J. clin. Path. (1962), 15, 200

Distribution of alkaline phosphatase in theserum proteins in hypophosphatasia

N. H. KORNER

From the Department of Medicine, St. Vincent's Hospital, Sydney, Australia

SYNOPSIS A patient with hypophosphatasia and her relatives, including an affected brother, were

studied with regard to serum alkaline phosphatase levels, phosphoethanolamine excretion, leucocytealkaline phosphatase, blood group genes, and alkaline phosphatase distribution in the serum proteinfractions.The pattern of alkaline phosphatase distribution in the serum protein fractions was normal both

in the clinically affected patients and in their relatives. Thus the electrophoretic mobility of serumalkaline phosphatases is not altered in hypophosphatasia.The mere presence of phosphoethanolamine in the urine was of no help in detecting heterozygotes

for the hypophosphatasia gene(s), as this substance was also found in control urines.

Hypophosphatasia is one of the group of inheritedconditions in which a metabolic defect is the con-sequence of an enzyme deficiency. In this diseaselow tissue alkaline phosphatase activity is reflectedin a low serum alkaline phosphatase level, and theoutstanding clinical feature is defective mineraliza-tion of bone with resultant skeletal deformities,e.g., genu valgus, Harrison's sulci. The histologicaland radiological picture of the affected bone re-sembles that of rickets.

Other clinical features of hypophosphatasia arethe premature loss of primary teeth, occasionallycraniostenosis, and thoracic skeletal deformitieswhich in severe cases may lead to death fromrespiratory infection (Engfeldt and Zetterstr6m,1954). Interstitial renal fibrosis and nephrocalcinosishave been described in badly affected infants but thedisease has also been discovered in apparentlynormal adults after a fracture (Beisel, Austen,Rosen, and Herndon, 1960).Apart from low levels of alkaline phosphatase

activity in the serum, bone, liver (Sobel, Clark, Fox,Robinow, 1953), kidney, small intestine (Harris andRobson, 1959), and leucocytes (Beisel, Benjamin,and Austen, 1959), the main biochemical abnor-mality is the excretion of excess phosphoethanol-amine in the urine (Fraser, 1957). The serumcalcium is usually normal, but in severe cases hyper-calcaemia has been observed and held responsiblefor nephrocalcinosis (Fraser, 1957) though renal

Received for publication 2 October 1961.

function has not been affected. Serum inorganic phos-phorus is usually normal but high levels can occur(Engfeldt and Zetterstrom, 1954). The probablepresence of adenosine monophosphate in the urinehas been described but not confirmed (McCance,Fairweather, Barrett, and Morrison, 1956).The mode of inheritance is believed to be by an

autosomal recessive gene (Fraser, 1957; Harris andRobson, 1959).The purpose of the present investigation was to

study the distribution of alkaline phosphataseactivity in the electrophoretically separated serumprotein fractions in two patients with hypophos-phatasia and their relatives. In normal subjects peakalkaline phosphatase activity is in the alpha 2globulins, and beta globulins have somewhat morethan half the alpha 2 activity. Albumin activity isoften more than 10% of the total (Korner, 1962),but alpha 1 and gamma activity is less than this(Rosenberg, 1959; Keiding, 1959). Any markedchanges in the electrophoretic mobility of serumalkaline phosphatases in hypophosphatasia mightthus be expected to cause deviation from the normalpattern of enzyme distribution.The phosphoethanolamine excretion, leucocyte

alkaline phosphatase, and blood groups of theaffected family are also described below, togetherwith the case history.

CASE REPORT

The patient, a girl aged 26 months, was admitted to St.Vincent's Hospital, Sydney, in June 1959. She was the

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Distribution ofalkaline phosphatase in the serum proteins in hypophosphatasia__C___E _,.s _l W. __ r ... - _l _';;t b. _

*.8jtti w | +

:, t X R,

P

FIG. IA FIG. lB

FIG. 1. A, the propositus, showing genu valgus, Harrison's sulci, anda squint; B, the propositus, showing loss of incisor teeth; C, bones ofthe legs, showing metaphyseal defects in femora, tibiae, and fibulae.The tibiae are bowed. FIG. IC

fourth child in the family, and pregnancy and deliveryhad been normal.Her head was thought to be somewhat abnormal in

shape since birth. The forehead was high, and a smalllump was present near the posterior fontanelle, whichwas later followed by the appearance of a hard lumpat the site of the anterior fontanelle, and a ridge runningbackwards along the sagittal suture. The mother thoughtthat a small anterior fontanelle was present at birth.The patient was breast fed for 10 days and was then

put on an adequate artificial feeding regime. She gainedweight and made satisfactory progress; at the age of Iyear she started to stand, but she did not walk till 19months. Medical advice was sought because she could notwalk as well as expected, and tended to overbalance.She was 'knock-knee'd' and 'walked on the insides of herfeet'.Her teeth appeared at the normal times, but she began

to lose some at the age of 13 months, and in all lost fiveteeth. Her ribs were noted to be unusual in shape, and aslight squint appeared when she was almost 2 years old.Prominent blue veins on her forehead had been presentsince the age of 12 months.

