Pergamon
Prog. LipidRes. Vol. 35, No. 2, pp. 93-132, 1996 Copyright © 1996 ElsevierScience Ltd. All rights reserved Printed in Great Britain 0163-7827(95)00014-3 0163-7827/96/$32.00
EFFECTS OF DIETARY FATTY ACID COMPOSITION ON PLASMA CHOLESTEROL Pramod Khosla*,:~ and Kalyana Sundramt *Foster Biomedical Research Laboratory, Brandeis University, Waltham, MA, U.S.A. and t Pa/m Oil Research Institute of Malaysia P.O. Box 10620, 50720, Kuala Lumpur, Malaysia
CONTENTS I. Introduction II. Dietary fa.tty acids Ill. Regression equations predicting the effects of dietary fatty acids on plasma lipids A. Effects of fatty acid classes on total cholesterol concentrations B. Effect:s of fatty acid classes on LDL-C and HDL-C concentrations C. Effects of individual fatty acids on total cholesterol - - human and animal studies D. Effects of individual fatty acids on LDL-C and HDL-C concentrations E. The non-linear response to linoleic acid IV. Mechanism of action of dietary fatty acids A. Animal studies - - hamsters B. Animal studies - - guinea-pigs C. Animal studies - - non-human primates D. Studies in tissue culture V. Review of human studies A. Laurie and myristic acid B. Palmitic acid C. Disparate effects of palmitic acid D. Stearic acid E. Oleic and linoleic acid VI. Trans-Fatty acids A. Consumption of trans-fatty acids B. Trans-Fatty acids and CHD - - epidemiological data C. Trans-Fatty acid effects on plasma lipids D. Mechanism of action of dietary trans-fatty acids VII. Summary References
93 95 96 96 97 97 100 100 102 102 110 111 113 115 119 119 122 124 124 124 125 125 125 127 128 128
ABBREVIATIONS AAD AHA apo CHD HDL-C LDL-C MUFA PUFA SFA
average American diet American Heart Association apolipopt otein coronary heart disease high density lipoprotein cholesterol low density lipoprotein cholesterol monounsaturated fatty acid(s) polyunsaturated fatty acid(s) saturated fatty acid(s)
TC VLDL-C 12:0 14:0 16:0 18:0
18:1 18:2
serum or plasma total cholesterol very low density lipoprotein cholesterol lauric acid myristic acid palmitic acid stearic acid oleic acid linoleic acid
I. I N T R O D U C T I O N D e a t h f r o m c a r d i o v a s c u l a r disease c o n t i n u e s to a c c o u n t f o r a m a j o r p r o p o r t i o n o f c h r o n i c - d i s e a s e r e l a t e d m o r t a l i t y in i n d u s t r i a l i z e d c o u n t r i e s . W h i l e t h e f a c t o r s c o n t r i b u t i n g to its m a n i f e s t a t i o n a r e m u l t i f a c t o r i a l , ~62"2°°o n e o f t h e r e a d i l y m e a s u r e d p a r a m e t e r s is t h e t o t a l s e r u m o r p l a s m a c h o l e s t e r o l ( T C ) c o n c e n t r a t i o n , w h i c h is p o s i t i v e l y r e l a t e d to i n c r e a s e d risk fi,om c o r o n a r y h e a r t disease ( C H D ) . 122'19°'199A d d i t i o n a l l y , as t h e i n t r i c a c i e s o f l i p o p r o t e i n m e t a b o l i s m h a v e u n r a v e l e d o v e r the last t w o d e c a d e s , a w i d e b o d y o f d a t a indicates that while a high concentration of low density lipoprotein cholesterol (LDL-C) SPresent address: Department of Nutrition and Food Science, Wayne State University, Detroit, MI, U.S.A. 93
94
P. Khosla and K. Sundram
is positively associated with risk, high density lipoprotein cholesterol (HDL-C) shows a negative association, i.e. a low level of H D L - C increases risk. 58'~5~'2°° The ratio of L D L - C / H D L - C (or TC/HDL-C), therefore, is an easily determined indicator of risk. 24'59 Since humans normally transport two to three times as much cholesterol in LDL as opposed to HDL, the primary focus of attention has been factors that affect LDL-C concentrations. Accordingly, the principal goal for any individual perceived to be at risk is to lower their LDL-C concentration. Although this objective can be achieved by drug therapy, one of the first lines of treatment is dietary intervention. For almost 90 years, it has been known that diet can affect TC levels and, in the case of laboratory animals, the ability to develop atherosclerosis. 9° Based on animal and human studies, numerous dietary factors have been implicated in their ability to affect TC levels. Perhaps the most scrutinized of all these factors is dietary fat (and its constituent fatty acids). The ability of dietary fats to influence TC has been studied extensively since the 1950s.3.5 7.18.22.23The classical studies by Hegsted, Keys and their colleagues 8'9'61-63,79,99-1°6 dissected the cholesterolemic effects of dietary fats into their constituent fatty acid classes, namely saturated, monounsaturated and polyunsaturated fatty acids (SFA, M U F A and PUFA, respectively). Extensive studies by these workers showed that SFA were twice as effective in raising TC as the P U F A were in lowering it, while M U F A were considered neutral. More detailed studies revealed that amongst the SFA, only those with 12, 14 and 16 carbons (lauric, myristic and palmitic) increased TC, while the 18 carbon, stearic acid, like M U F A was neutral. 79,j°6 Both groups of workers translated the results from their studies into simplified mathematical relationships which have been useful to scientists and clinicians in helping to formulate recommendations and dietary guidelines for the general public with a primary goal of lowering TC concentrations (specifically by decreasing LDL-C) and thereby reducing the risk from cardiovascular disease. Based on the studies from Keys, Hegsted and coworkers 8.9.6163.79.99106 on the efficacy of PUFA-containing diets to lower TC, as well as numerous epidemiological studies, 95'97'107'121'125'142'176which revealed positive associations between SFA intake and increased C H D risk, active recommendations were made to increase P U F A consumption at the expense of SFA. Accordingly SFA consumption decreased in the United States from the 1950s onwards and this was accompanied by a corresponding increase in P U F A consumption) 92 However, with the advent of newer methodologies, and an increased awareness of lipoprotein metabolism, attention has shifted from the effects of dietary fatty acid classes on TC, to the effects of specific fatty acids on specific lipoproteinfractions. As a consequence, results from several key animal 75:35,~63and human studies H8:95,2°~.224over the last decade have challenged two of the most fundamental tenets inherent in the Keys-Hegsted equations, namely the "equivalence" of the 12-16 carbon SFA and the "neutrality" of M U F A ) 38'146'2:1 Additionally, since we principally consume dietary fatty acids as part of triacylglycerols and not as fatty acids per se, increasing importance is being paid to the spatial arrangement of the fatty acids in the triglyceride molecule, and whether or not this affects the cholesterolemic response observed. Hg.17sFinally, over the last 5 years, some provocative epidemiological data ~2,2°8,2~3as well as some controlled human clinical trials 94:3H47'156'222has revived interest in dietary trans-fatty acids (t-FA) - - geometrical isomers of unsaturated fatty acids - - and how their consumption affects lipoprotein cholesterol concentrations. Since humans normally consume a mixture of different fats, and therefore mixtures of different fatty acids, the net effect on TC or individual lipoproteins will be the sum of, in all likelihood, numerous and possibly opposing effects that are going on in parallel. In order to decipher the "key players" in this situation it is necessary to have information on what each fatty acid does. The aim of this article, therefore, is to review recent studies which have advanced our knowledge on how plasma lipid and lipoprotein metabolism is affected by individual fatty acids, as opposed to classes of fatty acids. Our primary focus will be to provide an update on current thinking (encompassing the last 5 years) with regards to how individual fatty acids may impact plasma lipids, and how these effects may be modified by the presence or absence of other fatty acids. This review is divided into
Effects of dietary fatty acid composition on plasma cholesterol
95
three basic sections. In the first we review the various studies that have delineated regression equations predicting the effects of specific fatty acids on TC and lipoprotein cholesterol concentrations. In the second, we review the mechanism of action of dietary fatty acids on lipoprotein metabolism. The third section reviews the observed responses in plasma lipid concentrations in recent human studies assessing dietary fatty acid effects. It is not our purpose to provide an exhaustive review on dietary fats (or fatty acids) and plasma lipids/CHD. Several excellent reviews have recently covered some of these aspects in greater d e p t h , tv'43,~'6s'l~ II. D I E T A R Y F A T T Y A C I D S
These have ge,nerally been classified into four groupings: SFA, MUFA, n-6 PUFA and n-3 PUFA. The SFA can vary in chain length from 4 to 20 carbons with the 12-18 carbon ones predominating in the diet. Amongst these four, SFA, palmitic and stearic acid predominate with palmitic being the most abundant. SFA in the diet arise from the consumption of" dairy and animal products, as well as products derived from certain vegetable oils, namely coconut, palm and palm kernel oils. (Dairy and animal products also contain cholesterol, whilst all vegetable oils are essentially cholesterol-free.) The principal dietary M U F A is oleic acid. In fact in nature, of all fatty acids, palmitic and oleic are the most abundant in their respective classes. Linoleic acid is the predominant dietary n-6 PUFA. Pri:acipal dietary sources of M U F A and n-6 PUFA are derived from the consumption of vegetable oils (e.g. olive, soybean, corn and safflower oils). To keep this report within "manageable" proportions the n-3 PUFA will not be discussed further. In recent years, an additional "class" of fatty acids has come into the picture, the trans-fatty acids (t-FA). These are geometrical isomers of unsaturated fatty acids formed during biohydrogenation (in ruminant animals) and industrial hydrogenation. The principal trans-fatty acids in the diet are the t-MUFA. In the remainder of this report PUFA will generally refer to the 18:2 fatty acid, and M U F A will generally refer to oleic acid. t-MUFA will be specifiedL as indicated. Typical (i.e. average) diets in the U.S. supply 13-15%en (% energy) from SFA, 13-16%en from M U F A and 5-7%en from PUFA. t-MUFA intake ranges from 1 to 3%en. For a person consuming 2500 kcal/d this would translate into daily consumptions of -36-42 g/d of SFA, 36-45 g/d of MUFA, 14-19 g/d PUFA and 3-9 g/d of t-MUFA. Using the data of Ahrens and BoucheP for a simulated American diet, the specific contributions from individual fatty acids would be 1.5, 8.2, 4.3, 14.7 and 6.4%en for 14:0, 16:0, 18:0, 18:1 and 18:2, respectively (or 4, 23, 12, 41 and 18 g/d, respectively). While 14:0 intakes may appear unimportant especially when expressed as °/oen,~4'1~ this may not be the case when one considers the absolute intake (-4 g/d for a 2500 kcal/d diet). In addition, typical U.S. diets provide ~ 450 mg/d of cholesterol. Based on numerous clinical studies and several epidemiological reports various health agencies have recommended c,urtailing SFA and cholesterol intakes as a means of reducing risk from CHD. Thus the American Heart Association Step 1 diet calls for reducing fat and cholesterol intake to _< 30%en and ___125 mg cholesterol/1000 kcal/d, respectively. This prudent diet should comprise < 10%en from SFA (i.e. _< 28 g/d of SFA for a person with a caloric intake of 2500 kcal/d). The AHA Step II diet advocates further reductions in SFA ( _< 7%en or ~19 g/d SFA for a person with a caloric intake of 2500 kcal/d) and cholesterol ( _ 100 mg/1000 kcal/day). The above are average figures based on a 2500 kcal/d diet. However, it should be borne in mind that for given individuals and for specific segments of the population, there may be tremendous variation. Of the studies described in Table 5 (and discussed at a later point), caloric intakes ranged from 1300 to 5400 kcal/d. While for these two extreme ranges, the per cent energy from a specific fatty acid will be constant, the absolute amounts will vary four-fold. Similarly, expressing dietary cholesterol intake as mg/1000 kcal only reflects constant consumption across different diets if caloric intake is also constant.
96
P. Khosla and K. Sundram
TABLE 1. Regression Equations Predicting the Effect of Fatty Acid Classes on Serum Cholesterol Concentrations in Man Equation TI Equation T2 Equation T3 Equation T4 Equation T5
ASC = 2.74AS - 1.3lAP (ASC = 2.40ASt - 1.20AP + 1.5AC~/2) ASC = 2.16AS - 1.65AP + 0.065AC (ASC = 2.74AS* - 1.83AP + 0.071AC)~ ASC = 2.16AS* - 0.12AMb - 0.60AP ASC = 2.10AS - 1.16AP + 0.067AC ASC = 2.02AS* - 0.48AM - 0.96AP
(Keys et al., 1957)~ (Keys et al., 1965)~°~ (Hegsted et al., 1965)79 (Mensink and Katan, 1992)~48 (Hegsted et al., 1993)78 (Yu et al., 1995)221
ASC denotes the change in serum cholesterol in mg/dl, AS denotes changes in %en for all the SFA, AS* denotes changes in %en for SFA with 12, 14 and 16 carbons only, AM denotes changes in %en for MUFA (principally oleic acid), AP denotes the change in %en for PUFA (principally linoleic acid) and AC denotes the change in dietary cholesterol in mg/1000 kcal. aCalculated from the published data of Hegsted et al. 79 bMensink and Katan reported that the coefficient was not significantly different from zero. ~4s
III. R E G R E S S I O N E Q U A T I O N S PREDICTING THE E F F E C T S OF D I E T A R Y F A T T Y A C I D S ON P L A S M A L I P I D S A . Effects o f F a t t y A c i d Classes on T o t a l Cholesterol Concentrations
A series o f classic studies in the early 1950s r e p o r t e d on the cholesterol-lowering ability o f certain d i e t a r y fats o f vegetable origin. These studies subsequently p a v e d the w a y for the l a n d m a r k studies f r o m K e y s a n d c o w o r k e r s , ~'1°°'1°6 a n d H e g s t e d a n d colleagues. 79 It is p r o b a b l y safe to say t h a t the latter studies are some o f the m o s t frequently cited studies in the d i e t a r y fat a n d lipid field. Since these early reports, three a d d i t i o n a l g r o u p s have p u b l i s h e d regression e q u a t i o n s based on m e t a - a n a l y s e s o f p u b l i s h e d h u m a n studies 78'1'~a2~ (Table 1). A l l p u b l i s h e d e q u a t i o n s to d a t e are in a g r e e m e n t with r e g a r d s to the effects o f different fatty acid classes o n T C c o n c e n t r a t i o n s , n a m e l y t h a t replacing d i e t a r y c a r b o h y d r a t e , isoenergetically, with S F A increases T C whereas r e p l a c e m e n t with P U F A lowers TC. T h e b u l k o f this effect is m e d i a t e d by changes in L D L - C . (The m e t a - a n a l y s i s b y H e g s t e d et al. 78 b a s e d on 248 diets f r o m a total o f 47 p u b l i s h e d studies is the largest d a t a b a s e yet reported.) N o n e o f the e q u a t i o n s has been able to identify a n y specific role for M U F A , a l t h o u g h the recent e q u a t i o n by Y u et al. TM includes a small b u t significant negative coefficient for M U F A . W h i l e the original e q u a t i o n by K e y s et al.l°° did n o t include a t e r m for d i e t a r y cholesterol, s u b s e q u e n t e q u a t i o n s f r o m this g r o u p as well as r e p o r t s by H e g s t e d et al. 79 have included terms for d i e t a r y cholesterol. T h e analysis by M e n s i n k a n d K a t a n , ~48 a n d Y u et al. 221 selected diets with similar cholesterol contents a n d a c c o r d i n g l y c o u l d n o t define its role. (However, based on meta-analyses, 8°,m as well as a n extensive review o f the literature by M c N a m a r a , ~43 it is k n o w n t h a t d i e t a r y cholesterol itself c o n t r i b u t e s to increased TC, p r i n c i p a l l y L D L - C . ) R e c e n t e p i d e m i o l o g i c a l d a t a have identified d i e t a r y cholesterol to be a n i n d e p e n d e n t risk factor for C H D once o t h e r C H D risk factors have been a c c o u n t e d for. 77A89 Perusal o f T a b l e 1 reveals t h a t while there is close a g r e e m e n t with regards to the coefficient for S F A (specifically those with 12-16 C-coefficients o f 2.0-2.7 in the various regression equations), the coefficient for P U F A shows a l m o s t a three-fold v a r i a t i o n (0.60-1.65). One o f the reasons for this m a y reside in the fatty acid c o m p o s i t i o n o f the d a t a b a s e s used to generate the regression e q u a t i o n s (Table 2). T h e lowest coefficient for
TABLE 2. Dietary Fatty Acid Composition of Various Databases Study (n)
Ref. ~°°(42)
Ref. 79(38)
Ref. ~(65)'
Ref. 7s(248)~
Ref. 2:~(52)~
FAT SFA MONO PUFA P/S
30.9 _+ 11.9 12.0 _+ 6.8 12.9 _+6.1 6.0 _ 5.4 0.75 _+ 0.74
35.2 _+ 6.5 15.6 _+ 8.9 12.1 _ 6.3 7.5 + 7.4 1.03 _+ 1.54
33.1 _ 7.2 9.9 _+ 5.4 13,4 +_ 6.6 9,9 _+ 6.2 1.35 _ 1.20
34.3 _+ 7.0 13.7 _+ 6.2 12.8 _ 5.3 7.9 _+ 5.7 0.86 _+ 0.97
34.2 _+ 3.8 13.3 + 5.3 13.6 _+4.6 7.3 _+ 5.6 0.86 +_ i.18
Values (%energy) are the mean + SD. 'Meta-analyses of published studies - - 65 diets from 27 studies, 248 diets from 47 studies and 52 diets from 14 studies, respectively. The data set of Mensink and Katan (ref. ~), was characterized by lower SFA and higher PUFA than the other data sets.
