1. health and fitness
2. disease
3. cholesterol

CONTENTS
NEWS
4–5
There is only one boss. The customer
CARE FOR YOUR HEALTH
6–7
Positive aspects of fats
WORLD DIAGNOSTICS
Laboratory evaluation of lipid metabolism
Lipid metabolism disorders as a cardiovascular
risk factor. Interpretation of test results
8–12
13–15
DIAGNOSTICS UNDER MAGNIFYING
GLASS
Small Dense LDL Cholesterol and Coronary
Heart Disease
16–22
CLINICAL CASE
23
From case to case
Publisher:
PZ Cormay SA
ul. Wiosenna 22
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tel.: 22 751 79 10
faks: 22 751 79 11
e-mail: [email protected]
www.cormay.pl
2
Editing:
Chief Editor
– Monika Dziachan – PZ Cormay SA
Editing and proofreading – Agape
Whole truth in one drop
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Agape. Consultants and editors
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The editor reserves the right to shorten and edit
published materials.
CORMAY International Bulletin
EDITORIAL
Precious health
iving is as pleasurable as receiving. We love our
nearest and dearest and we want them to remain
healthy and fit for a long time. Let’s show them how
important they are to us and that we care for them every day.
We can give them an unusual present for less than 5 Euro!
G
Let’s get them a lipid profile.
An increased lipid level
is not a death sentence.
A change of diet and more
exercise can turn back the
biological clock by as much
as several years
Ask your loved ones to take the test; afterwards we
will involve more distant relatives. Let’s keep telling them
how important it is to themselves and to us – their next
of kin. High level of blood cholesterol remains
asymptomatic for a long time. We usually seek medical
assistance only when we notice symptoms such as
tingling sensation in fingers, cold hands or – even worse
– periodic heart conditions. Then it turns out that
changes to blood vessels are very advanced, sometimes
even irreversible. We can prevent them or slow them
down, but we cannot reverse them.
With your knowledge and experience you will be able
to convince your loved ones to take this simple, yet very
important test.
An increased lipid level is not a death sentence. A
change of diet and more exercise can turn back the
biological clock even by several years. Everyday exercise,
even only for 10 minutes improves physical condition
and makes us feel younger and healthier.
I hope you will enjoy this issue of our bulletin, which
will give you a number of interesting reasons for regular
lipid profile testing. I am sure they will encourage
heartfelt conversations with your loved ones, because
care and attention are the most precious things you can
offer them.
Tomasz Tuora
President of PZ CORMAY S.A.
President and CEO of Orphée S.A.
Nr 3 (25), autumn 2012
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3
NEWS
DOMINGO DOMINGUEZ – FACTS
Biolis 12i line
Domingo Dominguez is a Spanish Citizen and
French resident. Just after graduate in Engineering and Mechanics he started to work in ABX
(1986) as International Field Service Engineer
and travelled more than 200 days/year all around the world. From 1989 to 1991 he moved to
USA to be in charge of the US Technical Support and started to develop ABX Distributor Sales Network in Latin America.
In 1991, he came back to Montpellier and joined the ABX Headquarter of the company to
develop the International Sales Department,
and gradually climbed the scale to finally end
up in 2002 at the Head of the International
Sales, Service & Training Department of Horiba ABX.
In 2002 he left ABX to create with former President and Engineers of ABX, Orphée in Geneva and C2 Diagnostics in Montpellier. In 2010
Orphée has been acquired by Cormay and he
was appointed Chief Sales Officer of the Cormay Group.
All over those 25 years of activity, Domingo
Dominguez was mainly dedicated to International Business Activity spending more than
half of the year travelling in every part of the
world and exclusively in the IVD Market.
Domingo Dominguez enjoys very much his
professional activity and believes that the
most important to succeed in a professional
career is when you are doing something you like with people around you that share the same enthusiasm. Domingo Dominguez always
privileges the direct contact with people
when and where ever it is necessary. This is
why he spends most of his time travelling and
considers his job as a pleasant adventure
with many partners and friends all over the
world.
4
There is only one boss.
The customer.
Monika Dziachan : We have gathered here
today to tell our readers about Cormay
Group achievements this year. Some mi ght think that it is too early to do it in No vember.
Domingo Dominguez: Absolutely not. The
most important medical fairs of the year
– Medica, take place in November. That’s
where we meet the majority of our business
partners, summarise the past year and determine budgets and goals for the upcoming year. I think it is a perfect time.
M.D. : L e t u s s u m m a r i s e , t h e n . I w o u l d l i k e
to ask each of you to list the factors which
in your opinion were the most important to
the Group and / or to our clients. Domin go, would you like to start?
D.D.: For me, as the head of the Sales Department, the most important change is the
investment into the Audit-Orphee office in
Beijing. It is a very important market with
great and growing potential. We have terminated an agreement with the distributor
and we have opened our own office. From
the Group’s point of view, having our own
office has simplified logistics procedures
and enabled us to manage the level of customer service, distribution channels and
transport conditions. This in turn shortened the time of order fulfilment, enhanced
the availability of goods and lowered the
cost of transport which is covered by the
Whole truth in one drop
client. The Chinese office employees work
in accordance with procedures and standards elaborated in the head office in Switzerland. The procedures cover mainly the
areas of customer service and logistics. We
did not have to wait long for the effects of
this synergy. Now the clients can benefit
from the full range of European biochemistry and haematology products, POCT and
products for cytology testing LBC method,
all in one place. Since there is no time difference, communication has also improved.
Now our Chinese clients can call Beijing rather than Europe.
M.D. : A n d r z e j , i n y o u r o p i n i o n w h a t w a s
the key factor, considering that your
motto is „There is only one boss. The
customer”?
NEW REAGENTS
•
•
•
•
•
•
•
•
•
Biolis 50i line
Biolis 12i line
Biolis 15i line
Glucose HEX
Lactate new version
Total protein (not corrosive)
UA Plus
Bilirubin vanadate III generation
TG mono
CORMAY International Bulletin
NEWS
transportation. Our clients pay less, and
clearance time is shorter thanks to lack of
complicated customs clearance procedures. This year we resigned from Malloy-Evelyn method in the bilirubin test kit and
we replaced it with an excellent vanadium
method. It works very well for abnormal
samples, especially in newborns when doctor has to make a decision whether to irradiate the child or to perform blood transfusion. We also added a number of new parameters such as: glucose testing by hexokinase method, lactate, UA Plus and a reagent line for Biolis 12i analyser.
ANDRZEJ KENIK – FACTS
Graduate of Ternopil Academy of National Economy in Ukraine, Trade Management specialty, and
of Lazarski School of Commerce and Law, Foreign Trade specialty, M. Sc. in economics and management. Joined Cormay in 2008, currently working as Export Manager. From the beginning of his
professional career he has been working for niche
industries, where client relationship is the key factor which allows company development and maintaining the desired level of sales. His motto is:
“There is only one boss. The customer. The customer can make everyone from the chairman downwards redundant, simply by spending his money
elsewhere”. Andrzej implements his motto every
day on the North-European and emerging European, Central Asian and sub-Saharian markets.
M.D.: Andrzej, as per his motto, is focused
on the changes which improve customer
support. Pawe³, what was important to you?
Pawe³ Mirosz: I fully agree with Domingo.
Apart from customer support reorganisation mentioned by Andrzej, we have also
reorganised Orphee’s service support. We
have increased the number of field engineers, currently there are 4 of them, and each
of them is assigned to a geographic region.
This will speed up service reaction time and
provide the clients with additional comfort
of having an assigned field engineer.
There is one more factor, connected
with Audit Diagnostics. We have changed
the structure of the sales department,
which is now lead by Kamil Turonek. Some
of you will have a chance to meet him during Medica fairs. It has been almost
3 years since the acquisition of Orphée and
over a year since the acquisition of Audit
Diagnostics. Every month I watch how these companies learn best practice from each
PAWE£ MIROSZ – FACTS
Graduate of Technical University of Radom. Directly after graduation he started working in foreign
trade, at the beginning in glass product industry.
Since 2008 he has been working as Export Manager in Cormay. He delivers the information regarding the company and Cormay Group’s products
to clients in all corners of the world, mainly in Asia,
North Africa, Middle East and Central and South
America.
other. Usually we look at the best solutions
from one company and we implement them
throughout the Group. It is a continuous
process of self-improvement.
Thank you.
Monika Dziachan
Andrzej Kenik: Improvement in customer
support. I mean both service and application support at Cormay. At the moment
each of these areas are fully coordinated by
one person, so the client always knows who
he needs to call, and he is sure that his request is handled by the right person. And
we are sure that we will not miss anything.
Just to remind you: service matters are
handled by the Export Managers Assistant
– Alla Radzimovska and the person responsible for application issues is Pawel
Pastor. Our clients are very pleased with
that solution.
M.D. : W h a t e l s e ?
A.K.: During the past months our Research
and Development Department together
with the application team have improved
a number of kits, mainly when it comes to
reagent stability, sensitivity and linearity.
For example, the total protein test kit is not
corrosive anymore; it is now classified as an
irritant, which has a great advantages for
Nr 3 (25), autumn 2012
CMEF Exhibition in China
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5
CARE FOR YOUR HEALTH
Positive aspects
of fats
Heart conditions, diabetes, obesity on one hand; energy, good absorption of vitamins and health
on the other. Fats are associated with health advantages and risks at the same time. Therefore it is
important to maintain the necessary balance while consuming those organic compounds.
here are different types of fats – vegetable and animal fats, saturated
and unsaturated fats, solid and liquid fats. In chemistry fats are divided into
simple, complex and derived, and these
groups are further divided into subgroups
(e.g. wax, phospholipids, glycolipids). Fats
T
EXCESS FATS
•
•
•
•
•
•
•
6
Obesity
Heart conditions and vascular diseases
Sclerosis
Diabetes
Hypertension
Cancers
Other conditions
can also be classified in accordance to their
properties such as odourlessness or insolubility. Fats can be found in the human body
as well as in fruits and plants. Their diversity makes them a very important part of
a human life.
FATS IN EVERYDAY LIFE
Fats are an element of a healthy diet,
they are associated with proper development and functioning of the body. Recommended daily intake of fats varies according
to the gender and physical activity of a person, and may reach up to 120 g.
It is worth noting that vegetable fats are
considered healthier than animal fats, due to
components such as vitamin E or unsaturated
fatty acids which are present in vegetable oils.
Animal fats are the source of vitamin A and D
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which are also necessary for the proper functioning of the body so it is crucial for the health that a balanced dose of animal fats is supplied through certain products in our diet.
