glycogen storage diseases - journal of clinical...

J. clin. Path., 22, suppl. (Ass. clin. Path.), 2, 32-41

Glycogen storage diseasesROBERT MAHLER

From the Department of Metabolic Medicine, Welsh National School of Medicine, Cardiff

The glycogen storage diseases are disorders ofglycogen metabolism in which an excessive amountofglycogen accumulates in several tissues. The presenceof an excessive amount of glycogen may physicallyinterfere with the function of the tissue but manyof the clinical features of the various types ofglycogen storage diseases are due directly or in-directly to the impairment of carbohydrate metab-olism.Claude Bernard first isolated glycogen from the

liver in 1857 and described its chemical and physio-logical properties. A little over 70 years later, twoforms of glycogen storage disease were recognized:in one form, glycogen was present in large amountsin almost all the tissues of the body, including theheart and tongue, while in the other the excess ofglycogen was restricted to the liver and kidney. Inspite of much ingenious speculation, no headwaywas made in the understanding of these disordersuntil 1952 when Cori and Cori demonstrated theabsence of the enzyme glucose 6-phosphatase in theliver of children suffering from the hepatorenaltype of glycogen storage disease. Since then, atleast five other types of glycogen storage diseasehave been reliably recognized and classified on thebasis of specific enzyme defects related to glycogenmetabolism (Table I).

THE GLYCOGEN MOLECULE

The glycogen molecule is composed of over 120,000glucosyl units linked together in such a way that it

REDUC2NG END Ar 0z/z

CHO -R N How

:c0 c

FIG. 1. Formation ofbranched glycogen molecule.

assumes a tree-like appearance with relativelylong inner chains, consisting of up to 20 glucosylmoieties, and shorter outer chains of eight to 12glucosyl groups branching off from the inner chains(Fig. 1). The glycogen molecule is large enough to bevisualized under the electron microscope: it is roughlyspherical in shape with a molecular weight of20 x 106 and a volume of 200 cubic Angstroms but,depending upon the nutritional state, it may aggre-gate with other glycogen molecules into even largerparticles. Glycogen particles are usually present inthe cytoplasm, but they also occur in the lysosomes,and in certain pathological conditions may be seenin the nucleus and the mitochondria.

GLYCOGEN SYNTHESIS AND DEGRADATION

These occur by different pathways.The initial step is the conversion of glucose by

hexokinase to glucose 6-phosphate, which is thenre-arranged to form glucose 1-phosphate. The first

ILE ICLASSIFICATION OF MAJOR GLYCOGEN STORAGE DISEASES

Type Enzyme Defect Tissue Affected

Glucose-6-phosphatase

Acid maltase

[II (limit dextrinosis)

IV (amylopectinosis)

V (McArdle)

VI (Hers

'Debrancher'

'Brancher'

Muscle phosphorylase

Liver phosphorylase

Liver, kidney, gut Hepatomegaly, hypoglycaemia,ketosis, acidosis

Generalized, particularly heart, Cardiomegaly, heart failure,tongue, brain, WBC enlarged tongue, muscle weakness

death in infancyLiver, heart, muscle, RBC, WBC Hepatomegaly, moderate fasting

hypoglycaemia, muscle weakness,and wasting

Liver, spleen, heart, muscle, Cirrhosis, hepatic failureRBC, WBCSkeletal muscle only

Liver, WBC

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Pain, stiffness, weakness onexercise only; occasionallymyoglobinuriaHepatomegaly, moderate fastinghypoglycaemia

I (von Gierke)

II (Pompe)

Clinical Features

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Glycogen storage diseases

step of glycogen synthesis proper is the formation ofuridine diphosphoglucose (UDPG) from glucose 1-phosphate. This reaction is reversible, but the nextstep is the irreversible reaction of UDPG with'glycogen synthetase', which results in the formationof a chain in which glucose molecules are joinedto one another in a 1: 4-linkage (Fig. 1). This reac-tion is stimulated by glucose 6-phosphate and byinsulin. When the chains reach a length of about12 glucosyl units, three or four of the units aretransferred from the end of that chain to anotherchain but there they are attached in a 1: 6-linkage insuch a way that a branch point is formed (Fig. 1).The sequential interactions of 'glycogen synthetase'and 'branching enzymes' result in the typicalbranched glycogen molecule which has a character-istic colour and light absorption at 460 m,u whenstained with iodine.Glycogen breakdown is initiated by phosphoryl-

