Urinary ammonia content as a determinant of urinary pH during chronic metabolic acidosis

Urinary Ammonia Content as a Determinant of Urinary pH During Chronic Metabolic Acidosis Francis X. Schloeder

and Bobby J. Stinebaugh was

the urinary

During the course of metabolic acidosis, the urine pH initially falls but then rises to

observation

levels

monia

that

seem

gree of systemic the factors acidity

dosis,

using

and

acidosis.

responsible

nary sis

inappropriate

ments.

chronic the

by

The

rise

be greatest

acid

or

in

those

was

with

the

found

acidosis

and

least

milder

degrees.

This

paradoxical

ship

could

not be related

of systemic deficits,

acidosis,

acidification

of nonvolatile which

buffer

correlated

with

in

Addition

of ammonia

of acido-

nephron

with

to pro-

those

excretion.

duct

nephron

secretory

is the

shown

capacity,

acidosis.

to segments urine only

to have

pH.

segment limited

we propose

that

the

effect

of

ammonia

on

prolonged

acidosis

is due

addition

of basic

increases The

factor

the rise of urine

pH

duct by diffusion

Henle.

This

concept that mal tubule

view

is

NH, from

compatible

ammonia

entering

may be excreted

the

of the

H+

nixing

lecting

for

Since

during mented

of the

reserves

with

potassium or

large

by, the urinary

metabolic

not elevate

collecting tory

chronic

am-

determine,

relation-

to improvement

defects,

during

H+ should

supplefound most

external

be determined

aci-

alkali

pH

than

This

the urinary

may

concentration

acidity

bicarbonate

of ammonia.

that

uri-

metabolic

serum

in urine

rather

investigated

as the model

varying

concentration

We

de-

for the reduced

during fasting

to the

content

suggests

secrealkali-

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to

aug-

to the the

with the

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P

REVIOUS INVESTIGATIONS of the metabolic acidosis of fasting have shown that urinary pH (UpH) declined initially but rose after the fourth day despite the persistence of systemic acidosis. I-4 This paradoxical rise of UpH also occurs in other types of chronic acidosis5-” but has received little comment and has not been studied previously. The purpose of the present investigation was to evaluate various factors possibly responsible for the decreased urinary acidity. We studied this phenomenon in 13 subjects and documented that UpH rises regularly after the second to sixth day of acidosis. Furthermore, by varying the severity of acidosis with either alkali or acid supplements, we demonstrated that the UpH was highest in subjects with the most severe degrees of systemic acidosis and that the rise was least pronounced with milder acidosis. These data demonstrate that the urinary ammonium* concentration (UN,,) varied directly with the severity of systemic acidosis and that, in turn, UNu: correlated directly with urinary pH. We interpret this finding to indicate that UNH: is a determinant of UpH during chronic acidosis and suggest that diffu-

*In this discussion, ammonia NH;

to the protonated

will refer to the total

ammonia

(NH;

+ NHj).

ammonium

or

species, and NH3 to the dissolved gas.

From lhe Renal Secrion, Department of Medicine. Baylor College qf’ Medicine. and Ihe Methodisr and Vererans Administrarion Hospitals. Houston, Texas. Receivedjor publication January 25, 1977. Supported by research grants from the United States Army Research and Development Command and the National Science Foundation. This work was done at Gorgas Hospiral. Ancon, Canal Zone. Reprinr requests should be addressed to Dr. Bobby J. Slinebaugh. 6535 Fannin, Houston. Texas 77030. fi 1977 by Grune & Srrarron. Inc. ISSN 0026-0495.

Metabolism,

Vol. 26, No. 12 (December), 1977

1321

1322

SCHLOEDER

sion of ammonia from the loop of Henle to the collecting responsible for the increase of UpH. MATERIALS

