Development and validation of a reversed-phase high-performance liquid chromatographic method for the quantitation and stability of α-lipoic acid in cosmetic creams.

OBJECTIVE: To develop and validate a simple reversed-phase HPLC method for the quantitation and evaluation of stability of α-lipoic acid in cosmetics, according to International Conference on Harmonization (ICH) Guidelines. METHODS: The chromatography was performed on a reversed-phase Luna C18, analytical column (150 × 4.6 mm id, 5 µm particle size) with a mobile phase of potassium dihydrogen phosphate (pΗ 4.5; 0.05 M) and acetonitrile (60:40, v/v) and a flow rate of 1.0 mL min-1 with UV detection at 340 nm. Accelerated and long-term stability studies of α-lipoic acid in cosmetic cream were conducted under various degradation conditions including acid, basis, oxidation, and thermal and photolytic degradation, according to European Medicines Agency Guidelines CPMP/ICH/2736/99. RESULTS: The limit of detection (LOD) for the cosmetic cream was 0.9 µg mL-1 and the limit of quantitation (LOQ) was 2.8 µg mL-1 , while the retention time was 7.2 min. The method proved to be linear, precise and accurate. The stability results demonstrated the selectivity of the proposed method to the analysis of α-LA, and the degradation products were determined and evaluated in specific stress conditions in cosmetic creams. The applicability of the method was tested in two different developed cosmetic products (cream with 1.5 % w/w and emulsion with 1.0 % w/w of LA) and proved to be reliable. CONCLUSION: A reversed-phase HPLC-UV method was developed and fully validated for the analysis of α-lipoic acid in cosmetics. It is the first reported application on the quantitation of lipoic acid in cosmetic creams, while at the same time evaluates the stability in forced degradation conditions, in new cosmetic formulations. It proved to be suitable for the reliable quality control of cosmetic products, with a run time of <8 min that allows for the analysis of large number of samples per day.

OBJECTIF: Développer et valider une méthode HPLC (chromatographie en phase liquide à haute performance) simple en phase inversée pour la quantification et l'évaluation de la stabilité de l'acide α-lipoïque dans les cosmétiques, conformément aux Directives de la Conférence internationale sur l'harmonisation (ICH). MÉTHODE: La chromatographie a été réalisée sur une colonne analytique Luna C18 en phase inversée (150 × 4,6 mm id, taille des particules 5 µm) avec une phase mobile de dihydrogénophosphate de potassium (pH 4,5 ; 0,05 M) et d'acétonitrile (60:40, v/v) et un débit de 1,0 ml min−1 avec détection UV à 340 nm. Des études de stabilité accélérée et à longterme de l'acide α-lipoïque dans les crèmes cosmétiques ont été menées dans diverses conditions de dégradation, notamment en milieu acide, basique, par oxydation et dégradation thermique et photolytique, conformément aux lignes directrices de l'Agence européenne des médicaments CPMP/ICH/2736/99. RÉSULTAT: La limite de détection (LD) pour la crème cosmétique était de 0,9 µg ml et la limite de quantification (LQ) était de 2,8 µml−1 , tandis que le temps de rétention était de 7,2 min. La méthode s'est avérée linéaire, précise et exacte. Les résultats de stabilité ont démontré la sélectivité de la méthode proposée pour l'analyse de l'acide α-lipoïque et les produits de dégradation ont été déterminés et évalués dans des conditions de stress spécifiques dans les crèmes cosmétiques. L'applicabilité de la méthode a été testée dans deux produits cosmétiques différents développés (crème avec 1,5 % p/p et émulsion avec 1,0 % p/p d'acide lipoïque) et s'est avérée fiable. CONCLUSION: une méthode HPLC en phase inversée avec détection UV a été développée et entièrement validée pour l'analyse de l'acide α-lipoïque dans les cosmétiques. Il s'agit de la première application signalée concernant la quantification de l'acide lipoïque dans les crèmes cosmétiques et permettant en même temps d'évaluer la stabilité des conditions de dégradation forcée dans les nouvelles formulations cosmétiques. Cette méthode s'est avérée adaptée au contrôle de qualité fiable des produits cosmétiques, avec une durée d'exécution < 8 min qui permet l'analyse d'un grand nombre d'échantillons par jour.

