The data from this study demonstrate novel changes in serum amyloid A4, retinol binding protein, inter-alpha inhibitor H4, clusterin, and fibronectin plasma protein abundance with chronic, excessive alcohol self-administration in a non-human primate model. In a translational assessment, decreased plasma levels of fibronectin and increased levels of serum amyloid A4 were also evident in human subjects with excessive alcohol intake compared to controls. These data also provide evidence for the utility of combining plasma protein depletion technology and 2D-DIGE quantitation to identify biomarker candidates. In agreement with our recent demonstration that directed antibody-based (Luminex) and open discovery gel-based (2D-DIGE) proteomic technologies provide complementary rather than redundant data (VanGuilder et al., 2010b
) the proteins identified and confirmed in this study are novel and were not detected in our previous biomarker identification efforts (Freeman et al., 2010
). These proteins not only provide insight into the health effects of chronic, excessive alcohol consumption, but also may serve as biomarkers of alcohol intake.
Serum amyloid A4 (SAA4) is expressed in a number of tissues, with the liver being a primary organ source. Similar elevations of plasma SAA4 were observed in monkeys and humans (200–250% of respective controls). SAA4 is an acute phase response protein and a major component of high density lipoprotein (HDL) (Bausserman et al., 1980
). Circulating SAA4 levels are induced by a number of inflammatory factors (Akira et al., 1990
). Increased circulating levels of serum amyloid A have been reported in mice after high dose, but not low dose, ethanol administration (Pruett and Pruett, 2006
). In fact, this induction was highest at 24 hours after ethanol administration, providing both temporal and dose effect relationships that may aid in explaining the often contradictory evidence for ethanol as both an immunosuppressant and pro-inflammatory factor. Combined with the data from this study demonstrating elevated SAA4 levels in non-human primates and humans with high levels of ethanol consumption, this provides evidence of commonalities in the plasma proteomic response to ethanol across species.
Fibronectin is a ubiquitously expressed acute phase response glycoprotein with a wide range of functions from cell adhesion to blood coagulation. Alterations in fibronectin tissue levels with alcohol have been reported with a heterogeneous response depending on the tissue and dose of alcohol. Increased myocardial levels have been reported in rodents after low dose alcohol administration (Vendemiale et al., 2001
), while this protein is reported to increase in the lung with alcohol abuse (Burnham et al., 2007
). Moreover, increased hepatic levels may be an indicator of future cirrhosis in alcohol abusers (Junge et al., 1988
). However, there are no reports of circulating plasma fibronectin levels with excessive alcohol consumption. As fibronectin is produced in most major tissue types, including muscle and adipose, the decreased levels observed here may reflect global changes in a variety of organ systems.
The function of clusterin, also known as apolipoprotein J, is not known; however, sialylation levels of clusterin have been previously proposed as a biomarker of alcohol intake (Ghosh et al., 2001
). In this study, total levels of clusterin protein, but not protein modifications, were examined. Decreased levels of clusterin were evident in non-human primate samples, but were only statistically significant through within-subject, paired analysis. On the other hand, increased (20%) levels of total clusterin protein were evident in human subjects with excessive alcohol intake. The differences observed between the non-human primate and human data potentially arise from the difference of within-subject verses cross sectional measurements and will certainly warrant further investigation in future studies.
Inter-alpha inhibitor H4 (ITIH4) is a serine protease inhibitor produced in the liver and has characteristics of an acute phase response protein (Pineiro et al., 1999
). The increase in ITIH4 plasma levels illuminated by 2D-DIGE discovery was confirmed in non-human primate samples by immunoblotting, but did not translate to human subjects. This is not unexpected given that the human samples did not originate from a within-subject design. It is possible that some alcohol-responsive changes will become apparent only for within-subject assessments where individual differences in basal levels of expression can mask the effects of alcohol. While this reduces utility as a potential cross-sectional biomarker, applicability as part of within-subject biomarker panel remains. Additionally, it also contributes to the body of evidence suggesting changes in acute phase responses following ethanol self-administration.
Retinol binding protein 4 (RBP4) is the plasma isoform of the retinol binding protein family secreted by the liver and adipose tissue with the primary function of transporting retinol (vitamin A alcohol). Vitamin deficiencies in alcoholic patients is well described and is most likely due to both poor diet and impaired liver function (Hoyumpa, 1986
). While there is growing evidence for a role of RBP4 expression in obesity-induced diabetes (Yang et al., 2005
), no previous data regarding circulating levels of RBP4 with excessive alcohol intake is present in the literature. However, a relationship between vitamin homeostasis and regulation of lipid levels with excessive alcohol intake has been observed in this non-human primate model and may integrate the roles of vitamin status, lipoprotein levels, and inflammatory processes in alcohol related pathology (Lebold et al., 2010
). Like ITIH4, the changes in RBP4 levels observed in monkeys were not evident in the human subjects examined. Evaluation of RBP4, and the other plasma proteins, will need to be performed in larger subject populations to extend these findings.
This study expands upon our previous focused proteomic approach to biomarker discovery (Freeman et al., 2010
). The proteins examined with the 2D-DIGE technology did not overlap with those we have examined by antibody-based approaches, demonstrating a complementarity of these different proteomic approaches similar to that we have observed previously (VanGuilder et al., 2010b
). When the results of the present study are combined with previous data on plasma proteomic changes from this model (Freeman et al., 2010
), disruption of inflammatory homeostasis is evident, in agreement with a number of previous studies (Crews et al. 2006
). Further study is needed in both animal models and simpler in vitro
model systems to determine the interactions of classical inflammatory processes with lipoprotein metabolism, endocrine function, and nutritional status. Non-human primate self-administration models provide a valuable approach to define alterations in plasma proteins with alcohol abuse in a controlled setting and with a high relevance to the human condition (Grant and Bennett, 2003
As the primary goal of the present study was to identify novel biomarker targets, the validation of SAA4 and fibronectin as altered in human subjects indicates that they may have utility as analytes in a biomarker panel of alcohol intake. With some overlap in the distribution of protein abundance between Healthy and Drinking samples, the highest specificity will most likely be achieved adding these candidates to panels of biomarkers rather than as independent metrics. Changes in plasma abundance for these and other previously described alcohol-responsive plasma proteins have been reported with other conditions and disease states. Through combining multiple biomarkers into panels, as have been previously proposed for alcohol intake biomarkers, may also aid to in achieving disease specificity (Anton et al., 2002
; Freeman et al., 2010
; Korzec et al., 2005
). As well, determining alcohol-responsive changes in plasma proteins is important for understanding potential sources of false-positives in biomarker diagnostics of other diseases. Confounding variables such as inflammation and diabetes may impact the expression of these proteins, and must be considered when these biomarker candidates are implemented clinically as components of an alcohol consumption diagnostic test.
Further validation of these proteins as potential biomarkers will need to include, assessment in larger cross-sectional human studies, time-course examinations of abstinence from excessive alcohol consumption, and moderate drinking groups. Future development studies will aid in determining whether a longitudinal monitoring biomarker approach or cross-sectional diagnostic test achieves the highest sensitivity and specificity. Additionally, future studies with female subjects are required to examine commonalities and differences in the sex response to chronic excessive alcohol. Inclusion of the biomarker targets identified here with biomarker panel we have previously identified offers the potential to avoid issues of sensitivity and specificity associated with unitary biomarker diagnostics and ultimately increase the utility of these proteins as indices of alcohol use and abuse (Freeman and Vrana, 2010