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Journal of Lipid Research, Vol. 42, 1395-1401, September 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa

Correspondence to: Dennis R. Hoffman, To whom correspondence should be addressed., dhoffman{at}retinafoundation.org (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Many patients with X-linked retinitis pigmentosa (XLRP) have lower than normal blood levels of the long-chain polyunsaturated 3 fatty acid docosahexaenoic acid (DHA; 22:63). This clinical trial was designed to test whether down-regulation of DHA biosynthesis might be responsible for these reduced DHA levels. DHA biosynthesis was assessed in five severely affected patients with XLRP and in five age-matched controls by quantifying conversion of [U-¹³C]-linolenic acid (-LNA) to [¹³C]DHA. Following oral administration of [U-¹³C]-LNA, blood samples were collected at designated intervals for 21 days and isotopic enrichment of all 3 fatty acids was determined by gas chromatography/mass spectroscopy. Activity of each metabolic step in the conversion of -LNA to DHA was determined by comparison of the ratios of the integrated concentration of ¹³C-product to ¹³C-precursor in plasma total lipid fractions. The ratio of [¹³C]DHA to [¹³C]18:33 (the entire pathway) and that of [¹³C]20:53 to [¹³C]20:43 (⁵-desaturase) were significantly lower in patients versus controls (P = 0.03 and 0.05, respectively). The estimated biosynthetic rates of [¹³C]20:53, [¹³C]22:53, [¹³C]24:53, [¹³C]24:63, and [¹³C]22:63 were significantly lower in XLRP patients (42%, 43%, 31%, 18%, and 32% of control values, respectively; P < 0.04), supporting down-regulation of ⁵-desaturase in XLRP. The disappearance of ¹³C-labeled fatty acids from plasma was not greater in XLRP patients compared with controls, suggesting that XLRP was not associated with increased rates of fatty acid oxidation or other routes of catabolism.

Thus, despite individual variation among both patients and controls, the data are consistent with a lower rate of ⁵-desaturation, suggesting that decreased biosynthesis of DHA may contribute to lower blood levels of DHA in patients with XLRP. — Hoffman, D. R., J. C. DeMar, W. C. Heird, D. G. Birch, and R. E. Anderson. Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa. J. Lipid Res. 2001. 42: 1395;–1401.

Supplementary key words: omega-3 fatty acids, retinal degeneration, delta-5 desaturase, stable isotopes, lipid metabolism, linolenic acid

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Retinitis pigmentosa (RP) is a family of hereditary diseases characterized by photoreceptor degeneration with progressive night blindness and constriction of peripheral vision leading to functional blindness (1). Multiple inheritance patterns exist for RP, including autosomal dominant, autosomal recessive, and X-linked. Patients with the X-linked form of RP (XLRP) are among the most severely affected, with an onset of night blindness due to rod photoreceptor loss often detectable by age 5 years (2) (3) (4). Cone degeneration typically results in legal blindness by the second or third decade of life. With the exception of visual impairment, patients with RP including XLRP are commonly considered "healthy." However, a number of investigations have shown that many patients with RP have lower plasma and red blood cell (RBC) lipid levels of the -3 polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:63) than nonaffected individuals [reviewed in (5)]. In two studies, the majority of patients with XLRP had 30;–40% lower DHA levels in RBC lipids than normally sighted controls (6) (7).

Lower plasma levels of DHA and other 3 fatty acids also have been found in miniature poodles with progressive rod-cone degeneration (prcd), a model of inherited retinal degeneration that closely resembles human RP (8) (9) (10). Other animal models of RP including prcd dogs (11) and transgenic rats and mice with rhodopsin mutations (12) also have reduced DHA levels in lipid membranes of rod outer segments.

