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INVITED REVIEW
1Nephrology Section, Tulane University School of Medicine, New Orleans, Louisiana 70130-5927; and 2Nephrology Unit, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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 TOP ABSTRACT EVALUATION OF ACID-BASE BALANCE...A CONSIDERATION OF POSSIBLE...CHARGE BALANCE IN RELATION...RELATIONSHIP OF CA BALANCES...ACID-BASE BALANCE IN PATIENTS...REFERENCES |
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balances. Equivalently positive K+ and Ca2+ balances, and thus positive charge balances, accompany these negative acid imbalances. Similarly, positive Na+ balances, and thus positive charge balances, accompany these negative acid balances during the administration of NaHCO3. These charge balances are likely the result of the adsorption of onto the crystal surfaces of bone mineral. There do not appear to be significant errors in the measurements. acid production; acid excretion; acid balance; charge balance; calcium balance; potassium balance; sodium balance; organic anions; sulfate; ammonium
This report reviews the development of techniques of evaluating the sources and rates of fixed acid production in relation to renal net acid excretion, thus allowing assessment of acid balance in humans. The changes from control that occur when acid production is increased or decreased will be used to clarify the responses of both acid production and excretion and of acid balances and mineral balances. We also review the possible sources of error in the measurements of acid production and renal net acid excretion and the relationship of charge balance to acid balance.
The results demonstrate that renal net acid excretion does not increase as much as acid production when acid production is experimentally increased among healthy subjects, so that acid balances become positive. This imbalance is accompanied by equivalently negative charge balances that are the result of bone buffering of retained H+ and loss of Ca2+ from bone into the urine. The data also demonstrate that when acid production is experimentally reduced renal net acid excretion does not decrease as much so that acid balances become negative, or, in opposite terms, there are equivalently positive balances. This imbalance is accompanied by equivalently positive K+ and Ca2+ balances, and thus positive charge balances during the administration of KHCO3, or positive Na+ balances and thus positive charge balances during the administration of NaHCO3. These charge balances are likely the result of the adsorption of KHCO3 or of NaHCO3 onto the crystal surfaces of bone mineral.
The evaluation of potential errors in the measurements required to quantitate acid production and net acid excretion shows that the contribution of intestinal absorption of dietary acid or base must be directly evaluated by measuring dietary and fecal composition and cannot be indirectly assessed by measurements of urinary composition. These assessments also show that there are small errors, both positive and negative, that affect the measurement of urinary organic anions as a component of acid production. There is, however, no evidence for the existence of urinary cations, other than 
, that contribute to net acid excretion.
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EVALUATION OF ACID-BASE BALANCE IN HEALTHY HUMANS |
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TOPABSTRACTEVALUATION OF ACID-BASE BALANCE...A CONSIDERATION OF POSSIBLE...CHARGE BALANCE IN RELATION...RELATIONSHIP OF CA BALANCES...ACID-BASE BALANCE IN PATIENTS...REFERENCES |
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concentration 
and PCO2 (proportional to H2CO3) concentrations are stable. Thus blood pH or [H+] is also stable. Because the buffering capacity and acidity of body compartments with which the plasma is in equilibrium are steady in health, normal adults must be in balance (e.g., intake-output = 0) with respect to the daily production and excretion of hydrogen ions (or actual or potential ). The rate of production of the volatile acid H2CO3 from neutral fats, carbohydrates, and proteins is matched by an equivalent rate of excretion of CO2 by the lungs. Similarly, in health there is daily production of nonvolatile or "fixed" acids. When such acids are produced, the protons (H+) liberated by the dissociation of the acid react immediately with body buffers that can be represented by plasma 
, resulting in the formation of H2CO3 that is transported to the lungs and excreted as CO2. Obviously, ongoing consumption of body buffers by such a mechanism could not continue for any significant time without regeneration of . Therefore, it can also be assumed that the net rate of fixed acid (e.g., noncarbonic acid) input each day must be matched by an equivalent rate of acid removal so that net external (fixed) acid balance must equal zero. Such a process is analogous to the equivalence of metabolic H2CO3 (CO2) production and pulmonary excretion. Many studies during the first half of the 20th century led to an understanding that in health the kidneys excrete acid. These processes include the following:
1) The titration of filtered buffers, chiefly the titration of 
to 
(termed titratable acid), that are measured by titration of the urine from urinary pH to blood pH, usually pH 7.4 or estimated by calculation based on the urinary phosphate concentration, urinary pH, and the pKa2 = 6.8 for the dissociation 
and on the urinary concentration of creatinine (see below).
2) The production and excretion of ammonium 
by renal tubular cells from neutral precursors to form urinary (measured as total urinary plus the negligible amounts of NH3 when urine pH 
7.5). The protons (H+) thus added to the urine are, ultimately or in effect, derived from H2CO3 and result in the simultaneous addition of equivalent quantities of to renal venous blood. The excretion of is quantitatively equal to the net generated from the metabolism of glutamine to and . Thus the excreted is equivalent to the H+ excreted and the new regenerated (2).
3) Additionally, under most conditions, nearly all of the that appears in the glomerular filtrate is reabsorbed, thus preventing urinary loss of filtered (base).
