Galactose and Glucose Metabolism in the Isolated Perfused Suckling and Adult Rat Liver Wallace The

F. Berman,

development

perfusion

of the

us to characterize adult

galactose uptake

metabolic

rat livers.

differences

or was

4

the

in

Livers

rats were

the

glucose.

same

for

both

the first 35 min. The adult

tained

the initial

riod while up galactose

more

experimental uptake

by the

rapidly. on

young

liver

that of the adult.

Analysis

end of the 90 min perfusion

galactose

or glucose. than

In the immature stimulated

more

both

sugars

of glucose

By the end of the

adult,

weight

was

three

basis, times

of the livers at the showed

hepatic

adult

perfused

uridine-5’-diphosphogalactose cose perfused

output

in the

same perfu-

in young while

liver in the

than the media.

livers

contained

galactose

Galactose levels

each

In the adult,

media;

the level was lower

Galactose and

the

in

liver,

resulted

output.

than

higher

of

galactose

glucose

perfusion.

in glucose

a

observed

with either

being

after this pebegan

to be one-half

was

perfusion

did the glucose

however, levels

Segal

levels.

output

during

Galactose groups

media

Glucose

perfusion

liver main-

concentrations

circulating group

and Stanton

sion resulted

liver

period,

0. Bautista,

to take

rate of uptake

the immature

suck-

Jesusa

with 4 mM age

during

in

suckling

of fasted

perfused

mM

for the

liver has enabled

metabolism

ling and adult

R. Rogers.

technique

of the immature

carbohydrate versus

Shirley

of the

suckling

significantly

more

than

the glu-

livers of each age.

G

ALACTOSE IS a significant nutrient of almost all suckling mammals and, by contrast, is rarely present in the diets of the mature animals. The major organ of galactose disposition has been shown to be the liver.‘,’ As would be expected, developmental analyses of the major enzymes of liver galactose metabolism (galactokinase, galactose-l-phosphate uridyltransferase and uridine-5’diphosphate galactose-4-epimerase) showed that their peak specific activity was reached during the suckling period and then fell rapidly to adult levels.“-” Several studies have demonstrated the greater ability of the young animal, particularly man, monkey, and rat, to handle a given load of this hexose as compared to the adult.“-g Galactose tolerance tests or elimination capacities have been used as sensitive indicators of liver function.‘O.l’ Much has been elucidated about the galactose metabolic pathway (sugar nucleotide or Leloir pathway) over the last 25 yr.” Of major interest in most of these studies has been the desire to explain, biochemically, the clinical toxicity syndrome of the transferase deficient galactosemic individual. These patients may have mental retardation, severe derangement of liver function, and/or cataracts. Study of the physiologic operation of this pathway has been an important feature of the assessment of these abnormalities. Animal models have been produced that mimic the human toxic condition in some respects. Affected rat pups have been produced by feeding pregnant rats

From the Division of Biochemical Development and Molecular Diseases, Children’s Hospital of Philadelphia and the Departments of Pediatrics and Medicine, Medical School of the University of Pennsylvania, Philadelphia, Penn. 19104. Receivedjorpublication September 13, 1977. Supported by Program Project #HD 08536 from the National Institutes o/Health, Bethesda. Md. Address reprint requests to Stanton Segal, M.D.: The Children’s Hospital of Philadelphia, 34th Street and Civic Center Blvd.; Philadelphia, Penn. 19104. ‘c 1978 by Grune & Stratton, Inc. 0026~0495/7~/27/2~C~0003$02.00/0 Metabolism, Vol. 27, No 12 (December), 1978

1721

BERMAN

1722

ET AL

high galactose diets. ‘A,I4 Cataracts have also been produced by feeding young rat pups large amounts of galactose in their drinking water.‘” The chick model, in a like manner, shows a toxicity syndrome in which decreased brain glucose uptake has been demonstrated.“.” High levels of galactitol have been found in all tissues except livers in galactose toxic animals.‘X.‘” Galactitol has also been found in the urine of patients with defective galactose metabolism when exposed to galactose.z0.2’ In addition, it has been shown that galactose-l-phosphate is increased in the red blood cells of galactose-l-phosphate uridyltransferase dehcient individuals when challenged with galactose.22 The isolated perfused liver model has been used by several investigators to study galactose clearance since this organ accounts for about 90% of the metabolic capacity to dispose of this sugar.““.24 This model provides an intact organ in which to investigate complex interrelated events in a simulated physiologic state. The purposes of this paper are to report on the technique for perfusing suckling rat liver, the biochemical patterns of galactose and glucose metabolism in this preparation, and the comparison of this model with the isolated perfused adult rat liver. MATERIALS Male Sprague-Dawiey

