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Article

Pork Fat and Chicken Fat Similarly Affect Plasma Lipoprotein Metabolism in Cynomolgus Monkeys Fed Diets with Adequate Levels of Linoleic Acid^{1 ,2}

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

	ABSTRACT

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The effects on plasma lipoprotein metabolism of replacing pork fat (PF) with chicken fat (CF) (formulated as part of currently recommended prudent diets) was evaluated in 10 male cynomolgus monkeys. Monkeys were rotated through three dietary periods, (each of 10-wk duration), during which total cholesterol (TC), triacylglycerol (TG) and HDL-cholesterol (HDL-C) were measured (7, 8 and 9 wk) and in vivo lipoprotein metabolism evaluated (after 9 wk). Initially, all monkeys were fed a high-fat, high-cholesterol reference diet [38% of energy (en) from fat, 18%en saturated fatty acids (SFA), 10%en monounsaturated fatty acids (MUFA), 10%en polyunsaturated fatty acids (PUFA), 0.045 mg cholesterol/kJ diet]. Subsequently, monkeys were rotated through two test diets (30%en fat, SFA/MUFA/PUFA 1:1:1, 0.004–0.005 mg cholesterol/kJ diet), in which 80% of the fat was either PF or CF, with the remaining 20% derived from high-linoleic safflower oil. There was no significant difference between the two test diets for TG, TC, nonHDL-C, HDL-C or the ratio of TC/HDL-C. Lipoprotein composition, LDL apolipoprotein B pool size, fractional catabolic rate and transport rate were also not significantly different when monkeys consumed the two test diets. These data suggest that when incorporated into diets following current guidelines and containing adequate PUFA (~7–9%en), PF and CF similarly affect plasma lipids.

KEY WORDS: • pork fat • chicken fat • cynomolgus monkeys • LDL • HDL • kinetics

	INTRODUCTION

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For several years, health agencies have been advocating decreased dietary intake of total fat, saturated fat and cholesterol as an initial step in the management of hypercholesterolemia. The American Heart Association Step 1 diet (currently recommended for everyone over the age of 2 y) requires reducing total fat and cholesterol intake to 30% energy (%en) and <300 mg cholesterol/day, respectively. In this prudent diet, saturated fatty acids (SFA)⁴ should contribute <10%en. However, within the framework of the above guidelines, numerous permutations are possible, thereby leading to countless different diets. As long as the SFA content is below 10%en and the polyunsaturated fatty acids (PUFA) content does not exceed 10%en, the diets are technically Step 1-type diets. The rationale for these recommendations stems from the original studies of Keys et al. (1965) and Hegsted et al. (1965) which showed that SFA were cholesterol-elevating. Since these studies were first published, several groups have carried out meta-analyses which essentially confirm the original observations (Mensink and Katan 1992 , Yu et al. 1995 )

In order to facilitate consumer transitions to prudent diets, the simple "take-home" message has been to curtail the intake of meat (specifically "red meat") and dairy products (Krauss et al. 1996 ). Historically, pork has been "labeled" as a red meat along with beef and lamb. This may, in part, explain why the per capita consumption (since 1980) of beef and pork has decreased by 11 and 7%, respectively, while poultry and fish consumption has collectively increased by 46% in the same time period. In 1980 the per capita consumption of pork was 25% greater than that of poultry (chicken and turkey), whereas in 1995, poultry consumption exceeded pork consumption by almost 20%. Although pork consumption has been steady from 1992–1995, poultry consumption during the same period has still increased by ~2.7%. This is surprising given that the myoglobin and fat content of pork are similar to chicken. Based on a 3 oz (85 g) serving size of cooked meat (skinless or trimmed), the fat content of pork varies between 4.1–9.3 g and that of chicken between 3.1–9.3 g (USDA Handbook 1995 ).

