REVIEW ARTICLE

444

Ped

lot

tics

VOLUME 15 APRIL 1955 NUMBER 4

SPECIAL

SECTIONS

REVIEW

ARTICLE

THE METABOLISM

OF CARBOHYDRATES,

FATS AND

BILE

PIGMENTS

BY THE LIVER AND

THE ALTERATIONS

IN HEPATIC

DISEASE; A REVIEW OF

RECENT ADVANCES

By Victor A. Najjar*

T

HE ASTONISHING success of

chemothera-peutic agents in the treatment of

infec-tious diseases has rendered the

manage-ment of such cases relatively simple and

rewarding thereby shifting emphasis to less

common clinical problems in pediatrics. A

decade ago, metabolic diseases constituted a mere fraction of the clinical problems

en-countered; but now make up a large

seg-ment of those cases that call for unusual

skill and wide knowledge of disease. A

clear understanding of metabolic processes

has therefore become necessary for the

study and management of such cases.

The main factors concerned with the

normal function of an organ are its constitu-ent enzymes and the forces that influence

enzyme function. The latter are chiefly

From tile Department of Pediatrics, the Johns Flopkins University Medical School and the

Har-riet Lane home, Johns Flopkins Hospital, Balti-more.

The orginal work reported herein was supported

by a grant in aid [PHS, G-3289 (C 2)] from the

Division of Research Grants, National Institutes of

hleaith, U. S. Public ilealth Service.

(Submitted for publication December 16, 1954.)

o ADDRESS: The Johns Hopkins Hospital,

Balti-more 5, Md.

hormonal and nutritional influences. In

recent years, there has been a very rapid advance in our knowledge of the enzyme

systems involved in carbohydrate and fat

metabolism and the factors that affect the

function of those systems.

These advances have placed before the student of metabolism a pattern of stepwise

processes that determine the direction and

efficiency of metabolism in synthesis or deg-redation. These orderly processes make it possible to predict more or less accurately, the metabolic picture that may result from

a defect in any one particular step. It was with this in mind that a few metabolic

dis-eases were studied and precise biochemical

lesions discovered. In some instances these defects were enzymatic and in others essen-tionally hormonal. This is truly a reversal of the course of events more commonly

en-countered, because it is often the metabolic disease that furnishes the stimulus for the

advances in the physiology of metabolism.

Knowledge for knowledge’s sake has

REVIEW ARTICLE 445

This review will attempt to bring together the pertinent advances thus far made

rela-tive to the metabolic and excretory

func-tions of the liver with due emphasis on the disease process involved.

DISEASES OF CARBOHYDRATE

METABOLISM

These may be divided into 2 main cate-gories, those resulting from hormonal im-balance and those resulting from enzymatic

defects.

Diseases Due to Hormonal Influences

For a clear understanding of the

derange-ments in carbohydrate metabolism that give

rise to the various diseases known in pedi-atrics, it is necessary to review some of the

main enzymatic steps in the mobilization of glucose and glycogen in the liver.’ The influence of hormones on the pertinent

enzyme systems will be discussed in terms of their ability to regulate metabolism in

a seemingly purposeful manner. Diabetes

Diabetes is essentially a disease of

glu-cose metabolism. In Figure 1 are displayed

a series of steps showing, with necessary brevity, the alternate pathways the main

monosaccharides can follow once they enter

the body. Glucose is phosphorylated by adenosinetriphosphate (ATP) to form glu-cose 6-phosphate by the well-known glu-cokinase (hexokinase) enzyme.2 It is then converted to glucose 1-phosphate through the 2-step mechanism of

phosphoglucomu-tase recently elucidated.3 Finally glycogen

is formed by the combined action of phos-phorylase and the branching factor.4

HYPERGLYCEMIA AND ELECThOLYTE Loss: In the diabetic state glucose is not taken up readily by the liver and other organs due largely to the inhibition by the pituitary hormones. This results in a state of tissue starvation, oddly enough, in the face of a

plethora of glucose in the blood (hypergly-cemia). The production of the intermediates in the degradation of glucose to pyruvate (glycolysis) is therefore restricted. This

im-pairment of glycolysis, with minimal

amounts of intermediates, results not only in an inhibition of fat synthesis but also in

an acceleration of fat breakdown. The

com-ponents of the Krebs cycle which are

ordi-narily replenished tc a fair extent by the

glycolytic process must now be derived

largely from breakdown of cell proteins

through deamination of the component

amino acids. Unfortunately the amino acids

derived from ingested proteins are not well assimilated by the diabetic’s tissues due to

lack of insulin.5 These anomalous conditions go far to explain the picture of

gluconeo-genesis, the loss of nitrogen, potassium and phosphate incident to the tissue breakdown so often observed in diabetes. These

prod-ucts of tissue breakdown, in addition to the excess glucose and acetone bodies filtered

through the glomerulus, represent a large

increase of milliosmoles to be excreted. This

requires considerable amounts of water and

in turn results in dehydration and acidosis.

INQIEASED FORMATION OF ACETONE

BODIES, CHOLESTEROL AND Snmoms: The

in-ceased rate of fat breakdown results in

in-creased formatio,n of “active acetate,” the

acetyl derivative of coenzyme A

(acetyl-CoA). This greatly augmented “active

ace-tate” pool, cannot be drained sufficiently by the Krebs cycle or through fat synthesis as we have seen above. The other pathways where the “active acetate” may be utilized

can be seen in Figure 1: (a) It can form

acetone bodies by condensation of 2 mole-cules, eventually to form aceto-acetate. This latter, by reduction gives rise to l-hydroxy-but yrate and by decarboxylation to acetone. The excessive formation of these acetone bodies by the liver is beyond its limited ability to handle them effectively. It is also in excess of the amounts that the other more

efficient tissues can oxidize. This naturally

leads to accumulation of acetone bodies in

the blood stream and their appearance in the urine. (b) The “active acetate” (acetyl-CoA) by successive condensation results in the formation of cholesterol. Under

GALACTOPI NASE

GALACTOSE GALACTOSE-I-PHOSPHATE

ATP

GLUCOSE

AlP

GLYCOGEN

_____________________ I

GLUCOSE - I-PHOSPHATE

I (BLooD)

SPECIFIC

GLUCOSE-B-PHOSPHATE GLUCOSE

PHOSPHATA5C

I

FRUCTOSE-B-PHOSPHATE

_______________________

I

FRUCTOSE- I, 6-DI PHOSPHATE

TRIOSE PHOSPHATE AMINOACIDS

CITR’’” PIREBS

FATTY ACIDS

______________

ENERGY

KETONE BODIES _HOSPHOLIPID

K

_{CYCLE IYICLD I}

4HYDROXY-BUTYRATE

____

//

______

ACETATE

ACETOACETATE ACETOACETYL- C A .-ACETYL-CA PYRUVATE- (BLOOD) LACTATE

(ACTIVE ACETATE) ACETONE +CO,

ACETLATION (A) AROMATIC AMINESDETOXIFICATION

CHOLESTEROL

STEROID HORMONES (B) GLUCOSAMINE HYALURONIC ACID

(C) GALACTOSAMIN( CHONDROITIN

( CHOLINE ACETYLCHOLINE

FIG. 1.