ase, which successively hydrolyzes all the 1: 4-link-ages in a chain, each time producing glucose I -phos-phate, but is incapable of splitting the 1: 6-links atbranch points. To break through the 1: 6 linkanother enzyme is required, the 'debranchingenzyme', which yields a molecule of glucose andthrough its action exposes another chain of 1: 4-linkedglucosyl units to the action of phosphorylase. Thusthe degradation of glycogen, like its synthesis,requires the sequential interaction of two distinctenzymes and results in the formation of glucose andof glucose 1-phosphate which can be used by thecells for their metabolic requirements. Not all theglycogen is broken down by this mechanism: someof it finds its way into the intracellular lysosomeswhere it is hydrolysed by a-glucosidases to formmaltose and glucose.The identity and quantitative importance in

human metabolism of the separate pathways ofglycogen synthesis and degradation were onlyclarified when the specific enzyme defects of theglycogen storage diseases were recognized. Theirstudy emphasizes the contribution which clinicalmedicine can make to basic biochemistry and thehelp which can be gained from an understandingof the biochemical processes in the management ofpatients suffering from metabolic disorders. Ameasure of the wide interest which their study hasaroused is the number of reviews of this subjectwhich have appeared recently (Steinitz, 1967;Brown and Brown, 1968; Hers and van Hoof, 1968;Symposium, Amer J. clin Path., 1968; Mahler, 1969).

TYPE I GLYCOGEN STORAGE DISEASE (GLUCOSE6-PHOSPHATE DEFICIENCY)

In 1929 von Gierke described the excessive accumu-lation of glycogen in the liver and kidney in this

disease, and in 1952 Cori and Cori demonstratedthat there was no glucose 6-phosphatase activity inthe liver of affected cases. In the absence of theenzyme, the liver cannot produce glucose by eitherglycogenolysis or gluconeogenesis (Fig. 2) so thatpatients are liable to have attacks of profoundhypoglycaemia. Prolonged severe hypoglycaemiaresults in secondary disturbances of lipid metabolismwith increased mobilization of fat from adiposetissue and overproduction of cholesterol and ketonebodies in the liver. Normally the liver clears theblood of the lactic acid which has been produced bymuscles and other tissues with a high rate of glyco-lysis and converts it into glucose by the usualpathways of gluconeogenesis. In type I glycogenstorage disease the liver itself produces large amountsof lactic acid from glycogen, because when glyco-genolysis is stimulated, the glucose 6-phosphate isdiverted into the glycolytic pathway; the liver thusaccentuates an existing lactic acidosis and furtherdisturbs the acid-base balance of the body. Pro-longed severe lactic acidaemia may also lead toretention of uric acid by competition for transportat the renal tubule and may result in the appearanceof clinical manifestations of tophaceous gout.The accumulation of abnormal amounts of

glycogen in this disease is confined to the liver,kidney, and intestinal mucosa, ie, tissues whichnormally show glucose 6-phosphatase activity.Although all these tissues can also metabolizeglycogen by other pathways, it would seem that theenzyme is of importance in the control of theirglycogen content. In other tissues, such as muscle,which normally possess no glucose 6-phosphatase,there is no excess of glycogen.Absence of enzyme activity in this disorder has

been shown by direct assay of the enzyme in biopsies

GLYCOGEN---p g-)i-p - _|Glucagon[nolor+^oo I I _ ~~~~~4_

Fructose -.

_ ,- 9-11 -P

g- 6-P X4:

Pyruvate

0o2

_-* Glucose

Lactate

FIG. 2. Type I glycogen storage disease due to glucose6-phosphatase deficiency (X).

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from liver, kidney, and intestinal mucosa. Indirectevidence for the absence of glucose 6-phosphataseactivity in the liver can be obtained from variousfunctional tests: neither the administration ofglucagon or adrenaline, both of which stimulateglycogenolysis, nor the administration of galactoseor fructose, which are normally converted intoglucose by the liver, result in a rise in the bloodglucose level, but they produce an abnormal rise inblood lactate.The prognosis is poor in young children and

prevention of hypoglycaemia by frequent meals isimportant if damage to the brain is to be avoided.Administration of diazoxide, which interferes withthe release of insulin from islet cells, also helps tomaintain the blood sugar level within a clinicallysafe range, and rarely more heroic measures, suchas the anastomosis of the portal vein to the inferiorvena cava so that glucose from the gut bypasses theliver, have been used with some success for thispurpose.