One of the very popular options to provide fats in our diet is eating cakes, fried foods,
crisps or chocolate bars. They certainly con-
FATS DEFICIENCY
•
•
•
•
•
•
•
Diminished immunity
Higher risk of diseases and allergies
Lack of energy
Hair loss
Inflammation of the skin
Decreased vitamin supply
Others
CORMAY International Bulletin
CARE FOR YOUR HEALTH
tain fats, however the unhealthy ones which
have an adverse effect on our bodies. These
foods are not recommended, but not many people refrain from eating them.
Healthy fats which are necessary
in appropriate doses can be found
in plants, vegetables and fish.
Olive oil, nuts or tuna fish are
a great alternative for another
bag of chips or a packet of
crisps.
perature. Furthermore vitamins
A, D, E and K are fat soluble.
Undoubtedly fats have various properties which enable human bodies to
function properly.
Fat deficiency
leads to various
health conditions
and problems.
It prevents the
right amount
of fat soluble
vitamins to
enter the body and has an
adverse effect
on our fitness
due to low
energy. An insufficient level of
fats makes us more illness prone,
because the body’s
immunity decreases.
We are less immune to
bacteria and allergies. Lack
of fats also affects the function
of the heart, causing decrease of its
tension and promoting oedema. It also
affects the appearance, as it leads to inflammation of the skin, hair loss and growth
impairment in children and teenagers.
WHAT DO WE NEED
FATS FOR?
Fats, and mainly
unsaturated fatty acids,
have a great influence on
the human body. They
are a component of cell
membranes, they help maintain good condition of the
skin, enhance the healing
process and alleviate inflammations. They have a positive
effect on the body’s water balance, blood cholesterol level and gastric juices. They prevent internal organs from moving and therefore protect
their stability. Fats provide the necessary
energy and help maintain the body’s tem-
Healthy fats which
are necessary
in appropriate doses
can be found
in plants, vegetables
and fish
EVERYTHING IN MODERATION
HIGH FAT DIET – HEALTHY OR CONTROVERSIAL?
One of the best known diets is a high fat diet. It is supposed to improve your health and prevent illnesses. Nutrition based on balanced amounts of proteins, fats (mainly animal fats) and low amounts
of carbohydrates results in loss of weight and improves the physical condition. Despite being one
of the most popular diets it is also very controversial. The opponents emphasise the risk of sclerosis and the supporters show visible health improvement.
Nr 3 (25), autumn 2012
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Excess fat consumption also creates problems. It results in obesity and other conditions related to it such as diabetes, hypertension or cancers. Too much fat also enhances sclerosis, heart conditions and vascular diseases.
In order to prevent the negative effects of
excess fat it is important to maintain a healthy diet and physical activity. Instead of
unhealthy snacks it is better to eat lean grilled fish, fruits and vegetables and lead a more active lifestyle. Let’s remember that fats
are good, as long as we supply them in moderation.
7
WORLD DIAGNOSTICS
Laboratory
evaluation
of lipid metabolism
Lipids are a group of organic compounds soluble in nonpolar organic
solvents. They are divided into fats (triacyglycerols or triglycerides),
waxes, isoprene derived lipids (steroids and carotenoids)
and complex fats. Lipids are a part of cellular and tissue structure
and they play an important role in metabolism as they are a source
of energy and substrates for steroid hormone synthesis.
Bogdan Solnica, MD PhD
Diagnostics Department of the Jagiellonian
University Collegium Medicum, Cracow
riglycerides, free fatty acids and
lipids are the components of
serum.
As lipids are water-insoluble, they are
present in bodily fluids in the form of high
molecular complex compounds – lipoproteins (Table). All classes of lipoproteins have
a similar structure. They consist of a hydrophobic core which contains cholesterol esters
and triglycerides, surrounded by an amphiphilic or hydrophilic substance – specific proteins (apolipoproteins), phospholipids and
free cholesterol. Differences in chemical
composition of lipoproteins mainly concern
relative EC, TG, phospholipids and protein
content, which results in their various density and molecular size. Apolipoproteins, proteins specific for particular lipoprotein classes, apart from maintaining their structure,
also have a regulatory role in lipoprotein metabolism as receptor ligands and enzyme(1,2).
T
LIPID AND LIPOPROTEIN
TRANSFORMATION
Lipid metabolism in the human body
mainly involves transport of alimentary
8
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TG from intestines and endogenous TG
from the liver to the fatty tissue (where
they are accumulated) and to muscles
(where they form a source of energy) as
well as transport of cholesterol to bodily
tissues. It helps build cell membranes and
synthesise steroid hormones. Another important aspect of lipid metabolism is so
called reverse cholesterol transport
– from the tissues to the liver, where it is
used for biliary acid synthesis.
Mainly TG and free (non-esterified)
cholesterol is absorbed through diet. In
duodenum in the presence of pancreatic
lipase TG is hydrolysed to fatty acids, mono and diglycerides which are absorbed
into enterocytes together with cholesterol.
Chylomicrons (CM) consisting of resynthesised TG, cholesterol, phospholipids
and apoprotein B48 (apoB48), a fragment
of apoB100, are synthesised there. Due
to large molecular size CM are secreted
by intestinal lining to lymphatic vessels
and are transported to circulating blood
through thoracic duct. In blood CM molecules are attached to two apoproteins:
apoE and apoCII. Under the influence of
lipoprotein lipase (LPL), connected mainly
with capillary endothelium of fatty tissue
and skeletal muscles, activated by apoCII
TG present in CM are hydrolysed and free
CORMAY International Bulletin
WORLD DIAGNOSTICS
Tab. Lipoproteins
Class of lipoproteins
Chylomicrons (CM)
Very low density lipoproteins (VLDL)
Intermediate density lipoproteins (IDL)
Low density lipoproteins (LDL)
High density lipoproteins (HDL)
Lipoprotein (a) Lp(a)
fatty acids are released. Within about one
hour from ingesting food, TG content
drops significantly and CM remnants
(CMR) created as a result are uptaken by
hepatocytes, mainly through apoE binding
receptor, when they achieve appropriate
size (Fig.) Together with endogenous TG
and EC released CMR components in hepatocytes are inbuilt into very low density
lipoprotein molecules (VLDL), which are
secreted into blood. They contain less TG
than CM and apolipoproteins apoCII,
apoE and apoB100. ApoCII activated LPL
hydrolyses TG in VLDL molecules, causing relative increase of EC content in
them, decrease of molecule size and increase of density, which – together with
transfer of apoCII to high density lipoprotein molecules (HDL) – leads to formation
of intermediate lipoprotein (IDL).
Further hydrolysis of TG contained
in IDL molecules occurs in the presence
of hepatic lipase (HL), bound by glycosa-
Density (g/ml)
<0.93
0.93–1.006
1.006–1.019
1.019–1.063
1.063–1.210
1.040–1.090
Main lipid component
Density (g/ml)
100–500
30–80
25–50
18–28
5–15
25–30
Alimentary TG
Endogenous TG
Cholesterol esters and TG
Cholesterol esters
Cholesterol esters
Cholesterol esters
contents. The limiting factor of synthesis
rate is the reaction of b-hydroxy-b-methylglutaryl-coenzyme A reductase (HMG
CoA). Synthesis of this enzyme is inhibited by cholesterol. Endogenous cholesterol which is synthesised this way covers
about 70–80% of cellular demand
and 20–30% is covered by exogenous
cholesterol from LDL. Uptake of exogenous cholesterol is regulated by LDL receptor expression, dependant on cellular
cholesterol content.
In the process of transformation of lipoprotein to peripheral tissues free fatty
acids and cholesterol are provided (Fig.)
Balance of cholesterol distribution in
tissues in physiological conditions is ensured by reversible transport of cholesterol from peripheral tissues to the liver,
with involvement of HDL. Disk-shaped
HDL molecules which are synthesised in
enterocytes and hepatocytes and formed
from fragments of VLDL and IDL surface
As lipids are water-insoluble, they are present
in bodily fluids in the form of high molecular
complex compounds – lipoproteins
minoglycans with the surface of liver sinusoidal endothelial cells. Together with
transfer of apoE to HDL molecules, it leads to formation of low density lipoproteins (LDL) containing only one apolipoprotein – apoB100. Cholesterol comprises
approximately 50% of LDL molecular weight. This lipoprotein fraction containing
about 70% of the serum cholesterol pool
delivers it to peripheral tissues and replenishes its cellular supply by binding to
specific apoB100 recognising receptor.
Cells have the ability to synthesise
cholesterol, regulated by intracellular
Nr 3 (25), autumn 2012
and contain a small amount of phospholipids and apoAI, are referred to as nascent HDL (pre-b-HDL) and HDL3. These molecules bind with ABCA1 transporter present on peripheral cells, joining
apoAI. It allows the water phase absorption (diffusion, inflow) of free cholesterol
from cellular membranes to HDL molecules, where it gathers on their surface. Lecithin-cholesterol acyltransferase (LCAT)
activated by apoAI esterifies cholesterol
molecules, causing their movement to the
core part of HDL molecules and making
space for free cholesterol absorbed from
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Apolipoproteins
B48, CII, E
B100, CII, E
B100, E
B100
A, CII, E
B100, Glycoproteins
tissues. Gathering of cholesterol esters
changes the shape of HDL molecules into
spherical shape (a-HDL or HDL2). At the
same time HDL gathers apoCII and apoE,
transferred from VLDL and IDL molecules. Removal of cholesterol esters from
mature a-HDL molecules occurs directly
through selective reuptake through SR-B1
and receptors for LDL hepatocytes or
reuptake of complete HDL molecules in
the presence of apoE binding receptors
such as cubilin in proximal tubule cells of
a nephron. Another way is the transfer of
cholesterol esters from a-HDL to lipoprotein molecules containing apoB (VLDL
and LDL), through specific cholesterol
ester transporting protein (CETP) and
reuptake of such lipoprotein through hepatic receptors (Fig.)(1,2).
LABORATORY EVALUATION OF LIPID
AND LIPOPROTEIN TRANSFORMATION
Analytical methods enabling separation
and measurement of specific serum lipoproteins are preparative ultracentrifugation
used mainly for research purposes and
electrophoretic separation, hardly ever
used nowadays in diagnostic laboratories.
For routine diagnostics the content of
particular lipoproteins in blood is evaluated
indirectly on the basis of concentration of
their contents:
• total cholesterol (TC), HDL cholesterol
(HDL-C), LDL cholesterol (LDL-C) and
non-HDL cholesterol (non-HDL-C),
• triglycerides,
• apolipoprotein: apoA, apoB.
