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Lipid metabolism and retinoid therapy
Pharmac. Ther. Vol. 40, No. 1, pp. 55~5, 1989 Printed in Great Britain. All rights reserved
0163-7258/89 $0.00 + 0.50 Copyright © 1988 Pergamon Press plc
Guest Editor: R. M. MACKIE
LIPID
METABOLISM
AND
RETINOID
THERAPY
J. R. MARSDEN Department of Dermatology, University Hospital, Nottingham, U.K.
INTRODUCTION Awareness of a connection between vitamin A and lipid metabolism began as a result of descriptions of vitamin A poisoning (Sulzberger and Lazar, 1951; Elliott and Dryer, 1956). These were followed soon after by epidemiological studies describing the direct relationship between levels of vitamin A and serum cholesterol in adolescents (Bring et al., 1955; Hard and Esselburgh, 1960). When synthetic retinoids became available for clinical use in the mid 1970s, reports began to appear describing changes in serum lipids (Katz et al., 1980; Dicken and Conolly, 1980). Further investigations have produced remarkably congruent results, confirming the earlier studies, although there is still no direct information about the mechanism for retinoid hyperlipidaemia, its consequences, and its management. These are important questions because of the increasing use of retinoids: 112 patients were treated with isotretinoin or etretinate for a median period of 136 days in the Department of Dermatology at University Hospital, Nottingham, during 1986. However, a review of the available evidence together with a brief description of normal lipoprotein structure and function provides some clues to help answer these points. NORMAL LIPOPROTEIN METABOLISM Lipids in plasma are transported within particles called lipoproteins. These consist of a central oily core of water-insoluble triglyceride and cholesterol ester, which is surrounded by a protein 'shell' which has a hydrophobic interior and a hydrophilic exterior. These are called apolipoproteins and are designed apo A, B, C, and E; subtypes are referred to with a numerical suffix e.g. apo Aj, apo CH, apo B~00. In conjunction with the apoprotein shell and on the exterior of the particle there is a variable amount of hydrophilic detergent lipid, mainly cholesterol and phospholipid. As well as solubilising lipid, apoproteins perform two other vital functions. One is the activation of 3 enzymes which are pivotal to normal plasma lipid metabolism. The enzymes are lipoprotein lipase (LPL), hepatic triglyceride lipase (HTL), and lecithin-cholesterol acyl transferase (LCAT). The other function is to enable lipoprotein particles to bind to cells by acting as ligands for specific receptors, for instance apo B~00receptors. Lipoproteins are separated and classified according to their density. Chylomicrons are the largest and least dense particles, very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) becoming progressively smaller and denser (Table 1). Although this gives the impression of homogeneity, each lipoprotein class consists of a family of different sized particles. An outline of the pathways of lipoprotein metabolism is shown in Fig. 1. Dietary glyceride is hydrolysed in the small intestine to fatty acids; these are absorbed, together with cholesterol, into the small intestinal mucosal cell. Fatty acids are then re-esterified to triglyceride, and assembled together with cholesterol ester into chylomicrons. The apoproteins within newly synthesised chylomicrons are mainly apo At and apo B48. The latter is specific to chylomicrons and VLDL of intestinal origin, and is synthesised in the jejunum. Chylomicrons are released into the lymphatics and enter the blood via the thoracic duct; their function is to deliver fatty acids to sites of use (skeletal muscle) or 55
0.95-1.006
1.006-1.019
1.019-1.063 1.063-1.210
IDL
LDL HDL
<0.95
Chylomicrons
VLDL
Density (g/ml)
Particle
VLDL Liver, intestine
VLDL
Liver
Intestine
Source apo apo apo apo apo apo apo apo apo apo apo
A[, A . , A~v B4s, Ci, Cjl, Cm E Bi00, Cl, C]l, Cm E B~00, apo C E B~00 A~, AH Ci, C . , Cm E
Apoproteins
Cholesterol ester Cholesterol ester Phospholipid
Tfiglyceride and cholesterol ester
Mainly triglyceride
Mainly triglyceride
Core lipid content
Function
Transport of cholesterol from the liver to extra-hepatic cells Transport of cholesterol from extra-hepatic cells to the liver and other sites
VLDL-LDL intermediate
Transport of triglycerides synthesised in the liver
Transport of dietary fat as triglyceride and cholesterol esters
TABLE 1. Characteristics of Normal Human Plasma Lipoproteins
'7
t~
Lipid metabolism and retinoid therapy
57
ado BIO 0 receptor apo E receptor
LIVER
apo E receptor
nascent HDL
LPL
other sites
I-'1 , ~ o f catabolism
PL
LPL
intestine extrahepatic cell
FIG. 1. An outlin~fof normal lipoprotein metabolism (for details see text). C = chylomicrons; Crem = chylomicron remnants; LPL = lipoprotein lipase; C,PL = cholesterol and phospholipid; LCAT = lecithin cholesterol acyl transferase; LTP = lipid transfer protein; C,TG = cholesterol and triglyceride; ACAT = acyl cholesterol acyl transferase.
storage (adipose tissue). The vascular endothelium at these sites is rich in lipoprotein lipase, and this enzyme is activated by apo C. which the chylomicron acquires from H D L within the circulation. The result is that the core triglyceride is hydrolysed, and free fatty acids are released and taken up by muscle or fat cells. Consequently, the volume of the chylomicron progressively decreases and this produces excess surface components. These are mainly cholesterol, phospholipid and the A apoproteins, and are transferred to HDL. In exchange, the shrunken chylomicron remnant acquires apo E from HDL, which confers on it the ability to bind to liver cells which have specific receptors for chylomicron apo E. In this way the remnants are cleared from the blood and deliver dietary cholesterol to the liver. Lipid which is synthesised within the liver clearly requires a separate pathway for distribution. Triglyceride and cholesterol esters are packaged within VLDL particles, which are secreted by the liver into the blood. Newly secreted, or 'nascent', VLDL contains apo C, apo E, and apo B~00.As with chylomicrons, VLDL is metabolised at vascular endothelium in muscle and fat by lipoprotein lipase, and similarly this enzyme is activated by HDL-derived apo C,. As the VLDLs are depleted of triglyceride they become denser and smaller intermediate density lipoproteins (IDL) and the redundant surface phospholipid, cholesterol and apoproteins are transferred to HDL. However, the hydrophilic polar cholesterol molecule cannot be stored within lipoproteins unless it is esterified to make it hydrophobic. This function is carried out by the enzyme lecithin cholesterol acyl transferase (LCAT) which removes fatty acid from HDL phospholipid (lecithin) and transfers it to the newly-acquired cholesterol. LCAT is activated by the H D L protein apo A,. Some of the newly esterified cholesterol is transferred back to IDL and LDL by lipid transfer protein (LTP), a protein which redistributes and equilibrates lipids between lipoprotein
58
J . R . MARSDEN
classes. In this way triglyceride is removed from VLDL, and the cholesterol ester content of the products of VLDL catabolism is increased. Some of the products of catabolism, VLDL remnants and IDL, are taken up by apo E receptors on the surface of liver cells, and cleared from the blood. However, the majority of VLDL continues to be depleted of triglyceride until it enters the LDL density range. Low density lipoprotein predominantly contains cholesterol ester, and one molecule of apo B~00in each particle. Normally LDL is produced solely from VLDL catabolism. Most cells in the body contain apo B~0oreceptors on their surface to which LDL binds. The receptor-ligand combination is then internalised into lysosomes, and the cholesterol ester released; LDL can also enter the cell via a non-receptor pathway. Such large amounts of cholesterol are normally available to extra-hepatic cells that intracellular cholesterol production is virtually switched off by repression of HMG CoA reductase; this enzyme is the rate-limiting step in cholesterol synthesis. Cholesterol which is surplus to requirements for membrane and steroid synthesis has therefore to be returned to the liver, which is the only important site of catabolism. High density lipoproteins are derived from particles secreted by the liver and intestine. Nascent HDL are disc-shaped, consisting of little more than phospholipid membrane and apo Ax. These particles rapidly form into spherical, less dense HDL 3 and the larger HDL2 as they acquire the surplus surface components of chylomicrons and VLDL and become cholesterol ester enriched. To date, three major functions for HDL have been described. Firstly, it provides a sink for redundant surface components of the triglyceride rich lipoproteins and a source for apo C, and apo E, and secondly, it acts as a factory for production of cholesterol ester within plasma via the LCAT reaction. The third function is the movement of cholesterol from extra-hepatic cells to other sites of use, mainly the liver. This component of the plasma cholesterol flux is called reverse cholesterol transport. Recent evidence shows that specific HDL receptors exist on such tissue as smooth muscle cells and fibroblasts. These receptors bind apo E free HDL, and the ligand is probably apo A~. Binding of cholesterol poor HDL e.g. HDL 3, depletes cellular cholesterol, and binding is up-regulated in proportion to the cellular content of cholesterol. As a result of this HDL-induced cholesterol efflux from cells, more cholesterol ester enriched HDL 2 is produced via LCAT to add to that already produced from chylomicron and VLDL catabolism. Despite extensive research, the metabolic fate