Victor C. Gavino

Could the Quality of Dietary Fat, and Not Just Its Quantity, Be Related to Risk of Obesity?

This review focuses on the possible association between types of fatty acids and weight change. It examines the biological plausibility underlining these associations and the evidence obtained to date from clinical trials and epidemiological studies. Animal studies have shown that dietary short‐ and medium‐chain fatty acids compared to long‐chain fatty acids appear to promote weight loss. Similarly, monounsaturated fatty acids (MUFAs) appear to favor weight loss compared to saturated fatty acids (SFAs) in human studies. The structure of fatty acids seems to affect their degree of oxidation and deposition. Although results are conflicting, human studies follow the general trend reported in animal studies. These trials suggest that some fatty acids are prone to oxidation and some others lead to fat storage when comparing isocaloric diets. For instance, n‐3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic and docosahexaenoic acids are preferentially oxidizied to other PUFA but results remain inconsistent. Epidemiological studies concerning this issue reported that total dietary fat, which includes MUFA, PUFA, and SFA could increase the risk of obesity, but results are few and conflicting. The rising biological plausibility linking dietary fat quality and risk of obesity, together with the rather recent addition of fatty acids content in food composition tables, support the need for major epidemiological studies in that area.

Rat liver outer mitochondrial carnitine palmitoyltransferase activity towards long-chain polyunsaturated fatty acids and their CoA esters

The activity of the overt form of rat liver mitochondrial carnitine palmitoyltrasferase or CPT0 (EC 2.3.1.21) towards different fatty acid substrates was studied. The following non-esterified fatty acids (NEFA) and their CoA esters in the presence of 1% bovine serum albumin (BSA) were tested: 16∶0, 18∶0, 18∶1, 18∶2, 18∶3ω3, 20∶4, 20∶5ω3 and 22∶6ω3. The data fit a square hyperbolic model for enzyme catalysis (p<0.001, non-linear regression). Asymptotic Vmax and K0.5, substrate concentration at one-half Vmax, were calculated using total concentrations of acyl-CoA, or unbound concentrations of NEFA. BSA was found to act as a true substrate reservoir for NEFA in that the dissociation of the NEFA-BSA complex was 10–330 times faster than the CPT0 reaction. Regardless of form (NEFA or CoA ester), 18∶3ω3 gave the highest, while 22∶6ω3 and 18∶0 gave the lowest rates of acylcarnitine synthesis. Except for 18∶3ω3 and 18∶2, Vmax for NEFA was generally lower than for acyl-CoA, with the greates differences observed for 20∶4, 20∶5ω3 and 22∶6ω3, suggesting that acyl-CoA synthesis may also be important in the control of the entry of these fatty acids into the mitochondria. The data provide an enzymatic rationale for the relatively low content of 18∶3ω3 in esterified lipid.

Adipose Hormone-Sensitive Lipase Preferentially Releases Polyunsaturated Fatty Acids from Triglycerides

Rat adipose hormone-sensitive lipase-mediated release of fatty acids from triglycerides was studied in three model systems: i) cultured preadipocytes containing polyunsaturated fatty acid-enriched triglyceride; ii) perfused epididymal fat pads; and iii)in vitro incubations of crude preparations of hormone-sensitive lipase with synthetic triglyceride-analogues as substrates. We found that cultured preadipocytes challenged with 10μM norepinephrine tended to release more ω6 and ω3 polyunsaturated fatty acids than saturated fatty acids. Fat pads perfused with 10 μM norepinephrine preferentially released arachidonate and α-linolenate but tended to retain oleate and linoleate. Finally, crude preparations of hormonesensitive lipase released from the triglyceride-analogue substrates α-linolenate twice as fast as oleate. We conclude that rat adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. We suggest that this may be a mechanism by which these fatty acids are kept from being trapped in fat depots and maintained in the circulation.

