Metabolism of the Synthetic Cathinone Alpha-Pyrrolidinoisohexanophenone in Humans Using UHPLC--MS-QToF.

Alpha-pyrrolidinoisohexanophenone (α-PHiP/α-PiHP) is a synthetic drug structurally related to cathine, a natural psychoactive alkaloid, isolated from Khat plant. The α-PiHP is a structural isomer of α-PHP, and both α-PHP and α-PiHP could be considered an analog of α-PVP, a Schedule I drug under the Convention on Psychotropic Substances by the United Nations. This α-pyrrolidinophenone was first reported to European Monitoring of Drug and Drug Addiction by Slovenia in December 2016. In Hungary, it was initially reported in August 2016, and until 2021, it had been detected in seizures only twice and never been identified in biological samples. However, in 2021, its consumption became prevalent in Hungary. This study aims to investigate the α-PiHP metabolites by performing in vitro and in vivo metabolite identification studies of human liver microsome (pHLM), S9 fraction (pS9) and urine samples (from control and users), using liquid chromatography in conjunction with high-resolution mass spectrometry. Ten in vivo urinary metabolites of α-PiHP were tentatively identified and confirmed by in vitro metabolites detected in pHLM and pS9 samples. Among the eight Phase I and the two Phase II metabolites, five were more abundant in urine than the parent compound. The two major metabolites via reduction of the keto moiety (M01) and via oxidation of the pyrrolidine ring combined with aliphatic hydroxylation and keto reduction (M06) were identified. The metabolites via the combination of keto reduction and aliphatic hydroxylation (M04), via ring-opening followed by carboxylation (M09) and via glucuronidation of the keto reduced metabolite (M07) were also dominant. The minor metabolites were one Phase II metabolite (M08), two metabolites via aliphatic hydroxylation (M02 and M03), one metabolite via the combination of keto reduction and oxidation of the pyrrolidine ring (M05) and one metabolite via oxidation of the pyrrolidine ring (M10).

Influence of body height on aortic systolic pressure augmentation and wave reflection in childhood.

The enhanced wave reflection in small children is a well-known phenomenon. It is explained on the basis of differences in the body height and the timing of wave reflection. This hypothesis still has not been proved directly. The aim of our study was to determine the reference values of aortic augmentation index (Aix(ao)) and the simultaneously measured return time of the systolic pulse wave (RT) in relation to the body height to test this hypothesis. Aix(ao) and RT were measured by Arteriograph in a healthy population aged 3-18 years (n = 4619, 2489 males). The Aix(ao) decreased with increasing age in boys from 18.6 ± 8.4% to 4.7 ± 4.3% and in girls from 22.3 ± 9.2% to 8.1 ± 5.1%, whereas the RT increased from 115.5 ± 16.3 ms to 166.7 ± 20.8 ms in boys and from 106.7 ± 21.9 ms to 158.1 ± 15.5 ms in girls. These changes were constant during childhood, but they slowed down after the onset of puberty. Because aortic pulse wave velocity (PWV(ao)) measured in the same population was unchanged during childhood, the increase of RT can only be explained by the increase of aortic length due to growth. In the puberty PWV(ao) starts increasing indicating that RT (Aix(ao)) does not follow the increase (decrease) of aortic length proportionally.

Determination of main tetrahydrocannabinoids by GC-MS: impact of protein precipitation by acetonitrile on solid phase extraction of cannabinoids from human serum.

