PORPHYRINS AND HEME IN MICROORGANISMS

Jonas Fyrestam

Porphyrins and heme in

microorganismsPorphyrin content and its relation to phototherapy and antimicrobialtreatments in vivo and in vitro

Jonas Fyrestam

©Jonas Fyrestam, Stockholm University 2018 ISBN print 978-91-7797-079-8ISBN PDF 978-91-7797-080-4 Printed in Sweden by Universitetsservice US-AB, Stockholm 2017Distributor: Department of Environmental Science and Analytical Chemistry

List of papersThis thesis is based on the following papers, which will be referred to by Roman numerals,

I. Fyrestam, J., Bjurshammar, N., Paulsson, E., Johannsen, A., &Östman, C. (2015). Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry. Analytical and bioanalytical chemistry, 407(23), 7013-7023.

II. Fyrestam, J., Bjurshammar, N., Paulsson, E., Mansouri, N., Johannsen, A., & Östman, C. (2017). Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.Photodiagnosis and photodynamic therapy, 17, 115-123.

III. Fyrestam, J., & Östman, C. (2017). Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli. Analytical and Bioanalytical Chemistry, [Epub ahead of print].

IV. Fyrestam, J., & Östman, C. (2017). Escherichia coli is killed by 405 nm blue light due to its endogenous porphyrins induced by 5-aminolevulinic acid. Manuscript.

V. Bjurshammar, N., Malmqvist, S., Johannsen, G., Boström, EA., Fyrestam, J., Östman, C., & Johannsen, A. The Effect of Local Phototherapy on Gingival Inflammation - A Randomized Controlled Study. Manuscript.

The contribution of Jonas Fyrestam to the papers in this thesis was as follows:

I. The respondent was responsible for design of the study, parts of the experimental work, all data analysis, and significant parts of the writing.

II. The respondent was responsible for generating the idea, design of the study, parts of the experimental work, all data analysis, and significant parts of the writing.

III. The respondent was responsible for generating the idea, design of the study, all of the experimental work, all data analysis and significant parts of the writing.

IV. The respondent was responsible for generating the idea, design of the study, all of the experimental work, all data analysis and all of the writing.

V. The respondent was involved in generating the idea, parts of the design of the study and minor parts of the writing.

ContentsList of papers................................................................................................. vii

Abbrebrevations ............................................................................................11

Populärvetenskaplig sammanfattning ...........................................................13

Introduction and aims....................................................................................16

Part 1. Background .......................................................................................19 Phototherapy...............................................................................................................19 Porphyrins and heme ..................................................................................................19 Porphyrins as photosensitizers in phototherapy ..........................................................22 Biosynthesis of porphyrins and heme in bacteria ........................................................25 Heme acquisition as a potential drug target ................................................................30 Oral bacteria ...............................................................................................................31

Aggregatibacter actinomycetemcomitans ..............................................................31 Porphyromonas gingivalis......................................................................................32

Part 2. Determination of porphyrins and heme .............................................34 Samples and sampling................................................................................................34 Cell lysis......................................................................................................................35 Extraction and clean-up ..............................................................................................36

Solid phase extraction of porphyrins ......................................................................37 Optimization of heme extraction ............................................................................39 Protein precipitation ...............................................................................................42 Salting-out assisted liquid-liquid extraction ............................................................45

Liquid chromatography of porphyrins ..........................................................................45 HPLC columns.......................................................................................................46 Mobile phases .......................................................................................................48

Mass spectrometry of porphyrins ................................................................................50 Stability of porphyrins and heme in solution ................................................................53

Part 3. Applications .......................................................................................56 Photo inactivation of bacteria ......................................................................................56

Pilot study..............................................................................................................56 In vitro experiments ...............................................................................................57

Porphyrin content in microorganisms ..........................................................................59 Influence of culture conditions.....................................................................................60

Time of culturing ....................................................................................................61 Passage ................................................................................................................62 Cultivation medium ................................................................................................63

Influence of different additives on E. coli .....................................................................64 5-aminolaevulinic acid ...........................................................................................64

Hemin ....................................................................................................................67 Cobalt protoporphyrin IX........................................................................................67

Singlet oxygen production of porphyrins .....................................................................68 Clinical study...............................................................................................................71

Conclusions and future perspectives ............................................................74

References....................................................................................................77

Acknowledgements .......................................................................................86 HapPhy Days? A cohort study of the happiness during PhD studies ..........................86

11

Abbrebrevations5-ALA 5-aminolaevulinic acid

5-ALA-S 5-aminolaevulinic acid synthase

aPT Antimicrobial phototherapy

BOP Bleeding on probing

CCF Central composite face design

CFU Colony forming unit

COP-O Coproporphyrinogen oxidase

Co-PPIX Cobalt(III) protoporphyrin IX

EDTA Ethylenediaminetetraacetic acid

FECH Ferrochelatase

GC Gas chromatography

GI Gingival index

HILIC Hydrophilic interaction chromatography

His L-histidine

HMB Hydroxymethylbilane

HPLC High performance liquid chromatography

IC Internal conversion

ISC Inter system crossing

LED Light emitting diode

LLE Liquid-liquid extraction

LOD Limit of detection

logD Octanol-water partition coefficient at a specific pH

logP Octanol-water partition coefficient

LOQ Limit of quantification

m/z Mass-to-charge ratio

MS Mass spectrometry

MS/MS Tandem mass spectrometry

12

1O2 Singlet oxygen

OD600 Optical density at 600 nm

PBG Porphobilinogen

PBG-D Porphobilinogen deaminase

PDT Photodynamic therapy

PFP Pentafluorophenyl

PROT-O Protoporphyrinogen oxidase

PT Photo therapy

PI Plaque index

pI Isoelectric point

RNO N,N-Dimethyl-4-nitrosoaniline

ROS Reactive oxygen species

S0 Ground state

*Sn Excited singlet state

S/N Signal-to-noise ratio

SIM Selected ion monitoring

SPE Solid phase extraction

SRM Selected reaction monitoring

*T1 Excited triplet state

TPC Total porphyrin content

URO III-S Uroporphyrinogen III synthase

URO-D Uroporphyrinogen decarboxylase

UV-Vis Ultraviolet-visible

WHO World health organization

Quantum yield

Quantum yield of singlet oxygen

13

Populärvetenskaplig sammanfattning

Ett av de främsta hoten mot människans hälsa är bakteriers ökande

motståndskraft (resistens) mot antibiotika. Enligt Världshälsoorganisationen,

WHO, är situationen nu så allvarlig att den hotar hela den globala hälsan och

om resistensen fortsätter att öka så kan transplantationer och

cancerbehandlingar inte längre genomföras utan stora infektionsrisker. Ett av

de främsta sätten att begränsa spridningen av resistens är att minska

användandet av antibiotika, där kan förebyggande åtgärder för en ökad

hygien vara mycket effektivt. Om en infektion redan har brutit ut så kan

alternativa behandlingsmetoder minska antibiotikaanvändningen.

I slutet på 1800-talet fick fototerapi ett genombrott då dansken Niels

Ryberg Finsen upptäckte att man kunde behandla hudtuberkulos med UV-

ljus. För denna upptäckt fick han nobelpriset i medicin år 1903. I och med

Alexander Flemings upptäckt av penicillinet år 1928, försvann intresset för

för fototerapi helt, men har på senare år fått mer uppmärksamhet på grund av

den ökande antibiotikaresistensen. För att en fototerapeuptisk behandling ska

lyckas krävs tre huvudsakliga ingredienser: ljus, ett ämne som kan ta upp

ljus (fotosensiterare) och syre.

När en bakterie bygger en molekyl för att förvara järn inuti cellen så

tillverkar den en rad olika ämnen för vilka den egentligen inte har någon

biologisk användning av. Dessa kallas porfyriner och har en bra förmåga att

ta upp blått ljus och en hypotes är att dessa porfyriner kan användas för att

döda bakterier. Det övergripande målet med denna avhandling har varit att

bestämma förekomsten av dessa porfyriner i olika typer av bakterier, studera

hur väl de kan fungera som fotosensiterare och ifall mängden porfyriner kan

kopplas ihop med hur känslig en bakterie är för fototerapi.

14

Att bestämma förekomst och halt av ett kemiskt ämne som finns i en

bakterie är inte helt enkelt. Koncentrationen av porfyriner i bakterierna är

mycket låga. Ofta handlar det om några hundra pikogram i en normal

bakteriekultur, det vill säga miljarddelar av ett gram, eller 0,000000000001

gram. Man måste därför utveckla kemisk analytiska metoder för att kunna

hitta och vara helt säker på att det är rätt ämne som man analyserar, samt

mäta dessa låga halter och göra noggranna bestämningar av koncentrationen.

I den första publikationen utvecklades en metod för att kunna mäta

porfyriner i orala bakterier med hjälp av vätskekromatografi och

masspektrometri. Denna metod användes sedan i den andra publikationen

där vi lät bakterierna växa i olika miljöer och olika länge för att undersöka

om detta påverkade innehållet av porfyriner. Vi kunde där visa att

porfyrininnehållet inuti bakterierna varierar mycket beroende på hur

bakterierna odlas. Detta skulle kunna vara en förklararing till varför

resultaten från olika fototerapiexperiment beskrivna i litteraturen varierar. En

standardisering av hur bakterierna kultiveras behövs därför för att kunna dra

korrekta slutsatser från sina laborativa fototerapiexperiment.

I den tredje publikationen utvecklades en metod för analys av hem,

molekylen som förvarar järn inne i bakterien. Hem kan inte användas som

fotosensiterare, men eftersom det är en viktig molekyl för bakterierna så kan

ett lovande alternativ till antibiotika vara att man hindrar bakterien att få

tillgång till denna. När ett kemiskt ämne som är väldigt likt hem gavs till

bakterierna kunde man se att bakterierna tror att det är hem och tar upp detta

ämne, likt en trojansk häst, vilket medförde att hemkoncentrationen

minskade i bakterierna. Detta skulle därför kunna vara ett möjligt framtida

alternativ till antibiotika.

I det fjärde manuskriptet undersöktes hur bra bakterien E. coli kan dödas

med blått ljus. I dessa experiment analyserades bakteriernas

15

porfyrininnehåll, samt undersöktes hur bra olika porfyriner fungerar som

fotosensiterare. Resultaten visade att E. coli inte är känslig mot blått ljus och

att den tillverkar väldigt små mängder av egna porfyriner. När ett ämne som

bakterierna kan bygga porfyriner av tillsattes medförde detta till att E. coli

började producera mycket porfyriner. Bakterierna belystes efter tillsatsen av

detta ämne och det visade sig att relativt låga doser av blått ljus nu kunde

döda upp till 99,9999 % av alla bakterier.

