OxLDL ELISA

LDL (low density lipoproteins) are implicated in the development of the much dreaded atherosclerosis. But there is a more perilous condition that prevails prior to the development of atherosclerosis and which favours the formation of even smaller LDL. This is a condition called prolonged lipaemia.

Normal lipaemia follows ingestion of food. The lymph is milky and stays that way for about 4 hours before it clears up. The most important enzyme that clears a milky plasma after food ingestion (postprandial lipaemia) is lipoprotein lipase (LPL). LPL is an enzyme that appears stuck to fibrilar structures called heparan sulphate (like the heads of matchsticks).

What happens in prolonged lipaemia? In certain conditions, lipaemia remains for very long and is thus referred to as prolonged lipaemia. The plasma doesn't seem to clear up for a long long time. This is a clear sign of a danger that will be quite difficult to solve and resolve. Prolonged lipaemia is a clear sign of the inability of LPL to clear up plasma and make it clear again. Why LPL cannot clear up plasma as fast is another realm of research.

When prolonged lipaemia prevails, it gives LDL an opportunity to be made even smaller, so much smaller that they become highly penetrative, ie they easily penetrate the arterial intima and cause damage. What damage can small LDL possibly do in the underlying tissues? A lot of irreversible damages.

Small LDL are "dense" and are referred to as small dense LDL. Due to their small size (a continuum of sizes), they have a large surface area and are highly prone to oxidation. What is oxidised? What are the surface components of LDL which can be possibly oxidised? There is apolipoprotein B-100 (apoB-100) and some of the surface lipids (can be mono- or polyunsaturated fatty acids of the phospholipids). Cholesterol in the surface layer should not be changed (cholesterol itself is stable). So most probably apoB-100 and some of the unsaturated fatty acid components of the surface lipids can be oxidised. ApoB-100 itself is a huge protein that occupies practically the entire surface of small dense LDL. So it is most likely that in small dense LDL, apoB-100 are oxidised to such an extent that it renders the LDL "good enough" to be consumed by macrophages. So macrophages consume all the oxidised LDL and in turn die from over-consumption of oxidised LDL. This is referred to as the scavenger-receptor pathway, an unregulated pathway where macrophages eat up as much oxidised LDL as they possibly can till they die, becoming foam cells. Foam cells are a key cell found in atherosclerosis.

We can detect oxidised LDL (oxLDL) in the clinical research laboratory using enzyme-linked immunoassay (oxLDL ELISA). A high level of oxLDL indicates a high level of oxLDL in the plasma sample and in the real life situation. High oxLDL possibly equates to high macrophage activity in the underlying tissues of the common carotid artery (CCA), and possible changes/damages to the arterial intima, and intima media thickness (IMT).


OxLDL ELISA:
http://www.cellbiolabs.com/human-oxidized-ldl-elisa-kit
http://www.cellbiolabs.com/human-oxidized-ldl-elisa-kit
http://www.cellbiolabs.com/sites/default/files/STA-369-human-oxldl-elisa-kit.pdf

Vendor:
AXON SCIENTIFIC SDN. BHD.
No. 13-4, Jalan SP2/1,
Taman Serdang Perdana,
43300 Seri Kembangan,
Selangor DE, Malaysia
Tel: +603-89451482
Fax: +603-89419421
Email: info@axonscientific.com
Website: www.axonscientific.com

Diabetic Dyslipidaemia

Determinants of uncontrolled dyslipidaemia among adult type 2 diabetes in Malaysia: The Malaysian Diabetes Registry 2009
Original Research Article
Diabetes Research and Clinical Practice, Volume 96, Issue 3, June 2012, Pages 339-347
Boon How Chew*, Mastura Ismail, Ping Yein Lee, Sri Wahyu Taher, Jamaiyah Haniff, Feisul Idzwan Mustapha, Mohd Adam Bujang

* Corresponding author at: Department of Family Medicine, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89472520; fax: +60 3 89472328.
E-mail address: chewboonhow@yahoo.com (B.H. Chew).
Contents available at Sciverse ScienceDirect (institutional subscription)

What is CAD?

