Comenius University in Bratislava

Faculty of Mathematics, Physics and Informatics

Mgr. Ivana Lettrichová

Dissertation Thesis Overview

Saliva as diagnostic fluid: Its applicability to noninvasive evaluation

of oxidative stress status and ovulation detection

to obtain the academic degree Philosophiae doctor in the study

program:

4.1.12 Biophysics

Bratislava, 2015

The dissertation thesis was performed during the full-time PhD study program at the Department of

Nuclear Physics and Biophysics of the Faculty of Mathematics, Physics and Informatics, Comenius

University in Bratislava

Author: Mgr. Ivana Lettrichová

Department of Nuclear Physics and Biophysics

Faculty of Mathematics, Physics and Informatics

Comenius University

Mlynská dolina 842 48

Bratislava

Supervisor: doc. RNDr. Marcela Morvová, PhD.

Department of Astronomy, Physics of the Earth and Meteorology

Faculty of Mathematics, Physics and Informatics

Comenius University

Mlynská dolina 842 48

Bratislava

Consultant: prof. RNDr. Libuša Šikurová, CSc.

Department of Nuclear Physics and Biophysics

Faculty of Mathematics, Physics and Informatics

Comenius University

Mlynská dolina 842 48

Bratislava

Reviewers: …………………………………………………..

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Dissertation thesis defense will be held on …………… at ………. h with the dissertation committee in

the field of the PhD. study named by the committee chairman on ……………....

Study program 4.1.12. Biophysics

at the Faculty of Mathematics, Physics and Informatics, Comenius University in Bratislava, Mlynská

dolina, 842 48 Bratislava, room ………

Committee chairman

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prof. RNDr. Tibor Hianik, DrSc.

Department of Nuclear Physics and Biophysics

Faculty of Mathematics, Physics and Informatics

Comenius University, Mlynská dolina 842 48

Bratislava

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ABSTRACT

This work describes the possibilities of utilizing saliva as a non-invasive diagnostic

fluid, with emphasis on describing oxidative stress status in the oral cavity of young

healthy volunteers and detection of ovulation in humans and animal species. The

parameters analyzed to assess the salivary oxidative stress and antioxidant status

include: advanced glycation end-products (AGEs), advanced oxidation protein products

(AOPP), total antioxidant capacity (TAC) and ferric reducing ability of saliva (FRAS).

The interindividual and intraindividual variability of these parameters in the saliva of

young healthy individuals is described in this thesis. After identifying the sources of

variability, salivary markers of oxidative stress may be used to assess local health status

of the oral cavity, or help in disease monitoring.

In addition, basic salivary parameters (total protein concentration and pH), with the

level of sialic acid (free or bound to proteins), were assessed in an animal model

(lactating dairy cows) to find out if they can be used for noninvasive ovulation

detection. Further studies are planned to be aimed at the saliva of humans and

broadened to analysis of additional potential markers of ovulation as well.

ABSTRAKT

Predkladaná dizertačná práca opisuje možnosti využitia slín ako neinvazívneho

diagnostického prostriedku, s dôrazom na hodnotenie miery oxidačného stresu v ústnej

dutine mladých zdravých dobrovoľníkov a detekciu ovulácie u ľudí a zvierat. Parametre

analyzované v rámci oxidačného stresu a antioxidačného statusu slín zahŕňajú: koncové

produkty pokročilej glykácie (AGEs), produkty pokročilej oxidácie proteínov (AOPP),

celkovú antioxidačnú kapacitu (TAC) a schopnosť slín redukovať železité katióny

(FRAS). V práci je opísaná miera interindividuálnej a intraindividuálnej variability

týchto parametrov v slinách mladých, zdravých jedincov. Po identifikovaní zdrojov

variability môžu byť tieto slinné markery oxidačného stresu použité pre hodnotenie

lokálneho stavu ústnej dutiny, alebo pomôcť pri monitoringu ochorení.

Práca tiež opisuje analýzu základných parametrov slín (celkovú koncentráciu proteínov

a pH) a kyseliny sialovej (voľnej alebo viazanej na proteíny) u dojných kráv ako

zvieracieho modelu, s cieľom analýzy pre neinvazívnu detekciu ovulácie. V ďalších

štúdiách je plánované zameranie sa na ľudské sliny, s rozšírením o ďalšie potenciálne

markery ovulácie.

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1 Introduction

This dissertation thesis focuses on the diagnostic applications of saliva as a noninvasive

research medium. It summarizes the analysis of oxidative stress markers in saliva of

young healthy volunteers (performed in the laboratory of doc. MUDr. Ing. RNDr. Peter

Celec, DrSc., MPH (from the Institute of Molecular BioMedicine (IMBM) of the

Faculty of Medicine, Comenius University in Bratislava, Slovakia)), with a special

emphasis on the level of interindividual and intraindividual variability of these

parameters. In particular, advanced glycation end-products (AGEs), advanced oxidation

protein products (AOPP), total antioxidant capacity (TAC) and ferric reducing ability of

saliva (FRAS), with respective methods of their analysis are described.

As these parameters likely derive from local sources in the oral cavity, they can be

interpreted as valuable indices of oral health status. Besides measuring the

concentrations of individual biomarkers, characterizing and identifying the sources of

their variability is a very important step before they can be used for routine health status

or disease progress assessment and monitoring.

After many decades of extensive research, it is widely accepted at the present time that

a certain level of reactive oxygen species (and hence, oxidative stress) has an important

role in many physiological processes, including ovulation and fertility. The search for

an accurate and reliable ovulation detection marker is a high priority not only in the

field of research focused on the reproductive performance and economical profitability

of lactating dairy cows, but is extremely important to humans as well. As the fertile

window is only a few days long in the ovarian cycles of both species, robust markers

able to predict impending ovulation with a high level of sensitivity and specificity are of

tremendous importance for their reproductive management and fertility control.

