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Variation in the concentration of long chain free fatty acids in equine plasma over 24 hours
Br. vet.J. (1994). 150, 339
VARIATION IN THE CONCENTRATION OF LONG C H A I N FREE F A T T Y A C I D S I N E Q U I N E P L A S M A O V E R 24 H O U R S
C. E. ORME, M. DUNNETT and R. C. HARRIS Departnwnt of Physiology, Animal Health Trust, P.O. Box 5, Newmarket, Suffolk CB8 7DW, UK
SUMMARY The primary aim of this study was to examine the within-day variation in the concentration of total and individual long chain free fatty acids (C> 14) in normally fed horses. Plasma samples were collected over a 24 h period from 12 resting horses during three separate sessions (six horses in the first session and three in the second and third). Samples were analysed for individual long chain free fatty acids (FFA) and glucose. During normal feeding, the predominant FFA in plasma were palmitic (C16:0), linoleic (C18:2), oleic (C18:1), stearic (C18:0) and linolenic (C18:3). Together these acids constituted over 90% of the total concentration. Other FFA present were myristic (C14:0) and palmitoleic (C16:1) both of which constituted <5% of the total concentration. Ten out of the 12 horses sampled exhibited an early morning increase in FFA (P<0.001) localized around 0700 h and which was independent of feeding. The mean concentration of total FFA increased 4.5 fold (range 2.0-8.5) between 0400 and 1000 h. The predominant FFA showed the largest increase. KE~WORDS:Horse; free fatty acids; plasma.
INTRODUCTION In man and other species certain biochemical parameters and hormones exhibit regular variations throughout the day. These changes may be related to food intake, e.g. glucose in man, or may be independent of feeding and regulated by some other means, e.g. cortisol, growth hormone and iron (Zilva & Pannall, 1984). The day-night cycle usually imposes a diurnal rhythm on the feeding behaviour of an animal, sufficient fuel being stored during the active feeding period to provide for the metabolic demands of the period of sleep. Resting concentrations of plasma free fatty acids (FFA) influence the rate of FFA oxidation 0007/1935/94/040339-09/$08.00/0
© 1994Bailli&eTindall
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during subsequent exercise (Ravussin et al., 1986). As part of a wider investigation of the utilization of FFA as a fuel during exercise in the horse, it was necessary to ensure that studies would noi coincide with any diurnal increase in plasma FFA concentration. No previous investigations describing within-day variation in the concentration of long chain FFA in equine plasma could be found in the literature.
MATERIALS A N D M E T H O D S
The within day variation of total long chain (C>14) free fatty acids (FFA,) and individual long chain free fatty acids (FFA~) in equine plasma were measured over a 24 h period during three experimental sessions. Six horses designated Sn, Bd, He, Kj, Mr and Lb were sampled during the first session and three each during sessions 2 (Bo, Co and GI) and 3 (HI, Pn and Ls). All horses were fed normally and were not exercised during the study. The horses used consisted of eight geldings (Sn, Bd, He, Kj, Mr, Bo, Co and Ls) and four fillies (Lb, G1, HI, and Pn) weighing between 400-500 kg. Prior to the start of the study three of the horses (Kj, Mr and He) had been out at grass with little or no work whilst the remaining nine were boxed and in moderate work. Eleven of the 12 horses were fed Spillers Stud cubes (approximately 2 kg) and the twelfth, Bd, Spillers racehorse cubes (approximately 2 kg). Horses were fed at 0700, 1230 and 1630 h. The oil content of the cubed portion of the diets were 4.25 and 4.0% by fresh weight respectively. All horses were fed 2-3 kg of hay with the morning and evening feeds. Water was available ad libitum at all times. Venous blood samples were drawn at hourly intervals starting at 1700 h via a 13G catheter (Vygon, Benkat Instruments, UK) inserted into the left jugular vein under local anaesthesia. The horses were catheterized a minimum of 1 h before the first sample was take. Blood was dispensed into tubes containing Ethylenediamine tetracetic acid (EDTA) as an anticoagulant and centrifuged immediately. The plasma was aspirated and stored at -20°C for analysis of FFA,, FFAi and glucose. Concentrations of FFA~were measured by high performance liquid chromatography (HPLC). FFA, concentrations were calculated by the addition of FFA~ concentrations. EDTA was used as an alaticoagnlant because of the reported elevation in plasma FFA concentrations during storage of lithium heparin treated samples (Gleeson, 1987). EDTA inhibits the action of bacterial lipases and prevents the auto oxidation of FFA (Bachorik, 1982). Chemicals Methanol and acetonitrile (HPLC grade) were supplied by Romil Chemicals Ltd (Shepshed, Loughborough, Leicestershire, UK); ethanol was supplied by James Burrough (F.A.D.) Ltd (Witham, Essex, UK) and chloroform by Interchem (UK); potassium hydroxide was supplied by B.D.H. chemicals (Poole, Dorset, UK); 1,4,7,10,13,16,hexaoxacyclooctadecane (18 crown 6) by Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK), 4 bromophenacyl bromide, glucose and all FFA standards by Sigma Chemical Co. (Poole, Dorset, UK).
PLASMA FREE FATTY ACIDS
341
Analysis Plasma samples were analysed .for FF& using a Constametric 1 HPLC pump (LCD/Milton Roy, Stone, Staffordshire, UK), with a rheodyne 7125 injector and 50/11 loop (Rheodyne, Cotati, CA, USA) and LC-UV variable wavelength ultraviolet spectrophotometric detector (Phillips analytical, Cambridge, UK). An Apex 1 octadecylsilica analytical column (4.6 mm id/150 mm) protected by a Spherosorb ODS 2 (4.6 mm id/20 mm) guard column, with packing material of 5 a m was used (Jones Chromatography Ltd, Hengoed, Mid-Glamorgan, UK). Chloroform extracts of EDTA plasma were prepared according to Dawson et al. (1986). Extracts were dried under a stream of nitrogen and derivatized to produce p-bromophenacyl fatty acid esters using a modification of the method of Lim (1986). Heptadecanoic acid (margaric acid) was used as an internal standard. Ultra-violet absorption was measured at 260 nm using a mobile phase consisting of methanol-acetonitrile-water (81:9:10) at a flow rate of 1.5 ml min 1. The concentrations of FFA~ were calculated by comparison of sample peak heights to those of external standards of each acid. Single aliquots of each sample were analysed for FFA~. The within-day and between-day coefficients of variation for the HPLC method for FF& were 1.3 and 2.3% respectively. Plasma glucose was analysed on a Kone Specific auto analyser (Labmedics, Stockport, Cheshire). Statistical analysis The changes in FFA, and glucose observed were analysed by analysis of variance for repeated measures using the Fisher's protected least significant difference (PLSD) test. The FFA, data was then divided into two periods 1700-0100 h, 0100-1600 h (period 1) and 0200-0900 h (period 2) inclusive. This division was made on arbitrary grounds following analysis of the data by analysis of variance and of Fig. 2. A pooled estimate of the within-horse variance ~ was calculated using data from period 1 over the x~ values for each horse according to the following equation (Wonnacott & Wonnacott, 1972): ttt
=
(:qi- :~)z i=1 j=l
(r~- I) i=1
The estimate of ~ was subsequently used to calculate confidence limits about individual horse means during the same period using
(r~. - 1) degrees of freedom. i=1
RESULTS
Blood glucose concentration fluctuated throughout the 24 h sampling period. Analysis of variance showed a significant effect of time (P<0.01). Glucose concentration was significantly increased from 0800-1000 h, at 1400 h and 1600 h, and 2000 h (P<0.01), (Fig. 1). Glucose concentration was at its highest 1.5 h following the first feed (0730 h); 3.5 h following the second feed (1230 h) and 3.5 h follow-
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BRITISH VETERINARYJOURNAl,, 150. ,!
