ABSTRACT

Hypertriglycedidemia is the most important atherosclerotic

risk factor. Review of population based studies in India shows
increasing mean total TG levels. Recent studies have reported that
high cholesterol is present in 25–30% of urban and 15–20% rural
subjects. This prevalence is lower than high-income countries.
The most common dyslipidemia in India are borderline
high LDL cholesterol, low HDL cholesterol and high triglycerides.
Studies have reported that over a 20-year period total cholesterol, LDL
cholesterol and triglyceride levels have increased among urban
populations.(ELES [1])

OBJECTIVES
This study aimed a Comparison of Friedewald Formula with direct
LDL cholesterol measurement in patients with hypertriglyceridemia.
And to determine what extent LDL- cholesterol level is
underestimated when it is estimated by using calculation formulas
compared with LDL- C level measured by direct method

MATERIALS AND METHODS

Participants of the study group were selected from the
outpatients population of department of Biochemistry, Medical trust
Hospital, Ernakulam. All of 30 patients were included in the study
with hypertriglyceridemia .They ranged in age from 30 to 50 years.

For all the patients, 3-5ml of blood samples were collected

by venepuncture method into plain tube and The serum
samples were used for the analysis of lipid profile(TG,TC,LDL-
C,HDL-C)

RESULT

CONCLUSIONS
This study revealed that the LDL cholesterol measurement
by direct method is more accurate than original Friedewald’s
formula for the calculation of serum low-density lipoprotein
cholesterol in patients with TG levels more than 400mg/dl.
INTRODUCTION

A triglyceride (TG, triacylglycerol, TAG, or tri

acylglyceride) is an ester derived from glycerol and three fatty acids .
Triglycerides are the main constituents of body fat in humans and
other vertebrates, as well as vegetable fat. They are also present in the
blood to enable the bi directional transference of adipose fat and blood
glucose from the liver, and are a major component of human skin oils.
(2)
In the human body, high levels of triglycerides in the
bloodstream have been linked to atherosclerosis, heart disease[9] and
stroke.[8] However, the relative negative impact of raised levels of
triglycerides compared to that of
LDL:HDL ratios . The risk can be partly accounted for by a strong
inverse relationship between triglyceride level and HDL-cholesterol
level. But the risk is also due to high triglyceride levels increasing the
quantity of small, dense LDL particles.[10]

Asian Indians are known to have a unique pattern of

dyslipidemia with lower HDL cholesterol, increased TG levels and
higher proportion of small dense LDL cholesterol.[ 3]

More than 90%of laboratories of US estimate LDL-C levels using the

Friedwald formula (FF) .But the LDL.C level cannot be accurately
estimated if the TG value exceeds 400mg/dl as the TG : total
cholesterol ratio of VLDL cholesterol will differ..This formulas also
underestimate calculated LDL_C value than measured value.

OBJECTIVES OF THE STUDY

 Comparison of Friedewald Formula with direct LDL
cholesterol measurement in patients with
hypertriglyceridemia.
 To determine what extent LDL- cholesterol level is
underestimated when it is estimated by using calculation
formulas compared with LDL- C level measured by direct
method
 To compare the percentage of patients meeting LDL – C goal
using calculation formulas and direct method
Comparison of calculated LDLC with measured LDLC at various
increasing TG levels are presented in Fig. 6 which clearly
demonstrates that the underestimation of LDLC The Pearson’s
concordance correlation coefficients (Pc) of calculated LDLC with
measured LDLC were 0.9400 (95% CI: 0.9322 to 0.9469) for the
derived regression equation and 0.8613 (95% CI: 0.8459 to 0.8752) for
Friedewald’s formula. The bias correction factor (Cb), a measure of
accuracy was 0.9974 for the derived equation and 0.9369 for the
Friedewald’s formula. The accuracy (Cb) of the derived equation was
higher and closer to perfect (Cb = 1.0) compared to Friedewald’s
formula (0.9974 vs 0.9369).
Fig. 5. Mountain plot of calculated LDLC against measured LDLC
(measured LDLC – calculated LDLC). The solid line indicates the
derived regression equation and the broken line indicates the
Friedewald’s formula.
Fig. 6. Mean ±SD of LDLC at increasing TG levels
REVIEW OF LITERATURE

Among the noncommunicable diseases, cardiovascular

diseases (CVD) remain the biggest cause of death worldwide. Over the
last two decades cardiovascular mortality rates have declined in many
high-income countries. At the same time cardiovascular disease and
deaths have increased at an astonishingly fast rate in low- and middle-
income countries . Elevated serum low-density lipoprotein cholesterol
(LDLC) is one of the important independent risk factors for the
development of coronary heart disease . It is the recommended
primary basis for the correct classification in risk categories and
management of CVDs .[4]

