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(Investigative Ophthalmology and Visual Science. 2001;42:1707-1714.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

The Metabolism of Fatty Acids in Human Bietti Crystalline Dystrophy

June Lee1, Xiaodong Jiao1, J. Fielding Hejtmancik1, Muriel Kaiser-Kupfer1, William A. Gahl2, Thomas C. Markello2, Juanru Guo2 and Gerald J. Chader3

1 From the Ophthalmic Genetics and Clinical Services Branch, National Eye Institute and the 2 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; and the 3 Foundation Fighting Blindness, Hunt Valley, Maryland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the role of abnormal lipid metabolism in Bietti crystalline dystrophy.

METHODS. Cultured human lymphocytes and fibroblasts from patients with Bietti crystalline dystrophy (BCD) were incubated in the presence of [14C]18:3n-3 or [14C]18:2n-6. Incorporation into the cellular lipid pools and further metabolism by desaturation or elongation were monitored by thin-layer chromatography and HPLC. Results were compared with those in normal control subjects and patients with Wolman disease (WD).

RESULTS. Pulse–chase experiments with labeled fatty acids in all groups showed that, after 1 hour, radioactivity was largely confined to the triacylglyceride (TG) and choline phosphoglyceride (CPG) pools. However, after several hours, radioactivity was transferred from the TG and CPG pools, some going to the serine and ethanolamine phosphoglyceride (SPG and EPG) pools. Fibroblasts from all groups showed direct transfer of fatty acids (FAs) into CPG and EPG. Incorporation of labeled FAs into the EPG pool paralleled extensive desaturation and elongation of 18:2n-6 to 22:5n-6 and 18:3n-3 to 22:6n-3. Fibroblasts from patients with WD (a lysosomal acid lipase deficiency characterized by excessive lipid accumulation), showed higher incorporation of 18:2n-6 into TGs than did normal or BCD fibroblasts. Conversely, fibroblasts from patients with BCD showed lower conversion of 18:3n-3, but not of 18:2n-6, into polyunsaturated FAs (PUFAs) than those of normal subjects or patients with WD. This was true for total FAs, CPGs, and EPGs. Similar results were found in both fibroblasts and lymphocytes; however, unlike fibroblasts, lymphocytes from normal subjects showed similar levels of incorporation of FAs into EPGs and CPGs. In contrast, incorporation of 18:3n-3 into EPGs was decreased in lymphocytes from patients with BCD.

CONCLUSIONS. BCD is characterized by a lower than normal conversion of FA precursors into n-3 PUFA, whereas there is a higher than normal level of n-6 and n-3 FAs incorporation into TGs in cells from patients with WD. These findings raise the possibility that abnormal lipid metabolism associated with BCD is the result of deficient lipid binding, elongation, or desaturation in contrast to the lysosomal acid lipase deficiency found in Wolman disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bietti crystalline dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by multiple glistening intraretinal dots scattered over the fundus. These are associated with degeneration of the retina and sclerosis of the choroidal vessels, which ultimately results in progressive night blindness and constriction of the visual field. This condition is rare worldwide, having been reported in approximately 85 patients, but appears to be more common in individuals of Asian descent.1 2 From the frequency of first-cousin parents in his series, Hu1 estimated the frequency of the gene in China to be 0.005. Wilson et al.3 found crystals resembling cholesterol or cholesterol esters in the retina, and complex lipid inclusions in the cornea, conjunctiva, fibroblasts, and circulating lymphocytes, suggesting BCD may result from a systemic abnormality of lipid metabolism. More recently, histopathologic studies of the eye have demonstrated advanced panchorioretinal atrophy with crystals and complex lipid inclusions in choroidal fibroblasts.2

Wolman disease (WD) is a severe autosomal recessive disorder caused by mutations in lysosomal acid lipase,4 the same enzyme that is deficient in the clinically milder cholesterol ester storage disease.5 There is accumulation of triglycerides (TGs) and cholesterol esters (CEs) in the lysosomes of affected tissues, primarily the liver, adrenal glands, spleen, lymph nodes, bone marrow, and small intestine, but also the lung, thymus, skin, retina, and central nervous system.6 Two additional isozymes of lysosomal acid lipase exist, whose physiological role is unclear. Polyunsaturated fatty acids (PUFAs) are important constituents of cell membranes and play a significant role in cellular structure and function.7 Abnormalities of FA metabolism have been shown to cause several hereditary disorders, and disturbances of PUFA metabolism have already been described in a variety of pathologic conditions. These findings prompted us to analyze FA metabolism in human skin fibroblasts and lymphocytes from patients affected with BCD, a genetic disorder characterized by abnormal lipid deposition.

