Kathleen R. Ambrose

New radioiodinated methyl-branched fatty acids for cardiac studies

The effects of 3-methyl substitution on the heart retention and metabolism of 3-R,S-methyl-(BMIPP) and 3,3-dimethyl-(DMIPP) analogues of 15-(p-iodophenyl)-pentadecanoic acid (IPP) were studied in rats. Methyl substitution considerably increased the myocardial half-time values in fasted rats: IPP, 5–10 min; BMIPP, 30–45 min; DMIPP, 6–7 h. Because of the observed differences in the relative myocardial uptake and retention of these agents, an evaluation of the subcellular distribution profiles and the distribution of radioactivity within various lipid pools extracted from cell components was performed. Studies with DMIPP in food-deprived rats have shown high levels of the free fatty acid and only slow conversion to triglycerides. These data are in contrast to the rapid clearance of the straight chain IPP analogue and rapid incorporation into triglycerides, and suggest that the prolonged myocardial retention observed with DMIPP in vivo may result from inhibition of β oxidation. Subcellular distribution studies have shown predominant association of DMIPP and BMIPP with the mitochondrial and microsomal fractions, while IPP was primarily found in the cytoplasm. Because of the unique “trapping” properties and the high heart: blood ratios, [123I]DMIPP should be useful for evaluation of aberrations in regional myocardial uptake.

Evaluation of the metabolism in rat hearts of two new radioiodinated 3-methyl-branched fatty acid myocardial imaging agents.

The biological fate of two new radioiodinated 3-methyl-branched fatty acids has been evaluated in rat hearts following intravenous administration. Methylbranching was introduced in [15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPP) to inhibit β-oxidation. The goals of these studies were to correlate the effects of methyl-branching on the incorporation of these agents into the various fatty acid pools and subcellular distribution profiles, and to relate these data to the myocardial retention properties. The properties of BMIPP and DMIPP were compared with the 15-(p-iodophenyl)pentadecanoic acid straight-chain analogue (IPP). Differences in the heart retention of the analogues after intravenous administration in rats correlated with differences observed in subcellular distribution patterns. The dimethyl DMIPP analogue showed the longest retention and the highest association with the mitochondrial and microsomal fractions (34%, 38%) 30 min after injection. These data are in contrast to the rapid clearance of the straight-chain IPP analogue which showed much lower relative association with the mitochondria and microsomes (18%, 15%). The distribution patterns of each analogue in the various lipid pools appeared consistent with the expected capacity of the analogues to be metabolized by β-oxidation. In contrast to the rapid oxidation of the straight-chain IPP analogue, the 3-monomethyl BMIPP analogue appeared to undergo slower oxidation and clearance, whereas the dimethyl-branched DMIPP analogue was apparently not catabolized by the myocardium. All three analogues showed some incorporation into triglycerides. The metabolism patterns of the branched analogues reported here may provide useful information in the description of the mechanisms by which BMIPP and DMIPP are retained in rat myocardium.

INDUCTION OF TUMOR-SPECIFIC TRANSPLANTATION IMMUNITY IN HAMSTERS WITH CELL FRACTIONS FROM ADENOVIRUS AND SV40 TUMOR CELLS.

Summary The specific induction of transplantation immunity against adenovirus 31-and SV40-induced hamster tumor cell challenge employing either a crude membrane fraction of homologous adenovirus 31 tumor cells or SV40 tumor cell fragments is described. In each case immunity was equivalent or superior to that induced by whole, irradiated tumor cells previously described to contain TSTA, or to that induced by vaccination of mature hamsters with the live virus. Whole tumor homogenate of adenovirus 31 hamster cells, from which the membrane fraction was obtained failed to induce TSTA immunity. The possibility exists that a masking or enhancing component is present in the homogenate to interfere with TSTA immunity induction or detection. Addendum. Hamsters immunized with SV40 tumor cell ghost lysates were uniformly resistant to an initial cell challenge as shown in Table III. Several months later the survivors were challenged a second time and 42% of the hamsters developed tumors by day 50. One hundred percent of normal control hamsters similarly challenged developed tumors at this time. The authors are grateful to Mrs. Betty W. Harrell of Oak Ridge National Laboratory for the skillfully prepared electron micrographs of the cell ghosts, and to E. L. Candler, Oak Ridge National Laboratory, for technical assistance.

Effect of 3-methyl-branching on the myocardial retention of radioiodinated 19-iodo-18-nonadecenoic acid analogues

The effect of methyl-branching at the 3(beta-)-position on myocardial uptake and retention of fatty acids where radioiodide has been stabilized as a terminal trans-(E)-vinyl iodide has been evaluated in fasted rats. The syntheses of two new dimethyl-branched fatty acids, 17-iodo-3,3-dimethylheptadecanoic acid (14) and (E)-19-iodo-3,3-dimethyl-18-nonadecenoic acid (19), are described. Tissue distribution studies in fasted rats with [125I]-19 showed significant heart uptake (2 min, 4.56% dose/g), and prolonged retention (60 min 4.10% dose-g). These results suggest that [123I]-19 is a good candidate for further studies of regional myocardial fatty acid uptake patterns by SPECT.

Effects of fasting on the myocardial subcellular distribution and lipid distribution of terminal p-iodophenyl-substituted fatty acids in rats.

