Annex V
stock standard is stable for at least two
months at 4-7°C in a tightly sealed container. Prepare working standard freshly
each week and store in a brown glass
bottle at 4-7°C.
Apparatus
The procedure requires 16 x 125 mm screwcapped culture tubes, a mechanical rotator, a
vortex-type mixer, a water bath at 65 -70°C,
and a spectrometer or spectrophotometer to
measure absorbance at 405 nm.
Specimen Collection
Because serum triglyceride concentrations
are increased after an intake of most foods,
blood samples must be taken after a 10 -12 h
fast. This method requires 0.2 mL of serum,
but plasma from blood collected with ethylenediaminetetraacetate (EDTA) as an anticoagulant is also satisfactory. Triglycerides in
serum are stable at least three days when
stored at room temperature (24°C) or refrigerated at 4 - 7 0c,
Procedure
1. To a 16 x 125 mm screw-capped culture
tube (blank) containing 0.7 (± 0.1) g of
washed alumina, add 5.0 mL of isopropanol and 0.20 mL of water.
2. To another tube (standard) containing
0.7 (±O.l) g of washed alumina, add
5.0 mL of isopropanol and 0.20 mL of
working standard.
3. To another tube (unknown) containing
0.7 (± 0.1) g of washed alumina, add 5.0
mL of isopropanol and 0.20 mL of serum.
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4. Place all tubes on a mechanical rotator
for 15 min.
5. Centrifuge for approximately 10 min at
high speed to pack the alumina.
6. Transfer 2.0 mL of the clear supernatant
fluid to appropriately labelled test tubes.
7. Add 0.60 mL of saponification reagent to
a» tubes and mix with a vortex-type
mixer.
S. Let all tubes stand for at least 5 min, but
no longer than 15 min, at room temperature.
9. Add 1.5 mL of periodate reagent to all
tubes and mix with a vortex-type mixer.
10. Add 1.5 mL of acetylacetone reagent to
all tubes and mix with a vortex-type
mixer. Tightly cover each tube with Parafilm.
11. Place all tubes in a 65 - 70°C water bath
for approximately 15 min.
12. Remove all tubes from the water bath, allow to cool at room temperature for
10-15 min and vortex-mix for 5 s.
13. Within 1 h measure the absorbance of
standard and unknowns against the
blank at 405 min in 10- or 12-mm cuvettes.
14. Calculate values for unknowns by
comparing the absorbances (A) of the
unknown and standard as follows:
(Aunknown I Astandard) x 200
= triglycerides, mgldL
Clinical Interpretation
Normal concentrations of serum triglycerides for fasting (10-12 h) adults are approximately 30 -150 mg/dL.
stock standard is stable for at least two
months at 4-7°C in a tightly sealed container. Prepare working standard freshly
each week and store in a brown glass
bottle at 4-7°C.
Apparatus
The procedure requires 16 x 125 mm screwcapped culture tubes, a mechanical rotator, a
vortex-type mixer, a water bath at 65 -70°C,
and a spectrometer or spectrophotometer to
measure absorbance at 405 nm.
Specimen Collection
Because serum triglyceride concentrations
are increased after an intake of most foods,
blood samples must be taken after a 10 -12 h
fast. This method requires 0.2 mL of serum,
but plasma from blood collected with ethylenediaminetetraacetate (EDTA) as an anticoagulant is also satisfactory. Triglycerides in
serum are stable at least three days when
stored at room temperature (24°C) or refrigerated at 4 - 7 0c,
Procedure
1. To a 16 x 125 mm screw-capped culture
tube (blank) containing 0.7 (± 0.1) g of
washed alumina, add 5.0 mL of isopropanol and 0.20 mL of water.
2. To another tube (standard) containing
0.7 (±O.l) g of washed alumina, add
5.0 mL of isopropanol and 0.20 mL of
working standard.
3. To another tube (unknown) containing
0.7 (± 0.1) g of washed alumina, add 5.0
mL of isopropanol and 0.20 mL of serum.
191
4. Place all tubes on a mechanical rotator
for 15 min.
5. Centrifuge for approximately 10 min at
high speed to pack the alumina.
6. Transfer 2.0 mL of the clear supernatant
fluid to appropriately labelled test tubes.
7. Add 0.60 mL of saponification reagent to
a» tubes and mix with a vortex-type
mixer.
S. Let all tubes stand for at least 5 min, but
no longer than 15 min, at room temperature.
9. Add 1.5 mL of periodate reagent to all
tubes and mix with a vortex-type mixer.
10. Add 1.5 mL of acetylacetone reagent to
all tubes and mix with a vortex-type
mixer. Tightly cover each tube with Parafilm.
11. Place all tubes in a 65 - 70°C water bath
for approximately 15 min.
12. Remove all tubes from the water bath, allow to cool at room temperature for
10-15 min and vortex-mix for 5 s.
13. Within 1 h measure the absorbance of
standard and unknowns against the
blank at 405 min in 10- or 12-mm cuvettes.
14. Calculate values for unknowns by
comparing the absorbances (A) of the
unknown and standard as follows:
(Aunknown I Astandard) x 200
= triglycerides, mgldL
Clinical Interpretation
Normal concentrations of serum triglycerides for fasting (10-12 h) adults are approximately 30 -150 mg/dL.