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Keywords:

  • bone resorption;
  • vegetables;
  • urinary excretion;
  • acid;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Prevention of low bone mass is important to reduce the incidence of osteoporotic fractures. In man, the consumption of fruits and vegetables is associated with greater bone mineral density (BMD), an effect that is claimed to be caused by their base excess buffering metabolic acid, thought to dissolve bone. We showed previously that in the rat the consumption of several vegetables, salads, and herbs inhibits bone resorption and that onion increases bone mass. In this study we show that, although the intake of onion is associated with a decrease in urinary noncarbonic acid excretion and a concomitant inhibition of bone resorption of similar magnitude, the two findings are not causally related. Onion retains its bone resorption inhibitory activity in the rat even when added to a vegetarian diet with typical base excess. Onion and a mixture of vegetables, salads, and herbs retain their inhibitory activity even when metabolic acid is buffered with potassium citrate. In addition, neither the pH nor the potassium content of individual ashed vegetables, salads, and herbs correlates with inhibition of bone resorption. The effect of vegetables, salads, and herbs, which inhibit bone resorption in the rat, therefore is not mediated by their base excess but possibly by a pharmacologically active compound(s).


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BONE MASS in adult humans decreases with age, leading to an increased risk of fractures.(1) Osteoporotic fractures, besides causing suffering to the patient, are a major burden to health care because the direct expenditure for osteoporosis and associated fractures is around US$14 billion/year in the United States and exceeds US$10 billion/year in Europe.(2) Therefore, from a medical and economical view, it would be desirable if low bone mass could be prevented. A nutritional approach could represent an inexpensive means to achieve this goal. However, to date, no nutritional approach has been shown to be able to prevent osteoporosis; even the effect of calcium in milk on the relative risk of hip fractures seems to be restricted to the 10% of the female population with the lowest intake of calcium.(3) Thus, research into novel nutritional strategies preventing bone loss and their rational basis is needed.

The consumption of fruits and vegetables is associated with greater bone mineral density (BMD) in man, an effect that is claimed to be caused by their base excess buffering metabolic acid.(4,5) Dietary protein of animal origin rich in the sulfur-containing amino acids methionine and cystine metabolically generate sulfuric acid and it is held that this acid load is in part buffered by bone mineral, leading to bone dissolution.(6) Conversely, fruits and vegetables rich in potassium citrate(7) metabolically generate base, so buffering the acid produced by animal foodstuffs and, therefore, it is thought that they protect bone.(4,5) The finding that a vegetarian diet is associated typically with alkaline urine and low calciuria is taken as evidence to support this theory.

Previously, we have shown that some common vegetables, salads, and herbs, which are part of the normal human diet, inhibit bone resorption in the rat whereas foodstuffs of animal origin do not.(8) This evidence was interpreted by others to reflect the mechanism outlined previously.(9)

In this study we show that, although the inhibitory effect of vegetables on bone resorption correlates with their base excess, the two phenomena are independent.

Animals

Male Wistar Hanlbm rats (RCC Ltd., Füllinsdorf, Switzerland) were kept in standard animal facilities that comply with the Swiss and U.S. National Institutes of Health (NIH) guidelines for care and use of experimental animals. The experiments performed were approved by the State Committee for the Control of Animal Experimentation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Diets

Before the experiments, all rats had free access to tap water and were fed ad libitum pellets of a standard laboratory chow (Kliba 343; Kliba-Mühlen, Kaiseraugst, Switzerland) containing 1.1% calcium and 0.8% phosphate (wt/wt of dry food).

From the time when the rats were put into metabolic cages, all animals were given demineralized water to drink and the diets were given as wet food (deionized water was added to the food powder to give a wet but not a soupy consistency).

During the 10-day equilibration period in the metabolic cages and during the 10-day baseline urine collection, the animals were either group-fed a “vegetarian diet” containing 20% soy flour, 10% potato protein, and 2% casein as protein source (SoDi 2134; Kliba-Mühlen) or a “semipurified” diet with animal protein (20% casein) as the sole protein source (“casein-diet”; SoDi 2160, ibid; a AIN 76 based diet.(10))

The diets had a similar raw protein (17.4%), calcium (1.1%), and phosphate (1.2%) content. The calcium and phosphate content of the diets was verified in triplicate ashed samples dissolved in 1N HCl. Calcium was determined by atomic absorption spectrophotometry and phosphate was determined by photometry.(11,12) The values given by the manufacturer were confirmed.