DHA comprises a small percentage (1;–4%) of the total fatty acids of the membranes of most human tissues; however, it accounts for 30;–40% of fatty acids in rod photoreceptor outer segments of the human retina (13). The high concentration of this highly unsaturated fatty acid can increase membrane fluidity and, in turn, may modify the mobility of vital proteins and the activities of retinal enzymes (14) (15). Indeed, deficiencies of 3 fatty acids result in abnormal electroretinographic (ERG) responses in rats (16) (17) (18) (19), guinea pigs (20), and monkeys (21) (22) (23). Less mature ERG responses and/or lower visual acuity, as well as lower neurodevelopmental scores also have been associated with reduced blood lipid levels of DHA in preterm and term infants fed conventional formula versus breast milk or DHA-supplemented formula (24) (25) (26) (27) (28) (29). Significant correlations between RBC levels of DHA and ERG responses also have been reported such that patients with XLRP and low blood DHA levels have correspondingly poor ERG function (7). Thus, DHA may participate in optimizing the lipid microenvironment in photoreceptor membranes, thereby influencing enzyme and protein interactions.

DHA cannot be synthesized de novo by the human species, and therefore must be formed from shorter chain 3 precursors [e.g., -linolenic acid (-LNA), an essential fatty acid] or ingested intact (30). The currently accepted pathway for 3 fatty acid biosynthesis occurs though a series of desaturations (insertion of additional double bonds) and elongations (addition of two-carbon units). Thus, -LNA (18:33) is sequentially converted to 18:43, 20:43, 20:53, 22:53, 24:53, 24:63, and 22:63 (DHA). The respective enzymatic steps involved in -LNA conversion to DHA are desaturation (⁶-desaturase), elongation, desaturation (⁵-desaturase), elongation, desaturation (⁶-desaturase), and finally ß-oxidation. The desaturation and elongation steps occur in smooth endoplasmic reticulum (31), whereas the final ß-oxidation step occurs in peroxisomes (32). Liver is considered the primary site of DHA synthesis, but other tissues including retina, retinal pigment epithelium, and testes (33) (34) also convert -LNA to DHA via the 24-carbon intermediates.

There are three possible explanations for the low blood lipid DHA levels in patients with XLRP: 1) reduced synthesis of DHA; 2) increased catabolism of DHA; and 3) impaired transport, uptake, and/or trafficking of DHA, reducing the availability of DHA to target tissues. Tracing stable isotopically labeled -LNA (i.e., [U-¹³C]-LNA) through the 3 fatty acid intermediates to DHA in vivo permits critical examination of the first hypothesis and provides some insight into the possibility of increased catabolism of DHA associated with XLRP. In this clinical trial, ¹³C-enrichment of the 3 fatty acid intermediates in the plasma total lipid fraction of five patients with XLRP at various times following administration of [U-¹³C]-LNA was compared with enrichment of the same fatty acids at the same times in plasma lipids of five age-matched controls. The lower accumulation of ¹³C-label in DHA in patients with XLRP indicates that their metabolic conversion of -LNA to DHA is reduced. The lack of difference between groups both in expiration of ¹³CO₂ and disappearance of ¹³C-labeled fatty acids from total plasma lipids suggests that oxidation and other routes of catabolism of 3 fatty acids do not differ between XLRP patients and controls.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Subjects
All patients with XLRP had been diagnosed by retinal specialists and were in the advanced stages of disease. The trial included only males, as they are most severely affected by this disease. Each had an early onset of night blindness followed by significant loss of peripheral vision. All had nondetectable rod ERG responses, dark-adaptation values elevated by greater than three log units, and characteristically poor cone ERG function. Family histories of patients were consistent with the XLRP inheritance pattern; that is, presence of at least two affected male relatives, absence of male-to-male transmission, and expression of characteristics of carrier heterozygotes in either the patient's mother or daughter. Four patients with XLRP were Caucasian and one was Hispanic; ages ranged from 28 to 52 years (mean ± SD = 41 ± 8). One patient smoked about eight packages of cigarettes per week; no other patient smoked. None consumed more than one alcoholic drink per week. Body mass index (BMI; weight/height²) ranged from 26 to 36 (mean ± SD = 32 ± 3). All patients underwent a comprehensive physical examination including medical history, review of systems, review of diet and health habits, electrocardiograph, chest X-ray, blood chemistries, and baseline carbon dioxide production.