AND

METHODS

rats weighing 130 150 g were the adult animals used. By reducing litter size

to 8, we were able to obtain suckling rats weighing 30~ 50 g. All animals were deprived of food for 18 hr prior to use. Fasting animals had free access to water. were injected with 50 mg/kg

pentobarbital

quiet room with the temperature Glucose-free

galactose

was obtained Boehringer

from

Protein-free

adjusted to maintain euthermia

Pentex

(Kankakee.

(New York,

(Freehold, filtrates

the animals

and allowed to rest undisturbed

N.Y.),

III.).

Substrates

Calbiochem

and enzymes

(E. Rutherford,

were purchased

were prepared

Sigma,‘“-‘”

by coupled

enzyme

At the end of perfusion, clamped in Wollenberger determine

lactate

N.J.), Sigma (St. Louis, MO.),

using the sodium hydroxide-rinc

perchloric

&nicotinamide

the liver was rapidly as above. Glucose.

All of the phosphorylated

sulfate method of Somogyi.

dinucleotide

production

with

pyruvate kits

from

excised while the pump was running and freez.e galactose,

compounds

Somogyi filtrates

and galactitol

were used to

were analyzed

as well as glycogen were measured

by gas in 6%

acid filtrates:“,”

The alternate

method of Keppler and Decker:‘” for uridine-5’.diphosphoglucose

assay glucose-6-phosphate

(G-6-P),

glucose-l-phosphate

ing in sequence glucose-6-phosphate uridyltransferase (NADP).

adenine

tongs previously cooled in liquid nitrogen.“’

and pyruvate

chromatography.“”

V

from

N.J.).

Glucose and galactose were measured by their respective oxidases in kits from Worthington: and lactate

in a dark

in both agegroups.

was a product of Sigma (St. Louis, MO.). Bovine serum albumin fraction

Miles

Mannheim

and Worthington

Fifteen minutes before surgery,

intraperitoneally

dehydrogenase.

in the presence of excess UDPG

The uridylated

were determined

enzyme assays are those currently

Technique

(UDPG)

and galactose-I-P

phosphoglucomutase and nicotinamide

was used to

(Gal-l-P)

by add-

and galactose-l-phosphate

adenine dinucleotide

phosphate

hexoses were measured in a similar sequence.?’

The specific activities of galactokinase, galactose-4-epimerase

(G-l-P)

galactose-l-phosphate

uridyltransferase,

uridine diphospho-

in livers at 5, 90, and I35 min of perfusion. The methods for

used in our laboratory?

ofPerfusion

Perfusion of the adult livers was performed

by the techniques of Mortimore.:‘”

Differences

in the size

of vessels and fragility of the liver of the suckling rats made the small animals more difficult to perfuse than the mature accomplished

rats. These difficulties were overcome

in the following manner: The perfusions were

with the livers in situ by first opening the abdominal cavity along the linea alba and then

GALACTOSE

AND GLUCOSE

METABOLISM

1723

making bilateral incisions along the lowest ribs. The liver’s edge was then gently placed under the diaphragm and the intestines moved to the left exposing the porta hepatus. Silk ligatures, 5-0, were placed loosely around the portal vein superior to the entrance of the splenic vein and around the abdominal inferior vena cava (IVC) superior to the junction of the renal veins. A traction ligature was attached to the jejunum to straighten and immobilize the portal vein. Stainless steel 21-gauge cannulae on clear 20-gauge polyethylene tubing were used for infusion; to collect the media, 19-gauge cannulae on l8-gauge tubing were used. The remainder of the procedure was essentially that described by Mortimore for the adult. The perfusate consisted of Krebs-Ringer bicarbonate buffer pH 7.4 containing 3% bovine serum albumin. Each test substrate was added to the medium before the livers were cannulated. The total initial perfusate volume was 60 ml in all studies. All recirculating medium was oxygenated with premoistened, warmed 95% O,-5% CO, at 7 liter/min. The perfusate flow rate was adjusted to 2.0 ml/g liver/min. The six animals used for each experimental run differed in weight by less than 2 g and cannulation time was between 40 and 60 sec. Changes made in volume and concentration due to losses during cannulation, dilution by liver water, and removal of I ml aliquots for analysis were considered in the calculations. The calculations of uptake or output of any given compound were made based on the total water space of the system that included both the perfusate volume and the tissue glucose space. In order to establish the tissue water content under our experimental conditions, animals of both age groups (adult and I5 days) were analyzed in terms of changes of body weight, liver weight, and liver water content in the fasted and fasted perfused state. The amount of swelling in the livers after perfusion was also one of the parameters used to assess the viability of our red cell-free perfusion system. These test perfusions were carried out for 2 hr in a closed recirculating system using IO mM glucose as the standard substrate (Table I).