446 NAJJAR - METABOLISM IN THE LIVER

FRUCTOKINASE UCTOSC-I -PHOSPHATE

FRUCTOSE

AlP

for the “active acetate.” In diabetes as we

have seen, not only is this competition

re-duced by restricted fat synthesis but also the excessive fat breakdown serves to

aug-ment the pool further, favoring acceleration

of cholesterol synthesis. (c) In much the

same manner, we believe that the increased 1 1-oxycorticosteroid output in the severe diabetic is very likely due to excessive

syn-thesis in addition to any effect of the attend-ant manifestations of the alarm reaction, not

by direction and not in any way purposeful, but because of excessive availability of the steroid building block-”active acetate.” It

is noteworthy in this connection that the

in-creased steroid output comes about at the

height of ketosis,6 when the “acetate” pool

becomes markedly augmented. The net

effect of the increased 1 1-oxysteroids is to stimulate further increase in potassium loss beyond that accounted for by tissue break-down during active gluconeogenesis.

THE BIOCHEMICAL LESION IN DIABETES:

A few years ago it was clearly shown

that a piece of rat diaghragm, obtained

from an animal rendered diabetic, did not take up glucose from the medium in which

it was suspended as readily as a similar

sample derived from a normal animal.7’ 8

Upon the addition of insulin to the former,

the uptake o,f glucose by the muscle

im-proved considerably. This in essence

ap-pears to be a confirmation of what the clinician has known all along, as diabetic patients have high blood sugar levels be-cause the tissues are unable to take it up at a normal rate. The injection of insulin

improves the uptake and lowers the blood sugar. The experimental observation,

how-ever, goes far beyond confirming the clini-cal findings because the former was made on an isolated tissue. The greatest contri-bution that emerged from this, and which is not often appreciated, is that insulin as

such was directly responsible for the net

stimulation of glucose uptake by the iso-lated tissue. The effects on the patient o,r the diabetic animal could equally be true had insulin stimulated the output of some

447 this process bypasses the hexokinase reac-tion. However, glucose 6-phosphate is an

obligatory intermediate. Through the action of glucose 6-phosphatase22 glucose is formed which cannot readily re-enter the

cell. This acts as an effective drain on any glycogen that might be synthesized.

In-directly therefore, inhibition of glucose up-take is bound to inhibit the synthesis of

glycogen from pyruvate.

FORMS OF DIABErEs: From the preceding

discussion it is obvious that any lesion affecting the beta cells of the pancreas

deleteriously must give rise to deficiency of insulin and result in diabetes. This is the true type of diabetes one ordinarily associates with the usual patient. We have no assurance however, that such is the case.

Some patients with diabetes23 have been found to have normal or near normal insulin

content of the pancreas. However, the large majority of those studied had greatly

re-duced values even though no characteristic lesions of islet cells were observed.24 Never-theless other types of diabetes exist that

are well defined. The type associated with acromegaly is due to excessive secretion of the pituitary gland. The diabetes associated

with Cushing’s syndrome is the result of hyperplasia of the pituitary or the adrenal glands. Such hyperplasia produces excessive

hormones inhibitory to glucose uptake and

a picture of diabetes. Diabetes has been produced by excessive doses of ACTH and

cortisone. In contradistinction to the ordi-nary type of diabetes, this type is invariably associated with excessive deposition of fat.

Tumors or hypersecretion of the alpha cells of the pancreas or the argentophilic cells of

gastrointestinal mucosa would produce hyperglycemia, and possibly a diabetic pic-ture, due to hypersecretion of the

hyper-glycemic hormone glucagon.25. 26 No matter

what the particular cause of

hypergly-cemia and diabetes may be, these merely indicate that there is an imbalance in the

hormones affecting the blood sugar level; or essentially the tissue uptake of glucose

or; perhaps more precisely, though not alto-REVIEW ARTICLE

affected glucose uptake. As it now stands, the effect is that of insulin and is exercised

on the tissue itself. Insulin appears to have

2 effects : (a) Stimulation of glucose uptake

per se, and (b) counteracting the inhibitory effect of the pituitary growth hormone and

other inhibitory factors found in pituitary

92

The adrenal hormones also have an in-hibitory effect on the reaction and this is likewise nullified by the presence of insulin. The effect of the adrenal hormones seems to

be an indirect one, acting through

augmen-tation of the inhibitory effect of the pitui-tary hormones. Experiments on rats with pancreas, adrenals and pituitary removed,

singly or in various combinations, leaves

little doubt that these glands play a deter-mining role in the regulation of blood

32 These hormonal effects were also

studied in tissue extracts and found to act directly on the hexokinase reactions.’3 In that study hormonal extracts of the pituitary

gland were found to inhibit the hexokinase

reaction in the phosphorylation of glucose by ATP. Insulin, on the other hand acted

to relieve this inhibition, thus allowing hexokinase to catalyze the phosphorylation

of glucose at a normal rate even in the presence of the pituitary hormones.

Additional biochemical lesions in

dia-betes have been described.’4 Glucose 6-phos-phatasel5 16 and transaminase’T show

in-creased activity in livers of diabetic animals. It has been shown that the diffusion of sugars across the cell prior to phosphoryla-tion is impaired.’82#{176} This finding in itself

may explain all the biochemical sequelae

presented by the diabetic in much the same

way inhibition of hexokinase has been

called upon to explain them.

Multiple biochemical lesions have been

proposed. As evidence, it is pointed out that livers from diabetic animals cannot synthe-size giycogen from pyruvate at the normal rate.2’ This defect is corrected if the

448 NAJJAR - METABOLISM IN THE LIVER gether conclusively, affecting an enzyme

re-action. In clinical circumstances, the

mag-nitude of this imbalance is measured by a

high and prolonged glucose tolerance

curve.27 When there is excessive output of

inhibitory hormones, as in pituitary adenoma

or adrenal tumor (Cushing’s syndrome), re-moval of the tumor would reverse the

dia-betes. In all instances however, insulin ther-apy would restore the hormonal balance and result in effective control of most manifesta-tions of uncomplicated diabetes.

It has been estimated that 1 molecule of

insulin actmg on the diaphragm system28

mobilizes about 200 million molecules of glucose per hour. With the institution of insulin therapy there is also elimination of the electrolyte and nitrogen losses as all the

lesions discussed above are naturally

re-versed. Potassium accompanies glucose into

and out of the cells. When insulin drives

glucose into the cells there is concomitant

uptake of potassium from the blood. If

potassium is low as might occur following

diabetic acidosis, then a dangerously low

serum level of potassium may be

precipi-tated by the introduction of insulin. The manner in which potassium functions has been suggested by recent work29 which

indicates that like other metal ions it forms an integral part of the substrate complex upon which the enzyme acts.3#{176}

Hypoglycemia

The status in hypoglycemia is in general

the reverse of that in diabetes and hyper-glycemia. This is only true when one con-siders the mechanism which produces such a state. On the other hand, the circum-stances in a severe form of this condition

are very similar to those in diabetes. There is, in either case, tissue starvation for glu-cose. In the diabetic glucose is available but

cannot be utilized. Contrariwise, in hypo-glycemia glucose can be utilized yet is not

available because of the low blood level. Referring again to Figure 1 it may be seen that, as in diabetes, the net result of a

severe reduction in available glucose would

be to stimulate gluconeogenesis, loss of nitrogen, rapid breakdown of fatty acids

and excessive accumulation of acetoacetyl-CoA. The latter, by deacylation, results in acetoacetate precursor of the other 2 ace-tone bodies. The process then progresses as in diabetes from ketosis to acidosis and loss of electrolytes. The 2 conditions differ, however, in 1 major manifestation. In

hypo-glycemia convulsions commonly occur. In

diabetes, though tissue starvation is obvious to such a degree as to produce ketosis and acidosis, no convulsions have been recorded in the presence of hyperglycemia. This is

due most likely to the fact that the brain in diabetes can maintain an adequate rate of glucose utilization to serve its purpose but cannot do so in hypoglycemia of a certain severity.

