A high-performance liquid chromatography method for hypoxanthine determination in vitreous humour: application to estimation of post mortem interval.

We have developed a new technique to determine the concentration of hypoxanthine [Hx] in a reverse phase column using a modified high-performance liquid chromatography (HPLC) method that is faster and more reliable than those previously described. In this paper we present a formula for estimating the post mortem interval (PMI) based on this HPLC method by applying the inverse prediction method. The regression line obtained by changing the variables gives PMI = 0.183 [Hx] + 0.599 (PMI in hours, [Hx] in micromol/L, R2 = 0.531, P < 0.05).

The pathology of the heart conduction system in congenital heart block.

The incidence of congenital complete heart block is estimated in 1 of 2500-20,000 births. Many cases are isolated (found in an otherwise normal heart) and the pathology of the heart conduction system is variable. We report a 51-year-old man with the diagnosis of complete heart block, with a permanent pacemaker. No family history of rhythm disturbances was available. The patient presented and endocarditis after replacement of the pacemaker battery. The prognosis was poor and the patient died three months later. Autopsy examination showed signs of shock, of septic origin. The heart was hypertrophic (450g) and the left ventricle thickened. Histopathological examination of the heart conduction system showed that the sinus and atrioventricular nodes were normal, but the His bundle was interrupted and replaced by fibrous tissue. No inflammatory signs were present. Loss of conducting fibres and their replacement by fibrous tissue is the most common pathological process in complete heart block. In this case His bundle was mostly affected, different to Lev's disease where the process is more distal (branching atrioventricular bundle) and to Lenegre's disease, which shows a diffuse damage in the conducting system.

The isoelectric focusing of keratins in hair followed by silver staining.

An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.

Biochemical diagnosis of the intravital origin of skin wounds.

The performance of a spectrophotometric and a spectrofluorimetric method in the determination of histamine and serotonin in skin samples was compared. The spectrophotometric method was found to be superior in terms of reproducibility and sample stability, so it was chosen for subsequent use. An overall 30 rabbits were wounded at different times prior to and after death and the histamine and serotonin concentrations in the wounds were compared to those of control specimens. Vital wounds were found to result in increased concentrations of the amines. The spectrophotometric method was also applied to human skin samples from 12 corpses showing body signs of violence.

A silver staining method for the detection of polymorphic proteins in minute bloodstains after isoelectric focusing.

A silver staining method has been developed to study polymorphic proteins in bloodstains after isoelectric focusing. This method is highly sensitive and permits the detection of polymorphic proteins (i.e. alpha 1-antitrypsin, Gc and Tf C subtypes) in bloodstains as small as 0.2 microliter or less. The method is simple and reproducible and can be used after immunofixation. Blood stains can be identified after longer storage periods than is possible by using conventional staining methods.

The use of hybrid isoelectric focusing for the detection of polymorphic proteins in blood stains.

alpha1-Antitrypsin (Pi), transferrin (Tf) and orosomucoid (ORM) were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and also with a mixture of immobilines (HIEF). HIEF yields superior results from proteins typing in bloodstain extracts, since phenotypes are better distinguished and the bands are straighter and sharper. Also the sensitivity of HIEF is similar to IEF with CA.

Direct carboxyhemoglobin determination by derivative spectroscopy.

A fast and sensitive method for the determination of carboxyhemoglobin (COHb) in blood using derivative spectroscopy is described. The addition of sodium dithionite as a reducing agent is not required. The concentration interval studied was from 0.5% to 100% COHb.

Application of derivative spectroscopy to the determination of plasma theophylline in the presence of phenobarbital.

A rapid, simple, and sensitive procedure for the determination of theophylline in plasma using second derivative spectroscopy is described. Second derivative spectra of plasma extracts containing theophylline in concentrations ranging from 2 to 20 micrograms/mL were studied. The method is suitable for the measurement of plasma theophylline in patients treated for cardiorespiratory disorders. Interference from other compounds such as phenobarbital can be avoided.

Application of pelletized sodium borohydride in the spectrophotometric determination of arsenic.

The spectrophotometric determination of arsenic using sodium borohydride in pellet form, with excipients as the reducing agent and arseine former, has been studied. The use of pelletized sodium borohydride allows the formation of arsenic diethyldithiocarbamate and enables an accurate application of the method without the use of other reagents. The gradual reducing action of the pelletized sodium borohydride, together with the release of hydrogen thus forming arsine with As(III), is the main advantage of this procedure. The arsine thus formed bubbles through the chloroform solution of silver diethyldithiocarbamate to form the complex that allows the determination of the arsenic by visible spectrophotometry.

Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.

Isoelectric focusing patterns of some mammalian keratins.

An isoelectric focusing method followed by silver staining has been used for the study of keratins from a total of 97 individuals belonging to 17 families and 39 species. The method distinguishes perfectly between two different animal species. In addition, there are often considerable differences between breeds and even occasionally slight individual differences which in no way impede the identification of a particular species as such.

A new perspective in the estimation of postmortem interval (PMI) based on vitreous.

