Inherited enzyme defects: a review · ALKAPTONURIA This is a rare metabolic disease in which the...

J. clin. Path. (1963), 16, 293

Inherited enzyme defects: a reviewT. HARGREAVES'

From the Department of Chemical Pathology, St. George's Hospital Medical School,Hyde Park Corner, London, S. W.I

Sir Archibald Garrod in 1909 pointed out that fourdiseases, albinism, alkaptonuria. cystinuria, andpentosuria, were present from birth, lasted through-out life without worsening or improvement, andwere present in other members of the family (Garrod,1909). These diseases were described as 'inborn errorsof metabolism'. The original four diseases havebeen expanded to over 50 and the actual site of thebiochemical abnormality has been elucidated in manyof them. In this review we shall consider thosediseases which are either known or thought to bedue to single enzyme defects. We have not con-sidered those diseases that are thought to be due torenal tubular or intestinal abnormalities, forexample, cystinuria. It is not yet known whetherthis type of disease is due to a specific enzymedefect.The fundamental discovery of Mendel (1901) con-

cerning the inheritance of plant characters had justbeen resurrected and given prominence at the timewhen Garrod was most active. The transmission ofa single character as a governing agent for a singleenzyme and accordingly, as one step in intermediarymetabolism, seems implicit in Garrod's writings butis never stated as such. The concept attained cleardefinition in the 'one gene-one enzyme' principlefirst clearly stated by Beadle (1945). The formulationemerged gradually from studies of eye colour in thefruit fly Drosophila and received extensive supportfrom work done on induced mutants of Neurosporacrassa. The concept has been expressed as: 1 Allbiochemical processes in all organisms are undergenic control. 2 These biochemical processes areresolvable into series of individual stepwise reactions.3 Each biochemical reaction is under the ultimatecontrol of a different gene. 4 Mutation of a singlegene results only in an alteration in the ability of thecell to carry out a single primary chemical reaction.

It is now realized that the one gene-one enzymeconcept does not require a one enzyme-one genecorollary, and that the 'gene' is being replaced byprogressively smaller units of ultimate geneticcontrol.

IB.M.A. research scholar

The consequences of a genetic change and analteration in the quality or quantity of a protein willdepend on the role normally subserved by thatprotein; in the cases that we are considering this isderangement of enzymatic function. The derange-ments that can occur include a blocked metabolicsequence. In the reaction A---* B-- C--# D, ifC--# D is blocked then a clinical disorder mayresult from a failure to produce D, e.g., hypo-glycaemia due to an inability to form glucose fromglucose-6-phosphate in von Gierke's disease. Theprecursor C may accumulate; for example, bilirubinaccumulates in the absence of glucuronyl transferase.The remote precursers A and B may accumulate, forexample, glycogen in von Gierke's disease. The blockbetween C and D may result in an increased utiliza-tion of normally minor pathways, for example, inphenylketonuria. The application of these metaboliccycles will be reviewed in each section.The subject has been covered generally in recent

reviews, for example, that of Stanbury, Wyngaarden,and Fredrickson (1960).

DISTURBANCES IN AMINO-ACID METABOLISM

Recent advances in the past few years in the under-standing of the biochemistry of the amino-acidscoupled with the use of amino-acid chromatographyin urine has helped to elucidate problems in thedisturbance of amino-acid metabolism and todemonstrate new cases of disturbed amino-acidmetabolism.

PHENYLKETONURIA In 1934 F0oling described 10patients, some of them siblings, who excreted phenylpyruvic acid and were mentally deficient. Jervis(1953) demonstrated that phenylalanine hydroxylaseof the liver was inactive in these patients. Thecondition is inherited through a single autosomalrecessive gene. It is required that both parents of apatient have the defective form of one of the twogenes controlling phenylalanine hydroxylase; anapparent reduction of phenylalanine hydroxylasecan be detected in the parents. On the average one

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Albinism

Phenyl- Phenylalanineketonuria hydroxylase

?Tyrosinosis Tyrosinetransam nase

p hydroxy-Tyrosinosis phenylpyruvic(Medes) acid oxidase

Alkaptonuria Homogentisicacid oxidase

PhenylalanineI ,

\\-CH.CH COOH Tyrosinase

-\NHTyrosine 3,4 Dihydroxyphenylalanine 3,4 Dopaquinone--oMelanin

HO/ CHCHCOOH HO

NH. HO- -CH2.CHCOOH O= -CH2CH2COC

NH, NH.,

p-Hydroxyphenylpyruvic acid - 4 p-Hydroxyphenyllactic acid

HO/ -\ CH2C COOH HO-/ \-CH

\. O.-Ii0Homr entisic acid

HO/_-\\HOI\CH ,COOH

Maleylacetoacetic acid

IFumarylacetoacetic acid

Fumaric acid t- acetoacetic acid

i.,CH COOH

OH

FIG. 1. Metabolic blocks in thle oxida(tioni ofplhenylalanine ani(l tYrosine.

out of four offspring from two heterozygous parentshas both genes defective, and in these offspringphenylketonuria results. The disability is firstmanifest several weeks after birth, initially byelevation of the plasma phenylalanine level and bythe excretion of phenylpyruvic acid. After sixmonths, retardation of mental development isapparent. Many of the children have blond hair,blue eyes and fair skin, and a few show a tendencyto develop dermatitis or eczema. The majority ofpatients have an I.Q. of 30 or less.

Pathogenesis Phenylalanine is converted to tyro-sine by the enzyme system 'phenylalanine hydrox-ylase' in the liver alone; the reaction can be repre-sented as

L-phenylalanine.02±+N.A.D.P.H.-1 +H+--+-L-tyrosine+-N.A.D.P.++ H20-

'The following abbreviations are usedA.T.P. Adenosine TriphosphateN.A.D. Nicotinamide-adenine dinucleotideN.A.D.P. Nicotinamide-adenine dinucleotide phosphateU.D.P.G.A. Uridine diphosphate glucuronic acidU.D.P. Uridine 5'-pyrophosphate

Mitoma, Auld, and Udenfriend (1957) showed thattwo protein fractions are involved in this reaction, alabile fraction I found in liver and a more stablefraction II found in kidney and heart. The systemrequired N.A.D. or N.A.D.P. and iron together withan unknown but necessary coenzyme which can bereplaced by tetrahydrofolic acid. Fraction I is deficientin phenylketonuria.The deficiency of phenylalanine hydroxylase

causes an excessive accumulation of phenylalaninein the blood and cerebrospinal fluid. The excessivephenylalanine is converted by its transaminase tophenylpyruvic acid, and this in turn is converted tophenyllactic acid, phenylacetic acid, and phenyl-acetyl glutamine, which are excreted in the urine.o-Hydroxyphenylacetic acid, m-hydroxyphenylaceticacid, and indole products derived from tyrosine andtryptophan are also found in the urine of suchpatients. The excessive phenylalanine inhibits thenormal pathways of tyrosine metabolism, there is adecreased formation of melanine accounting forthe blond hair and blue eyes; plasma adrenaline

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Inherited enzyme defects: a review

levels are low in phenylketonurics. The excessivephenylalanine or one of its products possiblydamages the central nervous system; the patients arementally retarded and have epileptic seizures andabnormal electroencephalograms.The condition can be diagnosed by the presence

of phenylpyruvic acid in the urine and an increaseof phenylalanine in the plasma. Heterozygotes canbe detected by phenylalanine tolerance tests. Thehomozygous condition can be ameliorated by givingthe infants a diet low in phenylalanine, which has noeffect on older children, demonstrating that earlydiagnosis of the disease is essential.

TYROSINOSiS This is a very rare inborn error ofmetabolism described by Medes (1932). Her patientwas a male Russian Jew who excreted large quantitiesof p-hydroxyphenylpyruvic acid and other tyrosinemetabolites but had no clinical symptoms. Medes,Berglund, and Lohmann (1927) found an excess ofp-hydroxyphenylpyruvic acid in the urine of apatient with myasthenia gravis, and Felix, Leonhardi.and Glasenapp (1951) made similar observations ontwo patients with liver disease. The amount ofp-hydroxyphenylpyruvic acid excreted was consider-ably less than 50 mg. per day and it is probable thatthese cases are of a different type of metabolicabnormality from the case described by Medes.The metabolic defect is a deficiency of p-hydroxy-

phenylpyruvic oxidase which requires glutathioneand either ascorbic acid or dichlorophenolendo-phenol as cofactors. In the patient with tyrosinosisonly p-hydroxyphenylpyruvic acid is excreted butscorbutic guinea-pigs and scorbutic children(Huisman and Jonxis, 1957) excrete the p-hydroxy-phenyllactic acid as well. The patient described byMedes could convert the pyruvic derivative to thelactic derivative; one would assume then that if thetissues contained large amounts of p-hydroxy-phenylpyruvic acid the lactic acid would be formed,but the patient only excreted p-hydroxyphenyl-pyruvic acid. In various conditions in which p-hydroxyphenylpyruvic acid oxidase in the liver isimpaired the lactic as well as the pyruvic derivativesare found in the urine. It may be that in the case oftyrosinosis the defect is analogous to phenyl-ketonuria and the block is a defect of tyrosinetransaminase. It is difficult to explain the absence ofp-hydroxyphenyllactic acid in the urine with thepresence of a system converting p-hydroxyphenyl-pyruvic acid to p-hydroxyphenyllactic acid in thepatient.

ALKAPTONURIA This is a rare metabolic disease inwhich the enzyme homogentisic acid oxidase islacking. Homogentisic acid, produced in the meta-

bolism of phenylalanine and tyrosine, cannot bemetabolized; it accumulates and is excreted in theurine. If the urine is allowed to stand it becomesdark and this sign leads to early recognition of thedisease. Boedeker (1859) first diagnosed the diseasewith certainty and used the property of avid oxygenuptake to give the substance a name 'alkapton'.Garrod in 1909 wrote of the probable defect: 'Wemay further conceive that the splitting of the benzenering in normal metabolism is the work of a specialenzyme, that in congenital alkaptonuria this enzymeis wanting, whilst in disease its working may bepartially or even completely inhibited'. The con-dition is compatible with long life. Except for thedark-coloured urine there are no clinical manifesta-tions until the second or third decade whenochronosis begins to appear. Black pigments aredeposited in various parts of the body, and eventuallythe patients develop deforming arthritis in middleage.Garrod (1902) first described the inheritance of

alkaptonuria and presented evidence that the con-dition was congenital and familial. He suggestedthat it was inherited as a single recessive gene.A study of the cases reported since that time hassupported the view that the defect is due to a singlerecessive autosomal gene, and gains further supportby the finding that a single enzyme system is in-active in this condition and that only one clinicalform is known.

Since the feeding of homogentisic acid to alkapto-nurics results in almost quantitative recovery fromthe urine, while no such accumulation occurs innormal individuals, it is postulated that the con-dition represents a metabolic block at the stage ofhomogentisic acid. An analysis of enzymes involvedin tyrosine metabolism in normal and alkaptonuricliver showed that only homogentisic acid oxidase ismissing in alkaptonuric liver (La Du, Zannoni,Laster, and Seegmiller, 1958). Homogentisic acid isnot converted into maleylacetoacetate but accumu-lates in the blood and is excreted in the urine. Thehomogentisic acid is at least in part responsible forthe deposition of pigment in the tissues in thiscondition.

ALBINISM Albinism is an inherited defect of pigmentcells, melanocytes, to form melanin. Patients withthis condition have white or very pale yellow hairwhich is silky in texture. The pupils of the eye appearto be red and the iris is pink or bluish from reflectedlight. The intelligence of generalized albinos isnormal but no large series have been studied. Thecondition is transmitted as a simple recessive, whichis shown by the fact that 20 to 30% of the casesresult from cousin marriages.

