Analytical validation of an automated assay for the measurement of adenosine deaminase (ADA) and its isoenzymes in saliva and a pilot evaluation ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Clin Chem Lab Med 2021; 59(9): 1592–1599
Lorena Franco-Martínez, Fernando Tecles, Alberto Torres-Cantero, Enrique Bernal,
Indra San Lázaro, María José Alcaraz, María R. Vicente-Romero, Elsa Lamy,
Cristina Sánchez-Resalt, Camila P. Rubio, Asta Tvarijonaviciute, Silvia Martínez-Subiela*
and José J. Cerón
Analytical validation of an automated assay for
the measurement of adenosine deaminase (ADA)
and its isoenzymes in saliva and a pilot evaluation
of their changes in patients with SARS-CoV-2
infection
https://doi.org/10.1515/cclm-2021-0324 accurate way, and evaluate the possible changes of these
Received January 25, 2021; accepted April 15, 2021; analytes in individuals with SARS-CoV-2 infection.
published online April 28, 2021 Methods: The validation, in addition to the evaluation of
precision and accuracy, included the analysis of the effects
Abstract
of the main procedures that are currently being used for
SARS-CoV-2 inactivation in saliva and a pilot study to
Objectives: The aim of the present study was to validate a
evaluate the possible changes in salivary tADA and
commercially available automated assay for the measure-
isoenzymes in individuals infected with SARS-CoV-2.
ment of total adenosine deaminase (tADA) and its
Results: The automated assay proved to be accurate and
isoenzymes (ADA1 and ADA2) in saliva in a fast and
precise, with intra- and inter-assay coefficients of variation
below 8.2%, linearity under dilution linear regression with
R2 close to 1, and recovery percentage between 80 and 120%
Lorena Franco-Martínez and Fernando Tecles have contributed equally in all cases. This assay was affected when the sample is
to the manuscript.
treated with heat or SDS for virus inactivation but tolerated
*Corresponding author: Prof. Silvia Martínez-Subiela, Triton X-100 and NP-40. Individuals with SARS-CoV-2
Interdisciplinary Laboratory of Clinical Analysis Interlab-UMU, infection (n=71) and who recovered from infection (n=11)
Regional Campus of International Excellence Mare Nostrum, had higher mean values of activity of tADA and its
University of Murcia, Ed.16, 4th floor, Espinardo, Murcia 30100, Spain, isoenzymes than healthy individuals (n=35).
Phone: +34 868887168, E-mail: silviams@um.es. https://orcid.org/
Conclusions: tADA and its isoenzymes ADA1 and ADA2
0000-0002-1524-1558
Lorena Franco-Martínez, Fernando Tecles, Camila P. Rubio, Asta
can be measured accurately and precisely in saliva sam-
Tvarijonaviciute and José J. Cerón, Interdisciplinary Laboratory of ples in a rapid, economical, and reproducible way and can
Clinical Analysis Interlab-UMU, Regional Campus of International be analyzed after chemical inactivation with Triton X-100
Excellence Mare Nostrum, University of Murcia, Espinardo, Murcia, and NP-40. Besides, the changes observed in tADA and
Spain. https://orcid.org/0000-0003-3375-4274 (L. Franco-Martínez) isoenzymes in individuals with COVID-19 open the possi-
Alberto Torres-Cantero and Indra San Lázaro, Preventive Medicine,
bility of their potential use as non-invasive biomarkers in
Hospital Clínico Universitario Virgen de la Arrixaca, IMIB, Universidad
de Murcia, Murcia, Spain this disease.
Enrique Bernal and María José Alcaraz, Unit of Infectious Diseases,
Keywords: analytical validation; assay; biomarkers;
Hospital General Universitario Reina Sofía, Universidad De Murcia,
Murcia, Spain COVID-19; SARS-CoV-2.