of HDL 2 is still not entirely clear. Some of it is removed from the circulation by binding to specific HDL receptors on the liver. Some is further enriched with cholesterol ester to produce HDL~, also known as HDL E because it contains apo E and therefore binds to hepatic apo E receptors. Some of the cholesterol ester is exchanged with triglyceride in VLDL by lipid transfer protein producing triglyceride rich HDL2. This triglyceride is removed by apo A. activated hepatic triglyceride lipase, and so HDL 2 is converted back to HDL 3. As previously described, some cholesterol ester generated in HDL2 is transferred to IDL and LDL, and some of this finds its way back to the liver where it is taken up by the apo Bt00 receptor. From this description it is apparent that normal lipoprotein metabolism is complex and relies on many interdependent processes. Consequently, measurements of lipid, lipoprotein and apoprotein mass have limited value in defining the mechanisms underlying abnormal iipoprotein metabolism, although they provide useful clues. Such measurements provide the bulk of published information on retinoid hyperlipidaemia. The second part of this review describes these findings, and then attempts to use them to suggest possible explanations for retinoid hyperlipidaemia. ISOTRETINOIN Increases in concentration of serum triglyceride and cholesterol have been repeatedly described after treatment with isotretinoin (Katz et al., 1980; Lyons et aL, 1982; Zech et al., 1983; Marsden et al., 1984a and 1984b; Vahlquist et al., 1985b, Bershad et al., 1985). The changes are directly proportional to the dose used as shown in Fig. 2, and this data is consistent with the findings of Strauss et aL, 1984. This linear relationship continues
Lipid metabolism and retinoid therapy
59
triglyceride
cholesterol m
m
1.0
increase (mmol/I)
i I
I
I
I
I
I
_1
I
0
I
I
I
I
I I
dose ( m g / k g / d a y )
FXG.2. Relationship between dose of isotretinoin and increases in levels of serum cholesteroland triglyceride (0.05mg/kg/day n = 9, 0.2mg/kg/day n = 8, 0.4mg/kg/day n = 8, 0.8 mg/kg/day n = 12, 1.0 mg/kg/day n = 10). beyond the highest dose of 1 mg/kg/day shown in the figure, but adequate dose-response data above this point are not available. Pre-treatment levels of triglyceride and cholesterol which are within the reference range are unrelated to the increase during treatment (Marsden et al., 1984b; Vahlquist et al., 1985b). Increased triglyceride and cholesterol levels are apparent within 4 weeks of starting isotretinoin, and usually change little thereafter (Zech et al., 1983; Marsden et al., 1984a and 1984b). However, in a larger study of 60 patients, triglyceride levels in females continued to increase for 12 weeks and total serum cholesterol levels in males were greatest at 4 weeks with a 15% increase which declined to only a 5% increase at the end of treatment (Bershad et al., 1985). These increases are reversed within 2-4 weeks of stopping isotretinoin (Lyons et al., 1982; Marsden et al., 1984a and 1984b; Marsden, 1987). Separation of lipoproteins by ultracentrifugation shows that the largest increase in triglyceride mass is in the V L D L fraction, with smaller increases in LDL-triglyceride and a small increase or no change in HDL-triglyceride (Marsden et al., 1984a; Vahlquist et al., 1985a; Gollnick et al., 1985). In contrast, the largest increase in cholesterol mass is in LDL, with a small increase in VLDL-cholesterol, and a decrease in HDL-cholesterol (Zech et aL, 1983; Marsden et al., 1984a; Vahlquist et al., 1985b; Gollnick et al., 1985). These findings suggest that the number of V L D L and L D L particles is increased, although they do not exclude the possibility that the particles are also carrying more lipid. However it is now clear that concentrations of apo B are also increased after isotretinoin (Vahlquist et al., 1985b; Gollnick et al., 1985; Marsden, 1987), and that this correlates with total serum cholesterol and triglyceride (Laker et al., 1987). This strongly supports the idea that V L D L and L D L particle number is increased but gives no indication about which predominates. In one study of 13 subjects measurements of apo A~ levels show a small but consistent decrease (Laker et al., 1987). This difference did not reach statistical significance. Two other studies have not shown any difference (Vahlquist et al., 1985b; O'Leary et al., 1986) and so any change in apo A, is likely to be very small. Therefore, there is unlikely to be much difference in the number of H D L particles during treatment with isotretinoin, and recent evidence suggests that there is simply a reduction in cholesterol content of the HDL2 fraction with no difference in HDL3 cholesterol or H D L phospholipid (O'Leary et al., 1986). ETRETINATE Changes in serum lipids during treatment with etretinate are very similar to those described with isotretinoin. Mean levels of both triglyceride and cholesterol are increased (Zech et al., 1981; MichaElsson et al., 1981; Ellis et al., 1982; Vahlquist et al., 1985b; Marsden, 1987). The changes are apparent as early as 2 weeks after starting etretinate (MichaElsson et al., 1981; Marsden, 1987) and have usually reached their maximum by 8 weeks. Whilst these increases appear to be related to dose (Ellis et al., 1982), the precise
60
J.R. MARSDEN
dose-response relationship is unclear because of lack of comparison between groups of similar ages and pre-treatment lipid values. Like isotretinoin, the pre-treatment values for triglyceride and cholesterol do not predict the subsequent increase (Micha~lsson et al., 1981; Vahlquist et al., 1985b). Levels of both triglyceride and cholesterol return to normal after 4 weeks of stopping etretinate (Ellis et al., 1982; Marsden, 1987), confirming that its prolonged terminal elimination half-life does not delay reversal of hyperlipidaemia. Ultracentrifugation studies show that the largest increase in triglyceride mass is in the VLDL fraction, with smaller increases in LDL-triglyceride and no change in HDL-triglyceride (Micha~lsson et al., 1981; Vahlquist et al., 1985b; Gollnick et al., 1985). Although it is still unclear whether the largest increase in cholesterol mass is in VLDL (Micha~lsson et al., 1981; Zech et al., 1981; Gollnick et al., 1985) or in LDL (Vahlquist et al., 1985b), all of these studies show a small reduction in levels of HDL-cholesterol. There is no information about whether this reduction is in the HDL2 or HDL 3 fraction. Ellis et al. (1982) found no difference in HDL-cholesterol. It seems clear that there are small increases in apo B during treatment with etretinate (Micha~lsson et al., 1981; Vahlquist et al., 1985b; Gollnick et al., 1985) although there is no information about whether this increase is in VLDL or LDL apo B. However, levels of apo A~ and apo A. are unchanged, and so, like isotretinoin, the decrease in HDL-cholesterol presumably reflects alteration in HDL composition rather than a decrease in the number of HDL particles. The changes in lipids and lipoproteins resulting from treatment with etretinate are not as well characterised as they are after treatment with isotretinoin. Isotretinoin seems to be more potent, producing greater increases in triglyceride and cholesterol compared with an equimolar dose of etretinate (Vahlquist et al., 1985b). A minority of patients treated with both drugs have no changes in lipid levels during treatment. The explanation for this is unclear. However, the similarity of the changes suggests a common cause, and so it is easier to discuss both drugs together when considering possible mechanisms for retinoid hyperlipidaemia. MECHANISMS Explanations for retinoid hyperlipidaemia can be broadly classified as increased production or decreased clearance of either chylomicrons or of endogenously synthesised lipid or lipoproteins. It is extremely unlikely that the changes in serum lipids result from increased absorption of dietary lipid and increased chylomicron production, or from reduced chylomicron clearance. Chylomicrons are not present in the fasting serum samples of subjects with increased lipid levels due to retinoids because the serum is not lipaemic, and chylomicrons are not found on lipoprotein electrophoresis (Lyons et al., 1982; Marsden and Laker, unpublished observations). Also, the activity of lipoprotein lipase released into serum by intravenous heparin is no different during treatment with isotretinoin (van der Schroeff and Jansen, 1985; Bershad et al., 1985), and LPL activity in adipose tissue is no different during treatment with either isotretinoin or etretinate (Vahlquist et al., 1985b). A small decrease in skeletal LPL activity was found in this study but its relevance is unclear. Isotretinoin and etretinate bind extensively to lipoproteins in plasma (Zech et al., 1981), but this does not seem to affect the ability of LPL to hydrolyse triglyceride from VLDL (van der Schroeff and Jansen, 1985). The rate of removal from serum of an intravenous bolus of Intralipid (medium chain length triglyceride) may be used as a model for chylomicron clearance (Carlsson and R6ssner, 1972). There are small reductions in the elimination rate constant during treatment with isotretinoin (Vahlquist et al., 1985a; Gollnick et al., 1985) and etretinate (Vahlquist et al., 1985a). However, these observations are clearly not relevant to retinoid hyperlipidaemia because chylomicronaemia is absent. Alternatively, there could be increased synthesis of triglyceride and cholesterol in the liver. Retinoic acid produces increased synthesis of triglyceride from mevalonate in rat liver homogenates (Erdman et al., 1977), and total liver lipid is increased by feeding rats retinyl acetate (Prodouz and Navari, 1975) but in both cases liver cholesterol content is reduced.
