various groups. The results show the concentrations before and after the administration for 10 days of 4 g/day liquid paraffin, safflower oil or evening primrose oil (Epogam). The significance of changes occurring as the result of treatment was assessed by Student’s paired t-test. Tr = present at less than 0.1 mg/lOO mg. x p CO.l, xx p C 0.05, xxx p c 0.02, YXXXp < 0.01. Each value is the mean f SD Liquid Paraffin After Before
Safflower Oil Before After
Before
18:2n-6 18:3n-6 20:3n-6 20:4n-6
23.9 + 3.0 Tr 2.3 + 0.7 12.9 + 1.9
23.6 zk 3.4 Tr 2.1 -I 0.3 12.9 + 1.1
26.1 0.1 2.6 12.0
+ 3.7 + 0.2 + 0.6 _+ 1.9
23.8 + 3.4 Tr 2.5 + 0.6 12.2 + 1.9
18:3n-3 20:5n-3 22:5n-3 22:6n-3
Tr 1.1 t 0.5 1.2 f 0.3 4.2 f 1.8
Tr 1.2 “r 0.4 1.0 * 0.2 4.0 f 1.0
0.1 1.2 1.0 3.7
I!I 0.2 + 0.9 f 0.4 f 1.7
16:O 18:O 18: ln-9
26.0 t 2.3 10.1 f 2.8 15.8 f 2.2
25.8 + 1.8 10.7 + 1.7 16.4 k 1.7
26.1 + 2.6 9.3 f 2.8 14.9 + 2.8
28.3 z!z3.5”” Tr 2.6 + 0.5 13.1 + 1.6”“” 0.2 1.0 0.9 3.9
* 0.4 + 1.2 + 0.4 zk 1.5
0.3 1.1 0.8 4.0
Epogam After
+ + + t
0.5 0.6 0.5 1.5
25.6 k 2.3 27.5 + 2.7 8.6 + 2.3 9.0 f 2.6 13.2 + 1.4x”“” 15.6 f 2.2
23.4 0.2 3.0 12.4 0.3 1.0 1.0 4.0
+ 3.6 + 0.3 + 0.8”“” I? 2.3 + 0.6 + 0.8 ?I 0.6 f. 1.3
27.0 k 2.3 9.6 k 2.6 14.7 + 2.8
Table 3 Concentrations of fatty acids (in mg/lOO mg total lipid present) in the cholesterol ester fractions in the various groups. The results show the concentrations before and after the administration for 10 days of 4 g/day liquid paraffin, safflower oil or evening primrose oil (Epogam). The significance of changes occurring as the result of treatment was assessed by Student’s paired t-test. Tr = present at less than 0.1 mg/lOO mg. x p
Leukotriencs and Essential Group UK Ltd 1991
Fatty
Acids
(1991) 42. 245-249
The Effects of Evening Primrose Oil, Safflower Oil and Paraffin on Plasma Fatty Acid Levels in Humans: Choice of an Appropriate Placebo for Clinical Studies on Primrose Oil D. F. Horrobin,
K. M. Ells, N. Morse-Fisher
and M. S. Manku
Efamol Research Institute, POB 818, Kentville, Nova Scotia, B4N 4H8, Canada (Reprint requests to DFH) ABSTRACT.
In a number of diseases, plasma levels of iinoleic acid are normal or elevated while those of gamma-linolenic acid (18:3n-6, GLA) and further metabolites are below normal. Evening primrose oil (EPO), similar to safflower oil (SFO) except that it contains S-9% of 18:3n-6, has been proposed as a therapeutic agent in these diseases, such as atopic eczema. There is argument as to whether an appropriate placebo for clinical studies on EPO should be an inert material such as paraffin, or a linoleic acid - containing oil such as SFO. We have therefore compared in normal humans the effects on plasma fatty acids of administering EPO, SF0 and paraffm for 10 days. Paraffin had no effect on any fatty acid in any fraction. EPO raised the level of 20:3n-6 (dihomo-gamma-linolenic acid, DGLA) the immediate metabolite of GLA but had no significant effect on arachidonic acid. In surprising contrast, SF0 raised the levels of linoleic and of arachidonic acids, without raising those of DGLA. This suggests that linoleic acid may be rapidly converted to arachidonic acid by a tightly linked enzyme sequence: GLA, in contrast, may be rapidly converted to DGLA but then only slowly on to arachidonic acid. These results are consistent with recent in vitro observations by others on rat hepatocytes and human fibroblasts.
