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2 Clinica di Gastroenterologia ed Epatologia, Università degli Studi di Perugia, Perugia, Italy 06100; 3 Department of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center and Department of Pediatrics of the University of Cincinnati College of Medicine, Cincinnati, Ohio 45229; 4 Division of Internal Medicine, Department of Medicine, Surgery, Dentistry, San Paolo School of Medicine, University of Milan, Milan, Italy 20142; and 5 Clinica di Medicina Interna, Angiologia e Malattie da Aterosclerosi, Università degli Studi di Perugia, Perugia, Italy 06100
* To whom correspondence should be addressed. E-mail: kenneth.setchell{at}cchmc.org.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Most previous investigations aimed at understanding the relative cholesterol-lowering action of soy protein and isoflavones have compared purified soy proteins, such as isolated soy protein (ISP)6, with alcohol-washed soy protein, casein, or dairy products (3,10,11) as control diets and these latter matrices are considerably different from each other in terms of macro- and micronutrient composition (5,19). Results from such studies have yielded highly variable results and much confusion as to the relative merits of soy for cardiovascular health (13). It is debatable whether investigating the effects of foods made from ISP reflects the expected dietary effects of whole soy foods, especially by Asian adults. For example, dietary analysis of the types of soy foods consumed by Asians reveals that ISP, with almost exclusively isoflavone glucosides (20), is rarely a source of soy protein, whereas a high proportion of the isoflavone intake in Asians is in the form of aglycons, because fermented soy foods account for about one-third of the soy protein source (21,22).
In this study, we circumvented some of the above limitations by comparing the effects of a novel pasta that was enriched with isoflavones naturally sourced from the soy germ to those of a conventional soy pasta that lacked isoflavones for its effects on blood lipids in newly diagnosed patients with hypercholesterolemia. Utilization of the soy germ has the advantage that it is naturally milled from the soybean, is not subjected to chemical processing, and is highly concentrated in isoflavones (23). Furthermore, due to a unique interaction between the isoflavones in the soy germ and the semolina wheat, the final product delivered predominantly isoflavones in aglycon form and in this regard, this pasta had the compositional characteristics of a fermented Asian soy food. The resulting soy germ-enriched pasta delivered 33 mg/d of isoflavones, typical of isoflavone intakes by Asians (24), and the study afforded an assessment of the potential role of isoflavones in the absence of substantial amounts of soy protein.
Because isoflavones have been shown in numerous studies to have important effects on a number of surrogate markers of cardiovascular risk, including effects on lipid peroxidation (25,26), inflammation (27), and the vasculature (28–30), we also examined the effects of this isoflavone-enriched pasta on key elements of cardiovascular risk, including body weight, BMI, inflammation, antioxidant status, blood pressure, and endothelial function.
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Subjects and Methods |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Study design.
This 10-wk study was a single-crossover parallel-group, randomized controlled, blinded trial of 62 adult men and women newly diagnosed with hypercholesterolemia and consecutively recruited from outpatient referrals to the Lipid Clinic of the University of Perugia Hospital. Patients with a history of previous coronary heart disease, intestinal disease, or cancer were excluded from the study, as were smokers and anyone consuming >2 drinks of alcohol per day. Patients administered any form of lipid-lowering drugs or blood pressure medication, vegetarians, and anyone knowingly consuming soy foods regularly or taking antioxidant supplements were also excluded. The study was conducted at the University of Perugia School of Medicine (Perugia, Italy) and the study protocol conformed to the ethical guidelines of the Helsinki Declaration of 1975 for research on humans. The patients were counseled regarding the study design and were asked to adhere to the Italian Heart Association Step II diet throughout. This diet included a daily serving of pasta (80 g) and compliance to the study was facilitated by the fact that most Italians consume pasta on a daily basis. Prior to recruitment, all patients had been on a Step I diet for 
4 wk and after enrollment in this study were then placed on the Step II diet for the entire length of the study, including a run-in acclimatization period of 2 wk before the study commenced. At the end of the 2-wk run-in period, blood and urine were collected for baseline measurements of serum lipids and other markers of cardiovascular risk, including high sensitivity C-reactive protein (hsCRP) and urinary isoprostanes. Body weight, BMI, waist circumference, and blood pressure were measured and endothelial function was assessed by change in brachial artery flow-mediated vasodilatation at baseline. The 62 patients were then randomized to 2 groups; 31 patients were assigned to a group that was given 1 daily serving of soy germ-enriched pasta (80 g) for 4 wk and the other 31 patients (control group) consumed conventional pasta for the same period. The patients were unaware of which pasta they were given. After 4 wk, blood and urine were collected and the above tests repeated. After completion of this 4-wk dietary period, patients in the soy germ pasta group were switched to conventional pasta for a further 4 wk to determine the extent, if any, of rebound effect. The control group continued with conventional pasta for a further 4-wk period. After completion of the study, blood was collected and serum lipids were measured. Compliance to the study was assessed from counting any unused pasta returned after completion and from measurements of isoflavone concentrations in the serum from those patients assigned to the soy germ pasta group.
