LIN. CHEM. 35/12, 2285-2289
(1989)
3imultaneous Liquid-Chromatographic Determination of Vitamin K., and Vitamin E in Serum
L E. Cham,H. P. Reeser,and1. W. Kamet
Ve describe a high-performance liquid chromatographic
rocedure for the simultaneous measurement of vitamins K,
tnd E in human serum. Delipidated human serum (free of
tamins K, and E) was used to make standard solutions of
hese vitamins, and cetyl naphthoate and a-tocopheryl aceate were the internal standards for vitamin K, and vitamin E,
espectively.
A simple,novel separationmethod utilizing
quid-liquid partition chromatography was used as a preparttive “clean-up” procedure. Cetyl naphthoate and vitamin K,
after post-column reduction) were detected by fluorescence,
-tocopheryI acetate and vitamin E by ultraviolet absorption.
ensitivity (detection limit) of the assay was 30 pg for vitamin
and 5 ng for vitamin E per injection. The method is
pecific, precise, and more rapid than previously described
rocedures. Within- and between-assay CVs were 8.1% and
12.9%, respectively, for vitamin K1; 3.5% and 6.0%, respecively, for vitamin E. Analytical recoveries of vitamins K, and
E were 80% and 93%, respectively,
from serum and from
elipidated serum (standards). The average neonatal serum
oncentration of vitamin K, was 83 ng/L, 2.5 mg/L for vitamin
for normolipidemic adults, the values were 343 ng/L and
!9 mg/L, respectively, and for hyperlipidemic adults, 541
g/L and 11.1 mg/L, respectively.
ddItionaI Keyphrases: fat-soluble vitamins
lipids
iaphthoate
a-tocopheryl
acetate
fluorometry
)oms
hyperlipidemia . phylloquinone
.
cety!
new-
‘
(free of vitamins K1 and E) is used in preparing standard
solutions of these vitamins. A new internal standard (cetyl
naphthoate)
is used for the vitamin K1 assay; a-tocopheryl
acetate is used as the internal standard for the vitamin E
assay. A simple, novel, preparative
separation
method,
liquid-liquid
partition
chromatography,
separates
vitamins K1 and E and their respective internal standards from
other serum components. This clean-up procedure enables
the quantification
of vitamins
K1 and E in serum of
hyperlipidemic
patients. We have applied the method to
the determination
of vitamins
K1 and E in serum of
newborns, healthy adults, and hyperlipidemic
subjects.
Materials and Methods
Subjects
Blood samples were obtained
from 20 normal adult
subjects and 20 hyperlipidemic
(serum cholesterol
>5.5
mmoIIL or serum triglyceride
>1.75 mmol/L) subjects who
had fasted for 12 h.
Blood samples (10 mL) were taken from the antecubital
vein from fasting adults or from the umbilical cord vein
from 70 neonates.
The samples
were protected
from the
light and were centrifuged
at 4#{176}C.
Serum samples (up to 3
mL) were extracted immediately
or stored at -70 #{176}C
in
disposable polystyrene tubes with polyethylene
stoppers.
Serum pools were prepared from excess serum and were
used for the studies of within-day
coefficient
of variation
(CV), between-day
CV studies, and recovery studies. They
were also a source to prepare
The recent development
of sensitive techniques of mealuring vitamin
K1 in plasma has greatly assisted the
inderstanding
of factors influencing the availability,
meabolism, and functions
of vitamin
K in animals
and
iumans (1,2). However, methods (3-9) currently available
‘or determining
physiological concentrations
of vitamin K1
n biological samples by “high-performance”
liquid chromaography
(HPLC) require
several successive chromatoraphic
steps to separate this vitamin completely from
nterfering lipids. Thus they are not practicable for use in
‘outine analyses. For study of multiple plasma samples,
tuch as population groups, a simplified technique is needed.
?urther, because vitamin K and vitamin E share a lipidransport system in plasma (10, 11) and are known to have
number of biological interactions
(12-14), we have found
t useful and convenient
to monitor both vitamins
in
)lasma simultaneously
in various physiological
and pathoogical states. Specifically, this has helped us detect clinical
onditions involving changes in plasma phylloquinone
conentrations, the result of altered metabolism of the carrier
ipoproteins, rather than of vitamin K itself.
We report here a practical procedure for simultaneously
neasuring
the fat-soluble vitamins K1 and E in human
ierum by HPLC. A matrix of delipidated
human serum
Department
of Medicine,
University
of Queensland,
Royal Bris-
)ane Hospital, Brisbane, Queensland 4029, Australia.
