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© 2008 American Society for Nutrition J. Nutr. 138:431S-434S, February 2008

Supplement: Second International Acid-Base Symposium, Nutrition–Health–Disease

Food Composition and Acid-Base Balance: Alimentary Alkali Depletion and Acid Load in Herbivores1,2

Heidrun Kiwull-Schöne3,*, Peter Kiwull3, Friedrich Manz4 and Hermann Kalhoff5

3 Department of Physiology, Faculty of Medicine, Ruhr-University, 44780 Bochum, Germany; 4 Research Institute of Child Nutrition, 44225 Dortmund, Germany; and 5 Paediatric Clinic, 44137 Dortmund, Germany

* To whom correspondence should be addressed. E-mail: heidrun.kiwull-schoene{at}rub.de.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Alkali-enriched diets are recommended for humans to diminish the net acid load of their usual diet. In contrast, herbivores have to deal with a high dietary alkali impact on acid-base balance. Here we explore the role of nutritional alkali in experimentally induced chronic metabolic acidosis. Data were collected from healthy male adult rabbits kept in metabolism cages to obtain 24-h urine and arterial blood samples. Randomized groups consumed rabbit diets ad libitum, providing sufficient energy but variable alkali load. One subgroup (n = 10) received high-alkali food and ~15 mEq/kg ammonium chloride (NH4Cl) with its drinking water for 5 d. Another group (n = 14) was fed low-alkali food for 5 d and given ~4 mEq/kg NH4Cl daily for the last 2 d. The wide range of alimentary acid-base load was significantly reflected by renal base excretion, but normal acid-base conditions were maintained in the arterial blood. In rabbits fed a high-alkali diet, the excreted alkaline urine (pHu > 8.0) typically contained a large amount of precipitated carbonate, whereas in rabbits fed a low-alkali diet, both pHu and precipitate decreased considerably. During high-alkali feeding, application of NH4Cl likewise decreased pHu, but arterial pH was still maintained with no indication of metabolic acidosis. During low-alkali feeding, a comparably small amount of added NH4Cl further lowered pHu and was accompanied by a significant systemic metabolic acidosis. We conclude that exhausted renal base-saving function by dietary alkali depletion is a prerequisite for growing susceptibility to NH4Cl-induced chronic metabolic acidosis in the herbivore rabbit.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The rabbit is a common laboratory animal for medical basic research in respiratory and renal physiology. However, less attention has been paid in rabbits to the role of food mineral uptake for adaptive functions of acid-base balance. Laboratory rabbit pellet semipurified diet, adapted to the herbivore nutrition habit of this species, is characterized by an alkaline ash: the sum of fixed cations exceeds that of fixed anions, with the difference providing a measure of dietary alkalinity or potential bicarbonate (1,2).

Thus, the rabbit appears to be a suitable animal model for strict herbivore nutrition and may help our understanding of adverse effects caused by extreme base excretion and high urinary pH, e.g., for urinary carbonate stone formation (3). This may be of special interest when alkali-rich diets or therapies are recommended in human medicine to prevent the pathological consequences of nutritional acid load in humans in conditions with impaired renal function, e.g., immaturity in preterm infants (4) or regression in elderly persons (5).

Rabbits normally adapted to alkali-rich nutrition are often investigated for renal responses to chronic metabolic acidosis (6,7), which could only be achieved by ingestion of HCl or NH4Cl when normal feed was withheld, implying concomitant energy deficiency.

The aim of this study is 2-fold: first, to examine the role of high alkali load for urinary pH, bicarbonate excretion, and formation of precipitated carbonates, and second, to investigate the role of strongly reduced dietary alkali load at maintained energy intake for the development of metabolic acidosis induced by ingestion of ammonium chloride with the drinking water.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
As previously described in detail (2), healthy adult male conscious rabbits (Chinchilla Bastard; Charles River) were investigated, and the experiments were officially approved according to the "Principles of laboratory animal care." The rabbits were randomly selected for measurements from the central animal care unit of the department,fed an unchanged diet, and individually accustomed to a metabolism cage for at least 1 wk. Daily food consumption, water intake, and urine excretion were recorded. Data from 122–127 untreated rabbits under control conditions allowed better interpretation of the present results. These reference data were obtained from different series of experiments. They have been partly published elsewhere (2) or are still unpublished.

