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Markers within the regulatory region of the growth hormone receptor gene and their association with milk-related traits in Holsteins.

We studied sequence variations in the regulatory region of the bovine growth hormone receptor gene. A polymerase chain reaction (PCR)-based method for detecting AluI, AccI, and StuI restriction fragment length polymorphisms (RFLPs) in the 5' flanking region of the bovine growth hormone receptor gene was developed and tested for association with milk-related traits in Holstein bulls. Allele frequencies of the polymorphisms in two groups of Holstein progeny-tested bulls born from 1950 to 1970 and in the 1980s, respectively, were estimated. The allele frequency of the AluI(-) allele was 0.63 and 0.42 in the bulls from 1950 to 1970 and in the 1980s, respectively. The frequency of the StuI(-) allele was 0.14 and 0.07 in the two respective bull groups. Allele frequency for AccI(-) allele was about 0.22 in both bull groups. The differences in allele frequencies for the AluI polymorphism in the two bull groups were significantly different (P < or = .005). The AluI(+/+) bulls had a higher estimated breeding value (EBV) for fat (P < or = .016) than AluI(-/-) bulls. The average effect of allele substitution for the AluI polymorphism was +/- 8 for fat EBV. The AluI polymorphism could be further evaluated for use in marker-assisted selection in dairy cattle.

Alleles↗

Efficacy of enzyme supplementation after surgery for chronic pancreatitis.

Although surgical procedures that improve pancreatic drainage alleviate abdominal pain in the vast majority of patients with chronic pancreatitis, postoperative absorption and nutritional status are less predictable. The present study was designed to determine the efficacy of pancreatic enzyme supplementation in maintaining postoperative digestion and nutrition in patients who had received the local resection-longitudinal pancreaticojejunostomy (LR-LPJ) procedure for chronic pancreatitis. We evaluated nutritional status and intestinal absorption in 11 patients who had undergone LR-LPJ. The efficacy of postoperative pancreatic enzyme supplementation was studied by measurements of intestinal absorption and nutritional status at baseline, after 4 weeks of individualized daily dosage of pancreatin (Creon), and after an additional 4 weeks of randomization to receive another 4 weeks of pancreatin or placebo. All patients demonstrated abnormal digestion of fat, protein, and total energy at baseline 3 weeks after surgery. Pancreatin supplementation significantly improved the coefficients of absorption of dietary fat and total energy over the next 4 weeks. Between 4 and 8 weeks, pancreatin significantly improved protein absorption and nitrogen balance, whereas placebo substitution worsened the absorption of dietary fat and total energy. Nutritional status was not significantly altered over the 8-week study period, although four patients receiving pancreatin gained more than 3.6 kg body weight. The data suggest that long-term postoperative pancreatic enzyme supplementation is both efficacious and necessary in chronic pancreatitis patients after LR-LPJ.

Adult↗

Body composition determined by six different methods in women bilaterally adrenalectomized for treatment of Cushing's disease.

In 14 women, aged 47.2 +/- 10.5 yr, bilaterally adrenalectomized for Cushing's disease 13.6 +/- 7.7 yr before, all receiving conventional doses of glucocorticoid and mineralocorticoid substitution, body composition was studied with regard to body fat, body fat distribution, fat-free mass, and bone mineral density. The data were compared with those of 14 healthy women of similar age, body mass index, and menopausal state. Five different body composition measurement methods were compared, i.e. body densitometry by underwater weighing (UWW), total body water measurement by deuterium dilution (D2O dilution), dual energy x-ray absorptiometry (DXA), bioelectrical impedance spectrometry (BIS), and skinfold measurements, using a four-component model (4C-model) as the reference method. In the patients the percent body fat was significantly higher than that in the controls as determined by all methods, whereas fat-free mass was significantly lower when measured with the 4C-model, UWW, D2O dilution, DXA, or BIS. Measured by DXA, the percent trunk fat, estimated as [fat mass of the trunk (g)/total fat mass (g)] x 100%, was significantly higher in the patients than in the controls (39.3 +/- 6.4% and 29.9 +/- 7.8%, respectively; P < 0.001). Measured by DXA, there was no difference in total bone mineral density between the groups. Differences between the 4C-model, UWW, D2O dilution, and DXA for determination of percent body fat were rather small. Skinfold measurements and BIS results, however, deviated substantially from those obtained using the 4C-model. The study indicates that adrenalectomized patients receiving conventional glucocorticoid substitution have increased body fat percentages with a centripetal fat distribution and lowered fat-free mass, but normal bone mineral density. Furthermore, the study indicates that for clinical practice, DXA, D2O dilution, UWW, and the 4C-model can be used for determination of body composition in this patient group, whereas the more convenient bedside methods, BIS and skinfold measurements, did not give reliable results. We suppose that glucocorticoid overreplacement in adrenalectomized patients might be the cause of their abnormal body composition, although GH deficiency after pituitary surgery could have played a contributory role in some of the patients.

