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The safety of krill powder was assessed in a subchronic 13-week toxicity study where rats were fed krill powder or control diets. The krill powder inclusion in the test diet was 9.67% (w/w). There were no differences noted in body weight or food consumption in either gender. Differences in clinical chemistry values were noted in the krill powder-treated animals, but these findings were of no toxicological significance. A significant decrease in absolute heart weight, but not relative heart weight, was observed in both sexes given krill powder, although no corresponding histological changes were observed. Hepatocyte vacuolation was noted histologically in males fed krill powder. This finding was not associated with other indications of hepatic dysfunction. The no observed adverse effect level (NOAEL) for the conditions of this study was considered to be 9.67% krill powder.

Highlights • Krill oil is a safe dietary source of omega-3 fatty acids. • The no adverse effect level (NOAL) of krill oil was 5%. • Krill oil showed no mutagenic activity in bacteria., The safety of krill oil was assessed in a subchronic toxicity study and in a genotoxicity test. In a 13-week study, rats were fed krill oil or control diets. There were no differences noted in body weight, food consumption or in the functional observation battery parameters in either gender. Differences in both haematology and clinical chemistry values were noted in the krill oil-treated groups. However these findings were of no toxicological significance. Significant decreases in absolute and covariant heart weight in some krill oil-treated animals were noted although no corresponding histological changes were observed. In addition, periportal microvesicular hepatocyte vacuolation was noted histologically in males fed 5% krill oil. This finding was not associated with other indications of hepatic dysfunction. Given that the effects of the 13-week toxicity study were non-toxic in nature, the no observed adverse effect level (NOAEL) for the conditions of this study was considered to be 5% krill oil. The genotoxicity experiments documented no mutagenicity of krill oil in bacteria.

The growth rate ofGracilaria gracilis maintained in tanks at an abalone farm near Port Elizabeth, South Africa, was examined under various tank conditions and NH4-N pulse frequencies and concentrations. This was accompanied by analyses of the components of the internal nitrogen pool. A maximum growth rate of ca. 35% wk−1 was obtained at 1200 μM NH4-N. The alga was able to grow at non-nitrogen limited rates using only internal stored nitrogen to sustain growth for one week before the growth rate decreased to ca. 17% per week. NH4-N pulse frequency did not affect growth rate but one pulse per week led to a marked decrease in total-N, protein, phycoerythrin and chlorophyll-a content. An increase in pulse frequency to two pulses per week doubled the protein content from 2.351 ± 0.143 to 4.453 ± 0.090% (per unit dry mass). Carbohydrate content was inversely related to nitrogen storage. The growth rate in fouled tanks was always lower than in clean tanks. It seems likely that seaweeds and diatoms colonising the tank sides reduced light reflected off the inside of a tank, thereby reducing the growth rate.

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.

The rate of hemopexin synthesis in adult rat hepatocyte suspensions (0.17 ± 0.019 (6)) (mean ± SEM (n)) mg/g hepatocytes per hour) was found to be linear for 48 h. By contrast, the rate of synthesis of albumin and fibrinogen was close to linear for only 12 h after which it continued at a diminished rate. Supplementation of the incubation medium with insulin, cortisol, glucagon, triiodothyronine, and growth hormone effected no significant increase in the synthesis rate of hemopexin but by contrast did do so in that of albumin (22%) and of fibrinogen (123%) (although not to the point of causing these last to become linear). The pattern of hemopexin synthesis and its response to hormones is clearly different from that observed with other plasma proteins studied in this hepatocyte system. Hemopexin synthesis appeared to be at its maximum and to be independent of hormone supplementation, and it was continuing linearly at a time when the synthesis of other plasma proteins was falling.

