Consumption of Soymilk Reduces Lipid Peroxidation But May Lower Micronutrient Status in Apparently Healthy Individuals

Abstract

Consumption of soy products is speculated to reduce the risk and progression of some disease conditions. The underlying mechanisms mediating this effect are uncertain, but the lowering of oxidative stress has been suggested. This study was aimed at investigating the effect of soymilk on antioxidant status and lipid peroxidation in apparently healthy individuals. Five hundred milliliters of soymilk was taken daily by each of 39 apparently healthy individuals for a period of 28 days. Two sets of blood samples (baseline and after 28 days of soymilk intake) were collected and assayed for total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST), manganese (Mn) and zinc (Zn) levels, using standard methods. Consumption of soymilk significantly increased serum TAC and reduced serum MDA when compared with baseline values (P < .001, P < .001 respectively). The decrease in MDA concentration was significantly contributed by the women (P < .001). Mean serum Mn was also significantly reduced (P = .03) when compared with baseline values. There were no changes in serum Zn concentration and the activity of SOD enzyme. The serum GST activity was significantly increased in men (P = .02) and significantly reduced in women (P < .001) in comparison with their corresponding baseline values. Daily consumption of soymilk enhanced antioxidant status and this led to reduced lipid peroxidation. It also resulted in a significant reduction of Mn. The dietary use of soymilk as an adjuvant to supplement meals seems beneficial health wise. However, soymilk should be taken with caution as it could result in micronutrient deficiency.

Introduction

Soymilk is one of the soy products that has all the useful components of soy. It contains high-quality protein, dietary fiber, small quantities of saturated fatty acids, and no cholesterol and lactose.¹ Soy protein contains all amino acids essential for human nutrition, which makes soy products almost equivalent to animal sources in protein quality.² Soymilk also contains a significant amount of isoflavones that has antioxidant activity and phytic acids recognized as a chelating agent.³ There are divergent views on the role of soymilk consumption in health. Whereas some researchers have shown that it can have a profound effect on reduction of waist circumference,⁴ others^5
–7 reported an association between soy food consumption and reduced risk of prostate cancer in men and even in breast cancer recurrence in women. Handayami et al. ⁶ posited that the effect on the prostate is as a result of soy isoflavone, which modulates cell cycle progression and the expression of genes involved in cell cycle, metastasis, and angiogenesis.

One of the potential mechanisms mediating these beneficial effects might be attributed to soy's ability to reduce oxidative stress⁸ and consequently preventing and ameliorating diseases associated with it.

Oxidative stress is an imbalance between the rate of free radical production and mop up by the antioxidant defense system. It may arise as a result of lipid peroxidation leading to cellular injury and cellular dysfunction and has been implicated in the etiology of many disorders.⁹ The use of soy-based product in boosting total antioxidant capacity (TAC) and curbing the detrimental effects of oxidative stress is plausible. Studies have shown that soymilk might lower atherogenic index (low-density lipoprotein cholesterol) lipid fraction of cholesterol,¹⁰ blood pressure in men and women,¹¹ and consequently the risk of cardiovascular disease.¹² Although the potential mechanism mediating these effects might be reduced oxidative stress¹³ by the antioxidant activity of soy,¹⁴ the evidence for this is not well established, and existing data are inconsistent in supporting most of the suggested health benefits of soy. Zinc (Zn) is widely distributed in plant and animal tissues and occurs in all living cells. Manganese (Mn) is highly abundant in the mitochondrion. These micronutrients are essential cofactors in many enzymes, including superoxide dismutase (SOD).¹⁵

Soybeans are rich sources of minerals.¹⁶ Their use as adjuvant to supplement daily meals could have a boosting effect on trace minerals. However, phytic acid is a chelating agent. It binds and forms insoluble phytate–mineral complexes that are not readily absorbed by the human gastrointestinal tract with trace elements such as iron (Fe), Mn, copper, and heavy metals such as lead and cadmium.¹⁷

Some studies have reported beneficial effect of soy intake,^10,18,19 and in a few studies, however, no significant beneficial effect was found.^20,21 Numerous trials on the effect of soy intake on antioxidants were mostly on postmenopausal women and in obesity,²² in animal models^23,24 and in in vitro cultures²⁵ with paucity of information on apparently healthy individuals.^10,14

This study was, therefore, aimed at determining the serum levels of malondialdehyde (MDA), TAC, micronutrient trace metals such as Zn and Mn, and activities of SOD and glutathione S-transferase (GST) enzymes in some healthy Nigerian adults before and after 28 days of soymilk consumption and the possible role of gender in all of these.

