Physico-chemical characterization of bilirubin-10-sulfonate and comparison of its acid–base behavior with unconjugated bilirubin

Unconjugated bilirubin (UCB) is the end-product of heme catabolism in the intravascular compartment. Although beneficial for human health when mildly elevated in the body, when present at greater than a critical threshold concentration, UCB exerts toxic effects that are related to its physico-chemical properties, particularly affecting the central nervous system. The aim of the present study was to characterize bilirubin-10-sulfonate (ranarubin), a naturally occurring bile pigment, including determination of its mixed acidity constants (pKa*). Thanks to the presence of the sulfonic acid moiety, this compound is more polar compared to UCB, which might theoretically solve the problem with an accurate determination of the UCB pKa* values of its propionic acid carboxylic groups. Bilirubin-10-sulfonate was synthesized by modification of a previously described procedure; and its properties were studied by mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and circular dichroism (CD) spectroscopy. Determination of pKa* values of bilirubin-10-sulfonate and UCB was performed by capillary electrophoresis with low pigment concentrations in polar buffers. The identity of the synthesized bilirubin-10-sulfonate was confirmed by MS, and the pigment was further characterized by NMR, IR, and CD spectroscopy. The pKa values of carboxylic acid moieties of bilirubin-10-sulfonate were determined to be 5.02, whereas those of UCB were determined to be 9.01. The physico-chemical properties of bilirubin-10-sulfonate were partially characterized with low pKa* values compared to those of UCB, indicating that bilirubin-10-sulfonate cannot be used as a surrogate pigment for UCB chemical studies. In addition, using a different methodological approach, the pKa* values of UCB were found to be in a mildly alkaline region, confirming the conclusions of a recent critical re-evaluation of this specific issue.

Pathological Study of Facial Eczema (Pithomycotoxicosis) in Sheep

Facial eczema (FE) is a secondary photosensitization disease of farm ruminants caused by the sporidesmin A, produced in the spores of the saprophytic fungus Pithomyces chartarum. This study communicates an outbreak of ovine FE in Asturias (Spain) and characterizes the serum biochemical pattern and the immune response that may contribute to liver damage, favoring cholestasis and the progression to fibrosis and cirrhosis. Animals showed clinical signs of photosensitivity, with decrease of daily weight gain and loss of wool and crusting for at least 6 months after the FE outbreak. Serum activity of γ-glutamyltransferase and alkaline phosphatase were significantly increased in sheep with skin lesions. In the acute phase, edematous skin lesions in the head, hepatocytic and canalicular cholestasis in centrilobular regions, presence of neutrophils in small clumps surrounding deposits of bile pigment, ductular proliferation, as well as cholemic nephrosis, were observed. Macrophages, stained positively for MAC387, were found in areas of canalicular cholestasis. In the chronic phase, areas of alopecia and crusting were seen in the head, and the liver was atrophic with large regeneration nodules and gallstones. Fibrosis around dilated bile ducts, “typical” and “atypical” ductular reaction and an inflammatory infiltrate composed of lymphocytes and pigmented macrophages, with iron deposits and lipofuscin, were found. The surviving parenchyma persisted with a jigsaw pattern characteristic of biliary cirrhosis. Concentric and eccentric myointimal proliferation was found in arteries near damaged bile ducts. In cirrhotic livers, stellated cells, ductular reaction, ectatic bile ducts and presence of M2 macrophages and lymphocytes, were observed in areas of bile ductular reaction.

