Liver and Skeletal Muscle Lipids Have Differing Fatty Acid Profiles in Short-Gut Rats Fed Via Parenteral Nutrition

By McCowen, Karen C; Ling, Pei-Ra; Ollero, Mario; Maykel, Justin A; Et al

ABSTRACT. Background: In short-gut rats, we showed marked abnormalities in plasma lipid fatty acids using parenteral nutrition (PN) with lipid DS sham surgery rats. This suggests that either sensing or metabolism of parenteral lipid is abnormal in malabsorption. The goal of this study was to determine fatty acid profiles in skeletal muscle and liver in short-gut rats treated with PN compared with sham rats. Methods: Sprague-Dawley rats underwent laparotomy and massive small bowel resection (or sham surgery). Rats (n = 32, 16 sham, 16 short gut) were randomly assigned to PN with lipid or fat-free PN. After 5 days, weight loss was similar in all groups, and mixed hindlimb skeletal muscle and liver were biopsied. Results: We found marked differences between liver and skeletal muscle. In livers of short-gut animals, 22:4ω6, 22:5ω6, and 22:6ω3 were higher (all p < .05) than in sham. In skeletal muscle, short gut had no effect on fatty acid profiles. In liver, fat-free PN led to significant increases in 20:3ω6, 22:4ω6, 22:5ω6, 20:3ω9, 20:5ω3, 22:6ω3, and triene/tetraene ratio (all p < .05) compared with feeding PN with lipid, irrespective of short gut. In muscle, levels of the distal long-chain fatty acid metabolites and triene/tetraene ratio were minimally affected by nutrition. Serum glucose and insulin concentrations were similar in all 4 groups. Conclusions: Both the presence of short gut and type of PN led to increases in distal metabolites of fatty acids on ω:3 and ω:6 pathway in liver phospholipids but not in skeletal muscle during short-term PN feeding in rats. (Journal of Parenteral and Enterai Nutrition 30:27- 31, 2006)

Essential fatty acid deficiency (EFAD) is a rare but important clinical problem in patients with malabsorption.1 Classical biochemical findings of EFAD include reductions in the 2 essential fats, 18:2ω6 (linoleic acid) and 18:3ω3 (α-linolenic acid), with parallel elevations in 20:3ω9 (eicosatrienoic acid) in serum lipids2,3 and a triene/tetraene ratio (20:3ω9/ 20:4ω6) >0.2. Lipid emulsions currently in use in the United States are derived from soybean oil and prevent EFAD in patients receiving chronic parenteral nutrition (PN).4-10 Despite nutrition replacement, serum phospholipid fatty acid profiles remain abnormal in these patients, with features suggestive of up-regulation of hepatic desaturase activity.

In the present study, therefore, we set out to develop a rodent model of malabsorption using small bowel resection. We have previously published that lipid provided in PN to short-gut rats is not sensed normally, compared with sham-operated rats.12 Our principal findings in serum phospholipids were that arachidonic acid concentrations were excessively elevated in all rats fed fat-free diet for 5 days, suggesting up-regulation of hepatic desaturase enzymes in the face of a limiting supply of its precursor, linoleic acid. In shortgut rats fed with lipid, arachidonic acid was similarly high compared with sham, suggesting possible failure to down-regulate desaturase activity. In 1 published report, short-gut rats were noted to have elevated A6 desaturase (rate-limiting enzyme for conversion of linoleic acid into arachidonic acid) activity.13 However, arachidonic acid levels in these rats were not elevated,14 and the rats had not been given PN. In the present study, we set out to examine whether skeletal muscle and liver fatty acid profiles are influenced by either type of nutrition or the presence of short gut in our rat model.

METHODS

Male Sprague-Dawley rats (280-300 g) from Taconic Farms (Germantown, NY) were maintained on chow with access to water for 5 days before the study. Animal protocols were in compliance with NIH guidelines and approved by the Hospital Animal Use Committee. The animals (n = 32) were fasted overnight before the short bowel or sham surgery. For surgery, anesthesia was induced with xylazine 13 mg/kg and ketamine 87 mg/kg. Midline laparotomy in the short-gut rats allowed near-complete small bowel resection from 3 cm distal to the ligament of Treitz to 2 cm proximal to the ileocecal junction. End-to-end intestinal anastomosis was performed using interrupted 4.0 silk sutures. Sham animals underwent laparotomy and bowel manipulation and exteriorization for the typical length of time of intestinal resection. For analgesia at laparotomy, infiltration of bupivacaine (0.25%) around the site of the incision was performed, 3 mg/kg. Buprenorphine was administered postoperatively for pain every 6-8 hours, 0.1-0.5 mg/kg, subcutaneously.