Competency of the System The three major enzymes involved with galactose disposition were determined in both age groups at 5, 90, and 135 min of perfusion with IO mM glucose (Table 2). No changes in the activities of these enzymes were found in the perfused rat liver over the time interval studied. In order to test the metabolic function of our systems, we perfused adult livers with IO mM lactate for 55 min. During this perfusion, lactate gradually disappeared from the media (Fig. I). Lactate uptake was calculated to be 200 ~moles/lOO g body weight while the perfusate level fell from IO mM lactate to 5.77 mM. These fasted livers produced glucose while they were being perfused with lactate. The amount of glucose appearing in the media at the end of 55 min of perfusion was 4.62 rmoles/ 100 g. Bile collections were made in some animals by cannulating the common hepatic duct with PE IO tubing and allowing the bile to drain by gravity into collecting tubes. Cholic acid 0.056 PM was used as a cholegogue in these experiments only. The rate of bile production was 0.2 ml/hr/ 100 g body weight.

Table 1. Changes in Body and Liver Weights and Water Content Due to Fasting and Effect of Perfusion on the Fasted Liver of the 1 B-Day Old and Adult Rat 1B-Day-Old Fasted Condltlon Body wt (9)

Perfused 34.5 * .53

Liver wt (g) wet 0.95 f Liver wt (g) dry Liver % Body wt % Liver water

.03

0.22 f ,006

Adult Fasted

Fed

Nonperfused 34.6 f

1.04

Perfused 36.8 f 0.90

148.6 zt 1.7

Fed

Nonperfused 145.8 f 2.6

166.8 f 4.3

1.06 f .06

1.19 * .04

5.27 zt 0.19

5.06 f .09

6.85 zt 0.20

0.26 f .Ol

0.30 * ,009

1.32 f .04

1.40 * .03

1.91 * .05

2.75

3.06

3.23

3.55

3.46

4.10

76.90

75.20

74.50

75.10

72.30

72.10

% Fed wt: (1) Body (21 Liver Sprague-Dawley

94.00 79.80

89.10

87 40 76.90

73.90

rats were food deprived for 18 hr prior to perfusion with recirculating 10 mM glucose as

described under methods. Nonlittermates remained with the mother until fasting. Each datum is the mean f SEM of 8 determinations.

BERMAN

1724

Table 2.

Stability

of Young

and Adult

Rat Liver Enzymes

5

During

Perfusion

90 ml*

m,n

ET AL

135 ml,,

Galactokinase’ 4.9 * 1 0

Young

1 75

4.6 +

5.08 * 1 4

523

Young

154+28

15 4 * 3.34

Adult

2 99 *

73

167

Young

280

k

90

2.57 i

Adult

i

37 +

58

091

Adult Gal-l-P

i

57~i

.78

15

7 14

487i

uridyltransferase’ i

I

i79ai

39

58

2 94 + 1 02

UDP galactose-4-Epimerase’