Biomarkers of opiate use.

The interpretation of toxicological findings is critical for the thorough investigation of the use and abuse of psychoactive substances. A positive analytical result for a sample taken could usually result in criminal proceedings and a punitive outcome for the defendant whose sample was analysed. The detection of markers of illicit opiate misuse is important both in the management of substance misuse and in the postmortem identification of illicit opiate use. The aim of this study was to emphasise the role of opiate biomarkers available at the laboratory and in the clinical environment. Urine remains the biological tool of choice for qualitative detection of illicit drug use in a clinical setting, while quantitative accuracy remains strictly the domain of blood. Accurate interpretation of the screening tests within a clinical setting alongside other relevant information remains the key to the usefulness of any test. Moreover, the finding of a morphine/codeine concentration ratio in blood exceeding unity is a strong evidence that the person had used heroin, as opposed to having taken a prescription analgesic drug containing codeine.

Forensic investigation of submersion deaths.

CONTEXT: The diagnosis of the cause and the establishment of the manner of death in submersion cases are routine challenge for forensic pathologists as it presents considerable diagnostic difficulties. OBJECTIVE: To concisely review the main questions during a submersion death investigation and to give the respective documented answers. DATA SOURCES: The search strategy included a literature search of PubMed, Medline and Google Scholar databases, as well as a review of the cited references by the identified studies and a hand search of relevant textbooks and reference works. CONCLUSIONS: A complete autopsy, histopathological examination and full toxicological screening are important to determine whether death indeed followed submersion in the water, or to see whether any natural disease or substance use have contributed or caused death. In ambiguous situations, the co-estimation of circumstantial evidence may be of invaluable importance toward the conclusion concerning the cause and the manner of death. The thorough forensic investigation of the submersion deaths not only serves the justice administration, but it also presents considerable benefits for the public health.

Fundamental issues in forensic semen detection.

Sperm detection can be an important factor in confirming sexual assault in cases of rape. A large number of cases received in a forensic laboratory involve sexual offenses, making it necessary to examine exhibits for the presence of seminal stains. The objective of this paper is to provide an overview of the most important methods and tests used in the identification of spermatozoa or constituents of seminal fluid during the investigation of alleged sexual assault cases in forensic medical practice. Furthermore, this paper focusses on the basic knowledge that is necessary to the graduate students who wish to specialize in forensic sciences.

Health impacts of fire smoke inhalation.

Most fatalities from fires are not due to burns, but are a result of inhalation of toxic gases produced during combustion. Fire produces a complex toxic environment involving flame, heat, oxygen depletion, smoke and toxic gases. As a wide variety of synthetic materials is used in buildings (insulation, furniture, carpeting, and decorative items) the potential for severe health impacts from inhalation of products of combustion during building fires is continuously increasing. In forest fires the burning of biomass leads to smoke emissions, the composition of which, as well as the relative health impacts from their inhalation, depends on the ecosystem's chemical and physical features and the local environmental parameters. In the present review, health problems that appear to people exposed to fire smoke and especially to firefighters are described and suggestions for fire management and prevention are made.

Do clinically insignificant tumors of the prostate exist?

