Avarol-induced DNA strand breakage in vitro and in Friend erythroleukemia cells.

The hydroquinone-containing cytostatic compound avarol inhibits predominantly growth of those cell lines which have a low level of superoxide dismutase. The substrate of this enzyme, the superoxide anion, was found to be formed during the in vitro oxidation reaction of avarol to its semiquinone radical in the presence of oxygen. Under the same incubation conditions plasmid DNA (pBR322) was converted from the fully supercoiled circular form mainly to the nicked circular form, indicating that the compound causes primarily single-strand breaks. Using Friend erythroleukemia cells (FLC) it was found that avarol induces a dose-dependent DNA damage; the maximum number of DNA strand breaks was observed at 5 h after addition of the compound to the cells. Removal of avarol resulted in a rapid DNA rejoining with biphasic repair kinetics [first half-time, 8 min (90% of the breaks) and a second half-time, 40 min (10% of the breaks)]. When the degree of avarol-induced DNA damage in FLC was compared with the drug-caused inhibition of cell growth a close correlation was established. Avarol displayed no effect on dimethyl sulfoxide-induced erythrodifferentiation of FLC as determined by the benzidine reaction and by dot blot hybridization experiments. From incubation studies of FLC with [3H]avarol no hint was obtained for the formation of an adduct between DNA and the compound. The subcellular distribution of [3H]avarol was studied in liver cells after i.v. application of the compound. The predominant amount of the compound was present in the cytosolic fraction; little avarol was associated with plasma membranes, nuclei, and mitochondria. Using (a) oxidative phosphorylation and (b) oxygen uptake as parameters for mitochondria function, no effect of the compound on the activity of this organelle was determined. These results suggest that avarol forms superoxide anions (and in consequence possibly also hydroxyl radicals) especially in those cells which have low levels of superoxide dismutase. Moreover, evidence is provided that the active oxygen species cause DNA damage resulting in the observed cytotoxic effect.

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine during long-term parenteral nutrition in the growing rat.

Utilization of N-acetyl-L-tyrosine and glycyl-L-tyrosine as a source of tyrosine in infusion solutions was tested in rats receiving total parenteral nutrition for 4 wk. The four solutions tested were isonitrogenous and isocaloric. One of the solutions contained an adequate amount of L-phenylalanine; in the other three, two-thirds of the phenylalanine was replaced by a corresponding amount of either glycine, glycyl-L-tyrosine or N-acetyl-L-tyrosine. No differences in weight gain or N-balance could be detected as a result of administering either the solution with glycyl-L-tyrosine or with N-acetyl-L-tyrosine in place of the solution containing an adequate phenylalanine content. The solution in which two-thirds of the L-phenylalanine was replaced by glycine yielded only half of the weight gain and correspondingly reduced values for N-balance. Daily urinary excretion rates for N-acetyl-L-tyrosine and glycyl-L-tyrosine were 11% and 0.5%, respectively, of the infused amount. Plasma amino acid pattern was affected differently by the four solutions. The results indicate that both N-acetyl-L-tyrosine and glycyl-L-tyrosine are efficiently utilized by the rat during total parenteral nutrition.

Utilization of glutathione disulfide as cysteine source during long-term parenteral nutrition in the growing rat.

Utilization of intravenously administered glutathione disulfide was investigated during long-term parenteral nutrition in growing rats. In a series of cross-over studies, three solutions were tested against one another by recording weight gain, nitrogen balance, and plasma amino acid patterns. Solution 1 contained the required amount of methionine for rats, solution 2 had only one third of the required methionine, but was made isonitrogenous with glycine, whereas in solution 3, two thirds of the methionine was replaced by glutathione disulfide. Weight gain was about twice as high during infusion with either the required amount of methionine or the glutathione disulfide when compared with solution 2. Nitrogen retention was significantly higher during infusion with sufficient methionine or a corresponding amount of glutathione disulfide, when compared with the solution low in methionine. Plasma levels of cystine decreased significantly under the low methionine supply, but no difference was observed for the groups receiving sufficient methionine or the corresponding amount of glutathione disulfide. It is concluded that glutathione disulfide permits adequate cysteine supply in parenteral nutrition and may replace part of the methionine in the presence of an impaired conversion of methionine to cysteine.

