The measurement of acid mucopolysaccharides (glycosaminoglycans) in amniotic fluid and urine

Clinica Chimica Acta, 45 (1973) 73-83 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

73

CC*5357

THE

MEASUREMENT

OF ACID

(GLYCOSAMINOGLYCANS)

DENISE

M. DUNCAN,

R. W.

MUCOPOLYSACCHARIDES

IN AMNIOTIC

LOGAN,

FLUID

AND

M. A. FERGUSON-SMITH

URINE

AND

FRANCES

HALL

Departments ofBiochemistry and Medical Genetics, Royal Hospital for Sick Children and Queen Mother’s Hospital, Glasgow, G3 8.5 J (U.K.) (Received

August

II,

1972)

SUMMARY

The purpose of the investigation was to find a routine laboratory method potentially suitable for the prenatal detection of the mucopolysaccharidoses. Samples of amniotic fluid from therapeutic abortions were treated with pronase, dialysed and the glycosaminoglycans precipitated by cetyl pyridinium chloride (CPC). The precipitates were subjected to quantitative determinations of hexuronic acid and neutral sugar, and the glycosaminoglycans were separated qualitatively by electrophoresis on cellulose acetate and differential staining in alcian blue-magnesium chloride mixtures. The same techniques were applied to routine urine samples. Evidence is presented to support the contention that these combined investigations offer a high probability of detecting abnormalities in glycosaminoglycan metabolism.

INTRODUCTION

The mucopolysaccharidoses

form a group of similar disorders which are known

to be genetically distinct. With the exception of Hunter’s syndrome, which is Xlinked, they are transmitted by an autosomal recessive mode of inheritancel. All the conditions involve a metabolic defect of the acid mucopolysaccharides (glycosaminoglycans), thought to result from an abnormality in their clegradation2. The diagnosis of a mucopolysaccharidosis (MPS) is confirmed by the presence of an increased excretion of urinary glycosaminoglycans (GAG). Qualitative techniques can identify the various GAG present in excess, and this may assist in the diagnosis of a particular syndrome. As these conditions are genetically determined, it would be advantageous to have a routine laboratory method for antenatal diagnosis - thus ensuring that carrier parents of a particular MPS syndrome could have a normal family with respect to that syndrome. This would be accomplished either by therapeutic abortion of an affected foetus or possibly in the future by early treatment of an affected child. The latter possibility has been shown by the effect of plasma3 or leukocyte4 infusions giving long term clinical improvement5 in some mucopolysaccharidoses, most prob-

74

DUNCAN

et d.

ably attributable to the enzyme “replacement” therapy causing induced degradation of glycosaminoglycansa. This paper presents a scheme which is used routinely in this laboratory for the qualitative and quantitative estimations of GAG in samples of amniotic fluid and urine. METHODSANDMATERIALS

