Urinary mannose in mannosidosis

686

Brief clinical and laboratory observations

complicating pregnancy, J. Obstet. Gynecol. Br. Emp. 52: 235, 1945. 20. Fox, L. P., and Stature, W. J.: Krukenberg tumor complicating pregnancy, Am. J. Obstet. Gynecol. 92: 702, 1965. 21. Spadoni, L. R., Lindberg, M. C., Mottet, N. K., and Herrmann, W. L.: Virilizafion

Urinary mannose in

mannosidosis Nils E Nord6n, M.D., Per-Arne Ockerman, M.D., Lund, Sweden, and Lajos Szab6, M.D., Szeged, Hungary

The Journal o~ Pediatrics April 1973

co-existing with Krukenberg tumor during pregnancy, Am. J. Obstet. Gynecol. 92: 981, 1965. 22. Coignet, J., Rampal, M., Bernard, P. J., Piana, L., Unal, D., and Leon, R.: Pseudohermaphrodisme congenital par tumeur virilisante maternelle, Mars. Med. 103: 638, 1966.

definite diagnosis in suspected cases. The clinical symptoms and signs alone are not sufficient for a diagnosis. A diagnostic method using easily available material such as urine seems to be desirable. An analytic procedure has been worked out in order to find out whether increased amounts of mannosecontaining compounds are excreted in the urine in mannosidosis. This technique has now contributed to the discovery of two new cases of mannosidosis.

MATERIAL AND METHODS T ~ E F I R S T case of mannosidosis, described in 1967, * was characterized by psychomotor retardation, slightly gargoyl-like facial features, abnormal bone structure, gibbus, hepatosplenomegaly, cloudy areas in capsule of the lens, storage cells in bone marrow, vacuolized lymphocytes, storage of mannose-rich oligosaccharides in the tissues including brain and liver, and a low activity of a-mannosidase. No definite cases of mannosidosis in man have been published since the original description. A disease in cattle, pseudolipidosis, is similar to h u m a n mannosidosis with deficiency of c~-mannosidase and storage of mannose-containing compoundsY The lack of new case reports in man may be related to the difficulty in establishing a

From the Department of Clinical Chemistry, University Hospital, Lund, and the Department of Pediatrics, University Hospital, Szeged. Supported by The Swed;sh Medical Research Council (Grant No. 13X-2222), The University of Lurid, F6rstamajblommans Riks/6rbund, and Sti[telsen Solstickan. Reprint address: Klin. Kern. Lab., Lasarettet, S-22185, Lund, Sweden.

Urine samples. Samples of urine from the two patients (Cases 2 and 3) were mailed from H u n g a r y to Sweden. One h u n d r e d milliliter aliquots of urine, collected over a 24 hour period, from five boys and five girls without any known chronic diseases were used as control specimens; the control children were 3 to 10 years of age. All urine samples were collected without restrictions on food intake. Assay of lysosomal enzymes. The techniques used for liver biopsy specimens have been described previously. 3, 4 Gel chromatographic distribution of urinary carbohydrates. The urine samples were concentrated and applied to a column of Sephadex G-25 fine (5 x 140 cm.). The column was calibrated with Blue Dextran 2000 and known sugars, viz. glucose (Ka,, = 0.70), maltose (K,~ = 0.60), raffinose ( K ~ = 0.55), stachyose (K,v = 0.48), and maltopentaose (K,,, = 0.41). T h e effluent from the column was assayed according to the anthrone method. Material eluted from the column with Kay values less than 0.60 was pooled into fractions which were hydrolyzed, and the monosaccharides were as-

Volume 82 Number 4

sayed by gas chromatography. T h e procedure is described in detail by Norddn and associates. 5

CASE R E P O R T S Case 2 (Case 1 was the original patient1). The patient, a 7-year-old girl with moderate mental retardation, was the product of a normal pregnancy and birth. The face had moderately dysmorphic features, slightly resembling those of gargoylism. Only very discrete skeletal changes were seen. Moderate superficial opacities were found in the cornea. Height and weight were normal, and there was no tendency toward infections. She had moderate hepatomegaly, 2 cm. below the costal margin, but no splenomegaly. Urinary excretion of glycosaminoglycans included: 21 mg. of uronic acid per liter, 50 per cent of which was chondroitin-4-sulfate and chondroitin-6-sulfate and 25 per cent each of dermatan sulfate and heparan sulfate (normal values used were up to 15 rag. of nronic acid per liter and only traces of dermatan sulfate and heparan sulfate). In lymphocytes and leukocytes cytoplasmic inclusions were seen which stained metachromatically (red-brown) with toluidine blue, and showed red-brown granules when stained with periodic acid-Schiff and atcian blue. Case 3. The 4-year-old sister of Case 2 also was the product of a normal pregnancy and birth. She had repeated severe infections during infancy; hepatosplenomegaly and moderate growth and mental retardation were also present. The facial features were moderately dysmorphic but without typical signs of gargoylism. There were only very discrete skeletal changes and moderate superficial opacities of the cornea. The peripheral blood smears and the urinary excretion of glyeosaminoglycans were of the same pattern as in her sister. Electron microscopy of a liver biopsy sample 6 showed small and large vacuoles, filled with fine granular and reticular material, but also opaque pellets, elongated and spiral-formed membranes, electron-dense inclusion bodies, vesicles, paracrystalline structures, and osmiophilic, partly lamellar zones.