In this study, we characterized the metabolic flow of lipids in fibroblasts and lymphocytes of patients with BCD and compared the findings with those in normal control subjects and patients with WD. The metabolic lesion in WD is known, but the effect on processing of FAs has not been previously investigated. In BCD, the genetic lesion has not yet been determined. Although aberrations in the metabolic flow of lipids were present in both diseases, they were distinct, suggesting that, unlike WD, BCD may result from a deficiency of lipid binding or in FA desaturation or elongation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and Reagents
Reagents used for growth and maintenance of cells in culture were obtained from Gibco BRL Life Technologies, Inc. (Gaithersburg, MD) and fetal calf serum from Sigma Chemical Co. (St. Louis, MO). Lipid-deficient fetal calf serum (LDS) was prepared by the method of Bailey and Dunbar.8 Analysis of LDS by HPLC showed that more than 95% of the FAs had been removed. Protein recovery was in excess of 90%.

Patients
The clinical diagnosis of BCD was based on1 crystalline lysosomal material visible on histologic examination of lymphocytes and skin fibroblasts and2 clinical diagnosis based on the presence of superficial corneal crystals observed at the corneoscleral limbus by slit lamp biomicroscopy; abundant small, sparkling, yellow-white crystals in the posterior pole; and atrophy of retinal pigment epithelium, sclera, and choroid on funduscope examination.2 The diagnosis of WD was made on the basis of a severe deficiency of acid lipase and massive lysosomal storage of triacylglycerides and CEs, associated with hepatosplenomegaly and adrenal calcification. The study protocol adhered to the tenets of the Declaration of Helsinki, and informed consent was obtained from all participants.

Fibroblast and Lymphocyte Cultures
Fibroblasts from forearm skin specimens obtained by biopsy in three patients with BCD, one patient with WD, and three normal human female donors were routinely grown for approximately 4 weeks in an atmosphere of 95% air and 5% carbon dioxide at 37°C in Eagle’s minimal essential medium (EMEM) with Eagle’s salt solution supplemented with 20% fetal calf serum and containing L-glutamine (2.0 mM), penicillin (100 U/ml), and streptomycin (100 g/ml). Cells were subcultured at confluence (usually 1 week after inoculation) by removing the medium and washing twice with 2 ml Hanks’ Ca-Mg–free solution, followed by treatment with a 0.125% trypsin solution. After 10 minutes at 37°C, the cells had detached from the culture flask and were harvested by centrifugation for 5 minutes at 300g. Subcultures were prepared at 1:2 splits, as described by Mathers and Bailey9 and modified by Alberts et al.10 Similar studies were conducted on five transformed lymphocyte cultures from two normal subjects and three patients with BCD. The uptake of FAs by cultured human lymphocytes and fibroblasts did not appear to be influenced by the age of the donor within the limits of our experiment, nor by passage number. The growth rate or cellular morphology of cultured fibroblasts or lymphocytes did not vary with the disease status of the donor.

