Markedly inhibited 7-dehydrocholesterol-delta 7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes. S Shefer, … , T C Chen, M F Holick J Clin Invest. 1995;96(4):1779-1785. https://doi.org/10.1172/JCI118223. Research Article We investigated the enzyme defect in late cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome, a recessively inherited developmental disorder characterized by facial dysmorphism, mental retardation, and multiple organ congenital anomalies. Reduced plasma and tissue cholesterol with increased 7-dehydrocholesterol concentrations are biochemical features diagnostic of the inherited enzyme defect. Using isotope incorporation assays, we measured the transformation of the precursors, [3 alpha- 3H]lathosterol and [1,2-3H]7-dehydrocholesterol into cholesterol by liver microsomes from seven controls and four Smith-Lemli-Opitz homozygous subjects. The introduction of the double bond in lathosterol at C- 5[6] to form 7-dehydrocholesterol that is catalyzed by lathosterol-5-dehydrogenase was equally rapid in controls and homozygotes liver microsomes (120 +/- 8 vs 100 +/- 7 pmol/mg protein per min, P = NS). In distinction, the reduction of the double bond at C-7 [8] in 7-dehydrocholesterol to yield cholesterol catalyzed by 7-dehydrocholesterol-delta 7- reductase was nine times greater in controls than homozygotes microsomes (365 +/- 23 vs 40 +/- 4 pmol/mg protein per min, P < 0.0001). These results demonstrate that the pathway of lathosterol to cholesterol in human liver includes 7- dehydrocholesterol as a key intermediate. In Smith-Lemli-Opitz homozygotes, the transformation of 7-dehydrocholesterol to cholesterol by hepatic microsomes was blocked although 7-dehydrocholesterol was produced abundantly from lathosterol. Thus, lathosterol 5-dehydrogenase is equally active which indicates that homozygotes liver microsomes are viable. Accordingly, microsomal 7-dehydrocholesterol-delta 7-reductase is inherited […] Find the latest version: https://jci.me/118223/pdf Markedly Inhibited 7-Dehydrocholesterol-A7-Reductase Activity in Liver Microsomes from Smith-Lemli-Opitz Homozygotes Sarah Shefer,* Gerald Salen,** A. K. Batta,* A. Honda,* G. S. Tint,*" Mira Irons,§ Ellen R. Elias,§ Tai C. Chen,11 and M. F. Holick1l *UMD-New Jersey Medical School, Newark, NJ, 07103; tVeterans Affairs Medical Center, East Orange, NJ, 07019; §Tufts University Medical School, Boston, MA, 02155; and 1 Boston University Medical Center, Boston, MA, 02155 Abstract reduction of 3-hydroxy-3-methylglutaryl (HMG)' CoA (1, 2). The reaction is catalyzed by the microsomal enzyme, HMG- We investigated the enzyme defect in late cholesterol biosyn- CoA reductase, and is considered rate determining as the activ- thesis in the Smith-Lemli-Opitz syndrome, a recessively in- ity of the enzyme varies under feedback control. Unused sub- herited developmental disorder characterized by facial strate, HMG-CoA, can be converted to fatty acids and CO2 and dysmorphism, mental retardation, and multiple organ con- does not accumulate (3, 4). Subsequently, mevalonic acid is genital anomalies. Reduced plasma and tissue cholesterol transformed to isopentenyl pyrophosphate and two molecules with increased 7-dehydrocholesterol concentrations are bio- joined into geranyl pyrophosphate with a third molecule added chemical features diagnostic of the inherited enzyme defect. to form farnesyl pyrophosphate. Squalene arises from the con- Using isotope incorporation assays, we measured the trans- densation of two molecules of farnesyl pyrophosphate and is formation of the precursors, [3a-3H]lathosterol and [1,2- then cyclized into lanosterol, a 30 carbon sterol. In the reactions 3H]7-dehydrocholesterol into cholesterol by liver micro- from lanosterol to form cholesterol, three methyl groups are somes from seven controls and four Smith-Lemli-Opitz ho- eliminated at carbons C-4, C-4', and C-14, the double bonds mozygous subjects. The introduction of the double bond in in the side chain at C-24 [25], and in ring B at C-8 [9] are lathosterol at C-5[6] to form 7-dehydrocholesterol that is reduced, and a double bond inserted at C-5 [6] (5, 6). Latho- catalyzed by lathosterol-5-dehydrogenase was equally rapid sterol and 7-dehydrocholesterol are the last two precursors in in controls and homozygotes liver microsomes (120+8 vs the pathway before cholesterol. 