Processing of foodstuffs

Processing of foodstuffs was similar to that previously described.(8,13) Briefly, fresh vegetables, salads, and herbs were purchased locally, carefully washed, minced, air-dried at ∼50°C, and ground to a fine powder. Seven vegetables (broccoli, brussel sprouts, cauliflower, Chinese white and red cabbage, and potatoes) were cooked (as customary for human nutrition) before drying.(13) From commercially available dried onion flakes, Italian parsley, garlic powder, and heat-inactivated white hilum soybeans, the moisture was removed by adsorption over silica gel before grinding. Before freeze-drying, fresh eggs were whipped slightly and fresh meat was minced. Skimmed milk was purchased from a local retailer.

Dietary additions

Onion or a mixture of 14 vegetables (Mix 14, consisting of equal parts of arrugula, broccoli, cucumber, Chinese cabbage, red cabbage, dill, garlic, wild garlic, leek, lettuce, onion, Italian parsley, common parsley, and tomato) previously shown to significantly inhibit bone resorption(8) were mixed with the diet to provide a daily dose of 1 g/rat. Tripotassium citrate monohydrate (Fluka, Buchs, Switzerland) was mixed with food at the doses indicated. Appropriate amounts were added to batches of food sufficient for the 10 days of dietary intervention. These diets then were aliquoted into daily portions and kept frozen until use.

Urine collections

To collect urine, the rats were housed in individual metabolic cages. After 10 days of adaptation, 24-h urine collections were performed for 10 days to assess baseline values. Thereafter, the 10-day dietary intervention was started and 24-h urine collections were continued. To prevent bacterial growth 50 μl of a 5% (wt/vol) thymol solution in 2-propanol was added to each urine tube before use.

Measurement of ash pH

To mimic metabolic oxidation, 1 g of aliquots of the casein diet containing 7% vegetable or animal foodstuffs or the additions alone (100%) as well as the plain casein and vegetarian diets were ashed overnight at 600°C. The ash was mixed with 5 ml of CO2-free distilled water and tightly sealed and allowed to equilibrate overnight. The pH was measured immediately after removing the stoppers.

Measurement of potassium

Duplicate ashed samples of vegetables, salads, and herbs were dissolved in 1N HCl. Potassium was determined by flame photometry.

Measurement of urinary pH and noncarbonic acid excretion

Titratable acid (amount of base to reach pH 7.4) was measured with an automatic titrator coupled to an autoburette and a pH meter (Radiometer; Copenhagen, Denmark). Aliquots of 1 ml of urine were titrated. Acid urines were titrated with 0.1N NaOH and alkaline urines were titrated with 0.02N HCl to pH 7.4. In one experiment ammonium excretion was measured using a kit for ammonia determination, which was run on a Hitachi 911 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland).

Monitoring of bone resorption

The urinary excretion of3H-labeled tetracycline ([3H]-Tc) from chronically prelabeled rats, a method previously developed and validated for continual monitoring of bone resorption, was used.(11,14–16)

Briefly, to label the skeleton homogeneously, rats are injected subcutaneously from the first week of their life for 6 weeks (two times per week) with increasing amounts of a solution containing 10 μCi/ml of 7-[3H](N)tetracycline (New England Nuclear, Boston, MA, USA) dissolved in 0.15 M of NaCl. One week after discontinuation of the [3H]-Tc injections, the rats are housed in individual metabolic cages.

3H in urine was determined by liquid scintillation counting. Aliquots of 1 ml of urine were counted in 10 ml of Irga-Safe Plus scintillator (Packard International, Zürich, Switzerland) and the result (dpm) was multiplied by the urine volume.

Data presentation and statistical analyses

Results are given as individual values or as mean values ± SEM. The significance of differences were evaluated with the Student's t-test. Where appropriate, the 95% CI of the pertinent controls was calculated by multiplying the SEM with 1.96 and is given as a shaded box. Means of groups outside the 95% CI are significantly different from control (p < 0.05).(17) Furthermore, the significance of differences versus the hypothetical mean of 1.0 was calculated and is indicated by the suffixes. The correlation between the pH of ashed casein diet containing 7% vegetables and inhibition of bone resorption (Fig. 1B) as well as the potassium content of various ashed foodstuffs of vegetable origin and inhibition of bone resorption were analyzed by linear regression and the significance of the slopes was tested by ANOVA.