AND STINEBAUGH

duct is the mechanism

AND METHODS

Thirteen healthy female volunteers whose weight exceeded Metropolitan Life Insurance Company medium-frame weight tables by 5.6-20.8 kg were admitted to the Clinical Study Unit of Gorgas Hospital to fast for purposes of weight reduction and to cooperate in this study. The protocol was explained in detail to all participants, and signed Informed Consent forms were obtained. A standard short ammonium chloride loading test’* was done at least IO days before admission; all subjects suppressed urinary pH to ~5.3. This test was repeated at the end of the study in all patients whose final urine pH exceeded 5.5, again with a normal response. Initial evaluation included history and physical examination, complete blood count, urinalysis, chest x-ray, electrocardiogram, serum electrolytes and protein, endogenous creatinine clearance, colony count, and 24-hr protein excretion, all of which were within normal limits. These studies were conducted over a 4-day period during which the volunteers consumed a diet of 1800 calories containing 40 mEq of potassium daily. Total caloric restriction was then begun for IO days, all subjects received 50 mg of thiamin and 60 mEq of potassium chloride daily, and distilled water ad libitum. Five subjects received sodium chloride (NaCl) 102 mEq/day during both equilibration and fasting; four received NaCl 160 mEq/day during equilibration, but during fasting received this amount of sodium bicarbonate (NaHC03); four received 160 mEq/day of NaCl during equilibration and during fasting received this amount of ammonium chloride (NHdCI). Electrolytes* were administered in divided doses over a 14-l8-hr period under the direct observation of the nursing staff. Patients remained ambulatory in the air-conditioned unit for the duration of the study. All urine voided during the study period was collected under refrigeration at 4’C in 24-hr specimens in vessels containing thymol, phenyl mercuric nitrate, and mineral oil. Completeness of collection was verified by creatinine content. Venous blood was collected daily without stasis and before any intake or activity. Determinations of pH, pCO2, and titratable acidity (TA) were made immediately and portions of serum and urine were frozen at -2O’C for later determinations of other parameters. Balance Calculations Calculations of the external balance of potassium were limited to a comparison of measured intake with measured urinary excretion on a 24-hr basis. This method was felt to be reasonably accurate: stools were rare and previous studies have demonstrated that fecal electrolyte losses are negligible in fasting subjects receiving electrolyte supplements of similar magnitude,13 and Benedict has shown that the skin losses of chloride are <5 mEq/day during fasting.14

Analytical Methods Sodium and potassium were determined by the Instrumentation Laboratory (Lexington, Mass) model IL-143 flame photometer, chloride by a Buchler-Cotlove chloridometer (Buchler Instruments, Fort Lee N.J.), blood pH and blood and urine pCO2 by Instrumentation Laboratory model IL-113 blood gas analyzer, urine pH and titratable TA by Radiometer (Copenhagen) TTTIC pH Meter and Automatic Titrator, urinary ammonia (NH:) by the microdiffusion method of Conway,” creatinine and phosphate by standard AutoAnalyzer methods; and organic acids (B-OH-butyrate and acetoacetate) by enzymatic methods as described by Williamson and Mellanby.16 The serum bicarbonate concentration (HCOI) was calculated from the Henderson-Hasselbalch equation, using values of pK’ and S of 6.10 and 0.0306, respectively. Urine bicarbonate was calculated from the urine pH and pCO2 using an S factor of 0.0309 and pK’ of 6.33 OSdm.” Net acid was calculated as the sum of the concentrations of TA + NH: HCOT. Statistical analyses were those described by Snedecor.‘*

*NaCI and NaHC03 were administered as IOOO-or 600-mg tablets; NH4CI as 600-mg gelatin capsules. By analysis, these did not vary by more than 0.5 mEq. KC1 was administered as a I5 g/100 ml solution from a lo-ml graduated cylinder to within 0.1 ml.

URINARY

AMMONIA

1323

CONTENT

RESULTS

All patients tolerated the regime of fasting with electrolyte supplementation well, and none had vomiting or diarrhea. Two subjects vomited during the ammonium chloride loading test at the end of the study period; this was repeated on the next day with normal results. Serum Values The serum concentrations of sodium and potassium remained unchanged from prefast values in all subjects throughout the period of observation. The initial 103-104 mEq/liter serum chloride concentration changed as follows: the NaHCO, group decreased to loo-102 by the fifth to sixth day; the NaCl group rose to 106108 by the fifth day; and the NH4CI group rose to 110-l 13 by the fourth day. These levels persisted for the remainder of the study period. The serum concentration of bicarbonate, initially 25.8-27.2 mEq/liter, varied in rate and magnitude of depression in relation to the electrolyte supplements: the NaHCO, group reached lowest levels of 15.2-20.8 on the sixth to seventh day; the NaCl group reached lowest levels of 13.3-15.5 on the fifth to sixth day; and the NH4CI group reached lowest levels of 10.3-15.2 on the fourth to sixth day. Over the subsequent 3-6 days there was a slight upward trend of bicarbonate concentration, the mean rise by the last day being 1.4 + 0.4 (SEM) mEq/liter (range, - 1.2 to +3.1). Data for the NaCl group were the same as those reported previously for subjects fasting with only potassium supplements.* For the purposes of this study, systemic acidosis was considered stable (steady state) from the first 24-hr period, during which the serum bicarbonate concentration did not decrease by more than 0.5 mEq/liter, while the preceding period of falling bicarbonate concentration was defined as the developing stage.