Normal-sighted male volunteers of roughly the same age as the patients (±2 years) were recruited as controls (range = 29;–50 years; mean ± SD = 39 ± 7). Controls also underwent physical and ophthalmic examinations. All were Caucasian, nonsmokers, and consumed less than one alcoholic drink per week. BMI ranged from 26;–32 (mean ± SD = 28 ± 2; significantly less than patients, P = 0.04).

Participants were informed of the objectives and protocol of the study and written informed consent of each was obtained. The study was approved by the Institutional Review Boards of Presbyterian Hospital of Dallas, TX and Baylor College of Medicine and Affiliated Institutions, Houston, TX.

Experimental design
All subjects were provided a low fat, low 3 fatty acid diet for 1 week prior to and 2 weeks following tracer administration. Meals (breakfast, lunch, dinner, and snacks) were prepared to our specifications and delivered to each subject's home or work. The purpose of this "stabilization" diet was to reduce variability among participants due to differences in the individuals' typical diets and to stabilize fatty acid intake during the study, thus preventing "spikes" in blood levels of 3 fatty acids.

After admission to Presbyterian Hospital, baseline blood and breath samples were obtained and an in-dwelling catheter was placed in the subject's arm for blood sampling during a 30-h hospital stay. [U-¹³C]-LNA (0.5 g) obtained from Martek Biosciences Corporation, Columbia, MD, was sonicated into 3 oz of a low fat chocolate drink (Yoo-hoo, Carlstadt, NJ) and ingested immediately by all subjects. Subsequently, blood samples [collected in 5-ml Vacutainer^® (Becton Dickinson, Franklin Lakes, NJ) tubes containing EDTA as anticoagulant] were obtained at 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 h. Breath samples were obtained every half hour for the first 12 h, and then every hour for the next 12 h. These were collected in gas sampling bags and transferred to sterile 10-ml evacuated glass tubes for analysis of ¹³CO₂. The subjects were discharged but returned for blood samples at 2, 3, 4, 5, 7, 10, 14, and 21 days.

Plasma was separated by centrifugation (3,000 g x 10 min) and frozen at -80°C until analysis. Total plasma fatty acids were extracted according to the method of Bligh and Dyer (35) following addition of heptadecanoic acid (17:0) in phospholipid and triglyceride forms (Sigma-Aldrich Chemical Co., St. Louis, MO) as an internal standard for determination of 3 fatty acid concentrations. Fatty acids in the total lipid extracts were converted to methyl esters and pentafluorobenzyl derivatives using the methods of Morrison and Smith (36) and Hachey et al. (37), respectively. Methyl and pentafluorobenzyl esters were separated by gas chromatography (GC) on 30-m DB-225 (J & W Scientific, Folsom, CA) and 60-m SP-2380 (Supelco, Bellefonte, PA) capillary columns, respectively, using a Hewlett-Packard 5890 gas chromatograph.

Detection of GC-separated pentafluorobenzyl esters was accomplished by mass spectrometry (MS) using a Hewlett-Packard 5989A quadrupole mass spectrometer. Signals of the tracee ([M]) and tracer ([M + 18]) isotopomers of the 3 fatty acids were determined by selective ion monitoring for their corresponding molecular masses under negative chemical ionization (37). Peaks were identified by comparison with 3 fatty acid reference standards (Sigma-Aldrich Chemical Co.) or with authentic preparations of 24:53 and 24:63 (provided by Dr. H. Sprecher, Columbus, OH). Areas of the tracee and tracer MS signal peaks were converted to percentage enrichment of the tracer in its fatty acid pool (% enrichment = [tracer/(tracee + tracer)] x 100%).

