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Plasma fatty acids concentrations in postoperative patients with biliary atresia
APPLIED NUTRITIONAL INVESTIGATION
Nutrition Vol. 15, No. 10, 1999
Plasma Fatty Acids Concentrations in Postoperative Patients With Biliary Atresia HIDEO TAKAMATSU, MD, PHD,* HIROYUKI NOGUCHI, MD,* HIROYUKI TAHARA, MD,* TATSURU KAJI, MD,* RYUICHI SHIMONO, MD,* TAKAMASA IKEE, MD,* AND TETSUO ANDOH, PHD† From the *Department of Pediatric Surgery, and the †Department of Public Health, Kagoshima University Faculty of Medicine, Kagoshima, Japan ABSTRACT
The concentrations of plasma fatty acids in postoperative patients with biliary atresia (BA) were measured to clarify whether they had essential fatty acid deficiency. Thirty-eight fasting blood samples from 14 postoperative patients with BA were studied. All of them had the hepatic portoenterostomy without any stoma. Samples were divided into three groups on the basis of liver function. The concentrations of fatty acids in the plasma fat were measured quantitatively. Non-essential fatty acids levels were increased and -3 fatty acids levels were decreased with the progress of deterioration of hepatic function. Regarding -6 fatty acids, C18:2 and 20:4 did not show any significant difference between the three groups and the control, and only C20:3 increased with the deterioration of liver dysfunction. The ratio of C20:3 (-6) to C20:4 (-6) was increased significantly with the progress of liver dysfunction. The activity of ⌬-5 desaturase was suspected to be suppressed in BA patients with poor liver function. The BA patients with poor bile flow did not show any decrease of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency. Nutrition 1999;15:755–759. ©Elsevier Science Inc. 1999 Key words: biliary atresia, plasma fatty acids, fatty acid deficiency, desaturase
INTRODUCTION
In cases with cholestatic jaundice, such as biliary atresia (BA), it is thought that fatty acid deficiency and other nutrient deficiency is caused by the malabsorption of fat through the intestine. In fact, deficiency of essential fatty acids was reported in preoperative cases with BA.1,2 In cases of BA, hepatic portoenterostomy (Kasai operation) has relieved biliary obstruction in more than half of the infants. However, in some patients who undergo a Kasai operation, there is no marked restoration of bile flow, and intestinal deficiency of bile acids results, which cause profoundly defective fat digestion and absorption. Some authors report that essential fatty acid deficiency associated with a decrease in arachidonic acid content of serum is common after the Kasai operation for BA because of fat malabsorption.2,3 Yamashiro et al.4 recommends oral or enteral supplementation of essential fatty acid-rich powder for linoleic acid deficiency and disturbance of PGE1 biosynthesis in postoperative BA patients or ursodeoxycholic acid.3 The reason for the fatty acid deficiency is thought to be fat malabsorption. However, many postoperative patients with BA show high levels of total cholesterol, triacylglycerol and phospholipid, and low levels of HDL in serum. In this study, we measured the composition of plasma fatty
acids in the postoperative patients with BA to clarify whether they have essential fatty acid deficiency. MATERIALS AND METHODS
Fourteen postoperative patients with BA were studied. All of them had the hepatic portoenterostomy without any stoma. The age of the patients ranged from 2 mo to 8 y. They had been on age-appropriate diets. Thirty-eight fasting blood samples taken during a follow up study of liver function at the outpatient department were used for the analysis. Control blood samples were taken during routine preoperative examination of ten children with inguinal herniorrhaphy or other minor surgery. The samples were divided into three groups according to liver function, Group G (good liver function group: total bilirubin (TB) ⱕ1.0 mg/dL, GOT ⬍100 IU); Group M (moderate liver function group: 1.0 ⬍ TB ⱕ 4.0 mg/dL); and Group P (poor liver function group: TB ⬎4.0 mg/dL). Fatty acid concentrations in the plasma fat of the three groups and the control group were compared. Plasma Concentrations of Fatty Acids Heptadecanoic acid (100 mg) was added to the plasma (100 mL) in a test tube with a screw cap. The plasma was then
Correspondence to: Hideo Takamatsu, MD, PhD, Department of Pediatric Surgery, Kagoshima University Faculty of Medicine, 8-35-1, Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: [email protected]
Nutrition 15:755–759, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.
