Clinical Chemistry 45, No. 12, 1999
normal karyotype. Several studies have concluded that
the morphologic defect of hydrops rather than the chromosome abnormality may be associated with the positive
screen for Down syndrome and the subsequent identification of fetal Turner syndrome (5, 14 ). More recently,
Saller et al. (15 ) investigated maternal serum analyte
concentrations in euploid pregnancies and concluded that
nonimmune hydrops is associated with increased hCG. It
has been reported that two fetuses with nuchal thickening
and a subsequent probable diagnosis of Noonan syndrome were associated with a positive Down syndrome
screen related to decreased AFP and increased hCG (16 ).
It is not known whether those pregnancies with an
identifiable cystic hygroma would have subsequently
developed hydrops. Laundon et al. (7 ) included all fluid
collection in the category of hydrops, including hygroma
and cysts, and found increased free bhCG associated with
Turner syndrome. Our data combined with that of Saller
et al. (5 ), Knowles and Flett (4 ), and Laundon et al. (7 )
reveal that 30 of 34 fetuses with fluid collection (hydrops,
hygroma, and cysts) were screen positive. In our sample
of 19 fetuses, 4 (21%) had an increased Down syndrome
risk and 1 (5%) had an increased trisomy 18 risk without
abnormalities on ultrasound. Overall, 74% (14 of 19) of
our cases had a positive screen: 2 with a neural tube risk,
11 with a Down syndrome risk, and 1 with a trisomy 18
risk. Recently, inhibin A concentrations were moderately
decreased in cases of Turner syndrome without hydrops
but markedly increased in cases of Turner syndrome with
hydrops (17 ).
As has been reported previously, the maternal serum
AFP and uE3 concentrations were slightly reduced in
pregnancies affected with Turner syndrome. In addition,
hCG concentrations were increased in fetal Turner syndrome but more so in hydropic pregnancies. Based on
these findings, some cases of Turner syndrome may be
identified prenatally as a result of an increased risk for
Down syndrome when these markers are used. Thus,
women with an increased risk of Down syndrome based
on multiple-marker screening should be counseled that
Turner syndrome may be a possibility, even in the absence of fetal hydrops. The ascertainment of two fetuses
with Turner syndrome as the result of increased trisomy
18 risk suggests that Turner syndrome may be given as a
possible explanation for an increased trisomy 18 risk.
References
1. Canick JA, Palomaki GE, Osathanodh R. Prenatal screening for trisomy 18 in
the second trimester. Prenat Diagn 1990;10:546 – 8.
2. Greenberg F, Schmidt D, Darnule AT, Weyland BR, Rose E, Alpert E. Maternal
serum a-fetoprotein, b-human chorionic gonadotropin, and unconjugated
estriol levels in midtrimester trisomy 18 pregnancies. Am J Obstet Gynecol
1992;166:1388 –92.
3. Palomaki GE, Knight GJ, Haddow JE, Canick JA, Saller DN, Panizza DS.
Prospective intervention trial of a screening protocol to identify fetal trisomy
18 using maternal serum a-fetoprotein, unconjugated estriol, and human
chorionic gonadotropin. Prenat Diagn 1992;12:925–30.
4. Knowles S, Flett P. Multiple marker screen positively in the presence of
hydrops fetalis. Prenat Diagn 1994;14:403–5.
5. Saller DN, Canick JA, Schwartz S, Blitzer MG. Multiple marker screening in
pregnancies with hydropic and nonhydropic Turner syndrome. Am J Obstet
Gynecol 1992;167:1021– 4.
2261
6. Wenstrom KD, Williamson RA, Grant SA. Detection of fetal Turner syndrome
with multiple marker screening. Am J Obstet Gynecol 1994;170:570 –3.
7. Laundon CH, Spencer K, Macri JN, Anderson RW, Buchanan PD. Free bhCG
screening of hydropic and non-hydropic Turner syndrome. Prenat Diagn
1996;16:853– 6.
8. Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Royston P, Chard T, et al.
Maternal serum screening for Down syndrome in early pregnancy. Br Med J
1988;297:883–7.
