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CLINICAL CHEMISTRY > LEC LIPIDS PART 2 - LIPID DETERMINATION > Flashcards

LEC LIPIDS PART 2 - LIPID DETERMINATION Flashcards

1
Q

Disorders of lipids

A

Arteriosclerosis, obesity, hypertension, DIABETES MELLITUS and other abnormalities

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2
Q

Good prognosis if lipid

A

Early detection of deranged blood lipid profile

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3
Q

age for screening for lipid

A

20 years old, if the test is normal repeat again after 5 yrs

if not, depends on the physician

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4
Q
  • Indications for Lipid Profile include:
A
  1. ⁃ Screening for primary & secondary
    hyperlipidemias
  2. ⁃ Monitoring for risk of atherosclerosis
    3.⁃ Monitoring treatment of
    hyperlipidemias/Dyslipidemia
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5
Q

preanalytical considerations

biological variations such as

A
  1. Age: cholesterol levels increase with age.
  2. Sex: women have lower level than men except in childhood & after early 50’s.
  3. Season: cholesterol levels are slightly higher in cold periods.
  4. Food intake: daily intake of fat increases cholesterol levels.
  5. Medical conditions: thyroid, liver, and kidney diseases
  6. Acute illness
  7. Life-style: higher in sedentary and poor diet habits
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6
Q

explain the cholesterol level in terms of age

A

it increases with age

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7
Q

describe the gender relation of cholesterol

A

women have lower level than men except in childhood and after early 50`s

high in menopausal stage

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8
Q

can season affect cholesterol?

A

higher in cold cholesterol

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9
Q

why do menopausal women has high chole or high lipid profile?

A

because estrogen came from chole, if the cholesterol are can’t be converted into estrogen any longer, it will be deposited and stuck in the blood stream elevating lipids in the body

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10
Q

daily intake of fat increases cholesterol levels

true or false

A

true

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11
Q

in hyperthyroidism, chole is low because?

A

because of fast metabolism

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12
Q

if we have liver disease, do we have high or low chole?

A

low, 85% of chole is produced by the liver. If the liver is defected, there will be no organ that will produce it

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13
Q

describe the lipid profile of chronic alcoholic patients

A

low

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14
Q

describe the lipid profile of moderate alcoholic patients

A

high

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15
Q

components that clears the chylomicron

A

lipoprotein lipase that are activated by apo C2

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16
Q

Patient should fast for ___hours before sampling

A

12

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17
Q

Chylomicrons are cleared within__hrs and their presence
after 12hrs fast is abnormal.

A

6-9

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18
Q

Posture: Decreases of as much_________ of TC, LDL-C, HDL-C and apo-A-1 and B

A

10 %

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19
Q

correct position of getting sample

A

supine and lying position

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20
Q

Patient to be seated for __ prior to sampling to prevent hemoconcentration.

A

5min

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21
Q

Prolonged venous occlusion leads to increase in cholesterol conc by _______.

venous occlusion means tourniquet

A

10-15%

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22
Q

Exercise: Mild exercise produces a slight decrease in
conc of _______________.

A

chole and triglycerides

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23
Q

Those who walk for about 4 hours each week have an average cholesterol ___ lower and HDL-C ___ higher than inactive persons

A

5%; 3.4%

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24
Q

lipids that has higher concentration in the midcycle of menstruation

A

plasma Cholesterol and Triglycerides

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25
Q

A high fat diet increases serum __

A

triglyceride

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26
Q

-Ingestion of monounsaturated fat reduces __.

A

cholesterol

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27
Q

__ conc is reduced when sucrose intake is reduced.

A

Plasma triglyceride

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28
Q

A high carbohydrate diet decreases the serum conc. of___

A

VLDL-C, TG,
cholesterol and protein.

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29
Q

Large protein meals at lunch or in the evening also ___ the serum
cholesterol for atleast 1 hour after a meal.

A

increase

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30
Q

In vegeterian individuals, conc. of LDL & VLDL-C are reduced by ___ and ____

A

LDL 37%
VLDL 12%

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31
Q

Smoking:

The plasma cholesterol, triglyceride and LDL cholesterol conc. are higher by about ___respectively in smokers
than in non smokers

A

3 %, 9.1 % and 1.7 %

cholesterol, 3 %,
triglyceride 9.1 %
LDL cholesterol 1.7 %

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32
Q

__ cholesterol is lower in smokers than in non smokers

A

HDL

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33
Q

When MODERATE amount of alcohol is ingested
for 1wk, the serum TG conc is increased by more than _________.