The analysis of delta9-tetrahydrocannabinol (delta9-THC) and its main metabolites [11-hydroxy-delta9-tetrahydrocannabinol (11-OH-delta9-THC) and 11-nor-9-carboxy-delta9-tetrahydrocannabinol] in serum is a routine assay in forensic toxicology in the case of drivers influenced by Cannabis abuse and in other cases. Analysis of the specimen may involve protein precipitation, although there are authors who do not use this step. In this study we investigated the effect of acetonitrile as protein precipitant added to the serum on the absolute extraction recoveries of the analytes. This is very important not only from a forensic point of view, but also from the aspect of impact of delta9-THC therapy. Our results showed that in the case of spiked serum (2 ml), 80-87% extraction recovery can be achieved if 4 ml of acetonitrile is added before solid phase extraction. The second best result could be reached if no acetonitrile was added (64-73%). However, in the case of physiological sera of Cannabis consumers, no precipitation may be more advantageous in some cases. Matrix effects, which were studied by comparing the detectability and relative intensities of matrix peaks within the corresponding time windows of the analytes, were less influenced if the extraction was achieved with or without acetonitrile.

Detection of cannabigerol and its presumptive metabolite in human urine after Cannabis consumption.

Kemp et al. (1995) could detect delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol, three neutral cannabinoids, and the metabolites of delta9-THC in urine samples of Cannabis consumers. In this study we aimed to identify cannabigerol (CBG), which in its acid form is one of the main intermediate compounds of the biosynthesis of cannabinoids in hemp, in authority urine samples of proved Cannabis consumers. For this reason we applied the modified method of Kemp et al. to test for CBG, since enzymatic hydrolysis seems to be necessary for the formation of free neutral cannabinoids from conjugates. After extraction, derivatisation with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and GC/MS analysis, peaks of characteristic fragment ions (m/z 337, 391, 377 and 460) of bis-trimethylsilyl derivative of CBG appeared at 12.48 minutes in both real sample and the urine spiked with CBG. It shows that CBG enters the body during Cannabis smoking and is excreted with urine in a conjugated form, like other neutral cannabinoids. Analysing the chromatograms of hydrolysed and trimethylsilylated extracts we checked for the presence of CBG-metabolites based on the study of Harvey and Brown (1990). We detected a compound in the Cannabis consumers' urine extracts, having fragment ions at m/z 425, 465 and 479 at the retention time of 14.19 min which is presumed to be the 4"-hydroxy-CBG or 5"-hydroxy-CBG. However, it could not be identified completely by GC/MS. This peak was absent in non-hydrolysed urine samples, indicating that it is also excreted in glucuronated form.

Different reactivities of amphetamines with N-methyl-bis(trifluoroacetamide) in heated gas chromatographic injectors.

A fast gas chromatographic mass spectrometric method has been developed earlier for the determination of amphetamine derivatives in human serum and urine. For derivatization, N-methyl-bis(trifluoroacetamide) (MBTFA) was used. Derivatization was performed using an on-line mode, since 1 microl of MBTFA and 1 microl sample extract, dissolved in toluene were injected simultaneously. In this study, the reactivity of the several amphetamine type analytes with MBTFA was investigated. MBTFA used for flash derivatization was applied undiluted on the one hand and diluted 4--4096-fold with acetonitrile on the other hand. Studying several amphetamines in the test sample spiked at the same concentrations we found that they could be divided into 3 groups based on relative target ion peak areas as a function of MBTFA dilution. Group 1, containing only primary amines showed an early increase of the relative peak areas if we increased MBTFA concentration, where group 2 (mainly N-methyl secondary amines) showed that relative peak areas started to increase intensively at higher MBTFA concentrations. Finally, MDEA as an N-ethyl secondary amine, representing group 3, showed significant increase if only slightly diluted MBTFA was used as a flash reagent. This phenomenon can be explained mainly with the less and less reactivity of amine groups in the case of groups 2 and 3, compared to group 1. These findings could help to optimise analytical methods involving flash derivatization processes.

Piperazine-like compounds: a new group of designer drugs-of-abuse on the European market.