Utilization of methionine and N-acetyl-L-cysteine during long-term parenteral nutrition in the growing rat.

Utilization of methionine and N-acetyl-L-cysteine as a source of cysteine was tested in growing rats receiving total parenteral nutrition for four weeks. The three solutions tested were isonitrogenous and isocaloric. One of the solutions contained an adequate amount of L-methionine, in the other two, two thirds of the L-methionine was substituted by a corresponding amount of either glycine or N-acetyl-L-cysteine. Weight gain and N-balance were similar under the infusion with either the adequate amount of L-methionine or the N-acetyl-L-cysteine substituted. The solution in which two thirds of the L-methionine was replaced by glycine yielded only half of the weight gain and correspondingly reduced values for N-balance. The daily urinary excretion rate for N-acetyl-L-cysteine was 4.6% of the infused amount. Urinary excretion rates of the other amino acids and the plasma amino acid pattern was affected differently by the three solutions. The results indicate that cysteine is more rapidly available from N-acetyl-L-cysteine than from L-methionine when administered intravenously.

The metabolic status of internal intensive care patients as indicated by 3-methylhistidine excretion and nitrogen balance.

The metabolic status of 15 intensive care patients receiving a standardized total parenteral nutrition regimen was followed up to 15 days immediately after admission by measuring 3-methylhistidine, total nitrogen, and creatinine excretion. The average 3-methylhistidine excretion was within the normal range during the first 3 days, rising on day 4 and reached a maximum of 70% above normal values on day 5. It declined to within normal range thereafter in most of the patients. Mean values for creatinine excretion remained relatively constant within the normal range throughout the study. During all days 3-methylhistidine was negatively correlated with N-balance. It is concluded that these patients had increasing catabolism with a maximum on day 5 and that the catabolic condition was associated with an increased muscle protein breakdown.

Rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in human serum by isocratic adsorption HPLC.

The simultaneous determination of alpha-tocopherol and retinol in human serum is reported. The separation is carried out by means of isocratic HPLC on adsorption columns. UV-Detection is possible by using either one wavelength for both compounds (300 nm), or after a lambda change mode with typical wavelengths for alpha-tocopherol (292 nm) and retinol (325 nm). According to short retention times (10 min) and rapid extraction the method is useful for clinical research and allows about 50 analyses per day and operator. Blood from 176 human volunteers was collected and alpha-tocopherol and retinol levels in serum determined with this method. Statistical evaluation of different selected groups shows typical significant differences of alpha-tocopherol and retinol concentrations in smokers and oral contraceptive users.

PIP: An extraction and separation method for simultaneous vitamin A and E determination was developed to overcome problems of evaporation steps. The method did not involve an evaporation step and separation on adsorption columns by isocratic HPLC. It permits 50 analyses daily and operates with high sensitivity and reproducibility. Blood was collected from 115 healthy male and 63 healthy female volunteers ranging in age from 18-28 years. Retinol (25 mg) was dissolved in isopropanol and taken as stock solution. An aliquot of the stock solution was diluted and its absorbance measured at typical wave length against isopropanol and calculated. Alpha-Tocopherol was dissolved in isopropanol, an aliquot of this stock solution diluted its absorbance measured against isopropanol and calculated. Standard solutions were prepared by appropriate dilution of the measured stock solution. Retinol and a-tocopherol can be extracted in organic solvents after denaturation of the specific binding proteins by adding ethanol. A table shows that small amounts of serum and ethanol are sufficient to extract both vitamins quantitatively. Increasing a-tocopherol concentrations in ethanol can be extracted quantitatively in the hexane phase and, if added to hexane, the vitamin is not lost in the ethanol phase. The recovery study demonstrates different concentrations of the vitamin do not influence the quantitative extraction, and the recovery range indicates that the precision of the method is adequate within the linear range. The stock solution is stable more than 7 weeks when stored at -34 degrees Centigrade in glass vessels; the standard solutions should be prepared for each working day. The serum samples were stable more than 2 weeks when stored at -34 degrees Centigrade. The differences between the retinol values in selected groups are greater than the a-tocopherol differences. Retinol of females using oral contraceptives (OCs) was higher than of nonusers. This elevation was related to the estrogen content of the OCs. Cigarette smoking increased retinol levels slightly; in non-smokers the values were below the normal range. For OC users, the vitamin E levels did not show any significant differences. Cigarette smoking increased plasma vitamin A levels significantly but not vitamin E levels. The rapid simultaneous determination of retinol and a-tocopherol in plasma is useful in several clinical situations and in the nutritional assessment of normal subjects.

