.: . . . , A I OFT ORNL P I118 . : : ) . " 1. 3.6 EFFE 1.25 1.14 116 : MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDY - 1963 Duy ORNE-p-1118 APR 27 1996 For: P.S.E.B.M. CHANGE IN THE PARTTTION OF TOTAL-BODY NİTROGEN IN RADIATION CHIMERAS A. L. Kretchmar, W. H. McArtbur and C. C. Congdon . timisegravimeente n cre, RELEASED FOR ANNOUNCEMENT BEINING toimi - IN NUCLEAR SCIENCE ABSTRACTS CFSTI PRICES H.C. $ 6,00;MN.50 Medical Division, Oak Ridge Institute of Nuclear Studies, Oak Ridge, Tennessee Knoxville College, Knoxville, Tennessee Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Research sponsored by the United States Atomic Energy Commission under contract with the Union Carbide Corporation, the Oak Ridge Institute of Nuclear Studies, and Knoxville College. LEGAL NOTICE TH, raport me prepared una account of Government sponsored work, Nolther the Vallad Stalo, por Who Commision, por any porno acuing on baball of the Commission: A. Makes any warranty or reprivalaulon, expressed or implied, with respect to the accu- racy, complelopers, or whetuldous of the informativa contained in this report, or that ibo co of any information, appenaw, method, or proceso disclosed in to report may not infring. printoly omed richiajar 8. Ainmol way llabilluas with respect to the use of, or for damages rerulung from the use of Lay laformation, apparatus, Rothod, or proces, discloud lo walo report. As und la tenor, "porno actug og teball of the Commino" Includes hy oo. ploys or contractor of the Commission, or employer of such coolriclor, to the extent that Bucd employs or contractor of Use Commioloa, or employm of nuch contractor properes, dinnominatur, or provides Acces to, may Information purovant to tlo saployment or contract Will the commonloo, or all employment with such contractor. Running Title: Partition of Nitrogen in radiation Chimeras. Send proofs to: A. L. Kretchmar, M.D., Ph.D. Medical Division Oak Ridge Institute of Nuclear Studies P. 0. Box 117 Oak Ridge, Tennessee mi to . ... . Introduction While lethally irradiated mice can recover if they are given normal bone marrow cells after irradiation, they may succumb to a secondary mortality if the administered cells are not genetically compatible (1). This mortality is associated with a secondary syndrome (homologous disease), not related to the acute effects of irradiation, that has been considered to be in some respects a "metabolic starvation" (2). This imprecise phrase has been useful in that it directs uttention to metabolic parameters in the teradiation chimera for an explanation of the clinically prominent feature of wasting that accompanies the secondary syndrome. Es secondary loss in body weight occurs despite adequate intake of food comparable to that of normal or isologous marrow-treated irradiated mice (3). We have previously reported (4) that the nitrogen balance of radiation chimeras is positive and that these animals retain as much nitrogen as do Irradiated mice treated with isologous cel18. There latter animals do not show the secondary loss of weight that occurs in simlarly irradiated mice given homologous marrow. It was concluded that the nitrogen retained by 1sologous marrow-treated animals is utilized for regeneration of the hemopoietic system, other repair processes and growth. We speculated that since the homologous chimeras retained as much nitrogen but failed to gain in body weight, there was a shift in the distribution of body nitrogen in these mice with homologous marrow grafts, The present studies were undertaken to experimentally demonstrate such a shift and they indicate that nitrogen may be deposited in skin at the expense of the general tissues of the terradiation chimera. - 2 - welche * * Materials and Methods 1C3F, * Irradiated animals were al bylord male 16 D2/ mice to 24 weeks old. Donors of homologous bone marrow wereffet * - from 79-194 days old, DDAZTumle, male mice. The details of the bone marrow treatment have been m ab (IBM) THOM) reported (5,6); 20 x 10° isologous, or homologous cells were injected intravenously on the day of irradiation. There were 18 mice in eaed group (normal, IBM and HBM) at 4 and 25 days after treatment.. Nine hundred fifty r total-body irradiation was delivered with a constant potential X-ray machine; 250 kilovolts, 15 ma. Inherent filtration was 1 mm Al; half-value layer, 0.5 mm Cu; target-object distance 60 cm; dose rate in air approximately 157 r/min. Animals were kept 10 to a cage before and after Irradiation with free access to food and water. Some animals were maintained, in order to measure nitrogen balance, in the special metabolism cage previously described (4) for 48 or 96 hours before sacrifice. All animals were killed by decapitation, the blood being collected in a round bottomed flask (I), and after exsanguination the thoracic viscera were dissected and added to its contents. Similarly, the