María R. Vicente-Romero, Unit of Microbiology, Hospital General
Universitario Reina Sofía, Universidad De Murcia, Murcia, Spain
Elsa Lamy, Mediterranean Institute for Agriculture, Environment and Introduction
Development (MED), Advanced Research and Training Institute (IIFA),
University of Évora, Évora, Portugal. https://orcid.org/0000-0002-
Adenosine deaminase (ADA, EC 3.5.4.4) is an enzyme that,
9370-1337
Cristina Sánchez-Resalt, Sport Medicine Centre, University of Murcia, among other biological roles, catalyses the conversion of the
Espinardo, Murcia, Spain toxic molecules adenosine and/or deoxyadenosine to inosineFranco-Martínez et al.: Adenosine deaminase automatic analysis in saliva 1593
and deoxyinosine, respectively [1]. This enzyme is present in In this paper, it is hypothesized that tADA, ADA1, and
most tissues, especially in those with a lymphoid origin [2]. ADA2 might be quantified in saliva by an automated spec-
There are two ADA isoenzymes: ADA1 and ADA2, that can be trophotometric method and that, due to their relationship
differentiated by addition to the sample of erythro-9- with the immune system, they could potentially change in
(2-hydroxy-3-nonyl) adenine (EHNA). EHNA is a specific COVID-19 patients. Thus, the objectives of the present study
ADA1 isoenzyme inhibitor that, at a proper concentration, is were to validate an automated commercially available
able to completely inhibit ADA1 isoenzyme whereas remains spectrophotometric method for the measurement of tADA
ADA2 unaffected [3]. In humans, ADA1 is mainly present in and its isoenzymes in human saliva and to perform a pilot
lymphocytes and macrophages, whereas ADA2 is predomi- study to evaluate if salivary tADA, ADA1, and ADA2 change
nant in plasma [4–8]. In lymphoid tissues, ADA participates in diseased and convalescent COVID-19 patients compared
in mononuclear cell maturation from monocyte to macro- to healthy controls.
phage, as well as in the differentiation of B and T lymphocytes
[9–11]. Therefore, the level of this enzymatic activity in serum
reflects the cell-mediated immunity [12], being a biomarker of Materials and methods
chronic inflammation [13] and situations in which circulating
levels of T lymphocytes are high [14, 15]. Serum levels of ADA Participants and procedures
were shown to increase in many clinical conditions,
including infections [16, 17], inflammation [4, 13], immuno- Saliva samples from 13 healthy individuals (adults, six females) were
mediated disorders [6, 18, 19], and malignancies [20–24]. In used to characterize the ADA isoforms and establish the optimal
addition, ADA can also increase in serum due to leakage of concentration of EHNA to be used for ADA1 inhibition. For the
enzymes from damaged cells [25, 26]. calculation of imprecision and accuracy, samples from three healthy
individuals and three individuals infected with SARS-CoV-2 showing
Saliva is a biofluid with increasing medicine applica-
increases in tADA values were used.
tions since it is easy, safe, pain-free, and stress-free to Besides, saliva samples from 10 of the 13 healthy individuals used
sampling [27]. This non-invasive specimen can reflect the for ADA characterization (adults, five females) were used for studying
physiological and physiopathological state not only of the the influence of SARS-CoV-2 inactivation protocols in ADA analysis,
oral cavity but of all the organism [28]. Various biomarkers and saliva from 117 individuals (adults, 64 females) were used for the
of the immune system, inflammation, muscle damage, study of ADA changes in COVID-19.
In all cases, participants were asked not to exercise, eat, drink,
stress, oxidative status, or anaerobic metabolism, among
smoke, or brush their teeth for at least 1 h before saliva collection. An
others, have been studied in saliva, showing the potential of oral exploration of the subjects was performed to discard the presence
this biofluid. In the same line, total ADA (tADA) has been of inflammation or bleeding. All the studies were in accordance with
measured in saliva, and higher levels were observed in the the ethical standards of the Institutional and/or National Research
presence of oral malignancies [29], Sjögren’s syndrome [30], Committee (1349/2016) and with the 1964 Helsinki Declaration and its
later amendments or comparable ethical standards. All participants
and obesity [3]. However, no reports have been found in
were informed of the purpose and experimental procedures of the
which ADA activity of the different isoenzymes had been study and signed a written informed consent form prior to their
characterized in human saliva. participation. This study was approved by the Ethical Committee of the
The current COVID-19 pandemic caused by SARS-CoV-2 University of Murcia and the Ethical Committee of the IMIB-Arrixaca.
