The Determination
of
Esterase D
ın
MenstruaI Blood Stains
GL'R!"IDrDER KA UR ,
VrrAKK.
SHAR.\1AForensic Science, Departmenı of Chemisıry,
Punjabi Vniversity, Patiala -
147 002
IndiaMENSTRÜAL KAN LEKELERlsDE ESTERAZ D
AKTıVİTESİNİNTA
YİNtÖzet
Penjab Ün.ıvanites! öğrencisi olan 107 kııdan taze kan ve menstürasyon kanı alındı. Kan lekesı içeren materyal benzidin, fenolftalein, hemin kristalleri ve hemokromojen kristalleri testleri ile incelendi. Ayrıca, üreme kanalının glikojen içerigi, müsin ve epiıelya! hücreleri açılanndan da araştırıldı. EsD incelenmesinde 1 mm ka1ınlıgmda ni~asta jel elektroforezi yöntemi kullanıldı.
Tüm mensıürasyon kanı lekeleri örneklerinde yapılan benzidiıı, fen 01 ftalei:ıı, beınin kristalleri, hemokromojen kristaller, müsin, glikojen, müsin ve endometrial hücre testleri pozitif sonuç verdi. Nonnal kan ve menstruasyon kanı lekelerindeki EsD fenoüplerinin dağılımı incelendi ınce tabaka ııişasta jel elektroforoezi ile. EsD tiplerinin görülme sıkııgı 1-1 tipinde %37.38 , 2-1 tipinde %50.47 ve 2-2 tipinde %12.15 olarak bulundu. Gen frekandan EsD! için O.626±O.033, EsD2 için O.374tO.033 olarak hesaplandı.
Sunımary
Menstrual blood suills of 107 individuals were collccted and analysed by a thin layer st.arch gd clectrophoresis. The oC<.:Urrence of EsD types ı·1, 2-1 and 2· 2 was 37.38, 50.47 and 12.15 %, respectively. The estimated gene frequencies for EsD! and EsJ)2 were O.fi26 t 0.0:\1 and 0.374
t
0.033 respectively.Keywords .
Mens/rual b/aad - EsD - Palynwrphism - I:wzymes4 G. KAUR, V.K. SHARMA
INTRODUCTION
Menstrual
b
lood stains may be found on
th
e bed sheets, pillows and clothings of the
vict
im
or the accuse
d
. Its identification is b
a
sed on the detectio
n
of glycogen,
m
ucin,
and endometrial and vaginal epithelial cells
(1
-5). The me
n
strual blood is acidic
i
n
nature
due to the
p
rese
n
ce of lactic acid in vagina (6). The typ
i
ng o
f
genetic m
ar
kers
l
ike ccll
surface antige
n
s, proteins an
d
en
z
ymes can be ve
r
y
v
aluab
l
e
in the invcstiga
ti
on o
f
cri
m
es. Asano et al (7) repo
rt
ed that LDH-4 and LDH-5 fractions (especial
l
y the sum o
f
two) were h
i
ghly elevated in mens
tru
al blood stains. Whitehead and Divall (8) have
i
den
t
ificd menstrual blood stains by the imm
u
noelectrophoretic characterization of
so
l
uble fib
ri
n
ogen.
Polymorphism of human red cell esterase D was f
ir
st desc
ri
bed
b
y Hopkinson et al
(9). The enzyme, which is present in red cells and most other tissues (3-
10
), was shown
to be biochcm
i
ca
ll
y and genetically distinct from the previously recognize
d
red ec
U
esterases Al'
Az, A3, B and C (11-14).
it
was charac
t
erized by the abili
t
y to hydr
o
lyse
4-methyl u
m
belliferyl acetate and butyrate. The enzyme was detected
af
ter thi
n
laye
r
starc
h
gel e
l
ectrophoresis by the appearance of fluorescent bands due to the release of
u
mbeUiferone at the site of enzyme activity.
Hopkinson et al (9) discovered that EsD from human erythrocytes exh
i
bits
polymorphism viz., EsDI'I
,
EsD2.2 and EsD2.1 representing the homozygo
u
s and
hetero
z
ygous express
i
on of two allelie genes at an autosoma
l
locus. Three rare esle
r
ase
variants, EsD3.1, Es])4-1 and Es])4·2 have alsa been reported (15-16).
Esterase D is termed a non-specific esterase as its exact
m
etabo
li
c role in the
b
lood
i
s
unknown.
it
is reported, however, to be more active with esters of low mo
l
ecu
l
ar weight
fatty acids like acelates and butyrates. This enzyme oeeurs i
n
most tissues and its correct
phenotype can be easily recognized from their aqueous extracts (17). I
t
has not bccn
de
m
onstrated in seminal or vag
in
al secretions (17).