Early amniotic fluid samples were obtained from therapeutic abortions where no foetal abnormality was considered to exist and from cases suspected of suffering from a mucopolysaccharidosis. Later fluids were derived from routine cases of rhesus incompatibility, together with a few normal samples obtained for assessment of lecithin:sphingomyelin ratios in cases with previous bad obstetric histories. Urine samples were acquired from patients in whom the diagnosis of mucopolysaccharidosis was suspected, patients with cryptogenic mental retardation, normal surgical cases, neonates and normal adults. Thymol was used as a preservative in urine samples. Amniotic fluid samples are spun at IOOO rpm for 5 min to remove any cells and debris. This spun fluid can then be stored at -20'. A zo-ml aliquot of urine or a 5-IO-ml aliquot of amniotic fluid is heated for about 5 min in a boiling water bath to denature the protein material. The sample is allowed to cool and then treated with 4-5 mg pronase (Grade B Calbiochem)‘, dissolved in I ml 0.2 M Tris-HCl buffer, pH 8.0. The sample is then incubated in a 55” water bath for 24 h. Urine samples are removed after the 24 h, but amniotic fluid samples are subjected to an additional 24 h of pronase digestion with a further 4-5 mg pronase. The digested sample is then dialysed in Visking tubing8 (diameter, 2.5 cm). Dialysis is carried out at 4’ in three stages : (I) all day against distilled water; (z) overnight against 0.02 M Tris-HCl buffer, pH 8.0; (3) for 6 h against distilled water. The dialysed digest is then divided into two aliquots - one third of the total volume being used for qualitative tests and the remainder for quantitative estimations. One ml of 5% w/v cetyl pyridinium chloride (CPC) in 0.05 M sodium chloride is added dropwise with gentle agitation per IO ml of dialysed digest. The solution is allowed to stand for I h in a 37” water bath, then overnight at room temperature, and finally spun at 3000 rpm for 45 min. The supematant contains peptides and glycopeptides, but may also include keratan sulphate much of which redissolves in the presence of excess CPC9. If suspected of being present, the keratan sulphate may be extracted by running the supernatant through an ECTEOLA co1umn7. The CPC-GAG precipitate is washed with 10% w/v potassium acetate in ethanol at 4O and the precipitate dispersed in the ethanolic solution and allowed to stand for at least I h at 4O. The potassium-GAG precipitate is obtained by centrifugation at 3000 rpm for 45 min at 4” and then allowed to drain completely. When no excess ethanolic solution remains, the precipitate is dissolved in saturated benzoic acid. The precipitate for qualitative tests is normally dissolved in IOO ~1 benzoic acid and that for quantitative tests in 2.5 ml benzoic acid. ECTEOLA column. The ECTEOLA is prepared by washing I g of resin (Whatman ETII, capacity 0.5 mequiv/g) for a half hour in IOO ml of each of the following solutions: 0.5 M NaCl, 0.5 N NaOH, 3 M NaCI, 0.1 M NaCl: 0.1 N HCl (50:50, v/v) and finally in absolute ethanol (50 ml) lo. The washing solutions are removed by

GLYCOSAMINOGLYCANS

IN AMNIOTIC

FLUID

AND

URINE

75

suction-filtration between each successive wash. The resin is suspended in distilled water to form a thick sludge which is then degassed in vacua. A column of length 2 cm (internal diameter I cm) is sufficient for a preparation containing 200 pg hexuranic acid. A trial run using standard GAG is carried out to test the capacity of the resin. The supernatant from the dialysed, pronased sample is applied to the column, washed in with 5 ml distilled water, and the column is eluted with 2 x 5 ml 0.01 N HCl and 2 x 3 ml 2 M NaCl. The eluates are precipitated with ethanolic potassium acetate as described above, and carbazole and anthrone reactions carried out on the redissolved precipitate. Most of the keratan sulphate is eluted with 2 M NaCl but some may be present in the 0.01 N HCl eluate. Hexuronic acid. Hexuronic acid levels are measured by the carbazole method of Dischell as modified by Bitter and Muir12and Blackham and Raine13. In the case of urine samples, the ratio of hexuronic acid per gram creatinine is also calculated, thus allowing samples other than 24 h collections to be used for estimations14116. Neutral sugar. Neutral sugars are estimated by a modified method of Roe13p16. Optical densities of the solutions are, however, read at 625 nm. Optical densities are corrected as if equal amounts of material were used in the carbazole and anthrone methods and these are expressed in the form of a ratio. This result is used for comparison with those previously derived using solutions of various types of standard GAG. T&dine blue and alcian blue tests. Spot tests were performed on all urine samples prior to pronase digestion, to indicate the probable presence of a mucopolysaccharidosis. Quantitative and qualitative tests were carried out on all urines, whether the spot tests were positive or not. The spot tests consist of staining in alcian blue and toluidine blue. To stain in toluidine bluel’, a volume of urine containing 25 pg of creatinine is spotted, on Whatman No. I filter paper, in IO ,ul applications. To stain in alcian bluela, a volume of urine containing 5 ,ug of creatinine is spotted in 5 ~1 applications on Whatman No. I filter paper. Normal urines do not give a stain with either of the tests. Differential staining in alcian blue-M&l, solutions. Following the estimation of hexuronic acid content in the sample, differential staining in alcian blueI* is carried out using the concentrated solution of precipitate obtained from both amniotic fluid and urine specimens. Five yl applications of the solution containing IO pg GAG in total are spotted and dried as in the spot tests. Concentrated solutions of GAG standards were also differentially stained. Electrophoresis of GAG. For further evidence as to the types of GAG present in the sample, electrophoresis is performed on cellulose acetate membranes. The sample applied should contain at least 4 ,ug GAG to give adequate staining in alcian blue. A Shandon Celegram membrane is floated on the buffer in a Shandon electrophoresis tank until saturated and then blotted lightly to remove any excess buffer. The membrane is placed in position in the tank and Whatman No. 3 filter paper wicks saturated with buffer are placed on either edge of the membrane. The samples are applied at the cathode using a Hamilton syringe while the membrane is still moist. The buffer which gave the best separation, dependent on the structure of the polysaccharide backbone, was found to be 0.1 M barium acetate, pH 7.0~~.In this buffer the electrophoresis was run for 2 h at 12.5 V per cm (approx. 15 mA). To show the degree of sulphation of the GAG involved, electrophoresis may

