Essential fatty acid deficiency in four adult patients during total parenteral nutrition 2

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1 Essential fatty acid deficiency in four adult patients during total parenteral nutrition 2 Timothy J. Richardson and Demetrios Sgoutas ABSTRACT Four undernourished adults (15%-37% belo ideal body eight) received fat-free total parenteral nutrition for a period of 6 to 8 eeks. Chemical evidence of essential fatty acid deficiency (triene:tetraene ratio greater than.4) appeared in all patients during the first 3 eeks of treatment. Deficiency developed more rapidly in the to patients ho ere younger (ages 16 and 36) and more severely undernourished (26% and 37% belo ideal body eight) than in the to older patients (ages 62 and 76) ho ere less undernourished (15% and 16% belo ideal body eight). All patients continued to gain eight and maintain positive nitrogen balance throughout the course of total parenteral nutrition and developing essential fatty acid deficiency. Hepatomegaly and increased serum liver enzyme activities occurred in the to patients ith evidence of the most severe essential fatty acid deficiency. Liver biopsy, in the cases ith hepatomegaly, shoed hepatocytes containing fat and hat appeared to be enlarged, spherical mitochondria. There as no cellular infiltrate or significant degree of necrosis. Supplementation ith oral linoleic acid (as saffloer oil) reversed the essential fatty acid deficiency and the elevation of serum liver enzymes. Am. J. Clin. Nutr. 28: , In recent years, essential fatty acid (EFA) deficiency has been reported in humans in association ith the increasing use of fat-free intravenous nutrition. In infants receiving fatfree total parenteral nutrition (TPN) for 2 eeks or more, a clinical syndrome of scaly skin lesions, sparse hair groth, thrombocytopenia, and poor ound healing has been accompanied by the ell-knon chemical signs of EFA deficiency in plasma: lo content of linoleic and arachidonic acids and a high content of 5,8,1 t-eicosatrienoic acid (1-3). The triene:tetraene ratio, a chemical measure of the severity of EFA deficiency, as greater than 1 in symptomatic infants (2). Collins et al. (4) reported the case of an adult patient ith a skin rash, refractory anemia, and a triene:tetraene ratio beteen 2. and 3., after 14 eeks of fat-free TPN. Generally, hoever, very fe adult cases have been reported and it has been postulated that the deficiency is difficult to produce in adults because of large body stores of EFA. In this investigation, four undernourished adults that e studied during a 6- to 8-eek period of fat-free TPN developed EFA deficiency at a rate similar to that reported in infants (3). In to patients, oral saffloer oil supplementation alloed us to further define the linoleic acid requirement in adults, and to relate chemical and histological signs of hepatic dysfunction to EFA deficiency. Experimental methods The four patients studied ere referred by physicians associated ith Emory University. The pertinent clinical data ere as follos: EH: 36-year-old female in hom stage IIB carcinoma of the cervix had been diagnosed 18 months prior to admission. She had undergone multiple surgical procedures including a total pelvic exenteration. Her body eight as 37 kg or 74% of the ideal eight, calculated according to the Metropolitan Life Insurance Company actuarial tables (5). JM: 16-year-old male ith a 4-year history of regional enteritis. The patient as admitted for a course of TPN to manage an acute exacerbation and to prepare for segmental ileal resection. His body eight as 28 kg or 63% of ideal. WS: 76-year-old female ho had sustained a fracture of the left femoral neck in an automobile accident 14 months prior to admission. Multi- From the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia Supported by National Institutes of Health Grant RR TheAmerican Journal of Clinical Nutrition 28: MARCH 1975,pp Printed in U.S.A.

