Vol. 2X6 Ntr I I TAN(;IEK 1~1SE.\SF.-~:1.11~`1'05-1~1.I~~11 t:I' ,\I.. TANGIER DISEASE Report of a Case and Studies of Lipid Metabolism l'. ~:~.II;.I.~N-BI.IGII, M.R., M.R.A.C.P., B.Sc. (MEI>.), P. J. NESWL, M.D., F.R.A.C.P., ASD H. M. WHYTE, D.PHIL. (OXON.), F.R.A.C.P., F.R.C.P. Abstract ::J elucidaii: the deranged metabolism of Tangier dizase, 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 electrophorctic, immunologic and ultra- centrifugal means, was greatly reduced. Of a total plasma cholesterol of 59 mg per 100 ml (70 per cent esterified), only 7 mg was present in high- T ANCIEH disease \rras first described in two children by Fredrickson and his associates' in 1061. Ten other cases have since been descril>ed.2-x 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 cllolesterol in the macrophages of man) tissues,`," enlarged tortsils having a yellow-orange color, reflecting their high content of esterified cho- lesterol, frequentl) elevated plasma triglyceride concentrations and almormal quantities of circulat- ing chylomicrolls in tile fasting state. 40th parents of affected persons ha\.e IO\V plasma concentrations of HDL, indicating that the disorder is transmitted as an autosomal recessilre trait. In the additional cast reported below, since disor- dered metal)olism of esterified cholesterol is a fea- ture of the disease, the turnover of this class of lip- ids in the plasma was measured. Furthermore, HDL may be criticall>, connected with the activity of two enzymes, lecithin-cholesterol acyltransferase (LCAT) and lipoproteirl lipase, which are in\.olved respec- tively in the metabolisln of esterified cholesterol and triglyceride in plasma, and these enzymes were therefore studied. CASE REPORT 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 pmoles of free fatty acids released per minute per milliliter of plasma, compared to a normal value of 0.218. LCAT activity was 2.5 pg of cholesterol ester- ified per milliliter per hour of incubation, about half the normal value. clotting times rvere normal; an enlarged spleen \cac palpable. A blood transfusion rva\ given. Latet- in childhood. 2 teeth were removed and unusual hemorrhage again occurred. .\t the age of 6Y2 !ears. persistent tleafncss. thought to be due `to eustachian-tube bloc Lage. de\rllq)ed. and adenoidal [issue and tonsillar remnants \\ere remo\cd. The escised rissue had an unusual yellow appearance. microscopy of \\hich revealed increased numbers of large. pale. foam! macro- phages scatlerc(l dilTuwly thr-ou$~out the rissue. .Aftcr the `Ld operation the patient remained well. Ifer schoIa\tic achievemenrs were above average. \lensrru;trion had been normal since the menal-the in 196X. Octa~mnal spon'aneous nosebleeds of small volume had occurred r-e- centlv. There had hecn no symptoms of dial-r-hea or pares- thesia. high-density lipoprotein +I lecithin-cholesterol acyltransferase very-low-density lipoprotein The pa'ienl \+as I(i2.5 cm lall and \\eighed :ii..i kg; nutri- tion seemed normal. The blood pressure \vas IOiiO. Heart sounds Irere normal. There was no I~mphad~nol,ath\. and -the Iiwr and spleen were not palpable. The lundi \\ere normal. as \\ere the co,-neas on superficial examinnrion. The hard palate was high arched and associated wirh displace- ment of the secondar) teeth. Small. ol-angr->ellou. solid le- sions. I cm in diamctel, \\`ere presenr on the poswr-ior. a\- lxct of. the phar) nx. Esamination of the nerwws S\\I~III revealed no almorm,tlit~ UI-inalysir was negza'ive f.or- hlootl. protein, sugar and hilt. `I'hc Iiess ~$1 lbr- capill,rt \ I`ra~:il~t\ \vas negalive. A radin~ral~h of rhc c-hr\t and a pInill r,ldi- ograph 01. the abdomen beI-e nor-ma1. IIenio~Iol~in. \\ hicc- wll count. sewm alkaline l)hosl)h;ctaw. SC, um glutamic- oxalo- acetic tr;~1is;llllill;l~(`. lxw'hionil)in time. 1h1-omll1n clotting time and lk~srna fil~rinogcn H'~I-e alw normal. `I hr-onlbol~l.