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The biochemical lesion of albinism consists of afailure of melanocytes to synthesize tyrosinase, acopper-containing oxidase required for the conver-sion of tyrosine to melanin (Fitzpatrick, 1960).Tyrosine is oxidized via 3,4 dihydroxyphenylalanineto 3,4 dopa quinone by tyrosinase in the presence ofmolecular oxygen. Ultimately dopa quinone formsmelanin by a reaction which may or may not beenzymatic.

PARTIAL ALBINISM This is characterized by a con-genital absence of pigment in specific areas of thebody. Three varieties are known:- I Piebaldalbinism and 2 white forelock and spotting of theskin. These two forms are transmitted as simpledominants. 3 Albinism of the eye alone. This is asex-linked recessive characteristic and is characterizedby albinism of the fundus, hypoplasia of the macula,head nodding, nystagmus, and amblyopia. Thepathogenesis of these conditions is not known.

ABNORMALITITES OF AMINO-ACIDS IN THE UREA

CYCLE In the past few years inborn errors of meta-bolism involving the amino-acids of the urea cyclehave been described. The current concepts on theformation of urea are shown in Figure 2.

Argininosuccinic aciduria Argininosuccinic acidwas identified as the unknown ninhydrin-reactingmaterial on paper chromatograms of the urine oftwo mentally retarded children (Westall, 1960). Acase has also been reported by Levin, Mackay, andOberholzer (1961). The patients had normal bloodurea levels, suggesting that there is either another

CO. +

Carb

path of urea formation in these patients (Levin,Mackay, and Oberholzer, 1961) or that the defect islocalized to one tissue, for example, the brain. Insupport of this concept Allan, Cusworth, Dent, andWilson (1958) found higher concentrations ofargininosuccinic acid in cerebrospinal fluid than inplasma. Sporn, Dingman, Defalco, and Davies(1959) have shown that rat brain incorporateslabelled arginine into urea in vivo and can thussynthesize urea. Cusworth and Dent (1960) showedthat arginino-succinic acid, like ethanolaminephosphate and f-aminoisobutyric acid, is excretedin human urine without tubular reabsorption.

Citrullinuria McMurray, Mohyuddin, Rossiter,Rathbun, Valentine, Koegler, and Zarfas (1962) havedescribed the finding, during a routine amino-acidscreening programme, of citrulline in the urine of amentally retarded child aged 18 months. The con-centration of citrulline in the plasma and the cere-brospinal fluid was increased when compared with acontrol group and suggests that the defect is unlikelyto be of the renal tubules. A renal tubular defectmay be the cause of increased citrulline in the urineof patients with Hartnup disease and with theFanconi syndrome. Citrullinuria has previouslybeen described in cystinuria in humans and canines(e.g., Dent and Rose, 1951). Asatoor, Lacey,London, and Milne (1962) consider that the citrul-linuria in cystinurics is due to a small intestinalabsorption defect for dibasic amino-acids and isformed from arginine. Visakorpi (1962) suggestedthat the excretion of citrulline by a mentally defectivecystinuric was comparable to citrullinuria but this

NH3 ATP

)amyl phosphate

Aspartic acid

rbamyl Aspartic acid

ase Aspartic acid FlCi. 2. The utrea cYcle.

Citrulline

3 l1 Aspartic acid

Condensing enzyme

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is unlikely. In the case described by McMurray andhis colleagues (1962) the patient excreted urea inamounts comparable to those excreted by normalchildren, suggesting that the urea cycle is notseriously impaired in the liver. It may be thatcitrulline is produced in the brain as has beensuggested for argininosuccinic acid.Hyperammonaemia Russell, Levin, Oberholzer,

and Sinclair (1962) have described two cousins whowere mentally retarded and had cerebral degenera-tion. In both children the levels of ammonia in theblood and cerebrospinal fluid were elevated. Therewas no evidence of liver disease or excessive absorp-tion from the gut. Examination of the liver afterdeath showed a depression in the ornithine trans-carbamylase activity. The blood urea was withinnormal limits; this suggests that there may beanother method of urea production in these children.

MAPLE SUGAR URINE DISEASE In 1954 Menkes,Hurst, and Craig described a family in which fourout of six infants, one girl and three boys, diedduring the first weeks of life with what appeared tobe a congenital metabolic disease. The predominantsigns were vomiting, muscular hypertonicity, and amaple syrup odour to the urine. Paper chromato-graphy of the urine failed to reveal any abnormalityin the amino-acid pattern.

In 1957 Westall, Dancis, and Miller described a20-month-old infant who was mentally retarded, hadmuscular hypertonicity, and a maple syrup odour tothe urine. An excess of leucine was found in theurine. Further investigations suggested a metabolicblock in the degredative path of branched chainamino-acids. Further cases have been reported byMackenzie and Woolf (1959) and by Lane (1961).Lane suggested, on the basis of two possible cases

related to the patient, that the condition was in-herited as a simple recessive characteristic.The urine in this disease contains large amounts

of leucine, isoleucine, and valine; it also containsincreased amounts of the ox-keto derivatives of theseamino-acids (Dancis, Levitz, Miller, and Westall,1959). Evidence indicates a block at a point beyondtransamination (A) at, or beyond, oxidative decarbo-xylation (B).

AR-CH-COOHI- R-C-COOH R-C CO - S Co A

NH2 0 0 B

R-COOH

Transaminase was present in liver, brain, heart, andkidney at necropsy in the case described by Dancis,Levitz, and Westall (1960).

The urine also contains an amino-acid that wasthought to be methionine. Dent and Westall (1961)found that the plasma methionine was increasedwhen isoleucine was given to a patient with theabnormality. Norton, Roitman, Snyderman, andHolt (1962) consider that it is not methionine that isexcreted but probably alloisoleucine. The maplesyrup smell probably emanates from a-oxoisocaproicacid, a keto-acid derived from leucine.

HYPERGLYCINAEMIA Childs, Nyhan, Borden, Bard,and Cooke (1961) described a child, 3 years old, whowas investigated originally because of a failure tothrive, with acute exacerbations characterized byvomiting, lethargy, and irritability. The main findingswere of developmental retardation, ketosis, neutro-penia, hypogammaglobulinaemia, and periodicthrombocytopenia. The plasma levels of glycine,serine, alanine, isoleucine, and valine were raisedbut only glycine was present in excess in the urine.The toxic symptoms could be abolished by keepingthe protein in the diet to less than 0 5 g. per kilo-gram of body weight. Giving sodium benzoatelowered the level of glycine in the plasma causing aremission of the neutrophil leucopenia but it did notprevent the toxic symptoms. Leucine administrationin the patient increased the plasma threonine butnot in controls. It is not known whether the con-dition is genetically determined. The specific enzymedefect has not been localized. Kaser, Cottier, andAntener (1962) described a case of gluco-glycinuriain which the serum glycine level was normal, andit was thought that the glycinuria was due to atubular defect and was therefore different from thecase reported above.

HISTIDINAEMIA Ghadimi, Partington, and Hunter(1961) reported a case of a 3-year-old child who wasphysically small with retarded speech and hadexcessive amounts of histidine in his urine andplasma. The patient's sister also had a raised plasmahistidine level. Auerbach, DiGeorge, Baldridge,Tourtellotte, and Brigham (1962) have reportedanother case of histidinaemia and histidinuria in a2-year-old child. This child had been well until it hada febrile fit. Davies and Robinson (1963) recentlyreported a further case of histidinaemia. Thesechildren excrete histidine, imidazole pyruvic, imi-dazole lactic, and imidazole acetic acid. They do notexcrete urocanic acid, imidazole propionic, or formi-minoglutamic acid. Histidine administration causesan increase in the plasma histidine but urocanic acidadministration is accompanied by urinary excretionof urocanic acid, imidazole propionic acid, andformiminoglutamic acid. The enzyme deficiencyseems to be of histidase (histidine-ot-deaminase) but

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urocanase action appears to be normal. The defectis probably inherited as an autosomal recessivecharacter.

HYPERPROLINAEMIA Schafer, Scriver, and Efron(1962) have described hyperprolinaemia in a family,associated with nephropathy and deafness. Thepatient was a child admitted with fever, vomiting,and diarrhoea; he was deaf and had frequent clonicand tonic seizures, and the right kidney was hypo-plastic and the left kidney deformed. The plasmacontained an excess of proline and the urine an

excess of proline and hydroxyproline. The child'sfather was normal but the mother was deaf and hada poorly functioning left kidney but a normal amino-acid chromatogram. Three sisters of the patient hadabnormal electroencephalograms and hyperpro-linaemia; two of these three had haematuria andtwo other sibs were normal. It is probable thatglycine and hydroxyproline share a common tubulartransport mechanism with proline, accounting forthe occurrence of these two amino-acids with prolinein some of the afflicted individuals. No A-lpyrroline-5-carboxylic acid could be detected in the urine. Thedefect may be a lack of the enzyme proline oxidase.Hydroxyproline is formed from proline and this isincorporated in collagen; these subjects may alsohave collagen defects.

f-AMINOISOBUTYRIC ACIDURIA P-Aminoisobutyricacid was identified in some samples of human urineby Crumpler, Dent, Harris, and Westall (1951).Study of population groups has suggested that thehigh excretor gene originates in Asia. The abnorma-lity is familial with a recessive mode of inheritance.There is a marked difference in the excretion of/-aminoisobutyric acid in high and low excretorsafter intravenous administration of a test load,suggesting that the difference between high and lowexcretors lies in its metabolism; it may be thatthe acid is derived from thymine. An alternativemethod of formation from valine by way of a

transamination reaction of methyl malonyl-semi-aldelyde with glutamate has been suggested byKupiecki and Coon (1957).

,B-Aminoisobutyric acid is excreted by patientswith chronic myeloid leukaemia, liver disease, thenephrotic syndrome, and by very young infants.

CYSTATHIONINURIA Harris, Penrose. and Thomas(1959) described cystathioninuria in a mentallydefective patient in whose family two other memberswere found to secrete the substance. Increasedamounts of cystathionine were found in the kidneyand liver but it was not possible to tell if the amountin the brain was increased.

HYPOPHOSPHATASAEMIA In 1948 Rathbun describeda 3-week-old infant with rachitic bone lesions and adiminution in the alkaline phosphatase activity inbone, intestinal mucosa, and kidney despite adequatedoses of vitamin D; he suggested the condition becalled hypophosphatasaemia. Fraser (1957) hasreviewed the historical aspects. Sobel, Clark, Fox,and Robinow (1953) noted premature loss of teeth,observed that serum did not inhibit alkaline phos-phatase activity, and presented data suggestive ofhypersensitivity to vitamin D. Fraser, Vendt, andChristie (1955) and McCance, Morrison, and Dent(1955) discovered that the excretion of phos-phoethanolamine in urine is an integral feature ofthe disease. Patients with the disease have knockknees, bowing of the femurs and tibias, enlargedwrists, and a protuberant abdomen. There is poormineralization of bones and teeth, and at necropsythere is evidence of deficient calcification. Alkalinephosphatase is diminished or absent in serum, bone,and other tissues.

Hypophosphatasia is transmitted as an autosomalrecessive. The heterozygous carriers can be sometimesdetected by a decreased level of alkaline phosphatasein the serum and sometimes by an increased excretionof phosphoethanolamine in the urine.

It has been suggested that phosphoethanolamineis the true substrate of alkaline phosphatase andthat a block in the hydrolysis of the ester occursin this disease and this leads to a defect in calcification(Dent, 1956). As yet, there is no evidence in favouror against such a hypothesis.

PRIMARY HYPEROXALURIA AND OXALOSIS Primaryhyperoxaluria is a rare disorder characterized byprogressive bilateral calcium oxalate urolithiasisand nephrocalcinosis beginning in early childhood.The first reported case is probably that of Lepoutre(1925) who described bilateral urolithiasis in a44-year-old child with extensive crystalline depositsof calcium oxalate. In 1954 Aponte and Fetter des-cribed three cases of oxalate nephrocalcinosis inone family which were probably the first definiteexamples of oxalosis. The age of onset is usuallyabout 1 to 4 years old, and the disease is characterizedby repeated attacks of renal colic, haematuria. andthe passage of calculi. Excessive amounts of oxalateare excreted in the urine.