Additionally, the consumer has avoided pork because of its SFA and cholesterol content. The SFA content of pork fat (PF)(~39 g/100 g fat) is ~30% higher than the SFA content of chicken fat (CF)(~27 g/100 g fat). However of the ~39 g of SFA in 100 g PF, ~14 g is stearic acid (18:0) which does not raise LDL-cholesterol (LDL-C) (Bonanome and Grundy 1988 ), and almost 24 g is palmitic acid (16:0). The latter is the most abundant SFA and has generally been regarded as the biggest "culprit" in SFA-induced hypercholesterolemia (Grundy and Denke 1990 ). However, a considerable amount of recent data from Hayes and colleagues has challenged the notion that 16:0 is the principal cholesterol-raising SFA and proposes that the effects of 16:0 are "conditional," i.e., dependent on numerous other dietary and physiological factors (Hayes et al. 1995 ). Data from animal studies as well as controlled human feeding trials have shown situations in which 16:0 appears to be "neutral," i.e., has no effect on plasma total or LDL-C (Choudhury et al. 1995 , Hayes et al. 1991 , Khosla and Hayes 1992 and 1993 , Ng et al. 1992 , Sundram et al. 1995 ). In fact in a recent review of >130 human diets utilized in >30 different studies, no evidence was obtained for any cholesterol-elevating effects of 16:0 in normocholesterolemic populations consuming diets with moderate fat and cholesterol loads as well as adequate PUFA intakes (Khosla and Sundram 1996 ). Thus, in the best case scenario, 16:0 can behave like 18:1 and not influence LDL-C levels. This latter point is important regarding PF as 16:0 constitutes ~60% of the SFA content.

In addition to the lack of effects of 16:0 on plasma cholesterol, Hayes’s group identified myristic acid (14:0) and linoleic acid (18:2) as being the primary fatty acids that dictate the circulating lipoprotein profile (Hayes and Khosla 1992 ). Thus once 14:0 and 18:2 have been equalized across diets, one can manipulate 12:0, 16:0, 18:0 and 18:1 levels without impacting the plasma cholesterol level. Since the SFA (specifically the 12–16 C fatty acids) and cholesterol content of pork and chicken are comparable and the Step 1 diets provide optimal conditions [modest fat load, ~30%en, low-cholesterol content, <300 mg/d and adequate 18:2 intakes, ~5–6%en] for blunting any cholesterolemic effects of 16:0, we hypothesized that within this dietary scenario PF and CF would be comparable in terms of their effects on plasma lipoproteins.

To test this, we formulated pork- and chicken-based diets (in which essentially all fatty acids were balanced [specifically 14:0 and 18:2]), supplemented with vegetable oils (as neither animal fat provides adequate PUFA to satisfy the Step 1 dietary requirements) and evaluated their effects on plasma lipoprotein metabolism in the cynomolgus monkey. The latter has been widely used for investigating the effects of dietary fat and cholesterol on plasma lipoprotein metabolism (Brousseau et al. 1993 , Hunt et al. 1992 , Stucchi et al. 1995 and 1998 , Turley et al. 1995 ). Cynomolgus monkeys, though, are extremely sensitive to dietary cholesterol and ~14–20 times more sensitive than humans (Stucchi et al. 1998 ). In such situations, the dietary cholesterol-load becomes the major determinant of plasma LDL. However, when dietary cholesterol levels are low (~0.010 mg/kJ), dietary fatty acid-induced effects on plasma LDL are discernible (Hunt et al. 1992 ), and it has been suggested that dietary cholesterol levels of <0.2% be employed when evaluating the effects of individual fatty acids (Kris-Etherton and Dietschy 1997 ).

	MATERIALS AND METHODS

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Animals, diets and study design.

Ten male cynomolgus monkeys (Macaca fasicularis) were obtained from a registered supplier (Sierra Biomedicals, Sparks, NV). The animals, born between 1990–1993, had been originally imported from the the Philippines and China. In accordance with standard operating procedures set forth by Wayne State University’s Division of Laboratory and Animal Resources, all animals initially went through a 6–8-wk quarantine period during which a battery of tests was performed. They consumed Purina monkey (PM) nonpurified diet (Ralston Purina, St. Louis, MO) ad libitum. During the quarantine period, body weights were recorded at regular intervals, and each monkey’s caloric need evaluated, based on food consumption patterns. After the quarantine period, monkeys experienced a 2-wk adaptation period during which time they were fed 100% PM, 75% PM/25% purified diet, 25% PM/75% purified diet and finally 100% purified diet. Subsequent to the adaptation period, monkeys were fed purified diet exclusively. At all times monkeys had free access to tap water.