A

20 mg/dl

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33
Q

Prolonged moderate ingestion of alcohol may increase HDL-C conc

it will reduced the plasma concentration of

A

cholesterol ester transfer protein(CETP).

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33
Q

what sample can we use for lipid profile?

A

either plasma or serum

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34
Q

___ is preferred when lipoprotein are measured by ultracentrifugation and electrophoretic methods

A

Plasma

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35
Q

if plasma is used for lipid profile, what anticoagulant is the best choice?

A

edta

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36
Q

2 methods to detect lipoprotein using plasma

A

ultracentrifugation and electrophoretic methods

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37
Q

reference method for lipoprotein determination

A

ultracentrifugation

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38
Q

the only lipoprotein that is only in the origin during the electrophoresis

A

chylomicron

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39
Q

which sample tend to have lower concentration of lipids

venous or capillary?

and why?

A

capillary - same with glucose, because of water

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40
Q

an anticoagulant that will exert large osmotic effect

A

citrate

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41
Q

Citrate: exert large osmotic effect resulting in falsely __ plasma lipid and
lipoprotein concentration.

A

low

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42
Q

an anticoagulant that can alter electrophoretic mobility of
lipoproteins.

A

heparin

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43
Q

heparin can alter electrophoretic mobility of
lipoproteins because of its __

A

high molecular weight

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44
Q

electrophoresis is separation of components depending on the

A

sizes

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45
Q

EDTA plasma
are ___% lower than in serum.

A

3%

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46
Q

in terms of storing

TC, TG, HDL-C can be satisfactorily analyzed in ___ samples.

A

frozen

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47
Q

aside from total chole, hdl, and tg

-__ can also be measured in frozen samples.

A

Apolipoproteins

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48
Q

Cholesterol Estimation has 2 methods, what are they

A

chemical and enzymatic method

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49
Q

if we have a given serum and we need to convert it to plasma, what formula we should use?

A

serum x 3%

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50
Q

if we have plasma and we need to find the serum, what’s the formula

A

X 1.03

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51
Q

we store sample or plasma in what temperature for a long time

A
  • 70*
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52
Q

we store sample or plasma in what temperature for a short time (1-2 months)

A

-2 *

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53
Q

what is the former reference method for chemical estimation

A

abell kendall method

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54
Q

3 steps of abell kendall method

A
  1. Cholesterol is HYDROLYZED with alcoholic KOH
  2. Unesterified cholesterol is EXTRACTED with petroleum jelly/Hexane
  3. Measured using the L-B Reaction (COLORIMETRY)
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55
Q

RESULT OF HYDROLYZING CHOLESTEROL

A

cholesterol ester AND FATTY ACIDS

removing cholesterol from fatty acids is
free cholesterols

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56
Q

how Liebermann-burchardt reaction works?

A

the unsterified extracted will combined with sulfuric acid and acetic anhydride to form a bluish green solution

unsterified Cholesterol + Sulfuric acid + Acetic anhydride  bluish green solution

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57
Q

end product of liebermann-burchardt’s reaction

A

bluish green solution called Cholestadienyl monosulfonic acid

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58
Q

purpose of acetic anhydride and sulfuric acid

A

color developers

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59
Q

another chemical method for cholesterol estimation that only uses 2 steps

A

Bloors Method

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60
Q

Bloors Method: - Principle: 2 step

A
  1. Cholesterol is extracted using an alcohol ether mixture
  2. Measured using the L-B Reaction
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61
Q

in bloor’s method, the cholesterol is extracted using

A

alcohol ether mixture

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62
Q

in abell kendall method - the cholesterol is extracted using

A

petroleum jelly and hexane

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63
Q

in abell kendall method - the cholesterol is hydrolyzed using

A

alcoholic potassium hydroxide KOH

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64
Q

what is the principle of abell kendall method

A

3 step method - hydrolysis, extraction, colorimetry

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65
Q

what is the principle of enzymatic method

A

cholesterol oxidase method

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66
Q

Routine Lab - Assay of Choice for cholesterol

A

Cholesterol Oxidase Method

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67
Q

Cholesterol Oxidase Method is only specific for __

A

cholesterol esters

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68
Q

cholesterol esters are those cholesterols with ___

A

fatty acids

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69
Q

the cholesterol ester are hydrolyze by what enzyme to form free chole and fatty acids

A

cholesterol esterase

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70
Q

free cholesterol will be oxidized by what enzyme

A

cholesterol oxidase

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71
Q

free cholesterol will be oxidized by cholesterol oxidase to form

A

4 cholestene - 3 - one + h202

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72
Q

what is the goal of oxidizing free cholesterol in cholesterol oxidase method

A

to find the H202 which will be used in trinder’s reaction

73
Q

in trinders reaction we uses what enzyme

A

PEROXIDASE

74
Q

what specific peroxidase is used for trinder’s reaction

A

horse raddish peroxidase

75
Q

what dye is used to form quinoneimine dye in trinder’s reaction

A

4-aminophenazone

76
Q

a sequential reaction in trinder’s reaction

low chole shows
high chole show

A

low chole shows pink
high chole show red

77
Q

in trinder’s reaction or oxidase method, how many enzymes are used

A

3 - esterase , oxidase, peroxidase

78
Q

cholesterol oxidation or trinder’s method is read at what wavelength?