Effects of dietary fatty acid composition on plasma cholesterol
97
TABLE 3. Regression Equations Predicting the Effect of Fatty Acid Classes on LDL-C and HDL-C Concentrations in Man Equation T6 ALDL-C = 1.83AS* - 0.24AM' - 0.55AP (Mensink and Katan, 1992)~ Equation T7 ALDL-C = 1.74AS - 0.77AP + 0.044AC (Hegsted et al., 1993)78 (Yu et al., 1995)TM Equation T8 ALDL-C = 1.46AS' - 0.69AM - 0.96AP Equation T9 AHDL-C = 0.67AS~ + 0.34AM + 0.28AP (Mensink and Katan, 1992)~" (Yu et al., 1995)TM AHDL-C = 0.62AS* + 0.39AM + 0.24AP Equation TI0 ALDL-C and AHDL-C denote changes in LDL-C and HDL-C in mg/dl plasma. AS, AS*, AM, AP and AC as defined in legend to Table 1. "Coefficient not significantly different from zero. P U F A is that fl,om Mensink and Katan, ~48 whose database included the highest P U F A content and lowest SFA content (SFA:PUFA 1:1). The equation by Yu et al., TM assigning a cholesterol-lowering role for M U F A , is of interest as there ihas been much debate in recent years about the cholesterol-lowering ability of M U F A in relation to P U F A especially as several clinical studies have shown that replacement of SFA with M U F A specifically lowers LDL-C,13s'~46whereas substitution with P U F A lowers both L D L - C and H D L - C . ~38 The reason why M U F A appear hypocholesterolemic in the analysis of Yu et al. 22L is not clear, although one of the reasons the authors suggested was that in the various other databases, the C12-C16 SFA content was relatively ihigh (unlike their own database) and this may have " m a s k e d " the hypocholesterolemic effect of the M U F A . However, Mensink and K a t a n ~48 did not find a significant cholesterol-lowering role for M U F A and their database (Table 2) included a substantially lower SFA content.
B. Effects o f Fatty Acid Classes on L D L - C and H D L - C Concentrations
Three groups have published regression equations predicting fatty acid effects on L D L - C and H D L - C 78't48z21 (Table 3). All fatty acid classes appear to increase H D L - C , whereas only SFA and dieta:ry cholesterol increase LDL-C. P U F A lowers LDL-C, whilst only the analysis of Yu et al. 22~ assigns a L D L - C lowering role for M U F A . Interestingly, Hegsted et al. 78 failed to come up with a meaningful regression equation for H D L - C utilizing their extensive database.
C. Effects o f Itwdividual F a t t y Acids on Total Cholesterol - - H u m a n and Animal Studies
The early study by Hegsted et al. 79 was unique, in that it reported the entire fatty acid profile of the diets utilized, and not simply total SFA, M U F A and PUFA. Additionally, the study by Hegsted et at. 79 utilized an exceptionally large number of diets, which is in stark contrast to most studies undertaken today which frequently compare two or three diets. In this study, the workers evaluated the TC response to the consumption of a low-fat diet to which numerous test fats were added. Carried out in a mental institution with a group of chronic schizophrenic men, the study was carried out in a relatively controlled setting which ensured a high degree of compliance. The same men were fed over a three-year period a total of 36 different diets, each for a period of 4 weeks. The TC concentration obtained on a specific test fat was compared with the value obtained on a control diet. In the final analysis reported, the change in TC between a given test fat and the appropriate control was correlated with the per cent energy (%en) contributed by each dietary fatty acid. A series of equations were generated in an attempt to explain the change in TC in terms of the %en derived from specific fatty acid(s). The authors reported that the observed changes in TC reflected parallel changes in 13-1ipoprotein cholesterol (i.e. LDL-C). The salient points to emerge from this study were: 1. while 88% of the observed variation in TC could be explained by an equation
98
P. K h o s l a and K. S u n d r a m
utilizing the Zall SFA, PUFA and dietary cholesterol, i.e. seven of the eight dietary variables, an equation (equation 1) utilizing only four dietary variables, 14:0, 16:0, 18:2 and dietary cholesterol, explained 91% of the observed variation, i.e. ASC
= 8.45AEi4:0 + 2.12AEi6:0 -
1.87AEpuFA+ 5.64AC -- 6.24
( 1 ) 79
where ASC is the change in serum cholesterol (mg/dl), AE,4:0, AEI6:0, AEpuFArepresent the change in %en from 14:0, 16:0 and PUFA principally 18:2), respectively, and AC represents the change in dietary cholesterol (in 100 mg units). 2. Amongst individual fatty acids, myristic acid (14:0) alone appeared to be the most important (explaining 69% of the total variation in observed TC) and the most potent (a l%en increase in 14:0 increasing TC by 8 mg/dl). 3. Myristic acid was 4 x as cholesterolemic as palmitic acid (16:0). The PUFA (principally linoleic acid, 18:2) lowered TC in a linear fashion. Palmitic acid and 18:2 had roughly equal effects on TC, albeit in opposing directions. Additionally, 14:0 was roughly 4.5 x as effective in raising TC as 18:2 was in lowering it. The shorter chain fatty acids ( < 12 carbons), lauric acid (12:0), stearic acid (18:0) and oleic acid (18:1) appeared to have no effect on TC. Dietary cholesterol increased TC in a linear fashion and its effects were independent of the effects of the fatty acids themselves. Total serum cholesterol was not affected by the level of fat employed, which varied between 22 and 40% of total dietary energy. 4. The percentage composition of the dietary fat was more effective in predicting TC than the actual levels of the fatty acids consumed. As Hegsted et al. 79 wrote, "the proportions of fatty acids in the dietary fat rather than the percentage of calories they supply is thought to be of primary importance". In other words the presence or absence in the diet of a specific fatty acid(s) will dictate the response of other fatty acid(s) that are present. More than 30 years later, the same point was being emphasized by different workers.
7°,75
Based on multiple regression analysis, three subsequent reports have detailed the effects of specific fatty acids o n T C 7°:63'221 and have essentially confirmed the original study by Hegsted et al. 79 The fatty acid composition of the various databases is detailed in Table 4. One of the reports dealt with human subjectsy' while two dealt with animal studies. 7°''63 The two human data sets were remarkably similar in terms of the overall fatty acid composition of the analyzed diets as well as the total SFA, MUFA, PUFA and total fat. Notable exceptions included higher contents of short chain fatty acids ( < 12C), lauric and myristic acid in the diets used by Hegsted et al. 79 Additionally, in the latter study dietary cholesterol was an important variable. In contrast to the study by Hegsted and colleagues T A B L E 4. I n d i v i d u a l F a t t y Acid C o m p o s i t i o n o f Various D a t a b a s e s F a t t y acid (n) < 12:0 12:0 14:0 16:0 18:0 MUFA 18:2 18:3 ZSFA ZPUFA Fat Chol (mg) P/S
Ar9(36) 1.79 2.47 2.08 6.33 3.06 11.89 7.39 13.9 7.4 35.0 396 1.06
_ 3.01 _ 4.73 ___2.05" ___2.80 ___2.27" _ 6.40 _ 7.59 c + _ 444-
7.5" 7.6 6.6" 151 1.58
Bn2(41) NR 0.76 + 2.05 a~ 0.99 + 1.21 .b 7.19 ___2.85 3.84 _+ 2.98 b 13.90 + 4.79 7.86 _ 6.10 ~ 12.8 4- 5.3 b 7.9 +_ 6.1 34.5 + 3.6 b NI 0.98 4- 1.30
C7°(16) NR 3.92 ___6.48" 1.81 + 2.57 7.96 + 5.24 1.19 + 0.41 "~ 11.73 + 6.76 d 7.11 4- 7.20 0.28 + 0.30 14.9 _ 8.3 7.4 4- 7.3 34.0 ___4.4 ~ 0 1.07 4- 1.74
D'63(38) NR + 6.54 b + 2.66 b + 4.89 + 2.92 c + 6.56 d 4- 9.93 4- 0.10 + 8.7 "b 4- 9.9 ___ 1.2 ~ 0 1.17 _ 1.95
3.44 2.30 8.17 3.89 11.73 8.81 0.08 17.8 8.9 38.4
% e n e r g y from different fatty acids is s h o w n (mean + SD). A a n d B in h u m a n s , C in cebus m o n k e y s a n d D in gerbils. N R , N o t reported. NI, Dietary cholesterol was not included in the regression analysis. °Significantly different from each other. dExclusively 18:l. eDid not differentiate between 18:2 a n d 18:3.
Effects of dietary fatty acid composition on plasma cholesterol
99
(carried out in the same group of men), the report by Yu et al. was an analysis of 14 published human studies and was derived from 47 data points. All diets did include cholesterol (-300 mg/d), but as this was relatively uniform across studies, it was not included in the regression equation) = The complete regression equation from this study: TM ATC = 0.96AE12:0 + 5.58AE14:0 + 1.07AEI6:0 + 0.55AEIs:0 - 0.17AEMuFA -- 0.66AEpuFA, (2) TM explained 95% of the variation in total cholesterol. However, only the coefficients for 14:0, 16:0 and P U F A (principally 18:2) were significantly different from zero. The regression equation based solely on 14:0, 16:0 and P U F A was not reported, and in all likelihood the coefficients for 14:0, 16:0 and P U F A would change. However, as in the regression equation from Hegsted et al., 79 14:0, 16:0 and P U F A are the key fatty acids that impact cholesterol concentrations. The regression equations from the animal studies (equation 3, cebus monkeys; equation 4, gerbils; equation 5, hamsters) only include regression coefficients for 14:0 and 18:2. A TC = 10A E,4:0 - 48A (1ogEls:2)
(3) 7o
ATC = 7.6AE14:0 - 40A (logE,s2)
(4) '63
ATC = 5.0A El4:0 - 25A (logE18:z)
(5) ~,
ATC = 8.0AEi4:0 - 36A(logEls:2)
(6) 70
ATC = 5.2AE14:0 + 1.5AEi6:0 - 73A(logEts:2) + 0.18AC
(7) ,~,
Hayes and Khosla 7° showed that when the diets with cholesterol intakes > 306 mg/d were eliminated from the data set of Hegsted e t a / . , 79 the observed changes in serum cholesterol could be explained solely on the basis of 14:0 and 18:2 intakes (equation 6). When gerbil diets also contained cholesterol, the equation from Pronczuk et al. 163 also included coefficients for 16:0 and dietary cholesterol (equation 7). Amongst the SFA, all four studies (Table 4) identify a substantial hypercholesterolemic role for 14:0, whose coefficient ranges from 5.0 to 10 (i.e. replacing l % e n from carbohydrate with 14:0 would be predicted to increase TC by 5.0 to 10 mg/dl). None of the regressions assigns a significant coefficient for 12:0, 18:0 or 18:1. Both human equations assign a cholesterol-elevating role for 16:0, although its coefficient is 4-5 × less than that for 14:0, while 16:0 does not appear in the equations from the animal studies. With regards to other fatty acids, all regressions identify 18:2 to be cholesterol-lowering, although its coefficient varies almost three-fold in the human equations. By contrast, while the animal regression equations also ascribe cholesterol-lowering properties to 18:2, the use of a log term is indicative of a non-linear response (see discussion below). None of the regression equations includes a coefficient for MUFA. Finally, dietary cholesterol p e r se is hypercholesterolemic in the analysis of Hegsted et al. 79 and Pronczuk et al., ~63 but only at high intakes. Thus from these four reports it is clear that 14:0, 16:0, 18:2 and dietary cholesterol are important variables which directly affect TC. Finally, Mensink and Katan ~48provided regression coefficients for individual fatty acids based on an ana]ysis of 16 studies. From a total of 38 data points, the reported regression coefficients (in mg/dl) were ATC = 0.83A&2:0 + 4.79A&4:0 + 1.31AEi6:0 + 1.17AE,8:0 - 0.29AEls:l - 0.63AE~s:2 - 0.88dEls:3 (95%CI:12:0, - 2.2--+3.9;14:0, - 0.43 ~ 10.0;16:0,0.54--+2.1 ;18:0, - 1.1 --,3.5;18:1, -
0.8--+0.2;18:2, - 1.1~ - 0.15;18:3, - 3.5--,1.74)
However, the confidence intervals were wide (and with the exception of 16:0 and 18:2, included zero). Also in the database used by the authors, levels of 12:0 and 16:0 were strongly correlated. As in the case of the regressions from the other human studies,
100
P. Khosla and K. Sundram
myristic acid is the most potent fatty acid in terms of its ability to raise TC, although its coefficient is somewhat less than the value reported by Hegsted et al. 79 D. Effects o f Individual Fatty Acids on L D L - C and H D L - C Concentrations
To date, only one group has published a regression equation (equation 8) detailing individual fatty acid effects on HDL-C concentrations (in mg/dl). The equation from Yu et al. TM was generated from 14 studies and assigns significant cholesterol-elevating roles to 12:0, 16:0, M U F A and PUFA. The authors reported that the equation for LDL-C was similar to the one for total cholesterol (see equation 2). A HDL - C = 0.72AEi2:0 + 0.14AEl4:0 + 0.28AEt6:0 -- 0.15AE~8:0 + 0.29AEMuFA + 0.1 1AEpuFA (8) TM E. The Non-linear Response to Linoleic Acid
Regression analysis by Hayes and Khosla 7° and Pronczuk et a l ) 63 has revealed that the cholesterol-lowering effect of 18:2 in cebus monkeys, gerbils and hamsters, as well as certain published human studies (e.g. a sub-set of the data of Hegsted et al. 79 discussed by Hayes and KhoslaT°), may be non-linear. TC declines steeply as dietary 18:2 increases from - 1 % to 6%en, and above this level, the effect of 18:2 is generally less pronounced. Decreasing 18:2 from 6% to 1%en results in an independent cholesterol-raising effect, i.e. the cholesterol-elevation is not attributable to the simultaneous presence of high levels of 14:0 or 16:0. Furthermore, since 14:0 and 18:2 are the only dietary fatty acids of consequence in the regression analyses from these workers, 7°.163the TC response has been best described in terms of the dietary 18:2/14:0 ratio, which Leung et al. have referred to as the cholesterol-lowering ratio ~29 (this relationship is also non-linear). The break-point on the 18:2/TC curve has been referred to as the "18:2 threshold", and has been loosely defined as the level of 18:2 above which further increases in 18:2 have minimal impact on T C . 73 These observations have led these workers to postulate the "threshold h y p o t h e s i s " . 7°'71'73'74'1°8'1t3:63 Based on published human data (Western diets) the average threshold for 18:2 has been suggested to be ~5%en. TM According to this hypothesis, the non-linear response of 18:2 is more in keeping with empirical observations that TC (specifically LDL-C) generally cannot be lowered below a certain value. This value varies across different species and humans, and probably also differs in different human populations. Physiologically, the threshold level of 18:2 is thought to reflect the situation where LDL receptor activity and lipoprotein metabolism are maximally efficient. [In Tarahumara Indians, 3° a population characterized by low TC and low LDL-C concentrations - attributed to consumption of low fat (~10%en) and low cholesterol-containing diets (~70mg/d), 18:2 intake is ~4%en.] Any situation which results in LDL receptor suppression, or "stresses" lipoprotein metabolism, raises TC (and LDL-C) and accordingly increases the 18:2 requirement. Thus, an inherent assumption in this hypothesis is that amongst the fatty acids, 18:2 up-regulates LDL receptors. According to this hypothesis, for any of these normal or "abnormal" physiological situations, a certain threshold level of 18:2 is required to alleviate the burden and once this requirement for 18:2 is met, additional 18:2 becomes superfluous, i.e. represents "metabolic overkill", 75 and other dietary fatty acids, e.g. 18:1 or even 16:0, begin to "appear" neutral, i.e. can be exchanged for each other without affecting TC (or even LDL-C) concentrations. This 18:2 requirement can be met from either the diet or from 18:2 stored in adipose tissue reserves. In other words an individual with inadequate adipose reserves of 18:2 is unable to "compensate" when faced with a dietary onslaught of factors which increase TC, e.g. excess dietary cholesterol, 14:0 and in some cases 16:0, unless sufficient 18:2 is also present in the diet. By contrast, an individual with adequate reserves of 18:2 is generally refractory to 14:0 and dietary cholesterol, even when the diets have little 18:2. As a consequence, the
Effectsof dietaryfatty acid compositionon plasmacholesterol
101
