Attorney-at-Law, Montgomery, Alabama Technical Editor: Jack R. Kalin, PhD, DFTCB
Toxicology Discipline Chief, Alabama Department of Forensic Sciences
Introduction: Analysis of blood evidence in a DUI, traffic assault, or traffic homicide case is a critical element of the case for the investigating law enforcement officer as well as the prosecutor. Blood samples taken from the defendant are a key piece of evidence in establishing criminal culpability. This document is designed to give the non- scientifically trained law enforcement officer, prosecutor, or attorney sufficient information to understand the basic properties of alcohol and blood, a basic understanding of Alabama law regarding legal issues concerning the admissibility of blood sample evidence, and how blood samples are analyzed. Understanding Alcohol and Blood: The Basics
Alcohol: Alcohol1 is one of the oldest substances known to mankind, but its effects are continually being studied, re-studied, and analyzed. Beverage alcohol is commonly referred to as “ethanol” or “ethyl alcohol” as well as “alcohol.” Ethanol is one of a family of alcohols which includes methanol (methyl alcohol or “wood alcohol”), 1-Propanol (propyl alcohol), 1-Butanol (butyl alcohol), 2-Propanol (isopropyl alcohol or “rubbing alcohol”), and ethanediol (ethylene glycol or “antifreeze”). Common Alcohol Compounds
Common Name IUPAC Formula
Methyl alcohol Methanol CH3OH
Ethyl alcohol Ethanol CH3CH2OH
n-Propyl alcohol 1-Propanol CH3CH2CH2OH
Isopropyl alcohol 2-Propanol (CH3)2CHOH
n-Butyl alcohol 1-Butanol CH3 (CH2)2CH2OH Ethyl alcohol (ethanol) is a very small molecule that iscompletely soluble (miscible) in water. Ethanol is lighter than water (specific gravity = 0.789) and has a boiling point of 78 degrees Celsius. The fact that alcohol is both lighter than water and boils at a lower boiling point is essential in the distillation process. The main source of consumed alcohol is commercially prepared beverages: fermented alcoholic beverages and distilled alcoholic beverages. Beer and wine are typical fermented beverages. In both cases, a natural product (barley in the case of beer and grapes in the case of wine) is fermented by the addition of yeast microorganisms. The alcohol that is produced is the waste byproduct of the metabolism of the yeast’s or bacteria’s consumption of sugars found in the natural product. Throughout the remainder of this discussion, the terms ethanol and alcohol may be used interchangeably.
The Fermentation Process:
The understanding of alcohol must begin with the fermentation process. Fermentation of sugars by yeast is the oldest synthetic organic chemical produced by man. During fermentation, sugar is converted to drinking alcohol and carbon dioxide is released as gas bubbles. This chemical change was a great mystery to ancient man because the mixture appeared to be boiling without heat. It was not until the mid-19th Century when the noted French chemist and natural scientist Louis Pasteur discovered that alcoholic fermentation could occur only in the presence of small living “ferments” or, as they are known today, yeasts2.
Pasteur’s study on fermentation:
Louis Pasteur (1822-1895) was one of the most extraordinary scientists in history, leaving a legacy of scientific contributions which include an understanding of how microorganisms carry on the biochemical process of fermentation, the establishment of the causal relationship between microorganisms and disease, and the concept of destroying microorganisms to halt the transmission of communicable disease. These achievements led him to be called the founder of modern microbiology.
After his early education Pasteur went to Paris, studied at the Sorbonne, then began teaching chemistry while still a student. After being appointed chemistry professor at a new university in Lille, France, Pasteur began work on yeast cells and showed how they produce alcohol and carbon dioxide from sugar during the process of fermentation. Fermentation is a form of cellular respiration carried on by yeast cells; a way of getting energy for cells when there is no oxygen present. Pasteur found that fermentation could take place only when living yeast cells were present.
Pasteur was then called upon to tackle one of the most persistent problems plaguing the French beverage industry at the time, that of spoilage. Of special concern was the spoiling of wine and beer, which caused both great economic loss to the industry and tarnished France’s reputation for fine vintage wines. Vintners wanted to know the cause of l’amer, a condition that was destroying the best burgundies.
