Essential fatty acids deficiency in humans

Proo. Lipid Re.s, Vol. 19. pp. 187-215 C) Pergamon Press Ltd 1981. Printed in Great Britain

0163-7827/81/0701-0187505.00/0

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ESSENTIAL

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FATTY ACIDS DEFICIENCY

IN HUMANS

WILLIAM K. YAMANAKA, GORDON W. CLEMANS* and MARTHA L. HUTCHINSON* Division of Human Nutrition, Dietetics and Foods, University of Washington, Seattle, Washington 98195, U.S.A. and Division of Medical Oncoiogy) Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, U.S.A." CONTENTS I. INTRODUCTION

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II. Essential Fatty Acids (EFA) A. Definition of Essential Fatty Acids B. Dietary requirement for EFA C. Physiological functions of EFA

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Ill. ESSENTIALFATTYACIDD~HCI~CY (EFAD) A. Incidence in human nutrition B. Disease conditions that alter EFA status C. Biochemical changes in animal studies D. Biochemical changes in human studies E. Cellular effects of EFAD F. Treatment of EFAD

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IV. EVICT OF EFAD ON ORGANSvs'r~MS A. Effectof EFAD on growth B. Skin changes in EFAD C. Immunity and resistance to infection in EFAD D, Hematopoiesis in EFAD E. Liver changes in EFAD F. Pulmonary changes in EFAD O. Digestive system changes in EFAD H. Reproductive changes in EFAD I. Nervous system changes in EFAD J. Heart changes in EFAD

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CONCLUSION

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VI. REFERENCES

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I. I N T R O D U C T I O N

Essential fatty acid deficiency (EFAD), although discovered over 50 years ago, was rarely reported in humans prior to the introduction of long-term parenteral nutrition (PN) in 1968. Long-term PN has saved the lives of countless persons suffering from diseases which prohibit oral intake of nutrients for extended periods and has made feasible the use of medical treatments which had previously been inapplicable due to side effects which seriously compromised oral food tolerance. Since the introduction of long-term PN, reports of EFAD have accumulated rapidly because no source of essential fatty acids was approved for use in conjunction with PN in the United States prior to 1975 and adoption of the use of intravenous fat infusion did not follow immediately upon approval by the Food and Drug Administration. Continuous fat-free PN seems to provide optimal conditions for development of EFAD. It supplies no fat and inhibits the mobilization of the body's fat stores. EFAD has a wide range of potential consequences. They include effects upon skin morphology and permeability, pulmonary function, digestive function, liver morphology and function, hematopoiesis, immunity and infection resistance, reproductive function, 187 J,P.L.R. 19/3-4---F

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and nervous system function. One of the most interesting areas of research during the past few years is the relationship of EFAD and prostaglandin activity. The subject of EFA and polyunsaturated acids was reviewed in Volume 9 of Progress in the Chemistry of Fats and Other Lipids (precursor to this series). II. ESSENTIAL FATTY ACIDS A. Definition of Essential Fatty Acid

Fatty acids which relieve the symptoms produced in higher animals which have undergone prolonged treatment with fat-free, but otherwise adequate diets are considered to have essential fatty acid (EFA) activity. All recognized naturally occurring EFAs are polyunsaturated fatty acids (PUFA) having a carbon chain length of 18, 20, or 22 atoms and from 2 to 6 methylene-interrupted unsaturated bonds all in the cis configuration, a cis, cis. 1,4-pentadiene arrangement.~ 67 The two dietary fatty acids having significant EFA activity and acting as precursors from which all other naturally occurring EFAs can be derived by in vivo chain elongation and desaturation are linoleic acid and ~-linolenic acid. 1. Linoleic Acid and Alpha Linolenic Acid

Linoleic acid gives rise to the omega (co) 6 family of EFAs; ~-linolenic acid gives rise to 'he o)3 family, co Notation indicates the position of the unsaturated bond nearest the alethyl end of the fatty acid. Because in higher animals fatty acid chain lengthening occurs at the carboxyl end of the fatty acid molecule and desaturation occurs between the carboxyl end and the unsaturated bond nearest the carboxyl end, the number of carbon atoms between the methyl end of the molecule and the nearest double bond is fixed. Thus, the co number is unaffected by elongation and desaturation. The divinyl methane (methylene interrupted) pattern of unsaturated bonds is maintained, so the position of all unsaturated bonds relative to the methyl end of the fatty acid is invariant. Therefore, the structure of any PUFA naturally occurring in higher animals is fully described by three numbers: the number of carbons in the chain, the number of double bonds, and the number of carbon atoms between the terminal unsaturated bond and the methyl end of the fatty acid (the to number). Linoleic (18:2o)6) and linolenic (18:3o)3) acids have no endogenous precursors in animals or man. No significant synthesis of linoleic or linolenic acid occurs de novo in higher animals. 167 Thus, although there is some evidence that gut bacteria of some animals might be able to synthesize some linolenic acid, 15° these two fatty acids are referred to as the dietary EFAs. Of the naturally occurring fatty acids, it is those of the linolcic acid (o)6) family which are most active as EFAs. ~-Linolenic acid and its products are not capable of correcting all symptoms of EFA deficiency. For instance, it has been reported that linolenic acid stimulates growth but does not correct dermal symptoms or infertility in animals on a fat-free diet. 99 Tinoco et al. 2°4 suggest that linolenic acid may h a w limited function in the cerebral cortex or retina. Although no physiologically significant synthesis of linoleic acid occurs in higher animals, several studies have indicated that, at least under certain circumstances, some synthesis does occur in some animals. There arc reports of linoleic acid formation from a monounsaturated fatty acid by laying hens. ~s~ It has also been reported that fat deficient rats convert small amounts of 14:2oJ6 to linoleate, and that rats may convert rare 16:2o)6 to longer m6 polyunsaturated fatty acids.t93 • 2. Arachidonic Acid

Another member of the w6 family, arachidonic acid (20:4o)6), exhibits strong EFA

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activity. This fatty acid, which mammals readily form from linoleic acid and which is present in the diet in animal fats, appears to completely cure all fat deficiency symptoms,146It is aboutthree times as effective in stimulating growth in animals as is linoleic acid. l"e This has led to the suggestion that arachidonic acid might be the only truly E F A . 172 However, compelling evidence in conflict with this theory is provided by the fact that certain prostaglandins which appear to perform necessary physiological functions are formed from dihomo-7-1inolenic acid (20: 3co6), an intermediate in the synthesis of arachidonic acid. 17s Also, co6 fatty acids longer and more highly unsaturated than arachidonic acid, appear to he commonly found in membrane structural lipids. 99

3. Other Fatty Acids It has become increasingly clear that fatty acids other than those of the co6 and the co3 families can have EFA activity. Odd carbon chain number fatty acids of the oJ5 and co7 families have been shown to exhibit EFA activity, and it is possible that the to2 family also may do so. lsl'ls2 17:2co5 and 19:4c05 are active as EFAs even though they are not converted to arachidonate. 6 Experiments using synthetic fatty acids have resulted in the formation of several theories concerning the structural characteristics necegsary for EFAs in the performance of their apparent structural function in liver mitochondria 1o5 and their prostaglandin formation function. 2°7

4. Specific Roles of EFA in Synthesis of Essential Compounds At this time, it is not completely clear why fatty acids with specific structures exhibit EFA activity. Specifically, it is not certain what metabolic function(s) they perform in correcting fat deficiency symptoms. Nevertheless, it is known th&] EFAs act as precursors for prostaglandins 17s and it has been stated that "there is a stril~ing correlation between the: essential fatty acid activity of the precursor acids and the rate of formation of biologically active prostaglandins derived therefrom".2°7 Speculation is ongoing concerning whether prostaglandin synthesis is fully responsible for correction of EFAD symptoms in animals refed EFA. 2°7 But it is noteworthy here that less than: 0.01% of the linoleic acid which is normally taken in is excreted as prostaglandin metabolites 2°s and that administration of prostaglandins has as yet proven unsuccessful in correcting all EFAD symptoms. This has led to the suggestion that EFA must be required for other purposes in addition to prostaglandin synthesis.6 What these purposes are, if any exist, is uncertain, although it is known that PUFA have a role in formation of membrane phospholipids 99 and probably also in lipid transport. 9s PUFA are components of lipoprotein enzyme complexes and they may serve to hold enzymes in position in membranes. 2°s However, researchers have concluded that enzymatic reactions requiring PUFA-containing phospholipids may not specifically require EFAs, ~7 and that the membrane related and lipid transport functions of PUFA also may be performed by nonEFAs. 2°s

B. Dietary Requirement for EFA 1. Early Studies In 1918 Aron first suggested that fats have nutritional functions other than provision of food energy.98 In 1929 George and Mildred Burr succeeded in producing a fat-free experimental diet, which they used to demonstrate EFAD in rats. z3 The following year they showed that PUFA, especially linoieic acid, was responsible for dietary fat's health maintenance and restorative effect on fat deprived animals, '4 They coined the term "essential fatty acids." Because of the difficulty of producing a fat-free diet, the long period of time required to produce outwardly apparent deficiency symptoms in adult animals, 13 and the variability

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of the symptoms with different experimental conditions, 27 the early findings by Burr and Burr 2. were not consistently replicable. Hence, skepticism as to the significance and even the existence of EFAD persisted for a number of years.9a Not until the late 1950s, when accurate biochemical testing for EFAD became possible and humans were unequivocally demonstrated to require linoleic acid a2 did estimates of required levels of dietary EFA begin to appear in scientific literature. Since then estimates that 1-2% of total caloric intake as linoleic acid is required to avoid biochemical and clinical signs of EFAD in various higher animals have been widely supported. 95'a7

2. Requirements in Infants Human infants and children are generally believed to require a similar amount of dietary linoleate, 1-2~o of total calories as lin01eate, to avoid EFAD. 36,*°L-a5 This is somewhat less than is found in human milk. 33 However, one recent study concluded that the requirement may in fact be lower 39 while another indicates that a somewhat higher level of intake might be required to facilitate quick recovery in EFA deficient infantsJ s° Optimal intake level for infants has been estimated to be 4% of total calories, 2 or about 100mg of 18:2 per kg of body weight per day. .74

