Download THE PERACETIC ACID-SCHIFF STAIN The organic peracids, such

THE PERACETIC ACID-SCHIFF STAIN
RAYMOND BANGLE, JR., M.D.
Laboratory of Pathology and Pharmacology, National Institute of Arthritis and Metabolic
Diseases, National Institutes of Health, U. S. Public Health Service, Bethesda 14, Maryland
The organic peracids, such as perbenzoic, performic and peracetic acids, are
strong oxidizing agents. It may be considered that they are derived by the interaction of hydrogen peroxide and an organic acid with the formation of an acid
peroxide. Thus, the production of peracetic acid may be illustrated as follows:
CH.COOH + H2O2
glacial acetic
acid
* CH3COOOH +
peracetic
acid
H20
The present paper deals first with the known chemical reactions of organic
peracids with organic compounds. This provides one with a background for
understanding the chemical basis of the peracetic acid-Schiff reaction in tissue
sections. This histochemical method then is described, followed by the results
of the method in staining selectively various tissue elements. The use of blocking and extraction procedures and of additional staining reactions are presented
in order to substantiate the chemical basis of the peracetic acid-Schiff staining
in tissue sections.
T H E CHEMICAL REACTIONS OP T H E ORGANIC
PERACIDS
The organic peracids differ from periodic acid not only in their chemical structure but also in their inability to produce cleavage oxidation of vicinal glycols
in a polysaccharide complex or of vicinal hydroxy amino groups in hydroxyamino
compounds. The chemical reactions of the organic peracids are grouped according to the following classes of organic compounds: unsaturated lipids, amino
acids and protein-bound cystine.
Unsativrated lipids. Oxidation of unsaturated fatty acids with a peracid results
in a variety of reaction products depending upon the nature of the reagent, the
conditions of oxidation and the structure of the unsaturated fat.13 17 Oxidation
of unsaturated fatty acids occurs preferentially at the double bond. However,
the exact mechanism of peracid oxidation is obscured (1) if one does not know
if all peracids are structurally alike, if all peracids attack the double bond by
adding to it; (2) by furnishing 1 or 2 atoms of oxygen which in turn add to it;
or (3) for both reasons; and (4) if the peracid oxidation may be accompanied or
followed by secondary hydrolytic cleavage of the double bond. Assuming that
all these conditions exist with peracid oxidation of unsaturated acids, the intermediate and final reaction products would include epoxy or oxido acids, peroxido
acids, ketohydroxy acids, dihydroxy or polyhydroxy acids, aldehydo acids (semiReceived for publication October 2, 1953.
Dr. Bangle is Pathologist.
179
.180
VOL. 24
BANGLE
aldehydes) and aldehydes.13 In the case of performic acid oxidation of the double
bond, Lillie8 has illustrated the reaction as follows:
0
—HC=CH—
+
HCO,H
0
/ \ CH—
—HC
> —HC
CH— +
oxido group
OH
+
H20
HCOOH
OH
IICH—
• —HC
dihydroxy group
0—0
and —H C = C H
and by cleavage:
\- 2 H C 0 3 H
> —HC—CH— + 2 HCOOH
peroxido group
—HC=0 +
aldehyde
0=CH—
It should be emphasized that this reaction probably is oversimplified and should
not be construed as a general reaction for all unsaturated lipids. However, there
is evidence that the reaction does occur in certain instances under specific conditions, and it is the only plausible explanation for the mechanism of the peracetic
acid-Schiff staining in tissue sections.
By contrast, periodic acid, as usually employed histochemically, is capable of
producing cleavage oxidation of dihydroxy or polyhydroxy acids to yield aldehydes, though it is incapable of attacking the ethylenic group per se.
Amino acids. Toennies and Homiller20 showed that performic acid reacts only
with tryptophan, methionine and cystine, among the amino acids. The mechanism of the reaction in the case of tryptophan is not known. Methionine is oxidized to the sulfone level, whereas cystine is oxidized to 2 molecules of cysteic
acid.
Protein-bound cystine. Sanger18 has used performic acid to oxidize crystalline
insulin in order to split the disulfide bridges of the cystine residues. This procedure made possible, on the basis of solubility, the separation of 2 distinct types
of polypeptide chains in which the cystine residues had been converted to cysteic
acid residues.
