Genetic polymorphism of human parotid salivary amylase detected by isoelectric focusing electrophoresis and silver staining

In 332 samples of human parotid saliva collected at random from a Japanese population, the genetic polymorphism of salivary alpha-amylase was detected by isoelectric focusing electrophoresis in a pH range of 5.2-7.2 polyacrylamide gel followed by silver staining. This polymorphism, that was tentatively designated Amy1 S, consisted of extra three isozymes of a normal pattern (Amy1 N) and isoelectric points of these three isozymes were 5.5, 5.8 and 6.1, respectively. The inheritance was controlled by a dominant allele at an autosomal locus. The frequency of the genes determining these phenotypes were studied as follows: Amy1 S = 0.014 +/- 0.004, Amy1 N = 0.986 +/- 0.004.     

Review: The physiology of saliva and transfer of drugs into saliva

Although saliva or oral fluid “lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears”, quoting Mandel in 1990 [I.D. Mandel, The diagnostic uses of saliva, J. Oral Pathol. Med. 19 (1990) 119–125], it is now meeting the demand for inexpensive, non-invasive and easy-to-use diagnostic aids for oral and systemic diseases, drug monitoring and detection of illicit use of drugs of abuse, including alcohol. As the salivary secretion is a reflex response controlled by both parasympathetic and sympathetic secretomotor nerves, it can be influenced by several stimuli. Moreover, patients taking medication which influences either the central nervous system or the peripheral nervous system, or medication which mimic the latter as a side effect, will have an altered salivary composition and salivary volume. Patients suffering from certain systemic diseases may present the same salivary alterations. The circadian rhythm determines both the volume of saliva that will and can be secreted and the salivary electrolyte concentrations. Dietary influences and the patient's age also have an impact on composition and volume of saliva. The latter implies a wide variation in composition both inter- and intra-individually. Sampling must therefore be performed under standardized conditions. The greatest advantage, when compared to blood sample collection, is that saliva is readily accessible and collectible. Consequently, it can be used in clinically difficult situations, such as in children, handicapped and anxious patients, where blood sampling could be a difficult act to perform.     

唾液中滥用药物分析及其与血液中药物浓度的相关性

唾液是一种成分简单、易于采集的体液,某些药物在唾液中的浓度可以反映其血药浓度。本文分析了滥用药物进入唾液的机制和影响因素,综述了唾液中滥用药物分析时样品的采集、前处理和检测方法以及唾液与血液中药物浓度的相关性。认为唾液是临床和法医学方面很有价值的分析样品,用唾液中滥用药物浓度来推测血药浓度具有一定的法医学意义。     

Review: The physiology of saliva and transfer of drugs into saliva

Although saliva or oral fluid "lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears", quoting Mandel in 1990 [I.D. Mandel, The diagnostic uses of saliva, J. Oral Pathol. Med. 19 (1990) 119-125], it is now meeting the demand for inexpensive, non-invasive and easy-to-use diagnostic aids for oral and systemic diseases, drug monitoring and detection of illicit use of drugs of abuse, including alcohol. As the salivary secretion is a reflex response controlled by both parasympathetic and sympathetic secretomotor nerves, it can be influenced by several stimuli. Moreover, patients taking medication which influences either the central nervous system or the peripheral nervous system, or medication which mimic the latter as a side effect, will have an altered salivary composition and salivary volume. Patients suffering from certain systemic diseases may present the same salivary alterations. The circadian rhythm determines both the volume of saliva that will and can be secreted and the salivary electrolyte concentrations. Dietary influences and the patient's age also have an impact on composition and volume of saliva. The latter implies a wide variation in composition both inter- and intra-individually. Sampling must therefore be performed under standardized conditions. The greatest advantage, when compared to blood sample collection, is that saliva is readily accessible and collectible. Consequently, it can be used in clinically difficult situations, such as in children, handicapped and anxious patients, where blood sampling could be a difficult act to perform.     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

