THREE-STEP-REGULATION OF ACID-BASE BALANCE IN ...

THREE-STEP-REGULATION OF ACID-BASE

BALANCE IN BODY FLUID

AFTER ACID LOAD

Hisato YOSHIMURA, Mamoru FUJIMOTO, Osamu OKUMURA,

Jyunichi SUGIMOTO AND Tsutomu KUWADA*

Department of physiology, Kyoto Prefectural University of Medicine

At the beginning of this century, HENDERSON, HASSELBALCH, and VAN SLYKE clarified the concept of alkali reserve of blood and reported that in acidosis

and alkalosis blood bicarbonate i.e. alkali reserve, played an important role in

maintaining normal pH1). Recently, SWAN and PITTS2), SCHWARTZ3), and ELKINTON4) elucidated the quantitative role of extracellular buffering in the

metabolic acidosis of dogs and man, and emphasized that the intracellular buffering

should act in regulation of acid-base balance within the body . In 1953, DARROW and others5) studied this buffering mechanism and demon-

strated that hydrogen ions in the blood of acidotic rats could enter the cells in

exchange with intracellular cations, and found that serum bicarbonate was closely correlated with intracellular sodium and inversely with intracellular potassium . Such an ion exchange mechanism between extracellular and intracellular fluid was confirmed later by TOBIN6) in acidotic nephrectomized rats . In addition, it is well-known that pulmonary respiration is enhanced and urinary acidification is

potentiated during acidosis, while they are depressed in alkalosis. Thus, it may be stated that the following four functions are integrated in

acidosis as well as in alkalosis to maintain normal pH of body fluid:

1) Buffer action by extracellular fluid (ECF) , especially buffering in blood, 2) Buffer action by intracellular fluid (ICF), 3) Respiratory function , and 4) Renal excretion of excessive acid or alkali from the body .

Though many experiments were conducted in researches of the regulatory

mechanism of acid-base radicals in body fluid, quantitative relations among these

factors and their time sequence have not yet been fully clarified . To study these problems with dogs infused with acid is the aim of the

present investigation.

EXPERIMENTAL METHOD

Healthy adult female dogs weighing 5 to 15 Kg were fed on a diet which was rationed so as to be neutral , with a proper amount of water in metabolic cage the bottom

Received for publication September 15 , 1960*吉村寿人,藤本守,奥村修,杉本修一,桑田努

109

110 H. YOSHIMURA, et al.

of which was netted to separate feces from urine. Urine was collected every day, and its electrolyte contents were analysed. After the salts and water metabolisms of the dogs had attained an equilibrium, various amounts of hydrochloric acid from 0.6 to 9.2 mEq. per kilogram of body weight were infused intravenously under anesthesia. The acid-base balance in blood and water and salts distribution in body fluid were ex-amined every day for about a week after the acid infusion until changes due to acid infusion were almost restored. The daily diet taken by the animal was always kept constant throughout the experiment. Details of these experiments will be documented under separate headings.

Daily examination of acid-base metabolism. Collection of daily urine was made under liquid paraffine in the bottle to avoid

escape of CO2 . One or two milligram of quinone was added to the urine to prevent a decomposition of urea into ammonia. Feces were removed from the cage as quickly as possible to avoid contamination with the urine.

Items concerned with urine which were measured were pH, total CO2 , titratable acidity, concentrations of Na, K, Cl and NH3 and urine volume from which the excretion rate of salts was calculated. Dog's urine was usually acidified with a somewhat excessive excretion of acid radicals over basic ones, and thus titratable acid is produced. Excessive acid radical is, however, excreted largely in the form of ammonium salt which is believed to be ammonium chloride in mammals. Consequently, the total acid radicals excreted in excess can be represented by the sum of titratable acidity and ammonia excretion, and it is called base economy in this paper. The daily output of acid and base radicals for the duration of a week prior to acid infusion were averaged and designated as the control values. The excretion of those radicals or ions underwent changes after acid infusion, and their excess over the control values were calculated to see how disturbances in the acid-base balance in body fluid were regulated after the acid load. For the same purpose, total body water, extracel-lular space, circulating blood volume, hematocrit value and various electrolyte contents in serum were determined approximately 1 week before, at the end of acid infusion and 24 hours after the acid infusion.