2 ESSENTIAL FATTY ACID DEFICIENCY 259 ple orthopedic surgical procedures had failed and the patient had become anorexic and depressed during her extended period of debilitation and chronic hip discomfort. She as referred to the Clinical Research Facility for TPN preparation for insertion of a prosthetic femoral head. Her body eight as 42 kg or 84% of ideal. RW: 62-year-old male ith a long history of food intolerances folloing subtotal gastrectomy for peptic ulcer; admitted for evaluation of his unusual dietary habits and treatment of chronic undernutrition by TPN. His body eight as 55 kg or 85% of ideal. Total parenteral nutrition as performed in the Clinical Research Facility of Emory University according to Dudrick s method (6). Table 1 shos the contents of the infusion medium. At an average daily intake of 2.6 liters of infusion fluid, the patients received approximately 3,4 kcal. There as no oral intake except that to patients (EH and JM) received saffloer oil after EFA deficiency had appeared. Saffloer oil fatty acids contain 7% linoleic acid (7), and 1 2 g of linoleic acid ill provide 4% of the total daily caloric intake, assuming that 1 % of fat given absorbed. JM received 2 g of the oil (12.6 g of linoleic acid) during the sixth eek of TPN, hile EH received 3 g of saffloer oil during a comparable period. Blood as dran during the infusion. Specimens for lipid analysis ere collected in EDTA. Plasma as separated by centrifugation at 4C at 1, g for 1 mm and the erythrocytes ere isolated and studied separately (7). Aliquots of fresh plasma ere used for lipoprotein analysis by electrophoresis (8) and by ultracentrifugation (9). The remaining plasma as stored at -2C until cholesterol, lipid phosphorus, and triglycerides ere determined according to standard procedures (1-12). Total lipids ere extracted from 1. ml aliquots of plasma ith chloroform: methanol (2:1, v/v) according to Folch et al. (13) and evaporated to dryness under reduced pressure. The residue as then eighed and considered to represent the total lipid present in serum. Total lipid as applied to microcolumns of silicic acid (Unisil Clarkson TABLE 1 Composition per liter of the infusion fluid Dextrose (5% solution) 5 ml Freamine 5 ml Phosphorus 4 mg K 4OmEq Q 38mEq Na 5OmEq Mg2 l6meq lomeq % Freamine solution. Approximate concentration of amino acids (g/1 ml): L-isoleucine.59; L-leucine.77; L-lysine HC1.77; methionine.45; L-phenylalanine.48; L-threonine.34; L-tryptophan.13; L-valine.56; L-alanine.6; L-arginine.31; L-histidine.24; L-proline.95; L-serine.5; glycine 1.8; L-cysteine-HCI-H2O <.2. Chemical Company, Williams, Pa.); neutral lipids ere eluted ith chloroform and phospholipids ith methanol. Methyl esters of fatty acids ere prepared by transesterification using 4% sulfuric acid in methanol as previously described (7). Chromatographic peaks ere identified by comparison ith commercial standards (Applied Sciences Laboratories, State College, Pa.). 5,8,1 l-eicosatrienoic acid as identified by comparison to a chromatogram of hepatic phospholipid fatty acids of a rat rendered EFA deficient by dietary means. Quantification as by means of triangulation and the detector as calibrated using a standard containing methyl esters of saturated fatty acids ith even number of carbon atoms. Experimental values agreed ith calculated values ith an error of less than 4% for the major components. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GI5T) ere assayed spectrophotometrically by the method of Karmen et al. (14) and Henry et a!. (15), respectively. Creatinine phosphokinase (CPK) as measured according to Rosalki (16). Lactic acid dehydrogenase (LDH) as assayed according to Amador et al. (1 7) and alkaline phosphatase as determined by the method of Tietz and Green (18). Total protein, serum albumin, bilirubin, calcium, inorganic phosphorus, blood glucose and blood urea nitrogen (BUN) ere determined in a SMA 12/6 AutoAnalyzer. Electrolytes ere assayed by flame photometry (19) and magnesium by atomic absorption (2). Other tests performed ere complete blood count (CBC) (21) and serum osmolality (22). Results Body eights for the four patients shoed a continuous gain throughout the period of TPN. EH, JM, WS and RW had a final eight of 46, 37, 47, and 61 