Purified diets were pelleted by Dyets (Bethlehem, PA). The compositions of the diets are listed in Table 1 . The high-fat, high-cholesterol reference diet (38%en and 0.045mg/kJ diet, respectively) somewhat mimicked a typical average American diet. The two test diets were formulated according to current dietary guidelines (~30% en total fat, ~10%en SFA and ~4–5 mg cholesterol per kJ diet). The test diets differed in the fact that 80% of the fat was derived solely from PF or CF, with the remainder coming from high-linoleic safflower oil. Thus a pork/chicken substitution within the currently advocated dietary guidelines was achieved. The rendered animal fats used in this study consisted of pork adipose tissue from butcher weight hogs slaughtered in Illinois and Iowa and chicken adipose tissue from broilers slaughtered in Delaware and Arkansas. The final fats were analyzed for cholesterol and fatty acids (Table 2 ) by Covance Laboratories (Madison, WI). Fats arrived at Wayne State on dry ice and were shipped to Dyets for diet preparation. In addition to the animal fats, we supplied the diet manufacturer with refined, bleached, deodorized palm oil. All other dietary ingredients were provided by the diet manufacturer. The final fatty acids as fed to the monkeys are shown in Table 3 . Diets were prepared in a single batch for the entire duration of the study. Diets were stored at -20°C, and 5-kg batches 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 for throughout the study period. To account for the different energy densities of the reference and test diets, monkeys were fed a fixed amount of total energy during the entire study. The micronutrient content of the diets was adjusted such that all animals received the same quantities 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 protocols employed for similar studies in rhesus and cebus monkeys described elsewhere (Khosla and Hayes 1992 and 1993 , Khosla et al. 1997a and b ).

Plasma lipid determinations.

Following overnight food deprivation (14–16 h), monkeys were anesthetized with an intramuscular injection of Ketamine HCl (10 mg/100 µL; Vetalar®, Parke-Davis, Morris Plains, NJ) at a dose of 4–9 mg/kg body weight. 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, 20 min, 4°C. Total cholesterol (TC) and triacylglycerol (TG) concentrations were determined enzymatically (kits #352 and #336, respectively; Sigma Diagnostics®^TM, St. Louis, MO). HDL-C concentrations were determined in the supernatant of plasma samples following precipitation with phosphotungstic acid/Mg²⁺ (HDL cholesterol reagent, #352–4; Sigma Diagnostics®^TM). The difference between TC and HDL-C represented VLDL + LDL cholesterol.

Plasma lipoprotein isolation and characterization.

Sodium azide, gentamycin sulfate, benzamidine and EDTA (Edelstein and Scanu 1986 , Schumaker and Puppione 1986 ) were added to plasma samples prior to lipoprotein isolation.

At wk 7, lipoproteins were separated from individual monkey plasma using a five-step discontinuous salt gradient by density gradient ultracentrifugation as described by Goulinet and Chapman (1993) . After ultacentrifugation at 15°C, 35K for 48 h 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 fasting plasma samples by sequential ultracentrifugation (Havel et al. 1955 ) as detailed previously (Khosla and Hayes 1992 ).

Lipoprotein lipid compositions were determined enzymatically— free cholesterol; Free Cholesterol C kit CHOD-PAP, phospholipids; Phospholipids B kit CO-PAP—both from Wako Chemicals USA (Richmond, VA), while TC and TG were measured using the kits (#352 and #336, respectively) from Sigma Diagnostics®^TM. Lipoprotein protein concentrations were determined using the Markwell modification (Markwell et al. 1978 ) of the Lowry procedure (Lowry et al. 1951 ).

Preparation of lipoprotein tracers.

Six days prior to commencement of a turnover study, the monkeys were deprived of food for 16 h, and following sedation with ketamine, 10–12 mL blood was 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. Following dialysis (0.15 mmol/L NaCl, 1 mmol/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 ~5Bq/ng), and the intramolecular distribution of radioactivity was determined (Khosla and Hayes 1992 ). For the ¹³¹I-LDL, the proportion of total radioactivity associated with apolipoprotein B (apo B) was consistently >93%. Apo A1 accounted for >85% of the ¹²⁵I-HDL radioactivity and >90% of the HDL protein mass (Khosla and Hayes 1992 ).

Protocol for metabolic studies.

The procedure followed previously published protocols (Khosla and Hayes 1992 and 1993 , Khosla et al. 1997a ). 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 5-min plasma sample of each monkey (obtained following 16-h food deprivation) and the LDL apo B and HDL apo A1 concentrations determined (Khosla and Hayes 1992 ). 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 was bi-exponential, and analyzed in accordance with a 2-pool model (Matthews 1957 ) and the fractional catabolic rate (FCR) calculated (Kushwaha and Hazzard 1977 ). The assumptions and rationale for using the two-pool model, as well as the calculations for pool sizes (mg/kg body weight) and transport rates (TR) (mg · kg^-1 · d^-1) for each tracer, have been detailed previously (Khosla and Hayes 1992 and 1993). 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 (Khosla and Hayes 1993 ).