The second part of the experimental work, described in this thesis, focused on the

analysis of salivary sialic acid in lactating

dairy cows as a putative marker of

ovulation. This research project was part of

a research fellowship grant from the

National Scholarship Programme of the

Slovak Republic and took place in the

laboratory of prof. Stefan Ruhl, DDS, PhD.

(at the Department of Oral Biology of the

University at Buffalo, New York, United

States of America).

1.1 Saliva

Saliva is a complex biofluid that is being

produced by the major and minor salivary

glands, located in the oral cavity or in its

close proximity. The three major salivary

glands (parotid, submandibular and

sublingual) are found in pairs (Fig. 1.1). In

addition to components secreted from

Figure 1.1. Anatomical location of the

major salivary glands (parotid,

submandibular and sublingual) in

humans. Adapted from (Pfaffe, Cooper-

White et al. 2011).

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glandular sources, whole saliva contains other fluids, bacteria and products of their

metabolism, desquamated epithelial cells (in various stages of disintegration) and

extraneous debris that do not originate from the salivary glands (Malathi, Mythili et al.

2014).

Hundreds of minor salivary glands (labial, lingual, buccal, palatal) are located in the

submucosa of the oral cavity (Eliasson and Carlén 2010). Per day, approximately 500

ml to 1500 ml of whole saliva is being secreted (Lac 2001).

1.1.1 Bovine saliva

Similar to humans, bovine saliva is primarily secreted by the three pairs of major

salivary glands (parotid, submandibular and sublingual) (Fig. 1.2). Unlike humans

(which are monogastric), ruminants are polygastric species (having a four-compartment

stomach, composed of rumen, reticulum, omasum and abomasum). Ruminants, in

contrast to humans and other monogastric species, produce large quantities of saliva

(due to the generally coarse nature of their diet and in order to be able to maintain fluid

in their large-volume non-secretory rumen). The amount of bovine saliva secreted per

day has been reported to range from 98 to 190 liters in cows (Bailey 1961), and even

above 250 liters in high-yielding lactating dairy cows (Cassida and Stokes 1986).

Bovine saliva contains similar electrolytes as human saliva and mucosubstances as

primary organic components, which provide protection for the mucosal surface of the

oral cavity and rumen and are a nutrient source for rumen bacteria.

1.2 Characteristics of saliva

In contrast to humans, the pH of bovine saliva is in the alkaline range. Typical pH of

cow saliva is 8.2-8.5 (Bailey and Balch 1961). The pH of human unstimulated saliva is

in the neutral range (mean value pH 6.8). If the secretion is stimulated, the

Figure 1.2. Anatomical location of bovine major salivary glands. Adapted from (König and

Liebich 2007).

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concentration of bicarbonate rises too, resulting in higher pH (mean value pH 7.2)

(Bardow, Moe et al. 2000).

The major protein families of human saliva constitute around 95% of all salivary

proteins and they include: proline-rich proteins (PRPs; acidic and basic), amylase,

salivary mucins MUC5B (formerly MG1) and MUC7 (formerly MG2), agglutinins,

cystatins, histatins, statherin and other proteins (Helmerhorst and Oppenheim 2007).

Based on their size, salivary components can be divided into low-molecular weight and

high-molecular weight components. The low-molecular weight components include

electrolytes (bicarbonate, nitrate and nitrite, thiocyanate (SCN-) and hypothiocyanite

(OSCN-/HOSCN

-), sodium, potassium, chloride, calcium, phosphate, fluoride), glucose,

ammonia and urea. High-molecular weight components of saliva are mostly contributed

to by glycoproteins. Glycoproteins are post-translationally glycosylated proteins, that

contain oligosaccharide chains (glycans), covalently attached to polypeptide side chains.

Saliva harbors multpiple glycoproteins, such as proline-rich proteins, α-amylase,

lactoferrin, salivary peroxidase, kallikrein, secretory IgA and mucins (Levine, Reddy et

al. 1987).

There is a great variation in the composition of saliva not only between different

individuals, but also within the same individual at different timepoints during a day. The

patterns of salivary gland activity in a given individual are broadly similar, however,

there are some differences in the activity pattern of different glands (Ferguson and

Botchway 1980).

1.2.1 Viscoelastic properties of saliva

Although being composed of approximately 99% water, saliva is a non-Newtonian fluid

(with increasing shear, its viscosity decreases) (Lai, Wang et al. 2009). The rheological

properties of saliva are predominantly affected by the amount and characteristics of

mucins (Schenkels, Veerman et al. 1995).

1.2.1.1 Salivary mucins

Salivary mucins are a group of abundant, high molecular-weight polymeric salivary

glycoproteins that make up to 26% of salivary proteins (Amerongen, Bolscher et al.

1995) and contribute to its viscoelastic properties. As a consequence of their

glycosylation, mucins aquire a “bottle brush”-like structure (Fig. 1.3), with most

glycans termini negatively charged with a sialic acid (its carboxyl group) or a sulphate

group (Perez-Vilar and Hill 1999).

In human whole saliva, two major mucin populations have been identified, a large

MUC5B mucin (previously MG1, molecular weight >1 MDa) and a smaller, monomeric

MUC7 (previously MG2, molecular weight 200-250 kDa) with low viscoelastic

properties (Zalewska, Zwierz et al. 2000). MUC5B has counterparts in other mucous

sites in the body (trachea, gall bladder, endocervix) (Audie, Janin et al. 1993), while

MUC7 is found in salivary secretions (Amerongen, Bolscher et al. 1995) and lungs

(Biesbrock, Bobek et al. 1997). In addition to saliva, MUC5B is expressed in mucous

gland of tracheobronchial secretions, gall bladder and endocervix (Audie, Janin et al.

1993).

