C-II anapolipoproteinemia and severe hypertriglyceridemia

C-II Anapolipoproteinemia and Severe Hypertriglyceridemia Report of a Rare Case with Absence of C-II Apolipoprotein lsoforms and Review of the Literature

KEIJIRO

SAKU,

Cincinnati, CARMELO BRUCE

CEDRES,

M.D.

MCDONALD,

M.D.

Bridgeport, BARBARA BING LAXMI

W.

M.D.

Ohio

Connecticut A. LIU,

HYND,

MS.

M.D.

S. SRIVASTAVA,

MOT1 L. KASHYAP, Cincinnati,

Ph.D.

M.D.

Ohio

From the Apolipoprotein Research Laboratories, Departments of Medicine and Pathology, University of Cincinnati Medical Center, Cincinnati, Ohio, and the Department of Pediatrics, Bridgeport Hospital, Bridgeport, Connecticut. Requests for reprints should be addressed to Dr. Moti L. Kashyap, University of Cincinnati Medical Center, KPavilion, Mail Location 540, Cincinnati, Ohio 45267. Manuscript accepted February 28, 1984.

A new case of C-II anapolipoproteinemla (complete apollpoproteln C-II deficiency) as the cause of severe hypertriglycerldemia with chylomicronemia (type I llpoprotein phenotype) is described. The patient was a five-year-old boy living in Connecticut. He had spienomegaly, episodic abdominal pain, and bloody stools. Absence of apolipoprotein C-II (and Its isoforms C-Ill and C-IIn) was documented by a sensitive and specific radioimmunoassay, analytical isoelectric focusing, and in vitro lipolytic assay. Decreased levels of high- and low-density lipoprotein cholesterol and apolipoproteins A-l and A-II and increased levels of plasma triglycerides and apollpoproteln E were found. Post-heparln extra-hepatic lipoprotein lipase activity was within normal range. Incorporation of exogenous purlfled human apolipoprotein C-II to an incubation mixture of purified lipoproteln llpase and the patient’s triglyceride-rich llpoprotelns resulted in a dramatic increase in the catabolic rate of the defective trlglyceride-rich lipoproteins. The absence of the lsoforms of apolipoprotein C-II in this patient indicates that a common gene exlsts for the C-II isoprotelns, which appear to be necessary for normal triglyceride transport in humans. A literature review of 23 reported cases indlcates that xanthomas and hepatosplenomegaly are less common in C-II anapolipoprotelnemia than in llpoproteln lipase deficiency, the other major etiologlc cause of genetic chylomicronemia. Lipoprotein lipase is localized in the vascular endothelium in extrahepatic tissues and catabolizes triglyceride-rich lipoproteins. Apolipoprotein C-II, a protein constituent of triglyceride-rich lipoproteins and high-density lipoproteins, is a necessary cofactor for activation of lipoprotein lipase. A deficiency or absence of either lipoprotein lipase or apolipoprotein C-II results in severe hypertriglyceridemia. If not recognized, fatal pancreatitis can accompany these genetic defects. Thus, awareness of these disorders is important in the diagnostic work-up of episodic abdominal pain and/or established pancreatitis associated with hypertriglyceridemia. Compared with lipoprotein lipase deficiency, apolipoprotein C-II deficiency is a relatively newly described disorder, the first case being reported by Breckenridge et al [l] in 1978. Six kindreds have been found in the Caribbean, Japan, England, Holland, and Italy [l-8]. Interestingly, apolipoprotein C-II absence also affects plasma concentrations of other lipoproteins and apolipoproteins and serves to illustrate the role of apolipoprotein C-II in lipid metabolism.

