W.M. Nimal Ratnayake

Fat and fatty acid terminology, methods of analysis and fat digestion and metabolism: a background review paper.

Fats, oils or lipids consist of a large number of organic compounds including fatty acids, monoacylglycerols, diacylglycerols, triacylglycerols (TGs), phospholipids (PLs), eicosanoids, resolvins, docosanoids, sterols, sterol esters, carotenoids, vitamin A and E, fatty alcohols, hydrocarbons and wax esters. Classically, lipids were defined as substances that are soluble in organic solvents. This is a loose definition and could include a number of non-lipid organic compounds. A novel definition and comprehensive system of classification of lipids were proposed in 2005 [Fahy et al., 2005]. The novel definition is chemically based and defines lipids as small hydrophobic or amphipathic (or amphiphilic) molecules that may originate entirely or in part by condensations of thioesters and/or isoprene units. The proposed lipid classification system enables the cataloguing of lipids and their properties in a way that is compatible with other macromolecular data bases. Using this approach, lipids from biological tissues have been divided into 8 categories, as shown in table 1 . Each category contains distinct classes and subclasses of molecules [Fahy et al., 2005]. Published online: September 15, 2009

Influence of dietary protein and fat on serum lipids and metabolism of essential fatty acids in rats

A 120 d feeding study with adult rats was conducted to evaluate the influence of two protein sources (casein and gelatin), two protein levels (50 and 300 g/kg diet) and two fat levels (50 and 150 g/kg diet) on serum lipids (total cholesterol, HDL-cholesterol and triacylglycerols) and liver polyunsaturated fatty acid levels. In general, the concentrations of serum triacylglycerols and total cholesterol and liver phospholipid levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in rats fed on casein diets compared with those fed on the gelatin diets. These effects were more pronounced in rats fed on the high-casein (300 g/kg)-high-fat (150 g/kg) diet. Gelatin was hypocholesterolaemic and also suppressed the liver phospholipid levels of AA and DHA (reported for the first time). The difference in the amino acid composition between casein and gelatin may be responsible for the observed effects. Casein contains higher levels of glutamic acid, methionine, phenylalanine and tyrosine, while gelatin contains higher levels of arginine, glycine and hydroxyproline. It is suggested that a protein source which increases serum cholesterol may also increase the concentrations of AA and DHA in rat tissues.

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats.

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.

Concerns about the use of 15:0, 17:0, and trans-16:1n–7 as biomarkers of dairy fat intake in recent observational studies that suggest beneficial effects of dairy food on incidence of diabetes and stroke