Blood samples for full lipid panel (TC,
HDL-C, LDL-C, TG) are collected after
12–14 h within the previous meal. Total
cholesterol does not have to be measured in
a fasting patient. Measurements are carried
out in serum or plasma (serum concentration is ~3% higher than plasma). Maintaining the tourniquet for 3 minutes or standing for a long time before sample collection
can increase the concentration by 10% due
to blood thickening. Serum or plasma samples can be stored in +4°C for up to 4 days
or frozen in –20°C for a long time. HDL values can change during long storage. Serum
or plasma can be stored in –70°C.
9
WORLD DIAGNOSTICS
CHOLESTEROL
Cholesterol is a steroid alcohol, synthesised in tissues and delivered through diet.
It is transported by lipoproteins in blood: in
normal circumstances 70% of plasma part
of cholesterol is contained in LDL, 25% in
HDL and 5% in VLDL.
T OTAL
CHOLESTEROL
(TC)
Gas chromatography with Isotope dilution mass spectrometry (ID-MS) is
a definitive method of cholesterol measurement. In Liebermann and Burchard reference method cholesterol in the sample
or after extraction reacts with the mixture
of acetic acid anhydride and concentrated
sulphuric acid with formation of a coloured bicholestadien monosulphonate,
measured by spectrophotometry. In Abell
Kendall method Liebermann and Burchard
reaction is preceded by cholesterol
extraction with ethyl alcohol and ether
and saponification with KOH alcohol solution. Liebermann and Burchard reaction
is not fully specific for cholesterol and involves other sterols in the sample. It is
one of the limitations of this method as
compared to the definitive method, equal
to approximately 1.6%.
In routine diagnostic laboratories the
most commonly used methods are enzymatic methods with spectrophotometric
or fluorometric measurement, applied to
automated biochemistry analyzers.
As most of blood cholesterol is esterified, hydrolysis of cholesterol esters is
the first stage of measurement:
cholesterol
esterase
cholesterol ester + H2O
cholesterol + RCOOH
Drugim etapem oznaczenia jest utlenianie
cholesterolu:
cholesterol
oxidase
c h o l e s t e r o l + O2
4
D -cholestenon + H2O2
Measurement of amounts of products
formed in this reaction refers to D4-cholesterol solely or – more often – to third
reaction products with involvement from
hydrogen peroxide (H2O2) formed in amounts equimolar to oxidised cholesterol.
H2O2 can oxidize 4-aminoantipirin with
involvement of peroxidase in the presence of phenol. A product of that reaction
is quinone imine dye, measured by photometric method at 500–550 nm. Phenol
and 4-aminoantitriptin are the components of the Trinder’s reagent, used in
the CHOD-PAP method. Another option
is to oxidize ethanol by H2O2 in the presence of catalase with formation of acetate aldehyde, which is then oxidized in the
second reaction:
10
acetate aldehyde
dehydrogenase
+
acetate aldehyde+NADP
acetic acid+NADPH+H
The amount of formed NADPH is measured in the Wartburg’s optical test
at 340 nm.
In the presence of peroxidase H2O2
can oxidise substrates such as 10-acethyl-3.7-dihydroxyphenoxazine (ADHP)
with formation of fluorescence emitting
products – in this case resorufin.
Methods of cholesterol measurement
which use the oxidation leading to obtaining dyes or fluorochromes are exposed
to interference from substances which are
easily oxidisable by H2O2 and which use it
in the same way as ascorbic acid or hemoglobin. Other sterols in the sample can
also interfere with the reaction.
If the reagent kit does not contain
cholesterol esterase, free cholesterol is
measured. Following deduction from typical total cholesterol concentration, esterified cholesterol concentration is obtained.
HDL
CHOLESTEROL
(HDL-C)
HDL can be separated from other lipoprotein classes by ultracentrifugation. It is
considered to be the reference method, but
Whole truth in one drop
it is only used for research purposes. At
first diagnostic laboratories measured
HDL-C on the basis of precipitation of
other lipoprotein classes with the use of
polyanions and 2+ cations (heparin and
Mn2+, dextran and Mg2+ ions or phosphotungstic acid and Mg2+ ions) and measurement of cholesterol concentration in HDL
in supernatant obtained after centrifugation. Cholesterol measurements in this material are performed by immunoenzymatic
methods described above. Precipitation
methods are time consuming and also criticised for lack of precision and susceptibility to interference from high concentration
serum TG. It hinders precipitation of lipoproteins other than HDL and results in higher HDL-C results.
In mid nineties homogenous HDL-C
assays were introduced, based on various
reagents for selective availability and direct
measurement of cholesterol contained in
HDL. These methods are also referred to
as direct, as they do not require that other
lipoproteins are removed from the sample.
Homogenous HDL-C assays involve the
use of polyethylene glycol (PEG), a-cyclodextrins, synthetic polymers and detergents
as well as anti-apoB or anti-apoCIII antiboCORMAY International Bulletin
WORLD DIAGNOSTICS
LIVER
E
C
LPL
CMR
B48
BOWEL
A
C
E
CM
B48
WKT
CE
C
E
VLDL
CETP
LPL
B
A
TG
a--HDL
C
E
E
CETP
WKT
IDL
HL
B
WKT
CETP
KIDNEY
LCAT
LDL
B
CHOLESTEROL INFLOW
PERIPHERAL
TISSUE
PRE -BETA -HDL
Fig. Lipid and lipoprotein transformation in the human body. ---> (black) transport, secretion, reuptake; ---> (red) biochemical transformation;
CETP – cholesterol ester transporting protein, HL – hepatic lipase, LCAT – Lecithin-cholesterol acyltransferase, LPL – lipoprotein lipase
dies. These reagent cause precipitation
of CM, VLDL and LDL as soluble complexes, which precludes cholesterol contained
in them from entering into reactions catalysed by cholesterol esterase and cholesterol
oxidase. Only cholesterol contained in HDL
is ester hydrolysed and oxidised thereafter.
Another mechanism involves PEG modification of cholesterol oxidase and esterase
which do not react with cholesterol contained in CM, VLDL and LDL precipitated by
a-cyclodextrins. Direct methods of HDL-C
measurements are available as applications
for various biochemical analysers. Such
methods meet the accuracy and precision
criteria recommended by National Cholesterol Education Program and are commonly used in diagnostic laboratories(3,4).
LDL
CHOLESTEROL
(LDL-C)
Reference method is isolating an LDL
fraction through ultracentrifugation serum lipoproteins, precipitation of manganese ions with the use of heparin and me-
asurement of cholesterol. It is a time-consuming method and it requires a large sample. In everyday practice LDL-C
concentration is calculated on the basis
of Friedewald formula, knowing the analytical concentration of TC, HDL-C and
TG:
LDL - C = T C – HDL - C – T G / 5 ( w m g / d l )
or
LDL - C = T C – HDL - C – T G / 2 , 2
(w mmol/l)
At large TG concentrations (>4.6
mmol/l [400 mg/dl]) the results are not
accurate as there is a change of TG/cholesterol ration in VLDL.
Using Friedewald’s formula requires
additional testing of a sample collected
after 12–14 h from the previous meal, in
order to avoid postprandial chylomicronemia (hypertriglyceridemia). It must be
noted that calculated LDL-C concentration is encumbered with accumulated er-
rors of three measurements whose results
are used for calculation.
The first generation of analytical methods used for direct measurement
of LDL-C concentration was the methods
based on LDL precipitation with the use
of heparin, dextran or polivinyl sulphate.
Concentration of cholesterol contained in
precipitated LDL (LDL-Cprec) was assigned
as the difference between cholesterol
concentration in original testing material
(serum, plasma) and concentration in supernatant which does not contain LDL
was measured directly after precipitate
dissolution.
Further LDL-C testing methods are
based on the same principle as homogenous methods of HDL-C measurement.
They use reagents which contain various
detergents and agents which block or dissolve particular lipoprotein fractions, modify enzyme activity or remove H2O2 from
the reaction place. These methods are
used in automated analysers(3,5).
Whole truth in one drop
Nr 3 (25), autumn 2012
11
It should be mentioned that – despite
continuous work on improvement of analytical methods – Friedewald’s formula
remains the recognised method of measurement of LDL-C concentration. Use
of analytical methods is recommended
when LDL-C concentration cannot be calculated due to changes in lipoprotein
composition – mainly hypertriglyceridemia and in secondary dyslipidemias in
diabetes and in kidney diseases.
N ON - H D L C H O L E S T E R O L
(NON -HDL-C)
Non-HDL cholesterol reflects the
amount of cholesterol in all lipoprotein
fractions containing apoB. It is calculated
on the basis of the following formula:
well as possibly present free glycerol(3). In
diagnostic laboratories TG concentration
measurements are made in serum or plasma, usually by enzymatic methods and
automated biochemistry analysers. These
methods are based on reactions where
substrate is glycerol released during hydrolysis of TG and mono- and diacyloglycerols.
In one of the methods it is finally
transformed into phosphodihydroxyacetone:
TG+3H 2 O
lipase
glycerol+3 FFA
glycerol
kinase
glycerol+ATP
glycerophosphate+ADP
glycerophosphate
dehydrogenase
non - HDL - C = T C – HDL - C
TRIGLYCERIDES
Triglycerides (triacyloglycerol) are esters of glycerol and fatty acids, transported with blood mainly in chylomicrons
(exogenous alimentary TG) and VLDL
(endogenous TG). They are the basic
energetic substrate and are stored in the
fatty tissue.
The most definitive method of TG measurement is mass spectrometry with ID-MS isotope dilution. This method measures all glycerides present in the sample, e.g. mono- di- and trigrycerides as
12
glycerophosphate+NAD
phosphodihydroxyacetone+NADH+H +
The reaction rate is measured optically
at 340 nm wavelength.
Another option is the reduction of tetrazolium dye and colorimetric measurement:
NADH+H + + t e t r a z o l i u m d y e
formazan+NAD +
diaphorase
Another method uses Trinder’s reaction after prior oxidation of glycerophosphate by specific oxidase, with release of
Whole truth in one drop
H2O2. There is a risk of interference from
substances which are easily oxidated by
H2O2 (such as ascorbic acid).
APOLIPOPROTEINS
Concentration of apoB and apoAI reflects the concentration (number of molecules) of lipoproteins containing respectively CM, VLDL, LDL and HDL.
Concentrations of both apolipoproteins
are measured by immunochemical methods.
LIPOPROTEIN
ELECTROPHORESIS
Electrophoretic separation of particular classes of complete serum lipoprotein
molecules allows the evaluation of their
contents. Lipoprotein electrophoresis can
be carried out on different media such as
agarose gel, acetate cellulose, polyacrylamide gel and others. Chemical composition of lipoproteins allows to stein them
with lipophilic dyes. Bands formed by
particular lipoproteins have been named
in accordance with serum protein fractions of similar electrophoretic mobility:
a (HDL), pre-b (VLDL) and b (LDL). Due
to the possibility to measure cholesterol
in LDL and HDL, lipoprotein electrophoresis is nowadays rarely used.