1-Aryl-piperazine compounds are, depending on their substituents, selective for certain serotonin receptors and together with their easy availability and their so-called legal status, this group of psychoactive compounds are potential designer drugs-of-abuse. Internet in that respect is an important source of information and distribution facilities. Because this development may have consequences for the interpretation of future clinical and forensic toxicological case studies, some analytical aspects of 1-benzyl-piperazine (BZP), 1-[4-methoxyphenyl]-piperazine (pMeOPP) and 1-[3-trifluoromethylphenyl]-piperazine (TFMPP) were studied. BZP was not detected by the AxSYM FPIA technology designed to determine amphetamine-like compounds, but had showed some cross reactivity with EMIT d.a.u.. The cross reactivities at 300 and 12,000ng/ml (RS)-amphetamine equivalents were 0.4 and 1.3%, respectively. Although BZP was not identified directly by the REMEDi HS Drug Profiling System, it can be detected by this HPLC/UV scanning system. Using GC/NPD without derivatisation, BZP, pMeOPP and TFMPP can be analysed for and applying GC/MS without or with acetylation or trifluoroacetylation, these compounds can be identified unambiguously. The usefulness of GC/NPD and GC/MS in this respect was demonstrated by the quantitative and qualitative analysis of the content of a capsule with the synthetic stimulant A2, which proved to contain 86.4mg of BZP.

Apoptotic and mitotic activity in squamous cell carcinoma cells after combined modality treatment with gamma-irradiation and dibromodulcitol.

A-431 squamous cell carcinoma cells were treated in vitro with either 4 Gy radiation of 15 (or 45) microg/ml dibromodulcitol (DBD), as well as with combined 4 Gy irradiation and DBD, with the latter as either a pretreatment or post-treatment. DBD alone or in combination with radiation had a greater effect on cell proliferation than the effect of radiation alone. The difference is due to a higher level of apoptosis induced by DBD, especially in conjunction with radiation. Such a combination may therefore be useful in the treatment of squamous cell carcinoma, which in general responds poorly to radiation therapy.

Higher serum eosinophil cationic protein levels in children with cow's milk allergy.

BACKGROUND: In the pathogenesis of cow's milk allergy, abnormal immunologically mediated reactions play a basic role. Eosinophil activation also participates in the development of several allergies. The purpose of this study was to characterize the degree of this activation by measuring the serum level of eosinophil cationic protein (sECP) and establishing whether it is a useful parameter in monitoring oral cow's milk allergy. METHODS: The sECP level of 35 patients with previously confirmed cow's milk allergy (mean age, 16 months) was evaluated using a fluoroimmunoassay before the cow's milk rechallenge test and at 2 hours and 24 hours after cow's milk challenge. RESULTS: Of the 35 children with previously confirmed cow's milk allergy, 10 had positive clinical reactions after the milk rechallenge test, whereas 25 children had no reaction. The median sECP level of all the patients before the challenge test was significantly higher (12.4 microg/L) than that of the control group (4.3 microg/L) (P < 0.05). Two hours after the challenge, the median sECP of all patients (9.4 microg/L) was lower than the starting values. The median sECP levels were higher in children with positive challenge test results at all time points. However, this difference was not statistically significant. CONCLUSIONS: The normalization of sECP level may indicate the cessation of the cow's milk allergy. Therefore, the measurement of sECP may be helpful in determining the optimal time in which to repeat the challenge test, when the result will more likely be negative. The significant decrease of the sECP level 2 hours after the beginning of milk challenge test may be explained by the fact that this protein is excreted into the intestinal lumen.

[Serum eosinophil cationic protein (sECP) level after cow's milk challenge test in allergic children]. / A szérum eosinophil kationos protein szintjének változása tehéntejterhelést követóen allergiás gyermekekben.

