Urinary excretion of purines, purine nucleosides, and pseudouridine in immunodeficient children

BIOCHEMICAI.

MEDICINE

27, 37-45 (1982)

Urinary Excretion of Purines, Purine Nucleosides, and Pseudouridine in lmmunodeficient Children’ GORDON C. MILLS,

Department of Human and Department

FRANK C. SCHMALSTIEG, ROBERT J. KOOLKIN, RANDALL M. GOLDBLUM

AND

Biological Chemistry and Genetics, Division of Biochemistry, of Pediatrics, The University of Texas Medical Branch, Galveston. Texas 77550

ReceivedJuly 16. 1981

Although defects in some enzymes of purine catabolic and salvage pathways have been associated with immunodeficiencies, a metabolic defect has still not been demonstrated in most immunodeficient children. Since some enzymatic disorders that have been associated with these diseases (adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, and possibly purine 5’-nucleotidase deficiency) are involved in pathways of purine interconversion and breakdown, we have studied a number of children with immunodeficiency disease to evaluate the possibility that other metabolic abnormalities in purine metabolism might be involved. In previous studies from this laboratory, a markedly increased urinary excretion of deoxyadenosine was noted in a child with adenosine deaminase deficiency (l-5). There were also some minor alterations in excretion levels of other urinary purines and nucleosides. Other workers have shown that there is a markedly increased excretion of nucleosides (inosine, guanosine, and their deoxy analogs) in purine nucleoside phosphorylase deficiency (6). In the present study, we have analyzed the urine of seven other children with severe combined immunodeficiency @CID) and of six other children with other immunodeficiency disease for the presence of the following compounds: deoxyadenosine, adenosine, adenine, pseudouridine, 7-methylguanine, N*,N*-dimethylguanosine’, hypoxanthine, and I Supported in part by the following: NIH Grant DHEW RR-0073-14, General Clinical Research Centers Branch, Division of Research Facilities and Resources: and Grant 6-130 from the National Foundation-March of Dimes. * The N*,Nz-dimethylguanosine peak in the elution profile also contains some @-methylguanosine (1530%). Since these two compounds are closely related, they are considered together in our data summaries, although listed as dimethylguanosine. 37 0006-2944/82/010037-09$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproductxon I” any form reserved.

3x

MIl>I,S ET AI

xanthine. Pseudouridine, 7-methylguanine, and dimethylguanosine are all urinary catabolites of minor bases of nucleosides of nucleic acids. MATERIALS Chemicals

AND METHODS

and Solutions

Analytic grade ion-exchange resins (AGl-X4 and AC50-X4, 200/400 mesh) were obtained from Bio-Rad Labotatories. Chloroacetaldehyde was prepared from the dimethylacetal derivative as described previously (3). All components of buffers were reagent grade, and their pH values refer to pH at 23”. For sodium acetate buffers, the listed concentration refers to the Na’ concentration. The sources of the purines, pyrimidines, and nucleosides used as standards have been given previously (1). Patients Information regarding the immunodeficient children of this study is summarized in Table 1. Studies on cytosine and erotic acid excretion in urine (2) and on adenosylhomocysteine excretion in urine (5) in many of these same children have been reported previously. Other studies on the germ-free SCID child have also been reported (7). TABLE IMMUNODEFICIENT

CHILDREN

1 IN THIS STUDY .~

Age

Sex

Uric acid/ creatinine”

S.M.F.” D.V.C.

6 Months 6-7 Years

F M

I .52 0.44

J.R.G” A.E.E. B.T.H. D.V.I.” R.M.J.

4-5 Months 1-2 Weeks 2 Months Under 6 months Under 6 months

M F M M M

2.54 0.66

L.L.A.

4 Months to 3.5 years 8-9 Years 2.5 Years 8 Years 11 Years 2.5 Years 1.3 Years

M

1.12

M

1.12 0.50 0.28 0.49 0.60 0.64

Child

J.R.K. J.L.L. A.B.O. E.B.M. V.M.N. J.S.P.