Diagnostic tests are summarized in Table II.

BLOOD CHEMISTRY The classical biochemical fea-tures of type I glycogenosis are spontaneous hypo-glycaemia, persistent ketonaemia, and acidaemia.Hypoglycaemia causes secondary disturbances oflipid metabolism, manifested by raised plasmalevels of free fatty acids, cholesterol, and triglycer-ides; the last may be present in such large amountsthat they give the plasma a creamy appearance.Acidaemia is caused by the high concentration oflactic acid, which in turn is responsible for theretention of uric acid in blood.The glycogen content of red and white blood cells

is normal.

FUNCTIONAL TESTS A presumptive differentiationof type I glycogen storage disease from the othertypes can be made on the proper interpretationof a few, relatively simple, functional tests (TableII). Glucose 6-phosphatase is the enzyme whichcontrols the final common pathway for the releaseof glucose as the result of glycogenolysis andgluconeogenesis in the liver. In the absence of theenzyme stimulation of these processes cannotresult in a rise of the blood glucose level, butthe production of lactic acid proceeds freely (Fig. 2).

Glucagon Normally the intramuscular admini-stration of 0 5 mg glucagon, which acceleratesglycogen breakdown, causes the blood glucoseconcentration to increase within 15 to 30 minutes,with only a slight increase in the concentration oflactic acid. In the absence of the enzyme there is norise in the blood glucose level but a considerable

TABLE IISUMMARY OF THE CLINICAL AND FUNCTIONAL DIFFEREN-TIATION BETWEEN THE SIX MAJOR TYPES OF GLYCOGEN

STORAGE DISEASE

Tissue Response to Diagnostic TestAffected

Liver (andkidney)only

Muscle onlyMany

tissuesFastingSevere hypoglycaemiaModerate hypoglycaemiaNo hypoglycaemia

GlucagonNo increase inblood glucose Fasting

Normal increase inblood glucose f

No increase in Ptrdblood glucoseNormal increase in

blood glucoseNo increase in blood lactateNormal increase in blood lactateGross increase in blood lactate

Type of GlycogenStorage Disease

i, VIV

II, III, IV

IIII, IV, VIII, V

I, III, IV, Vlt

II, V

I, Vit

II, III, IVVIII, III, ivt, VI

Intravenous Galactose or FructoseNo increase in blood glucose INormal increase in blood glucose II, III, IV, V, VIAbnormal increase in blood lactate I

Ischaemic Forearm ExerciseNo increase in blood lactate III, Ivt, VNormal increase in blood lactate I, II, VI

increase in lactic acid, which may be sufficient toaggravate the existing acidosis.

Intravenous infusion ofgalactose orfructose Thesetwo sugars are converted in the liver through aseries of enzymatic reactions to glucose 6-phosphateand would then normally be dephosphorylated toglucose and raise the blood glucose level. In theabsence of glucose 6-phosphatase there is noincrease in blood glucose, but the lactic acid concen-tration in the blood increases as the result of themetabolism of glucose 6-phosphate through theglycolytic pathway.The failure of the blood glucose level to rise and

the abnormal rise in blood lactate after these twotests differentiates type I from all the other estab-lished types ofglycogen storage disease.

SPECIFIC INVESTIGATIONS Final proof of the diag-nosis, however, must rest on the demonstration ofa glycogen content more than 4% of wet weightand a very low glucose 6-phosphatase activity in asample of liver obtained by open or needle biopsy.

Estimation of glucose 6-phosphatase activity inintestinal biopsy material is an acceptable alternativeto liver biopsy and is the preferred technique for theinvestigation of relatives of affected patients, levels

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of about 50% normal being found in heterozygotes.Such studies have shown that the disease is trans-mitted as an autosomal recessive character.

TYPE II GLYCOGEN STORAGE DISEASE (LYSOSOMALO-GLUCOSIDASE DEFICIENCY)

Although this was one of the earlier types of glycogenstorage disease to be recognized as a clinical entity(Pompe, 1932), it is one of the most recent in which aspecific enzyme defect has been demonstrated(Hers, 1963). In this disease all the tissues of thebody contain large amounts of glycogen which maybe sufficient to interfere mechanically with thecontractility of the heart muscle and to causedeath from heart failure. On the other hand generaldisturbances of carbohydrate and lipid metabolismare only remarkable by their absence (Fig. 3).