Glycine is a significant precursor of oxalate(Crawhall, Scowen, and Watts, 1959) and in casesof primary oxaluria the oxalate was not appreciablyfurther oxidized to carbon dioxide but was excretedalmost quantitatively in the urine. These authorsfound a considerably increased incorporation oflabelled glycine into oxalate in hyperoxalurics; thesame proportion of urinary oxalate was derived from

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glycine. Overproduction of oxalate from glycine andother precursors was therefore best explained by a

block of glyoxalate degradation rather than byexcess formation of glyoxalate. The enzyme defectmay be a lack of glyoxylic glutamic transaminaseor of glyoxylic dehydrogenase. Administration ofsodium benzoate leads to an increased excretion ofglycine as hippurate and causes some improvement.The disease is possibly due to a rare recessive

character (Archer, Dormer, Scowen, and Watts,1958). The detection of heterozygotes is at presentimpossible. It may be that people who have recurrentoxalate stones represent carriers of this recessivecharacter, but the available evidence does not yetsupport this view.

DISTURBANCES OF CARBOHYDRATE METABOLISM

PENTOSURIA The presence of pentose in urine was

first described by Salkowski and Jastrowitz (1892).Much of our present knowledge of the clinicalaspects of the disease, its genetic transmission, andthe nature of the urinary sugar are due to the carefulobservations of Enklewitz and Lasker (e.g., Lasker,Enklewitz, and Lasker, 1936). The condition appearsto be harmless to health and wellbeing. It is found

D-glucose-6-phosphate 4

Uridine diphosphate glucuronic acid

D-glucuronic acid

NADPH

L-gulonic acid

NAD

3-ketogulonic acid

-CO.,

L-xylu losePresumed block

NADPHin pentosuria

Xylitol

|NAD

D-xyluloseATP

D-xylulose-5-phosphate

FIG. 3. The defect in pentosuria.

almost exclusively in Jews and causes no decrease inlife expectancy. It is probable that the condition istransmitted by a recessive gene. The pentose sugarthat is excreted in pentosurics is L-xylose. L-Arabitolis also excreted in the urine; this sugar may bederived from L-xylulose by reduction.

In 1936 Enklewitz and Lasker showed that thereis a direct relationship between the amount ofglucuronic acid administered and the amount ofL-xylulose excreted. Since feeding glucuronic acidto normal individuals does not produce this effect,they postulated a defect in the system that decarbo-xylates glucuronic acid to xylulose. Glucuronic acidis thought to be metabolized to D-xylulose by thepathway shown in Figure 3. The work of Touster(1960) suggests that the block in pentosuria is at thelevel of the N.A.D.P. linked L-xylitol-dehydrogenase.Hiatt (1958) demonstrated that labelled glucurono-lactone was converted to L-xylulose but not toribose, supporting this view, and Bozian and Touster(1959) that administration of glucuronolactone topentosuric individuals caused an increase in plasmaxylulose levels in contrast to the levels in normalsubjects. Freedberg, Feingold, and Hiatt (1959)used a specific enzymatic method to measureL-xylulose levels in serum and urine and by means ofa glucuronolactone load test they were able to detectheterozygotes.

FRUCTOsURIA Essential fructosuria is a rare errorof metabolism characterized by a congenital in-ability to utilize fructose completely. In this type ofabnormality the individuals show no clinicalsymptoms and the condition is harmless. The con-dition is transmitted as an autosomal recessive gene;the heterozygote condition cannot yet be detected.

In the normal individual when fructose is ingestedthere is a rapid rise of the respiratory quotient andelevation of the blood lactic acid. Patients withfructosuria do not show this response. At the presenttime the evidence is in favour of a defect of hepaticfructokinase. It is possible that the defect is in theutilization of fructose-l-phosphate. The enzyme forthe conversion of fructose-i-phosphate to fructose-6-phosphate is not present in liver and it may be thatthe fructose-1-phosphate accumulates and is excreted.

FRUCTOSE INTOLERANCE A congenital disease withsevere clinical manifestations was described byChambers and Pratt (1956). Administration offructose leads to an excessive and prolonged rise infructose in the blood, the blood glucose falls and thisis accompanied by nausea, haemorrhagic vomiting,trembling, profuse sweating, and somnolence. Aftercessation of the symptoms there is a slight icterus,albuminuria, and aminoaciduria. The inheritance of

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ATP ADP

Fructose - F

ATP ADP

Fructose-i-phosphate Fr

?DefectAldolase

Glyceraldehyde Dihydi

the disease seems to be of an autosomal recessivetype.

Froesch, Prader, Wolf, and Labhart (1959) noteda rapid and marked decrease in serum inorganicphosphate accompanying the decrease in the bloodglucose after intravenous fructose administration.The drop was attributable to binding of phosphatewith fructose to produce fructose-l-phosphate. Thedecreased utilization of fructose-l-phosphate wouldaccount for the slow release of inorganic phosphorus.They postulated a deficiency of fructose-I-phosphatealdolase, but this was not demonstrated by liverbiopsy, and that accumulation of fructose-I-phosphate inhibited utilization of fructose. Theauthors suggest that it is unlikely that the cases arehomozygotes and those with benign fructosuria areheterozygotes for the same defective gene. Dubois,Loeb, Ooms, Gillet, Bartman, and Champenois(1961) demonstrated a defect in the liver aldolase inthese patients. Nikkila, Somersalo, Pitkanen, andPerheentupa (1962) showed that the fructose-l-phosphatase aldolase was absent in the liver and thefructose-i, 6-diphosphate aldolase was 10% of thatfound in normal subjects. They suggested thatfructose releases homeostatic mechanisms andhypoglycaemia is a result of excessive insulinsecretion, but Froesch, Prader, Wolf, and Labhart(1959) suggested that the hypoglycaemia is due toinhibition of phosphoglucomutase by fructose-l-phosphate rather than by release of insulin.

GALACTOSAEMLA This is a hereditary conditioncharacterized by an inability to convert galactose toglucose in the normal manner. Infants with thiscondition appear normal at birth but after a fewdays of milk feeding they begin to vomit, becomelethargic, and fail to gain weight. Prolongedjaundice may occur in the neonatal period and the

ructos- 6-phosphate

ATP

-+ADP

ructose-1, 6-diphosphate

FIG. 4. The clefect infriclosuria.

DefectAIdolase

roxyacetone Glyceraldehydehosphate -3-phosphate

liver shows fatty infiltration which readily proceedsto cirrhosis if milk feeding persists. Ascites andoedema may develop and the child may die in theneonatal period. Those who survive are mal-nourished and dwarfed; at 2 to 3 months of age theyare mentally retarded and have cataracts. Thesepatients have a reducing substance, galactose, intheir urine together with aminoaciduria and albu-minuria. The aminoaciduria is primarily renal inorigin, and the amino-acids are mainly of the simplealiphatic chain type, e.g., serine, alanine, andglycine. The patients have high blood galactoselevels, and ingestion of galactose often depressesthe blood glucose, possibly by the release of insulin.The abnormality is transmitted as an autosomal

recessive. Carriers of the abnormal gene may befound on the basis of abnormal galactose tolerancetests and also by the presence of reduced amounts ofgalactose-l-phosphate uridyl transferase in erythro-cytes.The condition is due to a lack of galactose-l-

phosphate uridyl transferase. The site of the defectis based on the results of two types of experiment.Schwarz, Golberg, Komrower, and Holzel (1956)showed that when milk was given to a galactosaemicchild there was an abnormal accumulation ofgalactose-l-phosphate in the red cells; this meta-bolite was not found in normal children. Thissuggested that the block was beyond galactose-l-phosphate and that galactokinase was present inadequate amounts. Isselbacher, Anderson,Kurahashi, and Kalckar (1956) demonstrated thaturidine diphosphate galactose-4-epimerase anduridyl diphosphate glucose pyrophosphorylase, theenzymes needed for the third and fourth steps, werepresent in normal amounts. Galactose-l-phosphateuridyl transferase could not be demonstrated in theerythrocytes of galactosaemic children. Anderson,

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Galactose

Galactokinase Glucose-6

1 11Galactose-I-phosphate Glucose-l-

Uridyl UDP osetransferase

UDP galactose UDP galactose-4-e

Mucopolysaccharides Lactose

Kalckar, and Isselbacher (1957) demonstrated alack of the enzyme in the liver of two afflictedpatients. It has been suggested that there is analternative path of galactose metabolism bypassingthe transferase system, for example, U.D.P.-galactosepyrophosphorylase, allowing galactose-1-phosphateutilization and incorporation into U.D.P. galactose.The U.D.P.-galactose pyrophosphorylase is deficientat birth and matures with age, possibly accountingfor these patients' increased tolerance for galactosewith age. Support for the idea of an alternativepathway has come from the work of Elder, Segal,Maxwell, and Topper (1960), who demonstratedthat the ability of galactosaemics to oxidize a tracerdose of 14C galactose to 14CO2 was greatly increasedby menthol or progesterone. Maxwell and Topper(1961) observed that steroids exert a stimulatingeffect on galactose oxidation in liver slices.

GLYCOGEN STORAGE DISEASE This is an overallterm applied to a group of congenital and familialdiseases characterized by the deposition of abnor-mally large quantities of glycogen in the tissues.Cori (1957) has recommended a classification basedupon specific causative enzyme defects.

Type 1 Glucose-6-phosphatase deficiency hepato-renal glycogenosis von Gierke (1929) reported acondition characterized by excessive enlargement ofthe liver and other organs during early infancy dueto accumulation of glycogen. The condition ischaracterized by convulsions due to hypoglycaemia,failure to thrive, severe acidosis, and eruptivexanthoma. Growth and development is retarded anddeath frequently occurs in the first two years of life.The condition is transmitted as a simple autosomal

recessive. The heterozygous carrier can be detectedby raised glucose-6-phosphate and fructose-6-phosphate levels in the red cells. Cori and Cori

FIG. 5. The defect in galactosaemia.

(1952) first reported the abnormal biochemicalfindings in this type of glycogen storage disease.The glycogen in the liver is above 4% wet weight andhas a normal structure; phosphorylase, amylo-l, 6-glucosidase, phosphoglucomutase, U.D.P.G.-glyco-gentransglucosylase, and amylo (1,4-+1,6) trans-glucosidase are normal or only slightly reduced. Theglucose-6-phosphatase is absent or very muchreduced. Hers (1959) considers that only the com-plete absence of glucose-6-phosphatase can be heldresponsible for the disease. Schwartz, Ashmore, andRenold (1957) found that although affected infantsshow a normal response to intravenous glucose,galactose is excreted as lactic acid and not con-verted to glucose as in the normal infant. Thisindicates an inability to convert glucose-6-phosphateto glucose. The absence of glucose-6-phosphatasedeprives the patient of readily accessible glucose,thus causing hypoglycaemia, which in turn causesketosis, convulsions, increased gluconeogenesisfrom proteins, starvation diabetes, and poor responseto glucose tolerance tests. Giving adrenaline andglucagon to a well-nourished child with a well-glycogenated liver results in a 40 to 60% rise inblood glucose within 10 to 20 minutes by virtue ofthe depolymerization of stored glycogen, and itseventual conversion to glucose by means of thestimulating effect of adrenaline and glucagon onphosphorylase. There is increasing evidence that theabsence of glucose-6-phosphatase may not accountfor all the clinical features of the disease.The effect of glucagon on these children has been

studied intensively. Carson and Koch (1955) showedthat in classical glycogen storage disease onlyslightly diminished or normal results to glucagonadministration were found. This has been explainedby the effect of the debrancher enzyme, amylo-l,6-glucosidase, releasing free glucose from glycogen

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Type 4trAmylo (1,4ia1,6)transglucosidase (I

+1

UDPG glycogentransferase

UDPGpyrophosphatase

Type 5 muscleType 6 liver

PhosphorylasePhosphorylase bKinase

Phosphorylase

I-phosphate

(1,6)

Type 3Amylo-1, 6-glucosidase

(debrancher)

j l Type 1Glucose-6-

Glucose - 6 - phosphatephosphatase

Fructose-6-phosphate hosphogluconate

l IPyruvate Pentose phosphate

--Glucose

Lactate

Type I Glucose-6-phosphatase deficiency hepato-renal glycogenosisType 3 Debrancher deficiency limit dextrinosisType 4 Brancher deficiency amylopectinosisType 5 Myophosphorylase deficiency glycogenosisType 6 Hepatic phosphorlyase deficiency glycogenosis

without the need of a specific phosphatase. Lowe,Sokal, Mosovich, Sarcione, and Doray (1962) foundthat there was no correlation between glucose-6-phosphatase and fasting hypoglycaemia in a seriesof nine children with the abnormality. They foundmajor discrepancies between enzyme determina-tions in vitro and the clinical condition. The disorderhas recently been treated by giving glucagon afterfeeding (Lowe et al., 1962). Eberlein, Illingworth,and Sidbury (1962) have treated glucose-6-phosphatase deficiency with 9oa-fluor-1Ilf-hydroxy-17cx-methyl testosterone (Halotestin) and broughtabout clinical and biochemical improvement; theadrenaline response returned to normal under treat-ment. These findings suggest that the disease is likelyto prove more complicated than a simple deficiencyof glucose-6-phosphatase.