virus has implied two main changes of paradigm in the field Saliva was collected under supervision by passive drool using
of saliva analysis. One is the change in the way of dealing 5 mL polystyrene tubes with round bottoms [34]. Then, immediately
after the collection, the saliva was stored on ice until arrival at the
with saliva when used as a specimen for clinical analysis,
laboratory, weighed, centrifuged (4,000×g, 10 min, 4 °C), and the
since there is a need for inactivation of the SARS-CoV-2 virus supernatants were stored at −80 °C until further analysis. No samples
to minimize the risk of disease transmission. For this pur- showed blood contamination as determined by visual inspection.
pose, different inactivation protocols have been described,
including the inactivation with heat [31, 32] or chemical non- Analytical validation of the assay for ADA analysis
ionic procedures including NP-40 or Triton X-100 [31]. The
second one is the consideration of the saliva as a fluid that ADA was measured using a commercially available spectrophoto-
can be used for diagnosis and monitoring by the direct metric assay (Adenosine Deaminase assay kit, Diazyme Laboratories,
detection of the virus or antibodies, and also because Poway, California, USA) adapted to an automated analyser. In this
method, 50 µL of Reagent 1 containing 2 mM 4-aminoantipyrine
various biomarkers related to inflammation and immune
(4-AA), 0.1 U/mL purine nucleoside phosphorylase (PNP), 0.2 U/mL
system, including ADA [33], could be used in this disease. xanthine oxidase (XOD), and 0.6 U/mL peroxidase in 50 mM Tris-HCl
However, to the authors’ knowledge, there are no reports buffer (pH 8.0) is pipetted to the reaction cuvette with 10 µL of sample.
about the behaviour of ADA in saliva in COVID-19 patients. After 180 s, 25 µL of Reagent 2 containing 10 mM adenosine and 50 mM1594 Franco-Martínez et al.: Adenosine deaminase automatic analysis in saliva N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (EHSTP) in Group). To avoid inter-assay imprecision, all samples were measured in 50 mM Tris-HCl buffer (pH 4.0) is added. Adenosine is deaminated by the same run. ADA present in the sample to form inosine, which is then converted to hypoxanthine by purine nucleoside phosphorilase. Hypoxanthine is then oxidized by xanthine oxidase appearing hydrogen peroxide ADA in saliva in diseased and convalescent COVID-19 as a product of this reaction, which is further reacted with N-ethyl-N- patients and healthy controls (2-hydroxy-3-sulfopropyl)-3-methylaniline and 4-aminoantipyrine in the presence of peroxidase to generate quinine dye which is Three groups of individuals were used in this pilot study: kinetically monitored at 550 nm wavelength and 37 °C after 486 s [35]. – –Individuals that did not have any clinical sign of disease for at Manufacturer’s control solutions of two different values (Diazyme least 3 weeks prior to sampling and were negative to SARS-CoV-2 Adenosine Deaminase Control Set, DZ117A-CON 2 Level, Diazyme infection (n=35, 21 women, aged between 24 and 51 years old) Laboratories, Poway, California, USA) were used for the quality (healthy control group, HC). control analysis throughout the study. According to the data – Individuals that had mild clinical signs of the disease, recovered provided by the manufacturer of the kit, the clinical validation of the satisfactorily and were sampled at least 10 days after the cessa- assay in serum showed
Franco-Martínez et al.: Adenosine deaminase automatic analysis in saliva 1595
inhibition of ADA1 was achieved with 1 mM EHNA (0.12 mM Table : Intra- and inter-assay coefficients of variation (CV) of total
in the reaction mixture). ADA1 was the predominant isoform adenosine deaminase (tADA) and isoenzyme (ADA) in three pools
of saliva samples with different enzymatic activities.
in saliva, observing values below the lower limit of detection
for ADA2 in 2 out of 13 samples. Our results indicated that the
Assay Comparison Pool Mean, IU/L SD, IU/L CV, %
EHNA concentration of 1 mM was the most appropriate since
Intra . . .
it showed similar inhibition of ADA1 than the higher con-
tADA . . .
centrations of 4 mM. Using 1 mM EHNA, the median
. . .
