Effect of medium-chain triglycerides on liver fatty acid composition in alcoholics with or without cirrhosis

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The Ainerica,z Jourizal of’ Clinical Niitritio,z 26:JuLY 1973, pp. 738-743. Printed in U.S.A.

Effect

of

medium-chain

triglycerides

on

liver

fatty

acid

composition

in alcoholics

with

or without

cirrhosis1’2’3’4

J. R. Ma/age/ada,’ M.D., W. G. Linscheer,#{176} M.D., U. M. T. Houtsniuller,7 Ph.D.,

A. J. Vergroesen,8 M.D., M. Shah,9 M.D., and F. L. Iber,’#{176}M.D.

Animal experiments demonstrate that the incidence of nutritional cirrhosis in rats can be reduced by dietary fats of shorter carbon chain length (1). It has been shown that the composition of the liver lipids in rats (2) and in drinking alcoholics is determined in part by the chain length of exogenous fatty acids (3). However, the role of dietary lipids in the pathogenesis of alcoholic cirrhosis is un-known.

The present study is designed to evaluate whether or not the fatty acid composition of liver lipids in alcoholic patients without cir-rhosis is different from alcoholics with cirrho-sis, and secondly, to study the effect of me-dium-chain triglycerides (MCT) on liver lipid composition and liver functions in these pa-tients.

Material and methods

Twenty-nine patients, 4 females and 25 males, were selected for study. All had a history of heavy alcoholic intake for at least 10 years. The subjects were divided into two groups, those with and those without cirrhosis, on the basis of clinical and labo-ratory findings, including liver biopsy. The average patient age for the cirrhotics was 49, whereas the noncirrhotics averaged 45 years.

All patients were admitted to the Metabolic Wand and given a standard hospital diet (long-chain triglycerides, LCT diet). Then, within 1 week after alcohol withdrawal, the patients were ran-domly assigned to either continuation of the same (LCT) hospital diet or an MCT diet. Both diets were isocaloric (2,000 kcal). The MCT diet con-tained 70 g MCT” and 30 g LCT (80 g protein, 207 g carbohydrate), whereas the LCT diet contained 100 g LCT (90 g protein and 182 g carbohydrate).

The average percent fatty acid composition of

the MCT diet was as follows: 8:0, 36.3%; 10:0,

29.9%; 14:0, 0.3%; 16:0, 5.7%; 18:0, 2.7%; 14:1,

0.4%; 16:1, 0.9%; 18:1, 9.0%; 18:2, 14.8%. That of the LCT diet was: 14:0, 1.8%; 16:0, 26.1%; 18:0,

13.0%; 14:1, 0.3%; 16:1, 4.8%; 18:1, 39.3%; 18:2,

14.7%.

Both diets contained equal amounts of linoleic acid because Kaunitz et al. (2) have shown that changes in the liver lipid composition induced by MCT feeding in rats occur only if adequate

1From the Lemuel Shattuck Hospital, Tufts University School of Medicine, Boston, Massachu-setts, and the Unilever Research Laboratorium, Vlaardingen, Netherlands.

2A preliminary report of this investigation was

made at the annual meeting of the American Society for Clinical Nutrition in Atlantic City, New Jersey in May 1971.

a Address requests for reprints to Dr. Willem G. Linscheer, Veterans Administration Hospital, 800 Irving Ave., Syracuse, New York 13210.

4Supported in part by Public Health Service Research Grant AM-i 1566 and Training Grant AM-05424 from the National Institutes of Health, Bethesda, Maryland 20014.

5Fellow in Gastroenterology, Tufts University School of Medicine. Presently, Advanced Clinical Resident in Gastroenterology, Mayo Clinic, Ro-chester, Minnesota 55901. Recipient of a Juan March Foundation Fellowship. #{176}Assistant Profes-sor of Medicine, Tufts University School of Medi-cine. Presently, Associate Professor of Medicine, State University of New York College of Medicine:

and Chief, Gastroenterology, Veterans

Administra-tion Hospital, Syracuse, New York. Chief, De-partment of Chemistry, Unilever Research

Lahora-torium, Vlaardingen, Netherlands. Chief of the

Biology Department, Unilever Research

Laborato-rium. ‘ in Gastroenterology, Tufts

Uni-versity School of Medicine. ‘#{176}Professor of Medi-cine, Tufts University School of Medicine.

