UNIVERSITATEA DE MEDICIN I FARMACIE

IULIU HAIEGANU CLUJ-NAPOCA

REZUMATUL TEZEI DE DOCTORAT


Anemia cronic inflamatoare
din Poliartrita Reumatoid:
rolul hepcidinei





Doctorand: Adriana Anton
Conductor de doctorat: Prof. Dr. Horaiu D. Boloiu
Cluj-Napoca 2014















2
CUPRINS
INTRODUCERE

STADIUL ACTUAL AL CUNOATERII
1. Sindromul anemic din poliartrita reumatoid 17
1.1. Poliartrita reumatoid consideraii generale 17
1.2. Sindromului anemic din poliartrita reumatoid 17
1.2.1. Investigaii de laborator n sindromul anemic 18
1.2.2. Principalele tipuri de anemie din poliartrita reumatoid 19
1.3. Evaluarea activiii bolii la pacienii cu poliartrit reumatoid 19

2. Anemia cronic inflamatoare din poliartrita reumatoid 20
2.1. Rolul fierului in homeostazia organismului 20
2.2. Mecanismele patogenetice implicate n anemia cronic
inflamatoare 22
2.2.1. Anomalii la nivelul homeostaziei fierului 22
2.2.2. Eritropoietina 22
2.2.3. Anomalii ale hematopoiezei 23

3. Hepcidina 25
3.1. Introducere 25
3.2. Scurt istoric al descoperirii hepcidinei 25
3.3. Structura hepcidinei 25
3.4. Rolul biologic al hepcidinei 26
3.5. Factorii care reglez secreia de hepcidin 26
3.6. Dozarea hepcidinei 28

CONTRIBUIA PERSONAL
1. Ipoteza de lucru/obiective 33
2. Studiul 1 -Evaluarea variaiei hepcidinei serice la pacienii cu
poliartrit reumatoid n funcie de prezena anemiei i corelaia sa
cu activitatea bolii 35
2.1. Introducere 35
2.2. Ipoteza de lucru/obiective 36
2.3. Material i metod 36
2.4. Rezultate 43
2.5. Discuii 63
2.6. Concluzii 71
3. Studiul 2 -Legtura dintre reactanii de de faz acut i scorul de
activitatea al bolii (DAS28) la pacieni cu Poliartrit reumatoid
73
3.1. Introducere 73
3.2. Ipoteza de lucru/obiective 73
3.3. Material i metod 74
3.4. Rezultate 77
3.5. Discuii 86
3.6. Concluzii 87
3
4. Concluzii generale 89
8. Originalitatea i contribuiile inovative ale tezei 91

BIBLIOGRAFIE 93

Cuvinte cheie: Hepcidina, poliartrit reumatoid, anemie cronic inflamatoare,
anemia secundar deficitului de fier, receptori solubili de transferina (sTFR),
reactani de faz acut (VSH, CRP), scorul de activitate al bolii DAS28
























4
Introducere
Anemia este o manifestare frecvent la pacienii cu poliartrit reumatoid
(PR). n majoritatea situaiilor anemia din PR este multifactorial (secundar
inflamaiei, deficitului de fier asociat, terapiilor medicamentoase). Anemia
cronic inflamatoare (ACI), i anemia secundar deficitului de fier sunt cele mai
frecvente tipuri de anemie prezente la pacienii cu PR, cel mai adesea existnd
concomitent.
Tabloul periferic este de multe ori identic n ACI i n ACI cu deficit de fier
asociat. Diferite valori ale feritinei au fost propuse ca prag, pentru aprecierea
deficitului de fier asociat n prezena inflamaiei, ns aceste valori nu sunt
unanim acceptate. Utilizarea receptorilor solubili de transferina (sTfR), a fost
propus n ultimii ani ca metod de diagnostic diferenial al deficitului de fier n
prezena inflamaiei, specificitatea acestui test fiind crescut de calcularea
raportului sTfR/logferitina (sTfR-F index), ns acest test nu este accesibil n
practica medical curent datorit costurilor ridicate. Explorarea mduvei
osoase, testul gold standard, care face diagnosticul diferenial, al deficitului de
fier asociat la pacienii cu ACI, este un test invaziv, efectuat rar n practica
medical curent.
Gsirea unor metode de diagnostic accesibile, ieftine i neinvazive pentru
diagnosticul ACI i respectiv al deficitului de fier asociat are o mportan
deosebit pentru practica medical actual. Recentele descoperii legate de
intervenia hepcidinei n patogenia ACI, deschid noi perspective legate de
diagnosticul i tratamentul acestor pacienii. n conformitate cu datele teoretice
aceasta ar putea face diagnosticul ACI i respectiv al deficitului de fier n
prezena inflamaiei.

Contribuia personal

Studiul 1. Evaluarea variaiei hepcidinei serice la pacienii cu
poliartrit reumatoid n funcie de prezena anemiei i corelaia sa cu
activitatea bolii.

