Document NGg2pM8NOe2697yxyOkvzGQpQ
BIO-RAD\^crfkn<it<nie4.
MAY 1968
DIRECTIONS FOR PERFORMING THE DAVIS TEST* FOR URINARY ALA
The column- chromatography procedure which, you are about to perform for the determination of urinary delta-aminolevulinic acid (ALA) as an indicator
for early lead poisoning is divided into three se quential phases:
PHASE 1
Removal of porphobilinogen and urea from the urine which would inter fere with the analysis of ALA.
PHASE 2 Column elution of ALA.
PHASE 3 Colorimetric determination of eluted ALA.
Components of the Davis ALA Test Kit for 40 deter minations of urinary ALA. (Any of the three numbered catalog items listed below may be ordered separately....)
1* Catalog No. 95502, Support Rack with drain tray and numbered pipet guide (non-disposable).
2. Catalog No. 95004, disposable Piggy-back Col umn Units with following reagents and standard:
a. Bottle containing 500 ml of 1.0 M sodium acetate solution#(68 gm of CH^COONa.31^0 diluted with water to 500 ml). Refrigerate upon delivery.
b. Bottle containing 20 ml of acetylacetone (2,4-pentanedione)
c. Bottle containing 10 gm (pre-weighed) of reagent-grade ^-dimethylaminobenzaldehyde
d Bottle containing 10 ml of stock solution of ALA hydrochloride standard dissolved in acetate buffer, pH 4.6.** Refrigerate upon delivery. Each ml of stock solution
contains 125 pgr of A1A.HC1 which is equi valent to 100 jig of pure ALA.
3. Catalog No. 95501, one pound bottle of
perchloric acid (quantity sufficient for 200
tests*).
NOTE: 420 ml of glacial acetic acid is re quired but is not supplied with the kit be cause of postal shipping regulations.
BEGIN TEST PROCEDURE
STEP 1
TECHNIQUE
Remove numbered pipet guide and place piggy--back column units in support rack.
NOTE:
The piggy-back column units arrive with the top and bot tom resins in the correct position. This two column set is referred to as the column unit. Each urine sam ple requires one column unit for the determination of ALA.
*Davis, J.R. and Andelman, S.L., Arch. Environ. Health, 15:53-59 (1967)
STEP 2
TECHNIQUE
Apply 10 ml of distilled water to the column, unit and allow to drain through both columns into drain tray.
SUGGESTION
Use of a 10 ml Cornwall contin uous, syringe-type pipetter will permit fast, automatic de livery of the desired quantity of water to each column unit.
**0.8$ boric acid present as a preservative.
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STEP 3
V
TECHNIQUE
Replace numbered pipet guide. Apply 0.5tnl of* random urine sam ple to the top of the column unit by inserting pipet through the correctly numbered hole in pipet guide.
SUGGESTION
Disposable pipets have been found to be quite economical when test ing large numbers of urine samples Allowing the pipets to remain in the numbered, holes until all of the samples have been added has been found to eliminate the pos sibility of applying two urine samples to the same column unit.
STEP 4
TECHNIQUE
Remove pipets from column units. Remove numbered pipet guide. Ap ply 10ml of distilled water to each column unit and allow to drain through columns into drain tray. Repeat two more times for a total water wash of 30ml.
SUGGESTION
Xt is important that all column units are completely drained of the previous water wash before applying the next aliquot of 10ml of water to the column unit. This will prevent column overflowing.
STEP 5
TECHNIQUE
Remove and discard top dispos able column from piggy-back col umn unit. The 6 aliquots of ALA hydrochloride stock solution can now be pipetted into test tubes as working standards as indicated in the section on Preparation of a Standard Calibration Curve as shown on page 3- Subsequent steps for both the standards and unknowns are identical beginning with the addition of 1.0M sodium acetate.
STEP 6
4
TECHNIQUE
Remove bottom columns from sup port rack and place in empty numbered 20x150mm Pyrex test tubes held in a standard test tube rack. Add 7.0ml of 1.0M sodium acetate to each column and collect the drained liquid in the test tube*
SUGGESTION
2Oxl50mm disposable borosilicate glass culture tubes have been found to be quite economical when testing large numbers of urine samples.
STEP 7
TECHNIQUE
Remove and discard bottom dispos able column, from test tube. Add 0.2ml of acetylacetone to the contents of the test tube and mix
SUGGESTION
TJse of a 1 ml Repipet will per mit the rapid and precise deli very of acetylacetone. Use of a Vortex mixer will permit rapid and easy mixing of test tube con tents
Amounts of acetylacetone greatly in excess of 0*2 ml will inhibit final color development.
Xt is important that the test tubes be of the sug gested size in order to hold the column at idle cor rect height. Xt is also of importance that all column units be fully drained in order to completely elute the ALA.
STEP 8
Notes
TECHNIQUE
Place test tube rack containing test tubes in a boiling water bath (90C or greater) for 10 min., after which the rack should be removed from water bath and test tubes allowed to cool to room temperature.
SUGGESTION
Xn addition to the standard tem perature controlled water baths commonly available, a Bunsen burner or an electric hot plate can be used. Time for cooling of test tubes can be markedly decreased by immersing test tube rack in a pan of room temperature water.
Allowing test tubes to remain in boiling water bath greatly in excess of 10 minutes will inhibit final color development.
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STEP 9
TECHNIQUE
Add y.QmX of fresh Ehrlich1 a
`''FRESH Reagent to test tube and mix. h EHRLICH'S i Allow to stand for 15 tnin. for
REAGENT ' completion of* color development.