The myocardial lipid pool distribution and subcellular distribution of radiolabeled methyl-branched fatty acids in rats was evaluated under conditions of fasting (24 h) and feeding. With the unbranched iodophenyl fatty acid, fasting resulted in increased myocardial extraction and clearance time with a decrease in the incorporation into triglycerides and greater radioactivity in the mitochondrial fraction. With the monomethyl-branched analogue, the effects of fasting on lipid and subcellular distribution were minor except for a decrease in triglyceride incorporation. Like the unbranched analogue, the dimethyl-branched iodophenyl fatty acid showed increased myocardial extraction with fasting, however, this structurally-modified fatty acid showed increased rather than decreased incorporation into triglycerides.

Phase Specific Surface Autoantigens on Membranes of Fetus and Tumors

In the past two years, in collaboration with several colleagues, we have demonstrated the presence of antigenic determinants at the surface of SV40, adenovirus, polyoma tumors of hamsters, spontaneous and chemically induced tumors of mice (1–2) which are transiently present in fetal tissues but absent from adult tissues. In other laboratories chemically induced mouse tumors (3) and Maloney sarcoma tumor cells (4) also have demonstrable fetal or phase specific autoantigens (PSAA). The prospect has arisen that many and perhaps all cancers share antigens commonly present on the developing fetal membranes and that retrogenic phasing may be essential to the malignant process. Several major forms of human cancer have embryonic constituents reexpressed as exemplified by the alphafeto-protein of hepatomas or the carcinoembryomic antigen of cancers of endodermal origin.

Both total chain length and position of dimethyl-branching effect the myocardial uptake and retention of radioiodinated analogues of 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPP)

Introduction of geminal dimethyl-branching into the 3-position of 15-(p-iodophenyl)pentadecanoic acid (IPPA) significantly delays myocardial clearance in rats and dogs following intravenous administration. Several new analogues of DMIPP have been synthesized and evaluated in fasted rats. The effects of both the position of dimethyl-branching and the total chain-length of 3,3-dimethyl analogues on heart uptake and clearance kinetics have been studied. In the first series of compounds, two methyl groups were introduced into the 3-, 4-, 6-, or 9-position. Tissue distribution studies of the 15-(p-[I-125]iodophenyl)-analogues demonstrated that the position of dimethylbranching is an important factor affecting both myocardial specificity and retention. The [I-125]labeled 3,3- and 4,4-DMIPP analogues showed higher myocardial uptake and faster blood clearance than the 6,6- and 9,9-DMIPP analogues [heart, % dose/gm (heart : blood), 30 min: 3,3-DMIPP = 5.06 (12 : 1); 4,4-DMIPP = 8.03 (16.7 : 1); 6,6-DMIPP = 2.26 (3.1 : 1); 9,9-DMIPP = 3.06 (2.77)]. In the second series, the effects of total fatty acid chain length were evaluated with 3,3-dimethyl-substituted analogues with C11, C12, C13, C14, C15, and C19 chain lengths. The C14 and C15 chain length analogues showed the best properties [global heart uptake (heart : blood ratios): 30 min: C11, 0.70 (0.82); C12, 1.25 (0.68); C13, 0.47 (0.90); C14, 1.63 (3.54); C15, 5.06 (12); C19, 1.29 (0.82). These detailed studies have demonstrated that both total chain length and the position of geminal dimethyl-branching are important structural parameters which affect myocardial specificity and retention of ω-(p-iodophenyl)-substituted fatty acid analogues and that 3,3-DMIPP and 4,4-DMIPP are the best candidates with optimal properties for further study.

Evaluation of pancreatic lipase activity by simple urine analysis after oral administration of a new iodine-131-labeled triglyceride

A new iodine-131-labeled triglyceride analogue called “MIPAG” [1,2-dipalmitoyl-3-[(15-p-iodophenyl) pentadecan-1-oyl]rac-glycerol] has been prepared in which 15-(p-iodophenyl)pentadecanoic acid (IPPA) is attached to position-3. MIPAG has been developed for the evaluation of pancreatic exocrine function by simple urine analysis and has been evaluated in rats and humans. After oral administration, IPPA is released from the triglyceride by the action of pancreatic lipases followed by intestinal absorption and the principal IPPA metabolite (p-iodobenzoic acid, IBA) is primarily excreted in the urine. Excretion in the urine and feces was evaluated in rats, as well as the biodistribution in various organs over 21 days. Twenty patients without pancreatic disease (normals) and four patients with pancreatic insufficiency were also investigated. Following oral administration of 30 μCi of MIPAG, urine was collected for two successive 24-h periods. Blood samples were drawn and thin-layer chromatographic (TLC) analysis was performed on the serum lipid extracts. Urine from normals contained 44.9%±7.7% and 61.8%±8.4% of the administered activity after 24 and 48 h, respectively. The patients with pancreatic insufficiency excreted 13.1%±5.6% and 18.9%±6.2%, respectively, which was significantly decreased (P<0.001) compared with normals. The TLC profiles showed an increasing proportion of IBA with time. Urine analysis after oral administration of MIPAG thus appears to be an attractive new technique for the evaluation of pancreatic lipase activity by a simple urine analysis.