F M F F

1.60 1.21

Diagnosis SCID SCID, germ-free environment SCID SCID SCID SCID SCID. purine 5’-nucleotidase deficient SCID, adenosine deaminase deficient Wiscott-Aldrich

syndrome

Ataxia telangiectasia Cartilage-hair hypoplasia Pyruvate carboxylase deficiency

” The corresponding molar ratios for the 10 normal children were 0.65 5 0.32 (mean t SD). ’ These children died at 4-6 months of age.

PURINES

Ion-Exchange

Separative

AND

NUCLEOSIDES

IN URINE

39

Procedures

Prior to analytical ion-exchange separations, the urinary compounds were separated into two fractions on a short anion-exchange column at pH 10. Compounds with a negative charge at this pH are retained on the column while those with no charge pass through into the column wash (Fraction CW). The retained compounds are eluted (Fraction Ef with a small volume of acetate buffer, pH 5.2. This procedure has been described in detail previously (1). Adenosine, deoxyadenosine, and creatinine, in addition to other compounds, are found in fraction CW. Adenine, 7-methylguanine, and dimethylguanosine, in addition to other compounds, are in Fraction E. Uric acid is not eluted from the column by this procedure, so it is not present in either fraction. Most of the analytical separations of the components were carried out using either Fraction CW or Fraction E. Adenosine and deoxyadenosine (Fraction CW) were separated on a 0.5 x 20-cm cation-exchange column. H’ form, using sodium acetate buffers, 0.077 M, pH 4.8 and 5.2 as eluants. As a consequence of the column acidity, deoxyadenosine is converted to adenine and appears as the latter compound in the elution profile. Adenine (Fraction E) was separated in the same manner on a separate column. The adenine-containing peaks in elution profiles were detected by fluorescence assay after conversion to the etheno derivatives with chloroacetaldehyde. These separations have been described in detail previously (1) with a typical elution profile shown in Fig. 2 of that reference. 7-Methylguanine, dimethylguanosine, hypoxanthine, and xanthine were determined on a 0.5 x 40-cm cation-exchange column, H’ form, using an HCl gradient (O-l.5 N) for elution. 3-Methylxanthine, and 7-methylxanthine (derived from dietary or therapeutic methylxanthines) interfere with the xanthine determination by this ion-exchange procedure. When the presence of these methylxanthines in elution profiles was indicated by absorbance ratios, xanthine values were discarded. Adenine may also be determined in this separative procedure, but the fluorescence assay described above is a much more sensitive means of detection of adenine. This elution procedure, using HCl as eluant, has also been described in detail previously (1) with an example of an elution profile shown in Fig. 3E of that reference. Although there was a high adenine peak in that example, the adenine peak is barely visible in most urine specimens when ultraviolet absorbance measurements are utilized for detection of compounds. Pseudouridine was determined by anion-exchange analysis at a pH of ca. 10, after removal of other anionic and cationic components, using the procedure of Guri and Cole (8) with slight modifications. Additional details of this procedure, as applied in our laboratory, have also been

40

MILLS

ET

Al,

described previously (II. Uric acid was determined in the manner previously described (I). Creatinine in the CW fraction was determined either by elution from a cation-exchange column with HCI as described previously (1) or by elution from a cation-exchange column by sodium acetete buffer elution with absorbance measurements at 240 nm. The latter separation is illustrated in Fig. 3 of Ref. (2). In order to be assured of the correct identification of ultraviolet absorbing peaks in elution profiles, absorbance measurements were routinely made at 250, 260, 275, and 290 nm. The 250:260, 275:260, and 290:260 ratios provided a means of identification and an indication of purity for each of the peaks in elution profiles. In some instances we have also calculated the corresponding absorbance ratios at neutral pH or in NaOH for a more positive identification of eluted compounds. A millimolar absorbancy in HCI of 13.0 at 260 nm has been used for calculating amounts of N’,N’-dimethylguanosine (9). RESULTS