Lipid metabolismand retinoid therapy
61
However, large doses of retinol increases hepatic triglyceride and cholesterol (Misra, 1968). This increase in triglyceride production requires a supply of carbon skeleton. The likeliest source of this would be free fatty acids (FFA) delivered to the liver from adipose tissue. However, retinoic acid does not increase plasma FFA levels in the rat (Gerber and Erdman, 1979), and hypertriglyceridaemia is not mediated by adipose tissue lipolysis due to adrenaline. Isotretinoin does not increase plasma FFA levels in man (Laker et al., 1987) although this does not exclude increased flux of FFA into the liver or an increase in the proportion of FFA used for triglyceride synthesis rather than fl-oxidation. However, this seems unlikely because the same study shows no change in concentration of the products of fl-oxidation, and animal experiments show that this is not reduced by retinoids (Sherratt, personal communication). The closest analogy in man is familial hypertriglyceridaemia which is due to increased hepatic triglyceride synthesis (Brunzell et al., 1977). This results in increased VLDL and LDL triglyceride and decreased HDL cholesterol but, unlike isotretinoin and etretinate, no change in LDL cholesterol (Brunzell et al., 1983). Therefore there is no direct evidence to date to confirm that increased hepatic lipid synthesis is the primary cause of retinoid hyperlipidaemia in man. Instead of a primary increase in lipid synthesis, there could be an increase in synthesis of apolipoprotein, or a decrease in clearance of VLDL and/or LDL. As already described, both isotretinoin and etretinate increase levels of apo B, the major apoprotein in VLDL and the only apoprotein in LDL, and both VLDL and LDL are increased. Increased apo B synthesis would result in increased VLDL secretion, but not necessarily increased LDL apo B (Eaton et al., 1983). Because there is no data describing lipoprotein kinetics during retinoid treatment, the evidence in support of this hypothesis is indirect. Firstly, increasing production of VLDL would not be expected to alter its lipid composition and this is no different after isotretinoin (Marsden et al., 1984b). Secondly, there is close similarity between the changes in serum lipids during treatment with retinoids and those occurring in the disease familial combined hyperlipidaemia. This disorder is characterised by increases in both VLDL and LDL triglyceride and cholesterol and HDL triglyceride, and decreases in HDL cholesterol (Brunzell et al., 1983), and is due to increased apo B synthesis (Chait et al., 1980). Another characteristic of this disorder is its variable expression between individuals, a notable feature of retinoid hyperlipidaemia. Decreased clearance of VLDL and LDL seem unlikely because the accumulation of partially catabolised VLDL remnants would alter the lipid composition of VLDL by increasing the proportion of triglyceride. Also, there is no accumulation of remnants or IDL on lipoprotein electrophoresis (Marsden and Laker, unpublished observations). Thirdly, reduced clearance of VLDL in familial type Ill hyperlipoproteinaemia results in low levels of LDL, as opposed to the increased values found during retinoid treatment, and reduced clearance of LDL in familial hypercholesterolaemia is not usually associated with increased VLDL (Kachadurian and Utermann, 1973). Reduced levels of HDL2 cholesterol during treatment with isotretinoin could either mean reduced production from HDL 3 because of decreased catabolism of VLDL, or reflect greater cholesterol ester-triglyceride exchange between HDL2 and VLDL, IDL and LDL. Both LDL and HDL are triglyceride enriched during treatment with isotretinoin, which is consistent with the latter suggestion (Marsden et al., 1984b). Because several other drugs which increase serum triglyceride and cholesterol are inducers of hepatic microsomal cytochrome P-450 enzymes (Durrington, 1979; Miller and Nestel, 1973; Pelkonen et al., 1975; Bolton et al., 1980) it was suggested that isotretinoin might also have this effect. Isotretinoin does not increase clearance of antipyrine in man, a drug entirely dependent on metabolic clearance by the liver (Marsden et al., 1985) and slightly reduced the activities of several microsomal cytochrome P-450 enzymes (Finnen and Shuster, 1984). Further information is clearly required to identify the cause of retinoid hyperlipidaemia. The issue has been over-simplified in this discussion. For example, small increases in apo B can occur in response to increased hepatic triglyceride synthesis (Brunzell et al., 1983). J.P.T. 40/I--E
62
J.R. MARSDEN
The next step is to show whether or not VLDL, IDL, and LDL kinetics are changed by retinoid treatment and this investigation is now in progress. CONSEQUENCES OF RETINOID HYPERLIPIDAEMIA Although the changes in lipoprotein phenotype during retinoid treatment are well characterised, their consequences are not. There are two major theoretical risks. These are acute pancreatitis as a result of very high levels of serum triglyceride and increased atherogenesis. Neither has yet been reported. Pancreatitis is only likely as a consequence of severe hypertriglyceridaemia with fasting levels greater than l0 mmol/1. Values of this order have been found during treatment with isotretinoin (Lyons et al., 1982; Ellis et al., 1982; Dicken and Connolly, 1980; Katz et al., 1980) and all of these patients had increased pre-treatment levels. There is not enough information to know whether these patients had an underlying primary hyperlipoproteinaemia, but very large synergistic increases in serum triglyceride occur in patients with familial hypertriglyceridaemia who are then exposed to a secondary cause of hypertriglyceridaemia such as diabetes or alcohol (Chait and Brunzell, 1983). This is quite different from the additive response seen in patients who already have secondary hypertriglyceridaemia due to diabetes, alcohol or obesity. Treatment with isotretinoin and etretinate results in total levels which rarely exceed 3.5-4.0 mmol/1 (Gollnick et al., 1985; Marsden, unpublished observations). Similar small increments in triglyceride levels were found in relation to body mass index during treatment with isotretinoin by Bershad et al. (1985). The probability of increased atheroma and risk of ischaemic heart disease (IHD) is of much greater importance. Although it is a popular opinion that increased levels of triglyceride do not correlate with risk of IHD independently of other variables, there is increasing evidence to challenge this view. This largely stems from recent re-examination of the data relating triglyceride levels to IHD using more sensitive statistical techniques than multivariate analysis. The results show that serum triglyceride levels predict risk of IHD in males over 50 (Abbott and Carroll, 1984), and the correlation is particularly strong in both sexes when HDL cholesterol is low (Castelli, 1986). Comparison between risk factors in women over 50 shows that triglyceride levels are more powerful predictors of IHD than LDL levels. The association between levels of total serum cholesterol and LDL cholesterol with risk of IHD is much better known although its significance in the context of retinoid hyperlipidaemia has often been trivialised. This is largely because of the concept that cholesterol levels which lie within the 'normal range' can be ignored as far as IHD is concerned. This is incorrect. The relationship between cholesterol level and risk of IDH is log-linear, and therefore the gradient of risk is continuous throughout the reference range. This means that increments in cholesterol level produce increments in risk, and is well illustrated by the findings of the Whitehall study of 0.64 IHD deaths/100 males/10 years for an increase in cholesterol of 1 mmol/l (Rose and Shipley, 1986). However, when cholesterol concentrations rise above 6.5 mmol/l, risk of IHD increases much more rapidly (Martin et al., 1986). About 25% of western Europeans have values above this level, and are categorised as being at high risk of IHD by both the European Atherosclerosis Society (1987) and by the National Institutes of Health consensus panel (1985). Yet the upper limit of the reference range for serum cholesterol is 7.1-7.8 mmol/1 in most U.K. laboratories. It denotes the 95th centile and has no relevance to risk of IHD. As well as increasing levels of total serum cholesterol, retinoids alter the proportion of cholesterol in LDL and HDL. Concentrations of HDL cholesterol correlate indirectly and independently with risk of IHD (Kannel et al., 1979), especially HDL2 cholesterol which is the fraction reduced by retinoid treatment (Miller et al., 1981). The practical relevance of this is that although total serum cholesterol may not change during retinoid treatment, this could conceal considerable increases in LDL and decreases in HDL cholesterol. Measurement of the ratio L D L : H D L cholesterol before treatment and after 8 weeks on therapy would therefore be useful, and many clinical chemistry laboratories can now
Lipid metabolismand retinoid therapy
63