References on request.
CORMAY International Bulletin
WORLD DIAGNOSTICS
Lipid metabolism
disorders as a cardiovascular
risk factor. Interpretation of test results
In accordance with dyslipidemia definition, while interpreting
test results, one must associate them with desired values
rather than “reference value range”.
Bogdan Solnica MD, PhD
Diagnostics Department of the Jagiellonian
University Collegium Medicum, Cracow
D
yslipidemia is a clinical condition
when lipid and lipoprotein concentrations do not match the desired values. Traditional Fredrickson classification of dyslipidemia, based on lipoprotein electrophoresis (Table 1) is less
frequently used in everyday practice nowadays. However it is still relevant due to
the option of indirect evaluation of lipoprotein concentration changes on the basis of TC, HDL-C, LDL-C and TG test results.
There are primary and secondary dyslipidemias. Primary lipid metabolism disorders are due to genetic structure de-
fects and impairment of the function of
apolipoproteins, receptors and enzymes
involved in lipid metabolism (Tab. 2)(1,2).
Secondary dyslipidemias are associated
with various disorders. They can also be
caused by some medicines (Tab. 3). Atherogenic dyslipidemia which develops in
metabolic syndrome and type 2 diabetes is
of the largest clinical significance amongst
secondary lipid/lipoprotein metabolism disorders. This disorder (also known as lipid
triad) involves high TG concentration, low
HDL-C concentration and presence of abnormal LDL molecules (small dense LDL,
sdLDL) prone to modification, such as oxidation. The basic pathogenetic factor leading to changes in the lipid profile is insulin resistance of skeletal muscles and of
the liver. It leads to:
• increased TG and VLDL synthesis in
the liver and impaired VLDL catabolism
(decreased LPL activity),
• increased exchange of EC and TG between HDL and triglyceride-rich lipoprotein
(increased CETP activity) and increased
excretion of HDL (increased HL activity),
Table 1. Fredrickson classification
of dyslipidemias
Type
Change in lipoprotein and lipid concentration
I
IIa
IIb
III
IV
V
CM, TG, TC
LDL, TC, LDL-C
LDL, VLDL, TC, LDL-C, TG
CMR, VLDLR, TC, TG
VLDL, TG
CM, VLDL, TG, TC
Table 2. Primary dyslipidemias
Condition
Hyperchylomicronemia
Familial hypercholesterolemia
Familial hypercholesterolemia
Familial defective apolipoprotein B100
Polygenic hypercholesterolemia
Complex familial hypercholesterolemia
Dysbetalipoproteinemia
Nr 3 (25), autumn 2012
Fredrickson types
I
II a
II a
II a
II a
II b
III
Defect
Impaired LPL activity
LDL receptor defect (homozygous)
LDL receptor defect LDL (heterozygous)
Decrease o apoB100 affinity to LDL receptor
Complex gene polymorphism
Increased apoB synthesis
Changed apoE
Whole truth in one drop
Frequency of occurrence
Rarely
1:1 000 000
1:500
1:700–1:1000
1:10–1:20
1:100
1:10 000
13
WORLD DIAGNOSTICS
Table 3. The most common secondary dyslipidemias
Reason
Visceral obesity, metabolic syndrome
type 2 diabetes
Hypothyroidism
Nephrotic syndrome
Chronic kidney disease
Liver diseases with cholestasis
Medicines: progestagens, corticosteroids,
antiretroviral (protease inhibitors), thiazides,
some ß-blockers
Table 4. Interpretation of LDL-C,
TC and HDL-C results
according to NCEP-ATP III
Concentration, mg/dl (mmol/l)
Interpretation
Change of lipoprotein and lipid concentrations
TG. HDL-C, genotype B LDL sdLDL (Atherogenic dyslipidemia)
TC, LDL-C, TG
TC, LDL-C
TG
TC, LDL-C, LpX
TC, LDL-C
genes, including adhesion molecules: intercellular adhesion molecule (ICAM-1)
and endothelial cell adhesion molecule
(VCAM-1) as well as chemotactic factors
such as monocyte chemoattractant protein (MCP-1). These molecules attract monocytes to the vascular wall, and once the
monocytes bind to endothelium and migrate to subendothelial area they transform into macrophages. Oxidatively modified LDL, mainly sdLDL is uptaken by macrophages by a scavenger receptor, whose
expression is not regulated by cholesterol
The role of lipid disorders in pathogenesis
of atherosclerosis has been known and documented
by numerous epidemiological and clinical studies
LDL cholesterol
<100 (<2.6)
100–129 (2.6–3.5)
130–159 (3.5–4.1)
160–189 (4.1–4.9)
>
– 4.9)
– 190 ( >
optimum
close to optimum
borderline
high
very high
Total cholesterol
<200 (<5.2)
200–239 (5.2–6.2)
>
– 240 ( >
– 6.2)
desired
borderline
high
HDL cholesterol
<40 <1.0)
>
– 60 (>
– 1.6)
low
high
Table 5. Expected values of lipid
parameters according to NCEPATP III, mg/dl (mmol/l)
Change of lipoprotein
and lipid concentrations
Total cholesterol
<200 (<5.2)
LDL cholesterol
<100 (<2.6)
HDL cholesterol – females
>50 (>1.3)
males
>40 (1.0)
Non-HDL cholesterol
<130 (3.4)
Triglycerides
<150 (1.7)
Type
• increased exchange of EC and TG between HDL and triglyceride-rich lipoprotein and TG hydrolysis into LDL
with transformation into sdLDL.
It must be emphasised that dyslipidemias occur quite often and are not diagnosed in a number of people. Frequency
of polygenic hypercholesterolemia may
even reach 10% of the population and artherogenic dyslipidemia 20 to 30%(2).
14
THE ROLE OF LIPIDS
IN ATHEROGENESIS
The role of lipid disorders in pathogenesis of atherosclerosis has been known
and documented by numerous epidemiological and clinical studies. Escalated
atherosclerotic changes and increased
cardiovascular risk are in the clinical picture of primary and secondary hypercholesterolemia and dyslipidemia which is
atherogenic. Beginning with the famous
Framingham Heart Study, in many further
studies a relationship between certain types of dyslipidemia and incidence proportion and mortality due to circulatory disorders and as well as decrease of cardiovascular risk due to hypolipemic treatment.
Lipid metabolism disorders resulting in imbalance between
transport of lipids into tissues
and return transport of cholesterol esters to the liver,
cause accumulation of low
density lipoprotein molecules in the subendothelial area of artery walls.
These molecules take
part in initiation of various stages of atherogenesis.
Penetration of native
and oxidised LDL molecules
(ox-LDL) to endothelial cells
damages them. It activates transcription factors such as nuclear factor kB (NF-kB). They increase transcription of inflammatory response mediator
Whole truth in one drop
content in a cell. It leads to loading macrophages with cholesterol esters, forming
lipid droplets, which transforms them into
foam cells. Nevertheless, macrophages
activated by LDL and ox-LDL secrete
a number of proinflammatory cytokines.
Furthermore LDL and ox-LDL cause phenotypic modulation of vascular wall smooth muscle cells towards cells which proliferate and synthetise contents o intercellular matrix, such as collagen and glycosaminoglycans and collect cholesterol esters. Modulated macrophages and myocytes form atherosclerotic plaque. Atherosclerosis is considered an inflammatory
CORMAY International Bulletin
WORLD DIAGNOSTICS
SCORE risk card
females
smokers
non-smokers
non-smokers
smokers
180
19 23 26 31 36
36 41 47 53 59
180
10 12 14 17 20
160
14 17 19 23 27
26 31 35 41 46 65
160
7
8 10 12 14
14 17 19 23 27 65
140
10 12 14 16 19
19 22 26 30 35
140
5
6
7
8 10
10 12 14 16 19
120
7
8 10 12 14
14 16 19 22 26
120
3
4
5
6
180
13 15 17 20 24
24 28 32 37 43
180
5
6
7
9 10
160
9 10 12 15 17
17 20 24 28 32 60
160
4
4
5
6
140
2
3
4
4
120
2
2
2
3
180
3
3
4
4
160
2
2
3
140
1
2
2
120
1
1
180
2
7
20 23 27 31 36
7
8 10 12 14
10 12 14 17 20
7
9 10 12 14 60
5
5
6
7
8 10
3
3
4
5
6
5
5
6
8
9 11
3
4
4
4
5
6
7
2
3
3
3
4
4
5
1
2
2
2
2
3
3
4
2
2
3
3
3
4
4
5
6
7
9 10 12
12 14 17 20 23
120
5
6
9
9 10 12 14 17
180
8 10 11 13 16
16 19 22 26 30
160
6
7
140
4
5
6
7
8
11 13 16 18 22 55
8 9 11 13 16
120
3
3
4
5
5
6
180
5
6
7
9 10
160
4
4
5
6
7
7
1
1
2
2
2
2
3
3
4
4
3
3
4
4
5
5
9 10 12 14 50
6 7 9 10
160
140
140
1
1
1
1
1
2
2
2
3
3
120
2
2
2
3
4
4
4
5
6
7
120
1
1
1
1
1
1
1
1
2
2
180
2
2
3
3
4
4
5
5
6
8
180
1
1
1
1
1
1
1
1
2
2
160
1
2
2
2
3
3
3
4
4
5
160
0
0
1
1
1
1
1
1
1
1
140
1
1
1
2
2
2
2
3
3
4
140
0
0
0
0
0
1
1
1
1
1
120
1
1
1
2
2
1
2
2
2
3
120
0
0
0
0
0
0
0
0
1
1
4
5
6
7
8
4
5
6
7
8
4
5
6
7
8
4
5
6
7
8
8
7
9 11
7
8
9 11
10 12 15 17 20
45
systolic blood pressure (mmHg)
6
4
7
(mmol/l)
(mmol/l)
(mmol/l)
(mmol/l)
150 190 230 270 310
150 190 230 270 310
150 190 230 270 310
150 190 230 270 310
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
total cholesterol
55
age
7
140
age
systolic blood pressure (mmHg)
males
50
45
total cholesterol
10-year risk of cardiac death
>_ 15%
10–14%
5–9%
3–4%
2%
1%
< 1%
Fig. SCORE risc card (according to 2)
based condition. However lipid metabolism disorders leading to accumulation of
native and oxidised LDL are considered to
be the factor behind endothelial cell damage and initiation of inflammatory processes, covering the vascular wall(1,2).