Femte manuskriptet är en randomiserad klinisk studie där 48 personer

delades in i två grupper, en kontrollgrupp och en behandlingsgrupp. Under 8

veckor fick behandlingsgruppen borsta tänderna med en tandborste med en

inbyggd blå lampa i borsthuvudet. Kontrollgruppen använde samma

tandborste fast utan blått ljus. Flera olika odontologiska parametrar

undersöktes före, under och i slutet av studien. Man kunde notera en

minskning av mängden plack och hur lätt tandköttet börjar blöda vilket tyder

på en positiv effekt av det blå ljuset, men det gick inte att påvisa någon

statistisk skillnad mellan kontroll- och behandlingsgruppen. En förklaring till

detta kan vara att lysdioden i den tandborsten hade en våglängd på 450 nm

och inte 405 nm vilket är den våglängd där porfyriner har sin maximala

ljusabsorption.

16

Introduction and aimsOne of the greatest threats to the human health is increasing antimicrobial

resistance among pathogens. According to the World Health Organization

(WHO), this threatens the achievements of modern medicine [1]. Antibiotics

are for example used in major surgery, organ transplantation, treatment of

premature babies, and cancer chemotherapy. These are medical treatments

which cannot be performed in a safe way if antibiotics not are effective

enough [2]. Prevention of bacterial infections is one of the most efficient

ways to reduce antibiotic use and this can be done by increased hygiene and

sanitation improvements. However, preventive actions alone will not solve

the problem. Thus finding alternatives for treatment of bacterial infections is

of highest importance besides a more controlled use of antibiotics.

Major research efforts have been launched in recent years to identify

alternative antibacterial therapies. Phototherapy has been suggested as one of

these alternatives, where visible light can be used to inactivate a number of

different bacteria without the addition of an exogenous compound [3-5]. In

order for a phototheraputical treatment to be successful, three components

are necessary; light, oxygen and a photosensitizer. It is the photosensitizers

that have been the subject for attention in this thesis. Porphyrins,

intermediates of the biosynthesis of heme, are believed to work as

endogenous photosensitizers [6,7]. Porphyrins absorb light in the blue part of

the visible part of the electromagnetic spectrum and are capable to transfer

this energy to oxygen that will create reactive oxygen species (ROS).

However, the porphyrin content in bacteria has been sparingly investigated

and it is difficult to compare these studies since the cultivation conditions

among studies differ. Furthermore, the sensitivity for a specific

microorganism to blue light has not been correlated to the porphyrin content,

17

as well as to the singlet oxygen (1O2) production from different porphyrins

[8,9].

The primary aim of this thesis was to develop sensitive and selective

analytical methods for determination of porphyrins and heme in

microorganisms. These methods were then used to determine the heme and

porphyrin content in bacteria in order to better interpret the results from

phototherapy experiments.

In Paper I, a method was developed for the extraction, clean-up and

determination of porphyrins in oral bacteria using solid phase extraction with

subsequent analysis by high performance liquid chromatography coupled to

tandem mass spectrometry (HPLC-MS/MS). Several evaluation parameters

were determined, such as linearity, limits of detection and quantification,

accuracy, intra-day precision, recovery and matrix effects.

In Paper II, the method from Paper I was used to analyze two different

types of oral pathogens, Aggregatibacter actinomycetemcomitans and

Porphyromonas gingivalis, for their porphyrin content. The aim was to

investigate if the profile of endogenously produced porphyrins varies

according to changes in cultivation parameters.

In Paper III, a method for the determination of heme was developed by

using experimental design for the optimization of a liquid-liquid extraction

method for heme in Escherichia coli. The method was used to monitor the

heme content when different additives related to heme biosynthesis were

added to the cultivation broth. Further, a compound that is chemically

similar to heme was added to investigate if such a compound could interfere

with the heme acquisition mechanisms in E. coli. Heme acquisition

mechanisms in bacteria could be a potential target for drugs.

In Paper IV, E. coli was cultivated and treated with 405 nm light and

analyzed for its porphyrin content. The precursor of porphyrins, 5-

18

aminolevulinic acid, was used to bypass the negative feedback control of the

heme biosynthesis and enhance the porphyrin production in E. coli. The

photosensitivity and porphyrin content before and after administration of 5-

aminolevulinic acid was compared, and the relative singlet oxygen

production for three naturally occurring porphyrins was also determined.

Paper V is a randomized clinical trial in which a toothbrush equipped

with a 450 nm light-emitting diode (LED) was used for the subject’s daily

oral hygiene during 8 weeks. The control group was compared to the

treatment group for a set of inflammation markers and periodontal

parameters.

19

Part 1. Background

PhototherapyIn the late 19th century, phototherapy achieved a breakthrough when the

Dane Niels Ryberg Finsen (1860-1904) found that tuberculosis of the skin

could be treated with UV-light. These findings led to the Nobel Prize in

medicine in 1903 [10]. When Sir Alexander Fleming (1881-1955) in 1928

discovered penicillin [11] the interest in phototherapy was lost, but has

gained more attention in recent years due to increasing antibiotic resistance

[1]. To succeed with a phototherapy treatment, three components are needed:

light, a photosensitizer and molecular oxygen [12].

There are a number of publications where bacteria and other

microorganisms have been shown to be inactivated after illumination of

visible light [3-5,7,13-16]. There are generally two branches in

phototherapy. In photodynamic therapy (PDT) a photosensitizer is added

exogenously to the bacteria which then are illuminated with an appropriate

wavelength, often in the red light spectra [17,18]. The other one uses

endogenously produced photosensitizers which are naturally produced by the

bacteria and microbes, usually called antimicrobial phototherapy or

antimicrobial photodynamic therapy (aPT) [19].

Porphyrins and hemePorphyrins are macrocyclic compounds that occur naturally and have several

important representatives, such as chlorophyll and heme [20,21]. All

porphyrins share the same basic structure, a tetrapyrrole, where the four

, Figure 1.

20

Figure 1: Trivial names and structures for porphyrins, heme and hemin. Heme contains ferrous iron (Fe2+) while hemin is the oxidized form of heme containing ferric iron (Fe3+).

Naturally occurring porphyrins are characterized by their side chain

substituents and are often named by the number of carboxylic acid groups.

Other functional groups are methyl, ethyl and vinyl. Due to their common

NH

N HN

N

HOOC

COOH

COOHCOOH

HOOC

HOOC

COOH

COOH

NH

N HN

N

HOOC

COOH

COOH

H3C

HOOC

HOOC

COOH

COOH

NH

N HN

N

HOOC

COOH

COOH

HOOC

H3C

CH3

COOH

COOH

NH

N HN

N

HOOC

COOH

COOH

H3C

HOOC

HOOC

CH3

CH3

NH

N HN

N

HOOC

COOH

COOH

H3C

HOOC

H3C

CH3

CH3

NH

N HN

N

COOH

H3C

H3C

CH3

CH3

CH2

CH2

HOOC

N

N N

N

COOH

H3C

H3C

CH3

CH3

CH2

CH2

HOOC

Fe

N

N N

N

COOH

H3C

H3C

CH3

CH3

CH2

CH2

HOOC

FeCl

Uroporphyrin I Heptacarboxylic porphyrin I Hexacarboxylic porphyrin I

Pentacarboxylic porphyrin I Coproporphyrin I Protoporphyrin IX

Heme Hemin

21

basic structure, porphyrins have similar bands of absorbance in the region

between 390-750 nm. They have an intense band of absorbance between

390-425 nm, the Soret band, and two to four weaker bands in the region

between 480-700 nm, the Q bands [22]. In Figure 2 the absorption spectra of

coproporphyrin III is shown.

Figure 2: Absorbance spectra for coproporphyrin III showing the intense band of absorbance between 390-425 nm and four weaker bands between 480-700 nm.

The Soret band around 400 nm is often used for detection with UV-Vis due

to the high absorption in this region [23]. Most porphyrins also exhibit

characteristic fluorescence spectra in the region around 600 nm [22], and

since fluorescence detection has 1-3 orders of magnitude higher sensitivity

compared to UV-Vis it is the most frequently used detection method [24-27].

0

0.5

1

300 400 500 600 700

Nor

mal

ized

abs

orba

nce

Wavelength [nm]

22

Porphyrins as photosensitizers in phototherapyIn this work we have focused on the endogenous porphyrins role in aPT

experiments. It has been shown that light with an emission wavelength in the

Soret region of the absorption spectra of porphyrins is efficient for killing

bacteria [14,28-31]. The illumination wavelengths used in such studies range

between 405-460 nm [3,4,14-16,28-37]. Two studies have used different

wavelength in order to determine if some wavelengths are better at killing

bacteria. In those studies it was shown that illumination wavelengths > 420-

430 nm was not sufficient to inactivate certain bacteria [16,31]. However,

when using light covering the Soret spectral band of porphyrins, i.e.

wavelengths < 420-430 nm) the bacteria were inactivated. Only a few papers

have determined the porphyrin content together with the photosensitivity in

the selected microorganism. When this has been done, often nonspecific

detection methods have been used such as UV-Vis or fluorescence and the

analytical methods used often lack satisfactory validation [7,14,29,32,36].

Furthermore, the bacterial extracts are rarely subjected to separation with

HPLC for detection of the individual porphyrin species, instead the extracts

of the investigated bacteria are determined as a mixture [3,28,38]. The

fluorescence spectra of different porphyrins are rather similar, and the

porphyrin profile in these samples will be impossible to distinguish

accurately. This also results in that no conclusion can be drawn regarding

which porphyrin will have the major contribution to the photosensitivity of

the bacteria.

To be able to act as a photosensitizer, a compound must have a

-electron system which can absorb a photon of a certain

wavelength [39]. Upon absorption, a valence electron is raised from its

ground state (S0) to an excited singlet state (*S1 and *S2, Figure 3). By

internal conversion (IC) the photosensitizer will relax into the excited state

24

molecular oxygen [41]. Superoxide is not particular reactive to biological

systems, but can however react with itself to form more reactive species such

as hydrogen peroxide [41]. In the type 2 reaction the ground state triplet

oxygen accept the energy from the photosensitizer and become excited and

go into its higher energy state; singlet oxygen. Singlet oxygen is highly

reactive and will immediately react with its surroundings and e.g. oxidize

cellular components leading to cytotoxicity. The singlet oxygen is believed

to be the major damaging species in phototherapy [5,42-45]. Singlet oxygen

has a very short lifetime in aqueous solutions, around 4 μs [46-48], due to

waters high solvent deactivation properties via electronic-vibrational energy

transfer [49]. In that timeframe the singlet oxygen diffuses about 100 nm

which means that the photosensitizer needs to be close to a biologically

important system to cause photo toxicity [46].