provide this. The level of apo B also increases; apo B concentrations correlate well with proven IHD especially at lower levels of total serum cholesterol (Whayne et al., 1981) and so might provide a more accurate estimate of risk from retinoids. M A N A G E M E N T OF H Y P E R L I P I D A E M I A There is little published information about this. It is very rare for pre-treatment values which lie within the reference range to rise by more than 1-2 mmol/1, and so repeated measurements are not necessary, values obtained before treatment and after 4-8 weeks are sufficient to assess the response. However, patients with pre-existing hypertriglyceridaemia, especially those without evidence of a secondary cause, are at risk of very high triglyceride levels and possibly of pancreatitis. If pre-treatment values are 2-3 mmol/1, treatment can be started at low doses, but with measurements of triglyceride levels at weekly intervals. With fasting values in excess of 5 mmol/l, triglyceride levels can fluctuate enormously, and so treatment should be stopped. Although reducing the dose will probably reduce lipid levels, the amount by which they decrease is unpredictable. Dietary changes to reduce retinoid hyperlipidaemia are now being investigated. Supplementation with fish oil rich in eicosapentaenoic acid and docosohexaenoic acid (MaxEPA) produced large reductions in triglyceride levels, but much smaller changes in total cholesterol, and no increase in H D L cholesterol or decrease in apo B (Marsden, 1987). It is too early to say whether this has any therapeutic value but is most likely to be of value to patients with pre-existing hypertriglyceridaemia. Preliminary results of the effect of the low fat diet recommended by the WHO Expert Committee (1982) suggest that the response is small and due to coincidental weight loss in obese subjects rather than reduction of fat intake (Marsden and Marenah, unpublished data). However, until this is confirmed, it may be worth making dietary changes, especially if treatment is for more than a few months. Reduction of calories is effective in reducing triglyceride, and sometimes cholesterol also, in obese patients. In the non-obese, total calories should remain unchanged but total dietary fat can be reduced to about 30% of the calories, and energy from unrefined carbohydrate increased accordingly. Polyunsaturated fat should be increased to 10% of calories. Triglyceride levels decrease with avoidance of alcohol, sometimes dramatically. There is no information about the efficacy or safety of hypolipidaemic drugs in the treatment of retinoid hyperlipidaemia. CONCLUSIONS Both isotretinoin and etretinate cause hyperlipidaemia which is dose-related and reverses when treatment is stopped. The available evidence suggests that the likeliest explanation is increased production of VLDL but measurements of lipoprotein kinetics are necessary to confirm this. Large increases in triglyceride levels seem to be confined to subjects with pre-existing hypertriglyceridaemia, but smaller increases in triglyceride, cholesterol and apoprotein B and decreases in HDL cholesterol are very common. These changes in serum lipids and lipoproteins are likely to be associated with increased atherogenesis, and increased risk of IHD if treatment is continued for long periods. The best strategy to reduce this risk is to develop retinoid analogues which do not cause hyperlipidaemia. Acknowledgements--Much of the work referred to in this chapter was made possibleby financialsupport from Roche.
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BOLTON, C. H., JACKSON, L., ROBERTS, C. J. C. and Hm, TOG, M. (1980) Enzyme induction and serum and lipoprotein lipids: a study of glutethimide in normal subjects. Clin. Sci. 58: 419-421. BRING, S. V., WARNICK, K. P. and WOODS, E. 0955) Nutritional status of school children 15 and 16 years of age in three Idaho communities; blood biochemical tests. J. Nutr. 57: 29-45. BRUNZELL, J., CHAIT, A., GOLDBERG, A. and ALBERS, J. (1977) Monogenic familial hypertriglyceridaemia vs familial combined hyperlipidaemia', evidence for overproduction of triglyceride in familial hypertriglyceridaemia. Clin. Res.25, 3: 388A. BRUNZELL, J. D., ALBERS,J. J., CHAIT, A., GRUNDY, S. M., GROSZEK, E. and McDONALD, G. B. (1983) Plasma lipoproteins in familial combined hyperlipidaemia and monogenic familia] hypertriglyceridaemia. J. Lipid Res. 24: 147-155. CARLSON,L. A. and R6SSNER,S. (1972) A methodological study of an intravenous fat tolerance test with Intralipid emulsion. Scand. J. Clin. Lab. Invest. 29: 271-280. CASTELLI,W. P. (1986) The triglyceride issue: a view from Framingham. Am. Heart J. 112: 432-437. CHAIT, A. and BRUNZELL,J. D. (1983) Severe hypertriglyceridaemia; role of familial and acquired disorders. Metabolism 32: 209-214. CHAIT, A., ALBERS,J. J. and BRUNZELL,J. D. (1980) Very low density lipoprotein overproduction in genetic forms of hypertriglyceridaemia. Eur. J. Clin. Invest. 10: 17-26. DICKEN, C. H. and CONNOLLY,S. M. (1980) Eruptive xanthomas associated with isotretinoin (13-cis-retinoic acid). Archs Derm. 116: 951-952. DURRINGTON, P. N. (1979) Effect of phenobarbitone on plasma apolipoprotein B and plasma high density lipoprotein cholesterol in normal subjects. Clin. Sci. 56: 501-504. EATON, R. P., ALLEN, R. C. and SCHADE,D. S. (1983) Overproduction of a kinetic subclass of VLDL apo B, and direct catabolism VLDL apo B in human endogenous hypertriglyceridemia; an analytical model solution of tracer data. 3". Lipid Res. 24: 1291-1303. ELLIOTT, R. A. JR and DRYER, R. L. (1956) Hypervitaminosis A: report of case in adult. J. Am. Med. Ass. 161: 1157-1159. ELLIS, C., SWANSON, N., GREKIN, R. C., GOLDSTE1N, N. G., BASSETT, D. R., ANDERSON, T. F. and VOORHEES, J. J. (1982) Etretinate therapy causes increases in lipid levels in patients with psoriasis. Archs Derm. 118: 559-562. ERDMAN, J. W. JR, ELLIOTT, J. G. and LACHANCEP. A. (1977) The effect of three forms of vitamin A upon in vitro lipogenesis from three cholesterol precursors. Nutr. Rep. Intl. 16: 47-57. EUROPEANATHEROSCLEROSISSOCIETY (1987) Strategies for the prevention of coronary heart disease. Fur. Heart J. 8: 77-88. FINNEN, M. J. and SHUNTER, S. (1984) The effects of 13-cis-retinoic acid on hepatic and cutaneous monooxygenase activities: possible cancer protective mechanism. Br. J. Derm. 111: 704. GERBER, L. E. and ERDMAN, J. W. JR (1979) Effect of retinoic acid and retinyl acetate feeding upon lipid metabolism in adrenalectomized rats. J. Nutr. 109: 580-589. GOLLNICK, H., SCHWARTZKOPF,W., PROSCHLE,W., LULEY, C., SCHLEISING,M., MATTHEIS, E. and ORFANOS,C. E. (1985) Retinoids and blood lipids: an update and review. In: Retinoids: New Trends in Research and Therapy, pp. 445-460, SAURAT,J. (ed.) Karger, Basle. HARD, M. M. and ESSELBAUGH,N. C. (1960) Nutritional status of adolescent children IV. Cholesterol relationships. Am. J. Clin. Nutr. 8: 346-351. KACHADURIAN, A. K. and UTERMANN, S. M. (1973) Experience with the homozygous cases of familial hypercholesterolaemia. Nutr. Metab. 15: 132-142. KANNEL, W. B., CASTELLI,W. P. and GORDON, T. (1979) Cholesterol in the prediction of atherosclerotic disease. New perspectives based on the Framingham study. Ann. Intern. Med. 90: 85-91. KATZ, R. A., JORGENSEN,H. and NIGRA,T. P. (1980) Elevation of serum triglyceride levels from oral isotretinoin in disorders of keratinisation. Archs Derm. 116: 1369-1372. LAKER, M. F., GREEN, C., BHUIYAN,A. K. M. J. and SHUSTER,S. (1987) Isotretinoin and serum lipids: studies on fatty acid, apolipoprotein and intermediary metabolism. Br. J. Derm. in press. LYONS, F., LAKER, M. F., MARSDEN, J. R., MANUEL, R. and SHUNTER,S. (1982) Effect of oral 13-cis-retinoic acid on serum lipids. Br. J. Derm. 107: 591-595. MARSDEN,J. R. (1987) Effects of dietary fish oil on hyperlipidaemia due to isotretinoin and etretinate. Hum. Toxic. 6:219-222. MARSDEN, J. R., LAKER, M. F., FORD, G. P. and SHUNTER,S. (1984a) Effect of low dose cyproterone acetate on the response of acne to isotretinoin. Br. J. Derm. 110: 697-701. MARSDEN, J. R., TRINNICK, T. R., LAKER, M. F. and SHUNTER,S. (1984b) Effects of isotretinoin on serum lipids and lipoproteins, liver and thyroid function. Clin. Chim. Acta 143: 243-251. MARSDEN, J. R., SHUSTER,S. and DENNIS, J. 0985) Effect of isotretinoin on antipyrine clearance and levels of ~t-1 acid glycoprotein. Hum. Toxic. 4: 335-338. MARTIN, M. J., HULLEY, S. B., BROWNER,W. S. and KULLER, L. H. (1986) Serum cholesterol blood pressure and mortality: Implications from a cohort of 361,662 men. Lancet ii: 933-936. MICHAf~LSSON,G., BERGQVIST,A., VAHLQUIST,A. and VESSBY,B. (1981) The influence of 'Tigason' (Ro 10-9359) on the serum lipoproteins in man. Br. J. Derm. 105: 201-205. MILLER, N. E. and NESTLE, P. J. (1973) Altered bile acid metabolism during treatment with phenobarbitone. Clin. Sci. Mol. Med. 45: 257-262. MILLER, N. E., HAMMETT,F., SALTISSI,S., RAO, S., VAN ZELLER, H., COLTAUT, J. and LEWIS, B. (1981) Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins. Br. Med. J. 282: 1741-1744. MISRA, U. K. (t968) Liver lipids of rats administered excessive amounts of retinol. Can. J. Biochem. 46: 697-701. NATIONAL INSTITUTESOF HEALTH CONSENSUSCONFERENCE(1985) Lowering blood cholesterol to prevent heart disease. J. Am. Med. Ass. 253: 2080-2085.