A number of clinical types of hypogly-cemia have been suggestedu which taken as a group suggest that hypoglycemia as a general entity is by no means rare. It is

apparently more frequent than diabetes

and in most instances poses a clinical prob-lem in the differential diagnosis and in the

management.

There are patients with hypoglycemia caused by a deficiency of the adrenals such

as one encounters in Addison’s disease.

Here the balance between the hypogly-cemic and hyperglycemic hormones is tilted in favor of the former, because lesions of

the adrenals cause diminution of adrenal hormones which are hyperglycemic in func-tion, acting through the pituitary hormones

to counteract the insulin effect. Destructive lesions of the pituitary produce a similar type of hypoglycemia through the same mechanism of increased uptake of glucose

by the tissues. In office practice one

en-counters, not uncommonly, patients with simple isolated non-recurrent hypoglycemic

attacks that d not produce convulsions and that are self limited. These cases recover

PHOSPHORYL.ASE

GLYCOGEN

FORMAT I ON

AND

BREAKDOWN

%

PHOSPHOGLUCO-MUTASE

HEXOK I NASE/ \GLUCOSE- 6- PHOSPHATA SE

!3.-#{216}

- BLOOD GLUCOSE

ATP

REVIEW ARTICLE 449

REPRESENTS 2 OR 3 UNITS

FIG. 2.

Another type of hypoglycemia has

re-cently been described.32 Here the alpha

cells of the islets of Langerhans

of the

pan-creas are absent. These cells secrete the hor-inone glucagon, the hyperglycemic factor. It is also secreted by the argentophylic cells

of the gastrointestinal muco.sa.33

Much work has been done to elucidate the mechanism of action of glucagon.26’ This hormone appears to act through the

phosphorylase system. Phosphorylase (Fig.

2) initiates the reversible conversion of gly-cogen to glucose 1-phosphate. This com-pound is converted in turn to glucose

6-phosphate by phosphoglucomutase and

then by action of the specific phosphatase, glucose is formed. Thus stimulation of phosporylase in this direction favors the breakdown of glycogen to glucose. It has

been shown conclusively4’ that in the

presence of glucagon the activity of

phos-phoiylase ismore or less preserved. This can occur either through increased synthesis of the enzyme or through protection of the

al-ready synthesized enzyme from destruction.

What still remains to be learned is, how the hormone manages to direct the activity of phosphorylase toward the breakdown of

glycogen rather than towards its synthesis.

In vitro-work with rabbit liver slices shows that upon incubation with glucagon the phosphorylase activity in a slice is much higher than in a control slice not incubated with the hormone. This increase in

phos-phorylase activity can be demonstrated by

measurement of either glycogen synthesis or breakdown. None of the other enzymes

involved (Fig. 2) in the transformation of glycogen to glucose are affected in a similar manner by glucagon.

Another hormone, epinephrine, has the same net hyperglycemic effect as glucagon

and acts in apparently the same manner; by preserving the activity of liver phos-phorylase.26’

Deficiency of epinephrine would be

ex-pected to produce the same clinical picture of hypoglycemia as shown by deficiency of

450 NAJJAR - NIETABOLISM IN THE LIVER

This syndrome is one that includes a Glucagon is a protein of complicated

struc-ture and epinephrine is a simple organic

molecule. Each is from a different gland of

internal secretion, yet both act on the same

enzyme in the same manner.

The main types of hypoglycemia that can be explained on a hormonal or an enzymatic

basis are as follows:

1. Excessive insulin secretion.

a. Hyperplasia of the beta cells of the pancreas .

b. Tumor of the beta cells, insulomas. c. Functional over response through tile stimulus of low blood sugar.

2. Decreased secretion of adrenal

hor-mones.

a. Cortical, as in atrophy or tuberculo-sis of the adrenals affecting mainly the cortex such as occurs in Addison’s disease. The decrease in cortical hormones leaves the action of insulin on glucose uptake un-opposed.

b. Medullary, in lesions of the medulla, mainly resulting in decreased epinephrine secretion and diminished effect on phos-phorylase.

3. Decreased secretion of pituitary

hor-inones, such as follows destructive lesions

and occurs in some cases of pituitary

dwarf-i5ITfl. The decrease in the pituitary hormones

leaves the action of insulin on glucose up-take unopposed.

4. Decreased secretion of glucagon, due

to atrophy or absence of the alpha cells

of tile pancreas, which ordinarly increases l)lOOd sugar by its effect on phosphorylase.

5. Absence or inhibition of the specific

glucose 6-phosphatase, which is the only

enzyme known that can produce glucose

from the glycolytic intermediates. This con-dition obtains in certain forms of glycogen disease.

As can be summarized from the

fore-going, tile main types of hyperglycemia may

1)e analyzed on the same basis but the

causes are exactly the opposite of those re-ferred to above.

All tile disorders cited thus far affect the

liver function directly or indirectly or result

from a derangement thereof.

One major syndrome that cannot be

in-eluded among this group, due to lack of

any information regarding its cause, is

idiopathic infantile hypoglycemia. It is much more common than any of the other

causes of hypoglycemia. In a recent report31 it was found that out of 40 cases of hypo-glycemia, 23 fell into this category. In 2

patients who clinically were grouped in this syndrome the alpha cells in the islets of Langerhans were found to be absent. Most

of these patients were below 2 years of age and presented a high incidence as a familial

trait.32 One highly significant feature is the lack of any physical or physiological

anomalies except those immediately

con-cerned with the level of sugar in the blood such as insulin and glucose tolerance tests. Another characteristic is the favorable re-sponse to moderate therapy with

cortico-tropin. Such therapy may be started on the basis of 4 mg. per kilogram of body weight per day, divided into 4 equal doses for 4

days. Following this, one-fourth of this

dose is given in the form of corticotropin gel, night and morning for 1 week. If the fasting blood sugar remains normal with

this schedule, the morning dose is omitted during the suceeding weeks. Adjustment of

dosages for short or long periods of time must be guided by maintenance of fasting blood sugar levels above 40 mg. per 100 ml. and control of symptoms as adequate criteria. Four such patients treated in this manner are completely recovered, requiring

no further therapy. Other patients are well

maintained on about one-tenth of the origi-nal dose. Cortisone and hydrocortisone are not as effective as corticotropin.

Diseases Due to Enzymatic Defects

Other disorders of carbohydrate

metabo-lism involving the liver are due to enzymatic defects instead of hormonal influences as

discussed above.27

REVIEW ARTICLE 451

number of entities. It is necessary now, in

view of recent biochemical advances, to confine this term to the classical von Gierke’s disease. The symptomatology is

es-sentially that which results from hypogly-cemia including convulsions, ketosis and acidosis. Because of the increased amount

of glycogen deposition the liver becomes large and hard. It was found late1y3639 and since confirmed4#{176} that in cases of glycogen storage disease of the liver the enzyme

glucose 6-phosphatase is absent or nearly absent. It is evident from Figure 1, that all

the symptomatology of von Gierke’s disease can be deduced from this simple fact. Glucose is phosphorylated by hexokinase

thereby entering the glycolytic chain. It is

broken down to pyruvic acid then oxidized through the Krebs cycle if energy is needed

and work is performed by the liver.

