Clinical Chemistry 49, No.
6, 2003 983
tween free-hCG protein concentrations and the detection
rate for hCG transcripts, which provides an opportunity
to study the fetus at the molecular level.
This study demonstrates that tissue-specific fetal RNA
sequences can be detected in maternal blood in pregnant
women regardless of the genetic situation of the fetus
relative to the mother.
Such noninvasive access to the gene expression pattern
of fetal tissues offers new possibilities. The discovery of
additional fetal RNA markers (e.g., trophoblast-produced
human placental lactogen or placental growth hormones)
may open new opportunities for detecting and monitor-
ing aberrant or differentially expressed genes. Fetal RNA
analysis in maternal blood may therefore provide infor-
mation regarding gene expression in many physiologic
and pathologic conditions, especially those involving tro-
phoblastic tissue, even if the question of the role of such
transcripts remains to be elucidated.
We are indebted to Dr. Lavergne for reviewing this
manuscript.
References
1. Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, et al.
Fetal gender and aneuploidy detection using fetal cells in maternal blood:
analysis of NIFTY I data. Prenat Diagn 2002;22:609 15.
2. Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal D status
from maternal plasma: introduction of a new noninvasive fetal RHD geno-
typing service. Transfusion 2002;42:1079 85.
3. Costa JM, Benachi A, Gautier E, Jouannic JM, Ernault P, Dumez Y. First
trimester fetal sex determination in maternal serum using a real-time PCR.
Prenat Diagn 2001;21:1070 4.
4. Sekizawa A, Kondo T, Iwasaki M, Watanabe A, Jimbo M, Saito H, et al.
Accuracy of fetal gender determination by analysis of DNA in maternal
plasma. Clin Chem 2001;47:1856 8.
5. Costa JM, Benachi A, Gautier E. New strategy for prenatal diagnosis of
X-linked disorders. N Engl J Med 2002;346:1502.
6. Costa JM, Giovangrandi Y, Ernault P, Lohmann L, Nataf V, El Halali N, et al.
Fetal RHD genotyping in maternal serum during the first trimester of
pregnancy. Br J Haematol 2002;119:255 60.
7. Al-Mufti R, Howard C, Overton T, Holzgreve W, Gaenshirt D, Fisk ND, et al.
Detection of fetal messenger ribonucleic acid in maternal blood to determine
fetal RhD status as a strategy for non-invasive prenatal diagnosis. Am J
Obstet Gynecol 1998;179;210 4.
8. Poon LLM, Leung TN, Lau TK, Lo YM. Presence of fetal RNA in maternal
plasma. Clin Chem 2000;46:1832 4.
9. Rainen L, Oelmueller U, Jurgensen S, Wyrich R, Ballas C, Schram J, et al.
Stabilization of mRNA expression in whole blood samples. Clin Chem
2002;48:188390.
10. Giovangrandi Y, Parfait B, Asheuer M, Olivi M, Lidereau R, Vidaud M, et al.
Analysis of the human CGB/LHB gene cluster in breast tumors by real-time
quantitative RT-PCR assays. Cancer Lett 2001;168:93100.
11. Frendo JL, Vidaud M, Guibourdenche J, Luton D, Muller F, Bellet D, et al.
Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in
Downs syndrome. J Clin Endocrinol Metab 2000;85:3700 7.
12. Frendo JL, Therond P, Bird T, Massin V, Muller F, Guibourdenche J, et al.
Overexpression of copper zinc superoxide dismutase impairs human tropho-
blast cell fusion and differentiation. Endocrinology 2001;142:3638 48.
13. Hotakainen PK, Serlachius EM, Lintula SI, Alfthan HV, Schroder JP, Stenman
UE. Expression of luteinising hormone and chorionic gonadotropin -subunit
messenger-RNA and protein in human peripheral blood leukocytes. Mol Cell
Endocrinol 2000;162:79 85.
14. Taback B, Chan AD, Kuo CT, Bostick PJ, Wang HJ, Giuliano AE, et al.
Detection of occult metastatic breast cancer cells in blood by a multimo-
lecular marker assay: correlation with clinical stage of disease. Cancer Res
2001;61:884550.