The eosinophil cationic protein (ECP) level in the sera is higher in allergic diseases. The change of the ECP level in the sera of patients with cow milk allergy was examined during the cow's milk challenge test after a long cow's milk free diet period. The sECP level of 35 milk sensitive patients was determined by a fluoroimmunoassay (Pharmacia CAP System ECP FEIA) before the milk challenge test, as well as 2 and 24 hours after it. The average age of the patients was 16 (6-49) months. The basic sECP level of cow's milk allergic patients was significantly higher (12.2 micrograms/l vs. 7.0 micrograms/l, p < 0.05) than that of the control group (n = 20). The sECP level significantly decreased 2 hours after the milk challenge test (12.2 micrograms/l vs. 9.2 micrograms/l, p = 0.01), 24 hours after the challenge it was again on the basic level (11.2 micrograms/l, p = 0.26). Out of the 35 allergic children ten had positive clinical reactions after the milk challenge test (positive group), while 25 had no reaction after it (negative group). Comparing the sECP level of these two groups, no significant difference was found in the sECP level either before the milk challenge test or after it. The significant decrease of the sECP level 2 hours after the beginning of milk challenge test might be explained by the fact that this toxic protein is secreted into the bowel.

Carcinogenic alterations in murine liver, lung, and uterine tumors induced by in utero exposure to ionizing radiation.

The atomic bombing of Hiroshima and Nagasaki and the nuclear accident at Chernobyl raised the question of prenatal sensitivity to ionizing radiation-induced cancer. In this study, mice were exposed to single doses of gamma-radiation (0.2-2.0 Gy) at different embryonic stages. The tumor incidence increased with dose from 15% in control mice to 35% in mice irradiated with 2.0 Gy on 18 d of prenatal life. Various oncogenic events were investigated in lymphoid, liver, lung, and uterine tumors. We observed threefold to fivefold increases in myc expression in 25% of the lymphomas, and the expression of Ha-ras and p53 genes decreased in 40% and 60% of the lung tumors by twofold to fivefold. Point mutations were tissue specific: Ha-ras codon 61 mutations were found in about 40% of the liver adenocarcinomas, Ki-ras codon 12 mutations in about 17% of lung tumors, and p53 mutations in about 15% of the lymphomas. Amplification and rearrangement of the p53, myc, and Ha-, Ki- and N-ras genes were not detected. Loss of heterozygosity on chromosome 4 at the multiple tumor suppressor 1 and 2 genes was observed in all types of malignancies. Allelic losses on chromosome 11 at the p53 locus were found in lymphoid, liver, and lung tumors, but they were absent from uterine tumors. Multiple oncogenic changes were often detected. The frequency of carcinogenic alterations was similar in spontaneous and radiation-induced lymphoid, liver, and uterine tumors. In radiation-induced lung adenocarcinomas, however, the incidences of many oncogenic changes were different from those found in their spontaneous counterparts. This suggests that different oncogenic pathways are activated during spontaneous and in utero gamma-radiation-induced murine lung carcinogenesis.

Oncogenic changes in murine lymphoid tumors induced by in utero exposure to ionizing radiation.

We have investigated the oncogenic alterations in murine lymphomas induced by in utero exposure to gamma-radiation. The expression of the myc oncogene increased in 23% of the tumors. Alterations in the expression of the ras oncogenes and in the p53 tumor suppressor gene were not characteristic. The p53 gene was mutated in a low percentage of the tumors (12%). Ras mutations were not detected. Loss of heterozygosity (LOH) at the p53 locus was found in 30% of the tumors, and LOH at the mts tumor suppressor gene was detected in 23% of lymphomas. Multiple oncogenic changes were infrequent in the investigated tumors. There were no essential differences in the frequency of carcinogenic alterations in spontaneous and gamma-radiation-induced lymphomas.

[Simultaneous occurrence of ulcerative colitis and psoriasis in childhood]. / Colitis ulcerosa és psoriasis együttes elöfordulása gyermekkorban.

Authors report of a 10 years old girl who at age 2 showed signs of psoriasis and after 8 years typical symptoms of ulcerative colitis manifested. In connection with this case the association of two diseases is emphasized which may be explained by the similarity of genetic and immunological factors in both disorder. It is accentuated that at the observation of enteral symptoms in patients with psoriasis the possibility of non specific inflammatory bowel disease has also to be considered.

Palmar lunate transtriquetral fracture dislocation.

