Abstract:
PREGNANCY TOXAEMIA IN SHEEP - A REVIEW D.B. LINDSAY* and V.H. ODDY Summary -a(1) Origin of the ketonaemia. The review is presented in three parts. Quantitative estimates are presented for rates of ketone production at various The current view is presented of the body sites and in differing conditions. mechanism initiating ketogenesis - by de-repression of carnitine acyl Utilisation of ketones and possible adverse effects ,of transferaseI. Factors affecting ketonaemia are discussed (2) Origin of the hypoglycaemia. glucose production are discussed and it is argued that hypoglycaemia stemming from undernutrition arises primarily because endogenous reserves may be depleted even in the fed state; and an animal has only a limited capacity to However, uterine glucose mobilise body reserves for glucose production. utilisation may also play some part in the induction of hypoglycaemia. (3) It is argued that hypoglycaemia alone is an Aetiology of the symptoms. insufficient explanation of the symptoms. Synergistic factors which might play some part are discussed. Finally, other possible and quite different explanations are considered. Such possibilities are purely speculative, but are at least experimentally testable. I. INTRODUCTION It is appropriate to recall in this review that probably the classic study of the symptoms of this disorder was that of McClymont and Setchell (19%). The metabolic background was largely developed by Reid (e.g. 1968). Since then there has been some investment of energy, especially at the fringes of the subject, but little additional illumination, particularly at the centre. characteristic biochemical indices are a hypoglycaemia and The It is proposed to devote two sections to the origin of these hyperketonaemia. biochemical changes, and a final section which will indicate how inadequate still is our knowledge of the aetiology. 8 II. KETONAEMIA In earlier work, estimation of blood ketone bodies was not very accurate Modern enzymatic because of rather unsatisfactory analytical methods. techniques of estimation have shown that there is very little free acetone in fresh blood; that in all ruminants there is a mild ketonaemia, even in dry non-pregnant animals; and that the ratio of 3-hydroxybutyric/acetoacetic acids [3HB/AcAc] is higher in ruminants than in other species, especially Man. In Table 1, taken from recent (unpublished) studies of sheep fitted with portal and hepatic sampling catheters, there is some increase in arterial ketone concentrations even in pregnant sheep fed ad libitum. The increase however is small compared with that seen in alloxan-diabetic, and toxaemic pregnant sheep. The mild ketonaemia of dry animals is derived from ketone production by the gastro-intestinal tract, the liver actually removing ketones, although incompletely. AFRC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT UK * Present address: CSIRO Division of Tropical Animal Science, Tropical Cattle Research Centre, Rockhampton, Queensland 4702 Mean concentrations and rates of production of ketones by the Table 1. gastrointestinal tract (GIT) and liver of sheep. Number of animals in parenthesis. Negative values indicate uptake Arterial concentration (mM) 3HB 0.49 0.54 1.08 2.46 Net output (mmoles/hour) Liver GIT 6.5 9.9 4.6 7.0 Non-pregnant Single pregnant (10) (5) AcAc 0.03 0.04 0.14 0.44 3HB AcAc 1.0 -1.5 7.1 30.5 34.3 3HB AcAc -0.4 -3.9 1.3 2.3 3.6 -0.3 3.5 Twin pregnant (3) Fed diabetic (6) pregnant, toxaemic 2-10 0.2-1.0 - - - The mildly increased ketonaemia of twin-pregnant sheep is due to a substantial production of 3HB by the liver - there is little systematic variation in gastro-intestinal ketone production, and AcAc is generally taken up by the liver. In contrast to this, in the diabetic animal, there is a substantial elevation of both ketone bodies. Although there is a change - an output of acetoacetate - the production of 3HB is not markedly increased. Thus a 2-3 fold rise in ketone concentration is a result of only 1045% increase in It is commonly held that ketonaemia is due to increased production. production, not decreased utilisation. The findings shown here however, suggest this is only true for a moderate ketonaemia - up to about 1 .SmM 3HB and Ketogenesis by the liver is maximal at about these arterial O.l-0.2mM AcAc. concentrations, and the high ketonaemia of undernourished and toxaemic pregnant ewes is due to limited peripheral utilisation. This was shown in earlier work in which 3HB production