Received
December 16, 1989; accepted August 9, 1989.
up standard
solutions
delipidated
serum
for making
of the vitamins.
Instrumentation
HPLC was performed
with a system incorporating
a
Model M-45 Solvent Delivery System, a Model 6000A
Solvent Delivery System to supply the reducing agent, a
Lambda-Max
Model 481 LC spectrophotometer,
a GuardPak Resolve C18 precolumn, a 5-nm-particle
spherical C18
Resolve column (3.9 mm i.d. x 15 cm), a dual-channel
Servogor 10 mV recorder (all from Waters Associates, Inc.,
Milford, MA 01757); a Rheodyne 7125 injection valve; an
F-bOO Hitachi
fluorescence
spectrophotometer;
and a
stainless steel coil (0.8 mm i.d. x 1 m), which was used as
a reactor.
Reagents
Methanol,
isopropanol,
and hexane (all HPLC purity)
were purchased from Waters Associates, Brisbane, Australia. Ethanol (absolute) was purchased from CSL (Brisbane).
Phylloquinone
(all trans-vitamin
K1), a-tocopherol,
a-tocopheryl acetate, and sodium borohydride were from Sigma
Chemical Co., St. Louis, MO. y-Tocopherol (biochemical
grade) was from Fluka Chemie A. G., Buchs, Switzerland.
1-Naphthoic acid was from the Aldrich Chemical Co., Inc.,
Milwaukee, WI, and cetyl alcohol was from Merck, Darmstadt, F.R.G. Tritiated
phylloquinone
(all tran,s, specific
activity
1.4 x 1012 dpmlg)
was
kindly
mann-La Roche, Basle, Switzerland.
water was used throughout.
donated
Distilled,
by Hoff-
de-ionized
CLINICAL CHEMISTRY, Vol. 35, No. 12, 1989 2285
Standards.
Pooled normal adult human serum was delipidated with a mixture of butanol-diisopropyl
ether as
previously described (15-17). This procedure removes lipids, including the fat-soluble vitamins (18), but does not
precipitate
proteins
(including
apolipoproteins)
(15-18).
The delipidated serum was extracted (see below, extraction
of vitamins K1 and E) and the sample extract was injected
onto the column. Neither vitamin K1 nor vitamin E was
present in this extract.
A stock standard solution of vitamin K1 was made up in
ethanol (approximately
10 mg/L). The precise concentration was calculated
by using a molar absorptivity
coefficient of 19 900 L mol’
cm’ at 248 nm for phylloquinone in ethanol. The concentration
and purity of vitamin
K1 were routinely checked by multi-wavelength
spectrometry with a Hitachi U-3200 UV-visible spectrophotometer
(GEC, Brisbane,
Australia)
and by HPLC. Immediately
before use, dilutions were made with isopropanol and added
to delipidated serum to obtain aqueous standards (vitamin
K1 working standards,
0.0625
4.0 pg per liter of delipidated serum).
A stock standard solution of a-tocopherol (400 mg/L) was
made up in isopropanol. The concentration
and purity of
vitamin E were routinely
checked by multi-wavelength
spectrometry
and by HPLC. Appropriate
portions of this
solution were added to delipidated serum (which now also
contained
the vitamin K1 standard)
to obtain aqueous
standards
(a-tocopherol
working standards,
0.625
40.0
mg per liter of delipidated
serum). Working standards in
delipidated
serum were stored at -70 #{176}C
and were stable
for at least nine months. All solutions containing vitamin
K1 were shielded from light.
‘
-
-
-
Procedures
Synthesis of cetyl naphthoate. We esterified 24.2 g of cetyl
alcohol with 17.2 g of 1-naphthoic
acid in 200 mL of
benzene, adding five drops of concentrated
sulfuric acid as
a catalyst.
The
mixture
was
refluxed
for 3 h, and
the
benzene was subsequently
removed by distillation.
The
remaining
solid material
was dissolved in 200 mL of
hexane and transferred
to a separating
funnel. We added
100 mL of de-ionized water to the hexane, inverted the
mixture
several times, then removed the water layer. After
washing the hexane layer twice more with de-ionized
water, we removed the hexane by evaporation in a vacuum
chamber.