Pasteur examined wine under the microscope and noticed that when aged properly the liquid contained little spherical yeast cells. But when the wine turned sour, there was a proliferation of bacterial cells which were producing lactic acid. It was the run-away production of lactic acid that caused the spoilage. Pasteur suggested that gradually heating the wine to a temperature range of 120 - 130 degrees Fahrenheit would kill the bacteria that produced lactic acid and allow the wine to age properly. Pasteur’s book, Etudes sur le Vin, published in 1866 revolutionized the wine industry.
In his work with yeast, Pasteur also found that air should be kept from fermenting wine. In the presence of oxygen, yeasts and bacteria break down alcohol into acetic acid - vinegar. Pasteur carried on many experiments with yeast. He showed that fermentation can take place without oxygen (anaerobic conditions), but that the process still involved living micro-organisms such as yeast.
Pasteur’s discoveries of the spoilage inherent in the natural fermentation process allowed him to develop the fundamental concept of the “germ” theory of disease transmission. While performing his experiments dealing with yeasts, and later with the silk-worm industry, Pasteur had also developed what has come to be known today as sterile technique, or the boiling or heating of instruments and food to prevent the proliferation of microorganisms. Pasteur’s theories of transmission of micro-organisms were gradually accepted by medical science during the decade 1870 - 1880, after work by noted British medical doctor and surgeon Joseph Lister confirmed the germ-transmission theory of disease control in relation to infection rates in sterile and non- sterile operating settings.
In 1897, scientist Edward Buchner reported that yeasts could be broken up and that the cell-free yeast juice could ferment sugar. Later, it was found that the yeast juice contains the enzymes necessary for the conversion of sugars to alcohol and carbon dioxide. As a consequence of isolating the enzymes necessary for fermentation, mass production of beer and wine products was greatly facilitated.
The basic understanding of the potential effects of naturally occurring yeasts and other microorganisms and the subsequent collection, preservation, and testing of blood samples cannot be overstated. As will be explained later in this material, any naturally occurring yeast or micro-organism present during the collection phase of the whole blood sample can have a significant effect on the resulting reported blood alcohol concentration.
ETHANOL IN BEVERAGES
Fermented Beverages: Wine ethanol concentrations generally range from 12-15 % from the fermentation of crushed grapes, but may be “fortified” by the introduction of additional alcohol during the production process. Most table wine sold in the state of Alabama is 12.5 % ethanol by volume3. Most commonly, beer with a 3.2-5 % ethanol concentration is sold within the state. Beer ethanol concentrations when fermented can range from 3 % to as high as 15 %, but are regulated by state law to not exceed 6 % alcohol by volume4.
Distilled Beverages: Production of distilled alcoholic beverages begins with the fermentation of one or more natural grains such as corn, wheat, rye and barley. These grains are the source of carbohydrates (sugars) necessary for the process. The result is a wort (fermented fluid) containing up to 12 % ethanol by volume, which is then distilled by heating. Alcohol (ethanol), which evaporates at 78 degrees Celsius, travels into a cooling apparatus (condenser) where it re-liquefies. The now-concentrated ethanol can be collected in a storage container, and given flavorings. Whiskey, vodka, gin, and a variety of other alcohol beverages are produced in this manner. What distinguishes the various beverage types is the carbohydrate source (grain). Homemade distilled ethanol, commonly referred to as “moonshine”, while generally having no flavoring added, possesses a fruit-like odor. The ethanol concentration in “moonshine” can range from the low 60-proof range (30% ethanol) to as high as 120-proof (60% ethanol). The name “moonshine” is derived from the nocturnal, clandestine nature of this illicit beverage production5.
