Liver glycogen phosphorylase deficiency - Europe PMC

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Liver glycogen phosphorylase deficiency. G. 1. Drummond, ph.d., D. F. Hardwick, m.d. and S. Israels, m.d., Vancouver, B.C.. Deficiencies of enzymes involved in.
Liver glycogen phosphorylase G. 1. Drummond, ph.d., D. F. Hardwick,

m.d. and

S.

deficiency

Israels, m.d., Vancouver, B.C.

Deficiencies of enzymes involved in glucagon test, when the fasting blood the catabolism of glycogen such as sugar was 43 mg. per 100 ml., was per¬ glucose-6-phosphatase, phosphory- formed (Fig. 2) and this produced an flat response. The pH of the lase,amylo-1,6-glucosidase(debranchequally have and acid maltase blood was 7.46 with a bicarbonate ing enzyme) attracted considerable clinical atten¬ content of 22 mEq./l. Serum choles¬ tion in recent years.1 Although these terol was 177 mg. and the uric acid diseases are characterized by abnor¬ 3.7 mg. per 100 ml. The urine was mal glycogen deposition in the affected negative for reducing substances and tissues, diagnosis of a particular gly- there was no ketonuria. In view ofthe cogenosis has usually been made with hepatomegaly and the poor response certainty only by direct assay of bi¬ to glucagon and in the absence of any opsy specimens for the enzymes in¬ growth disturbance, it seemed prob¬ volved. More recently, Hulsmann, able that this was a form of glycogen Oei and Van Creveld2 and Williams storage disease. A liver biposy was and Field3 have shown that a defi¬ obtained, frozen immediately in dry ciency of liver phosphorylase is re¬ ice and used for the studies described flected in the phosphorylase activity below. of leukocytes. This observation has raised the suggestion that leukocyte Histopathological examination phosphorylase activity determinations The hepatic pathology seen in hepatic may be of diagnostic value in liver phosphorylase deficiency is similar to phosphorylase deficiency. Van Cre¬ that observed in glucose-6-phosphatase veld and Huijing4 have extended this deficiency and amylo-1,6-glucosidase observation and shown that determi¬ deficiency.5 All three conditions are nation of amylo-1,6-glucosidase activ¬ characterized by the coexistence of ity in leukocytes can be diagnostic substantial lipid with excess stored also. This report concerns a case glycogen in hepatocytes, together with diagnosed as liver phosphorylase de¬ a fine fibrosis in the periportal zones. following assay of leukocyte Sections of the biopsy specimen em¬ ficiency bedded in paraffin showed ballooned and liver enzymes. hepatocytes which contained glycogen Clinical observations on periodic acid-Schiff staining (Fig. G.S., aged 17 months, was admitted 3A). Lipid, present in small (0.2 to to the Health Center for Children, 1.0;u)globules diffusely spread through Vancouver, because of an enlarged ab¬ the hepatocytes, especially those in domen. She had had no significant the centrilobular region, was demonillnesses. Her father and mother were not consanguineous, were in their mid-twenties and had had no serious illnesses. One sibling, aged 4 years, was in excellent health. On examina¬ tion her weight was 9.6 kg. and her height was 47 cm., both within the normal range. The only positive find¬ ing was a enlarged abdomen (Fig. 1) resulting from hepatomegaly, for the liver could be palpated 11 cm. below the right costal margin. lt was firm but not tender or nodular. The spleen was not palpable. Fasting blood sugar

(modified Somogyi-Nelson technique) was 59 mg./lOO ml.; following the in¬ jection of 1 mg. glucagon it rose to 72 mg. per 100 ml. in 60 minutes (Fig. 2) and then returned during the next hour to the

fasting level.

A second

FIG. 1.G. S. showing enlarged pable area of liver.

740 C.M.A. JOURNAL/APRIL 11, 1970/VOL. 102

abdomen and

pal¬

GLUCAGON TOLERANCE

0

15

FIG. 2.Blood

30

45 MINUTES

glucose response

60 to

90

120

glucagon.

strated by Oil Red 0 staining of frozen liver (Fig. 3B.) A fine periportal fibro¬ sis was noted. These findings, though not differential, are indicative of hepatic glycogen storage disease.

Liver glycogen The amount of glycogen was deter¬ mined by the enzymatic method of Johnson, Nash and Fusaro.6 By this specific method, the glycogen content of the biopsy sample was determined to be 15.5% (wet weight), a value far above the normal (3-5% wet weight) and in the range characteristic of de¬ ficiencies of glucose-6-phosphatase, and phos¬ amylo-l,6-glucosidase, phorylase.1 In the case of amylo-1,

6-glucosidase (debranching enzyme) deficiency, glycogen structure is known to be abnormal, with short outer chains of a-l,4-glucose residues,and similar in structure to phosphorylase limit dextrin. In phosphorylase and glucose6-phosphatase deficiencies, glycogen, although elevated in quantity, is nor¬ mal in structure. Some information regarding the structure of such poly¬ saccharides can be obtained by their colour formation in the presence of iodine.8,9 The iodine-absorption spec¬ trum of glycogen isolated from the biopsy sample when performed by the method of lllingworth and Cori8 gave a maximum absorption at460 m/x, G. 1. Drummond, ph.d., Professor of Pharmacology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia. D. F. Hardwfck, m.d., Associate Professor of Path¬ ology, Faculty of Medicine, University of British

Columbia. S. Israels, m.d., Professor and Head, Department of Pediatrics, Faculty of Medicine, University of British Columbia. Reprint requests to: Dr. George 1. Drummond, De¬ partment of Pharmacology, University of British Columbia, Vancouver, British Columbia.

