Short title - page header:oxidative Hemolysis Induced by Various Vitamins

	ABSTRACT
	INTRODUCTION
	MATERIALAS AND METHODS
	RESULTS AND DISCUSSION
	REFERENCES

	ABSTRACT

    Hemolytic effect of some water-soluble vitamins (niacin B5, pyridoxine B6, thiamine B1 and ascorbic and acid C) on erythrocytes was studied spectrophotometrically at relatively high concentration. The oxidation mechanism of hemoglobin was the same for the used vitamins. Vitamin C was the strongest hemolytic agent in comparison with the other vitamins, while vitamin B1 is the weakest one. The results were confirmed by studying the variation in conductivity of erythrocytes with temperature in the range 20-40 °C for the used vitamins at a concentration of 2 mM and after two hours from adding each vitamin to the erythrocytes suspension. The conductivity measurements show that the conductivity for the used vitamins is lower than that for control (without adding vitamin) due to hemoglobin oxidation , also may be due to the electrical reorganization of the erythrocyte membrane after the interaction of the used vitamin with it. The obtained results insure the oxidizing effect of the used vitamins on hemoglobin and consequently their hemolytic effect on erythrocytes.

KEY WORDS: hemolysis; erythrocytes; niacin; pyridoxine; thiamine; ascorbic acid; conductivity

	INTRODUCTION

Oxidative damage to biological systems is the basis of a number of physiological and pathological phenomena (1). For that it has been presented in great number of studies. Erythrocytes have often been used as a convenient model for these studies. The oxidation process in erythrocytes affects the over all cell structures, hemoglobin and membrane. Several hypotheses have been proposed to explain the mechanism of erythrocytes hemolysis following oxidative stress in vivo and vitro (2). Hemoglobin appears to be the main site of damage when various oxidative drugs are used (3). Under other oxidative conditions, the membrane appears to be the target of injury leading to hemolysis (4).

Oxidative denaturation of hemoglobin is a process in which oxidative changes in the molecule cause its disruption. This process takes place when the concentration of oxidants in a red cell is increased. The intracellular aggregates of denatured hemoglobin produced when oxidative denaturation occurs in the red cell are called Heinz bodies (5). The formation of Heinz bodies is directly associated with the induction of hemolytic anemia. Many chemical compounds cause the oxidation of hemoglobin, yet underlying molecular mechanisms remain unknown. Many vitamins are commonly believed to be nontoxic although toxicity can appear when large amounts of these vitamins are consumed (6). Several authors have developed different methods to measure the antioxidant activity of many compounds: pulse radiolysis (7), inhibition of the oxidation of chemical and biological targets (8, 9). Among them, the hemolysis assays have already been used for a long time in measuring free radical damages. Previous studies (10-12) have demonstrated the hemolytic effect of ascorbic acid (Vitamin C) on erythrocytes and they attributed it to an oxidative damage (oxidative hemolysis).

The study of the passive electrical properties of the cell membrane is an area of active interest, yielding a lot of information on the structure and physiology of cells and different cell compartments (13). The aim of the present work is to study the hemolytic effect of some water-soluble vitamins (niacin B₅, pyridoxine B₆, thiamine B₁ and ascorbic acid C) on erythrocytes when added with relatively high concentration.

	MATERIALS AND METHODS

Preparation of erythrocytes suspension

Human blood from healthy donors, ant coagulated with heparin, was stored at 4°C and used for experiment in the same day. Erythrocytes were isolated by centrifugation (10 min, 1500 revolution/min), plasma and buffy coat removed and the cells washed three times with isotonic saline (0.15 M NaCl) at room temperature. Washed erythrocytes were suspended in saline to reach a concentration of about 3 × 10⁵ cells/ml.