Type 2 Glycogen storage disease of the heartThis disease has been extensively reviewed by di

FIG. 6. The glycogen storage diseases.

Sant' Agnese, Andersen, and Mason (1950). Thecondition becomes manifest in early infancy; theinfant feeds poorly, becomes listless, and fails togain weight. This is followed by episodes of inter-mittent cyanosis, especially during feeding, leadingto attacks of dyspnoea with no specific cause. Theinfants usually die of cardiac failure or broncho-pneumonia. The heart is enlarged and appearsglobular on x-ray examination. At necropsy it weighstwo to five times the normal for the age. Histo-logically there is massive infiltration with glycogen.Sometimes excessive amounts of glycogen aredeposited in smooth and striated muscles andsometimes excessive infiltration with a basophilicsubstance is found in skeletal muscle. The conditionis probably transmitted as a single recessive auto-somal gene. No enzymic defect has been advancedto account for this disease. Hers (1961) recentlydescribed a defect in the hydrolysis of maltose to

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glucose (x-glucosidase) in liver and heart. Thisfinding would suggest a role for the hydrolytic pathof glycogen breakdown through the oligo-saccharides. An alternative explanation involving atransglycosylation reaction immediately before thedebranching enzyme has been suggested. At pre-sent we do not know the site of the enzyme defectwith certainty.

Type 3 Debrancher deficiency limit dextrinosisThe clinical descriptions of this disease are similar tothose found in glucose-6-phosphatase deficiency butthey tend to be milder and difficulties with hypo-glycaemia and development are less severe. It ispossible that early cases reported as von Gierke'sdisease were actually deficiency of the debranchingenzyme.

In the original case described by Forbes (1953)abdominal enlargement was found at the age of 1

year, fasting blood sugars were slightly below nor-mal, and there was a diminished response to adrena-line. The fasting blood sugars were in the normalrange by the age of 12, when the abdominal dis-tension decreased. At this age the liver functiontests became abnormal. Liver biopsy showed thatparenchymal cells were packed with glycogen, anda definite increase in the periportal connective issue.

ltlingworth, Cori, and Cori (1956) showed thatthe content of muscle and liver glycogen wasincreased and that the glycogen had an increasednumber of branch points and short external chains.There was an absence of detectable amylo-1,6glucosidase activity with normal amounts ofphosphorylase and glucose-6-phosphatase. Hers(1961) used a new method for measuring the enzymebased on the observation that the enzyme catalyseda limited reincorporation of 14C-labelled glucoseinto the phosphorylase limit dextrin. As a result itwas shown that the disease was present with enzyme

defects in liver and muscle in one variant or with adefect in liver but not in muscle in another variant.

Information regarding the genetics of the conditionis meagre. It is probable that inheritance is througha single recessive autosomal gene.

Type 4 Brancher deficiency amylopectinosis In1956 Andersen reported on a 17-month-old whitemale infant whose liver showed glycogen which on

analysis was less branched and had larger inner andouter branches than normal. The complaints were

primarly hepatic, with oedema, ascites, and bleedingtendencies. Liver function tests were abnormal butthe blood sugar was normal and no acidosis was

present. A glucose tolerance test showed a moderaterise and a slow fall of the blood glucose level. Theresponse to adrenaline was moderate, but less thanin normal individuals. Necropsy showed glycogeninfiltration of liver, spleen, lymph nodes, and

intestinal mucosa together with nodular cirrhosis ofthe liver. No glycogen was found in the kidneys.The glycogen has different characteristics to thatfound in von Gierke's disease in that it was sticky,hard to handle, and slow to precipitate.

Illingworth and Cori (1952) studied the abnormalglycogen and found a decreased number of branchpoints and a greater chain length than normal. Itseems that there are fewer branch points andsignificantly longer chains. Cori (1957) postulatedthat these defects could be explained by a deficiencyof the brancher enzyme amylo (1,4 > 1,6) trans-glucosidase, but unfortunately not enough tissue wasavailable for direct enzymatic measurement.

Sidbury, Mason, Burns, and Ruebner (1962) havedescribed a second case of abnormal glycogenstructure. The liver, kidney, muscle, and bloodcontained an unusual polysaccharide with an in-creased average outer chain length.The hereditary transmission of the condition is

not known, for although the sibling of the casedescribed by Andersen died of 'glycogen storagedisease' it is not known which type of the disease itwas.

Type S Myophosphorylase deficiency glycogenosisAt least eight patients with this abnormality havebeen described. McArdle (1951) described a 30-year-old man who had pain, weakness, and stiffnessof muscles after exercise; the symptoms disappearedat rest. On examination it was found that 10 to 20%of the normal work could be performed by theforearm and hand muscles in the ischaemic statebut maintenance of the contracted state gavelocalized swelling. There was a fall in the lactate andpyruvate levels in the venous return in blood comingfrom an ischaemic muscle, in contrast to the sharpincrease in these metabolites in the blood fromnormal muscle. There was a normal hyperglycaemicresponse to adrenaline but a smaller rise in bloodlactate than produced in a normal person. The bestconfirmation of the abnormality is the inability todetect an increased blood lactate level after exercise,especially under ischaemic conditions. Histologicallyexcessive amounts of diffuse granular glycogen arefound in muscle. Homogenates of biopsy materialcannot form lactate but have an intact glycolyticcapacity, as shown by normal lactate productionafter the addition of glucose-i-phosphate. There isno detectable phosphorylase a and b activity. ThePR enzyme catalysing the conversion of phos-phorylase a to b and phosphorylase kinase arenormal. It is not known why there is not a greateraccumulation of glycogen in phosphorylase-deficientmuscles. It may be due to breakdown by hydrolyticenzymes or to a decreased extraction of blood glucoseby muscle. It is also not known whether there is a

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deficiency of phosphorylase in cardiac muscle. Sucha deficiency may not be detectable since cardiacmuscle can use substrates other than glucose forenergy. Hepatic phosphorylase activity is intact inthis disease.Immunological studies have shown that muscle

phosphorylase is either completely lacking or so

modified that it does not react immunologically.There is insufficient evidence to determine the modeof inheritance but the absence of a single enzyme inan otherwise complete chain suggests that the defectis a result of a single genetic determination. The fallin lactate would seem to result from its being used atthe same time as its production is curtailed. Schmidand Hammaker (1961) have described a family studyand, using the failure of the level of the blood lactateto rise after exercise, suggest a recessive mode ofinheritance.

di Sant 'Agnese, Andersen. and Metcalf (1962)described a child with excessive muscle glycogen.They pointed out that McArdle's syndrome usuallyappears in the second decade and this was the firstrecorded instance of it being observed in a child.

Type 6 Hepatic phosphorylase deficiency glyco-genosis Hers (1959) first described glycogen storagedisease associated with a diminished hepatic phos-phorylase activity. In one family three children were

affected: one had decreased phosphorylase with a

normal level of glycogen; the other two had phos-phorylase activities that were not decreased but hadincreased liver glycogen. In the first case it may bethat glycogen breakdown via a non-phosphorolyticpathway might be sufficient to limit the accumulationof glycogen, and in the second two cases it may bethat the system is not behaving in vivo as in vitro.Myophosphorylase is apparently normal.

U.D.P.G. glycogen transferase deficiency Lewis,Stewart, and Spencer-Peet (1962) described a case ofinfantile hypoglycaemia in which there was a

deficiency of U.D.P.G.-glycogen 1,4-transglucosylase(U.D.P.G. glycogen transferase). A pair of identicaltwins was investigated to determine the cause ofprofound hypoglycaemia. Glucose tolerance testsshowed a reduced capacity to assimilate glucose and arise in blood glucose following the administration ofglucagon suggested an impaired ability to storeglycogen. A liver biopsy showed that while normalamounts of phosphorylase, U.D.P.G. pyrophos-phorylase, and glucose-6-phosphatase were present,there was a complete absence of U.D.P.G.-glycogen1,4-transglucosylase. The father and three siblingshave abnormalities of carbohydrate metabolism,suggesting that the condition is an inborn error ofmetabolism.The glycogen storage diseases illustrate some of the

problems in the investigation of inborn errors ofmetabolism. It is an oversimplification ofthe problemto suggest that every single-enzyme defect demon-strated in vitro is necessarily the cause of the illness.Isolated enzyme reactions do not occur in livingtissue but are part of an integrated whole. For someyears, however, each enzyme defect was equatedwith a more or less well-defined clinical type.Eberlein, Illingworth, and Sidbury (1962) describea family in which a male child had a deficiency ofglucose-6-phosphatase although the muscle glycogenwas normal. A younger sister was investigated twoyears later for a very similar clinical picture. Herglucose-6-phosphatase level was only 'moderately'decreased but she had a deficiency of the debranchingenzyme, amylo-1,6-glucosidase. Both children hadincompletely degraded glycogen, or 'limit dextrin'in the red cells. The father and a third sibling hadtwice the normal amount of glycogen in the red cells.

Other multiple defects already reported include thecase of Sokal, Lowe, Sarcione, Mosovich, and Doray(1961) who noted a deficiency of both phosphorylaseand glucose-6-phosphatase in one patient. Calder-bank, Kent, Lorber, Manners, and Wright (1960)described a glucose-6-phosphatase defect in the siblingof a patient previously shown to have an amylo-1,6-glucosidase defect. Before any generalizations aremade it must be conclusively proved that these aregenetic and not post-genetic defects.

INTESTINAL GLYCOSIDASE DEFECTS Holzel, Schwarz,and Sutcliffe (1959) have described two children inone family in whom there was defective lactoseabsorption causing malnutrition in infancy. Thechildren had chronic diarrhoea. Oral lactosetolerance tests failed to increase the blood glucoseand led to watery stools of low pH containing lacticand other acids. The good state of health of a 10-year-old child with lactose deficiency is in keepingwith endogenous formation of galactose. Diarrhoea,in which thin, foamy, bulky stools were produced,due to a lack of invertase, was described by Weijers,van de Kamer, Dicke, and ljsseling (1960). Therewas an elevation of blood glucose after oral admin-istration of maltose but no elevation of bloodglucose after an oral sucrose tolerance test. Thissuggests that the two disaccharides are dealt with bydifferent enzymes. The same authors have alsoreported diarrhoea due to a deficiency of maltose.Patients with invertase deficiency do not toleratedextrin and show a decreased elevation of bloodsugar following starch loads.

Holzel, Mereu, and Thomson (1962) have de-scribed a case of severe lactose intolerance associatedwith lactosuria. Six cases have now been recordedwith this condition. It is probable that these children

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cannot metabolize lactose whereas in the cases withdefective lactose absorption the defect is limited tothe hydrolytic enzymes of the gut.