Bovine submaxillary mucin (BSM) is an important component of bovine saliva that

contains diverse sialic acid modifications (Blix and Lindberg 1960). It was found that

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gene and protein similarity of MUC5B and MUC7 between cows and humans is 23.6%

and 38.4%, respectively (Hoorens, Rinaldi et al. 2011).

1.2.1.2 Sialic acid

Sialic acids are a group of 9-carbon monosaccharides (Fig. 1.4), commonly attached at

the terminal position of oligosaccharide residues (in glycoproteins, glycolipids and

glycosphingolipids), that vary in the type of their substituents and the degree of N-

glycolylation and O-acetylation. Taken together, there are more than 50 structurally

different molecules of sialic acids known to this day, owing to the multitude of possible

modifications (Angata and Varki 2002).

Figure 1.4. Diversity of sialic acids based on the many types of chemical substitutions. The

nine-carbon backbone of sialic acids is shown in α configuration. Adapted from (Varki,

Cummings et al. 2009).

Figure 1.3. Major biochemical features of gel-forming mucins. A. Oligomeric gel consists

of several mucin monomers. B. Dimers are linked through the disulfide bonds between

disulfide-rich (D) domains. C. Along each fiber, there are interspersed “naked” globular

protein regions, stabilized by multiple disulfide bonds. D. Attached glycans (N-linked or O-

linked) are mostly decorated by negatively charged sialic acid or sulphate group residues.

These structural features enable the mucins to act as protective barriers for cells. Adapted

from (Cone 2005).

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N-acetyneuraminic acid (Neu5Ac) is the most abundant and the best studied

modification of sialic acid in nature (Fig. 1.5).

Salivary sialic acid is mainly bound to glycoproteins (predominantly mucins (Klein,

Carnoy et al. 1992, Thomsson, Prakobphol et al. 2002, Thomsson, Schulz et al. 2005,

Karlsson and Thomsson 2009) and secretory IgA (Carpenter, Proctor et al. 1997)) and

can be utilized by oral bacteria as a nutrient or they can transfer it to their own cell

surface (thus protecting themselves from activating the host's innate immune response)

(Severi, Hood et al. 2007).

1.3 Saliva as diagnostic fluid

In recent years, the advent and improvement of new techniques (such as proteomics,

metabolomics, genomics and bioinformatics) enabled detailed identification of salivary

constituents. Some of them are being studied for their possible use as biomarkers

(Schipper, Silletti et al. 2007). The current challenge is therefore to identify and

translate markers to be used with simpler methods, in a form of reliable diagnostic kits,

and most preferentially for home use.

In addition to protein and bacterial markers, saliva is a source of DNA (of human and

bacterial origin) and can therefore be used in other fields (such as forensics) as well

(Walsh, Corey et al. 1992). Compared to plasma as the traditionally used biofluid for

diagnostic purposes, analytes in saliva are very variable and are usually found in

considerably lower concentrations; while some constituents are only present in plasma

and not in saliva (or vice versa). By comparing human salivary and plasmatic

proteomes, it was found that around 27% of the proteins in whole saliva are found in

plasma (Loo, Yan et al. 2010). If possible to substitute, using saliva over plasma or

other biofluids is advantageous because of the easiness of collection, generally lower

associated anxiety of the subjects, lower cost of collection and virtually no health

hazard for the person performing the collection procedure (Cuevas-Córdoba and

Santiago-García 2014).

Steroid hormones can be analyzed in saliva, specifically their free fraction (as they are

small lipophilic molecules, able to diffuse into saliva secretion). In women of

reproductive age and with normal cycles, the pattern of estradiol and progesterone in

saliva closely resembles the menstrual cycle-related changes observed in blood

(Gandara, Leresche et al. 2007).

Figure 1.5. N-acetylneuraminic acid (Neu5Ac). Keto-enol tautomerism is shown. Molecular

formula: C11H19NO9. Adapted from (Achyuthan and Achyuthan 2001).

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When using saliva as a diagnostic medium, a multianalyte system is more informative

than assessing a single biomarker. Probably the biggest burden to incorporate salivary

analysis into routine dental and medical practice is the lack of normal reference values,

due to the great variability of secretion, composition and flow of saliva (Quintana,

Palicki et al. 2009, Thomas, Branscum et al. 2009).

1.4 Glycosylation pattern of the cervical mucus throughout the

menstrual and estrous cycle

Change in the mucin glycosylation pattern of the cervical mucus is the main alteration

at the time of ovulation in humans (specifically, the O-glycosylation pattern of the

MUC5B mucin) (Fig. 1.6) (Argüeso, Spurr-Michaud et al. 2002, Andersch-Björkman,

Thomsson et al. 2007). MUC5B is the predominant mucin of the human endocervix

(together with MUC4) (Yurewicz, Matsuura et al. 1987, Gipson, Spurr-Michaud et al.

1999) and was shown to peak at midcycle (Gipson, Moccia et al. 2001). Also, the cyclic

variation of L-fucose and sialic acid levels in human cervical mucus is connected to the

activity level of respective glycosyltransferases, responsible for the addition of these

sugars (Scudder and Chantler 1982). Similarly, in bovine cervical mucus, the greatest

activity of β-galactosidase and sialidase was observed 12 hours after estrus (Pluta, Irwin

et al. 2011).

1.5 Lactating dairy cows

Lactating dairy cows (or milk cows) are adult female cows (generally of the species Bos

taurus; Holstein breed), bred for the ability to produce large quantities of milk. To

maintain in lactation, a dairy cow must be bred and produce calves. Reproductive

efficiency has a major direct impact on profitability of livestock operations. Purely for

the reasons of profitability, each dairy cow should complete an average of at least four

Figure 1.6. Human cervical mucus oligosaccharides, increased relative to other sequences during

the menstrual cycle. Proposed structures based on mass spectrometry data. During ovulation, a

relative increase in neutral fucosylated oligosaccharides was found (an increase of Glc-NAc-6-

GalNAcol Core 2 structures and a relative decrease of NeuAc residues), while the NeuAc-

6GalNAcol and NeuAc-3Gal- epitopes are typical for the non-ovulatory phases. Adapted from

(Andersch-Björkman, Thomsson et al. 2007).