(25th–75th percentiles) for ADA1 and ADA2 in saliva were Inter . . .
4.82 (3.25–6.50) and 0.49 (0.14–2.12) IU/L, respectively. . . .
For tADA, the intra- and inter-assay CVs were lower than . . .
5.0 and 6.2%, respectively (Table 1). Spiking recovery ranged Intra . . .
ADA . . .
from 100.20 to 105.74%. Linearity under dilution of the saliva
. . .
samples in ultrapure water resulted in a linear regression Inter . . .
equation with R2 close to 1 in the three samples analysed . . .
(Figure 2). The lower limit of detection of the assay was . . .
0.08 U/L and the lower limit of quantification was 0.19 U/L. SD, standard deviation.
For ADA2, the intra- and inter-assay CVs were below 7.6
and 8.2%, respectively (Table 1). Spiking recovery ranged
from 96.67 to 99.60%, and linearity under dilution showed NP-40 and Triton X-100 treatments did not show signifi-
correlation coefficients of R2>0.98 in all cases (Figure 2). The cant changes in the measured ADA activities.
lower limit of detection for ADA2 was 0.07 U/L and the lower
limit of quantification was 0.11 U/L.
ADA in saliva in diseased and convalescent
COVID-19 patients and healthy controls
Influence of SARS-CoV-2 inactivation
protocols in salivary ADA Results of the pilot study with healthy, COVID-19 convales-
cent, and COVID-19 diseased individuals on salivary ADA
Results of the SARS-CoV-2 inactivation protocols on sali- levels are summarized in Figure 3. The groups of diseased
vary ADA levels are summarized in Table 2. When the individuals and convalescents had significantly higher
effects of the different inactivation protocols were evalu- tADA values compared to healthy, with median (25th–75th
ated, three of them (H65, H92 and SDS) showed a marked percentiles) of 10.3 (3.78–18.8), 10.5 (4.75–17.3), and 2.8
reduction in tADA and its isoenzymes, with values close to (1.75–3) IU/L, respectively. Also, for ADA1, values were
zero. On the other side, the samples inactivated using higher for diseased and convalescents, with values of 3.7
(1.4–8.8) and 4.03 (2.84–7.56) IU/L, respectively, compara-
tively to healthy individuals with values of 1.5 (0.8–2) IU/L.
For ADA2, values were 6.7 (2.42–10.1), 4.6 (1.73–8.73), and
0.88 (0.38–1.13) IU/L, respectively for diseased, convales-
cent, and healthy groups. No significant differences were
observed between diseased and convalescents.
Discussion
To the authors’ knowledge this is the first report in which
ADA isoenzymes are characterized in human saliva sam-
ples, and an automated assay for their measurement is
Figure 1: Adenosine deaminase (ADA) activity in saliva from 13
described and analytically validated. In addition, it is
healthy humans before and after adding erythro-9-(2-hydroxy-
3-nonyl) adenina (EHNA) to the samples at different concentrations. described that this assay can be performed in saliva sam-
Asterisks indicate statistically significant results vs. the original ples treated with NP-40 or Triton X-100 for inactivation of
samples (***p1596 Franco-Martínez et al.: Adenosine deaminase automatic analysis in saliva
Figure 2: Representative graph of linearity
under dilution of total adenosine
deaminase (tADA) and ADA2 in saliva.
Table : Median (th–th percentile) ADA (tADA) and its isoforms (ADA and ADA) in saliva after the different inactivation SARS-CoV-
treatments.