The study sample size was based on the following anticipations of diet-induced changes during the first 4 wk: mean change of total cholesterol (TC) concentrations was expected to be in the order of 5% of a mean baseline value of 6.98 mmol/L (270 mg/dL) in the enriched pasta group [i.e. a difference of 0.39 mmol/L (15 mg/dL), with a SD of the same magnitude as this difference; no change and the same SD as above were expected in the control group]. Assuming a risk of type I error of 0.05 (2-sided), it was calculated that to attain a power of 0.90 in detecting such a difference between the changes occurring in the 2 groups, we would need to allocate at least 22 subjects in each group.
Anthropometrics. Body weight and height were measured and BMI calculated by standard techniques.
Lipids. Serum TC, triglycerides (TG), and HDL cholesterol (HDL-C) were determined by enzymatic colorimetric method (Dimension Autoanalyser; DADE). LDL cholesterol (LDL-C) was calculated by the Friedewald equation (31). Lipids were expressed as concentration (millimoles per liter) of TC, LDL-C, HDL-C, and TG.
Other markers.
Serum hsCRP levels were measured using the Latex-Enhanced CRP assay (Dade Behring High Sensitivity CRP Assay) on a nephelometer. Urinary 8-isoprostane (8-epi-prostaglandin F2
) analysis was measured by previously published methods (32). Serum total and individual isoflavones were measured by GC-MS as described previously (33).
Endothelial function. Brachial arterial blood pressure was measured with a mercury sphygmomanometer after patients sat rested for 10 min or longer. The mean value of 3 measurements was calculated. Endothelial function was measured from brachial artery flow-mediated vasodilatation with B-mode ultrasound imaging of the brachial artery and by assessing the increase in artery diameter during reactive hyperemia (34).
Isoflavone analysis of soy germ pasta. The isoflavone composition of both the soy germ used in the manufacture of the isoflavone-enriched pasta and the final pasta product was determined by reverse-phase HPLC-MS as described previously (20).
Data analysis. Continuous variables were summarized as mean values ± SEM. The significance of baseline comparisons between group means was assessed using the Student's t test. Changes that occurred during the study were calculated by subtracting the value determined after 4 or 8 wk from the baseline value. The significance of changes that occurred within each study group was analyzed by a paired t test of the hypothesis that the difference had a mean of 0. To compare between groups the changes that occurred in a continuous variable, we used multiple regression analysis to adjust for patients' baseline values and thus control for potential imbalance of that variable between the groups at baseline (35). We built regression equations by including a follow-up value as the dependent variable, the baseline value for that variable, and a binary variable coding for group. The regression coefficient for this group variable was used to estimate the adjusted difference between the mean changes of each group. The same statistical methods were used in the analysis of the 29 patients who consumed isoflavone-enriched pasta and were compared according to equol producer status, the binary variable coding for study group being equol producer status instead of type of pasta. Logarithmic (serum HDL-C and LDL-C, TG, hsCRP, and urinary isoprostane), reciprocal (TC), or square root (flow-mediated dilation) transformations were applied to some variables to achieve homogeneity of variances or approximate normal distributions. Statistical analyses were 2-sided and performed using Stata Statistical Software (version 8.0, Stata). Differences with a P-value < 0.05 were considered significant.
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Results |
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BMI. The BMI and waist circumference did not change during the study in the 2 patient groups. BMI was 26.6 ± 0.8 kg/m2 in both groups at baseline. After 4 and 8 wk, BMI was 26.3 ± 0.8 kg/m2 and 26.5 ± 0.8 kg/m2 in the enriched pasta group and 26.3 ± 0.8 kg/m2 and 26.5 ± 0.8 kg/m2 in the control group. At both time points, the 2 groups did not differ in the changes from baseline after adjustment for patients' baseline values (P = 0.72 and P = 0.97, respectively).
Serum isoflavones. In 29 of the 31 patients consuming soy germ pasta, compliance was confirmed by the marked increase in serum isoflavone concentrations in 29 of the 31 patients; there was insufficient sera to perform the isoflavone measurement in 2 patients. A diet incorporating soy germ pasta with 33 mg isoflavones boosted fasting serum total isoflavone levels from 42 ± 4 nmol/L (10.5 ± 0.9 µg/L) at baseline in all patients to a steady-state concentration of 222 ± 21 nmol/L (55.5 ± 5.2 µg/L) (P < 0.001) (Fig. 3A).