Introducere
ACI din PR este rezultatul unui proces imun care este mediat de citockine.
Hepcidina este o protein reglatoare a metabolismului fierului sintetizat n
special n ficat
1
, sub forma unui precursor (pre-prohepcidin), compus din 86 de
aminoacizi
2
. Forma biologic activ (hepcidin 25), este secretat n plasm. Pn
n acest moment, au fost identificate dou izoforme, hepcidin20 (n ser i urin),
i hepcidin22 (urin), dar semnificaia biologic acestor izoforme nu este inc pe
deplin cunoscut
3
. Hepcidina acioneaz prin legarea de exportatorul celular al
fierului (ferroportin), determinnd degradarea i internalizarea acestuia,
rezultatul fiind sechestrarea fierului n macrofage, hepatocite i eritroblati
4
.
Anemia, hipoxia i inflamaia regleaz expresia hepcidinei
5
. ACI and anemia
secundar deficitului de fier sunt tipurile de anemie cele mai frecvente ntlnite
n PR, cel mai adesea coexistnd, fcnd diagnosticul diferenial extrem de dificil.
Examinarea mduvei osoase este considerat metoda gold standard pentru
diagnosticarea deficitului de fier asociat n prezena inflamaiei, dar fiind o
manoper invaziv nu este utilizat dect rar n practica medical curent.
Feritina, este utilizat ca marker al deficitului de fier n absena inflamaiei, dar
5
valori normale i chiar crescute ale feritinei nu exclud absena deficitului de fier
n prezena inflamaiei, feritina fiind un reactant de faz acut
6
. Att n ACI ct i
n anemia secundar deficitului de fier, concentraia seric a fierului i saturaia
transferinei (TfS%) sunt reduse, reflectnd deficiena absolut de fier din anemia
secundar deficitului de fier i hiposideremia secundar ncorporrii fierului n
macrofage din ACI. STfR-este forma trunchiat a receptorilor de transferin aflai
la suprafaa celulelor care necesit fier, iar concentraia lor seric nu este
crescut de inflamaie sau infecie, ca i n cazul feritinei
6
.
Ipotez de lucru/Obiective
Obiectivele principale ale acestui studiu au fost: evaluarea hepcidinei la
pacieni cu PR i diferite profile anemice i la un lot de control i corelaiile
acesteia cu parametrii profilul anemic, corelaiile hepcidinei cu reactanii de faz
acut (VSH, CRP) i scorul de activitate al bolii DAS28. Obiectiv secundar-
evaluarea profilului anemic, reactanii de faz acut i hepcidina n funcie de
valorile testului pentru depistarea sngerrilor digestive oculte (FOBT).
Material i metod
Subieci
n acest studiu prospectiv au fost nrolai 69 de subieci de ras
caucazian dintre care 54 de pacieni cu PR i 15 persoane sntoase n grupul
de control. Toi cei 54 de pacieni au ntrunit criteriile revizuite din 1987 ale
Colegiului American de Reumatologie pentru diagnosticul PR
7
iar subiecii din
lotul de control au fost recrutai din personalul spitalului, fr istoric de boal
inflamatorie. Criteriile de excludere au fost: macrocitoza, suplimentarea cu fier,
sngerarea ori transfuziile n ultimele trei luni. Afeciunile hepatice, renale,
endocrine, maligne, bolile limfoproliferative, precum i infeciile acute sau
cronice au fost criterii de excludere. Evaluarea clinic a inclus datele demografice
ale subiecilor (vrst, sex) i durata bolii pacienilor. Medicaia curent
(antiinflamatorii nesterioidine, sterozi, DMARD (difuse modyfing drug therapy)
i terapia biologic au fost notate pentru toi pacienii.
Probe
Toate probele de snge fost recoltate ntre 7:00 - 9:00 AM, pe nemncate
n aceeai zi n care s-a fcut examinarea articular. VSH (viteza de sedimentare a
hematiilor) i hemograma complet au fost efectuate n primele dou ore de la
recoltare, n timp ce pentru restul testelor serul a fost stocat la -80 de grade C.
Testele de laborator pentru evaluarea anemiei
Anemia a fost definit conform criteriilor din 1968 ale OMS (Organizaia
Mondial a Sntii). Brbaii care are au avut valori ale hemoglobinei (Hb)<13
mg/dl i femeile care au avut valori ale Hb<12mg/dl au fost considerai anemici
8
.
Hemograma complet, inclusiv Hb, Ht (hematocritul), numrul de leucocite i
trombocite au fost efectuate. Indicii eritrocitari: VEM (volumul eritrocitar
mediu), HEM (hemoglobina eritrocitar medie), CHEM (concentraia medie de
hemoglobina eritrocitar), RDW (distribuia de mrime a eritrocitelor) au fost
efectuate pe un analizor automat Abacus junior. Testul ELISA a fost folosit pentru
determinarea cantitativ a STfR i a hepcidinei, iar msurtorile au fost efectuate
pe Elisa Reader 230 S. Determinarea seric a hepcidinei a fost efectuat utiliznd
un kit comercial ELISA pentru hepcidin (Uscn Life Science Inc.Whuan, China),
iar determinarea concentraiei serice a sTfR s-a fcut utiliznd un test comercial
de la R&D Systems Inc (Minneapolis, SUA). Valorile STfR au fost convertite din
nmol/l n mg/l utiliznd greutatea molecular a STfR
9
. Dup cum a fost sugerat
6
de studiile anterioare, raportul dintre sTfR i log de feritin (sTfR-F index) a fost
utilizat pentru diagnosticul diferitelor profile anemice
10,11,12
, valoarea de 1.5
asTfR-F index, descris de studii anterioare
13,14,15
a fost utilizat pentru
caracterizarea profilului anemic la pacienii notri. Pacienii care au avut, sTfR-F
index <1.5 au fost clasificai n grupul ACI iar pacienii cu TfR-F index >1.5, au
fost clasificai n grupul ACI+deficien de fier. Valorile feritinei serice au fost
determinate prin metoda de electrochemiluminescen imunologic (ECLIA) pe
un analizor automat Elecsys1010. Determinarea cantivativ a fierului seric a fost
efectuat pe un analizor automat Cobas Mira plus, utiliznd metoda
colorimetric. Transferina (Tf) a fost determinat prin metoda nefelometric pe
un analizor semi-automat. Capacitatea total de legare a fierului (CTLF) i
(TfS%) au fost calculate utiliznd urmtoarele formule: CTLF (mol/L)=25.1 x Tf
(g/ L)
16
and TfS (%)=Fe(mg/L) x 70.9/Tf (g/L)
17
. Pacienii anemici au fost testai
pentru depistarea sngerrilor oculte FOBT, utiliznd un test imunologic rapid
(PreventID

, Preventis GmbH-Bensheim Germany).