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Figure FIG. 1. (A) pH of various ashed foodstuffs of vegetable origin (circles with numbers corresponding to 2, cauliflower; 3, broccoli; 4, wild garlic; 5, shallot; 6, dill; 8, endive; 10, garlic; 11, potato; 12, leek; 14, Italian parsley; 15, common parsley; 16, carrot; 17, red cabbage; 18, arrugula; 19, soy; 20, lettuce; 21, tomato; 22, white cabbage; and 24, onion) or of animal origin (squares: 7, egg; 9, meat; and 13, skimmed milk) as well as the plain casein diet (large square with 1) and the vegetarian diet (large circle with 23). (B) pH of various ashed aliquots of the casein diet containing 7% of vegetable or animal foodstuffs (same symbols and numbers as in panel A). The results are plotted against the in vivo inhibition of bone resorption shown earlier.(8)

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RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The role of the base excess of various vegetables, salads, and herbs on bone metabolism was investigated in vitro and in vivo.

The ash pH of the casein diet is lower than that of the vegetarian diet (Fig. 1A); the ash pH of milk, meat, and egg is similar to that of the casein diet and is more acid than most of the vegetables. However, the pH of the latter varies within a broad range of 3.5 pH units, whereas that of the diets containing 7% of various foodstuffs varies within a narrower range of 1.5 pH units (Fig. 1B). Furthermore, the higher pH of the ashed casein-diet containing 7% vegetables does not correlate with the inhibition of bone resorption.

Onion was chosen for in depth in vivo investigations. Rats fed the vegetarian diet have an alkaline urine (Fig. 2A), a slightly negative titratable acid (Fig. 2B), and a very low ammonium excretion (Fig. 2C), reflecting the basic nature of vegetable nutrition. Under the vegetarian diet, the daily excretion of noncarbonic acid, as calculated from the titratable acid and ammonium excretion, was −0.024 ± 0.031 mM of hydrogen ions. On switching rats to the casein diet, the urine pH becomes acid, the titratable acid and ammonium excretion increasing substantially. Thus, the 10-day cumulative noncarbonic acid excretion increased to 16.3 ± 0.4 mM of protons, reflecting the metabolic acid generation from the casein diet and the powerful acid excretory capacity of the kidneys. Addition of 7% onion to the casein diet slightly increased urinary pH and decreased slightly both titratable acid and ammonium excretion. Treatment with onion led to a decrease of the cumulative proton excretion by 17 ± 3% (p < 0.01) and of bone resorption by 18 ± 2% (p < 0.001), indicating an effect on bone resorption of a similar magnitude to the effect on acid excretion induced by the casein diet.

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Figure FIG. 2. In vivo effect of the vegetarian (squared background) and the casein diet (dotted background) on (A) urinary pH, (B) titratable acid, and (C) ammonium excretion. All rats were switched to the casein diet on day 3. Control plain casein diet (full circles; n = 5), treated (open squares; casein-diet with 7% onion; n = 5).

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If onion was to inhibit bone resorption by the base excess outlined previously, one would predict that onion should have no effect on bone resorption in rats fed the vegetarian diet. To test this hypothesis, onion was administered to rats on both diets. However, bone resorption was significantly inhibited in rats fed the vegetarian diet, indicating that the effect of onion on bone resorption is not mediated by the base excess (Fig. 3).

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Figure FIG. 3. Effect of treatment with 7% onion on bone resorption in rats fed the vegetarian (A) and the casein diet (B). Bone resorption is given as the cumulative amount of3H excreted into urine over 10 days. The values of the treated groups (n = 5 each) are given as the ratio treated/untreated control. (Values below 1.0 indicate inhibition of bone resorption.) The 95% CI (95% CI = 1.96 × SEM) of the respective control groups (n = 6) is given as a box. The box with squared background indicates the 95% CI of the vegetarian diet and the box with a dotted background indicates the 95% CI of the casein diet. Full squares indicate diets with 7% onion. Mean values outside the 95% CI are significantly different from control (p < 0.05).(17) The significance of differences versus the hypothetical mean of 1.0 is indicated by the suffixes (++p < 0.01;+++p < 0.001).