One subgroup of rabbits (n = 10) received high-energy alkali-rich standard pellets under control conditions, whereby the mean daily energy intake and alkali load were ~395 kJ·kg–1 and ~65 mEq·kg–1, respectively. During the experimental period of 5 d these rabbits were given a 1% ammonium chloride solution instead of drinking water, corresponding to a mean daily NH4Cl uptake of 15.4 ± 1.4 mEq·kg–1. The other subgroup (n = 14) was fed energy-reduced standard pellets with normal alkali content for control, whereby the mean daily energy intake was ~250 kJ·kg–1 and the alkali load ~40 mEq·kg–1. To achieve approximately the same energy intake but reduced alkali load during the experimental period, the standard food was changed for 5 d into a commercial high-energy/low-alkali mixture consisting of peanuts, cornflakes, and carob-tree-fruit skin for choice ad libitum. During the last 2 d, this group was given NH4Cl with water, corresponding to a mean daily NH4Cl uptake of 4.2 ± 0.4 mEq·kg–1. The composition of the 2 standard feed pellets and the low-alkali mixture [Table 1 of Kiwull-Schöne et al. (2)] was determined by analysis of ash in cooperation with the Research Institute of Child Nutrition, Dortmund, and the Institute for Animal Health and Food Quality, Kiel.

    Blood analysis. Arterial blood samples were taken from the central ear artery under superficial local anesthesia. Arterial pH (pHa) and blood gas values were measured by conventional electrodes (ABL 5 Radiometer) at 38°C.

    Urine analysis. The excreted 24-h urine was collected under paraffin oil to prevent the loss of carbon dioxide. Because the alkaline urine of rabbits eating standard food contained a considerable amount of precipitate, a stirred aliquot was centrifuged for separate analysis of the clear supernatant and the precipitate. The acid-base status of the supernatant was investigated titrimetrically, e.g., for actual pH (pHu) and concentrations of bicarbonate, and the precipitate was dried at 60°C, weighed, and analyzed for carbonate as loss of CO2 after addition of HCl and back-titration with NaOH (2,8).

    Statistical analysis. Presented data are group means ± SD. After corroborating normal distribution and equality of variances, significant differences between group means were tested by unpaired t tests. The limit of significance was at P < 0.05. Reference values from rabbits not treated with NH4Cl (see above) underwent regression analysis to obtain relations between selected variables and 95% mean confidence intervals. Statistical analysis was in part carried out using SPSS 8.0 for Windows software.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Effects of different diets on urinary pH and excretion of insoluble compounds. Rabbits fed species-adapted alkali-rich standard pellets excreted a highly alkaline urine (pHu > 8.0), but those fed a low-alkali diet or that spontaneously fasted sharply decreased urinary pH values to the range of 6.0 (Fig. 1). Additionally, within the range of high alkali load, the 95% mean confidence interval of pHu for untreated rabbits (n = 127) elucidates urinary acidification both on NH4Cl ingestion during high-alkali load and on dietary alkali depletion alone. As a result, the alkaline urines contained large amounts of insoluble compounds, up to a daily mean of >1.5 g/kg body weight (2), which became progressively smaller with urinary acidification because of alimentary alkali depletion (Fig. 2), but remained rather unaffected by additional ingestion of NH4Cl. In comparison, data of an untreated reference group (n = 124) reveal strong reduction of precipitate on urinary acidification because of dietary alkali depletion, but there were no additional effects of NH4Cl ingestion. According to Flatt and Carpenter (9), rabbit urinary precipitates consist mainly of calcium carbonates (CaCO3 and CaCO3·H2O). On the basis of the average molecular weight of the 2 components, the proportion of carbonates decreased together with the total amount of precipitate, from ~85% on alkali-rich to ~30% on alkali-reduced diets. This is consistent with increasing proportions of phosphate (data not shown).


Figure 1
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  		FIGURE 1  Effect of alimentary acid-base load on renal acidification in rabbits. Ordinate: Urinary pH (pHu) values. Abscissa: Dietary acid-base load calculated from the feed's ash cation-anion difference and daily food intake ad libitum (2). Mean values ± SD of the subgroups ingesting ammonium chloride (NH4Cl) either with high-alkali food (n = 10) or with low-alkali food (n = 14) differ significantly (P ≤ 0.01).

 

Figure 2
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  		FIGURE 2  Effect of renal acidification on excretion of insoluble compounds in rabbit urine. Ordinate: Daily excreted amount of precipitate. Abscissa: Urinary pH (pHu) values. Mean values ± SD of the subgroups ingesting ammonium chloride (NH4Cl) either with high-alkali food (n = 10) or with low-alkali food (n = 14) differ significantly (P ≤ 0.01).

 
    Acid-base regulation during alimentary alkali depletion and/or acid load. In rabbits, a wide range of dietary alkali load can be achieved by varied electrolyte composition of the food and individually different food intake (2). Against this variable nutritional background, acid-base regulation is illustrated for a large number of otherwise untreated rabbits serving as reference groups (Figs. 1–3). The renal response to alimentary alkali depletion comprised urinary acidification and effectively maintained systemic acid-base balance in terms of pHa.


