Abstract
Extract: Peripheral blood lymphocytes from a 33-year-old male with citrullinemia were established in long term, continuous culture. This lymphocyte line was designated UM-21 (University of Michigan, lymphoid line no. 21), and the behavior of the cells of this line was studied under selective conditions in vitro. When arginine was removed from the medium and replaced with citrulline, the UM-21 lymphocytes were unable to enter logarithmic growth phase. This contrasted with lymphocyte lines from normal donors which were able to utilize the citrulline readily and to grow logarithmically. We interpreted this as presumptive evidence that, unlike normal lymphocytes, the citrullinemic lymphocytes were deficient in argininosuccinic acid synthetase (AS). Despite the initial inability of the citrullinemic lymphocytes to grow logarithmically in a medium with 200 mg/liter citrulline substituted for the normally used arginine (Cit+, Arg− medium), after 4 weeks of continuous incubation some cells did begin to divide and a variant of the UM-21 line was subsequently established.
Cells from the parental UM-21 line, the variant line, and two normal lymphocyte lines were incubated with ureido-14C-citrulline to determine their relative uptake and incorporation of citrulline. On autoradiography, the UM-21 cells showed no label. Cells from the variant line showed a diverse labeling pattern: 11.7% of cells were heavily labeled, 32.4% were lightly labeled, and 55.9% showed no label. The cells from the normal donors were uniformly heavily labeled. Further, the incorporation of label into trichloroacetic acid-precipitable cellular material was examined. UM-21 incorporated 360 cpm, the variant line 1,951 cpm, and the two normal lines 2,544 and 2,427 cpm, respectively. The counts per minute are here expressed per 105 cells, or 3.3–3.5 × 10−3 mg protein.
Speculation: Although a more sensitive assay for AS will have to be developed for lymphocyte lines, it is apparent from the data presented here that the citrullinemic defect is expressed in cultured lymphocytes under proper selective conditions. The basic defect in this disease is likely to be either a regulatory or structural gene mutation, and the variant line may represent a reverse mutation. It may prove possible to study each of the four known genetically determined disorders of urea metabolism by use of the cultured lymphocyte.
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Spector, E., Bloom, A. Citrullinemic Lymphocytes in Long Term Culture. Pediatr Res 7, 700–705 (1973). https://doi.org/10.1203/00006450-197308000-00005
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DOI: https://doi.org/10.1203/00006450-197308000-00005
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