This resulting
crystalline
residue (cetyl naphthoate, approximately
37 g), when chromatographed
under
the conditions described in this paper, gave a single peak
by fluorometric detection, but was not detectable by ultraviolet absorption.
For the stock solution we used cetyl naphthoate
(78 g)
and a-tocopheryl
acetate (100 mg) dissolved in 1 L of
isopropanol.
Extraction of vitamin K1 and vitamin E. Serum specimens (usually 0.5-3 mL, depending on whether the specimen was from hyperlipidemic
or newborn subjects) and
working standards
in delipidated
serum were transferred
to a borosilicate
glass screw-cap
(Teflon-lined)
16 x 125
mm culture tube; 7.8 ng of cetyl naphthoate
and 10 pg of
a-tocopheryl acetate in isopropanol (0.1 mL stock solution)
were added as internal
standards.
Serum proteins were
denatured
by adding ethanol (2 mL per milliliter of serum),
and the lipids were extracted into 5 mL of hexane. After
mixing this solution in the dark for 15 mm, we separated
the two phases by centrifugation
(3500 x g, 5 mm). The
2286
CLINICAL CHEMISTRY, Vol. 35, No. 12, 1989
upper (hexane) layer, containing
vitamin K1, vitamin E,
the internal standards,
and other lipids (not present in
delipidated
serum containing
the standards),
was transferred to a borosilicate
glass screw-cap
(Teflon-lined)
16 x 125 mm culture tube.
Clean-up of vitamin K1, vitamin E, and internal standards. The hexane layer was “washed” by mixing with 5
mL of methanol:water
(9:1 by vol) in the dark for 10 mm.
The two phases
were separated
by centrifugation
(3500 x g, 5 mm). The upper (hexane) layer was transferred to a disposable pointed borosilicate glass screw-cap
(Teflon-lined)
tube (17 x 125 mm), and the hexane was
evaporated
under a stream of nitrogen. The residue obmined after extraction was dissolved by addition of 120 pL
of isopropanol; 100 L of this was injected onto the column.
Analysis.
For HPLC analyses of the vitamin K1 and
vitamin E, we injected 100 L of the sample extract onto
the column. The column was eluted isocratically
with
ethanol:water
(92:8, by vol) at a flow rate of 0.9 mL/min at
room temperature.
The effluent from the column was mixed
with the reducing agent (800 mg of sodium borohydride in
1 L of ethanol) and fed directly into a post-column reaction
system at 55#{176}C
in an SE Grant waterbath
(Grant Instruments, Barrington,
Cambridge,
CB2 50Z, U.K.). This reduced the vitamin K1, which we detected with the internal
standard
cetyl naphthoate
by fluorescence
spectrophotometry at an excitation wavelength
of 320 nm, an emission wavelength of 430 nm, and a sensitivity setting range
at x 20. The flow-rate
of the reducing
agent
was 0.6 mL/
mm. The effluent from the fluorescence detector was fed
directly into the LC spectrophotometer,
where vitamin E
and the internal standard
a-tocopheryl
acetate were detected at an absorbance
wavelength
of 292 mm and a
sensitivity
setting range at 0.1 A. Peak heights of the
vitamins and their internal standards
were recorded on a
dual-channel
chart recorder. Vitamin K1 and vitamin E
concentrations
of the sample were measured
by peakheight ratios with their corresponding
internal standards
and calculated from a calibration curve.
Recovery experiments. Known amounts of vitamin K1
and a-tocopherol were added to delipidated serum samples
and to serum samples that contained known amounts of
endogenous
vitamin K1 and vitamin E. Total amounts
were then measured and the analytical
recovery was assessed.
To calculate
percentage
recovery,
the
amount
of
endogenous
vitamin was subtracted
from the measured
total amount, divided by the added amount, and multiplied
by 100.
We also assessed extraction
recoveries
of [3H]phylloquinone (4800 counts/mm)
added directly to 1 mL of
delipidated
or nondelipidated
sera in a scintillation
vial.
Control sera containing no radioactively
labeled phylloquinone were also included. The serum samples were extracted and “cleaned up” as described above, the washed
hexane layer was transferred
into a scintillation
vial and
evaporated under a stream of nitrogen, and the residue was
mixed with 10 mL of “Ready Safe” scintillant.
We counted
the radioactivity
in the samples with a scintillation
counter
(Beckman), using external standardization
and counting
for 5 mm at 35-50% efficiency, then calculated the recovery
of the extracted radiolabeled
phylloquinone.
