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400
M .
A. BARRADAS et
al.
controls had a history of diabetes, vascular disease or
hypertension.
The patients studied were attending the diabetic and
vascular out-patient clinics at Th e Royal Free Hospi-
tal. The ID D M group consisted of 23 patients (1 1 men,
12 women); m edian age 50 years (range 23-72); m edian
duration
of
diabetes 6 years (range 2-25); median
blood glucose 11.5 mm ol I (range 4.0-28.3); median
glycosylated haemoglob in 9.5 (range 5.8-1 1.3). Th e
NIDDM group consisted of 23 patients (15 men, 8
women), median age 66 years (range 40-86); median
duration of diabetes 10 years (range 1-15); median
blood glucose 1 .4 mmol 1
I
(rang e 5.2- 13.7);median
glycosylated haemoglobin 10.1O o ( range 7 - 5 1 .1 ).
The PVD group consisted of 29 patients (18 men, 11
women); median age 73 years (range 47-79). All the
patients in the PVD group had: (i) intermittent
claudication for more than 6 months with (ii)
ank1e:arm systolic blood pressure (SBP) ra tio < 0.85.
Patients were defined as hypertensive
if
they had a
diastolic blood pressure
>
95 mmHg on two separate
occasions.
Study
2
For this study, a similar but smaller population of
patients and healthy subjects were sampled. The
control gr oup consisted of 10 healthy subjects (7 men,
3 women); median age 50 years (range 25-68). The
IDDM group consisted of 11 patients 6 men, 5
women); median age 53 years (range 23-77); median
duration
of
diabetes 11 years (range 3-40). The
NI DD M grou p consisted of 14 patients (7 men, 7
women); median age 62 years (range 51-83); median
duration of diabetes
11
years (range 2 months-30
years). The PV D group consisted of 13 PVD patients
10
men, 3 women); median age 68 years (range 56-83).
Patients were diagnosed as PVD according to the
criteria described in stud y
1.
Drugs
Healthy subjects denied taking drugs for at least 2
weeks prior to sampling. Diabetic patients were
on
standard treatment regimens with insulin/oral hypog-
lycaemic agents and diet. Hypertensive patients (DM
and PVD) were
on
a combination of nifedipine and
bendrofluazide.
Blood
sample collection and processing
Blood was taken from the antecubital vein of patients
and volunteers with minimal stasis, and nine parts of
bIood were added to one part
of
3.8% w/v trisodium
citrate (B DH , Poole, Dorset, U .K.). T o each 1 ml of
blood 100 pg of acetylsalicylic acid (B D H) were added
to prevent an y release of intraplatelet constituen ts [4]
Plasma for serotonin measurements was prepared by
centrifuging blood for 20 min a t 1500x g at 22C. The
supernatant was frozen immediately and kept at
-40C until assay. Platelet rich plasma ( PR P) was
prepared as previously described 151 Platelet counts
were then performed using a Coulter
ZM
counter
(Coulter Electronics Limited, Luton, Bedfordshire,
U.K.). Following counting, the PRP was centrifuged
a t
1000
x g
for
10
min t o prepare platelet pellets. The
pellets were washed with Is oton
I1
(Co ulter Electronics
Limited) an d stored at 0C until analysis.
Platelet serotonin
assay
Platelet pellets were resuspended in physiological
saline 0.9 w/v) and a platelet lysate prepared by
ultrasonicating the platetet pellet for 3
x
10 sec a t a n
amplitude of 18 microns using an MSE-Soniprep
sonicator
MSE,
Sussex, U .K.).
In
order to ensure that
our sonication procedure fully disrupts platelets, we
counted, sized and plotted platelet populations before
and after sonication. F or this purpose, platelets were
obtained from healthy subjects and the above para-
meters were assessed using a Co ulter Z M counter with
a Channelyzer C-1000 and X-Y Recorder (Coulter
Electronics Limited). Following sonication, the plate-
let count was reduced to
< 5
of the original, the
mean platelet volume became unmeasurable and the
graphical plot obtained with the sonicated platelets
became indistinguishable from the plot ob tained with
platelet diluent (Isoton 11). The serotonin content in
the platelet lysates was assayed by a modification of the
spectrofluorimetric method of Drummond & Gordon
[6,7]. Briefly, 6 M trichloroacetic acid (B DH ) was added
to the platelet lysates to precipitate the proteins. T he
samples were then centrifuged at 10
000
x g for 4 min.
A portion of the supernatants was removed and
transferred to glass test-tubes, and to each of these
o-
phthaldialdehyde (Sigma Chemical
Co.,
Poole, Dor -
set,
U.K.)
in HC1 was add ed, and the m ixture heated
in boiling water fo r 10min. T he tubes were then cooled
in ice and washed twice with chloroform Analar
(BDH) to remove any traces
of
trichloroacetic acid [8].
Th e aqueous phase was removed a nd the fluorescence
was read in a Perkin-Elmer MPF-3 (Hitachi Ltd,
Tokyo, Japan) fluorescence spectrophotometer, with
excitation and emission wavelengths of 360 and 475
nm, respectively. Sta nda rds and blanks were processed
in the same way a s the platelet lysates. Th e intra-assay
coefficient of variation (CV) for the whole procedure
was 4.0% n 20) and the interassay CV for the whole
procedure was 12% (n= 8). As an added precaution,
samples fro m each o f the grou ps studied were included
in each assay.
Plasma serotonin
ss y
Plasma serotonin concentrations w ere estimated using
a radioimmunoassay. A ntisera, stan dard s and re-
agents were purchased from Im munodiagnostics Ltd
(Wash ington, Tyne and W ear, U.K.). T he intra-assay
CV for this method was 3.1% n =
10)
and the
interassay CV was 5.1
%
n 10).
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INTRAPLATELET SEROTONIN IN DIABETES MELLITUS AND VASCULAR
DISEASE
401
Blood glucose determination
Blood glucose measurements were carried out in the
clinic at the time
of
blood sampling, using the YSI
Model 23 AM glucose analyser (Yellow Springs
Instruments, Yellow Springs, U.S.A.).
Glycosylated HbAl determination
Glycosylated HbAl measurements (electrophoretic
technique) were performed on whole blood using the
German-Hawksley glycosylated haemoglobin kit
(German-Hawksley, Northampton, U.K.).
Statistical analysis and expression
of
results
Platelet 5-HT content is expressed as nmol per
lo9
platelets. Since the distribution of the data was non-
parametric, the results are expressed as medians with
the range in parentheses.
The Mann-Whitney U-test (two-tailed) was used for
comparing unpaired data . Details of comparisons are
included in the appropriate tables. The Chi-square test
was used for the analysis of frequency. Linear regres-
sion analysis was carried out using a validated com-
puter program in use in the Department of Chemical
Pathology and Human Metabolism at The Royal Free
Hospital.
Results
Study I
Comparison between
young
and elderly healthy
subjects.
In healthy subjects platelet 5-HT content
decreased with increasing age n 34,
r
.45,
P