9. Palomaki GE, Haddow JE, Knight GJ, Wald NJ, Kennard A, Canick JA, et al.
Risk-based prenatal screening for trisomy 18 using a-fetoprotein, unconjugated oestriol and human chorionic gonadotropin. Prenat Diagn 1995;15:
713–23.
10. ACOG Committee. ACOG Committee opinion on Down syndrome screening.
Int J Gynecol Obstet 1994;47:186 –90.
11. Wald NJ, Cuckle HS, Densem JW, Nanchahal K, Canick JA, Haddow JE, et al.
Maternal serum unconjugated oestriol as an antenatal screening test for
Down’s syndrome. Br J Obstet Gynecol 1988;95:334 – 41.
12. MacDonald ML, Wagner RM, Slotnick RN. Sensitivity and specificity of
screening for Down syndrome with a-fetoprotein, hCG, unconjugated estriol
and maternal age. Obstet Gynecol 1991;77:63– 8.
13. Saller DN, Carpenter MW, Canick JA. A prenatal screening program using
AFP, UE3, and HCG: detection of pregnancies complicated by hydropic
changes [Abstract]. Am J Obstet Gynecol 1991;164:355.
14. Wenstrom KD, Boots LR, Cosper PC. Multiple marker screening test:
identification of fetal cystic hygromas, hydrops, and sex chromosome
aneuploidy. J Matern Fetal Med 1996;5:31–5.
15. Saller DN Jr, Canick JA, Oyer CE. The detection of non-immune hydrops
through second-trimester maternal serum screening. Prenat Diagn 1996;
16:431–5.
16. Aranguren G, Garcia-Minaur S, Loridan L, Uribarren A, Vargas LM, RodriguezSoriano J. Multiple marker screen positive results in Noonan syndrome.
Prenat Diagn 1996;16:183– 4.
17. Lambert-Messelian GM, Saller DN Jr, Tumber MB, French CA, Peterson CJ,
Canick JA. Second trimester maternal serum inhibin A levels in fetal trisomy
19 and Turner syndrome with and without hydrops. Prenat Diagn
1998;1061–7.
What Happens to Vitamin K1 in Serum after Bone
Fracture? Bill E. Cham,1* Jeffery L. Smith,2 and David M.
Colquhoun3 (1 The Curacel Institute of Medical Research,
14/1645 Ipswich Rd., Rocklea Queensland 4106, Australia; 2 Lipid Metabolism Laboratory, Department of Surgery, University of Queensland, Royal Brisbane Hospital,
Herston Queensland 4029, Australia; 3 Wesley Medical
Centre, Auchenflower Brisbane Queensland 4060, Australia; * author for correspondence: fax 61-7-3274-4453)
We wished to investigate the mechanism of decreased
serum vitamin K1 after bone fractures. Vitamin K1 plays a
role in bone formation because it is required as a cofactor
for the transformation of glutamic acid (Glu) residues on
proteins to g-carboxyglutamic acid (Gla) residues. The
double carboxy group on Gla residues has high affinity
for the binding of calcium. Bone formation involves
vitamin K-dependent small peptide osteocalcin (boneGla-protein) that is secreted by osteoblasts.
Serum concentrations of vitamin K1 reflect in part the
capacity of the serum to carry the vitamin. Vitamin K1 is a
lipid, and little is known about the binding of this vitamin to
proteins other than that it is transported in serum by the
lipoproteins. It is not known whether there is a specific
“vitamin K1-binding protein” in tissues as has been suggested for vitamin E as a “tocopherol-binding protein” (1).
Deficiency of vitamin K will lead to defective g-carboxylation of vitamin K-dependent proteins and will be
2262
Technical Briefs
manifested by the failure of these proteins to function
normally (2– 8 ). Low serum concentrations of vitamin K1
have been reported to occur in patients with traumatic
bone fractures (4 – 6 ), although others have reported nonsignificant decreases in vitamin K1 in the immediate 48 h
after low-energy trauma hip fracture (9 ). These serum
concentrations were considered pathological because they
were significantly lower than values in age-matched control subjects. Recent studies, however, have shown that
even very low absolute concentrations of vitamin K1 in
serum do not reflect the vitamin K status (3, 10, 11 ). The
use of a relative measure of serum vitamin K1, the ratio of
vitamin K1 to lipids (11 ) or to apolipoproteins (3, 12 ), has
become essential in evaluating vitamin K1 nutritional
status as has been shown for vitamin E (11 ). Under
routine conditions, the ratios of vitamin K1 and vitamin E
to other plasma lipoproteins components are relatively
constant. Changes in the metabolism of lipoproteins such
as are the case with hypercholesterolemia do not seem to
affect such ratios (3, 11 ).