‘ MCT were given as a combination of 25.5 g in MCT margarine (“Genes” margarine kindly sup-plied by Unilever Research Laboratonium, Via-ardingen, Netherlands, containing 20% of octanoic

acid and 80% of decanoic acid), and the rest of the

MCT (44.5 g) was incorporated into a

com-mercially prepared formula (Portagen, Mead John-son and Co., Evansville, Indiana, the MCT content of which was 71% octanoic acid and 22% decanoic acid).

at PENNSYLVANIA STATE UNIV PATERNO LIBRARY on September 17, 2016

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amounts of linoleic acid are included in the expeni-mental diets. Linoleic acid is an essential fatty acid not present in the MCT.

A liver biopsy was performed, as indicated for

diagnostic purposes, on each patient on the last day of the experimental diet. Biopsy specimens were obtained with a Menghini needle 1.4 mm in di-ameter. Two-thirds of the biopsy specimen was used for histology, and the remaining third was washed in cold isotonic saline (4 C), dried with filter paper, and weighed. The mean weight of these fragments was 16 mg. The specimen was lyophilized and stored under nitrogen for further lipid analysis.

1-Jistological procedures

Sections of liver tissue were stained and cx-amined by a pathologist who knew only that the patients were chronic alcoholics. Cirrhosis of the liver was diagnosed in 13 patients; 16 patients were classified as noncirrhotics.

Lipid analysis

Total transmethylation of all lipids in the lyophilized liver biopsies was accomplished by re-fluxing at 65 C in methanolic HCI (HC1 = 7%, w/w) for 1 hr as verified by thin-layer chromatog-raphy. After cooling, methanol and HC1 were evaporated under reduced pressure. The residue was redissolved in chloroform and washed with water. Chloroform and traces of water were evaporated under reduced pressure and the result-ing material, containing the methyl esters of all the fatty acids of the specimen, were analyzed by gas-liquid chromatography (GLC). The more volatile medium-chain fatty acids, if present, are partially lost during this procedure. However, the

metabolism of these fatty acids is extremely fast

and biopsies were taken from fasting patients.

Un-der these conditions practically no medium-chain fatty acids can be expected in liver tissue.

The gas-liquid chromatograph was a Carlo Erba chromatograph type (Biochemical). A 5% DEGS column in Gaschrom Q was used. Tempera-ture range was 100 to 210 C, temperature increases 2 C/mm. Detector: flame ionization. The relative error of our GLC technique was 0.3%.

Twenty-six fatty acids ranging from 10- to 24-carbon chain length were identified in the liver tis-sue. Each was expressed as a percentage of all fatty acids measured. The amount of tissue was usually not sufficient for the determination of total lipids.

Serum analysis

Fasting blood samples were obtained from all patients, both shortly before and at the end of the study period. Hematocrit, bilirubin, prothrombin time, glutamic-oxalacetic and glutamic-pyruvic transanlinases, alkaline phosphatase, total protein,

and albumin were determined in all serum samples by the routine laboratory methods.

On the 12th day of the study, fasting serum samples were obtained from all patients and pre-served frozen. The fatty acid composition was de-termined on five of the patients selected because one or more of their liven fatty acids was well above the mean value. These studies were done to de-termine if exceptionally high liver fatty acid con-centrations corresponded with serum fatty acid levels. Fatty acids from these serum samples were determined following the same method utilized for the liver tissue.

Stool fat analysis

Stools were collected from the 8th to the 13th day of the study (5-day stool collections). Total stool fat was measured using a modification of the method of Jover and Gordon (4). Medium-chain fatty acids (MCFA) as well as long-chain fatty acids (LCFA) are completely extracted and measured by this technique.

Results

Liver fatty acid composition

Liver fatty acid composition of alcoholic

patients with and without cirrhosis on LCT

diet. The relative amounts of liver fatty acids

in these patients, expressed as a percentage of all fatty acids measured, are presented in Table 1. High concentrations of palmitic (1 6 : 0), stearic (1 8 : 0), oleic (1 8 : 1), and linoleic (1 8 : 2) acids were present in all pa-tients. They accounted for more than 70% of the total liver fatty acids. Fourteen fatty acids, present in more than 50% of the liver biopsy specimens, were used for statistical analysis. No significant differences were found between the fatty acid composition of alcoholic patients with or without cirrhosis on a normal hospital diet (LCT diet).

