To analyze the changes in vitamin B6 status in women following slightly hypocaloric diets based on the relative increase consumption of foods whose intakes are below those recommended, and to study how these changes influence the proportion of fat-free mass.
Intervention study of two slightly hypocaloric diets: diet V (increased consumption of vegetables), or diet C (increased consumption of cereals, especially breakfast cereals).
A total of 49 women with a body mass index (BMI) of 25–35 kg/m2.
Dietetic, anthropometric and biochemical data were collected at the start of the study and at 2 and 6 weeks.
Both the C and V subjects showed a reduction in their energy intake, body weight, BMI and fat mass. Pyridoxine intake increased in both groups and plasma pyridoxal phosphate (PLP) levels increased only with diet C. An association was found between the increase in plasma PLP at 6 weeks and the increase in pyridoxine intake (r=0.451; P<0.01). Multiple regression analysis showed a positive association between the increase in PLP at the end of the study and the increases in the pyridoxine intake, B6 density or B6/protein ratio. At the end of the study, and only in those women whose PLP levels were increased, the higher the increase in PLP level, the higher the increase in fat-free mass percentage (r=0.4426, P<0.05)
Interventions aimed at weight control should also try to maintain or improve nutritional status. A diet rich in cereals (especially fortified breakfast cereals) appears to be useful in improving vitamin B6 status. Such an improvement could help maintain fat-free mass during periods of weight loss.
Pyridoxine, or vitamin B6, is involved in numerous metabolic reactions as it activates a number of coenzymes.1 It is needed for the metabolism of proteins, due to the fact that aminotransferases require pyridoxal phosphate (PLP) as cofactor.2 The maintenance of an adequate vitamin B6 status in persons following weight loss diets may help maintain their proportion of fat-free mass, which is at risk when following such diets.3
Unfortunately, women who are concerned about their weight frequently embark on poorly controlled diets and sporadic fasting,4, 5 which could lead to their developing vitamin deficiencies, including that of vitamin B6.4, 6
The aim of the present work was to determine the vitamin B6 status of overweight/mildly obese women concerned about their weight, to analyze the changes in the status of this vitamin, and to study the influence of plasma PLP on the proportion of fat-free mass, when these women followed weight control diets that approximated food intake to the theoretical ideal, that is, by increasing the relative consumption of either cereals or vegetables.
Materials and methods
The study subjects were 49 women aged 20–35 years (mean 28.2±4.6). Most were university students. All were enrolled through a public offer to take part in a study on ‘The assessment of nutritional status and improvement of weight control’. The study was publicized using posters, radio announcements and via publications directed towards young, female university students.
Initially, all interested parties were interviewed by telephone to ensure that they met the inclusion criteria, which were:
Age 20–35 years.
Body mass index (BMI) 25–35 kg/m2.
In the case of ex-smokers, not having quitted in the previous 2 months.
To be free of all disease that might interfere with the results, such as diabetes, hyperthyroidism, metabolic disease, hypertriglyceridemia, lactose or gluten intolerance (celiac disease) and food allergies.
To not be currently involved in a weight loss program.
To have not lost more than 4.5 kg in the 2 months before the study.
To have not lost or gained more than 3 kg between the first interview and the start of the study.
To have a regular menstrual cycle.
To take no more than two alcoholic drinks per day.
To be neither pregnant nor breastfeeding.
Those interested in taking part and who declared themselves to meet all inclusion criteria were invited to the Department of Nutrition at the Universidad Complutense de Madrid. Here, their weights and heights were recorded, and questionnaires were completed to collect personal, health and dietary information. All persons who were confirmed as meeting the inclusion requirements were informed of the aim of the study, of the clinical tests they would undergo, and of the number and type of interviews and tests to which they would be subject. According to the requirements of the Ethics Committee of the Faculty of Pharmacy (University Complutense), all subjects signed a witnessed form of consent to be included.
The final number of aspirants was 193; 134 were either excluded or finally decided not to take part. Of the remaining 59, 49 concluded the 6-week dietary intervention period; these 49 made up the final study population.
The subjects included were randomly assigned to one of two dietary intervention groups:
Diet C—weight control measures based on increasing the proportional consumption of cereals (especially breakfast cereal) (n=25).
Diet V—weight control measures based on increasing the proportional consumption of greens and vegetables (n=24).