Reactanii de faz acut
VSH-ul a fost determinat prin metoda Westergreen iar CRP-ul a fost
determinat prin nefelometrie, utiliznd un kit comercial Turbox CRP.
Scorul DAS 28
Urmtorii parametrii sunt inclui n formula matematic pentru
calcularea scorului DAS28: numrul articulaiilor tumefiate i dureroase, VSH
sau CRP, evaluarea global a activitii bolii de ctre pacient (scala analog
visual)
18
. n cadrul studiului nostru am folosit VSH-ul pentru calcularea scorului
DAS28.
Rezultate
Evaluarea profilului anemic al pacienilor cu PR
Din cei 54 de pacieni cu PR, 43 au fost femei (79.7%) i 11 brbai
(20.3%). Grupul de control a avut o distribuie similar, 13 femei (86.7%) and 2
brbai (13.3%). Din cei 54 de pacieni cu PR, 38 pacieni (70.4%) au avut
anemie (grupul anemic) i restul de 16 pacieni (29.6 %) au format grupul non-
anemic (NA). Pacienii din grupul anemic cu sTfR-F index <1.5 au fost clasificai
ca aparinnd grupului ACI (n=19), iar pacienii cu sTfR-F index >1.5 (n=19), au
fost considerai ca avnd deficit de fier asociat (grupul ACI+deficit de fier
asociat). Vrsta pacienilor a fost, 58.2614.24 ani pentru grupul anemic,
52.2515.96 ani pentru grupul NA i 5714.64 ani pentru grupul de control.
Durata bolii nu a fost diferit ntre diferitele subgrupe de pacieni cu PR
(10.6810.89 ani, pentru grupul anemic i 9.375.40 ani, pentru grupul NA
(p>0.05). n concordan cu clasificarea anemiei n funcie de valorile Hb
(anemie uoar (Hb=9.5-12g/dl), moderat (Hb=8-9.5g/dl), sau sever
(Hb<8g/dl)10, pacienii notri au prezentat anemie uoar. Chiar dac pacienii
din grupul ACI (n=19) au avut valori mai mari ale Hb dect pacienii din grupul
ACI+deficien de fier (n=19) (11.11.5 vs. 10.21.6), diferena nu a fost
semnificativ din punct de vedere statistic. Indicii eritrocitari (VEM, HEM,
CHEM), nu au prezentat valori semnificative statistic intre cele dou grupe de
pacieni anemici (ACI, ACI+deficien de fier) sugernd faptul c acetia nu pot fi
folosii n diagnosticul diferenial al deficitului de fier asociat la pacienii anemici
cu PR. Grupul ACI a avut valori mai crescute comparativ cu grupul ACI+deficien
de fier pentru urmtorii parametrii: fierul seric (59.936.7 vs 36.5, p=0.007),
feritina (176.815.1 vs. 523.2, p=0.002), Tf (3.40.6 vs. 3.70.7, p=0.04. Grupul
7
ACI a avut valori semnificative sczute ale fierului (59.936.7 vs. 82.945.8,
p=0.009) comparative cu grupul NA, n timp ce valorile Tf nu au fost diferite,
TfS% a fost semnificativ crescut n grupul ACI. Nu s-au evideniat diferene
semnificative ale parametrilor caracteristici metabolismului fierului pentru
grupele (ACI vs. Control) i (NA vs. Control). n cadrul studiului nostru, dei
indicii eritrocitari i RDW nu au fost diferii din punct de vedere al semnificaiei
statistice pentru cele dou grupe anemice (ACI i ACI+deficien de fier),
parametrii clasici folosii n determinarea deficitului de fier au prezentat valori
diferite din punct de vedere statistic.
Analiza valorilor hepcidinei n populaia studiat
Testul Anova efectuat a artat c ntre cele 4 grupuri de studiu (grupul
ACI, grupul ACI+deficien de fier, grupul NA, grupul de control), exist diferene
statistic semnificative pentru un nivel de risc acceptat de 5%, p<<0.05. Mediile
hepcidinei difer statistic, pentru urmtoarele perechi de grupe ACI-NA
(p=0.006), ACI-Control (p<0.001), ACI+deficien de fier-NA (p=0.002),
ACI+deficient de fier-Control (p<0.001). De remarcat faptul c, pacienii din
grupele anemice au valori supraunitare ale hepcidinei, n timp subiecii din
grupul NA i grupul de control au valori subunitare ale valorilor serice ale
hepcidinei. Pacienii anemici au valori semnificativ crescute ale hepcidinei
comparativ cu pacienii din grupul NA i grupul de control. Compararea celor
dou grupe de pacieni anemici, ACI i ACI+deficien de fier a evideniat faptul
c, dei grupul ACI prezint valori serice ale hepcidinei mai crescute comparativ
cu grupul ACI+deficien de fier, acest diferen, nu a atins pragul semnificaiei
statistice (p=0.85). Nu s-au observat diferene ntre grupul NA i grupul de
control.
Valorile medii ale Hb pacienilor cu PR sunt mai mici comparativ cu
grupul de control (11.51.94 vs. 14.11.1), aceeai situaie fiind prezent i
pentru fierul seric (58.637.8 vs. 77.3 22). Valorile medii ale hepcidinei serice
sunt mai mari comparativ cu grupul de control (1.000.62 vs. 0.450.19). Analiza
corelaiilor hepcidinei cu variabilele studiate evideniaz prezena unor corelaii
negative, semnificative ntre hepcidin i Hb (R=-459, p=0.000<0.05), i
respectiv hepcidin i fier (R=-357, p=0.004<0.05), pentru un nivel de risc de
acceptat 5% la pacienii cu PR. Pentru grupul ACI+deficien de fier, s-au
evideniat corelaii bune, semnificative statistic ntre hepcidin i TfS% (R=-0.46,
p<0.05) i ntre hepcidin i fier. ntre hepcidin i fier a existat o dependen
invers proporional, cu o corelaie bun (R=-0.43), semnificativ statistic
(p=0.04). Pentru grupul NA i grupul de control, nu s-au evideniat valori
semnificative statistic. De menionat c, nu exist diferene statistic ntre valorile
serice ale hepcidinei ntre grupul NA i grupul de control.
Corelaia hepcidinei cu reactanii de faz acut (VSH, CRP) i scorul de
activitate al bolii DAS 28
Analiza corelaiilor dintre hepcidin i reactanii de faz acut (VSH, CRP),
pentru pacienii cu PR a artat faptul c hepcidina se coreleaz pozitiv cu VSH-ul
(R=0.352), semnificativ statistic (p=0.009<0.05) precum i cu CRP, (R=0.369
p=0.006<0.05) i scorul DAS28. Valorile VSH-ului i ale CRP-ului, precum i ale
scorului DAS 28 nu au fost diferite statistic ntre cele dou grupe anemice ACI i
ACI+deficien de fier. Valori semnificative statistic ale VSH-ului, s-au nregistrat
ntre grupele cu anemie (ACI i ACI+deficien de fier) i grupul NA. Pentru
grupul NA s-a observat c hepcidina se corelaz bine, pozitiv (R=0.515) cu VSH,
8
(p=0.041<0.05). Pentru lotul de control nu s-au evideniat existena unor
corelaii dintre hepcidin i reactanii de faz acut (VSH, CRP)
Rezultatele testelor pentru determinarea sngerrilor digestive oculte
(FOBT)
Din cei 38 de pacieni cu anemie, 34 de pacieni au fost testai pentru
depistarea sngerrilor oculte. Din cele 34 de teste, 9 pacieni au fost testai
pozitiv, restul de 25 de pacieni au fost testai negativ. Pacienii care au avut teste
negative au avut valoarea medie a fierului mai mare dect cei cu valori pozitive
(55.5229.95 vs. 32.6717.25), (p=0.039). Valori semnificative statistic s-au
nregistrat pentru sTfR (2.561.03 vs. 3.481.15) (p=0.033) i TfS (11.435.75
vs. 7.073.92) (p=0.044). Dei valorile medii ale VSH-ului i CRP-ului au fost mai
ridicate n grupul de pacieni cu test pozitiv, comparativ cu pacienii care au avut
test negativ, aceste valori nu au fost statistic semnificative. Ceilali parametrii
studiai (Hb, Ht, VEM, HEM, CHEM, RDW, Tf, CTLT) nu au fost modificai
semnificativ ntre cele dou grupe de pacieni. Rezultatele obinute de noi
sugereaz c faptul c n condiiile existenei unor semnale conflictuale in ceea ce
privete reglarea sintezei de hepcidin, inflamaia este elemental dominant.
Discuii
Hepcidina reactant de faz acut
Informaiile referitoare la hepcidina din literatura ultimilor ani au crescut
exponeial, dar numrul studiilor care au determinat valoarea hepcidinei la
pacieni cu PR este nc foarte mic, numrul pacienilor pe care s-au efectuat
aceste studii este de asemenea sczut, iar rezultatele raportate sunt
contradictorii.
Un studiu din 2008 care a studiat valorile pro-hepcidinei la pacieni cu PR
i Lupus eritematos systemic (LES) a artat c valorile acesteia nu s-au corelat cu
scorurile de activitate ale bolii, valorile Hb, i nici cu parametrii care
caracterizeaz metabolismul fierului ori nivelul citokinelor
19
, n timp ce un
studiu efectuat pe pacieni cu boli inflamatorii intestinale a artat c s-au
observat corelaii semnificative ntre hepcidin i feritin i ntre prohepcidin i
feritin, iar valorile hepcidinei s-au corelat cu activitatea bolii, cu CRP, valorile
hepcidinei i prohepcidinei fiind semnificativ crescute comparativ cu grupul de
control
20
. Alte studii care au explorat relaia dintre CRP i hepcidin pe diferite
categorii de pacieni, pacieni care au suferit intervenii chirurgicale la ficat i
respectiv pacieni care faceau hemodializ nu au observat nici o corelaie ntre
CRP i valorile hepcidinei
21,22
. Studiul nostru care a fost efectuat pe pacieni cu
PR cu funcie renal i hepatic normal a artat existena unei corelaii
semnificative statistic ntre reactanii de faz acut (VSH, CRP), similar cu
rezultate raportate anterior
23
.
Corelaia hepcidinei cu scorul de activitate al bolii -DAS28
n cadrul studiului nostru a fost observat o corelaie pozitiv ntre
hepcidin i scorul de activitate al bolii DAS28 pentru pacienii cu PR, observaie
facut i de alte studii anterioare
23
.
Hepcidina i intervenia acesteia n homeostazia fierului n PR
Hepcidina s-a corelat negativ cu fierul pentru lotul PR, grupul anemic i
pentru grupulACI+deficien de fier. Nu s-au observat corelaii ntre hepcidin i
fier la pacienii din grupul de control. Aceste observaii sugereaz faptul c
prezena inflamaiei din PR joac un rol important in homeostazia fierului, prin
intermediul hepcidinei.
9
Analiza hepcidinei pe loturile de studiu
Analiza valorilor serice ale hepcidinei pentru loturile de studiu, a
evideniat diferene semnificative statistic ntre valorile hepcidinei la pacienii cu
ACI i pacienii cu ACI+ deficien de fier comparativ cu lotul de control. De
asemenea exist o diferen statistic semnificativ ntre pacienii cu diferitele
tipuri de anemie i pacienii cu PR far anemie. Testul Elisa folosit de noi nu a
evideniat nici o diferen semnificativ statistic grupele ACI i ACI cu deficit de
fier asociat. Consttrile noastre sunt similare cu cele prezentate de un studiu
anterior care nu a putut s separe diferitele subtipuri de anemie pe baza
concentraiei serice a hepcidinei
24
. Rezultate contrare celor prezentate de noi au
fost prezentate de alt studiu, unde valorile prohepcidinei au fost semnificav
sczute la pacienii cu deficien de fier asociat inflamaiei cronice la pacienii
cu PR
25
.
Concluzii
1. Pacienii cu ACI i respectiv ACI+deficien de fier, au valori mai crescute ale
hepcidinei comparativ cu subiecii din grupul NA i grupul de control;
2. Pacienii cu ACI au valorii medii ale hepcidinei mai mari dect pacienii cu
ACI i deficit de fier asociat, dar acest acesta diferen nu a fost semnificativ
din punct de vedere statistic;
3. Hepcidina seric se coreleaz semnificativ statistic, invers proportional, cu
valorile Hb i a fierului seric la pacienii cu PR;
4. Hepcidina este un reactant de faz acut;
5. Hepcidina se coreleaz cu reactanii de faz acut (VSH, CRP) i scorul de
activitate al bolii DAS28;
6. n prezena unor semnale concomitente i contradictorii n reglarea
hepcidinei, inflamaia este elementul dominanat.