FIG. 3. Type II glycogen storage disease. Glycogenaccumulates within lysosomes (heavily outlined) due toabsence of 'acid maltase' (X).

In normal cells lysosomes contain a variety ofhydrolytic enzymes active at a low pH, whichdegrade macromolecular compounds such as glyco-gen, proteins, and lipids which have been engulfedby these organelles (De Duve, 1959). In type IIglycogenosis Hers demonstrated the absence ofan ac-: 4-glucosidase, which normally splits anyglycogen enclosed within the lysosome into maltoseand glucose. In the absence of the enzyme glycogenaccumulates in increasing amounts and with theelectron microscope a large aggregation can be seensurrounded by the lysosomal membrane. Thestructure of the glycogen molecule isolated from thetissues is normal.

It was shown experimentally (Cuthbertson andFleming, 1964) that a glucosidase derived fromAspergillus niger was capable of lowering liver,3

muscle, and heart glycogen in the rat after intra-peritoneal administration. Baudhuin, Hers, andLoeb (1964) made use of this observation to treat apatient with type II glycogenosis and showed thatintramuscular administration of purified fungala-glucosidase decreased the glycogen content of theliver from 115 to 8% (normal level up to about5%).


BLOOD CHEMISTRY There are no recognizableabnormalities of carbohydrate or lipid metabolism.

FUNCTIONAL TESTS All the usual functional testsemployed in the investigation of glycogen storagediseases produce normal responses.

SPECIFIC TESTS The diagnosis may be suspectedfrom the unusually high concentration of glycogenfound in several tissues (not uncommonly exceeding10% of the fresh weight of the tissue). Examinationof the tissue under the electron microscope showsthat the lysosomes are filled with glycogen granulesand that this glycogen persists in the lysosomes whenthe cells have been exposed to glucagon or adrena-line, although their cytoplasm is depleted of itsglycogen content by this treatment.The diagnosis is confirmed by demonstrating the

absence of x-l,4-glucosidase in liver or in leucocytesusing a biochemical assay devised by Hers (1963).In heterozygous carriers the leucocyte enzyme isreduced.

TYPE III (,LYCOGEN STORAGE DISEASE ('DEBRANCHINGENZYME DEFICIENCY)

This disease is characterized by the absence of the'debranching enzyme', amylo-l: 6-glucosidase, andan accumulation of a dextrin-like glycogen withabnormally short outer chains. In the absence ofthe debranching enzyme, phosphorylase acts onsuccessive 1: 4-glucosyl linkages in the outer chainsof the glycogen molecule, but reaches the limit ofits action close to a 1: 6-linked branch point, leavingpolysaccharide whose structure resembles that of apollarded tree and is described as a 'limit dextrin'(Fig. 4). The disease is therefore called 'limitdextrinosis'.The enzyme occurs in most tissues of the body,

and accumulations of limit dextrin have been foundin many tissues as well as in erythrocytes andleucocytes. Hypoglycaemia occurs on fasting becauseglycogen can be degraded only to a limited extent,but even then it is rarely severe because glucosecan still be formed by gluconeogenetic pathwaysfrom amino-acids and other precursors.

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FIG. 4. Type III glycogen storage disease. A limit FIG. 5. Type IVglycogen storage disease. A starch-likedextrin accumulates due to limited degrading ofglycogen as polysaccharide with few branch points accumulates as thea result of 'debranching enzyme' deficiency (X). result of 'branching enzyme' deficiency (X).

Detailed studies of the enzyme itself suggestthat it consists of a transferase as well as a glucosi-dase. Two clinical forms of type III glycogenosishave also been described, one affecting the liverand other organs including muscle, the otheraffecting mainly muscle but not liver, but there is asyet no evidence to show that the clinical distinctionis related to the biochemical properties of theenzyme.The prognosis in this type of glycogen storage

disease is much better than in type I and manycases are known to have survived into adult lifeor have only been discovered accidentally at a laterage. It is important to make an accurate diagnosisin order to be able to give an optimistic prognosiswith some confidence.


