ENZYMATIC ASSAY BLOOD TESTING
By: Mark Thiessen
The Thiessen Law Firm
1017 Heights Blvd.
Houston, Texas 77008
1. Whole blood is drawn from the arm (great for GC)
2. Need Plasma or Serum for Enzymatic Assay testing, so strip the proteins from the blood with TCA (Trichloroacetic Acid)
a. And centerfuge the whole blood with the proteinizing agent (TCA)
b. That leaves you with:
i. Supernate – top stuff (plasma) and alcohol
ii. Precipitate – red corpuscles, white corpuscles, and platelets
- TCA protein pellet
- Red blood cells
3. Pipette or pour off the Supernate (called aspirating)
a. The Precipitate is thrown out
4. Put the aspirated Supernate into the analytical device or Autoanalyzer – Siemens Dimension Vista Platform
5. In order to get alcohol to react, add a known quantity of an enzyme (NAD+) and ADH (Alcohol Dehydrogenase) which catalyzes metabolism of alcohol to acetaldehyde
ETOH + NAD+ --------à Acetaldehyde + NADH + H+
6. Spectrometry (a colormetric response) is used to analyze light going in v.s. light coming out.
a. Beer’s Law aka Lambert – Beer Law (also same law in Intoxilyzer 5000
b. Spectrophotometer is a device that measure light intensity (photometer) as a function of a color or a wavelength of light.
i. You need to know the spectral bandwidth and linear range of absorption measurement of the spectrophotometer
c. So, use a source light and shine the light into the monochromator, which picks the particular wavelength
d. And that wavelength is beamed at the sample – 340 nanometers for NADH
e. Sample absorbs that light (specifically NADH absorbs it at 340)
f. Then photodetector on the other end measures how much actually made it through
g. SO, if the analyte or NADH is present then it interferes with the energy emitted
h. The photodetector will detect less energy
i. And a comparison is made between expected and what was measured
7. Get a colormetric response: whether is lighter or darker based upon the concentrate of the analyte or NADH
PROBLEMS – NADH is not specific just for ETOH to the exclusion of others.
1. Acute Fluid Loss – pump someone full of Lactated Ringers Solution
a. used to combat acidosis, which is a chemical imbalance with acute fluid loss or renal failure
2. Lactate – compound formed as a result of trauma and hypoxia (tissue deprived of oxygen)
3. LDH – Lactate Dehydrogenase – is in the muscle cells to breakdown lactate formed after anaerobic exercise
-but, also released into blood stream after trauma or car crash
ETOH + ADH + NAD + Ringer, Lactate or LDH ----à Acetaldehyde + NADH (but way more) + H
8. Measures more NADH than if Ringers, Lactate, or LDH not present. The machine can’t differentiate NADH from alcohol or the others!
9. = FALSE HIGH SERUM ETHANOL RESULT
(Many thanks to Justin McShane for his edits)