79
Q

cholesterol oxidase method has a linearity up to

A

600 - 700mg/dL (15.54 - 18.13mmol/L)

80
Q

normal value of cholesterol

81
Q

ADVANTAGE of enzymatic method

A

 Precise and accurate
 Lesser interferences -bilirubin, ascorbic acid, Hb
Smaller sample quantity
Rapid; does not require preliminary extraction step
 Can be used to measure unesterified cholesterol by omitting de-esterification step
 Mild reagents; better suited for automated analyzers

82
Q

DISADVANTAGE of enzymatic reaction

A

They are not absolutely specific for
cholesterol.
Cholesterol oxidase reacts with other sterols e.g plant sterol
Ascorbic acid and Bilirubin interfere by consuming H202
Bilirubin interference can produce falsely high or low values
Significant only at conc >5mg/dL decreasing Cholesterol values by 5 -15%

83
Q

INCREASED CHOLESTEROL are found in

A
  1. Biliary cirrhosis
  2. Hyperlipoproteinemia types
    II, III, V
  3. Nephrotic syndrome
  4. Uncontrolled DM
  5. Alcoholism
  6. Primary Hypothyroidism
84
Q

DECREASED CHOLESTEROL

A
  1. Severe hepatocellular
    disease (alcoholic liver
    disease)
  2. Malnutrition
  3. Severe burns
  4. Hyperthyroidism
  5. Malabsorption syndrome
85
Q

limit of interferences of bilirubin for chole

A

not greater than 200 mg/dl

86
Q

limit of interferences of hemoglobin for chole

A

not greater than 20 mg/dl

87
Q

more than 5 mg/dl in bilirubin will interfere to chole increasing the value of _- %

88
Q

the reference method for cholesterol

A

gas chromatography/mass spectrometry

gc ms

89
Q

Cholesterol Desirable level:

A

: < 200mg/dL (< 5.2mmol/L);

90
Q

in a modified abell kendall method, they use hexane, chloroform, and silisic acid

what is the purpose of chloroform in the process?

A

remove phospholipid

91
Q

the color of the end product of the modified abell kendall method

91
Q

what kind of lipid is affected in a fasting specimen?

A

LDL and Triglyceride

91
Q

in a modified abell kendall method, they use hexane, chloroform, and silisic acid

what is the purpose of silisic acid?

A

adsorption of phospholipid via chromatography

91
Q

a post prandial lipemia

A

chylomicron

91
Q

in non fasting sample, what type of triglyceride carrying lipoprotein is found

A

chylomicron

91
Q

we use plasma - the preferred sample for measuring lipoprotein.

In ultracentrifugation, what substance do we use __ to separate lipoproteins based on density

A

potassium bromide

91
Q

what apoprotein that can inhibit the function of apo c2?

A

apo protein c3

91
Q

if we go under fasting, what type of triglyceride carrying lipoprotein can we observed on greatest amount?

91
Q

what is the specific

91
Q

polyanions

A

heparin sulfate, dextran sulfate and phosphotungstate

91
Q

divalent cations

A

Mg, Ca and Mn

92
Q

Most commonly for HDL and is reasonably specific

A

POLYANION PRECIPITATION:

92
Q

the cdc reference method

A

abell kendall

92
Q

what’s in it inside the cholesterol making them possible to be measured using colorimetric assay/chemical method

A

the presence of double bonds and hydroxyl groups of the esterols

92
Q

what is the end product of the salkowski method

A

cholestadienyl disulfonic acid - red solution

92
Q

Precipitating reagents such as ___ and ____ are used to remove all lipoproteins except
HDL

A

divalent cations and polyanions

93
Q

how can ascorbic acid affect the result of cholesterol determination

A

falsely decrease

94
Q

how can hemoglobin and bilirubin affects the result of cholesterol determination

A

falsely increase

95
Q

how can high level of triglyceride affect hdl determination

A

elevated triglyceride results to incomplete sedimentation which is as well will cause the hdl result elevated