Thus during the steady state in health, the daily net rate of acid excretion by the kidneys can be quantitated as urinary titratable acid plus less any filtered escaping renal tubular reabsorption and excreted into the urine. This quantity is termed renal net acid excretion. The overall excretion of net acid is now known to be accomplished by multiple transport processes along the nephron, including Na+/H+ exchange in both the proximal and distal tubules (7) and by both an H+-ATPase and an H+-K+-ATPase as well as by secretion in distal nephron segments (14, 27, 57).
Because protons (H+) are always available throughout body fluids and thus cannot be directly traced, the techniques for identifying and quantifying the sources of fixed acid production are, necessarily, indirect. Measurements of metabolic products (conjugate bases, anions) that, by biochemical reasoning, must have been associated with the production or consumption of H+ are thus required. Early in the 20th century, several sources of fixed acid production had already been recognized; these can be detected based on urinary constituents:
1) The urinary excretion of organic acid anions reflecting either the ingestion and absorption of nonmetabolizable free organic acids in the diet, the formation of acids as end products of metabolism such as uric and oxalic acids; or the incomplete oxidation to CO2 + H2O of acids normally produced during metabolism, such as lactic and citric acids (and acetoacetic and 
-hydroxybutyric acids during starvation or diabetic ketoacidosis, etc.), accompanied by the production, retention and buffering of an equivalent quantity of H+ (73). Urinary organic anions have also been referred to as representing "loss of potential base," but this is correct only with respect to those urinary organic anions such as citrate, lactate, pyruvate, etc. that could have been metabolized to if retained within the body. Those urinary anions that reflect the formation of acid end products of metabolism such as oxalate, urate, etc. identify acid production.
2) The urinary excretion of 
, reflecting the oxidation of sulfur contained in the neutral amino acids methionine, cysteine, or cystine of dietary protein or endogenous tissue proteins during starvation (28, 41, 71) that is also accompanied by the generation of an equivalent quantity of H+. Additionally, it was known that normal foods in the diet contain bases, as actual or potential as inorganic cationic salts (principally K-salts) of metabolizable organic anions such as citrate, acetate, etc. (71).
Whether acid or base might be excreted into the feces in health had not been clarified, although large fecal losses of actual or potential with severe diarrhea as in cholera had been known for a century or more (70). However, techniques had not yet been developed that might permit, among healthy adults in the steady state, both identification of the sources of acid production as well as a quantitative comparison of the daily rate of acid production to the daily rate of renal net acid excretion.
The original impetus for such studies arose from observations among patients with advanced chronic kidney disease who exhibited low, but stable, serum associated with low rates of renal net acid excretion related to the low rate of urinary excretion that accompanies advanced kidney diseases (67). By contrast, healthy subjects eating comparable diets had normal serum and much higher rates of and, hence, net acid excretion. These disparate observations led to consideration of two alternative explanations: 1) the stabilization of serum among the patients with chronic kidney diseases resulted from a reduction in the daily rate of fixed acid production equivalent to the limitation in net acid excretion caused by the kidney disease; or 2) normal rates of fixed acid production continued, and during acidosis additional routes for acid excretion or disposal (buffering) might become operative, serving to stabilize the low serum . A review of the acid balances and bone among patients with renal failure is presented in a subsequent section of this review.
Assessment of the Sources of Acid Production and Acid Balances Among Healthy Adults Using Liquid-Formula Diets
The original technique of quantitating acid production compared with net acid excretion among healthy adults utilized a liquid-formula diet (65). That diet contained glucose, cornstarch hydrolysate (dextrin), corn oil, and a purified soy protein. Na+ was provided as NaCl. K+ was provided as KCl. The soy protein was prepared at its isoelectric point and contained essentially no Na+, K+, Ca2+, Mg2+, or Cl-. However, the protein did contain PO4 (17 mmol/100 g), presumably mostly as esters of the hydroxylated amino acids serine and threonine. For some of the original studies (65), the metabolism of this esterified PO4, effectively to H3PO4, was considered to quantitatively represent acid production when H3PO4 was subsequently present as and in blood in a ratio of 4:1 at pH = 7.4, at which pH the valence of PO4 = 1.8. For a few of the original studies (65) and all subsequent studies using soy protein formulas, 0.45 mmol = 0.9 meq of Ca(OH)2 and 0.45 mmol = 0.9 meq Mg(OH)2/mmol PO4 in the protein were added to the formula, thus neutralizing to pH 7.4 the potential acid from the PO4 in the protein. Because the other mineral salts in these diets were neutral (NaCl, KCl), the estimated unmeasured anion of these soy formulas was approximately zero, both by calculation and based on analyses of the diets {e.g, 
(Na+ + K+ + Ca2+ + Mg2+), meq/day - [(Cl-) + (1.8 · PO4), meq/day] = 0}. Moreover, because the formulas did not contain any fiber, the subjects defecated on average only every 2.5 days, and average fecal weight was only 45 g/day. Consequently, for those studies possible excretion of acid or base into the feces was considered minimal and was neglected.
