Chicken Liver Homogenates Research Articles

Effects of early nutrition and sanitary conditions on antibody levels in early and later life of broiler chickens

Immune maturation of broiler chickens may be affected by management, such as early life feeding strategy (early versus delayed nutrition) or by low or high sanitary conditions (LSC versus HSC). We compared systemic maternal (MAb), natural (NAb), natural auto- (NAAb), and antigen specific antibody (SpAb) levels (IgM, IgY) between broilers (n = 48 per treatment) that received early (EN) or delayed nutrition for 72 h (DN) housed in either low (LSC) or high sanitary conditions (HSC) between 7 and 35 d of age. We found minimal interactions between feeding strategy and sanitary conditions. At 7 d of age, broilers receiving EN compared with DN, had elevated levels of IgM binding keyhole limpet hemocyanin (KLH), phosphoryl-conjugated ovalbumin (PC-OVA), and muramyl dipeptide (MDP), whereas effects of feeding strategy diminished at later ages. In LSC compared with HSC broilers, levels of NAb agglutinating RRBC and sheep red blood cells (SRBC) were already elevated from 14 d of age onwards. At 33 d of age, antibody levels (NAb, NAAb, anti-LPS, anti-MDP) were all elevated in LSC, compared with HSC broilers, for both IgM and IgY, but not IgM against KLH. Western blotting revealed different binding patterns of NAAb against chicken liver homogenate, which may indicate that the NAAb repertoire is affected by antigenic pressure. Our data suggest that antibody levels are affected for an important part by environmental conditions (feeding strategy and sanitary conditions), but minimally by their interaction. However, it remains to be further studied whether the enhanced levels of antibodies as initiated by EN and LSC contribute to enhanced resistance to infectious diseases.

Transfer of natural auto-antibodies via egg yolk in chickens divergently selected for natural antibodies binding keyhole limpet hemocyanin

Barcodes of natural auto-antibody (NAAb) profiles based on staining intensity of isotypes binding numbers of self-(tissue) antigen fragments were suggested as parameters for immune diversity, and related to genetic background and health status in man, rodents and poultry. Here, hens, eggs and hatchlings from chicken lines divergently selected and bred for high (H line) or low (L line) total natural antibodies (NAb) levels in plasma binding keyhole limpet hemocyanin (KLH) at 16 weeks of age were tested for their NAAb repertoire binding chicken liver homogenate (CLH) fragments using quantitative Western immunoblotting. The aims of this study were 1. to detect line differences between the H and L line adult hens, eggs and hatchlings for the IgM and IgG isotypes binding CLH fragments, 2. study the presence of NAAb of both isotypes in yolk and albumen, as well as in hatchlings to detect a maternal NAAb transfer route via the egg to the hatchling, and 3. study whether new self-antigen binding isotypes and idiotypes are present in the hatchling.NAAb binding CLH fragments were found in plasma of adult hens (both IgM and IgG), in yolk (IgG only), and hatchlings (mostly IgG, but low levels of IgM). Auto-profiles of IgM showed homogeneity, while IgG profiles were heterogenic between individual hens and individual hatchlings. Significant higher levels as indicated by staining intensity and number of stained CLH fragments were found in plasma of hens genetically selected for high levels of NAb binding KLH. Lines could be clustered based on their auto-profiles indicating that profiles of self-binding IgM and IgG antibodies are genetically based. Visual comparison, clustering and correlation of hens and their hatchlings showed similarities for the IgG, but not the IgM isotype, indicating maternal transfer of IgG NAAb via the yolk. The IgM profile in the hatchlings on the other hand might represent neonatal self-binding antibody formation. As a consequence, hatchlings initially depend for self-binding antibodies on maternal IgG provision during early life.

Effect of Zinc Sulfate and Zinc Glycine Chelate on Concentrations of Acute Phase Proteins in Chicken Serum and Liver Tissue