Other methods. Serum cholesterol and triglyceride
were
measured by established
procedures (19).
Treatment of data. For comparative statistical analysis o
groups, we used Student’s t-test.
lesu Its
0.0 4-
Precipitating
serum proteins with ethanol facilitated the
xtractability
of lipids by hexane. However, direct applicaion of these extracted lipids to the HPLC system failed to
eparate vitamin
K1 from other lipids (Figure 1, left).
Washing” the hexane layer, which contains the extracted
ipids, with a mixture
of methanol/water
(9/1, by vol)
liminated
the interference
of lipids and allowed
good
[PLC separation
and quantification
of vitamin K1 (Figure
,right) and vitamin E (Figure 2). Lipid extracts from the
era of patients did not contain any substances that intersred with the detection of the internal
standards
(not
hown). Cetyl naphthoate
(retention
time 7.0 mm) was
luted just before vitamin K1 (retention
time 8.5 miii)
Figure 1, right), whereas a-tocopheryl
acetate (retention
Line 6.4 mm) was eluted just after vitamin E (retention
Line 4.8 miii) (Figure 2). Figure 1 (right) shows a chromatoram of a serum extract of vitamin K1 from a normal
ubject (concentration
500 ng/L) with added cetyl naphhoate (7.8 zgfL). A large peak with a retention time of 26
iin was present in the serum extract. The total HPLC time
r quantifying
vitamin K1 and vitamin E was 30 mm.
‘igure 2 illustrates
a chromatogram
of vitamin E (9.0
sg/L) and a-tocopheryl
acetate (10 mg/L) in the same
erum extract.
The reduction of vitamin K1 by sodium borohydride was
emperature
dependent
(Figure 3), there being a linear
elationship
in the peak-height
ratio of vitamin K1 to the
sternal standard cetyl naphthoate
and temperature
up to
0#{176}C.
We thus chose a reaction temperature
of 55#{176}C
ecause this enabled appropriate
reduction of vitamin K1.
The delipidated
serum used to make up the vitamin
tandards contained no traces of endogenous vitamin K1
nd vitamin E (Figure 4, right). It was feasible to get both
cod signal-to-noise
ratio and selectivity towards the backround signal from serum samples containing at least 30
ig of vitamin K1 on column. Increasing the length of the
eaction coil to up to 2 in resulted in broadening
of the
eaks and did not improve the sensitivity of the method. In
w
0
z
0
U)
0
6
9 12
Fig. 2. HPLC chromatogram of normal human serum extract after the
preparative methanol/water (9/1, by vol) wash clean-up step
Peak 1, vItamin E; peak 2, a-tocopheryl acetate. Conditions of the mobile
phase as for Fig. 1. Detection: Ultraviolet absorbance at 292 nm, attenuation
at 0.1 absorbanceunits full scale (AUFS)
3.4S
#{149}
#{149}
3.0-
.
.
S
“I
‘.4-
.
0
‘.3.
4
#{149}
0
‘
20
30
TEMPERATURE
00
3
MIN
‘0
00
00
70
SO
.
Fig. 3. Effect of temperature on the reduction of vitamin K, by sodium
borohydride
After columnchromatography the effluent(flow rate 0.9 mL/min)from the
column was mixed wIththe reducingagent (sodiumborohydflde,800 mg/I. In
ethanol; flow rate 0.6 mL/min) and fed directly Into a post-column reaction
system (stainlesssteel coIl, 0.8 mm id. x 1 m) kept In a waterbath at
temperaturesfrom 10 to 80 C. Vitamin K, and the Internal standard cetyl
naphthoatewere detected by fluorescence spectrophotometryas in Fig. 1
0
‘-So
4
S
#{149}1
z
0
#{149}I0
S.
S
S
U
S
S.
C
0
-I
tOO
0
MIHU1S
3
IC
20
30
MINUTES
ig. 1. HPLC chromatogram of normal human serum extract (left)
iithout added intemal standard and without the preparativemethaal/water (9/1, by vol) wash clean-up step, and (right) after the
reparativemethanol/waterwash clean-upstep
‘oak 1, vitamin K1; peak 2, cetyl naphthoate. Conditions: mobile phase
thanol/water (92/8, by vol), flowrate0.9 mL/min, post-columnreductionwith
odium borohydride (800 mg/L in ethanol), flow rate 0.6 mUmin. Detection:
xcitation wavelength 320 nm, emission wavelength 430 nm, fluorescence
ensitivity setting range x20. Unmarkedpeaks are unidentified
the absence of sodium borohydride, no peak was detectable
at a retention time of 8.5 miii.