Acid infusion. Thirty experiments were performed with twenty five trained mongrel dogs. The

animals were anesthetized with an intravenous injection of 50-100 mg/Kg sodium methyl-hexabarbiturate, and were kept supine under loose restraint. Following an adequate control period, the infusion of 0.16 normal hydrochloric acid was started and maintained for 1 to 3 hours at a constant rate of 1-2 ml/min. with a calibrated constant infusion pump. A urine sample was collected from indwelling catheter of urinary bladder at intervals of 30-120 minutes before, during and after infusion. A blood sample was withdrawn from the jugular vein or carotid artery at the midpoint of each sampling period of urine.

For the survey of respiratory gas metabolism, expired air was collected in a Douglas bag from trachea catheter every 30 min. during the experiment, and the

gas was analysed with a Roken gas analyser (a modification of Haldane's method) or with Scholander's method7).

Method of analysis.

Blood was centrifuged under liquid paraffine to separate the plasma. The specific

ACID-BASE BALANCE IN BODY FLUID 111

gravity of whole blood as well as of serum was determined every time by means of

the copper sulfate method, and hematocrit was calculated from these values.

The pH of blood and urine was determined at 37°C as soon as possible after

collection without exposure to air with a syringe type glass electrode which was

devised by one of the authors8). In a few experiments, a glass electrode for circulating

blood9) was used. In these instances, the pH of circulating arterial blood was recorded

during the experiment with a Shimadzu electronic balancing recorder10).

The total CO2 content of blood and urine was determined by Van Slyke's mano-

metric method modified by SAITO11).

From pH and total CO2 content in serum or urine, its pCO2 was calculated by

means of the Henderson-Hasselbalch equation, adopting 6.10 for serum pK' and

pK'=6.33-0.5ƒÊ for urine, where ƒÊ is ionic strength calculated from total ionic

concentration. Bunsen coefficients of 0.550 for serum and of 0.560 for urine were

adopted.

Blood buffer which is utilized to neutralize the administered acid was calculated

from Hastings and Singer's nomogram12) for human blood.

Sodium and potassium in serum as well as in urine were measured with Lange's

flame photometer13), while chloride was determined by a modified method of SCHALES

and SCHALES14). Ammonia was determined by Conway's method15), while titratable

acidity was estimated electrometrically by the method of ASADA in our laboratory").

The expired air was analyzed for its volume with gasmeter and for its contents

of O2, CO2 and N2 with the gas analyzer mentioned above.

The total body water was estimated, as usual, by intravenous injection of 1%

N-acetyl-amino-antipyrine (NAAP) solution at the rate of 2 ml/Kg. The blood and

urine were collected 60 and 180 min. after injection and the concentration of NAAP

was analysed by Brodie's method17). The measurement was made about a week before

the acid infusion.

For the determination of ECF, continuous inulin infusion method or thiocyanate

method was utilized. The circulating blood volume was also measured by the congo-red

method21). Details of experiments are as follows:

Inulin space: After a priming injection of 100 mg/Kg of 4% inulin solution, inulin

infusion was performed at the rate of 1 mg/min./Kg sustained during the subsequent 2

hours. Urine and blood samples were collected four times at intervals of 30 minutes dur-

ing infusion and inulin concentration was analysed by means of Schreiner's method18).

Calculation of inulin space was made as usual, and the average value from these 4

measuring periods was used as a representative of extracellular space. The inulin

space was measured before and at the end of acid infusion period. (In the latter case,

the inulin was dissolved in the acid solution which was infused.) After 24 hours,

it was measured again in most cases.

Thiocyanate space: In some experiments, 5% sodium thiocyanate was infused at the

rate of 0.8 ml/Kg and thiocyanate space determined by the CRANDALL and ANDERSON

method19) was taken as extracellular space. Since the thiocyanate space was always

somewhat larger than the inulin space, either of these two methods was used for the mea-

surement of extracellular space with each respective animal throughout the experiments.