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O'LEARY,T. T., KANIGSBERG,N., SIMO,I. E., WALKER,J., GOODALL,J. C. and Ooi, T. C. 0986) Changes in serum lipids and high density lipoprotein composition during isotretinoin (Accutane) therapy. IX International Symposium on Drugs Affecting Lipid Metabolism, Florence, Italy. Abstract p. 115. PELKONEN, R., FOGELHOLM,R. and NIKKILA,E. A. (1975) Increase in serum cholesterol during phenytoin treatment. Br. Med. J. 258: 85. PRODOUZ,K. N. and NAVARI,P. M. (1975) Effects of vitamins A and E on rat tissue lipids. Nutr. Rep. Int. 11: 17-28. ROSE, G. and SHIPLEY,M. (1986) Plasma cholesterol concentration and death from coronary heart disease: 10 year results of the Whitehall study. Br. IVied. J, 293: 306-308. STRAUSS,J. S., RAPINI, R. P., SHALITA,A. R., KANECKY,E., POCHI, P. E., COMITE,H. and EXNER,J. H. (1984) Isotretinoin therapy for acne: results of a multicenter dose-response study. J. Am. Acad. Derm. 10: 490-496. SULZBERGER,M. B. and LAZAR,M. P. (1951) Hypervitaminosis A: report of case in adult. J. Am. med. Ass. 146: 788-793. VAHLQUIST,C., LITHELL,H., MICHAfLSSON,G., VAHLQUIST,A. and VESSBY,B. (1985a) Lipoprotein metabolism during sequential treatment with etretinate and isotretinoin in man. In: Retinoids: New trends in research and therapy, pp. 472-475, SAURAT,J. (ed.) Karger, Basle. VAHLQUIST,C., MICHAf~LSSON,G., VAHLQUIST,A. and VESSBY,B. (1985b) A sequential comparison of etretinate (Tigason) and isotretinoin (Roaccutane) with special regard to their effects on serum lipoproteins. Br. J. Derm. 112: 69-76. VAN DER SCVIROEFF,J. G. and JANSEN,H. (1985) Post-heparin lipolytic activity and in vitro lipolysis of serum triglycerides during treatment with isotretinoin. In: RetMoids: New Trends in Research and Therapy, pp. 466-471, SAURAT,J. (ed.) Karger, Basle. WHAYNE,T. F., ALAUPOVlC,P., CURRY,M. D., LEE, E. T., ANDERSON,P. S. and SCHECHTER,E. (1981) Plasma apolipoprotein B and VLDL-, LDL- and HDL-cholesterol as risk factors in the development of coronary artery disease in male patients examined by angiography. Atherosclerosis 39:411-424. WHO EXPERTCOMMITTEE(1982) Prevention of coronary heart disease. WHO Tech. ReD. Set. no. 678. ZECH, L. A., GROSS,E. G., MCCLEAN,S. W., PECK, CJ. L. and BREWER,H. B. (1981) Lipoprotein metabolism in patients receiving synthetic retinoids isotretinoin and etretinate. Arteriosclerosis 1: 390a. ZECH, L. A., GROSS,E. G., PECK, G. L. and BREWER,H. B. 0983) Changes in plasma cholesterol and triglyceride levels after treatment with oral isotretinoin. Archs Derm. 119: 987-993.
0163-7258/89 $0.00 + 0.50 Copyright © 1988 Pergamon Press plc
Guest Editor: R. M. MACKIE
LIPID
METABOLISM
AND
RETINOID
THERAPY
J. R. MARSDEN Department of Dermatology, University Hospital, Nottingham, U.K.
INTRODUCTION Awareness of a connection between vitamin A and lipid metabolism began as a result of descriptions of vitamin A poisoning (Sulzberger and Lazar, 1951; Elliott and Dryer, 1956). These were followed soon after by epidemiological studies describing the direct relationship between levels of vitamin A and serum cholesterol in adolescents (Bring et al., 1955; Hard and Esselburgh, 1960). When synthetic retinoids became available for clinical use in the mid 1970s, reports began to appear describing changes in serum lipids (Katz et al., 1980; Dicken and Conolly, 1980). Further investigations have produced remarkably congruent results, confirming the earlier studies, although there is still no direct information about the mechanism for retinoid hyperlipidaemia, its consequences, and its management. These are important questions because of the increasing use of retinoids: 112 patients were treated with isotretinoin or etretinate for a median period of 136 days in the Department of Dermatology at University Hospital, Nottingham, during 1986. However, a review of the available evidence together with a brief description of normal lipoprotein structure and function provides some clues to help answer these points. NORMAL LIPOPROTEIN METABOLISM Lipids in plasma are transported within particles called lipoproteins. These consist of a central oily core of water-insoluble triglyceride and cholesterol ester, which is surrounded by a protein 'shell' which has a hydrophobic interior and a hydrophilic exterior. These are called apolipoproteins and are designed apo A, B, C, and E; subtypes are referred to with a numerical suffix e.g. apo Aj, apo CH, apo B~00. In conjunction with the apoprotein shell and on the exterior of the particle there is a variable amount of hydrophilic detergent lipid, mainly cholesterol and phospholipid. As well as solubilising lipid, apoproteins perform two other vital functions. One is the activation of 3 enzymes which are pivotal to normal plasma lipid metabolism. The enzymes are lipoprotein lipase (LPL), hepatic triglyceride lipase (HTL), and lecithin-cholesterol acyl transferase (LCAT). The other function is to enable lipoprotein particles to bind to cells by acting as ligands for specific receptors, for instance apo B~00receptors. Lipoproteins are separated and classified according to their density. Chylomicrons are the largest and least dense particles, very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) becoming progressively smaller and denser (Table 1). Although this gives the impression of homogeneity, each lipoprotein class consists of a family of different sized particles. An outline of the pathways of lipoprotein metabolism is shown in Fig. 1. Dietary glyceride is hydrolysed in the small intestine to fatty acids; these are absorbed, together with cholesterol, into the small intestinal mucosal cell. Fatty acids are then re-esterified to triglyceride, and assembled together with cholesterol ester into chylomicrons. The apoproteins within newly synthesised chylomicrons are mainly apo At and apo B48. The latter is specific to chylomicrons and VLDL of intestinal origin, and is synthesised in the jejunum. Chylomicrons are released into the lymphatics and enter the blood via the thoracic duct; their function is to deliver fatty acids to sites of use (skeletal muscle) or 55
0.95-1.006
1.006-1.019
1.019-1.063 1.063-1.210
IDL
LDL HDL
<0.95
Chylomicrons
VLDL
Density (g/ml)
Particle
VLDL Liver, intestine
VLDL
Liver
Intestine
Source apo apo apo apo apo apo apo apo apo apo apo
A[, A . , A~v B4s, Ci, Cjl, Cm E Bi00, Cl, C]l, Cm E B~00, apo C E B~00 A~, AH Ci, C . , Cm E
Apoproteins
Cholesterol ester Cholesterol ester Phospholipid
Tfiglyceride and cholesterol ester
Mainly triglyceride
Mainly triglyceride
Core lipid content
Function
Transport of cholesterol from the liver to extra-hepatic cells Transport of cholesterol from extra-hepatic cells to the liver and other sites
VLDL-LDL intermediate
Transport of triglycerides synthesised in the liver
Transport of dietary fat as triglyceride and cholesterol esters
TABLE 1. Characteristics of Normal Human Plasma Lipoproteins
'7
t~
Lipid metabolism and retinoid therapy
57
ado BIO 0 receptor apo E receptor
LIVER
apo E receptor
nascent HDL
LPL
other sites
I-'1 , ~ o f catabolism
PL
LPL
intestine extrahepatic cell
FIG. 1. An outlin~fof normal lipoprotein metabolism (for details see text). C = chylomicrons; Crem = chylomicron remnants; LPL = lipoprotein lipase; C,PL = cholesterol and phospholipid; LCAT = lecithin cholesterol acyl transferase; LTP = lipid transfer protein; C,TG = cholesterol and triglyceride; ACAT = acyl cholesterol acyl transferase.