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TABLE I

Laboratory Data in Patient with C-ii Anapoiipoproteinemia

White blood cell count (/mm3) Hemoglobin (g/dl) Hematocrit (percent) Platelets (X 105/mm3) Serum glutamic oxaloacetic transaminase (mU/ml) Serum glutamic pyruvic transaminase (mu/ml) Lactic dehydrogenase (mu/ml) Urea nitrogen (mg/dl) Uric acid (mg/dl) Glucose (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dl) Urinalysis

4,700 13.8 32 2.34 18 10 93 15 5.4 81 2,500 200 Negative

In this report, a new case of C-II anapolipoproteinemia (apolipoprotein C-II deficiency) is described. To the best of our knowledge, this is the first such case found in the United States. The literature is reviewed briefly, and differences in clinical features between C-II anapolipoproteinemia and lipoprotein lipase deficiency as etiologic causes of hypertriglyceridemia are discussed. CASE REPORT The patient was five years and five months old, male, and of Puerto Rican origin. He was referred to the Bridgeport Hospital Pediatric Clinic for investigation of hepatosplenomegaly. He was born prematurely, with a birth weight of 1,600 g and required mechanical ventilation at birth because of significant respiratory distress. At 11 months, he underwent surgery for bilateral inguinal hernia. At the age of two and a half, he was found to have a hepatosplenomegaly and an elevated plasma cholesterol level. A history of episodic bouts of abdominal pain, vomiting, and bloody stools was also documented. The family history was negative for heart disease or diabetes mellitus. There was no known consanguinity in the family. On physical examination, his weight was 14.3 kg and his height was 99.1 cm; he looked slightly small for his age. Blood pressure was 90/65 mm Hg. The liver was palpated 1 to 2 cm below the right costal margin, and the spleen was hard, firm and palpable, approximately 6 cm below the left costal margin. The cardiovascular system, lungs, and neurologic findings were within normal limits. There were no cornea1 arcus or xanthomas. The results of the laboratory studies on an outpatient basis are summarized in Table I. They are unremarkable except for elevated plasma triglyceride level (2,500 mg/dl). Upper abdominal ultrasound examination revealed significani splenomegaly with a homogeneous spleen and normal liver. Unfortunately, the rest of the family members were not available for study. METHODS Lipid, Lipoprotein, and Apoiipoprotein Analysis. Plasma triglyceride and total cholesterol levels were determined in duplicate by standardized autoanalyzer II procedure at the

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University of Cincinnati Lipid Research Center Laboratory [9]. Lipoprotein cholesterol was measured as follows. Blood plasma samples were ultracentrifuged at density (d) = 1.006 g/ml at 45,000 rpm for 24 hours using a Beckman type Ti 50.3 rotor and Beckman Model L5-50 ultracentrifuge. The infranate was quantitatively recovered and total cholesterol measured. Highdensity lipoprotein cholesterol was taken as the d = 1.063 infranate cholesterol (C) concentration. Lowdensity lipoprotein cholesterol was derived by subtraction of highdensity lipoprotein cholesterol from total cholesterol in the d = 1.006 infranate. Apolipoproteins A-l, A-II, and B were quantified by electroimmunoassay, which was carried out in 1 percent agarose, 2 percent polyethylene glycol with barbital buffer at pH 6.6, using monospecific antibodies according to Laurell’s [lo] technique and as described previously [ 111. Plasma apolipoprotein C-II concentration was determined by specific double-antibody radioimmunoassay described earlier [ 121. Triglyceride-rich lipoprotein, which was isolated by preparative ultracentrifugation d less than 1.02 g/ml, was lyophilized and delipidated with acetone: ethanol (1:l volume for volume) at 20°C [ 131, then dissolved in 0.5 ml of 0.01 MTris/hydrogen chloride, pH 6.0, containing 6 M urea. Triglyceride-rich lipoprotein urea-soluble protein was applied to analytical isoelectric focusing gels with a pl range between 3.5 and 5.0 as described previously [ 141 to show apolipoprotein C subspecies. Post-Heparin Plasma Lipoiytic Activity. Post-heparin lipolytic activity was determined essentially according to the method of Krauss et al [ 151 using the modification of Huttunen et al [ 161. Instead of 10 units of heparin per kilogram of body weight, 100 units of heparin was injected intravenously into one arm and venous blood was collected 15 minutes later. The plasma was separated by low-speed centrifugation at 3,000 rpm for 15 minutes at 4’C and stored at -7O’C. Post-heparin plasma (30 ~1) was incubated in duplicate at 37’C for 10 minutes with 70 ~1 of heparin (15 units/ml) or protamine sulfate (60 mg/ml). After this incubation, 650 ~1 of substrate and 50 ~1 of normal human plasma were added, and incubation was carried out at 37’C for one hour. The substrate was prepared as follows: 300 mg of cold triolein, 45 &i of tritiated triolein, 600 mg of fatty acid-free albumin, 1.6 ml of 1 percent triton X-100, and 34.2 ml of buffer (0.194 M Trisihydrogen chloride plus 0.1 percent sodium chloride, pH 8.6) were sonicated for four minutes. Incubation was terminated by the addition of 4 ml of isopropanol/3 Nsulfuric acid (40: 1 v/v), 2 ml of water, 5 ml of heptane; the free fatty acids were extracted into 0.1 M potassium hydroxide and their radioactivity was determined for calculation of fatty acid liberation. Kinetics of Lipoiysis of Triglyceride-Rich Lipoproteins. Apolipoprotein C-II-absent triglyceride-rich lipoproteins were labeled with dansyl, 5dimethylaminonaphthalene-1 sulfonyl phosphatidyl-ethanolamine (DPE) (DPE/triglyceride = 1:40; 3 mg of triglyceride per milliliter). Incubation was then carried out at 37’C for one hour with 11 pg of purified C-II per milligram of triglyceride. After incubation, the triglyceride-rich lipoproteins were m-isolated, and a mixture of 600 ,ug of this triglyceride-rich lipoprotein, fatty acid-free bovine serum albumin, and bovine lipoprotein lipase in 1.0 ml of 50 mM Trislhydrogen chloride, 0.9 percent sodium chloride, pH 7.6,