Abnormalities of various cholesterol lipoprotein lipids such as high

total cholesterol, low density lipoprotein (LDL) cholesterol, very low
density lipoprotein (VLDL) cholesterol and triglycerides and low high
density lipoprotein (HDL) cholesterol are important coronary heart
disease (CHD) risk factors. There is a strong pathophysiological
association of raised LDL cholesterol with initiation and progression
of coronary atherosclerosis[2]
Global Burden of Metabolic Risk Factors Study
reported trends in total TG levels in different countries and various
regions of the world from the years 1980–2008. 5 It was estimated that
total TG levels increased in India as well as many other low-income
and lower-middle income countries over this period. 6 On the other
hand, TG levels declined in most high-income countries.(ELS 2)

HYPERTRYGLYCERIDEMIA
Hypertryglyceridemia denotes high blood levels of TG ,the most
abundant fatty molecules in most organism. Elevated levels of TG are
associated with atherosclerosis ,even in the absence of
hypercholestermia and predispose to cardiovascular disease .very high
TG levels also increase the risk of avite
pancreatitis .hypertryglycredimeia itself idsusually
symptomless ,although high levels may be associated with skin lesions
known as xanthomas

Causes

 Obesity
 Overeating
 Diabetes mellitus and insulin resistance - it is one of the defined
components of metabolic syndrome (along with central obesity,
hypertension, and hyperglycemia)
 Excess alcohol consumption
 Kidney failure, nephrotic syndrome
 Genetic predisposition; some forms of familial hyperlipidemia
such as familial combined hyperlipidemia i.e. Type II
hyperlipidemia
  Lipoprotein lipase deficiency -Deficiency of this water-soluble
enzyme, that hydrolyzes triglycerides in lipoproteins, leads to
elevated levels of triglycerides in the blood.
 Lysosomal acid lipase deficiency or
 Cholesteryl ester storage disease
 Certain medications e.g. isotretinoin,
blockers, protease inhibitors
 Hypothyroidism (underactive thyroid)
 Systemic Lupus Erythematosus and associated autoimmune
responses
 Glycogen storage disease type 1.
 HIV medications

Treatment

Lifestyle changes including weight loss and dietary modification may

improve hypertriglyceridemia. The decision to treat
hypertriglyceridemia with medication depends on the levels and on the
presence of other risk factors for cardiovascular disease. Very high
levels that would increase the risk of pancreatitis is treated with a drug
from the fibrate class. Niacin and omega-3fatty acids as well as drugs
from the statin class may be used in conjunction,with statins being the
main drug treatment for moderate hypertriglyceridemia where
reduction of cardiovascular risk is required.[1]
LIPID PROFILE

TG
There are many different types of triglyceride, with the main division
between saturated and unsaturated types. Saturated fats are "saturated"
with hydrogen — all available places where
hydrogen atoms could be bonded to carbon atoms are occupied. These
have a higher melting point and are more likely to be solid at room
temperature.
Unsaturated fats have double bonds between some of the
carbon atoms, reducing the number of places where hydrogen atoms
can bond to carbon atoms. These have a lower melting point and are
more likely to be liquid at room temperature.
The American heart association recommends an optimum TG
level of 100mg/dl(1.1mmol/l) or lower to improve heart health(wiki)

Total cholesterol
Total cholesterol is the total amount of cholesterol in your blood.our
total cholesterol includes lowdensity lipoprotein cholesterol and high
density lipoprtien cholesterol cholesterol is waxy,fat like substance
foundin every cell in our body
Total cholesterol = LDL+HDL+TG/5
High-density lipoprotein

High-density lipoprotein (HDL) is one of the five major groups

of lipoproteins.[1] Lipoproteins are complex particles composed of
multiple proteins which transport all fat molecules (lipids) around the
body within the water outside cells.
They are typically composed of 80–100 proteins per particle
(organized by one,two or three ApoA; more as the particles enlarge
picking up and carrying more fat molecules) and transporting up to
hundreds of fat molecules per particle.(5 chattarje)

HDL particles remove fat molecules from cells which need to

export fat molecules. The lipids carried include cholesterol,
phospholipids, and triglycerides, amounts of each are quite variable.
Increasing concentrations of HDL particles are strongly associated
with decreasing accumulation of atherosclerosis within the walls of
arteries. This is important because atherosclerosis eventually results in
sudden plaque ruptures, cardiovascular disease, stroke and other
vascular diseases. HDL particles are sometimes referred to as "good
cholesterol’’ because they can transport fat molecules out of artery
walls, reduce macrophage accumulation, and thus help prevent or even
regress atherosclerosis, but studies have shown that HDL-lacking mice
still have the ability to transport cholesterol to bile,suggesting that
there are alternative mechanisms for cholesterol removal.

