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Articles

Palmitic and Stearic Acids Similarly Affect Plasma Lipoprotein Metabolism in Cynomolgus Monkeys Fed Diets with Adequate Levels of Linoleic Acid¹

Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202

³To whom correspondence should be addressed. E-mail: pkhosla{at}sun.science.wayne.edu

	ABSTRACT

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This study was designed to evaluate whether the exchange of specific saturated fatty acids [SFA; palmitic acid (16:0) for stearic acid (18:0)] would differentially affect plasma lipids and lipoproteins, when diets contained the currently recommended levels of total SFA, monounsaturated fatty acids and polyunsaturated fatty acids (PUFA). Ten male cynomolgus monkeys were fed one of two purified diets (using a cross-over design) enriched either in 16:0 (palmitic acid diet) or 18:0 (stearic acid diet). Both diets provided 30% of energy as fat (SFA/monounsaturated fatty acid/PUFA: 1/1/1). The palmitic acid and stearic acid diets were based on palm oil or cocoa butter (59% and 50% of the total fat, respectively). By adding different amounts of sunflower, safflower and olive oils, an effective exchange of 16:0 for 18:0 of 5% of energy was achieved with all other fatty acids being held constant. Monkeys were rotated through two 10-wk feeding periods, during which time plasma lipids and in vivo lipoprotein metabolism (following the simultaneous injection of ¹³¹I-LDL and ¹²⁵I- HDL were evaluated). Plasma triacyglycerol (0.40 ± 0.03 vs. 0.37 ± 0.03 mmol/L), plasma total cholesterol (3.59 ± 0.18 vs. 3.39 ± 0.23 mmol/L), HDL cholesterol (1.60 ± 0.16 vs 1.53 ± 0.16 mmol/L) and non-HDL cholesterol (2.02 ± 0.26 vs. 1.86 ± 0.23 mmol/L) concentrations did not differ when monkeys consumed the palmitic acid and stearic acid diets, respectively. Plasma lipoprotein compositional analyses revealed a higher cholesteryl ester content in the VLDL fraction isolated after consumption of the stearic acid diet (P < 0.10), as well as a larger VLDL particle diameter (16.3 ± 1.7 nm vs. 13.8 ± 3.6 nm; P < 0.05). Kinetic analyses revealed no significant differences in LDL or HDL transport parameters. These data suggest that when incorporated into diets following current guidelines, containing adequate PUFA, an exchange of 16:0 for 18:0, representing 11 g/(d·10.46 mJ) [11 g/(d·2500 kcal)] does not affect the plasma lipid profile and has minor effects on lipoprotein composition. Whether a similar effect would occur in humans under comparable dietary conditions remains to be established.

KEY WORDS: • palmitic acid • stearic acid • cynomolgus monkeys • low density lipoprotein • high density lipoprotein • kinetics

	INTRODUCTION

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As an initial step in the management of hypercholesterolemia, most health agencies advocate a diet that restricts total saturated fatty acids (SFA)⁴ and/or dietary cholesterol. In this prudent diet, SFA should contribute < 10% of energy (%en). The rationale for these recommendations stems from classic studies (1 ,2) that showed that SFA were cholesterol elevating, while polyunsaturated fatty acids (PUFA) lowered cholesterol. Since these studies were first published, several groups have carried out meta-analyses that essentially confirm the original observations (3 4 5) .

With regards to individual SFA, Hegsted et al. (2) showed that myristic acid (14:0) was four times more potent than 16:0 in raising total plasma cholesterol (TC), whereas 18:0 had no effect. Although subsequent regression analyses (3 ,5) as well as human-feeding studies (6) have assigned a lower cholesterol-raising potential to 14:0 than was originally proposed by Hegsted et al. (2) , 14:0 is still believed to be the most powerful cholesterol-raising SFA. However, typical American diets contain very little 14:0. In such diets, 18:0 (a major constituent of red meat and confectionery products) contributes 3.5–4% total energy and represents 10% of the dietary energy derived from fat and 25% of the energy derived from the SFA, whereas 16:0 accounts for >60% of the SFA. Because the latter is the most abundant SFA in the diet, it is frequently regarded as the major contributor to SFA-induced hypercholesterolemia. Despite these differences among the individual SFA, they are still frequently classified as a single group.

