GROUP 8
BSMT-3D
Sadie, James Karl
Estacio, Katrina
Pineda, Anna Criselda
What
is Alcohol?
“Alcohol" has been synonymous with "spirituous"
liquids for the past years. The history of its consumption, along with codes or
laws restraining its consumption was way back in 1700 B.C. There are four known
types of alcohol: ethyl alcohol, methyl alcohol, isopropyl alcohol and butyl
alcohol or butanol. Ethyl Alcohol, or ethanol (C2H5OH),
is commonly used in making alcoholic beverages or liquor. The remaining types,
if ingested can result in blindness and death, even in relatively small doses.
Ethyl
alcohol or Ethanol is the intoxicating agent used as an ingredient in making
beer, wine and liquor. It is created by fermentation of yeast, sugars, and
starches. Grapes and grains like
barley and wheat are most commonly used in making the alcoholic beverages.
Ethyl Alcohol simply called as alcohol comes second to
caffeine as the most widely utilized drug in the society. Alcohol, being a
legal substance many employers do not often test for it as part of their urine
drug testing program. Typically, a urine alcohol assay is done using a
biochemical method where in alcohol, oxidizes acetaldehyde in the presence of
alcohol dehydrogenase if it is present in the specimen. Simultaneously after
oxidation, Nicotinamide adenine dinucleotide (NAD) is reduced to NADH. Upon
spectro-photometric analysis of NADH the concentration of alcohol can now be
obtained. Gas chromatography with flame ionization detection is the most common
confirmatory method used. The screening and confirmation cut-off values for
Alcohol are 0.02 or 0.04 g/dL.
How do we measure the concentration of Ethyl Alcohol in the body?
Just a “nice to know” facts, alcohol is metabolized in the body by
our liver. It is first converted to an aldehyde, specifically ethanol to acetaldehyde,
by the enzyme Lactate Dehydrogenase isoenzyme-6 or also known as the Alcohol
Dehydrogenase. Then it is furthermore converted into an acid, acetaldehyde to
acetic acid, by the enzyme Aldehyde Dehydrogenase present in our liver. Acetic
acid is soluble in water and eliminated in the body as a waste via urine,
perspiration, and even in our breath.
Ethanol concentration can be measured using blood, urine, saliva
or breathe tests. Toxic concentration merely depends on the individual’s
tolerance and usage although levels greater than 300-400 mg/dL can be fatal due
to respiratory depression. Conversion unit: one millimole of ethanol per liter
of blood is equal to 4.61 milligrams of ethanol per 100 milliliters of blood.
There are three methods known to measure the
level of Ethyl alcohol in the body. It could be measured using Blood tests,
Urine tests and Breath tests.
Below are specific methods used in the
determination of Ethyl alcohol levels:
BLOOD ALCOHOL TESTS
WHAT IS A BLOOD ALCOHOL?
Blood
alcohol (also known as blood alcohol content) is the concentration of ethanol
on a person’s blood stream and is usually measured as weight per volume and is
converted to percentage. This blood alcohol content is used to determine if the
person is legally drunk with alcohol for law enforcement or medical purposes.
Because alcohol is absorbed in the stomach and the small intestine, it will go
then into the bloodstream and will travel throughout the body and brain which
can affect the cognitive functioning of a person that can lead to injuries and
unnecessary act.
Analysis of urine and blood samples for ethyl
alcohol levels is one of the most frequently performed tests for
drugs-of-abuse. The results of these tests are utilized for medico-legal
purposes that include identifying impairment or legal intoxication, and
diagnosis and treatment of alcohol abuse and poisoning.
WHAT TEST COULD BE DONE
TO DETERMINE THE CONCENTRATION OF ALCOHOL IN BLOOD?
There are several
methods in determining the concentration of ethanol in the BLOOD, these
includes:
- Headspace
Gas Chromatography-Flame Ionization Detector (HSGC-FID)
This method provides a
procedure for the quantitation of ethyl alcohol in blood by FID with
simultaneous confirmation by Mass Spectroscopy and can also be utilized as an
identification method for inhalants such as 1,1-difluoroethane.
