Pigments and accumulations

Necrosis v. Apoptosis Decreased ATP production Increased mitochondrial permeability Leakage of Ca++ Ca-activated hydrolases degrade lipid, protein, RNA, DNA Denatured nucleic acid and protein may be large or small After initial “blebbing” of ER or plasma membranes, membranes burst ATP depletion initiates necrosis Inflammation elicited Bax/Bak channels open Increased mitochondrial permeability Leakage of CytC CytC/Apaf-1-activated caspases cascade to activate DNase Condensed nucleosomal DNA is cut into 200 bp fragments After initial “blebbing,” condensed apoptotic bodies form ATP required for apoptotic enzyme activity No inflammation

Hemoglobin

Oxyhemoglobin Oxygen-bound hemoglobin, Hb or HbO2, is red Mucous membranes and nail beds look pink Lack of Hb is called anemia  Too few erythrocytes Hemmorhage or i.v.cell-free fluids (Ringer’s) Quantified as hematocrit (packed cell volume)  Erythrocytes are hypochromic Dietary iron deficiency  Anemic tissues are pale; less red, more colorless

Hypochromic anemia

Deoxyhemoglobin Reduced hemoglobin, HHb is dark purple-red Mucous membranes and nail beds look blue (cyan) Presence of HHb is called cyanosis  Poor systemic circulation due to shock, heart disease  Poor oxygenation of Hb due to low oxygen tension, pneumonia, COPD Acrocyanosis is limited to distal extremities (acro = extreme)  Cold-induced vasular constriction  Reynaud’s syndrome Primary may be instigated by extreme temperature or emotional stress Secondary to other disorders, CREST syndrome –calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia

Pallor and cyanosis

Methemoglobin Ferrous (oxidized) hemoglobin, Hb(III), is brown Mucous membranes and nail beds look brown or darkly cyanotic with chocolate-colored arterial blood Cyanosis absent cardiopulmonary symptoms caused by elevated circulating Hb(III) is called methemoglobinemia  Hb(III) is normally <2% total; normally produced  Hb(III) reduced to HHb with oxidation of NADH by one of three isotypes of cytochrome b5 reductase (cytb5r, EC )  Hb(III) has altered conformation and does not bind O2  Symptoms appear at >30% total hemoglobin  Death occurs at around 70% Hines bodies within erythrocyes consist of precipitated Hb(III) Scabs (and old meat) look brown due to oxidized Hb(III)

Methemoglobinemia Certain drugs and nitrites oxidize HHb to Hb(III)  antimalarials (chloroquine, primaquine), topical anesthetics (benzocaine, lidocaine, prilocaine), inhaled nitric oxide, sulfonamides, etc. Hereditary Hb mutations that stabilize iron in ferric state Hereditary cytochrome b5 reductase deficiency  Type-I deficiency is limited to red cell isoform  Type-II affects all isoforms in all tissues and presents as severe encephalopathy, microcephaly, generalized dystonia, movement disorders and mild cyanosis Toxic methemoglobinemia is treated with low dose of methylene blue  NADPH-dependent cyb5r (slow) is activated by methylene blue Blue dye reduces NADP+ to NADPH Enzyme reduces Hb(III) to HHb with oxidation of NADPH to NADP+

Signs of methemoglobinemia

Carboxyhemoglobin Carbon monoxide-bound hemoglobin, COHb, is bright red Mucous membranes and nail beds look cherry red, along with veinous blood COHb does not transport oxygen  CO is essentially permanently bound to the heme 200-fold greater binding affinity than oxygen  Chronic exposure leades to hypoxia  Acute exposure may be deadly Symptoms start at 20% saturation Death may occur at 70% saturation

Hemoglobinemia  RBCs break apart releasing Hb into plasma  Plasma looks pink Hematuria  Whole RBCs in urine Hemoglobinurea  Lysed RBCs and Hb in urine  Hb is nephrotoxic  Affected tubules show reddish-orange crystals of Hb upon autopsy Hb imbibition (post-mortem only)  Heart and vessels appear pale pink due to post-mortem RBC rupture

Hemolysis and Hemoglobinuria

Hemoglobin recycling Ruptured RBCs in tissues are phagocytosed by macrophages  spleen, liver Old RBCs accumulate in spleen and liver to be processed by macrophages and Kuppfer cells Iron is removed to ferritin which may oxidize to hemosiderin Iron-free heme is converted to biliverdin (green) Biliverdin converted to bilirubin (yellow) Bilirubin is insoluble, transported to liver through plasma bound to albumin Hepatocytes conjugate bilirubin with glucuronic acid making it water soluble Conjugated bilirubin is excreted in bile

Hepatic bile ducts

Colors of bruising Initial hemorrhage of RBCs into tissue is cleared by macrophages, which process Hb  Oxyhemoglobin and Deoxyhemoglobin  Deoxyhemoglobin and Biliverdin  Biliverdin and Bilirubin  Bilirubin and Hemosiderin  Hemosiderin When iron is completely cleared, tissue resumes normal color Accumulation of hemosiderin is hemosiderosis Hemochromatosis is severe, chronic accumulation

Colors of bruising

Excessive accumulation of iron Accumulation of hemosiderin is hemosiderosis Hemosiderin is normally found in marrow, spleen, liver Hemosiderosis in tissues is secondary to:  iron intake oveload (enteral or parenteral), long-term hemodialysis or transfusions, blood disorders Hemochromatosis is severe, chronic accumulation in liver, pancreas, myocardium  Primary, genetic hemochromatosis most frequent in men of northern European descent Hemochromatosis results in oxidative damage and inflammation