She started to speak at the age of 2 years, and develop-ment as regards eating, toilet training, intelligence, andemotional responses seemed quite normal. She hadsuffered no fractures, or other relevant illnesses.

PHYSICAL EXAMINATION She was a child of normal size(height 82 5 cm., weight 12-7 kg.), with a high, prominentskull vault, and a ridge of bone running from the site ofthe anterior fontanelle back along the sagittal suture,giving the appearance of craniostenosis. The veins overthe forehead were prominent, and there was a minordegree of convergent strabismus. The head circumferencewas 46-5 cm. Her gait was somewhat awkward, but theoptic fundi and the remainder of the central nervoussystem were normal.Genu valgus deformity, Harrison's sulci, and a rachitic

rosary were present (Fig. 1). One upper and four lowerincisor teeth were missing. No other abnormality wasdetected clinically.

PROGRESS Details of biochemical and radiologicalinvestigations are given in Table I.

It was decided to treat the craniostenosis surgically,and a left parietal cranioplasty was performed. Anincision in the left parietal bone extending for three-quarters of the circumference of an ellipse was lined withpolyethylene film. The child recovered well, and sixmonths later was re-admitted for operation on the rightparietal bone. She had made good progress (height 87 cm.,weight 12-3 kg.) but her movements and gait remainedsomewhat clumsy. The rachitic rosary was no longer evi-dent, but physical examination was otherwise unchanged.

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N. H. Korner

TABLE IINVESTIGATIONS OF PROPOSITUS

Serum alkaline phosphatase (King-Armstrong units/100 ml.)

Serum calcium (mg./100 ml.)Serum phosphorus (mg./l100 ml.)Urine phosphoethanolamineBlood urea (mg./l00 ml.)Serum electrolytes (mEq./l.)Serum proteins (g./100 ml.)Electropherogram

Radiographs

Other enzymes

HaematologicalW.R., Eagle, Kline testsUrine

A right parietal cranioplasty was performed, this timewith a full circumferential incision in the bone.

Convalescence was prolonged somewhat by woundinfection, but after a course of chloromycetin she madea complete recovery, and has continued to develop well.

FAMILY HISTORY

Sixteen members of the family were seen, but in only14 was the serum protein alkaline phosphatasedistribution studied. There was no history of anybone disease, nor were the parents consanguineous.All four grandparents were well. There were fourother children, aged 7 (boy), 6 (boy), 4 (girl), and7 months (boy), all of whom were clinically well. Theyoungest boy, who had a very low serum alkalinephosphatase level, started to lose some teeth soonafter they erupted, and was therefore considered tobe affected by the disease, though radiographs ofthe skull and the right knee, taken at the age of3 weeks, were normal. The eldest boy had a slightchest deformity, suggestive of residual Harrison'ssulci. All the children had serum alkaline phosphatasevalues (15 to 25 King-Armstrong units/100 ml.,Table 1II) well below the normal range for children.

3 2 15 23(Repeated estimations)10-7 10 2 10-0 9*550 50 57 62Grossly increased29 27Na 144 K 48 HCO3 2506-1 7-4Within normal limits(slight increase in alpha 2 and beta globulins)Metaphyseal defects in lower ends of the femora, also in the fibulae, lowerright ulna and upper ends of humeri, craniostenosisSerum acid phosphatase 2-4 Gutman and Gutman units/100 ml. S.G.O.T.26 units/100 ml.Normal haemoglobin, leucocyte count, E.S.R.NegativeNormal cytology, no albuminuria, no glucose

PHOSPHOETHANOLAMINE EXCRETION Phosphoethanol-amine excretion was studied in 14 members of thefamily. In all of them urine extracts examinedchromatographically showed the presence of phos-phoethanolamine. The estimations were not quanti-tative, and the extracts chromatographed representedlarger volumes of urine than those used by Harrisand Robson (1959). There appeared to be a grossincrease in phosphoethanolamine excretion in thepropositus and her father, and the three brothers andthe paternal grandfather also seemed to excrete morethan the other members of the family. Phospho-ethanolamine was found in the urines of six out ofseven controls, and thus under the test conditions thepresence of this substance in the urine was of nohelp in detecting heterozygotes for the hypo-phosphatasia gene(s).