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Serum cholesterol and lipids. At baseline evaluation, patients in the enriched pasta group tended to have lower levels of TC (P = 0.129), HDL-C (P = 0.086), and TG (P = 0.132) than those in the control group (Table 1). After 4 wk, serum levels of both TC and LDL-C decreased from baseline levels (P < 0.001) in the isoflavone-enriched pasta group, whereas levels did not change in the control group. TC decreased by 0.47 ± 0.13 mmol/L (18 ± 5 mg/dL) more in the isoflavone-enriched pasta group than in the control group (P = 0.001) after adjustment for patients' baseline values. Similarly, LDL-C decreased by 0.36 ± 0.1 mmol/L (14 ± 4 mg/dL) more in the isoflavone-enriched pasta group than in the control group (P = 0.002).
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After 4 wk, serum HDL-C concentrations did not change significantly from baseline levels in either the isoflavone-enriched pasta group or the control group. Serum TG concentrations during consumption of either type of pasta did not change (P = 0.301). Overall, isoflavone-enriched pasta lowered serum TC by 7.3% (P = 0.001) and LDL-C by 8.6% (P = 0.002) beyond the effect of a 2-wk run-in on a National Cholesterol Education Program Step II diet (Fig. 4). This improvement in serum lipids was lost when this group reverted to consuming conventional pasta.
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concentrations, a surrogate marker for antioxidant status, decreased from 58 ± 6 ng/L at baseline to 39 ± 4 ng/L after 4 wk in patients consuming soy germ pasta (P < 0.001). Measurements were not performed on the control group. Endothelial function. At baseline, flow-mediated vasodilation was similar in the 2 study groups (Fig. 6). After 4 wk, flow-mediated vasodilation increased 2 ± 0.8% (P = 0.012) in the isoflavone-enriched pasta group, whereas there was no change in the control group. Consistent with improved endothelial function after consuming soy germ pasta, flow-mediated vasodilation increased by an estimated 2.3 ± 0.8% more in the enriched pasta group than in the control group (P = 0.003) after adjustment for patients' baseline values. Blood pressure did not change in the 2 groups during the study period. After 4 wk and 8 wk, the isoflavone-enriched pasta group and the control group did not differ in the changes from baseline for systolic or diastolic blood pressures after adjustment for patients' baseline values (data not shown).
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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15% of soy flour, which distorts the sensory properties of the pasta, provides relatively low levels of isoflavones, and results in a product that is easily distinguishable from conventional pasta. By contrast, the soy germ pasta used in this study was unique in being indistinguishable from conventional pasta, thereby making a randomized blinded study possible and furthermore, allowing conventional pasta with similar nutritional qualities to be used as an ideal control. Importantly, the use of this novel food afforded a means of evaluating the hypocholesterolemic effect of isoflavones in the virtual absence of soy protein. Compliance was established from measurement of serum isoflavone concentrations, which showed steady-state serum concentration of 222 ± 21 nmol/L, consistent with the known pharmacokinetics of isoflavones (33,38). For those patients given conventional pasta, compliance was judged by the number of packages of pasta returned at the end of the study and was facilitated by the fact that most Italians consume pasta daily. After randomization of the patients, the baseline serum TC, HDL-C, and TG concentrations were slightly higher, although not significantly so, in patients assigned to the control group compared with those in the soy germ pasta group. Any cholesterol-lowering effect of the pasta diets should therefore have been more achievable in the control group. Despite this, patients who consumed conventional pasta experienced no change from baseline in serum TC and LDL-C during the entire 8-wk period (Fig. 4). By contrast, isoflavone-enriched soy germ pasta led to significant reductions from baseline in mean serum TC and LDL-C beyond that observed from the Step II diet alone (Fig. 4) and the extent of change after 4 wk was highly significant when compared with patients assigned to conventional pasta. Sex differences or differences between pre- and postmenopausal women in the observed effects were not examined, because the sample size lacked sufficient statistical power. Our thought that this effect was a function of the presence of isoflavones in the pasta rather than the food matrix was supported by the rebound effect observed when the soy germ pasta group was switched to regular pasta for a further 4 wk. After 8 wk, the serum TC and LDL-C concentrations in this patient group returned to near baseline levels and the relative percent change in serum lipids was not significant, and similar to that of the control group. The effect of both pastas on HDL-C and TG was minimal, due perhaps to the fact that these lipids were not abnormal and also because of the large inter-individual variabilities. This may be expected based on meta-analyses of soy and cholesterol showing HDL-C is largely unaffected by soy protein, or at best marginally increased, whereas TG are typically lowered 7–10% in hypertriglyceridemia (3,10,11).