The mechanism of FID
is based on the detection of electrically charged particles (ions) produced
during combustion of organic compounds in a hydrogen flame. The
generation of these electrically charged particles or ions is equal to
the amount of organic species in the sample gas stream.
- Direct
Blood-Injection Method for Gas Chromatographic Determination of Alcohols
This method is a
simple and rapid method that is described for determining different alcohol
levels in the blood. This method uses less than 1 microliter of blood, mixed
with an internal standard, and is injected directly into a gas chromatograph
equipped with flame-ionization detector. This method is sensitive to less than
10 micrograms per ml.
This method has the
same principle with Headspacer gas chromatography FID but has one notable
difference. It uses less than 1 µl of blood, mixed with an internal
standard, and is injected directly into a gas chromatograph equipped with a
flame-ionization detector.
· High Performance
Liquid Chromatography
(c) analyticalprofessional.blogspot.com
·
High-performance
liquid chromatography (HPLC) is a method based on chromatography is widely used
in forensic science. The “HP” acronym is sometimes referred to the words high
pressure (versus high performance), but it refers to the same analytical
system. HPLC is useful in drug analysis, toxicology, explosives analysis, ink
analysis, fibers, and plastics to name a few forensic applications.
·
A solvent that flows
under high pressure over coated beads (mobile phase) with the solid stationary
phase. While flowing through the column, molecules contained in the sample
separate selectively between the stationary and the mobile phase. Those that
interact more with the stationary phase will wrap behind those molecules that
separate with the mobile phase. As a result, the sample established at the
front part of the column and will emerge in separate bands (called peaks),
bands emerging first being the components least interacted with the stationary
phase and as a result flows quicker through the column. The components that
appear last will be the ones with most interaction with the stationary phase
and thus moved the slowest through the column. A detector is placed at the end
of the column to identify the elute components.
Ethanol
was quantified by measurement of the succeeding raise in acetaldehyde level
after reaction with alcohol dehydrogenase and nicotinamide adenine dinucleotide
(ADH-NAD) using the fluorimetric HPLC method. Ethanol standards ranging 0.3 to
200 mg/dl were examined and the boundary of quantification of the fluorimetric
HPLC method was found to be 6 mg/dl. Assessment of the HPLC method accuracy was
evaluated by testing blood samples containing 6-200 mg/dl of ethanol and
comparing its results to those of the ADH-NAD enzymatic method (r2 = 0.993).
The blood ethanol concentrations of a volunteer after a pulse of 0.3 g/kg of
ethanol was determined with the described HPLC method and were compared to the
results from the ADH-NAD enzymatic method (r2 = 0.986). Thus, the fluorimetric
HPLC method for the measurement of ethanol is of potential clinical utility.
EFFECTS AT A SPECIFIC CONCENTRATION OF BLOOD ALCOHOL
CONTENT (BAC)
(The following concentration of BAC may vary
depending on the method or test that is used)
0.02-0.03 BAC
|
Euphoria,
loss of shyness,
|
0.04-0.06 BAC
|
Warmth
sensation, Euphoria, exaggeration of behavior and emotional intensified
|
0.07-0.09 BAC
|
Impairment
of balance, speech, vision and hearing. Euphoria, reason and memory are impaired
|
0.16-0.19 BAC
|
Dysphoria,
nausea
|
0.25 BAC
|
Severely
impaired mental, physical and sensory functions,
|
0.30 BAC
|
May
pass out and difficult to awaken
|
0.35 BAC
|
Possibility
of coma. This is the level of surgical anesthesia
|
0.40 BAC and up
|
Coma,
possible death due to respiratory arrest
|
URINE ALCOHOL TESTS
Enzymatic Method
(c) enwikipedia.org
This
is a method utilized for the quantitative determination of chemical substances
in solution using enzymes. Enzymatic methods quantify substances that have the
capability of taking part in chemical reactions catalyzed by enzymes, as well
as substances that activate or inhibit enzymes. This method is highly sensitive
and specific, since enzymes catalyze the substance conversion with rapidly and
selectively.
In
the determination of the substrate in an enzyme-catalyzed reaction, addition of
an enzyme and other components to the sample is essential. Upon completion of
the reaction, the reaction product quantity in the solution is to be measured.
