The esterification of cholesterol in the liver of the chick embryo.

The development of the chick embryo was characterised by the accumulation of large droplets of lipid in the cytoplasm of the embryonic liver, as revealed by electron microscopy. Analysis of the lipid composition of the livers indicated that this accumulation resulted from a dramatic increase in the cholesteryl ester content of the tissue during the the latter part of the embryonic period. This lipid is apparently derived from yolk cholesterol and may be taken up by the liver in the form of lipoprotein remnants. Significant levels of acyl-CoA: cholesterol acyltransferase (ACAT) activity were expressed in the liver throughout the second half of the developmental period, and this activity was maximal at the time when lipid transfer from the yolk was most intensive. The activity of microsomal cholesterol ester hydrolase (CEH) was very low throughout development, and no CEH activity was detected in the cytosolic fraction. In addition, substantial amounts of a cytosolic protein which inhibits CEH activity were present. Thus the relative activities of these enzymic systems are consistent with the net accumulation of cholesteryl ester which occurs in the liver during development.

Tissue-specific changes in lipid composition and lipoprotein lipase activity during the development of the chick embryo.

Lipoprotein lipase was present at a high specific activity in adipose tissue and heart of the chick embryo at the 14th day of development. The enzyme was also present in skeletal muscle but was absent from brain and liver. Major increases in the activity of lipoprotein lipase in adipose tissue and heart occurred from day 12 of development, concomitant with the beginning of the period of lipid uptake from the yolk. These results suggest that lipoprotein lipase may be involved in the utilisation of yolk-derived lipid by the tissues of the embryo. Relatively high levels of docosahexaenoic acid (22:6(n--3)) were present in the triacylglycerol isolated from plasma, adipose tissue, heart and liver. The relative proportions of this fatty acid in the triacylglycerol of the different tissues may be explicable in terms of the substrate specificity of lipoprotein lipase.

The transfer of docosahexaenoic acid from the yolk to the tissues of the chick embryo.

Changes in the amounts of the major fatty acids present in the lipids of the yolk complex and the embryo were delineated during embryogenesis of the chicken. The rates of transfer of palmitic, oleic, linoleic, linolenic and arachidonic acids from the lipids of the yolk complex were essentially identical. In contrast, docosahexaenoic acid (DHA) was preferentially transferred from the yolk complex at a rate which was significantly higher than that exhibited by the other major fatty acids. The rates of accumulation of both arachidonic acid and DHA in the lipids of the whole embryo were significantly greater than the rates observed for the C16 and C18 fatty acids, particularly between days 12 and 16 of the 21-day embryonic period. Analysis of the fatty acid composition of plasma lipid throughout development indicated that the triacylglycerol fraction contained relatively high proportions (up to approx. 14% w/w of total fatty acids) of DHA, but much lower proportions (approx. 3%) of arachidonic acid. In contrast, plasma phospholipid was enriched in arachidonic acid (up to approx. 18%), but contained much lower proportions (generally less than 3%) of DHA. A considerable amount of DHA was incorporated into adipose tissue triacylglycerol, so that by the time of hatching, the tissue represented a major store of this fatty acid. Over the hatching period, the amount of DHA in adipose triacylglycerol decreased dramatically, by up to 85%, whereas there was little or no change in the amounts of the other major fatty acyl components in this tissue. The amount of DHA as a component of brain phospholipid increased continuously throughout the developmental period studied. However, by the time of hatching, the amount of DHA in brain phospholipid represented less than 10% of the amount of this fatty acid originally present in the lipids of the yolk.

Tissue-specific differences in antioxidant distribution and susceptibility to lipid peroxidation during development of the chick embryo.

