Freeman, an assistant professor of pharmacology at Penn State College of Medicine, said the challenge in determining alcohol abuse versus substance abuse (such as cocaine, heroin, or PCP) is that alcohol is a legal substance for those over 21.

"Unlike routine testing for illicit drugs, you can’t just look for a trace of alcohol because many people enjoy a drink in a responsible manner, and alcohol is very quickly metabolized. Discriminating between excessive and responsible levels of drinking makes this a greater challenge," said Freeman, the lead investigator.

Penn State Hershey researchers have been working for two-and-a-half years with Kathleen A. Grant at the Oregon National Primate Research Center on the project and have identified a set of 17 proteins in the blood that accurately predict alcohol usage 90 percent of the time in the primates.

Freeman said the team is interested in learning how the brain works, especially when it comes to alcohol abuse and drug addiction.

"What is it that changes in the brain with substance abuse, addiction, that makes it so hard for people to stay sober or stay clean to achieve that goal," he said. Freeman said tests that are currently available, such as breathalyzers, are used for acute intoxication at the time a person is given the test. Because alcohol quickly metabolizes, the current tests are not sensitive or specific enough to serve as diagnostics, he said.

"It (the tests) won’t tell us what people did yesterday or over the past two weeks or the past three months," he said, adding that researchers have been able to measure a set of 17 proteins in monkeys’ blood for alcohol use and separate usage into three categories: no alcohol use, drinking up to two drinks per day, and drinking at least six drinks per day.
"What we want to be able to do is take someone who is in treatment and make sure they have not relapsed," he said.

Most of the proteins they looked at relate to liver, immune and nervous system functions in the body, he said. "Alcohol is this little tiny molecule, and it binds to all sorts of stuff throughout the body. It’s not like a lot of other drugs. Long periods of heavy drinking are known to have adverse impacts on the liver, the heart, on the nervous system and on immune function," he said.

Freeman said the team found that the levels of some proteins increased or decreased with as little as one or two drinks a day. "These same changes occurred with heavier levels of drinking. We also found other proteins that responded only to heavy levels of drinking. Combined, these proteins allow us to classify subjects into non-drinking, alcohol-using and alcohol-abusing groups," he said.

Freeman said the next step in the research is to do blood tests on humans. "We’re working with a few different centers around the U.S. that have inpatient treatment facilities for alcoholism," he said. "We’ll collect blood throughout their stay to see if the patients’ protein pattern reverts from an excessive drinking pattern to a pattern that’s indicative of alcohol abstinence."

The researchers also are trying to determine whether the changes measured return to normal levels with cessation of drinking, and looking for additional proteins to both increase accuracy and provide alternates if some of the initial 17 do not work in humans.

He said the goal is to create a diagnostic test for alcohol consumption that may be used in areas of public safety, such as aviation or national security, for parole conditions or for helping physicians determine if a patient may have an alcohol-abuse problem.

The findings have been published in Biological Psychiatry, the journal of the Society of Biological Psychiatry.

Others working on the project are Kent Vrana, chairman, Department of Pharmacology; Anna C. Salzberg, Penn State Hershey Cancer Institute; and Steven W. Gonzales, Biotic Micro Inc.

The research project is funded by the National Institutes of Health and the National Institute on Alcohol Abuse and Alcoholism.

"The challenge in alcohol abuse as opposed to substance abuse—things like cocaine or heroin or PCP—is that alcohol is a perfectly legal substance for those over 21," said Willard M. Freeman, Ph.D., department of pharmacology and lead investigator. "Unlike routine testing for illicit drugs, you can’t just look for a trace of alcohol because many people enjoy a drink in a responsible manner and alcohol is very quickly metabolized. Discriminating between excessive and responsible levels of drinking makes this a greater challenge."

Penn State Hershey researchers, working for two-and-a-half years in cooperation with Kathleen A. Grant, Ph.D., at the Oregon National Primate Research Center, identified a set of 17 proteins in the blood that accurately predicted alcohol usage 90 percent of the time in non-human primates. Researchers were able to separate usage into three categories—no alcohol use, drinking up to two drinks per day, and drinking at least six drinks per day. Protein levels rose and declined depending on alcohol consumption.

"We observed that the levels of some proteins increased or decreased with as little as one or two drinks a day," Freeman said. "These same changes occurred with heavier levels of drinking. We also found other proteins that responded only to heavy levels of drinking. Combined, these proteins allow us to classify subjects into non-drinking, alcohol-using, and alcohol-abusing groups."

The researchers are continuing their work, first by determining whether the changes measured return to normal levels with cessation of drinking. Second, they are looking for additional proteins to both increase accuracy and provide alternates if some of the initial 17 do not work in humans.

Working with groups around the world, Penn State Hershey researchers—led by Freeman and Kent Vrana, chair, department of pharmacology—plan to collect blood from people undergoing inpatient treatment for alcohol abuse.

"We’ll collect blood throughout their stay to see if the patients’ protein pattern reverts from an excessive drinking pattern to a pattern that’s indicative of alcohol abstinence," Freeman said.