A palmar lunate transtriquetral fracture dislocation with a concomitant radial styloid avulsion fracture has not been described before in the literature. This injury represents an interesting variation of stage IV perilunar instability. Treatment was complicated by persistent scapholunate dissociation (rotary subluxation of the scaphoid) after attempted closed reduction and percutaneous pinning. At open reduction, the proximal half of the triquetrum, which had been dislocated palmarward with the lunate and which had been thought to be reduced after our attempted closed reduction was indeed returned to its normal position. However, it was rotated 180 degrees on its transverse axis. Restoration of the normal scapholunate interval was not possible until the triquetral fracture was reduced.

Protein phosphorylation and kinase activities in tumour cells after hyperthermia.

Phosphorylation of various proteins and the activities of specific kinases were studied in tumour cells after hyperthermia. P388 lymphoid tumour cells were treated at 40-45 degrees C for 1 h in vitro. Immediately after heat treatment, particulate and cytosol cell fractions were isolated, phosphorylated proteins separated and various kinase activities were measured. Hyperthermic treatment of the cells caused a significant decrease in protein kinase C activity while the activity of calcium-ion and phospholipid-independent protein kinases increased. Phosphorylation of cytosol proteins of 120, 80, 33, 25 and 14 kDa increased significantly after hyperthermia, and protein kinase C selectively phosphorylated the last three of these proteins. The phosphorylation of three heat shock proteins (44, 70 and 85 kDa) was not changed after hyperthermic treatment. Four tyrosine kinase activities were separated. The protein tyrosine kinase activity decreased to one-tenth of the control value after 45 degrees C for 1 h hyperthermia. The changes in kinase activities and protein phosphorylation induced by hyperthermia proved to be temperature- and time-dependent.

Effect of hyperthermia and X-irradiation on survival and occurrence of metastases in mice bearing P388 tumor.

Survival of P388 lymphoid tumor-bearing mice and the occurrence of metastasis was studied after combined modality treatment with hyperthermia and X-irradiation. P388 ascites tumor cells were treated at 42 degrees C or 43.5 degrees C for 1 hr in vitro and transplanted on B6D2F1 mice intraperitoneally (i.p.) or intramuscularly (i.m.). Hyperthermic treatment at 43.5 degrees C increased the median survival time (MST). Increased life-span (ILS) was found after i.p. transplantation (54%) and after i.m. transplantation (30%). During the life-span of tumor-bearing animals, significantly fewer metastases were observed in liver and spleen after hyperthermia and 5-10% metastasis occurred after transplantation of ascites tumor cells treated at 43.5 degrees C in vitro compared with 90% in the untreated control animals. The lower occurrence of metastasis could not be ascribed to the higher cell-killing effect of hyperthermia. When both modalities were combined the best tumor growth retardation effect was obtained when ascites tumor cells were treated at 43.5 degrees C for 1 hr before being transplanted i.m. and 1 day later locally X-irradiated with 6 Gy. In this case, 77% ILS was found demonstrating a synergistic effect of the two modalities. While X-irradiation alone did not change the occurrence of metastasis, after combined modality treatment it was as low as with hyperthermia alone (5-10%). In connection with the significantly lower occurrence of metastasis, the possible alterations of P388 tumor cell membrane and surface proteins induced by in vitro hyperthermic treatment are discussed.

Transcription of human ribosomal DNA may terminate at multiple sites.

The termination of human pre-rRNA transcription has been investigated. The most abundant possible termination site was detected 360 bp downstream from the 28S gene, in front of the first SalI box of the rDNA spacer. This site, however, is partially bypassed during transcription, and three additional termination points were detected inside the heterogeneous region of the rDNA spacer. Later sites were mapped about 930, 1030 and 1110 bp downstream from the 3' end of the 28S rRNA gene. The authors suggest that the T clusters and pyrimidine-rich regions play an important role in the termination processes. They either may influence the efficiency of the SalI boxes in terminating the synthesis of pre-rRNAs or may serve as independent signals for the fail-safe termination of readthrough transcripts. In both cases transcription of human rDNA ceases at multiple sites.

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.