Studiul 2. Legtura dintre reactanii de faz acut (VSH, CRP) i scorul de
activitatea al bolii (DAS28) la pacieni cu Poliartrit reumatoid.

Introducere
Anemia, manifestare extra-articular frecvent n PR, este rezultatul
producerii deficitare a eritropoietinei, scderii aciunii acesteia la nivelul
mduvei osoase, a eliberrii deficitare a fierului la nivelul celulelor sistemului
reticulo-endotelial, secundar pierderilor de snge la nivel gastrointestinal,
precum i a toxicitii medicaiei imunosupresive. n practica clinic, CRP i VSH
sunt utilizate pentru monitorizarea activitii bolii. VSH-ul poate fi considerat
mai puin specific n msurarea rspunsului de faz acut dect CRP, pentru c
este influenat de diferii factori alii dect inflamaia sistemic, inclusiv vrsta,
sexul, concentraia Hb, valoarea imunoglobulinelor serice i a factorului
rheumatoid
26
. Scorul DAS28 este un test simplu i obiectiv pentru determinarea
activitii bolii, monitorizarea activitii bolii i rspunsul la terapie. HAQ-DI este
un test pe care pacientul il completeaz singur i pe baza rezultatelor obinute se
face o apreciere asupra impactului pe care boala l are asupra strii de sntate i
a calitii vieii
27
.
Ipoteza de lucru/Obiective
Obiectivul principal al acestei cercetri a fost studierea reactanilor de
faz acut (VSH, CRP) la un grup de pacieni cu PR cu sau far ACI, i a corelaiile
reactanilor de faz acut cu scorul DAS28 precum i cu testele autoadministrate
10
de ctre pacient (HAQ-DI) pentru determinarea efectului pe care boala asupra
calitii vieii pacientului. Obiectivul secundar a fost evaluarea corelaiilor dintre
reactanii de faz acut (VSH, CRP) i feritina.
Material si metod
n acest studiu au fost nrolai 54 de pacieni cu PR, cu vrsta peste 18 ani,
care au ntrunit criteriile de diagnostic pentru PR din 1987 ale Colegiului
American de Reumatologie
7
. Anemia a fost definit ca fiind reprezentat de
concentraia Hb<12 mg/dL pentru femei i a Hb <13 mg/dL pentru brbai.
Pacienii au avut anemie normocrom, normocitar sau anemie microcitar.
Macrocitoza, alte tipuri de anemie, hemoragia recent, afeciunile hepatice,
renale, antecedente de afeciuni maligne, infecii acute i cronice au fost
considerate criterii de excludere. Sngele a fost colectat pe nemncate, ntre 7.00
AM-9.00 AM. VSH-ul i hemoleucograma complet au fost efectuate n aceeai zi,
serul pentru restul testelor fiind separat i depozitat la -20C pentru
determinarea ulterioar.VSH-ul a fost determinat prin metoda Westergen iar
CRP a fost determinat prin nefelometrie utiliznd Turbox CRP Orion.
Determinarea feritinei serice a fost efectuat utiliznd un kit de feritin, pe un
aparat Elecsys 2010. Scorul DAS28 a fost calculat folosind urmtoarele variabile-
numrul articulaiilor dureroase i tumefiate, VSH, i evaluarea pacientului
asupra activiii bolii (VAS, mm)
18
. HAQ-DI ntreab pacienii despre capacitatea
lor de a efectua diferite activiti precum i dac au nevoie de ajutor pentru
efectuarea lor.
Rezultate
n acest studiu au fost nclui 54 de pacieni cu PR. n funcie de
prezena/absena anemiei pacienii au fost clasificai n grupul anemic i
respective grupul non-anemic (NA). Nu s-au nregistrat diferene semnificative
statistic ntre vrsta pacienilor i durata bolii ntre grupul anemic i grupul NA.
Analiza parametrilor studiai pentru grupele anemic i NA a evideiat faptul c
exist o diferen semnificativ a valorilor VSHului (p = 0.001 < 0.05), CRPului
(p=0.008<0.5), i a scorului DAS 28, dar nu s-au observat diferene semnificative
pentru feritina i HAQ-DI.O corelaie pozitiv puternic s-a observant ntre
scorul DAS28 i CRP (R=0.417, p=0.009<0.01) n grupul anemic. Trendul
reprezentat de norul de puncte care reprezint CRP i DAS28 este cel mai bine
descris de o funcie cubic. In grupul NA, s-a observant o corelaie pozitiv
puternic ntre scorul DAS28 i VSH ((R = 0.724, p = 0.002 < 0.001), statistic
semnificativ, i o corelaie semnificativ ntre HAQ-DI and ESR (R = 0.621, p =
0.01<0.05). Pentru aceste dou perechi de variabile s-a studiat funcia de
regresie care exprim dependena lor i s-a observant c exist o dependen
liniar ntre VSH i DAS28 (coeficient de determinare = 0.525) i de asemenea
ntre VSH i HAQ-DI (coeficient de determinare = 0.386). Analiza corelaiilor
colaterale dintre reactanii de faz acut (VSH i CRP) i feritina pentru lotul
anemic evideniat faptul c exist o corelaie bun cu CRP, (R=0.532), puternic
semnificativ statistic p=0.001<0.05, precum i o corelaie slab cu VSH,
(R=0.304), semnificativ statistic, p=0.043<0.05. Funcia de regresie care
exprim legtura dintre CRP-feritin este destul de bine determinat, R2=0.29.
Pentru lotul NA analiza corelaiilor dintre reactanii de faz acut (CRP i VSH) i
feritin a artat c feritina se corelaz foarte bine, pozitiv cu VSH (R=0.751),
semnificativ statistic (p=0.001<0.05). Dreapta de regresie descrie o bun
aproximare a tendinei legturii dintre cele doua mrimi (coef de determinare
11
R2=0.564). Feritina se corelaz foarte bine, pozitiv cu CRP (R=0.841),
semnificativ statistic (p<<0.05). Dreapta de regresie descrie o bun aproximare a
tendinei legturii dintre cele dou mrimi (coef de determinare R2=0.708).
Discuii
Corelaii foarte puternice pozitive s-au inregistrat ntre scorul DAS28 i
CRP pentru grupul anemic i DAS28 i VSH pentru grupul NA. Corelaii pozitive
ntre scorul DAS28 i CRP i VSH, confirm constatri fcute de studii
anterioare
28,29,30
. De remarcat corelaiile feritinei cu reactanii de faz acut
(VSH, CRP) pentru pacienii din grupul anemic i grupul NA. Valorile feritinei
serice se coreleaz cu rezervele de fier n condiii normale precum i la pacienii
cu deficiene de fier sau n condiiile existenei unei exces de fier n organism, dar
feritina este i un reactant de faz acut care crete n diferite infecii, inflamaii
precum i n diferite cancere
31
. La pacienii din cadrul studiului nostru toate
aceste afeciuni au fost criterii de excludere astfel nct putem spune c valorile