Hepatogenous Photosensitization in Steer by Brachiaria decumbens

Background: Although the etiology of hepatogenous photosensitization has not yet been fully elucidated, it is known that hepatotoxic substances (saponins) present in grasses of the genus Brachiaria spp. are responsible for intoxication of several species, causing great economic losses in the whole world. Since this grass is the source of food for the herd in Brazil, and other countries of the world, the aim of this paper is to describe the epidemiological, clinical, laboratory and anatomopathological aspects of a steer with this disease.Case: A 3-year-old Nellore steer was referred to veterinary care at a property in Bahia, with a 3-week history of swelling, loss of cutaneous tissue in the ear and scrotum region, and dry faeces. The animal was raised in pasture with Brachiaria decumbens along with five animals of different age and sex; however, it was the only one to present symptoms. Although the animal had been treated at the farm, there was no clinical improvement. On clinical examination, the steer was apathetic with jaundiced mucous membranes, nasal and ocular discharge, epiphora, and ulcers on the labial and gum commissure. The steer had leukocytosis with neutrophilia, anemia, and hyperfibrinogemia. The body condition score (BCS) was 3 (BCS ranges from 1 to 9), and the skin lesions observed were bedsores, necrosis and scabs in several regions. The increase in liver enzymes (GGT, AST) indicated hepatic impairment, suggesting a case of hepatogenous photosensitization. The therapeutic protocol instituted was enteral hydration, electrolyte replacement, topical application of ointment in the injured areas. In addition, it was recommended to maintain the animal in the shade, supply of good quality grass, and a new clinical evaluation in seven days. On new examination, it was observed that there was no satisfactory clinical improvement of the animal, and persistence of laboratory changes. Despite the poor prognosis, treatment was continued for another month with the same recommendations. However, in view of the severe clinical condition and unfavorable prognosis, the animal was submitted euthanasia. Necropsy revealed extensive areas of bedsores, erythema, severe jaundice in the mucous membranes, eyeballs and opaque corneas. The liver had an enlarged volume with bulging edges and a greenish color. The kidneys had a pale brownish color, with an irregular and mottled subcapsular surface, with blackened and depressed spots. Histologically, the cytoplasm of the hepatocytes was finely vacuolated, sometimes refringent and with an abundant presence of bile pigment. It was also observed in the middle of the liver parenchyma, multiple foci of accumulation of macrophages filled with vacuoles of different sizes containing saponins and crystals of saponins inside bile ducts. Furthermore, it was possible to observe hypertrophy and hyperplasia of Kupffer cells, disarrangement of hepatocytes with individual necrosis of hepatocytes.Discussion: The diagnosis of hepatogenous photosensitization was based on history, clinical, laboratory and anatomopathological findings. Serum biochemistry was important to measure hepatic impairment and possible secondary lesions, which were confirmed by the necropsy. Although hepatogenous photosensitization is less common in adult cattle, it must be considered in the differential diagnosis of skin lesions, reduced appetite, and jaundice. Since it was a sporadic case, individual predisposition is probably a preponderant factor. Keywords: cattle, photodermatitis, intoxication, liver injury, skin lesions.

Biliverdin Chiral Derivatives as Chiroptical Switches for pH and Metal Cation Sensing

A series of six optically active derivatives of the bile pigment biliverdin, namely (βS,βʹS)-dimethylmesobiliverdin-XIIIα cyclic esters of linear diols [HO(CH2)nOH] where n = 1-6, have been investigated by vibrational circular...

Analisis Komposisi dan Distribusi Batu Empedu di Laboratorium Fakultas Kedokteran Universitas Indonesia (FKUI) Jakarta

Abstrak Batu empedu merupakan penyakit yang banyak ditemukan di Indonesia. Analisis komposisi batu empedu penting untuk mengetahui penyebab, dasar metabolisme pembentukannya, faktor risiko, serta untuk edukasi diet terhadap pasien. Komposisi batu empedu tersebut dapat dipengaruhi oleh berbagai faktor termasuk usia dan jenis kelamin. Sampai saat ini belum terdapat data analisis komposisi dan distribusi batu empedu di Jakarta. Dengan demikian, penelitian ini bertujuan untuk menganalisis komposisi dan distribusi batu empedu yang dihubungkan dengan usia dan jenis kelamin. Penelitian ini merupakan penelitian cross-sectional menggunakan 230 data sekunder hasil analisis komposisi batu empedu di Laboratorium Biokimia dan Biologi Molekuler FKUI dalam periode 3 tahun terakhir (tahun 2017 – 2019). Analisis batu dilakukan dengan uji Salkowski untuk menilai kolesterol serta uji kalsium, karbonat, Fe, pigmen empedu, dan fosfat. Hasil penelitian menunjukkan bahwa batu empedu sebesar 1,3% merupakan batu murni dan 98,7% merupakan batu campuran. Komposisi terbanyak pada batu campuran yaitu kolesterol (83,91%). Batu empedu paling banyak ditemukan pada perempuan kelompok usia 40—49 tahun (27%). Terdapat hubungan komposisi kolesterol dengan usia (p<0,05) namun tidak terdapat hubungan (p>0,05) dengan jenis kelamin (p>0,05). Kesimpulan dari penelitian ini yaitu kolesterol merupakan penyusun komposisi batu empedu yang terbanyak yang berhubungan dengan usia, namun tidak berhubungan dengan jenis kelamin. Kata kunci: Batu Empedu, Distribusi, Komposisi, Kolesterol Abstract Gallstones are a disease that is commonly found in Indonesian. Analysis of the composition of gallstones is important to determine the etiology, the metabolic basis of its formation, risk factors, and to educate for the patient's diet. The composition of gallstones can be influenced by various factors including age and sex. Until now there is no data analysis of the composition and distribution of gallstones in Jakarta. Thus, this study aims to analyze the composition and distribution of gallstones that are associated with age and sex. This study is a cross-sectional study using 230 secondary data from the analysis of the composition of gallstones in the Laboratory of Biochemistry and Molecular Biology of FMUI in the last 3 years period (2017 - 2019). Stone analysis was carried out with the Salkowski test to assess cholesterol and also calcium, carbonate, Fe, bile pigment, and phosphate assays. The results showed that 1.3% gallstones were pure stones and 98.7% were mixed stones. The most composition in mixed stones is cholesterol (83.91%). Gallstones are most commonly found in women in the age group of 40-49 years (27%). There is a correlation between cholesterol composition with age (p <0.05) but there is no relationship (p> 0.05) with gender (p> 0.05). This study concluded that cholesterol was the most common constituent of gallstone. Its levels increased with age and did not differ significantly between genders. Keywords: Gallstone, Composition, Distribution, Cholesterol