Materials and Methods

Subject selection

This study was carried out in Ekiadolor, Edo State, Nigeria. A total of 39 apparently healthy male and female volunteers between the ages of 18 and 35 years started and completed the study. They comprised 18 men and 21 women who drank soymilk.

Participants were excluded from the study if found to be:

(1) outside the age range of 18 and 35 years.

(2) on medication including multivitamins.

(3) regular alcohol consumers.

(4) smokers.

(5) women who were pregnant, lactating, or using oral pills.

(6) on any special diet.

The use of questionnaire was employed in the collection of data relevant to the study from participants after their consents were duly sought and obtained through the consent form.

This study protocol was approved by Nnamdi Azikiwe University Teaching Hospital Ethics Committee.

Soymilk was prepared from soybeans using a cold extraction method as described by Oloye²⁶ but with slight modifications. Before the commencement of this research, a pilot study on soymilk preparation was carried out, which resulted in the modification of Oloye's cold extraction method. Five hundred milliliters of soymilk was given to each participant (test subjects). The soymilk was given to them daily for a period of 28 days. The participants were closely monitored for compliance and they were advised not to vary their diet. Before and after soymilk intake, the body weight of each participant was measured using a clinical weighing scale in kilograms, whereas the height was measured in meters using a stadiometer.

Blood sample collection

Two sets of fasting venous blood samples (baseline and 28 days) were collected from the subjects. At the commencement of the study, 5 mL of blood was collected from each participant (baseline sample). This served as control. After 4 weeks, another 5 mL of blood was also collected from each participant (28 days samples). The blood samples were allowed to clot at room temperature, retracted, and centrifuged at 805 g for 10 min and the serum was separated and stored at −20°C until used for analysis of biochemical parameters.

Analytical methods

Total antioxidant activity was determined by a spectrophotometric method.²⁷ The principle is based on the reduction of ferric iron (Fe³⁺) by the antioxidant (ferric reducing ability) to ferrous tripyridyltriazine at a low pH to form a violet color, read spectrophometrically at the wavelength of 593 nm.

MDA, a lipid peroxidation product, was also determined spectrophotometrically using a method described by Gutteridge and Wilkins.²⁸ This method is based on the pink colored product formed, having an absorption maximum at 540 nm wavelength when MDA is heated with 2-thiobarbituric acid under an alkaline condition.

SOD and GST enzyme activities were determined using the methods of Misra and Fredovich²⁹ and Habig et al.,³⁰ respectively. Superoxide anions generated by xanthine oxidase reaction is known to cause the oxidation of adrenaline to adrenochrome, the yield of adrenochrome produced by superoxide anion introduced increases with increasing pH. The ability of SOD to inhibit the autoxidation of adrenaline at pH 10.2 forms the basis for these reactions. GST in contrast catalyzes the conjugation of l-glutathione to 1-chloro-3-4 dinitrobenzene through the thiol group of the glutathione. The formation of the glutathione conjugate is proportional to the enzyme activity, which is read at 340 nm. Zn and Mn concentrations were both estimated by atomic absorption spectrophotometry (AAS).³¹ In AAS, the amount of light absorbed by the atomized element when the sample is aspirated into the flame is proportional to the concentration of the element in the sample. Since metals have their characteristic absorption wavelength, a source lamp composed of the element is used, making the method relatively free from spectral or radiation interference.

Statistical analysis

Data obtained were subjected to statistical analysis. The mean, standard deviation (SD), and paired Student's t-test were computed using statistical package for social science version 20 (SPSS, IL, USA). Results are reported as mean ± SD and regarded as significant if P < .05.

Results

After 28 days of soymilk intake (Table 1), the mean serum concentration of TAC was significantly increased (P < .001) compared with the baseline TAC value, whereas the mean serum concentrations of MDA and Mn, respectively, were significantly reduced (P < .001 and P = .03). There were no significant changes in Zn, SOD, GST, and body mass index (BMI).

Table 1.