The goal is to create a diagnostic test for alcohol consumption that may be used in areas of public safety like aviation or national security, for parole conditions and for helping physicians determine if a patient may have an alcohol abuse problem. Currently there are tests that try to address this issue, but Freeman said these tests are not sensitive and specific enough to serve as diagnostics.
"Many of these tests rely on just one protein," he said. "The limitation to this approach is that these tests often look at proteins produced by the liver. While these proteins increase with excessive alcohol intake, they also increase with any type of injury to the liver. For example, a lot of prescription drugs are hard on the liver. These tests let us know that the liver is being stressed but can’t discriminate between excessive drinking and other conditions, which therefore reduces the utility of these tests.

"That’s where we see the promise in this panel of proteins. The proteins are produced by a number of organs including the liver, the muscle, and the brain. This unique fingerprint that is indicative of alcohol abuse is less likely to be produced by unrelated conditions."
Freeman stresses, a diagnostic test would not be testing for alcoholism, but rather, alcohol intake.

"In a strictest use of the words, alcoholism is a psychological diagnosis as opposed to a level of drinking," he said. "The Diagnostic and Statistical Manual really classifies alcohol abuse and alcoholism based on how alcohol is interfering with your life. Obviously we can’t use a blood test to say yes, your drinking is interfering with your home life. But the amount of drinking and the amount of problems it causes in your life are tightly correlated.

"We envision, a number of years down the line if this becomes a diagnostic test, that if the test indicates that you’re drinking a lot, it would prompt a referral to a specialist in alcohol abuse and alcoholism. This test could provide an objective indicator to help people begin addressing what may really be a problem in their lives."

Also contributing to this research are Anna C. Salzberg, Penn State Hershey Cancer Institute and Steven W. Gonzales, Biotic Micro, Inc. The researchers’ findings are published online in Biological Psychiatry. The National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism supported this work.

"This paper assesses the validity of the severity ranking of the 11 criteria and the overall severity score with respect to known AUD correlates, including alcohol consumption, psychological functioning, family history, antisociality, and early initiation of drinking, in a representative population sample of U.S. past-year drinkers (n=26,946). The unadjusted mean values for all validating measures increased steadily with the severity threshold score, except that legal problems, the criterion with the highest score, was associated with lower values than expected.

“After adjusting for the total number of criteria endorsed, this direct relationship was no longer evident. The overall severity score was no more highly correlated with the validating measures than a simple count of criteria endorsed, nor did the two measures yield different risk curves. This reflects both within-criterion variation in severity and the fact that the number of criteria endorsed and their severity are so highly correlated that severity is essentially redundant," wrote D.A. Dawson and colleagues of the National Institute on Alcohol Abuse and Alcoholism.

The researchers concluded: "Attempts to formulate a scalar measure of AUD will do as well by relying on simple counts of criteria or symptom items as by using scales weighted by IRT measures of severity."

"Cytokines are proteins which mediate and regulate the inflammatory reaction in infectious and autoimmune diseases," explained Pilar A. Sáiz, associate professor of psychiatry at the University of Oviedo, Spain and corresponding author for the study.

"Clinical observation of increased circulating levels of pro-inflammatory cytokines such as IL-1 and TNFα in patients with alcohol liver disease suggests that they might play a role on the pathogenesis of the disease. Additionally, these cytokines act in the central nervous system affecting the functionality of the serotoninergic and dopaminergic systems, which have been also related to the pathophysiology of AD, and with the brain reward systems involved in alcohol reinforcement."

Sáiz and her colleagues recruited 200 (169 males, 31 females) AD patients from an outpatient detoxification unit, as well as 420 (216 males, 204 females) healthy individuals without a history of drug/alcohol/psychiatric problems (known as "controls"), from the north of Spain. All of the Spanish Caucasian participants were genotyped for four polymorphisms—IL-1α -889 C/T, IL-1ß +3953 C/T, IL-1RA (86bp)n, and TNFα -308A/G—and assessed at baseline and again at six months for alcohol intake, addiction severity, and biomarkers of alcohol intake.

"We found that AD male patients and healthy control male patients differed in the genotype frequencies of the IL-1RA polymorphism owing to an excess of the A1/A1 genotype in the AD males," said Sáiz. "On the other hand, analysis of the IL-1 gene complex revealed a higher frequency of the IL-1a -889C / IL-1b +3953C / IL-1RA A2 haplotype in the control group than among the AD patients, as well as in the abstainers after six months of follow-up compared to the non-abstinent patients."

A haplotype is a combination of alleles at multiple loci that are transmitted together on the same chromosome, Sáiz explained. Prior research has suggested that the study of single polymorphisms may miss more complex haplotype effects, she added.

"In short, this haplotype seemed to exert a protective effect and was related to better outcomes," said Sáiz. "These findings provide further tentative evidence of the role of the IL-1 gene complex in AD as well as evidence that the nature of the associations may be direct, gender-specific, or involve haplotype effects."

She cautioned readers to remember, however, that AD is influenced by both genetic and environmental factors. "We are talking about a multifactorial polygenic disorder caused by a combination of small variations in different genes, often in concert with environmental factors," she said. "In spite of great efforts, the exact genes related to the pathophysiology of alcoholism are yet unknown. Our work represents a small piece of the puzzle."