Since it appears likely that there are multiple causes of severe combined immunodeficiency, we have shown results on individual subjects in Table 2 with excretion values expressed in relation to creatinine excretion. Four of the seven SCID children had excretion values for at least one compound greater than 2 SD above the mean for the normal children. Of these SCID children, one child (J.R.G.) had significantly increased excretion of five of the compounds. In contrast, the germ-free SCID child (D.V.C.) excreted less than 60% of the normal mean value for each of the six compounds shown in these tables. Excretion levels of all compounds in the other immunodeficient children fell within the normal range. For comparison, we have also shown excretion levels of these same compounds in an adenosine deaminase-deficient child (L.L.A.) with SCID, and also in an adenosine deaminase-deficient adult female (N.T.U.), who was not immunodeficient. Much of the data for the SCID child, L.L.A. have been published previously (I), while other studies on the adenosine deaminase deficient adult will be presented in a separate communication.3 Since the values in Table 2 are expressed in relation to creatinine excretion, and the latter is related to muscle mass, we have expressed excretion values for the SCID children in a different manner in Fig. I. In this figure, the values are expressed in relation to excretion of uric acid plus creatinine. This reduces part of the possible bias introduced by the fact that normal children are healthier than SCID children and hence often have a greater muscle mass. We have also included data for ’ F. C.

Schmalstieg

ct ul..

in

preparation

5.5 15.1 5.8 5.9 12.3 14.2

16.5 k 2.7

5.3 9.5 2 6.1

Other immunodeficient J.R.K. (2) J.L.L. A.B.O. E.B.M. V.M.N. J.S.P.

SCID-ADA deficient L.L.A. (7)

ADA deficient adult (NTU)’ Normal children (8)

PURINE

TABLE

39.5 66.7 e 22.2

125 -t 14

84 88 34 36 80 95

193” 36 157 50 88 104 79

4dJ/Cr

OF PURINES.

2

4.0 7.6 k 5.9

29.5”

7.7 6.3 4.5 7.1 8.6

12.6 2.7 13.8 10.3 12.9 17.2 13.7

DiMeGuoiCr

NWLF.OSIDES,

AND

1.95 2.25 2 2.44

5.83 2 3.80

1.52 0.47 0.53 3.03 3.31

3.71 0.10 9.72” 0.81 0.36 5.41 2.73

Ade/Cr

PSE~WIIRIDINF:

0.12 1.04 2 0.42

3.96 2 1.19

0.89 1.25 0.42 1.17 0.80

1.19 0.55 2.00h 1.80 1.84 2.52” 1.48

AdoiCr

Note. Numbers in parentheses indicate number of urine specimens analyzed. Results are expressed as nmole compound/kmole Abbreviations: Cr, creatinine; ADA, adenosine deaminase; SCID. severe combined immunodeficiency. For others see Fig. 1 legend. * Change of > 3 SD from mean for normal children. h Change of 2-3 SD from mean for normal children. ’ Studies made prior to a transfusion treatment regimen; only three samples were analyzed for DiMeGuo. ’ Not immunodeficient.

19.3 2.4 36.3 23.2b 20.3 23.4h 13.8

7-MeGua/Cr

EXCRETION

SCID-ADA normal S.M.F. D.V.C. (2) J.R.G. (2-3) A.E.E. B.T.H. D.V.I. (2) R.M.J. (3)

Subjects

URINARY

creatinine.

1.41 0.76 t 1.13

45.3 2 16.6

0.31 0.71 0.86 0.55 0.55 0.51

0.57 0.26 6.80” 0.53 0.57 1.18 0.47

dAdo/Cr

P

42

I

c

‘23456c

1

Ill ‘6c

FIG. 1. Excretion of pyrimidine and puke nucleosides and purines in six children with severe combined immunodeficiency. The excretion values were first calculated as Z(nmole compound)/(bmole creatinine + kmole uric acid). The figure shows excretion values for each SCID subject expressed as percentage of the excretion noted in normal children. The SCID children were: 1, S.M.F.; 2, D.V.C.: 3, J.R.G.; 4, A.E.E.: 5, D.V.I.: and 6, R.M.J. The mean value (n = 8-10) with 1 SD is indicated for the control subjects (designated by C). Abbreviations: 7-MeGua, 7-methylguanine; Di-Me-Guo. dimethylguanosine; Ade. adenine; Ado, adenosine; dAdo, deoxyadenosine; AdoHcy. adenosylhomocysteine; Hx, hypoxanthine; Cyt. cytosine.