~\- rin gcncr-afion xi,\ onI> .56 per ~cnl of' normal. .I-he platelet coltnt W;I\ X2.000. I`h e Io\v piatelct count and I-cduc cc1 Ihi-oml~ol)l;lrtin gwwacion wcw no1 e\al~l;ktctl lur ther-. Plasma Lipids and Lipoproteins Blootl wits tdc~i1 in tlicb fxsliiig 5t;ltc \3,itll tliso- dium EKW4 ;IS antico;ii:illn,lt, and tllcx plkisma lippro- teins HUL, low-tleusit)~ iipoprotein (LIIL) and very- low-density iipoproteili (\`LI>L) were sepurated I>> the preparative nletiuik: of Fredrkkson and his CO- workers'O; p-chi0ron~c:~~~ i~riphen)~is ~!fonnte \viis adci- ed to the piusmu in i; j+l concent: ,;.ion Of 2 In>1 to inhibit the activity of I CAT during handling of the plasma." The conte:it of ohoiesteroi'~ and of triglycerideI was measured in each lipoprotein frac- tion. Plasma was submitted to eiectrophoresis on 1 per cent agarose after prestaining of the lipoproteins with Sudan Black B.14 Plasma from the patient and both her parents was tested against antihuman HDL antiserum (Behringwc::-ke) in 1 per cent agarose iI>. the method of double diffusion. Esterified Cholesterol Turnover in Vitro Approximateiy 110 PC of 3H-DL-nlevaionic! acid* (Radiochemical Centrr, Amersham) was injected in- travenously illto 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 intenais after injection for as long as 72 hours. Specific activ- ity-time curves were constructed, and the turno\,er of esterified choiesterc~i was then calculated with the method of Sestel aid Monger.15 Plasma LCAT Activity (Ro?a of Cholesterol Esterification in Vitro) The method of Glo;;:yet and \\`right was `used.16 Plasma sim~ples of the patient with Tangier disease and her parents and a riormal person's plasma \vere heated at 56oC for 4,s minutes to ai~oiisi~ enzyme activity and used us Ihe sollrce of substrate iipo- proteins. Post-Heparin Lipoprotein Lipase Activity Two methods were used: that of Fredrickson et al." and that of Bobei-g anti Carlson.' Hepariu was given intravenously to the patient, her father and a normal person in R dose of 0.1 mg per kilogram of body weight. Blood was tilken 10 and 20 minutes after injectioll and imnlediateiy ciliiled, and the plasma frozen at --10oC: until ready for use as the source of enzyme. Plasma Lipids and Lipoprsteins The concentr;ltim)s of cholesterol anti triglyceride in whole plasma ailtl iii indi\.idliiii lipoproteiils iire shown in Tal~ic 1. The c~l~oiesterol content of the whole piasnl;i of the p!ticllt was 59 mg per 100 ml (70 per cent esterifieti), ;ciiti Of this oni}. 7 infi per 100 1111 M'ilS in tile 1 IDL fraction, ;Ibout l/8 the normni value for ff3n:!les.19 The choi~sterol content of the IllII, in Ijot paretlts WilS lower tha11 nOrm;il.2 The plasma trigi~u!ritie concentration for the p;l- Cent was nt the extreme upper limit Of the norlij;tl range expected for a person of her age mcl sex.1~ \Vhen pinsnm from the patient was sul>jected II I eiectrophoresis on 1 per cent ngarose, no alpha I (high-dellsity) or prebeta (very-low-density) iipoprt,- tein was seen. No precipitin line was seen when plasma from the patient was diffused against auti- IIDL untiserlmi in agnrose. Precipitin lines \\rere seen when the parents' and a normal person's play- ma were used. Esterified Cholesterol Turnover in Vivo Specific activity time curves