Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.

Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing β-amino acid residues - a lesson about the importance of animal experiments

Neurotensin(8-13) (NTS(8-13)) analogs with C- and/or N-terminal β-amino acid residues and three DOTA derivatives thereof have been synthesized (i.e., 1-6). A virtual docking experiment showed almost perfect fit of one of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives, 6a, into a crystallographically identified receptor NTSR1 (Fig.1). The affinities for the receptors of the NTS analogs and derivatives are low, when determined with cell-membrane homogenates, while, with NTSR1-exhibiting cancer tissues, affinities in the single-digit nanomolar range can be observed (Table 2). Most of the β-amino acid-containing NTS(8-13) analogs (Table 1 and Fig.2), including the (68) Ga complexes of the DOTA-substituted ones (6; Figs.2 and 5), are stable for ca. 1 h in human serum and plasma, and in murine plasma. The biodistributions of two (68) Ga complexes (of 6a and 6b) in HT29 tumor-bearing nude mice, in the absence and in the presence of a blocking compound, after 10, 30, and 60 min (Figs. 3 and 4) lead to the conclusion that the amount of specifically bound radioligand is rather low. This was confirmed by PET-imaging experiments with the tumor-bearing mice (Fig.6). Comparison of the in vitro plasma stability (after 1 h) with the ex vivo blood content (after 10-15 min) of the two (68) Ga complexes shows that they are rapidly cleaved in the animals (Fig.5).

Acute kidney injury in three dogs after ingestion of a descaling agent containing maleic acid

Maleic acid (MA) is a common component of descaling products and is widely used in daily life. Accidental ingestion in relevant amounts does not play a major role in human beings; however, it seems to be highly toxic for dogs. It has been commonly used experimentally to induce Fanconi syndrome in dogs or small rodents. Two dogs were presented for acute kidney injury (AKI) after accidental ingestion of a descaling agent containing MA at an estimated amount of 70 mg/kg each. The third dog involved was euthanased by the referring veterinarian, and postmortem pathological analysis revealed severe acute tubular necrosis consistent with toxic nephropathy. The other dogs received symptomatic therapy for AKI including treatment with haemodialysis and showed complete normalisation of serum creatinine at a follow-up after five months. Renal damage can be very severe, but seems to be at least partially reversible and an attempt to treatment is warranted.

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

Fatty acid composition, element concentration and isotope ratios of sea ice algae sampled in Rijpfjorden, Svalbard

The accelerating decrease of Arctic sea ice substantially changes the growth conditions for primary producers, particularly with respect to light. This affects the biochemical composition of sea ice algae, which are an essential high-quality food source for herbivores early in the season. Their high nutritional value is related to their content of polyunsaturated fatty acids (PUFAs), which play an important role for successful maturation, egg production, hatching and nauplii development in grazers. We followed the fatty acid composition of an assemblage of sea ice algae in a high Arctic fjord during spring from the early bloom stage to post bloom. Light conditions proved to be decisive in determining the nutritional quality of sea ice algae, and irradiance was negatively correlated with the relative amount of PUFAs. Algal PUFA content decreased on average by 40 % from April to June, while algal biomass (measured as particulate carbon, C) did not differ. This decrease was even more pronounced when algae were exposed to higher irradiances due to reduced snow cover. The ratio of chlorophyll a (chl a) to C, as well as the level of photoprotective pigments, confirmed a physiological adaptation to higher light levels in algae of poorer nutritional quality. We conclude that high irradiances are detrimental to sea ice algal food quality, and that the biochemical composition of sea ice algae is strongly dependent on growth conditions.

(Table 2) Fatty acid profile of blubber from harp seals and hooded seals from the Greenland Sea and Denmark Strait

Sub-Arctic marine ecosystems are some of the most productive ecosystems in the world's oceans. The capacity of herbivorous zooplankton, such as Calanus, to biosynthesize and store large amounts of lipids during the short and intense spring bloom is a fundamental adaptation which facilitates the large production in these ecosystems. These energy-rich lipids are rapidly transferred through the food chain to Arctic seals. The fatty acids and stable isotopes from harp seal (Phoca groenlandica) and hooded seal (Cystophora cristata) off East Greenland as well as their potential prey, were analysed. The results were used to describe the lipid dynamics and energy transfer in parts of the East Greenland ecosystem. Even if the two seal species showed considerable overlap in diet and occurred at relatively similar trophic levels, the fatty acid profiles indicated that the bases of the food chains of harp and hooded seals were different. The fatty acids of harp seals originate from diatom-based food chain, whereas the fatty acids of hooded seals originate from dinoflagellate and the prymnesiophyte Phaeocystis pouchetii-based food chain. Stable isotope analyses showed that both species are true carnivores on the top of their food chains, with hooded seal being slightly higher on the food chain than harp seal.