LACTATE METABOLISM DEFECT Hartmann, Wohlt-mann, Purkerson, and Wesley (1962) described a

child that appeared to have an abnormal carbo-hydrate metabolism. The child had a persistentacidosis and secreted an acid urine. The lactic acidlevel in the blood was persistently elevated. Theliver glycogen was decreased, the glucose-6-phosphatase was increased in the liver, and the redcells had an increased rate of glycolysis. The bio-chemical explanation of these findings is still indoubt but it seems from the published case thatthere is a defect in the N.A.D.: N.A.D.H. systemassociated with pyruvate metabolism. The acetylationof other substances may be decreased, but theprecise abnormality in pyruvate metabolism has notyet been demonstrated.

HEREDITARY SPHEROCYTOSIS This is a congenitalhaemolytic disorder characterized by the presence

of spherocytes in the peripheral blood. The patientshave a mild degree of anaemia, varying from patientto patient, but it is rarely severe. There is a slightincrease in unconjugated bilirubin in the serum, an

increased excretion of stercobilinogen in the stools,and a mild to moderate reticulocytosis. From time totime the patients developed a sudden crisis, char-acterized by malaise, anorexia, mild fever, and boutsof jaundice. There is sudden destruction of thecirculating erythrocytes and a decrease of erythro-poiesis with maturation arrest in the bone marrow.The cause of these crises is not well understood, butthey are frequently associated with infections.

Laboratory studies show an increased osmoticfragility of red cells and an increased mechanicalfragility. The cells in hereditary spherocytosis under-go lysis five or 10 times as rapidly as normal cellswhen incubated at body temperature for 48 hours.This abnormal degree of autohaemolysis can belargely prevented by the addition of glucose.The overall rates of glucose consumption and

phosphate exchange in hereditary spherocytes are

normal but when the cells are incubated in plasmacontaining 32P a disproportionately small amount of32p is recovered from the intracellular and stromalphosphate esters and a proportionately greateramount from the inorganic phosphate pool of thecell. It appears that the movement of phosphateinto various intermediates deviates from normal butthe exact nature of the abnormality is at present notclear; possibly there are abnormalities of enolase or

a phosphokinase. It has been suggested that themaintenance of the biconcave shape of red cells

depends on an adequate provision of energy andthis is deficient in hereditary spherocytosis. Adeno-sine, which restores the phosphate changes inaffected cells to normal, appears to replace thedeficit. The red cells in the spleen may be deprivedof glucose and are thus more readily removed fromthe circulation. Splenectomy does not alter themetabolism of the red cell.

Hereditary spherocytosis is transmitted as anautosomal dominant with most of the afflictedindividuals being heterozygotes. It is not yet certainwhether the dominance is complete.

HEREDITARY NON-SPHEROCYTIC HAEMOLYTIC ANAEMIAPatients with this disease usually present withjaundice and anaemia. There may be a history ofweakness, malaise, and abdominal pain with hepaticor splenic enlargement. In children it may be con-fused with haemolytic disease of the newborn butthe Coombs test is negative. Examination of the redcells shows no stippling, sickling, or fragmentationbut there is a reticulocytosis, hyperbilirubinaemia,and other signs of excessive haemolysis.The condition has been reported in about a dozen

families and it appears to be transmitted as an auto-somal dominant, with a variable degree ofpenetrance. It is quite possible that the disease isheterogenous because glucose affects autohaemo-lysis in some families but not in others. In somecases there is a normal rate of glycolysis and 32pexchange but increased amounts of 2,3-diphospho-glycerate in the red cell. In others there is a lowcontent of ATP, deficient glucose consumption, anda decreased 32P-orthophosphate exchange.Newton and Bass (1958) found that some patients

with non-spherocytic haemolytic anaemia hadunstable reduced glutathione and diminished glucose-6-phosphate dehydrogenase activity. The Heinzbodies formed after incubation with acetyl-phenyl-hydrazine were identical to those seen in drug-sensitive subjects, but these patients differ fromdrug-sensitive individuals in that in them the redcell survival time is shortened.

HEREDITARY DRUG-INDUCED HAEMOLYTIC ANAEMIACordes (1926) reported haemolytic anaemia associ-ated with pamaquin (an 8-aminoquinoline com-pound) therapy. The introduction of primaquine, atherapeutically more effective 8-aminoquinolinederivative in 1950, re-opened the study of thisproblem and in 1952, Hockwald, Arnold, Clayman,and Alving reported that primaquine inducedintravascular haemolysis in 10% of Negroes butrarely in Caucasians. Two years later it was demon-strated that sensitivity to haemolytic anaemiainduced by these drugs was a property of the red

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Dihydr

Glucose

NADP NADPH

Glucose-6-phosphate ll T +6-phosF

Fructose-6-phosphate,,

Fructose-1, 6-diphosphate

oxyacetone lyceraldehyde<--phosphate -3-phosphate

iLactate

blood cell due to the presence of abnormal erythro-cytes (Dem, Weinstein, Le Roy, Talmage, andAlving, 1954).The haemolytic cycle consists of three phases. The

first is an acute haemolytic phase, characterized by a

fall in haemoglobin, a rise in the reticulocytes, anda darkening of the urine; it has been shown thatabout half the original red cell population is des-troyed in this time. The second phase is of recovery,

lasting three to four weeks, in which the haemo-globin concentration rises and the reticulocyte countfalls. This passes to a phase of equilibrium which isfollowed by a maintenance of normal haemoglobinlevels and a normal reticulocyte count.The disease is also induced by naphthalene, nitro-

furantoin, and fava beans. In fava bean sensitivitythere is another defect, because in every case offavism the defect associated with primaquinesensitivity is present but patients with the latterdefect can eat fava beans without haemolysis. Thedefect is transmitted as a sex-linked dominant trait.There is a marked degree of variability in the degreeof penetrance, especially amongst females.

Carson, Flanagan, Ickes, and Alving (1956)showed that there is a deficiency of glucose-6-phosphate dehydrogenase in the erythrocytes fromprimaquine-sensitive individuals. This can be shownby the fact that dilute haemolysates of drug-sensitiveerythrocytes are unable to promote the reduction ofoxidized glutathione when glucose-6-phosphate isthe substrate. Oxidized glutathione is normallyreduced when N.A.D.P. or 6-phosphogluconate issupplied. The enzyme deficiency is particularly

FIG. 7. The defect in drug-induced Ihaemolytic anaemia.

Dhogluconate

6-phospho-3-ketoglucoriate

ilRibulose-5-phosphate

'. Ribose-5-phosphate

severe in older red cells. There is evidence that astromal factor inactivates the enzyme in drug-sensitized cells but not in normal cells. The glucose-6-phosphate dehydrogenase activity in leucocytes isnormal in primaquine-sensitive Negroes but isdecreased in Caucasian subjects. The red cells fromsensitive subjects are deficient in reduced glutathioneand in total glutathione. The content of N.A.D.P. isreduced and possibly that of N.A.D.; the glutathionereductase is increased. The catalase is decreased inthe sensitive red cells but the aldolase is increased.The deficiency of glucose-6-phosphate dehydro-genase results in an abnormality of the oxidation ofglucose in sensitive red cells via the hexose mono-phosphate shunt, resulting in a failure to reduceoxidised glutathione and this in turn is responsiblefor the low glutathione levels in sensitive individuals.The defect can be diagnosed by a deficiency ofglucose-6-phosphate dehydrogenase in red cells andby the glutathione stability test.

DISTURBANCES IN ENDOCRINE METABOLISM

THYROID The biosynthesis of thyroid hormonetakes place through a series of separate steps. Iodideis absorbed through the gastrointestinal tract asinorganic iodide and is rapidly distributed through-out the extracellular fluid of the body. Some of theiodide is excreted through the kidneys but most istrapped within the thyroid gland and is oxidized toelemental iodine. Iodide trapping is oxygen de-pendent and is inhibited by cyanide, azide, andsulphydryl inhibitors; it is enzymically controlled

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Intracellular

Gastrointestinal tract

1112

Plasma iodide oThyroid iodide

I \

Renal excretion IV Thyroid iodine

?V

Plasma proteinbound iodide ThyroglobulinTri-iodothyroni ne Mono- and di-Thyroxine iodotyrosine

III

ThyroglobulinTri-idothyronineThyroxinf.(

and oxidative phosphorylation is involved. Thenormal resting thyroid maintains a 20:1 concentra-tion ratio of iodide against the plasma. Thiocyanateand perchlorate ions prevent iodide trapping and re-

lease iodide; their site of action is not known. Acongenital defect of iodide trapping and of a failurein the formation of organic iodide have been de-scribed.The next step in thyroid hormone synthesis is the

incorporation of iodine into the 3 position in tyrosylresidues to form mono- and di-iodotyrosines. Theiodination may be linked with the oxidation ofiodine and although it is thought that the oxidationrequires peroxidase, the nature of the mechanism isnot yet known.The next step is the condensation of some of the

iodinated tyrosyl residues to form iodinated thyro-nines, thyroxine, and tri-iodothyronine; coupling isthought to occur when the amino-acids are linkedto peptides and is accelerated by oxygen. Thereaction may depend on the formation of freeiodine or on a more specific mechanism. Althoughno enzyme has been demonstrated for this step,coupling does not proceed in cell-free homogenatesbut it does in slices, suggesting that the formation isenzymically controlled or requires a high degree ofcellular organization. A defect in this step results in

FIG. 8. Iodine metabolism.

cretinism from a failure of iodotyrosines to couple.The remainder of the iodinated tyroxyl residues

are dehalogenated by the enzyme 'iodotyrosinedehalogenase' and returned to the thyroid iodidepool. A defect in this system results in cretinism froma failure of iodotyrosines to deiodinate.Lack ofiodide-concentrating mechanism Stanbury

and Chapman (1960) have described a patient lackingthe iodide concentrating mechanism who was unableto concentrate iodide in the thyroid, saliva, or gastricjuice. The gland contained a small amount of protein-bound iodide which probably entered by diffusion.The patient was treated with potassium iodide.This case appears to indicate that there is a differ-ence between the concentration of the iodide and theconversion to elemental iodine in the thyroid gland(stage 1, Fig. 8).

Familial goitre from failure to form organiciodine In 1950 Stanbury and Hedge reported a

case of cretinism and goitre in a girl of 15. She haddeveloped normally until the age of 6 months andthereafter showed marked retardation. Signs ofcretinism were noted at the age of 1 year and a

goitre was noticed at the age of 7. The patient was

one of a family of seven children; the three olderchildren were normal, the next four were cretins.Other cases have been described. Schultz, Flink,

Extracellular

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Kennedy, and Zieve (1957) described a 34-year-oldcretin with a huge lobular thyroid who had the sameenzymatic defect. The defect is probably transmittedas an autosomal recessive.

All the patients described will release labellediodine from the thyroid gland when given potassiumthiocyanate; the majority have been severe cretinswith retarded structural and mental development.Normally no accumulated iodine can be releasedfrom the thyroid by potassium thiocyanate. Thedefect in these patients is one of failure to formorganic iodine (stage II, Fig. 8) and then there iscompensating hypertrophy of the gland. McGirr(1960) classified this as a defect in peroxidase.

Cretinism with failure of condensation of iodo-tyrosines Stanbury, Ohela, and Pitt-Rivers (1955)described a 25-year-old woman who showed re-tarded development and had a goitre. Evidence ofcretinism had appeared at the age of 4 years withretarded growth and thick, dry skin. At the time ofher admission the thyroid gland was enlarging.Histology of the gland removed at a subtotalthyroidectomy showed a multinodular goitre withextreme hyperplasia and no colloid formation.There were two consanguineous marriages in thefamily and a sister four years older suffered from anidentical condition. Labelled iodine rapidly accumu-lated in the gland but was not discharged by potas-sium thiocyanate. Chromatographic analysis of theserum showed thyroxine and triodotyrosine in theblood but not in the thyroid gland. Excessiveamounts of mono- and di-iodotyrosine were foundin the gland. The simplest explanation of the defectis that there was a failure of coupling to form tri-iodothyronine and thyroxine in the thyroid gland(stage III, Fig. 8).

