Lipids Flashcards

1
Q

Lipids in the Body

A

Lipids exist in the body in various forms with each form having a different structure and function. Lipids include:

  • Individual fatty acids.
  • Triglycerides.
  • Phospholipids in every cell membrane.
  • Cholesterol and steroid based compounds ( e.g. oestrogen).
  • Sphingolipids found in nerve cell membranes, e.g. myelin.
  • Glycolipids involved in cell identity (like a cell ‘passport’).
  • Cerebrosides glycosphingolipids found in the brain.
  • Fat soluble vitamins A, D, E, K.
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2
Q

Phospholipid

A

phosphate + fatty acids

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3
Q

Glycolipid

A

carbohydate + lipid

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4
Q

Cerebroside

A

waxy lipid + sugar

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5
Q

Sphingolipid

A

long chain amino alcohol + fatty acid + sugar

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6
Q

Function of Lipids

A
  • Energy (ATP) production - each gram of fat supplies the body with about nine calories.
  • Storage of energy reserves - fats are a more efficient form of storage energy than carbohydrates or proteins, so the body stores any excess energy as fat.
  • Cell membrane structure - phospholipids and cholesterol stabilise cell membranes, whilst allowing a degree of fluidity which is crucial to the function of every cell.
  • Thermal insulation in subcutaneous tissue and protection around organs.
  • Steroid hormones - progestogens, androgens, glucocorticoids, mineralocorticoids and oestrogens are derived from cholesterol.
  • Formation of eicosanoids - signalling molecules involved in a range of processes such as blood coagulation and inflammation.
  • Growth and development - the brain is rich in arachidonic acid (AA) and docosahexaenoic acid (DHA).
  • Constituents of nervous tissue structure (sphingomyelin).
  • Aid to cell signalling processes.
  • Required for the absorption of fat soluble vitamins.
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7
Q

Fatty Acids

A

Fatty acids are hydrocarbon chains with an acid group at one end and a methyl group at the other.

  • Short chain fatty acids (up to 5 Cs) and medium chain fatty acids (6-12 Cs) travel directly to the liver where they can be used to create energy or ketones. Medium chain triglycerides (MCTs) can be used as a source of energy before exercise (e.g. 1 tbsp).
  • Long chain fatty acids (14-22 Cs) and very long chain fatty acids (> 22 Cs) are used to build cell membranes.
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8
Q

Short
Chain Fatty Acids

A

Short chain fatty acids (SCFAs) have fewer than six carbon atoms.
• SCFAs are produced when dietary fibre is fermented in the colon.
• Acetate, propionate and butyrate are the most common SCFAs.
• Butyrate is particularly important for colon health because it is the primary energy source for colonocytes. It supports the intestinal tight junctions .
• SCFAs are speculated to have a role in the microbiota gut brain axis crosstalk.
• Butyrate is thought to have an anti-inflammatory effect on the colon.

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9
Q

Hydrogenation

A
  • Unsaturated fats can be saturated by the addition of hydrogen as in hydrogenation when oils are made into solid spreads.
  • Hydrogenation turns the natural fatty acid into unnatural forms (i.e. trans fats ) which are damaging to health
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10
Q

Saturated fatty acids

A

Contain no C-C double bonds. All the carbons are completely saturated with hydrogen bonds. Solid at room temperature.

Saturated fat intake has been a fiercely debated topic.

  • A lot of studies about high saturated fat diets and health, have reported on dietary intake of saturated fats from junk foods.
  • A recent review by the Journal of the American College of Cardiology found that there was inadequate scientific evidence to keep advising against foods high in saturated fats, including coconut, unprocessed meat, eggs and dark chocolate.
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11
Q

Types of Saturated Fats

A

Butyric acid 4-C Caprylic acid 8-C Lauric acid 12-C Palmitic acid 16-C Stearic acid 18-C

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12
Q

Butyric acid 4-C

A

Butter, Dairy Produced in the gut

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13
Q

Caprylic acid 8-C

A

Coconut

Palm Kernel

Breast milk

Anti-fungal properties

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14
Q

Coconut oil

A

Coconut oil contains medium chain triglycerides (MCTs) which the body uses as a source of fuel or turns them into ketones. • MCTs increase the number of calories burned compared to longer chain fatty acids. • Coconut oil contains 50% lauric acid. Monolaurin is formed from lauric acid. Both substances have antibacterial , antiviral and antifungal properties. • ↑ HDL cholesterol, ↓ LDL • Preliminary studies show positive outcomes in epilepsy and Alzheimer’s disease.