storage (adipose tissue). The vascular endothelium at these sites is rich in lipoprotein lipase, and this enzyme is activated by apo C. which the chylomicron acquires from H D L within the circulation. The result is that the core triglyceride is hydrolysed, and free fatty acids are released and taken up by muscle or fat cells. Consequently, the volume of the chylomicron progressively decreases and this produces excess surface components. These are mainly cholesterol, phospholipid and the A apoproteins, and are transferred to HDL. In exchange, the shrunken chylomicron remnant acquires apo E from HDL, which confers on it the ability to bind to liver cells which have specific receptors for chylomicron apo E. In this way the remnants are cleared from the blood and deliver dietary cholesterol to the liver. Lipid which is synthesised within the liver clearly requires a separate pathway for distribution. Triglyceride and cholesterol esters are packaged within VLDL particles, which are secreted by the liver into the blood. Newly secreted, or 'nascent', VLDL contains apo C, apo E, and apo B~00.As with chylomicrons, VLDL is metabolised at vascular endothelium in muscle and fat by lipoprotein lipase, and similarly this enzyme is activated by HDL-derived apo C,. As the VLDLs are depleted of triglyceride they become denser and smaller intermediate density lipoproteins (IDL) and the redundant surface phospholipid, cholesterol and apoproteins are transferred to HDL. However, the hydrophilic polar cholesterol molecule cannot be stored within lipoproteins unless it is esterified to make it hydrophobic. This function is carried out by the enzyme lecithin cholesterol acyl transferase (LCAT) which removes fatty acid from HDL phospholipid (lecithin) and transfers it to the newly-acquired cholesterol. LCAT is activated by the H D L protein apo A,. Some of the newly esterified cholesterol is transferred back to IDL and LDL by lipid transfer protein (LTP), a protein which redistributes and equilibrates lipids between lipoprotein
58
J . R . MARSDEN
classes. In this way triglyceride is removed from VLDL, and the cholesterol ester content of the products of VLDL catabolism is increased. Some of the products of catabolism, VLDL remnants and IDL, are taken up by apo E receptors on the surface of liver cells, and cleared from the blood. However, the majority of VLDL continues to be depleted of triglyceride until it enters the LDL density range. Low density lipoprotein predominantly contains cholesterol ester, and one molecule of apo B~00in each particle. Normally LDL is produced solely from VLDL catabolism. Most cells in the body contain apo B~0oreceptors on their surface to which LDL binds. The receptor-ligand combination is then internalised into lysosomes, and the cholesterol ester released; LDL can also enter the cell via a non-receptor pathway. Such large amounts of cholesterol are normally available to extra-hepatic cells that intracellular cholesterol production is virtually switched off by repression of HMG CoA reductase; this enzyme is the rate-limiting step in cholesterol synthesis. Cholesterol which is surplus to requirements for membrane and steroid synthesis has therefore to be returned to the liver, which is the only important site of catabolism. High density lipoproteins are derived from particles secreted by the liver and intestine. Nascent HDL are disc-shaped, consisting of little more than phospholipid membrane and apo Ax. These particles rapidly form into spherical, less dense HDL 3 and the larger HDL2 as they acquire the surplus surface components of chylomicrons and VLDL and become cholesterol ester enriched. To date, three major functions for HDL have been described. Firstly, it provides a sink for redundant surface components of the triglyceride rich lipoproteins and a source for apo C, and apo E, and secondly, it acts as a factory for production of cholesterol ester within plasma via the LCAT reaction. The third function is the movement of cholesterol from extra-hepatic cells to other sites of use, mainly the liver. This component of the plasma cholesterol flux is called reverse cholesterol transport. Recent evidence shows that specific HDL receptors exist on such tissue as smooth muscle cells and fibroblasts. These receptors bind apo E free HDL, and the ligand is probably apo A~. Binding of cholesterol poor HDL e.g. HDL 3, depletes cellular cholesterol, and binding is up-regulated in proportion to the cellular content of cholesterol. As a result of this HDL-induced cholesterol efflux from cells, more cholesterol ester enriched HDL 2 is produced via LCAT to add to that already produced from chylomicron and VLDL catabolism. Despite extensive research, the metabolic fate of HDL 2 is still not entirely clear. Some of it is removed from the circulation by binding to specific HDL receptors on the liver. Some is further enriched with cholesterol ester to produce HDL~, also known as HDL E because it contains apo E and therefore binds to hepatic apo E receptors. Some of the cholesterol ester is exchanged with triglyceride in VLDL by lipid transfer protein producing triglyceride rich HDL2. This triglyceride is removed by apo A. activated hepatic triglyceride lipase, and so HDL 2 is converted back to HDL 3. As previously described, some cholesterol ester generated in HDL2 is transferred to IDL and LDL, and some of this finds its way back to the liver where it is taken up by the apo Bt00 receptor. From this description it is apparent that normal lipoprotein metabolism is complex and relies on many interdependent processes. Consequently, measurements of lipid, lipoprotein and apoprotein mass have limited value in defining the mechanisms underlying abnormal iipoprotein metabolism, although they provide useful clues. Such measurements provide the bulk of published information on retinoid hyperlipidaemia. The second part of this review describes these findings, and then attempts to use them to suggest possible explanations for retinoid hyperlipidaemia. ISOTRETINOIN Increases in concentration of serum triglyceride and cholesterol have been repeatedly described after treatment with isotretinoin (Katz et al., 1980; Lyons et aL, 1982; Zech et al., 1983; Marsden et al., 1984a and 1984b; Vahlquist et al., 1985b, Bershad et al., 1985). The changes are directly proportional to the dose used as shown in Fig. 2, and this data is consistent with the findings of Strauss et aL, 1984. This linear relationship continues
Lipid metabolism and retinoid therapy
59
triglyceride
cholesterol m
m
1.0
increase (mmol/I)
i I
I
I
I
I
I
_1
I
0
I
I
I
I
I I
dose ( m g / k g / d a y )
