The effects of dietary supplementation with isoflavones from red
clover on the lipoprotein profiles of post menopausal women with
mild to moderate hypercholesterolaemia
J.B. Howes
b, D. Sullivan
c, N. Lai
c, P. Nestel
d, S. Pomeroy
d, L. West
e, J.A. Eden
b,
L.G. Howes
a,*
aDepartment of Clinical Pharmacology,St George Hospital,Uni6ersity of New South Wales,Kogarah,NSW, Australia 2217 bDepartment of Obstetrics and Gynaecology,St George Hospital,Uni6ersity of New South Wales,Kogarah,NSW, Australia 2217
cDepartment of Clinical Biochemistry,Royal Prince Alfred Hospital,Camperdown,NSW, Australia dBaker Medical Research Institute,Prahran,Vic., Australia
eNo
6ogen Pty. Ltd.,Wicks Road,North Ryde,NSW, Australia
Received 11 May 1999; received in revised form 28 September 1999; accepted 15 October 1999
Abstract
The effects of dietary isoflavone supplementation using a purified extract of red clover containing approximately biochanin A 26 mg, formononetin 16 mg, daidzein 0.5 mg and genistein 1 mg per tablet at doses of one or two tablets per day were compared to placebo in a three-period, randomised, double blind, ascending dose study in 66 post menopausal women with plasma cholesterol levels between 5.0 and 9.0 mmol/l. Each treatment period lasted 4 weeks and a further nine women received placebo for the full 12-week period. All women consumed a low isoflavone diet for 2 weeks preceding the commencement of the study and for the 12-week study period. Urinary isoflavone excretion was very low in subjects receiving placebo but increased in a dose-dependent manner during therapy with one and two of isoflavone tablets. Dietary supplementation with isoflavones did not significantly alter total plasma cholesterol, LDL cholesterol, HDL cholesterol or plasma triglyceride levels. However, inverse correlations were found between urinary genistein excretion and plasma triglyceride levels and between urinary O-DMA excretion (an isoflavone metabolite) and plasma triglyceride levels in subjects receiving one isoflavone tablet, suggesting a weak relationship between isoflavone intake and plasma triglycerides which may be influenced by individual differences in isoflavone absorption or metabolism. The results suggest that isoflavone phytoestrogens from red clover in the proportions and quantities studied do not significantly alter plasma lipids in post menopausal women with moderately elevated plasma cholesterol levels. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Isoflavones; Phytoestrogens; Cholesterol; HDL; LDL
www.elsevier.com/locate/atherosclerosis
1. Introduction
There has been increasing interest in the effects of soy on cardiovascular risk factors because of the low incidence of cardiovascular disease in populations that consume diets high in soy. A study of 4838 Japanese men and women showed that serum total cholesterol levels decrease with increasing consumption of soy products in men but not in women [1]. Studies such as these suffer from difficulties in measuring the amount
of protein consumed and the possibility that other dietary or lifestyle factors may influence cholesterol levels. A number of interventional studies have substi-tuted soy protein for protein consumed in the subjects’ regular diet to determine whether the apparent reduc-tion in plasma cholesterol levels that accompanies diets that are high in soy is due to an effect of soy protein or merely due to the replacement of saturated fat with polyunsaturated fat [2]. These studies in humans have produced mixed results, although the results of similar studies in animal models of hyperlipidemia have been more consistent and more impressive [3,4]. While a meta-analysis of the human trials supported a
choles-* Corresponding author. Tel.:+61-2-9350-3964; fax:+ 61-2-9350-2633.
terol lowering effect [2], exclusion of several well con-ducted but negative studies may have reduced its valid-ity [5,6]. It has been hypothesised that isoflavones present in soy protein are responsible for or contribute to low cholesterol levels in populations consuming high soy diets [7], although it is possible that other compo-nents of soy protein contribute to this effect [3]. There have been very few, if any, studies examining whether dietary supplementation with purified isoflavones alter plasma lipoprotein profiles in humans. We have studied the effects of a purified extract of isoflavones from red clover in tablet form given over a 10-week period in increasing doses, to examine the effects on total choles-terol, triglycerides, HDL and LDL cholesterol. Isoflavones from red clover differ from soy in that the principal isoflavones in red clover are biochanin A and formononetin, while those in soy consist solely of genis-tein and diadzein. However, biochanin A and for-mononetin are metabolised extensively in vivo to genistein and daidzein respectively (Fig. 1).