Schematic of whiskey “Still” as used in production of “moonshine” whiskey:
ETHANOL IN BLOOD
Ethanol is classified as a ‘Central Nervous System’ depressant (CNS) whose impairing effects are in proportion to its presence in the CNS. However, blood rather than brain tissue is the preferred representative specimen for a chemical test for impairment because blood delivers ethanol to the CNS and thereby accurately reflects CNS exposure to ethanol. A large body of research exists which relates ethanol concentrations in whole blood with human performance. Whereas any biological specimen may be analyzed for ethanol (blood, plasma, serum, urine, ocular fluid, tissues), results for whole blood provide an accepted, uniform standard for interpretations. For these reasons, statutes typically base per se limits for ethanol content in whole blood (or breath, which is a related, but different subject, and is not addressed in this publication). Forensic ethanol analyses are conducted with whole blood when it is available. Determining a subject’s blood alcohol concentration (BAC) is the single most important issue in establishingcriminal and civil liability in a judicial proceeding where alcohol is alleged to have been an element of the offense or the cause of action.
Absorption Principles: Whereas the entire gastro-intestinal tract (GI) is capable of alcohol absorption, almost 90% takes place in the small intestine where structural microvilli greatly increase the surface area of the gut available for absorption. With its small molecular size, ethanol readily crosses the GI tract membranes via passive diffusion and enters the circulation, mixing completely with the fluid portion of blood (blood is approximately 85% water). Ethanol then distributes throughout the body where it rapidly crosses back through membranes into the tissues and, most significantly, across the blood-brain barrier.
Blood: The adult human contains approximately five liters of blood, constituting about 8% of the total body weight. Whole blood is a complex, heterogeneous mixture of solid material and fluid. The solid material comprises red blood cells (erythrocytes), platelets (thrombocytes), and white blood cells (leucocytes - lymphocytes and phagocytes). Each cell type has a specific function:
• Red blood cells contain hemoglobin which binds oxygen and transports it throughout the body.
• Platelets participate in forming blood clots
• White blood cells are responsible for cell-mediated immune responses to foreign organisms
There are approximately 500 times more erythrocytes than leukocytes. The volume portion of whole blood occupied by red cells is the hematocrit (HCT), which is defined as the volume of red cells divided by the total blood volume. An average HCT for adult males is 40% to 50% and for adult females, 35% to 45%. The HCT changes with age. A low HCT indicates a relatively lower content of red blood cells in whole blood, which may be due to anemia, blood loss (internal or external) or other disease conditions.
The fluid portion of whole blood is called plasma, which may be prepared by removing the cellular solids from unclotted blood (typically by centrifugation). Serum is the fluid portion of whole blood remaining after the blood has clotted and the clot is removed. Because plasma and serum contain no cellular solids, they contain a relatively greater content of water than does whole blood. This is significant because ethanol distributes into the various body compartments in proportion to their water content. In that regard, plasma and serum, with a water content of 95% to 97 % will contain more ethanol than the whole blood from which they are derived (approximately 85% water). This difference, 10% to 15%, highlights the importance of establishing what specimen - whole blood or plasma - was tested for ethanol before making any interpretations of the results. This issue will be discussed further in this publication.
Blood alcohol concentration: Results of forensic analyses are typically expressed as a grams of ethanol per 100 mL of specimen or grams percent (g %) or simply percent (%). That a blood ethanol concentration was reported to be 0.080 g/100 mL, however, does not imply that 100 mL of blood was analyzed and 0.080 grams of ethanol were detected. The Alabama Department of Forensic Sciences (ADFS) analyzes 100 microliters (0.10 mL or 100 millionths of a liter) of specimen. From this volume of specimen, the actual mass of ethanol detected is on the order of 500 nanograms (500 billionths of a gram). While this may seem miniscule, it represents an astronomical 6 million billion molecules or the equivalent of some 1 million times the population of the earth and is sufficient to provide reliable and accurate results in a properly calibrated analytical system. ETHANOL IN THE BRAIN
Alcohol affects various centers in the brain, both higher and lower order:
Ethanol is a ‘Central Nervous System’ depressant (CNS) thataffects the brain and nervous system quickly after it enters the blood stream. The effects of ethanol are continuous and progressive, meaning the overall effect on the CNS and on subject performance increases as the concentration of ethanol in the CNS increases. However, all centers of the brain are not equally affected by the same BAC - the higher-order centers are more sensitive than the lower-order centers. As the BAC increases, more and more centers of the brain are depressed until all centers are depressed. The order in which alcohol affects the various brain centers is as follows:
Hypothalamus and pituitary gland
Medulla (brain stem)
The cerebral cortex is the part of the brain responsible for the highest functions of human performance. The cortex processes information from the senses, performs “thought” processing and consciousness (in combination with a structure called the basal ganglia), initiates most voluntary muscle movements and influences lower-order brain centers. In the cortex, the effects of alcohol are commonly recognized:
• Depresses the behavioral inhibitory centers - The person becomes more talkative, more self-confident and less socially inhibited.