crescute ale feritinei la pacienii notri sunt datorate exclusiv inflamaiei
secundare boli reumatologice. HAQ-DI, nu a fost semnificativ diferit pentru
pacienii anemici i cei din grupul NA, constare care pledeaz pentru faptul c
aceste teste sunt subiective n ceea ce privete acvititate bolii, acestea trebuie
ntotdeuna nsoite de parametrii obiectivi (VSH, CRP) cnd se fac aprecieri
asupra activitaii bolii la pacieni cu PR cu sau far anemie. Importana testelor
obiective (VSH, CRP) folosite pentru diagnosticul i monitorizarea activitii
pacienilor cu PR este susinut i de noile criterii pentru diagnosticul PR
aprobate n 2010 de Colegiul American de Reumatologie i Liga European
contra Reumatismului
32
, aceste teste fcnd parte dintre noile criterii de
diagnostic.
Concluzii
1. Pacienii din grupul anemic au boal mai activ comparativ cu pacienii din
grupul NA;
2. Valorile HAQ-DI din grupul anemic nu au diferite semnificativ comparativ cu
pacienii din grupul NA;
3. Testele autoadministrate de ctre pacient (HAQ-DI), trebuie ntotdeuna
nsoite de teste obiective pentru aprecierea activitii bolii;
4. Feritina, reactant de faz acut, se coreleaz cu CRP i VSH la pacienii cu PR,
independent de statutul lor anemic.
Concluzii generale
1. Pacienii cu PR i anemie au boal mai activ comparativ cu pacienii cu PR
fr anemie;
2. Testele autoadministrate de ctre pacient (HAQ-DI) nu sunt semnificativ
diferite ntre pacienii anemici i non-anemici, fiind necesar ca aceste teste s
fie ntotdeuna nsoite de teste obiective pentru aprecierea activitii bolii;
3. Hepcidina este un reactant de faz acut;
4. Hepcidina se coreleaz cu reactanii de faz acut (VSH, CRP) i scorul
DAS28;
5. Pacienii cu PR i anemie prezint valori crescute ale hepcidinei comparativ
cu pacienii far anemie i subiecii din lotul de control;
6. Indicii eritrocitari, RDW i severitatea anemiei nu pot fi utilizai n dignosticul
deficitului de fier asociat la pacienii cu ACI;
7. Hepcidin se coreleaz cu Hb i fierul la pacienii cu PR;
12
8. Pacienii cu ACI au tendina de a avea valori mai crescute ale hepcidinei
comparativ cu pacienii cu ACI+deficit de fier asociat , dar determinarea
seric a hepecidinei efectuat de noi nu a putut face diagnosticul diferenial al
deficitului de fier asociat la pacienii cu ACI;
9. n condiiile existenei unor semnale concomitente conflictuale n reglarea
hepcidinei, inflamaia este stimulul dominant.
Originalitatea i contribuiile inovative ale tezei
Informaiile despre hepcidin i rolul acesteia n homeostazia fierului n
organism n condiii normale i patologice sunt foarte numeroase n literature
ultimilor ani i cu toate acestea datele obinute n urma unor studii clinice pe
pacieni sunt relativ puine, iar numrul studiilor care au determinat valorile
hepcidinei i au incercat s fac o analiz a acestora la pacienii cu PR i ACI i la
un lot de control sunt i mai puine, uneori cu rezultate contradictorii.
Originalitatea acestei teze este dat de tematic abordat, modul n care acest
tez a fost conceput precum i de concluziile obinute. Hepcidina a acionat n
studiul nostru ca un reactant de faz acut, pacienii anemici avnd valori mai
crescute, comparativ cu grupul non-anemic i grupul de control. Chiar dac
aceast cercetare nu a putut s demonstreze c hepcidina poate s fac
diagnosticul diferenial al deficitului de fier n prezena inflamaiei, noile
generaii de teste standarizate precum i stabilirea unor protocoale speciale
legate de recoltarea i pstrarea probelor biologice vor reui s fac asta n
viitorul apropiat. Unele aspecte legate de acest cercetare, au fost publicate
pentru prima data n literatur ca urmare a acestei cercetri. Concluzia original
obinut n urma studierii valorilor hepcidinei la pacienii cu diferite profile
anemice, este c n prezena unor semnale concomitente i contradictorii legate
de reglarea hepcidinei inflamaia este elementul dominat. Rezultatele obinute
de noi n urma acestei cercetri se adaug informaiilor relativ puine publicate
pn n acest moment care au studiat hepcina la pacieni cu PR, impreun cu
acestea aducnd argumente legate utilitatea practic n viitor a determinrii
hepcidinei la pacienii cu PR i ACI.














13
IULIU HATIEGANU UNIVERSITY OF MEDICINE AND
PHARMACY, CLUJ-NAPOCA






ABSTRACT OF THE DOCTORAL DISSERTATION

The Chronic Inflammatory
Anemia in Rheumatoid
Arthritis: the hepcidin role






Doctoral Candidate Adriana Anton
Scientific Coordinator Prof. Dr. Horaiu D. Boloiu
Cluj-Napoca 2014
















14
TABLE OF CONTENTS
INTRODUCTION



CURRENT STATE OF KNOWLEDGE

1. Anemic syndrome in Rheumatoid Arthritis
17
1.1. Rheumatoid arthritis general considerations
17
1.2. Clinical features of anemic syndrome in rheumatoid arthritis
17
1.2.1. Laboratory tests investigation in anemic syndrome
18
1.2.2. Types of anemia in rheumatoid arthritis
19
1.3.Evaluation of disease activity in patients with rheumatoid arthritis and anemia
19

2. Chronic inflammatory anemia in rheumatoid arthritis
20
2.1. The role of iron in the body homeostasis
20
2.2. Pathogenesis of chronic inflammatory anemia
22
2.2.1. Iron homeostasis abnormalities
22
2.2.2 Erythropoietin
22
2.2.3.Hematopoiesis abnormalities
23

3. Hepcidin
25
3.1. Introduction
25
3.2. Short history of hepcidin discovery
25
3.3. Hepcidin structure
25
3.4. The biological role of hepcidin
26
3.5. Factors that regulate hepcidin secretion
26
3.6. Hepcidin determination
28

PERSONAL CONTRIBUTION

1. Hypothesis/objectives
33
2. Study 1 - Evaluation of serum hepcidin variation in patients
with rheumatoid arthritis according to the anemia profile and its
correlation with disease activity
35
2.1. Introduction
35
2.2. Hypothesis/objectives
36
2.3. Material and methods
36
2.4. Results
43
2.5. Discussions
63
2.6. Conclusions
71
3. Studiul 2 - The relationship between the acute-phase
reactants (ESR, CRP) and disease activity score (DAS28) in patients
with RA.