18:2 threshold wLries across species and different human populations, and furthermore the threshold can shift in a given species depending on the metabolic circumstance. How a given individual or animal utilizes the 18:2 provided is dictated by his "metabolic setpoint ''73. For any given individual, lipoprotein metabolism is dependent on several factors which include LDL receptor activity, body mass index, age, insulin status, etc. These factors collectively establish a "lipostat" for that p a r t i c u l a r metabolic status (metabolic set-point). Hence, feeding high levels of 18:2 to an obese, elderly subject results in a different cholesterolemic response than feeding the s a m e levels of 18:2 to an active, young individuali of ideal body weight, etc. Thus certain individuals respond by decreasing their TC in response to 18:2-intakes only up to 3-4%en, whereas others continue to show substantial decreases up to 7-8%en before the impact of dietary 18:2 subsides. This may relate to differences in EFA status at the time of dietary intervention. The variability of the threshold level in humans may be "masked" in most human studies, since most diets supply 18:2 at irLtakes (3-10%en) where the response appears linear. Additionally, unless one assesses sew~ral diets in the same study, in which 18:2 intakes covers a broad range (which includes levels below and above threshold), the non-linear response may be missed. There is at present (for reasons discussed above) few data in the human literature to support the idea of a non-linear response to 18:2. However, Hegsted e t al. 78 in their meta-analysis, did note that the observed variation in TC utilizing their regression equation (equation T4, T~tble 1) was not significantly improved by use of a non-linear term for 18:2 - - in other words a non-linear response was as g o o d as a linear response. In a recent human study, Howard et al. 84 progressively increased 18:2 from 3% to 12.8%en (at the expense of 18:1) as part of diets in which total fat accounted for ~30%en. Total cholesterol concentrations decreased from 229 to 224 mg/dl. These changes were considerably less than those that would be anticipated if the effect of 18:2 was linear. The only human study at present that we are aware of that provides direct support for the importance of the dietary 18:2/14:0 ratio was recently reported by Leung e t al. ~29 The study was designed to assess possible "lifestyle" risk factors which may contribute to increased concentrations of plasma lipids. Since there had been a significant migration of Chinese from Jiangmen (a city in Guandon Province in mainland China) to Hong Kong in the late 1940s, and people in the latter city were following a more affluent lifestyle, Leung et al. assessed fat intakes and plasma lipid concentrations in second-generation (age, weight and height-matched) children in these two cities. TC (176 _+ 32 vs 161 _+ 24 mg/dl, n = 94 and 99, respectively) and all other plasma lipid parameters (TG, HDL-C, LDL-C, VLDL-C, Apo A1 and ApoB) were significantly higher in the Hong Kong children. Fat intake was asses,;ed by chemical analysis of the total diet consumed on two successive week days from 20 children randomly selected from each cohort. Total fat inteLke as well as SFA, and MUFA intake (but not PUFA intake) was significantly higher in the Hong Kong children. Although 18:2 intake was significantly higher in the Hong Kong group (7.94 vs 6.56 g), the 18:3 intake was significantly higher in the children from Jingmen (1.17 vs 0.57 g). There was a significant non-linear correlation between the measured TC and the 18:2/14:0 ratio (P = 0.0001). The TC in the Hong Kong children (200 _+ 46 mg/dl) reflected 18:2/14:0 ratios of generally < 10, while the TC concentrations in the Jingmen children (150 + 7 mg/dl) reflected ratios which ranged from 10 to 45. Differences in cholesterol intakes between the populations were not reported and would have been expected to make some contribution to the observed responses, especially as the authors stated that milk and beef were major sources of SFA in the diets of the Hong Kong group. Additionally, from the information provided, it was not possible to assess whether the wide variation in the 18:2/14:0 ratio in the Jingmen children was due to variations in 14:0, 18:2 or both. Nevertheless, the results provide preliminary evidence that at least in young (age 7) growing humans, with presumably little 18:2 deposition in their adipose tis:sue, 18:2 and 14:0 intakes may play a significant role in determining TC concentrations. According to Hayes e t al. TM the public health implication of the 18:2 threshold concept is that in order to maximize reductions in TC and LDL-C, individual and population 18:2
102
P. Khosla and K. Sundram
thresholds need to be evaluated in order to establish the optimum level of dietary 18:2 needed, if maximal dietary lowering of total cholesterol and LDL-C is to be achieved. In people with severe hypercholesterolemia (and presumably the "worst" LDL receptors and lipoprotein metabolism) a higher level of 18:2 may be needed than the currently advocated level of 10%en. IV. MECHANISM OF ACTION OF DIETARY FATTY ACIDS These have been discussed in great detail by Grundy and Denke u and McNamara) ~ Briefly, dietary fatty acids can affect plasma LDL-C concentrations by a multitude of mechanisms, which include effects on LDL receptors, VLDL and LDL transport rates, effects on enzymes involved in lipid metabolism as well as fatty acid induced changes in cell membranes. No all encompassing mechanism exists and in all likelihood several mechanisms are involved. Much of the research in recent years has focused on the ability of dietary fatty acids to affect LDL receptors, and the bulk of the work is in animals. There have been no human studies of consequence since the above two reviews were published and, therefore, we will only discuss the animal studies. A. A n i m a l Studies - -
Hamsters
The golden Syrian hamster has been used extensively in recent years by Spady, Dietschy and coworkers as a model for human sterol synthesis and LDL metabolism. 3s,s3.~s~ls~,217-22° Utilizing this model, these workers have provided detailed information on how dietary cholesterol and fatty acids interact to affect cholesterol synthesis and LDL metabolism in vivo (see reviews by Dietschy et al. 35-37and Spady et al)S6). The studies are based on measurement of LDL-C and receptor-mediated LDL uptake in various tissues (specifically liver) following a particular dietary intervention and correlating the observed uptake with the uptake measured in chow-fed animals (i.e. animals fed low-fat, low-cholesterol control diets) at the s a m e LDL-C concentration. To achieve this, receptor-mediated and receptor-independent LDL uptake at ~ifferent plasma LDL-C (up to 500 mg/dl) were established by infusing various amounts of LDL over 4 h. During this time period, the amount of LDL infused was adjusted to maintain a constant plasma LDL specific activity. Collectively, the above studies have established several important principles with regards to in vivo cholesterol and LDL metabolism) 5 These include the observations that animals with low levels of hepatic cholesterol synthesis (e.g. hamsters and humans) respond to alterations in cholesterol flux across the liver, principally by altering LDL metabolism, whereas animals with high levels of hepatic cholesterol synthesis (e.g. rat) readily adjust their cholesterol synthetic capacity. Accordingly, cholesterol feeding induces marked changes in plasma LDL-C in the hamster, whereas it is relatively unaffected in the rat. The majority of whole body LDL uptake in hamsters occurs in the liver (-80%) with > 90% of this being receptor-mediated. As plasma LDL-C is increased (up to 500 mg/dl), receptor-mediated LDL uptake increases in a saturable fashion, whereas receptor-independent uptake increases linearly. High plasma LDL-C are invariably associated with a decreased clearance of LDL (reflected as a decrease in LDL FCR) and do not necessarily indicate impaired receptor activity. This can only be established (as indicated above) by ascertaining LDL uptake in control and experimental animals with the s a m e LDL concentration. Bearing the above in mind, the hamster studies have shown that feeding 0.12% cholesterol + 20% w/w SFA (hydrogenated coconut oil) increased LDL-C more than two-fold (175 vs 69 mg/dl) by suppressing LDL receptor activity -88% (hepatic LDL clearance of 5 vs 42 ~tl/h/g liver), as compared to feeding the sarrle diet with 20% PUFA (safflower oil)) s~ Additionally, hepatic cholesteryl ester concentration was decreased two-fold with SFA consumption (7 vs 13 mg/g liver), while hepatic cholesterol synthesis was unaffected. Studies with the same dietary fat sources but with three different levels of dietary cholesterol revealed that increasing dietary cholesterol (0.06, 0.12 and 0.24%
Effects of dietary fatty acid composition on plasma cholesterol
103
w/w) suppressed LDL receptors and raised LDL-C in a dose-dependent manner (LDL clearance rates 77, 50 and 38 ~tl/h/g and LDL-C of 40, 63 and 86 mg/dl, respectively), whereas these changes were exacerbated by SFA (LDL clearance 32, 17 and 14 lal/h/g and LDL-C of 86, 156 and 198 mg/dl, respectively), but prevented by PUFA (LDL clearance 103, 54 and 41 ~tl/h/g and LDL-C of 32, 57 and 71 mg/dl, respectively)2s2 By contrast, in the absence of exogenous dietary cholesterol, feeding a low-fat (5% w/w) chow diet or 20% w/w fat diets, enriched in SFA, M U F A (olive oil) or PUFA resulted in similar LDL c,learance rates (115, 116, 114 and 138 I~l/h/g, respectively), and generally low LDL-C concentrations (25, 18, 20 and 41 mg/dl, respectively)?s2 The increase in LDL-C with the SFA, therefore, presumably reflected increased production of LDL-C. The changes induced by feeding hydrogenated coconut oil in the above studies 's'''s2 could be attributed tc, the long chain fatty acids (12-18 carbons) since feeding of 0.12% cholesterol + 20% MCT (77% as the 8:0 fatty acid and 23% as the 10:0 fatty acid) produced essentially similar values to those obtained with cholesterol-feeding by itself? ~s Since the above studies utilized a single fat source, Woollett et al. investigated the effects of feeding mixtures of SFA and PUFA (again by manipulating hydrogenated coconut oil and safflower oil). 22° LDL-C levels decreased from 190 to 50 mg/dl as SFA were systematically replaced by PUFA. This decrease in LDL-C was accompanied by increased LDL receptor activity, decreased LDL production rates and increases in hepatic cholesteryl ester content. Furthermore, by manipulating the actual amount of fat fed to the animals, this study also showed that reducing total fat intake (from 45 to 20%en by decreasing SFA) was not as effective in lowering LDL-C as maintaining the same fat intake (45%en) but replacing SFA with PUFA. To ascertain the effects of specific SFA in the hamster model, Woollett et al. fed hamsters commercially available triglycerides consisting of a single SFA (from 6 to 18 carbons in length)) '9 Animals were fed cholesterol-containing diets (0.12% w/w) formulated with 80% w/w chow, 10% w/w olive oil and 10% w/w of the test triglyceride + 0.12% cholesterol. Feeding of the 6:0, 8:0, 10:0 and 18:0 triglycerides maintained LDL-C -65 mg/dl, whilst the 12:0, 14:0 and 16:0 triglycerides elevated LDL-C to -100 mg/dl. The increased LDL-C could be attributed to significant reductions in hepatic LDL receptor activity as well as increased LDL production rates. The 12:0 and 14:0 triglycerides lowered hepatic cholesterol levels in comparison to the levels induced by the 6:0, 8:0 and 10:0 triglycerides. Surprisingly, hepatic cholesterol levels with the 16:0 and 18:0 triglycerides (lower than the 12:0 and 14:0 triglycerides) were similar even though LDL-C was significantly lower with the 18:0 triglyceride as compared to the 16:0 triglyceride. A recent study by Horton et al. has delineated the SFA effect observed in the above studies ~s~''s2'22°in more detail? 3 Utilizing hamsters fed semisynthetic diets with a background 2% w/w corn oil (to alleviate EFA-deficiency), effects of feeding cholesterol containing diets (0, 0.06 and 0.12% w/w) rich in either SFA (hydrogenated coconut oil) or PUFA (safflower oil) on hepatic LDL receptor protein and mRNA levels have been reported. The results from this study, namely that SFA increase LDL-C by decreasing LDL receptor activity, whilst :in agreement with the earlier reports, ~s~'~s2,22°additionally show that the decrease in LDL receptor activity is accompanied by parallel changes in hepatic LDL receptor protein levels and mRNA levels. Thus this study provides strong evidence that the SFA-induced effects on LDL metabolism is primarily at the mRNA level. Based on data from the above studies, LDL-C concentrations in vivo are thought to be determined by two processes - - a receptor-dependent process which depends on the LDL receptor and is saturable, and a receptor-independent process, which is not saturable. Dietschy 34and colleagues35have calculated and reported the various rate constants, which determine LDL-C concentrations in vivo. The receptor-dependent process is defined by (a) Jd, the rate of receptor-dependent LDL-C transport; (b) Jm, the maximal rate of LDL-C that can be achieved when all receptors are occupied; (c) J , the LDL-C production rate; (d) C,, the plasma LDL-C concentration; (e) Kin, the apparent affinity of the LDL-C particle for its receptor. Km also corresponds to Jm/2, the LDL-C concentration needed to achieve half-maximal LDL-C transport. The receptor-independent process is defined by
104
P. Khoslaand K. Sundram
(a) J~, the rate of receptor-independent LDL-C transport; (b) Cj; (c) P, a proportionality constant which describes the relationship between J~ and G. In the whole animal, total LDL-C removal from the plasma, Jr, equals the sum of the receptor-dependent and receptor-independent transport, i.e. Jt = Jd + J~. Furthermore, in the steady state Jt equals the rate of LDL-C production. Dietary cholesterol and fatty acids have been shown to exert their effects, principally, by altering, Jt and Jm (the rate of LDL-C production and the rate of LDL-C removal by the receptor-dependent process). Studies in tissue culture by Goldstein and Brown revealed that the LDL receptor is designed to function maximally at plasma LDL-C concentrations of ~25 mg/dl,~6 a level approximating the observed value, in newborn human infants,2°7various animal species not susceptible to atherosclerosis, tSz and several animal species maintained on low-fat, low cholesterol (i.e. chow) diets. 34'35From the studies of Dietschy and colleagues, the affinity of the LDL-C particle for its receptor is relatively uniform in rabbits, rats, hamsters and humans (-90 mg/dl)) 4'35Thus in humans with LDL-C concentrations of 90 mg/dl, the rate of LDL-C removal (or uptake) via LDL-receptors would be expected to be 50% of the maximum that can be achieved (i.e. the rate when LDL-C concentrations are ~25 mg/dl). Furthermore, LDL-C concentrations generally change little until LDL receptors are suppressed > 50% .34.134Thus based on the hamster model, humans can readily "sustain" a 50% loss of LDL-receptor uptake (when LDL-C approaches 90 mg/dl) and yet not be at risk from CHD, provided LDL-C production does not increase dramatically. (Note that in terms of CHD risk, desirable levels are < 130 mg/d168.) However, in Western man LDL-C concentrations are seldom < 90 mg/dl. (Of the various studies encompassing 148 different diets described in Table 5, LDL-C concentrations of 90 mg/dl were apparent in only 16 of these.) Interestingly, in Tarahumara Indians,3° a population characterized by a myriad of characteristics associated with low risk of CHD, the LDL-C concentration is 87 mg/dl (cf. Km of 90 mg/dl). The mechanism for the differential effects of dietary cholesterol and fatty acids on LDL-C metabolism has been postulated to operate by shifting the equilibrium between a putative small regulatory pool of free cholesterol and the pool of cholesteryl esters) 4 When there is net cholesterol delivery to the liver, part of the excess cholesterol is esterified by the enzyme ACAT (whose activity is driven by the availability of free cholesterol and the acyl-CoA derivative of oleic acid) and both the regulatory pool of free cholesterol and cholesteryl esters expands. The increase in the former is then conveyed to the nucleus via a series of transcription factors2°,2~2which ultimately decreases the mRNA for the LDL receptor and accordingly decreases LDL receptor activity. In this situation LDL receptor activity is inversely related to liver CE concentrations. Since both the regulatory pool of free cholesterol and the cholesteryl ester pool change in concert with each other, the ratio of FC/CE is unaltered. By contrast, in situations in which net cholesterol delivery to the liver is zero, the activity of LDL receptors is dependent on a shift in the equilibrium between the regulatory pool of free cholesterol and the cholesteryl ester pool) 4 Thus SFA (specifically 12:0, 14:0 and 16:0) shift the equilibrium and expand the free cholesterol regulatory pool, thereby decreasing receptor activity,3L219whereas MUFA (18:1) shifts the equilibrium to expand the cholesteryl ester pool (and deplete the free cholesterol pool) and thereby increases LDL receptor activity.3t Stearic acidTM and the SFA of < 10 carbons 31,219have no affect on this equilibrium and therefore appear neutral (i.e. do not affect LDL receptor activity or LDL-C concentrations). There are as yet no data on 18:2. Although the above studies31,s3.18°-185,2tT-22°have provided detailed information, forwarding our understanding of the mechanisms regulating LDL metabolism, certain limitations need to be kept in mind. Firstly, all studies have utilized one hamster strain, whereas it is known that lipoprotein and bile acid metabolism is not uniform amongst different hamster strains?°4'2°5 Secondly, these studies provide little if any information on how fatty acids impact on other lipoproteins (e.g. VLDL and HDL), which is important considering that the hamster is essentially an HDL animal,6°.Tz76,lTj-~73and they do not take into account how LDL production might be affected by fatty acid-induced changes in VLDL production.