However, a series of studies starting in the early 1990s by Hayes and colleagues has challenged the above simplistic notion by focusing attention to the fact that the putative effect of any fatty acid is dependent both on the metabolic status of the individual as well as the other dietary fatty acids present. They showed that in normocholesterolemic cebus, rhesus and squirrel monkeys consuming very low cholesterol-containing or cholesterol-free diets, 16:0 did not impact plasma cholesterol or LDL-C levels, and in such situations its effects were similar to those of oleic acid (18:1) (7 8 9 10) . Only when LDL receptors were suppressed (as in the situation when 0.3% cholesterol was fed) did 16:0 show a hypercholesterolemic effect when compared with 18:1 (10) . In a meta-analyses of data from cebus monkeys, Hayes and Khosla (11) showed that in normocholesterolemic animals consuming low cholesterol diets (reflecting unimpaired LDL receptor activity), TC (and LDL-C) was dependent solely on the dietary content of 14:0 and linoleic acid (18:2). Furthermore, above a threshold level of 18:2 of 5–6%en, other fatty acids appeared essentially equivalent. A reappraisal of the data of Hegsted et al. (2) by Hayes and Khosla (11) suggested that a similar situation may pertain to humans, especially when dietary fat intake is moderate (30–33%en) and dietary cholesterol intakes are below 300 mg/d. This has been reported subsequently in several human studies (12 13 14 15) that found dietary 16:0 to be equivalent to 18:1 under the appropriate experimental settings (i.e., low total fat intake of 30%en, low dietary cholesterol intake of < 300 mg/d). A meta-analysis by Khosla and Sundram (16) of > 30 different studies evaluating > 130 diets yielded similar results. Thus, there are sufficient data to justify the argument of Hayes that 16:0 can be viewed as a fatty acid that is "conditionally hypercholesterolemic" (17) or, for that matter, conditionally neutral.

Most of the above studies evaluating the neutrality of 16:0 have compared it with the monounsaturated 18:1. There has been no formal test of the putative neutrality of 16:0 with 18:0, a SFA, which is universally regarded as neutral. To test the effects of the above exchange on plasma lipoprotein metabolism, we fed cynomolgus monkeys cholesterol-free diets with similar contents of SFA, monounsaturated fatty acids and PUFA (1/1/1). The major dietary fat used was either palm oil or cocoa butter (59% or 50% of the total fat, respectively). These fats represented major sources of 16:0 and 18:0, respectively. To satisfy the conditions outlined in Hayes’ threshold hypothesis described above, total dietary fat was restricted to 30%en and both diets were supplemented with adequate safflower oil to satisfy the 18:2 requirement for both a step 1 diet as well as the threshold hypothesis. Consequently, we were able to directly compare a 5%en exchange between 16:0 and 18:0. (In the typical American diet, this would represent a 100% increase in 18:0 intake, from 25% of total SFA to 50%.) The cynomolgus monkey has been widely used for investigating the effects of dietary fat and cholesterol on plasma lipoprotein metabolism and is currently considered one of the desirable animal models for evaluating the effects of individual fatty acids on plasma lipids (18) .

	MATERIALS AND METHODS

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Animals, diets and study design.

Ten male cynomolgus monkeys (Macaca fasicularis) born between 1990 and 1993 were used for the current study. All monkeys were fed purified diets (Table 1 ). The purified diets were pelleted by Dyets (Bethlehem, PA). The two test diets were formulated according to current dietary guidelines for total fat and SFA (30%en total fat, 10%en SFA). No exogenous cholesterol was added to the diets. For the 16:0-rich diet [palmitic acid (PA) diet], 59% of the test fat was derived from palm oil, whereas for the 18:0-rich diet [stearic acid (SA) diet], 50% of the test fat was derived from cocoa butter. Additional vegetable oils were used to ensure that the final diets fed to the monkeys contained similar contents of MUFA and PUFA (Table 2 ). In terms of gross composition, an exchange of 16:0/18:0 of 5%en was achieved. The amounts of palm oil and cocoa butter used represented the maximum amount that could be incorporated into the diets, while still satisfying the criteria for total SFA in a step 1 diet setting. All ingredients were provided by the diet manufacturer. Diets (prepared in a single batch for the entire duration of the study) were stored at -20°C, and 5-kg batches were removed as needed for daily feeding to the monkeys. These batches were kept at 4°C.