A compound that will generate singlet oxygen upon illumination is not a

good photosensitizer per se. It is important that the photosensitizer is

efficient in producing singlet oxygen relative to the number of absorbed

photons. Einstein introduced the concept of quantum yield ( ) for a

photochemical reaction, also known as the Stark-Einstein Law of

Photochemical Equivalence [50]:

( ) =

The quantum yield at a specific wavelength is the fraction between the

number of events and the number of photons absorbed [51]. The event may

be different kinds of photophysical processes (e.g., intersystem crossing,

fluorescence and phosphorescence) or photochemical reactions (e.g.,

photosynthesis and singlet oxygen production). In an ideal situation for aPT

25

applications, one absorbed photon will be able to generate one singlet

oxygen molecule. Therefore the quantum yield has a value between 0 and 1.

In Paper IV, the quantum yield of singlet oxygen formation for three

different naturally occurring porphyrins was determined using a singlet

oxygen probe.

Biosynthesis of porphyrins and heme in bacteriaIn this section the well-studied biosynthetic pathway of heme is explained

briefly. For a more thorough review of heme biosynthesis, see [52]. The

biosynthesis of heme has different biological pathways depending whether

heme is synthesized in prokaryotic (C5-pathway) [53] or in eukaryotic

organisms (Shemin pathway) [54]. In both these pathways 5-aminolaevulinic

acid (5-ALA) is produced, but in different ways. The compound 5-

aminolaevulinic acid is the first building block of porphyrins and heme,

Figure 4.

Figure 4: Formation of 5-ALA by the Shemin and C5 pathway.

SCoA

O

HOOC+

COOH

H2N

Succinyl-CoA Glycine

Glutamyl-tRNA HOOC

NH2

H

O

Glutamate-1-semialdehyde

SHEMIN PATWAY

C5 PATHWAY

H2NO

COOH

5-ALA5-A

LA-S

26

In the C5 pathway, 5-ALA is formed from glutamate and glutametyl-tRNA,

and in Shemin pathway from succinyl coenzyme A and glycine where it is

catalyzed by the enzyme 5-aminolaevulinic acid synthase (5-ALA-S). The

formation of 5-ALA is the rate-determining enzymatic step in heme

biosynthesis and is tightly regulated by negative feedback control of heme

[38,55]. Pure 5-ALA can be added to the bacterial cultures in order to bypass

this negative feedback control [42,56]. If a bacterium has all the necessary

enzymes for the continuous synthesis of heme, the bacteria will start an

excessive production of porphyrin intermediates. Addition of 5-ALA to

bacteria was performed in Paper IV in order to increase the intracellular

content of porphyrin and investigate how the photosensitivity of E. coli was

affected.

Two 5-ALA molecules are enzymatically catalyzed by 5-aminolaevulinic

acid dehydratase to condense with each other and form porphobilinogen

(PBG), a monopyrollic precursor, Figure 5.

Figure 5: Formation of porphobilinogen from two 5-ALA molecules.

Porphobilinogen is the corner stone of porphyrins and heme and by

condensation of four PBG molecules, the linear and symmetrical

hydroxymethylbilane (HMB) is formed, Figure 6. Porphobilinogen

deaminase is the enzyme that catalyzes the formation of HMB.

HOOC

O

NH2

COOH

O

H2N

5-ALA dehydratase

NH

COOHCOOH

H2N

+

5-ALA PBG

27

Figure 6: Biosynthesis of hydroxymethylbilane from PBG.

Hydroxymethylbilane can react in two ways; enzymatically catalyzed by

the enzyme uroporphyrinogen III synthase (URO III-S) to form the

asymmetrical uroporphyrinogen III, or if URO III-S is absent, the unstable

HMB will form the physiologically unimportant uroporphyrinogen I by

spontaneous ring closure, Figure 7 [22]. At this stage of the heme

biosynthesis, and also in the upcoming steps, porphyrinogens are formed, not

porphyrins. Porphyrinogens are non-fluorescent compounds which cannot

act as photosensitizers themselves, but the oxidation products of

porphyrinogens are the corresponding porphyrin [22]. Porphyrins are with

one exception byproducts in the heme biosynthesis and they will not be

further metabolized. Uroporphyrinogen III contains four acetic acid groups

which are subjected to stepwise decarboxylation into methyl groups by the

enzyme uroporphyrinogen decarboxylase (URO-D), forming hepta-, hexa-

and pentacarboxylic acid porphyrinogens of type III isomer. However, the

URO-D enzyme is not specific at which pyrrole ring this decarboxylation

starts, leading to a complex mix of different isomers of these

porphyrinogens. Nor are URO-D specific to uroporphyrinogen III, and the

NH

COOHCOOH

H2N

PBG

NH

NH HN

HNHO

HOOC

HOOC

COOHHOOC

COOH

COOH

HOOC

HOOC

HMB

PBG-D

28

same decarboxylation process can be seen for uroporphyrinogen I [22]. In

the last decarboxylation step, catalyzed by URO-D, the coproporphyrinogen

III is formed, containing four propionic acid groups. Coproporphyrinogen

oxidase (COP-O) converts two of the propionic acid groups into vinyl

groups by oxidative decarboxylation, forming protoporphyrinogen IX. COP-

O is highly specific to the coproporphyrinogen III isomer, so

coproporphyrinogen I will not further be subject for enzymatic

transformation [22]. By oxidation, protoporphyrinogen IX is converted to

protoporphyrin IX, the only porphyrin that is actually part of the biosynthetic

pathway of heme. This transformation is catalyzed by the enzyme

protoporphyrinogen oxidase (PROT-O). The final step in the heme

biosynthesis is the insertion of ferrous iron into the protoporphyrin IX ring

structure by the enzyme ferrochelatase (FECH). Heme is rather toxic to cells

so its synthesis is tightly regulated by a negative feedback control where

heme inhibits the production of 5-ALA as mentioned above [38,55].

The killing efficiency in different aPT experiments varies and one

possible explanation can be that the different culture conditions vary among

studies [57]. If porphyrins make the major contribution to photosensitivity

and are synthesized endogenously by the bacteria, the outcome of aPT

experiments could be highly dependent on the bacterial intracellular

concentration of porphyrins. In Paper II we investigated the intracellular

concentrations of porphyrins when oral bacteria were cultivated in vitro

using different culture conditions, such as, time of culturing, passage and

different agar compositions.

30

Heme acquisition as a potential drug targetIron is an important cofactor in many biological systems and it is necessary

for respiration in bacteria [58,59]. During the initial state of an infection,

vertebrates have developed different iron withholding mechanisms that

reduce the concentration of free iron that is available for the bacteria to

negligible concentrations, < 10-18 M [60]. For example the two proteins

lactoferrin and transferrin, are produced in excess [61]. Even though the

concentration of free iron is low, a large reservoir of iron is present bound in

heme molecules. Heme occurs in various hemoproteins in vertebrates, such

as hemoglobin and myoglobin. Pathogens have developed strategies to

scavenge heme from these proteins and then either transport heme into the

cell or extract the iron from heme and transport it into the cell [60,61].

Since iron is essential for almost all bacteria, the heme acquiring

mechanisms in pathogens have been suggested to be a potential drug target

[62-67]. Limiting the access to iron

could have an antimicrobial effect

and one way could be to use a

compound that has high affinity for

the heme acquiring receptors on the

bacterial cell wall. Adding this

compound to the site of treatment

could block the bacterial uptake of

heme and bacterial replication is

reduced. Since the bacteria have a

high affinity for heme, a compound

that will mimic heme could be useful.

In Paper III we investigated

N

N

N

N

OOH OHO

Co

Figure 8: Structure of cobalt(III)protoporphyrin IX.

31

cobalt(III) protoporphyrin IX (Co-PPIX), Figure 8, for the ability to mimic

real heme and reduce the intracellular concentration of heme in E. coli.

Cobalt protoporphyrin IX has previously been shown to reduce the growth of

Porphyromonas gingivalis in both planktonic state and in biofilms [68]. The

Co-PPIX molecule is chemically similar to heme with the difference that the

essential iron (II) is replaced with the biologically insignificant cobalt (III).

The hypothesis is that bacteria will take up Co-PPIX and this will potentially

disrupt the heme metabolism in the bacteria.

Oral bacteria

Aggregatibacter actinomycetemcomitansA. actinomycetemcomitans, (prev. known as Actinobacillus

actinomycetemcomitans) is a facultative anaerobic gram negative bacterium

[69]. In 2006, A. actinomycetemcomitans was reclassified when it was

shown that the bacterium is not dependent on exogenous sources of heme to

be able to grow [70]. Boyce and coworkers [71] had previously categorized

A. actinomycetemcomitans as an X-factor dependent bacterium (requires

heme) since they could see that the bacterium was able to grow significantly

better in the presence of heme. The few bacteria that were able to grow

without the presence of heme were seen as minor variants of A.

actinomycetemcomitans. However, A. actinomycetemcomitans has all the

necessary enzymes for the de novo synthesis of heme, which is lacking in X-

factor dependent bacteria [70]. Investigations of the iron source preferences

for A. actinomycetemcomitans have shown that this specie has the possibility

to utilize heme, hemin, FeCl3 and hemoglobin as external iron sources [72-

75]. Differences between strains are not uncommon, e.g., not all strains are

32

able to use hemoglobin as iron source [74]. Overall the uptake and utilization

of iron, including the heme biosynthesis, has earlier not been well studied in

A. actinomycetemcomitans.

A. actinomycetemcomitans has been shown to be sensitive to blue light

[3,76]. Cieplik et al. [3] illuminated A. actinomycetemcomitans with 460 nm

light and observed a decrease of viable cells 10 steps after a light

dose of 150 J/cm2. In Paper I and II, A. actinomycetemcomitans were lysed

and analyzed for porphyrin content using HPLC-MS/MS.

Porphyromonas gingivalisP. gingivalis is an anaerobic, gram negative bacterium that is X-factor

dependent and requires external sources of iron for growth and virulence

[73,77]. This specie can acquire iron from both organic sources, e.g., heme,

hemoglobin, myoglobin, cytochrome C, and transferrin) as well as from

inorganic iron sources, e.g., FeCl3 and Fe(NO3)3 [78]. P. gingivalis lacks the

enzyme 5-ALA-S which catalyzes the formation of 5-ALA. It also lacks

most of the enzymes that are involved in the de novo synthesis of porphyrins

and heme [79]. If 5-ALA is added to cultures of P. gingivalis, no excessive

production of porphyrins can be seen [38].