The purpose of this study was to determine the tissue-specific acquisition of antioxidant capacity during chick embryo development and to assess the effectiveness of this process in the prevention of lipid peroxidation. The transfer of alpha-tocopherol, carotenoids and ascorbic acid from the yolk/yolk sac membrane (YSM) to the developing chick embryo and the distribution of these antioxidant compounds between the embryonic tissues were investigated. The concentrations of alpha-tocopherol and carotenoids in the yolk decreased between day 15 of development and hatching at day 21, concomitant with an increase in the levels of these antioxidants in the YSM. The concentration of both these lipid-soluble antioxidants in the liver increased dramatically between day 18 of embryonic development and day 1 after hatching. The adipose tissue content of alpha-tocopherol also increased markedly during the late embryonic/early neonatal period. However, the levels of alpha-tocopherol in the liver were far higher than in any other tissue with particularly low levels observed for the brain. Also, carotenoids were undetectable in the developing brain. Ascorbic acid was not present in the initial yolk but high levels of this water-soluble antioxidant were detected in the YSM, particularly at the early stages of development. The concentration of ascorbic acid in the embryonic brain was far higher than in any other tissue. Homogenates of brain tissue were extremely susceptible to lipid peroxidation during incubation in vitro whereas extracts of liver, yolk and YSM were relatively resistant to lipid peroxidation, particularly in the absence of exogenous Fe2+.

The preferential mobilisation of C20 and C22 polyunsaturated fatty acids from the adipose tissue of the chick embryo: potential implications regarding the provision of essential fatty acids for neural development.

The aim of this study was to determine the relative mobilisation of the different fatty acyl components of the triacylglycerol (TAG) of the chick embryo's adipose tissue in the light of the specific requirements of the developing neural tissues of the embryo for C20-22 polyunsaturated fatty acids. Pieces of adipose tissue, obtained from embryos at various developmental stages, were incubated in vitro in Dulbecco's Medium containing serum albumen. The fatty acid compositions of the initial tissue TAG and of the free fatty acid (FFA) mobilised from the tissue during 1 h of incubation were determined and compared. The composition of the FFA released into the medium under conditions of basal (i.e., unstimulated) lipolysis was markedly different in several respects from that of the TAG from which it originated. The polyunsaturated fatty acids, 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3, were consistently found to be preferentially released into the medium, whereas the major fatty acyl constituents of the tissue, 16:0 and 18:1n-9, were selectively retained in the TAG. For example, at day 18 of development, the proportions (% w/w of fatty acids) of 20:5n-3 and 22:6n-3 released into the incubation medium were respectively 6.5 and 7.5 times higher than in the original tissue TAG. Glucagon stimulated the overall rate of mobilisation by approx. 2-fold and also partially suppressed the preferential mobilisation of C20-22 polyunsaturates. These results may be relevant to the elucidation of the means by which essential polyunsaturates are delivered from the yolk to the neural tissues of the embryo, with the implication of a mediatory role for the embryonic adipose tissue in this transfer.

Effect of 2-mercaptoethanol on some metabolic indices of ageing of CBA/Ca inbred mice.

It has been shown in several studies that 2-mercaptoethanol (2-ME) improves the life span and aspects of life performance of laboratory animals. To obtain further details on the beneficial effects of 2-ME, a long-term study has been performed on male CBA/Ca inbred mice treated with this antioxidant. Four month-old mice were each given 4 micrograms of 2-ME in physiological saline via intraperitoneal (i.p.) injection 3 x per week. Measurements were made of the following: cold tolerance (heat performance), apparent total body protein turnover (T1/2), changes in the major lipid and fatty acid compositions of the liver, superoxide dismutase activity and formation of malondialdehyde and observations on a range of pathological changes. It was found that the basal rectal temperatures of the treated mice were higher and in the oldest group, heat performance capacity was better than those of the controls. After about 1 year of age the apparent biological half-life time of total body protein (T1/2) was observed to be shorter in the treated mice. Significant increases were observed to occur in the proportions of the polyunsaturated fatty acids in the lipids of the liver in the mice injected with 2-ME. Although no differences were observed in the superoxide dismutase activities, malondialdehyde concentrations in the livers of the experimental mice were significantly increased. Autopsy data showed that Dunn-sarcomas associated with amyloidoses occurred more frequently in the untreated mice.

Effect of aging on cold tolerance and thyroid activity in CBA/Ca inbred mice.