For example, the quantification of ethanol in a specimen using alcohol
dehydrogenase is carried out in the presence of the enzyme’s coenzyme,
nicotinamide adenine dinucleotide (NAD). Through the enzyme-catalyzed reaction
it is converted quantitatively into reduced NAD. Establishing the concentration
of the reduced NAD and calculate the concentration of ethanol is possible if
absorption is measured. This method allows determinations of 1 microgram (xg)
of alcohol in 1 milliliter of solution.
EMIT® Ethyl Alcohol Assays
EMIT® Ethyl Alcohol Assays have been intended
to accurately quantify ethyl alcohol in urine, serum, and plasma. In clinical
studies, EMIT tests have been proven to be accurate, and they also correlate
well with other assays of ethyl alcohol measurement such as, gas
chromatography.
Due to the evaporative property of ethyl
alcohol, testing for its level needs particular precautions and procedures
during collection of sample, handling, and analysis, to ensure the accuracy of
the results.
METHODOLOGY
The Emit® II Plus Ethyl Alcohol Assay is
based on an enzymatic reaction. Reagent 1 contains the buffering system.
Reagent 2 contains alcohol dehydrogenase (ADH), the coenzyme nicotinamide
adenine dinucleotide (NAD), buffer, preservatives, and stabilizers. The ADH
catalyzes the oxidation of ethyl alcohol to acetaldehyde. During this reaction,
NAD is reduced to NADH. The increase in absorbance at 340 nm is proportional to
the concentration of alcohol in the specimen.
REAGENTS
Precautions
• For in vitro diagnostic use.
• Do not leave alcohol-containing solutions
exposed longer than necessary. Tightly cap upon storage.
• Do not use expired calibrators.
Preparation of Reagents
The Emit® II Plus Ethyl Alcohol Assay
reagents are ready to use.
Storage of Assay Components
• Store reagents upright at 2–8°C and tightly
capped when they are not to be used.
• Unopened reagents are stable until the
expiration, if stored at 2–8°C.
• Do not freeze reagents nor store them to
temperatures above 32°C.
SPECIMEN COLLECTION AND PREPARATION PRECAUTIONS
• The assay requires serum, plasma, or urine.
• Avoid use of alcohol as disinfectant in
collecting or storing blood specimens.
(Use aqueous zephiran (benzalkonium
chloride), iodine, or other suitable aqueous
Disinfectants)
• Make sure the sample tube is tightly closed
to avoid evaporation of alcohol.
• Fluoride/oxalate are the preferred for
storing blood prior to analysis of plasma specimens. The anticoagulants
citrate, EDTA, fluoride/oxalate, and heparin may be used.
• Urine samples should be stored refrigerated
with the sample container. Before opening, invert it gently several times.
• If not tested immediately, specimens may be stored
refrigerated at 2–8°C for up to 3 days. After 3 days, specimens should be
stored frozen. Avoid repeated thawing.
• Frozen specimens must be thawed and
thoroughly mixed before analysis.
• High turbidity specimens must be
centrifuged prior to analysis.
• Urine samples with the pH 3.0–11.0 do not
require adjustment of pH.
• Specimens should be treated and handled as
potentially infectious.
LIMITATIONS OF THE PROCEDURE
• In
diluting specimens having high alcohol levels, the following factors may affect
the results: diluting with the appropriate fluid (Emit® Ethyl Alcohol Negative
Calibrator or deionized, distilled water), and precision of the dilution.
• Other
substances or factors (eg, technical or procedural errors) may as well
interfere with the test and cause inaccurate results.
EXPECTED VALUES
The
Emit® II Plus Ethyl Alcohol Assay quantifies alcohol levels in human urine,
serum, or plasma having levels 10–600 mg/dL (0.01–0.60%, 0.1–6.0 g/L) alcohol.
Note:
To convert mg/dL to g/L ethyl alcohol, multiply by 0.01.
Toxic
dosage for children is at 3 g/kg body weight, but a more little amount can be
fatal in the presence of induced hypoglycemia or drug intake. Alcohol-tolerant
adults have been observed to endure blood levels of 1500 mg/dL (1.50%, 15 g/L)
provided with treatment.