Low-density lipoprotein

Small particle LDL has been associated with the progression of

atherosclerosis and blockage the artery lumen, because it can carry
cholesterol into smaller vessels. But LDL is also essential for carrying
lipids that keep the human body alive, including in those small vessels.
Low-density lipoprotein (LDL) is one of the five major groups of
lipoprotein which transport all fat molecules around the body in the
extracellular water.[1] These groups, from least dense to most
dense, are chylomicrons (aka ULDL by the overall density naming
convention),
very low-density lipoprotein (VLDL) intermediate-density
lipoprotein (IDL),low-density lipoprotein and high-density lipoprotein
(HDL). LDL delivers fat molecules to cells. LDL can contribute to
atherosclerosis if it is oxidized within the walls of arteries. It is
important to note that LDL is not "bad cholesterol". It is an essential
transport system for lipids the human body needs to survive, including
cholesterol.
There is both "large" and "small" particle LDL, and while
only small is associated with cholesterol-related issues, neither is
"bad". Even "small" LDL is necessary to conduct nutrients to vessels
that "large"LDL cannot reach. Lipoproteins transfer lipids (fats)
around the body in the extracellular fluid, making fats available to
body cells for receptor-mediated endocytosis.[2][3] Lipoproteins are
complex particles composed of multiple proteins, typically 80–100
proteins/particle (organized by a single apolipoprotein B for LDL and
the larger particles). A single LDL particle is about
220–275 angstroms in diameter, typically transporting 3,000 to 6,000
fat molecules/particle, and varying in size according to the number and
mix of fat molecules contained within.[4]
The lipids carried include all fat molecules with cholesterol,
phospholipids, and triglycerides dominant; amounts of each
varying considerably. The conventional interpretation ofcholesterol
levels holds that higher levels of LDL particles pose increased risk of
cardiovascular disease. LDL particles are thought to invade the
endothelium and become oxidized, since the oxidized forms would be
more easily retained by the proteoglycans

LIPID PROFILE
DESIRABLE BODERLINE HIGH
RISK

HDL CHOLESTEROL 60mg/dl 35-45 mg/dl <35

mg/dl

LDL CHOLESTEROL 60-130 130-159 mg/dl 160-189

mg/dl mg/dl
TRIGLYCERIDE <150 150-199 mg/dl 200-499
mg/dl mg/dl

<200 200-239 mg/dl 240

TOTALCHOLESTEROL mg/dl mg/dl

FRIEDWALD EQUATION
Low-density lipoprotein cholesterol (LDL-c) is the major
measured parameter for cardiovascular risk assessment. The generally
accepted formula (LDL-F) for estimating LDL-c developed by
Friedewald and colleagues in1972 using data from 448 individuals
suffers from known inaccuracies at extremes of triglyceride (TG) and
total cholesterol (TC) values. Low-density lipoprotein cholesterol
(LDL-c) is a frequently used and major laboratory parameter for
cardiovascular risk assessment.
More than 40 years ago, William T Friedewald et al.1
developed a formula for LDL-c estimation
using a database of 488 individuals based on fasting plasma
measurements of total cholesterol (TC), high-density lipoprotein
cholesterol (HDL-c) and triglycerides (TG): LDL-c ¼
total cholesterol 2 HDL-c 2 0.2 _ TG, and it has enjoyed
widespread acceptance since that time. Although accurate
in most cases, Friedewald’s formula applies poorly to a
number of atypical situations, such as extremes of TG
and TC values.2(ACCURATE
MATERIALS AND METHODS
Participants of the study group were selected from the outpatients population
of department of Biochemistry, Medical trust Hospital, Ernakulam. All of 30
patients were included in the study with hypertriglyceridemia .They ranged in age
from 30 to 50 years.

For all the patients, 3-5ml of blood samples were collected by venepuncture
method into plain tube and The serum samples were used for the analysis of lipid
profile(TG,TC,LDL-C,HDL-C)

STUDY DESIGN

Retrospective study

STUDY PERIOD

May2 to august30 2019

AGE GROUP

30 – 50 years

SAMPLE SIZE

The study population consist of total 30subjects.

INCLUSION CRITERIA
 Patients having age group between 30 – 50 years ,with hypertriglyceridemia.
The ranged tg value from 400-1000mg/dl
 Both male and female are included
EXCLUSION CRITERIA

 Pregnant and lactating women.