Fatty acid content of POM, sediment, foraminifer and peracarid samples obtained during POLARSTERN cruise ANT-XXIV/2 (PS71)

In order to investigate the diversity of diet composition in macrobenthic peracarid crustaceans from the Antarctic shelf and deep sea, the fatty acid (FA) composition of different species belonging to the orders Isopoda, Amphipoda, Cumacea and Tanaidacea was analysed. Multivariate analyses of the FA composition confirmed general differences between the orders, but also distinct differences within these orders. To gain information on the origin of the FAs found, the potential food sources sediment, POM and foraminiferans were included in the study. Most of the analysed amphipod species displayed high 18:1(n-9)-18:1(n-7) ratios, widely used as an indicator for a carnivorous component in the diet. Cumaceans were characterised by increased phytoplankton FA markers such as 20:5(n-3) (up to 29% of total FAs), suggesting a diet based on phytodetritus. High values of the FA 20:4(n-6) were found in some munnopsid isopods (up to 21% of total FAs) and some tanaidacean species (up to 19% of total FAs). 20:4(n-6) also occurred in high proportions in some foraminiferan samples (up to 21% of total fatty acids), but not in sediment and POM, possibly indicating the ingestion of foraminiferans by some peracarid crustaceans.

Amino acid geochronology of foraminifera test from north Queensland margin

In this study, we demonstrate the utility of amino acid geochronology based on single-foraminiferal tests in Quaternary sediment cores from the Queensland margin, Australia. The large planktonic foraminifer Pulleniatina obliquiloculata is ubiquitous in shelf, slope, and basin sediments of north Queensland as well as pantropical oceans. Fossil tests are resistant to dissolution, and retain substantial concentrations of amino acids (2-4 nmol/mg of shell) over hundreds of thousands of years. Amino acid D and L isomers of aspartic acid (Asp) and glutamic acid (Glu) were separated using reverse phase chromatography, which is sensitive enough to analyze individual foraminifera tests. In all, 462 Pulleniatina tests from 80 horizons in 11 cores exhibit a systematic increase in D/L ratios down core. D/L ratios were determined in 32 samples whose ages are known from AMS 14C analyses. In all cases, the Asp and Glu D/L ratios are concordant with 14C age. D/L ratios of equal-age samples are slightly lower for cores taken from deeper water sites, reflecting the sensitivity of the rate of racemization to bottom water temperature. Beyond the range of 14C dating, previously identified marine oxygen-isotope stage boundaries provide approximate ages of the sediments up to about 500,000 years. For this longer time frame, D/L ratios also vary systematically with isotope-correlated ages. The rate of racemization for Glu and Asp was modeled using power functions. These equations can be used to estimate ages of samples from the Queensland margin extending back at least 500,000 years. This analytical approach provides new opportunities for geochronological control necessary to understand fundamental sedimentary processes affecting a wide range of marine environments.

Seasonal fatty acid and fatty alcohol composition of Pseudocalanus minutus and Oithona similis in Kongsfjorden (Svalbard)