0899-9007/99/$20.00 PII S0899-9007(99)00150-1
756
PLASMA FATTY ACIDS IN BILIARY ATRESIA TABLE I. CONCENTRATIONS OF FATTY ACIDS IN PLASMA OF THE POSTOPERATIVE PATIENTS WITH BILIARY ATRESIA
Palmitic acid (C16:10) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2 -6) Bis-homo-␥ linolenic acid (C20:3 -6) Arachidonic acid (C20:4 -6) ␣-Linolenic acid (C18:3 -3) Eicosapentaenoic acid (C20:5 -3) Docosapentaenoic acid (C22:5 -3) Docosahexaenoic acid (C22:6 -3) Total fatty acid C20:3 -6/C20:4 -6
Control (n ⫽ 10)
Group G (n ⫽ 14)
Group M (n ⫽ 12)
Group P (n ⫽ 12)
665.8 ⫾ 29.6 g/mL 67.6 ⫾ 5.6 g/mL 237.5 ⫾ 12.2 g/mL 705.8 ⫾ 26.4 g/mL 892.9 ⫾ 50.7 g/mL 33.2 ⫾ 2.4 g/mL 157.7 ⫾ 7.6 g/mL 21.9 ⫾ 3.0 g/mL 22.7 ⫾ 5.8 g/mL 17.6 ⫾ 1.7 g/mL 78.3 ⫾ 8.1 g/mL 2988.2 ⫾ 132.9 g/mL 0.21 ⫾ 0.017
655.1 ⫾ 44.6 g/mL 85.9 ⫾ 8.9 g/mL 222.8 ⫾ 13.0 g/mL 706.8 ⫾ 61.7 g/mL 980.1 ⫾ 65.7 g/mL 35.7 ⫾ 4.1 g/mL 163.9 ⫾ 13.6 g/mL 12.3 ⫾ 4.2 g/mL 28.5 ⫾ 2.6 g/mL 28.6 ⫾ 7.7 g/mL 92.4 ⫾ 10.0 g/mL 2924 ⫾ 190.6 g/mL 0.22 ⫾ 0.018
884.3 ⫾ 106.3 g/mL 134.6 ⫾ 17 g/mL 251.7 ⫾ 27.6 g/mL 880.7 ⫾ 95.9 g/mL 1160.7 ⫾ 147.1 g/mL 46.2 ⫾ 6.9 g/mL 166.3 ⫾ 25.4 g/mL 13.3 ⫾ 2.3 g/mL 25.1 ⫾ 4.7 g/mL 14.8 ⫾ 2.1 g/mL 77.8 ⫾ 11.4 g/mL 3655.5 ⫾ 426.7 g/mL 0.28 ⫾ 0.016
965.2 ⫾ 96.1 g/mL 214.7 ⫾ 13.7 g/mL 235.2 ⫾ 24.2 g/mL 934.0 ⫾ 82.4 g/mL 940.0 ⫾ 107.4 g/mL 54.2 ⫾ 7.2 g/mL 149.3 ⫾ 13.7 g/mL 16.8 ⫾ 3.5 g/mL 12.5 ⫾ 2.3 g/mL 19.3 ⫾ 4.7 g/mL 52.3 ⫾ 7.5 g/mL 3592.1 ⫾ 322.1 g/mL 0.36 ⫾ 0.037
All data are expressed as mean ⫾ standard error (mg/mL). Total fatty acid is the sum total of measured fatty acids. C20:3 -6/C20:4 -6 is the ratio of bis-homo-␥-linolenic acid/arachidonic acid. Group G, good liver function group; Group M, moderate liver function group; Group P, poor liver function group.
saponified with 3 mL of 10% KOH-methanol under nitrogen at 80°C for 2 h. Heptadecanoic acid was added as an internal standard to allow plasma fatty acid concentrations to be determined as absolute values. The saponified matters in the test tube were treated with 1 mL of 6N-HCl and then transmethylated at 80°C for 2 h. The lipid fraction was separated by thin layer
chromatography on Kieselgel HF254⫹366 with HexaneDiethylether (95:5), and the methylesterified fatty-acid fractions were analyzed by gas liquid chromatography on 5% Shinchrom E7-1.5 The peaks of the fatty acids were identified by relative retention time with an internal standard, and then quantified using an integrator. Fatty acids, such as palmitic acid (16:0), palmitoleic
FIG. 1. The plasma concentrations of non-essential fatty acids, C16:0, C16:1, and C18:1 are shown. The concentrations of these three fatty acids increase with the progress of the hepatic dysfunction and those of Group P are significantly higher than those of Group G and the control (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
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FIG. 2. The plasma concentrations of essential fatty acids, C18:2 -6, C20:3 -6, and C20:4 -6 are shown. There is no significant difference in the concentration of C18:2 -6 and C20:4 -6 between the four groups. The concentration of C20:3 -6 increases with the deterioration of liver function, and that of Group P is significantly higher than control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), ␣-linolenic acid (18:3), bishomo-␥-linolenic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5), and docosahexaenoic acid (22:6) were detected on gas liquid chromatography. All the data were expressed as mean ⫾ standard error (mg/ mL). The total of the measured fatty acids was also expressed as total fatty acid concentration. Statistical analysis with unpaired Student’s t test was performed by Statview II, Abacus (Berkeley, CA, USA). Statistical significance was considered to be achieved at P ⬍ 0.05. RESULTS
Non-essential Fatty Acids The concentrations of fatty acids in the plasma fat are shown in Table I. The concentrations of total fatty acids in Groups M and P were higher than those in control and Group G but there was no significant difference. Regarding C16:0, C16:1, and C18:1, Group G showed the same levels as control. The concentrations of these three fatty acids increased with the progress of hepatic dysfunction and Group P was significantly higher than Group G and control (Fig. 1). Regarding C18:0, there was no significant difference between the four groups. Essential Fatty Acids Regarding C18:2 -6, Group M showed the highest concentration among the four groups whereas Group P was almost the same as Group G and control. There was no significant difference in the concentration of C18:2 -6 between the four groups. The