In subjects receiving NaCl or NH,Cl, there was a prompt fall in the UpH, lowest values occurring on the second to fourth day (NaCl, 5.10-5.37; NH,Cl, 4.9555.12). In those receiving NaHCO,, in whom the serum bicarbonate concentration declined more slowly, UpH fell less abruptly; both parameters generally reached lowest values (pH 5.16-5.51) within a l-day period (fifth through seventh days). Thereafter, the pH of the urine of all patients rose. The increments by day 10 were as follows: NaHCO,, 0.10-0.43; NaCl, 0.30-0.76; NH,Cl, 0.60-0.88. The mean increase for the entire group between the day of greatest urinary acidity and the final day of the study was 0.51 f 0.07 (p < 0.001). Observations of serum bicarbonate and UpH in a representative subject of each group are presented in Fig. 1. Acid Excretion Net acid excretion increased as systemic acidosis developed, maximum values generally occurring at the time serum bicarbonate reached its lowest point and remaining essentially stable during the subsequent steady state. Both components of net acid (TA and NH:) increased in parallel during the developing stage of acidosis, but thereafter TA excretion diminished while ammonia excretion (UNHtV) remained stable for the rest of the study. Thus, the fraction of

1324

ScHLoEDER

26

24

F

16

2b-

6.2-

Zb-

6.0-

20-

5.6-

16 -

5.2 -

14

5.0

AND

SWNEEIAUGH

tt 12L BLOOD wcoj

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I

2

3

4

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6

7

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pH

'

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6

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DAYS

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Fig. 1. Serum bicarbonate concentration in mM/liter and UpH during 10 days of fasting in representative subjects receiving electrolyte supplements ( NaHCOs, 160 mEq/day; NaCI, 102 mEq/day; NH,CI, 160 mEq/day) and KCI 60 mEq/day. Values not shown for subject 1 are blood bicarbonate 26.1 on day 3, and UpH 7.35 on day 1,7.41 on day 2, and 6.23 on day 3. Initially, both blood HCO; and UpH declined (developing stage), but thereafter blood HCO; became stable and UpH rose. The greatest increment of UpH occurred in the subjects with the most profound acidosis, and the least rise in those with the mildest degree of acidosis.

net acid excreted as NH: was greatest during steady state acidosis and was related to the electrolyte supplement: the NaHCO, group excreted 70% of net acid as NH$, while the NaCl and NH4Cl groups excreted 84% and 92%, respectively. These data are presented in Table 1, and Fig. 2 shows observations for representative subjects of each group. Urine volume (V) varied from 465 to 2880 ml/24 hr (mean 1240 f 108) during the developing stage, and from 410 to 2215 m1/24/hr (mean 1110 f 130) during the steady state (p < 0.5). Urinary osmolality was greater than serum in all subjects throughout both periods, averaging 868 f 21 and 585 + 18 mOsm/kg, respectively. Potassium Balance The abrupt increase in potassium intake from 40 to 60 mEq/day at the inception of fasting resulted in a positive potassium balance in the first few days and diminished total body potassium deficits. Although a kaliuresis of fasting oc-

URINARY AMMONIA

1325

CONTENT

Table 1. Excretion of Net Acid, Titratable in mM/24 Urine

Measurement

and Test

Group

Net acid NaHC03

Acidity, and Ammonia

hr + SEM in Each Group on Indicated Days

First

Day

DQY

Lowest

Day

UpH

SerumHCO;

of Lowest

Last Day

126 + 22

126 +28

NaCl

38 zt 4

123 zt 21

165 zt 24

176 +26

NH,CI

63 f 7

111 f

165 f 16

136 1-6

Tit&able

-2*2

of

12

100 + 16

acidity

NaHC03

47 * 5 (37)*

47 zt 7 (37)