Uptake and metabolism of retinoic acid induces inhibition of cell growth: a study in a rat rhabdomyosarcoma cell line (BA-HAN-1C) using nonlinear theoretical models.

Retinoic acid causes a significant inhibition of cell growth of the tumor cell line BA-HAN-1C. This growth inhibition is the same whether the cells are treated with a pulse dose of retinoic acid (RA) or continuously expand to RA. The determination of RA and its degradation products within the culture medium and in the cells showed that after 24 hours 13-cis-RA was the major retinoid in all cells (96 ng/10(6) cells); all-trans-RA represented 56 ng/10(6) cells. After 48 hours 4-hydroxy-RA and a small amount of 5,6-epoxy-RA was found in the cells and also in the culture medium. 4-hydroxy-RA increased up to 96 hours, whereas 13-cis- and all-trans-RA were not detectable in the cells after 96 hours. We conclude that the BA-HAN-1C cells take up and metabolize RA. Nonlinear fit analysis of the time behavior of the RA concentration in medium demonstrates that the RA uptake unexpectedly follows a mono-exponential time function. Discussion of the experimental results in connection with a proper compartment model shows that uptake and metabolism of RA cannot be described really by a first order kinetics. The mathematical analysis leads to a more complicated kinetic model with certain restrictions for the corresponding rate constants.

[L-tryptophan and the eosinophilia-myalgia syndrome--consequences for artificial nutrition?]. / L-Tryptophan und das Eosinophilie-Myalgie-Syndrom--Konsequenzen für die künstliche Ernährung?

Present knowledge on the possible cause of the tryptophan-induced eosinophilia-myalgia syndrome is discussed on the basis of a literature survey. The initially favored hypothesis of metabolites of a deranged tryptophan metabolism in some persons as cause of the syndrome has no plausibility for several reasons discussed in this paper. In the meantime trace backs of implicated tryptophan lots have led to one manufacturer who has changed his production procedure. The implicated lots contain a variety of impurities detectable by HPLC. Whether these impurities are the immediate cause of the syndrome or just markers remains to be established. An animal model suitable to clarify this question has recently been developed. Taking all these measures to identify and eliminate suspicious lots, there is no reason to withhold live saving artificial nutrition with tryptophan-containing preparations.

On the problematic nature of vitamin E requirements: net vitamin E.

The requirement for vitamin E is closely related to the dietary intake of polyunsaturated fatty acids (PUFA). By the protective mechanism to prevent PUFA from being peroxidized, vitamin E is metabolically consumed. In addition, PUFA impair the intestinal absorption of vitamin E. Therefore PUFA generate an additional vitamin E requirement on the order of 0.6, 0.9, 1.2, 1.5, and 1.8 mg vitamin E (RRR-alpha-tocopherol-equivalents), respectively, for 1 g of dienoic, trienoic, tetraenoic, pentaenoic, and hexaenoic acid. For this reason, the gross vitamin E content of food containing PUFA does not allow an evaluation of this food as a source of vitamin E. A suitable measure is the net vitamin E content, i.e., gross vitamin E minus the amount needed for PUFA protection. Therefore, some food-stuffs generally considered as vitamin-E sources, as concluded from their gross vitamin E content, cause in reality a vitamin E deficiency if not sufficiently compensated by other vitamin E supplying food constituents. Examples of the net vitamin E content of some fats and oils, fish and nuts are shown. Consequences for food composition data and food labeling and the problem of meeting the vitamin-E requirements are discussed.

Use and abuse of high dosages of vitamin B6.

