May 20, 1992 - The biodistribution and metabolism of[8-14C]-octacosanol in rats were investigated to understand the mechanism of in- creased physical ...
Annals of Nutrition and Metabolism
Original Paper
Main-Editor: G. Wolfram, Miinchen
Separatum Publisher: S. Karger AG, Basel Printed in Switzerland
Yearul Kabir Shuichi Kimura Laboratory of Nutrition, Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan
Ann Nutr Metab \993;37:33-38
Biodistribution and Metabolism of Orally Administered Octacosanol in Rats
KeyWords
Abstract
Octacosanol Biodistribution Metabolism Adipose tissue Physical exercise
The biodistribution and metabolism of[8-14C]-octacosanol in rats were investigated to understand the mechanism of increased physical exercise and motor endurance by octacosanol. After 14C-octacosanol administered, radioactivity of octacosanol was mainly found in adipose tissue, especially in brown adipose tissue. Absorption of octacosanol is very low and mainly excreted through feces. The radioactivity of octacosanol was also partly expired as 14C02. About 49% of the administered dose were excreted through different pathways. Metabolites of octacosanol are present in the urine. Octacosanol may be partly oxidized and degraded to fatty acids through ~-oxidation_
Introduction
A good deal of evidence has accumulated to support the hypothesis that motor endurance and physical performance are increased by octacosanol [1-3]- It is shown in a series of controlled experiments that octacosanol improves some aspects of athletic performance in healthy volunteers in combination with physical training [4]. In the rat it was also found that the amount of voluntary exercise
Received: May 20,1992 Accepted: August 25, 1992
was significantly increased by supplementation of octacosanol in the diet [5]. Recently it has been reported that octacosanol stimulated the conversion of lipids into energy [6]. Patients with mild parkinsonism appeared to obtain great benefit from octacosanol supplementation [7]. A rationale for this benefit is that these long-chain alcohols are present in the central nervous system but absent in the usual diet [8]. Hormone-like activities of long-chain alcohols in animals
Dr. Yearul Kabir Associate Professor Department of Biochemistry University of Dhaka Dhaka-I 000 (Bangladesh)
© 1993 S. Karger AG, Basel 025(}-6807/931 0371-0033$2.75/0
•••
Air at 20-22 °C
t Glass metabolic cage
I
1 N NaOH solution
Fig. 1. Collection system of expired 14C02 and excreta (diagrammatic
and plants have been reported [9]. Although the increased motor endurance and physical performance from octacosanol are known, nothing is known about the biodistribution, absorption and metabolism of administered octacosanol in the body. The present study was undertaken to investigate the biodistribution and metabolism of octacosanol fed to rats. The knowledge of biodistribution and metabolism of long-chain alcohols like octacosanol is of interest because the alcohol moiety of wax could be a valuable natural raw material for this purpose.
Materials and Methods Experimental Animals Twelve young (4-week-old) male Wi star rats (purchased from Funabashi Farm, Japan) weighing 63-65 g, maintained on an ad libitum supply of a pellet diet (Funabashi F2, Japan) and drinking water were used for the study. [8_14C]-octacosanol was given orally through a stomach tube. Three animals were killed each at 1, 2, 3, and 7 days after an administration of 10 ~Ci [8_14C]-octacosanol in 0.5 ml solution in tricaproyl-glycerol. All rats were killed by ethyl ether anesthesia and the blood was collected from the abdominal
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Kabir/Kimura
aorta in a disposable plastic rin. Blood was centrifuged and the plasma was stored analysis. The rats were then
representation).
syringe coated with hepaat 3,000 rpm for 15 min, in a vial at 0 °C for later rapidly dissected.