kg, respectively, indicating an increase in body mass of an average of 7.5 kg. Nitrogen balance remained positive (+3 to +8 g N/day) in all patients in spite of the developing EFA deficiency. There as no significant decline in the patients anabolic response to the perfusate. The lipid classes folloed during TPN included plasma phospholipid fatty acids, tnglycenides, lipoproteins, and cholesterol. Figure 1 shos plasma phospholipid fatty acids in patients EH and JM before, during TPN, and after the oral supplementation ith saffloer oil. Changes during TPN included a decrease of approximately 8% in linoleic acid and 6% in arachidonic acid, small increases in palmitic and oleic acids and a large increase in 5,8, 1 1-eicosatrienoic acid. After saffloer oil supplementation began (denoted by arro), the concentrations of the fatty acids ere restored to pretreatment values: linoleic and arachidonic

3 26 RICHARDSON AND SGOUTAS a. e C acids ithin 2 eeks, and the 5,8,1 1-eicosatrienoic acid ithin 5 eeks. Figure 2 gives the ratio of 5,8,1 t-eicosatnienoic acid (tniene) to arachidonic acid (tetnaene) against time. Within 3 eeks of the start of TPN all four patients had a ratio above.4, hich is C) >- F F indicative of a developing EFA deficiency (23). I. 6 8 WEEKS FIG. 1. Changes in the major phospholipid fatty acids of plasma of EH (solid points) and JM (liollo points) during TPN. The treatment started the first day after the first sample as analyled ( time). At the time indicated by an arro, saffloer oil supplementation as initiated and continued until the eighth eek hen TPN as discontinued and a regular oral diet began.., o, Palmitic acid, #{149}, o, oleic acid;#{149}, o, linoleic acid;,o, 5,8,1 l-eicosatrienoic acid; and &, A, arachidonic acid. Each point represents the mean of to determinations. Weeks FIG. 2. Plot of triene:tetraene ratio of serum phospholipid fatty acids versus time of hyperalimentation. EH,.; RW, A; WS, ; and JM, o. Hyperalimentation as discontinued after 6 eeks in WS and RW and after 8 eeks in EH and JM. Saffloer oil supplement as initiated in EH and JM during the 6th eek. 8 EH and JM reached a maximum value of 2.6 and 2.3, respectively, just prior to saffloer oil supplementation, hereas RW and WS reached ratios of 1.3 and I., respectively, at the time that TPN as discontinued and a regular diet as initiated. The rate of the development of the deficiency (indicated by the slope of the plotted line of the tniene:tetnaene ratio) as more rapid in patients EH and JM than in patients RW and WS. Serum lipid analyses for EH and JM are shon in Table 2. Triglycerides began to increase as soon as TPN as initiated and they continued to rise until saffloer oil supplementation restored them to their pret reatment values. Lipoprotein electrophoresis indicated a continuing increase in the pne-3-lipoprotein fraction until saffloer oil as administered, hen this class of lipoproteins promptly returned to preinfusion levels. In describing similar results, Collins et al. (4) provided evidence indicating that the high level of triglycerides as not transported by the plasma very lo density lipoproteins. Expeniments in this laboratory, hich ill be communicated separately, shoed that the extra load of the triglycerides as carried by an abnormal lipoprotein of the sinking type (26). During the course oftpn, plasma concentrations of electrolytes, magnesium, calcium, BUN, and phosphorus remained ithin normal range. There as no significant change in the CBC or prothrombin time. The only apparent change during the developing EFA deficiency involved the liver function tests. Although bilirubin, total protein, and alkaline phosphatase values did not change, a marked elevation of GOT, GPT, CPK, and LDH as observed in patients EH and JM. Figure 3 shos that changes in GOT activity in patients EH and JM paralleled changes in the tniene:tetraene ratio. The saffloer oil supplement, given to EH and JM corrected the deficiency and the level of GOT simultaneously returned to normal. LDH and GPT levels (not shon) closely paralleled the increases and decreases in the tniene:tetraene ratio similar to GOT. CPK concentrations, hoever, ere not altered by the saffloer oil supplementation. Scaly skin rash, a classical clinical feature of EFA deficiency, as not observed in any of the patients in spite of chemical evidence of EFA deficiency. EH experienced a septic episode near the end of the TPN period. Changes in