These diets were designed to provide a mean of approximately 20% less than the theoretical energy requirements of the subjects. Theoretical energy expenditure (kJ per day) was established by taking into account the body weight, age and physical activity of all subjects using equations proposed by the WHO (World Health Organization).7 For an age range of 18–30 years, the corresponding equation is:
((15.3 × weight (kg))+679) × physical activity coefficient
With this diet, the weight control measures were based on restricting the consumption of energy-rich foods and increasing the consumption of cereals—especially breakfast cereals (30–40 g per serving).8 This intervention is justified because this food group is underrepresented in the Spanish diet; the quantities normally consumed are below those recommended (about two servings per day compared to a recommended minimum of six servings per day).9, 10 Therefore, increasing the proportional consumption of these foods to approximate the diet to the theoretical ideal seems advisable.9, 10 Breakfast cereals and cereal bars were selected for the intervention as, apart from carbohydrate, they also provide fiber, vitamins and minerals. The cereal chosen was Special K (Kellogg España, Madrid, Spain) because of its particularly high mineral and vitamin content per unit weight; this product provides 1.7 mg vitamin B6 per 100 g. However, the subjects were also advised to eat other cereals, for example, bread, rice and pasta.
With this diet, the weight control measures were based on restricting the consumption of energy-rich foods and increasing the intake of greens and vegetables. This diet is also justified by the notable difference in the recommended and observed consumption of these foods (about 1–1.5 servings per day compared to the recommended minimum of 3 servings per day).9, 10 To establish the weight of a vegetable serving, the weights given by United States Department of Agriculture have been used: dark green leafy vegetables, 80.8 g; deep yellow vegetables, 72.7 g; starchy vegetables, 84.3 g and others, 84.2 g.11
Compliance with diet rules
Over the entire intervention period (a total of 6 weeks), the subjects attended a weekly appointment to record anthropometric data and to discuss (and solve) any difficulties in following the diet assigned.
The following data were collected from all subjects during the preintervention stage, and again at 2 and 6 weeks.
The subjects completed a questionnaire on their normal physical activity. This information was used to calculate their energy expenditure.13 Subjects indicated the length of time spent sleeping, eating, playing sport and so on during both working days and weekends. An activity coefficient was established for each subject by multiplying the time spent in each activity by established coefficients7, 14—1 for sleeping and resting, 1.5 for very light activities (those that can be done sitting or standing up such as ironing, typing or painting), 2.5 for light activities (for example, walking), 5 for moderate activities (for example, playing tennis, skiing, dancing) and 7 for intensive activities (for example, felling trees, playing basketball)—and then dividing the sum by 24 h.
Weight and height were determined using a Seca Alpha digital electronic balance (range 0.1–150 kg) and a Harpenden digital stadiometer (range 70–205 cm), respectively. For both measurements, subjects were barefoot and wore only underwear. All data were collected at the Department of Nutrition by trained personnel following norms set out by the WHO.15 The BMI was calculated from the body weight and height figures (BMI=weight (kg)/height (m)2).
The percentage of body fat (%BF) was determined from the body density using the equation of Siri:16 %BF=(495/body density)−450. Body density was calculated from the formulae of Durnin and Womersley:17 body density=1.1567−0.0717 × log(sum of skin fold thickness: biceps+triceps+subscapular+suprailiac). Using the value for %BF and subject body weight, the fat mass and fat-free mass were calculated:
Information was collected on any disease problems, the consumption of medications, supplements and the consumption of manufactured diet foods. These data showed whether the subjects met the inclusion criteria and whether there were any differences between the groups that might modify the results. None of the subjects suffered any disease.
A ‘food and drink record’ was used to register all intakes (both at home and away) for 3 days, including a Sunday.18 Subjects were instructed to record the weights of consumed food if possible, and to use household measurements (spoonfuls, cups and so on) if they could not. The aim was to have as true a record as possible; subjects were asked to record all intakes, even though they broke the ‘rules’ of their diet.