Body Mass Index, Serum Total Antioxidant Capacity, Malondialdehyde, Manganese, Zinc, Superoxide Dismutase, and Glutathione S-Transferase at Baseline and After 28 Days of Soymilk Intake (Mean ± Standard Deviation)

Parameters	Baseline (n = 39)	28 days (n = 39)	t	P
BMI (kg/m²)	22.58 ± 6.70	21.98 ± 4.02	0.736	.47
TAC (μmol/L)	688.85 ± 123.10	819.74 ± 118.70	8.337	<.001^*
MDA (nmol/mL)	3.43 ± 0.75	2.75 ± 0.88	3.909	<.001^*
Mn (nmol/L)	15.83 ± 7.82	13.10 ± 6.73	2.279	.03^*
Zn (μmol/L)	18.11 ± 2.59	17.24 ± 2.91	0.279	.15
SOD (U/mL)	19.51 ± 6.20	20.72 ± 5.90	0.467	.38
GST (U/mL)	20.94 ± 8.51	18.27 ± 8.71	1.229	.28

Results significant at P < .05, asterisk (^*) indicates significant results.

BMI, body mass index; GST, glutathione S-transferase; MDA, malondialdehyde; Mn, manganese; SOD, superoxide dismutase; TAC, total antioxidant capacity; Zn, zinc.

When the subjects were categorized according to gender, women who took soymilk (Table 2) had a significant increase (P < .001) in their mean serum concentration of TAC. However, the serum concentrations of MDA and GST were significantly decreased (P < .001 and P < .001, respectively) when compared with their corresponding baseline values. No significant changes were observed in their BMI and serum concentrations of Mn, Zn, and SOD before and after soymilk consumption.

Table 2.

Body Mass Index, Serum Total Antioxidant Capacity, Malondialdehyde, Zinc, Manganese, Superoxide Dismutase, and Glutathione S-Transferase at Baseline and After 28 Days of Soymilk Intake in Women (Mean ± Standard Deviation)

Parameters	Baseline (n = 21)	28 days (n = 21)	t	P
BMI (kg/m²)	21.89 ± 4.50	21.57 ± 4.50	0.992	.33
TAC (μmol/L)	628.86 ± 118.80	770.81 ± 112.70	7.042	<.001^*
MDA (nmol/mL)	3.39 ± 0.74	2.26 ± 0.68	4.753	<.001^*
Mn (nmol/L)	14.01 ± 7.64	12.19 ± 6.37	1.375	.18
Zn (μmol/L)	18.02 ± 2.80	17.50 ± 2.71	0.790	.44
SOD (U/mL)	20.70 ± 6.21	20.14 ± 5.70	0.266	.80
GST (U/mL)	27.53 ± 4.42	16.26 ± 6.91	7.444	<.001^*

means significant result.

Consumption soymilk by male subjects, in contrast, revealed significantly increased serum concentrations of TAC and GST (P < .001 and P = .02 in each case), but there were no significant changes in the mean BMI, MDA, Mn, Zn, and SOD on comparison with baseline values (Table 3)

Table 3.

Parameters	Baseline (n = 18)	28 days (n = 18)	t	P
BMI (kg/m²)	23.39 ± 8.92	22.45 ± 3.32	0.533	.60
TAC (μmol/L)	758.83 ± 84.40	876.83 ± 100.72	4.747	<.001^*
MDA (nmol/L)	3.48 ± 0.79	3.33 ± 0.73	0.788	.44
Mn (nmol/L)	18.02 ± 7.70	14.20 ± 7.10	1.815	.09
Zn (μmol/L)	18.22 ± 2.39	17.02 ± 3.20	1.233	.23
SOD (U/mL)	18.13 ± 6.02	21.41 ± 6.10	2.087	.05
GST (U/mL)	13.24 ± 5.01	20.63 ± 3.31	2.528	.02^*

means significant results.

Discussion

There is growing interest in the intake of soy-based products due to its claimed health benefits, which includes risk reduction of some diseases (such as cancer, atherosclerosis, and diabetes mellitus).

Results obtained from this study showed that dietary consumption of soymilk significantly increased TAC in both men and women. This is similar to the findings of Peter et al. ¹⁴ These authors reported that TAC was significantly increased by soy intake in both genders, with the greatest increase was observed in men. Soy products are rich in isoflavones and phytic acids that are known to have antioxidant activities.