N-acetylgalactosamine (GalNAc)

galactose (Gal)

N-acetylglucosamine (GlcNAc)

fucose (Fuc)

N-acetylneuraminic acid (NeuAc), sialic acid

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lactations (Knaus 2009). Despite great efforts in breeding programs and improved

management of dairy cows (that substantially increased the milk yield since the 1950s),

the reproductive performance decreased as there is a negative genetic correlation

between milk yield and fertility (Spalding, Everett et al. 1975). The management of

lactating dairy cows is plagued by poor reproductive efficiency due to low fertility and

low rates of estrus detection (Pursley, Kosorok et al. 1997).

1.5.1 Bovine estrous cycle

In terms of ovarian functions and hormone fluctuations, the bovine estrous cycle is

similar to the human menstrual cycle (Mihm, Gangooly et al. 2011). Both species are

monovular (ovulating one dominant follicle per cycle) and polycyclic (with recurring

cycles, interrupted by pregnancy, lactation and pathological conditions). There is also

apparent similarity in size and morphology of the ovaries in cows and women. The size

of preovulatory (Graafian) follicles is about 15 and 20 mm (for cows and humans,

respectively) (Adams and Pierson 1995). Therefore, cows as large animal models have

been used in research of the follicular dynamics during the estrous cycle by

ultrasonography (Adams and Pierson 1995, Adams, Singh et al. 2012).

In contrast to the human menstrual cycle, the bovine estrous cycle follicular phase is

much shorter (3-4 days), with the luteal phase spanning from 16 to 18 days (Adams,

Singh et al. 2012).

1.6 Oxidative stress

Oxidative stress is defined as an imbalance between the production of free radicals and

reactive species and the antioxidant defense mechanisms, in favor of the prooxidants

(Sies 1991). Free radicals are highly reactive molecules (or molecular fragments) with

one or more unpaired valence electrons and they include reactive oxygen species (ROS)

or reactive nitrogen species (RNS) (derived from oxygen or nitrogen, respectively).

However, not all ROS and RNS are free radicals.

The majority of ROS and RNS are generated in the respiratory chain of mitochondria

(Figueira, Barros et al. 2013). Considering the endocrine system, the synthesis of

steroids from cholesterol contributes to their generation as well, through the action of

cytochrome P450 superfamily (Agarwal, Aponte-Mellado et al. 2012).

Antioxidants are molecules capable of inhibiting the oxidation of other molecules.

Overall, the effects of oxidative stress are considered not to be exclusively harmful, as a

certain level of oxidative stress in indispensable for normal function of various

metabolic processes and signaling pathways (Lyu, Lyu et al. 2007). However, excessive

and chronic oxidative stress (generated during elevated metabolism or various

pathological conditions) can cause serious damage to lipids, nucleic acids and proteins.

Carbonyl compounds are produced downstream of ROS under hyperglycemic

conditions, by autoxidation of carbohydrates, lipids and amino acids. Carbonyl stress

is thus an irreversible form of non-enzymatic oxidation, when reducing sugars (or other

carbonyl substances) react with amino groups of proteins and other molecules (Dalle-

Donne, Rossi et al. 2006), resulting in structural and functional changes (Berlett and

Stadtman 1997). They include carbonylated proteins, where the side chains are oxidated

to their respective aldehyde and ketone derivatives.

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Elevated oxidative or carbonyl stress and diminished antioxidant capacity have been

implicated in the pathogenesis of several diseases and their complications (Valko,

Leibfritz et al. 2007), including oral pathologies, such as caries (Ahmadi-Motamayel,

Goodarzi et al. 2013), gingivitis and periodontitis (Celec, Hodosy et al. 2005, Pradeep,

Ramchandraprasad et al. 2013). Salivary markers of oxidative and carbonyl stress might

be thus useful in dentistry.

Advanced glycation end-products (AGEs), a marker of carbonyl stress, represent a

heterogenous group of compounds. Their sources are endogenous reactions, diet

(Uribarri, Cai et al. 2005) and smoking (Cerami, Founds et al. 1997). Autooxidation of

glucose results in the formation of free radicals and hydrogen peroxide, thus further

potentiating the formation of AGEs (Wolff, Jiang et al. 1991).

Advanced oxidation protein products (AOPP) were identified as a marker of protein

oxidation in 1996 in uremic patients (Witko-Sarsat, Friedlander et al. 1996). They are

carried by oxidized proteins in plasma (especially albumin) and do not have oxidant

properties themselves (Witko-Sarsat, Friedlander et al. 1996). AOPP are formed mainly

by chlorinated oxidants (i.e. hypochlorous acid, chloramines) as a result of

myeloperoxidase activity of neutrophils (Capeillère-Blandin, Gausson et al. 2004), thus

providing a marker of their inflammatory response (Witko-Sarsat, Friedlander et al.

1998).

The concentration of individual antioxidants can be measured in various biofluids

separately, but this is labor-intensive, costly and time-consuming. An alternative

approach is to measure the total antioxidant capacity (TAC). TAC is contributed to

mostly by the total proteins (their -SH or thiol groups) and uric acid, in addition to total

bilirubin and vitamins C and E (Erel 2004). Another parameter for estimating the

cumulative antioxidant capacity is the ferric reducing ability of saliva (FRAS) that

was modified from the FRAP (ferric reducing ability of plasma) assay, developed in

1996 (Benzie and Strain 1996) to measure the “antioxidant power” of human plasma. In

addition to uric acid, the FRAP assay estimates other non-enzymatic antioxidants (e.g.

vitamin C and E, bilirubin) (Ergun, Troşala et al. 2011), but it cannot detect antioxidants

containing –SH groups such as proteins and reduced glutathione (Vassalle, Masini et al.

