Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.     

Randomly amplified DNA polymorphisms in dogs are reproducible and display Mendelian transmission

Many inherited diseases occur in pure-bred dogs, but diagnosis at the level of DNA is impossible because the canine genome is largely unknown. Random amplification of polymorphic DNA (RAPD) provides many polymorphisms, but the reproducibility and Mendelian inheritance are not beyond doubt. An optimized polymerase chain reaction (PCR) was developed for canine DNA with respect to the annealing temperature and the concentrations of MgCl₂, template DNA and primers. RAPD amplification products were in the range of 100–1500 base pairs. With six primers, 21 different reactions with different electrophoretic patterns were obtained, yielding 9–29 products per reaction. In DNA from dogs of 16 different breeds, 14% of the products were polymorphic; when only beagles were included the rate of polymorphism was 10%. All of the reaction products were completely reproducible in 16 DNA samples. Mendelian transmission was analysed in six beagle families (42 dogs). The segregation of polymorphic amplification products in 21 reactions performed on DNA from all beagles was nearly complete; in only two of the 630 reactions was there a product that could not be traced back to either of the parents. The reproducibility and Mendelian behaviour of polymorphisms generated by RAPD in dogs makes this tool very suitable for development of DNA markers of canine inherited diseases.     

DNA polymorphism of human HLA-linked complement C4 allotypes, including C4 null alleles, in the Finnish population

Human HLA-linked complement C4 gene products, C4A and C4B, show extensive genetic polymorphism. In both loci, an allele without a gene product, C4 null, is also observed. We have performed a restriction enzyme analysis of genomic DNA samples from individuals having all common (frequency over 1%) C4 protein allotypes observed in the Finnish population. Only one allotype-specific RFLP marker was observed. With some enzymes a DNA polymorphism was observed, which was not detectable by C4 protein typing. Analysis of 10 different C4B null haplotypes and 4 C4A null haplotypes suggested that only one haplotype, HLA-B8 C4A0 B1, carried a C4A gene deletion. This was observed in all 4 unrelated individuals homozygous for this haplotype.     

Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.     

C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.     

Rapid detection of the 46C --> T polymorphism in the factor XII gene, a novel genetic risk factor for thrombosis, by melting peak analysis using fluorescence hybridization probes

Factor XII (FXII) level is an important intermediate phenotype associated with thrombotic disease. The 46C --> T transition in the exon 1 of the Factor XII (F12) gene is a significant, prevalent, and independent genetic risk factor for thrombotic disease. It is also associated with interindividual variation of plasma FXII zymogen levels. The aims of this study were to develop a rapid, reproducible, and easy method for 46C --> T genotyping and to compare its reliability with the classical endonuclease digestion methodology. DNA samples from 100 subjects were genotyped for the 46C --> T transition using the classical endonuclease digestion method with Sfna I. The genotypes of three of them (each with a different 46C R T genotype) were confirmed by direct sequencing analysis. We then set out to construct a LightCycler PCR protocol to detect the 46C --> T polymorphism. This protocol was designed to combine a rapid-cycle polymerase chain reaction (PCR) with an allele-specific fluorescent probe melting for mutation detection. In the three sequenced samples, as well as in the remaining 97, the LightCycler PCR procedure unambiguously resulted in the same genotype previously observed by sequencing and endonuclease digestion. Characteristic fluorescent curves were obtained for each genotype; the first derivative of these curves had a maximum at an apparent hybridization temperature (Tm) that was specific for each probe/allele duplex. The whole process took less than 40 min. Thus, if this method is used with a rapid DNA extraction, the genotypes would be obtained within 60 min after receiving a blood sample. In conclusion, the technique presented allows for easy, reliable, and rapid detection of this polymorphism, and is suitable for typing both small and large numbers of DNA samples.     

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.     

An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.     

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.     

Development of a novel telomerase assay using the PPDK–luciferin–luciferase detection system

Telomerase participates in malignant transformation or immortalization of cells, and has attracted attention as an anticancer drug screening and diagnostic tumor marker. We developed a novel telomerase assay called the PPDK–luciferin–luciferase system bioluminescence assay (PLLBA) using pyruvate phosphate dikinase (PPDK). In this assay, pyrophosphate produced by the telomerase reaction and polymerase chain reaction (PCR) is converted to ATP by PPDK, and ATP is detected by the firefly luciferin–luciferase reaction. In this work, telomerase substrate was obtained in accordance with the telomeric repeat amplification protocol (TRAP). Telomerase‐positive (500 cells/assay), ‐inactive (heated for 10 min at 85 °C) and ‐negative (only Chaps lysis buffer) samples were used. As a result, the findings clearly showed that the signal‐to‐noise (S/N) ratio of the positive cells was 39.5. After the telomerase reaction and PCR, PLLBA was completed ~ 120 s later. A high level of reproducibility was obtained with ‐ coefficient of variation (CV) of 4.1% (positive cells). The detection limit for cells using telomerase was one cell per assay. This assay for telomerase activity was also shown to be adaptable to human cancer‐derived cell lines. Copyright © 2013 John Wiley & Sons, Ltd.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

