Pigmentation in Jaundice

Pigmentation in Jaundice

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My textbook says that the yellow appearance of a jaundiced person is due to the accumulation of bile pigments in the skin. I am unable to understand why these pigments are not deposited in a healthy person? Please help.

The yellow color comes from the accumulation of Bilirubin in the body. Bilirubin is a breakdown product of hemoglobin (contained in erythrocytes, also known as red blood cells, and responsible for the oxygen transport in the body). (This is also the reason why bruises get colored, as the blood which was released into the skin gets removed.) As erythrocytes live only for around 120 days, the hemoglobin needs to be recycled at the end of the life cycle, as free hemoglobin is a pretty problematic substance.

The recycling of the erythrocytes happens in the liver, the iron is recycled, and the hemoglobin is split in the globin (which is further broken down into the amino acids) and the heme which is oxidized by the heme oxidase to biliverdin and further by the biliverdin reductase to bilirubin.

As bilirubin is not water soluble it is bound to albumin and released into the bloodstream for the transport to the liver. In the liver this process goes on further until the bilirubin is water soluble and can be released with the bile into the intestine where it is finally excreted. See the image (from here) for an illustration:

If your liver is not working properly (for example due to an infection) the break-down process of the albumin-bound bilirubin is reduced or doesn't work and the yellow bilirubin accumulates in the body until the skin gets yellow. So this indicates a liver malfunction and is usually nothing to take easy.


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Neonatal jaundice (Infant Jaundice). So just to run through what we're discuss go through Definitions, the causes of neonatal jaundice, clinical features, investigations and management.

So why do we need to learn about neonatal jaundice well over 60% of newborns become Dauntless this may be physiological or it may be due to underlying disease and this may have serious consequences as high unconjugated serum bilirubin is neurotoxic and can cause deafness connectors and athetoid cerebral palsy. Just to run through some definitions a neonate is defined as between birth and until 28 days postnatally and jaundice is when the serum bilirubin is over 30 millimoles per liter.

So what are the causes of neonatal jaundice well it's best to divide these into causes according to the age of onset of jaundice as this is a useful guide to the likely cause. So if the baby develops darkness within 24 hours it's usually a hematological condition such as recess hemolytic disease ABO incompatibility g6pd deficiency or even it could be a congenital infection between 24 hours and 2 weeks could be physiological jaundice which we'll discuss in more detail later or breast milk.

Jundice are also infection again hemolytic disorders or breathing if the baby develops jaundice when they're over two weeks old it's important to divide these inter unconjugated bilirubin and conjugated bilirubin. The unconjugated causes are again physiological and breast milk jaundice again infection. But also hypothyroidism and the hemolytic disorders. If the bilirubin is conjugated then the causes could be sepsis, TPN, Neonatal hepatitis, Cystic fibrosis or bile duct obstruction. So just to discuss in a bit more detail physiological jaundice is very common so often starts when the baby is 24 hours old it peaks at a few days but always resolves by day 14 the mechanism is due to the immaturity of hepatic bilirubin conjugation an action is required.

If the serum bilirubin is over 260 millimoles per liter breast milk .Jaundice is when jaundice is exacerbated just due to the contents of what's in the breast milk the cause for this is unknown infection is also an important cause of jaundice and this is due to a number of mechanisms. Firstly poor fluid intake by the infant hemolysis reduced hepatic function or an increase in entero hepatic circulation conjugated jaundice is suggested by dark urine and pale stools. Billary atresia is an important cause to remember so the clinical features of neonatal jaundice are firstly that the jaundice progresses in a cough alack caudal direction. It's observed most easily by directly blanching the skin and in direct sunlight the manifestations of connectors are lethargy poor feeding irritability and increased tone.

So to run through the investigations that you do for a neonate that was doing this firstly you could just do a urine dip to rule out a urinary tract infection and send this off the culture if necessary the investigations done within the Bloods would be that you had checked the level of the serum bilirubin. There are now a lot of transcutaneous bilirubin meters in hospitals. But these are often inaccurate so best to take a venous sample you can check the hemolytic disorders by doing an FBC in film checking the blood group and doing a direct Coombs test to check for infection a capillary blood gas is always useful and you can consider a septic screen. Such as lfts a tort screen TFT is a beaker to rule out hypothyroidism.

You can also do hepatitis serology our red blood cell deflects and tests for cystic fibrosis imaging is required to rule out biliary atresia this could be a liver ultrasound scan or a liver biopsy or even nuclear medicine liver scans.

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The composition and function of bile

The hepatobiliary system comprises the liver, bile duct and gall bladder. Bile is synthesized and secreted by polarized hepatocytes into bile-canaliculi, flows through bile ducts, stored in the gall bladder and is finally drained into the duodenum. The main physiological function of bile is to emulsify the lipid content of food, and this lipid emulsion facilitates lipid digestion and the absorption of lipid-soluble substances. Additionally, bile secretion is an important route to regulate cholesterol homeostasis, hemoglobin catabolism, and the elimination of drugs or drug metabolites [1].

Bile is a yellow-to-greenish amalgam of water, bile acids, ions, phospholipids (phosphatidylcholine), cholesterol, bilirubin, proteins (such as glutathione and peptides) and the other xenobiotics [1]. The yellow-to-greenish color of bile is caused by bilirubin and its derivative, which are also the origin of stool color. Bilirubin is the end catabolite of hemoglobin and other heme-containing proteins, such as myoglobin. The heme molecule is oxidized to biliverdin in hepatocytes and then reduced to unconjugated bilirubin. Unconjugated bilirubin is conjugated with one to two molecules of glucuronic acid via Uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1). Bilirubin conjugation increases water solubility and reduces cytotoxicity of bilirubin. Hepatic and intestinal UGT1A1 are functionally reduced in neonatal stages, and hence, unconjugated hyperbilirubinemia is commonly found in human neonates [2]. Conjugated bilirubin, or direct bilirubin, is the major form of bilirubin in bile and is eliminated in stool. Jaundice, a yellowish pigmentation of the skin and sclera, is caused by the disrupted excretion of bilirubin and biliverdin. Interestingly, some studies involving neonates or adults have shown that hyperbilirubinemia is protective against diseases, including metabolic syndrome and asthma, [2, 3] suggesting that bilirubin may play a role as an antioxidant [4].

Bile acids are colorless and are the most abundant organic components of bile. Bile acids, a group of detergent-like molecules, are synthesized from cholesterol and are typically associated with sodium or potassium ions in the form of bile salts. Bile salts mediate lipid emulsion and act as signaling molecules to regulate gene expression [5,6,7]. Phospholipids and cholesterol, the second and third most abundant organic components of bile, protect against injury of the biliary epithelium from bile acids [1].

Biosynthesis and enterohepatic circulation of bile acids

Bile acids can be synthesized from cholesterol via two pathways in hepatocytes to generate two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), through cytochrome P450 (CYP) enzymes, including CYP7A1, CYP8B1, and CYP27A1. Primary bile acids are conjugated with glycine or taurine (glyco- or tauro-conjugated CA and CDCA), with increased solubility and reduced cytotoxicity. In the intestines, gut-resident microbiota deconjugate bile salts to generate the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA) [8, 9]. In human livers, de novo synthesized bile salts are 500–600 mg daily [10]. More than 90% of bile acids are reabsorbed at the distal ileum and transported back to the liver through circulation systems for the next cycle, called the enterohepatic circulation. Bile salts cycle 6- to 10-times daily. The total amount of bile salt in the body is called bile acid pool, which is approximately 2–3 g. In contrast to bile acids, only trace amounts of conjugated bilirubin will enter the enterohepatic circulation. The blockage of enterohepatic circulation to enhance bile salt elimination has been applied in surgical and medical treatments for cholestasis (Fig. 1).

The enterohepatic circulation, homeostasis of bile acids and treatment targets for cholestasis. The grey arrows indicate the route of enterohepatic circulation of bile acids. Bile acids are synthesized from cholesterol in hepatocytes to generate the primary bile acids CA and CDCA. After conjugation with glycine or taurine, bile acids (BAs) are transported from hepatocytes into the bile canaliculi via BSEP. Intestinal microbiota converts primary bile acids into the secondary bile acids DCA and LCA. Most of BAs reabsorbed by the enterocytes through ASBT in the apical membrane and then delivered into the portal circulation system via BA efflux transporter OSTα/β in the basolateral membrane. BAs are re-absorbed into hepatocytes. Hepatocytes secrete these BAs along with the de novo synthesized bile acids enter the next cycle. Bile acids also play roles in signaling to regulate the homeostasis of bile acids. The nuclear receptor FXR is the bile acid receptor to regulate bile acid homeostasis at the synthesis and the elimination levels, acting in the hepatocytes and enterocytes. The figure also shows different therapeutic targets at hepatocellular transport or enterohepatic circulations. 1°BAs, primary bile acids 2°BAs, secondary bile acids 4-PB, 4-phenylbutyrate ASBT, apical sodium dependent bile acid transporter BAs, bile acids BSEP, bile salt export pump CA, cholic acid CDCD, chenodeoxy cholic acid DCA, deoxycholic acid FGFR4, fibroblast growth factor receptor 4 FXR, farnesoid X receptor G(T)CA, glyco- or tauro-cholic acid G(T)CDCA, glyco- or tauro-chenodeoxy cholic acid LCA, lithocholic acid MRP3, multidrug resistance-associated protein 3 MRP4, multidrug resistance-associated protein 4 NTCP, sodium/taurocholate co-transporting polypeptide OATP1B1/3, organic-anion-transporting polypeptide 1B1 and 1B3 OSTα/β, organic solute transporter-α/β RXRα, retinoid X receptor α SHP, small heterodimer partner UDCA, ursodeoxycholic acid

In human fetuses after 22 and 26 weeks of gestation, taurine-conjugated di-hydroxyl bile acids can be detected in the gallbladder. After 28 weeks, small amounts of glycine conjugates are synthesized. In postnatal stages, the ratio of CA to CDCA declines from 2.5 to 1.2 [11]. Infant livers are under development, have a small bile acid pool, and have a limited capacity for bile excretion and reabsorption. Therefore, neonates and infants, particularly premature infants, are prone to cholestasis caused by various insults, such as ischemia, drugs, infection, or parenteral nutrition.

Hepatocellular transporters mediating bile flow (Fig. 2)

Bile flow is generated by osmotic forces associated with the amount of bile salts secreted into bile canaliculi. Bile secretion from hepatocytes is mediated by a group of transport proteins, particularly ATP-binding cassette (ABC) containing proteins. The bile salt export pump (BSEP encoded by ABCB11) is the pivotal transporter mediating bile acid transport into bile canaliculi. BSEP is exclusively expressed in the apical/canalicular membrane of hepatocytes. After secreted into the small intestine, bile salts are absorbed into intestinal cells via the apical sodium-dependent bile acid transporter (ASBT encoded by SLC10A2) and then secreted into the circulation system through the basolateral heterodimeric transporter OSTα-OSTβ (encoded by OSTA and OSTB, respectively) [12,13,14].

