Oxidation of butyrate to ketone bodies and CO2 in the rumen epithelium, liver, kidney, heart and lung of camel (Camelus dromedarius), sheep (Ovis aries) and goat (Carpa hircus)

Comp. Biochem. Physiol., Vol. 65B, pp. 699 to 704

0305-0491/80/0401-0699502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

OXIDATION OF BUTYRATE TO KETONE BODIES AND C O 2 IN THE RUMEN EPITHELIUM, LIVER, KIDNEY, HEART AND LUNG OF CAMEL (CAMELUS DROMEDARIUS), SHEEP (OVIS ARIES) AND GOAT (CARPA ItIRCUS) B. EMMANUEL Department of Animal Biology, School of Veterinary Medicine, University of Shiraz, Shiraz, Iran

(Received 23 July 1979) Abstract--l. The oxidation of butyrate to ketone bodies and CO2 was studied in tissues (rumen epithelium, liver, kidney, heart and lung) of camel (Camelus dromedarius), sheep (Ovis aries) and goat (Carpa hircus). 2. The rumen epithelium and the liver of the goat and the sheep oxidized considerable quantities of butyrate to ketone bodies and CO2; whereas, in camel both tissues converted negligible amounts. 3. The kidney of the camel metabolized more butyrate than that of the sheep or the goat. 4. In all species studied, the heart and the lung oxidized very little butyrate.

INTRODUCTION

Volatile fatty acids including acetate, propionate and butyrate are fermentation products of carbohydrate metabolism by micro-organisms in the rumen (Hungate et al., 1961). Two pathways were proposed for synthesis of butyrate from acetyl-CoA by rumen microflora, namely the reversal of fl-oxidation and a mechanism involving the utilization of malonyl-CoA (Leng, 1970). Ruminants fed on different rations have ruminal butyrate concentrations of 5.4-22.9mM (Church, 1971b). The production rate of butyrate in the rumen is 37-55 mmols/hr per sheep in the fed state (Annison et al., 1967; Leng & West, 1969). Butyrate is oxidized (Pennington, 1954; Goosen, 1976), and is converted to ketone bodies by the rumen epithelium (alimentary ketogenesis) (Pennington, 1952; Annison et al., 1957, 1963; Hird & Weideman, 1964). Previous work in this lab had shown that plasma concentrations of ketone bodies, and the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in camel were much lower than respective values in sheep (Chandrasena et al., 1979b). It was concluded that the low plasma levels of ketone bodies are due to: 1, low activity of 3-hydroxybutyrate dehydrogenase in the rumen epithelium and the liver; 2, the absence of papillae in the rumen epithelium of the camel (Vallenas et al., 1971) which greatly reduces the surface area available for metabolic activity and 3, camel lacks the third compartment of the stomach (omasum) (Hansen & Schmidt-Nielsen, 1957) which is reported to produce some ketone bodies (Pennington, 1952; Hird & Symons, 1961). In contrast to monogastrics in which the liver produces ketone bodies (hepatic ketogenesis), in the fed ruminant the liver plays a minor role in ketogenesis (Leng & West, 1969). Hungate et al. (1959) and Williams (1963) reported that the rumen function in camel yields the same products at rates and in proportion comparable to those in true ruminants. Therefore, the fate of butyrate in

camel raised some speculations. In view of previous observations (Chandrasena et al., 1979b) and literature data, the possibility of the oxidation of butyrate by the camel rumen epithelium was considered. The present study was aimed to investigate the oxidation of butyrate to ketone bodies and CO2 in rumen epithelium, liver, kidney, heart and lung of camel. The same tissues which are known to metabolize butyrate in true ruminants were utilized from the sheep and goat for comparison. MATERIALS A N D M E T H O D S