96
Q

Similar to the HDL-C precipitation method but uses a
precipitant that is complexed to magnetic particle

A

MAGNETIC METHOD

97
Q

This sediments and does not require centrifugation

A

MAGNETIC METHOD

98
Q

Has been adapted for use in automated clinical chemistry
analyzers

A

MAGNETIC METHOD

99
Q

It allows the supernatant to be analyzed without the
need to remove it from the sedimented complex

A

MAGNETIC METHOD

100
Q

homogenous assay or direct hdl c assay is a type of what method

A

enzymatic method

101
Q

HOMOGENOUS ASSAY (Direct HDL-C Assay):

Enzymatic method: First reagent - “___” non-HDLs

102
Q

blocker for homogenous assay

A

Use of Antibodies or Polymers or complexing agent
e.g Cyclodextrin

103
Q

HOMOGENOUS ASSAY (Direct HDL-C Assay) steps

A

Enzymatic method: First reagent - “blocks” non-HDLs

Use of Antibodies or Polymers or complexing agent
e.gCyclodextrin

Modification of cholesterol esterase and oxidase enzymes which makes them selective for HDL-C

Use of blanking step that selectively consumes cholesterol from non-HDL species

104
Q

Reference method for HDL-C
estimation is

A

Three-step Procedure

105
Q

comment about three step procedure

A

tedious and expensive

106
Q

2 types of ULTRACENTRIFUGATION

A

Preparative Ultracentrifugation
Density gradient method

107
Q

a type of centrifugation that Uses sequential density adjustments of serum to FRACTIONATE MAJOR and MINOR classes of LP

A

Preparative Ultracentrifugation

108
Q

(non-equilibrium or equilibrium
techniques) permits fractionation or several or ALL classes of LPs in a
single run

A

Density gradient methods -

109
Q

Use antibody-coated plates specific for epitopes on apolipoproteins
both in routine and research lab

A

IMMUNOCHEMICAL METHODS

110
Q

INDIRECT METHODS of LDL c estimation

A

friedewald equation

111
Q

what is the equation of friedewald

A

LDL - TC - HDL - TG/2.175 in mmol/L
LDL - TC - HDL - TG/5 in mg/dl

112
Q

Tedious -reserved for samples where Friedewald
equation is inappropriate

A

Beta-Quantification

113
Q

(Reference method) for ldl estimation

A

Beta-Quantification

114
Q

2 steps of Beta-Quantification

A
  1. Ultracentrifugation to remove VLDL leaving behind LDL and HDL as well as IDL and Lp(a)
  2. Chemical Precipitation of HDL-C
115
Q

in the ultracentrifugation, we are removing ___ to keep __ and ___ as well
as IDL and Lp(a)

A

vldl; ldl and hdl

116
Q

Limitations of ldl c estimation in indirect or friedwald method

A

not appropriate in:

  1. Samples with TG > 400mg/dL
  2. Patients with suspected Dysbetalipoproteinaemia
117
Q

Other limitations of ldl estimation ; indirect or friedewald equation

A
  1. Does not account for cholesterol associated with IDL and Lp(a)
  2. Underestimate LDL-C in chronic alcoholics
  3. Unsuitable for monitoring
  4. Mis-classifies 15 - 40 % of patients when TG levels are between 200 to 400 mg/dL
118
Q

Selectively measures LDL after masking non
LDL cholesterol, OR By selectively solubilizing LDL

A

Homogenous method

119
Q

Advantages of homogenous method

A

Does not involve measurement of TGs thus
non-fasting samples can be used

Comparable to calculated LDL results from
beta-quantitation on normal lipemic
specimens

120
Q

May be useful in evaluation of type Ill
hyperlipoproteinemia

A

VLDL-C/Plasma TG ratio

121
Q

VLDL-C/Plasma TG ratio is expressed in

A

mol/mol or mass/mass

122
Q

> 0.689 in VLDL-C/Plasma TG ratio means

A

hyperlipoproteinemi

123
Q

VLDL-C/Plasma TG ratio ref range

A

0.689- 0.091

124
Q

VLDL-C/Plasma TG ratio

Ranges ___ in samples without beta VLDL

A

0.230-0.575

125
Q

give the arrangement of the lipoprotein from top to bottom arranged using electrophoresis

A

origin - chylomicron
ldl - beta
vldl - pre beta
hdl - alpha

126
Q

the most anodic lipoprotein in electrophoresis

A

hdl - alpha lipoprotein

127
Q

arrange the density of the lipoproteins out from ultracentrifugation

A

chylomicrons - 0.95 g/ml
vldl - 1.006 g/ml
IDL - 1.019 g/ml
LDL - 1.063 g/ml
HDL - 1.090 g/ml
fatty acids + albumin