A sensitive and simple assay of saliva on stamps

Identification of saliva on stamps or envelope flaps remains yet a not widely studied problem. In most forensic laboratories it is seldom carried out, but this fact does not reduce the importance of the assay. Most authors consider amylase a sufficiently specific marker of the presence of saliva; really, the only other human body fluid that contains high amounts of this enzyme is the pancreatic juice (and therefore feces). Here we present a simple and sensitive assay for the determination of alpha-amylase that uses a commercially available and well-known substrate. It is hydrolyzed by amylase with the production of soluble blue fragments, that can be measured by photometry, obtaining objective results. The presented assay identifies 1 X 10(-6) diluted saliva or that present on 0.5 mg of a stamp; 16-year-old samples can also be identified. Intra-assay and day-to-day CV resulted in 10.8% and 13.7%, respectively. Owing to the high sensitivity of the test, handling samples or reagents can introduce contamination with saliva traces, giving false-positive results. Addition of EDTA 0.1 mol/l to the incubation mixture, lowering the sensitivity to 1 X 10(-3) diluted saliva, overcomes this problem.     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

人唾液中苯丙胺类药物检测方法进展

目前人唾液中苯丙胺类药物的检测研究较少,鉴于唾液检材具备很多的实用价值和优势,本文综述了国内外对唾液检材中苯丙胺类药物的检测研究进展,重点包括免疫分析筛选方法、液-液萃取法、固相微萃取法等萃取方法以及气相色谱/质谱联用法、液相色谱/质谱联用法等确证方法的发展情况。     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

唾液检材在毒品检测中的应用

近年来,毒品滥用问题日益突出,提高生物样品中毒品检测技术的性能是法庭毒物学研究的重点。相比于血液和尿液样品,唾液在的样品采集和毒品检测中具有诸多优势,因而逐渐受到重视。本文对近年来国内外唾液样本用于毒品检测方面的研究成果进行综述,介绍唾液毒品检测的发展情况以及相关的代谢动力学研究状况,旨在为相关研究和实践提供参考。     

Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples

The purpose of the present study was to identify salivary molecules carrying the ABH blood group antigens in Koreans and to investigate the changes in these antigens according to processing and storage of saliva samples. Secretor or non-secretor phenotypes and salivary components carrying the ABH antigens were identified in 90 subjects, 30 subjects in each ABO blood group, by SDS-PAGE and immunoblotting. Saliva samples were then obtained from 12 secretors-two males and two females in each ABO blood group and aliquots of both fresh saliva samples and their supernatants after centrifugation were stored at room temperature, 4, -20 and -70 degrees C. The same experiments were performed after 1, 3 and 6 months to investigate changes in the blood group antigens. In all 68 secretors, high-molecular-weight salivary mucin (MG1) was found to be the primary carrier of the ABH antigens. A salivary component of approximately 80 kDa also carried H antigen in seven saliva samples of 22 blood type O secretors. The blood group antigens were better detected in centrifuged samples. In saliva samples preserved at room temperature and 4 degrees C, the blood group antigens were either not detected or detected as degraded molecules. No change was found in the blood group antigens in saliva samples preserved at -20 and -70 degrees C for 6 months.     

Addressing the alternate hypothesis: Transfer and persistence of saliva beneath fingernails

This study investigated the transfer and persistence of salivary DNA under fingernails. This was performed to address a common alternate hypothesis presented to scientists in court, asserting that a relatively large quantity of DNA detected beneath the fingernails, typically from a victim of crime, originates from innocuous transfer of saliva in a casual setting.It was determined through these studies that contact with liquid saliva was an effective way to transfer foreign DNA beneath fingernails. However, when saliva was dried, DNA did not readily transfer through casual contact.When liquid saliva was placed directly beneath fingernails the amount of DNA detected from the saliva donor twenty-four hours later was several hundred-fold lower than the amount detected when sampling occurred immediately following deposition. Furthermore, when the recipients’ hands were washed immediately following the deposition of liquid saliva beneath fingernails, the majority of foreign DNA was removed following one hand washing and all detectable foreign DNA was removed from most recipients’ hands after three or six hand washings.This study demonstrates that casual contact with wet saliva can result in the transfer of substantial quantities of DNA beneath fingernails but that it does not typically persist for extended periods of time and is mostly removed if the hands are washed soon after deposition.     

Further evidence for phenotypes and gene frequencies of nine salivary polymorphisms in Japanese population

Nine salivary polymorphic systems (Pa, Pb, Pr, Db, PmF, PIF, Ph, Amy1 and s-AcP) were examined using parotid and whole saliva from random Japanese individuals. The gene frequencies obtained were: Pa+ = 0.221, Pb1 = 1.000 Pr1 = 0.741, Db+ = 0.033, PIF+ = 0.715, Ph+ = 0.029, Amyv1 = 0.013 and s-AcPA = 0.217, respectively.     

