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PIGMENT METABOLISM
PRESENTER - Dr SHREYA PRABHU
MODERATOR - Dr ANISHA T S
1
INTRODUCTION
PIGMENTS are colored substances, some of which are normal
constituents of cell, whereas others are abnormal and accumulate
in cells only under special circumstances.
They absorb visible light within a narrow band between 400-800
nm.
Thus pigments greatly differ in origin, chemical constitution, and
biological significance.
They can be organic or inorganic compounds that remain insoluble
in most solvents
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CLASSIFICATION
A)ENDOGENOUS PIGMENTS
1) HEMATOGENOUS PIGMENTS
a. Hemosiderin
b. Hemoglobin
c. Bilirubin
d. Porphyrins
2) NON HEMATOGENOUS PIGMENTS
a. Melanin
b. Lipofuscins
c. Chromaffin
d. Pseudomelanosis
e. Dubin-Johnson pigment
f. Ceroid-type lipofuscins
g. Hamazaki-Weisenberg bodies
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B)EXOGENOUS PIGMENTS
Inhaled pigments
Ingested pigments
Injected pigments
C)ARTIFACT PIGMENTS
Formalin
Malaria
Schistosome
Mercury
Chromic oxide
Starch
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ENDOGENOUS
PIGMENTS
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HEMOSIDERINS
Hemoglobin derived, GOLDEN YELLOW to BROWN granular intracellular
pigments.
They contain iron in the form of ferric hydroxide that is bound to a protein
framework
Formed by aggregates of ferritin (iron complexed to apoferritin) found
especially within the phagocytes of the bone marrow, spleen, liver where the
break down of senescent RBC takes place.
Excessive storage of hemosiderin(hemosiderosis) occurs in situation where
there is excessive breakdown of red cells or systemic overload of iron
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HEMOSIDEROSIS
LOCALISED GENERALISED
LOCAL TISSUES PARENCHYMAL DEPOSISTS
(Macrophages, fibroblasts, endothelial (Liver, Kidney, Pancreas. Heart, Skin)
cells and alveolar cells) RED CELL DEPOSISTS
(Liver, Spleen, Bone marrow)
Examples: Examples:
1.Hemorrhage in tissues 1.Acquired Hemosiderosis
2.Black eye 2.Hereditary Hemosiderosis
3.Brown induration lung 3.Excessive dietary intake (Bantus
4.Infraction disease)
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DEMONSTRATION OF HEMOSIDERIN AND
IRON
PERLS PRUSSIAN BLUE REACTION FOR FERRIC IRON:
Considered to be first classical histochemical reaction.
Treatment with an acid ferrocyanide solution will result in the unmasking of
ferric iron in the form of the hydroxide, Fe(OH)3, by dilute hydrochloric acid.
The ferric iron reacts with a dilute ferrocyanide solution to produce an insoluble
blue compound, ferric ferrocyanide (prussian blue)
FIXATION:
Avoid the use of acid fixatives. Chromates will also interfere with the
preservation of iron
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10SECTIONS:
Works well on all types of section, including resin
FERROCYANIDE SOLUTION:
1% aqueous potassium ferrocyanide 20 ml
2% aqueous hydrochloric acid 20 ml
Freshly prepared just before use
METHOD:
Take a test and control section to water
Treat sections with the freshly prepared acid ferrocyanide solution for 10-30 minutes
Wash well in distilled water
Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red
Wash rapidly in distilled water
Dehydrate, clear, and mount in synthetic resin
RESULTS:
Ferric iron Blue
Nuclei Red
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A SECTION OF LIVER FROM A PATIENT WITH HEMOCHROMATOSIS
STAINED FOR FERRIC IRON WITH PERLSMETHOD. FERRIC IRON IS
STAINED BLUE
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LILLIES METHOD FOR FERRIC AND FERROUS IRON
Ferric iron dark Prussian blue
Ferrous iron dark Turnbulls blue
Nuclei Red
HUKILL AND PUTTS METHOD FOR FERROUS AND
FERRIC IRON
Ferrous iron Red
Nuclei Blue
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A SECTION OF PLACENTA TREATED WITH LILLIES METHOD FOR
FERROUS IRON. FERROUS IRON IS STAINED DARK BLUE
HEMOGLOBIN
HEMOGLOBIN is a basic conjugated protein bound to globin and is the red
pigment component, responsible for the transportation of oxygen and carbon
dioxide.