ma linoleic acid levels are normal or above. In contrast it may be possible to correct both the biochemical abnormality and the clinical illness by providing GLA directly in the form of oils containing GLA, such as evening primrose oil (EPO). One such oil, Epogam, derived from strains of evening primrose selected and bred to give material of constant composition has recently been approved by the UK Department of Health for use in the UK National Health Service for the treatment of atopic eczema. In almost all diseases, including even those which are life threatening, it is now recognised that a substantial placebo response is likely to occur. As a result of this placebo response, a patient may experience substantial subjective and objective improvement in clinical status, even though the material administered is completely pharmacologically inert. The proof that GLA-containing oils are effective in any disease therefore requires randomised, double-blind, placebo-controlled trials to be carried out in several hundred patients in several centers. In most countries, before a claim can be made that a product is effective in treating a particular disease, organisations such as the FDA in the
INTRODUCTION In order to be fully effective as an essential fatty acid, linoleic acid must be metabolised to gammalinolenic acid (18:3n-6, GLA) and to further metabolites. In several diseases, notably atopic eczema and premenstrual syndrome, plasma phospholipid linoleic acid levels are somewhat above normal, while concentrations of GLA and further metabolites are below normal (l-3). There are a number of possible explanations for these observations, but perhaps the most likely one is that conversion of linoleic acid to GLA is slower than normal. There is also evidence that the diabetes and in association with excess alcohol intake there is impaired conversion of linoleic acid to GLA (4-6). If GLA formation is impaired, then there may be a functional deficiency of derived essential fatty acids #in spite of a normal intake of linoleic acid. Administration of linoleic acid in this situation would seem to have little merit especially when plas-
Date received 13 July 1990 Date accepted 16 November 1990 24.5
246
ProstaglandinsLeukotrienesand EssentialFatty Acids
USA, the Dept. of Health in the UK, or Health and Welfare in Canada, must be satisfied that the trials have been well conducted, that the results are reliable and that an appropriate placebo has been used. We have been involved in organising clinical trials on EPO and have encountered disagreements as to what should be used as the placebo. Most of these trials have involved the administration of about 4 g/day of EPO whose fatty acid composition is shown in Table 1. Four g per day of EPO contains about 2.5 g of linoleic acid and 320 mg of GLA. Possible placebos include other linoleic acid-co@aining oils which contain no GLA such as safflower oil, or inert materials which contain no essential fatty acids such as liquid paraffin. Many, but not all,, non-medical scientists in the lipid field have strongly advocated the use of linoleic acid-containing bils as placebos in trials of EPO in conditions such as atopic eczema. They have been interested, not so much in the clinical result of whether or not patients improve over their baseline state, but in the difference between effects of linoleic acid and GLA. Such scientists have therefore urged us to use safflower oil as placebo. This is in spite of the fact that linoleic acid is unlikely to be of clinical benefit since the patients concerned clearly have no dietary deficiency of linoleic acid and have plasma levels of linoleic acid which may be sqmewhat above normal. In contrast, many, but, not all, physicians and regulatory officials have expressed different concerns. They are not interested in the details of essential fatty acid metabolism but in whether patients given EPO improve or not as compared with what happens with a truly inert placebo. They have pointed out that there is no evidence that safflower oil, or any other linoleic acid-containing oil is an inert placebo. Linoleic acid is known to be a precursor of many substances, which could theoretically have pro-inflammatory actions. Some physicians therefore argued that linoleic. acid might make atopic eczema worse. In this situation, if EPO had no effect at all, making patients neiiher worse nor better, it would appear to be better than the linoleic acid placebo because it was doing less harm. This might be a result which tiould interest lipid biochemists but it would be of no interest to physicians whose concern is with clinical improvement over baseline. The physicians and some regulatory officials therefore argued for the use of a truly inert substance as placebo. Liquid paraffin was suggested. At a dose of 4 g/day this has no pharmacological actions. Fifteen to 30 g per day are required to produce a laxative effect. We took the view that these theoretical arguments could be resolved only by an experimental study. We have therefore compared the effects of
administering 4 g/day of Epogam EPO, safflower oil and liquid paraffin for 10 days to normal individuals. In some respects the results were surprising and indicated that the regulators and physicians were correct to be suspicious of possible pharmacological effects of safflower oil.