Registration of hemolysis

Hemolysis or kinetics of hemoglobin breakdown in erythrocytes exposed to the used vitamins with different concentrations [B₅ (0.25, 0.5, 1.0, and 2.0 mM), B₆ (1.0, 2.0, 4.0, and 8.0 mM), B₁ (0.25, 0.5, 1.0 and 2.0 mM) and C (0.5, 0.8, 2.0 and 4.0 mM)] were registered spectrophotometrically (Jenway spectrophotometer model 6300) at 577 nm (spin state band of iron- hem), where the decrease in the intensity of the peak at 577 nm represents the degree of hemoglobin breakdown or degree of hemolysis. The cuvette of the spectrophotometer was filled by 5.8 ml of the suspension and 0.2ml of the used vitamin of certain concentration, then the value of the absorbance at 577nm was recorded after shaking gently and carefully before each measure.

In this part, the measured values of absorbance are a representative experiment and error bars cannot be calculated on raw absorbance values due to day to day variations in total spectrometric readings.

Conductivity measurements

The used apparatus was a conductance bridge (Griffen and George Ltd. London) with glass tube filled by the suspension, containing two platinum disc electrodes separated by a fixed distance. For recording the variations in conductivity with temperature, 0.2 ml of the used concentration of vitamin was added to 5.8 ml of erythrocytes suspension and the mixture was left 2 hours till complete hemolysis is fulfilled , then the variation in conductivity with temperature is recorded, by warming the glass tube holding the mixture in a water bath.

RESULTS AND DISCUSSION

The kinetics of hemoglobin breakdown in erythrocytes exposed to different concentrations of ascorbic acid (0.5, 0.8, 2.0 and 4.0 mM) is shown in Fig. 1, in which the absorbance decreases with time for each concentration, this means that the degree of hemolysis (degree of hemoglobin breakdown) increases with time. It was shown also that the degree of hemolysis depends on the concentration of ascorbic acid, in which it increases by increasing concentration. From this figure there is a time interval during which the variations in absorbance with time is nearly linear, the slope of this interval gives the hemolysis rate (H.R); the rate at which the number of cells in the suspension decreases. Fig. 2 shows the variation of hemolysis rate (H.R) with different concentrations of vitamin C. From this figure it is clear that the hemolysis rate increases by increasing the concentration. The obtained results indicate that the number of erythrocytes decreases rapidly at higher concentrations (hemolysis). The hemolysis was believed to be due to the interaction of ascorbic acid with erythrocytes causing lipid peroxidation of membrane and oxidation of hemoglobin (oxidation of Fe⁺² to Fe⁺³) (10-12). This process leads to hemolysis of erythrocytes if the concentration of ascorbic acid is relatively high (low concentrations of ascorbic acid act as antioxidants).

The same effect can be observed for pyridoxine B₆ of concentrations: 1.0, 2.0, 4.0 and 8.0 mM as shown in Fig. 3, for niacin B₅ of concentrations: 0.25, 0.5, 1.0 and 2.0 mM as shown in Fig. 4 and for thiamine B₁ of concentrations: 0.25, 0.5, 1.0 and 2.0 mM as shown in Fig. 5. Comparing the four figures, it may be concluded that the mechanism of hemoglobin breakdown and consequently hemolysis of erythrocytes is the same for the four used vitamins and as described before for ascorbic acid. Fig. 6 shows the effect of the four vitamins at concentration of 2mM on erythrocytes suspension. From figure it is clear that vitamin C is the strongest hemolytic agent in comparison with the other vitamins, in which the hemolysis is fulfilled at about 20 minutes from adding the used concentration (2.0 mM), while vitamin B₁ is the weakest one.

The variation in conductivity of erythrocytes with temperature in the range 20-40 °C is shown in Fig. 7 for control (without adding vitamin ) and for the four used vitamins at a concentration of 2mM and after two hours from adding vitamin. From figure it is clear that the conductivity for the used vitamins is lower than that for control due to hemoglobin oxidation, also for all curves the conductivity increases by increasing temperature. The figure also shows that the conductivity of vitamin C is the higher one in comparison with other vitamins. Vitamins C and B₆ are known to cross cell membranes easily and to be taken into the red blood cell , oxidizing hemoglobin to methemoglobin (14), so decreases the conductivity of the suspension (12). Also, the change in electrical conductivity due to the addition of vitamin in comparison with control may be due to the electrical re-organization of the erythrocyte membrane after the interaction of such vitamin with it (15).The difference in the values of conductivity- after adding the used vitamins to erythrocytes suspensions-from one vitamin to another maybe due to the difference in conductivity of such vitamins themselves.