Hepatocellular transporters, enzymes, and regulators involving in bile transport, metabolism, and secretion. A1AD, alpha-1 antitrypsin deficiency A1AT, alpha-1 antitrypsin ALG, Alagille syndrome BAs, bile acids BSEP, bile salt export pump Canalicular, canalicular membrane CF, cystic fibrosis CFTR, cystic fibrosis transmembrane conductance regulator DJ, Dubin-Johnson syndrome FIC1, familial intrahepatic cholestasis 1 FXR, farnesoid X receptor JAG1, jagged 1 MDR3, multidrug resistance protein 3 MRP2, multidrug resistance-associated protein 2 MRP3, multidrug resistance-associated protein 3 MRP4, multidrug resistance-associated protein 4 MYO5B, myosin VB NTCP, sodium/taurocholate co-transporting polypeptide OATP1B1, organic-anion-transporting polypeptide 1B1 OATP1B3, organic-anion-transporting polypeptide 1B3 OSTα/β, organic solute transporter-α/β PC, phosphatidylcholine PFIC, progressive familial intrahepatic cholestasis PS, phosphatidylserine Sinusoidal, sinusoidal membrane SHP, small heterodimer partner TJP2, tight junction protein 2

The basolateral/sinusoidal membrane of hepatocytes contains several bile acid transporters to absorb bile acids from sinusoidal blood, including Na + -taurocholate co-transporting polypeptide NTCP (encoded by SLC10A1), OATP1B1 and OATP1B3 (encoded by SLCO1B1 and SLCO1B3, respectively) [12, 15]. OATP1B1 and OATP1B3 also function in the uptake of bilirubin into hepatocytes [16]. Conjugated bilirubin and organic anions are transported via canalicular multidrug resistance-associated protein 2 MRP2 (encoded by ABCC2) and, to a lesser extent, via ABCG2 into bile. Under physiological or cholestatic conditions, conjugated bilirubin may be excreted via MRP3 (encoded by ABCC3) across sinusoidal membranes into blood, to a lesser extent, and reabsorbed by OATP1B1 and OATP1B3 [3, 16].

Lipids are also important components of bile. The heterodimeric transporter ABCG5/8 mediates cholesterol across canalicular membranes. Phosphatidylcholine (PC) is flopped by the floppase multidrug resistance P-glycoprotein 3 (MDR3, encoded by ABCB4) to the outer lipid leaflet and then extracted by bile salts into bile to form micelles. The combination of cholesterol and sphingomyelin makes membranes highly detergent resistant [17, 18]. The flippase FIC1(ATP8B1) is required to flip phosphatidylserine (PS) back from the outer lipid leaflet to the inner lipid leaflet of the canalicular membrane to stabilize the integrity of the canalicular membrane [19]. Additionally, FIC1 is required for the functional expression of MDR3 [20]. Thus, hepatocytes and biliary epithelium are protected from bile acid toxicity through the efflux of bile acids mediated by BSEP and the functions of MDR3 and FIC1.

Homeostasis of bile acid pools

The homeostasis of bile acids is tightly controlled by the de novo synthesis of bile acids and the expression of transporters that affect hepatocellular bile acid levels. The key regulating molecules are farnesoid X receptor (FXR, NR1H4) and membrane-bound Takeda G protein-coupled receptor (TGR5) [6]. FXR is a nuclear receptor that is highly expressed in hepatocytes and enterocytes in the distal small intestine and colon. TGR5 is expressed in enteroendocrine cells, gallbladder cells and cholangiocytes. FXR forms heterodimers with other nuclear receptors to mediate its transcriptional activity [21,22,23,24]. Upon binding with bile acids as its natural ligands, FXR downregulates the expression of bile acid synthesis enzymes (mainly CYP7A1) and the sinusoidal uptake transporter of NTCP but upregulates the expression of the bile acid efflux transporter BSEP to reduce intracellular bile acid concentrations [25,26,27,28,29]. When bile acids are accumulated in hepatocytes, activated hepatic FXR increases sinusoidal bile acid efflux via MRP4 and heterodimeric OSTα/β [30, 31]. FXR also inhibits the expression of the ileal bile acid transporter ASBT to reduce the enterohepatic circulation of bile acids [32, 33]. Activation of FXR induces enterocytes to release FGF19. Through enterohepatic circulation via the portal vein, FGF19 translocates to the liver and inhibits the expression of CYP7A1 in the hepatocytes [34]. Through FXR, bile is controlled via a negative feedback loop at the transcriptional level via transporters and bile acid synthesis systems.


Cholestasis is defined as disturbances in bile flow caused by diseases either in the hepatocytes, intrahepatic bile ducts or extrahepatic biliary system. Cholestatic liver disease is one of the most common forms of liver disorders resulting from inherited or acquired liver diseases. Inadequate bile flow of any causes results in accumulation of bile contents, including bilirubin, bile acids, and lipids in the liver, and consequently cause elevated levels of bilirubin and bile salts in the liver and blood, as well as dysregulated lipid metabolisms. Clinically, patients usually manifest jaundice as a result of hyperbilirubinemia. Other symptoms include clay stool, pruritus, or infrequently, bleeding episodes such as intracranial hemorrhage. Chronic cholestatic liver disease may progress to liver cirrhosis and liver failure and is the leading cause of pediatric liver transplantation. According to the anatomical location of its occurrence, cholestasis is divided into extrahepatic and intrahepatic cholestasis. Extrahepatic cholestasis is caused by structural abnormalities of the biliary tract including the obstruction of bile ducts and the gallbladder. Surgical treatments are typically applied to restore the physiological function. However, intrahepatic cholestasis is more complicated and typically requires sophisticated investigations. The common causes of extrahepatic and intrahepatic cholestasis are shown in Fig. 3.

Etiologies of intrahepatic and extrahepatic cholestasis of inherited or secondary causes. dis: disorders

Etiologies of inherited bilirubin metabolism disorders causing indirect hyperbilirubinemia

Disturbances in the bilirubin metabolisms result in accumulation of bilirubin in the liver and blood, and consequently cause hyperbilirubinemia detected by routine serum biochemistry test, or called jaundice clinically. Gilbert syndrome is a benign clinical condition usually present mild intermittent jaundice in children or adult. TA repeat polymorphism (UGT1A1*28) in the promoter of UGT1A1 gene is the most commonly affected region. Gilbert syndrome can be identified in the general population, and many are identified by blood test of a health exam [35].

Crigler-Najjar syndrome is also cause by mutations in the UGT1A1 gene. Type I is a rare autosomal recessive disorder with complete loss of enzymatic function that cause extremely high bilirubin levels (above 20 mg/dL) and may lead to encephalopathy due to kernicterus. Treatments include phototherapy, exchange transfusion, or liver transplantation. Crigler-Najjar syndrome Type II manifests medium levels of hyperbilirubinemia (around 5–20 mg/dL), with retention of some enzymatic activity. Phenobarbital can be used intermittently to reduce bilirubin levels below 10-15 mg/dL.

Genetic variations in the UGT1A1 gene, especially 211 G to A (G71R in exon 1) mutation, as well as variations in the glucose-6-phosphate dehydrogenase (G6PD) and OATP2 genes, also contribute to the occurrence of neonatal jaundice and breast-feeding jaundice [36,37,38]. Homozygous 211 G to A mutation has been reported to be associated with severe neonatal jaundice.

Etiologies of inherited cholestasis causing direct hyperbilirubinemia

Inherited cholestatic liver diseases may manifest early in life. The presenting age ranges from infancy to young adulthood. In the last 20 years, there has been tremendous progress in understanding the genetic background of cholestatic liver disease [39,40,41,42,43]. Table 1 lists the categories and genes involved in inherited genetic disorders. Up to now, more than 100 inherited diseases are identified to cause cholestatic liver diseases with the initial presentation of jaundice. Some disorders may be associated with congenital anomalies or with multiple organ involvement. We have previously investigated the genetic background of pediatric patients in Taiwan with BSEP, FIC1, MDR3 defects [44,45,46,47]. We have also reported adaptive changes of hepatocyte transporters associated with obstructive cholestasis in biliary atresia, an important extrahepatic cholestatic liver disease with common symptom of prolonged neonatal jaundice [48, 49]. The distribution of disease types in Taiwanese infants with intrahepatic cholestatic liver diseases is shown in Fig. 4.

Distributions of final diagnosis of intrahepatic cholestasis in infancy in 135 Taiwanese infants 2000–2012. (Adapted from Lu FT et al., J Pediatr Gastroenterol Nutr 201459: 695–701). ALG, Alagille syndrome GGT, gamma-glutamyl transpeptidase IEBAM, inborn error of bile acid metabolism NH, neonatal hepatitis NICCD, neonatal intrahepatic cholestasis caused by citrin deficiency PFIC, progressive familial intrahepatic cholestasis

Progressive familial intrahepatic cholestasis (PFIC) is a clinical syndrome with features of chronic intrahepatic cholestasis that typically begin in infancy and progress to biliary cirrhosis and hepatic failure by the first or second decade of life [40, 46, 50]. The first three types of genetic defects identified are commonly referred to as PFIC1, PFIC2, and PFIC3. PFIC1 and PFIC2 are characterized by low serum γ-glutamyltransferase (GGT) levels. PFIC1 (Byler’s disease) patients have FIC1 gene mutations, and PFIC2 patients have mutated BSEP gene. PFIC3 is characterized by high serum GGT levels and is caused by genetic mutations in the MDR3 gene [51, 52]. BSEP plays a pivotal role in bile physiology as it mediates canalicular bile salt export and is the main driving force of bile flow [53].

With the advances in genetic technologies in recent years, novel disease-causing genes for PFIC have been reported. FXR, the key regulator of bile acid metabolism, have been implicated in a novel form of infant cholestasis with liver failure in two European families [54]. We also identified a fatal case of infant cholestasis with liver failure occurring before 3 months of age [55]. Additionally, TJP2 and MYO5B have been found to cause PFIC. TJP2 is an important component of tight junctions, and a deficiency of TJP2 disrupts the tight-junction structure in the liver [56]. MYO5B is associated with low GGT infant cholestasis. MYO5B is an actin-based motor protein and an effector of Rab11a/b. MYO5B mutations result in the dysregulation of Rab proteins and further disrupt the trafficking of BSEP [57, 58]. Doublecortin domain containing 2 (DCDC2), a tubulin-binding protein, is associated with renal-hepatic ciliopathy and neonatal sclerosing cholangitis [59,60,61]. The mitochondrial transcription factor TFAM is associated with mitochondrial DNA depletion syndrome [62]. Recently, a homozygous single nucleotide deletion in organic solute transporter-β (OSTβ/SLC51B) was demonstrated to cause congenital diarrhea and cholestasis [63].

Dubin-Johnson and Rotor syndrome are two inherited disorders manifesting direct hyperbilirubinemia but with normal or minimally elevated alanine transaminase (ALT) levels, clinically manifesting as jaundice. Dubin-Johnson syndrome is caused by disruption of MRP2 and characterized by grossly black livers and pigment deposition in hepatocytes. Neonatal cholestasis caused by Dubin-Johnson syndrome has been reported in Taiwan and Japan [64, 65]. Our group has identified patients recovered from neonatal cholestasis had re-emergence of jaundice in young adulthood after long-term follow-up [64]. Rotor syndrome has recently been identified to be caused by genetic disruption of both SLCO1B1 and SLCO1B3 genes [66, 67]. These two disorders are benign and do not require specific treatment.

Genetic cholestasis not only causes pediatric liver disease but may also be present in adult liver disease. Additionally, adult liver diseases may result from genetic liver diseases. In general, protein functional disturbances are less detrimental and are typically caused by missense genetic mutations or multifactorial disorders. Cholestasis in pregnancy has been associated with genetic variants/mutations in ABCB4, ABCB11, ATP8B1, ABCC2 and TJP2 [68]. Adult benign recurrent intrahepatic cholestasis (BRIC) is also associated with PFIC-related genes and may have mutations that are less damaging [69,70,71,72]. Acquired forms of cholestasis, such as drug-induced liver disease, have also been associated with genetic variants [73, 74].

