Effects of dietary cholesterol on plasma lipoproteins in smith-lemli-opitz syndrome

ABSTRACT Smith-Lemli-Opitz syndrome is a condition of impaired cholesterol synthesis that is caused by mutations in _DHCR7_ encoding 7-dehydrocholesterol-Δ7 reductase. Birth defects and

mental retardation are characteristic. Deficient plasma and tissue cholesterol and excess cholesterol precursors 7 and 8 dehydrocholesterol (7DHC and 8DHC) contribute to the pathogenesis.

Cholesterol is transported to tissues _via_ lipoproteins. We measured the effect of dietary cholesterol (egg yolk) on plasma lipoproteins to evaluate this potential treatment. We used the

enzymatic method to measure total sterols in lipoproteins (_n_ = 12) and plasma (_n_ = 16). In addition, we analyzed individual plasma sterols by a gas chromatographic method. Samples were

evaluated after 3 wk of a cholesterol-free diet and after 6–19 mo of dietary cholesterol. We also analyzed the distribution of sterols in lipoproteins and the apolipoprotein E genotype.

Dietary cholesterol significantly increased the total sterols in plasma (2.22 ± 0.13 to 3.10 ± 0.22; mean ± SEM; _p_ < 0.002), in LDL (0.98 ± 0.13 to 1.52 ± 0.17 mM), and in HDL (0.72 ±

0.04 to 0.92 ± 0.07). Plasma cholesterol increased (1.78 ± 0.16 to 2.67 ± 0.25 mM; _p_ < 0.007) and plasma 7DHC decreased in 10 children, but the mean decrease was not significant. The

distribution of individual sterols in each lipoprotein fraction was similar to the distribution in plasma. The baseline cholesterol and the response to dietary cholesterol was the same in

children with 3/3 and 3/4 apolipoprotein E genotypes. Dietary cholesterol increased total sterols in plasma, LDL, and HDL in children with Smith-Lemli-Opitz syndrome. These favorable

increases in the lipoproteins are potentially therapeutic for this condition. SIMILAR CONTENT BEING VIEWED BY OTHERS EFFECTS OF PHYTOSTEROL SUPPLEMENTATION ON LIPOPROTEIN SUBFRACTIONS AND

LDL PARTICLE QUALITY Article Open access 15 May 2024 METABOLISM OF TRIGLYCERIDE-RICH LIPOPROTEINS IN HEALTH AND DYSLIPIDAEMIA Article 22 March 2022 METABOLOMIC PROFILING OF A CHOLESTEROL

LOWERING PLANT-BASED DIET FROM TWO RANDOMIZED CONTROLLED FEEDING TRIALS Article 22 April 2025 MAIN The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition with multiple

malformations and mental retardation (OMIM #270400). SLOS is caused by mutations in the gene encoding the final enzyme in the cholesterol biosynthetic pathway 7-dehydrocholesterol

Δ7-reductase (DHCR7; E.C 1.3.1.21). Although the syndrome was initially described in 1964 (1), it was not until 30 y later that deficiency of a cholesterol biosynthetic enzyme was identified

as the cause of the syndrome (2–4). The identification and cloning of the gene defective in SLOS, _DHCR7_, were reported in 1998 (5–7). Currently, >80 different mutations in _DHCR7_ have

been found to be associated with SLOS (8,9). SLOS is characterized clinically by microcephaly, cleft palate, dysmorphic facies, limb abnormalities (especially syndactyly of toes 2 and 3),

genital anomalies, endocrine malfunction, cataracts, malformation in the heart and kidney, mental retardation, feeding difficulties, and growth retardation (10). Clinical features are

variable in severity, presumably reflecting the specific causative mutations and the extent of the decrease in enzyme activity. Some mildly affected patients exhibit only a few of the

described features. The biochemical consequences of the enzyme deficiency include abnormally low or low-normal cholesterol concentrations along with accumulation of the precursor that is the

substrate of DHCR7, 7-dehydrocholesterol (7DHC), and its derivative 8-dehydrocholesterol (8DHC). Treatment is sought to correct the biochemical imbalance and improve physical and

psychomotor development. Several studies have been conducted to evaluate the effects of supplemental and dietary cholesterol as a potential treatment for this condition (11–19). These