3. Requirements in Older Children and Adults Children probably require more linoleic acid as a percentage of total daily caloric intake than do adults because growth increases the demand for cell membrane components. Nevertheless, 1-2~ of total calories as linoleate is also the most common estimate of the adult linoleate requirement.~ In patients who have suffered EFAD as a result of absorptive dysfunctions or who are receiving total parenteral nutrition, 2 ~ of calories as linoleate might not be sufficient. One study of an adult whose small intestine had been partially excised reported that administration of parenteral fat emulsion providing 2.29/0 of total calories as linoleate failed to eliminate biochemical evidence of EFAD. al The authors point out that removal of major portions of the small intestine may decrease the effectiveness of bile resorption, increasing the required linoleate intake. They conclude that "the requirements in man are close to those of animals," and that a n adult man may require about 7.5 g of linoleate per day. More recently, it has been reported that administration of 100 g of linoleate per week ~ 14 g/day) as parenterai fat emulsion failed to prevent the development in an adult of skin lesions characteristic of EFAD.I 1, In another study in which EFA deficient patients were treated using parenteral fat, one patient's daily linoleate infusion was decreased to 2% of calories from 10.5% with subsequent biochemical evidence of an increase in the severity of deficiencyJ ~4 The authors concluded that "the parenteral fat requirement to prevent essential fatty acid deficiency, particularly during the phase of rapid anabolism, has not been clearly established." Determination of the minimum EFA requirement and of optimal intake level is complicated by the fact that dietary intakes of other nutrients may affect the EFA requirement. For instance, it has been shown that inclusion of saturated fats, cholesterol and probably medium chain triglycerides in the diet may increase the EFA requirement.98,~ 19,197

C. Physiological Functions of EFA As recently as 1970 it was stated in an extensive review of literature concerning EFAs that "it is not known why EFA are dietary requirements."77 Though it remains true that the underlying causes of many of the clinical symptoms of EFAD are not well understood, there appears to be agreement that EFA fulfil structural functions, as part of membrane lipoprotein complexes. EFA may also for%n necessary components of plasma lipid transport complexes. Vigorous exploration of the function of EFA in the formation

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of prostaglandins, thromboxanes and endoperoxides is now underway. (See Volume 20 of this series for discussions of these subjects,) Evidence is accumulating that poor performance of one or more of these three functions in EFAD states may be causally linked to clinical manifestations of deficiency. 1. Membrane Function

The first function to be widely attributed to EFA, or to PUFA, was that of an essential component of the phospholipids which serve as structural units of biomembranes. Extensive circumstantial evidence suggests that EFA are necessary for normal membranes. Compared with triglycerides, phospholipids have a greater affinity for EFA, indicating that EFA are preferred structural lipid components. 16° The fl position of structural phospholipids is preferentially esterified with EFA-family fatty acids. 2~° The physical properties (e.g. fluidity)of phospholipids are in large part determined by the chain length and degree of unsaturation of their component fatty acids. The physical properties, in turn, affect the phospholipids' ability to perform structural functions, such as maintenance of the normal activity of membrane-bound enzymes,s4 The location of double bonds within fatty acids also affects the membrane properties, 52 suggesting the possibility that EFA, rather than PUFA in general, might be required for normal membrane function. Changes in levels of dietary lipid are capable of affecting the properties of membranes of mammalian tumor cells, 116 With inadequate intake of EFA, 5,8,1 l-eicosatrienoic acid appears partially replacing EFA in the fl position of phospholipids. Membrane malfunction follows. 1°5 Perhaps the strongest evidence that membrane dysfunction is a direct result of changes in the fatty acid pattern of structural phospholipids is the mitochondrial swelling which seems to occur with EFAD, but which has been studied primarily in vitro. 9~ Mitochondria are normally high in EFA and no prostaglandin synthesis is known to occur within them. 1°5 The growth of mouse LM cells in suspension culture has been shown to be inhibited by media which were high in long chain, saturated fatty acids and lacking EFA. 4a When the percentage of unsaturated fatty acids in membrane phospholipids was reduced to below 50%, cell growth was severely curtailed. This suggests that normal membrane fluidity may be necessary for optimal cell growth. Several studies suggest that membrane permeability is affected by changes in its fatty acid composition which occur in EFAD. The calcium permeability of sarcoplasmic membranes may be diminished in EFA deficient rats. ~a6 A study of L1210 murine leukemia cells indicates that changes in membrane fluidity caused by varying the lipid content of the host diet affect the extent to which methotrexate, an antineoplastic drug, is able to enter intact leukemia cells. '2 It has also been shown that smooth endoplasmic reticulum proliferation in response to phenobarbitol is reduced in EFA deficient rats, 26 suggesting reduced penetration of cell membranes by the drug. 2. Prostaglandin Synthesis

Prostaglandins (PGs)are a group of potent hormone-like substances with very short half-lives which are produced in a number of tissues in response to a variety of stimuli. PGs act near the site of their synthesis to produce varied physiological effects. Endoperoxides, intermediates in the synthesis of PGs, also have important metabolic effects. EFA derivatives with 20 carbon atoms are the direct precursors of all common prostaglandins, 2°a though PGs can be formed from other fatty acids. 1°'2°7 In higher animals PG synthesis and lipoxidation, a reaction of uncertain functions, are the only enzymecatalysed reactions known to require EFA as substrates. Arachidonic acid serves as the precursor for prostaglandin E2(PGE~) and PGF~. l~a Dihomo-~-linolenic acid (20:3ta6), an intermediate product in the conversion of linoleic acid' to arachidonic acid, is the precursor of PGEI 2°s and PGFI~. 17s

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These are formed from fatty acids which are freed from the fl position of membrane phospholipids. The first step in formation is the splitting of the fatty acid from the phospholipid by phospholipase A2. It appears that factors limiting the availability of direct precursors of PG play a role in controlling the rate of PG formation,a: Dietary EFA intake affects PG synthesis. EFAD has been shown to lead to decreased levels of pG.2O~ Homeostatic mechanisms may operate in EFAD to minimize its effect on PG levels. EFAD appears to trigger increased synthesis of PG from available substrate 114 and decreased PG catabolism, s7 Conversely, increasing dietary linoleic acid 51'1°v or arachidonic acid lss has been shown to increase PG levels. Another way in which fatty acids are involved in the regulation of PG metabolism is through their ability to inhibit PG synthesis. Oleic, linoleic and 0Minolenic acids have been shown to decrease the conversion of arachidonic acid to PGE2.1sa It is thought that this might be an instance of competitive inhibition. The 5,8,11,14-cicosatetraynoic acid also can inhibit PG synthesis. 19° The extent of EFAD or supplementation which is required to affect PG synthesis is uncertain. But variations in the makeup of dietary fat which might occur within normal human diets apparently do not affect PG synthesis. However, biosynthesis is increased when the dietary ratio of linoleic acid to saturated fatty acids exceeds five and is curtailed when biochemical EFAD is present, xzs It has been suggested that in rats it is necessary to produce serum 20:3m9/20:4m6 ratios ~ six before significant decreases in platelet PG synthesis consistently result. ~s9 A study of three stressed low birth weight infants whose mean 20:3o~9/20:4~o6 ratio was 2.7, found significantly decreased levels of urinary PGE metabolite when compared with controls and levels after EFA repletion. 67 In contrast, a study of rabbits maintained on a fat-five diet for up to eight weeks reported decreased levels of PGE, E2, and F2~ in lung, skin, and eye tissue, as This occurred despite the absence of a decrease in the relative levels of PG-precursor fatty acids in liver or red cell phosphoglycerides. 3. Lipid Transport The possibility that EFA is necessary for normal lipid transport from the liver is suggested by the development of fatty liver which is often noted in EFAD. However, it is currently uncertain whether EFA have a specific function in lipid transport or if the fatty liver apparently characteristic of EFAD represents a failure in prostagiandin synthesis or in some unknown function of EFA.

ilI. ESSENTIAL FATTY ACID DEFICIENCY (EFAD) A. Incidence in Human Nutrition It has long been felt that the probability of EFAD developing in the general infant population of Western countries is very low, although premature infants might be an exception,s° Very few cases of EFAD have been confirmed in infants free from disease, although it is possible that the once popular use of skim milk-based infant formulas might have precipitated EFAD in some infants, a5 A long series of experimental studies beginning in 1919 clearly demonstrates the susceptibility of human infants to EFAD, given proper conditions. 1. Early Studies in Infants The first published report attempting to consider the effects of EFAD in human infants appears to be that of yon Gr'6er. 21°a Two infants on skim milk diets for nine months were found to have slightly retarded growth, poor appetite and possible respiratory infections. One of the two suffered from exudative diathesis. Both infants were considered

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to be healthy and normal when examined several months after an EFA containing diet was reinstituted. Holt et al. ~°3 are responsible for the next significant report concerning EFAD in infants. Three infants fed low fat diets for short periods were monitored. O n e developed bronchitis and marked eczema which disappeared when fat was added to the diet and reappeared upon resumption of the low fat diet. EFAD has been experimentally produced in infants on numerous subsequent occasions, s2,9s Hansen et al. s2 extended the findings of von Gr6er by demonstrating that symptoms of EFAD could be consistently produced in a relatively short period (one month) in otherwise healthy infants be feeding skim milk as the only calorie source. The results of the study by Hansen suggest that EFAD might occasionally occur amongst normal infants. Other reports indicate that E F A D is a relatively common resuIt of treatment for various disease states in infants. Several reports deal with the effects of malabsorption on infants and children. For instance, one infant treated for chylous ascites using a low fat diet manifested clinical symptoms attributable to EFAD. a4 Infants suffering from steatorrhea who were treated with a low fat diet developed dermal EFAD symptoms, s2

2. E F A D in Adults

It is generally concluded that adults are less susceptible to EFAD than are children, due to the former group's lower EFA requirement, which probably results from cessation of growth and development of larger adipose EFA stores. However, a number of studies indicate that adults with malabsorptive conditions, often resulting from bowel resection, can develop EFAD while on an oral diet. 1~3'211