concentration of C20:3 -6 increased with the deterioration of liver function and was significantly higher in Group P than in control or Group G. The concentrations of C20:4 -6 were almost the same in each of the four groups (Fig. 2). The concentrations of C18:3 -3 in the three BA groups were lower than control and increased slightly with the deterioration of liver function, but only Group M showed a significant difference from the control group. The concentrations of C20:5 were higher in Groups G and M than in control. Group P was the lowest of the four groups and significantly different from Groups G and M. The concentration of C22:5 in Group G was higher than the other groups, and that of Group P was almost the same as control. The concentration of C22:6 in Group G was higher than control, but Group M was almost the same as control, and Group P was significantly lower (Fig. 3). The ratio of C20:3 -6 to C20:4 -6 in Groups M and P was significantly higher than in control and Group G (Fig. 4). DISCUSSION
The possibility of malabsorption of triacylglycerols contained in the diets of children with cholestasis suggests a deficiency of essential fatty acids. In BA or syndromatic paucity of interlobular bile ducts, linoleate deficiency was present as shown by low 18:2 fatty acids in plasma lipids.2 In a prospective study on infants with BA, Handa et al.1 measured the composition of the plasma fatty acids to determine the requirement of fat emulsion. In 14 infants who underwent initial operation for treatment of BA, a trend toward essential fatty acid deficiency was evident before surgery. Gourley et al.6 reported the fatty acid composition of total lipids extracted from plasma and erythrocytes of five patients who
758
PLASMA FATTY ACIDS IN BILIARY ATRESIA
FIG. 3. The plasma concentrations of essential fatty acids, C18:3 -3, C20:5, and C22:6 are shown. Only Group M shows significant difference of concentration of C18:3 -3 from control. The concentration of C20:5 in Group P was the lowest among four groups and was significantly different from Groups G and M. The concentration of C22:6 in Group P was significantly lower than control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
had received a hepatic portoenterostomy for treatment of extrahepatic BA. Three of these patients, including one with successful surgery, demonstrated evidence of essential fatty acid deficiency, including decreased levels of linoleic and arachidonic acids with concomitant increases in palmitoleic and oleic acids. In two of these, the ratio of 5, 8, 11-eicosatrienoic acid to arachidonic acid (“triene/tetraene”) exceeded 0.3, suggesting essential fatty acid deficiency. Gourley et al. concluded that even patients with successful hepatic portoenterostomy risked developing essential fatty acid deficiency. Yamashiro et al.7 also reported that essential fatty acid deficiency in BA, which is associated with a decrease in the arachidonic acid content of the serum, is common after the Kasai operation for BA because of fat malabsorption. Linoleate deficiency was present as shown by low 18:2 fatty acids in plasma lipids. Patients with BA may have deficiency of docosahexaenoic acid which is believed to be an important long-chain polyunsaturated fatty acid, possibly essential for neurofunction in infants, secondary to fat malabsorption. In our study, the plasma fatty acids in the postoperative BA patients with good liver function were at almost the same levels as control. The deterioration of the liver function caused the increase of non-essential fatty acids and the decrease of -3 fatty acids of eicosapentaenoic acid and docosahexaenoic acids. Although the concentrations of ␣-linolenic acid in the three BA groups were lower than control, only Group M showed a significant difference. Regarding non-essential fatty acids and -3 fatty acids, our findings are similar to those of other authors. However, the concentrations of -6 fatty acids, linoleic acid, and arachidonic acid in the three BA groups were almost as same as the control, and the concentration of bis-homo-␥-linolenic acid was increased with the progress of liver dysfunction and in Group P was significantly higher than both control and Group G (Fig. 2). Thus, the results regarding -6 fatty acids seem to conflict with those reported by
FIG. 4. The ratio of C20:3 -6 to C20:4 -6 in Groups M and P were significantly higher than in control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
PLASMA FATTY ACIDS IN BILIARY ATRESIA
759
