Pigment metabolism

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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

    3

  • 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)

  • 8

  • 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

  • 11

    A SECTION OF LIVER FROM A PATIENT WITH HEMOCHROMATOSIS

    STAINED FOR FERRIC IRON WITH PERLSMETHOD. FERRIC IRON IS

    STAINED BLUE

  • 12

  • 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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  • 15

    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

    17

  • 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.

    18

  • 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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  • 20

    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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  • 28

  • 29

    Intracellular

    Cholestasis,

    Bile pigments in

    The cytoplasm

    Fig: CHOLESTASIS

  • 30

    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

  • 35

  • 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