Hemosiderosis, hemochromatosis

Jaundice, icterus Bilirubin build-up in tissues  Prehepatic or hemolytic: due to excessive hemolysis build-up of unconjugated bilirubin  Hepatic or hepatocellular: due to failure of at least 80% of liver function both conjugated and unconjugated bilirubin accumulates  Posthepatic or obstructive: due to failure of bile to drain into GI Conjugated bilirubin accumulates

Porphyrins Porphyra = purple Porphyrins, aka tetrapyrroles, are intermediates of heme synthesis Conjugated bonds in ring structures are photoactive, fluorescent Porphyrias result from inborn errors of heme metabolism Seven separate types of porphyria Most common disorder, acute intermittent porphyria, is an autosomal dominant deficiency of the third enzyme in the heme synthesis pathway (porphobilinogen deaminase) Several intermediates in the pathway, including the first, aminolevunlinic acid, are neurotoxic Symptoms of accumulation include UV sensitivity and neurological dysfunction

Variegate porphyria Autosomal dominant acute hepatic porphyria Protoporphyrinogen oxidase gene on bands 1q22-23 Termed “South African” as it occurs in South Africans of Boer descent with an incidence of 1 case per 330 All are descendants of Gerrit Jansz and Ariaantje Jacobs who married at the Cape of Good Hope in 1688 Similar founder effects explain the high prevalence in the houses of Stuart, Hanover, and Prussia (British royal family) Symptoms begin in the second or third decade of life Cutaneous manifestations are present in 80% of patients Acute attacks are seen in approximately 50% of patients causing neuropsychiatric, gastrointestinal, and cardiovascular manifestations as a result of the increase in porphobilinogen (PBG) and aminolevulinic acid (ALA)

Symptoms of porphyria

Lipofuscin Fuscus = brown Inclusions of lipid peroxides, phosphates, proteins Not harmful Colors ear wax Accumulates in liver, heart from normal “wear and tear” Pigment accumulates near nucleus Indicative of age, oxidative damage Does not stain blue with Prussian blue or Perl’s iron

Lipofuscin

Melanin Melas = black Synthesized from tyrosine by tyrosinase Reaction confined to melanosome compartment of melanocytes in dermis Whole melanosome is transferred to keratinocytes in epidermis Blocks UV radiation Benign accumulations (freckles, moles) are called nevi (pl., singular: nevus or naevus = from birth; birthmark) Alternative name, lentigo, like a lentil or pea

Histological accumulations Endogenous pigments  Hemoglobin, bilirubin, biliverdin, porphyrin, iron, hemosiderin, melanin, lipofuscin Compounds that absorb histological stain  Lipid and cholesterol  Hyaline  Amyloid  Proteins  Calcium complexes (soaps, CaPO4) Exogenous pigments

Hyaline Histologic appearance of pale, glassy,diffuse pink in H&E staining Eosine binds free amino groups  N-ends, Lys, Arg  May indicate protein breakdown or influx of plasma proteins Accumulation of staining, not itself a pigment

Amyloid Amyl = starch Misfolded proteins in B-pleated sheet formation that resist digestion and accumulate as inclusions Amyloid accumulations in glomeruli stain brown with iodine, similar to the iodine reaction with glycogen Amyloid and glycogen stain pink under white light with Congo red, but polarized light bounces off amyloid and looks green

Protein accumulations Pink in H&E staining Proteins may accumulate as aggregates in vacuoles or in extracellular spaces Protein droplets may stain brightly in proximal tubules Secretory granules budded from ER may stain brightly as Russell bodies Misfolded proteins aggregate, such as  1- antitrypsin in hepatocytes

Dystrophic calcification Associated with necrosis, aging or damaged heart valves Precipitated calcium salts look white Basophilic when stained with H&E Blue granular crystals

Calcified bicuspid

Metastatic calcification Associated with imbalances in phosphorous equilibrium or hypercalcemia  Hyperparathyroidism stimulates resorption of Ca from bone  Accelerated bone turnover due to immobility, metastatic cancer, leukemia, Paget disease  Vitamin-D intoxication, sarcoidosis  Renal failure Phosphate retention Hyperparathyroidism Principally affects acid secreting cellls  Gastric mucosa, kidneys, lungs, systemic arteries, pulmonary veins

Sarcoidosis

Exogenous pigment accumulations  Foreign matter, colored or refractive in microscope slides Anthracosis – coal miner’s black lung (anthrako = charcoal, carbon) Silicosis – another occupational hazzard Accumulations of matter that stains  Proteins Extracellular matrix and cytoskeletal fibers  Glycogen  Fat, lipid, cholesterol Actually a void in H&E due to solvent

Exogenous pigment accumulations  Foreign matter, colored or refractive in microscope slides Anthracosis – coal miner’s black lung (anthrako = charcoal, carbon) Silicosis – another occupational hazzard Accumulations of matter that stains  Proteins Extracellular matrix and cytoskeletal fibers  Glycogen  Fat, lipid, cholesterol Actually a void in H&E due to solvent

Reversible damage – fatty change Intracellular accumulations of a variety of materials can occur in response to cellular injury. Here is fatty metamorphosis (fatty change) of the liver in which deranged lipoprotein transport from injury (most often alcoholism) leads to accumulation of lipid in the cytoplasm of hepatocytes.