BACKGROUND: The discrepancy between minimal disease on biopsy and disease found in the subsequent prostatectomy specimen, in terms of the size and grade of tumor, extracapsular extension or positive margins, led several authors to dispute the existence of clinically insignificant impalpable tumors of the prostate. However, considering that prostate-specific antigen (PSA) is an indicator of prostate malignancy and since many impalpable prostatic carcinomas (PCs) are detected by a combination of PSA, transurethral ultrasound and needle biopsy (T1c), in the era of PSA screening, it is expected that most of the impalpable tumors found incidentally at transurethral resection of the prostate (stage T1a/b), could be clinically insignificant. AIM: The aim of this study was to identify the characteristics of latent, impalpable PCs and to analyze the incidence of clinically insignificant PCs among hypothetical stage T1 prostate cancers in tumors found incidentally at postmortem examination. METHODS: We examined 40 cases of impalpable PCs found in 212 prostate autopsy specimens of men between 30 and 98 years of age who died of diseases other than carcinoma of the prostate and related conditions. RESULTS: Most of T1 histological PCs (57.5%) had a Gleason score between 2 and 4, while 30% had Gleason score between 5 and 6. Only 5 (12.5%) had a Gleason score above 7. Twenty-nine of 40 stage T1 histological cancers (67.5%) had volume of <1 cm(3). The highest volume tumors were those of intermediate and high grade (Gleason sums 5-8). Among tumors with volumes of <1 cm(3), 96.55% were confined within the prostatic capsule. CONCLUSIONS: The majority of impalpable PCs were low-volume, well-differentiated tumors corresponding to clinically insignificant neoplasms. Similar characteristics could be attributed to most of the impalpable carcinomas detected after prostatectomy in clinical practice.

An evaluation of dental methods by Lamendin and Prince and Ubelaker for estimation of adult age in a sample of modern Greeks.

Teeth can be used as accurate tools in age-at-death estimation in forensic cases. No previous data exist on estimating age from teeth in a modern Greek population. The aim of this study was to evaluate Lamendin's and Prince and Ubelaker's ageing methods on a modern Greek skeletal sample. In total, 1436 single-rooted teeth from 306 adult individuals (161 males and 145 females) were examined. Only measurements of periodontosis and translucency showed positive correlation with age. Results showed a bias - an overestimation for ages under 40 years and an underestimation over this age. However, the use of wider age groups proved to be more appropriate. Low values of error were observed for the group of middle-aged individuals. In conclusion, both methods can be considered accurate in estimating age-at-death of middle-aged individuals. This study provides more information about the accuracy and applicability of these dental methods on modern European populations.

Environmental aspects of VOCs evolved in the early stages of human decomposition.

In the present study, the time profile, measured as "accumulation", of volatile organic compounds (VOCs) produced during the early stages of human decomposition was investigated. A human cadaver was placed in a sealed bag at approximately the 4th day after death. Evolved VOCs were monitored for 24 h by sampling at different time intervals. VOCs produced were analyzed by thermal desorption/gas chromatography/mass spectrometry (TD/GC/MS). Over 30 substances were identified in total. These included mainly aliphatic and aromatic hydrocarbons, oxygenated compounds (alcohols, aldehydes, ketones) and organic sulfides. The last were the most prominent class of compounds identified. Eleven compounds were present in all the sampling cycles and constitute a "common core": ethanol, 2-propanone, dimethyl disulfide, methyl benzene, octane, 2-butanone, methyl ethyl disulfide, dimethyl trisulfide and o-, m- and p-xylenes. The last sampling cycle yielded the most abundant compounds in number and quantities. Inorganic gases such as CO2, CO, NH3 and H2S were also determined. The fundamental physicochemical properties of the evolved VOCs were used for evaluating their environmental impacts. It appears that the decay process, which is a dynamic procedure, can provide chemical signals that might be detected and properly evaluated by experts in the fields of forensic sciences, search and rescue units and environmental scientists.

Genotype-phenotype assessment in autosomal recessive arrhythmogenic right ventricular cardiomyopathy (Naxos disease) caused by a deletion in plakoglobin.