other authors. The reason for this discrepancy may be found in the method of expressing the composition of the fatty acids in the plasma lipid. Other reports have expressed the composition of plasma lipid fatty acids as a percentage. The increase in concentrations of non-essential fatty acids and no change in concentrations of -6 fatty acids with the progress of hepatic dysfunction resulted in the increase of total fatty acids and decrease of percentage of -6 fatty acids in total fatty acids. Thus, the BA patients with poor bile flow, but without severe liver dysfunction, did not show any decrease in concentration of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency. The liver plays an important role in fatty acid metabolism, especially in the -oxidation of fatty acids which is disturbed with the progress of liver dysfunction. If liver function is impaired, there may be no marked change of fatty acid synthesis, but there may be significant influence on -oxidation of fatty acids. Nonessential fatty acids accumulated with the progress of hepatic dysfunction because of disturbed -oxidation of fatty acids. Regarding -6 essential fatty acids, there may be malabsorption of fat in cases with hepatic dysfunction, but depression of the -oxidation of fatty acids keeps the concentration of -6 fatty acids within normal levels. The low concentration of -3 fatty acids is caused by malabsorption and possibly also a diet rich in linolenic acid which is common in Japan at present. Depression of the -oxidation of fatty acids prevents such low concentration of -3 fatty acids. Biagi et al.8 reported on ⌬-6-desaturase (⌬6D) activity of human liver microsomes from patients with different types of liver injury. They observed a significant decrease in ⌬6D activity, evaluated by a radiochemical technique using 1-[14C]-linolic acid as a substrate, in cirrhotic patients with no correlation with the etiology of the cirrhosis. The ⌬6D activity within the pathologic group was quite similar. Thus, liver disease seems to be the main
cause of the decreased enzyme activity regardless of its etiology. In our study, the ratio of C20:3 -6 to C20:4 -6 increased significantly with the progress of liver dysfunction. The ratio of C20:3 -6 to C20:4 -6 is thought to indicate the activity of ⌬-5 desaturase (⌬5D) which is necessary to convert the C20:3 -6 to C20:4 -6. Although there is no report regarding the correlation between ⌬5D and liver injury, this enzyme activity seemed to be suppressed in BA patient with poor liver function. It can be inferred from the results of the present study that patients with good liver function do not need to have any supplementation of essential fatty acids, but do need a fat-balanced diet (adding perilla oil or fish oil). If the patients with poor liver function have intravenous supplementation of fat emulsion, it will result in the increase of the concentration of -6 fatty acids and non-essential fatty acids. Patients with poor liver function should have a fat-balanced diet which includes 3–5 calorie percent of -3 fatty acids with perilla oil or fish oil, but those with severe liver dysfunction should receive intravenous supplementation of fatbalanced fat emulsion. It has been reported that sesamin derived from sesame seed improves the hypercholesterolemia and low concentration of high density lipoprotein cholesterol.9,10 In our preliminary experience, sesame seed paste administration to a BA patient with poor liver function improved not only hypercholesterolemia but also unbalanced fatty acid composition (high concentration of non-essential fatty acid and the low concentration of eicosapentaenoic acid).11 SUMMARY
The concentrations of plasma fatty acids in postoperative patients with BA were measured. Patients with poor bile flow but without severe liver dysfunction did not show any decrease in the concentration of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency.
REFERENCES 1. Handa N, Suita S, Ikeda K, et al. Requirement of parenteral fat in infants with biliary atresia. JPEN 1985;9:685 2. Dupont J, Ame´de´e-Manesme O, Pepin D, et al. Eicosanoid synthesis in children with cholestatic disease. J Inherit Metab Dis 1990;13:212 3. Miyano T, Yamashiro Y, Shimizu T, et al. Essential fatty acid deficiency in congenital biliary atresia: successful treatment to reverse deficiency. J Pediatr Surg 1986;21:277 4. Yamashiro Y, Ohtsuka Y, Shimizu T, et al. Effects of ursodeoxycholic acid treatment on essential fatty acid deficiency in patients with biliary atresia. J Pediatr Surg 1994;29:425 5. Andoh T, Uda H, Yoshimitsu N, et al. The sex differences in cord-blood cholesterol and fatty-acid levels among Japanese fetuses. J Epidemiol 1997;7: 226
6. Gourley GR, Farrell PM, Odell GB. Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia. Am J Clin Nutr 1982;36:1194 7. Yamashiro Y, Shimizu T, Ohtsuka Y, et al. Docosahexaenoic acid status of patients with extrahepatic biliary atresia. J Pediatr Surg 1994;29:1455 8. Biagi PL, Hrelia S, Stefanini GF, et al. ⌬-6-desaturase activity of human liver microsomes from patients with different types of liver injury. Prostaglandins Leukot Essent Fatty Acids 1990;39:39 9. Sugano M, Inoue T, Koba J, et al. Influence of sesame lignans on various lipid parameters in rats. Agric Biol Chem 1990;54:2669 10. Hirose N, Inoue T, Nishihara K, et al. Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 1991;32:629 11. Kaji T, Takamatsu H, Noguchi H, et al. Administration of sesame paste for hyperlipidemia in postoperative patient with biliary atresia (case report). Jap J Surg Metab Nutr 1998;32:9