2004, Huang, Ou et al. 2005).

There is no “universal” marker of oxidative stress as there are many causative oxidative

agents and various molecules that are prone to oxidative damage. Due to the complex

nature of oxidative stress, a wide palette of biomarkers has to be evaluated. This enables

the understanding of the role of oxidative stress in pathogenesis of diseases and may be

potentially used for disease screening or monitoring (Čolak 2008). Concerning the

salivary parameters of oxidative stress, very little data exist on these markers in healthy

populations (aside for the particular control groups used in studies aimed at different

oral pathologies).

1.6.1 Effect of oxidative stress on human reproduction

The production of ROS is high in reproductive tissues, due to their active metabolism

and steroidogenesis. Reproductive tissues are thus under considerable levels of

oxidative stress (Fujii, Iuchi et al. 2005). Reactive oxygen species produced by the pre-

ovulatory follicle are considered important and even indispensable (Shkolnik, Tadmor

et al. 2011) inducers of ovulation (Ruder, Hartman et al. 2009). Possible sources of

ROS for initiation of the ovulatory process may be leukocytes localized around the pre-

ovulatory follicle (Brännström and Enskog 2002).

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Oxidative stress has been identified to play a key role in the pathogenesis of subfertility,

in both males (as adverse effects on sperm quality and functions) and females (Chandra,

Surti et al. 2009, Agarwal, Aponte-Mellado et al. 2012). Excessive oxidative stress has

been linked to a number of reproductive diseases (e.g. endometriosis, polycystic ovary

syndrome and unexplained infertility) and complications during pregnancy (such as

spontaneous abortion, recurrent pregnancy loss, preeclampsia and intrauterine growth

restriction) (Agarwal, Aponte-Mellado et al. 2012).

One of the factors might be maternal lifestyle and environmental exposure to pollutants.

Pregnancy itself may be associated with increased oxidative stress as a result of

increased metabolic activity (Ruder, Hartman et al. 2008). Late secretory phase human

endometrium has been demonstrated to have elevated lipid peroxide concentrations and

decreased superoxide dismutase concentrations (Agarwal, Gupta et al. 2008).

1.6.1.1 Mammalian ovulation as an inflammatory event

The first coherent hypothesis that ovulation might be considered as an inflammatory

event or a localized, acute sterile inflammation, was proposed in the 1980s (Espey

1980). This hypothesis emerged due to consistent observations that certain aspects of

the ovulatory process resemble an acute inflammatory reaction and ovulatory follicles

have many features similar to the characteristics of an inflamed tissue. Vascular changes

occuring in the ovulating ovary (increased vascular permeability and blood flow) are

similar to the ones found at sites of inflammation, as well as increased extravasation and

activation of leukocytes (Oakley, Frazer et al. 2011).

1.7 Ovulation detection

As ovulation can only be confirmed (without any doubt) either by successful

fertilization or by direct microscopic examination of the ovaries, there is a great need to

find alternative markers of ovulation and easy methods of their evaluation (ideally

suitable for home use), and with good specificity and sensitivity. The rationale for

knowing the exact timepoint of imminent ovulation is either to use this information for

desired conception or to aid contraconception.

1.7.1 Salivary markers of ovulation

Saliva, the most accessible body fluid, is a good candidate to search for an ovulation

detection marker, as the well-described hormonal patterns of progesterone and estradiol

of serum during the menstrual cycle are occurring in saliva, too (Lu, Bentley et al. 1999,

Gandara, Leresche et al. 2007). Multiple salivary constitents have been studied in the

past to determine their relation to the menstrual cycle and the timepoint of ovulation.

They include proteins (Tenovuo, Laine et al. 1981, Alagendran, Saibaba et al. 2013),

amino acids (Alagendran, Rameshkumar et al. 2009, Nithya, Alagendran et al. 2010),

sialic acid (Oster and Yang 1972, Calamera, Vilar et al. 1986) and carbohydrates

(Alagendran, Archunan et al. 2010, Al-Khafagy, Al-Ani et al. 2012), electrolytes

(Puskulian 1972, Tenovuo, Laine et al. 1981, Alagendran, Archunan et al. 2010) and

various enzymes (peroxidase (Tenovuo, Laine et al. 1981), α-amylase, alkaline

phosphatase, β-glucuronidase, arylsulfatase; all derived from salivary leukocytes (Boyer

and France 1976, Cockle and Harkness 1978)). Other examined parameters in saliva in

relation to the menstrual cycle include glucose (Davis and Balin 1973) or phosphate

(Ben-Aryeh, Filmar et al. 1976).

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2 Thesis aims The experimental part of this dissertation thesis is divided into two parts: evaluation of

the variability of salivary oxidative stress status in young healthy individuals and

investigating sialic acid as a putative marker of ovulation in saliva of lactating dairy

cows.

2.1 Variability of salivary markers of oxidative stress and antioxidant

status in young healthy individuals - aims

We aimed to assess the interindividual and intraindividual variability of advanced

glycation end-products (AGEs), advanced oxidation protein products (AOPP), total

antioxidant capacity (TAC) and ferric reducing ability of saliva (FRAS) daily over 30

consecutive days in young healthy volunteers.