OBJECTIVES: The purpose of this study was to examine the genotype-phenotype relation with respect to penetrance, age and severity of expression, disease progression and prognosis in a recessively inherited arrhythmogenic right ventricular cardiomyopathy (ARVC). BACKGROUND: Naxos disease is a recessively inherited ARVC caused by a mutation in the gene encoding plakoglobin (cell adhesion protein) in which the cardiac phenotype is associated with palmoplantar keratoderma and woolly hair. METHODS: Twelve families with Naxos disease underwent cardiac and molecular genetic investigation. Serial cardiac assessment with annual resting 12-lead and 24-h ambulatory electrocardiogram (ECG) and two-dimensional echocardiography was performed during 1 to 16 years, median 7 +/- 6 years in all 78 surviving members. RESULTS: Twenty-eight surviving members were homozygous and 40 were heterozygous for the mutation. All adults who were homozygous (n = 26) fulfilled the diagnostic criteria for ARVC, the youngest by the age of 13 years. In eight who were heterozygous, minor ECG or echocardiographic abnormalities were observed. Of the 26 subjects who were affected homozygotes, 92% showed ECG abnormalities, 92% ventricular arrhythmias, 100% right ventricular structural alterations and 27% left ventricular involvement. During follow-up (10 +/- 6 years), 16 (62%) developed structural progression, 12 (46%) arrhythmic events and 7 (27%) heart failure. The annual disease-related and sudden death mortality was 3% and 2.3%, respectively. CONCLUSIONS: Autosomal recessive ARVC caused by a mutation in plakoglobin was 100% penetrant by adolescence. Affected subjects who were homozygous experienced progressive disease with adverse prognosis. A minority of subjects who were heterozygous showed minor ECG/echocardiographic changes, but clinically significant disease did not develop.

Adverse effects of anabolic steroids in athletes. A constant threat.

Anabolic-androgenic steroids (AAS) are used as ergogenic aids by athletes and non-athletes to enhance performance by augmenting muscular development and strength. AAS administration is often associated with various adverse effects that are generally dose related. High and multi-doses of AAS used for athletic enhancement can lead to serious and irreversible organ damage. Among the most common adverse effects of AAS are some degree of reduced fertility and gynecomastia in males and masculinization in women and children. Other adverse effects include hypertension and atherosclerosis, blood clotting, jaundice, hepatic neoplasms and carcinoma, tendon damage, psychiatric and behavioral disorders. More specifically, this article reviews the reproductive, hepatic, cardiovascular, hematological, cerebrovascular, musculoskeletal, endocrine, renal, immunologic and psychologic effects. Drug-prevention counseling to athletes is highlighted and the use of anabolic steroids is must be avoided, emphasizing that sports goals may be met within the framework of honest competition, free of doping substances.

A study of volatile organic compounds evolved from the decaying human body.

Two men were found dead near the island of Samos, Greece, in the Mediterranean sea. The estimated time of death for both victims was 3-4 weeks. Autopsy revealed no remarkable external injuries or acute poisoning. The exact cause of death remained unclear because the bodies had advanced decomposition. Volatile organic compounds (VOCs) evolved from these two corpses were determined by thermal desorption/gas chromatography/mass spectrometry analysis (TD/GC/MS). Over 80 substances have been identified and quantified. The most prominent among them were dimethyl disulfide (13.39 nmol/L), toluene (10.11 nmol/L), hexane (5.58 nmol/L), benzene 1,2,4-trimethyl (4.04 nmol/L), 2-propanone (3.84 nmol/L), 3-pentanone (3.59 nmol/L). Qualitative and quantitative differences among the evolved VOCs and CO2 mean concentration values might indicate different rates of decomposition between the two bodies. The study of the evolved VOCs appears to be a promising adjunct to the forensic pathologist as they may offer important information which can be used in his final evaluation.

Analysis of volatile organic compounds released from the decay of surrogate human models simulating victims of collapsed buildings by thermal desorption-comprehensive two-dimensional gas chromatography-time of flight mass spectrometry.

Field experiments were devised to mimic the entrapment conditions under the rubble of collapsed buildings aiming to investigate the evolution of volatile organic compounds (VOCs) during the early dead body decomposition stage. Three pig carcasses were placed inside concrete tunnels of a search and rescue (SAR) operational field terrain for simulating the entrapment environment after a building collapse. The experimental campaign employed both laboratory and on-site analytical methods running in parallel. The current work focuses only on the results of the laboratory method using thermal desorption coupled to comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (TD-GC×GC-TOF MS). The flow-modulated TD-GC×GC-TOF MS provided enhanced separation of the VOC profile and served as a reference method for the evaluation of the on-site analytical methods in the current experimental campaign. Bespoke software was used to deconvolve the VOC profile to extract as much information as possible into peak lists. In total, 288 unique VOCs were identified (i.e., not found in blank samples). The majority were aliphatics (172), aromatics (25) and nitrogen compounds (19), followed by ketones (17), esters (13), alcohols (12), aldehydes (11), sulfur (9), miscellaneous (8) and acid compounds (2). The TD-GC×GC-TOF MS proved to be a sensitive and powerful system for resolving the chemical puzzle of above-ground "scent of death".