INTERPRETATION OF LIPID TEST
RESULTS
Lipid profile covers serum total cholesterol, HDL and LDL cholesterol as well as
triglycerides. All or a portion of these tests are performed with aim to detect dyslipidemia and afterwards to monitor hypolipemic treatment. Diagnosis of dyslipideNr 3 (25), autumn 2012
mia must include how it affects the cardiovascular risk and risk of other disorders (like pancreatitis in hypertriglyceridemia).
It must be stressed that in accordance
to dyslipidemia definition, in order to interpret the results, they must be referred
to desired (target) values, rather than to
„reference value ranges”. In accordance
to the current state of knowledge, desired
values of lipid profile tests, mainly LDL-C,
HDL-C and TG are not associated with increased cardiovascular risk (Tab. 4,5)(2,3).
Lipid profile test results exceeding desired
values indicate the need of therapeutic intervention (change of lifestyle, change of
Whole truth in one drop
diet, medications) as a part of primary or
secondary preventive measures against
cardiovascular episodes. The aim of dyslipidemia treatment is to obtain desired values of lipid profile tests.
Dyslipidemia is not the only cardiovascular risk factor so lipid test results, mainly LDL-C, TC and HDL-C should be
analysed in context of other risk factors.
Cardiology societies recommend
SCORE card (Systematic Coronary Risk
Evaluation) as a tool for cardiovascular
risk evaluation(2,4).
References on request.
15
DIAGNOSTICS UNDER MAGNIFYING GLASS
Small Dense LDL
Cholesterol and
Coronary Heart Disease
Results from the Framingham Offspring Study
Masumi Ai1, Seiko Otokozawa1, Bela F. Asztalos1,
Yasuki Ito2, Katsuyuki Nakajima1, Charles
C. White3, L. Adrienne Cupples3, Peter W. Wilson4,5
and Ernst J. Schaefer1*
BJECTIVE: Wesought to establish
reference values for a new direct
assay for small dense LDL cholesterol (sdLDL-C) and to measure sdLDL-C
concentrations in patients with established
coronary heart disease (CHD) vs controls.
METHODS: Direct LDL-C and sdLDL-C
were measured in samples from 3188 male
and female participants of the Framingham
Offspring Study, including 173 men and 74
women with CHD.
RESULTS: Postmenopausal status and
male sex were associated with higher sdLDL-C concentrations (P < 0.0001). Cholesterol-lowering medication use was more frequent (P < 0.0001) in CHD patients than in
controls (46.8% vs 11.4% in men; 35.1%
vs 8.8% in women). In men, mean LDL-C
was lower in CHD than in controls (3.22
O
were found in 10.4% and 22.0% of men and
in 24.3% and 27.8% of women with CHD,
respectively.
CONCLUSIONS: Despite 4-fold greater
cholesterol-lowering therapy use, CHD patients had mean LDL-C concentrations above the LDL-C goal of <2.6 mmol/L (<100
mg/dL). Although women with CHD had higher sdLDL-C concentrations than controls,
this difference was not seen in men. These
findings may explain some of the high residual risk of future CHD events in CHD patients.
Increased plasma LDL cholesterol (LDL-C)(1)
concentrations have been shown to be a significant risk factor for coronary heart disease (CHD), and the use medications that
decrease LDL-C concentrations has been
shown to reduce heart disease risk(1)(2). LDL
is comprised of a variety of different subfractions that can be separated by ultracentrifugation, gradient gel electrophoresis,
nuclear magnetic resonance, and specific
precipitation methods(3)(4)(5)(6)(7). Gradient gel
Increased plasma LDL cholesterol (LDL-C)(1)
concentrations have been shown to be a significant
risk factor for coronary heart disease
vs 3.51 mmol/L, P < 0.0001), whereas mean sdLDL-C concentrations were similar
(0.83 vs 0.84 mmol/L, P = 0.609). In women, mean LDL-C was similar in CHD and
controls (3.53 vs 3.46 mmol/L, P = 0.543),
but mean sdLDL-C was higher (0.83 vs 0.68
mmol/L, P = 0.0015). The mean percentage of LDL-C as sdLDL-C was higher in both
men and women with CHD than controls
(P < 0.01). Increased LDL-C and sdLDL-C
16
Whole truth in one drop
electrophoresis and nuclear magnetic resonance imaging are semiquantitative methods. For nuclear magnetic resonance analysis a substantial number of assumptions
are required to estimate concentrations
of LDL subfractions as well as total LDL
particle number(8)(9). Hirano and colleagues
have developed a method that uses heparin
sodium salt precipitation followed by centrifugation for measuring small dense LDL
CORMAY International Bulletin
DIAGNOSTICS UNDER MAGNIFYING GLASS
Table 1.
Characteristics and plasma lipid concentrations in men and women participating in the FOS (cycle 6) without
CHD, diabetes mellitus, cholesterol-lowering medications, and hormone-replacement therapies.a
Men
(n = 1080)
Variable
Age, years
Body mass index, kg/m2
Total cholesterol, mmol/l
Triglycerides, mmol/l
HDL-C mmol/l
Calculated LDL-C, mmol/l
Direct LDL-C, mmol/l
sdLDL-C, mmol/l
sdLDL-C jako % LDL-C
Large LDL-C mmol/l
LDL-C <2.6 mmol/L, %
LDL-C >4.2 mmol/L, %
sdLDL-C <0.5 mmol/L, %
sdLDL-C >1.0 mmol/L, %
57.1 (9.7)
28.2 (4.3)
5.21 (0.92)
1.29 (0.91–1.81)
1.16 (0.32)
3.38 (0.82)
3.54 (0.84)
0.82 (0.39)
22.9 (8.9)
2.72 (0.69)
12.2
23.8
24.1
26.5
Women
(n = 1012)
57.4 (10.5)
27.1 (5.6)
5.47 (1.02)
1.13 (0.82–1.60)
1.48 (0.39)
3.39 (0.92)
3.49 (0.94)
0.67 (0.41)
18.6 (8.7)
2.82 (0.75)
16.9
22.7
43.5
13.6
P
0.4851
<0.0001
<0.0001
<0.0001b
<0.0001
0.7218
0.1540
<0.0001
<0.0001
0.0013
0.0024c
0.5631c
<0.0001c
<0.0001c
a V Values are expressed as mean (SD), median (25th–75th percentile), or percentage.
b P obtained by x2 test.
c P obtained after log transformation of the data.
Table 2.
Characteristics and plasma lipid concentrations in premenopausal and postmenopausal women participating
in the FOS (cycle 6) without CHD, diabetes mellitus, cholesterol-lowering medications, and hormone-replacement
therapies.a
Variable
Premenopausal
(n = 313)
Postmenopausal
(n = 698)
P
Age, years
Body mass index, kg/m2
Total cholesterol,mmol/l
Triglycerides, mmol/l
HDL-C mmol/l
Calculated LDL-C, mmol/l
Direct LDL-C, mmol/l
sdLDL-C, mmol/l
sdLDL-C jako % LDL-C
Large LDL-C mmol/l
LDL-C <2.6 mmol/L, %
LDL-C >4.2 mmol/L, %
sdLDL-C <0.5 mmol/L, %
sdLDL-C >1.0 mmol/L, %
46.4 (5.0)
26.7 (5.5)
5.03 (0.94)
1.07 (0.72–1.35)
1.47 (0.39)
3.05 (0.88)
3.17 (0.91)
0.55 (0.38)
17.0 (9.1)
2.62 (0.73)
28.1
12.5
60.7
8.6
62.4 (8.3)
27.2 (5.6)
5.68 (0.99)
1.20 (0.89–1.73)
1.48 (0.39)
3.55 (0.89)
3.63 (0.93)
0.72 (0.41)
19.3 (8.4)
2.92 (0.74)
11.9
27.4
35.9
15.6
<0.0001
0.1622
<0.0001
<0.0001b
0.6906
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001c
<0.0001c
<0.0001c
0.0025c
a V Values are expressed as mean (SD), median (25th–75th percentile), or percentage.
b P obtained by x2 test.
c P obtained after log transformation of the data.
cholesterol (sdLDL-C) directly. Their results
with this method correlated highly with the
measurement of cholesterol in sdLDL at
a density between 1.044 and 1.063 g/mL
as isolated by ultracentrifugation(10)(11)(12)(13).
We used this assay along with direct lipoprotein cholesterol to measure sdLDL concentrations in participants in cycle 6 of the
Framingham Offspring Study (FOS). Our
goals were to determinine reference values
for this new assay and identify possible differences in results in CHD cases vs controls.
M ATERIALS
AND
METHODS
STUDY PARTICIPANTS
Participants in the FOS, a long-term
community-based prospective observational
study of risk factors for CHD, are the offspring and their spouses of the original Framingham Heart Study cohort(14)(15)(16). During
cycle 6 of the FOS (1995–1998) standardized medical history data were collected
from all participants and they underwent
Nr 3 (25), autumn 2012
a physical examination including measurement of fasting lipid concentrations. All
samples were stored in our laboratory at 80°C and were not thawed until we used
them for analysis. Selection criteria for the
CHD patients at cycle 6 included a history
of myocardial infarction, acute coronary insufficiency, or angina pectoris. None of the
participants had acute coronary syndrome
at the time of the examination. We performed our analyses on all available plasma
samples from male and female participants
in cycle 6. To determine a reference value
for sdLDL-C, we selected male and female
participants of FOS (cycle 6) without CHD
or diabetes and not taking cholesterol-lowering medications or hormonal replacement
therapy (1080 men and 1012 women). We
determined sex differences in the healthy
population. In healthy women, we also
determined differences between premenopausal (n = 313) and postmenopausal
(n = 698) women. We also determined differences between CHD cases and controls
Whole truth in one drop
(individuals with no evidence of CHD at
cycle 6) of each sex. There were 173 male
CHD cases and 1335 male controls,
and 74 female cases and 1606 female controls. We did not exclude patients on cholesterol-lowering medication from this analysis laboratory measurements.
L ABORATORY MARKERS
Total cholesterol, triglyceride, and HDL
cholesterol (HDL-C) were determined by
standard enzymatic methods. HDL-C was
measured after isolation of the HDL supernatant following dextran sulfate magnesium
precipitation(17). LDL-C was calculated by
using the Friedewald formula(18). For the
purposes of this study, we used archived
plasma samples that had been frozen at 80°C and never previously thawed for the
assessment of direct LDL-C and sdLDL-C
by automated standardized enzymatic analysis on a Hitachi 911 automated analyzer.