Polyunsaturated fatty acid contents of some traditional fish and shrimp paste condiments of the Philippines

Abstract The fatty acid composition as well as the water, NaCl, ash and fat contents of six types of shrimp and fish paste condiments of the Philippines were determined. The condiments were prepared by incubating the fish or shrimp in high concentrations of salt and under high humidity at ambient temperature over several months. The primary objective was to assess the contents of polyunsaturated fatty acids, and in particular the content of all- cis -4,7,10,13,16,19-docosahexaenoic acid (DHA) in these products. The product derived from small shrimp fry ( Acetes spp.) had the highest content of DHA. The contents of polyunsaturated fatty acids, including DHA, of this particular condiment, were not significantly different from that of fresh untreated shrimp fry, indicating the presence of mechanisms that protect against polyunsaturated fatty acid autoxidation during the preparation process. Also, the DHA contents of the condiment derived from juvenile Siganus approached that of the Acetes condiment because of its relatively high fat content. In conclusion, two of the fish and shrimp paste condiments tested may be sources of dietary DHA for those who consume this type of food.

The role of dietary choline in the beneficial effects of lecithin on the secretion of biliary lipids in rats

Earlier studies showed that dietary soybean lecithin increases biliary lipid secretion, which mainly comes from the contribution of high density lipoprotein (HDL) and hepatic microsomal pools of phosphatidylcholine and cholesterol. In addition, a lecithin diet enhances bile secretion and prevents bile acid-induced cholestasis. This study evaluated the contribution of choline, a component of lecithin, to the observed effect of lecithin on biliary secretory function. Rats were fed either a control diet (CD), a choline diet (ChD) or a lecithin-enriched diet (LD) for 2 weeks. Results showed that like LD, ChD induced an increase in bile flow and bile acid secretion rate when compared with the control diet. However, unlike LD, ChD did not significantly increase biliary phospholipids and cholesterol output. An increase of hydrophilic bile acids (i.e. ursodeoxycholic and muricholic acids) in bile of rats fed choline could explain why the biliary phospholipid and cholesterol secretion was not increased. During taurocholic acid infusion, both experimental diets increased bile flow and the bile acid secretion rate maximum (BASRm). The cholestasis usually observed after the BASRm is reached was inhibited by ChD and LD. Both diets induced a decrease in plasma cholesterol (total and HDL), however, only LD induced statistically significant changes. Analysis of total cholesterol and phospholipid content of microsomes and canalicular membranes indicated no statistically significant difference between control and experimental groups either under basal conditions or after bile acid infusion. Similarly, the phospholipid classes and fatty acid composition of biliary phosphatidylcholine were not altered by feeding ChD and LD. We conclude that choline contributes to the beneficial effect of a lecithin diet on bile secretion. It is postulated that this effect may be attributed to modulation of HDL and an enhancement of the cholesterol and phospholipid pools destined for biliary secretion.

Kinetic analysis of the selectivity of acylcarnitine synthesis in rat mitochondria

Mitochondrial acylcarnitine synthesis is an obligatory step in the transport of cytosolic long-chain FA into the mitochondria. It is an important control point in the partitioning of cytosolic fatty acids to synthetic pathways or to mitochondrial β-oxidation. Mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) is the enzyme that catalyzes the transformation of long-chain fatty acylCoA esters to acylcarnitine. Additionally, the isoform of acylCoA synthetase (EC 6.2.1.3) found in mitochondria, which is in close proximity to CPT I on the outer membrane, may act in concert with CPT I to form acylcarnitines from cytosolic nonesterified FA (NFFA). The mitochondrial acylcarnitine synthesis pathway is exposed to multiple fatty acid substrates present simultaneously in the cell milieu, with each fatty acid present at varying pool sizes. The selectivity of this pathway for any particular fatty acid substrate under conditions of multisubstrate availability has not yet been tested experimentally. Our objective was to develop mathematical equations that make use of kinetic constants derived from single-substrate experiments to predict the selectivity of the acylcarnitine synthesis pathway under conditions in which two or more substrates are present simultaneously. In addition, the derived equations must be verifiable by experiment. Our approach was to begin with a Michaelis-Menten model that describes the initial rates of an enzyme system acting on multiple and mutually competitive substrates. From this, we derived equations expressing ratios of reaction rates and fractional turnover rates for pairs of substrates. The derived equations do not require assumptions concerning the degree of enzyme saturation. Using rat mitochondrial preparations and the NEFA substrate pairs, linolenic-oleic acids and palmitic-linoleic acids, we showed that the shape of the experimentally derived data on acylcarnitine synthesis fits the predictions of the derived model equations. We further validated the derived equations by showing that their predictions calculated from previously published kinetic constants were consistent with data from actual experiments. Thus, we are able to conclude that with respect to acylcarnitine synthesis, the fractional turnover rate of the linolenic acid pool would always be 2.9-fold faster than that of the oleate pool regardless of the pool size of either fatty acid. Similarly, the fractional turnover rate of the palmitate pool would always be 1.8-fold faster than that of the linoleate pool regardless of pool size. We extended our kinetic model to more than two mutually competitive substrates. Using previously published rate constants for eight physiologically relevant fatty acids, the derived model predicts that regardless of pool size of any of the fatty acids, the linolenate pool, whether as NEFA or as a CoA ester, would always have the highest fractional turnover rate with respect to acylcarnitine synthesis. Conversely, the stearate pool whether as NEFA or as CoA ester will have the lowest fractional turnover rate relative to all the other fatty acids.