CAD is the most common complication and a major cause of mortality in T2DM (type 2 diabetes mellitus). Patients with T2DM have a higher prevalence of small and dense LDL particles, which are more susceptible to oxidation (ie become oxLDL), therefore increasing atherogenic risk (high oxLDL, high risk CAD) even when there is not a high concentration of LDL cholesterol. This is true of the Indians who usually have low LDL-C but still have high risk for CAD.

What is LDL-C? 

LDL cholesterol (LDL-C) is a measure of the cholesterol carrying capacity of the lipoprotein constituents in blood. Generally, a high total cholesterol (high TC) correlates with a high LDL-C. When LDL-C is high, the opposite occurs to HDL-C. When LDL-C is high, HDL-C is low and this is true is most populations which have been studied except in the Indians. In the case with Indians, they have low LDL-C and low HDL-C. The low HDL-C generally is attributed to endothelial dysfunction, a prerequisite for atherosclerosis and impending CAD. So when we detect Indians with low LDL-C and low HDL-C, it is a tell tale sign and hallmark of an impending CAD. There is no doubt about it.

What is HDL-C?

If LDL carry cholesterol to peripheral tissues (extrahepatic tissues, ie not the liver; tissues apart from the liver), the HDL carry excess cholesterol in the reverse direction, ie back to the liver. For this role, HDL activity is described as reverse cholesterol transport (RCT). What can go wrong with HDL activity. HDL formation and cholesterol uptake are significant initial steps in RCT. When there is prolonged lipaemia, LPL activity is arrested (inhibited). So very little nascent HDL are formed by lipolysis (the other sources of HDL being hepatic production and intestinal production).

When there is disruption to the endothelial surface, cholesterol efflux is affected (less efficient or inefficient). Because choleserol uptake by nascent discoidal HDL is affected, HDL cannot 'grow big', ie they cannot imbibe the excess cholesterol from endothelial cells. Hence, these nascent or small HDL do not contain sufficient cholesterol for the next step to occur - cholesterol esterification by an enzyme called lecithin:cholesterol acyltransferase (LCAT). We must remember that the spherical structure of HDL itself is only possible when the ratio of its lipid components is correct (there is a formula for this). Otherwise the HDL remain discoidal (flattened) and not spherical. Since LCAT activity is limited, the HDL size cannot grow and thus it should be expected there would be a lot of tiny HDL particles in those who have high risk of CAD.

What lipid ratios and indices or combinations are safe or unsafe?

There are so many reports and findings today, which have not been properly tabulated for easy reference. It takes a lot of knowledge to understand what the data means and to be able to interpret correctly. It does take practice and every case is different. Generally,

1. high LDL-C: high risk CAD
2. high HDL-C: low risk CAD
3. high LDL-C, low HDL-C: high risk CAD
4. low-C, low HDL-C: high risk CAD
5. diabetics: high risk CAD
6. diabetic dyslipidaemia: high risk CAD

What is diabetic dyslipidaemia?

When lipaemia is prolonged, a lot of things go wrong intravascularly (in the blood itself). In diabetics, there is prolonged lipaemia; the blood does not rapidly clear up after we eat. Why? This is because a lot of the apparatus to clear up the blood has gone bonkers. I'm talking about lipolysis by lipoprotein liase (LPL). Because of prolonged lipaemia, it gives time for LDL to transform to very small and dense particles called small dense LDL. These little particles are dangerous not only because of their tiny sizes but because they are prone to oxidation. Tiny LDL are oxidised to oxLDL. Tiny LDL can penetrate the arterial intima and lodge in the underlying tissues. They are oxidised and gorged by macrophages. The oxLDL maybe gone but the macrophages now become laden and die to become foam cells. That is the beginning of all the problems related to arterial stiffening etc. Then CAD is the ultimate happening.