P. gingivalis has been a subject for aPT experiments. Soukos and

coworkers [9] investigated several black pigmented oral bacteria and their

susceptibility to light in the region 380-520 nm. P. gingivalis was the least

affected bacterium and also had the lowest concentration of porphyrins in the

subsequent HPLC analysis. Only one porphyrin could be detected in P.

gingivalis, coproporphyrin. In another study where P. gingivalis was grown

with hemoglobin as an iron source instead of hemin, the sensitivity to

irradiation decreased [8]. P. gingivalis is a black pigmented bacterium that

becomes heavily blackened when grown in heme rather than hemin. The

33

-oxo bisheme [80].

This compound has been proposed to act like an oxidative buffer, keeping an

anaerobic environment inside the cell [80]. Since this compound also

absorbs light in the blue region, a plausible assumption could be that the -

oxo bisheme present in the cell walls protects the interior of the cell from

blue light, and thus no singlet oxygen can be produced. P. gingivalis was

analyzed for porphyrin content in Paper I and III.

34

Part 2. Determination of porphyrins and heme

Samples and samplingThe samples that have been studied in this work are mainly bacterial cultures

(Paper I-IV), but for method development purposes baker’s yeast was also

used (Paper I and III). Sampling is one of the most crucial parts when

analyzing porphyrins and heme in bacteria. If it is performed incorrectly, this

will have severe impact on the accuracy in, as well as the precision of the

reported concentrations. Even if a thorough sampling procedure is

performed, there are some difficulties in sampling of bacteria, of which the

estimation of sample weight is one. In most cases bacteria are cultivated on

agar or in liquid broth, and there are differences in the sampling procedures.

When grown in broth the cell density is often estimated by the optical

density at 600 nm (OD600) using a spectrophotometer. This technique does

not use the absorption of light that is normally the case when using a

spectrophotometer. Instead it uses the scattering of light when a photon hits a

particle or a cell. This scattering is proportional to the biomass concentration

in the sample. Extra care should be addressed when dealing with samples

with high density, where a multiple scattering effect may occur and the

response is no longer linear to the biomass. In those cases the samples

should 600 may then be calibrated to

the dry weight of the investigated bacteria. Due to variations in size for

different bacteria, this calibration should be performed for every species

investigated. When growing bacteria in broth, sampling by centrifugation is

the most commonly used method [6,32,77]. In Paper III and IV the sample

weight of E. coli was determined by using OD600 and then centrifuged.

35

Problems arise when determinations of sample weight for bacteria grown on

agar plates is necessary. In this case the bacteria form colonies which are

large aggregates of many individual bacteria. The bacteria in those

aggregates sticks together, therefore it is not possible to remove the bacteria

from the agar plate and then place it in a liquid and determine the OD600,

since this will give inaccurate estimations of the sample weight. A common

way is to gravimetrically determine the sample weight by scraping off the

bacteria from the agar plate and then put it in a pre-weighed test tube [81]. In

this way the wet weight of the bacteria can be determined, but moisture

differences may still give variations in the determined weight. In Paper I

and II the sample sizes were determined gravimetrically from agar plates.

The determination of sample weight is rarely mentioned in studies where

the intracellular concentrations of porphyrins have been determined.

Samples are instead normalized to the protein content or the optical density

[9,32,82]. This complicates the comparison of the concentrations given in

different studies. However, it can partly be solved by determining the

porphyrin composition by using the percent distribution of the individual

porphyrins [6,9,32,83], but gives no information about the absolute

concentrations.

Cell lysis In order to release the intracellular content and make it available for

chemical analysis, it is essential to lyse the cells. In Paper I-IV a method

using a combination of a lysis buffer and ultrasonication was employed. The

harvested microbial cells were first put in a Tris-EDTA buffer at pH 7.2 for

one hour, protected from light. Tris/EDTA buffer has previously been shown

to be effective in lysis of A. actinomycetemcomitans [84]. Tris has the

36

possibility to interact with lipopolysaccharides in the cell membrane, which

causes a destabilization [85]. Divalent cations, such as Mg2+ and Ca2+,

stabilize the membrane by crosslinking carboxylated and phosphorylated

lipids. The chelating agent EDTA in the lysis buffer binds to these metal

ions and destabilizes the cell membrane [86,87].

In many non-quantitative bioanalytical applications, e.g. protein

purification and DNA isolation, a lysis efficiency of 100% is not of highest

importance. When the aim is to perform quantification of intra cellular

contents, it is important with a lysis efficiency as close to 100% as possible

in order to have high accuracy in the reported concentrations A physical cell

disruption method, using an ultrasonication probe, was added to the

chemical lysis procedure to increase the efficiency. High frequency

ultrasound energy (20-50 kHz) is applied to the sample medium through a

metal probe. It creates cavities which implode and create shock waves

causing the cells to rupture. To avoid excessive heating of the sample, the

ultrasonication is performed in multiple short bursts and the sample should

be placed in an ice-water bath.

In Paper I the above mentioned method for cell lysis was investigated for

its ability to lyse A. actinomycetemcomitans. After treatment with buffer and

ultrasonication no viable bacteria could be seen on agar plates compared to

99.98%.

Extraction and clean-upTwo commonly used clean-up methods used for porphyrins and heme are

solid phase extraction (SPE) and liquid-liquid extraction (LLE). The latter is

the most commonly used method for the extraction of porphyrins from

37

plasma and red blood cells, as well as or studying bacterial extracts

[6,22,32,44,88-90]. Solid phase extraction has mostly been used for urine

samples and in a few studies of porphyrins in bacterial extracts, in all cases

using C18 as the stationary phase [22,44].

Solid phase extraction of porphyrinsSolid phase extraction was used for clean-up of bacterial extracts in Papers I

and II. The major advantages of using SPE for porphyrin extraction is that it

consumes low volumes of organic solvents, yields cleaner samples by

removing interfering compounds, and the possibility to concentrate the

analytes without excessive evaporation of extraction solvents [91]. Most

microorganisms produce porphyrins in very low concentrations (ng/mg) [9].

In order to detect porphyrins in bacterial extracts it is necessary to

concentrate the samples or use large sample sizes. Since many

microorganisms are difficult to culture, it is often beneficial to concentrate

the samples instead of using large sample sizes.

End-capped reversed phase C18 SPE column was used for clean-up of

bacterial extracts in Papers I and II, due to the good retention for

porphyrins. Naturally occurring porphyrins have a wide range of polarity due

to differences in the number of carboxylic acid groups attached to the

macrocyclic porphyrin structure. Uroporphyrin, with its eight carboxylic

acid groups, is the most polar with a logP value of 3.51 at pH 1.7

(Chemspider, Chemicalize). Protoporphyrin IX, the most nonpolar porphyrin

on the other hand has a logP of 6.43 at pH 4.5 which means a difference in

mass transfer to octanol by a factor of around 1000. Since porphyrins are

zwitterions, the partition coefficient changes dramatically at more

physiologically pH. For example the logD values for uroporphyrin and

protoporphyrin IX at pH 7.4 are -21.22 and 2.29 respectively (Chemspider,

38

Chemicalize). This difference makes it challenging to develop a clean-up

method with recoveries close to 100% for all porphyrins. It is also difficult

to find a suitable internal standard that will compensate for losses during the

clean-up. To reduce the difference in the polarity of the compounds, the

sample should be acidified prior to extraction and in Papers I-II 6M formic

acid was used. Recoveries were investigated by passing a porphyrin standard

through the clean-up steps. They ranged between 55-99%. With the lowest

recoveries observed for uroporphyrin and protoporphyrin IX, 67 and 55 %

respectively, Figure 9. Standard deviations were less than 10% for all

investigated porphyrins, and the clean-up using SPE was considered to be

acceptable for further studies.

Figure 9: Recoveries for porphyrins using the SPE clean-up in Paper I. Error bars show the standard deviation for triplicate experiments (n=3). UP: uroporphyrin; 7P: 7-carboxylporphyrin; 6P: 6-carboxylporphyrin; 5P: 5-carboxylporphyrin; CP: coproporphyrin; MP: mesoporphyrin IX; PPIX: protoporphyrin IX.

0

20

40

60

80

100

120

UP 7P 6P 5P CP MP (IS) PPIX

Rec

over

y [%

]

40

Experimental design can be used to find optimal conditions by systematic

alternation of the experimental settings [93]. By using experimental design

the number of experiments can be reduced, and also account for variables

that might be correlated. Each variable is given a minimum and maximum

value, which are coded to -1 and +1 respectively. By varying each variable

one at a time, a full factorial design is created, and this is illustrated by the

red circles in Figure 10. Using only a full factorial design will not yield any

information about the optimal settings of variables, it will only provide

information about the significance of each variable [93]. There are a number

of optimization designs that introduce a quadratic term into the model which

will find an optimum within the experimental space. One of the most basic

ones is central composite design which combines a full factorial design with

a star design. The star design used in Paper III is a CCF design where the

star points are at a coded distance of one from the center point and can be

seen in Figure 10 as green circles. The blue circle in the middle of the design

shows the center point which was analyzed in triplicate to minimize the risk

of missing non-linear relationships in the middle of the intervals and

allowing for determination of confidence intervals [93].

In a first experiment the relative yield of heme using three different

organic solvents; acetone, acetonitrile and methanol was evaluated. Acetone

and HCl have been used in previous studies for the extraction of heme from

different plants [94-97] and the extraction efficiency using this solvent was

set to 1. Acetone and acetonitrile showed the same extraction properties,

while methanol was only able to extract 66% of the heme relative to acetone.

Pure acetonitrile and 1.7 M HCl were also evaluated and they could only

. Thus the optimization of the heme extraction was

made with acetonitrile as the extractant.

42

compositions can be used for clean-up purposes without losing any heme

from the sample. By using pure acetonitrile, and thus not extract any heme,

lipophilic interfering substances in the lysate can be removed and provide

cleaner samples. It is also possible to produce analyte free matrices that can

be used for validation proposes e.g. evaluation of matrix effects.

Protein precipitationMany bioanalytical applications utilize protein precipitation as a fast sample

clean-up to remove proteins from biological matrices [98]. By changing the

surrounding environment, in which the proteins are stable, they can be

precipitated. Protein solubility depends on ionic interaction with salts, polar

interactions with aqueous solvent and electrostatic forces between charged

molecules [98]. The different protein precipitation techniques are; use of

organic solvents, changing the pH, metal ion and salting out [98].

By using organic solvents, the dielectric constant of the protein solution

will decrease, which increases the attraction between charged molecules and

the electrostatic interactions between proteins are enhanced. Organic

solvents also remove the water surrounding the protein surface which

decreases the hydrophobic interactions between proteins, thus enhancing

protein aggregation by increasing the electrostatic interactions [99].

