Use of the Oximeter-539 WTW, equipped with a sensor Trioxmatic-300, for the functional analysis of dilute solutions of human Hb-A.

Dissociation curves for oxygen of dilute samples of human adult Hb-A were evaluated on this occasion, by using the Oximeter-539 WTW with its sensor, and a suitable spectrophotometer. At this purpose, Hb samples were desaturated in oxygen upon given experimental conditions, by bubbling pure nitrogen in them, and their re-oxigenation in air was followed, step by step, by multiple oximetries. In addition, all the spectrophotometric measurements of the saturation of Hb-O2%, corresponding to each individual oximetry, were carried out parallely but separately. Dilution of Hb-A was maintained at 0.1 mM in heme. The p50 at pH 7.3 was 4.435 +/- 0.299 Torr, with the n-value of 2.7 +/- 0.2; Bohr effect was -0.55 +/- 0.08, within a pH range between 6.8, 7.3 and 7.8, whereas chloride and DPG effects at pH 7.3 (the most useful value) were 0.42 +/- 0.44 and 0.453 +/- 0.0187 respectively. In conclusion, these results are similar to those obtained with automated procedures, upon comparable experimental conditions, but do not require expensive and sophisticated instruments. Such a technique could be very useful in the hemoglobinopathies, which are common in Italy, and it could be easily adapted to perform comparative studies on animal hemoglobins not far from human species.

Studies on the oxygen transport in the clinical association between Hb-S and Hb-C.

The whole blood oxygen affinity of a Negro carrier of SC disease was found to be characterized by some right-shifted p50 and clearly increased Bohr effect, whereas the isolated and purified Hb-S and Hb-C exhibited slight deficiencies mainly of the Bohr effect. The right-shifted p50 from whole blood can be easily explained by the mild anemia with a parallel increase of 2,3-diphosphoglycerate (DPG), whereas the functional discrepancies between whole blood function and that of the purified Hb-S and C could be due, at least in part, to the presence in vivo of consistent amounts of hybrid Hb tetramers of the type alpha alpha beta S beta C. Unfortunately, the mechanism promoting the formation (or dissolution) of hybrids are fundamentally unknown; so, either their presence and functional properties are very difficult to be explored.

Hemoglobin Agenogi [beta 90 (F6) Glu-->Lys] found in Piedmont. Case report.

An electrophoretically slow-moving hemoglobin, with abnormal beta chains, has been found in a young woman and in three members of her family. This variant amounted to 41% of the total Hb, and did not cause important clinical manifestations, although characterized by decreased oxygen affinity. Structural and aminoacid analyses revealed the mutation of Hb-Agenogi: 90 (F6) Glu-->Lys, a rare variant so far detected in unrelated racial and ethnic groups. This is the first affected family of ascertained Piedmontese ancestry.

Direct DNA analysis in a new Italian carrier of Hb-Belfast: beta 15 (A12) Trp-->Arg.

A young woman aged 21 was found to be a new carrier of Hb-Belfast: beta 15 (A 12) Trp-->Arg, and the characteristics of her hemoglobinopathy were not different from those of the four cases so far described: mild hemolysis with molecular instability of the abnormal Hb, red cells inclusion bodies, and slight alterations of some functional parameters of whole blood. On this occasion, direct DNA analysis indicated the genomic nucleotide replacement of the disease: TGG-AGG. This was inherited by the mother, originating from Bari (Apulia).

[Functional analysis of diluted solutions of human hemoglobin by using Oximeter-3000 WTW equipped with Trioxmatic-300 electrode]. / Analisi funzionale delle soluzioni diluite di emoglobina umana con l'Ossimetro-3000 WTW, munito di elettrodo Trioxmatic-300.