3. E F A D in Parenteral Nutrition

Probably the most common cause of EFAD in all age groups is the long-term intake of fat-free parenteral nutrition (PN). Early work on parenteral administration of fats was reviewed by Freeman in Volume 3 of this series. Long-term fat-free PN was developed in 1968. 50 It was not until 1975 that the FDA approved a fat emulsion (Intralipid) for clinical use in PN. Its use is presently being steadily but gradually adopted. The advent of fat-free PN has meant that many patients who are unable to tolerate oral food for extended periods, and therefore were difficult to treat, now survive. But these patients often become EFA deficient. Generally patients on fat-free PN are much more prone to EFAD than those who are treated with oral fat-free feedings for similar time periods. PN is commonly administered as a continuous infusion of glucose-containing solution. Continuous glucose infusion results in the constant elevation of serum insulin. This depresses the release of fats, including EFA, from adipose fat stores. 1m3.127 Adipose fat is normally about 10% EFA.I ~s A 70 kg man has about 12 kg of adipose tissue or over 1000 g of EFA. Assuming that his requirement is about 7.5g per day, he could meet his EFA needs for more than 130 days with no dietary EFA. If release of adipose EFA is suspended through the use of continuous glucose infusion, either enteral or parenteral, plasma EFA depletion commences almost immediately. It hasbeen shown that after as few as three days of continuous glucose definite biochemical evidence of EFAD may appear in a healthy adult and that deficiency symptoms are ameliorated by a 24-hr fast. 2~3 Reports of EFAD in infants undergoing long-term fat-free P N began appearing in the early 1970s. t6~ Collins et al: 32 reported one of the first studies of EFAD with PN in a human adult. This report concerned a patient who received continuous drip PN for 100 days following major resection of the small intestine. Both biochemical and clinical symptoms of EFAD were noted. Numerous studies subsequently have appeared demonstrating the prevalence of EFAD in both infants and children ~'2~'~60'226 and adults receiving prolonged fat-free PN. 2t,49,~2.~ 1~,~3s.~~a.~74.22o

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4. EFAD in Free-livina Populations

An unusual report noted the existence of biochemical evidence of EFAD in free-living Rhodesians. The authors, who intended to use this group in their study as controls, noted that "perhaps (EFAD is) not so unusual on the traditional local maize diet.''2~ B. Disease Conditions that Alter EFA Status

Relatively little is known about the function of EFA in human metabolism. However, evidence suggestive of possible roles of EFA in human physiology may be provided by reports of EFA deficiency-like syndromes produced concomitant to a variety of disease conditions where no causal relationship between the EFA deficiency-like alterations and other manifestations of the disease have been proven. •1. Chronic Diseases

Numerous chronic illnesses are characterized by EFA deficiency-like syndromes. Red blood cells of persons with abetalipoproteinemia have been found to exhibit a fatty acid pattern characteristic of EFAD. ~6z It has been hypothesized that red cell changes in abetalipoproteinemia might be the result of malabsorption resulting from the disease and of low fat intake prescribed as treatment rather than being primary results of the disease. However, it has also been suggested that these changes in fatty acid levels could be the result of genetically induced metabolic abnormalities. ~92 In Refsum's disease, a hereditary defect of ot hydroxylation leading to accumulation of phytanic acid, changes in the myelin sheath and early death, linoleic acid levels have been observed to be substantially depressed in various organs. 6 In cystic fibrosis, the fatty acid pattern was altered reflecting mild EFAD. However, the ratio of triene to tetraene was not considered abnormal. ~°~ 2. Alcoholism

One report indicates that in a majority of alcoholic patients treated with diets high in EFA, fatty livers tend to normalize, 1x0 and patients with alcoholic cirrhosis have been noted to have decreased levels of serum linoleic acid. tg~ 3. Malignancies

Patients with gynecological malignancies have been shown to have decreased serum linoleic acid and increased palmitic and stearic acid.4z Cholesteryl ester fraction in serum may contain depressed levels of linoleic acid in liver carcinoma patients.~25 It has been noted that malignant tissues tend to be unusually high in arachidonic acid, ~s4 leading to speculation that decreased linoleic acid observed in cancer patients might be the result of rapid chain lengthening and desaturation of linoleate by tumor tissue. 4~ 4. Biliary Disease

Several investigators have reported that patients with gallstones manifest fatty acid pattern changes characteristic of EFADY s Serum arachidonic acid levels m a y be depressed. Changes in liver fatty acids appear to include increases in 16:1, 18:1 and 20: 3to9, and decrease in arachidonic acid, a pattern similar to that seen in EFAD. 5. Kwashiorkor

Persons suffering from kwashiorkor 2°° or recovering from kwashiorkor ~aO have shown serum lipid patterns similar to those of EFAD.

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6. Multiple Sclerosis i

The possibility that EFA insufficiency is involved in multiple sclerosis (MS) has been widely discussed. Baker and associates 1| found the brain lipids of MS patients to differ from those of normal patients in the relative proportions of saturated and unsaturated fat. It has been observed that the serum, red cells, and platelets of some patients have a lower linoleate level as a percentage of total fatty acid than does the serum of controls, despite unimpaired absorptionJ 5.7a Following linoleic acid administration, the linoleate concentration remains higher in the serum of controls than in that of MS patients.IS Supplementation of MS patients' diets with linoleic acid or oleic acid in a large, longterm, double blind experiment resulted in the linoleate-treated patients suffering milder and shorter relapses than patients fed oleate, although the overall rate of degeneration was not unequivocally affectedJ 45 Clausen and Moiler 3° found that breeding rats on diets low in unsaturated fat yielded increased susceptibility to experimentally induced allergic encephalitis, sometimes regarded to be similar to MS. Several researchers have gone so far as to suggest that insufficient levels of dietary EFA may be a factor leading to development of MS.1 s,6o

7. Hospitalized Patients A serum fatty acid profile similar to that found in EFA deficiency has been reported in hospital patients suffering from various acute nonneurological illnesses. 125 All 35 patients studied had reduced serum linoleic acid. Persons suffering from infectious disease exhibited an elevated level of monounsaturated fatty acids similar to that seen in EFAD. Linoleic acid levels showed no significant difference between the infectious and non-infectious disease groups. The authors assert that decreased serum levels of linoleic acid in acute illness might be a reflection of increased need for linoleic acid as a consequence of increased free thyroxine level. However, they also note significant decreases in the linoleate intake of the acute nonneurological illness patients. Patients recovering from certain traumas may appear to be EFA deficient. For instance, persons recovering from severe burns manifest red cell fatty acid patterns similar to those seen with EFAD and correctable with EFA-containing infusions. 92

C. Biochemical Chanoes in Animal Studies It has long been known that animals fed fat-free or EFA-free diets experience changes in lipid metabolism characterized primarily by alterations in the fatty acid patterns of various tissues and blood products long before they manifest any clinical signs of EFAD.

1. Early Studies Using lsomerization Technique Early research, using the isomerization technique of fatty acid analysis, indicated that deficient animals have decreased dienoic and tetranoic acid levels and increased trienoic acid levels as a percentage of total fatty acids, s3 This was demonstrated in plasma and a wide variety of tissues. 9s It was shown that in mitochondria and especially in microsomai cell fractions, EFAD resulted in rapid decrease in dienes, tetraenes and pentaenes, and an increase in trienes. Supplementation with linoleic acid resulted in increased tetranoic acid levels, whereas feeding linolenic acid yielded increased pentaenoic and hexanoic acids. In 1960, Holman suggested what has evolved to become the most common biochemical measure of EFA status. 97 He introduced the concept of the trienoic/tetraenoic acid ratio as an indicator of the severity of EFAD in rats, and later demonstrated its applicability in other species. 146 A ratio of 0.4 or greater was considered indicative of EFAD. More recently, using data obtained by gas chromatography, the upper.limit of normality was found to be 0.2 for humansJ °2 The triene/tetraene ratio has limitations because it is affected by dietary fatty acids other than linoleic acid. 1°°a'146

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2. EFA Studies Using Gas-liquid Chromatography Commencing in the late 1950s and the early 1960s, earlier findings were replicated and expanded using gas-liquid chromatography, which is capable of more specific determination of fatty acid structure, including chain length and double bond positions. It was determined that the increase in triene as a percentage of total fatty acids which occurs in EFAD was accounted for almost entirely by 5,8,11-eicosatrienoic acid (20: 3o~9), while the decrease in dienoic and tetraenoic acids was mostly accounted for by decrease in linoleic and arachidonic acids, respectively, and that these changes occurred in virtually all tissues of all higher animals tested. 9a Palmitoleic acid (16:1) and oleic acid (18:1) were found in increased concentrations due to increased synthesis of these fatty acids during EFAD. Levels of myristic (14:0), palmitic (16:0) and stearic acid (18:0) were found to be unaffected by EFAD. ~46 Linoleic acid was always present in liver lipids, even when none had appeared in the diet for many days. 191 The 8,11,14-eicosatrienoic acid (20: 3co6) was absent regardless of presence or absence of dietary EFA, in contrast with human tissues which contain significant 20:3oJ6. All 20:3 which was present was in the form of 5,8,11-eicosatrienoic acid (20:3oJ9). Very little linolenic acid (18:3) was present unless included in the diet. Male rats seemed to experience greater decreases in linoleic acid and greater increases in 5,8,11-eicosatrienoic acid than female rats fed EFA deficient diets for the same length of time. ~47 Supplementation of the diet with EFA markedly reduced the 20:3t09 level, with arachidonic being the most effective EFA, followed by linolenic acid and linoleic acid. 9a Thus, linolenic acid intake can decrease biochemical evidence of EFAD even though it cannot reverse all clinical symptoms of deficiency. When 18:3ta3 is a significant component in the diet, the triene/tetraen¢ ratio is not a reliable indicator of EFA status.

3. Mechanism of S,8,I1-Eicosatrienoic Acid Synthesis Fulco and Mead 7° demonstrated that 5,8,11-eicosatrienoic acid, which is synthesized in animals fed diets deficient in linoleic acid, is formed by chain elongation and desaturation of oleic acid. The results of an experiment conducted by Mohrhaner and Holman 146 suggest that linolenic acid, linoleic acid and oleic acid may compete as substrate for enzymes involved in desaturation, with linolenic acid having the greatest affinity for the enzymes, followed in order by linoleate and then oleate. Thus, competitive inhibition would account for the absence of 20:3co9 when linoleic or linolenic acid appear in the diet. Other findings indicate that, in rats, linolenic acid has an affinity ten times as strong for the elongation and desaturation enzyme systems as linoleic acid, which in turn has three times the affinity of oleic acid. laa Arachidonic acid and other highly polyunsaturated members of the to6 family are more powerful in suppressing certain fatty acid conversions than is linoleic acid. It has been shown that by supplying oleic acid as 15% of calories the triene/tetraene ratio in animals may be increased by a factor of four. ~7 Mead aa concludes that relative affinity for the enzyme systems and relative concentrations of precursor fatty acids which appear at enzyme active sites combine to determine what fatty acids are formed, with affinity being maximal for trienes of increasing carbon chain length to about C 18. He suggests that the structure of the carbon chain also has some importance because20:3o~9 is not further elongated or de,saturated in significant amounts, whereas 20:3a~6 and 20:3o~3 are. Evidence exists that fatty acid dehydrogenation reactions are more strongly affected than are elongation reactions by the inclusion of dietary components which create competitive inhibition. 1as But microsomal chain elongation has also been shown to be inhibited by fatty acids causing competitive inhibition. Arachidonic acid, and not linoleic acid, appears to limit the appearance of 20:3oJ9 in animal tissues by competing for position in glycerophospholipids.13a Homeostatic mechanisms appear to operate during EFAD to maintain a pool of normal unsaturated fatt), acids. Fat deprivation increases the rate at which arachidonic