Detection of GC-separated methyl esters was accomplished by flame ionization. Identities were confirmed against GC of 3 fatty acid standards. Although 20:43 was detectable in the plasma lipid extracts by GC/MS, it could not be quantified directly by GC; thus, its concentration was estimated by comparison of its MS signal area with that of 20:46 (arachidonic acid). All subjects had plasma lipid concentrations of 3 fatty acids determined at 0, 8, 24, 168, and 504 h. Because the concentrations differed minimally, individual averaged values were used in all subsequent calculations.

Breath samples were analyzed for the ¹³C/¹²C ratio of CO₂ by isotope ratio MS using a Europa Scientific 20-20 Stable Isotope Analyzer mass spectrometer (Franklin, OH). Atom percentage enrichments for ¹³CO₂ in breath samples were converted to ¹³CO₂ expiration rates (nmol/min/kg) and cumulative percentage expiration (% of [U-¹³C]-LNA dose) using CO₂ production rates (ml/kg/min) measured at baseline.

Data analysis
Data are expressed as nmol [¹³C]fatty acid/ml plasma (i.e., the product of the ¹³C-enrichment of each fatty acid and the plasma concentration of the fatty acid). Total accumulations of ¹³C-fatty acids are reported as a function of time after isotope administration. Area under the curve (AUC; nmol [¹³C]fatty acid/ml plasma/504 h) was calculated by trapezoidal integration using IGOR software (Wavemetrics Inc., Lake Oswego, OR). An estimate of the initial biosynthetic rate (nmol [¹³C]fatty acid/ml plasma/h) of each intermediate fatty acid was established by linear regression of the leading edge of the ¹³C-accumulation curve. Rates of fatty acid disappearance were estimated as the rate of decline in [¹³C]fatty acid concentrations from the time of peak enrichment to 504 h. Product/precursor ratios of individual metabolic steps in conversion of -LNA to DHA were established from AUC data. -LNA oxidation was estimated by respiratory expiration of ¹³CO₂ and expressed as percentage of [U-¹³C]-LNA administered. Statistically significant differences (P < 0.05) in mean values of each variable between patients and controls were detected by Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mean baseline blood chemistry values of the two groups of subjects are summarized in Table 1. As expected, patients with XLRP had significantly lower RBC lipid DHA content than controls (Table 1). Neither plasma lipoprotein profiles nor other blood chemistries differed significantly between groups; however, alanine aminotransferase activity was somewhat higher in XLRP patients versus controls (P = 0.06). Baseline carbon dioxide production of the control group was 300 ± 50 ml/min; that of the XLRP group was 340 ± 60 ml/min (P = 0.28).

Fig 1 shows the concentrations of [¹³C]-LNA and [¹³C]DHA in plasma lipids of individual control subjects and XLRP patients, as well as the mean concentrations of both groups. Although these values were variable in both groups, the difference in maximal mean peak concentration of [¹³C]-LNA in plasma lipids between controls and patients was not statistically significant (see Fig 1A and Fig B; 42 ± 24 nmol/ml and 51 ± 18 nmol/ml, respectively; P > 0.5). The time to reach the maximal peak concentration also did not differ between groups (4 vs. 3.5 h). In contrast, there were marked differences between controls and XLRP patients in both mean plasma lipid [¹³C]DHA concentration and the time to reach peak concentration (Fig 1C and Fig D). The mean concentration of [¹³C]DHA in plasma lipids of controls peaked at 0.11 nmol/ml, approximately 144 h after tracer administration, whereas the mean peak plasma lipid concentration of [¹³C]DHA was lower in patients with XLRP (0.06 nmol/ml; P = 0.21), and occurred 96 h later (240 h; P = 0.03) than controls.

