Davorka Sutlović

Interaction of humic acids with human DNA: Proposed mechanisms and kinetics

Human DNA quantification by quantitative real‐time PCR (QRT‐PCR) has gained great importance in forensic DNA and ancient DNA studies. However, in such samples, DNA quantification is impaired by the frequently present humic acid (HA). We have previously shown that the addition of synthetic HA inhibits QRT‐PCR. In this study we investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT‐PCR inhibition. In QRT‐PCR with pure human DNA and no HA added, VMAX was 40. With DNA sample containing 4 μg/mL of HA, VMAX was 30.30 while the addition of extra Taq polymerase to the same sample changed VMAX into 38.91, amplifying between 80 and 90% of input DNA. The KM/VMAX ratio in all the samples remained constant, indicating that the mechanism of HA inhibition of QRT‐PCR is uncompetitive by nature. Moreover, HA shifts the human DNA melting temperature point (Tm) from 75 to 87°C and inhibits DNase I‐mediated DNA cleavage, most probably affecting the enzymes activity.

Acute, food-induced moderate elevation of plasma uric acid protects against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans.

We examined the effects of acute, food-induced moderate increase of plasma uric acid (UA) on arterial stiffness and markers of oxidative damage in plasma in healthy males exposed to 100% normobaric oxygen. Acute elevation of plasma UA was induced by consumption of red wine, combination of ethanol and glycerol, or fructose. By using these beverages we were able to separate the effects of UA, wine polyphenols and ethanol. Water was used as a control beverage. Ten males randomly consumed test beverages in a cross-over design over the period of 4 weeks, one beverage per week. They breathed 100% O(2) between 60(th) and 90(th)min of the 4-h study protocol. Pulse wave augmentation index (AIx) at brachial and radial arteries, plasma antioxidant capacity (AOC), thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides (LOOH) assessed by xylenol orange method, UA and blood ethanol concentrations were determined before and 60, 90, 120, 150 and 240 min after beverage consumption. Consumption of the beverages did not affect the AIx, TBARS or LOOH values during 60 min before exposure to hyperoxia, while AOC and plasma UA increased except in the water group. Significant increase of AIx, plasma TBARS and LOOH, which occurred during 30 min of hyperoxia in the water group, was largely prevented in the groups that consumed red wine, glycerol+ethanol or fructose. In contrast to chronic hyperuricemia, generally considered as a risk factor for cardiovascular diseases and metabolic syndrome, acute increase of UA acts protectively against hyperoxia-induced oxidative stress and related increase of arterial stiffness in large peripheral arteries.

Comparison of acute effects of red wine, beer and vodka against hyperoxia-induced oxidative stress and increase in arterial stiffness in healthy humans

OBJECTIVE We determined and compared acute effects of different alcoholic beverages on oxygen-induced increase in oxidative stress plasma marker and arterial stiffness in healthy humans. METHODS Ten males randomly consumed one of four tested beverages: red wine (RW), vodka, beer (0.32 g ethanol/kg body wt) and water as control. Every beverage was consumed once, a week apart, in a cross-over design. The volunteers breathed 100% normobaric O(2) between 60th and 90th min of 3h study protocol. Plasma lipid peroxides (LOOH) and uric acid (UA) concentration, blood alcohol concentration (BAC) and arterial stiffness (indicated by augmentation index, AIx) were measured before and 30, 60, 90, 120 and 180 min after beverage consumption. RESULTS Intake of all alcoholic beverages caused a similar increase of BAC. The oxygen-induced elevation in AIx was similarly reduced in all three groups relative to the control (3.4 ± 1.3%, 5.4 ± 2.2% and 0.2 ± 1.6% vs. 13.7 ± 2.6% for red wine, vodka, beer and control, respectively, 60 min after intake). Exposure to oxygen resulted in increased plasma LOOH in all groups. However, in RW group this increase was lowest (1.1 ± 0.5) in comparison to the vodka (2.1 ± 0.5), beer (1.6±0.3) and control (2.5 ± 0.4μM/L H(2)O(2)). 60 min after intake of RW and beer plasma UA significantly increased (34 ± 4 and 15 ± 3) in contrast to vodka and control (-6 ± 2 and -8 ± 2μmol/L). CONCLUSION All three alcoholic beverages provided similar protection against oxygen-induced increase in arterial stiffness, probably due to central vasodilatatory effect of alcohol itself, but only RW provided protection against oxygen-induced oxidative stress.