 Smoking and alcoholic patients.
 Patients with other systemic d/s

COLLECTION OF BLOOD SAMPLES

For all the patients, 3-5ml of blood samples were collected by

venipuncture method into plain tube and serum was separated by
centrifugation .The serum samples were used for the analysis of lipid
profile(TG,TC,LDL-C,HDL-C)

.
Fig; Plain blood collection bottle
INSTRUMENT

Estimation of Lipid profile (TG,TC,LDL-C,HDL-C)is done in SIEMENS

Dimension EXL200.An integrated chemistry and immunoassay analyzer with 47
assays onboard and up to 627 tests per hour.

Figure : SIEMENS Dimension EXL 200

METHOD OF ESTIMATION

Detection of TC

Principle

Cholesterol esters cholesterol esterase (CE) cholesterol +fatty acid

Cholesterol+O2 cholesterol oxidase(CO) cholest-4-ene-3-0ne+H2O2

2H2O2+DEA-HCL/AAP Horse radish peroxidase(HPO) 4-H2O+Oxidised

(N,N diethylaniline –HCL/4-amino antipyrine)

DEA-HCL/AAP

The absorbance due to oxidized DEA-HCL/AAP is directly proportional to the

total cholesterol concentration and is measured using a polychromatic
(452,540,700nm)end point technique.

Reagents

CE 0.7U/L

CO 0.1U/L

HPO 2.4U/L

AAP 4.5 µmol

Buffer

Cholate

DEA 5.8µmol

Surfactant

Procedure
REAGENTS VOLUME
Sample size 3µl
Reagent 1volume 88µl
Reagent 2 volume 26µl
Diluent volume 241µl
Temperature 37oC
Wavelength 452,540,700nm
Type of measurement Polychromatic end point

Reference Range

150-200 mg/dl

Detection of TG

Principle

 TG Lipoprotein lipase (LPL)

Glycerol + fatty acid
 Glycerol +ATP Glycerol kinase (GK)
Glycerol 3 phosphate +ADP
Glycerol 3 phosphate +O2 Glycerol 3 phosphate oxidase(GPO)
Dihydroxyacetone
phosphate +H2O2
 2H2O2 +Aminoantipyrine + Peroxidase (POD
Quinoneimine+HCL+4H2O
4- chlorophenol

The change in absorbance due to the formation of quinoneimine is directly

proportional to the total amount of glycerol and its precursors in the sample and is
measured using a bichromatic (510 ,700 nm)endpoint technique.

Reagents
Lipoprotein lipase 7.5 KU/L

ATP 3 mmol/L

Glycerol kinase 0.5 KU/L

Glycerol 3 phosphate oxidase 2.2KU/L

4-amino antipyrine 0.75 mmol/L

4-chlorophenol 6 mmol/L

Peroxidase 5 KU/L

Mg 2+ 22.5 mmol/L

Buffer Ph 7.2 50 mmol/L

Procedure

REAGENTS VOLUME
Sample size 4µl
Reagent 1 volume 133µl
Temperature 370C ± O.10C
Wavelengths 510 and 700 nm
Type of measurement Bichromatic end point

Reference Range

30- 180 mg/dl

Detection of HDL

Principle

HDL,LDL,VLDL, Dextran sulphate Non reactive LDL,VLDL,

Chylomicrons Magnesium sulfate Chylomicrons +HDL

cholesterol

Esters

HDL Cholesterol esters+ polyethylene glycol(PEG) HDL

Cholesterol

H2O Cholesterol esterase +RCOOH

HDL Cholesterol+O2 PEG Cholesterol oxidase 4 Cholestenone +H2O2

-N-(2 hydroxy 3 sulfopropyl)-3 -5- Peroxidase color development+5H2O

Dimethoxyaniline(HSDA)+H++H2O

Reagents

HEPES buffer 10.07 mmol/lpH 7.4

2(N-cyclohexylamino)ethanesulfonic acid 96.95mmol/l

Dextran sulfate 1.5g/l

Magnesium nitrate hexahydrate ≥11.7mmol/l

N(2-hydroxy-3-sulfopropyl)3,5dimethoxyaniline 0.96 mmol/l

Ascorbate oxidase ≥50µkat/l

Peroxidase preservative ≥16.7µkat/l

HEPES buffer 10.07mmol/l

PEG cholesterol esterase ≥3.33µkat/l

PEG cholesterol oxidase ≥127µkat/l

Peroxidase ≥333µkat/l

4 aminoantipyrine 2.46mmol/l

Preservative

NaOH 1.00M

Procedure

REAGENTS VOLUME
Sample volume 3µl
Reagent 1 volume 300µl
Reagent 2 volume 100µl
Temperature 37oC ±0.1oC
Reaction time 8.6 minutes
Wavelength 600 and 700 nm
Type of measurement Bichromatic end point