BLOOD CHEMISTRY Hypoglycaemia on fasting, ele-vated plasma free-fatty acid and occasionally raisedplasma cholesterol levels may be observed. Theglycogen content of erythrocytes is raised (normalvalues 20 to 130 ,ug/g Hb).

FUNCTIONAL TESTS These are summarized in Figure5.

Glucagon In a fasting patient the outer chainsof the glycogen molecule have already been'cropped' as far as a branch point by phosphorylase,so that further stimulation of phosphorylaseactivity by glucagon remains ineffective and thereis no rise in the blood glucose level. After a meal,however, the outer 1: 4-linked chains lengthen andnow provide a substrate on which phosphorylasecan act, so that an injection of glucagon under theseconditions can produce an increase in the bloodglucose level.

Infusions of galactose or fructose These resultin a normal rise in the blood glucose level becausethere is no interference with the conversion toglucose of the glucose 6-phosphate, which is formedfrom these sugars in the liver. There is no abnormalincrease in blood lactate.

SPECIFIC INVESTIGATIONS Glycogen isolated fromtissues or red blood cells can be shown to possessshort outer chains of 1,4-linked glucosyl units.When stained with iodine, the limit dextrin has alight-absorption maximum at 460 m,u.The diagnosis is confirmed by carrying out an

assay for the debranching enzyme, for which anelegant technique has recently been developeddepending upon the release of 14C-glucose fromlabelled glycogen (Hers and van Hoof, 1968).

Heterozygous carriers of the disease can bedetected by assay of amylo-1, 6-glucosidase activityin leucocytes or erythrocytes, in which the activityis about 50% of normal.

TYPE IV GLYCOGEN STORAGE DISEASE ('BRANCHINGENZYME' DEFICIENCY)

Type IV glycogen storage disease is caused by adeficiency of the 'branching enzyme', oc-1,4-glucan--*1,6-transglucosidase, and is characterized by theaccumulation of a glycogen with abnormally longouter chains. The outer chains lengthen beyondthe normal 8 to 10 glucosyl units, because unitscontinue to be joined in 1,4-linkages through theaction of 'glycogen synthetase' without the formationof branch points (Fig. 5). The accumulation of thismaterial in the liver is associated with the develop-ment of cirrhosis, progressing to portal hypertensionand hepatic failure early in life.

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It appears to be a rare form of glycogen storageand so far only six cases have been described in theliterature (Andersen, 1952; Sidbury, Mason, Bums,and Ruebner, 1962; Brown and Brown, 1966;Holleman, van der Haar, and de Vaan, 1966;Fernandes and Huijing, 1968; Levin, Burgess, andMortimer, 1968). The clinical features of thedisease are predominantly those of portal hyper-tension and cirrhosis. Deposits of the abnormalglycogen occur also in several other tissues, includingthe spinal cord, and in leucocytes and erythrocytes.The structure of the glycogen in this disease

resembles that of starch and, like starch, it isrelatively insoluble in water and stains blue withiodine. The disease is therefore sometimes called'amylopectinosis'.

All the patients described have died early in lifewith a cirrhotic liver. An attempt has been madeto prevent the development of cirrhosis by treatmentwith steroids (Sidbury et al, 1962). More recentlyin another patient liver glycogen was reduced bythe intramuscular administration of a purifiedfungal oa-glucosidase (Fernandes and Huijing, 1968),but in this case cirrhosis was already far advancedbefore treatment with the enzyme was begun.


BLOOD CHEMISTRY There are no disturbances ofcarbohydrate or lipid metabolism and the patternof liver damage dominates the picture. Red cell andwhite cell glycogen concentration is raised.

FUNCTIONAL TESTS These (Fig. 5) are distortedto a varying degree by the liver damage, but theresult of stimulation of glycogenolysis by glucagonand of gluconeogenesis from galactose and fructoseis probably normal, as would be expected.

SPECIFIC TESTS Glycogen, isolated from tissues orblood cells, gives a lavender colour when stainedwith iodine and can be identified spectrophoto-metrically by its peak absorption of light at 550 mjtin contrast to that of the normally branchedglycogen-iodine complex which has an absorptionpeak at 460 mp,.

Confirmation of the diagnosis is obtained bydetermining 'branching enzyme' activity which ismeasured by the extent to which glucose 1-phosphateis polymerized when it is incubated with a tissuehomogenate.

