1 Renal Division, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada M5B 1A6; and 2 Renal Division, Tri-Service General Hospital, National Defense Medical Center, Taipei 100, Taiwan
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ABSTRACT |
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Our objective was to describe the process
of alkali disposal in rats. Balance studies were performed while
incremental loads of alkali were given to rats fed a low-alkali diet or
their usual alkaline ash diet. Control groups received equimolar NaCl
or KCl. Virtually all of the alkali was eliminated within 24 h
when the dose exceeded 750 µmol. The most sensitive response to
alkali input was a decline in the excretion of NH
acid-base balance; ammonium; bicarbonate; citrate; net acid excretion; organic anions; 2-oxoglutarate
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INTRODUCTION |
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TO ACHIEVE ACID-BASE balance, the rate of appearance and elimination of H+ must be equal (11, 26). One can readily identify metabolic pathways that produce H+, because the overall valence of all end products other than H+ is more anionic than all of its substrates (11, 13). In this analysis, cofactors such as adenine nucleotides and redox couples are ignored because they are present in catalytic amounts and are both formed and removed in a metabolic process (13). Said another way, for the net accumulation of H+, there would have to be an equivalent rise in the concentration of the more anionic form of these catalysts. Nevertheless, this cannot produce a large H+ load, because these catalysts are below the millimolar range in the body.
In quantitative terms, the net appearance of H+ is
equal to the number of milliequivalents of SO
Acid production and net acid excretion do not explain mass
balance of H+, because these terms do not include
production and elimination of dietary alkali (8, 22).
One's diet contains organic anions ingested largely as their
K+ salts (8, 24). The daily alkali load is the
result of metabolism of many of these anions to neutral end products
(13). This alkali input can be removed by titrating some
of the daily H+ production, or it can be eliminated
directly (bicarbonaturia) or indirectly (involving a metabolic and
renal process; Fig. 1). The indirect
removal of alkali begins with H+ and organic anion
production from neutral precursors such as glucose. Next, these organic
anions are converted into end products of metabolism by being excreted
into urine as their Na+ or K+ salts (i.e., not
with H+ or NH" in the urine (Ref.
17; Eq. 1). In fact, organic anion excretion in
rats consuming their usual diet is an order of magnitude larger than
net acid excretion (22, 25)
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(1) |
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The present study was designed to evaluate mechanisms that eliminate a progressively increasing alkali load. Two groups of rats were studied; one group consumed a regular alkaline ash diet, and the second group consumed a low-alkali diet. Pair feeding was an essential component of the protocol. The incremental exogenous alkali load was given by the intraperitoneal (IP) route to bypass the uncertainties of absorption in the gastrointestinal tract (see METHODS). With respect to total unmeasured anion excretion, two were singled out for more detailed evaluation. Our rationale was that each one represented a significant proportion of the total unmeasured anions when alkali was provided and, also, being trivalent (citrate) or divalent [2-oxoglutarate (2-OG)], they could chelate ionized Ca2+ in the urine (6).
Our results indicate that the mechanisms to remove lower doses of
alkali decreased the rate of excretion of NH
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METHODS |
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Rats
Rats were cared for in accordance with the guidelines of the Canadian Council on Animal Care. St. Michael's Hospital Animal Care Committee approved the study protocol.Procedure
Adult male Wistar rats (300-400 g) were housed in individual metabolic cages and given 20 ml of 5% sucrose to drink at 0900 and 1600 on the experimental days. The amounts of food given and remaining were weighed daily. The completeness of urine collection was assessed by examining the creatinine excretion rate (5); thymol was added as a urine preservative. Blood was drawn under light anesthesia before and after the balance period.Experimental Protocols
Because the regular laboratory rodent diet has more listed cations than anions (Table 1), it contains an appreciable unknown anion load. Many of these unknown anions are absorbed and converted by metabolism to HCO
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Low-alkali diet. The experimental period (2 days) was preceded by a 2-day accommodation to the diet. One control group (n = 6) continued to consume it without supplements on each of the 2 experimental days. Two other groups of 6 rats each received 3,000 µmol KCl or NaCl in 2 equally divided doses by the IP route to control for Na+ or K+ inputs.