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acid is formed from linoleic acid by rat liver microsomes in vitro. 9~ The increased synthesis o f arachidonate coincides with increased importance of a secondary pathway for intermediate metabolism from linoleate. EFAD may lead to decreased turnover of polyunsaturated fatty acids 0PUFA), 133 a l t h o uhg at least one study has found evidence of increased linoleic acid turnover in phospholipidof EFA deficient rats. 3~

4. Synthesis of Isomers of Linoleic Acid Positional isomers of linoleic acid (I8:2o,'6) arise during EFAD in rats, 179 These include 18:2o~10 and w9, normally absent from carcass: fat, and 18:2o~7, normally present in very small amounts. 18:2oJ7 and wO may appear within seven days on a fat-free diet. After 95 days on a diet free of EFA but containing hydrogenated coconut oil, 18:2o~10 accounted for 6.8% of all 18:2, 18:2o~9 accounted for 8.9% and 18:21,o7 accounted for 37.3%. Only 47.0°/0 of 18:2 was linoleic acid (18:2oJ6). These isomers do not appear to be derived from linoleic acid but from shorter chain and/or less unsaturated precursors. This rise in positional isomers may help to account for the consistent failure of 18:2 to disappear from tissues of animals fed fat-free diets for extended periods. A positional isomer of arachidonic acid also arises in EFA deficient rats. In advanced EFAD, it has been noted to account for one-fourth of all 20:4.146 The presence of these positional isomers might lead to the underestimation of the severity of biochemical EFAD using the gas-liquid chromatography method which cannot distinguish isomers of 20:4.

5. Fatty Acid Composition in Various Lipid Classes Variability of the fatty acid composition is greater between the major lipid classes than Within lipids of the same class isolated from different cellular fractions of the same tissue, es Biochemical indications of EFAD in rats are more pronounced in some major lipid classes than others. For instance, in liver, the differences in the percentage of fatty acids accounted for by linoleic acid between animals fed cottonseed or safflower oil and those fed coconut oil are greatest in triglycerides, next greatest in steryl esters and least in phospholipids. Roughly the same is true in plasma, although phospholipids and steryl esters are quite similar in the extent of changes in 18:2 level elicited by EFAD. In red cells, where the lipid is predominantly phospholipid, steryl esters and phospholipids are more strongly affected by changes in fatty acid intake than are triglycerides. These findings contrast somewhat with the pattern found in humans, Synthesis and concentrations of the major lipid classes are affected by EFAD and/or a fat-free diet in animals. The total plasma lipid level has been reported t o be approximately 65% of normal in EFA deficient rats. TM But reports vary concerning the mechanisrns, extent, and direction of changes ~in concentration of the major lipid classes which are induced by insufficient fat intake. This is the case, in part, because the levels of the various lipid classes respond differently to fat deprivation depending on the species of animal which is e x a m i n e d . 212

6. Effect ofF, FAD on Lipid Synthesis It appears that liver linoleate concentration plays a role in regulation of lipogenesis, s It is generally agreed that in rats EFAD leads to increased concentrations of triglycerides in the liver. But the extent to which this is the result of decreased triglycerides secretion is not well established, 69'19j although there does appear to be agreement that very low density lipoprotein (VLDL), the primary carrier of triglycerides in serum, is found in decreased concentrations, possibly due to decreased synthesis, and that plasma triglycerides are depressed. Evidence also suggests that triglyceride degradation may be accelerated in EFAD. 99 In EFAD, triglycerides With two monounsaturated acids and a saturated one become more common as diunsaturated fatty acids decline and disappear, tee EFAD also leads to accumulation of phospholipids in the livers of.experimental ani-

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mals and to decreased concentrations in the serum. ~47 Radioactive tracer experiments using 32p indicate that phospholipid turnover is increased in EFAD. 31 Inclusion of 20:4 in the phospholipid seems to decrease turnover. Phospholipids of EFA deficient animals contain increased proportions of relatively saturated fatty acids. 169 The 5,8,11-eicosatrienoic acid appears primarily in the/~ position of phosphatidylcholine and phosphatidyl ethanolamine, replacing arachidonic acid. z8 Rat liver mitochondrial phospholipids containing arachidonic acid have been shown to bind slower but more firmly to mitochondrial proteins than do those containing other fatty acids, including fatty acids characteristic of EFAD. 99 Decreased binding of phosphotipid to transport proteins resulting from EFAD could yield a decreased ability to transport triglycerides out of the liver and thus produce the fatty liver seen in EFAD. 7. Effect of EFAD on Cholesterol Homeostasis

Cholesterol synthesis appears to be substantially decreased in livers of EFA deficient rats, whether deficiency is produced by a fat-free diet or a diet which contains 30% hydrogenated coconut oil. 149 Nevertheless, cholesterol collects in the liver, primarily as cholesteryl esters, even though cholesterol esterification in the liver may be decreased in EFAD. Perhaps feedback inhibition of cholesterol synthesis is stimulated in these animals. It has been speculated that the relationship between liver cholesteryl ester concentration and dietary EFA may be curvilinear, with high concentrations of liver cholesteryl esters occurring with both EFA deficient and high linoleate diets, and low concentrations with normal diets. Cholesterol esterification in plasma has been shown to increase in EFADJ 95 In the absence of EFA, cholesterol is found more commonly to be esterified with monosaturated and saturated fatty acids than is the case in the presence of dietary EFA. 5 This leads to speculation that cholesteryl esters collect in the livers of deficient rats because these more saturated esters do not adequately perform transport functions. Serum cholesterol in rats is lowered by EFAD. However, rabbits fed a fat-free diet show increased serum cholesterol. 8. Effect of EFAD on Triolyceride Synthesis

Fatty acid synthesis is often increased in the livers of EFA deficient rats, but whether the increase in fatty acid synthesis is a result of EFAD, as such, or is the result of a fat-free diet is controversial. The prevailing explanation appears to be that it is EFAD which causes the increased synthesis. 1°9 Investigators have reported that when fatty acids are fed to rats which have been fasted and then fed a fat-free high sucrose diet, EFA decreases the activity of liver enzymes involved with the synthesis of saturated and monounsaturated fatty acids to a greater extent than do other fatty acids. 152 But other findings, which show non-EFA to be equally as effective in depressing liver fatty acid synthesis, suggest that increased liver lipogenesis is not a result of EFAD as such. t91 Liver lipolysis in rats is substantially decreased in EFAD. ~76 Serum free fatty acids in rats appear to be increased by EFAD even when the diet contains saturated fatJ 91 M o r e than 50% of total fatty acid synthesis in rats occurs in adipose tissue. 2s9 Effects of EFAD on adipose fatty acid synthesis have received little attention. However, one study indicates that safflower oil-fed rats exhibit greater adipose lipogenesis than do tallow-fed: rats. 212 This finding was replicated in pigs. EFAD has been found not to cause disproportionate EFA uptake by or removal from adipose. 9' D. Biochemical Chanoes in Human Studies 1. Plasma Fatty Acid Trends in EFAD in Humans

Less is known concerning the effects of EFAD on human fat metabolism, but evidence suggests that deficiency affects humans in a way similar to other higher animals. Much

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attention has been focused on the changes caused by EFAD in plasma fatty acid patterns because these changes are easily determinalole indicators of the development of EFAD. As in other higher animals, human EFAD results in characteristic alterations in the relative levels of plasma fatty acids. In all plasma lipid classes of both adults and ~infants, EFA deficient diets commonly result in decreased levels of linoleic acid and increased palmitoleic and oleic acid and 5,8,11-¢icosatrienoic acid. 3~'~33'~6°,164 A decrease in arachidonic acid level occurs ,with less regularity. 49'111 Although some researchers have looked at total plasma, ~4 investigators often separate the plasma lipids into lipid classes by thin-layer chromatography prior to quantification of fatty acids by gas-liquid chromatography. It is probably the plasma phOspholipid which provides the earliest unequivocal evidence of EFAD. ~s9 It contains the largest proportion of arachidonic acid and the long chain PUFAs, including 5,8,11-eicosatrienoic acid, whose concentrations are affected by EFA status. 16° Nevertheless, clear evidence of EFAD can also be obtained from analysis of plasma total fat, ~74 cholesteryl esters,3 ~,~o triglycerides,3 ~.~6o and free fatty acids. 6s Ten-fold changes in levels of 18:2, 16:1 and 20:4 and somewhat smaller changes in 18:1 levels are common with parenteral administration of diets devoid of EFA for six or more weeks. 1~°'~74 With humans, both adults 2~3 and infants, z~ as noted in other higher animals, plasma linoteate values often tend to stabilize not long after the inception of a fat-free diet, within 1-2 weeks for both children and adults. Researchers have suggested several possible aRernative hypotheses to explain this occurrence, including the release of tissue linoleate during tissue catabolism, hydrolysis of biliary phospholipids and substitution of isomers of linoleate not distinguishable by fatty acid analysis techniques commonly employed.s so2~3 Until recently, relatively little information has been available concerning normal values for fatty acid levels in plasma lipid classes. ~°° The customary research technique has compared the subjects' levels prior to experiencing an BFA-POor diet with lipid values following such a diet. Some reliable normal values for human plasma phospholipid fatty acids have recently emerged. 1°°'~°2 They indicate that sex and age after the first four months of life have little affect o n plasma fatty acid composition. 1°°'~°2 2. Fatty Acid Composition of Human Tissues

