Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function. For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated. The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2. A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation. It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.

Human liver slices were prepared with a Krumdieck slicer from macroscopically healthy surgical waste after partial hepatectomy. They were incubated without or with the addition of the inducer beta-naphthoflavone (BNF) (25 mum) either immediately after preparation (fresh slices) for up to 24 hours or after cryopreservation in liquid nitrogen (thawed slices) for up to 6 hours. Potassium concentration was well maintained in fresh and thawed slices over 24 and 6 hours, respectively, but at lower levels than in rat liver slices. Albumin secretion showed relatively large interindividual differences. Both parameters were lower in thawed slices than in fresh ones, but indicated a certain number of viable cells. In untreated fresh slices CYP1A1-mRNA was not detectable; however, it increased distinctly within 6 hours of exposure to BNF. The amounts of induced CYP1A1-mRNA differed by a factor of more than 100 among six human livers and were lower than in fresh rat liver slices. Even in thawed human slices, CYP1A1-mRNA expression could be induced in vitro by BNF, although at a very low level and preferentially in those specimens with comparably high inducibility already before freezing.

Principle steps necessary for cryopreservation of precision-cut liver slices as currently applied by different groups are summarized including own results concerning mode of freezing. Now we use rapid freezing by immersion in liquid nitrogen after exposure to 10% DMSO as the cryoprotectant for rat liver slices. The results indicate well-maintained cytochrome P450 (CYP)-dependent deethylation rates in slice homogenate after short-term incubation. ECOD rate in intact thawed slices was even higher than in fresh ones after 2 h incubation. In contrast to fresh slices all parameters except protein content decreased to marginal levels during long-term incubation of thawed slices for 24 h. The first preliminary experiments on albumin secretion by thawed rat liver slices, measured between the 2nd and the 4th hour of incubation, showed partial maintenance of this liver specific differentiated function. Trials to induce CYP1A1 in thawed rat liver slices in vitro by beta-naphthoflavone (BNF) resulted in increased expression of CYP1A1-mRNA within 6 h as shown by RT-PCR and quantified by competitive RT-PCR. The decline of deethylation rates, determined in slice homogenates, and of viability within 24 h incubation was not prevented by exposure to BNF or DMSO. The results derived from one sample of cryopreserved human liver slices indicate a quite acceptable maintenance of function up to 6 h, if the same protocol as developed for rat liver slices was used.

Zinc intake of adults in Germany was determined from 1988 to 1996 by the duplicate portion method. Altogether, 14 test populations each consisting of at least 7 women and 7 men collected their daily consumed food and beverages over 7 subsequent days. Furthermore, 10 female and 10 male vegetarians took part in a duplicate study in 1996. Our results show that food dry matter consumption of persons with mixed diets did not change from 1988 to 1996. Vegetarians (women: 390 g/day, men: 479 g/day) consumed 32% more dry matter than non-vegetarians. However, energy intake was not influenced by kind of diet. Zinc intake of adults with mixed diets decreased from 1988 to 1996 statistically saved by 20 to 25%. In 1996, women took in 6.0 mg Zn/day and men 7.5 mg Zn/day. The zinc intake of men amounted to about 25% more than that of women. This sex-depending difference vanished when zinc consumption was related to body weight. Possible reasons for the declining zinc supply might be changes in food preference, a higher consumption of processed foodstuffs, for example refined sugar, polished rice and flour with low extraction rate, and a decreased intake of meat. Compared to this, zinc intake of vegetarians (women: 8.6 mg Zn/day, men: 9.5 mg Zn/day) was 34% higher than that of non-vegetarians. These findings were caused by the enhanced dry matter intake of vegetarian persons. It seemed that the higher zinc consumption of vegetarians might compensate the lower bioavailability of zinc due to phytate-richer diet. Both non-vegetarian and vegetarian test persons were healthy and showed no visible signs of zinc deficiency. Mild zinc deficiency could not be excluded, especially for persons consuming less than 5 mg Zn/day. In conclusion, zinc intake of adults in Germany is marginal and should be improved.