INFORMATION FROM USAF - SCHOOL OF AVIATION MEDICINE

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP75B00285R000300090024-4
• Release Decision: RIPPUB
• Original Classification: S
• Document Page Count: 10
• Document Creation Date: December 15, 2016
• Document Release Date: October 6, 2003
• Sequence Number: 24
• Publication Date: February 16, 1970
• Content Type: MF

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S-E C R E T
16 February 1970
MEMORANDUM FOR: Chief, Clinical Division, OMS
SUBJECT Information From USAF - School of
Aviation Medicine
1. Lipo-Protein Determination
Use Beckman Microzone with Gelman's Sepraphore III Paper
and cellulose acetate.
Uses of Selman's Procedure using cyclo-hexanone mixed
with ethanol.
Procedure is in Gelman's Book on Clinical Pathology.
Gelman is at P.O. Box 1448, Ann Arbor, Michigan. Latest
article on this technique is in A.M. Society Clinical
Pathologists (ASCP) Journal, January 7, 1970,
(Electrophoresis).
2. Triglyceride Determination
Apparently there have been many changes in techniques
in past few years.
Mrs. Dorothy Wease, who has been at the SAM Laboratory
for many years, is-somewhat of an authority on this procedure.
Her procedure is her own modification using the Technicon
Analyzer (Technicon Procedure # 78). (This has been found very
satisfactory over past few years.)
The enclosed Blue Book has the 1966 method for extraction.
After this first step, it is all done in auto-analyzer. (The
old method utilized a zealite mixture in extracting which is
not. satisfactory.)
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S E C R E T
At present time manifold tube on analyzer will not
handle ether; therefore, change procedure to use isopropanol.
Isopropanol does not get all lipoproteins back in solution;
therefore, a small amount of ether is added at the end to
accomplish this.
The phospho-lipids are lost in this procedure. Cholesterol
and triglycerides are run on same sample. The method for
this procedure is attached.
. The method for determining phospho-lipids is now in print
and Mrs. Wease will send it to me as soon as completed.
3. If necessary, it can be easily arranged to have a
tech go to San Antonio and spend whatever time is necessary
at their laboratories.
25X1
FS OMS
Attachments
1 - Copy of Lipoprotein Electrophoresis Procedure
2 - Copy of Triglyceride Procedure
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 Approved For Rsiease 2003/11/25: CIA-RDP75B0028500300090AUACHMENT I
LIPOPROTEIN AND GLYCOP,ROEIN ANALYSIS
LIPOPR0T= ELECTROP TORE;STS
'ary small molecules are transported in blood in combi-
nation with plasma proteins. Lipoprotein is an example
of such a complex. The lipoproteins contain such lipids
as free cholesterol, cholesterol esters, phospholipids,
and mono-, di- and tri-glycerides.
Electrophoresis is a valuable aid in diagnosing several
specific lipoprotein deficiency states and in character-
izing lipoproteinemias. It is much more convenient and
economical than ultracentrifugation and is adaptable to
the screening of many subjects at relatively low cost.
Visual inspection of the electrophoregram is presently
employed to check for normals and some abnormals of spe-
cific types. The Gelman densitometric procedures now
allow quantitation of lipoprotein electrophoregrams in
only a slightly longer time.
The normal fasting plasma lipoprotein electrophore gram.
should contain three bands. These bands are located in
the a pre-5 and B-globulin region. A fourth band will
be logAted at the origin if chylomicrons are present.
Two staining procedures are presented. One involves lipid
staining with Oil Red 0. The alternate method involves
lipid oxidation followed by staining with a modified
Schiff's reagent as proposed by Kohn (1961). This method
gives stained bands that coincide with those obtained by
the Oil Red 0 method.
A. Equipment and Supplies
Same as for the basic serumrotein procedure
Oil Red 0 Linid Stain Procedure:
Oil Red 0, C.I. (Fisher Chemical No. NA-689;
un>1 --- IT_ ^,1 nr
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l J. i:r Tl c ,. c :.. .:M
Ozone-Schii'1 Stain P r o c e d
Schiff's Reagent-Fuchsi.. Sulfurous Acid for the de-
tection of aldehydes,(fiarleco No.. 2818)
Nitric Acid
Sulfuric Acid
Barium Dioxide (Ba02) .`_
ED.A (tetra-sodium salt o`
ettiylenediaminetetra-acetic
acid)--.--,
Beaker, 2000 ml
Aluminum Foil
B. Specimen
Use a fasting-state blood sample. Collect 5 ml blood
and gently mix in a tube containing 1.0 ml of 0.001 M
EDTA in 0.15 M NaCl. Use low speed centrifugation to
separate the plasma. Remove plasma and store at 10 C
to 30 C. Under these conditions lipoproteins are
stable for at least 2 weeks as judged by electrophore-
gram reproducibility (Kelleher, 190"8).' EDTA is recom-
mended to prevent the formation of oxidation products
from the normally present lipoproteins: This oxi-
dation occurs very rapidly in the presence of. catalytic
concentrations of divalent cations, giving an electro-
phoregram showing six bands rather than the normal four
bands.