DUNCAN et al.

76

also be run using 0.1 N HCI, pH 4.0 for 1.5 h at 4 V per cm and 15 mA*. Both of these buffers can be used only once. Standard GAG samples are run with each test sample. After the run is complete, the membrane is blotted dry and stained for 5 min in 0.5% alcian blue in a methanol-acetic acid-water solution of 5 :I :4, v/v/v. The membrane is then given three washes in methanol-acetic acid-water, 50 : 5 : 45, v/v/v, until most of the background colour has been removed. It is given a final wash in 95% ethanol for 30 set and finally cleared in a solution of cyclohexanoneeg5°/o ethanol, 30:70, v/v, for 30 set, before mounting on a clean glass plate. The plate is drained and air dried. When completely dry, the strip is removed from the plate and mounted on a white cardlO. RESULTS

The method described (Method A) was compared with the method of O’Brien cetyltrimethylammonium bromide (CTAB) as a precipitating agent after overnight dialysis in Visking tubing against running tap water (Method B). CPC and CTAB were used as precipitating agents in both methods and CPC was found to give a better recovery of GAG 14. Method B was found to give a higher estimate of hexuronic acid content than Method A, but this was found to be due to non-specific material which could be eliminated by digestion with pronase. Treatment by pronase for a 24-h period was found to be sufficient for urine samples, but a further 24-h treatment was found to be necessary for amniotic fluid samples. When GAG standards were added to urines and then subjected to either Method A or B, recovery was found to be 66% from Method A and 50% from Method B. A certain amount of material was found to be lost during dialysisZ1 but most of the loss occurred at the stage of precipitation. In all the results shown no allowance for such losses has been made. Pronase treatment prior to Method B did not improve the recovery. Amicon ultrafiltration was tried as an alternative to Visking tubing dialysis, but the recovery was only 40%. In Table I are shown the results for the ratio of optical densities produced when equal amounts of GAG standard were subjected to the carbazole and anthrone techniques. In Table II are shown the comparable ratios obtained from 80 samples of amniotic fluid and 67 specimens of urine. In the differential staining it was found that at least IO ,ug of total GAG et a1.20, using

TABLE RATIO

I

OFOPTICALDENSITIES(CARBAZOLE:ANTHRONE)

STANDARDS

Standard Keratan suluhate Chondroitin-sulphate Chondroitin s&hate Heparitin sulphkte Hyaluronic acid Galactose Glucuronolactone

Ratio A B

Cl.?:1 B-:1 8 :I II

:I

17 :I 0.4: I 17 :I

OBTAINED

USINGEQUALAMOUNTS

OF

GAG

GLY~OSAMI~OGLYCA~S

TABLE

IN AMNIOTIC

FLUID AND URINE

77

II

RATIO

OF

URINE

SAMPLES

OPTICAL

DENSITIES

(CARBAZOLE:ANTHR~NE)