ing the final feed (1630 h). G l u c o s e h o m e o s t a s i s was m a i n t a i n e d overnight. Ghtcose c o n c e n t r a t i o n i m m e d i a t e l y b e f o r e the first feed was n o t significantly d i f f e r e n t f r o m the c o n c e n t r a t i o n at 1700 h (P<0.01). T h e p e r c e n t a g e c o n t r i b u t i o n o f FFA~ to the total c o n c e n t r a t i o n was calculated. As s h o w n in Fig. 2, the m o s t a b u n d a n t FFA~ was palmitic acid (C16:0). At 1700 h, the n e x t m o s t a b u n d a n t acids were linoleic (C18:2), oleic (C18:1), stearic (C18:0), a n d linolenic (C18:3) in d e c r e a s i n g o r d e r o f m a g n i t u d e . T o g e t h e r these five acids c o n s t i t u t e d 94.5% o f the total c o n c e n t r a t i o n a n d individually 26.1, 24.9, 19.2, 17.0 a n d 7.3% respectively at 1700 h. O t h e r FFA i n c l u d e d myristic a n d palmitoleic acids b o t h o f w h i c h individually c o n s t i t u t e d less t h a n 5% o f the total. T h e pre-
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"'"....,,,. ......................... .
5.0 -. ..........' 4.5
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7001900 21002300 01000300 05000700 09001100 13001500 Time (h) Fig. 1. Variation in plasma glucose concentration (retool -1, mean+_sn) over 24 h in ll horses. Using Fisher's protected least significant difference test the glucose concentration at 0900 h was greater than at time points marked a, b, c or d (P<0.01); at 1000 and 2000 h was greater than at time points marked b, c or d (15<0.01); and at 1600 h was greater than at the time point marked d (P<0.01).--Q--mean; ..... mean+st~; ..... mean-sD 35 25 20@ 15 5-
0170019002100230001000300050007000900110013001500 Time (h) a Lin01enic zx Stearic
• Oleie • Linoleic •Myristic • Palmitoleic o Palmitic
Fig. 2. Variation in the mean percentage contribution of individual FFA to the total concentration over 24 h.
PLASMA FREE FATTY ACIDS
343
70 00
~- 50 1
Feed 2
Feed 3
a~ 3o 9.0 10
i i i i i i i 1700190021009.3000100030005000700
i
i i i i 0900110013001500
Time (h)
Fig. 3. Variation in total plasma FFA (mean, gmol I-I, C>14) over 24 h in 11 horses. Using Fisher's protected least significant difference test the total FFA concentration at 0700 h was greater than that at the ti,ne points marked a, b or c (P<0.001); at 0500 h was greater than at the time points marked a or b (P<0.001); and at 0600 h was greater than at the time points marked a (P<0.001 ).
d o m i n a n c e o f the main FFA was m a i n t a i n e d over tlle 24 h, however, there were small fluctuations ill their respective c o n t r i b u t i o n to the total c o n c e n t r a t i o n . Between 0400 a n d 1000 h t h e r e was an increase in the p e r c e n t a g e c o n t r i b u t i o n o f oleic acid to the total c o n c e n t r a t i o n with s i m u l t a n e o u s d e c r e a s e in that o f stearic acid. FFA, s h o w e d m i n i m a l variation d u r i n g p e r i o d 1 (Fig. 3). Mlalysis o f variance s h o w e d a significant effect o f time (P<0.001) a n d revealed a p e r i o d in which FFA, c o n c e n t r a t i o n was significantly elevated (P<0.001). T e n o u t o f the 12 horses exhibited all increase in FFA, in tile early h o u r s o f tile m o r n i n g (Fig. 4). T h e increase was localized a r o u n d 0700 h a n d r e p r e s e n t e d a m e a n 4.5-fold increase ( r a n g e 2.0-8.5) above individual horse m e a n s over p e r i o d 1. C h a n g e s in FFA~ c o n c e n trations s h o w e d tile same t r e n d as that o f FFA, (Table 1, Fig. 3). T h e m a g n i t u d e a n d tile d u r a t i o n o f the elevation in FFA, varied b e t w e e n horses (Fig. 4). Analysis o f variance indicated that the inean FFA, c o n c e n t r a t i o n at 0500, 0600 a n d 0700 h were significantly i n c r e a s e d (P<0.001). This statistical analysis, however, implies that all horses s h o w e d an increase a n d d o e s n o t illustrate tile variation b e t w e e n horses. For this reason fllrther statistical analysis was c a r r i e d o u t as d e s c r i b e d previously. T h e estimated within subject variance for p e r i o d 1 was 4.5/2mol I-'. T e n o u t o f the 12 horses studied s h o w e d a rise in FFA, I)etween 0200 a n d 1000 h. In all these horses the p e a k c o n c e n t r a t i o n e x c e e d e d the 99% c o n f i d e n c e limits calculated a b o u t the individual h o r s e m e a n s d u r i n g p e r i o d 1, i.e. :~ll,,.~,,,, 1~, a n d in seven o u t o f the 10 horses the p e a k c o n c e n t r a t i o n e x c e e d e d the 99.9% c o n f i d e n c e limits. In five o u t o f 10 horses the p e a k c o n c e n t r a t i o n r e m a i n e d outside the 99.9% confid e n c e limits for f o u r o r m o r e consecutive hours. T h e increases in FFA, o b s e r v e d were m u c h g r e a t e r than c o u l d be a c c o u n t e d for by r a n d o m variation r e c o r d e d d u r i n g p e r i o d 1.