The energy and nutrient contents of these foods were then calculated using food composition tables.8 The values obtained were compared to those recommended19 to determine the adequacy of the diets. Special attention was paid to energy and vitamin B6, intakes. Dial software (Alce Ingeniería 2004) was used to process all data.20
Blood biochemical study
Blood samples were obtained in the morning after an overnight fast of 10 h. A qualified person collected the blood sample from the cubital vein in the arm. The patients were in sitting position during the extraction. PLP is preincubated for 30 min with purified tyrosine decarboxylase apoenzyme in acetate buffer and is then incubated with L-tyrosine for 60 min. The decarboxylated metabolite, tyramine, is extracted into ethyl acetate, air dried, dissolved in borate buffer and reacted with fluorescamine to form a fluorophor (395/475 nm). The radioactivity emitted by this fluorophor is measured. This allows the plasma PLP concentration to be determined (C.V.=8.5%).21
Means and standard deviations (s.d.) were calculated for all variables and the normality of the data was checked. Analysis of variance (ANOVA) for paired samples was used to analyze the change in variables over time in each diet group. The results for group C and group V were then compared using the Student's t-test (or the Mann–Whitney test if the distribution of results was not homogeneous). Linear correlation coefficients were calculated using the Pearson's test. At the end of the study, associations of PLP increase with changes in vitamin B6 intake, B6 density and B6/protein were assessed by multiple regression analysis. Comparisons between proportions were performed using the χ2-test. All calculations were made using RSigma Babel Software (Horus Hardward, Madrid). Significance was set at P<0.05.
The initial dietetic results showed no differences in the food intake between the subjects. During the intervention, group C subjects showed an increased intake of cereals and group V subjects showed an increased intake of vegetables at both 2 and 6 weeks. Fruit intake increased and meat/fish/eggs intake decreased in both groups at both time points (Table 1).
Both the V and C diets were associated with a reduced energy intake and both induced a similar loss of weight and reduction in the BMI and BF mass (%); both diets were therefore effective in this respect (Tables 2 and 3).
Pyridoxine intake, the contribution of this intake to the coverage of the recommended intake, pyridoxine density, the index of nutritional quality (pyridoxine density/density recommended) (INQ) of this vitamin in the diet, and the vitamin B6/protein ratio increased with both diets at 2 and 6 weeks, although this increase was significantly greater in diet C than in diet V subjects (Tables 3 and 4). Plasma PLP increased only with diet C at 2 and 6 weeks (Tables 3 and 4). Plasma PLP variation after 2 weeks of the intervention with diet C was 151.3±114.8% and with diet V 34.4±54.2% (P<0.001). After 6 weeks the variation was 120.9±137.8 and 28.3±73.9% with diet C and V, respectively.
An association was found between the increase in plasma PLP at 6 weeks (compared to initial data) and the increase in dietary pyridoxine intake (r=0.451; P<0.01), when all the women were considered. The increase in PLP negatively correlated with initial levels of PLP (r=−0.6888, P<0.001). Multiple regression analysis showed a positive association between the increase in PLP at the end of the study and the increase in the pyridoxine intake (adjusting for the initial PLP). Thus, by increasing the relative intake of B6 by 1 mg per day, the expected PLP increase would be 18.2±6.5 nmol l−1 (adjusted R2=0.559, P<0.001). The same was found for an increase in 1 mg B6/MJ (β=99.6±42.5; R2=0.537, P<0.001) and for an increase of 1 mg B6 per g protein (β=1310±476; R2=0.556, P<0.001).
Body fat-free mass (%) increased with both diets, although fat-free mass (kg) only increased with diet C (Table 2). At the end of the study the women who increased their plasma PLP by more than 17.6 nmol l−1 (the mean for the increase in PLP) increased their proportion of fat-free mass (%) more than those whose PLP levels increased by less than 17.6 nmol l−1 (3.9±2.9 vs 2.0±2.3%; P<0.05). Similarly, increasing the plasma PLP by more than 17.6 nmol l−1 (the mean the increase in PLP) allowed the proportion of fat-free mass to be increased by more than 2.71% (the mean for the increase in fat-free mass; odds ratio=0.143 (0.038–0.540); P<0.01).
At the end of the study, and only in those women whose PLP levels were increased, the higher increase in PLP level, the higher increase in fat-free mass percentage (r=0.4426, P<0.05).