The alkali load was given as a hypertonic solution (300 mM) by the IP route on each of the 2 experimental days. The doses were (in µmol) 750 (n = 12), 1,125 (n = 8), 1,500 (n = 12), 2,250 (n = 6), or 3,000 (n = 10). The 1,125-, 1,500-, and 2,250-µmol doses were divided into two equal portions and given at 0900 and 1600 to minimize pulse increase in the plasma HCORegular laboratory rat diet. The design of these experiments was similar; pair-fed rats consumed 18 ± 2 g of regular chow (n = 9). Rats in the experimental groups were given (in µmol) 750 (n = 6), 1,500 (n = 6), or 3,000 (n = 6) of NaHCO3 by the IP route as described for the previous protocol. An additional group (n = 6) received 3,000 µmol of NaCl to control for the Na+ load.
Analytic techniques.
Na+ and K+ in plasma and urine were determined
by flame photometry, whereas Cl was determined by
electromimetic titration. Blood-gas analysis was performed at 37°C
with a digital pH/blood-gas analyzer (model 178 blood/pH analyzer;
Corning). The concentration of HCO
Calculations
Total unmeasured anions.
This total includes citrate and 2-OG and was calculated as the sum of
the excretion of the measured cations (Na+ + K+ + Ca2+ + Mg2+ + NH + HCO
Urinary unmeasured anion excretion rate that reflects elimination of alkali load. This excretion rate was the total unmeasured anion excretion rate per day with an alkali supplement minus the daily excretion of total unmeasured anions with no alkali supplement while the low-alkali diet was consumed.
Total alkali excretion. Net acid and total unmeasured anion excretions were measured in rats that did and did not receive alkali supplements. The difference in these excretion rates compared with alkali-supplemented rats was used to calculate how much alkali was eliminated by the process of a decrease in net acid excretion and by an increase in the excretion of unmeasured anions.
Fractional excretion of citrate and 2-OG.
The fractional excretion of citrate and 2-OG was measured in urine
collected over 4 h and midpoint blood collected from an identically treated subgroup to avoid studying anesthetized rats. The
blood acid-base parameters were comparable at 2 and 24 h. The
fractional excretion was calculated as
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Statistical Analysis
Results are reported as means ± SE. Statistical analysis was performed by using an unpaired Student's t-test. A P value <0.05 was considered to be statistically significant. ![]() |
RESULTS |
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Rats fed the low-alkali diet had a plasma pH,
PCO2, and HCO
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Rats consuming the low-alkali diet had the highest rate of
excretion of net acid and the lowest rate of excretion of total unmeasured anions, citrate, and 2-OG. With the lower doses of alkali,
the decline in net acid plus the rise in the daily total unmeasured
anion excretion were somewhat lower than the rise in alkali intake
(Fig. 2). At these lower doses, alkali
was eliminated primarily by lowering the excretion of
NH
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The remaining studies were carried out in rats that consumed their
usual diet. These rats had a higher excretion of citrate and total
unmeasured anions than did alkali-loaded rats fed the low-alkali diet.
The rise in citrate excretion was in part due to an increased filtered
load and a rise in its fractional excretion (peak ~70% of its
filtered load; Table 5). With the 750 µeq of NaHCO3 load,
the major pathway for alkali elimination was a further rise in the
excretion of total unmeasured anions. With 1,500 µeq of
NaHCO3, there was a large increment in 2-OG excretion
(total 800 ± 64 µeq/day) to values approaching those of citrate
(976 ± 124 µeq/day; Table 4; Fig.
3). Interestingly, the fractional excretion of 2-OG approached 400%, and there was also an increase in
the plasma level of 2-OG (Table 5). The
acid-base status in plasma reported in Table 1 was similar in rats used
to obtain plasma levels of citrate and 2-OG. Again, bicarbonaturia
became prominent with the largest alkali dose.
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DISCUSSION |
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There have been many studies in which acid or alkali loads were given to rats to measure the rates of excretion of net acid and total unmeasured anions in general as well as citrate in particular (e.g., reviewed in Refs. 3, 8, 19, 25). What was not addressed in previous studies was the sensitivity of each of these response elements to an alkali load while a low-alkali diet was consumed. Furthermore, it was not clear what renal mechanisms (secretion and/or reabsorption) participated in the excretion of individual organic anions, because these processes were obscured in 24-h urine collections. Another emphasis of the present study was to evaluate how the defense of acid-base balance would affect the composition of urine with respect to the risk of formation of kidney stones.