Seasonal lipid dynamics of various developmental stages were investigated in Pseudocalanus minutus and Oithona similis. For P. minutus, the dominance of 16:1(n?7), 16:4(n?3) and 20:5(n?3) fatty acids indicated a diatom-based nutrition in spring, whereas 22:6(n?3), 16:0, 18:2(n?6) and 18:1(n?9) pointed to a flagellate-based diet during the rest of the year as well as omnivorous/carnivorous low-level feeding during winter. The shorter-chain fatty alcohols 14:0 and 16:0 prevailed, also reflecting biosynthetic processes typical of omnivores or carnivores. Altogether, the lipid signatures characterized P. minutus as an opportunistic feeder. In contrast, O. similis had consistently high amounts of the 18:1(n?9) fatty acid in all stages and during all seasons pointing to a generally omnivorous/carnivorous/detritivorous diet. Furthermore, the fatty alcohol 20:1(n?9) reached high percentages especially in adult females and males, and feeding on Calanus faecal pellets is suggested. Fatty alcohols, as wax ester moieties, revealed significant seasonal variations in O. similis and a seasonal trend towards wax ester accumulation in autumn in P. minutus. P. minutus utilized its lipid deposits for development in the copepodite stages III and IV and for gonad maturation in CV and females during the dark season. However, CVs and females depended on the spring phytoplankton bloom for final maturation processes and reproduction. O. similis fueled gonad maturation and egg production for reproduction in June by wax esters, whereas reproduction in August/September co-occurred with the accumulation of new depot lipids. Both species revealed significantly higher wax ester levels in deeper (>50 m) as compared to surface (0-50 m) dwelling individuals related to a descent prior to overwintering.

Biometry, and fatty acid and fatty alcohol composition of the Arctic ctenophore Mertensia ovum from Kongsfjorden

Lipids of the Arctic ctenophore Mertensia ovum, collected from Kongsfjorden (Svalbard) in 2001, were analysed to investigate seasonal variability and fate of dietary lipids. Total lipids, lipid classes and fatty acid and alcohol compositions were determined in animals, which were selected according to age-group and season. Changes in lipids of age-group 0 animals were followed during growth from spring to autumn. Total lipids increased from May to September. Lipids as percentage of dry mass were lowest in August indicating their use for reproduction. Higher values occurred in September, which may be due to lipid storage for overwintering. Wax esters were the major lipid class accounting for about 50% of total lipids in age-group 0 animals from July and August. Phospholipids were the second largest lipid fraction with up to 46% in this age-group. The principal fatty acids of M. ovum from all age-groups were 22:6(n-3), 20:5(n-3) and 16:0. Wax ester fatty alcohols were dominated by 22:1(n-11) and 20:1(n-9) followed by moderate proportions of 16:0. The unique feature of M. ovum lipids was the high amount of free fatty alcohols originating probably from the dietary wax esters. In May, free alcohols exhibited the highest mean proportion with 14.6% in age-group 0 animals. We present the first data describing a detailed free fatty alcohol composition in zooplankton. This composition was very different from the alcohol composition of M. ovum wax esters because of the predominance of the long-chain monounsaturated 22:1 (n-11) alcohol accounting for almost 100% of total free alcohols in some samples. The detailed lipid composition clearly reflected feeding of M. ovum on the herbivorous calanoid species, Calanus glacialis and C. finmarchicus, the abundant members of the zooplankton community in Kongsfjorden. Other copepod species or prey items seem to be less important for M. ovum.

Station characteristics and copepod fatty alcohol, fatty acid and lipid composition during MSM02/4 in Fram Strait

The biodiversity of pelagic deep-sea ecosystems has received growing scientific interest in the last decade, especially in the framework of international marine biodiversity initiatives, such as Census of Marine Life (CoML). While a growing number of deep-sea zooplankton species has been identified and genetically characterized, little information is available on the mechanisms minimizing inter-specific competition and thus allowing closely related species to co-occur in the deep-sea pelagic realm. Focussing on the two dominant calanoid copepod families Euchaetidae and Aetideidae in Fram Strait, Arctic Ocean, the present study strives to characterize ecological niches of co-occurring species, with regard to vertical distribution, dietary composition as derived from lipid biomarkers, and trophic level on the basis of stable isotope signatures. Closely related species were usually restricted to different depth layers, resulting in a multi-layered vertical distribution pattern. Thus, vertical partitioning was an important mechanism to avoid inter-specific competition. Species occurring in the same depth strata usually belonged to different genera. They differed in fatty acid composition and trophic level, indicating different food preferences. Herbivorous Calanus represent major prey items for many omnivorous and carnivorous species throughout the water column. The seasonal and ontogenetic vertical migration of Calanus acts as a short-cut in food supply for pelagic deep-sea ecosystems in the Arctic.