Introduction

Osteoporosis is a significant public health problem worldwide, which can result in fractures and increased morbidity and mortality in both men and women. The risk of developing osteoporosis in later life may be reduced by maximising peak bone mass (PBM) which, depending on the skeletal site, is believed to occur between late adolescence and adulthood in girls and boys (Bonjour et al, 1991; Anderson & Rondano, 1996; Nguyen et al, 2001) with over 90% of bone mineral accretion completed by 18-y of age in boys and 16 y of age in girls (Bonjour et al, 1994). While it is recognised that there is a strong genetic influence on PBM, there are several modifiable environmental factors of which the most important are weight-bearing activities (Bailey et al, 1999) and diet (Heaney et al, 2000; Eastell & Lambert, 2002).

Predominant emphasis has been placed on ensuring an adequate calcium intake and vitamin D status for mineralisation of the skeleton. However, there is increasing evidence supporting the direct and indirect effects of nutrients such as vitamin K (Knapen et al, 1989; Szulc et al, 1994; Luukinen et al, 2000), vitamin C (Gunnes & Lehmann, 1995; Hall & Greendale, 1998; Simon & Hudes, 2001), and potassium (Tucker et al, 1999; New et al, 2000) on bone metabolism and calcium balance. Higher intakes of fruit and vegetables have been shown to be associated with higher bone mineral density (Tucker et al, 1999; New et al, 2000) and lower rates of bone loss in both men and women (Tucker et al, 1999). This may be due to the lower urinary calcium excretion in response to reduced acid load (Buclin et al, 2001). A higher dietary acid load is believed to result in bone mineral dissolution and increased bone resorption, resulting in the release of carbonate, citrate, together with calcium, sodium, potassium to buffer the acid load (Bushinsky, 2001). As a result of increased bone resorption and possibly impaired renal reabsorption, urinary calcium excretion is increased in response to dietary acid load (Barzel & Massey, 1998; Massey, 1998). The role of the skeleton in acid-base homeostasis has been discussed in detail by Barzel (1995) and New (2002).

The principal factors contributing to acid load include sulphur from the catabolism of sulphur amino acids (methionine and cysteine), which are highest in animal protein, nuts and cereals; phosphorus, which is mainly supplied by meat and dairy products and chloride. Determinants of alkali load include potassium, magnesium, sodium and calcium. Two methods for estimating dietary acid–base balance have been published and it is of interest to compare these methods. The protein/potassium ratio (Frassetto et al, 1998) gives an indication of the acid–base balance of the diet and has been shown to account for 71% of the variation in renal net acid excretion (Frassetto et al, 1998). However, this method only takes into consideration one component from each side of the acid–base equation. The method of Remer and Manz takes into account the range of nutrients previously described and it adjusts for nutrient absorption and body size (Manz et al, 1984). This method has also been validated against renal net acid excretion in adults and adolescents (Remer & Manz, 1994; Remer et al, 2003).

To achieve a greater awareness of diet composition in context of bone health of adolescents, the main objectives of this study were (1) to examine the contribution of major food groups to the intake of bone related nutrients in 16–18-y old girls and boys; (2) to compare two methods for determination of acid–base balance: an indirect estimate of net acid excretion, NAE_ind and the protein/potassium ratio (Remer & Manz, 1994; Frassetto et al, 1998); (3) and finally to investigate the associations between NAE and diet composition.

Methods

Subjects and data collection

The subjects were from the 150 male and 144 female students who volunteered to take part in the Cambridge Bone Studies (Prentice et al, 2002; Stear et al, 2003). The female students were recruited from two Cambridge sixth-form colleges. The male students were recruited from the two sixth-form colleges, a comprehensive school and two independent schools, one of which was boarding. Seven-day food diaries were completed by 70% of the girls (n=101) and 77% of the boys (n=111) in the overall data set; details of these subjects are shown in Table 1. Diaries were completed by the girls between September 1996 and March 1997 and by half the boys between September 1997 and July 1998 and the other half, between October 1998 and April 1999. Portion sizes were matched against food photographs and quantities were described in household measures. They were also asked some supplementary questions about their diet such as the type of milk they usually drank, the type of fat used for cooking or spreading, whether they ate meat, and the type of water they drank.