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15
Q

Lauric acid 12-C

A

Coconut

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16
Q

Palmitic acid 16-C

A

Coconut Palm Palm kernel

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17
Q

Stearic acid 18-C

A

Beef, Pork, Lamb, Mutton Cocoa and shea butter Butter

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18
Q

Unsaturated fatty acids

A

Contain one or more double bonds between carbons. Liquid at room temperature.

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19
Q

Monounsaturated fatty acids

A

Have one double bond in the chain.

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20
Q

Monounsaturated Fats

A

Palmitoleic acid Omega - 7

Oleic acid Omega - 9

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21
Q

Polyunsaturated fatty acids

A

Have several double bonds.

The more double bonds there are in a fatty acid, the less stable it is, increasing susceptibility to oxidation.

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22
Q

Omega System

A
  • The omega system uses the number of carbon atoms, the number of double bonds, and the number of carbons from the omega end to the first carbon in the double bond.
  • The omega 6 fatty acid, arachidonic acid , is referred to as 20:4 w6.

20 = no. of carbons

4= no. of double bonds

6= no. of carbons to first double bond

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23
Q

Polyunsaturated Fats Omega 3

A

Alpha linolenic acid (ALA) Stearidonic acid (SDA) EPA and DHA

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24
Q

Palmitoleic acid Omega - 7

A

Sea buckthorn berries Coconut Coconut, palm kernel Macadamia nut

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25
Q

Oleic acid Omega - 9

A

Olive, avocado Almond, peanut, pistachio Brazil nuts, pecan, cashew Hazelnuts, neem, macadamia Animal fat, butter

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26
Q

Alpha linolenic acid (ALA)

A

Flaxseeds (richest source 50% of its fatty acids are ALA) Chia seeds, hemp seeds, dark green leaves Pumpkin seeds, soybean, rapeseed (canola) Walnuts, wheat germ

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27
Q

Stearidonic acid (SDA)

A

Blackcurrant seeds

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28
Q

EPA and DHA

A

Cold water fish oil Salmon, trout, tuna, anchovies, mackerel Sardines, herring Spirulina, chlorella

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29
Q

Polyunsaturated Fats Omega-6

A

Linoleic acid (LA) Gamma linolenic acid (GLA) Arachidonic acid (AA)

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30
Q

Linoleic acid (LA)

A

Safflower Sunflower, hemp, soybean, walnut Pumpkin seed, sesame, almond, chia, cashew Rapeseed, wheat germ, avocado, Brazil nut Borage oil Evening primrose and hemp oil Blackcurrant seed oil Meat Other animal products

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31
Q

Gamma linolenic acid (GLA)

A

Borage oil Evening primrose and hemp oil Blackcurrant seed oil

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32
Q

Arachidonic acid (AA)

A

Meat Other animal products

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33
Q

Unnatural Trans Fats

A

Unnatural trans fatty acids are produced by high temperatures and hydrogenation.
• They are found in margarine, processed foods and refined vegetable oils.
• Trans fats stiffen cell membranes, making them prone to oxidation. This also alters their protective action and permeability, impeding normal cell function.
• Trans fats alter blood triglyceride and cholesterol profiles and are linked to an increased risk of cardiovascular disease, insulin resistance and cancer.

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34
Q

Cis and Trans Fatty Acids

A

At each double bond, two possible isomeric forms exist.
– Cis configuration = the H atoms are on the same side of the double bond. The majority of natural fats are cis.
– Trans configuration = the H atoms are on separate sides of the double bond.
• Conjugated linoleic acid (CLA) is a natural trans fat found in grass fed meat and dairy products. Studies
indicate CLA helps increase lean muscle mass and decrease body fat.

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35
Q

Essential Fatty Acids (EFAs)

A

There are two fatty acids that cannot be made in the body and so are essential in the diet. They are: 1. Linoleic acid (an omega-6 fatty acid). 2. Alpha linolenic acid (an omega 3 fatty acid). • Arachidonic acid was once thought to be essential in the diet, but we now know it can be made from linoleic acid. • Humans lost the ability to introduce double bonds into fatty acids between the carbon atoms 6‒7 and 3‒4, making LA and ALA essential in the diet.