The aim of the study was to determine how inorganic and organic forms of zinc affect the concentrations of C-reactive protein (CRP), serum amyloid A (SAA), alpha-1-acid glycoprotein (α-1-AGP), haptoglobin (Hp), and transferrin (TRF) in the blood and liver tissue of 450 1-day-old Ross 308 chicken. Four experimental groups received one the following: inorganic zinc (ZnSO4), a zinc phytase enzyme supplement (ZnSO4-F), organic zinc in combination with glycine (Zn-Gly), or organic zinc supplemented with phytase (Zn-Gly-F). The chicken serum and liver homogenates were assayed using an ELISA kit. The results of the study showed statistically significantly higher serum and liver concentration of SAA in the group of birds that received zinc sulfate in comparison to the group of birds receiving zinc in organic form. A statistically significantly higher serum concentration of CRP and α-1-AGP was also noted in the group receiving zinc sulfate as compared to the Zn-Gly group. Comparison of the serum concentration of TRF between the supplemented groups showed a statistically significant increase in this parameter in the Zn-Gly-F group as compared to the ZSO4-F group. The increase in the serum concentration of Hp in all groups in comparison to the control may indicate stimulation of local immune mechanisms. The results of this study showed an increase in the concentrations of APPs such as AGP and TRF following the administration of zinc glycine chelates, which may demonstrate their effect on metabolic processes in the liver and on immunocompetent cells that regulate the intensity of the immune response.

ANTIOXIDANT AND HEPATOPROTECTIVE POTENTIALS OF BAUHINIA VARIEGATA SEEDS AGAINST FERRIC CHLORIDE-INDUCED LIPID PEROXIDATION IN CHICKEN LIVER HOMOGENATE

Background: Iron accumulation in the liver is customary in pathological conditions associated with oxidative stress. Iron is also an essential element for organisms at its physiological levels. This study was aimed at investigating the antioxidant and hepatoprotective potential of acetone and methanol defatted seed extracts of Bauhinia variegata Linn (Leguminosae) against ferric chloride (FeCl3)-induced lipid peroxidation in chicken liver in vitro.
 Materials and Methods: Antioxidant capacity and hepatoprotective potential of acetone and methanol defatted seed extracts of B. variegata were evaluated using the established in vitro models, FeCl3-induced lipid peroxidation in chicken liver homogenate and thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH). Ascorbic acid, and acetone and methanol seed extracts of milk thistle (Silybum marianum) were used as antioxidant and hepatoprotective controls. 
 Results: An overall of 80% acetone and absolute methanol defatted seed extracts of B. variegata [B. variegata acetone extract (BVAc) and B. variegata methanol extract (BVMe)] revealed to possess antioxidant and free radical scavenging capacity, which can protect the liver from FeCl3-induced lipid peroxidation in a concentration-dependent manner when compared with controls, ascorbic acid and extracts of milk thistle. At concentrations of 250 µg/mL and 300 µg/mL, thiobarbituric acid reactive species (TBARS) inhibitions were 80.5% and 97.6% for BVAc; and 84% and 98.7% for milk thistle acetone extract (MSAc). At concentration of 300 µg/mL, BVMe and milk thistle methanol extract (MSMe) TBARS inhibitions were 89.0% and 93.8%, respectively. These findings may confirm the presence of antioxidant compounds with hepatoprotective potential in both defatted seed extracts of B. variegata. 
 Conclusion: The findings of this study suggest acetone and methanol extracts of B. variegata defatted seeds may serve as good sources of natural antioxidant and hepatoprotective agents.

Analisis Kromatografi Lapis Tipis (KLT) dan Aktivitas Antihiperurisemia Ekstrak Rebung Schizostachyum brachycladum Kurz (Kurz) pada Mencit Putih Jantan

Ekstraksi dan analisis KLT dari ekstrak rebung Schizostachyum brachycladum Kurz (Kurz), serta uji aktivitas antihiperurisemia pada mencit putih jantan telah dilakukan. Proses ekstraksi dilakukan dengan cara maserasi. Penentuan golongan senyawa dilakukan dengan berbagai reagen warna pada plat KLT. Pengukuran kadar asam urat dilakukan pada hari ke-8 setelah pemberian dosis harian homogenat hati ayam sebagai penginduksi asam urat bersamaan dengan suspensi ekstrak pada dosis 25, 50 dan 100 mg/kg dan allopurinol sebagai pembanding. Kadar asam urat serum mencit putih jantan diukur dengan menggunakan spektrofotometer. Hasil pengukuran memperlihatkan pemberian ekstrak rebung dengan dosis 25, 50 dan 100 mg/kgBB dapat menurunkan kadar asam urat mencit putih jantan dan berbeda secara signifkan dibandingkan dengan kontrol (p<0,05). Ketiga dosis ekstrak tidak memperlihatkan perbedaan yang nyata dalam menurunkan kadar asam urat (p>0,05). Penelitian ini menyimpulkan bahwa ekstrak rebung memiliki aktivitas antihiperurisemia.