Relatively little information exists concerning the effects of a fat deficient diet on the fatty acid patterns of human tissues other than the red blood cell. This is the case, in part, because EFAD has rarely proceeded to the point of mortality in humans. Using the red cell, Horwitt and associates 1°4 first demonstrated that human tissue fatty acid patterns are affected by dietary fatty acid intake. Subsequently, it was shown that patients treated for hypercholesterolemia with a fat-free diet for 4-II weeks had unusually low linoleic and arachidonic acid levels in their red cells. 5s These levels normalized more slowly than did plasma fatty acid values upon feeding a complete diet, but they did so quickly enough to indicate that fatty acids can be incorporated into red cells after the cells have been released from the bone marrow. Effects of dietary fatty acid intake appeared to occur sooner in red cells than in human adipose. Red cells have been reported to show less pronounced changes in 5,8,11-eicosatrienoate levels in response to EFA deficient diets than does plasma lipid. 9~ But the decrease in red cell 18:2 has been shown to be prompt and steady with fat-free intravenous alimentation. T M Biopsy samples from the liver and muscle of one adult following 193 days of fat-free parenteral nutrition indicated that phospholipid fatty acid concentrations of both tissues were probably affected by EFAD. 31 Liver phospholipid fatty acid consisted of 8.0% 18:2o6, 14.8% 20:406 and 1.5% 20:3oJ9, while muscle phospholipid was 18.6% 18:2o)6, 10.8% 20:406 and 1.9% 20:30"9. These data are difficult to interpret in the-absence of information concerning normal values. A study reported by Paulsrud et al. 16° includes autopsy data on an infant who died during severe EFAD resulting from prolonged

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parenteral fat-free alimentation. Again, analysis of fatty acid concentration data is hampered by the absence of normal values for comparison. But based on comparison with values obtained from normal and deficient animals, it seems evident that tissue fatty acid levels were severely affected by the absence of dietary EFA. Linoleic acid was very depressed and 5,8,1 l-eicosatrienoic acid was present in high concentration. Phosphatidylcholine fatty acids from various sources were quantified, and 5,8,11-eicosatrienoic acid was found to constitute 13.1% of the phosphatidylcholine fatty acid in serum, 11.0% in the adrenal glands, 9.9% in liver, 7.8% in ventricles, 7,0% in kidneys, 4.8% in pancreas and colon, and 1.1% in spinal cord. The relative 5,8,11-eicosatrienoic acid concentrations are probably proportional to fatty acid turnover rates in the various tissues. No tissue showed higher levels of deficiency than did serum. This fact, along with the rapid depletion of serum EFAs which was noted, led the authors to conclude that tissue stores may be affected only after serum EFA has been depleted. ~6° 3. Effect of EFAD on Cholesterol Homeostasis Although numerous reports indicate that intake of linoleate decreases serum cholesterol,~92 human plasma concentration of total lipids and of the various lipid classes does not appear to be greatly affected by EFAD or a fat-free diet. As with other animals, there is controversy as to the direction and mechanism of effects seen in EFAD. White and associates 2~6 detected some moderate plasma lipid elevations in EFA deprived infants. Cholesteryl esters were significantly elevated. Total lipid and triglyceride were somewhat increased after one week of fat-free alimentation, but after five to seven weeks little additional change was noted. 4. Effect of EFAD on Fatty Acid, Triglyceride and Lipoprotein Levels Increased total serum fatty acid, VLDL, and serum triglycerides have been observed in patients receiving fat-free parenteral feeding.31,1:3"2°3'22~ Levels normalized upon resumption of EFA intake. However, Collins 3~ has argued that the increase in plasma triglycerides which was observed concurrent with increased VLDL in two adults fed via continuous drip parenteral infusion may have resulted from the high carbohydrate concentration in the solution. He suggests that it may be an example of carbohydrateinduced hypertriglyceridemia and not the result of EFAD. He. also noted a substantial decrease in the proportion of total plasma triglycerides carried by VLDL in two patients. This, he hypothesized, might be directly attributable to EFAD. No effect was noted of an EFA deficient vs lipid infused condition on serum cholesterol in a patient on a cholesterol-free diet. Serum cholesterol was consistently low. By contrast, orally fed malabsorption patients who manifested EFAD often showed a decrease in fasting plasma concentrations of cholesteryl ester, triglycerides and phospholipids, ls9 similar to what has been noted in rats. This study also noted that, whereas in normal subjects a higher concentration of plasma arachidonic acid was found in the lecithin of HDL than in that of LDL or VLDL and less linoleic acid was found in cholesteryl esters of VLDL than in those of LDL or HDL, with EFA depletion these concentration differences were absent. This, the authors argue, might indicate that the difference between the cholesteryl ester composition of the various lipoprotein classes is due to preferential incorporation of linoleic acid into low and high density lipoproteins. This conclusion is supported by the findings of a study in which the fate of linoleic acid administered as a soybean oil emulsion to an EFA deficient patient was monitored. 2°3 5. Definition of Biochemical EFAD--Triene/Tetraene Ratio Interpretation and comparison of fatty acid profile~ in EFAD has been simplified by the widespread adoption of the most recently evolved form of Holman's triene/tetraene ratio, the 20:3t09/20:4
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EFAD in humansJ 92 Numerous studies have employed this ratio to estimate the extent of EFAD. For instance, Jn the previously mentioned report of EFAD in infants by Paulsrud et al., ~6° this ratio was used as an index of comparative severity of the deftciency found in different tissues. It was noted that the highest value obtained for the ratio in plasma phospholipids was 18.3, whereas in cholesteryl esters, a maximum of 5.1 was attained. It was felt that 20:3~9 and 20:4o~6 are not present in sufficient concentrations in serum triglycerides to make the ratio of their concentrations meaningful. When the patient attaining these high values later died, ratios for tissue phosphatidylcholine were determined, and showed levels that ranged between 6.0 in pancreas and 1.1 in spinal cord. After the period of EFA repletion, the highest value obtained for plasma phospholipids for the surviving patients was 2.2 and the lowest was less than 0.03. In several cases, repletion proceeded for only a few days before plasma lipid analysis was performed. The relatively low values attained illustrate the speed with which plasma fatty acids return to normal upon commencement of linoleate intake. A recent study claims to have detected the highest 20:3oJ9/20:4o~ ratio ever noted in humans. ~°7 The analysis by this study of the serum phospholipids of neonates receiving fat-free parenteral alimentation for periods averaging about 24 days yielded ratios as high as 146, largely due to extremely low concentrations of arachidonic acid. In contrast, a study of one adolescent and two adults who received fat-free parenteral feeding for about 6 weeks to 6 months reported ratios in plasma~total fatty acid _ranging between 2.7 and !3.3.174 Numerous other studies including those in which patients took in oral food 163'211'213 and those in which feeding was by P N have reported substantially elevated 20:3o~9/20:4¢o6 ratios. 31'62'139'173'199'213'216 A few studies of patients receiving seemingly fat deficient diets have found no 20:3~o9 or only traces of the fatty acid.l 11.138.221 All of this serves to illustrate both the high variabifity of outcomes within the literature and the seemingly increased susceptibility of neonates to EFAD. The literature concerning human EFAD which employs current lipid analysis techniques is predominantly limited to reports of the preceding type, and can be characterized basically as attempts to follow the biochemical and, to a lesser extent, the clinical course of EFA deficient persons. No study has systematically varied EFA intake and analyzed resultant serum lipid patterns using gas chromatography 100 and little has been done to study fatty acid patterns of normal persons. However, studies of the fatty acid patterns of serum total lipid, phosphofipid, cholesteryl ester, triglyceride and free fatty acid of a large group of normal persons of both sexes and widely varying ages have now been reported. 1°°'1°2 Based on the findings of these studies, which show that relative fatty acid levels vary tittle from subject to subject, Holman now concludes that the boundary separating a normal 20:3o~9/20:4o~6 ratio from one indicative of EFAD should be lowered from 0.4 to 0.2.1oo This value roughly equals his sample's mean value plus one standard deviation. Attainment of this value is believed to precede, not to coincide with appearance of known clinical symptoms. Others have supported Holman. :9s Holman also suggests that the algebraic sum 18:2o.~6 + 2 0 : 4 ~ 6 - 20:3o~9 and the proportion of fatty acids accounted for by oJ6 acids are useful indicators of EFA status, l°°a The latter is currently the preferred index of EFA l°°b status. E. Cellular Effects of EFAD Mitochondria are the ceUuhr organelles which are most notably affected by EFAD. EFAD results in swelling of mitochondria isolated from laboratory animals. 219 It has been suggested that this may be the best available indication that EFA is a necessary structural component of membranes because mitochondria are membranous structures and no PC} synthesis has been demonstrated within mitochondria. 1°5 Isolated liver mitochondria from EFA deficient animals are unusually fragile 122 and have increased numbers of cristae. 219 Similar structural alterations have been noted in myocardial mitochondria.l 9,

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Mitochondrial function is disrupted by EFAD. In isolated rat liver mitochondria4~'117 and in yeast mitochondria, s9 oxidative phosphorylation is partially uncoupled from electron transport, resulting in decreased ATP production. This is not surprising given the fact that the oxidative phosphorylation system is located within the mitochondrial membrane. The uncoupling apparent in EFAD may result from a defect in the NAD linking segment of oxidative phosphorylation. 4~a.7 Evidence concerning mitochondrial changes in EFA deficient humans appears to be limited to one study. Richardson and Sgoutas *Ta report liver biopsies from two patients which found evidence of enlarged, spherical mitochondria following about six weeks of fat-free parenteral nutrition. F, Treatment o f E F A D

EFAD may be treated by either or both of two basic approaches: (1) EFA may be administered; and (2) metabolic conditions may be fostered which permit the utilization of adipose EFA stores, assuming such stores are present. EFA may be administered orally, intravenously, by cutaneous application, or in transfused blood products, although the practicality of the last route of administration is questionable. Oral administration is the method of choice when the patient is capable of normal intestinal absorption. Numerous reports document an uncomplicated course of recovery produced in humans of all ages by oral intake of EFA-containing oils either alone or as part of a mixed diet. 62'92'1~°'173'213 No definite time-table for recovery is inferable based on available studies but it appears that fat-free parenteral feeding may precipitate EFAD in somewhat less time than a normal diet can correct the deficiency.160'213 It has been suggested that EFAD treatment should include a diet containing at least 4% of calories as linoleic acid. 173 Intravenous EFA is now administered in the United States primarily in the form of an aqueous fat emulsion consisting of soybean oil, fractionated egg yolk lecithin, glycerol and water. This product has been shown to be effective in treating EFAD. 2~'32"92"***'.38".64'1~4"2°3 The most commonly administered concentration contains 10% soybean oil. The manufacturer recommends a maximum daily lipid dosage of 2.5 g/kg of body weight for adults and 4 g/kg for pediatric patients. 9 The minimum dosage sufficient to prevent or cure EFAD in persons with no other EFA source is not known at this time, but evidence suggests that more EFA be provided intravenously than would be required as oral intake. 32'** 1,174 This preparation has been used for a number of years in Europe with only sporadic reports of negative side effects other than those commonly encountered with any regimen of hyperalimentation. Serious problems occasionally reported include overloading syndrome, hepatomegaly and splenomegaly, thrombocytopenia and leucopenia.9 Deposition of brown pigment in the reticuloendothelial system has also been reported, but its impor, tance is uncertain. Intravenous emulsion is contraindicated in patients with hyperlipemia, lipoid nephrosis, of acute pancreatitis accompanied by hyperlipemia. Caution is recommended in its administration in the presence of severe liver damage, pulmonary disease, anemia, blood coagulation disorders, or potential for development of fat embolism. The effectiveness of both cutaneous application of EFA and EFA administration as a normal component of infused blood products is questionable. Studies of cutaneous EFA application for the treatment of EFAD have resulted in contradictory outcomes. Interest in the use of cutaneous EFA was stimulated by a report by Press et al. .63 that cutaneous sunflower seed oil corrected the abnormal serum lecithin fatty acid patterns of three EFA deficient adults. Such small amounts of sunflower seed oil (250 mg per day) were required to produce this effect that the authors hypothesized that cutaneously applied linoleic acid might be more efficiently utilized than orally administ.ered linoleic acid. Two weeks after commencement of treatment, serum lecithin fatty acids, which had been 10-127/o 5,8,11-eicosatrienoic acid, had nearly normalized. Transepidermal water loss was de-