Reference Range

40 -60 mg/dl

DETECTION OF LDL

Priniciples of Procedure
Detection of LDL

The ALDL Cholesterol assay is a homogenous method for directly measuring

LDL-C levels in human serum or plasma, without the need for any off-line
pretreatment or centrifugation steps
The method is in a two reagent format and depends on the properties
of detergent 1which solubilizes only non-LDL particles. Cholesterol released is
consumed by cholesterol esterase and cholesterol oxidase in a non- color forming
reaction .Detergent 2 solubilizes the remaining LDL particles.The soluble LDL-C
is then oxidised by the action of cholesterol esterase and cholesterol oxidase
forming cholestenone and hydrogen peroxide (h2O2). The enzymatic action of
peroxidase on H2O2 produces color in the peresence of
N,N-bis(4-sulfobutyl)-m-toludine,disodium salt (DSBmT) and 4-aminoantipyrine
(4-AA) that is measured using a bichromatic (540,700nm) endpoint
technique .The color produced id directly proportional to the amount of LDL-C
present in the sample .

 Nonsoluble LDL-C ,VLDL-C, DETERGENT1

solubleNonLDL-C
HDL-C,Chylomicron DSBmT+peroxidase (VLDL-C,HDL-C
chylomicrons)

 Soluble Non LDL-C CHOLESTEROL ESTRASE

non colour forming
Cholesterol oxidase

 Non solubleLDL-C detergent 2

soluble LDL-C
  Soluble LDL-C + O2 CHOLESTEROL ESTERASE
cholesterone + H2O2
CHOLESTEROL OXIDASE

 H2O2 ++DSBmT+4-AA PEROXIDASE

Colour development

Wells FORM INGREDIEN CONCENTARTI SOURCE

 TS ON

1,2,3 LIQUI MES BUFFER

D

Reagen DETERGENT 1 CELLULOMONA

t1 S sp

CHOLESTER PH6.3 PESUDOMONAS

OL- sp
ESTERASE
HORSERADISH

CHOLESTER
OL-OXIDASE

PEROXIDASE

4-AA

ASCORBIC CUCURBITA sp
ACID

OXIDASE

PRESERVATI
VE

4,5,6 LIQUI MES BUFFER, PH6.3

D
Reagen DTERGENT
t2
DSBmT

PRESEVATIV
 E

TEST CONDITIONS
SAMPLE SIZE 3µL

REAGENT 1 VOL 300µL

REAGENT 2 VOL 100µL

TEMPRATURE 37O C

WAWELENGTH 540nm AND 700nm

TYPE OF MEASURMENT BIOCHROMATIC ENDPOINT

FRIEDEWALD FORMULA

The friedewald formula (FF) is the main method for evaluvating low – density
lipoprotein cholesterol(LDL-C)
The friedewald formula (FF) is an estimation of LDl-C level that uses the
following levels of total cholesterol (TC0,triglyceride TG and high density
lipoprotein cholesterol HDL-C ,The measurement to be applied in the FF , of
TC HDL-C and TG must be in mg/dl ;the estimation differs and was not
performed for the mmol/L. TheFF became the standard method forLDL-C
 assessment because it is economilcal and simpler than direct assays ,the most
accurate LDL-C measurment metods

LDL-C (mg/dL) = TC(mg/dL) - HDL-c(mg/dL) –TG(mg/dL/5

RESULT
Our Study have shown that the accuracy of Fredwald formula declines as TG
increases beyond 400mg/dl.
DISCUSSION
SUMMARY AND CONCLUTION
Strategies for treatment of lipid abnormalities are primarily based on LDL-C
concentration. Therefore, LDLC must be accurately determined to establish a
personal CHD risk profile in order to initiate dietary adjustments, drug therapy and
to monitor their effects
40 years ago, Friedewald et al. created a formula to estimate LDL-c from
directly measured TC, HDL-c and TG values. This formula is relatively accurate
for the majority of individuals in modernized nations. However, it has since
become apparent that Friedewald’s formula has important limitations,
including inaccurate LDL-c estimation in patients with TG greater than 4.5
mmol/L,(abd 2012 6)
LDL-C should be measured by direct homogeneous assay in routine clinical
laboratories, as the calculated methods did not have a uniform performance for
LDL-C estimation at different TG levels
BIBILOGRAPHY