Læknablaðið : fylgirit - 03.01.2017, Blaðsíða 23

X V I I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 9 1
LÆKNAblaðið/Fylgirit 91 2017/103 23
Allopurinol treatment effectively reduces DHA excretion and prevents
renal complications. The aim of this study was to develop and optimize
an UPLC-MS/MS assay for quantitative measurement of the active all-
opurinol metabolite, oxypurinol in human plasma, utilizing design of
experiments (DoE). Additionally, an UPLC-MS/MS assay to assess APRT
enzyme activity was developed.
Methods: Experimental screening was performed to reveal significant
UPLC-MS/MS factors influencing the peak area for oxypurinol. Significant
variables were optimized utilizing partial least square regression.
Following optimization, a sample preparation method was developed
for oxypurinol in plasma from 2 individuals with APRT deficiency taking
allopurinol 300mg/day and 2 healthy controls. The APRT enzyme activity
was evaluated by measuring the formation of AMP in erythrocyte lysates
from 11 patients, 3 heterozygotes and 10 controls using an UPLC-MS/MS
assay. Zygocity was confirmed with genetic testing (APRT sequencing).
Results: Significant difference was observed in plasma oxypurinol
concentration between the 2 patients (94.9 and 150.9µg/ml) and normal
genotype healthy controls (BLQ). Further, significant changes were obser-
ved in the formation of AMP between patients, heterozygotes and normal
genotype controls confirming the diagnosis of APRT deficiency.
Conclusions: An UPLC-MS/MS assay for quantitative measurement of
oxypurinol in human plasma was successfully developed and optim-
ized utilizing DoE. The plasma measurement method may be used for
pharmacotherapy monitoring and evaluation of DHA tissue stores. Both
UPLC-MS/MS assays may be used for the diagnosis of APRT deficiency.
E 52 Markers for Increased Thrombotic Risk Within the Complete
Blood Count
Sæmundur Rögnvaldsson1, Sigrún H. Lund1, Malin Hultcrantz2, Guðný
Eiríksdóttir3, Tamara B. Harris4, Vilmundur Guðnason3, Sigurður Y. Kristinsson1
1Faculty of Medicine, University of Iceland, 2Dept. of Hematology, Karolinska Institutet, 3The
Icelandic Heart Association, 4National Institute of Aging
s.rognvaldsson@gmail.com
Introduction: The complete blood count (CBC) provides the parameters
of hematocrit (Hct), white blood cell count (WBC), and platelet count
(PLT). Studies have shown an increased thrombotic risk in healthy
individuals with elevated Hct and HGB. However these studies did not
include sufficient clinical data to adjust for confounders. We aim to assess
whether elevation of these markers increase thrombotic risk in the general
population.
Methods and data: CBC and baseline characteristics were obtained from
5755 elderly participants of the Reykjavik-AGES study at enrollment.
Incidences of venous and arterial thrombosis 10 years before and after
enrollment were acquired from the National Health Service. Hct, WBC
and PLT in quintiles were used to determine exposure with the second
quintile as reference. Cox proportional hazard regression was used for
statistical analysis and adjusted for risk factors of thrombosis. Individuals
with grossly abnormal measurements were excluded.
Results: Crude analyses of Hct revealed a dose dependent increased
risk of arterial (HR1.2, CI[1.08-1.33], p<0.001) and venous (HR1.2, CI[1.8-
1.34], p=0.001) thrombosis. After adjusting for confounders there was no
association. Analysis of PLT did not show an effect on risk of thrombosis.
Analysis of WBC showed a dose dependent increase in the risk of arter-
ial (HR1.31, CI [1.18-1.45], p<0.001) and venous (HR1.16, CI[1.02-1.32],
p=0.027) thrombosis.
Conclusions: In this large population based cross-sectional cohort study
we observed no association of elevated Hct or PLT and risk of thrombosis.
We found a dose-dependent increase in thrombotic risk with elevated
WBC. We therefore conclude WBC to be a marker of thrombotic risk in the
general population.
E 53 A unique IL-10 driven defect involving transitional B cells
in IgA deficiency
Andri L. Lemarquis1, Rakel Natalie Kristinsdóttir2, Helga Kristín Einarsdóttir2,
Björn R. Lúðvíksson2
1Department of Immunology, and Rheumatology Research, Landspítali-The National
University Hospital of Iceland, 2Department of Immunology, Faculty of Medicine/ University
of Iceland
aleofifi@gmail.com
Introduction: Selective IgA deficiency (IgAD) is the most common
primary immune deficiency in the western world. It´s pathogenesis is due
to a largely unknown deregulation in B cell maturation.
Methods: Different B and T cell populations from IgAD individuals and
healthy controls (HC) were analysed and their function assessed using
FACS and ELISA.
Results: IgAD individuals demonstrated a significant defect in IgA+
B cells and significantly lower number of transitional B cells (TC,
CD19+CD24hiCD38hi) compared to HC. Both in peripheral blood and
after CpG induced expansion. Furthermore a significantly higher fraction
of IgAD TC was IL-10+ compared to HC following stimulation. Following
IgA class switching promoting cultures of isolated B cells a unique
population of CD20+IgD-IgM-IgG-IgA- B cells being “stuck” in its differ-
entiation pathway was revealed in IgAD individuals. This specific defect
was not due to dysfunctional iTregs since their numbers and function
was found to be normal in IgAD. A major function of TC is their IL-10
secretion. Therefore the long lived IgA production was tested with and
without the exogenous addition of IL-10 in a model mimicking the long
lived induction and maintenance of IgA production. Long lived IgA prod-
uction was achieved up to 3 weeks in HC as measured by ELISA but failed
completely in IgAD individuals.
Conclusions: IgAD may be caused by a maturation defect in transitional
cells or to antecedent maturation stages related to TLR9 stimulation,
leading to an increase in IgG-IgA-IgD-IgM- B cells which may be due to
proliferation without developmental progression.
E 54 Development of a Cyclodextrin-based Aqueous 0.2% (w/v)
Cyclosporin A Eye Drop Formulations
Sunna Jóhannsdóttir1, Zoltán Fülöp1, Einar Stefánsson2, Þorsteinn Loftsson1
1Faculty of Pharmaceutical Science, University of Iceland, 2Faculty of Medicine, University of
Iceland
suj1@hi.is
Purpose: Cyclosporin A (CyA) is a lipophilic, cyclic polypeptide with
anti-inflammatory properties. It is used in topical treatment of dry eyes
and available in oil-based surfactant containing eye drops. Surfactants
can irritate the eye surface and oil-based drops can result in blurred
vision. Our aim was to develop aqueous oil-free CyA eye drops free of
surfactants and prepare the eye drops for toxicological evaluation in
rabbits.
Methods: The CyA/CD aggregation was studied using dynamic
light scattering (DLS) and by solid drug fraction measurements.
Physicochemical properties of the formulation were also determined.
Toxicological studies in 16 rabbits were initiated.