2.2 Salivary sialic acid as a putative marker of ovulation in domestic

cows - aims

Based on the multitude of studies, performed on cycle-dependent changes in mucin

glycosylation profile and relative carbohydrate composition of cervical mucus of

different species, we were interested to find out if similar changes occur in saliva as

well. Specifically, we focused on investigating the level of sialic acid (free, bound and

total) in saliva of lactating dairy cows. Cows were chosen as an animal model because

the hormonal changes in their estrous cycle closely resemble the hormonal profile of

human menstrual cycle. Furthermore, currently used reproduction management

protocols enable to synchronize cycles of multiple individual cows, housed at the same

barn.

3 Materials and methods

3.1 Variability of salivary markers of oxidative stress and antioxidant

status in young healthy individuals – materials and methods

Unstimulated saliva was collected from 34 young healthy indivudals (16 females, mean

age 23.4±3.0 years and 18 males, mean age 25.4±3.1 years), daily over a period of 30

consecutive days. The recruited volunteers were instructed to collect whole

unstimulated saliva into 2 ml tubes in the morning, 10 minutes after brushing their teeth

and before eating. After collection, the samples were frozen immediately and stored at

-20 ºC until analysis. All participants filled in questionnares to assess their health status

(including acute or chronic illness) and in order to exclude subjects who smoke,

consume alcohol or medications and artificial antioxidant supplements or perform

sports activities that might influence the levels of oxidative stress markers.

Prior to analysis, saliva samples were centrifuged (3000 x g, 10 minutes at 4 °C) and

aliquotes of clarified saliva were transferred into 96-well microtitration plates. All

studied parameters (AGEs, AOPP, TAC and FRAS) were assayed by established and

previously described spectrophotometric and spectrofluorometric microplate-based

methods (as further detailed in the thesis).

12

3.2 Salivary sialic acid as a putative marker of ovulation in domestic

cows – materials and methods

Twelve lactating dairy cows (2-5 years old, Holstein breed, housed at a Cornell

University barn) were treated for estrous cycle synchronization (following a standard

herd OvSynch protocol with timed hormonal injections (Pursley, Mee et al. 1995)) and

ovulation was confirmed by ultrasound examination of the ovaries, behavioral signs and

analysis of estradiol and progesterone levels. Whole saliva and blood samples were

collected longitudinally over a 40-day period (Fig. 3.1). Sampling of saliva was

performed with a cellulose sponge (1 cm x 1 cm x 5 cm, pre-wetted with distilled

water), that was placed into the cow’s mouth and the content was subsequently pressed

out using a syringe into collection vials. Before and during collection of saliva the

access to food was not permitted.

Saliva was stored at -80 ºC until analysis. After thawing, samples were centrifuged

(17,000 x g, 1 min., 4 ºC). First, a collaboration with a private company (Oratel

Diagnostics LLC, Hammondsport, New York, United States of America) included

evaluating a kit for estrus detection. Due to a confidentiality disclosure agreement with

Oratel Diagnostics LLC, details about the procedure and outcome of those experiments

are not shown in the thesis. Basic salivary parameters (total protein concentration and

pH), together with the level of sialic acid, were assessed to see if they correspond to the

stage of the estrous cycle. Details of all experimental procedures are described in the

thesis.

Figure 3.1. Study design in relation to the hormonal profile of bovine estrous cycle. OvSynch

treatment (intramuscular injections for ovulation synchronization), stages of the estrous cycle

based on hormonal profiles and wave pattern of follicular growth. Days shown on the timeline

are the days of saliva sampling. Abbreviations: LH – luteinizing hormone, FSH – follicle-

stimulating hormone, GnRH – gonadotropin-releasing hormone, PGF2α – prostaglandin F2α.

Estrous cycle hormonal profile figure adapted from (Forde, Beltman et al. 2011).

13

4 Results

4.1 Variability of salivary markers of oxidative stress and antioxidant

status in young healthy individuals – results (Lettrichová, Tóthová et al.

2005)

Mean values for all analyzed oxidative stres parameters (advanced glycation end-

products – AGEs, advanced oxidation protein products – AOPP, total antioxidant

capacity – TAC and ferric reducing ability of saliva – FRAS), with their corresponding

standard deviations and the coefficients of variation (for interindividual variability and

the range of intraindividual variability) are summarized in Table 4.1. Mean salivary

levels of AGEs, AOPP, TAC and FRAS varied significantly between males and females

(Fig. 4.1A, p < 0.05 for AGEs and Figs. 4.1B-4.1D, p < 0.001 for AOPP, TAC and

FRAS, respectively).

Repeated measures two-way ANOVA showed significant influence of sampling day as

a factor affecting the variability of salivary AOPP (F = 1.764, p < 0.01), but not AGEs,

TAC or FRAS (Table 4.2). Gender as a factor affecting variability was found to be

statistically significant only for salivary FRAS (F = 6.743, p < 0.05).

All assessed salivary parameters (AGEs, AOPP, TAC and FRAS) were significantly

higher in men than in women (p < 0.05 for AGEs and p < 0.01 for AOPP, TAC and

FRAS). The interindividual variability was approximately 60% for AGEs and AOPP

and 30-40% for TAC and FRAS in both genders. (The interindividual variability of

FRAS was significantly higher in men, compared to women (p < 0.01)). The

intraindividual variability ranged (in both genders) from 20% for TAC (Fig. 4.2C) to

30% for AGEs (Fig. 4.2A) and FRAS (Fig. 4.2D) and 45% for AOPP (Fig. 4.2B). The

gender differences for the intraindividual variability were not statistically significant.

Table 4.1. Descriptive statistics of all measured parameters of oxidative stress status. Inter-

dividual and intraindividual variability. AGEs – advanced glycation end-products, AOPP –

advanced oxidation protein products, TAC – total antioxidant capacity, FRAS – ferric reducing

ability of saliva, CV – coefficient of variation.

14

Figure 4.2. Intraindividual variability of AGEs (A), AOPP (B), TAC (C) and FRAS (D). AGEs

– advanced glycation end-products, AOPP – advanced oxidation protein products, TAC – total

antioxidant capacity, FRAS – ferric reducing ability of saliva. Data are presented as median ±

interquartile range.