Nutrition Vol. 15, No. 10, 1999
Plasma Fatty Acids Concentrations in Postoperative Patients With Biliary Atresia HIDEO TAKAMATSU, MD, PHD,* HIROYUKI NOGUCHI, MD,* HIROYUKI TAHARA, MD,* TATSURU KAJI, MD,* RYUICHI SHIMONO, MD,* TAKAMASA IKEE, MD,* AND TETSUO ANDOH, PHD† From the *Department of Pediatric Surgery, and the †Department of Public Health, Kagoshima University Faculty of Medicine, Kagoshima, Japan ABSTRACT
The concentrations of plasma fatty acids in postoperative patients with biliary atresia (BA) were measured to clarify whether they had essential fatty acid deficiency. Thirty-eight fasting blood samples from 14 postoperative patients with BA were studied. All of them had the hepatic portoenterostomy without any stoma. Samples were divided into three groups on the basis of liver function. The concentrations of fatty acids in the plasma fat were measured quantitatively. Non-essential fatty acids levels were increased and -3 fatty acids levels were decreased with the progress of deterioration of hepatic function. Regarding -6 fatty acids, C18:2 and 20:4 did not show any significant difference between the three groups and the control, and only C20:3 increased with the deterioration of liver dysfunction. The ratio of C20:3 (-6) to C20:4 (-6) was increased significantly with the progress of liver dysfunction. The activity of ⌬-5 desaturase was suspected to be suppressed in BA patients with poor liver function. The BA patients with poor bile flow did not show any decrease of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency. Nutrition 1999;15:755–759. ©Elsevier Science Inc. 1999 Key words: biliary atresia, plasma fatty acids, fatty acid deficiency, desaturase
INTRODUCTION
In cases with cholestatic jaundice, such as biliary atresia (BA), it is thought that fatty acid deficiency and other nutrient deficiency is caused by the malabsorption of fat through the intestine. In fact, deficiency of essential fatty acids was reported in preoperative cases with BA.1,2 In cases of BA, hepatic portoenterostomy (Kasai operation) has relieved biliary obstruction in more than half of the infants. However, in some patients who undergo a Kasai operation, there is no marked restoration of bile flow, and intestinal deficiency of bile acids results, which cause profoundly defective fat digestion and absorption. Some authors report that essential fatty acid deficiency associated with a decrease in arachidonic acid content of serum is common after the Kasai operation for BA because of fat malabsorption.2,3 Yamashiro et al.4 recommends oral or enteral supplementation of essential fatty acid-rich powder for linoleic acid deficiency and disturbance of PGE1 biosynthesis in postoperative BA patients or ursodeoxycholic acid.3 The reason for the fatty acid deficiency is thought to be fat malabsorption. However, many postoperative patients with BA show high levels of total cholesterol, triacylglycerol and phospholipid, and low levels of HDL in serum. In this study, we measured the composition of plasma fatty
acids in the postoperative patients with BA to clarify whether they have essential fatty acid deficiency. MATERIALS AND METHODS
Fourteen postoperative patients with BA were studied. All of them had the hepatic portoenterostomy without any stoma. The age of the patients ranged from 2 mo to 8 y. They had been on age-appropriate diets. Thirty-eight fasting blood samples taken during a follow up study of liver function at the outpatient department were used for the analysis. Control blood samples were taken during routine preoperative examination of ten children with inguinal herniorrhaphy or other minor surgery. The samples were divided into three groups according to liver function, Group G (good liver function group: total bilirubin (TB) ⱕ1.0 mg/dL, GOT ⬍100 IU); Group M (moderate liver function group: 1.0 ⬍ TB ⱕ 4.0 mg/dL); and Group P (poor liver function group: TB ⬎4.0 mg/dL). Fatty acid concentrations in the plasma fat of the three groups and the control group were compared. Plasma Concentrations of Fatty Acids Heptadecanoic acid (100 mg) was added to the plasma (100 mL) in a test tube with a screw cap. The plasma was then
Correspondence to: Hideo Takamatsu, MD, PhD, Department of Pediatric Surgery, Kagoshima University Faculty of Medicine, 8-35-1, Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: [email protected]
Nutrition 15:755–759, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.