15. Hautkappe ALA, Lu M, Mueller H, Bex A, Harstrick A, Roggendorf M, et al.
Detection of germ-cell tumor cells in the peripheral blood by nested reverse
transcription-polymerase chain reaction for -fetoprotein-messenger RNA
and human chorionic gonadotropin-messenger RNA. Cancer Res 2000;
60:3170 4.
16. Hara I, Yamada Y, Miyake H, Hara S, Gotoh A, Fujisawa M, et al. Detection
of -human chorionic gonadotropin expressing cells by nested reverse-
transcriptase-polymerase chain reaction in the peripheral blood stem cells
of patients with advanced germ cell tumor. J Urol 2002;167:148791.
17. Ng EKO, Tsui NBY, Lam NYI, Chiu RWK, Yu SCH, Wong SCC, et al. Presence
of filterable and nonfilterable mRNA in the plasma of cancer patients and
healthy individuals. Clin Chem 2002;48:12127.
18. Lambert NC, Lo YMD, Erickson TD, Tylee TS, Guthrie KA, Furst DE, et al.
Male microchimerism in healthy women and women with scleroderma: cell
or circulating DNA? A quantitative answer. Blood 2002;100:284551.
19. Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, et al.
Quantitative analysis of fetal DNA in maternal plasma and serum: implica-
tions for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768
75.
Functional Hyperhomocysteinemia in Healthy Vegetar-
ians: No Association with Advanced Glycation End
Products, Markers of Protein Oxidation, or Lipid Peroxi-
dation after Correction with Vitamin B12, Katarna Se-
bekova,1* Marica Krajcovicova-Kudlackova,1 Pavol Blazcek,2
Vojtech Parrak,3 Reinhard Schinzel,4 and August Heidland4
(1 Institute of Preventive and Clinical Medicine, 2 Hospital
of Ministry of Defense of Slovak Republic, and 3 St. Cyril
and Method Hospital, 833 01 Bratislava, Slovakia; 4Uni-
versity of Wuerzburg, 97070 Wuerzburg, Germany; * ad-
dress correspondence to this author at: Institute of Pre-
ventive and Clinical Medicine, Limbova 14, 833 01
Bratislava, Slovakia; fax 421-2-59369-170, e-mail
sebekova@upkm.sk)
Vegetarians are at risk of developing hyperhomocysteine-
mia (HHcy). The predominant or selective consumption
of proteins of plant origin shifts homocysteine (Hcy)
metabolism to the remethylation pathway (1 ), which
requires vitamin B12 as a cofactor and methyltetrahydro-
folate as a substrate. In the vegetarian diet, the intake of
folic acid exceeds the recommended dietary allowance
(RDA), whereas intake of vitamin B12 is inadequate or
even absent (2 ).
HHcy represents an independent risk factor for cardio-
vascular disease (3 ). Autooxidation of Hcy produces
reactive oxygen species (ROS) (4 ), which may stimulate
lipid peroxidation and formation of advanced oxidation
protein products (AOPPs) and advanced glycation end
products (AGEs). Interaction of AGEs with their specific
receptor, RAGE, induces formation of ROS (5 ). In mice,
HHcy was shown to enhance the expression of RAGE (6 ).
AGEs, AOPPs, and lipid peroxidation products are impli-
cated in the pathogenesis of degenerative and inflamma-
tory diseases, including atherosclerosis (7 ).
In vegetarians, plasma concentrations of AGEs are
mildly but significantly increased compared with popu-
lations on a Western mixed diet (8 ). We therefore inves-
tigated (a) whether there is an association between Hcy
and plasma AGE concentrations or markers of protein
oxidation and lipid peroxidation, and (b) whether supple-
mentation of vitamin B12 affects the mentioned analytes in
vegetarians with HHcy produced by a potential vitamin
B12 deficit.
The study was approved by the Institutional Ethics
Board and was conducted according to the Declaration of
984 Technical Briefs

Helsinki. All participants gave written consent to partic-
ipate.
We investigated 63 healthy vegetarians in whom HHcy
had been revealed previously. The normohomocysteine-
mic (NHcy; Hcy 12.0 mol/L) subgroup (with plasma
folate and vitamin B12 concentrations within the appro-
priate reference intervals) was compared with the sub-
group with functional HHcy (Hcy 12.0 mol/L) attrib-
utable to vitamin B12 deficiency (plasma B12 220
pmol/L) who had plasma folate and iron concentrations
and blood values within the appropriate reference inter-
vals. This subgroup was then administered intramuscular
doses of vitamin B12 (Leciva), with an initial dose of 1000
g and four 300-g doses over the next 14 days.