Table 1 - Characteristics of the boys (n=111) and girls (n=101).

Full table

Coding and nutrient analysis

The diet records were coded using the in-house program, DIDO (Diet In Data Out) (Price et al, 1995) and nutrient analysis performed using the in-house suite of programs based on the nutrient data base of McCance and Widdowson edition 5 (Holland et al, 1991). The vitamin K₁ content of a wide range of foods was provided by Bolton-Smith from published data (Bolton-Smith et al, 2000) and unpublished data (C Bolton-Smith and MJ Shearer). Intakes of sodium were calculated from food and drinks only, there was no estimate of salt used in cooking or at table. Intakes of calcium included calcium from the water drunk where it had been recorded. This was coded as Cambridge tap water unless otherwise specified.

The nutrient analysis program allowed certain related items of foods to be grouped together in order to estimate their relative contribution to total nutrient intake. Further information on group composition is given at the bottom of Table 3. The percent nutrient intake (e.g. average calcium intake from milk and cream as a percentage of total calcium intake) for each food group was calculated for each subject. The average percentage contribution for boys and girls was then determined.

Table 3 - Contribution of foods and food groups to percent intake of major bone-related nutrients in boys (n=111) and girls (n=101).

Full table

Acid–base balance

Net acid excretion (NAE_ind) of the subjects was estimated indirectly from the diet by the method of Remer and Manz (Remer et al, 2003):

where S is Sulphur calculated from the dietary protein (g/day) 0.49; P, phosphorus (mg/day) 0.037; K, potassium (mg/day) 0.021; Mg, magnesium, mg/day 0.026.

The factors take into account average intestinal absorption and convert the cations and anions into milliequivalents (mEq).

(Manz et al, 1984)

Body surface area was calculated from height and weight according to the formula:

(DuBois and DuBois, 1916)

Sodium and chlorine were not included in the calculation as salt intake was not measured and these elements tend to balance each other. Calcium was also omitted as it has the smallest impact on acid–base balance (Remer et al, 2003). The subjects were grouped in fifths of NAE_ind (both sexes combined), in order to examine the other characteristics of their diet for each fifth.

In order to rank the foods eaten according to dietary acid load, the potential renal acid load (PRAL) was calculated for 17 food groups consumed by the boys and the girls. Having analysed the nutrient intake from each of the food groups, the calculation of PRAL was the same as NAE_ind but endogenous organic acids was omitted (Remer & Manz, 1995).

A separate estimate of acid–base balance was arrived at using the method of Frassetto et al (1998), which calculates the protein/potassium ratio of the diet expressed as g/mEq.

Data analysis

Data analysis was performed using SPSS for MS Windows 10. NAE_ind and most nutrient intakes were found to be normally distributed with the exception of vitamins C, D and K, which were transformed into natural logs before analysis to normalise the data. Analysis of variance and simultaneous multiple regression analysis was used to examine differences between groups. The food groups chosen to go into the regression model were those that have previously been shown to have an effect on acid–base load (Remer & Manz, 1995). As a linear relationship between variables could not be assumed, Spearman rank correlation coefficients were used to investigate the association between energy adjusted nutrient intakes and NAE_ind. Significant differences were taken as P<0.05. In order to assess the likely validity of the reported energy intakes, the intake/basal metabolic rate (EI/BMR) was calculated (Goldberg et al, 1991) with a ratio of less than 1.1 taken to indicate low energy reporters. In all, 12% of the boys (n=13) and 8% of the girls (n=8) were found to be potential under-reporters. The mean NAE_ind was not significantly different with and without under-reporters and they were included in the statistical analysis.

Ethical approval

Written informed consent was obtained from both the subjects and their parents or guardians. Approval for the study was given by the Ethical Committee of the MRC Dunn Nutrition Unit (of which MRC Human Nutrition Research was formerly a part).