was determined from the isotope dilution of labelled 3HB (Leng 1965) and from the limited capacity of muscle to use both ketones It should be appreciated however that the isotope (Pethick and Lindsay 1982). dilution technique over-estimates ketogenesis, because in muscle there is some interconversion of AcAc and 3HB (Pethick and Lindsay 1982) and also some intramuscular ketogenesis from long-chain fatty acids (Pethick et al. 1983) although this does not apparently result in a net release of ketones from muscle. (a) Mechanism of ketogenesis The means by which ketone formation is induced in ruminants is not quite settled, although much has been clarified in the last few years. The mechanism currently favoured from studies in rats is shown in Figure 1. One view, the source of much controversy in the period 1950-1970, was that the rate of tricarboxylic acid (TCA) cycle, activity determined formatiqn of ketones from Acetyl CoA. McGarry and Foster (1971) however, showed that there could be large variation in ketone production with little variation in CO2 formation from the TCA cycle. It was subsequently shown that the rate of transfer of long-chain fatty acids into mitochondria was the rate-limiting factor. This occurred via a carnitine ester and the catalysing enzyme (carnitine acyl tranferase, CAT ) was shown to be powerfully inhibited by malonyl CoA. The rise in ketone tormation in fasting and diabetes was a consequence of a fall in malonyl CoA, and consequently de-repression of CAT1 [see review by McGarry and Subsequent studies have firmly strengthened this hypothesis Foster 19801. indeed the sensitivity of the response to malonyl CoA seems to be increased with a fall in the malonyl CoA concentration, amplifying the effect (Robinson and Zammit 1982). Malonyl CoA is formed in the initiation of fatty acid synthesis, suggesting a strong inverse relation between fatty acid synthesis and ketone In ruminants, the liver is not regarded as a quantitatively formation. important site for fatty acid synthesis (e.g. Ingle et al. 1972) and there has been some doubt as to whether the malonyl CoA mechanism applied., Brindle et al. (1985) have shown that in sheep, in contrast to rats (and other species) Since methyl malonyl CoA is an effective inhibitor of CAT in the yM range. an intermediate of propiona z e metabolism, this would methyl malonyl CoA is offer a plausible explanation of the anti-ketogenic effect of propionate in sheep (and presumably, cattle). However, in our studies of hepatic metabolism in pregnant and diabetic sheep, there was no correlation between hepatic propionate uptake, and 3HB output which throws some doubt on the physiological Recently Zammit significance of propionate as a controller of ketogenesis. hepatic variation malonyl communication) has found CoA (personal concentration is related to ketogenesis in sheep liver, as in other species. Although the amount of fatty acid synthesised is limited, he argues that it is One curious sufficient for malonyl CoA to function as a rate determinant. feature of the biochemistry of ketogenesis in ruminant liver, that is so far unexplained, is the virtually complete absence of 3HB dehydrogenase from liver mitochondria. Despite some initial scepticism there is clearly a soluble This differs from the typical enzyme present (Watson and Lindsay 1972). mitochondrial enzyme in not being stimulated by lecithin. However, the maximum activity is barely sufficient to account for rates of 3HB production observed in vivo. b) Significance of ketone production Ketones are used as a significant energy source by skeletal (Pethick and In fasting Lindsay 1982) and cardiac muscle (Lindsay and Setchell 1976). pregnant ewes free fatty acids (FFA) and ketones are of about equal importance for skeletal muscle as energy sources and together are able to account for more However, with a mild than 80% of oxygen consumption (Pethick et al., 1983), ketonaemia glucose uptake (corrected for lactate output) was found to be inversely related to FFA but not 3HB concentration (Oddy and Lindsay 1986) suggesting that it is predominantly FFA rather than ketones that exert a sparing action on glucose, by inhibiting non-glycolytic glucose utilisation. 