Another
Enzymatic Method similar to EMIT is DrugCheck Alcohol rapid test.
Gas Chromatography
(c) wwwp.cord..edu
A highly
rapid and sensitive gas chromatographic method for ethanol determination in
various biological specimens (human whole blood, serum, urine, and fecal
supernatants) was developed. This method uses direct injection of the specimen
into the gas chromatograph, without pretreatment. Injection was done in between
the glass beads. 50 or more injections of the specimens could be done before
replacement of the liner by a new one. This injection technique permits direct
injection of large sample volumes of up to 10 μL without troubling the gas
chromatographic separation. Sensitivity of the method was made by the injection
of these samples. The detection boundary for ethanol is 0.1 mg/L (2 μmol/L)
when an injection volume of 5 μL was used.
Principle:
Gas chromatography
(GC) is a method widely used for the identification, quantitation and
separation of mixture components. In this technique, a sample is converted to
the vapor state and a flowing stream of carrier gas (often helium or nitrogen)
sweeps the sample into a thermally-controlled column.
In the case of gas-liquid chromatography, the
column is filled with solid particles coated with a non-volatile liquid, called
as the stationary phase. As the sample mixture travels through the column, the
sample components which act together with the stationary phase use up more time
in the stationary phase vs. the moving gas phase and thus it requires more time
to flow through the column.
The
main goal of GC is the detection and separation of components of a mixture and
it is thus necessary to be able measure the efficiency of a chromatographic
column. One way to express column efficiency is through calculating the height,
equivalent to a theoretical plate (HETP) which is then the column length
divided by the number of theoretical plates.
Ethyl Glucuronide (EtG) is a direct
metabolite of beverage alcohol (ethanol). Its presence in the urine may aid
in detection of alcohol consumption, even it is no longer quantifiable.
EtG in the urine may indicate that alcohol was ingested.
Conventional laboratory methods identify the
actual alcohol in the system, which reflects current consumption within the past
few hours. The presence of EtG in urine means that ethanol was ingested within
the past 3 to 4 days, or about 80 hours after ethanol is eliminated from the
body.
Through ETG alcohol
testing, it can be detected in the urine for up to 3-5 days following
consumption of alcohol. ETG assays are extremely helpful for the measurement of
younger people’s alcohol consumption as well as people under supervision of the
criminal justice system when they tend prohibit alcohol consumption.
Studies show that alcoholics who abstain will
show undetectable levels of EtG in their urine after more or less 80 hours of
detoxification.Studies state that EtG ‘s stability is more
than 4 days at room temperature. Recent experiments also show that upon heating
urine at 100 degrees C stability of ETG increases.Thus, heat does not yield to
the breakdown of EtG.
Methods used in detection of EtG include
immunoassay (EIA or ELISA), gas chromatography/mass spectrometry (GC/MS),
liquid chromatography/mass spectrometry (LC/MS), and liquid chromatography/mass
spectrometry/mass spectrometry (LC/MS/MS). Immunoassay have limited
specificity and sensitivity with a limit of detection of approximately 1000
ng/mL. GC/MS and LC/MS methods are known to be more specific than
immunoassay, and offer detection limits of approximately 500 ng/mL.
DRI Ethyl Glucoronide Assay
DRI
Ethyl Glucoronide Assay is an enzyme immunoassay intended for the
semi-quantitative and qualitative determination of ETG in human urine at 500 –
1000 ng/ml.
DRI ETG
Assay is a liquid ready-to-use homogenous enzyme immunoassay. This assay uses
specific antibodies that can detect ETG without any significant cross
reactivity to other glucoronide compounds. It is also based on competition
between a free drug from the urine with fixed amount of antibody binding sites
and drug labeled with Glucose-6-Phosphate Dehydrogenase (G6PDH). In the absence
of free drug from the sample, the specific antibody binds the drug labeled with
G6PDH and causes a decline in the enzyme activity. A Direct relationship is
created by this phenomenon in urine and enzyme activity. This active enzyme has
the capability to convert NAD to NADH yielding to change in absorbance that can
be measured spectrophotometrically at 340 nm.
Precautions
and Warnings:
1. This
test is for in vitro diagnostic use only. Reagents are harmful if ingested.
2.