0899-9007/99/$20.00 PII S0899-9007(99)00150-1
756
PLASMA FATTY ACIDS IN BILIARY ATRESIA TABLE I. CONCENTRATIONS OF FATTY ACIDS IN PLASMA OF THE POSTOPERATIVE PATIENTS WITH BILIARY ATRESIA
Palmitic acid (C16:10) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2 -6) Bis-homo-␥ linolenic acid (C20:3 -6) Arachidonic acid (C20:4 -6) ␣-Linolenic acid (C18:3 -3) Eicosapentaenoic acid (C20:5 -3) Docosapentaenoic acid (C22:5 -3) Docosahexaenoic acid (C22:6 -3) Total fatty acid C20:3 -6/C20:4 -6
Control (n ⫽ 10)
Group G (n ⫽ 14)
Group M (n ⫽ 12)
Group P (n ⫽ 12)
665.8 ⫾ 29.6 g/mL 67.6 ⫾ 5.6 g/mL 237.5 ⫾ 12.2 g/mL 705.8 ⫾ 26.4 g/mL 892.9 ⫾ 50.7 g/mL 33.2 ⫾ 2.4 g/mL 157.7 ⫾ 7.6 g/mL 21.9 ⫾ 3.0 g/mL 22.7 ⫾ 5.8 g/mL 17.6 ⫾ 1.7 g/mL 78.3 ⫾ 8.1 g/mL 2988.2 ⫾ 132.9 g/mL 0.21 ⫾ 0.017
655.1 ⫾ 44.6 g/mL 85.9 ⫾ 8.9 g/mL 222.8 ⫾ 13.0 g/mL 706.8 ⫾ 61.7 g/mL 980.1 ⫾ 65.7 g/mL 35.7 ⫾ 4.1 g/mL 163.9 ⫾ 13.6 g/mL 12.3 ⫾ 4.2 g/mL 28.5 ⫾ 2.6 g/mL 28.6 ⫾ 7.7 g/mL 92.4 ⫾ 10.0 g/mL 2924 ⫾ 190.6 g/mL 0.22 ⫾ 0.018
884.3 ⫾ 106.3 g/mL 134.6 ⫾ 17 g/mL 251.7 ⫾ 27.6 g/mL 880.7 ⫾ 95.9 g/mL 1160.7 ⫾ 147.1 g/mL 46.2 ⫾ 6.9 g/mL 166.3 ⫾ 25.4 g/mL 13.3 ⫾ 2.3 g/mL 25.1 ⫾ 4.7 g/mL 14.8 ⫾ 2.1 g/mL 77.8 ⫾ 11.4 g/mL 3655.5 ⫾ 426.7 g/mL 0.28 ⫾ 0.016
965.2 ⫾ 96.1 g/mL 214.7 ⫾ 13.7 g/mL 235.2 ⫾ 24.2 g/mL 934.0 ⫾ 82.4 g/mL 940.0 ⫾ 107.4 g/mL 54.2 ⫾ 7.2 g/mL 149.3 ⫾ 13.7 g/mL 16.8 ⫾ 3.5 g/mL 12.5 ⫾ 2.3 g/mL 19.3 ⫾ 4.7 g/mL 52.3 ⫾ 7.5 g/mL 3592.1 ⫾ 322.1 g/mL 0.36 ⫾ 0.037
All data are expressed as mean ⫾ standard error (mg/mL). Total fatty acid is the sum total of measured fatty acids. C20:3 -6/C20:4 -6 is the ratio of bis-homo-␥-linolenic acid/arachidonic acid. Group G, good liver function group; Group M, moderate liver function group; Group P, poor liver function group.
saponified with 3 mL of 10% KOH-methanol under nitrogen at 80°C for 2 h. Heptadecanoic acid was added as an internal standard to allow plasma fatty acid concentrations to be determined as absolute values. The saponified matters in the test tube were treated with 1 mL of 6N-HCl and then transmethylated at 80°C for 2 h. The lipid fraction was separated by thin layer
chromatography on Kieselgel HF254⫹366 with HexaneDiethylether (95:5), and the methylesterified fatty-acid fractions were analyzed by gas liquid chromatography on 5% Shinchrom E7-1.5 The peaks of the fatty acids were identified by relative retention time with an internal standard, and then quantified using an integrator. Fatty acids, such as palmitic acid (16:0), palmitoleic
FIG. 1. The plasma concentrations of non-essential fatty acids, C16:0, C16:1, and C18:1 are shown. The concentrations of these three fatty acids increase with the progress of the hepatic dysfunction and those of Group P are significantly higher than those of Group G and the control (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
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FIG. 2. The plasma concentrations of essential fatty acids, C18:2 -6, C20:3 -6, and C20:4 -6 are shown. There is no significant difference in the concentration of C18:2 -6 and C20:4 -6 between the four groups. The concentration of C20:3 -6 increases with the deterioration of liver function, and that of Group P is significantly higher than control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), ␣-linolenic acid (18:3), bishomo-␥-linolenic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5), and docosahexaenoic acid (22:6) were detected on gas liquid chromatography. All the data were expressed as mean ⫾ standard error (mg/ mL). The total of the measured fatty acids was also expressed as total fatty acid concentration. Statistical analysis with unpaired Student’s t test was performed by Statview II, Abacus (Berkeley, CA, USA). Statistical significance was considered to be achieved at P ⬍ 0.05. RESULTS
Non-essential Fatty Acids The concentrations of fatty acids in the plasma fat are shown in Table I. The concentrations of total fatty acids in Groups M and P were higher than those in control and Group G but there was no significant difference. Regarding C16:0, C16:1, and C18:1, Group G showed the same levels as control. The concentrations of these three fatty acids increased with the progress of hepatic dysfunction and Group P was significantly higher than Group G and control (Fig. 1). Regarding C18:0, there was no significant difference between the four groups. Essential Fatty Acids Regarding C18:2 -6, Group M showed the highest concentration among the four groups whereas Group P was almost the same as Group G and control. There was no significant difference in the concentration of C18:2 -6 between the four groups. The