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creased at the site of application even though initial water losses were not elevated. |~s Scaly lesions disappeared. Treatment increased epidermal lecithin linoleic acid but did not decrease epidermal lecithin 5,8,11-eicosatrienoic acid, nor was arachidonic acid concentration changed. Olive oil application had no effect. Friedman et al. 64"~s performed a similar experiment with two EFA deficient infants, but using greater amounts of sunflower seed oil (about 1400 mg/kg per day). After only a few days of this treatment, substantial increases had occurred in the arachidonic acid concentration of plasma phospholipids and 5,8,11-eicosatienoic acid was decreased. In one subject, treatment quickly led to increases in linoleic acid but this failed to occur in the other subject. Less pronounced changes occurred in red cell phospholipids. Adipose triglycerides persistently exhibited depressed linoleic acid levels after treatment in the patient tested. This, the authors suggest, may indicate that prolonged cutaneous treatment may be necessary for full normalization. Hunt et al. ~°~ report contrasting findings. When six neonates receiving fat-free parenteral nutrition were administered 100 mg/kg of body weight per day of cutaneous sunflower seed oil, improvement in EFAD was not apparent. In fact, in five of six subjects, EFAD worsened. The dosage administered was greater than that administered by Press et al. ~63 but less than that given by Friedman et al. 6s Because the EFA in this dosage level represents only about 1% of total calories, Hunt et al. 1°~ tested four additional infants, who were administered 740-1100 mg/kg per day for 76 days. They report that at the end of this period all showed signs of severe biochemical EFAD. One had clinical signs of EFAD. Thus, it appears at this time that there can be no certainty as to the effectiveness of cutaneous EFA application in the treatment of EFAD in humans. The use of plasma infusions to supply EFA to deficient persons has met with little success. The relatively low concentration of EFA found in plasma mitigates against the effectiveness of this therapy. Plasma from fasting individuals contains approximately I l mEq of fatty acid per liter. 1') This provides about 825 mg of linoleic acid per liter. ~°° Thus, an adult would require daily infusion of almost 10 liters of plasma to satisfy the EFA requirement. Given this fact, it is to be expected that researchers who have administered moderate dosages of blood products in an attempt to ameliorate EFAD have not reported beneficial effects. 2s,~65,216 Even exchange transfusion in infants has failed to significantly improve the course of EFAD. 6s EFAD is also treatable by establishing conditions which facilitate the release of EFA from the patient's fat stores during parenteral alimentation. This is accomplished by temporarily suspending the infusion of glucose. 2~3 Insulin levels then fall, allowing the release of fatty acids from adipose tissues. The patient may be fasted 2|3 or amino acids may be infused during the suspension of glucose administration. ~27 Both techniques have been shown to result in correction of biochemical EFAD in persons with normal fat stores.127,213 However, neither technique has been widely adopted. The wisdom of partial or complete fasting of already undernourished patients has been questioned as has the advisability of producing abrupt changes in the blood glucose level. ~2 IV. E F F E C T O F E F A D O N O R G A N SYSTEMS

A, Effects o f E F A D on Growth

Diminished growth has been a consistent consequence of EFAD in young animals, as discussed by Holman. 98 This decreased weight gain, not equally shared by the various organs, becomes apparent in growing animals prior to the onset of cutaneous lesions that are symptomatic of EFAD. The thyroid gland is disproportionately small, whereas the brain, adrenals, liver, heart and kidneys each constitute an increased proportion of total body weight in EFAD. Infant studies performed during the early investigations of EFAD, as discussed by Soderhelm et al., 192 showed a decreased growth rate in EFAD. Later investigations of ~.P.L.L

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infants on long term parenteral nutrition without EFA support the early reports of growth retardation in EFA deficient infants. 27 There is evidence which suggests that development of cutaneous changes may precede the development of growth retardation in human infants. ~6° A persistent finding in EFAD, which may indirectly affect growth, is the increased requirement for energy, as indicated by an increased BMR. 2~4 The biochemical change associated with the increased BMR appears to be a defect in mitochondrial energy production. 2° In EFA deficient animals fed ad libitum, a 40-60% increase in food intake over the normal controls has been noted concomitant with a substantial decrease in nitrogen retention. ~53 Recent evidence shows a correlation of dietary protein with the kind and severity of cutaneous and blood lipid related EFAD symptoms, suggesting that protein metabolism is altered in EFAD. 94 Several other factors probably contribute to decreased growth. Holman ~°° has suggested that the body heat loss in EFA deficient animals due to increased water permeability of the skin may be a major contributing factor. Also, a change in growth hormone (GH) in EFAD has recently been rePorted in chicks. 56 EFAD depressed energy efficiency and the hypophyseal GH level. Since the authors also have shown depressed hypophyseal gonadotrophic activity in EFAD, they suggest that EFAD has a more general effect of depressing all pituitary functions. The administration of exogenous G H to EFAD rats apparently does not increase weight gain unless the dietary insult is corrected. 44 A relationship between other nutrients and the effect of EFAD on growth has been suggested by numerous investigators. Deuel et ai. 43 reported that feeding saturated fat to EFA deficient animals further decreased growth. B. Skin Chanoes in E F A D

Skin rash is probably the most frequent externally observable symptom of EFAD in both humans and other animals. It has been reported in numerous studies of EFAD in humans, at,6a,l~t,16°.t6a'174'21a In infants receiving fat deficient diets, obvious rash has been observed within 3 months. 16° Skin rash has been observed in an adult after as few as 46 days of fat-free parenteral nutrition. 147 The appearance and microscopic structure of the rash seen in EFAD have been discussed in detail for the rat 9a and human. 2~7 Absence of normal keratinization seems to be a central feature. The tendency to manifest the rash is quite variable, depending upon humidity, with 40-50% relative humidity being optimal for rash development in animals; growth rate, with slower growing animals showing less severe rash, availability of drinking water, 9a micronutrient intake, 25 protein intake, 94 and other factors. Dudrick and his associates, who were responsible for developing long-term parenteral nutrition, report finding no skin manifestations of EFAD in any of the approximately 1,300 persons which they have treated using fat-free parenteral nutrition. 49 Dudrick speculates that their patients might receive more fat soluble vitamins than have those of researchers reporting EFAD-related rashes. Vitamin E might cause an increase in the half-life of arachidonic acid, thus avoiding rash development, Dudrick suggests. His group asserts that skin rash, and clinical EFAD symptoms in general, are invariably absent when arachidonic acid levels are not depressed, x29 However, at least one report appears to contradict this assertion.t 1 The etiology of cutaneous symptoms is as yet uncertain. The epidermis of EFA deficient rats shows characteristic alterations in the fatty acid pattern. 22a Early studies appeared to implicate altered membrane structure. 77 More recently, it had been noted that DNA synthesis in the epidermis is increased in EFAD. ~26 Epidermal proliferation is hastened.~ ao Lipid metabolism is disturbed, with increased synthesis and accumulation of sterol ester in the skin. 2~a Triglyceride synthesis is moderately increased and the activity • of the pentose cycle appears to be increased. In the early 1970s, it became clear that biosynthesis of PGE2 from arachidonic acid occurs in animal skin and in the microsomal

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fraction of human skin Ce]JS. 112'222 The skin of EFA deficient animals was found to be almost totally lacking in P G s . 2°7 Skin lesions of EFAD showed improvement at the treatment site when topical P G E 2 w a s a p p l i e d . PGE 2 was shown to act to inhibit biosynthesis of excessive sterol esters by the Skill. 223 However, the researchers did not demonstrate a causal relationship between inhibition of sterol ester synthesis and clearance of the cutaneous lesions. 20:3W3 was found to inhibit PG synthesis in human skin but 20: 3W9, which increases during EFAD, was not tested. Intradermal inject.ion of PGE2 has been found to increase epidermal DNA synthesis in humans. 126 A number of animal experiments suggest that the balance of PGs in the skin may be an important determinant of epidermal proliferation and differentiation. Houtsmuller l°5 has noted in a recent review that only fatty acids which are precursors of bioactive PGs act to relieve cutaneous symptoms of EFAD and that the in vitro rate of PG formation from a fatty acid is highly correlated with the fatty acid's ability to normalize the skin's water permeability. Increased permeability to water is another cutaneous symptom of EFAD. 9s Although it .seems almost certain that P G insufficiency mediates at least some of the effects of EFAD on the skin, the mechanism of that mediation is currently open to speculation. There exist at least two studies which suggest that PG depletion might not mediate the increased water permeability of skin seen with EFAD. This holds open the possibility that EFAs are necessary here as structural phospholipid components or for elaboration of intercellular barrier lipids. Hartop and Prottey ss have found that cutaneous application of linoleic acid or ~,-linolenic acid, but not P G precursors dihomo-y-linolenic acid or arachidonic, decrease the rate of transepidermal water loss in EFA deficient rats. Hartop and Prottey claim also to have noted that cutaneous application of neither PGE~ nor PGE2 esters normalized transepidermal water loss. They assert that dermatosis and impaired barrier function of the skin which are seen with EFAD may not be causally linked. They note that with EFAD, onset of scaly dermatitis precedes by several weeks the onset of skin permeability changes, whereas with EFA refeeding, skin permeability normalizes much quicker than does skin appearance. Another study found elaboration of normal numbers of epidermal lamellar bodies but insufficient intercellular lipid deposition from these organelles to seal intercellular spaces in the epidermal barrier layer of the skin of EFA deficient mice. 5a Topical application of linoleic acid as triglyceride has been shown to be capable of ameliorating skin rash and decreasing water loss to subnormal levels at the location of application in three EFA deficient humans. ~67 There was no apparent effect on epidermal lecithin, arachidonic acid or 20:3W9 levels but there was normalization of the levels of these fatty acids in serum lecithin. Although the findings of this study were similar to those of animal experiments, they have not been consistently replicable in humans. 62 Recently, a note of caution has appeared in the literature concerning the diagnosis and treatment of skin rash coincident with EFAD resulting from fat-free parenteral nutrition.IS3 EFA deficient parenterally fed patients are also prone to zinc deficiency, which can result in a rash similar to that resulting from EFAD but responsive to administration of zinc. Thus, a zinc deficiency might in some cases be the actual cause of what is interpreted to be an EFAD symptom. It appears to be possible that zinc has a role in the metabolism of EFA to PGs.