Metabolic Studies
For uptake studies, cells were plated on 60-mm petri dishes (surface area, 28 cm2) in 2 ml EMEM supplemented with 10% fetal calf serum at inoculum densities ranging from 1.4 to 2.2 x 104 cells/cm2. After incubation for 24 hours at 37°C, the medium was removed, and 2 ml experimental medium, consisting of EMEM supplemented with 10% LDS and containing 2 µCi [14C]18:2n-6 (56.9 mCi/mmol) or 2 µCi [14C]18:3n-3 (59.6 mCi/mmol; both from New England Nuclear Corp., Boston, MA), complexed to albumin as described by Yavin et al.,11 was added to the cells. Cell cultures were incubated at 37°C. At designated times, the radioactive medium was removed and cells were washed twice with 3 ml 0.15 M NaCl containing 80 mg/100 ml FA-free albumin (Pandas’ Miles Laboratories, Kankakee, IL,) and then twice with 3 ml EMEM.12 The cells derived from two to four petri dishes were scraped off with a rubber policeman, suspended in EMEM, pooled, and harvested by centrifugation for 5 minutes at 300g.

Pulse–Chase Studies
For pulse–chase studies, cells were incubated for 24 hours with medium containing [14C] FA. The radioactive medium was removed, and the cells were washed as described for metabolic studies. Nonradioactive EMEM (2 ml) supplemented with 10% LDS and antibiotics was added, and the incubation was continued at 37°C for indicated times. During the chase period, cultures were refed every 2 to 3 days. The results were derived from pooled cell extracts from two to four plates for each data point. When duplicate analyses were performed, the values were essentially identical with the results for which different cell lines were used.

Extraction and Separation of Cellular Lipids
Lipid analysis was performed on a mixture of whole and broken cells. Cultures in which there was a large proportion of broken cells were discarded. The washed cell pellets from two to four combined petri dishes were homogenized with 1 ml methanol using a mechanized Potter-Elvehjem–type homogenizer (Corning, Corning, NY) with a Teflon pestle. Lipids were extracted by the addition of 2 ml chloroform-methanol (2:1, by volume) to the homogenate, which was then rehomogenized in the organic solvents. Portions were removed for protein determination by the method of Bradford.13 After the homogenate was allowed to stand for 30 minutes at 4°C, the lipid extract was filtered through a glass–wool plug in a Pasteur pipette that had previously been washed with chloroform-methanol (2:1). The filtrate was evaporated under nitrogen, resuspended in 4 ml chloroform-methanol (2:1), and mixed with 1 ml water. After mixing, the two phases were allowed to separate, the upper aqueous phase was removed, and the organic phase was evaporated to dryness and stored in a freezer under nitrogen.

Lipid Analysis
Lipids were analyzed as described previously.14 Briefly, neutral lipids were separated from phospholipids by one-dimensional thin-layer chromatography (1D-TLC) on silica gel H plates with a solvent system of hexane-ethylether-acetic acid (80:20:2). Individual phospholipids were separated by two-dimensional thin-layer chromatography (2D-TLC).15 16 Spots of phospholipids were scraped off the plates and transferred to vials for scintillation counting.

Preparation of FA Phenacyl Esters
FA phenacyl esters (FAPEs) were prepared as described previously.14 Briefly, FAPEs were visualized by UV absorption, according to the method of Hanis et al.17 Extracted lipids were evaporated to dryness, immediately suspended in 1 ml 3.3% KOH in ethanol, and saponified by heating for 40 minutes at 55 o C, after which 1 ml H2O was added. The samples were acidified to pH 2.0, and the free FAs (FFAs) were extracted with hexane, dried under nitrogen, resuspended in 50 µl acetone containing 10 mg/ml bromoacetophenone, and mixed with 50 µl acetone containing 10 mg/ml triethylamine. The sample was heated in a boiling water bath for 5 minutes and cooled, and 70 µl acetone containing 2 mg/ml acetic acid was added. The sample was then evaporated to dryness with nitrogen and resuspended in 100 µl acetonitrile.14

HPLC Analysis of FAPEs
FAPEs were analyzed as described previously.14 FAPEs were separated by HPLC with an LC-18 reversed-phase column eluted with a gradient from 80:20 acetonitrile-water to 90:10 acetonitrile-water over 95 minutes monitored by UV absorbance at 254 nm. Radioactivity was measured by scintillation counting.18 The purity of the radiolabeled FAs used was 96% for 18:3n-3 and 97% for 22:6n-3, determined by HPLC, as previously described.19