View larger version (33K): [in this window] [in a new window]   Figure 1. Concentrations of 13C-labeled -LNA and DHA in plasma lipids of five controls and five patients with XLRP as a function of time following [U-13C]-LNA administration. Mean (solid black line) and individual data (solid symbols and dashed lines) are given for the [13C]-LNA plasma concentration in controls (A) and in XLRP patients (B). Data for [13C]DHA plasma concentrations of controls are shown in C, and those of patients with XLRP are shown in D.

A comparison of the integrated concentrations of ¹³C-labeled 3 fatty acids in plasma lipid of patients with XLRP and controls is shown in Fig 2A. Concentrations of [¹³C]18:3, [¹³C]18:4, and [¹³C]20:4 were 46%, 82%, and 136% higher, respectively, in patients with XLRP than in controls, whereas the integrated concentrations of [¹³C]20:5, [¹³C]22:5, [¹³C]24:5, [¹³C]24:6, and [¹³C]22:6 were 3%, 2%, 2%, 38%, and 46% lower, respectively, in patients with XLRP than in controls. These data, although not statistically significant (P 0.06), are consistent with a block in conversion of 18:33 to 22:63 at the ⁵-desaturation step (i.e., 20:4320:53).

View larger version (57K): [in this window] [in a new window]   Figure 2. A: Integrated plasma concentrations of 13C-labeled 3 fatty acids, and (B) estimated [13C]fatty acid biosynthetic rates in XLRP patients compared with controls. Bars represent mean (±SD, vertical black lines) values of integrated [13C]fatty acid concentrations (AUC) from XLRP patients as a percentage of control. Control AUC values (nmol/ml plasma/504 h) for [13C]18:33 were 720 ± 340; [13C]18:43, 29 ± 12; [13C]20:43, 82 ± 61; [13C]20:53, 120 ± 58; [13C]22:53, 62 ± 18; [13C]24:53, 7 ± 2; [13C]24:63, 10 ± 4; and [13C]22:63, 37 ± 20. Estimated biosynthetic rates of 13C-labeled fatty acids were determined by the linear regression slope of the leading edge of [13C]fatty acid accumulation curves as a function of time up to peak enrichment. Estimated biosynthetic rates for XLRP patients are given as percentage of control; control values (pmol/ml plasma/h) for [13C]18:43 were 258 ± 139; [13C]20:43, 277 ± 181; [13C]20:53, 72 ± 38; [13C]22:53, 22 ± 12; [13C]24:53, 5.1 ± 1.4; [13C]24:63, 5.6 ± 2.8; and [13C]22:63, 0.82 ± 0.51. Significant differences are identified by P values. Data for [13C]18:33 is omitted, as it results from accumulation rather than synthesis.

The estimated biosynthetic rates of formation of each intermediate fatty acid were determined by the slope of the ¹³C-enrichment curve as a function of time up to the peak ¹³C concentration. Estimated biosynthetic rates of 18:4 and 20:4 were nearly identical in XLRP patients and controls (Fig 2B), whereas in patients, the rates of 20:5, 22:5, 24:5, 24:6, and 22:6 biosynthesis were 58%, 76%, 69%, 82%, and 69% lower than in controls, respectively (all differences were significant, P < 0.05). These data also are consistent with down-regulation at the ⁵-desaturase step.

Further assessment of conversion activity was made by comparing product-to-precursor ratios. Thus, ratios of the integrated concentration of the ¹³C-product of each step in conversion of 18:33 to 22:63 to the ¹³C-precursor of each step were calculated ( Fig 3). The ratio of [¹³C]DHA (product of the pathway) to [¹³C]18:33 (precursor) in patients with XLRP was roughly half that of the control group (P = 0.029). The ratio of the integrated concentration of [¹³C]20:53 to that of [¹³C]20:43, the step catalyzed by ⁵-desaturase, also was significantly lower in patients with XLRP versus controls (P = 0.05). The integrated concentrations of ¹³C-product/¹³C-precursor of other steps in conversion of 18:33 to 22:63 (e.g., desaturation, elongation, and ß-oxidation) did not differ significantly between patients and controls.