Hcy (9 ), vitamin B12, and folate concentrations (Elecsys
2010 System; Boehringer); AGE-associated fluorescence
(350/450 nm) (10 ); carboxymethyllysine (CML; compet-
itive ELISA; Roche Diagnostics) (11, 12 ); AOPPs (13 ); and
lipid conjugated dienes (CDs) (14 ) were measured in all
participants before and in the treated group 4 weeks after
the last intervention. Plasma concentrations of -carotene
(15 ); vitamins A (15 ), C (16 ), and E (15 ); thyrotropin
(IRMA); triiodothyronine (RIA); and thyroxine (RIA; all
assays from Immunotech) were determined. Routine
chemistries were performed on a COBAS Integra 700
analyzer (Roche), and blood profiles were determined on
a Sysmex KX-21 analyzer.
The participants nutritional regimens were evaluated
by use of dietary interviews and food frequency question-
naires. The intake of vitamin B12 was determined with use
of the Alimenta database (Food Research Institute).
The results of the above analyses are presented as the
mean (SE) in Table 1. For statistical evaluation, we used
unpaired and paired Wilcoxon tests. Regression analysis
was performed. P 0.05 was considered significant.
For the whole group, plasma Hcy (by 15%) was slightly
increased, whereas plasma vitamin B12, folate, and iron
concentrations and the blood profiles (data not given)
were within the appropriate reference intervals. Mean
(SE) daily intake of vitamin B12 was 2.41 (0.11) g (RDA
2.0 g/day). All participants had normal kidney and
thyroid gland function (data not given). Plasma albumin
and vitamin concentrations indicated a balanced diet.
Stepwise multiple regression analysis revealed that
plasma vitamin C, vitamin B12, and iron concentrations
correlated significantly with Hcy (ANOVA, F 12.12;
P 0.0001).
NHcy vegetarians had significantly higher vitamin B12
concentrations than the HHcy group because the latter
were vitamin B12-deficient. Other investigated variables
did not differ significantly. Stepwise multiple regression
analysis suggested that CML, vitamin B12, and folate
concentrations (inverse relationship) correlated signifi-
cantly with Hcy (ANOVA, F 7.399; P 0.007).

Table 1. Pertinent data for the group as a whole, the NHcy vegetarians, and those with HHcy attributable to vitamin B12
deficiency before and after vitamin B12 administration.a
Functional HHcyc

All NHcyb Basal Posttreatmentd

(n 63) (n 36) (n 14) (n 14)
Gender, F/M 38/25 25/11 5/9
Age, years 38.5 (1.8) 39.8 (2.6) 42.1 (3.0)
BMI,e kg/m2 22.2 (0.4) 22.1 (0.5) 22.5 (1.0)
Time on diet, years 8.2 (0.6) 8.3 (0.8) 7.6 (1.0)
Albumin, g/L 53.4 (0.4) 53.6 (0.5) 52.2 (0.7) 52.6 (0.8)
Creatinine, mol/L 96.4 (1.8) 95.6 (2.4) 101.0 (4.4) 98.2 (4.6)
Iron, mol/L 17.3 (0.8) 16.6 (1.1) 17.8 (1.3) 17.7 (1.0)
Hcy, mol/L 13.8 (0.8)f 9.7 (0.3) 18.6 (1.6)f 10.9 (0.8)h
Vitamin B12, pmol/L 243.5 (12.5)g 287.2 (16.7) 151.7 (9.7)f 281.1 (18.3)h
Folate, nmol/L 29.7 (1.3) 31.7 (1.8) 30.3 (2.7) 31.3 (2.3)
AGE-Fl, AU 213.6 (6.0) 218.6 (6.0) 231.5 (17.8) 246.3 (32.9)
CML, g/L 403.5 (13.1) 408.3 (17.0) 364.5 (31.7) 384.8 (22.8)
Vitamin C, mol/L 67.4 (2.3) 70.5 (3.3) 64.9 (4.0) ND
Vitamin E, mol/L 26.0 (0.8) 26.3 (1.2) 25.2 (1.30) ND
Vitamin A, mol/L 1.78 (0.06) 1.77 (0.73) 1.89 (0.13) ND
-Carotene, mol/L 0.56 (0.05) 0.61 (0.06) 0.56 (0.12) ND
AOPPs, mol/L 43.9 (2.0) 42.3 (2.4) 43.7 (4.3) 44.2 (5.4)
CDs, A233 1.15 (0.06) 1.12 (0.07) 1.11 (0.13) 1.30 (0.16)
a
Data are given as the mean (SE).