Utilizing these formula diets, we observed that among healthy subjects, studied when their serum [] was normal and stable, mean daily net fixed acid production, estimated as urinary + urinary organic acids, was, on average, identical to the mean daily rate of net acid excretion. Thus the net external fixed acid balance was zero, the subjects being in balance with respect to fixed acid production and excretion (65). Additional studies using formula diets were also carried out to assess the effects of experimental alterations in acid production on acid balance achieved by either increasing net fixed acid production among healthy subjects by the administration of NH4Cl or reducing net fixed acid production by the administration of NaHCO3 (36). Those studies showed that NH4Cl administration caused systemic acidosis but that the consequent increase in net acid excretion was less than the increase in acid production (= + organic acids + the NH4Cl load) so that acid balances became positive (36). Moreover, NaHCO3 administration reduced fixed acid production, but the decrease in renal net acid excretion was not as large so that acid balance became negative (i.e., there was positive base balance) (36). Serum was steady during these studies of NH4Cl or of NaHCO3 loading.
In summary, studies using neutral liquid-formula diets demonstrated that fixed acid production was identified by the urinary excretion of + organic anions, the sum of these two matching the simultaneous rate of renal net acid excretion so that healthy adults with normal and stable serum were in acid balance. Furthermore, the failure of net acid excretion to increase as much as did acid production during NH4Cl loading or to decrease as much as did acid production during NaHCO3 loading implied the retention of acid or base (HCO3), respectively, at sites outside the ECF or the apparent space of distribution for HCO3.
Assessment of the Sources of Acid Production and Acid Balances Among Healthy Adults Using Normal Whole-Food Diets
During the original development of the methods to assess the components of acid production and excretion (41, 65), it again became evident that normal whole-food diets contain base as actual or potential as inorganic cationic salts (principally K-salts) of metabolizable organic anions such as citrate, acetate, etc. The precise identity and quantity of such base(s) ingested each day in the diet could not be directly measured. However, that quantity could be indirectly estimated as the difference between the dietary content of inorganic cations ( Na+ + K+ + Ca2+ + Mg 2+, meq) and of inorganic anions Cl- + (1.8 · PO4), meq] (43). It was also observed that the feces similarly contain actual or potential base, known to be principally acetate, propionate, and butyrate (43). Once again, the precise identity and quantity of such fecal base(s) could not be directly measured. However, the daily fecal excretion of such base could similarly be estimated using the same calculation applied to the analyses of the diet (43). The difference between the dietary intake of unmeasured anion and the fecal excretion of unmeasured anion thus represented the net intestinal absorption of unmeasured anion, generally a positive number, reflecting addition of actual or potential to the body. During the original studies of this type (43), it was observed that for a group of 16 healthy adults fed various diets that fixed acid production measured in this manner averaged 59 ± 30 (SD) meq/day and net acid excretion averaged 60 ± 21 meq/day. Accordingly, mean acid balance for this group was not different from zero (P > 0.7), averaging -1 ± 12 meq/day (43). The individual data for net acid excretion in relation to fixed acid production are compared among these subjects in Fig. 1 (43).
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Effects of Base or Acid Administration to Healthy Human Subjects: Results of Paired Studies During Control Periods and During Acid or Base Administration Periods in the Same Subjects
Obviously, multiple analyses of diets, feces, and urine are required for the measurement of acid balance, and multiple analytic errors might significantly bias the results. However, when groups of subjects are studied while eating individually constant diets during both control conditions and during the continuous administration of additional acid or base, the analytic variations should be minimized. Therefore, the changes from control (
) in the components of the acid balance should clarify the processes involved in the consequent increases or decreases in acid production, the accompanying increases or decreases in renal net acid excretion, the directional changes in acid balance, and the distribution (buffering) of retained base or acid. Moreover, the resulting changes from control in the balances of other minerals and of charge balance can also be evaluated.
Effects of increasing acid production using NH4Cl. Fourteen healthy adults were studied while eating individually constant diets during control conditions and during the administration of NH4Cl in varying but individually constant doses ranging from 138 to 384 meq · day-1 · subject-1 and averaging for the group 231 ± 69 (SD) meq/day (3.23 ± 0.61 meq · kg body wt-1 · day-1) (1, 37, 74). The control observations were obtained during a 6-day period after 10 days of adaptation to each subject's constant whole-food diet. The observations during NH4Cl administration were also obtained during a 6-day period that began 12 days or longer after NH4Cl was begun. As shown in Table 1, fecal Cl- excretion did not change from control rates during the administration of NH4Cl, indicating that all of the administered Cl- was absorbed. Fecal excretion of unmeasured anion also did not change. Thus on average for the group, the decrease in net intestinal absorption of base (unmeasured anion) was equivalent to the quantity of NH4Cl administered. The consequent alteration in systemic acid-base balance and renal net acid excretion during NH4Cl (see below) had no effect on the fecal excretion of actual or potential base (as unmeasured anion). Because the sum of urinary excretion of and organic anion was nearly unchanged during the administration of NH4Cl, net fixed acid production increased by a quantity equivalent to the increased intestinal absorption of the NH4Cl administered (in effect reflecting the metabolism of the administered NH4Cl to urea + HCl). However, renal net acid excretion did not increase to an equivalent degree. Thus the average acid balance for the group became significantly more positive by +24 ± 22 meq/day; P = 0.001. As shown in Fig. 2, these 14 subjects were, on average, in a steady state with respect to body weight, serum , and net acid excretion during control and with respect to serum and net acid excretion during NH4Cl acidosis. However, during induced acidosis these subjects exhibited a decline in body weight, averaging for the group -0.046 kg/day. Accompanying the retention of acid, Ca2+ balances (Table 1) became negative by an average of -16 ± 12 meq/day, P < 0.001, because of increased excretion of Ca2+ into the urine without any detectable change in net intestinal absorption of dietary Ca2+. Additionally, K+ balances also were slightly, but significantly, more negative by -4 ± 6 meq/day; P = 0.033, and Mg2+ balances were marginally more negative by -4 ± 8 meq/day; P = 0.052. PO4 balances were more negative by -6 ± 3 mmol/day; P < 0.001. The cumulative quantity of Ca2+ lost during the 6-day balance period averaged 96 meq, an amount that could only have been derived from bone, the sole significant body store of Ca2+. In view of the loss in weight accompanying the retention of H+ during stable NH4Cl acidosis, it seems reasonable to assume that the K+ and Mg2+ losses and some of the PO4 losses derived from cells, likely muscle cells but possibly including bone, as a result of the adverse effects of acidosis on protein metabolism (reviewed in Ref. 54), whereas most of the Ca2+ was lost from bone mineral together with 
(8, 11-13). Urinary hydroxyproline excretion also increases during NH4Cl acidosis, providing further evidence for increased bone resorption (33). The effects of metabolic acidosis on bone and bone cells in vitro are well known and reviewed elsewhere (10, 24).