Statistical analyses.

All statistical analyses were performed using a Power Macintosh 6100®^TM computer (Apple Systems, Cupertino, CA) with the Statview 512^+TM (Brain Power, Calabasca, CA) statistical package. Significant differences between the PF- and CF-based diets were calculated using repeated measures ANOVA. Results are presented as the means ± SEM, n = 10.

	RESULTS

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Monkeys consumed their diets generally without incident. When monkeys consumed the PM nonpurified diet, plasma lipids were 0.56 ± 0.05, 2.12 ± 0.13, 1.06 ± 0.08, 1.03 ± 0.08 mmol/L, for TG, TC, HDL-C and non HDL-C concentrations, respectively. The TC/HDL-C ratio was 2.03 ± 0.15. During the course of the entire study, monkey energy intakes averaged 2478 ± 609 kJ/d, and their body weight averaged 6.14 ± 1.39 kg. There was no significant difference between the PF- and CF-based diet periods for energy intake (2285 ± 756 vs. 2436 ± 626 kJ/d) or body weight (6.16 ± 1.76 vs. 6.01 ± 1.13 kg).

Plasma lipid concentrations.

When monkeys were fed the high-fat, high-cholesterol reference diet, TC averaged 5.72 ± 0.65 mmol/L with almost 69% of the cholesterol being transported in the non-HDL (i.e., VLDL + LDL) fraction (Table 4 ). Both test diets produced significant reductions in TC (37% decrease for PF and 44% decrease for CF), which could be attributed solely to significant reductions in non-HDL-C (48% for PF and 53% for CF) because HDL-C was not affected [although the latter values tended to be somewhat lower (P = 0.17) with the two test diets]. Additionally the test diets resulted in significant reductions in TG concentrations (29% for PF and 30% for CF) although the absolute magnitude of the changes was small, reflecting the generally low baseline plasma TG concentrations. Even though the test diets reduced the TC/HDL-C ratio by ~30%, these changes were not significant (P = 0.13, probably reflecting the large variation seen when monkeys consumed the high-fat, high-cholesterol diet). There was no significant difference between the PF- and CF-based diet periods, for any of the lipid variables.

Two major peaks, centered around fractions 6–7 and fraction 15, were discernible and represented LDL and HDL, respectively (Fig. 1 ). Feeding of the reference diet resulted in a LDL peak that was substantially larger than the HDL peak. Both diets resulted in significant reductions in cholesterol transported in lipoproteins of d < 1.053 kg/L (fractions 1–7), although there was no significant difference between the two test diets. The cholesterol distribution in lipoproteins of 1.054 < d < 1.16 kg/L (HDL) was essentially the same when all three diets were fed. Based on the data from Figure 1 , fraction 1 (referred to as VLDL), fractions 3–10 (designated LDL) and fractions 11–25 (designated HDL) were pooled from individual monkeys and analyzed further for lipoprotein composition (see below).

View larger version (29K): [in this window] [in a new window]   Figure 1. Line plot of the cholesterol distribution in plasma lipoproteins from monkeys fed the reference, CF and PF-based diets. Lipoproteins were isolated by density gradient ultracentrifugation of plasma obtained after 7 wk of dietary treatment. Each point is the means ± SEM, n = 8. There was no significant difference in lipoprotein cholesterol distribution between monkeys fed the test diets.

In comparison to the high-fat, high-cholesterol reference diet, both test diets significantly changed lipoprotein particle composition (Table 5 ). The percentage cholesterol ester decreased significantly (by 58% in VLDL, and 50% in LDL and HDL). This was accompanied by significant increases in the percentage phospholipid (20–33% increase in VLDL, 24–38% increase in LDL and 25% increase in HDL). The percentage protein increased when monkeys consumed the test diets (100–133% increase in VLDL, 47–63% increase in LDL and 14–30% increase in HDL). The percentage TG was unchanged except for HDL, the latter particles exhibiting a significant 50% decrease in percentage TG when monkeys consumed test diets. Again, there was no significant difference between the two test diets.