RESULTS Lysosomal enzymes. One liver biopsy was performed in Case 3. Assay of lysosomal

Brief clinical and laboratory observations

68 7

Table I. Activity of acid hydrolases in a liver biopsy specimen from Case 3 ~"

Enzyme a-Mannosidase P-Glucosidase fl-Galactosidase N-acetyl-fi-glucosarninidase fi-Glucuronidase a-Fueosidase Acid phosphatase

Control subjects Mean (observed range) 5.2 (1.1-14.7)t 0.64 (0.20-0.95):~ 4.8 (1.7-11.7)t

Case 3 0.1 0.34 13.8 30.4 11.5 18.7 63.6

13.3 4.3 3.1 22.0

*All values given in International protein.

(7.6-29.9)t (2.2-9.2) t (1.8-5.1)w (11.5-40.1)-~

units per gram of

fFrom Lundqulst and Ockerman.~ ++From Hultberg and Ockerman. 4 Recalculated by a factor 5 to obtain International units per gram of protein, assuming a protein content of 20 per cent of the wet weight. w

results.

acid hydrolases (Table I) demonstrated the presence of increased activities of fl-galactosidase, N-acetyl-/?-glucosaminidase, p-glucuronidase, a-fucosidase, and acid phosphatase. /?-Glucosidase was within the normal range, and a-mannosidase had no measurable activity. U r i n a r y carbohydrate-contalning compounds. The gel chromatographic distribution was used to estimate the anthrone-reacting material excreted in the urine samples. T h e amount of anthrone-reacting material was considerably greater in the urine from the two patients than in that from the control subjects. This increase was most pronounced between K,v = 0 . 1 7 and 0.50. T h e results of the gas liquid chromatographic analyses of the gel chromatographic fractions from the first two samples of urine from Cases 2 and 3 and the ranges observed in urine from the ten control subjects are shown in Table I. The total excretion of mannose in the material having K,~ values less than 0.60 was approximately 25- to 100fold higher in the two patients as compared to the control subjects (Table I I ) . DISCUSSION T h e clinical findings in the two patients are similar to but not identical to those of

688

Brief clinical and laboratory observations

The Journal of Pediatrics April 1973

Table II. Urinary excretion of carbohydrate-containing compounds e

Sugar

Fr. I

Mannose Fucose Galactose Glucose Ribose Arabinose Xylose

65 60 180 35 . 3 20

Fr. 1 =

Case 2 [ Fr. 2 I Fr. 3 I250 96 100 75 . . 15 7

1350 61 50 70 . 15 5

[Total 2665 217 330 180 33 32

Case 3 Fr. 1 I Fr. 2 I Fr. 3 I Total 150 90 330 60 2 3 15

2200 170 150 170 -25 13

1450 30 55 100 -19 9

3800 290 535 330 2 47 37

Control subjects (N = 10) Total observed range 43- 99 79-203 78-306 81-152 0- 17 0-

9

34- 62

material excluded from the gel: Kay < 0.08.

Fr. 2 = medium-sized molecules; Kay = 0,08 to 0.95. Fr. 3 = small molecules: Kay = 0.25 - 0,60. Total = all material with K ~ < 0.60 including material excluded from the gel. mAll values given as micromoles of sugar per liter of urine.

the first case of mannosidosis. They are not specific enough for a definite diagnosis. T h e electron microscopic picture supports the existence of a lysosomaI storage disorder. The enzyme activities found in the liver biopsy specimen make mannosidosis a probable but not proved diagnosis. T h e gel chromatographic pattern of urinary anthrone-reacting compounds is quite abnormal in both patients. The sugar analyses reveal a high excretion of mannose, indicating a pathologic excretion of at least one urinary mannose-containing compound. Only material eluted earlier than maltose is ineluded in the assayed fractions. Although molecular weight estimates using the Sephadex are not always reliable, the pathologic substance or substances probably have a molecular weight higher than a disaccharide but lower than 5,000 (exclusion limit for Sephadex G-25). It is reasonable to believe that this pathologic urinary compound or compounds results from an altered catabolism of endogenous mannose-containing molecules, e.g., glycoproteins due to a decreased ~-mannosidase activity. SUMMARY

Two sisters, 7 and 4 years of age, with clinical symptoms and signs slightly resem-

bling gargoylism were studied. T h e activity of c~-mannosidase was extremely low in a liver biopsy specimen from one of the patients, whereas other acid hydrolases had abnormally high activities. I n both patients a markedly increased excretion of urinary mannose-containing compounds was found. The authors wish to thank Mrs. B. Boman and Mrs. B.-L. Oberg for skillful technical assistance. REFERENCES

I. Ockerman, P. A.: A generalized storage disorder resembling Hurler's syndrome, Lancet 2: 239, 1967. 2. Hocking, J. D., Jolly, R. D., and Batt, R. D.: Deficiency- of a-mannosidase in Angus cattle, Biochem. J. 128: 69, 1972. 3. Lundquist, A., and Ockerman, P. A.: Fineneedle aspiration biopsy of the liver in healthy adults. Activity of acid hydrolases, Enzymol. Biol. Glin. 10: 300, 1969. 4. Hultberg, B., and Ockerman, P, A . : fi-Glucosidase activities in human tissues. Findings in Gaucher's disease, Clin. Chim. Acta 28: 169, 1970. 5. Nord6n, N. E., Eriksson, O., Hultberg, B., and Ockerman, P. A.: The gel chromatographic distribution of urinary carbohydrate compounds, Clin. Chim. Acta. In press. 6. Ormos, J.~ Monus, Z., Szab6, L., and Csapo, Z.: Die Bedeutung der elektronenmikroskopischen Untersuchung der Leberbiopsle bel der Beurteilung der verschiedenen Mucopolysaccharidosen, Verb. Dtsch. Ges. Pathol. 55: 1971.