Alkali is present in the diet, primarily as K+ salts of
organic anions; its source is usually fruits and vegetables. Therefore, to remove alkali from the diet, one must reduce its K+
content. To control for the separate effects of K+
depletion and alkali removal, two control groups were employed. The
first received KCl in equimolar amounts to KHCO3. The
second control group received NaCl instead of KCl to control for the Cl load. In all cases, the acid-base composition of the
urine of rats receiving KCl or NaCl was similar to that in the
non-alkali-treated rats fed the low-alkali diet. Hence, the effects of
KHCO3 administration were likely due to its alkali rather
than its K+ load.
The traditional view of acid-base balance focuses on the production of
acids (sulfuric and phosphoric acids) and H+ removal by the
renal excretion of net acid (14). This view ignores the
large net alkali load of the diet despite the fact that all the urinary
indicators of net H+ production
(SO
All alkali supplements were eliminated in the 24-h period of
observation, except in rats fed the low-alkali diet and given <1,500
µmol of KHCO3 (Fig. 2). This small positive alkali
balance might be due to a titration of H+ that was retained
when the diet was devoid of its alkali supplements. In rats fed the
low-alkali diet, the most sensitive major response to the alkali load
was a decline in the excretion of NH
When rats were fed their regular chow, the intake of alkali was large
enough to have a near-maximal rate of excretion of citrate (948 µeq/day). Two mechanisms participated in this response. First, there
was an increased net production of citric acid, as evidenced by the
near doubling of its concentration in plasma. Second, the reabsorption
of filtered citrate was inhibited because its fractional excretion rose
to ~70% (Table 5). Even larger alkali loads were required to augment
the excretion of 2-OG (800 µeq/min; Table 4). Again, two mechanisms
were involved in this process. First, more 2-OG was produced because
there was a twofold rise in its concentration in plasma. Second, there
was a net renal secretion of 2-OG because its fractional excretion was
~400% (Table 5). Bicarbonaturia rose by an appreciable amount in
rats that received the largest doses of alkali (Tables 3 and 4). This
was not simply due to an expansion of extracellular fluid volume
(7), because rats given equimolar NaCl did not have an
appreciable rise in their rate of excretion of HCO
There is a much smaller daily rate of excretion of total unmeasured
anions in humans than in rats; in general, this excretion rate is
quantitatively similar to that of NH
Physiological Perspectives
We identified two advantages of minimizing bicarbonaturia with an alkali load. First, having a urine pH ~6 could decrease the likelihood of nephrolithiasis by diminishing the risk of precipitation of Ca3(PO4)2 and uric acid in the urinary tract (1, 6). To form Ca3(PO4)2, ionized Ca2+ must react with divalent inorganic HPOThe second potential role for minimizing bicarbonaturia and/or having a
higher urine pH concerns the regulation of the excretion of
K+ (12). Kaliuresis is augmented when the
urine pH is high and aldosterone acts (4). The main reason
for a higher rate of excretion of K+ in this setting is a
higher concentration of K+ in the lumen of the cortical
collecting duct (4, 30). Lin et al. (23)
demonstrated that the rate of excretion of K+ and the
transtubular K+ concentration ratio rose appreciably when
bicarbonaturia and/or a more alkaline luminal pH was present during
prolonged fasting. This high transtubular K+ concentration
ratio could not be attributed to enhanced excretion of Na+
or anions that were not reabsorbed in the cortical collecting duct
(ketoacid anions). Lin et al. speculated that distal delivery of
HCO
Concluding Remarks
The traditional analysis of acid-base balance focuses primarily on acids and largely ignores the disposition of dietary alkali; the excretion of total unmeasured anions (potential HCOWith either an acid or an alkali load, the overall purpose seems to be
avoiding large swings in urine pH (6). Minimizing bicarbonaturia despite large changes in alkali intake could be important in lessening the risk of kidney stone formation and permitting HCO
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Kamel S. Kamel for helpful discussions and suggestions during the preparation of this manuscript. We are also indebted to Stella Tang and Chee Keong Chong for technical assistance and Jolly Mangat for secretarial assistance.
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FOOTNOTES |
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This research was supported by Canadian Institutes for Health Research Grant MT-15485.
Address for reprint requests and other correspondence: M. L. Halperin, Lab No. 1, Research Wing, St. Michael's Hospital, 38 Shuter St., Toronto, Ontario, Canada M5B 1A6 (E-mail: mitchell.halperin{at}utoronto.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 8, 2002;10.1152/ajprenal.00006.2001
Received 9 January 2001; accepted in final form 18 December 2001.
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