Alterna-tively, it can be deposited as glycogen.

Glycogen can always supply the liver with

energy through degradation to pyruvic acid. However, when the other organs of the

body need a supply of energy, it becomes

necessary to replenish the blood stream with

glucose. In von Gierke’s disease the liver

is unable to do so because of the deficiency of glucose 6-phosphatase. The glucose

pres-cut in the blood is drawn upon exhaustively and hypoglycemia ensues. The presence of

hypoglycemia results in increased fatty acid breakdown and poor fat synthesis

culminat-ing in ketosis.

It is our opinion that the ketone bodies in this disease must originate in organs other than the liver as those tissues do suffer from

lack of sufficient glycolytic intermediates because of the hypoglycemia, resulting in increased fatty acid breakdown. The liver

on the other hand, is not affected in any way by the hypoglycemia. It has an

abun-dance of glycogen and presumably can con-vert glycogen to pyruvic acid furnishing an abundance of giycolytic intermediates.

These should keep fatty acid metabolism within the normal range and no ketosis should result. In most cases of glycogen

storage disease of the liver glucose

6-phos-phatase is not completely absent. The effect of ACTH on raising31 the blood sugar may be due to stimulation of the defective

glu-cose 6-phosphatase. Complete recovery after

puberty has been observed27 followed by

normal development.

Other types of glycogen disease of the liver are due to abnormality in the structure of glycogen.36’ 4’ Glycogen is a branched

polysaccharide and the molecular structure resembles that of a tree, Figure 2. The stem

and branches are made up of rows of glu-cose units. The first carbon of one molecule

is attached to the fourth carbon of another and the first carbon of the latter molecule is in turn attached to the fourth carbon of still another and so on until 6 to 8 such glucose units are arranged lengthwise in an amylose chain. At this point a branch is

formed by a detachment of the first carbon of a glucose molecule from an amyiose

chain at a 1-4 linkage, followed by attach-ment onto the sixth carbon of a glucose

molecule 6 to 8 glucose units farther along from the point of the previous branch. This

actually amounts to the formation of a 1-6 branch linkage at the expense of a 1-4 chain linkage from an amylose chain. Additional 1-4 carbon linkages can now be built on to lengthen the branches until sufficient units are built, whence a new branch is again

formed. Phosphorylase is the enzyme that

forms as well as breaks the 1-4 linkages.

Transglucosidase is the enzyme that forms

the 1-6 linkages and amylo-1, 6-glucosidase is the enzyme that breaks them. These 3 enzymes together build up and break down glycogen molecules. Normally they exist in

a balance which produces normal gly-cogen.4’ 41 When for some reason this

bal-ance is upset abnormal glycogen may form, with more branches or fewer branches than normal as the case may be.36 Figure 2 shows

the shape of a glycogen molecule and the enzymes that are involved in its synthesis

452 NAJJAR - METABOLISM IN THE LIVER of glycogen disease of the liver36 the

gly-cogen had 15 glucosidic units per branch and in this respect resembled corn starch. These forms of glycogen storage disease affect the musculature of the heart resulting

in enlargement and failure. The skeletal muscles, kidneys and white blood cells may

show abnormally high accumulations of

glycogen.

The only form of dietary therapy is to

institute frequent feedings of high protein

27 This provides glucose frequently

and in a steady stream from conversion of protein. Corticotropin has been used with success in a few cases.31

Galactosemia (Galactosuria)

Galactosemia is a disease of infancy in which galactose is not metabolized but

ac-cumulates in the blood and urine. The in-fant obtains galactose from the lactose of milk. Galactose by its very nature is toxic to some tissues,42 particularly to the brain, the lens of the eye and the liver.43 At least these are the organs that show damage in

the afflicted infant leading to the develop-ment of mental retardation, cataract and

cirrhosis of the liver, with jaundice and splenomegaly. Happily, this is a disease that can be prevented altogether by the alert

pediatrician who is likely to discover the

anomaly by the simple expedient of a rou-tine analysis for sugar in the urine. In an infant who is not acutely ill, the finding of a

reducing sugar in the urine without acidosis and ketosis should alert the physician to a search for the reducing non-fermentable sugar galactose.

The exact site of the anomaly has not as yet been located. However, it is most likely in 1 of 3 known steps in the metabolism of galactose. As can be seen in Figure 1

galactose is phosphorylated by a specific galactokinase to form galactose 1-phosphate.

An enzyme then catalyzes a reaction

be-tween galactose 1-phosphate and uridine-diphosphate-glucose to form uridine-diphos-phate-galactose, giving off glucose

1-phos-phate, which is then metabolized in the

regular manner. The newly formed

uridine-diphosphate-galactose is now converted by

the enzyme waldenase into

uridine-diphos-phate-glucose. The latter again repeats the

cycle to give off glucose 1-phosphate and take in its place a molecule of galactose

1-phosphate. The net result of all this is the

change of galactose 1-phosphate to glucose 1-phosphate.44’

If there were in galactosemia a defect

in gaiactokinase, galactose would accumu-late in the blood. If the other steps were missing there would be an accumulation of galactose 1-phosphate or

uridine-diphos-phate-galactose in the liver and galactose need not accumulate in the blood. This

de-duction is not unreasonable unless there are specific phosphatases for the above

galac-tose derivatives, in which case galactose would form and reenter the blood. The most

likely defect is, however, in the

galacto-kinase.

It has lately been shown4#{176}that the

over-all conversion of galactose to glucose is

rather slow in the infant on milk diet to an

extent that an appreciable amount (40 per

cent) of the blood sugar may be galactose.

The uptake of galactose from the blood,

which may be a galactokinase function, is

depressed in infants recovering from

diar-rhea when intake of galactose has been

interrupted. The uptake of glucose is not similarly affected and one wonders whether gaiactokinase may not be more amenable to adaptive stimulation than the

giuco-kinase.

The only form of therapy is complete

elimination of milk and milk products from

the diet by the use of lactose free milk substitutes. Galactose appears to be better

tolerated later on in childhood and it may

be possible to reinstitute it then without untoward effects.

Fructosemia (Fructosuria)

This condition is rare but nevertheless

encountered in practice. Here fructose is not metabolized, accumulates in the blood

REVIEW ARTICLE 453

*

dietary sources of fructose are sucrose (table sugar) and free fructose (in fruits).

Fructose is phosphorylated in the liver by

fructokinase to form fructose 1-phosphate

as indicated in Figure 1. This is either

phos-phorylated again on the 6-carbon47 then

split by aldolase to 2 phosphorylated 3-carbon fragments, or split directly by

aldo-lase into 2 3-carbon fragments only 1

of which is phorphorylated.48 This latter is

dihydroxyacetone phosphate and is common

to both types of fructose breakdown.

The defect in fructosemia is almost cer-tainly one affecting fructokinase. No other defect further along in the metabolism will

result in fructosemia and fructosuria and

allow viability. As described above there are

2 alternative pathways after fructose

1-phosphate is formed, both of which

in-volve enzymes essential for the maintenance

of life and therefore cannot be implicated. Patients with fructosemia are symptom

free since fructose per se appears to be ex-creted rather rapidly and has no toxic effect at the concentration ordinarily encountered

in the blood.