The thiocyanate space was measured about a week prior to the acid infusion and the

measurement after acid infusion was omitted. The extracellular space at the end


of acid infusion was calculated by means of Darrow-Yannet's equation20). The value taken after 24 hours was the same as that before the infusion. Results of calculation were compared with those obtained by inulin infusion, and it was verified that the changes of distribution of water and salts in ECF calculated by these two methods were essentially the same. Circulating blood volume: Circulating blood volume was determined by the congo-red method reported by YAMAMOTO21) who ascertained that the values measured by his method were essentially the same as those measured by the Evans blue method22). One per cent congo-red solution was injected intravenously at the rate of 0.4 ml/Kg and the dye concentration in serum 4 min. after injection was measured with a colorimeter, and the circulating serum volume was calculated as usual. Calculation of ionic contents in the extracellular fluid.

In order to calculate ionic content in ECF, concentration in the interstitial fluid

(ISF), should be calculated at first. For this purpose, the electrolyte concentration in serum was converted to those in serum water and were corrected for the Donnan effect. Content of serum water was calculated from the specific gravity of serum by means of the following equation of FUKUYAMA and SATO23),

Serum water(g/dl)=100-386-(SG-1.000)-1.39/

SG,

where S, represents the specific gravity of serum.

The Donnan membrane factor for anions was adopted uniformly as to be 1.05,

while it was 0.95 for cation. By multiplying the ionic concentration in ISF with its

space which corresponds to the extracellular space minus total serum volume, the

total quantity of the ion in ISF was calculated. The ionic content in ECF corresponds

to the sum of the ion content in ISF and that in the total circulating serum.

Calculation was made before and after the acid infusion and the difference

between the two was calculated. When the measurement of ECF after acidification

was omitted, the following Darrow-Yannet's procedure was adopted for the calculation.

Let T equal the total body water before acid infusion; I, the volume of infusing

solution; E, the extracellular volume before acid infusion; m, the osmolal concentration

in body fluid before acid infusion; and i, the effective osmolal concentration of in-

fusing solution; the extracellular volume after acid infusion, E', can be calculated by

solving the following equations simultaneously:

(T-E)･m=(T+I-E')･x,

E･m+I･i=E'･x.

x is the osmolal concentration after acid infusion and is cancelled from these equations

by calculation. As the value of in, 310 mOsm/l was usually adopted.

RESULTS

I. Acid- base balance during acid infusion;

1. Changes of blood and urine during acid infusion: FIG. 1 illustrates one of the data obtained in the experiments with the normal dog. Infusion of acid

into the experimental animal provokes a gradual decrease of blood pH and of


FIG. 1. Change of Acid-Base Balance in Body Fluid after Acid Infusion.

total CO2 concentration in serum accompanied with a considerable hyperventila-

tion. Increased expiration due to hyperventilation continued for more than

three hours after the end of acid infusion. Nevertheless, the total CO2 con-

centration in serum shows a rapid but slight restoration after the end of acid

infusion, as seen in FIG. 1. Thus CO2 expired originates not only from blood

but also from other compartments of the body fluid. Serum pH was also restored

rapidly to a certain extent after the end of acid administration, and thus a buffer

action by body fluid compartments except blood may be involved in the

physiological regulation of acidosis. Urine pH was initiated to decrease after a short latent period, accompanying an augumented renal excretion of ammonia

and titratable acidity. As these changes appear gradually, they can , by no means, explain the rapid restorations in acid-base balance in blood after the acid infusion . The reason will be discussed below.

The decrease of blood pH and of serum total CO2 from respective control

values was calculated at the end of acid infusion and is plotted in FIG . 2 in relation to the acid load. Good positive correlations exist and the correlation coefficient

for the pH decrease is 0.74 and that for the total CO, decrease is 0 .84.


FIG. 2. Rate of Acid Infusion and the Decreases of Blood pH and

Serum Total CO2 Concentration.

The rectilinear regression line calculated from the available data presents a

slope of 0.028 for pH decrease, and 1.5 for CO2 decrease. It means that the

pH of blood decreases by 0.028 and the total CO2 content in serum decreases with the rate of 1.5 mEq/l to an acid load of 1 mEq/Kg.

On the other hand, detailed observation revealed that the relations presented

in the figure were not exactly rectilinear. The magnitude of pH decrease and of CO2 decrease tended to attain their respective constant levels as the acid load

increased above the certain level. (See dashed lines in the figure.) The fact

suggests that the physiological regulation of acid-base balance was accentuated

as the acidosis became severe.