Effects of dietary fatty acid composition on plasma cholesterol
105
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0.1 1.1 0 l.l 0 0.1 1.1 0.0.4 0.0.4 0.1 0 19.4 0 0 0 0 0 0 0 1.2 0.7 0.9 0.5 3.4 3.4 0.6 0.8 0 0 0 0 0 0 0 0.7 0.5 0 0 0 1.3 0.5 1
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
Friday et al., 52 4M, 1F Age 40, SC 187, 34%en Zock and Katan, 223 age 25 26M, 30F, SC 187, 41%en Mata e t al. ~7 21F, age 43, SC 191 36%en Barr et al., 13 48M, age 25, SC 183 37 and 30%en
Mattson and G u n d y (138) 20M, age 59, SC 263 40%en, IF Reiser et al. ~ 19M, age 26, SC 156 35%en Bonanome and G r u n d y j9 I I M , age 64, SC 227 40%en; LF McDonald et a l ) 4° 8M, age 19-32, SC 171 36%en Mensink and K a t a n ~ 27M, 3IF, age 25, SC 186 37%en Mensink and Katan, ~.7 age 25 25M, 34F, SC 184, 40%en Ginsberg et al. ~ 36M, age 23, SC 183 38 and 30%en Chan et al. 2s 8M, age 20-34, SC 173 34%en
12:0
No. (ref. and study characteristics) 0.3 0 3.5 0 0.3 0 3.5 0.4 0.04 0.1 0.7 7 0.1 0.4 0.04 0.04 1.3 0.4 0.4 3.2 1.4 1.3 0.5 2.7 2.2 0.9 0.8 1.5 0.5 0.5 0.5 0.5 3.2 0.1 0.9 1 0 0 0 1.8 0.9 1.6
14:0 6.5 3.4 9.8 1.9 6.5 3.4 9.8 17.4 2.0 2.6 8.6 2.8 2.2 18.0 3.3 2.2 8.2 3.1 3.9 9.3 6.8 6.2 4.7 8.1 5.9 4.8 4.8 8.1 4.4 3.3 4.9 4.0 9.3 2.6 5.8 5.7 12.3 6.3 5.8 7.4 5.1 6.1
16:0 0.6 !.06 3.7 1.4 0.6 1.06 3.7 2.0 1.0 1.1 6.5 0.6 0.6 1.9 17.2 0.9 4.2 1.1 2.2 4.1 3.2 3.4 3 3.5 2.7 2.3 0.4 4.2 1.6 1.3 1.8 1.9 4.7 1 2.8 11.8 3.1 2.5 2.6 3.2 2.2 2.8
18:0
%en
17.3 12.4 7.5 5.1 17.3 12.4 7.5 15.8 29.3 5.8 17 1.9 3.8 15.5 15.8 31.9 15 20 7 11.5 15.1 10.8 23.0 12.8 11 10.4 17.2 13.1 19.0 18.4 8.4 10.1 10.2 4.5 14.7 15.4 13.5 21.4 12.4 14.5 13.2 10.8
18:i
T A B L E 5. C o n t i n u e d
4.7 10.7 0.2 21.2 4.7 l 0.7 0.2 3.9 7.0 29.1 0.7 0.5 28.1 3.8 3.2 4.8 7 10 22 4.6 7.9 12.7 4.6 3.4 8.9 9.7 9.9 4.1 7.4 6.6 15.3 12.2 1.6 25 12.5 4.3 3.2 3.3 12.5 8 7.8 6.5
18:2
140" 150" 148" 146" 141" 420 241 215 100 100 100 100 100 150" 150" 140" 136" 405 410 415 469 276 295
300 300 400 300 300 300 400 0 0 0 450 450 450 100 100 100
Chol 221 221 272 200 219 226 260 224 197 191 155 168 141 202 173 181 173 143 142 199 171 178 172 193 175 168 173 172 140 139 143 140 188 141 183 189 204 186 183 181 171 176
TC
39 38 35 40 46 40 42 40 44 55 50 50 54 49 52 55 55
44 45 44 45 44 52 47 57 55 55 58 52 45 45 47
115 90 86 94 90 116 81 109 116 133 108 114
HDL-C
mg/dl
143 ll9 120 98 110 90 140 110 119 115 87 86 128 105 111 103 121
LDL-C
Continued
92 65 84 70 67 73 62 84 92 84 93 84
70 77 95 269 114 78 80 259 249 231 88 78 72 128 129 122 91 73 73 82 83 73 72 83
TG
~r
o~
5.4 0.5 0.2 2.3 0.6 0.9 1.0 1.9 1.8 0.1 0.04 0.04 0.4 0.1 0.2 3.5 0.11 0.7 0.2 0.7 3.3 0.8 0.2 0.4 0.4 0.4 0.4 0.4 0.9 0.7 16.5 0.4 11.3 1.1 0.8 3.5 1.7 0.4 1.3
12.5 0.3 0.1 0.6 0.2 0.3 0.7 0.8 3.8 0 0.04 17.6 0.12 0.01 0.01 0.80 0.01 0.4 0.02 0.2 0.8 0.3 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.9 0 0 0.4 0.3 0.3 10.7 0.007 0.1 1.5
89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 !13 114 115 116 117 118 119 120 121 122 123 124 125 126 127
Tholstrup et al., ~°' 12M Age 24, SC 143, 40%en Zock et a l ? 24 23M, 36F, age 29, SC 196 39%en Tholstrup et al. 2°2 15M, age 25, SC 155 40%en Judd et al., ~ 29M, 29F
Sabate et al., '69 18M, Age 30, SC 198, 30%en Lichtenstein et al. '33 7M, 8F, age 61, SC 221 30%en Lichtenstein et al. ~2 8M, 6F, age 63, SC 223 31%en
N g et al. 'sT 20M, 13F, age 29, SC 194 31%en Nestel et al. ' ~ 27M, age 49, SC 22i 37%en Sundram e t a / . , 197 age 36 38M, SC 198, 41%en C h a n g and Huang, 26 14M Age 23, SC 157, 30%en Denke and G r u n d y 33 14M, age 63 40%en, LF Kris-Etherton et al. "~ 19M, age 26, SC 120--,205 37%en
14:0
12:0
No. (ref. and study characteristics) 4.9 13.4 6.3 8.4 5.2 9.8 11.7 8.6 3.8 4 1.9 | 17.4 4.5 9.3 9.3 4.5 9.2 9.4 9.2 9.5 5.5 3.8 3 4.3 4.4 4.3 4.7 8.4 7.6 1.7 16.8 4.7 14.9 5 2.9 13.6 15.4 7.8
16:0 1.4 1.8 1.9 3.8 2.3 2.3 2.8 3.5 1.3 1.3 0.9 0.9 1.7 1.4 11.4 4.5 1.7 10.3 11.4 10.3 4.7 2.2 1.6 !.5 1.6 1.6 1.6 1.8 3.8 3.1 1.2 1.9 4.3 4.1 3.8 1.1 15.7 1.8 2.8
18:0
%en
3.9 14 21.8 8.4 i7.8 12.9 15.9 15.3 5.8 18.4 30.3 17.8 16.0 27.2 13.2 10.1 10.1 12.1 13.3 12.3 10.4 8.8 7.4 13.7 8.4 16.2 8.4 8.6 10.6 10.1 16.3 16.4 10.9 11.6 20.9 14.2 15.6 13.5 16.7
18:1
T A B L E 5. C o n t i n u e d
.9 3.7 2.9 3.4 5.3 5.7 5.9 5.2 10.2 4.7 6.2 2.5 4.0 2.3 2.1 1.7 17.8 1.8 2.1 1.7 1.6 9.5 16.5 4.9 10.7 3.3 10.7 11.7 2.4 3 4.1 4.2 3.8 4.4 4.1 2.2 2.5 3.9 6.1
18:2 200 200 200 113* 64* 73* 193 259 304 304 0 0 0 360 360 360 360 360 360 360 360 237 125 81" 85* 84* 85* 197" 109" 226* 59* 63* 345 359 352 63* 62* 63* 130"
Chol 234 195 197 228 215 226 191 190 151 155 172 191 200 152 165 176 139 172 170 176 189 182 160 194 194 205 194 205 218 229 135 133 201 192 175 171 123 158 203
TC 165 133 134 ! 63 151 161 110 113 84 90 128 143 152 92 103 113 83 108 109 115 125 112 94 126 125 132 124 135 140 156 85 87 119 115 101 i19 84 114 129
LDL-C 40 37 37 38 38 42 51 48 54 50 32 37 35 48 44 45 45 54 52 51 52 47 45 44 44 46 44 47 45 46 43 39 64 59 58 46 33 38 55
HDL-C
mg/dl
Continued
143 125 126 139 135 128 85 89 69 83 93 94 94 84 87 88 73 70 67 74 80 114 103 109 108 112 110 100 116 131 66 63 88 89 84 66 65 62 91
TG
O
0
8
t~
e-L
@
rn
3.6 4.6 0.8 0.2 0.2 0.2 0.6 5.0 0 0 0 0.2 0.2 0 0 0 0 0.3 0.2 0.3 2.9
128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148
2.3 2.7 1.2 0.7 0.6 0.7 1.9 2.5 0.5 0.5 0.3 0.6 0.6 0.8 1.5 0.8 2.2 0.9 1.0 1.1 1.3
14:0 10.3 6.9 11.1 4.8 4.6 8.0 11.4 7.2 11.2 3.9 8.2 5.1 10.1 6.1 8.0 7.0 9.1 5.0 4.6 5.5 3.3
16:0 3.1 1.1 1.5 1.9 1.8 2.0 2.7 2.7 1.4 1.2 1.5 1.8 2.1 2.9 4.6 3.7 4.2 1.9 2.1 2.4 1.4
18:0
%en 10.9 10.3 10.8 16.9 20.3 17.6 12.6 10.4 12.8 16.9 11.8 18.9 11.6 16.5 15.1 15.0 12.2 14.3 12.1 8.5 5.7
18:1 6.1 3.2 3.5 2.1 2.7 2.7 4.4 4.9 3.5 6.4 7.3 3.4 4.3 8.2 5.4 9.3 4.4 3.0 4.2 7.0 12.8
18:2 130" 199 205 70* 64* 66* 216 211 197 194 201 194 175 176 203 603 635 150" 150" 150" 150"
Chol 217 170 154 222 216 224 194 190 176 172 174 179 180 160 165 186 194 229 228 227 224
TC 141 105 93 157 150 157 114 113 99 94 93 132 129 104 112 125 134 160 160 159 156
LDL-C
45 45 44 45
57 46 42 43 43 44 59 59 48 48 56 31 35
HDL-C
mg/dl 95 89 96 113 112 115 77 77 75 83 65 84 86 72 101 81 83 127 125 121 119 >
TG
The above data set is from 36 studies encompassing 148 diets. The studies utilized 998 subjects (715 males and 283 females). For each study, the sex and number of subjects used and their mean age (or range in ages) in years is provided. Also the mean entry level serum cholesterol (SC) concentration is listed. For some studies the SC represents the value attained on a c o m m o n pre-experimental diet. Also denoted is the percentage of total energy from fat. The percentage energy for each fatty acid consumed is listed. Cholesterol intakes are in mg/d or mg/1000 kcal (denoted by an asterisk). In compiling the table we have not differentiated between serum or plasma concentrations. Blank entries denote either no information in the paper, or that the information was provided in a m a n n e r not allowing calculation of the absolute values (e.g. data in graphical form or mean changes reported). In selecting the above studies we utilized the same criteria as those outlined by Mensink and K a t a n ~.8 and Y u e t al. TM In addition, only those studies that reported the entire fatty acid profile have been selected.
Howard e t al. 84 34M, 29F, age 46, SC 235 27%en
Age 43, SC 205, 40%en Sundram e t al. j95 17M, age 21, SC 167, 30%en Nestel e t al. TM 34M, age 49, SC 225 37%en Schwaab e t a l ? 7° 15F, age 24, SC 188, 38%en Sundram e t a l ) ~ 23M, age 22, SC 174 31%en C h o u d h u r y e t al., 2s age 27 10M, 9F, SC 214, 31%en Fielding e t al. 5~ 84M, age 29, SC 172 37%en
12:0
No. (ref. and study characteristics)
T A B L E 5. C o n t i n u e d
::3" C)
Effectsof dietaryfatty acid compositionon plasmacholesterol
109
Seldom have plasma triglyceride concentrations been reported, although it is known that the hamster frequently develops hypertriglyceridemia.~6t:7~ This is an important issue as recently discussed by Lin et al.~34 According to these workers, the increase in LDL receptor activity induced by PUFA relative to SFA in the hamster studies may be overestimates. SFA and cholesterol feeding in the hamster produces hypertriglyceridemia, consequently VLDL-C also increases. As a result there is increased conversion of VLDL to LDL resulting in an expansion of the LDL-C pool. As a consequence when LDL uptake (which does not discrirrfinate between VLDL, VLDL remnants or radiolabeled LDL for hepatic receptors) and LDL-C are correlated with LDL-C and uptake in the chow-fed hamster at the same LDL-C concentration, LDL-receptor activity appears more suppressed than might actually be the case. Consequently, PUFA-feeding "appears" to be associated with increased LDL receptor activity. By contrast in the guinea-pig, which does not develop hypertriglyceridemia, PUFA feeding does not reverse the LDL-receptor down-regulation induced by dietary cholesterol. TM Thirdly, the bulk of the information from the hamster studies, detailing fatty acid effects on lipid metabolism has been obtained in animals fed diets containing 0.12% w/w dietary cholesterol. Studies by other workers72,76,t72 have documented a major expansion of the h a m s t e r V L D L ''2'76J24J72 and HDL p o o l s , 72'76'124 as well as LDL pool when diets contain cholesterol. The: observed effects on LDL metabolism in the hamster studies may be of secondary importance to the changes in the dynamics of the other lipoproteins. Fourthly, in several of the above mentioned hamster studies the authors assert that dietary cholesterol is needed to clearly document a fatty acid effect. This seems to imply prior knowledge: of what the effect is. While there is no doubt that the fatty acid effect can be accentuated by the presence of dietary cholesterol, using cholesterol-free diets, fatty acid-induced effects on plasma lipoproteins have been documented in several species including hamsters, ~35'~s2non-human primates, 27'7°,75'~°9'H°,H2,159gerbils, ~63guinea-pigs44,45and humans. ~6'33'm If anything, the hamster data clearly show dietary cholesterol to be the primary determinant of LDL-C metabolism, and what is attributed to be a fatty acid effect, is in fact, a cholesterol x fat interaction. In fact, Spady and Dietschy noted that in humans, the "detrimental effect of saturated triglycerides on LDL cholesterol metabolism could be minimized if the intake of dietary cholesterol could be reduced to essentially zero. ''~s2 However, the authors dismiss this on the basis that "it is nearly impossible to accomplish this through dietary means." While this may be true in Western man, since it would necessitate a major change in dietary habits, the advice to minimize cholesterol intake may be a key nutritional guideline in certain developing countries where fat and cholesterol intake seem to be on the upswing as a consequence of increased economic prosperity. Finally, the ,dietary fat sources utilized in the hamster studies have been chosen to represent extrerae intakes, either exclusively SFA (by use of hydrogenated coconut oil) or PUFA (exclusively safflower oil) and as discussed by Horton et al., s~ the mechanism operating under such extreme conditions may not be relevant when less drastic alterations in dietary fatty acids are involved. Furthermore, it is not possible at present to decipher whether the detrimental changes observed when hydrogenated coconut oil is the sole fat source are exclusively attributed to the presence of the SFA or to the relative absence of PUFA. Additionally, in some of the studies31.219"artificial triglycerides" containing only one fatty acid have been utilized, which is clearly at odds with conventional human diets. It is of interest that the reported equivalence of the C 12-C1 6 SFA in hamsters on plasma LDL 2j9 is analogous to an early human study which reached the same conclusion) 4~In this latter study, subjects were also fed diets containing trilaurin, trimyristin and tripalmitin. Similarly Zock et al. TM and Tholstrup et al. 2°L2°2 found 14:0 to be less potent when fed as part of a structured synthetic triglyceride, whereas studies using naturally occurring fats have clearly shown myristic acid to be the most potent in terms of its effects on LDL-C both in human 79'HsJ57`t66.lg5and animal studiesY 5 It would appear that feeding myristic acid as part of trimyrsitin "mutes" its hypercholesterolemic potential and as a consequence its cholesterol-raising ability is lowered, and, accordingly myrsitic acid, appears similar to lauric and palmitic acid.
110
P. Khosla and K. Sundram B. A n i m a l Studies - - Guinea-pigs
The guinea-pig model has been utilized extensively in recent years by McNamara and colleagues to study the effects of dietary fat saturation on lipid and lipoprotein metabolism. 2'~9'~34 Several characteristics of the guinea-pig have made it a particularly useful model for studying dietary fat-mediated changes on lipoproteins, including the fact that, like humans, guinea-pigs have a L D L / H D L ratio > 2, an active plasma CETP, and the ratio of their hepatic free to esterified cholesterol is similar to the ratios found in humans. 2 In addition, dietary fatty acid effects are observed even in the absence of dietary cholesterol, and like humans, the observed effects are generally in the LDL fraction. One problem with the guinea-pig model is the fact that TC concentrations are generally two-fold less than the values observed in humans (usually < 100 mg/dl plasma). Additionally, the above studies suffer from the drawback that in all cases, dietary fat was derived from a single fat source (an obvious contrast to the human situation), and accordingly the observed responses cannot necessarily be attributed to specific fatty acids. Furthermore, the failure to control tightly all the dietary fatty acids (by blending different fats), has meant that it has not been possible to define a range of concentrations over which specific fatty acids exert their effects. Nevertheless, these studies have provided invaluable information on the action of different fatty acid classes, especially the PUFA. In guinea-pigs fed low-fat, low cholesterol-containing semisynthetic diets (19%en fat and < 0.01% w/w cholesterol), a PUFA-enriched diet - - corn oil (2.3%en SFA, 4.2%en 18:1 and 9.2%en 18:2), lowered TC and LDL-C compared to either a MUFA-enriched diet - - olive oil (2.3%en SFA, 15%en 18:1 and 1.4%en 18:2) or a SFA-enriched diet - lard (7.5%en SFA, 8.1%en 18:1 and 2%en 18:2). *~ PUFA feeding was associated with a lower LDL CE/P ratio, higher receptor-mediated binding of LDL to membranes from PUFA-fed animals, while the affinity of the apo B/E receptor for LDL was unaffected. By contrast, SFA and M U F A feeding produced similar plasma lipid concentrations and hepatic receptor number. Essentially the same responses were observed when the diets were formulated with dietary fat contributing 35%en. 45 The decreased LDL-C and higher hepatic apoB/E receptor number observed with PUFA feeding was associated with a lower LDL apoB pool size, higher receptor-mediated LDL FCR and a lower LDL apoB flux rate. 47Additionally, differences in LDL composition and size contributed to the differential rates of LDL turnover observed in vivo. These three studies 44'45'47clearly showed that even at very low intakes of dietary cholesterol, dietary fat saturation affected lipid metabolism at various points, and these changes were apparent at both low and high levels of dietary fat intake (19 and 35%en). In addition to the effects on LDL metabolism, dietary fat saturation affected whole body and hepatic cholesterol synthesis rates, 49 hepatic H M G -CoA reductase activities45 as well as H D L binding to hepatic membranes. 46 Lin et al. TM evaluated the effects of the same three dietary fat sources detailed above 44'45'47 in the presence of different levels of dietary cholesterol (0, 0.08, 0.17 and 0.33% w/w). The levels of dietary cholesterol were chosen to correspond to absorbed amounts representing 6, 50, 100 and 200% of the daily endogenous cholesterol synthesis in the guinea-pig. Regardless of dietary fat, TC and LDL-C were unaffected until the dietary cholesterol load was >_ 100% of the daily endogenous synthesis. As in previous studies, the PUFA-enriched diet resulted in lower TC and LDL-C in comparison to the M U F A and SFA-enriched diets. While dietary fat type and cholesterol both acted independently as well as interactively to affect LDL concentrations, LDL composition and hepatic apo B/E receptor number, dietary PUFA were not effective in preventing receptor down-regulation induced by dietary cholesterol. In fact 50% receptor activity was lost with the P U F A + 0.17% cholesterol-containing diet, whereas the same degree of down-regulation (i.e. 50%) was achieved when the M U F A and SFA diets contained 0.33% cholesterol. At 0.17% w/w dietary cholesterol (equivalent to 100% of endogenous cholesterol synthesis), receptor number (Bmax) was 1470, 1800 and 1220 ng/mg membrane protein for the SFA, M U F A and PUFA diets, respectively, while these figures were 1320, 1160 and 1140 ng/mg with 0.33% w/w dietary cholesterol (twice the level of endogenous cholesterol synthesis).