C. Immunity and Resistance to Infection in EFAD

Increased susceptibility to infection is a well documented consequence of EFAD in animals, 77'81 and has been reported in human infants. 1,.~ Infection is a common clinical problem for persons undergoing fat-free hyperalimentation. I t has been asserted that infection is the most common complication encountered in prolonged use of a n intrav e n o ~ catheter 3' and that patients undergoing fat-free hyperalimentation seem unusually susceptible to infection, s4

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Much remains to be clarified concerning the role of EFA in immunity. The cause of increased susceptibility to infection in EFAD is unknown, although decreased lymphocyte levels have been reported in infection-prone, EFA deficient rats. 96 One theory suggests that altered membranes might be in some way implicated. 77 Substantial evidence suggests that EFAD may actually stimulate some aspects of immune response. EFA deficient mice exhibit accelerated skin allograft rejection and decreased tumor incidence and growth. ~3¢'1¢3 Conversely, administration of EFA might depress some components of the immune response. Injection of arachidonic, at-linolenic and linoleic acids, repectively, in order of decreasing strength of effect, have been shown to prolong skin allograft survival in mice receiving a standard, apparently non-EFA deficient diet. 141 Both primary and secondary cell-mediated immune responses in vivo seem to be affected. Also, both intraperitoneal injection and oral administration of linoleic acid have resulted in significantly prolonged skin aUograft survival in rats. 17s The injections supplied 0.6 g linoleic acid per kg of body weight, while dietary treatment consisted of 1.1 g of linoleic acid per kg of body weight. Composition of the control diet was not revealed. The two treatments resulted in similar increases in allograft survival time. This study and another with similar results using mice ~¢° have been criticized because the control group apparently was not submitted to trauma similar to that resulting from treatment. However, one group of investigators which has criticized these studies has recently published research which adds credibility to the notion that EFA may suppress immune response and that this may occur in humans. ~37 A double-blind experiment involving 89 recipients of cadaveric renal transplants indicated that functional graft survival rate was significantly better at 3--4 months (but not at 6 months) post transplant for persons who received daily supplements of about 3.6 ml of oil from the evening primrose plant, containing about 74~ linoleic acid and about 8% 7-1inolenic acid than for persons receiving an olive oil placebo T M of the kidneys rejected, mean duration of function was significantly longer, almost three times as long, in the PUFA-receiving group vs the placebo group. Composition of the diets was not mentioned, but they apparently were not EFA deficient. It seems remarkable that such small quantities of supplement could be shown to have significant effects. However, it had previously been demonstrated that primrose oil may be substantially more potent in its immunity-related effects than is pure linoleic acid, 59 and the dosage administered here had been shown to be quite effective in slowing lymphocyte response to antigen, as measured by macrophage electrophoretic mobility. 2°6 McHugh et al.t 31 hypothesize that the delayed rejection may result from a direct effect of the fatty acids on the immune system and that prostaglandins might also be involved. They suggest further that variations in platelet aggregation might be important, as aggregation is known to occur in severe allograft rejection, and platelet aggregation is known to be affected by EFA intake. They speculate that a higher dose of the PUFA might possibly provide longer lasting benefit. Nevertheless, it should be noted that a study of skin and heart grafts in rats found a "negligible effect" from linoleic acid on allograft survival, t77 Several other studies indicate that EFA may suppress human immune response. Lymphocytes from normal subjects and especially from persons suffering from or closely related to persons suffering from multiple sclerosis show evidence of suppressed antigen recognition in vitro in the presence of physiological levels of linoleic acid. 6° Arachidonic acid produced somewhat greater suppression while oleic had little effect. ~44 Intraperitoneal injection of a soybean oil emulsion high in linoleic acid has been found to impair bacterial clearance in mice, often with fatal consequences.6~ In vitro chemotaxis of human neutrophils was also found by these !nvestigators to be impaired. However, the methods employed by this study have been criticized.4° Not all studies have found EFAD to potentiate or EFA intake to suppress the immune response. A study has recently been conclu,'~ed which focused on humoral immunity.4~ This study appears to be the first attempt ,0 determine the relationship between EFA intake and this type of immune response. Findings c6ntrast sharply with those of Mertin and others who have focused on cell-mediated immunity. Humoral immunity in mice

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was found to be adversely affected by EFAD, while high doses of EFA had no effect when compared with a diet containing 2% corn oil. Deficiency related humeral immunity decreases preceded growth or appearance changes. Challenge by both T-cell dependent and T-cell independent antigens produced a significantly reduced humeral immune response in deficient animals. EFAD generally resulted in decreased spleen weight with a proportional decrease in splenic lymphocyte production. Reactivity of lymphocytes to antigen was also decreased. The authors suggest that changes in membrane fatty acid composition may have been a causal factor in this decreased reactivity. A study testing the in vitro effects of a soybean oil emulsion on human lymphocyte transformation found that emulsion levels similar to those in serum during its administration enhanced both Varidase and phytohemaglutinin-stimulated lymphocyte transformation. 157 There are some questions concerning this finding because neither lower nor somewhat higher concentrations of Intralipid produced statistically significant effects. One study involving mice indicates that some aspects of the lymphoid system react to subcutaneous PUFA injection as they would to antigen stimulation, t a6 Also, a significant increase in phagocytosis of bacteria was noted when mouse macrophages grown in cell culture were cultivated in the presence of cis-unsaturated fatty acids.IS4 Arachidonic acid was particularly effective but linoleic and linolenic acid containing media also stimulated phagocytosis. The authors speculate that this effect was mediated by increases in the fluidity of membranes resulting in an increased ability of cell components to move and diffuse. However, a study of the effects of saturated and unsaturated fatty acids bound to albumin found no significant effect by any fatty acid on phytohemaglutinin transformation of human lymphocytes cultured in human AB serum and concluded that the earlier findings of immuneregulatory effects from unsaturated fatty acids may have been experimental artifacts. 2°s The mechanisms by Which EFAs exert their apparent immunity related effects remain uncertain. Lending support to the notion that particular effects may be PG mediated is a report of the effects in vitro of 0.08 mg per ml infusions of linoleic acid, arachidonic acid, oleic acid, PGE1 and PGE2 on the response of lymphocytes to protein antigens.IS6 The description of this study is lacking in statistical detail, but it appears to indicate that all five substances caused some degree of response inhibition with arachidonic acid being most effective. Other investigators have found PGE~ to have a regulatory effect on lymphocyte function ~7~ and have noted that a number Of PGs can suppress both the immediate and delayed hypersensitivity of in vitro preparations of human cells. 124 I n conflict with evidence that PG activity is important in suppression of lymphocyte function is a report indicating that not only linoleic and arachidonic acids but also non-PG precursor acids such as oleic, palmitic and stearic acids bound to bovine serum albumin produce in vitro inhibition of human lymphocyte function. 21s It was also noted that when palmitic or stearic acid was introduced along with arachidonic or linoleic acid, the inhibition decreased. The authors assert that, up to the time of their study, all in vitro work concerning EFA and immunity had used nonesterified fatty acids. They suggest the inhibition noted in their experiment may result from "a specific biochemical action" and not from a physiological effect, and warn that the findings might not be relevaent to in rive situations. Another study, one which compared the effects of fatty acids on antigen stimulated vs nonstimulated cells, found that 18:0 as free fatty acid had a stronger inhibitory effect on in vitro lymphocyte transformation than did either 18:2 or 20:4.~'~2 However, only PUFA showed a clear difference between their inhibitory effects on antigen stimulated vs nonstimulated cells, their effects being primarily limited to activated lymphocytes. This suggests that EFA may have a unique role in regulating lymphocyte function, possibly mediated by PGs. However, this study has received criticism for its use of fatty acids dissolved in ethanol and its findings have not been replicable with fatty acids bound to albumin. 2°s D. Hematopoiesis in E F A D

F~tion of various components of blood appears to be adversely affected by EFAD. In EFA deficient rats, a decrease in small l ~ p h o c y t e s has been noted along with

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concurrent increased incidence of infection. 96 Findings in at least two studies suggest that EFA deficient humans might also experience decreases in blood component synthesis. Collins et al. 32 report a case of severe refractory anemia in an adult receiving fat-free parenteral feedings. Tests showed the anemia not to be due to iron, B12 or Colic acid deficiencies. Following commencement of lipid infusions, erythropoeisis approached n o r m a l levels. Another group has reported finding thrombocytopenia in an EFA deficient infant. 27 The condition disappeared following administration of a soybean oil emulsion. This group asserts that evidence exists that thrombocytopenia seen in EFAD results from a failure of separation of megakaryocyte buds. Logically, thrombocytopenia may be involved in the causation of the retardation in wound healing which has been reported in EFA deficient mice and of which there is preliminary evidence in a deficient human infant. 27 However, this possibility has not been investigated.