The decay curves for ¹³¹I-LDL and ¹²⁵I-HDL are shown in Figure 2 . The decrease in the percentage of initial radioactivity was significantly faster for LDL than HDL (~19–20 h for LDL radioactivity to decline to 50% of the initial value, while for HDL the time was almost 48 h). Both tracers exhibited a bi-phasic decay. Kinetic parameters for the ¹³¹I-LDL data are presented in Table 6 . Both the PF- and CF-based diets significantly reduced the LDL apoB pool size by 70% relative to values when monkeys consumed the reference diet. This reduced pool size reflected both accelerated catabolism and decreased LDL apo B TR. Consistent with the similar plasma lipids and lipoprotein composition, there was no difference in LDL kinetic parameters between the two test diets. Comparable to the similar HDL-C concentrations observed for all three diets (Table 4) , kinetic parameters for HDL were unaffected by dietary treatment and averaged: 130 ± 14 mg/kg body weight for apo A1 pool size, 0.282 ± 0.009 pools/d for FCR and 34 ± 4 mg · kg^-1 · d^-1 for apo A1 transport rate.

View larger version (21K): [in this window] [in a new window]   Figure 2. Line plot of the decline in plasma radioactivity following the simultaneous injection of 131I-LDL and 125I-HDL into monkeys fed the reference diet. Values are means ± SEM, n = 8.

	DISCUSSION

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In comparison to the high-fat, high-cholesterol reference diet, the test diets had decreased total fat, decreased SFA and decreased cholesterol content. Application of human regression equations (Clarke et al. 1997 , Howell et al. 1997 , Mensink and Katan 1992 , Yu et al. 1995 ) to our data predicts a decrease in LDL-C concentration of 0.30–0.38 mmol/L (in going from the high-fat, high-cholesterol diet to either of the two test diets), based on the fatty acid and cholesterol contents of the diets utilized. Of this 0.30–0.38 mmol/L reduction, almost 80% (0.25–0.30 mmol/L) would have been predicted based solely on the SFA, monounsaturated fatty acid (MUFA) and PUFA content (Mensink and Katan 1992 , Yu et al. 1995 ), while the remaining 20% (0.05–0.08 mmol/L) would have been predicted based on the cholesterol content (Clarke et al. 1997 , Howell et al. 1997 ). However, we observed much larger decreases (of 1.89–2.07 mmol/L) in non-HDL-C (essentially LDL-C, as the cynomolgus monkey has very small amounts of VLDL-C). The cynomolgus monkey is known to be extremely sensitive to dietary cholesterol (Brousseau et al. 1993 , Hayes and Khosla 1996 , Hunt et al. 1992 , Stucchi et al. 1995 and 1998 ), being ~14–20 times more sensitive than humans (Stucchi et al. 1998 ). Additionally, even though our reference diet had a cholesterol content (189 mg/4200 kJ) which was close to what humans might consume, the smaller size of cynomolgus monkeys (6 kg body weight vs. 70 kg for humans) coupled with its lower energy intake (~2500 kJ/d in this study vs. 10, 500 kJ/d for humans), resulted in an absolute cholesterol intake of ~19 mg · kg body^-1 · d^-1—which is almost three times the corresponding figure for humans (~7 mg · kg body^-1 · d^-1). Therefore, the large decrease that we observed in LDL-C levels following feeding of the test diets is in all likelihood due to their lower cholesterol content. A recent regression equation for cynomolgus monkeys (Stucchi et al. 1998 ) predicts a decrease in LDL-C of ~1.29 mmol/L for each 100 mg dietary cholesterol reduced per 4200 kJ. Application of this equation to our data predicts a decrease in LDL-C of 2.2 mmol/L, a figure more in line with what we observed. Thus, although our test diets lowered TC and LDL-C (in comparison to the high-fat, high-cholesterol reference diet), the decrease was attributable mainly to the removal of cholesterol. This is in contrast to the situation in humans, where under comparable dietary settings, the major effect appears to be related to decreasing SFA intakes.