SUGGESTION
Use of a lOnil Repipet- or an auto matic 10ml syringe pipet will per mit the rapid and easy delivery of fresh Ehrlich's Reagent. Use of a Vortex mixer will permit the rapid and pasy mixing of teat tube con tents
STEP 10
TECHNIQUE
Read optical density of final reaction mixture at 553 mp in a standard laboratory colorimeter.
SUGGESTION
Normal values of urinary ALA produce a faint yel low to faint pink color while abnormally elevated values of urinary ALA produce a distinct red color. It is therefore possible in a large screening pro gram to immediately eliminate all non-red colors by visual inspection. A very accurate quantitation of the rag$ ALA in the red samples can then be ob tained by plotting the optical density of the red samples against known concentrations of pure ALA standard carried through the identical procedure.
INSTRUCTIONS FOR PREPARING FRESH EHRLICH'S REAGENT
Add 420 ml of glacial acetic acid to an empty, amber stoppered bottle.
Add entire io>gra of reagent-grade p-dimetbylaminobenzaldehyde (DMAB) to the glacial acetic acid and mix. If all 40 determinations of ALA are to be performed on the same day, then add 80 ml of 72$ perchloric acid to the entire acetic acid solution of DMAB. This final solu tion is fresh Ehrlich's Reagent and will remain stable up to 6 hours.
3* Note; If all 40 determinations are not to be performed on the same day, then add 1<? ml of 72$ perchloric acid to every 100 ml aliquot
of acetic acid solution containing the added \ DMAB in order to prepare the desired quantity
of fresh Ehrlich's Reagent. The remainder of the acetic acid solution of DMAB not contain ing pei'chloric acid will remain stable for several months if`kept in a refrigerator where it will solidify. Subsequent thawing on the day of the analysis is performed by running warm water over the outside of the bottle fol lowing which perchloric acid can then be added as outlined above to prepare fresh Ehrlich's Reagent.
PREPARATION OF A STANDARD CALIBRATION CURVE
X. Six aliquots of the ALA hydrochloride stock solution (0.01 ml, 0.05 ml, 0,10 ml, 0.20 ml, 0.30 ml and 0,50 ml) are used as working
standards.*
Vol. of Standard pg of ALA per Corresponding mg$ of
Stock Solution total reac urinary ALA for a 0.5
(1 i = 125 pg
tion volume ml sample of urine
of ALA.HCl)
applied to the column
0.01 ml
0.05 ml 0.10 ml 0.20 ml 0.30 ml 0.50 ml
1 pg
5
10 jug
20 g
* 30 ' 5 Mg
0.20 mg$ 1.00 mg$ 2.00 mg$
l*.00 mg$ 6.00 mg$ 10.00 mg$
7. Set a colorimeter to 100$ transmission at 553 mp using the reagent blank*
8. Read the optical density, of the standards and unknowns.
9. Prepare a standard curve by plotting the con centrations of the standards against optical density*
10.
Read the concentration of urinary ALA in the unknowns (0*5 ml urine sample applied to the column unit) in terms of mg$ from the stand
ard calibration curve obtained.
2. Dilute each of the six working standards to 70 ml mark with 1.0 M sodium acetate using graduated test tubes.
3. Pipet 7.0 ml of 1.0 M sodium acetate into an empty test tube to be used as a reagent blank.
4. Add 0.2 ml of acetylacetoue to each tube and mix.
5. Place tubes in a boiling water bath for 10 minutes, remove and cool to room temperature.
6. Add 7.0 ml of fresh Ehrlich's Reagent to each tube, mix and let stand for 15 minutes for complete color development.
Note: 125 pg ALA.HC1 corresponds to 100 pg ALA
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INTERPRETATION OF URINARY ALA VALUES
Urinary ALA Range (mg$)
Urinary ALA Code
Relationship to Lead
Exposure
0.00 - 0.54
0.55 - 0.99 1.00 - 1.49 1.50 - 1.99 2.00 - 2.99 3.00 - 5-99 6.00 -10.00
Normal Trace 1 plus 2 plus
3 plus 4 plus 5 plus
None None Moderate Heavy Severe Critical Overwhelming
*
EXPECTED RESULTS FOR THE DAVIS TEST FOR XJRINART ALA
Limit ot detectability in urine... 0.03mg$ Sensitivity of test........................................... 0.15 pg Variation between duplicate samples + 2$ Recovery of ALA from column........ 97$
ORDERING INFORMATION
Catalog No.
Description
Pricing
TEST KITS 95002
20 Piggy-back Column Units (Complete with reagents* and standard)
95004
40 Piggy-back Column Units (Complete with reagents* and standard)
95020
200 Piggy-back Column Units (Complete with reagents* and standard)
95501
* Note-. Perchloric acid {72%) is not supplied with the kit but is available in one pound bottles sufficient for 200 tests.
$ 34.50 60.00
207.00 4.00
SUPPORT RACK FOR HOLDING DISPOSABLE COLUMNS DURING TEST PROCEDURES
95502
Support rack for 40 Column Units with drain tray and numbered pipet guide.
13.75
REPIPETS FOR EXTRA CONVENIENCE
95801
1 ml, with 950 ml amber bottle
95802
10 ml, with 950 ml amber bottle
47.50 47.50
All prices are FOB shipping point, either Richmond, California, or New York City. Terms: Net 30 days.
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BIO-RAD'
Main Office and Laboratories
32nd & Griffin Avenue Richmond, California 94804 Telephone: (415) 234-4130 Telex: 033-658
Eastern Sales and Warehouse
22 Jones Street New York, N.Y. 10014 Telephone: (212) WA 4-4232 Telex: 01-26108
European Distribution
MUnchen-Grafelfing Am Kirchenholzi 6 West Germany Telephones: 833698 Telex: 528014
833699
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