Gl. Vol. 286 No. II TANGIER DISEASE —CLIFFON-BLIGH EP AL. 567 TANGIER DISEASE Report of a Case and Studies of Lipid Metabolism P. Curron-Bricu, M.B., M.R.A.C.P., B.Sc. (Mep,), P. J. Nesrer, M.D., F.R.A.C.P., anp H. M. Wuyrte, D.PHi. (Oxon.), F.R.A.C.P., F.R.C.P. Abstract :: elucidaic the deranged metabolism of Tangier disease, the turnover of esterified choles- terol, and the activities of lipoprotein lipase and of lecithin-cholesterol acyltransferase (LCAT) were ‘studied in a patient with typical findings. The plas- ma concentration of high-density lipoproteins, as- sessed by electrophoretic, immunologic and ultra- centrifugal means, was greatly reduced. Of a total plasma cholesterol of 59 mg per 100 ml (70 per cént esterified), only * mg was present in high- ANGIER disease was first described in two children by Fredrickson and his associates? in 1961. Ten other cases have since been described.?8 The features of the disease are a very low content of high-density lipoprotein (HDL) in the circulating plasma, the accumulation of a large quantity of esterified cholesterol in the macrophages of many tissues,'® enlarged tonsils having a yellow-orange color, reflecting their high content of esterified cho- lesterol, frequently clevated plasma triglyceride concentrations and abnormal quantities of circulat- ing chylomicrons in the fasting state. Both parents of affected persons have low plasma concentrations of HDL, indicating that the disorder is transmitted as an autosomal recessive trait. In the additional case reported below, since disor- dered metabolism of esterified cholesterol is a fea- ture of the disease, the turnover of this class of lip- ids in the plasma was mneasured. Furthermore, HDI may be critically connected with the activity of two enzymes, lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase, which are involved respec- tively in the metabolism of esterified cholesterol and triglyceride in plasma, and these enzymes were therefore studied. CASE REPORT A girl, born on November 30, 1955, at full term and weighing 2.3 kg had no apparent physical abnormality at birth. She appeared healthy and fed well, and growth was normal. Walking and the development of speech were also normal. Growth of permanent teeth was irregular, this fact being attributed to the distorting influence of a high arched palate. Between the ages of 5 months and 2 years she had 3 convulsions, each associated with a raised temperature. At the age of 2¥2 vears the tonsils were found to be greath enlarged and were removed at operation. The surgeon ¢x- perienced difliculty in securing hemostasis, and during the 24 hours after operation, extensive hemorrhage from the tonsillar beds occurred. Madiiple bruises appeared sponta- neously on the arms and legs within a few hours of the op- eration. The platelet count was 108.000, and bleeding and From the Depariment of Clinical Science, John Curtin School of Medical Research, Australian National University, Canberra, Austra- lia, where reprint requests should be addressed to Dr. Nestel density lipoprotein. In vivo turnover of plasma es- terified cholesterol, measured after injection of ra- diolabeled mevalonic acid, was 48 mg per hour, similar to values obtained in normal subjects. Post- heparin plasma lipoprotein lipase activity was 0.071 umoles of free fatty acids released per minute per milliliter of plasma, compared to a normal value of 0.218. LCAT activity was 2.5 ug of cholesterol ester- ified per milliliter per hour of incubation, about half the normal value. clotting times were normal; an enlarged spleen was palpable. A blood transfusion was given. Later in childhood, 2 teeth were removed and unusual hemorrhage again occurred. At the age of 6¥2 years, persistent deafness, thought to be due to eustachian-tube blockage, developed. and adenoidal tissue and tonsillar remnanis were removed. The excised tssue had an unusual yellow appearance, microscopy of which revealed increased numbers of large, pale. foamy macro- phages scattered diffusely throughout the ussue. After the 2d operation the patient remained well. Her scholastic achievements were above average. Menstruation had been normal since the menarche in 1968. Occasional spontaneous nosebleeds of small volume had occurred re- cently. There had been no symptoms of diarrhea or pares- thesia. Abbreviations Used HDL: high-density lipoprotein LCAT: lecithin-cholesterol acyltransferase LDL: low-density lipoprotein VLDL: very-low-density lipoprotein The patient was 162.5 cm tall and weighed 55.5 kg: nutri- tion seemed normal. The blood pressure was 105/70. Heart sounds were normal. There was no lymphadenopathy. and the liver and spleen were