Popula
ti
on varia
n
ts of other esterases
Aı'
Az,
A3 ' B and C have proven
t
o be eit
h
e
r
exceedingly rare or
p
ractically non-existent, and conseque
n
tly have no forensic
appliea-tion. Esterase D
,
because of its high genetic po
l
ymorphism
i
n
E
uropea
n
, African and
Asian Indian populations, has a
poıentialuse as another important parameter i
n
forensic
serology (10
,
17-21). Since not much work appears to have been done on EsD in
men-strual blood Slains, the present study was undertaken.
Paired fresh blood and menstnıal blood stains were collected from 107 girls of Punjabi University,
Paıiala. The washed packed red cells were hremolysed by freezing and thawing, whereas, nonnal blood
stains and menstnıal blood stains were dried and stored at room temperatnre until analysed.
The pH of all menstnıal blood stains was detennined with a pH paper and was 4 - 4.5. The blood stained material was examined by benzidine (1, 22), phenolphtalein (22, 23), hremin crystal (22, 24), hremochromogen crystal (22, 25) tests. These were also tested for glycogen, mucin and epithelial cells of reproductive tracı (26).
The species origin was detennined by immunodiffusion (27) and counter-immunoelectrophoretic (28) techniques using anti-human, anti-fowl, anti-goaı, anti-cow and anti-dog serum. The electrophoresis of EsD was conducted in 1 mm thick starch gel (19). Whatrnan No. 3 filter paper strips (7 mm x 1 mm) were soaked in the hremolysates and eluates from the blood stained materials and were loaded in the gel. The samples were treated with O.OSM Cleland's reagent for 10-15 minutes.
Gel: A 1 mm thick gel, containing 10% hydrolysed starch in gel buffer, was prepared by the
method of Wraxall and Culliford (29). A gel plate of 100 x 120 x 1 mm dimensions was used throughout.
Gel bu ffer: Gel buffer was made by dissolving 0.818 gm Tris, 0.136 gm boric acid and 0.378 gm citric acid in 500 mL distilled water; pH was adjusted to 7.2.
Tank buffer: Tank buffer consisted of 27.210 gm boric acid and 1.680 gm lithium hydroxide in one litre of distilled water; pH was adjusted to 7.2.
Elec/rophoresis: Electrophoretic separation was carried out by applying constant voltage of 10 V /
cm in a refrigerator at 4"C for 5 hours.
Reac/ion bu ffer: This comprised of 0.05 M sodium acetate adjusted to pH 6.9 with acetic acid. The reaction buffer was always prepared fresh and was used immediately.
De/ec/ion: Five milligram of 4-methylumbelliferyl acetate was dissolved in 2-3 drops of acetone
and then immediately mixed with 10 mL of the reaction buffer. This mixture was soaked on to a piece of 1 mm Whatman fılter paper and laid on the gel. The plate was incubated for 5-10 minutes at room temperature. The paper overlay was removed and the bands of esterase activity were observed fluorescing brightly under a long-wave ultraviolet lamp.
RESUL Tand DISCUSSION
All samples gaye positive results with benzidine, phenolphtale
i
n,
hıemincrystal and
hıemochromogencrystal tests
confırmingthe presence of blood in the stains. All
6 G. KAUR, V.K. SHARMA
sam
pl
es were alsa posit
i
ye for m
u
ein, glyeogen and endometria
l
, bas
al
and preeomified
ee
ll
s confirming the stains to be of menstrual b
l
ood (Table
I
).
T
he normal blood stai
n
s
gaye negatiye resu
lt
s when tested similarly as they laek
m
ucin, glycogen and
yaginal
epithel
i
al as well as endometrial cells (2, 3). Af ter identification of menstrual blood
s
t
ains, tests for the species Ofigin by immunodiff
u
sion and
counter-immuno-eleetrophoresis proyed them to be positiye for humans as only
anti-human
serum gaye
positiye reactions, confirming the stains to be of human origin (T
a
ble
II
).
Table I. Results of tests for identification of menstrual blood stain
Sr. Test No. +ve
no. S.T.*
Benzidine 107 107 (100%)
2 Phenolphthalein 107 107 (100%)
3 HlEmın crystal 107 107 (100%)
4 HlEmochromogen 107 107 (100%) 5 Test for mucin 107 107 (100%) 6 Test for glycogen 107 107 (100%) 7 Test for endometrial cells 107 107 (100%) 8 Test for precomified cells 107 107 (100%) 9 Test for basal cells 107 107 (100%)
(Figures in parenthesis indicate percentage values)
Table II. Results of the tests for species origin
Sf. Technique no.