Pentosuria

Pentosuria is a condition in which pentose

is excreted regardless of the dietary intake. Ingested pentose is excreted to some extent but is undoubtedly metabolized to a

sigriifi-cant degree. In view of this, it is difficult to suggest even a possible abnormality of

pentose metabolism. Tile pentose ordinarily excreted by patients with pentosuria is the ketopentose, xylulose. The metabolism of

ribose and ribulose in mammalian tissue has

been thoroughly studied of late49 but no

significant work has been done with other pentoses such as arabinose and xylose and xylulose.5#{176} There is a suggestion that

glu-curonic acid may be a precursor in that the administration of this compound increases

the excretion of xylulose. Pentosuria is

symptom free and is of interest to the stu-dent of human genetics.5052 It is not known whether this anomaly involves the liver

but the possibility is not remote.

DISEASES OF FAT METABOLISM

The liver is the major organ concerned

with lipid and phospholipid metabolism.lI The capacity of the liver to synthesize and break down fat is so great that it still pro-ceeds normally in humans in such condi-tions as hepatitis with liver necrosis and cirrhosis of the liver, and in animals in experimental cirrhosis produced by carbon tetrachloride poisoning.’45#{176}

Liver fat, like most tissue fat, is composed of 3 main types of compounds: (1) The

neutral lipids, in which 3 fatty acids form esters with each of the alcoholic groups of glycerol. This type may be repre-sented by the neutral fat, tristearin, a trigly-ceride of the following composition

0

112C-0-C-c171-135

0

HC-0--C-C17H3.

0

H2C-0-C---C17H35

The neutral fats obtained from animals

gen-erally contain an even number of carbon

atoms in the fatty acid radicles such as

palmitic C16, stearic C18, and linoleic C1+, the last 2 being unsaturated. Triglycerides are ordinarily composed of more than 1 type of fatty acid.

There are enzymes in the liver that

hydrolyse neutral fats to the constituent acids and glycerol. From a molecule of tristearin the products of lipase action would be 3 molecules of stearic acid and 1 molecule of glycerol. Such enzymes may well be able to synthesize neutral fats from glycerol and fatty acid in vivo as was shown

in vitro.57 Another form of esterification would be to activate the fatty acid with

co-enzyme A (CoA) in which case 3 molecules

454 NAJJAR - METABOLISM IN THE LIVER

ill the synthesis of tile dipalmityl ester of glycerophosphate (dipalmityl phosphatidic acid) which upon the addition of choline

forms the corresponding phospholipid.58’ (2) Phospholipids are another major com-ponent of liver lipids. These are also tn-esters of glycerol like neutral fat except that the third alcoholic group of glycerol is

esterified with phosphorylcholine. This type of lipid is synthesized from alpha glyc-erophosphate, 2 fatty acid-CoA esters and

phosphorylcholine or choline59’ 60 by the

action of the necessary enzyme systems. These phospholipids are broken down by a variety of phospholipases. Below is shown a typical phospholipid, lecithin, and the

sites of action of the various phospholipases

(phospholipase A, B, C, and D).

-A 0

.1’

H

--H

1IC---0- -C---I1

I12C---()---- P-0---Uhoiine

/

\

N

/“ 0 0 \

II

1) C

(3) Cholesterol esters form the third

major group of liver lipids. In this case

cholesterol is esterified by a fatty acid. Such

a synthesis has been shown to occur in

vitro, catalyzed by pancreatic lipase and

serum lipase. Bile salts are essential for this synthetic activity.57 This is particularly interesting in view of the fact that lipases

are essentially hydrolytic rather than

syn-thetic enzymes. However, in the body they

may well carry out both functions.

The steps for breakdown and synthesis of

neutral fats and phospholipids and cho-lesterol esters have been fairly well studied. Knowledge of these steps is helpful in

enabling one to formulate theories that are

necessary prerequisites for investigations into the mechanisms of accumulation of

any of the various lipids in the liver or

blood in pathological conditions. From the

breakdown of neutral fat and phosphoiipid one obtains glycerol, which is oxidized as a carbohydrate, and fatty acids, which are

then estenified with CoA for further break-down.61’ 62 The steps in the breakdown of

such a CoA ester, presented schematically below, may be outlined as follows :

Dehy-drogenation giving rise to an unsaturated bond between carbons 2 and 3 (c and

)

followed by addition of water to form the

1-hydroxyacid and further dehydrogenation to produce a keto-acid. The keto-acid CoA ester now reacts with a free CoA molecule so

as to split off a 2-carbon fragment,

yield-ing acetyl CoA and a fatty acid CoA ester

shorter by 2 carbons. The same chain of

reactions now operates repeatedly on sue-cessively shortened fatty acid-CoA esters until all are broken down to acetyl CoA

fragments.

The acetyl CoA suffers any one of a

number of fates: (1) It can condense with oxalacetate to form citrate and become oxi-dized in the Krebs cycle. (2) It can also condense with another molecule of acetyl

CoA t form acetoacetyl CoA, which then loses its CoA through the action of an active liver enzyme, deacylase. The acetoacetate so

formed accumulates as 1 of 3 ketone bodies,

on is reduced to form another ketone body,

13-hydroxybutyrate, or decarboxylated to form acetone, the third ketone body. These,

as we observed earlier, accumulate in the blood in diabetes because they are oxidized poorly in that disease. (3) Another pathway for acetyl CoA is to polymerize and form

cholesterol and other sterols. In conditions such as diabetes or starvation where there

is accumulation of acetyl CoA due to in-creased fatty acid oxidation and breakdown as well as poor fat synthesis the only

0

+2H

±1120

Acetone + CO2

I

Acetoacetate

±9211

/

/

/

$ Hydroxybutyrate

± CoA

Krebs cycle

Cholesterol, steroids

metabolism in childhood. Tile pictrire pre-sented might be expected to afford helpful guidance for the investigation of such dis-eases. As a good illustration of the difficul-ties, it should be noted that experimental fatty liver can be produced by a diet

(lefi-REVIEW ARTICLE 455

RCII2-CH2-C112----C-CoA

Jr

RCII2-CH- -CH-C-CoA

ll.c?

RCH2-CHOH-CH2--C---CoA

0 0

RCH2-C--CH2---C--CoA

0 0

RCH2-C---CoA CH3-C-CoA

Fatty Liver

It was deemed necessary to present tile steps of lipid, phospholipid and fatty acid

breakdown in some detail because this

in-formation should form the basis of any

de-tailed investigation of the diseases of fat

N-Cl!

II

II

TIC-C-N

CLI

/

N=C-N

+ IIC-CII0H-CHOH-CH-CH2-S-CH2-CHNH2---COOH

0 CH3

Adenosine

It may well be that fatty infiltrations of the increase in blood fat and cholesterol esters liver not responding to methionine are due in cases of nephrosis, nephritis,

hypothy-Methionine (Sulfonium)

456 NAJJAR - METABOLISM IN THE LIVER

cient in methyl donors, yet in human liver disease with fatty infiltration, no such

die-tary history is obtainable and no amount of

treatment with choline or methionine is of

any value. It is not known whether the

de-feet is in tile lipases that breakdown their re-spective lipids, in the amount of CoA

avail-able, in the adenosinetriphosphate (ATP)

necessary to esterify fatty acids with CoA,

in the hydrogen acceptors necessary for the oxidative breakdown of the fatty acid, or in

any one of the enzymes involved in the

series of steps outlined above. It is now

within the reach of the clinical investigator to study these possibilities since methods and adequate technics are available.