Monkeys were fed a fixed amount of diet daily (110–220 g/d: divided equally between a morning and an afternoon feed) designed to maintain constant body weights throughout the study. All procedures and protocols were in accordance with, and ratified by, the University’s Health Physics Department (radioactivity compliance) and the Animal Investigation Committee.

All procedures used were minor modifications to a detailed protocol recently reported for these animals (19) .

Plasma lipid determinations.

After overnight food deprivation (14–16 h), monkeys were anesthetized with an intramuscular injection of Ketamine HCl (Vetalar; Parke-Davis, Morris Plains, NJ; 10 mg/100 µL) at a dose of 4–9 mg/kg body. Blood was obtained from the saphenous or femoral vein and placed into EDTA-wetted tubes kept on ice. Plasma was isolated by centrifugation at 1000 x g for 20 min at 4°C. TC and triglycerides (TG) concentrations were determined enzymatically (kits 352 and 336, respectively; Sigma Diagnostics, St. Louis, MO). HDL-C concentrations were determined in the supernatant of plasma samples after precipitation with phosphotungstic acid/Mg²⁺ (PTA/Mg²⁺; HDL cholesterol reagent 352–4; Sigma Diagnostics). The difference between TC and HDL cholesterol represented VLDL + LDL cholesterol.

Plasma lipoprotein isolation and characterization.

Sodium azide, gentamycin sulfate, benzamidine and EDTA (20 ,21) were added to plasma samples before lipoprotein isolation.

At wk 7, lipoproteins were separated from individual plasma samples using a five-step discontinuous salt gradient by density gradient ultracentrifugation as described by Goulinet and Chapman (22) . After ultacentrifugation at 15°C, 35,000 x g for 48 hrs in a SW 41.1 rotor, 30 fractions (first fraction, 500 µL + 29 subsequent fractions of 400 µL each) were collected. The cholesterol content of each was measured. From a plot of fraction number, density and cholesterol content, two major peaks and one minor peak were discernible. Based on this cholesterol distribution, lipoprotein fractions were pooled to yield fraction 1, designated VLDL (d < 1.019 kg/L), fraction 2, designated LDL (1.019 < d < 1.063 kg/L), and fraction 3, designated HDL (1.063 < d < 1.21 kg/L). At week 9, VLDL, LDL and HDL of the same density cuts detailed above were isolated from plasma samples from food-deprived monkeys by sequential ultracentrifugation (23) as detailed elsewhere (19) .

Lipoprotein lipid compositions were determined enzymatically: free cholesterol, Free Cholesterol C kit CHOD-PAP; phospholipids, Phospholipids B kit CO-PAP—both from Wako Chemicals (Richmond, VA), while TC and TG were measured using the kits (352 and 336, respectively) from Sigma Diagnostics. Lipoprotein protein concentrations were determined using the Markwell modification (24) of the Lowry procedure (25) . Particle diameters of each lipoprotein class were estimated from the core-to-surface volume ratios (26) .

Preparation of lipoprotein tracers.