Cretinism from failure of iodotyrosine to de-iodinate In 1953 McGirr and Hutchison reported12 patients who were cretins and hypothyroid.Patients with this type of disease develop myxoedemaor goitre before the age of 3, and they are mentallyand physically retarded. The thyroid shows variablepathological changes; some areas are characteristicof colloid goitre, other areas have no colloid, andyet other areas show fibrosis. The subjects haveexcessive amounts of mono- and di-iodotyrosine inthe blood. If these substances are given in a labelledform to patients after thyroidectomy or receivingthyroid the labelled iodotyrosine can be recoveredquantitatively from the urine. The failure to deiodi-nate the iodotyrosines has been demonstrated inthyroid tissue (stage IV, Fig. 8) removed at opera-tion, and also in liver, kidney,. and other organs.The loss of iodine from continuous excretion issufficient to prevent the formation of adequateamounts of thyroxine.

It has been suggested that there is also a defectof coupling in these patients but significant amountsof labelled tri-iodothyronine and thyroxine can bedetected in their blood. The intensely hyperplasticglands are thought to secrete hormone precursors asfast as they are formed. The defect can be overcomeif sufficient iodine is provided (Stanbury, 1961).The genetics of this condition have been studied

thoroughly in a group of itinerant Scottish tinkersin which intermarriage within the group wasextremely frequent. A study of the family tree showsthat this form of cretinism with goitre behaves as asimple autosomal recessive gene. It is probable thatthe cases are homozygous; relatives of cases de-scribed in Holland had defective di-iodotyrosinedehalogenase activity and were probably hetero-zygotes.

Cretinism with iodinated polypeptides in the serumThese patients are characterized by the appearancein blood of iodinated amino-acids which are linkedto peptides and are not extractable in acid butanolas are the iodinated thyronines which normallyappear in peripheral blood. Stanbury and McGirr(1957) have described two sisters who were mentallyretarded, hypothyroid, and had large goitres. Sincethen other cases have been described. The patientsshow hypothyroidism and no butanol extractable1311-labelled component in peripheral blood, al-though in some cases it was found in urine.

Hydrolysis of the circulating component gavemono and di-iodotyrosines, tri-iodothyronine, andthyroxine. It may be that there is a defect in thesynthesis of thyroglobulin, which may be com-parable to the genetic control of haemoglobinsynthesis, a defect in the degradation of thyro-globin or in the enzymes controlling the degradation.At present it is not possible to define the abnormality.This may be an abnormality of stage V (Fig. 8).

There are insufficient cases to define the mode ofinheritance but the reported occurrence in twobrothers and in two sisters suggests that the diseaseis hereditary.

THE ADRENOGENITAL SYNDROME The adrenogenitalsyndrome is usually manifested by pseudoherma-phroditism of females and premature virilization ofmales and may occur spontaneously or as result ofadrenal or gonadal tumours. The syndrome iscaused by heritable defects in the biosynthesis ofsteroids by the adrenal glands. These defects resultdirectly and indirectly in the overproduction ofcertain steroids, some of which are androgenic. Thecardinal sign is virilization but the clinical manifesta-tions depend on the genetic sex, the age at onset, andthe type and degree of the metabolic defect.The patients can be divided into four groups:

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FIG. 9. Pathways of steroid nietabolism.

dehydrogenase

I11 -deoxycorticoste(compound S)

C

Androsterone 11 fl-hydroxy-lase

AB21 -hydroxylase

(compound F)

A Virilism, partial block 21-hydroxylaseB Virilism with adrenocortical insufficiency, more complete block 21-hydroxylaseC Virilism with hypertension, block 11 ,-hydroxylase

1 virilization, 2 virilization with adrenocorticalinsufficiency, 3 virilization with hypertension, and4 virilization with adrenocortical insufficiency andhypertension.

Virilism The age of onset of virilization is mostcommonly prenatal but is probably not before thefifth month of development. Females exhibit a

variable degree of malformation of the clitoris;virilization likewise takes place in males. Bothsexes show accelerated somatic development; theepiphyses close early, and a deficit in growth results.Pubic hairmay appear in the second year; in the malethe testes remain small and immature. The femalesusually have amenorrhoea accompanying progressivevirilization marked by male muscular development,male hair distribution, growth of beard and oftentemporal or frontal baldness. Breast development isslight and the voice is lowered in pitch. When the

adrenogenital syndrome is not present at birth butoccurs in the prepubertal period it is almost alwaysdue to an adenoma or adenocarcinoma of the adrenalcortex.The adrenogenital syndrome can be distinguished

from other forms of pseudohermaphroditism andvirilism by the presence of diffuse adrenocorticalhyperplasia and large quantities of androgenicketosteroids in the urine. Initially treatment wasdirected against these, for example, by partialadrenalectomy.

Wilkins, Lewis, Klein, and Rosemberg (1950)administered cortisone to a female hermaphroditewith virilizing adrenal hyperplasia and found thatthe urinary ketosteroids fell. Bartter, Albright,Forbes, Leaf, Dempsey, and Carroll (1951) foundthat A.C.T.H. stimulated the production of 17-ketosteroids in these patients but did not produce

Cholesterol

srone

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an immediate potassium loss or sodium retention,and as a result they suggested that the primarydefect is decreased production of the sugar hormoneby the adrenal cortex, thus the amounts of A.C.T.H.and of androgen were increased.

In 1953 Jailer suggested that hydroxylation of17-hydroxyprogesterone at C-II and C-21 to hydro-cortisone was impaired. All evidence now indicatesthat in the most common form of the syndrome thereis a partial block in the hydroxylation of C-21.Blood 21-hydroxycorticosteroids and urinary meta-bolites of hydrocortisone are decreased. The bloodA.C.T.H. is increased and this stimulates the glandto produce large quantities of steroids that cannotbe hydroxylated at C-21. Since pregnanetriol is themajor corticosteroid metabolite in the urine of un-treated patients, it has become a valuable asset indiagnosing the adrenogenital syndrome. Pregnane-triolone is always present in the urine of congenitaladrenal hyperplasia, and unlike pregnanetriol, isabsent in cases of adrenal carcinoma so far examined.The unusual urinary steroids are products of

alternative routes of metabolism of compounds ofwhich the major routes are blocked in this syndrome.It is likely that part of the increased 17-ketosteroidsin this syndrome is derived from 170x-hydroxypro-gesterone. Pregnanetriolone reflects the large quanti-ties of 21-deoxycortisol which accumulate belowthe block. The virilization characteristic of thisdisease is due to C19 steroids which can be derivedfrom 17-hydroxyprogesterone. Most of the patientsdo not have adrenal insufficiency, as increasedstimulation by A.C.T.H. probably provides sufficienthydrocortisone. Further evidence that 21-hydro-xylation is not totally impaired is shown by theincrease in blood and urine corticoids after stimula-tion with A.C.T.H.

In view of the large amounts of pregnanetriol and1 7-ketosteroids excreted relative to decreased ornormal amounts of hydrocortisone, it seems there isimpaired conversion of 17x-hydroxyprogesterone tohydroxycortisone. Bongiovanni (1958) showed thatadrenal glands from two patients with the adreno-genital syndrome contained neither compound S (17-hydroxy-1 1-desoxycorticosterone) nor cortisol butthere was increased 17-hydroxyprogesterone. Homo-genates of these glands did not form 21-hydroxylatedsteroids. The ability to convert to 11-hydroxylate17-hydroxy-11-desoxycorticosterone (compound S)to cortisol was not impaired. These observationssupport the concept of defective 21-hydroxylationand unimpaired 11-hydroxylation in this disease.The inheritance is consistent with an autosomal

recessive gene, which, when homozygous, producesthe syndrome. The fact that the disease affects asingle generation is also consistent with a homo-

zygous recessive character. Females appear to bepredominant over males but this could be explainedby the greater ease of diagnosis in females.

Virilism with adrenocortical insufficiency In ap-proximately half the patients with congenital adrenalvirilism there are associated disturbances of salt andwater metabolism. Characterized by urinary loss ofsodium and chloride, these abnormalities appear bythe age of 7 weeks in almost all the affected infants.The infants are apathetic, they vomit, they havediarrhoea, and they are dehydrated. They tend togo into circulatory collapse. It may be difficult todiagnose the condition in males because they mayhave normally sized genitalia at birth.

In this group no tetrahydrocortisol is excretedand there is more urinary pregnanetriol than in theprevious group. This indicates that the block in21-hydroxylation is more nearly complete. Theadrenal gland cannot produce enough 21-hydro-xylated steroids to prevent salt loss and signs ofadrenocortical insufficiency. In some patients saltloss only occurs in superimposed stress such asinfection.An alternative explanation is that there is a salt-

diuretic hormone produced by the overstimulatedgland. Klein, Taylor, Papadatos, Laron, Keele,Fortunato, Byers, and Billings (1958) have shownthe presence of a substance in the urine of infantswith congenital adrenal hyperplasia that causessodium excretion in rats. It may be that, on thebasis of synthetic steroid studies, salt metabolismcan be affected by a progesterone-like compound.

Virilism with hypertension Another group ofpatients with the adrenogenital syndrome have, inaddition to progressive virilization, hypertensionwhich can be severe enough to produce cardio-megaly and heart failure. The major virilizing formsand the hypertensive forms appear to be inheriteddifferently. Both forms were never observed simul-taneously in one family in a survey of 18 families.The defect in the hypertensive form is different

from the major virilizing form. Eberlein andBongiovanni (1955) found an excess of 17-hydroxy-11-desoxycorticosterone (compound S) and thetetrahydroderivative in the blood but no 11-oxygenated 17-ketosteroids were detected and therewas a small amount of pregnanetriol excreted relativeto the 17-ketosteroids. The defect can be explainedby a defect in hydroxylation of C-11. It is generallythought that deoxycorticosterone is responsible forthe hypertension in this disease.The above descriptions associate the clinical

syndromes with specific enzyme defects but evidenceis accumulating that obscures the individual natureof each syndrome. Bartter and Pronove (1960)studied three infants with the adrenogenital syn-

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drome associated with adrenal insufficiency andhypertension. This would suggest that there may bea single genetically determined defect resulting inthe absence of a cofactor or enzyme required forboth 21- and 11-hydroxylation. It is probable thatthere are several substrate specific enzymes whichdetermine which of several 11-deoxy substrates willbe hydroxylated. The oxygen used comes from theatmospheric oxygen and N.A.D.P. and is involvedin the system. The N.A.D.P. may be used onlysecondarily, for example, to reduce a metal in theenzyme after its oxidation with oxygen. ThusII -hydroxylation is a complex and as yet unknownprocess. It is probable that 21-hydroxylation is alsocomplex; the genetically determined defect isprobably in one of the cofactors for the reaction.Another type of defect has been reported by

Bongiovanni (1961) in which there was a deficiencyof 3 P-hydroxy dehydrogenase. The condition was

lethal and a group of 3 /-hydroxy A-5-steroids pre-

dominated in the urine.

DISTURBANCES IN PIGMENT METABOLISM

THE HEREDITARY METHAEMOGLOBINAEMIAS This is a

rare condition, of which the major feature is a

diffuse persistent slate-gray cyanosis which is presentfrom early infancy and unassociated with clubbing ofthe fingers or significant cardiac murmurs. Methae-moglobin comprises 20 to 50% of the total haemo-globin. Compensating polycythaemia and associatedreticulocytosis are occasionally seen. The blood ischocolate coloured and fails to turn red on oxygena-

tion due to excessive amounts of methaemoglobin.The increased concentration of methaemoglobinoccurs in the absence of any exogenous methaemo-globin-producing substances. Methaemoglobinaemiais not a single disease but a group of diseases whichmay be due to formation of an abnormal methaemo-globin resistant to reduction; a deficiency in theerythrocyte reducing system with accumulation ofnormal methaemoglobin; or abnormal endogenousproduction of oxidizing substances.Methaemoglobinaemia associated with abnormal

methaemoglobins In 1948 Horlein and Weberreported a type of methaemoglobinaemia which was

transmitted as a dominant. The methaemobloginhad a different spectral absorption curve which theyshowed was due to a globin abnormality. Gerald(1958) has pointed out that electrophoresis ofhaemolysates in this condition shows the presence

of abnormal Hb M-type pigments. These have beenpurified and studied spectroscopically and have beenclassified as Hb MB (Boston), Hb Ms (Saskatoon),and Hb MM (Mineapolis). The methaemoglobins are

oxidation products of these abnormal haemoglobins.