Incubation Tissues (rumen epithelium, liver, kidney, heart and lung) were obtained from Shiraz city abattoir immediately after slaughter, kept in ice and transferred within 45 rain to the laboratory. Rumen epithelium was stripped from the underlying tissue, washed with physiological saline a few times while stirring (to remove food particles and bacteria) and then blotted. It has been reported that the epithelium was considerably more active in butyrate metabolism than the underlying tissue (muscle) (Pennington, 1952). Tissue slices (0.5 mm thick) were cut by means of a tissue slicer (Arthor H. Thomas Co., Philadelphia). The incubation was carried out in an Erlenmeyer flask with a rubber stopper and a removable central glass well. This well contained 0.25 ml of hyamine hydroxide to trap the released 14C02, and a filter paper to increase surface area. The incubation medium contained 45#mols KC1, 6/~mols EDTA, 15/~mols MgCI 2, 150/amols Tris, 37.5#mols KEHPO4, 37.5 pmols KH2PO4, 30~mols n-butyric acid, and l#Ci n-[1-14C]butyrate (24mCi/mmol; Radiochemical Centre, Amersham, Bucks, U.K.) in a total volume of 3 ml and at pH 7.1. To this medium 0.5 g of stripped rumen epithelium or 0.5 g of tissue slices were added. The flask was then alternately evacuated and refilled with oxygen gas for 30 sec, using hypodermic needles inserted through the stopper. The reaction mixture was incubated at 37°C for 1 hr. At the end of incubation time, 2 ml of 10% perchloric acid (w/w) were injected to terminate the reaction and to release 14CO2. The reaction mixture was shaken for an extra 2 hr 699

700

B. EMMANUEL

to trap the released 14CO2. In each experiment, two control samples containing all the compounds, but tissue were used.

Butyrate

(1) t

Counting of 14CO2

The glass well containing hyamine hydroxide, the filter paper, and the trapped a4CO2 was transferred into 10 ml of toluene containing 30 mg PPO, and 100/~g POPOP, and counted in a liquid scintillation counter (Packard, Model 3330} with a counting efficiency of 85~, for the labelled carbon atom. Correction was made for quenching by applying the channel-ratio procedure of Bruno & Christian (1961).

Fatty acids ~-- ~-- .--- ~- Butyryl-CoA

Crotonyl-CoA

(3t [

Determination of ketone bodies

3-Hydroxybutyryl-CoA

The acidified reaction mixture was transferred to a test tube, and centrifuged at 20000 for l0 min. Then 3 ml of the supernatant were neutralized with 20°/,) (w/v) of KOH. Concentrations of 3-hydroxybutyrate and acetoacetate were measured enzymatically as described by Williamson & Mellanby (1963), and Mellanby & Williamson (1963), respectively. NAD, NADH and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) were products of Boehringer Mannheim GmbH, Mannheim.

(4) j

RESULTSANDDISCUSSION The oxidation of butyrate to ketone bodies and C O / i n various tissues of camels, sheep and goats was considered in the present studies. Metabolic pathways showing butyrate utilization are shown in Scheme 1. The enzymes of ketogenesis have been shown in the rumen epithelium. The presence of butyryl-CoA synthetase (EC 6.2.1.2) was reported (Ash & Baird, 1973). High activity of L-(+)-3-hydroxybutyryI-CoA dehydrogenase (EC 1.1.1.35) was found in the rumen epithelium of the sheep (Emmanuel et al., unpublished data). Three different mechanisms have been reported for the conversion of acetoacetyl-CoA to acetoacetate, namely deacylation, the transferase reaction and the 3-hydroxy-3-methyl glutaryl-CoA route (Baird et al., 1970; Bush & Milligan, 1971). The activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been shown by several workers (Lahunta, 1965; Koundakjian & Snoswell, 1970; Watson & Lindsay, 1972; Weeks, 1974a,b; Chandrasena et al., 1979b). The operation of Krebs cycle in the rumen epithelium was suggested by Annison et al. (1963). Studies of Seto & Umezu (1959) and Seto et al. (1970) provided experimental evidence for a functional tricarboxylic acid cycle in this tissue. The results on conversion of butyrate to ketone bodies are presented in Table I. The rumen epithelium of the goat produced high quantities (23.4 #mols/hr per g wet wt), which is 77% conversion. This conversion is slightly lower than the value (80-85~o) reported previously (Fell & Weeks, 1975). The results might have been underestimated in the present work. The amount of butyrate used in the reaction mixture was 30pmols of which 23.4/~mols was converted to ketone bodies (Table 1), and 5.4/~mols to CO2 (Table 2). Therefore, the level of substrate could have limited the production rate. A comparison of the appearance of butyrate in portal blood of the sheep with its net production rates in the rumen suggested that as much as 80-90% of the butyrate absorbed was metabolized in the rumen wall (Annison et al., 1957; Fell & Weeks, 1975). The values

CO2 + H20 ~-- ~-- ~-- ~ Acetoacetyl-CoA (Krebscycle)