73
3.1. Introduction
73
3.2. Hypothesis/objectives
73
3.3. Material and methods
74
15
3.4. Results
77
3.5. Discussions
86
3.6. Conclusions
87
4. General conclusions
89
8. The originality and the innovative contributions of the
thesis 91


REFERENCES
93

Key words: Hepcidin, rheumatoid arthritis, anemia of chronic inflammation, iron
deficiency anemia, solubile transferring receptors (sTFR), acute phase reactants
(ESR, CRP), disease activity score (DAS28)



































16
Introduction
Anemia is a common morbidity in patients with rheumatoid arthritis (RA).
Anemia of chronic disease (ACD), also called anemia of inflammation (ACI) that
develops in RA patients, is an immune-mediated process that involves cytokine
synthesis. ACD and anemia secondary to iron deficiency (IDA) are the most
common types of anemia present in RA patients and often occur concurrently.
Peripheral picture is often identical in ACD and ACD associated with iron
deficiency. Ferritin is used as a marker of iron deficiency but even normal or
increased values do not exclude the presence of iron deficiency, ferritin being an
acute phase reactant, which increases during inflammatory process. Using of
soluble transferrin receptor (sTfR) has been proposed in recent years as a
method of differential diagnosis of iron deficiency in the presence of
inflammation. The specificity of the test is increased by calculating the ratio
sTfR/logferitina (sTfR -F index), but this test is not available in clinical practice
due to high costs. Bone marrow biopsy is considered, the gold standard method
for diagnosing of IDA in the presence of inflammation but has disadvantage of
being an invasive and expensive diagnostic tool.

Hepcidin intervention in the pathogenesis of ACD has opened new perspectives
for the diagnosis and treatment of patients with RA. According to some data
hepcidin could made the diagnosis of IDA in the presence of inflammation. In our
research we present the results of two studies that have studied different aspects
of ACI in patients with RA.

Personal contribution

Study 1. Evaluation of serum hepcidin variation in patients with
rheumatoid arthritis according to the anemia profile and its correlation
with disease activity

Introduction
ACD is the result of immune response that is mediated by citockine. Hepcidin is
an iron regulatory protein produced mainly in the liver
1
, as a precursor (pre-
prohepcidin) consisting of 86 amino acids
2
. Biologically active form (hepcidin
25) is secreted into the plasma. At the present, two isoforms have been
identified, hepcidin20 (serum and urine), and hepcidin22 (urine), but the
biological significance of these two forms is not clear
3
. Hepcidin acts by binding
to cellular iron exporter (ferroportin), inducing its internalisation and
degradation, resulting in trapping of iron in macrophages, hepatocytes and
erythroblasts
4
. Anemia, hypoxia and inflammation regulate hepcidin gene
expression
5
. ACD and IDA are frequently associated with PR and, although
similar, their pathogenetic mechanism is different and sometime difficult to
differentiate. Moreover, ACD may coexist with IDA. The examination of bone
marrow is considered the "gold standard" for diagnosing IDA the presence of
inflammation, but being an invasive test is rarely used in clinical practice.
Ferritin is used as a marker of iron deficiency but even normal or increased
values do not exclude the presence of iron deficiency, ferritin being an acute
phase reactant
6
. In both the ACD and IDA, serum iron and transferrin saturation
(TfS) are reduced, reflecting iron deficiency anemia secondary to absolute iron
17
deficiency from IDA and hypoferremia secondary to iron incorporation in
macrophages. STfR - is a truncated form of the tissue transferrin receptor
present on the surface of cells that require iron, and their concentration is not
increased by of inflammation or infection, unlike ferritin
6
.
Objectives
The main objectives of this study were: to assess serum hepcidin levels in
patients with PR and various anemia profiles and controls and its correlation
with parameters of anemic profile and hepcidin correlations with acute phase
reactants (ESR, CRP) and DAS28 score. Secondary objective assessment of
anemic profile, acute phase reactants and serum hepcidin levels based on tests
for occult digestive bleeding.
Material and Methods
Subjects
In this prospective study were enrolled 69 Caucasian subjects of which, 54
patients with RA and 15 healthy subjects (control group). All 54 patients met the
1987 revised criteria of the American College of Rheumatology for the diagnosis
of PR
7
, and control group subjects were recruited from hospital staff, without any
history of inflammatory disease. Exclusion criteria were: macrocytosis, blood
transfusions and iron supplementation in the last 3 months and recent acute
blood loss. Moreover, patients were excluded if they had any signs of symptoms
of renal, hepatic, endocrine, hematological, lymphoproliferative or malignant
disease. Patients with acute or chronic infections were also excluded. The same
exclusion criteria were applied for control group. Clinical assessments included
demographics (age, gender) and disease duration of patients, current medication
(anti-inflammatory nesterioidine, steroids, DMARDs (Difuse modyfing drug
therapy) and biological therapy were noted for all patients.
Probes
All blood samples were collected between 7:00-9:00AM, on the same day when
clinical and joint examination was made. ESR (erythrocyte sedimentation rate)
and complete blood counts were performed within two hours of collection the
blood. For the rest of the tests, the serum was stored at -80 C for subsequent
testing.
Laboratory tests for the assessment of anemia
Anemia was defined according to WHO (World Health Organization) criteria
from 1968. Males with hemoglobin values (Hb) < 13 mg / dl and women with Hb
values of < 12mg/dl were considered anemic
8
. Complete blood count including
Hb, Ht (hematocrit), WBC and platelet counts were performed. Red cell indices
such as: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin),
MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution
width) were performed on an automatic analyzer Abacus junior. ELISA was used
for the quantitative determination of sTfR and hepcidin, and the measurements
were carried out on ELISA Reader 230 Determination of serum hepcidin levels
was carried out using a commercial ELISA kit for hepcidin (USCN Inc. Whuan Life
Science, China). sTfR serum determination was performed by using
commercially available kit from R & D Systems Inc. (Minneapolis, USA). STfR
values were converted from nmol/l to mg/STfR it using molecular weight of
sTfR
9
.

