Liver uptake of amino acids and carbohydrates during a single circulatory passage.

AMERICAN JOURNAL OF PHYSIOLOGY Vol. 228, No. 4, April I 975. Printed in U.S.A.

Liver

uptake

during

of amino

a single

acids and carbohydrates

circulatory

passage

WILLIAM

M. PARDRIDGE AND LEONARD S. JEFFERSON Physiology, College of Medicine, The Milton S. Hmhey Medical The Pennsylvania State University, Hershey, Pmn.sylvania 17033

Department

of

PARDRIDGE, WILLIAM M., AND LEONARD S. JEFFERSON. Liuer uptake of amino acids and carbohydrates during a single circulatory passage. Am. J. Physiol. 228(4) : 1155-I 161. 1975.-The uptake of i4Clabeled amino acids and carbohydrates by liver following rapid injection into the portal vein was measured relative to a simultaneously injected highly diffusible reference, tritium-labeled water, (3HOH). A 0.25-ml bolus of buffered Ringer solution contest substance and 3-6 PCi of taining l-2 @i of the 14C-labeled 3HOH was administered by rapid portal injection in anesthetized rats. Circulation was terminated after a single passage of the bolus through the hepatic microvasculature and the tissue was immediately macerated, solubilized, and subjected to liquid scintillation counting. Liver uptake indices (LUI) were calculated from the ratio of 14C to 3H in liver tissue relative to the same ratio in the injection mixture. LUI’s of five carbohydrates were measured : sucrose (24.3 yO), inulin (27.7y0), D-mannitol (80.27,), Dglucose (96.8c/;,) and L-glucose (26.7 yO). The LUI for cholic acid was 127.1 (7c. Among 18 amino acids tested, the LUI’s were the highest for the acidic ones (L-aspartic acid, 100.0% and L-glutamic acid, 86.4y0) and lowest for the basic ones (L-arginine, 37.4y0 and L-lysine, 3 1 .4yQ). Stereospecificity for glucose and alanine uptake, saturation kinetics for glutamic acid (Km = 4.8 mM) and aspartic acid (k', = 2.7 mM), and cross-inhibition among uptake of the acidic amino acids were observed. These findings confirmed the applicability of a technique which was originally developed for studies of amino acid uptake in brain to characterization of transport systems in liver. liver transport mannitol; cholic

mechanisms; acid; tritiated

glucose water;

uptake; rat liver

sucrose;

inulin;

HEPATIC UPTAKE of radiolabeled amino acids and carbohydrates has been studied in tissue slices (6, 12, 20, 23, 30) and in the isolated perfused liver (9, 13, 16, 21, 22, 29). Characterization cf specific transport systems, hcwever, has been hampered by rapid metabolism cf the labeled ccmpound subsequent to transport. Furthermore, measurements of the kinetic parameters of liver transport prccesses have proven dimcult since compounds such as glucose (33) and glutamic acid (12) equilibrate between blocd and liver very rapidly with half-times between 0.5 and 1 min. The effects of metabolism can be minimized and accurate rate measurements of transport, as opposed to equilibrium measurements, can be made if methods are used that measure liver uptake over very short time intervals such as required for a single pass through the hepatic vasculature. Recently liver uptake cf radiolabeled amino acids and carbohydrates after a single circulatory pass was measured with an indicator-dilution technique (2). The indicator-dilution tech-

Center,

nique was adapted to liver transport processes after having been demonstrated by several investigators (7, 8, 34) to be an effective method for studying transport processes in brain. In additian to the indicator-dilution technique, a method using tritiated water as an internal standard has recently been developed by Oldendorf (24) for studies of hrain uptake of radiolabelcd compounds. The Oldcndorf technique shares the advantage of the indicator-dilution method in that uptake is measured over a pericd of seconds in a relatively undisturbed in situ liver preparation. In both methods, the uptake of a given 14C-labeled test substance is measured relative to that of a reference tracer (a freely diffusible 3HOH reference in the case of the Oldendorf technique or an extracellular marker, *3Na- or 3H-labcled inulin, in the case of the indicator-dilution technique) following the simultaneous injection of the test and reference isotopes into the majcr vascular supply of the organ. The two methods have yielded comparable results in brain uptake studies. For cxample, both methods have demonstrated the presence of a neutral amino acid transport system in the blood-brain barrier (25, 34). With his technique, Oldendorf has recently been able to identify several other separate transport systems in the blocd-brain barrier, namely basic amino acid (26), hexose (26), and monccarboxylic acid (27) transport systems. In view cf the fact that the Oldcndorf technique has proven successful in brain transport studies and that the liver has proven amenable to studies using the indicator-diluticn technique, the present study was undertaken to confirm the adaptability of the Oldendorf technique to liver. The applicability of this technique to the study of liver transport systems was judged first by confirmation of data obtained by other methods and second by the demonstration of the characteristics of carriermediated transport : saturation, ccmpetition, and stereospecificity. METHODS