b
Hcy 12.0 mol/L.
c
Hcy 12.0 mol/L
d
Four weeks after treatment.
e
BMI, body mass index; AGE-Fl, AGE-associated fluorescence; AU, arbitrary units; ND, not determined.
f,g
Compared with NHcy: f P 0.01; g P 0.05.
h
P 0.01 vs pretreatment values.
 Clinical Chemistry 49, No. 6, 2003
Hcy concentrations were 12.0 mol/L (HHcy) in 27 of
the vegetarians. HHcy was not associated with a vitamin
B12 deficiency in 6 of these individuals, whereas in the
remaining 21, the daily intake of vitamin B12 was 1.48
(0.09) g. Seven individuals were not evaluated in the
clinical study (refusal of participation or exclusion be-
cause of concomitant anemia and iron deficiency). Plasma
albumin and vitamin concentrations and body mass index
were within the appropriate reference intervals, thus
allowing the exclusion of protein energy malnutrition as a
source of HHcy.
In vegetarians with HHcy attributable to vitamin B12
deficiency, HHcy was not associated with increases in
plasma AGE, AOPP, or CD concentrations. However, the
pretreatment Hcy concentrations did correlate with AOPP
and CD concentrations (r 0.598; P 0.03 and r 0.575;
P 0.03, respectively).
In individuals with a vitamin B12 deficiency, plasma
vitamin B12 concentrations normalized and Hcy concen-
trations decreased to within reference values by 4 weeks
after initiation of vitamin B12 treatment. The higher the
pretreatment Hcy value, the more profound the observed
decrease (r 0.872; P 0.001). None of the other investi-
gated variables was affected significantly. Posttreatment
Hcy concentrations and changes in Hcy during treatment
did not correlate with any of the investigated data.
In a general population on a Western mixed diet, folate
deficiency represents the main risk factor for develop-
ment of HHcy. Its supplementation produces only a mild
decrease in Hcy (17 ). In long-term vegetarians, vitamin
B12 deficiency plays a key role in the pathogenesis of
HHcy because it is lacking in the vegan diet (2 ). In vegans,
the only sources of vitamin B12 are bacteria in the lower
intestinal tract. Intake of food of animal origin (milk, dairy
products, and eggs) contributes to vitamin B12 concentra-
tion in lacto-ovo-vegetarians. In HHcy vegetarians, the
estimated intake of vitamin B12 (74% of RDA) was insuf-
ficient to maintain a balance of this vitamin.
The high plasma folate observed in vegetarians might
be attributable to higher folate intake. Moreover, the
methyl folate trap might be a contributing factor. In
vitamin B12 deficiency, 5-methyltetrahydrofolate and folic
acid may accumulate (18 ). Cobalamin deficiency renders
folate largely biologically ineffective, although its plasma
concentrations and distribution appear sufficient. In the
present study, plasma folate concentrations did not differ
between the NHcy and pre- and posttreatment HHcy
individuals. Treatment with vitamin B12 decreased Hcy
by 41.8%, much more than might be expected in mild
HHcy. Thus, the methyl folate trap explains the rapid and
substantial correction of HHcy after vitamin B12 supple-
mentation in vegetarians with vitamin B12 deficiency.
In NHcy vegetarians with sufficient plasma vitamin B12
and folate, CML appeared to correlate with Hcy concen-
trations. This relationship is of particular interest because
this chemically defined AGE results not only from the
classic pathway of AGE formation via Amadori products
but also from autooxidation of glucose and from lipid
peroxidation (5 ). The direct relationship between CML
985
and Hcy could be of interest with regard to the mild
increase in circulating AGE in healthy vegetarians (8 ).