Absorptiometry, Photon↗

Similar serum lipoprotein cholesterol concentrations in healthy subjects on diets enriched with rapeseed and with sunflower oil.

A double-blind cross-over study was conducted during two 3-week periods to compare the effects of rapeseed oil and sunflower oil, enriching a normal diet, on the lipoprotein and fatty acid composition in healthy subjects. It was carried out in randomized order at residential schools, comprising 101 persons (mean age 29.2 years). The dietary fats used for cooking and as table margarine were prepared from rapeseed oil during one period and from sunflower oil during the other. No changes were made in the total fat content or other dietary nutrients. During both treatment periods the serum cholesterol (-4%, P < 0.001), LDL cholesterol (-5% to -7%, P < 0.01 and 0.001) and apolipoprotein B (-5%, P < 0.001) concentrations decreased significantly and to the same extent, while serum triglycerides, HDL cholesterol, apolipoprotein A-1 and lipoprotein (a) remained virtually unchanged. The content of 18:2 n-6 serum phospholipids was increased after the sunflower oil-enriched diet, and the contents of oleic acid (18:1 n-9), alpha-linolenic acid (18:3 n-3), and eicosapentaenoic acid (20:5 n-3) were increased after the rapeseed oil-enriched diet. The concentration of alpha-tocopherol increased and gamma-tocopherol decreased after the sunflower oil-enriched diet, less so after the rapeseed oil-enriched diet. It is concluded that substitution of mono- and polyunsaturated fats for saturated fats without any other dietary changes causes a significant improvement of the lipoprotein profile in healthy subjects. The rapeseed oil and sunflower oil fats were equally effective in this respect. The results also indicate that humans have a certain capacity to elongate and desaturate alpha-linolenic acid to 20:5 n-3 in vivo. Dietary fats based on rapeseed oil seem to be attractive alternatives to the more commonly used oils and fats rich in linoleic acid. Financial support from the Swedish Council for Forestry and Agricultural Research and the Swedish Margarine Industrial Association for Nutritional Physiological Research is gratefully acknowledged.

Adult↗

Effects of dietary fat saturation on plasma lipoprotein(a) and hepatic apolipoprotein(a) mRNA concentrations in cynomolgus monkeys.

Plasma lipoprotein(a) (Lp(a)) concentration is an independent risk factor for the development of premature coronary artery disease. Although the majority of available data indicates that circulating Lp(a) levels are under strict genetic regulation, there is some evidence that this parameter may be subject to dietary modification as well. The effects of dietary fat saturation on plasma Lp(a) levels and hepatic apolipoprotein(a) (apo(a)) mRNA abundance were examined in ten unrelated cynomolgus monkeys which were fed each of three experimental diets enriched in saturated (SAT), monounsaturated (MONO), or polyunsaturated (POLY) fatty acids in a crossover design consisting of three 13-week periods. Each diet contained 30% of calories as fat with 0.1% dietary cholesterol by weight and differed solely by the isocaloric substitution of fatty acids as 60% of total fat calories. The mean Lp(a) level for these animals during the SAT diet (13.4 +/- 2.4 mg/dl) was significantly greater as compared with those observed during the MONO (8.6 +/- 2.2 mg/dl, P < 0.0003) and POLY (9.3 +/- 2.1 mg/dl, P < 0.002) diets, while the difference noted between the MONO and POLY diets was nonsignificant. Hepatic apo(a) mRNA abundance was decreased in these animals during the MONO diet relative to both the SAT and POLY diets, with only the difference between the SAT and MONO diets achieving statistical significance (P < 0.02). Our data demonstrate that the substitution of dietary SATs with either MONOs or POLYs result in significant reductions of Lp(a) levels in these monkeys. However, only the MONO diet significantly decreased hepatic apo(a) mRNA levels relative to the SAT diet, suggesting that dietary MONOs and POLYs may differ in the manner by which they regulate plasma Lp(a) levels.