Immunodiffusion 2
Counter-immunoelectrophoresis
(Figures in parenthesi.s inmeate percentage values) No. S.T.* +ve 107 107 (100%) 107 107 (100%) -ve -ve * No. T.S. = Number of samples tested
Samples EsD phenotypes 1-1 2·1
NB
L
40 54 (37.38%) (50.47%) 40 54MBS
(37.38%) (50.47%)NBL = normal blood lysates,
MBS
=
menstrual bl00d slains. 2·2 13 (12.15%) 13 (12.15%)Gene frequencies
EsDl=0.626±0.033
EsD2=0.374±O.033
EsD1=D.626±O.033
EsD2=0.374±O.033
Table III
presents the distribution
o
f EsD phenotypes in
107 normal and menstrua}
blood samples.
The EsD types 1-1
,
2-1 and
2-2
oceurred to
the extent of 37.38,5
0
.47
and
12.15 respectively, both
İnthe
normal blood and
menstrual blood
stains. All the
samples showed
the
commonly known
EsD
types
only
(Fig
.
1).
Both EsDl and EsD2
consists of
three reg
u
larly spaced
isozyme
bands which show
deer
e
asing
staining
intensity
with
i
n
ereasi
n
g a
n
odal electrophoretie
mobilities. Heterozygote, EsD2-1,
is
represented by three prominen! bands and two fast moving faint bands. The slowest band
of EsD2
has
the
same m
o
bility as that
o
f the fastest band
of EsDl type,
wh
i
le t
h
e
s
lo
west band of
EsDl and that of EsD2r1 are of exactly the same mobility.
M.B. N.B. M.B. N.B. M.B. N.B.
L..--
_
---'i
i
L-
_
--'
(:·;·:>·;':::
·
;
·
·;::
·
;
·:>·;·:>1
r
·;·
·;>
·
;.:::·;··
;::
·;·:>·;··
;::
)
i
1 i
i
r·:·:·;··;·:·:··;·:·;
·
·;
·
:·:··;·.::}
r·;·:·;··;·:·;··;·:·;··;·>;··:·.:;·}i
111
1
11
111111
I11111111111
_
_
f%2i?/iJ
~_ _
_
_
[:
.
.
: ... :
.. :
...
:
... :J
i:··:.··:.··:.··:.··:) ıı:-':: "ı : . : ' , : : "ı :,' "ı::
1 _
"
.:
'.::
ii:: LO:: "ı :ıl "ı ::~EJDEJ
EJ
D
D
M.B.= Menstrual blood ; N.B.= Nonnal blood
+
i
Figure 1. Diagrammatical represemalion of electrophoretic patterns of phenotypes of EsD (by
8 G. KAlJR, V.K. SHARMA
The gene frcquencies estimated for EsDl and EsD
2were 0.626
±
0.033
and 0.374
±
0.033 respectively.
The frequencies ohserved
by
us are well comparahle to the frequencies reported
by
Saclıdeva eLal (17).
The prescnt results indicate that the menstrual hlood stains can be
iderııified byusing
tests for mucin, glycogen and presence of endometria! and vaginal epithetial cens. The
origin of
ıhesamples can also be determined. EsD, one of the
polyınorphicgenetic
markers, can also be
demonstraıedin menstrual blood stains. The identification of
ABO(H), Rh(D) GLO-I, POM and EAP were also
reporıedearlier (5, 30). The
identificalion
of ABOCH), Rh(D), GLO-I, PGM, EAP and EsD phenolypes is expectcd
to be of great help as an important
corroboraıiveevidence in identifying the victim or
the cıııprit
Ackn owledgcments
We are thankrul to Dr. S.M.S. Chalıa/, LeeLUfer in Human Biology, for supplying us varjous chemicals for the presenı study. One of us (GK) IS also u13nkful to BPRD (Minisıry of Home Affaus) foı ıhe award of fellowship. Special thanks are als o due to various donms who very generously donated various samples.
REFERENCES
1- Cullilord, RJ., Nickolls, L.C (1964) J. Foreıı.'>ic Sci., 9, 175·191. 2- Rhodes, P. (1969) J & A OlUrchill Ltd., London.
3-Shaw, W. (1971) J & A ehurchill Ltd., London.
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14· Tashian. R.E. (1969) New York, London, 307-336. 15-Bender, K., Frank, R. (1974) lIumangenelik,23, 315·318.
16· Berg, K., Schwanfislıcr,
F.,
Wi,cherath, H. (1976)Hum.
Geneı., 32, 8l-83. 17 - Sachdeva, M.P., Arora, V.K., Bhalla, V. (1984) Med. Scl. Law, 24, 142 145. 18· Haywaıd, J.W., Bo.won.h, A.L (l975) J Forensic Sci. Soc., 15, 289-292.19- Parkin, B.H., Adaıns, E.G. (1975) Med. Sci. Law, 15, 102·105.