The oxygen carrying capacity of dilute solutions of hemoglobin from normal human adults was examined, by using the above indicated Oximeter. The results show that, if the pO2s are compared with those drawn from the Oximeter-539 WTW (a simpler instrument) carrying the same oxygen electrode, there is a good correspondence between the data drawn from both the instruments. The advantage of the former is that pO2s are measure in mBr, whereas the latter measures the oxygen of the aqueous solutions in mg/l; then mg must be converted into Torr pO2. Since both instruments are usually employed in the oxygen measurement of waste waters of earth, another conclusion is that their sensitivity also allows the use in the bio-medical (and zoological) field. In fact, the data obtained agree with those of recent literature on the subject, which are mainly drawn from automated and sophisticated apparatuses specifically built at this purpose.

[Environmental pollutants and karyologic effects. Usefulness of environmental and biological monitoring]. / Inquinanti ambientali ed effetti cariologici. Utilità del monitoraggio ambientale e biologico.

The results of a study carried out on a group of workers exposed to numerous pollutants (HCl, NaOH, ZnCl2, Sn, Pb, ecc.) in an industrial environment are reported. The research was broken down into two parts: 1) drawing up of a map of environmental pollution; 2) evaluation of the biological exposure of individuals submitted to control. The blood level of Pb was assessed in chosen subjects as a biological indicator of exposure and, at the same time, lymphocyte cultures were prepared in order to highlight any increase in chromosome exchanges and chromosome aberrations.

Molecular characterization of a rare hemoglobin variant: Hb-Hinsdale: beta139(H17)ASN-->Lys.

The authors describe the characteristics of a rare hemoglobin mutant found in a young female. She carries the aminoacid replacement of Hb-Hinsdale:beta139(H17)ASN-->Lys, which was identified at molecular level. This case, clinically and hematologically symptomless, is identical, but genetically independent, to the cases first described in an American family.

[Three-dimensional facial reconstruction of computerized tomography images by computer-aided design: example of an anthropologic study]. / Reconstitution faciale tridimensionnelle d'images tomodensitométriques par conception assisté par ordinateur: l'exemple d'une étude anthropologique.

Thanks to the medical scanning techniques and the reliability of some applications programs in 3D reconstruction, we are now able to explain an observation methodology based on the example of an anthropology study on Egyptian mummies'heads through a non-destructive proceeding. We have managed to demonstrate the exploration of the cervicofacial anatomy in the three dimensions of space and the possibility of dissociating the different anatomical tissues. The authors would like to point out the various consequences such a methodology may engender in any field.

Age estimation in subadult Egyptian remains.

In anthropological analyses of past populations, it is very important to be able to accurately reconstruct the palaeodemographic profile in order to interpret infant mortality as an indicator of the environmental, social and cultural conditions. There are various methods to evaluate the age of immature individuals but some of these methods are strongly influenced by the different rates of skeletal development observed in populations from various geographical areas and/or from various time periods, as well as between the sexes. Clearly, there is a need for adopting a method of estimation of age at death, which will be the one most suitable for analysing the particular skeletal sample. In this study we investigated subadults from the Egyptian osteological collection housed in the Museum of Anthropology and Ethnography of the University of Turin. For each individual, the age at death was estimated based on the degree of eruption and mineralisation of the teeth. Then the estimated age at death was correlated with the measurements of the long bones and ilium. We showed that greater regularity and constancy of rates of skeletal growth could be assessed with measurements, alternative to using maximum length of diaphysis. Moreover, using alternative characters, it was possible to markedly increase the number of individuals whose age at death could be estimated. Our study also showed the need to use a reference sample consistent with the sample being analysed and, which was derived from similar biological-environmental context. Therefore, our proposed method can be used for the estimation of age at death in pre/protohistorical populations from the Mediterranean region.

Surname analysis in biological anthropology: Alpine populations in the 17th and 18th centuries.