Dear Editor: It is with interest that I read the articles by Santaren et al. (1) and Yakoob et al. (2) about the relation of circulating concentrations of pentadecanoic acid (15:0), heptadecanoic acid (17:0), and trans-palmitoleic acid (trans-16:1n–7) with the incidence of diabetes and stroke. Santaren et al. (1) reported that serum concentrations of pentadecanoic acid are associated with insulin sensitivity and β cell function, as well as a 27% decreased risk of type 2 diabetes. Yakoob et al. (2) reported no significant associations of total plasma or red blood cell pentadecanoic acid, heptadecanoic acid, and trans-palmitoleic acid with risk of stroke. Because several previous studies implicated pentadecanoic acid, heptadecanoic acid, and trans-palmitoleic acid in serum, plasma, red blood cells, and adipose tissue as valid biomarkers for dairy intake (3–9), Santaren et al. (1) suggested that their findings may contribute to future recommendations regarding the benefits of dairy products on type 2 diabetes, and Yakoob et al. (2) concluded that circulating biomarkers of dairy fat are not significantly associated with stroke. A commentary written by Arne Astrup in the same issue of the Journal (10) stated that “there is no evidence left to support the existing public health advice to limit consumption of dairy to prevent CVD [cardiovascular disease] and type 2 diabetes.” I am concerned with the use of pentadecanoic acid, heptadecanoic acid, and trans-palmitoleic acid as biomarkers of dairy fat intake. It is true that these are present in dairy fat, although at very low amounts (pentadecanoic acid at 1.0%, heptadecanoic acid at 0.6%, and trans-palmitoleic acid at 0.3%) (11). These 3 fatty acids, however, are not limited to dairy fat. In particular, fat from beef, veal, lamb, and mutton also contains all of these fatty acids at amounts similar to those found in dairy fat (12, 13). The presence of pentadecanoic acid and heptadecanoic acid, at amounts comparable to dairy fat, has also been reported in many other common dietary fats and foods, including chicken, lard (13), marine and freshwater fish (14), marine oils (15), some vegetables (cabbage and cucumber) (16), and seaweeds (17). Several common vegetable oils also contain small amounts of heptadecanoic acid (18). Rapeseed (canola) oil contains both pentadecanoic acid and heptadecanoic acid (19). These data suggest that pentadecanoic acid and heptadecanoic acid are widely distributed in nature and present in many common foods, including dietary fats, albeit in small amounts. Unfortunately, this information is not commonly available because many scientific publications on fatty acid composition of dietary fats and foods focus only on the major and nutritionally important fatty acids and do not show data for pentadecanoic acid and heptadecanoic acid because these fatty acids are minor components and have no known nutritional or biological significance. Another factor that needs to be considered in choosing a fatty acid as a biomarker is that it should not be endogenously synthesized. Many previous studies made the assumption that circulating trans-palmitoleic acid is solely derived from the consumption of dairy fat (9). However, it was recently found that circulating trans-palmitoleic acid is not exclusively diet derived but may also be endogenously produced by the partial β-oxidation of dietary vaccenic acid (trans-18:1n–7) (20). Vaccenic acid is the major trans fatty acid isomer in dairy fats but is also present in partially hydrogenated oils. In Canadian dairy products, vaccenic acid accounts for 22–43% of total trans-18:1 isomers (21). Partially hydrogenated vegetable oils also contain considerable amounts of vaccenic acid: proportions ranging from 15% to 24% of total trans-18:1 isomers have been found in partially hydrogenated canola and soybean oil samples (22). Trace amounts of pentadecanoic acid and heptadecanoic acid are synthesized in leaves (23) and are present in common vegetables as noted above (16). It is not known whether animals and humans have the capability to synthesize pentadecanoic acid and heptadecanoic acid, but this should not be ruled out until it has been examined. A further concern is the uncertainty of correct identification of pentadecanoic acid, heptadecanoic acid, and trans-palmitoleic acid in the gas chromatography (GC) analysis of fatty acid mixtures. These fatty acids are always found in very low concentrations in blood samples and dietary fats and very often coelute with other fatty acids in GC analysis. For example, pentadecanoic acid overlaps with 9-cis-tetradecenoic acid (9c-14:1), trans-palmitoleic acid overlaps with iso-heptadecanoic acid and 3-trans-heptadecanoic acid (3t-16:1; a common trans fatty acid in plants), and heptadecanoic acid elutes close to 11-cis-hexadecenoic acid (11c-16:1) and 13-cis-hexadecenoic acid (13c-16:1) (24). Thus, if the GC conditions are not optimized, it is possible that the concentrations of pentadecanoic acid, trans-palmitoleic acid, and heptadecanoic acid may be exaggerated due to inclusion of the overlapping components. Because the fatty acid analytic methods used were not described by Santaren et al. (1) and Yakoob et al. (2), it is not known whether they have encountered any such fatty acid analysis problems. Considering these possible uncertainties of the dietary origin and the analysis of pentadecanoic acid, heptadecanoic acid, and trans-palmitoleic acid, we should be cautious in making conclusions about the role of dairy fats in diabetes and stroke.