G. for 10 minutes). The results are shown in Fig. 4. The velocity of the Glycogen catabolizing reaction as determined by the increase enzyme profile in absorption at 340 m/x was complete¬ Patient Normal] G.S. Levels ly linear over the experimental period Enzyme to 40 minutes in the case of the (up Gkicose-6-phosphatase 3.75 3.5-5 re¬ Q1M. Pi/g./min.) biopsy), thus constituting a highly Acid maltase liable determination. From the reac¬ 0.525 0.5-0.7 (JJLM. glucose/g./min.) tion velocities it was calculated that Amylo-1,6-glucosidase 0.74 rat liver produced glucose-1-phos¬ 0.7-1.0 (/XM. glucose/g./min.) phate at the rate of 5.18 /xM./g./min., Phosphorylase kinase 38.6 38-49 (units/g.) while the biopsy sample was only 9% Phosphorylase of this (0.462 /xM. glucose-l-P04/g./ 2.86 25 (JIM. Pi/g./min.) min.). The relative activity of the 31.1 Rat liver phosphorylase this biopsy material and rat liverwellin with The assays are described in the text. thus rcmarkably assay agreed Normal levels except for phosphorylase the assay used in Table I, and served kinase are those given by Hers/1 Normal levels for phosphorylase kinase are from to confirm the disease as hepatic this laboratory and represent the range

TABLE I

FIG. 3 Histology ofliver biopsy. A. Note the dark, large PAS-positive glycogen gran¬ ules in the ballooned hepatocytes. (PAS stain, x 640.) B. Note the deposits oflipid in different-sized globules in the hepatocytes ofthe centrilobular zone. (Oil Red 0, Frozen section, 40.)

identical to that of rabbit-liver gly¬ cogen. Under identical conditions, phosphorylase limit dextrin (charac¬ teristic of amylo- 1,6-glucosidase defi¬ gave a maximum at 430 m/x. ciency) This evidence pointed to a normal glycogen structure in the biopsy ma¬ terial and because of the mildness of the clinical symptoms indicated hepatic phosphorylase deficiency rather than

glucose-6-phosphatase deficiency.

Liver enzyme determinations A sample of the biopsy material

was

homogenized in 10 volumes of 20 mM Tris-HCl, 2 mM EDTA(edetateethy¬ lene diaminetetraacetic acid) pH 7.5 in a Potter-Elvehjem homogenizer. Glucose-6-phosphatase, acid maltase, amylo-1,6-glucosidase, and phos¬ phorylase were assayed by the methods described by Hers.1 Phosphorylase kinase was assayed at pH 8.2 by the method of Drummond, Duncan and Hertzman,10 using a 20-minute kinase incubation period and employing rab¬ bit skeletal muscle phosphorylase-/?

the substrate. The results are shown in Table I. All activities were in the normal range except that of phos¬ phorylase, which was about 11 % of normal. In these studies a sample of fresh rat liver was simultaneously as¬ sayed for each enzyme activity, and its phosphorylase activity is also given in Table I. The data strongly indicated

from six

biopsy samples.

phosphate. A more specific and sensi¬ tive spectrophotometric assay involves determination of activity in the physio¬ logical direction of glycogen degrada¬ tion. In this method phosphorylase activity is determined by measuring the rate of triphosphopyridine nucleotide (TPN+) reduction in the presence of excess glycogen, inorganic phos¬ phate, phosphoglucomutase and

glucose-6-phosphate dehydrogenase. The rate of TPN+ reduction is limited by the amount of phosphorylase activ¬ ity added (centrifuged extract). The assay was conducted essentially as de¬ scribed by Hiilsmann, Oei and Van Creveld2 and the biopsy extract was again compared with the rat liver ex¬ tract (in this case centrifuged at 5000

as

phosphorylase deficiency (Type VI glycogenosis) and filled all the criteria defined by Hers1 for this disease, name¬ ly normal glucose-6-phosphatase and amylo-1,6-glucosidase activities, high liver glycogen and phosphorylase activity below 15 /xM Pi/g./min. In the above assay, phosphorylase is de¬ termined in the direction of glycogen of inor¬ synthesis, i.e. the liberation ganic phosphate from glucose-1a

G.S.