128
Q

in electrophoresis, we used the most sensitive type of gel or medium

A

agarose gel or polyacrylamide

129
Q

stain used in electrophoresis for lipoprotein

A

Oil red O
Sudan
Fat Red 7b

130
Q

former
Reference Method) for triglyceride

A

Van Handel and Zilversmith Method

131
Q

in Triglycerides/TAG:
chemical method

  1. Lipids are extracted using chloroform and
    phospholipids and removed by __ absorption
132
Q

Principle or steps of van handel zilversmith method

A

1.
TAG – > alcoholic KOH Glycerol + Fatty
acids
2. Glycerol + periodic acid –> Formaldehyde
3. Formaldehyde + Chromotropic acid ——> Blue
solution

133
Q

in Van Handel and Zilversmith Method

what is added in the formaldehyde?

A

Chromotropic acid

134
Q

in Van Handel and Zilversmith Method

what is the end product color

A

blue solution

135
Q

a modification of in Van Handel and Zilversmith Method that will not use colorimetry but instead fluorimetry

A

hantzsch condensation method

136
Q

hantzsch condensation method

steps or principle

A

1.TAG – > alcoholic KOH –>Glycerol + Fatty
acids
2. Glycerol + periodic acid –> Formaldehyde
3. Formaldehyde + acetone + ammonia ——> yellow
solution

137
Q

in hantzsch condensation method

what is added in formaldehyde

A

acetone and ammonia

138
Q

in hantzsch condensation method

what is the end color

139
Q

principle of enzymatic method of triglyceride is based on

A

glycerol kinase method

140
Q

chromogen used in trinder’s reaction in tryglyceridee determination

A

4 amino antipyrine

141
Q

in chemical method - glycerol kinase method

the end color is

142
Q

Glycerol Kinase Method Read absorbance at ___ wavelength

143
Q

Glycerol Kinase Method Read absorbance at 550 nm wavelength and linear up to
___

144
Q

INCREASED TAG are seen in

A
  1. Hyperlipoproteinemia type i,
    iib, iii, iv, v
  2. Alcoholism
  3. Nephrotic syndrome
  4. Hypothyroidism
  5. Pancreatitis
145
Q

DECREASED TAG are seen in

A
  1. MALABSORPTION
    SYNDROME
  2. Malnutrition
  3. Brain infarction
  4. Hyperthyroidism
146
Q

an indicator of combined LDL and VLDL
concentration

147
Q

major protein of HDL

148
Q

the variant of LDL, an independent indicator
of CHD risk

149
Q

reference method or rnage of Apo A

A

120 - 160mg/dl

150
Q

reference method or rnage of Apo B

A

< 120 mg/dl

151
Q

reference method or range of Lp A

A

< 30 mg/dl

152
Q

Hyperlipoproteinemias have been classified using the
system, which is not commonly used today.

A

Fredrickson-Levy classification

153
Q

Mostcommonmethod for apoprotein

A

IMMUNOTURBIDIMETRY

154
Q

easily adapted spectrophotometric analyzers allows the use of commercially available antisera and reference sera

A

IMMUNOTURBIDIMETRY

155
Q

apoprotein that has indirect relation to coronary disease

156
Q

other test we can conduct for apolipoprotein

A

ELISA , radial immunodiffusion, keme keme

157
Q

Type I hyperlipoproteinemia: has an Elevated ___

A

chylomicrons

158
Q

Type IIa hyperlipoproteinemia: has an increased

A

Increased LDL

159
Q

Type lIb hyperlipoproteinemia: Increased ___

A

LDL and VLDL

160
Q

Type III hyperlipoproteinemia: Increased ___

161
Q

Type IV hyperlipoproteinemia: Increased ___

162
Q

Type V hyperlipoproteinemia: Increased ___

A

VLDL with increased chylomicrons

163
Q

Total cholesterol level very low,
triglyceride level nearly undetectable, LDL and Apo B-100
absent

A

Abetalipoproteinemia

164
Q

Unable to synthesize apo B-100
and apo B-48, low total cholesterol level and normal to low
triglyceride level

A

Hypobetalipoproteinemia

165
Q

Severely elevated triglyceride
level and low HDL level

A

Hypoalphalipoproteinemia

166
Q

HDL absent, apo A-I and apo A-II very low
levels, LDL low, total cholesterol level low, triglyceride level
normal to slightly increase

A

Tangier disease

CLINICAL CHEMISTRY (24 decks)

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