The finding that nicotinamide is

methyl-ated in the body has been successfully ap-plied as a means of draining the methyl pool in the liver.63 A photomicrograph of liver

from a rat on a diet adequate for growth,

containing 20 per cent casein and 0.5 per cent nicotinamide is shown in Figure 3.

Methylation is an essential metabolic proc-ess as well as a detoxifying one. With excess nicotinamide the methylation required for

detoxification of this vitamin exhausted the

labile methyl pooi and fatty liver was the

result.

The coenzyme responsible for

methyla-tion has just been identified. It is a product

of the reaction between methionine and

ATP designated as “active methionine,” S-adenosylmethionine; its formula fol-lows6’60:

to an inability to form “active methionine” because methionine as such cannot donate its methyl group to an acceptor molecule. Although methionine can be formed without

the help of ATP from betaine#{176}7 and related compounds68 it remains the principal agent for transmethylation; such as in the

forma-tion of creatine from guanidoacetic acid, the

methylation of nicotinamide and probably

other detoxifying reactions, e.g., methyla-tion of pyridine.

It is still not clear how a deficiency of the methyl pool, or essentially of “active

methionine,” can produce accumulation of fat in the liver. It is even less clear how

fatty infiltration occurs under pathological conditions.

Lipemia

As we stated previously, it is a function of the liver to mobilize fat for use by the tis-sues of the body. When damage occurs to the liver, such as in poisoning by chemical or toxic agents, diabetes, severe

malnutri-tion, biliary cirrhosis and glycogen storage disease, fats are not mobilized and accumu-late in the blood giving rise to lipemia or

hyperlipemia. The real cause of the accumu-lation of fat in the blood is still obscure. In

some instances the lipemia is associated with a high level of cholesterol esters. This is particularly prominent in

REVIEW ARTICLE 457

roidism and obstructive jaundice is due to

depression of liver function.

Idiopathic Familial Lipemia

This entity described first in a child69’ #{176}

and subsequently in adults71’ 72 is

charac-terized by a marked increase in neutral

fats of the blood and to a lesser extent an

increase in phospholipids and total choles-terol, enlargement of the liver, enlargement of the spleen, recurrent attacks of abdominal pain and xanthomata of the skin. Although

other explanations have been advanced697’

as a cause for this disease an impairment of liver function possibly affecting heparin

metabolism is quite likely. The reason for

this postulate is that injection of heparin

produces clearing of the serum turbidity

due to the high fat content and also a

de-crease in the level of serum fat.71

This effect of heparin on the

physico-chemical nature of fat has been studied

ex-tensively during the past few years.73 This role may not be limited to serum lipids but

may well be active in the various organs.

Heparin is most abundant in the liver and its main function may be in that organ but it is present to some extent in various

tis-sues. Any disturbance of the liver that

af-fects tile synthesis of heparin is bound to

affect fat metabolism and serum fat. This

may well be the reason why most cases of abnormal lipemia show some evidence of impairment of the liver.

The effect of heparin on serum lipids

is exerted through the “clearing factor.”

When heparin is either injected into a pa-tient or added in vitro to the serum there

results a clearing of the serum of the

turbidity due to fat and hence the name

“clearing factor.”74’ This factor apparently acts much like lipase, splitting the lipids into glycerol and fatty acids; at the same

time there is a change in the lipoproteins

of the serum from a low to a high density type. However, it has been shown conclu-sively that it is distinct from serum lipase.73

Because injection of heparin into hyper-lipemic patients results in stimulation of the clearing factor it is reasonable to assume

that in such states heparin production is

de-fective. This has not yet been confirmed by heparin measurements. It also may be con-jectured that heparin acts on the clearing factor as a hormone controlling this aspect of fat metabolism, much like insulin acts on glucose uptake and epinephrine and

glu-cagon act on the phosphorylase system to regulate carbohydrate metabolism.

It is evident from the foregoing, that in

the disorders of fat metabolism one cannot locate the biochemical lesion with any measure of confidence, as one is able to do in some diseases of carbohydrate

metabo-lism. The reason is obvious as we have only just begun to uncover the steps in the metabolism of fat. There is little doubt that the future holds exciting and perhaps life

saving possibilities if only the clinician will endeavor tc remain well informed of the biochemical advances in this field. “Active methionine” (S-adenosylmethionine) is not

available commercially. Judging by its chemical structure it should penetrate cell

membranes with little difficulty. When it becomes available, there should be exten-sive trials for possible beneficial effect on fat deposition in the liver and other tissues

including atheromatous patches of arterio-sclerosis and diseases of lipid metabolism (Gaucher, Niemann-Pick, Hand-Sch#{252}ller-Christian). This may be over optimistic but

few advances in medicine have not been sparked by some measure of optimism.

BILE PIGMENT METABOLISM

Only a few steps in the formation and excretion of bile pigment are known. How-ever, knowledge of these steps is sufficient

for an understanding of the behavior of bile pigments in liver disease. Bilirubin arises from the heme moiety of hemoglobin. The protoporphyrin ring of heme undergoes an

oxidative rupture76 freeing the iron from its center and yielding biliverdin, an oxidized form of bilirubin. Biliverdin is in turn

attach-458 NAJJAR - METABOLISM IN THE LIVER

ITleilt to tile serum proteins. It is difficult to

ascertain tile exact nature of the protein to wilich it is attached in serum under physio-logical conditions. Alcohol77 and ammonium sulfate fractionation of serum indicates that

it is attached to the globulin fraction78’ 79

whereas with electrophoresis8#{176} it appears in tile albumen fraction. Further analysis

with paper electrophoresis8’ shows that

above pH 7.0 the indirect bilirubin moves

with the albumen fraction. Below this pH, progressively more dissociates from the pro-tein becoming electrostatically neutral and

precipitating on the parer until at pH 5.0

there is little or no motion in the electric

field. It was this pro)erty that made it

pos-sible to extract the pigment from serum

with ether and subsequently crystallize it

through rigid pH control.7#{176} In any event, the protein to which this indirect bilirubin

becomes attached seems to depend on the

conditions used. There is little doubt,

how-ever, that bilirubin can combine in vitro

with albumen and globulins, except gamma

globulin.79’ 82

Regardless of the Proteill that carries the pigment, the liver cell is able to dissociate it and, through a specific trallspOrt mechanism, excrete it into the bile ducts. The

mecha-nism by which bilirubin is excreted by the

liver cell has been investigated lately to

some extent.81 Fresh rabbit liver slices were used for this study. The ability of the liver

slice to take up and concentrate the

pig-ment from a bathing fluid would essentially be a direct measure of the ability of the liver cells to transport bilirubin from the extra-cellular fluid through the cell substance and into tile bile canaliculi, where the pigment

accumulates. In Table I it may be seen that rat and rabbit liver slices are indeed able

to take up tile pigment and concentrate it

within the slice-expressed as intracellular bilirubin. This concentration can either take place in the cytoplasm of the cell and/or, more reasonably, in tile callaliculi and bile ducts. This is an active process requiring energy, rather than a mere diffusion across a membrane. It does not occur at 0#{176}C.

ex-cept to a limited extent which can be ac-counted for by the process of diffusion into

the extracellular fluid; no actual concentra-tion of pigment takes place at 0#{176}C.