Figure 4.1. Salivary AGEs (A), AOPP (B), TAC (C) and FRAS (D) concentrations in females

and males. All analyzed samples are plotted. AGEs – advanced glycation end-products, AOPP

– advanced oxidation protein products, TAC – total antioxidant capacity, FRAS – ferric

reducing ability of saliva. * denotes p < 0.05, *** denotes p < 0.001 when compared to

females. Data are presented as median ± interquartile range.

15

4.2 Salivary sialic acid as a putative marker of ovulation in domestic

cows - results

Eight out of the twelve cows responded to the OvSynch treatment by developing

a characteristic hormonal profile of normal estrous cycle. Representative results for one

responding cow of salivary total protein concentration, pH, sialic acid (free, relesead by

mild acid hydrolysis and de-O-acetylation followed by mild acid hydrolysis), hormonal

profile of estradiol and progesterone from blood and pattern of follicle growth (as

assessed by ultrasound) are shown in Fig. 4.3.

Based on individual differences in responses to the OvSynch protocol treatment and

variable estrous cycle length, we identified representative samples throughout the cycle,

in order to be able to perform statistical analyses (Figs. 4.4 and 4.5). Data were analyzed

in GraphPad Prism (version 6.01, USA) using one-way ANOVA (α = 0.05) with

Tukey’s post hoc multiple comparison test.

The criteria for assesment of estrous cycle stages were:

Ovulation (OV): based on ultrasound data and hormonal profile

Proestrus (PE): peak level of estradiol

Diestrus (DE): peak level of progesterone

Note: due to missing data, it was not possible to reliably recognize the phase of

metestrus for most of the cows and therefore it was not considered for the statistical

analyses.

Summarized data for all 8 responding cows of progesterone, estradiol, total salivary

protein concentration and pH are shown in Fig. 4.4. The mean total protein

concentration of bovine saliva was 0.44±0.29 mg/ml and mean pH 9.00±0.47. No

statistically significant difference for pH and protein cocentration during the estrous

cycle stages was observed.

Summarized data for sialic acid measurements of all eight responding cows are shown

in Fig. 4.5. Data are expressed in µg sialic acid per ml of saliva and in µg sialic acid per

mg of salivary total protein amount. No statistically significant difference for the level

of sialic acid in bovine saliva between the estrous cycle stages was found.

Table 4.2. Repeated measures two-way ANOVA - selected components of variability. AGEs

– advanced glycation end-products, AOPP – advanced oxidation protein products, TAC –

total antioxidant capacity, FRAS – ferric reducing ability of saliva, F – F-ratio, p – p value, ns

denotes – not significant,* denotes p < 0.05 and ** denotes p < 0.01.

16

Figure 4.3. Representative result for a responding cow (cow 8176). Salivary total

protein concentration, pH, sialic acid (free, hydrolyzed, and de-O-acetylated and

hydrolyzed); blood hormonal profile of estradiol and progesterone and follicle size

data from ultrasound examination of the ovaries. Day of ovulation marked with a red

arrow, OvSynch treatment injections with blue arrows.

.

Correlation analyses of salivary sialic acid levels (free and released by mild acid

hydrolysis and de-O-acetylation followed by mild acid hydrolysis) with total protein

concentrations for all cows (n = 12) and all analyzed samples are plotted in Fig. 4.6.

Free as well as total sialic acid levels in bovine saliva correlated with the respective

total protein concentrations.

17

Figure 4.5. Summarized data for all 8 responding cows, showing salivary sialic acid content,

expressed in µg per ml saliva (left panel) and in µg per mg protein. Free sialic acid (top), mild

acid hydrolysis (middle) and de-O-acetylation and mild acid hydrolysis treatment (bottom).

Data are shown as median of 8 means ± interquartile range. No statistically significant

difference for sialic acid levels in relation to estrous cycle stages was found.

Figure 4.4. Summarized data for 8 responding cows: blood hormonal profile of progesterone

and estradiol (left panel) and salivary total proteins and pH (right panel), are grouped based on

the individual stages of the estrous cycle. Data shown as median of 8 means ± interquartile

range. No statistically significant difference for pH and protein concentration was observed.

18

In summary, basic salivary parameters (total protein concentration and pH) varied

throughout the cycle and were not correlated with the estrous cycle phases. Salivary

sialic acid levels (free, bound and total) correlated with the total protein concentration,

but not with the respective cycle phases. Our pilot study did not show evidence that the

levels of salivary sialic acid can be used as predictors of ovulation.

Figure 4.6. Correlation analysis of salivary sialic acid and total protein concetrations for all

12 cows (free sialic acid, mild acid hydrolysis and de-O-acetylation and mild acid hydrolysis).

All analyzed samples plotted.

19

5 Discussion

5.1 Variability of salivary markers of oxidative stress and antioxidant

status in young healthy individuals - discussion

The results presented in this thesis are to the best of our knowledge, the first attempt to

characterize the variability of advanced glycation end-products (AGEs), advanced

oxidation protein products (AOPP), total antioxidant capacity (TAC) and ferric reducing

ability of saliva (FRAS) in young healthy volunteers (Lettrichová, Tóthová et al. 2005).

The high intraindividual and interindividual variability of all salivary parameters,

observed in our study makes the interpretation of individual values difficult and

indicates that the use of these parameters is limited to studies on population level or

possibly for disease monitoring.

In our study, lower values of TAC and FRAS were observed in females compared to

males. Gender differences in markers of oxidative stress and enzymatic antioxidant

defense are usually attributed to differences in sex hormones, especially estradiol.