4 ESSENTIAL FATTY ACID DEFICIENCY 261 TABLE 2 Lipid composition of plasma in hyperalimented patients EH JM Weeks of Cholesterol, Triglycerides, Phospholipids, Cholesterol, Triglycerides, Phospholipids, Treatment mg/1 ml mg/too ml mg/1 ml mg/1 ml mg/1ml mg/1 ml ito Saffloer oil as given orally after the 5th eek. Normal values ith their standard deviations are: cholesterol 23 ± 15; triglycerides 11 ± 12; and phospholipids 228 ± 16. a z a I- z Li a WEEKS FIG. 3. Plot of triene:tetraene ratio of serum phospholipid fatty acids-eh,.; JM, o and serum GOT activity-eh, ; JM, A versus time of TPN. Saffloer oil supplementation began during the 6th eek, as indicated by the arro. serum liver enzymes ere associated ith clinically evident hepatomegaly in both EH and JM. Histological examination of the liver by percutaneous biopsy (EH) and at laparotomy (JM) revealed no evidence of mass necrosis but it did sho some minor structural alterations. In patient EH during the sixth eek of TPN (triene:tetraene ratio of 2.5) the biopsy revealed fatty infiltration of hepatocytes particularly in the region of the central vein. There as no cellular infiltrate but occassional parenchymat cells had degenerated. Many hepatocytes contained large eosinophilic cytoplasmic spheres hich ere thought to be enlarged, sollen mitochondria similar to those seen in advanced EFA deficiency in mice (27). JM shoed a similar histologic pattern although the fatty infiltration as more marked. Discussion The rapid development of EFA deficiency in EH and JM parallels the findings in infants (1, 3) and contrasts ith a previously reported adult case (4). The deficiency observed in RW and WS developed more sloly and as less severe. This contrast of EH and JM ith RW and WS may be related to to factors: the younger age of EH and JM and the more severe pretreatment undernutrition of EH and JM. Ordinarily one ould not expect an age difference alone to influence susceptibility to EFA deficiency. The severe undernutrition, hoever, of JM and EH may have depleted their body stores of EFA to a level comparable to that of infancy (26). In addition, the anabolic response to TPN by EH and JM (average daily eight gain of 21 g and 2 g, respectively) as greater than that by RW and WS (average daily eight gain of 12 g and 15 g, respectively), and this more rapid tissue groth may have increased utilization of EFA. The daily requirement for linoleic acid in the adult, particularly in the settling of rapid anabolism, has not been ell established. Collins et al. (4) suggested that hen infused intravenously more than 7.5 g linoleic acid ould be needed daily to prevent EFA deficiency. We found that hen taken orally g of linoleic acid prevented further deficiency from developing, and it reversed the deficiency already present. In caloric intake, the results ould suggest that if linoleic acid provides 4% of the daily calories, any EFA deficiency ill be very minor and certainly subclinical. Any

5 262 RICHARDSON AND SGOUTAS supplement providing more than this percentage ill probably prevent EFA deficiency from occurring during fat-free TPN. It should be noted, hoever, that because the patients had intestinal diseases, the amount of linoleic acid given orally cannot meaningfully be expressed as a percentage of the total caloric intake. The present results also seem to suggest that EFA deficiency may be a primary etiologic factor in the liver dysfunction associated ith fat-free TPN. In patients EH and JM, moderate EFA deficiency of brief duration as associated ith structural alterations in hepatic cells and marked elevation of serum enzyme activities. Prompt return of enzyme levels to normal after administration of saffloer oil, suggested a causative role of the EFA deficiency. Presently, e are studying several patients using TPN in order to document the relationship beteen EFA deficiency and liver changes. It is of interest to note that contrary to experiments in rats in hich EFA deficiency caused fatty liver and hypotriglyceridemia (28), our experiments and those of others (4) in man during fat-free TPN have shon