Research on Glybera Gene Therapy

Scenario
Gene therapy has ventured out of the clinical research lab and is now available for treating patients. Glybera is used for patients with fat problems, especially those who have hypertriglyceridaemia and therefore face problems of clumped fat leading to pancreatitis-like symptoms and other high-fat related problems (problems with lipases). With Glybera, the virus infects cells and genetic reconstruction leads to normal function of lipase(s) and fat usage. The blood is cleared of excess fatty material and build-up. Prolonged hyperlipidaemia is halted. There is less chances of pancreatitis occurring following high fat intake (eg after makan kenduri). Patients no longer have to rush to A&E, complaining of excruciating pain of supposedly pancreatitis. Reduced occurrence of prolonged hyperlipaemia means less chances for formation of oxidised LDL (ox-LDL). Less ox-LDL means less chances for macrophages to foam cell conversion in the arterial intima, and therefore reduced atherosclerosis, hence reduced possibilities of ischaemic heart disease (IHD). So, overall, a priori, gene therapy is good for patients with genetic problems such as fat-related problems.

Other applications
Glybera should also be good for diabetics since diabetics have prolonged lipaemia. Prolonged lipaemia is a hallmark of those who have IGT (impaired glucose tolerance) before they progress to full blown diabetes. It is a big hope that Glybera can help IGT patients. IGT patients are difficult to find or trace since they don't come to hospital (they are not ill or sick and don't have diabetes). IGT is a pre-diabetic state. The other category of patients is those who have IFG (impaired fasting glucose). When fasted, IFG patients have high blood glucose. I have not seen clinical trials of Glybera in IGT and IFG patients. So we have to wait till someone does the research and the research findings are out. That's the future.

More on gene therapy at BBC News:
http://www.bbc.co.uk/news/health-20179561

Endogenous Lipoprotein Pathway

SEQ: Describe the endogenous lipoprotein pathway. (10 minute/10 marks)
(Every sentence will receive a mark. Can also add a diagram and label it.)

Model Answer:

1. Endogenous pathway happens all the time.

2. It includes VLDL-LDL metabolism, LDL cascade and LDL receptor-mediated uptake.

3. The liver synthesizes VLDL. Hepatocytes releases it into the Space of Disse --> sinusoid --> enters systemic circulation.

4. VLDL has several fates.

a. VLDL carries triglycerides in liver to peripheral tissues for utilization.

b. When VLDL enters vascular beds, it undergoes hydrolysis (lipolysis) where its contents of triglycerides are hydrolysed to free fatty acids (FFA).

c. VLDL remnants are taken up into liver by LDL receptors and LDL receptor -related lipoproteins.

d. The VLDL is reduced in size and is converted into VLDL remnants and IDL, as a result of lipoprotein lipase (LPL) activity.

5. Unesterified FFA has several fates.

a. During lipolysis, some FFA can be carried by plasma albumin and dispersed in plasma for delivery to other cells. Not much of this happens.

b. Normally, FFA can enter underlying adipocytes by simple diffusion. Inside the adipocytes, the FFA are re-esterified to form triglycerides (TG) once more. Adipocytes store TG until required (as an energy souce during fasting or starvation). A majority of FFA are stored in adipocytes following lipolysis.

c. In times of starvation, when blood glucose is low and glycogen reserves are low, triglycerides stored in adipose tissues are hydrolysed by hormone sensitive lipase (HSL) and the FFA are released from adipose tissue. FFA then attach to circulating albumin and brought to liver for beta-oxidation, for continued energy supply.

6. IDL has several fates.

a. IDL can be converted into LDL by LPL in blood (intravascular).

b. IDL can be taken up by hepatic receptors.

c. IDL can be converted into LDL by hepatic lipase (HTGL) in liver.