Protein precipitation can also be carried out by changing the pH of the

protein solution. A protein has an isoelectric point (pI), i.e. the pH where the

protein has a net charge of zero, and therefore it has poor solubility in

aqueous solvents. At a pH less than the pI the proteins will have a positive

net charge and acidic agents will form insoluble salts with the positively

charged amino acids, leading to protein precipitation [98].

Salting out is also a common way to precipitate proteins in bioanalytical

procedures. Only a few proteins are soluble in pure water and most require a

43

small concentration of ions (< 0.5 M) to remain stable. At higher

concentrations of salts, the protein surface will become highly charged so

proteins will interact with each other and precipitate [100].

In Paper III, methanol, acetonitrile and acetone were evaluated for their

efficiency to precipitate proteins from S. cerevisiae lysate. The precipitating

solvents were a mixture of organic solvents and HCl, in the same proportions

that were optimized for heme extraction using acetonitrile as mentioned

earlier. Pure acetonitrile as well as 1.7 M HCl were evaluated for their

efficiency to precipitate proteins.

For determination of protein concentration in solution, the simplest and

most direct assay is to use the light absorption of peptide bonds below 210

nm [101]. One may also measure the absorbance at 280 nm, were amino

acids containing aromatic side chains, i.e. tyrosine, tryptophan and

phenylalanine, exhibit a strong UV-light absorption. A major disadvantage

using this direct determination is that not all proteins contain these aromatic

amino acids and will skew the determination of protein concentration [102].

Several reagent based protein assays have been developed instead. The

method of choice for protein quantification in Paper III was the Bradford

method [103]. The Bradford assay is a colorimetric protein assay based on

the formation of a complex between the dye Brilliant Blue G, and proteins in

the solution. When the dye is bound to the protein it will cause a shift in

absorbance from 465 to 595 nm, and the amount of absorption is

proportional to the protein concentration [103]. Bovine serum albumin was

used as a standard to check the linear response of the spectrophotometer. The

Bradford method has many advantages. It is simple to perform, cheap,

relative high sensitivity, and it will measure the absorbance in the visible

light region. A major disadvantage in using the Bradford method is that it is

not always compatible with some detergents, buffers and organic solvents.

45

Salting-out assisted liquid-liquid extractionCombining the results from optimized extraction of heme and the ability of

different solvents to precipitate proteins, acetonitrile:HCl was chosen to be

the solvent for extraction of heme and porphyrins in Paper III and IV. Due

to its low extraction efficiency of heme and its good protein precipitation

properties, pure acetonitrile was used for clean-up purposes. By first adding

pure acetonitrile to the bacterial lysate, 100% precipitation of all proteins

was performed with no extraction of heme. The sample was centrifuged to

form a pellet of precipitated proteins and by discarding of the organic

supernatant, unwanted hydrophobic compounds could be removed. By

adding acetonitrile:HCl to the protein pellet, heme was extracted from the

precipitated proteins. To remove hydrophilic interfering compounds,

saturated MgSO4 solution and NaCl were added to the samples. By

increasing the ionic strength of the solution, acetonitrile was no longer

miscible with the aqueous solvent and a two phase liquid-liquid system was

formed. Heme is hydrophobic and is partitioned into the upper acetonitrile

layer. The evaluation of the matrix effects using this method for heme

showed that the matrix effect was low, and the signal obtained for a spiked

analyte free matrix was 106 ± 3 % compared to a standard dissolved in pure

solvent.

Liquid chromatography of porphyrinsPorphyrins are macrocyclic structures with inherent low volatilities and

vapor pressures, which makes porphyrins unsuitable to analyze with gas

chromatography (GC) [22], even though some studies have used GC to

separate both metallo- and free base porphyrins [105-107]. However, the

46

recent development of high efficiency columns has made HPLC the

technique of choice for porphyrin separation [22].

HPLC columnsAs mentioned above, porphyrins have varying arrangements and kinds of

side chain substituent groups around the porphyrin’s macrocycle which gives

these compounds varying degrees of hydrophobicity. These differences in

hydrophobicity are suitable for the separation by reversed-phase

chromatography [20]. The hydrophobicity of the side-chain substituents of

porphyrins increases in the following order: CH=CH2 > CH2CH3 > CH3 >

CH2CH2COOH > CH2COOH [22].

Porphyrins have been able to be separated on reversed phase columns

ranging from octadecyl carbon chain bonded silica (C18) to the least

retentive of all alkyl group bonded phases, trimethylsilyl (C1) [22].

C1 columns allow fast separation of naturally occurring porphyrins, but the

chromatographic resolution between porphyrins with 4-8 carboxylic acid

groups is poor. C18-columns are more retentive than C1 columns and leads to

longer analytical runs, but the resolution between porphyrins and some of

their isomers will on the other hand be enhanced. The separation of a

porphyrin mixture on a C18 column is shown in Figure 13.

A number of different HPLC columns from a variety of manufacturers

were evaluated for their ability to separate porphyrins, e.g. C18 (ACE),

biphenyl (Restek), C18-PFP (ACE), Poroshell C18 (Agilent), Core-shell C18

(Phenomenex), ZORBAX HILIC Plus (Agilent), and C18-Phenyl (ACE).

The biphenyl column was able to separate two isomers of 6-carboxyl

porphyrin when using methanol as an organic modifier in the mobile phase

which none of the other columns were able to do. When the biphenyl column

was used with acetonitrile as the organic modifier in the mobile phase, these

47

two isomers co-eluted and the retention was similar to traditional C18

stationary phases. By using different organic solvents in the mobile phase,

the biphenyl column can be switched between two different separation

mechanisms, hydrophobic interactions using acetonitrile and - interactions

using methanol. Acetonitrile will significantly weaken the - interactions

between the porphyrins and the phenyl groups in the stationary phase, and

when used with methanol an almost orthogonal elution compared to C18 can

be achieved depending on the type of analytes [108].

Time [min]

0

100

1.00 2.00 3.00 4.000.00

Rel

. Int

. [%

]

50

UP7P 6P

5P

CP

Hemin

MPIX

PPIX

Figure 13: Total ion chromatogram from the HPLC-MS/MS analysis of a standardmixture of porphyrins and hemin. UP: uroporphyrin; 7P: 7-carboxylporphyrin; 6P:6-carboxylporphyrin; 5P: 5-carboxylporphyrin; CP: coproporphyrin; Hemin;MPIX: mesoporphyrin IX; PPIX: protoporphyrin IX. Flow rate: 1 mL/min (figureand caption from Paper III).

48

Another stationary phase that demonstrated better separation abilities than a

regular C18, was the C18-PFP column. In this stationary phase, the octadecyl

carbon chain is attached to the silica particles like a regular C18 column, but

at the end a pentafluorophenyl (PFP) group is attached. This phase has

several separation mechanisms; C18 hydrophobic separation mechanisms, -

interactions with the aromatic ring, dipole-induced dipole and/or hydrogen

bonding interaction with the electronegative fluorine atoms of the PFP-

group. The porphyrin standard used in Paper I-IV contained small quantities

of impurities of porphyrin isomers, in particularly for pentacarboxylic acid

porphyrin. These isomers could be used to see how well the column

separates naturally occurring porphyrin isomers from bacterial extracts. The

C18-PFP column exhibited the best isomer separation properties under the

same chromatographic conditions, where three isomers were visible in the

chromatogram. A 75 mm C18-PFP (75mm) was used for the HPLC porphyrin

separation in Paper I and II, while in Paper III and IV a regular C18 column

was used.

Porphyrins were not well separated on HILIC columns, with extensive

band broadening and co-elution as a result. Neither could the porphyrins be

well separated on the Core shell C18 column, where the first four eluting

compounds, uroporphyrin to pentacarboxylporphyrin, co-eluted with an

extensive peak broadening and bad peak symmetry. Porphyrins are well

separated on regular C18 stationary phases so this problem is most likely due

to the core shell design of the particles in this type of column.

Mobile phasesA commonly used mobile phase composition for porphyrin separation is the

1 M ammonium acetate-acetic acid buffer with a pH between 5.1 and 5.2,

together with methanol/acetonitrile [14,25,109]. This mobile phase has

49

demonstrated good separation properties of different porphyrin isomers.

Porphyrins are zwitterions, meaning that they may have both a positive and a

negative charge depending on the pH. On reversed phase columns the

ionization state of porphyrins will highly affect the chromatographic

resolution, especially between isomers. At low pH, all carboxylic acids are

fully protonated, but both the amine groups get protonated, leading to a

positive net charge. At high pH the carboxylic acid groups will be

deprotonated to a varying degree, leading to a negative net charge. A

complete ionization of a porphyrin on a reversed phase column will result in

poor retention, while a neutral porphyrin will have an excessive retention

leading to long elution times. In the pH between 5.1 and 5.2 the optimal

separation between porphyrin isomers can be achieved, especially for

porphyrins with 4 to 8 carboxylic acid groups [22]. However, during

experimental work for Paper I high signal suppression in the electrospray

ion source could be observed using this buffer. It decreased the signal/noise-

ratio (S/N) and increased the limit of detection (LOD) for all the analytes.

HPLC-MS/MS analyses using a lower concentration of ammonium acetate

(10 mM) did not reduce the ion suppression to acceptable levels. Bacteria

contain porphyrins in low concentrations and sample sizes are often small.

Generally a sample weight between 50 and 100 mg is possible to collect

from five agar plates, therefore low LODs and LOQs are necessary rather

than the possibility to separate different isomers. For these reasons the

ammonium buffer was discarded as a mobile phase.

Other mobile phases were evaluated such as ion pairing with

triethylamine, acetonitrile and methanol as organic modifiers. Triethylamine

resulted in faster elution but the chromatographic resolution was poor. No

resolution between mesoporphyrin and protoporphyrin could be achieved.

Acetonitrile was finally selected as the organic modifier due to higher

50

elution strength compared to methanol, which resulted in shorter run times

(Paper I-IV).

Mass spectrometry of porphyrinsThe fragmentation of naturally occurring porphyrins mainly takes part at the

carboxylic acid groups [22,110]. The macrocycle structure itself is very

stable and does not fragment at reasonable collision energies [110]. The loss

of m/z 59 Da corresponds to the benzylic cleavage of the propionic acid side

chain (loss of •CH2COOH radical), and the loss of 46 Da corresponds to the

fragmentation of the acetic acid side chain (loss of HCOOH). The loss of 46

Da is specific to acetic acid substituents and can therefore be used for

structural determinations [22]. Losses of 58 and 45 Da are results of the

rearrangement of a proton between fragments and product ions [22]. Two

other fragmentation patterns are often seen as well; the loss of water (m/z 18

Da) from the quasi-molecular ion and cleavage of methyl groups followed

by rearrangement of a proton (loss of 14 Da) [22]. There is possible to see

losses of 73 Da and the literature have contradictory explanations of the

origin of these fragments. Fateen [111] explain those by loss of•CH2CH2COOH radical, while Lim [22] argues that these more likely are

formed by losses of •CH2COOH and •CH3 followed by protonation of the

resulting product ion. In Figure 14 the product ion spectrum is shown for

pentacarboxylporphyrin I containing four propionic- and one acetic acid

groups.