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BRITISH VETERINARYJOURNAL, 150, 4 200
A --~Bd -o- H e Mr Bo - ~ G1 -o- Co
150 o
I00
0 200 - -
15o
_
7._ ioo
B
/I
--o- H o
+
L~
II ~\ l
+
Li
l V /11
-
50 0 I I I I I I I I I I I 1700 1900 2100 2300 0100 0300 0~00 5 0700 0900 1100 1300 1500 Timc (h) Fig. 4. Variation in total FFA (].tmol/I) in individual horses. Graph (A) shows the six horses which showed the smallest increase in total FFA in the early hours of the morning. Graph (B) shows the five horses which showed the largest increase in free fatty acids during the early hours of the morning. Session 1 (Sn, Bd, He, Lb and Mr); session 2 (Co, Bo and GI); session 3 (HI, Pn and Ls).
T h e d a t a f r o m the twelfth h o r s e (Kj) has n o t b e e n i n c l u d e d in the statistical analysis. Difficulties were e x p e r i e n c e d d u r i n g c a t h e t e r i z a t i o n a n d the FFA, a n d FFA~ f o r this h o r s e were elevated for the first 12 h o f sampling. T h e increase in FFA c o n c e n t r a t i o n following c a t h e t e r i z a t i o n p r o b a b l y resulted f r o m a d r e n a l i n release. A d r e n a l i n stimulates a d i p o s e tissue lipolysis a n d this c o u l d explain the increases in p l a s m a FFA o b s e r v e d in this h o r s e ( A n d e r s o n & Aitken, 1977).
DISCUSSION D u r i n g n o r m a l f e e d i n g the m o s t a b u n d a n t FFA f o u n d in this g r o u p o f horses were palmitic, linoleic, oleic, stearic a n d linolenic acids in d e c r e a s i n g o r d e r o f m a g n i tude. L u t h e r et al. (1981) r e p o r t e d that palmitic, oleic, stearic a n d myristic acids were the m o s t a b u n d a n t FFA in e q u i n e p l a s m a in o r d e r o f o c c u r r e n c e , constituting 96.1% o f the total p l a s m a c o n c e n t r a t i o n . In contrast, Rose a n d
PLASMA FREE FATTYACIDS
345
Table I Individual FFA concentrations ( m e a n + s v , / a m m o l 1-~) over 24 h in 11 horses Time (h)
Linolenic
Myristic
1700 1800 1900 2000 2100 2200 2300 2400 0100 0200 0300 0400 0500 0600 0700 0800 0900 1000 1100 1200 1300 1400 1500 1600
2.2_+2.0 1.8_+1.0 1.7_+1.1 1.9_+1.3 1.4_+0.8 1.6_+1.1 1.4_+1.0 1.5_+0.8 1.5_+1.1 1.8+1.3 2.3+2.1 2.3_+1.6 4.4_+3.5 4.4_+4.1 6.5+5.2 2.8+1.4 2.5_+1.8 1.7_+0.7 1.3_+0.6 1.6_+1.0 1.4+0.7 1.6+1.6 1.3+0.9 1.3_+0.7
1.0_+0.6 0.7_+0.4 0.7_+0.3 0.8_+0.2 0.7_+0.4 0.7_+0.3 0.8_+0.3 0.8_+0.3 0.7_+0.4 0.8-+0.3 0.9_+0.4 1.0_+0.4 1.9_+2.0 1.5_+0.9 1.8_+1.2 1.0_+0.7 1.1_+0.9 0.9_+0.4 0.5_+0.4 0.7+0.3 0.8_+0.8 0.7-+0.5 0.8_+0.4 0.9_+0.3
Palmitoleic
Linoleic
Palmitic
Oleic
0.6_+0.6 6.0_+2.0 6.7_+3.4 5.2+2.8 0.5_+0.8 5.9_+1.2 6.2_+2.7 4.3+1.2 0.3_+0.3 5.4+1.4 5.7_+2.3 3.6_+1.2 0.4_+0.8 5.6+1.5 5.4_+1.5 3.8_+0.8 0.4_+0.4 5.3+1.2 6.3_+2.1 3.9_+0.8 0.4_+0.4 5.3_+1.1 5.6_+1.3 3.4_+0.8 0.3_+0.3 5.5_+1.4 5.6+1.4 3.8_+1.1 0.3_+0.5 5.1_+0.7 5.6+1.0 3.6_+0.7 0.3_+0.3 5.6_+1.5 6.3+1.8 4.2_+1.7 0.3+0.3 6.3_+2.5 7.2-+3.2 5.2_+2.9 0.5_+0.5 6.9_+4.1 7.6_+4.2 5.9_+4.3 0.6_+0.6 7.0+3.4 7.9_+4.0 6.0_+3.7 2.0_+2.5 14.5+11.4 15.6_+12.1 13.5_+11.6 1.3_+1.3 12.8+8.2 13.9_+9.8 13.1_+9.7 1.8-+1.5 16.7_+10.5 17.5-+12.1 16.3_+12.4 0.6+0.7 8.1_+4.4 8.4_+3.9 7.0_+4.0 1.0+1.5 9.3_+9.7 9.9+9.7 8.6__.11.2 0.4+0.4 5.8-+1.9 6.1+2.9 4.4-+2.8 0.1+0.2 4.7_+1.0 5.0-+1.0 3.3_+0.5 0.1_+0.3 5.6_+2.8 5.4_+1.6 3.8_+2.1 0.2-+0.4 4.9+1.8 5.0_+1.2 3.4_+1.2 0.2_+0.4 5.8+3.6 5.8_+4.5 4.2-+4.2 0.4_+0.3 8.0+5.6 6.1_+2.7 4.2_+2.2 0.4_+0.4 4.7_+1.4 5.8_+2.2 3.5_+0.8
SteaTic
4.3_+1.8 3.9_+1.7 4.1_+2.3 3.7+1.0 4.7+2.6 3.8_+1.0 3.8_+0.9 3.6_+0.9 4.2+0.7 4.6-+1.4 4.8_+1.7 4.5_+1.0 6.4_+2.3 6.9_+3.7 8.0-+4.0 5.4_+2.2 5.1_+3.3 4.5_+1.8 3.4_+0.8 3.5+0.8 3.7+1.1 3.8+2.0 5.2+2.7 4.6+2.7
Using Fisher's protected least significant difference test FFA concentration was significantly different (P<0.05) at any given time when the numerical difference between two sample times exceeded 1.5, 0.6, 0.7, 3.9, 4.2, 4.2 or 1.7 /.tmol I-I for linolenic, myristic, palmitoleic palmitic, oleic or stearic acids respectively. Sampson 1982) r e p o r t e d that d u r i n g exercise and food deprivation the predominant FFA in o r d e r of o c c u r r e n c e were oleic, palmitic, linoleic and linolenic which together constituted 88% o f the total. T h e FFA~ profile o f plasma in non-ruminants is influenced by diet and will therefore vary according to the composition o f the diet. S h o r l a n d et al. (1952) r e p o r t e d that the adipose tissue o f grass-fed horses contains 17% linolenic acid and 4% linoleic acid as c o m p a r e d with 2% linolenic acid and 22% linoleic acid f o u n d in the adipose tissue of horses fed oats. These variations agree with the fact that grass lipids consist p r e d o m i n a n t l y of linolenic acid, whereas the lipids of oats are rich in linoleic acid (Shorland, 1962). It is likely that the differences in the plasma FFA profile observed between these studies probably reflect differences in the respective diets o f the animals. Blood glucose concentrations showed a significant increase between 1.5-3.5 h post feeding. Plasma glucose c o n c e n t r a t i o n has been previously r e p o r t e d to peak between 4-8 h following a meal (Frape, 1986). T h e time at which a peak in blood glucose following a meal is observed will d e p e n d on the nature o f the carbohydrate c o m p o n e n t o f the diet i.e., the relative a m o u n t s of complex carbohydrate to simple sugars, since the latter are digested and absorbed m u c h m o r e rapidly. T h e observed peaks in plasma glucose c o n c e n t r a t i o n following all feeds in this