LEUCOCYTE ALKALINE PHOSPHATASE This was esti-mated histochemically by a slide method (Leonard,Israels, and Wilkinson, 1958). In all except two of the14 relatives (a maternal aunt, 60% weakly positive,940% negative, and a distant cousin, 20% stronglypositive, 15% weakly positive, 830% negative) thereaction was either 100% negative or 1 % weakly

TABLE IIBLOOD GROUPS

Subject

PropositusAffected brotherSisterBrother 1Brother 2MotherFatherPaternal grandmotherPaternal grandfatherMaternal grandmotherMaternal grandfatherMaternal auntMaternal great-aunt (grandfather's sister)Maternal great-aunt's daughter

Al BBAl BBBBAl0

AlBAl BAl BAl BB

MNMNNNNNMNMNMNMNNMNNN

CC DeeCc DeeCc DeeCc DeeCc DeeCc DeeCC DeeCc DeeCC DeeCc DeeCc DeeCc DeeCc Deecc ddee

S negativeS negativeS positiveS positiveS positiveS negativeS positiveS positiveS negativeS negativeS negativeS negativeS positiveS positive

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Distribution of alkaline phosphatase in the serum proteins in hypophosphatasia

AIb. Alpha Alpha2 Ibe t a

60

50

40

30

20

10

Affected SiblingsUnaffected Siblings

- -- Parents

FIG. 4

C1(

60

50

40

30

20

FIGS. 2, 3, 4, and 5. Alkaline phosphatase distributionin the serum protein fractions, expressed as apercentage ofthe total serum alkaline phosphatase level.Fig. 2, in normal adults; Fig. 3, in normal children;Fig. 4, in affected siblings, in unaffected siblings, and inparents; Fig. 5, as in Fig. 4.

0

Affected SiblingsUnaffected Siblings a

--- Parents

FIG. 5

0/c

60o

i040j

30

20-0

0O

Gamma

. I 11iii

Al b. Aiphal Alpha2 Beta ammo

III'IiItII

III''I,litIIIIII

III

:. :.i.!ii

I.L..LJ.FIG. 2

Alib. AAlghal

60

50

40 -

A

30

20

10

AIII I2 i l-i11 1 iFIG. 3

_1 .

- - - _ --

11111141111[tijilLHUMII[LllltlilUllllltit il-11111111

3 2-I--

.......LI I............LLL-

-1IlAlpha

.LLLL .

B

.

203

beta |Gamma

,HIII,.I- -----------I 11

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204 N. H. Korner

O,b AI b|B eta Gomma

70

60

50

40

30

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GRANDPARENTS & OTHER RELATIVESFIG. 6

FIG. 6. Alkaline phosphatase distribution in the serumprotein fractions, expressed as a percentage of the totalserum alkaline phosphatase level, in grandparents andotherrelatives.

positive. In the propositus it was once 1% weaklypositive but when measured three days after aconsiderable leucocytosis (36,000 W.B.C./c.mm.)associated with a wound infection, the reaction was100% negative. Of two normal controls, one alsohad a 100% negative reaction and one had a 40%weakly positive reaction, 96% negative. A positivecontrol had 80% strongly positive, 20% weaklypositive leucocytes.

BLOOD GROUPS Genotyping was done by the RedCross Blood Transfusion Service, Sydney (Table II).The results were recorded for possible use in futuregenetic studies.

ALKALINE PHOSPHATASE DISTRIBUTION IN SERUM

PROTEIN FRACTIONS The alkaline phosphatase acti-vity in the electrophoretically separated serumprotein fractions was studied by a method describedin a separate paper (Korner, 1962).The normal pattern of distribution is shown in

Figs. 2 and 3.

THE FINDINGS IN HYPOPHOSPHATASIA

THE PROPOSITUS AND HER AFFECTED BROTHER Sera

collected on two different occasions were studied.The results are set out in Table III and Figs. 4 and 5.In the propositus, levels in alpha 2 and beta globulinswere equal in the first specimen, but in the second

TABLE IIIALKALINE PHOSPHATASE LEVELS EXPRESSED AS A PERCENTAGE OF THE TOTAL SERUM

ALKALINE PHOSPHATASETotal AlbuiminAlkalinePhosphatase(K.-A. u./100 ml.)

I Alpha 1 2 Alpha 2 3 Beta 4 Gamma Blank

Propositus and Affected Brother1 2i Propositus1 7 mth. Affected brother2 3 Propositus2 1 yr.