30 It 2 (30)

NaCl

-12

18 f 2(47)

* 3

50+8(41)

46 i 6 (28)

29 %6(16)

NH,CI

17 * 2(27)

29 i 3 (26)

26 f 1(16)

12 %2(8)

Ammonia NaHC03

10 f 2(a)

79 f

NaCl

20 f

73 zt 15(59)

119*20(72)

147 zt 21 (84)

NH,CI

46 f 7 (73)

81 *8(74)

139 zt 15 (84)

124%5(92)

1 (53)

18 (63)

79 i

16 (63)

70 + 16 (70)

*Numbers in parentheses indicate percentage of net acid

200

r

SUBJ

1

It+

NItI' NH.,+

TA I

200

Fig. 2. Representative studies showing urinary excretion of net acid (t-l+), ammonia (NH:), and titratable acidity (TA) in mm/ 24 hr in subjects receiving electrolyte supplements during 10 days of fasting. Values for H+ and TA are not shown for subject 1 on days 1 and 2, since there was excretion of net alkali on these days. The quantity of net acid increased in all subjects, but the proportion excreted as NH: ‘was lowest in the least acidotic group ( NaHCOs) and greatest in the most acidotic group (NH4CI), the NaCl group being intermediate.

I

I

I

I

I

SUBJ 10 NH4CI

Ii’ NH4’ 100

0

12345618

9 DAYS

10

1326

SCHLOEDER

AND STINEBAUGH

curred,3~4,9-2’ with mean potassium excretion rising from 38.9 f 7.1 mEq/24 hr on the first day to 7 1.1 A 8.9 mEq/24 hr on the fourth day, daily negative potassium balance was minimal. Cumulative potassium deficity was limited to less than 50 mEq in all patients with a single exception.* With the subsidence of kaliuresis, most subjects developed a neutral cumulative external potassium balance by the sixth day and were in positive cumulative potassium balance by the final day (mean 78.9 + 10.3 mEq). Similar prevention of total body potassium deficits by potassium supplementation during fasting has been reported previously. ‘9-2’ Correlations

Previous workers, in the studies of the early stage of metabolic acidosis, have correlated UNH:V with pH, but have not commented on the relationship of UN”: to pH. *-” The correlation coefficients for both UNHz and UNHzV to pH during the developing stage were identical (r = -0.7506, p < 0.001 and r = -0.7562, p > 0.001, respectively). The relationship of V to pH during this period was not significant (r = +0.1730, p > O.l), indicating that variations in V (within the range observed in this study) exerted little influence on UpH. On more significantly with the other hand, during the steady state, UN”,+ correlated pH (r = +0.5814, p < 0.001) than did UNmV (r = 0.3445, p < 0.01). This difference was due to a negative correlation of V with pH during this period (r = -0.3091,~ < 0.01). The relationship of UNH: to pH is plotted on a scatter diagram in Fig. 3 and the regression equations for UNm to pH during the developing stage and steady state acidosis are shown in Fig. 4. DISCUSSION

During the course of systemic metabolic acidosis, the urinary pH initially falls but subsequently rises to levels that appear inappropriately high, despite continuation or even progression of acidosis. ’ I2 Our results confirmed this pat-

.: 3 . ‘. : l ::. . . . : l\**, . . l* s..** . l8 l

l

8

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Relationship between the urinary conFig. 3. centration of ammonia and UpH during steadystate acidosis in fasting subjects receiving electrolyte supplements as indicated. There was a direct and significant correlation of UpH with ammonia concentration, described by the equation: UpH = 4.5610 + 0.5066 log UN”: (r = +0.5814, p < 0.001).