adenosylhomocysteine (5) and cytosine (2) and have expressed the data in this figure in percentage of control values, in order to give an excretion profile for each of the SCID children. The values are qualitatively similar to those noted in Table 2, although the significance of several values is reduced. SCID child, J.R.G., shows elevated excretion levels for 7-methylguanine, deoxyadenosine, adenosylhomocysteine, and cytosine; S.M.F. and D.V.I. have elevated values for pseudouridine and cytosine; A.E.E. and D.V.I. have elevated values for adenosine; and S.M.F. has an elevated value for adenosylhomocysteine. The germ-free SCID child (D.V.C.) again shows consistently low values for excretion of these compounds. In Table 3, we have considered excretion values for the SCID children as a group and have made statistical comparisons with excretion levels found in normal children. Excretion values in this table are again related to the sum of uric acid and creatinine excretion values. The germ-free SCID child (D.V.C.) has been omitted from these statistical evaluations for reasons that will be discussed subsequently. Although mean excretion values in SCID children are higher than normal for six of the eight compounds, only 7-methylguanine is excreted in significantly increased amounts when the data are expressed in this manner. The six children with other immunodeficiencies did not show significant variation from the normal mean in excretion levels of the eight compounds summarized in Table 3.

PURINES

AND NUCLEOSIDES TABLE

EXCRETION

IN URINE

OF PURINES.

Pseudouridine 7-Methylguanine Dimethylguanosine Deoxyadenosine Adenosine Adenine Hypoxanthine Xanthine

103.2 19.0 11.14 1.15 1.48 3.15 19.3 21.6

3

PURINE NWLEOSIDES.

SCID 2 36.5 (5) 2 1.70b(S) I 1.75 (5) + 1.22 (5) 2 0.56 (5) 2 1.66 (5) 2 4.7 (5) ? 11.9 (5)

43

IN URINE

AND PSEUDOURIDINE

Other immunodeficiencies 69.5 12.3 8.9 0.75 1.06 2.21 19.3 24

-c t IT k 2 + t -

27.2 (6) 5.1 (6) 1.7 (5) 0.35 (6) 0.40 (5) 1.60(5) 6.3 (6) 30 (2)

Normals 79.4 10.2 8.5 0.81 1.19 2.40 18.7 23

t + 2 c 2 2 ?z -

16.2 (IO) 5.7 (IO) 5.0 (8) 1.02 (8) 0.31 (7) 2.35 (9) 10.5 (8) 32 (3)

” The ADA-deficient child (L.L.A.) and the germ-free child (D.V.C.) have not been included in these comparisons. Values are expressed as 2 @mole compound)/(pmole creatinine + pmole uric acid) t SD. Numbers in parentheses indicate the number of children in each group. b Significantly different from normal with a P value of 0.005: other values do not differ significantly (P > 0.05).

DISCUSSION The types of urinary components that we have studied may be grouped as follows: (a) those that are excretory products formed as a consequence of breakdown of minor nucleosides of ribonucleic acids (pseudouridine, 7-methylguanine, and dimethylguanosine); (b) products preceding uric acid in the normal catabolic pathway for purines of nucleotides and nucleic acids (hypoxanthine and xanthine); and (c) adenine or adeninecontaining compounds that are involved in salvage pathways or in interconversion reactions between various metabolic pathways (adenine, adenosine, deoxyadenosine) (cf. Fig. 4 of (1)). We have previously studied excretion levels of three other compounds in urine of immunodeficient children. Cytosine excretion was found to be elevated in a number of children with severe combined immunodeficiency, while erotic acid excretion values were not appreciably altered from normal (2). Adenosylhomocysteine, an intermediate of the adenosylmethionine pathway, has also been studied in these same SCID children, with three of the children showing significantly increased urinary excretion levels (5). In order to provide a more complete picture of excretion patterns in the SCID children, we have included adenosylhomocysteine and cytosine data in the profiles of Fig. 1. SCID child, J.R.G., showed the most dramatic increase above normal in excretion levels of the various compounds that have been studied. We suspect that these increased excretion levels may have been due to an increased turnover of nucleic acids, possibly as a consequence of infection, since J.R.G. had disseminated cryptococcosis. In contrast, excre-

44

MILLS

ET AL.