Previous evidence that portal blood insulin is an hepatotrophic factor led to this study of its effect on hepatocytes, isolated from fasted rats, in suspension culture. Control hepatocytes (C), noninsulin-treated, and those infused continuously at low (LI) and high (HI) levels of insulin were compared concurrently with regard to their survival, glucose transport, and intracellular concentrations of glucose, glycogen, and adenine nucleotides, over a 48-hr period of incubation. Low insulin was adjudged to be comparable to portal insulin concentrations in fasted animals and HI to those in fed animals. All hepatocytes had been depleted of glucose, glycogen, and adenine nucleotides at the start of the study by prior fasting of the rat. For the first 6 hr of culture, there was little difference between C, LI, and HI with reference to the above parameters. In contrast, after 48 hr of incubation, cell survival, as judged by the DNA content, was signficiantly lower in C compared with LI and HI. The transport of 3--0-[methyl-3H] D-glucose was also significantly lower in C compared with LI and HI. The higher uptakes in both LI and HI were reduced by phloridzin, which had little effect on C. Correspondingly, the intracellular glucose concentrations in C were significantly lower than the extracellular glucose concentrations in contrast to those in LI and HI, which were comparable. For intracellular concentrations of glycogen and adenine nucleotides, the results of LI and HI were amalgamated as they were not significantly different from each other at 48 hr. Upon analysis, glycogen values were significantly higher for insulin-treated cells. Similarly, the total adenine nucleotide p-ol (ATP + ADP + AMP) also was clearly higher in HI + LI than in C. These results indicate that contrary to findings in studies with the perfused liver that have been of a short-term nature, insulin is necessary in the longer term (greater than 12 hr) for maintaining the transport of glucose into hepatocytes; thereby insulin promotes the maintenance of intracellular glucose, glycogen, and adenine nucleotide concentrations, and also enhances cell survival.

Hormone effects on the synthesis of alpha(1) (acute-phase) glycoprotein and of albumin by isolated rat hepatocytes in suspension were examined. Insulin, glucagon, cortisol, somatotropin (bovine growth hormone) and tri-iodothyronine were added to achieve physiological concentrations in the medium [Jeejeebhoy, Ho, Greenberg, Phillips, Bruce-Robertson & Sodtke (1975) Biochem. J.146, 141-155]. After periodic additions, there were increases (compared with values for non-hormone-treated suspensions) in the concurrent absolute syntheses of alpha(1) (acute-phase) glycoprotein and of albumin. Trends were detectable after 24h, and significant increases were demonstrated after 48h of incubation (219 and 119% respectively of control values). Manipulation of hormones, by omission from the mixture or by addition of only one or two hormones in various combinations, indicated that for alpha(1) (acute-phase) glycoprotein (which may be representative of some other acute-phase proteins), cortisol was one of the most important hormones involved in the stimulation of synthesis, with glucagon enhancing the effect of cortisol but not being stimulatory by itself. Addition of actinomycin D inhibited this stimulation, suggesting that cortisol might have acted through promotion of RNA synthesis. For albumin, cortisol alone did not stimulate synthesis, but its absence from a hormone mixture significantly decreased synthesis compared with that observed with the complete hormone mixture. Our findings support the possibility that following tissue injury, synthesis of alpha(1) (acute-phase) glycoprotein may be stimulated by the hormonal response to this injury (which response includes elevated blood concentrations of cortisol and glucagon).

A protein of the Ig G family has been isolated from the serum of a patient with a tentative diagnosis of a plasma cell neoplasm. The protein has a lower sedimentation constant (5.4) and a lower molecular weight (125,000) than normal immunoglobulins of the G family. The protein has heavy-chain determinants of type G and light-chain determinants of the κ-type. Heavy and light chains have been prepared by reductive cleavage followed by gel filtration. The heavy-chain preparation is homogeneous in starch gels in acidic buffer containing urea but has a faster mobility than normal Ig G heavy chains. The light-chain preparation is resolved into two components in electrophoresis, and both have slower mobility than normal Ig G light chains. The heavy- and light-chain preparations cross react with normal Ig G heavy and light chains in immunodiffusion analysis. Sedimentation equilibrium studies suggest that both the heavy and light chains have lower molecular weights than their normal counterparts.