Figure 3
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  		FIGURE 3  Effect of alimentary alkali depletion and acid load on systemic acid-base balance in rabbits. Ordinate: Arterial pH (pHa) values. Abscissa: Dietary alkali load defined as in Figure 1. Mean values ± SD of the subgroups ingesting ammonium chloride (NH4Cl) either with high-alkali food (n = 10) or with low-alkali food (n = 14) differ significantly (P ≤ 0.01).

 
As long as the high-alkali nutritional background prevailed, a high cumulative NH4Cl load by as much as 75 mEq·kg–1 over 5 d significantly decreased pHu to 7.00 ± 0.32 (n = 10, P < 0.001) compared with 8.09 ± 0.30 (n = 58) in the reference group (2) but did not impair the arterial pH values (Fig. 3). On low alkali intake, however, a much smaller cumulative NH4Cl challenge of only ~0.1 (8 mEq·kg–1 for 2 d) did cause a significant acidosis in the arterial blood (P < 0.01), decreasing pHa to 7.342 ± 0.052 (n = 14) compared with 7.418 ± 0.031 (n = 19) on alkali reduction alone. Under the latter condition with and without NH4Cl load, urinary pH values did significantly (P < 0.01) but not greatly differ between 5.69 ± 0.45 and 6.32 ± 0.58, respectively. Furthermore, the 95% mean confidence interval of pHa for a large range of dietary alkali load in untreated rabbits (n = 122) (Fig. 3) illustrates that additional NH4Cl ingestion does not impair arterial pH homeostasis as long as high alkali supply is provided, but considerable systemic acidosis develops as a result of (even much smaller amounts of) ingested NH4Cl on concomitant dietary alkali depletion.

Thus, in the rabbit it is only possible to elicit a manifest NH4Cl acidosis against the background of low-alkali nutrition.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
    The impact of herbivore alkali-rich nutrition on kidney function. Rabbits on species-specific alkali-rich standard semipurified diet excrete highly alkaline urines but develop significant urinary acidification on dietary alkali depletion. Consequently, alkaline urines of rabbits contain large amounts of insoluble compounds, mainly consisting of calcium carbonates (9), which progressively disappear at lower values of pHu. The precipitated carbonate must be considered a portion of total base excretion (2), e.g., for quantitative estimation of urinary net acid excretion during NH4Cl challenges.

Interestingly, large amounts of calcium carbonate crystals are not harmful to the urinary tract of the rabbit because they form a soft chalk-like matter and are covered with mucus that is excreted by specialized glands (10). In contrast to these adaptations in the herbivore rabbit, vegetarian nutrition in omnivore species such as humans may enhance the risk of calcium carbonate stone formation, at least under extreme conditions such as progredient bone demineralization, e.g., from immobilization during prolonged bed rest or weightlessness in space labs (11).

    The role of nutritional alkali background for the manifestation of an experimental ammonium chloride acidosis. In the herbivore rabbit, systemic acid-base balance is normally maintained over a wide range of nutritional alkali load by appropriate renal base reabsorption (2). The present results have shown that it is not possible to induce a systemic metabolic acidosis in rabbits even by high-dose application of ammonium chloride under normal herbivore nutrition. The resulting distinct renal acidification, which likely is a base-saving response, appears rather unexpected in view of persisting high alkali intake but may explain systemic acid-base homeostasis. However, prior stress on renal acid-base control by alimentary alkali depletion, leading to nearly complete base reabsorption (2), elicits growing susceptibility to chronic metabolic acidosis in the rabbit. In agreement with observations on preterm infants (12), and based on supplementary yet unpublished observations, our data predict significant reduction of arterial pH (and base excess) for net acid excretion values above 0 at a pHu level below ~6.5.

Because of the diet's high energy content, our approach has the further advantage of sufficient energy supply in addition to alkali depletion, so that adverse effects on respiration and metabolism, also involved in acid-base balance, can be avoided. Up to now, investigators attempting hyperchloremic metabolic acidosis in rabbits performed concomitant food deprivation (6,7).

There is general agreement that Formula reabsorption by proximal tubules is the leading process in renal acid-base regulation (13). At the molecular level, many functions of isolated kidney tubules have been studied in the rabbit (7,14), which has shown that carbonic anhydrase II and IV in different nephron segments are stimulated during chronic metabolic acidosis. Likewise, the sodium-proton exchanger subtype NHE3 is profoundly involved in proximal tubular bicarbonate retention and abundance and activity of the NHE3 protein are also regulated, lowered by alkali load (15) and enhanced by NH4Cl-induced acidosis (16,17). Recently, we have shown that chronic metabolic acidosis in rabbits did cause NHE3 mRNA up-regulation not only in the kidney but also in brainstem regions involved in central respiratory control (18).