3HB, but not AcAc is also able to provide energy for the oxidative requirements Utilisation appears to be of the gravid uterus (Pethick and Lindsay 1982). proportional to concentration, and up to 25% of energy needs can be met in this way in fasted pregnant ewes, since only a small and variable proportion may be released as AcAc. The fetus does not appear to utilise ketones (Alexander et Morriss et al. 1974) so utilisation may be that of the myometrium al. 1969; and placenta, which together account for nearly half the oxygen consumption of the pregnant uterus (Meschia et al. 1979). In contrast to evidence from studies in fasting humans, neither 3HB nor appears to be used by the sheep brain (Lindsay and Setchell 1976); nor do elevated concentrations appear to interfere in any way with glucose utilisation by the sheep brain. ACAC There is no basis for the older view that ketosis is pathological. We now recognise that ketonaemia is merely a sign of increasing dependence on fatty acids as an energy source, and there is no evidence that even a severe Indeed it has been suggested that ketone formation is ketonaemia is toxic. essentially protective - that ketones act weakly to prevent the toxic effects of high concentrations of long chain fatty acids, by limiting lipolysis (Williamson and Hems 1970). Nevertheless, there are some adverse effects of an elevated ketonaemia, since it leads to an acidosis. Pethick and Lindsay (1982) found in fasting pregnant ewes there was a significant negative regression between blood 3HB and blood bicarbonate. It has been reported that in humans, ketonaemia may partly Such a response would obviously be harmful in pregnant suppress appetite. ewes, but so far has no experimental support. III. HYPOGLYCAEMIA In all animals fasting results in a fall in the blood glucose concentration as a result of a decline in exogenous glucose or glucose precursors. In pregnancy the fall is more marked and this has been attributed to the sustained uptake of glucose by the fetus despite decreasing glucose production. This view was developed early in studies of pregnancy,toxaemia and on the whole more recent work has tended to give quantitative support. w Glucose production Judson and Leng (1968) showed that nutrient energy intake was an important determinant of rate of glucose production (GPR), From the observations of Steel and Leng (1973) it was clear this was also true for pregnant sheep - any specific effect of pregnancy was small, relative to the effect of food intake. Oddy et al. (1985) studied sheep through the dry, pregnant and lactating states and obtained a relation independent of physiological state between GPR and ME intake and fleece-free body weight (FFBW): Wilson et al. (1981, 1983) however, have suggested that there is a marked stimulus to glucose production in pregnancy which is particularly striking in twin-bearing ewes. They argued that rate of glucose production was better correlated with lamb birth weight than with ME intake. However, I if we apply the above equation of Oddy et al. to the values of the study by Wilson et al. (1983), (although we can only approximately estimate fleece-free body weight) estimate of GPR (95=100mg/min) is only a little less than measured (108 mg/min), If we also apply the equation of Oddy et al. to our own (unpublished) study with twin-bearing ewes, the estimated rate of GPR (103 mg/min) was almost exactly that measured (104 mg/min). Specific differences in GPR may depend on prior body condition. Hough et al. (1985a) have recently reported an appreciable (25%) stimulus to GPR in pregnancy after allowing for the effect of increased food intake. (b) Glucose precursors The importance of propionate as a glucose precursor in pregnancy has been studied, using isotope dilution techniques, by Steel and Leng (1973) and Wilson Both groups suggested there was some improvement in pregnancy et al. (1983). in the proportion of propionate produced which was used for glucose synthesis. Even so, only 25.40% of GPR was derived from propionate. They suggested therefore the contribution of amino acids to gluconeogenesis was substantial. In recent, (unpublished) studies, we (Lindsay, Barker and Northrop) have made with pregnant sheep chronically catheterised to enable quantitation of hepatic nutrient exchange, we were surprised to find that on average the uptake of gluconeogenic precursors was about 25% greater than was needed to account for the glucose output. Propionate uptake alone was sufficient to account for 67% of glucose output, whereas total aNH2-N uptake, The if all was available for gluconeogenesis, could account for only 12%. other major potential contributors were glycerol (17%) and lactate' (28%). The latter two values are in the same range as those