0.462yumoles glucose- l-P04/g/min. =

30 40 50 60 MINUTES FIG. 4.Spectrophotometric assay of liver phos¬ phorylase. The assay mixture consisted of Tris-HCl buffer, pH 7.5, 150 fxM.; NaCl, 40 juM.; potassium phosphate, pH 7.5, 30 juM; glycogen, 2 mg./ml.; 5'AMP, 4 jiiM; TPN+, 1.5 ;uM.; magnesium acetate, 30 juM.; glucose-6-phosphate dehydrogenase, 50 ng ; phosphoglucomutase, 50 /xg. in a final volume of 3.0 ml. Liver homogenate (0.1 ml.) was added to start the reaction which was followed by measuring the in¬ crease in absorption at 340 m/u. The light path was 1 cm.; the temperature was 30°C

phosphorylase deficiency. Leukocyte phosphorylase activity Because of^ the previously mentioned observations2'3 that studies of leuko¬ cytes may be diagnostic in cases of hepatic phosphorylase deficiency, leukocyte phosphorylase determina¬ tions were performed. Heparinized blood (10-ml. samples) was obtained from thepatientG.S., from her mother, her father and one other individual (G.I.D.) and leukocytes were isolated by the method of Hiilsmann, Oei and Van Creveld2 as modified by Huijing.11 The leukocytes from each sample were homogenized in 0.4 ml. of 10 mM. Tris-HCl buffer, pH 7.0, in a small Potter-Elvehjem homogenizer and centrifuged at 4°C. at 10,000 G. for 10 minutes. Phosphorylase determi¬ nations were performed in duplicate at 30°C. by the spectrophotometric method under the conditions specified in Fig. 4, the increase in absorption at 340 m/x being recorded at two-minute intervals for 120 minutes. The results in Table II show that the leukocytes from the patient possessed only about 40% ofthe phosphorylase activity of the father and of G.I.D. The mother's leukocyte phosphorylase activity was even lower than that of the patient. These results, although surprising, are strikingly similar to those several cases reported by Hiilsmann, Oei and Van Creveld2 and Williams and Field3 in which the mother in each instance was deficient in leukocyte phosphory¬ lase. The parents described in these reports, like the mother studied here,

had no symptoms of glycogenosis. This probably results from the rela¬ tively mild symptomatology being present in the mother a generation ago at a time when glycogen storage diseases were not well understood and were rarely diagnosed. Williams and

C.M.A. JOURNAL/APRIL 11, 1970/VOL. 102 741

thus not possible to compare the activities directly with those reported in the literature. In spite of the limiting P/:osphorylase nature of the data, we believe (mJIM glucose-i -P04 that the results further confirm the formed/hr./O.i mL extract) Subject disease in the patient G.S. as phos3.15 Mr. S. (father) phorylase deficiency. This case adds 2.98 G.I.D. another to those previously reported24 1.25 Patient, G.S. which suggest that examination of 0.44 Mrs. S. (mother) leukocyte enzyme activity can be diagnostic in cases of hepatic glycogenoField3 expressed leukocyte phospho- sis Type VI. The case presented here seems typirylase activity on the basis of the leukocyte count. In our study no such cal of Type VI glycogenosis,"5 being determinations were made, and it is characterized by mild hypoglycemia, TABLE ii Leucocyte glycogen phosphorylase

prominent hepatomegaly, poor response to glucagon, and low (but not absent) hepatic and leukocyte phosphorylase. These observations, and the fact that the patient has not required dietary management, indicate a good prognosis, as has also been found in other reported cases."4'5 The technical assistance of Miss C. Anne Powell is acknowledged.

References I. HERS, H. G.: Glycogen storage disease. In: Advances in metabolic disorders, vol. 1, edited by R. LEVINE and R. LUFT, Academic Press Inc., New York, 1964, p. 1. 2. HULSMANN, W. C., OEI, T. L. and VAN CREVELD, 5.:

Lancet, 2: 581, 1961. 3. WILLIAMS, H. E. and FIELD, J. B.: J. Cli,,. h,s'e.st., 40: 1841, 1961. 4. VAN CREVELD, 5. and HUIJINO, F.: Amer. J. Mcd.,

38: 554, 1965. 5. FIELD, R. A.: Glycogen deposition diseases. In: Metabolic basis of inherited disease, 2nd ed., edited by J. B. STANBURY, J. B. WYNGAARDEN and D. 5.

FREDRICKSON, McGraw-Hill Book Company, New York, 1966, p. 141. 6. JoHNsoN, J. A., NASH, J. D. and FusARo, R. M.: Anal. Bioc/,cm., 5: 379, 1963.

74t44ta*04L,

7. WAsHKo, M. E. and RICE, F. W.: Cl/n. Chem.,

7: 542, 1961.

C'49.uiCte4

8. ILLINOWORTH, B. and CORI, G. I.: J. B/of. C/win.,

199: 653, 1952. 9. 5.osuRv, 3. B., et ol.: Pet//air/cs, 27: 103, 1961. 10. DRUMMOND, G. I., DUNCAN, L. and HERTZMAN, F.:

J. B/al. Chem., 241: 5899, 1966. 11. HUIJINO, F.: Cl/ti. C/i/rn. Ada, 9: 269, 1964.

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742 C.M.A. JOURNAL/APRIL 11, 1970/VOL. 102