2.15: Bilirubin (Waters and Gerande – DMSO Method)
- Page ID
- 38643
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)RELATED READING: Chapters 27, 36. See Methods in CD-ROM for Bilirubin.
OBJECTIVES
Upon completion of this exercise, appropriate discussion, and related reading, the student will be able to:
- Perform direct and total bilirubin analysis on 2 samples with 90% accuracy.
- Explain the effect of free hemoglobin on bilirubin determinations in this method.
PRINCIPLE
Bilirubin will react with diazotized sulfanilic acid to yield a purple compound, azobilirubin. This product can be quantitated spectrophotometrically at 560 nm. This procedure is used to measure the soluble conjugated (direct-reacting) bilirubin alone or in combination with the less soluble unconjugated (indirect-reacting) bilirubin. To measure total bilirubin, a suitable solvent that allows for the measurement of both direct and indirect—reacting bilirubin is used. The unconjugated bilirubin concentration is the difference between direct-reacting and total bilirubin values. Care should be taken to protect samples from strong or prolonged light because bilirubin pigments are light sensitive.
MATERIALS
- Total bilirubin reagent
- Spectrophotometer
- Direct-reacting bilirubin
- Cuvettes
- Sodium nitrite
- Pipets
- Standard
- 13 x 100 mm
- Test Tubes
- Controls
- Samples
PROCEDURE
Total Bilirubin
- Label a sufficient number of 13 x 100 mm test tubes as “Test” and “Blank” for each standard, control, and serum sample to be tested.
- Add 3 mL of total bilirubin reagent to all “Blank” and “Test” tubes.
- Using a Pasteur pipet, add 1 drop of sodium nitrite reagent to each “Test” tube.
- At precise one minute intervals, add 200 \(\mu\)L of the appropriate standard/control/sample to each “Test” and “Blank” tube.
- Mix well.
- Incubate at room temperature for exactly 5 minutes.
- Read the absorbance of the first sample against it’s own blank at 560 nm; repeat for each subsequent sample at 1 minute intervals.
- Record your results on the data sheet.
Direct Bilirubin
- Label a sufficient number of 13 x 100 mm test tubes as “Blank” and “Test” for the patient samples only.
- Add 3 mL of direct bilirubin reagent to each “Blank” and “Test” tube.
- Using a Pasteur pipet, add 1 drop of sodium nitrite reagent to each “Test” tube.
- At precise one minute intervals, add 200 \(\mu\)L of serum to the “Blank” and “Test” tube for each sample and mix.
- Incubate at room temperature for exactly 5 minutes.
- Read the absorbance of the first sample against it’s own blank at 560 nm; repeat for each subsequent sample at 1 minute intervals.
- Record your results on the data sheet.
OPTIONAL EXERCISE
- Prepare red blood cells washed free of plasma. Resuspend the cells with distilled water to 5 times the original blood volume.
- Add 0.05 mL of this hemolysate to one of the controls or samples run in the above procedure described for total bilirubin.
- Record the results on the data sheet.
| DATA SHEET, EXERCISE #15 |
NAME: ___________ DATE: ___________ |
RESULTS
| Absorbance (total) | Total Bilirubin (mg/L) | Absorbance Direct | Direct (Conjugated) Bilirubin (mg/L) | Indirect (Unconjugated Bilirubin (mg/L) | |
|---|---|---|---|---|---|
| Standard | |||||
| Normal Control | |||||
| Abnormal Control | |||||
| Sample # | |||||
| Sample # |
CALCULATIONS
Calculate the total bilirubin for the controls and serum samples. Calculate the direct and indirect bilirubin for the patient serum samples. Use the absorbance/concentration proportion method.
\[\text{Total bilirubin} = \text{conjugated (“direct”) bilirubin} - \text{unconjugated (“indirect”) bilirubin}\]
\[\text{Unconjugated bilirubin} = \text{Total} - \text{Direct bilirubin}\]
Optional Exercise
| Absorbance | Total Bilirubin (mg/L) | |
|---|---|---|
| Hemoglobin-spiked sample | ||
| Unspiked sample (# ) |
Discussion Questions
- What substances are known to cause interference with this method?
- Why is it necessary to run a serum blank with each sample?
- What is the purpose of the sodium nitrite in the reaction?
- Is the type of blank employed in this exercise a reagent or sample blank?
- What compound was employed in the “total” reagent to solubilize the unconjugated bilirubin? What other solubilizers were also used?