reported in pregnant sheep, using isotope dilution techniques - for glycerol, 18% (Ford et al. 1980) and for lactate, 23% (Faichney et al. 1981). Despite earlier suggestions (Lindsay the isotope dilution technique probably does underestimate the 1978) Fractional contribution of propionate (from contribution of propionate. hepatic uptake) is similar in dry and pregnant sheep (Lindsay et al. unpublished), Extra glucose production in pregnancy is probably derived mainly from endogenous sources even in sheep with a substantial food intake. One consequence of this mobilisation of tissue reserve even in fed animals, is that when food intake falls, there is little capacity to meet continuing need for glucose by mobilising of additional reserves - there is a fixed limit to this capacity, because mobilising of tissue protein reserves is known to be limited by fat, or some product of fat metabolism (Goodman et al, Fat can only contribute (apart from a small amount of odd chain-number 1984). fatty acids, which can provide 1 mole propionyl CoA per mole fatty acid) through its glycerol content. Triacyl glycerols are released from adipose any re-esterification whether in tissue as FFA and glycerol in a 31 ratio. adipose tissue, in the liver ('fatty' liver) or elsewhere has to be in the form of acyl glycerols. Thus glycerol can be used for glucose synthesis only while the corresponding fatty acids are being oxidised. A limit to this is set when the energy from fatty acid oxidation approaches the total metabolic requirement. Thus, one factor at least in the more marked hypoglycaemia of undernourishment in pregnancy is a sharper fall in glucose production, because of the restricted ability to switch to endogenous precursors. (c) Glucose utilisation (1) Uterus It is now well recognised that in assessing total demands on the mother, account must be taken of myometrial and placental, as well as fetal metabolism, that is, in the present context, total uterine glucose utilisation. Hay et al. (1983) found total uterine uptake, in well fed singlepregnant sheep (130.14Od gestation) was 30.40% of total glucose production, and this proportion did not change appreciably in sheep that were off feed, or They found uterine uptake was significantly deliberately fasted for 7 days. related to maternal glucose concentration, although the slope was markedly less than that relating GPR and glucose concentration. tidy et al. (1985), who also measured uterine glucose uptake in their studies, reported mean values for two differing diets. At the glucose concentrations reported, uterine uptakes were roughly comparable to those of Hay et al. (1983). However, it is striking that uterine uptake was not affected by diet. These sheep were, however, chronically adapted to the diet, in contrast to the acute changes imposed by Hay et al. In our study with twin-pregnant ewes, uterine uptake was not related to maternal glucose concentration, perhaps because measurements were made within a more limited range of maternal glucose concentration. Moreover estimated mean uterine glucose uptake was not significantly different from the However, the mean maternal glucose mean value obtained by Hay et al. concentration was substantially lower, and it may be calculated that at this concentration uterine uptake of glucose was appreciably greater for the twinbearing, compared to the single-bearing ewes. (ii) utilisation. muscle was 'remainder' uterine and particularly Other tissues Oddy et al. affected bY glucose, that skeletal mus in sheep on a Skeletal muscle is (1985) noted that diet, but not by is, the fraction cle uptake, was mu low energy intake. a significant site for glucose glucose utilisation by hindlimb physiological state, whereas remaining after subtraction of ch reduced in late pregnancy, We have observed (see, for example, Pethick (1980)) and confirmed by more recent (unpublished) studies that a significant (P<O.OS) linear relationship can be demonstrated between net glucose utilisation by skeletal muscle, and the arterial glucose concentration. (This does not imply other factors are not also important, particularly in well-nourished animals, where net glucose utilisation in muscle may change in pregnancy without alteration in the mean glucose concentration (Hough et al. 1985b)). In recent studies of splanchnic glucose utilisation (which probably constitutes the major fraction of 'remainder' glucose utilisation as defined by Oddy et al. (1985) there is possibly also a