Extraction and Analysis The organs and tissues, liver, kidneys, spleen, heart, lungs, brain, gastrointestinal tracts, epididymal adipose tissue, perirenal adipose tissue, brown adipose tissue and gastrocnemius muscle, were quickly removed and washed in cold 0.9% NaCI. After blotting with a filter paper, their weights were recorded. Then a portion of the fresh samples (about 50-100 mg) of each organ and tissue were taken in a counting vial. To solubilize the samples, 1.0 ml of tissue solubilizer (soluene350) (Packard Chemical Co.) was added and the samples were kept for 8-12 h at 50 ° C. After decolorization with H202, 10 ml of aqueous counting scintillator (ACS-II) (Amersham, USA) were added and the radioactivity was counted in an Aloka LSC-903 liquid scintillation counter (Packard Instrument, Ill., USA). The counts were corrected for quenching with external standards and computation was carried out using a digital computer. Collection of Expired 14C02 and Excreta Soon after the administration of 14C-octacosanol the animals were kept individually for 7 days in a metabolic glass cage designed to trap expired carbon dioxide and to collect the excreta (fig. 1). Urine and feces were separately collected. The 14C02 expired was trapped in large test tubes containing 1 N NaOH ar-
Octacosanol, Biodistribution Metabolism
and
ranged serially as shown in figure I. Every 2 h the tubes were replaced with a new set of tubes without interrupting the run. The first sampling period was preceded by a 2-hour wait and followed by 2-hour intervals over a period of 24 h. The inside temperature of the cage was kept at 20-22°C. The radioactivity in NaOH solution was counted as described previously after neutralizing 1.0 ml NaOH solution by adding an appropriate amount ofHCI to the vial. Extraction and Analysis of Radioactivity in Feces and Urine Feces from the animals were collected every day over the 7-day period. Samples of feces were prepared for the analysis of radioactivity by overnight freezedrying and powdering in a mortar and pestle. A known amount of feces powder (about 50 g) was put in a vial and 0.2 ml concentrated HN03 was added. The mixture was kept at 50 °C for I h, then heated for 8 hat 45 °C after adding 0.3 ml of 30% H202 + 70% HCI04 (1: I). After solubilizing the samples in 1.0-ml tissue solubilizer Soluene-350 and adding 10 ml of counting scintillator (ACS-II), the radioactivity was counted. The urine was also collected for 7 days and analyzed for radioactivity. In a vial 1.0 ml urine was taken and heated for 1-2 h at 45 °C after adding 0.5 ml of 30% H202 + 70% HCI04 (1: 1). Then after dissolving the samples in 1.0 ml Soluene-350 and adding 10 ml ACS-II counting scintillator, the radioactivity was counted. Extraction of Urinary Metabolites A urine sample (1.0 ml), after thawing and filtration, was extracted separately with 3.0 ml of chloroform-methanol (2: I; v/v) and 3.0 ml of water. The mixture was vigorously shaken. After an additional extraction of the metabolites with chloroform-methanol and water, portions (1.0 ml) from the combined chloroform-methanol as well as the water extracts were taken in vials for radioactivity counting. Extraction of Radioactivity from Fecal Metabolites Samples of feces were prepared for extraction by overnight freeze-drying and powdering in a mortar. All feces were powdered in a mortar. A known weight of feces powder (about 50 mg) was extracted separately with 5.0 ml of chloroform-methanol (2:1, v/v) and 5.0 ml of water by the method described above and the radioactivity was counted in each extract.
Results
The biodistribution of radioactivity in various organs after administration of 14C-octacosanol is presented in table 1. Following an oral dose of [8_14C]-octacosanol to the rats, the highest concentration of plasma radioactivity was reached in 1 h, indicating that the compound was quickly absorbed. The amount of radioactivity from octacosanol was very low in various organs after the administration and among the organs the highest amount of radioactivity was present in the liver (table 1). But when expressed per gram of tissue, the largest amount of radioactivity was found in the adipose tissues, especially in the brown adipose tissue. The excretion of radioactivity through feces was about 32% of the administered dose (fig. 2). About 26% was excreted within 24 h. In comparison with feces, the excretion through urine was very small. The expiration of 14C02 over 7 days was about 15% of the dose (fig. 3). Also in this case, more than 50% of the expiration occurred within the first 24 h. Afterwards the expiration of CO2 decreased markedly. The excretion of radioactivity through three different pathways over 7 days was about 49% of the administered dose (fig. 4). Total recovery of radioactivity after 7 days was 32.14% in feces, 1.31 % in urine, and 15.12% in expired CO2. The 14C-radioactive counts of chloroformmethanol (2: 1, v Iv) extracts of feces and urinary metabolites prepared after oral administration of 14C-octacosanol are given in table 2. The radioactivity excreted through feces was almost completely extracted by chloroform:methanol (2:1, v/v), whereas very little radioactivity was present in chloroform:methanol extract of urine, where the radioactivity was mainly present in the water layer (table 2).
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Table 1. Biodistribution of radioactivity administration of 14C-octacosanol
in rats at various times after one dose of oral
Time (days) after administration,
Organ
% dose/g tissue
2 liver BAT PAT EAT Digestive tracts Spleen Kidney Heart Lung Brain
0.98 1.96 1.31 1.17 0.38 0.19 0.17 0.14 0.15 0.04 0.06 0.12
I
Muscle? Plasma/ml
0.57 1.05 1.28 0.88 0.27 0.16 0.15 0.13 0.14 0.05 0.06 0.08
(3.56) (0.21) (0.20) (0.30) (1.83) (0.05) (0.15) (0.04) (0.10) (0.06) (1.89)
(1.87) (0.13) (0.20) (0.26) (1.44) (0.04) (0.13) (0.04) (0.09) (0.06) (2.15)
3
7
0.32 (1.07) 0.57 (0.08) 1.12 (0.13) 0.69 (0.17) 0.14(0.75) 0.12 (0.03) 0.13 (0.11) 0.11 (0.04) 0.13 (0.07) 0.04 (0.05) 0.05 (1.69) 0.05
0.08 0.26 0.75 0.65 0.06 0.05 0.06 0.06 0.06 0.05 0.04 0.01
(0.34) (0.05) (0.12) (0.25) (0.46) (0.02) (0.06) (0.02) (0.04) (0.07) (1.50)
Four-week-old male Wistar rats were given [8-14C]-octacosanol through a stomach tube. At various times the animals were killed and rapidly dissected. Values are the means of 3 rats and values in parentheses indicate % dose/organ. BAT = Brown adipose tissue; PAT = perirenal adipose tissue; EAT = epididymal adipose tissue. I Including contents. 2 Calculated from an estimated muscle mass equal to 45% of body weight.