OBTAINED

FROM

AMNIOTIC

Number examined

Diagnosis

Carbazole:anthrone _.._ Range Mean

g-14 weeks weeks rg---40 weeks

39

2.1

14

Normal amniotic fluid Normal amniotic fluid Normal amniotic fluid

Neonate
9 7 35 8

Normal Normal Normal Normal

2.6 1.3 1.9

0.5-4-9 0.4-4.2 0.5-2.8 0.7-3.0

r-13 years 5- 7 years

4 3

I year

I

Hurler Sanfilippo Morquio

6.0 3.1 1.3

5.0-8.0 2.7-3.3 -

I 5-x 8

27

_--

TABLE

STAINING

IN

Present results. b. Scott

ALCIAN

I.2

BLUE/&i&&

& Dorlir@.

b

0.80 0.35 0.60 0.60 0.35 0.60 0.20

0.60 0.10

0.20

0.25

0.80 0.50

1.00

0.5a

0.30 0.60 0.10 0.20

OF HEXURONIC

ACID

IN

SAMPLES

OF AMNIOTIC

FLUID

AND

URINE

URINE

Number examined

Diagnosis

9 7 35 8

Normal


4 3 1

Neonate

(b)

DECOLOURISATION

IV

EXCRETIOK (8)

SOLUTIONS

c. Bake?.

a

Keratan sulphate Chondroitin sulphate A Chondroitin sulphate B Chondroitin sulphate C TIeparitin sulphate Heparin Hyaluronic acid DNA

TABLE

0.3-6.6 0.8-4.4 0.4-3.9

2.5 1.2

III

DIFFERENTIAL a.

ratio

AMNIOTIC

Age

FLUID

Number

Normal Normal Normal

__.-

Hurler Sanfilippo Morquio

mg hexuronic

acid 1s creatinine

Mean + S.D.

Range

47 zt 13 34i 11 15f 5 2 5%

31- 70 19- 45 g- 26 28

27 19

78-140 63- 93

102

*

87 f 22

--_-

Diagnosis

mg hexuronic acid/loo ml fluid Mean f S.D. Range 0.66 f 1.70 log transformation 1.33 i 0.50 1.00 & 0.35

examined

g-14 weeks

39

Normal

15-r 8 weeks 19-40 weeks -.-

27 =4

Normal Normal

0.08-3.20 0.25-2.60 0.52-X.39

PATTERN

FLUID

AND

material were required to maintain visualisation as the progressive decolourisation of the GAG occurred. Pronase treatment prior to dialysis and precipitation was also found to be necessary or staining occurred only up to 0.2 &I M&l,. The results obtained during the present study differ slightly from those of Scott and Dorling18, and Bakerz2 as shown in Table III. Figs. I and 2 show the results obtained with GAG standards using cellulose acetate electrophoresis with 0.x M barium acetate and 0.1 N HCI, respectively. As

Fig. I. GAG standards-electrophoresis using 0.1 M barium acetate buffer, pH 7.0. C.S.A., chondroitin sulphate A; C.S.C., chondroitin sulphate C; KS., keratan sulphate; C.S.B., chondroitin sulphate B; H.A., hyaluronic acid; KS., heparitin sulphate.

Fig. 2. GAG s~ndards~lectrophoresis

using 0.1 N HCl. Abbreviations:

see legend to Fig. I.

GLYCOS~~INOGLYC~~NS IN AXNIOTIC FLUID AND URINE

79

can be seen, the only material obtained commercially (heparin) proved to be impure. In Figs. 3 and 4 are shown the findings using similar procedures with samples of amniotic fluid, urine and mixed standards applied. Using the electrophoretic separation obtained, an estimate was made of the relative percentage contributed by the various types of GAG in amniotic fluid. These findings are shown in Fig. 5.