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36
Q

Essential Fatty Acids Conversion

A

ALA and LA have to be obtained from foods so are ‘essential’ From ALA (omega 3) and LA (omega 6), the next in the sequence is manufactured in the body from the preceding fatty acid in the chain, with the help of special enzymes. The most important enzyme that catalyses the chemical reaction to produce GLA and EPA is Delta-6 desaturase

37
Q

Omega-3 Pathways

A
38
Q

Omega 6 Pathway

A
39
Q

EFAs: Western Diet

A

A typical western diet is abundant in omega-6 fatty acids (plant oils, grain fed meat and dairy), and low in omega 3 fatty acids from ALA sources (flaxseeds, pumpkin seeds) and EPA / DHA sources (oily fish). • Human beings evolved on a diet with an omega 6:omega 3 of 1. • In Western diets the ratio is generally around 16:1. • The relatively low rate of conversion of ALA to EPA / DHA suggests that EPA and DHA are conditionally essential nutrients. • To achieve the EFSA recommended intake of 250 mg EPA / DHA, consume 2 3 portions of oily fish per week or from an algal source.

40
Q

EFAs: Functions

A

• EFAs are vital components of all cell membranes and help to maintain membrane fluidity . The fluidity of the membrane must be maintained within a certain range for the cell to function properly. • They act with cell membrane proteins thereby affecting the transport of substances into and out of the cell. • EFAs are key components of organelle membranes such as those of the mitochondria. • EFAs are necessary for cell to cell communication. • They are essential for foetal and child brain development • EFAs are precursors of eicosanoids , which are ‘local’

41
Q

EFAs: Roles

A

Skin Endocrine system Reproductive system Circulatory Musculoskeletal Immune Neurological

42
Q

Alpha Linolenic Acid (ALA)

A

ALA is an omega 3 fatty acid, 18:3 n 3. • Food sources include flaxseeds, hempseeds, soybeans, and walnuts. It is also found in dark green leaves • Many edible plants produce this 18 carbon polyunsaturated fatty acid.

43
Q

Alpha Linolenic Acid (ALA): Therapeutic Uses

A

Cardiovascular disease (CVD) Neurological Anti-inflammatory

44
Q

Alpha Linolenic Acid (ALA): Drug Interactions

A

• Blood thinning medications: – Omega 3 fatty acids may increase the anti-coagulant effects of blood thinning medications, e.g. warfarin and aspirin. – While the combination of aspirin and omega 3 fatty acids may actually be helpful, under certain circumstances (such as CVD), these should only be taken together under GP supervision. • Cholesterol lowering medications (i.e. statins): – May have an agonist effect when combined with statins.

45
Q

EPA and DHA

A

• Formed from alpha linolenic acid (ALA). • Eicosapentaenoic acid (EPA) is an omega 3 fatty acid, 20:5 n 3. • Docosahexaenoic acid (DHA) is an omega 3 fatty acid, 22:6 n 3. • Main food sources include oily fish and human breast milk. Marine algae are a rich source of DHA.

46
Q

EPA and DHA: Therapeutic Uses

A

Cardiovascular Disease Anti-inflammatory Neurological Health Foetal Health

47
Q

EPA and DHA: Foetal Health

A

Functions: • Support foetal brain development (language, visual, motor functions) • There is evidence that mothers who supplement EPA and DHA during pregnancy and breastfeeding may protect their children against allergies. Therapeutic Uses • Pregnancy support (for foetal health) Dose: EPA 800mg, DHA 400mg/day.

48
Q

EPA and DHA for Vegetarians and Vegans

A

A vegetarian or vegan diet can meet EPA / DHA needs: • Include good sources of alpha linolenic acid in the daily diet, such as flaxseed and hempseed • Support EFA conversion through increasing dietary intake of enzyme co-factors (zinc, magnesium and B6) • Moderate the use of oils rich in omega-6 fatty acids, and avoid processed foods rich in these oils. • Consider algal EPA / DHA supplements.