Gas–Liquid Chromatography
FA methyl esters were identified with a gas chromatograph (5890 series II; Hewlett-Packard, Palo Alto, CA) equipped with a cyanopropil column of 50-m length (Sil 88; Chrompack, Middelburg, The Netherlands). The running conditions were as follows: initial temperature, 60°C, followed by an increase (at 20°C/min) to 190°C for 1 minute, followed by another increase (at 25°C/min) to 230°C for 31 minutes. The column head pressure was 1.5 bar. Identification of FAs was verified by comparison with authentic standards (Nu-Chek Prep, Elysian, MN) and by mass spectrometry (5971 MSD; Hewlett-Packard). FA results were expressed as a percentage (wt/wt) of all FAs detected with a chain length of between 12 and 22 carbon atoms.

Other Methods
Protein concentrations were determined by the method of Bradford.13 Analytical reagent grade FA standards and lipid standards were purchased from Sigma. The LC-18 column was calibrated with phenacyl derivatives of the FA standard.

Statistical Analysis
Data were analyzed by ANOVA, factoring for age group, and by a comparison-among-means test (Tukey-Kramer method). Results were considered statistically significant at P < 0.05. Data are expressed as mean ± SEM. In all figures, each bar represents the mean of four replicates of three samples, each with the SEM indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of Incorporation of FAs into Cellular Lipid Pools in Fibroblasts
Human skin fibroblasts incubated for 24 hours with [14C]18:2n-6 incorporated into the cellular lipids 40% to 50% of the total disintegrations per minute (dpm) added to the medium. During the initial phases of incubation with labeled 18:2n-6, TGs and choline phosphoglycerides (CPGs) were labeled preferentially (Fig. 1) . After 1 hour, 70% to 75% of the total radioactivity was recovered in the TG and CPG fractions. By 6 hours of incubation, the amount of radioactivity in the TG fraction began to decline, reaching approximately 5% of the total radioactivity by the end of the 24-hour incubation in normal control subjects and BCD, whereas in WD, it decreased to 14%. In contrast, uptake into the cerebroside (CER), CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) pools increased progressively in all samples. Very little radioactivity remained in the FFAs, and there was little change in the diacylglycerol (DG), or CE pools (Fig. 1) . Incorporation of labeled 18:2n-6 into the TG fraction was significantly increased over that in control subjects in WD, ranging from approximately 125% at 1 hour, to 170% at 6 hours, to 250% at 24 hours (P < 0.038). Incorporation into CEs also increased in WD samples (P < 0.036). Incorporation of label into the TG and CE fractions in the BCD samples was similar to that in WD samples and higher than that in control samples (P < 0.041).



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  		Figure 1. Distribution of radioactivity among lipid fractions from cultured fibroblasts of patients with BCD or WD and age-matched control subjects, after incubation with [14C]18:2n-6. Data are a percentage of the total radioactivity incorporated at each time point. The total disintegrations per minute per milligram protein at (A) 1, (B) 6, and (C) 24 hours were 99.9, 98.9, and 99.9 in control subjects; 98.2, 106.6, and 100.2 in patients with BCD; and 127.5, 153, and 157.3 in patients with WD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each).

 
Overall, the distribution of radioactivity after incubation of human fibroblasts with [14C]18:3n-3 resembled that obtained with [14C]18:2n-6. The TG fraction contained approximately 31% of total radioactivity after 1 hour’s incubation, but only 4% in control and 14% in WD samples at the end of 24 hours, with BCD samples intermediate. Incorporation into CPG and EPG fractions increased progressively in all samples between 1 and 24 hours of incubation (Fig. 2) . The levels of radioactive 18:3n-3 incorporation into the SPG, CER, and CE fractions were similar to those of [14C]18:2n-6 (compare Figs. 1 and 2 ). After similar labeling of TGs in all three samples at 1 hour, the turnover of [14C]18:3n-3 was fastest in the control cells and lowest in the WD cells.



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  		Figure 2. Distribution and comparison of radioactivity among lipid fractions from cultured fibroblasts of patients with BCD, patients with WD, and age-matched control subjects after incubation with [14C]18:3n-3. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at (A) 1, (B) 6, and (C) 24 hours were 99.8, 97.5, and 99.9 in control subjects; 99.2, 123.3, and 111.9 in patients with BCD; and 117.3, 174, and 166.7 in patients with WD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each).