Diseases related to ductal plate malformation are an important group of developmental disorders that lead to a paucity or malformation of intrahepatic or interlobular bile ducts. Alagille syndrome, first described by Alagille et al., is based on clinical diagnostic criteria including a characteristic face a paucity of interlobular bile ducts in liver pathology and cardiac, eye, and vertebral anomalies [75]. The JAG1 mutation accounts for > 90% of cases of Alagille syndrome, and mutations in NOTCH2 have been described in a minority of patients [76]. Other syndromic disorders and polycystic liver/kidney diseases may also present with infant cholestasis as the first symptom.

Cholestasis is a common manifestation of hepatic metabolic disorders, including carbohydrate, amino acid, and fat metabolism, as well as mitochondrial and endocrine anomalies. Most of these diseases are rare disorders, and the disease incidence largely depends on ethnic background. For example, neonatal cholestasis caused by citrin deficiency (NICCD) is an important cause of cholestasis in East Asian children [77, 78]. We have previously identified facial features and biochemical characteristics for the phenotypic diagnosis of NICCD [79, 80]. Alpha 1-antitrypsin (A1AT/SERPINA1) deficiency and cystic fibrosis are important causes in western countries but how lower incidences in Asian populations.

Inborn errors of bile acid metabolism constitute a group of important metabolic disorders causing infant cholestasis. Notably, oral primary bile acid supplementation is effective and can avoid patient deterioration and the need for liver transplantation upon timely treatment [81, 82].

Neonatal hemochromatosis is an important cause of neonatal liver failure that manifests as early onset cholestasis. However, recent studies have elucidated this condition as a disorder of gestational alloimmune liver diseases instead of hereditary hemochromatosis [83]. Treatment involves exchange blood transfusion and intravenous immunoglobulin applied as early as when the neonate is born.

Other congenital anomalies, such as chromosomal anomalies, endocrine disorders, and developmental disorders may also cause cholestasis. Liver disease is typically a multi-organ manifestation of congenital anomalies.


Clinical history

A careful clinical history is important to investigate common secondary causes of jaundice and cholestasis, including hemolytic anemia, G6PD deficiency, hereditary spherocytosis and other red cell membrane disorders, prematurity, sepsis, drug-induced liver injury, parenteral nutrition-associated liver diseases, ischemia, and pregnancy. Ethnic background and parental consanguinity are clues for certain types of inherited liver disorders.

Phenotypic diagnosis

The traditional phenotypic diagnosis includes low GGT as a signature of PFIC1 (FIC1 defect) and PFIC2 (BSEP defect). GGT levels, Byler’s bile in electron microscopy, and duodenal biliary bile content can be used as clinical markers to indicate further genetic confirmation [84, 85]. Syndromic cholestasis, including Alagille syndrome, can be diagnosed by phenotypic criteria [75]. Patients with NICCD have phenotypic features, and we have developed a clinical scoring system to aid in diagnosis [79, 86]. Importantly, investigating the involvement of extrahepatic organs is important for differential diagnosis.

Biochemical diagnosis

For patients suspecting jaundice or cholestasis, routine liver biochemistry tests include total and direct bilirubin levels, aspartate transferase levels, ALT levels, GGT and alkaline phosphatase (ALP) levels. Low serum GGT level disproportionate to severity of cholestasis is a clinical clue for inherited cholestasis such as PFIC and inborn errors of bile acid synthesis. Some disorders with metabolic signatures can be diagnosed with biochemical analysis. Diseases, such as inborn error of bile acid metabolism (IEBAM), [87] and metabolic disorders, such as NICCD, [86] require analysis by mass spectrometry.

Genetic diagnosis

Genetic diagnosis is a definitive diagnosis for inherited genetic liver diseases, as many of these diseases lack adequate biomarkers. Genetic tests have largely evolved in the past two decades due to the tremendous progress of genetic analysis technologies. Conventional genetic diagnosis uses direct sequencing for selected genes based on the phenotype of the patient. High-throughput methods have subsequently been developed, such as a resequencing chip that detects 5 genes for genetic cholestasis (SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4) in 2007 [88]. Denaturing high-performance liquid chromatography and high-resolution melting analysis have been used to detect single-gene variants in large numbers of patients [46, 79]. Recent next generation sequencing (NGS) panels in liver diseases have incorporated a limited number of genes, particularly PFIC [65, 89]. Expanded panel-based NGS involving more than 50 genes has been used in clinical patients with promising results [55, 90]. Whole exome sequencing has been applied to identify novel disease-causing genes [57, 63].


Nutritional support

Bile mediates the intestinal absorption of fat and fat-soluble vitamins. In cholestatic liver diseases, the defective absorption of fat and fat-soluble vitamins (vitamins A, D, E, and K) is commonly observed but clinically obscure. Fat malabsorption results in calorie insufficiency and failure to thrive, especially in early childhood. Patients are advised to use formulas containing medium-chain triglycerides or add oils containing medium-chain triglycerides to their food. Deficiency in fat-soluble vitamins may result in multiple organ dysfunctions, including rickets, coagulopathy, and defective neurological, immunological and visual functions. Without supplementation, symptoms of deficiency, such as coagulopathy, osteoporosis, fracture, growth failure and life-threatening hemorrhage, may occur in patients. In addition, deficiencies in fat-soluble vitamins may also cause inadequate anti-oxidation, which is frequently overlooked in clinical patients.

Medical treatment

Although jaundice is the common manifestation of the highly variable etiologies, treatment does not target only to jaundice improvement (to reduce serum bilirubin level), but to target the underlying disorders that may cause hepatobiliary injury and progressive fibrosis and cirrhosis, which is usually associated with elevated bile acid levels or abnormal metabolites. Additional treatment goals are to improve nutritional status, pruritus and life quality, to prevent or to treat cirrhosis related complications.

PFICs, Alagille syndrome, and inborn errors of bile acid synthesis are the most devastating disorders that cause cirrhosis and may need liver transplantation. Effective treatment options for PFICs and Alagille syndrome are limited. Several drugs are under investigation and clinical trial. Here we will discuss about the standard treatment and several newly developed therapeutic strategies for these disorders.

Ursodeoxycholic acid (UDCA) has widely been used to treat cholestatic liver disease and is effective to improve biochemical parameters and pruritus [91]. However, UDCA is not an ideal therapeutic option for PFIC2 patients with BSEP defects. In animal models, UDCA may aggravate liver injury due to the inability of BSEP to export UDCA from hepatocytes [92]. There is a need to develop new drugs targeting BSEP defects. Missense mutations in BSEP/ABCB11 impair protein translation or intracellular trafficking, which reduce canalicular expression of BSEP and eventually cause cholestasis. Recent studies have indicated that 4-phenylbutyrate (4-PB, Buphenyl), a clinically approved pharmacological chaperone, can be used to restore the canalicular expression of BSEP. By using MDCK II cells and SD rats, Hayashi et al. reported that 4-PB significantly relocalizes and enhances the cell surface expression of both wild-type and mutated rat Bsep [93]. Besides its effect on Bsep expression, 4-PB treatment significantly increased hepatic MRP2 and decreased serum bilirubin level in patient with ornithine transcarbamylase deficiency (OTCD) [94]. Moreover, Gonzales et al. applied 4-PBA to PFIC2 patients and successfully restored the hepatic secretion of bile acids and decreased total serum bilirubin via the re-localization of mutated BSEP to canalicular membranes [95]. In addition to 4-PB, steroids are a therapeutic option to enhance BSEP expression. Cell culture experiments have suggested that dexamethasone upregulates Bsep and Mrp2 at the mRNA level in rat primary hepatocytes [96, 97], and treatment with glucocorticoids induces the expression of Bsep, Mrp2, and cytochrome P450 oxidase in rat livers [98]. Additional animal experiments and clinical tests have shown that steroid treatment improved bile homeostasis. For example, dogs receiving a high dosage of hydrocortisone (5 mg/kg) showed a significant increase in bile flow [99]. Engelmann et al. reported that steroids effectively ameliorated cholestatic itches and reduced the serum level of bile salts and bilirubin in two PFIC2 patients carrying missense mutations in BSEP [100].

Blocking enterohepatic circulation has been recently shown as a promising strategy to reduce the hepatic accumulation of bile acids in PFIC2 patients. After secretion from the gallbladder into the intestine, a majority of bile acid is absorbed by enterocytes via ASBT and recycled to liver via enterohepatic circulation. Two independent animal studies have shown that small molecule ASBT inhibitors, SC-425 and A4250, effectively reduced the enteric uptake of bile acid, decreased serum total bilirubin levels, and improved liver fibrosis and inflammation in Mdr2 knockout mice, an animal model of PFIC3 [101]. Moreover, on March 2018, A4250 successfully passed clinical phase II trials ( Identifier: NCT02630875).

The recently developed FXR agonist (Obeticholic acid) has been demonstrated to improve the ALP level in primary biliary cirrhosis [102], and has also been investigated for the treatment of nonalcoholic steatohepatitis (NASH) [103,104,105].

Certain types of the inborn errors of bile acid metabolism are treatable [81]. Oral cholic acid therapy is indicated for 3β-Hydroxy-Δ(5)-C27-steroid oxidoreductase (HSD3B7) deficiency, Δ (4)-3-oxosteroid 5β-reductase (SRD5B1, AKR1D1) deficiency, and Zellweger spectrum disorders [106]. CDCA has also been reported to be effective for oxysterol 7α-hydroxylase (CYP7B1) deficiency, cerebrotendinous xanthomatosis, and other forms of bile acid synthetic defects [107]. After treatment, patients may recover from liver dysfunction, free of jaundice, and avoid liver transplantation. Life-long therapy is indicated for the oral supplementation. Early diagnosis and treatment is important to improve outcome.

Many patients with cholestatic liver disease suffer from pruritus, except patients with inborn errors of bile acid synthesis. Alagille syndrome, PFIC1 and 2 commonly cause disturbing pruritus, which affects daily life quality. Antihistamines, rifampin, and cholestyramine have been used to partially improve the symptoms of this condition. UV-B phototherapy is an alternative therapy to treat pruritus.

Biliary diversion and nasogastric drainage

Palliative treatment with biliary diversion surgery by the disruption of enterohepatic circulation may relieve pruritus and improve liver biochemical profiles. Several strategies have been used, including external biliary diversion or ileal exclusion [108,109,110].

Liver transplantation

Liver transplantation is considered a curative treatment for various liver diseases [111]. However, for PFIC2 patients, the recurrence of the BSEP defect has been reported due to circulating BSEP antibodies [112, 113]. Anti-CD20 antibody and plasmapheresis have been reported to treat recurrent BSEP deficiency [114]. The outcomes in BSEP defects of common European mutations, such as D482G, are better than those of other mutation types [85]. In addition, patients with multi-organ manifestations, such as diarrhea and pancreatic insufficiency in PFIC1, cannot be treated by liver transplantation.

Liver tumor surveillance

The disruption of bile acid transport not only causes PFIC but has also been associated with hepatocellular carcinoma and cholangiocarcinoma [115, 116]. Patients with BSEP deficiency and tyrosinemia are of greater risk of developing hepatocellular carcinoma (HCC). It is mandatory that patients with PFIC be screened for liver tumors on a regular basis. Alpha-fetoprotein is not typically elevated. Some patients were found to have HCC in the explanted liver. Thirty-eight out of 175 pediatric HCC patients receiving liver transplantation were diagnosed with inherited liver diseases [117].