The ability to take up tile pigment is also a function of time, arriving eventually at a state of equilibrium in about 30 minutes at

37#{176}C. under the conditions described in Table I (cf. Fig. 4). The amount of pigment concentrated in the slice depends on the

bilirubin concentration in the medium. Figure 5a shows such a relationship. When such values are plotted, using the Lime-weaver-Burk (Fig. 5b) plot, a Michaelis constant value is obtained which shows that

this mechanism is working at a half-maxi-mum rate, when bilirubin concentration is around 1 mg. per 100 ml. and when the theoretical maximum occurs at infinite

pig-ment concentration. This value is within the

normal range of rabbit serum. From a physi-ological standpoint, this indicates that the

ability of rabbit liver to excrete bilirubin as studied by this technic is rather poor. If this

analogy can be extended to the human it

would tend to explain why jaundice develops

so readily in such a variety of diseases and the serum level of bilirubin rises rapidly fol-lowing hemolysis.

Other tissue slices were subjected to the

same type of study. Fresh kidney, muscle

and brain slices revealed no ability to take up and concentrate bile pigment from serum under the same conditions. The only uptake was slight and limited to that occur-ring by diffusion alone, comparable to that obtained at 0#{176}C.where no active metabo-lism occurs. This mechanism was also stud-ied using liver homogenates. Although this

system is necessarily highly artificial it can serve to study transport mechanisms

satis-factorily. A cellophane bag is used to repre-sent the cell wall. The reaction mixture

in-side the bag represents the cytoplasm which contains the necessary enzymes. The

me-dium in which the bag was bathed functions as the extracellular bile capillary space. Into the bag is placed: 0.5 ml. of 20 per cent

ser-I0

L&J

1-8

a-z6

-J

2

0

0 15 30 45 60 75 90

REVIEW ARTICLE 459

INCUBATION

TIME

-

MINUTES

Fic. 4.Liver slices were incubated at 37#{176}C.in a medium containing 100 y of bilirubin in 0.5 ml. of serum buffered at pH 7.4. Samples were taken out at various intervals and assayed for bilirubin uptake

(details provided in Table I).

um containing 200 ‘L of bilirubin and 0.5 ml. of 0.2 \‘I phosphate buffer pH 7.6. The bag

is placed in a flask containing 1.0 ml. of 5

per cent crystalline human albumen and 1.0

ml. of 0.1 s’I phosphate buffer pH 7.6. An

identical control was run at 0#{176}C.The flasks were shaken at 25#{176}C.for an hour. It was thought that if liver homogenate contained

the necessary elements of the excretion

mechanism (enzymes, cofactors, etc.), the

bilirubin inside the bag would be detached

from the protein, as a first step in its

trans-port. If this happened then the pigment

having been freed would pass at random

through the pores of the membrane and,

once outside the bag, become trapped by

forming a complex with albumen. The

pur-pose of the albumen then is to prevent

for-mation of an equilibrium with free

pig-ment. This would be similar to removing

the pigment from the system, much as would be accomplished by drainage through

the biliary ducts.

Using this technic, it was found that bili-rubin passed to the outside increasingly with time, but only a small fraction, 20-30 , was recovered. It was found that the pig-ment inside the bag was simultaneously

be-ing destroyed by the liver homogenates,

presumably by oxidation, so that at the

end of an hour very little bilirubin was left in the bag for transportation. There was negligible diffusion at 0#{176}C.or at 25#{176}C.

using brain and muscle homogenates. How-ever, with kidney homogenates a definite

effect was obtained, approaching 20 to 50

per cent of that shown by comparable

amounts of liver homogenates.

mecha-

20-15

I0-5.

a

5

10

15

20

25

30

?‘/ML.

OF

MEDIUM

X102

20

15

I

‘V

o.

BI LIRUBIN

CLEARANCE

IN

VITRO

T.37#{176}C.

V

)/ML.

I.C.F./30

M IN.

b

25

-J

U

-j

5

10

IS

20

25

30

35

XIO2

Fics. 5a (Upper) and b (Lower). Liver slices were incubated for 30 minutes at 37#{176}C.in a medium

containing various amounts of bilirubin in 0.5 ml. of serum and buffer at pH 7.4 and assayed for

biliru-bin uptake (details provided in Table I). Figure 5b shows the same values as in Figure 5a but in a

reciprocal plot. V = velocity (y bilirubin uptake in 30 minutes per ml. of intracellular fluid). S =

TABLE I

CONCENTRATION OF BlLInumN -y/ml.

REVIEW ARTICLE 461

nism or the result of the proteolytic action

of cathepsins present in tissues. These are particularly abundant in kidney and liver tissue. If the latter reaction is operative, it may well function as an integral part of the transport mechanism, by destroying the

pro-tein of the pigment protein complex and thereby freeing the pigment for entry into

or exit from the cell.

The observations presented above

empha-size the value of in vitro as compared to in

vivo experiments. It would be impossible with the latter to determine whether the process of bile pigment excretion is by sim-pIe equilibrium diffusion or whether it

oc-curs actively with energy requirement. With tile tissue slice technic, it is clear that the

latter is the case and that in all aspects

the process obeys the laws of energy

requir-ing processes, the rate of which is a func-tion of temperature, time and concentra-tion. Liver slices poisoned with fluoride, iodoacetate, or subjected to autolysis by preincubation at 37#{176}C.for 15 to 30 minutes were rendered useless for experiments

de-signed to inhibit the energy yielding reac-tions. Such slices took up pigment rapidly

from the medium at 0#{176}C.This showed that such treatment must have ruptured or in some manner damaged the cell wall. The

pigment also readily leaked out when the

slices were washed with ice cold water.

This emphasizes the importance of using fresh slices from healthy livers.

Once the pigment is excreted into the bile ducts, or possibly during the process, the bilirubin becomes direct, in the form of a metal chelate complex under the conditions obtaining in bile regarding pH and metal ion concentration. The metal complex

dis-sociates when the pH is lowered. However, it may be rendered quite stable by binding

to protein through the metal of the complex, resulting in what is termed a ternary com-plex (Pigment-Metal-Protein). A good

il-lustration of this has recently been re-ported.84 An uncharged azopyridine dye (Pyridine-2-azo-p-dimethyl-aniline) which

shows little or no association with albumen

Tissue Slices

Reaction Mixture

Intracellular Fluid

Rat liver (37#{176}C.) 18

Rabbit liver (0#{176}C.) 18

Rabbit liver (37#{176}C.) 18

I

Q7 Q8 67

Liver slices about 0.5 to I .0 mm. thick were

incu-bated to 0 to 37#{176}C.in a medium consisting of 0.5 ml.

of serum containing 727 of indirect bilirubin and 3.5 ml. of phosphate buffer pH 7.4, 0.05 M and shaken in a Warburg bath for 30 minutes. Subsequently the slices were washed with I liter of water at 0#{176}C.,homogenized

in 1 ml. of 0. M acetate buffer pH 4.0, then extracted with S ml. of acetone. The acetone extract was clarified

by centrifugation followed by addition of one-half

vol-ume of 95 per cent ethanol. Bilirubin was then

meas-ured in the Beckman model DU Spectrophotometer at

440 mu.

or pepsin becomes strongly bound by either protein when a metal ion is added, such as

zinc, copper, cobalt or manganese. A simi-lar process appears to involve the suggested metal-bilirubin complex.7#{176} In instances of obstruction of the bile duct system there is a re-entry of the metal chelate complex from bile into the blood stream where it is stabilized by serum albumen. This complex

has a high stability constant with the equi-librium being far in the direction of the

complex. The bilirubin-metal-albumen corn-plex represents the direct acting bilirubin of serum. There are suggestions that other

biirubin compounds may exist and8587 that

salt and ester forms of bilirubin are also

direct. The protein involved with this

pig-ment is now definitely established by salt fractionation7#{176} and electrophoresis,8’ to be albumen.