In the present study the mean initial intake of vitamin B6 was adequate; in fact the intake of most women surpassed the recommended intake of 1. 3 mg per day19, 22 (Table 3). The coverage of the recommended intake and the plasma PLP concentrations recorded were higher than in other studies involving young women.23, 24, 25 However, despite the good mean intake figures recorded, at the beginning of the study 12.3% of the women showed PLP values of <25 nmol l−1, the lower normal limit established by some authors26 (Table 3). Such a situation can worsen when women follow unbalanced diets, and many women who are concerned about their weight frequently follow restrictive, poorly controlled diets,5 and run the risk of negatively affecting their vitamin status.4
The initial dietetic and anthropometric results (Tables 1, 2, 3) are similar to those obtained for other groups of overweight women;9, 10, 14, 27 Although weight and height in C and V subjects were significantly different, BMI did not differ between these groups at the beginning of the study (Table 2). The initial consumption of meat/fish/eggs was somewhat higher than that recommended (2–3 servings per day), whereas those of cereals/legumes, greens/vegetables and fruits were lower (the recommended number of servings per day is 6–10 for cereals and pulses, 3–5 for greens and vegetables, and 2–4 for fruits); similar results have been reported in other studies9, 10 (Table 1). The hypocaloric diets followed in this study were designed to approximate the diet to the theoretical ideal, either by increasing the consumption of cereals or vegetables—the observed intakes of which were the most removed from those recommended. It has been shown that the following of balanced hypocaloric diets not only favors weight loss but helps to improve the status of some nutrients.11, 28, 29, 30
An increase was seen in plasma PLP at 6 weeks in diet C subjects (Tables 3 and 4). This group showed the greatest increase in pyridoxine intake at this time, as well as the greatest increases in dietary pyridoxine density, the coverage of the recommended intake, the INQ (pyridoxine density/density recommended) and the vitamin B6/protein ratio (Tables 3 and 4). Although these also increased in the group V subjects, no improvement in plasma PLP was seen (Tables 3 and 4).
The better results seen among the group C subjects might be explained by the fact that the breakfast cereals making up part of their diet were enriched in vitamin B6; certainly they are richer in this vitamin than vegetables.8 In addition, the availability of this vitamin in vegetables is lower because of losses produced in cooking. Further, in vegetables this vitamin is often found as conjugated pyridoxine glycoside, the bioavailability of which is reduced.31
Several studies report that breakfast cereals are nutrient dense and make a substantial contribution to the intake of B vitamins for the accompanying energy provided.4, 32, 33, 34, 35 Thus, those who consume large amounts of breakfast cereals may have a better status with respect to several vitamins. Indeed, an association has been reported between vitamin B6 status and cereal consumption.33, 34, 35 This agrees with the better vitamin B6 status seen in the present diet C subjects; although vitamin B6 intake increased with both diets, the values of the indicator variables were all higher in the C subjects (Table 4).
As PLP is a coenzyme required by transaminases that take part in protein metabolism (in the synthesis of essential amino acids and the catabolism of most amino acids), protein intake conditions vitamin B6 requirements.36 The recommended intake of vitamin B6 is 0.016 mg g−1 of protein;37 in the present study, the mean intake of the women in both diet groups was greater both at the start and the end of the study. The increase in the intake was, however, greater among the C subjects (Tables 3 and 4). Some authors report that for every 14 g increase in protein intake the plasma PLP concentration is reduced by 23 nmol l−125 and that the intake of vitamin B6 therefore conditions plasma PLP levels;38 this agrees with the results of the present study.
The fact that PLP levels increased with diet C (Tables 3 and 4) might be considered positive as, during slimming, there is a risk of a deterioration in the status of some nutrients.4 The maintenance of an adequate vitamin B6 status is important as the body's proteins are catabolized more strongly when energy intake is restricted.3 In the present study the women who most increased their plasma PLP concentrations were those who most increased their proportion of fat-free mass (%); the greater the increase in PLP achieved, the greater the improvement seen.
Some studies report that plasma homocysteine and methionine concentrations increase in relation to the proportion of fat-free mass.39 However, no previous studies have reported a relationship between the concentration of PLP and fat-free mass. Further work is needed to corroborate the present results.
Although, the energy restriction provided by the two diets was similar, both provoked weight loss and a reduction in BMI and fat mass. However, diet C more strongly increased the intake and blood concentration of vitamin B6. Therefore, in the context of a slightly hypocaloric diet, the consumption of breakfast cereals could be of use in weight control and in the improvement of vitamin B6 status. Finally, it would appear that an increase in plasma PLP (an indicator of vitamin B6 status) is related to an increase in the proportion of fat-free mass during weight loss, although this needs to be further studied.
This work was financed by Kellogg España via the Universidad-Empresa project 362/2003. RMO, AMLS and PA contributed to the study design, and ARN, LMB, and ERR performed the data collection. RMO, AMLS, ARN, ERR, PA and LMB were involved in data analysis and the interpretation of results. ERR, RMO and AMLS contributed to the writing of the paper.
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Conflict of interest/Disclosure
None of the authors have any personal or financial conflicts of interest.
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