Effect of age on hepatic cytochrome P450 of Ross 708 broiler chickens

Age has significant impact on hepatic cytochrome P450 (CYP450) systems in animals. Ross 708 broiler chicken is a breed of chicken with fast growth characteristics. Cytochrome P450 in the livers of Ross 708 broiler chicken of different ages has been investigated. The birds were raised under standard husbandry conditions. A certain number of chickens was randomly sampled weekly for liver collection from d 1 to 56 posthatch. The chicken body and liver weights were recorded. The chicken livers were processed for liver microsomes though a multiple-step procedure at low temperature. Total CYP450 content in chicken liver homogenates and liver microsomes was measured using a UV/visible spectroscopic method. The enzymatic activities of CYP450 in the chicken liver microsomes were determined through incubation of CYP450 isoform substrates followed by measurement of formation of their metabolites. The chicken showed an opposite age pattern in hepatic CYP450 content and activities compared with most mammals. The hepatic CYP450 content and activities of chicken at d 1 posthatch were higher than at other ages. The total hepatic CYP450 content in chickens at d 1 posthatch was more than twice the average hepatic value of the chickens at d 7 to 28. This high CYP450 fell quickly in the first week posthatch and slightly rose from d 28 to 56. Hepatic CYP450 activities of CYP1A, 3A, 2C, 2D, and 2H were much higher in the chicken at d 1 posthatch. The differences of these enzymatic activities between d 1 and other ages of chicken were CYP450 isoform dependent. This result suggests that embryonic development of chicken livers has a significant impact on the age profile of hepatic CYP450 content and activities of posthatch chickens.

Marked Endotheliotropism of Highly Pathogenic Avian Influenza Virus H5N1 following Intestinal Inoculation in Cats

Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

Hand-raising a spectral tarsier (Tarsius tarsier) at the Ueno Zoological Gardens

On February 10, 2008, a newborn male spectral tarsier (Tarsius tarsier) was found on the floor of the indoor exhibit room in the Small Mammal House of the Ueno Zoological Gardens. The dam showed no signs of providing maternal care and therefore we decided to hand-raise the infant. Its birth weight was 18.7 g. We placed the dam and infant in an incubator and gave 12.5-25% formula (for kittens), until the 145th day after birth. We limited the volume of formula intake to avoid excessive intake and to prevent diarrhea. For nutrition enrichment, we added a chicken liver homogenate to the formula 1-3 times per day. The infant was given a sunbath for 10 min on the 28th day. He showed no serious decline in health, except for diarrhea that occurred during the first few days after birth. He ate a small cricket for the first time on the 50th day and easily caught mealworms on his own on the 105th day. Gradual changes in feeding times, formula concentration, and the nutritionally enriched formula were essential for successfully hand-raising the tarsier.

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate.

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.

Biogenesis and function of lipolysosomes in developing chick hepatocytes

Proliferation of lipolysosomes is one of the characteristic aspects of embryonic chick hepatocytes. Formation of lipolysosomes is observed in the well-developed trans-Golgi network, with the highest frequency occurring from 11 to 14 days of incubation. The lipolysosomes usually contain a small and electron-dense lipid inclusion; however, during development, they gradually enlarge with an accompanying reduction in the electron density of the inclusion. Lipolysosomes isolated from neonatal chick liver homogenates were mainly composed of esterified cholesterol and showed considerably high activity of lysosomal enzymes. Moreover, the lipolysosome fraction is clearly shown to be a function of intralysosomal lipolysis via acid lipase. This accumulation of esterified cholesterol within lipolysosomes might be attributed to an excessive uptake and conversion of plasma lipoproteins to lipolysosomes. This concept is supported by the appearance of an abundance of coated pits and both “early” and “late” endosomes. The major components of plasma lipoprotein are low density lipoprotein (LDL) and high density lipoprotein (HDL), the cholesterol-rich lipoproteins, whose cholesterol content increases during the last week of incubation when the lipolysosomes quickly enlarge. Plasma lipoprotein particles are produced in the yolk sac epithelium from yolk very low density lipoprotein (VLDL) and transferred via the vitelline circulation to the chick liver. After hatching, when the supply of nutrients from the yolk sac is terminated, lipolysosomes immediately decrease in size and number. The cholesterol and fatty acids released are useful as an energy source and lipid metabolism in general, especially after hatching. Food intake induces the use of and accelerates the disappearance of lipolysosomes. Instead of lipolysosomes, lipid droplets appear and increase in number and size with concomitant increases of triglyceride concentrations in the liver homogenates, suggesting that lipogenesis has begun in the chick hepatocyte. Microsc. Res. Tech. 39:444–452, 1997. © 1997 Wiley-Liss, Inc.