Three dogs of five experiments, in which decrease of blood pH and serum total CO2 concentration reached more than 0.2 pH and 15 mEq/l respectively,

died soon after the end of acid infusion. The lethal amount of acid load varied within the range of 5.3 to 9.2 mEq/Kg of body weight.

2. Buffer action of body fluid administered with acid: To clarify quantitative

relations of buffer action in various compartments of body fluid, the amount of

acid neutralized with blood buffer and that with interstitial buffer, and urinary

excretion of excessive acid radicals were calculated. Neutralization with blood

buffer was estimated by Hastings and Singer's nomogram. As the buffer in the interstitial fluid is almost exclusively the bicarbonate, its reduction due to

acid infusion corresponds approximately to the acid neutralized in the interstitial fluid.

Table 1 is a summary of the calculations of acid neutralized in various com-

partments of body fluid (with dogs) of which ECF was measured by the inulin infusion method. The acid load was 3.6-4.8 mM/Kg in these cases. The figures

in parenthesis represent the percentage neutralizations of loaded acid by various


TABLE 1

Buffer Action of Body Fluid upon the Infused Acid

and its Excretion by Kidney.

compartments, or its percentage excretion in urine which was calculated from

the base economy. It is demonstrated that an average of 56% of the total acid

load is neutralized in ECF at the end of acid infusion, and the portion neutralized by blood buffer is only 15% or so. The excretion of excess acid in urine is

negligible at this stage. Thus the rest of acid load (44% ) is assumed to be buffered

by the intracellular buffer. This latter factor may also be responsible for the

rapid restoration of blood pH immediately after acid infusion. The restoration

of depressed blood pH was 0.09 and that of serum total CO2 concentra-tion was 5.5 mEq/l on the average in a few hour after completion of acid infusion. This increase of CO2 corresponds to an increase of 0.2-3.0 mEq of

bicarbonate in ECF per kilogram of body weight. While the pulmonary output

of CO2 was rather enhanced, shivering or any other movement, whereby CO2

production would increase, was not observed throughout this period. Therefore, the CO2 increase in extracellular fluid and pulmonary output may be regarded

as the result of the neutralization of acid by intracellular bicarbonate which fur-


nishes excess CO2 into ECF.

The percentage neutralization of infused acid by the intracellular buffer was calculated over the whole range of acid load, i.e. 0.6-9.2 mEq/Kg. It varied

from 8% to 67% at the end of acid infusion, and the mean value was 39%, i.e.

two fifths of the acid load. 3. Changes of ionic distribution in body fluid after acid infusion: FIG . 3 illustrates changes of Na, Cl, and K concentrations in serum and urine during acid infusion.

The serum concentration of Na decreased gradually during acid infusion, while

the serum Cl and K concentration tended to increase slowly. The decrease of

Na concentration and the increase of Cl concentration are explicable as a direct

effect of HCl infusion. On the other hand, the increase of K may be a result of migration from intracellular fluid. Output of Na, K and Cl in urine all increased

during and after administration of HCl. The fact was regarded as a result of

neutralization of infused HCl by bicarbonate in the body.

In order to examine changes of ionic distribution in body fluid quantitatively, total contents of Na, K, Cl and HCO3 in ECF before and at the end of acid

infusion were calculated, and changes in contents of respective ions due to acid

FIG. 3. Change of Ionic Concentrations in Body

Fluid after Acid Infusion.


administration were estimated. Ionic excretions in urine during acid infusion

being measured, their differences from normal excretions during the corresponding

duration before acid load were also calculated.

These are all summarized in TABLE 2. The negative sign in the table means

a decrease of ionic excretion or of ionic quantity in body fluid. The sum of the change actually determined in ECF and the change in excretion for respective

ions corresponds to the total changes in ECF due to acid infusion. The table

demonstrates that Na, K and Cl (indicated by B1, B2 and C respectively) were

all increased in ECF by acid infusion, while the bicarbonate was decreased. This decrease of bicarbonate corresponds largely to the amount of the acid neutralized

by ISF which prevails to the extent of over two thirds of extracellular buffering,

as predicted from TABLE 1. Increases of Na and K in ECF are probably due

to migration from ICF, since the dog was in a fasting state in the experiment.