concentration of C20:3 -6 increased with the deterioration of liver function and was significantly higher in Group P than in control or Group G. The concentrations of C20:4 -6 were almost the same in each of the four groups (Fig. 2). The concentrations of C18:3 -3 in the three BA groups were lower than control and increased slightly with the deterioration of liver function, but only Group M showed a significant difference from the control group. The concentrations of C20:5 were higher in Groups G and M than in control. Group P was the lowest of the four groups and significantly different from Groups G and M. The concentration of C22:5 in Group G was higher than the other groups, and that of Group P was almost the same as control. The concentration of C22:6 in Group G was higher than control, but Group M was almost the same as control, and Group P was significantly lower (Fig. 3). The ratio of C20:3 -6 to C20:4 -6 in Groups M and P was significantly higher than in control and Group G (Fig. 4). DISCUSSION
The possibility of malabsorption of triacylglycerols contained in the diets of children with cholestasis suggests a deficiency of essential fatty acids. In BA or syndromatic paucity of interlobular bile ducts, linoleate deficiency was present as shown by low 18:2 fatty acids in plasma lipids.2 In a prospective study on infants with BA, Handa et al.1 measured the composition of the plasma fatty acids to determine the requirement of fat emulsion. In 14 infants who underwent initial operation for treatment of BA, a trend toward essential fatty acid deficiency was evident before surgery. Gourley et al.6 reported the fatty acid composition of total lipids extracted from plasma and erythrocytes of five patients who
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PLASMA FATTY ACIDS IN BILIARY ATRESIA
FIG. 3. The plasma concentrations of essential fatty acids, C18:3 -3, C20:5, and C22:6 are shown. Only Group M shows significant difference of concentration of C18:3 -3 from control. The concentration of C20:5 in Group P was the lowest among four groups and was significantly different from Groups G and M. The concentration of C22:6 in Group P was significantly lower than control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
had received a hepatic portoenterostomy for treatment of extrahepatic BA. Three of these patients, including one with successful surgery, demonstrated evidence of essential fatty acid deficiency, including decreased levels of linoleic and arachidonic acids with concomitant increases in palmitoleic and oleic acids. In two of these, the ratio of 5, 8, 11-eicosatrienoic acid to arachidonic acid (“triene/tetraene”) exceeded 0.3, suggesting essential fatty acid deficiency. Gourley et al. concluded that even patients with successful hepatic portoenterostomy risked developing essential fatty acid deficiency. Yamashiro et al.7 also reported that essential fatty acid deficiency in BA, which is associated with a decrease in the arachidonic acid content of the serum, is common after the Kasai operation for BA because of fat malabsorption. Linoleate deficiency was present as shown by low 18:2 fatty acids in plasma lipids. Patients with BA may have deficiency of docosahexaenoic acid which is believed to be an important long-chain polyunsaturated fatty acid, possibly essential for neurofunction in infants, secondary to fat malabsorption. In our study, the plasma fatty acids in the postoperative BA patients with good liver function were at almost the same levels as control. The deterioration of the liver function caused the increase of non-essential fatty acids and the decrease of -3 fatty acids of eicosapentaenoic acid and docosahexaenoic acids. Although the concentrations of ␣-linolenic acid in the three BA groups were lower than control, only Group M showed a significant difference. Regarding non-essential fatty acids and -3 fatty acids, our findings are similar to those of other authors. However, the concentrations of -6 fatty acids, linoleic acid, and arachidonic acid in the three BA groups were almost as same as the control, and the concentration of bis-homo-␥-linolenic acid was increased with the progress of liver dysfunction and in Group P was significantly higher than both control and Group G (Fig. 2). Thus, the results regarding -6 fatty acids seem to conflict with those reported by
FIG. 4. The ratio of C20:3 -6 to C20:4 -6 in Groups M and P were significantly higher than in control and Group G (GC, control; GG [Group G], good liver function group; GM [Group M], moderate liver function group; GP [Group P], poor liver function group).