One explanation for the profound effects of this novel food on serum lipids may be related to an interaction between the food matrix and the isoflavones. Soy germ contains primarily the glucoside conjugates of daidzein and glycitein, whereas the proportion of aglycons is typically <3% (23). However, due to the natural occurrence of ß-glucosidases in semolina wheat (39), hydrolysis of the isoflavone glucosides occurred, leading to a pasta product that contained primarily aglycon forms of isoflavones (Figs. 1 and 2). In this regard, the food used in our study differs from all previous dietary intervention studies of soy and cholesterol. In the original meta-analysis on soy and cholesterol (3), the majority of the studies used products containing almost exclusively isoflavone glucosides (20) and none included a fermented soy food or a food in which the aglycon content was high. Furthermore, isoflavone supplement studies that have shown variable effects on serum lipids (28,40–43) also deliver primarily the glucoside conjugates. Dietary analysis of soy foods consumed by Japanese adults revealed that fermented soy foods comprise a relatively high proportion of the total soy protein source, whereas ISP is rarely consumed. Based on typical dietary intakes of isoflavones by Asians, which range from 20 to 50 mg/d (24), and from the known composition of isoflavones in different soy foods, we estimate that 20–30 mg/d of isoflavone aglycons are consumed by Japanese adults from fermented soy foods, similar to the daily intake of isoflavone aglycons from the isoflavone-enriched pasta.
The probable advantage of delivering isoflavones in the aglycon form is that they are more rapidly absorbed than the isoflavone glucosides (33,44,45) that require hydrolysis for bioavailability (46). Furthermore, the predominance of daidzein in the soy germ pasta, coupled with a possible prebiotic effect of a hard wheat food matrix, could facilitate the intestinal biotransformation of daidzein to equol (47,48), suggested to be a key factor in the efficacy of soy (36). In this regard, 69% of the hypercholesterolemic patients consuming soy germ pasta were equol producers. This is much higher than the expected 20–30% of equol producers typically observed in Western populations consuming a mixed diet (36,37) but is comparable to the frequency of equol producers in Japanese adults (45,49,50). When the lipid data were analyzed according to equol producer status, the equol producers showed significant reductions from baseline in serum TC and LDL-C compared with those who did not produce equol (Fig. 7). This finding lends support to the theory that equol may be a key metabolite in the clinical efficacy of soy (36).
Independent of cholesterol, cardiovascular risk is also associated with other surrogate markers and in this respect we examined the effect of the soy germ pasta on a number of key elements of cardiovascular risk. Atherosclerosis leading to cardiovascular disease is now well recognized as a chronic inflammatory process (51). CRP is a nonspecific marker of inflammation that is often elevated in patients with atherosclerosis and cardiovascular disease (52,53). Several clinical studies have measured a number of inflammatory markers, including serum CRP levels in response to dietary intervention with soy protein products containing isoflavones (26,54–58). The findings have been inconsistent, although the consensus is that unlike estrogen therapy, which increases CRP levels (59,60), the presence of isoflavones either reduces or has no effect on CRP. In our study, serum hsCRP concentrations at baseline in both groups of patients were close to the upper limit of the normal range and did not differ between the groups. Soy germ pasta reduced serum hsCRP concentration by 42% after 4 wk (P < 0.001) and this improvement was abolished when these patients switched to conventional pasta for a further 4 wk. By contrast, serum hsCRP did not change in patients assigned to conventional pasta. The difference in hsCRP levels between the soy germ pasta and conventional pasta groups was highly significant. Consistent with earlier studies, this observation confirms the opposing action of isoflavones compared with estrogens. Consistent with the findings for cholesterol, the reduction in serum hsCRP was greater (P = 0.001) in equol producers than equol nonproducers.
Isoflavones have antioxidant properties (61), with equol reportedly possessing the highest in vitro antioxidant activity (62,63). Several clinical studies (25,26,64,65), although not all (66–68), in which soy isoflavones have been used reported significant reductions in ex vivo lipid peroxidation. We did not determine the extent of lipid peroxidation but did measure urinary isoprostanes as a surrogate marker of antioxidant status in the patients assigned to the soy germ pasta group and found significantly decreased urinary isoprostanes after 4 wk. However, because we did not measurement urinary isoprostanes in patients consuming conventional pasta, it is not possible to know if this decrease was influenced by the presence of isoflavones in the pasta or would have occurred independent of isoflavones.