Induction of metallothionein in the liver of carbon tetrachloride intoxicated rats: an immunohistochemical study.

Metallothioneins (MTs), are low molecular weight proteins, mainly implicated in metal ion detoxification. In the present study, we investigated the expression of hepatic MT in a rat model of injury and regeneration, induced by carbon tetrachloride (CCl(4)) administration. A single intraperitoneal injection of 1 ml CCl(4)/kg body weight was performed in male Wistar rats, killed at different time points post-administration. The enzymatic activities of aspartate and alanine aminotransferases in serum were determined, in addition to the liver histological findings, to estimate hepatotoxicity. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase in liver tissue and the assessment of the mitotic index in hepatocytes, were used as indices of regeneration. MT was detected immunohistochemically in liver tissue sections. CCl(4) administration caused severe hepatic injury, followed by regeneration. MT expression became prominent as early as 12 h after the administration of CCl(4), in the nuclei of hepatocytes, while at 24 and 36 h intense cytoplasmic staining for MT appeared in the hepatocytes in the vicinity of necrotic areas. The peak of hepatocyte proliferative capacity, occurring at 48 h post-CCl(4) administration coincides with the maximum nuclear and cytoplasmic MT expression. At further time points MT expression presented a decreasing trend. Induction of MT expression was observed in the liver after a single administration of CCl(4), being more prominent at the time of maximum hepatocellular proliferation, participating actively in the replication of hepatocytes.

Tumor histology and stage but not p53, Her2-neu or cathepsin-D expression are independent prognostic factors in breast cancer patients.

BACKGROUND: Several factors are currently employed for prognosis assessment and treatment determination in breast cancer. An array of molecular parameters, such as p53, Her2-neu (c-erbB 2) and Cathepsin-D, are also examined to improve clinical patient management. We have conducted a statistically powerful study of the prognostic value of conventional factors and of the investigational factors p53, Her2-neu and Cathepsin-D in patients with invasive breast carcinoma, in order to compare their significance. Our analysis was extended to determine the associations of p53 and Her2-neu with risk of death and relapse among patients with and without lymph node metastases. MATERIALS AND METHODS: In a set of 125 primary breast tumors, p53 and Her2-neu expression were immunohistochemically evaluated. Cathepsin-D, estrogen and progesterone receptor concentrations were determined in cytosols by a standard immunoradiometric assay. RESULTS: Over a mean of 62 months, 49 patients (39%) had a relapse and 29 patients (23%) died. Overexpression of p53, Her2-neu and Cathepsin-D was observed in 31%, 46% and 88% of cases, respectively. Overall survival was associated with histology (hazard ratio 0.04, 95% confidence interval: 0.01, 0.49 for lobular tumors) and stage (hazard ratio 5.94, 95% confidence interval: 1.30, 27.15 for stage III samples). Disease-free survival was also related to histology (hazard ratio 0.23, 95% confidence interval: 0.08, 0.73 for lobular tumors) and stage (hazard ratio 4.27, 95% confidence interval: 1.36, 13.36 for stage III tumors). Patients with both negative nodal status and Her2-neu overexpression tended to display an elevated risk of death. CONCLUSION: Our results support the prognostic power of tumor histology and stage and emphasize the need for further studies on the prognostic impact of p53. Her2-neu and Cathepsin-D in breast cancer. Additionally, our analysis indicates that deregulation of Her2-neu might characterize a subgroup of node-negative patients with poor prognosis who could benefit from an aggressive adjuvant therapy.

Liver metallothionein expression in thioacetamide-intoxicated rats.