Male Sprague-Dawley rats, weighing approximately 300 g, were anesthetized by an intraperitoneal injection of sodium pentobarbital (Nembutal, 4.5 mgjkg). The abdomen was opened and the portal vein exposed. The hcpatic artery was ligated with care to ensure the patency of the underlying supericr mesenteric artery. A mixture of approximately 0.5 &i of 14C-labeled test substance and 1.5 &i of 3HOH in 0.25 ml of Ringer solution (Na+ 147, K+ 4, Ca++ 4, Cl- 155 meq/liter) buffered to pH 7.4 with 4 mM HEPES buffer (Calbiochem) was injcctcd rapidly into the exposed portal vein through a 25-gauge needle. 1155

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W.

1156 This size needle did not occlude the vessel and free flow of portal blood past the needle persisted throughcut the procedure. The injection was sufficiently rapid that the test solution entered the liver as a bolus, thereby minimizing mixing of the injection solution with blocd during the initial passage through the hepatic vasculature (24). Injection of the mixture as a bolus allowed far an isolated exposure of the test substance to liver transport sites with minimal competition from substances in free solution in blood. After injection, the needle was left in the vein to prevent bleeding. At various times following the injection, portal blood flow was terminated by transection of the vein. The left major lobe cf the liver was removed quickly and the tissue was macerated by successive extrusions through 15-gauge and 18-gauge needles. Triplicate samples of lOO250 mg of the macerated wet liver were digested in 1.0 ml of KCS solubilizer (Amersham/Searle) by shaking in a 50°C water bath for approximately 2 h. A sample of the injection solution was diluted in distilled water and treated with NCS solubilizer in the same manner prior to liquid scintillation counting. To each of the samples was added 10 ml of scintillation fluid consisting of 0.6 % PPO in toluene. Standard quench curves for converting counts per minute far each isotope (‘“C, 3H) to disintegrations per minute (dpm) were determined according to the method of Williams et al. (32). A Beckman LS- 100 scintillation counter was used and all isotopes were purchased from New England Nuclear Corporation. The ratio of 14C-dpm/3H-dpm in liver tissue was divided by the same ratio in the injection mixture. The result was multiplied by 100 to yield the liver uptake index (LUI), an expression of the net uptake of the test substance as a percentage of the net water uptake. The calculation is as follows: LUI

=

14C-dpm/“H-dpm 14C-dpm/3H-dpm

and is in accordance with for brain uptake (24). Rearranged algebraically 14C-dpm 3H-dpm

(liver) (injection)

x

original

the LUI

is equivalent

(injection) (injection)

PARDRIDGE

AND

L.

S. JEFFERSON

RESULTS

Standard &z&ion time. Before measuring the LUI’s for the selected 14C-labeled test substances, it was necessary to determine the time required for the injection bolus to make a single passage through the hepatic vasculature. These experiments involved determining the rate of clearance of two extracellular space markers, [14C]inulin and [14C]sucrose, from liver tissue following portal injection. The LUI for each compound versus the time elapsed between portal injection and termination of blood flow was determined and the results are presented in Fig. 1. The time between injection of the test mixture and transection of the portal vein was varied from 2 to 30 s. By 18 s the LUI’s fcr both of these extracellular markers had fallen to a value of about 25 % and was unchanged at 30 s. These findings indicated that substances shown to be extracellular markers (10) were for the most part carried out of the hepatic vascular space within 18 s. The sucrose and inulin persisting in liver beyond 18 s presumably represented residual tracer not yet washed out of the interstitial space. Both washout of 3HOH from liver cells and persistance of residual amounts of the extracellular markers in the interstitial space presumably accounted for the maintenance of the LUI’s for these campounds during the 18- to 30-s period. Subsequent studies were terminated at 18 s following portal in-jection of the test mixture since it was considered that this time period

2: g -

!z iz 3 u W > -

Oldendorf’s

(liver)/14C-dpm (liver) / 3H-dpm

100

M.