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Effects of decreasing acid production using KHCO3. The effects of KHCO3 administration are presented in Table 2 (34). Ten healthy adults were observed during a control 6-day balance period that began after they had eaten their constant diet for 10 days. They were then observed while continuing to eat the same diet during a 6-day experimental balance period that began 6 days after the ongoing administration of KHCO3, 61 ± 1 mmol/day (0.90 ± 0.11 meq · kg body wt-1 · day-1) was begun. As shown in Table 2, fecal K excretion did not change from control rates during the administration of KHCO3, and the fecal excretion of unmeasured anion also did not change. Thus on average for the group, all of the administered KHCO3 was absorbed, and the increase in net intestinal absorption of base (unmeasured anion) was equivalent to the quantity of KHCO3 administered. The consequent alteration in systemic acid-base balance and renal net acid excretion had no effect on the fecal excretion of actual or potential base (as unmeasured anion). Because the sum of urinary excretion of and organic anion was nearly unchanged during the administration of KHCO3, net fixed acid production decreased by a quantity equivalent to the increased intestinal absorption of the administered as KHCO3. However, renal net acid excretion did not decrease to an equivalent degree. Thus the average acid balance for the group became significantly more negative by -13 ± 11 (SD) meq/day; P = 0.005. Equivalently, but in opposite terms, balance became more positive by +13 ± 11 meq/day during KHCO3 administration. Accompanying the retention of HCO3, K+ balances became almost equivalently more positive by +11 ± 10 mmol/day; P = 0.006. Additionally, Ca2+ balances became slightly, but significantly, more positive by 1.8 ± 1.6 meq/day; P = 0.009. The changes in K+ + Ca2+ balances during KHCO3 administration were matched by an equivalently more positive charge balance or, in other words, a more negative acid balance (equivalent to retention). Thus it seems reasonable to conclude that KHCO3 was retained. As shown in Fig. 3, these subjects were, on average, in a steady state with respect to body weight, morning fasting serum , and daily renal net acid excretion during both control conditions and during KHCO3 administration. Moreover, average body weight for the group did not differ during KHCO3 administration compared with control. Mean fasting serum was slightly, but not significantly, higher during KHCO3 administration in these studies, although during other studies, when 90 mmol of KHCO3 were administered each day, serum was observed to increase significantly when measured 90 min after the ingestion of one-third of the daily KHCO3 dose (30 mmol) (39). Serum K+ concentrations did not increase (not shown). Body weight was measured to the nearest 0.01 kg daily in each subject and was stable. That observation indicates that intracellular water was not retained in osmotic proportion to the cumulative retention of 66 mmol K+ over the 6-day balance period because retention of such a quantity of K+ within cells (with an anion) would have been expected to obligate retention of water, resulting in an increase body weight averaging 
0.5 kg, an amount that should have been detectable. Thus it seems reasonable to propose that the KHCO3 was retained in an osmotically inactive form. Speculatively, KHCO3 was retained as a solid phase adsorbed or secreted onto the vast surfaces of apatite in bone (11, 15).
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Effects of decreasing acid production using NaHCO3. The same 10 healthy adults were also studied, using the same control period and the same individually constant diets, during a 6-day period that began 6 days after the continuous administration of NaHCO3 (60 ± 1 meq/day, 0.88 ± 0.11 meq·kg body wt-1·day-1) was begun (34). As shown in Table 3 and Fig. 3, the effects of NaHCO3 were, for the most part, similar to those of KHCO3 administration in that urinary net acid excretion did not decrease as much as did acid production so that acid balances became more negative by -12 ± 11 meq/day; P = 0.011. Accompanying this retention of , Na+ balances became almost equivalently more positive by +11 ± 14 mmol/day; P = 0.033 without any change in body weight, suggesting the retention of NaHCO3 in an osmotically inactive form as a solid phase adsorbed or secreted onto the surfaces of apatite in bone (11). Unlike the effect of KHCO3 administration, the administration of NaHCO3 was not accompanied by a reduction in urinary Ca2+ excretion or more positive Ca2+ balances. The failure of NaHCO3 to cause Ca2+ retention is the result of the additional Na load, causing extracellular volume expansion and increased urinary Ca2+ excretion when the diet also contains NaCl. Increasing dietary NaCl intake is known to increase urinary Ca2+ excretion (5, 39, 53, 56). Additionally, the ongoing administration of mineralocorticoid when the dietary intakes of NaCl and Ca2+ are constant is accompanied by NaCl retention, weight gain, and increased urinary Ca2+ excretion (44). Other studies have also shown that the administration of NaHCO3 when the diet also contains NaCl does not result in a sustained reduction in urinary Ca2+ excretion (34, 39). However, when dietary Na intake is kept constant by substituting NaHCO3 for part of the NaCl in the diet, urinary Ca2+ excretion does decline (50).