The relation between the potassium concentration in the vitreous humor, [K+], and the postmortem interval has been studied by several authors. Many formulae are available and they are based on a correlation test and linear regression using the PMI as the independent variable and [K+] as the dependent variable. The estimation of the confidence interval is based on this formulation. However, in forensic work, it is necessary to use [K+] as the independent variable to estimate the PMI. Although all authors have obtained the PMI by direct use of these formulae, it is, nevertheless, an inexact approach, which leads to false estimations. What is required is to change the variables, obtaining a new equation in which [K+] is considered as the independent variable and the PMI as the dependent. The regression line obtained from our data is [K+] = 5.35 + 0.22 PMI, by changing the variables we get PMI = 2.58[K+] - 9.30. When only nonhospital deaths are considered, the results are considerably improved. In this case, we get [K+] = 5.60 + 0.17 PMI and, consequently, PMI = 3.92[K+] - 19.04.

Stature estimation from radiographically determined long bone length in a Spanish population sample.

The estimation of stature from of a variety of bones is an important aspect of forensic work. In order to obtain reliable results, it is important to have comparative data obtained from the same population group as the skeletal remains. However, lack of up to date information on the population groups of Southern Europe makes the estimation of stature from bones in this area subject to possible error. In this study, the stature of 104 healthy adults from Spain was measured, and an anteroposterior teleradiograph of the right lower and the right upper limb of every subject in the study was made in order to measure the lengths of the femur, tibia, fibula, humerus, cubitus and ulna. Pearson's regression formulae were obtained for both limbs. In males, we found the femur to be the most accurate predictor of stature (R = 0.851), whereas in females best results were obtained with the tibia (R = 0.876).

Immunohistochemical analysis of P-Selectin as a possible marker of vitality in human cutaneous wounds.

Immunohistochemical detection of mediators of inflammation, such as P-Selectin, has been proposed to assess vitality of wounds. Forty-five incised cutaneous wounds (24 vital, 14 post-mortem, seven with induced autolysis/putrefaction) were immunostained with antibodies against P-Selectin and CD31. The percentage of stained lumina for P-Selectin out of the total of CD31 positive vessels (P-S/CD31 index) was calculated at both edges of every specimen. In vital samples, the P-S/CD31 index ranged from 10.7% to 71.4% at the wound edge, and was 12.5-58.8% for the opposite margin, with a ratio between both indices of 0.37-1.77 (mean: 0.94). In post-mortem cases, the index ranged from 22.5% to 69.2% at the wound edge, and was 28-89.5% at the opposite margin, with a ratio between both indices of 0.76-1.9 (mean: 0.96). Differences between ratios were not statistically significant and thus precluded any assessment of vitality. The analysis of P-Selectin/CD31 immunoreactivity in skin wounds was not useful for the diagnosis of vitality when evaluating both edges for each specimen. Moreover, P-Selectin has been detected in post-mortem injuries and it is not specific to vital injuries. Microscopic evaluation becomes difficult after autolysis/putrefaction.

No cash no whiplash?: Influence of the legal system on the incidence of whiplash injury.

Whiplash injury has been a continuous source of controversy due to its association with litigation. We studied the incidence of whiplash associated disorder (WAD) in two similar socioeconomic areas and carried out a retrospective study based on the hypothesis that the Spanish law 30/1995 might have an affect on the incidence and duration of cervical symptoms and the persistence of impairment. More than 10,000 patients injured in traffic accidents were studied over a period three years. Of these, only patients with an initial diagnosis of whiplash injury were included in the study. Patients with other injuries were excluded. The patients were classified into two groups: Galicia-Spain and North-Central Portugal (depending on where the accident took place and the medico-legal evaluation procedure in force). Statistical analysis was made using SPSS 13.0 and Statistix 8.0. We found a statistically significant difference between Spain and Portugal in the incidence of WAD and in the duration of symptoms. The incongruities caused by the compulsory application of Spanish law arise from the fact that evaluation on a points scale of impairment does not always reflect the functional state of the injured person.

Distribution of the Pi, TfC, and Gc subtypes in Galicia (North West Spain).

Alpha 1-antitrypsin (Pi), Gc, and TfC subtypes were determined by isoelectric focusing in thin layer agarose (AGIF) and polyacrylamide gels (PAGIF) in a total of 480 individuals from Galicia. The following gene frequencies were observed: for Pi:PiM1:0.660; PiM2:0.115; PiM3:0.060; PiS:0.149; PiZ:0.009; PiF:0.005; PiI:0.001; for Gc:Gc1S:0.572; Gc1F:0.120; Gc2:0.308; for TfC: TfC1:0.778; TfC2:0.180; TfC3:0.041; TfC6:0.001. A rare variant TfC6-2 was found and the intrafamilial distribution of the TfC6 allele studied. The use of these systems for forensic purposes and the peculiar distribution of some of their alleles in the Galician population are discussed.

PGM1 subtypes in Galicia (NW Spain).

A total of 1,086 individuals from Galicia have been typed for red cell PGM1 using isoelectric focusing in polyacrylamide and agarose gels. The frequency of the four alleles at the PGM1 locus was found to be PGM11+ : 0.6211, PGM11- : 0.1137, PGM12+ : 0.2109, and PGM12- : 0.0543.

Genetic markers in alcoholic liver cirrhosis.

11 genetic markers were typed in 157 individuals suffering from alcoholic cirrhosis, and compared with a random sample of healthy individuals. No significant differences were found for transferrin, specific group component, orosomucoid, esterase D, phosphogluconate dehydrogenase and adenylate kinase. Strong associations between alcoholic cirrhosis and alpha-1-antitrypsin PI*Z allele, haptoglobin HP*1 allele and acid phosphatase ACP AC phenotype were observed. The biological significance of these associations and their relationships with the development of alcoholic cirrhosis are also discussed.