for esterified and unesterified cholesterol in whole plasma are show11 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 choiestcroi is an esti- FATHER 0 UNESTERIFIED CHOLESTERO . ESTERIFIED CHOLESTEROL 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- DI -Movalonic Acid. Table 1. Cholesterol and Triglyceride Content of Lipoprotein Fractions Derived from Whole Plasma. -- SUUECI AGE (Y R) CHOLESTEROL CONTENT (Mc/lOO ML) TRIGLYCERIDE CONTENT (MoilOO ML) WHOLE PLASMA HDL LDL VLDL WHOLE PLASM.4 HDL LDL "LDL Father 47 167 (75 % csterilied) 31 I13 23 86 14. 30 42 Mother 38 200 (71 % esterified) 26 I51 23 31 12 Ihghter (patient) IS 59 (70 % eslerified) 7 47 5 2 7 49 2 Mde conlrol 31 166 60 98 8 56 16 23 17 m;lte of the turnover rate* of the esterified choles- low value of 1.7 wg was also obtained for the pa- tel.01 pool in question. A measure of the turnover tient when plasma from a normal person was used rate and turnover of the plasma esterified cholester- as substrate, indicating that the additional Iipopro- 01 pool can be obtained by means of the method of tein substrate provided did not enhance the rate of' calculation used by Nestel and Monger'J and ester&&ion. Xloutafis and Myant. 2o The values are shown in Tahle 2 for the whole-plasma esterified cholesterol Post-Heparin Lipoprotein Lip&e Activity pool and for individual plasma lipoprotein pools. The results are shown in Table 4. Xlasimum \.a]- Since a direct measure of the turnover rate of ues for lipoprotein lipase were seen in each case at Table 2. Turnover of Esterified Cholesterol in Vivo. SUBJECT Father Mother Patient ESTERIFIED CHOLwrEROL POOL TURNOVER RATE TURNOVER WG) (Hit-`) Wc/Hd "HOLE "LDL LDL HDI. WHOLE "LDL l.DL HDL WHOLE "LDL LDL "DL PLASMA PLASM* PLASMA 2913 I89 1872 558 0.015 0.022 0.026 0.024 44 4 49 4213 229 3218 465 0.022 0.02 1 0.029 0.022 93 5 93 I 639 90 537 120 0.075 0.039 0.043 I .750' 48 4 23 `L `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.15 Turn- over rate for 1lDL was then calculated by division of turnover by the pool size. Table 2 shows that the turnover rates for esterified ch:;iesterol in the patient's whole plasma and in the individual lipopr&eins were higher than the corresp:: iding values for her parents. The difference is i)articularly striking for the HDL, in which the tu. !:over rate for the Tangier patient was estimated at I.750 per hour. However, the turnover of esterified c~~olesterol in whole plasma, 48 mg per hour, was similar to values reported by NesteP in normal persons. The turnover for the patient's HDL esterified chc!esterol appears higher than for her parents, but it should he emphasized thqt this value was calculated indirectly and that the true magni- tude of difleerc;nce 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 25 bg of cholesterol esterified per milliliter of ac- tive plasma I>,:" hour of incubation was obtained for the patient W~:EII her own plasma was used as sub- strate: this VX.; 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 sampIing time, 10 minutes after the injec- tion of heparin. with both assay methods, the acti\-- ity of the patient's plasma was consider&l>. 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 20 o UNESTERIFIED CHOLESTERO . ESTERIFIED CHOLESTEROL II) 1500 MOTHER I 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-Mevalontc Acid. 570 `l-HE NEW EN(;l.AKD ,]C)UKNAI. 