(5)/~6\\, S 17) Acetoacetate

(S) [ 3-Hydroxybutyrate Scheme I. Metabolic pathways utilizing butyrate. The numbers in brackets refer to: 1 = Butyryl-CoA synthetase (EC 6.2.1.2); 2 = Butyryl-CoAdehydrogenase (EC 1.3.99.2); 3 = Crotonase (EC 4.2.1.17); 4 = L-(+)-3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35); 5 = Deacylase (EC 3.1.2.11); 6 = 3-Oxo a c i d CoA transferase (EC 2.8.3.5);7 = 3-Hydroxy-3-methylglutaryl-CoAsynthase (EC 4A.3.5) plus 3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4); and 8 = 3-Hydroxybutyrate dehydrogenase (EC l.l.l.30). in sheep were about twice lower than in goat, and are related to the data of Goosen (1976) performed on cows. Other workers found higher conversion rates in the rumen epithelium of cattle (Bush et al., 1970), and lamb (9-10 weeks old) (Giesecke et al., 1979). On the other hand, the production rates in the rumen epithelium of adult sheep and cattle were 6.2 and 5.5/~mols/hr per g wet wt, respectively (Stangassinger et al., 1979). The values were converted from dry weight to wet weight basis considering the information given by Weeks (1971). It appears then that the conversion of butyrate to ketone bodies in ruminants is influenced by factors including species, diet and developmental stage of the rumen epithelium. In addition, experimental conditions such as availability of oxygen in the incubation medium and the pH (Pennington & Sutherland, 1956) may affect greatly the results. The production of ketone bodies in camel was negligible (42 and 81 times less than that of sheep and goat, respectively). This finding agrees with previous observations showing very little activity of 3-hydroxybutyrate dehydrogenase in the rumen epithelium, and low plasma levels of ketone bodies in camel (Chandrasena et al., 1979b). The results of the present studies with respect to the capacity of camel rumen epithelium to produce ketone bodies are more pertinent than earlier data since it measures the complete reactions leading from butyrate to ketone bodies (Scheme 1) as compared to that of one enzymic reaction (3-hydroxybutyrate dehydrogenase). Ratios of 3-hyd-

6;O.lO)

0.41( 0.46( 0.89

0.18(

6;0.09) 6;O.lO)

0.28( 0.36( 0.78

0.40( 0.28( 8;0,10) 1.42

BHB

AcAc

BHB/AcAc

BHB

AcAc

BHB/AcAc

0.51

0,38( 6;0,03)

6;O.Ol)

Camel

0.40

I,ll( 9;0,22)

0.44(

9;0.13)

6;0.07)

0.86( 1.07

6;0.25)

O-92(

1.08

0,74(13;0,11)

0,80(13;0,33)

Kidney

6;0.30)

2.86(

and acetoacetate, respectively.

0.72

0.17( 6;0,05) 0.26

0.12( 1,23(10;0.22)

6;0.01)

6;2.34) lb32

6;2.95)

13.31( 10,08(

1.10

5.85(13;1.83)

6.42(13;0.72)

Epithelium

per g wet wt)

0.32(10;0.04)

1.05

6;0.74)

3.00(

0.85

1.53(13;0.32)

1.30(13;0.33)

Liver

* The values in brackets are number of observations, and &SD, respectively. BHB, and AcAc are 3-hydroxybutyrate, The reaction mixture is described in the Materials and Methods section.

S;O.OS)

6;0.05)

1*03

1*03

BHB/AcAc Goat

0.35(12;0.03)

0.36(12;0,12)

0.29(11;0.11)

*

Sheep

Lung

0,30(11;0.10)

Heart

BHB

Tissue

AcAc

Product

Table 1. Conversion of butyrate to ketone bodies by various tissues of the sheep, the goat and the camel (pmols product/m

B. EMMANUEL

702

Table 2. Conversion of butyrate to CO: by tissues of the sheep, the goat and the camel (#mols butyrate used/hr per g wet wt)

Tissue Heart

Lung

Kidney

Liver

Epithelium

Sheep 0o10(11;0.02)

0o29(12;0.04)

1.34(13;0.23)

0o86(13;0.09)

2.76(13;0.50)

1.68(6;0.32)

5.43(6;1.25)

0.43(10;0o07)

0.04(6;0.01)

Goat

0.48(6;0.09)