The obtained results insure the oxidizing effect of the used vitamins on hemoglobin and consequently their hemolytic effect on erythrocytes.  

DISCUSSIONS

On the basis of the obtained results, adopting recommendations by Hodge and Sterner as per the OECD (22), the LD₅₀ for Maca-GO for oral applications was established as 15 g/kg body weight – a way above the OECD 2g/kg limit. According to obtained results it is reasonable to suppose that Maca-GO doesn’t present health hazard for humans consuming this product at the dose up to 15g/kg (which is the equivalent to approximately 1 kg Maca-GO for 66kg body weight man/women or nearly 1.3kg for 85kg person).

The results obtained in this study confirm previously obtained observations on rats (12) exposed to various levels of Maca-GO (0.75g/kg and 7.5g/kg body weight of rats) where no detectable negative physiological, clinical, history-pathological nor toxic effects existed which could be attributed to the used doses of Maca-GO and administered to rats during either 28 days or 90 days experimental periods.

From reported in the literature (13, 22) long list of physiological and physical benefits of Maca traditionally used for centuries as vegetable with “medicinal properties” by natives of Peru and now gaining popularity as a dietary supplement in the USA and Europe, the followings appear to be of importance for menopausal women: balancing hormonal secretion, stimulation of body metabolism, increase in energy and vitality (32) stress reduction, antidepressant activity, memory improvement, enhancement of sexual drive (7, 21, 33). Since hormones affect entire spectrum of metabolic and psychological responses of women during pre-and postmenopausal stages, in an adopted laboratory animal model, both ovariectomized rats – in their, surgically-induced physiological status resembling post-menopause and non-ovariectomized, sexually experienced rats with active ovaries – as pre-menopausal animals were used. Analyzing biochemical blood indices and results from pharmacodynamic tests, an attempt has been made to identify metabolic axis, along which Maca-GO as a dietary supplement, may exhibit its action on post- and pre-menopausal rats.

Therapeutic properties of Maca roots are linked to alkaloids identified in 1960s by Chacon (1) and later confirmed by other researchers, however in recent years other groups of active constituents were reported such as polyunsaturated acids and their amides (“macaine” and “macamide”), sterols (campesterol, stigmasteroland beta-sitosterol, and aromatic glucosinolates (benzyl and p-methoxybenzyl glucosinolates and their derived isothiocyanates) (9, 13, 14, 15, 16). There is no single “active” component identified which would be agreed to as a “functional” or “active” marker for Maca, therefore in this and follow-up study, Maca in its entirety – as traditionally Maca has been referred to, is being used after an application of a standard pre-gelatinization process and referred to as pre-gelatinized organic Maca – Maca-GO.

Previously published results from preliminary study (11) demonstrated that Maca-GO used for extended period of time (8 months) in postmenopausal women significantly increased progesterone (PRG) and only slightly reduced E2 level. A shorter time of Maca-GO administration (2 months) significantly reduced PRG in relation to placebo level while the E2 was not affected. In this study, Maca-GO significantly reduced both E2 and PRG in “postmenopausal” rats, while there was a significant decrease in E2 at significantly increased PRG in “pre-menopausal” rats. This effect of Maca-Go on rats with active ovaries may be extremely valuable when extrapolating results from the laboratory model to potential responses in perimenopausal women, who are subjected to fluctuations in E2/progesterone ratio resulting in relative increase in E2 level due to progressive decline in secretion of PRG by corpus luteum (21). The observed in this study significant reduction in both E2 and Progesterone after administration of Maca-GO to ovariectomized rats needs to be interpreted with caution since it is reasonable to suppose that such effect in women with inactive ovaries would not be physiologically-beneficial.