Effect of MCT on alcoholic patients with

cirrhosis. In the cirrhotic population, MCT

feeding induced significant differences in the fatty acid composition of the liver lipids. The relative concentration of oleic acid decreased from 28.3 to 20.4% (P < 0.01). The mean percentage of stearic acid decreased from 16.4 to 14.4%, but the difference was not statistically significant (P < 0.1).

Two fatty acids of shorter chain length,

myristic (14:0) and pentadecanoic acid

(15:0), were higher in the cirrhotic patients fed the MCT diet than in those fed the LCT

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LCT diet MCT diet Without cirrhosis n = 10 With cirrhosis n = 6 0.25 25.10 5.35 1 .05 15.35 23.73 12.25 1.50 4.48 0.67 .23 1 .45 0.17 0.81 0.15 28.01 4.5 0.89 17.99 25.81 10.27 1 .42 3.85 0.15 0.23 .56 ± 0.08 ± 0.17 ± 0.04 ± 1.68 ± 0.33 ± 0.14 ± 1.10 ± 1.81 ± 0.69 ± 0.31 ± 1.44 ± 0.06 ± 0.10 ± 0.54 0.38 2.35 0.55 25.8 5.15 1 .52 14.45 20.40 12.13 .67 4.98 0.38 0.22 3.12 ± 0.16 ± 0.52 ± 0.14 ± 2.10 ± 0.88 ± 0.42 ± 0.71 ± 1.82 ± 1.10 ± 0.30 ± 1.01 ± 0.27 ± 0.18 ± 1.09 28 12:0 14:0 15:0 16:0 16:1 16:2 18.0 18:1 18:2 20:3 20:8 20:5 22:5 22:6

diet (P < 0.02). No significant differences were found between the other liver fatty acids. The percentage of linoleic acid, an essential fatty acid, in liver tissue was similar in both groups of patients on the MCT diet (12. 1 %

)

and patients on the LCT diet (11.2%).

Effect of MCT diet on alcoholics without

cirrhosis. The MCT diet failed to have any

significant influence on the liver fatty acid

composition of the noncirrhotic patients as

no difference was found between any of the fatty acids for patients without cirrhosis on either the LCT or MCT diet. As the patients with and without cirrhosis had a similar liver lipid composition on LCT diets, the propor-tion of both stearic and oleic acids was lower in the cirrhotics fed MCT than noncirrhotics on the same diet (P < 0.02). The percentages of myristic and pentadecanoic acids in the liver were lower in noncirrhotic patients on the MCT diet than in cirrhotics on the same diet (P < 0.02).

Serum fatty acid composition

Five patients had unusually high values of one or more fatty acids in the liver (20:3 and 26:6 in four patients and 20:4 in one patient). Their serum fatty acid composition was determined to assess whether or not the high values of the liver fatty acids correlated with the corresponding serum levels.

Ca a LI >. I-Li LU U) 2 8 4 y:O.2 + Q.99x r 0.95 6 TABLE 1

Liver fatty acid composition of alcoholic patients, percent of the total fatty acids

Fatty acid

Ut d: SE) Without cirrhosis With cirrhosis

n=6 0.22 ± 0.10 0.49 ± 1.10 ± 0.35 1.03 ± ± 0.07 0.19 ± ± 1.26 24.4 ± ± 0.95 4.27 ± ± 0.28 1.17 ± ± 1.91 16.46 ± ± 3.0 28.34 ± ± 1.09 11.26 ± ± 0.33 1.10± ± 1.16 4.59 ± ± 0.30 0.10 ± ± 0.60 0.69 ± ± 0.55 1.03 ± 0.16 0.16 0.06 1.44 0.35 0.67 .89 .73 0.81 0.30 0.61 0.07 0.29 0.21

LIVER FATTY ACIDS

FIG. 1. Serum versus liver fatty acids. Fatty acids are expressed as a percentage of the total fatty acids in serum (ordinate) and in liver tissue (abscissa). 0 = Fatty acids present in unusually high concentrations in the liver. #{149}= Fatty acids present in “normal” concentrations in the liver.