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study may have been slightly earlier than previously reported due to differences in the nature of the carbohydrate portion of the diet. T h e sharp rise in FFA, observed in the early hours of the m o r n i n g was mucla greater than could be a c c o u n t e d for by r a n d o m variation r e c o r d e d during period 1. It is possible that the increase in FFA, was the result of a reduction in plasma glucose concentration d u r i n g the night. In man it has been r e p o r t e d that mobilization o f FFA can be elicited by a fall in blood glucose concentration o f 25% or more (Newsholme & Leech, 1983). Figure 1, however, shows that an adequate supply of forages ensured that glucose homeostasis was maintained overnight thus eliminating a fall in the plasma glucose concentration as the cause o f the increase in FFA, in the early hours o f the morning. Forages which are rich in fibre e x t e n d the absorptive period. Horses, although essentially non-ruminants have the ability to digest structm'al carbohydrates such as celhdose and hemicellulose in the large intestine, mainly the caecum. Symbiotic bacterial colonization of the caecum enables their digestion with the subsequent p r o d u c t i o n of volatile fatty acids (VFA). These VFA provide a substantial energ3, source which ensures that glucose homeostasis is not c o m p r o m i s e d overnight ( H i n ~ et al., 1971). An increased plasma cortisol concentration may have had a secondary effect on plasma FFA. Cortisol increases the sensitivity of the adipose tissue to the lipolytic h o r m o n e s so that for a given concentration o f h o r m o n e , the rate o f lipolysis is laigher in the presence of cortisol (Fernandez & Saggerson, 1978). Cortisol has been reported to exhibit a diurnal rhythm in the horse, its concentration reaching a maximuln in the early hours of the m o r n i n g prior to waking (Hoffsis et al., 1970; Larsson et al., 1979). T h e time at wlaich the peak in cortisol concentration is reported to occur, however, varies. Larsson et al. (1979) r e p o r t e d that the peak o c c u r r e d at 0600 h and Hoffsis et al. (1970) at 0800 h. In humans, cortisol has a latent effect on fat mobilization, the response taking several hours to be elicited (Guyton, 1992). If cortisol has a latent effect on fat mobilization in horses it is unlikely that it was responsible for the a f o r e m e n t i o n e d increase in FFA obsei'ved in this study. At 0500 h there was already a significant increase in plasma FFA in most o f the horses, which if due to an earlier rise in cortisol would have m e a n t an increase in cortisol at about 0300 h. Cortisol c a n n o t be dismissed as the cause of the increase in FFA since it was not lueasured. T h e energy mobilizing actions of the lipolytic hormones, either in the presence of cortisol or not, were probably responsible for the increase in FFA, in preparation for the increased energy d e m a n d s o f wakefidness. Stavvation, sustained exercise, stress and hypoglycaemia are all situations which result in an elevation in plasma FFA due to an increase in their rate of mobilization. H o r m o n e sensitive or adipose triglyceride lipase catalyses the flux generating step in the release of FFA fi-otn adipose tissue. In man, adrenaline, noradrenaline, thyroid stimulating horm o n e and parathyroid h o r m o n e are all lipolytic h o r m o n e s (Newsholme et al., 1983). T h e glucocorticoids, cortisol and thyroxin have a secondary effect on lipolysis. It can be c o n c l u d e d from this study that a percentage of horses show an early m o r n i n g increase in FFA which c a n n o t be explained by normal variation throughout the rest of the day. T h e cause of this increase is unknown and requires further investigation.
PLASMA FREE FATTYACIDS
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ACKNOWLEDGEMENTS T h e authors gratefully acknowledge the part f u n d i n g of this study by Dalgety Agriculture (Spiller's speciality feeds, Bristol) and are indebted to J o a n n e Keeley for her veterinary assistance.
REFERENCES ANDERSON, M. G. & AITKEN,M. M. (1977). Biochemical and physiological effects of catecholamine adminisla-ation in the horse. Research in Veterinary Science22, 357-60. BACHORm,P. S. (1982). Collection of blood samples for lipoprotein analysis. Clinical Chemi.~try 28, 1375-8. DAWSOY, R. M. C., ELLWOT,D. C., EWOT, W. H. & JONES, K. M. (1986). Biochemical procedures. In Data for Biochemical Research, 3rd edn. p. 551. New York: Oxford University Press. FEm~ANDEZ,B. M. &SAC,GERSO~,E. D. (1978). Alterations in response of rat white adipocytes to insulin, nor-adrenalin, corticotropin and glucagon after adrenalectomy. Biochemical • Journal 174, 111-18. FP..xP~:, D. (1986). The utilization of absorbed nutrients for maintenance, work and growth. In Equine Nutrition and Feeding, ed. D. Frape, p. 21. New York: Churchill Livingstone. GLI-'ESON, (1987). Effect of heparin and storage on human plasma free fatty acid concentration. Clinica Chimica Acta 169, 315-18. Guvro,~, A. C. (1991). Textbook of Medical Physiology, 8th edn. pp. 847-8. Philadelphia: W. B. Saunders. Hls'rz, H. T., ARGENZIO,R. A. & S('HR'IVER,H. F. (1971). Digestion coefficients, blood glucose levels, and molar percentage of volatile acids in intestinal fluid of ponies fed varying forage-grain ratios. Journal of Animal Science 33, 992. Howsls, G. F., MVRDXCK,P. W., TH.~PE, V. L. & AULT, K. (1970). Plasma concentrations of cortisol and corticosterone in the normal horse. American Journal of Vetelqnary Research 31, 1379-87. LARSSON,M., EDq~qs-r, L. E., EmtaN, L. & PERSSON,S. (1979). Plasma cortisol in the horse, diurnal rhythm and effects of exogenous ACTH. Acta Vetennaria Scandinavica 20, 16-24. LIM, C. K. (1986). Lipids. In HPLC of SmaU Molecules, A Practical Approach. pp. 69-102. Oxford: IRL press. Lu'rH~:R, D. G., Cox, H. U. & DI,~IOPOULLOS,G. T. (1981). Fatty acid composition of equine plasma. American Journal of Veterinary Research 42, 91-3. NI:.WSHOLME,E. A. & LEI':CH,A. R. (1983). Integration of carbohydrate and lipid metabolism. In Biochemistry for the Medical .Sciences. pp. 336-55. Chichester:John Wiley & Sons Ltd. R.~xvussiy, E. et al. (1986). Effect of elevated FFA on carbohydrate and lipid oxidation during prolonged exercise in humans.Journal of Applied Physiolog), 60, 893-900. SHORmND, F. B. (1962). The comparative aspects of fatty acid occurrence and distribution. In Comparative Biochemist13, Volume I11, Constituents of Life - Part A, eds M. Floorkin & H. S. Mason, pp. 1-102. New York: Academic Press. SHORL.XNt), F. B., BRUt:E, L. W. &JE~oP, A. W. (1952). The component fatty acids of lipids fi'om fatty tissues, muscle and liver. Biochemical Journal 52, 400-7. W,~'~N,.xcorr, T. H. & WANNACOlq',R.J. (1972). h~troductmy Statistic.s, 2nd edn. pp. 221. New York: Wiley International. ZtLVA,J. F. & PARNALL,P. R. (1984). Clinical Chemistry in DiagTwsis and Treatment. London: Lloyd-Luke (Medical Books) Ltd.