Cl-increase in ECF may largely be explained by administration of HCl. As Cl-increase in ECF (C) is always a little larger than the administered HCl, which

is indicated by A in the table, the excess Cl (E in the table) should be shifted

from ICF, being accompanied by Na and K from ICF. The alkali shifted from

TABLE 2 Changes of Ion Distribution in Body Fluid By Acid Infusion (at the End of Infusion).


ICF is, however, always in excess of Cl from ICF (F in the table), and it is verified that this excess alkali holds a good correlation with the acid load. There-

fore, it is presumed that the excess alkalis, Na and K, migrate from ICF in com-

bination with HCO3 , and are utilized for acid neutralization as was pointed out

in the previous section. The percentage ratio of this alkali shift to acid load (100 X F/A) was calcu-

lated and was plotted in relation with the percentage neutralization by ICF (100 X

G/A) which was previously calculated in TABLE 1. FIG. 4 reveals that a close

correlation exists between the two. The correlation coefficient is 0.764 and the

regression coefficient is 0.692. It follows that about 70% of intracellular buffering

is effected by this intracellular bicarbonate during and at the end of acid infusion.

Y =0.692X•~13.30

r -0.764

Pr<0.005

FIG. 4. Comparison of Percentage Ratio of Excess Alkali Shifted from ICF to Acid Load (F/A) with Percentage neutralization of Loaded Acid by ICF Buffer (G/A).

In short, when an excess of acid is infused in the body fluid, about three

fifths is neutralized by ECF and the remaining two fifths by ICF at the end

of acid administration. The intracellular buffering is mainly effected by the

neutralization of acid with bicarbonate which migrated from ICF into ECF. II. Metabolism of acid-base radicals; Acidotic dogs were fed with the same

ration of diet as that given before acid administration. Daily restoration from

acidosis was observed by measuring acid-base balance in blood and urine. -

A part of the results is illustrated in FIG. 5. Serum pH and total CO,

content were restored completely on the next day of acid infusion.

On the other hand, urinary pH and bicarbonate (or total CO2) excretion

in urine were still decreased 24 hours after acid load and were gradually restored to the normal value about 3 or 4 days thereafter. Ammonia excretion increased

remarkably for a couple of days after infusion, while the increase of titratable

acidity was not so remarkable. In the figure, the sum of ammonia and titratable

acidity, i.e. the base economy, is drawn with a solid line, and the mean level of control value before acid infusion is indicated with a dashed line. The base

economy is clearly demonstrated as increasing after the acid load. The increase


FiG. 5. Diurnal Alteration of Acid-Base

Balance in Blood and of Base Economy of Urine

before and after Acid Infusion in Normal Dog.

of base economy indicates an excessive elimination of acid radicals from the

kidneys.

To examine quantitative relation with respect to acid neutralization in body

fluid compartments and acid elimination from kidney, similar calculations, which were made at the end of acid infusion, were performed with the data after 24

hours under the postulation that the intermediate metabolism in the body is

always kept constant.

A part of the result of calculation is presented in the lower half of TABLE 1. Since serum pH and total CO2 content were restored to the control value, as

shown in FIG. 5, the percentage neutralization by ECF after 24 hours decreased

nearly to zero. On the other hand, the percentage excretion of excess acid, which

is calculated from the base economy in excess of the control value, amounted

to about 24% , as an average, though it varied widely from dog to dog. Thus the remaining three-fourths of loaded acid should have been neutralized by ICF.

It follows that the acid infused almost disappears from ECF in 24 hours and

is largely neutralized by ICF and partly excreted through the kidneys. It is

worth-while to mention that the portion of acid neutralized by ICF increases

gradually as the time elapses after acid infusion.


FIG. 6. Time Sequence of Acid-Base Rugulation after Acid Infusion.

The portion of acid excreted in urine also increase with the passage of time,

and finally the excess of acid radicals which corresponds to the infused amount was eliminated completely from the body fluid (c.f. FIG. 6). The time of

complete excretion is indicated in TABLE 1 and was found to be 6 days as an

average. The range of variation in the whole experiments is from 2 to 8 days.

Discussions on time sequence of acid neutralization and excretion, and on

the mechanism involved in buffer action of ICF will be made in the following

section.

DISCUSSION

Similar calculations to that made in TABLE 1 were performed with data on successive days subsequent to acid administration under the postulation of con-

stant intermediate metabolism with respect to acid-base balance in the body.

