Interesterification and lipoproteins
Based on previous data concerning TG-MS affecting lipid metabolism [
5,
6], a primary objective of this study was to compare the relative response by plasma lipoproteins to structural differences in dietary fatty acids and TG-MS introduced by partial hydrogenation (production of TFA) or interesterification with 18:0. In the final analysis, 15% of TG-MS in IE had 18:0 at sn2, compared to none of the TG-MS in POL having sn2-18:0. Furthermore, IE had 2.5-times more SFA at sn2 than POL; and although we were not able to make the assessment with our assay, many of the sn2-FA in PHSO are reportedly
trans 18:1 with essentially no SFA at sn2 (24). Thus, our data support previous observations that structural differences between fatty acids and TG-MS can perturb lipoprotein metabolism, eg. when TFA or 16:0 are introduced at sn2 in fat molecules where MUFA or PUFA normally reside [
9‐
11,
26]. Similarly, our data confirm that compared to a naturally structured fat, random interesterification of a polyunsaturated dietary fat with 18:0 alters its TG-MS as well as the lipoprotein metabolism encountered during its consumption [
9,
27].
Both partial hydrogenation and IE represent substitutes for naturally saturated dietary fats. Our study directly addressed the efficacy of those substitutions by comparing unmodified TG-MS in a naturally occurring saturated fat, POL, with PHSO and IE. Approximately equal amounts of total SFA (14–18 %en) were provided during the two saturated fat test periods, ie. with POL and IE diets, while the TFA-rich diet provided about 12 %en as SFA plus TFA. Both modified fats included specific alterations in sn2-FA. Although total plasma cholesterol was minimally affected by the 3 diets, the distribution of cholesterol among lipoproteins was altered. PHSO (representing unnatural incorporation of
trans 18:1 at sn2) elevated LDL; and both PHSO and IE (with 18:0 atypically present at sn2) lowered HDL relative to the naturally-structured POL with mainly
cis 18:1 and a lesser amount of 16:0 at sn2. The increased LDL/HDL ratio following consumption of PHSO was also significant and typical of the pattern reported during
trans-fat consumption [
8,
9,
28‐
30]. It is noteworthy that in at least two of those studies when 18:0 was interesterified into unsaturated oils and compared to
trans 18:1,
cis 18:1, or IE fat rich in 18:2 [
8,
9], both the added 18:0 in IE fat as well as
trans 18:1 from partial hydrogenation were found to raise LDL and lower HDL. Such results are similar to our findings, where the naturally occurring fat structure in POL served as the control fat.
The high proportion of test fat (>70%) in our diets was coupled with specific exchanges between fatty acids and modified TG-MS. The comparison was focused on 16:0 in a natural fat, 18:0 from a randomized fat, and 16:0 plus about one-third the amount of TFA from partial hydrogenation. This comparison between relatively high intakes of specific SFA and TFA enhanced the potential to detect an effect induced by the concomitant change in dietary TG-MS. Despite the fact that the TFA diet provided 50% more 18:2 than POL, and favorably increased 18:2 among plasma TG fatty acids, the TFA diet still exerted a more negative impact on plasma lipoproteins. One would predict that reducing 18:2 in the TFA diet to that of the control diet would have rendered the TFA diet even less desirable [
9]; or alternatively, had the POL diet been adjusted to contain an equivalent amount of 18:2 as the other two diets, it should have performed even more favorably [
31]. To this point, it is noteworthy that POL, with its superior metabolic effect, had the lowest total dietary 18:2 content and only 1% of its TG molecules having sn2-18:2 with most 18:2 at sn1,3. These collective data imply that a lack of 18:2 at sn2 is not as problematic as the abnormal insertion of SFA (or TFA) in that location.
These data also support the previous conclusion [
30] that TFA are more detrimental than either of the two main SFA (16:0 and 18:0) that they were designed to replace in food products, at least gram for gram when considering lipoprotein metabolism. It is also clear that SFA as 16:0 in the TG-MS of natural POL was an improvement (even with less 18:2 present) compared to an approximately equal mass of 18:0 randomly inserted into SBO in the form of the IE fat, leading to a significant amount of 18:0 at sn2. This indicates that 18:0 should not be considered a neutral SFA, at least when randomized into sn2 or if it becomes the major dietary SFA. By the same token, others have shown that 16:0 randomized into the sn2 position raises LDL cholesterol in men when compared to natural palm oil [
10], a fat in which sn2 is largely
cis 18:1, similar to the situation with POL here. Thus, as these several studies demonstrate, manipulating dietary TG-MS can negatively influence plasma lipoproteins, even though the exact degree of TG modification required and the mechanism are unclear.