similar, albeit weak, <relationship between glucose utilised and arterial glucose concentrations (r=O.41, It is likely that many tissues may show such a (weak) relationship P = 0.07). when the glucose concentration varies over a substantial range, as can occur in One notable exception however, pregnancy (and to some degree, in lactation), is the brain, where utilisation is constant, independent of concentration, at least down to capillary glucose concentrations well below 1mM (Pappenheimer and However, it must be emphasised the total glucose utilisation Setchell 1973). by the sheep brain is small, relative to use by other tissues, simply because of the small weight of brain in sheep, relatively to body size. (d) Cause of hypoglycaemia It is readily possible to show a correlation between glucose production and concentration in pregnant (Hay et al. 1983; Oddy et al. 1985; Lindsay et al. unpublished) but not in non-pregnant sheep (see Oddy and Annison 1979). This is consistent with the view that the prime determinant of glucose concentration in pregnancy is the input of dietary precursors since, as discussed earlier, the mobilising of tissue reserves in the fed state permits little additional endogenous mobilisation when dietary input falls. The slope of the Tissue utilisation does however also play some part. line relating net glucose utilisation and concentration for muscle, is quite Thus, utilisation in similar to that relating production and concentration. muscle will fall, more or less in proportion to fall in dietary input. For the pregnant uterus, arid for the gastro-intestinal tract the corresponding slope is more shallow. Thus utilisation does not decline proportionately for a given fall in glucose concentration, and this in itself will intensify the decrease in concentration. In all tissues There is a further feature which should be mentioned. In discussing using glucose there is generally some lactate production. glucose conservation, glycolysis to lactate and its resynthesis to glucose in liver (and kidney) are merely a means of transferring energy from liver and kidney to peripheral tissues, with no net loss of glycogenic carbon. In discussing muscle glucose utilisation, net glucose used (i.e., after correction However, for the gastrointestinal for lactate production) was emphasised. In the pregnant tract and uterus, gross glucose utilisation was considered. uterus, about 20% of glucose is glycolysed to lactate and returned to maternal circulation (that transferred to the fetal side must be regarded as an irreversible loss). In contrast, lactate production can on average account for Thus, as a net user of glucose, the almost all the glucose used by the gut. uterus is much more significant than the gastrointestinal tract. There has been much dispute whether glucose production is reduced in toxaemic ewes. Kronfeld and Simesen (1961) claimed reduced production, while The latter finding is supported by Oddy Ford (1965) observed no difference. 25 and (unpublished) - rates of production were 32 and 34 mg/min in toxaemic; Wastney et al. (1983) 27 mg/min in starved asymptomatic twin-pregnant sheep. found feeble capacity for gluconeogenesis with hepatocytes from toxaemic compared to asymptomatic ewes. This might however be a consequence of general liver damage, which Gallagher (1959) emphasised was common in toxaemic ewes. Wastney et al. (1982) have also found some differences in glucose tolerance, with a higher insulin-resistance index in 'susceptible' compared to What we must bear in mind with all non-susceptible starved pregnant ewes. these findings however, is whether they are prior to, or consequences of, the pathological state. IV AETIOLOGY OF THE CLINICAL SYMPTOMS The major difficulty with pregnancy toxaemia lies in explaining how the clinical symptoms develop. It is often claimed the condition simply stems from undernourishment in multi-parous ewes. One of US (~3. Lindsay) however has been consistently unsuccessful in inducing clinical signs by starving or underfeeding sheep, and D.W. Pethick in the same laboratory was equally unsuccessful with a combination of fasting and stressing (by trucking), although he was readily able to induce the condition in Australia. Wastney et al. (1983) in New Zealand have recently distinguished between 'susceptible1 and *non-susceptible' ewes, the latter being those developing no symptoms after 10 days of fasting, but still having live foetuses. There is little doubt the symptoms a're derived