studies evaluated the effect of supplemental and/or dietary cholesterol on plasma sterol (cholesterol, 7DHC, 8DHC) concentrations. In the current study, in addition to measuring plasma

sterols, we analyzed lipoproteins (LDL, HDL, VLDL, and triglyceride) to test the hypothesis that dietary cholesterol will increase total sterols in LDL. A second objective was to determine

the distribution of cholesterol, 7DHC, and 8DHC in lipoproteins because this has not been reported previously. We measured lipoprotein sterols at baseline (after 3 wk of an essentially

cholesterol-free diet) and after 6–19 mo of dietary cholesterol primarily from egg yolk. A third objective was to determine whether apolipoprotein E (apoE) genotype affects the response to

dietary cholesterol. METHODS SUBJECTS AND STUDY DESIGN. Sixteen children with SLOS were enrolled; two of the children were siblings. We have previously reported data on plasma sterol levels,

sterol synthesis, urinary mevalonate, and DNA mutations in as many as 14 of these children (19–22), but we have not previously reported lipoprotein data. All study children had clinical

features of SLOS and elevated 7DHC and 8DHC plasma concentrations diagnostic of SLOS. The children (Table 1) were admitted to the Oregon Health & Science University General Clinical

Research Center for 1-wk periods. Parents were instructed to feed their child an essentially cholesterol-free diet (baseline diet) at home for 3–4 wk before the first admission to establish

baseline conditions. The cholesterol content of these diets ranged from 0.4 to 3.3 mg cholesterol · kg−1 · d−1; one child was breast fed and consumed 14 mg cholesterol · kg−1 · d−1 contained

in breast milk. The baseline diet period was repeated when the initial study was unsuccessful as a result of illness, nonadherence to diet, difficulties with sample collection, or other

reason. During these admissions, the baseline diet was continued under metabolic ward conditions. On discharge, children were prescribed dietary cholesterol as egg yolk and/or butter fat. In

a few cases, crystalline cholesterol in oil or emulsion was prescribed. For all other admissions, the children continued to receive cholesterol. Children returned every 3–12 mo for

monitoring. Blood samples were evaluated during the last week of the baseline diet and after 6–19 mo of dietary cholesterol. Details of the diets are described in previous reports (18,19).

In summary, children aged 0–6 mo were fed one half egg yolk/d; children aged 6–12 mo were fed 1 egg yolk/d; and children aged 12 mo or older were fed 1.5 to 2 egg yolks/d. On the basis of an

estimate of 210 mg of cholesterol per egg yolk, the children received ∼18–60 mg · kg−1 · d−1 of cholesterol. Eggs were hardboiled, and the yolks removed and mixed with formula, breast milk,

or solid food, according to the child's regular diet. At times, egg yolk was cooked in formula or milk. Egg yolks were also provided as powdered egg yolk (subjects 5 and 9) or as whole

eggs (in subject 8 for 46% of the duration of the dietary cholesterol period). Powdered egg yolk was stored at 4°C to prevent oxidation of its cholesterol. Six children received butter fat,

heavy cream, crystalline cholesterol in soybean oil (16), or crystalline cholesterol in an aqueous suspension (23) instead of egg yolk during some or all of the supplemental cholesterol

period. Table 2 describes the source and duration of the supplemental cholesterol (other than egg yolk) for these children. Feeding difficulties are common in SLOS, and seven of the 16

children in our study required tube feeding. The same dietary protocols were used for children who were tube fed or orally fed. These studies were approved by the Oregon Health & Science

University (Portland, OR) Institutional Review Board; the parents gave informed consent for the participation of their child. LIPOPROTEIN AND PLASMA STEROL ANALYSIS. Total sterols in each

lipoprotein fraction and in plasma as well as plasma triglycerides were measured by the standard clinical method (enzymatic method) with a Hitachi 704 Chemistry Analyzer, in compliance with

the standardization and surveillance programs of the Centers for Disease Control and Prevention Laboratory (Atlanta, GA) (24,25). This enzymatic method uses cholesterol esterase and

cholesterol oxidase and overestimates plasma cholesterol concentrations from children with SLOS because dehydrocholesterols are substrates for cholesterol oxidase and contribute to the

resulting value (26). In plasma, the sum of the sterols (measured individually by gas chromatographic method) was similar to the total sterols (measured by enzymatic method) at baseline and

after dietary cholesterol (_R_2 = 0.98). Therefore, we assumed for subsequent analyses by the enzymatic method that each value represented “total sterols,” that is, cholesterol, 7DHC, and