Relationships between cold tolerance, serum levels of thyroxine (T4), thyronine (T3), and thyrotropine (TSH), and thyroid morphometry have been investigated in male CBA/Ca inbred mice at various ages through their life span. From the data obtained it appeared that there was an age-related decrease in cold tolerance up to 18 months of age which was followed by an increase, the age effect being most apparent in relation to cold resistance and cold tolerance during the recovery period following cold exposure. The age-related changes in cold tolerance appeared to be associated with changes in the serum concentrations of T3, T4, and TSH. In contrast to the T3 serum levels which showed a decrease at 36 months, the thyroxine contents showed a perceptible decrease from the age of 12-18 months onwards. A similar pattern was observed for the TSH levels, with a peak at 21 months, followed by a decline at 30 months. A relationship with age between serum T4 level and thyroid weight was indicated together with structural changes in the thyroid gland, particularly during senescence, for example the size and number of thyroid epithelial cells had become enlarged by 30 months of age.

The role of the placenta in the supply of essential fatty acids to the fetal sheep: studies of lipid compositions at term.

A study has been made of the comparative distribution and fatty acid compositions of the major lipid fractions of maternal plasma, placenta and fetal plasma of the sheep at approximately 120 days of gestation. Cholesteryl esters and phospholipids constituted the major lipid fractions present in both maternal and fetal plasmas. In the placenta, phospholipids accounted for some 60 per cent of the total lipid present. Within the phospholipids of the maternal and fetal plasmas and placenta, phosphatidylcholine comprised the largest component. Whereas maternal plasma contained high levels of linoleic and arachidonic acids, fetal plasma contained a low proportion only a linoleic acid and high proportions of delta 5,8,11 eicosatrienoic acid and arachidonic acid. In the maternal plasma the arachidonic acid:linoleic acid ratio was only 0.17, but in the fetal plasma the ratio was 3.32. The differences in the lipid and fatty acid compositions between the maternal plasma, fetal plasma and placenta are discussed in relationship to the distinctive polyunsaturated fatty acid metabolism of the fetal and newborn lamb.

Ineffectiveness of annual chest roentgenograms in tuberculin skin test-positive hospital employees.

Annual routine chest x-ray examinations have been recommended for many years for tuberculin skin test-positive hospital employees who have not received chemoprophylaxis. To examine the case-finding effectiveness of this policy in a university teaching hospital, employee health records covering an 18-year period were reviewed. Pulmonary tuberculosis was diagnosed in four employees during that time period. None of the patients were identified through the routine annual chest x-ray examination. In the study institution the policy requiring routine annual chest x-ray examinations for tuberculin-positive hospital employees who had not received preventive therapy was rescinded. Currently, the first chest x-ray examination is required at the time of skin test conversion, the second 1 year after conversion, and the third 2 years after conversion. Health teaching and counseling regarding the symptoms of tuberculosis have been initiated for tuberculin-positive hospital employees.

Pseudomonas stutzeri infection. A review of hospital isolates and a review of the literature.

Pseudomonas stutzeri is uncommonly isolated from patients and rarely causes disease. Clinical isolates of P. stutzeri from a university hospital were reviewed over a 16-year period. In the hospital review, only three patients were identified with P. stutzeri infection, and in only one of these was the organism present as the sole isolate. A review of the literature shows that P. stutzeri is most frequently isolated from blood, wounds, the respiratory tract, and urine. Patients with P. stutzeri infections often have serious underlying disease but generally respond to treatment with antibiotics including the aminoglycosides, the antipseudomonal penicillins, trimethoprim-sulfamethoxazole, and the third-generation cephalosporins.

Ocular involvement in Yersinia enterocolitica infection presenting as Parinaud's oculoglandular syndrome.

Ocular involvement in Yersinia enterocolitica infection presenting as a Parinaud's oculoglandular syndrome occurred in a 77-year-old woman with diabetes. Yersinia enterocolitica was recovered from cultures of the conjunctiva, cornea, fistula tract, and blood. The patient responded to parenteral and topical administration of gentamicin and a corneal transplant. While hospitalized, she developed peritonsillar inflammation and enlarged, tender lymph nodes in the preauricular, submaxillary, and submandibular areas. The combination of the unilateral granulomatous conjunctivitis and enlarged regional lymph nodes was consistent with the diagnosis of Parinaud's oculoglandular syndrome. Yersinia enterocolitica may be another cause of Parinaud's oculoglandular syndrome.