Alexander, Fox and Hudson1 investigated the oxidation products of the disulfide bond in degreased wool keratin. They found, on the basis of cation-exchange
experiments, that on oxidation of wool with peracetic acid, free sulfonic acid
groups were not produced. However, on subsequent acid hydrolysis, cysteic acid
was produced almost quantitatively. To explain this, they postulate the formation of an intermediate combined heterocyclic mixed imide of a carboxylic and
sulfonic acid, respectively. Further data obtained by the same authors provide
evidence that a limited oxidation product from wool cystine (possibly a sulfoxide)
was formed by a side reaction. There is no mention that sulfinic acids or aldehydes were produced.
FEB. 1954
181
PERACETIC ACID-SCHIFF STAIN
THE PERACETIC ACID-SCHIFF
METHOD
The performic acid and the peracetic acid-Schiff methods were introduced
independently by Pearse16 and by Lillie.8
The performic acid and the peracetic acid solutions used by Pearse contained
an excess of formic acid and of acetic anhydride, respectively. It has been observed that the excess acid in these solutions occasionally is detrimental to tissue
sections, causing swelling and dissolution of certain structures and loss of sections from the slide.2 Lillie8, 9 used solutions of performic acid and of peracetic
acid prepared according to Greenspan's specifications.6 These solutions contained
an excess of hydrogen peroxide and did not result in loss of sections even at 16
hours.
Though performic acid and peracetic acid are equally satisfactory for the
histochemical reaction, the use of peracetic acid has one definite advantage.
Greenspan's peracetic acid reagent is more stable than his performic acid reagent. The latter should be prepared fresh for each batch of sections, whereas
peracetic acid is effective for 1, 2 or more weeks depending upon the amount
of use.
Following oxidation in peracetic acid at room temperature, the tissue sections are washed for 10 minutes in running tap water and then transferred to
standard "cold Schiff" reagent manufactured according to Lillie's specifications.7
Three rinses in 0.5 per cent aqueous Na 2 S 2 05 totaling 5 minutes are used directly
after the 10 minutes of Schiff treatment. The sections then are washed in water,
dehydrated, cleared and mounted in balsam or polystyrene. In this procedure,
nuclei usually are rendered Schiff-positive owing to a Feulgen-type nucleal reaction. This source of confusion is overcome readily by use of an iron hematoxylin
counterstain.
The time interval for oxidation of tissue sections apparently is not critical.
Usually 1 or 2 hours are employed, though 5 to 10 minutes often are equally effective. Prolonging the oxidation interval beyond 6 hours may lead to a gradual
decrease in intensity of the Schiff coloration, possibly due to destruction of aldehyde groups.
The Schiff reaction step should be limited to 10 or 15 minutes. Within this
interval, the Schiff reagent stains, so far as is known, only free aldehyde groups.
Ketones in vitro occasionally give a typical positive reaction with Schiff's reagent,
though the time required for the color change is longer than that with soluble or
insoluble aldehydes.3
APPLICATION OF THE PERACETIC ACID-SCHIFF
METHOD
A positive peracetic acid-Schiff reaction in tissue elements is indicated by a
deep pink, red or purple-red color, providing that these elements are not stained
similarly by 10 minutes of Schiff treatment without prior oxidation. The nuclear
staining has been mentioned above and is based upon the principle of the Feulgen
nucleal reaction. Tissue elements that have been examined in this laboratory
and found to be stained more or less consistently by the peracetic acid-Schiff
method are: myelin sheaths; red blood cells; retinal rod acromere lipid; hard
keratin (hair shafts, nails); lipid granules within the epithelium of eccrine sweat
182
BANGLE
VOL. 24
glands; specific lipid granules (lipofuscin) within the ovary, testis and adrenal
gland; subcutaneous fat cells; and ceroid of the choline-deficiency hepatic cirrhosis of rats. All the tissue structures listed are stained both by the peracetic
acid-Schiff method and by oil-soluble dyes, except hard keratin, which is not
sudanophilic at room temperature. Paraffin sections may be used providing the
lipid material resists extraction during dehydration.
The above tissue structures, after staining with oil-soluble dyes, can be decolorized readily by appropriate dye solvents and restained in undiminished
amounts, providing that the lipid remains insoluble, in the solvents employed.