Molecular bases for human complement C7 polymorphisms,C7*3 and C7*4

Complement C7 is one of the components of membrane attack complex (MAC) generated by the terminal complement cascade. C7 protein is polymorphic and most of its polymorphisms have been identified using isoelectric focusing (IEF), which detects protein charge differences. To date, the molecular bases of the polymorphisms detected by IEF have not been determined. In this paper, we describe the structural bases of two C7 IEF-detected polymorphisms, C7*3 and C7*4, both of which are common in Asian populations. C7*3 resulted from substitution of cysteine (Cys) at amino acid residue 106 by charged arginine (Arg; C106R), while charged lysine (Lys) at amino acid residue 398 was replaced by neutral glutamine (Gln; K398Q) in C7*4. As C7*3 is hypomorphic, it is important to study its possible associations with diseases such as immunological disorders and infections. We present genetic bases for this C7 polymorphism, which we determined using polymerase chain reaction (PCR)-based genotyping, a simple and accurate method suitable for large-scale studies.     

吉林省延边地区丙型肝炎病毒基因型分布

为了解延边地区不同丙型肝炎病毒(HCV)基因型的分布特点,应用逆转录-聚合酶链反应(RT-PCR)技术,检测了48例本地区抗HCV阳性血清,结果有29例出现HCVRNA阳性.再利用限制性片段长度多态性(RFLP)技术,对29例RT-PCR阳性标本进行HCV基因分型.结果延边地区主要流行HCVⅡ型,其次是HCVⅢ型,也存在HCVⅡ/Ⅲ混合型,且HCVⅢ型的感染率较高.     

The eighth component of human complement: molecular basis of C8A (C81) polymorphism

Using an exon-specific polymerase chain reaction (PCR) followed by direct DNA sequence analysis we have analyzed the polymorphism of the -chain of the eighth component of human complement (C8) at the DNA level. We found that two common alleles, C8A^*A and C8A^*B, are characterized by the substitution of a single amino acid (Gln to Lys), which is caused by a point mutation of a single nucleotide (C to A) in exon 3 at position 187 of the mature C8 cDNA sequence. Based on this mutation, an allele-specific PCR was designed detecting the two alleles of C8A. We applied this method to type the C8A polymorphism using DNA samples from a Chinese Han population. The comparison with the data of protein typing of the same samples proved that the described method is efficient and reliable for the identification of C8A genotypes and may be valuable for further application in population studies and forensic science.     

PCR detection method of Clostridium scindens and C. hiranonis in human fecal samples

Clostridium scindens and C. hiranonis have high bile acid 7alpha-dehydroxylating activity. Direct PCR and nested PCR with the specific primers for C. scindens and C. hiranonis were developed and tested for the detection of them in human fecal samples. Nested PCR detected C. scindens in all samples, but direct PCR detected it in 27 of 34 samples. Regarding C. hiranonis, nested PCR detected it in only 18 of 34 samples although nested PCR has a high level of sensitivity. Nested PCR was a specific and sensitive method for the detection of C. scindens and C. hiranonis in fecal samples.     

Long distance PCR in detection of inversion mutations of F8C gene in hemophilia A patients

In the present paper, the experience with detection of intron 22 inversion of F8C gene in severe hemophilia A patients using a recently described long-distance PCR (LD-PCR) method was reported. To test the sensitivity and the specifity of the LD-PCR, analysis of 46 DNA samples of patients and their family members, previously tested by Southern hybridization, was performed. In addition, 16 DNA samples of severe hemophilia A patients in which causative mutation was unknown, were included in analysis. Four-primers, P, Q, A&B, which are able to differentiate between the affected males with or without the inversion, and in female carriers, were used in LD-PCR. Two primers, P&Q, are located within the F8C gene flanking int22h1. Two further primers, A&B, flank int22h2 and int22h3, extragene homologs of int22h1. Nine combinations of four primers were used to identify the optimal one. Four-primers (P, Q, A&B), three-primers (P,Q & B;P, A & B; A, B & Q;P, Q & A) and two-primers (A & B; P & Q; A & Q; P & B) PCR amplifications were performed in the hemophilia A patients as well as in obligate carriers DNA samples. Successful amplification required introduction of some modifications of the original protocol. The most reproducible and uniform results were obtained using two-primers PCR, performed in four single reactions. Thus, a total of 46 DNA samples, 22 were hemizygous for inversion, 6 without the inversion, 14 carriers and 4 non-carriers of inversion. Perfect correlation between genotypes determined using Southern hybridization and LD-PCR was achieved. The optimalized two-primers LD-PCR protocol was used for analysis of 16 DNA samples of severe hemophilia A patients with unknown mutation. Ten cases of inversions and six cases without them were detected. Thus in additional 10 severe hemophilic patients DNA diagnosis was completed. The most successful and reproducible results were obtained performing four single LD-PCR reactions with combinations of two-primers A & B; P & Q; A&Q, and P&B in each DNA sample and this approach is recommended for routine using in clinical practice.     