On the other hand, observed in this study antidepressive effect of Maca-GO in ovariectomized rats may add to difficulty in interpretation of trends in changes of the two hormones, particularly in view of lack in relationship between antidepressive effect and lowered E2 level, since reduced level of E2 has been shown in clinical study on laboratory animals as responsible for development of the depressive symptoms (34). Also, observed reduction in both ACTH and Cortisol levels which was accompanied by the demonstrated antidepressive effect of Maca-GO on ovariectomized rats is similar to reported by DeMoranville and Jackson (35) and Sapolsky (36), who, in people suffering from illnesses and in clinical models to study depression, observed an increase in Cortisol with progression of depressive state. From this point of view, Maca-GO appears to have positive effect on alleviation of depression with a simultaneous sedative effect as well. This could be ascribed to high daily dose of Maca-GO applied to rats in this study, since doses of 15 and 7.5mg/kg used over 15 days, resulted in a stimulation of motoric function in male rats (37). There maybe a difference in motoric responses to Maca-Go by male and female rats, but this aspect has not been reported yet in literature available to authors’ to-date.

Observed in this study improved long-term cognitive ability in ovariectomized rats receiving Maca-GO is worthy to be emphasized, since changes in mood and disturbance of cognitive functions, which are frequently considered as first signs of ovarian dysfunction in menopausal women, may be alleviated, reduced or delayed by use of Maca-GO prior-to, during transition and at postmenopausal stage. This may be further supported by the fact of very low toxic status of Maca-GO, with its LD₅₀ determined at >15g/kg body weight, hence, safe for oral use and being well tolerated as dietary supplement (equivalent to approximately 1kg oral intake of Maca-GO per day by women of 66kg BW). Supporting this assumption is the dose of 500mg/kg BW per day used in this four week long study (1/20 of LD₅₀), which has not affected morphologic blood parameters, remaining within the ranges considered as normal for rats (30, 38). Long-term administration of Maca-Go to ovariectomized rats resulted in slight reduction of blood cholesterol and triglycerides which may indicate its positive effect on metabolism of lipids, also observed by other authors (39).

Results obtained in this model laboratory study on Maca-GO are encouraging enough to justify setting up further laboratory and/or clinical study on postmenopausal women to validate positive effects of this preparation as a dietary supplement on human subjects under average everyday situation. In this study, Maca-GO was administered at one, arbitrary-chosen level only, which eliminated detection of possible variation in daily dose-related individual responses to different level of intake, as well as due to potential different sensitivity of rats to Maca, which was observed by Muller (6) in her clinical practice on women. This would need to be studied further. Also, it will be essential to compare observed in this study antidepressive and sedative effect of Maca-GO, with known antidepressants such as fluoxetine (40), which is recommended in treating menopausal women for depression symptoms (41, 42). Fluoxetine, through inhibition of reverse uptake of serotonin, is believed to mimic action of estrogen (43), responsible for elevation in level of serotonin, an important mood elevating neurotransmitter in the brain.

Interpreting behavior of rats in pharmacodynamic model as applied in this study, Maca-GO possesses typical antidepressant-like profile when administered to ovariectomized rats. This anti-depressive effect was associated with lowering of Cortisol and ACTH levels which may indicate a sedative effect of Maca-GO on ovariectomized rats. In study reported by Lowicka et al. (44), where the effect of administering gelatinized Maca to ovariectomized rats was compared with fluoxetine – a known antidepressant (40), the observed effects of Maca on spontaneous activity and antidepressive-like activity were different from those induced by fluoxetine. Therefore, it may be assumed that mechanisms other than serotoninergic system are involved in pharmacological action of Maca, which also, differently affected ovariectomized and non-ovariectomized rats. Previously reported results obtained on laboratory animals with intact ovaries (12) showed similar effect as observed in this study on both ovariectomized and non-ovariectomized rats, that Maca-GO have positive effect on both, reduction in blood cortisol and therefore, lowering susceptibility of rats to stress factors and its sedative effect on laboratory animals, the properties also reported by Lopez Fondo et al. (45).