SUBJECTS AND METHODS Subjects were university students and laboratory personnel who appeared to be in good health. A blood sample was withdrawn from each subject prior to the start of the study. Each subject then took 4 x 500 mg capsules each morning and evening for 10 days (4 g/day in total) and another blood sample was taken on the 11th day. Forty-two subjects took Epogam EPO, 24 subjects took safflower oil (SFO), and 10 subjects took liquid paraffin. Each group contained both males and females but separate analysis showed no sex differences in the results: the two sexes were therefore grouped together. The lipids were extracted from the plasma, the lipid fractions separated by thin layer chromatography, the separate fractions transmethylated and analysed by gas chromatography according to methods previously described (7). Table 1 Compositionof the fatty acids in the safflower oil and evening primrose oil used in the studies described in this paper Safflower Linoleic (18:2n-6) Gamma-linolenic (18:3n-6) Oleic (18: ln-9) Stearic (18:0) Palmitic (16:0) Alpha-linolenic (18:3n-3) Others
79.0 10.7 2.1 6.9 0.6 0.7
Evening Primrose 70.2 8.7 12.7 1.8 5.8 0.8
RESULTS The before and after treatment concentrations of fatty acids in the phospholipid, triglyceride and cholesterol ester fractions are shown in Tables 24. There were no changes in the total amounts of phospholipid, cholesterol esters or triglycerides. There were no changes in response to paraffin in any fatty acid in any lipid fraction, indicating that paraffin was a truly inert placebo with regard to fatty acid levels. EPO, in contrast, produced significant elevations of dihomo-gamma-linolenic acid (DGLA, 20:3n-6), the immediate elongated metabolite of GLA, in the plasma phospholipid fraction. Oleic acid concentrations fell significantly in triglyceride and cholesterol
Choice of an Aooronriate
Placebo for Clinical Studies on Primrose Oils
Table 2 Concentrations of fatty acids (in mg/lOtl mg total lipid present) in the total phospholipid fractions in the various groups. The results show the concentrations before and after the administration for 10 days of 4 g/day liquid paraffin, safflower oil or evening primrose oil (Epogam). The significance of changes occurring as the result of treatment was assessed by Student’s paired t-test. Tr = present at less than 0.1 mg/lOO mg. x p CO.l, xx p C 0.05, xxx p c 0.02, YXXXp < 0.01. Each value is the mean f SD Liquid Paraffin After Before
Safflower Oil Before After
Before
18:2n-6 18:3n-6 20:3n-6 20:4n-6
23.9 + 3.0 Tr 2.3 + 0.7 12.9 + 1.9
23.6 zk 3.4 Tr 2.1 -I 0.3 12.9 + 1.1
26.1 0.1 2.6 12.0
+ 3.7 + 0.2 + 0.6 _+ 1.9
23.8 + 3.4 Tr 2.5 + 0.6 12.2 + 1.9
18:3n-3 20:5n-3 22:5n-3 22:6n-3
Tr 1.1 t 0.5 1.2 f 0.3 4.2 f 1.8
Tr 1.2 “r 0.4 1.0 * 0.2 4.0 f 1.0
0.1 1.2 1.0 3.7
I!I 0.2 + 0.9 f 0.4 f 1.7
16:O 18:O 18: ln-9
26.0 t 2.3 10.1 f 2.8 15.8 f 2.2
25.8 + 1.8 10.7 + 1.7 16.4 k 1.7
26.1 + 2.6 9.3 f 2.8 14.9 + 2.8
28.3 z!z3.5”” Tr 2.6 + 0.5 13.1 + 1.6”“” 0.2 1.0 0.9 3.9
* 0.4 + 1.2 + 0.4 zk 1.5
0.3 1.1 0.8 4.0
Epogam After
+ + + t
0.5 0.6 0.5 1.5
25.6 k 2.3 27.5 + 2.7 8.6 + 2.3 9.0 f 2.6 13.2 + 1.4x”“” 15.6 f 2.2
23.4 0.2 3.0 12.4 0.3 1.0 1.0 4.0
+ 3.6 + 0.3 + 0.8”“” I? 2.3 + 0.6 + 0.8 ?I 0.6 f. 1.3
27.0 k 2.3 9.6 k 2.6 14.7 + 2.8
Table 3 Concentrations of fatty acids (in mg/lOO mg total lipid present) in the cholesterol ester fractions in the various groups. The results show the concentrations before and after the administration for 10 days of 4 g/day liquid paraffin, safflower oil or evening primrose oil (Epogam). The significance of changes occurring as the result of treatment was assessed by Student’s paired t-test. Tr = present at less than 0.1 mg/lOO mg. x p