(Acceptedfor publication 28 October1993)
VARIATION IN THE CONCENTRATION OF LONG C H A I N FREE F A T T Y A C I D S I N E Q U I N E P L A S M A O V E R 24 H O U R S
C. E. ORME, M. DUNNETT and R. C. HARRIS Departnwnt of Physiology, Animal Health Trust, P.O. Box 5, Newmarket, Suffolk CB8 7DW, UK
SUMMARY The primary aim of this study was to examine the within-day variation in the concentration of total and individual long chain free fatty acids (C> 14) in normally fed horses. Plasma samples were collected over a 24 h period from 12 resting horses during three separate sessions (six horses in the first session and three in the second and third). Samples were analysed for individual long chain free fatty acids (FFA) and glucose. During normal feeding, the predominant FFA in plasma were palmitic (C16:0), linoleic (C18:2), oleic (C18:1), stearic (C18:0) and linolenic (C18:3). Together these acids constituted over 90% of the total concentration. Other FFA present were myristic (C14:0) and palmitoleic (C16:1) both of which constituted <5% of the total concentration. Ten out of the 12 horses sampled exhibited an early morning increase in FFA (P<0.001) localized around 0700 h and which was independent of feeding. The mean concentration of total FFA increased 4.5 fold (range 2.0-8.5) between 0400 and 1000 h. The predominant FFA showed the largest increase. KE~WORDS:Horse; free fatty acids; plasma.
INTRODUCTION In man and other species certain biochemical parameters and hormones exhibit regular variations throughout the day. These changes may be related to food intake, e.g. glucose in man, or may be independent of feeding and regulated by some other means, e.g. cortisol, growth hormone and iron (Zilva & Pannall, 1984). The day-night cycle usually imposes a diurnal rhythm on the feeding behaviour of an animal, sufficient fuel being stored during the active feeding period to provide for the metabolic demands of the period of sleep. Resting concentrations of plasma free fatty acids (FFA) influence the rate of FFA oxidation 0007/1935/94/040339-09/$08.00/0
© 1994Bailli&eTindall
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during subsequent exercise (Ravussin et al., 1986). As part of a wider investigation of the utilization of FFA as a fuel during exercise in the horse, it was necessary to ensure that studies would noi coincide with any diurnal increase in plasma FFA concentration. No previous investigations describing within-day variation in the concentration of long chain FFA in equine plasma could be found in the literature.
MATERIALS A N D M E T H O D S
The within day variation of total long chain (C>14) free fatty acids (FFA,) and individual long chain free fatty acids (FFA~) in equine plasma were measured over a 24 h period during three experimental sessions. Six horses designated Sn, Bd, He, Kj, Mr and Lb were sampled during the first session and three each during sessions 2 (Bo, Co and GI) and 3 (HI, Pn and Ls). All horses were fed normally and were not exercised during the study. The horses used consisted of eight geldings (Sn, Bd, He, Kj, Mr, Bo, Co and Ls) and four fillies (Lb, G1, HI, and Pn) weighing between 400-500 kg. Prior to the start of the study three of the horses (Kj, Mr and He) had been out at grass with little or no work whilst the remaining nine were boxed and in moderate work. Eleven of the 12 horses were fed Spillers Stud cubes (approximately 2 kg) and the twelfth, Bd, Spillers racehorse cubes (approximately 2 kg). Horses were fed at 0700, 1230 and 1630 h. The oil content of the cubed portion of the diets were 4.25 and 4.0% by fresh weight respectively. All horses were fed 2-3 kg of hay with the morning and evening feeds. Water was available ad libitum at all times. Venous blood samples were drawn at hourly intervals starting at 1700 h via a 13G catheter (Vygon, Benkat Instruments, UK) inserted into the left jugular vein under local anaesthesia. The horses were catheterized a minimum of 1 h before the first sample was take. Blood was dispensed into tubes containing Ethylenediamine tetracetic acid (EDTA) as an anticoagulant and centrifuged immediately. The plasma was aspirated and stored at -20°C for analysis of FFA,, FFAi and glucose. Concentrations of FFA~were measured by high performance liquid chromatography (HPLC). FFA, concentrations were calculated by the addition of FFA~ concentrations. EDTA was used as an alaticoagnlant because of the reported elevation in plasma FFA concentrations during storage of lithium heparin treated samples (Gleeson, 1987). EDTA inhibits the action of bacterial lipases and prevents the auto oxidation of FFA (Bachorik, 1982). Chemicals Methanol and acetonitrile (HPLC grade) were supplied by Romil Chemicals Ltd (Shepshed, Loughborough, Leicestershire, UK); ethanol was supplied by James Burrough (F.A.D.) Ltd (Witham, Essex, UK) and chloroform by Interchem (UK); potassium hydroxide was supplied by B.D.H. chemicals (Poole, Dorset, UK); 1,4,7,10,13,16,hexaoxacyclooctadecane (18 crown 6) by Aldrich Chemical Co. Ltd (Gillingham, Dorset, UK), 4 bromophenacyl bromide, glucose and all FFA standards by Sigma Chemical Co. (Poole, Dorset, UK).