The association of a cystatin C gene polymorphism with late-onset Alzheimer's disease and vascular dementia

A polymorphism in the cystatin C (CST3) gene was suggested to associate with Alzheimer's disease (AD). In the present study we attempted to determine the association between CST3 polymorphism and AD or vascular dementia (VD), and whether such effects are dependent of the APOE4 allele. The polymorphisms of CST3 genotype were determined using polymerase chain reactions (PCR) followed by gel electrophoresis in 124 AD, 70 VD, and 115 control individuals. No statistical difference in CST3B allele frequencies was observed among all three groups. Associations between CST3B/B genotype and AD patients older than 75-year-old, or VD patients younger than 75-year-old were evident. The APOE4 allele alone significantly increased the odds for the developing AD, but not VD. A logistic regression analysis revealed that either CST3 or its interaction with APOE4 were not significant predictors of AD. However, a synergistic association of CST3 and APOE4 alleles was observed in predicting VD patients. These results suggest that CST3 might interact with APOE4 on conferring vascular pathologies.     

Reactions of 4 methylphenyl isocyanate with amino acids

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lung sensitization and asthma. Protein adducts of isocyanates might be involved in the aetiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4 methylphenyl isocyanate 4MPI with the tripeptide valyl glycyl glycine and with single amino acids yielding N 4 methylphenyl carbamoyl L valyl glycyl glycine 4MPI Val Gly Gly , N 4 methylphenyl carbamoyl L valine 4MPI Val , N 4 methylphenyl carbamoyl L aspartic acid 4MPI Asp , N acetyl S 4 methylphenyl carbamoyl L cysteine 4MPI AcCys , N acetyl N 4 methylphenyl carbamoyl lysine 4MPI AcLys , N acetyl O 4 methylphenyl carbamoyl tyrosine 4MPI AcTyr and N acetyl O 4 methylphenyl carbamoyl D,L serine 4MPI AcSer . The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4 methylaniline 4MA . The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 C with 6 M HCl in and or 0.1 M NaOH at room temperature, following methods applied for the analyses of biological samples of arylisocyanate exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 C. The hydrolysates were analysed using HPLC with UV detection and quantified against the internal standard, 4 fluoroaniline or 4 chloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; after 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI Val and 4MPI Asp yielded the respective hydantoins 3 4 methylphenyl 5 isopropyl 1,3 imidazoline 2,4 dione and 2 1 4 methylphenyl 2,5 dioxoperhydro 4 imidazolyl acetic acid. For future studies, we propose to hydrolyse biological samples with 0.3 M NaOH at 100 C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also result from arylamine exposure. Therefore, we propose to analyse the N terminal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.     

A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field‐collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real‐time polymerase chain reaction (RT‐PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme‐linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP‐ELISA). However, because of the relatively high costs associated with equipment and reagents, RT‐PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA‐based nested PCR protocol, modified from a loop‐mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP‐ELISAs in field‐collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4‐fold higher sensitivity than the CSP‐ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium‐positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.     

Reactions of 4 methylphenyl isocyanate with amino acids

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lung sensitization and asthma. Protein adducts of isocyanates might be involved in the aetiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4 methylphenyl isocyanate 4MPI with the tripeptide valyl glycyl glycine and with single amino acids yielding N 4 methylphenyl carbamoyl L valyl glycyl glycine 4MPI Val Gly Gly, N 4 methylphenyl carbamoyl L valine 4MPI Val, N 4 methylphenyl carbamoyl L aspartic acid 4MPI Asp, N acetyl S 4 methylphenyl carbamoyl L cysteine 4MPI AcCys, N acetyl N 4 methylphenyl carbamoyl lysine 4MPI AcLys, N acetyl O 4 methylphenyl carbamoyl tyrosine 4MPI AcTyr and N acetyl O 4 methylphenyl carbamoyl D,L serine 4MPI AcSer. The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4 methylaniline 4MA. The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 C with 6 M HCl in and or 0.1 M NaOH at room temperature, following methods applied for the analyses of biological samples of arylisocyanate exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 C. The hydrolysates were analysed using HPLC with UV detection and quantified against the internal standard, 4 fluoroaniline or 4 chloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; after 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI Val and 4MPI Asp yielded the respective hydantoins 3 4 methylphenyl 5 isopropyl 1,3 imidazoline 2,4 dione and 2 1 4 methylphenyl 2,5 dioxoperhydro 4 imidazolyl acetic acid. For future studies, we propose to hydrolyse biological samples with 0.3 M NaOH at 100 C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also result from arylamine exposure. Therefore, we propose to analyse the N terminal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.