A possible explanation for the lower total antioxidant capacity and ferric reducing

ability of saliva in women, observed in our and other studies, may be a lower

unstimulated salivary flow in females due to smaller size of salivary glands (based on

their smaller body size) (Inoue, Ono et al. 2006).

One of the limitations of our study is its cross-sectional design. It is, thus, difficult to

characterize the causes of increased oxidative stress and antioxidant status in males

compared to females. Our study is purely observational, describing the variability of

oxidative stress markers in young healthy individuals.

Further studies are needed and anticipated to explore the mechanism and causes of the

existing variability, as well as the exact origin of oxidative stress markers in saliva.

Gender differences should also be taken into account in future clinical studies.

5.2 Salivary sialic acid as a putative marker of ovulation in domestic

cows - discussion

Sialic acid in relation to menstrual cycle was investigated in human saliva only in two

studies so far (Oster and Yang 1972, Calamera, Vilar et al. 1986). Oster and Young

reported a minimum in salivary sialic acid bound to mucins, occurring at midcycle

(Oster and Yang 1972). In the study by Calamera and et al. (Calamera, Vilar et al.

1986), sialic acid concentrations showed a peak either 4 or 5 days before the LH surge

preceding ovulation. In addition, the lowest value of sialic acid observed corresponded

to the ovulatory period. The authors speculated that this preovulatory peak of salivary

sialic acid could be related to the maximal activity of sialyltransferase, occurring around

the same time (Calamera, Vilar et al. 1986).

On the contrary, our pilot study did not find evidence that the level of salivary sialic

acid can be used as a predictor of ovulation in lactating dairy cows. Total protein

concentration and pH of saliva varied throughout the cycle, but were not correlated with

the individual estrous cycle phases. The levels of salivary sialic acid (free and bound)

were correlated with the total protein amount, but not with the respective estrous cycle

stages. Further studies are aimed at testing salivary glycoproteins as possible candidates

for ovulation detection in domestic cows and humans.

20

6 Conclusions

In conclusion, saliva is continuously gaining importance and the interest of dentists and

physicians as a valuable diagnostic fluid, mostly because of its non-invasive and easy

collection. The variability in flow, composition and rheological parameters complicates

the application of diagnostic tests into every day healthcare practice. Identifying and

minimizing the sources of variation is therefore of great importance. The search for

accurate and robust biomarkers continues, in an effort to identify reliable analytes for

assessment of multiple physiological or pathological conditions.

In this thesis, we aimed to describe the inter-individual and intra-individual variability

of advanced glycation end-products, advanced oxidation protein products, total

antioxidant capacity and the ferric reducing ability of saliva in a group of young healthy

volunteers. To the best of our knowledge, we were the research group first to do so.

Further studies should be aimed at identifying the respective sources of this variability,

in order to possibly utilize these markers in the assessment of the local health status of

the oral cavity, or in the monitoring of disease states.

In addition, we studied the levels of sialic acid in saliva of lactating dairy cows as a

putative marker of ovulation. We were not able to confirm a correlation with the

respective estrous cycle stages in our study. Undeniably, investigating markers of

ovulation in saliva of various species is a very interesting research avenue that should

be continued in the future. Bovine as well as human reproductive physiology share

common research interests – to reliably predict the timepoint of ovulation and

understand the factors determining and influencing their fertility. Identification, analysis

and validation of alternative, robust biomarkers of ovulation in noninvasive fluids such

as saliva should be the focus of further studies.

Analyzing ovulation biomarkers in a noninvasive specimen (such as saliva) has an

additional benefit of simplicity and convenience of handling. Following this research

path has a promising potential for the development of salivary ovulation kits suitable for

home use that are envisioned to be sold in the near future.

21

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ADC Publications in international indexed journals

Lettrichová I, Tóthová L, Hodosy J, Behuliak M, Celec P. (2015). "Variability of

salivary markers of oxidative stress and antioxidant status in young healthy

individuals." Redox Rep. [Article in press].

ADE Publications in international non-indexed journals

Foltin, V, Foltinova, J, Neu, E, Morvova, M, Lettrichova, I (2011). "Placenta - organ

important for fetus and interesting for the rise of the Attention Deficit Hyperactivity

Disorder Syndrome - interdisciplinary study. " Neuro Endocrinol Lett. 32(1):44-50.

cited by:

Ptáček R, Kuželová H, Stefano GB. (2011). "Dopamine D4 receptor gene DRD4

and its association with psychiatric disorders." Med Sci Monit. 17(9):RA215-20.

AED Publications in peer-reviewed Slovak collections, scientific monographs

Šimera, M, Foltin, F, Lettrichová, I, Grolmusová, Z, Morvová, M, Neu, E, Foltinová, J

(2007) "Výsledky interdisciplinárneho výskumu olova v placente indikujú

hyperkinetický syndróm u detí." Aktuality súčasného biomedicínskeho výskumu I.,

Bratislava : Asklepios, 2007. ISBN 978-80-7167-120-6. S. 101-105.

AFH Published abstracts from Slovak scientific conferences

Foltinová, J, Morvová, M, Foltin, V, Neu, E, Šimera, M, Lettrichová, I (2009) "Lead -

A dangerous metal for the developing fetus, an interdisciplinary study". ACTA

HISTOCHEMICA. 111(5): ABSTRACTS FROM MORPHOLOGY 2007 HELD IN

BRATISLAVA, 9-12 SEPTEMBER 2007 Abstracts (2009), p. 433

Active participation at international conferences

43rd Annual Meeting and Exhibition of the American Association for Dental Research

(AADR) in Charlotte, North Carolina, USA, March 12-14, 2014 – poster presentation

Gordon Research Conference (Salivary Glands & Exocrine Biology) in Galveston,

Texas, USA, February 15-20, 2015 – poster presentation

Meeting of the International Association for Dental Research (IADR) in Boston,

Massachusetts, USA, March 11-14, 2015 – poster presentation