The interplay of ion-exchange proteins and enzymes for acid-base homeostasis in rabbits exposed to NH4Cl against the background of high and low nutritional alkali load remains to be clarified.


   FOOTNOTES
 
1 Published in a supplement to The Journal of Nutrition. Presented as part of the Second International Acid-Base Symposium, Nutrition–Health–Disease, held in Munich, Germany, September 8–9, 2006. Financial support for this symposium was provided by Protina Pharmaceutical Company. Guest Editors for the supplement publication were Thomas Remer and Juergen Vormann. Guest Editor disclosures: J. Vormann is a consultant to Protina Pharmaceutical Company; T. Remer received an unrestricted research grant from Protina Pharmaceutical Company. Back

2 Author disclosures: H. Kiwull-Schöne, P. Kiwull, F. Manz, and H. Kalhoff, no conflicts of interest. Back


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Emmett M, Alpern RJ, Seldin DW. Metabolic acidosis. In: Seldin DW, Giebisch G, editors. The kidney: physiology and pathophysiology, New York: Raven Press; 1992. p. 2759–2836.

2. Kiwull-Schöne H, Kalhoff H, Manz F, Kiwull P. Food mineral composition and acid-base balance in rabbits. Eur J Nutr. 2005;44:499–508.[Medline]

3. Hesse A, Heimbach D. Causes of phosphate stone formation and the importance of metaphylaxis by urinary acidification: a review. World J Urol. 1999;17:308–15.[Medline]

4. Kalhoff H, Manz F. Nutrition, acid-base status and growth in early childhood. Eur J Nutr. 2001;40:221–30.[Medline]

5. Frassetto L, Morris RC Jr, Sellmeyer DE, Todd K, Sebastian A. Diet, evolution and aging. The pathophysiologic effects of the post-agricultural inversion of the potassium-to-sodium and base-to-chloride ratios in the human diet. Eur J Nutr. 2001;40:200–13.[Medline]

6. Richardson RMA, Goldstein MB, Stinebaugh BJ, Halperin ML. Influence of diet and metabolism on urinary acid excretion in the rat and the rabbit. J Lab Clin Med. 1979;94:510–8.[Medline]

7. Winkler CA, Kittelberger AM, Schwartz GJ. Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis. Am J Physiol. 1997;272:F551–60.[Medline]

8. Lüthy C, Moser C, Oetliker O. Dreistufige Säure-Basen-Titration im Urin. Med Lab (Stuttg). 1977;30:174–81.[Medline]

9. Flatt RE, Carpenter AB. Identification of crystalline material in urine of rabbits. Am J Vet Res. 1971;32:655–8.[Medline]

10. Atkinson DE, Bourke E. Metabolic aspects of the regulation of systemic pH. Am J Physiol. 1987;252:F947–56.[Medline]

11. Pfeifer-Glatz U. Immobilisation und Urolithiasis im Erwachsenenalter. Thesis, Faculty of Medicine, Friedrich-Wilhelms-University, Bonn, Germany, 1993.

12. Kalhoff H, Manz F, Kiwull P, Kiwull-Schöne H. Food mineral composition and acid-base balance in preterm infants. Eur J Nutr. 2007;46:188–95.[Medline]

13. Giebisch G, Windhager E. Transport of acids and bases. In: Boron WF, Boulpaep EL, editors. Medical physiology. Philadelphia: WB Saunders; 2003. p. 845–60.

14. Tsuruoka S, Kittelberger AM, Schwartz G. Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis. Am J Physiol. 1998;274:F259–F67.[Medline]

15. Eladari D, Leviel F, Pezy F, Paillard M, Chambrey R. Rat proximal NHE3 adapts to chronic acid-base disorders but not to chronic changes in dietary NaCl intake. Am J Physiol. 2002;282:F835–43.

16. Wu MS, Biemesderfer D, Giebisch G, Aronson PS. Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis. J Biol Chem. 1996;271:32749–52.[Abstract/Free Full Text]

17. Ambuhl PM, Amemiya M, Danczkay M, Lotscher M, Kaissling B, Moe OW, Preisig AP, Alpern RJ. Chronic metabolic acidosis increases NHE3 protein abundance in rat kidney. Am J Physiol. 1996;271:F917–25.[Medline]

18. Kiwull-Schöne H, Kiwull P, Frede S, Wiemann M. Role of brainstem sodium/proton exchanger 3 for breathing control during chronic acid-base imbalance. Am J Respir Crit Care Med. 2007;176:513–9.[Abstract/Free Full Text]





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