2. Methods and materials
Ninety-three post menopausal women were recruited from The St George Hospital, Kogarah, NSW, The Royal Prince Alfred Hospital, Camperdown, NSW and The Baker Institute, Melbourne, Vic. (31 from each centre). The women had total cholesterol levels between 5.0 and 9.0 mmol/l and were free of clinically apparent cardiovascular disease. All had at least 6 months of amenorrhea and a plasma FSH of \40 IU/l.
The subjects had a medical examination and fasting blood was taken for a lipid profile. A full dietary
history was taken and vegetarians and subjects who regularly consumed soy were excluded. Subjects were also excluded if they had been on any form of lipid lowering drug therapy, estrogen, or ‘natural’ therapy (herbal, vitamin or isoflavone supplementation) within the last 6 weeks. Smokers were also excluded. Twenty-four-hour urine was collected for isoflavone levels and a 7-day dietary diary was completed prior to collection. After fulfilling the entry criteria, subjects were ran-domised to receive double-blind therapy with either placebo or increasing doses of isoflavones. Patients were randomised to active or placebo therapy in an 6:1 ratio. All subjects received placebo for the first 3 – 4 weeks. During the last week of this period another 7-day dietary diary was completed and fasting blood samples were collected on two consecutive days for the measurement of lipid profiles. Subjects randomised to active therapy received one isoflavone tablet daily for 5 weeks while those randomised to control therapy tinued to receive placebo. The isoflavone tablets con-tained approximately biochanin A 26 mg, formononetin 16 mg daily, daidzein 0.5 mg and genis-tein 1 mg.
At the end of this treatment period, the subjects randomised to active therapy received two tablets of isoflavones daily for a further 5 weeks while those randomised to control therapy continued to receive placebo. At the end of each treatment period fasting blood was collected on two consecutive days for the measurement of plasma lipids. In addition, a 24-h urine collection was performed at the end of each treatment phase for the measurement of urinary isoflavone excretion.
Isoflavones in urine were assayed by high perfor-mance liquid chromatography with UV detection; 10-ml aliquots of urine were mixed with 100 ml of
glucuronidase and the mixture was incubated for 24 h at 37°C after which it was extracted on a C-18 solid phase extraction column (Waters, Sydney, Australia). Isoflavones were diluted with 3 ml of methanol and 10
ml of the extract was injected into the HPLC system.