 
Samples from patients with BCD incorporated slightly higher amounts of [14C]18:3n-3 and [14C]18:2n-6 into neutral lipids (TGs and CEs) overall in 6 and 24 hours than did samples from control subjects. Samples from patients with WD showed higher incorporation into neutral lipids than did those from control subjects, especially at 24 hours. Between 5% and 15% of labeled 18:2n-6 and 18:3n-3 was also found in an unidentified lipid (UL; Figs. 1 2 ). This substance migrated between the CPGs and EPGs in all systems used. Incorporation into the ULs did not vary significantly among the samples studied.

Metabolism of Labeled 18:2n-6 and 18:3n-3 into Specific FAs in Fibroblasts
The distribution of label among the individual FAs of total cellular lipids after 6 and 24 hours of incubation of fibroblasts from patients with BCD or WD and age-matched control subjects with [14C]18:2n-6 is shown in Figure 3 . After 24 hours, approximately 50% of the radioactivity remained as 18:2n-6 in control, BCD, and WD samples, and only 18% of the label was found in the higher PUFAs, mostly in 20:4n-6, but also to some degree in 20:2n-6, 20:3n-6, 22:4n-6, and 22:5n-6. The conversion of 18:3n-3 to its higher derivatives proceeded more rapidly (Fig. 4) . At the end of 24 hours, 55% to 61% of the radioactivity incorporated into cellular lipids remained as 18:3n-3, whereas approximately 30% was incorporated in 20:3n-3, 20:4n-3, 20:5n-3, 22:5n-3, and 22:6n-3. There was no significant difference among fibroblasts from normal control subjects and patients with WD or BCD in 18:2n-6 elongation and desaturation. However, incorporation of [14C]18:3n-3 into all PUFAs was significantly lower in fibroblasts from patients with BCD, with values ranging from 53% to 86% of that in control subjects. Samples from patients with WD showed similar 18:3n-3 elongation and desaturation to those from control subjects. Conversely, at 24 hours there was similar incorporation of [14C]18:2n-6 into 16:0, and 16:1n-7 FAs in BCD, WD, and control samples, whereas incorporation of [14C]18:3n-3 into these pools increased to levels roughly twice as high in BCD samples as those in control and WD samples (Figs. 3 4) .



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  		Figure 3. Incorporation of labeled [14C]18:2n-6 into specific FAs in cultured fibroblasts from patients with BCD, patients with WD, and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at (A) 6 and (B) 24 hours were 175.2 and 170.2 in control subjects, 169.6 and 178.1 in patients with BCD, and 193.6 and 281.6 in patients with WD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each). The FAs 20:5n-3, 22:5n-3, and 22:6n-3 were also measured and found not to differ significantly from 0.

 


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  		Figure 4. Incorporation of labeled [14C]18:3n-3 into specific FAs in cultured fibroblasts from patients with BCD or WD and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at (A) 6 and (B) 24 hours were 150.6 and 155.8 in control subjects, 63.2 and 45.1 in patients with BCD, and 143.4 and 168 in patients with WD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each). The FAs 18:1n-9, 18:1n-7, 20:0, 22:0, 24:1n-9, 20:4n-6, and 22:4n-6 did not differ significantly from 0.

 
Pulse–Chase Studies
The distributions of radioactivity among individual FAs of the total cellular lipid fraction, CPGs, and EPGs after pulse–chase studies of fibroblasts with [14C]18:2n-6 are shown in Figure 5 . During the chase period, 18:2n-6 was readily converted to higher polyenic FAs in all fractions, mainly to 20:4n-6 and 22:4n-6. Desaturation and elongation of 18:2n-6 to 22:4n-6 was slightly more extensive in the total lipid and EPG fractions. There were no significant differences in incorporation of [14C]18:2n-6 into specific FAs among samples from normal control subjects and those from patients with BCD or WD during the chase period. There was little recycling of two carbon units from [14C]18:2n-6 into 16:0 or 18:0 in any group.