18
As suggested by previous studies, the ratio of sTfR and ferritin log (sTfR - F
index) was used for the diagnosis of various anemic profiles in our
patients
10,11,12
. A cut-off of 1.5 for sTfR-Findex as described by previous
studies
13,14,15
was used. Patients with sTfR -F index <1.5 were classified as ACD
group and patients with sTfR -F index> 1.5 were classified as ACD+IDA group.
Serum ferritin values were determined by electrochemiluminescence
immunoassay on an automatic analyzer Elecsys1010 using a commercial kit.
Quantitative serum iron determination was carried out on a Cobas Mira
automated analyzer plus using the colorimetric method. Transferrin (Tf) was
determined by nephelometric method using a Transsferin Turbox kit on a semi-
automatic analyzer. The total iron binding capacity (TIBC) and (TfS%) were
calculated using the following formulas: TIBC (micromol/L) = 25.1 x Tf (g/L)
16

and TfS% (%)=Fe (mg/L) x 70.9/Tf (g/L)
17
. Anemic patients were tested for fecal
occult bleeding (FOBT) using a rapid immunoassay.
Acute phase reactants
ESR was determined by the Westergreen and CRP was determined by
nephelometry using a commercial kit Turbox CRP.
DAS 28 score
DAS 28 score is used in medical practice and clinical research to evaluate disease
activity. 28 specific joints that are most commonly affected in PR are examined.
The number of swollen and tender joints count, ESR or CRP, patients global
assessment of disease activity (visual analogue scale) are introduced in a
mathematic formula for score calculation
18
. In our study we used ESR to
calculate the DAS28 score.
Results
Assessment profile anemic patients with PR
In the RA group (n=54), 43 women (79.7%) and 11 (20.3%) were enrolled. The
control group had a similar distribution 13 women (86.7%) and 2 males
(13.3%). In the RA group, 38 patients (70.4%) had anemia (anemic group) and
the rest 16 patients (29.6%) did not, the non-anemic (NA). Patients from the
anemic group with sTfR-F index<1.5, were sub-classified as ACD group (n=19)
and patients with sTfR-F index> 1.5 (n=19) were sub-classified as ACD+ IDA
group. The age of patients age were 58.26 14.24 for anemic group, 52.25
15.96 for NA group and 57 14.64 for the control group. Disease duration was
not different between different subgroups of patients with RA (10.68 10.89
years for anemic group 9.37 5.40 years for the NA group (p > 0.05). According
to the classification of anemia based on Hb values (mild anemia (Hb=9.5-12g/dl),
moderate (Hb=8-9.5g/dl), or severe (Hb<8g/dl) 10, our patients had mild
anemia. Even though patients in the ACD group (n=19) had higher Hb values
than patients from ACD+IDA group (n=19) (11.110.21.6 1.5 vs), the difference
was not statistically significant. In ACD +IDA group Hb values were not normally
distributed. Red cell indices indices (MCV, MCH, MCHC) were not different
between ACD and ACD+IDA groups, suggesting that these cant be used as a
differential diagnosis of iron deficiency in anemic patients with PR. The ACD
group had higher values compared to ACD+IDA group for following parameters:
serum iron (59.936.7 vs 36.5, p=0.007), ferritin (176.815.1 vs. 523.2,
p=0.002), Tf (3.40.6 vs 3.70.7, p=0.04). The significant values were observed
for parameters (iron, ferritin, TfS%, CTLF, sTfR) when ACD+IDA group the group
was compared with ACD and NA groups. There were not statistically significant
19
differences in iron metabolism parameters for following groups (ACD vs Control)
and (NA vs. Control). Although, red cell indices and RDW were not different in
terms of statistical significance for ACD and ACD+IDA groups, classical
parameters used in determining iron deficiency showed statistically different
values.
Hepcidin analysis in the studied population
Anova test showed that among the four study groups (ACD, ACD+IDA, NA, and
the control group), there were statistically significant differences to an
acceptable risk level of 5%, p<<0.05. ACD and ACD+IDA groups had significantly
higher serum hepcidin concentrations than controls (p<0.001, p<0.001) and NA
group (p=0.006, p=0.002).No difference was observed between ACD and
ACD+IDA groups (1.180.68 vs1.150.51, p=0.85) and between NA group and
controls (0600.48 vs0.450.19, p=0.66).
The mean of serum hepcidin values were higher compared to the control group
(1.000.620.19 vs. 0.45), and Hb levels for RA patients and serum iron were
lower than controls (11.51.94 vs. 14.11.1), (58.637.8 vs. 77.322). In RA
group (N=54), the serum hepcidin concentrations correlated significantly
negatively with Hb (R=-459, p=0.000<0.05) and serum iron (R=-357,
p=0.004<0.05). In ACD+IDA group there was a good correlation between
hepcidin and TfS% (R=-0.46, p<0.05) and between hepcidin and iron (R=0.43,
p=0.04). No correlations were observed for NA and Controls groups.
Hepcidin correlation with acute phase reactants (ESR, CRP) and DAS 28
Acute phase reactants (ESR, CRP) and DAS28 were evaluated. Positive
correlations were noted between hepcidin and acute phase reactants: ESR
(R=0.352, p=0.009<0.05) and CRP (R=0.369 p=0.006<0.05), and DAS28 score
(R=289, p<0.05). ESR, CRP and DAS28 score, were similar between ACD and
ACD+IDA groups. A positive correlation was observed between hepcidin and ESR
(R=0.515, p=0.041<0.05) for NA group. No correlations were observed for
controls.
Test results for determining occult digestive bleeding (FOBT)
Patients form anemic group (n=38) were investigated for FOBT. A total of 34
probes were collected: 24 patients had tested negative and 9 had tested positive.
Patients tested positive had significantly higher hepcidin level than those tested
negative (1.510.34 vs. 0.990.59). A significant difference in serum iron
(55.5229.95 vs. 32.6717.25, p=0.039), sTfR (2.561.03 vs. 3.481.15 p=0.033
and TfS% (11.435.753.92 vs. 7.07, p=0.044), was observed. No differences
were observed for Hb, Ht and red cell indices between patients tested positive
and negative for FOBT.
Discussions
Hepcidin - acute phase reactant
The information about hepcidin has increased greatly in the last few years, but
the number of studies that have studied hepcidin in patients with RA is still low.
The number of patients that included in these studies is also low, and the results
reported are not consistent.
A study published in 2008, showed that pro-hepcidin levels in patients with RA
and Systemic Lupus Erythematosus (SLE) did not correlate with disease activity
scores, Hb values, or serum iron status and cytokine level
19
, while a study on
patients with inflammatory bowel disease have showed significant correlations
between hepcidin and ferritin, prohepcidin and ferritin, and the serum hepcidin
20
levels correlate with disease activity and CRP values
20
. Other studies that have
explored the relationship between CRP and hepcidin in various categories of
patients, patients with liver surgery and hemodialysis patients that did not
observe any correlation between CRP and values of hepcidin
21,22
. Our study
performed on patients with RA with normal renal and hepatic function showed
the existence of a statistically significant correlation between acute phase
reactants (ESR, CRP) and hepcidin, similar to the results previously reported
23
.
In our study, acute and chronic infections, and malignant disease that could
interfere with serum hepcidin values were exclusion criteria.
Hepcidin correlation with disease activity score DAS28
In our study, we observed a positive correlation between hepcidin and disease
activity score DAS28 for RA patients, observation that had been made by
previous studies
23
.
Hepcidin correlation with serum iron
Hepcidin was negative correlated with serum iron in RA patients, anemic group
and ACD+IDA group. No correlation was observed in control group. The results
showed that inflammation has an important role in iron homeostasis and
hepcidin is the key element in this action.
Hepcidin analysis in the study groups
Analysis of serum hepcidin for study groups, revealed statistically significant
differences between the hepcidin values in patients with ACD and ACD+IDA
groups compared with the controls. The ELISA test we used for serum hepcidin
determination did not find a significant difference between ACD and ACD+IDA
groups, results that are similar with previous reported results
24
. Results contrary
to those reported by us were presented by another study
25
.
Conclusions
Patients from ACD and ACD+IDA groups had higher serum hepcidin values
compared with NA group and control group;
ACD group had mean serum hepcidin leve higher than patients form
ACD+IDA group, but this difference was not statistically significant;
Serum hepcidin was negative correlated, with Hb and serum iron:
Hepcidin is an acute phase reactant in patients with RA;
Serum hepcidin leves correlate with the acute phase reactants (ESR, CRP) and
the disease activity score DAS28;
In the presence of simultaneous and conflicting signals that are involved in
the hepcidin synthesis, inflammation seems the dominant signal

Study 2. The relationship between the acute-phase reactants (ESR,
CRP) and disease activity score (DAS28) in patients with RA.

