658
Technical Briefs
percentage can be expressed only as 0.02%, 0.2%, 2%, or
20%. As expected, the male DNA percentages deviated
further from the expected (median, 0.2%; mean, 24.3%;
IQR, 0.2–20%), with the skewness becoming more severe
(Fig. 1). These data could be interpreted in completely
opposite ways depending on whether the median (much
lower than the true value of 4.5%) or the mean (much
higher than the true value of 4.5%) was chosen for
presentation.
The issue associated with analytical imprecision was
equally applicable to the estimation of fetal DNA percentages. Repeated measurements of the two second-trimester
maternal plasma samples by RQ-PCR yielded a median of
3.9%, mean of 3.7%, and IQR of 2.1–5.3% and a median of
5.2%, mean of 5.0%, and IQR of 2.8 –7.2%, respectively.
However, results for the fivefold serial dilution method
were as follows: median, 4.8%; mean, 11.1%; IQR, 1.6 – 8%;
and median, 1.6%; mean, 3.4%; IQR, 1.6 – 8%, respectively
(Fig. 1).
We therefore conclude that, in our hands, gentle centrifugation did not confer any observable advantage and
formaldehyde addition did not yield the previously reported dramatic increases in fractional concentrations of
fetal DNA (7 ). The latter could have resulted from the
imprecise estimation of the fetal DNA percentages in
maternal plasma when the previously reported serial
dilution method was used.
This work was supported by an Earmarked Research
Grant (CUHK4395/03M) from the Research Grants Council of the Hong Kong Special Administrative Region,
China. Y.M.D.L. and R.W.K.C. hold patents and patent
applications on aspects of fetal nucleic acid analysis from
maternal plasma.
References
1. Bianchi DW. Circulating fetal DNA: its origin and diagnostic potential-a
review. Placenta 2004;25(Suppl A):S93–101.
2. Chiu RWK, Lau TK, Leung TN, Chow KCK, Chui DHK, Lo YMD. Prenatal
exclusion of ␤ thalassaemia major by examination of maternal plasma.
Lancet 2002;360:998 –1000.
3. Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al.
Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:
485–7.
4. Lo YMD, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, et al.
Prenatal diagnosis of fetal RhD status by molecular analysis of maternal
plasma. N Engl J Med 1998;339:1734 – 8.
5. Lo YMD, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, et al. Increased
fetal DNA concentrations in the plasma of pregnant women carrying fetuses
with trisomy 21. Clin Chem 1999;45:1747–51.
6. Zhong XY, Hahn S, Holzgreve W. Prenatal identification of fetal genetic traits.
Lancet 2001;357:310 –1.
7. Dhallan R, Au WC, Mattagajasingh S, Emche S, Bayliss P, Damewood M, et
al. Methods to increase the percentage of free fetal DNA recovered from the
maternal circulation. JAMA 2004;291:1114 –9.
8. Chiu RWK, Poon LLM, Lau TK, Leung TN, Wong EMC, Lo YMD. Effects of
blood-processing protocols on fetal and total DNA quantification in maternal
plasma. Clin Chem 2001;47:1607–13.
9. Lo YMD, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative
analysis of fetal DNA in maternal plasma and serum: implications for
noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768 –75.
10. Angert RM, LeShane ES, Lo YMD, Chan LYS, Delli-Bovi LC, Bianchi DW. Fetal
cell-free plasma DNA concentrations in maternal blood are stable 24 hours
after collection: analysis of first- and third-trimester samples. Clin Chem
2003;49:195– 8.
11. Lam NYL, Rainer TH, Chiu RWK, Lo YMD. EDTA is a better anticoagulant than
heparin or citrate for delayed blood processing for plasma DNA analysis. Clin
Chem 2004;50:256 –7.
DOI: 10.1373/clinchem.2004.042168
Quantification of Thiol-Containing Amino Acids
Linked by Disulfides to LDL, Angelo Zinellu, Salvatore
Sotgia, Luca Deiana, and Ciriaco Carru* (Chair of Clinical
Biochemistry, University of Sassari, Viale San Pietro 43/B,
07100 Sassari, Italy; * author for correspondence: fax 39079228120, e-mail carru@uniss.it)
Several studies have indicated that plasma proteins interact with homocysteine (Hcy) to form stable disulfidelinked products. Hcy in plasma is mainly bound to
albumin, but interactions with ceruloplasmin, fibrin, annexin II, and transthyretin have been also reported (1– 4 ).
In 1991, Olszewski and McCully (5 ) described the presence of Hcy in lipoproteins in patients with hypercholesterolemia. Because the analysis was performed after acidic
hydrolysis of apoprotein, the Hcy measured by these
authors was the sum of (a) Hcy incorporated in the
primary structure of apolipoprotein B-100 (5 ), (b) Hcy
thiolactone bound to lysine residues of protein by amide
or peptide linkages and converted to Hcy by acidic
conditions after release (6 ), and (c) Hcy linked to apolipoprotein B-100 (apoB-100) by a disulfide bond.