49
Q

EPA and DHA: Factors that determine Omega Fish oil quality:

A

Factors that determine Omega Fish oil quality: • It is important to assess the sustainability practices of a company when selecting a fish oil. Check if it is made from sustainably caught fish approved by the Marine Stewardship Council, where bycatch of non targeted species is minimised. • Check that the oil is independently tested for purity and toxins, This will ensure minimal levels of toxic chemicals such as dioxins, PCB’s and heavy metals. • When selecting a fish oil, make sure that the EPA and DHA content is listed on the label.

50
Q

EPA and DHA: Factors that determine vegan omega oil quality

A

• Extracted from microalgae of the schizochytrium species using water extraction methods (instead of hexane, alcohol and other solvents), to provide DHA. • Extracted from echium seed oil which contains stearidonic acid (SDA) which is easily converted to EPA and DHA. • Free from carrageenan which may induce inflammation in colonic cells. • Cold pressed, organic.

51
Q

EPA / DHA drug interactions

A

• Anticoagulants EPA may increase bleeding time, so fish oil could make the effects of these drugs stronger. • Aspirin in combination with aspirin, fish oil could be helpful in the treatment of some forms of coronary artery disease. However, this combination may also increase the risk of bleeding. • Diabetes medications fish oil supplements may lower blood glucose levels and could make effects of diabetes drugs stronger. • Blood pressure medication DHA may lower blood pressure (so monitor).

52
Q

Linoleic Acid (LA)

A

LA is an omega-6 fatty acid, 18:2 n 6. • Food sources include vegetable oils safflower, sunflower, soybean, and corn oils . It is found in nuts, seeds and some vegetables • Conversion of LA to GLA requires vitamin C, B3, B6, magnesium and zinc.

53
Q

Gamma Linolenic Acid (GLA)

A

GLA is an omega-6 fatty acid, 18:3 n 6. • Main food sources include evening primrose oil, blackcurrant seed oil, hemp and borage oils

54
Q

Gamma Linolenic Acid (GLA): Therapeutic Uses

A

Rheumatoid arthritis ADHD Eczema

55
Q

Evening Primrose Oil (EPO)

A

EPO is abundant in LA, and contains GLA which is also present in borage, blackcurrant seed and hemp seed.

56
Q

Evening Primrose Oil (EPO): Therapeutic Uses

A

Premenstrual syndrome (PMS) Cyclical mastalgia (breast pain) Female fertility •GLA is a precursor to PG1, which inhibits prolactin (↑ in women with •Dosage: 1500 mg daily for three months. •GLA forms PG1 which inhibits the synthesis of arachidonic acid metabolites (= anti inflammatory •Dosage: 1000 mg 3 x daily for four to six months.

57
Q

Evening Primrose Oil (EPO): Cyclical mastalgia (breast pain)

A

• GLA is a precursor to PG1, which inhibits prolactin (↑ in women with • Dosage: 1500 mg daily for three months. • GLA forms PG1 which inhibits the synthesis of arachidonic acid metabolites (= anti inflammatory • Dosage: 1000 mg 3 x daily for four to six months.

58
Q

Evening Primrose Oil (EPO): Female fertility

A

• GLA forms PG1 which inhibits the synthesis of arachidonic acid metabolites (= anti inflammatory • Dosage: 1000 mg 3 x daily for four to six months.

59
Q

Gamma Linolenic Acid (GLA): Drug interactions

A

Borage seed oil, and possibly other sources of GLA, should not be used during pregnancy. • Dosages of greater than 3,000 mg / day may increase AA production. Drug interactions: • Ceftazidime it may increase the effectiveness of this antibiotic. • Chemotherapy it may increase treatment effects. • Cyclosporine it may increase the immunosuppressive effects. • NSAIDs NSAIDs may counteract the effects of GLA. • Phenothiazines they may increase the risk of seizures.

60
Q

Arachidonic Acid (AA)

A

Arachidonic acid (AA) is an omega-6 fatty acid, 20:4 n-6. • Arachidonic acid is primarily found in animal products such as meat, eggs and dairy, especially when those animals are intensively raised on grain. • Dihomo gamma linolenic acid (DGLA) can be converted to AA using delta 5 desaturase. However, this enzyme is used preferentially for the omega-3 pathway, so the majority of AA in the diet is from animal products.

61
Q

Arachidonic Acid (AA): Inflammation

A

Arachidonic acid is often seen as inflammatory, but: • Inflammation is a key part of the immune system’s response to injury and infection. • AA is metabolised by COX-1 and COX-2 enzymes to the inflammatory prostaglandin series 2. • This causes inflammatory effects including fever, vascular permeability and vasodilation, pain and oedema. • However, to prevent excessive inflammation PG2 induces 15-LOX activity that leads to the formation of lipoxins (anti inflammatory).