Fig. 3. Amniotic fluids, urines and mixed GAG standards-electrophoresis acetate buffer, pH 7.0.

4. AmmotIc

flu1~1s,urmes and mixed crxcr standards-electrophoresis

using 0.1 M barium

using 0.1 N HCl.

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rx

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XI-.

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SAMPLE

+ * *bzgh)gg

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COMPOSITION

GLYCOSA~INOGLYCA~S

IN AiXNIOTIC FLUID AND URINE

81

Fig. 6 illustrates the hexuronic acid concentration in the amniotic fluid and urine samples. In particular it shows the peak of concentration in amniotic fluid which occurs just prior to 20 weeks of gestation, and illustrates the general trend through pregnancy and following birth. Table IV depicts the hexuronic acid content of the various specimens but presents them in a different fashion, in that the data has been arranged in suitable age groupings and the ratio of hexuronic acid to creatinine has been employed for urine specimens. The results for all groups except those involving fluids of 9 to 14 weeks showed normal dist~butions. The latter showed a near normal distribution following logarithmic transformation, a fact which could be explained by two comparatively high values obtained. DISCUSSION

From the results obtained with the standards it was thought that the carbazole:anthrone ratio obtained for specimens would offer a valuable diagnostic aid. Unfortunately as can be seen from the results, neither amniotic fluid nor urine showed any tendency to follow the pattern exhibited by the standards. This could be explained by the effect of pronase on the polysaccharide-protein link. The carbohydrate of the polysaccharide is linked to serine through a galactosylgalactosylxylose groupZ3. After proteolytic treatment this linkage group almost certainly remains attached to the polysaccharide a4. The linkage is known to occur in the chondroitin sulphates, heparin and heparitin sulphate. In keratan sulphate and hyaluronic acid the nature of the linkage has not been definitely ascertained. The inclusion of the galactose in the GAG isolated by our method would reduce the carbazole: anthrone ratio, whereas the standard GAG is reputed to be galactose-free. In addition, shorter polysaccharide chains and, therefore, the presence of extra linkage groups may also contribute to a reduction in the expected ratio. In amniotic fluids of later gestational age where hyaluronic acid is not predominant, the staining in the presence of 0.2 M MgCl, is as strong as in the earlier fluids where hyaluronic acid is predominant, suggesting that DNA, which also stains to 0.2 M MgCl, could possibly be present in relatively greater amounts in the later fluids where the hyaluronic acid has decreased. From the differential staining technique it was shown that keratan sulphate is a constituent in many amniotic fluids. This does not show by electrophoretic separation, as keratan sulphate and the chondroitin sulphates do not give discrete bands on cellulose acetate. The remaining GAG separate on cellulose acetate electrophoresis in either 0.1 N HCl or 0.1 M barium acetate. In fluids of less than 30 weeks gestation, hyaluronic acid constitutes more than 40% of the total GAG, thereafter it falls sharply to about 10% and only appears in trace amounts in postnatal urines. The slow moving bands, the upper of which (slow band 2) has a mobility similar to heparitin sulphate, increase in amount throughout pregnancy and are seen to be present in neonatal, child and adult urines. The lower of the two slow bands (band I) is most marked in fluids of later gestation. The chondroitin sulphates do not appear to vary very much in relation to total GAG between birth and adulthood. Unlike the results of Danes et dz6, where the total hexuronic acid per IOO ml

82

DUNCAN

et

ai.