That a major portion of the direct

pig-ment is a metal complex has conclusively

been shown recently.81 This was done by converting direct bilirubin into the indirect

type by treatment of the serum at pH 10 with versene

(ethelene-diamine-tetraace-tate) followed by prolonged dialysis to

462 NAJJAR - METABOLISM IN THE LIVER manner, but with no added versene, or

ver-sene already chelated with lead or cop-per, showed little or no formation of in-direct pigment. The direct pigment was obtained from serum of cases of jaundice due to atresia of the bile ducts. It was characterized by the van den Bergh test, mobility with the albumin fraction by paper

electrophoresis at all pH values between

5 and 9, insolubility in ether at pH 5.0, and by salt fractionation. The indirect pigment formed in the above experiment was

char-acterized by the van den Bergh test, paper electrophoresis as discussed earlier, its

solu-bility in ether at pH 579 and by crystalliza-tion. There is little doubt then that the di-rect pigment complex is of a molecular spe-cies different from the indirect, structurally

and in its physical and chemical behavior, although the pigment itself is the same in its

organic chemical constitution.

These fundamental differences between the 2 forms of bilirubin parallel the different experiences that each undergoes in its trans-port via the liver. Because of this, it is our

opinion that the van den Bergh test which differentiates the two, should lend itself eminently to exact interpretation when

dealing with hepatic or hemolytic disease. There are very few chemical tests that pin

point a lesion so accurately. The mere pres-ence of direct reacting bilirubin in fresh serum, indicates unequivocally that the pig-ment has gone through the liver substance to the bile ducts andsubsequently diffused back into the blood. This back diffusion of

the pigment can take place either because of (a) obstruction of the bile ducts, due to

congenital anomalies, bile calculi, chole-dochus cyst, etc., or an inflammatory

proc-ess in the duct system, either extrahepatic or intrahepatic, or (b) damage tc the liver cells, due to hepatitis, chemicals, toxins (bacterial or viral) or cirrhotic changes. Such damage also suppresses the excretory mechanism and causes accumulation of in-direct bilirubin as well. This effect has been also shown to occur in vitro. With tissue

slices that have been damaged by slight

autolysis, indirect bilirubin diffuses into the substance of the tissue quite readily. The same was shown to be true using direct

bilirubin from sera of obstructive jaundice. The conditions affecting liver function

that are ordinarily reflected by the type of bilirubin accumulating in the serum are

(a) those disturbing the excretory mecha-nisrn within the liver cells, (b) those altering the permeability of the cells, and (c) those

obstructing flow of the bile in the duct sys-tern whether intrahepatic or extrahepatic.

(1) In obstructive jaundice with uncorn-plicated biliary occlusion, such as in early cases of congenital obstruction, the bilirubin

that accumulates is purely of the direct

type. Routine laboratory measurements al-most invariably show a small amount of

in-direct. This is usually a false value because

the extinction of the az dye is greater in

alcoholic solutions. When the

indirect

value

is over 10 per cent of the total, then it is real and indicates advanced involvement of

the liver cells with cirrhosis. In this case the

excretory mechanism is inhibited and the

in-direct pigment accumulates.

(2) In hepatitis, whether toxic or

in-fectious in origin, there is definite damage

to the parenchymal cells as judged by cephalin flocculation, thymol turbidity and

glucose tolerance tests. This causes a sup-pression of the excretory mechanism with a substantial accumulation of indirect

bill-rubin. The damage to the liver cells also allows diffusion of the

direct

pigment into the blood. The usual picture in hepatitis is

therefore an accumulation of both pigments in about comparable quantities, the

magni-tude of which depends on the extent of

damage to the liver cells.

(3) In hemolytic jaundice, the increased rate of hemoglobin breakdown results in

larger quantities of

indirect

bilirubin avail-able for excretion. If the amount is greater than the excretory mechanism of the liver

can accommodate, the pigment accumulates in the serum and is only of the indirect type. If damage of the liver is associated with the

REVIEW ARTICLE 463 the indirect bilirubin will be accentuated.

The direct pigment, by diffusion back through damaged liver tissue would also

appear in significant amounts in the serum. In this situation the flocculation tests would be found to be positive.

(4) The bilirubin excretory system within

tile cell may be diminished not only as a result of pathological processes, as we have seen above, but also through a defect in the

excretory system of congenital origin. Two

such entities have been described:

Fa;iz ilial non-hemolytic /aundice is

usual-ly found in young adults but probably

ap-Pears at 1)irth. Jaundice is of the indirect

type, is commonly mild, and a bilirubin

tol-erance test shows delayed excretion.88 The other entity, congenital familial

non-hemo-lytic anndice wit/i kernicterusi9 is a much more severe type of defect. This disease has

a strong familial tendency, is always associ-ated with a high level of bilirubin in the serum, also of the indirect type as would be expected. There is delayed excretion of an injected dose of bilirubin.#{176} This is the only positive finding with reference to the liver

other than a high level of bilirubin in the

serum. There was an associated kernicterus in 6 out of 8 cases so far described. The

dis-ease was first noted89 in 3 families (M.H. and T.) with definite consanguinity. Since

then, a fourth family (H), closely related to the previous (H), has been encountered with 1 affected child, a girl 12 months old. She is entirely free of symptoms except for the obvious jaundice. The fathers and

moth-ers of both (H) families are brothers and

sisters respectively. The mothers are in turn related to their husbands.9’

There are to date, 2 children who have survived the disease. Neither shows any

signs of kernicterus. One is the 12 months old girl referred to above. The other J.D.H.,

a boy previously reported, is at present 4

years old and is completely free of

symp-toms except for jaundice due to a high

in-direct bilirubin (18 to 24 mg. per 100 ml.) in the serum. This child shows no itching or

other skin manifestations, no clubbing of

the fingers, no enlargement of the liver or

spleen, and no evidence of anemia. Since this boy came to our attention 4 years ago, periodic measurements of serum bilirubin have been obtained. The level of bilirubin has remained more or less constant since

the beginning of the second month of life. This indicates that the excretory function

of the liver has neither improved nor di-minished with age. It is also evident that high levels of indirect bilirubin did not show any progressive toxicity in these 2

children. However, 6 children with this dis-ease succumbed with all the manifestations of kernicterus. It is not known why some of these patients survive and others do not.

The naturally occurring indirect bilirubin may be toxic under special circumstances. If so, the special circumstances in the 6 in-stances mentioned may be similar to those

observed in kernicterus secondary to blood group incompatibilities.92 It has been

sug-gested that indirect bilirubin may be toxic to the brain only during the first few hours or days after birth. Brain respiration has been shown in vitro to be inhibited by high levels of bilirubin and the inhibition was counteracted by large amounts of cyto-chrome C. Whether this is the mechanism

of bilirubin toxicity, if it is toxic, is at pres-ent still uncertain.93

This review of recent developments in metabolism in the liver concerning carbo-hydrates, fats and bile pigments, is in no

way all-inclusive. Such is not its aim.

Re-search in this field has recently become so

extensive that one falters in his endeavor to absorb the multitude of facts appearing in the literature. This review is an attempt to ferret these, piece them together and bring

the clinical and biochemical facets in proper

apposition and perspective. Despite daring thrusts into the fields of enzymology and

biochemistry, no attempt is made to go be-yond the facts.

REFERENCES