Adult

and 15.day-old

rat livers were perfused with

assays at trme intervals indicated. Experimental

* Expressed

as n moles product formed/mg

1 17

35112

25

10 mM glucose

1 08 i

Lwers were removed

64 for enzyme

condrtions are descrrbed under Materrals and Methods protem/mln

RESULTS

Perfusion of Young Liver 4 mM Glucose. Fasted 15day old rat livers were perfused for 90 min with recirculating media initially containing 4 mM glucose. This perfusate was used to establish a basis for comparing the results of galactose perfusion. The amount of glucose appearing in the perfusate increased in a linear fashion during this study as shown in Fig. 2. Although no lactate had been added to the perfusate, lactate appeared in the media at the start of the perfusion and was subsequently taken up. The level of lactate present at 15 min was 10 ~moles/lOO g and rapidly decreased to 2 cl.moles,/lOO g by 90 min of perfusion. The concentrations of intrahepatic metabolites were determined in the 15-day old rat liver after a 90-min perfusion. Glycogen content was 0.2 mg/g compared with 0.9 mg/g in the unperfused fasted suckling liver. The levels of phosphorylated and uridylated intermediates of glucose and galactose metabolism are given in Table 3. These values show little variation from those obtained in our hands in the fasted unperfused livers. Lactate and pyruvate were 1.3 and 0.45 pmoles/g respectively. Table 4 is a comparison of hexose levels in perfusate and liver. The level of glucose found in the liver was less than the level of perfusate glucose. The concentration of glucose perfusing through the liver at 90 min was 5.35 mM while the liver taken at that time point contained 3.27 pmoles/g wet weight.

r 2200

1

0 GLUCOSE OUTPUT

-

0 LACTATE

UPTAKE

/,/o

2q i

% Y g 150 f I2 c

100 -

8 #

I

50

s z -0

i

-

e--y,

Fig.

I

,

jy

i

1.

Glucose

uptake

by fasted

fusion

with

line represents liver perfused

IO

20

30 MINUTES

10

40

50

the mean

output

adult mM

lactate.

glucose

output

with substrate.

of six determinations.

of all points

is within

*

and

lactate

rat liver during The

per-

dashed

of the adult Each point

is

The SEM

10 ~moles/lOO

g.

GALACTOSE

AND GLUCOSE

1725

METABOLISM

Fig. 2. Glucose output by 15-day-old and adult rat livers perfused with 4 mM glucose or 4 mM galactose. Each point represents the mean of eight assays. The SEM of all points is within & 10 ~moles/lOO g. The 15-min values were adjusted to zero to take into consideration residual and variable glycogen remaining in the fasted livers since Mortimore has shown that in the red cell-free perfusion system glycogen stores are rapidly depleted.3w The observed glucose output at 15 min during perfusion with 4 mM glucose was 32 fimoles/lOO g adult rat and 142 pmoles/lOO g 15-day old rat. During perfusion with 4 mM galactose. these values were 92 ~moles/lOO g adult rat and 42 pmoles/lOO g 15-day-old rat.

2

200

o_

n

4mM

GLUCOSE

0

4mM

GALACTOSE

---

5

15 DAY ADULT

OLD

P

IOO-

E 0 50

-

! 0 _)

I

LO

d

15

35

55

75

95

MINUTES

4 mM Galactose. After the initial background experiments with 4 mM glucose the investigation of galactose uptake and metabolism was undertaken. Galactose uptake proceeded slowly during the first 35 min (Fig. 3). Uptake was 15 pmoles/lOO g at 35 min and increased 1 l-fold during the ensuing 55 min of perfusion to 165 pmoles/ 100 g. As galactose was taken up, glucose was put out into the perfusate. These data, shown in Fig. 2, were corrected for glucose released in the first 1.5min of perfusion when a minimal amount of galactose was taken up and glucose would be derived Table 3.

lntrahepatic

Levels of Metabolites

in the Perfused Young and Adult Rat Livers pm&&g

Wet Weight

Young

Adult

4mM

4mM

4mM

Galactose

GllKlXe

Gal.XtOSe

Galactose

.9 l .09

Glucose

1.62

0

+ .19

3.27

.73 & .I7

f .58

f

.25

.13 z!z .07

.2

.75 f

.95 f .08

.8 zt .2

G-6-P

.08 f .02

.07 * .02

.04 l .Ol

.07 f

01

G-l-P

.ll

.05 f

,009

:04 It .Ol

.06 f

,006

Gal-l-P

.23 f .oa

.15+

.02

.ll

.09 f

,007

UDPG

.06 i- .02

03 f

,006

.Ol * ,001

.009$

03 f

,006

.19

1 3 * .28

.08 f 1.73

Galactitol Gal/Gal-

l

0 1 -P

.46 f

.18

ATP

zk.04

.06

3.29

04zk

Lactate

.2 f

0

213zk.49

Glycogen’

UDPGal

.01t

4mM GlUCOSe

1.25

f

* .02

.Ol It ,003

.05 * .Ol t

.02 f

113h.16

0

,004

1.04*.11

0

3.9

.2

0

6.6

Gal-l-P/ UDPGal

2.9

UDPGNDPGal

.75

UDPG/G-1-P

.54

G-l-P/G-6-P

Fasted clamped * mg/g tp

15-day

and adult

liver.

