BAC figure Flashcards
What were the experimental conditions for the BAC study?
Ogg1 mice were treated i.p. with EtOH (4 g/kg or 2 x 2.5 g/kg EtOH, 4 hours apart). Mice were anesthetized using isoflurane for cardiac puncture blood sampling. Blood samples were collected from 4-5 male mice in heparinized vacutainers (lithium heparin 68 USP units per tube; Becton–Dickinson, Oakville, ON, Canada) at 0, 0.5, 1, 3, 5 and 7 hours. Blood EtOH concentrations were measured by the British Columbia Provincial Toxicology Centre using high-performance liquid chromatography with flame ionization detection267.
What were the BAC results in the Ogg1 mice?
To determine a maternal EtOH dose that caused sufficient oxidative DNA damage in fetal brains, without causing apparent maternal or fetal toxicity, we tested 2 doses based on the literature. In the Ogg1 strain, a single i.p. dose of 4 g/kg produced a peak blood EtOH concentration of 504 + 89 mg/dL (Fig. 4-2). Administration of 2 doses of 2.5 g/kg EtOH, 4 hours apart, produced a peak of 265 + 40 mg/dL.
How was the BAC measured?
Principles of HS-GC-MS
A. Headspace Sampling (HS)
* Analyzes the volatile fraction of a sample without injecting the sample itself.
* The sample is placed in a sealed vial and heated to allow volatile components to partition into the gas phase.
* A portion of the headspace gas is injected into the GC system.
B. Gas Chromatography (GC)
* Separates volatile components based on their boiling points and polarity.
* Uses a carrier gas (e.g., helium or nitrogen) to transport the sample through a capillary column.
* A stationary phase within the column interacts with the analytes, causing separation based on retention time.
C. Mass Spectrometry (MS)
* Identifies and quantifies compounds based on their mass-to-charge ratio (m/z).
* The analyte molecules are ionized, fragmented, and detected by the mass spectrometer.
* Provides a unique mass spectrum for each compound, aiding in identification.
D.Detection with FID
* The eluted ethanol enters the FID detector, where it is burned in a hydrogen-air flame.
* The combustion ionizes ethanol molecules, generating a measurable electrical current.
* The signal intensity is proportional to the ethanol concentration in the sample.
What are the advantages and disadvantages of HS-GC with flame ionizing detection?
Advantages of HS-GC-MS
✅ No Sample Preparation Needed – Directly analyzes volatile compounds without complex extractions.
✅ Minimizes Contamination – Avoids direct injection of liquids or solids, reducing instrument fouling.
✅ Selective and Sensitive – HS-GC-MS can detect trace levels of analytes with high specificity.
✅ Automated and Reproducible – Many modern HS systems allow for automated, high-throughput analysis.
✅ Non-Destructive – The sample remains largely intact, allowing further analysis if needed.
Limitations of HS-GC-MS
❌ Only Volatile Compounds Can Be Analyzed – Non-volatile analytes require different techniques (e.g., LC-MS).
❌ Limited Sensitivity for Some Analytes – Some compounds have low headspace partitioning.
❌ Optimization Required – Parameters like temperature, equilibration time, and vial pressure must be optimized for each sample type.
Discuss the impact of the mouse strain on BAC
Alcohol studies and mouse strains
* Blood alcohol concentration (BAC) in mice varies significantly across different strains, reflecting genetic influences on alcohol consumption and metabolism.
* Strain-Specific BAC Variations:
Research comparing multiple inbred mouse strains has revealed substantial differences in both ethanol intake and resultant BACs. In a study examining 12 inbred strains, C57BL/6J mice exhibited the highest ethanol consumption and BAC levels. Conversely, strains such as DBA/2J consumed less ethanol and consequently had lower BACs. These findings underscore the role of genetic factors in alcohol consumption behaviors and metabolism.
pubmed.ncbi.nlm.nih.gov
1. Key Factors Affecting BAC in Mice
Several factors influence how BAC differs among mouse strains:
A. Genetic Background
* Different inbred and outbred mouse strains exhibit distinct responses to ethanol due to genetic variability in alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the enzymes responsible for ethanol metabolism.