Stepwise linear regression methods have determined
that serum concentrations of vitamin K1 could best be
predicted by using equations excluding lipids but containing only apolipoprotein A1 and B concentrations:
Vitamin K1 (mg/L) 5 369 3 apolipoprotein B (g/L) 3
apolipoprotein A1 (g/L). The correlation coefficient between the calculated values of vitamin K1 using serum
concentrations of apolipoproteins A1 and B and the measured (HPLC) values of vitamin K1 was 0.83 (3 ).
It has been suggested that the low serum concentrations
of vitamin K1 observed in patients with traumatic bone
fractures was a consequence of sequestration of this
vitamin from the circulation for use at the fracture site
where it is required for the Gla transformation of special
bone peptides (4 ).
LDL, which contains apolipoprotein B, is a negative
acute phase reactant and is induced by inflammation
(13, 14 ) such as with traumatic bone fractures. A decrease
in synthesis and an increase in degradation of apolipoprotein B have been shown to occur. The reduction in
apolipoprotein B concentration is reflected by a reduction
in serum LDL concentrations as has been shown after
acute myocardial infarction (13, 14 ). Consequently, because of the relationship of vitamin K1 and apolipoprotein
B (3 ), this may reflect the reduction in serum vitamin K1
concentrations observed in patients soon after fracture.
Thus it is possible that the lower concentration of vitamin
K1 in serum observed after bone fracture is attributable to
a generalized lipoprotein carrier phenomenon (the negative acute phase response of LDL), which is an alternative
explanation to the sequestration of vitamin K1 from its
carrier to bone fracture site.
To test whether the metabolism of vitamin K1 in patients with bone fractures is altered independently of the
lipoprotein carrier system, we measured vitamin K1 (15 )
and apolipoproteins A1 and B (3, 16 ) in sera from eight
patients admitted for treatment of traumatic bone fractures in the pelvis. The sera were collected from all male
patients (mean age, 45.0 years; range, 15– 64 years) within
10 days (range, 1–9 days) of sustaining the fracture (4 ).
The exclusion criteria were blood transfusions or surgical
procedures before the period of sample collection and
past and present illnesses related to bone metabolism. No
subjects had been treated for osteoporosis, and none
received medications before or during the study that
might have affected calcium metabolism.
Serum samples were also taken from 12 healthy male
subjects (mean age, 46.4 years; range, 19 –55 years) for
analyses of vitamin K1, apolipoprotein A1, and apolipoprotein B. There was no statistical difference (Student
t-test) between the ages of controls and patients with fractures. Informed consent was obtained from all participants.
The procedures used for these human studies were in accord
with the Helsinki Declaration of 1975, as revised in 1996.
Serum vitamin K1 concentrations from all subjects were
also calculated by applying derived equations containing
apolipoproteins A1 and B concentrations (3 ).
The mean serum concentration of vitamin K1 was significantly lower in the fracture patients than in the control
subjects, but the concentrations of apolipoproteins A1 and B
were not significantly different in the two groups (Table 1).
When vitamin K1 concentrations in serum were calculated by the equation: Vitamin K1 (mg/L) 5 369 3
apolipoprotein B (g/L) 3 apolipoprotein A1 (g/L) (3 ), the
obtained mean value for the control subjects was 348
mg/L, similar to the value of 361 mg/L (Table 1). The ratio
of corresponding individual measured/calculated values
of vitamin K1 in serum was 1.04 (SD 5 0.04).