Recent studies have demonstrated that there are at least
nine free sulfhydryl groups (⫺SH) in the apoB-100 primary structure that could potentially bind plasma free
aminothiols by disulfide linkage (7 ). Because lysyl residues of apoB-100 could react in vivo with plasma Hcy
thiolactone by an amide bond, the number of free apoprotein sulfhydryl groups could increase considerably,
thus increasing the number of sites that may be bound
with plasma aminothiols (6 ). These LDL modifications are
accompanied by an increase in density and in electrophoretic mobility of lipoprotein and are associated with
functional alterations that make Hcy-LDL more susceptible to aggregation and to spontaneous precipitation.
Moreover, higher uptake of Hcy-LDL by membrane receptor and by phagocytosis and a higher accumulation of
intracellular cholesterol have been observed in cultured
macrophages, suggesting that homocysteinylation could
increase the atherogenicity of LDL (8, 9 ). Thus, to study
the association between Hcy and lipid metabolism, a
highly sensitive method to measure Hcy and other thiols
bound to apoB-100 is required.
Here we describe a simple capillary electrophoresis
method with laser-induced fluorescence detection to measure physiologic thiols bound to apoprotein by a disulfide
linkage. We assessed the performance of this analytical
method by measuring apoB-100-bound thiols in 16 volunteers. Participants were not receiving dietary supplements
of vitamin B6, B12, or folate or statin therapy as deter-
Clinical Chemistry 51, No. 3, 2005
mined by medical interviews. Blood was collected into
Vacutainer Tubes containing EDTA. Plasma was prepared
by centrifugation at 2000g for 10 min at 4 °C. LDL was
isolated by ultracentrifugation according to the methods
of Himber et al. (10 ) and McDowell et al. (11 ). Recovered
LDL was passed through Sephadex PD-10 column equilibrated with phosphate-buffered saline to remove salts,
EDTA, and other interfering compounds. Lipoprotein (a)
was isolated as described by Fless et al. (12 ).
Because the aim of our work was to measure thiols
bound to apoB-100 only, it was important to verify that
the LDL subfraction obtained by ultracentrifugation did
not contain other lipoprotein types. We therefore checked
the purity of the isolated LDL by capillary electrophoresis
with diode array detection, using our previous method to
monitor the oxidative state of the lipoproteins (13 ). The
obtained electropherograms showed that the LDL fraction
was free from the other lipoproteins. This result was
confirmed by classic lipoprotein agarose electrophoresis
of the isolated subfractions.
For quantification of apoB-100-bound thiols, we delipidated 200 ␮L of the LDL fraction (1 g/L protein) by
adding 2 mL of methanol– chloroform (2:1 by volume)
and centrifuging the sample at 2000g for 5 min. After
protein precipitation, we discarded the supernatant; we
then washed the protein pellet three times with 50 g/L
5-sulfosalicylic acid and successively dissolved it in 200
␮L of 0.2 mmol/L NaOH at 60 °C for 15 min. We then
reduced the disulfide bonds by incubation with 20 ␮L of
100 mL/L tributyl-n-phosphate (TBP) in dimethyl formamide for 10 min. We mixed 50 ␮L of the resulting sample
with 100 ␮L of 150 mmol/L sodium phosphate buffer (pH
12.5) and 10 ␮L of 0.8 mmol/L 5-iodoacetamidofluorescein. After letting the mixture react for 15 min, we diluted
the derivatized samples 100-fold in water and analyzed
them by capillary electrophoresis. ApoB-100-bound thiols
were analyzed by a P/ACE 5510 capillary electrophoresis
system equipped with a laser-induced fluorescence detector (Beckman), as described previously with some modifications (14 ). Briefly, we used an uncoated fused-silica
capillary 57 cm long (50 cm to the detection window) with
a 75-␮m i.d. Analysis was performed with 35 nL of
sample under nitrogen pressure (0.5 psi) for 5 s with an
electrolyte solution containing 18 mmol/L sodium phosphate, 14.5 mmol/L boric acid, and 75 mmol/L N-methyld-glucamine (pH 11.4). The separation conditions (22 kV,
150 ␮A at normal polarity) were reached in 20 s and held
at a constant voltage for 8 min. Separations were carried
out at 40 °C and monitored by fluorescence detection with
excitation at 488 nm and emission at 520 nm. As seen in
Fig. 1A, use of the above electrolyte solution enabled
baseline separation of cysteinylglycine (Cys-Gly), Hcy,
and Cys calibrators in ⬍8 min.
After LDL isolation by ultracentrifugation, we extracted
the lipids from the lipoprotein by methanol– chloroform
and then discarded that layer. The sample containing
apoB-100 was successively washed three times with a
solution of 5-sulfosalicylic acid to remove the watersoluble small residual components in the extracted sam-
659
Fig. 1. Electropherograms of thiol calibrators (A) and thiols released
from apoB after TBP treatment (B).
RFU, relative fluorescence units.
ples. We reduced the disulfide bond between the thiols
and apoB-100 by adding TBP to the apoprotein solution.
