Triglyceride Clearing in Glycogen Storage Disease

Pediat. Res. 8: 114-1 19 (1974) Hepatic glycogenosis postheparin lipoprotein lipase hyperlipidemia triglyceride

Department of Pediatrics, Sophia Children's Hospital and Neonatal Unit, Medical School, Erasmus University, Rotterdam, The Netherlands

Extract Peripheral uptake of triglyceride from plasma was investigated by intravenous fat tolerance tests and by postheparin lipoprotein lipase measurements in children with different types of glycogen storage disease. The patients with a glucose 6-phosphatase deficiency were characterized by a [significantly diminished triglyceride elimination rate (5.79 f 2.78%/min) and 5-min postheparin lipoprotein lipase activity (40.2 f 23 pEq fatty acid (FA)/liter/ min). The patients with a deficiency of debranching enzyme showed a significantly diminished triglyceride elimination rate (4.84 f 1.61 yo/min) whereas the 5-min postheparin lipoprotein lipase activities did not significantly differ from the control values (49.6 f 27.7 pEq FA/liter/min). The patients with a deficiency of the phosphorylase system showed neither a significantly diminished triglyceride elimination rate (7.34 =t 2.65yo/min) nor a diminished 5-min postheparin lipoprotein lipase activity (61.7 f 30.1 ,LE~FA/liter/ min) . .t ' G Triglyceride elimination rates were correlated positive~y'g&i%hplasma lipoprotein lipase activities (r = 0.55, P < 0.05). ,I> ,. ‘‘I

, ? Speculation ,,

" I ./ The dietary treatment of hyperlipidemia in hepatic glycogenosis might be based on the outcome of the intravenous fat tolerance test, a low fat clearance being an Indica- tion for a low fat, high carbohydrate diet, a normal fat tolerance for a higb$aiLt,.low carbohydrate diet. , .

Introduction fasting state, conversion of glucose 6-phosphate to glu- Little is known about the causes of hyperlipidemia in cose is blocked, so that glycogen can only be degraded patients with glycogen storage disease (GSD) [4, 5, 81. to pyruvate through the glycolytic pathway. Part of The highest lipid levels are found in patients with a the excess pyruvate is converted into lactate, another glucose 6-phosphatase deficiency. They have the high- part into acetyl-CoA, and thence into fatty acids, pre- est fatty acid cholesterol ratios and sometimes even a dominantly palmitate [4, 121. pre-p-band on the electropheretograms. De novo fatty Hyperlipidemia is less striking in children with a acid synthetizing activity from citrate is high in liver deficiency of debranching enzyme or of the phospho- biopsies of these patients 171. The pathogenesis of the rylase system. In the latter deficiencies, the hyperlipi- hypertriglyceridemia in patients with a glucose 6-phos- demia is characterized by a dense p-lipoprotein band, phatase deficiency is presumed to be as follows: in the no pre-p-lipoprotein band being present on the elec- Triglyceride uptake in glycogen storage disease 115 tropheretograms; the glycolysis is enhanced postpran- 40-min blood samples in most GSD patients, but in 5- dially, which leads to an increased lactate production min blood samples only in most control subjects. As and an increased liponeogenesis [4]. we concur with Rossner et al. [13] that the 5-min Although enhanced lipid production in the liver postheparin LPL activity probably gives more infor- probably plays a causative role in the pathogenesis of mation about fat elimination than the 40-min value, GSD hyperlipidemia, it may not be the only factor. A we have chosen the 5-min value as representative of pathologically low triglyceride clearance rate might LPL activity. The LPL activity was determined by the also be an important factor. By measuring the triglyc- method of Kelly [ll] as modified by Jansen and Hiils- eride elimination rate and the serum postheparin lipo- mann [9]. The following minor modifications were protein lipase activity, we tried to gain more insight introduced: the incubation mixture consisted of 1.1 ml into the apparently complex causes of the hyperlipide- Intralipid emulsion containing approximately 3.5 &i mia. (14C)trioleate, 3.0 ml human plasma freed of p-lipoproteins as described by Jansen and Hiilsmann [9], 6.0 ml Materials and Methods 10% solution of bovine albumin in 0.2 M Tris buffer pH 8.5, and 1.9 ml 0.2 Tris buffer, pH 8.5. This The study seven patients with a glucose 6- mixture was kept at 37' for 40 min. After this pretreat- phosphatase seven patients with a de- ment, 0.8 ml incubation medium was mixed with 0.2 branching enzyme and six patients with a ml plasma to be investigated. The incubation time was deficiency of the phosphorylase system. Enzymatic 35 min. studies had confirmed the diagnosis in all patients (Tables 1-111). Ten patients without hyperlipidemia in whom an intravenous fat tolerance test had been Results performed served as control subjects (Table IV). They Intralipid Elimination Constant in Patients and Con- all had a normal nutritional status. Informed consent trol Subjects was obtained in each case. An intravenous fat tolerance test was performed in The Intralipid elimination constants of the patients all GSD patients and control subjects. Heparin-re- with a glucose 6-phosphatase deficiency and of the leased plasma lipoprotein lipase (LPL) activity was patients with a debranching enzyme deficiency were measured in most GSD patients and in some control significantly lower than those of the control patients subjects. Both tests were performed on consecutive (P < 0.05). For the patients with a deficiency of the days. The patients and the control subjects did not phosphorylase system, the difference was not signifi- fast before the tests in order to prevent hypoglycemia cant (Tables I-V). in the patients. The meals during the 12-hr period before each test did not contain fat. The last meal was Heparin-releaired LPL Activity in Patients and Con- always given 2 hr before the test. The diet in the 2 trol Subjects weeks preceding the tests contained about 25% of total The heparin-released LPL activity was higher in the calories at fat, mainly polyunsaturated, for all pa- 5-min sample than in the 40-min sample in the major- tients. ity of GSD patients. Three out of six glucose 6-phos- The intravenous fat tolerance test [2] was carried phatase-deficient patients, and one out of four de- out according to the method of Carlson and Rossner branching enzyme-deficient patients, however, showed [3]. For all tests, a 20% Intralipid emulsion [15] was 5-min activities lower than the 40-min activities. Most administered intravenously in a dose of 0.1 g/kg body patients with a glucose 6-phosphatase deficiency wt. Capillary blood samples were collected every 5 showed lower 5-min LPL activities as compared with min. The Intralipid concentration was determined by the control subjects, the difference between both nephelometry. For each disappearance curve a mini- groups being significant (P < 0.05). Some patients mum of four consecutive points was used. The loga- with a deficiency of debranching enzyme or a defi- rithms of the Intralipid concentrations were plotted ciency of the phosphorylase system showed decreased against time. Straight lines were obtained from which LPL activities as well, but the LPL activities of the the correlation coefficients were calculated. latter two groups as a whole did not differ significantly The LPL activities were measured after the intrave- from the LPL activities of the control subjects (Tables nous injection of 100 U heparinlkg body wt in 5- and I-V). Table I. Data for seven patients with a glucose 6-phosphatase deficiency'