80 t 60 40

calculation

x

to 100

and is, therefore, equivalent to the percent extraction, or net uptake, of the 1Glabeled test substance divided by the percent extraction, or net uptake, of the 3HOH reference. The percent extraction of the ‘G-labeled test substance could, of course,, be computed directly by careful measurements of weight of liver tissue analyzed as well as volumes of test solution injected. These tedious manuevers were avoided by the use of 3HOH as an internal standard which allowed calculation of a normalized liver uptake index for each ‘G-labeled test substance. Neither the volume of injection solution nor weight of liver tissue digested and counted need to be known in this method. The use of the method described here, therefore, provides an index of tissue extraction as opposed to a direct measure of tissue extraction obtained by venous sampling techniques such as the indicator-.dilution technique.

01-

IO

20 SECONDS AFTER INJECTION

30

FIG. 1. Time required for injected bolus to make a single passage through hepatic microvasculature. Liver uptake indices were determined on tissue samples taken at various times following portal injection of a 0.25-ml bolus containing either 0.5 &i of [Wlinulin, or 0.5 &i of [‘“Cl sucrose, and 1.5 &i of 3HOH. Each point represents the mean of at least 3 determinations on individual livers. Vertical lines above or below each point represent 1 SE.

TABLE 1. Liver uptake of 14C-labeled carbohydrates and 14C-labeled cholic acid

Sucrose Inulin p-Mannitol n-Glucose L-Glucose Cholic acid Liver uptake ber of observations ‘were terminated

Injected Concn, mM

Liver

.479 .149 .121 .008 .250 .062

24.3 27.7 80.2 96.8 26.7 127.1

Uptake

Index

& 3.2 (5) & 2.7 (3) zt 6.1 (3) zt 0.8 (3) zt 0.8 (3) & 10.9 (3) --~~ indices are means + SE. For each mean the numis the number in parentheses. All experiments at 18 s following rapid portal injection.