62
Q

Eicosanoids

A

Eicosanoids are made by the oxidation of omega-3 and 6 fats. They are locally acting hormone like signalling molecules. • They have a short life span and are involved in: – Inflammation. – Blood vessel permeability and constriction. – Blood coagulation. – Immune cell behaviour. – Lipid accumulation. – Central nervous system signalling • Eicosanoids include prostaglandins, leukotrienes, thromboxanes, resolvins and protectins

63
Q

Eicosanoids: Formation

A

Fatty acids are released from the membrane phospholipids by the enzyme phospholipase A2. • These are converted to eicosanoids by cyclooxygenase (COX) and lipoxygenase (LOX) this is dependent on the starting fatty acid and an outside stimulus. • Eicosanoids can be made from arachidonic acid (AA), eicosapentaenoic acid (EPA) and dihomo y linolenic acid (DGLA). • They can have both pro and anti inflammatory effects.

64
Q

Eicosanoids: Prostoglandins

A

Prostaglandins fall into three families or series, depending on which fatty acid they are made from: Series 1 Prostaglandins (PG1) made from DGLA Series 2 Prostaglandins (PG2) made from AA Series 3 Prostaglandins (PG3) made from EPA

65
Q

Eicosanoids: Series 1 Prostaglandins (PG1) made from DGLA

A

Keep blood platelets from sticking together. Remove excess sodium and water from the body. Relax blood vessels promoting circulation. ANTI- INFLAMMATORY

66
Q

Eicosanoids: Series 2 Prostaglandins (PG1) made from AA

A

Mostly promote platelet aggregation. Promote sodium and water retention (↑ BP) Oppose functions of series 1 prostaglandins. PRO-INFLAMMATORY

67
Q

Eicosanoids: Series 3 Prostaglandins (PG1) made from EPA

A

Some have weak platelet aggregating properties. Prevent the release of AA from cell membranes. EPA is the most important factor limiting PG2 production. ANTI-INFLAMMATORY

68
Q

Eicosanoids: Inflammatory Cycle

A

Eicosanoids made from arachidonic acid produce initial inflammation. This is ‘shut off’ by the introduction of eicosanoids made from DGLA and EPA.

69
Q

EFA Metabolism

A

Cell membrane fatty acid composition determines which prostaglandins will predominate; e.g. a diet rich in arachidonic acid leads to the formation of more pro-inflammatory PG2. • The more abundant fatty acids will occupy the enzyme active sites, which highlights the importance of omega 3 and 6 balance. • A high consumption of EPA and DHA from omega 3 means that a higher proportion of fatty acids resides in the cell membrane at the expense of AA. • This can result in immune suppression. Hence, it is all about balance.

70
Q

EFA Metabolism: Genetic variability

A

Genetic variability affects the synthesis of EPA and DHA. Polymorphisms are common in the genes coding for delta-6 and delta-5 desaturase. • Other omega 6 and omega 3 fatty acids can be synthesised from ALA and LA respectively. • By desaturation addition of a double bond between two carbon atoms and / or elongation addition of two carbon atoms. • Both LA and ALA compete for the same desaturase and elongase enzymes. • Only 1-20% of ALA is converted to EPA. • Women of reproductive age convert ALA 2.5 times better than men

71
Q

EFA Metabolism Map

A
72
Q

Inhibitors of EFA Metabolism: Delta-6 Desaturase

A

Magnesium, B6, zinc deficiency Insulin resistance Viruses Refined sugars Alcohol Stress hormones, e.g. cortisol High intake of EPA DHA Excess trans fats and cholesterol

73
Q

Inhibitors of EFA Metabolism: Delta-5 Desaturase

A

Insulin resistance Zinc deficiency Alcohol Excess trans fats and cholesterol Stress hormones, e.g. cortisol High intake of EPA / DHA

74
Q

Omega Testing

A

EFA testing includes: • Omega 3 index a marker for cardiovascular risk. • Omega 6:3 ratio a marker for chronic illness. • AA:EPA ratio a marker of ‘silent’ inflammation. Maintaining the balance of omega 3 and omega 6 can be addressed by supplementing EPA / DHA, along with addressing any co factor deficiencies needed for interconversion. • Fatty acid profile testing options: – Genova essential and metabolic fatty acids test (blood test). – Wiley’s Finest omega 3 index test (blood spot). – Igennus Opti O-3 (blood spot).