TYPE V GLYCOGEN STORAGE DISEASE (MUSCLE PHOS-PHORYLASE DEFICIENCY)

Type V glycogenosis is confined entirely to skeletalmuscle which contains a moderate excess of normally

- Lactate

FIG. 6. Type V glycogen storage disease. In muscleglycogen accumulates and lactic acid is not producedby adrenalin or ischaemia as a result of muscle phos-phorylase deficiency (X).

structured glycogen. It is caused by a deficiency ofphosphorylase (Schmid and Mahler, 1959; Mom-maerts, Illingworth, Pearson, Guillory, and Seray-darian, 1959) (Fig. 6).A thorough clinical description of the first case

and the demonstration of the defect in glycolysisin the patient's muscles was given by McArdle(1951) and at least 30 more cases have been reportedin the literature in the last 10 years. The strikingclinical features are the development of pain andstiffness in the limbs on exercise, in some cases alsothe appearance of 'electrically silent' contractures inthe muscles, and in several cases myoglobinuriaafter fairly vigorous exercise. It was believed that thestiffness and contractures on exercise were due to afailure of the production of adenosine triphosphate(ATP) because of impaired glycogenolysis, asoccurs in rigor mortis. However, ATP levels in thecontracted muscle show little reduction (Rowland,Araki, and Carmel, 1965) and the most recentstudies by McArdle and his colleagues (Gruener,McArdle, Ryman, and Weller, 1968) indicate thatthe contractures may be due to a partial failureby the sarcoplasmic reticulum to reaccumulatecalcium during vigorous exercise. 'Blebs' of glycogenare present just internal to the sarcolemma andelectron microscopy shows accumulation of glycogenwithin distorted mitochondria (Gruener et al, 1968).

Phosphorylase activity is the final expression ofthe integrated action on the enzyme of severalactivating and inactivating processes (Sutherlandand Robison, 1966), but type V glycogenosis hasbeen shown to be due to the absence of the enzymeitself, and not to an inhibition of its action. Absenceof the enzyme protein, as opposed to inability to

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detect enzyme activity, was confirmed by usingan antiphosphorylase serum (Robbins, 1960); theonly other type of glycogen storage disease in whichabsence of the specific enzyme protein has beendemonstrated in this way is that due to phospho-fructokinase deficiency but it is reasonable toassume, though not proven, that in the other typesof glycogen storage disease the specific enzymeprotein is also absent.

BLOOD CHEMISTRY At rest, there is no abnormalityofcarbohydrate or lipid metabolism.

FUNCTIONAL TESTS After exercise, and especiallyif it has been performed under relatively ischaemicconditions, there is a complete absence of a riseof lactic acid concentration in venous blood drainingthe ischaemic muscle (Fig. 6). After the exercise theextraction of glucose and fatty acids from bloodflowing through the exercised muscles is greatlyincreased.

Glucagon The blood glucose concentration risesin a normal manner after the injection of glucagonbecause it stimulates liver phosphorylase, which isimmunologically distinct from the muscle enzyme(Henion and Sutherland, 1957) and therefore maybe presumed to be under separate genetic control.There is a normal rise in the blood glucose

concentration after an intravenous infusion offructose or galactose.

SPECIFIC TESTS Muscle biopsies contain a moderateexcess of glycogen of normal structure. Phosphoryl-ase assay in muscle shows that there is no enzymeactivity, even after the addition of adenosine 5-monophosphate which activates the inactive form ofphosphorylase. No lactic acid is formed when amuscle homogenate is incubated without addedsubstrate, and the block can only be at a level aboveglucose 1-phosphate because lactic acid is producedfrom all the phosphorylated intermediate compoundsof carbohydrate metabolism.

Patients can undertake a fair amount of additionalexercise if, beforehand, their plasma glucose orfree fatty acid levels are raised by suitable meansso that these substances can provide the fuelnecessary for the exercise.