not palpable. The lundi were normal, as were the corneas on superficial examination. The hard palate was high arched and associated with displace- ment of the secondary teeth. Small, orange-vellow. solid le- sions. } cm in diameter, were present on the posterior as- pect of, the pharynx. Examination of the nervous system revealed no abnormality. Urinalysis was negative for blood, protein, sugar and bile. The Hess test for capillary fragility was negative. A radiograph of the chest and a plain radi- ograph of the abdomen were normal. Hemoglobin, white- cell count, serum alkaline phosphatase. serum glutamic oxalo- acetic transaminase, prothrombin time. thrombin clotting time and plasma fibrinogen were also normal. Chromboplas- tin generation was only 56 per cent of normal. The platelet count was 82,000. The low platelet: count and reduced thromboplastin generation were not evaluated further. Vhe father, 47, and the mother, 38 years of age. were well. The mother’s maternal grandmother and the patient's father shared the same surname, but no consanguinity between the 2 families was known. The families of both facher and moth- er were of Scottish origin. General medical examination of the patient’s father and mother was negative. METHODS Plasma Lipids and Lipoproteins Blood was taken in the fasting state with diso- 568 THE NEW ENGLAND JOURNAL OF MEDICINE Mar. 16, fa7y dium EDTA as anticoagulant, and the plasma lipopro- teins HDL, low-density lipoprotein (LDL) and very- low-density lipoprotein (VLDL) were separated by the preparative method of Fredrickson and his co- workers”; p-chloromerciriphenylse!fonate was add- ed to the plasma in fis.) concentr:iion of 2 mM to inhibit the activity of .CAT during handling of the plasma." The content of cholesterol? and of triglyceride’? was measured in each lipoprotein frac- tion. Plasma was submitted to electrophoresis on ] per cent agarose after prestaining of the lipoproteins with Sudan Black B."4 Plasma from the patient and both her parents was tested against antihuman HDL antiserum (Behringwerke) in 1 per cent agarose by the method of double diffusion. Esterified Cholesterol Turnover in Vitro Approximately 140 ue of %H-pi-mevalonic acid* (Radiochemical Centre, Amersham) was injected in- travenously into the patient and into each of her parents, and the specific activity of unesterified and esterified cholesterol in whole plasma and in HDL, LDL and VLDL was measured at frequent intervals after injection for as long as 72 hours. Specific activ- ity-time curves were constructed, and the turnover of esterified cholesterol was then calculated with the method of Nestel and Monger.3 Plasma LCAT Activity (Rote of Cholesterol Esterification in Vitro) ; The method of Glonset and Wright was used.'6 Plasma samples of the patient with Tangier disease and her parents and a normal person’s plasma were heated at 56°C for 45 minutes to abolish enzyme activity and used as the source of substrate lipo- proteins. Post-Heparin Lipoprotein Lipase Activity Two methods were used: that of Fredrickson et al”? and that of Boberg and Carlson.'8 Heparin was given intravenously to the patient, her father and a normal person in a dose of 0.1 mg per kilogram of body weight. Blood was taken 10 and 20 minutes after injection and immediately chilled, and the plasma frozen at —10°C until ready for use as the source of enzyme. RESULTS Piasma Lipids and Lipoproteins The concentrations of cholesterol and triglyceride in whole plasma and in individual lipoproteins are shown in Table 1. The cholesterol content of the whole plasma of the patient was 59 mg per 100 ml (70 per cent esterified), and of this only 7 mg per 100 ml was in the HDL fraction, about 1/8 the normal value for females. The cholesterol content of the HDL in both parents was lower than normal.2 *p-mevalonic acid is the biologic isomer, and the radioactivity asso-- ciated with the L-isomer is excreted in the urine, The plasma triglyceride concentration for the pa tient. was at the extreme upper limit of the nonnal range expected for a person of her age and sex.'4 When plasma from the patient was subjected ty electrophoresis on 1 per cent agarose, no alpha } (high-density) or prebeta (very-low-density) lipopro- tein was seen. No precipitin line was seen when plasma from the patient was diffused against anti- HDL antiserum in agarose. Precipitin lines were seen when the parents’ and a normal person’s plas- ma were used. Esterified Cholesterol Turnover in Vivo Specific activity time curves for esterified and unesterified cholesterol in whole plasma are shown in Figure 1. Similar kinds of curves were obtained for LDL and VLDL. The curves for HDL obtained for each parent are shown in Figure 2. The HDL curves for the patient could not be obtained be- cause of low levels of radioactivity in the HDL cho- lesterol. The steepness of the rise of a given specific activ- ity-time curve for esterified cholesterol is an esti- FATHER 2000) © UNESTERIFIED CHOLESTEROL @ ESTERIFIED CHOLESTEROL 1500} wooo}? 