Postheparin LPL activity, Glucose 6-phosphatase Cholesterol, Triglycerides, Kz,.%/ ,. pEq FA/liter/min Case Sex 'get yr mmol/liter mmol/liter min2 in liver, nmoles S min 40 min Pi/min/mg protein

1 F: female; M: male; LPL; lipoprotein lipase: FA: fatty acid. 2 Intralipid elimination constant [3]. 8 Normal range 24.0-93.0.

Table 11. Data for seven patients with a debranching enzyme deficiency1

Postheparin LPL activ- Debranching enzyme, nmol Sex Age, yr Cholesterol, Triglycerides, Kz, %/ ity, pEq FA/liter/min glucose produced from limit Case mmol/liter mmol/liter min dextrin/min/mg protein S min 40 min Leucocytes Liver

Normal range n m u

1 F: female; M: male; LPL: lipoprotein lipase; FA: fatty acid. 2 The assay was modified and performed at a higher concentration of limit dextrin (normal range for leucocytes 2.07-5.04 (n = 8), for liver 2.13-9.09 (n = 3).

Leuels of Triglyceride aind Cholesterol in Blood in nificant positive correlation (r = 0.55, P < 0.05, Fig. Patients and Control Subjects 1). The Intralipid elimination constants and the The triglyceride levels were significantly higher (P plasma trigl~cerides, On the One hand (Fig. 2), and the < 0.05) in the three patient groups as with LPL activities 5 min after heparin injection and the the control subjects. The cholesterol values were sig- plasma trigl~ceridesyOn the hand (Fig. 3)9 were nificantly increased in the patients with a debranching not enzyme deficiency as compared with the control subjects (Tables I-V). Discussion Much work has been done to find the cause of the Correlations of Intralipid Elimination Constants, LPL deficient LPL activity in disturbances of the triglycer- ~ctiuities,and Levels of Trigl~ceride in ide elimination system. The synthesis of LPL activity of GSD Patients in adipose tissue taken from rats appears- - to be under The Intralipid elimination constants and the LPL hormonal control. Insulin has a positive effect, activities 5 min after heparin injection showed a sig- whereas catecholamines, glucagon, growth-stimulating Triglyceride uptake in glycogen storage disease 117