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Figure 1. The appearance of the fasting plasma from a normal subject (left) and the patient with C-II anapolipoproteinemia (right).

TABLE II

Plasma Concentrations of Llplds, Lipoprotein Cholesterol, and Apollpoprotelns (mg/dl) In Patient

Total cholesterol Total triglycerides High-densitylipoproteincholesterol Low-densitylipoproteincholesterol Apolipoproteins A-l A-II E B C-II

156 1,194 11 36 50 15 17 92 Undetectable

was monitored continuously for an increase in fluorescence at 37’C against time according to the method of Johnson et al [ 171. Apolipoprotein C-II-absent triglyceride-rich lipo-

proteins labeled with DPE as just described were incubated with saline, re-isolated, and used as a control. RESULTS The fasting plasma of the patient (Figure 1, rlght) showed a thick sticky cream layer on the top after standing at 4’C overnight. The infranates were clear in both the patient and a normal subject (Flgure 1, left). The plasma concentration of lipids, lipoprotein cholesterol, and apolipoproteins are shown in Table II. On

an unrestricted diet, plasma triglyceride concentration averaged 1,194 mg/dl and total cholesterol averaged 156 mg/dl. High-density lipoprotein cholesterol, lowdensity lipoprotein cholesterol, and plasma apolipoprotein A-l and A-II levels were markedly reduced in this patient, 11, 36, 50, 15 mg/dl, respectively, whereas apolipoprotein E was increased (17 mg/dl). Apolipoprotein C-II was undetectable in plasma by radioimmunoassay. Figure 2 shows the analytical isoelectric focused gels of triglyceride-rich lipoprotein urea-soluble apolipoprotein Cs from the patient (Figure 2, left) and a normal subject (Figure 2, right). Approximately 200

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TABLE Ill

Plasma Post-Heparin Lipolytlc Activity @M/ml/hour free fatty acids) In Patlent

Hepatic triglyceride lipase Extra-hepatic lipoprotein lipase Total post-heparin lipolytic activity l

7.0

9.8 (17-26)’ 19.4 (14-27) 29.2 (31-53)