LIVER

UPTAKE

OF

AMINO

ACIDS

AND

1157

CARBOHYDRATES

would be early enough to minimize washout of aHOH and at the lower end of the spectrum, while histidine behaved late enough to assume that most of the nonextracted sub- very differently having the highest LUI (106 %) of the stance had passed out of the liver. amino acids tested. The aromatic amino acids, L-tryptoCarbohydrates and cholic acid. As listed in Table 1, the five and L-tyrosine as well as the @an, L-phenylalanine, i4C-labeled carbohydrates selected for study exhibited a branched-chain aliphatic amino acids, L-leucine, L-isoleuwide range of liver uptake indices. The nonmetabolizable tine, and L-valine, were all grouped near the upper end of extracellular markers, sucrose and inulin, defined the lower the uptake spectrum. Glycine, L-alanine, L-threonine and end of the uptake spectrum around 25%. Campounds L-proline were grouped near the lower end of the spectrum shown by other investigators to be taken up rapidly by with the basic amino acids. The LUI for n-alanine was liver, D-ghCOSe (3) and cholic acid (31), occupied the op- 24 =t 2 %, which in comparison with the LUI of L-alanine posite end of the uptake spectrum exhibiting liver uptake at 46 f 2 % was indicative of a stereospecific mechanism indices of 97 f 1 and 127 % 10 %, respectively. The data mediating liver uptake of alanine. for n-mannitol confirmed other reports (2, 10) that this To establish that the LUI’s observed for the amino acids compound, considered an extracellular space marker in a were due to a saturable transport system, competitive inhibinumber of tissues, is readily extracted by liver. The LUI for tion of the uptake of ‘“C-labeled amino acid was tested by L-glucose of 27 & 1% supported previous reports of a adding unlabeled amino acid to the injection mixture. The stereospecific mechanism for hepatic uptake cf glucose (33). data from a self-inhibition screen of six amino acids are The fact that the LUI for cholic acid was greater than 100 % shown in Fig. 2. The LUI for r.-histidine, which was 106% indicated that the net uptake of this substance by liver at when the injection solution contained only a tracer amount 18 s following portal injection was greater than the net of 14C-labeled amino acid, was determined in the presence hepatic uptake of the 3HOH reference. The greater net of 60 mM unlabeled L-histidine. A considerable inhibition uptake of cholic acid is probably due to minimal efflux of was observed as the LUI for a tracer dose of histidine was this compound as compared to water ef3ux during the 18-s depressed 79 % where 100 % inhibition represents depression circulation period. Table 1 also lists the concentration of of the LUI down to that of sucrose. The LUI for sucrose is each compound in the injection mixture. This represents shown for comparison, The fact that the LUI for histidine the amount that was present in the labeled material ob- was not depressed to the sucrose level indicated that either tained from the manufacturer and ranged from 8 to 479 PM. 60 mM was not a saturating concentration, or that a fraction AK&O acids. The liver uptake indices for eighteen i4C- of histidine entry was mediated by a nonsaturable mechalabeled amino acids are presented in Table 2. The tracer nism. In the case of similar studies for L-aspartic acid and concentration of each amino acid injected is also listed. The L-glutamic acid, 100 and 92 % inhibition, respectively, was amino acids presented a wide spectrum of liver uptake in- observed when 60 mM unlabeled amino acid was added to dices ranging from values similar to those observed for the a tracer concentration of the 14C-labeled amino acids. These extracellular space markers to values greater than 100%. data indicated that virtually all the uptake of acidic amino As, a class the acidic amino acids, L-aspartic acid and Lacids was mediated by a saturable process. In the case of phenylalanine, valine, and alanine, variable degrees of glutamic acid, demonstrated the greatest net uptake having LUI’s of 100 and 86 %, respectively. The basic amino acids, inhibition were observed in, the presence of 60 mM unlabeled amino acids. An inhibition of the LUI’s of 36, 61, and L-a&nine at 38 % and L-lysine at 3 I%, shared a position TABLE

2. Liver uptake of W-labeled

amino acids

120

T C2C:ZM

Histidine Aspartic acid Glutamic acid Tryptophan Phenylalanine Tyrosine Leucine Isoleucine Valine Alanine Glycine Threonine Arginine Proline Lysine n-Aianine Cycloleucine cr-Aminoisohutyric

acid

.008 .012 .009 .053 .005 .I25 .I25 .125 .OlO .060 .125 .I25 .125 .I25 .I25 .070 .284 .485

Liver

105.8 100.0 86.4 81.8 80.5 80.2 78.2 77.8 63.3 45.7 43.8 40.3 37.4 32.0 31.4 23.5 23.8 22.0

Uptake

i f f i f f i f i f f i f f f i f i

10.3 5.5 1.9 8.4 5.3 3.7 1.8 1.5 2.4 1.5 6.5 2.6 1.8 6.3 0.8 1.5 2.3 0.8

Index

(6) (7) (3) (4) (6) (3) (3) (3) (6) (5) (5) (3) (3) (3) (3) (6) (3) (5)

Liver uptake indices are means f SE. For each mean the number of observations is the number in parentheses. Unless otherwise stated all stereoisomers for the amino acids listed were the L configuration. All experiments were terminated at 18 s following rapid portal injection.

100 22 s-

80

[3 Tracer cmino acid only

t

‘1

i L1

T

ITI Tracer

DIUS 60mM

unlobelcd

k? 3 E >

40

i 20

iit

600

[

FIG. 2. Inhibition of uptake of a number of W-labeled amino acids by unlabeled amino acids. Liver uptake index for each amino acid was measured when ‘injection mixture contained either tracer concentration only (open bars), or tracer concentration plus corresponding unlabeled amino acid at a concentration of 60 mM (shaded bars). LUI for [Wlsucrose is shown for comparison. Abbreviations used are as follows: L-histidine, L-HIS; L-aspartic acid, L-ASP; Lglutamic acid, L-GLU; n-phenylalanine, L-PHE; L-valine, L-VAL; r.-alanine, L-ALA. Data presented for each condition represent means of at least 5 determinations on individual livers. Vertical lines above bars represent 1 SE.