The test diets utilized in the current study had very low cholesterol contents and as such represented "extreme" Step 1 test diets. Given great sensitivity of cynomolgus monkeys to dietary cholesterol (discussed above), as well as the fact that we wished to evaluate pork and chicken per se, exogenous cholesterol was not added. In the absence of exogenous cholesterol, fatty acid-induced effects on plasma lipids are discernible in this animal model. Hunt et al. (1992) observed TC concentrations of 4.3 and 3.0 mmol/L (LDL-C 2.4 and 1.7 mmol/L, respectively) when cynomolgus monkeys were fed diets containing 0.01 mg/kJ cholesterol and varying P/S ratios. Thus the low endogenous cholesterol content of our test diets (~0.005 mg/kJ) allowed us to compare the PF- and CF-based diets in terms of their fatty acid content. Our data show that within this dietary setting (when PF or CF represented 80% of the dietary fat), neither fat conferred any distinct advantages in terms of the plasma lipoprotein profile. In fact, no differences were apparent between effects of the PF- and CF-based diets for any of the variables that were measured.

Both test diets contained 10.98 g test fat/100 g diet (Table 1) . This translates to ~67 g test fat for a human consuming 2500 kcal (10460 kJ). Since a 3-oz (85 g) serving size of pork has 4.1–9.3 g fat, a consumption of 7–16 oz (198–454 g) of pork per day would be needed to achieve the amount consumed in the current study. [The corresponding value for chicken would be 7–22 oz (198–624 g).] These values are somewhat higher than what is currently recommended for humans [the Food Guide Pyramid advocates consumption of two to three servings per day from the meat group—which includes lean meat, poultry or fish (USDA 1995 )]. Even with these higher intakes, we did not detect any discernible differences in effects on plasma lipids between the two test diets.

There are at least two explanations for the similar effects of the PF- and CF-based diets on plasma lipids. Firstly, the PF- and CF-based diets had similar contents of cholesterol (0.0048 mg/kJ vs. 0.0040 mg/kJ, respectively), total SFA (9.1 and 8.0%en, respectively) and specifically the 12–16C SFA (6.1 and 6.4%en, respectively). MUFA content was the same in the two diets (~10%en) while the PUFA content was about 20% higher in the CF-based diet (8.9%en vs. 7.4%en). Using either of the recently published human regression equations (Hegsted et al. 1993 , Mensink and Katan 1992 , Yu et al. 1995 ) predicts that this difference in PUFA would at best have contributed to a nonsignificant (and nondetectable) cholesterol lowering of 0.08 mmol/L (3 mg/dL) with the CF-based diet. Thus, the diets produced comparable plasma lipid profiles.

An alternative and as yet still somewhat controversial explanation is that stemming from the work of Hayes and colleagues. These workers showed that in normocholesterolemic subjects (cebus, rhesus and squirrel monkeys) consuming very low-cholesterol-containing or cholesterol-free diets, palmitic acid (16:0) did not impact plasma cholesterol or LDL-C levels, and in such situations its effects were similar to those of oleic acid (Hayes et al. 1991 , Khosla and Hayes 1992 and 1993 , Pronczuk et al. 1991 ). Only when LDL receptors were suppressed (as when 0.3% cholesterol was fed) did 16:0 show a hypercholesterolemic effect compared to oleic acid (Khosla and Hayes 1993 ). In a meta-analyses of data from cebus monkeys, Hayes and Khosla (1992) showed that in normocholesterolemic individuals consuming low-cholesterol diets (reflecting unimpaired LDL receptor activity), TC (and LDL-C) was dependent solely on the dietary content of 14:0 and 18:2, and 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. (1965) by Hayes and Khosla (1992) 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 in several recent human studies (Chodhury et al. 1995 , Ng et al. 1992 , Sundram et al. 1995 and 1997 ) which 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-analyses by Khosla and Sundram (1996) of >30 different studies evaluating >130 diets yielded similar results. Thus, there are sufficient data to justify Hayes’ argument that 16:0 can be viewed as a fatty acid which is "conditionally" hypercholesterolemic (Hayes et al. 1995 ).

With the above arguments in mind, it should be noted that in the current study, the dietary cholesterol content of the PF- and CF-based diets was negligible, a situation favoring maximal LDL receptor expression. Therefore, the palmitic acid content, based on Hayes’ data, would have been predicted to make no contribution to plasma LDL cholesterol. The 18:2 content (7.2 and 8.6%en, for the PF- and CF-based diets, respectively) was above the threshold level for 18:2 (of 5–6%en) proposed by Hayes and Khosla (1992) , implying that this level of 18:2 was sufficient to counter the cholesterol-elevating effect of any other dietary component. Finally, 14:0, generally regarded as the most potent cholesterol-elevating fatty acid (Hegsted et al. 1965 , Mensink and Katan 1992 , Yu et al. 1995 ), was present in small amounts (0.1–0.3%en) to have any impact on LDL levels. Thus, the two diets produced essentially similar effects on plasma lipids.