Biogenesis and function of lipolysosomes in developing chick hepatocytes

Proliferation of lipolysosomes is one of the characteristic aspects of embryonic chick hepatocytes. Formation of lipolysosomes is observed in the well-developed trans-Golgi network, with the highest frequency occurring from 11 to 14 days of incubation. The lipolysosomes usually contain a small and electron-dense lipid inclusion; however, during development, they gradually enlarge with an accompanying reduction in the electron density of the inclusion. Lipolysosomes isolated from neonatal chick liver homogenates were mainly composed of esterified cholesterol and showed considerably high activity of lysosomal enzymes. Moreover, the lipolysosome fraction is clearly shown to be a function of intralysosomal lipolysis via acid lipase. This accumulation of esterified cholesterol within lipolysosomes might be attributed to an excessive uptake and conversion of plasma lipoproteins to lipolysosomes. This concept is supported by the appearance of an abundance of coated pits and both "early" and "late" endosomes. The major components of plasma lipoprotein are low density lipoprotein (LDL) and high density lipoprotein (HDL), the cholesterol-rich lipoproteins, whose cholesterol content increases during the last week of incubation when the lipolysosomes quickly enlarge. Plasma lipoprotein particles are produced in the yolk sac epithelium from yolk very low density lipoprotein (VLDL) and transferred via the vitelline circulation to the chick liver. After hatching, when the supply of nutrients from the yolk sac is terminated, lipolysosomes immediately decrease in size and number. The cholesterol and fatty acids released are useful as an energy source and lipid metabolism in general, especially after hatching. Food intake induces the use of and accelerates the disappearance of lipolysosomes. Instead of lipolysosomes, lipid droplets appear and increase in number and size with concomitant increases of triglyceride concentrations in the liver homogenates, suggesting that lipogenesis has begun in the chick hepatocyte.

CDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

Capacity in the Liver of the Broiler Chick for Conversion of Supplemental Methionine Activity to L-Methionine

The objective of the present experiments was to determine whether the levels of supplemental methionine sources currently used in practical diets exceed the capacity of the chick to convert the supplement to L-methionine. Supplemental sources examined included DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMB, Alimet®, or MHA®) and DL-methionine (DL-Met). Two approaches were taken: first, the amount of enzyme activity available for conversion of the two supplemental methionine sources was determined using optimum reaction conditions for each and chick liver homogenate as the enzyme source. These experiments showed that total liver enzyme conversion activity was 564 μmol/h for DL-HMB and 529 μmol/h for DL-Met. The total activities for the two sources were not different when measured at saturating substrate concentration. Second, to address the question of whether the enzyme is limiting for either source under practical feeding conditions, birds were fed starter diets supplemented with DL-HMB or DL-Met at .25% of the diet for 3 wk. When hepatic levels of free HMB and methionine were determined, birds fed DL-HMB contained 7.6 nmol HMB/g of liver and 84.7 nmol methionine/g of liver. Birds fed DL-Met had levels of 7.6 nmol HMB/g liver and 80.3 nmol methionine/g liver. These results indicate no accumulation of HMB or DL-Met, which might occur if conversion capacity were saturated. By calculation, a bird consuming 100 g/day of a diet supplemented with .25% activity would need to convert about 70 μmol/h, indicating a sevenfold excess of enzyme in the liver alone.

In vitro metabolism of testosterone on hepatic tissue of chicken (gallus domesticus)

Among the subcellular fractions of chicken liver homogenates, the microsomal and cytosol fractions were most active in metabolism of testosterone with mutually different enzymological features. On the other hand, the nuclear and mitochondrial fractions had far lower activity of metabolizing the steroid. Metabolism by the cytosol fraction: the following steroids were identified as the metabolites of testosterone. 5β -Dihydro testosterone (17β-hydroxy-5β-androstan-3-one), 5β-androstane-3α,17β-diol and its 3β-epimer, 3α-hydroxy-5β-androstan-17-one and its 3β-epimer and 5β-androstanedione. Metabolism by the microsomal fraction: from testosterone under aerobic condition, androstenedione was obtained as the major metabolite, besides the minor polar metabolites, production of which diminished when incubated in the atmosphere of carbon monoxide. From the results, testosterone was accepted to be firstly converted by the cytosol fraction into 5β-dihydrotestosterone which was then reduced to 5β-androstane-3α,17β-diol and its 3β-epimer. These diols were further converted partially to 3α-and 3β-hydroxy-5β-androstan-17-ones. These pathways were supported by the results of our incubation study with 5β-dihydrotestosterone and 5β-androstanedione as substrates. By the microsomes, testosterone was aerobically and anaerobically transformed to androstenedione as the major metabolite. Throughout our incubation experiments, no 5α-reduction of a Δ 4-3-oxo-steroid was detected in the chicken liver.