5 mth. Affected brotherParents and Clinically Normal Siblings

1 7 Brother A1 6 Brother B1 4 Sister1 32 Mother1 32 Father2 8 Brother A2 7 Brother B2 5 Sister2 32 Mother2 32 Father

Other Relatives1 67 Paternal grandfather1 66 Paternal grandmother1 60 Maternal grandfather1 61 Maternal grandmother1 28 Maternal aunt1 59 Maternal great-aunt1 17 Maternal great-aunt's daughter

32

2-3

1999 S

5

76

7

25

213031

7

816

212620

8

77

10S

3

5

4

2

6 0 3 0 44 16 32 0 0 0

5

45

5

3585

42 5

7

435

666

30

218214116167

121810157

11

32230

360

30

26l

24

5

41010687290

4967846

7S

25

1076246

173

5

5

43

41525319253726642849

75493446353833

1817308

2510111128

12111013171912

13100

15191517151932

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91611162017

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24446

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0

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450

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2382

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Speci- Age Subjectmen (yr.)

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Distribution of alkaline phosphatase in the serum proteins in hypophosphatasia

specimen there was the usual peak in alpha 2.Albumin had a relatively high proportion of totalactivity in specimen 1 (19%) and less in specimen 2(9%).The affected brother had the usual peak in alpha 2

in both sera, with slightly less activity in the betaband.

PARENTS AND THREE OTHER SIBLINGS The results onthe two sets of sera are set out in Table III and Figs.4 and 5. The pattern of distribution of enzymeactivity was normal.

REMAINDER OF THE FAMILY Four grandparents, amaternal aunt, and a maternal great-aunt and herdaughter were available for study (Table Ill and Fig.6). The pattern of alkaline phosphatase distributionwas normal in all.

DISCUSSION

The distribution of alkaline phosphatase activity inthe serum protein fractions in patients with hypo-phosphatasia and their relatives appears to benormal, with the usual peak in alpha 2 globulins,somewhat lower beta activity, and low activity in thealbumin and the alpha 1 and gamma globulins. Theserum alkaline phosphatases in this condition thusresemble normal alkaline phosphatases in electro-phoretic mobility, and this is in keeping with theconcept of hypophosphatasia as a quantitativeenzyme deficiency affecting all the tissues whichnormally contain alkaline phosphatase.Most of the physiological processes associated

with the action of alkaline phosphatase do not seemto be detectably deranged in hypophosphatasia, e.g.,the renal re-absorption of glucose, and defective

mineralization of bone and the loss of primary teethare the only abnormalities directly attributable tothe enzyme deficiency.

It is of interest that all five siblings had low serumalkaline phosphatase levels confirmed by repeatedestimations. The most likely explanation is that thethree clinically normal children were heterozygouscarriers of a recessive gene and consequently dis-played some biochemical abnormality. On the dataavailable the possibility of inheritance by means ofa dominant gene with variable expressivity could notbe excluded.The presence of phosphoethanolamine in the

urine, measured under our test conditions, was ofno help in detecting heterozygotes, as this substancewas also found in normal urines.

This work was supported by a grant from the MedicalResearch Committee, St. Vincent's Hospital, Sydney.My thanks are due to Miss L. Silvester for performing

the estimations of phosphoethanolamine excretion, andto Dr. R. D. Rothfield for help and advice. I also wishto thank Dr. W. J. Burke, Sir Douglas Miller, and Mr.J. L. Dowling for permission to publish details of thecase.

REFERENCES

Beisel, W. R., Austen, K. F., Rosen, H., and Herndon, E. G. (1960).Amer. J. Med., 29, 369.Benjamin, N., and Austen, K. F. (1959). Blood, 14, 975.

Engfeldt, B., and Zetterstrom, R. (1954). J. Pediat., 45, 125.Fraser, D. (1957). Amer. J. Med., 22, 730.Harris, H., and Robson, E. B. (1959). Ann. hum. Genet., 23, 421.Keiding, N. R. (1959). Scand. J. clin. Lab. Invest., 11, 106.Korner, N. H. (1962). J. clin. Path., 15, 195.Leonard, B. J., Isracls, M. C. G., and Wilkinson, J. F. (1958). Lancet,

1, 289.McCance, R. A., Fairweather, D. V. I., Barrett, A. M., and Morrison,

A. B. (1956). Quart. J. Med., 25, 523.Rosenberg, I. N. (1959). J. clin. Invest., 38, 630.Sobel, E. H., Clark, L. C., Fox, R. P., and Robinow, M. (1953).

Pediatrics, 11, 309.

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