NT H H SDS NP Triton
tADA, IU/L . (.–.) . (. –.) b a
. (. –.)
b a
. (. –.)
b b a
. (.–.) . (.–.)
ADA, IU/L . (.–.) .b (.b–.)a .b (.b–.)a .b (.b–.)a . (.–.) . (.–.)
ADA, IU/L . (.–.) (–.)a (–.)a (–.)a . (.–.) . (.–.)
NT, no inactivation treatment (control); H, °C heat; H, °C heat; SDS, .% SDS; NP, .% NP-; Triton, .% Triton X-.
a
Differences of statistical relevance vs. NT measurements. bLower limit of detection has been indicated since no value was obtained.
150
**** **** *** ****
50 60
ADA2 activity (IU/L)
tADA activity (IU/L)
ADA1 activity (IU/L)
**** **** 40
100 40
30
20 Figure 3: Adenosine deaminase (tADA) and
50 20
its isoforms (ADA2 and ADA1) activities in
10
saliva in healthy (HC), 10 days after positive
0 0 SARS-CoV-2 PCR (CONV) and COVID-19
0
symptomatic individuals (DIS).
A
A
A
2
2
2
1
1
1
D
D
D
A
A
A
A
A
A
Asteriks indicate differences of statistical
tA
tA
tA
D
D
D
D
D
D
A
A
A
A
A
A
IS
V
C
V
C
IS
relevance between the groups (***pFranco-Martínez et al.: Adenosine deaminase automatic analysis in saliva 1597
usually leads to denaturation and/or aggregation and the additional studies to gain knowledge about the possible
denaturing effect of SDS [39, 40]. On the other side, NP-40 application of these enzymes in different clinical situations.
and Triton X-100 inactivation protocols did not cause al-
terations in ADA or its isoenzymes in saliva and could be
Acknowledgments: The authors thank all participants that
used in routine settings.
agreed to participate in this study. We also want to
The increases found in tADA and their isoenzymes in
particularly acknowledge the patients and the the Biobank
patients with COVID-19 could be related to the presence of T
of Region of Murcia Network, IMIB Biobank (PT17/0015/
lymphocyte activation and increased monocytes/macro-
0038) integrated in the Spanish National Biobanks
phage activity, since this enzyme has been previously asso-
Network for their collaboration.
ciated with these cells [41]. Increases of ADA in serum have
Research funding: LFM was granted with a predoctoral
been reported for tuberculosis [42, 43]. As well, serum in-
contract ‘FPU’ from the University of Murcia, Spain. AT was
creases of the isoenzyme ADA2 has been described in other
granted a post-doctoral fellowship “Ramón y Cajal” by
viral diseases such as immunodeficiency virus infection [44].
“Ministerio de Economía y Competitividad”, Spain. This work
It was also interesting to observe that ADA levels remain
was supported by a grant from the Program for Research
elevated, comparatively to control, even during convales-
Groups of Excellence of the Seneca Foundation, Murcia,
cence, suggesting that assessment of ADA in saliva can be
Spain (grant 19894/GERM/15) and CEECIND/04397/2017 from
useful in COVID-19 management in different phases of the
the Portuguese Science Foundation.
disease. It would be of interest in the future to perform studies
Author contributions: All authors have accepted
involving a larger number of patients in which it could be
responsibility for the entire content of this manuscript and
explored the behaviour of ADA in individuals with different
approved its submission.
severity of clinical signs and also to evaluate if there could be
Competing interests: The authors report no other conflicts
a relationship between this enzyme and the prognosis of the
of interest. The authors alone are responsible for the
disease. In addition, the relation of ADA with other bio-
content and writing of the paper.
markers of inflammation and changes in lymphocyte subsets
Informed consent: Informed consent was obtained from all
population would be of interest to be explored. If the results of
individuals included in this study.
these trials were satisfactory, the measurements of ADA in
Ethical approval: Research involving human subjects
saliva could be used as marker of lymphocyte T activation.