Table 111. Data for six patients with a deficiency of phosphorylase system1

Choles- Postheparin LPL activity, Phosphorylase in leucocytes, Phosphorylase b Cases Sex Age, yr zgto mol/ K%.%/min pEq FA/liter/min nmol/min/mgprotein cytes,kinase U inactivated/ leuco- liter liter 5 min 40 min -AMP +AMP min/mg protein

Normal range n 6 6 m 5.28 1.87 u 0.68 1.17

1 M: male; LPL: lipoprotein lipase; FA: fatty acid. 2 Patients I and 2 (brothers) are phosphorylase deficient; phosphorylase activity in the liver 0.0 and 0.0 nmoles/min/mg protein, respec- tively; patients 3-6 are phosphorylase b kinase deficient; liver biopsy, performed in one patient (patient 4), confirmed the diagnosis.

Table ZV. Data for 10 control children1

Postheparin LPL activity, peq Cholesterol, Triglycerides, Ke, %/ FA/li ter/min Case Sex Age, yr mmol/liter mmol/liter min 5 min 40 min

- 1M: male; F: female; LPL: lipoprotein lipase; FA: fatty acid. hormone, and adrenocorticotropic hormone have been low levels of glucose and insulin in blood [6]. As for shown to have a negative effect on the LPL activity our patients with this enzyme defect, not only LPL [lo]. Further studies on the factors regulating LPL ac- activities, but also Intralipid clearance rates were sig- tivity were described in 1966 by Salamon and Robin- nificantly reduced as compared with the control pa- son [14], who incubated intact epidymal fat bodies tients. Our patients deficient in debranching enzyme taken from rats that had been starved for 48 hr and in or the phosphorylase system, however, in whom symp- which the activity of LPL was low, in a medium de- toms of hypoglycemia are mild and rare, showed LPL signed to promote enzyme activity. They found that, activities that did not differ significantly from those of as expected, the LPL activity increased to a level char- the control patients. Nevertheless, the patients with a acteristic of the tissue of intact animals in the fed debranching enzyme deficiency showed Intralipid state. Glucose and insulin were important constituents clearance rates significantly lower than the control sub- of the incubation medium, insofar as the omission of jects. We have no explanation for the discrepancy be- either markedly reduced the increase in enzyme activ- tween the normal LPL activity and decreased fat elim- ity. Therefore, LPL synthesis in patients with a glu- ination in debranching enzyme-deficient patients. cose 6-phosphatase deficiency may be impaired by the Thus, we may explain the hyperlipidemia of GSD Table V. Intravenous fat tolerance test (IVFTT) and 5-min postheparin lipoprotein lipase (LPL) in glycogen storage disease patients and control sub-jects

~ Glucose 6-phos- Debranching en- Phosphorylase Control subjects Glucose 6-phos- Debanching en- phatase deficiency zyme deficiency deficiency phatase deficiency zyme deficiency Phdok)z~pControl subjects

I FA: fatty acid.

carbohydrate-induced liponeogenesis [4], as impaired fat elimination probably plays a significant role in debranching enzyme deficiency only. (Discrepant results of fat elimination tests in debranching enzyme deficients do not permit a firm conclusion.) The dietary implications should be that for children deficient

X x x PATIENTS CONTROLS I I I I I I I 20 40 60 80 100 120 140 LPL ACTIVITY peq ~~/l/min

Fig. I. Relation between lipoprotein lipase (LPL) activities measured 5 min after the intravenous injection of 100 U heparinlkg body wt and Intralipid elimination constants of patients with glycogen storage disease and control subjects. FA: fatty add. patients as follows. The hypertriglyceridemia of patients with a glucose 6-phosphatase deficiency is due to X GSD PATIENTS a combination of a decreased triglyceride elimination CONTROLS rate and increased endogenous liponeogenesis [7, 121, the former factor being the more important one. We I did, indeed, observe that a high fat diet worsens the I I I I I 2 4 6 8 10 1'2 hypertriglyceridemia, which thus seems to be fat-in- INTRALIPID ELIMINATION CONSTANT(%min) duced (data not shown). The hyperlipidemia of pa- Fig. 2. Relation between Intralipid elimination constants and tients with a deficiency of debranching enzyme or the levels of triglyceride (TG) in plasma (log values) of patients with phosphorylase system, however, is mainly caused by glycogen storage disease (GSD) and control subjects. Triglyceride uptake in glycogen storage disease 119

A positive correlation was found between the Intra- lipid elimination constant and the postheparin LPL activity in the patients.