The HPLC system consisted of a 25-cm, 5 mM, C-18
stationary phase column (Symmetry, Waters, Sydney, Australia) and a gradient acetonitrile/water mobile phase. The limit of detection of the assay for each of the isoflavones measured was 5 ng/ml (signal-to-noise ratio of greater than 3.1). Isoflavones were quantified using UV detection at 283mM. The between-day
coeffi-cients of variation for the assay were daidzein 19%, genistein 19%, formononetin 8% and biochanin 15% (n=8) while the within-day coefficients of variation were daidzein 2%, gensitein 2%, formononetin 1% and biochanin 2% (n=6). All samples from the one subject were assayed in the same assay run. Six tablets from the batch of isoflavone tablets used for the pharmacoki-netic study were assayed for isoflavone content. The
Table 1
Urinary excretion of isoflavones (mg/24 h) during dietary supplementation with isoflavones from red clovera
0 mg 40 mg 80 mg P-value
Placebo
Daidzein 0.2390.34 0.3590.38 0.9791.74 NS
1.1491.94 5.9493.85
Active 26.639105.56 NS
Placebo
Genistein 0.0290.04 0.0390.06 0.1890.38 NS
Active 0.2890.54 2.1692.19 4.7193.82 B0.001
0 0
Placebo 0
Formononetin 0
Active 0.0390.14 0.1190.38 1.1591.00 B0.001
0 0
Biochanin A Placebo 0 0
0.8490.29 1.0591.04
Active 2.3691.31 B0.001
Placebo
O-DMA 0.1690.35 0.0190.01 0.3790.92 0.0185
Active 0.1790.53 2.0892.02 6.1396.76 B0.05
0.2690.35 0.3890.40
Placebo 1.2092.14
Total isoflavones NS
Active 1.4492.43 10.0796.63 22.47912.02 B0.001
aIsoflavone supplementation produced dose dependant increases in urinary isoflavone excretion.P-value, level of statistical significance of the
effect of dose (ANOVA); NS, not significant.
data were analysed by repeated measures analysis of variance and all results are expressed as the mean9
S.D.
All subjects provided written and informed consent. The protocol was approved by the respective ethics committees of the three participating institutes.
3. Results
Of 93 patients recruited, 75 completed the study (66 women on active treatment and nine on placebo). The mean age of the women was 5897.3 years and their FSH levels were 78927.4 IU/l. Urinary isoflavone results are presented in Table 1. All patients had either undetectable levels or very low levels of isoflavones in urine during the run in phase and placebo phases of the study. Patients receiving one and two isoflavone tablets had dose-dependent increases in urinary isoflavone lev-els. Urinary formononetin and biochanin A levels were low in patients receiving active therapy while genistein and daidzein levels increased substantially, reflecting the extensive metabolism of formononetin and biochanin A to daidzein and genistein respectively.
The results of plasma lipids are presented in Table 2. There were no significant changes in total cholesterol, triglycerides HDL cholesterol or LDL cholesterol in either the active or control groups. However, there was a significant correlation between plasma triglyceride levels and urinary genistein excretion (b= −0.256, P=0.028) and a similar correlation between plasma triglyceride levels and urinary excretion of the isoflavone metabolite O-DMA in patients receiving one isoflavone tablet daily (b= −0.277, P=0.017). How-ever, no relationship between plasma triglyceride levels and isoflavone excretion was apparent when the two isoflavone tablets per day were consumed. The isoflavone constant of the six tablets assayed (mean9
S.D.) were daidzein 0.4490.02 mg, genistein 1.299
0.06 mg, formononetin 15.790.64 mg and biochanin A 26.2091.13 mg (total 43.6391.48 mg), all present as the aglycone form.
The body weights of the subjects did not change during the study (68.8911.1 and 68.3911.4 kg at commencement and end of the study for the active group, 67.699.2 and 67.899.4 kg respectively for the control group) and blood pressures did not differ sig-nificantly between the active and control patients.
4. Discussion
There is continued debate about the effects of isoflavone phytoestrogens on plasma lipids. A meta-analysis of studies of dietary supplementation with soy protein (which contains isoflavone phytoestrogens) con-cluded that soy protein has a modest effect in lowering plasma cholesterol levels but that this action is largely restricted to subjects with high cholesterol levels [2]. If
Table 2
Plasma cholesterol and triglyceride levels (mmol/l) during dietary supplementation with an isoflavone extract from red clovera
0 mg 40 mg 80 mg
Placebo
Cholesterol 6.4691.2 6.3791.30 6.4791.20 Active 6.3491.0 6.1391.0 6.1691.0 Placebo 1.7390.81
Triglycerides 1.7490.67 1.4090.52 Active 1.4490.61 1.3790.56 1.6290.93
1.2490.22 1.3090.29
Placebo 1.3090.22
HDL
cholesterol Active 1.5290.35 1.5490.33 1.5390.32 Placebo 4.2591.14 4.1291.14 4.2491.04 LDL
cholesterol Active 4.2690.96 4.2090.98 4.2490.99
aIsoflavone supplementation did not significantly alter plasma
soy protein lowers plasma cholesterol levels it remains uncertain whether this effect is due to estrogen-like activity of isoflavones such as genistein and daidzein or due to another component of soy protein.