This indicates that the material stained was, at least in part, lipid. Furthermore,
on the basis of chemical analyses, certain of the above tissue structures (ceroid,
myelin, hair shafts and subcutaneous fat) are known to contain lipids possessing
a varying degree of unsaturation. In view of what has been stated before in regard to the chemical reactions of organic peracids, and in view of the selectivity
of the Schiff reagent for aldehyde groups, if limited to 10 minutes' use, it seems
likely that the peracetic acid-Schiff reaction in the tissue elements listed is due
to oxidation of unsaturated lipids with the production of insoluble aldehyde
groups and other products. To substantiate this, it is necessary to employ specific
blocking procedures, extraction procedures, and additional histochemical
methods.
THE USE OF SPECIFIC BLOCKING PROCEDURES
1. Halogenation. Chlorine and bromine add readily to unsaturated acids to
yield saturated compounds. There are several factors that control the rate and
completeness of the reaction.4 For this reason, the failure of halogenation in
blocking the peracetic acid-Schiff staining of a tissue structure may not mean that
unsaturated lipid is absent. However, the finding that halogenation prevents
the subsequent staining, whereas the solvent for the halogen does not, is strong
presumptive evidence that unsaturated lipid is present. For the procedure of
halogenation, treatment of tissue sections in bromine:carbon tetrachloride (1:39
volume dilution) at room temperature for 1 to 2 hours has been employed in this
laboratory. This procedure blocks completely the subsequent peracetic acidSchiff reaction of myelin sheaths, retinal rod acromere lipid, hair shafts, lipid
granules within the eccrine sweat gland epithelium and ceroid. The bromine
solvent alone does not affect the subsequent peracetic acid-Schiff staining of
these structures, indicating that the reaction is not based upon dissolution of the
lipid material in carbon tetrachloride.
2. Blocking of carbonyl groups, particularly aldehyde groups. Though it is probable that a 10-minute Schiff reaction in tissue elements is not due to reactive
groups other than aldehydes, the fact that carbonyl blocking reagents abolish
the subsequent Schiff reaction is confirmatory evidence. The blocking reagents
include phenylhydrazine, aniline chloride and dimedone. A 5 per cent aqueous
solution of phenylhydrazine-HCl condenses readily with aldehydes and many
ketones at room temperature. 6 An M / l aqueous solution of aniline chloride condenses more readily with aldehydes than with ketones at room temperature. 6
FEB. 1954
PERACETIC ACID-SCHIFF
STAIN
183
A saturated solution of dimedone in water reacts only with aldehydes.19 The
procedure for blocking is as follows: tissue sections are oxidized in peracetic acid,
washed in water, treated in 1 of the blocking reagents at room temperature for
varying time intervals, washed in water and then transferred to the Schiff reagent. That the Schiff coloration in a tissue element is prevented by the prior use
of one or all of the blocking reagents, but not by the aqueous solvent alone, is
indicative that carbonyl (aldehyde or ketone) groups are produced as the result
of oxidation. The failure of blocking does not mean necessarily that carbonyl
groups are absent, but suggests either that such groups are in some manner unavailable to the blocking reagent or that the conditions of blocking are not optimum.
The Schiff staining of peracetic acid oxidized myelin sheaths, ceroid and lipid
granules within eccrine sweat gland epithelium is abolished readily by application of phenylhydrazine or aniline chloride at room temperature, thus indicating
that aldehyde groups are produced as the result of oxidation. The blocking in
the case of hard keratin is less effective unless the procedure is carried out under
increased temperature.
3. Blocking of hydroxyl and amino groups. Acetylation or benzoylation readily
blocks these groups.9 Since vicinal glycols or vicinal hydroxy amino groups are
responsible for the periodate Schiff reaction, acetylation or benzoylation blocking procedures would be expected and are known to prevent this histochemical
reaction.9 Since these groups are not attacked by peracetic acid oxidation, the
blocking procedures would not be expected to affect the peracetic acid-Schiff
reaction. In practice, this is found to be so.
LIPID EXTRACTION
PROCEDURES
The solubility of lipids varies markedly according to the nature of the solvent,
the temperature of the solvent and the structure of the fat. In addition, fixation
of tissue by formalin or chromate fixation, or both, renders certain lipids, such
as those in myelin, less soluble. Therefore, the stainability of a particular tissue
structure after the use of a lipid-extraction procedure may not mean that the
material stained is other than fat. One method for determining if lipids are removed completely from tissue by an extraction procedure is to analyze the extracted residue chemically for fat content. Another method for checking the
effectiveness of a lipid-extraction procedure makes use of the application of an
oil-soluble dye to tissue before and after extraction.