In contrast, the calculated mean concentration of vitamin K1 from the bone fracture patients, 369 mg/L, was
Table 1. Concentrations of measured and calculated vitamin K1 and measured apolipoproteins A1 and B in the sera of
healthy subjects and patients after bone fracture.a
Healthy controls
(n 5 12)
Patients
(n 5 8)
Vitamin K1 assayed,
mg/L
Apolipoprotein B,
g/L
Apolipoprotein A1,
g/L
Vitamin K1 calculated,
mg/L
Vitamin K1 assayed/
calculated
361 6 77b
(296–572)
221 6 81d
(100–390)
0.73 6 0.12
(0.53–0.91)
0.93 6 0.24
(0.62–1.33)
1.60 6 0.29
(1.23–1.95)
1.40 6 0.41
(1.00–2.03)
348 6 78
(281–561)
369 6 91e
(307–585)
1.04 6 0.04c
0.60 6 0.13f
a
The results are expressed as mean 6 SD; the concentration ranges are shown in parentheses. Vitamin K1 concentrations were assayed by an established liquid
chromatographic procedure (15). Apolipoproteins A1 and B were measured by immunoassays (16). Vitamin K1 was calculated by the formula: Vitamin K1 (mg/L) 5
369 3 apolipoprotein B (g/L) 3 apolipoprotein A1 (g/L) (3). The vitamin K1 ratios of assayed and calculated values were from corresponding individual subjects.
b–f
Pairs differing significantly from each other at P ,0.01: b vs d, d vs e; at P ,0.001: c vs f.
Clinical Chemistry 45, No. 12, 1999
significantly different (P ,0.01) from the measured mean
concentration of 221 mg/L (Table 1). The ratio of corresponding individual measured/calculated values of vitamin K1 in serum of bone fracture patients was 0.60 (SD 5
0.13). This ratio was significantly different (P ,0.001)
from that of the control subjects (Table 1).
The current results confirm that the circulating serum
vitamin K1 concentrations are reduced shortly (within 10
days) after bone fracture (4 –7 ). It is known that vitamin
K1 deficiency occurs in elderly subjects (6 ). However, in
our study, because all the subjects were males and their
ages were not relatively old, together with the fact that the
age range of the controls was similar to that of the
patients, it is unlikely that age had an effect on our
observations. In control subjects, vitamin K1 concentrations in serum can be calculated from the concentrations
of apolipoproteins A1 and B (3 ). However, in patients
after bone fracture, the changes in serum vitamin K1
concentrations are not paralleled with changes in their
carrier systems, the lipoproteins. There is no evidence that
after bone fracture apolipoprotein A1 and B concentrations in serum are altered. It appears that serum vitamin
K1 is utilized independent of its lipoprotein carriers in
serum. These observations support the concept that this
vitamin is sequestered from lipoproteins in the circulation
for use, perhaps at the fracture site (4 ). The current results
indicate that the mode of sequestration of vitamin K1 is
independent of the metabolism of lipoproteins, an observation that has not previously been reported. In patients
without bone fractures, the vitamin K1 concentration can
be predicted from the concentrations of apolipoproteins
A1 and B, whereas it cannot be predicted by the same
equation in patients with bone fractures. The mechanism
of vitamin K1 delivery to the fracture site remains to be
elucidated, but it could conceivably operate via a putative
receptor in which the vitamin K1 is selectively taken up,
analogous to the interaction of HDL with cells without the
loss of its apolipoprotein components. This study further
indicates a need to validate the use of equations containing the serum concentrations of apolipoproteins A1 and B
to calculate vitamin K1 concentrations in serum (3 ) in
patient groups where changes in vitamin K1 metabolism
are in question.
This work was supported by grants from Curacel International Pty Ltd and the National Health and Medical Research Council of Australia. We thank Peter Roeser for
supplying the serum from the patients. We gratefully acknowledge technical assistance from Karim Cham, Tania
Chase, Annette Miles, Mark Jones, and Kelly Kingston.
References
1. Traber MG, Soko RJ, Burton GW, Ingold KU, Papas AM, Huffaker JE, Kayden
HJ. Impaired ability of patients with familial isolated vitamin E deficiency to
incorporate–tocopherol into lipoprotein secreted by the liver. J Clin Investig
1990;85:397– 407.