In the present study, we examined the effects of dietary supplementation with purified isoflavones derived from red clover added to a diet low in isoflavones in post menopausal women with moderately elevated plasma cholesterol levels. We used a standard-ised extract containing the isoflavones biochanin A, formononetin, genistein and daidzein in quantities of approximately 26, 16, 0.5 and 1 mg respectively. Previ-ous studies have indicated that more than 60% of biochanin A and formononetin are metabolised to genistein and daidzein, respectively, in humans [8].
Dietary supplementation with the isoflavone extract produced dose-dependent increases in urinary isoflavone excretion, mostly genistein and daidzein. The urinary levels achieved were similar to those character-istically found in populations consuming high isoflavone diets [9]. The low levels of urinary isoflavone excretion during the placebo phase of the study and in the patients who continued on placebo therapy for the duration of the trial indicate a high level of compliance of the patients to the low isoflavone diet. Despite achieving urinary isoflavone excretion levels similar to those in populations consuming high isoflavone diets, we did not observe any significant changes in total cholesterol, LDL cholesterol, HDL cholesterol or plasma triglycerides during supplementation with one or two isoflavone tablets. We did observe a statistically significant inverse correlation between urinary genistein excretion and plasma triglyceride levels and between the urinary excretion of the isoflavone metabolite O-DMA and plasma triglyceride levels during treatment with one tablet of isoflavone daily. This suggests that there may be a weak relationship between the consumption of some isoflavones and plasma triglyceride levels which is dependent upon individual variation in the absorp-tion or metabolism of the isoflavones. However, these relationships were not apparent during dietary supple-mentation with two isoflavone tablets, suggesting that the relationships may have been spurious.
The results of our study do not suggest that isoflavones contribute significantly to a cholesterol low-ering effect of soy protein. However, the results do not exclude the possibility of such an effect being present in subjects with very high plasma cholesterol levels (as the mean plasma cholesterol level of our population was 6.5 mmol/l) or following a longer period of treatment. Further studies are required to investigate the effects of isoflavone supplementation in patients with very high cholesterol levels, the group that appears to be most responsive to the hypocholesterolemic effects of nutri-tional changes in general. Furthermore, the nature of the isoflavones present in the extract from red clover
that we studied differed from that present in soy protein. It is possible that isoflavones differ in their effects on plasma lipoproteins and that the composition of the supplement that we chose to study was not optimal for plasma cholesterol reduction. However, the extensive metabolism of biochanin A and formononetin to genistein and daidzein produced urinary isoflavone excretion profiles that would be expected to be fairly similar to these observed during soy protein supplemen-tation. Our results are consistent with a previous study which found no effects of dietary supplementation with soy isoflavones on plasma lipid profiles [10], and the results of a previous study which suggested that the isoflavone content of diets that include soy protein supplementation is not a major determinant of their effects on plasma lipid profiles [11].
In conclusion, despite achieving isoflavone intakes equivalent to populations with high isoflavone diets who characteristically have low cholesterol levels, we found no significant effect of isoflavone supplementa-tion on plasma lipids, with the excepsupplementa-tion of a possible weak relationship between genistein or O-DMA excre-tion and plasma triglyceride levels. The results of the study suggest that isoflavone supplementation using the extract from red clover chosen does not have a signifi-cant impact on plasma lipid profiles of post menopausal women with moderately elevated plasma cholesterol levels.
Acknowledgements
Supported by a grant from Novogen Pty. Ltd., Australia.
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