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During growth, daily rates of renal net acid excretion among infants have been observed to exceed rates of fixed acid production, so that daily acid balances are negative (30) as during the administration of KHCO3 or NaHCO3 to adults. That effect necessarily must accompany the deposition of alkaline mineral into the growing skeleton, largely as Ca10(PO4)6(OH)2/Ca10(PO4)6CO3, a process that may, in opposite terms, be thought of as an additional source of acid production.
Overview of acid production and excretion. Figure 4 summarizes the relationship between the changes from control in daily urinary net acid excretion and the changes in net endogenous fixed acid production for the individual subjects given NH4Cl (1, 37, 74; summarized in Table 1), the subjects given KHCO3 (34; summarized in Table 2), and among 4 additional subjects in which acid production was increased by increasing dietary protein intake as egg white (1). The relationship is net acid excretion, meq/day = 3.81 ±[(SE)2.77] + 0.90667 ±(SE)[0.0161] · acid production, meq/day. The slope for this relationship is significantly <1, but the intercept is not different from zero. Because net acid excretion did not increase as much as acid production when acid production was increased using NH4Cl or egg white, the data points for those studies lie below the identity line. By contrast, the data points for the subjects given KHCO3, with one exception, lie above the identity line because net acid excretion did not decrease as much as did acid production. In other words, the data viewed in this manner also indicate that acid balances become positive when acid production is increased and become negative, reflecting retention, when acid production is reduced.
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To summarize, net fixed acid production among healthy adults eating normal diets can be estimated by the sum of urinary + organic anions less the difference between the sum of inorganic cations and anions in the diet and their sum in the feces. This quantity matches urinary net acid excretion so that healthy adults with normal and stable serum are in acid balance. When acid production is experimentally increased, acid balances become positive and Ca2+ balances become negative, reflecting buffering by bone. When acid production is experimentally reduced, acid balances become negative, reflecting equivalent retention together with K+ and Ca2+ retention during the administration of KHCO3 and of Na+ retention when dietary Na+ intake is kept constant during the administration of NaHCO3, either also reflecting buffering of base by bone.
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A CONSIDERATION OF POSSIBLE ERRORS IN THE ESTIMATES OF FIXED ACID PRODUCTION AND RENAL NET ACID EXCRETION |
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TOPABSTRACTEVALUATION OF ACID-BASE BALANCE...A CONSIDERATION OF POSSIBLE...CHARGE BALANCE IN RELATION...RELATIONSHIP OF CA BALANCES...ACID-BASE BALANCE IN PATIENTS...REFERENCES |
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Urinary sulfate. Oxidation of organic sulfur to sulfate, as reflected by the daily urinary excretion rates of , is the first major component of fixed acid production. The measurements of urinary using either gravimetric (63) or turbidometric (4) methods appear to be adequately specific and precise and therefore not a significant source of error. The possibility exists that after its production significant quantities of might be excreted via other routes, thereby escaping detection and leading to an underestimation of fixed acid production. However, even during methionine loading, when production and urinary excretion rates were markedly increased, fecal excretion was negligible (41). Losses of sulfate via the skin would also appear to be negligible even with moderate visible sweating.
Urinary organic anions. Urinary organic anion excretion in humans reflects the second major component of fixed acid production. Presently, various precise methods do not appear to be available that allow individual identification and quantitation of all these organic anions. Thus their estimation remains dependent on an estimate derived from the titration of urine from pH 2.7 to urinary pH, after removal of urinary PO4 by incubation of the urine with solid Ca(OH)2 and reacidification of the filtrate to pH 2.7, a method devised early in the last century (73).
Studies in rats have shown that the urinary excretion of organic acids falls with acid loading and rises with alkali loading (6, 17, 61). However, as shown in Fig. 5, using the individual data for the subjects whose studies are summarized in Tables 1, 2, 3, the daily urinary excretion of total organic acids, as titrated between pH 2.7 and 7.4, did not change significantly as urinary pH or daily net acid excretion varied during the administration of NH4Cl, KHCO3, or NaHCO3. Thus unlike rats, in which urinary excretion rates of organic anions decrease markedly during acid loading and increase markedly during the administration of loads (6, 17, 61), humans do not exhibit either an evident decrease in total organic acid excretion in response to acid administration or an increase in response to the administration of base.