Uptake and metabolism of structured triglyceride by Caco-2 cells: reversal of essential fatty acid deficiency

Structured lipids have been proposed as efficient vehicles for the supplementation of essential fatty acids (EFA) to patients with malabsorption. We investigated how a novel structured triglyceride (STG), containing purely octanoic acid in the sn-1/sn-3 and [14C]linoleic acid in the sn-2 positions, was incorporated into different lipid classes in Caco-2 cells. We also evaluated the contribution of gastric lipase in the uptake and metabolism of [14C]linoleic acid from the STG. We furthermore determined the potential of the STG to correct EFA deficiency induced in Caco-2 cells. The absorption of STG by Caco-2 cells was significantly greater compared with that of triolein. The addition of human gastric lipase significantly enhanced cellular uptake of the labeled substrate, reflecting the stereoselectivity of gastric lipase to hydrolyze medium chain FA. Analysis of the intracellular lipids synthesized revealed a predominance of phospholipids-monoglycerides. Most of the radioactivity in the lipoproteins isolated from Caco-2 cells was recovered in TG-rich lipoproteins (45%) and to a lesser extent in the high-density lipoprotein (36%) and low-density lipoprotein (17%) fractions. The administration of STG to Caco-2 cells rendered EFA deficient produced a marked increase of the cellular level of linoleic and arachidonic acids. This resulted in a lower ratio of 20:3(n-9) to 20:4(n-6), reflecting the correction of EFA deficiency in Caco-2 cells. Our data demonstrate that STG, in the presence of gastric lipase, have beneficial effects on lipid incorporation, lipoprotein production, and EFA status, utilizing Caco-2 cells as a model of EFA deficiency.Structured lipids have been proposed as efficient vehicles for the supplementation of essential fatty acids (EFA) to patients with malabsorption. We investigated how a novel structured triglyceride (STG), containing purely octanoic acid in the sn-1/ sn-3 and [14C]linoleic acid in the sn-2 positions, was incorporated into different lipid classes in Caco-2 cells. We also evaluated the contribution of gastric lipase in the uptake and metabolism of [14C]linoleic acid from the STG. We furthermore determined the potential of the STG to correct EFA deficiency induced in Caco-2 cells. The absorption of STG by Caco-2 cells was significantly greater compared with that of triolein. The addition of human gastric lipase significantly enhanced cellular uptake of the labeled substrate, reflecting the stereoselectivity of gastric lipase to hydrolyze medium chain FA. Analysis of the intracellular lipids synthesized revealed a predominance of phospholipids-monoglycerides. Most of the radioactivity in the lipoproteins isolated from Caco-2 cells was recovered in TG-rich lipoproteins (45%) and to a lesser extent in the high-density lipoprotein (36%) and low-density lipoprotein (17%) fractions. The administration of STG to Caco-2 cells rendered EFA deficient produced a marked increase of the cellular level of linoleic and arachidonic acids. This resulted in a lower ratio of 20:3(n-9) to 20:4(n-6), reflecting the correction of EFA deficiency in Caco-2 cells. Our data demonstrate that STG, in the presence of gastric lipase, have beneficial effects on lipid incorporation, lipoprotein production, and EFA status, utilizing Caco-2 cells as a model of EFA deficiency.