Regardless of which of the above explanations apply, an important point to note is that we were evaluating PF and CF with added 18:2 [to satisfy the PUFA requirement for both a Step 1 diet and for the threshold level of 18:2 discussed by Hayes (1995) ]. Perusal of Table 2 reveals that if we had fed PF or CF by themselves (without any added safflower oil), the SFA content would have been 10.6 and 9.2%en, respectively, while the PUFA content would have been 3.2 and 4.9%en, respectively. At this level of PUFA, neither fat would have satisfied the Step 1 or threshold requirement. Therefore, for PUFA levels to approach the suggested Step 1 maximum of ~10%en, 20% high-linoleic safflower oil was added to the test fats. Additional studies from our laboratory (Gupta and Khosla, unpublished data) have shown that within this same dietary framework, diets with 59% of the fat from palm oil or 50% of the fat from cocoa butter (with added vegetable oils such that PUFA levels approach ~10%en) resulted in similar plasma lipids as the PF- and CF-based diets employed in the current study.

Whether the same response would be observed in humans must await direct experiments. Our data suggest that within the Step 1 framework (with its inherent restrictions on SFA and cholesterol intake in the presence of adequate PUFA), PF should produce comparable effects on plasma lipoproteins as chicken fat. A preliminary report in humans (Bales et al. 1995 ) found that plasma lipoproteins did not significantly differ between men and women fed diets containing skinless chicken or lean pork (~10 oz/8400 kJ diet) with 25%en from total fat. In a recently reported study (Davidson et al. 1999 ), the effect of a diet containing lean red meat on serum lipids was compared with a diet containing lean white meat in 190 hypercholesterolemic free-living people. The lean red meat group consumed 80% of their meat in the form of beef, veal or pork, whereas the lean white meat group consumed 80% of their meat from poultry or fish. Both groups consumed ~170 g (6 oz) of the appropriate meats, 5–7 d/wk. Both groups exhibited similar LDL-C concentrations when these meats were consumed as part of a Step 1 diet for up to 36 wk (total fat ~30% en, 12–16C SFA ~ 6%en, PUFA intakes ~6%en with a cholesterol intake of ~200–240 mg/d).

Finally, our data suggest 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 diet framework. Additional reductions may require altering one or more other nutrients. Further reductions in total fat (e.g., going to a Step 2-type 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. While, in theory the lowest LDL-C that could be achieved in cynomolgus monkeys (or any other species for that matter) is ~0.6 mmol/L [the concentration at which LDL receptors function maximally (Brown and Goldstein 1986 ) when the rate of LDL uptake is maximal (Dietschy et al. 1993 )], the more realistic value may be closer to ~1 mmol/L, obtained after feeding the PM nonpurified diet. It will be interesting to establish in future experiments, the maximum %en from fat (and/or the fatty acid mix) that will sustain an LDL-C of 1 mmol/L.

	ACKNOWLEDGMENTS

 
We thank Eric J. Hentges of the National Pork Producers Council for his input into the design of the study. We are indebted to Karen Rossman and her colleagues for their expert care of the nonhuman primates. We are grateful to Carolena Curry for periodic assistance with blood sample collection. We thank Kalyana Sundram from the Palm Oil Research Institute of Malaysia for the RBD palm oil used in formulating the control diet. We also thank Thomas V. Fungwe and David Klurfeld for helpful discussions.

	FOOTNOTES

 
¹ Supported in part by a grant from the National Pork Producers Council (NPPC #1905) and start-up funds from the College of Science, Wayne State University.

² Presented in part at the Federation of the American Societies for Experimental Biology meeting in Washington, D.C., 1999. Abstract published in the FASEB J. 13: A561 (1999).

³ To whom correspondence should be addressed.

⁴ Abbreviations used: CF, chicken fat; en, energy; FCR, fractional catabolic rate; LDL-C, LDL-cholesterol; MUFA, monounsaturated fatty acids; PF, pork fat; PM, Purina monkey; SFA, saturated fatty acids; TC, total cholesterol; TG, triacylglycerol; TR, transport rate.

Manuscript received March 2, 1999. Initial review completed June 23, 1999. Revision accepted January 3, 2000.

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