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  		Figure 5. Pulse–chase studies of comparison of [14C]18:2n-6 in metabolites among FAs of the total lipid (A, D, and G), CPG (B, E, and H), and EPG (C, F, and I) fractions in cultured fibroblasts from patients with BCD or WD and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at 1, 4, and 12 days were, respectively, 465.1, 300.7, and 212.9 into total lipids; 272.4, 182.2, and 66.9 into EPGs; and 53, 56.6, and 85.4 into CPGs in control subjects; 420.3, 259.4, and 208.7 into total lipids; 257.6, 169.0, and 65.6 into EPGs; and 53, 52.6, and 82.2 into CPGs in patients with BCD; and 470.1, 329.3, and 230.8 into total lipids; 286.2, 189.1, and 68 into EPGs; and 53.0, 57.2, and 86.6 into CPGs in patients with WD. Each bar (±SE) represents the mean of four replicates (three cell lines each).

 
Pulse–chase studies with [14C]18:3n-3 are shown in Figure 6 . Similar to 18:2n-6, 18:3n-3 was extensively converted into higher polyenic FAs, mainly 22:5n-3 and 22:6n-3, but also 20:5n-3, after 1 and 4 days. The proportions of radioactivity in 22:5n-3 and 22:6n-3 were greater in the total lipid and EPG fractions than in the CPG fraction. In contrast, the proportion of radioactivity recycled into 16:0 and 18:0 carbon atom FAs was particularly high in the CPG fraction, especially at 1 and 4 days (e.g., 15% in CPGs vs. 5% in total FAs and 4% in EPGs after 1 day). Compared with normal control samples, samples from patients with BCD elongated and desaturated smaller amounts of 18:3n-3 into 20:4n-3, 20:5n-3, 22:5n-3, and 22:6n-3 after 1 (92% overall), 4 (85%), and 12 days (82% of chase). This difference was most marked in the CPG and EPG fractions. After 4 and 12 days, fibroblasts from patients with WD also showed reduced elongation and desaturation, in most cases at levels intermediate between fibroblasts from patients with BCD and normal control subjects. Once more, this was most marked for 22:5n-3 in the CPG and EPG fractions. Incorporation of [14C]18:3n-3 into other FAs was concomitantly increased in these fractions in both BCD and WD samples compared with control samples. In contrast, there was no significant difference in the amount of recycling of two carbon units from 18:3n-3 into 16:0 or 18:0 among normal control subjects and patients with BCD or WD.



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  		Figure 6. Pulse–chase studies of comparison of [14C]18:3n-3 in metabolites among FAs of the total lipid (A, D, and G), CPG (B, E, and H), and EPG (C, F, and I) fractions in cultured fibroblasts from patients with BCD, patients with WD, and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at 1, 4, and 12 days were, respectively, 385.7, 249.5, and 149.8 into total lipids; 149.5, 85.3, and 47.9 into EPGs; and 77.8, 101.5, and 80.8 into CPGs in control subjects; 238.7, 171.9, and 111 into total lipids; 120.1, 69.1, and 44.5 into EPGs; and 47.9, 62.3, and 58 into CPGs in patients with BCD; and 337.9, 223.3, and 139 into total lipids; 134.1, 74.4, and 46.8 into EPGs; and 70.8, 92.2, and 72 into CPGs in patients with WD. Each bar (±SE) represents the mean of four replicates (three cell lines each).