Effectsof dietaryfatty acid compositionon plasmacholesterol
111
In order to de]Lineatepossible differences induced by different SFA, the above workers have also reported the results of studies in which guinea-pigs were fed fats with differing SFA composition?8Thus in animals fed diets (fat 35%en) in which the fat source was either palm kernel oil (rich in 12:0 and 14:0), palm oil (rich in 16:0) or beef tallow (rich in 16:0 and 18:0), feeding of the 12:0 + 14:0-ri,ch diet (18.3%en 12:0, 6.3%en 14:0, 0.5%en 18:1 and 0.5%en 18:2) elicited higher TC and LDL-C then feeding the 16:0-rich diet (15.2%en 16:0, 14%en 18:1 and 3.4%en 18:2) or the 16:0 + 18:0-rich diet (8.2%en 16:0, 4.9%en 18:0, 16.5%en 18:1 and 0.6%en 18:2). As can be seen, all fatty acids were not adequately controlled (e.g. with regards to the 18:1 and 18:2 contents) such that the observed effects may not be attri'butable solely to the SFA. Regardless, these studies have shown that the increase in LDL-C associated with palm kernel oil feeding was associated with a lower Bm~xas compared to beef tallow (2110 vs 3710 ng/mg), a higher LDL apo B pool size, higher LDL flux rate and a lower receptor-mediated LDL FCR. The LDL parameters associated with palm oil feeding were either similar to those observed with beef tallow, or intermediate. Interestingly, the beef tallow diet resulted in a higher Bmaxthan the value reported when animals were fed the PUFA-enriched diet (3710 vs 2590 ng/mg). TM In addition to the differential effects of different SFA on LDL metabolism, Abdel-Fattah et al. also investigated SFA-induced effects on VLDL metabolism.2 Palm kernel oil (rich in 12:0 + 14:0) elevated TC and LDL-C as compared to beef tallow (rich in 16:0 + 18:0). Between lard and palm kernel oil, the former significantly increased VLDL-TG secretion, while the latter significantly increased VLDL apoB secretion. Collectively, the above studies suggest that PUFA (18:2) induced lowering of TC and LDL-C relative to MUFA and SFA is associated with increased hepatic apoB/E receptor number, increased LDL clearance as well as alterations in the size and composition of LDL. Unlike the dietary studies in humans, MUFA-feeding is not as effective in lowering TC and LDL-C as PUFA-feeding. Amongst the SFA, the most detrimental effects on LDL metabolism are those induced by 12:0 + 14:0, and the latter fatty acids suppress LDL receptors more than 16:0 + 18:0. However, as alluded to above, these studies did not control for all dietary fatty acids, and accordingly several questions remain unanswered, e.g. are 12:0 + 14:0 exerting their effects because the diets were also low in 18:2? Are the beneficial effects of 18:2 dose-dependent or only manifest at very high 18:2 concentrations? Are the effects of 18:1 species specific, or is the source of the 18:1 (in the above case olive oil) an important consideration? Additionally, if 18:0 is neutral as suggested by others, then can the observed effects be attributed to the 16:0 content? The answers to these questions must await future studies.
C. Animal Studies - - Non-human Primates
In a study utilizing three species of monkeys (rhesus, squirrel and cebus) raised from birth on corn oil or coconut oil, Pronczuk et al.165 investigated the cholesterolemic response to short term feeding (8 weeks) of different "saturated" fats - - lard, tallow and butter. An additional diet utilized 1/3 of the "parent" fat (coconut oil or corn oil) and 2/3 fish oil. Since the latter contained cholesterol this was added to all diets at 0.05-0.11 mg/kcal (which represents 125-275mg cholesterol/d human equivalent based on a daily consumption o1" 2500 kcal). Surprisingly, none of the saturated animal fats were as hypercholesterolemic as the 14:0-rich, 18:2-poor coconut oil. The cholesterolemia observed with the 16:0 anLd 18:0-rich fats (lard and tallow) was not significantly different from that observed during corn oil (high 18:2) consumption. Furthermore, in animals reared on coconut oil, both lard and tallow lowered SC and LDL-C without affecting HDL-C, and thereby lowered the LDL/HDL ratio. In monkeys raised on corn oil, only butterfat (rich in 12:0 + 14:0 a:ad like coconut oil, low in 18:2) increased the LDL/HDL ratio attributable to increased LDL-C. In addition to postulating that 16:0 may be "neutral", the authors speculated that concentrations of specific fatty acids stored in adipose tissue may play a role in explaining the observed effects. Rhesus monkeys, relatively insensitive to dietary
112
P. Khoslaand K. Sundram
fat saturation, maintain higher concentrations of PUFAs in adipose tissue, whereas in the fat sensitive cebus, 12:0 + 14:0 account for more than 50% of the adipose tissue fatty acids. To test the neutrality of 16:0, Hayes e t al. 7~ fed the same three species of monkeys cholesterol-free diets in which different vegetable oils were blended to control for specific fatty acids. Three of the diets provided equal amounts of total SFA, MUFA and PUFA. However, amongst these three diets 12:0 + 14:0 were systematically replaced by 16:0. A highly saturated fat diet ( > 80% SFA) and an AHA-type diet (equal amounts of SFA, MUFA and PUFA) were also utilized as "positive" and "negative" controls. As expected these two control diets produced the worst and best lipid profile, respectively. The neutrality of 16:0 was confirmed on three counts. (1) As 12:0 + 14:0 were replaced by 16:0, SC and LDL-C decreased. (2) Replacing half of the PUFA from the AHA diet with 16:0 failed to significantly affect the plasma lipid concentrations, whereas replacing half the PUFA from the AHA diet with 12:0 + 14:0 significantly increased SC, LDL-C, apoB and the LDL/HDL ratio. (3) The observed SC showed an essentially "perfect" fit with the predicted SC based on the regression equations of Keys and Hegsted if 16:0 was considered neutral. The authors proposed that "thresholds" of PUFA and/or MUFA can be made available from the diet or adipose tissue reserves to counterbalance the consumption of a given amount and type of SFA. Rudel e t alJ 67 examined the effects of dietary SFA, MUFA and PUFA in African Green monkeys fed cholesterol-enriched diets (0.8 mg/kcal cholesterol; 35%en fat). The animals were fed their respective diets for up to 66 weeks. A high level of dietary cholesterol was fed in order to elevate TC into the 300 mg/dl range (levels which constitute high risk for humans) in order to evaluate atherosclerosis p e r s e . All diets provided at least 5%en from 18:2. Two experimental periods were utilized, a mixed fat period and a pure fat period. Feeding of SFA (very high concentrations of 16:0) resulted in the highest TC concentrations while PUFA (18:2-rich) feeding produced the lowest level, MUFA (rich in 18:1) were intermediate. During the second feeding period, lipoprotein profiles assessed after 9 weeks of feeding revealed that as compared to SFA, both MUFA and PUFA lowered TC and LDL-C. In addition PUFA also lowered HDL-C. The decreases in LDL-C and HDL-C were reflected by appropriate decreases in apoB and apoA1 concentrations. An interesting observation in this study was that when cholesterol was accidentally omitted from the SFA-enriched diet, the TC plummeted from -390 to -200 mg/dl in 2 weeks. The authors suggested that dietary fatty acids exhibit a threshold, and their respective effects can only be demonstrated once that threshold level has been exceeded. Hunt e t al. evaluated the long term effects (83 weeks) of feeding purified diets (-43%en from fat and P:S ratios 0.5 and 0.9) and different levels of dietary cholesterol (0.01, 0.06 and 0.50 mg/kJ) on LDL transport in Cynomolgus macaques. 87The desired P:S ratios were achieved by blending four different fats. Oleic acid was equalized across diets and the change in P:S ratio was achieved by replacing SFA with 18:2. TC and LDL-C increased with increasing dietary cholesterol, while replacing SFA with 18:2, at any given cholesterol intake, lowered TC and LDL-C. Receptor-mediated LDL FCRs decreased with increasing dietary cholesterol, but were not affected by the P:S ratio. Similarly LDL protein synthetic rates tended to increase with increasing dietary cholesterol, and these rates were generally lowered as SFA were replaced with 18:2. At low cholesterol intake (0.01 mg/kJ) hepatic LDL uptake was higher with the higher P:S ratio. Consistent with the LDL turnover data, hepatic LDL uptake was suppressed with cholesterol feeding. Nicolosi e t al. 159 assessed the effects on LDL metabolism of long term feeding (3-10 years) of SFA or PUFA-enriched diets (exclusively coconut oil and corn oil, respectively), both in the absence and presence ( + 0.1% w/w) of dietary cholesterol. Cebus monkeys were fed semipurified diets with a moderate fat load (-31%en). PUFA feeding was associated with lower TC, HDL-C and LDL-C, regardless of the dietary cholesterol load. In the presence of dietary cholesterol, corn oil fed animals displayed significantly higher plasma cholesterol concentrations than animals fed corn oil alone. By contrast, plasma cholesterol concentrations in coconut oil-fed animals were not affected by the
Effectsof dietaryfatty acid compositionon plasmacholesterol
113
addition of dieta:ry cholesterol. The observed changes in LDL-C were reflected in parallel changes in LDL apoB concentrations and could be explained solely on the basis of changes in LDL clearance, since LDL apoB transport rates were unaffected. However, in sharp contrast to other studies,87SFA and cholesterol feeding decreased both receptor-mediated and receptor-independent LDL FCRs, and furthermore, the major determinant of the observed respon,;e was the degree of dietary fat saturation and not the dietary cholesterol load. In an associated study by Kuo et al., 123mononuclear cells were isolated from the animals utilized by Nicolosi et al) 59 Cells from the corn-oil fed animals exhibited an increased affinity as well as. increased maximal binding of the LDL receptor when homologous LDL was used as the ligand. Generally, increased mononuclear cell degradation of LDL was associated with an increase in cellular cis-unsaturated fatty acids, an increase in membrane fluidity and a lowering of plasma TC. It was proposed that in addition to changes in LDL receptor number, PUFA exerted their effects by altering LDL receptor structure and conformation. In rhesus monkeys fed cholesterol-free diets (31%en fat) rich in either 12:0 + 14:0 or 1 6:0 + 18:1, plasma lipids were unaffected by the fatty acid exchanges) °9 However, feeding of the 16:0 + 18:l-rich diet resulted in a higher VLDLapoB pool size attributed to a three-fold higher VLDL apoB transport rate, whereas the 12:0 + 14:0-rich induced a five-fold increase in the transport rate of LDL apoB derived from VLDL-independent sources (i.e. so called "direct" production of LDL apoB) resulting in a two-fold increase in the circulating LDL pool and slight decrease in HDL relative to 16:0 + 18:1. This result is consistent with the interpretation that 16:0 + 18:1 enhanced triglyceride synthesis and VLDL production, whereas 12:0 + 14:0 reduced VLDL output but increased "direct LDL" production. Similar rates of receptor-independent and receptor-mediated LDL clearance were observed in normocholesterolemic cebus and rhesus monkeys fed cholesterol-free diets (40%en fat), in which the dietary fat was derived from a single oil rich in 16:0, 18:1, or 1 8:2.~l° Plasma lipid concentrations were identical in animals fed the 16:0-rich and 18:1-rich diets. The TC was lower in cebus fed the high 18:2 intake, but this decrease was due entirely to a reduction in HDL-C (putatively apo A1 production). Interestingly, the 16:0-rich diet produced the lowest LDL/HDL ratio (significantly better than the 18:2-rich diet). This result was in stark contrast to that obtained in African Green monkeys (see above) fed cholesterol-enriched diets utilizing the same dietary fat source, t67 Khosla and Hayes "2 fed cebus monkeys cholesterol-free diets (33%en fat) in which a stepwise exchanlge of 10%en between 16:0 and 18:1 was achieved, while 14:0 and 18:2 levels were kept cons~:ant. Also the same diets were fed with 0.3% w/w dietary cholesterol. Plasma lipid concentrations and lipoprotein metabolism (LDL and HDL kinetics) was unaffected when cholesterol-free diets were used, whereas 16:0 was hypercholesterolemic compared to 18:1 when diets contained cholesterol. In the latter case, the increased LDL-C pool was associated with an elevated LDL apoB pool, a lower clearance rate of LDL and higher plasma triglyceride concentrations (indicative of increased VLDL production). However, it is clearly apparent from this study, that when fatty acids were tightly controlled, even though 16:0 increased LDL-C more than 18:1, the major effector of the increase in LDL-C was the dietary cholesterol itself. In a follow up study in cebus monkeys, again fed cholesterol-free diets (31%en fat) in which 14:0 and 18:2 were tightly controlled, identical plasma lipid concentrations (TC, HDL-C and VLDL + LDL cholesterol) resulted when 8%en was exchanged between 12:0 and 16:0) 64 Furthermore, there were no differences in the composition of the lipoproteins following isolation by density-gradient ultracentrifugation. D. Studies in Tissue Culture
Two recent sl:udies are noteworthy. Rumsey et al) 68 have provided preliminary in vitro evidence which is in agreement with the in vivo hamster data 31'34 that fatty acid-induced
114
P. Khoslaand K. Sundram
changes in LDL receptor activity are mediated by changes in ACAT activity. Using human skin fibroblasts, Hep G2 cells and J774 macrophages, these workers ~68 showed that incubation with oleic acid (oleic acid/BSA molar ratio of 2:1), increased cellular cholesteryl ester accumulation, ACAT activity and LDL cell binding and degradation. Incubations with different fatty acids revealed that cellular free cholesterol levels were unaltered, and that the observed effects on LDL degradation were directly correlated with the cellular CE/FC ratio. These results are consistent with the notion that fatty acids alter the distribution of FC and CE, and an increase in cellular CE (mediated by ACAT) stimulates LDL receptor activity. Amongst the various fatty acids tested, incubations with oleic acid resulted in the highest LDL degradation and the highest CE/FC ratio. Amongst the saturated fatty acids LDL degradation was highest with 16:0 and lowest with 8:0 and 18"0, although the data with the SFA were quite varied (e.g. no definitive effect of 14:0 could be established). Interestingly, 16:0 (like 18:1) stimulated LDL receptor activity and CE accumulation, the latter being in agreement with the observation of Goodman et al? 7 reported some 30 years earlier. In contrast to the above effects noted with 16:0, Srivastava et al. ~Ss found 16:0 inhibited LDL receptor activity in Hep G2 and Caco2 cells. However, 16:0 did not affect the mass of the LDL receptor, LDL receptor mRNA levels or the rates of transcription of LDL receptor mRNA, consistent with the notion that 16:0 was affecting LDL receptor gene expression by a post-transcriptional mechanism. In the presence of 25-hydroxycholesterol (analogous to the in vivo situation of feeding 16:0 + cholesterol), 16:0, suppressed LDL receptor activity (relative to oleic acid) by decreasing LDL receptor mRNA. The effects of 16:0 were apparent at different concentrations as well as different palmitate/BSA ratios. Additionally, the deleterious effects of 25-hydroxycholesterol and 16:0, on LDL receptor activity, were additive and in general agreement with various pieces of in vivo data that dietary cholesterol augments the effect of dietary SFA. A possible mode of action of 16:0 may be related to the observation that 16:0 increases the cellular accumulation of phospholipids, which may alter LDL receptor cycling. 2°9 Regardless, this study suggests that 16:0 administered in the presence of 25-hydroxycholesterol enhances the regulatory effect of cholesterol, and although no data were reported for intracellular FC and CE, 16:0 was proposed to be exerting its action by affecting the equilibrium between these two pools. ~88 Collectively, the animal studies and the in vitro data suggest that different physiological situations (dependent on LDL receptor status) dictate the response of specific fatty acids to cholesterolemia. In situations in which LDL receptor expression is < 50% of the maximal receptor expression (as measured in vivo by rates of liver LDL uptake), and LDL-C concentrations are < 90 mg/dl, dietary fatty acids (namely 12:0, 14:0, 16:0, 18:0, 18:1 and 18:2) have minimal effect on the circulating LDL-C concentration. Under these conditions, high concentrations of SFA ( > 10%en) may effect LDL-C primarily by affecting the rates of LDL production. An increase in LDL production may result from two different pathways, either (1) an increase in the conversion of VLDL remnants to LDL or (2) the so-called direct production or secretion of LDL. The latter has been described (based on kinetic analyses) in normal human subjects, 98 familial homozygous and heterozygous hypercholesterolemic patients, 9L~79 patients with familial combined hypertriglyceridemiaH6 and hypertriglyceridemiafl3 as well as various animal species including rats, 5° rabbits, 1~5.127 miniature pigsfl5 baboons, ~26 rhesus 1°9 and cynomolgus monkeys?5 Although it has been argued that the kinetic data can be interpreted without postulating the direct production of LDL, TM several studies have shown that LDL-like particles may be directly secreted by the liver in 1)itro. T M Direct production of LDL is thought to depend on the hepatic availability of cholesterol relative to triglycerideg6'"6 and occurs when there is an increased demand for the transport of hepatic cholesterol at the expense of triglyceride-rich VLDL. In the case of increased conversion of VLDL remnants to LDL, ll7 this may depend on either increased lipolytic activity and/or increased production of VLDL by the liver. Dietary fatty acids can affect both gross VLDL transport (i.e. production rates), as well as the composition of the secreted lipoprotein. For example,
Effectsof dietary fatty acid compositionon plasma cholesterol
115
secretion of a TG-poor, CE-rich VLDL may be a poorer substrate for LPL as opposed to a TG-rich, CE-poor VLDL. Regardless of the manner by which VLDL transport increases, in situations of impaired LDL receptor activity, the net effect is manifest as an increase in the transport rate of L D L . 64'67 The above effects of dietary SFA are also manifest when diets contain cholesterol. However, in this situation, the effect of dietary cholesterol per se becomes a major determinant of the observed response. In such instances, both net cholesterol flux across the liver as well as the fatty acids delivered to the liver (as part of the lipoprotein particle) dictate the observed response. The end result is that LDL receptor saturation and suppression results, mediated by a redistribution of intracellular FC and CE which is dependent on the activity of A C A T ) 4 As LDL receptor activity decreases, LDL transport rates invariably increase due to the inability to clear VLDL remnants. Thus in the presence of dietary cholesterol, SFA increase LDL-C by both attenuating the effects of dietary cholesterol on L D L receptors as well as by increases in LDL transport rates. The hamster s t u d i e s 3t:24'22° a s well as the tissue culture data ~88suggest that 14:0 and 16:0 are the principal fatty acids responsible for the observed effects. However, since t~'ese studies have invariably only assessed one concentration of 16:0, it is not clear whether the same effects are manifest across the broad range of 16:0 intakes that are encountered in everyday human diets, and these studies do not provide information on how the effects are modified by the presence of other dietary fatty acids (e.g. 18:1 and 18:2). While there are abundant data from animal studies detailing how M U F A and PUFA lower LDL-C levels in comparison to SFA, the mechanistic data in humans are scant (see reviews by Grundy and Denke, 64 and McNamara~44).