Amylase levels in semen and saliva stains

Amylase levels were determined for 148 semen samples and 20 saliva samples as well as for their corresponding stains. The effect of aging on the detectability of amylase activity in these stains was also investigated. The Phadebas amylase test was used for the quantitative assay of amylase. High levels of amylase in fluid saliva resulted in high levels being detected in saliva stains. Lower levels present in most seminal fluids produce little or no detectable amounts of amylase in stains. Interpretations are made as to the possible sources of amylase activity found in stains from laboratory casework based on both the amylase concentration and the elapsed time between collection and analysis. The evidential value of the presence or absence of amylase activity in casework stains is also discussed.     

家兔唾液与血液中氯胺酮浓度的相关性

目的研究家兔唾液中氯胺酮及代谢物去甲氯胺酮浓度与血药浓度的相关性。方法实验家兔分为氯胺酮灌胃组（6只）、静脉注射组（6只）和对照组（6只）,分别于染毒前、后不同时间点收集唾液和血液。采用气相色谱/质谱联用（GC/MS）全扫描定性、气相色谱（GC）定量分析样品中氯胺酮及去甲氯胺酮的浓度。采用双变量Pearson相关分析研究唾液中药物浓度和血药浓度的相关性。结果氯胺酮灌胃组和静脉注射组给药后各时间点氯胺酮及去甲氯胺酮在唾液和血液中的浓度相关系数（r）范围为0.80-0.95。结论氯胺酮及去甲氯胺酮在唾液和血液中的浓度均有良好的相关性,根据唾液药物浓度推断血药浓度可用于氯胺酮滥用的法医学鉴定。     

Detection of amphetamine in bloodstains,semen, seminal stains,saliva, and saliva stains

Low nanogram quantities of amphetamine were detected in 100μl samples of dried bloodstains using radioimmunoassay. Saliva, saliva stains, semen, and seminal stains also contained measurable quantities of the drug.     

Diagnostics of the AB group of AB0(H) system in saliva traces

On the basis of the mixed agglutination reaction of the saliva spots, obtained from persons with groups A, B, and AB (extractors) as well as mixtures of saliva obtained from the above subjects in various combinations, were experimentally studied. It was shown to be possible to diagnose the AB group both in the "pure" saliva traces and as an admixture of the AB group saliva to the groups A and B saliva obtained by using the anti-A and anti-B hetero-immune gemagglutinating sera. Our results confirm the previously published opinion on that antigens A and B of the ABse group saliva are positioned in the cis-acting mode, i.e. they are located on one molecule of surface structure of the epithelial (buccal) cell.     

A novel method for the identification of saliva by detecting oral streptococci using PCR

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.     

唾液血型抗原分泌时限变化及温度对抗原活性的影响

探讨人唾液中ABH血型抗原不同时限的分泌量,以及保存温度对血型抗原活性的影响。应用时间决定性荧光免疫测定法（TR．FIA）对O型分泌型10例和非分泌型5例人在不同条件下唾液中H抗原量进行检测。唾液血型抗原的分泌量随时间而波动,进餐后降低明显,但不干扰分泌型的判定。37℃保存48h抗原活性完全丧失,6℃保存1周抗原活性几乎没有变化。结果表明,唾液分泌时段不影响分泌型判定,将唾液制成斑痕可长期保存样品。     

A rapid method to detect dried saliva stains swabbed from human skin using fluorescence spectroscopy

Saliva on skin is important in forensic trace evidence. If areas where saliva is present can be outlined, this may lead to DNA analysis and identification. This study describes a rapid and non-destructive method to detect dried saliva on the surface of the skin by fluorescence spectroscopy. Eighty-two volunteers deposited samples of their own saliva on the skin of their ventral forearm. A control sample of water was deposited at three different sites on the contralateral arm. Saliva and water control were then allowed to air-dry. Swab samples were taken from dried saliva and control sites and were dissolved in 0.1M KCl solution. Emission spectra were obtained from the solution and were characterized by a principal maximum at 345-355nm with excitation at 282nm. The fluorescence emission intensity was greater than background readings obtained from the control swab site in 80 of 82 volunteers (approximately 97.6%). The fluorescence profile of saliva samples were similar to those obtained from aqueous samples of pure amylase and tryptophan, an endogenous fluorophore in alpha-amylase. The presence of an emission peak at 345-355nm with excitation at 282nm could provide a strong presumptive indication of saliva deposition.