2. Suttie JW. Recent advances in hepatic vitamin K metabolism and function.
Hepatology 1987;7:367–76.
3. Cham BE, Smith JL, Colquhoun DM. Interdependence of serum concentra-
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Bitensky L, Hart JP, Catterall A, Hodges SJ, Pilkington MJ, Chayen J.
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Hodges SJ, Pilkington MJ, Stamp TC, Catterall A, Shearer MJ, Bitensky L,
Chayen J. Depressed levels of circulating menaquinones in patients with
osteoporotic fractures of the spine and femoral neck. Bone 1991;12:387–9.
Hodges SJ, Akessin K, Vergnaud P, Obrant K, Delmas PD. Circulating levels
of vitamin K1 and K2 decreased in elderly women with hip fracture. J Bone
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Roberts NB, Holding JD, Walsh HP, Klenerman L, Helliwell T, King D, Shearer
M. Serial changes in serum vitamin K1, triglyceride, cholesterol, osteocalcin
and 25-hydroxyvitamin D3 in patients after hip replacement for fractured
neck of femur in osteoarthritis. Eur J Clin Investig 1996;26:24 –9.
Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz GA. Vitamin
K intake and hip fractures in women: a prospective study. Am J Clin Nutr
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Takahashi M, Kushida K, Hoshino H, Aoshima H, Ohishi T, Inoue T. Acute
effects of fracture on bone markers and vitamin K. Clin Chem 1998;44:
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Mandelbrot LM, Guillaumont M, Leclercq M, Lefrere JJ, Gozin D, Daffos F,
Forestier F. Placental transfer of vitamin K and its implications in fetal
haemostasis. Thromb Haemost 1988;60:39 – 43.
Cham BE, Smith JL, Colquhoun DM. Correlations between cholesterol,
vitamin E, and vitamin K1 in serum: paradoxical relationships to established
epidemiological risk factors for cardiovascular disease. Clin Chem 1998;
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Kohlmeier M, Salomon A, Saupe J, Shearer MJ. Transport of vitamin K to
bone in humans. J Nutr 1996;126(Suppl 4): 1192S– 6S.
Avogaro P, Bittolo Bon G, Cazzalato G, Quinci GB, Sanson A, Sparla M,
Zagatti GC. Variations in apolipoproteins B and A1 during the course of
myocardial infarction. Eur J Clin Investig 1978;8:121–9.
Pfohl M, Schreiber I, Liebich HM, Haring HU, Hoffmeister HM. Upregulation
of cholesterol synthesis after acute myocardial infarction–is cholesterol a
positive acute phase reactant? Atherosclerosis 1999;142:389 –93.
Cham BE, Roeser HP, Kamst TW. Simultaneous liquid-chromatographic
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Pruvot I, Flevet C, Durieux C, Vu Dac N, Fruchart JC. Electroimmunoassay
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Molecular Forms and Ultrastructural Localization of
Prostate-specific Antigen in Nipple Aspirate Fluids,
Ferdinando Mannello,1* Manuela Malatesta,1 Maurizio Sebastiani,2 Serafina Battistelli,1 and Giancarlo Gazzanelli1 (1 Istituto di Istologia & Analisi di Laboratorio, Facoltà di
Scienze Matematiche, Fisiche e Naturali, Università Studi,
Via E. Zeppi, 61029 Urbino-PS, Italy; 2 Centro di Senologia, AUSL 1, Pesaro, Italy; * author for correspondence:
fax 39-0722-322370, e-mail [email protected])
Prostate-specific antigen (PSA) is a member of the human
kallikrein family of serine proteases that for a long time
was thought to be produced exclusively by the epithelial
cells of the prostate gland (1 ). Because of its tissue
specificity, PSA has been widely used as a marker for
diagnosing and monitoring prostate cancer (2 ). However,
recent studies have demonstrated the widespread distribution of PSA in a variety of tumor types, healthy tissues,
and biological fluids [reviewed in Ref. (3 )]. Although the
physiological role and the biological significance of extraprostatic PSA currently are unknown, it has been suggested that this serine protease should be regarded as a
growth factor regulator produced by cells bearing steroid
hormone receptors (4, 5 ). Several studies have biochemi-