74 • A. Ascenzi, A. Bellelli, M. Brunori, G. Citro, R. Ippoliti, E. Lendaro, and R. Zito 



% I 



lUO 200 



Antigen (ng) 



300 



Figure 1 . Reaction of antiglobin rabbit antiserum against 

 liuman alpha and beta chains. 



dards. Quantification was achieved by integration of dot in- 

 tensity using a LKB Laser Scanner densitometer and digital 

 integration. 



Results 



Apohemoglobin (globin), alpha chains and beta chains were 

 used to standardize rabbit antisera raised against human 

 globin. The results of one experiment illustrating this pro- 

 cedure are shown in Figure 1 . 



Lumbar vertebrae extracts were tested with the same anti- 

 serum and the content of hemoglobin in the skeletal remains 

 estimated by comparison with the standard curves. As al- 

 ready shown qualitatively (Ascenzi et al. 1985), hemoglobin 

 or hemoglobin fragments can be determined quantitatively in 

 bone extracts with this technique. The average hemoglobin 

 content of lumbar vertebrae from adults buried for 1 5 years is 

 0.7 to 1 fig/ 100 gram of dry powder (Table 1 ). 



A larger amount of titratable material is extracted from the 

 lumbar vertebrae of younger individuals. As a general rule, 

 the younger the individual, the higher the hemoglobin con- 

 tent of the bone extracts, as may have been anticipated. 



Skull and other bones were used to determine the correla- 

 tion between hemoglobin content and anatomy and physiol- 

 ogy of the skeleton. It was found that, for the same individu- 

 al, the absolute amount of hemoglobin detected from bone 

 powder varies markedly with the type of bone examined. 

 Highly vascularized bones, whose marrow exhibits high 

 erythropoietic activity, are (as expected) much richer in titra- 

 table hemoglobin than bones with low or absent erythropoie- 

 tic activity. Data in Table 1 report one example by comparing 

 the hemoglobin content of the lumbar vertebrae and the skull 

 of an infant. 



Table L Content of hemoglobin (or hemoglobin 

 fragments) in lumbar vertebrae extracts (except D2 = 

 skull); quantitation with antiglobin rabbit antiserum 



Sample Age of Time elapsed Hb ng/lOO g bone 

 subject pom death () = No. of tests 



A senescent 15 years 0.16-0.2 (3) 



B adult 15 years 0.9-1.0(3) 



C adult 15 years 0.7-1.0(3) 



Dl infant 15 years 1.1-1.6(3) 



D2 infant 15 years 0.2-0.4 (3) 



Table 2. Content of hemoglobin (or 

 hemoglobin fragments) in lumbar vertebrae 

 of adults at different times after death (in 

 years) 



Sample Time elapsed Hb iig/100 g bone 

 from death () = No. of tests 



These results are in complete agreement with the known 

 correlation between the age of the subject and erythropoietic 

 activity, since in very young individuals all the skeleton 

 houses active marrow. Surprisingly, the amount of hemo- 

 globin detected in lumbar vertebrae of adults seems to be 

 essentially independent of the time elapsed from burial, at 

 least for the homogeneous set of data reported in Table 2. 

 Because very ancient samples are rare and some bones are 

 more easily destroyed, the oldest specimens consisted only 

 of lumbar vertebrae and skulls; nevertheless similar depen- 

 dence of hemoglobin content on age at death and anatomy 

 was demonstrated (data not shown). 



The reactivity of antialpha and antibeta rabbit antisera was 

 standardized as described above, and the content of alpha 

 chains in bone extracts was determined. Due to some residual 

 cross-reactivity, only a semiquantitative estimate is reported; 

 however, it was clearly observed that both chains can be 

 detected in ancient skeletal remains, and are present in signif- 

 icant amount. 



This is particularly important in the case of the alpha 

 chains, in view of the known sensitivity of these polypeptides 

 to proteolytic degradation (Chalevelakis et al. 1975), which 

 had raised concern that it might be difficult to determine 

 quantitatively the content of each chain in ancient skeletal 

 specimens. 



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