20 10L50

*The cumulative potassium on the final day of the study.

balance

in patient

12 was -77

mEq on day 5 and it was + 12 mEq

URINARY

AMMONIA

CONTENT

1327

Fig. 4. Change in the relationship of urinary ammonia concentration and pH between the stages of falling serum bicarbonate concentration and subsequent stable systemic acidosis.

tern of UpH change: lowest values occurred on the second through sixth days of acidosis and subsequently rose by 0.10 to 0.88 pH units. During steady state acidosis, UpH correlated directly with the depression of serum bicarbonate: the NH,Cl group was most acidotic but had the highest UpH, while the least acidotic NaHCOJ group had the most acid urines (Fig. 1). These data are quantitatively similar to other reports of prolonged acidosis due either to exogenous acid loads or to fasting, and we conclude that a rise of UpH is characteristic of prolonged metabolic acidosis and is not peculiar to fasting. This phenomenon has received little comment and has not been subjected to detailed investigation. A rise of UpH during continuing metabolic acidosis might result from (1) diminution of acidosis, (2) a defect in hydrogen ion (H+) secretion, (3) decreased delivery of nonreabsorbable anions to the distal nephron, or (4) an increased rate of entry of base into the tubule. Improvement of systemic acidosis was not a factor, since the blood pH did not change and the mean serum bicarbonate concentration for all subjects on the day of lowest UpH (18.2 + 0.6 mEq/liter) was higher than on the final day of the study (16.3 f 0.6), while mean UpH rose from 5.19 f 0.4 to 5.71 + 0.05 (p < 0.001). A defect of proximal tubular H+ secretory capacity can be excluded on the basis of previous reports from this laboratory” and distal renal tubular acidosis because of the normal response to the short NH&l loading test at the conclusion of the study. A decline in distal H+ secretion and a rise in UpH might occur during the course of chronic metabolic acidosis if there were a decrease in the delivery of relatively nonreabsorbable anions to the distal nephron. 22 Since the excretion of organic acids was diminished by the administration of NH,Cl but augmented in the NaHCOj group (Schloeder and Stinebaugh, unpublished observations), it is possible that the greater distal delivery of these relatively nonreabsorbable anions was responsible for the low UpH in the NaHCO,-loaded patients. This seems unlikely for two reasons. First, in the NaCl group, both the organic acid excretion and pH of the urine rose concurrently from the day of lowest UpH to the last day of the study. Second, UpH rises regularly during the second week of experimental NH4Cl acidosis, in which there is no increased excretion of nonreabsorbable anions.5m” The UpH would also be expected to rise if there were an increase in the urinary content of H + acceptors. Figure 2 illustrates that the excreted quantities of nonvolatile buffers was decreasing during steady-state acidosis while the excretion of NH: rose and accounted for an increasing fraction of net acid excretion. Furthermore, the relative amount of net acid excreted as NH: was

1320

SCHLOEDER

AND STINEBAUGH

highest in the high-pH, most acidotic NH.,CI group and least in the low-pH, least acidotic NaHCO, (Table 1). Thus, the relative availability of only one buffer, NHj, could account for the diminishing urinary acidity and the difference of UpH between groups. We conclude from these observations that the availability of the buffer NH3 is determined by the duration and severity of systemic acidosis, and that the increase in ammonia production is responsible for the rise in urinary pH (Fig. 3). The conclusion that UNH: content is a factor in the determination of UpH is not implicit in the concept that urinary acidity augments ammonia excretion by “trapping” the nondiffusible protonated species, ammonium.23*24 When the relationship of UpH and UN”: is compared during developing acidosis and steady-state acidosis, as in Fig. 4, it is apparent that the inverse relationship of the “trapping” hypothesis exists only during early acidosis. The direct correlation during prolonged acidosis cannot be explained on the basis of improved pH-related transport mechanisms. Our conclusion that the augmented ammonia production of chronic acidosis is responsible for the rise in UpH is in agreement with Tannen, who reported that the acidification response to an acute acid challenge was diminished by high ammonia excretion25 and that the decreased ability to acidify the urine of potassium depletion states could also be explained by an elevated ammonia production.26 Further support for this concept, albeit in the nonacidotic state, is provided by the effect of the administration of ammonia precursors, following which the increased ammonia excretion correlated directly with UPH.~~*~* Critical examination must be given to the mechanism by which increased ammonia production causes a rise in UpH. Hydrogen ion secretion in both proximal and distal tubules is thought to be controlled by gradient limitations and were to exhibit a substantial secretory reserve. 29 If large quantities of ammonia to enter segments of the nephron with unlimited H+ secretory capacity, the added buffer would be titrated to the limiting pH gradient and tubular fluid pH would not change.* Furthermore, since only 0.1% of the total ammonia (pK 9.03) entering the collecting duct from the distal tubule would exist as basic NHj, it is unlikely that this quantity would be sufficient to overwhelm collecting duct H+ secretory capacity and elevate the pH of the final urine. For these reasons, addition of increased quantities of ammonia into either the proximal or distal tubules should not elevate UpH. In order for ammonia to cause a rise in pH, it would have to enter the tubule at a rate exceeding the H + secretory capacity at the point of entry. Since the