PLASMA FREE FATTY ACIDS
341
Analysis Plasma samples were analysed .for FF& using a Constametric 1 HPLC pump (LCD/Milton Roy, Stone, Staffordshire, UK), with a rheodyne 7125 injector and 50/11 loop (Rheodyne, Cotati, CA, USA) and LC-UV variable wavelength ultraviolet spectrophotometric detector (Phillips analytical, Cambridge, UK). An Apex 1 octadecylsilica analytical column (4.6 mm id/150 mm) protected by a Spherosorb ODS 2 (4.6 mm id/20 mm) guard column, with packing material of 5 a m was used (Jones Chromatography Ltd, Hengoed, Mid-Glamorgan, UK). Chloroform extracts of EDTA plasma were prepared according to Dawson et al. (1986). Extracts were dried under a stream of nitrogen and derivatized to produce p-bromophenacyl fatty acid esters using a modification of the method of Lim (1986). Heptadecanoic acid (margaric acid) was used as an internal standard. Ultra-violet absorption was measured at 260 nm using a mobile phase consisting of methanol-acetonitrile-water (81:9:10) at a flow rate of 1.5 ml min 1. The concentrations of FFA~ were calculated by comparison of sample peak heights to those of external standards of each acid. Single aliquots of each sample were analysed for FFA~. The within-day and between-day coefficients of variation for the HPLC method for FF& were 1.3 and 2.3% respectively. Plasma glucose was analysed on a Kone Specific auto analyser (Labmedics, Stockport, Cheshire). Statistical analysis The changes in FFA, and glucose observed were analysed by analysis of variance for repeated measures using the Fisher's protected least significant difference (PLSD) test. The FFA, data was then divided into two periods 1700-0100 h, 0100-1600 h (period 1) and 0200-0900 h (period 2) inclusive. This division was made on arbitrary grounds following analysis of the data by analysis of variance and of Fig. 2. A pooled estimate of the within-horse variance ~ was calculated using data from period 1 over the x~ values for each horse according to the following equation (Wonnacott & Wonnacott, 1972): ttt
=
(:qi- :~)z i=1 j=l
(r~- I) i=1
The estimate of ~ was subsequently used to calculate confidence limits about individual horse means during the same period using
(r~. - 1) degrees of freedom. i=1
RESULTS
Blood glucose concentration fluctuated throughout the 24 h sampling period. Analysis of variance showed a significant effect of time (P<0.01). Glucose concentration was significantly increased from 0800-1000 h, at 1400 h and 1600 h, and 2000 h (P<0.01), (Fig. 1). Glucose concentration was at its highest 1.5 h following the first feed (0730 h); 3.5 h following the second feed (1230 h) and 3.5 h follow-
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BRITISH VETERINARYJOURNAl,, 150. ,!
ing the final feed (1630 h). G l u c o s e h o m e o s t a s i s was m a i n t a i n e d overnight. Ghtcose c o n c e n t r a t i o n i m m e d i a t e l y b e f o r e the first feed was n o t significantly d i f f e r e n t f r o m the c o n c e n t r a t i o n at 1700 h (P<0.01). T h e p e r c e n t a g e c o n t r i b u t i o n o f FFA~ to the total c o n c e n t r a t i o n was calculated. As s h o w n in Fig. 2, the m o s t a b u n d a n t FFA~ was palmitic acid (C16:0). At 1700 h, the n e x t m o s t a b u n d a n t acids were linoleic (C18:2), oleic (C18:1), stearic (C18:0), a n d linolenic (C18:3) in d e c r e a s i n g o r d e r o f m a g n i t u d e . T o g e t h e r these five acids c o n s t i t u t e d 94.5% o f the total c o n c e n t r a t i o n a n d individually 26.1, 24.9, 19.2, 17.0 a n d 7.3% respectively at 1700 h. O t h e r FFA i n c l u d e d myristic a n d palmitoleic acids b o t h o f w h i c h individually c o n s t i t u t e d less t h a n 5% o f the total. T h e pre-
~8.5
"7
..... .....
~ 6 0
.
.
0
..'
~
5.5 ,,..'"''..,.,.,"'...,..*'""
"'"....,,,. ......................... .
5.0 -. ..........' 4.5
I
I
,.'.,..~o.."
"..................../ I
I
I
I
I
I
I
I
I
I
7001900 21002300 01000300 05000700 09001100 13001500 Time (h) Fig. 1. Variation in plasma glucose concentration (retool -1, mean+_sn) over 24 h in ll horses. Using Fisher's protected least significant difference test the glucose concentration at 0900 h was greater than at time points marked a, b, c or d (P<0.01); at 1000 and 2000 h was greater than at time points marked b, c or d (15<0.01); and at 1600 h was greater than at the time point marked d (P<0.01).--Q--mean; ..... mean+st~; ..... mean-sD 35 25 20@ 15 5-
0170019002100230001000300050007000900110013001500 Time (h) a Lin01enic zx Stearic
• Oleie • Linoleic •Myristic • Palmitoleic o Palmitic
Fig. 2. Variation in the mean percentage contribution of individual FFA to the total concentration over 24 h.
PLASMA FREE FATTY ACIDS
343
70 00
~- 50 1
Feed 2
Feed 3
a~ 3o 9.0 10
i i i i i i i 1700190021009.3000100030005000700
i
i i i i 0900110013001500
Time (h)
Fig. 3. Variation in total plasma FFA (mean, gmol I-I, C>14) over 24 h in 11 horses. Using Fisher's protected least significant difference test the total FFA concentration at 0700 h was greater than that at the ti,ne points marked a, b or c (P<0.001); at 0500 h was greater than at the time points marked a or b (P<0.001); and at 0600 h was greater than at the time points marked a (P<0.001 ).