The kits used for these tests (LDL-C and
sdLDL-C) were provided by Denka Seiken,
Tokyo, Japan. The precipitation reagent
(0.1 mL) contained 150 U/mL of heparin-sodium salt (Sigma) and 90 mmol/L of
MgCl2 (Nakarai), and was added to 0.1 mL
of plasma, mixed, and incubated for 10 min
at 37°C(10)(11). The samples were then placed
in an ice bath for 15 min, and then the precipitate was collected by centrifugation
at 21000g for 15 min at 4°C(10)(11). Aliquots
of the supernatant were used for measurement of the cholesterol concentration.
Within- and between-run CVs for the direct LDL-C assay were 0.77% and 1.30%
and for sdLDL-C were 4.99% and 4.67%,
respectively. We calculated large LDL-C
concentrations as: direct LDL-C – sdLDL-C,
and we also calculated the percentage
of LDL-C as sdLDL-C based on direct measurements. Assays for direct LDL and
sdLDL-C have previously been calibrated
and directly compared with concentrations
obtained after isolation of LDL and sdLDL
by ultracentrifugation, and very similar results were obtained(10). When we compared
concentrations obtained for direct LDL-C
and sdLDL-C in fresh plasma (n = 20) vs
concentrations obtained in plasma stored
at -80°C for 3 months, we obtained virtually identical results. All laboratory personnel were blinded with regard to the clinical
status of study participants. In addition we
have not previously observed any effects on
plasma measurements of either LDL or
HDL particles from use of frozen samples
provided the samples were stored at -80°C
and never thawed until just before use, and
then thawed rapidly at 37°C in a water
bath(16)(19). Moreover, this issue has been
checked and verified by the manufacturer
of the sdLDL-C assay, Denka-Seiken. In addition, we received a direct communication
17
DIAGNOSTICS UNDER MAGNIFYING GLASS
Table 3.
Means and selected percentiles of direct LDL-C, calculated LDL-C, small dense LDL-C, large LDL-C, and sdLDLC/LDL-C ratio in participants of the FOS (cycle 6) without CHD, diabetes mellitus, cholesterol-lowering
medications, and hormone-replacement therapies, according to sex and menopausal status for women.a
Mean
Variable
Direct LDL-C, mmol/l
Men
Women
Premenopausal
Postmenopausal
Calculated LDL-C, mmol/l
Men
Women
Premenopausal
Postmenopausal
sdLDL-C mmol/l
Men
Women
Premenopausal
Postmenopausal
Large LDL-C, mmol/l
Men
Women
Premenopausal
Postmenopausal
% of LDL-C as sdLDL-C
Men
Women
Premenopausal
Postmenopausal
Percentiles
10.
25.
50.
75.
90.
1080
1012
313
698
3.54 (0.84)
3.49 (0.94)
3.17 (0.91)
3.63 (0.92)
2.49
2.32
2.15
2.51
2.96
2.85
2.47
3.00
3.51
3.41
3.04
3.50
4.10
4.08
3.74
4.22
4.67
4.74
4.24
4.86
1074
1008
313
694
3.38 (0.82)
3.39 (0.92)
3.04 (0.88)
3.55 (0.89)
2.35
2.29
2.08
2.44
2.81
2.78
2.41
2.98
3.33
3.32
2.99
3.47
3.93
3.90
3.54
4.08
4.43
4.63
4.10
4.72
1080
1011
313
697
0.82 (0.39)
0.67 (0.41)
0.55 (0.38)
0.72 (0.41)
0.36
0.27
0.23
0.30
0.53
0.38
0.32
0.42
0.75
0.57
0.45
0.64
1.05
0.86
0.65
0.91
1.36
1.17
0.99
1.21
1080
1011
313
697
2.72 (0.69)
2.83 (0.75)
2.62 (0.73)
2.92 (0.74)
1.89
1.90
1.76
1.98
2.24
2.34
2.06
2.44
2.70
2.75
2.59
2.83
3.14
3.32
3.11
3.40
3.56
3.82
3.51
3.92
1080
1011
313
697
22.9 (9.0)
18.6 (8.7)
17.0 (9.1)
19.3 (8.4)
12.4
9.3
8.7
9.7
16.3
12.7
11.5
13.5
21.8
16.8
14.6
17.7
28.4
23.2
20.7
23.9
35.1
29.5
27.4
30.4
a Calculated LDL-C calculated by Friedewald formula; % of LDL-C as sdLDL-C calculated by using direct measurements.
from the developer of this assay (Tsutomu
Hirano, personal communication, January 23, 2010), who reported that results
obtained on EDTA plasma samples frozen
at -80°C and never thawed until analysis
were virtually identical to those obtained
using fresh plasma.
Results of experiments carried out at
Denka Seiken, the assay manufacturer, indicated that storage of serum at 4°C and use
for the assay within 7 days of sampling resulted in no significant change in the concentrations obtained. Changes in the results
began to be seen with sera stored longer
than 7 days at 4°C. Studies on frozen sera
and plasma were also conducted, and results confirmed that whereas frozen EDTA
plasma was suitable for this assay, serum or
heparinized plasma were not. The manufacturer generated data on the stability of EDTA plasma over 2 years when stored at 80°C. The manufacturer has noted, as have
we, that quick freezing of the
samples following collection is most important
for future sdLDL-C
measurement
using heparin-magnesium
precipitation.
All plasma
samples from
the FOS were
collected in
18
EDTA and were quick frozen, stored at 80°C, and never thawed until used in this
investigation.
STATISTICAL ANALYSIS
Descriptive statistics, mean (SD) for continuous variables or proportions for categorical variables, were computed for all study
variables and all study groups. The distribution of the variables was compared between
individuals with or without prevalent CHD,
by using 2-sample t-tests (with log-transformed data, if necessary) for continuous variables and x2 tests for categorical variables.
R ESULTS
Data on male and female participants in
the FOS (cycle 6) without CHD or diabetes
and not taking cholesterol-lowering medications or hormone replacement therapy are
provided in Table 1. Men and women had
similar ages; however, body mass index, waist circumference, systolic and diastolic blood pressure, prevalence of use of antihypertensive treatment and aspirin, and drinking
more than 1 alcoholic beverage per week
were all significantly higher (P < 0.0001) in
men than in women (see Table 1 in the Data Supplement that accompanies the online
version of this article at http://www.clinchem.org/content/vol56/issue6).
Women had significantly higher concentrations of total cholesterol and HDL-C
than men (P < 0.0001), whereas men had
Whole truth in one drop
significantly higher concentrations of triglyceride and higher total cholesterol/HDL-C
ratios than women (P < 0.0001). Men and
women had similar concentrations of non-HDL-C and LDL-C, but men had significantly higher sdLDL-C concentrations and
higher LDL as sdLDL-C percentages than
women (P < 0.0001). Men were less likely
to have an LDL-C concentration in the optimal range of <2.6 mmol/L (<100 mg/dL)
as defined by the National Cholesterol Education Program(20). Men also were less likely to
have sdLDL-C concentrations <0.5 mmol/L
(<20 mg/dL) than women. This threshold
was based on approximate 25th-percentile concentrations in control individuals.
Moreover, men were more likely to have
increased sdLDL-C concentrations in
excess of 1.0 mmol/L (40 mg/dL) than
women. This threshold was based on approximate 75th-percentile concentrations
in the control male population. In Japan
the cutpoint for an increased sdLDL-C
CORMAY International Bulletin
DIAGNOSTICS UNDER MAGNIFYING GLASS
Table 4.
Characteristics and plasma lipid concentrations in male FOS (cycle 6) participants with and without CHD.a
Variable
(n = 1335)
Age, years
Body mass index, kg/m2
Body mass index >30, %
Waist (cm)
Waist >102 cm, %
Systolic Blood Pressure (mmHg)
Diastolic blood pressure, mmHg
Hypertension, %
Hypertensive treatment, %
Taking aspirin regularly, %
Diabetes mellitus, %
Oral glycemic-control drug users, %
On insulin treatment, %
ß-Blocker users, %
Cigarette smokers, %
Alcohol use >1 drink/week, %
Cholesterol-lowering drug users, %
Total cholesterol, mmol/L
Triglycerides, mmol/L
HDL-C, mmol/L
Total cholesterol/HDL-C ratio
Non-HDL-C, mmol/L
Calculated LDL-C, mmol/L
Direct LDL-C, mmol/L
sdLDL-C, mmol/L
% of LDL-C as sdLDL-C
Large LDL-C, mmol/L
Fasting glucose, mmol/L
LDL-C < 2.6 mmol/L, %
LDL-C >4.2 mmol/L, %
sdLDL-C < 0.5 mmol/L, %
sdLDL-C >1.0 mmol/L, %
Triglyceride >1.7 mmol/L, %
Non-HDL-C < 3.4 mmol/L, %
Non-HDL-C >4.9 mmol/L, %
58.0 (9.7)
28.5 (4.4)
30.1
101.3 (10.9)
42.6
129.6 (17.1)
77.7 (9.3)
41.9
27.7
31.1
11.8
4.4
1.4
9.4
14.5
54.5
11.4
5.17 (0.93)
1.33 (0.94–1.87)
1.16 (0.32)
4.66 (3.78–5.64)
4.02 (0.93)
3.32 (0.82)
3.51 (0.84)
0.84 (0.41)
23.7 (9.5)
2.67 (0.70)
5.89 (1.49)
13.4
22.4
23.3
27.6
31.1
23.0
15.6
(n = 173)
65.3 (7.9)
28.7 (4.3)
31.8
102.4 (10.9)
46.2
129.4 (17.6)
73.4 (9.5)
66.5
58.5
78.0
29.5
12.2
4.6
54.9
12.7
46.8
46.8
4.81 (0.95)
1.43 (1.05–2.11)
1.04 (0.28)
4.72 (3.85–5.47)
3.75 (0.85)
2.99 (0.78)
3.22 (0.81)
0.83 (0.39)
26.1 (10.0)
2.39 (0.69)
6.45 (1.89)
22.0
10.4
15.0
22.0
38.2
31.4
5.3
P for differences
<0.0001
0.6249
0.6542b
0.1922
0.3669b
0.8633
<0.0001
<0.0001b
<0.0001b
<0.0001b
<0.0001b
<0.0001b
0.0029b
<0.0001b
0.5190b
0.0563b
<0.0001b
<0.0001
0.0127c
<0.0001
0.5369c
0.0002
<0.0001
<0.0001
0.6094c
0.0019c
<0.0001
0.0003
0.0026b
0.00033b
0.0141b
0.1157b
0.0607b
0.0151b
0.0003b
a V Values are expressed as mean (SD), median (25th –75th percentile), or percentage.
b P obtained by x2 test.
c P obtained by using log-transformed data.
concentration has been established as
>0.9 mmol/L (>35 mg/dL), similar to
what we have observed in Framingham.