A good correlation was found between the

liver fatty acids present in normal amounts and their paired plasma levels (r = 0.95 ±

0.65, correlation coefficient ± SE, b = 0.99)

(Fig. 1). The fatty acids present in higher concentrations in the liver were all located below the regression line indicating that

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o(I.8) #{149}=Cirrhotic NonclrrhotiC E 8 z -i LU U, z LU (:5 z I U

FIG. 2. Change in serum albumin concentration

in chronic alcoholics with and without cirrhosis. Patients were given either an LCT or an MCT diet for 2 weeks.

-0.8

LCT Diet (2 weeks) MCT Diet (2 weeks)

high liver fatty acid concentrations were not a reflection of elevated serum fatty acid levels.

Liver function tests

Patients with impaired liver function tests at the time of their inclusion in the study showed a slight improvement after 2 weeks regardless of diet. No significant differences were found between those subjects on the MCT diet and those on the LCT diet in relation to hematocrit, bilirubin,

transami-nases, alkaline phosphatase, and total

pro-tein in serum.

The only exception was the serum albumin concentrations. They increased more in pa-tients fed MCT than in those fed the standard LCT diet (P < 0.05) (Fig. 2). The serum albumin levels of the two groups on MCT diet were combined as they were not dif-ferent at the beginning of the study period. No difference was noted in serum albumin concentration after 2 weeks on the LCT diet.

Analysis of fat in the stools

Daily stool fat excretion reflected no significant differences between patients on the MCT diet (6.16 ± 0.62 g/24 hr, mean ±

SE) as compared with those on the LCT diet

(5.15 ± 0.70 g/24 hr). Steatorrhea was mild

in the cirrhotics (5) (6.74 ± 0.81 g/24 hr), probably due to the preselection of patients as only subjects with a prothrombin time of less than 3 sec above the control were chosen for the subsequent study in connection with the liver biopsy to be performed at the end of the study. The prothrombin time is a sensitive test for parenchymal function, and therefore bile salt production was probably adequate for normal fat absorption in most patients.

Discussion

Billing et al. (6) have demonstrated that the liver lipid composition of one needle biopsy specimen correlates well with multiple biopsy samples taken from the same liver. Lieber and his co-workers (3) have shown that the fatty acid spectrum of the liver lipids in man changes during heavy alcohol intake as exogenous fatty acids are

incorpo-rated into liver lipids. Our patients were de-prived of any alcohol for at least 2.5 weeks prior to the liver biopsy. During this time, the fatty infiltration of the liver probably diminished as only four patients had a fatty liver at the time of biopsy. To our knowledge, there are no comparable studies of normal subjects from a similar population using the same technique of fat analysis.

In the present study, the fatty acid com-position of cirrhotic patients on essentially a normal hospital diet (LCT diet) was similar to that of noncirrhotic alcoholics on the same diet. By histological examination, fatty infil-tration was not found to be grossly different between the two groups. It is therefore highly probable that alcohol induces deposition of lipids of the same composition regardless of the presence of cirrhosis. This assumption is supported by Biomstrand’s studies (7) demon-strating a normal pattern of fatty acid syn-thesis from acetate in liver slices from cir-rhotic patients.

While in the hospital our patients, de-prived of alcohol and fed high protein diets, probably were metabolizing their liver lipid depots. It is notable that during this phase of catabolic fat metabolism, the liver lipid composition changed in response to MCT intake in the cirrhotic patients, but not in the noncirrhotic subjects. This may be ex-plained by the differences in transport and metabolism of the MCT by the cirrhotics as compared with the noncirrhotics. We have

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demonstrated in our laboratory that MCT administration to cirrhotic patients leads to higher and more persistent serum levels of MCFA than in noncirrhotics (8). Thus, it is possible that high serum concentrations of MCFA maintained over longer periods are responsible for the shift of LCFA to shorter chain length fatty acids in the liver of the cirrhotic patients. The noncirrhotic alcoholic patients may show changes when

MCT are fed over longer periods. Kaumtz et al. (2) demonstrated that feeding of MCT to noncirrhotic rats for a period of 8 weeks leads to a decrease of total liver lipids with a relative increase of fatty acids of shorter chain length (from C10 to C14).