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other authors. The reason for this discrepancy may be found in the method of expressing the composition of the fatty acids in the plasma lipid. Other reports have expressed the composition of plasma lipid fatty acids as a percentage. The increase in concentrations of non-essential fatty acids and no change in concentrations of -6 fatty acids with the progress of hepatic dysfunction resulted in the increase of total fatty acids and decrease of percentage of -6 fatty acids in total fatty acids. Thus, the BA patients with poor bile flow, but without severe liver dysfunction, did not show any decrease in concentration of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency. The liver plays an important role in fatty acid metabolism, especially in the -oxidation of fatty acids which is disturbed with the progress of liver dysfunction. If liver function is impaired, there may be no marked change of fatty acid synthesis, but there may be significant influence on -oxidation of fatty acids. Nonessential fatty acids accumulated with the progress of hepatic dysfunction because of disturbed -oxidation of fatty acids. Regarding -6 essential fatty acids, there may be malabsorption of fat in cases with hepatic dysfunction, but depression of the -oxidation of fatty acids keeps the concentration of -6 fatty acids within normal levels. The low concentration of -3 fatty acids is caused by malabsorption and possibly also a diet rich in linolenic acid which is common in Japan at present. Depression of the -oxidation of fatty acids prevents such low concentration of -3 fatty acids. Biagi et al.8 reported on ⌬-6-desaturase (⌬6D) activity of human liver microsomes from patients with different types of liver injury. They observed a significant decrease in ⌬6D activity, evaluated by a radiochemical technique using 1-[14C]-linolic acid as a substrate, in cirrhotic patients with no correlation with the etiology of the cirrhosis. The ⌬6D activity within the pathologic group was quite similar. Thus, liver disease seems to be the main
cause of the decreased enzyme activity regardless of its etiology. In our study, the ratio of C20:3 -6 to C20:4 -6 increased significantly with the progress of liver dysfunction. The ratio of C20:3 -6 to C20:4 -6 is thought to indicate the activity of ⌬-5 desaturase (⌬5D) which is necessary to convert the C20:3 -6 to C20:4 -6. Although there is no report regarding the correlation between ⌬5D and liver injury, this enzyme activity seemed to be suppressed in BA patient with poor liver function. It can be inferred from the results of the present study that patients with good liver function do not need to have any supplementation of essential fatty acids, but do need a fat-balanced diet (adding perilla oil or fish oil). If the patients with poor liver function have intravenous supplementation of fat emulsion, it will result in the increase of the concentration of -6 fatty acids and non-essential fatty acids. Patients with poor liver function should have a fat-balanced diet which includes 3–5 calorie percent of -3 fatty acids with perilla oil or fish oil, but those with severe liver dysfunction should receive intravenous supplementation of fatbalanced fat emulsion. It has been reported that sesamin derived from sesame seed improves the hypercholesterolemia and low concentration of high density lipoprotein cholesterol.9,10 In our preliminary experience, sesame seed paste administration to a BA patient with poor liver function improved not only hypercholesterolemia but also unbalanced fatty acid composition (high concentration of non-essential fatty acid and the low concentration of eicosapentaenoic acid).11 SUMMARY
The concentrations of plasma fatty acids in postoperative patients with BA were measured. Patients with poor bile flow but without severe liver dysfunction did not show any decrease in the concentration of -6 fatty acids in the plasma, but were at risk of developing -3 fatty acid deficiency.
REFERENCES 1. Handa N, Suita S, Ikeda K, et al. Requirement of parenteral fat in infants with biliary atresia. JPEN 1985;9:685 2. Dupont J, Ame´de´e-Manesme O, Pepin D, et al. Eicosanoid synthesis in children with cholestatic disease. J Inherit Metab Dis 1990;13:212 3. Miyano T, Yamashiro Y, Shimizu T, et al. Essential fatty acid deficiency in congenital biliary atresia: successful treatment to reverse deficiency. J Pediatr Surg 1986;21:277 4. Yamashiro Y, Ohtsuka Y, Shimizu T, et al. Effects of ursodeoxycholic acid treatment on essential fatty acid deficiency in patients with biliary atresia. J Pediatr Surg 1994;29:425 5. Andoh T, Uda H, Yoshimitsu N, et al. The sex differences in cord-blood cholesterol and fatty-acid levels among Japanese fetuses. J Epidemiol 1997;7: 226
6. Gourley GR, Farrell PM, Odell GB. Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia. Am J Clin Nutr 1982;36:1194 7. Yamashiro Y, Shimizu T, Ohtsuka Y, et al. Docosahexaenoic acid status of patients with extrahepatic biliary atresia. J Pediatr Surg 1994;29:1455 8. Biagi PL, Hrelia S, Stefanini GF, et al. ⌬-6-desaturase activity of human liver microsomes from patients with different types of liver injury. Prostaglandins Leukot Essent Fatty Acids 1990;39:39 9. Sugano M, Inoue T, Koba J, et al. Influence of sesame lignans on various lipid parameters in rats. Agric Biol Chem 1990;54:2669 10. Hirose N, Inoue T, Nishihara K, et al. Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 1991;32:629 11. Kaji T, Takamatsu H, Noguchi H, et al. Administration of sesame paste for hyperlipidemia in postoperative patient with biliary atresia (case report). Jap J Surg Metab Nutr 1998;32:9