Modifying sn2 fatty acids, particularly by introducing a saturated fatty acid at this site, would seem a likely candidate for distorting lipoprotein metabolism. Caprenin, an artificial fat with randomized behenic acid (22:0), exerts a negative impact on human LDL/HDL metabolism similar to that seen with TFA [
32], and both 22:0 and TFA elicited effects similar to randomized 18:0 observed in this study and in two previous studies where IE fat was fed [
8,
9]. Long chain (18-22C) saturated fatty acids are relatively uncommon in natural fats, with 18:0 representing the most prevalent at intakes <2–4% daily energy. Furthermore, 18:0 in natural fats is usually esterified at sn1 and sn3 on glycerol, as in beef tallow and cocoabutter [
33]. Artificial insertion of 18:0 at sn2 during random interesterification, along with the shear mass of 18:0 consumed (12 %en), may have been problematic in the current study and previously [
8,
9]. The mass of 18:0 consumed may be critical because a large intake (7 %en), from sheanut butter was found to depress HDL similar to Caprenin [
34]. Sheanut butter is reported to have 18% of sn2 as SFA (mostly 18:0), or 3× the amount of sn2-SFA present in cocoabutter [
33], a fat that does not appear to affect lipoprotein metabolism adversely [
4,
12]. Although consumption of 12 %en as 18:0 would not be feasible from natural fats, it is possible to envision an exaggerated intake via structurally modified fats resulting from the growing impetus to eliminate TFA from the diet. The incorporation of 18:0 has seemed especially appealing since 18:0 from natural fats often has been considered neutral in its impact on cholesterol metabolism [
2‐
4,
35].
Glucose perturbations
Among studies on the metabolic effects of randomized fat involving 18:0, metabolism of glucose and insulin has not been addressed. Considering global trends in obesity, insulin resistance, and diabetes, which are often associated with the metabolic syndrome, our observation that altered dietary TG-MS may adversely influence glucose metabolism warrants attention. From epidemiological data on the association between TFA intake and diabetes [
36] one might have anticipated the TFA-induced rise in glucose observed after 4 wk. Even though previous experiments involving TFA in humans found no effect on glucose metabolism other than postprandial insulin hypersecretion [
37,
38], those studies reported essentially no change in the fasting lipoprotein profile either, which is somewhat atypical. In our study IE fat had less effect than the TFA-rich PHSO on lipoproteins, but proved more deleterious for glucose metabolism, suggesting that modified TG-MS, from either trans-rich or IE fats, was a factor.
After 4 wk on each diet, fasting insulin was inversely related to glucose, ie. insulin was moderately lower after PHSO (-10%, ns) and substantially lower after IE (-20%, p < 0.001) compared to POL, while glucose was significantly elevated by both modified fats (about 5% and 20%, respectively). The elevated 4 wk fasting glucose following IE was foreshadowed by the 40% greater glucose IAUC observed during the 8 h postprandial challenge with the IE test meal. The patterns of lower plasma insulin (IE, PHSO) and C-peptide (IE) postprandially following the test meal challenge with the two modified fats relative to POL, suggests that reduced insulin secretion, rather than insulin resistance, accounted for the higher glucose values observed with modified fats. The 20% rise in fasting glucose with IE was clinically important, as well, since it rose to a range that could be considered prediabetic after only 4 wk [
39]. These results appear to be in contrast to the elevated serum insulin and insulin resistance typical of obesity and type 2 diabetes associated with the metabolic syndrome [
40,
41].
Our findings suggest that altered dietary fat composition or TG-MS influenced insulin secretion to impact glucose metabolism negatively. Others have reported initially lower blood glucose coupled with sharply higher initial insulin secretion 20–60 min postprandially in subjects fed a single meal of IE palm oil compared to natural palm oil [
11]. Postprandial insulin hypersecretion also occurred in diabetic subjects fed extreme intakes of TFA or SFA compared to cisMUFA [
38]. Thus, a connection seems to exist between dietary fat composition and insulin secretion. The link may include the n-3 PUFA content of fats. For example, it is noteworthy that dietary 18:3n-3 and long chain n-3 PUFA have been linked to production of intestinal GLP-1 [
42], which enhances insulin secretion. It may prove ironic that partial hydrogenation of vegetable oils, or interesterification with 18:0, are implemented in part to remove 18:3n-3 and improve product shelf life, even though fats modified in this manner may inadvertently suppress insulin secretion after their prolonged consumption. Interesterification resulting in high intake of 18:0, including sn2-18:0, appeared to accentuate the negative effect observed with the TFA diet. In reference to insulin metabolism, both 18:0 and TFA fed at high levels (8% en) also induced inflammatory markers [
43] that are associated with diabetes [
44].
It is not apparent whether the observed alteration in glucose and lipoprotein metabolism represents a fatty acid effect per se or modification of TG-MS. For example, earlier work in gerbils traced saturated fat intake and inositol deficiency to a lack of PUFA needed structurally for phosphatidyl inostitol (PI) synthesis required for fat secretion by intestinal mucosal cells [
45]. Since PI is also involved in beta-cell secretion of insulin [
46], PI structure or availability may be a consideration in the mechanistic aspect of our current clinical observations. Extreme SFA consumption (usually to the exclusion of dietary PUFA) is typically linked to insulin resistance [
47], while other studies have reported that dietary SFA enhances insulin secretion more than dietary PUFA [
48], a possibility suggested by our findings where POL diet had the lowest P/S ratio. Clarification is needed, but careful scrutiny of the dietary PUFA load as it affects dietary TG-MS and phospholipid synthesis related to insulin dynamics is warranted [
6].
In past investigations, glucose metabolism has not received the intense scrutiny afforded lipoproteins in most studies involving dietary fat. The present results linking TFA and 18:0- rich IE fats with abnormal metabolism of glucose suggest that it would be prudent to determine the biologically tolerable mass of 18:0 that can be incorporated in diets as IE fats.