6.0

5.0

P4.D LL

pg of very-low-density lipoprotein protein was loaded, and complete absence of apolipoprotein C-II isoforms (both C-II, and C+) was found. Extra-hepatic lipoprotein lipase activity in post-heparin plasma (free fatty acids 19.4 PM/ml/hour) was within the normal range, whereas hepatic triglyceride lipase was decreased (Table III). The time course of the fluorescence increase during lipoprotein lipase-mediated hydrolysis of DPE-labeled triglyceride-rich lipoprotein in both absence and presence of exogenous human pure apolipoprotein C-II is shown in Figure 3. Re-isolated triglyceride-rich lipoprotein into which purified apolipoprotein C-II was incorporated had a remarkable effect on the lipolytic curves. As shown in Table IV, apolipoprotein C-II increased the kinetic parameters of triglyceride-rich lipoprotein lipolysis.

:i

2.0

1 .o 15

20

25

30

incubation Time (Min.)

Figure 3. Effect of apolipoprotein C-l! on the hydrolysis o DPE-labeled triglyceride-rich lipoproteins from the patient with C-II anapolipoproteinemia (details in the Methods section).

undetectable apolipoprotein G-II in their kindred from the Caribbean region. In a later study, apolipoprotein C-II was undetectable in 11 homozygotes and was intermittently detectable by electroimmunoassay in one patient in this kindred [3]. A faint band in the position of apolipoprotein C-II (by isoelectric focusing) was also noted in two of the patients [3]. In other studies, apolipoprotein C-II was absent by double immunodiffusion, electroimmunoassay, or gel electrophoresis. The term “apolipoprotein C-II deficiency” has been used to describe this condition, although it is likely that almost all patients described so far had an absence of apolipoprotein C-II. The reason for this is two-fold. First, double immunodiffusion and electroimmunoassay procedures lack the sensitivity of radioimmunoassays, and it was therefore impossible for previous investigators to be certain that apolipoprotein C-II was absent. Second, although isoelectric focusing and polyacrylamide gel electrophoresis are reasonably sensitive

With a very sensitive (capable of detecting nanogram quantities) radioimmunoassay, no apolipoprotein C-II was detected in the plasma of our patient. Analytical isoelectric focusing of delipidated isolated triglyceride-rich lipoproteins also failed to show apolipoprotein C-II isoforms (C-Ill and C+) even when the gels were overloaded with triglyceride-rich lipoprotein protein. Thus, by these two powerful and sensitive criteria, this patient had an absence of apotipoprotein C-IL AIthough 22 patients have been previously described with this condition [i-8], the radioimmunoassay technique showing apolipoprotein C-U absence in whole plasma has been used only in one previously reported kindred of two patients, also from our group [4,18]. Breckenridge et al [l] and Cox et al [2] used double immunodiffusion, polyacrylamide gel electrophoresis, and an in vitro lipoprotein lipase activator assay and found

Klnetlc Parameters of Llpolysls: Effect of Apollpoproteln C-II on Apollpoproteln C-II-Absent TrlglycerldeRich Llpoproteln Labeled wlth Fluorescence Probe lnltialVelocity (unltslmlnufe)’

Patient’s native triglyceride-rich lipoprotein (control) Patient’s triglyceride-rich lipoprotein with incorporation of human apolipoprotein C-II

4.7 12.1

Arbftrary fluorescence units.

460

10

5

COMMENTS

l

+Apo C,

ll

Reference values.

TABLE IV

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IncreaseIn FlwrsscenceoverBasslIne’ 20 Minutes 30 Mlnufer 2.3 6.7