Hepatocyte transplantation and gene therapy

Liver transplantation is often an ultimate option for patients with severe cholestasis, but the rarity of organ sources is an important issue. Hepatocyte transplantation might be an alternative therapy to use efficiently donor tissue in a less invasive manner. Cell therapy has been investigated in animal models with various extents of hepatocyte repopulation, including models of PFIC3 (Mdr2 knockout mice), PFIC2 (Abcb11 knockout mice) and hereditary tyrosinemia [118,119,120]. In previous studies, we found that UDCA can provide a selective growth advantage to donor hepatocytes in Abcb11 knockout mice and enhance the repopulation of donor hepatocytes and partially correct the bile acid profile [92]. However, insufficient long-term substitution ratio of donor hepatocyte in the livers of recipients, and the lack of donor cell sources limits the wide application of UDCA to treat clinical patients. For the past two decades, more than 20 patients with inherited liver-based metabolic disorders have received hepatocyte transplantation. Most of these patients showed only partial and transient improvements in metabolic function for several months and finally underwent liver transplantation [121,122,123]. Among these individuals, two patients with PFIC2 showed no obvious benefits after hepatocyte transplantation, as the existing fibrosis impaired the engraftment of the transplanted hepatocytes [122]. Recently, glyceryl trinitrate have been shown to enhance the efficacy of the transplanted hepatocyte repopulation in Mdr2 knockout mice [124]. With additional treatment to boost donor cell repopulation, hepatocyte transplantation might be refined and benefit patients with cholestasis.

Few studies on experimental gene therapy for cholestatic liver diseases have been reported. The adenoviral transfer of the aquaporin-1 gene has been shown to improve bile flow in rats with estrogen-induced cholestasis, but the effect in inherited cholestatic disease has not been validated [125].


Melanin is produced by cells called melanocytes in a process called melanogenesis. Melanin is made within small membrane–bound packages called melanosomes. As they become full of melanin, they move into the slender arms of melanocytes, from where they are transferred to the keratinocytes. Under normal conditions, melanosomes cover the upper part of the keratinocytes and protect them from genetic damage. One melanocyte supplies melanin to thirty-six keratinocytes according to signals from the keratinocytes. They also regulate melanin production and replication of melanocytes. [7] People have different skin colors mainly because their melanocytes produce different amount and kinds of melanin.

The genetic mechanism behind human skin color is mainly regulated by the enzyme tyrosinase, which creates the color of the skin, eyes, and hair shades. [11] [12] Differences in skin color are also attributed to differences in size and distribution of melanosomes in the skin. [7] Melanocytes produce two types of melanin. The most common form of biological melanin is eumelanin, a brown-black polymer of dihydroxyindole carboxylic acids, and their reduced forms. Most are derived from the amino acid tyrosine. Eumelanin is found in hair, areola, and skin, and the hair colors gray, black, blond, and brown. In humans, it is more abundant in people with dark skin. Pheomelanin, a pink to red hue is found in particularly large quantities in red hair, [13] the lips, nipples, glans of the penis, and vagina. [14]

Both the amount and type of melanin produced is controlled by a number of genes that operate under incomplete dominance. [15] One copy of each of the various genes is inherited from each parent. Each gene can come in several alleles, resulting in the great variety of human skin tones. Melanin controls the amount of ultraviolet (UV) radiation from the sun that penetrates the skin by absorption. While UV radiation can assist in the production of vitamin D, excessive exposure to UV can damage health.

Loss of body hair in Hominini species is assumed to be related to the emergence of bipedalism some 5 to 7 million years ago. [16] Bipedal hominin body hair may have disappeared gradually to allow better heat dissipation through sweating. [10] [17] The emergence of skin pigmentation dates to about 1.2 million years ago, [18] under conditions of a megadrought that drove early humans into arid, open landscapes. Such conditions likely caused excess UV-B radiation. This favored the emergence of skin pigmentation in order to protect from folate depletion due to the increased exposure to sunlight. [8] [9] A theory that the pigmentation helped counter xeric stress by increasing the epidermal permeability barrier [19] has been disproved. [8]

With the evolution of hairless skin, abundant sweat glands, and skin rich in melanin, early humans could walk, run, and forage for food for long periods of time under the hot sun without brain damage due to overheating, giving them an evolutionary advantage over other species. [7] By 1.2 million years ago, around the time of Homo ergaster, archaic humans (including the ancestors of Homo sapiens) had exactly the same receptor protein as modern sub-Saharan Africans. [17]

This was the genotype inherited by anatomically modern humans, but retained only by part of the extant populations, thus forming an aspect of human genetic variation. About 100,000–70,000 years ago, some anatomically modern humans (Homo sapiens) began to migrate away from the tropics to the north where they were exposed to less intense sunlight. This was possibly in part due to the need for greater use of clothing to protect against the colder climate. Under these conditions there was less photodestruction of folate and so the evolutionary pressure working against the survival of lighter-skinned gene variants was reduced. In addition, lighter skin is able to generate more vitamin D (cholecalciferol) than darker skin, so it would have represented a health benefit in reduced sunlight if there were limited sources of vitamin D. [10] Hence the leading hypothesis for the evolution of human skin color proposes that:

  1. From about 1.2 million years ago to less than 100,000 years ago, archaic humans, including archaic Homo sapiens, were dark-skinned.
  2. As Homo sapiens populations began to migrate, the evolutionary constraint keeping skin dark decreased proportionally to the distance north a population migrated, resulting in a range of skin tones within northern populations.
  3. At some point, some northern populations experienced positive selection for lighter skin due to the increased production of vitamin D from sunlight and the genes for darker skin disappeared from these populations.
  4. Subsequent migrations into different UV environments and admixture between populations have resulted in the varied range of skin pigmentations we see today.

The genetic mutations leading to light skin, though partially different among East Asians and Western Europeans, [20] suggest the two groups experienced a similar selective pressure after settlement in northern latitudes. [21]

The theory is partially supported by a study into the SLC24A5 gene which found that the allele associated with light skin in Europe "determined […] that 18,000 years had passed since the light-skin allele was fixed in Europeans" but may have originated as recently as 12,000–6,000 years ago "given the imprecision of method" , [22] which is in line with the earliest evidence of farming. [23]

Research by Nina Jablonski suggests that an estimated time of about 10,000 to 20,000 years is enough for human populations to achieve optimal skin pigmentation in a particular geographic area but that development of ideal skin coloration may happen faster if the evolutionary pressure is stronger, even in as little as 100 generations. [5] The length of time is also affected by cultural practices such as food intake, clothing, body coverings, and shelter usage which can alter the ways in which the environment affects populations. [7]

One of the most recently proposed drivers of the evolution of skin pigmentation in humans is based on research that shows a superior barrier function in darkly pigmented skin. Most protective functions of the skin, including the permeability barrier and the antimicrobial barrier, reside in the stratum corneum (SC) and the researchers surmise that the SC has undergone the most genetic change since the loss of human body hair. Natural selection would have favored mutations that protect this essential barrier one such protective adaptation is the pigmentation of interfollicular epidermis, because it improves barrier function as compared to non-pigmented skin. In lush rainforests, however, where UV-B radiation and xeric stress were not in excess, light pigmentation would not have been nearly as detrimental. This explains the side-by-side residence of lightly pigmented and darkly pigmented peoples. [19]

Population and admixture studies suggest a three-way model for the evolution of human skin color, with dark skin evolving in early hominids in Africa and light skin evolving partly separately at least two times after modern humans had expanded out of Africa. [20] [24] [25] [26] [27] [28]

For the most part, the evolution of light skin has followed different genetic paths in Western and Eastern Eurasian populations. Two genes however, KITLG and ASIP, have mutations associated with lighter skin that have high frequencies in Eurasian populations and have estimated origin dates after humans spread out of Africa but before the divergence of the two lineages. [26]

The understanding of the genetic mechanisms underlying human skin color variation is still incomplete, however genetic studies have discovered a number of genes that affect human skin color in specific populations, and have shown that this happens independently of other physical features such as eye and hair color. Different populations have different allele frequencies of these genes, and it is the combination of these allele variations that bring about the complex, continuous variation in skin coloration we can observe today in modern humans. Population and admixture studies suggest a 3-way model for the evolution of human skin color, with dark skin evolving in early hominids in sub-Saharan Africa and light skin evolving independently in Europe and East Asia after modern humans had expanded out of Africa. [20] [24] [25] [26] [27] [28]

Dark skin Edit

All modern humans share a common ancestor who lived around 200,000 years ago in Africa. [29] Comparisons between known skin pigmentation genes in chimpanzees and modern Africans show that dark skin evolved along with the loss of body hair about 1.2 million years ago and that this common ancestor had dark skin. [30] Investigations into dark skinned populations in South Asia and Melanesia indicate that skin pigmentation in these populations is due to the preservation of this ancestral state and not due to new variations on a previously lightened population. [10] [31]

MC1R The melanocortin 1 receptor (MC1R) gene is primarily responsible for determining whether pheomelanin and eumelanin is produced in the human body. Research shows at least 10 differences in MC1R between African and chimpanzee samples and that the gene has probably undergone a strong positive selection (a selective sweep) in early Hominins around 1.2 million years ago. [32] This is consistent with positive selection for the high-eumelanin phenotype seen in Africa and other environments with high UV exposure. [30] [31]

Light skin Edit

For the most part, the evolution of light skin has followed different genetic paths in European and East Asian populations. Two genes however, KITLG and ASIP, have mutations associated with lighter skin that have high frequencies in both European and East Asian populations. They are thought to have originated after humans spread out of Africa but before the divergence of the European and Asian lineages around 30,000 years ago. [26] Two subsequent genome-wide association studies found no significant correlation between these genes and skin color, and suggest that the earlier findings may have been the result of incorrect correction methods and small panel sizes, or that the genes have an effect too small to be detected by the larger studies. [33] [34]

KITLG The KIT ligand (KITLG) gene is involved in the permanent survival, proliferation and migration of melanocytes. [35] A mutation in this gene, A326G (rs642742 [36] ), has been positively associated with variations of skin color in African-Americans of mixed West African and European descent and is estimated to account for 15–20% of the melanin difference between African and European populations. [37] This allele shows signs of strong positive selection outside Africa [28] [38] and occurs in over 80% of European and Asian samples, compared with less than 10% in African samples. [37] ASIP Agouti signalling peptide (ASIP) acts as an inverse agonist, binding in place of alpha-MSH and thus inhibiting eumelanin production. Studies have found two alleles in the vicinity of ASIP are associated with skin color variation in humans. One, rs2424984 [39] has been identified as an indicator of skin reflectance in a forensics analysis of human phenotypes across Caucasian, African-American, South Asian, East Asian, Hispanic and Native American populations [40] and is about three times more common in non-African populations than in Africa. [41] The other allele, 8188G (rs6058017 [42] ) is significantly associated with skin color variation in African-Americans and the ancestral version occurs in only 12% of European and 28% of East Asian samples compared with 80% of West African samples. [43] [44]

Europe Edit

A number of genes have been positively associated with the skin pigmentation difference between European and non-European populations. Mutations in SLC24A5 and SLC45A2 are believed to account for the bulk of this variation and show very strong signs of selection. A variation in TYR has also been identified as a contributor.