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By contrast, as shown in Fig. 6 (1, 33, 34, 37, 41, 65, 74), the percentage of urinary organic acid anions that are titrated between pH 2.7 and urinary pH and that constitute a component of net fixed acid production increases, in a manner resembling the upper segment of a sigmoid titration curve, from 70 to 80% of total urinary organic acids (titrated from pH 2.7 to 7.4) at a urinary pH of 5 to nearly 100% above a urinary pH of 7. By contrast, the percentage of total urinary organic acids that are excreted as free acids (titrated between urinary pH and 7.4) necessarily decreases from 20 to 30% of the total at pH 5 to only a few percentage points or zero at a urinary pH above 7. These free organic acids do not contribute to fixed acid production. Thus, as shown in Fig. 7, which uses the individual studies that are summarized in Tables 1, 2, 3 (1, 34, 37, 74), the daily urinary excretion rates of organic acid anions decrease as urinary pH falls and tend to increase as urinary pH rise, whereas their excretion rates fall as net acid excretion increases and increase as net acid excretion decreases.
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The pKa2 for the dissociation of 
is 1.7. Accordingly, it has been suggested that the titration of organic anions between pH 2.7 and urinary pH will overestimate organic anions because of the titration of some 
, theoretically resulting in the titration of about 10% of urinary . A reevaluation of measured mean daily organic anion excretion rates for five subjects observed during control, methionine loading, and recovery (41) demonstrates that organic anion excretion rates do increase slightly above control rates as excretion rates increase during the administration of methionine and on the first recovery day when urinary SO4 excretion rates remain above control. This relationship is shown in Fig. 8. On average, organic anion increases by 3.5 meq · day-1 · 100 meq 
. Extrapolation of that relationship to the wide normal range of daily rates of urinary excretion, which may range from 15 meq/day during the ingestion of a low-protein diet to 75 meq/day during the ingestion of a high-protein diet, would indicate that the titration could overestimate urinary excretion rates of organic anions by 1-3 meq/day.
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Urinary citrate is one of the components of urinary organic anions. Citrate excretion in humans is well known to fall with acid loading and rise with alkali loading. Some data illustrating these effects are presented in Fig. 9 (33, 34, 39). As shown in Fig. 9A, daily urinary citrate excretion falls exponentially as net acid excretion rises. Among subjects being given KHCO3 or NaHCO3, citrate excretion averaged 15.3 meq/day when net acid averaged -1 meq/day. Among subjects eating only control diets, citrate excretion averaged 11.2 meq/day when net acid averaged 57 meq/day. Among subjects being given NH4Cl, citrate excretion averaged only 0.4 meq/day when net acid averaged 264 meq/day. As shown in Fig. 9B, evaluation of the changes from control in urinary citrate excretion in relation to the changes from control in net acid excretion among subjects given KHCO3, NaHCO3, or NH4Cl demonstrates that urinary citrate excretion falls linearly as net acid excretion rises.
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It has also been suggested that the treatment of urine with excess solid Ca(OH)2 to precipitate 
before organic acid titration may cause the loss of citrate, possibly by the precipitation of calcium citrate. To asses this issue, we measured the citrate concentrations in urine specimens from seven healthy subjects before and after that treatment. We found that citrate concentration decreased from 5.02 ± 3.07 to 0.87 ± 0.87 meq/l or by an average of -83%. This methodological error indicates the loss of citrate due to Ca(OH)2 before organic acid titration would result in the underestimation of urinary anion excretion. When acid production exceeds 100 meq/day, urinary citrate falls progressively below 5 meq/day (Fig. 9A) so that in such circumstances the loss of citrate as a consequence of the Ca(OH)2 treatment before the determination of organic anions would cause an underestimation of the later value by 1-4 meq. Urinary citrate excretion rises when acid production decreases (Fig. 9A). Urinary citrate excretion was measured before and during the administration of KHCO3 and NaHCO3 in 9 of the 10 subjects given those salts (Tables 2 and 3; Ref. 34) and rose on average 4.8 ± 2.2 meq/day (P < 0.001) during the administration of KHCO3 and rose 3.0 ± 3.0 meq/day (P < 0.025) during the administration of NaHCO3 (Lemann J, unpublished observations). If 80% of those increments were lost as a consequence of the Ca(OH)2 treatment before determination of organic anions, the resulting decrease in the organic anion estimate would have been only 2-4 meq/day.
Collectively, the overestimation of organic anions resulting from the titration of and the underestimation of organic anions resulting from the loss of citrate are thus small and nearly quantitatively equal, leading to the conclusion that there are no major or significant errors in evaluating by titration the contribution of urinary organic anions to acid production.
Net Intestinal Absorption of Actual or Potential Base or Acid Ingested in the Diet
Net intestinal absorption of dietary actual or potential is estimated by calculating the difference between measured dietary [(Na + K + Ca + Mg) - (Cl + 1.8 · PO4)] and measured fecal dietary [(Na + K + Ca + Mg) - (Cl + 1.8 · PO4)]. Some of the dietary anions are ingested as free unionized acids and would not be identified by this calculation but could be a source of acid production. However, if such acids were completely metabolized to CO2 + H2O, they would not contribute to fixed acid production. Furthermore, if they were absorbed and buffered and not metabolized and excreted into the urine, they would be identified as a source of acid production by their inclusion in the measurement of urinary organic anions and would equivalently increase the urinary excretion of net acid. These effects as well as the effects of other possible substances on the estimation of fixed acid production and net acid excretion are summarized in Table 4.
