A.P. De Leenheer

Variation of ascorbic acid content in different live food organisms.

Abstract Ascorbic acid (AA) is an essential nutrient both in particle and live aquafeeds. In order to better assess the needs for this nutrient during larviculture the AA content of algae, rotifers and Artemia was studied with respect to their suitability at startfeeding. In general, the microalgae evaluated were rich in AA (1000–4000 μg AA/g DW), but showed a considerable variability among the different species: e.g. the concentration in Isochrysis and Chlorella reached values 3-to 4-fold the percentage of Tetraselmis (0.11 % of DW). Brachionus routinely cultured on Chlorella contained 2300 μg AA/g DW. Cysts of various batches and strains of Artemia differed considerably in ascorbic acid-2-sulphate (AAS) concentration (296–517 μg AA/g DW). The amount of AA available in the freshly-hatched nauplii reflected exactly the AAS reserve present in the cysts, what evidences the complete conversion of AAS to free AA during completion of embryonic development into nauplii. Boosting techniques both for Brachionus and Artemia using ascorbyl palmitate (AP) as the vitamin C source were established. The addition of 20% AP in the diet of Brachionus enhanced their AA content 10-fold over 3 days of culture. Supplementation of the enrichment emulsion for Artemia with 20% AP increased the AA content up to 2000 μg/g DW after 24 h enrichment. This lipophilic derivative of AA appeared to be a stable form of vitamin C for enhancing AA levels in the live diets during culture and/or enrichment. This bioencapsulation method provides a tool for hatcheries to build up high AA concentrations in the live prey administered to first feeding larvae of aquaculture organisms in case of specific requirements (e.g. with respect to handling stress, deformities). A survey of commercial hatcheries indicated that a wide range of products is used for the cultivation and boosting of rotifers, which consequently affect their AA levels. In general, the AA content in the algae and, consequently, the algal enrichment of Brachionus tended to score lower in the hatcheries than under lab conditions.

Plasma concentrations of vitamin K1 and PIVKA-II in bottle-fed and breast-fed infants with and without vitamin K prophylaxis at birth

Plasma vitamin K1 and proteins induced by vitamin K absence (PIVKA) were assayed simultaneously 1–4 days and 29–35 days after delivery in three groups of infants: breast-fed not receiving vitamin K at birth (n=12), bottle-fed without vitamin K administration at birth (n=7) and breast-fed receiving 1 mg vitamin K1 administered by intramuscular injection at birth (n=13). The bottle-fed infants had a significantly higher vitamin K1 plasma level than breast-fed infants who did not receive vitamin K1 at birth. Extremely high levels of vitamin K were obtained 1–4 days after intramuscular administration. At the age of 1 month, breast-fed infants had the same plasma vitamin K1 concentration whether or not they had received vitamin K1 supplements. Decarboxy prothrombin (PIVKA-II) a reliable indicator of biochemical vitamin K deficiency, was found in 5 out of 12 breast-fed and in 2 out of 6 bottle-fed infants who had not received supplemental vitamin K1 after birth. In a separate study, we followed up to 90 days after birth a larger group if infants. PIVKA-II was found with significantly greater frequency in breast-fed infants receiving no vitamin K than in breast-fed infants receiving 1 mg vitamin K intramuscularly at birth, or in bottle-fed infants without extra vitamin K1. These data form a strong argument for routine vitamin K prophylaxis after birth for all breast-fed infants. The optimum dose and manner of administration require further study.

An oral challenge for turbot larvae with Vibrio anguillarum

M. Chair 1, M. Dehasque 1, S. Van Poucke 2, H. Nelis 2, P. Sorgeloos ~* and A.P. De Leenheer 3 1 Laboratory of Aquaculture and Artemia Reference Center, University of Ghent, Rozier 44, B-9000 Ghent, Belgium 2Laboratory for Pharmaceutical Microbiology, University of Ghent, Hare/bekestraat 72, B-9000 Ghent, Belgium 3Laboratory of Medical Biochemistry and Chemical Analysis, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium

Sensitive gas chromatographic--mass spectrometric screening of acetylated benzodiazepines.

GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

Simultaneous determination of fifteen low-dosed benzodiazepines in human urine by solid-phase extraction and gas chromatography–mass spectrometry

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia beta-glucuronidase for 1 h at 56 degrees C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

High-performance liquid chromatographic assay of vitamin K in human serum.

A sensitive combined adsorption and reversed-phase high-performance liquid chromatographic procedure is described that permits the determination of endogenous vitamin K1 levels in serum. The separation of vitamin K1 (cis- + trans-isomers), vitamin K1 epoxide and the menaquinones MK-2, MK-4 and MK-9 by adsorption, reversed-phase and cyano-bonded phase chromatography is discussed. The methodology was further developed for the quantitative measurement of vitamin K1 in human serum. Concentrations as low as 500 pg/ml in serum could be detected. The identity of the vitamin K1 peak was confirmed by UV absorption spectrophotometry and re-chromatography. The method has been applied to the determination of serum levels in normal healthy individuals and patients treated with vitamin K1.