8DHC. We measured the concentrations of individual sterols (cholesterol, 7DHC, and 8DHC) in each lipoprotein fraction (LDL, HDL, and VLDL) after separation by ultracentrifugation (24) (_n_ =

2). This ultracentrifugation step requires more blood than is routinely obtained in these small children, so we were able to do this only with two children. Both children were receiving

dietary cholesterol at the time of measurement. In all of the children, we measured the concentrations of individual sterols in plasma. These individual plasma sterol concentrations were

measured by capillary-column gas chromatography with a CP-Wax 57 column (25 M, 0.32 mm ID; Chrompack Co., Raritan, NJ) (5,20). Internal standard (5α-cholestane) and an authentic standard of

the cholesterol were used for calibration. In the two children, the distribution of individual sterols in each lipoprotein was the same as the distribution in plasma. Assuming that this was

the case for all of the children, we calculated the concentrations of cholesterol, 7DHC, and 8DHC in each lipoprotein fraction. For example, the concentration of cholesterol in LDL equals

the ratio of cholesterol to total sterols in plasma sample (gas chromatographic method) multiplied by the total sterol concentration in LDL (enzymatic method). Data are expressed as means ±

SEM. Statistical analysis of differences between means was determined using a two-tailed _t_ test for paired samples. APOE GENOTYPING. The apoE gene has three alleles, ε2, ε3, and ε4, coding

for the three major isoforms (E2, E3, and E4) of this protein. The alleles were identified by PCR amplification of nucleotides 2823–3093 followed by restriction endonuclease digestion with

_Hha_I. The six possible genotypes were determined from the sizes of the DNA fragments (27). RESULTS EFFECT OF DIETARY CHOLESTEROL ON LIPOPROTEINS AND TOTAL STEROLS. By the enzymatic method,

we measured total sterols in plasma (16 children) and total sterols in each lipoprotein fraction (12 children). Measurements that were taken at baseline (after 3 wk of an essentially

cholesterol-free diet) were compared with measurements after 6–19 mo of dietary cholesterol. The total sterols in plasma increased from 2.22 to 3.10 mM (_p_ < 0.002; Table 3). Six

children received alternative sources of cholesterol instead of egg yolk during some or all of the supplemental cholesterol period. The total sterols in this subgroup increased from 2.38 ±

0.28 to 3.26 ± 0.28 mM. Total sterols in LDL (enzymatic method) increased significantly (from 0.98 to 1.52 mM; _p_ < 0.02) in parallel with the increase in total plasma sterols. HDL

increased similarly from 0.72 to 0.92 mM (_p_ < 0.03). There were no changes in total sterols in VLDL (0.49 to 0.58 mM; _p_ = 0.48) or in plasma triglyceride concentrations (1.11 to 1.24

mM; _p_ = 0.49) in response to the dietary cholesterol. Under normal conditions, changes in dietary cholesterol do not affect VLDL cholesterol or plasma triglyceride concentrations (28). In

children 5–9 y of age, the normal triglyceride concentration in fasting plasma ranges from 31 to 104 mg/dL (29). Our values for triglycerides are slightly higher, likely because fasting

samples could not be obtained from infants. EFFECT OF DIETARY CHOLESTEROL ON PLASMA CONCENTRATIONS OF CHOLESTEROL, 7DHC, AND 8DHC. To determine whether the increase in total sterols resulted

specifically from an increase in the cholesterol component of plasma, we measured plasma cholesterol, 7DHC, and 8DHC concentrations in 16 SLOS children at baseline (after 3 wk of

essentially cholesterol-free diet) and after 6–19 mo of dietary cholesterol. Dietary cholesterol raised plasma cholesterol from 1.78 to 2.67 mM (_p_ < 0.007; Table 4). Not only did the

concentration of plasma cholesterol increase, but also the ratio of cholesterol relative to the sum of all sterols increased from 0.79 to 0.84. Dietary cholesterol did not significantly