100+7 pmol/mg protein per min, P = NS). In distinction, Recently, we have described an abnormality in late choles- the reduction of the double bond at C-7 [8] in 7-dehydro- terol biosynthesis in subjects with the Smith-Lemli-Optiz syn- cholesterol to yield cholesterol catalyzed by 7-dehydro- drome (7, 8). Homozygotes with this recessively inherited con- cholesterol-A7-reductase was nine times greater in controls dition cannot form cholesterol efficiently, and accumulate the than homozygotes microsomes (365±23 vs 40+4 pmol/mg final precursor, 7-dehydrocholesterol, in plasma and tissues protein per min, P < 0.0001). These results demonstrate (Fig. 1). As a result of deficient biosynthesis, organs are de- that the pathway of lathosterol to cholesterol in human liver prived of cholesterol and contain substantial quantities of 7- includes 7-dehydrocholesterol as a key intermediate. In dehydrocholesterol so that development is hindered and critical Smith-Lemli-Opitz homozygotes, the transformation of 7- functions are impaired. Smith-Lemli-Opitz homozygotes mani- dehydrocholesterol to cholesterol by hepatic microsomes fest facial dysmorphic features and congenital anomalies affect- was blocked although 7-dehydrocholesterol was produced ing multiple organs particularly the brain, where cholesterol abundantly from lathosterol. Thus, lathosterol 5-dehydroge- normally constitutes 25% of the myelin, and substantial amounts nase is equally active which indicates that homozygotes liver are present in gray matter. All are mentally retarded (9, 10). microsomes are viable. Accordingly, microsomal 7-dehydro- We postulated the inherited enzyme defect involves 7-dehydro- cholesterol-A7-reductase is inherited abnormally in Smith- cholesterol-A/7-reductase, that catalyzes the conversion of 7- Lemli-Opitz homozygotes. (J. Clin. Invest. 1995. 96:1779- dehydrocholesterol to cholesterol (Fig. 1) which is the penulti- 1785.) Key words: abnormal cholesterol biosynthesis - 7- mate reaction in the biosynthetic pathway (7, 8, 11-13). In dehydrocholesterol * Smith-Lemli-Opitz syndrome support of this reasoning, rats treated with BM 15.766, an inhib- itor of 7-dehydrocholesterol-L\7-reductase became deficient in Introduction cholesterol and accumulated 7-dehydrocholesterol in all tissues reproducing the biochemical abnormalities as seen in Smith- According to current theory, (Fig. 1) mevalonic acid is the first Lemli-Opitz homozygotes (14, 15). precursor in the cholesterol biosynthetic pathway that is almost To prove that 7-dehydrocholesterol-A 7-reductase is abnor- totally committed to cholesterol synthesis. It is formed from the mal in Smith-Lemli-Opitz homozygotes, the final two reactions in the cholesterol biosynthetic pathway were investigated (Fig. Address correspondence to Sarah Shefer, UMD-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. Phone:201-982- 1. Abbreviations used in this paper: GLC, gas-liquid chromatography; 5043; FAX:201-982-6761. HMG, hydroxymethyl glutaryl; TMS, trimethylsilyl. The following Received for publication 2 March 1995 and accepted in revised names are also used: cholesterol, 5-cholesten-3/3-ol; 7-dehydrocholes- forn 9 June 1995. terol, 5,7-cholestadien-3f3-ol; 8-dehydrocholesterol, 5,8-cholestadien- 3,3-ol; lathosterol, 7-cholesten-3,8-ol; 8-cholestenol, 8-cholesten-3,f- The Journal of Clinical Investigation, Inc. ol; lanosterol, 4,4,14-trimethyl 8(9), 24(25)-cholestadien-3,/-ol. Volume 96, October 1995, 1779-1785 24-dihydrocholanosterol, 4,4,14-trimethyl-8 (9) -cholesten-3,6-ol. Abnormal Cholesterol Synthesis in Smith-Lemli-Opitz Homozygotes 1779 ACETATE Solvent HMG-CoA front *HMG-CoA reductose MEVALONATE LANOSTEROL Figure 1. Flow diagram showing key intermedi- ates in the pathway for 'if the formation of choles- terol from acetate. HMG- CoA reductase is consid- * VII ered the rate-controlling enzyme that regulates the * VI HO formation of mevalonic acid, the first almost to- Figure 2. The separation Lat hosterol tally committed precur- V of precusors from cho- sor in the pathway. La- lesterol on Silicia Gel G nosterol the first cyclized * IV plates containing Ag Lotho -5-dehydrogenose sterol is also noted. Two NO3. The following Rf key reactions in late cho- S ||| values were observed: I, lesterol biosynthesis are 7-dehydrocholesterol, "I emphasized: the conver- 0.09; II, 8-dehydrocho- sion of lathosterol to 7- * 11 lesterol, 0.14; III, dehydrocholesterol cat- desmosterol, 0.27; IV, HO alyzed by lathosterol-5- cholesterol, 0.31; V, dehydrogenase, and the lathosterol, 0.37; VI, 7- Dehydrocholesterol transformation of 7-de- lanosterol, 0.51; and VII, hydrocholesterol to cho- uctOse . OriginO 24(25)-dihydro- 7- DCH- A- red' lesterol catalyzed by 7- lanosterol, 0.56. Solvent dehydrocholesterol-L\7- system/chlorform/ reductase. The suspected acetone, 57:2, vol/vol. abnormal reaction be- tween 7-dehydrocholest- erol and cholesterol in the HO Smith-Lemli-Opitz syn- drome is denoted by a clinical phenotype that included microcephaly, sloping forehead, ptosis, Cholesterol cross. flat nasal bridge, low set posteriorly rotated ears, micrognathia, polydac- taly, and syndactyly of the second and third