Heme is composed of protoporphyrin, a substance built up from pyrrole rings
and combined with ferrous iron.
Histochemical demonstration of the ferrous iron is only possible if the close
binding in the heme molecules is cleaved
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As Hb is normally present within red blood cells its
demonstration is not necessary.
Outside its normal position in RBC, Hb may be found free in
areas of recent hemorrhage, in macrophages.
The pathological conditions like casts in the lumen of renal
tubules in cases of hemoglobinuria or active
glomerulonephritis.
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DEMONSTRATION OF HEMOGLOBIN
Methods demonstrate the enzyme, Hemoglobin peroxidase,
which is reasonably stable and withstands short fixation and
paraffin processing.
This peroxidase activity was demonstrated by the Benzidine-
nitroprusside methods ( Lepehne-Pickworth Benzidine
Trchnique), but because of the carcinogenicity of benzidine, these
methods are not recommended.
Tinctorial method, The amido black technique and the Kiton
red-Almond green technique are worth noting
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LEUCO PATENT BLUE METHOD
Hemoglobin peroxidase Dark Blue
Nuclei Red
BILE PIGMENTS
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24Heme Biliverdine Bilirubin(unconjugated)
Bilirubinalbumin complex
(Uptake by liver)
Conjugated
bilirubin Bilirubin-diglucuronide
in intestine Urobilinogen
StercobilinogenUrobilinogen
In kidney
Urobilin
Excretion in urine
Stercobilin
Excretion in feces
20% absorptionEnterohepatic
circulation
80% Intestine
oxygenase
Bilirubin
reductase
Heme
Glucuronyl
transfersae
BILE PIGMENTS
Bilirubin (conjugated+unconjugated), biliverdine, hematoidin-
together refered to as Bile pigments
They are chemically and physically distinct with solubility in
water and alcohol
Bilirubin is the orange-yellow pigment, a toxic waste product
in the body.
It is extracted and biotransformed mainly in the liver, and
excreted in bile and urine.
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HEMATOIDIN-
Virchow first described in sites of old hemorrhage
Related to bile pigments but differ
Thought that heme has undergone a chemical change within these areas- led to it being trapped- preventing transportation to liver
Extracellular yellow-brown crystals and amorphous masses within old hemorrhagic areas
Microscopically- appear as bright yellow pigment in sections of old splenic infarcts, old hemorrhagic areas of brain or infarcted tissues
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Microscopical examination of any liver sections that contains bile
pigments will almost certainly reveal a mixture of biliverdine and
both conjugated and unconjugated bilirubin
In H&E stained sections- bile if present-
seen as small yellow brown globules within bile canaliculi- indicating
obstruction
Within hepatocytes (they need to be distinguished from Lipofuscin)
Conditions- Prehepatic/ Hepatic/ Post hepatic
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Intracellular
Cholestasis,
Bile pigments in
The cytoplasm
Fig: CHOLESTASIS
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BILE PLUG (arrow) showing expansion of bile canaliculus by bile
DEMONSTRATION OF BILE PIGMENTS:
Need arises in the histological examination of liver where
distinguishing from lipofuscin is of significance
Both appear yellow-brown in H&E paraffin sections
Bile pigments are not autofluorescent and fail to rotate the
plane of polarized light, whereas Lipofuscin is autofluorescent
Most common method- Modified Fouchet Technique
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MODIFIED FOUCHETS (HALL) TECHNIQUE
(FOR LIVER BILE PIGMENTS)
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RESULTS-
Bile pigments emerald to blue green
Muscle yellow
Collagen red
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OTHER TECHNIQUES
GMELIN TECHNIQUE-
Only method that shows identical result with liver, gallbladder bile and
hematoidin.
Method- Deparaffinized sections of tissue treated with nitric acid and
changing color spectrum is produced around pigment deposits
Red Purple Green
KUTLIKS TECHNIQUE-
Method-Sections treated with ferric iron solution
Result- Bilirubin- Green on pale yellow background
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PORPHYRIN PIGMENTS
Normally occur in tissues in small amounts.
Considered to be precursor of the heme portion of Hb
PORPHYRIAS are rare pathological conditions that are
disorders of the biosynthesis of porphyrins and heme
Found most abundantly in liver