TYPE VI GLYCOGEN STORAGE DISEASE (LIVER PHOS-PHORYLASE DEFICIENCY)

This type of glycogen storage disease is not asclearly defined as any of the preceding types interms of its enzymatic defect. It is probably bestconsidered as a group of disorders (Fig. 7) in whichan excess of glycogen in the liver is associated with

/ GLYCOGEN \

Galactsj.:~ < I-|Glucagon[Gaiact g_ _p

g-6-p Glucose

|Fructo7se|------>Pyruvate Lactate

FIG. 7. Type VI glycogen storage disease. In liveglycogen accumulates as a result of diminished livephosphorylase activity (X).

diminished liver phosphorylase activity (Hers andvan Hoof, 1968)). It is noteworthy that in this typeenzyme activity is diminished but not totallyabsent. In this respect type VI glycogenosis differsfrom all the other types.About a third of all cases of glycogen storage

disease affecting primarily the liver appear to fallinto this rather ill-defined group in which hepato-megaly and moderate hypoglycaemia on fastingare the main clinical features. In several patientswho had attacks of severe hypoglycaemia, an ad-ditional enzyme defect was found, such as glucose6-phosphatase deficiency (Sokal, Lowe, Sarcione,Mosovich, and Doray, 1961) or glycogen synthetasedeficiency (Parr, Teree, and Lamer, 1965). Leucocytephosphorylase is similar to liver phosphorylase inmany respects and this too is low in patients withhereditary deficiency of liver phosphorylase, butactivity of the immunologically distinct muscle phos-phorylase is not affected.

Like muscle phosphorylase, liver phosphorylaseactivity is the end result of activating and inactivatingreactions affecting the enzyme, and in at leastone patient low 'phosphorylase activity' was duenot to a defect of the enzyme itself but to a defect ofphosphorylase-kinase (Hug, Schubert, Chuck, andGarancis, 1967) which plays a part in convertingphosphorylase from the inactive to the active form.

BLOOD CHEMISTRY Moderate hypoglycaemia onfasting is the only notable deviation from normal.

FUNCTIONAL TESTS These consisted of the responsesto glucagon and galactose infusion.

Glucagon A poor hyperglycaemic response is usu-ally obtained but there are many exceptions to thisrule and even in the same patient the response maybe poor on some occasions and normal in others.

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Galactose infusion A normal hyperglycaemicresponse is obtained unless an additional defectof glucose 6-phosphatase is present.

SPECIAL TESTS High glycogen content and lowphosphorylase activity in the liver provides reason-able grounds for making a diagnosis of typeVI glycogenosis. However, as there is no sharpupper limit for a normal liver glycogen con-tent, nor a sharp lower limit for phosphorylaseactivity, it is difficult to define precisely the limits oftype VI glycogen storage disease.

OTHER GLYCOGEN STORAGE DISEASES

In a multienzyme system such as that concernedwith carbohydrate metabolism it is not surprisingthat several other metabolic blocks should have beendiscovered associated with an abnormal accumu-lation of glycogen.The two diseases which are convincingly docu-

mented are phosphofructokinase deficiency (Tarui,Okuno, Ikura, Tanaka, Suda, and Nishikawa, 1965;Layzer, Rowland, and Ranney, 1967) and phospho-glucomutase deficiency (Illingworth and Brown,1964).

PHOSPHOFRUCTOKINASE DEFICIENCY The clinicalmanifestations of this disease are similar to thoseof type V glycogenosis and are presumably theresult of diminished energy production due tointerference with the normal glycolytic pathway(Fig. 8). This is one of the few inherited metabolicdiseases in which absence of the enzyme proteinhas been demonstrated by the use of an antiserumto the enzyme (Layzer et al, 1967).Blood chemistry There is no abnormality at

rest.Response to glucagon There is no interference

with glucose production by the liver and thereforethere is a normal hyperglycaemic response after aninjection of glucagon.

Galactose infusion results in a normal rise ofblood glucose level.

Ischaemic forearm exercise Glycolysis cannotproceed beyond fructose 1-phosphate, thereforeno lactate is formed when ischaemic exercise isundertaken.

Special tests Confirmation of the diagnosis isobtained by measuring the activity of the enzymeand by demonstrating that lactic acid cannot beformed from fructose 1-phosphate or any higherphosphorylated intermediate of the Embden-Meyerhof pathway, but that it can be formed fromfructose 1,6-diphosphate and subsequent inter-mediate metabolites. These tests prove the absence

GLYCOGEN

GLUCOSE -I -PHOSPHATE

11GLUCOSE -6-PHOSPHATE

11FRUCTOSE-1 -PHOSPHATE

Fructose Diphosphatase 1 [ Phospho-fructokinase

FRUCTOSE -1, 6-DIPHOSPHATE

DIHYDROXYACETONE x GLYCERALDEHYDE-3-PHOSPHATE

PYRUVATE -==LACTATE

FIG. 8. Phosphofructokinase deficiency (X) preventslactic acidformation.

of enzyme activity; absence of the enzyme itselfcan be demonstrated with an immunological tech-nique employing an antiserum to the enzyme.