*This statement is inherent in the assumption that the proximal and distal tubules display kinetics and possess large secretory reserves. The gradientgradient-limited H + secretory limited concept has resulted primarily from the microperfusion studies of Malnic and deMelloThey perfused isolated tubular segments with various Airesj’ and Malnic and Giebisch.3’ concentrations of bicarbonate and phosphate. They noted that the reabsorptive half-times were the same and that the final pH of the perfusates were similar, regardless of the initial buffer concentration of the perfusate. Presumably, the kinetics of protonation of the buffer, ammonia, in the proximal and distal tubules would not be different (i.e., the final tubular pH would be determined by the limiting gradient and be independent of the ammonia concentration).

URINARY

AMMONIA

CONTENT

1329

collecting duct is the only segment of the nephron that has been shown to have a significant secretory limit, it may be concluded that for ammonia to affect UpH it must enter the collecting duct in its basic form, NH3. Ammonia is produced primarily in the renal cortex and enters the lumen of both the proximal that enters the and distal tubules by nonionic diffusion. 32Part of the ammonium proximal tubular fluid diffuses directly from the loop of Henle into the collecting duct.33.34 Gaseous ammonia that enters the collecting duct by this route is strongly basic, while that arriving by way of the distal tubule is primarily acidic (NH,+). Therefore, only the ammonia entering the collecting duct from the loop of Henle could lead to alkalinization of the urine. Augmentation of ammonia ,production by prolonged metabolic acidosis could therefore result in a rise in UpH by increasing the magnitude of this flux. It is of interest to consider why we find a direct correlation between pH and urinary ammonia in chronic acidosis while Pitts reported an inverse correlation.23 This difference results from the difference in experimental design of the two investigations. In Pitts’ experiment the UpH was elevated acutely by sodium bicarbonate infusion into a chronically acidotic dog. Therefore, the renal ammonia production was likely to be either constant or decreasing during NH, will distribute the course of the experiment. 35 Under these circumstances, according to its concentration gradient and the UpH will be a significant factor in the control of ammonia excretion by the process of nonionic diffusion. By contrast, in our experiments, the renal ammonia production was increasing daily. Thus, ammonia production, in addition to pH-related transport mechanisms, was a significant factor in the urinary excretion of ammonia. If the increase in ammonia production had no effect on UpH, then the inverse correlation of ammonia to UpH predicted by the nonionic diffusion hypothesis and demonstrated during early acidosis should have been observed during chronic acidosis. The direct correlation between ammonia and UpH during the later stages of chronic acidosis observed in our studies was not predictable from the nonionic diffusion hypothesis and suggests that other mechanisms must be responsible for the observed correlation. The observation that the ammonia concentration (UN”:) of the urine correlated more significantly with UpH than did total ammonia excretion (UNH:V) during steady-state acidosis deserves examination. If the rise in UpH was due to augmented ammonia production, one might assume that the pH should correlate best with I_J,,:V, since the I_JNH:V is dependent on ammonia production. Evaluation of our results reveals that the superior correlation of UpH with UNH: was due to a negative correlation between pH and V (r = -0.3091, p < 0.01). The physiologic implication of this finding may be that factors that determine V during prolonged acidosis also affect the quantity of NH3 diffusing into the collecting duct from the loop of Henle. Since medullary blood flow is directly proportional to V,36 reduction of the quantity of blood perfusing the vasa rectae during periods of low urinary volume might decrease the quantity of NH, removed from the medulla and increase medullary NH, content and the flux ofNH3 into the collecting duct. Consequently, the relative proportion of total urinary ammonia entering the collecting duct lumen by this route would increase under conditions of hydropenia. Since only this portion of the urinary