The serum concentration of the lipid peroxidation biomarker, MDA, was significantly decreased in women, whereas there was no significant change in men. In a similar study by Peter et al.,¹⁴ increased soy intake had no significant effects on thiobarbituric acid reacting substances (TBARSs) levels (a lipid peroxidation marker) in women but significantly increased TBARSs levels in men. However, these authors also reported a significant decrease in protein marker of oxidative stress in women, whereas levels in men had no significant difference. An experimental work carried out by Ibrahim and co-workers³² in male mice models suggests that isoflavone, a key component of soy, protects against peroxidative damage to membrane lipids in vivo by possibly promoting the activities of antioxidant enzymes, SOD, and catalase. However, there was no significant change in the activity of SOD enzyme in this study. A similar study by Chang et al. ³³ in diabetic subjects did not also record any significant change in the enzyme activity. Lee et al.,²³ in a separate study, revealed a significant decrease in SOD activity on diabetes-induced mice. SOD catalyzes the spontaneous dismutation and breakdown of superoxide radical to either oxygen or hydrogen peroxide and they are among the first line of defense in the detoxification of products of oxidative stress.³⁴

In this study, gender differences were observed with the intake of soymilk on mean GST concentration. The levels of GST were significantly increased in men, whereas in women, GST levels were significantly reduced. Activities of GST enzymes are hormonally regulated, with gender-specific expression of the enzyme activity seen in humans. GST levels are generally higher in women than in men,³⁵ which is evident in the findings of this study; the mean GST level in women before soymilk supplementation was higher than the mean GST level in men. Hayes and Pulford³⁶ are of the opinion that GST activities can be induced or increased by phenolic antioxidants. Soymilk contains phenolic antioxidants, which could have been responsible for the induction of GST in men in this study. However, the reason for the reduction of GST activities in women after soymilk supplementation in this study is unclear. GST is a phase II enzyme responsible for the detoxification of toxin and carcinogens. Thus increasing the activity of this enzyme seems beneficial in cancer prevention.³⁴ Studies have shown that soy genistein protects human mammary epithelial cells from carcinogen and genotoxic substance by inducing gluthathione/GST system; however, this was demonstrated in in vitro cultures²⁵ and animal models.^37,38

The serum concentration of Mn was significantly reduced, whereas there was no significant change in serum Zn concentration. The significant decrease in Mn concentration might be the result of the binding effect of soy phytic acids.¹⁷ The major concern about the presence of phytate in the diet is its negative effect on mineral uptake.^39,40 Phytate forms an insoluble phytate–mineral complex with trace metals that are not readily absorbed by the human gastrointestinal tract and, therefore, results in the reduction of the bioavailability of minerals,⁴¹ consequently leading to deficiencies especially in diets already low in minerals. It was suggested that phytic acid exerts these overwhelming health benefits in the gastrointestinal tract and on other target tissues through its chelating ability.¹⁷

Also in this study, consumption of soymilk had no significant effect on BMI. This corroborates the findings of a similar study by St-Onge et al. ⁴² that soy protein-rich food supplementation does not enhance weight loss and, therefore, could not give credence to the emerging notion that soy protein-rich foods might be considered potential foods for weight management, in the quantities consumed in their study. In contrast, Anderson et al. ⁴³ demonstrated that soy protein was associated with weight loss in obese women. The insignificant decrease in BMI in this study might be due to the quantities of soy protein consumed and/or the duration of the study.

In conclusion, this study revealed that soymilk intake in some Nigerians enhanced total antioxidant status, which led to reduced lipid peroxidation. The dietary use of soymilk to supplement meals seems beneficial to health. The reduction of serum concentration of Mn could be caused by binding of soy phytate to the minerals, thereby forming insoluble phytate–mineral complex. This could be of public health importance as this would result in mineral deficiency in societies that rely solely on their diet for trace minerals. However, the formation of insoluble complexes has been attributed as one of the potential mechanisms through which soy achieves its health benefits. Also, consumption of soymilk enhanced GST levels in men, whereas there was a depletion of GST levels in women. The depletion of GST levels observed in women after soymilk consumption cannot be explained. There was no change in BMI and SOD levels. It is, therefore, recommended that in light of the many health benefits of soy products, consumption of soymilk is recommended but should be taken with caution. Further studies are required to fully elucidate the underlying mechanisms of GST enzyme reduction in women after soymilk consumption.

Footnotes

Acknowledgments

We wish to sincerely acknowledge the secretarial assistance rendered by Mrs. Nwanneka and the efforts of the volunteers of the soymilk study who patiently adhered to all the instructions given to them.

Author Disclosure Statement

No competing financial interests exist.

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