View larger version (30K): [in this window] [in a new window]   Figure 3. Product/precursor ratios of 13C-labeled 3 fatty acids. Values are means (±SD, vertical black lines) and derived from integrated AUC values for [13C]fatty acid concentrations in plasma of controls (solid bars) and patients with XLRP (hatched bars). Significant differences are identified by P values.

The rate of disappearance of each intermediate [¹³C]fatty acid was estimated as the slope of the plasma fatty acid curve beginning at the peak of ¹³C-label enrichment through 504 h ( Table 2). There was no evidence of a greater rate of disappearance of any [¹³C]fatty acids in patients with XLRP. Rather, the rate of disappearance of most labeled intermediates beyond the ⁵-desaturase step, including DHA, appeared to be slower in XLRP patients.

Oxidation of [U-¹³C]-LNA was assessed by differences in breath ¹³CO₂ of patients with XLRP versus controls following administration of the tracer ( Fig 4). Breath ¹³CO₂ (nmol/min/kg body weight) of both patients and controls peaked 4 h post-dose and returned to baseline within about 36 h; peak ¹³CO₂ enrichment of the two groups did not differ significantly (P = 0.37). Cumulative breath ¹³CO₂ excretion, expressed as percentage of the [U-¹³C]-LNA dose administered, also did not differ significantly between groups (P > 0.3).

View larger version (28K): [in this window] [in a new window]   Figure 4. Expiration rate of 13CO2 following tracer administration in controls (solid triangles and solid lines) and in patients with XLRP (open squares and dashed lines). Inset: cumulative expiration of 13CO2, given as percentage of tracer dose.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This clinical trial was conducted to assess activity of the DHA biosynthetic pathway in patients with XLRP. The reduced accumulation of [¹³C]DHA and lower estimated initial rates of DHA synthesis in patients with XLRP implicate a defect in the metabolic conversion pathway of -LNA to DHA. Furthermore, the statistically significant differences in product/precursor ratios between controls and patients are highly suggestive that down-regulation of ⁵-desaturase contributes to the lower circulating pools of DHA associated with XLRP. The reduced blood lipid DHA levels in XLRP patients do not appear to be attributable to higher rates of oxidation of 3 fatty acids, as estimated by expiration of ¹³CO₂ in the breath. Although by no means conclusive, the [¹³C]fatty acid disappearance rates reported here are not consistent with an elevated catabolism of DHA as a cause for the lower circulating levels of DHA in XLRP patients. Further evaluation of this possibility will require studies with in vivo [¹³C]DHA administration.

Nevertheless, a number of assumptions and confounding variables may affect the interpretation of these data. The major such assumption is that the plasma pool is homogeneous with other lipid pools and that periodic sampling of plasma reflects the metabolic activity in the whole body. Although this appears to be true generally, plasma is a dynamic medium with numerous tissues releasing and absorbing varying amounts of fatty acids; this makes kinetic analysis of metabolism tentative at best. Another potential problem is intersubject variability; however, despite this, accumulation of [¹³C]DHA was consistently lower in patients with XLRP than in controls.

A break in metabolic activity at the ⁵-desaturase step was evident in XLRP patients from integrated [¹³C]fatty acid concentrations, biosynthetic activities, and product/precursor ratios. By examination of both integrated [¹³C]24:5 and 24:6 concentrations and biosynthetic rates, it appears that the metabolic flux of ¹³C-label through these 24-carbon intermediates also was attenuated in both controls and patients with XLRP (Fig 2). This may be associated with slower transport of these fatty acids into and out of peroxisomes prior to the final ß-oxidation step to produce DHA and subsequent esterification to phospholipid (38).