1330

SCHLOEDER

AND STINEBAUGH

ammonia has an alkalinizing effect, the observed negative correlation between V and UpH might be anticipated. Conversely, during acute acidosis, with unstimulated renal ammonia production and minimal diffusion of NH, from the loop of Henle into the collecting duct, variations in V would not be expected to correlate with changes in urinary acidity. In summary, the available evidence is most compatible with the concept that ammonia diffuses from the loop of Henle into the collecting duct during chronic metabolic acidosis. Our demonstration that the UpH correlates directly with the urinary ammonia concentration supports this hypothesis and suggests that the quantity of ammonia diffusing from the loop of Henle into the collecting duct is a significant factor in the determination of the final UpH during prolonged acidosis. ACKNOWLEDGMENT The authors would technical assistance.

like to express

their

gratitude

to Maria

Isabela

Vasquez

for her excellent

REFERENCES I. Gamble JL, Ross GS, Tisdall FF: The metabolism of fixed base during fasting. J Biol Chem 57:633-695, 1923 2. Lennox WG: Chemical changes in the blood during fasting in the human subject. Arch Intern Med 38:553-565, 1926 3. Rapoport A, From GLA, Husdan H: Metabolic studies in prolonged fasting. I. Inorganic metabolism and kidney function. Metabolism 14:31-46, 1965 4. Schloeder FX, Stinebaugh BJ: Defect of urinary acidification during fasting. Metabolism l5:17-25, 1966 5. Ryberg C: On the formation of ammonia in the kidneys during acidosis. Acta Physiol 15: 114-122, 1948 6. Wood FJY: Ammonium chloride acidosis. Clin Sci 14:81-89, 1955 7. Lemann J Jr, Litzow JR, Lennon EJ: The effects of chronic acid loads in normal man: Further evidence for the participation of bone mineral in the defense against chronic acidosis. J Clin Invest 45: 1608-1614. 1966 8. Sartorius OW, Roemmelt JC, Pitts RF: The renal regulation of acid-base balance in man. IV. The nature of the renal compensations in ammonium chloride acidosis. J Clin Invest 28:4233439, 1949 9. Clarke E, Evans BE, MacIntyre I, Milne MD: Acidosis in experimental electrolyte depletion. Clin Sci 14:42ll440, 1955 10. Elkinton JR, Huth EJ, Webster GD Jr, et al: The renal excretion of hydrogen ion in renal tubular acidosis. Am J Med 29:554-575, 1960

I I. Henneman PH, Wallach S, Dempsey EF: Metabolic defect responsible for uric acid stone formation. J Clin Invest 41:537-542, 1962 12. Wrong 0, Davies HEF: The excretion of acid in renal disease. Q J Med 28:259-3 13, I959 13. Haag BL, Reidenberg MM, Schuman CR, Channic BJ: Aldosterone l7-hydroxy-corticosteroid, l7-ketosteroid, and fluid and electrolyte responses to starvation and selective refeeding. Am J Med Sci 254:652-658, 1967 14. Benedict FG: A Study of Prolonged Fasting. Washington, DC, Carnegie Institute of Washington Pub1 No 203. 1915 15. Conway EJ: Micro-diffusion and Volumetric Error (ed 4). London, Lockwood, 1967, p 98

Analysis Crosby,

16. Williamson DH, Mellanby J: Acetoacetate and D-(-)-B-hydroxybutyrate, in Bergmeyer H (ed): Methods of Enzymatic Analysis (ed 2). New York, Academic, 1965, p 454 17. Hastings AB, Sendroy J Jr: The effect of variations in the ionic strength on the apparent first and second dissociation constants for carbonic acid. J Biol Chem 65:445-455, 1925 IS. Snedecor GW: Statistical Methods Applied to Experiments in Agriculture and Biology (ed 5). Ames, Iowa, Iowa State University Press, 1956 19. Stinebaugh BJ, Schloeder FX: Glucoseinduced alkalosis in fasting subjects. Relationship to renal bicarbonate reabsorption during fasting and refeeding. J Clin Invest 51: l3261336, 1972 20. Schloeder

FX, Stinebaugh

BJ: Electrolyte

URINARY

AMMONIA

CONTENT

excretion in subjects fasting in tropical

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