Research indicates the selection for the light-skin alleles of these genes in Europeans is comparatively recent, having occurred later than 20,000 years ago and perhaps as recently as 12,000 to 6,000 years ago. [26] In the 1970s, Luca Cavalli-Sforza suggested that the selective sweep that rendered light skin ubiquitous in Europe might be correlated with the advent of farming and thus have taken place only around 6,000 years ago [22] This scenario found support in a 2014 analysis of mesolithic (7,000 years old) hunter-gatherer DNA from La Braña, Spain, which showed a version of these genes not corresponding with light skin color. [45] In 2015 researchers analysed for light skin genes in the DNA of 94 ancient skeletons ranging from 8,000 to 3,000 years old from Europe and Russia. They found c. 8,000-year-old hunter-gatherers in Spain, Luxembourg, and Hungary were dark skinned while similarly aged hunter gatherers in Sweden were light skinned (having predominately derived alleles of SLC24A5, SLC45A2 and also HERC2/OCA2). Neolithic farmers entering Europe at around the same time were intermediate, being nearly fixed for the derived SLC24A5 variant but only having the derived SLC45A2 allele in low frequencies. The SLC24A5 variant spread very rapidly throughout central and southern Europe from about 8,000 years ago, whereas the light skin variant of SLC45A2 spread throughout Europe after 5,800 years ago. [46] [47]

East Asia Edit

A number of genes known to affect skin color have alleles that show signs of positive selection in East Asian populations. Of these only OCA2 has been directly related to skin color measurements, while DCT, MC1R and ATRN are marked as candidate genes for future study.

), [64] melanocortin 1 receptor (MC1R) Arg163Gln (rs885479 [65] ) [66] and attractin (ATRN) [20] have been indicated as potential contributors to the evolution of light skin in East Asian populations.

Tanning response Edit

Tanning response in humans is controlled by a variety of genes. MC1R variants Arg151Sys (rs1805007 [67] ), Arg160Trp (rs1805008 [68] ), Asp294Sys (rs1805009 [69] ), Val60Leu (rs1805005 [70] ) and Val92Met (rs2228479 [71] ) have been associated with reduced tanning response in European and/or East Asian populations. These alleles show no signs of positive selection and only occur in relatively small numbers, reaching a peak in Europe with around 28% of the population having at least one allele of one of the variations. [31] [72] A study of self-reported tanning ability and skin type in American non-Hispanic Caucasians found that SLC24A5 Phe374Leu is significantly associated with reduced tanning ability and also associated TYR Arg402Gln (rs1126809 [73] ), OCA2 Arg305Trp (rs1800401 [74] ) and a 2-SNP haplotype in ASIP (rs4911414 [75] and rs1015362 [76] ) to skin type variation within a "fair/medium/olive" context. [77]

Albinism Edit

Oculocutaneous albinism (OCA) is a lack of pigment in the eyes, skin and sometimes hair that occurs in a very small fraction of the population. The four known types of OCA are caused by mutations in the TYR, OCA2, TYRP1, and SLC45A2 genes. [78]

In hominids, the parts of the body not covered with hair, like the face and the back of the hands, start out pale in infants and turn darker as the skin is exposed to more sun. All human babies are born pale, regardless of what their adult color will be. In humans, melanin production does not peak until after puberty. [7]

The skin of children becomes darker as they go through puberty and experience the effects of sex hormones. [ citation needed ] This darkening is especially noticeable in the skin of the nipples, the areola of the nipples, the labia majora in females, and the scrotum in males. In some people, the armpits become slightly darker during puberty. The interaction of genetic, hormonal, and environmental factors on skin coloration with age is still not adequately understood, but it is known that men are at their darkest baseline skin color around the age of 30, without considering the effects of tanning. Around the same age, women experience darkening of some areas of their skin. [7]

Human skin color fades with age. Humans over the age of thirty experience a decrease in melanin-producing cells by about 10% to 20% per decade as melanocyte stem cells gradually die. [79] The skin of face and hands has about twice the amount of pigment cells as unexposed areas of the body, as chronic exposure to the sun continues to stimulate melanocytes. The blotchy appearance of skin color in the face and hands of older people is due to the uneven distribution of pigment cells and to changes in the interaction between melanocytes and keratinocytes. [7]

It has been observed that females are found to have lighter skin pigmentation than males in some studied populations. [10] This may be a form of sexual dimorphism due to the requirement in women for high amounts of calcium during pregnancy and lactation. Breastfeeding newborns, whose skeletons are growing, require high amounts of calcium intake from the mother's milk (about 4 times more than during prenatal development), [80] part of which comes from reserves in the mother's skeleton. Adequate vitamin D resources are needed to absorb calcium from the diet, and it has been shown that deficiencies of vitamin D and calcium increase the likelihood of various birth defects such as spina bifida and rickets. Natural selection may have led to females with lighter skin than males in some indigenous populations because women must get enough vitamin D and calcium to support the development of fetus and nursing infants and to maintain their own health. [7] However, in some populations such as in Italy, Poland, Ireland, Spain and Portugal men are found to have fairer complexions, and this has been ascribed as a cause to increased melanoma risk in men. [81] [82]

The sexes also differ in how they change their skin color with age. Men and women are not born with different skin color, they begin to diverge during puberty with the influence of sex hormones. Women can also change pigmentation in certain parts of their body, such as the areola, during the menstrual cycle and pregnancy and between 50 and 70% of pregnant women will develop the "mask of pregnancy" (melasma or chloasma) in the cheeks, upper lips, forehead, and chin. [7] This is caused by increases in the female hormones estrogen and progesterone and it can develop in women who take birth control pills or participate in hormone replacement therapy. [83]

Uneven pigmentation of some sort affects most people, regardless of bioethnic background or skin color. Skin may either appear lighter, or darker than normal, or lack pigmentation at all there may be blotchy, uneven areas, patches of brown to gray discoloration or freckling. Apart from blood-related conditions such as jaundice, carotenosis, or argyria, skin pigmentation disorders generally occur because the body produces either too much or too little melanin.

Depigmentation Edit

Albinism Edit

Some types of albinism affect only the skin and hair, while other types affect the skin, hair and eyes, and in rare cases only the eyes. All of them are caused by different genetic mutations. Albinism is a recessively inherited trait in humans where both pigmented parents may be carriers of the gene and pass it down to their children. Each child has a 25% chance of being albino and a 75% chance of having normally pigmented skin. [84] One common type of albinism is oculocutaneous albinism or OCA, which has many subtypes caused by different genetic mutations. Albinism is a serious problem in areas of high sunlight intensity, leading to extreme sun sensitivity, skin cancer, and eye damage. [7]

Albinism is more common in some parts of the world than in others, but it is estimated that 1 in 70 humans carry the gene for OCA. The most severe type of albinism is OCA1A, which is characterized by complete, lifelong loss of melanin production, other forms of OCA1B, OCA2, OCA3, OCA4, show some form of melanin accumulation and are less severe. [7] The four known types of OCA are caused by mutations in the TYR, OCA2, TYRP1, and SLC45A2 genes. [78]

Albinos often face social and cultural challenges (even threats), as the condition is often a source of ridicule, racism, fear, and violence. Many cultures around the world have developed beliefs regarding people with albinism. Albinos are persecuted in Tanzania by witchdoctors, who use the body parts of albinos as ingredients in rituals and potions, as they are thought to possess magical power. [85]

Vitiligo Edit

Vitiligo is a condition that causes depigmentation of sections of skin. It occurs when melanocytes die or are unable to function. The cause of vitiligo is unknown, but research suggests that it may arise from autoimmune, genetic, oxidative stress, neural, or viral causes. [86] The incidence worldwide is less than 1%. [87] Individuals affected by vitiligo sometimes suffer psychological discomfort because of their appearance. [7]

Hyperpigmentation Edit

Increased melanin production, also known as hyperpigmentation, can be a few different phenomena:

    describes the darkening of the skin. describes skin discolorations caused by hormones. These hormonal changes are usually the result of pregnancy, birth control pills or estrogen replacement therapy. , also known as "liver spots" or "senile freckles", refers to darkened spots on the skin caused by aging and the sun. These spots are quite common in adults with a long history of unprotected sun exposure.

Aside from sun exposure and hormones, hyperpigmentation can be caused by skin damage, such as remnants of blemishes, wounds or rashes. [88] This is especially true for those with darker skin tones.

The most typical cause of darkened areas of skin, brown spots or areas of discoloration is unprotected sun exposure. Once incorrectly referred to as liver spots, these pigment problems are not connected with the liver.

On lighter to medium skin tones, solar lentigenes emerge as small- to medium-sized brown patches of freckling that can grow and accumulate over time on areas of the body that receive the most unprotected sun exposure, such as the back of the hands, forearms, chest, and face. For those with darker skin colors, these discolorations can appear as patches or areas of ashen-gray skin.

Melanin in the skin protects the body by absorbing solar radiation. In general, the more melanin there is in the skin the more solar radiation can be absorbed. Excessive solar radiation causes direct and indirect DNA damage to the skin and the body naturally combats and seeks to repair the damage and protect the skin by creating and releasing further melanin into the skin's cells. With the production of the melanin, the skin color darkens, but can also cause sunburn. The tanning process can also be created by artificial UV radiation.

There are two different mechanisms involved. Firstly, the UVA-radiation creates oxidative stress, which in turn oxidizes existing melanin and leads to rapid darkening of the melanin, also known as IPD (immediate pigment darkening). Secondly, there is an increase in production of melanin known as melanogenesis. [89] Melanogenesis leads to delayed tanning and first becomes visible about 72 hours after exposure. The tan that is created by an increased melanogenesis lasts much longer than the one that is caused by oxidation of existing melanin. Tanning involves not just the increased melanin production in response to UV radiation but the thickening of the top layer of the epidermis, the stratum corneum. [7]

A person's natural skin color affects their reaction to exposure to the sun. Generally, those who start out with darker skin color and more melanin have better abilities to tan. Individuals with very light skin and albinos have no ability to tan. [90] The biggest differences resulting from sun exposure are visible in individuals who start out with moderately pigmented brown skin: the change is dramatically visible as tan lines, where parts of the skin which tanned are delineated from unexposed skin. [7]

Modern lifestyles and mobility have created mismatch between skin color and environment for many individuals. Vitamin D deficiencies and UVR overexposure are concerns for many. It is important for these people individually to adjust their diet and lifestyle according to their skin color, the environment they live in, and the time of year. [7] For practical purposes, such as exposure time for sun tanning, six skin types are distinguished following Fitzpatrick (1975), listed in order of decreasing lightness:

Fitzpatrick scale Edit

The following list shows the six categories of the Fitzpatrick scale in relation to the 36 categories of the older von Luschan scale: [91] [92]

Type Also called Sunburning Tanning behavior Von Luschan's chromatic scale
I Light, pale white Always Never 0–6
II White, fair Usually Minimally 7–13
III Medium white to light brown Sometimes Uniformly 14–20
IV Olive, moderate brown Rarely Easily 21–27
V Brown, dark brown Very rarely Very easily 28–34
VI Very dark brown to black Never Never 35–36

Dark skin with large concentrations of melanin protects against ultraviolet light and skin cancers light-skinned people have about a tenfold greater risk of dying from skin cancer, compared with dark-skinned persons, under equal sunlight exposure. Furthermore, UV-A rays from sunlight are believed to interact with folic acid in ways that may damage health. [93] In a number of traditional societies the sun was avoided as much as possible, especially around noon when the ultraviolet radiation in sunlight is at its most intense. Midday was a time when people stayed in the shade and had the main meal followed by a nap, a practice similar to the modern siesta.