< .05.

$p < .Ol.

4.5

.2

.5

.25

.17

.7l

20.2

1 .o

46.7

rat livers were

and assays are as described

2.2

1 .6

1.4

Glut/G-6-P

5

perfused

under methods.

for 90 min with initial substrates Results

.86

53.2

are means

f

47 listed.

Livers were freeze

SEM for eight animals.

BERMAN

1726

Table 4.

Concentration

of Hexoses in Liver

and Perfusate in Young and Adult Rat Livers

Tissue

HexoseConcentration

ImM)

Young Perfused

sugar

Media Tissue

samples

rected

young were

derived

GIU GIU

2 48

1 .?O

0 73

Fasted

from

and

adult

obtained the

rat

2.33

livers

at the

end

mterahepatic

for extracellular

Adult

Gal Gal

were

sugar

hexose

study

levels

(perfusatel

Gal

Gill

GIU

GlU

5 37

2 64

3 74

6 04

0 44

as described as prevmusly

(Table

3) and

concentration

GlU

Gal

3 29

perfused

of each

ET AL

in the

text

described.

adjusted

Tissue

for

2 09 for

Tissue tissue

concentrations

90

min

hexose

water

and

perfusate

concentrations

space

are those

3 18 Lwer

(Table

1) and

for intracellular

were corfluld

from rapid early glycogenolysis. The rate of glucose output during galactose perfusion was twice that observed during glucose perfusion. The total amount of galactose that was removed from the original 60 ml vol of perfusate was 70 pmoles. The same levels of lactate were observed in the media during perfusion with galactose as had been found during the glucose study. Although the concentration of galactose perfusing through the liver at the end of the study was 2.5 mM, the amount present in the liver at the same time was 0.9 pmoles/g (Tables 3 and 4). The level of recirculating glucose was I .70 mM while the amount found in the young liver was not markedly different at I .62 pmoles/g. Thus, at 90 min, hepatic galactose values were lower than perfusate galactose values while hepatic glucose levels were similar to perfusate glucose levels. As can be seen in Table 3 there is significantly greater U DPGalactose (U DPHGal) in the galactose perfused as compared to the glucose perfused livers (p < 0. I ), while the other galactose metabolites are not different between these two groups. This finding suggests that in these studies epimerase is the least active of the galactose metabolizing enzymes. Perfusion

of Adult Liver

4 rrlM Glucost~. Adult rat livers were perfused with 4 mM glucose under similar conditions to the sucklings. An increase in perfusate glucose levels was noted throughout the perfusion. Figure 2 shows the additional glucose produced above the 4 mM glucose base line. As the levels of perfusate glucose rose during 3 200 8 \ P

1 150 -

-

15 DAY OLD

---

ADULT

0

g Y

100

-

50

-

2 2 3 g

/

_-

fl :: d W

/ s_----~

0

15

0 f-J_---

__--

0

--

I

30

I

I

I

1

45 60 MINUTES

75

90

Fig. 3. Galactose uptake by 15-day-old and adult rat livers during perfusion with 4 mM galactose. N and SEM are the same as those given in Fig. 2.