Released thiols were successively derivatized by the selective thiol laser-induced fluorescence-labeling agent
5-iodoacetamidofluorescein and separated by capillary
electrophoresis, as shown in Fig. 1B.
Calibration curves for a water solution of Cys-Gly (y ⫽
24.5x ⫹ 0.22 ␮mol/L), Hcy (y ⫽ 33.3x ⫹ 1.18 ␮mol/L),
and Cys (y ⫽ 15.8x ⫹ 0.98 ␮mol/L) show good correlation
(r2 ⬎0.99 for all thiols), demonstrating the linear response
over the concentrations tested (20 – 8000 nmol/L). We
calculated the reproducibility of the injections by injecting
the same calibration solution 10 times consecutively. We
evaluated the within-run (intraassay) imprecision of the
method by injecting the same extracted sample 10 times
consecutively and the between-run (interassay) imprecision by injecting the same extracted sample on 10 consecutive days. Precision tests indicated good repeatability
of our method for both migration times (CV ⬍0.7%) and
areas (CV⬍3.1%). Moreover, we obtained good intra- and
interassay reproducibility (CV ⬍7% and CV ⬍10%, respectively).
We determined the recovery of the thiols by adding
pure thiols to apoB-100 samples. In particular, we added
oxidized thiols after isolation and purification of apopro-
660
Technical Briefs
Table 1. Thiol forms in the studied volunteers.
Mean (SD)
LDL-Cys-Gly, ng/mg of apoB
LDL-Hcy, ng/mg of apoB
LDL-Cys, ng/mg of apoB
Total Cys-Gly, ␮mol/L
Total Hcy, ␮mol/L
Total Cys, ␮mol/L
Reduced Cys-Gly, ␮mol/L
Reduced Hcy, ␮mol/L
Reduced Cys, ␮mol/L
49.12 (10.67)
5.05 (1.83)
109.4 (39.3)
29.76 (4.15)
10.19 (2.86)
248.7 (25.2)
4.15 (0.57)
0.27 (0.05)
11.96 (1.34)
tein from LDL, before the TBP addition. The analytical
recoveries, evaluated at four different concentrations for
each thiol, were 95.6 –103.5% for Cys-Gly, 95.1–104.5% for
Hcy, and 94.8 –102.6% for Cys. The limit of detection,
calculated based on 35-nL injections of a solution of
calibrators (after dilution 100-fold in water), was 25
pmol/L, corresponding to an injected quantity of ⬃0.5
amol, with a signal-to-noise ratio of 3. The limit of
quantification, also calculated by 35-nL injections of sample, was ⬃15 nmol/L.
We tested the performance of our capillary electrophoresis method by measuring apoB-100 in 16 volunteers.
Reduced and total thiols were also measured by capillary
electrophoresis, as described previously, and the data are
reported in Table 1. Cys was the most abundant thiol
bound to apoB-100 [mean (SD), 109.4 (39.3) ␮mol/L], but
smaller amounts of Cys-Gly [49.12 (10.67) ␮mol/L] and
Hcy [5.05 (1.83) ␮mol/L] were detected. To verify
whether the Hcy linked to apoprotein is proportional to
Hcy plasma concentrations, we performed the univariate
Pearson correlation between these variables in the samples from our volunteers. ApoB-bound Hcy was correlated with both total (r ⫽ 0.69; P ⬍0.02) and reduced
plasma Hcy (r ⫽ 0.78; P ⬍0.01).
In conclusion, by modifying our previous assay, we
have developed a method that is able to simultaneously
measure all thiols linked to apoB-100 by a disulfide bond.
We obtained evidence for the method suitability by measuring the concentrations of different thiols in 16 volunteers. For the first time we demonstrate that LDL carries
not only Hcy but also Cys-Gly and Cys and that the latter
is present largely in the LDL fraction. Moreover, we show
that there is a positive relationship between plasma Hcy
concentrations (both total and reduced) and Hcy linked to
apoprotein. Even if the mechanism by which Hcy exerts
its deleterious effects is unknown, several findings have
suggested that oxidation of LDL is a key step in atherogenesis. The etiology of vascular disease resulting from
hyperhomocysteinemia has been attributed to thiol autooxidation, a process that generates reactive oxygen
species such as superoxide and hydrogen peroxide. It has
been suggested that these damaging species are generated
under conditions of hyperhomocysteinemia and that they
lead to oxidative damage of LDL (15 ). The integrated
study of hyperhomocysteinemia and lipoprotein metabolism may help us to better understand the deleterious
effects of increased Hcy concentrations as they relate to
the atherogenic thiolated LDL.
This study was supported by the “Assessorato dell’Igiene
e Sanità Regione Autonoma della Sardegna”, by the
“Ministero dell’Istruzione, dell’Università e della
Ricerca”, and by the “Ministero della Sanità (Attività di
Ricerca Finalizzata–2002)” (Italy). We greatly appreciate
the assistance of Maria Antonietta Meloni with the language in the manuscript.
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DOI: 10.1373/clinchem.2004.043943