TABLE I

Composition of soybean oil lipid emulsion used in Intralipid

A silicone catheter (ID 0.025 in., OD 0.047 in.; Helix Medical Inc, Carpinteria, CA) was placed in the internal jugular vein and tunneled to the interscapular region, then exteriorized and sutured to a swivel (Instech Laboratories, Plymouth Meeting, PA). Four hours postoperatively, PN was started. Short-gut and sham animals (n = 16 per group) were randomized to receive PN with lipid or fat-free PN. Only tap water was allowed orally. PN contained amino acids, dextrose, and essential micronutrients at 200 kcal/kg/d with 2 g nitrogen/kg/d, in 210 mL/kg/d. Rats randomized to lipid (n = 8 per group) got 30% nonprotein energy as fat, using 20% Intralipid (Table I). PN was continued for 5 days, a time that has been shown to alter desaturase activity in response to dietary changes in rodents.15,16

After discontinuation of PN, animals were sedated, blood samples were drawn into plain tubes by cardiac puncture and immediately placed on ice, and euthanasia was achieved using CO2. Biopsies of liver and gastrocnemius were performed immediately, and these tissues were immediately frozen in liquid nitrogen, then stored at – 80C. For analysis, approximately 0.3 g tissue was homogenized in 2 mL saline.

Tissue lipids were extracted by using 6 vol of chloroform- methanol (2:1, vol/vol), centrifuged at 800 g for 3 minutes, and the resulting lower phase was aspirated. Heptadecanoic acid was added to all samples as internal standard in the form of diheptadecanoyl phosphatidylcholine (30 g from a chloroform:methanol [1:1, vol/vol] stock solution; Nu-Chek Prep, Elysian, MN) before extraction. Lipid extracts from the different sample preparations were fractionated into phospholipids by solid-phase chromatography using an aminopropyl column, as described elsewhere.17 We chose to examine phospholipids only, because these more appropriately reflect tissue membranes. The resulting fractions were evaporated to dryness under nitrogen. Fatty acids were transmethylated by alkaline methanolysis using the BF^sub 3^ reagent kit (Supelco, Bellefonte, PA). Dry fractions were resuspended in 0.5 mL of methanolic base, vortexed, and incubated at 100 C for 3 minutes. Subsequently, boron trifluoridemethanol (0.5 mL) was added, and samples were vortexed and incubated at 100C for 1 minute before addition of hexane (0.5 mL), repeat vortexing, incubation at 100C for 1 minute, and addition of 6.5 mL of saturated NaCl. Samples were finally centrifuged at 800 g for 2 minutes. The hexane upper layer was transferred to a new glass tube and an aliquot injected in a Hewlett Packard 5890A gas chromatograph. A Supelcowax column of 30-m length and 0.5-mm internal diameter was used. Initial temperature was 150C and final temperature 260C. Detector temperature was 300C and the total running time, 27 minutes. Fatty acid methyl ester peaks were identified by comparison of retention times of standard mixtures (Nu- Chek-Prep) and quantified in comparison with the internal standard (methylheptadecanoate) detector response.

Results are expressed as nmol percent of total fatty acid content in the sample and total nmol per gram of tissue. Large variations in different fatty acid amounts result in data presentation with differing decimal points, but only 3 significant figures are presented.

Insulin was measured in serum using a commercial radioimmunoassay (RIA) kit (ICN, Costa Mesa, CA). Glucose was measured in serum using glucose oxidase method.

Statistical Analysis

Data are presented as means SEM. Groups were compared for differences using 2-way ANOVA, examining the effects of short-gut vs sham surgery and type of PN. Bonferroni correction was used as a post hoc test (SigmaStat 2.0, SSPS Inc, Chicago, IL). Significance is defined by the 95% CI.

RESULTS

All groups of rats lost some weight after 5 days of PN, with no significant differences between groups as previously reported. All groups of rats were euglycemic at the end of the feeding, with similar plasma insulin concentrations (Table II).

Liver

Rodents fed PN + lipid, irrespective of sham or short-gut assignment, had increases in both percent molarity (Table III) and total amount per g (Table IV) of the 2 essential fatty acids, linoleic (18:2ω6) and α linolenic (18:3ω3) acids in liver phospholipids, reflecting their provision in the nutrition solution. In parallel, total liver fat was higher in the PN + lipid groups. The presence of a short gut increased the distal elongation steps of both ω6 and ω3 pathways (higher 22:4ω6, 22:5ω6 and 22:6&#\969;3), irrespective of diet assignment.

Despite this increased supply of precursors in PN + lipid groups, inhibition of distal elongation and desaturation was found in all 3 families of polyunsaturated fatty acids (PUFA). Thus, 20:3ω9, 20:5ω3, 22:6ω3, 22:4ω6, and 22:5ω6 were all significantly decreased (Table III) in PN/fat groups. In addition, feeding PN/fat inhibited de novo lipogenesis such that both 16:1ω7 and 16:0, which are prominent products of this process, were higher in rats fed fat-free PN.