The times sequence of acid neutralization in various compartments of body fluid,

in relation with renal excretion of excess acid, were examined. A part of the

results of calculation is presented in FIG. 6. The amount of acid infused being

taken on the ordinate, the acid induced is drawn with a solid line (A) in relation with the time element. The top of line A represents the total amount of acid

infused and the horizontal dashed line is drawn at this level for reference. The

acid neutralized by ISF being taken downward from the level of infused acid,

the line B' is drawn and, from B', the acid neutralized by whole blood is taken

downward to draw B. The distance between A and B represents the portion of

acid neutralized with the buffers in the total extracellular fluid. As the renal excretion of excess acid was not appreciable for a couple of hours after the

acid infusion, the gap between B and the base line should represent the acid neutralized by the intracellular buffer. On and after the first day after acid

infusion, the excess acid radical was excreted in the form of base economy in excess of the control value as already mentioned. The additive sum of daily

output of excess base economy is represented by a line C. As the base economy


was composed of ammonium salt and titratable acidity, the line D is drawn to

represent the additive sum of titratable acidity. Therefore, the gap between C

and D is the additive sum of excess acid radical which is excreted in the form

of ammonium salt. The acid neutralized by the intracellular buffer corresponds to the gap between B and C this time.

It is demonstrated in the figure that the acid infused is at first neutralized

largely by the extracellular buffer, then the neutralization by the intracellular buffer proceeds gradually until most of acid is moderated in ICF, and finally the

acid is excreted by the kidneys in the form of base economy in excess, especially

in the form of ammonium chloride. Thus it is verified that the physiological

disposal of excess acid is carried out in three steps in succession.

The excretion of excess acid is completed on the second day (48 hours

after acid loading) in this case, and thereafter normal fluctuation of renal excre-tion of base economy follows. Usually, however, the time of complete excretion

is around a week as previously mentioned, so far as the load of acid is moderate.

The sequence of this three-step-regulation is schematically represented in

FIG. 7. As indicated in the figure, acceleration of CO2 output appears in asso-

ciation with both extracellular and intracellular bufferings and thus the respiratory

regulation of acid-base balance is implicated in the first and the second phase of

FIG. 7. Scheme of three-step-regulation of

acid-base balance in body fluid after acid load,


regulatory mechanism. The fact of intracellular buffering was already pointed out by previous

authors, e.g. SWAN and PITTS2), TOBING) and SCHWARTZ3) and the percentage neutralizations by ICF at the end of acid infusion which they described of similar magnitude to those represented in TABLE 1. The new points which were revealed by the present investigation are that the neutralization by ICF progresses as the time elapses after the acid administration, and is associated with the other two

physiological mechanisms in a certain sequence to eliminate the effect of excess acid in body fluid. As an objection to the present analysis, it may be mentioned that the intermediate metabolism might not be kept constant after acid infusion. As its answer, it is emphasized that the dog is fed with the constant food throughout the experiment, and the cumulative sum of excess base economy is maintained constant at the level of acid load in most cases after completion of acid elimination as is shown by the curve C in Fig. 6. Thus, there is no indication of alteration in intermediate metabolism.

In the previous section, the mechanism of intracellular buffering was explained as being due largely to migration of alkali bicarbonate from cells into ECF on the basis of ionic distribution at the end of acid infusion. On the other hand,

TABLE 3

Changes of Ion Distribution in Body Fluid

24 Hours After Acid Infusion.


the ionic distribution 24 hours after the infusion may be different from that at

the end of infusion. Thus, a similar calculation to that made previously was

performed with the data 24 hours after the infusion. Results are summarized in TABLE 3, and will be explained in relation with the data at the end of acid

infusion.

Reduced bicarbonate content in ECF after acid load has been restored

completely in 24 hours, while the increased Cl content in ECF is decreased partly

and Na content is not changed appreciably from that at the end of acid infusion.