However, the other markers associated with oxidative
stress (fluorescent AGEs, AOPPs, and CDs) showed no
correlation with Hcy concentrations. There are limited
data available on the association of Hcy and oxidative
stress in the NHcy population. Powers et al. (19 ) found a
correlation between plasma Hcy and malondialdehyde in
young men, but because a methionine-load test produced
a significant increase in Hcy but not malondialdehyde,
they considered it unlikely that the oxidative stress could
be a direct effect of Hcy.
In contrast to NHcy vegetarians, in the subgroup with
functional HHcy, Hcy correlated directly with AOPPs and
CDs. These data agree with the findings of Voutilainen et
al. (20 ), who reported an association between Hcy and
F2-isoprostanes in men with HHcy. In spite of its long
duration, HHcy in our study group was not associated
with increased AOPP or CD concentrations. It is conceiv-
able that the high plasma concentrations of antioxidants
in vegetarians may partially abrogate the potentially
enhanced formation of ROS induced by HHcy and AGEs.
Even the short-term intervention with vitamin B12 ef-
fectively normalized Hcy concentrations in vegetarians
with functional HHcy. This action was not associated
with a change in plasma AGE, AOPP, or CD concentra-
tions, indicating that high Hcy concentrations in vegetar-
ians do not support the concept of a causal link to markers
of oxidative stress. It should be emphasized that long-
term maintenance of normohomocysteinemia in long-
term vegetarians may be achieved only by their adherence
to a recommended consumption of food of animal origin
or by supplementation with vitamin B12 preparations
sufficient to saturate the body. Otherwise, vitamin B12
concentrations decrease and hyperhomocysteinemia is
reestablished within the next 6 months (21 ).
It must be considered that the HHcy state is a reflection
of metabolic events within a cell. Thus, our in vivo
investigation carries limitations in that measurements in
plasma may not accurately reflect intracellular oxidative
modifications.
In summary, this study provides the first data indicat-
ing that, in vegetarians with functional HHcy attributable
to vitamin B12 deficiency, increases in Hcy may be paral-
leled by increases in markers of lipid peroxidation and
oxidation of proteins, without their overt increase. Addi-
tional studies are needed to elucidate the relationship
between Hcy and AGEs, AOPPs, and lipid oxidation
products in a population on a standard Western mixed
diet and with various disease states.
We wish to acknowledge support from Leciva SK
(Bratislava, Slovakia) and the Verein zur Bekampfung der
Hochdruck-und Nierenkrankheiten (Wuerzburg, Ger-
many) as well as the excellent assistance of Andre Klassen
in preparing the manuscript. A portion of this study was
presented as a poster at the 2002 AACC Annual Meeting
(Orlando, FL).
986 Technical Briefs
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1. Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem 1990;
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2. Herbert V. Vitamin B12, plant sources, requirements and assay. Am J Clin
Nutr 1998;48:852 8.
3. Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovas-
cular disease. Annu Rev Med 1998;49:31 62.
4. Lang D, Kredan MB, Moat SJ, Hussani SA, Powell CA, Bellamy MF, et al.
Homocysteine-induced inhibition of endothelium-dependent relaxation in
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2000;20:4227.
5. Fu MX, Requena JR, Jenkins AJ, Lyons TJ, Baynes JW, Thorpe S. The
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6. Hofmann MA, Lalla E, Lu Y, Gleason MR, Wolf BM, Tanji N, et al.
Hyperhomocysteinemia enhances vascular inflammation and accelerates
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7. Stitt AW, Bucala R, Vlassara H. Atherosclerosis and advanced glycation:
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8. Sebekova K, Krajcovicova-Kudlackova M, Schinzel R, Faist V, Klvanova J,
Heidland A. Plasma levels of advanced glycation end products in healthy,
long-term vegetarians and subjects on a Western mixed diet. Eur J Nutr
2001;40:275 81.
9. Vester B, Rasmussen K. High performance liquid chromatography method
for rapid and accurate determination of homocysteine in plasma and serum.
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Determination of advanced glycation end products in serum by fluorescence
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11. Mellinghoff AC, Reininger AJ, Wuerth JP, Founds HW, Landgraf R, Hepp KD.