1158

W.

M.

PARDRIDGE

AND

L.

S. JEFFERSON

“C-L-ASP

100

CONC , l25)lt.l /‘*C-TRACER cl f w :: k

3

60

ASPARTATE

40

L

El ; 20 s

600 :;..

GLUTAMATE

to 20 10 40 50 60

0 K) 20 30 40 50 60 70. 80 90 100 CONCENTRATION OF UNLABELED AMINO ACID (mM)

FIG. 3. Self-inhibition of uptake of [%]aspartic acid (leftpuael) and [“C]glutamic acid (right ponsl). Liver uptake index for each labeled amino acid was determined in presence of progressively greater concentrations ofcorresponding unlabeled amino acid in injection mixture. In insets, self-inhibition data are converted to a double-reciprocal plot yielding approximate K,,, values for aspartate and glutamate uptake. Abscissas of the insets represent reciprocal of concentration of unlabled amino acid in injection mixture. Ordinates of insets represent reciprocal of difference in liver uptake of labeled amino acid in a tracer concentration (LUI,) and at a self-inhibiting concentration processes cannot be computed from ordinate WI.). vm., of uptake intercept since LUI is an index of transport rate, not an absolute measure. Each point represents mean of at least 3 determinations on individual livers. Vertical lines through each point represent 2 SE.

77 % was observed for phenylalanine, valine, and alanine, respectively. A closer look at the saturation kinetics for the acidic amino acids is presented in Fig. 3. The LUI’s for [r4C]aspartic acid and [i*C]glutamic acid are plotted against the concentration of unlabeled amino acid in the injection mixture, ranging from 2.5 to 60 mM in the case of aspartic acid and from 2 to 100 mM in the case of glutamic acid. The LUI’s for tracer concentrations of each amino acid are also indicated. Double-reciprocal plots of these data indicated that the half-maximal saturation concentrations were 2.7 mM for aspartic acid and 4.8 mM for glutamic acid. Having demonstrated saturation of uptake for the acidic amino acids, cross-inhibition of uptake between aspartic acid and glutamic acid was investigated. These data are presented in Fig. 4. When the injection solution containing a tracer quantity of [r4C]aspartic acid was made 60 mM with unlabeled glutamic acid, a 92 % inhibition of the LUI for aspartic acid was observed. Similarly, a 97 % inhibition of the LUI for a tracer quantity of [14C]glutamic acid was observed when unlabeled aspartic acid was added to the insection solution at a concentration of 60 mM. Although the data in Fig. 4 indicated that aspartic and glutamic acid may share a common transport system, conclusive evidence depends upon demonstrating that the K, determined from self-inhibition studies is equal to the Ki determined from cross-inhibition studies for both aspartic acid and glutamic acid (4). Starvation ejects. Figure 5 illustrates the effect of a 48-h fast on the hepatic uptake of [14C]alanine, [14C]glutamic acid and [(u-r4C]aminoisobutyric acid (AIB) as compared to

‘.C-L-GLU

iii y

60

2 a =1:

40 80 I

E :

20 0 t-

FIG. 4. Cross-inhibition of uptake between acidic amino acids. Liver uptake indices for [r’C]aspartic acid (ASP) and [“C]glutamic acid (GLU) were measured when injection mixture contained either tracer concentration only (open bars), or tracer concentration plus competitive unlabeled amino acid at a concentration of 60 mM (shaded bars). LUI for [iC]sucrose is shown for comparison. Data presented for each condition represent means of at least 5 determinations on individual livers. Vertical lines above bars represent 1 SE.