A reduction in the synthesis of DHA in patients with RP is also consistent with previous studies (7). A crude comparison of product/precursor ratios of RBC concentrations of 3 fatty acid intermediates of the individual desaturation and elongation reactions in the pathway revealed significant reductions in the final steps of DHA biosynthesis in patients with XLRP versus controls. These results are supported by a dietary supplementation study in patients with autosomal dominant RP (39). In this study, both patients and controls received a low 3 fatty acid "stabilization" diet followed by 3 weeks of supplementation with eicosapentaenoic acid (20:53). Plasma lipid eicosapentaenoic acid content increased similarly in both patients and controls. However, plasma lipid DHA content of controls increased by 26% over the 3-week period of supplementation, whereas that of the RP patients did not change, which is consistent with decreased metabolic production of DHA.

Mutations of retina-specific genes are considered the primary cause of RP. The focus has been on mutations in genes controlling enzymes involved in the phototransduction cascade and proteins of structural significance in the photoreceptor. Despite persistent observations of low blood lipid levels of DHA associated with retinal dysfunction, it is not currently known whether alterations in 3 fatty acid metabolism are involved in the pathophysiology of RP. Because the lipid anomaly occurs in various genetic forms of RP, it may be a secondary phenomenon (10). Anderson et al. (10) hypothesized that the genetic mutations resulting in retinal degenerations in patients with RP also produce a metabolic stress that invokes adaptive structural and biochemical modifications in retinal photoreceptors so as to reduce stress-induced damage to the photoreceptors. Because DHA has numerous unsaturated bonds and may be a target of oxidative stress, reduction of tissue levels of DHA may serve to limit intracellular damage. Such a mechanism also may explain local regulation of DHA levels within the retina. However, a reduction in circulating DHA would supposedly be regulated by the liver. Bazan and Rodriguez de Turco (40) proposed recently that the retina generates some signal to the liver, resulting in regulation of hepatic DHA production or circulatory transport mechanisms. The current finding of apparently lower ⁵-desaturase activity in XLRP is consistent with this concept. ⁵-Desaturase is known to be influenced by a number of circulatory and environmental factors including insulin, glucocorticoids, and dietary fatty acids (41) (42) (43). Thus, we conclude that ⁵-desaturase activity is lower in patients with XLRP, and speculate that regulation of ⁵-desaturase activity by factors originating from the retina or by stress-induced factors may account for down-regulation of hepatic production and/or release of DHA by the liver, resulting in diminished blood lipid levels of DHA in patients with XLRP.

	ACKNOWLEDGMENTS

This study was funded by the Foundation Fighting Blindness. Investigators were supported by the National Eye Institute (R.E.A., D.G.B.), National Institute for Child Health and Human Development (J.C.D.), Research to Prevent Blindness (R.E.A.) and the Food and Drug Administration (D.R.H.). This work has been funded, in part, with federal funds from the U.S. Department of Agriculture, Agricultural Research Service, under Cooperative Agreement No. 38-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does the mention of trade names, commercial products, or organizations imply endorsement by the United States Government. We appreciate the organizational and technical assistance of Dr. David Hachey, Beverly Romero, Deidre Capper, Malou Arnold, and Jennifer Sayne. We are also grateful to Dr. Tony Babb and Darren DeLorey of the Institute for Exercise and Environmental Medicine, Dallas, TX for determination of baseline CO₂ production, and for medical assistance from Dr. Rand Spencer (Texas Retina Associates, Dallas, TX) and from Drs. Robert Harris, Jeffrey Phillips, and Mark Fleschler of Texas Internal Medicine Associates, Dallas, TX. We are also grateful to Tyler's Restaurant of the Cooper Aerobics Center, Dallas, TX for preparation of meals. We thank our patients and control volunteers for participating in this clinical trial.

Manuscript received January 19, 2001; and in revised form May 3, 2001

Abbreviations: AUC, area under the curve; DHA, docosahexaenoic acid; ERG, electroretinographic; GC, gas chromatography; LNA, linolenic acid; MS, mass spectrometry; prcd, progressive rod-cone degeneration; RBC, red blood cell; RP, retinitis pigmentosa; XLRP, X-linked retinitis pigmentosa

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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