Approximately 10% of the variance in skin color occurs within regions, and approximately 90% occurs between regions. [94] Because skin color has been under strong selective pressure, similar skin colors can result from convergent adaptation rather than from genetic relatedness populations with similar pigmentation may be genetically no more similar than other widely separated groups. Furthermore, in some parts of the world where people from different regions have mixed extensively, the connection between skin color and ancestry has substantially weakened. [95] In Brazil, for example, skin color is not closely associated with the percentage of recent African ancestors a person has, as estimated from an analysis of genetic variants differing in frequency among continent groups. [96]

In general, people living close to the equator are highly darkly pigmented, and those living near the poles are generally very lightly pigmented. The rest of humanity shows a high degree of skin color variation between these two extremes, generally correlating with UV exposure. The main exception to this rule is in the New World, where people have only lived for about 10,000 to 15,000 years and show a less pronounced degree of skin pigmentation. [7]

In recent times, humans have become increasingly mobile as a consequence of improved technology, domestication, environmental change, strong curiosity, and risk-taking. Migrations over the last 4000 years, and especially the last 400 years, have been the fastest in human history and have led to many people settling in places far away from their ancestral homelands. This means that skin colors today are not as confined to geographical location as they were previously. [7]

According to classical scholar Frank Snowden, skin color did not determine social status in ancient Egypt, Greece or Rome. These ancient civilizations viewed relations between the major power and the subordinate state as more significant in a person's status than their skin colors. [97] [ page needed ]

Nevertheless, some social groups favor specific skin coloring. The preferred skin tone varies by culture and has varied over time. A number of indigenous African groups, such as the Maasai, associated pale skin with being cursed or caused by evil spirits associated with witchcraft. They would abandon their children born with conditions such as albinism and showed a sexual preference for darker skin. [98]

Many cultures have historically favored lighter skin for women. Before the Industrial Revolution, inhabitants of the continent of Europe preferred pale skin, which they interpreted as a sign of high social status. The poorer classes worked outdoors and got darker skin from exposure to the sun, while the upper class stayed indoors and had light skin. Hence light skin became associated with wealth and high position. [99] Women would put lead-based cosmetics on their skin to whiten their skin tone artificially. [100] However, when not strictly monitored, these cosmetics caused lead poisoning. Other methods also aimed at achieving a light-skinned appearance, including the use of arsenic to whiten skin, and powders. Women would wear full-length clothes when outdoors, and would use gloves and parasols to provide shade from the sun.

Colonization and enslavement as carried out by European countries became involved with colorism and racism, associated with the belief that people with dark skin were uncivilized, inferior, and should be subordinate to lighter-skinned invaders. This belief exists to an extent in modern times as well. [101] Institutionalized slavery in North America led people to perceive lighter-skinned African-Americans as more intelligent, cooperative, and beautiful. [102] Such lighter-skinned individuals had a greater likelihood of working as house slaves and of receiving preferential treatment from plantation owners and from overseers. For example, they had a chance to get an education. [103] The preference for fair skin remained prominent until the end of the Gilded Age, but racial stereotypes about worth and beauty persisted in the last half of the 20th century and continue in the present day. African-American journalist Jill Nelson wrote that, "To be both prettiest and black was impossible," [104] and elaborated:

We learn as girls that in ways both subtle and obvious, personal and political, our value as females is largely determined by how we look. … For black women, the domination of physical aspects of beauty in women's definition and value render us invisible, partially erased, or obsessed, sometimes for a lifetime, since most of us lack the major talismans of Western beauty. Black women find themselves involved in a lifelong effort to self-define in a culture that provides them no positive reflection. [104]

A preference for fair or lighter skin continues in some countries, including Latin American countries where whites form a minority. [105] In Brazil, a dark-skinned person is more likely to experience discrimination. [106] Many actors and actresses in Latin America have European features—blond hair, blue eyes, and pale skin. [107] [108] A light-skinned person is more privileged and has a higher social status [108] a person with light skin is considered more beautiful [108] and lighter skin suggests that the person has more wealth. [108] Skin color is such an obsession in some countries that specific words describe distinct skin tones - from (for example) "jincha", Puerto Rican slang for "glass of milk" to "morena", literally "brown". [108]

In Pakistan & India, society regards pale skin as more attractive and associates dark skin with lower class status this results in a massive market for skin-whitening creams. [109] Fairer skin-tones also correlate to higher caste-status in the Hindu social order—although the system is not based on skin tone. [110] Actors and actresses in Indian cinema tend to have light skin tones, and Indian cinematographers have used graphics and intense lighting to achieve more "desirable" skin tones. [111] Fair skin tones are advertised as an asset in Indian marketing. [112]

Skin-whitening products have remained popular over time, often due to historical beliefs and perceptions about fair skin. Sales of skin-whitening products across the world grew from $40 billion to $43 billion in 2008. [113] In South and East Asian countries, people have traditionally seen light skin as more attractive, and a preference for lighter skin remains prevalent. In ancient China and Japan, for example, pale skin can be traced back to ancient drawings depicting women and goddesses with fair skin tones. [ citation needed ] In ancient China, Japan, and Southeast Asia, pale skin was seen as a sign of wealth. Thus skin-whitening cosmetic products are popular in East Asia. [114] Four out of ten women surveyed in Hong Kong, Malaysia, the Philippines and South Korea used a skin-whitening cream, and more than 60 companies globally compete for Asia's estimated $18 billion market. [115] Changes in regulations in the cosmetic industry led to skin-care companies introducing harm-free skin lighteners. In Japan, the geisha have a reputation for their white-painted faces, and the appeal of the bihaku ( 美白 ) , or "beautiful white", ideal leads many Japanese women to avoid any form of tanning. [116] There are exceptions to this, with Japanese fashion trends such as ganguro emphasizing tanned skin. Skin whitening is also not uncommon in Africa, [117] [118] and several research projects have suggested a general preference for lighter skin in the African-American community. [119] In contrast, one study on men of the Bikosso tribe in Cameroon found no preference for attractiveness of females based on lighter skin color, bringing into question the universality of earlier studies that had exclusively focused on skin-color preferences among non-African populations. [120]

Significant exceptions to a preference for lighter skin started to appear in Western culture in the mid-20th century. [121] However a 2010 study found a preference for lighter-skinned women in New Zealand and California. [122] Though sun-tanned skin was once associated with the sun-exposed manual labor of the lower class, the associations became dramatically reversed during this time—a change usually credited to the trendsetting Frenchwoman Coco Chanel (1883-1971) presenting tanned skin as fashionable, healthy, and luxurious. [123] As of 2017 [update] , though an overall preference for lighter skin remains prevalent in the United States, many within the country regard tanned skin as both more attractive and healthier than pale or very dark skin. [124] [125] [126] Western mass media and popular culture continued [ when? ] to reinforce negative stereotypes about dark skin, [127] but in some circles pale skin has become associated with indoor office-work while tanned skin has become associated with increased leisure time, sportiness and good health that comes with wealth and higher social status. [99] Studies have also emerged indicating that the degree of tanning is directly related to how attractive a young woman is. [128] [129]

Neonatal jaundice: aetiology, diagnosis and treatment

A significant proportion of term and preterm infants develop neonatal jaundice. Jaundice in an otherwise healthy term infant is the most common reason for readmission to hospital. Jaundice is caused by an increase in serum bilirubin levels, largely as a result of breakdown of red blood cells. Bilirubin is conveyed in the blood as 'unconjugated' bilirubin, largely bound to albumin. The liver converts bilirubin into a conjugated form which is excreted in the bile. Very high levels of unconjugated bilirubin are neurotoxic. Phototherapy is a simple and effective way to reduce the bilirubin level. Most term babies have 'physiological' jaundice which responds to a short period of phototherapy, and requires no other treatment. A few babies have rapidly rising bilirubin levels which place them at risk of kernicterus. Current management of jaundice in the UK is guided by the NICE guideline. Any infant with high serum bilirubin or a rapidly rising bilirubin level needs to be treated urgently to avoid neurotoxicity. High levels of conjugated bilirubin in a term baby can indicate biliary atresia, and babies with persisting jaundice must have their level of conjugated bilirubin measured. Preterm infants on long-term parenteral nutrition may develop conjugated jaundice which generally improves with the introduction of enteral feed and weaning of intravenous nutrition.

Keywords: Neonatal jaundice conjugated jaundice exchange transfusion kernicterus phototherapy.

Research Article

1 Directorate of Veterinary, Veterinary Hospital, Sulaimani City, Kurdistan Region, Northern Iraq
2 College of Veterinary Medicine, University of Sulaimani, Sulaimani Nwe, Street 27, Zone 11, Sulaimani City, Kurdistan Region, Northern Iraq
3 Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43300UPM, Serdang, Selangor, Malaysia

*Corresponding author: Hazhaow Muhamad Murad, College of Veterinary Medicine, University of Sulaimani, Sulaimani Nwe, Street 27, Zone 11, Sulaimani City, Kurdistan Region, Northern Iraq, Tel: + 9647701595663 E-mail: [email protected]

The colour of meat and fat of carcasses have a great effect on consumers demand especially beef carcasses. The majority of beef carcasses have creamy-white to cream fat colour. Yellowness of fat is due to the presence of carotenoid pigments within adipocyte or due to certain diseases like jaundice. The significance of this study was to differentiate between normal and abnormal beef yellow carcasses by using chemical laboratory tests and scientific basics. For this purpose, sixty yellow carcasses had been examined through the period end of September 2013 to the end of March 2014. The fat samples obtained from different parts of the carcasses and through the Rimmingtone and Fowri chemical tests, the actual cause of yellowness was determined. These results approved that the sex has a major effect on carotenoid deposition in the majority of the carcasses. Therefore, jaundice cases were determined for the first time in Sualaimani slaughter house chemically. Miss diagnosis of carotenoid carcasses as jaundice case causes a greater economic loss and its necessary to make Rimmingtone and Fowrie Tests as a routine laboratory tests in Sulaimani Slaughter houses for differentiation.

Jaundice Chemical test Sulaimani slaughterhouse Beef carcass

Red meat contains high biological value proteins, range of fats and important micronutrients that are needed for good health throughout life. Purchased red meat usually consists of both lean tissue (muscle) and fat tissue, which can be either distributed throughout the muscle as marbling (internal fat) or surrounding the muscle meat as selvage or external fat [1].

The colour of bovine subcutaneous adipose tissue (carcass fat) is an important component of beef carcass quality and thus, beef carcass grading systems [2]. In most beef markets, excessive yellowness in bovine carcass fat colour is undesirable [3].

Consumers usually prefer white fat in finished beef, and some export markets place high value on colour of fat in cuts of beef [4]. Beef fat normally has white to creamy white colour [5]. Morgan Everitt [6] approved that yellowness of fat is due to the presence of carotenoid pigments within adipocytes or due to certain diseases. Beta carotene, is a major precursor of vitamin A, and is the main contributing carotenoid pigment in beef fat although trace amounts of alpha-carotene and xanthophylls have also been found. According to Herenda et al. [7] icterus is the result of an abnormal accumulation of bile pigment, bilirubin, or haemoglobin in the blood. Yellow pigmentation is mainly observed in the skin, internal organs, sclera (the white of the eye), tendons, cartilage, arteries, joint surfaces, which is a clinical sign of a faulty liver or bile duct malfunction, but it may be also caused by diseases in which the liver is not impaired and divided into three main categories Pre-hepatic jaundice (haemolytic icterus), hepatic jaundice (toxic icterus) and post-hepatic jaundice (obstructive icterus).

In Sulaimani slaughterhouse, the routine inspection for the suspected yellow carcasses depends on naked -eye and experience of the inspector. So the aim of this study was to differentiate between the yellow carcasses which may be due to carotene accumulation or icterus by using chemical tests as laboratory examination for confirmation.