0. Reagent
1. Buffer-300 ml (100 ml for tray and 200 ral for
SepraTek Chamber). Dissolve 1 packet of High-
Resolution Buffer in distilled water and dilute
to 670 ml. Some clinicians add 0.685 gm EDTA to
the buffer (0.001 M concentration) to prevent
problems with divalent cations.
2.. Stain-1.0 gm.Oil Red 0 in 1000 ml warm 70% methyl
alcohol. Bring to a boil for 5 minutes. Then,
store at 370 C until used.
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D. Electrophoresis Procedure
0
1. Carry out electrophoresis as outlined for the
serum protein procedure (Steps 1-15) except make
3 or 4 applications by repeating Step 12.
2. Electrophorese at 200 volts for 30 minutes.
When the run is over, turn off and disconnect the
Power Supply. Remove Sepraphore strip holding it
horizontally taut. Do not allow buffer to run over
the strip.
E. Oil Red 0 Staining Procedure
Follow equipment, reagents, and specimen preparation
given above.
1. Place damp electrophoregram into 100 m1 of the
Oil Red 0 stain for 1L-24 hours at 370 C. If
more than one strip is stained at one time place
the strips into the staining tray in such a way
as to prevent one strip from overlapping any
other. Maintain the 370 C temperature by placing
the staining tray in an incubator.
2. After 2L hours, rinse the strip(s) in 100 ml of
distilled water.
3.
Decolorize background in a hypochlorite solution
composed as follows: 1 ml ca nercial laundry
bleach (5.25% sodium hypochlorite) to 200 ml
aqueous, 5% acetic acid. Five to ten minutes is
sufficient to lighten the background.
Store in aqueous 51"01 acetic acid until ready for
clearing and quantitation.
F. Alternate Procedure (Modified Schiff's Reagent)
1. Ozonation-DO IN A WELL VENTILATED HOOD. Wrap a
piece of insulated wire around the periphery of
a 2000 ml beaker and near the top. String wires
across the top of the beaker to form "clothes
lines" (a "6d" shape) using the periphery wire as
anchor points.
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ce gm a 2 into a 250 cc side aria flask
(Pyrex). Connect a piece of tubing to the side
arm. Have a base of sufficient length that the
free end can be placed into the beaker.
Suspend the electrophoresed strip from the ozonator
wire rack and clamp with a paper clip. Cover the
top of the beaker with a piece of alum-1: un, foil.
Add 20 to 25 ml concentrated H2SOh to the side arm
flask and stopper immediately. Ozone is carried
over to the beaker via the tubing. Continue for
10 minutes. Additional H2SO! may be added should
gas production decrease before 10 minutes has
passed.
3.
2. Remove the strip(s) from ozonator. Place into
0.001 N HC1 rinse solution for one minute. If
more than one strip is stained at one time, do
not allow strip(s) to overlap, because some of
the lipoprotein may diffuse onto or contaminate
another strip, causing irregular band shapes and
patterns.
Staining and Decolorizing. Place the strip(s)
into the Schiff's reagent trays. (Do not allow
extra strips to overlap.)
Keep the stain-containing trays covered with
aluminum foil to exclude light. The stain reagent
may be used a number of times. It should be dis-
carded when it begins turning pink.
Stain for 30 to 60 minutes to develop maximum blue-
purple color.
Wash. Carefully remove the stained electrophoregram
and wash in 3 successive baths of 0.5% aqueous
nitric acid.
Store in the final rinse until ready for clearing
and quantitation.
G. Clearing and Quantitating
1. After employing either the Schiff's Reagent or
the Oil Red 0 staining.tecanique, remove the
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electrophorogram from the final rinsing solution.
Blot the strip(s) between two dry blotter pads
until only the excess moisture is removed. The
strip(s) should still feel damp to the touch. It
is imperative, particularly with the Schiff's
Reagent technique, that the strip not be completely
dry since not only will the bands fade, but also,
the clearing solution will destroy the membrane.
2. Place the damp membrane into a clearing solution
composed of either 30% dimethyl sulfomide (DSO)
in water or 30% aqueous dimethyl formamide (DMT)
for 5 minutes.
3.