The colorimetric estimation of protein by the Lowry technique using a liquid scintillation counter.

A comparison has been made of the use of the spectrophotometer and liquid scintillation counter for the colorimetric quantification of protein. The attenuation of photon detection from sealed miniature 14C radioactive standards enabled protein quantification over a concentration range far in excess of that achievable by the use of a spectrophotometer. Accuracy of quantification was high over the entire range of protein concentration. The ability of the technique to provide quick and accurate protein estimations is discussed.

Developmental changes in the levels of molecular species of triacylglycerol that contain docosahexaenoic acid in adipose tissue of the chick embryo.

Triacylglycerols from the initial yolk and from the adipose tissue of the chick embryo were subjected high-performance liquid chromatography in the silver ion mode to resolve molecular species of triacylglycerol on the basis of the degree of unsaturation. A total of 12 such species was resolved from the yolk samples, with only one of these species containing docosahexaenoic acid. In contrast, 17 species were resolved from adipose tissue at day 12 of embryo development, of which 8 contained docosahexaenoic acid. The major species containing this fatty acid consisted of docosahexaenoic acid in combination with palmitic and oleic acids. The amounts of the species consisting of C16 and C18 fatty acids in the adipose tissue increased continuously from day 12 until just before hatching at day 19 and then decreased slightly over the hatching period. In contrast, the amounts within the adipose depot of the species containing docosahexaenoic acid reached a maximal level by day 16 and then decreased dramatically to almost undetectable levels during the hatching period. The possibility that adipose tissue triacylglycerol in the chick embryo may function as a temporary store of docosahexaenoic acid for subsequent delivery to the developing neural tissues is suggested.

The fatty acid composition of brain phospholipids from chicken and duck embryos.

The effects of differences in the fatty acid composition of the lipids of egg yolk on the subsequent levels of arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) in the total phospholipids and in the isolated phospholipid classes of the embryonic brain were investigated by a comparison of two domesticated avain species, the chicken and the duck. The yolk phospholipids of chicken eggs contained similar proportions of 20:4n-6 and 22:6n-3 (approx. 6% wt/wt of total fatty acids). In marked contrast, the yolk phospholipids of commercially produced duck eggs contained an overwhelming preponderance of 20:4n-6 over 22:6n-3 (approx. 10% cf.1%). These differences between the yolks of the two species were only partly reflected in the fatty acid compositions of the total phospholipids of the embryonic brains at equivalent developmental stages. Typically, the chicken brain phospholipids contained approximate proportions of 20:4n-6 and 22:6n-3 of 8% and 17%, respectively, whereas both these polyunsaturates were present at approx. 11% in the duck samples. The brain phospholipids were resolved into their component phospholipid classes by high performance liquid chromatography. In both species, phosphatidylcholine contained only low levels of 20:4n-6 and 22:6n-3, whereas phosphatidylethanolamine displayed a high content of 22:6n-3. Phosphatidylserine was also rich in 22:6n-3 whereas phosphatidylinositol exhibited a high proportion of 20:4n-6. The results suggest that the relatively low level of 22:6n-3 in the yolk of duck eggs is partly compensated for by an enhanced efficiency in the incorporation of this fatty acid into the brain phospholipids, in comparison with the chicken.

The effects of dietary supplementation with docosahexaenoic acid on the phospholipid fatty acid composition of avian spermatozoa.

This study represents an attempt at the dietary manipulation of the fatty acid composition of chicken spermatozoa in order to enhance the levels of n-3 polyunsaturates at the expense of the n-6 fatty acids, which normally predominate in the lipids of avian semen. Male chickens were provided with either a control diet supplemented with maize oil or the test diet supplemented with fish oil (Tuna Orbital Oil) from 10 weeks of age. Semen samples were collected from the birds after 30 and 48 weeks of supplementation. The fish oil diet induced a significant but limited increase in the proportion of 22:6n-3 in the spermatozoan phospholipid in parallel with an equivalent decrease in the proportions of 20:4n-6 and 22:4n-6. However, since the maximal level of 22:6n-3 in the phospholipid that was achieved by fish oil feeding was less than 10% (wt/wt of fatty acids), these changes fell far short of representing a switch from the typical avian pattern to that more characteristic of the n-3 enriched mammalian semen. Analysis of the fatty acid compositions of the constituent classes of phospholipid in the spermatozoa indicated that, in both dietary states, the phosphatidylethanolamine fraction contained much greater proportions of n-6 and n-3 C20-22 polyunsaturates than the phosphatidylcholine fraction. The results indicate that the typical fatty acid profile of the spermatozoa of domesticated poultry, characterised by the predominance of C20-22 n-6 polyunsaturates, displays a considerable degree of resistance to manipulation by dietary means and does not adopt the "mammalian" type of profile following supplementation with n-3 fatty acids.