decrease the mean plasma 7DHC (Table 4) or 8DHC (0.23–0.21 mM and 0.19–0.20 mM, respectively). At baseline, there was no correlation between the concentrations of cholesterol and 7DHC. After

dietary cholesterol, however, there was a significant negative correlation (_R_2 = 0.62, _p_ < 0.01; Fig. 1). Children who responded to the dietary cholesterol with the greatest increase

in plasma cholesterol concentrations tended to obtain the lowest 7DHC concentrations. DISTRIBUTION OF STEROLS WITHIN EACH LIPOPROTEIN FRACTION. The increase in total sterols in LDL and HDL

could result from an increase either in cholesterol or in the precursor sterols 7DHC or 8DHC. The plasma samples from two children had an adequate volume to separate the VLDL, LDL, and HDL

by ultracentrifugation. We then measured the individual sterol concentrations (cholesterol, 7DHC, and 8DHC) by gas chromatographic method in each fraction. We compared the distribution of

individual sterols in each lipoprotein with the distribution of individual sterols measured in plasma (Fig. 2). The concentrations of total sterols in each of these two children (subjects 7

and 9) were different from one another (2.43 and 1.24 mM, respectively). In addition, the distribution of cholesterol relative to the total sterols in plasma was different in these two

children (90 and 70%, respectively). For each child individually, however, the distribution of cholesterol, 7DHC, and 8DHC (relative to total sterols) in each lipoprotein fraction was the

same as the distribution in plasma (Fig. 2). Assuming that this is true for all cases, we calculated the concentration of cholesterol in LDL and HDL for each child (_n_ = 12). Results showed

that LDL cholesterol increased from 0.73 ± 0 0.13 to 1.31 ± 0.18 mM (_p_ < 0.02) and HDL cholesterol increased from 0.57 ± 0.05 to 0.79 ± 0.08 mM (_p_ < 0.03). The concentrations of

7DHC in LDL (0.10 mM and 0.11 mM) or 8DHC in LDL (0.09 and 0.10 mM) did not change. LACK OF EFFECT OF APOE GENOTYPE ON PLASMA CHOLESTEROL AND ON EFFICACY OF DIETARY CHOLESTEROL. Plasma

sterols in SLOS children varied widely at baseline and after dietary cholesterol. Of the three isoforms of apoE, the E4 isoform is associated with increased concentration of plasma

cholesterol (30). We determined whether apoE genotype could account for some of this variability. Three of the SLOS children had the 3/4 apoE genotype, whereas nine had the 3/3 genotype.

Plasma cholesterol concentrations, however, were similar in SLOS children with either genotype at baseline (1.74 _versus_ 1.80 mM; _p_ = 0.90) and after 6–19 mo of dietary cholesterol (2.62

_versus_ 2.81 mM; _p_ = 0.80; 3/4 and 3/3, respectively). DISCUSSION Cholesterol deficiency is characteristic of SLOS and may be a major contributor to the characteristic clinical features.

Cholesterol is an essential component of cell membranes and nervous system neurons and myelin, as well as serving as the substrate for important biologic compounds, including bile acids and

steroid hormones. Recently, cholesterol was shown to be an essential component of the lipid rafts involved in intracellular trafficking of proteins and lipids (31) and cell surface signaling

proteins (32). In addition, cholesterol has been shown to promote CNS synaptogenesis (33). Cholesterol also plays a critical role during fetal development. The cholesterol molecule is a

covalently bound component of active hedgehog proteins, a family of embryonic signaling proteins (34). Cholesterol deficiency could alter the function of the hedgehog signaling pathway. In