Modulation of GTP-dependent fusion by linoleic and arachidonic acid in derivatives of rough endoplasmic reticulum from rat liver

The effect of modulation of the content of unsaturated free fatty acids on GTP-dependent fusion of stripped rough microsomes from rat liver was determined. Cytidine monophosphate, CDP and CTP were all observed to be able to stimulate free fatty acid accumulation and coincident membrane fusion. GTP was required for membrane fusion in the presence of cytidine nucleotide but was not required for free fatty acid accumulation. In the presence of GTP and cytidine nucleotide, the addition of ATP and CoA led to the synthesis of triacyglycerol and marked inhibition of both free fatty acid accumulation and membrane fusion. Delipidated bovine serum albumin also inhibited both free fatty acid accumulation and membrane fusion. Analysis by gas chromatography indicated that linoleic acid and arachidonic acid were the most actively fluctuating of the accumulated free fatty acids. Comparison by quantitation indicated a high correlation between GTP-dependent membrane fusion and changes in amount of unesterified linoleic acid and arachidonic acid. The results suggest that polyunsaturated free fatty acids may be required for GTP-dependent membrane fusion.

Effect of 4,7,10,13,16,19-docosahexaenoic acid on triglyceride accumulation and secretion in rat hepatocytes in culture.

We have investigated the effect of oleic (18∶1) and 4,7,10,13,16,19-docosahexaenoic (22∶6ω3) acids on triglyceride (TG) accumulation, secretion and reuptake in rat hepatocytes in culture. We also calculated the percentage of total TG, TG-esterified 18∶1 and TG-esterified 22∶6ω3 that were secreted relative to the total accumulation (intra+extracellular TG). Both fatty acids were incorporated mainly in the intracellular TG fraction. Treatment with 18∶1 but not with 22∶6ω3 increased the quantity of TG secreted into the culture medium relative to controls. Treatment with 22∶6ω3 caused a greater accumulation of intracellular TG than 18∶1. This arises in part from the preferential retention of 22∶6ω3-enriched TG by the hepatocytes. At 24 hr, there was no longer any difference in the net secretion of TG by 18∶1 and 22∶6ω3-treated cells, which may be a consequence of the reuptake of TG-esterified 18∶1. There was no reuptake of TG-esterified 22∶6ω3. We conclude that inhibition of hepatocyte TG synthesis is not obligatory for 22∶6ω3-induced diminution of TG secretion.

Malondialdehyde-modified high-density lipoprotein cholesterol: plasma removal, tissue distribution and biliary sterol secretion in rats

Evidence has been accumulating for the putative role of chemically or oxidatively altered lipoproteins in accelerating events in the atherogenic process. In this study, the movement of free cholesterol from native high density lipoprotein (HDL) and malondialdehyde (MDA)-modified HDL to the liver for biliary cholesterol secretion and bile acid transformation was examined in vivo. To this end, HDL from normal donor rats was isolated, conjugated with MDA, labelled with [14C]cholesterol and injected i.v. into rats with bile diversion. While the 6 h collection revealed no substantial differences in bile flow, less 14C excretion was recovered in the fresh bile of animals receiving MDA-modified HDL. Bile analysis indicated that a significant decline in labelled bile acid secretion characterized these rats. Compared with controls, MDA-modified HDL also caused an enhanced accumulation of [14C]cholesterol in the liver and the kidneys, with reduced delivery to the sites of steroidogenesis, i.e., the adrenals and testes. No plasma removal differences were noted in the HDL of both groups of rats. Thus, modification of HDL by MDA seems to impair the tissue distribution of its cholesterol moiety, particularly in the liver, where it accumulates at the expense of bile acid transformation.