abdominal viscera (II), skin, tail and head (III), carcass (IV), and excreta (v) were put into appropriately labeled vessels. Fifty to 150 ml of 6N H2SO4 were added, a reflux condenser was adjusted over each and digestion at boiling temperature carried overnight or to completion. The fat which overlay the digested specimens was extracted with chloroform and measured gravimetrically. The extracted samples were then diluted to give approximately 1 mg N/ml and nitrogen was determined in aliquots by the Technicon Autodigester and Autoanalyzer system (7). The total nitrogen of the animal was thus partitioned into four gross compartments: I, blood and thoracic viscera; II, abdominal viscera; III, skin, tall and head; IV, carcass. + pоr саn gе зн/Aug eum ]ғ, ** [C57BL/6 cum ex DBA/z Cum ou]F, Results 20 Nitrogen balance was variable and with only 1 (48 hours) or 2 (96 hours) periods no significant difference between normal, isologous, or homologous marrow-treated irradiated mice was noted; the data are therefore not included. The most significant findings were in the nitrogen of "skin" (III) and "carcass" (IV). Figure 1 (a and b) 11lustrates the correlation between body weight and "carcass nitrogen" in unirradiated control mace 4 and 25 days after 1 ml Tyrode's solution injected intravenously. There is 12.5 £ 1.25 mg nitrogen per gram body weight in this compartment. In irradiated mice 4 and 25 days after to x 106 isologous bone marrow cells (Fg. 1, c and a) the same relationship fits the data. If irradiated mice are given x 106 homologous cells, however, (Fig. 1, e and f) a significant number of the animals has less nitrogen in the "carcass". This low carcass nitrogen, while it occurs in some of the 4-day mice, characterizes the majority of the animals 25 days after homologous marrow. The data also show that about half of the mice in this group have not recovered body weight and weigh less than 18 grams. Figure 2 (a and b) shows that "skin nitrogen" 18 also correlated with body weight in normal mice and that approximately the same amount of nitrogen per gram of body weight is contained in this compartment as in the "carcass." Again, the data for irradiated mice given 18ologous bone marrow cells is comparable to normal animals. Four animals in the 1sologous marrow-treated series had skin nitrogen content that was higher than expected for their body weight. When the ratio of skin/carcass nitrogen content was computed, however, the ratio was normal, indicating that relative distribution of body nitrogen was not abnormal. Among the irradiated mce given homologous marrow, 7 of 18 at 4 days and 5 of 8 of the animals below 18 grams in body weight at 25 days had high skin nitrogen content. 4 . In Fig. 3 there is no correlation between the ratio skir/carcass nitrogen and body weight in normal (Fig. 3 a and b) and irradiated mice given isologous bone marrow cells (Fig. 1 c and a). This ratio is slightly over 1.0. However, in irradiated mice given homologous cells (Fig. 3 e and 1), the ratio skin/carcass nitrogen 18 greater than 1.0 in most and there 18 a negative correlation with body weight. This trend, present as early as 4 days after treatment, 18 especially clear at 25 days. Thus, in irradiated mice given homologous marrow, low carcass nitrogen 18 associated with increased skin nitrogen and this association 18 especially characteristic of animals weighing less than 18 grams. Discussion When skin/carcass ratios were computed for the homologous marrow-treated animals the shift in distribution of body nitrogen from carcers to skin was clearly demonstrated. There is, indeed, a definite negative correlation betveen this ratio and body weight in chimeras at 25 days. This indicates that the animals with severest secondary disease (as judged by body weight) show the greatest shift in nitrogen distribution but that the tendency is present in all the animals over the whole range of clinical severity present in this experiment, The method used to divide the whole animal had the advantage of technical simplicity but the disadvantage of grossness. This disadvantage 18 particularly important with regard to skin where no attempt was made to separate dermis, epidermis and fur. Since absent or delayed hair growth is a part of the abnormality in irradiation chimeras, it seems likely that the amount of nitrogen included in our "skin" that is due to the protein content of the fur would be less in animals given homologous marrow than in control or isologous marrow-treated mice. Despite this factor tending to decrease the nitrogen content of "skin", the results show that the degree of shift of nitrogen from the general tissues to skin is sufficient to be detectable with a simple and relatively crude partition of the animals. may be These experiments constitute biochemical evidence that skin sa major target organ of the antihost activity of grafted