Results

Contribution of major food groups to the intake of bone-related nutrients

Table 2 shows the mean daily intake of protein, calcium, phosphorus, potassium, magnesium, and vitamins C, D and K and Table 3 shows the principal food sources. Average calcium intakes of both boys and girls were above the UK reference nutrient intake (RNI) (Department of Health, 1991) and the EU population reference nutrient (PRI) (Commission of the European Communities, 1993) with only 3% of the boys and girls having calcium intakes below the Lower Reference Nutrient Intake (LRNI). Predictably, the principal source of calcium in the diet was milk and milk products, which accounted for 42% of total intake in both the boys and girls. Cereal foods were also important contributors of calcium, 20% for the boys and 22% for the girls, due to the consumption of fortified breakfast cereals and bread. The contribution to calcium intake from water was 4.5% in boys and 6.7% in girls.

Table 2 - Mean daily intake of energy and nutrients of boys (n=111) and girls (n=101) and corresponding UK RNI values.

Full table

Average phosphorus intakes were higher than the RNI; only 3% of the boys and 5% of the girls had a calcium/phosphorus ratio 1. The principal sources of calcium also provided most of the phosphorus. In addition, meat and meat dishes further contributed 19% of total phosphorus in boys and 13% in girls. Potatoes contributed 7% of the phosphorus in boys and girls and beverages contributed 4.5% (boys) and 3.2% (girls).

Potassium and magnesium intakes of the boys were close to the UK RNI, but the mean intakes of the girls were below the RNI. Potatoes contributed around 25% of total potassium and 11–12% of total magnesium. The intake of sodium from foods, 4.0 g/day (boys) and 3.0 g/day (girls) was higher than the RNI (1.6 g/day) and the UK target for adult sodium intake of 2.4 g/day. (Scientific Advisory Committee on Nutrition, 2002).

The intakes of vitamins were all positively skewed with some very high intakes, particularly of vitamin C, the median intake of which was double the RNI. Beverages were the highest contributors to vitamin C intakes (40%) followed by potatoes (20%) and fruit (13–14%).

Mean vitamin K intakes, expressed in relation to body weight, were close to the 'safe and adequate' recommendation of 1 g/kg body weight/day (Department of Health, 1991), but 39% of the boys and 36% of the girls had intakes lower than this. The main source of vitamin K was green leafy vegetables, 24% (boys) and 27% (girls). Other vegetables, including green beans and peas contributed 13–14% of the vitamin K. Potatoes and potato products appeared to be a good source of vitamin K, but this was due to the fats and oils used in manufacture and cooking. Similarly, cakes and biscuits contributed 9–10% from the fats used in their manufacture.

There is no RNI for vitamin D for this age group in the UK; the intakes of this sample appear to be close to the average intake of dietary vitamin D by British adults, 3 g/day (Department of Health, 1991) and that reported in the National Diet and Nutrition Survey of 3.3 g/day (boys, 15–18 y) and 2.2 g/day (girls,15–18 y) (Gregory et al, 2000).

Comparison of two estimates of acid–base balance

Table 4 shows the mean NAE_ind and protein/potassium ratio for the 110 boys and the 101 girls in the study. A wide range of values were found for both boys, NAE_ind = 33.4–114 mEq/day, and girls, NAE_ind = 21.8–93.2 mEq/day. Regardless of the method used, the mean dietary acid–base balance was significantly higher in the boys compared to the girls, P<0.001 for NAE_ind (67.8 mEq/day for boys vs 53.8 mEq/day for girls) and P=0.001 for protein/potassium ratio (boys, 1.35 g/mEq/day for boys vs 1.25 g/mEq/day for girls). There was a significant correlation between the two methods of calculation of acid–base balance. However, at higher values of NAE_ind and protein/potassium ratio, there was less concordance between the two measures as shown in Figure 1. Examination of the subjects with NAE_ind > 100 (n=8) compared to those with protein/potassium ratio > 1.38 (n=8) showed a clear separation by body weight (mean of 75 kg compared to 58 kg). NAE_ind includes a factor (endogenous acid excretion) calculated from body weight and height, but when this body size factor was taken out of the calculation, the divergence still remained as the same individuals remained separated by their phosphorus intake, a mean of 2388 mg/day compared with 1307 mg/day. When the values for NAE_ind of all the subjects were divided into fifths, of those in the highest fifth of NAE_ind there was a very small group (n = 5) who were also in the lowest fifth of calcium intake. These had a lower consumption of milk and milk products and a higher consumption of meat and meat dishes than average, and a low consumption of fruit, but vegetable and potato intake were close to average.