fluid was seen to fall throughout pregnancy, we find that the hexuronic acid content rises until about 17 weeks' gestation and then falls. Any result for amniotic fluid would, therefore, have to be related to the relevant age group before presence of excess GAG could be established. It would appear that 14 weeks is a suitable time for amniocentesis to be performed, offering the possibility of repeating this at 17 weeks if required. Where localisation of the placenta by ultrasonic means is available, the risk to both mother and foetus is minimised. As seen from Table IV the normal range of hexuronic acid concentration in amniotic fluids is 0.08-3.20 mg/roo ml. This is comparable to the range reported by Matalon et ccLz6 of 0.6-3.5 mg/roo ml and by Danes et al.a5 of 0.9-3.3 mg/roo ml. Unlike Matalon et al. we find that a substance with mobility similar to heparitin sulphate in barium acetate electrophoresis is present in ail normal amniotic fluids. The presence of heparitin sulphate alone does not, therefore, constitute an abnormaiity. In amniotic fluids we feel that a particular GAG must be present in excess before the foetus can be said to be abnormal. Thus, some quantitative estimate of the individual components is indispensable before diagnosing an affected foetus. Matalon et aLz7 have indicated the possible unreliability of chemical analysis of amniotic fluids taken prior to 16 weeks’ gestation. For this reason they favour examination of cultured cells. Whilst we agree that the ultimate diagnosis depends on the demonstration of the specific enzyme defect involved, we still consider that examination of the fluid in suspect cases before and after 16 weeks’ gestation offers a more practical means of diagnosis. Two amniotic fluids have been received from patients reputed to have had previous children affected with a mucopolysaccharidosis. Both these fluids yielded rest&s which were qualitatively and quantitativeIy normal for their respective gestational ages. One patient delivered a child with a normal GAG pattern although the child was mentally retarded from an unassociated cause. Subsequent tests on urine from the original sibling also showed a normal pattern both qualitatively and quantitatively, although this child had been thought to be suffering from Sanfilippo’s syndrome. She is now believed to have a mucolipidosisz8 where increased urinary GAG is not a constant feature-z. The other fluid is from the fifth pregnancy of a woman with one normal child and two children affected with Hurler’s syndrome. This patient has not yet delivered. Of the patients examined for urinary mucopolysac~harides, nine urines showed positive spot tests and an elevated hexuronic acid content when expressed as a ratio of hexuronic acid:creatinine for the relative age group. In five of these cases the GAG present in excess was shown to be chondroitin sulphate B and heparitin sulphate by electrophoresis and this gave confirmation of a diagnosis of Hurler’s syndrome. i)ne patient was presented as Morquio’s syndrome and this was confirmed by a slightly elevated hexuronic acid content and by the isolation from an ECTEOLA column of a considerable amount of keratan sulphate. The latter was also shown to be present by differential staining (decolourised by 0.8 M MgCI,). Only one third of keratan sulphate present in the sample was precipitated by CPC and the remaining keratan sulphate was recovered by running the supernatant through the ECTEOLA column. The three other patients with a high hexuronic acid:creatinine ratio showed an excess of heparitin sulphate which suggested the diagnosis of Sanfilippo’s syndrome. One patient with normal quantitative results showed an electrophoretic pattern

GLYCOSAMINOGLYCANS

IN AMNIOTIC

FLUID

AND

URINE

83

intermediate between normal and Hurler’s syndrome, where chondroitin sulphate B was present in a similar amount to chondroitin sulphate A. Together with the clinical picture, these results suggest a forme fruste of Hurler’s syndrome or perhaps a double heterozygote for Hurler’s and Scheie’s syndromes. This implies that we can expect to find qualitative abnormalities with quantitatively normal total hexuronic acid content. Both the quantitative and qualitative results obtained in this study for normal urines are comparable with those reported by several authors1472Q--34. ACKNOWLEDGEMENTS