* Selectively bred strains, such as High Alcohol Preferring (HAP) and Low Alcohol Preferring (LAP) mice, demonstrate extreme differences in voluntary ethanol consumption and BAC levels.
B. Metabolic Rate and Alcohol Clearance
* Mice metabolize ethanol at different rates based on strain-specific enzyme activity.
* Strains with faster alcohol clearance exhibit lower peak BAC, while strains with slower metabolism maintain higher BAC levels for longer durations.
C. Drinking Behavior and Ethanol Intake
* Voluntary ethanol consumption impacts BAC significantly. Some strains drink large volumes of ethanol readily, leading to higher BAC, while others avoid it, resulting in lower levels.
D. Sex Differences
* Male and female mice of the same strain can have different BACs due to sex-dependent metabolic rates, hormonal influences, and differences in body composition (fat-to-water ratio).
What are some examples of different mouse strains and how the differ in their BAC?
A. C57BL/6J (B6) Mice
* High ethanol preference and voluntary consumption.
* Achieve high BAC levels (often exceeding 100 mg/dL or 1.0 mg/mL) under the Drinking in the Dark (DID) paradigm.
* Have a high tolerance to alcohol-induced sedation.
* Used extensively in alcohol preference and dependence studies.
✅ Genetic Traits:
* High expression of ADH1 and ALDH2, leading to efficient ethanol metabolism.
* Mutations in the GABA-A receptor (GABRA2) reduce sensitivity to ethanol-induced sedation, allowing for higher intake.
* Higher expression of dopamine D2 receptors (DRD2), increasing ethanol reinforcement and voluntary drinking.
* NPY system is altered, leading to increased alcohol preference.
✅ Effects on BAC:
* High voluntary ethanol consumption and high BAC levels (~100–150 mg/dL in the DID paradigm).
* High tolerance to sedative effects of ethanol.
* Used extensively in studies of alcohol preference and dependence.
B. DBA/2J Mice * Low ethanol preference and significantly lower voluntary consumption. * Exhibit rapid sedation and strong aversion to alcohol due to genetic sensitivity. * Achieve much lower BACs despite access to ethanol. * Often used in studies investigating genetic resistance to alcohol consumption. ❌ Genetic Traits: * Lower expression of ADH1, leading to slower ethanol metabolism. * Higher activity of GABA-A receptors, making the strain highly sensitive to alcohol-induced sedation. * Lower levels of dopamine D2 receptors, reducing reinforcement and alcohol-seeking behavior. * Higher NPY activity, which suppresses ethanol intake. ❌ Effects on BAC: * Low voluntary ethanol consumption and low BAC levels (~20–40 mg/dL). * Strong aversion to ethanol due to rapid sedation. * Used in studies of genetic resistance to alcoholism. C. FVB/NJ Mice * Moderate ethanol preference, but show a strong sedative response. * Often used for transgenic studies where alcohol metabolism is a factor. D. Swiss Webster Mice * Show moderate ethanol consumption and BAC levels. * Commonly used as a general-purpose strain in toxicology and pharmacokinetics studies. 🔷 Genetic Traits: * Moderate ADH1 activity but variable ALDH2 function, leading to variability in alcohol metabolism. * Some lines show higher GABA receptor sensitivity, leading to sedation at lower doses. 🔷 Effects on BAC: * BAC levels range from 40–80 mg/dL depending on drinking conditions. * Commonly used as a general-purpose strain in pharmacokinetics and alcohol toxicology. E. High Drinking in the Dark (HDID) Mice * Selectively bred to reach high BACs (>1 mg/mL) after binge-like ethanol drinking. * Exhibit increased ethanol consumption without changes in alcohol preference. Used for modeling binge drinking behavior and its neur