Reagents used in the assay contain sodium azide which may react to lead or
copper plumbing. When disposing reagents always flush a large volume of water
to prevent azide build up.
3. Do
not use expired reagents.
BREATH ALCOHOL ANALYSIS
As stated earlier, ethanol levels in the body can be
expelled in the air that we breathe out. Scientists and researchers made another alcohol assay which makes use of breath as
specimen in testing alcohol levels in the body. Breath alcohol analysis started
in the year 1950’s wherein there is only limited knowledge in pulmonary
physiology. Only little amount of alcohol is present in the breath of a person
and the estimated ratio of breath and blood alcohol content is 2100:1.
Therefore, breath analyzers are usually used for screening tests especially
used by police in screening “drunk driving” or drivers who are under the
influence of alcohol. Still the best specimen of choice in testing alcohol
concentration of the body is blood.
TYPES OF BREATH ANALYZERS
· Analyzers
Based on Color Change
(c) www.breathalyzeralcoholtester.com
These
types of breath analyzers are the oldest types. They are based on the principle
of color change which uses chemicals that alter in color in the presence of
alcohol. Chemicals present in the analyzer include Silver nitrate, Potassium
dichromate, and Sulfuric acid. Alcohol from breath is absorbed by the Sulfuric
acid, which also provides acidic medium in order to facilitate the process,
turning to orange in color and then converts Potassium dichromate into Chromium
sulfate which is green in color. The Silver nitrate catalyzes the reaction.
Reaction of color change is measured by photocells connected to a meter.
Breathalyzer
was the first machine invented by Smith and Wesson.
·
Analyzers
Based on Infrared Spectroscopy
(c) electronics.howstuffworks.com
These
types of breath analyzers follow the Beer-Lambert’s Law but uses Infrared
light. Assay is done wherein a continuous spectrum of IR light is passed
through the specimen allowing light to be absorbed and transmitted.
Beer-Lambert’s Law states that the concentration of a substance is directly
proportional to the absorbed light and inversely proportional to the
transmitted light. Therefore, the greater the absorbed light, the greater the
amount of alcohol present in the breath and vice versa; the greater the
transmitted light, the lesser the alcohol present.
The
Intoxilyzer was a brand of breath analyzer functioning based on Infrared
Spectroscopy developed by Omicron in Palo Alto, California.
·
Analyzers
Based on Fuel Cell Technology
(c) www.intox.com
These
types of analyzers contains fuel cell which is a porous disk coated with
platinum oxide on both sides. Acidic solutions with various salts are contained
within the porous layer allowing charged particles to travel along the medium. It
also contains platinum wires which connects the 2 sides of the disk containing
platinum oxide. If alcohol is present on the person’s breath, alcohol is
converted by the platinum oxide, into acetic acid, hydrogen ions, and
electrolytes on the top surface. Hydrogen, which is a charged particle,
combines with oxygen in the air converting to water and removing electrons from
the platinum oxide. This results to excess of electron on top while deficit of
electron on the bottom, allowing electrons to flow through the platinum wire
creating an electric current. This electric current is proportional to the
amount of alcohol present from the air exhaled. The current is converted to
equivalent blood alcohol by the microprocessor of the machine.
Alcotest
and Alco-Sensor are example of machine which utilizes Fuel Cell Technology.
INTERFERENCES OF BREATH ANALYZERS
·
High amounts of substances which are
structurally similar with alcohol metabolites being tested
·
High amount of acetone in the air
·
Acetone is converted to isopropyl
alcohol by the Alcohol dehydrogenase
·
Methanol poisoning
·
Exposure to toluene or xylene
Negative effects of Alcohol Abuse
Long-term
and frequent alcohol consumption is associated with diseases of the liver and
heart, damage to the nervous system, psychiatric problems like depression and
anxiety and even cancer.
Furthermore,
Alcohol consumption can cause sudden changes in behavior. Even at low doses may
impair judgment and coordination. It can also induce feelings of relaxation,
suppression of anxiety, and in some, it may even increase self-confidence. Nevertheless,
as the dose is increased, normally beyond six ounces of 100 proof alcohols.
These euphoric feelings it causes begin to pave way to feelings of depression.
