Colipase enhances hydrolysis of dietary triglycerides in the absence of bile salts.

This study explores how dietary lipids are digested when intraduodenal bile salts are low or absent. Long-chain triglycerides emulsified with phosphatidylcholine were found to be hydrolyzed very slowly by pancreatic lipase alone, as if the surface layer of phospholipids enveloping the triglycerides impeded the action of the enzyme. Colipase enhanced triglyceride hydrolysis severalfold, both when added before or after the lipase. Hydrolysis became even more rapid when the emulsion was first incubated with pancreatic phospholipase. Hydrolysis of long-chain triglycerides was also severely impeded when other proteins were added to the system, probably because they adsorbed to the oil-water interface of the emulsion droplets. It was previously known that bile salts can relieve such inhibition, presumably by desorbing the adsorbed proteins. Colipase was found to enhance hydrolysis severalfold in a dose-dependent manner even in the absence of bile salts, i.e., it could partially or completely relieve the inhibition depending upon the amount and the type of inhibitory protein added to the system. Prior exposure of a protein-coated triglyceride emulsion to another lipase also enhanced the rate at which pancreatic lipase could then hydrolyze the lipids. Most dietary triglycerides are probably presented for intestinal digestion in emulsions covered by proteins and/or phospholipids. These emulsions would be hydrolyzed slowly by pancreatic lipase alone. However, through the action of the lipase in stomach contents and of pancreatic phospholipase and through the lipolysis-promoting effects of collipase, these triglycerices can be rather efficiently hydrolyzed, even in the absence of bile salts.

The effects of pH and salt on the lipid binding and enzyme activity of lipoprotein lipase.

This paper demonstrates a striking difference between the effects of salt and pH on the activity of lipoprotein lipase against two different substrates: Intralipid and bovine milk fat droplets. With the former substrate 1 M NaCl caused only a slight reduction in enzyme activity and the stimulation by apolipoprotein C-II was the same from 0.1 to 1.1 M NaCl. In contrast, 0.5 M or more NaCl virtually abolished the enzyme activity in the milk system. In this system the salt also abolished binding of the enzyme to the lipid droplets, whereas in the Intralipid system most of the enzyme remained bound even at 1 M NaCl. A similar picture was obtained with respect to effects of pH. In the milk system the activity decreased sharply at pH values above 8.5, whereas in the Intralipid system it continues to rise to pH 10, and the stimulation by activator protein is the same at all pH values. Correlating with this, the binding of the enzyme to the lipid droplets was highly dependent on pH values in the milk systems, with optimum binding around pH 8, whereas in the Intralipid system most of the enzyme remained bound to the lipid droplets at all pH values. These studies demonstrate that apolipoprotein C-II can activate lipoprotein lipase at a wide range of salt concentrations and of pH. They suggest that the well-known effects of high salt concentrations and of high pH to decrease lipoprotein lipase activity are exerted primarily on the enzyme itself.

On the pH dependency of lipoprotein lipase activity.

The relation between pH and activity for lipoprotein lipase against emulsions of long-chain triacylglycerols has previously been studied in several laboratories and found to be a bell-shaped curve with optimum activity between pH 8 and 9. In contrast, using short-chain triacylglycerols or monoacylglycerols as substrates we had found that the activity rises continuously with pH to at least pH 10.5. This suggested that some factor other than the active site mechanism limited the activity at high pH in traditional assay systems. We, therefore, reinvestigated the activity against long-chain triacylglycerols under conditions where binding of the enzyme to the emulsion droplets and enzyme stability was not limiting. Under these conditions the activity continued to rise from pH 8 to pH 10, and the degree of stimulation by apolipoprotein C-II was found to be the same over the whole range studied (pH 6.5-10.5).

Immunochemical properties of lipoprotein lipase. Development of an immunoassay applicable to several mammalian species.

The reaction of bovine lipoprotein lipase with its antibodies was found to be conformation-dependent. One aspect of this was that most antisera were more reactive with denatured than with native 125I-labeled lipoprotein lipase. Another aspect was that denatured lipase did not compete effectively with native lipase for those antibodies which caused inhibition of the enzyme's activity. This latter observation leads to the conclusion that the inhibiting antibodies recognize conformation-dependent determinants on the native enzyme. Fab fragments prepared from an inhibiting antiserum blocked the binding of the lipase to triacylglycerol/phospholipid droplets. This suggests that the inhibition results from reaction of the antibodies with the enzyme as it exists in solution, either covering the lipid-binding site on the enzyme or making it impossible for the enzyme to go through the conformational transitions necessary for binding to lipid. Most rabbit antisera did not react with rat or mouse lipoprotein lipase, but some sera showed a weak cross-reaction. Antisera raised in hens showed a much stronger cross-reaction, enough to be useful for heterologous immunoassays. An immunoassay for the bovine lipase was developed. For reproducible results it was necessary to have tracer, standard and samples in denatured form. This was accomplished by heating them in SDS, and running the immunoreaction in a Triton X-100-containing medium.