Six days before beginning a turnover study, the monkeys were deprived of food for 16 h, and after sedation with ketamine, 10–12 mL blood were withdrawn from the femoral vein of individual monkeys into EDTA-wetted tubes using a 22-gauge needle. LDL (1.019 < d < 1.063 kg/L) and HDL (1.063 < d < 1.21 kg/L) were isolated from pooled homologous plasma by sequential ultracentrifugation. The isolated LDL and HDL were washed and concentrated by recentrifugation at their appropriate densities. After dialysis (0.15 mmol/L NaCl/1mmol/L EDTA pH 7.4), lipoprotein protein concentration was determined as detailed above. LDL and HDL were labeled with Na¹³¹I and Na¹²⁵I (Amersham, Chicago, IL), respectively (to specific activities of 5 Bq/ng), and the intramolecular distribution of radioactivity was determined (9) . For the ¹³¹I-LDL, the proportion of total radioactivity associated with apo B was consistently > 95%. Apo A1 accounted for > 85% of the ¹²⁵I-HDL radioactivity and > 90% of the HDL protein mass (9) .

Protocol for metabolic studies.

The procedure followed was identical to our most recently published protocol (19) . Briefly, monkeys were injected simultaneously with 185–555 kBq of homologous ¹³¹I-LDL and ¹²⁵I-HDL. A 5-mL blood sample was collected from a femoral vein at 5 min postinjection, and additional 1-mL samples obtained periodically up to 6 d. (The amount of blood taken from each monkey over the 6 d averaged < 10% of the estimated blood volume.) LDL and HDL were isolated by sequential ultracentrifugation from the plasma sample of each monkey obtained at 5 min (after 16 h of food deprivation) and the LDL apo B and HDL apo A1 concentrations determined (9) . The blood sample obtained at 5 min also represented the 9-wk data point.

Kinetic analyses.

Plasma ¹³¹I and ¹²⁵I radioactivity data for both tracers were biexponential and analyzed in accordance with a two-pool model (27) and the fractional catabolic rate (FCR) calculated (28) . The assumptions and rationale for using the two-pool model, as well as the calculations for pool sizes (mg/kg body) and transport rates [TR (mg. kg^-1.d^-1)] for each tracer have been detailed previously (9 ,10) . The TR (or production rate) was calculated as the product of the FCR and the pool size. The limitations in the analyses in using HDL apo A1 concentrations and the FCR for whole HDL, as a measure of HDL apoA1 TR, have also been discussed previously (10) .

Statistical analyses.

All statistical analyses were performed using a Power Macintosh 6100 computer (Apple Systems, Cupertino, CA) with the Statview 512 (Brain Power, Calabasca, CA) statistical package. Significant differences between the PA and SA diets were calculated using repeated-measures analysis of variance. Results are presented as means ± SD.

	RESULTS

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Monkeys consumed their diets without incident. Among the 10 monkeys, body weights and energy intakes ranged from 5.2–8.4 kg to 1200–3400 kJ/d. However, consistent with the design of the study, there was no significant differences between the PA and SA diet periods for body weight (6.77 ± 1.08 vs. 6.68 ± 1.03 kg) or energy intake (1855 ± 813 vs. 1840 ± 807 kJ/d). In addition, the body weights of the 10 monkeys at the end of the 20-wk study (6.70 ± 0.98 kg) were not significantly different from the respective values at the beginning of the study (6.66 ± 1.42 kg).

TC concentrations (means of the 7-, 8- and 9-wk samples) were not significantly different between diet periods (Table 3 ). Of the TC, 44% was distributed in the HDL fraction, while the remainder was associated with the apo B containing lipoproteins. Neither diet affected HDL-C, nonHDL-C, TG or the TC/HDL-C ratio. This trend was also apparent in the isolated lipoprotein fractions, which revealed no differences in the percent composition of any of the constituents, with the exception of the percent cholesterol esters in VLDL, which tended to be higher in the VLDL isolated from the monkeys when they consumed the SA diet (0.05 < P < 0.10; Table 4 ). Based on the lipoprotein composition data, particle diameters were estimated. When monkeys were fed the SA diet, VLDL particles had 18% larger diameters (P < 0.05; Table 5 ).