Men also were significantly more likely to
have increased total triglyceride concentrations above 1.7 mmol/L (150 mg/dL)
and less likely to have non–HDL-C concentrations of <3.4 mmol/L
Nr 3 (25), autumn 2012
(130 mg/dL) (see online Supplemental
Table 1). These latter cutpoints were identified by the National Cholesterol Education Program Adult Treatment Panel III(20).
Information on the differences between
premenopausal and postmenopausal women is provided in Table 2. Postmenopausal
women had significantly higher total cholesterol, triglyceride, total cholesterol/HDL-C
ratio, non-HDL-C, calculated LDL-C,
direct LDL-C, and
Whole truth in one drop
sdLDL-C concentrations than did premenopausal women (P < 0.0001). Postmenopausal women also had significantly higher
large LDL-C and higher fasting glucose
concentrations. Postmenopausal women
had significantly greater waist circumferences and higher systolic blood pressure, and
they were more likely than premenopausal
women to have a history of hypertension
and to be on antihypertensive therapy as
well as to be taking aspirin (P < 0.0001)
(see online Supplementary Table 2).
19
DIAGNOSTICS UNDER MAGNIFYING GLASS
Table 5.
Characteristics and plasma lipid concentrations in female FOS (cycle 6) participants with and without CHD.a
Variable
Age, years
Body mass index, kg/m2
Body mass index 30, %
Waist, cm
Waist >102 cm, %
Systolic blood pressure, mmHg
Diastolic blood pressure, mmHg
Hypertension, %
Hypertensive treatment, %
Taking aspirin regularly, %
Diabetes mellitus, %
Oral glycemic-control drug users, %
On insulin treatment, %
ß-Blocker users, %
On estrogen therapy, %
Postmenopause, %
Cigarette smokers, %
Alcohol use >1 drink/week, %
Cholesterol-lowering drug users, %
Total cholesterol, mmol/L
Triglycerides, mmol/L
HDL-C, mmol/L
Total cholesterol/HDL-C ratio
Non-HDL-C, mmol/L
Calculated LDL-C, mmol/L
Direct LDL-C, mmol/L
Small dense LDL-C, mmol/L
% of LDL-C as sdLDL-C
Large LDL-C, mmol/L
Fasting glucose, mmol/L
LDL-C 2.6 mmol/L, %
LDL-C 4.2 mmol/L, %
sdLDL-C 0.5 mmol/L, %
sdLDL-C 1.0 mmol/L, %
Triglyceride 1.7 mmol/L, %
Non-HDL-C 3.4 mmol/L, %
Non-HDL-C 4.9 mmol/L, %
P for differences
58.1 (9.6)
27.3 (5.7)
25.8
93.9 (14.8)
26.3
126.5 (19.6)
73.9 (9.1)
35.8
22.9
19.8
8.3
2.5
1.2
8.8
26.3
76.5
15.5
30.5
8.8
5.48 (0.99)
1.25 (0.88–1.84)
1.48 (0.41)
3.71 (2.99–4.64)
3.98 (1.03)
3.31 (0.89)
3.46 (0.93)
0.68 (0.42)
19.0 (8.9)
2.79 (0.73)
5.54 (1.42)
17.3
21.8
42.3
15.3
29.5
27.3
17.5
66.1 (8.0)
29.1 (5.4)
35.1
101,1 (13.3)
39.7
140.4 (24.6)
73.6 (11.6)
83.8
73.0
62.2
29.7
10.8
9.5
47.3
23.0
94.6
18.9
21.6
35.1
5.58 (1.02)
1.53 (1.07–2.05)
1.40 (0.42)
4.09 (3.47–4.70)
4.18 (0.99)
3.33 (0.82)
3.53 (0.87)
0.83 (0.44)
23.6 (12.8)
2.70 (0.78)
6.54 (2.48)
13.5
24.3
27.0
27.8
40.5
13.5
13.7
<0.0001
0.0082
0.0757b
<0.0001
0.0110b
<0.0001
0.8502
<0.0001b
<0.0001b
<0.0001b
<0.0001b
<0.0001b
<0.0001b
<0.0001b
0.5307b
0.0003b
0.4322b
0.1035b
<0.0001b
0.3887
0.0030c
0.0725
0.0233c
0.1177
0.9241
0.5430
0.0015c
0.0003c
0.3420
0.0009
0.4040b
0.6068b
0.0090b
0.0045b
0.0417b
0.0089b
0.3999b
a V Values are expressed as mean (SD), median (25th –75th percentile), or percentage.
b P obtained by x2 test.
c P obtained by using log-transformed data.
Selected percentile concentrations for direct LDL-C, calculated LDL-C, and sdLDL-C
for healthy men and women are presented in
Table 3. The Adult Treatment Panel of the National Cholesterol Education Program has selected approximate 75th-percentile concentrations (4.15 mmol/L or 160 mg/dL) for LDL-C
as being associated with high CHD risk.
The 75th percentile for direct LDL-C
was 4.10 mmol/L in men and 4.08 mmol/L in
women, with somewhat lower values for calculated LDL-C. The 75th percentile for
sdLDL-C was 1.05 mmol/L (40 mg/dL) in
men, 0.91 mmol/L (35 mg/dL) in postmenopausal women, and 0.65 mmol/L (25 mg/dL)
in premenopausal women. These concentrations are very similar to reference values measured in fresh plasma or serum that we have generated in the US and that have been
generated in Japan.
Data comparing male CHD cases and
controls are presented in Table 4. Men
with CHD had significantly higher mean
20
age and diastolic blood pressure, and were more likely to have hypertension, to be
on therapy for hypertension, to be taking
aspirin, to have diabetes, to be on oral
glycemic control medication or insulin, to
use beta blockers, and to be on cholesterol-lowering medications than controls (P
< 0.0001). The percentage of men taking cholesterol-lowering medications
was 11.4% in controls and 46.8% in men
with CHD. Among all individuals on cholesterol-lowering therapy, 88% were on
statin monotherapy, 5% were on statins
plus another agent (mainly a fibrate), and
the remainder (7%) were on a fibrate, niacin, or resin monotherapy. Very similar
distributions were seen in male and female study participants with CHD, as well as
in male and female controls on cholesterol-lowering treatment. Therefore, overall, 93% of CHD patients who were receiving cholesterol-lowering medication were receiving some form of statin therapy.
Whole truth in one drop
The recommended goal for patients with
established CHD, based on the National
Cholesterol Education Program, is an LDL-C <100 mg/dL or 2.6 mmol/L(20). The mean direct LDL-C in men with CHD was
3.2 mmol/L, and only 22% of men with
CHD had an LDL-C below the recommended target. Direct LDL-C concentrations
were used for this calculation; however, very similar percentages were obtained when
calculated LDL-C was used (e.g., 25%).
Male CHD patients had significantly lower
mean total cholesterol, but their mean triglyceride concentrations were higher and
their mean HDL-C concentrations significantly lower than those in controls, as were
their mean calculated LDL-C and direct LDL-C. Despite these results, sdLDL-C
did not differ significantly between men with
CHD and controls, and men with CHD had
higher percentages of LDL-C as sdLDL-C
and significantly higher fasting glucose concentrations than controls.
A very similar pattern was observed for
the women, but the differences between cases and controls were even greater than for
the men (Table 5). Compared with controls,
women with CHD were significantly older
had a greater mean body mass index and waist circumference and a higher systolic blood
pressure. Women with CHD were also more
likely than controls to have hypertension and
to be on antihypertensive treatment, to be
taking aspirin regularly; to be diabetic and
be taking medications for diabetes; to be on
ß blockers, to be on cholesterol-lowering
medication, and to be postmenopausal.
Despite the fact that about 4 times as
many women were receiving cholesterol-lowering medication in cases than in controls
(35.1% vs 8.8%), the total cholesterol concentrations were similar between cases and
controls, and the mean triglyceride concentration was significantly higher, as was the
mean total cholesterol/HDL-C ratio. Calculated LDL-C and direct LDL-C concentrations were similar, whereas sdLDL-C concentrations were significantly higher in female CHD cases than in controls. Substantially higher numbers of women with CHD
had sdLDL-C >1.0 mmol/L compared to
controls. Only 13.5% of female CHD cases
were at the recommended LDL-C goal of
<2.6 mmol/L.
Because there were significant differences between CHD cases and controls in rates of the use of cholesterol-lowering medication, we sought to determine whether the
differences we observed in lipid concentrations persisted when we excluded study participants who were on cholesterol-lowering
medications, whether they were controls or
CHD patients. For men, only 92 CHD cases and 1181 controls remained, and for
women 48 cases and 1464 controls remaCORMAY International Bulletin
DIAGNOSTICS UNDER MAGNIFYING GLASS
ined. In these analyses no significant differences between cases and controls were observed for either men or women with regard
to calculated or direct LDL-C or sdLDL-C
concentrations. It should be noted, however, that physicians are less likely to put
CHD patients on cholesterol-lowering medications if they are at or close to their LDL-C goal, and the sample size was small. Prospective studies to be carried in this population in the future will provide better insight with regard to the utility of this assay for
CHD risk assessment.
DISCUSSION
SdLDL can be assessed by ultracentrifugation, gradient gel electrophoresis, or nuclear magnetic resonance spectroscopy, and
studies indicate that patients with CHD have higher concentrations than healthy individuals. However, these methods are labor-intensive and available only in advanced lipid-testing laboratories, and they have not
been well standardized. In this study, we
evaluated sdLDL-C for the first time in a US
population with a method that uses precipitation followed by centrifugation and the
measurement of cholesterol concentrations
on an automated analyzer. We have generated reference values that agree well with
concentrations obtained with fresh plasma.
A major limitation of our study was that
we used plasma stored at -80°C, and there
is no guarantee that we would have obtained
the same results had we used fresh plasma.
However, results of comparison studies performed by us and in Japan indicate virtually
identical results with the use of fresh vs frozen plasma for sdLDL-C. Moreover, the reference values that we obtained were similar
to those obtained in the Japanese population
by using fresh plasma or serum samples. Results of case-control studies have demonstrated that CHD cases are more likely than
controls to have increased plasma triglyceride and sdLDL concentrations and decreased
HDL and large HDL concentrations
(3)(4)(5)(6)(7)(8)(9)(12)(13)(16)
.The sdLDL-C assay studied here has been applied to Japanese
CHD cases and controls, and increased sdLDL-C has been linked to the
presence of CHD and to the severity
of coronary disease as assessed by angiography(12)(13). For LDL particles as
assessed by gel electrophoresis, the
presence of increased sdLDL has been associated with increased CHD
risk in the prospective Quebec Cardiovascular Study(21). In addition,
increased total LDL particle number as determined by nuclear magnetic resonance was associated
with increased carotid intimal-medial
wall thickness in the Multi-Ethnic Study
of Atherosclerosis(22). Moreover, increased
Nr 3 (25), autumn 2012
sdLDL and total LDL particle numbers predicted recurrent CHD events in the Veteran
Affairs HDL Intervention study, and these
parameters were favorably affected by gemfibrozil treatment(23). Most recently, the use
of a novel ion mobility assessment of lipoprotein subspecies revealed 3 different lipoprotein patterns that have been linked to
CHD risk in a Swedish population: (a) increased LDL, (b) decreased HDL, and (c) increased triglycerides and sd LDL and decreased
large HDL(24).