Remove the strip from the clearing solution and
place on a 2.25" x 2.75" glass slide. Place the
strip and the glass slide into an oven set at 100?
C for 5 to 10 minutes. Remove the strips as they
clear. Prolonged overheating yields brittle, curled
strips that are difficult to handle.
11. Scan at 515-555 nm for the Schiff's technique and.
525 nm for the Oil Red 0 technique.
References
1. Cawley, L. P. and B. Minard, "The Serum Lipoproteins,,"
Lab Synopsis (Certified Blood-Donor Service, Inc.,
1400 Crossways Park Drive, Woodbury, N.Y. 11797.
Formerly Hoechst Pharmaceutical Co.), 251-56 (1967).
2. Chin, H. P. and D. H. Blankenhorn, "Separations and
Quantitative Analysis of Serum Lipoproteins by Means
of Electrophoresis on Cellulose Acetate," Clin. Chico.
Acta., 20 (1968), 305-314.
4.
Fallon, H. J. and J. W. Woods, "Response of Hyper -
proteinemia to Cholesty::ramine Resin," JAMA, Vol. 201,
No. 13, 1161-11614, June 24, 1968.
Fredrickson, D. S., R. I. Levy and '. S. Lees, "Fat
Transport in Lipoproteins---An Integrated Approach to
Mechanisms and Disorders," New Lngland Journal of
Medicine 267, 32-44, 914-103, 143-156, 215-226, January
5, 1967.
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 5.Ap(p @g@.j,FcprjieaW 2~Q03~1, / 5~jG~A- P 5Bp02 e0~Q300090024-4
"Lipoprotein E.l ectrophoresis", presented at the 1968
A.S.C.P. Neotir_S, Miami, Florida.
6. Hatch, F. T., "The Serum Lipoproteins," 223-243, and
"Serun Lipoproteins in Coronary Heart Disease,"
422-1[29; in F. W. Sunderman and F. W. Sunderman, Jr.,
Serum Proteins and the Dysproteinemias, J. B. Lippicott
Co., Philadelphia and Montreal (1964).
7. Kelleher, P. C., Univ. of Vermont College of Medicine,
Burlington, Vt., (personal communication) 1968.
8. Kohn, J., "A Lipoprotein Staining i~athod for Zone
F .ectrophoress," Nature, 189, 312-313, 1961.
9. Kohn, J., "Cellulose Acetate Electrophoresis and
Immunodiffusion Techniques" in I. Smith, Chromato-
graphic and Electrophoretic Techniques. Vol. II-Zone
Electrophoresis, W. Heinemann, Ltd., London, and
Interscience Publishers, New York, 59-60 (1960).
10. Kroman, H. S., J. Nodine, S. R. Bender, and A. N.
Brest, "Lipids in Normals and Patients with Coronary
Artery Disease," Amer. J. Med. Science, Vol. 248,
No. 5, November 19611.
11. Kroman, H. S., S. R. Bender, A. N. Brest and M. L.
Maskovitz, "The Interrelationship of Blood Lipids
and Estrogens," J. Atherascler Res., 6 (1966) pp.
2117-255.
12. Martin, R., Miami Valley Hospital, Dayton, Ohio,
(Personal Communication).
13. Owano, L. R., "Serum Lipids," The University Mich.
Med. Center Journal, Vol. 31, No. 1, January-February
1965.
14. Wessler, S. and L. A. Avioli, "Classification and
Management of Familial Hyper lipopr oteinemia," JAMA.,
Vol. 207, No. 5, 929-933, February 3, 1969.
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 ATTACHMENT 2
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Triglyceride Procedure
1. KReaz.^zzt Silicic Acid (BIO-SIL HIA minus 325 mesh)
2% KO U
Acetyl Acetone
Periodate with 3% Acetic Acid
Isopropyl Alcohol (Redistilled)
2. Procedure i
(3) Let stand 15 minutes.
(4)
Into 50 ml round bottom pyrex tubes (with glass stoppers)
1 ml standards, controls, serum.
Add 2.5 g silicic acid to each tube and stopper.
Nit tubes sharply on padded surface to shake silicic acid
and serum loose from the sides of the tubes
(Do
not break tubes.).
(5) Add 25 ml ether; stopper, shake once by hand, and remove
pressure.
(6) Shake in horizontal action shaker 10 minutes.
(7) Centrifuge standards for 1 minute at 5000 rpm. (Standards
take longer to settle out than do-other samples because of less
water).
(8) Pipette a 5 ml aliquot from each tube into another 50 rol tube.
(9) Remove stopper and dry in 60?C water bath. ---
((10) Remove and cool at room temperature.. V,.;,-4- rLe