Figure 1.

Comparison of estimates of acid–base balance using NAE_ind (Remer & Manz, 1994) and protein/potassium ratio (Frassetto et al, 1998).

Full figure and legend (105K)

Table 4 - Mean estimates of dietary acid–base balance of boys (n=111) and girls (n=101).

Full table

Relationship between NAE and diet composition

Table 5 shows the contribution of the weights of foods eaten to the variance in NAE_ind. In boys and girls there was a significant positive correlation between NAE_ind and the weight of milk, cheese, meat and cereal foods consumed and a negative correlation with the weight of potatoes and fruit consumed. In girls, NAE_ind was also negatively correlated with vegetable consumption. In all, 58% of the variance in NAE_ind could be explained by this model in the boys and 51% in the girls.

Table 5 - Multiple regression analysis of NAE_ind against weights of foods eaten.

Full table

Table 6 shows the food groups consumed ranked according to their potential renal acid load (PRAL). For both boys and girls, potatoes were the main contributor to dietary alkali load, while meat was the acidic food group most consumed by the boys and cheese and yoghourt were most consumed by the girls.

Table 6 - PRAL (Mean, s.d.) of each food group and ranking according to intake for boys and girls.

Full table

Nutrient inter-relationships

Table 7 shows the Spearman rank correlation coefficients between energy adjusted (nutrient/MJ) nutrient intakes and between NAE_ind and energy adjusted (nutrient/MJ) nutrient intakes for boys and girls together. Vitamin K intake was significantly positively correlated with intakes of potassium, magnesium and vitamin C and negatively correlated with NAE_ind. Calcium intake was significantly positively correlated with all nutrients and NAE_ind except for vitamin K and vitamin C. Sodium intake from foods was significantly positively correlated with NAE_ind as it was with protein and phosphorus (both of which were included in the calculation of NAE_ind).

Table 7 - Spearman rank correlation between nutrients and NAE_ind for boys and girls combined.

Full table

Discussion

The results of this study should be considered in the context of the background of the students who volunteered to participate. They were all continuing to receive secondary education and the majority of their parents were in nonmanual occupations. Although extensive work has been carried out on the effects of calcium intake on bone mineral status in adolescents, few studies have included an evaluation of subject's usual diet. The results of this dietary assessment of 16–18-y-old adolescent boys and girls showed that, on average, their nutrient intakes met the current UK RNI. Calcium intake was significantly positively correlated with all nutrients and NAE_ind except for vitamin K and vitamin C. This multicollinearity is in agreement with the findings of Holbrook & Barrett-Connor (1991) in 900 men and women aged 50–79 y and indicates that the effects of dietary calcium on bone health cannot be considered in isolation.

Phosphorus intakes were higher than UK RNI, resulting in a mean calcium/phosphorus ratio lower than 1. The implications of this for bone metabolism and mineral accretion in adolescents are currently unknown. It has been suggested that a ratio of more than 1 may be associated with greater bone mineral content in young women (Teegarden et al, 1998) and a lower phosphorus intake would reduce NAE_ind. However, increasing the ratio would be difficult, as the principal sources of calcium were also rich sources of phosphorus. Interestingly, less than 1% of total phosphorus intake came from soda type beverages, while 12–15% came from the milk (and cream), which also provided 19–25% of calcium intake. This contrasts with the finding that the changing food habits of teenagers in the US involve substitution of milk by phosphorus rich beverages (Calvo, 1993). However, due the high consumption of fruit juices and fortified fruit squashes by the Cambridge teenagers the greatest contribution to vitamin C intake came from beverages rather than the 'traditional' sources of fruits and vegetables.