GALACTOSE

AND

GLUCOSE

METABOLISM

1727

the experimental period the amount of lactate fell from its peak of 8 pmoles/ 100 g at 15 min to 1 ~mole/lOO g at 90 min. Intrahepatic levels of metabolites measured after a 90 min (Table 3) glucose perfusion of the fasted adult rat livers revealed the glucose to be 3.29 pmoles/g. The initial concentration of perfusate glucose presented to these livers was 4mM, yet the final perfusate level increased to 6.04 mM (Table 4). Only half this value was found in the liver. The amount of glycogen found in the adult livers was 0.13 mg/g compared with 0.26 mg/g found in the fasted unperfused adult rat liver. While no galactose was detected in the adult liver, there were 0.09 pmoles Gal-lP/g, and almost no UDPG or UDPGal were present. The lactate value was 1.04 pmoles/g while pyruvate was 0.21 pmoles/g liver. 4 mM Galactose. When 4 mM galactose was perfused, the amount of galactose taken up by the adult liver in the first 15 min was 9 rmoles/lOO g (Fig. 3). Galactose uptake continued at a linear rate during the course of perfusion. At the end of the 90 min experimental period, the amount of galactose taken up by the adult liver was 50 pmoles/lOO g leaving 2.64 mM galactose in the perfusate. The rate of the uptake process was 33 pmoles/ 100 g/hr. The curve of glucose production was the same as the curve obtained during glucose perfusion as drawn in Fig. 2. The lactate values in this set of perfusions remained the same as those during perfusion with 4 mM glucose perfusate. Twice the amount of UDPGal was present in livers perfused with galactose (p < .05). All of the other metabolites measured had values close to those obtained in the glucose experiments. At the end of this experiment, the level of galactose remaining in the perfusate, 2.64 mM, exceeded that found within the livers, 0.73 pmoles/g. The same trend was observed between tissue and media glucose. The concentration of perfusate glucose was 3.75 mM while the liver contained 2.13 pmoles/g (Tables 3 and 4). Comparison of Young Versus Adult Perfusions Glucose output was observed from the livers of animals of both age groups perfused with glucose. The rates of this production at each age, however, were different. A 0.95 g liver of the 15-day old rat put out glucose at twice the rate of a 5.27 g liver of the adult. The levels of hepatic glucose and glycogen (Table 3) were the same for each age and, with the exception of Gal-l-P, no differences in levels of the other metabolites were noted. When galactose was perfused, widely divergent results were found in the suckling versus adult rat livers. Galactose uptake was the same for both age groups during the first 35 min. After this period, however, while the adult liver maintained the initial rate of uptake, the immature liver increased its rate of galactose uptake. By the end of the 90-min experimental period, uptake by the young liver was three times that of the adult. Thus, the 15-day old rats weighing l/4 of an adult rat took up to 165 hmoles galactose/lOO g when perfused with galactose compared to 50 pmoles/ 1OOgfor the adult animals. No galactitol was found either in the perfusate or within the liver tissue at any time. Livers of both ages perfused with galactose contained less galactose than was present in the media (Table 4). At the same time, galactose perfusion of sucklings resulted in higher glucose levels within the livers compared to the media, while in

1728

BERMAN

ET AL

the adult the tissue level was lower than the media. Although there were differences in levels of individual hexoses, the total hexose (gal + glu) concentration within the liver was the same for both age groups: 3.28 mM in the young versus 3.81 mM in the adult. Immature and adult livers perfused with galactose contained significantly greater amounts of UDPGal compared with their glucose perfused counterparts. DISCUSSION

In vitro analyses of the developmental patterns of Leloir pathway enzymes in liver have demonstrated that they are most active during the suckling period when galactose is a component of the diet. 3--d The specific activities of these enzymes fall rapidly after the suckling period to low adult levels. Because of these findings, a model system of the isolated perfused ICday old suckling rat liver was developed. At this age, the liver of the rat is essentially free of hematopoietic tissue, large enough to be readily connected to the perfusion apparatus, and still immature in that the galactose metabolizing pathway, relative to liver size, is 2 to 33fold more active than the adult. In order to avoid the possible interpretive problems presented by red blood cell galactose metabolism, these studies were carried out using red cell-free perfusate. Hems36 and Williamson3’ have shown that red cell-free liver perfusion could be performed with high oxygen and perfusion flow rates in which optimal gluconeogenesis occurred. Despite these findings, Mortimore,:%” in studying glycogen homeostasis, has shown that oxygen extraction from the perfusate is greater with added red blood cells for any given flow rate and that glycogen stores cannot be maintained in th absence of red cells. Since galactose uptake is minimal during the initial 15 min of study, we assumed that the early and variable glucose release was from the breakdown of residual glycogen in the livers. The lti-min values of glucose output were adjusted as noted in Fig. 2. Our parameters were optimized in the adult by measuring the rate of gluconeogenesis from lactate and the amount of glucose released into the perfusate after glucose perfusions. The results reported here compare favorably with those of ROSS”” and Exton’” using red blood cell-containing media. In addition, the observed bile flow rate was essentially the same as that previously reported in the literature. This finding has been used as an indicator of the viability of the preparation. *‘.‘2 Optimal bile flow reflects the adequacy of oxygenation and intrahepatic perfusate flow, as well as net liver energy level. The isolated perfused liver model is physiologically similar to the intact animal liver and, therefore, suitable for these studies. In an earlier study using rat liver slices, Tygstrup”” observed galactose uptake to be only a fraction of that found in the perfused liver. An in vivo experiment was carried out by Keiding*’ using fed adult rats in which the maximum galactose elimination capacity was determined to be similar to that found using isolated liver perfusion. Keiding’” and Sparks,‘” studying galactose clearance in isolated perfused adult fed pig and rat livers, respectively, have reported results similar to those found here. Several important differences are apparent between adult and suckling animal metabolism of both glucose and galactose. The present studies have shown that hepatic galactose uptake on a unit weight basis was 3 to 4-fold greater in the suck-