Intoxication occurs when the liver is loses its ability to metabolize more than
one ounce of alcohol every hour. Thus, if a person consumes alcohol exceeding
the body’s ability to metabolize, intoxication occurs. Intoxication may last
from one to 12 hours, and its after-effects (“hang-over”) for more or less 24 hours.
Repeated
consumption of alcohol may lead to tolerance that in turn leads to greater doses
required to attain its desired effects. If a person develops dependence to
alcohol, a sudden cessation of its intake will most likely produce withdrawal
symptoms.
Alcohol can be toxic if its amount reaches a
conc1entration above 460 milligrams of alcohol per 100 milliliters of blood
(0.46 g/dL). Respiratory depression can cause death upon severe alcohol
intoxication, and this can be accelerated if alcohol is to be taken with CNS
depressant medications.
Positive effect of Drinking Alcohol
Looking at the brighter side, drinking alcohol still has
a benefit in an individual’s body. Some say that it is good for the heart.
Well, a lot of studies were already conducted and showed that it reduces one’s
risk of death from Coronary Heart Disease or CHD, but according to the
researchers that only those individuals that drink moderately are qualified in
this good news. Defining “moderate drinking,” in some studies, refers to less
than one drink per day. And defining “one drink,” in the US, considers
approximately 12 ounces of beer, 5 ounces of wine, or 1½ ounces of spirits
(hard liquor such as gin or whiskey). Each sums up at approximately 12 to 14
grams of alcohol. Due to these studies, researchers came up to a hypothesis
that alcohol may prevent the risk from a condition of having an impaired blood
flow to the extremities resulting from narrowed blood vessels or Peripheral
Vascular Disease. But according from the data from Framingham Heart Study,
there is no significant relationship between alcohol and PVD, but further
studies from Physicians’ Health Study showed that there is a 26% risk reduction
from PVD among “moderate drinkers.” But still, comparing these positive effects
of drinking alcohol from its negative effects, in our opinion, it is not
beneficial to drink alcohol since a lot of negative effects arise in consuming
such drinks.
References:
Michael L. Bishop: Clinical
Chemistry Techniques, Principles, Correlations 6th edition copyright
2010 by
Lippincott Williams & Wilkins, Wolters Kluwer business, pp. 624-627
Porter WF, Moyer TP:
Clinical toxicology. In Tietz Textbook of Clinical Chemistry. 4th edition.
Edited
by CA Burtis, ER Ashwood. Philadelphia, WB Saunders Company, 1993, pp
1155-1235
The Role of the
Laboratory in the Investigation of Solvent Abuse Hum Exp Toxicology 1982; v. 1,
p.299-
311.
The Science of
Drinking: How Alcohol Affects Your Body and Mind By Amitava Dasgupta
http://www.hsph.harvard.edu/nutritionsource/alcohol-full-story/
“Alcohol: Balancing Risks and
Benefits”
http://pubs.niaaa.nih.gov/publications/arh24-1/05-11.pdf “Tenth Special
Report to the United States
Congress on Alcohol and Health: Health Risks
and Benefits of Alcohol Consumption”
“DRI Ethyl Glucoronide Assay”
“Liquid chromatographic
determination of ethyl alcohol in body fluids”
“Quantifying ethanol by high
performance liquid chromatography with precolumn enzymatic conversion and
derivatization with fluorimetric detection”
“High-Performance
Liquid Chromatography”
“Highly
sensitive gas chromatographic analysis of ethanol in whole blood, serum, urine,
and fecal supernatants by the direct injection method”
encyclopedia2.thefreedictionary.com/Enzymatic+Methods+of+Analysis
“Enzymatic Methods of Analysis”
“Drug Check Alcohol
Rapid Test”
“Ethanol Analysis
by Headspace Gas Chromatography with Simultaneous Flame-Ionization and Mass
Spectrometry Detection”
“EMIT Ethyl Alcohol
Assays: Answers to Frequently Asked
Questions”
“Ethyl
Glucoronide (ETG) Alcohol Testing”
“Alcohol ETG Urine Testing
Resources”
“Emit II Plus Ethyl
Alcohol Assay”
“Gas
Chromatography: Principles and Determination of Percent Alcohol”