7. LDL has several fates.

a. Normally, LDL is taken up by hepatic LDL (B,E) receptors. The contents of LDL are broken down into FC, CE, PL, TG/DG/MG/FFA and amino acids. These are kept within the hepatocytes or recycled for use by other cells. Hepatic contents of FC and CE are regulated by ACAT. Few things can happen if cholesterol is low, specifically in the cell or in the blood. If there is low cellular FC, stored cholesterol (as CE) is broken down to free cholesterol (FC) by ACAT. If there is low plasma FC, the liver cell makes more FC via HMG CoA reductase. The 2 enzymes, ACAT and HMG-CoA reductase, are sensitive to cellular and blood cholesterol levels, and combined, they regulate cholesterol levels in cells and blood.

b. LDL can also deliver its contents directly to cells, eg adrenal glands, for synthesis of steroid hormones.

c. Under abnormal levels of LDL in the blood (eg hyperlipidaemia), LDL can be taken up by the scavenger receptors present at extrahepatic tissues (EHT).

i. LDL apoB will be oxidized due to the prolonged presence of LDL in the blood. Also, the LDL particle is now smaller and apoB is not stable at this stage. ApoB becomes easily oxidised. Oxidised apoB has higher affinity for macrophages. Thus, oxidised LDL will be taken up by macrophages in EHT.

ii. Macrophages contain scavenger receptors on their surface. Macrophages are present on blood vessel walls. Oxidised LDL will attach and be internalised.

iii. The oxidised LDL are hydrolysed into component cholesterol, fatty acids, glycerol and amino acids, which are stored within the macrophages.

iv. This uptake of oxidised LDL is unregulated (ie, not controlled). The macrophages take up as much LDL until they stop functioning and die off, becoming foam cells.

LDL and atherogenesis

Taken from: http://ethesis.helsinki.fi/

2.2.1. LDL and atherogenesis

LDLs transport about 75% of the total cholesterol in blood circulation. Evidence exists that LDL cholesterol is a critical atherogenic factor (Grundy 1995,1997, Frishman 1998). A large number of epidemiologic studies have demonstrated a strong positive correlation between elevated LDL cholesterol levels and the development of coronary artery disease (CAD) (Kannell et al 1979, Krauss 1987, Genest and Cohn 1995, Frishman 1998). Genetic studies have also documented that inheritable hypercholesterolemias (familial hypercholesterolemia, familial defective apoB-100), mainly with elevated levels of LDL cholesterol, are the primary cause of premature CAD (Goldstein et al. 1973, Tybjaerg-Hansen et al. 1992). In addition, apoB and LDL particles have been identified in atherosclerotic plaques (Hoff et al. 1979a, Hoff et al. 1979b) and in vitro studies have shown that elevated LDL levels damage endothelial cell (EC) layer and penetrate into the arterial intima. The accumulation of LDL in the arterial wall initiates monocyte and smooth muscle cell migration and transforms macrophages and smooth muscle cells into cholesterol-loaded foam cells, which are the major cell components found in the plaque (Goldstein et al 1979, Brown and Goldstein 1983). Furthermore, pathological studies have demonstrated that the lowering of LDL-cholesterol is associated with reduced severity of atherosclerotic lesion and improvement of cardiac functional parameters (Zambon and Hokanson 1998). For example, reduction in cholesterol levels may reduce the susceptibility of LDL to oxidation which is a causal factor for the initiation and progression of atherosclerosis. Protection of LDL from oxidation could increase nitric oxide bioavailability and improve endothelium-dependent vasomotor, anti-inflammatory, and anticoagulant properties of the endothelium (Guetta and Cannon 1996). Finally, clinical studies have shown that lowering of LDL cholesterol has been associated with the reduction of CAD morbidity and mortality (Gotto 1995). The Scandinavian Simvastatin Survival Study (4S) showed that the lipid-lowering agent simvastatin significantly reduced the risk of coronary death and major coronary events in 4444 patients with coronary disease over the median follow-up period of 5.4 years (Scandinavian Simvastatin Survival Study Group 1994). These effects were presumed to be due to the beneficial reduction of serum lipids and lipoproteins, in which LDL cholesterol was reduced by 35%. The best evidence supporting lipid-lowering therapy for primary prevention comes from the West of Scotland Study (Shepherd et al. 1995). In this study, treatment with pravastatin resulted in significant reduction in nonfatal myocardial infarctions and death due to CAD. Taken together, these studies strongly support the importance of LDL in atherogenesis.