FXG.2. Relationship between dose of isotretinoin and increases in levels of serum cholesteroland triglyceride (0.05mg/kg/day n = 9, 0.2mg/kg/day n = 8, 0.4mg/kg/day n = 8, 0.8 mg/kg/day n = 12, 1.0 mg/kg/day n = 10). beyond the highest dose of 1 mg/kg/day shown in the figure, but adequate dose-response data above this point are not available. Pre-treatment levels of triglyceride and cholesterol which are within the reference range are unrelated to the increase during treatment (Marsden et al., 1984b; Vahlquist et al., 1985b). Increased triglyceride and cholesterol levels are apparent within 4 weeks of starting isotretinoin, and usually change little thereafter (Zech et al., 1983; Marsden et al., 1984a and 1984b). However, in a larger study of 60 patients, triglyceride levels in females continued to increase for 12 weeks and total serum cholesterol levels in males were greatest at 4 weeks with a 15% increase which declined to only a 5% increase at the end of treatment (Bershad et al., 1985). These increases are reversed within 2-4 weeks of stopping isotretinoin (Lyons et al., 1982; Marsden et al., 1984a and 1984b; Marsden, 1987). Separation of lipoproteins by ultracentrifugation shows that the largest increase in triglyceride mass is in the V L D L fraction, with smaller increases in LDL-triglyceride and a small increase or no change in HDL-triglyceride (Marsden et al., 1984a; Vahlquist et al., 1985a; Gollnick et al., 1985). In contrast, the largest increase in cholesterol mass is in LDL, with a small increase in VLDL-cholesterol, and a decrease in HDL-cholesterol (Zech et aL, 1983; Marsden et al., 1984a; Vahlquist et al., 1985b; Gollnick et al., 1985). These findings suggest that the number of V L D L and L D L particles is increased, although they do not exclude the possibility that the particles are also carrying more lipid. However it is now clear that concentrations of apo B are also increased after isotretinoin (Vahlquist et al., 1985b; Gollnick et al., 1985; Marsden, 1987), and that this correlates with total serum cholesterol and triglyceride (Laker et al., 1987). This strongly supports the idea that V L D L and L D L particle number is increased but gives no indication about which predominates. In one study of 13 subjects measurements of apo A~ levels show a small but consistent decrease (Laker et al., 1987). This difference did not reach statistical significance. Two other studies have not shown any difference (Vahlquist et al., 1985b; O'Leary et al., 1986) and so any change in apo A, is likely to be very small. Therefore, there is unlikely to be much difference in the number of H D L particles during treatment with isotretinoin, and recent evidence suggests that there is simply a reduction in cholesterol content of the HDL2 fraction with no difference in HDL3 cholesterol or H D L phospholipid (O'Leary et al., 1986). ETRETINATE Changes in serum lipids during treatment with etretinate are very similar to those described with isotretinoin. Mean levels of both triglyceride and cholesterol are increased (Zech et al., 1981; MichaElsson et al., 1981; Ellis et al., 1982; Vahlquist et al., 1985b; Marsden, 1987). The changes are apparent as early as 2 weeks after starting etretinate (MichaElsson et al., 1981; Marsden, 1987) and have usually reached their maximum by 8 weeks. Whilst these increases appear to be related to dose (Ellis et al., 1982), the precise
60
J.R. MARSDEN
dose-response relationship is unclear because of lack of comparison between groups of similar ages and pre-treatment lipid values. Like isotretinoin, the pre-treatment values for triglyceride and cholesterol do not predict the subsequent increase (Micha~lsson et al., 1981; Vahlquist et al., 1985b). Levels of both triglyceride and cholesterol return to normal after 4 weeks of stopping etretinate (Ellis et al., 1982; Marsden, 1987), confirming that its prolonged terminal elimination half-life does not delay reversal of hyperlipidaemia. Ultracentrifugation studies show that the largest increase in triglyceride mass is in the VLDL fraction, with smaller increases in LDL-triglyceride and no change in HDL-triglyceride (Micha~lsson et al., 1981; Vahlquist et al., 1985b; Gollnick et al., 1985). Although it is still unclear whether the largest increase in cholesterol mass is in VLDL (Micha~lsson et al., 1981; Zech et al., 1981; Gollnick et al., 1985) or in LDL (Vahlquist et al., 1985b), all of these studies show a small reduction in levels of HDL-cholesterol. There is no information about whether this reduction is in the HDL2 or HDL 3 fraction. Ellis et al. (1982) found no difference in HDL-cholesterol. It seems clear that there are small increases in apo B during treatment with etretinate (Micha~lsson et al., 1981; Vahlquist et al., 1985b; Gollnick et al., 1985) although there is no information about whether this increase is in VLDL or LDL apo B. However, levels of apo A~ and apo A. are unchanged, and so, like isotretinoin, the decrease in HDL-cholesterol presumably reflects alteration in HDL composition rather than a decrease in the number of HDL particles. The changes in lipids and lipoproteins resulting from treatment with etretinate are not as well characterised as they are after treatment with isotretinoin. Isotretinoin seems to be more potent, producing greater increases in triglyceride and cholesterol compared with an equimolar dose of etretinate (Vahlquist et al., 1985b). A minority of patients treated with both drugs have no changes in lipid levels during treatment. The explanation for this is unclear. However, the similarity of the changes suggests a common cause, and so it is easier to discuss both drugs together when considering possible mechanisms for retinoid hyperlipidaemia. MECHANISMS Explanations for retinoid hyperlipidaemia can be broadly classified as increased production or decreased clearance of either chylomicrons or of endogenously synthesised lipid or lipoproteins. It is extremely unlikely that the changes in serum lipids result from increased absorption of dietary lipid and increased chylomicron production, or from reduced chylomicron clearance. Chylomicrons are not present in the fasting serum samples of subjects with increased lipid levels due to retinoids because the serum is not lipaemic, and chylomicrons are not found on lipoprotein electrophoresis (Lyons et al., 1982; Marsden and Laker, unpublished observations). Also, the activity of lipoprotein lipase released into serum by intravenous heparin is no different during treatment with isotretinoin (van der Schroeff and Jansen, 1985; Bershad et al., 1985), and LPL activity in adipose tissue is no different during treatment with either isotretinoin or etretinate (Vahlquist et al., 1985b). A small decrease in skeletal LPL activity was found in this study but its relevance is unclear. Isotretinoin and etretinate bind extensively to lipoproteins in plasma (Zech et al., 1981), but this does not seem to affect the ability of LPL to hydrolyse triglyceride from VLDL (van der Schroeff and Jansen, 1985). The rate of removal from serum of an intravenous bolus of Intralipid (medium chain length triglyceride) may be used as a model for chylomicron clearance (Carlsson and R6ssner, 1972). There are small reductions in the elimination rate constant during treatment with isotretinoin (Vahlquist et al., 1985a; Gollnick et al., 1985) and etretinate (Vahlquist et al., 1985a). However, these observations are clearly not relevant to retinoid hyperlipidaemia because chylomicronaemia is absent. Alternatively, there could be increased synthesis of triglyceride and cholesterol in the liver. Retinoic acid produces increased synthesis of triglyceride from mevalonate in rat liver homogenates (Erdman et al., 1977), and total liver lipid is increased by feeding rats retinyl acetate (Prodouz and Navari, 1975) but in both cases liver cholesterol content is reduced.