As part of an interdisciplinary research program on Alpine populations, we studied the biodemographic evolution of two populations of the Dauphiné in the period 1690-1799. We analyzed several indexes derived from surname analysis to infer the genetic structure of the populations. Although situated in the same area of the Dauphiné, the two communities of Vallouise and Chiomonte had different biodemographic characteristics. Vallouise was heavily populated but genetically homogeneous, whereas Chiomonte was less populated but more heterogeneous. The two districts also differed in geographic position: Vallouise was a glacier-enclosed valley that did not attract new inhabitants; Chiomonte was situated in an open valley served by important roads and thus was able to attract many new inhabitants. The demographic differences between the two populations explain the differences in genetic structure. The index of isonymous relationship (R(i)) being different from 0 is due to the rare marriages between members of the two populations. Because R(i) is based on surnames, which are mostly polyphyletic, it can overestimate the genetic relationships between the populations, as in the case of consanguinity assessed by matrimonial isonymy.

Functional properties of dilute Hb solutions studied without tonometric techniques.

The authors found that, by continuously bubbling pure nitrogen within a dilute Hb solution, the latter releases its oxygen with an intensity which is quite proportional to the duration of bubbling. Such a procedure can be employed instead of the common tonometric technique, to measure functional properties of Hb. In fact, not only dissociation curves can be obtained, but also a correct evaluation of the affinity changes upon pH variations (Bohr effect). The results obtained by this technique are not far from those recently reported by using tonometry, although the method is less precise and still not suitable for routine purposes. Nevertheless, it can be substantially improved.

Functional studies on nine different haemoglobins with high oxygen affinity.

Nine carriers of beta-abnormal haemoglobins with increased oxygen affinity (Hb X) were examined. Their oxygen dissociation curves from whole blood were more or less left-shifted, with six of nine also characterized by biphasism. This refers to an 'inflection point' usually positioned at about 50-60% of Hb O(2) saturation, commonly believed to be a limit between oxygenation of the normal and abnormal components. In effect, the inflection does not always correspond to the Hb X level, which sometimes is much lower than 50% of the total Hb. Moreover, the upper half segment of the dissociation curve could not only be an expression of Hb A oxygenation, since it is always left-shifted. However, a high Hb A level is commonly believed to be the main compensatory factor of these subjects, but many indications suggest that often they have at least three, and not only two, main haemoglobin species: Hb A, Hb X plus hybrids of the type alpha(2)beta(A)beta(X). These would oxygenate after Hb X, but before Hb A. Finally, the interaction of 2,3-diphosphoglycerate with Hb X and/or hybrid tetramers must be altered, and the releasing of oxygen from both is more or less reduced. Unfortunately, it is difficult to demonstrate the presence of hybrids directly, i.e. with amino acid analysis of the abnormal beta-globin.

Use of an enzymatic mixture against met-Hb excess during oximetry of dilute Hb-A solutions.

A mixture of NADH, cytochrome-C-reductase and methylene blue was employed to antagonize ferri(met)-Hb formation during oximetries of dilute samples of human Hb-A. Its efficacy is clear at any pH between 6.8, 7.3 and 7.8, in the presence of 100 mM NaCl. In these cases, ferri(met)-Hb decreases of about 2/3, compared with the enzyme-free controls, and p50 increases. On the contrary, when samples are examined at pH 7.3 with 600 mM NaCl and/or 100 mM NaCl plus 1 mM DPG, ferri(met)-Hb of the samples containing the enzymes also decreases, but not more than 1/2 of its initial value, whereas also p50 decreases. Finally, Bohr effect is lower in the samples containing the enzymes. In conclusion, the enzymatic mixture is very useful, but not easy to be handled; so, some observations for its correct use are required.

Catalase against met-Hb excess during oximetries of dilute Hb-A samples.

Functional parameters of diluted Hb-A have been determined before and after addition of catalase and disodium-EDTA to the samples. There are no important differences between the results drawn from catalase added samples and catalase free ones, except for the fact the met-Hb level at pH 7.8 is significantly lower in the samples containing catalase. On the contrary, catalase is almost ineffective against met-Hb at pH 6.8, whereas its activity at pH 7.3 is rather modest. Another limitation is that catalase remains active against met-Hb for not more than 15-20 minutes after addition to the sample, which is just the time necessary for one complete (manual) oximetry.