Concepts of vitamin B6 megatherapy are classified in three categories: 1. megatherapy with well-known mechanism of action; 2. megatherapy with hypothetical or unknown, but nevertheless plausible mechanism of action; 3. megatherapy on the basis of pure speculation. Examples of all three categories are shown. The dosages applied extend from 100 mg up to several grams; the duration of treatment varies from weeks to several years. After long-term intake of high doses toxic effects can occur in the form of peripheral sensory neuropathy, in two reported cases combined with a subepidermal vesicular dermatosis. The threshold above which toxic effects can occur appears to be somewhat between 300 and 500 mg/day; however, systematic investigations in this dose range have never been performed. Furthermore, there appears to be an inverse relationship between the dose and the time up to the occurrence of toxic symptoms. The danger consists less in controlled application by a physician as far as he is aware of the clinical picture of sensory neuropathy. Doses in excess of 500 mg are rarely necessary in specific indications. Unlike this situation, self-medication by laymen on the basis of promises in obscure health magazines is much more risky because the dose is frequently raised more and more up to several grams per day when the expected effect does not occur.

[Usefulness of fat infusion within the frame of parenteral feeding]. / Zur Zweckmässigkeit von Fettinfusionen im Rahmen der parenteralen Ernährung

The interactions of fat and carbohydrate metabolism are surveyed. The posttraumatic metabolism is characterized by a stress induced high lipolytic rate and a high concentration of non-esterified fatty acids in the blood. The increased fatty acid oxidation causes by effects of metabolites a catabolic situation with enhanced ketogenesis, gluconeogenesis and protein breakdown. High levels of fatty acids and ketone bodies reduce peripheral glucose utilization. In such a situation, infusions of fat emulsions are disadvantageous, since fatty acids, set free from the triglycerides, would aggravate the catabolic metabolism and change the nitrogen balance for the worse. In normal metabolic situations with high energy need, or in long term parenteral nutrition, fat infusions are necessary to meet the needs of energy and of essential fatty acids. Fat infusions shoudl be combined with carbohydrates and amino acids in an appropriate relation.

Quantitative aspects of the metabolism of glucose, fructose, sorbitol and xylitol.

There is no formula to calculate practicable application rates for carbohydrates and polyols. Maximum turnover rates, elimination constants, half-life, and total clearances are rough criteria only to marke out a possible range. Suitable and safe infusion rates have to be established empirically for each substance, considering carefully desired and undesired effects. Recommendations for safe infusion rates are the safer, the most parameters have been considered. Present knowledge is compiled in Table 2.

[Biochemistry and metabolism of sugar substitutes]. / Biochemie und Stoffwechsel von Zuckeraustauschstoffen

The metabolisms of the sugar substitutes fructose, sorbitol and xylitol and their interdependence with the metabolism of glucose are demonstrated. The metabolic characteristics of these substitutes are discussed with regard to therapeutical utilization. Differences between oral and parenteral administration are shown and finally dosage guidelines for parenteral administration are established on the basis of the different metabolic effects depending on the administered dose.

[Significance of vitamins in parenteral nutrition]. / Die Bedeutung der Vitamine in der parenteralen Ernährung.

Parenteral nutrition is incomplete without vitamins. Marginal vitamin deficiency under parenteral nutrition is certainly more common than generally recognized. Even marginal and undiscernable vitamin deficiency interferes with healing processes and increases the rate of complications since vitamins are involved in a variety of ways in wound healing, regeneration processes and immune function. If total parenteral nutrition is necessary for longer periods of time, exceeding 5 days, vitamins should be substituted in the recommended doses. The assessment of a marginal vitamin deficiency is difficult to perform and extremely expensive. It is therefore easier, safer and cheaper to substitute vitamins in total parenteral nutrition from the beginning, if preceeding malnutrition is likely. Recommendations for dosage and mode of application are reported and explained.

Enzymatic and pharmacokinetic studies on the metabolism of branched chain alpha-keto acids in the rat.

Michaelis-constants and enzyme activities for dehydrogenation and transamination of the three branched chain alpha-keto acids in liver, kidney, skeletal muscle, and brain of rats are reported. After oral load only 11-22% of the keto acids pass the liver unchanged. Blood levels in pharmacokinetic and absorption studies are related to the Michaelis-constants. At the low keto-acid concentrations after oral application, dehydrogenation in the non-hepatic tissues is supposed to prevail over transamination. Data on feed efficiency of branched chain alpha-keto acids reported in the literature support this view. The chance for transamination is better after intravenous administration. The transferability of our data to humans, and various factors influencing the efficiency of branched chain alpha-keto acids are discussed in connection with data reported in the literature.