75
Q

Cholesterol

A

Cholesterol is an important compound for cell structure and function. Beneficial properties are often overlooked because of negative perceptions around cardiovascular disease risk.

76
Q

Cholesterol: Functions

A

• Cholesterol is essential for the synthesis or action of: – Vitamin D and calcium metabolism. – Cortisol and related hormones. – Aldosterone for mineral and fluid balance. – Sex hormones oestrogen, progesterone and testosterone. – Bile salts and acids needed for digestion. – Membrane integrity , especially in the – Lipoproteins , needed for triglyceride transport.

77
Q

Cholesterol: Digestion

A

A diet rich in triglycerides stimulates cholesterol synthesis in the liver and small intestine. • It is excreted in the stool intact, mostly as bile products. • The excretion is increased by absorption onto non-digestible carbohydrates (fibre). • Gut bacteria from healthy microbiomes metabolise cholesterol = less reabsorption. • Dietary cholesterol does not significantly affect plasma cholesterol levels in most people as they are primarily influenced by genetic and nutritional factors.

78
Q

LDL and HDL

A

Doctors and other health officials often refer to cholesterol as ‘good’ and ‘bad’, especially in reference to heart • LDL and HDL cholesterol are in fact carriers . Cholesterol sits within these lipoproteins to be transported to wherever needed. • A lot of other substances are carried within them including CoQ10, beta carotene and vitamin E. • LDL (low density) takes cholesterol from the liver to cells. • VLDL (very low density) takes triglycerides to cells. • HDL (high density) collects cholesterol from cells to transport back to the liver.

79
Q

Increases in cholesterol

A

Increases in cholesterol may indicate an increased demand for cholesterol’s anti inflammatory function or an increased need for cholesterol to repair membranes, make hormones, etc. • Atherosclerosis requires LDL cholesterol to deposit in the arterial wall and become oxidised. • Atherosclerosis is an inflammatory disease. In the absence of inflammation or injury to the endothelium, cholesterol does not deposit. • There are varying sizes of LDL cholesterol. Measuring particle size rather than total cholesterol is a better health indicator.

80
Q

Cardiovascular Markers: LDL particle size

A

LDL particle size: people whose LDL particles are predominantly small and dense have a threefold greater risk of coronary artery disease, whereas the large and fluffy type may be protective.

81
Q

Cardiovascular Markers: HDL particle size

A

HDL particle size: larger HDL particles are more effective at removing cholesterol from the blood. Larger particles better exert anti inflammatory and anti thrombotic effects, as well as promoting nitric oxide production in endothelial cells.

82
Q

Cardiovascular Markers: Lipoprotein (a)

A

Lipoprotein (a) lipoprotein (a) is a blood clotting agent. It appears to be a key genetic risk factor in coronary artery disease. Higher levels are associated with greater risk.

83
Q

Cardiovascular Markers: Lp-PLA2

A

Lp PLA 2 an enzyme that plays a role in endothelial inflammation and atherosclerosis.

84
Q

Cardiovascular Markers: Fibrinogen

A

Fibrinogen - raised levels are a risk factor for clot formation

85
Q

Cardiovascular Markers: C-reactive protein

A

C-reactive protein inflammatory marker associated with CVD.

86
Q

Cardiovascular Markers: Lipid peroxides

A

Lipid peroxides raised levels reflect oxidative damage to membranes.

87
Q

Phospholipids

A

Phospholipids are the structural basis of all cell membranes. Different types of phospholipids perform roles in cellular function, such as insulin signalling. • Phosphatides contain glycerol, two long chain fatty acids, a phosphate group, and either inositol, choline or serine. • Phosphatidylcholine predominant phospholipid in the body. • Lecithin synthesised by the liver and plays a role in emulsification (fat digestion). Lecithin increases the solubility of cholesterol and helps improve cognitive function.

88
Q

Phospholipids: Lecithin

A

• Lecithin synthesised by the liver and plays a role in emulsification (fat digestion). Lecithin increases the solubility of cholesterol and helps improve cognitive function.