Sensitivity
of the assay for vitamin E was 5 ng on
column. Standard
curves for peak-height
ratios for the
pairs vitamin K1:cetyl naphthoate
and a-tocopherol:a-tocopheryl acetate are shown in Figure 5. The regression
equations of the standards made up in delipidated
serum,
calculated
from peak-height
ratios, were as follows: for
vitamin K1, y = 2.46x
0.19, r = 0.995, n = 7; for vitamin
E, y = 3.97x + 0.18, r = 0.999, n = 7.
The results in Table 1 show the analytical
recovery of
vitamin K1 and vitamin E from delipidated
serum and
nondelipidated
serum. The recovery of the extraction
of
[3H]phylloquinone
in delipidated
and nondelipidated
serum is also shown. Table 2 shows the results of our studies
of precision, carried out with low and high serum vitamin
K1 and E concentrations.
We also applied the proposed
-
CLINICAL CHEMISTRY, Vol. 35, No. 12, 1989 2287
Table 2. PrecisIon of Assay of Phylloquinone and
Vitamin E In Serum
0
PC
N
Phylloqulnone,
VItamIn E,
mg/L
ng/L
I-SO
4
Within-run (n
Mean±SD
CV,%
Si
0)
z
0
5) 50
S.
Day-to-day (n
Si
‘I
Mean ± SD
CV, %
1:
12)
=
680
=
±50
8.1
8.89 ± 0.62
3.5
98.2 ± 13.3
12.9
12.07 ± 0.72
6.0
8)
Ui
0
z
method to measure vitamins K1 and E in sera from necnates, normal adults, and hyperlipidemic
subjects (Table
3).
U)
0
U)
U)
Discussion
0
5
O
20
036912
30
MI N
Fig. 4. HPLC chromatogram of butanol-diisopropyl ether delipidated
humanserumextract:(left) conditions as for Fig. 1, with fluorescence
detection; (right) conditions as for Fig. 2, with absorbancedetection
MINUTES
Neither vitamin K, nor vitamin E was present.This serum was used to make
up the working standard solutions
S
I54
ZO
I
lx
0
01
-I-.
I#{149}
U.
U
0
I-
4
S
8
C
2
S
0
Previously described HPLC assays required several successive tedious chromatographic
steps to completely sepa#{149}
rate vitamin K1 from interfering lipids and are not practicable for use in routine analysis
(3-9). The proposee
method specifically separates
and quantifies vitamin K1
accurately and rapidly. Sample preparation
by deproteun.
ization with ethanol and extraction
of the vitamins
is
hexane, followed by a simple “washing” procedure with a
mixture of methanol-water
to eliminate interfering lipids,
simplifies and speeds the assay. The preparative clean-ut
step with the methanol-water
wash before HPLC reducei
the lipid load injected onto the LC column, so that we
replaced the precolumn after
100 injections, the analyti.
cal column after --700 assays.
Under the described HPLC conditions,
a-tocopherol
it
not separated
from y-tocopherol. This combined peak it
represented
here as vitamin E. a- and ‘y-tocopherol repro.
sent over 90% of vitamin E concentration
in plasma, the
ratio of the concentrations
of a- to ‘y-tocopherol in serun
being approximately
5 to 1 (20). The molar absorptivit3
coefficients (at wavelength
292 nm) for a-tocopherol in the
mobile phase is 3058 L mol’
cm1; for y-tocopherol
3645. If required, complete separation of a-tocopherol froir
y-tocopherol can be obtained by altering the composition oi
the mobile phase to ethanol-water
(85/15, by vol). The
retention times of a- and y-tocopherol then become 10.8 anc
8.6 mm, respectively. Under these conditions, the retentior
times of cetyl naphthoate
and vitamin K1 are 17 and 2
miii, respectively,
and are quantifiable.
However, the tot
HPLC running time then takes over 1 h, which is not
feasible for routine analyses.
A newly described internal standard, cetyl naphthoate
.
1
WEIGHT
2
10
CETYL
20
RATIO.
4
P NY LI. OOU
RATIO,
WEIGHT
3
ISO
NE
NAPHTHOATE
30
-T OC
-TOC0PH#{128}RYL
40
OP
HE 0 0.