In the liver, MCFA are oxidized in a much higher proportion than LCFA (9). Scheig (10) showed that one-third of the two-car-bon fragments formed from the oxidation of the MCFA are used in the elongation of pre-existing fatty acids. It is possible that this process is impaired in patients with parenchymal insufficiency leading to a rela-tive increase of shorter chain fatty acids and a decrease of LCFA as observed in the cir-rhotic group. Linoleic acid, an essential fatty acid, which cannot be synthesized in the liver, was probably not affected because the two diets contained equal amounts.

Alcoholic patients admitted to our hospital usually have a low serum albumin level. This is probably due to the effect of the alcohol and inadequate protein intake. Twenty-two of the twenty-nine patients selected for study had serum albumin concentrations below 3.5 mg/100 ml. No change was observed in the two groups of patients on a regular hos-pital diet (LCT diet) regardless of a high pro-tein intake. When MCT was substituted for LCT, a significant improvement in plasma albumin concentration was seen in 16 pa-tients treated, probably reflecting hepatic syn-thesis. Correction of the hypoalbuminemia in cirrhotic patients is usually considered to correlate with recovery of parenchymal func-tion (11). During MCT treatment, patients with fat malabsorption syndromes (12) showed an increase in serum albumin. Mean fat excretion in the patients selected for the

present study was close to normal; no

change was observed during MCT treat-ment. Thus, correction of fat malabsorption

cannot be considered a causal factor in the

improvement of serum albumin levels

ob-served in both groups of patients on an MCT diet.

Cirrhosis occurred less frequently in ani-mal experiments when isocaloric amounts of LCT (corn oil) were replaced by lipids of shorter chain length (coconut oil) in cir-rhotogenic diets (protein- and choline-de-ficient diets) (1). In contrast to LCT, a high proportion of exogenous MCT is oxidized in the liver (9). Free energy liberated in the process is available for cell metabolism. The lower incidence of nutritional cirrhosis in rats may be explained on the basis of in-creased cell resistance or decreased protein requirement. In our opinion, the same hy-pothesis may be applicable to the problem of parenchymal necrosis in active alcoholic cirrhosis. If MCFA exert a protecting effect on the parenchymal cells, progression of cirrhosis may be arrested. This hypothesis can only be tested through controlled clinical trials with repeat biopsies.

Summary

Dietary fatty acids are known to be de-posited in liver lipids of alcoholic patients, but their role in the pathogenesis of alcoholic cirrhosis is uncertain. In the present study, the fatty acid composition of liver biopsies obtained from seven patients with alcoholic cirrhosis on an essentially normal hospital diet (100-g fat diet for 2 weeks) was com-pared with those of noncirrhotic alcoholics (six patients) on the same diet. No significant difference was found in the relative amounts of any of 14 fatty acids ranging from C12 :0

tO C22.6.

Substitution of MCT for LCT in similarly selected alcoholics with and without cirrhosis for 2 weeks changed the liver lipid com-position of the cirrhotic patients (6 patients), but not in alcoholics without cirrhosis (10 patients). The shorter chain fatty acids (C14 :0

and C15 :0) increased and the long-chain fatty acids (C18:0 and C18:i) decreased. It is most likely that the persistently elevated se-rum levels of the medium-chain fatty acids in the cirrhotic patients was responsible for the shift towards the shorter chain fatty acids.

Twenty-two of the twenty-nine alcoholic

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patients (13 had cirrhosis) selected for the study had serum albumin levels below 3.5 mg/i 00 ml. No change was observed in the two groups on the LCT diet. However, a significant increase of serum albumin levels was observed in the patients fed MCT for a 2-week period. This is usually considered to correlate with recovery of parenchymal function. The data presented suggest a po-tential value of MCT in the treatment of cir-rhosis.

(

The authors extend grateful thanks to Miss Pat

Burns and Miss Sheila Lieberman for technical assistance, to Mrs. Theresa Bulman and the nursing staff for care of the patients on the Metabolic Ward, to Miss Abigail Kerr for dietary planning and management, to Dr. Hugo Muench for as-sistance in the statistical analysis, and to Dr. George Curtis for examination of the liver biopsies. References

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