complied with all relevant national regulations,
This could complement the assays that are currently used in
institutional policies and is in accordance with the tenets
this disease for the pathogen detection and the quantification
of the Helsinki Declaration (as revised in 2013), and has
of antibodies produced, with the advantage that all these
been approved by Ethical Committee of the University of
measurements could be performed in saliva [33]. The present
Murcia and the Ethical Committee of the IMIB-Arrixaca.
study presents some limitations. First, although no saliva
sample showed blood contamination by visual inspection and
none of the participants showed apparent oral diseases, an
objective criterion such as haemoglobin or transferrin mea-
References
surement was not employed. Second, the results of tADA and
1. Iizuka H, Koizumi H, Kamigaki K, Aoyagi T, Miura Y. Two forms of
its isoenzymes evaluation in patients with COVID-19 should adenosine deaminase in pig epidermis. J Dermatol 1981;8:91–5.
be considered as preliminary due to the limited number of 2. Adams A, Harkness RA. Adenosine deaminase activity in thymus
samples and confirmed in a larger number of individuals. and other human tissues. Clin Exp Immunol 1976;26:647–9.
Three conclusions can be summarized from the present 3. Chielle EO, Bonfanti G, De Bona KS, Moresco RN, Moretto MB.
study. First, tADA and its isoenzymes ADA1 and ADA2 can Adenosine deaminase, dipeptidyl peptidase-IV activities and lipid
peroxidation are increased in the saliva of obese young adult. Clin
be measured accurately and precisely in saliva samples in a
Chem Lab Med 2015;53:1041–7.
rapid, economical, and reproducible way. Second, ADA was 4. Santosh UP, Renukananda GS, Abhilash S. Role of adenosine
not affected by NP-40 and Triton X100 SARS-CoV-2 inacti- deaminase in common chronic ENT infections. J Clin Diagn Res
vation protocols, opposing 65 and 92 °C heating and SDS 2016;10:MC01–2.
treatments that reduced ADA activity. Last, higher values in 5. Muraoka T, Katsuramaki T, Shiraishi H, Yokoyama MM. Automated
enzymatic measurement of adenosine deaminase isoenzyme
tADA and its isoenzymes were found in diseased and
activities in serum. Anal Biochem 1990;187:268–72.
convalescent COVID-19 patients. Overall, the automated 6. Sari RA, Taysi S, Yilmaz Ö, Bakan N. Correlation of serum levels of
assay presented in this report can contribute to a broader use adenosine deaminase activity and its isoenzymes with disease
and application of the measurement of tADA and its iso- activity in rheumatoid arthritis. Clin Exp Rheumatol 2003;21:
enzymes in human saliva and to the performance of 87–90.1598 Franco-Martínez et al.: Adenosine deaminase automatic analysis in saliva
7. Stancíková M, Lukác J, Istok R, Cristalli G, Rovensky J. Serum 24. Ebrahimi-Rad M, Khatami S, Ansari S, Jalylfar S, Valadbeigi S,
adenosine deaminase activity and its isoenzyme pattern in Saghiri R. Adenosine deaminase 1 as a biomarker for diagnosis
patients with systemic lupus erythematosus. Clin Exp Rheumatol and monitoring of patients with acute lymphoblastic leukemia. J
1998;16:583–6. Med Biochem 2018;37:128–33.
8. Aghaei M, Karami-Tehrani F, Salami S, Atri M. Adenosine 25. Galanti B, Nardiello S, Russo M, Fiorentino F. Increased
deaminase activity in the serum and malignant tumors of breast lymphocyte adenosine deaminase in typhoid fever. Scand J Infect
cancer: the assessment of isoenzyme ADA1 and ADA2 activities. Dis 1981;13:47–50.
Clin Biochem 2005;38:887–91. 26. Bansal SK, Singh RP, Narang RK, Joshi LD, Bansal A, Agrawal AK.