SLOS, some of the pathologic characteristics may also be due to accumulation of cholesterol precursors. For example, accumulation of 7DHC in the brain has been associated with impaired

learning in rats (35). 7DHC has also been shown to be teratogenic in rat embryos (36). Therefore, treatment for SLOS that normalizes plasma and tissue cholesterol levels while decreasing

accumulation of potentially toxic cholesterol intermediates and metabolites is sought. Dietary cholesterol has been considered a potential treatment. Lipoproteins are essential for transport

of dietary cholesterol from the intestines to the other tissues of the body as well as for cellular uptake of cholesterol. We measured total sterols in each lipoprotein fraction in 12

children. The total sterols significantly increased in LDL and HDL in response to dietary cholesterol. The ratios of noncholesterol sterols to cholesterol in serum do not always predict the

ratios in the different lipoproteins (37). Therefore, in two children, the concentration of each sterol within each lipoprotein was measured to determine the distribution of the sterols in

lipoproteins. In these two children, the distribution of individual sterols within each lipoprotein fraction was shown to be the same as the distribution in plasma. We calculated the

concentration of each sterol in each lipoprotein fraction in 12 children using the assumption that plasma sterol distribution equaled lipoprotein sterol distribution. These results showed

that after dietary cholesterol, _1_) the cholesterol concentrations in LDL and HDL increased, and _2_) there was not a preferential increase of 7DHC or 8DHC in either LDL or HDL. The

increases in LDL and HDL cholesterol in these children in response to dietary cholesterol could provide a larger pool of cholesterol available for tissue uptake. It has been shown that

125I-HDL2 from plasma of patients with abetalipoproteinemia was bound, taken up, and degraded by cultured human fibroblasts (37a). This suggests that HDL in addition to LDL can transport

cholesterol to tissues. Kinetic studies will be needed to determine whether there is a corresponding increase in LDL and HDL cholesterol uptake by cholesterol-deficient tissues in SLOS. Only

two published studies have reported lipoprotein data in SLOS patients. Pierquin _et al._ (38) described one severely affected child. This child had low concentrations of total sterols in

LDL (13 mg/dL) and HDL (8 mg/dL) and a low concentration of apoA-I (67 mg/dL), the major protein of HDL, as compared with normal ranges. Behulova _et al._ (39) performed similar measurements

in eight children who had SLOS and were not receiving supplemental or dietary cholesterol. Five of the children were severely affected, and three had less severe clinical features. All

eight children studied had low concentrations of total sterols in LDL (range: 0–1.18 mM) and HDL (0.13–0.84 mM) and low concentrations of apoA-I (<0.12–0.94 g/dL). The concentrations of

total sterols in VLDL were normal or above normal, and the concentrations of apoB were normal or borderline low. Neither the effect of dietary cholesterol on lipoproteins nor the

distribution of the individual sterols (in lipoprotein fractions or in plasma) was measured in these two studies. The effect of dietary cholesterol on plasma sterols was similar to our

previous report (19). After 6–19 mo of dietary cholesterol with egg yolk (in most cases), the mean plasma cholesterol in children with SLOS significantly increased when compared with the

mean concentration at baseline. The increase in cholesterol (57%, _n_ = 16) found in the current study was similar to the increase in cholesterol (55%, _n_ = 7) after 1–3 mo of dietary

cholesterol in our earlier study (19). These percentage increases were greater than those increases seen in adults who were given high-cholesterol diets (40). After dietary cholesterol,

plasma cholesterol rose to above the fifth percentile in four children of the current study, 3.18 mM for normal children aged 5–9 y (29). Although 10 children showed a decrease in plasma

7DHC (range: 8–77%), six children had an increase or no change. After dietary cholesterol, the lowest concentration of 7DHC in the SLOS children, 0.04 mM, was still higher than normal,

0.00026 mM (41). The bioavailability of egg yolk cholesterol _versus_ crystalline cholesterol has not been reported recently. We chose to use egg yolk as the source of cholesterol in most

subjects because there is some evidence that it is more effective in raising plasma cholesterol concentrations. In normal adults, dietary egg yolk increased plasma cholesterol concentrations

more readily than crystalline cholesterol supplementation (69 _versus_ 19 mg/dL), even though the subjects consumed lower amounts of cholesterol in the form of dietary egg yolks (475–1425

mg/d) than the amounts consumed as cholesterol in the crystalline form (1200–3600 mg/d) (42). In this present study, the increases in plasma concentrations were similar in both groups.