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Because the effect of onion on bone resorption was larger (18 ± 3%) in the rats fed the casein diet compared with the rats receiving the vegetarian diet (13 ± 2%), the question arose whether in rats on the casein diet, the base excess of onion had possibly contributed to the larger effect. To mimic the base excess of the vegetarian diet, rats receiving the casein diet were given potassium citrate at a dose buffering the metabolic acid load from the casein diet. As shown in Fig. 4 (left of panel A) a daily dose of 252 mg of potassium citrate mixed with food fulfills this requirement. When this dose of potassium citrate is added to the casein diet containing onion or a mixture of the 14 active vegetables, the effect on bone resorption is, if anything, slightly larger (Fig. 4B), suggesting that the reduced response to onion in rats fed the vegetarian diet is unlikely to be caused by the base excess of the vegetarian diet. In addition, a dose of potassium citrate, which leads to an even larger decrease in titratable acid excretion compared with the vegetarian diet, has no effect on bone resorption (Fig. 4A).

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Figure FIG. 4. Effect of buffering the acid load induced by the casein diet by treatment with potassium citrate on urinary titratable acid excretion (left of panels A and B) and on bone resorption (right of panels A and B). Titratable acid excretion and bone resorption are given as the cumulative amount of H+ and3H, respectively, excreted into urine over 10 days. The values of the treated groups (n = 5) are given as the ratio treated/untreated control. (Values below 1.0 indicate inhibition.) The 95% CI of the respective control groups fed the plain casein diet (n = 6 each) is given as the box with dotted background. (A) A dose-response for potassium citrate performed in two separate experiments. (B) The effect of a daily dose of 252 mg of potassium citrate on the effect of 1 g of onion or 1 g of a mixture composed of equal parts of the 14 vegetables (Mix 14) previously shown to be active.(8) Same statistical analysis as described in Fig. 3 (+p < 0.05;++p < 0.01;+++p < 0.001 versus 1.0).

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To explore whether possibly other organic anions from potassium salts can explain the failure of potassium citrate to mimic the inhibitory effect of vegetables, salads, and herbs on bone resorption, their potassium content was measured. As shown in Fig. 5, although the potassium content of vegetable foodstuffs varies over a wide range, it does not correlate with the inhibition of bone resorption (p = 0.164).

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Figure FIG. 5. Potassium content of various ashed foodstuffs of vegetable origin. For numbers indicating the vegetables investigated see Fig. 1A. The results are plotted against the in vivo inhibition of bone resorption shown earlier.(8)

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study shows that, although the intake of vegetables, salads, and herbs is associated with a decrease in urinary titratable acid excretion, reflecting their base excess, and a concomitant inhibition of bone resorption of similar magnitude, the two findings are not causally related. Hence, these results contrast with the interpretation from some earlier studies in humans.

Also, in humans, a vegetarian diet results in alkaline urine, reflecting the base excess, and is associated with lower calciuria,(18) presumably reflecting decreased bone resorption compared with omnivores. Therefore, a causal relationship has been inferred. It is claimed that part of the calciuria is derived from bone as a consequence of the neutralization by bone mineral of the acid load generated by metabolism of animal proteins rich in sulfur-containing amino acids, methionine, and cystine, thus leading to bone dissolution.(6) That the intake of animal protein correlates with hip fracture(19) and an increased risk for forearm fracture is associated with animal protein intake, but no such association for vegetable protein(20) is taken as evidence in support of the “acid hypothesis.” On the other hand, there is recent epidemiological evidence for a negative association of hip fracture with animal protein intake,(21) and a study using an animal protein supplement to correct for the protein malnutrition often occurring in elderly persons provides solid evidence of an attenuation of proximal femur bone loss in patients with a recent hip fracture.(22) Thus, the issue whether animal protein intake is deleterious to bone health is controversial.

Our results, using an extensively validated model to monitor bone resorption sensitive to various inhibitors of bone resorption, used clinically(11,14–16) contrast with the acid hypothesis outlined previously but are supported by the following evidence: although urine from vegetarians is alkaline, omnivores excrete about 60 mmol of hydrogen ions per day into their urine and the acid excretion in subjects on a high-meat diet can exceed 150 mmol/day.(23) However, young human adults with intact renal function can excrete as much as 1000 mmol of hydrogen ions per day.(23) Thus, the acid hypothesis would come into play only in the aging adult with strongly impaired renal function and/or diminished capacity to manufacture ammonium ions. In addition, it recently has been suggested that the organic material in bone and not the mineral itself is responsible for the acute buffering of the additional hydrogen ions during metabolic acidosis.(24) This suggests that the calciuria observed in humans consuming a high animal protein, acidogenic diet is possibly not the consequence of bone mineral dissolution by the acid but could be coincidental and thus similar to what we report here.