-Sodium hydroxide (Merk, Germany)
-Di ethyl Ether (PARS Chemi, India)


Fat samples were obtained from different location of (60) suspected beef carcasses (male and female), in 15 days through the period (End of September/2013 to the end of March/2014) from Sulaimani slaughterhouse. Samples were placed in a close glass containers, then transferred to the laboratory in Sulaimani Slaughter house immediately for analysis.

Two gram of fat samples was placed in test tube and 5 mL of a 5% of NaOH were added. The mixture was boiled for about 1 minute, and shacked frequently until the fat was dissolved. Then the test tubes were cooled under the tap water till it was comfortably warm to the hand. An equal volume of ether was added and mixed gently. The mixer allowed settling down. The solution settled out in to layers [8].

Interpretation of the test

a. The bile salt is soluble in water, so if the color was due to jaundice the bottom layer will be colored yellow.

b. If the color was due to carotene, the top layer colored yellow, as these are soluble in ether. The judgment was easier when test tube viewed against a white back-ground.

Table 1 shows the percentage number of cases according to the carotene and jaundice. The result revealed that at the (day 4 and day 13) of the study the percentage of slaughtered animals with carotene case were high, while jaundice cases were high at the (day 12). Figure 1 shows the percentage number of carotenoid cases according to the breed. The number of local breeds (Sharabi-Sub breed Karadi) slaughtered in Sulaimani slaughterhouse were higher than foreigners one, where Figures 2a and 2b which shows the percentage of cases with carotene and jaundice results according to the animal sex. In carotene cases a high percentage recorded in females, while in jaundice cases male percentage were high. In Table 2, the results revealed both of carotenoid and jaundice cases were high in outdoor feeding animals.

Table 3 shows the regression analyses of four factors on carotenoid and jaundice yellow beef carcasses. It was found that there is negative relationship between all included independent variables (sex, breed, age and diet) on the one hand, and the carotenoid in the other.

While Figure 3 shows positive result for icterus (jaundice), by using Rimmingtone and Fowrie tests, the result showed that the bottom layer had yellow colour, while the top had white. Where Figure 4 shows positive result for carotene by using Rimmingtone and Fowrie tests, and the result indicated that the top layer had yellow colour, while the bottom layer had white colour. Figure 5 shows the carotenoid cases features among other cases in Sulaimani Slaughterhouse, whereas Figure 6, shows the features of jaundice, carotenoid, and normal carcasses. In the icteric cases, all part of the carcass had yellow colour (body fat, tendon, internal organ, white tissue), while in carotenoid carcass, only body fat had yellow colour. In Figure 7 which show ictric (jaundice) carcasses with internal organ, and illustrate the yellow colour in internal organ (liver, kidney, lungs, heart fat). Then Figure 8 shows the foreign breed of beef (Brahman) in Sulaimani slaughterhouse and Figure 9 demonstrates the local breed of beef (Sharabi –Sub Karadi).

In the routine postmortem examination in Sulaimani slaughterhouse, animals with yellow colour were treated as “suspects” on examination judgment depends on inspector choice by naked eye and experience. The percentage of cases according to the carotene and jaundice, had been shown in Table 1, which revealed that the carotenoid carcasses had high value in day (4 and 13) of inspection (22/10/2013 and 5/3/2014), those two days marked the October and March in this study. In our province, it has been well known that March is the beginning of the spring season, and the pastures had been developed, so the outdoor feeding started again. For November the same results could be true, as reported by Yang et al. [9] Strachan et al. [10] inclusion of grazed or conserved grass in cattle diets leads to yellow coloured carcass fat because such forages are rich sources of the compounds responsible for yellowness, namely β-carotene. While in winter the animals spent longer period on a conventional indoor ration and become less yellow in colour [11]. The jaundice cases usually occur as a disease case not depending on the season, this is mainly due to causative agent. Carotenoids are relatively unstable and as soon as grass appears to have dried, their concentration much reduced. Most grains contain only small concentrations of carotenoids. This is why colour decreases when cattle are fed in feedlots fat. Betacarotene is only a minor component (about 5-8%) of the total carotenoids in plants. However, it is selectively absorbed, accounting for more than 80% of the yellow pigments present in beef fat with carotenoid cases [12].

Figure 1: The percentage of carotenoid cases according to the breed

Table 1: The percentage of examined cases according to the carotene and jaundice

Figure 2: The percentage of cases according to the sex. (a) Carotene, (b) Jaundice

Table 2: The percentage of carotene and jaundice cases according to the feed source

Table 3: The regression analysis of carotenoid and jaundice yellow beef carcasses according to variable factors

Figure 1 shows that the number of carotenoid cases, is higher in local breed than foreigners, which is well distinguished in Figures 8 and 9, this could be due to that the local breed (Sharabi) has the ability to deposit β-carotene in there fat more than that of the foreign breed (Brahman). The intensity of fat depend on breed [6] Morgan and Everitt, 1969 also agreed with that in their study, where they claimed that fat colour intensity and carotene levels were higher in the Jersey than in Friesian and Aberdeen Angus cattle. While Kruk et al. [13] discovered that pure Jersey -cows had higher β-carotene concentrations in their subcutaneous adipose tissue than either Jersey Limousin or pure Limousin cows. Figure 2a shows number of cases with carotene according to the animal sex, which in carotene cases a high percentage recorded in females. Walker et al. [3] also reported that females had yellower subcutaneous fat than steers. Barton and Pleasants, [14], who compared different breeds of steers rose on pasture and slaughtered at 30 months of age, and found that beef breeds had significantly more carcasses with white fat than dairy breed carcasses and the Jersey breed had more with yellow fat carcasses than any other breed. There is a great effect of lipid metabolism on the colour of bovine carcass fat during lactation. Lactogenesis in ruminants is a lipid metabolism where by lipolysis increases and lipogenesis decreases in adipose tissue [15]. Dunne et al. [16], claimed that the high concentration of carotene in the dairy cows was due to accumulation of carotenoid through the life of cow coupled with intermittent periods of lipid depletion coincident with lactations and produce the yellow adipose tissue of old dairy cows. Where Figure 2b showed that the numbers of jaundice cases were high in male (67%) than female, this could be due to that jaundice is a disease case which may relate to parasitic, hepatic or obstructive condition. According to Table 2, there was a high value of carotenoid cases in outdoor beef, as mentioned before the outdoor feed contain grass and green forage, while indoor feeding mainly contain grain, maize silage would decrease the yellowness of adipose tissue and have little effect on the sensory characteristics of beef as approved by [17], who used cereal based concentrate instead grass silage in the ration of beef cattle. Feeding concentrates produces subcutaneous adipose tissue which is less yellow than that from cattle fed diets containing forage [10,18]. The same fact was also recommended by Yang et al. [19] who reported that cattle predominantly pasture fed tended to accumulate β-carotene in subcutaneous and intramuscular fat. Simonne et al. [20] reported Japanese Black steers are fed roughage containing little β-carotene to avoid producing yellow body fat, especially subcutaneous fat. Because not all of the ingested and absorbed carotene is transformed into vitamin A, the surplus is present first in the blood and then deposited in adipose and hepatic tissues, where it accumulates. As a result, yellow fat is frequently observed in the carcasses of pasture-fed animals [6,10,19,21].

Figure 3: Positive result for icterus (jaundice) (Rimmingtone and Fowrie tests)

Figure 4: Positive result for carotene (Rimmingtone and Fowrie tests)

Unfortunately, the result revealed that more female are slaughtered in Sulaimani slaughterhouse than male local breed, which is against meat lows and legislations except in certain cases such as sterility or emergency [22]. The regression analyses of four factors on carotenoid and jaundice yellow beef carcasses, showed that there was negative relationship between the all included independent variables (sex, breed, age and diet) on the one hand and the carotenoid in the other, as shown in Table 3. More specifically, male sex variable has inverse impact on the dependent variable (carotenoid). So it is expected high status among females as been approved in Figure 2a, it’s to be noted that according to (T-test) this variables is the only significance variables, apart from intercept coefficients. It was clear that the significance of other variables (except sex) can be attributed to the reality of the data which obtained from owners and size of data.

The positive result for jaundice, as been showed in Figure 3 which revealed the yellow coloration in bottom layer while the upper layer was white, this is due to that, bile salt are not soluble in water. Whereas Figure 4 revealed the positive result for carotene, with yellow top layer because carotene soluble in ether and that’s why the bottom was white [8].

At daily routine inspection after slaughtering, the carotenoid cases appeared obviously among other carcasses due to specific yellow colour as shown in Figure 5. Icterus is the result of an abnormal accumulation of bile pigment, bilirubin, or haemoglobin in the blood. Yellow pigmentation is observed in the skin, internal organs, tendons, cartilage, arteries, joint surfaces [7], as shown in Figure 6, both jaundice and carotenoid and normal- white carcass appeared with quite differential between these two cases just at the slaughtering line of the slaughterhouse. In icteric case, the whole carcass white tissue colours were become yellow. While in carotenoid carcasses only the adipose tissue were have yellow colour [19]. Also Figure 7, revealed that the ictric carcass in Sulaimani slaughterhouse, the yellow colouration of the whole body and internal organ appear.

Figure 5: The caroteneoid cases among normal cases in Sulaimani slaughterhouse

Figure 6: The jaundice, carotenoid, normal carcasses in Sulaimani slaughterhouse

Figure 7: Ictric carcass with internal organs in Sulaimani slaughterhouse

What is Unconjugated Bilirubin?

Approximately 80% of the bilirubin originates from the breakdown of hemoglobin released by the degradation of obsolete erythrocytes in the monocyte-macrophage system. This happens predominantly in the spleen and to a lesser extent in the bone marrow and the liver (Kupffer cells).

Heme is derived from hemoglobin after separating the globin. After removal of the iron from the heme, biliverdin is formed. Under the action of biliverdin reductase, biliverdin is converted to α-bilirubin – unconjugated bilirubin (indirect). It is not soluble in water. 95% of it is transported from the monocyte-macrophage system to the liver associated with blood serum albumin. The non-albumin-bound bilirubin is fat soluble, has an affinity to the nervous tissue. It is highly toxic and, in large quantities, can cause brain damage – bilirubin encephalopathy.


yellowness of skin, sclerae, mucous membranes, and excretions due to hyperbilirubinemia and deposition of bile pigments . It is usually first noticeable in the eyes, although it may come on so gradually that it is not immediately noticed by those in daily contact with the jaundiced person. Called also icterus.

Jaundice is not a disease it is a symptom of a number of different diseases and disorders of the liver and gallbladder and of hemolytic blood disorders. One such disorder is the presence of a gallstone in the common bile duct, which carries bile from the liver to the intestine. This may obstruct the flow of bile, causing it to accumulate and enter the bloodstream. The obstruction of bile flow may cause bile to enter the urine, making it dark in color, and also decrease the bile in the stool, making it light and clay-colored. This condition requires surgery to remove the gallstone before it causes serious liver injury.

The pigment causing jaundice is called bilirubin . It is derived from hemoglobin that is released when erythrocytes are hemolyzed and therefore is constantly being formed and introduced into the blood as worn-out or defective erythrocytes are destroyed by the body. Normally the liver cells absorb the bilirubin and secrete it along with other bile constituents. If the liver is diseased, or if the flow of bile is obstructed, or if destruction of erythrocytes is excessive, the bilirubin accumulates in the blood and eventually will produce jaundice. Determination of the level of bilirubin in the blood is of value in detecting elevated bilirubin levels at the earliest stages before jaundice appears, when liver disease or hemolytic anemia is suspected.