Antimicrobial Effects of Wine: Separating the Role of Polyphenols, pH, Ethanol, and Other Wine Components

While the antimicrobial effectiveness of wine is well documented, relative contributions of the wine components to its antimicrobial activity is controversial. To separate the role of wine phenolics, ethanol, and pH from other wine constituents, the antimicrobial effects of intact wine were compared to that of phenols-stripped wine, dealcoholized wine, ethanol, and low pH applied separately and in combination, against 2 common foodborne pathogens, Salmonella enterica serovar Enteritidis and Escherichia coli. All samples were biochemically characterized with respect to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, and pH. Antioxidative activity of the samples corresponded to their total phenolics content. Except for respective controls, pH and ethanol content were similar in all samples. The order of antibacterial activity of the samples was: intact wine > phenols-stripped wine > dealcoholized wine > combination of ethanol and low pH > low pH > ethanol. Separate application of ethanol or low pH showed negligible antibacterial activity while their combination showed synergistic effect. Antibacterial activity of the samples could not be related to their total phenolics and resveratrol content, antioxidant capacity, ethanol content, or pH. Our study indicates that antimicrobial activity of complex solutions such as intact wine cannot be exclusively attributed to its phenolic or nonphenolic constituents, nor can the antimicrobial activity of wine be predicted on the basis of its particular components.

Discovering the 60 years old secret: identification of the World War II mass grave victims from the island of Daksa near Dubrovnik, Croatia.

Aim To describe the organization, field work, forensic anthropological examination, and DNA analysis conducted to identify the victims from a World War II mass grave found on the Dalmatian island of Daksa near Dubrovnik (Croatia) in 2009. Methods Excavation of the site was performed according to standard archeological procedures. Basic anthropological examination was made to determine the minimum number of victims, sex, age at death, and height. The bones with pathological and traumatic changes were identified. DNA was extracted from powdered bones and relatives’ blood samples. Y-chromosome and autosomal short tandem repeats (STR) were used to establish the relationship of the remains with the putative family members. Results The remains were found to belong to at least 53 distinctive victims. All were male, mostly with gunshot wounds to the head. DNA analysis and cross-matching of the samples with relatives resulted in 14 positive identifications using the Y-chromosomal STRs and 4 positive identifications using the autosomal STRs. Conclusions This study showed that even in cases of more than 50-year-old, highly degraded human remains from mass graves, Y-chromosomal and autosomal STRs analysis can contribute to identification of the victims.

Suicide by fentanyl.

Suicide by Fentanyl Fentanyl is a potent, short-acting narcotic analgesic widely used as surgical anaesthetic. This article presents a case in which fentanyl was self-injected by a 41-year old nurse, an employee at the hospital emergency department, who was found dead at home. She had no known history of drug and alcohol abuse. Two syringes, one empty and one filled with a clear liquid, were found near the body, while a needle was stuck into her hand. Toxicological analysis showed fentanyl poisoning. Fentanyl overdose was declared the cause of death and the manner of death was classified as suicide. To our knowledge, death due to the intravenous injection of fentanyl has not previously been reported in Croatia. Samoubojstvo Fentanilom Fentanil je snažan narkotik u velikoj mjeri upotrebljavan kao anestetik pri kirurškim zahvatima. U ovom radu prikazan je slučaj samoubojstva upotrebom fentanila. 41-godišnja medicinska sestra, zaposlena u hitnoj službi pronađena je mrtva u svom stanu. Nije bilo podataka o njezinu zaživotnom uživanju alkohola niti drugih sredstava ovisnosti. Dvije šprice, jedna prazna i druga ispunjena prozirnom tekućinom, pronađene su kraj njezina tijela, dok je igla bila ubodena u njezinu ruku. Toksikološka analiza dala je pozitivan rezultat na fentanil. Uzrok smrti bio je predoziranje fentanilom u svrhu samoubojstva. Prema našim saznanjima, ovo je prvi dokazani slučaj samoubojstva u Hrvatskoj koji je počinjen fentanilom.

Drug analysis in necrophagous flies and human tissues.

Drug Analysis in Necrophagous Flies and Human Tissues Necrophagous insects may provide useful information about the time, place and cause of death. In addition, they can serve as reliable alternative specimens for toxicological analysis in cases where human tissue and fluids, normally taken during autopsies, are not available, due to decomposition of the corpse. This paper reports the results of drug analysis of the larvae of two fly families, Calliphoridae and Sarcophagidae, collected from the body of a middle-aged man who had committed suicide approximately three weeks before his corpse was found. Multiple samples of decomposed human tissue, of the blowfly, and of the larval flesh were analysed using gas chromatography/mass spectrometry (GC/MS), and amphetamine was detected in all samples. While the screening results were beyond doubt, the quantitative analysis was less clear, and further research is needed in this area. Analiza Droga U Nekrofilnim Muhama I Ljudskim Tkivima Insekti strvinari mogu pružiti korisne informacije o vremenu, mjestu i uzroku smrti. Dodatno, oni mogu poslužiti kao pouzdani zamjenski uzorci za toksikološke analize u slučajevima kada ljudska tkiva i tekućine, koji se uobičajeno uzimaju tijekom obdukcije, ne postoje zbog raspadanja trupla. Ovaj rad prikazuje rezultate analize droga u uzorcima ličinki dviju porodica muha (Diptera: Calliphoridae i Sarcophagidae), prikupljenima s tijela muškarca srednje Životne dobi koji je počinio samoubojstvo otprilike 3 tjedna prije pronalaska njegova mrtvog tijela. Višestruki uzorci raspadajućeg ljudskog tkiva i ličinki muha analizirani su uporabom plinske kromatografije uz detekciju spektrometrijom masa (GC/MS) i u svima su otkriveni amfetamini. Dok su rezultati preliminarne kvalitativne analize bili izvan svake sumnje, kvantitativni su rezultati bili manje jasni te se preporučuje daljnje istraživanje u ovom području.