from a dysfunction of the central nervous system (CNS), with the higher centres being among the first to be affected, McClymont and Setchell (1955) suggested a sustained hypoglycaemia was the origin of the condition. This was based on two findings (a) very similar symptoms could be induced by insulin hypoglycaemia (b) affected ewes had a significantly lower blood glucose concentration. However, the actual difference was very small (non-symptomatic 18-25 Moreover, mg/lOOml [mean 21.13 and symptomatic 15-23 mg/l00 ml [mean 18.11). from studies by Pappenheimer and Setchell (1973) and Lindsay and Setchell (1976) these concentrations would not be sufficient to lead to a significant fall in cerebral glucose utilisation. One might argue that in the older work blood glucose concentrations were measured by methods not entirely specific for glucose, but modern enzymatic methods do give similar results for glucose concentrations e.g. Oddy (unpublished) observed values of plasma glucose of 24, 27 mg/l 00 ml (equivalent to blood glucose values of 18-20 mg/l00 ml) in two cases of pregnancy toxaemia, while in asymptomatic 'animals, plasma glucose concentrations were 21-25 mg/l00 ml. If hypoglycaemia per se is insufficient as an explanation, either we have to consider some additional factor which exacerbates the effect of hypoglycaemia on the nervous system, or we consider some other factor affecting the brain. Reid (see e.g. review, 1968) agreed that acid-base disturbance may exacerbate the effect of hyperglycaemia. However, in several animals studied by Lindsay and Setchell (1976), there was a severe ketonaemia, with a corresponding fall in bicarbonate, coupled with a hypoglycaemia; but in no case was there evidence of interference' with cerebral glucose utilisation. Reid (1968) has also suggested that elevated cortisol is a feature of many interfere with cerebral toxaemic animals, and could perhaps glucose utilisation. There is no evidence so far for such an effect. Moreover Saba et al. (1966) showed there was no general elevation of cortisol in affected ewes and indeed ACTH injections could prevent the appearance of clinical signs. We may also note that there are other hormonal changes known to occur in pregnant sheep (see, e.g. Vernon et al. 1981) in particular there is a fall in plasma insulin, and increase in plasma growth hormone, placental lactogen and progesterone. These would in sum be expected to lead to a 'diabetogenic state! as postulated by Reid many years earlier. However, whether these changes are maintained, or magnified, and what effect this might have on cerebral carbohydrate metabolism, is quite unknown. CNS We should finally consider other possible factors that might affect the l stimula fasted effects Bannist (2 neurosi Pethick Biotin Kempton et al. (1978) have shown that biotin addition can 1 te glucose synthesis (but not elevate plasma glucose concentrations) in pregnant ewes. Biotin deficiency can result in a hypoglycaemia, with on the CNS in the fatty liver and kidney syndrome of chickens (see e.g. .er 1979). Thiamine Deficiency is known to be implicated in cerebrocortical 1 S8 and hypoglycaemia and ketonaemia may also occur (see Lindsay and 1983 for a review). It is known that ruminants destroy most dietary choline in (3 ) Choline the ga stro-intestinal tract (Neil1 et al. 1981), and carefully conserve choline In rats at least, choline uptake by brain is known to be important in (1 l cerebral metabolism (Wurtman and Fernstrom 1976). (4) Tyrosine/phenylalanine/tryptophane These amino acids are precursors of CNS transmitters and cerebral uptake has been suggested as important in affecting brain metabolism (Wurtman and Fernstrom, 1976). The branched-chain amino acids (leucine, isoleucine valine) could alsc ) be significant since they are thought to compete with the aromatic amino acids for uptake by'the brain. (51 NH3 Elevated ammonia concentrations we know can affect the CNS. it is also recognised (see e.g. Reid 1968) that normal metabolism by liver and kidney may be affected in pregnancy toxaemia, and this could result in elevated blood NH3. Possible involvement could These possibilities are purely speculative. however be tested experimentally, by suitable comparison of toxaemic and asymptomatic cases - probably of these putative factors only blood NH3 would be difficult to measure in field conditions. Even for blood NH3, a suitable technique might be possible. 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