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.

Rapid removal to the liver of intravenously injected lipoprotein lipase.

Lipoprotein lipase was purified from bovine milk and labeled with 125I. After intravenous injection to rats the labeled lipase rapidly disappeared from the blood. The initial half-life was about 1 min and more than 70% of the radioactivity was found in the liver at 10 min. 30 min after the injection about 10% of the injected radioactivity was present in acid-soluble form in blood, indicating that the enzyme had been rapidly degraded. Injection of asialofetuin, ribonuclease B or mannan in amounts known to block the hepatic receptors for glycoproteins with exposed galactose, N-acetylglucosamine or mannose residues did not retard the removal of the lipoprotein lipase. Thus, some other, as yet undefined, receptor is implicated. Lipoprotein lipase is known to bind to heparin and some related polysacchrides. Heparin injected before the enzyme delayed its removal and heparin injected after the enzyme caused an immediate increase in blood radioactivity, signifying return from tissues to blood of labeled enzyme. Lipoprotein lipase is present at the endothelium in several extrahepatic tissues and is rapidly turned over. Its presence in blood in appreciable amounts would cause a derangement of lipid transport. The efficient hepatic removal of the enzyme may thus serve an important physiological purpose in keeping the blood levels of this enzyme low.

Demonstration of hepatic heparin-releasable lipase in the guinea-pig.

It was recently reported by Yamada et al. (Yamada, N., Murase, T., Akanuma, Y., Ikakura, H. and Kosaka, K. (1979) Biochim. Biophys. Acta 575, 128-134) that the guinea-pig has no hepatic heparin-releasable lipase. We have, however, found a low but definite lipase activity in guinea-pig post-heparin plasma with the characteristics of the hepatic lipase. This activity, as measured in our assay, is only about one-tenth of that in rat post-heparin in plasma. Although the activity is thus much lower than in some other animals, its presence demonstrates that the guinea-pig is not qualitatively but only quantitatively different in this respect.

Purification and properties of lipoprotein lipase in guinea pig milk.

Lipoprotein lipase was purified from guinea pig milk by chromatography on heparin-Sepharose followed by chromatography on an immobilized preparation of heparin that had been N-desulphated and then acetylated. This second step was necessary to separate a plasma protein, presumably antithrombin, from the lipase. The guinea pig enzyme turned out to be quite similar to lipoprotein lipase from bovine milk with respect to composition and molecular size. Furthermore, the specific activities and the dose-response relations for activation by apolipoprotein C-II were quite similar for the two enzymes. Antibodies raised against the guinea pig milk enzyme inhibited not only this enzyme but also the lipoprotein lipase activity in post-heparin plasma and in homogenates from adipose tissue and heart.

Activity of lipoprotein lipase in thyroidectomized rats.

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue pieces. There were no differences between control and hypothyroid rats in the disappearance of intravenously injected 125I-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of 125I-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.

Immunohistochemical localization of lipoprotein lipase in human adipose tissue.

The distribution of lipoprotein lipase (LPL) was studied in needle biopsies of human adipose tissue. Antibodies against bovine milk LPL react with and inhibit the activity of the human enzyme. These antibodies were used for immunohistochemical studies of the distribution of LPL in human adipose tissue. Immunoreactive enzyme was observed in adipocytes and connective tissue cells resembling preadipocytes. It was also seen in perivascular cells, in capillaries and in larger vessels. Intravenous administration of heparin led to a substantial decrease of immunodetectable LPL in vessels, whereas the enzyme in adipocytes and connective tissue cells was unaffected.

Low-dose oral methotrexate as second-line therapy for persistent trophoblast after conservative treatment of ectopic pregnancy.

The aim of this study was to evaluate the efficacy of methotrexate as second-line treatment for ectopic pregnancy. Oral methotrexate was used in 15 patients with evidence of persistent trophoblast after conservative laparoscopic surgery for tubal pregnancy. The treatment was successful in 14 of 15 cases, and the mean time for decline of serum hCG to nonpregnant levels was 24 days. In the remaining case, hCG continued to rise. Side effects were noticed, even at a low dosage, but only in those subjects not receiving citrovorum rescue. As an alternative to a second operation, oral methotrexate appears to be an effective and well-tolerated therapy for persistent trophoblast.

Persistent toxic chemicals: more than Stockholm persistent organic pollutants.

Schafer and Kegley bring up the important issue of excessive chemicals exposure of children. However, they do not consider in depth the "cumulative and simultaneous exposures faced by children, (.) moving beyond the chemical-by-chemical approach of the past", as quoted from the US Environmental Protection Agency national agenda to protect children from environmental health threats. Existing evidence for contamination by many substances beyond those dealt with in the article calls for additional protective measures. These could include an extra margin of exposure by a factor of 10 to cover cumulation of chemicals, for adults and children alike.