increased serum triglyceride levels to accompany the development of fatty liver. Possibly, a severe and prolonged EFA deficiency, as in the rodent experiments is required before hepatic secretion of triglycerides ould become impaired. fl The authors are very much indebted to Dr. W. J. Millikan for performing the catheterizations and for his help ith the patients. Dr. D. Rudman made available the patients and provided valuable assistance. The excellent technical assistance of Mrs. R. Jones is acknoledged. References 1. CALDWELL, M. D., AND H. T. JOHNSON. Essential fatty acid deficiency in an infant receiving prolonged parenteral alimentation. J. Pediat. 81: 894, PAULSRUD, J. R., L. PENSTER, C. F. WHIT- TEN, S. STEWART AND R. T. HOLMAN. Essential fatty acid deficiency in infants induced by fat-free intravenous feeding. Am. J. Clin. Nutr. 25: 897, WHITE, H. B., M. D. TURNER, A. C. TURNER AND R. C. MILLER. Blood lipid alterations in infants receiving intravenous fat-freealimentation. J. Pediat. 83: 35, COLLINS, F. D., A. J. SINCLAIR, J. P. ROYLE, D. A. COATS, A. T. MAYNARD AND R. F. LEONARD. Plasma lipids in human linoleic acid deficiency. Nutr. Metabol. 13: 15, GOODHART, R. S., AND M. E. SHILS. Modern Nutrition in Health and Disease. Philadelphia: Lea & Febiger, 1973, p DUDRICK, S. J. Parenteral hyperalimentation. Ann. Surg. 176: 259, SGOUTAS, D. S., AND R. JONES. The effect of intravenous hyperalimentation on erythrocyte lipids. Proc. Soc. Exptl. Biol. Med. February NOBLE, R. P. Electrophoretic separation of plasma lipoproteins in agarose gel. J. Lipid Res. 9: 693, HAVEL, R. J., H. A. EDER AND J. H. BRAGDON. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34: 1345, ZLATKIS, A., B. ZAK AND A. J. BOYLE. A ne method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 41 : 486, BARTLETT, G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 234: 466, VAN HANDEL, E., AND D. B. ZILVERSMIDT. Micromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 5: 152, FOLCH, J., M. LEES AND G. H. SLOANE STANLEY. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497, KARMEN, A., F. WOROBLEWSKI AND J. S. LADUE. Transaminase activity in human blood. J. Clin. Invest. 34: 126, HENRY, R. J., N. CHIAMORI,. J. COLUB AND S. BERKMAN. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminal glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am. J. Clin. Pathol. 34: 381, ROSALKI, S. B. An improved procedure for serum creatine phosphokinase determination. J. Lab. Clin. Med. 69: 696, AMADOR, E., L. E. DORFMAN AND W. E. C. WACKER. Serum lactic dehydrogenase activity: an analytical assessment of current assays. am. Chem. 9: 391, TIETZ, N. W., AND A. GREEN. An automated procedure for the determination of phosphorine and alkaline phosphatase in serum. Clin. Chem. Acta 14: 566, l HENRY, R. J. Clinical Chemistry: Principles and Techniques. Ne York, Hoeber Medical Division: Harper & Ro, WILLIS, J. B. The determination of metals in blood serum by AAS. II. Magnesium. Spectrochim. Acta 16: 273, DAVIDSOHN, I., AND J. B. HENRY. Clinical Diagnosis by Laboratory Methods. Philadelphia: Saunders, JOHNSON, R. B., AND H. HOCHS. Osmolality of serum and urine. In: Standard Methods of amnical Chemistry, edited by S. Meites. Ne York: Academic, 1965, vol HOLMAN, R. T. Biological activities of and requirements for polyunsaturated acids. In: Prog-

6 ESSENTIAL FATTY ACID DEFICIENCY 263 ress in the Chemistry of Fats and Other Lipids. Oxford: Pergamon, 1971, vol. IX. 24. HANSEN, A. E.,. BECK AND H. F. WIESE. Susceptibility to infection manifested by dogs on a lo fat diet. Federation Proc. 7: 289, 1948 (abstr). 25. HANSEN, A. E., H. F. WIESE, A. N. BOELSCHE, M. E. HAGGARD, D. J. D. ADAM AND H. DAVIS. Role of linoleic acid in infant nutrition. Clinical and chemical study of 428 infants fed on milk mixtures varying in kind and amount of fat. Pediatrics 31: Suppl. 1, 171, Memorandum by World Health Organization: Classification of hyperlipidemias and hyperlipoproteinemias. Bull. World Health Organ. 43: 891, WILSON, J. W., AND E. H. LEDUC. Mitochondrial changes in the liver of EFA deficient mice. J. Cell Biol. 16: 281, FUKAZAWA, T., AND. 5. PRIVETT. Biosynthesis of protein relative to the accumulation of fat in the liver of EFA deficient rats. Lipids 7: 387, 1971.