Comparison of spectral characteristics in human and pig biliary system with hyperspectral imaging (HSI)

Injuries to the biliary tree during surgical, endoscopic or invasive radiological diagnostic or therapeutic procedures involving the pancreas, liver or organs of the upper gastrointestinal tract give rise to the need to develop a method for clear discrimination of biliary anatomy from surrounding tissue. Hyperspectral imaging (HSI) is an emerging optical technique in disease diagnosis and image-guided surgery with inherent advantages of being a non-contact, non-invasive, and non-ionizing technique. HSI can produce quantitative diagnostic information about tissue pathology, morphology, and chemical composition. HSI was applied in human liver transplantation and compared to porcine model operations to assess the capability of discriminating biliary anatomy from surrounding biological tissue. Absorbance spectra measured from bile ducts, gall bladder, and liver show a dependence on tissue composition and bile concentration, with agreement between human and porcine datasets. The bile pigment biliverdin and structural proteins collagen and elastin were identified as contributors to the bile duct and gall bladder absorbance spectra.

Bile pigment in small-bowel water content may reflect bowel habits: a retrospective analysis of a capsule endoscopy imaging series

Background: Pigmented bile salts darken the small-bowel lumen and are present with bile acid, which is involved in the development of bowel habits. The small-bowel water content (SBWC) in the ileum could represent the colonic environment, but no studies have focused on this feature. However, measurement of crude SBWC can be challenging because of the technical difficulty of the endoscopic approach without preparation. Our aim was to evaluate optically active bile pigments in the SBWC of patients with abnormal bowel habits using capsule endoscopy (CE) to investigate the impact of bile acid on bowel habits. Methods: The study population included 37 constipated patients, 20 patients with diarrhea, and 77 patients with normal bowel habits who underwent CE between January 2015 and May 2018. Patients with secondary abnormal bowel habits were excluded. In addition to conventional imaging, we used flexible spectral imaging color enhancement (FICE) setting 1 imaging, in which the effects of bile pigments on color are suppressed. Intergroup color differences of SBWC in the ileum (ΔE) were evaluated from conventional and FICE setting 1 images. Color values were assessed using the CIE L*a*b* color space. Differences in SBWC lightness (black to white, range 0–100) were also evaluated. Results: The ΔE values from the comparison of conventional images between patients with constipation and with normal bowel habits and between patients with diarrhea and with normal bowel habits were 12.4 and 11.2, respectively. These values decreased to 4.4 and 3.3, respectively, when FICE setting 1 images were evaluated. Patients with constipation and diarrhea had significantly brighter (34.4 versus 27.6, P < .0001) and darker (19.6 versus 27.6, P < .0001) SBWC lightness, respectively, than patients with normal bowel habits. The FICE setting 1 images did not reveal significant differences in SBWC lightness between those with constipation and with normal bowel habits (44.1 versus 43.5, P = .83) or between those with diarrhea and with normal bowel habits (39.1 versus 43.5, P = .20). Conclusions : Differences in SBWC color and darkness in the ileum appear to be attributable to bile pigments. Therefore, bile pigments in SBWC may reflect bowel habits.

Gallensteine – interdisziplinäre Behandlung

Gallstones develop in the gallbladder or the bile ducts. According to their chemical composition, gallstones can be divided into cholesterol stones, which are common, and the rare bile pigment stones. Altogether, up to 20 % of all adults develop gallstones and more than 20 % of them symptoms or complications. Female sex, age, pregnancy, physical inactivity, obesity, overnutrition and genetic factors such as ABCB4 deficiency of the hepatic lecithin transporter are kown risk factors for gallstone formation. In about one half of all patients biliary symptoms precede the three common and potentially life-threatening complications (acute cholecystitis, acute cholangitis and biliary pancreatitis). Although our knowledge about the genetics and pathophysiology of gallstones has improved, current treatment algorithms are predominantly invasive (ERC and surgery). Thus, better strategies are needed to prevent the formation of gallstones in general.