Natural history of patients with untreated liver metastases from colorectal cancer.

One hundred fifty-five patients, laparotomized because of colorectal cancer, were retrospectively evaluated with special attention given to the natural course of untreated synchronous liver metastases. The median survival time for patients with synchronous liver metastases was 4.5 months. The survival time was mainly influenced by the extent of tumor involvement in the liver. Patients with elevated levels of serum-alkaline phosphatase at the time of operation had a significantly shorter survival time than those with normal values. Serum alkaline phosphatase levels are a good indication of prognosis. The incidence of synchronous liver metastases was 16 percent. This low rate is partly explained by the development of metachronous liver metastases in five patients within 1 year. Comparison with previous reports, often more than 10 years old, reveals that the poor prognosis of patients with untreated liver metastases from colorectal cancer has remained unchanged.

Introduction: present knowledge on the effects of radioactive contamination on pregnancy outcome.

This introduction gives a brief review of the effects on pregnancy outcome that might follow radioactive contamination of the environment. These include miscarriages, congenital anomalies, damage to the central nervous system expressed through reduced intelligence and a risk of tumours late in life. Knowledge is fragmentary and field studies are difficult, which lends weight to the attempts at studying the effects of the Chernobyl accident in Europe which are the subject of the present symposium.

Provitamins and vitamins D2and D3in Cladina spp. over a latitudinal gradient: possible correlation with UV levels.

Provitamin D2, vitamin D2 and vitamin D3 were identified in the thallus of a lichen species, Cladina arbuscula (Wallr.) Hale and W.L. Culb. The identification of vitamin D3 was supported by: (1) co-chromatography in both reverse and straight phase HPLC (high performance liquid chromatography), (2) ultraviolet absorption spectrum, and (3) molecular ion peaks demonstrated by ESI (electrospray ionisation) mass spectrometry. The contents of vitamin D3 range from 0.67 to 2.04 µg g⁻¹ dry matter in the thalli of C. arbuscula specimens grown under different natural conditions, while provitamin D3 could not be detected. The ranges for provitamin D2 and vitamin D2 were 89-146 and 0.22-0.55 µg g⁻¹ dry matter, respectively, while the contents of provitamin D3 were below the detection limit (0.01 microg g(-1) dry matter). When C. arbuscula thalli collected at different latitudes from northern Finland to Greece were compared, a positive correlation of vitamin D2 and D3 contents with modelled UV-B radiation at the collection sites was found. A single sample of C. rangiferina from northern Finland gave much higher values for the vitamins. A possible reason could be the lower content of UV-B absorbing pigment in the latter species.

What is a reasonable cost for protection against radiation and other risks?

In Sweden, the National Audit Bureau is encouraging attempts at discussing the costs of protection. For many years, a government authority considering new regulations must provide the government with background material illustrating the costs, the expected benefits, and other aspects of the decision. Direct costs should be stated together with nonquantifiable factors indicated by a valuation of whether their consequences are positive or negative. Applied to radiation protection, this includes discussion of the resources worth spending in order to prevent a case of serious radiation injury. If the marginal cost for a protective measure is < 5 million Swedish crowns (5 MSEK: approximately 1 million U.S. dollars) per prevented case, the radiation protection authority considers the measure to be strongly justified. If the cost exceeds 25 MSEK per case, then very strong reasons are required for implementation of the measure. In the intermediate interval, measures are particularly justified if the marginal costs are in the lower end and the total societal cost of the protection is of little concern at the national economical level. The interval 5-25 MSEK per case corresponds to 0.4-2 MSEK person-sievert-1. Resource allocation for health protection in general, ethical aspects, and practical difficulties in assessing costs and risks are briefly discussed in the paper. Priorities in Swedish radiation protection are presented and a case study for the use of carbon fiber cassettes in x-ray diagnostics is given as an example.

Post-deposition transport of radionuclides in fruit.

This paper considers two main pathways for contamination of fruit by radionuclides: (i) absorption after deposition directly to exposed fruit surfaces, and (ii) absorption after deposition to other exposed plant surfaces followed by translocation to fruit. The aim is to collect the available information on fruit from temperate regions, identify the factors affecting post deposition processes in fruit plant systems, identify gaps in knowledge and give recommendations for future work. The majority of information available on above-ground absorption and further translocation to fruit concerns 134Cs and 85Sr in soluble form in apple, strawberry and grapevine. In general, 85Sr is absorbed and translocated to a lesser extent than is 134Cs. The rate of absorption and translocation depends on the physiological stage and age of the plant, and varies between different plant species and varieties.