Pigment metabolism

PIGMENT METABOLISM

PRESENTER - Dr SHREYA PRABHU

MODERATOR - Dr ANISHA T S

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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