The Role of Alcohol in Road Traffic Accidents With Fatal Outcome: 10-Year Period in Croatia Split–Dalmatia County

Objective: The aim of the study was to investigate the relationship between alcohol consumption and fatal road traffic accidents that took place in Split–Dalmatia County (Croatia) during a 10-year period (from August 2001 to August 2011). Methods: A retrospective analysis of 474 autopsy reports was performed, with an emphasis on 337 toxicologal findings of blood alcohol concentration (BAC) and the mode of participation in traffic (driver, passenger, pedestrian, or cyclist). Relations between the BAC, the weekday on which death occurred, victims age and gender, and differences according to 3 successive legislation periods within the observed time frame were analyzed. The BAC was measured by gas chromatography with headspace and flame ionization detection. Results: Alcohol was regarded as a contributive death factor if BAC was positive; that is, higher than 0.5 g/kg. BAC was positive in 177 cases. Most often it was the car driver who had a positive BAC. Victims of traffic accidents were mostly male drivers, and those accidents were more often associated with alcohol consumption. Consumption of alcohol produced a significant increase in culpability. Conclusion: The results of our study show that alcohol remains one of the main contributing factors of traffic accidents in Split–Dalmatia County. Unfortunately, legislation changes regarding the allowed BAC did not cause any reduction in casualties among drivers driving under the influence of alcohol (DUIA). Chi-square test of the number of total driver victims and driver victims who were DUIA revealed no significant difference (P = .173).

Blood alcohol stability in postmortem blood samples.

AbstractThe effect of long-term storage on alcohol stability in postmortem blood samples was investigated. Seventy-nine blood samples were used in this study. For the first time, blood alcohol concentrations were measured within 1 to 4 days after being taken at the Laboratory of Forensic Toxicology. After storage (ranging from 191 to 468 days), each sample was reanalyzed for blood alcohol concentration, and results were compared with the original analysis.Results showed good agreement between the experimental measurements. About 90% of the results lie within 95% limits, and 10% were outside. Despite these positive correlations, the results of some blood samples showed a high variation in ethanol concentration. Deviation in alcohol concentration between replicates of up to 10% (observed in 39% samples) is not acceptable as precise forensic evidence.

Fatal Poisoning By Alcohol and Heroin

Fatal Poisoning By Alcohol and Heroin Drug abuse with alcohol consumption have been on the rise in Split-Dalmatian County for a while now. This article reports two separate cases with three deaths due to fatal combinations of heroin and alcohol. The first case of poisoning is related to a young couple, a 30-year-old man and a 28-year-old woman, who were found dead in a car, surrounded by cans of a variety alcoholic drinks. Two needles were found beside the bodies as well. The victims were registered drug abusers who had been in withdrawal programs. The second case was a 29-year-old man who was found dead in a house. Three fresh injection marks were visible on his right arm, and two needles were near his body. He was not known as a drug addict, but he had tried to commit suicide recently. Carboxyhaemoglobin was found in blood samples of both victims from the first case. The concentration was 25 % and that could contribute to their death. In both described cases blood alcohol concentration was higher then 1.60 g kg-1. Toxicology tests were positive for heroin, meconin, acetaminophen, 6-acetylmorphine, codeine, noscapine and papaverine. Ethanol, being a respiratory depressant, combined with morphine drastically increases the risk of rapid death due to respiration failure. Kobna Intoksikacija Alkoholom I Heroinom U posljednje vrijeme u Splitsko-dalmatinskoj županiji konzumiranje opojnih droga uz alkohol je u porastu. Ovim radom prikazali smo dva slučaja s tri smrtno stradale osobe koje su bile kobne žrtve kombinacije alkohola i heroina. U prvom slučaju, mladi par, u dobi od 30 i 28 godina, pronađen je u automobilu okružen limenkama raznih alkoholnih pića. Dvije šprice nađene su kraj tijela. Žrtve su bili registrirani ovisnici i na programima odvikavanja u organiziranim tretmanima. Drugi slučaj bio je 29-godišnji muškarac pronađen mrtav u napuštenoj kući. Na njegovoj desnoj ruci primijećene su tri svježe ubodne rane, a dvije šprice pronađene su kraj tijela. Nije bio poznat kao ovisnik o drogama, ali je nedavno pokušao samoubojstvo. Karboksihemoglobin nađen je u uzorcima krvi obiju žrtava iz prvog slučaja. Koncentracija je bila 25 %, što je moglo pridonijeti njihovoj smrti. U oba opisana slučaja maseni udjel alkohola u krvi bilo je veći od 1.60 g kg-1. Toksikološke analize bile su pozitivne na heroin, mekonin, acetaminofen, monoacetil morfin, kodein, noskapin i papaverin. Alkohol, i sam kao depresor respiracije, zajedno s heroinskom smjesom značajno povisuje rizik od nagle smrti kao posljedice zatajenja disanja.