Patient Care . Assessment of the patient with jaundice includes observations of the degree and location of yellowing, noting the color of urine and stools, and the presence of itching. Since jaundice can be accompanied by severe itching, frequent skin care is important to preserve skin integrity. Tepid sponge baths can help reduce discomfort and promote rest.

Patients with severe jaundice are at risk for encephalopathic changes that produce confusion, impaired mentation, and altered levels of consciousness. The potential for injury is increased and demands vigilance and safety measures to protect the patient.

The jaundice of the cell

“Fair is foul and foul is fair” (so say the witches in the opening scene of Macbeth), and examples of this paradox abound in biology. Life-giving oxygen is also the source of cytotoxic oxidative stress lethal gases like carbon monoxide are neurotransmitters that, among other things, facilitate reproduction (1). Another example is that of bilirubin, formerly dismissed as a waste product, whose image has been burnished by evolving evidence for a role in protecting cells from injury (2, 3). The latest development in this story, which focuses on the bilirubin-synthesizing enzyme biliverdin reductase (BVR), is the subject of an article by Barañano in this issue of PNAS (4).

The case has been made for a neuroprotective effect of bilirubin.

Bilirubin is best known as a yellow pigment contained in bile, which is released from the gall bladder into the duodenum to aid digestion. In Hippocratic medicine, yellow bile was one of four bodily fluids, or humors, that promoted health when in balance and illness when not. Humoral imbalances could also explain differences in personality: an excess of yellow bile made one choleric black bile, melancholic blood, sanguine and phlegm, phlegmatic. It was not until the middle of the last century that the chemical structure of bilirubin was determined and the molecule was synthesized, by Hans Fischer, a German chemist. Fischer received the Nobel Prize in chemistry in 1930 for his work on the bilirubin precursor heme. He killed himself in 1945 when his Munich laboratories were destroyed in an Allied bombing raid on Nazi Germany.

Bilirubin is produced as a by-product of the degradation of hemoglobin from senescent or hemolyzed red blood cells. The heme moiety of hemoglobin, in which iron is coordinated to each of four pyrrole rings of protoporphyrin, enters the blood and is transported to the liver. There, and in most other tissues, heme is metabolized by the microsomal enzyme heme oxygenase 1 (HO1) in the presence of nicotinamide-adenine dinucleotide phosphate (NADPH) and oxygen, to produce biliverdin, carbon monoxide, and iron. In other tissues, including the brain, another isoform of heme oxygenase, HO2, is involved. Following its synthesis by HO1 or HO2, biliverdin is converted by the phosphoprotein BVR (5), in the presence of NADPH, to bilirubin (Fig. 1). The crystal structures of the principal fetal (biliverdin IXβ reductase, or BVRB) and adult (biliverdin IXα reductase, or BVRA) forms of BVR have been reported recently (6, 7).

Synthesis of bilirubin from heme. HO, heme oxygenase BVR, biliverdin reductase CO, carbon monoxide.

The clinician confronts bilirubin in several settings. It is responsible for yellow discoloration of the skin in physiological jaundice of the newborn (Fig. 2) and in erythroblastosis fetalis, which usually results from Rh blood group incompatibility between an Rh-negative mother and an Rh-positive neonate. In this disorder, bilirubin accumulates as a result of antibody-induced hemolysis, which releases hemoglobin that is sequentially converted to heme, biliverdin, and bilirubin. Treatment involves phototherapy and exchange transfusion to prevent kernicterus, in which high concentrations of bilirubin gain access to the basal ganglia and other brain structures, resulting in lethargy, rigidity, seizures, death, and, in long-term survivors, choreoathetosis, hearing loss, and Parinaud's syndrome (paralysis of upward gaze ref. 8). Jaundice is also seen in hepatocellular disorders and extrahepatic biliary obstruction, which lead to increased bilirubin levels in the blood and its subsequent deposition in elastin-rich tissues, such as skin and sclera. Finally, because bilirubin is derived from hemoglobin, it is seen at sites of recent bleeding, such as hematomas under the skin. A special case is xanthochromia, or yellow coloration, of the cerebrospinal fluid, which every medical student learns to recognize as a sign of subarachnoid hemorrhage.

Physiological jaundice of the newborn (photo courtesy of Maeve Greenberg).

The example of kernicterus, in particular, suggests that bilirubin is cytotoxic. Several studies have shown such toxicity (9), which typically occurs at micromolar concentrations of bilirubin. Bilirubin causes death of cultured neurons (10) and cerebral microvascular endothelial cells (11) in vitro, and this has features of apoptotic cell death, including DNA fragmentation, release of cytochrome c, activation of caspase-3, and cleavage of poly(ADP)ribose polymerase. N-methyl- d -aspartate receptor antagonists can protect cultured neurons from bilirubin toxicity (12), implicating this class of glutamate receptors in pathogenesis. Bilirubin may also be an indirect cause of neuronal death after subarachnoid hemorrhage. Bilirubin oxidation products, derived from blood that enters the subarachnoid space when aneurysms rupture, cause cerebral vasospasm (13), and this leads, in turn, to morbidity and mortality from delayed cerebral ischemia or infarction.

Accumulating evidence points to a protective role of bilirubin as well. Stocker et al. (14) showed that bilirubin is an antioxidant that can scavenge peroxyl radicals, and bilirubin has been reported to protect against a variety of pathological processes, including complement-mediated anaphylaxis (15), myocardial ischemia (16), pulmonary fibrosis (17), and cyclosporin nephrotoxicity (18).

In a series of papers from the Snyder laboratory, the case has been made for a neuroprotective effect of bilirubin. Doré et al. (19) showed that hydrogen peroxide toxicity was increased in hippocampal neuron cultures from HO2-knockout mice, and that addition of free bilirubin (25–50 nM), or bilirubin conjugated to albumin (10–250 nM), improved survival. They later reported that after focal cerebral ischemia induced by occlusion of the middle cerebral artery followed by reperfusion, or intracerebral injection of the excitotoxic amino acid N-methyl- d -aspartate, injury was more extensive in HO2-knockout (but not HO1-knockout) than in wild-type mice, which is consistent with a role for the products of HO2 in protection from ischemic injury (20). The failure of HO1-knockouts to show increased injury may be related to the nonneuronal location of HO1. In a follow-up study, the form of cell death against which HO2 protects was investigated (21). Death was induced in cultured cerebellar granule neurons by withdrawing serum and reducing the extracellular concentration of potassium. In both wild-type and HO2-knockout mice, cell death was predominantly apoptotic, as judged by morphological features including nuclear fragmentation and apoptotic bodies. However, cell death was approximately two-fold greater in cultures from HO2 knockouts, and this difference was accounted for almost completely by apoptosis. Focal cerebral ischemia in HO2-knockout mice or wild-type mice treated with the HO2 inhibitor tin protoporphyrin IX also increased the number of cells with apoptotic morphology in the ischemic penumbra. In addition to these results implicating HO2 in protection from cerebral ischemia, an intriguing connection to Alzheimer's disease has been made. Takahashi et al. (22) used yeast two-hybrid analysis and coimmunoprecipitation studies to identify proteins that interact with HO2. They found that one such protein was amyloid precursor protein (APP), which is the source of β-amyloid in Alzheimer's disease, and which is mutated in some familial forms of the disorder. Interaction with wild-type APP inhibited the activity of HO2, but the Swedish, Dutch, and London APP mutations had about twice the inhibitory effect of wild-type APP. Moreover, cortical neuron cultures from mice expressing the Swedish mutation showed defects in bilirubin production and enhanced toxicity from hydrogen peroxide. The authors concluded that HO may help to regulate oxidative injury in Alzheimer's disease, which is one way that heme deficiency could impact the disease, as proposed recently (23). These and related studies have been reviewed (24).

In this issue of PNAS, Barañano et al. (4) ask, “As biliverdin is water-soluble and readily excreted, why should mammals have evolved the energetically expensive, potentially toxic, and apparently unnecessary capacity to reduce biliverdin (to bilirubin)?” The simple answer, from their own prior work, is that bilirubin is neuroprotective, but the low (nanomolar) concentrations of bilirubin present in cells, and the high concentrations of oxidants against which they protect, must be reconciled. To explain this discrepancy, Barañano et al. hypothesize that a mechanism must exist to amplify the antioxidant effect of bilirubin.

The mechanism they propose involves redox cycling (Fig. 3). Biliverdin is reduced to bilirubin through the action of BVR: bilirubin interacts with reactive oxygen species (ROS), which neutralizes their toxicity and oxidizes bilirubin, thereby regenerating biliverdin. As this cycle is repeated, the antioxidant effect of bilirubin is multiplied. This scheme gives rise to at least two testable predictions. First, ROS should promote the synthesis of biliverdin. Second, depletion of BVR should increase levels of ROS and their toxic effects. To test the effect of ROS on biliverdin synthesis, Barañano et al. treated HeLa cells with a compound that generates ROS, and found that biliverdin was produced. To determine the effect of BVR depletion on ROS activity, they used RNA interference and succeeded in reducing BVR activity in HeLa cells to 5–10% of normal levels. Oxidative activity, measured by a fluorescence assay, increased by ≈200%. Similar results were obtained with cultured cortical neurons. BVR-depleted cells were also more susceptible to caspase-dependent death from hyperoxia and to hydrogen peroxide toxicity, which is consistent with an inability to recycle bilirubin and amplify its antioxidant effect. Finally, the antioxidant activity of BVR was quantitatively comparable to that of glutathione, leading the authors to suggest that BVR and glutathione may be the principal endogenous antioxidants associated with the membrane and cytoplasmic compartments, respectively.

Amplification of the neuroprotective effect of bilirubin by redox cycling. Biliverdin is reduced to bilirubin by biliverdin reductase (BVR) and is regenerated when the detoxification of reactive oxygen species (ROS) oxidizes bilirubin back to biliverdin. In this manner, low concentrations of bilirubin can be recycled to neutralize large amounts of ROS.

A common feature of endogenous neuroprotective systems is that many of them are transcriptionally induced by the injury states against which they protect. In cerebral ischemia, for example, increased expression in the ischemic penumbra, a region that can be salvaged by endogenous or exogenous neuroprotectants, is observed for such diverse protective proteins as heat-shock proteins, growth factors, hypoxia-inducible factor-1 and its targets, and anti-apoptotic Bcl-2-family gene products (25, 26), as well as the recently identified oxygen-binding protein, neuroglobin (27). It is of interest, therefore, that BVR expression is also increased in the penumbra after focal cerebral ischemia (28), which may be further evidence of its neuroprotective role.

An additional function for BVR has been identified. The protein contains a leucine zipper DNA-binding motif and, in homodimeric form, binds to a region of the HO1 promoter that contains two AP-1 sites (29). Therefore, BVR may protect cells not only by catalyzing the formation of bilirubin, but also by transcriptional activation of HO1, which promotes the efflux of potentially toxic iron from cells exposed to oxidative stress (24).

In a 1990 review titled “Is bilirubin good for you?”, McDonagh (30) summarized the evidence for a protective role of bilirubin, concluding that “the biochemical path from red (heme) to green (biliverdin) to yellow (bilirubin) may defend as well as degrade.” Barañano et al. (4) have provided important additional evidence for this view by demonstrating a mechanism that makes it quantitatively plausible, thereby spotlighting a potential therapeutic target in stroke and other disorders associated with oxidative injury.