Crocidolite asbestos fibers in smoke from original Kent cigarettes.

The original version of the Kent Micronite cigarette filter used crocidolite, a form of asbestos, from 1952 until at least mid-1956. Cigarettes from intact, unopened packs of the brand from this period were examined. One filter contained approximately 10 mg of crocidolite. Crocidolite structures were found in the mainstream smoke from the first two puffs of each cigarette smoked. At the observed rates of asbestos release, a person smoking a pack of these cigarettes each day would take in more than 131 million crocidolite structures longer than 5 microns in 1 year. These observations suggest that people who smoked the original version of this cigarette should be warned of their possible substantial exposure to crocidolite during the 1950s.

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion.

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.

Dietary fat assimilation and bile salt absorption in the killifish intestine.

Radiolabeled taurocholate (TC) and triolein were used to study fat assimilation and bile salt absorption in the stomachless saltwater killifish, Fundulus heteroclitus. Fat absorption occurred primarily in the proximal intestine with approximately 87% of a single dose (9 mg fat/8 g fish) absorbed in 2 h. Luminal triolein hydrolysis and enterocyte triolein resynthesis were tightly coupled. Killifish gallbladder bile contains taurocholate and cholate in an equal molar ratio at a combined concentration of 237 +/- 25 mM (n = 10) in 24-h-fasted fish. During fat assimilation luminal bile salt and fatty acid concentrations ranged between 10 and 30 mM. Between and during meals the total concentration of bile salts in the intestinal tissue remained roughly constant (4-6 mM) with the proximal one-third of the intestine containing 40% of the total and the remainder equally distributed between the mid and distal regions. All three regions of the intestine rapidly incorporated ingested TC in vivo, with the amount incorporated proportional to the pool size. In contrast, in vitro at low TC concentrations (60 nM), the distal one-third of the intestine incorporated 10 times as much TC in 2-min uptake experiments as the proximal and mid regions. Although there are many similarities between fat and bile salt assimilation in killifish and mammals, overall the processes are much simpler in killifish.

The use of the Iatroscan TH-10 analyzer to quantify total lipids in a variety of sample types and lipid classes in human gallbladder bile.

Two methods for the measurement of total lipid weight in biological and geological samples and the major lipid classes in human gallbladder bile using the Iatroscan TH-10 analyzer are described. Total lipid determination involves the application of small (5 microliter) volumes to Chromarods, focusing of the sample into one band by partial development in chloroform-methanol (1:1), and quantification by flame ionization detection (FID). The response variation between different sample types did not affect the linearity of response, allowing a reproducibility of +/- 10% of the mean or better for samples ranging from 0.5 to 32 micrograms. Total lipid determinations in 10 samples could be performed in 30 min. The three major components of human gallbladder bile (cholesterol, phospholipids and bile acids) also were quantified with the Iatroscan. Samples focused on Chromarods were separated using a double development scheme in two solvent systems. All three components exhibited a linear response over the range of 0.25 to 8 micrograms. The repeated scanning of rods required at concentrations greater than 3 micrograms did not affect linearity of response. Samples from 10 patients could be processed in less than one hr. Several techniques are discussed to increase reproducibility when performing quantitative lipid analysis with the Iatroscan.

Intramembranous particles are clustered on microvillus membrane vesicles.

Many intramembranous particles in pig jejunal microvillus membranes cluster during cell disruption and membrane vesiculation with the MgCl2 aggregation technique (Hauser, H., Howell, K., Dawson, R.M.C. and Bowyer, D.E. (1980) Biochim. Biophys. Acta 602, 567-577). Isolated brush borders and purified microvillus membrane vesicles were jet-frozen and examined by freeze-fracture electron microscopy. From 30 to 60% of purified vesicles exhibited no intramembranous particles on their fracture face and 22-39% exhibited clustered or aggregated intramembranous particles. Only 6-15% of the vesicles exhibited the random distribution of intramembranous particles that is characteristic of intact enterocytes. Aggregation was not reversed after dialysis to remove divalent cations. Prior freezing of tissue or vesicles (-70 degrees C) gave the same results as fresh unfrozen material. Heterogeneity of microvillus vesicles may occur among the vesicles generated from a single microvillus.