9. Sullivan JL, Osborne WRA, Wedgwood RJ. Adenosine deaminase Serum adenosine deaminase in pulmonary tuberculosis,
activity in lymphocytes. Br J Haematol 1977;37:157–8. malignancy and non-tubercular respiratory diseases. Indian J
10. Macdermott RP, Tritsch GL, Formeister J. Adenosine deaminase Chest Dis Allied Sci 1991;33:189–93.
and nucleoside phosphorylase activities in normal human blood 27. Yoshizawa JM, Schafer CA, Schafer JJ, Farrell JJ, Paster BJ, Wong
mononuclear cell subpopulations. Clin Exp Immunol 1980;42: DTW. Salivary biomarkers: toward future clinical and diagnostic
303–7. utilities. Clin Microbiol Rev 2013;26:781–91.
11. Burnstock G. Purinergic signalling – an overview. In: Novartis 28. Lee Y-H, Wong DT. Saliva: an emerging biofluid for early detection
Foundation symposium; 2006:26–48 pp. of diseases. Am J Dent 2009;22:241–8.
12. Fontes Baganha M, Pego A, Lima MA, Gaspar EV, 29. Rai B, Kaur J, Jacobs R, Anand SC. Adenosine deaminase in saliva
Robalo Cordeiro A. Serum and pleural adenosine deaminase. as a diagnostic marker of squamous cell carcinoma of tongue.
Correlation with lymphocytic populations. Chest 1990;97: Clin Oral Invest 2011;15:347–9.
P605–10. 30. Tvarijonaviciute A, Zamora C, Martinez-Subiela S, Tecles F, Pina F,
13. Mishra OP, Gupta BL, Ali Z, Nath G, Chandra L. Adenosine Lopez-Jornet P. Salivary adiponectin, but not adenosine
deaminase activity in typhoid fever. Indian Pediatr 1994;31: deaminase, correlates with clinical signs in women with Sjögren’s
1379–84. syndrome: a pilot study. Clin Oral Invest 2019;23:1407–14.
14. Köse K, Yazici C, Aşçioǧlu Ö. The evaluation of lipid peroxidation 31. Patterson EI, Prince T, Anderson ER, Casas-Sanchez A, Smith SL,
and adenosine deaminase activity in patients with Behçet’s Cansado-Utrilla C, et al. Methods of inactivation of SARS-CoV-2
disease. Clin Biochem 2001;34:125–9. for downstream biological assays. bioRxiv 2020. https://doi.org/
15. Calis M, Ates F, Yazici C, Kose K, Kirnap M, Demir M, et al. 10.1101/2020.05.21.108035.
Adenosine deaminase enzyme levels, their relation with disease 32. Pastorino B, Touret F, Gilles M, de Lamballerie X, Charrel RN.
activity, and the effect of colchicine on adenosine deaminase Evaluation of heating and chemical protocols for inactivating
levels in patients with Behçet’s disease. Rheumatol Int 2005;25: SARS-CoV-2. bioRxiv 2020. https://doi.org/10.1101/2020.04.11.
452–6. 036855.
16. Ungerer JPJ, Burger HM, Bissbort SH, Vermaak WJH. Adenosine 33. Ceron J, Lamy E, Martinez-Subiela S, Lopez-Jornet P, Capela-
deaminase isoenzymes in typhoid fever. Eur J Clin Microbiol Infect Silva F, Eckersall P, et al. Use of saliva for diagnosis and
Dis 1996;15:510–2. monitoring the SARS-CoV-2: a general Perspective. J Clin Med
17. Vijayamahantesh, Amit A, Dikhit MR, Pandey RK, Singh K, 2020;9:1491.
Mishra R, et al. Elevated serum ADA activity as a marker for 34. Contreras-Aguilar MD, Escribano D, Martínez-Subiela S,
diagnosis and prognosis of visceral leishmaniasis and post Martínez-Miró S, Rubio M, Tvarijonaviciute A, et al. Influence
Kala-Azar Dermal leishmaniasis in Indian patients. PLoS One of the way of reporting alpha-amylase values in saliva in
2016;11:e0154117. different naturalistic situations : a pilot study. PLoS One 2017;
18. Hitoglou S, Hatzistilianou M, Gougoustamou D, Athanassiadou F, 12:1–13.
Kotsis A, Catriu D. Adenosine deaminase activity and its isoenzyme 35. Delacour H, Bousquet A, Fontan E, Ceppa F. Ammonia does not
pattern in patients with juvenile rheumatoid arthritis and systemic interfere with the Diazyme adenosine deaminase test. Clin Chem
lupus erythematosus. Clin Rheumatol 2001;20:411–6. Lab Med 2013;51:225–6.