500} a £ = MOTHER © 2000) » = 1500] | 2 G 1000] < 2 500} ae Vu mw a “ DAUGHTER TANGI DISEASE 20001 ANGIER DIS 1500) 1000) e 500] 10 20 30 40 50 60 70 TIME -HOURS Figure 1. Specific Activity-Time Curves for Unesterified and Esterified Cholesterol in the Whole Plasma of the Pa- tient and Her Parents after an Intravenous Injection of H- pni-Mevalonic Acid. Vol 286 No. 11 TANGIER DISEASE—CLIFTON-BLIGH ET AL. 569 Table 1. Cholestero! and Triglyceride Content of Lipoprotein Fractions Derived from Whole Plasma. Supsect AGE (YR) CHOLESTEROL CONTENT (MG/S00 M1) TRIGLYCERIDE CONTENT (MG/100 Mx) WHOLE PLASMA HDL LDL VLDL WHOLE PLASMA HDL LDL VLDL Father 47 167 (75 % esterified) 31 113 23 86 14 30 42 Mother 38 200 (71 % esterified) 26 151. 23 88 37 12 39 Daughter (patient) 15 59 (70 % esterified) 7 47 5 136 1 49 80 Male control 31 166 60 98 8 56 16 23 17 mate of the turnover rate* of the esterified choles- terol pool in question. A measure of the turnover rate and turnover of the plasma esterified cholester- ol pool can be obtained by means of the method of calculation used by Nestel and Monger'® and Moutafis and Myant2° The values are shown in Table 2 for the whole-plasma esterified cholesterol pool and for individual plasma lipoprotein pools. Since a direct measure of the turnover rate of low value of 1.7 wg was also obtained for the pa- tient when plasma from a normal person was used as substrate, indicating that the additional lipopro- tein substrate provided did not enhance the rate of ~ esterification. Post-Heparin Lipoprotein Lipase Activity The results are shown in Table 4. Maximum val- ues for lipoprotein lipase were seen in each case at Table 2. Turnover of Esterified Cholesterol in Vivo. SUBJECT EsTeRiFiED CHOLESTEROL POOL (Mo) WHOLE = VLDL LDL HDL WHOLE PLASMA PLASMA Father 2913 189 1872 558 0.015 Mother 4213 229 3218 465 0.022 Patient 639 90 $37 12* 0.075 TurRNOvER RATE TURNOVER (Hr-!) (Ma /Hr) VLDL LDL HDL WHOLE YLDL LDL HDL PLASMA 0.022 0.026 0.024 44 4 49 13 0.021 0.029 0.022 93 5 93 10 0.039 0.043 1.750* 48 4 23 21 *Estimate. esterified cholesterol in HDL could not be obtained in the patient with Tangier disease, an estimate was made by subtracting the values for the turnover for LDL and VLDL from that of, whole plasma."® Turn- over rate for IDL was then calculated by division of turnover by the pool size. Table 2 shows that the turnover rates for esterified chciesterol in the patient’s whole plasma and in the individual lipoproteins were higher than the corresposding values for her parents. The difference is particularly striking for the HDL, in which the tu:nover rate for the Tangier patient was estimated at !.750 per hour. However, the turnover of esterified cholesterol in whole plasma, 48 mg per hour, was similar to values reported by Nestel?! in normal persons. The turnover for the patient’s HDL esterified chc!esterol appears higher than for her parents, but it should be emphasized that this value was calculated indirectly and that the true magni- tude of difference between the parents and the pa- tient is uncertain. On the other hand, the turnover in the daughter's LDL is considerably less than that in her parents. ° LCAT Activity The results are shown in Table 3. The value of 2:5 wg of cholesterol esterified per milliliter of ac- tive plasma per hour of incubation was obtained for the patient wien her own plasma was used as sub- strate: this wis much lower than the values ob- tained for her parents and for a normal person. A *Turnover rate is the fraction of the plasma pool replaced per hour. the first sampling time, 10 minutes after the injec- tion of heparin. With both assay methods, the activ- ity of the patient’s plasma was considerably less than the activity obtained with her father’s plasma or with the plasma of a normal person. DISCUSSION The case reported satisfies the requirements for FATHER 2000) © UNESTERIFIED CHOLESTEROL e@ ESTERIFIED CHOLESTEROL » 15004 E E 1000; “S ws. ° ° E500 oo — >. o y MOTHER y 20004 = % 1500] XC & 3 1000) 500} ° —s 10 20 30 40 50 60 70 TIME-HOURS Figure 2. Specific Activity-Time Curves for Unesterified and Esterified Cholesterol in the HDL of the Parents of the Patient after an Intravenous Injection of 3H-Mevaionic Acid. 570 THE NEW ENGLAND JOURNAL OF MEDICINE Table 3. LCAT Activity (in Vitro Cholesterol Esterification Rate) ACTIVE