-

ALA

i. GLU

Al6

SUCROSE

FIG. 5. Effect of fasting on amino acid uptake by liver. Liver uptake index for alanine (ALA), glutamic acid (GLU), and or-aminoisobutyric acid (AIB) was determined in normal fed rats (open bars) and in rats deprived of food for 48 h (shaded bars). LUI for sucrose is shown for comparison. Data presented for each condition represent mean of at least 5 determinations on individual livers. Vertical lines above bars represent 1 SE.

that for [r4C]sucrose. Following 48 h of feed deprivation, net liver uptake of alanine, glutamic acid, and AIB was increased by threefold, 66 %, and tenfold, respectively, over that of controls when all values were corrected for the LUI of the extracellular marker, sucrose. No effect on the liver uptake of sucrose after starvation was observed indicating that the enhanced uptake of amino acids was not due to an increased extracellular volume of distribution. The fact that the LUI for AIB increased as well indicates that the increased uptake is probably due to increased transport rather than increased utilization. These results are in accord with the increase in liver uptake af [r”C]cycloleucine after starvation in the rat observed by Nallathambi et al. (23). These investigators attributed the increased uptake to enhanced influx as opposed to decreased efflux of the labeled amino acid. Increased exchange diffusion due to an increase in free intracellular amino acid did not appear to be a plausible


LIVER

UPT/\KE

OF

AMINO

ACIDS

A4ND

CARBOHYDRiiTES

explanation for the increased uptake of label since the hepatic content of all 14 amino acids studied, including glutamic acid and alanine, was decreased below normal in liver of starved rats ( 1). An increase in portal blood flow due to starvation might possibly explain the increased amino acid uptake. However, the low LUI for AIB was indicative of a low penetrability of the liver cell membrane which inferred that the flux of AIB across the cell membrane was limited by membrane permeability and was, therefore, only a minimal function of blood flow.

The technique used in the present studies was developed in recent years by Oldendorf (24-28) for investigating uptake mechanisms across the blood- brain barrier. It has proven to be a highly successful method for studying carriermediated transport systems localized in the blood-brain barrier. Oldendorf selected 3HOH as a freely diffusible internal reference, since the blood-brain barrier is so permeable to water that the brain uptake of water from blood is essentially flow limited (25). In assessing the potential of other organs for investigation with the Oldendorf technique, no organ appeared more suited, with the possible exception of the placenta, than the liver. Goresky (11) has shown that since the liver sinusoids have no basement membrane or other capillary lining the plasma is contiguous with the space of Disse and therefore the only kinetically significant permeability barrier in the uptake of blood constituents by liver cells is the cell membrane. Since the hepatic cell membrane is so permeable to water (19), the uptake of 3HOH is flow limited in liver as it is in brain. In the present study the applicability of the Oldendorf technique to liver has been investigated. The liver uptake index of a number of 14C-labeled compounds has been determined using 3HOH as an internal reference. The LUI is a measure of the net uptake of a given compound relative to the net uptake of the 3HOH reference. The use of an internal reference normalizes the liver uptake of the 14C-labeled compound relative to the uptake of the 3HOH reference. Since the hepatic uptake of water is a function of portal blood flow when the hepatic artery is ligated, factors affecting portal blood flow will alter the uptake of the 3HOH reference and therefore will change the LUI. Although ligation of the hepatic artery does not alter portal blood flow (14), both hemorrhage or ligation of the mesenteric or splenic arteries would decrease portal blood flow and artifactually alter the LUI. Bv avoiding such complications in the surgical exposure of the portal vein, the portal blood flow, and, therefore, the liver uptake of the 3HOH reference can be maintained within a narrow range. The maintenance of a constant liver uptake of the 3HOH reference ensures that changes in the LUI reflect changes in the net uptake of the 14C- labeled compound. In that the LUI is an index of net uptake for a given labeled compound by liver, the LUI reflects the balance between influx and efflux of the radiolabeled compound between liver and blood during the 18-s circulation period. The influx of the compound is a direct function of blocd concentration, cell membrane permeability, and the rate of blood flow. Efflux of the tracer subsequent to influx during