• Impedes the processing of information from the senses - Vision can be affected at low levels of alcohol. Depth-of-field and peripheral vision are affected at BAC levels as low as 0.03% to 0.04%. General reflex response is slowed and fine motor skills are impaired at low levels of alcohol. Also, the threshold for pain is raised.
• Inhibits thought a process - The person does not use good judgment or think clearly. These effects become more pronounced as the blood alcohol concentration increases.
The limbic system consists of areas of the brain called the hippocampus and septal area. The limbic system controls emotions, learning and memory. As alcohol affects this system, the person is subject to exaggerated states of emotion (anger, aggressiveness, withdrawal) and memory loss.
The cerebellum coordinates the movement of muscles. The brain impulses that begin muscle movement originate in the motor centers of the cerebral cortex and travel through the medulla and spinal cord to the muscles. As the nerve signals pass through the medulla, they are influenced by nerve impulses from the cerebellum. The cerebellum controls fine movements. For example, a sober individual can normally touch finger to their nose in one smooth motion with their eyes closed; if the cerebellum is not functioning, the motion would be extremely shaky or jerky. As alcohol affects the cerebellum, muscle movements become uncoordinated6. It is at the approximate level of 0.08 % to 0.10 % blood alcohol concentration that noticeable impairment can be determined through the use of properly administered field sobriety tests.
In addition to coordinating voluntary muscle movements, the cerebellum also coordinates the fine muscle movements involved in maintaining balance. As alcohol affects the cerebellum, a person loses his or her balance frequently. At this stage, this person might be described as “falling down drunk.”
Hypothalamus and Pituitary Gland
The hypothalamus is an area of the brain that controls and influences many automatic functions of the brain through actions on the medulla, and coordinates many chemical or endocrine functions (secretions of sex, thyroid and growth hormones) through chemical and nerve impulse actions on the pituitary gland.
The medulla (or brain stem) controls or influences involuntary functions such asbreathing, heart rate, temperature and consciousness. As alcohol depresses upper centers in the medulla, such as the reticular formation, a person will start to feel sleepy and may eventually become unconscious as BAC increases. If the BAC gets high enough to influence the breathing, heart rate and temperature centers, a person will breathe slowly or stop breathing altogether, and both blood pressure and body temperature will fall. These conditions can be fatal.
Stages of Alcoholic Influence/Intoxication: Kurt M. Dubowski, Ph.D., The University of Oklahoma Department of Medicine, a noted authority on alcohol and the dynamics of ethanol distribution and the effects on the human body, developed a chart describing the clinical signs and symptoms resulting from the ingestion of alcohol and which is based on the blood alcohol concentration measured in grams/100 mL. Because not all centers of the brain are affected at the same blood alcohol concentrations, different subject behaviors may be visible at similar alcohol levels. This gives rise to the myth that “everyone reacts differently to alcohol”. Actually, everyone reacts the same to alcohol; their CNS becomes depressed. What is different, however, is the degree to which each function of the CNS is depressed in each subject. The sum of these depressed functions results in the behaviors visible among subjects, which may be different. That blood alcohol concentrations overlap for each clinical sign demonstrates this phenomenon. CLINICAL SIGNS/SYMPTOMS