19. Ziaee V, Amiran A, Moradinejad M, HaghiAshtiani M. Evaluation 36. Administration, U.S. Department of Health and Human Services
of serum adenosine deaminase changes before and after Food and Drug, Center for Drug Evaluation and Research (CDER) C
treatment in patients with systemic lupus erythematosus, for VM (CVM). Guidance for Industry bioanalytical method
Henoch-Schonlein Purpura and juvenile Idiopathic arthritis. Ann validation [Internet]; 2001. Available from: https://www.fda.gov/
Paediatr Rheumatol 2013;2:21–6. downloads/Drugs/Guidance/ucm070107.pdf.
20. Kurata N. Adenosine deaminase. Nippon Rinsho Jpn J Clin Med 37. Tvarijonaviciute A, Martínez-Subiela S, Ceron JJ. Validation of 2
1995;53:1178–83. commercially available enzyme-linked immunosorbent assays
21. Pirinççi N, Geçit I, Güneş M, Yuksel M, Kaba M, Tanık S, et al. for adiponectin determination in canine serum samples. Can J Vet
Serum adenosine deaminase, catalase and carbonic anhydrase Res 2010;74:279–85.
activities in patients with bladder cancer. Clinics (Sao Paulo). 38. Cossio MLT, Giesen LF, Araya G, Pérez-Cotapos MLS, Vergara RL,
2012;67:1443–6. Manca M, et al. Schalm’s veterinary hematology, 6th ed., vol.
22. Mahajan M, Tiwari N, Sharma R, Kaur S, Singh N. Oxidative stress XXXIII; 2012:81–7 pp.
and its relationship with adenosine deaminase activity in various 39. Lyubarev AE, Kurganov BI, Orlov VN, Zhou HM. Two-state
stages of breast cancer. Indian J Clin Biochem 2013;28:51–4. irreversible thermal denaturation of muscle creatine kinase.
23. Ghaderi B, Amini S, Maroofi F, Jalali C, Javanmardi M, Roshani D. Biophys Chem 1999;79:199–204.
Adenosine deaminase activity in chronic lymphocytic leukemia 40. Yan YB, Wang Q, He HW, Hu XY, Zhang RQ, Zhou HM. Two-
and healthy subjects. Iran J Cancer Prev 2016;9:e5069. dimensional infrared correlation spectroscopy study ofFranco-Martínez et al.: Adenosine deaminase automatic analysis in saliva 1599
sequential events in the heat-induced unfolding and aggregation 43. Soedarsono S, Prinasetyo K, Tanzilia M, Nugraha J. Changes of
process of myoglobin. Biophys J 2003;85:1959–67. serum adenosine deaminase level in new cases of pulmonary
41. Ungerer JPJ, Oosthuizen HM, Bissbort SH, Vermaak WJH. Serum tuberculosis before and after intensive phase treatment. Lung
adenosine deaminase: isoenzymes and diagnostic application. India 2020;37:126–9.
Clin Chem 1992;38:1322–6. 44. Tsuboi I, Sagawa K, Shichijo S, Mitsuo M, Yokoyama M, Ou DW,
42. Kumabe A, Hatakeyama S, Kanda N, Yamamoto Y, Matsumura M. et al. Adenosine deaminase isoenzyme levels in patients with
Utility of ascitic fluid adenosine deaminase levels in the human T-cell lymphotropic virus type 1 and human
diagnosis of tuberculous peritonitis in general medical practice. immunodeficiency virus type 1 infections. Clin Diagn Lab Immunol
Can J Infect Dis Med Microbiol 2020;2020:5792937. 1995;2:626–30.You can also read