Animals↗

[Liberalized diabetic diet for type I diabetics under intensive insulin injection therapy. Follow-up observations for over 2 years].

The dietary habits and metabolic status of 14 selected ambulant type-1 diabetics, who were previously treated with conventional insulin therapy, were investigated over a period of 2 years under intensified insulin injection therapy. In this, multiple doses of regular insulin in combination with intermediate-acting insulin were injected daily and the regular metabolic controls as well as adjustment of the insulin dosage were undertaken by the patients themselves. Whereas conventional insulin therapy made exclusive use of intermediate-acting insulin, 41% of the daily dose in the intensified insulin injection therapy was regular insulin. The total daily insulin dose of 51 +/- 12 U/d was therefore not significantly different from that in the conventional insulin therapy (49 +/- 10 U/d). During the intensified insulin injection therapy the usual diabetic diet was relaxed in several aspects: neither the daily intake of fat, protein and calories, nor substitution in the diet using exchange lists was specified. Despite this liberalization HbA1c values decreased significantly from 9.3 +/- 1.2% to 7.8 +/- 0.7% (P less than 0.05) and blood lipids remained in the normal range. The results of this retrospective investigation lead to the conclusion that despite some measure of diet liberalization, selected, trained, type-1 diabetics of normal body weight and without primary dyslipoproteinaemia can attain good metabolic control under intensified insulin injection therapy.

Activities of Daily Living↗

Effect of dietary lipid on benzo(a)pyrene metabolism by perfused rat liver.

Male Holtzman albino rats weighing 60-75 gm were fed purified synthetic diets containing casein (18%), alphacel (4%), wheat starch (44%), sucrose (26%), Jones Foster salt mix (4%), and vitamin mix (4%) for three weeks. The diet for group I contained no fat, whereas corn oil (10%) substituted for 10% sucrose in the diet for group II. All animals were administered 3-methylcholanthrene [20 mg/kg intraperitoneally (ip) in 0.5% methylcellulose solution] for two successive days prior to sacrifice in order to increase the activity of the mixed function oxidase (MFO) responsible for metabolizing benzo(a)pyrene [B(a)P]. After 21 days on diet, livers were perfused with benzo(a)pyrene in blood-free Krebs Henseleit bicarbonate buffer containing 100 mg glucose plus 200 units of heparin per 100 ml. Aliquots of perfusate were removed at five, ten, 20, 30, 40, and 60 minutes for analysis of B(a)P metabolites. Higher concentrations of B(a)P-9, 10-dihydrodiol; B(a)P-4,5-dihydrodiol; B(a)P-7,8-dihydrodiol; and 3-hydroxy B(a)P were recovered from livers of rats fed the diet containing 10% corn oil than from livers of rats fed the fat-free diet. The concentrations of 9-hydroxy B(a)P except the 9,10-dihydrodiol were recovered from bile along with two unidentified metabolites. Following the perfusion period, homogenized and extracted livers from rats fed the diet containing corn oil retained significantly higher concentration of the three dihydrodiols and two phenols than livers from rats fed the fat-free diet.

Animals↗

Growth hormone (GH) substitution for one year normalizes elevated GH-binding protein levels in GH-deficient adults secondary to a reduction in body fat. A placebo-controlled trial.