Retention mechanisms of tetracyclines on a C8 reversed-phase material

Abstract The influence of eluent parameters on the retention of tetracyclines on a C8 reversed-phase column has been investigated. Optimal performance was obtained using eluents containing organic acids at relatively low pH, where the solutes mainly exist in their cationic forms. In contrast to earlier studies, which suggested that ionpair formation was the separation mechanism for tetracyclines, a more complex model is presented. Retention behaviour in the present system was not consistent with one single mechanism but was obviously caused by a mixture of phenomena. Essentially, several ionic interactions are likely to predominate over purely hydrophobic effects.

The effect of supplemental ascorbic acid in enriched live food for Clarias gariepinus larvae at startfeeding

Abstract The effect of three dietary ascorbic acid (AA) concentrations, each applied via two feed types, on production characteristics and physiological condition of African catfish ( Clarias gariepinus ) larvae has been assessed in two 10-day culture trials. Three treatments received only Artemia nauplii enriched with an experimental emulsion containing 0, 10, or 20% ascorbyl palmitate (AP) and yielding 530, 1200 and 1600 μg AA g −1 DW Artemia , respectively; the other three treatments were fed the same Artemia diets which were partially substituted by an artificial diet containing no vitamin C (ratio 20:80). No differences in survival could be observed; however, from day 6 onwards the 20%-AP group showed significantly better growth compared to the 0%- and 10%-AP treatments. For the cofeeding series, the same positive, but not significant, influence of vitamin C on dry weight was found. Moreover, the animals receiving the highest vitamin C supplementation displayed a considerably lower stress sensitivity than those of the 0%- and the 10%-AP groups, for both the 100%- and the 20%- Artemia series. These differences had occurred by day 2, which might be indicative of the importance of AA in early development. A second trial, which was a repetition of the first one, revealed the same tendencies; however, growth differences were smaller, probably due to the higher incorporation levels of AA obtained in the live diet (530, 1700 and 2300 μg AA g −1 DW) and in the catfish larvae. Growth results of both experiments were supported with data from the ultrastructural evaluation of the hepatocytes; i.e. a more organized cell compartmentation and better-structured cell organelles in the 20%-AP group of the Artemia series compared to the control are indicative of a more active metabolism. The slow growth in the cofeeding series was documented by the poor condition of the hepatocytes. In a third experiment it was verified that the growth effect of the 20%-AP boosted Artemia diet was the result of the extra AA incorporation and not of the concomitant palmitic acid (PA), which was set free after hydrolysis of AP in the Artemia nauplii, and which could possibly be used as a supplemental energy source. The three treatments were fed Artemia nauplii enriched with 0% AP, 12% PA and 20% AP, respectively. Growth and stress resistance of the latter group were significantly higher compared to the control and the PA-supplemented fish. To our knowledge this is the first evidence for the positive role of high dietary vitamin C levels (more than 1500 μg AA g −1 DW) on larval development of an aquaculture species, and more specifically of C. gariepinus .

Evaluation of vitamin C-enriched Artemia nauplii for larvae of the giant freshwater prawn

The effect of high levels of ascorbic acid (AA) delivered through enriched live food has been verified through the successful culture of larval giant freshwater prawn, Macrobrachium rosenbergii. Two successive feeding trials were set up using a control (550 Μg AA g−1 DW) and two different AA-enrichment levels in Artemia (1300 and 2750 Μg AA g−1 DW). Under standard culture conditions, no differences in growth nor survival could be observed demonstrating that the nutritional requirements are below 550 Μg AA g−1 DW, which is the normal level occurring in freshly-hatched Artemia. However, a significantly positive effect could be demonstrated on the physiological condition of the postlarvae, measured by means of a salinity stress test, when vitamin C-boosted live food was administered. Since the AA levels in the predator larvae are linked with the enrichment levels in the live prey, it may be assumed that a positive influence on stress resistance was caused by feeding vitamin C-enriched Artemia. It is expected that under suboptimal conditions, supplementation of high vitamin C levels might also enhance production characteristics.

Liquid chromatographic determination of efficacy of incorporation of trimethoprim and sulfamethoxazole in brine shrimp (Artemia spp.) used for prophylactic chemotherapy of fish.

The brine shrimp Artemia, an excellent live food source in aquaculture, has been studied as a carrier to deliver selected chemotherapeutic agents to fish for prophylactic treatment of infectious diseases. To monitor the efficiency of incorporation of trimethoprim and sulfamethoxazole in Artemia franciscana, a sensitive and specific analytical method was developed. It is based on homogenization of Artemia nauplii in methanol, extraction of lipids with hexane, solid-phase cleanup on C18 cartridges, and reversed-phase liquid chromatography with detection at 210 nm. The method is sensitive (detection limit, on the order of 3 micrograms/g with a sample quantity of 30 mg [dry weight]) and reproducible (coefficients of variation, 2.2 and 1.8% for trimethoprim and sulfamethoxazole at levels of 79.6 and 257 micrograms/g of body weight, respectively). Preliminary quantitative data indicated excellent uptake and persistence of both therapeutic agents in A. franciscana, with levels of 115 micrograms/g for trimethoprim and 277 micrograms/g for sulfamethoxazole.