V. REVIEW OF HUMAN STUDIES Table 5 details. 36 human studies (25 from 1990 onwards) which we will refer to in order to illustrate specific points about fatty acids and their impact on plasma lipid concentrations. 'These studies, selected according to the criteria listed by Mensink and Katan, ~48and Yu et al., TM provided sufficiently detailed information on the individual fatty acids in the diets as f e d (generally determined by direct analysis as opposed to calculations from published ~autrient data-bases). In studies which utilized diets enriched in n-3 fatty acids or trans-falty acids, the respective diets are not shown (the effects of trans-fatty acids is discussed separately). In all, the 36 studies listed represent the data from 148 different diets fed under both metabolic ward and outpatient settings. Twenty-one studies assessed diets in which fat contributed 35~,1 °/oen 16,19,33,51,66,94,118,137,138,140,146,147,154,156,166,170,197,201,202,222,224while 12 studies assessed diets with fat representing 30--35°/0en. 14'25'26'28'52'84'131-133'157'169'195'196Two studies compared the effects of an AAD (38%en) with a Step 1 diet (30%en from fat) within the same study, ~3,54while the original report by Hegsted et al. 79 assessed diets in which fat contributed 22-40%en. The studies represent data from a total of 998 subjects (715 men and 283 women) and amongst each study, the number of subjects varied from five (4 males and 1 f e m a l e ) 137 t o 84 (all men). 5~The original study by Hegsted et al. 79 utilized 36 different diets, fed for 4 week periods to the same group of 10 men over a 2-3 year period. Since this study, the maximum number of diets assessed in a single report is eight. "s Most studies used a rotating crossover design, such that each subject was fed each diet and, therefore, acted as his/her own control. All studies used solid-food diets except for five reports in which liquid-formula (LF) diets were used either in whole 16:9'33'13sor in part. 66 Finally, in 10 reports the TC levels of the subjects at entry into the study was 220-250 mg/d119"79'84'94'131-133"154"156 o r > 250 m g / d l , 14'138 while it was < 200 mg/dl in the remaining studies. The data by Hegsted et al. 79 (diets 1-3t ,re provided as a frame of reference. Within these 36 diets, several points are notewort,.. Firstly, diets with high levels of 14:0 and cholesterol induced the highest TC concentrations (diets 27-33, 36). (The authors reported that TC changes reflected changes in LDL-C.) Invariably, some of these diets were
116
P. Khosla and K. Sundram
also low in 18:2 (diets 30-33, 36). The same was true when diets contained low (116 mg/d) to moderate (306 mg/d) cholesterol contents (diets 3, 7, 13). Secondly, at any given intake of dietary cholesterol, a direct exchange of 18:1 for 18:2 lowered TC (e.g. diets 1, 2 @ 116 mg cholesterol/d; diets 9, 10 or 11, 12 @ 306 mg cholesterol/d; diets 34, 35 @ 686 mg cholesterol/d). The TC for the worst (diet 36) and best (diet 2) diets was 287 and 168 mg/dl, respectively. This reduction in TC (of 109 mg/dl) was achieved by decreasing dietary cholesterol (686 to 116 mg/d), 12:0 + 14:0 (20.8 to 0.12%en) and increasing 18:2 (from 1.6 to 25.8%en). The collective change in 16:0, 18:0 and 18:1 was < 4%en. Since the study was carried out over a 3-year period (and utilized 10 men rotating through 36 diets), time-dependent drifts in the measured parameters would almost certainly have occurred, nevertheless, careful perusal of the data reveals that dietary cholesterol, 18:2 and 12:0 + 14:0 were playing major roles (it was not possible to differentiate between 12:0 and 14:0 in this study as the intakes of the two fatty acids were directly correlated). Harris e t al. 66 using 12 normocholesterolemic men and women, found that LDL-C decreased by 12 mg/dl when 8.4%en from 16:0 + 18:0 and 5.6%en from 18:1 was replaced with 14%en from 18:2 (diets 37, 38). Of the 8.4%en in the SFA, 16:0 only represented 2.8%en, and therefore the bulk of the effect may be attributed to the 18:2 increase. Becker e t al. ~6 using 12 normocholesterolemic men fed LF diets evaluated the effects of replacing 16:0 + 18:0 (diet 41) with either 18:1 (diet 40) or 18:2 (diet 39). Both exchanges lowered LDL-C, with a more pronounced effect of 18:2. The 18:2-rich diet did not lower HDL-C. Of interest is that even though the baseline cholesterol concentrations were low (166 mg/dl), all three cholesterol-free diets, produced significant changes in TC. Since none of the diets contained any 14:0, the results can be explained solely on the basis of the 18:2 exchanges. Thus increasing 18:2 from 4.3 to 6.8%en decreased TC from 133 to 127 mg/dl and as the 18:2 was increased further (to 18.3%en and presumably well above threshold levels) TC decreased marginally further to 123 mg/dl. Baudet e t a L 14 evaluated the response of 24 Benedictine nuns fed four different diets rich in either 18:2, 18:1, 18:1 + 18:2 or 14:0 + 16:0. The 24 nuns were stratified into three groups, 12 normocholesterolemic nuns (TC 188 mg/dl) and 12 hypercholesterolemic nuns (TC 266 mg/dl). A further stratification of the hypercholesterolemic nuns into two groups of six was necessary based on different plasma cholesteryl ester profiles. Within each cohort, the 14:0 + 16:0-rich diets (diets 45, 49, 53) resulted in the highest TC concentrations (217, 272 and 260 mg/dl), while the 18:2-rich diet (diets 42, 46, 50) resulted in the lowest TC levels (163, 175 and 200 mg/dl). (Although extensive information was provided on lipoprotein compositions, lipoprotein cholesterol concentrations were not reported.) An interesting observation from this study is that when one compares diets 44 vs 43 (or 48 vs 47, or 52 vs 51) in the normocholesterolemic and two groups of hypercholesterolemic nuns, TC either decreased by 7 mg/dl (diets 44 vs 43, 52 vs 51) or was unaffected (diet 48 vs 47) even though diets 43, 47 and 51 provided less 18:0, more 18:1, less 18:2 and t w i c e a s m u c h 16:0 as diets 44, 48 and 52. These data, therefore, do not support the notion that 16:0 is hypercholesterolemic. Accordingly, the high TC observed with the 14:0 + 16:0-rich diets was in all probability a consequence of the high levels of 14:0 coupled with the very low levels of 18:2. Mattson and Grundy ~38evaluated 20 hypercholesterolemic men (TC 263 mg/dl) fed LF diets with 40%en from a single fat-source rich in 16:0 + 18:1, 18:1 or 18:2 (diets 54-56). Each diet utilized extreme shifts in fatty acids, which would be difficult to achieve using solid-food diets. None the less, replacing ~15%en from 16:0 (diet 54) with 18:1 (diet 55) lowered TC and LDL-C. When ~22%en from 18:1 (diet 55) was additionally replaced with 18:2 (diet 56) TC and HDL-C were further lowered. Distinct differences were observed between the normotriglyceridemic and hypertriglyceridemic subjects. Although these diets approximated the typical American fat content (40%en), they did not mimic the typical fatty acid distribution. This was probably the first study to show a potent
Effectsof dietaryfatty acid compositionon plasmacholesterol
I 17
hypercholesterolemic effect of 16:0, which was almost certainly magnified by the use of hypercholesterolemic subjects (see below). In the study by Reiser et a l ) 66 the highest TC (168 mg/dl) was observed with the diet (58) with a high 14:0 and low 18:2 content (7 and 0.5%en, respectively). Replacement of the 12:0 + 14:0 with 16:0, 18:0 and 18:1 (diet 57) or 18:2 (diet 59) lowered TC, LDL-C and HDL-C. The low TC and LDL-C with diet 59 may simply have been a consequence of the very high levels of 18:2 (28%en). When this 18:2 was replaced with 16:0, 18:0 and 18:1 (diet 57), TC and LDL-C increased. This may have reflected the higher 16:0 content or alternatively the very low 18:2 content ( < l%en). The former interpretation is consistent with tlhe observation of Baudet et a l ) 4 Consequently, when 16:0 and 18:1 were replaced with 17',:0+ 14:0 (but 18:2 levels were unchanged), TC and LDL-C increased to 168 and 110 mg/dl, respectively (diet 58). Additionally, HDL-C also increased. Thus the data from this study can also be explained solely on the basis of changes in 14:0 and 18:2. Bonanome and Grundy ~9compared the effects of replacing 16:0 with either 18:0 or 18:1, in 11 mildly hypercholesterolemic (TC 227 mg/dl) male subjects fed LF diets, in a metabolic ward setting. In order to formulate a diet with a high 18:0 content, fully hydrogenated soybean oil and high oleic sunflower oil were chemically hydrolysed and randomly reesterified (a process that would have resulted in "atypical" triglyceride moieties). Replacing 15%en from 16:0 (diet 60) with either 18:0 (diet 61) or 18:1 (diet 62) lowered both TC and LDL-C. Although neither the 18:0- or 18:l-rich diets significantly affected HDL-C concentrations, the 18:0-rich diet significantly improved the LDL-C/HDL-C ratio as compared to the 16:0-rich diet. As with the study by Mattson and Grundy 138 using LF diets, the baseline TC concentration was lowered by all diets, regardless of fat'Ly acid composition. McDonald et a l ) 4° evaluated the effects of replacing a mixed fat diet (64) with either a canola oil-based 18:l-rich diet (64)or a sunflower oil-based 18:2-rich diet (65), in eight normocholesterolemic male subjects. This is one of the few studies in which the control diet (65) approximated the AAD in terms of both the total SFA, MUFA and PUFA content as well the individual SFA content. In fact the fatty acid content of the mixed fat diet (1.3%en, 14:0; 8.2%en, 16:0; 4.2%en, 18:0; 15%en, 18:1; 7%en, 18:2) was in remarkably good agreement with the simulated AAD reported by Ahrens and Boucher" (1.5%en, 14:0; 8.2%en, 16:0; 4.3%en, 18:0; 14.7%en, 18:1; 6.4%en, 18:2). Unfortunately, tlhe cholesterol content of the diets was not reported by McDonald et al. Using these dietary exchanges, replacing -9%en from the SFA with 5%en from 18:1 and 3%en from 18:2 (diet 64) lowered TC, LDL-C and HDL-C by 30, 25 and 5 mg/dl, respectively. When both the SFA (9%en) and MUFA (8%en) were replaced by 15%en from 18:2 (diet 65), the same reductions in TC, LDL-C and HDL-C were noted. The results can be interpreted in one of two ways - - either 18:1 and 18:2 are equally effective in decreasing cholesterol concentrations when they replace SFA or once 14:0 has been removed (diets 64, 65) and the threshold requirement for 18:2 has been met (diet 64), additional 18:2 i~; superfluous (diet 65) and in such a situation 18:1 "appears" as effective as 18:2. A similar argument pertains to the data of Chan et al. 25 where once 14:0 was removed from the diet (diets 75, 78) and the 18:2 requirement was met (diets 75, 76), additional 18:2 (diets 77, 78) conferred no benefit on TC concentrations and again 18:1 appeared as effective as 18:2. Mensink and Katan, 146 evaluated the effects of replacing SFA (diet 66) with either a mixture of MUFA and PUFA (diet 67) or PUFA alone (diet 68), in normocholesterolemic subjects. Both exchanges were equally effective in reducing TC and LDL-C, and unlike the data from lVlattson and Grundy ~38 the PUFA-enriched diet did not adversely affect HDL-C concentrations. This may have been due to the fact that in the current study ~46 the PUFA diet only supplied 12%en from 18:2 whereas in the former study, m 29%en was derived from 18:2. It should be pointed out that in the current study, when SFA were replaced with the mixture of MUFA and PUFA (cf. diet 66 with 67) 14:0 decreased by
! 18
P. Khoslaand K. Sundram
1.8%en and 18:2 increased by 3.3%en, and these exchanges alone could account for the bulk of the observed decrease in TC. Furthermore, diet 67 supplied ~8%en from 18:2, a level close to or above the threshold value predicted by Hayes et al. TM Accordingly, increasing the 18:2 content further, to 12.7%en (diet 68) failed to improve the plasma cholesterol significantly and, again, 18:1 appeared as effective as 18:2. Careful scrutiny of the dietary fatty acid profile reveals that a similar explanation can be forwarded to account for the observations of Ginsberg e t al., 54 Barr e t al. ~3 and Friday e t al. 52 By contrast, the data from Kris-Etherton et al. H8 are of interest as 18:1 and 18:2 were exchanged for each other below the 18:2 threshold. In this study, normocholesterolemic men were fed solid-food diets and the results of two different studies (Study 1, diets 102-105; Study 2, diets 106-109) were reported. In the first study, feeding of a 18:2-poor (1.7%en) but SFA-enriched diet (3.5%en from 14:0 and 9.3%en from 16:0, diet 104) produced the highest TC and LDL-C concentration (176 and 113 mg/dl, respectively). Replacing the SFA with 18:1 (diet 102), while keeping 18:2 constant (-2%en), lowered TC and LDL-C to 152 and 92 mg/dl, respectively. When the SFA were replaced with 18:2 (diet 105), TC and LDL-C values (139 and 83 mg/dl) were even lower. The low levels of 18:2 in diets 102 and 104, were in all probability below the threshold requirement for 18:2, and supplying extra 18:2 was more effective in lowering TC and LDL-C than supplying extra 18:1. TM Also in this study, replacing 14:0 (diet 104) with 18:0 (diet 103) but maintaining a constant level of 16:0 and 18:2, lowered TC and LDL-C, clearly suggesting that 14:0 was hypercholesterolemic compared to 18:0. The results from Study 2 essentially paralleled the results from Study 1. Mata et al. J37fed diets devoid of any 12:0 and 14:0, with background cholesterol intakes of-400 mg/d to 21 normocholestrerolemic females. TC values on the control diet (diet 83) averaged 204 mg/dl. Replacing 16:0 with 18:1 (diet 84) or 18:2 (diet 85) effectively reduced TC and LDL-C concentrations to the same degree. Since 14:0 was not a factor in these diets, the reduction in plasma cholesterol concentrations can be attributed to the decrease in 16:0. As discussed earlier, the various regression equations 7°'79'~63identify 16:0 to be an important hypercholesterolemic factor when diets contain > 300 mg cholesterol. Thus in this study, removal of the 16:0, would have removed the potent 16:0 x cholesterol synergism, and accordingly would account for the reduction in TC and LDL-C that was observed. In the study by Friday e t al., 52 5 normocholestreolemic subjects (4 males and 1 female) were fed a 14:0 + 16:0-rich, 18:2-poor diet (diet 79). Decreasing the SFA (14:0 by 3%en, 16:0 by 7%en) and MUFA (by 6%en) and replacing these with 23%en from 18:2, lowered TC, LDL-C and HDL-C by 47, 35 and 5 mg/dL, respectively (diet 80). In a study utilizing 84 normocholesterolemic men from different ethnic backgrounds, Fielding e t a l ? ~ compared the effects of SFA and PUFA-enriched diets at two levels of dietary cholesterol intake. At low cholesterol intakes (-200 mg/d), TC concentrations (165 mg/dl) on the SFA-enriched diet (diet 142: 1.5%en from 14:0, 8%en from 16:0 and 5.4%en 18:2) were not significantly different (160 mg/dl) from the PUFA-enriched diet (diet 141: 0.8%en from 14:0, 6.1%en from 16:0 and 8.2%en from 18:2). At high cholesterol intakes (~600 mg/d), TC concentrations (194 mg/dl) on the SFA-enriched diet (diet 144: 2.2%en from 14:0, 9.1%en from 16:0 and 4.4%en 18:2) were significantly higher (186 mg/dl) than the PUFA-enriched diet (diet 143: 0.8%en from 14:0, 7%en from 16:0 and 9.3%en from 18:2). TC concentrations reflected changes in LDL-C concentrations, and distinct differences were noted amongst the various ethnic groups. However, the dietary cholesterol, itself accounted for the principal increase in TC and LDL-C, regardless of whether the diet contained SFA or PUFA. Howard et al. 84 evaluated the effects of systematically replacing 18:1 with 18:2 as part of a AHA Step 1 diet (30%en from fat and 150 mg cholesterol/1000 kcal) in mildly hypercholesterolemic males and females (TC 235 mg/dl). Dietary 18:1 was sequentially decreased (from 14 to 6%en) and replaced with 18:2 (3 to 13%en). TC progressively decreased from 229 to 224 mg/dl, while LDL-C decreased from 160 to 156 mg/dl). HDL-C was not affected.