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To avoid the need to analyze diets and to analyze feces collected over periods of sufficient duration to ensure that the fecal collections are accurately timed, it has been proposed that the measurement of urinary [(Na+ + K+ + Ca2+ + Mg) - (Cl- + 1.8 · PO4)] would provide a more convenient estimate of the net intestinal absorption of actual or potential base or acid ingested in the diet (58). Figure 10A compares the changes from control in urinary [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8 · PO4)] to the changes from control in the directly measured net intestinal absorption of unmeasured {diet - fecal [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8 · PO4)]} for the individual subjects summarized in Tables 1, 2, 3 (1, 34, 37, 74). The slope for the relationship shown in Fig. 10A is 0.903 ± 0.013, a value that is significantly <1. Thus the estimate based on urine overestimates the changes from control of measured absorption during the administration of NH4Cl and underestimates the changes from control of measured absorption during KHCO3 and NaHCO3 administration. During NH4Cl administration, the mean of the changes from control dietary unmeasured anion absorption based on urine overestimates the directly measured mean of the changes from control absorption by an average of 16 ± 15 meq/day (P = 0.008), because the measurement based on urine does not take into account the ongoing negative Ca2+ balances (Table 1). During KHCO3 administration, the mean of the changes from control of dietary unmeasured anion absorption based on urine underestimates the mean of the changes from control of directly measured absorption by an average of -10 ± 10 meq/day (P = 0.011), because the urine measurement does not take into account the ongoing positive K and Ca2+ balances (Table 2). Similarly, during NaHCO3 administration, the mean of the changes from control absorption based on urine underestimates directly measured mean of the changes from control absorption by an average of -11 ± 12 meq/day (P = 0.015), because the urine measurement does not take into account the ongoing positive Na+ balances (Table 3). Alternatively, the changes from control in charge balances become positive as the changes from control in acid balance become negative during the administration of KHCO3 or NaHCO3, whereas the changes from control in charge balances become negative as the changes from control become positive during the administration of NH4Cl (see below).
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When Oh (58) suggested that the measurement of net intestinal absorption of potential base or acid could be indirectly assessed by measurement of urine [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8 · PO4)], that view was based on a reanalysis of earlier data (36, 39, 43, 45, 65) that are inadequate for such an evaluation. Except for the data for five subjects given NH4Cl (39), the other data were for studies utilizing liquid-formula diets (36, 43, 63) during which Mg2+ was not measured. Moreover, during the latter studies the duration of fecal collections was too short to adequately evaluate daily excretion rates of fecal Na+, K+, Ca2+, Cl-, and PO4 as well as Mg, had it been measured. Figure 10B shows urinary [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8 · PO4)] in relation to directly measured net intestinal absorption of unmeasured anions {diet - fecal [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8* PO4)]} using the absolute measurements during control and during either the administration of NH4Cl, KHCO3, or NaHCO3 for the studies summarized in Tables 1, 2, 3, as proposed by Oh (58). When the data are viewed in this manner, the slope for the relationship is 0.987 ± 0.029, a value that is not different from one. Thus the changes from control (Fig. 10A) become undetectable, and the urinary and directly measured estimates of intestinal absorption of cations or anions do not differ. Consequently, during the administration of NH4Cl, that estimation of intestinal absorption of unmeasured anions based on urinary composition does not, in the absence of Ca2+ balance data, reveal that the increase in net acid excretion is less than the amount of potential acid fed, indicating H+ retention, nor that the increase in urinary Ca2+ is derived from body stores (bone), not enhanced intestinal Ca2+ absorption (see below). Similarly, the use of the measurement of urinary composition [(Na+ + K+ + Ca2+ + Mg2+) - (Cl- + 1.8 · PO4)] to estimate intestinal absorption of unmeasured anions during the administration of KHCO3 or NaHCO3 does not, in the absence of K+ or Na+ balance data, reveal that the decrease in net acid excretion is less than the amount of base () administered nor the retention of K+, Ca2+, or Na+, as previously described. Thus, unfortunately, urinary measurements and analyses alone do not adequately reflect important changes in acid-base and electrolyte balance with changes in dietary acid-base content.
In summary, the components of fixed acid production appear to be adequately measurable without significant errors as the sum of urinary + organic anions less the difference between the sum of inorganic cations and anions in the diet and their sum in the feces.
Components of Urinary Net Acid Excretion
Ammonium. The measurements of urinary ammonium, whether by microdiffusion and titration (19) or an automated method (49), appear to be specific and sufficiently precise and thus not subject to significant error.