Mean vitamin K intakes of the Cambridge subjects were close to 1 g/kg/day and were comparable to intakes reported from other age groups in the UK (Price et al, 1996; Thane et al, 2002) and in the US for various age groups (Booth & Suttie, 1998). There is no current UK RNI for vitamin K but, based on requirements for hepatic synthesis of the vitamin K-dependent coagulation factors, the safe and adequate intake is 1 g/kg body weight/day (Department of Health, 1991). More recently, the US has set an adequate intake of 125 and 90 g/day for men and women, respectively (Institute of Medicine, 2001), to take into account the higher requirement for carboxylation of bone osteocalcin. Various studies suggest that current intakes of vitamin K in the US (Feskanich et al, 1999; Binkley et al, 2000; McKeown et al, 2002) and the Netherlands (Vermeer et al, 1995) are associated with a high proportion of under-carboxylated osteocalcin. However, it is not known whether adolescent vitamin K requirements are also higher since bone turnover is approximately 10–20 times that found in healthy adults (Szulc et al, 2000). Despite the fact that the Cambridge sample was an elite group in socioeconomic terms, their consumption of leafy green vegetables was very low. Low intakes of vitamin K were associated with low intakes of potassium, magnesium and vitamin C, and thus increased NAE_ind. At least 20% of the vitamin K intake came from fats in cakes and biscuits and, particularly in the boys' diet, potato products such as chips and crisps. It is generally recommended that consumption of these types of foods, which are generally also high in salt, should be limited as they can contribute to a high total energy intake and obesity but this might also reduce vitamin K intakes. There is also limited and conflicting evidence that the vitamin K from fats is better absorbed than that from vegetables (Gijsbers et al, 1996; Booth et al, 1999).

A wide range of acid–base balance values were found using both the Remer and Frassetto methods, that is, NAE_ind and protein/potassium ratio, respectively. These results have shown that the Frassetto method may underestimate the acid load of those with larger body size and high phosphorus intakes. The lowest NAE_ind was found in girls with a very high fruit and vegetable consumption and little or no meat consumption. However, there were others with low NAE_ind who only consumed chips, baked beans, crisps, chocolate, peanuts and lager, all contributing to a high intake of potassium and thus a lower acid load. Examination of these diets indicates that a low acid load is not necessarily associated with current concepts of a healthy diet (a diet low in fat and providing 5 servings of fruit and vegetables). In this sample, NAE_ind was positively related to sodium intake from foods. Higher sodium intake has been shown to increase urinary calcium excretion in adults and children (Shortt & Flynn, 1990; Matkovic et al, 1995); therefore, it is possible that the combined effects of a high salt intake, a low calcium intake and a high NAE_ind may compromise optimal bone mineral accretion.

NAE_ind was positively related to phosphorus intake, as would be expected, and nearly half of the phosphorus was derived from meat and dairy products (two subjects with high NAE_ind ate no meat but consumed very large quantities of cheese). Since dairy products were also the main source of calcium, a reduction in these foods might be considered to be counter-productive (although it is not known whether a more alkali diet reduces calcium requirement). Care may need to be taken in interpreting the apparent benefits of a low NAE_ind for bone health, in that subjects with a low NAE_ind generally had little or no meat intake and those with a high NAE_ind had a high consumption of milk products. Interpretation of the effects of NAE_ind on bone should be coupled with an evaluation of overall diet and nutrient intake.

In the wider population of teenagers and adults, those following a weight loss regime that advocated high protein, low carbohydrate diets (Reddy et al, 2002) could be at increased risk of incurring a negative effect on bone mineral, particularly since such diets often do not include potatoes that are an important factor for lowering NAE_ind.

Although most of the CABS subjects were living at home, at this age adolescents are reaching a stage of some independence in relation to diet. The diets of these teenagers were characteristic of the modern Western diet being high in protein, fat, sodium and phosphorus. Average calcium intakes were above the UK RNI, but there were several subjects whose diet was characterised by a low calcium and a high sodium content combined with a high acid load. More research is needed to determine whether such a dietary profile compromises achievement of optimal peak bone mass. The findings of this study indicate that it is important to consider overall diet quality when interpreting the effects of single nutrients or NAE_ind on bone. Further analysis on the effects of nutrient intake and dietary patterns on bone mineral status will shed light on the relative importance of these findings.

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Acknowledgements

The work was funded by the Medical Research Council, as an addition to work supported by awards from the Department of Health/Medical Research Council Nutrition Research Initiative (boys study) and the Mead Johnson Research Fund (girls study). The views expressed in this publication are those of the authors and not necessarily those of the sponsors.