 
Comparison of Incorporation of Labeled 18:2n-6 and 18:3n-3 into the Cellular Lipid Pools of Cultured Lymphocytes
Incorporation of [14C]18:2n-6 into cellular lipid pools of cultured lymphocytes was similar to that in fibroblasts and showed no differences between control and BCD samples. There was little incorporation of [14C]18:2n-6 into EPGs in control or BCD samples (data not shown). After incubation of lymphocytes from patients with BCD and age-matched control subjects with [14C]18:3n-3 for 24 hours, incorporation into the cellular lipids of human lymphocytes ranged from 40% to 50% of the total radioactivity added to the incubation medium (2.5 x 10 4 dpm). During the initial phases of the incubation, EPGs, CPGs, and TGs were labeled preferentially, and this pattern continued throughout the 24-hour incubation period (Fig. 7) . Lymphocytes from control subjects showed markedly greater incorporation into EPGs than did fibroblasts from control subjects, with levels approaching those of CPGs. In contrast, samples from patients with BCD showed only 10% to 20% as much incorporation into EPGs, whereas they showed 75% to 80% as much incorporation into CPGs as in normal control subjects. Incorporation into TGs was significantly higher in lymphoblasts from patients with BCD than those from control subjects, from 1 hour to 24 hours. The TG fraction labeling was approximately twice as high in BCD as in control samples.



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  		Figure 7. Incorporation of labeled [14C]18:3n-3 into specific lipids in cultured human lymphocytes from patients with BCD and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein at (A) 1, (B) 6, and (C) 24 hours were 159.8, 131.5, and 111.2 in control subjects and 131.3, 95.3, and 93.1 in patients with BCD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each).

 
Comparison of Metabolism of Labeled 18:3n-3 into Specific FAs in Lymphocytes
The metabolism of [14C]18:2n-6 into specific FAs in lymphocytes was similar to that in fibroblasts, with no significant differences between BCD and control samples (data not shown). The distribution of label among the individual FAs of the total cellular lipids of patients with BCD and age-matched control subjects after 1, 6, and 24 hours of incubation with [14C]18:3n-3 is shown in Figure 8 . After 24 hours, in control subjects 53% of the radioactivity remained as 18:3n-3 and only 25% of the label was found in the higher PUFAs, initially in 20:3n-3 but, by 24 hours, in 20:4n-3, 20:5n-3, 22:5n-3, and 22:6n-3 as well (Fig. 8) . Small amounts of 16:0, 16:1, 18:0, and 24:0 were also observed at 6 and 24 hours. Compared with normal control samples, BCD samples show decreased uptake (65% to 75% of control) and correspondingly lower elongation and desaturation of 18:3n-3, although this represented a higher percentage conversion of the 18:3n-3 taken up (33% vs. 25%). This slight decrease was not specific to any single metabolite (Fig. 8) . There was also an increase in the recycling of [14C]18:3n-3 into unsaturated FAs (22% vs. 10%). These results were similar to [14C]18:3n-3 incorporation in fibroblasts.



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  		Figure 8. Incorporation of labeled [14C]18:3n-3 into specific FAs in cultured human lymphocytes from patients with BCD and age-matched control subjects. Data are a percentage of the total radioactivity incorporated at each time point. The disintegrations per minute per milligram protein (A) 1, (B) 6, and (C) 24 hours were 123.8, 126.7, and 119 in control subjects and 71.7, 39.6, and 37.4 in patients with BCD, respectively. Each bar (±SE) represents four separate experiments (three cell lines each). The FAs 18:1n-9, 18:1n-7, 20:0, 22:0, 20:4n-6, 22:4n-6, and 22:5n-6 did not differ significantly from 0.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we compared FA metabolism of lipid classes in human lymphocytes and fibroblasts from patients with BCD or WD and age-matched control subjects. After approximately 6 hours, incorporation of FAs into the TG fraction in WD samples and to a lesser extent in BCD samples rose above that in normal control samples. However, in contrast to the WD samples, the incorporation of [14C]18:3n-3 into n-3 PUFAs was significantly decreased in BCD. Because the same desaturases and elongases were involved in desaturation and elongation of both n-6 and n-3 FAs, the decreased incorporation of [14C]18:3n-3 relative to [14C]18:2n-6 may have been due to increased catabolism, perhaps caused by differential binding by cellular FA-binding proteins.20 For example, the lower level of 18:3n-3 label found in PUFAs after 24 hours may be attributable to faster oxidation in BCD. Both 18:2 and 18:3 were actively oxidized in mitochondria, resulting in loss of precursor from measured PUFA products. This decrease held for FAs in the CPG and EPG fractions as well as total FAs. These findings suggest that abnormal lipid storage in BCD occurs by a different mechanism than in WD.