TABLE II

Serum concentrations (mean SEM) of insulin and glucose in short- gut rats (n = 32) after 5 days of PN*

TABLE III

Profile (mean SEM, n = 32) of liver phospholipids (nmol %) in short-gut rats fed with PN for 5 days

Skeletal Muscle

The findings were quite different in muscle phospholipids. Short- gut rats differed from sham rats both in molar percent and absolute amounts of 18:3ω3, with increases in the sham-operated compared with short gut. However, short gut and sham did not differ consistently for the other fatty acids. Fatty acid profiles reflected mostly dietary supply, with increases in oleic (18:1ω9), linoleic (18:ω6), and α-linolenic (18:3ω3) acids in PN + fat groups (Tables V, VI). Similar to liver, higher palmitoleic (16:1ω7) acid in fat-free PN groups is related most likely to de novo lipogenesis. Distal long-chain PUFA derivatives of these precursors were similar in all 4 groups so that neither diet nor presence of short gut influenced the desaturation/elongation steps.

TABLE IV

Profile (mean SEM, n = 32) of liver phospholipids (nmol per gram tissue) in short-gut rats fed with PN

DISCUSSION

Our results show that short-term PN has little influence on skeletal muscle phospholipid fatty acid profiles in the postoperative rat, whether intact or with short gut. In contrast, hepatic phospholipids are substantially altered even after just 5 days of different PN regimens in the postoperative rat with a short gut. The presence of a short gut led to increases in the amounts of 22:4ω6, 22:5ω6, and 22:6ω3, which are the distal products of elongation and desaturation of linoleic and α linolenic acid. Our methodology does not permit determination of the etiology of these changes, because any number of processes may contribute. Direct measurement of hepatic desaturase activity will allow determination of whether the presence of a short gut results in enzyme up-regulation. We have not performed any direct assessment of hepatic desaturases. Other researchers have shown significant increases in both Δ5 and Δ6 desaturase activities in liver microsomes after 75% intestinal resection.13 However, that study differed from ours in that nutrition support was not given, and a probable state of EFAD was present.14 Clearly, EFAD itself is associated with increased activity of those desaturases, so the 2 models are not directly comparable.

TABLE V

Profile (mean SEM, n = 32) of muscle phospholipids (nmol %) in short-gut rats fed with PN for 5 days

It is likely that future experiments with this shortgut model will demonstrate enhanced Δ5 and Δ6 desaturase activity in hepatic microsomes. A plausible mechanism for the unexpected changes found in the short-gut rat liver involves delivery of nutrients to the liver. In addition to resection of considerable portions of intestine, the operation we performed involved ligation of major tributaries of the portal vein. Therefore, short-gut and sham rats differ in route of PN supply to the liver. In the intact organism, nutrition that is administered through a central vein reaches the liver through both the portal vein and hepatic artery. One potential explanation for the results from the present study is that reduced delivery of lipid via the portal vein in the short-gut rat leads to a perceived dietary deficiency of fatty acids and thus up- regulation of hepatic desaturases. For skeletal muscle, where delivery of nutrition is not altered by the presence of short gut, no differences between these rats and sham rats would be expected.

TABLE VI

Profile (mean SEM, n = 32) of muscle phospholipids (nmol per g tissue) in short-gut rats fed with PN for 5 days

Additional findings from this study were that this short-term duration of fat-free PN was sufficient to enhance turnover of ω- 3, ω-6, and ω-9 precursors into their longer-chain derivatives. This suggests some combination of fatty acid elongases and Δ6/Δ5 desaturase activation in the face of relative substrate deficiency and is not a surprising result.

These data suggest that diet-induced changes in plasma lipid fatty acid profiles, which are sometimes used as a surrogate for tissue phospholipid fatty acids, may not reflect at least short- term tissue responses to dietary manipulations. Another example of a dichotomy between fatty acid profiles in liver and muscle has been found in patients with end-stage liver dis ease where impaired production of very-long-chain PUFA by the liver is not mirrored by skeletal muscle changes.18 In addition, and most relevant to the hospitalized patient receiving postoperative PN, different tissues metabolize the supplied essential fatty acids at quite different rates, with liver responsiveness being far more rapid than skeletal muscle. These data also add to the literature that suggests that different tissue desaturases are active and regulate local fatty acid production.19

ACKNOWLEDGMENTS

KCM was supported by a grant from the American Liver Foundation.

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Karen C. McCowen, MD, MRCPI; Pei-Ra Ling, MD; Mario Ollero, DVM, PhD; Justin A. Maykel, MD; Paola G. Blanco, MD; and Bruce R. Bistrian, MD, PhD*

From the Beth Israel Deaconess Medical Center, Boston, Massachusetts

Received for publication April 12, 2005.

Accepted for publication October 4, 2005.

Correspondence: Bruce R. Bistrian, MD, PhD, Beth Israel Deaconess Medical Center, West Campus, 1 Deaconess Rd, Boston, MA 02215. Electronic mail may be sent to bbistria@bidmc.harvard.edu.

Current author affiliations: Mario Ollero, DVM, PhD, Universit Ren Descartes, Paris, France; Justin A. Maykel, MD, University of Minnesota, Department of Surgery, Minneapolis, Minnesota.

Copyright American Society for Parenteral and Enteral Nutrition Jan/ Feb 2006