Publisher Summary This chapter discusses the effect of ethanol on albumin and fibrinogen synthesis in vivo and in hepatocyte suspensions. An important and specific function of the endoplasmic reticulum of the hepatocyte is the synthesis of plasma proteins so that a specific hepatotoxic effect of ethanol likely ought to be reflected by a concurrent change in plasma protein synthesis. Furthermore, as the synthesis of these proteins occurs on ribosomes attached to the rough endoplasmic reticulum, changes in RNA metabolism and in the structure of the endoplasmic reticulum seem likely to be associated with alterations of plasma protein synthesis. Experimental alteration or prevention of any hepatotoxic effects of ethanol on RNA metabolism, on the structure of the endoplasmic reticulum, and on the synthesis plasma proteins may suggest means of treating or preventing ethanol-induced hepatic injury.

A system using hepatocyte suspensions in vitro was developed for studying the synthesis of albumin, fibrinogen and transferrin. Conditions for optimum survival of the hepatocyte and for synthesis of these plasma proteins were defined for this system. These conditions included the use of horse serum (17.5 percent, v/v, heat-inactivated), an enriched medium (Waymouth's MB 752/1), an O2 tension of between 18.7 times 10(3) and 26.7 times 10(3) Pa and constant stirring. Albumin, fibrinogen and transferrin synthesis rates were obtained of 0.32 p 0.094(10), 0.12 p 0.030(11) and 0.097 p 0.017(10) [mean p S.D. (n)]mg/h per g of hepatocytes respectively. These rates were maintained for the first 12h of study and synthesis continued at a diminished rate up to 48h. The synthesis of albumin was decreased in a medium containing less amino acids and glucose, but that of fibrinogen was substantially unaffected. ATP concentrations up to 12h and RNA/DNA ratios up to 24h were comparable with values in vivo. The ability to study cells up to 48h permitted us to find that the addition of a mixture of hormones consisting of glucagon, cortisol, tri-iodothyronine and growth hormone enhanced fibrinogen synthesis. Addition of insulin to the above mixture resulted in increased synthesis for albumin and transferrin but not for fibrinogen.

Very low density lipoprotein (VLDL) synthesis was studied using suspensions of isolated rat hepatocytes prepared and incubated as described previously (1). These hepatocytes synthesized and secreted VLDL over a 24-hr period, in quantities permitting its isolation, without using carrier, for determining absolute synthesis rates and analysing the peptide pattern. The mean secretion of triglyceride, cholesterol and rat VLDL protein was 410 ± 46.6, 36.6 ± 0.1 and 34.9 ± 5.4 (mean ± SEM, n = 5) μg/g hepatocyte/hr over 24 hr, during which incorporation of 3H-valine into VLDL protein approached linearity. Suitable polyacrylamide gel electrophoresis showed the hepatocytes secreted the peptides found in circulating rat VLDL but with a different proportion of the fast to the slowly migrating ones.

Decrease of absolute synthesis of albumin and fractional synthesis of transferrin was observed within 3h of orally administering ethanol (4ml/kg) to rats maintained on a 40%-protein diet. In contrast, absolute synthesis of fibrinogen was unaffected. With this ethanol intake, the changes in protein synthesis occurred without significant ultrastructural change in the liver. When the ethanol intake was greater (8ml/kg) ultrastructural disruption was observed. However, both the decrease of plasma protein synthesis and the ultrastructural alterations could be prevented by the simultaneous administration of a mixture of amino acids with the ethanol. The latter findings, not reported hitherto, suggest that ethanol may interfere with hepatic plasma protein synthesis and ultrastructure more through a disturbance of amino acid metabolism than through direct physical damage to the hepatocyte. An Appendix outlines the deconvolutional method used to correct for losses of labelled protein in the period during which measurements were made. The principle may also be applied to labelled plasma urea. The details of the calculations are given in a supplementary paper that has been deposited as Supplementary Publication 50007 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.

A method described by McFarlane (1963a) for the measurement of the absolute rate of synthesis of liver-made plasma proteins was used to show that, within a few hours of giving bovine growth hormone to rats, fibrinogen synthesis increased significantly without a change in albumin synthesis.