Ultrastructural and biochemical studies of lipolysis by lipolysosomes in chick hepatocytes.

Lipolysosomes in chick hepatocytes were studied by biochemical and electron-microscopic techniques. Their lipolysis was investigated in vitro using the floating lipid fraction isolated from chick liver homogenates. Many lipid droplets were noted to be enveloped by a unit membrane with a partially thickened electron-dense layer containing some myelin-like structures, electron-dense material or electron-lucent droplets, close to the inner leaflet of the membrane. Cytochemically, the dense layer showed acid phosphatase activity, and thus the lipid droplets were identified as lipolysosomes. The lipolysosomes isolated in a floating lipid fraction retained the characteristic dense layer and the surrounding membrane, and the fraction showed little contamination by other organelles. Free fatty acids were released in the fraction after incubation at 37 degrees C, but not at 0 degree C. The reaction was completely inhibited by HgCl2, p-chloromercuribenzoate and esterastin, a potent inhibitor of acid lipase. These results suggest an involvement of acid lipase in lipolysis of the lipolysosomes.

53 LOCALIZATION OF XANTHINI DEHYDROGENASE IN LIVER

Xanthine dehydrogenase has been reported to be present in an endothelial cell in beef or rat liver and also in peroxisome of a hepatic cell. However, as the most enzyme was recovered in 100,000xg supernatant of rat or chicken liver homogenate, cellular and subcellular localization in liver are still obscure. We reexamined localization of xanthine dehydrogenase in the tissue of chicken liver by imrnunohistochemical method, or by the method of isolation of hepatic cells. Chicken liver xanthine dehydrogenase purified by the method of Nishino was used for the immunization of rabbit. The antisera obtained from the rabbit gave a distinct single precipitin line in agar against chicken liver xanthine dehydrogenase or crude extract. This antisera were used for immunohistochemical experiments. Chicken liver were perfused with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin and sectioned. They were stained with the indirect method using enzyme labeled antibody. The positive staining was observed in the cytoplasm of hepatocytes, Kupffer cells and endothelial cells, suggesting that these type cells contain xanthine dehydrogenase. After isolation of hepatocytes most of xanthine dehydrogenase was recovered in 100,000xg supernatant fraction from homogenate of hepatocytes and the specific activity of enzyme was similar to that in 100,000xg supernatant fraction from homogenate of liver.

The reduction of aromatic alpha-keto acids by cytoplasmic malate dehydrogenase and lactate dehydrogenase

This study demonstrates that cytoplasmic malate dehydrogenase (MDH-s) catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, that the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; EC 1.1.1.96) is identical to MDH-s, and that the reduction of aromatic alpha-keto acids is due predominantly to a previously unrecognized secondary activity of MDH-s and the remainder is due to the previously recognized activity of lactate dehydrogenase (LDH) toward aromatic keto-acids. MDH-s and KAR have the same molecular weight, subunit structure, and tissue distribution. Starch gel electrophoresis followed by histochemical staining using either p-hydroxy-phenylpyruvic acid (HPPA) or malate as the substrate shows that KAR activity comigrates with MDH-s in all species studied except some marine species. Inhibition with malate, the end product of the MDH reaction, substantially reduces or totally eliminates KAR activity. Genetically determined electrophoretic variants of MDH-s seen in the fresh water bony fish of the genus Xiphophorus and the amphibian Rana pipiens exhibited identical variation for KAR, and the two traits cosegregated in the offspring from one R. pipiens heterozygote studied. Both enzymes comigrate with no electrophoretic variation among several inbred strains of mice. Antisera raised against purified chicken MDH-s totally inhibited both MDH-s and KAR activity in chicken liver homogenates. There is no evidence to suggest that any protein besides MDH-s and LDH catalyzes this reaction with the possible exception of the situation in Xiphophorus, in which a third independent zone of HPPA reduction is observed. In most species the activity formerly described as KAR appears to be due to a previously unsuspected activity of MDH-s toward aromatic monocarboxylic alpha-keto acids. In all species examined the KAR activity is associated only with MDH-s; in tissue homogenates the mitochondrial form of MDH (MDH-m) is not detected after electrophoresis using HPPA as a substrate.