foreign bone marrow cells. Three antihost activity constitutes a significant drain on the animal's metabolic resources as is evident by the decreased fraction of the total nitrogen of the chimera that is present in the carcass. The shift of nitrogen to a non-cellular integumentary compartment from muscle could result in loss of weight without loss of nitrogen from the animal. The evidence 18, therefore, consistent with this speculative explanation of the positive nitrogen balance without weight gain or in the presence of actua), weight loss (cf Fig. ID, ref. 4), exhibited by radiation chimeras. The molecular form of the extra nitrogen deposited in "skin" might be the proteins of antigen-antibody complexes. Hager et al. (8) bave presented direct evidence for antibody protein on the surface of cells of a homograft during rejection and have further indicated that antibody is also present on the host's cells as well. There is clear evidence for location of transplantation antigens on cell surfaces (9). The antibody bound to cells by surface antigens could constitute a large reservoir for extracellular protein. It would not be surprising if the grafted Immunological apparatus of the chimere, were reacting present they against skin since histopathologic evidence of damage to integumentary structures 18 frequently observed (10). Summary Irradiated mice treated with homologous bone marrow cells have a decreased fraction of their total-body nitrogen in the carcass and an increased fraction in skin. This shift in nitrogen distribution is detectable - 6 - as early as 4 days after irradiation and marrow treatment, but more definite at 25 days. The results are biochemical evidence that some integumentary I may be structure or structures a major target of the graft-versus-host reaction in radiation chimeras. Acknowle zment The interest and operated suggestions of Dr. J. R. Totter of the staff of the Division of Biology and Medicine, U. S. Atomic Energy Conmission, is gratefully acknowledged. REFERENCES 1. Congdon, C.C., in Tocantins, L.M., ed., Progress in Hematology, New York Grune, 1959, v. 2, 21. 2. Urso, I.S., Congdon, C.C., Owen, R.D., Proc. Soc. Exp. Biol. Med., 1959, v. 100, 395. 3. McArthur, W.8., Kretchmar, A.I., Congdon, C.C., Blood, 1963, v. 22, 503. 4. McArthur, W.8., Kretchmer, A.L., Congdon, C.C., Proc. Soc. Exp. Biol. Med., 1964, v. 117, 171, 5. Kretchmar, A.L., Congdon, C.C., Am. J. Physiol., 1961, v. 200, 102. 6. Suu, V-T., Congdon, C.C., Kretchmar, A.L., Proc. Soc. Exp. Biol. Med., 1963, v. 113, 481. Some 7. Ferrari, A., Ann. N.Y. Acad. Sci., 1960, v. 87, 792. 8. Hager, E.B., DuPwy, M.P., Wallach, D.F.H., Ann, N.Y. Acad. Sci., 1964, v, 120, 447. 9. Davies, D.A.L., in C.N.R.S. Conference on the Transplantation of Allogeneic Hemopoietic Cells, 1964, in press. 10. De Vries, M.J., in C.N.R.S. Conference on the Transplantation of Allogeneic Hemopoietic cells, 1964, in press. Fig. 1. Plot of nitrogen content of "carcass" against body weight for normal (a and b) and irradiated mice given isologous (c and a) or homologous bone marrow cells (e and f). In 6 groups (6 mice per group) of normal mdce the carcass content was 38.3 - 39.2 per cent of the total nitrogen of the animal. The lines in all the graphs are drawn with a slope of 1/11.25 and 1/13.75 g fat free body weight/mg. nitrogen. These lines indicate the area on the graph corresponding to the statement that' 12,5 4 1.25 mg of nitrogen per gram body veight 18 contained in the carcass of normal mice. BODY WEIGHT vs. CARCASS NITROGEN 4- DAY 25-DAY an . :) * NORMAL MICE NORMAL MICE 300 FAT-FREE BODY WEIGHT (grams) IBM MICE IBM MICE do 100 -300 HBM MICE HBM MICE 7% MICE to tattoo o NITROGEN (mg) NITROGEN (mg) AEC BIOMED.-ORINS Fig. 2. Plot of nitrogen content of "skin" against body weight for normal (a and b), and irradiated mice given 1Bologous (c and a) or homologous bone marrow cells (e and 1). Refer to legend of Fig. I for explanation of lines. Nitrogen content of "skin" was 38.5 - 40.2 per cent of the total nitrogen of the normal animals - 12.53 + 1.38 mg per gram of Mrithi body weight. BODY WEIGHT VS. SKIN NITROGEN 4-DAY 25-DAY NORMAL MICE FAT-FREE BODY WEIGHT (grams) s opě Ře or Repair IBM MICE 4100-300 нем MAM MICE MICE orta O 300 NITROGEN (mg) NITROGEN (mg) AEC BIOMED - ORINS Fig. 3. Plot of the ratio of "skin" to "carcass" nitrogen against body weight for normal (a and b), and irradiated mice given isologous (c and a) or homologous bone marrow cells (e and f). The lines indicate that this ratio 18 1.03 + 0.13 in normal mice. BODY WEIGHT VS. SKIN/CARCASS NITROGEN RATIO 4-DAY 25-DAY 26% * **** NORMAL MICE NORMAL L6 2.0 LLLL 1.0 .6 2.0 BODY WEIGHT (grams) bomino IBM MICE IBM MICE o 184 ...** : HBM MICE MICE HBM MICE : Lehe 416 20 140 1.6 20 SKIN/CARCASS N RATIO SKIN/CARCASS N RATIO AEC BIOMED. - ORINS END DATE FILMED 5 / 20 / 66 ST - _ - . - - * . . . . 1 . . .