Introduction
Anemia is a frequent extra-articular manifestation in RA. Defective
erythropoietin production, reduced bone marrow response to erythropoietin,
increased the level of inflammatory cytokines, increased the hepcidin level and
the defective release of iron from reticulo-endothelial cells, and also
gastrointestinal blood loss secondary to nonsteroidal anti-inflammatory drugs
(NSAIDs), and bone marrow toxicity secondary to immunosuppressive
medication, are the most important causes for anemia. In clinical practice CRP
and ESR are used inmonitoring disease activity and response to the treatment.
21
ESR may be considered less specific in measuring acute phase response than CRP
because it is influenced by various factors other than systemic inflammation,
including age, sex, Hb concentration, the serum immunoglobulins levels and
rheumatoid factor
26
.
DAS28 score is a simple and objective test for evaluation of disease activity and
therapy response.HAQ-DI is a self-administered patient completed
questionnaire. The disability index of these instruments raises a series of
questions in patients about their function, for as much as they can do or cannot
do, and also what additional aids they need to complete certain task
27
.
Hypothesis / Objectives
The main objective of this research was to study the acute phase reactants (ESR,
CRP) in a group of patients with PR with or without anemia and acute phase
reactants correlations with DAS28 score and self-administered by the patient
tests (HAQ -DI). The secondary objective was to assess the correlations between
acute phase reactants (ESR and CRP) that are commonly used in medical practice
and ferritin.
Material and method
Our study involved 54 patients with RA, diagnosed according to the 1987 revised
criteria of the American College Of Rheumatology
7
. Anemia was defined as
represented by the concentration of Hb<12mg/dl for women and Hb<13mg/dL
for men. All anemic patients had normochromic, normocytic to microcytic
anemia. Patients with macrocitosis, history of recent acute blood loss, acute
hemolytic anemia, and iron therapy were excluded from this study. Also patients
with known malignancies, renal and hepatic failure, acute or chronic infections
were excluded. Blood was collected in fasting state, between 7:00 AM and 9:00
AM. ESR and CBC (complete blood count) were performed in the same day, for
the remaining tests serum was separated and stored in at 20C, for ulterior
determination. ESR was determined by the Westergen method and serum CRP
was determined by nephelometry, using the Turbox CRP Orion Diagnostica.
Quantitative serum ferritin determination was performed using Ferritin kit on a
Elecsys 2010. DAS28 score was calculated using the following variables: tender
and swollen joints count, ESR, and patient assessment of disease activity (VAS,
mm)
18
. HAQ-DI asks patients about their ability to perform various activities and
whether need help to perform these activities.
Results
A total of 54 patients were included in this study. According to their anemic
status, patients were classified in anemic group and non-anemic group (NA). No
differences were observed between age and disease duration between the two
groups. There is a statistically significant difference between anemic group and
NA group in ESR (p = 0.001 < 0.05) and CRP values (p = 0.008 < 0.5), and DAS28
score, but no difference was observed for ferritin and HAQ-DI. Positive strong
correlation between DAS28 score and CRP (R=0.417, p =0.009<0.01) was
observed in RA anemic group. For those two variables (DAS28 and CRP) in RA
anemic group we studied regression function that describes their correlation.
The trend represented by the cloud of points studied for CRP and DAS28 in RA
anemic group is the best described by a cubic function. In The NA group, there is
a positive strong correlation between DAS28 and ESR, statistically significant
(R=0.724, p=0.002<0.001). Also, there is a positive correlation, statistically
significant, between HAQ-DI and ESR (R=0.621, p=0.01<0.05). There is a linear
22
dependence between ESR and DAS28 score (determination coefficient = 0.525),
and also between ESR and HAQ-DI (determination coefficient = 0,386). In anemic
group a positive correlation was observed between CRP and ferritin (R=0.532,
p=0.001<0.05 and between ESR and ferritin (0.304, p=0.043<0.05). In NA group,
ferritin was correlated with ESR (R=0.751), statistically significant
(p=0.001<0.05) and CRP (R=0.841), statistically significant (p<<0.05).
Discussion
A positive strong correlation between DAS28 score and CRP was observed in
anemic group, and between DAS 28 score and ESR in NA group. Positive
correlations between DAS28 score and CRP and ESR were similar with those
reported by previous studies
28,29,30
. Positive correlation was observed for anemic
and NA groups between ferritin and acute phase reactants (CRP and ESR). Serum
ferritin values correlate with the iron stores in the normal patients, but ferritin in
an acute phase reactant and it increase in various infections, inflammations and
malignancy
31
. These diseases were considered exclusion criteria in our study.
HAQ-DI were not different between anemic and NA groups. The HAQ-DI is a
subjective test for evaluation of disease activity and they must always be
accompanied by objective parameters (ESR, CRP) in making assessments on
disease activity in patients with RA with or without anemia. The importance of
objectives tests (ESR, CRP) used for the diagnosis and monitoring of patients
with RA is supported by the new criteria for the diagnosis of RA approved in
2010 by the American College of Rheumatology and the European League against
Rheumatism
32
these tests being a part of the new diagnostic criteria.
Conclusions
Patients with anemia have more active disease than non-anemic patients;
HAQ-DI scores did not differ significantly between anemic and non-anemic
patients;
Self administrated tests (HAQDI), must always be accompanied by objective
tests to assess disease activity;
Ferritin correlates with CRP and ESR in RA patients, independent of their
anemic status.
General conclusions
Patients with RA and anemia have more active disease than non-anemic
patients;
Self-administered test (HAQDI) is not significantly different between anemic
and non-anemic patients and have to be always be accompanied by objective
tests to assess disease activity;
Hepcidin is an acute phase reactant;
Hepcidin correlates with acute phase reactants (ESR, CRP) and DAS28 score;
RA patients with anemia have higher serum hepcidin level than patients
without anemia and subjects in the control group;
Red cell indices, RDW and severity of anemia could not be use for diagnosis of
iron deficiency in the presence of inflammation
Hepcidin correlates with Hb and serum iron
Serum hepcidin levels were higher in patients with ACD, than patients from
ADC+IDA group, but the difference was not statistically significant
Our data suggest that in the presence of concurrent conflicting signals in the
hepcidin synthesis, inflammation is the dominant factor.
Originality and innovative contributions of the thesis
23
Information about hepcidin and its role in iron homeostasis in normal and
pathological conditions are very numerous in the literature of recent years.
However the data obtained from clinical trials in patients is a few, and the
number of studies that have determined the values of serum hepcidin in patients
with RA and ACD and a control group are even fewer, sometimes with conflicting
results. The originality of this thesis is given by theme, how this thesis has been
developed and the conclusions reached. In our study of hepcidin acted as an
acute phase reactant and the intervention in pathogenesis of anemia in patients
with RA was obvious. Even though this research could not demonstrate that
hepcidin can make differential diagnosis of iron deficiency in the presence of
inflammation, the new generation of standardize tests will be able to do that in
the near future. Some aspects of this research were published for the first time in
the literature.


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