Formation of plasma advanced glycosylation end products (AGEs) has no
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AT, Zingraff J, et al. Advanced oxidation protein products as a new marker of
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17. Brattstrom LE, Israelsson B, Jeppson JO, Hultberg BL. Folic acidan
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18. Herbert V. Experimental nutritional folate deficiency in man. Trans Assoc Am
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19. Powers RW, Majors AK, Lykins DL, Sims CJ, Lain KY, Roberts JM. Plasma
homocysteine and malondialdehyde are correlated in age- and gender-
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Bilirubin in Amniotic Fluid Does Not Interfere with the
Abbott TDx FLM II Assay, Rohit Cariappa, Curtis A.
Parvin, and Ann M. Gronowski* (Department of Pathology
and Immunology, Division of Laboratory Medicine,
Washington University School of Medicine, 660 South
Euclid Ave., Box 8118, St. Louis, MO 63110; * author for
correspondence: fax 314-362-1461, e-mail gronowski
@pathology.wustl.edu)
Bilirubin, a breakdown product of lysed red blood cells, is
present in amniotic fluid in very small concentrations
relative to serum (1 ). In an uncomplicated pregnancy,
bilirubin in amniotic fluid peaks at 19 22 weeks of
gestation at concentrations of 1.6 1.8 mg/L (1 ). Rh iso-
immunization and its severe manifestation, erythroblas-
tosis fetalis, are associated with intrauterine hemolysis,
which leads to increases in amniotic fluid bilirubin con-
centrations (2 ) up to 9.6 mg/L (3, 4 ). The assessment of
fetal lung maturity (FLM) in these pregnancies is some-
times necessary, but the ability to utilize current methods
in the presence of increased bilirubin concentrations is
unclear. The commercial TDx FLM II assay (Abbott Lab-
oratories), which is based on fluorescence polarization
technology and the dye PC-16 (5 ), is the most commonly
used method to assess FLM (6 ). Although the manufac-
turer does not recommend testing icteric amniotic fluid
samples, nothing has been published about the nature or
quantification of their interference with the TDx FLM II
assay. Our objective was to examine the effect of bilirubin
on TDx FLM II concentrations.
Frozen leftover amniotic fluid samples sent to the
laboratory for physician-ordered FLM testing were sorted
and combined posttesting to obtain six pools with FLM
values of 23, 31, and 36 mg/g (immature, 39 mg/g); 44
mg/g (intermediate); and 60 and 77 mg/g (mature, 55
mg/g). Each pool contained amniotic fluid from at least
four women. Amniotic fluid samples with even minimal
visual evidence of hemolysis or meconium were excluded
from the study. Each pool was analyzed for total bilirubin
concentration on the Hitachi 747 analyzer. The analytical
sensitivity for bilirubin measurements in amniotic fluid is
poor on this analyzer at concentrations 1.0 mg/L (Table
1 in the Data Supplement that accompanies the online
version of this Technical Brief at http://www.clinchem.
org/content/vol49/issue6/); therefore, this assay is not
used routinely to measure bilirubin in amniotic fluid. This
method was suitable, however, for the purpose of con-
firming that none of the pooled amniotic fluid samples
had pre-addition bilirubin concentrations 1.0 mg/L.
Human studies committee approval was obtained for this
study.
A stock solution of unconjugated bilirubin (2500 mg/L)
was prepared by dissolving bilirubin (cat. no. 101018; ICN
Biomedicals Inc.) in laboratory-grade dimethyl sulfoxide
(DMSO; cat. no. D-5879; Sigma Chemical Company; final
concentration, 40 mL/L) at alkaline pH, and then neutral-
izing the solution with acid. The DMSO concentration in
the sample with the highest concentration of bilirubin in
our experiments (15 mg/L) was 1 mL/L. Addition of 2
mL/L DMSO to amniotic fluid samples caused no change
in FLM concentrations.
The stock solution of unconjugated bilirubin was added
to an aliquot of pooled sample, vortex-mixed, and serially
diluted to create samples with decreasing concentrations
of bilirubin. The FLM concentrations of these samples and
the pre-addition pool were measured in duplicate in the
TDx FLM II assay. Experiments were performed on 2
separate days (three pools each day). Validation studies
were performed with serum to establish that there was no
matrix effect when measuring bilirubin at this concentra-