Effect of alcohol consumption and acetaldehyde on blood cells and molecules: pathogenic and diagnostic implications

Abstract

Väitöstutkimuksessa selvitettiin etanolin ja sen ensimmäisen hajoamistuotteen, asetaldehydin, veren soluille ja molekyyleille aiheuttamia muutoksia. Tutkimuksilla on merkitystä alkoholin aiheuttamien kudosvaurioiden mekanismien selvittelyssä sekä kehitettäessä uusia menetelmiä alkoholin suurkulutuksen toteamiseksi aikaisempaa varhaisemmassa vaiheessa.

Asetaldehydi on erittäin reaktiivinen molekyyli, joka voi muodostaa sidostuotteita monien erilaisten valkuaisaineitten kanssa. Alkoholin suurkuluttajien verenkierrossa havaittiin tällaisia sidostuotteita sekä niitä vastaan muodostuneita, erityisesti IgA-luokan vasta-aineita. Koe-eläimissä tuotetuilla asetaldehydin ja proteiinien välisiä sidoksia tunnistavilla vasta-aineilla selvitettiin myös mahdollisuuksia uusien alkoholin suurkulutusta kuvaavien testien käyttöönottoon. Esimerkiksi asetaldehydin ja VLDL-kolesterolin välisiin sidostuotteisiin kohdistuva vasta-aine osoittautui tehokkaaksi pienillä asetaldehydipitoisuuksilla valmistettujen kondensaattien tunnistamisessa. Tämä havainto voi avata uusia mahdollisuuksia alkoholin liikakäytön toteamiseksi. Myös alkoholin käytön aiheuttamat autoimmuunivasteet voivat tutkimuksen mukaan osoittautua hyödyllisiksi esimerkiksi erilaisten maksasairauksien erottelussa.

Paljon alkoholia käyttävillä henkilöillä havaittiin veressä ja luuytimessä punasolujen ja niiden esiasteiden sisältävän asetaldehydin muovaamia rakenteita. Ne saattavat olla vaikuttamassa punasolujen poikkeavuuksien syntyyn alkoholisteilla. Alkoholin suurkuluttajille on tyypillistä että verenkierron punasolut ovat kooltaan tavanomaista suurempia ja punasolujen keskitilavuus (MCV) on suurentunut. Myös veren muiden solulinjojen poikkeavat määrät ovat tavallisia. Vähäiset verihiutaleitten määrät olivat alkoholisteilla yleisempiä kuin raittiilla verrokeilla tai kohtuukäyttäjillä. Luuytimessä alkoholin suurkuluttajilla on usein poikkeavaa rautakertymää sisältäviä punasoluja sekä poikkeavaa morfologiaa, mm. ylimääräistä sytoplasmista vakuolisaatiota, punasolujen ja verihiutaleitten esiasteissa voimakkaan päihtymyksen jälkeen. Tämän perusteella potilailta, joilla nämä arvot ovat poikkeavia, tulisi aina kysyä alkoholin käyttötottumuksista.

Alcohol abuse is known to have a wide array of adverse effects on serum proteins, blood cells and their progenitors in bone marrow. The mechanisms by which it does so have not yet been estab-lished, however. Recent studies have suggested that acetaldehyde, the first metabolite of ethanol, is responsible for many of its harmful effects, being a highly reactive molecule and capable of forming stable condensates with proteins and cellular constituents. Acetaldehyde-derived protein conden-sates have also been suggested as possible new markers of alcohol abuse.

The present work is focused on the effects of ethanol and acetaldehyde on blood cells and pro-teins. The formation of acetaldehyde-derived protein modifications in vivo was studied using anti-bodies recognizing acetaldehyde-modified epitopes in proteins independently of the nature of the carrier protein. The subjects were patients representing a wide range of ethanol consumption and abnormalities in haematological parameters, alcoholics admitted for detoxification and patients with alcoholic liver disease. The controls were individuals without any history of excessive alcohol con-sumption.

Routine haematological examinations of patients presenting with cytopenia and a history of haz-ardous drinking showed the incidence of anaemia to be lower than that in the corresponding non-alcoholic controls. Abnormal platelet and leukocyte levels were common, especially in the anaemic alcoholics. Both an elevated mean cell volume of erythrocytes (macrocytosis) (67% vs. 18%) and elevated mean cell haemoglobin (63% vs. 22%) were more frequent in the alcoholics. Reticulocyto-sis, thrombocytopenia and combined cytopenias were all common findings in the alcoholic patients. Blood smears from the alcoholics typically showed round macrocytes, stomatocytes and knizocytes. Bone marrow aspirates revealed vacuolization of pronormoblasts and megakaryocytes, especially in individuals with recent intoxication.

Immunocytochemical analyses of peripheral blood erythrocytes indicated that acetaldehyde-derived epitopes occurred both on the cell membrane and inside the erythrocytes. Bone marrow aspirates also showed positive staining for acetaldehyde adducts in erythropoietic cells. Separation of erythrocyte proteins by high-pressure liquid chromatography revealed the formation of fast-eluting haemoglobin fractions which reacted with antibodies against acetaldehyde adducts.

Antibodies raised in rabbits against condensates with acetaldehyde and lipoproteins (LDL, VLDL) or bovine serum albumin reacted with protein adducts generated at physiologically relevant concentrations of acetaldehyde in vitro. The antibody prepared against the acetaldehyde-VLDL condensate was found to provide the most effective in vivo detection of acetaldehyde adducts in erythrocyte and serum proteins from alcoholic patients.

The generation of autoimmune responses to the acetaldehyde-derived epitopes was studied in heavy drinkers admitted for detoxification and in patients with alcoholic liver disease. The mean anti-acetaldehyde adduct IgA levels in the alcohol abusers, who had mild or no clinical and bio-chemical signs of liver injury, were significantly higher than those in the moderate drinkers or ab-stainers. The highest titres occurred in patients with alcoholic liver disease. IgM antibodies were also significantly higher in the alcohol consumers without liver disease, whereas IgG levels were high only in the patients with alcoholic liver disease. The cytokine profiles of the alcoholic patients also showed significant differences, with the highest levels of both pro-inflammatory cytokines (IL-2, IL-6, IL-8, TNF-᩠and the anti-inflammatory cytokine (IL-10) occurring in the patients with alcoholic liver disease.

These results suggest that alcohol abuse is an important risk factor for abnormalities in a number of haematopoietic cell lines and in circulating proteins. Acetaldehyde-erythrocyte adducts appear to be formed in vivo in the blood and bone marrow of patients with excessive alcohol consumption, which may contribute to the generation of erythrocyte abnormalities. Acetaldehyde adducts are also immunogenic. Antibodies raised against acetaldehyde-VLDL adducts could provide the basis for a new diagnostic assay to assess excessive alcohol consumption. The data also show autoimmune responses to acetaldehyde-modified proteins in alcoholics, which may also prove to be of value in the differential diagnosis of alcohol-related diseases.