These specific fragmentation patterns make porphyrins suitable to analyze

with tandem mass spectrometry in selected reaction monitoring (SRM)

mode. In general the LOD and LOQ are lower when using SRM, but the

opposite was seen for most of the porphyrins. Calculating the S/N from

51

seven injections of a standard in both selected ion monitoring (SIM) and

SRM mode showed that the average S/N was only higher in SRM for

protoporphyrin IX and coproporphyrin (data not published). This is probably

because of fewer carboxylic acid groups that can stabilize the molecule

during fragmentation. Even though the S/N was lower in SRM this was

selected as detection mode in Paper I-IV due to the higher selectivity.

53

Stability of porphyrins and heme in solution

One of the most crucial problems to overcome in porphyrin quantification is

the tetrapyrrole’s tendency to aggregate in aqueous solutions [112]. Not

accounting for this aggregation will most likely result in large quantification

errors. There are several hypothesizes why porphyrin structures aggregate.

The commonly accepted one is that porphyrins form aggregates by -

stacking mechanisms [113]. The bonds in the porphyrin ring structure can

be stacked in different ways and form large porphyrin complexes, stable

enough to sustain a chromatographic run [114]. If using nonspecific

detectors, such as UV-Vis and fluorescence, this will give multiple peaks

when these dimers and higher aggregates have different retention times in

the column. When porphyrins are analyzed with MS/MS, these aggregates

are not visible as peaks (if not scanned for) but are manifested as a loss in

peak area of the monitored porphyrin. Especially hydrophobic porphyrins

with only two or less carboxylic acid groups such as heme and

protoporphyrin have been shown to be extra sensitive to aggregation

[38,112,114,115]. In this work a number of different experiments

investigating the aggregation in different solvents have been performed (data

not published). In an experiment heme and coproporphyrin were extracted

from baker’s yeast and the content of porphyrins and heme in the extract was

determined during 12 hours while stored in an autosampler tray protected

from light. The peak area for coproporphyrin was stable during these 12

hours, while the peak area for heme drastically decreased. After 12 hours,

88% of the original peak area for heme was lost. In another experiment a

porphyrin standard was dissolved in different solvents at equal concentration

and the peak area from the HPLC-MS/MS analysis compared. All areas of

the analytes were relative to areas given by a standard solved in 6 M formic

54

acid. Fairly concentrated hydrochloric acid (1.5 M) in general gave smaller

peak areas compared to 6 M formic acid and more concentrated HCl (6 M)

was even worse, where the response for uroporphyrin was below the limit of

detection. When taking the same solvent and diluting it further with tris

buffer, uroporphyrin could be detected once again. This demonstrates that

HCl is not destructive to uroporphyrin; it only pushed the equilibrium

towards aggregate formation. Once the solvent was changed, the equilibrium

was shifted back in favor to uroporphyrin in free form. Lower concentration

of formic acid gave lower relative peak areas due to be the porphyrins not

being protonated to the same extent and therefore having reduced solubility

in aqueous solvent. The solution with a concentration of 6 M formic acid

was approximately 1:4 (v/v) formic acid:H2O, meaning that it has some

characteristics of an organic solvent, which also increases the solubility,

especially for the more hydrophobic meso- and protoporphyrin IX. In

general, the area for more hydrophobic porphyrins increases with higher

organic solvent composition, while it decreases for the more hydrophilic

porphyrins.

Due to tetrapyrrole’s tendency to aggregate in different solvents it is

necessary to investigate if this artefact is present before doing any

quantitative analysis. This is most easily done by preparing standards of the

analytes in both organic and aqueous solvents, at an equal concentration, and

to compare the peak areas. If the areas deviate from each other more than the

instrumental precision, extra care should be addressed. If the method allows

it, the use of ion pairing could be used to stabilize the porphyrins. A

common ion pairing agent is tetrabutylammonium ion [114]. Another

approach is to always keep the solvent composition in the sample the same

as for standards. In this way the detector response factor will be the same for

sample and standard and accurate quantification can be done. This latter

56

Part 3. Applications

Photo inactivation of bacteria

Pilot studyOur research group has previously shown that single colonies of A.

actinomycetemcomitans may emit red fluorescence when illuminated with

blue light [116]. The intensity of this fluorescence increased during growth,

but did not correlate to the size of the colony. This indicates that the

bacterium accumulates fluorescent compounds probably due to the

porphyrin content.

During the work with Paper I intracellular porphyrins in the bacterium A.

actinomycetemcomitans were identified. This, together with the support from

the literature, led to cooperation with a Swedish company marketing

toothbrushes with two incorporated 465 nm LEDs in the brush head. A set of

brushes with 410 nm LEDs were manufactured in order to overlap the Soret

band of porphyrins. A. actinomycetemcomitans was cultivated on blood agar

plates and two toothbrushes were placed side by side and let to illuminate the

center of an agar plate for 10 min in the morning and in the afternoon (total

time of 20 min). Reference agar plates where no illumination was performed

were treated the same way as the illuminated plates. In Figure 16 a reference

plate and an illuminated plate are shown.

58

voltage power supply. By adjusting the voltage output the illumination

energy reaching the bacterial cultures could be adjusted. Each column of

LEDs in the matrix was used for illumination of bacteria in 10 mm cuvettes.

In this way the illumination could be performed at relatively high cell

number densities, serial diluted, and subsequently streaked on agar plates.

Colony forming units (CFU) can be calculated on light treated bacteria and

compared to reference samples where no illumination has been performed.

In Paper IV E. coli was cultivated and illuminated. E. coli is a bacterium

that has shown poor sensitivity for blue light, but the results from different

studies vary [3,33]. In this study E. coli was illuminated with light doses

ranging from 4.8–172.8 J/cm2 and no antimicrobial activity could be seen,

Figure 17. Even a small increase in CFU during the illumination procedure

was observed and E. coli was suspected to still grow during the illumination.

To investigate if the porphyrin content could have an impact on the photo

sensitivity of E. coli it was grown in broth supplemented with 5-ALA. As

mentioned earlier, the addition of 5-ALA can lead to high concentrations of

intracellular porphyrins if the bacteria have all necessary enzymes for the de

novo synthesis of heme. E. coli sensitivity for blue light increased

remarkably. By using a light dose of 4.8 J/cm2 the number of viable bacteria

was reduced with more than 3 log10 steps (99.9% reduction). A light dose of

86.4 J/cm2 gave a reduction of viable bacteria > 6 log10 steps and thus the

level to work as a disinfectant of E. coli was passed.

59

Porphyrin content in microorganismsWhen the porphyrin content was analyzed in three different microorganisms;

S.cerevisiae, P. gingivalis and A. actinomycetemcomitans it was revealed

that the porphyrin content differs between these microorganisms.

Differences both in the total amount of porphyrins and their respective

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

0 30 60 90 120 150 180

Surv

ival

frac

tion

Illumination light dose [J/cm2]

E. coli

E. Coli + ALA

Limitanitimicrobialactivity

Limitdisinfectant

Figure 17: Comparison of the photosensitivity for E. coli grown in presence andabsence of 5-ALA. A survival fraction of 1 corresponds to references where noillumination of bacterial culture has been performed. Values are means for threeindependent experiments and error bars represent the standard deviation. Redmarks are single experiments using bacterial cultures with an OD600 > 0.1 (Figureand caption from Paper IV).

60

concentrations were observed. The total amount of porphyrins was highest in

A. actinomycetemcomitans, more than 4 times higher concentration than in

S. cerevisiae. On the other hand, S. cerevisiae had the largest number of

porphyrins detected and pentacarboxylic porphyrin was the only one of the

naturally occurring porphyrins that not could be detected. In P. gingivalis

only coproporphyrin and protoporphyrin IX were detected. As stated earlier

P. gingivalis lacks crucial enzymes for the de novo synthesis of heme,

among others all of the enzymes involved in the formation of 5-ALA is

lacking so theoretically it should not be possible for P. gingivalis to

synthesize any of the porphyrins. The findings were therefore not expected,

but coproporphyrin and protoporphyrin IX have been detected in P.

gingivalis previously [8,9], which is in agreement with our results. The

presence of protoporphyrin IX could be due to P. gingivalis requiring heme

to grow and has an uptake up heme from the growth medium. By enzymatic

removal of iron from heme, protoporphyrin IX can be formed as been

observed in Haemophilus influenzae [117]. Detection of coproporphyrin in

P. gingivalis could not be explained by these mechanisms since there are

currently no known enzymatic pathways that convert protoporphyrin IX to

coproporphyrin. One possibility is that the growth medium contains

coproporphyrin and P. gingivalis heme acquiring mechanisms also have an

uptake of other tetrapyrroles such as coproporphyrin.

Influence of culture conditionsResults from aPT experiments found in the literature differ greatly; one

pathogen may in one study have an entirely different sensitivity for blue

light than in another one. For example has E.coli been shown to possess low

sensitivity for blue light in some studies [3,33] while in another E. coli could

61

be killed with blue light [30]. One explanation is that different light sources

are used and they emit light of different wavelengths. Another observation

was that the cultivation procedure for the same pathogen varied between

studies. Since porphyrins are synthesized by the bacteria themselves, the

way they are cultivated could affect the intracellular composition of

porphyrins and hence their sensitivity to blue light. Three cultivation

parameters were selected that often varies between studies and that could

have a high impact on the biosynthesis of porphyrins; the cultivation time,

passage number and the kind of cultivation media.

Time of culturingThe two oral pathogens, A. actinomycetemcomitans and P. gingivalis were

selected as model organisms for investigating the effect of culture time on

the intracellular porphyrin profile in Paper II. A. actinomycetemcomitans is

able to synthesize porphyrins endogenously while P. gingivalis is not. A.

actinomycetemcomitans was grown and analyzed for porphyrin content at

each day for 3 to 9 days, and P. gingivalis was grown and analyzed at 4, 7

and 11 days, Figure 18.