2.5 6.9

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methods (but do not approach the sensitivity of radioimmunoassays) for protein detection, the techniques are restricted to assessment of apolipoprotein C-II in triglyceride-rich lipoproteins. Thus, investigators who based their diagnosis on these electrophoretic techniques could not be absolutely certain that apolipoprotein C-II was absent in other lipoproteins (highdensity lipoproteins) or in plasma. Thus, the term “apolipoprotein C-II deficiency” was appropriate, given the lack of certainty regarding its absence. However, in our patient, the most sensitive techniques in the world for apolipoprotein C-II detection failed to reveal any. Thus, we propose that the term “C-II anapolipoproteinemia” is more appropriate for this condition. The biochemical and clinical features in the previously described patients, some of their kindreds, and our patient are summarized in Table V. There are some similarities between them not only in clinical features, but also in laboratory data. C-II anapolipoproteinemia is an autosomal recessive disorder [l-4]. Almost all homozygotes have severe hypertriglyceridemia and reduced levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and apolipoproteins A-l, A-II, and B. Increased levels of apolipoprotein C-III (Kindreds 1, 2, 3, and 5 in Table V) and apolipoprotein E (Kindreds 1,3, and 7) have been described. The patients with “apolipoprotein C-II deficiency” have latent post-heparin lipolytic activity that appears after the addition of apolipoprotein C-II or plasma as a co-factor to post-heparin plasma. Three homozygotes with “apo C-II deficiency” (Kindreds 1, 2, and 3) were found to have a marked elevation of post-heparin extra-hepatic lipoprotein lipase. A 20-fold increase in lipoprotein lipase activity in adipose tissue was found in a patient from Kindred 3 [5,19,20]. These observations suggest a compensatory increase in lipoprotein lipase as a homeostatic mechanism for triglyceride catabolism in some patients. However, in our patient, lipoprotein lipase activity was within the normal range for adults, as also in the two patients from Kindred 6. Decreased hepatic triglyceride lipase was observed in five kindreds, including our patient. A review of clinical features shows that 75 percent of homozygotes had episodic abdominal pain, whereas concurrent coronary heart disease or diabetes mellitus was rare. Five patients described by Cox et al [2] and one by Stalenhoef et al [6] (combined with lipoprotein lipase deficiency) had pancreatitis. In Kindred 4 (combined lipoprotein lipase and apolipoproteins C-II and E3 deficiency), four homozygous siblings had marked hepatosplenomegaly with lipemia retinalis at a very young age. In fact, the clinical features of familial lipoprotein lipase deficiency summarized by Frederickson and Levy [22] in 1972, and other kindreds with lipoprotein lipase deficiency described recently showed

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that eruptive xanthomas are common in this condition and more than 75 percent of the patients also have hepatosplenomegaly, often found in infancy. However, only our patient with C-II anapolipoproteinemia and two homozygotes described by Fellin et al [8] had marked hepatosplenomegaly, and only one patient in Kindred 6 had eruptive xanthomas, indicating these physical findings are less frequent in uncomplicated C-II anapolipoproteinemia. The age at which lipoprotein lipase deficiency is detected in most instances is less than five years, whereas that of apolipoprotein C-II deficiency is five to 62 years old, as shown in Table V, suggesting that a history of eruptive xanthomas and the presence

of hepatosplenomegaly make lipoprotein lipase deficiency more likely, especially in a young patient. Plasma triglyceride concentrations in “apolipoprotein C-II deficiency” are relatively lower than the values seen in patients with typical familial lipoprotein lipase deficiency. Thus, it appears that lipoprotein lipase deficiency results in more overt clinical symptoms than the enzyme co-factor (apolipoprotein C-II) deficiency. In this study, both isoforms of apolipoprotein C-II (C-II, and C-lip) were absent. This observation lends strong evidence that a common gene exists for the C-II isoproteins, which appear to be necessary for normal triglyceride transport.