0~51(6;0.10)

1.82(6;0.40) Camel

Oo13(8;0.02)

0.04(6;0o01)

4.16(9;0.45)

* The values in brackets are number of observations, and +SD, respectively.The reaction mixture is described in the Materials and Methods section. roxybutyrate to acetoacetate were 0.72, 1.10 and 1.32 for camel, the sheep and goat, respectively. Weigand et al. (1975) and Bush et aL (1970) reported ratios of 0.46 and 2.63, respectively. The results on relative amounts of acetoacetate and 3-hydroxybutyrate should be treated with precaution. The acetoacetate formed during incubation can be decarboxylated by the rumen epithelial acetoacetate decarboxylase (Seto et al., 1964), and through a non-enzymatic reaction (spontaneous decarboxylation) (Emmanuel et al., 1979). Furthermore, as mentioned above, the availability of oxygen has an important bearing on the relative proportions of ketone bodies formed. The total ketone production in the liver was 1.55, 2.83 and 5.86 #mols/hr per g wet wt for camel, sheep and goat, respectively. The pattern was similar to the data on the rumen epithelium. Results of other studies calculated on the same basis showed similar values (Pennington, 1952; Bush et aL, 1970). The result on the kidney of sheep is in accord with earlier reports (Pennington, 1952). The values for camel were comparable to other species. In all three species, the heart and the lung produced very little ketone bodies from butyrate. The results of the kidney, heart and lung might have been lower than the true values due to possible utilization of ketone bodies formed during the incubation period by these tissues. The disappearance of ketone bodies by the kidney, heart and lung of sheep has been reported (Leng & Annison, 1964; Kaufman & Bergman, 1971). The results on the oxidation of butyrate to CO2 (Table 2) show that the rumen epithelium and the liver of the goat had a higher capacity than sheep. These tissues of the camel oxidized very little butyrate. On the other hand, the kidney of camel oxidized more than other two species. In all species tested, the heart and lung converted very little butyrate to C 0 2 . Hird & Symons (1961) in studying the mode of formation of ketone bodies from butyrate in the rumen epithelium and the omasum of sheep used four types of butyrate labelled separately in each of its carbon atoms. They concluded that carbon atoms 1 and 2 are

oxidized at greater rates than carbon atoms 3 and 4. Similarly, Lindsay & Ford (1964), using [1-14C]ace tate and [2-14C]acetate in sheep concluded that the oxidation rates of carbon 1 was 1.58 times that of carbon 2. In the present studies, [1-14C]butyrate was utilized, and therefore, correction was made accordingly. The results of butyrate oxidation support earlier investigations, showing little conversion of butyrate to CO2 by various tissues including sheep liver (Leng & Annison, 1963), sheep rumen epithelium and omasum (Hird & Symons, 1959, 1961), and cow rumen epithelium (Goosen, 1976). The concentrations and proportions of volatile fatty acids in camel rumen is the same as that of true ruminants (Williams, 1963). Therefore, the fate of butyrate metabolism in camel raises interesting speculations. Present studies show that the rumen epithelium oxidizes negligible quantities of butyrate either to ketone bodies or CO2. Some butyrate is metabolized in the kidney providing energy for this organ which is known to be very active in this animal (Schmidt-Nielsen, 1964). It is possible that butyrate is metabolized to a greater extent by other tissues not tested in the present work. It can also serve as precursor for fat synthesis in the adipose tissue (hump) (Scheme 1). Volatile fatty acids, in particular butyrate, stimulate papillae growth (Church, 1971a; Feel & Weeks, 1975). The rumen epithelium of camel is devoid of papillae. This may suggest that butyrate is probably not the cause, but an intermediary metabolite leading from butyrate to ketone bodies, and its complete oxidation to CO2, in the tricarboxylic acid cycle, yields energy for metabolic activities of the rumen epithelium. In camel, other fermentation end products of the rumen or glucose may have a significant role in supplying energy for this tissue. It is concluded that in contrast to true ruminants, in camel negligible quantities of butyrate are oxidized to ketone bodies and COz by the rumen epithelium, The kidney metabolizes some of this compound. This v,ork confirms further, earlier studies carried out on

Oxidation of butyrate in tissues of ruminants camel in this laboratory (Emmanuel & Emady, 1978; Chandrasena et al., 1979a; Chandrasena et al., 1979b).

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