01; ~IEDI<:INE Mar. llj l'r';:! Table 3. LCAT Activity (in Vitro Cholesterol Esterification Rate) ACTIVE PLASMA SOURCE OF ~TERlFlCATlON SUBSTRATE RATE' A: Normal person Normal pcrson 4.5 f 0.4 Father Father 4.1 rf: 0.1 Mother Mother 7.0 * 0.3 Patient Patient 2.5 f 0.2 B: Father Normal person 4.5 rt 0.1 Mother Normal person 6.1 _+ 0.3 Patient (Tangier disease) Normal person 1.7 + 0.3 o pg -of cholesterol esterified/ml of fresh test plasmajhr of incubation (mean 91 SD). the diagno$s 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 IIDL 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 \`LDL 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 nppear- ante to lesions in the pharynx described in other tonsillectomized patients \vitl> Tangier disease.3*5 Also, microscopical examination of adenoidal tissue in our patient showed large numbers of pale macro- phages with foamy cytoplasm assumed to contain lipid material hut not proved to be esterified choles- terol. The patient had a low platelet count on two occasions, as previously described by Hoffman and Fredrickson3 and by Kummer et al.6 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,2' and therefore the pathogenesis of the dep- osition of esterified cholesterol in macrophages can- not be linked with abnormal plnsma turnover. The normal turnover in the patient was brought about by a high turnover rate in a]1 lipoproteins, especial- ly in the IIDL. Furman et al.*2 found that labeled human IIDL disappeared more rnpidl~~ from the cir- culation when the IIDL pool size was reduced. Table 4. Plasma Lipoprotein Lipase Activity 10 and 20 Minutes after Heparin. SUl?JECI LWOPHOTFIN LIPASE AC-~~VITY* COCONVT OIL SUtSTHATEt lNraAt.lPlo S"LwH*TEf at IO nh a, 20 min (rt 10 nrin (I, 20 min Normal person 0.218 0.160 0.080 0.02 I Father 0.234 0.201 0.111 0.061 Patient 0.071 0.040 0.033 0.009 o rmoles of fret fz11ty ;tcids released/ml of pl.usmo/min of incubntion. tin the form of Ediol; method of Fredrickson et ;~l." ;Mclhod of Bobura B G~rlson." One of the factors thought to regulate ~IIC tliiI,. over of esterified cholesterol in plasma is the C~IX! I,,(' LCA'I', the activity of which has been x+soci,i$c,(] with the presence of HDL in the plasm;~.~~~' P.,. tients with genetic deficiency of LCAT ;!]so il.:\ c low plasma HDL concentrations.2s.2fi The activit\ of LCAT has been positively correlated with the WI,- centration of unesterified cholesterol in the p];,+ ma.*' In our patient, low HDL concentration or lo\\. unesterified cholesterol concentration, or both, ma!. have contributed to the low LCAT activity. Ijo\v- ever, the LCAT activity in the patient's plasma did not rise when additional substrate was provided I)!- use of normal plasma, suggesting that the enz)mc concentration itself was diminished. Because in vi\-o turnover of total plasma esterified cholesterol uxs normal, it seems likely that factors in addition to LCAT are responsible for the turnover of esterified cholesterol in the plasma. The postheparin lipoprotein lipaqe activity of our patient's plasma was found to be much lower than that in her father and in a normal person. LOW ill:,+ ma postheparin lipase activity has previousl\, 1:: r`.! described in a patient with LCAT and 1 ;. , deficiency. 2fi Lipoprotein lipase appears to rz.! .i:' HDL, or at least one apoprotein common to I-: i;! and \`LDL as a cofactor, to achieve maximal a~:~.~ ity,28-33 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 li- pase activity. We are indebted to blrs. C. Foxman. Mrs. A. Lynch and Xlrs. G. Porter for technical assistance. I. 2. 3. 4. 5. 6. 7. REFERENCES Fredrickson DS. Altrocchi PH, Avioli LV, et al: Tangier disease: combined clinical staff conference at the National Institute< ~;f Health. 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