Since Na and Cl excretion in urine decreased during these 24 hours, a part of

Na and Cl in ECF should have entered ICF. The fact is actually verified by

calculating the shifts of Na, K and Cl from ICF to ECF as was performed in TABLE 2, i.e. the Cl-shift (e) and the sum of alkali shifts (b1+b2) thus calculated

are always negative. Furthermore, the fact that the magnitude of (e) is always

larger than (b1+b2) indicates that some Cl ion has penetrated from ECF into ICF without accompanying alkali ions but probably with H. Thus, the Cl-

shift in excess of alkali (f in the table) may correspond to the amount of HC1

which has entered the cells, HCl migrated into cells should be neutralized by

protein and some other buffers in the cells. The intracellular neutralizations 24 hours after acid infusion in TABLE 1 being corrected to the values per Kg

unit of body weight (g), they were lined at the bottom of TABLE 3 in order to compare with the portion of acid migrated into ICF (f) . The comparison revealed that f and g coincide well within the scope of experimental error. Thus

it is evidenced that the intracellular buffering is effected almost entirely by migra-

tion of HCl into cells 24 hours after the acid load.

Summarizing the results obtained above, the mechanism involved neutraliza-

tion by intracellular buffer is presented diagrammatically as follows:

In the early period after acid infusion, the intracellular bicarbonate shifts to

ECF as indicated by the reaction (1) , and is neutralized with HC1 to form H2CO3

which is expired by enhanced respiration. The intracellular buffering is mainly due to this reaction. After a lapse of time the infused acid, HCl, begins to

enter ICF and is neutralized by protein and other intracellular buffers, whereby

the bicarbonate in ECF tends to be restored (the reaction (2)) . While the reaction (2) predominates absolutely 24 hours after acid infusion, the two reactions

may exist in the middle period between the end of acid infusion and 24 hours

later. It is not certain at the present study whether or not the penetration of

HCl may invariably occur even when the acid administered is not HCl. Factors

which influence the rate of shift of bicarbonate and HCl are also unknown. These

124 H. YOSHIMURA, et el.

points should be clarified by future studies.

SUMMARY

Twenty five mongrel adult dogs were infused with isotonic HC1 solution (0.6-

9.2 mEq/Kg), and the changes of acid-base balance in the blood and the excretion

of excessive acid in the urine were observed until the normal acid-base balance in body fluid was restored. Results obtained are outlined as follows:

1. The pH and bicarbonate in the blood decreased in proportion to the acid

load at the end of acid infusion, and depression of pH and bicarbonate in the blood of 3 dogs out of 5 which died of the acidosis was over 0.2 pH and 15

mEq/l respectively.

2. The acid infused was neutralized mainly (about 60%) by the extracellular

buffer (especially by bicarbonate), and the rest (about 40%) by the intracellular buffer at the end of infusion. After 24 hours, however, the large part of acid

load (about 3/4 of the total) was buffered by the intracellular fluid, a part (1/4)

was excreted in urine, and thus the alkali reserve in extracellular fluid was completely restored. The excess acid was gradually excreted after a lapse of

days mainly as ammonium chloride and partly titratable acid, or inclusively as

the base economy. After 2-8 days, the excretion was completed. Thus the regulation of acid-base balance in body fluid after acid loading

was carried out in three steps, i.e. neutralization by extracellular buffer, neutraliza-

tion by intracellular buffer and renal excretion as the base economy.

3. By measuring the distribution and the amounts of Na, K, Cl and HCO3 in

extracellular fluid, it was confirmed that neutralization by intracellular buffer after

acid administration was effected mainly by an alkali shift from the cells in the

early period, and by the penetration of the acid radical (Cl) into the cells thereafter.

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13) YOSHIMURA, H. AND INOUE, T.: Jap. J. Med. Prog. 46: 1, 1959, (Japanese). 14) ASPER, S. P., O. SCHALES AND SCHALES, S. S.: J. Biol. Chem. 168: 779, 1947. 15) CONWAY, E. J.: Microdiffusion Analysis and Volumetric Error, 1950, London, England. 16) ASADA, T.: Jap. J. Med. Prog. 43 : 513, 1956, (Japanese). 17) BRODIE, B. B.: Proc. Soc. Exp. Biol. & Med. 29: 1266, 1951. 18) SCHREINER, G. E.: Proc. Soc. Exp. Biol. & Med. 74: 117, 1950. 19) CRANDALL, L. A. AND ANDERSON, M. X.: Am. J. Dig. Dis. & Nut., 1: 126, 1934. 20) WELT, L. G.: Clinical Disorder of Hydration and Acid-Base Equilibrium, 21, 1955,

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THREE-STEP-REGULATION OF ACID-BASE BALANCE IN ...

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