Similar bioavailability and lymphatic transport of benzo(a)pyrene when administered to rats in different amounts of dietary fat.

This study assessed the effect of concomitant lipid absorption on the bioavailability and lymphatic transport of benzo(a)pyrene (BP), a carcinogenic polycyclic aromatic hydrocarbon (PAH). Conscious, male Sprague-Dawley rats, equipped with biliary and mesenteric lymphatic catheters received intraduodenally a dose of 0.4 mumoles 3H-labeled BP completely dissolved in either 50 mumoles or 500 mumoles of olive oil. Diversion of mesenteric lymph allowed biliary and urinary excretion of 3H to be used as an indirect measurement of relative 3H portal transport. Total radiolabel recovered in a 24-hr period in each group was 20.0 +/- 2.6% of the 3H dose given in 50 mumoles of oil, and 17.0 +/- 1.0% of the 3H dose administered in 500 mumoles of oil. In animals receiving the low-fat test meal, 79.4 +/- 1.4% of the recovered radiolabel was found in bile; the corresponding value for the high fat dose was 78.5 +/- 2.6%. Thus a tenfold variation in the mass of the carrier vehicle (triglyceride oil) did not significantly effect the disposition of BP, and portal, not lymphatic transport, was the major route of post-absorptive transport. Although the chylomicrons produced from both fat doses were initially contaminated with BP, within 1-1.5 hr the radioactivity in lymph began to drop such that by 3 hr in the animals fed high fat, the chylomicrons were essentially free of BP. These results show that the rat enterocyte quickly adapts to PAH-contaminated dietary fat, even during the assimilation of a single dose of fat. Presumably, during the post-absorptive synthesis of chylomicrons from pre-chylomicrons, BP is metabolized and removed from the triglyceride oil droplets.

A simple inexpensive cryogenic storage device for microscopy specimens.

Cryogenic storage devices for microscopy specimens are costly and may require a particular size or type of dewar. Described here is a simple inexpensive storage container for small specimens (less than 5 mm in diameter) which can be constructed in less than 15 min from common laboratory materials. The unit is modular in design, can fit into any thermos type dewar and is reusable.

The production of liquid crystalline product phases by pancreatic lipase in the absence of bile salts. A freeze-fracture study.

The hydrolysis of gum arabic-stabilized trioleylglycerol emulsions by pancreatic lipase was examined by freeze-fracture electron microscopy in the absence of bile salts. A sequence of liquid crystalline product phases was produced during the non-equilibrium conditions of hydrolysis. The morphology of the product phases were pH- and droplet size-dependent. At pH 8.3 the initial product phase was composed of homogeneous spherical vesicles regardless of trioleylglycerol drop size. As the reaction progressed the partially hydrolyzed droplets showed a crystalline 'crust' and a true lamellar phase which was often swollen, giving an isotropic appearance to this phase. Some droplets demonstrated a possible transitory hexagonal phase composed of tubular-lamellar elements in close association with the oil phase. These tubular-lamellar elements graded into a lamellar phase at the aqueous/product interface. A cubic phase was not discernible. At pH 7.0 a single phase was seen which covered the drop surface with an amorphous layered 'crust'. The significance of these phases is discussed in relation to those produced by pure and mixed lipids under equilibrium conditions.

Hydrolysis of triacylglycerol emulsions by lingual lipase. A microscopic study.

The effect of lingual lipase on four different triacylglycerol emulsions was observed by light microscopy at pH 5-6. The extent of hydrolysis on the microscope slide was determined with the aid of radioactive emulsions or by analyzing the products by gas-liquid chromatography. Artificial emulsions that had been stabilized with amphiphilic lipids gradually coalesced during the unstirred lipase reactions. Gum arabic-stabilized emulsions and human milk fat droplets did not stick to each other or coalesce during lingual lipase hydrolysis. No visible liquid-crystalline product phases, as are seen with pancreatic lipase (Patton, J.S. and Carey, M.C. (1979) Science 204, 145-148), were observed with lingual lipase. The products of lingual lipase activity, protonated fatty acid and diacylglycerol, appear to remain dissolved in the oil phase of the triacylglycerol particle.