However, the number of children is too small to determine whether the source of cholesterol made a difference in the plasma sterol response. The absorption of cholesterol from the intestine

is complex (43) and may be dependent on many factors, including emulsification, micelle formation (44), and cholesterol transporters (45,46). Dietary cholesterol presumably would lower

plasma 7DHC and 8DHC in SLOS by inhibiting hepatic hydroxymethyl glutaryl (HMG) CoA reductase through feedback inhibition of this rate-limiting enzyme. We have shown that dietary cholesterol

(for 2 mo or 2 y) decreased HMG CoA reductase activity as measured by the amount of mevalonate in 24-h urine samples. Only after 2 y, however, was there a decrease in plasma 7DHC (_n_ = 4)

(19). These results and the wide range of responses of plasma 7DHC in our current study reflect the complex regulation of the cholesterol biosynthetic pathway. CONCLUSION This is the first

report of the effect of dietary cholesterol on sterols in lipoproteins in children with SLOS and of the distribution of cholesterol, 7DHC, and 8DHC in lipoproteins. Increase of total sterols

in plasma in response to dietary cholesterol was paralleled by increases of total sterols in LDL and HDL. Calculated concentrations of cholesterol in LDL and HDL also increased. This bodes

well for potential therapy of SLOS, because the increased LDL and HDL cholesterol provides a larger pool of cholesterol for tissue uptake. The plasma 7DHC concentrations did not return to

normal in the current study. Although dietary cholesterol inhibits HMG CoA reductase in SLOS (19), further inhibition, perhaps by statin drugs, may be necessary to normalize the 7DHC.

ABBREVIATIONS * apoE: apolipoprotein E * DHCR7: 7-dehydrocholesterol Δ7-reductase * HMG: hepatic hydroxymethyl glutaryl * SLOS: Smith-Lemli-Opitz syndrome * 7DHC: 7-dehydrocholesterol *

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ACKNOWLEDGEMENTS We thank Gary Sexton, Ph.D., for assistance with statistical analysis and Pam Smith and Carol Marsh for lipoprotein separation. We thank the children and families for

participation in the study and the staff of the Oregon Health & Science University General Clinical Research Center. We thank Sandra Banta-Wright, R.N., M.S.N., N.N.P., Daniel Marks,

M.D., and numerous other health care providers who assisted in the care of these children. We thank the many health care providers who kindly referred these children to us. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Pediatrics, Department of Medicine, OR Health & Science University, Portland, 97239, OR Louise S Merkens & Robert D Steiner *

Division of Endocrinology, Department of Medicine, Metabolism and Nutrition, OR Health & Science University, Portland, 97239, OR William E Connor, Don S Lin & Donna P Flavell *

Department of Genetics, Kaiser Permanente Northwest, Portland, 97227, OR Leesa M Linck * Department of Molecular and Medical Genetics, Child Development and Rehabilitation Center,

Doernbecher Children's Hospital, OR Health & Science University, Portland, 97239, OR Leesa M Linck & Robert D Steiner Authors * Louise S Merkens View author publications You can

also search for this author inPubMed Google Scholar * William E Connor View author publications You can also search for this author inPubMed Google Scholar * Leesa M Linck View author

publications You can also search for this author inPubMed Google Scholar * Don S Lin View author publications You can also search for this author inPubMed Google Scholar * Donna P Flavell

View author publications You can also search for this author inPubMed Google Scholar * Robert D Steiner View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHOR Correspondence to Robert D Steiner. ADDITIONAL INFORMATION Supported by grants from the National Heart, Lung, and Blood Institute (PHS HL-64618 and HL-073980); National

Center for Research Resources (PHS 5 M01 RR000334 and PHS 3 M01 RR003344-33S3); National Institute for Child Health and Human Development, OR Child Health Research Center (PHS 5P30

HD33703-04); the American Academy of Pediatrics Section on Genetics and Birth Defects; the Collins Foundation; and the Smith-Lemli-Opitz Advocacy and Exchange. R.D.S. is a Clinical Associate

Physician Investigator of the Oregon Health & Science University General Clinical Research Center. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Merkens, L., Connor, W., Linck, L. _et al._ Effects of Dietary Cholesterol on Plasma Lipoproteins in Smith-Lemli-Opitz Syndrome. _Pediatr Res_ 56, 726–732 (2004).

https://doi.org/10.1203/01.PDR.0000141522.14177.4F Download citation * Received: 20 January 2004 * Accepted: 07 July 2004 * Issue Date: 01 November 2004 * DOI:

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