E. Liver Changes in E F A D

Several changes occur in the liver of EFA deficient animals. The most commonly observed of these is the development of fat deposits. 7'9a'191 These deposits are produced more readily in male than female rats. 14a They have been demonstrated to be the result of insufficient linoleic acid intake and probably are not simply the result of a high carbohydrate, fat-free diet. 191 Their development coincides in time with the exhaustion of liver EFA, which occurs after about 10 weeks in rats receiving EFA free oral ad libitum diets. Deposits consist primarily of triglycerides, although liver phospholipid concentration is also increased. Saturated cholesteryl esters also accumulate in the liver. Resting levels of liver bile acids are lowered by EFAD. 97 Conflicting results appear in the literature concerning whether triglyceridc secretion from the liver is impaired by EFAD. 6a,19~ However, impaired secretion does provide a reasonable explanation of the low plasma levels of triglycerides and very low density lipoproteins often reported.in EFAD, as peripheral uptake seems not to be stimulated by the deficiency.69 An increased dency to form gallstones has also been reported in rats undergoing fat free feeding.9s FA feeding provided protection against cholesterol stone formation. Though there is little information available concerning liver changes in EFA deficient humans, some evidence exists that fat deposits might also develop in livers of EFA deficient humans. Liver biopsies performed on a 36 year old female who had experienced fat free parenteral feeding, for 9 months revealed fatty changes in centrolobular hepatocytes which were corrected after emulsion was administered.Ill Also, liver enlargement was noted in two of four adults who underwent 6-8 weeks of fat free parenteral feedings.~ 73 Biopsy revealed fat deposits within hepatocytes and possible enlarged mitochondria. Serum samples showed elevated levels of liver enzymes including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase, creatine phosphokinase (CPK) and lactate dehydrogenase. Oral administration of safflower oil was followed by normalization of all serum enzyme levels except CPK. The authors concluded that "The present results.., seem to suggest that EFAD may be a primary etiologic factor in the liver dysfunction associated with fat free TPN." Of interest here is the fact that fatty livers coexisted with elevated serum trigiyceride levels, in contrast with results of animal experiments, which often find decreased serum triglycerides. It should be noted that another study monitoring liver function in EFA deficient subjects found no consistent increase in serum GOT.I ~l The mechanism by which EFAD leads to fatty liver remains to be elucidated, although fatty acid synthesis seems to .be increased by EFAD t52 or by a fat free diet. 191 The possibility that PGs mediate EFAD's liver effects is suggested by the finding that PGE~ inhibits cholesteryl ester synthetase in rat l i v e r . 223 However, Fukazawa's group has theorized that the metabolic defect caused by EFAD which results in fatty liver may involve disruption of the synthesis of membrane structural elements.69

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F. Pulmonary Changes in EFAD Changes in pulmonary morphology and function have been demonstrated in EFA deficient animals. 9s Pulmonary edema has been found in deficient rats, During the course o f EFAD in rats, lung changes have been noted to occur culminating in the formation of large cholesterol-containing lipid granulomas. These granulomas disappeared when mobilized as a result of linoleic acid intake. Concurrent with these changes, there may be an increased susceptibility to pulmonary infection, although evidence here seems to be primarily anecdotal. Cells of the lungs of deficient dogs appear to have a lowered resistance to water influx resulting from infusion of various aqueous solutions. Results of a case study of an EFA deficient premature infant suffering from chronic bronchopulmonary dysplasia indicate that palmitic acid was reduced and levels of other fatty acids were abnormal in pulmonary surfactant. 66 Dipalmitoyl phosphatidylcholine is important for the surface tension lowering function of pulmonary surfactant. Surfactant composition normalized and clinical improvement was noted upon treatment of the EFAD. It has been suggested that the alterations in surfactant composition seen in this study might arise from increased A9-desaturase activity, which has been demonstrated in lung tissue from EFA deficient animals, ~2 PGs have been implicated in pulmonary function and pathophysiology unrelated to EFAD. 65 It has been shown that the lungs are the main site of degradation of primary PG's E and F. However, the possible role of PG level changes in the production of the pulmonary symptoms of EFAD has yet to be elucidated.

G. Dioestive System Changes in EFAD Relatively little is known concerning the effect of EFAD on t h e digestive system. However, EFA deficient rats have been shown to have decreased absorptive capacity coincident with structural changes in their intestinal mucosa. °s'99 Malabsorption of fats, amino acids and sugars results. This may be partially responsible for the decreased weight gain noted in EFA deficient young animals. A large study of infants fed skim milk based diets concluded that EFAD resulted in loose stools, s6 Why macronutrient malabsorption occurs in EFAD is not known: However, it is known that PGs are capable of affecting smooth muscle tone, and specifically, intestinal contraction. Recently it has been demonstrated that EFA restriction suppresses vitamin D dependent calcium absorption by rat gut.sac preparations. 9° Contrary to expectation, treatment with indomethacin, an inhibitor of PG synthesis, resulted in increased calcium absorPtion by gut-sacs from animals fed vitamin D and EFA. The effect of EFAD on weanling rat salivary gland function has been studied by Alam and Alam.* They noted that EFAD results in a decreased rate of flow of whole saliva. N o change occurred in the concentration of protein or in salivary amylase activity.

H. Reproductive Changes in EFAD Some of the earliest studies on EFAD in animals produced adverse effects on the reproductive system. "4 The changes associated with EFAD in pregnancy and reproduction in rats are discussed by Holman. 98 The offspring are often malformed and poorly developed when the pregnant female rat is deficient in EFA and these offspring generally fail to survive weaning. Apparently, EFA is not necessary for conception but is necessary for normal fetal development and the prevention of fetal resorption. "s Both linoleic acid and arachidonic acid will prevent the changes associated with EFAD, but linolenic acid will not.170 Male rats become sterile in EFAD and refuse to mate s7 or, in moderate EFAD, do mate but frequently are infertile,93 There is evidence that testicular damage may be irreversible,~ 10 while others have shown some recovery following administration of linoleic acid or gonadotropin. 76 Ultrastructural change of the endoplasmic reticulum of the

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Sertoli cells of the testes of rats has been reported in EFAD. 93 The administration of PGE2 intrascrotal!y did not reverse these subcellular structural changes but did produce some repair. Others showed substantial normalization of EFA deficient male rat fertility following administration of P G E 2 . 79 The same report also showed a return of female rat reproductive function to near normal following PGE2 administration. T h e mechanism of reproductive organ degeneration in EFAD is not fully understood, Van Dorp et al. 209 showed that EFAD in rats produced a decrease in prostaglandin synthesis and this decrease may be associated with reproductive failure. Others have reported the effect of EFAD on gonad-related hormones. In cockerals, the total pituitary gonadotropic activity was greatly reduced in EFAD, and spermatogenesis showed only a partial recovery on refeeding with corn oil. 55 In rats, EFAD did not seen to hamper the production of testosterone, which led some investigators to conclude that androgen production was not the cause of testicular degeneration. 3 Another possible mechanism for testicular degeneration is the subcellular damage to the Sertoli cells by EFAD, irrespective of the secondary absence of prostaglandin. 93 I. Nervous System Changes in EFAD

The adult brain appears to be resistant to the changes seen in other organs that are associated with EFAD. Relatively small changes are seen in the lipid composition of the brain in EFAD. In animals on EFA deficient diets, the fatty acid pattern changes, but much slower than other tissues of the body. 72'14e The major changes are decreases in 20:4W6 and 22"4W6 and increases in 20:3W9 and 22:3W9 in the phospholipid fraction. 71 There is evidence that experimental allergic encephalmyelitis (EAE) is associated with EFAD in animals ~87 but a recent study failed to support this. ~23 In the developing brain, more profound changes are attributable to EFAD. In rodents exposed to EFAD during gestation, the newborn showed decreased brain growth and reduced brain DNA, RNA and protein. 7s,132 Another group showed similar changes in EFA deficient rats, with an increase in saturated and monoenoic acid and a decrease .in the polyunsaturates in newborns. ~2~ These investigators noted that animals that suffered gestational EFAD were irreversibly impaired in learning behavior, while EFAD during the post weaning period had no effect on learning performance. Others found similar learning disorders in EFAD. 7~,~2° In a human infant with severe EFAD, the indications are that the brain is one of the last and least affected organs in the body. ~ ° In refeeding studies of animals on previously EFA deficient diets, the brain lipid changes associated with EFAD were very slow in returning to normal following adequate dietary EFA. 74 J. Heart Changes in EFAD

A number of changes in EFA deficient animals have been reported in both the morphology and the function of the heart. Upon histological evaluation of cardiac muscle increased vascularity has been noted. 98 In electrocardiographic studies, EFA deficient animals showed a notching in the QRS complex, indicating changes in ventricular conduction. 29 These changes occurred before any skin changes were evident, and were consistently reported in 100 animals. Other investigators reported a decrease in myocardial contractability in EFA deficient rats. 2°1 In short term infusion studies on dogs, the polyunsaturated fatty acids, including EFA, caused an increase in coronary flow which was apparently not due to prostaglandin (PG) synthesis.19 The heart changes associated with EFAD are not fully understood, but PG has been reported to play a regulatory role in cardiac function. In studies on isolated heart tissue, very rapid synthesis and release of PG occurred which regulated coronary flow and other heart functions. 155 Administration of PGE2 led to increased heart rate and a decrease in blood pressure, ~ls while PGF2 increased the contractile force of perfused heart muscle. ~7 In a series of tests using different forms of PG, TenHoor and Vergroesen2°2 found that PGEI, PGE2, PGFt~ and PGF2~ not only increased heart contractability but also de-

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creased the tendency to arrythmia. All of these studies of the effect of PG on heart function emphasize the importance of EFA as a precursor to PG and of the fact that in severe EFAD 5,8,1 l-eicosatrienoic acid, which is not a PG precursor, becomes the predominant fatty acid in heart phospholipid, t35 V. C O N C L U S I O N

Research in EFA metabolism during the past decade has added further detail to the understanding of the mechanisms by which the EFAs exert their profound biochemical and subcellular effects. Especially noteworthy has been the growth in knowledge concerning the role of EFAs as precursors of the prostaglandins. It has become increasingly clear that many of the symptoms of EFAD may result from insufficient biosynthesis of prostaglandins and related compounds. Most of the EFAD seen today is secondary to other diseases or is a side-effect of the treatment of another disease. Probably the most frequent cause is the long term administration of fat-free parenteral nutrition. EFAD has rarely been witnessed to progress to life threatening severity and the degree to which EFAD adversely affects the clinical course of concurrent illnesses is a topic for debate. However, due to its diverse effects, there is little question that EFAD may significantly complicate the course of other disease conditions. EFA is necessary for optimal functioning of the lungs, heart, and liver. The development of a normal immune response may depend on the availability of EFA. In fact, most of the major organ systems are adversely affected by EFAD, Fortunately, the availability of fat emulsions now presents an effective and generally innocuous treatment for EFAD. A need remains for further research concerning the desired level of EFA intake for normal cell functioning in healthy individuals and in those suffering from disease. Continued development of indices used to identify optimal plasma and cellular EFA levels also seems in order. Recent research indicates that EFA functions and requirements are greatly influenced by various minerals and by protein intake; these findings need to be expanded and further clarified.

(Received 27 January 1981) REFERENCES

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