PLASMA SOURCE OF ESTERIFICATION SUBSTRATE Rate*® A: Normal person Normal person 4.54 0.4 Father Father 41+ 0.1 Mother Mother 7.0 + 0.3 Patient Patient 2.5 + 0.2 B: Father Normal person 4540.1 Mother Normal person 6.1 + 0.3 Patient (Tangier disease) Normal person 1.7+03 *yug -of cholesterol esterified/ml of fresh test plasma/hr of incubation (mean + SD), the diagnosis of Tangier disease in the following respects:“a low total plasma cholesterol in the pa- tient with a normal proportion esterified; near ab- sence of HDL from the plasma as assessed by ultra- centrifugal, electrophoretic and immunologic tech- nics; a high plasma triglyceride concentration but an absence of pre-beta migrating VLDL on electro- phoresis; and a low HDL cholesterol concentration in the plasma of both parents. In the present case lymphoid tissue was not di- rectly analyzed for esterified cholesterol content. However, tissue of a yellow-orange color observed in the pharynx of our patient was similar in appear- ance to lesions in the pharynx described in other tonsillectomized patients with Tangier disease.3* Also, microscopical examination of adenoidal tissue in our patient showed large numbers of pale macro- phages with foamy cytoplasm assumed to contain lipid material but not proved to be esterified choles- terol. The patient had a low platelet count on two occasions, as previously described by Hoffman and Fredrickson? and by Kummer et al.® in this disease. The explanation for her episodes of unusual bleed- ing is unknown. The turnover of esterified cholesterol in the whole plasma for the patient and her father was normal,?! and therefore the pathogenesis of the dep- osition of esterified cholesterol in macrophages can- not be linked with abnormal plasma turnover. The normal turnover in the patient was brought about by a high turnover rate in all lipoproteins, especial- ly in the HDL. Furman et al.2? found that labeled human HDL disappeared more rapidly from the cir- culation when the HDL pool size was reduced. Table 4. Plasma Lipoprotein Lipase Activity 10 and 20 Minutes after Heparin. SupEecT Lipoprotein Lipase Activity® COCONUT OIL SUBSTRATE Tf INTRALIPID SUBSTRATE atlOmin at 20 min atlOmin at 20 min Normal person 0.218 0.160 0.080 0.021 Father 0.234 0.201 O.tit 0.061 Patient 0.071 0.040 0.033 0.009 *ymoles of free fatty acids released /m! of plasma/min of incubation, tin the form of Ediol; method of Fredrickson et al. }Method of Boberg & Carlson.!# Mar. If. layy One of the factors thought to regulate the tinn- over of esterified cholesterol in plasma is the enzyme LCAT, the activity of which has been associated with the presence of HDL in the plasma?! P,. tients with genetic deficiency of LCAT also nave low plasma HDL concentrations.45 The activity of LCAT has been positively correlated with the con- centration of unesterified cholesterol in the plas- ma? In our patient, low HDL concentration or low unesterified cholesterol concentration, or both, may have contributed to the low LCAT activity. How- ever, the LCAT activity in the patient’s plasma did not rise when additional substrate. was provided by use of normal plasma, suggesting that the enzyme concentration itself was diminished. Because in vivo turnover of total plasma esterified cholesterol was normal, it seems likely that factors in addition to LCAT are responsible for the turnover of esterified cholesterol in the plasma. The postheparin lipoprotein lipase activity of our patient’s plasma was found to be much lower than that in her father and in a normal person. Low plas- ma postheparin lipase activity has previously heen described in a patient with LCAT and 2 ts, deficiency. Lipoprotein lipase appears to recite HDL, or at least one apoprotein common to Hi) and VLDL as a cofactor, to achieve maximal actis ity,2®33 and the low enzyme activity in the plasma of our patient with Tangier disease is consistent with this view. The frequently observed increase in the plasma triglycerides in patients with Tangier dis- ease may be related to diminished lipoprotein [i- pase activity. We are indebted to Mrs. C. Foxman, Mrs. A. Lynch and Mrs. G. Power for technical assistance. REFERENCES 1. Fredrickson DS, Altrocchi PH, Avioli LV, et al: Tangier disease: combined clinical staff conference at the National Institutes of Health. Ann Intern Med 55:1016-1031, 196] 2. Fredrickson DS: The inheritance of high density lipoprs cia deficiency (Tangier disease). J Clin Invest 43:228-236, 1964 3. Hoffman HN, Fredrickson DS: Tangier disease (familial density lipoprotein deficiency): clinical and genetic features 1 adults. Am J Med 39:582-593, 1965 4. 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