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However, large doses of retinol increases hepatic triglyceride and cholesterol (Misra, 1968). This increase in triglyceride production requires a supply of carbon skeleton. The likeliest source of this would be free fatty acids (FFA) delivered to the liver from adipose tissue. However, retinoic acid does not increase plasma FFA levels in the rat (Gerber and Erdman, 1979), and hypertriglyceridaemia is not mediated by adipose tissue lipolysis due to adrenaline. Isotretinoin does not increase plasma FFA levels in man (Laker et al., 1987) although this does not exclude increased flux of FFA into the liver or an increase in the proportion of FFA used for triglyceride synthesis rather than fl-oxidation. However, this seems unlikely because the same study shows no change in concentration of the products of fl-oxidation, and animal experiments show that this is not reduced by retinoids (Sherratt, personal communication). The closest analogy in man is familial hypertriglyceridaemia which is due to increased hepatic triglyceride synthesis (Brunzell et al., 1977). This results in increased VLDL and LDL triglyceride and decreased HDL cholesterol but, unlike isotretinoin and etretinate, no change in LDL cholesterol (Brunzell et al., 1983). Therefore there is no direct evidence to date to confirm that increased hepatic lipid synthesis is the primary cause of retinoid hyperlipidaemia in man. Instead of a primary increase in lipid synthesis, there could be an increase in synthesis of apolipoprotein, or a decrease in clearance of VLDL and/or LDL. As already described, both isotretinoin and etretinate increase levels of apo B, the major apoprotein in VLDL and the only apoprotein in LDL, and both VLDL and LDL are increased. Increased apo B synthesis would result in increased VLDL secretion, but not necessarily increased LDL apo B (Eaton et al., 1983). Because there is no data describing lipoprotein kinetics during retinoid treatment, the evidence in support of this hypothesis is indirect. Firstly, increasing production of VLDL would not be expected to alter its lipid composition and this is no different after isotretinoin (Marsden et al., 1984b). Secondly, there is close similarity between the changes in serum lipids during treatment with retinoids and those occurring in the disease familial combined hyperlipidaemia. This disorder is characterised by increases in both VLDL and LDL triglyceride and cholesterol and HDL triglyceride, and decreases in HDL cholesterol (Brunzell et al., 1983), and is due to increased apo B synthesis (Chait et al., 1980). Another characteristic of this disorder is its variable expression between individuals, a notable feature of retinoid hyperlipidaemia. Decreased clearance of VLDL and LDL seem unlikely because the accumulation of partially catabolised VLDL remnants would alter the lipid composition of VLDL by increasing the proportion of triglyceride. Also, there is no accumulation of remnants or IDL on lipoprotein electrophoresis (Marsden and Laker, unpublished observations). Thirdly, reduced clearance of VLDL in familial type Ill hyperlipoproteinaemia results in low levels of LDL, as opposed to the increased values found during retinoid treatment, and reduced clearance of LDL in familial hypercholesterolaemia is not usually associated with increased VLDL (Kachadurian and Utermann, 1973). Reduced levels of HDL2 cholesterol during treatment with isotretinoin could either mean reduced production from HDL 3 because of decreased catabolism of VLDL, or reflect greater cholesterol ester-triglyceride exchange between HDL2 and VLDL, IDL and LDL. Both LDL and HDL are triglyceride enriched during treatment with isotretinoin, which is consistent with the latter suggestion (Marsden et al., 1984b). Because several other drugs which increase serum triglyceride and cholesterol are inducers of hepatic microsomal cytochrome P-450 enzymes (Durrington, 1979; Miller and Nestel, 1973; Pelkonen et al., 1975; Bolton et al., 1980) it was suggested that isotretinoin might also have this effect. Isotretinoin does not increase clearance of antipyrine in man, a drug entirely dependent on metabolic clearance by the liver (Marsden et al., 1985) and slightly reduced the activities of several microsomal cytochrome P-450 enzymes (Finnen and Shuster, 1984). Further information is clearly required to identify the cause of retinoid hyperlipidaemia. The issue has been over-simplified in this discussion. For example, small increases in apo B can occur in response to increased hepatic triglyceride synthesis (Brunzell et al., 1983). J.P.T. 40/I--E
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The next step is to show whether or not VLDL, IDL, and LDL kinetics are changed by retinoid treatment and this investigation is now in progress. CONSEQUENCES OF RETINOID HYPERLIPIDAEMIA Although the changes in lipoprotein phenotype during retinoid treatment are well characterised, their consequences are not. There are two major theoretical risks. These are acute pancreatitis as a result of very high levels of serum triglyceride and increased atherogenesis. Neither has yet been reported. Pancreatitis is only likely as a consequence of severe hypertriglyceridaemia with fasting levels greater than l0 mmol/1. Values of this order have been found during treatment with isotretinoin (Lyons et al., 1982; Ellis et al., 1982; Dicken and Connolly, 1980; Katz et al., 1980) and all of these patients had increased pre-treatment levels. There is not enough information to know whether these patients had an underlying primary hyperlipoproteinaemia, but very large synergistic increases in serum triglyceride occur in patients with familial hypertriglyceridaemia who are then exposed to a secondary cause of hypertriglyceridaemia such as diabetes or alcohol (Chait and Brunzell, 1983). This is quite different from the additive response seen in patients who already have secondary hypertriglyceridaemia due to diabetes, alcohol or obesity. Treatment with isotretinoin and etretinate results in total levels which rarely exceed 3.5-4.0 mmol/1 (Gollnick et al., 1985; Marsden, unpublished observations). Similar small increments in triglyceride levels were found in relation to body mass index during treatment with isotretinoin by Bershad et al. (1985). The probability of increased atheroma and risk of ischaemic heart disease (IHD) is of much greater importance. Although it is a popular opinion that increased levels of triglyceride do not correlate with risk of IHD independently of other variables, there is increasing evidence to challenge this view. This largely stems from recent re-examination of the data relating triglyceride levels to IHD using more sensitive statistical techniques than multivariate analysis. The results show that serum triglyceride levels predict risk of IHD in males over 50 (Abbott and Carroll, 1984), and the correlation is particularly strong in both sexes when HDL cholesterol is low (Castelli, 1986). Comparison between risk factors in women over 50 shows that triglyceride levels are more powerful predictors of IHD than LDL levels. The association between levels of total serum cholesterol and LDL cholesterol with risk of IHD is much better known although its significance in the context of retinoid hyperlipidaemia has often been trivialised. This is largely because of the concept that cholesterol levels which lie within the 'normal range' can be ignored as far as IHD is concerned. This is incorrect. The relationship between cholesterol level and risk of IDH is log-linear, and therefore the gradient of risk is continuous throughout the reference range. This means that increments in cholesterol level produce increments in risk, and is well illustrated by the findings of the Whitehall study of 0.64 IHD deaths/100 males/10 years for an increase in cholesterol of 1 mmol/l (Rose and Shipley, 1986). However, when cholesterol concentrations rise above 6.5 mmol/l, risk of IHD increases much more rapidly (Martin et al., 1986). About 25% of western Europeans have values above this level, and are categorised as being at high risk of IHD by both the European Atherosclerosis Society (1987) and by the National Institutes of Health consensus panel (1985). Yet the upper limit of the reference range for serum cholesterol is 7.1-7.8 mmol/1 in most U.K. laboratories. It denotes the 95th centile and has no relevance to risk of IHD. As well as increasing levels of total serum cholesterol, retinoids alter the proportion of cholesterol in LDL and HDL. Concentrations of HDL cholesterol correlate indirectly and independently with risk of IHD (Kannel et al., 1979), especially HDL2 cholesterol which is the fraction reduced by retinoid treatment (Miller et al., 1981). The practical relevance of this is that although total serum cholesterol may not change during retinoid treatment, this could conceal considerable increases in LDL and decreases in HDL cholesterol. Measurement of the ratio L D L : H D L cholesterol before treatment and after 8 weeks on therapy would therefore be useful, and many clinical chemistry laboratories can now
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provide this. The level of apo B also increases; apo B concentrations correlate well with proven IHD especially at lower levels of total serum cholesterol (Whayne et al., 1981) and so might provide a more accurate estimate of risk from retinoids. M A N A G E M E N T OF H Y P E R L I P I D A E M I A There is little published information about this. It is very rare for pre-treatment values which lie within the reference range to rise by more than 1-2 mmol/1, and so repeated measurements are not necessary, values obtained before treatment and after 4-8 weeks are sufficient to assess the response. However, patients with pre-existing hypertriglyceridaemia, especially those without evidence of a secondary cause, are at risk of very high triglyceride levels and possibly of pancreatitis. If pre-treatment values are 2-3 mmol/1, treatment can be started at low doses, but with measurements of triglyceride levels at weekly intervals. With fasting values in excess of 5 mmol/l, triglyceride levels can fluctuate enormously, and so treatment should be stopped. Although reducing the dose will probably reduce lipid levels, the amount by which they decrease is unpredictable. Dietary changes to reduce retinoid hyperlipidaemia are now being investigated. Supplementation with fish oil rich in eicosapentaenoic acid and docosohexaenoic acid (MaxEPA) produced large reductions in triglyceride levels, but much smaller changes in total cholesterol, and no increase in H D L cholesterol or decrease in apo B (Marsden, 1987). It is too early to say whether this has any therapeutic value but is most likely to be of value to patients with pre-existing hypertriglyceridaemia. Preliminary results of the effect of the low fat diet recommended by the WHO Expert Committee (1982) suggest that the response is small and due to coincidental weight loss in obese subjects rather than reduction of fat intake (Marsden and Marenah, unpublished data). However, until this is confirmed, it may be worth making dietary changes, especially if treatment is for more than a few months. Reduction of calories is effective in reducing triglyceride, and sometimes cholesterol also, in obese patients. In the non-obese, total calories should remain unchanged but total dietary fat can be reduced to about 30% of the calories, and energy from unrefined carbohydrate increased accordingly. Polyunsaturated fat should be increased to 10% of calories. Triglyceride levels decrease with avoidance of alcohol, sometimes dramatically. There is no information about the efficacy or safety of hypolipidaemic drugs in the treatment of retinoid hyperlipidaemia. CONCLUSIONS Both isotretinoin and etretinate cause hyperlipidaemia which is dose-related and reverses when treatment is stopped. The available evidence suggests that the likeliest explanation is increased production of VLDL but measurements of lipoprotein kinetics are necessary to confirm this. Large increases in triglyceride levels seem to be confined to subjects with pre-existing hypertriglyceridaemia, but smaller increases in triglyceride, cholesterol and apoprotein B and decreases in HDL cholesterol are very common. These changes in serum lipids and lipoproteins are likely to be associated with increased atherogenesis, and increased risk of IHD if treatment is continued for long periods. The best strategy to reduce this risk is to develop retinoid analogues which do not cause hyperlipidaemia. Acknowledgements--Much of the work referred to in this chapter was made possibleby financialsupport from Roche.
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