1159 the 18-s circulation period is directly related to membrane permeability and blood flow and inversely related to the size of the existing intracellular pool of free amino acid or carbohydrate. Assuming efflux of the labeled compound is small relative to influx during the 18-s circulation period, then differences in the LUI reflect differences in the permeability of the hepatic cell membrane for the various compounds under study, providing portal blood flow is maintained within the normal range. The assumption that efflux is small during the 18-s circulation period may not be justified for those amino acids whose intracellular concentrations are low. In liver of fed rats intracellular concentrations of amino acids such as leucine, isoleucine, valine, histidine, tryptophan, phenylalanine, and tyrosine are in the range of 0.1-0.2 pmol/g ( 1) and are approximately the same as the concentration in the For these compounds dilution of the injection mixture. injected 14C-labeled amino acid subsequent to influx will be less than that for those amino acids whose intracellular concentrations are high and substantial efflux may occur during the 18-s circulation period. Therefore, the LUI for these compounds underestimates unidirectional influx and underestimates cell membrane permeability. However, for such amino acids as aspartic acid, glutamic acid, alanine, and glycine, efflux during the 18-s circulation period is probably very small relative to influx since the free intracellular pool of these amino acids is large, 2-3 pmol/g, in liver of fed rats ( 1). Therefore, the LU I approximates the unidirectional influx and differences in the LUI among these amino acids reflect differences in the liver cell mcmbrane permeability. The fact that the LUI for glutamic acid or aspartic acid is approximately the same as the LUI for tyrosine or phenylalanine does not indicate that the permeability of the liver cell membrane for these compounds is the same. In fact the membrane permeability for compounds, such as tyrosine cr phenylalanine, that presumably exhibit substantial efflux by virtue of their low intracellular free pools, could be higher than that for compounds such as aspartic acid or glutamic acid, that presumably exhibit little efllux due to large intracellular pools. The permeability of the hepatic cell membrane for a given 14C-labeled carbohydrate or r4C-labeled amino acid that traverses the membrane by a facilitated mechanism will be a saturable process which will be a function of the Michaelis-Menten constant, K,, and the maximal velocity, V rnaxp of the specific membrane-bound carriers. Since differences in the LUI among compounds that exhibit minimal efflux during the 18-s circulation period reflect differences in cell membrane permeability for each compound, the LUI reflects the Vma,/K, ratio for the carrier-mediated transport of each compound. The major advantage of the technique used in the present studies is the sensitivity of the method for detecting saturable uptake processes from which the as demonstrated by Oldendorf’s K, may be calculated, characterization of several transport systems in the bloodbrain barrier (2547). Two other characteristics of carriermediated transport, cross-inhibition and stereospecificity, are also readily detected with the use of the Oldendorf technique. For example, similarly to the results observed in studits of the liver uptake of glucose (Table l), stereospecific differences were observed in the case of L- and D-alaninc


1160 was 46 % as compared (Table 2). The LUI f or L-alanine to that for n-alanine at 24 %, which was virtually the same as the LUI for sucrose. T‘hese data in conjunction with the 77 $1 inhibition of the LUI for L-[14C]alanine at a 60 mM concentration of unlabeled L-alanine were consistent with a carriermediated transport of alanine in liver. Analogous mechanisms must also exist for liver transport of glutamic acid and aspartic acid. An apparent K, of 2.7 mM in the case of aspartic acid as well as a Km of 4.8 mM for glutamic acid may be calculated from the self-inhibition data in Fig. 3. The Kn values calculated in Fig. 3 probably slightly overestimate the physiological Km. Since some mixing with plasma occurs during passage of the bolus through the liver, the concentration of substrate in the space of Disse may differ somewhat from the substrate concentration in the injection solution. The LUI for [14C]glutamic acid of 86 %, which was indicative of a high permeability of liver cells for this amino acid, was not incompatible with the low distribution ratio of 0.5 observed in liver slices (12). This result was expected if the transport of glutamic acid across the cell membrane was rapid, but uptake was limited to only a few intracellular The low LU I for a-[14C]aminoiso butyric compartments. acid of 24% was comparable to that of sucrose and was indicative of a low permeability of liver cells for this compound during a single circulatory pass. This result was consistent with similar data obtained with the rapid indicator-dilution technique (2) in which the liver uptake of AIB after a single circulatory pass was barely above that of 22Na the extracellular space marker. However, such a low uptake of AIB by liver cells was not incompatible with observations in liver slices (6, 30) in which AIB distribution ratios far in excess of unity were obtained after several hours of incubation. These results were expected if liver transport of AIB, albeit slow, was a concentrative process. The results of the studies presented here provide a basis for at least three areas of further investigaticn. First, the rate of efflux subsequent to liver uptake of the 3HOH reference or “C-labeled test compound within the 18-s circulation period must be quantitated. Efflux rates may be determined in vivo or in the isolated. perfused organ by following nonmetabolizable amino acid the loss of injected 14C-labeled or hexose from liver with time. The efflux rate constant for the 3HOH reference or for any nonmetabolizable 14C-labeled compound may then be determined similarly to efflux

W.

