Purine metabolism in adenosine deaminase deficiency.

Abstract

Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides.Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), an enzyme that is widespread in animal tissues, plays a key role in (a) the normal catabolic pathway for adenine nucleotides, and (b) the production of hypoxanthine for use in the purine salvage pathway (Fig. 1). Intense interest has centered on this enzyme since Giblett et al. (1) described the association of its deficiency with an autosomal recessive immunodeficiency. The disease is characterized by serious infections, severe thymusdependent (T) lymphocyte and variable bone marrow-dependent (B) lymphocyte deficiencies, and characteristic skeletal abnormalities. This disease is fatal unless the immune system of affected patients is reconstituted. While the clinical and genetic aspects of this disorder have been well described (2-6), the relationship between the enzyme deficiency and the immunodeficiency is not understood. Although recent evidence suggests a causal relationship (7), elucidation must ultimately depend upon the measurement of key metabolites and enzyme activities in the affected purine and pyrimidine pathways.The identification and quantitation of adenine and adenosine in blood has been difficult because the amounts of these substances are low and adenine nucleotides are relatively high within erythrocytes. The recent development of a sensitive fluorescence assay for adenine-containing compounds has enhanced the detection of these compounds in red cells. In the present study, various ion exchange procedures were used for preliminary separations, and adenine-containing compounds were identified and quantitated using either ultraviolet absorption or fluorescence spectra. With these techniques, we have studied changes in purine and pyrimidine pathways in an adenosine deaminase deficient individual. MATERIALS AND METHODS

Purine metabolism in cultured human fibroblasts derived from patients deficient in hypoxanthine phosphoribosyltransferase, purine nucleoside phosphorylase, or adenosine deaminase.

“…Additional experiments need to be designed which will allow alterations in the intracellular concentrations of potentially inhibitory nucleotides independently from changes in the concentration of PP-ribose-P, so that the relative importance of these two factors in controlling the rate of purine synthesis de novo may be evaluated. Our finding that neither methylene blue nor inosine increased the rate of ["4C]formate incorporation into purines in Lesch-Nyhan cells is consistent with Hershfield's (29) findings that indicate that there may be conditions where the concentration of PP-ribose-P is not rate-limiting for purine synthesis de novo (12,27 broblasts is consistent with the observation that patients who lack ADA do not overproduce purines de now (20) and suggests that adenosine can be effectively metabolized via adenosine inase. In conclusion, we feel that purine overproduction in the above mutations can best be demonstrated by the resistance of purine synthesis de novo to potentially inhibitory purine nucleosides and bases, rather than by the absolute rate of incorporation of ["4C]formate into purines.…”

“…Fibroblasts were grown from foreskins obtained from apparently normal male infants or from skin biopsies obtained from normal volunteers or from patients deficient in HPRT, ADA (20), or PNP (3). Fibroblast cultures were routinely maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 ,ug/ml).…”

“…ADA deficiency leads to a severe combined immunodeficiency with T-cell and usually B-cell dysfunction (19). No purine overproduction has been found in patients lacking ADA (20).…”

Severe Combined Immunodeficiency in a Child with a Healthy Adenosine Deaminase Deficient Mother

“…The mother had less than 1% of normal ADA activity in both erythrocyte and lymphocyte extracts, but her whole peripheral blood lymphocytes demonstrated about 6% of normal activity. (7,8,10,18,19,21,24). Individuals with this form of SCID invariably have profound deficiencies of ADA activity in both erythrocytes and lymphocytes (1 2); however, there are reports of individuals with partial deficiencies of this enzyme who apparently have normal or near normal function of the immune system (3,11,13).…”

Plasma deoxyadenosine, adenosine, and erythrocyte deoxyATP are elevated at birth in an adenosine deaminase-deficient child.

“…ADA-deficient children accumnulate the substrate adenosine in their plasma and erythrocytes, and excrete moderately increased amounts of adenosine and markedly increased amounts of deoxyadenosine, another substrate of ADA, in their urine (4)(5)(6)(7)(8)(9)(10)(11). Additionally, deoxyATP (dATP), a metabolite of deoxyadenosine, is markedly increased in erythrocytes and lymphocytes (7,(10)(11)(12)(13).…”