	DISCUSSION

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A threefold increase in the 18:0 content (1.2%en to 5.2%en, PA SA diet) resulted in the SA diet having > 50% of its total SFA from 18:0. Despite this manipulation, plasma lipids were essentially the same in the PA diet, in which 16:0 accounted for 85% of the total SFA. In the typical American diet, to achieve a 18:0 content similar to that used in our SA diet would necessitate a doubling of 18:0 intakes. It may be argued that what we observed in the current dietary setting was not a true reflection of what the fatty acids were doing but simply a reflection of the insensitivity of our model to detect any fatty acid exchanges (especially because the diets were cholesterol-free). Although cynomolgus monkeys are extremely sensitive to dietary cholesterol (30 31 32) , 14–20 times more sensitive than humans (32) , fatty acid-induced effects on plasma lipids are discernible in this animal model even in the absence of dietary cholesterol. Extreme manipulation of the P/S ratio produces changes in LDL-C of 0.7 mmol/L (30) . The reason why we did not note any changes in our study was that our P/S ratios were comparable.

The only dietary effects that we noted were on VLDL composition and particle size. VLDL isolated after feeding of the SA diet tended to have a higher content of cholesterol esters (0.05 < P < 0.10) and had a significantly larger estimated particle diameter. These compositional changes affect the metabolic fate of plasma VLDL (33) . The enrichment in VLDL cholesteryl esters may reflect increased hepatic incorporation and/or differential remodeling in the plasma compartment, reflecting differences in the activities of various enzymes (e.g., acyl cholesterol acyl transferase, cholesteryl ester transfer protein and lecithin cholesterol acyl transferase). Additionally, the size of the VLDL particle will determine both its rate of removal via the liver, as well as the amount that is converted to LDL (34 35 36 37) . We cannot distinguish between these possibilities because we did not evaluate any of the above-mentioned enzymes or VLDL transport parameters. However, neither of the above possibilities is likely to be of major importance in the current study because the VLDL pool (both in terms of cholesterol and triglyceride content) following feeding of the SA and the PA diets was relatively small.

For more than four decades, it has been known that 18:0 does not raise TC compared with other SFA. The initial observations from Keys et al. (1) and Hegsted et al. (2) have been confirmed in several metabolic ward studies (38 ,39) that have used extremely divergent fatty acid profiles, frequently reflecting the use of a single fat source. From these metabolic ward studies, 18:0 typically behaved like 18:1 and did not affect TC or LDL-C but was significantly hypocholesterolemic when compared with 16:0. The mechanistic basis for why 18:0 does not raise TC or LDL-C is not completely clear. It has been argued that 18:0 is not as well absorbed as the other SFA. However, absorption > 90%, and comparable to that seen for 16:0, has been reported in humans (38 39 40 41) . Alternatively, it has been suggested that 18:0 is desaturated to 18:1 in vivo and, accordingly, does not affect LDL-C (42) . However, a recent study in humans using ¹³C-labeled 16:0 or 18:0 showed that the in vivo desaturation of 18:0 to 18:1, although faster than the desaturation of 16:0 to 16:1, was relatively low and would be anticipated to have little impact on plasma lipids (43) . Although we cannot distinguish between the above possibilities (because we observed similar lipid profiles and lipoprotein transport parameters), they illustrate the point that there is still no consensus to explain the neutrality of 18:0.

When more realistic fatty acid exchanges have been used, using solid, whole-food diets, the effects of the 16:0/18:0 exchanges have been less clear-cut. Nestel et al. (44) noted no effects on plasma lipids of a 5%en 16:0/18:0 exchange in hypercholesterolemic subjects. In normocholesterolemic healthy women, the same 5%en exchange resulted in a significant increase in TC following the 16:0-rich diet but reflected a significant increase in HDL-C and apo A1 concentrations (45) , while LDL-C tended to be higher. Storm et al. (46) noted significantly higher TC concentrations in noninsulin-dependent diabetic subjects fed a 16:0-rich diet vs. a 18:0-rich diet (5.3 ± 1.3 5.0 ± 1.2 mmol/L) but found no significant effects on LDL or HDL cholesterol levels. A 14%en exchange between 16:0 and 18:0 resulted in a significant lowering of TC and LDL-C following the 18:0-rich diet in healthy women (47) , although the fats used were synthetic triglycerides. When 5%en from SFA (12:0, 14:0 and 16:0) was replaced with 18:0, in 15 healthy females, plasma lipids and lipoproteins were not significantly affected (48) .