d o m i n a n c e o f the main FFA was m a i n t a i n e d over tlle 24 h, however, there were small fluctuations ill their respective c o n t r i b u t i o n to the total c o n c e n t r a t i o n . Between 0400 a n d 1000 h t h e r e was an increase in the p e r c e n t a g e c o n t r i b u t i o n o f oleic acid to the total c o n c e n t r a t i o n with s i m u l t a n e o u s d e c r e a s e in that o f stearic acid. FFA, s h o w e d m i n i m a l variation d u r i n g p e r i o d 1 (Fig. 3). Mlalysis o f variance s h o w e d a significant effect o f time (P<0.001) a n d revealed a p e r i o d in which FFA, c o n c e n t r a t i o n was significantly elevated (P<0.001). T e n o u t o f the 12 horses exhibited all increase in FFA, in tile early h o u r s o f tile m o r n i n g (Fig. 4). T h e increase was localized a r o u n d 0700 h a n d r e p r e s e n t e d a m e a n 4.5-fold increase ( r a n g e 2.0-8.5) above individual horse m e a n s over p e r i o d 1. C h a n g e s in FFA~ c o n c e n trations s h o w e d tile same t r e n d as that o f FFA, (Table 1, Fig. 3). T h e m a g n i t u d e a n d tile d u r a t i o n o f the elevation in FFA, varied b e t w e e n horses (Fig. 4). Analysis o f variance indicated that the inean FFA, c o n c e n t r a t i o n at 0500, 0600 a n d 0700 h were significantly i n c r e a s e d (P<0.001). This statistical analysis, however, implies that all horses s h o w e d an increase a n d d o e s n o t illustrate tile variation b e t w e e n horses. For this reason fllrther statistical analysis was c a r r i e d o u t as d e s c r i b e d previously. T h e estimated within subject variance for p e r i o d 1 was 4.5/2mol I-'. T e n o u t o f the 12 horses studied s h o w e d a rise in FFA, I)etween 0200 a n d 1000 h. In all these horses the p e a k c o n c e n t r a t i o n e x c e e d e d the 99% c o n f i d e n c e limits calculated a b o u t the individual h o r s e m e a n s d u r i n g p e r i o d 1, i.e. :~ll,,.~,,,, 1~, a n d in seven o u t o f the 10 horses the p e a k c o n c e n t r a t i o n e x c e e d e d the 99.9% c o n f i d e n c e limits. In five o u t o f 10 horses the p e a k c o n c e n t r a t i o n r e m a i n e d outside the 99.9% confid e n c e limits for f o u r o r m o r e consecutive hours. T h e increases in FFA, o b s e r v e d were m u c h g r e a t e r than c o u l d be a c c o u n t e d for by r a n d o m variation r e c o r d e d d u r i n g p e r i o d 1.
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A --~Bd -o- H e Mr Bo - ~ G1 -o- Co
150 o
I00
0 200 - -
15o
_
7._ ioo
B
/I
--o- H o
+
L~
II ~\ l
+
Li
l V /11
-
50 0 I I I I I I I I I I I 1700 1900 2100 2300 0100 0300 0~00 5 0700 0900 1100 1300 1500 Timc (h) Fig. 4. Variation in total FFA (].tmol/I) in individual horses. Graph (A) shows the six horses which showed the smallest increase in total FFA in the early hours of the morning. Graph (B) shows the five horses which showed the largest increase in free fatty acids during the early hours of the morning. Session 1 (Sn, Bd, He, Lb and Mr); session 2 (Co, Bo and GI); session 3 (HI, Pn and Ls).
T h e d a t a f r o m the twelfth h o r s e (Kj) has n o t b e e n i n c l u d e d in the statistical analysis. Difficulties were e x p e r i e n c e d d u r i n g c a t h e t e r i z a t i o n a n d the FFA, a n d FFA~ f o r this h o r s e were elevated for the first 12 h o f sampling. T h e increase in FFA c o n c e n t r a t i o n following c a t h e t e r i z a t i o n p r o b a b l y resulted f r o m a d r e n a l i n release. A d r e n a l i n stimulates a d i p o s e tissue lipolysis a n d this c o u l d explain the increases in p l a s m a FFA o b s e r v e d in this h o r s e ( A n d e r s o n & Aitken, 1977).
DISCUSSION D u r i n g n o r m a l f e e d i n g the m o s t a b u n d a n t FFA f o u n d in this g r o u p o f horses were palmitic, linoleic, oleic, stearic a n d linolenic acids in d e c r e a s i n g o r d e r o f m a g n i tude. L u t h e r et al. (1981) r e p o r t e d that palmitic, oleic, stearic a n d myristic acids were the m o s t a b u n d a n t FFA in e q u i n e p l a s m a in o r d e r o f o c c u r r e n c e , constituting 96.1% o f the total p l a s m a c o n c e n t r a t i o n . In contrast, Rose a n d
PLASMA FREE FATTYACIDS
345
Table I Individual FFA concentrations ( m e a n + s v , / a m m o l 1-~) over 24 h in 11 horses Time (h)
Linolenic
Myristic
1700 1800 1900 2000 2100 2200 2300 2400 0100 0200 0300 0400 0500 0600 0700 0800 0900 1000 1100 1200 1300 1400 1500 1600
2.2_+2.0 1.8_+1.0 1.7_+1.1 1.9_+1.3 1.4_+0.8 1.6_+1.1 1.4_+1.0 1.5_+0.8 1.5_+1.1 1.8+1.3 2.3+2.1 2.3_+1.6 4.4_+3.5 4.4_+4.1 6.5+5.2 2.8+1.4 2.5_+1.8 1.7_+0.7 1.3_+0.6 1.6_+1.0 1.4+0.7 1.6+1.6 1.3+0.9 1.3_+0.7
1.0_+0.6 0.7_+0.4 0.7_+0.3 0.8_+0.2 0.7_+0.4 0.7_+0.3 0.8_+0.3 0.8_+0.3 0.7_+0.4 0.8-+0.3 0.9_+0.4 1.0_+0.4 1.9_+2.0 1.5_+0.9 1.8_+1.2 1.0_+0.7 1.1_+0.9 0.9_+0.4 0.5_+0.4 0.7+0.3 0.8_+0.8 0.7-+0.5 0.8_+0.4 0.9_+0.3
Palmitoleic
Linoleic
Palmitic
Oleic
0.6_+0.6 6.0_+2.0 6.7_+3.4 5.2+2.8 0.5_+0.8 5.9_+1.2 6.2_+2.7 4.3+1.2 0.3_+0.3 5.4+1.4 5.7_+2.3 3.6_+1.2 0.4_+0.8 5.6+1.5 5.4_+1.5 3.8_+0.8 0.4_+0.4 5.3+1.2 6.3_+2.1 3.9_+0.8 0.4_+0.4 5.3_+1.1 5.6_+1.3 3.4_+0.8 0.3_+0.3 5.5_+1.4 5.6+1.4 3.8_+1.1 0.3_+0.5 5.1_+0.7 5.6+1.0 3.6_+0.7 0.3_+0.3 5.6_+1.5 6.3+1.8 4.2_+1.7 0.3+0.3 6.3_+2.5 7.2-+3.2 5.2_+2.9 0.5_+0.5 6.9_+4.1 7.6_+4.2 5.9_+4.3 0.6_+0.6 7.0+3.4 7.9_+4.0 6.0_+3.7 2.0_+2.5 14.5+11.4 15.6_+12.1 13.5_+11.6 1.3_+1.3 12.8+8.2 13.9_+9.8 13.1_+9.7 1.8-+1.5 16.7_+10.5 17.5-+12.1 16.3_+12.4 0.6+0.7 8.1_+4.4 8.4_+3.9 7.0_+4.0 1.0+1.5 9.3_+9.7 9.9+9.7 8.6__.11.2 0.4+0.4 5.8-+1.9 6.1+2.9 4.4-+2.8 0.1+0.2 4.7_+1.0 5.0-+1.0 3.3_+0.5 0.1_+0.3 5.6_+2.8 5.4_+1.6 3.8_+2.1 0.2-+0.4 4.9+1.8 5.0_+1.2 3.4_+1.2 0.2_+0.4 5.8+3.6 5.8_+4.5 4.2-+4.2 0.4_+0.3 8.0+5.6 6.1_+2.7 4.2_+2.2 0.4_+0.4 4.7_+1.4 5.8_+2.2 3.5_+0.8
SteaTic
4.3_+1.8 3.9_+1.7 4.1_+2.3 3.7+1.0 4.7+2.6 3.8_+1.0 3.8_+0.9 3.6_+0.9 4.2+0.7 4.6-+1.4 4.8_+1.7 4.5_+1.0 6.4_+2.3 6.9_+3.7 8.0-+4.0 5.4_+2.2 5.1_+3.3 4.5_+1.8 3.4_+0.8 3.5+0.8 3.7+1.1 3.8+2.0 5.2+2.7 4.6+2.7