It has been postulated that a new reactive groupoccurs in the globin which is capable of complexingferric iron; this could be at a point of alteration inthe amino-acid sequence, as has been described forthe abnormal haemoglobins.No abnormality has yet been described in the

erythrocyte enzyme systems. The life span is normaland there is no reticulocytosis.Methaemoglobinaemia associated with a deficiency

of the methaemoglobin-reducing system Gibson(1954) studied several families with methaemo-globinaemia and demonstrated a defect in theenzyme system which is normally responsible formaintaining haemoglobin in the reduced state. Inthe normal red cell methaemoglobin reduction isprobably linked to glycolysis through N.A.D.Certain autoxidizable dyes, such as methylene blue,are capable of accelerating the reduction of methae-moglobin. In the presence of the proper substrate,methylene blue activates the hexose monophosphateshunt. Most investigators believe that the reactionbetween methaemoglobin and the reduced pyridinenucleotides is an enzymatically controlled step.This has been termed 'methaemoglobin reductase'and it may catalyse the reduction of methaemo-globin with N.A.D.P.H. and N.A.D.H. Scott andGriffith (1959) have shown that there is an enzymein normal red cells which reduces methaemoglobinand has a preference for N.A.D. Gibson (1954)considered that there was a deficiency of the enzymecatalysing the reduction of methaemoglobin byN.A.D. in this type of congenital methaemoglo-binaemia. Scott and Griffith (1959) have shown thatthere is a deficiency of an N.A.D-dependent enzymein haemolysates from patients with hereditarymethaemoglobinaemia on the basis of the reactionbetween 2,6-dichlorobenzenone-indophenol andN.A.D.H. This type of congenital methaemo-globinaemia is transmitted as a recessive character.

Congenital methaemoglobinaemia due to excess ofendogenous oxidizing substances Fishberg (1948)described a case which may be of this type. Thepatient excreted large amounts of benzoquinone-acetic acid in the urine, the amount excreted varyingroughly with the methaemoglobin level in the blood.Administration of ascorbic acid abolished themethaemoglobinaemia and the excretion of benzo-quinoneacetic acid.

THE PORPHYRIAS Porphyria has aroused interestamong clinicians and laboratory workers becauseof the unusal clinical manifestations with which it isassociated and the relative ease with which theporphyrins can be identified, even in small amounts.Recent reviews on the porphyrias include those ofSchmid (1960) and Waldenstrom (1957). In this

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Glycine

Succinate

a-Ketoglutarate FIG. 10. Thedefect itl erythropoieticporphyria.

S-aminolaevulinic acid

Porphobilinogen

Porphobilinogen deaminase

Polypyrryl methane

Uroporphyrinogen Porphobilinogen deaminasedeaminase

Uroporphyrinogen III Uroporphyrinogen I

Uroporphyrin III Uroporphyrin I

Decarboxylation Decarboxylation

Coproporphyrinogen III Coproporphyrinogen I

Coproporphyrin III Coproporphyrin I

Protoporphyrinogen III (9a)

Protoporphyrin 111

Haem

discussion we will consider those forms of porphyriafor which a hereditary basis has been clearly esta-blished.

Congenital erythropoietic porphyria This hasoften been confused with the hepatic types ofcutaneous porphyria. The first sign suggesting thedefect is the excretion of red urine containinguroporphyrin but no porphobilinogen; this may beat birth or during the first few years of life. Photo-sensitivity appears in the first few years of life whichis manifest by a vascular or bullous eruption whichcan lead to scarring. Hypertrichosis is found and

the teeth may show a red or brownish coloration.Splenomegaly is present and in the majority of casesthere is increased haemolytic activity, which maybe intermittent. The urine contains uroporphyrinmainly type I but some type III with small amountsof coproporphyrin, mainly type I.

The most likely site of the metabolic defect is inthe enzymatic mechanism that converts porpho-bilinogen to uroporphyrinogen. Porphobilinogendeaminase converts porphobilinogen to uropor-phyrinogen I but uroporphyrinogen III formationrequires the combined action of porphobilinogen

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deaminase and uroporphyrinogen isomerase. Itwould appear that either overactivity of porphobi-linogen deaminase or a defect in uroporphyrinogenisomerase would account for the defect. The primarydefect involves the production of porphyrins of thetype I isomer for which the body has no use. Thedefect appears confined to the haemopoietic systemand may involve only part of the erythroid cells.

Its occurrence in more than one child in the samefamily and the ratio of 1:3 for affected and un-affected siblings of both sexes suggest that theabnormality is inherited as a Mendelian autosomalrecessive trait. The penetrance of the abnormal geneappears complete. The lack of increased porphyrinexcretion in parents and normal siblings of patientssuggests that individuals who are heterozygous forthe trait do not exhibit the metabolic abnormality.

HEPATIC FORMS In congenital erythropoietic por-phyria the metabolic defect can be traced to thedeveloping red cells, but in the other forms of humanporphyria no such defect is found. In these formsthe liver is the only organ containing large amountsof porphyrins and porphyrin precursors; hepaticfunction is frequently impaired.

In this review we shall consider the different typesof hepatic porphyria as they appear in geneticallyhomogenous groups. This classification does nottake into account the precise metabolic defect as it isnot yet known.

The Swedish type This was extensively investi-gated by Waldenstrom in 1937. The syndrome ischaracterized by abdominal and neurologicalsymptoms which are frequently intermittent. Theabnormality is believed to be inherited as a Mendeliandominant but afflicted individuals may be free ofsymptoms. Present methods fail to show an abnor-mality in asymptomatic persons who are geneticallycertain carriers.The major biochemical defect in this form is the

overproduction of the porphyrin precursors &-aminolaevulinic acid and porphobilinogen. There islittle information to support the idea that the bio-synthesis of some haem-containing compound isblocked and the increases in the pyrroles attempt tocompensate for this. A second hypothesis whichsuggests a defect of formation or metabolism of&-aminolaevulinic acid is consistent with the findingsstudying the pool size and disposition of &-amino-laevulinic acid and porphobilinogen. The primarydisturbance probably does not involve porphyrinmetabolism but occurs at the level of pyrrole bio-synthesis which is interrelated with the tricarboxylicacid cycle.

South African type Dean and Barnes (1959)4

studied a large group of porphyric patients in thewhite population in South Africa. These patientscan be traced to a pair of early settlers in SouthAfrica. Many of the patients have skins sensitive tosunlight and slight mechanical trauma. Cutaneousmanifestations are often the only symptoms in menwho feel well throughout life. In some membersacute attacks of abdominal pain occur; women aremore likely to have abdominal pain and skin sensiti-vity is usually slight. Acute attacks can occur,perhaps with peripheral and bulbar paralysis whichmay produce respiratory failure. In many personsthe disease is latent throughout life. In this form thecharacteristic finding is the excretion of largeamounts of coproporphyrin and protoporphyrin inthe faeces. Faecal excretion is elevated even thoughpatients may be in clinical remission and may beasymptomatic throughout life. During acuteepisodes urinary porphyrin is increased and theurine contains large amounts of &-aminolaevulinicacid and porphobilinogen while during remissionthe excretion of these compounds returns to normallimits. This type is different from the Swedish typeby the fact that large amounts of copro-and proto-porphyrin are excreted in the faeces. The SouthAfrican studies indicate that the defect is inheritedas a Mendelian dominant trait.Schmid (1960) reviewed the other reported

cases of porphyria in which the metabolic defect andthe mode of inheritance is less clear. It is difficult toclassify these families at present; for example, Holti,Rimington, Tate. and Thomas (1959) have describeda porphyric family in which the defect is a dominanttrait. The disease was noticed in a young woman whodeveloped bullous skin lesions after delivery, andnine other members of the family had chemicalevidence of porphyria in that the faecal excretion ofcoproporphyrin and protoporphyrin was increased.The urinary porphyrins were normal or only slightlyincreased. This also differs from the South Africantype in that in the South African type large amountsof 8-aminolaevulinic acid and porphobilinogen wereexcreted coinciding with the acute abdominal andneurological manifestations. It is probable that thisfamily is different from the South African andSwedish types.As can be seen the nature of the metabolic defect

of the hepatic types has not yet been determined andit is in this field that future study should lie.

HYPERBILIRUBINAEMIA Conjugation of bilirubin bythe liver is essential for excretion of the pigment. Thepassage of bilirubin through the liver cell involvesseveral stages, and various type of congenitaljaundice have been described, related to each ofthese stages.

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Jaundice due to disturbance of the transportmechanism This syndrome has been called Gilbert'sdisease, hereditary non-haemolytic bilirubinaemia,familial non-haemolytic jaundice, or constitutionalhepatic dysfunction. The syndrome is characterizedby a mild, chronic, apparently non-haemolytichyperbilirubinaemia with unconjugated pigment inthe blood (Meulengracht, 1947). This syndrome hasa variable course but often the jaundice is observedcoincidentally with a routine examination or duringsome other illness.

In this syndrome bilirubin uptake by the liver(Klatskin, 1961) is defective. The pigment in theblood is unconjugated and the bile pigment contentof the bile is similar to normal bile. Arias, Lowy,and London (1958), using needle biopsy specimensand a very sensitive assay method with 4-methylumbelliferone, claimed to show a decrease in theglucuronyl transferase in the liver. The glucuroni-dation system using N-acetyl-p-aminophenol andmenthol in vivo is normal. Schmid and Hammaker(1959) believed the abnormality to be due to defectivebilirubin transport from the plasma to the site ofconjugation in the liver cell. In Britain 'Gilbert'ssyndrome' describes those patients who have a defectin bilirubin uptake.The occurrence ofunexplained hyperbilirubinaemia

in more than one member of a family has repeatedlybeen reported, and the investigations suggest thatthe syndrome is genetically controlled and may betransmitted as a Mendelian dominant. It has beensuggested that this syndrome represents the heter-ozygous form of congenital non-haemolytic jaundice(Crigler-Najjar syndrome) or that Gilbert's syn-drome represents a milder degree of the same defectas the Crigler-Najjar syndrome. Against this is theobservation that patients with Gilbert's syndromeform glucuronides at normal rates and siblings ofpatients with congenital non-haemolytic jaundicedo not have mild hyperbilirubinaemia.

Jaundice due to disturbance of bilirubin conjugationBefore bilirubin can be excreted in the bile it mustbe converted to a water-soluble conjugate withglucuronic acid, bilirubin diglucuronide; this pro-cess takes place in the liver. The glucuronic acidcomes from uridine diphosphate glucuronic acid;the reaction is catalysed by glucuronyl transferase, amicrosomal enzyme. Glucuronyl transferase isdeficient in the newborn animal (Lathe and Walker,1958); as the newborn matures the activity of theenzyme increases, ultimately attaining adult levelswithin a few weeks. A deficiency of glucuronyltransferase can be a cause of neonatal jaundice.

Congenital familial non-haemolytic jaundice(Crigler-Najjar syndrome) was described by Criglerand Najjar in 1952. In three related families six

infants were found in whom severe jaundice ap-peared about two days after birth and persistedthroughout life. All but one developed severe changesin the central nervous system resembling kernicterusand five died before they were 15 months old. Othercases have been described by Schmid (1960).Investigations show that these patients clear brom-sulphthalein normally and that the gall bladder canbe visualised normally by intravenous cholangio-graphy. Only unconjugated bilirubin is found in theserum; the bilirubin tolerance is impaired and thereis no evidence of excessive haemolysis.