tion levels of these compounds in the germ-free SCID child, D.V.C.. were generally much below mean normal values. A comparison of excretion in these two subjects suggests that microorganisms may, directly or indirectly, contribute to the urinary components. Death of microorganisms, either in the tissues or in the gastrointestinal tract, could produce some breakdown of nucleic acids. Some of the products in the gastrointestinal tract could be absorbed, metabolized, and excreted in the urine. Another possible consequence of tissue infections would be increased cell death of the host tissue, with a corresponding increase in rate of breakdown of tissue nucleic acids. In addition. increased metabolic activity in tissues due to the infection could result in greater production of these purine breakdown products. In our statistical evaluations of SCID children as a group (Table 3). the germ-free child (D.V.C.) has been omitted since his excretion values were consistently lower than those of normals and of other SCID children. This means that our comparisons in Table 3 were between healthy normal children and SCID children who were subjected to repeated infections despite the use of antibiotics. Consequently, the significantly elevated value for 7-methylguanine excretion noted in the SCID children as a group may be reflection of an increased RNA turnover due to the presence of infection. 7-Methylguanine is derived primarily from the breakdown of messenger RNA and transfer RNA. The children with other immunodeficiencies. as a group, did not have as severe problems with infection as the SCID children, and excretion levels for the former did not differ from the normal values (Table 3). The degree of change in excretion levels noted in this study does not appear to be great enough to suggest a metabolic defect in purine metabolism in the SCID children other than the adenosine deaminase deficiency noted in subject L.L.A. The studies do suggest a word of caution, when infected children are compared with healthy children, regarding the interpretation of excretion data for these catabolic products of nucleic acids. SUMMARY Studies have been carried out using ion-exchange analysis for determination of urinary purines, pyrimidines, and nucleosides in children with immunodeficiency disorders. Using cation and anion-exchange techniques, the following compounds of urine have been quantitatively determined: deoxyadenosine, adenosine, adenine, pseudouridine, 7-methylguanine, N2,N2-dimethylguanosine, hypoxanthine, and xanthine. Excretion levels of these compounds did not differ significantly from normal values in six children with various immunodeficiency diseases, excluding severe combined immunodeficiency. However. of the seven children

PURINES AND NUCLEOSIDES

IN URINE

45

studied with severe combined immunodeficiency disease (normal adenosine deaminase), four showed increased excretion levels for one or more of the compounds studied. A germ-free child with severe combined immunodeficiency had lower excretion levels than the mean normal value for most of these same compounds. The possibility is considered that the increased excretion levels noted may be a consequence of repeated episodes of infection in most children with severe combined immunodeficiency . ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Katherine Newkirk for a portion of these studies; also the cooperation of Drs. Rafael Wilson and Buford Nichols, Texas Childrens Hospital, Houston. in securing urine from two of the SCID children.

REFERENCES 1. Mills. G. C., Goldblum. Med.

R. M., Newkirk.

K. E.. and Schmalstieg, F. C.. B&hem.

20, 180 (1978).

2. Mills, G. C., Schmalstieg, F. C., Newkirk. K. E.. and Goldblum, R. M.. C/in. Chem. 25, 419 (1979). 3. Mills, G. C., Schmalstieg, F. C., Trimmer, K. B.. Goldman. A. S., and Goldblum, R. M., Proc. Nat. Acad. Sci. USA 73, 2876 (1976). 4. Schmalstieg, F. C.. Mills, G. C., Nelson, J. A., May, L. T., Goldman, A. S.. and Goldblum, R. M., J. Pediat. 93, 597 (1978). 5. Mills, G. C., Foster, N. G.. and Goldblum, R. M., Biochem. Med. 26, 90-105 (1981). 6. Cohen, A., Doyle, D.. Martin, D. W.. and Ammann. A. J.. N. Engl. J. Med. 295, 1449 (1976). 7. Mukhopadhyay, N., Richie, E., Mackler, B. F.. Montgomery, J. R.. Wilson, R.. Fernbath, D. J., and South, M. A., Exp. Hematol. 6, 129 (1978). 8. Guri, C. D., and Cole, L. J., C/in. Chem. 14, 383 (1968). 9. Hall, R., “The Modified Nucleosides in Nucleic Acids.” Columbia Univ. Press, New York, 1971.