Using Fisher's protected least significant difference test FFA concentration was significantly different (P<0.05) at any given time when the numerical difference between two sample times exceeded 1.5, 0.6, 0.7, 3.9, 4.2, 4.2 or 1.7 /.tmol I-I for linolenic, myristic, palmitoleic palmitic, oleic or stearic acids respectively. Sampson 1982) r e p o r t e d that d u r i n g exercise and food deprivation the predominant FFA in o r d e r of o c c u r r e n c e were oleic, palmitic, linoleic and linolenic which together constituted 88% o f the total. T h e FFA~ profile o f plasma in non-ruminants is influenced by diet and will therefore vary according to the composition o f the diet. S h o r l a n d et al. (1952) r e p o r t e d that the adipose tissue o f grass-fed horses contains 17% linolenic acid and 4% linoleic acid as c o m p a r e d with 2% linolenic acid and 22% linoleic acid f o u n d in the adipose tissue of horses fed oats. These variations agree with the fact that grass lipids consist p r e d o m i n a n t l y of linolenic acid, whereas the lipids of oats are rich in linoleic acid (Shorland, 1962). It is likely that the differences in the plasma FFA profile observed between these studies probably reflect differences in the respective diets o f the animals. Blood glucose concentrations showed a significant increase between 1.5-3.5 h post feeding. Plasma glucose c o n c e n t r a t i o n has been previously r e p o r t e d to peak between 4-8 h following a meal (Frape, 1986). T h e time at which a peak in blood glucose following a meal is observed will d e p e n d on the nature o f the carbohydrate c o m p o n e n t o f the diet i.e., the relative a m o u n t s of complex carbohydrate to simple sugars, since the latter are digested and absorbed m u c h m o r e rapidly. T h e observed peaks in plasma glucose c o n c e n t r a t i o n following all feeds in this
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study may have been slightly earlier than previously reported due to differences in the nature of the carbohydrate portion of the diet. T h e sharp rise in FFA, observed in the early hours of the m o r n i n g was mucla greater than could be a c c o u n t e d for by r a n d o m variation r e c o r d e d during period 1. It is possible that the increase in FFA, was the result of a reduction in plasma glucose concentration d u r i n g the night. In man it has been r e p o r t e d that mobilization o f FFA can be elicited by a fall in blood glucose concentration o f 25% or more (Newsholme & Leech, 1983). Figure 1, however, shows that an adequate supply of forages ensured that glucose homeostasis was maintained overnight thus eliminating a fall in the plasma glucose concentration as the cause o f the increase in FFA, in the early hours o f the morning. Forages which are rich in fibre e x t e n d the absorptive period. Horses, although essentially non-ruminants have the ability to digest structm'al carbohydrates such as celhdose and hemicellulose in the large intestine, mainly the caecum. Symbiotic bacterial colonization of the caecum enables their digestion with the subsequent p r o d u c t i o n of volatile fatty acids (VFA). These VFA provide a substantial energ3, source which ensures that glucose homeostasis is not c o m p r o m i s e d overnight ( H i n ~ et al., 1971). An increased plasma cortisol concentration may have had a secondary effect on plasma FFA. Cortisol increases the sensitivity of the adipose tissue to the lipolytic h o r m o n e s so that for a given concentration o f h o r m o n e , the rate o f lipolysis is laigher in the presence of cortisol (Fernandez & Saggerson, 1978). Cortisol has been reported to exhibit a diurnal rhythm in the horse, its concentration reaching a maximuln in the early hours of the m o r n i n g prior to waking (Hoffsis et al., 1970; Larsson et al., 1979). T h e time at wlaich the peak in cortisol concentration is reported to occur, however, varies. Larsson et al. (1979) r e p o r t e d that the peak o c c u r r e d at 0600 h and Hoffsis et al. (1970) at 0800 h. In humans, cortisol has a latent effect on fat mobilization, the response taking several hours to be elicited (Guyton, 1992). If cortisol has a latent effect on fat mobilization in horses it is unlikely that it was responsible for the a f o r e m e n t i o n e d increase in FFA obsei'ved in this study. At 0500 h there was already a significant increase in plasma FFA in most o f the horses, which if due to an earlier rise in cortisol would have m e a n t an increase in cortisol at about 0300 h. Cortisol c a n n o t be dismissed as the cause of the increase in FFA since it was not lueasured. T h e energy mobilizing actions of the lipolytic hormones, either in the presence of cortisol or not, were probably responsible for the increase in FFA, in preparation for the increased energy d e m a n d s o f wakefidness. Stavvation, sustained exercise, stress and hypoglycaemia are all situations which result in an elevation in plasma FFA due to an increase in their rate of mobilization. H o r m o n e sensitive or adipose triglyceride lipase catalyses the flux generating step in the release of FFA fi-otn adipose tissue. In man, adrenaline, noradrenaline, thyroid stimulating horm o n e and parathyroid h o r m o n e are all lipolytic h o r m o n e s (Newsholme et al., 1983). T h e glucocorticoids, cortisol and thyroxin have a secondary effect on lipolysis. It can be c o n c l u d e d from this study that a percentage of horses show an early m o r n i n g increase in FFA which c a n n o t be explained by normal variation throughout the rest of the day. T h e cause of this increase is unknown and requires further investigation.
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ACKNOWLEDGEMENTS T h e authors gratefully acknowledge the part f u n d i n g of this study by Dalgety Agriculture (Spiller's speciality feeds, Bristol) and are indebted to J o a n n e Keeley for her veterinary assistance.
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(Acceptedfor publication 28 October1993)