The high affinity growth hormone binding protein (GHBP) in human serum derives from the extracellular domain of the GH receptor. It is well known that fat mass correlates positively to GHBP levels, but it is uncertain whether GH secretory status influences GHBP levels. Since body composition is known to change during GH substitution in adult GHD patients, we determined the relation between GHBP and body composition during GH substitution in GHD adults. Twenty-five GHD adults aged 45.0 +/- 1.8 years, were examined before and after 12 months of placebo-controlled GH substitution (2 IU/m2) in a parallel design. A group of 27 healthy age- and gender-matched normal-weight adults provided reference data. The participants underwent anthropometric measurements [body mass index (BMI), waist/hip ratio (W/H)], computer-tomography (CT-scan) of femoral and abdominal regions, dual-energy X-ray absorptiometry (DEXA-scan), and bioimpedance (BIA), as well as blood sampling. At baseline, the GHBP levels were increased compared to controls (1.63 +/- 0.14 nmol/l vs 1.12 +/- 0.1 nmol/l, P = 0.01). During 12 months of GH substitution, GHBP levels decreased to the levels of the control subjects. GHBP correlated positively to indices of adiposity in GHD patients at baseline: intra-abdominal fat (r = 0.54, P = 0.005), subcutaneous abdominal fat (r = 0.59, P < 0.002), body fat (BIA) (r= 0.41, P= 0.044), BMI (r= 0.58, P = 0.002), and total body fat (DEXA scan) (r= 0.61, P < 0.001). After 12 months of GH substitution, different estimates of body fat were significantly decreased in the GH treated group, but the positive relationship between GHBP and these estimates of body fat was maintained. In multiple linear regression analyses, fasting insulin levels were also a significant determinant of GHBP levels. We conclude that GHBP levels are increased in GHD patients and decrease to normal levels during 12 months of GH substitution. Furthermore, GHBP is predominantly correlated to indices of adiposity also in GHD patients.

Adipose Tissue↗

Influence of dietary fish on fatty acid composition of the erythrocyte membrane in coronary heart disease patients.

This study examines the effects of dietary fish oil on the fatty acid composition of the erythrocyte membrane in persons with coronary heart disease. Thirty men with myocardial infarction were studied during three consecutive periods, during which they consumed 1) their habitual diet, 2) a diet specially designed to prevent ischaemic cardiopathy, and 3) a diet rich in polyunsaturated fat, in which fish was substituted for meat. At the end of each diet, fatty acid concentrations in the erythrocyte membrane were measured, and a 48 h recall dietary questionnaire was used to determine the types of foods consumed. Despite the increase in the intake of (n-3) polyunsaturated fatty acids (PUFA), the fatty acid composition of the erythrocyte membrane failed to reflect the greater dietary supply of eicosapentaenoic acid, and showed increased concentrations of stearic (C16) and palmitic (C18) acid. The increase in dietary (n-3) polyunsaturated fatty acids markedly increased 22:4 (n-6), 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) fatty acids.

Adult↗

Vitamin E in foods from high and low linoleic acid diets.

As part of a human diet study, vitamin E activity was estimated in foods used in seven daily menus. Each menu was designed to contain 35% fat calories with either 10 or 30 gm/day of linoleic acid (18:2) and 500 mg/day of cholesterol. To estimate vitamin E activity, each food used in the menus was analyzed for alpha and gamma tocopherol content by high-pressure liquid chromatography with fluorescence detection. This article reports the alpha and gamma tocopherol contents of those foods, tocopherol contributions from each food in one sample 2,400-kcal menu, and the mean daily vitamin E activity (milligram alpha tocopherol equivalents) of all seven menus at five caloric levels. Major sources of alpha tocopherol (greater than 10% of the RDA) common to both diets (10 and 30 gm linoleic acid) were olive oil and a few fruits and vegetables. Additional major sources in the 30-gm linoleic acid diets were polyunsaturated fatty acid (PUFA) vegetable oils and margarine. Contrary to a common assumption, increasing the level of PUFA in the menus did not necessarily result in higher milligram equivalents of alpha tocopherol because soybean oil, with a tocopherol composition that is predominantly gamma tocopherol, was the major source of linoleic acid in the diets. Thus, vitamin E activity was not necessarily increased when soybean oil was substituted for a less saturated fat such as olive oil, which has mostly alpha tocopherol.

Chromatography, High Pressure Liquid↗

[Digestion of fat and protein and its relation to the activity of faecal enzymes in mucoviscidosis infants under substitution therapy with pancreatic enzymes (author's transl)].