Metallothioneins (MT), a group of ubiquitous low molecular weight proteins, implicated primarily in metal ion detoxification, are known to be expressed during hepatocellular proliferation after partial hepatectomy in rats. In the present study, we investigated the expression of MT in a rat model of liver injury and regeneration, induced by intraperitoneal administration of thioacetamide (TAA). The animals were killed at 0, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours after TAA administration. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of thymidine kinase, and the assessment of the mitotic index in hepatocytes were used as indices of liver regeneration. Liver MTs were detected immunohistochemically. TAA administration caused severe hepatic injury, followed by regeneration. MT expression became prominent in hepatocytes as early as 12 hours post-TAA administration. At 24 and 36 hours post-TAA administration intense nuclear and cytoplasmic staining of hepatocytes was found in the vicinity of necrotic areas. The maximal nuclear and cytoplasmic MT expression coincides with the peak of hepatocyte proliferative capacity, occurring at 48 and 60 hours post-TAA administration. MT expression correlated positively with the Zn content of liver tissue, but negatively with serum one, at the time of maximum hepatocyte proliferative capacity. This study suggests that MT participates in hepatocyte replication after toxin-induced liver injury.

The arterial communication between the gastrocnemius muscle heads: a fresh cadaveric study and clinical implications.

The purpose of this investigation was to describe the anatomy of the communicating (anastomotic) vessels between the gastrocnemius muscle heads and to record the extent of their supply potential. Ensuing clinical implications are discussed. Fourteen fresh cadaveric gastrocnemius muscles were examined. Detailed dissections of the communicating vessels were facilitated after injections of methylene blue or cadaveric blood solutions through the medial, lateral, or both sural arteries. The extent of the arterial cross-supply between the muscles' heads through these vessels was determined in-eight specimens after methylene blue perfusions through the lateral sural arteries, while one specimen was examined after injection of methylene blue and yellow ink through the lateral and medial sural arteries, respectively. Communicating vessels were detected in all 14 specimens. A mean number of 5.8 vascular bundles and single vessels was found. The bundles consisted of arterioles and, as all indications suggested, of concomitant venules as well. Regarding arterial cross-supply, it was clearly evident that each head could be vascularized solely from the contralateral one, mostly through these bundles. However, even if only a part of the bundles was preserved intact, vasculature was not affected.

The venous communication between the gastrocnemius muscle heads.

This investigation establishes the venous communication between the gastrocnemius muscle heads. Fourteen fresh (with the exception of one) cadaveric specimens were examined. Anastomotic veins were dissected along the raphe after perfusion of each muscle with 0.1 % methylene blue solution. A distally based musculocutaneous perforating vein of the medial head was the site of perfusion in 13 specimens, whereas one specimen received injection through the accompanying vein of the medial sural cutaneous nerve. Additional perfusion of dye from different sites was required in two specimens. Communicating veins were dissected in all 14 muscles. Direct anastomotic veins were detected in eight specimens; whereas in six the accompanying vein of the medial sural cutaneous nerve that had been included proved to be the intermediate pathway of venous communication between the muscle heads. Obstructive valves were encountered in most cases; nevertheless anastomotic veins were always recognized. The role of valves and the clinical implications are discussed.

The inferiorly based gastrocnemius muscle flap: anatomic aspects.

The arterial communication between the gastrocnemius muscle heads through their lowest anastomotic arteriole bundle alone was examined in specimens from 14 fresh cadavers. In 3 specimens, the larger vessels in close vicinity to the lowest vessels were preserved as well. Distinct communication between the arterial networks of the heads was demonstrated in all cases after injecting dyes through both sural arteries or into the lateral sural artery and the lowest anastomotic arteriole in 11 and 3 specimens, respectively. Therefore, it seems that one head can be adequately supplied from the contralateral one through their lowest anastomotic arteriole(s); nevertheless, the location of this vessel varies significantly and cannot be detected preoperatively. Measurements demonstrated that although this vessel is not found at a constant level, it is invariably detected in the lower third of the medial gastrocnemius head's length and, in 93 percent of cases, in the lower fourth. Thus, rough preoperative planning becomes feasible. Given that the venous communication between the heads has been documented as well, the authors think that an inferiorly based flap of the medial gastrocnemius head for defects of the middle third of the tibia might be both reliable and applicable; however, for reasons of safety, the muscle heads should remain attached along their lower third.