Observed in this study, a decrease in E2 and an increase in E2-to-PRG ratio in ovariectomized rats, as a result of Maca-GO ingestion (ratio from 33 to 44), contradict recorded antidepressive effect of this preparation in a laboratory model on rats. However, in non-ovariectomized rats, the same ratio was reduced (from 10 to 6), which may explain slight positive antidepressive effect of Maca-Go and support observations reported in the literature (43, 46). Also Lucille (47) emphasized that the balance between progesterone, estradiol and thyroid function is one of the key factors in female maintaining the hormonal balance during the reproduction years and in menopause. It is a key function of progesterone to control estradiol and prevent negative effects of its dominance as well as to support thyroid function in maintaining growth, healthy bone metabolism and balancing psychological equilibrium in female organism during their reproductive and then in a menopausal stage. In this study such a relationship has been confirmed in non-ovariectomized rats in relation to E2 and PRG only, but has not been confirmed in ovariectomized animals, since in those animals Maca-GO administration resulted in both estradiol and PRG reduction with no significant effect on thyroid profiles.

Maca-GO reduced serum Iron level in non-ovariectomized rats and had no effect on ovariectomized animals which does not support observations obtained in earlier laboratory trial on male and female rats (12) nor confirmed the results reported in the literature (38), suggesting that Maca may stimulate the absorption of dietary Iron from the digestive tract.

In the next part of this paper (Part II), blood biochemistry and pharmacodynamics in a laboratory model on rats as reported here, are follow-up by clinical study on early-postmenopausal women volunteers, who self-administered Maca-GO in various length and time intervals intermittently with placebo in a double blind, coordinated multi-centre study in which similar biochemical measurements and testing of physical and psychological menopausal symptoms were conducted in “human clinical model”.

View larger version : [in a new window] Figure 1: Kinetics of hemoglobin breakdown in erythrocytes exposed to different concentrations of ascorbic acid.

 

 

View larger version : [in a new window] Figure 2: Variation of the hemolysis rate (H.R) with concentration of vitamin C.

 

View larger version : [in a new window] Figure 3: Absorbance variation of erythrocytes with time due to the effect of different concentrations of B6.

 

View larger version : [in a new window] Figure 4: Absorbance variation of erythrocytes with time due to the effect of different concentrations of B5.

 

View larger version : [in a new window] Figure 5: Absorbance variation of erythrocytes with time due to the effect of different concentrations of B1.

 

View larger version : [in a new window] Figure 6: Kinetics of hemoglobin breakdown in erythrocytes due to the effect of the used vitamins at concentration of 2mM.

 

View larger version : [in a new window] Figure 7: Variation of conductivity with temperature for control and vitamins C, B6, B5 and B1 at concentration of 2mM.

	CONCLUSIONS

1. In comparison to placebo, After 2 months of using Maca-GO capsules by perimenopausal women, serum levels of FSH, E2, PG and ACTH substantially increased.
2. Results of interviews conducted by a gynecologist after two months of using Maca-G supplement, majority of women (15 of 18 which concluded the four month trial) observed reduction in general feeling of discomfort, typically observed in the early postmenopausal stage.
3. According to responses given in Kupperman’s questionnaire for assessment of Menopausal Index, frequency of hot flushes and incidence in night sweating, interrupted sleep pattern, nervousness, depression and heart palpitations, were most pronounced symptoms in improving quality of life of perimenopausal women exposed to Maca-GO administration.
4. Preliminary observations outlined in this paper justify further clinical study on use of Maca-GO in perimenopausal women, so as to assess effectiveness of Maca as a potential non-hormonal therapeutic supplement which may help women to reduce discomfort associated with perimenopause as an alternative to, or lessening dependence on HRT program.

	ACKNOWLEDGEMENT

This study was conducted and jointly supported as a part of a long-term joint R&D program which commenced in 1999 by the Research Institute of Medicinal Plants in Poznan, Poland and School of Health Study, Charles Sturt University, Australia and since 2002 continued by the Therapeutic Research, TTD International Pty Ltd Sydney, Australia on therapeutic properties of novel therapeutic preparations and medicinal plants originated from Australasia, Oceania and South America. A supply of a commercial batch of standardized Maca-GO powder used in this study by NatureCorp Pty Ltd, Australia, is gratefully acknowledged.

	DISCLAIMER

Reference to a company and/or product named in this paper is only for purpose of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. Some results in a preliminary form, were presented at the 7th Congress of European Association of Clinical Pharmacology and Toxicology in Poznan ( Poland), 25-29 June 2005.

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