The article reports on a study to determine the influence of different dosage schedules of a pancreas enzyme preparation on the digestion of fat and protein and on the lipolytic, tryptic and chymotryptic activity of the faeces in children suffering from mucoviscidose. After administration of the pancreatin preparation at the low dose level, the quantity of faeces, the excretion of fatty acids and of nitrogen are reduced and the absorption coefficient is improved. The faecal tryptic and chymotryptic activity increases, whereas the lipase activity in the faeces merely shows a tendency to rise. Increasing the pancreatin dose up to a still tolerable value by doubling the dosage does not result in further reduction of the excretion of fat and nitrogen although it enhances the proteolytic activity of the faeces while the lipase activity remains unaffected. Determination of the lipolytic, tryptic or chymotryptic activity is, therefore, unsuitable for assessing the fat and protein digestion and for estimating the required dose of pancreatic enzyme. No increase in uric acid excretion was seen in the urine collected for 24 hours after administration of both enzyme doses.

Adolescent↗

Influence of carbohydrate and fat intake on diet-induced thermogenesis and brown fat activity in rats fed low protein diets.

Voluntary intake of protein, fat and carbohydrate (CHO) was modified by feeding young rats either a control purified diet [% metabolizable energy (ME): protein 21, fat 7, CHO 72], a control diet plus sucrose solution (20%) to drink (final intakes 17, 6 and 77% ME as protein, fat and CHO, respectively) or a low protein diet substituted with either CHO (8, 7 and 85% ME as protein, fat and CHO, respectively) or fat (8, 20 and 72% ME as protein, fat and CHO, respectively). Total ME intakes corrected for body size were similar for all rats, but body weight, energy gain and net energetic efficiency were lower in both low protein-fed groups than in the control group. The acute thermogenic response (% rise in oxygen consumption) to a standard balanced-nutrient meal was higher (12%) in sucrose-supplemented and in low protein groups (15-16%) than in control rats (8%). Brown adipose tissue protein content and thermogenic capacity (assessed from purine nucleotide binding to isolated mitochondria) were greater than control values in sucrose-fed and protein-deficient animals, and the greatest levels of activity were seen in low protein-fed rats with a high fat intake. The results demonstrate that the changes in energy balance, thermogenesis and brown adipose tissue activity that result from protein deficiency cannot be ascribed to changes in the level of energy intake or to a specific increase in the amount or proportion of either CHO or fat. They suggest that the protein-to-energy ratio must be the primary influence on thermogenesis and brown fat activity in these animals.

Adipose Tissue, Brown↗

Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women.

BACKGROUND: The rates of colon cancer in various countries are strongly correlated with the per capita consumption of red meat and animal fat and, to a lesser degree, inversely associated with the consumption of fiber. METHODS: We conducted a prospective study among 88,751 women 34 to 59 years old and without a history of cancer, inflammatory bowel disease, or familial polyposis who completed a dietary questionnaire in 1980. By 1986, during 512,488 person-years of follow-up, 150 incident cases of colon cancer had been documented. RESULTS: After adjustment for total energy intake, animal fat was positively associated with the risk of colon cancer (P for trend = 0.01); the relative risk for the highest as compared with the lowest quintile was 1.89 (95 percent confidence interval, 1.13 to 3.15). No association was found for vegetable fat. The relative risk of colon cancer in women who ate beef, pork, or lamb as a main dish every day was 2.49 (95 percent confidence interval, 1.24 to 5.03), as compared with those reporting consumption less than once a month. Processed meats and liver were also significantly associated with increased risk, whereas fish and chicken without skin were related to decreased risk. The ratio of the intake of red meat to the intake of chicken and fish was particularly strongly associated with an increased incidence of colon cancer (P for trend = 0.0005); the relative risk for women in the highest quintile of this ratio as compared with those in the lowest quintile was 2.49 (95 percent confidence interval, 1.50 to 4.13). A low intake of fiber from fruits appeared to contribute to the risk of colon cancer, but this relation was not statistically independent of meat intake. CONCLUSIONS: These prospective data provide evidence for the hypothesis that a high intake of animal fat increases the risk of colon cancer, and they support existing recommendations to substitute fish and chicken for meats high in fat.

Adult↗

Determination of optimum fat intake.