The binding of FAs and their transport during the cell cycle is of central importance in normal cellular metabolism. Our data demonstrate that in the BCD samples more 18:3n-3 was incorporated into TGs than in control samples, despite an initial lag period. In experiments with each FA precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG fractions, apparently moving into more complex lipids, especially the CPG pool in patients with BCD or WD and age-matched control subjects. In part, this may represent conservation of the labeled PUFAs in the CPG and EPG pools, whereas they were actively oxidized and thus lost from the FFA pool. In BCD, WD, and control fibroblasts, significantly more precursor was incorporated into CPGs than EPGs in both 18:2n-6 and 18:3n-3 studies. This suggests that a minimal transfer of these FAs from TGs into EPGs is likely to exist in fibroblast cultures, as opposed to control lymphocytes in which incorporation into EPGs is similar to incorporation into CPGs. In contrast, lymphocytes from patients with BCD showed little incorporation into EPGs, appearing similar to fibroblasts in this regard. This suggests the possibility of a block in this pathway in BCD lymphocytes.

Mammals are unable to synthesize FAs unsaturated in the n-3 or n-6 position. Thus, longer chain FAs such as 22:6n-3 are either acquired directly from the diet or synthesized from their respective precursors, 18:3n-3 in the case of 22:6n-3, by sequential elongation and desaturation along well-established metabolic pathways.21 The major lipid components in fibroblasts and lymphocytes are CPGs and EPGs.22 Incorporation of 18:2n-6 and 18:3n-3 into the EPG pool during the pulse–chase experiments in the present study occurred concomitantly with extensive desaturation and elongation of 18:2n-6 into 22:5n-6 and of 18:3n-3 into 22:6n-3. Thus, any defect in the uptake, transport, or elongation and desaturation of PUFAs may result in a wide variety of metabolic abnormalities. It is noteworthy that 18:3n-3 uptake, elongation, and desaturation were significantly lower in cells from patients with BCD than in those from patients with WD or from normal control subjects. {omega}-3 FAs are not interconvertible with {omega}-6 FAs and are known to serve a metabolic role distinct from them.23

Incorporation of FAs into the TG fraction was significantly higher in lymphocytes of patients with BCD than in normal control subjects, whereas incorporation of FAs into the CPG fraction was low and even lower into the EPG fraction. This suggests that a direct transfer of FAs between these phospholipid pools is defective in BCD lymphocytes. The decreased incorporation of labeled 18:3n-3 into EPG appeared to be specific for lymphocytes because this incorporation was low in both control and BCD fibroblasts. Transfer of FAs between these two pools could occur by successive hydrolysis and activation to the FA coenzyme A analogues and/or elongation and desaturation when the FAs are esterified to the glycerol moiety of CPGs.24 Thus, the decreased transfer could be due to a specific defect in transfer of FAs between these pools or in the desaturation or elongation of the FAs that is concomitant with this transfer. Alternatively, differential labeling of the TG, EPG, and CPG fractions also may reflect differences in their de novo synthesis in the different diseases through elongation and {Delta}6 desaturation followed by ß-oxidation.25

These experiments give us our first insight into PUFA metabolism in WD and BCD. Abnormally high levels of TG and cholesterol storage were seen in cultured cells from patients with WD or BCD, whereas metabolism of labeled FA precursors into n-3 PUFA was decreased in BCD. A deficiency or dysfunction of an FA-binding protein could cause decreased uptake and transfer between lipid pools. Also, deficient elongation and desaturation of FA precursors could cause the deposit of lipids within disease cells in crystalline form. This initial characterization of lipid and FA metabolism in BCD suggests that a closer examination of both lipid-binding proteins and enzymes active in desaturation and elongation of FAs is warranted in BCD.


   Footnotes
 
Submitted for publication December 13, 2000; accepted January 25, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: J. Fielding Hejtmancik, OGVFB/NEI/NIH, Building 10, Room 10B10, 10 Center Drive MSC 1860, Bethesda, MD 20892-1860. f3h{at}helix.nih.gov


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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