GALACTOSE

AND

GLUCOSE

METABOLISM

1729

lings as compared to the adult rat. This finding parallels the observations of Segal et al.‘j who studied galactose disappearance and oxidation during incubation of minced liver tissue from rats of varying ages. These data appeared to reflect the 2 to 3-fold higher specific activity of the galactose metabolizing enzymes in immature rat liver compared to the adult.“-5 The isolated perfused near term and adult monkey livers showed similar differences. y Concomitant with galactose uptake, there is glucose production. The rate of production from the 15day old liver is three-fold greater than from the adult. This would be expected based on our previous knowledge of the activity of the galactose metabolizing pathway in the young. When glucose is perfused through livers from rats of both ages, no glucose uptake is observed. On the contrary, net glucose production was found and for the adult livers, the amount of glucose released into the media was comparable to levels found by other investigators. 3R-39Glucose output in response to glucose perfusion from the immature liver was greater than that seen from adult livers, but less than observed from galactose perfused suckling rat liver. These findings might suggest that gluconeogenesis is a more active function in young versus adult rat and that this process is less responsive to suppression by exogenous glucose in the young animal. The hepatic concentrations of free hexose differed during galactose perfusion in the young compared to the adult. The observed disequilibrium after galactose perfusion for the concentration of free hexose between tissue and media raises several unanswered questions. Although the tissue glucose concentration is nearly the same as the perfusate glucose the tissue levels of galactose were approximately 12 of those circulating for both young and adult. This might imply that the rate of metabolism of galactose is greater than the rate of its entry into the liver cell. The fact that in the young, the level of hepatic glucose is twice the media concentration is consistent with a rapid conversion of galactose to glucose at a rate that exceeds the exit capacity of the hepatocyte membrane. The adult liver metabolizing galactose, on the other hand, generates a media to tissue glucose ratio similar to that observed when glucose itself is perfused. KeidingZ3 studying adult pig and adult rat have suggested that galactose Williams-‘” and Goresky*’ investigating metabolism was rate limiting, but this is not consistent with the present data. The tissue levels of galactose and gal-l-P suggest that the kinase and transferase are not rate limiting in this system. In both immature and adult livers perfused with galactose, a significant build-up of UDPGal was observed so that in these studies epimerase appears to be a limiting step in galactose disposition. This finding is consistent with the relatively low activity of this enzyme that led to the postulate that it is the rate limiting step in galactose metabolism.“:‘.“” The present studies were not specifically designed to evaluate the etiology of galactose liver toxicity. Such studies evaluating metabolic effects of high galactose levels are currently in progress. The formation of galactitol, especially in the lens of the eye, appears to be the etiologic factor in galactose induced cataracts and may contribute to toxicity in other tissues.1x-21 In these experiments, no galactitol was found either in the liver or perfusate. This finding, supported by prior observation that no galactitol is found in the liver of galactose-fed, toxic rats, makes it likely that hepatotoxicity is due to some other factor.

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ADDENDUM

Since this paper was submitted, a study of gluconeogenesis in the suckling rat has been published: Beaudry M, Chiasson J, Exton J: Gluconeogenesis in the suckling rat. Am J Physiol233:E175-E180, 1977. REFERENCES I.

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