In a 14-day experiment, adult rats weighting 250-260 g were given ad libitum diets with a constant protein content [casein - 10 %] and a mounting fat content [margarine - 0, 10, 20, 30 and 40 % - added at the expense of carbohydrates]. The growth parameters of protein biological value - PER and NPR - were determined and together with weight increments in correlation to protein an fat intake [a linear correlation] they were used on the basis of their regression equations to calculate the reciprocal relationships of fat and protein intake [for equality y]. By substituting the optimum protein intake [43.3 g/14 days - according to NPU], the optimum fat intake for adult rats was found to correspond to dietary fat concentrations of 9.72 % [y = weight increments], 12,82 % [y = PER] and 11.83 % [y = NPR]. These results wee verified in a biological experiment in which adult animals, for 14 days, were given a limited amount [31 g/animal per day] of diets containing 10 % protein [casein] an d9, 10, 11, 12, and 13 % fat [margarine - at the expense of carbohydrates] with the aim of studying the effect of a mounting fat intake on optimum protein intake [43.4 g/14 days]. The correctness of the mathematical method for determining the optimum fat intake was verified by a biological method [the optimum net protein utilization , NPU, which is highest in diets containing 11 % fats, i.e. 22.5 % of the total energy value of the diet]. It was also found that this method, both along and in conjunction with other methods [especially the one using the NPR parameter], was suitable for the determination of optimum nutrient values.

Animals↗

Dietary sources of fats and cholesterol in US children aged 2 through 5 years.

OBJECTIVES: This study of lipid intakes among preschool children (1) analyzed the contributions of 38 food groups to fat, saturated fat, and cholesterol intakes; (2) estimated the effects of food substitutions on intakes; and (3) examined demographic differences in food group intake and food group sources of these lipids. METHODS: The sample consisted of 547 children, aged 2 to 5 years, from the US Department of Agriculture's 1985 and 1986 Continuing Surveys of Food Intakes by Individuals. Dietary information for 4 nonconsecutive days throughout a year was used. All foods were classified into groups and the lipids contributed from each group were computed. RESULTS: Over 80% of the children consumed more total fat, saturated fats, and cholesterol than is recommended. The major source of total fat and saturated fats was whole milk; the major sources of dietary cholesterol were eggs and whole milk. Children's food consumption patterns differed by region of the country and race/ethnicity, providing opportunities to refine nutrition education interventions and evaluations. CONCLUSIONS: By substituting lower-fat foods for the major sources of saturated fats, significant reductions in preschool children's intakes of saturated fats, fat, and dietary cholesterol could be achieved.

Child Nutritional Physiological Phenomena↗

[Medium chain triglycerides in the feeding of praemature infants. Fat balance studies (author's transl)].

18 72-H Fat balance studies were performed in 9 premature infants with birthweights between 1650 and 2400 gm. At the age of two and three weeks, they received alternatively, one of two formulas identical in composition except for the type of fat: 30% of the vegetable fat in the first formula had been substituted by medium chain triglycerides (MCT) in the second formula. On the MCT-containing formula, the fat absorption significantly improved in the same infants as compared to their fat absorption capacity on the vegetable fat formula. The absorption of individual fatty acids, calculated by gaschromatographic analysis of pooled stool samples, also improved for most of the fatty acids with the MCT-formula. There were no adverse changes in blood glucose concentrations or in acidbase status of the infants when being fed the MCT-containing formula.

Acid-Base Equilibrium↗

Trans fatty acids and cardiovascular risk.

Trans fatty acids are found in partially hydrogenated vegetable oil, in meats, and in dairy products. Their effect on blood cholesterol concentrations was examined decades ago, but recently there has been renewed interest in understanding how trans fatty acids affect blood lipids and lipoprotein cholesterol concentrations. Current advice to reduce cardiovascular disease (CVD) risk includes decreasing the consumption of saturated and total fat to help manage blood cholesterol concentrations. Saturated fat contributes significantly to total fat intake and markedly raises blood cholesterol concentrations. Trans fatty acids, which are consumed in much smaller quantities, have been shown to be modestly hypercholesterolemic in studies that have substituted hydrogenated vegetable oils for unhydrogenated oils. In contrast, when partially hydrogenated vegetable oils containing trans fatty acids are substituted for cholesterol-raising saturated fats, blood cholesterol levels are reduced. Partially hydrogenated vegetable oils are used in place of saturated fat in many food products. These foods can help consumers lower their saturated fat intake to achieve dietary recommendations. The following review critically examines the role of hydrogenated fats in the food supply, the metabolism of trans fatty acids, and the scientific literature surrounding the effects of partially hydrogenated vegetable oils and trans fatty acids on blood cholesterol concentrations and cardiovascular disease risk.

Cardiovascular Diseases↗