The so-called "fat solvents" include petroleum ether, hexane, diethyl ether,
chloroform, benzene, ethanol, methanol, acetone, gasoline, carbon tetrachloride,
pyridine and others. These solvents may be combined. Thus, the use of boiling
chloroform: methanol (1:1 or 2:1) is an effective extraction procedure and,
furthermore, is capable of fixing thin blocks of tissue.
The solubility of the lipid in the various tissue structures stained by the peracetic acid-Schiff method varies. Thus, the reactive lipid in the subcutaneous fat
cell is extracted readily by ethanol at room temperature, whereas the reactive
lipid of ceroid is extremely difficult to extract even by use of boiling solvents.
184
VOL. 24
BANGLE
H I S T O C H E M I C A L METHODS F O R FATTY ACID
PEROXIDES
Peracid oxidation of unsaturated acids results in a variety of products, among
which are peroxides. The histochemical demonstration of peroxides, as well as
aldehydes, in a tissue element that previously was oxidized by peracetic acid is
supportive evidence that such an element contained unsaturated lipid.
The aldehyde groups are detected by the Schiff reagent, whereas the peroxides
are detected by the methods described below.
1. The ferric ferricyanide reduction test. Lillie and Burtner 10 found that a variety
of substances are capable of reducing ferric ferricyanide mixtures to a blue or deep
green, often insoluble compound (Prussian blue). Hydrogen peroxide gave a
prompt reaction. Cod liver oil and linseed oil reacted within 10 minutes. They
suggest that the reaction with polyunsaturated fats may be due to the presence
of fatty acid peroxides rather than to the ethylenic groups per se.
Peracetic acid oxidation of unsaturated acids accelerates the production of
peroxides as compared to noncatalyzed atmospheric oxidative rancidity. For
example, fresh ceroid, fresh hair shafts or fresh subcutaneous fat gives a weak
(faint blue or spotty) ferric ferricyanide reduction reaction, although after peracetic acid oxidation the same structures are stained intensely (dark blue). A
direct reaction occurs in rancid subcutaneous fat.
2. The indophenol blue synthesis test (the Winkler-Schultze or "M-nadi oxidase"
reaction)}2 In the presence of atmospheric oxygen, oxidase or peroxide, an insoluble dark blue compound (indophenol blue) is synthesized from a mixture
of a-naphthol and dimethyl-p-phenylenediamine-HCl. If the histochemical reaction is carried out under anaerobic conditions or is limited to less than 5
minutes in air, the production of indophenol blue in a lipid material (sudanophilic material) may be considered as being due to the presence of fat peroxides.
This is found to occur in the case of fresh ceroid, fresh hair shafts and fresh subcutaneous fat, only after previous oxidation with peracetic acid. A direct reaction occurs in rancid subcutaneous fat.
T H E P E R A C E T I C A C I D - S C H I F F R E A C T I O N O F HARD
KERATIN
This subject is discussed separately because there is controversy in regard to
the mechanism of the reaction. As has been mentioned before, peracetic acid
oxidation of hard keratin (hair shafts, nails) gives rise to a product which is
stained red with Schiff's reagent. Pearse16, 16 believes that this reaction in tissue
sections is due to the oxidation of protein-bound cystine with the subsequent
formation not only of cysteic acid (alanine-beta-sulfonic) but also of another
acid (alanine-beta-sulfinic). The latter is thought by him to be responsible for
the positive reaction with Schiff's reagent. Lillie and Bangle11 have shown that
Pearse's hypothesis probably is incorrect. This is based upon the experimental
results obtained from use of specific blocking methods (e.g., bromination), of
prolonged lipid extraction procedures, of reagents capable of oxidizing sulfinic
acids, and of in vitro reactions with sulfinic and sulfonic acids and with insulin.