Comparison of type I 5′-deiodination of thyroxine and of reverse-triiodothyronine in rat and chicken liver homogenates

The characteristics of 5′-deiodination in the chicken liver have been compared to those in the rat. Using 6- n-propyl-2-thiouracil (PTU) at 1 m M in vitro, it was shown that, in accordance with former in vivo studies, only type I 5′-deiodinase exists in the liver of the chicken. When PTU was used in a concentration (10 μ M) close to its K i species differences could be demonstrated as for PTU sensitivity. 5′-Deiodinase in the chicken liver was more susceptible against the inhibitor. The early thiol-independent phase of deiodination was longer in the chicken than in the rat. Reverse-triiodothyronine (r-T 3) seemed to be a more suitable substrate for this enzyme in the chicken when compared to either T 4 degradation or r-T 3 degradation in the rat. It is concluded that major characteristics of 5′-deiodination in the chicken are similar to those in the rat; however, the slight variances observed might explain some species differences found earlier.

In-vitro conversion of thyroxine to tri-iodothyronine by chicken hepatic 5'-deiodinase: kinetic studies.

In-vitro studies with chicken liver homogenates demonstrate that the conversion of thyroxine (T4) to tri-iodothyronine (T3) is dependent upon tissue concentration, time of incubation, pH, temperature, the presence of dithiothreitol (DTT) and the concentration of substrate (T4), and is heat-labile. The generation of T3 is inhibited by iopanoic acid and 6-n-propyl-2-thiouracil. The kinetics of conversion of T4 to T3, determined by Lineweaver-Burke analysis, indicated an apparent Michaelis-Menten constant (Km) of 1.16 mumol/l with a maximum velocity (Vmax) of 44.57 pmol T3 generated/mg protein per min from T4. Dithiothreitol appears to behave as a co-substrate for this system with an apparent Km of 98.5 mumol/l and a Vmax of 1.41 pmol T3 generated/mg protein per min at a T4 concentration of 5 mumol/l. These data suggest that the conversion of T4 to T3 in fowl proceeds by means of an enzymatic system, probably 5'-monodeiodinase, and is responsible for maintaining T3 levels in vivo.

Drug-Induced Changes in Selenium-Dependent Glutathione Peroxidase Activity in the Chick

The ability of aurothioglucose and D(-)-penicillamine hydrochloride to inhibit selenium-dependent glutathione peroxidase (SeGSH-Px) in vitro and to increase exudative diathesis in vitamin E-deficient chickens was studied. Aurothioglucose and penicillamine competitively inhibited SeGSH-Px in inverse proportion to the concentration of hydrogen peroxide and reduced glutathione, respectively, in chick liver postmitochondrial supernatant assay preparations. Neither drug inhibited glutathione reductase or superoxide dismutase at the concentrations tested; however, both inhibited catalase in a semilogarithmic fashion. This was true for both the purified bovine enzyme and chick liver homogenate. Aurothioglucose and penicillamine injected subcutaneously at the back of the neck increased exudative diathesis in vitamin E-deficient chickens fed 0.1 ppm Se, and effectively overcame the protective effect of selenium 72 h after injection in chicks fed vitamin E-free, low selenium diets supplemented with 0.0–0.1 ppm Se. Assays of plasma and of liver, lung and kidney postmitochondrial supernatants indicated that all observed reductions in SeGSH-Px activity preceded increases in exudative diathesis. Plasma and liver SeGSH-Px activities were lower at early times (6–24 h) after treatment with high doses of either drug. Lung SeGSH-Px activities were only lower in chicks receiving 240 mg penicillamine/kg 6 h after treatment; kidney SeGSH-Px activities were only lower in chicks treated with the highest dose of aurothioglucose 48 h after treatment. Brain SeGSH-Px activities were unaffected by drug treatment and the heart had higher SeGSH-Px activities only at 6 h after treatment with the highest dose of either drug compared to saline controls. Catalase activities in liver homogenates were only significantly altered by penicillamine; the highest dose caused the activity to be higher than that in salinetreated chicks. The cause of the lower SeGSH-Px activities could be either lower enzyme concentrations in tissues of the drug-treated groups and/or direct inhibition. Whatever the mechanism, it is concluded that exudative diathesis can be used to determine which drugs reduce SeGSH-Px activity in the chick.