P. gingivalis had almost the same porphyrin pattern during all the days of

incubation; protoporphyrin IX being the major porphyrin species and

coproporphyrin III at a relative low concentration level. For A.

actinomycetemcomitans the pattern changed drastically during the

incubation. At day 3 coproporphyrin III was the most abundant porphyrin

making up 80% of the total porphyrin content (TPC), followed by

protoporphyrin IX (16% TPC) and coproporphyrin I (4% TPC). At day 6 this

pattern had reversed; the most abundant porphyrin was now protoporphyrin

IX with 88% of TPC and coproporphyrin III was 9% of TPC.

63

actinomycetemcomitans was cultivated for 4 and 7 days with and without

passage. At day 4 the porphyrin profile was more or less the same between

passaged and non-passaged bacteria. However, at day 7 there was a large

difference. Total porphyrin content increased 28 times upon passage. There

was also a difference in the porphyrin profile, where except from

coproporphyrin I, coproporphyrin III and protoporphyrin IX, also

uroporphyrin and heptacarboxylic porphyrin could be detected in the

passaged bacteria.

This significant difference in TPC between passaged and non-passaged

bacteria could have a high impact on the results from in vitro illumination

experiments. In a real case scenario where bacteria in the oral cavity are

illuminated, the bacteria will most likely have a high passage number. It is

therefore recommended to use passaged bacteria in aPT experiments in vivo.

Cultivation mediumThere is a large variety of cultivation media. There are two main types, solid

(agar) and liquid media (broth). Broth has traditionally been used in

illumination experiments. This is due to the ease in performing dilution

series and to calculate killing efficiency. Agar is a better choice when

mimicking biofilms.

P. gingivalis is an X-factor dependent bacterium, meaning that it requires

heme to be able to grow. Heme can be added in pure chemical form, or by

addition of blood to the agar. P. gingivalis were grown both on blood and

blood free agar in Paper II and the bacteria was analyzed for porphyrin and

heme content. The colonies were black pigmented and contained 5 times

higher concentrations of heme when grown on blood compared to

transparent when grown on blood free agar. Also the porphyrin profile was

different. Coproporphyrin III could not be detected and the protoporphyrin

64

IX concentration was lower in bacteria grown on blood free medium. This

further emphasizes the hypothesis that P. gingivalis has an uptake of

coproporphyrin from the growth medium since blood contains

coproporphyrin.

Influence of different additives on E. coliE. coli was used as a model organism when different additives related to

heme biosynthesis were added to the growth medium. The primary focus

was to analyze the bacterial extracts for heme content in order to see if these

additives could change the heme concentration. The porphyrin content was

also analyzed as they are intermediates in the heme biosynthesis.

5-aminolaevulinic acid5-aminolaevulinic acid was added to the cultivation broth and after 48 h E.

coli was analyzed for porphyrins and heme content, Figure 19 (Paper IV).

The cultures became colored light purple after addition of 5-ALA. The

chemical analyses revealed different porphyrin species in high

concentrations compared to bacterial culture where no 5-ALA was added. In

the control sample only coproporphyrin I and protoporphyrin IX could be

detected, but with concentrations below LOQ. Addition of 5-ALA resulted

in the production of uro-, hepta-, hexa-, penta-, copro-, and protoporphyrin

IX. The total porphyrin content increased from 1.5 to 1882 nmol/g after

administration of 5-ALA. The most abundant porphyrin was uroporphyrin

followed by protoporphyrin IX. The broth, in which the bacteria were

grown, was also analyzed and it contained up to 20 times higher

concentrations of porphyrins than the bacterium itself. The only exception

was protoporphyrin IX where only small amounts could be quantified in the

65

broth. This indicates that the bacterium excretes porphyrins when the

intracellular concentration becomes too high. Extra caution should be taken

if illumination experiments are carried out after administration of 5-ALA

since the broth itself may produce singlet oxygen and have an antimicrobial

effect. Bacteria should therefore be washed in new broth prior to

illumination experiments. The reason why protoporphyrin IX not is excreted

is unknown, but it is possible that if E. coli was allowed to grow for a longer

period of time, it would start to excrete protoporphyrin IX as well.

66

Figure 19: Total ion chromatograms from the HPLC-MS/MS analysis of E. coli grown on LB broth (A) and after addition of 5-ALA (B). Chromatograms have been enlarged and normalized to CP I in the range 0.00-1.80 min. UP, uroporphyrin; 7P, 7-carboxylporphyrin; 6P I, 6-carboxylporphyrin I; 6P III*, 6-carboxylporphyrin III (tentatively identified), 5P, 5-carboxylporphyrin; CP I, coproporphyrin I; CP III,coproporphyrin III; PPIX, protoporphyrin IX (Figure and caption from Paper IV).

67

HeminAs mentioned earlier some bacteria are X-factor dependent and require heme

to grow. These kinds of bacteria, such as P. gingivalis lack the enzymes for

the de novo biosynthesis of heme. But how about bacteria that have all these

necessary enzymes for heme biosynthesis? Can and will they acquire heme

from their environment? E. coli was grown in broth spiked with hemin and

after 24 h the bacteria were harvested and analyzed (Paper IV). The heme

content in E. coli was found to increase 4.5 times in the treated culture

compared to the control. E. coli is able to bind and transport heme through

its cell walls. Whether E. coli synthesizes heme itself or takes it up from the

environment is still unknown, but the higher concentrations of intra cellular

heme are an indication that E. coli prefers to acquire heme from its

environment when available in free form. The high affinity for heme could

be a potential drug target for treatment of bacterial infections, utilizing

structural analogs to heme.

Cobalt protoporphyrin IXHeme-acquiring systems in pathogens have been proposed to be candidates

for a drug target [62]. Cobalt protoporphyrin IX (Co-PPIX) is a structural

analog to heme where the iron (II) in heme has been replaced with cobalt

(III). The macrocyclic ring structure and the side chain groups are identical.

To investigate if E. coli could mistakenly recognize Co-PPIX as heme, the

bacteria were analyzed after administration of Co-PPIX to the cultivation

media in Paper IV. In one experiment Co-PPIX was added to the broth and

E. coli was analyzed for heme content. If E. coli takes up Co-PPIX, the heme

concentration should be lower than in a control where no Co-PPIX has been

added. The result showed that heme concentration was reduced by 48%. In

another experiment hemin and Co-PPIX were added in equimolar

68

concentrations. It was observed that the heme concentration once again was

reduced by 52% compared to an experiment where only heme had been

added to the broth. The lysate was additionally analyzed in fullscan and the

m/z for [Co-PPIX+H]+ was extracted. It showed that only bacteria spiked

with Co-PPIX contained this compound in high concentrations. These

experiments show that Co-PPIX does mimic heme and that E. coli has an

equal affinity for Co-PPIX as for heme. No antimicrobial effect could

however be seen for Co-PPIX to E. coli cultures and the total cell number

was equal after the administration. We believe that the capacity of E. coli to

synthesize heme de novo is the reason that no antimicrobial effect was seen.

Singlet oxygen production of porphyrinsIn Paper IV the 1O2 production for three naturally occurring porphyrins were

determined by using a probe that reacts with oxidation products caused by

reaction with singlet oxygen. Reaction with the probe will cause a shift in

absorbance, and the loss of absorbance of the probe can be observed with

UV-Vis. We chose one of the most commonly used protocol, which is highly

specific for 1O2 and also suitable for aqueous systems [46]. The probe used in

this work was N,N-dimethyl-4-nitrosoaniline (RNO) and L-histidine (His)

was used for reaction with 1O2. L-histidine reacts with 1O2 and produces a

trans- annular peroxide product [HisO2]. If RNO is present [HisO2] can react

with RNO causing a loss in absorbance of RNO which can be monitored at

440 nm [118].

1O2 + His [HisO2] (1)

[HisO2] + RNO RNO2 + products (2)

69

The porphyrins were dissolved individually in 0.01 M solutions of histidine

and then 50 μM of RNO was added. The illumination was performed in 10

mm path length cuvettes and analyzed with UV-Vis after illumination with

varying light doses. It is important to perform experiments where no

photosensitizer has been added to the solution in order to rule out that the

probe is not sensitive for the light. The slope of the plot of bleached RNO vs

the irradiation time is proportional to the rate of production of singlet oxygen

produced by the photosensitizer [46]. Figure 20 shows the photo bleaching

of RNO using protoporphyrin IX as a photosensitizer and a reference sample

where no porphyrin has been added.

Figure 20: UV-Vis spectra for the photo bleaching of RNO, with (A) and without (B)protoporphyrin IX as a photosensitizer. The cuvette was illuminated for a total time of 60 min in (A), and in (B) for 120 min (figure from Paper IV).

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

300 350 400 450 500 550

Abs

orba

nce

nm

RNO + PPIX0 - 60 min

300 350 400 450 500 550

nm

RNO

0 - 120 min

A B

70

To determine the quantum yield of singlet oxygen ( ) generated by a

porphyrin it is necessary to determine how many singlet oxygen molecules that

are produced as a function of absorbed photons by the porphyrin molecule.

Doing this in absolute terms is very complex. A more common, alternative way

to perform these kinds of determinations is by using a compound with a well

determined . By matching the absorbance (at a specific wavelength) of both

the reference compound and the photosensitizer of interest, one can assure that

the same numbers of photons are absorbed in each case. The for the porphyrin

of interest can be calculated by the following equation:

=

Where Unknown and Std denote the quantum yield of singlet oxygen

produced by the investigated photosensitizer and the standard, respectively,

and k is the slope of the loss in absorbance of RNO vs the irradiation time.

Determining the of photosensitizers using this method will only explain in

what ratio an absorbed photon will generate one singlet oxygen molecule. It does

not tell how well a photosensitizer absorbs photons, i.e. the compounds extinction

coefficient. By taking the concentration needed for each photosensitizer to reach

the same optical density in consideration, a relative of each photosensitizer

may be determined. This was done in Paper IV for uroporphyrin,

coproporphyrin and protoporphyrin IX and shown in Figure 21.

71

Figure 21: Relative photo bleaching of RNO by 405 nm irradiation for three different porphyrins. A0 and A is the absorbance of RNO measured at 440 nm at time 0 and the specific irradiation time respectively (figure from Paper IV).

Uroporphyrin III was the porphyrin that was most efficient in producing

singlet oxygen, followed by coproporphyrin III and protoporphyrin IX. It

should be stressed that the of the different porphyrins is not the only

parameter that determines if a porphyrin is efficient at killing bacteria. Depending

on the different physical properties of porphyrins, they may concentrate at

different subcellular locations in bacteria. The location of a photosensitizer is of

highest importance since the lifetime of singlet oxygen is only 4 μs in aqueous

solutions and in that time it will only diffuse about 100 nm [46].