It is probable that the defect in these cases is alack of the enzyme, glucuronyl transferase. Jervis(1959) showed that a liver homogenate from apatient with the syndrome could not conjugatebilirubin in vitro. This would support the theorythat the condition is due to a lack of, or an impair-ment of, glucuronyl transferase.

Crigler and Najjar (1952) showed that there wasa definite familial pattern as six cases occurred inthree related families. It is probable that the defectis inherited as a recessive trait. Childs, Sidbury, andMigeon (1959) fed sodium salicylate to the relationsof three cases and found that glucuronide excretionwas impaired. It may be that the gene is incompletelydominant for other glucuronide-forming compounds.

Jaundice due to disturbances ofconjugated bilirubinexcretion In 1954 Dubin and Johnson described achronic benign intermittent jaundice in youngpeople characterized by a raised serum bilirubinlevel which is mainly in the conjugated form. Theliver is greenish black due to an excess of blackgranules in the liver cell. This pigment may be alipofuscin and the granules may be altered lysosomes.The syndrome can be familial (Wolf, Pizette,

Richman, Dreiling, Jacobs, Fernandez, and Popper,1960). Wolf and his colleagues found that the degreeof liver pigmentation varied but the mode of inheri-tance is not yet known.

Rotor, Manahan, and Florentin (1948) describeda type ofjaundice similar to the Dubin-Johnson typebut the liver was not pigmented. As in the Dubin-Johnson type of jaundice there is an increase in theconjugated bilirubin in the serum and the 210-minute value for the level of bromsulphthalein isgreater than the 45-minute value. The results suggestthat the excretion of the conjugated pigment isimpaired. The jaundice occurs in several membersof a family but the evidence is insufficient to deter-mine whether the defect is genetically controlled.

ABNORMALITIES OF PURINE METABOLISM

GOUT Primary gout is a disorder of purine meta-bolism, the cardinal feature of which is hyperuric-

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aemia. It is pre-eminently a disorder of adult malesand is characterized both by recurrent attacks ofacute arthritis of unknown cause and by a chronicarthritis associated with deposits of urates in jointsand other tissues.The heritable nature of gout has been recognized

since the time of Galen but it has only been includedas an inborn error of metabolism in the last thirtyyears. The hesitancy was due to the idea that goutwas not an error of metabolism but was based on analternative and slightly divergent path of meta-bolism met with in a large part of the total popu-lation. Now that it has been classified as an inbornerror of metabolism it can be seen that gout con-forms to a well-defined clinical and metabolicpattern. These considerations apply to primary goutand not to the secondary types.A family history of primary gout has been

recorded in only 6 to 18% of cases but is enlargedwhen a study of hyperuricaemia is made. Asympto-matic hyperuricaemia is approximately five times ascommon as clinical gout in families of gouty per-sons. It is thought that penetrance of the presumedautosomal dominant is variably incomplete indifferent families; expressivity ranges from a re-latively early appearance of fulminating symptomsto a long-delayed appearance of mild symptoms.There is a striking preponderance of males (95%).The considerations that apply to gout apply to

inborn errors of metabolism in general. The spectrumof expression is presumed to be the result of acomplex interaction of genetic, metabolic, andenvironmental influences (Gutman, 1959); theappearance of gout does not depend on the geneticendowment alone since it is the predisposition togout, not gout itself, that is inherited.Although inborn, no sign of gout is detectable

until hyperuricaemia is found at puberty, especiallyin males, persisting in variable degree. Acute goutyarthritis appears at sporadic intervals, then topha-ceous deposits form and the patient has jointsymptoms between the acute attacks. Eventuallychronic gouty arthritis, chronic gouty bursitis, renalimpairment, and sometimes renal failure ('goutykidney') develop.Among other inborn errors of metabolism not

apparent at birth, galactosaemia and alkaptonuriaappear some days or weeks after birth and glycogenstorage disease can occur some months after birth.Genetically transmitted hypercholesterolaemia canbe considered similar to gout in that the cholesterollevel does not rise until after puberty and symptomsdo not appear until middle life.The precipitation of attacks of acute gouty

arthritis may be analogous to the crises in sickle cellanaemia or the abdominal crises in acute porphyria.

It is doubtful whether uric acid itself is the precipit-ating agent. The storage of uric acid in tophi isanalogous to the storage of lipids, carbohydrate, andpigments in other 'storage' diseases; why cartilage isthe site selected is unknown, but it also occurs inochronosis. Uric acid is precipitated in the kidneyitself and as stones in the lower parts of the renaltract; the factors causing precipitation include anincreased concentration in the urine, infection, and adrop in the urinary pH.The cause of hyperuricaemia has been debated for

many years and it seems probable that in goutysubjects there is an increased urate generation anda decreased urinary excretion (Wyngaarden, 1960).Gutman and Yu (1957) showed that 30% of goutysubjects excrete excessive amounts of urate in theurine. These authors considered that there is noevidence for excessive reabsorption from the kidneyand that hyperuricaemia is due to excessive pro-duction of uric acid. In the hyperexcretors of uricacid overproduction of uric acid must occur but inmost cases large amounts are excreted in the gut(Sorensen, 1959), also implying an overproductionof uric acid.

In the diseases previously considered, e.g.,phenylketonuria and alkaptonuria, the abnormalmetabolites are formed from intermediary com-pounds but uric acid is an end-point of metabolism.It was formerly thought that uric acid originatedsolely or largely from preformed exogenous orendogenous nucleic acids and that the hyperuric-aemia of gout was due to excessive nucleic aciddegradation.

Overincorporation of glycine or other precursorsinto uric acid is demonstrable in many cases ofgout, and those subjects who excrete large amounts ofuric acid have shown overincorporation of glycine.It is probable that overincorporation is more fre-quent than can be demonstrated by turnover studies.Overincorporation of precursors into uric acidappears to be due to a shunt pathway whereby apercentage of newly formed pyridine nucleotides isbroken down to purine bases which are then oxidizedto uric acid. It may be that there is a defect in thecontrol of the production of phosphoribosylamineJones, Ashton, and Wyngaarden (1962) demon-strated an increased specific activity in the ribosemoiety of imidazoleaceticacid riboside after admin-istration of 14C glucose in certain gouty subjects.There was an increased turnover of phosphoribosylpyrophosphate, suggesting a failure in control at anearly site in purine synthesis. The hypothesis mustaccount for an excessive production of urate andthis is best explained by the idea that precursors ofpurines are deflected from competing pathways.The concept implies that the lack of enzyme in

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Transferase

Di hyd rooratase

NH3+ CO+ ATP FIG.ll. I

Carbamyl Phosphate Aspartic acid

Carbamyl aspartic acid

Dihydroorotic acidDihydrooroticdehydrogenase I

Orotic acidOrotidylicpyrophosphorylase

Orotidylic acicProbable site Orotidylicof defect decaboxlase

Uridylic acidUridylickinase I

Uridine triphc

Cytidine triph

Cytidylic acidprimary gout does not reside in the sequentialreaction of purine biosynthesis but elsewhere.The heredity factor in the hyperuricaemia of gout

is well established; whether the hyperuricaemia isdetermined by an autosomal dominant with com-plete penetrance or by cumulative gene action is notyet known. It seems likely that different patterns ofinheritance may occur.

XANTHINOSIS This is a rare disorder characterizedby the excretion of very large amounts of xanthine inurine and the tendency to form renal stones.Dent and Philpot (1954) described a girl of41 years

with a clear renal stone. Analysis showed that thestone contained xanthine but no detectable uricacid and the patient had no detectable uric acid inthe blood. Dickinson and Smellie (1959 reinvesti-gated the child, and, using specific enzymaticmethods, showed that the plasma oxypurines wereincreased. Other cases of xanthine stones have beendescribed but this is the only case that has beenfully investigated biochemically.Dent and Philpot suggested that gross xanthinuria,

together with low levels of uric acid in the plasmaand urine, may be due to a block in the oxidation ofxanthine to uric acid or to the excessive excretion ofxanthine preventing its conversion to uric acid. Thismay be due to a renal tubular defect preventing itsreabsorption. Dickinson and Smellie (1959) sug-gested that both defects are present. It was suggestedthat the enzyme xanthine oxidase is almost entirely

The defect in oroticacicdturia.

lacking, it would be expected that hypoxanthinewould also be present in the plasma.The genetic defects in xanthinuria are obscure,

but investigation of the relatives has precluded anautosomal dominant mode of inheritance.

OROTICACIDURIA Huguley, Bain, Rivers, andScoggins (1959) described a child in whom there wasa refractory megaloblastic anaemia associated withthe excretion of orotic acid in urine. From the ageof 3 months the child was chronically ill, had re-peated respiratory infections and diarrhoea, thebone marrow was megaloblastic, and the urinecontained crystals of orotic acid. There was someimprovement in all features of the disease after theadministration of steroids and almost completeclearing of evidence of the disease after the adminis-tration of uridylic and cytidylic acids.The findings implied a defect in the pyrimidine

synthetic pathway between orotic acid and orotidylicacid. A lack of orotidylic phosphorylase or orotidylicdecarboxylase would account for the accumulationof orotic acid. The fall in orotic acid excretion afteruridylic and cytidylic acid administration could beascribed to a negative feedback mechanism.The parents were related and reduced levels of

both orotidylic pyrophosphorylase and orotidylicdecarboxylase in erythocytes of both parents and oftwo surviving siblings were found. It may be that thisdefect is of a recessive type.

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PSEUDOCHOLINESTERASE DEFICIENCY

Suxamethonium (succinyl dicholine) is a short-acting muscle relaxant. Its removal depends on theconcentration of serum pseudo-cholinesterase.Lehmann and Ryan (1956) described a familialincidence of low pseudo-cholinesterase. Kalow(1959) has reviewed work in which it was shown thatthe serum enzymes of such patients differed fromthose of normals as determined by substrate affinitiesas well as by lessened sensitivity to inhibitors. Thedecreased sensitivity to dibucaine (nupercaine) was

determined; the results fell into three groups whichbest fit the model of two autosomal allelic geneswithout dominance, yielding normal, atypical, andmixtures of the two enzymes. It was found that an

inhibitor in potato peel distinguished the threegroups and related well to the dibucaine inhibitions.Inhibition studies indicate that the difference at theanionic site could be because of a relatively weakeffective charge in the atypical enzyme. In this inbornerror the cholinesterase is not present in smalleramounts but has rather different catalytic propertiesthan normal.

CONCLUSION

In this review we have indicated those diseases thatcan be called inborn errors of metabolism. This listis not complete because we have left out thosediseases which are either due to defective tubularreabsorption in the kidney or to impaired absorptionfrom the gut. These hereditary diseases demonstratethe importance of the biochemical approach inclinical medicine. The hereditary make-up of theorganism determines the potentiality, but otherfactors, for example the environment, play a part.For instance, the haemolytic anaemia due to glucose-6-phosphate dehydrogenase deficiency only appears

when some factor intervenes to oxidize the cellglutathione; the galactosaemic infant becomes illwhen fed on galactose. This combination of geneticand environmental factors may play an importantpart in our understanding of other disease, forexample, diabetes. Although the diseases we havedescribed are for the most part rare, it is probablethat in the next decade others will be found, and,more important, some of the commoner diseaseswill be better understood by the application ofmethods of investigation similar to those outlined.

ADDENDUM

HOMOCYSTINURIA Carson, Cusworth, Dent, Field,Neill, and Westall (1963) have described homo-cystinuria in two mentally defective sibs. Homo-

cystine was also found in the plasma. The children'smother and the two children have an impairedmethionine tolerance, probably indicating an in-herited defect of methionine metabolism.

I should like to thank Professor N. H. Martin for hishelp and advice in the preparation of this review, andalso to acknowledge the help given by the British EmpireCancer Campaign.

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