Furthermore, there is no chemical evidence that sulfinic acid groups arise from
peracetic acid oxidation of wool keratin. 1 It seems unlikely that the peracetic
FEB. 1 9 5 4
PERACETIC ACID-SCHIFF STAIN
185
acid-Schiff reaction in hair shafts is due to adsorption of peracetic acid with subsequent oxidative recolorization of the Schiff reagent.2 A possible explanation
of the chemical basis of this reaction as applied to hair shafts is that unsaturated
substances are attacked, yielding aldehydes, peroxides and probably other products. Nicolaides14 has shown that human hair shafts possess many unsaturated
acids, alcohols and sterols, as well as squalene—a highly unsaturated hydrocarbon. The iodine value of the total fat was 50 to 60. The lipid content of hair
shafts is extremely difficult to extract completely without destroying the hair.
SUMMARY
The peracetic acid-Schiff method is based chemically upon the oxidation of
double bonds in unsaturated lipids with the production of insoluble aldehyde
groups. Epoxides and peroxides also are produced. The aldehydes are detected
by the Schiff reagent, whereas the peroxides are detected by the ferric ferricyanide reduction test or by the indophenol blue synthesis test. These reactions
are blocked by prior halogenation (bromination) which converts unsaturated
acids to saturated compounds. The latter are nonreactive to peracetic acid oxidation.
The application of the peracetic acid-Schiff method to various lipid-containing
tissue structures is described.
REFERENCES
1. ALEXANDER, P : , F O X , M . , AND HUDSON, R . F . : T h e reaction of oxidizing agents with
wool. 5. T h e oxidation products of t h e disulphide bond and t h e formation of a sulphonamide in the peptide chain. Biochem. J., 49: 129-138, 1951.
2. BANGLE, R., J R . : Unpublished observations.
3. Ciiu, C. H . U . : A histochemical s t u d y of staining t h e axis cylinder with fuehsinsulfurous acid (Schiff'$ reagent). Anat. R e c , 108: 723-745, 1950.
4. D E U E L , H . J., J R . : T h e Lipids: Their Chemistry and Biochemistry. E d . 1. New York:
Interscience Publishers, I n c . , p p . 153-155, 1951.
5. GREENSPAN, F . P . : T h e convenient preparation of per-acids. J . Am. Chem. S o c , 68:
907, 1946.
6. HICKINBOTTOM, W. J . : Reactions of Organic Compounds. E d . 2. London: Longmans,
Green & Co., 1948, 481 p p .
7. L I L L I E , R . D . : Simplification of t h e manufacture of Schiff reagent for use in histochemical procedures. Stain Technol., 26: 163-165, 1951.
8. L I L L I E , R. D . : Ethylenic reaction of ceroid with performic acid and Schiff reagent.
Stain Technol., 27: 37-45, 1952.
9. L I L L I E , R . D . : Histopathologic Technic. E d . 2. New Y o r k : Blakiston Co., scheduled
to appear in J a n . , 1954.
10. L I L L I E , R . D . , AND BURTNER, H . J . : T h e ferric ferricyanide reduction test in histochemistry. J . Histochem. & Cytochem., 1: 87-92, 1953.
11. L I L L I E , R. D . , AND B A N G L E , R., J R . : Manuscript in p r e p a r a t i o n .
12. LISON, L . : Histochiniie et Cytochimie Animales. E d . 2, P a r i s : Gauthier-Villars, p p .
403-106, 1953.
13. MARKLEY, K . S.: F a t t y Acids: Their Chemistry and Physical Properties. E d . 1. New
York: Interscience Publishers, I n c . , 1947, p p . 387-477.
14. NICOLAIDES, N . : Personal communication.
15. P E A R S E , A. G. E . : T h e histochemical demonstration of keratin by methods involving
selective oxidation. Quart. J. Micr. So., 92: 393-402, 1951.
16. P E A R S E , A. G. E . : Histochemistry, Theoretical and Applied. E d . 1. Boston: Little,
Brown and Co., 1953.
17. RALSTON, A. W . : F a t t y Acids and Their Derivatives. New Y o r k : John Wiley & Sons,
Inc., p p . 917-920, 1948.
18. SANGER, F . : Fractionation of oxidized insulin. Biochem. J., 44: 126-128, 1949.
19. SMITH, G. F . : Analytical Applications of Periodic Acid (HsIOo) and Iodic Acid (HIO3)
and Their Salts. E d . 5, Champaign, 111.: T h e Garrard Press. 1950.
20. T O E N N I E S , G., AND HOMILLER, R. P . : T h e oxidation of amino acids by hvdrogen peroxide in formic acid. J. Am. Chem. S o c , 64: 3054-3056, 1942.