The present results, confirming the difference in ash pH between the casein diet and the vegetarian diet(6,25) are supported by the acid excretion in the rat, suggesting that in vitro oxidation is a valid means of predicting metabolic acid generation from foodstuffs. Because some organic acids contained in vegetables, salads, and herbs, such as benzoic, quinic, and hippuric acid, are likely to be incompletely metabolized in vivo, the ash pH probably will underestimate somewhat the in vivo acid status.(25,26) Nevertheless, our data do not support the view that vegetables, salads, and herbs with more alkaline ashes inhibit bone resorption to a larger extent than foodstuffs with more acid ashes. Thus, ash pH and bone resorption do not appear to be related causally, suggesting that also in vivo the base excess of vegetables and the effect on bone resorption possibly are not related.

As shown with onion and a mixture of 14 active vegetables, salads, and herbs (Mix 14), the base excess of vegetable foodstuffs and the effect on bone resorption, although they occur simultaneously in our animal model, must be independent. If this were not so, no significant effect of onion in rats on the vegetarian diet would have been observed and potassium citrate would have blunted the effect of onion and the Mix 14. Moreover, the potassium content of vegetables, salads, and herbs used to assess other organic anions possibly metabolically providing base does not correlate with the effect of vegetables, salads, and herbs on bone resorption. Consequently, the effect of foodstuffs of vegetable origin on bone resorption must stem from another mechanism.

In our previous work, we showed that a variety of common vegetables, salads, and herbs are potent modulators of bone metabolism in the rat.(8) The activity from onion can be extracted; it inhibits osteoclastic resorption in vitro(27) and loss of bone in an osteoporosis model in aged rats.(28) Thus, the activity in this onion extract is likely to reflect the effect of a pharmacologically active compound(s), an assumption that is strongly supported by this work. The nature of this compound(s) is not yet known. Very recently, it has been found that rutin, a flavonoid abundant in onion, inhibits ovariectomy-induced osteopenia in growing rats. Unfortunately, however, a single pharmacologic dose was used,(29) which was much higher than that contained in the 1 g of vegetables we used. Therefore, it is open whether rutin can partly explain the activity of vegetables on bone metabolism.(30,31) Nevertheless, these results show that common vegetables consumed by humans in western countries contain pharmacologically active compounds modulating bone metabolism. Moreover, other compounds occurring in plant-derived foodstuffs such as vitamins (K and C) and phytoestrogens (coumestrol, zearalenol, isoflavones, and humulone) have been identified to be potentially important for bone health.(32–35)

As outlined previously, the validity of the acid hypothesis to explain the effect of fruits and vegetables on bone health in humans is not compelling. If so, another mechanism must come into play. An alternative to the acid hypothesis could be that in humans, as in the rat, bone resorption is inhibited directly by pharmacologically active compounds from vegetables, independently of their base excess. Such a hypothesis also could explain the cross-cultural association between animal protein consumption and hip fracture,(19) because populations on a very low intake of animal protein must instead consume large amounts of vegetables. Moreover, there is no compelling evidence to suggest that the recently published positive association between vegetable and fruit consumption and greater BMD would not fit our hypothesis.(4,5) Finally, the association between net endogenous noncarbonic acid production (NEAP) calculated from protein and potassium intake(7) assessed by dietary recalls and low bone mass and high bone resorption(36) does not prove a causal relationship and could as well be explained by the higher intake of vegetables and fruits, providing inhibitors of bone resorption, in women with a low NEAP.

The aim of this study was to investigate the mode of action of vegetables, salads, and herbs to inhibit bone resorption in the rat. From the results of these short-term studies, we conclude that the base excess of vegetables, salads, and herbs is not responsible for their inhibitory effect on bone resorption. Whether this also holds true in the long-term remains to be established. Long-term alkali administration increased bone formation in rats on a low calcium diet.(37) Whether the base excess of vegetables, salads, and herbs would be sufficient to stimulate bone formation cannot be answered with this investigation. Nevertheless, the available evidence in humans does not suggest an association between fruit and vegetable consumption and bone formation as measured with serum osteocalcin.(5) Thus, it may be inferred that, yet to be identified, a pharmacologically active compound(s) from plant-derived foodstuffs was responsible for the inhibition of bone resorption and not their base excess.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Peter Aeby for skillful technical assistance and Gerd Printzen, Central Clinical Chemical Laboratory, University Hospital Bern, for ammonium determination. We also thank Carol Lim for correcting the English typescript and Gerda Mühlbauer for the literature retrieval. This investigation was supported in part by the Swiss National Science Foundation (grant 32-53740) and by Novartis Consumer Health (Nyon, Switzerland).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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