An important point to note in our study is that we were evaluating 16:0 vs. 18:0 as part of diets with adequate levels of 18:2. In some of the above studies [e.g., Snook et al. (47) ], PUFA contributed < 3%en, and as such the lack of 18:2 would have negatively impacted the LDL-C pool. The values of 10%en from 18:2 that we used represents the upper limit for a step 1 diet and is 30% higher than what is consumed in the United States. It is possible that the direct equivalence of 16:0 and 18:0 that we observed reflects that fact. A 16:0/18:0 exchange at lower 18:2 intakes may produce different results; 18:2 up-regulates LDL-receptors (49) and lowers LDL-C. The up-regulation is mediated via sterol regulatory element binding protein 1 (50 ,51) . The latter is an intracellular nuclear transcription factor protein, synthesized as a 125-kDa precursor that is attached to the endoplasmic reticulum and the nuclear envelope. In sterol-depleted cells, proteolytic cleavage of the membrane bound protein produces a soluble 68-kDa fragment that translocates to the nucleus. Here the fragment recognizes and binds to a specific sequence of the LDL receptor gene and stimulates transcription. Recently, it was shown that PUFA feeding suppresses sterol regulatory element binding protein 1 (52) by accelerating its decay to the soluble fragment.

Thus, a decrease in 18:2 content would be expected to down-regulate LDL receptors. In such a situation, 16:0 is believed to promote hypercholesterolemia, due to its propensity to increase VLDL production (10 ,11) . If 18:0 is truly neutral, then a 16:0/18:0 exchange at low 18:2 intakes would be expected to show a hypercholesterolemic effect of 16:0, as observed by Snook et al. (47) . We would, therefore, suggest that the equivalence of the PA and SA diets seen in the current study is in part a reflection of the high levels of 18:2 used.

The practical implication of the above concept is that within the framework of the step 1 diet, which restricts SFA and provides for 6–10%en from PUFA, it is highly unlikely that any significant effects on blood lipids would result as a consequence of a 16:0/18:0 exchange. In such situations, other factors must determine the efficacy of one fatty acid over another. In this regard, 16:0 would appear to have certain advantages because preliminary data suggest that 18:0 may be thrombogenic and may increase plasma Lp(a) levels (53) . Direct verification of the efficacy of 16:0/18:0 exchanges within the step 1 setting must await future human studies.

Finally, the current data [as well as evaluation of four different diets in these same animals (Gupta and Khosla, unpublished data)] provide additional support for the idea expressed earlier (19) that in cynomolgus monkeys, a LDL-C of 2 mmol/L may be the lower limit that can be obtained by manipulating fatty acids within a step 1 framework. Additional reductions may require altering one or more other nutrients. Additional reductions in total fat (e.g., a diet with 25%en and even lower SFA content) may be only marginally beneficial, although the optimal fat level to achieve this remains to be determined. Although in theory the lowest LDL-C that could be achieved in the cynomolgus monkey (or any other species for that matter) is 0.6 mmol/L [the concentration at which LDL receptors function maximally (54) ] when the rate of LDL uptake is maximal (55) , a more realistic value may be closer to 1 mmol/L, which is obtained after feeding a nonpurified diet (19) .

	ACKNOWLEDGMENTS

 
We thank Karen Rossman and her colleagues for their expert care of the nonhuman primates, and Thomas V. Fungwe for helpful discussions.

	FOOTNOTES

 
¹ Supported in part by a grant from the Palm Oil Research Institute of Malaysia and start-up funds from the College of Science, Wayne State University.

² Present address: Oxford Biomedical Research Inc., Rochester Hills, MI.

⁴ Abbreviations used: FCR, fractional catabolic rate; PA, palmitic acid; %en, percent of energy; PUFA, polyunsaturated fatty acid; SA, stearic acid; SFA, saturated fatty acid; TC, total plasma cholesterol; TG, triglycerides; TR, transport rate.

Manuscript received March 8, 2001. Initial review completed March 30, 2001. Revision accepted May 7, 2001.

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