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Breckenridge WC, Little JA, Steiner G, Chow A, Poapst M: Hypertriglyceridemia associated with deficiency of apolipoprotein C-II. N Engl J Med 1978; 298: 12651273. 2. Cox DW, Breckenridge WC, Little JA: Inheritance of apolipoprotein C-II deficiency with hypertriglyceridemia and pancreatitis. N Engl J Med 1978; 299: 142 I- 1424. 3. Breckenridge WC, Alaupovic P, Cox DW, Little JA: Apolipoprotein and lipoprotein concentrations in familial apolipoprotein C-II deficiency. Atherosclerosis 1982; 44: 223. 4. Yamamura T, Sudo H, lshikawa K, Yamamoto A: Familial type I hyperlipoproteinemia caused by apolipoprotein C-II deficiency. Atherosclerosis 1979; 34: 53-65. 5. Miller NE, Rao SN, Alaupovic P, et al: Familial apolipoprotein C-II deficiency: plasma lipoproteins and apolipoproteins in heterozygous and homozygous subjects and the effects of plasma~infusion. Eur J Ctin Invest 1981; 11: 69-76. 6. Stalenhoef AFH. Casoarie AF. Demacker PNM, Stouten JTJ, Lutterman JAI Van’t Laar Ai Combined deficiency of apolipoprotein C-II and lipoprotein lipase in familial hyperchylomicronemia. Metabolism 1981; 30: 919-926. 7. Catapano AL, Mills GL, Roma P, LaRosa M, Capurso A: Plasma lipids, lipoproteins and apoproteins in a case of apo C-II deficiency. Clin Chim Acta 1983; 130: 317-327. a. Fellin R, Baggio G, Poli A, et al: Familial lipoprotein lipase and aooliooprotein C-II deficiency. Atherosclerosis 1983; 49: 5’5-68. ’ 9. Lipid Research Clinics Program (publication NIH 75-628). Bethesda, Maryland: National Institutes of Health, Education and Welfare, 1974; Publication no. (NIH) 75-g-50. 10. Laurel1 CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biothem 1986; 15: 45-52. 11. Mendoza SG, Zerpa A, Carrasco H, et al: Estradiol. testosterone, apolipoproteins, lipoprotein cholesterol, and lipolytic enzymes in men with premature myocardial infarction and coronary occlusion. Artery (in press). 12. Kashyap ML, Srivastava LS, Chen CY, et al: Radioimmunoassay of human apolipoprotein CII. A study in normal and hypertriglyceridemic subjects. J Clin Invest 1977;60: 171-180.

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Kashyap ML, Barnhart RL, Srivastava LS, et al: Effects of dietary carbohydrate and fat on plasma lipoproteins and apolipoproteins CII and Clll in healthy men. J Lipid Res 1982; 23: 877-886. Kashyap ML, Hynd BA, Robinson K, Gartside PS: Abnormal preponderance of sialylated apolipoprotein Clll in triglyceride rich lipoproteins in type V hyperlipoproteinemia. Metabolism 1981; 30: 11 l-l 18. Krauss RM, Levy RI, Fredrickson DS: Selective measurement of two lipase activities in post heparin plasma from normal subjects and patients with hyperlipoproteinemia. J Clin Invest 1974; 54: 1107-l 124. Huttunen JK, Ehnholm C, Keki M, Nikkila EA: Postheparin plasma lipoprotein lipase and hepatic lipase in normal subjects and in patients with hypertriglyceridemia: correlations to sex, age and various parameters of triglyceride metabolism. Clin Sci Mel Med 1976; 50: 249-260. Johnson JD, Taskinen MR, Matsuoka N, Jackson RL: Dansyl phosphatidylethanolamine-labeled very low density lipoproteins. A fluorescent probe for monitoring lipolysis. J Biol Chem 1980; 255: 3461-3465. Matsuoka N, Shirai K, Johnson JD, et al: Effects of apolipoprotein C-II (apo C-II) on the lipolysis of very low density lipoproteins from apo C-II deficient patients. Metabolism 1981; 30: 818-824. Brunzell J, Miller N, Alaupovic P: Increased lipoprotein lipase in subiects with apo CII deficiency and familial serum,. If. poprotein lipase ‘inhibitor (abstrj. Clin Res 1980; 28: 517A. Brunzell JD, Miller NE, Alaupovic P, et al: Familial chylomicronemia due to circulating inhibitor of lipoprotein lipase activity. J Lipid Res 1983; 24: 12-19. Lamberts SW, Casparie AF, Miedema K, Hennemann G, Hulsmans HAM: Thyroxine binding globulin deficiency in a family with type I hyperlipoproteinemia. Clin Endocrinol 1977: 6: 197-206. Fredrickson DS, Levy RI: Familial hyperlipoproteinemia. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, eds. The metabolic basis of inherited disease, 3rd ed. New York: McGraw-Hill, 1972; 545-614.