Effects of dietary fatty acid composition on plasma cholesterol
119
A. Lauric and Myristic Acid
Three studies have compared 12:0 "head to head" with other fatty acids. Using cholesterol-free LF diets, Denke and Grundy33compared the effects of 12:0 with either 16:0 or 18:1 in 18 men. The 12:0-rich diet (17.6%en from 12:0) raised TC by 9 mg/dl compared to a diet with 17.4%en from 16:0. The increase occurred exclusively in LDL-C. Both diets supplied 16-18%en from 18:1 and 2.5-4%en from 18:2. In comparison to the 18:l-rich diet (30.3%en 18:1, 0.04%en 12:0) the 12:0-rich diet (17.6%en 12:0, 17.8%en 18:1) increased TC, LDL-C and HDL-C by 28, 15 and 5 mg/dl, respectively. TG levels were unaffected. Using 15 normocholesterolemic women (SC 188 mg/dl) fed solid-food diets (with about 200 mg/d choles'terol), Schwab et al. 17°failed to find any difference on plasma lipid levels following a 4%en exchange between 12:0 and 16:0 with background 18:1 and 18:2 levels of 10 and 5%en, respectively. In a preliminary report, 3s exchanging 5%en between 12:0 and 16:0 also f~.iled to elicit any changes in plasma lipids in normolipemic people fed solid-food diets. Similarly, a 10%en exchange between 12:0 and 16:0 (with all other fatty acids held constant) failed to elicit any change in plasma lipid concentrations in gerbils or cebus monkeys fed cholesterol-free diets) 64 Replacing 10%en from 14:0 with 16:0 lowered TC by 9 mg/dl in normocholesterolemic subjects (TC 19'6 mg/dl). 224 Reductions were apparent in both LDL-C (4 mg/dl) and HDL-C (5 mg/dl). However, the response to feeding the 14:0-rich diet was considerably less than anticipated based on the human regression equations. Replacing 16%en from 14:0 with 16:0 in normolipemic men TM resulted in similar TC levels (135 and 133 mg/dl), but again the response to 14:0 was considerably less than would have been predicted from the human regression equations.
B. Palmitic Acid
Mattson and Grundy ~3scompared the effects of replacing 15%en from 16:0 with either 18:1 or 18:2 in hypercholesterolemic men (TC 263 mg/dl) fed cholesterol-free LF diets which provided a background level of 16%en from 18:1 and 4%en from 18:2 (Table 6). The 16:0-rich diet decreased TC by 39 mg/dl compared to entry level values. However, when 16:0 was :replaced with 18:1 or 18:2 TC decreased further to 197 and 191 mg/dl, respectively. The decreases occurred in both LDL-C (23-24 mg/dl) and HDL-C (1-4mg/dl). Additionally TG decreased by 10-28 mg/dl. Bonanome and G r u n d f f compared the effects of replacing 14-16%en from 16:0 with either 18:0 or 18:1 in mildly hypercholesterolemic men (SC 27 mg/dl) fed cholesterol-free LF diets with background 18:1 and 18:2 levels of 15 and 4%en, respectively. Again, the 16:0-rich diet lowered SC by 25 mg/dl compared to entry levels. However, replacement of 16:0 with 18:0 or 18:1 elicited further decreases of 29 and 21 mg/dl, respectively. Reductions were again apparent in LDL-C. Denke and Grundy33compared the effects of replacing 15%en from 16:0 with 18:1 in 18 men :fed cholesterol-free LF diets with background 18:1 and 18:2 levels of 16 and 4%en, respectively. The 16:0-rich diet raised SC by 28 mg/dl compared to the 18:1 diet, with LDL-C increasing by 24 mg/dl and HDL-C by 3 mg/dl. No effects were noted on TG. The entry level lipid characteristics of the subjects were not reported. The above three metabolic-ward studies t9'33:38 were characterized by (1) the use of liquid-formula diets, (2) high levels of total fat intake -40%en, (3) relatively "older" male subjects with moderate to severe hypercholesterolemia based on their entry level SC concentrations, ,(4) the feeding of atypical diets (with 16:0 representing -45% of the total fatty acids in the palmitic acid enriched diets and 18:1 accounting for -75-80% of the total fatty acids in the oleic acid enriched diets) which would be very difficult to achieve in the everyday human experience. Cobb and Risch29 noted that the most significant predictors of LDL-C responsiveness were age and the degree of change in dietary fat saturation. Older subjects required smaller changes in dietary fat saturation than did less-responsive subjects to achieve a comparable reduction in LDL-C levels. Furthermore, the latter subjects
0.4 0.04
0
0
0.12 0.04
0 0
0.2 0.3
0.3 0.1
0.2 0.2
0.3 0.3
0 0
0.2 0.2
Bonanome and Grundy 19 I l M , age 64; SC = 227; 40%en; LF
Denke and Grundy 33 14M, age 63; SC = NR; 40%en; LF
Mata et al. ~37 2IF, age 43; SC = 191; 36%en; SF
Nestel et al. ~ 27M, age; SC = 221; 37%en; SF
Ng et al. ~57 20M, 13F, age; SC = 194; 31%en; SF
Nestel et al. ~ 34M, age 49; SC = 225; 37%en; SF
Zock et al. 224 23M, 36F, age 29; SC = 196; 39%en; SF
Sundram et al. t~ 23M, age 22; SC = 174; 31%en; SF
Choudhury et al. :s 10M, 9F, age 27; SC = 214; 31%en; SF
14:0
5.1 10.1
3.9 8.2
14.9 5
4.6 8.0
13.4 6.3
5.2 9.8
12.3 6.3
17.4 1.9
2.2
18.0
17.4 2
16:0
18:0
1.8 2.1
1.2 1.5
4.1 3.8
1.8 2.0
1.8 1.9
2.3 2.3
3.1 2.5
1.7 0.9
1.9 0.9
2.0 1.0
18.9 11.6
16.9 11.8
11.6 20.9
20.3 17.6
14 21.8
17.8 12.9
13.5 21.4
16.0 30.3
15.5 31.9
15.8 29.3
18:1
18:2
3.4 4.3
6.4 7.3
4.4 4.1
2.7 2.7
3.7 2.9
5.3 5.7
3.2 3.3
4.0 6.2
3.8 4.8
3.9 7
194 175
194 201
359 352
64 66
200 200
64~ 73 ~
405 410
0 0
100 I00
0 0
C
LDL-C
133 134
151" 161"
133" 108~
179 180
172 174
192a 175a
132 129
94 93
115" 10P
216 a 150~ 224 ~ 157a
195 197
215" 226"
204 a 186a
200 a 152a 172~ 128~
202 a 140a 181" 119"
224" 143 a 197" 119"
TC
31 35
48 ~ 56"
59 58
43 44
37 37
38 a 42 a
55" 58"
35 32
42 44
39 38
84 86
83 65
89 84
112 115
125 126
135 128
84 ~ 93 ~
94 93
128 122
259 249
HDL-C TG
mg/dl L/H
4.3 3.7
2.15 a 1.68 a
1.85 ~ 1.61 a
3.49 3.57
3.74 3.71
4.0 3.8
1.9
2.4
4.3 4.0
3.6 3.0
3.7 3.1
Study characteristics include the number and sex of the subjects, mean age, starting serum cholesterol (SC) concentration, the %en from fat and whether the study used liquid formula (LF) or solid food (SF) diets. The SC is either the entry level value or the value attained on a basal or habitual diet. For the L/H ratio, values in italics were obtained by dividing LDL-C/HDL-C. "Mean values for a given parameter sharing a common superscript are significantly different.
0.6 0.6
0.5 0.3
1.1 0.8
0.6 0.7
0.5 0.2
0.6 0.9
0 0
0.4 0.04
0.4 0.04
12:0
0.04 0.04
Ref./study characteristics
Mattson and Grundy m 20M, age 59; SC = 263; 40%en; LF
Dietary fatty acids %en
TABLE 6. H u m a n Studies Directly Comparing Dietary Palmitic vs Oleic Acid
o
Effectsof dietaryfattyacid compositionon plasmacholesterol
121
tended to be younger. In fact changes in LDL-C concentrations could be described by a multiple regression equation based on the change in %en from the SFA and the age of the subjects. It is clear that given the a b o v e conditions, high concentrations of palmitic acid raise TC relative to high concentrations of 18:1. However, an unheralded aspect of the above studies is that they help define the "boundary limits" of the maximal TC lowering benefit that can be derived from dietary fatty acid induced changes. In other words, for subjects with high risk (in terms of baseline TC, age and levels of fat consumed) under the strictest dietary conditions, the maximal TC and LDL-C lowering that can be anticipated with dietary fatty acid exchanges is -25 mg/dl. Based on the above, it is no surprise that when solid-food diets are utilized with more realistic fattty acid exchanges and mildly hypercholesterolemic to normocholesterolemic younger subjects are used, the hypercholesterolemia attributed to palmitic acid is either muted or disappears. In two studies from Nestel and coworkers, 154'156replacing -4%en from 16:0 with 18:1 lowered LDL-C by -7-10 mg/dl in mildly hypercholesterolemic male subjects (SC 220 mg/dl), consuming solid-food diets, when other fatty acids were tightly controlled and cholesterol intakes were low ( < 100 mg/1000 kcal). When solid-food diets have been utilized and young, normocholesterolemic subjects have been used, Lhe situation is quite different. Ng et al. j57 exchanged 7%en between 16:0 and 18:1 in 33 normocholesterolemic men and women (entry SC 190 mg/dl) in diets which provided < 200 mg/d cholesterol and 3%en from 18:2. SC, LDL-C, HDL-C and TG concentrations were identical on both diets. Sundram et al. 196 exchanged 4%en between 16:0 and 18:1 in 23 normocholesterolemic men (SC 172 mg/dl) consuming diets with < 200 mg/d cholesterol and at least 6%en from 18:2. Again there were no differences in SC and LDL-C concentrations between the two diets, although HDL-C was elevated on the 16:0-rich diet. In a preliminary report from Sundram e t a / . 198 replacing 5%en from 16:0 with 18:1 in 27 normocholesterolemic men and women (SC 180 mg/dl) consuming diets with < 200 mg/d cholesterol, resulted in identical plasma lipid concentrations. Choudhury et al. 28 managed a 5%en exchange between 16:0 and 18:1 in 19 men and women with starting SC of 213 mg/dl. Dietary cholesterol was again low, < 200 mg/d, and diets provided 3--4% en from 18:2. Both diets resulted in similar plasma lipid concentrations. By contrast, replacing 10%en from 16:0 with 18:1 in normocholesterolemic young subjects (SC 196 mg/dl, age 29) lowered LDL-C by 14 mg/dlY This latter study by Zock e t al. TM is the only study that we are aware of which has shown a hypercholesterolemic effect of palmitic acid in young, normocholesterolemic subjects consuming solid-food diets with moderate cholesterol intakes. This study differs from the three studies2s,157'196which found 16:0 to be, equivalent to 18:1, in that the level of total fat was 39%en, whereas in the three former studies it was -31 a/oen.28As7't96 Although it is not possible as yet to attribute the observed difl'erences to the level of fat p e r se, two points are worth noting. Firstly, in the Dutch study.,TM the diets fed did not utilize natural fat sources. The 18:l-rich diet was prepared by blending high oleic sunflower oil, fully hydrogenated sunflower oil, high linoleic acid sunflower oil, fractionated palm oil and an interesterified mixture of various oils. The 16:0-rich diet was formulated by blending fractionated palm oil, cotton seed oil and fully hydrogenated sunflower oil. The feeding of atypical triglyceride moieties may have been partly responsible for the observed effects. By contrast when the habitual Dutch diet was maximally replaced with palm oil, SC and LDL-C were unaffected.197 One other human study has compared the effects of 16:0 and 18:1 with all other fatty acids being tightly controlled. Mata et al. 137 fed normocholesterolemic females (SC 191 mg/dl) 36%en fat-containing solid food diets. Replacing 6%en from 16:0 with 18:1 lowered SC andL LDL-C by 18 and 25 mg/dl, respectively. The relatively pronounced reduction in SC and LDL-C may have been attributed to the fact that diets contained relatively high amounts of cholesterol (400 mg/d) and according to the regression
122
P. Khoslaand K. Sundram
equations of Hegsted et al. 79 and Pronczuk e t al., 163 the high SC observed on the 16:0-rich diet, may have been partly attributed to a 16:0 × cholesterol interaction. In support of this latter argument, in cebus monkeys, a stepwise exchange of 10%en between 16:0 and 18:1, with all other fatty acids held constant, resulted in identical plasma lipid values and LDL and HDL kinetic parameters when fed as part of a cholesterol-free diet, whereas 16:0 appeared hypercholesterolemic relative to 18:1 in the presence of high levels of dietary cholesterol) ~2 Thus, in direct comparisons 16:0 has been found to be hypocholesterolemic224 or similarTM relative to 14:0, hypocholesterolemic relative to 12:0+ 14:0, 75'135'157'195'197 hypercholesterolemic33 or similar38'~64:° relative to 12:0, hypercholesterolemic relative to 18:0~9and hypercholesterolemic~9'33:38'224or similar28'112'157:96:98 relative to 18:1. The response relative to 18:1 seems to be a function of the cholesterolemia of the subjects used, with 16:0 appearing hypercholesterolemic in hypercholesterolemic~9'33'mand mildly hypercholesterolemic subjects, ~54:56but similar to 18:1 in normocholesterolemic subjects. 28'157'~96'198Based on these observations, the assertion that 16:0 is the major cholesterol-raising fatty acid becomes less clear cut, and as argued by Hayes and colleagues, one can actually begin to make the case that, in several situations, 16:0 may in fact be "neutral", like 18:170'71"73 75,108 113,135,163-165 o r at least, "conditionally cholesterolemic".74 C. Disparate Effects of Palmitic Acid
Why does palmitic acid appear to show such disparate effects when the human regression equations79:48'221and a vast body of animal data suggest otherwise? One reason may pertain to the metabolic status of the subjects studied (as discussed above). Secondly, as discussed elsewhere, the animal studies have frequently examined the effects of 16:0 at very high concentrations and the results have been extrapolated for all concentrations of 16:0. Thirdly, even though regression equations using individual fatty acids show 14:0 to be the most potent, when regression equations based on total SFA are utilized (usually for convenience) they invariably assign the major cholesterol-raising effect to 16:0 since it is the most abundant fatty acid in the diet and accordingly overestimate its potency. In this regard the analysis by Hayes and Khosla is of interest. 7° After a retrospective analysis of the data of Hegsted e t at., 79 these authors suggested that, in situations in which dietary cholesterol is low ( < 300 mg/d) and lipoprotein metabolism is not compromised, dietary 14:0 and 18:2 may explain the bulk of the changes in TC levels.7° Additionally, these workers proposed that in normocholesterolemic subjects ( < 225 mg/dl SC?) and when cholesterol intakes are low ( < 300 mg/d?) the levels of palmitic acid may be irrelevant. Thus when utilizing the 16 diets of Hegsted et al. 79 in which cholesterol intake was < 306 mg/d, these authors showed that 85% of the observed variation in TC could be explained by equation (6) (detailed elsewhere). To test the above concept, we compared the observed TC with that predicted by equation (6), based on the dietary 14:0 and 18:2 content, as well as the baseline TC of the subjects in the various studies listed in Table 5. In all, 137 observations from 33 studies were evaluated (Fig. la). As can be seen, 65% of the variation in the observed TC could be explained on the basis of the 14:0 and 18:2 content alone. It is of interest that two of the main outliers in this analysis were 14:0-rich diets in which the high level of 14:0 was achieved by using artificially generated fats. 2°L224 Upon elimination of diets with > 400 mg/d (a more conservative figure than the 300 mg/d suggested by Hayes and Khosla 7°) and those that used artificially generated fats, this value increases to 80% (Figure 1b). Further elimination of diets that used liquid formulas as well as studies in which the starting cholesterol concentration of the subjects was > 235 mg/dl, increases the value to 870 (Figure lc). Thus it is clear from this analysis that in 27 different studies utilizing 83 diets, the level of 16:0 appeared to be "irrelevant", i.e. 16:0 appeared "neutral" like 12:0, 18:0 and 18:1. An analysis of the frequency distribution revealed that in almost 7 0 o of the diets, 14:0 represented < 1.0%en, 16:0 accounted for 6-9%en, 18:1 levels were 1O-17 %en and 18:2 levels were 1-10 %en. Whether 16:0 is neutral per se, or simply appears
Effects of dietary fatty acid composition on plasma cholesterol
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Pred TC (mg/dL plasma) Fig. I. Comparison of observed and predicted TC concentrations. Predicted TC was calculated on the basis of equation 67°,and is a function of the starting cholesterol concentration (SC) as well as the per cent dietary energy from 14:0 and 18:2. Studies presented in Table 5 were used for the calculations. (a) Correlation for 137 observations from 34 studies. Does not include data from references 33 and 118 in which the m e a n SC was not reported. (b) Correlation for 104 observations from 31 studies. Excludes diets with > 400 mg dietary cholesterol and those that used artificially generated triglycerides. (c) Correlation for 83 observations from 27 studies. Excludes data in which the subject's SC was >235 mg/dl as well as studies that used liquid-formula diets, artificial triglycerides or > 400 mg cholesterol per day. JPLR
35/2--B
123
124
P. Khoslaand K. Sundram
so when fed in diets with low levels of 14:0 and adequate 18:2 cannot be resolved, but a study in cebus monkeys"2 fed cholesterol-free diets (14:0 and 18:2 were held constant), in which 16:0 was increased sequentially from 6 to 16%en at the expense of 18:1 revealed no differences in plasma lipids, supporting the idea that in such situations, the level of 16:0 is immaterial. It should though be stressed that while there may be subtle differences in any given study, overall a model that only requires knowledge of the 14:0 and 18:2 content goes a long way in explaining the observed TC.