Use of internal vial for determining quench correction in scintillation counting of heterogeneous media.

An insert vial situated at the center of the counting medium and containing a standard count has been used for quench correction in the liquid scintillation counting of gels containing suspensions of silicic acid scrapings from thin layer chromatoplates. The method has provided a simple and direct means of routine count correction for a range of 14-C-labeled lipid classes following separation on thin layer chromatoplates. The use of insert vial provides several advantages over other methods for the count correction of these suspensions.

The metabolism of 18:0 and 18:2(n-6) by the ovine placenta at 120 and 150 days of gestation.

The effects of approach of parturition in the sheep on the incorporation of 18:0 and 18:2(n-6) into the placenta lipids and on the activities of the delta 9- and delta 6-desaturase enzymes of placental tissue have been studied in vitro. The incorporation of 18:0 into the esterified lipids of placental tissue between the 120th and 150th days of gestation declined markedly; the high level of incorporation of 18:2(n-6) into the esterified lipids of the placenta (some 2-fold higher than 18:0) remained constant over the gestational period. While placental delta 9-desaturase activity was the same at 150 days as at 120 days of gestation, the activity of the delta 6-desaturase enzyme increased significantly. These results are discussed in relation to the fetal demand for fatty acids near term and the differences that exist between the mechanisms of maternal-fetal transfer of 18:0 and 18:2(n-6) in the sheep.

A unique lipid pattern associated with the gall bladder bile of the chick embryo.

A study has been made of the lipid and fatty acid composition of the gall bladder bile of the chick embryo during the last week of incubation. The lipids and their fatty acid composition showed a unique pattern when compared to other animal species. Of the total lipid present, phospholipid accounted for less than half, and there were substantial proportions of both cholesteryl ester and triglyceride. In the cholesteryl ester, the proportion of which increased significantly over the last week of incubation, there was a very high level of oleic acid. The phospholipid contained a high level of arachidonic acid. The results are discussed in relation to observations on the biliary lipids of other animal species and the major features of the lipid metabolism of the chick embryo during the last week of incubation.

A single-step method for determination of the specific radioactivity of lipids.

The use of a liquid scintillation counter to measure both the mass and the radioactivity content of charred 14C-labeled lipid bands from thin layer chromatoplates has been evaluated. Following lipid mass determination from a measurement of the external standard channels ratio, a suitable choice of counting parameters enabled a reproducible and efficient 14C count to be obtained over virtually the whole range of lipid concentrations tested. Although the charring procedure resulted in some loss of radioactivity, the efficiency of counting remained high enough for accurate dpm measurements.

Unsaturated fatty acid compositional changes and desaturation during the embryonic development of the chicken (Gallus domesticus).

An investigation has been made to correlate the activities of the delta 9- and delta 6-long chain fatty acid desaturation systems with the increased levels of oleic and arachidonic acids in the liver relative to the yolk sac membrane of the chick embryo during the last week of development. The membrane exhibited high levels of both stearic and linoleic acid desaturation in the early stages of yolk lipid mobilization, the activities of both enzyme systems decreasing with the approach of hatching. Stearic acid desaturation in the liver also decreased with the approach of hatching, but linoleic acid desaturation increased. The observed levels of desaturation in the yolk sac membrane are capable of making a considerable contribution to the accumulations of mono- and polyunsaturated fatty acids in the embryonic liver, the requirement for which does not appear to be satisfied by the yolk lipids. With the approach of hatching and the functional regression of the yolk sac membrane, the role is taken over by the embryonic tissues.