Numerous lines of evidence indicate that isoflavones exert effects in the vasculature. Prompted by the early observation that a soy diet fed to monkeys improved vascular reactivity, Clarkson et al. (70) showed that acute administration of genistein has potent vasodilatory effects (69) similar to estrogen. The potency of genistein (aglycon) was subsequently confirmed in human studies where it produced an NO-dependent vasodilatation in men and women, comparable to that observed with agents such as acetylcholine. Oral administration of 54 mg/d pure genistein for 1 y led to an improvement in brachial artery flow-mediated dilatation in healthy postmenopausal women with a mean improvement of 5.5% (71). Dietary intervention studies have yielded mixed results regarding the effects of soy isoflavones on endothelial function (30,43,58,66,72–75). However, the inconsistency among the findings may be because these studies used different forms of isoflavones, mostly isoflavone glycosides; some studies used soy protein, whereas some were without soy protein and provided either as supplements or as components of the diet. Other than the 1 study of pure genistein (71), no previous study to our knowledge has examined the potential of isoflavones delivered as aglycons. In our study, endothelial function was measured by the change in brachial artery flow-mediated vasodilatation (34), considered a gold-standard measure for arterial reactivity. Isoflavone-enriched pasta increased brachial arterial flow-mediated dilatation by 2.3 ± 0.8% (P = 0.003) over just a 4-wk period. By contrast, no change was observed with conventional pasta. The mechanism by which isoflavones improve endothelial function has been postulated to be through a number of possible channels. Genistein, the isoflavone most studied, may act through gene expression to alter endothelial-dependent vasodilatation (29,71), or its effects could be through endothelial-independent actions such as calcium channel antagonism, which has been shown in rabbit aorta (76). It appears not to act through estrogen receptor , but whether estrogen receptor ß, which is found in abundance in the vasculature (77), plays a role is uncertain. The soy germ pasta delivered mainly daidzein, rather than genistein, resulting in a high proportion of equol producers. Interestingly, equol was recently shown to increase endothelial NO synthase gene expression (78), activate endothelial NO synthase, and increase NO production in human aorta and umbilical vein endothelial cells (79). We speculate that the high proportion of equol producers in the soy germ pasta group may explain the vasodilatory effects of the isoflavone-enriched pasta.
Despite the improved endothelial function from consuming soy germ pasta, we did not observe an effect on blood pressure. Several studies have alluded to small improvements in blood pressure with isoflavone intake (80–82) and epidemiological data of Chinese women show an inverse relationship between long-term soy food intake and blood pressure, which is strongest in older subjects (16). It is possible that the duration of dietary intervention with soy germ pasta may have been too short to discern beneficial effects on blood pressure.
Body weight, BMI, and waist circumference were unaffected by daily consumption of pasta with or without isoflavones for 8 consecutive weeks. There was a slight reduction in BMI in both groups, but this was not significant, an observation of relevance given that concerns over weight gain has been one of the main reasons for a marked reduction in commercial sales of pasta in recent years.
In conclusion, in this study of newly diagnosed hypercholesterolemic adults, we have shown that an isoflavone-enriched soy germ pasta delivering levels of isoflavones consistent with those consumed in Asian countries (24) but in the virtual absence of soy protein has important beneficial effects beyond that of a Step II diet on serum TC and LDL-C, serum hsCRP, urinary 8-isoprostanes, and brachial artery flow-mediated vasodilatation. Such effects are important in reducing the long-term risk of cardiovascular disease. These changes rebounded to baseline values when patients reverted to pasta that did not contain isoflavones and no lipid-lowering effects were observed in patients consuming conventional pasta lacking isoflavones. The improvement in serum lipids and other cardiovascular risk markers was greatest in patients who were equol producers, suggesting a possible role for equol in the mechanism of action. It is proposed that the effectiveness of this pasta is accounted for by the unique chemical composition of the isoflavones, which by interaction with ß-glucosidases naturally present in Durum wheat results in a predominance of isoflavone aglycons and a food product more characteristic of an Asian fermented soy food. We speculate the predominance of aglycon forms of isoflavones and the possible prebiotic effect of semolina wheat explains the high proportion of equol producers and the overall clinical effectiveness of this novel food.
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FOOTNOTES |
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6 Abbreviations used: HDL-C, HDL cholesterol; HsCRP, high sensitivity C-reactive protein; ISP, isolated soy protein; LDL-C, LDL cholesterol; TC, total cholesterol; TG, triglyceride.
Manuscript received 17 January 2007. Initial review completed 20 February 2007. Revision accepted 16 July 2007.
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LITERATURE CITED |
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