D. Stearic Acid Several studies have established that stearic acid does not affect SC and is accordingly considered neutral like CHO and oleic acid. 16't9,32'79`tls,166 A recent supplement to the American Journal of Clinical Nutrition published several papers devoted solely to stearic acid. ~9~There seems to be a general consensus that stearic acid, like the short chain fatty acids ( < 10 carbons) and oleic acid does not affect lipid concentrations in an adverse manner. However, a recent report by Tholstrup et al., has presented evidence that in comparison to 16:0 and 12:0 + 14:0, stearic acid increased plasma levels of the atherogenic lipoprotein, Lp(a), in human subjects. 2°3 Generally, Lp(a) levels have been shown to be insensitive to dietary manipulation, although in recent years, dietary trans-fatty acids have been shown to adversely affect Lp(a) concentrations in some studies (see section VI). If the findings of Tholstrup et al. are verified by other workers, this may become a point for future concern.
E. Oleic and Linoleic Acid Mattson and Grundy t38 were the first to show that 18:1 was as effective in decreasing LDL-C (almost 30 mg/dl) as 18:2, and unlike the latter did not lower HDL-C when replacing SFA. Though the study utilized cholesterol-free LF diets with very high concentrations of 18:1 and 18:2 (almost 30%en in a diet providing 40%en from fat), it paved the way for several reports investigating the efficacy of 18:l-enriched diets as a means for lowering L D L - C . 14'16'25'51'54'66'79'118'140't46'147'166"222 The cholesterol-lowering properties of 18:2 have been known for more than four decades. 3"22,23'79It is not our intention to review the effects of 18:1 and 18:2. These have been reported elsewhere in great depth,~7'64'65'~44and we have alluded to the role of 18:2 as part of the threshold concept throughout this review. The importance of dietary 18:1 and 18:2 as part of cholesterol-lowering regimens is not a contentious issue. vI. TRANS-FATTY ACIDS Recently, dietary trans-fatty acids have been thrust into the forefront of both public and scientific scrutiny, trans-Fatty acids are geometrical isomers of unsaturated fatty acids. Although trans-fatty acids occur naturally in animal (ruminant) products (up to 5% of total fatty acids), the principle dietary source of trans-fatty acids results from the consumption of products utilizing hydrogenated vegetable oils. While numerous trans isomers result during chemical hydrogenation, the major focus of attention has been the trans-monounsaturated fatty acids. Prior to 1990, collective evidence suggested that trans-fatty acid consumption was not a health hazard in terms of hypercholesterolemia,4~'~39'~5"~s7and extensive reviews42,43 found scant evidence that trans-fatty acids adversely affect the plasma lipid profile. However, starting with the study of Mensink and Katan, ~47 several reports have surfaced94'~3~'~5°a55'~56'2~5'2~6 which suggest that trans-fatty acids have an adverse effect on blood lipids [either increasing LDL and/or total plasma total cholesterol, decreasing HDL cholesterol and/or increasing Lp(a) concentrations]. Additionally, epidemiology data from Willett and coworkers ~2.~,~.m have sparked tremendous controversy in both the scientific and popular press with regards to trans-fatty acid consumption.~°'~'.~3°,~58.223Several recent reviews have dealt with the topic of trans-fatty acids, tS.39.4°.~.~2°'~49.2~t.2~4
Effectsof dietary fatty acid compositionon plasma cholesterol
125
A. Consumption of Trans-Fatty Acids trans-Fatty acid consumption varies heavily with socioeconomic factors. The average per capita con:~umption in the U.S. has been estimated to be 7.6g/person/d g8 to 15.2 g/person/d, '4° while recent estimates put the figure at 8.1 g/person/d 89 (approximately 6% of the total U.S. fat consumption). Average trans-fatty acid consumption in the U.K. has been estimated to be 7 g per day translating to 6% of the total fat intake. 2~ However, these average figures may be misleading, conservative estimates suggest that certain populations may consume as much as 27 g/person/d, 4° which can amount to 24% of the total fat intake. Litin and Sacks TM calculated that 9.6 g of TFA were ingested in a 1800 calorie diet trarLslating to 5% of the total energy intake. B. Trans-Fatty Acids and CHD - - Epidemiological Data Using responses to a semiquantitative food frequency questionnaire, Willett and colleagues 12,2°6,2°s'2~3 have provided epidemiological evidence that trans-fatty acid consumption (specifically, trans-fatty acids produced by chemical hydrogenation) shows a positive association with a plasma lipid profile that is conducive to premature CHD. Although the epidemiology data do not provide for a "cause and effect" explanation, it has nevertheles,; served to increase awareness of trans-fatty acid consumption. Briefly, these three epidemiological reports have detailed that (a) trans-fatty acid consumption is positively correlated with TC, LDL-C and negatively correlated with HDL-C; 2°6 (b) trans-fatty acid consumption shows a positive association with the risk of CHD; ~°8'2'~(c) trans-fatty acid intake may be directly related to risk of a first myocardial infarction. ~2 Additionally, both total trans-fatty acids as well as specific trans isomers (e.g. trans-palmitoleic acid) have been found to be significantly higher in the plasma of patients with angiographically documented coronary artery disease in comparison to normal matched control subjects. 177Collectively, the epidemiological data support the thesis that dietary trans-fatty acids may a potential and significant risk factor for CHD.
C. Trans-Fatty Acid Effects on Plasma Lipids Using 59 normocholesterolemic young men and women, Mensink and Katan 147 compared the effects of exchanging 10% dietary energy between an oleic, elaidic or a saturated fatty acid-rich diet in which fat provided 40% of the dietary energy. The level of trans-fatty acids consumed in the elaidic acid-rich diet averaged 33 g/d (11%en) - - a figure that is four-fold higher than the 8 g/d consumed in a typical American diet. 89 Of the 11%en derived from trans-fatty acids, the trans-C 18:1n-9, trans-C 18:1n- 10 and the trans-C-18:ln-ll isomers contributed 3.2, 2.5 and 2.3% en, respectively. Compared to the oleic acid-rich diet, feeding of the elaidic acid-rich diet resulted in significantly higher TC, LDL-C and plasma TG and significantly lower HDL-C (54 of 59 subjects). Compared to the elaidic acid-rich diet, the saturated fatty acid-rich diet significantly increased TC, LDL-C and HDL-C, while TG was unaffected. As a combination of these changes, consumption of the trans diet produced significantly higher ratios of LDL-C/HDL-C (10% higher compared to the saturated fatty acid-rich diet, and 28% higher corapared to the oleic acid-rich diet) and TC/HDL-C (7.6% higher compared to the saturated[ fatty acid-rich diet and 20% higher compared to the oleic acid-rich diet). A subsequent analysis of frozen serum samples from this study ~5° revealed that the trans diet also resulted in significantly higher concentrations of Lp(a) in comparison to the levels observed with the oleic acid-rich diet. However, the saturated fatty acid-rich diet produced significantly lower Lp(a) concentrations than the oleic acid-rich diet. Collectively, recent clinical studies (Table 7) have shown trans-fatty acid consumption (3.8-11.2%en) to be associated with increased concentrations of TC, 94'131'147"155'1~'215 LDL-C, ~'13~''55"'::6'~5'222and, where detected, decreased plasma HDL-C concentrations. ~'~'7"222 These changes in plasma lipoprotein cholesterol, collectively resulted in increased ratios
s a t u r a t e d fat
oleic acid-rich moderate trans high t r a n s SFA-rich
trans
p a l m i t i c acid-rich baseline corn oil b a s e d
trans
habitual oleic acid-rich
trans
linoleate-rich stearate-rich
1.5 1.5 1.0 3.6
0.6 0.2 0.3 0.3 0.4 0.1 0.1
0.7 0.5 0.5
0.5 0.4 3.4
12:0
1.3 1.3 1.1 2.3
2.3 0.6 0.8 0.9 1.5 0.4 0.3
0.9 1.0 1.0
0.5 0.7 2.7
14:0
7.8 7.2 7.4 10.3
8.4 5.2 4.9 9.8 6.9 3.9 4.2
5.8 5.7 4.8
4.7 4.3 8.1
16:0
2.8 3.1 3.2 3.1
3.8 2.3 3.7 2.3 3.3 1.6 2
2.8 11.8 3.0
3.0 3.6 3.5
18:0
16.7 14.1 11.4 10.9
8.4 17.8 15.2 12.9 10.4 9.6 7.5
14.7 15.4 14.6
23.0 12.6 12.8
c18:1
D i e t a r y fatty acids ( % e n )
0.8 3.8 6.7 0.7
< 1 1.4 5.7 < 1 0.8 0.4 4.2
0.1 0.3 7.7
0 10.9 1.8
t18:l
6.1 6.0 6.2 6.1
3.4 5.3 6.6 5.7 7.0 8.5 7.9
12.0 3.9 3.8
4.6 4.6 3.4
18:2
135 135 135 135
113 64 68 73 128 83 77
140 136 140
146 133 141
Cc
203 ~bc 211 ~d 213 ~ 217 ~d~
228 215 ~b 229" 226 b 223 194~ 205 a
183 a8 189 a 189 b
172 a8 183 ac 193 ~
TC g
129 abc 137 ad 139 b 141 ~
163 151 ab 165 a 1618 153 125 b 135 b
109 a8 116 a 119 b
103 ab 118 "c 121 ~
LDL-C
mg/dl
55 ~b 54 c 53 ~b 57 ~
38 38 a 38 b 42 ~b 48 44 43
57 ~b 55 ~ 538
55 ab 48 ac 55 b~
HDL-C
91 a8 98 • 103 ~ 95 c
139 135 142 128 108 110 114
84 a 92 a 89
72 a~ 83 ~ 83 b
TG h
3.96 abc 3.9 ~d 4.29 ~¢ 3.8P ~
4.29 3.97 4.34 3.83 4.83 4.58 ~ 5.54 a
1.91 2.11 2.25
2.02 ~b 2.58 ac 2.34 ~
L/H i
Study characteristics include n u m b e r a n d sex of subjects, s t a r t i n g s e r u m or p l a s m a cholesterol (SC) in mg/dl, a n d either the subjects' m e a n age or the age range. ~-~In each study, m e a n values, sharing a c o m m o n superscript were significantly different. Statistics n o t c a l c u l a t e d for the b a s e l i n e / h a b i t u a l diets, rDietary cholesterol (mg/1000 kcal), gTotal p l a s m a or serum cholesterol. hTriglyceride, iFor refs ~ a n d ~3~ this is T C / H D L - C .
J u d d et al. ~ 58 (29M, 29F) SC = 205 Age: 25-65 y
Lichtenstein et al. ~3~ 14 (6M, 8F) SC = 238 Age: 44-78 y
Nestel et al. ~56 27M, SC = 221 Age: 30-63 y
Z o c k a n d K a t a n 222 56 (26M, 30F) SC = 187 Age: M, 25 y; F, 24 y
oleic acid-rich
M e n s i n k and K a n t a n ~.7 59 (25M, 34F) SC = 184 Age: M, 25 y; F, 26 y
trans
Diet
Ref./Study characteristics
T A B L E 7. R e c e n t Clinical Studies E v a l u a t i n g the Effects o f D i e t a r y t r a n s - F a t t y A c i d s o n P l a s m a L i p i d s
e~
e-
::r Q
Effectsof dietaryfatty acid compositionon plasmacholesterol
127
of LDL-C/HDL-C, 94`131.~sS'j56`215`222as well as increased ratios of TC/HDL-C. 94.13ta55.156,21s'222 Three of the studies reported increases in plasma Lp(a) concentrations)47a56'222 An additional study found that replacement of the habitual Dutch diet (containing trans-fatty acids) with palm oil lowered Lp(a) concentrations:2 Utilizing the data from these clinical studies, Zock et al. 223 have provided evidence that the observed effects of trans-18:l on LDL-C and HDL-C show a dose-dependent relationship, with every 1%en increase from dietary trans-18:l raising LDL-C by 1.55 mg/dl and decreasing HDL-C by 0.5 mg/dl. In carrying out their analyses, the authors adjusted for differences in the other dietary fatty acids using their recent regression equation) 4s Khosla and Hayes TM compared the observed LDL-C and TC in the various clinical studies ~3~a47'~56":23 with those predicted by the equations of Heg:~ted et al.,Ts and found significant correlations when trans-fatty acids were treated as cholesterol-raising SFA but not when trans-fatty acids were considered to be neutral. However, while trans-fatty acids appear to behave like SFA in terms of their LDL-C raising ability, their HDL-C lowering effect seems to be opposite to that of SFA. Emphasizi~ag this latter point, a preliminary report by Sundram et al. 198 found similar plasma lipid levels in normocholesterolemic subjects fed diets rich in either 12:0 + 14:0 or trans-18:l. However, these values were significantly higher than the values observed when the subjects were fed diets rich in either 16:0 or cis 18:1. This suggests that trans-fatty acids behave like 12:0 + 14:0 in increasing LDL-C. Additionally, trans-fatty acids appear to be unique amongst dietary fatty acids in that they increase Lp(a).S2.147.15° In contrast to the results from the human studies, a detailed metabolic study utilizing the hamster model has proposed that trans-18:l is in fact biologically neutral. Based on the data from this study as well as a re-evaluation of the human clinical studies, it was proposed ~Ssthat trans-fatty acids are biologically neutral, and "appear" cholesterol-elevating since they replaced eis-mono and -PUFA in the various clinical studies cited above. While trans-fatty acids may indeed be biologically neutral in the hamster, the empirical human data and the analyses of Zock et al., 223 and Khosla and Hayes"" do not support this tenet in humans. Most of the studies to date have compared trans-fatty acids with their cis isomers, this may not be a legitimate comparison since in the practical everyday setting, trans-fatty acids replace SFA. In other words, since most recommendations advocate reducing SFA and replacing them with MUFA, the "real" test should be to compare trans-fatty acids with SFA. The limited data that are available on this are inconclusive and although some studies show a favorable', reduction in plasma TC when SFA are replaced with trans-18:l, most of the studies show an adverse lipid profile with the trans diets.
D. Mechanism of Action of Dietary trans-fatty Acids While several animal studies have attempted to evaluate the mechanism of action of dietary trans-fatty acids, there is as yet scant information from human studies to resolve this point. Severa.l workers in an attempt to explain the adverse effects of trans-fatty acids on plasma lipids and lipoprotein concentrations, have concluded that trans-fatty acids behave like SFA "4'131'147and accordingly increase plasma LDL levels by a down-regulation of LDL receptors. However, while this standpoint is convenient, it invariably leads to the question - - whicrh SFA(s)? In order to explain the mechanism of trans-fatty acid effects on plasma lipids, one has to consider mechanisms that increase LDL-C, decrease HDL-C and possibly increase triglycerides and VLDL-C. Two reports ~'2~°have detailed higher plasma CETP activity in subjects fed trans-1 8:1-rich diets as opposed to subjects fed the corresponding cis diets. This is consistent with in vitro observations showing that elaidic acid, but not its cis counterpart, oleic acid, increased cholesteryl ester transfer from HDL to LDL. ns Studies in hamsters69 suggest that part of the effect of trans-18:l is to down-regulate LDL receptors, thereby delaying LDL clearance and consequently elevating plasma LDL-C concentrations. However, in this latter hamster study, dietary fatty acids were poorly
128
P. Khosla and K. Sundram
controlled and the observed effects cannot be directly attributed to the trans-fatty acids. As detailed above an additional hamster study has shown that trans-18:l is biologically neutral since it behaves in an analogous manner to stearic acid, although this conclusion is at odds with the human literature. VII. SUMMARY
It should be clear from the preceding sections that the effects of dietary fatty acids on plasma lipids get more complicated the more we try to simplify them! We have presented one argument as to how different fatty acids may interact to impact human plasma lipids. This is by no means an endorsement that ours is the only argument. Nevertheless, a strong case can be made for 14:0 and 18:2 as being the key players in this scenario. The role of palmitic acid seems to be the most controversial. While clearly certain studies do indeed reveal 16:0 to be hypercholesterolemic relative to 18:1, the data from studies suggesting that it behaves similarly to 18:1 are equally compelling. What is certain is that it is erroneous to assume that 16:0 is the major cholesterol-raising SFA simply because it is the most abundant SFA in the diet. Clearly, 18:0 cannot be considered cholesterol-elevating. One is therefore left with the 12-16C SFA. However, 12:0 and 14:0 are only of concern if diets contain palm-kernel, coconut oil or dairy products as major dietary constituents. Accordingly one is left with 16:0 and its response is highly dependent on the metabolic status as well as the age of the subjects being used. While "elderly" hypercholesterolemic humans clearly benefit from decreased 16:0 (and all SFA) consumption, "younger" normocholesterolemic subjects fail to show such clear-cut effects. Additionally, the concomitant levels of dietary cholesterol and 18:2 also have a major bearing on the cholesterolemic response of 16:0 As far as guidelines for the general public are concerned, clearly for people with TC > 225 and LDL-C > 130 mg/dl and/or those who are overweight (i.e. those percieved to be at high risk), the primary emphasis should clearly be on reducing total fat consumption. Decreasing saturated fat consumption will invariably also lower dietary cholesterol consumption. The latter manouver will generally lower TC and LDL-C. Whether the reduction occurs because of the removal of 14:0, or 16:0 and/or dietary cholesterol is a mute point, since most dietary guidelines advocate curtailing intake of animal and dairy products, which will result in reductions of all the SFA. It remains to be established whether life-long adherence to the above dietary guidelines in those subjects with normal cholesterol levels and an absence of the other conventional risk factors for CHD, will result in a subsequent decrease in CHD risk. In the latest NCEP report 39 million Americans were targeted as those who would benefit from reductions in LDL-C, principally by dietary means. This is indeed a very high number. But that leaves almost 220 million Americans! For them the age old recommendation to consume a moderate fat load, maintain ideal body weight and eat a varied and balanced diet would still appear to be the most powerful advice. Acknowledgments--We are grateful to all our colleagues for their generous comments and suggestions during
the preparation of this review. We are indebted to PORIM for their financial support for the human and animal studies undertaken bY the authors.
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