We should like to express our appreciation to Professor A. Dorfman and Dr. J. A. Cifonelli for the gift of pure GAG standards, and the obstetricians, paediatricians and members of the nursing staff without whose cooperation the study could not have been undertaken. Thanks are also due to Dr. P. Johansen and Mrs. C. Wallace for assistance during the early stages of this work. REFERENCES I V. MCK~SICK, Heritable Disorders of Connective Tissue, C. V. Mosby Co., St. Louis, 1966, p. 325. 2 E. F. NEUFELD AND J. FRATANTONI, Science, 169 (1970) 141. 3 N. DI FERRANTE, B. NICHOLS, P. DONNELLY, G. NERI, R. HRGOVCIC AND R. BERLUND, PYOC. N&l. Acad. %., U.S., 68 (1971) 303. 4 A. G. KNUDSON, N. Dr FERRA~TEAND J. E. CURTIS, Proc. Nat. Acad. Sci. U.S., 68 (197r) 1738. 5 E. F. NEUFELD. Hospital Practice, (Feb. 1972) 107. 6 E. F. NEUFELD AND M. J. CANTZ, Ann. N. Y. Acad. Sk., 179 (1971) 580. 7 E. WESSLER, C&z. C&m. Acta, 16 (1967) 235. 8 E. WESSLER, Acta Univ. Ups. Abstr. Up+. Diss. Med., 85 (1970). 9 C. A. ANTANOPOULOS, E. BORELIUS, S. GARDELL, B. HAMNSTR~M AND J. E. SCOTT, B&him. Biophys. Acta, 54 (1961) 213. IO P. JOHAXSEN, Personal communication. II J. DISCIIE, J. Biol. C&m., 167 (1947) 189. 12 T. BITTER AND H. M. MUIR, Anal. Biochem., 4 (1962) 330. 13 G. A. BLACKHAM AND D. N. RAINE, Ann. CEin. &o&em., 6 (1969) 49. 14 L. ROSENFELD, CL&. Chim. Acta, 31 (1971) 263. zg J. S. MAYES AND R. G. HANSEN, Pvoc. Sac. Exp. Biol. Med., 122 (1966) 927. 16 J. H. ROE, J. Biol. Ckem., 2iz (1955) 335. 17 S. MEITES AND W. R. FAULKNER, ManuaE of Practical Micro and GeneraE Procedures in Clinical Chemistry, Charles C. Thomas, Springfield, Ill., 1962, p. 225. 18 J. E. SCOTT AND J. DORLING, Histochenzie, 5 (1965) 221. 19 E. WESSLER, Anal. Biochem.. 26 (1968) 479. 20 D. O’BRIEN, F. A. IRBOTT AND D. 0. RODGERSON, Laboratory Manual of Pediatric MicroBiochemical Techniques. HarDper & Row, New York, 4th ed., 1968, D. I 211. ZI N. DI FERRANTE, Anal. B&em., ZI (1967) 98. 22 J. R. BAKER, S.S.E.I.M. Symposium 3, E. S. Livingstone, Edinburgh, 1968, p. 143. 23 A. DORFXAN, Med. Prisma, 3 (rq7x). 24 N. TASIGUCHI, C&n. Ckim. Acta, 37 (1972) 225. 25 B. S. DANES, J. T. QUEENAN, E. Ga~ow AND L. L. CEDERQUIST, Lam&, i (1970) 947. 26 R. IMATALON, A. DORFMAN, C. B. JACOBSON AND H. L. NADLER, Lance& i (1970) 83. 27 R. MATALON, A. D~RFMAN AND H. L. NADLER, Lancet. i (1972) 798. 28 J. W. SPRANGER AND H.-R. WIEDEMAN, NeuropZdiatrie, ; i;9jd)-3. 29 J. W. SPRANGER, H. TODT AIVD H.-R. WIEDEMAN, Clin. Chim. Acta, 17 (1967) 142, 30 W. M. TELLER, E. C. BURKE AND J. W. ROSEVEAR, 13. F. MCKENZIE, J. Lab. Clin. filed., 59 (1962) 95. 71 C. RICH, N. DI FERRANTIS AND R. ARCHIBALD, 1. Lab. C&z. Med.. 50 (1957) 686. 32 D. KAPLAN, V. MCICUSICIC, S. TREBACH AND 6. LAZARUS, J. Lab.-Cl&:-Ged., 71 (1968) 48. 33 G. MANLEY, M. SEVERN AND J. HAWKSWORTH, J. Clin. Pa&l., 21 (1~68) 3x9. 34 D. VARADI, J. CIFO~UELLI AND A. DORFMAN, Biochinz. Riophys. Acta, 141 (1967) 103.