Table 2. Radioactivity of chloroform-methanol over 7 days after administration of 14C-octacosanol
Sample
Replicates
Radioactivity
(Chl:Meth) and water extracts of feces and urine collected
in extract
Chl:Meth (2: 1)
Dist. water
cpm"
% recovery
cpm-
Feces powder, cpm/50 mg 253,426 1 2 3 mean
187,935 202,080 243,050 211,022
99.71 99.71 99.85 99.76
547 580 352 493
Urine, cpm/ml 11,535 1 2 3 mean
238 244 459 314
a
36
2.44 2.22 4.33 3.00
9,530 10,748 10,138 10,139
% recovery
0.29 0.29 0.15 0.24 97.56 97.78 95.67 97.00
Total radioactivity recovery cpm
Recovery %
188,482 202,660 243,402 211,515
74.37 79.97 96.04 83.46
9,768 10,992 10,597 10,453
84.68 95.29 91.87 90.60
Feces: cpm in extract of 50 mg, urine: cpm in extract of 1.0 ml.
Kabir/Kimura
Octacosanol, Metabolism
Biodistribution
and
7.0 26.0 Q) U)
22.0
0
"0
*'
Q) U)
0
"0
'0
*'
5.0
'0
18.0
14.0 3.0 10.0
6.0
2.0
2
3
4
5
Time after administration,
6
2
7
Fig. 2. The excretion of radioactivity through feces over 7 days after administration of 14C-octacosanol.
3
4
5
Time after administration,
days
6
7
days
Fig. 3. The expiration of 14C02 over 7 days after administration of 14C-octacosanol.
Discussion
The persistence of radioactivity in tissues and feces of rats through to the 7th day after a single dose of 14C-octacosanol administration (table 1, fig. 2) suggests that the radioactivity had accumulated in the tissues and that the observed effect of octacosanol in ongoing energy metabolism processes during exercise is genuine, since enhanced physical performance in humans and animals occurs after daily ingestion of octacosanol [1-5]. Catabolism of octacosanol was speculated upon from the radioactivity expired as 14C02 (fig. 3). The results suggested that some of the 14C-octacosanol might be converted into a fatty acid which supplied 14C02 and energy by the process of oxidation. The other possibility is that all the free acid produced from octacosanol did not undergo a direct oxidation and
40
30 Q) U)
o "0
20
'0
*' 10
o Feces
CO2
Urine
expired
Excretion
Fig. 4. Excretion of radioactivity through three diferent pathways over 7 days after administration of 14C-octacosanol.
37
part of it was stored in the fat pool of the adipose tissue. This is in accordance with the findings of Neptune et al. [10], who reported that the conversion of long-chain fatty acids to 14C02 constituted only a small fraction of the total CO2 in the rat diaphragm. The amount of radioactivity in the lipid layer of the feces (table 2) extract might be due to free octacosanol, whereas in urine it might be mainly due to the metabolites of octacosanol.
The presence of radioactivity in adipose tissues was of particular interest since they are related to energy mobilization and utilization. It is possible that octacosanol in adipose tissue might be related to a direct stimulation of the effect of catecholamines on free fatty acid release in adipose tissue cells or to other hormones on fat mobilization. Further work is rquired to elucidate the role of octacosanol.
References Passwater R: Octacosanol: Its name really increased. Health Q 1982;7: 14-15. 2 Katahira R, Shichigo I: Octacosanol. Seiyakukojo 1984;4:461-466. 3 Saint-John M, McNaughton L: Octacosanol ingestion and its effects on metabol ic responses to sub maximal cycle ergometry, reaction time and chest and grip strength. Int Clin Nutr Rev 1986;6:81-87. 4 Cureton TK Jr: Improvements in physical fitness associated with a course of US avy underwater trainees, with and without dietary supplements. Res Q 1963;34:440-453.
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5 Kabir Y, Kimura S: Biochemical effects of octacosanol on voluntary exercise in rats. Dhaka University Studies Part E 1991 ;6:77-85. 6 Shimura S, Hasegawa T, Takano S, Suzuki T: Studies on the effect of octacosanol on motor endurance in mice. Nutr Rep Int 1987;36: 10291038. 7 Snider SR: Octacosanol in parkinsonism. Ann Neurol 1984; 16:723. 8 Pakkala SG, Fillerup DL, Mead JF: The very long-chain fatty acids of human brain sphingolipids. Lipids 1966; I :449-450.
Kabir/Kimura
9 Kolattukudy PE: Enzymatic synthesis of fatty alcohols in Brassica oleracea. Arch Biochem Biophys 1971; 142:701-709. 10 Neptune EM Jr, Sudduth HC, Foreman DR, Fash FJ: Phospholipid and triglyceride metabolism of exercised rat diaphragm and the role of these lipids in fatty acid uptake and oxidation. J Lipid Res 1960; 1:229235.
Octacosanol, Metabolism
Biodistribution
and