PHOSPHOGLUCOMUTASE DEFICIENCY The only patientin whom this enzyme defect has been demonstrateddirectly by enzyme assay had an enlarged liver witha high glycogen content (Illingworth and Brown,1964). Deficiency of this enzyme poses interestingand as yet unsolved problems concerning the path-ways of glycogen synthesis, because the enzyme isessential for the formation of glucose 1-phosphatewhich is usually held to be the immediate sub-strate for intracellular glycogen synthesis.

GENERAL IMPLICATION OF STUDIES ON GLYCOGENSTORAGE DISEASES

In his monograph on 'Inborn errors of metabolism'Garrod (1909) developed the concept that in suchdisorders the normal course of metabolism isblocked by the congenital deficiency of a specificenzyme. In human disease the first direct proof ofthis concept was the demonstration by Cori andCori (1952) of a deficiency of glucose 6-phosphataseas the cause of type I glycogen storage disease. Thisdiscovery initiated wide interest in enzymologicalstudies of human tissue and has led to the recog-nition of an ever increasing number of inherited

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metabolic diseases (Stanbury, Wyngaarden, andFredrickson, 1966). The Coris' discovery led directlyto a new and better understanding ofthe whole groupof glycogen storage diseases and made it possible togive an accurate prognosis and institute appro-priate therapeutic measures based on an under-standing of the underlying metabolic abnormality.With the discovery of the enzymatic abnormality

in type IL glycogen storage disease, the concept of'lysosomal disease' has received serious attention.van Hoof and Hers (1964) have postulated thatHurler's disease may also be an example of aninborn lysosomal disease, in which the metabolismof mucopolysaccharides is primarily affected, andrecent experimental work has shown that damageto lysosomes by drugs during foetal developmentcan lead to congenital malformations. Thus studiesin glycogen storage disease have cast light on a newapproach to the study of disease in general.The remarkable observation that an enzyme,

when injected intraperitoneally into an experimentalanimal, or intramuscularly in patients with type IIand type III glycogen storage disease, can reducethe amount of intracellular glycogen, may have far-reaching consequences for the treatment of manydiseases, particularly those in which a specificenzyme defect can be demonstrated. There are stillmany general problems to be solved, such as themechanisms whereby the enzyme gains entranceto the cell in an active form, and the suppressionof the immune response to the enzyme protein, buteven the moderate success achieved by this techniquein the glycogen storage diseases encourages furtherwork on these lines.The recognition of the enzyme defect in type V

glycogenosis is a particularly striking illustrationof the way in which clinical medicine and bio-chemistry depend upon each other. McArdle'sdisease is the first example of a myopathy which hasbeen shown to be clearly related to the absence of asingle, specific enzyme. On the other hand, thepresence of excess glycogen in the muscle provedthat the then recently discovered pathway ofglycogen synthesis via 'glycogen synthetase' is ofimportance in man, that glycogen synthesis andglycogen degradation proceed largely by separatepathways and that the action of phosphorylasein vivo is irreversible for all practical purposes.This cleared up the hitherto inexplicable observationthat stimulation of phosphorylase activity in vivoby glucagon or adrenaline inevitably leads to lossof glycogen, instead of reaching an equilibriumas would be expected on the basis of reversibleenzyme kinetics.The study of the glycogen storage diseases has

been of importance in genetic investigations, and

the development of accurate and sensitive enzymeassays has made it possible to identify clinicallyunaffected heterozygous carriers of a recessive gene.In the last few years a number of cases have beenreported in which more than one enzyme appearsto be missing. If these observations are correctand not due to a technical fault in the estimationof the enzymes involved, then further thought mustbe given to the 'one gene, one enzyme' theory.There are a number of possible explanations forthese observations, based on genetic mechanismsor on protein polymorphism, but further researchin this field is needed.The glycogen storage diseases, like other inherited

metabolic diseases, may be natural experimentsin evolution and, because they upset the usualcomplex self-regulatory mechanisms of the cell,they present a unique opportunity for studyingintracellular biochemical processes.

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