References and Notes 1. BOBERG,J.: Heparin released blood plasma lipoprotein lipase activity in patients with hyperlipoproteinemia. Acta Med. Scand., 191: 97 (1972). 2. BOBERC,J., CARLSON,L. A., AND HALLBERG,D.: Application of a new intravenous fat tolerance test in the study of hypertriglyceridemia in man. J. Atheroscler. Res., 9: 159 (1969). 3. CARLSON,L. A., AND ROSSNER,S.: A methodological study of an intravenous fat tolerance test with intralipid emulsion. Scand. J. Clin. Lab. Invest., 29: 271 (1972). 4. FERNANDES,J., AND PIKAAR,N. A,: Hyperlipemia in children with liver glycogen disease. Amer. J. Clin. Nutr., 22: '617 (1969). 5. HOWELL,R. R., ASHTON,D. N., AND WIJCAARDEN,J. B.: Glu- cose-6-phosphatase deficiency glycogen storage disease: Studies x G S D PATIENTS on the interrelationships of carbohydrate, lipid, and purine a CONTROLS abnormalities. Pediatrics, 29: 553 (1962). 6. HUG, G., AND SCHUBERT,W. K.: Serum insulin in type I glycogenosis: Effect of galactose or fructose administration. Diabetes, 16: 791 (1967). 7. HULSMANN,W. C., EIJKENBOOM,W. H. M., KOSTER,J. F., AND 20 40 60 80 100 120 FERNANDES,J.: Glucose 6-phosphatase deficiency and hyper- LPL ACTIVITY ~.~eq/l/min lipemia. Clin. Chim. Acta, 30: 775 (1970). Fig. 3. Relation between lipoprotein lipase (LPL) activities mea- 8. JACOVIC,S., KHACHADURIAN,A. K., AND HSIA, D. Y. Y.: The sured 5 min after the intravenous injection of 100 U heparinlkg hyperlipidemia in glycogen storage disease. J. Lab. Clin. body wt and levels of triglyceride (TG) in plasma (log values) of Med., 68: 769 (1966). patients with glycogen storage disease (GSD) and control subjects. 9. JANSEN,H., AND H~~LSMANN,W. C.: Long-chain acyl-COA hydrolase activity in serum: Identity with clearing factor in glucose 6-phosphatase, a low fat, high carbohydrate lipase. Biochim. Biophys. Acta, 296: 241 (1973). 10. JEANRENAUD,B., HEPP, D.: Adipose Tissue Regulation and diet is indicated, whereas for children with phospho- Metabolic Functions (Academic Press, New York, 1970). rylase deficiency and possibly also those with a de- 11. KELLY,T. F.: Rapid assay of labelled free fatty acids in a branching enzyme deficiency, carbohydrate rather than mixture of labelled lipids. J. Lipid Res., 9: 799 (1968). fat restriction is indicated. 12. PIKAAR,N. A., AND FERNANDES,J.: Palmitic acid as an index of carbohydrate induced lipogenesis (unpublished data). 13. R~SSNER,S., LARSSON-COHN, W., CARLSON,L. A., AND BOBERC, Summa y J.: Effects of an oral contraceptive agent on plasma lipids, The hyperlipidemia of 20 patients with GSD was stud- plasma lipoproteins, the intravenous fat tolerance and the post-heparin lipoprotein lipase activity. Acta Med. Scand., ied by the intravenous Intralipid tolerance test and 190: 301 (1971). the determination of the plasma postheparin lipopro- 14. SALAMON,M. R., AND ROBINSON,D. S.: Clearing factor lipase tein lipase activity. Patients with a glucose 6-phospha- in adipose tissue: A medium in which the enzyme activity of tase deficiency were characterized by abnormally low tissue from starved rats increases in vitro. Biochem. J., 99: results for both tests as compared with control chil- 640 (1966). dren. Patients with a deficiency of debranching enzyme 15. Batch 292174, Vitrum, Stodtholm. showed a decreased Intralipid elimination constant, 16. The technical assistance of Miss N. de Kort is gratefully ac- knowledged. whereas the LPL activities did not significantly differ 17. Requests for reprints should be addressed to: P. P. FORGET, from control values. Patients with a deficiency of the M.D., Sophia Childreus Hospital, Erasmus University, Rotter- phosphorylase system showed normal results for both dam, The Netherlands. tests. 18. Accepted for publication October 1, 1973.