The possibility has been suggested that urine may contain other cationic buffer(s) capable of carrying secreted H+ into the urine in addition to , perhaps histidine or a similar substance (60). On theoretical grounds, histidine is unlikely to serve in this manner. Whereas pKa for the dissociation of the iminazole group of histidine is 6.0, so that one-half of the histidine present at an average urine pH of 6.0 would buffer a proton, the average urinary excretion of histidine among adults is in the range of 0.5 to 1.0 mmol/day, so that histidine would carry only a very small amount of H+ secreted along the nephron into the final urine. Further evidence for the absence of significant quantities of unmeasured cation as well as unmeasured anion is summarized in Fig. 11. The mean sum of the directly measured urinary anions for a large number of studies (1, 34, 36, 37, 40, 42, 51; Lemann J, unpublished observations), estimated as + Cl- + + + + organic anions, averaged 298 meq/day. That value was not significantly different from the simultaneously measured sum of the urinary cations, estimated as Na+ + K+ + Ca2+ + Mg2+ + + creatinine+, that averaged 303 meq/day. Furthermore, when the relationship between the sum of the anions for each subject was evaluated in relation to the sum of the cations for each subject, the slope of the relationship was not different from unity and the intercept was not different from zero. Additionally, 76 of these studies were carried out while the subjects ate control diets (mean urinary pH = 6.05 ± 0.44; mean urinary net acid excretion = 49 ± 28 meq/day), 26 studies while the subjects received NH4Cl (mean urinary pH = 5.40 ± 0.13; mean urine net acid excretion = 272 ± 61 meq/day), and 20 studies while the subjects received KHCO3 or Na HCO3 (mean urinary pH = 6.67 ± 0.18; mean urinary net acid excretion = 6 ± 13 meq/day). Regardless of variations in fixed acid production and both net acid excretion rate and urinary pH, the data points are similarly distributed above and below the identity line. Thus experimental variation of acid-base balance does not reveal the urinary excretion of presently unrecognized or unidentified urinary cations or anions.
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Titratable acid. Historically, titratable acid has most often been directly determined by titration of urine from urinary pH to blood pH, usually pH = 7.40. The titration measures the contribution of the major urinary buffers, phosphate and creatinine, to urinary proton excretion and, in addition, the contribution of urinary organic anions that are, effectively, not ionized at urinary pH. The latter fraction of urinary organic acids, that are titrated between urinary pH and blood pH, does not represent acid excretion because they are excreted as free acids, just as they were produced. Moreover, these free urinary organic acids are not a component of net acid excretion as they do not identify regeneration. Furthermore, it has been known for almost a century (21), and subsequently confirmed (35), that when urine contains increasingly large concentrations of ammonium, titratable acid, as measured by direct titration, is increasingly overestimated because of the titration of 
. Additionally, when urine contains increasingly large concentrations of Ca2+ together with 
, there is precipitation of CaHPO4/Ca3(PO4)2(OH)2/Ca10(PO4)6CO3 from solution when the titration reaches a pH of 
7.0, resulting in the simultaneous release of H+ that also causes overestimation of titratable acid. Accordingly, it would appear preferable to estimate titratable acidity by calculation using urinary pH, the urinary contents of PO4, and a pKa2 = 6.8 for the reaction , and the urinary content of creatinine and a pKa = 4.97 for the reaction creatinine+ 
creatinine + H+ (35).
. The measurement of urinary excretion requires the collection of urine under a layer of mineral oil or toluene to minimize the loss of dissolved CO2. The concentration of is then calculated from measurements of urinary pH and total CO2 concentration, as employed in the studies reviewed in this report, or of urinary pH and PCO2. Figure 12 illustrates the exponential relationships between urinary and urinary pH and between daily urinary excretion rates and pH for a large number of 24-h urinary measurements (34, 39, 40, Lemann J, unpublished observations). When urinary pH is in the range of 6.0-6.4 or less, are mostly 5 mmol/l, but daily urinary excretion rates may reach 10-15 mmol/day, quantities that significantly reduce renal net acid excretion. When urinary pH rises to levels above pH 6.4, urinary and excretion rates increase progressively. The urinary loss of becomes negligible when urine pH is 6.0.
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To summarize, renal net acid excretion appears to be accurately measured, without significant errors, by the sum of urinary plus titratable acid as calculated from the urinary content of PO4 and creatinine together with urinary pH minus urinary HCO3.
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CHARGE BALANCE IN RELATION TO ACID BALANCE |
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TOPABSTRACTEVALUATION OF ACID-BASE BALANCE...A CONSIDERATION OF POSSIBLE...CHARGE BALANCE IN RELATION...RELATIONSHIP OF CA BALANCES...ACID-BASE BALANCE IN PATIENTS...REFERENCES |
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Figure 13A summarizes the changes from control of charge balances in relation to the changes from control of acid balances for the subjects given NH4Cl (Table 1), KHCO3 (Table 2), or NaHCO3 (Table 3). As shown in Fig. 13B, negative Ca2+ balances primarily account for the negative charge balances during the administration of NH4Cl, whereas positive K+ + Ca2+ or positive Na+ balances primarily account for the positive charge balances during the administration of KHCO3 or NaHCO3, respectively. Accordingly, as shown in Fig. 13C, charge balances become negative during the administration of acid, as Ca2+ balances become negative via urinary Ca2+ losses as acid balance become positive in relation to bone buffering of H+. Charge balances become positive during the administration of base as acid balances become more negative due to retention with K+ + Ca2+ during the administration of KHCO3 or with Na+ during the administration of NaHCO3.
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) in relation to daily net fixed acid production, urinary (
} - fecal {(
) - 
} among 16 healthy adults eating constant whole-food diets (
), soy protein-formula diets (
), or diets containing egg yolk (
); y = 21.659 + 0.67075x; r2 = 0.888; P < 0.001. The slope for the line is not different from 1, and the intercept is not different from 0; P > 0.8. Redrawn from the original data in Ref. 
) and then during the administration of NH4Cl (
); y = 1.6 + 0.002x; r2 = 0.00, not significant. Adapted from data in Refs. 
(meq/day) to the sums of the cations, estimated as 
(meq/day) during control (