Lower serum magnesium concentration is associated with diabetes, insulin resistance, and obesity in South Asian and white Canadian women but not men

Background A large proportion of adults in North America are not meeting recommended intakes for magnesium (Mg). Women and people of South Asian race may be at higher risk for Mg deficiency because of lower Mg intakes relative to requirements and increased susceptibility to diabetes, respectively. Objective This study compared serum Mg concentrations in South Asian (n=276) and white (n=315) Canadian women and men aged 20–79 years living in Canadas Capital Region and examined the relationship with diabetes, glucose control, insulin resistance, and body mass index. Results Serum Mg concentration was lower in women of both races and South Asians of both genders. Racial differences in serum Mg were not significant after controlling for use of diabetes medication. A substantial proportion of South Asian (18%) and white (9%) women had serum Mg <0.75 mmol/L indicating hypomagnesemia. Use of diabetes medication and indicators of poorer glucose control, insulin resistance, and obesity were associated with lower serum Mg in women, but not in men. Conclusions These results suggest that the higher incidence of diabetes in South Asians increases their risk for Mg deficiency and that health conditions that increase Mg requirements have a greater effect on Mg status in women than men.

Effects of dietary protein and fat on cholesterol and fat metabolism in rats

Abstract The influence of two fat sources (soybean oil and a 4:1 mixture of coconut oil and soybean oil) fed at three different levels (5, 10 and 20% by weight) and two protein sources (casein and gelatin supplemented with limiting amino acids) on cholesterol and fat metabolism in rats was determined. Fat or protein type had a significant ( P P de novo fat synthesis and cholesterol kinetics. De novo fat synthesis was highest in animals fed gelatin-soybean oil diets. De novo cholesterol synthesis followed the same patterns observed for fat synthesis. Since overall serum cholesterol levels were lower in rats fed gelatin-soybean oil diets, cholesterol clearance rates must have been higher to compensate for the increased synthesis observed in rats fed these diets.

Mandatory trans fat labeling regulations and nationwide product reformulations to reduce trans fatty acid content in foods contributed to lowered concentrations of trans fat in Canadian women's breast milk samples collected in 2009–2011

BACKGROUND Recent efforts in Canada to reduce industrial trans fatty acids (TFAs) in foods include mandated inclusion of TFA content on food labels and recommendations by Health Canada that encourage the food industry to voluntarily limit TFA content in all vegetable oils and soft margarines and in all other prepackaged foods to <2% and <5% of total fat, respectively. OBJECTIVE To assess the impact of these efforts, we measured the concentration of TFAs in human breast milk samples. DESIGN The TFA content in 639 breast milk samples collected in 2009, 2010, and 2011 from breastfeeding mothers in 10 major cities across Canada was analyzed by gas chromatography. RESULTS The mean (±SD) TFA contents were 2.7 ± 0.9% (n = 153, range: 1.4-7.2%), 2.2 ± 0.7% (n = 309, range: 1.0-6.8%), and 1.9 ± 0.5% (n = 177, range: 0.9-3.4%) of total milk fat for samples collected in 2009, 2010, and 2011, respectively. These values are considerably lower than the value of 7.2 ± 3.0% (range: 0.1-17.2%) found previously for Canadian human milk in 1992. On the basis of a linear correlation between the percentage of TFAs in the diet and human milk fat established by Craig-Schmidt et al, and assuming that 30% of energy of a lactating mothers diet is derived from fat, we estimated from the TFA human milk fat data that TFA intake of Canadian breastfeeding mothers was 0.9%, 0.5%, and 0.3% of total energy in 2009, 2010, and 2011, respectively. These estimated values are lower than the WHOs maximum recommended intake of 1% of total energy for a healthy diet. CONCLUSIONS The results suggest that the trans fat labeling regulations introduced in 2003 and recommendations by Health Canada in 2007 instructing the food manufacturers and restaurants to limit TFAs in foods have resulted in significant reductions in TFAs in the diets of Canadian breastfeeding mothers and their breast milk.

Impact of dietary protein on lipid metabolism in hamsters is source-dependent and associated with changes in hepatic gene expression.