M.

PARDRIDGE

AND

L.

S. JEFFERSON

measurements made in brain (28) or in muscle (17). Efflux data will complement the uptake data presented here in two ways: (i) the extent to which the LUI is an index of the maximal fractional extracticn and, therefore, unidirecticnal influx, may be quantitated; ii) possible transport symmetry across the liver cell membrane may be analyzed similar to the transport symmetry demonstrated fcr glucose transport across the blood-brain barrier (28). Second, the preliminary data on the increased uptake of amino acids by liver with starvation presented in Fig. 5 must be expanded with special emphasis on hormonally mediated mechanisms. For example, growth hormcne has been shcwn to stimulate amino acid transport across the liver cell mcmbrane (13) and preliminary data (29) indicate that growth hormone increases the Vmuzcf amino acid transport in liver. With regard to hormonal mechanisms, the data in Tables 1 and 2 indicate that the liver cell membrane is highly permeable to glucose and several amino acids. The rate of transport of these ccmpounds across the liver cell membrane may be a function cf the rate cf portal blood1 flow as well as cell membrane permeability. Therefore, in addition to increasing cell membrane permeability, a hormone could enhance the rate of influx of a compound into liver by increasing hepatic blood flow. Effects on blood flow versus pcrmeability must clearly be segregated when studying the effects on influx rate of such hormones as glucagon, which has been shown to nearly double portal blood flow socn after intravenous administration ( 15). Third, transport systems for the basic and neutral amino acids must be characterized independently cf that shcwn for the acidic amino acids. Special emphasis cn possible transport heterogeneity is essential in light of Christensen’s (4, 5) studies for neutral amino acid uptake by the Ehrlich ascites tumor cell. We are grateful for the technical assistance of Mr. -James W. Robertson and the many valuable discussions with Dr. Howard E. Morgan. This study was supported by Public Health Service Grant AM13499 from the National Institutes of Health. A portion of this work was presented at the Fifty-Seventh Annual Meeting of the Federation of American Societies for Experimental Biology, April 1973, ,4tlantic City, N.J. Present University

address Medical

Received

for

of W. M. Pardridge: Center, Boston, Mass.

publication

4 March

University 02 118.

Hospital,

Boston

1974.

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Multicomponent cascade reactions of unprotected carbohydrates and amino acids.

Uptake of amino acids and thymidine during the first cell cycle of synchronized hamster cells.

Cerebral uptake of nicotine and of amino acids.

The release and uptake of excitatory amino acids.

Utilization of amino acids and carbohydrates by Salmonella serotypes at 22 degrees and 12 degrees.

Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease.

Branched-chain amino acids in liver diseases.

Letter: High affinity uptake of neurotransmitter amino acids.

Chirality sensing of amines, diamines, amino acids, amino alcohols, and α-hydroxy acids with a single probe.

Human secretory component--III. Carbohydrates, amino acids and N-terminal sequence.

Ion-exchange chromatography of sulfur amino acids on a single-column amino acid analyzer.

Boronic acids for fluorescence imaging of carbohydrates.

Stimulation of erythropoietin secretion by single amino acids.

Placental amino acid uptake. III. Transport systems for neutral amino acids.

Effects of somatotropin and duodenal infusion of amino acids on nutrient passage to duodenum and performance of dairy cows.

Administration of branched-chain amino acids during sustained exercise--effects on performance and on plasma concentration of some amino acids.

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids.

Tracking single-particle rotation during macrophage uptake.

Single-Stranded DNA Uptake during Gonococcal Transformation.

Hepatic uptake and release of glucose, lactate, and amino acids in acutely uremic dogs.

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components.

Inhibition of stomatal opening during uptake of carbohydrates by guard cells in isolated epidermal tissues.

Decrease of uptake and exchange of neurotransmitter amino acids after depletion of their synaptosomal pools.

Uptake and metabolism of carbohydrates by epidermal tissue.