Many studies have confirmed the link
between increased triglycerides, decreased
HDL, and increased total and sdLDL, especially in individuals with metabolic syndro-
stance, visceral adiposity, CRP, triglycerides,
and sdLDL-C(25). Dietary trans fatty acids also increase sdLDL-C significantly(26). Placebo-controlled trials of primary and secondary prevention by use of statin treatment
have demonstrated great benefit in CHD
risk reduction associated with reductions
in LDL-C concentrations, but very substantial residual CHD risk remains(1)(2). Recently
in a large randomized placebo-controlled
trial in individuals with normal LDL-C and
increased C-reactive protein concentrations, study participants who received rosuvastatin at a dose of 20 mg/day and who
got their LDL-C concentrations to <70
mg/dL and their CRP concentrations to
Many studies have confirmed the link between
increased triglycerides, decreased HDL,
and increased total and sdLDL
me(8). Such individuals also frequently have
increased waist circumference and insulin
resistance, and increased concentrations
of C reactive protein. Giving individuals
high fructose diets increases insulin resi-
Whole truth in one drop
<1.0 mg/L had the greatest reduction in
CHD risk vs placebo(27). Rosuvastatin has also been shown to promote regression of
coronary atherosclerosis(28).
Investigators have made substantial efforts to subfractionate lipoprotein particles
to identify those individuals who retain enhanced residual risk of CHD despite being
on statin therapy. It remains to be determined whether assays of lipoprotein particle
fractions will be superior to the standard lipid profile in large-scale prospective studies. We have previously shown that HDL
particles, as assessed by 2-dimensional gel
electrophoresis, provide superior CHD
risk prediction compared to routine measurement of HDL-C when tested in a case-control fashion, as well as in the prospective Veteran Affairs HDL Intervention Trial(29)(30). Treatment with
the simvastatin/niacin combination
to increase concentrations of large
HDL has been shown be predictive
of regression of coronary atherosclerosis(31). The same may be the
case for sdLDL-C vs LDL-C; however, this effect awaits confirmation by prospective data analysis.
We have reported that rosuvastatin at a dose of 40 mg/day is
even more effective than atorvastatin at 80 mg/day in lowering sdLDL-C by more
than 50%, along with lowering LDL-C by a similar percentage(32). Rosuvastatin is also more effec-
21
DIAGNOSTICS UNDER MAGNIFYING GLASS
tive in raising HDL-C and large HDL particles than is atorvastatin(33).
Our data suggest that sdLDL-C has
promise as a new test for assessment of
heart disease risk. The advantage of this
test is that it has excellent reproducibility,
and after sample pretreatment, sdLDL-C
can be run on high-throughput analyzers,
which makes this test much more user-friendly and more applicable than specialized tests such as gradient gel electrophoresis, nuclear magnetic resonance, and
gradient ultracentrifugation. These latter
tests require shipping samples to specialized laboratories. The sex differences we
observed are interesting, as are the differences in sdLDL-C in premenopausal vs
postmenopausal women. We have previously reported similar findings for LDL-C
and apolipoprotein B (apoB) concentrations in this population(34). Aging and menopause are associated with a significant
increase in LDL related to delayed clearance(35)(36). Compared with premenopausal
women, postmenopausal women also have
increased apoB production into VLDLs,
which are converted to LDL(36). It is well
known that CHD risk markedly increases
with aging in men and, after menopause, in
women, and alterations in LDL clearly contribute to this increased risk(20).
Our data also indicate that, despite
4-fold higher cholesterol-lowering medication use (mainly statins) in cases than controls, mean sdLDL-C concentrations were
very similar in male cases vs controls, and
were significantly higher in female cases
than controls. In prospective data from Framingham we have documented that apoB is
superior to calculated LDL-C and non-HDL-C in CHD risk prediction, but that
the apoB/apoA-I ratio does not provide information about CHD risk that is superior
to the total cholesterol/HDL-C ratio(37). It
remains imperative to carry out prospective analysis to determine whether sdLDL-C
is an independent predictor of CHD, and
how it compares with apoB concentrations.
Ultimately the question remains as to whether clinicians should measure lipoprotein
subclasses and apolipoproteins in their patients to optimize prediction of CHD risk.
In our view emerging data indicate that these parameters do add information about residual risk, especially in patients with established CHD, but future analyses must be
carried out in large prospective cohort studies and intervention studies.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation
22
of data; (b) drafting or revising the article for
intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures of Potential Con flicts of Interest: Upon manuscript submission, all authors completed the Disclosures
of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: Y. Ito, Denka Seiken.
Consultant or Advisory Role: K. Nakajima, Denka Seiken; L.A. Cupples, Denka
Seiken; P.W. Wilson, Liposcience.
Stock Ownership: None declared.
Honoraria: P.W. Wilson, Liposcience (immediate family member).
Research Funding: M. Ai and S. Otokozawa, Denka Seiken and Kyowa Medex; E.J.
Schaefer, Denka Seiken; P.W. Wilson, Liposcience. B.F. Asztalos and E.J. Schaefer were supported by grants R01 HL-60935,
HL 74753, and PO50HL083813 from NIH
and contract 53-3K-06 from the United
States Department of Agriculture Research
Service. L.A. Cupples and C.C. White were
supported by NHLBI N01-HC 25195 and
HL 60935 from NIH.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study,
choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Acknowledgments: The Denka Seiken
Corporation, Tokyo, Japan, provided the direct LDL and sdLDL-C assay kits used in
this study.
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CORMAY International Bulletin
CLINICAL CASE
From case
to case
Every reader can become involved in this
section. We are willing to publish any interesting
and non-standard cases you may have
encountered.
We hope that the published cases will become
a useful educational tool for mastering your
diagnostic skills.
Cardiovascular risk evaluation
and revision of treatment.
Primary dyslipidemia.
Clinical case
No 1
Clinical case No 2
Clinical information:
A 62-year-old obese woman with type 2 diabetes, hypertension
and ischemic heart disease (stable angina), for the past two
years treated with atorvastatin due to dyslipidemia, admitted for
cardiovascular risk evaluation and revision of treatment.
Does not smoke or drink alcohol.
Physical examination showed blood pressure of 130/74 mmHg,
steady heart rate of 64/min, stable circulatory system,
weight 75 kg, height 163 cm (BMI 27.8 kg/m2),
waist circumference 102 cm.
normal ECG result.
Clinical information:
A 31-year-old male admitted to A&E department due to burning chest pains continuing for two
days. The patient has not been previously diagnosed with ischemic heart disease or any other
cardiovascular disease. He was not diabetic, has never smoked, drunk alcohol or taken any
medications continuously. The patient’s father died of myocardial infarction at the age of 41.
On the basis of an ECG and cTnI test result the patient was diagnosed with ST segment elevation
myocardial infarction. Percutaneous coronary intervention has been performed in order to
remove the occlusion in the infarct-related artery.
Physical examination showed obesity (BMI = 31 kg/m2, waist circumference 102 cm).
Blood pressure 124/72. No heart failure traits were detected. It was noted that the patient
had xanthelasma on both eyelids (see picture).
Laboratory tests:
TC – 175 mg/dl (4.5 mmol/l)
HDL-C – 35 mg/dl (0.9 mmol/l)
LDL-C – 94 mg/dl (2.4 mmol/l)
non-HDL-C – 140 mg/dl (3.6 mmol/l)
TG – 230 mg/dl (2.6 mmol/l)
Fasting glycemia – 110 mg/dl (6.1 mmol/l)
HbA1C – 7.5 %
Figure. Xanthelasma of the eyelids.
Source: http://www.atlasdermatologico.com.br
Cardiovascular risk has been assessed as very high. It is due to
a number of factors: obesity, type 2 diabetes, hypertension,
dyslipidemia and diagnosed ischemic heart disease. Patients
with circulatory system diseases and/or diabetes are
considered to be at a high cardiovascular risk, regardless of
other factors. Despite statin treatment the patient’s lipid profile
is abnormal (certain parameters are out of normal range).
Concentration of LDL-C which is the primary goal of treatment
in high risk patients) should not exceed 70 mg/dl (1.8 mmol/l).
Also non-HDL-C and TG needs to decrease and HDL-C should
increase.
In order to decrease cardiovascular risk the patient’s
medicine to statins. The patient was also suggested to change
her lifestyle (controlled physical exercise) and to modify her
diet in order to lose weight.
Nr 3 (25), autumn 2012
Laboratory tests carried out after the acute phase of illness showed the following:
Fasting glycaemia – 90 mg/l (5.0 mmol/l)
Glycaemia 2hrs OGTT – 110 mg/dl (6.1 mmol/l)
TC – 230 mg/dl (5.9 mmol/l)
LDL-C – 168 mg/dl (4.3 mmol/l)
HDL-C – 24 mg/dl (0.6 mmol/l)
TG – 124 mg/dl (1.4 mmol/l)
Liver and kidney function tests results were normal.
Living family members had their lipid profile tested.
The results from the mother were as required, and brother’s results were as follows:
TC – 215 mg/dl (5.6 mmol/l)
LDL-C – 156 mg/dl (4.0 mmol/l)
HDL-C – 32 mg/dl (0.8 mmol/l)
TG – 118 mg/dl (1.3 mmol/l))
The patient has been diagnosed with primary dyslipidemia – a heterozygous form of familial
hypercholesterolemia. He has also been diagnosed with visceral obesity, which was not
accompanied by any disorders which could indicate metabolic syndrome. Nevertheless
the obesity might have been the reason for CRP levels equal to an indirect cardiovascular risk.
Whole truth in one drop
23
Now, I am 19-parameters analyzer
RDW-SD – Additional morphological information:
• Shows a statement of erythrocytes distribution without examination of blood smear under
the microscope;
• RDW-SD is a more sensitive parameter with the presence of a minor population
of macrocytes or microcytes, because it measures the lower part of RBC distribution curve.
At the same time, the rate will change at high reticulocytosis due to their large size, which
extends the base curve of the distribution of red blood cells;
• This measurement is performed at a relative height of 20% above the baseline. The wider
the curve is spread by erythrocytes of different sizes, the higher the RDW-SD value will be.