This study tested the hypothesis that protein source is a factor determining the impact of the diet on lipid metabolism in hamsters. Twenty-eight hamsters of similar body weight were assigned for a period of 8 weeks to one of the following four diets (seven per group) containing either 20 % (w/w) casein (CAS), beef protein (BF), wheat gluten (WG) or soya protein (SOY). The fat composition of the diet was the same (15.5 % w/w) in all groups and provided SFA, MUFA and PUFA representative of the average Canadian diet. After an overnight fast, blood and liver were collected for the measurement of serum lipids, fatty acid composition of liver phospholipids and mRNA levels of selected genes involved in lipid metabolism. WG resulted in lower total cholesterol, HDL-cholesterol and non-HDL-cholesterol but, along with SOY, in higher mRNA levels of cholesterol 7 alpha-hydroxylase and LDL receptor. Furthermore, both WG and SOY resulted in lower 18 : 3n-3, 20 : 4n-6, total n-6 PUFA, 18 : 1n-9 and total MUFA, but higher 22 : 6n-3, total n-3 PUFA, 22 : 6n-3/18 : 3n-3 and 22 : 5n-3/18 : 3n-3 ratios in liver phospholipids, and higher hepatic Delta6-desaturase mRNA levels. These results show that the impact of dietary protein on lipid metabolism is source-dependent and associated with changes in mRNA abundances of key hepatic enzymes and receptors.

Effects of supplemental cystine or methionine on growth and lifespan of stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive (SHRSP) rats are considered a suitable model for studying the effects of dietary and other environmental factors on human essential hypertension and haemorrhagic stroke. To investigate the suitability of a control diet for this strain of rats, we studied the effects of supplementing casein and soya protein isolate (SPI) with two sulphur amino acids (methionine and cystine) on the growth and lifespan of SHRSP rats. The source of dietary protein and the type of supplemental sulphur amino acid had significant (P < 0.05) effects on food intake and weight gain measured after 31 d of the feeding study, while only the type of supplemental sulphur amino acid had significant effects on mean survival times and the survival rates. On average, the casein groups had higher food intake and weight gain compared with the SPI groups. The methionine-supplemented groups had lower food intake but higher weight gain than the cystine-supplemented groups. Similarly, the methionine-supplemented groups had higher mean survival times and survival rates compared with the cystine-supplemented groups. The data would suggest that a control diet based on cystine-supplemented casein (as recommended for normal healthy rats by the American Institute of Nutrition), may not meet the sulphur amino acid requirements for SHRSP rats, and that the methionine-supplemented casein would be an appropriate control diet for this animal model.

LONGEVITY OF THE STROKE-PRONE HYPERTENSIVE RATS IS INFLUENCED BY THE SOURCE AND AMOUNT OF DIETARY PROTEIN

Longevity was studied in the Stroke-Prone Spontaneously Hypertensive (SHRSP) rats fed diets containing either three levels of casein protein (10, 20 and 40%) or two sources of protein (casein and soybean protein isolate, SPI, each providing 40% protein). All diets contained 10% soybean oil and required levels of other nutrients. The diets and water (containing 1% NaCl) were provided ad libitum during the entire study. Animals were observed for the following symptoms leading to death such as laboured respiration (dyspnoea), development of edema, lethargy and low response to stimuli, as well as nasal and ocular secretions (red). If an animal was in pain or judged to be unable to live for 24 h, euthanasia was performed. Death or severe symptoms leading to euthanasia were attributed to stroke, confirmed by necropsies. Mean survival rates of SHRSP rats fed 10% casein, 20% casein, 40% casein and 40% SPI diets were 77.2 ± 5.5, 95.9 ± 9.3, 98.8 ± 20.9 and 140.0 ± 37.3 days, respectively. Death due to stroke was significantly (P <0.001) earlier in the animals fed 10% casein protein diet compared to those fed the other diets. The death rates of the SHRSP rats fed the 20 and 40% protein casein diets were not different but the survival rate of the animals fed the SPI diet (40% protein) was significantly (P <0.001) longer than of those fed the casein diets. Since the SHRSP rat is one of the most suitable models for stroke in humans, our data would suggest that dietary protein may also play a role in the incidence of stroke in humans.