Clinical studyA clinical study was performed at Karolinska Institutet (Stockholm,

Sweden). Forty eight first year students completed an 8-week study, where

0

0.2

0.4

0.6

0.8

1

0 10 20 30

ln (A

0/A)/[

C]

Irradiation time [min]

Uroporphyrin III

Coproporphyrin III

Protoporphyrin IX

72

toothbrushes with and without incorporated 450 nm blue LED were

compared, Figure 22.

The participants in the study were divided into two control groups and two

intervention groups. Persons that were smokers had used antibiotics less than

three month prior the study and/or

having periodontitis were excluded from

the study. Three clinical parameters

were chosen to be monitored during the

study: bleeding upon probing (BOP),

gingival index (GI) and plaque index

(PI). Several inflammation biomarkers

were also analyzed such as: Interleukin-

-6, IL -8, and tumor necrosis factor alpha in saliva and gingival

crevicular fluid.

After 8 weeks there were no statistical differences at level p=0.05 in BOP,

GI and PI, between the group using the 450 nm blue light toothbrush and the

group without blue light, Figure 23.

The overall trend is that the clinical parameters for the group using blue

light have decreased more from baseline than the control group. The

decrease in PI was significantly different from the control group at a

significance level of p=0.06.

When inflammation markers were analyzed in saliva and/or gingival

crevicular fluid, the concentrations were close to or below LOD for several

markers. Markers that could be detected and quantified were IL-1B and IL-8

in saliva, and MMP-8, and TIMP-1in gingival crevicular fluid. There was no

statistically significant difference in any of these markers between the

control and intervention group (p 0.44). However, within the intervention

group, inflammation markers were significant reduced after 8 weeks

Figure 22: Toothbrush, with the incorporated 450 nm LED turned on, used in the clinical study.

74

Conclusions and future perspectivesPorphyrins have been shown in a number of different studies to be a

promising class of compounds to use for inactivation of bacteria, either by

their photosensitizing abilities or by their biological importance. This field of

science, however, suffers from insufficient information about the

intracellular porphyrin content in bacteria in order to draw accurate

conclusions. Most of the research in aPT carried out today is focused on in

vitro illumination of bacteria and the results from the studies differ.

This thesis presents new analytical methods for accurate determinations

of porphyrins and heme, together with novel insights in the porphyrin

content in bacteria and also the connection between porphyrin content and

the bacterial sensitivity for blue light.

A method for determination of porphyrins in oral bacteria has been

developed and used to detect porphyrins in two oral pathogens, P. gingivalis

and A. actinomycetemcomitans. It was shown that both these pathogens

contained porphyrins in varying amounts and the porphyrin profile is highly

dependent on the culture conditions, such as time of culturing, passage, and

type of agar. At most the total porphyrin content increased 28 times, only by

performing a single passage of the bacteria. For obvious reasons cultivation

conditions could have an impact on aPT experiments if the photosensitizer

concentration varies to such a high degree. This could explain why

variations are observed in the results from aPT experiments and standardized

cultivation procedures are required to be able to draw accurate conclusions

from aPT experiments. However, there is a need for further investigations in

how the porphyrin profile in different pathogens differs depending on the

culture conditions.

In the aPT field there is not yet any consensus if any porphyrin species is

better as a photosensitizer than another. In fact, the porphyrin content has not

75

been able to be correlated to the photosensitivity for blue light. The relative

quantum yield for three naturally occurring porphyrins were determined and

it could be seen that there is quite a large difference in singlet oxygen

production when using a 405 nm light source. By using 5-ALA a change in

the porphyrin content of E. coli could be seen. E. coli is normally non-

sensitive to blue light, but after administration to 5-ALA the photosensitivity

increased considerably together with the porphyrin content. This shows that

porphyrins clearly affect the photosensitivity of bacteria. However, due to

the design of these experiments we were not able to draw any definite

conclusions of the contribution of the individual porphyrins on the

photosensitivity. It is not only the quantum yield of singlet oxygen that

defines how well a porphyrin acts as a photosensitizer inside bacteria. The

hydrophobicity will most likely determine where it is located. If a

photosensitizer is positioned close to biologically important systems it can

be an efficient photosensitizer even if it has a low quantum yield of singlet

oxygen. Interesting experiments can be carried out in the future where 5-

ALA is administered to bacteria at different periods of time. This will cause

different porphyrin profiles and aPT experiments can be performed to

determine the photosensitivity at these times. In this way it should be

possible to alter the porphyrin content in the same microorganism and the

photosensitivity could be correlated to a specific porphyrin.

It is of course important to perform in vitro studies to understand the basic

mechanisms of aPT experiments, but it is essential that aPT also work in

vivo. In the clinical study we could see a weak trend that the 450 nm LED

toothbrush possessed a phototherapeutic effect for three clinical indices. All

indices were decreased in the intervention group, but there were no

statistically significant difference compared to the control group. However,

four inflammation markers were significantly decreased in the intervention

76

group while only one decreased significantly in the control group. In future

studies a toothbrush with a LED emitting wavelengths close to 405 nm

should be used for better targeting the porphyrin absorbance. It could also be

possible to use high powered LEDs emitting light with higher intensity to

achieve a phototherapeutic effect.

Despite the biological importance of heme, there are no evaluated

analytical methods for the determination of heme in microorganisms. We

developed and evaluated an extraction and clean up method for heme using a

fast and simple salting-out LLE, solving one of the most crucial problems

with the determination of heme, aggregation. The method was developed for

the simultaneous determination of porphyrins and all analytes were baseline

separated within a runtime of 4 min using HPLC-MS/MS system. Addition

of a heme lookalike compound had high affinity for the heme acquiring

mechanisms of E. coli and decreased the intracellular content of heme. Since

heme is the major iron source for pathogens during an infection in

vertebrates, limiting the access for this compound could be promising for

future antimicrobial treatments. If one is allowed to speculate freely; the

heme acquiring mechanisms in pathogens may be used in future applications

to transport antimicrobial compounds across the bacterial envelope. By using

a compound that chemically looks like heme, “Trojan horse” compounds, it

could be possible to exploit the heme acquisition pathways to selectively

introduce antimicrobial compounds.

77

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HapPhy Days? A cohort study of the happiness during PhD studies Jonas Fyrestam*

A B S T R A C T

Objectives: This work aims to test the hypothesis that PhD students are more at risk of large fluctuations in the normalized happiness index.

Methods: A retrospective cohort study of one male PhD student born in 1981 was con-ducted. The normalized happiness index of the PhD student was monitored during five years and compared to a control group where none of the objects had been exposed to PhD studies.

Results: It could be seen that the normalized happiness index fluctuated significant (p=0.0001) for the PhD student when compared to the control group. However, area un-der a curve was well above the area over a curve.

Conclusions: These data suggest that suffering a PhD program will most likely cause high fluctuations in normalized happiness index. Mental stress during publication pro-cedures, failed experiments and thesis writing may well explain low normalized happi-ness index values while awesome colleagues and a magnificent working environment correlates well with high ones.

1. Introduction

It is commonly accepted that PhD stu-dents from the unit of analytical chemistry (ACESk) at Stockholm University are ex-traordinary. However, rarely is the happi-ness during the PhD studies mentioned after the successful dissertation proce-dure. Normalized happiness index (NHI) is a novel tool to estimate the happiness over time and was used in the present study.

2. Methods

This was a retrospective cohort study with no ethical approval at all.

2.1 Participants

All males (n=1) working at ACESk, during 2012-2017, whose body weight is most eas-ily measured in metric tons (0.1 ± 0.01)

were enrolled. In order to limit the num-ber of confounders only Swedish PhD stu-dents born in May 1981 were selected. To maintain the full integrity of the PhD stu-dent, he was anonymized with the code: JF_81. As a control group all other males in Sweden, not finished a PhD, were se-lected (n= 5 043 523).

2.2 Study design

JF_81 was first assigned a main supervi-sor with excellent skills in chromatog-raphy and analytical chemistry in gen-eral1. With a free hand and encouraging words he let JF_81 evolve as a researcher and as a person, something that he will forever be thankful.

Subsequently, an assistant supervisor was allotted to JF_812. Fruitful discus- 1 C. Östman 2 G. Thorsén

87

sions about research and teaching took place and JF_81 was always amazed by his broad knowledge in so many fields of science. JF_81 was placed in an office together with several other PhD students3. Due to them the working days for JF_81 became a real joy and he is grateful for their friendship and support. Additionally, all past and present PhD students and staff at ACESk made the working days so much easier to go through. Between the years 2012-2017 numer-ous questions and problems not related to research turned up for JF_81. Thanks to the administrative and technical staff4 these problems were easily solved. These persons, together with some others5, made the nutrition intake at 11:00 a.m. one of the highlights of the day. Regarding the research JF_81 had the pleasure to work with several highly skilled scientists and diploma workers6. Without them the scientific work would have been much more challenging. At the end of 2017, JF_81 had to summarize the past five years by writing the thesis. Due to the kindness and inval-uable comments from several people the manuscript was highly improved7.

2.3 Chemicals

Ethanol (purity between 2.1 to 95%) was used occasionally. Nicotine and caffeine was used on a daily basis and obtained from various manufacturers. Garlic (puri-

3 F. Mashayekhy, F. Idaresta, R. Avagyan and A. Saleh 4 L. Elfver and J. Rutberg 5 R. Westerholm, U. Nilsson, C. Östman and K. Lidén 6 N. Bjurshammar, E. Paulsson, N. Mansouri and J. Carlsson. 7 C. Östman, U. Nilsson, R. Westerholm, C. de Wit, L. Ilag, G. Thorsén, J. Aasa, F. Idaresta, R. Avagyan, E. Paulsson and H. Lim

ty > 99.9% was used the first half of the PhD studies).

2.4 Normalized happiness index

JF_81 was instructed to rank every work-ing day with a number between -100 to +100 where -100 correspond to low happi-ness (sadness) and +100 correspond to high happiness. By doing super advanced statistical treatment these numbers were converted into NHI values ranging from -1 to +1. JF_81 was also instructed to take notes on specific events that happened during the PhD studies. The control groups NHI values for each day were nor-malized to 0.

3. Results and discussion

In Figure 1 the NHI values during JF_81 PhD study is plotted together with some specific events that took place. Area under a curve (AUC) is highlighted in green, while area over a curve (AOC) is high-lighted in red. AUC is 63% and AOC is 37%, so most of the time JF_81 was happy compared to the control group. However, it is clearly visible that there are high fluc-tuations in NHI values. Most of the time JF_81 is very happy or very sad (NHI val-ues > 0.3 and < -0.3 respectively), rarely in between. These peaks and valleys corre-spond well with specific events that took place during the PhD study.

4. Conclusion

JF_81 wants to thank everybody at ACESk

for making this a fantastic time!

Acknowledgement

The Swedish research council and Elin Paulsson are acknowledged for financial support.