ACETATE
Fig. 5. Standard curves for peak-height ratio vs weight ratio for
phylloquinone:cetyl
naphthoateand a-tocopherol:a-tocopheryl acetate
Conditionsas in Figs. 1 and 2
-
Table 1. AnalytIcal Recove ry of Phylloqulnone and a&Tocopherol Added to Patients’ Delipidated and
Nondelipidated Sera (n = 4)
Phylloqulnone,
A, Standardsadded
B, Delipidated serum
C, Delipidatedserum + standards
% recovery [(C B)/A] x 100
-
E, Serum
F, Serum + standards
% recovery [(F E)/A1x 100
-
Mean
1.00
1tg/L
SD
0.10
0
0.83
83
0.19
0.98
0.10
80
4
2288 CLINICAL CHEMISTRY, Vol. 35, No. 12, 1989
6
0.05
0.10
r-TocopheroI,
Mean
8.01
0
7.47
93
7.36
15.40
100
mg/I.
H1Phylloquinone,
dpm
Mean
SD
249.
4.
207.
5
SD
0.24
14479
0.08
3
12 790
88
34.5
0.32
1.17
12
39.4
12251
84
5A
180.
4
Table 3. Serum Phylloqulnone and Vitamin E Concentrations In Newborns, Normal Adults, and Subjects with
Hyperllpldemla
VitamIn E, mgJL
Phylloqulnone, ng/L
n
Mean
SD
Cord blood
70
67
46
Range
Adults
22
343
Hyperllpldemla
20
541
Cord blood
20
2.5
155
197
204-910
1.0
0.3-5.3
24-571
49-590
N Serum cholesterol concentration >5.5 mmol/L, or serum triglyceride >1.75 mmolIL.
which is eluted just before vitamin K1, contributes
to the
accuracy of the method. Internal
standards
used in the
assay of vitamin K1 by other investigators,
e.g., menaquinone -6, are present in biological material
(21). Cetyl
naphthoate was synthesized chemically and we regard it as
superior as an internal standard to endogenous compounds
in biological materials.
Another important new approach is the solvent that we
used for vitamin K1 and a-tocopherol
standards.
Delipidated serum retains the endogenous carriers of lipid and
fat-soluble vitamins,
the apolipoproteins,
but is devoid of
the lipids and vitamins
themselves
(15-17). Thus the
preparation
of the standard curves and the assay of serum
samples could follow an identical procedure,
with the
standard curve passing through the origin. Recovery studies showed that vitamin K1 and vitamin E were extracted
from delipidated
serum and from patients’ serum at equal
rates. The absolute recovery of vitamin K1 was -80% and
for vitamin E was -93%.
Intra- and interassay
repeatability
studies have shown
that the method is precise and reliable. Recovery studies
show that the assay is accurate.
Values quoted for vitamin K1 concentrations
in plasma
have tended to become lower as the sensitivity
of the
method used has increased
(22). Our results (Table 3)
closely agree with recent data for adults determined
with
electrochemical
detection methods (0.2
0.7 1ag/L, n = 45)
(22). Conflicting data have been presented on vitamin K1
concentrations
in plasma from neonates. Values equivalent
to adult concentrations
have been quoted (6), but usually
results have been much lower, ranging from 4 to 98 ng/L
(22,23). Our data (Table 3) yielded a value of 67 ± 46 ng/L
(mean ± SD, n = 70), supporting the validity of the lower
concentrations
reported. Comparison
of data from hyperlipidemic subjects-both
vitamin K1 and vitamin E-is
rendered difficult by the heterogeneity
of the tested population, but we saw a clearcut trend towards higher concentrations of both vitamins
in these subjects, which is in
accord with previous reports (10, 22).
-
We acknowledge
the financial
support of the National
Health
and Medical Research Council of Australia.
References
1. Stenflo J, Fernlund
P, Egen W, Roepstorff P. Vitamin K
dependent modifications of glutamic acid residues in prothrombin.
Proc Natl Acad Sci USA 1974;71:2730-3.
2. Suttie JW. Recent advances in hepatic vitamin K metabolism
and function. Hepatology 1986;7:367-76.
3. Lefevere MF, De Leenheer AP, Claeys AE, Claeys W, Steyaert
H. Multidimensional liquid chromatography: a breakthrough in
the assessment of physiological vitamin K levels. J Lipid Res
1982;23:1068-72.
Adults
21
7.9
1.5
5.5-11.7
Hyperllpldemlaa
21
11.1
3.0
6.0-17.7
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